Development of a concentration-enhanced mobility shift assay platform for aptamer-based biomarker detection and kinase profiling

摘要

New methods to quantify rare biomarkers from patient samples are critical for developing point-of- care diagnostic platforms. To be compatible with resource limited settings, these assays have to provide fast and accurate results without sacrificing ease of use. Biosensing in homogeneous fashion is the preferred format which satisfies these criteria, but the lack of amplification method is a bottleneck that limits their use for sensitive applications. To address this issue, this thesis explores physical signal amplification means to increase the sensitivities of homogeneous assays. We identified several key applications where the use of these technologies could make a positive impact in improving medical diagnostics systems and advancing biological research. We first outline the use of electrokinetic concentration to realize a continuous signal amplification scheme that increases the sensitivity of homogeneous mobility shift assays. By simultaneously concentrating and separating reacted and unreacted species (with different mobilities) in this device, we can perform sensitive, quantitative and ratiometric measurement of target biomarkers. Using this platform, we improved the sensitivity of aptamer affinity probe capillary electrophoresis to achieve pM detection limit of IgE and HIV-RT in simple buffer and serum sample. This work is timely and impactful as it directly addresses the sensitivity shortcomings of using aptamers as low cost and robust substitutes for antibodies in point-of-care applications. Next, we presented a herringbone nanofilter array device which can perform continuous sizes-elective concentration of biomolecules based on their direct interaction with nanostructures with comparable critical dimensions. We demonstrated the use of this platform to perform a novel homogeneous immunoassay for detecting a cardiac biomarker, C-reactive protein, at clinically relevant concentrations. Finally, we demonstrated that the concentration-enhanced mobility shift assay platform is a powerful tool for probing biological activities such as cellular kinase activities. We have developed technology to isolate, grow and lyse single cells, and used our platform to measure kinase activities from single cells. Through rational design of peptide substrates and spacers, this platform has the ability to simultaneously concentrate and separate multiple analytes. This enables users to obtain simultaneous measurements of multiple cellular kinase activities that could reveal important information about their functional relationships.