Mu Xiaomei, Wang Xin, Qin Yuxin, Huang Yong, Tian Jianniao, Zhao Shulin
State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin, 541004, China.
State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin, 541004, China.
Talanta. 2023 Jan 1;251:123795. doi: 10.1016/j.talanta.2022.123795. Epub 2022 Jul 31.
The development of a biosensing platform with high sensitivity, high specificity, and low cost for the detection of biomarkers, especially one that is programmable and universal, is critical for disease surveillance and diagnosis, yet it remains a difficulty. Herein, we combined the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system with a fluorescent label-free biosensor platform for sensitive and specific detection of disease-related protein, small molecule and nucleic acid. In this strategy, we designed an exonuclease III-mediated target cycle and released a universal trigger chain to stimulate the enzyme activity of CRISPR/Cas12a for additional signal amplification. The hydrolysis of ssDNA-templated silver nanoclusters (ssDNA-Ag NCs) as the reporter probe resulted in a significant decrease of fluorescence intensity. This biosensing platform can be flexibly used to the sensitive and specific determination of protein, small molecule, or microRNA in biological samples by simply transforming the target recognized sequences in the DNA hairpin. In this work, a new label-free sensing system used the fluorescent ssDNA-Ag NCs as the signal output does not need to be marked in advance and has no background signal. In addition, the method has the advantages of low cost, simple operation and high speed, and provides an innovative idea for the development of a powerful clinical diagnosis tool.
开发一种用于生物标志物检测的具有高灵敏度、高特异性和低成本的生物传感平台，尤其是一种可编程且通用的平台，对于疾病监测和诊断至关重要，但仍然是一个难题。在此，我们将成簇规律间隔短回文重复序列（CRISPR）/Cas系统与无荧光标记的生物传感器平台相结合，用于灵敏且特异的检测疾病相关蛋白质、小分子和核酸。在该策略中，我们设计了一种核酸外切酶III介导的靶循环，并释放一条通用触发链以刺激CRISPR/Cas12a的酶活性进行额外的信号放大。作为报告探针的单链DNA模板化银纳米簇（ssDNA-Ag NCs）的水解导致荧光强度显著降低。通过简单改变DNA发夹中的靶识别序列，该生物传感平台可灵活用于灵敏且特异的测定生物样品中的蛋白质、小分子或微小RNA。在这项工作中，一种新的无标记传感系统使用荧光ssDNA-Ag NCs作为信号输出，无需预先标记且无背景信号。此外，该方法具有成本低、操作简单和速度快的优点，为开发强大的临床诊断工具提供了创新思路。
