A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia.
Biosensors (Basel). 2023 Aug 17;13(8):824. doi: 10.3390/bios13080824.
CRISPR/Cas12-based biosensors are emerging tools for diagnostics. However, their application of heterogeneous formats needs the efficient detection of Cas12 activity. We investigated DNA probes attached to the microplate surface and cleaved by Cas12a. Single-stranded (ss) DNA probes (19 variants) and combined probes with double-stranded (ds) and ssDNA parts (eight variants) were compared. The cleavage efficiency of dsDNA-probes demonstrated a bell-shaped dependence on their length, with a cleavage maximum of 50%. On the other hand, the cleavage efficiency of ssDNA probes increased monotonously, reaching 70%. The most effective ssDNA probes were integrated with fluorescein, antibodies, and peroxidase conjugates as reporters for fluorescent, lateral flow, and chemiluminescent detection. Long ssDNA probes (120-145 nt) proved the best for detecting Cas12a trans-activity for all of the tested variants. We proposed a test system for the detection of the nucleocapsid (N) gene of SARS-CoV-2 based on Cas12 and the ssDNA-probe attached to the microplate surface; its fluorescent limit of detection was 0.86 nM. Being united with pre-amplification using recombinase polymerase, the system reached a detection limit of 0.01 fM, thus confirming the effectiveness of the chosen ssDNA probe for Cas12-based biosensors.
基于 CRISPR/Cas12 的生物传感器是用于诊断的新兴工具。然而，它们在异构格式中的应用需要有效检测 Cas12 活性。我们研究了附着在微孔板表面并被 Cas12a 切割的 DNA 探针。比较了单链 (ss) DNA 探针（19 种变体）和具有双链 (ds) 和 ssDNA 部分的组合探针（8 种变体）。dsDNA 探针的切割效率表现出对其长度的钟形依赖性，切割最大值为 50%。另一方面，ssDNA 探针的切割效率单调增加，达到 70%。最有效的 ssDNA 探针与荧光素、抗体和过氧化物酶缀合物集成作为荧光、侧向流动和化学发光检测的报告器。对于所有测试的变体，长 ssDNA 探针（120-145nt）被证明最适合检测 Cas12a 的反式活性。我们提出了一种基于 Cas12 和附着在微孔板表面的 ssDNA 探针的检测 SARS-CoV-2 核衣壳 (N) 基因的测试系统；其荧光检测限为 0.86 nM。与使用重组酶聚合酶进行预扩增相结合，该系统达到了 0.01 fM 的检测限，从而证实了所选 ssDNA 探针对基于 Cas12 的生物传感器的有效性。
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