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<p>Though pathogenic strains represent a small portion of the total variety of existing <em>Escherichia coli </em>strains, they contribute extensively to human morbidity and mortality. Disease outbreaks caused by enterohaemorrhagic <em>E. coli</em> of the serotype O157:H7 and the “Big Six” serotypes (i.e., O26, O45, O103, O111, O121 and O145) have driven the development of assays for pathogen detection. From culture-based assays requiring several days for confirmation of target organisms, to quantitative PCR (qPCR) tests that provide pathogen identification in several hours’ time, the sensitivity, specificity and speed of bacterial diagnostics have seen improvements that increased the efficacy of assays used to detect pathogens at clinically relevant levels. One relatively unexplored field of diagnostics is the use of conserved signature insertion/deletions (CSIs) as stable genetic markers for pathogen detection. This thesis presents two qPCR assays that target an <em>E. coli</em> O157:H7-specific insertion in a CSI. In a more preliminary study, an EvaGreen-based qPCR assay was developed that had a detection limit of 16 <em>E. coli</em> O157:H7 genome equivalents. An improved format of the O157:H7-specific CSI assay, using TaqMan probes, was later established. TaqMan probes are sequence-specific, while DNA-intercalating EvaGreen dye is sequence-independent. Though the TaqMan probe-based assay had a higher detection limit of 100 genome equivalents, the assay maintained detection sensitivity in presence of genetically similar (<em>E. coli</em> K-12) and dissimilar (fish sperm) DNA in excess amounts (1000-fold and 800-fold excess of target DNA, respectively), demonstrating its potential for pathogen detection in environmental samples where the presence of background flora may influence detection. These assays thus represent an exploration into the use of CSIs as diagnostic tools. This thesis also provides a guide for future developments of pathogen detection using CSIs, such as those that may be present in toxigenic species of Cyanobacteria and human pathogens, including <em>Vibrio</em> and <em>Campylobacter</em>.</p> / Master of Science (MSc)

The development of a waterborne pathogen detection method that is rapid, multiplex, sensitive, and specific, would be of great assistance for water treatment facilities and would help protect water consumers from harmful pathogens. Here we have utilized surface enhanced Raman spectroscopy (SERS) in a sensitive multiplex pathogen detection method. Two strategies are proposed herein, one that utilizes SERS antibody labels and one that measures the intrinsic SERS signal of organisms. For the SERS label strategy, gold nanoparticles are conjugated with antibodies specific to Cryptosporidium parvum and Giardia lamblia and with organic dye molecules. The dye molecules, rhodamine B isothiocyanate (RBITC) and malachite green isothiocyanate (MGITC) were surface enhanced by the gold nanoparticles resulting in unique fingerprint SERS spectra. The SERS label method was successful in detecting G. lamblia and C. parvum simultaneously. The method was subsequently coupled with a filtration step to both concentrate and capture cysts on a flat surface for detection. Raman mapping across the filter membrane detected ~95% of the spiked cysts in the optimized system. In the second type of strategy, intrinsic virus SERS signals were detected with silver nanoparticles for enhancement. Principal component analysis performed on the spectra data set resulted in the successful differentiation of MS2 and PhiX174 species and also for the differentiation of viable virus samples and inactivated virus samples. / Ph. D.

The focus of the work has been on computational approaches to solving biological problems. First, microarray analysis was used to study the role of PLDα1 in drought stress in Arabidopsis. Second, a tool for designing and in-silico testing of primers for PCR-based pathogen detection will be discussed. Phospholipase D (PLD) has been implicated in a variety of stresses including osmotic stress and wounding. PLDα 1-derived phosphatidic acid interacts with ABI1 phosphatase 2C and promotes abscisic acid signaling. Plants with abrogated PLDα 1 show insensitivity to ABA and impaired stomatal conductance. My goal is to identify PLDα-mediated downstream events in response to progressive drought stress in Arabidopsis. <i>Arabidopsis thaliana</i> (Col-0) and antisense-PLDα 1 (Anti-PLDα) were drought stressed by withholding water. Anti-PLDα experienced severe water stress at the same time period that Col-0 experienced less water stress. Diurnal leaf water potential (LWP) measurements showed that Anti-PLDα had lower LWP than Col-0 under drought stress conditions. qRT-PCR revealed up to 18-fold lower values for PLDα transcripts in stressed Anti-PLDα plants when compared to stressed Col-0. Microarray expression profiles revealed distinct gene expression patterns in Col-0 and Anti-PLDα. ROP8, PLDδ and lipid transfer proteins were among the differentially expressed genes between the two genotypes. Different microarray analyses methods (TM4 and Expresso) were also compared on two different data sets. The results obtained from Expresso analysis were more accurate when compared with quantitative RT-PCR data. Rapid diagnosis of disease-causing agents is extremely important since delayed diagnosis can result in disease spread and delayed prophylaxis. It is even more important in an era where disease-causing agents are used as bioterrorism agents. Rapid advances in sequencing technology have resulted in the sequencing of thousands of microorganisms in recent years. Availability of genomic sequences has made it possible to identify and characterize microorganisms at the molecular level. PCR-based detection is powerful for pathogen diagnostics since it is rapid and sensitive. We have developed a tool, PathPrime, that can design primers, computationally test them against target genes, and potential contaminant sequences, and identify a minimum set of primers that can unambiguously detect a given list of sequences. / Ph. D.

