multi-material paper-disc devices for low cost biomedical

MULTI-MATERIAL PAPER-DISC DEVICES
FOR LOW COST BIOMEDICAL DIAGNOSTICS
E. Vereshchagina, K. Bourke, L. Meehan, C. Dixit, D. Mc Glade, and J. Ducrée
Biomedical Diagnostics Institute, National Centre for Sensor Research,
School of Physical Sciences, Dublin City University, Ireland
ABSTRACT
In this work we demonstrate novel, low cost, hybrid,
polymer-paper disc devices for carrying out biomedical
tests in resource-poor settings. The system combines the
advantages of two major effects – sample transport in a
paper membrane by capillary force and active rotational
flow control by centrifugal force. Capabilities of the paper
disc system include but are not limited to spatio-temporal
flow control, pre-storage of reagents and integrated
colorimetric indicators all suitable for performing
multiple-step bioassays.
INTRODUCTION
Recently hybrid paper microfluidic systems have
drawn great scientific interest and are considered one of the
main breakthroughs in low-cost medical diagnostics for the
developing world [1]. Paper-based systems have proved to
be simple, straightforward and robust engineering
solutions for a variety of existing challenges in biomedical
diagnostics enabling bio-analytical tests such as rapid
ELISA [2], extraction [3] and identification of genetic
material of pathogens [4], multiplexed detection of
disease-specific antibodies [5], blood typing [6] and many
others in resource-poor settings.
Paper is a cheap, easy-to-process, biocompatible, and
renewable material suitable for reagents pre-storage in dry
and liquid forms [7],[8], reagent transport and mixing [9],
separation and blood filtration [6], [10]. It can be integrated
into both flexible and rigid polymeric microfluidic
housings, patterned with a hydrophobic wax to control the
wetting properties [11] and inks to form the electrodes
[12]. This all makes paper a very attractive substrate for
low cost rapid manufacturing of biodiagnostic devices.
Most of the state-of-the-art paper devices only allow a
single step analytical test protocol with limited quantitative
capabilities. So the common objective is to improve the
functionality and accuracy of the quantitative readout of
the paper-based systems and to permit multi-step medical
tests at reduced costs.
In this contribution we present multi-material,
low-cost, hybrid paper-disc devices. These are versatile
microfluidic platforms for a variety of point-of-care
medical tests that need to be carried out quickly and
conveniently by unskilled personnel in resource-limited
environments. Our paper-disc concept opens very
promising opportunity towards the integration of
multiple-step assays, spatio-temporally controlled
transport, storage and release of sample and reagents,
ultimately leading to the next generation of low cost
biomedical diagnostic systems. In addition, the integration
of paper on a disc enables the implementation of important
fluidic unit operations which may be impossible or difficult
to achieve in polymer-only systems, e.g. transport of
978-1-4673-5655-8/13/$31.00 ©2013 IEEE
sample towards the center of the disc by capillary action,
sample recirculation along the direction of centrifugal
force, incorporation of passive colorimetric readout,
bio-functionalization, etc. Previously, Hwang [11] and
Godino [12] pioneered this research area and demonstrated
the high potential of paper-disc platforms.
In this work we investigated the feasibility of using a
whole paper disc and paper elements for different fluidic
unit operations on a centrifugal platform to leverage the
integration of a multiple step bioassay. This work yields
several new aspects of paper integration on a centrifugal
platform:
1) Spatio-temporal control of the sample and
reagents by incorporation of papers with different
flow properties;
2) Flow guiding using hydrophobic paper-wax
restrictors;
3) Pre-storage of dry and liquid bio-reagents;
4) Innovative manufacturing and assembly process.
Figure 1. Overview of the three-layer assembly of the
polymer – paper disc: top and bottom PMMA layers can be
cleaned and reused, middle paper-disc with wax printed
fluidic structures is disposed after a test is completed.
