5. Micropatterning and microelectrochemical characterisation of

Transcription

MICROPATTERNING AND MICROELECTROCHEMICAL
CHARACTERISATION OF BIOLOGICAL RECOGNITION
ELEMENTS
Dissertation
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
der Fakultät für Chemie der
Ruhr-Universität Bochum
Vorgelegt von
Eugen Florin Turcu
Bochum, July 2004
This work was carried out between April 2001 and July 2004 at Lehrstuhl für Analytische
Chemie, AG Elektroanalytik & Sensorik under the supervision of Prof. Dr. W. Schuhmann.
Tag der mündlichen Prüfung
20. Juli 2004
Referent
Prof. Dr. W. Schuhmann
Korreferent
Prof. Dr. W. S. Sheldrick
Prüfer
Prof. Dr. C. Wöll
Contents
1. Introduction: Why going smaller?
2. Methods microstructuring
2.1 Lithography
4
2.1.1. Photolithography
4
2.1.2. Soft-lithography
6
2.1.3. Other methods for microstructuring
8
2.2. Ink-jet printing/lithography – the flow through piezo microdispenser
9
3. Tools for probing microstructures
3.1 Scanning Probe Microscopy (SPM)
12
3.2 Scanning Electrochemical Microscopy (SECM)
14
3.3 Notes
25
4. Electrodes for electrochemistry in small volumes
4.1 Integrated working/reference assembly
27
4.1.1. Preparation of precursor electrode
28
4.1.2. Chemical deposition of silver onto
30
the body of precursor electrodes
4.1.3. Application of the coaxial Pt-µWE/Ag-RE in SECM
4.2 Miniaturised Ag/AgCl reference electrode
34
39
5. Micropatterning and microelectrochemical
characterisation of biological recognition elements
5.1 Enzyme microstructures
42
5.1.1 About enzymes
44
5.1.2 Glucose oxidase (GOD)
51
5.1.3 Patterning of GOD by means of piezo microdispenser
53
5.1.4 Visualisation of GOD microstructures by SECM
62
5.2 Defined adhesion/growth of living cells
5.2.1 Introduction
71
72
5.2.2 What is available so far?
73
5.2.3 Results and discussion
74
5.2.4 Conclusions
79
5.3 DNA microstructures
80
5.3.1 DNA microarrays
82
5.3.2 Detection of DNA hybridisation –
87
What are the options?
5.3.3 The repelling mode of SECM –
97
A new and promising assay for imaging
DNA microarrays and detecting DNA
hybridisation
5.3.4 Detection of DNA hybridisation in
118
the repelling mode of SECM
5.3.5 Conclusions and outlook
124
5.4 Notes
127
6. Experimental
131
7. Conclusions
136
8. Acknowledgment
139
9. References
140
10. Curriculum vitae
157
PUBLICATIONS
•
Florin Turcu, Albert Schulte, Gerhard Hartwich, Wolfgang Schuhmann. “Label-free
electrochemical recognition of DNA hybridisation by means of modulation of the
feedback current in SECM”, Angew. Chem. Int. Ed., 2004, 43, 3482-3485.
•
Florin Turcu, Karla Tratsk-Nitz, Solon Thanos, Wolfgang Schuhmann, Peter
Heiduschka. “Ink-jet printing for micropattern generation of laminin for neuronal
adhesion” J. Neurosci. Methods, 2003, 131, 141-148.
•
Albert Schulte, Mathieu Etienne, Florin Turcu, Wolfgang Schuhmann. “High
resolution constant distance scanning electrochemical microscopy on immobilised
enzyme micropatterns” G.I.T. Imaging and Microscopy, 2003, 5, 46-49.
•
Florin Turcu, Albert Schulte, Gerhard Hartwich, Wolfgang Schuhmann. “Imaging
immobilised ss-DNA and detecting hybridisation by means of the repelling mode of
scanning electrochemical microscopy (SECM)”, Biosens. Bioelectron., 2004, accepted.
•
Florin Turcu, Albert Schulte, Wolfgang Schuhmann. “Scanning electrochemical
microscopy (SECM) in nanolitre droplets using an integrated working/reference
electrode assembly“, Anal. Bioanal. Chem., 2004, submitted.
•
Anh Nguyen, Jane Hübner, Florin Turcu, David Melchior, Hans-Willi Kling, Siegmar
Gäb, Oliver J. Schmitz. “Analysis of alkyl polyglicosides by capillary electrophoresis
with pulsed-amperometric detection”, Electrophoresis, 2004, submitted.
Why going smaller?
Micropatterning and microelectrochemical
characterisation of biological recognition elements
1.
Introduction: Why going smaller?
A question that often comes in mind when reading the title of a new book or journal article,
is “Why this one? Does it say enough about the content of that writing?” Well, most of the
books have very general titles. Let us consider two of them that are widely spread:
“Analytical Chemistry”, “History of …” and many others that have something in common:
they all speak about something, which somehow can be related to this general topic.
Analytical Chemistry explains what an analysis means and how many types we know, how
to perform precise measurements and how to get rid of different errors that could appear
besides, many theoretical and technical insights. Each book has its main content, which
follows the most important ideas of the subject it is speaking about. Authors have found an
open field to express their own thoughts and views about all the things they are writing
about. This gives the explanation why book’s contents look so different even if they are
dealing with the same topic.
This thesis is entitled “Micropatterning and microelectrochemical characterisation of
biological recognition elements” and may suggest to the reader a survey of all kinds of
biological microscopic structures: how to make and how to use them. What, however,
should be expected from this piece of work? In fact, it is tackling the micropatterning of
biological recognition elements. In particular, these were an enzyme, nucleic acids, and
living cells (in particular neurons). A piezoelectric microdispenser has been used as
microprinting device to create complex enzyme structures on different materials and also
was
used
to
prepare
oligonucleotide
microarrays. Furthermore,
the Scanning
Electrochemical Microscope (SECM) has been chosen as the main tool for exploring the
obtained microstructure and for imaging their local (electro)chemical properties with high
spatial resolution.
Before going into details I would like to raise a question: Why do we need
microstructures? If you answer: because we need miniaturisation, my questionnaire moves
further to the next and most important question: Why do we need miniaturisation? There
are unlimited possibilities to answer. However, a clear example for the need of
1
Why going smaller?
miniaturisation can be found with developing computers, tools that everyone loves when
they are small and very efficient in doing lots of work for us.
The long history of mechanical calculators has its roots in the Antiquity when the “abacus”
was the computing tool. Top leading scientists were later involved in the development of
these sophisticated machines: Leonardo da Vinci, Napier (the inventor of logarithms),
Wilhelm Schickard, Blaise Pascal, Gottfried Wilhelm von Leibniz, just to mention the
most famous of them.
Pascal built several mechanical machines, named “Pascalines” for helping his father in
adding and subtracting large sequences of numbers (he was a tax collector). Instead of
abacus and “Neper’s bones“ all other machines were quite big. In the 20th century the
basics of electronic computing systems were about to be discovered and developed by
Alan Turing and John von Neumann. The first representative electronic computer able to
carry out general-purpose computations was borna
in USA in 1946. Its name is ENIAC (Electronic
Numerical Integrator And Computer) (Figure 1).
Just by judging its dimensions and capability one
can conclude that it had been a real “Dinosaur” as
it occupied a large surface (about 1000 square
feetb) for a low computational power (for instance
5000 additions per second). Nevertheless, I
suppose it had been made in the smallest format
possible at that time. About 50 years later, the
new generation of computers became more
compact (less than 1 square feet) due to the
achievements with the miniaturisation of their
Fig. 1 ENIAC – an early digital
computer. It was characterised by a
huge volume and poor computational
speed (Image courtesy of the Computer
History Museum).
components. Something else changed, too: the
computing speed increased about many hundred thousand times. A laptop with more than 2
GHz clock frequency is a common tool these days.
In conclusion, miniaturisation means smaller and smarter tools for tomorrow! This is a
„sine qua non” condition of all future technological development in order to improve our
lives. As Euclid used to finish his (successful) mathematical demonstration: “Quod Erat
Demonstrandum” (QED)!
a
J. Mauchly was the chief consultant and J. P. Eckert the chief engineer of team involved in the construction
of ENIAC.
b
1m2 = 10.76 square feet; thus, ENIAC required a room of about 10x10 m2!
2
Why going smaller?
Confession:
It happened in summer 2000, that I received a phone call from Prof. Dr. Elisabeth Csöregi
(Lund University, Sweden). About a year before, I contacted her and asked for help in
elucidating the structure of a polymer, which I accidentally prepared in 1989. Having this
in my mind, I thought that she would, although a bit late, discuss with me about my
request. Well, I was wrong. Instead, she mentioned that I could go to Wolfgang in
Germany. What a surprise! I am lucky! I can go abroad and study. But where is this place
“Wolfgang”? I though it must be a small town because I never heard about it! This were
my thoughts as E. Csöregi went on explaining me I have to send her a CV (curriculum
vitae and not a cyclic voltammogram!) and the details of the passport, a document that I did
not even have at that time. It was days after this event when I realised Wolfgang is not a
place but the name of my future boss!!! Ups! In the next period of time some e-mails were
exchanged between Wolfgang and me, thus I was informed about what I could do in
Germany, particularly, in the Biosensors laboratory at Ruhr University of Bochum.
Biosensors? I never heard about this, but I knew a little about chemical sensors! Pdf files
of publications describing the topics and research directions in Wolfgang’s group were sent
by e-mail. Unfortunately, I couldn’t read them because the suitable software for opening
such files had never been installed on the only computer of the Chemistry Department at
North University of Baia Mare. However, some bits of information were displayed on the
website of the ELAN-group (Electroanalytik, Biosensors) and gave me a rough idea about
my future colleagues and research topic.
As mentioned above, my PhD work is dedicated to micropatterning of biological
recognition elements. Furthermore, a novel coaxial microelectrode for advancing
microelectrochemical measurements in small electrolyte volumes was developed. The
major part of the dissertation, however, was focusing on the development of a simple
strategy to detect hybridisation of nucleic acids on DNA chips. Working on the technology
of DNA microarrays was enjoyable to me not only because it is a hot-topic these days but
also because I had the opportunity to work on something that was completely new to our
lab. The entire work presented in the following chapters is based on the concept of
miniaturisation.
3
Methods for microstructuring
2.
2.1.
Lithography
“As tiny as possible” seems to be the present day trend of science and technology with
respect to any kind of device. I would like to present here, the microfabrication, not as a
dead point of miniaturisation but as a reliable multi-purpose technique for fabrication of
chemical and biological analysis tools. Many ways of microstructuring are derived from
common devices used every day. For instance stamps or printer-like “ink” dispensers
occupy an important place among the microstructuring tools; or one can “write” with a
“nano-pencil”. Scaling down structures originated in the field of electronics where devices
became smaller in size and the individual components of the integrated electric circuitries
had to fit on limited space. Nowadays, microfabrication procedure has a strong impact in
most of the areas of contemporary science and technology and the knowledge and
experimental procedures for miniaturisation were transferred from electronics also to
chemistry and biochemistry for creating sensors with better performances. The ability to
generate patterns of biomolecules on different material surfaces is important for biosensor
technology, tissue engineering, and fundamental studies in cell biology. There are several
well established ways to pattern biomolecules onto substrates, such as photolithography,
soft lithography, nano-pen lithography, and spotting techniques. Although this is the
accepted classification of micro/nano-structuring techniques, to my opinion the key word
“lithography”, that itself means “writing on materials”a, is in fact sufficient to cover all the
existing methods. Thus, no matter how the patterned substance reached the substrate, the
underlying method is a lithographical one! However, the following part that is dedicated to
a succinct presentation of each micropatterning approach; the common categorisation will
be used.
2.1.1. Photolithography
Photolithography is the process of copying geometric shapes from a mask to the surface of
a substrate. The working principle resembles the formation of the “positive image” from
the negatives on to a developed film in photography (Figure 2). The steps involved in the
photolithographic processes are: wafer cleaning, barrier layer formation, photoresist
application, soft baking, mask alignment, exposure and development, hard baking.
a
Lithography is a combination of the Greek words „lithos“ and „graphein“ with the meaning of stone and
write respectively. It denoted, in earlier times, various items, one of which being “art of engraving on
precious stones”.
4
The wafer is initially coated by spin-coating with a photoresist. The desired pattern is then
projected onto for example a wafer in a machine called a stepper. The stepper functions
similarly to a slide projector and creates high-contrast monochromatic images. Light from
the source is focussed through some lenses onto a "mask" (reticle), containing the desired
image in order to produce it on the wafer. Unlike a slide projector, the stepper does not
enlarge the image but actually reduces it in a similar way than sunlight is generating a
shadow of a cloud that is smaller than it actual size. When the image is projected onto the
wafer, the photoresist material undergoes some light-induced chemical reactions, which
cause the regions exposed to light to be either more or less susceptible to chemical etch. If
the exposed regions become more susceptible to the etch, the material is called a positive
photoresist, while it is a negative photoresist if it becomes less susceptible.
Fig. 2
Comparison between the photography (left) and photolithography (right). In
photography, the negative image on the film is transferred onto the photo-paper in such
a way that the final positive image is larger as the picture captured on the photo-film. In
contrast, in the photolithographic processes, the image grafted on the mask is reduced
in size. For simplicity, no lenses are shown.
The resist is finally "developed" by exposing it to the chemical etchant, which removes
either the exposed (positive photoresist) or the unexposed (negative photoresist)
photoresist. The substrate then has a patterned polymer coating on its surface. This pattern
can then be etched into the underlying wafer by either a wet chemical etch or a plasma
etch. The ability to project a clear image of a very small feature onto the wafer is limited
5
by the wavelength of the light that is used and the aperture of the lense (Rayleigh
diffraction). With ultraviolet light, features of 100-200 nm can be obtained.
The major disadvantages of photolithographical methods are listed below:
-
the smallest achievable dimension is limited by the optical diffraction;
-
not simply adopted for patterning non-planar surfaces;
-
tolerates only little variations in the material that can be used (there is not a large
selection of available photoresists on one hand, and on the other hand common
wafers such as glass or ceramics are not that suitable as substrates);
-
provides very poor control over the chemistry of patterned surfaces (especially
when complex organic functional groups are desired at the patterned surface);
-
high cost due to the necessary sophisticated facilities and technologies.
2.1.2. Soft-lithography
Alternatives to photolithography were developed in the last 2-3 decades and showed their
potential for micro- and nano-patterning1,2. There are some procedures resorting to
stamps/moulds made of flexible polymers rather made hard materials. "Soft lithography" is
a new high resolution patterning technique developed at Harvard by Prof. G. M.
Whitesides in which the stamp is made from an elastomeric material namely
polydimethylsiloxane - PDMS. Members of the “soft-lithography” family are microcontact
printing (µCP), replica moulding (REM), microtransfer moulding (µTM), micromoulding
in capillaries (MIMIC) and solvent-assisted micromoulding (SAMIM).
Microcontact printing uses a PDMS stamp (copied from a master that is previously
prepared by photolithography) to transfer molecules onto surfaces. In a first step, the stamp
is covered with the desired molecules (thiols, proteins, nucleic acids, enzymes). The stamp
is allowed to dry and afterwards pressed onto the surface to be patterned. The soft rubberlike stamp provides a large-area contact on the molecular scale, even on rough or slightly
curved surfaces and molecules are transferred directly from the stamp to the surface.
With replica moulding, a liquid precursor of PDMS is pressed against a patterned surface
with nanometer-sized relief structures. After curing, the cross-linked PDMS is cautiously
peeled off the structure perfectly copying the morphology of what was called the original
master3. The nanostructures present on the PDMS replica are, in turn, re-replicated using a
rigid organic polymer, for example, an photochemically curable polyurethane (PU), to
produce polymeric nanostructures very similar to (or indistinguishable from) those on the
surface of the original master. A great advantage of this approach is that it can produce
6
solid copies with smaller or larger
features as the original master! This is
achievable because the PDMS copy can
be mechanically bent (Figure 3).
In microtransfer moulding4, the mould
must be prepared before being used for
patterning. Accordingly, the recessed
regions of a stamp are filled with a
solution or pre-polymer and the exceeding
liquids are removed away from the crests.
The filled mould is then brought into
contact with the substrate. This technique
allows formation of both interconnected
and isolated microstructures and is also
suitable for building up 3D-structures
layer by layer.
With micromoulding in capillaries, the
Fig. 3 The soft elastomeric structure can be
mechanically bend and thus making possible
the preparation of copies with larger (l1) or
smaller (l2) features as the original master.
dried and clean mould is positioned over
the substrate. In the next step the precursor of the polymer is forced to enter the
microscopic channel formed by capillary forces. Once the polymer is hardened, the mould
is removed to reveal newly prepared 3D-structures. If the mould is filled for instance with
a solution of an alkane thiol in ethanol, thiol microstructure are available by this procedure.
In contrast, to all the above-described soft-lithographic procedures, solvent-assisted
micromoulding does not deposit a particular polymer or self-assembled monolayer at the
surface, but removes material from the substrate in an etching process. The mould is placed
on the substrate and a solvent able to corrode the substrate is filled in the capillaries. When
the solvent is completely evaporated, the mould is removed. Quasi-3D-structures are
possibly created.
An intrinsic problem with any lithography based on an elastomeric mould originates from
the material properties of the mould. The softness of the mould leads to mould deformation
in the process of patterning or mould preparation and, of course, the deformation gets
worse as the pattern size becomes smaller, typically for feature sizes smaller than several
hundred nanometers. To overcome this problem, an amorphous fluoropolymer material
7
was used as a mould material. The unique properties of the mould material made it
possible to pattern densely populated extremely fine features (80 nm line-width)5.
2.1.3. Other methods for microstructuring
A promising alternative is the LIGA process that was developed at the Institute of
Microstructure technology (IMT) in the early eighties under the leadership of Dr. W.
Ehrfeld6. LIGA made possible the mass-production of microcomponents at low-cost. The
steps involved are X-ray lithography, electroformation and plastics moulding.
•
Deep X-ray lithography: LIGA requires a highly penetrating, intense, and parallel
X-radiation, typically supplied by a synchrotron. The application of X-radiation
imposes the use of specific materials for the mask with the „transparent” part made
of very thin foil of metals such as titanium or berylliumb, and the absorbers
consisting of a comparatively thick layer of gold. The lateral structural information
is transferred by illumination of the mask with deep X-ray radiation into a plastics
layer, normally polymethylmethacrylate (PMMA, Plexiglas), by „shadowing”.
Exposure to radiation modifies the plastic material in such a way that it can be
removed with a suitable solvent, leaving behind the structure of the unexposed
plastic (the „shadowed areas”) as the primary structure. Of note, high aspect ratio
structures with heights of up to 1 mm and a lateral resolution down to 0.2 µm are
obtainable.
•
Electroforming: the microcavities generated by the removal of the irradiated plastic
material can be filled with metal by electroforming processes. In this way, the
negative pattern of the plastics structure is generated as a secondary structure out of
metals, such as nickel, copper and gold, or alloys, such as nickel-cobalt and nickeliron.
•
Plastics moulding: Plastics moulding is the key to low-cost mass production by the
LIGA process. The metal microstructures produced as mentioned above are used as
moulding tools for the production of reliable replicas of the primary structure in
large quantities and at low cost.
Mask-free methods for micropatterning surfaces were established using the tip of scanning
probe microscopes to locally modify surfaces down to nm range. The way of inducing
b
Due to its poor electronic structure, Be is also used at large scale for X-ray transparent windows in nuclear
reactors and in radiation detectors. As a matter of fact, its low atomic weight recommends it as moderator for
slowing down rapid neutrons in a nuclear reactor and hence promoting the self-sustaining nuclear reaction.
8
controlled alteration of substrates by scanning probe lithography (proximal probe
lithography) relies on the following interactions:
-
electrical: a scanning tunnelling microscope (STM) tip generates local electrical
fields that modify the surface underneath;
-
electrochemical: a microelectrode, the SECM tip, is used to locally generate a
reagent that is able to etch the substrate;
-
mechanical: an atomic force microscope (AFM) with its sharp tip operated in the
contact mode scratches the surface or transfers material from the tip to the surface
(dip-pen nanolithography, DPN7-9; Figure 4);
-
optical: the optical fibre probe of a near-field scanning microscope (NSOM)
exposes the photoresist at local areas underneath the tip.
Fig. 4 “Writing” with a nanoscopic ink-pen that is the AFM tip.
The “ink“ molecules that are dissolved in the solution underneath
meniscus bind due to their high affinity to the substrate.
2.2. Ink-jet printing/lithography - the flow-through piezo-microdispenser
Simple but cost-effective lithographic processes that can be applied even to irregular
substrates under ambient conditions have been developed. Ink-jet lithography is a valuable
tool providing a non-destructive and localised surface modification technology in which
small droplets of ink (or other liquids) are jetted from a small aperture (nozzle) directly to a
specified position of the substrate.
Professor Hertz from the Department of Electrical Measurements, University of Lund
developed about 25 years ago continuous ink-jet techniques that had the ability to
modulate the ink-flow characteristics for grey-scale ink-jet printing on different media. The
idea of obtaining grey-scale printing was to control the number of droplets deposited in
each pixel (spot) so that the amount of ink volume in each pixel was adjusted to create the
desired grey tone10. May be inspired by the work of Hertz, Thomas Laurell from the same
9
Department developed a microdispenser11 capable to shoot and deposit picolitre-sized
droplets of solutions on substrate surfaces.
An obvious advantage of this printing procedure over stamp-based methods is that tiny
droplets can be deposited in small cavities12. In contrast to a deposition from a spotting
needle, a microdispenser can be used with much more viscous solutions because it is
actively forcing the solution out of the reservoir using the piezo-actuator. As shown later,
this was helpful when, for instance, an enzyme–polymer mixture that certainly is more
viscous than pure water had to be micropatterned on a chosen substrate. A schematic
representation of a microdispenser is shown in Figure 5.
Fig. 5 Schematic representation of the piezo-microdispenser
head. Alternative voltage pulses applied to a thin silicon
membrane eject picoliter droplets of solution (yellow) towards
the substrate.
In brief, the microdispenser is capable to emit ultra small droplets of solutions of choice
based on rapid movements of a flexible silicon membrane. The membranec is coupled to a
piezoelectric ceramic actor (3x3x2 mm3) and fastened by in a plastic frame (Figure 6),
which expands and contracts upon the excitation with suitable alternating voltages. That
way the system is able to generate a series of short pulses of pressure each of them ejecting
a single droplet with a volume of roughly 100 picolitres.
c
Two silicon plates, obtained by anisotropic etching (one for the nozzle and another for inlet/outlet of
solution and membrane), are glued and sealed with waterproof epoxy. The inner chamber is then enclosed by
the two plates to give a volume of few microlitres down to hundreds of nanolitre.
10
The droplets are released through the specially designed, pyramidal nozzle with an orifice
size of 40×40 µm and directed perpendicular towards the target surface where the
deposition typically make spots with diameter of about 70 µm. If the droplets are shot in a
horizontal plane, they can travel without any change of the trajectory up to several
centimeters, at least if there is not strong air motion. Certainly, droplets emitted
downwards can be delivered with high precision from the nozzle to a chosen area of the
opposing substrate. A precise movement of the target substrate relative to the
microdispenser nozzle during microdispensing can be used to deposit either rows of
individual spots or lines and grids from overlapping lines.
Fig. 6 Two versions of a piezo-microdispenser head: nozzle (1),
piezo actuator (2), etched silicon chips (3).
A pump is connected to the chamber via short silicon tubes. The excess of the filling
solution is drained out through the outlet consequently avoiding an exposure of the
membrane to overpressure inside the piezo chamber. Any time when a voltage pulse is
applied to the piezo, one droplet is hurled through the nozzle.
The flow-through microdispenser has proven to be ideal for applications in biochemical
and analytical chemistry where small sample volumes and rapid sample handling are key
features.
11
Tools for probing microstructures
3.
3.1.
Scanning Probe Microscopy (SPM)
Microscopes are representing powerful tools for extracting and mapping detailed
information about the microstructure of an object13 and helped to unveil secrets of nature
that were long time hidden to observers since human eyes are limited to a resolution in the
order of 100 µm, which is just about the thickness of a hair14. First scientific observations
using microscopes to explore the microcosmos were reported by R. Hooke (1667) and A.
Leeuwenhoek (1697)a. The design of the optics of microscopesb changed their look
dramatically over time. At the early stage of development, simply a drop of water, oil,
honey or glass fixed on a small round cut in a metallic plate or a tube containing two lenses
fixed at its extremities were the plain devices to observe things otherwise invisible to the
naked eyes. We are used to think about microscopes as a holder that contains a tube having
two lenses: the eyepiece (ocular) and an objective (H. and Z. Janssen, 1590). This is the
more “classical” picture of a microscope. Over the years, microscopes developed into
sophisticated instruments with improved magnification and resolution (maximum allowed
by Abbe diffraction). These were accomplished by using not only visible light but also
electromagnetic waves with a shorter wavelength such as UV or fascicle of accelerated
electrons. Up till the 80’s of last century, visualisation techniques typically generated
secondary images arising from the interaction of light with structural parts of objects under
study. Then a new set of techniques appeared that provided images of an object without
depending on the diffraction of light. Going along with changing the principles of image
generation, the tubular form of the old-fashioned microscopes turned into new designs.
Scanning-probe microscopy (SPM) refers to a large number of techniques that employ
needle-like probes with physically small tip dimension that are mechanically scanned back
and forth across a surface. The probe tip can be made sensitive to a variety of surface
properties and the measure of distance-dependent interactions between the probe tip and
surface is actually used for imaging the interface. The most widely used high-resolution
imaging tools certainly are the atomic force microscope (AFM) and the scanning
tunnelling microscope (STM)15,16. As a matter of fact, these crucial tools, AFM and STM,
a
Robert Hooke (Freshwater, 1635 – London, 1703) British scientist known for his work in physics,
astronomy and mathematics; studied various microscopic objects and published the in “Micrographia”;
Antony van Leeuwenhoek (Delft, Nederland 1632 – id. 1723,) self-educated, he made lenses with great
curvature, built microscopes and was the first to observe yeast, bacteria, and other tiny creatures in a drop of
water and the circulation of blood corpuscles in capillaries.
b
The word microscope comes from Greek language: “micro” = small and “skopein” = look at.
12
were invented by the same scientist, namely G. Binnig (he and H. Rohrer were awarded the
Nobel Prize for Physics in 1986 and shared the prize with E. Ruska who first presented the
electron microscope in 1931 in Berlin)c,17. AFM is taking advantage of force interaction
between the sample surface and a sharp tip that is attached to a flexible cantilever.
Recording the deflection of the cantilever as a function of lateral position provides
topographical images with up to nanometer resolution. With STM, on the other hand, a
voltage is applied between a nanometre-sized metal tip and a conducting surface. With the
STM tip being in extreme proximity to surface and scanned, a distance-dependent
tunnelling current is flowing and may alter laterally with changes in the tip-to-sample
separation. STM gives access to imaging the topography of conductors with atomic
resolution and has been successful for example even in imaging individual atoms laying on
compact surfaces. As it is not restricted to conducting surfaces, AFM is more suitable for
biological imaging. This has been demonstrated by employing an AFM for watching
biological macromolecules in motion or simply biopolymers such as DNA that are coiled
or stretched on a substrate18,19.
Although, the different SPM devices (Table 1) have many technical components in
common (i.e. the systems that are used for precise micropositioning of the probe tip), the
working principles behind the instruments differ quite a lot depending on the specific
interaction between the tips and sample. In general, SPM is applicable for “in-situ”
investigation of samples. Indeed, studying the target in its ambient environment is a great
advantage because no vacuum is needed as for example with a scanning electron
microscope (SEM). This and their high spatial resolution made scanning probe
microscopes so popular in a relatively short time.
Table 1
Probe Microscopy
Acronym
Appearance year
Notes
Scanning Tunnelling Microscopy
STM
1982
in-situ technique
Scanning Near-Field Optical
Microscopy
SNOM
1985
Atomic Force Microscopy
AFM
1987
Magnetic Force Microscopy
MFM
1987
Scanning Electrochemical
Microscopy
SECM
1989
c
Although the first (transmission) electron microscope was presented by M. Knoll and E. Ruska in 1931, the
scanning electron microscope was in fact discovered by M. Ardenne in 1938. The first commercial available
SEM came up in 1966 (Cambridge Instruments Comp.).
13
Scanning Ion Conductance
Microscopy
SICM
Scanning Capacitance Microscopy
SCM
Scanning Chemiluminescence
Microscopy
SCLM
Scanning …. Microscopy
…
1989
…
…
Among the numerous SPM devices, only one is based on an important technological
advancement in modern electrochemistry: the voltammetric and potentiometric
microelectrode. Approximately at the same time, W. Engstrom20 and A. J. Bard21
introduced voltammetric microelectrodes as new type of scanning probes and paved the
way for so-called scanning electrochemical microscopy (SECM). Being attached to highprecision positioning devices, the micrometre-sized tip of a disk-shaped microelectrode
enabled local electrochemical measurements while being scanned across the sample
surface. As will become clear below, the spatial resolution for SECM imaging is somewhat
limited and much lower than for AFM/STM. SECM, on the other hand, can more easily
provide images of variations in interfacial (electro) chemical activity.
3.2.
