Acta Chim. Sinica ›› 2017, Vol. 75 ›› Issue (11): 1087-1090.DOI: 10.6023/A17090433 Previous Articles     Next Articles

Special Issue: 纳米传感分析



胡正利a, 杜冀晖b, 应佚伦a, 彭岳一a, 曹婵a, 龙亿涛a   

  1. a 华东理工大学化学与分子工程学院 结构可控先进功能材料及其制备教育部重点实验室 上海 200237;
    b 深圳大学附属南山医院中心实验室 深圳 518052
  • 投稿日期:2017-09-22 发布日期:2017-11-07
  • 通讯作者: 龙亿涛
  • 基金资助:


Single-Molecule Analysis of Colorectal Cancer-associated MicroRNAs via a Biological Nanopore

Hu Zhenglia, Du Jihuib, Ying Yiluna, Peng Yueyia, Cao Chana, Long Yi-Taoa   

  1. a Key Laboratory for Advanced Materials & School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China;
    b Central Laboratory, Affiliated Nanshan Hospital, Shenzhen University, Shenzhen 518052, China
  • Received:2017-09-22 Published:2017-11-07
  • Contact: 10.6023/A17090433
  • Supported by:

    Project supported by the National Key R&D Program of China (No. 2017YFC0906500), the National Natural Science Foundation of China (No. 21505043), Innovation Program of Shanghai Municipal Education Commission (No. 2017-01-07-00-02-E00023) and the Fundamental Research Funds for the Central Universities (Nos. 222201714012, 222201718001, 222201717003).

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 (tD-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 (tD-S1), possibly indicating the differences in probe-α-HL interaction. Therefore, miRNA 92, miRNA 21 and miRNA 16 could be well identified by tD-L1 (signal shape) and tD-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