(11d) Determination of Binding Interactions Using Optical Microcavities

Determination
of Binding Interactions Using Optical Microcavities

Carol E. Soteropulos^{1, 2},
Heather K. Hunt^{2, 3}, Andrea M. Armani^1,3,4

¹Department of Biomedical Engineering,
University of
Southern California,
Los Angeles, California
90089, USA

²Department of Biological Engineering,
University of
Missouri,

³Mork Family
Department of Chemical Engineering and Materials Science, University of
Southern           

California,
Los Angeles, California
90089, USA

⁴Ming Hsieh Department
of Electrical Engineering-Electrophysics,
University of
Southern California,

Los Angeles, California
90089, USA

Optical
detection techniques, ranging from fluorescent-based assays to label-free
surface plasmon resonance (SPR) sensors, enable researchers not only to detect
trace substances and perform diagnostics, but also to probe the fundamental
nature of numerous biological systems. 
For example, SPR sensors are routinely used to determine
substrate-enzyme binding kinetics.  The
determination of accurate binding kinetics for molecular systems is fundamental
to the understanding of the interactions between biomolecules within a binding
pair.  For example, the way in which
individual protein molecules of an enzyme interact (association / dissociation
equilibria, molecular positioning, etc.) within enzyme-substrate complexes is
the key to their specificity and ability to catalyze a reaction.  This information is subsequently used to
design improved and more targeted therapeutics. 

An
emerging label-free optical detection technique is the silica optical
microresonator, which, while originally designed for telecommunications, has
shown great applicability for detection and diagnostics.  These evanescent-field devices have very low
optical losses, and are therefore able to confine light for long periods of
time at specific resonant wavelengths.  When
a molecule binds to the surface of the cavity, the effective refractive index of
the optical field is modified, and the resonant wavelength changes, thus
enabling the sensing capabilities of these devices.  The overall sensitivity (detection limit) of
this platform is determined by the optical loss or the photon lifetime of the
microcavity.  Due to their low optical
loss, microcavity devices have demonstrated very high sensitivities towards a
variety of biological and chemical targets, including single molecules, single
viruses, and single nanoparticles.[3-5] 

Although
much work has been done demonstrating the sensing and diagnostic capabilities
of these devices, they have not been explored for their applicability as probes
for kinetics measurements.  One of the
primary hurdles in using optical microcavities to study binding kinetics is the
development of a robust and stable surface chemistry that can immobilize one
half of the binding pair without degrading the optical performance of the
device.  A covalent attachment is
necessary to determine binding kinetics because the probe molecule must be
reliably attached to the surface in order to obtain consistent information
regarding the probe-ligand system. 
Covalent attachment of probe molecules using silane
coupling agents, in conjunction with vapor deposition techniques, has been
shown to address these requirements for on-chip optical resonators, such as the
microtoroid.[6-7]  Here, we adapt these facile protocols to
generate silica microsphere optical resonators that present with a uniform
surface coverage of biotin probe molecules. 
This method ensures that the microsphere resonators can be used as
highly sensitive (strept)avidin sensors, due to the high biotin / (strept)avidin binding
affinity.  Specifically, we modify the
protocols to better maintain the optical sensitivity of microsphere resonant
cavities.  We demonstrate the maintenance
of optical performance of a functionalized spherical resonant cavity before
examining the detection capabilities towards streptavidin of the functionalized
optical cavities in aqueous solution and analysis of the binding kinetics.

The
surface chemistry (uniformity, biological activity) was verified using
fluorescent microscopy.  Before and after
functionalization, the optical performance of the microcavity was characterized
to determine the impact of the chemistry on the device.  The photon lifetime or quality factor (Q) of
the device was > 1 million after probe attachment, well above the Q-threshold
necessary to detect single viruses or particles.  Finally, the site stability was determined
after long-term storage (>60 days). 
After characterizing the surface functionalization, the functionalized
devices were used to detect 1 nM streptavidin in an
aqueous solution.  Subsequently, we
determined the binding kinetics constants based on the resonant wavelength shift
during detection, using a reversible bimolecular reaction model for the binding
pair to calculate the dissociation constant (k_d) through a simple exponential function
describing the dissociation phase [8].  
Our measured constants agreed with those previously determined through
other methods for the immobilized biotin-streptavidin system.  We also showed the limiting impact of mass
transport on the determination of the association constant using such devices.  Similar mass transport limitations have been
commented upon by many researchers in the surface plasmon resonance community
[8]. 

As
a result of the covalent attachment of biotin to the microsphere surface, we
are able not only to detect low concentrations of biotin, but also to determine
binding kinetics of the biotin-streptavidin pair.  Through this type of highly sensitive analysis,
we can better quantify the interaction between two molecules, potentially at
the single molecule level, and gain important information regarding interaction
strengths, as well as the specific way in which molecules interact during a
chemical reaction.  In order to calculate
an association constant (k_a), we would need to take into account a
number of separate issues which arise as a result of one molecule of the pair
being immobilized on a surface, such as steric hindrance and mass transport
limitations [9].  This demonstration
marks the first example of the use of such novel optical platforms for probing
fundamental biological questions.

References

1.   H.
K. Hunt, and A. M. Armani, "Label-Free Biological and Chemical
Sensors," Nanoscale 2,
1544-1559 (2010).

2.   X.
D. Fan, I. M. White, S. I. Shopoua, H. Y. Zhu, J. D. Suter, and Y. Z. Sun,
"Sensitive optical biosensors for unlabeled targets: A review,"
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(2008).

3.   A.
M. Armani, R. P. Kulkarni, S. E. Fraser, R. C. Flagan, and K. J. Vahala,
"Label-Free, Single-Molecule Detection with Optical Microcavities,"
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4.   F.
Vollmer, S. Arnold, and D. Keng, "Single virus detection from the reactive
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5.   J.
Zhu, S. K. Ozdemir, Y.-F. Xiao, L. Li, L. He, D.-R. Chen, and L. Yang,
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ultrahigh-Q microresonator," Nature Photonics 4, 46-49 (2009).

6.   H.
K. Hunt, and A. M. Armani, "Recycling Microcavity Optical
Biosensors," Optics Letters 36
(2011).

7.   H.
K. Hunt, C. Soteropulos, and A. M. Armani, "Bioconjugation to Optical
Microresonators," Sensors 10,
9317-9336 (2010).  

8.  
S. Zhao, and W. M. Reichert, "Influence of Biotin Lipid Surface
Density and Accessibility of Avidin Binding to the Tip of an Optical Fiber
Sensor," Langmuir 8, 2785-2791
(1992).

9. 
P. Schuck, and A. P. Minton, "Analysis of Mass Transport-Limited
Binding Kinetics in Evanescent Wave Biosensors," Analytical Biochemistry 240, 262-272 (1996).