Development and Application of SERS Based Immunoassay using a Gold Plated Membrane for use in Flow-Through Immunoassay

Rapid detection for influenza virus is vital for human healthcare and illness prevention. Influenza A is the most common viral pathogen that infects humans. A common strain of influenza A is H1N1. This seasonal pathogen is so ubiquitous seasonal vaccines contain a killed strain of seasonal H1N1 virus. In 2009, a pandemic of H1N1 broke out worldwide, which resulted in the hospitalizations and death of thousands of people in the United States. Current influenza diagnostic methods clinics use are limited by their sensitivity/detection, or lack of rapid analysis. Immunoassays (IA) are a universal standard for viral detection. The high binding affinity and specific recognition of antibodies (Ab) for antigens (Ag) is the premise behind IA. A common method used in laboratories and medical clinics is the lateral flow assay, which sacrifices sensitivity and low limit-of-detection (LOD) for rapid results. Another method, enzyme-linked immunosorbant assay (ELISA) sacrifices time for low LOD and high sensitivity. Studies have shown the rate-limiting step of the Ab-Ag binding reaction is diffusion limited. Low LODs are achieved through long incubation periods. Mass transport of the analyte to the surface is slow due to diffusion limitations. A novel method is needed to overcome diffusion limitations while not sacrificing current analytical standards. A rapid IA was developed to enhance binding kinetics via flow to overcome diffusion limitations. Ab-modified filters were explored to function as a capture substrate and flow rate was investigated as a means to increase mass transport. Surface-enhanced Raman spectroscopy (SERS) analysis was employed for its multiple advantages over other analytical detection methods. SERS is simple to use, offers rapid analysis, high sensitivity, low LOD, portability through hand-held Raman spectrometers, and the ability for multiplexed detection. The polycarbonate track etched (PCTE) filters were gold plated for reduction in background fluorescence, increased plasmonic coupling between the substrate and Raman label and ease of Ab adsorption. Electrode-less deposited gold filters were fabricated and modified with antibody to serve as the capture substrate. A syringe pump systematically and actively transported the analyte and SERS tag through the filter. Analyte detection was performed with a Raman spectrometer for analysis via SERS. Extrinsic Raman labels (ERLs), i.e., SERS tags, served as the Raman label and were composed of gold nanoparticles modified with a Raman reporter molecule and an Ab. The nanoparticle served as the SERS enhancing substrate, the reporter molecule provided a unique signal and the Ab imparted biospecificity. Model mouse-IgGs (Ag) and goat anti-mouse IgG (Ab), were used as the template model for development and optimization of the protocol. Upon successful modification of the gold plated filters, passive assays demonstrated capability of the filters to function as capture substrates. A syringe pump was used to control and investigate flow rate as a means to overcome diffusion limitations. Binding kinetics for model antibody were investigated and it was revealed that active transport was a viable means to expedite mass transport of the analyte to the capture substrate. In this study, the active flow procedure was able to reduce a 24 hour passive assay to a 10 minute flow assay, while enhancing the limit of detection 10-fold over passive assays. Optimization of the protocol was investigated to decrease assay time, and/or reagents used. Rinse steps thought to be necessary were determined unnecessary, thus these steps could be eliminated to reduce assay time without loss of analytical performance. Stint assays were performed that directed the flow of sample and ERL through a small area on the filter surface. Signal intensities for stint assays were increased while using the same volume of reagent as non-stint assays. The protocol developed in this thesis provides the foundation for an active, rapid flow through immunoassay using SERS for detection