EXPERIMENTAL
Fabrication of paper disc devices
The paper-disc prototypes were designed in AutoCAD
2012 and Solid Works 2011 and manufactured by polymer
rapid prototyping technologies using 1.5-mm thick
poly(methylmethacrylate) (PMMA) (Radionics, Ireland),
86-μm and 56-μm thick pressure sensitive adhesive layers
(PSA, Adhesive Research, Ireland), chromatographic
paper #1, #2, #20, #31ET WHATMAN® (GE Healthcare,
UK). The following prototyping techniques were applied:
1049
MEMS 2013, Taipei, Taiwan, January 20 – 24, 2013
CO2 laser ablation (Zing 16 Laser, Epilog, USA) to
structure the PMMA sheets, a standard knife plotter
(ROBO Pro cutter/plotter, Graphtec, USA) for cutting of
PSA and paper on PSA, MDX-40A CNC-milling machine
for the definition of chambers in PMMA. The fluidic
structures were printed on paper using ColorQube 8570
DN (Xerox Corporation, USA) color printer, subsequently
covered with a tin foil and heated up from both sides using
a hotplate at 150 ̊ C to ensure melting of the wax. Then
paper structures were cut to the shape of the disc or
segments ready for the embedment into the polymeric
support.
centrifugal test stand using a computer controlled motor
(Faulhaber Minimotor SA, Switzerland) to spin the discs
(REF 22 CASCADE). High-resolution imaging, even
during fast rotation, was achieved by a sensitive camera
with short exposure time (Sensicam qe, PCO, Germany)
and mounted on a motorized, 12x zoom lens (Navitar,
USA).
Model bioassay systems
Two types of model bioassay systems were used: a
horseradish peroxide (HRP)-based enzymatic assay where
liquid TMB was employed to yield a color change from
blue (370 nm) to yellow (450 nm) and, and ELISA-based
immunoassay by using mouse IgG and its detection with
anti-mouse IgG-conjugated to HRP, where addition of
TMB substrate yields a color change indicating successful
binding of two specific antibodies. The HRP-TMB assay
consisted of one blocking, and one washing step, and
subsequent addition of TMB substrate. The enzymes
(HRP) were pre-stored in paper and dried prior to the test,
bovine serum albumin (BSA, 1%), washing buffer and
TMB substrate were added in liquid form and transported
according to the assay protocol. The ELISA contained two
more steps as additional antibody was added and washed.
All tests were carried out for at least ten different dilutions
to yield a range of colorimetric responses along with
appropriate positive and negative controls.
ImageJ software was used to analyze the colorimetric
response of the assays; all intensities were calculated
relative to a blank intensity of paper with a negative
control.
RESULTS AND DISCUSSION
Figure 2. Overview of the five layer assembly containing
three PMMA layers – top, bottom and middle layer with
embedded contour-cut paper segments with printed wax
fluidic structures.
Two alternative types of disc assemblies were
engineered. The first assembly is a 3-layer system (Fig. 1)
consisting of a top PMMA layer bonded with transparent
PSA to define reagent chambers, fluidic channels and
venting holes, a middle paper layer patterned with wax to
form fluidic structures, and a base PMMA layer. All layers
were aligned and connected before the test with metal
screws. The PMMA layers can be cleaned and reused, and
the paper layer is disposed after the test is completed.
The second type of assembly consists of five layers
(Fig. 2): three PMMA layers forming chambers, areas for
papers and a base, and two PSA layers used to connect the
structure irreversibly and cut channels. The paper segments
are cut to a design shape and manually aligned on PSA
between PMMA layers.
The first assembly approach permits multiple uses of
the PMMA layers, easy access to test areas and
disposability of the paper disc. The second assembly
approach is easier if complex assays are required with each
layer adding to the overall functionality of the system.
Experiments were performed on a custom made
Flow control in paper
Transport of reagents in paper channels is governed by
capillary and centrifugal forces. Parameters of control for
the flow rate are the rotational frequency, properties of
integrated paper (fiber density / porosity, thickness) in
combination with reagents (viscosity, surface tension).
Figure 3. Active control of liquid flow rate in paper
networks shown for four types of WHATMAN® papers (#1,
#2, #20, #31ET, flow rate and volumes depend on type of
paper and frequency; the most suitable paper is selected
for a desired frequency protocol.
1050
At low rotational frequencies capillary force dominates
and transport of reagents occurs towards the center of the
disc. When two forces are balanced, the reagent transport is
stopped. At higher frequencies the dominating centrifugal
force guides the sample radially outwards. Given the same
frequency and reagent properties, the flow rate is set by the
paper type. Based on this, paper networks can be built with
constant flow rates.
As an example, in Figure 3A we show the dependence
of wicked distance on frequency for #1 paper. Figures 3B
and 3C show flow rate regimes achieved in four different
papers at 1500 rpm and 2500 rpm, respectively, and
Fig. 3D shows the top view of the channel and liquid
transport in time. At 1500 rpm the flow through #20 paper
channels is the slowest; however, it is the fastest when the
frequency increases to 2500 rpm.