Scanning Electrochemical Microscopy (SECM)
The development of ultramicroelectrodes was one of the most important contributions to
electroanalytical chemistry. As their name implies, ultramicroelectrodes are extremely
small, with dimensions in the order of micrometers or less. This small size and the
electrode characteristics that come with it (low capacitive currents, low IR drop, enhanced
mass transfer - see note 1 - and high faradaic current densities) can be exploited in a
number of unique applications22. Among them are measurements in highly resistive media,
high-speed voltammetry and electroanalysis in very small volumes. The integration of
ultramicroelectrodes into a scanned probe instrument led to the invention of scanning
electrochemical microscopy which allows observing local electrochemical activity at the
liquid/solid interface or at microscopically small object.
How does SECM work?
A little information about the basic principle of SECM will be introduced because this is
necessary to understand the following parts of this work. With SECM, amperometric or
potentiometric ultramicroelectrodes with radii, r, in the order of a few µm or even less are
employed as electrochemically active scanning probes (SECM tips). In case of an
amperometric SECM tip, an electric current will flow if a redox active compound is
14
present and the tip polarised at proper potential. Tip interaction with a target surface via an
electroactive species results in distance-dependent variation of the tip current.
One tool – multiple operational modes
The SECM23,d as a whole and self-containing instrument helps in different modes to
visualise electrochemically active targets or to modify surfaces exposed to electrolyte
solutions. These are the amperometric feedback, the generation/collection, penetration, and
the surface modification mode24. In general, the amperometric tip current (iT) is plotted as
a function of its horizontal coordinates (X and Y) for imaging superficial electrochemical
activity of a sample. In the following, modes I to IV are briefly described and possible
applications discussed.
Fig. 7 Feedback mode of SECM: for a given redox reaction Ox→Red carried out
at the SECM tip, a steady-state current is recorded in bulk of the solution I=Ilim
(A); in the vicinity of an electrically conductive surface the Ox species are
regenerated with the consequence of local rise of Ox concentration and hence a
higher current recorded at the SECM tip (B); thus an insulating substrate
obstructs the diffusion of Ox to the tip, thus lowering the measured faradic current
(C). Generation-collection mode of SECM: for instance, surface confined species
generate an electroactive compound that is detected at the SECM tip (D).
I. The amperometric feedback mode of SECM
Let us imagine a disk-shaped microelectrode (active diameter smaller than about 50 µm)
that is immersed in a solution containing a redox species (e.g. ferri- or ferrrocyanide).
Shortly after the potential of the electrode is set to a value suitable to oxidise or reduce the
d
The acronym SECM stands for the instrument itself - scanning electrochemical microscope - as well as for
the specific field of probe microscopy that is scanning electrochemical microscopy.
15
electroactive compound, a steady-state faradaic current is observed. The magnitude of this
current depends, besides the rate of electron transfer, on the concentration and diffusion
coefficiente of the electrochemically active substance and on the diameter of the
electroactive disk of the microelectrode. For an unstirred solution, the steady-state current
(i∞) (Figure 7A) is purely controlled by diffusional mass transport of the electroactive
species and given by the well-known equation:
i∞ = 4 ⋅ n ⋅ F ⋅ D ⋅ c ⋅ a
eqn. 1
where n denotes the number of electrons taking part in the electrode process, F the Faraday
number (1F = 96484.6 C⋅mol-1), D the diffusion coefficient (cm2⋅s-1)f, c the concentration
of electroactive species (mol⋅cm-3), a the radius of the disk-shaped electrode (cm); i∞ is
thus measured in ampere (A).
Probing a target with SECM clearly requires the microelectrode tip to be brought close to
the target surface since otherwise the surface-specific information can not be collected. In
the vicinity of a surface, however, the current measured by the SECM tip changes
significantly dependent on the tip-to-sample separation and the nature of the approached
surface25,26. How? and why? are the two questions to be answered in the following. We
should consider two cases:
1. the surface is electrochemically active (electric conductor, enzyme modified).
2. the surface is electrochemically inactive (electric insulator, enzyme-free).
1. An electrochemically active surface: the positive feedback
We are bound to a great degree by our own innately human way of thinking and judging
phenomena around us which is the common sense! We just simply have learned that some
things happen and have a certain course. This helps us to understand new items by
comparing the new facts with old schemes in our brain. Quite often, in the
physics/chemistry (especially the quantum mechanic, relativistic theory, quantum
chemistry…) the common sense deplorably fails! During my PhD, I had the chance to
introduce several students to the basics of SECM, and therefore I noticed that typically the
answer to the question “What should the current be if the SECM microelectrode is brought
close to a conductive surface?” is wrong. The students anticipated that the current will drop
to zero when the electrode is approaching a surface; their common sense suggested them
e
As obtained from the Fick’s first equation, the diffusion coefficient is a linear function of temperature:
D = k⋅R⋅T, where k is a constant, R is the gas constant and T is the temperature of solution.
f
D has typical values ranging within 10-5 – 10-6 cm2⋅s-1 that depends strongly upon the composition of the
electrolyte.
16
that the current must decrease irrespective of whether the surface is conductive or not, the
surface will decrease the mass flow of the reacting species towards the electrode’s disk.
Against the common belief, the current increases (see Figure 7B and 8). Why? We shall
remember that any electroconductive surface will gain a superficial, negative or positive
net charge when it is in contact with a solution of a redox active species.
Fig. 8
Cyclic voltammograms recorded at a 10 µm Pt microelectrode in bulk (blue line) and at about 15 µm above a gold
surface (dark line). Not only is the steady-state current higher
close to the surface but the form of CV changes from hysteresis
to a perfect S-shape; 5 mM [Fe(CN)6]3- in 0.1 M phosphate
buffer and 3 M NaCl and 0.05 M NaOH; 500 mV/s scan rate;
Ag/AgCl 3 M KCl reference electrode.
In SECM practice, the solution contains typically an electroactive species that can undergo
reversibly redox transformation. A redox compound in solution will electrically charge the
inert metallic interface (Pt or Au) to a potential of which polarity and amplitude are
controlled by the ratio of the Red/Ox concentrations of the redox species. The potential of
a gold surface, for instance, in a supporting electrolyte containing only Fe3+ and no other
redox species (especially no Fe2+) is controlled by the electroactive species present in the
bulk of electrolyte (see note 2). According to the Nernstg equation, the open circuit
potential the gold surface, at least under this experimental conditions, is expected to be
significantly more positive than the E0 of the Fe3+/Fe2+ redox couple (see below), and thus
able to oxidise Fe2+ back to Fe3+ (equation 2):
g
Walther Nernst (Briesen, West Prussia 1864 - Berlin 1941), German physicist and chemist with
contributions in thermodynamics; Nobel Prize for Chemistry in 1920.
17
0
+
E Fe3+ / Fe 2 + = E Fe
3+
/ Fe 2 +
RT a Fe3+
lg
nF a Fe 2 +
eqn. 2
where E (V) is the actual electrode potential, E0 is the standard electrode potential
measured against a normal hydrogen electrode (NHE) at 25° C, and 1 M activity, a, of the
two ions; R is the gas constant (8.314 JK-1mol-1), T is the absolute temperature (K); n is the
number of electron transferred in the redox reaction. If T = 298 K, the constant in front of
logarithm is 0.059 V. Here, E0 = +0.356 V.
In SECM, a redox mediator is amperometrically reduced (or oxidised) at the SECM tip
with a potential ensuring diffusional control of the tip current. With the SECM tip far
above a surface, tip- generated species simply diffuses into the bulk without undergoing
any further reaction. When the tip is brought close to a contacting surface, however, tipgenerated species has a chance to interact with the charged interface and, due to its
polarisation (see above) it will be oxidised (or reduced). Consequently, the local
concentration of Ox in the gap between the microelectrode tip and surface increases
compared to bulk concentration and hence the amperometric current measured at the tip
increases, too. This effect is called the positive feedback, and represented schematically in
Figure 1B. Important to obtain the negative feedback is to position the SECM tip into the
nearfield. The variation of the positive feedback SECM tip current with tip-to-sample
distance is given by equation 327:
iT
0.78
 1.07 
+ 0.33 exp −
( L) = 0.68 +
eqn. 3
i∞
L
 L 
where L = distance between SECM tip and sample ( µm ) .
radius of active area ( µm )
A plot of iT versus L is known as approach curve and can be used to calculate and adjust
the tip position of a microelectrode of known dimensions.
Note: Surface-immobilised enzyme can act in the similar way as a bare metallic surface
and regenerate redox species that is consumed by the SECM. This process is known as
“enzyme-mediated positive feedback”28.
2. An electrochemically inactive surface: the negative feedback
The surface of insulating target is inert to the redox species and at small tip-to-sample
separation mechanically hinders the free diffusion of the reacting species towards the
electrode’s tip. Due to the lower flux, the amperometric tip current decreases compared to
18
the value obtained with the tip in bulk (Figure 7C). This observation is regarded as
negative feedback. Obviously, the tip-to-sample distance has a major impact on the
hindrance of diffusion and the extent of negative feedback. The degree of negative
feedback also is influenced by the ratio of the total diameter of the microelectrode and the
diameter of the active area. For a diffusion-controlled electrochemical reaction at an
SECM tip, and a disk-shaped electrode with 1:10 disk to insulation ratio, the relative
faradaic current variation obeys with good accuracy (1.2%) equation 4:
iT
( L) =
i∞
1
1.51
 2.40 
0.29 +
+ 0.66 exp −
L
 L 
eqn. 4
General note:
A SECM tip is said to be positioned within the working distance when the measured
current is experiencing the influence of the conductive or insulating surface. This criterion
must be achieved in order to allow SECM measurements.
3. SECM imaging in the negative and positive feedback mode
Negative and positive amperometric feedback are highly distance-dependent, and approach
curves (I/I∞ vs. tip-to-sample separation d) permit to place the SECM tip at appropriate
working distances within the regime of the nearfield (at a height of about a few times the
SECM tip radius or less). Typically, image acquisition is achieved by scanning the SECM
tip laterally at a user-defined fixed height and simultaneously recording the tip current as a
function of position (constant-height feedback mode imaging). Figure 9 is showing in a
schematic the current profiles that one expects when a SECM tip is scanned at a constant
height across samples exposing lateral variations in topography and conductivity.
To give you an idea about SECM imaging in the amperometric feedback mode, I would
like to present a SECM image of a fingerprint that was scanned with a Pt microdisk
electrode in a solution containing 5 mM [Ru(NH3)6]3+ as the active mediator. A sample
with alternating conducting and non-conducting areas was made by firmly pressing a
finger tip against a chemically deposited layer of silver on glass. This contact led to local
removal of silver at areas where the papillae of the skin was touching the silver film. As
can be seen from Figure 10, the variation in conductivity and/or heterogeneities and
variation of the flatness of the sample were nicely revealed.
19
Fig. 9
Schematic representation of SECM tip reduction current
profiles expected when scanning the tip in amperometric feedback
over (A) electrically conductive, (B) electrically non-conductive
surfaces that have topographic heterogeneities; a topographically
homogenous but electrically heterogeneous surface can also be
image in the feedback mode (C).
Fig. 10 SECM micrograph of a fingerprint. The imaging was
performed with a 10 µm Pt disk electrode in the feedback mode
of SECM. Dark areas correspond to low currents and indicate
where the finger’s papillae touched the thin layer of chemically
deposited silver on glass surface. Scanning solution: 5 mM
rutheniumhexamine chloride in 0.1 M phosphate buffer(pH 6.7).
20
II. The generation - collection mode of SECM
As the name suggests, it involves the production of an electrochemically active species at
one place, which after a short diffusion through solution is immediately detected at another
place. If the microelectrode is positioned within the working distance, and operated
amperometrically, it generates a concentration profile by consuming a substance available
in solution, and the properly polarised substrate collects the products. Hence, it is said the
SECM works in the tip-generation/substrate-collection mode (TG/SC).
Opposite to the case above mentioned, the generation process can be carried out by the
substrate and the products gathered at the microelectrode, a situation that is described as
substrate-generation/tip-collection mode of SECM29 (Figure 7D) Typical examples where
the tip is used as a collector are studies of the diffusion of metabolites released from living
cells30, corrosion31 of different materials32-34.
III. Penetration mode of SECM
This mode involves the movement of the SECM tip along a single direction that is
perpendicular to the target (Z-axis). Of course, any medium that allows the microelectrode
to break through without harming it is of interest for this SECM mode. Thus, gels,
biological tissues, solutions with a concentration gradient, or even soft films are
appropriate for being investigated with a tip in the penetration mode of SECM.
IV. Surface modification by means of the SECM
The SECM has been successfully used for modifying numerous surfaces35. This is
achievable, on the one hand due to the possibility of accurately moving microelectrodes
over a substrate and on the other hand, by taking advantage of the SECM tips to generate
reagents for directed chemical transformation of the substrate. For instance, inorganic
microstructures were created by tip-induced reduction of different metallic cations36,37.
Also, a SECM-based fabrication of microstructures of conductive polymers38 such as
polypyrrole39 and polyaniline40 and polythiophene41 or polybithiophene42 derivatives by
oxidative formation of polymerisable radical-cations from their monomers has been
described. Self-assembled monolayers (SAM) of alkanethiolates patterned on gold43 by
means of SECM allowed the local formation of biologically active microstructures
containing for instance glucose-oxidase44. Micromachining is a controlled alteration of
surfaces for creating microstructures. It can also be achieved with SECM tip-generated
etching reagents, for instance bromine45 that act as curving tool and has found application
in wet etching of silicon46, some semiconductors47 or metals like copper48.
21
The item writing mode of SECM is occasionally accounted in papers regarding surface
modification by SECM but it is in fact nothing more than the above mentioned
techniques49,50.
Constant distance mode of SECM
Most often SECM experiments are performed with the SECM tip kept at “constant-height”
above the substrate. However, there are some inherent limitations especially on tilted
samples, on surfaces with larger variations in topography and at surfaces that display both,
variation in topography and conductivity. For instance, a particular substrate has a rough
surface with gaps or heights of sizes not far from the total diameter of the scanning tip. If
the tip is positioned in the working distance, and scanned over the structure, it may collide
with the surface protuberances. In the case of a tilt surface, depending upon the scanning
direction, the electrode tip could either crash on the surface or retreat from the working
distance. In both cases, a clear interpretation of the measurements would be difficult
because changes in the tip current arising from distance variations cannot easily be
differentiated from those originating from alterations in surface activity. Moreover, since
the tip-to-sample distance has to be in the orders of few tip radii of the UME (within the
nearfield) it is obvious that with a decreasing electrode size scanning in this mode becomes
hard51. Constant height mode is only applicable when changes of the sample height or the
overall surface tilt do not exceed the tip-to-sample distance.
The introduction of the “constant-distance” SECM employing an optical detection scheme
for shear forces between the electrode tip and the surface permitted the operation of probe
tips with electroactive diameters below a micrometer and simultaneous imaging52,53. The
integrated shear force-sensitive and computer-controlled feedback loop of this mode forces
the SECM tip to follow the contours of the surface during the entire time of scanning and
imaging. This not only allows simultaneous acquisition of the electrochemical tip response,
the sample topography but also effectively prevents against tip crash even with smallest
SECM tips.
The very strict experimental requirements (perfect alignment of the laser source with the
mirrors, photodiode detector and tapered capillary of the ultramicroelectrode) required
with the optical read out of the damping of shear forces, led to efforts for improving the
“constant-height” methods. A non-optical shear force-based distance control was
established by replacing the laser beam and the light-sensitive diode with a piezo
receiver54.
22
SECM measuring tips
At the beginning, most of the SECM tips were fabricated with noble metals or graphite in
disk-shaped micro- and nano-electrodes with active diameters of 1-25 µm55,56. Glass pulled
capillaries constituted the body and the insulation of the electrodes. In addition to diskshaped microelectrodes, ring57,58 or hemispherical59,60 electrodes have gained recognition
and found applications in SECM.
New electrode materials allowed direct measurement of pH61,62, or potentiometric
monitoring of anions (chlorine)63. Besides potentiometric, SECM tips were also
manufactured from pulled capillaries filled with ion-selective ionophors64. Carbon fibre
microelectrodes (CFMEs), insulated by anodic electrophoretic deposition of paint
(EDP)65,66 were used as SECM-tips for low-noise recordings of neurotransmitters release67.
A remarkable electrode for simultaneous electrochemical, optical and topographical
images was obtained from an optical fiber surrounded by a gold ring electrode and an
electrophoretic insulating sheath68.
Drawback
Spatial resolution of SECM is primarily determined by the size of the SECM tip and for
that reason not on atomic scale as for instance with scanning tunnelling microscopy (STM)
and atomic force microscopy (AFM). However, the electrochemical nature of the scanning
probe offers an exceptional chemical selectivity and SECM therefore serves as an excellent
tool for studying interfacial (electro)chemical properties and reactions.
23
Home build SECM set-up
All SECM experiments gathered in this volume were carried out with the following set-up
(Figure 11).
Fig. 11 Scheme of representing the scanning electrochemical microscope (left) and photo of
the SECM used in this work (right). The microelectrode is positioned close to the sample with
the Z stepper motor. When the working distance is achieved, X and Y motors move the sample
according to the given scanning parameters: speed, distance between data points and time for
data acquisition. Unlike the Faraday cage that is useful anytime when recording low
amperometric currents, the vibration-free platform is useless if the SECM tip’s height is not
controlled by shear-forces. Scale bar on the right image represents 35 cm.
24
3.3.
1.
Notes
Short after being introduced to preparation of microelectrodes by Dr. E. Bonsen, (2001) I
had difficulties in understanding, without resolving mathematical equations, why it is
believed the mass transfer towards a microelectrode is enhanced compared to a
macroelectrode! I felt something is missing to me, something like a qualitative
representation of the diffusional processes that take place around a macro- and microelectrode the surface. Quite a while after that moment I have got in mind a clear picture of
a reasonable explanation showing why “the mass transfer is improved”. The mass transfer
is improved because the resource of the fresh material that is carried towards the
electrode, by diffusion, is much higher for a microelectrode as compared to a
macroelectrode (Figure a). Let us name the as unit volume (orange square), the volume of
electrolyte of which content of redox species is fully emptied after the potential step was
applied to the electrode. Once the first species were consumed, fresh material is pushed
towards the electrode by forces derived from the chemical potential of the present species.
In the in the case of the macroelectrode a number of unit-volumes are emptied (9 white
squares in this example) and 13 units are in vicinity to supply the mass transfer (blue
squares), whereas, in the case of the microelectrode one white square has only 5 blue
squares around. I think is possible and didactically useful to compare the ratios of the blue
square to white square, because as big are the resulting numbers as efficient is the mass
transfer! For the above example, where the macroelectrode has 1.44 and the
microelectrode 5, it becomes clear why the last mentioned is better in enhancing the
diffusion of reactive species in direction of electrode surface. This model is valid for
spherical geometries as well.
Fig. a
Qualitative model explaining why the mass transfer of active species
towards an electrode surface is more efficient for microelectrodes as compared to
macroelectrodes.
25
2.
Example: a portion of a noble metal is immersed in [Fe(CN)6]3- aqueous solution. Thus, the
potential of the surface is polarised at approximately +400 mV versus the Ag/AgCl 3M KCl
reference electrode (potential measurement at zero current, so-called open circuit potential
(OCP) of noble metal - reference electrode couple). An electrolyte containing [Fe(CN)6]4lowers the open circuit potential to about -200 mV. These two compounds are partners of a
redox couple. If an electrochemical in relation is carried out at the tip of a SECM that is
positioned in the working distance, for instance the reduction of ferricyanide, the minute
quantity of ferrocyanide will be quickly consumed at the conductive substrate and this
before being able to affect the perturb the OCP of the metal – solution interface. This
means that the species in high excess (usually in bulk) are controlling the potential of the
substrate while the traces of the partner species are thermodynamically instable (Figure b).
The OCP is hence a critical number for it determines what species can exist at a certain
potential (circa 300 mV in the mentioned case).
Fig. b The stability potential of the redox couple ferro/ferricyanide.
These domains are revealed in a cyclic voltammogram performed
with a 10 µm Pt disk electrode in a solution of 5 mM ferricyanide, 0.1
M phosphate buffer, 3 M NaCl at a scan rate of 100 mV/s; Ag/AgCl
3M KCl reference electrode.
26
Electrodes for electrochemistry in small volumes
4. Electrodes for electrochemistry in small volumes
4.1. Integrated working/reference assembly
The information about SECM probes given before dealt with conventional type of diskshaped microelectrodes. In the following, an electrode will be described that is well
suitable for electrochemical measurements in small volumes of electrolytes and especially
for SECM applications in nanoliter droplets. The motivation to work on the development
of such electrode originated from an observation that a colleague of mine complained
against the difficulty to operate a three-electrode configuration in the tiny vials of a
microtitre plate (Figure 12).
Fig. 12 Working electrode small, … but the reference?
When aiming on applying precisely positionable microelectrodes for measurements in
ultra-small electrolyte volumes it is crucially important that the total diameter of their
sensing tips including the insulating material is sufficiently small. Microelectrodes
fulfilling this prerequisite have been constructed either by sealing carbon fibres or thin
metal wires into tapered tips of pulled glass micropipettes56,69,70 or by coating carbon fibres
with a thin layer of an electrochemically depositable polymer 65,66,72, and chemical vapourdeposited quartz73, respectively. However, these needle-type electrodes have to be jointly
used with a reference electrode (RE) to accomplish at least a 2-electrode configuration as
required for voltammetry.
27
Clearly, a completion of such a 2-electrode assembly in highly restricted space is a
challenging task. Recently a miniaturised dual electrochemical probe for voltammetry and
SECM in nl droplets was introduced74. A carbon fibre was sealed with epoxy into one of
the two tapered channels of pulled theta glass capillaries while the other kept a
macroscopic Ag/AgCl wire in a chloride solution. The reference system was separated
from the analyte solution with an agar salt bridge that was placed in the tip opening. This
probe design obviously had an advantage for measurements in extremely small volumes
since a combined µWE/RE assembly is more easily to position, needs not as much space,
only a single micro-positioning device and avoids the risk of collision, which can occur
when the tips of two separated mobile electrodes are brought closely together and moved
autonomously from each other. However, the preparation of the proposed electrode system
is difficult and needs a lot of hands-on experience to reach a sufficient success rate.
Furthermore, the tapered tips of the double-barrelled microelectrodes are fragile and can
easily break during operation.
In principle, a technically easier alternative for the construction of dual probe tips is
offered by depositing a metal onto the outer surface of the (glass) insulation of disk-shaped
microelectrodes. Polishing the tip of metal-coated microelectrodes would lead to the
exposure of the active disk that is concentrically surrounded by the insulation and an outer
ring of metal and some used this approach for the fabrication of a coaxial microelectrode
designed for in vivo measurements of nitric oxide75. Pt wires were sealed into the tips of
tapered glass capillaries and then silver was sputtered on the glass surface to form a
Ag/AgCl reference electrode. Concentric layers of metal were deposited on the insulation
of microelectrodes by sputtering76, chemical vapour or electroless deposition (also used for
preparation of substrates in microcontact printing77) were also used to establish an active
electrical shield for reducing the stray capacitances and improving the microelectrode
noise78-81. Note: spatial resolved surface analysis (potentiostatic, galvanostatic techniques)
of non-wetting substrates can be also achieved with the scanning droplet cell method82
where the substrate under investigation must act as the working electrode.
4.1.1. Preparation of precursor electrode
Disk-shaped Pt microelectrodes served as precursors for the fabrication of coaxial Pt/Ag
electrode assemblies. They were constructed from 10 µm-diameter Pt wires that were
tightly sealed into the tapered ends of pulled borosilicate glass capillaries (O.D. 1.5 mm,
I.D. 0.75 mm, L 100 mm). In contrast to the well established method of back-connecting
the electrodic material to external copper wire with bi-component silver epoxy glue, which
28
is awkward an alternative and easy method was developed. The Pt and a Cu wire (Ø 0.5
mm) were attached to each other by filling the capillaries with a small amount of Zn
powder and crushed tin solder, which after placement was melted by careful heating in an
coiled filament at high currents. The melting solder entrapped the Zn particles and the
wires ensuring good electrical contact between all components. Smooth Pt micro disks
were exposed by polishing the tip at 90° on emery paper (grade 320 to 2000) and then on a
polishing cloth wetted with alumina suspension (particle sizes: 3 µm, 1 µm, and 0.3 µm).
The ohmic response does not change over a wide range of scan rates; therefore these
electrodes can successfully replace the epoxy back-connected electrodes. Table 2 outlines
the advantages of zinc-based electrodes over epoxy ones.
Tabel 2
Bi-component silver epoxy glue
Zinc powder
1. most part of the glue is wasted (it
1. no single zinc particle is wasted
remains onto the preparation dish, gloves)
2. requires a oven for fastening the two
2. no oven necessary (ready made
components (800 C, 1 hour)
electrode)
3. no efficient control during electrode
3. every preparation step is under control
preparation
4.
glue
–
platinum/copper
wires
connection is fragile (once the glass
4. zinc – tin is strongly binds against glass
(because of the instant wetting resin)
capillary breaks, the glue-wires contact is
usually lost)
5. expensive
5. inexpensive
This method was applied first for thicker platinum wires without using zinc. In this case,
the wires are stiff enough not to bend when adding the copper wire and there was a good
contact. However, for thin platinum wires this method is not suitable because the delicate
wire can break or turn away as the copper is inserted and hence no electrical contact is
established (see Figure 13 A, B). To overcome this problem, the narrow space between the
copper wire and the capillary tip was filled with zinc powder (Figure 13 C, D).
The ohmic resistivity of the Zn-to-Pt connection was found to be low and the electrode
typically worked reliable.
29
Fig. 13
Only solder alone can not connect the copper lead to the thin Pt wire (A, B).
Zinc microparticles can establish an intimate contact between the external copper lead
and the the glass-embedded platinum wire (C, D).
4.1.2. Chemical deposition of silver onto the body of precursor electrode
Well-adhering layers of Ag were accessible only when the glass insulation of Pt
microelectrodes was rubbed with emery paper (grade 320), wiped with soft tissue paper
and rinsed with a stream of acetone in order to remove the grinding dust. Obviously, this
treatment increased the roughness of the substrate herewith offering a better physical
support for anchoring the Ag deposit to the glass through microscopic fissures in its
surface.
In a small tube, 2 ml of 5% AgNO3 solution were then treated with 10% NaOH solution
until no more dark-coloured precipitate (Ag2O) was formed upon addition. The Ag2O was
converted into soluble [Ag(NH3)2]OH by slowly adding 28% NH4OH until the solution
became clear and colourless. Tollens’ reaction and formation of metallic silver was
induced by adding a few ml of a reducing agent, here, it is 10 % glucose. To enhance the
rate of Ag deposition, the body of the Pt microelectrode was preheated with a heat gun, and
then quickly immersed into the freshly prepared silvering bath for only a few seconds,
taken out and immediately dried with the handheld dryer. For uniform and compact Ag
layers the treatment had to be repeated multiple times. For further Ag deposition, the
microelectrodes were rinsed with distilled water before heating (Figure 14). As can be seen
from the Figure 14, the chemically deposited silver appeared as a uniform coating of white
colour. The deposits typically had a morphology reflecting to some extent the irregularities
of the abraded surface of the underlying glass and were mechanically sufficiently stable
not to be damaged by handling of the electrodes and performing electrochemical
30
measurements. However, to enhance the stability of the silver layer and extend its lifetime, the electrode surface can be covered with ordinary nail varnish.
Fig. 14
The transformation of the precursor electrode (left) into the silver coated electrode
(right) requires three steps: blowing hot air on the precursor (1), dipping into the Ag+ solution
(2), rinsing with pure water (3).
Finally, a copper wire was firmly attached to the silver layer using a heat shrinking tube
(Figure 15).
Optionally, thicker layers of silver could be deposited on pre-coated microelectrodes by
galvanic Ag deposition from a thiosulphate-based silvering bath (pH 10) (Figure 16). At
room temperature, a current density of 0.5-0.6 A/dm2 was applied to form well-adhering
and stable Ag deposits. Tips of Ag-coated microelectrodes were carefully polished on
emery paper (grade 2000) and polishing cloth soaked with alumina to re-establish a clean
electroactive Pt disk.
Reproducibly, a flat tip geometry was obtained with the 10-µm-diameter Pt wire well
centred in the glass insulation and the thin layer of chemically deposited Ag, respectively.
In the particular case shown, the total tip diameter was about 200 µm giving a RG value
(dtotal/dPt disk) of about 20. However, tips with diameters down to about 50 µm were
obtained by successively polishing them at an angle in order to reduce the thickness of the
glass insulation at the apex and then upright to expose the Pt disk.
A top view of the tip of a polished Pt/Ag electrode assembly is shown in the scanning
electron micrograph of Figure 17. The following experiments were performed with an
electrode identical to the one in Figure 18.
31
Fig. 15 The external connection of the coaxial silver layer.
Fig. 16 The thickness and morphology of the chemically deposited
silver layer can be adjusted by electroplating of silver in an
electrolysis cell with a silver anode.
32
Fig.17 Scanning electron micrograph of the Ag-chemically coated electrode.
Fig. 18 The completed coaxial working/reference electrode.
33
In order to verify ability of this electrode assembly to perform electrochemical
measurements a series of experiments were designed to address this problem. For instance,
Figure 19 compares two cyclic voltammograms of 5 mM [Ru(NH3)6]Cl3 that were
recorded in a single well of a 384-well microtitre plate (Figure 20) at a coaxial PtµWE/Ag-RE electrode assembly with either the Ag layer of the assembly as internal
pseudo reference (A) or a miniaturised Ag/AgCl 3 M KCl system external reference
electrode (B). In both cases, the CVs displayed the expected sigmoidal shape (steady state)
that is characteristic for microelectrodes. The half-wave potential (E1/2) for the reduction of
Ru3+ measured vs. the internal pseudo reference was shifted by about -60 mV as compared
with the external reference electrode. However, the shape of the CVs did not change
notably over time indicating that the coaxial Ag electrode is sufficiently stable to be used
as an integrated pseudo reference for voltammetric measurements, even without
chloridisation.
