Figure 2. Right. Schematic of TIR setup for immunoassay

Application of Fluorescent Nanodiamonds for
Detection of Molecular Binding
H. Doan, J. Kimbal, T. Nguyen, D. Shumilov, S. Raut*, T. W. Zerda, B. P. Maliwal*, I. Gryczynski*, Z. Gryczynski
Physics –Astronomy Department - Texas Christian University, * UNT Health Science Center, Fort Worth, Texas
Abstract
Total Internal Reflection Fluorescence (TRIF) for a long time has been utilized for detecting molecular binding to the dielectric (glass) surface. Highly
enhanced and surface confined evanescent field excites fluorophores close to the surface (up to 100 nm) very effectivel and can be scattered by
Nanoparticles. Recently developed new fluorescent probes, Fluorescent Nanodiamonds (FNDs) have excellent biocompatibility, almost perfect (~100%)
quantum yield, infinite photostability, and long fluorescence lifetime. In addition diamond material has very high refracting index (n=2.37) resulting in very
high scattering of diamond particle. These two attributes, fluorescence and scattering, offers new potential uses of FNDs particles as markers in
biomedical assay applications. The study’s goal is to functionalize FNDs surface with various functional groups and attach such FNDs to various
biomolecules. FNDs are small enough (5-100nm) to be bound to proteins or DNA molecules. By monitoring fluorescence and scattering of evanescent
field we can monitor binding of FNDs labeled biomolecules to the activated glass surface. Because of unprecedented photostability of FNDs, we can use
ratio between fluorescence and scattering signals to increase detection sensitivity. In our approach we will use FNDs for detecting two types of molecular
binding: (1) Immunoassay – binding of FNDs labeled antigen to surface immobilized antibody, and (2) DNA hybridization binding FNDs labeled as DNA to
the complementary strands immobilized to the glass surface.
Introduction
Development of fluorescent probes has been a subject of intense effort for many practical biomedical
applications ranging from tracking of cellular processes like endocytosis and exocytosis to physiological markers
detection and cancer diagnostics. Tens of fluorescence probes are developed every year to be used for
proteins/DNA/RNA labeling, tissue imaging, but still after many years of significant effort we are still missing “perfect”
probes. The best available fluorescent dyes may have high brightness but show short fluorescence lifetimes
(typically only few nanoseconds or less that are difficult to separate from background), have low photostability
(dramatically limiting the observation time), and exhibit significant blinking (severely perturbing practical single
molecule applications). Fluorescent Nanodiamonds (FNDs) are new emerging probes with amazing fluorescent
properties where point defects embedded in the diamond crystal lattice result in exceptional luminescent properties.
The nitrogen-vacancy defects fluoresce in red or green. Fluorescent NDs in the strong evanescent field as shown in
Figure 1 will: (1) significantly scatter the evanescent field to form the scattered light of the same frequency as the
excitation light, and (2) be effectively excited and produce strong fluorescence signal. Both, scattering and
fluorescence can be independently used for a very sensitive detection. Utilizing both processes simultaneously, in
the form of a ratio metric detection, has a high potential to further improve detection sensitivity.
Figure 2. Right. Schematic of TIR setup for immunoassay
measurements. Left. Actual photography of the stage.
Total Internal Reflection (TIR)
To limit the background signal, excitations confined to a very small volume or to a thin
slice are frequently utilized. Excitation with the evanescent field induced by the total internal
reflection (TIR) is one of the most effective and frequently used approaches. It occurs when
the angle of incidence is greater than the critical angle, θc=sin-1(n2/n1), see Figure 2(the
photography in Figure 2 shows the home-build TIRF system stage). Then a wave penetrates
the less dense medium [1]:
E(x,z,t)=Eoexp(-z/d)exp[i(kxxn1sinθ1/n2 – ωt)]
where kx=2p/l and d is the penetration depth d=l(n12sin2θ –n22)-1/2/2p.
The disturbance propagates along the x direction as an evanescent wave. The y-direction is
perpendicular to the xz plane of incidence.
