Stephen Arnold

Whispering-gallery-mode biosensing: label-free detection down to single molecules

Optical label-free detectors, such as the venerable surface plasmon resonance (SPR) sensor, are generally favored for their ability to obtain quantitative data on intermolecular binding. However, before the recent introduction of resonant microcavities that use whispering gallery mode (WGM) recirculation, sensitivity to single binding events had not materialized. Here we describe the enhancement mechanisms responsible for the extreme sensitivity of the WGM biosensor, review its current implementations and applications, and discuss its future possibilities.

Protein detection by optical shift of a resonant microcavity

We present an optical biosensor with unprecedented sensitivity for detection of unlabeled molecules. Our device uses optical resonances in a dielectric microparticle (whispering gallery modes) as the physical transducing mechanism. The resonances are excited by evanescent coupling to an eroded optical fiber and detected as dips in the light intensity transmitted through the fiber at different wavelengths. Binding of proteins on the microparticle surface is measured from a shift in resonance wavelength. We demonstrate the sensitivity of our device by measuring adsorption of bovine serum albumin and we show its use as a biosensor by detecting streptavidin binding to biotin.

Shift of whispering-gallery modes in microspheres by protein adsorption

Biosensors based on the shift of whispering-gallery modes in microspheres accompanying protein adsorption are described by use of a perturbation theory. For random spatial adsorption, theory predicts that the shift should be inversely proportional to microsphere radius R and proportional to protein surface density and excess polarizability. Measurements are found to be consistent with the theory, and the correspondence enables the average surface area occupied by a single protein to be estimated. These results are consistent with crystallographic data for bovine serum albumin. The theoretical shift for adsorption of a single protein is found to be extremely sensitive to the target region, with adsorption in the most sensitive region varying as 1/R(5/2). Specific parameters for single protein or virus particle detection are predicted.

Single virus detection from the reactive shift of a whispering-gallery mode

We report the label-free, real-time optical detection of Influenza A virus particles. Binding of single virions is observed from discrete changes in the resonance frequency/wavelength of a whispering-gallery mode excited in a microspherical cavity. We find that the magnitude of the discrete wavelength-shift signal can be sufficiently enhanced by reducing the microsphere size. A reactive sensing mechanism with inverse dependence on mode volume is confirmed in experiments with virus-sized polystyrene nanoparticles. By comparing the electromagnetic theory for this reactive effect with experiments, the size and mass (≈5.2 × 10−16 g) of a bound virion are determined directly from the optimal resonance shift.

Multiplexed DNA quantification by spectroscopic shift of two microsphere cavities

We have developed a novel, spectroscopic technique for high-sensitivity, label-free DNA quantification. We demonstrate that an optical resonance (whispering gallery mode) excited in a micron-sized silica sphere can be used to detect and measure nucleic acids. The surface of the silica sphere is chemically modified with oligonucleotides. We show that hybridization to the target DNA leads to a red shift of the optical resonance wavelength. The sensitivity of this resonant technique is measured as 6 pg/mm(2) mass loading, higher as compared to most optical single-pass devices such as surface plasmon resonance biosensors. Furthermore, we show that each microsphere can be identified by its unique resonance wavelength. Specific, multiplexed DNA detection is demonstrated by using two microspheres. The multiplexed signal from two microspheres allows us to discriminate a single nucleotide mismatch in an 11-mer oligonucleotide with a high signal-to-noise ratio of 54. This all-photonic whispering gallery mode biosensor can be integrated on a semiconductor chip that makes it an easy to manufacture, analytic component for a portable, robust lab-on-a-chip device.

Excitation of resonances of microspheres on an optical fiber

Morphology-dependent resonances (MDRs) of solid microspheres are excited by using an optical fiber coupler. The narrowest measured MDR linewidths are limited by the excitation laser linewidth (<0.025 nm). Only MDRs, with an on-resonance to off-resonance intensity ratio of 10(4), contribute to scattering. The intensity of various resonance orders is understood by the localization principle and the recently developed generalized Lorentz-Mie theory. The microsphere fiber system has potential for becoming a building block in dispersive microphotonics. The basic physics underlying our approach may be considered a harbinger for the coupling of active photonic microstructures such as microdisk lasers.

Whispering gallery mode carousel – a photonic mechanism for enhanced nanoparticle detection in biosensing

Individual nanoparticles in aqueous solution are observed to be attracted to and orbit within the evanescent sensing ring of a Whispering Gallery Mode micro-sensor with only microwatts of driving power. This Carousel trap, caused by attractive optical gradient forces, interfacial interactions, and the circulating momentum flux, considerably enhances the rate of transport to the sensing region, thereby overcoming limitations posed by diffusion on such small area detectors. Resonance frequency fluctuations, caused by the radial Brownian motion of the nanoparticle, reveal the radial trapping potential and the nanoparticle size. Since the attractive forces draw particles to the highest evanescent intensity at the surface, binding steps are found to be uniform.

Perturbation approach to resonance shift of whispering gallery modes in a dielectric microsphere as a probe of a surrounding medium

A first-order perturbation theory similar to the one widely used in quantum mechanics is developed for transverse-electric and transverse-magnetic photonic resonance modes in a dielectric microsphere. General formulas for the resonance frequency shifts in response to a small change in the exterior refractive index and its radial profile are derived. The formulas are applied to two sensor applications of the microsphere to probe the medium in which the sphere is immersed: a refractive index detector; and a refractive index profile sensor.

Label-free detection of single protein using a nanoplasmonic-photonic hybrid microcavity.

Recently we reported the detection and sizing of the smallest RNA virus MS2 with a mass of 6 ag from the resonance frequency shift of a whispering gallery mode-nanoshell hybrid resonator (WGM-h) upon adsorption on the nanoshell and anticipated that single protein above 0.4 ag should be detectable but with considerably smaller signals. Here, we report the detection of single thyroid cancer marker (Thyroglobulin, Tg) and bovine serum albumin (BSA) proteins with masses of only 1 ag and 0.11 ag (66 kDa), respectively. However, the wavelength shifts are enhanced beyond those anticipated in our earlier work by 240% for Tg and 1500% for BSA. This surprising sensitivity is traced to a short-range reactive field near the surface of our Au nanoshell receptor due to intrinsic random bumps of protein size, leading to an unanticipated increase in sensitivity to single protein, which grows larger as the protein diminishes in size. As a consequence of the largest signal-to-noise ratio in our BSA experiments (S/N ≈ 13), we conservatively estimated a new protein limit of detection for our WGM-h of 5 kDa.

Plasmonic enhancement of a whispering-gallery-mode biosensor for single nanoparticle detection

We describe and demonstrate a physical mechanism that substantially enhances the label-free sensitivity of a whispering-gallery-mode biosensor for the detection of individual nanoparticles in aqueous solution. It involves the interaction of dielectric nanoparticle in an equatorial carousel orbit with a plasmonic nanoparticle bound at the microparticle’s equator. As the dielectric particle parks to hot spots on the plasmonic particle we observe frequency shifts that are enhanced by a factor of 4, consistent with a simple reactive model. Once optimized the enhancement by this mechanism should exceed several orders of magnitude, putting individual protein within reach.