State Key Laboratory for Organic Electronics and Information Displays, Jiangsu Key Laboratory for Biosensors, Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, Nanjing 210023, China.
Anal Chem. 2023 May 2;95(17):6810-6817. doi: 10.1021/acs.analchem.2c05165. Epub 2023 Apr 19.
Membrane protein dimerization regulates numerous cellular biological processes; therefore, highly sensitive and facile detection of membrane protein dimerization are very crucial for clinical diagnosis and biomedical research. Herein, a colorimetric detection of Met dimerization on live cells via smartphone for high-sensitivity sensing of the HGF/Met signaling pathway was developed for the first time. The Met monomers on live cells were recognized by specific ligands (aptamers) first, and the Met dimerizations triggered the proximity-ligation-assisted catalytic hairpin assembly (CHA) reaction to generate large amounts of G-quadruplex (G4) fragments which can further combine hemin to form G4/hemin DNAzymes possessing the horseradish-peroxidase-like catalytic activity for catalyzing the oxidation of ABTS by HO and producing the colorimetric signal (i.e., color change). The colorimetric detection of Met on live cells was then achieved by image acquisition and processing via a smartphone. As a proof-of-principle, the HGF/Met signaling pathway based on Met-Met dimerization was facile monitored, and the human gastric cancer cells MKN-45 with natural Met-Met dimers were sensitively tested and a wide linear working range from 2 to 1000 cells with a low detection limit of 1 cell was obtained. The colorimetric assay possesses a good specificity and high recovery rate of MKN-45 cells spiked in peripheral blood, which indicates that the proposed colorimetric detection of Met dimerization can be used for convenient observation of the HGF/Met signaling pathway and has extensive application prospects in point-of-care testing (POCT) of Met-dimerization-related tumor cells.
膜蛋白二聚化调节着许多细胞生物学过程；因此，对膜蛋白二聚化的高灵敏度和简便检测对于临床诊断和生物医学研究至关重要。在此，首次开发了一种通过智能手机对活细胞中 Met 二聚化进行比色检测的方法，用于高灵敏度检测 HGF/Met 信号通路。活细胞上的 Met 单体首先被特异性配体（适体）识别，Met 二聚化触发邻近连接辅助的催化发夹组装（CHA）反应，产生大量 G-四链体（G4）片段，这些片段可以进一步与血红素结合形成具有辣根过氧化物酶样催化活性的 G4/血红素 DNA 酶，用于催化 HO 氧化 ABTS 并产生比色信号（即颜色变化）。然后通过智能手机进行图像采集和处理来实现活细胞上 Met 的比色检测。作为原理验证，基于 Met-Met 二聚化的 HGF/Met 信号通路被简便地监测，并且天然具有 Met-Met 二聚体的人胃癌细胞 MKN-45 被灵敏地检测到，并获得了从 2 个到 1000 个细胞的宽线性工作范围和低至 1 个细胞的检测限。比色测定具有良好的特异性和外周血中掺入的 MKN-45 细胞的高回收率，这表明所提出的 Met 二聚化比色检测可用于方便地观察 HGF/Met 信号通路，并在与 Met-二聚化相关的肿瘤细胞的即时检测（POCT）中具有广泛的应用前景。
