Photonic tools for advanced sensing and imaging at the nanoscale.

Abstract

This dissertation reports a novel bio-sensing strategy based on single-mode, electro-active, integrated optical waveguide (SM-EA-IOW) platforms. It also reports the development of a super-resolved far-field optical imaging tool to enable optical, electronic, and spectroelectrochemical investigations at the nanoscale. SM-EA-IOW platforms with its outstanding sensitivity for spectroelectrochemical interrogation was combined with a sandwich bioassay for the development of a novel immunosensing based strategy for label-free detection of infectious pathogens. The strategy begins with the functionalization of the electroactive waveguide surface with a capturing antibody aimed at a specific target analyte. Once the target analyte is bound to the photonic interface, it promotes the binding of a secondary antibody that has been labeled with a redox active reporter. This labeled antibody reporter forms the analytical signal, which is linked uniquely to both the spectral and electrochemical properties of the redox probe designed to specifically recognize a target analyte. Based on this novel detection strategy experimental results in the interrogation of influenza A (H5N1) HA protein have reached an outstanding level of detection in the picomolar range. In addition, the novel label-free SM-EA-IOW bio-sensing strategy was successfully demonstrated for detection of gram-negative bacteria in present authentic clinical eye samples. Such demonstration has also shown the flexibility and ability of the new strategy to probe samples in in the microliter volume range, without any prior processing or pre-enrichment steps. As the groundwork towards the optimal operation of the novel sensing strategy, the effects of the adsorption process and the rate of electron transfer of redox bound species to the electrode surface were thoroughly studied. For each interface of a multilayer immunoassay assembly the surface density, the adsorption kinetic, and the electron-transfer rate of bound species of a redox-active protein were investigated using an optical impedance spectroscopy (OIS) technique based on measurements obtained with the SM-EA-IOW platform. Such methodology and acquired knowledge are crucial for the rational development of novel and advanced immuno-biosensors. Electrochemically modulated fluorescent molecules to be conjugated with relevant antibodies for creating an electroactive probe at the nanoscale was also investigated. Such capability has the potential to enable the development of an arrayed immunosensing technology. Fluorescence emission at the nanoscale suffers from two main restrictions, diffraction limit and photobleaching effects. To address these hinders, a modulated stimulated emission depletion microscope (STED) that is capable of achieving far-field super-resolved images was developed and used to reduce the power of the applied excitation and depletion laser beams diminish photobleaching effects in single-molecule sub-diffraction STED imaging. These two photonic devices provide new approaches for bio-sensing from…