Quintela Irwin A, Wu Vivian C H
Produce Safety and Microbiology Research Unit, US Department of Agriculture, Agricultural Research Services, Western Regional Research Center Albany California USA
RSC Adv. 2020 Sep 30;10(59):35765-35775. doi: 10.1039/d0ra06223e. eCollection 2020 Sep 28.
Immuno-based biosensors are a popular tool designed for pathogen screening and detection. The current antibody-based biosensors employ direct, indirect, or sandwich detection approaches; however, instability, cross-reactivity, and high-cost render them unreliable and impractical. To circumvent these drawbacks, here we report a portable sandwich-type bacteriophage-based amperometric biosensor, which is highly-specific to various Shiga toxin-producing (STEC) serogroups. Environmentally isolated and biotinylated bacteriophages were directly immobilized onto a streptavidin-coated screen-printed carbon electrode (SPCE), which recognized and captured viable target cells. Samples (50 μL) were transferred to these bacteriophage-functionalized SPCEs (12 min, room temp) before sequentially adding a bacteriophage-gold nanoparticle solution (20 μL), HO (40 mM), and 1,1'-ferrocenedicarboxylic acid for amperometric tests (100 mV s) and analysis (ANOVA and LSD, < 0.05). The optimum biotin concentration (10 mM) retained 94.47% bacteriophage viability. Non-target bacteria ( and Typhimurium) had delta currents below the threshold of a positive detection. With less than 1 h turn-around time, the amperometric biosensor had a detection limit of 10-10 CFU mL for STEC O157, O26, and O179 strains and values of 0.97, 0.99, and 0.87, respectively, and a similar detection limit was observed in complex matrices, 10-10 CFU g or mL with values of 0.98, 0.95, and 0.76, respectively. The newly developed portable amperometric biosensor was able to rapidly detect viable target cells at low inoculum levels, thus providing an inexpensive and improved alternative to the current immuno- and laboratory-based STEC screening methods.
基于免疫的生物传感器是一种用于病原体筛选和检测的常用工具。当前基于抗体的生物传感器采用直接、间接或夹心检测方法；然而，不稳定性、交叉反应性和高成本使其不可靠且不实用。为了克服这些缺点，我们在此报告一种便携式夹心型基于噬菌体的安培生物传感器，它对各种产志贺毒素大肠杆菌（STEC）血清群具有高度特异性。将环境分离并生物素化的噬菌体直接固定在链霉亲和素包被的丝网印刷碳电极（SPCE）上，该电极可识别并捕获活的靶细胞。将样品（50 μL）转移到这些噬菌体功能化的SPCE上（室温下12分钟），然后依次加入噬菌体 - 金纳米颗粒溶液（20 μL）、HO（40 mM）和1,1'-二茂铁二甲酸进行安培测试（100 mV s）和分析（方差分析和最小显著差异法，<0.05）。最佳生物素浓度（10 mM）可保持94.47%的噬菌体活力。非靶细菌（和鼠伤寒沙门氏菌）的电流变化低于阳性检测阈值。该安培生物传感器的周转时间不到1小时，对STEC O157、O26和O179菌株的检测限为10 - 10 CFU mL，分别为0.97、0.99和0.87，在复杂基质中也观察到类似的检测限，即10 - 10 CFU g或mL，分别为0.98、0.95和0.76。新开发的便携式安培生物传感器能够在低接种水平下快速检测活的靶细胞，从而为当前基于免疫和实验室的STEC筛选方法提供了一种廉价且改进的替代方法。