Fig. 19 Cyclic voltammograms recorded vs. the inner reference (A) and
an external Ag/AgCl reference electrode (B); 5 mM [Ru(NH3)6]Cl3 in 0.1
M phosphate buffer; 100 mV/s.
4.1.3. Applications of the coaxial Pt-µWE/Ag-RE in SECM
It was thought that this electrode could also be turned to the development of a robust and
easy to use (micro-)electrode arrangement supporting straightforward SECM imaging in
nanoliter volumes of solutions. To demonstrate the suitability of the coaxial Pt-µWE/ AgRE electrode assembly for SECM applications, it has been attached to the Z-positioning
element of the SECM and used as a scanning probe with integrated reference electrode in
electrolytes containing 5 mM [Ru(NH3)6]Cl3.
34
Fig. 20
Electrochemistry performed with the integrated Pt working / Ag
reference electrode assembly in a single well of a 384-well microtitre plate.
The Pt-µWE, was operated in the amperometric feedback mode and scanned at constant
height. To induce diffusion-limited reduction of the dissolved redox mediator, it was kept
at -350 mV vs. the integrated coaxial Ag-RE or, in control measurements, vs. the external
Ag/AgCl reference electrode. In a large-volume electrochemical cell, tip approach curves
were recorded in both configurations on insulating (glass) and conducting (gold) surfaces.
The resulting negative (positive) feedback curves were properly overlapping and in good
accordance with the curves from theoretical calculations (not shown). Furthermore,
approach curves were performed on gold using either an ordinary Pt disk microelectrode or
a Pt-µWE/ Ag-RE electrode assembly with the ring of silver as SECM tips. As can be seen
in Figure 21, the obtained positive feedback curves were almost perfectly lying on top of
each other. This clearly demonstrates that a recycling of tip-induced Fe2+ at the conducting
outer ring of silver does not disturb significantly the positive feedback interaction between
the Pt-µWE and a conductive sample surface.
For the given electrode geometry this was expected, since the distance between the Ag ring
and the Pt-µWE is about 100 µm which is far too large to be within the regime of the
electrochemical feedback of a 10 µm diameter microelectrode. In contrast, an alteration of
the feedback behaviour cannot be excluded when reducing the total tip diameter of PtµWE/Ag-RE assemblies below about 20-30 µm. SECM test trials were then carried out in
small droplets (< 1µl) of the mediator solution that was placed on top of an array of four Pt
band microelectrodes (length: 1 mm; width: 25 µm; spacing: 25 µm) (Figure 22).
35
Fig. 21 Approach curves that were recorded on a gold surface with (∆) the tip of
a coaxial 10 µm-diameter Pt-µWE/Ag-RE electrode assembly and (O) a 10 µm
diameter Pt disk microelectrode of similar RG value and with no silver deposited
on the insulation; scan speed: 1 µm/s; electrolyte: 5 mM [Ru(NH3)6]Cl3/0.1 M KCl.
Fig. 22 Schematic of the SECM experiment designed to scan with the coaxial electrode in
a 500 nl solution and of the Pt array band used as a substrate.
As usual, tip approach curves were used to position the tip of the Pt/Ag electrode assembly
in close proximity to the sample surface. Typically, a working distance was chosen at
36
which the amperometric tip current dropped off to about 50 % of the value measured in
bulk solution. Figure 23 shows a representative 3-dimensional feedback image of the Pt
microstructure that was acquired with the coaxial Pt-µWE/Ag-RE electrode assembly in a
droplet of 500 nl volume of mediator solution. The Pt bands are clearly visible with
negative feedback observed over insulating areas in the neighbourhood of the Pt bands and
positive feedback due to the regeneration of consumed mediator molecules above the
electrochemically active Pt bands.
Fig. 23
3-D SECM image acquired by operating a coaxial Pt-µWE/Ag-RE as dual
electrode scanning probe in the amperometric feedback mode on a Pt band microarray;
scan speed: 5 µm/s; 5 mM [Ru(NH3)6]Cl3) in 0.1 M phosphate buffer.
Successfully, imaging the Pt band microstructure provided the first evidence about the
operability of the coaxial SECM tip arrangement for measurements in small volumes.
However, we observed that solution evaporation became a critical parameter for image
acquisition due to rapid changes in the volume of the nl droplets. Accordingly, a raise in
mediator concentration took place, ultimately increasing the SECM tip currents in due
course of imaging. This effect is confirmed by Figure 26A showing a set of fifty line-scans
that were obtained on the Pt band microarray one after the other at scan speeds of about 5
µm/s. Within the one hour of the experiment, the amperometric tip current changed
virtually by a factor of two. Especially, with low scan speeds for SECM imaging, the
experiment will take considerable time and hence, a strategy to prevent solution
37
evaporation is of key importance when aiming for long-term SECM measurements in
microvolumes. Various methods have been proposed previously to stabilise small sample
volumes, among them, regulating the humidity in their environment by maintaining a
water-saturated atmosphere in specially designed closed chambers, adding glycerine to the
electrolyte to lower the water vapour pressure, or covering the solution with mineral oil8385
. Here, the tiny droplets of ruthenium solution were protected with a reasonably thick
layer of paraffin oil against evaporation. As shown in the schematic representation in
Figure 24 and 25, a large-diameter O-ring of appropriate thickness was used to keep the
paraffin oil in place and hinder it to spread that thin that the droplet would be exposed to
air and hence start to fade away.
Fig. 24 Schematic representation of SECM in nanoliter droplet. A layer of paraffin
oil serves as a protection against solution evaporation and the O-ring keeps the
electrolyte in the proper place.
38
500 nl of electrolyte
Pt band array
Fig. 25 Photographs showing the aqueous droplet over the four Pt band, all covered by paraffin oil
(left) and the immersed electrode positioned at the working distance over the Pt array (right).
This experimental design is simple, and primarily was chosen to gain maximal spatial
freedom for tip movements in X and Y direction. Certainly, the Pt-µWE was first placed
into the droplet before adding the paraffin oil to the cell to avoid a contamination of the
surface of the tip electrode with the oil.
Figure 26B is a clear proof that paraffin oil indeed offered an effective diffusion barrier for
water molecules and did not permit nl-sized droplets to evaporate and change their volume
significantly throughout SECM imaging. Again, fifty line-scans were taken successively
on the Pt band microarray at a scan speed of 5 µm/s but at this time taking advantage of the
aforementioned strategy of evaporation protection. Although the line scans were not fully
superimposed, the current values of the first and fiftieth scans differed from each other by
only 3-4 %. This was clearly indicating that the droplet volume and concentration of the
mediator could be kept reasonably stable for the duration of the experiment when using
coverage with paraffin oil, a prerequisite for successful carrying out long-term SECM
measurements in nl volumes.
4.2. Miniaturised Ag/AgCl reference electrode
SECM experiments performed in a conventional electrochemical cell required the use of a
pseudo-Ag reference electrode considering the bulky Ag/AgCl 3M KCl reference electrode
available on the market. It turned out that a pseudo Ag/AgCl reference can not be used in
oxidising media (such as ferricyanide solutions), because the electrode potential is greatly
affected. For this reason, a reference electrode had to be made, which is stable and small
enough to fit in the available holder of the SECM tip. To build a trustworthy Ag/AgCl 3M
KCl reference electrode, a Pasteur pipette was used as specified (Figure 27) together with:
39
a ceramic frit with a diameter about 1mm mm, shrinking tube, 3-4 cm of silver wire. The
short cylindrical frit (about 2 mm length) is sealed into the narrowed part of the Pasteur
pipette with the flame of a portable piezo-torch.
A
B
Fig. 26
Droplet SECM carried out on a Pt band microarray in 500 nl droplets of a 5 mM
[Ru(NH3)6 ]Cl3 in 0.1 M phosphate buffer solution with a coaxial Pt-µWE/Ag-RE as scanning probe.
50 line scans in (A) were recorded with the droplet exposed to air while the ones in (B) were
obtained using a layer of paraffin oil to protect the droplet against solution evaporation.
The chloridisation of the silver wire was carried out in a typical solution of 3M KCl and
0.1M HCl in water (1:1 volumes) with a Pt coil as counter electrode. A relative thick and
adherent layer of AgCl is obtained in two potential steps: 1 min at 5 V and then 10 min at
15 V.
The shift of the electrode potential measured versus a commercial Ag/AgCl electrode is
within few mV. The reserve of Cl- (3M KCl) inside the capillary is high enough keep the
potential constant and to sustain low currents for long time in a two electrodes set-up as
usually used. A similar reference electrode was chosen as reference electrode for all
experiments described in the next chapters.
40
Fig. 27
Simple, four-steps procedure for manufacturing of a Ag/AgCl 3M KCl reference
electrode; Pasteur pipette (1), storing tube (2), electrode (3), frit (4), glass cutter and the cutting
line (5), complete electrode (6). The syringe shows the way of refilling the capillary with fresh
KCl. This operation can be done easily at anytime.
41
Enzyme microstructures
5. Micropatterning and microelectrochemical characterisation
of biological recognition elements
5.1.
Enzyme Microstructures
A biosensor is intended to be specific to a unique substrate (analyte). However, real
samples (physiological fluids, waste waters, wine etc.) are usually made up of multiple
components and monitoring all of them would require a bundle of biosensors. The size and
large number of different biosensors necessary to perform a complex analysis is a hurdle
for small amounts of samples (Figure 28). The actual technology of micropatterning
Biological Recognition Elements (BRE) such as photolithography, soft-lithography, selfassembled monolayers, piezo-microdispensing permits the creation of multi-analyte arrays
with densely packed biosensors by immobilising BREs onto different substrates and
preserving the activity of these molecules. Thus, an array is a promising candidate for
becoming an ideal tool for detecting a large spectrum of analytes with a miniscule device.
The successful marriage of biology and microelectrochemistry will result in improved
biosensing devices that can have wide applications. This marriage, however, will require
microfabrication methods that effectively assemble sensitive biological components (e.g.
enzymes, nucleic acids, proteins, and viable cells) onto suitable substrates. The work
described throughout this subchapter was focussing on the application of polymers as
structural materials used to entrap enzymes as recognition element.
One example that illustrates our approach is the use of a copolymer of ethylene and vinylacetate (Vinnapas® EP16 W1) and glucose oxidase (GOD) for the assembly of
micropatterned enzymes. The EP16 offers thin-film properties that allow it to be locally
deposited onto different surfaces in response to an applied voltage on a piezo
microdispenser. Once the enzyme and the polymer are mixed, the enzyme can be locally
assembled onto a given surface. SEM (Scanning Electron Microscope) and SECM
micrographs have demonstrated that micro-deposition allows enzyme assembly to be
performed with high spatial selectivity while the deposition conditions are sufficiently mild
to retain the molecular structure of the enzyme (the activity of trapped enzyme had been
high enough to produce detectable amounts of hydrogen peroxide).
1
Wacker Polymer Systems GmbH & Co. KG, Burghausen, Germany
42
Fig. 28
Schematic showing the power of miniaturisation in reducing the
number of individual sensors and laboratory space.
43
About enzymes
5.1.1. About enzymes
Due to its unique 3-dimensional structure, each enzyme is specific to its substrate or to
structurally similar compounds. Enzymes and substrates need proper circumstances to
interact with each other.
In the second half of the XIXth century, E. Buchnera had
investigated the alcoholic fermentation of sugars and proved the yeast cells are not
necessary to ferment glucose, fructose or maltose. A juicy extract of brewer’s yeast is able
to perform this task. His belief was that a protein, named by him zymase, must be
responsible for this process. Inspite of XIXth century dynamism, lack of adequate
information and minimal equipments made the separation of enzymes from cells and the
structural analysis complicated and thus explaining why Buchner had doubts in
considering the zymase as a member of enzyme family. It was a fact that “there are
important differences between fermenting action and the action of ordinary enzymes.
The latter is solely hydrolytic and can be imitated by the simplest chemical means86”.
Well, he was wrong but this example intended to briefly give a view of the problems and
ideas that scientists had encountered about 100 years ago working on enzymes.
The biological processes that occur within all living organisms are chemical reactions, and
enzymes regulate most. Without enzymes, many of these reactions would not take place at
a perceptible rate. Enzymes are remarkable catalysts! Not only do they effectively
accelerate the rate of the reaction, but they also limit the potential side reactions so that the
yield of a reaction is essentially 100%. The overall energy of the reactant interaction with
the active site can be determined from the equilibrium constants of the reactant-enzyme
complexes [ES]. The equilibrium constants are usually in the order of 102 – 1010 M which
corresponds to free energies of interaction on the order of -12 to -60 kJ/mol. As can be
noticed, these energies are approximately 10 to 15 times lower than those associated with
covalent bonding.
Enzymes catalyse all aspects of cell metabolism. This includes the digestion of food, in
which large nutrient molecules (such as proteins, carbohydrates, and fats) are broken down
into smaller molecules; the conservation and transformation of chemical energy; and the
construction of cellular macromolecules from smaller precursors. Many inherited human
diseases, such as phenylketonuria87 (PKU - a genetic disorder that results in deficiency of
an organism to convert phenylalanine to tyrosine), albinism (a genetic circumstance which
a
Eduard Buchner (Munich, Germany 1860 – 1917, Munich) won the Nobel Prise in 1907 for “his
biochemical
researches
and
his
discovery
of
cell-free
fermentation”
(see:
http://www.nobel.se/chemistry/laureates/ 1907/buchner-bio.html)
44
About enzymes
results in a lack of melanin pigment in the eyes, skin and hair of both animals and humans)
and many others, result from a deficiency of a particular enzyme.
Enzymes have valuable industrial applications, the fermenting of wine, leavening of bread,
curdling of cheese, or brewing of beer have been practiced from earliest times, but not until
the 19th century were these reactions understood to be the result of the catalytic activity of
enzymes. Medical applications of enzymes make the metabolism products monitoring
easier and less stressful to the patients (one example to be mentioned is the biosensor for
glucose). In the early stages of the studies on fermentation, the notions “enzyme” and
“ferment” coexisted88. Latin and Greek inspired innumerable scientists over time when
looking for a name to be given to a new substance, living creature or phenomenon. And
this is exactly what happened to enzymes: enzumos is a Greek word meaning “yeast” and
fermentum i.e. a Latin word having the same meaning.
Monitoring health-relevant molecules in body fluids became accomplishable in a simple
manner due to the high (sometimes absolute) specificity of enzymes that make them
perfect tools able to function in complex environment such as blood.
Enzyme classification
A close view on the functional structure of enzymes shows proteins are not sole
components but often accompanied by other groups like FADH2, NADH, porphyrins.
Some enzymes made of pure proteins are able to attach and convert the substrate into
product. However there are a large number of enzymes that use special non-proteic units to
establish a catalytic reaction. This is the basis of an enzyme classification (Figure 29) as
briefly depicted below:
1. Holoproteinsb
- are enzymes entirely built from aminoacids
2. Proteides
- contain beside a protein (apoenzyme) that is inactive but specific to a substrate, a
chemical part that undergoes enzyme specific reaction namely cofactorc. Upon
attachment type, the cofactor is called a prosthetic group, if binds covalently and
is called coenzyme if binds loosely (see note 1).
b
c
From Greek “holos” that means “whole”
The cofactor apart from enzyme is able to catalyse the reaction of numerous substrates.
45
About enzymes
Fig. 29 Enzyme classification. Enzymes only made of proteins are called holoproteides
whereas enzyme containing proteins and other compounds are named as proteides. An
apoenzyme is inactive as long as the cofactor is apart. The cofactor is covalently or
loosely linked to the apoenzyme and thus it is named prosthetic group or coenzyme
respectively.
Enzyme coding
“Enzyme Committee” (EC)89 that is a subdivision of “Nomenclature Committee of the
International Union of Biochemistry and Molecular Biology” (NC-IUBMB) established
the enzyme nomenclature. The large variety of enzymes determined EC to assign each
enzyme a code made up of four numbers (say A.B.C.D.). “A” indicates one of the 6 major
classes (see note 2). “B” and “C” codes the affiliation to a sub- and a sub-subclass of
enzymes. “D” is just a numerator that shows the place of enzyme in the list with enzymes
of the same sub-subclass. According to EC nomenclature, one can refer to glucose oxidase
as EC 1.1.3.4. Commonly, the glucose oxidase is known as GOD. This name will be used
in the following parts of this work.
Enzyme activity
Enzyme activity means enzyme ability to transform a substrate into a product. Either a
product or a reagent (even the substrate) is experimentally detected in order to measure,
under controlled conditions, the enzyme activity. It is influenced by many factors
(substrate, solvent, temperature, inhibitors, ionic strength, pH and cofactor). Two kinds of
disturbing factors of enzyme activity: 1) specific to each individual enzyme as substrate,
cofactor, and inhibitor. For most of enzymes, some substrates can react with but at
different rates. This justification is valid for the cofactor and inhibitor too; 2) factors that
control the protein conformation as solvent, ionic strength, pH and temperature affects the
protein backbone shape. Temperature influences both the kinetic and the protein
46
About enzymes
conformation. The enzyme activity has to be measured at the optimal temperature of the
given enzyme (see note 3)! Dissolved oxygen must be taken into account for the enzymes
that use it during the substrate transformation.
Enzyme activity related definitions
The amount of enzyme that is involved in a biochemical reaction is difficult to determine
in terms of grams of enzyme, as its purity is often low and not to mention that parts of it
might be in an inactive state. Parameters that are more relevant are the activity of the
enzyme preparation. These activities are typically measured in terms of the activity unit
(U), which is defined as the amount of substrate (micro-, nano- or pico- moles) converted
by a certain enzyme quantity within a unit time. An international unit (I.U.) corresponds to
a unit time of 1 minute. Another unit, which is used but did not receive widespread
acceptance is the katal (kat) even though recommended by International System of Units
(SI). One katal will catalyse the transformation of 1 mol of substrate per second. The old
unit (U) was generally nameless for approximately 30 years and only occasionally called
“international unit.“ It uses minutes instead of seconds as its unit of time, which is not
keeping with SI or with the usual way of expressing rate constants in chemical kinetics.
Sometimes non-standard activity units are used such as Soxhet, Anson (AU – Anson unit),
Kilo Novo, which are based on physical changes for instance lowering viscosity, and
supposedly better understood by industry. These units are gradually falling into disuse. Of
a practical interest is the specific activity defined as the number of units corresponding to 1
milligram of enzyme preparation (U/mg).
Enzyme kinetics
Enzyme catalysed reactions are basically chemical reactions and a kinetic study is
meaningful when aiming to understand the reaction mechanism or calculating the yields.
“Don’t waste clean thinking on dirty enzymes” said E. Rackerd and he meant that
detailed studies on how enzyme catalyses the conversion of one substance to another is
generally waste of time until the enzyme has been purified from other substances that make
up a crude cell extract. The mixture of thousands of different proteins released from a
disrupted cell that typically contains several that direct other rearrangements of the starting
material and the product of the particular enzyme’s action. Only when one has purified the
enzyme to the point that no other enzymes can be detected can one feel assured that a
single type of enzyme molecule determines the conversion of substrate to product and does
nothing more90.
d
Efraim Racker (1913 Neu-Sandez, Poland – 1991 Syracuse, New York, USA) American scientist that
isolated, from mitochondria, the ATP synthase in 1960.
47
About enzymes
Experiments show that:
a. for low substrate concentrations S, the reaction rate is proportional to S,
v = k⋅⋅S
(first order reaction);
b. for high substrate concentrations S, the reaction rate is independent on S,
v=k
(zero order reaction);
Herein, E stands for the enzyme and S for the substrate. Conclusion: although the net
reaction is simply S
P, it must actually have a mechanism with steps that involve the
enzyme reaction. Enzyme kinetics obeys Michaelis-Menten equation (L. Michaelis and M.
Menten, 1913)e. They proposed the following reaction scheme:
k1
S+E
k-1
k2
[SE]
P+E
In the conversion of S to product P, the enzyme undergoes no net change.
Symbols have the following meanings: S – substrate; E – enzyme; [SE] – activated
complex; P – product; k
1
– constant of activated complex formation; k
activated complex break-up; k
2
-1
constant of
– constant of activated complex consumption. From the
above listed experimental data and assuming that the formation and breakdown of the [SE]
has reached a value that is no more changing during reaction (steady-state approximation),
than the following equation can be derived (equation 5):
vP = k2
[E0 ][S ]
K M + [S ]
Eq. 5
Accordingly, the rate of the enzymatic reaction depends linearly on the amount of enzyme
added and on the amount of substrate as well.
Particular cases:
1.
[S ] >> K M
→ vP = vP max = k 2 [E0 ]
2.
[S ] << K M
→ vP = k 2
3.
[S ] = K M
→ vP =
[E0 ][S ]
KM
v P max
2
e
Nowadays, both more general and more specific models of enzyme kinetic exist for the enzymes that do not
follow the mechanism described by Michaelis-Menten equation.
48
About enzymes
The third equation can be used to define KM as substrate concentration at which the
reaction rate is half of the maximum rate. KM (normally expressed in mM) is a short hand
k −1 + k 2
and therefore has no dimension.
k1
of:
Note:
-
the fundamental characteristic of the Michaelis-Menten mechanism is a transition
from first-order to zero-order kinetics near a critical substrate concentration that is
KM;
-
KM gives quick information about enzyme affinity to the substrate (the lower the
value of KM, the stronger is the binding between substrate and enzyme and hence
more product is obtainable in a given time);
k −1
as KS (the dissociation constant of [ES]);
k1
-
it is useful to refer to
-
KM indicates the required substrate concentration to achieving half of the maximum
reaction rate;
-
when determining KM, the optimal pH and temperature must be set;
-
for the majority of enzymatic systems k2 is small compared to k-1 and thus KM ≅
KS;
-
the steady-state approximation, i.e. [SE] = ct., is true as long as the enzyme
concentration is small compared to the concentration of the substrate.
Artificial enzymes
Surprisingly, it has been possible to design assemblies of inorganic or organic molecules
that have remarkable catalytic properties and high specificity to substrate. Enzymes are
isolated and purified from plants or animals and due to abundance of other biomolecules
this process is complex and time consuming. A great advantage could be given by
possibility of on-demand straightforward synthesis of enzyme-like materials. This issue
already raised lot of attention in the last decades and achievements till date are
encouraging. Briefly, three approaches towards the synthesis of such non-natural catalyst
that mimic real enzymes are presented.
Inorganic
An artificial enzyme analogue of Nafion/lead-ruthenium oxide pyrochlore (Py) chemically
modified electrode (NPyCME) was synthesized by in situ precipitation through blocking of
Nafion’s hydrophilic zones91.
49
About enzymes
Organic
The plausible translation of the principle of enzymatic catalysis to artificial catalysts has
attracted also organic/macromolecular scientists since the 60’. One should not forget that
the exclusive properties of enzyme rely on their polymeric structure! Synthetic polymeric
molecules are attractive chiefly because of their chemical and physical properties (can
withstand heat and chemical attacks)92. Moreover, such polymers are obtainable with little
effort for industrial production. In principle, a cross-liked polymer is structured around a
molecule that acts as a template (for best performances of the artificial enzyme, it should
be a transition state analogue of the aimed enzymatic reaction). The monomers are bearing
functional groups that can later interact with the template through covalent or non-covalent
binding. After removal of the template, an imprint containing the functional groups in a
certain spatial orientation remains. The polymer has to have a high degree of cross-linking
in order to preserve the shape of the template. Otherwise the cavity will shrink and the
substrate will not fit in.
Biochemical
It is well known that antibodies are protein complexes used by the immune system to
identify and neutralise foreign entities (called antigens) that accidentally enter a living
organism. Each antibody is binding only a single type of antigens in a similar way as an
enzyme binds its substrate. This feature lead to the idea of creating an artificial antibody
(also: abzyme, catalytic antibody or catmab – from catalytic monoclonal antibodies). So,
how is this accomplished? We shall imagine one is aiming to generate a catalyst for a
reaction having a transition state [TS]∗. In brief, the following steps are required:
1. a substance resembling the structure of the transition state is synthesised (known as
hapten/haptenef;
2. the hapten is then transformed into an antigen by covalent binding of a polypeptide
(usually bovine serum albumin, BSA);
3. subsequently, the newly formed antigen ([TS]∗-BSA) is injected in small portions
in a mouse;
4. the antibody is isolated from the serum of the mouse and used as a catalyst.
The first abzymes were reported in 1986 and since then various types were described93,94.
f
Hapten (from the Greek word “haptein” - to fasten) is, formally, an incomplete antigen that can stimulate
the antibody production only in combination with a particular protein.
50
Glucose oxidase
5.1.2 Glucose oxidase (GOD)
It is a flavoenzyme1 that catalyse the following reaction:
β-D-Glucose + GOD-FAD
δ-D-gluconolactone + GOD-FADH2
Once a GOD molecule had converted a single glucose molecule into gluconolactone, it is
not any longer able to transform more glucose. The redox moiety of GOD changes its
redox state from oxidised to reduced as glucose (reduced form) turns into gluconolactone
(oxidised form). However, GOD can transform an unlimited number of glucose molecules
by the supportive action of dissolved molecular oxygen. It is present in any solution unless
it is purposely removed (physically or chemically) and re-oxidise GOD back to its active
structure with concomitant formation of hydrogen peroxide:
GOD-FADH2 + O2
GOD-FAD + H2O2
The dissolved molecular oxygen is a co-reagent in this case. A glucose biosensor with
improved electrode kinetics is calling for controllable concentration of the co-reagent and
high diffusion towards the core of GOD. New developments in glucose monitoring of
blood are aiming at finding a replacement for the natural co-reagent with electrochemically
active complexes of transition metals such as osmium or ruthenium.
Since the GOD´s discovery as an “antibiotic”, (shown subsequently to be due to peroxide
formation) there has been a wide interest in GOD chiefly because of its value in glucose
determination. Although specific to β-glucose, it is employed to determine total glucose
because as a result of the consumption of β-glucose, the α- form from the equilibrium is
converted to the β- form by mutarotation. Structurally, glucose oxidase consists of two
identical polypeptide chain subunits each of them containing one Fe atom and one FAD
(Figure a). It works in a wide range of pH, but optimal at pH 5.5. As inhibitors Ag+, Cu2+,
Hg2+ are the most important. The GOD activity is colorimetrically detected by the increase
in absorbance at 460 nm resulting from the oxidation of o-dianisidine (highly toxic
compound which can cause skin irritation and sensitisation) through a peroxidase coupled
system. An orange colour is generated in this two steps reaction. One unit of glucose
oxidase causes the oxidation of 1 µmol o-dianisidine per minute at 25oC, pH 6. The KM is
about 33 mM and the turn-over number is approximately 2.3x104 molsubstrate /second ⋅
molenzyme.
1
FAD is the acronym of Flavine-Adenine Dinucleotide; FAD is a member of the adenilic acid derivates that
play a key role in biochemical processes as cofactors.
51
Glucose oxidase
Glucose oxidase is perfect to work with as a trial-enzyme because of its high stability in
solution. Dry preparations having a yellowish colour, are stable for years when stored cold.
However, it is incompatible with strong oxidising agents and is better to keep it away from
moisture. Besides these, the low cost, makes it affordable by many laboratories (104 units
costs 20 € or 106 costs 900 €; from Sigma Product Catalogue for Germany 2002-2003).
Only glucose oxidase isolated from Aspergillus niger2 was used in the present experiments,
described in chapter 5.
About FAD
FAD’s (Flavin Adenine Dinucleotide) molecular structure and redox behaviour are shown
in Figure a. FAD could be seen as being made up of riboflavin residue (dark blue) and an
ADP (adenosine diphosphate, shown in red). The redox active parts are coloured in
magenta and located on the isoalloxazine ring (blue, on the right side) of FAD. R denotes
the ADP-[CH2-(CHOH)3-CH2)].
NH2
ADP residue
N
O
O
H2
P O C
O
O
N
OH
P
OH
OH OH
CH2
FAD
N
O
oxidate state
R
N
H3C
N
H3C
N
O
N
N
H
O
CHOH
-
2H+ /
-
+ 2H+ / + 2e-
2e-
CHOH
R
H
H3C
N
N
H3C
N
CHOH
CH2
H3C
H3C
N
O
N
N
N
H
H
O
FADH 2
O
N
H
O
reduced state
Riboflavin (Vitamine B2) residue
Fig. 30 The molecular structure of FAD. On the left is shown how the riboflavin
residue undergoes redox reaction.
2
Aspergillus niger is a fungus, non-pathogenic to humans, causing the black mould on certain types of fruit
and vegetables.
52
Patterning GOD by means of piezo microdispenser
5.1.3 Patterning GOD by means of piezo microdispenser
5.1.3.1. Aim
The main goal of the effort is directed to enzyme micropatterning and to demonstrate the
possibility of constructing a complex self-containing biosensor array for multiple
applications: monitoring of drugs, pollutants, or any other compound of interest in water,
food or physiological fluids. Before proceeding to present specific details of this work, the
reader should be acquainted with the enzyme micropatterning techniques available up to
date and their advantages/disadvantages.
Since biosensors are wished to become simply tools for home-monitoring of glucose, urea
and other substances, increasing attention has been given to the formation of localised
biomolecule microstructures in the last decade. Therefore, simple tools coordinated by
hand (capillaries)95 or sophisticated machinery controlled by computers (laser confocal
microscope96,97, SECM, microdispenser) are used to create basic (spots) or complex (lines
or grids) microstructures. The spatial resolution of such micro/nano structures is
determined by precision in controlling the distance between two neighbouring spots.
