Abstract
The identification of short nucleic acids and proteins at the single molecule level is a major driving force for the development of novel detection strategies. Nanopore sensing has been gaining in prominence due to its label-free operation and single molecule sensitivity. However, it remains challenging to detect small molecules selectively. Here we propose to combine the electrical sensing modality of a nanopore with fluorescence-based detection. Selectivity is achieved by grafting either molecular beacons, complementary DNA, or proteins to a DNA molecular carrier. We show that the fraction of synchronised events between the electrical and optical channels, can be used to perform single molecule binding assays without the need to directly label the analyte. Such a strategy can be used to detect targets in complex biological fluids such as human serum and urine. Future optimisation of this technology may enable novel assays for quantitative protein detection as well as gene mutation analysis with applications in next-generation clinical sample analysis.
Highlights
The identification of short nucleic acids and proteins at the single molecule level is a major driving force for the development of novel detection strategies
Alignment was achieved by mounting the nanopipette on a coverslip inserted into a custom sample holder on a highresolution motorised stage and using an electron multiplying charge coupled device camera (Fig. 1b) to visually align the x−y axes
The z height was finetuned by scanning this axis in 10 nm step sizes until scattering was observed using an avalanche photodiode (APD)
Summary
The identification of short nucleic acids and proteins at the single molecule level is a major driving force for the development of novel detection strategies. We show that the fraction of synchronised events between the electrical and optical channels, can be used to perform single molecule binding assays without the need to directly label the analyte Such a strategy can be used to detect targets in complex biological fluids such as human serum and urine. The most widely used commercial technologies for detection of given molecular targets are based on immunoassays or gel electrophoresis These methods tend to lack the required sensitivity especially when one needs to probe low biomarker concentrations in complex fluids such as serum. It is not always trivial to distinguish between the signal arising from a target analyte bound to the carrier and a carrier that translocates in a folded state To address these challenges, the advantages of both nanopore sensing and single-molecule fluorescence spectroscopy can be combined to enable an efficient strategy for small molecule detection using nanopores. Fluorescent probes can be used to target molecules that are difficult to detect using conventional nanopore sensing, while the combined electrical and optical signals can be used to quantify binding affinities, as well as to selectively confirm the presence of a particular biomarker
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