School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China.
Sensors (Basel). 2024 Sep 23;24(18):6138. doi: 10.3390/s24186138.
Direct detection of miRNA is currently limited by the complex amplification and reverse transcription processes of existing methods, leading to low sensitivity and high operational demands. Herein, we developed a CRISPR/Cas13a-mediated photoelectrochemical (PEC) biosensing platform for direct and sensitive detection of miRNA-21. The direct and specific recognition of target miRNA-21 by crRNA-21 eliminates the need for pre-amplification and reverse transcription of miRNA-21, thereby preventing signal distortion and enhancing the sensitivity and precision of target detection. When crRNA-21 binds to miRNA-21, it activates the trans-cleavage activity of CRISPR/Cas13a, leading to the non-specific cleavage of biotin-modified DNA with uracil bases (biotin-rU-DNA). This cleavage prevents the biotin-rU-DNA from being immobilized on the electrode surface. As a result, streptavidin cannot attach to the electrode via specific biotin binding, reducing spatial resistance and causing a positively correlated increase in the photocurrent response. This Cas-PEC biosensor has good analytical capabilities, linear responses between 10 fM and 10 nM, a minimum detection limit of 9 fM, and an excellent recovery rate in the analysis of real human serum samples. This work presented an innovative solution for detecting other biomarkers in bioanalysis and clinical diagnostics.
目前，直接检测 miRNA 受到现有方法中复杂的扩增和逆转录过程的限制，导致灵敏度低且操作要求高。在此，我们开发了一种基于 CRISPR/Cas13a 的光电化学（PEC）生物传感平台，用于直接灵敏地检测 miRNA-21。crRNA-21 对靶 miRNA-21 的直接特异性识别消除了 miRNA-21 的预扩增和逆转录的需要，从而防止了信号失真，增强了目标检测的灵敏度和精度。当 crRNA-21 与 miRNA-21 结合时，它会激活 CRISPR/Cas13a 的反式切割活性，导致带有尿嘧啶碱基的生物素修饰 DNA（biotin-rU-DNA）的非特异性切割。这种切割阻止了生物素-rU-DNA 固定在电极表面上。因此，链霉亲和素无法通过特异性的生物素结合附着在电极上，减少了空间电阻并导致光电流响应呈正相关增加。这种 Cas-PEC 生物传感器具有良好的分析能力，在 10 fM 至 10 nM 之间具有线性响应，检测限低至 9 fM，在分析真实的人血清样本时具有出色的回收率。这项工作为生物分析和临床诊断中检测其他生物标志物提供了一种创新的解决方案。
