Nucleic Acid Amplification by Template-Dominated Click Chemistry for Ultrasensitive DNA/RNA Detection on an Electrochemical Readout Platform

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.