Electroactive Interface for Enabling Spectroelectrochemical Investigations in Evanescent-Wave Cavity-Ring-Down Spectroscopy.

In this study, we report the development of an electrically active solid-liquid interface for the evanescent-wave cavity-ring-down spectroscopic (EW-CRDS) technique to enable spectroelectrochemical investigations of redox events. Because of a high-quality transparent conductive electrode film of indium tin oxide (ITO) coated on the interface of total internal reflection of the EW-CRDS platform, a cavity ring-down time of about 900 ns was obtained allowing spectroelectrochemical studies at solid-liquid interfaces. As a proof-of-concept on the capabilities of the developed platform, measurements were performed to address the effects of an applied electric potential to the adsorption behavior of the redox protein cytochrome c (Cyt-C) onto different interfaces, namely, bare-ITO, 3-aminopropyl triethoxysilane (APTES), and Cyt-C antibody. For each interface, the adsorption and desorption constants, the surface equilibrium constant, the Gibbs free energy of adsorption, and the surface coverage were optically measured by our electrically active EW-CRDS tool. Optical measurements at a set of constant discrete values of the applied electric potential were acquired for kinetic adsorption analysis. Cyclic voltammetry (CV) scans under synchronous optical readout were performed to study the effects of each molecular interface on the redox process of surface-adsorbed protein species. Overall, the experimental results demonstrate the ability of the electro-active EW-CRDS platform to unambiguously measure electrode-driven redox events of surface-confined molecular species at low submonolayer coverages and at a single diffraction-limited spot. Such capability is expected to open several opportunities for the EW-CRDS technique to investigate a variety of electrochemical phenomena at solid-liquid interfaces.

Detection of influenza virus by electrochemical surface plasmon resonance under potential modulation.

In this study we report the development of a novel viral pathogen immunosensor technology based on the electrochemical modulation of the optical signal from a surface plasmon wave interacting with a redox dye reporter. The device is formed by incorporating a sandwich immunoassay onto the surface of a plasmonic device mounted in a micro-electrochemical flow cell, where it is functionalized with a monoclonal antibody aimed to a specific target pathogen antigen. Once the target antigen is bound to the surface, it promotes the capturing of a secondary polyclonal antibody that has been conjugated with a redox-active methylene blue dye. The methylene blue displays a reversible change in the complex refractive index throughout a reduction-oxidation transition, which generates an optical signal that can be electrochemically modulated and detected at high sensitivity. For proof-of-principle measurements, we have targeted the hemagglutinin protein from the H5N1 avian influenza A virus to demonstrate the capabilities of our device for detection and quantification of a critical influenza antigen. Our experimental results of the EC-SPR-based immunosensor under potential modulation showed a 300 pM limit of detection for the H5N1 antigen.

Influenza virus immunosensor with an electro-active optical waveguide under potential modulation.

Here we report the development of a novel immunosensor-based strategy for label-free detection of viral pathogens by incorporating a sandwich bioassay onto a single-mode, electro-active, integrated optical waveguide (EA-IOW). Our strategy begins with the functionalization of the electro-active waveguide surface with a capture antibody aimed at a specific virus antigen. Once the target antigen is bound to the photonic interface, it promotes the binding of a secondary antibody that has been labeled with a methylene blue (MB) dye. The MB is a redox-active probe whose optical absorption can be electrically modulated and interrogated with high sensitivity by a propagating waveguide mode. In this effort, we have targeted the hemagglutinin (HA) protein from the H5N1 avian influenza A virus to demonstrate the capabilities of the EA-IOW device for detection and quantification of an important antigen. Our initial results for the HA H5N1 influenza virus show a remarkable limit of detection in the pico-molar range.

Adsorption Properties and Electron-transfer Rates of a Redox Probe at Different Interfaces of an Immunoassay Assembled on an Electro-active Photonic Platform.

Physical and chemical properties of a redox protein adsorbed to different interfaces of a multilayer immunoassay assembly were studied using a single-mode, electro-active, integrated optical waveguide (SM-EA-IOW) platform. For each interface of the immunoassay assembly (indium tin oxide, 3-aminopropyl triethoxysilane, recombinant protein G, antibody, and bovine serum albumin) the surface density, the adsorption kinetics, and the electron-transfer rate of bound species of the redox-active cytochrome c (Cyt-C) protein were accurately quantified at very low surface concentrations of redox species (from 0.4 to 4% of a full monolayer) using a highly sensitive optical impedance spectroscopy (OIS) technique based on measurements obtained with the SM-EA-IOW platform. The technique is shown here to provide quantitative insights into an important immunoassay assembly for characterization and understanding of the mechanisms of electron transfer rate, the affinity strength of molecular binding, and the associated bio-selectivity. Such methodology and acquired knowledge are crucial for the development of novel and advanced immuno-biosensors.

Photobleaching reduction in modulated super-resolution microscopy.

Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases in photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.