Selective single molecule sensing in nanopore

Nanopores have emerged as one of the powerful tool for single molecule detection as it offers advantages such as label free and minimal sample preparation; this stimulates many exciting applications in biophysics and molecular sensing. The sensing principle can be achieved by electrophoretically driving biomolecules in solution through a nanometer pore. The ability to detect and measure abundance of many proteins precisely and simultaneously is an important and predictive biological research. To date, quantification of multiple proteins in buffer or purified samples have been successfully demonstrated in ELISA or tagged with fluorescence probes to detect specific molecules however, they rely on sophisticated and expensive diagnostics platform, as well as long assay time which hinder the process of simple and parallel screening multiple proteins at the single molecule level. Perhaps the biggest challenge for nanopore protein detection is the lack of selectivity, making it challenging to differentiate between multiple analytes; let alone trying to obtain meaningful data from complex biological samples such as human serum. Therefore, there is a need to develop strategies whereby such detection modalities can be used with unprocessed biological samples where thousands of different background proteins exists, often at much higher concentrations than the target analyte making detection exceptionally challenging. We present in this thesis some novel strategies that can improve the selectivity of the nanopore, allowing efficient detection and analysis of biomolecules in solutions accurately. Specifically, detection of multiple proteins via the use of aptamers attached onto a DNA carrier and in particular utility in detecting in biological sample in a low costs, scalable and sensitive manner. We were able to detect multiplex proteins, differentiate different protein size as well as accurately locate the proteins bound to the carrier without the need for extensive sample preparation and amplification, allowing direction sensing of proteins in unmodified samples. This thesis also introduces a process where having a control over the pore dimensions to ensure the dimensions or probe molecules match the pore size allowing good signal to noise ratio and enhancement in sensing ability. We show that Al2O3 atomic layer deposition (ALD) modified nanopipettes is capable to reduce the pore diameter down to 7.5 nm while allowing batch production of reproducible pipettes. Importantly, the sensing abilities were not affected by the ALD deposition. The other strategies demonstrate precise opening of the nanopore by electroetching the graphene nanoflakes (GNFs) that coated the nanopipette. The pore opening process enable in situ nanopore size control while performing DNA translocation, broadly functioning the nanopore devices. Overall the combined findings from the above strategies provided an incredible insight on the sensing and molecular biophysics of proteins and DNA at the single molecule level. The use of aptamer modified carrier increases the sensitivity and selectivity of the nanopore platform and enable potential applications for sensing of multiple proteins biomarker with single molecule sensitivity.Open Acces