MicroRNAs (miRNAs), 18~22 nucleotides in length, are a class of single-strand noncoding short RNAs and have been used as biomarkers for diagnosis and prognosis of cancers. Herein, an α-hemolysin (α-HL) nanopore was adapted for the colorectal cancer-associated miRNAs analysis, with the merits of high-throughput, ultra-sensitivity and no requirements of amplification/labelling. DNA probes, consisting of a signal tag in each end and a response element in the middle section, were designed. The response element could be well-matched with miRNA and utilized for specific recognition of the target miRNA, while the signal tag increased the capture rate of the miRNA·probe complex. Due to the poor stacking of thymine residues, poly(dT)_n need to overcome a high entropic barrier when traversing through the α-HL nanopore confined space, resulting in distinct double-level blocked events, which contributes to the visualized differences in signal shape and prolonged duration. Thus, poly(dT)_n was selected as the signal tag of probe. Added in the cis side of α-HL, miRNA·probe was forced to traverse across the nanopore confined space under the potential of 140 mV through a pair of Ag/AgCl electrodes (cis grounded). Typical three-stage blocked event was observed, reflecting the translocation process:capture and dissociation of miRNA·probe, translocation of probe, temporarily residence and translocation of miRNA. Stage 1 (S1) represented the process from capture of miRNA·probe complex to translocation of the entire probe. The typical blocked events of miRNA 92·probe 92 showed a two-level S1, where Level 1 (L1) with a current blockage of 0.57±0.01 was generated mainly by translocation of the poly(dT)₄₀ signal tag. As the duration is associated with DNA length, probe 21 with smaller poly(dT)₂₀ signal tag was designed to detect miRNA 21, resulting in a shorter L1 of miRNA 21·probe 21 whose duration (t_D-L1) was 1/3 of that for miRNA 92·probe 92. As the signal shapes vary with DNA sequences, probe 16 with signal tag of poly(dC)₄₀ was used to sense miRNA 16, with miRNA 16·probe 16 producing a different single-level S1 with miRNA 92·probe 92 and miRNA 21·probe 21. The statistical results demonstrated that the three kinds of miRNA·probe produced different durations for S1 (t_D-S1), possibly indicating the differences in probe-α-HL interaction. Therefore, miRNA 92, miRNA 21 and miRNA 16 could be well identified by t_D-L1 (signal shape) and t_D-S1 (duration). Moreover, the serum sample have been tested. Hence, α-HL nanopore can be applied to build ultrasensitive single molecule biosensor for miRNA.

Key words: microRNA, α-hemolysin, nanopore confined space, single molecule biosensor