Applying CRISPR-Cas12a as a Signal Amplifier to Construct Biosensors for Non-DNA Targets in Ultralow Concentrations
Applying CRISPR-Cas12a as a Signal Amplifier to Construct Biosensors for Non-DNA Targets in Ultralow Concentrations
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应用 CRISPR-Cas12a 作为信号放大器构建超低浓度非 DNA 靶标生物传感器
DOI：
10.1021/acssensors.9b02305
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发表时间：
2020-04-24
期刊：
影响因子：
8.9
通讯作者：
Xie, Guoming
中科院分区：
文献类型：
--
作者：
Li, Junjie;Yang, Shuangshuang;Xie, Guoming
有效的信号放大是必不可少的，以构建超灵敏的生物传感器的生物相关物种与丰富的伴随干扰。在这里，我们应用LbaCas 12 a作为信号放大器来开发多功能CRISPR-Cas 12 a平台，以检测超低浓度的各种分析物。该平台依赖于LbaCas 12 a的无差别单链DNA酶活性，其识别由非DNA靶标产生的单链DNA中间体，低至飞摩尔浓度，随后增强荧光信号输出。在功能性核苷酸（DNAzyme和适体）的帮助下，已经设计了Pb 2+和鲍氏不动杆菌的超灵敏生物测定，检测限分别低至0.053 nM和-3 CFU/mL。它还允许在皮摩尔浓度下同时检测四种microRNA（miRNA），而不会受到其他对应物的显著干扰，这表明了高通量多重miRNA表达谱分析的潜力。鉴于CRISPR-Cas 12 a平台的多功能性和通用性，我们预计目前的工作将推动基于CRISPR-Cas的平台在生物分析中的应用，并为超灵敏生物传感器设计提供新的见解。
Efficient signal amplification is essential to construct ultrasensitive biosensors for biologically relevant species with abundant concomitant interferences. Here, we apply LbaCas12a as a signal amplifier to develop a versatile CRISPR-Cas12a platform to detect a wide range of analytes in ultralow concentrations. The platform relies on the indiscriminate single-stranded DNase activity of LbaCas12a, which recognizes single-stranded DNA intermediates generated by non-DNA targets down to femtomolar concentrations and subsequently enhances the fluorescence signal output. With the help of functional nucleotides (DNAzyme and aptamer), ultrasensitive bioassays for Pb2+ and Acinetobacter baumannii have been designed with a limit of detection down to similar to 0.053 nM and -3 CFU/mL, respectively. It also allows simultaneous detection of four microRNAs (miRNAs) at a picomolar concentration without significant interferences by other counterparts, suggesting the potential of multiplexed miRNA expression profiles analysis in high throughput. Given the versatility and generality of the CRISPR-Cas12a platform, we expect the current work to advance the application of CRISPR-Cas-based platforms in bioanalysis and provide new insights into ultrasensitive biosensor design.