Worldwide, 3.575 million people die each year from water-related diseases. The water and sanitation crisis claims more lives than any warfare and is predicted to be one of the biggest global challenges of this century. The rapid, accurate detection of viral pathogens from environmental samples is an ongoing and pertinent challenge in biological engineering. Currently employed methods are lacking in either efficiency or specificity. Here we explore a novel method for virus detection and concurrently use this method to learn more about the very early stages of the virus infection process. The method combines Fourier transform infrared (FTIR) spectroscopy, a method of visualizing molecules based on changes in vibration of particles, and mammalian cells as the biosensor. This method is used to detect and investigate viruses from the family picornaviridae, chosen due to their public health burden and their widespread presence in environmental samples, especially water sources. This family includes the Polioviruses, echoviruses and Coxsackieviruses, among others, many of which are human pathogens.The research outlined in this dissertation is aimed at developing and implementing a new cell-based biosensor that combines the advantages of FTIR spectroscopy with the ability of buffalo green monkey kidney (BGMK) cells to sense diverse stimuli, including infective enteroviruses. The goal of developing this biosensor is outlined in the first paper. The second paper focuses on the application of advanced statistical methods to analyze the spectra to discriminate different viral infections in BGMK cells. Finally, we designed a non-reactive metal biochamber to use with attenuated total reflectance-FTIR. This allowed near-continuous acquisition of real-time spectral data for the study of biochemical changes in mammalian cells caused by poliovirus (PV1) infection. This system is capable of tracking changes in cell biochemistry in minute intervals for many hours at a time.This work demonstrates the feasibility of FTIR spectroscopy in combination with the broad sensitivity of mammalian cells for potential use in the detection of infective viruses from environmental samples. We envision this method being extended to high throughput, automated systems to screen for viruses or other toxins in drinking water systems and medical applications.

Previous studies have provided evidence that endothelial cell-based potassium ion selective electrodes possess the ability to quantify substances that have permeability-altering effects on those endothelial cells. The capability of these so-called biosensors to detect elevated concentrations of certain chemical agents found following tumor formation make them useful in the application as an alternative tumor angiogenesis assay. In this study an epithelial cell line, human colon adenocarcinoma epithelial cells (Caco-2), was used to fabricate membranes that were used to test concentrations of these chemical agents, known as cytokines, mimicking the concentrations that have been observed in the serum of healthy individuals as well as the higher concentration found in individuals with cancer. Additionally background information is provided related to the development of whole cell-based biosensors, metabolic pathways related to tumor angiogenesis and the subsequent increase in cytokine concentration, properties of the Caco-2 cell line that make them useful for the application in cell-based biosensors, and the ultimate effect the cytokines have on the permeability of the cells.

The contamination of the environment, accidental or intentional, in particular with chemical toxins such as industrial chemicals and chemical warfare agents has increased public fear. There is a critical requirement for the continuous detection of toxins present at very low levels in the environment. Indeed, some ultra-sensitive analytical techniques already exist, for example chromatography and mass spectroscopy, which are approved by the US Environmental Protection Agency for the detection of toxins. However, these techniques are limited to the detection of known toxins. Cellular expression of genomic and proteomic biomarkers in response to toxins allows monitoring of known as well as unknown toxins using Polymerase Chain Reaction and Enzyme Linked Immunosensor Assays. However, these molecular assays allow only the endpoint (extracellular) detection and use labels such as fluorometric, colorimetric and radioactive, which increase chances of uncertainty in detection. Additionally, they are time, labor and cost intensive. These technical limitations are unfavorable towards the development of a biosensor technology for continuous detection of toxins. Federal agencies including the Departments of Homeland Security, Agriculture, Defense and others have urged the development of a detect-to-protect class of advanced biosensors, which enable environmental surveillance of toxins in resource-limited settings. In this study a Surface-Enhanced Raman Spectroscopy (SERS) immunosensor, aka a SERS-linked immunosensor assay (SLISA), has been developed. Colloidal silver nanoparticles (Ag NPs) were used to design a flexible SERS immunosensor. The SLISA proof-of-concept biosensor was validated by the measurement of a dose dependent expression of RAD54 and HSP70 proteins in response to H2O2 and UV. A prototype microchip, best suited for SERS acquisition, was fabricated using an on-chip SLISA to detect RAD54 expression in response to H2O2. A dose-response relationship between H2O2 and RAD54 is established and correlated with EPA databases, which are established for human health risk assessment in the events of chemical exposure. SLISA outperformed ELISA by allowing RISE (rapid, inexpensive, simple and effective) detection of proteins within 2 hours and 3 steps. It did not require any label and provided qualitative information on antigen-antibody binding. SLISA can easily be translated to a portable assay using a handheld Raman spectrometer and it can be used in resource-limited settings. Additionally, this is the first report to deliver Ag NPs using TATHA2, a fusogenic peptide with cell permeability and endosomal rupture release properties, for rapid and high levels of Ag NPs uptake into yeast without significant toxicity, prerequisites for the development of the first intracellular SERS immunosensor.