Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Environmental and Operational Medicine, Tianjin, 300050, China.
Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Environmental and Operational Medicine, Tianjin, 300050, China.
Biosens Bioelectron. 2022 Dec 15;218:114792. doi: 10.1016/j.bios.2022.114792. Epub 2022 Oct 8.
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems exhibit significant potential in developing biosensing technology due to their collateral cleavage capabilities. Herein, we introduced the collateral cleavage activity of CRISPR/Cas14a to activate DNA hydrogel for ultrasensitive detection of the myocardial infarction biomarker creatine kinase MB (CK-MB). In this strategy, the designed CRISPR/Cas14a system can be activated by introducing complementary DNA (cDNA) derived from competitive dissociation and exponential amplification (EXPAR), which is positively correlated with creatine kinase isoenzyme (CK-MB) concentration. Then the activated Cas14a protein can be utilized to indiscriminately cleave the DNA hydrogel cross-linker strand, leading to the degradation of the gel matrix and thus releasing the pre-encapsulated PtNPs/Cu-TCPP(Fe). PtNPs/Cu-TCPP(Fe) can trigger the TMB reaction, leading to an increase in absorbance value at 450 nm, thus enabling the quantitative detection of CK-MB. The proposed strategy combines CRISPR/Cas14a with DNA hydrogel for the first time, improving the programmability of DNA hydrogel and providing a reliable, sensitive, and versatile detection platform for trace non-nucleic acid targets.
成簇规律间隔短回文重复序列（CRISPR）/Cas 系统由于其旁切活性而在开发生物传感技术方面具有显著的潜力。在此，我们介绍了 CRISPR/Cas14a 的旁切活性，用于激活 DNA 水凝胶，以超灵敏检测心肌梗死生物标志物肌酸激酶 MB（CK-MB）。在该策略中，通过引入与肌酸激酶同工酶（CK-MB）浓度呈正相关的竞争解离和指数扩增（EXPAR）衍生的互补 DNA（cDNA），可以激活设计的 CRISPR/Cas14a 系统。然后，激活的 Cas14a 蛋白可以随机切割 DNA 水凝胶交联链，导致凝胶基质降解，从而释放预先封装的 PtNPs/Cu-TCPP(Fe)。PtNPs/Cu-TCPP(Fe)可以引发 TMB 反应，导致 450nm 处吸光度值增加，从而实现 CK-MB 的定量检测。该策略首次将 CRISPR/Cas14a 与 DNA 水凝胶相结合，提高了 DNA 水凝胶的可编程性，为痕量非核酸靶标提供了可靠、灵敏、多功能的检测平台。