The TIR excitation has significant advantages when applied to samples deposited on a
surface. The magnitude of the evanescent field decays rapidly in the z-direction and
becomes negligible at a distance greater than ½ of the excitation wavelength, typically ~200
nm. In addition, the evanescent field is about 3 fold enhanced as compared to the impinging
beam [2]. These properties of TIR highly suppress background allowing for sensitive
detection in dense biological media, such as blood or serum.
We realized that combining fluorescent NDs with enhanced evanescent excitation will bring
many new advantages that can greatly improve detection sensitivity. New FNDs bring
exceptional qualities to the fluorescence based detection. The most significant are excellent
(almost perfect) photostability, high quantum yield (~100%), large Stoke’s shift, and low
sensitivity to external conditions (solvent, pH, salt, etc). Another important feature of diamond
particles is a very large refractive index and excellent scattering properties. Typically the
presence of high scattering is considered a problem in fluorescence detection. But in view of
NDs’ excellent fluorescent properties we realized that we could use scattering to our
advantage. Using an evanescent type of illumination (the dark field illumination)
nanodiamonds will scatter the excitation light and at the same time emit fluorescence signal
at a considerably different wavelength. Since scattering of diamond particles is orders of
magnitude stronger than that due to DNA or any biological macromolecules, the scattering
signal from a thin layer sample will be dominated by scattering from NDs. Using both,
scattering intensity at the excitation wavelength and fluorescence intensity at the emission.
Application of Fluorescent Nanodiamonds for
Detection of Molecular Binding
H. Doan, J. Kimbal, T. Nguyen, D. Shumilov, S. Raut*, T. W. Zerda, B. P. Maliwal*, I. Gryczynski*, Z. Gryczynski
Physics –Astronomy Department - Texas Christian University, * UNT Health Science Center, Fort Worth, Texas
Preparation and Functionalization of NDs
We have been testing different size of nanodimonds (from 5-125 nm) and their response in TIR
evanescent field. By refluxing NDs in 1:1 mixture of concentrated H2SO4 : HNO3 for 24 Hr at 90 deg
C and subsequently, in 0.1M NaOH for 2 Hr at 90 deg C and then in 0.1M HCl for 2 Hr at 90 deg C we
were able to obtain first functionalized NDs. Washing extensively with water and centrifugation at
12000 rpm and drying allow us to obtain COOH covered NDs. This is manifested by greatly enhanced
solubility. Figure 3 shows suspension of raw NDs and functionalized with COOH after 1 hour. Non
functionalized NDs precipitate on the bottom while functionalized stay in the solution.
Figure 3.
Binding of FNDs to the Surface
Scattering of NDs
Scattering of NDs
Scattering of NDs with Fluororescent
Conclusion
Recent efforts in the field of immunoassays and immunosensors have been directed toward smaller sample
volumes, shorter assay times, cost reduction, simpler assay protocol, and most importantly high sensitivity. Different
miniaturization methods based on dye labels and spatial resolution are exploited for fluorescence sensing. Presented
TIRF based fluorescence detection takes advantage of recent development of new fluorescent nanodiamonds (FNDs).
These probes will add to extra sensitivity, simplicity, and practicality of the detection; allowing ultrasensitive detection
down to a single molecule (nanoparticle) level and enabling development of new type of simple detection devices for
individual use by broader community.
Future Works
High photostability of FNDs and very high refractive index of diamond material will open new possibilities for utilizing
new emerging technologies that take advantage of high field enhancement like surface plasmons resonance (SPR)
technology and metal enhanced fluorescence (MEF). After testing developed NDs with TIR evanescent field we will test
new probes with evanescent field generated by surface plasmons polaritrons excited in thin (~ 50 nm) metal film. Such
evanescent field is known to be 20 – 30 folds stronger than evanescent field generated by TIR. This for example will
make possible to develop DNA hybridization assay based on very limited number of DNA copies. Possible applications
include genomics and forensic and investigative genetics.
References
1. E. Hecht, Optics, Addison-Wesley, Reading, 1990
2. Axelrod, D., E.H. Hellen, R.M. Fulbright. Total internal reflection fluorescence. In: Topics in Fluorescence Spectroscopy. Vol. 3:
Biochemical Applications, (Lakowicz, J.R., Ed.) Plenum Press, New York, 1992, pp. 289-343.