State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, Jilin, P. R. China.
School of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei 230026, Anhui, China.
ACS Sens. 2023 Jul 28;8(7):2852-2858. doi: 10.1021/acssensors.3c00806. Epub 2023 Jul 4.
Rapid and accurate detection of biomarkers was very important for early screening and treatment of diseases. Herein, a sensitive and amplification-free electrochemiluminescence (ECL) biosensor based on CRISPR/Cas12a and DNA tetrahedron nanostructures (TDNs) was constructed. Briefly, 3D TDN was self-assembled on the Au nanoparticle-deposited glassy carbon electrode surface to construct the biosensing interface. The presence of the target would activate the trans-cleavage activity of Cas12a-crRNA duplex to cleave the single-stranded DNA signal probe on the vertex of TDN, causing the Ru(bpy) to fall from the electrode surface and weakened the ECL signal. Thus, the CRISPR/Cas12a system transduced the change of target concentration into an ECL signal enabling the detection of HPV-16. The specific recognition of CRISPR/Cas12a to HPV-16 made the biosensor have good selectivity, while the TDN-modified sensing interface could reduce the cleaving steric resistance and improve the cleaving performance of CRISPR/Cas12a. In addition, the pretreated biosensor could complete sample detection within 100 min with a detection limit of 8.86 fM, indicating that the developed biosensor possesses the potential application prospect for fast and sensitive nucleic acid detection.
快速准确地检测生物标志物对于疾病的早期筛查和治疗非常重要。在此，构建了一种基于 CRISPR/Cas12a 和 DNA 四面体型纳米结构（TDN）的灵敏、无需放大的电化学发光（ECL）生物传感器。简而言之，3D TDN 自组装在沉积有金纳米粒子的玻碳电极表面上，以构建生物传感界面。靶标的存在会激活 Cas12a-crRNA 双链体的转切割活性，从而切割 TDN 顶点上的单链 DNA 信号探针，导致 Ru(bpy)从电极表面脱落，减弱 ECL 信号。因此，CRISPR/Cas12a 系统将目标浓度的变化转化为 ECL 信号，从而实现对 HPV-16 的检测。CRISPR/Cas12a 对 HPV-16 的特异性识别使该生物传感器具有良好的选择性，而 TDN 修饰的传感界面可以降低切割空间位阻，提高 CRISPR/Cas12a 的切割性能。此外，预处理后的生物传感器可以在 100 分钟内完成样品检测，检测限低至 8.86 fM，表明所开发的生物传感器具有用于快速灵敏核酸检测的潜在应用前景。
