The Central Laboratory, Fujian Key Laboratory of Precision Medicine for Cancer, Key Laboratory of Radiation Biology of Fujian Higher Education Institutions, the First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China.
Department of Pharmaceutical Analysis, Higher Educational Key Laboratory for Nano Biomedical Technology of Fujian Province, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350122, China.
Anal Chem. 2023 Mar 28;95(12):5331-5339. doi: 10.1021/acs.analchem.2c05421. Epub 2023 Mar 16.
As an enzyme-free exponential nucleic acid amplification method, the click chemistry-mediated ligation chain reaction (ccLCR) has shown great prospects in the molecular diagnosis. However, the current optics-based ccLCR is challenged by remarkable nonspecific amplification, severely hindering its future application. This study demonstrated that the severe nonspecific amplification was generated probably due to high random collision in the high DNA probe concentration (μM level). To solve this hurdle, a nucleic acid template-dominated ccLCR was constructed using nM-level DNA probes and read on an electrochemical platform (cc-eLCR). Under the optimal conditions, the proposed cc-eLCR detected a low-level nucleic acid target (1 fM) with a single-base resolution. Furthermore, this assay was applied to detect the target of interest in cell extracts with a satisfactory result. The proposed cc-eLCR offers huge possibility for click chemistry-mediated enzyme-free exponential nucleic acid amplification in the application of medical diagnosis and biomedical research.
作为一种无酶的指数核酸扩增方法，点击化学介导的连接链反应（ccLCR）在分子诊断中显示出了广阔的前景。然而，目前基于光学的 ccLCR 受到显著非特异性扩增的挑战，严重阻碍了其未来的应用。本研究表明，严重的非特异性扩增可能是由于在高 DNA 探针浓度（μM 级）下高随机碰撞产生的。为了解决这个难题，使用 nM 级别的 DNA 探针构建了一种核酸模板主导的 ccLCR，并在电化学平台上进行读取（cc-eLCR）。在最佳条件下，所提出的 cc-eLCR 以单碱基分辨率检测到低水平的核酸靶标（1 fM）。此外，该检测方法还应用于细胞提取物中目标物的检测，取得了令人满意的结果。所提出的 cc-eLCR 为点击化学介导的无酶指数核酸扩增在医学诊断和生物医学研究中的应用提供了巨大的可能性。