Consequently, hands-on devices can not compete with the highly precise micropositioning
stepper motor or piezo elements for creating biosensor architectures with high spatial
resolution. However, two ways of placing biological recognition elements onto a surface
are available so far:
Active placement
Active placement refers to the procedure of delivering small droplets of biomolecule
directly at the point of interest (capillary, microdispensers).
Indirect methods
These methods require a chemical pre-modification of the surface aiming at the localised
immobilisation of biomolecules. The affinity of surface is tuned with an appropriate
functionality to facilitate the specific binding of the biomolecules (for instance, self
assembled monolayers of thiols).
It was already shown (M. Mosbach, S. Gaspar98), by means of the substrate generation –
tip collection mode of SECM, that the enzymes are preserving their activity after being
shot through the microdispenser nuzzle (withstanding the shear forces and elevated
pressure). Excellent 3-dimensional SECM images of different surface confined enzymes
were achieved. Nevertheless, the enzyme concentration was not optimised. Conversely, the
polymer matrix used to entrap the enzymes at a certain surface had not the optimal
concentration.
53
Following their foot steps, I sought to further develop the complexity of the enzymatic
patterns and to study the possibility to increase the dynamic rangea of the microsensor
arrays.
Note
An enzyme that is interacting with the substrate in bulk will generate a certain amount of
product. If the same quantity of enzyme is by any way grafted to a surface, then the speed
of the product formation is smaller as in bulk. Now, when one reduces the dimensions of
the catalyst patch, one also decreases the amount of product that can be produced within a
given time, and therefore, one increases the difficulty in analysing the product. Typically, a
spot of GOD preparation pasted at a surface by means of piezo microdispenser contains
6.25 x 10-16 mol of GOD (for 1 mg/ml GOD in the spotting mixture and a droplet of 100
pl). In ideal case, with the enzyme free in a large volume of solution, 6.25 x 10-16 mol of
GOD will generate roughly 40 million product molecules per second (and of course will
consume the same number of glucose molecules). If these molecules diffuse away into a
volume of 2 ml (of the electrochemical cell used in SECM measurements), then there is an
analytical difficulty in measuring the product in the resulting ≅ 2 x 10-10 M solution. As
already mentioned, this minute quantity represents an apex for the given amount of GOD
and reaction time. The available product is even less if the enzyme is immobilised into a
thin film of polymer. Several factors merge their contributions to make the reaction
sluggish:
a. the diffusion of the substrate towards the enzyme molecules is hindered by
the film and the surface on which the enzyme lies;
b. enzyme is not free to accommodate its position with regard to the incoming
substrate (as would happen in bulk where it can rotate and translate in any
direction);
c. as D-glucono-1,5-lactone is formed and is accumulating in the film, the
enzymatic reaction is slowed down because this product is (a weak)
inhibitor of GOD.
Taking into account these facts, it becomes clear why a local electrochemical probe must
be placed close to the enzymatic patches in order to be able to measure the enzyme
activity/substrate concentration through the redox active products. A large disk-shaped
electrode is not appropriate for investigating micrometric structures (because it could
a
The dynamic range is the ratio of the maximum value versus the lowest detectable value of a specific
parameter. Typically, is measured in B (Bel) or dB (decibel), in a logarithmic scale: B=lg(value1/value2).
54
record signal from multiple enzyme microspots at the same time) and hence
microelectrodes were preferred (active diameter 10 µm). Obviously, the microelectrode is
moved across the enzymatic microarray with a micropositioning system (see chapter 2).
We shall find in chapter 3 and 4 a detailed description of the microelectrodes
properties/manufacturing.
5.1.3.2. General consideration about GOD microdispensing
Home-made microdispensing set-up
The schematic view of a home-made microdispenser control set-up is depicted in Figure
31. The trigger signal generated in the computer was send to the wave generator where the
power output of the power supply was modulated in order to produce on-demand-release
of droplets. The rely card made possible the control of two stepper motors via a single
motor-card. A home-made electronic board combined the carrier signal (from the wave
generator) with the power applied to the piezo actuator.
Fig. 31 Schematic of the control set-up of the microdispenser.
The surface chosen for patterning glucose oxidase was gold sputtered on silicon wafer.
Improved adherence of the gold layer to the silicon support was typically attained by using
an intermediate layer of titanium between Au and silicon. Such plates were cut in
rectangular pieces with appropriate dimensions, 0.5 x 0.5 cm2 (chip). A plastic holder
allowed the placement of 5 chips in a row, at a distance of 1 cm from the head of the
microdispenser. Once a chip was spotted, the next chip was positioned in the shooting
direction of the microdispenser. In this way, it was possible to overcome the drying of the
enzymatic mixture at the nozzle. This unwanted clogging had to be avoided because
normally it compromised the chips by shooting droplets out of the line. Repairing/cleaning
the dispenser is not easy and successful any time. The mixture containing the enzyme was
prepared by adding 1 mg GOD and 2 mg EP16 in 1 ml tri-distilled water. The yellowmilky solution did still contain polymer aggregates with an overall size that could block the
55
microdispenser. Such big particles have to be removed prior to shooting. It was noticed
that a filter unit of 5 micrometers pore-diameter retained not only large amounts of the
polymer but even enzyme. Alternatively, centrifugation of the enzyme-polymer mixture
was found to be a better choice. Centrifugation for half a minute at a speed of 700
rotation/min (for a normal lab centrifuge) was enough to settle the large aggregates. The
clear supernatant had been carefully sucked into a 1 ml syringe and further used to fill the
chamber of the microdispenser. Although the droplets are visible to naked-eye, while
shooting they have to be well illuminated with a fiber optic light from a side in order to see
if they are ejected from the microdispenser. Once a droplet touches the surface of the gold
it appears as a glittery spot for 1-2 seconds till it gets dried. The polymer matrix is
hydrophilic and thus is capturing a lot of water around. In this case the GOD is rather
mobile (swimming between polymer chains) and could dissolve away from the polymer if
not allowed to cure for 20-24 hours before being further used. This GOD preparation had
enough activity for months even after keeping it at room temperature. However, to avoid a
probable loss of enzyme from the polymeric matrix all enzyme micropatterns herein
presented were cured 24 hours.
4.1.3.3. Simple GOD microstructures
A PC in combination with Windows software programmed in Microsoft Visual Basic 3.0
(Microsoft, Unterschleißheim, Germany) was used for the control of all system parameters
Fig. 32 Snapshot of the software interface used for micropatterning with
the piezo microdispenser.
56
(like: number of droplets per spot, distance between and the number of lines; number of
spots per line), and also for data acquisition. A snapshot of the user-friendly interface of
the mentioned software is shown in Figure 32.
With this automated device one could simply create a variety of microstructures of GOD
captured in the Vinnapas EP16® polymer dispersion. The SEM micrographs shows, arrays
of spots (Figure 33A) with different enzyme concentration or even grids (Figure 33B) with
variable load of enzyme were achieved. The polymer film is thinner in the central part than
at the rims (see Figure 33C) where apparent heights of 1-2 µm were measured for with the
electron-beam parallel with the surface of the microstructure (the apparent heights was
used because the enzyme-polymer film was not metalised by sputter-coating prior to
imaging). Figure 33D gives a closer look at the nanoscale structure of the sputter coated
gold. As plainly visible, the gold surface is not smooth but full of cracks and gaps. It is
possible that these features have a certain role in stabilising the polymeric layer.
Fig. 33 SEM micrographs of spots and grid of piezo-dispenser patterned GOD
containing polymer. The dispensing mixture contains 1 mg/ml enzyme and 2
mg/ml EP16 W (A, B, C). Number of droplets / spot is increasing from the upper
right corner to the lower left corner of the image (A, B). Close-up of a single spot
(C). Gold sputtered surface appears as broken up. This feature could explain
why EP16 adheres so well to this gilded surface (D). Scale bar represent: 200
µm (A, B), 20 µm (C) and 1 µm (D).
57
Gold is a material that has a certain affinity to many compounds, especially to organic
functional groups. Is it possible to immobilise glucose-oxidase on a gold surface without
resorting to any polymers? To address this question, two aqueous solutions were prepared
one containing only polymer (2 mg/ml) and other only enzyme (1 mg/ml). The idea was to
dispense the droplets from each preparation as spots and then to compare the morphology
of the corresponding films. A well adhering and rather homogenous covering was obtained
with the polymer suspension in water (Figure 33A). Surprisingly, the enzyme alone formed
solitary accumulations and hence indicating low natural affinity for gold (Figure 34B)
Fig. 34 SEM images corresponding to 2 mg/ml pure polymer spot (A) and pure 1
mg/ml GOD spot (B). Scale bars are identical and represent 20 µm. Both were
aqueous solutions.
A versatile tool for micropatterning biological molecules is the microdispenser, it offers a
straightforward procedure to create complex geometrical structures automatically, or semiautomatically or to generate as already mentioned, enzyme gradients. For this, multiple
droplets having identical composition are directed towards the same area of the target
surface. Now, precautions must be taken owing the fact that 1) the overall enzyme activity
is not increasingly proportional to the number of droplets/spot and 2) the diameter of the
spots is directly proportional to the number of droplets/spot; these two issues will be
discussed in more detail.
58
Discussion
Enzyme exposure to the substrate and its relation to diameter/number of droplets
In order to optimise the glucose biosensor array, the enzyme concentration in the spots was
varied. Two approaches could be used to change the biological recognition element spot
concentration; firstly, it can simply be achieved by increasing its concentration in the
shooting solution, but the disadvantage is the necessity to refill the dispenser every time
and for each concentration. Secondly, the target place can be shot multiple times and the
advantage is that the complex arrays can be created without refilling the microdispenser. A
series of SEM micrographs shows the spots with variable diameters obtained by increasing
the number of droplets deposited on each target place. For the same composition of the
enzyme solution (1 mg glucose oxidase and 2 mg polymer per ml of tri-distilled water), the
diameter of the spots increases with the number of droplets (say n) as depicted bellow
(Table 3):
Tab. 3
n
1
2
3
Diameter (µm)
75.7
97.0
133.3
Note: the numbers on this table are valid only for the mixture composition described above.
Is the enzyme activity increases with n? (It is about activity of a spot as active entity not
about the specific enzyme activity, which is not affected by n). A negative answer could
suggest a misuse of the enzyme, or in other words that is useless to try to increase spots
activity in this way. Theoretically, a larger volume will produce a larger diameter spot and
considering a constant film height one can calculate the ratio of the droplet/spot radius. Let
us assign the following parameters for droplet and spot (which is in fact a disk) (Table 4):
Tab. 4
Droplet
Spot (Disk)
V
volume of a single droplet
volume of a single spot
R
radius of a single droplet
-
R'
-
radius of a single spot
h
-
height of the spot
59
The volume is the only parameter that has equal values for both droplet and spot, and
hence the possibility to find a relation between the droplet volume and the corresponding
spot radius (Figure 35).
Fig. 35 The droplet and its corresponding spot (disk).
Radius of the disk is a function of the droplet volume.
Similar ideas lead to an equation describing the relation of any two spot radii when their
volume ratio is known.
R2'  V2 
=  
'
R1  V1 
1/ 2
n
=  2
 n1



1/ 2
The ratio of the volumes is equal to the ratio of the number of droplets and this explains the
second term of the upper equation. For instance, if the volume is doubled then the radius
ratio is 2 = 1.41 . For a triple volume the resultant ratio is 3 = 1.73 . These are theoretical
values and will be used as a reference to calculate the deviation of the real spot diameters
and to explain why the “spots activity” is not proportionally increasing with parameter n
(Table 5). Rn' is the radius of a spot made of n droplets.
Tab. 5
Theoretical
Experimental
Remarks about film
R2' R1'
1.41
1.28
ticker as single drop
R3' R1'
1.73
1.76
expected thickness
Remarks presented here are roughly valid for bigger droplets. Indeed, a larger droplet will
splash and spread more around the impact area as compared to a small one. However, for a
double sized droplet, the film is a bit thicker as for a single droplet of the same solution.
Consequently the active substance in the mixture is less exposed to the solution in the case
of a double droplet as for single one (Figure 36).
60
This behaviour will decrease the response signal of a biosensor (an amperometric current
in this case) because of the “hidden” enzyme in the thick film. A well permeable polymer
film usually is enough to make the active compound reachable from the bulk of the
substrate solution. Vinnapas EP 16 polymer dispersion had a good behaviour in terms of
permeability for water and glucose.
Fig. 36 The exposure of the enzyme in dependence
from polymer film thickness; (A) thin film exposes it
better as a tick film (B). Light coloured molecules stand
for “hidden” enzyme.
61
Visualisation of GOD microstructures by SECM
5.1.4 Visualisation of GOD microstructures by SECM
An indication that surface immobilisation of GOD is possible with the use of a piezomicrodispenser is only half the way showing that the construction of a biosensor could be
done in an easy and flexible manner by ink-jet printing. It is important to demonstrate that
this approach would preserve the natural and main important feature of an enzyme: its
catalytic properties. Thus, it was proceeded to probe the activity of the surface confined
glucose oxidase by measuring the released hydrogen peroxide from a GOD microstructure
in the presence of glucose. The SECM was the chosen instrument for demonstrating a set
of quantitative and qualitative measurements of micropatterns of glucose oxidase.
Microlines with variable enzyme content
The microdispenser chamber was carefully filled with a mixture of GOD (1 mg/ml) and
Vinnapas® EP16 polymer dispersion (2 mg/ml). To obtain continuous lines, each new
droplet was shot out of the nozzle along the X axis with a displacement of 50 µm (for an
average spot diameter of 75 µm). Typically, two lines were spaced at 500 µm to each
other. The freshly dispensed microstructures were allowed to cure overnight at ambient
temperature, before being studied by SECM. Although such an arrangement of
enzyme/polymer microstructures can still be observed with the naked eye, it is better to
inspect them with the aid of a light-microscope to check for possible errors (such as
unwanted satellite droplets or discontinuities in the pattern). Integrated scale bars along the
length were represented by metallic wires with known diameter (either 500 µm Ag, or 10
µm Pt) (Figure 37).
Fig. 37 Optical photographs of GOD/EP16 polymer microstructured on gold
surface. The spotting solution contained 1 mg/ml GOD and 2 mg/ml EP16. Metallic
wires (Pt 10 µm and Ag 500 µm) were used as scale bars.
62
To facilitate the SECM measurements these enzyme modified gold chips had to be first
placed into an electrochemical cell (EC cell). For this experiment, it could be a simple Petri
dish, a small cup, or a cell screwed from the top. Once the chip (5x5 mm2) is in place, the
two electrodes (a 10 µm Pt disk-shaped ultramicroelectrode and an Ag/AgCl pseudoreference electrode) necessary to perform electrochemical measurements are positioned
inside the EC cell. The ultramicroelectrode (UME) is brought manually to about 1 mm
above a clean area of the gold chip and in the vicinity of the enzyme pattern. In this
position, the working microelectrode is too far from the chip to detect the minute
concentration of anything released from the enzyme preparation. The refinement of the
height of electrode was typically achieved in the feedback mode of SECM. In this
particular case, the approach was done in a 5 mM solution of [Ru(NH3)6]3+ in 0.1 M
phosphate buffer pH 6.7, until the tip-current increased with 30% of the bulk value.
Subsequently, the solution of the mediator was removed and the chip was thoroughly
rinsed with pure water. A solution of glucose in 0.1 M phosphate buffer was then poured
into the cell over the GOD micropattern. As the glucose and GOD are reacting, the
products (gluconolactone and H2O2) start to diffuse away into the bulk of the solution.
Now, if the scanning ultramicroelectrode of the SECM is kept at constant potential, namely
+600 mV vs. the reference electrode, the oxidation of hydrogen peroxide occurring at the
Pt disk can be monitored (Figure 38).
Fig. 38 Schematic showing the underlying idea of the measurement of released
hydrogen peroxide in the generation-collection mode of SECM. The incoming
glucose is converted at the enzyme microstructure into gluconolactone and
hydrogen peroxide, which is subsequently detected at the Pt disk electrode.
63
The obtained amperometric current is proportional to the concentration of the substrate if
the enzyme is not in the saturation regime. Because of the motion of the tip, the entire
microstructure is screened and the currents corresponding to specific locations within the
studied area are recorded by a computer.
Calibration curves obtained in the generation-collection mode of SECM
Optimal concentration of enzyme in a microstructure cannot merely be calculated because
the KM value changes when the enzyme is immobilised onto a film at a surface. For this
reason an uncomplicated enzyme micropattern was designed and investigated to find the
favourable concentration of GOD. The spotting mixture included 1 mg/ml GOD and 2
mg/ml polymer Vinnapas® EP16 in 0.1 M phosphate buffer pH 6.7.
The produced
microstructure consisted of three parallel lines with 500 µm spacing. The enzyme
concentration was varied through varying the number of droplets deposited per target spot.
Accordingly, the number increased from line 1 (A) to line 3 (C). A 25 µm Pt disk electrode
as SECM probe was scanned over the three lines of the microstructures at different
substrate concentrations (Figure 39). It turned out that the enzyme was used in excess
compared to the amount of available glucose. Therefore, in the amperometric recording
three current peaks with roughly equal heights can be noticed. Nevertheless, the maximal
current response in each case (A, B, C) is proportional to the glucose concentration in bulk.
Fig. 39
Line-scans over a GOD microstructure obtained in generationcollection mode of SECM. Line A was prepared by dispensing 1 droplet/spot
whereas lines B and C were prepared with 2 and respectively 3 droplet/spot.
Glucose concentration were (mM) 5 – yellow, 7 – red, 10 – cyan, and 20 – for
dark blue. Supporting electrolyte 0.1 M phosphate buffer; 25 µm Pt SECM tip;
30% current increase.
64
These plots are, in fact, calibration curves. Thus, to obtain calibration curves, the enzyme
concentration should not be raised to more than 1 mg/ml because the analytical signal
(amperometric current from the oxidation of H2O2) is not improved.
A GOD concentration of 0.1 mg/ml and 2 mg/ml Vinnapas® EP16 in the dispensing
solution, and high concentration of glucose (100 mM) in the bulk, resulted in a saturation
of the enzyme. In this case, a micro-grid with increasing concentration of GOD from top to
bottom and left to right was scanned again with a 25 µm Pt tip in the generation-collection
mode of SECM. Figure 40 shows the SECM (A – bird view and B – three dimensional)
images of this grid. Note: if the enzyme is working in the saturation range, then the kinetic
of the GOD enzymatic reaction depends upon the diffusional transfer of O2 from outside
gas atmosphere.
Fig. 40 SECM images of a microgrid with increasing GOD concentration from top to bottom and
from left to right. The spotting solution contained 0.1 mg/ml enzyme and 2 mg/ml polymer
dispersion. Imaging achieved in the generation-collection mode of SECM. (A) bird-view of the grid;
white areas indicate higher concentration of hydrogen peroxide; (B)
three-dimensional
representation of the grid; elevated areas stand for increased enzyme concentration. Glucose
concentration was 100 mM. Supporting electrolyte 0.1 M phosphate buffer; 25 µm Pt SECM tip; 30%
current increase.
High-resolution constant-distance SECM on immobilized enzyme micropatterns
Up to this point, enzymatic microstructures were imaged only with the SECM tip moved at
constant height above the surface. This mode of scanning has the advantage of being fast
and straightforward. Furthermore, it can be performed with standard Pt-glass
microelectrodes. However, the constant-height mode of SECM has certain limits on
heterogeneous surfaces displaying variations in both, conductivity along with topography.
On these samples, changes in the tip current arising from distance variations for principal
reasons cannot easily be differentiated from ones originating from alterations in
conductivity. In addition, with the constant-height mode the tip crash is at high risk on
tilted or rough surfaces, especially when decreasing the size of the SECM tip for imaging
65
at higher resolution. On the contrary, constant-distance SECM offers the advantage of a
accurate control of the tip-to-sample distance since the integrated shear force-sensitive and
computer-controlled feedback loop of this mode forces the SECM tip to follow the
contours of the surface during the entire time of scanning/imaging. This does not only
allow simultaneous acquisition of the electrochemical tip response and the sample
topography but also effectively prevents against tip crash even with smallest SECM tips.
The foundation of the constant-distance SECM, a mode in which the scanning tip follows
the profile of the sample at constant distance, is an oscillating measuring tip that is forced
to vibrate, in the electrolyte solution, at its resonance frequency. This frequency comes
from superposition of the hydrodynamic friction force and the excitation mechanic force.
Any object that happens to be close to (under or aside) the vibrating tip will increase the
friction because it stops the solvent molecules oscillating around the probe tip. Hence, a
change in the vibration properties of the tip appears and is noticed as a shift in vibration
amplitude and phase. This is the heart of shear-force constant distance mode of SECM.
In the non-optical shear force based distance control54, two piezoelectric plates are glued to
the upper part of the highly flexible, needle-like ultramicroelectrodes just above the region
where the pulled capillary starts to get thinner (Figure 41). The upper plate vibrates the
SECM tip at its resonance frequency while the lower serves as a piezoelectric detector of
the amplitude of tip oscillation and frequency, respectively.
Fig. 41
Schematic representation of the set-up as used for constantdistance mode SECM with an integrated non-optical (piezoelectric)
detection of the distance-dependent shear forces.
66
During the approach of the SECM tip towards the sample, shear forces start to appear in
close proximity of the surface, which leads to a damping of the tip vibration and a phase
shift, both effectively registered by connecting the detecting piezoelectric plate to a dualphase analogue lock-in amplifier. In this way, the tip-to-sample distance is usually
automatically stopped at a user-defined degree of damping of the vibration amplitude, for
example at 70 - 80 % of the unaffected value with the tip far above the surface. Once the
tip is in the working-distance, the tip can be scanned in X and Y directions. The positive or
negative shift in the vibration amplitude and phase as recorded by the lock-in amplifier
provides an output signal for the Z-axis stepper motor that can move the electrode up or
down unless they reach the user-defined set-point value resulted from the electrode
approach curve. Continuous communication between the lock-in-amplifier, computer and
Z positioning system makes up the computer-assisted feedback loop that accurately
controls the tip-to-sample distance all through the scanning experiment.
A first experiment showed how efficient this technique is in guiding the vibrating
microelectrode across a rough surface without colliding the tip with the protuberances on
the surface. A microscopic spot of an enzyme/polymer mixture was dispensed on a glass
surfaces and visualised in air using the unpolished tips of a pulled Pt-nanoelectrode as
scanning probe. The unpolished tip was used because it offers much smaller total tip
dimension as a polished, disk-shaped nanoelectrode. Figure 42A presents a typical
scanning electron micrograph (SEM) of a 70-µm-diameter glucose oxidase/Vinnapas
EP16 spot on glass. The morphology of the spot appears non-homogeneous and displays
small local variations in the density of the enzyme/polymer film. Obviously, most of the
Fig. 42 Scanning electron micrograph (A) and shear-force topography image (B) showing a nonhomogeneous distribution of an enzyme/polymer mixture inside the circular area of a microscopic
spot of glucose oxidase/ Vinnapas EP 16. The diameter of the spot is about 76 µm.
67
two compounds deposits upon drying in a thin rim at the edge of the spot. Figure 42B
illustrates the topography of the same spot as imaged using the constant-distance mode of
SECM with an integrated piezoelectric detection for shear forces. As with SEM, local
micrometer-sized topographical variations are clearly visible in the shear-force image
underlining unequivocally the ability of the shear-force based constant-distance mode of
SECM to resolve surface topography at high resolution.
The topographic resolution of constant-distance SECM depends on size the sharpest part at
the very end of the probe tips, which is responsible for the shear force contact while the
current resolution is related to the size of the electro active area of the tip. To visualise the
activity of the enzyme microspot, a quartz glass-insulated Pt-disk nanoelectrode had to be
used for the visualisation of the prepared enzyme-containing polymer microstructures.
Such a nanoelectrode can be made by simultaneously pulling a quartz glass capillary
together with an inserted platinum wire using a laser-based micropipette puller56. Careful
grinding the tips of the pulled capillaries on polishing pads leads to the exposure of active
Pt-disks with diameters of far below one micrometer.
With the encouraging output achieved in air, the application of the non-optical shear-force
constant distance SECM was then supposed to be focused on the simultaneous imaging of
topography and enzymatic activity of a polymer-enzyme micropreparation in aqueous
solution (Figure 43).
In order to fulfil this requisite the following strategy was considered: a number of polymer
(P) and polymer-enzyme (P&E) microstructures (such as lines) patterned onto a surface
should present both topographic and chemical activity features when immersed in a
solution containing the substrate of the given enzyme. Thus it should be possible to scan
with the vibrating tip of the SECM positioned within the near field, over these polymeric
microstructures and to detect variations in the samples topography and the amperometric
current image of the mentioned arrangement of lines.
To practically verify the above mentioned assumptions, a polymer micropattern consisting
of three lines (70 µm in width each) made of Vinnapas® EP16 and with glucose oxidase
was microstructured by means of the piezo microdispenser. The enzyme was entrapped
only in the middle line (2 mg/ml polymer and 1 mg/ml GOD) whereas the outer lines
contained pure polymer (2 mg/ml). With this arrangement, high-resolution constantdistance SECM imaging of the topography and localized glucose oxidase activity was
performed in solutions containing 50 mM glucose. A disk-shaped Pt nanoelectrode with tip
diameters of about 500 nm was used as scanning probe for the measurements. SECM
68
imaging in the constant-distance mode of operation was carried out at scanning speeds of
0.1 - 1 µm s-1 for X and Y displacements, while the stable feedback loop guaranteed a
constant tip-to-sample distance of about 100 - 200 nm. To allow the local detection of
enzymatically generated H2O2 in the generator/collector mode the SECM tip was poised to
a potential of 600 mV vs. an Ag/AgCl pseudo reference electrode. As clearly visible in
Figure 44A, line scans of topography (blue) and amperometric SECM tip current (red)
were both simultaneously acquired by scanning across the enzyme/polymer linemicrostructure (Figure 44B). Although the topographical resolution with disk-shaped Pt
nanoelectrodes was found to be not as good as with the tapered tips of unpolished
electrodes, small lateral variations in the topography of the enzyme/polymer structure are
still visible. On the other hand, an increase in the amperometric tip current was observed
only just above the middle polymer line. This was expected as only the middle line
contained active enzyme and thus, the local production of H2O2 is limited to the area
covered by that line.
Fig. 43 Trajectory of a SECM tip scanned over a 3-dimensional microstructure
made of a polymer (P) in which the central part contains a biological recognition
element such an enzyme (E). The shift of the resonance frequency of the vibrating
tip due to the shear forces is used to obtain the topographic image of the studied
microstructure (blue line). Polarisation of the SECM tip at proper potential could
be used to achieve an image of the electrochemical activity of the surface by
detecting, for instance, a product of the enzymatic reaction that takes place within
the polymer matrix (red line).
69
Fig. 44 (A) Line scans of the topography (blue) and amperometric SECM tip
current (red) simultaneously acquired in solution containing 50 mM glucose by
scanning across a polymer microstructure consisting of three lines of Vinnapas®
EP16, and with GOD immobilised only in the central line (B). Measurements
performed in the constant-distance mode of SECM with a 500 nm Pt-disk
nanoelectrode. Scale bar: 100 µm.
Conclusion
The combination of nanometre-sized SECM tips with the non-optical shear force
positioning allowed simultaneous imaging of the topography and of the local chemical
activity of, for instance, enzyme-containing polymer microstructures with high spatial
resolution. With such achievements, SECM is widening its application field to objects with
nanoscopic surface features and (electro) chemical inhomogeneities.
70
Defined adhesion/growth of living cells
5.2.
In the beginning of the year 2004, I had an opportunity to listen to a lecture by Professor P.
Fromherz, at the Ruhr University of Bochum. He is the director of Max Plank Institute for
Biochemistry in Martinsried (near Munich) and world-wide known as the initiator of the
research towards binding the neurons to electronic devices, the so-called neuroelectronic
interfacing. According to his confession, the idea of developing such devices came up in an
unexpected way. Getting irritated while using a personal computer (a Macintosh) about 20
years ago, he dreamt to replace the keyboard with an electronic implant in the brain that
could possibly transfer all the wished commands directly to computera. Hearing all this I
was thinking that if this would have been realised by now, I would have wrote my PhD
thesis faster and enjoyed the springtime! Ok, this was only a minor thought. Actually, I
realised how complicated it is to fulfil this taskb, and that it is a complementary part of the
work directed to the growing of neurons on artificial microstructures – it was done in
cooperation with Dr. P. Heiduschka from the Department of Experimental Ophthalmology,
University Eye Hospital Münster. Indeed, the electrical interfacing of semiconductors and
neurons necessitate micropatterning techniques to control neuronal networks.
Artificial networks of living neurons are supposed to facilitate the understanding of the
properties and functions of neurons. Sooner or later they will be grown in-purpose in
damaged tissues of the human/animal body in order to repair locally the connection of the
nerve cells. In the framework of an ELMINOS project, the task was to prepare
microstructures of laminin on different substrates for growing chicken embryo neurons. All
the following results are shared with Karla Tratsk-Nitz, who prepared the neurons, the
special buffers and also incubated neurons over the laminin microstructures (for details see
chapter 6 “Experimental”). The microdispensing work was mostly done at the Eye
Hospital, Münster. For this, I travelled several times to Münster together with a heap of
equipment (computer, microdispenser, holders, wave generator, power supplies and
obviously lots of connectors, plugs and replacements for the microdispenser) (Figure 45).
