Heine Viktoria, Kremers Tom, Menzel Nora, Schnakenberg Uwe, Elling Lothar
Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Pauwelsstraße 20, D-52074 Aachen, Germany.
Chair of Micro- and Nanosystems and Institute of Materials in Electrical Engineering 1, RWTH Aachen University, Sommerfeldstraße 24, D-52074 Aachen, Germany.
ACS Sens. 2021 Mar 26;6(3):1003-1011. doi: 10.1021/acssensors.0c02206. Epub 2021 Feb 17.
Monitoring glycosyltransferases on biosensors is of great interest for pathogen and cancer diagnostics. As a proof of concept, we here demonstrate the layer-by-layer immobilization of a multivalent neoglycoprotein (NGP) as a substrate for a bacterial fucosyltransferase (FucT) and the subsequent binding of the fucose-specific lectin (AAL) on an electrochemical impedance spectroscopy (EIS) sensor. We report for the first time the binding kinetics of a glycosyltransferase in real-time. Highly stable EIS measurements are obtained by the modification of counter and reference electrodes with polypyrrole: polystyrene sulfonate (PPy:PSS). In detail, the -acetyllactosamine (LacNAc)-carrying NGP was covalently immobilized on the gold working electrode and served as a substrate for the FucT-catalyzed reaction. The LacNAc epitopes were converted to Lewis (Le) and detected by AAL. AAL binding to the Le epitope was further confirmed in a lectin displacement and a competitive lectin binding inhibition experiment. We monitored the individual kinetic processes via EIS. The time constant for covalent immobilization of the NGP was 653 s. The FucT kinetics was the slowest process with a time constant of 1121 s. In contrast, a short time constant of 11.8 s was determined for the interaction of AAL with the modified NGPs. When this process was competed by 400 mM fucose, the binding was significantly slowed down, as indicated by a time constant of 978 s. The kinetics for the displacement of bound AAL by free fucose was observed with a time constant of 424 s. We conclude that this novel EIS biosensor and the applied workflow has the potential to detect FucT and other GT activities in general and further monitor protein-glycan interactions, which may be useful for the detection of pathogenic bacteria and cancer cells in future biomedical applications.
在生物传感器上监测糖基转移酶对于病原体和癌症诊断具有重大意义。作为概念验证，我们在此展示了一种多价新糖蛋白（NGP）作为细菌岩藻糖基转移酶（FucT）底物的逐层固定化，以及随后岩藻糖特异性凝集素（AAL）在电化学阻抗谱（EIS）传感器上的结合。我们首次实时报道了糖基转移酶的结合动力学。通过用聚吡咯：聚苯乙烯磺酸盐（PPy:PSS）修饰对电极和参比电极，获得了高度稳定的EIS测量结果。详细来说，携带N-乙酰乳糖胺（LacNAc）的NGP被共价固定在金工作电极上，并作为FucT催化反应的底物。LacNAc表位被转化为Lewis（Le）表位，并通过AAL进行检测。在凝集素置换和竞争性凝集素结合抑制实验中，进一步证实了AAL与Le表位的结合。我们通过EIS监测了各个动力学过程。NGP共价固定的时间常数为653秒。FucT动力学是最慢的过程，时间常数为1121秒。相比之下，AAL与修饰后的NGP相互作用的时间常数较短，为11.8秒。当该过程被400 mM岩藻糖竞争时，结合明显减慢，时间常数为978秒。观察到游离岩藻糖取代结合的AAL的动力学，时间常数为424秒。我们得出结论，这种新型EIS生物传感器和所应用的工作流程有可能检测FucT和其他一般的糖基转移酶活性，并进一步监测蛋白质-聚糖相互作用，这在未来的生物医学应用中可能对检测病原菌和癌细胞有用。