Paper-wax flow barriers
Local modification of the papers hydrophilic
properties was used as a means of timing and spatial
control of reagent transport. The hydrophobic wax barriers,
valves, channels were printed on paper to alternate the
liquid path. Figure 4 shows the hydrophobic wax barriers
on paper channels allocated at different radial positions on
a rotating disc. Liquid release occurs sequentially for the
three structures when a burst frequency is reached. Several
wax barriers or valves can be printed along one channel to
separate the regions where liquid or dry reagents are
pre-stored. The wax barriers are suitable for allocating
various volumes; delivering the reagent at specific times,
i.e. controlled by the frequency of rotation; spatially
separating various reagent compartments; and are reusable.
Figure 4. The burst frequencies of the three radially
staggered wax barrier valves depend on the radial
position. This valving scheme is used for temporal and
spatial control of sequential reagent delivery.
Reagent pre-storage for colorimetric assays
Paper channels were made out of four paper types chromatographic paper #1, #2, #20, #31ET WHATMAN ®
- enzymes and antibodies were spotted (prior to CD
assembly) and the bioassay protocol was optimized to
achieve the minimum detection limit while introducing
less steps in the procedure. The BSA blocking step was
employed to minimize the background, i.e. to increase the
sensitivity of the colorimetric response. The highest color
intensity was obtained with the #31ET paper. This paper
substrate is the thickest (0.5 mm) and suitable for storage
of heavier reagent loadings compared to other.
1.996 x 10 -6 mg/ml was determined as the detection
limit for colorimetric detection of HRP in paper.
Liquid reagents were introduced into the paper
directly before the test, allowed to soak into areas separated
by wax restrictors. Dimensions of the area were selected
based on the volume to be transported according to the
assay as each paper type can hold only a defined amount of
liquid. The compartments with pre-stored liquid reagents
were allocated at different radial positions on the disc to
ensure delivery in sequence.
Figure 5. Top view on one of the manufactured devices:
structure for studying Coriolis effect: depending on
frequency and direction of rotation liquid is split between
two arms.
Reusable disc concept
For clinical analysis devices should comply with
sterility regulations and thus a single-use is preferred. For
that a disposable low-cost system was designed and is
shown in Figure 1 and Figure 5. It consists of polymeric
discs containing loading and/or storage chambers,
channels and a wax-treated paper disc sandwiched in
between. The latter is disposed after analysis and the
polymeric housing containing fluidic interfaces can be
cleaned and reused. This allows easier access to the test
areas as the paper disc can be taken out at each time.
The structure for the study of the Coriolis effect shown
in Figure 5B was implemented. The Coriolis force acts in a
direction perpendicular to the axe of rotation and alternates
the sample path either left or right depending if rotation is
clockwise or counter clockwise. Depending on the
direction and frequency of rotation (1000 rpm was used for
most of the experiments) the sample is transported to one
of the split paper channels and can be extracted in the outer
chamber.
Integration of bioassays
Alternatively, a five layer assembly was implemented
suitable for integrating multiple step assays. In Figure 6A
the microfluidic prototype is shown containing liquid
reagent storage in green and detection zones with
1051
dry-stored antibodies indicated in red. All reagent delivery
steps, according to the bioassay protocol, are implemented
in a controlled manner, i.e. using a combination of
wax-printed fluidic structures and various paper materials.
After an assay is completed, a colorimetric signal is
recorded by a camera. As shown in Figures 6B and 6C for
two model assays the color intensity is correlated with the
concentration of enzyme/antibody in the detection zone.
Figure 6. Top view on one of the manufactured disc with
indicated areas of paper chambers used for storage and
delivery of assay reagents and detection zone (A), example
of HRP – TMB assay in the detection zone on #31ET
WHATMAN® paper: color intensities indicate different
concentrations of HRP enzyme (B), Mouse IgG assay in the
detection zone on #1 WHATMAN® paper: actual test
results in higher color intensity, while controls result in
less / no color change (C).
CONCLUSIONS
In this work we investigated capabilities of novel, low
cost, hybrid, polymer-paper disc devices for biomedical
applications. We demonstrated examples of temporal and
spatial control of sample and reagent transport by
integrating paper networks of various porosity, printed
hydrophobic wax fluidic structures, reagent pre-storage
(enzymes, antibodies and other assay reagents in liquid
form) and integrated colorimetric indicators on paper disc.