A close-up of the microdispenser and positioning system is shown in Figure 46.
a
P. Fromherz, “Neuroelectronic Interfacing: semiconductor chip with ion channel, nerve cells, and brain”,
Nanoelectronics and Information Technology, Editor: Rainer Waser, Wiley-VCH Berlin, 2003, 781-810.
b
This is a considerable technical challenge because an ionic current in the cell has to interact with the
electronic current in the silicon chip and between these two parts (cell–chip) is gap of several tents of nm that
blocks the current flow.
71
Fig. 45 Microdispenser set-up as mounted and used at the Eye Hospital,
Münster. (A) wave generator; (B) rely card; (C) power supplies; (D) piezo
microdispenser and positioning system; (E) laminar flow bench.
Fig. 46 Close-up of the active area: (A) the microdispenser head; (B)
reservoir of the spotting mixture; (C) cold-light lamp; (D) sample positioning
stepper motors; (E) microdispenser leads.
The aim of the present study was to check whether laminin patterns can be created by
the microdispensing technique and if such patterns are suitable for patterned adhesion of
neurons.
5.2.1. Introduction
Study of properties and function of neurons belongs to the major research fields in life
sciences. Connections between neurons are established by synapses between axons and
72
dendrites. In order to study the properties of neuronal network (signal propagation, or
neurotransmitters release), they should be grown in vitroc in a defined pattern, where
contacts between neurons can easily be traced99,100. Otherwise, such investigation might be
difficult owing the complicated structure of naturally developed neuronal networks.
5.2.2. What is available so far?
A spectrum of different technologies for the directed growth of living cells is available.
Different approaches have been tried to achieve defined adhesion of neurons on artificial
substrates. They include modifications of surface topography by grooves, wells or similar
structures101-105 or spatially dissolved chemical modification of the surface106,107, which can
be achieved by microcontact printing (stamping)108-112, photolithography99,114-116 or plasma
polymerisation117. An alternative method having the ability to create well defined and
reproducible surface microstructures at the micron level is using an excimer laser beam to
micro-sculpture a polymeric film118. If microstructured electrodes are present on the
substrate, they can be coated selectively as it has been demonstrated for adhesion of
neuroblastoma cells on platinum stripes coated with laminin-derived peptides119 and axonal
outgrowth on laminin-coated platinum stripes120. Electroactive structures were also created
by microcontact printing, and a patterned attachment of two different cell types could be
achieved after coupling an RGD peptide to quinone groups121.
Laminin is a large, multi-domain protein122 with many binding cites for cellular
receptors123,124. It plays a crucial role in the development and maturation of the nervous
system125,126. Laminin has been used for many years as substratum for in vitro cultivation
of neurons127-129, and adhesion and growth promoting properties of laminin could be
achieved by adsorptive deposition of a polylysine layer prior to adsorption of laminin130.
There are also reports of patterned deposition of laminin by photolithography131,132 and
microcontact printing133-135.
Many of the methods of patterned surface modification are restricted to certain material
properties of the substrate and/or require expensive equipment and complicated
procedures. On the search for a simple and inexpensive method, which should be suitable
for a variety of substrate materials, we choose the so-called ink-jet printing11. This method
has been known by commercial printers for several years. For this study, we used a piezoceramic actuated dispenser developed at the Lund Institute of Technology (for details see
chapter 2). Initially developed for the handling of very small volumes of nano- or picoc
„In vitro“ experiments/processes are taking place out of a living organism (in a test tube, for instance).
Normally they occur in living organisms that means “in vivo”. From the Latin word “vitreum” – glass /
vitreus – made of glass.
73
litres in microscaled analytical devices, e.g. for MALDI-TOF MS136,137, the microdispenser
can also be utilised for the creation of substance patterns on substrates, with a high
variability regarding the geometrical shape of the pattern. This has been demonstrated
already by the creation of microstructures glucose oxidase and other enzymes on gold
substrates98. Moreover, several substrate materials can be used.
5.2.3. Results and discussion
In the further described experiments with neurons, continuous lines with one droplet/spot
were made, with a width of 100 µm and a distance of usually 500 µm. The solution shot
onto the substrates contained 2-4 mg/ml Vinnapas® and 20 µg/ml of laminin diluted in
Hank’s balanced salt solution. Since agglomerates of the polymer could clog the
microdispenser nozzle, they were, prior to use, centrifuged (10·g) for 30 seconds and the
agglomerates settled down at the bottom of an Eppendorf tube.
Note: Special circumstances as handling living cells requires a sterile environment.
Therefore, all instrumentation and materials involved in this project were sterilised prior
use with a 70% aqueous solution of ethanol. To circumvent any contamination of the
equipment, the microdispensing set-up (micropump, positioning system, and glass cover
slips) were placed into a laminar flow bench after being disinfected. A constant stream of
filtered and sterilised air was continuously flowed outwards the hood. Accordingly, no
impurities could enter the space where the microstructures are obtained. In addition, one
has to wear rubber gloves all the time and has to clean them with alcoholic solution as
often as one retracts the hands from the hood. The successful growth of neurons depends
partially on the degree of sterility of gloves and equipment.
At the very beginning of this common project, the idea was to check whether the aqueous
solution of laminin is proper for use in this form or it needs a support. The result is shown
in Figure 46A. It can be seen that the liquid in the lines contracts; as a result, single
irregular dots are formed. This behaviour can be attributed to the properties of laminin that
possesses strong intermolecular interactions. In order to obtain continuous lines on the
glass substrate, a mixture of laminin and Vinnapas® was used. Although the contraction is
still visible, continuous lines could be created now (Figure 46B). Therefore, this mixture of
laminin and Vinnapas® was used in the experiments with the neurons.
74
As the next step, it was checked, if the laminin molecules entrapped in the polymer are still
accessible to surface binding molecules so that neurons could bind to them. For this
purpose, Vinnapas® lines with and without laminin were created and stained with an antilaminin antibody. It could be seen, by fluorescent microscopy, that the antibody was really
bound to the lines containing laminin in a dot-like manner (Figure 47), whereas no staining
can be seen if the anti-laminin antibody or laminin were omitted.
Fig. 46 Comparison of lines shot on glass slides with: (A) laminin solution and
®
(B) a mixture of laminin and Vinnapas . Scale bar: 200 µm.
®
Fig. 47 Staining of laminin–Vinnapas lines using the anti®
laminin antibody and Cy2 -conjugated secondary antibody.
As depicted in the above images, there are also some fluorescent spots visible
outside the lines. This could mean that some laminin molecules might have been
washed out of the polymer matrix during the long procedure of immunochemical
staining. Nevertheless, it should have no significant effect on the patterned adhesion
of neurons, because there is still enough laminin present on the lines.
75
Moreover, neurons adhering outside the lines are not able to outgrow neurites, so that they
do not become part of a neural network developing on the lines.
A suspension of neurons was applied onto the substrates with the lines. Examples of the
behaviour of neurons on glass cover slips are shown in Figures 48. It is obvious that
adhesion of neurons follows the laminin-Vinnapas® lines.
Fig. 48 Examples of neurons cultured on laminin–Vinnapas
glass cover slips.
®
lines on
Coverage of lines with neurons is not perfect at some places indicating some
inhomogeneities in the composition of the polymer lines. Neuronal adhesion is not
restricted solely to the lines, as several cells were found also outside the lines.
In order to verify that adhered cells are neurons, a new set of experiments were carried out
in Bochum this time. Immunocytochemical staining was performed with an antineurofilament antibody. The vast majority of cells were found to be stained indicating that
neurons were attached to the laminin-Vinnapas® lines (Figure 49).
To obtain this fluorescent image, a Vinnapas®-laminin mixture was patterned onto glass
cover slips. For this experiment a solution containing 4 mg polymer and 14 mg laminin/ml
was used. The microstructures were subjected to the following incubating and rinsing
steps:
a) 1 hour incubation in 1% BSA phosphate buffer;
b) 1 hour incubation with α-laminin antibody that previously dissolved in BSA
phosphate buffer;
c) three-times gently rinsing with phosphate buffer;
d) 1 hour incubation with the fluorescent tagged antibody dissolved in 1% BSA
phosphate buffer;
76
e) rinsing with phosphate buffer;
f) 30 minutes immersion in 5% glutaraldehyde;
g) rinsing with phosphate buffer.
h) drying and preserving in a cold, dark place.
®
Fig. 49 Neurons on a laminin–Vinnapas line on a glass cover
slip. The cells are visualised by means of immunocytochemically
staining with an anti-neurofilament anti-body.
Steps d) and g) have to be carried out in the absence of intense light.
It was also checked whether laminin-Vinnapas® lines were suitable for neuronal adhesion
on gold, silicon and glassy carbon. Figure 50 shows the result of a negative control
performed on glassy carbon substrate, where lines were made only with Vinnapas® i.e.
without laminin. No adhesion of neurons occurs on Vinnapas® lines that are lacking
laminin (Figure 50A). Small and faint fluorescent dots were seen on the polymer lines on
all opaque substrates, possibly by non-specific inclusion of the fluorescent dye DiI into the
polymer matrix. Glassy carbon is a material which possesses a variety of heterogeneities in
its structure. They could provide sites for non-specific cell adhesion. Indeed, it was found
that there were several cells on the glassy carbon surface outside the lines. A good
adhesion of neurons onto the polymer lines on glassy carbon could be observed, if laminin
was present in the Vinnapas® (Figure 50B).
77
®
Fig. 50 Behaviour of neurons on: (A) Vinnapas lines and (B) laminin–
®
Vinnapas lines shot onto glassy carbon. The broken lines indicate position
and width of the polymer lines. Both scale bars represent 100 µm.
More cells adhere non-specifically outside the lines, some of them forming dense clusters.
The reason for this behaviour could be leaching of laminin out of the polymer lines and
subsequent local adsorption of laminin at the glassy carbon surface, thus providing
additional potential adhesion sites for the neurons. The cluster-like gathering of the
neurons indicates that pure glassy carbon surface is not appropriate for neuronal adhesion.
A completely unexpected finding was observed on gold substrates. The whole surface was
populated more or less densely with neurons, whereas no cells could be found on the polymer lines (Figure 51A). The bright dots of fluorescent dye visible on the lines represent
most probably stained cellular debris attached to the lines. At the moment, we cannot
explain this behaviour, and further experiments should be performed.
On silicon substrates, only a very weak adhesion of neurons could be found at all (Figure
51B). Although the lines seemed to be preserved, only few neurons adhered to them.
Nevertheless, they exhibited normal neuronal appearance and a good growth of neurites.
Due to small number of adhering cells, individual cells and their connections can easily be
traced.
®
Fig. 51 Behaviour of neurons on laminin–Vinnapas lines shot onto: (A)
gold and (B) silicon substrates. The broken lines indicate position and
width of the polymer lines. Both scale bars represent 50 µm.
78
5.2.4. Conclusions
It could be demonstrated that the adhesion of the neurons followed the prepared
micropatterns. Primary embryonic neurons are able to attach to lines made from a mixture
of laminin and Vinnapas®, though to a different extent depending on the substrate. It is not
yet understood why neurons adhered to the pure gold surface instead of the polymer lines,
which contained the adhesion-supporting laminin. It may be that sub-micrometric features
of the surfaces can promote better the cell adhesion and growth on smooth supports rather
than rough ones. Vinnapas® turned out to be a useful polymer matrix for laminin in order
to achieve adhesion of neurons and even outgrowth of neurites. It swells in an aqueous
environment, exhibiting hydrogel-like properties. These findings open the route for the
generation of complex small neuron arrays and for the electrochemical investigation of the
obtained neuron matrix.
79
DNA microstructures
5.3.
DNA microstructures
Many metaphoric names have been given to the XXth century. The scientific and
technological revolution have created numerous new research fields and the achievement
rates were so high that the century itself was named after key words specific to the new
found disciplines. The century of speed, of electronics, space era and not to mention the
most recent one: the century of genetic engineering. With every new idea and great
discovery, scientists around the world were seeking to contribute with their best to the new
challenge. Deoxyribonucleic acid (DNA), the “super-molecule” encrypting the secrets of
life within its long chain made of four basic units adenine (A), thymine (T), guanine (G),
and cytosine (C) (Figure 52) could not stay apart of this phenomenon. DNA represents a
cellular library with the complete information required to form complex organisms. The
information storage is achieved by a genetic code made up of sequences of the A, T, G and
C units. The problems to be solved had been different in the last decades and it became
time to take advantage of the enormous amount of information encoded in DNA and used
it to get better tools for detecting and curing genetic diseases, inherited or acquired.
New improvements in life care must pay attention to the detection of genetic vectors as
DNA strands which can give a great deal of information about the incipient phase of a
disease development. Each period of mankind history had its particular implements or
methods to identify and to cure diseases. In this century, the application of highly selective
genetic tools in a parallel manner will play a significant role to perform multiple analyses
Fig. 52 The four nitrogenous bases present in the structure of DNA.
at once. In this context, DNA chips or microarrays represent the foundation of the DNAbased high-throughput analysis. It is a currently developing technology that is reshaping
molecular biology. Some ideas about important aspects of DNA microarray are introduced
in the following.
80
DNA microstructures
The DNA
Any cell of an organism contains in form of DNA molecules the complete set of genetic
information necessary for building a fully functional new similar organism. The nuclear
DNA that is the physical support of this information is called genome. The size of the
genome is usually measured in terms of base pairs (bp) and largely varies from as little as
50 thousand (Phage λa) to billions (human genome has about 3 billions bp). In a cell
nucleus, the long DNA moleculesb are found within entities called genes that are defined as
the physical and functional units of heredity. The unique arrangement of the four
constituents, the nucleotides (A, T, G, C), in a gene ensures the
distinctive features of individual organisms. To a higher level of
organisation, the genes are coupled in an array of genes that are
known as chromosomes. A chromosome is the self-replicating
genetic material during cell division process.
DNA as biopolymer
The structure of a DNA polymer consists of repeating chain
sugar (β-D-deoxyribose) residues linked by phosphate units.
The four organic bases are attached to the side of the chain
(Figure 53). Condensation of a purine or pyrimidine base with
deoxyribose produces a nucleoside. An ester bound between a
phosphate group and the hydroxyl of deoxyribose generates a
nucleotide. Two single DNA strands are able to bind to each
other forming double stranded DNA obeying the base-pairing
rules of Watson and Crick:
Fig. 53 A fragment
of DNA. B1-B3 are
nitrogenous bases.
•
bases of one strand are bound by H-bonds with bases of the other strand;
•
purine bases bind pyrimidine bases; thus A pairs with T, and C with G;
•
A and T are hold together by two H-bonds, while G and C are hold by three Hbonds (Figure 54).
Three secondary structures were identified by X-ray crystallography for double stranded
DNA: A-DNA and B-DNA are two geometries of the standard right-handed double
helices, while Z-DNA is the left-handed double-helical structure that is only stable at high
concentrations of NaCl or MgCl2.
a
b
A phage (or bacteriophage) is a virus that contaminates only bacteria. Phage λ specifically infects E. coli.
Stretching the DNA molecule from a single human cell, leads to a DNA string as long as 1.8 meter.
81
DNA microstructures
5.3.1. DNA microarrays
A microarray138 is a spatially ordered and miniaturised arrangement of a large number of
surface immobilised reagents. Typically, the spot sizes are less than 200 µm in diameter.
Roger Ekins was the first who described a “microspot multianalyte immunoassay”139,140 in
1989. His team thought this new tool would be of value for the analysis of complex protein
mixtures deriving from recombinant DNA technologies. Nevertheless, E. Southern had the
idea of using nucleic acids molecules to interrogate other nucleic acid molecules attached
to a solid support141. Since 1995, the term “microarray” got slowly in wide spread use.
Fig. 54
Formation of hydrogen bonds (red doted lines) between adenine-thymine (AT) and
cytosine-guanine (CG) as postulated by Watson-Crick base-pairing rules.
A microarray is named according to the reagent that is confined at the surface rather than
to the analytes they aim to detect. Figure 55 is exemplifying different types of microarrays
together with possible applications142.
Fig. 55
Example of microarrays and their possible
applications.
82
DNA microstructures
It is common use to call the surface-immobilised reagent the probe and the analyte in the
sample the targetc (Figure 56). This nomenclature will be used throughout the chapter
about DNA microarray which also are frequently called DNA chips. It should be
mentioned that the name GeneChip® is owned by Affymetrix Incorporation and refers to
their high density, oligonucleotide-based DNA array. However, the term “gene chip” is
often used as a general terminology within the microarray technology.
Fig. 56 Hybridisation at a DNA microarray (chip). DNA targets, from the bulk of the
solution, bind to their complementary DNA probes (match) but do not hybridise to the
non-complementary ones (mismatch).
DNA microarrays exploit the preferential binding of complementary single-stranded
nucleic acid sequences. The underlying principle is the same for all microarrays, no matter
how they are made. The unknown sample (target) is hybridised to the ordered array of
immobilised DNA strands whose sequence is known (probes)143-145. Due to the large
number of different probes, the microarray can identify thousands of DNA fragments
simultaneously which offers the chance to perform genetic analysis on a huge scale. This is
a prerequisite to gain profit from the mountain of information resulting from the
completion of the Human Genome Project146-149 (HGP, formally started in 1990) which
otherwise would be of no use but simply a collection of data.
Typical applications of DNA microarrays are150-153:
c
In the literature one can find two confusing nomenclature systems for referring to hybridisation partners,
but both commonly used "probes" and "targets". According to the nomenclature recommended by Bette
Phimister (Nature Genetics Supplements, 1999, 21, 1) a "probe" is the tethered nucleic acid with known
sequence, whereas a "target" is the free nucleic acid sample whose identity/abundance is being detected.
83
DNA microstructures
1. identification of the sequence and gene mutation (such as Single Nucleotide
Polymorphism – SNPd);
2. determination of the expression level (abundance) of a genee;
3. comparison of gene expression in different populations of cells (for instance
healthy versus diseased cells, or the evolution of gene expression during the
embryonic development; disease diagnosis);
4. drug discovery (pharmacogenomics) which is aiming to find correlation between
therapeutic responses to drugs and the genetic profiles of patients;
5. toxicological research (toxicogenomics) which is aiming to find the correlations
between toxic responses to poisons and changes in the genetic profiles of the
organisms exposed to such pollutants.
Hopes
The beginning of the era of molecular detection of cancer started last year, when a team of
cancer specialists recruited a group of women for clinical test for breast cancer
investigations. Researchers are convinced that they understand gene expression patterns
now well enough to use them in life-altering treatment decisions. Thus, the first ever
clinical study to assign patients to a standard or aggressive therapy based on a gene scan is
expected to lead to the implementation of DNA microarrays as routine clinical tools in
hospitals within the next years154.
5.3.1.1. The preparation of DNA microarrays
Various materials can be used for grafting DNA strands, but most used are glass, silicon,
plastic or gold-covered slides. The immobilisation of artificial155 or natural
deoxyribonucleic acid on solid supports is a crucial step for any application in the field of
DNA microarrays. It determines the efficacy of the hybridisation and influences the signal
strength for the detection. Thousands of spots of natural or synthesised156 single-stranded
DNA probes in the form of cDNAf / oligonucleotidesg (see note 5) are fabricated by highspeed robotics using contact or non-contact printing methods157-159.
Owing to the fact that DNA probes can be easily synthesised “in situ”160,161, nucleic acid
microarrays are currently dominating the field. Unlike DNA, there is no chemical
d
SNP is a DNA sequence variation that occurs when a single nucleotide in the genome sequence is changed.
About 2 of every 3 SNPs involve the replacement of C with T.
e
The expression (transcription) level of a gene is the amount of its corresponding mRNA present in the cell.
f
The term cDNA denotes a complementary DNA that is a single-stranded DNA molecule complementary in
base sequence to a RNA strand.
g
Oligonucleotide is a DNA molecule usually composed of 25 or fewer nucleotides; sometimes even 80-mers
are called oligonucleotides maybe because they look small compared to a typical cDNA that has 500-5000
bases.
84
DNA microstructures
possibility yet to build proteins at a microarray surface and thus protein molecules must be
delivered to the surface for the formation of protein chips. “In-situ” synthesis of a library
of oligonucleotides consists of stepwise building a number of different DNA strands from
its particular nucleotides by using ink-jet printing162 or photolithographic methods similar
to those used in the semiconductor industry/silicon technology (Affymetrix, Figure 57)163.
A remark about the DNA spot diameter: photolithographic-based in-situ synthesis can
produce extremely tiny spots with diameters down to 2 µm which is much smaller than the
100 µm obtainable by ink-jet spotting.
CombiMatrix is applying Lab-on-a-Chip technology for in-situ simultaneous synthesis of
thousands of DNA probes. Individual addressable microelectrodes of an array locally
generate, via an electrochemical reaction, reagents that facilitate the in-situ synthesis of
DNA fragments. A great advantages of this approach is the highly parallel synthesis of
hundred of thousands molecules since it saves time and reduces costs.
Fig. 57 Affymetrix is fabricating the high-density probes GeneChip® through a
combination of photolithography and combinatorial chemistry. The protected
probes of a DNA film are rendered ready for chemical reaction only in the areas
exposed to ultraviolet light (1-2). Deprotected DNA probes react with one of the
protected monomers (A, T, G, or C) and increase the length of the addressed
probes (3). This process is repeated until the desired DNA chip is ready (4-6).
Thiol-modified DNA was patterned with an atomic force microscope (AFM) on a resistcovered gold surface with line widths as small as 15 nm164. However, patterning with an
AFM tip is not a largely accepted method for DNA microarrays preparation due to the low
production rates.
85
DNA microstructures
The most commonly used technology is based on the localised immobilisation of complete
DNA strands that are prepared by solid phase synthesish,165. In this case, the DNA probes
must be modified at one end with an appropriate functional group (thiol166 or amino167) for
establishing a covalent bond between the probe and chip surface.
Fast and precise delivery of the DNA at the substrate surface is typically achieved by
means of a spotting machine or arrayer (Figure 58). A robotic platform transfers the DNA
sample from the wells of a microplate to the slide in the form of a spot under careful
control of the temperature and humidity. Evidently, an automation of the DNA chip
fabrication gives more reproducibly, accurately and quickly access to high density DNA
microarrays. A key element of an arrayer is the spotting pen that is shown schematically in
Figure 59 (see note 6).
Fig. 58
Schematic of a
spotting machine (arrayer).
The precise delivery of the
substance at the chip surface
occurs by contacting the
spotting pen with the chip in a
computer-assisted
process.
The spotting solutions are kept
in microtitre plates; washing
and
drying
stations
are
cleaning and drying the pens in
between spotting steps.
Fig. 59
Examples of split-pins. (1)
Normal slot filled with the spotting
solution; (2) Slot ended at the upper
part with a larger cut used for storing
the larger quantities of spotting
solution.
h
R. B. Merrifield (FortWorth, Texas, USA 1921), Chemistry Nobel Prize Laureate in 1984, „for his
development of methodology for chemical synthesis on a solid matrix“.
86
DNA microstructures
5.3.2. Detection of DNA hybridisation – What are the options?
The above discussion on DNA microarrays intended to give a brief description of this
revolutionary tool, but did not show anything about the way a DNA microarray could be
used. As a consequence of the Watson and Crick base pairing rules (see note 7) the kind of
interaction between the DNA probe and target is the formation of the double helix, a
process known as hybridisation of DNA. If the DNA targets to be identified are part of a
complex mixture (such as blood or tissue extract), the compatibility/match of the purines
and pyrimidines would guarantee that preferentially the wished targets will be measured.
Accordingly, the fundamental scheme that researchers are looking for is a method to
discriminate between a certain single stranded DNA (ss-DNA) and its corresponding
double strand (ds-DNA) when both of them are possibly present at the microarray surface.
Detection of DNA hybridisation is performed in multiple ways but optical, mass sensitive
and electrochemical techniques are dominating. A concise review of the established
recognition methods of DNA hybridisation is provided in the following. They are
discussed considering their working principle (optical, mass sensitive or electrochemical
methods) or in concordance with the features of the attached active groups/reporters (label
and label-free methods).
5.3.2.1. Optical detection
Either the DNA targets or the probes are functionalised with a fluorescent dye. If the
targets are tagged then the corresponding spots on the chip are not appearing fluorescent
unless the DNA probes are hybridised. If, however, the probes are labelled one has to take
advantage of a quenching of fluorescence. This is only observed if the labelled probe
strands are flattened on the surface of a metal. Following the hybridisation with the
unlabeled targets, the probes will stretch upwards placing the label far above surface and
thus increasing the detected fluorescence (TIFI168 – target induced fluorescence increase,
Figure 60).
In general, sophisticated instrumentation is required for reading out the weak light signals
emitted by the DNA labelled spots169. Laser scanning fluorescence microscopy using for
example fluorophor-labelled target DNA, is known for its excellent sensitivity and
reliability and turned into the standard for the detection of hybridisation. Nevertheless,
thinking about a widespread application the size, price and complexity of fluorescencebased DNA detection systems as well as the dependence of the method on the long-term
stability of the fluorescent dyes are major drawbacks.
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DNA microstructures
Fig. 60 Target induced fluorescence increase (TIFI) detection of hybridisation is based
on the fact that the fluorescence of the label of a single stranded DNA probe is much
lower as compared to the fluorescence signal of the hybridised probe.
5.3.2.2. Mass sensitive detection
A widespread and methodically investigated representative of this sensor type is the quartz
crystal micro balance (QCM). A quartz crystal is forced to oscillate at its own resonance
frequency (5 to 20 MHz). If the power used to vibrate the quartz is kept constant, the
oscillation frequency will changes upon mass addition to its surface. The QCM response is
directly related to mass variations and thus easy to interpret (see note 8). This simple
principle is used to monitor hybridisation of nucleic acids by measuring the frequency shift
that follows the formation of the double strand170-172.
Another integrated biosensor technology based on thin-film bulk acoustic resonators has
been introduced. As the above, the detection principle of these sensors is label-free and
relies purely on a resonance frequency shift caused by mass loading of an acoustic
resonator. The sensor has been proved to be suitable to detect proteins as well as DNA
molecules, with a mass sensitivity being 2500 times higher than for a 20 MHz quartz micro
balance173. Such methods are not only label-free but also able to detect in real-time DNA
hybridisation and hence can provide kinetic information. A limitation of this mechanical
approach to detect hybridisation is the difficulty to use it for large number of samples
(parallelisation) in comparative genome studies with the goal to identify hundred or
thousands DNA fragments in relatively short time.
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DNA microstructures
5.3.2.3. Electrochemical detection of hybridisation
The complexity of fluorescence-based detection systems as well as the limited
dissemination of mass sensitive techniques is hindering the development of independent
medical points-of-care devices based on DNA microarrays. In view of that, a remarkable
number of electrochemical (EC) hybridisation assays have been proposed as practical
alternatives to optical readouts. As emphasised in a series of recently published review
articles174-179 electrochemical DNA chips can be much simpler in instrumentation and are
easier to miniaturise, since the fabrication of electrochemical devices actually is well
compatible to micro- and nanofabrication. In fact, the recent appearance of commercially
available systems such as Toshiba’s Genelyzer™ (Toshiba, 2003) and Motorola’s
eSensor™ 180 is a good sign that an EC-based technology indeed has the potential to offer
relatively cheap, easy-to use and portable analytical platforms for high-throughput DNAbased diagnostics. Such sandwich-like hybridisation assay are used for determining
hybridisation or even point mutations via electrochemical redox signals arising from
ferrocene-tagged signalling probes (AMBERi)181,182. Figure 61 gives a typical example of a
sandwich-like DNA chip where generally three DNA fragments are involved: a capture
probe that is confined at the chip surface and binds the target; another DNA fragment,
signalling probe, is labelled and complementary to a part of the target, other as used to
binding the capture probe. With this complicated stepwise procedure, a redox or
fluorescent label is transferred at the chip surface where is afterwards detected and thus
giving a proof for the hybridisation.
In general, electrochemical detection of base pairing benefits from differences in the
intrinsic electrical properties of single (ss) and double (ds) stranded nucleic acids183 and/or
employs easily oxidisable or reducible hybridisation indicators and redox labels. Stacked
arrays of aromatic heterocyclic base pairs in the core of immobilised ds-DNA, for instance,
are strongly supporting long-range electron transfer184-186 through the duplex (π way)
towards conductive carriers, an effect, however, that is worse in the presence of disruptive
mismatches and not observed with ss-DNA187,188. Barton, Hill and co-workers made use of
this observation for establishing sensitive (see note 9) schemes for the electrocatalytic
detection of hybridisation and mismatch recognition189,190.
i
AMBER is the acronym for amperometric bioelectronic reporter.
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DNA microstructures
Fig. 61 Sandwich-like assay for detection of hybridisation. A capture DNA probe bind first the
non-labelled target (1). The signalling DNA probe is immobilised at the chip surface via the
target, and thus allowing the redox label to exchange electrons with the electrically polarise chip.
A self assembled monolayer containing molecular wires facilitate the current flow. (Motorola
eSensor™).
There is a category of compounds known as intercalatorsj that are appreciated by the DNA
biosensor community because they opened a new route for the detection of ds-DNA. This
approach is simple on the one hand and on the other hand is highly selective, hence
offering good discrimination rates between ss- and ds-DNA (the binding affinities of metal
complexes with DNA are generally in the following order: intercalation, K > 106 M-1 >
hydrophobic interaction, K >105 M -1 > electrostatic interaction, K > 103 M -1)191,192. It had
been shown that the interaction between DNA and some transition metal complexes
changes from electrostatic to intercalative with increasing ionic strength193. Threading
intercalators are derivatives of polyaromatic systems bearing an electroactive label on the
side arms (naphthalene diimide194,195). They intersperse within the ds-DNA thus allowing
EC detection of DNA on arrays (Figure 62). Methylene blue (MB) has good voltammetric
behaviour and an appropriate structure for binding to ds-DNA. Thus, MB could be
employed as signalling tag for ds-detection196. Natural compounds, such as hemin, were
also used as intercalators197. Osmium198, ruthenium or cobalt199,200 complexes have found
applications as electroactive intercalators too, but they are toxic and their synthesis is time
consuming. Electrode surface modification with functionalised multi-walled nanotubes
(MWNT-COOH) turned to be a suitable intermediate for DNA immobilisation with
j
An intercalator is a type of DNA ligand that inserts or intercalate between adjacent base pairs of ds-DNA.
