CRISPR/Cas12a system has attracted extensive concern in biosensing due to its high specificity and programmability. Nevertheless, existing Cas12a-based assays mainly focus on nucleic acid detection and have limitations in non-nucleic acid biomarker analysis. To broaden the application prospect of the CRISPR/Cas technology, a cascade Cas12a biosensing platform is reported by combining dual-functionalized gold nanoparticles (FGNPs)-assisted rolling circle amplification (RCA) and Cas12a -cleavage activity (GAR-Cas) for ultrasensitive protein and exosome analysis. FGNPs serve as a critical component in the transduction of protein or exosome recognition information into nucleic acid amplification events to produce Cas12a activators. In the GAR-Cas assay, by integrating the triple cascade amplification of FGNPs-assisted transduction, RCA, and Cas12a signal amplification, ultralow abundance of target molecules can arouse numerous concatemers to activate Cas12a -cleavage activity to release intense fluorescence, allowing the ultrasensitive detection of as low as 1 fg/mL (∼41 aM) cTnI and 5 exosomes per μL. Furthermore, the presented strategy can be applied to detect exosome levels from clinical samples, showing excellent performance in distinguishing cancer patients from healthy individuals. The GAR-Cas sensing platform exhibits great potential in clinical diagnosis and enlarges biosensing toolboxes based on CRISPR/Cas technology for non-nucleic acid target analysis.

中文翻译：

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基于滚环扩增辅助的 CRISPR/Cas12a 策略的超灵敏蛋白质和外泌体分析

CRISPR/Cas12a系统因其高特异性和可编程性而引起了生物传感领域的广泛关注。然而，现有的基于Cas12a的检测主要集中于核酸检测，在非核酸生物标志物分析方面存在局限性。为了拓宽CRISPR/Cas技术的应用前景，结合双功能化金纳米粒子（FGNPs）辅助滚环扩增（RCA）和Cas12a切割活性（GAR-Cas）超灵敏蛋白的级联Cas12a生物传感平台和外泌体分析。 FGNP 在将蛋白质或外泌体识别信息转导至核酸扩增事件以产生 Cas12a 激活剂的过程中发挥着关键作用。在GAR-Cas检测中，通过整合FGNPs辅助转导、RCA和Cas12a信号放大的三重级联放大，超低丰度的目标分子可以激发大量多联体激活Cas12a裂解活性，释放强烈的荧光，从而实现超灵敏检测每 μL 含有低至 1 fg/mL (∼41 aM) cTnI 和 5 个外泌体。此外，所提出的策略可用于检测临床样本中的外泌体水平，在区分癌症患者与健康个体方面表现出优异的性能。 GAR-Cas传感平台在临床诊断中展现出巨大的潜力，并扩大了基于CRISPR/Cas技术的非核酸靶点分析的生物传感工具箱。