Marchlewicz Kasper, Ostrowska Iga, Oszwałdowski Sławomir, Zasada Aleksandra, Ziółkowski Robert, Malinowska Elżbieta
Warsaw University of Technology, Faculty of Chemistry, The Chair of Medical Biotechnology, Noakowskiego 3, 00-664, Warsaw, Poland.
Faculty of Chemistry, The Chair of Analytical Chemistry, Warsaw University of Technology, 00-664, Warsaw, Poland.
Talanta. 2021 May 15;227:122161. doi: 10.1016/j.talanta.2021.122161. Epub 2021 Feb 8.
The presented study is focused on the development of electrochemical genosensor for detection of tox gene fragment of toxigenic Corynebacterium diphtheriae strain. Together with our previous studies it fulfils the whole procedure for fast and accurate diagnostic of diphtheria at its early stage of infection with the use of electrochemical methods. The developed DNA sensor potentially can be used in more sophisticated portable device. After the electrochemical stem-loop probe structure optimization the conditions for real asymmetric PCR (aPCR) product detection were selected. As was shown it was crucial to optimize the magnesium and organic solvent concentrations in detection buffer. Under optimal conditions it was possible to selectively detect as low as 20.8 nM of complementary stand in 5 min or 0.5 nM in 30 min with sensitivity of 12.81 and 0.24 1⋅μM respectively. The unspecific biosensor response was elucidated with the use of new electrode blocking agent, diethyldithiocarbamate. Its application in electrochemical genosensors lead to significant higher current values and the biosensor response even in conditions with magnesium ion depletion. The developed biosensor selectivity was examined using samples containing genetic material originated from a number of non-target bacterial species which potentially can be present in the human upper respiratory tract.
本研究聚焦于开发用于检测产毒白喉棒状杆菌菌株tox基因片段的电化学基因传感器。与我们之前的研究一起，它完成了使用电化学方法在感染早期对白喉进行快速准确诊断的整个过程。所开发的DNA传感器有可能用于更复杂的便携式设备。在对电化学茎环探针结构进行优化后，选择了用于检测真实不对称PCR（aPCR）产物的条件。结果表明，优化检测缓冲液中的镁和有机溶剂浓度至关重要。在最佳条件下，能够在5分钟内选择性地检测低至20.8 nM的互补链，或在30分钟内检测0.5 nM，灵敏度分别为12.81和0.24 1⋅μM。使用新型电极封闭剂二乙基二硫代氨基甲酸盐阐明了非特异性生物传感器响应。它在电化学基因传感器中的应用导致电流值显著更高，并且即使在镁离子耗尽的条件下生物传感器也有响应。使用含有源自一些可能存在于人类上呼吸道的非靶细菌物种的遗传物质的样品来检测所开发的生物传感器的选择性。
