The Center for Clinical Molecular Medical Detection , The First Affiliated Hospital of Chongqing Medical University , Chongqing 400016 , China.
Department of Laboratory Diagnosis, The Third Affiliated Hospital of Shenzhen University , Shenzhen University , Shenzhen 518000 , China.
ACS Sens. 2020 Feb 28;5(2):557-562. doi: 10.1021/acssensors.9b02461. Epub 2020 Feb 12.
A rapid and sensitive isothermal method is crucial for point-of-care (POC) nucleic acid testing. Recently, RNA-guided CRISPR/Cas12a proteins were discovered to exhibit target-triggered nonspecific single-stranded deoxyribonuclease (ssDNase) activity. Herein, the ssDNase cleavage capacity of the CRISPR/Cas12a system for interfacial hairpin DNA (hpDNA) and linear DNA was investigated in detailed. A novel electrochemical DNA biosensor was then developed via target-induced Cas12a cleaving interfacial hpDNA. In this strategy, the RNA-guided target DNA binding activates the robust Cas12a ssDNase activity. The immobilized hpDNA electrochemical reporters with a low surface coverage and incompact morphological structure present accessible substrates for highly efficient Cas12a cleavage, leading to a highly sensitive electrochemical DNA biosensor. Under the optimal conditions, as low as 30 pM target DNA was detected in about 60 min with 3.5 orders of magnitude dynamic range from 50 pM to 100 nM. Furthermore, the practical application ability of the established sensing method for detecting the target in complex matrices was also demonstrated. The proposed strategy enables rapid and sensitive DNA determination, providing a potential tool for POC molecular diagnostics.
一种快速且灵敏的等温方法对于即时检测（POC）核酸检测至关重要。最近，人们发现 RNA 引导的 CRISPR/Cas12a 蛋白具有靶向触发的非特异性单链脱氧核糖核酸酶（ssDNase）活性。在此，详细研究了 CRISPR/Cas12a 系统对界面发夹 DNA（hpDNA）和线性 DNA 的 ssDNase 切割能力。然后，通过靶标诱导 Cas12a 切割界面 hpDNA，开发了一种新型电化学 DNA 生物传感器。在该策略中，RNA 引导的靶 DNA 结合激活了强大的 Cas12a ssDNase 活性。固定化的 hpDNA 电化学报告分子具有低表面覆盖率和不紧凑的形态结构，为高效 Cas12a 切割提供了可及的底物，从而实现了高灵敏度的电化学 DNA 生物传感器。在最佳条件下，在约 60 分钟内可以检测到低至 30 pM 的靶 DNA，动态范围为 50 pM 至 100 nM，达到 3.5 个数量级。此外，还证明了所建立的传感方法在复杂基质中检测靶标的实际应用能力。该策略可实现快速灵敏的 DNA 测定，为即时分子诊断提供了一种潜在工具。
