<p>Research in paper-based analytical devices has been increasing in recent years. Before technology transfer and market acceptance, these paper-based sensors have to be validated with field samples. In this study, we have made an attempt to evaluate the effectiveness of paper-based sensors to detect pesticides in real world samples. Generation 1 biosensor has been modified to be user friendly. There is no difference in the performance of generation 2 sensors; they detect pesticides based on colorimetric assay. The assay protocol involves first introducing the sample to the sensing zone by pipetting the water sample. Following an incubation period of 15min, the substrate end of the sensor is dipped into the sample to move paper bound indoxyl acetate (IDA) to the sensing region to initiate the enzyme-catalyzed hydrolysis of the substrate, resulting in the development of blue color. The presence of pesticide is indicated by either a decrease in color intensity or with no color development at all.</p> <p>To evaluate the effectiveness of biosensor in detecting pesticides in real world samples, a field study was conducted in four villages of southern India. Water samples from different aquatic environment including both surface water and ground water, were tested using generation 2 paper-based sensors. The paper-based sensors were capable of detecting organophosphorus pesticides in real world samples. The results were confirmed using GC-MS.</p> <p>The presence of higher concentration of dibutyl phthalate (in the range of 100uM to 10mM) in water can be a potential interference for the paper-based assay for the detection of pesticides in water. The paper-based biosensor assay platform can detect pesticides in the environmental samples and results have been validated by GC-MS. But for transfer of technology to the industry, further optimization is required to improve the stability of substrate to withstand atmospheric temperature fluctuations thus allowing the storage and shipment of the biosensor strips. Additionally to conduct reliable assays and obtain consistent results, the fabrication of biosensor strips needs to be improved to maintain the consistent volumes of bioinks impregnated on paper support.</p> / Master of Applied Science (MASc)

Functional nucleic acids (FNAs), which include both DNA/RNA aptamers and DNA enzymes, have emerged as promising biological recognition elements for biosensors. These species typically require immobilization on or within a solid support, which is usually interfaced to some kind of signal transducer and readout system when use in biosensor. Our group has successfully immobilized several functional nucleic acids in the past, including fluorescence-signalling DNA enzymes, DNA aptamers and RNA aptamers by entrapping them into porous silica or organosilica materials prepared by the sol-gel method using percursors such as sodium silicate (SS), diglyceryl silane (DGS), tetrametylorthosilicate (TMOS) and trimethoxymethysilane (MTMS). While the earlier work established the ability of entrapped FNAs to retain binding and catalytic activity, only limited information was obtained on how different factors affect the performance of immobilized FNAs, and no information was obtained on the effects of aging and storage conditions on FNA performance. The initial objective of this thesis was to employ advanced fluorescence methods to better understand the nature of immobilized DNA and RNA aptamers, and in particular how entrapment in different sol-gel based materials affected FNA performance for detection of small molecule analytes. It was found that the ability of the entrapped aptamer reporters to remain fully hybridized was the most important factor in terms of signalling capability for both DNA and RNA aptamer reporters. It was also observed that more polar materials derived from SS were optimal for both types of aptamer reporters, since these allowed the entrapped aptamers to remain hydridized to their complementary strands and still retain the dynamic motion needed to undergo structure switching, while providing a minimum degree of leaching. The second objective of my research was to develop a paper-based biosensing device incorporating immobilized DNA and RNA aptamers that could be used in the fields of point-of-care diagnostics to further expand the utility of structure-switching aptamer reporters to real world application. A dual response (fluorescence / colorimetric) paper-based sensor utilized printed graphene oxide to immobilize both a RNA and a DNA aptamer in a recognition zone. Upon target addition, the aptamer desorbed and eluted to an amplification zone where rolling circle amplification was used to generate a colorimetric output. This sensor could function with clinical samples such as serum and stool, and allowed detection of key bacterial markers (ATP and glutamate dehydrogenase) at clinically relevant levels. / Thesis / Doctor of Philosophy (PhD)

Carbon nanotubes (CNTs) and gold nanoparticles (AuNPs) are widely used for sensing applications due to their distinctive electrical and optical properties, and we have explored the development of methods that enable the incorporation of these nanomaterials into new and improved sensing devices. As a means for fabricating simple, low-cost and fast detection platforms for various applications, we have developed paper-based electrochemical detection platforms based on CNTs or platinum nanoparticle (PtNP)-CNT composite materials. We describe the use of a paper-based, low density, a three-dimensional thin film of interconnected CNTs as an electrode material. We studied the electrochemical properties of these paper-based CNT electrodes and demonstrated their use as an electrochemical sensor for the sensitive detection of guanine-based nucleotides. We further describe the functionalization of this paper-based electrode by fabricating a PtNP-SWCNT hybrid film via a vacuum filtration-based method. The interconnected PtNP structure formed on top of the CNT-coated paper was directly used as an electrocatalyst for methanol oxidation. Compared to paper-based PtNP-SWCNT hybrid films formed by electrochemical deposition, hybrid films formed by vacuum filtration showed a higher electrochemical surface area and enhanced electrocatalytic response to methanol oxidation. We have also developed methods based around DNA-modified AuNPs, which offer an excellent colorimetric platform for target detection. The DNA density on the surface of modified AuNPs affects enzymatic activity, colloidal stability of AuNPs, the orientation of the probe DNA and its hybridization efficiency. The combination of all these factors ultimately dictates the reaction time and sensitivity of colorimetric assays. We demonstrate the use of DTT as a modulator to control DNA surface coverage on the surface of AuNPs. Using this DTT treatment and a novel probe for exonuclease III activity, we have developed a colorimetric assay based on DTT-treated, DNA-modified AuNPs that can achieve more sensitive and rapid detection of DNA and enzymes relative to existing sensor platforms.

Point-of-care testing (POCT) is defined as medical testing at or near the site of patient care and has become a critical component of the diagnostic industry. POCT has many advantages over tests in centralized laboratories including small reagent volumes, small size, rapid turnaround time, cost-effectiveness, low power consumption and functional integration of multiple devices. Paper-based POCT sensors are a new alternative technology for fabricating simple, low-cost, portable and disposable analytical devices for clinical diagnosis. The focus of this dissertation was to develop simple, rapid and low cost paper-based POCT sensors with high sensitivity and portability for disease biomarker detection. Lateral flow strips (LFS) were used as the basic platform as it provides several key advantages such as simplicity, fast response time, on site and cost-effectiveness, and it can be used to detect specific substances including small molecules, large proteins and even whole pathogens, in a sample by immunological reactions. Earlier designs of paper strips lacked the quantitative information of the analyte concentration and could only provide single analyte detection at a time. In this study, a series of modifications were made to upgrade the platform to compensate for these limitations. First, we developed a gold nanoparticle based LFS for qualitative colorimetrical detection of bladder cancer related biomarkers in standard solutions and in urine samples. Second, by incorporating an image processing program “ImageJ”, a semi-quantitative LFS platform was established. The capability of the strip was evaluated by testing a small DNA oxidative damage biomarker in urine and cell culture models. Third, we combined the electrochemical method and colorimetrical method for quantitative biomarker detection. Finally, we integrated a commercialized blood glucose meter to quantitatively detection of two non-glucose biomarkers by converting their signals to that of glucose. The upgraded sensor could provide a noninvasive, rapid, visual, quantitative and convenient detection platform for various disease biomarkers. In addition, this platform does not require expensive equipments or trained personnel, deeming it suitable for use as a simple, economical and portable field kit for on-site biomarker monitoring in a variety of clinical settings.