The extent of devastation caused by a biological warfare attack is highly correlated to the time from release to detection. As a step towards lowering the detection time the international project TWOBIAS was launched. Here, the main goal is to develop an automated, specific and sensitive combined detection and identification instrument capable of identifying a biological threat within an hour. The identification unit is comprised of a sample preparation module, an amplification module and a detection module and utilizes a proximity ligation assay in combination with circle-to-circle amplification in order to detect a biological threat. This thesis describes the automation of the sample preparation steps of the assay and the integration with the downstream units. The functionality of the sample preparation module was verified by subjecting it to biological samples in a laboratory and at a real-life location. The results showed that the sample preparation module was capable of preparing a sample collected in a complex environment with the same results as a sample prepared in a laboratory.

Doctor of Philosophy / Department of Chemistry / Jun Li / Developing new and rapid methods for pathogen detection with enhanced sensitivity and temporal resolution is critical for protecting general public health and implementing the food and water safety standards. In this research vertically aligned carbon nanofiber nanoelectrode arrays (VACNF NEAs) have been explored as a sample manipulation tool and coupled with fluorescence, surface enhanced Raman scattering (SERS) and impedance techniques for pathogen detection and identification. The key objective for employing a nanoelectrode array is that the nano-Dielectrophoresis (nano-DEP) at the tip of a carbon nanofiber (CNF) acts as a potential trap to capture pathogens. A microfluidic device was fabricated where nanofibers (~ 100 nm in diameter) were placed at the bottom of a fluidic channel to serve as a ‘point array’ while an indium tin oxide coated glass slide acted as a macroscale counter electrode. The electric field gradient was highly enhanced at the tips of the CNFs when an AC voltage was applied. The first study focused on the capture of the viral particles (Bacteriophage T4r) by employing a frequency of 10.0 kHz, a flow velocity of 0.73 mm/sec, and a voltage of 10.0 Vpp. A Lithenburg type of phenomenon was observed, that were drastically different from the isolated spots of bacteria captured on VACNF tips in previous study. At the lowest employed virus concentration (1 × 10[superscript]4 pfu/mL), a capture efficiency of 60% was observed with a fluorescence microscope. The motivation of the second study was to incorporate the SERS detection for specific pathogen identification. Gold-coated iron-oxide nanoovals labeled with Raman Tags (QSY 21), and antibodies that specifically bound with E.coli cells were utilized. The optimum capture was observed at a frequency of 100.0 kHz, a flow velocity of 0.40 mm/sec, and a voltage of 10.0 Vpp. The detection limit was ~210 CFU/mL for a portable Raman system with a capture time of 50 seconds. In the final study, a real-time impedance method was employed to detect Vaccinia virus (human virus) in the nano-DEP device at 1.0 kHz and 8.0 Vpp giving a detection limit of 2.51 × 10[superscript]3 pfu/mL.

<p>Swift recognition of disease-causing pathogens at the point-of-care enables life-saving treatment and infection control. However, current rapid diagnostic devices often fail to detect the low concentrations of pathogens present in the early stages of infection, causing delayed and even incorrect treatments. Rapid diagnostics that require multiple steps and/or elevated temperatures to perform have a number of barriers to use at the point-of-care and in the field, and despite efforts to simplify these platforms for ease of use, many still require diagnostic-specific training for the healthcare professionals who use them. Most nucleic acid amplification assays require hours to perform in a sterile laboratory setting that may be still more hours from a patient’s bedside or not at all feasible for transport in remote or low-resourced areas. The cold-chain storage of reagents, multistep sample preparation, and costly instrumentation required to analyze samples has prohibited many nucleic acid detection and antibody-based assays from reaching the point-of-care. There remains a critical need to bring rapid and accessible pathogen identification technologies that determine disease status and ensure effective treatment out of the laboratory.</p> <p>Paper-based diagnostics have emerged as a portable platform for antigen and nucleic acid detection of pathogens but are often limited by their imperfect control of reagent incubation, multiple complex steps, and inconsistent false positive results. Here, I have developed mechanisms to economically improve thermal incubations, automate dried reagent flow for multistep assays, and specifically detect pathogenic antigens while improving final output sensitivity on paper-based devices. First, I characterize miniaturized inkjet printed joule-heaters (microheaters) that enable thermal control for pathogen lysis and nucleic acid amplification incubation on a low-cost paper-based device. Next, I explore 2-Dimensional Paper Networks as a means to automate multistep visual enhancement reactions with dried reagents to increase the sensitivity and readability of nucleic acid detection with paper-based devices. Lastly, I aim to create a novel Reverse-Transcription Recombinase Polymerase Reaction mechanism to amplify and detect a specific region of the Spike protein domain of SARS-CoV-2. This will allow the rapid detection of SARS-CoV-2 infections to aid in managing the current COVID-19 pandemic. In the future, these tools could be integrated into a rapid diagnostic test for SARS-CoV-2 and other pathogens, ultimately improving the accessibility and sensitivity of rapid diagnostics on multiple fronts.</p>