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DNA microstructures
Fig. 62 Schematic showing the application of intercalators for detecting
DNA hybridisation. The intercalator has a polyaromatic core with labels
linked to its side chains. In this case, the redox label is detectable at the
electrically polarised chip surface only if hybridisation took place. Intrinsic
electroconductive properties of the DNA double helix allows electrons to
be wired from the label towards chip or vice versa.
improved rates of the electron transfer between the electrode and daunomycin as redox
intercalator201.
Other approaches for transduction of hybridisation take advantage of differences in the
binding affinities of dissolved redox active metal complexes for ss-DNA and
ds-DNA202-204, the oxidation of guanine (Figure 63) and adenine moieties205-208 (only these
two are used because the redox potential is suitable) or amplification strategies with metal
nanoparticles209-212. When guanine bases are oxidised at the surface of the microarray
itself, the high potentials involved could hydrolyse the water. The water oxidation can be
overcome by a suitable modification of the DNA chip surface, for instance with indium tin
oxide (ITO)k,213. It has been reported that modifications of the chip/electrode surface with
polyelectrolytes such as poly(allylamine hydrochloride) and poly(styrenesulfonate) and
above-mentioned ITO, lead to notable improvement of signal-noise (S/N) ratio when gold
nanoparticle probes and silver enhancement are used to detect hybridisation214. One should
mention that target-modified gold nanoparticles have another advantage because they
k
Indium tin oxide (ITO) is in fact indium oxide doped with tin oxide (In203:Sn02); it is used to prepare
transparent conductive coatings by electron-beam evaporation or sputtering. It has numerous applications in
display devices (such as flat panels, field emission), photovoltaic devices and heat reflecting mirrors; high
melting point: ~1900° C.
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DNA microstructures
induce instability in the duplex. This significantly alters the melting profile of the
hybridised DNA, allowing single base mismatch to be perceived215. Silver nanoparticles
labelled with DNA probes and Anodic Stripping Voltammetry (ASV) of Ag+ were used as
ds-DNA detection assay216. Nevertheless, these approaches require extra amounts of noble
metals and supplementary chemical steps for enhancement of signal.
Fig. 63 Oxidation of guanine bases of single stranded probe
DNA can be achieved with tris(2,2’-bipyridyl) ruthenium(III)
electrogenerated at the surface of the DNA chip. Hybridised
DNA has the guanine bases hidden inside the core of the
double helix.
A technique for detecting DNA hybridisation has been reported that is using “electroactive
beads”. It is sensitive but relies on immobilising both the targets and the probes at the
surface of magnetically and electrochemically active microspheres217.
Instead of using dissolved redox active hybridisation indicators, marker molecules can also
be covalently bound to strands of the DNA probe, the target or oligonucleotides that are
used as autonomous signalling probes. An example is the enzyme-amplified hybridisation
test from Heller’s group, which enables detection of duplex formation between surfaceanchored target strands and enzyme-tagged signalling probes by amperometrically
monitoring the product of the enzymes action as it is exposed to the substrate218-222. A
merge project of several German companies, namely Infineon Technologies, Siemens,
Fraunhofer Gesellschaft, November and Eppendorf Instrumente, is employing enzymes in
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DNA microstructures
detection of DNA hybridisation in an interdigitated array of gold electrodes223. In brief,
two neighbouring microelectrodes E1 and E2 are polarised at opposite potentials. They
cycle the redox active compounds released by the enzyme-labelled target upon
hybridisation enabling the detection and quantitation of hybridisation amperometrically
(Figure 64).
Fig. 64 Microelectrode array for amperometric detection and quantitation
of hybridisation. Redox active compounds released by an enzymelabelled target are consequently cycled between the interdigitated
electrodes E1 and E2 while the corresponding current is monitored.
An interesting technique based on light-induced electron transfer from a DNA probe label
to the chip has been developed by FRIZ Biochem (Light Addressable Direct Electrical
Readout, LADER). An electron donor/acceptor complex label of a DNA probe is
selectively illuminated with ultraviolet light. If the probe bearing the label is single
stranded, no current is measured, whereas in the case of hybridisation, the ds-DNA can
wire electrons along its π-way. Even if multiple probes labelled with the same light
sensitive complex are simultaneously present at the chip surface, this method permits the
particular DNA probes to be identified by directing the UV beam in the right spot. It
becomes clear that a microelectrode array is not necessary. In addition, a high sensitivity is
achieved due to the ability of the complex to amplify the current by pushing up to 106
charges/second in the measuring electrical circuit (a mediator in bulk ensures that the
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DNA microstructures
electron donor/acceptor complex is permanently brought back to initial state subsequent to
UV-label interaction) (Figure 65).
Fig. 65 LADER resorts to the use of light induced electron donors/acceptors to pump
charges up/down only along π-way. Thus, ss-DNA generates no current while the dsDNA can wires an amplified number of charges due to the regeneration of the label by
the electrochemically active mediator in the bulk of solution.
From the same company, FRIZ Biochem, a chronocoulometric procedure, Electrically
Detected Displacement Assay (EDDA), uses short strands (4 bases only) of redox-labelled
DNA as reporter to probe the statue of the surface immobilised DNA. This method
requires two chronocoulometric measurements, one prior and another subsequent to
hybridisation. Firstly, the immobilised DNA probes are hybridised with a number of the
labelled reporter. The redox active labels are then kept relatively close to the conducting
chip surface and detectable by means of a potential step that is applied to the chip (working
electrode). The charge transport through the electrical circuit due to the electrochemical
transformation of the label is recorded as a fast decaying charge-time curve. A similar
experiment is then performed after hybridisation of the probes with their complementary
targets. Displacement of the short labelled reporter DNA by the longer targets is reducing
the concentration of redox labels in the vicinity of the chip surface (Figure 66). Therefore,
the integral of the charge-time curve is smaller as in the first case with the difference being
the indication of hybridisation. This detection scheme does not call for target labelling as
many of the other electrochemical assays but still requires the use of labelled signalling
DNA.
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DNA microstructures
Capacitance measurements224 (dependence of the differential capacitance C of the
electrode double layer on potential E, derived from C-E curves), electrochemical
impedance spectroscopy225-227 (frequency response of the impedance Z of the electrode
double layer-EIS) and constant current chronopotentiometry (dependence of dE/dt on the
potential at constant current) are also used to electrochemically study the interaction of
different redox labels with ss- and ds-DNA228.
Fig. 66 With the electrically detected displacement assay (EDDA), neither the capture
probe nor target is labelled. Contrary, short signalling strands of DNA are tagged with a
redox active compound and used as a reporter. The reporter that is hybridised with
probe strands is displaced when the chip is exposed to the target. In a potential step
experiment the replacement of signalling probes with targets is detected as a significant
drop of the charge integral.
Janata and co-workers proposed a new approach for simple and direct electrochemical
detection of a hybridisation, which is based on the electrostatic modulation of the flux of
chloride ions through a polypyrrole film into which the DNA probes are entrapped229.
Interestingly, the transport of Cl- ions is controlled by the status of the DNA probes with
higher values of diffusion observed in case of ss-DNA (Figure 67).
Recently, studies were reported on using electrochemical DNA biosensor as a screening
tool for environmental pollution monitoring230. Lucarelli231 and co-workers, for example,
presented a disposable electrochemical biosensors based on ds-DNA that was immobilised
on the surface of a screen-printed graphite electrode (SPGE). Voltammetry was employed
to investigate the electrode surface and changes in the DNA redox properties (oxidation of
guanine base) were monitored in order to study the interactions between DNA and the
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DNA microstructures
analytes. Although yet not specifically addressed to the detection of hybridisation, the
principles behind the strategy may be helpful as well to discriminate between ss- and dsDNA.
Fig. 67 Modulation of Cl- fluxes by polypyrrole (PPy) film for label-free
detection of DNA hybridisation.
5.3.2.4. Other methods
Besides the above mentioned schemes for detecting the hybridisation of DNA, a series of
papers suggest surface plasmon resonance232 (SPR) as a label-free and/or real-time
hybridisation assay. The last decade witnessed a remarkable development of SPR use in
biomedical applications233 and it seems that DNA chip technology is on its way to taking
advantage of this reliable and sensitive technique234.
5.3.2.5 Concluding remarks
A number of schemes for detecting hybridisation events on the surface of DNA
microarrays have been developed ranging from optical readouts using a sophisticated and
expensive instrumentation to rather simple electrochemical assays. However, hybridisation
detection is still an active field of ongoing research and development.
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DNA microstructures
5.3.3. The repelling mode of SECM: A new and promising assay for
imaging DNA microarrays and detecting DNA hybridisation
5.3.3.1. SECM and DNA microarrays
Recently, the visualisation of oligonucleotides and polynucleotides (poly[G], calf thymus
DNA) immobilized onto aldehyde-modified glass substrates was achieved in the
generation/collection mode of SECM through the oxidation of guanine residues by tipgenerated [Ru(bpy)3]3+-molecules235 (Figure 68). This ruthenium complex is a strong
oxidising agent and able to oxidise guanines. Compared to non-hybridised DNA spots, an
increased current is observed over an area with hybridised DNA due to the larger number
of guanine bases present in the double strands.
Fig. 68
Detection of DNA hybridisation via SECM tip generated
[Ru(BiPy)3] (III). This complex can oxidise guanine bases while itself is
reduced to the initial state. The oxidation current of [Ru(BiPy)3] (II) is
thus higher above the ds-DNA as compared to areas carrying ss-DNA.
A silver-enhanced SECM imaging of DNA hybridisation was demonstrated236. Capture
probes attached to insulating glass slides were hybridised with biotinylated targets and only
regions where sequence-specific hybridisation had occurred could be developed by the
adsorption of streptavidin-gold nanoparticles followed by electroless silver particle
deposition. The silver staining procedure formed locally conductive regions at which the
SECM tip current was amplified due to the appearance of positive feedback (Figure 69).
The related increase in the measured tip current made hybridised spots visible. In addition,
DNA duplex regions were successfully visualised by SECM using ferrocenyl naphthalene
diimide as intercalating hybridisation indicator237.
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DNA microstructures
Fig. 69
Detecting hybridisation with feedback mode SECM. A biotinylated target is
hybridised to the DNA probes (1,2). In a further step, gold nanoparticles are bound to the
double strand via streptavidin – biotin interactions and than a silver reduction is carried out
over the gold nanoparticles layer unless a compact film of silver is obtained (3). A ruthenium
complex mediator is recycled only above the hybridised area where the silver layer could be
produced.
5.3.3.2. Aim
The abovementioned methods that were designed to address a key issue of present genetic
research, the detection of hybridisation on DNA chips, have inherent drawbacks: they are
either complicated due to a high number of steps necessary for delivering the final result,
or they need of sophisticated machinery. Often miniaturisation that is essential for the
fabrication of individual medical point-of-cares instruments is difficult to achieve.
The idea of using a negatively charged redox compound to probe the status of DNA
strands immobilised at a chip surface appeared very exciting and was thought to offer a
straightforward alternative to the present hybridisation detection assays especially to those
based on electrochemistry. This detection scheme turned out to be not only a tool for
detecting hybridisation, but also could be used as an unconventional approach for
inspecting the quality of spotted nucleic acids microarrays.
5.3.3.3. Imaging and detection principle
At pH values above about 5, the phosphate groups of nucleic acids are likely to be
deprotonated. As illustrated in Figure 70, the diffusion of an anionic species An- towards
DNA-modified regions of a surface will therefore be effectively hindered due to
electrostatic repulsion. This coulomb interaction and modulation of diffusional mass
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DNA microstructures
transport provided the basis for establishing a novel label-free electrochemical detection of
hybridisation.
Fig. 70 Schematic representation of the influence of coulomb interaction on
the diffusion of An- towards a DNA-modified surface. Electrostatic repulsion
between An- and the phosphate groups at the backbone of the immobilised
DNA strands hinders the diffusion of the anionic species to the underlying
surface. For ss-DNA, the flux of An- (Iss) is higher than for the ds-DNA (Ids) due
to the increase in negative charges through formation of aggregates between
the probe and unlabeled target.
At a properly polarised SECM tip, [Fe(CN)6]3- can be reduced to [Fe(CN)6]4-. Far above a
conducting DNA chip surface (Au), a cathodic tip current arises from the diffusion limited
reduction of ferricyanide ions at the tip electrode, and a steady-state current value is
measured. However, close to the chip surface, the tip-generated [Fe(CN)6]4- is diffusing
towards the Au surface and an oxidised back to [Fe(CN)6]3-. This electrochemical
recycling is increasing the tip current compared to the value in bulk (positive feedback of
SECM). Due to the influence of repulsion, the diffusion of tip-generated [Fe(CN)6]4- is
hindered above surfaces carrying DNA and virtually unaffected in the DNA-free areas.
Additional negative charge is introduced by hybridising the probe strands with a
complementary target. Hybridisation thus becomes detectable thanks to an enhancement of
the effect of coulomb interaction on the diffusional flux of electroactive species.
Because the phosphate groups of nucleic acids and a negatively charged mediator are
presenting a repelling force to each other, a DNA chip with the recognition element spotted
in a regular pattern on a conducting surface is electrochemically highly heterogeneous. For
a given density of DNA probes, number of bases per individual strand, ionic strength of the
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DNA microstructures
Fig. 71 Schematic of the principle of DNA visualisation in the repelling
mode of SECM.
electrolyte solution and concentration of the mediator, the electrostatic interaction and
corresponding modulation of the diffusional transport of [Fe(CN)6]4- to the surface is
mainly determined by the ss/ds status of DNA strands within the probe spots, with fluxes
of the [Fe(CN)6]4- -species being higher in case of ss-DNA. The determination of the
relevant fluxes of anions can be achieved by subjecting DNA chips to local
electrochemical measurements. As shown in Figure 71, these spatially resolved
measurements can be achieved by performing SECM in the amperometric feedback mode.
Moving the SECM tip at fixed height above an oligonucleotide spot gives reason for a
sudden drop in the SECM tip current since here tip-generated [Fe(CN)6]4- molecules are
repelled, transfer rates for Au-induced recycling diminished in turn leading to a decreased
redox amplification by the positive feedback effect.
5.3.3.4. Oligonucleotides and the substrate
To prove the abovementioned hypothesis right, experiments have been carried out on DNA
microarrays of synthetic 20-mer oligonucleotides. They were supplied by FRIZ Biochem
GmbH, Munich, Germany. Immobilisation of the 20mers was accomplished using the selfassembly of the 3'-thiol modified strands that were spotted from a 250 mM phosphate
buffer aqueous solution on Au sputtered glass surfaces (2.5 x 7.5 cm2) with a professional
microarrayer (see Materials and Methods). Typically, the DNA chips contained DNA spots
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DNA microstructures
of two different 20-mer sequences, denoted as Oligo8 and Friz12 (O8 and F12
respectively). The base sequences are shown in Figure 72.
Fig. 72 The base sequence of the probes and targets used for spotting/hybridisation.
Fluorescent tags were necessary to provide comparative fluorescence measurements of the
hybridisation events. As can be noticed from this figure, the two DNA probe strands
include all the four bases. Freshly prepared DNA chips were post-assembled with 1 mM
propanethiol in deionised water, overnight, and at room temperature. This increased
hybridisation efficiency and helped to avoid unspecific adsorption of target strand. The
layout of the DNA chip is depicted in Figure 73A. A thin film of gold is necessary for the
specific anchoring of the thiol-modified DNA probes to the chip, but its adherence to glass
slides is poor. For this reason, an intermediate layer of chromium or titanium is applied on
the glass before gold is sputtered (Figure 73B).
Fig. 73A The layout of the DNA chip.
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DNA microstructures
Fig. 73B
The sandwich structure of the DNA chip.
DNA spots consist of a monomolecular film of DNA molecules and, hence, are not visible
with naked eyes. In order to facilitate the positioning of an SECM tip near to a DNA
microstructure and to shorten the scanning time of the DNA chip by choosing only a
particular area of the chip, a series of marks were stained or engraved at the surface of the
chip. There are two types of marks: temporary and permanent.
Marking the DNA chip
1. Temporary marks – are polycrystalline deposits of potassium phosphates and NaCl
grown at the hydrophilic DNA modified surface of the chip; they are obtained by
rinsing the chip with some drops of 1M NaCl PBS and air drying. The DNA-free
areas are not noticeably changed by this treatment owing the hydrophobic
properties of the thiol-modified surface. Although they are meaningful for the
positioning of the electrode, the crystals are dissolved when the electrolyte is filled
in the electrochemical cell. However, with the help of these momentary marks, the
chip was marked further with permanent marks.
2. Permanent marks – are fine scratches engraved with a scalpel on the gold surface.
Figure 74 indicates these marks on the chip.
The SECM tip is positioned over the DNA microarray in two steps. First, the tip of the
microelectrode is manually placed close to the “L”-shaped scratch (point 2 in Figure 74)
and roughly 1 mm above the surface. Then, the reference electrode is attached to its holder
and the electrolyte is filled into the cell. Subsequently, a computer-assisted precise
electrochemical approach of the electrode is performed to position the electrode tip well
within the feedback distance. For both, electrochemical approach curve and line scan
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DNA microstructures
measurements, the SECM tip is polarised at 0 mV versus the Ag/AgCl 3 M KCl reference
electrode in order to reduce ferricyanide under diffusion-limited conditions. For the chosen
10 µm diameter platinum disk microelectrode, a tip-to-sample distance of 10-15 µm
corresponded to an increase in the amperometric tip response of about 50%. Though the
gold surface was in most of the cases post-assembled with propane-thiol, the SECM
feedback remained to be positive. Of course, longer alkane-thiols (C6 or C12) would
gradually turn the feedback to negative as the access of the anions to the gold surface is
hindered.
Fig. 74 Marks on the DNA chip as coordinates
for the positioning and imaging; vertical line (1),
approximate start point of the scan (2),
horizontal line (3), exact start point (4).
5.3.3.5. The electrochemical cell and set-up for measurements on DNA microarrays
Numerous electrochemical cells were and could be designed for SECM applications, but
here are presented those that have been used in the experiments of this PhD work.
1. Conventional electrochemical cell
The rectangular DNA chips were too large to be fixed in the standard electrochemical cells
available in the laboratory. Accordingly, a cell as shown schematically in Figure 75 was
made of Plexiglas. The width was 28, the length 55 and height 15 mm. The inner
cylindrical hole was 15 mm in diameter but had a larger opening at the bottom to allow an
O-ring to be placed. Four screws pressed down the O-ring onto the chip surface in order to
make a water-tight seal. The electrochemical cell with the DNA microarray fixed to the
bottom was mounted on the two-axis translation stage of the SECM. Thus, the
electrochemical cell and the microarray could be driven by computer-controlled stepper
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DNA microstructures
motors in X, Y direction with a nominal resolution of 0.625 µm per half step. Tip approach
(Z-movement) was achieved with a third stepper motor that was mounted perpendicular to
the ones moving the cell and kept the Pt disk microelectrode that was used as SECM tip.
Fig. 75 Drawing of a typical SECM screwed-from-the-top electrochemical cell; the body of the cell
is made of Plexiglas (1); O-ring (2); substrate (3); translation stage (4).
2. Simplified cell
In addition to the conventional electrochemical cell, a simplified cell was constructed with
little efforts and only an O-ring defining the electrolyte volume (see Figure 76).
Fig. 76
Simplified electrochemical cell for SECM measurements.
Vacuum fat was used to keep the O-ring in place and obtain a water-tight seal to the chip
surface. The advantage of this simple arrangement is that it tip placement and positioning
is easier for the user since no walls of a chamber are hindering the optical observation of
the microelectrode and the sample. Furthermore, lower volumes of electrolyte are needed
which could be an advantage when one is aiming on monitoring the hybridisation of the
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DNA microstructures
probe strands with their complementary targets in real time while the duplex formation
takes place. Since the chip has to be exposed to a certain concentration of targets, a lower
volume would help to save on the expensive synthetic target oligonucleotides. The solvent
evaporation is considerably reduced if it is ensured that the surface of the solution is not
having a convex curvature but plain or concave (Figure 77).
Fig. 77
The curvature of the surface of the electrolyte in the simplified
electrochemical cell has a high impact on the evaporation rate of the solvent:
convex (A) > plain (B) > concave (C).
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DNA microstructures
5.3.3.6. Imaging ss-DNA in the repelling mode of SECM
The preliminary experiments were performed in a ferrocyanide solution in 1M PBS. The
SECM tip was polarised at +400 mV vs. Ag/AgCl 3 M KCl in order to oxidise the
mediator. For this reason, the DNA spots appeared in the SECM images as areas
displaying a sudden drop of the anodic tip current.
Figure 78 represents a SECM image of a DNA microarray that was recorded at the very
beginning of this project. It is a single line scan carried out in 5 mM ferrocyanide in 1M
PBS with a 10 µm Pt disk electrode scanned at 5 µm/s. Obviously this was a remarkable
result because it demonstrated that the underlying idea of the proposed new detection
protocol was right: the downwards peaks as visible in this SECM image are a visualisation
of the DNA spots. Indeed, repelling forces between the negatively charged mediator and
the phosphate groups of the DNA strands on surface seemed to have a strong impact on the
amperometrically feedback current of the SECM. When larger areas of a DNA chip were
scanned, colour bird-view or 3-D plots could be obtained by converting the raw data into
the desired image files (here not shown, see below).
Fig. 78 The first SECM scan over a ss-DNA microarray indicated the
position of some DNA spots (lower parts of the curve); 5 mM [Fe(CN)6]4in 0.1 M phosphate buffer and 1 M NaCl; scan rate 10 µm/s.
Proof of principle
In order to prove that electrostatic repulsion is responsible for the creation of contrast
between the DNA-modified and bare chip surface, experiments were carried out in the
presence of a negatively and positively charged mediator. The latter should not experience
a repelling force from the phosphate groups of the immobilised ss-DNA and the feedback
response of the SECM tip thus not be disturbed. In Figure 79, the upper curve displays a
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DNA microstructures
Fig. 79
Line scans acquired by scanning the tip of a 10 µm diameter Pt
microelectrode at a fixed height of 10 µm across a spot of single stranded
oligonucleotide. Negatively charged [Fe(CN)6]3- (5 mM in 0.1 M potassium
phoshate/3 M sodium chloride, pH 6.5) was used as the mediator and the SECM
tip was polarised to 0 mV vs. Ag/AgCl/3 M KCl. Positively charged [Ru(NH3)6]3+ (5
mM in 0.1 M potassium phoshate/3 M sodium chloride, pH 6.5) was the mediator
and the SECM tip was polarised to –400 mV vs. Ag/AgCl/3 M KCl.
representative amperometric recording taken in a [Fe(CN)6]3- solution during a complete
move over an individual oligonucleotide spot. The current to the left and right are
indicative of the unbiased positive feedback. As expected, the DNA strands drastically
lowered the current values above the DNA spot. This was as expected and verifies the
reduced rates of redox recycling due to electrostatically hindered diffusion of tip generated
species. The drop of the tip current almost completely disappeared when substituting
[Fe(CN)6]3- by [Ru(NH3)6]3+. With [Ru(NH3)6]3+, only a negligible decrease in the SECM
tip response was observed (Figure 79, lower curve, and inset) which most probably is due
to a pure steric hindrance of diffusion. The different behaviour of the two mediators gave
evidence that really an electrostatic repelling force is responsible for a DNA-induced
modulation of the tip response.
SECM measurements and the evaporation of solvent
A typical DNA microstructure had 24 spot within a 1200 µm x 1500 µm square. Applying
the typical parameters for imaging in the SECM feedback mode (scan speed: forward scan
5-10 µm/s, backward scan 500 µm/s; distance between two measuring points in X
direction: 5-10 µm; 0.5 s waiting time before data acquisition is performed; 25 µm
distance between two neighbouring X-line scans), 10 to 20 hours are needed to complete a
full image of the DNA array.
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DNA microstructures
Fig. 80 CVs measured before (black) and after (red) recording
of a complete SECM image of a DNA chip in the imaging
solution. Image acquisition took 16 h. Amperometric current
increase due to the loss of solvent is ignorable; 5 mM
ferricyanide in 0.1 M phosphate buffer and 3 M NaCl; scan rate
100 mV/s.
At this time scale, evaporation of water from the measuring buffer was expected to lead to
a raise in the concentration of the mediator, and hence to an increase of the faradic current
at the SECM tip. However, cyclic voltammograms recorded at the beginning and the end
of the acquisition of a full SECM image of the 24-spot DNA microarray did not display
significant differences in the diffusion-controlled amperometric currents (see Figure 80).
From this it was assumed that solution evaporation at the given experimental conditions
did not influence the experiments on the DNA chips notably.
Non-specific adsorption
Imperfections in the arraying process may lead to inhomogeneities in the deposition of the
DNA spotting solution and the formation of areas with a certain degree of non-specific
adsorbed DNA strands that surround the desired DNA spots. A practical and
straightforward method for lowering the influence of non-specifically adsorbed ss-DNAs is
to post-assembly the DNA chip with alkane-thiols directly after spotting the DNA probes.
As can be clearly seen in the Figure 81, the gloom accompanying the ss-DNA 20-mer spots
(A) is totally vanished for a chip subjected to a post-assembly with hydroxy-propanethiol
(B) since alkane-thiol molecules are able, in a dynamic equilibrium, to displace loosely
bound (physisorbed) DNA probe strands. On the other hand, the presence of a dense
alkane-thiol monolayer is reducing the contrast between the background signal (DNA free
108
DNA microstructures
surface) and DNA spots and thus decreases the sensitivity of the SECM imaging in the
repelling mode of SECM.
Fig. 81 The effect of thiol post-assembly on the non-specific ss-DNA adsorption: without
post-assembly (A) and with hydroxyl-propanethiol (B); 5 mM ferrocyanide in 0.1 M phosphate
buffer and 1 M NaCl. Spot size: 100 µm in length.
Removal of DNA strands that are physically adsorbed at the chip surface can also be
achieved by soaking the chip in saline buffer for few hours (Figure 82A, B). A strong jet of
buffer directed towards the chip surface can be used as well, without fearing that the shear
forces caused by the liquid flow could damage the self-assembled film!
Fig. 82
The effect of saline buffer soaking on the non-specific
adsorption: no buffer soaking (A) and with soaking (B) in 0.1 M
phosphate buffer and 1 M NaCl.
Furthermore, the so-called edge effect (“doughnut” effect; see note 10) is contributing to a
heterogeneity in the distribution of the DNA strands within an individual spot. The
doughnut effect results from a capillary flow of the bulk of a drying droplet outwards the
edge that can carry any dispersed material to the margin (see Figure 83). That a ring-like
deposition of the biological recognition element takes place when spotting a DNAcontaining solution becomes evident from the SECM image shown in Figure 84. The
higher density of probe strands at the edge of the spot leads to an enhanced repelling which
is visible in the profile of this image.
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DNA microstructures
Fig. 83 Formation of a ring-like-deposition (RLD)
Fig. 84 The ring-like deposit (RLD) of a ss-DNA spot (30 µM probe
concentration) visualised in the repelling mode of SECM; 5 mM
ferricyanide in 0.1 M phosphate buffer and 1 M NaCl.
Certainly, such phenomena have to be considered when aiming at quantitative
measurements of ss- or ds-DNA. The repelling mode of SECM is very sensitive to
variations in the density of probes, and thus could be an excellent tool for inspecting
the quality of the spotting procedure.
The microdispenser as an alternative tool for the preparation of DNA microarrays
Most of the experiments for the evaluation of the repelling mode of SECM as a tool to
detect hybridisation on DNA microarrays were carried out on commercially available chips
that were supplied by FRIZ Biochem, Munich, Germany. These microarrays were prepared
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DNA microstructures
with a pin-spotting instrument. Alternatively to this routinely used method of patterning
DNA chips, a piezo microdispenser (GeSIM mbh, Germany, Figure 85) was used to
fabricate in-house DNA microarrays. Although the functioning of this particular piezomicrodispenser is similar to the one presented in Chapter 2 and 5, there are little
differences between them in the electronic part of the set-ups This commercially available
dispenser is capable to deposit spots about 230 µm in diameter. Typically, the spotting
solutions contained 3’-thiolated adenine 20-mer in 250 mM phosphate buffer (pH 6.7).
Before spotting, the gold surface has been immersed 5 minutes in Piranha mixture and than
rinsed thoroughly with tri-distilled water. Longer contact between the gold-covered glass
slide and the corrosive mixture could peel off the gold film, especially if the metal layer
underneath is exposed and attacked.
Fig. 85 Ink-jet set-up used for micropatterning spots 230 µm diameter of
DNA microarrays on gold surfaces. The piezo-dispenser head is shown in
the inset.
Figure 86 displays a home-made DNA array in which the probe spots are still covered with
the spotting solution. After 4 hours of curing time in which the thiolated DNA strands were
chemisorbed onto the gold surface, the chip was rinsed with 1 M NaCl phosphate buffer
and used for scanning in the repelling mode of SECM. The imaging of the single stranded
DNA probes was carried out in 3 M NaCl in 0.1 M phosphate buffer with 5 mM
ferricyanide as mediator.
