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, Columbia, Missouri, 65211, USA

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

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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).