Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-111, Tehran, Iran.
Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Science, P.O. Box 1411713137, Tehran, Iran.
Mikrochim Acta. 2018 Mar 12;185(4):217. doi: 10.1007/s00604-018-2749-y.
The authors describe an electrochemical assay for fast detection of Escherichia coli (E. coli). It is based on a dual signal amplification strategy and the use of a screen-printed carbon electrode (SPCE) whose surface was modified with a polyaniline (PANI) film and gold nanoparticles (AuNPs) via cyclic voltammetry (CV). In the next step, avidin was covalently immobilized on the PANI/AuNP composite on the SPCE surface. Subsequently, the biotinylated DNA capture probe was immobilized onto the PANI/AuNP/avidin-modified SPCE by biotin-avidin interaction. Then, DNA of E.coli, digoxigenin-labeled DNA detector probe and anti-digoxigenin-labeled horseradish peroxidase (HRP) were placed on the electrode. 3,3',5,5'-Tetramethylbenzidine (TMB) and HO solution were added and the CV electrochemical signal was generated at a potential of -0.1 V (vs. Ag/AgCl) and a scan rate 50 mV.s. The assay can detect 4 × 10 to 4 CFU of E. coli without DNA amplification. The biosensor is highly specific over other pathogens including Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis, Staphylococcus haemolyticus and Pseudomonas aeruginosa. It can be concluded that this genosensor has an excellent potential for rapid and accurate diagnosis of E.coli inflicted infections. Graphical Abstract Schematic of an electrochemical E. coli genosensor based on sandwich assay on a polyaniline/gold nanoparticle-modified screen printed carbon electrode (SPCE). The biosensor can detect 4 × 10 to 4 CFU of E. coli without DNA amplification.
作者描述了一种用于快速检测大肠杆菌（E. coli）的电化学分析方法。它基于双信号放大策略和使用通过循环伏安法（CV）在丝网印刷碳电极（SPCE）表面修饰的聚苯胺（PANI）膜和金纳米粒子（AuNPs）。在下一步中，通过共价键将亲和素固定在 SPCE 表面的 PANI/AuNP 复合材料上。随后，通过生物素-亲和素相互作用将生物素化的 DNA 捕获探针固定在 PANI/AuNP/亲和素修饰的 SPCE 上。然后，将大肠杆菌的 DNA、地高辛标记的 DNA 检测探针和抗地高辛标记的辣根过氧化物酶（HRP）放置在电极上。添加 3,3',5,5'-四甲基联苯胺（TMB）和 HO 溶液，并在 -0.1 V（相对于 Ag/AgCl）的电势和 50 mV.s 的扫描速率下产生 CV 电化学信号。该测定法无需 DNA 扩增即可检测到 4×10 到 4 CFU 的大肠杆菌。该生物传感器对其他病原体（包括肺炎克雷伯菌、奇异变形杆菌、粪肠球菌、溶血葡萄球菌和铜绿假单胞菌）具有高度特异性。可以得出结论，这种基因传感器具有快速准确诊断大肠杆菌感染的巨大潜力。
