The capability to selectively identify and quantify small nucleic acids or proteins in low abundance in the clinical sample has become a major driving force for the development of next-generation diagnostic strategies. To this end, nanopores have been a promising analytical tool for sensing biomolecules at the single-molecule level, directly in biofluids, without the need for sample processing. Nanopores, however, generally the lack of selectivity and accuracy to discriminate small molecules that have similar size but different structure and biological function, and this, in turn, has limited their applications in diagnostics.
In this thesis, I present a novel opto-electronic strategy combining the electrical sensing modality of a nanopore with fluorescence-based detection to address these challenges. By engineering a molecular beacon (MB) attached to a DNA carrier, I was able to show that target molecules could be selectively and accurately identified through reading a synchronised opto-electrical signal originating from the carrier and bound target that was translocated through the nanopore. By quantifying the fraction of synchronisation, one can predict the target concentration as well as determine the target binding affinity without directly labelling of the target itself. The method is based on aptamer binding and is applicable for a wide panel of targets, including proteins with known aptamer recognition sequences or DNA and RNA with known sequences. It was also possible to discriminate the latter with single-nucleotide resolution.
In addition, it was possible to detect more than one target simultaneously and increase the throughput by using DNA carriers of a specific length for each target. In this thesis, it is shown that multiple miRNAs (miR-141, miR-375) that have been implicated in prostate cancer can be quantified simultaneously with only one test, performed directly in the clinical sample. I demonstrate the ability of this strategy to distinguish single nucleotide polymorphism (SNP) within the same miRNA family. Notably, this strategy can simultaneously profile the small aberrations in multiple miRNA expression in patients with different stages of prostate cancer.
Overall, the present findings in this thesis have pushed the nanopore sensing a step closer to the practical biomedical diagnosis and provide insights for developing next-generation clinical sample assays.