Interdisciplinary Institute for Technological Innovation (3IT), Laboratory for Quantum Semiconductors and Photon-based BioNanotechnology, CNRS UMI-3463, Université de Sherbrooke, 3000, boul. de l'Université, Sherbrooke, Québec, Canada J1K 0A5; Department of Microbiology and Infectiology, Centre de Recherche du CHUS, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12th Avenue North Sherbrooke, Québec, Canada J1H 5N4.
Interdisciplinary Institute for Technological Innovation (3IT), Laboratory for Quantum Semiconductors and Photon-based BioNanotechnology, CNRS UMI-3463, Université de Sherbrooke, 3000, boul. de l'Université, Sherbrooke, Québec, Canada J1K 0A5.
Talanta. 2019 Jan 15;192:270-277. doi: 10.1016/j.talanta.2018.09.043. Epub 2018 Sep 14.
Antibiotic resistant bacteria have become a threat to world health. An advanced method of detection, based on matrix assisted laser desorption ionization time-of-flight mass spectroscopy can identify bacteria relatively rapidly, but it is not suitable to measure bacterial antibiotic resistance. Biosensors may be able to detect resistance by monitoring growth after capture on sensor surfaces but this option has not been addressed adequately. We have evaluated the growth of Escherichia coli after capture in 96 well microplates and observed that growth/capture efficiency was relatively similar for antibody-based techniques, but non-specific capture varied considerably. We confirm that neutravidin binds E. coli non-specifically, which limited its use with biotinylated antibodies or aptamers. Centrifugation enhanced bacterial growth/capture considerably, indicating that procedures enhancing the interaction between bacteria and surface-bound antibody have the potential to improve growth efficiency. Capture and growth required larger numbers of bacteria than capture and detection on biosensor surfaces. Previously, we reported that the minimum concentration of live E. coli required for initiating growth on a GaAs/AlGaAs biosensor was ~ 10 CFU/mL (Nazemi et al., 2018), and we speculated that this could be related to the poisonous effect of Ga- and As-ions released during dark corrosion of the biosensor, however in the present report we observed that the same minimum concentration of E. coli was required for growth in an ELISA plate. Thus, we argue that this limitation was related rather to bacterial inhibition by the capture antibodies. Indeed, antibodies at titres designed to capture bacteria inhibited bacterial growth when the bacteria were added to growth medium at titres less than 10 CFU/mL, indicating that antibodies may be responsible for the higher limits of sensitivity due to their potential to restrict bacterial growth. However, we did not observe E. coli release after 6 h following the capture indicating that these bacteria did not degrade antibodies.
耐药细菌已成为全球健康的威胁。基于基质辅助激光解吸电离飞行时间质谱的先进检测方法可以相对快速地识别细菌，但不适合测量细菌的抗生素耐药性。生物传感器可以通过监测在传感器表面捕获后的生长来检测耐药性，但这种方法尚未得到充分解决。我们评估了在 96 孔微孔板中捕获后大肠杆菌的生长情况，发现基于抗体的技术的捕获/生长效率相对相似，但非特异性捕获差异很大。我们证实链霉亲和素非特异性结合大肠杆菌，这限制了它与生物素化抗体或适体的使用。离心显著增强了细菌的生长/捕获效率，表明增强细菌与表面结合抗体之间相互作用的程序有可能提高生长效率。与在生物传感器表面捕获和检测相比，捕获和生长需要更多数量的细菌。以前，我们报道了在 GaAs/AlGaAs 生物传感器上启动生长所需的活大肠杆菌最小浓度为~10 CFU/mL（Nazemi 等人，2018 年），我们推测这可能与生物传感器暗腐蚀过程中释放的 Ga 和 As 离子的毒性有关，然而在本报告中，我们观察到大肠杆菌在 ELISA 板中生长所需的最小浓度相同。因此，我们认为这种限制与捕获抗体对细菌的抑制作用有关。事实上，当将细菌以低于 10 CFU/mL 的效价添加到生长培养基中时，设计用于捕获细菌的抗体效价会抑制细菌的生长，这表明抗体可能由于其限制细菌生长的潜力而导致更高的灵敏度限制。然而，我们没有观察到捕获后 6 小时内大肠杆菌的释放，这表明这些细菌没有降解抗体。