These are essential fluid unit operations which can be
combined for engineering systems for multiple-step
analytical tests.
Our current work addresses reproducibility and
accuracy of colorimetric assay readout.
We envision that the next generation of paper-disc
devices will enable more complex and precise low cost
bio-diagnostic analysis.
ACKNOWLEDGEMENTS
This research has been partially supported by the FP7
ENIAC project CAJAL4EU, ERDF and Enterprise Ireland
under Grant No IR/2010/0002.
REFERENCES
[1] X. Mao, T.J. Huang, “Microfluidic diagnostics for the
developing world”, Lab Chip, vol. 12, 1412-1416, 2012.
[2] C.M. Cheng, A.W. Martinez, J. Gong, C.R. Mace, S.T.
Phillips, E. Carrilho, K.A. Mirica, G.M. Whitesides,
“Paper-based ELISA”, Angew. Chem., vol.122, pp. 4881
–4884, 2010.
[3] A.V. Govindarajan, S. Ramachandran, G.D. Vigil, P.
Yager, K.F. Bohringer, “A low cost point-of-care viscous
sample preparation device for molecular diagnosis in the
developing world; an example of microfluidic origami”,
Lab Chip , vol. 12, pp. 174-181, 2012.
[4] B. Veigas, J.M. Jacob, M.N. Costa, D.S. Santos, M.
Viveiros, J. Inácio, R. Martins, P. Barquinha, E. Fortunato,
P.V. Baptista, “Gold on paper–paper platform for
Au-nanoprobe TB detection”, Lab Chip, vol. 12, pp.
4802–4808, 2012.
[5] L. Lafleur, D. Stevens, K. McKenzie, S.
Ramachandran, P. Spicar-Mihalic, M. Singhal, A. Arjyal,
J. Osborn, P. Kauffman, P. Yager, B. Lutz, “Progress
toward multiplexed sample-to-result detection in low
resource settings using microfluidic immunoassay cards”,
Lab Chip, vol. 12, 1119-1127, 2012.
[6] M.S. Khan, G. Thouas, W. Shen, G. Whyte, G. Garnier,
“Paper Diagnostic for Instantaneous Blood Typing”, Anal.
Chem., vol. 82, pp. 4158–4164, 2010.
[7] R. Pelton, “Bioactive paper provides a low-cost
platform for diagnostics”, Trend. Anal. Chem. vol. 28,
pp.926-942, 2009.
[8] P.J. Bracher, M. Gupta, G.Whitesides “Patterning
precipitates of reactions in paper”, J.Mater.Chem., vol. 20,
pp. 5117-5122, 2010.
[9] J.L. Osborn, B. Lutz, E. Fu, P. Kauffman, D.Y.
Stevens, P. Yager, “Microfluidics without pumps:
reinventing the T-sensor and H-filter in paper networks”,
Lab Chip, vol. 10, pp. 2659-2665, 2010.
[10] S. J. Vella, P. Beattie, R. Cademartiri, A. Laromaine,
A.W. Martinez, S.T. Phillips, K.A. Mirica, G.M.
Whitesides, “Measuring markers of liver function using a
micropatterned paper device designed for blood from a
fingerstick”, Anal. Chem. vol. 84, pp. 2883−2891, 2012.
[11] H. Hwang, S.H. Kim, T.H. Kim, J.K. Park, Y.K. Cho,
“Paper on a disc: balancing the capillary-driven flow with a
centrifugal force”, Lab Chip, vol. 11, pp. 3404-3406, 2011.
[12] N. Godino, E. Comaskey, R. Gorkin III, J. Ducrée,
“Centrifugally enhanced paper microfluidics”, in Proc.
MEMS 2012, Paris, January 29 – February 2, 2012.
[13] W. Dungchai, O. Chailapakul, C.S. Henry, “A
low-cost, simple, and rapid fabrication method for
paper-based microfluidics using wax screen-printing”,
Analyst, vol. 136, pp.77-82, 2011.
[14] N. Godino, R. Gorkin III, K. Bourke, J. Ducrée,
“Fabricating electrodes for amperometric detection in
hybrid paper/polymer lab-on-a-chip devices”, Lab Chip,
vol. 12, pp.3281–3284, 2012.
CONTACT
*J. Ducrée, tel: +353-1-700-5377; jens.ducree@dcu.ie
1052