CRISPR/Cas12a介导的模块化纳米孔转导放大策略用于铜离子的检测OA

Due to the advantages of label-free operation,high sensitivity and high throughput,solid-state nanopore-based sensing technologies exhibit broad application prospects for analyte characterization at the single-molecule level.To address the insufficient resolution and selectivity of solid-state nanopores for direct detection of small-sized,non-nucleic-acid targets,a modular signal amplification strategy integrating DNAzymes,clustered regularly interspaced short palindromic repeats(CRISPR),hybridization chain reaction(HCR),and solid-state nanopores was constructed in this work,using Cu2+as a model target for validation.In this strategy,a Cu2+-responsive DNAzyme first converted the small-molecule input into a nucleic acid signal.Subsequently,the trans-cleavage activity of the Cas12a/crRNA system was employed to achieve intermediate signal amplification,and HCR was further used to generate long-chain nucleic acid assemblies that could be efficiently resolved by solid-state nanopores.Compared with conventional strategies that required direct sequence coupling between the recognition module and the amplification module,the introduction of the CRISPR module in this method reduced the coupling between upstream and downstream nucleic acid sequence design,thereby improving the editability and scalability of the system.Real-time fluorescence results showed that the minimum distinguishable concentration of Cu2+reached 1 μmol/L,and the system also exhibited good anti-interference performance.Agarose gel electrophoresis and nanopore results further confirmed that Cu2+could be stepwise transduced into HCR products,yielding nanopore event distributions that were clearly distinguishable from those of the negative control and interfering ions.This method provided a new signal amplification strategy for nanopore-based detection of small size non-nucleic-acid targets.