Liu Mengyue, Zhang Fengjuan, Dou Shouyi, Sun Jiashuai, Vriesekoop Frank, Li Falan, Guo Yemin, Sun Xia
College of Agricultural Engineering and Food Science, Shandong University of Technology, No. 266 Xincun Xilu, Zibo 255049, Shandong, China.
Shandong Provincial Engineering Research Center of Vegetable Safety and Quality Traceability, No. 266 Xincun Xilu, Zibo 255049, Shandong, China.
Anal Methods. 2023 Mar 30;15(13):1661-1667. doi: 10.1039/d2ay01822e.
In this work, aptamers against with better performance were obtained cell systematic evolution of ligands by exponential enrichment (cell-SELEX) and dissociation constants (Kd) of aptamers were estimated to range from 133.87 to 199.44 nM. Furthermore, the selected aptamer was employed for label-free colorimetric detection of using gold nanoparticles (AuNPs) with peroxidase-like activity to catalyze the oxidation of tetramethylbenzidine (TMB) by hydrogen peroxide (HO) to produce color development. This colorimetric apta-assay started with an aptamer-bacteria binding step, and the concentration of residual aptamers after binding depended on the amount of target bacteria. Then, the amount of separated residual aptamers determined the degree of cetyltrimethylammonium bromide (CTAB)-inhibited catalytic activity of AuNPs, which resulted in a color change from dark blue to light blue. Owing to the excellent peroxidase activity of AuNPs, they could emit strong visible color intensity in less than 1 minute to improve visual detection sensitivity. Under optimized conditions, the sensitivity of detection was 5 × 10 CFU mL visually and 75 CFU mL using the UV-vis spectrum with a linear range from 5 × 10 to 1 × 10 CFU mL. And it had shown a good recovery rate in real samples of water, juice and milk compared with classical counting methods.
在本研究中，通过指数富集配体的细胞系统进化技术（cell-SELEX）获得了性能更佳的抗[具体物质未明确]适配体，并估计适配体的解离常数（Kd）范围为133.87至199.44 nM。此外，所筛选出的适配体被用于利用具有过氧化物酶样活性的金纳米颗粒（AuNPs）对[具体物质未明确]进行无标记比色检测，该活性可催化过氧化氢（HO）氧化四甲基联苯胺（TMB）以产生显色反应。这种比色适配体分析始于适配体与细菌的结合步骤，结合后残留适配体的浓度取决于目标细菌的数量。然后，分离出的残留适配体的量决定了十六烷基三甲基溴化铵（CTAB）对AuNPs催化活性的抑制程度，这导致颜色从深蓝色变为浅蓝色。由于AuNPs具有出色的过氧化物酶活性，它们能够在不到1分钟的时间内发出强烈的可见颜色强度，从而提高视觉检测灵敏度。在优化条件下，视觉检测灵敏度为5×10 CFU/mL，使用紫外可见光谱时为75 CFU/mL，线性范围为5×10至1×10 CFU/mL。并且与传统计数方法相比，在水、果汁和牛奶的实际样品中显示出良好的回收率。
