Harvard Brigham
Vollmer Laboratory of Nanophotonics and Biosensing

Optical resonator biosensors

Label-free biosensing down to single molecules

A resonance is most easily experienced by observing a pendulum or by listening to a violin player. Less intuitive is the example of an optical resonance where coherent light is brought to interfere with itself. We achieve this feat by confining light in micro- and nanoscale optical cavities. Immune to damping in a liquid, these resonant optical devices are ultra-sensitive biosensors: For example, the binding events of single Influenza virus particles are observed from discrete resonance-frequency shifts.

We are developing such optical resonator biosensors to a) detect single molecules b) manipulate macromolecules with optical forces c) enhance sensitivity with plasmonic nano-antennas d) provide biosensors for POC, for chip-scale devices, for lab-on-chips e) integrate micro-sensors for organ-on-chips and engineered tissue f) study interaction of evanescent biosensor interface with novel materials such as graphene.

In particular, we are interested in optical resonance excited in glass microspheres. When the light enters into a dielectric microsphere, it can remain trapped inside the sphere due to the total internal reflection. The circulating light results in self-interference producing an optical resonance phenomenon, so-called whispering gallery modes (WGMs). WGMs were originally discovered for sound waves in the whispering gallery of St Paul's Cathedral 1878 by Lord Rayleigh. One of the most interesting properties of optical WGMs are their high quality factor (Q factor) which can reach up to 109. Such high-Q resonances are ideal for sensitive detection of biomolecules, even in solution. The Q factor is proportional to the decay time of the waves and depends on both the surface scattering loss and the absorption loss in the medium. WGMs are characterized by two polarization ( TE and TM) and three mode numbers ( radial n, angular l, and azimuthal m ). For efficient coupling light in or out of the microsphere, a phase-matched optical waveguide mode, a tapered optical fibers or a prism are most widely used as coupling elements. Also far-field illumination and detection is also possible although with less efficiency. A common method to fabricate microspheres in relies on melting the tip of an optical fiber using an oxygen-propane torch or a CO2 laser. Microspheres sensors with small ellipticity (~2%) can be easily manufactured due to the surface tension of glass.

Photonic biosensor array chip for cell response to chemicals

Development of the Label-free optical micro-resonator biosensors for high-throughput, real-time analysis of cell response to chemical libraries is the primary biosensor project in Dr. Frank Vollmer's Lab at Harvard / Brigham Women's Hospital. In this project, the microfabrication technique of an array of microwells will be combined with the high-Q optical resonator system. The high-Q WGM based sensing mechanism has the capability to monitor a single molecule binding event without the use of labels, which surpasses the sensitivity of other commercially available techniques such as surface Plasmon resonance sensors or ELISA assay. Our approach will use microspheres in polymer microwells to read-out cell response in high-throughput experiments. Caspase-3 antibody conjugated microspheres is used to detect the secretion of apoptotic protein markers in response to a toxic chemical, for example released from a printed microgel. This approach will be further developed for sensors in organ-on-chip applications, and for developing a commercial label-free biosensor platform.

WGM biosensors for single biomolecule detection using tapered fiber coupling

Highly sensitive and label-free biosensors can be implemented using a silica sphere and a tapered optical fiber. The tapered fiber excites resonance in a microsphere sensor (on-a-stem) which is used to probe for the binding of biomarkers to surface-immobilized antibodies. Self-interference gives rise to the WGMs when the resonant conditions are satisfied. Any small perturbation, for example of a single molecule binding on the equatorial optical path on the sphere surface, will change the resonant condition resulting in a quantitative change of the optical signal. We monitor the resonance wavelength shift to determine the number of binding events on the sphere surface, in real-time.

Microwell array fabrication using PDMS

Microscale technologies such as microfabrication and soft lithography are used to integrate microsphere sensors with single cells in taylored micro-environments and engineered tissue. For this, we fabricate an array of microspheres using a PDMS stamping method. This array of microspheres can be placed on top of the microfabricated microwell structures. PEG microwells will be fabricated using the micro-molding approach. PDMS mold with defined microstructures will be first fabricated by soft-lithography, which will be used to mold photocurable PEG solution. Various molecules can be encapsulated within photocrosslinked PEG. Due to the inertness of PEG molecules, drugs or other proteins can be released from PEG microgells based on themolecular diffusion rate as well as the pore diameter of the polymeric network.

Microsphere surface functionalization

In order to detect the apoptotic proteins (caspase-3 proteins), microsphere surface will be modified with caspase-3 specific antibody. At least three strategies for the immobilization of caspase-3 specific antibodies to microspheres will be explored. In the first approach, microsphere surface will be coated with biotinylated dextran polymer which will immobilize the streptavidin molecules. Then the biotinylated caspase-3 antibody will bind to the streptavidin molecules. Tetraphenyl-easter(TFP) polyethylene oxide(PEO) biotin can be used to immobilize the streptavidin instead of using the dextran polymer as a second method. TFP reacts with primary amines which will be introduced on the microsphere surface through aminosilanization. Then the TFP-PEO biotin will be coupled to the biotinylated antibody through the streptavidin molecule. Lastly, a high pH carbonate coating buffer can simply used. The antibodies are diluted in the buffer and coat on the microspheres for a few hours or overnight.

Surface plasmon resonance

Since their introduction nearly 30 years ago, biosensors based on surface plasmon resonance (SPR) have become one of the most popular tools used interrogate bimolecular interactions. Despite this, and somewhat surprisingly, there are still many phenomena in this system which are not well understood. In our research, we look at the influence of focussed beams on plasmon excitation using the Kreschmann attenuated total reflection (ATR) configuration. The effect of such a focussed beam is many-fold. First, if one uses (for example) an incident Gaussian beam at the SPR resonance angle, the far field pattern is a familiar "notched Gaussian" -- where the notch is coincident with surface plasmon polarition excitation on the metal film. Curiously, this pattern is not static but evolves spatially with propagation into the far field. This effect is a spatial manifestation of the metal's casual response to incident light and can be understood directly with Fourier optics principles. Second, focussed beams cause an increase in the amount light directionally emitted from the system. This directional emission is due to surface rougness modifiying the in-plane momentum of SPPs and the result is a hollow cone of light about (nearly) the SPR resonance angle. Embedded in this cone is information related to the fundamental mesoscopic transport mechanisms of surface plasmon polaritons themselves. Here we study such phenomena in the context of highly sensitive SPP biosensing.