Due to the fact that the resolution in X and Y direction was not the same (10 µm distance
between two data point along X-axis and 25 µm along Y-axis), the circular spots of
patterned DNA probes were imaged in the SECM micrograph as squares.
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DNA microstructures
Fig. 86 Example of a home-made DNA microarray. The photographic image to
the left displays a freshly prepared microarray where the droplets of solution are
not yet dried. The image on the right is a SECM micrograph of a small area of the
same microarray. White spots represent the DNA modified regions of the Au
surface (low feedback current). Note: no shadows around the spots and no
heterogeneities within the spots are observed!
Sequence specificity
Two oligonucleotides with equal number of bases but different sequences should offer the
same repelling force when interacting with negatively charged redox species. Although
only two different DNA strands (O8 and F12) were studied, it appeared in the course of all
experiments involving these entities that one type of DNA (F12) blocked more the
diffusion of ferricyanide ions than the other (O8). Figure 87 undoubtedly visualises this
significant difference between the two oligonucleotides. If would be possible to prove
beyond any doubts that strands having the same number of nucleotides but different
sequence are distinguishable by the repelling mode of SECM, this could lead to a
powerful, cheap and uncomplicated assay for comparative genomic studies.
Fig. 87 Repelling mode of SECM is a method possibly sensitive to the sequence of the
oligonucleotides immobilised at the chip. Two 20-mers (F12 and O8) oligonucleotides with
different sequence can be clearly distinguishable in these SECM micrographs; 5 mM
ferricyanide in 0.1 M phosphate buffer and 3 M NaCl; DNA probe concentrations were 100 (first
line) and 30 µM (second line) for both F12 and O8.
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DNA microstructures
5.3.3.7. Factors affecting the imaging quality in the repelling mode of SECM
Imaging of ss-DNAs in the repelling mode of SECM is influenced by a set of parameters.
The conformation of a single flexible polyelectrolyte molecule with a hydrophobic
backbone in aqueous solution is effected by the interplay of the short-range intramolecular
attraction and the long-range Coulomb repulsion238. Besides, electrostatic attraction
between charged cylindrical polyelectrolytes in aqueous medium can be induced by
multivalent counterions239. Any quantitative model wished to describe completely the
interactions between the DNAs or other polyelectrolytes and ions in solution call for an
accurate description of the potential of the electric field around the charged backbones. The
modified Poisson-Boltzmann (MPB) equations together with the Booth’s theory of water
dielectric saturation and an experimental dependence of water dielectric constant on ionic
concentrations are normally used to calculate the mean electrostatic potential and ionic
distributions around a DNA-like highly charged cylindrical polyion240.
Of course, many factors could be identified as important, but accidentally (effect of tip-tosample distance) or in purpose, there were four parameters found during the investigations
that were considered critical and sufficient for the proper study of DNA hybridisation.
These achievements are presented in the following.
Effect of DNA probe concentration
At first, in a set of experiments a number of oligonucleotide spots that were different from
each other only in the density of the immobilised probes were examined. Variations in
surface density of the probes were accomplished by spotting equal volumes of probe
solutions, however, with probe concentrations ranging from 100 µM to 1 µM. Although
not exactly known, the surface density was expected to be correlated to the concentration
of the probe in the spotting solution. For this reason the following results will be discussed
in terms of the concentration of the spotting solutions that were used to produce the studied
DNA chips. Figure 88 illustrates in a series of representative SECM line scans and images
that the modulation of the SECM tip current due to repulsion between the mediator and
probe strands is well correlating with the concentration of spotting solution.
Typically, the contrast (defined as the difference of the signals above a probe spot and the
neighbouring surface) was significantly improved and better SECM images of the single
stranded oligonucleotide were achieved at higher probe concentrations. With the lowest
concentration (1 µM), almost no effect was observed and the spots were almost invisible
for the repelling mode of SECM. This was seen as an indication that the strands in fact
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DNA microstructures
were so loosely arranged that their anionic phosphate groups could not really hinder
[Fe(CN)6]4- molecules to approach the Au surface and undergo redox recycling. In
contrast, oligonucleotide strands in dots spotted from the highest concentrated solution
(100 µM) effectively blocked the diffusion of [Fe(CN)6]4- towards the gold surface and
offered good contrast. However, the “30-µM” spots nearly equally influenced the tip
current.
Fig. 88
Representative SECM line scans (top) and bird-view SECM images (bottom)
obtained by imaging 120 µm diameter spots of a 20 base oligonucleotide at concentration
ranging from 1 to 100 µM by means of the repelling mode of SECM. Measuring solutions: 5
mM ferricyanide in 3 M NaCl/0.1 M phosphate buffer, pH 5.7 (for the line scans) or 5 mM
[Fe(CN)6]3- in 1 M NaCl/0.1 M phosphate buffer, pH 6.3 (for the bird-views). SECM tip: 10
µm Pt disk microelectrode. Scan speed 10 µm/s.
This suggested that above a certain density the electrical field produced by closely
arranged probe strands is well protecting the spot region against passage of free-diffusing
anions. This, however, explains why the impact of coulomb repulsion on the diffusion of
the [Fe(CN)6]3-/[Fe(CN)6]4- couple reaches saturation at a critical probe concentration.
Oligonucleotide spots were detectable and could be successfully visualised with the
repelling mode of SECM in a wide range of concentrations. Nevertheless, from the
observed saturation of the influence of electrostatic interaction on the SECM signals at
higher concentration it became obvious that choosing a suitable superficial density of
probes is a prerequisite when aiming on the imaging of DNA strands and detecting
hybridisation. This is confirmed by a qualitative model, which is schematically depicted in
Figure 89.
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DNA microstructures
Due to the presence of anionic phosphate groups in their backbones, individual probe
strands can be considered as thin negatively charged rods that, in first approximation, form
a cylindrical electric field with a radius r1 around themselves. Within r1, anionic species
will be repelled by coulomb interaction and hence will not find access to the surface.
Fig. 89 Qualitative model representing schematically the coulomb interaction between
ss-DNA and a negatively charged redox species indicated by the arrows. The circles
represent the “forbidden” area at which anions do not find access to the chip surface.
Obviously, this assumption leads to the consideration that probe strands should not be
arranged too close to each other to avoid overlapping of their electric fields. In this case,
the entry for anions within the probe spot would be fully blocked. The observed feedback
current would then become independent from the number of negative charges localised
within the spot thus preventing to distinguish between ss- and hybridised DNA probes at
higher concentration. On the other hand, the distance between two adjacent strands should
not be too large to allow for a significant modulation of the diffusional flux of the redox
species to the underlying Au surface, even after duplex formation.
Effect of mediator concentration
The influence of the redox mediator concentration on the performance of the repelling
mode of SECM for imaging single-stranded nucleic acids was investigated in electrolyte
solutions containing either 5 mM or 50 mM [Fe(CN)6]3-. A selected spot of probe
oligonucleotide was sequentially imaged in these electrolytes with the electrochemical cell
being rinsed many times with phosphate buffer and the scanning solution of choice to
ensure accuracy of the [Fe(CN)6]3- concentration. Since, the diffusion-limited current at a
disk-shaped microelectrode is proportional to the bulk concentration of the redox species
according to the equation
i = 4⋅ n ⋅ F ⋅ D ⋅ r ⋅ c
115
DNA microstructures
with n = number of transferred electron; F = Faraday constant, D = diffusion coefficient,
c = bulk concentration of the redox active compound, and r = radius of the electroactive
electrode surface, a 10 fold increase in the concentration of the redox mediator is leading to
a 10 fold increase in the amperometric tip current in bulk solution. As a matter of fact, also
the current that is observed due to redox recycling at the same tip-to-sample distances is
proportionally higher in solutions of the higher mediator concentration because the
feedback current is directly related to the number of tip-generated species available. As
shown in Figure 90, however, the ratios of tip currents obtained in the vicinity of an
imaged spot and just above it were apparently not dependent on the concentration of the
chosen mediator. Hence, the contrast for imaging oligonucleotides in the repelling mode
can not be enhanced by varying this parameter.
Fig. 90 Influence of the mediator concentration on the visualisation of
ss-DNA in the repelling mode of SECM; probe concentration: 10 µM;
5 mM (top curve) or 50 mM (bottom curve) [Fe(CN)6]3- in 3 M NaCl/0.1
M phosphate buffer, pH 5.7; SECM tip: 10 µm Pt disk microelectrode;
scan speed 10 µm/s.
Effect of ionic strength
It is well-known that two charged particles are only facing electrostatic interaction when
their individual electric double layers start to overlap. Since the extension of the electric
double layer is changing with the ionic strength of a surrounding solution, the repelling
force between phosphate groups at immobilised DNA strands and free-diffusing negatively
charged redox species should be strongly affected by the composition of the electrolyte.
Oligonucleotide spots were therefore subjected to repelling-mode SECM measurements in
solutions with significantly different total ion concentrations. The line scans presented in
116
DNA microstructures
Figure 91 demonstrate that the contrast of SECM imaging indeed was enhanced when
lowering the ionic strength of the measuring solution from 3 to 0.02 M.
In fact, the extension of the electrical double layer expands when lowering the ionic
strength. Regarding the model, this corresponds to an increase of r1 and thus to a reduction
in the surface area available for redox recycling. Worth mentioning that the enhancement
in contrast achieved in solutions of low ionic strength is of little benefit for the detection of
hybridisation since the aggregates of probe and target strands are not stable under this
condition. A compromise must be found to ensure on the one hand good SECM imaging
and on the other hand to provide a suitable environment for the stabilisation of doublestranded DNA after hybridisation has occurred.
Fig. 91 Effect of the ionic strength of the electrolyte solution
on the visualisation of ss-DNA in the repelling mode of SECM.
Line scans were obtained at an ionic strength of 0.02 M (left) or
3 M (right). Probe concentration: 10 µM; 5 mM [Fe(CN)6]3- in
phosphate buffer of the given ionic strength. SECM tip: 10 µm
Pt disk microelectrode; scan speed 10 µm/s.
Effect of tip-to-sample distance
One and the same oligonucleotide spot was imaged sequentially in the repelling mode with
the SECM tip positioned at working distances of 6 and 15 µm. As expected, the closer the
SECM tip was scanned across the DNA microstructure, the better was the contrast (Figure
92). The reason for this observation is an improved collection of tip-generated [Fe(CN)6]4at the chip surface and recycled [Fe(CN)6]3- at the tip electrode, respectively. Apparently,
the probability for loosing the redox species through lateral diffusion is lower at a smaller
spacing between the microelectrode tip and chip surface. However, even though it is
meaningful to scan in close proximity, precaution must be taken when applying SECM in
constant-height mode. For large scan lengths, variations of the tip-to-sample distance due
to e.g. a surface tilt could easily disturb the current response or even lead to tip crash.
117
DNA microstructures
Fig. 92
Influence of the tip-to-sample distance on the
visualisation of ss-DNA in the repelling mode of SECM. Line
scans were obtained at a tip to sample distance of 6 µm (left) or
15 µm (right); probe concentration: 10 µM; 5 mM [Fe(CN)6]3- in
3 M NaCl/0.1 M phosphate buffer, pH 5.7; SECM tip: 10 µm Pt
disk microelectrode; scan speed 10 µm/s.
5.3.4. Detection of DNA hybridisation in the repelling mode of SECM
So far, merely the visualisation of single stranded oligonucleotides in the repelling mode of
SECM was demonstrated. Nevertheless, the major challenge of work was to develop a
straightforward electrochemical approach for detecting the hybridisation of DNA in a truly
label-free manner. Being aware of the multiple factors that are affecting the imaging of
ss-DNA, the investigations were proceeded to study the influence of the probe density on
the recognition of hybridisation.
It was an outcome of the first experiments on detection of DNA hybridisation in the
repelling mode of SECM that the way the hybridisation and imaging were carrying out was
not optimal. For instance, the DNA chip subjected to hybridisation and the one designed to
be the control chip were not one and the same. With this approach, a few µl droplet of the
target solution were placed over all the DNA spots of the microarray and covered with a
glass cover slip which basically meant that hybridisation could not be selectively
performed at a given area of the chip. Several questions can arise from such a situation:
-
Are observed differences in the current peaks of the ss-DNA and ds-DNA induced
by the hybridisation or are variations in the probe density responsible for the effect?
-
Is the SECM tip positioned at the same height over the hybridised and control
chips? Little variations possibly could have an impact on the obtained results?
Well, the difficulties described above were plainly solved in an extremely easy manner,
namely, the spots of one DNA microarray was divided in two sub-areas by means of the
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DNA microstructures
vertical scratch (see Figure 93). In this way, the control and hybridisation experiments can
be performed on the same chip. This ensures that the probe spots should be of fairly
identical properties and that the tip-to-sample distance above the ss- and ds-DNA spots are
not varying too much since they are only a fraction of 1 mm apart.
Fig. 93 Layout of the DNA chip after hybridisation on the right side
with DNA target (A’) complementary only for one type of probe (A).
The status of individual DNA spots was worked out by measuring and comparing the
current response of the SECM tip (a 10 µm Pt disk polarised at 0 mV vs. Ag/AgCl 3 M
KCl and positioned at about 15 µm above the chip surface) that was scanned across two
otherwise identical microstructures one of which, however, had been exposed to the
hybridisation solution containing the complementary target and the other to target-free
hybridisation solution (control). It had been shown in the previous pages that a
considerable number of DNA probe strands is washed off, while soaking the chip in saline
buffer. To be able to perceive the hybridisation event, the control spots and hybridised
areas must be equal times in contact with buffer solutions. Hence, a “blind” hybridisation
had to be carried out only on the control area, whereas the hybridisation solution should
not touch the control region. The vertical scratch defining the control and hybridisation
regions of the chip was found helpful because it is acting as a barrier stopping the solution
wetting the forbidden area at the opposite side (Figure 94).
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DNA microstructures
A typical hybridisation protocol consists of a series of steps:
1. scratch;
2. 2 hours blind hybridisation on the left side (in the hybridisation cell);
3. 2 hours hybridisation on the right side (in the hybridisation cell);
4. chip rinsing with 3 M PBS;
5. SECM measurement with 5 mM ferricyanide in 3 M PBS.
Hybridisation experiments were performed at room temperature with the following
solutions prepared in phosphate buffer (pH 6.5):
Blind hybridisation
Real hybridisation
-
2
Buffer (M)
0.1
0.1
NaCl (M)
1
1
SDS (%)
0.01
0.01
Target (µM)
Before and after hybridisation the chips were rinsed with 3 M PBS. Owing to the affinity
of gold for many compounds, it is recommended to filter all buffer solutions through filter
units with a pore size of 5 µm. Otherwise, the gold surface will be gradually covered with
impurities from solutions. This could result in unwanted effects on the amperometric
feedback behaviour of the chip, disturb the electrochemical approach of the electrode and
lead to noisy SECM images.
A Petri dish was used as the hybridisation cell (Figure 94) in which the DNA chip and a
small plate filled with the same buffer as used for hybridisation were placed. This closed
system helped to minimise the evaporation of the 5 µl droplet solution of target DNA, that
otherwise will dry out before the completion of the hybridisation.
A synopsis of the results of a typical hybridisation experiment obtained for 100 µM, 10
µM, 5 µM, 3 µM, and 1 µM probe concentration is shown in Figure 95 and 96. Except for
the lowest probe concentration, the DNA spots could be well visualised but a visible
change in the tip current upon hybridisation was only observed at the 10 µM DNA probe.
In theory, binding of the target strands is increasing the net charge and enhancing the
electric field within the probe spot. Accordingly, the “forbidden” area for free-diffusing
redox-active anions is expanded upon hybridisation (r1→r2; Figure 97). For hybridisation
detection, probe strands should not be too close to each other to avoid that the sphere of
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DNA microstructures
Fig. 94 The DNA hybridisation cell was a simple Petri
dish in which the DNA chip and small water container
were placed.
their electrical fields overlap. In that case, already for single strands the flux of anions
towards the chip surface would be blocked to a high extent, making it difficult for the
hybridisation of target to have an additional effect and become detectable. In principle, the
separation between two single strands needs to be approximately 2⋅r2 (or ε) for optimal
detection of base pairing (see note 11).
By means of proper selection of a set of parameters as can be clearly seen in Figure 95,
which are expected to have a significant impact on the possibility to detect DNA
hybridisation with repelling-mode SECM, a good current contrast between probe spots and
hybridised spots can be obtained.
121
DNA microstructures
Fig. 95 Detection of hybridisation on an oligonucleotide
microarray by means of
repelling mode of SECM.
Single line scans were
carried out over pairs of DNA
spots. The current traces to
the left correspond to nonhybridised strands (control)
whereas the traces to the
right were acquired on
hybridised
spots. Probe
concentration in spotting
solution: 1 to 100 µM. 5 mM
[Fe(CN)6]3- in 3 M NaCl/0.1
M phosphate buffer, pH 5.7.
SECM tip: 10 µm Pt disk
microelectrode. Scan speed
10 µm/s.
Fig. 96
A close-up of the 10 µM ss-DNA and ds-DNA amperometric
current peaks as measured at the scanning tip in the repelling mode of
SECM after hybridisation with the complementary target.
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DNA microstructures
Fig. 97 Qualitative model representing schematically the coulomb interaction between dsDNA and negatively charged redox species An-. Upon duplex formation the forbidden area
increases with radii changing from r1 (rss) to r2 (rds).
Non-specific adsorption of target during hybridisation process
DNA has a high affinity to gold and therefore is able to forms stable films non-specifically
adsorbed targets on the substrate. Thus, an exposure of a gold surface to a solution
containing DNA molecules is altering its feedback behaviour that way that it appears less
conductive. This is confirmed by the observations shown in Figure 98 (to the left). The
current observed above the control region (not exposed to any dissolved DNA) was much
higher due to positive feedback than in the region which was exposed to target DNA.
In order to diminish the amount of non-specifically adsorbed targets and enhance the
detection of double helix, it is recommended to use detergent additives such as sodium
dodecylsulphate (SDS). Typical SDS concentrations of 0.01% in the hybridisation solution
are of great help since the target could not be any longer observed by SECM at the gold
plane. It should be mentioned that SDS is not efficient at very high target concentration
(10-20 µM) and it is impossible to overcome unwanted physical adsorption of targets by
increasing the SDS concentration because its solubility in saline buffers is rather low.
Interestingly, 2 M NaOH solutions can, in a few minutes, wash off the excessive target.
Moreover, as a side effect, loosely bound DNA probes are removed as well (Figure 98, to
the right). This observation shows that the surface density of DNA probes will change
when the chip is dehybridised in NaOH, which in principle restrains the number of
hybridisation-dehybridisation cycles of a certain microarray.
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DNA microstructures
Fig. 98 Non-specifically adsorbed DNA target on a hybridised DNA chip after hybridisation
with solution lacking SDS (left). The same chip, after a short 2 M NaOH treatment (right).
5.3.5. Conclusions and outlook
A truly label-free electrochemical method for visualising immobilised nucleic acids and
their hybridisation on microarrays was developed. The method is based on the electrostatic
repulsion between the phosphate groups of DNA and free-diffusing [Fe(CN)6]4- ions and is
straightforward and reliable. The coulomb interaction is modulating the diffusion
properties of the electroactive species in the vicinity of probe strands and thus had a strong
impact on the transport of the negatively charged species towards the conducting chip
surface in DNA-modified regions. Using SECM in the amperometric feedback mode,
significant decreases in positive feedback currents are observed above spots of nucleic
acids due to the local appearance of repulsion between the active components of the assay.
The density of the capture probe, the ionic strength of solution and the tip-to-sample
distance are influencing the capability of so-called repelling mode SECM to visualise DNA
while the concentration of the chosen mediator has not effect on the contrast of imaging.
Miniaturisation of the detection set-up
Verification of the developed detection scheme was performed by local electrochemical
measurements in a sophisticated SECM set-up that obviously is not suitable for common
use in medical diagnostics. However, the obtained results promote, in principle, the design
of a simplified electrochemical device that could be produced using microfabrication
technology. A device could, for example, consist of an array of immobilised probes in a
base plate and an array of individually addressable Pt microelectrodes in a cover plate able
of being revolved upon a lateral hinge as suggested in Figure 99 (similar Pt microelectrode
arrays have already found many applications especially in biosensors).
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DNA microstructures
Using specially designed spacer and alignment elements, the microelectrodes and DNA
spots could be arranged on top of each other and kept at proper working distance. The
difference in the response of an individual Pt microelectrode before and after exposure to a
sample would evaluate the status of the opposite capture probe and indicate hybridisation
in case the complementary target had been present in the sample solution.
Fig. 99
Miniaturised clamping device for reading out DNA hybridisation from a
multielectrode array.
Label-free, instrument-free detection of DNA hybridisation
The preparation of the DNA chips for hybridisation and SECM measurements required
rinsing-steps for removing unwanted compounds off the chip surface. For instance, saline
buffers were used before marking the DNA chip. During those operations, it was noticed
that salt deposits are formed on the surface of the chip exactly in the positions of the DNA
spots. Could such phenomena be used for specific imaging of immobilised ss- or dsDNAs? Theoretically, special circumstances may lead to crystalline deposits of which
dimensions are correlated with the electrical charge of the polyanions because as many
deprotonated phosphate groups are present at a spot, as many counterions would be
necessary to compensate their electrical charge. The preliminary experiments showed that
the amount of salts trapped at DNA-modified areas is proportional to the concentration of
the saline buffer (Figure 100) and that high density spots grow bigger deposits as small
ones, to a certain extent (Figure 101). Hybridisation could not be yet detected. Apparently,
the relation between the concentration of DNA probes and size of the salt deposit was not
as precise as would be needed for detection of DNA hybridisation. However, preliminary
results in “imaging” DNA chips by means of the appearance of salt deposits are
125
DNA microstructures
encouraging because the differences between DNA probes of various surface densities are
clearly noticeable within the salt patterns (Figure 100). Future work has to be directed
towards finding the appropriate conditions (substances, solvents) for inducing specific
crystal/deposit formation around DNA spots.
Fig. 100
First step to a label-free instrument-free
detection of hybridisation? The concentration of the
rinsing buffer determines the thickens of the salt
deposit at the ss-DNA spots! The yellow rectangular is
pointing out a printing error.
Fig. 101 Another suggestive
example of dependence between
the size of the salt deposit and
concentration of ss-DNA probes
for two 20-mers with different
sequence (F12 and O8).
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Micropatterning and microelectrochemical characterisation of biological recognition elements
5.4
Notes
1. The term “holoenzyme” denotes an apoenzyme linked to its corresponding cofactor.
2. The six major classes of enzymes according to EC:
1.
EC 1 Oxidoreductases
To this class belong all enzymes catalysing oxido-reductions. The common name
is “dehydrogenase” but “oxidase” is used when oxygen is an electron acceptor.
2.
EC 2 Transferases
Are enzymes transferring a group from one compound (generally regarded as
donor) to another (acceptor).
3.
EC 3 Hydrolases
These enzymes catalyse the hydrolysis of various bonds.
4.
EC 4 Lyases
These enzymes cleave C-C, C-O, C-N and other bonds by other means than by
hydrolysis or oxidation.
5.
EC 5 Isomerases
They catalyse changes within one molecule.
6.
EC 6 Ligases
Ligases are enzymes that catalyse the joining of two molecules with concomitant
hydrolysis of the diphosphate bond in ATP or a similar triphosphate.
3. Optimal working temperature of an enzyme is to be associated with the conditions the
source of enzyme is living under. Aspergillus niger, the fungus used to produce citric acid
and gluconic acid in industry is a accessible source of glucose oxidase. The optimal growth
temperature of this specific organism is 35-37 °C so that any enzyme obtained from this
fungus best works at 35-37 °C. Special application in biochemistry, one great example
being Polymerase Chain Reactiona (PCR) would not become possible without enzymes
that normally function at elevated temperatures. DNA-polymerase that is isolated from
Thermus aquaticus, a bacteria living in hot springs, needs a hot medium (about 72 °C) to
work properly. Hence, its DNA-polymerase is suitable to using in PCR for mass-copying of
DNA excluding cloning techniques.
Directed growth of living cells
4. Laminin is a major component of basement membranes of living cells. It has numerous
biological activities including promotion of cell adhesion, migration, growth, and
differentiation, including neurite outgrowth. Owing these functions it is used as a thin
coating on tissue/cells-culture surfaces or as a soluble additive to culture medium. Laminin
a
Kary B. Mullis (1944, USA) American scientist that received the Nobel Prize for „his invention of the
polymerase chain reaction (PCR) method” in 1993. The discovery was first presented in 1985.
127
has been shown in culture to stimulate neurite outgrowth, promote cell attachment,
chemotaxis, and cell differentiation.
DNA microstructures
5. Complementary DNA is synthesized in laboratory from a messenger RNA template (mRNA
– messenger RNA). The cDNA are used as probes in DNA microarrays. To understand
better why this is important for the hybridisation experiments, let us follow the steps of a
virtual experiment for the study of a comparative gene expression.
i. choosing the cell population: two different kind of cells of an organism are
selected;
ii. extraction of mRNA and the reverse transcription: genes which code for
protein are transcribed into messenger RNA’s in the cell nucleus. After
being released in the cytoplasm, the mRNA is translated into proteins by
ribosome. Although, this RNA could be used for grafting it onto a
microarray as probes, this is not practical. mRNA is prone to being
destroyed especially by RNA-digesting enzymes that normally are
everywhere where our fingers touched a surface. To prevent the loss of
mRNA, it is preferred to reverse-transcribe the mRNA into more stable
DNA that is the complementary cDNA.
iii. fluorescent labelling of cDNA’s;
iv. hybridisation at the surface of the DNA microarray;
v. scanning the hybridised microarray.
To obtain cDNAs libraries, vital for the mass production of DNA microarrays, the cDNA is
cloned into a plasmid (an extra-chromosomal circular DNA molecules, distinct from the
normal bacterial genome) and then transferred in Escherichia coli where the plasmid is
replicated many times. At last, the cDNA is extracted and purified from the bacterial
content.
6. A preferred method for deposition of very small (1-2 nanolitre) quantities of DNA-probe
solution onto the slide involves the of use pins. The pins of the spotting machines are
made of stainless steel, chromium, titanium or other metals that have sufficient resistant
against corrosion and certain hardness to minimise the blunting of the sharp tips. These
may be "solid" pins, which dispense just once per sample collection action, or "split" pins,
which pick up much more sample at a time and are capable of a multi-dispensing mode of
operation. In either case, sample is transferred passively by means of surface tension as
the tip gently touches the slide surface. Electrical Discharge Machining (EDM) is normally
used to cut the fine slots in the tip of the pins.
7. Several scientists were at the same time in run for elucidating the structure of nucleic acids
in the early 1950s
241
. Simultaneously, ongoing research programmes in the physical,
organic and biological chemistry were intersected in order to elucidate a mysterious
process: DNA replication. In these circumstances, teams of foremost scientist as L. Pauling
and R. B. Corey, Wilkins et all242, and R. E. Franklin and R. S. Gosling understood the DNA
128
molecule is a helix. Pauling build a wrong model of the double helix while R. Franklin did
not bother at all about the possible arrangements of the DNA constituents243,244 even
though she was very close to get it245. It was J. D. Watson and F. H. C. Crick, who
suggested a model explaining that: DNA has two anti-parallel and complementary strands
in a double-helix arrangement246. Furthermore, the novel DNA structure even suggested a
possible copying mechanism for the genetic material247. The two DNA chains are held
together by interactions between the nitrogenous bases on opposite strands. These
interactions are called base pairing. The famous DNA Base Pairing Rules, are :
i. adenine (A) and guanine (G) are purines and cytosine (C) and thymine (T)
are pyrimidines. Purines base pair with pyrimidines;
ii. A pairs with T and vice versa while C pairs with G and vice versa;
iii. there are 2H-bonds holding A and T together and 3H-bond for C and G;
iv. because of the additional hydrogen bond, C and G base pair is stronger as
A and T.
8. The relation between the frequency-shift of a quartz crystal microbalance and the change
in the mass of the film attached to the crystal is given the next equation:
 ∆m 
∆ν = − 2.3 ×10 6 ×ν 0 × 

 A 
where ∆ν denotes the shift of the frequency corresponding to ∆m; ν0 is the resonant
frequency of the bare quartz surface; A is the surface of the exposed quartz crystal. For a
given working-frequency of 10-15 MHz, frequency shifts of 10-2 or even lower can be
detected. Consequently, a mass as small as 10-11 g is measurable by QCM248.
9. It is frequent that scientist are reporting extremely low detection limits of ds-DNA, namely
550 amol (J. J. Gooding, “Electrochemical DNA hybridisation biosensors”, Electroanalysis,
2002, 14, page of interest is 1152). I find this is a misused technical detail because such
limits were achieved only with very long DNA chains. For instance, in the above mentioned
case was used a 1497 bases DNA strand and if one takes into account that there are the
same number of reporters (guanines) on each strand, this is equivalent to 1497 of
individual and simple species. If such ideas are accepted, then one should agree the
human being is also able to see with naked eyes single molecules: a big mono-crystal of
diamond is a single molecule, or?
10. The edge (doughnut) effect is a ring-like deposit (RLD)249 which results from a capillary
flow of the bulk of a drying droplet outwards the edge that can carry any dispersed material
to the margin. Example: a coffee drop falls on the table and by splashing and drying out
creates a brownish disk. After the complete drying of the drop, a ring is observed at the
margins. Note: this ring will not be noticed if the coffee contains sugar. Sugar makes the
liquid viscous and the outwards flow is slowed down. Consequently, the ring is less visible.
