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<p>MicroRNA (miRNA), a crucial regulatory factor for maintaining homeostasis, is closely linked to the pathogenesis of numerous diseases. However, traditional miRNA detection technologies exhibit limitations, including low selectivity and sensitivity. In this study, a dual-mode self-calibrated nanopore sensing system based on rolling circle amplification (RCA) and hydrophobic modification is proposed for the highly sensitive detection of miRNA-21. Binding of miRNA-21 activates the DNAzyme catalytic activity, which triggers the stepwise release of a fluorescent group. Concurrently, residual sequences on the nanopore surface catalyze the RCA reaction, increasing the density of negative charges and amplifying the current signal, with enhancements reaching up to 384%. Fitting the ratio of electrical and fluorescence signals to the Hill equation enables self-calibrated detection of miRNA-21, thereby effectively eliminating systematic errors. This system achieves a linear detection range for miRNA-21 from 1 pM to 100 nM in fluorescence assays and from 1 fM to 1 nM in electrochemical assays, with detection limits of 0.16 pM and 0.28 fM, respectively. Furthermore, this innovative approach enables sensitive detection of clinical serum samples with elevated miRNA-21 expression levels. The system holds potential applications in the field of biomedicine, particularly in advancing disease diagnosis.</p>

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Dual-Mode Self-Calibrated Strategy Based on Synergistic Amplification Effects of in Situ Rolling Circle Amplification and Hydrophobic inner Wall of the Nanopores for Highly Sensitive Detection of microRNA

  • 期刊论文

  • 发表日期：

    2025年10月22日

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  • Yilin Zhang,
  • Jiangxue Dong,
  • Yufan Zhang,
  • Saimei Zhang,
  • Yajie Fan,
  • Yanlei Li,
  • Qun Ma,
  • Hongyuan Yan,
  • Shigang Shen,
  • Zhongfeng Gao

摘要

MicroRNA (miRNA), a crucial regulatory factor for maintaining homeostasis, is closely linked to the pathogenesis of numerous diseases. However, traditional miRNA detection technologies exhibit limitations, including low selectivity and sensitivity. In this study, a dual-mode self-calibrated nanopore sensing system based on rolling circle amplification (RCA) and hydrophobic modification is proposed for the highly sensitive detection of miRNA-21. Binding of miRNA-21 activates the DNAzyme catalytic activity, which triggers the stepwise release of a fluorescent group. Concurrently, residual sequences on the nanopore surface catalyze the RCA reaction, increasing the density of negative charges and amplifying the current signal, with enhancements reaching up to 384%. Fitting the ratio of electrical and fluorescence signals to the Hill equation enables self-calibrated detection of miRNA-21, thereby effectively eliminating systematic errors. This system achieves a linear detection range for miRNA-21 from 1 pM to 100 nM in fluorescence assays and from 1 fM to 1 nM in electrochemical assays, with detection limits of 0.16 pM and 0.28 fM, respectively. Furthermore, this innovative approach enables sensitive detection of clinical serum samples with elevated miRNA-21 expression levels. The system holds potential applications in the field of biomedicine, particularly in advancing disease diagnosis.