CRISPR/Cas12a linked sandwich aptamer assay for sensitive detection of thrombin,Analytica Chimica Acta

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CRISPR/Cas12a linked sandwich aptamer assay for sensitive detection of thrombin
Analytica Chimica Acta ( IF 5.7 ) Pub Date : 2023-12-06 , DOI: 10.1016/j.aca.2023.342106
Fengxi Zhu ₁ , Qiang Zhao ₂

Affiliation

Background

Thrombin is a serine protease and hemostasis regulator with multiple functions and recognized as an important biomarker for diseases, and sensitive detection of thrombin is of significance for clinical diagnostics and disease monitoring. Recently, the target-triggered nonspecific single-stranded deoxyribonuclease activity of CRISPR/Cas system is discovered, making it become a powerful tool in assay developments due to the ease of signal amplification. In the short period of development, many CRISPR based nucleic acid detection methods have already played a critical role in clinical diagnostics. However, the application of CRISPR/Cas system for protein biomarkers remains limited.

Results

Here we describe a CRISPR/Cas12a linked sandwich aptamer assay for detection of thrombin, which was based on the formation of a sandwich complex of target by using a capture aptamer or antibody coated on the microplate and a well-designed detection DNA strand. The detection DNA strand contained an anti-thrombin aptamer and an active DNA of Cas12a, thus the sandwich complex was labeled with the active DNA. The active DNA triggered activity of Cas12a in indiscriminately cleaving fluorophore and quencher labeled DNA reporters, causing significant fluorescence increase. Our method enabled sensitive detection of thrombin down to 10 pM, and it showed high selectivity for thrombin. The assay exhibited good performance in diluted serum samples, demonstrating the applicability for thrombin analysis in the real media.

Significance

This assay combines the merits of high affinity of aptamer, trans-cleavage activity of Cas12a, high selectivity of sandwich format analysis, and high-throughput detection of microplate assay, and it shows promise in applications.

中文翻译：

用于灵敏检测凝血酶的 CRISPR/Cas12a 连接夹心适体测定

背景

凝血酶是一种具有多种功能的丝氨酸蛋白酶和止血调节剂，被认为是疾病的重要生物标志物，灵敏检测凝血酶对于临床诊断和疾病监测具有重要意义。最近，CRISPR/Cas系统的靶标触发非特异性单链脱氧核糖核酸酶活性被发现，由于易于信号放大，使其成为检测开发中的强大工具。在短短的发展过程中，许多基于CRISPR的核酸检测方法已经在临床诊断中发挥了关键作用。然而，CRISPR/Cas系统在蛋白质生物标志物方面的应用仍然有限。

结果

在这里，我们描述了一种用于检测凝血酶的 CRISPR/Cas12a 连接夹心适体测定法，其基于通过使用微孔板上包被的捕获适体或抗体和精心设计的检测 DNA 链形成靶标的夹心复合物。检测DNA链含有抗凝血酶适体和Cas12a活性DNA，因此夹心复合物被活性DNA标记。活性 DNA 触发 Cas12a 不加区别地切割荧光团和猝灭剂标记的 DNA 报告基因的活性，导致荧光显着增加。我们的方法能够灵敏地检测低至 10 pM 的凝血酶，并且对凝血酶表现出高选择性。该测定在稀释的血清样品中表现出良好的性能，证明了在真实培养基中凝血酶分析的适用性。