Yuan Guolin, Xia Xianru, Zhang Jicai, Huang Jian, Xie Fei, Li Xiandong, Chen Dongliang, Peng Chunyan
Department of Laboratory Medicine, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, 442000, China; Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Shiyan, 442000, Hubei, PR China.
Department of Laboratory Medicine, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, 442000, China.
Biosens Bioelectron. 2023 Feb 1;221:114424. doi: 10.1016/j.bios.2022.114424. Epub 2022 May 27.
Vascular endothelial growth factor (VEGF) plays an important role in atherosclerosis, and the detection of VEGF is critical for the prevention, monitoring, and diagnosis of cardiovascular diseases. Here, a novel "signal on-off-super on" sandwich-type aptamer sensor with a triple signal amplification strategy was developed for the first time. Based on the capture aptamer was labeled with methylene blue (MB) on the internal bases, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-coupled voltage enrichment was used to amplify the electrochemical signal. To improve the analytical performance of the aptamer sensor, gold nanoparticles@TiCT-Mxene (AuNPs@TiCT-Mxene) were synthesized through the electrodeposition of AuNPs on the TiCT-Mxene surface, providing active sites for the immobilization of the aptamer and amplifying the electrochemical signals. The excellent trans-cleavage activity of the CRISPR-Cas12a system was harnessed to cleave signal probes. The cleaved signal probes were enriched using an electrochemical signal instead of complicated target amplification steps before detection. Hence, we report a simplified detection process for amplifying electrochemical signals. Under optimal conditions, the aptamer sensor exhibited high sensitivity, acceptable stability, and reproducibility with a wide linear range from 1 pM to 10 μM (R = 0.9917) and an ultralow detection limit of 0.33 pM (S/N = 3). Therefore, we propose a novel strategy of CRISPR-Cas12a-based protein detection that opens a new window for the diagnostic applications of various biomarkers.
血管内皮生长因子（VEGF）在动脉粥样硬化中起重要作用，VEGF的检测对于心血管疾病的预防、监测和诊断至关重要。在此，首次开发了一种具有三重信号放大策略的新型“信号开-关-超开”夹心型适体传感器。基于捕获适体在内侧碱基上标记有亚甲基蓝（MB），利用成簇规律间隔短回文重复序列（CRISPR）-Cas12a偶联的电压富集来放大电化学信号。为了提高适体传感器的分析性能，通过在TiCT-Mxene表面电沉积金纳米颗粒（AuNPs）合成了金纳米颗粒@TiCT-Mxene（AuNPs@TiCT-Mxene），为适体的固定提供活性位点并放大电化学信号。利用CRISPR-Cas12a系统出色的反式切割活性来切割信号探针。在检测前，利用电化学信号而非复杂的靶标扩增步骤富集切割后的信号探针。因此，我们报道了一种简化的电化学信号放大检测过程。在最佳条件下，适体传感器表现出高灵敏度、可接受的稳定性和重现性，线性范围宽，为1 pM至10 μM（R = 0.9917），检测限超低，为0.33 pM（S/N = 3）。因此，我们提出了一种基于CRISPR-Cas12a的蛋白质检测新策略，为各种生物标志物的诊断应用打开了一扇新窗口。
