Ivanov Aleksandr V, Safenkova Irina V, Zherdev Anatoly V, Dzantiev Boris B
A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, Leninsky Prospect 33, 119071, Moscow, Russia.
A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, Leninsky Prospect 33, 119071, Moscow, Russia.
Biosens Bioelectron. 2022 Jul 15;208:114227. doi: 10.1016/j.bios.2022.114227. Epub 2022 Mar 30.
CRISPR-Cas12-based biosensors are a promising tool for the detection of nucleic acids. After dsDNA-target-activated Cas12 cleaves the ssDNA probe, a lateral flow test (LFT) is applied for rapid, simple, and out-of-laboratory detection of the cleaved probe. However, most of the existing approaches of LFT detection have disadvantages related to inverted test/control zones in which the assay result depends not only on the cleavage of the probe but also on the second factor: the binding of the non-cleaved probe in the control zone. We proposed a novel platform for the detection of trans-cleaved DNA using a universal DNA-IgG probe and LFT with the sequential direct location of test and control zones. The advantage of the platform consists of the assay result depending only on the cleaved probe. For this, we designed a composite probe that comprise two parts: the DNA part (biotinylated dsDNA connected to ssDNA with fluorescein) (FAM), and the antibody part (mouse anti-FAM IgG). The Cas12, with guide RNA, was activated by the dsDNA-target. The activated Cas12 cleaved the probe, releasing the ssDNA-FAM-IgG reporter that was detected by the LFT. The sandwich LFT was proposed with anti-mouse IgG adsorbed in the test zone and on the surface of gold nanoparticles. We called the platform with direct location zones and direct analyte-signal dependence the DNA-Immunoglobulin Reporter Endonuclease Cleavage Test (DIRECT). Therefore, this proof-of-concept study demonstrated that the combination of the proposed DNA-IgG probe and direct LFT opens new opportunities for CRISPR-Cas12 activity detection and its bioanalytical applications.
基于CRISPR-Cas12的生物传感器是用于核酸检测的一种很有前景的工具。在双链DNA靶标激活的Cas12切割单链DNA探针后，应用侧向流动检测（LFT）对切割后的探针进行快速、简单且无需实验室设备的检测。然而，现有的大多数LFT检测方法都存在与测试/对照区倒置相关的缺点，即检测结果不仅取决于探针的切割，还取决于第二个因素：未切割探针在对照区的结合。我们提出了一种使用通用DNA-IgG探针和LFT检测反式切割DNA的新型平台，测试区和对照区顺序直接定位。该平台的优点在于检测结果仅取决于切割后的探针。为此，我们设计了一种复合探针，它由两部分组成：DNA部分（生物素化双链DNA连接到带有荧光素的单链DNA）（FAM）和抗体部分（小鼠抗FAM IgG）。带有向导RNA的Cas12被双链DNA靶标激活。激活的Cas12切割探针，释放出单链DNA-FAM-IgG报告分子，通过LFT进行检测。我们提出了一种夹心LFT，在测试区和金纳米颗粒表面吸附抗小鼠IgG。我们将具有直接定位区和直接分析物信号依赖性的平台称为DNA免疫球蛋白报告核酸酶切割测试（DIRECT）。因此，这项概念验证研究表明，所提出的DNA-IgG探针与直接LFT的结合为CRISPR-Cas12活性检测及其生物分析应用开辟了新机遇。