Diagnosis plays a very crucial role in medicine and health care, which makes biosensors extremely important in modern technological context. Till date, various types of biosensors have been developed that are capable of detecting a wide range of biologically important species with great sensitivity and selectivity. However, most of these sensing units require highly sophisticated instrumentation and often lack the desired portability. Liquid crystal (LC) droplets, on the other hand, are a new type of functional material that are finding increasing research attention as a new sensing unit due to their tunable optical property, high surface area, portability and cost-effectiveness. In this dissertation, functionalized LC droplets for biosensing at aqueous-LC interface are highlighted. Chemically functionalized LC droplets dispersed in aqueous solution were prepared by the self-assembly of amphiphilic molecules at the aqueous/LC interface. These functionalized LC droplets showed a well-defined director of configuration and a specific optical pattern when observed with a polarizing light microscope. It was discovered that the interaction of chemically functionalized LC droplets with an analyte triggers transition of the director of configuration of the LC within the droplets, providing a simple and unique optical sign for the detection of the analyte. Moreover, the director of configuration transition happened in a concentration dependent manner, allowing both qualitative and quantitative detection of the analyte. The sensitivity of chemically functionalized LC droplets depends not only on the nature of amphiphilic molecules but also the size and number of the droplets. iv The dissertation essentially deals with the application of these chemically functionalized LC droplets in detecting several biologically important species. It was observed that the adsorption of charged macromolecules (dendrimers, proteins, and viruses) on polyelectrolyte functionalized LC droplets triggered a bipolar-to-radial configuration transition based on the polar verses nonpolar interaction. By using a simple optical microscope, microgram per milliliter concentrations of bovine serum albumin, cowpea mosaic virus, and tobacco mosaic virus could be detected in aqueous solution. The detection limit of Mastoparan X polypeptide decorated LC droplets in detecting E. coli could reach to approximately 10 bacteria per milliliter. In this case, the high affinity of the polypeptide towards the bacterial causes the former to detach from the LC droplets, triggering the director of configuration transition of the LC inside the droplets. Finally, surfactant decorated LC droplets were used to detect lithocholic acid (LCA), a toxic bile acid used as a specific biomarker for colon cancers. In this case, the director of configuration transition of the LC inside the droplets is a result of the replacement of the surfactant from the aqueous/LC interface by LCA. The microgram per milliliter concentration of LCA, a clinically significant concentration, could be easily detected by changing the length of surfactants. These studies highlight the novel use of surface functionalized LC droplets to detect biologically important species. Due to their tunable optical property, coupled with high surface area and portability, surface functionalized LC droplets have great potentials in the design of next generation biosensors

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Surface enhanced Raman spectroscopy (SERS) is highly promising analytical technique for trace detection of analytes. It is particularly well suited for environmental analyses due to its high sensitivity, specificity, ease of operation and rapidity. The detection and characterization of environmental contaminants, using SERS is highly related to the uniformity, activity and reproducibility of the SERS substrate. In this thesis, SERS substrates were produced by gold nanoparticle formation on wax patterned chromatography paper. In situ reduction of hydrogen tetrachloroaurate (gold precursor) by trisodium citrate dihydrate (reducing agent) was used to produce gold nanoparticles within a paper matrix. These gold nanoparticle based SERS substrates were analyzed by FE-SEM, UV-Vis and Raman spectroscopy. This work discusses the SERS signal enhancements for Raman active MGITC dye for a series of substrates prepared by in situ reduction of gold salt and pre-produced gold nanoparticles. UV-Vis analysis was performed to understand the effect of different molar ratio (reducing agent to gold precursor) and reaction time on the size and shape of the localized surface plasmon resonance (LSPR) band that dictates the SERS enhancements. It was concluded that lower molar ratio (1:1 and 2:1) of citrate-to gold produced better SERS signal enhancements and broader LSPR band. Therefore, use of lower molar ratio (MR) was recommended for paper-based substrates using in situ-based reduction approach. / Master of Science