Date on Master's Thesis/Doctoral Dissertation

8-2020

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Physics and Astronomy

Degree Program

Physics, PhD

Committee Chair

Mendes, Sergio B.

Committee Member

Sumanasekera, Gamini

Committee Member

Smadici, Serban

Committee Member

O'Toole, Martin G.

Author's Keywords

electrochemically; plasmon waves; nano-scale; applications of redox

Abstract

In this thesis, an electrochemically-modulated surface plasmon resonance (EC-SPR) technique was developed for challenging investigations and applications in different redox assemblies. The principle of the technique is based on the nano-scale confined and sensitive interplay between a surface plasmon wave and a redox-active probe featuring an optical transition when undergoing a faradaic redox process. Such faradaic process is electrochemically controlled by modulating the surface electric potential of the SPR platform to create an optical output signal that is substantially immune to the negative impacts of background environment and enabling the identification and quantification minute properties linked to a molecular event of interest. First, the structure of the EC-SPR platform and the ideal experimental conditions to optically interrogate it were investigated. Different strategies were examined for the ideal SPR configuration to enhance the spectroelectrochemical response imprinted into the optical signal. A redox active probe, the cytochrome c protein, was used as a model system to report the performance of the EC-SPR device and to confirm its suitability for spectroelectrochemical measurements. Experimental measurements of SPR reflectance curves and numerical analysis of the experimental data based on a developed Mathematica code were implemented to support the rationale design of SPR configurations aiming to maximize the optical response linked to a redox process and to ultimately reach a high performance for the EC-SPR platform. Next, a combination of the EC-SPR device and the optical impedance spectroscopy technique (OIS) was demonstrated to provide an effective analytical path to study electron transfer rates in redox species. When compared to competing technologies, the major advantages of this combination are the ability to implement the device over a broad range applied electric potentials and to reach high modulation frequencies. These features increase the capability of the EC-SPR technology for investigations and applications of fast reaction rate constants for a wider range of surface-adsorbed redox species. The capability of the EC-SPR device for determining electron-transfer rates was experimentally demonstrated here. The electron-transfer rate of a redox probe, cytochrome c protein, immobilized at different functionalized assemblies on the plasmonic interface were investigated. The results show that each functionalized layer has a strong impact on the electron-transfer rate of a redox probe interacting with the functionalized electrode of the EC-SPR platform. The quantification and understanding of the electrochemical behavior of these functionalized layers and the ability of the EC-SPR platform to monitor the electron-transfer rate of biomolecular assemblies provide the groundwork for creating a novel biosensing strategy of high performance, which is described below. Using the EC-SPR device, a novel biosensing methodology that uses redox reactions, controlled by a surface electrode, to electrically modulate the optical output of a molecular probe featured in a sandwich-style bioassay was developed and demonstrated here. A monoclonal antibody was bound to the EC-SPR platform to create an interface that was prepared to recognize and capture a target protein. Once these antigens were captured on the device surface, they promoted the immobilization of a polyclonal secondary antibody that has been labeled with a methylene blue (MB) dye. A hemagglutinin (HA) protein from the H5N1 avian influenza A virus was used to demonstrate the capability of the EC-SPR device for the detection and quantification of a critical influenza antigen. In addition, the capability of the EC-SPR device to directly detect and reproducibly identify the presence of gram-negative bacteria in complex samples (whole blood and plasma) was successfully demonstrated. The experimental results with viral and endotoxin biomaterials have produced outstanding limits of detection in the pico-molar range. Using this approach, only the modulated photon output is surface-coupled, offering a means to significantly reduce the effects of background signal that comes from the substrate or fluid media. Overall, the EC-SPR technology provides an inexpensive and easy-to-use transduction platform that offers a simpler path towards I) investigating a variety of redox-transduced events with extremely low surface densities and a small difference in their molar absorptivities, II) high sensitivity detection of multiplexed targets with a small footprint that only requires a small amount of sample material at low concentrations of a target antigen.

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