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 whisperinggallery 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 (Ϸ5.2 ؋ 10 ؊16 g) of a bound virion are determined directly from the optimal resonance shift.biosensor ͉ influenza ͉ optical resonance V irus particles are a major cause for human disease, and their early detection is of added urgency since modern day travel has enabled these disease agents to be spread through populations across the globe (1). Fast and early detection on site of an outbreak requires biosensors where ideally individual viral particles produce a quantitative signal. Here, we report the observation of discrete changes in frequency of whispering gallery modes (WGMs) as Influenza A virions bind to a microsphere cavity. A ''reactive'' perturbation of the resonant photon state is confirmed in measurements with similar-sized polystyrene (PS) particles near a wavelength of 1,310 nm: The frequency/ wavelength shift signal follows a strong dependence on cavity curvature near the predicted ϳR Ϫ5/2 scaling (2), providing a mechanism for increasing signal by limiting modal volume. By reducing the microsphere radius to just Ͻ40 m and operating at a more favorable wavelength near 760 nm where reduced water absorption is expected to enhance sensitivity (3), binding steps of individual Influenza A (InfA) virions are seen that easily exceed the experimental noise level. Analytic equations are derived that relate discrete changes in resonance wavelength to the size and mass of adsorbed virions. Although field effect techniques using nanofibers (4) and interferometric approaches based on light scattering (5) have demonstrated single virion sensing in the past, reactive WGM sensing adds new dimensions to what can be learned: The measured wavelength shift enables one to quantitatively identify the virion size and mass.Experimental Approach WGM resonances are perturbed toward longer wavelength as particles with polarizability in excess to that of water adsorb to a microsphere cavity. Individual binding events have been theorized to produce discrete steps in a time-trace of the wavelength shift signal (2). To probe for single binding events we immerse a silica microsphere in a suspension of polystyrene particles (PS) with radius a ϭ 250 nm (Fig. 1). The PS particles are diluted in PBS to final concentrations Ϸ10-50 fM. A tunable distributed feedback laser (DFB) (Ϸ1,311 nm nominal wavelength) excites WGMs by evanescent coupling from a tapered optical fiber. A WGM mode is detected as a Lorentzian-shaped trough in ...

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.

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.

The authors present an approach for specific and rapid unlabeled detection of a virus by using a microsphere-based whispering gallery mode sensor that transduces the interaction of a whole virus with an anchored antibody. They show theoretically that this sensor can detect a single virion below the mass of HIV. A micro-fluidic device is presented that enables the discrimination between viruses of similar size and shape.