Micro and Nanoscale Aptasensors for Detection of Low Molecular Weight Biomarkers Towards Clinical Diagnostic Applications

Biosensors have been developed for their potential applications to clinical diagnostics, particularly for detection of disease-relevant biomarkers. As affinity biosensors have emerged for the application, aptamers, i.e., oligonucleotide receptors, have gained much attention due to their ability to offer high affinity, specificity, stability, and rapid, low cost production. While aptame based biosensors, called aptasensors, have shown great promise as a clinical assay tool, their sensitive detection of low molecular weight biomarkers is challenging. In this thesis, we present microfluidic aptasensors for label free and sensitive detection of low molecular weight analytes by focusing on arginine vasopressin (AVP), an oligopeptide hormone and a clinically important biomarker. We first present an integrated microfluidic aptasensor for label free detection of AVP by mass spectrometry. The integrated device selectively extracts AVP from human plasma ultrafiltrate samples and then repeatedly deposits the AVP on a MALDI plate for further analyte enrichment, thereby enabling highly sensitive AVP measurements. To further explore aptamer based detection of AVP, we have developed an optomagnetic aptasensor capable of detecting a low molecular weight analyte using magnetic nanoparticles (MNPs). In this aptasensor, second to be presented in the thesis, an inhibition assay principle is used, in which degrees of MNP clustering depend on the ATP concentration. The clustering state is then measured by an optomagnetic readout system that provides information about the distribution of cluster sizes, thus enabling us to relate the signal to the analyte concentration in a simple mix and read manner. A proof of concept demonstration of the sensor operation is provided using adenosine triphosphate (ATP) as a model small molecule analyte. We next exploit surface enhanced Raman spectroscopy (SERS) for detection of AVP. A SERS active substrate with aptamer functionalized leaning nanopillars is used for sensitive and specific detection of AVP labeled with a Raman tag. Large area Raman mapping on the substrate enables reliable SERS based AVP quantification, and microfluidic integration allows rapid and efficient analyte detection. Lastly, a competitive binding assay format is employed for label free detection of AVP. We finally present a microfluidic aptasensor that integrates aptamer based selective analyte preconcentration with conductance based graphene nanosensing for detection of AVP. In the integrated device, low abundance AVP is enriched via solid-phase aptamer based selective preconcentration, and then measured by a graphene field effect transistor (FET) based nanosensor through aptamer based competitive binding, allowing sensitive and label free detection of AVP. We conclude the thesis by a discussion of directions for future work, proposing strategies for pursuing technological advancements to ultimately enable highly sensitive and rapid detection of AVP in human bodily fluids in clinical diagnostic settings.