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Many efforts are being made in translating the nanopore into an ultrasensitive single-molecule platform for various genetic and epigenetic detections. However, compared with current approaches including PCR, the low throughput limits the nanopore applications in biological research and clinical settings, which usually requires simultaneous detection of multiple biomarkers for accurate disease diagnostics. Herein we report a barcode probe approach for multiple nucleic acid detection in one nanopore. Instead of directly identifying different targets in a nanopore, we designed a series of barcode probes to encode different targets. When the probe is bound with the target, the barcode group polyethylene glycol attached on the probe through click chemistry can specifically modulate nanopore ion flow. The resulting signature serves as a marker for the encoded target. Therefore counting different signatures in a current recording allows simultaneous analysis of multiple targets in one nanopore. The principle of this approach was verified by using a panel of cancer-derived microRNAs as the target, a type of biomarker for cancer detection.

The nanopore can electrophoretically trap single DNA/RNA molecules for genetic/epigenetic detections. However, low selectivity for complex samples (extracts from plasma) remains the challenge to clinical applications. We report an novel biophysical mechanism--Carrier-guided-Nanopore-Dielectrophoresis(CND)--for selective nucleic acids detection. We invented a polycationic micro-carrier. Upon hybridization with the target, the target•carrier forms a moment-tunable dipole, which can be attracted into the nanopore by dielectrophoresis from a huge electric field gradient (107V/m-per-nm) outside the nanopore entrance. In contrast, any non-target species without carrier hybridization carries negative charge and would electrophoretically migrate away from the nanopore. Consequently only the target•carrier nanopore signatures can be identified; any interference signal from non-target nucleic acids is completely eliminated. Unlike electrophoresis that lacks selectivity, the nanopore dielectrophoresis can selectively drive target nucleic acids of any size by using a universal micro-carrier. This represents the first and substantial step in translating the nanopore-sensor into a clinically-usable tool for molecular diagnostics. We demonstrate how to utilize this mechanism to accurately quantify cancer-derived microRNA biomarkers in patient plasma.Nat.Nanotech._DOI:10.1038/NNANO.2011.147.ACS_Nano_DOI:10.1021/nn305789zView Large Image | View Hi-Res Image | Download PowerPoint Slide