For a funny explanation please read the book of Robert L. Wolke “Was Einstein seinem
Friseur erzählte”, Piper Verlag GmbH, München, 2001, 255-257.
129
11. At some point in elaborating this qualitative model of DNA-DNA interaction, I sought to
establish an equation between R1 and the optimal separation distance ε for a given set of
DNAs (target and probes) and negative charged redox mediator. Despite the fact that
describing the overall electrostatic interaction between involved parts is not a challenge,
the problem could not be solved because of turning an theoretical equation valid in vacuum
condition to complex environment where the electrical permittivity of solution differs in bulk
and around the polyelectrolyte strands, or where the DNA’s charges are distributed not
along a straight -road but coiled as function of the ionic strength of the solution are only two
examples of significant obstacles not easy surmountable. Nevertheless, the problem is
solvable, and finding the solution could be of great deal of help for mass production of DNA
microarrays, when knowing the optimal surface density could save expensive probes! It
may worth finding the ε distance only if the repelling-like detection techniques will raise
enough attention and have a market among the already commercialised DNA detection
kits.
130
Experimental
6.
Experimental
Chemicals and Materials for the preparation of microelectrodes
Solutions were prepared with triply-distilled water. Chemicals were purchased from Sigma
Aldrich (Deisenhofen, Germany). Tollens’ reagent for the electroless deposition of silver
consisted of a mixture of 5% AgNO3, 10% NaOH, 28-30% NH4OH, and 10% Glucose.
The Ag electroplating bath consisted of 40 g/L Ag2SO4, 380 g/L Na2S2O3 ⋅5H2O, 20 g/L
Na2B4O7 ⋅10H2O (borax) and 20 g/L Na2SO4 ⋅10H2O. Cyclic voltammetry and SECM were
performed in solutions containing 5 mM [Ru(NH3)6]Cl3 and either 0.1 M KH2P04/ K2HP04
buffer or 0.1 M KCl as supporting electrolyte. Paraffin (mineral) oil was from Sigma (EC
number 232-455-8).
Pt wire was from Goodfellow/Germany and glass capillaries from glass capillaries
Hilgenberg/Germany. Alumina polishing suspension, 3 µm, 1 µm and 0.3 µm was from
Leco Co., Lakeview Ave., USA. Zinc powder was from Fisher Chemicals and soldering
wire from Conrad/Germany. Polishing cloth (red) was from Heraeus Kulzer, Wehrheim,
Germany.
Chemicals and Materials for the preparation of enzyme microstructures
Glucose oxidase, type X-S from Aspergilus niger activity of 119,000 units/g solid, was
purchased from Sigma Chemical Co., St. Louis, USA. The polymer dispersion Vinnapas®
EP16 W was kindly provided by Wacker Polymer Systems GmbH & Co. KG, Burghausen,
Germany. Solutions of glucose oxidase and Vinnapas® EP16 W were prepared with triplydistilled water. Filter units FP 30/5,0 CN (black rim) were purchased from Schleicher &
Schuell, Dassel, Germany.
Chemicals and Materials used in the experiments regarding the defined
adhesion/growth of living cells
Laminin from the mouse Engelbreth–Holm–Swarm sarcoma was from Boehringer
Mannheim, Germany. DiI was applied as fluorescent dye (1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate) was from Molecular Probes Europe, Leiden, The
Netherlands.
Four different substrates were used for the experiments: glass cover slips, pure silicon
samples coated with a 130 nm gold layer (Chemistry Department at the Ruhr University,
Bochum), pure silicon samples provided by the Institute of Thin Film and Ion Technology,
131
Experimental
Research Centre Jülich, Germany, and glassy carbon samples (Hochtemperatur-Werkstoffe
GmbH, Thierhaupten, Germany).
Neurones were prepared from embryonic chicken forebrain under sterile conditions.
Chemicals and Materials for the preparation of DNA microstructures
KH2PO4, K2HPO4⋅3H2O, NaCl, K3[Fe(CN)6] and [Ru(NH3)6]Cl3 were from Sigma,
Deisenhofen, Germany and SDS (sodium dodecylsulphate) from Merck, Darmstadt,
Germany.
FRIZ Biochem, Munich, Germany provided oligonucleotide microstructures (DNA chips)
along with the complementary targets.
Instrumentation
Basic components of the SECM set-up
Experiments were carried out with a home-built SECM in a one-compartment cell in twoelectrode configuration. Faraday cage was home build and the low-noise VA10 amplifier
from npi electronics GmbH, Tamm, Germany. Potentials were measured against the
chemically deposited Ag coating or an independent, miniaturised Ag/AgCl/3M KCl
reference electrode. Stepper motors with a resolution of 0.625 µm per half step were from
Owis, Staufen, Germany. The personal computer used software programmed in Microsoft
Visual Basic 3.0 (Microsoft, Unterschleißheim/Germany). High current power supply (32
V and 24 A) was from Statron Elektronik, Fürstenwalde, Germany.
Microdispenser set-up
The microdispenser was operated together with a wave generator from Hewlett Packard,
type 33120A. Stepper motors for precice movements of the substrate had a resolution of
0.625 µm per half step and were from Owis, Staufen, Germany. The personal computer for
controlling the microdispensing procedure used software programmed in Microsoft Visual
Basic 3.0 (Microsoft, Unterschleißheim/Germany).
Microspotter for DNA microarray fabrication
For spotting a microarrayer from Cartesian Technologies, Inc., USA equipped with a
printhead (ChipMaker™) and microspotting pins (120 µm diameter) from Telechem
International, Inc. was used. Electrochemical measurements were carried out with a low132
Experimental
noise VA10 amplifier from npi electronics GmbH, Tamm, Germany. Piezo microdispenser
with 700 nl droplet size, was from GeSIM mbh, Germany.
Other instrumentation
A fluorescence microscope (Axiophot, Zeiss, Germany) was used for the observation of
stained neuronal cells.
Experiment protocols
Preparation of glass Pt-disk microelectrodes
Borosilicate glass capillaries (O.D. 1.5 mm, I.D. 0.75 mm, L 100 mm) were tapered down
to form a closed sharp tip, process carried out with a classical capillary puller. The open
end of the capillary is connected to a water vacuum pump all along the heating process.
This reduces the risk of getting air bubbles between the Pt wire and glass insulation, and
additionally, shortens the embedding time of Pt in the molten glass tip. The particular
coiled filament in this case used a current of 17 A to heat glass tube.
Platinum wires with diameters of 10 µm were tightly sealed into these tapered ends of
pulled capillaries. Smooth Pt micro disks were exposed by carefully polishing the tip at 90°
on emery paper (grade 320 to 2000) and then on a polishing cloth wetted with alumina
suspension (particle sizes: 3 µm, 1 µm, and 0.3 µm). The back connection between the Pt
wire and copper lead was established by filling the capillaries with Zn powder (10 µm
particles) unless the protruding Pt is fully covered and then with crushed tin solder, which
after placement was melted by careful heating with the coiled filament at 9 A. The Cu wire
was fastened at the upper part of the capillary with shrinking tube I order to avoid breaking
the contact between Cu and Pt. Note: in the case of Pt wires with diameter bigger as 50
µm, the Zn powder is non necessary but only the solder.
Silver electroplating
The Ag electroplating bath consisted of 40 g/L Ag2SO4, 380 g/L Na2S2O3 · 5H2O, 20 g/L
Na2B4O7· 10H2O (borax) and 20 g/L Na2SO4· 10H2O in tri-distilled water. The compounds
are not all easily dissolved in water (i.e. AgSO4) and for this reason the solution has to be
kept in the ultrasonic bath for few minutes. One should know that the colour of the solution
133
Experimental
is dark green or even black even if all substances used to prepare the mixture are
colourless. So, do not waste it!
At room temperature, a current density of 0.5-0.6 A/dm2 is the ideal. A lower current will
take too long time for preparing a reasonable thick layer of silver whereas a higher current
leads to formation of Ag dendrites. The appearance of dendrites is a sign that the
electrochemical reaction is kinetically limited and thus if, however, high current are
allowed to flow through he electrolysis cell, the solution must be stirred to enhance the
mass transport towards the cathode.
Neurons preparation
For the preparation of prepare the neurons, fertilised chicken eggs were incubated at 37.7
0
C and 60% humidity for 8–10 days. Dissociated cells were prepared from the forebrain of
the chicken embryo. After decapitation, the brain was dissociated by incubation in 1 mg/ml
trypsin in HBSS for 10 min. at 37 0C. In order to reduce DNA-mediated aggregation of
cells, 100 µl solution of DNAse were added. The cell suspension was washed by
centrifugation at 550 × g for 6 min in HBSS containing trypsin inhibitor solution (1 mg/ml)
and subsequently incubated with trypsin inhibitor (3 min. at 37 0C). After a second
washing step by centrifugation, the cells were re-suspended in the so-called S4-medium,
and cell suspensions were given onto the substrates. The serum-free S4-medium was
prepared starting from Dulbecco’s modified Eagle’s medium (Gibco BRL Life
Technologies, Scotland) according to the procedure described literature1 with the
modification that a higher glucose concentration was applied. After 2 days of cultivation
in the S4 medium in an incubator at 37 0C and with 5% CO2, the cultured neurones were
washed shortly with phosphate buffer saline (PBS), pH 7.4, and fixed with a solution of
4% paraformaldehyde in PBS overnight at 4 0C. The samples were washed again with
PBS. Cells on glass cover slips could be inspected with usual phase contrast microscopy.
The cells located on opaque substrates were stained for 5 s in a solution of DiI in 70%
ethanol. The lipophilic dye is incorporated very quickly into the membranes of cells and
neurites, and cells and neurites can be visualised with a fluorescence microscope2.
1
L. K. Needham, G. I. Tennekoon, G. M. McKhann, “Selective growth of rat Schwann cells in neuron- and
serum-free primary culture”, J. Neurosci., 1987, 7, 1-9.
2
P. Godement, J. Vanselow, S. Thanos, F. Bonhoeffer, “A study in developing visual systems with a new
method of staining neurones and their processes in fixed tissue”, Development, 1987, 101, 697-713.
134
Experimental
Immunochemistry
For immunochemistry, neuronal cultures were washed and fixed as described above. Then,
they were washed with PBS three times in order to remove the paraformaldehyde and
blocked with 1% bovine serum albumin (BSA) in PBS for 1 h. Antibody against
neurofilament (Sigma) was added in a dilution of 1:400 in 1% BSA in PBS overnight at
4 0C. The samples were washed again three times with PBS, and a secondary antibody
labelled with Cy2® (Jackson Immuno Research Laboratories, West Grove, PA) was added
in a dilution of 1:200 in 1% BSA in PBS for 1 h. After three washing steps with PBS, the
cell cultures were inspected with a fluorescence microscope. The same procedure was
applied for the immunochemical staining of the polymer lines on glass cover slips. In order
to check whether laminin being entrapped in the polymer could be recognised, an antilaminin antibody (Sigma) was used for the detection of laminin.
DNA microstructures
Phosphate buffers
The 0.1 M phosphate buffer was prepared from 0.05M KH2PO4 and 0.05 K2HPO4· 3H2O in
one litter of tri-distilled water (pH 6.7).
Measuring buffer
With the o.1 M phosphate buffer a solution of 1M NaCl was prepared for hybridisation
purposes (pH 6.5). SECM measurements were performed in a phosphate buffer containing
3 M NaCl and 5 mM ferricyanide (pH 5.7).
Dehybridisation solution
The dehybridisation solution contained only 2M NaOH in NaOH. This solution should be
used only fresh and filtrated through at least 5 µm filter unit prior to use.
135
Conclusions
7.
Conclusions
The aims of this PhD thesis, some of them being defined from the very beginning, others
coming up during the course of this work, are strongly tuned to lab experiments but have
also had to do with development of theoretical aspects of microstructures. Investigations of
microstructures of various components of living cells, which are key elements of the
chemical/biochemical sensor technologies, and their possible applications, ask first of all,
for manufacturing of the microstructures by means of specific tools, a piezomicrodispenser in this particular case.
It was shown that with the ink-jet printing technique, complex geometries are
microstructured in a straightforward manner. When it was planned to work on the directed
growth of neurons, it turned out that even patterning laminin is a challenge due to its
structural fragility. Although, the experiments were performed in the laboratory of Eye
Hospital, Münster, that was not that familiar to me, it was of great help to have a modular
spotting set-up that could be adapted to the special conditions (clean rooms, flow benches)
of that laboratory. Special care had been taken to avoid laminin getting damaged and to
minimise contaminations of the instrumentation and chips and to preserve the vitality of
neurons. The output was very encouraging giving that the cells clearly followed our
microstructures. These results open a new route to, for instance, development of neuron
networks useful in studying cell growth or for coupling microelectronics to living
organisms. The microdispenser has found another interesting application in the field of
biosensor microarrays. Multi-analyte sensors will offer a great deal of help in monitoring
metabolites or pollutants, and thus, we sought to add study the conditions in which an
enzyme base microarray could be used to monitor glucose concentration. This study
involved the SECM, owing its abilities to probe electrochemically the course of, in this
case enzymatic reactions. It turned out that calibration curves for glucose could be obtained
in a broad domain of concentrations by varying the content of enzyme within the
microstructures.
The tremendous advance of microarrays, especially of those based on nucleic acids, is
mostly directed towards the invention of a sensitive, reliable, simple, and cost-effective,
miniaturised device for the detection of DNA hybridisation on DNA chips. Along with
long-ago established fluorescence detection of hybridisation, the electrochemical methods,
and especially the label-free approaches are given much attention these days because they
fulfil all the requirements for developing individual medical point-of-care devices. With
background knowledge in SECM, I sought to make use of one of the intrinsic property of
136
Conclusions
DNA, namely the negative electrical charges of the DNA backbones, to establish a method
for a label-free electrochemical procedure to visualise surface confined DNAs strand and
to detect their hybridisation. Investigating a number of DNA microarrays in the newly
introduced repelling mode of SECM, the main factors that have an essential impact on the
quality of this mode of DNA imaging were revealed. Moreover, it was noticed that in order
to improve the sensitivity of this technique, the superficial concentration of DNA probes
must have a special value as resulting from a qualitative model described in this work. Part
of the DNA chapter, dedicated to the detection of hybridisation explains the difficulties
that had been overcome before it could be proved that it is possible to distinguish the
double stranded DNA signal from those belonging to the single stranded DNA.
Microelectrochemistry in small volumes is needed when the amount of available analyte is
reduced or for combinatorial electrochemistry in micro- or nano-titre plates. Two or three
electrodes can be easily fit in a single vial of microtitre plate due to the novel electrode
assembly developed in the framework of this PhD. Its fabrication, possible applications
and advantages as well as disadvantages were described. A great advantage of this coaxial
Pt working/Ag reference electrode comes from its easy manufacture with common
instruments. Thus it can be prepared in-house in laboratories lacking sophisticated
instruments for coating by sputtering or vapour deposition of metals. Additionally, a
miniaturised Ag/AgCl x M reference electrode preparation was presented, and as the above
mentioned electrode system, this can be produced with available material in any
laboratory.
In conclusion, by shortly mentioning some of the significant results of this PhD it was
sought to outline the alternative methods or improvements that could be of use in analytical
chemistry, developed at some points in the course of my PhD time.
Personal note
Thanks to the variety of fields this PhD covered over the last few years, I had the
opportunity to learn/understand concepts, theories related to that part of science regarded
as electrochemistry, and to gain practical experience in manipulating specific
instrumentation. I have found a proper atmosphere, in this group, for developing my own
ideas and checking whether there are valid or not. However, the different research topics I
was involved in matched nicely with each other and thus allowed me to unify the results
within a broad title such as “Micropatterning and microelectrochemical characterisation of
biological recognition elements”.
137
Conclusions
There are several items I would like to continue in one way or another. For instance, I
believe that the micropatterning of biomolecules can still be further improved and
performed with common instrument in a reliable and reproducible way; the detection of
DNA hybridisation should end up with a miniaturised and trustworthy device based either
on electrostatic repulsion between an anion and the deprotonated phosphate groups, or
based on the DNA-induced salt crystallisation (DISC) at the DNA spots that theoretically
could differentiate ss- from ds-DNA. Manufacturing of integrated electrode assemblies is a
funny and relaxing habit that could deliver more useful SECM tips or just simple
miniaturised electrodes.
I hope I will find a chance to go on with these and other scientific plans!
138
Acknowledgment
8.
Acknowledgment
I would like to thank all those who have been generous with their time and helped me by
any mean over the last three years. I am in particular thankful to:
My esteemed supervisor Prof. Dr. Wolfgang Schuhmann
-
for his guidance along electrochemistry, constant encouragement, patience and
understanding;
Dr. Albert Schulte
-
for the sleepless nights in the lab and his helping from the very beginning and all
along my PhD time; he also guided me in writing this thesis.
Dr. Thomas Erichsen
-
for his useful software and daily computer troubleshooting;
Dr. Gerhard Hartwich
-
for providing DNA chips and knowledge about DNA microarrays.
Dr. Peter Heiduschka
-
for the successful collaboration on neuron growth on micropatterned surfaces.
Kalathur Ravi
-
for making the English readable and for the pleasant teamwork in the lab.
Dr. Rolf Neuser
-
for the great SEM images and his friendliness.
Gerhard Hartwich and Wolfgang Schuhmann. Düsseldorf, October 2002.
139
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on various aminosilane layers”, Langmuir, 2002, 18, 1764-1769.
168.
http://www.frizbiochem.de/
169.
F. Perraut, A. Lagrange, P. Pouteau, O. Peyssonneaux, P. Puget, G. McGall, L. Menou, R.
Gonzalez, P. Labeye, F. Ginot “A new generation of scanners for DNA chips”, Biosens.
Bioelectron., 2002, 17, 803-813.
170.
F. Caruso, E. Rodda, D. N. Furlong, “Quartz crystal micro-balance study of DNA
immobilisation and hybridisation for nucleic acid sensor development”, Anal. Chem., 1997,
69, 2043–2049.
171.
I. Willner, F. Patolsky, Y. Weizmann, B. Willner, „Amplified detection of single-base
mismatches in DNA using microgravimetric quartz-crystal-microbalance transduction”,
Talanta, 2002, 56, 847-856.
172.
T. Livache, E. Maillart, N. Lassalle, P. Mailley, B. Corso, P. Guedon, A. Roget, Y. Levy,
“Polypyrrole based DNA hybridisation assays: study of label free detection processes
versus fluorescence on microchips”, Journal of pharmaceutical and biomedical analysis,
2003, 32, 687-696.
173.
R. Gabl, H.-D. Feucht, H. Zeininger, G. Eckstein, M. Schreiter, R. Primig, D. Pitzer, W.
Wersing, „First results on label-free detection of DNA and protein molecules using a novel
integrated sensor technology based on gravimetric detection principles”, Biosens.
Bioelectron., 2004, 19, 615-620.
174.
P. de-los-Santos-Alvarez, M. J. Lobo-Castanon, A. J. Miranda-Ordieres, P. Tunon-Blanco,
“Current strategies for electrochemical detection of DNA with solid electrodes” Anal.
Bioanal. Chem., 2004, 378, 104-118.
175.
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technology” Meas. Sci. Technol., 2004, 15, R1-R11.
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Factor V Leiden mutation using disposable pencil graphite electrodes”, Anal. Chem., 2003,
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assay based on a silver nanoparticle label”, Analyst, 2002, 127, 803-808.
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detection”, Langmuir, 2003, 19, 989-991.
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copies of DNA in a 10-muL droplet at 0.5 fM concentration”, Anal. Chem., 2003, 75, 32673269.
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amperometric sandwich test for RNA and DNA”, Anal. Chem., 2002, 74, 158-162.
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amperometric detection of a 38-base oligonucleotide at 20 pmol L-1 concentration in a 30µm L droplet”, Anal. Bioanal. Chem., 2002, 374, 1050-1055.
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10.
CURRICULUM VITAE
FAMILY NAME
TURCU
FIRST NAME
EUGEN FLORIN
ADDRESS
Markstrasse 329
44801 Bochum, Germany
Tel.
0049-234-3384897 (private)
0049-234-3226202 (work)
E-mail
eugen.turcu@rub.de
DATE OF BIRTH
1973.10.18
PLACE OF BIRTH
Baia Mare, Romania
NATIONALITY
Romanian
MARITAL STATUS
Married
Florin Turcu
ACADEMIC DEGREE
•
Diploma in Physics and Chemistry, from the Faculty of Science,
North University of Baia Mare, Romania; Diploma thesis: “Active
protection against corrosion“, July 1997.
EDUCATION
• Since 01.04.2001, PhD student with Prof. Dr. Wolfgang Schuhmann,
Lehrstuhl für Analytische Chemie, Abteilung Elektroanalytik und
Sensorik, Ruhr Universität Bochum. The topics of the PhD thesis
include the creation of enzyme microstructures and micropatterns of
living cells, as well as the development of a new, truly label-free
electrochemical detection of DNA hybridisation.
• 1999 – 2000 Research assistant at Department of Chemistry, North
University of Baia Mare; Chairman and organizer of the Chemistry
Club that was designed for participants from the Faculty of Science
and the local high schools. Also, I taught Chemistry at my high
school.
• 1997 - 1999 Technical assistant at Chemistry Department, North
University of Baia Mare.
• 1992 – 1997 Studies in Physics and Chemistry at the North
University of Baia Mare, Romania. Graduation: July 1997.
RESEARCH EXPERIENCE
• Basic knowledge about (micro-) electrochemistry.
• Scanning electrochemical microscopy (SECM) and its application for
the visualisation of variety of samples (enzyme microstructures, DNA
spots, patterns of conductive polymers).
Bochum, 05.07.2004
158
Florin Turcu
• DNA microarrays and electrochemical-based label-free detection of
DNA hybridisation.
• Fabrication and characterisation of enzyme microstructures.
• Fabrication of micropatterns of living (neuronal) cells.
• Microelectrode fabrication, characterisation and application.
• Four-probe conductivity measurements.
ADDITIONAL QUALIFICATIONS
•
Soft
Skills
I
&
II:
“Communicating
with
Audience
&
Giving
Presentations” at Graduate School of Chemistry and Biochemistry,
Ruhr Universität Bochum, 11th-17th July 2003.
•
Fluent in spoken English and good in written English.
•
German (good in understanding)
Bochum, 05.07.2004
159
Florin Turcu
PUBLICATIONS
•
Florin
Turcu,
Schuhmann.
Albert
Schulte,
“Label-free
Gerhard
electrochemical
Hartwich,
recognition
Wolfgang
of
DNA
hybridisation by means of modulation of the feedback current in
SECM”, Angew. Chem. Int. Ed., 2004, 43, 3482-3485; Angew.
Chem., 116, 3564-3567.
•
Florin Turcu, Karla Tratsk-Nitz, Solon Thanos, Wolfgang Schuhmann
Peter Heiduschka. “Ink-jet printing for micropattern generation of
laminin for neuronal adhesion” Journal of Neuroscience Methods,
2003, 131, 141-148.
•
Albert Schulte, Mathieu Etienne, Florin Turcu, Wolfgang Schuhmann.
“High
resolution
constant
distance
scanning
electrochemical
microscopy on immobilised enzyme micropatterns” G.I.T. Imaging
and Microscopy, 2003, 5, 46-49.
•
Florin
Turcu,
Schuhmann.
hybridisation
Albert
Schulte,
“Imaging
by
Gerhard
immobilised
means
of
the
Hartwich,
ss-DNA
repelling
and
mode
of
Wolfgang
detecting
scanning
electrochemical microscopy (SECM)”, Biosens. Bioelectron., 2004, in
press.
•
Florin Turcu, Albert Schulte, Wolfgang Schuhmann. “Scanning
electrochemical microscopy (SECM) in nanolitre droplets using an
integrated working/reference electrode assembly“, Anal. Bioanal.
Chem., 2004, submitted.
•
Anh Nguyen, Jane Hübner, Florin Turcu, David Melchior, Hans-Willi
Kling,
Siegmar
Gäb,
Oliver
J.
Schmitz.
“Analysis
of
alkyl
polyglicosides by capillary electrophoresis with pulsed-amperometric
detection”, Electrophoresis, 2004, submitted.
Bochum, 05.07.2004
160
Florin Turcu
CONFERENCES
Florin Turcu, Dominik Schäfer, Albert Schulte, Gerhard Hartwich,
Wolfgang Schuhmann. “Repelling mode of SECM: a new approach for
visualising DNA microarrays and detecting hybridisation” The 3rd
Workshop on Scanning Electrochemical Microscopy (SECM), 11-12th
June 2004, Dublin City University, Dublin, Ireland, Poster presentation.
Anh Nguyen Minh Nguyet, Florin Turcu, Jane Hübner, Oliver J. Schmitz,
Siegmar Gäb. “Optimisation of the analysis of alkyl polyglycosides by
MEKC-PAD” 17th International Symposium on Microscale Separation and
Capillary Electrophoresis, 8-12th February 2004, Salzburg, Austria,
Poster presentation, P166.
Florin Turcu, Albert Schulte, Gerhard Hartwich, Wolfgang Schuhmann.
„Label-free electrochemical detection of hybridisation events on DNA
chips”, 4th EMBL PhD Student International Symposium, 20-22nd
November 2003, Heidelberg, Germany, Poster presentation.
Florin Turcu, Albert Schulte, Wolfgang Schuhmann, Gerhard Hartwich,
Herbert Wieder, Thomas Kratzmüller. „Ink-jet dispensing of DNA
microarrays
and
label-free
electrochemical
detection
of
DNA-
hybridisation events“ Workshop on new trends in nucleic acid based
biosensors, University of Florence, Polo Scientifico di Sesto Fiorentino,
25-28th October 2003, Oral presentation, Book of Abstracts p. 30.
Florin Turcu, Albert Schulte, Wolfgang Schuhmann. „Coaxial referenceworking electrode assembly for electrochemistry in small volumes“
ELACH-6 Conference, Vienna, Austria, 14-17th of September, 2003,
Oral presentation, Book of Abstracts V13.
Wolfgang Schuhmann, Albert Schulte, Mathieu Etienne, Florin Turcu,
Ingrid Fritsch. „High-resolution shear-force dependent constant-distance
Bochum, 05.07.2004
161
Florin Turcu
mode SECM“ 226th ACS National Meeting, New York, USA, 7-11th
September 2003.
Florin Turcu, Herbert Wieder, Thomas Kratzmüller, P. Frischmann, G.
Hartwich, W. Schuhmann. “Label-freie electrochemische Detektion von
Oligonukleotid-Hybridisierung mittels electrochemischer Rastermikroskopie (SECM)”, 3. BioSensorSymposion (BSS), 30. März / 1. April 2003
Potsdam, Germany, Oral Präsentation, Tagungsbuch
p. 28.
Florin Turcu, Wolfgang Schuhmann. “Lokale Detektion von Oligonukleotidspots mittels elektrochemischer Rastermikroskopie (SECM)”, INCOMSondersymposium / 74. AGEF-Seminar, 26 März 2003, Düsseldorf,
Germany, Oral Präsentation.
Mathieu Etienne, Florin Turcu, Albert Schulte, Wolfgang Schuhmann.
High Resolution SECM imaging of complex enzyme microstructures“,
53rd Annual Meeting of the International Society of Electrochemistry,
ISE
2002,
15-20
September,
Düsseldorf,
Germany,
Poster
presentation, Book of Abstracts p. 76.
Peter Heiduschka, K. Tratsk-Nitz, S. Thanos, Florin Turcu, Wolfgang
Schuhmann.
“Defined
adhesion
and
growth
of
neurones
on
microstructured polymer patterns made by ink-jet printing”, 53rd Annual
Meeting of the International Society of Electrochemistry, ISE 2002,
15-20 September, Düsseldorf, Germany, Oral Presentation, Book of
Abstracts p. 187.
Florin Turcu, Mathieu Etienne, Bernardo Ballesteros Katemann, Marcus
Mosbach, Thomas Erichsen, Wolfgang Schuhmann. “Formation of
chemically active microstructures as a basis for novel miniaturised
analytical devices”, 9th International Conference on Electroanalysis,
Bochum, 05.07.2004
162
Florin Turcu
9-13 June 2002, Cracow, Poland, Oral presentation O 46 in the Book of
Abstracts.
Florin Turcu, Mathieu Etienne, Bertrand Ngounou, Thomas Erichsen,
Wolfgang
Schuhmann.
recognition
elements
“Non-manual
as
a
immobilisation
prerequisite
for
the
of
biological
preparation
of
miniaturised biosensor systems”, The Seventh World Congress on
Biosensors, Kyoto, Japan, 15-17 May 2002, Poster presentation
P2-3.22 in the Abstracts Book.
Florin Turcu, Karla Tratsk, Peter Heiduschka, Albert Schulte, Wolfgang
Schuhmann. “Polymere Mikrostrukturen als Basis zur lokalisierten
Zelladhäsion“, ELMINOS, 26 April 2002, Düsseldorf, Germany, Poster
Präsentation.
Florin Turcu „Polymer receptor for a methanol chemical sensor“, The
XXVth Chemistry and Chemical Engineering National Conference, 6-8th of
October 1999, Calimanesti-Caciulata, Romania, Poster presentation PS
3, Book of Abstracts pp. 266.
Florin Turcu „Study on the chemical separation of silver from silvercopper electrotechnical alloy”, Chemistry and Chemical Engineering
National Conference, 16-18th of October 1997, Bucharest, Romania,
Poster presentation, Book of Abstracts vol. 1, pp. 85-86.
Bochum, 05.07.2004
Bochum, 05.07.2004
163

5. Micropatterning and microelectrochemical characterisation of

Transcription

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