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Electrochemical Impedance Spectroscopy Biosensor Enabling Kinetic Monitoring of Fucosyltransferase Activity
ACS Sensors ( IF 8.2 ) Pub Date : 2021-02-17 , DOI: 10.1021/acssensors.0c02206 Viktoria Heine 1 , Tom Kremers 2 , Nora Menzel 1, 2 , Uwe Schnakenberg 2 , Lothar Elling 1
ACS Sensors ( IF 8.2 ) Pub Date : 2021-02-17 , DOI: 10.1021/acssensors.0c02206 Viktoria Heine 1 , Tom Kremers 2 , Nora Menzel 1, 2 , Uwe Schnakenberg 2 , Lothar Elling 1
Affiliation
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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 Aleuria aurantia 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 N-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 Lewisx (Lex) and detected by AAL. AAL binding to the Lex 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.
中文翻译：
电化学阻抗光谱生物传感器实现岩藻糖基转移酶活性的动力学监测。
监测生物传感器上的糖基转移酶对于病原体和癌症诊断非常重要。作为概念的证明，我们在此证明了多价新糖蛋白（NGP）作为细菌岩藻糖基转移酶（FucT）的底物的逐层固定以及随后的岩藻糖特定的Aleuria aurantia lectin（AAL）结合电化学阻抗谱（EIS）传感器。我们首次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）修改对电极和参比电极，可以获得高度稳定的EIS测量值。详细来说，N携带乙酰乙酰乳糖胺（LacNAc）的NGP共价固定在金电极上，并用作FucT催化反应的底物。将LacNAc表位转化为Lewis x（Le x），并通过AAL检测。AAL绑定到Le x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该表位。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 s。FucT动力学是最慢的过程，时间常数为1121 s。相反，对于AAL与修饰的NGP的相互作用，确定了11.8 s的短时间常数。当此过程与400 mM岩藻糖竞争时，结合时间明显延长，如978 s的时间常数所示。观察到游离岩藻糖置换结合的AAL的动力学，时间常数为424 s。我们得出的结论是，这种新颖的EIS生物传感器及其应用的工作流程具有检测总体上FucT和其他GT活性并进一步监控蛋白质-聚糖相互作用的潜力，
更新日期：2021-03-26
中文翻译：
电化学阻抗光谱生物传感器实现岩藻糖基转移酶活性的动力学监测。
监测生物传感器上的糖基转移酶对于病原体和癌症诊断非常重要。作为概念的证明，我们在此证明了多价新糖蛋白（NGP）作为细菌岩藻糖基转移酶（FucT）的底物的逐层固定以及随后的岩藻糖特定的Aleuria aurantia lectin（AAL）结合电化学阻抗谱（EIS）传感器。我们首次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）修改对电极和参比电极，可以获得高度稳定的EIS测量值。详细来说，N携带乙酰乙酰乳糖胺（LacNAc）的NGP共价固定在金电极上，并用作FucT催化反应的底物。将LacNAc表位转化为Lewis x（Le x），并通过AAL检测。AAL绑定到Le x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该表位。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 s。FucT动力学是最慢的过程，时间常数为1121 s。相反，对于AAL与修饰的NGP的相互作用，确定了11.8 s的短时间常数。当此过程与400 mM岩藻糖竞争时，结合时间明显延长，如978 s的时间常数所示。观察到游离岩藻糖置换结合的AAL的动力学，时间常数为424 s。我们得出的结论是，这种新颖的EIS生物传感器及其应用的工作流程具有检测总体上FucT和其他GT活性并进一步监控蛋白质-聚糖相互作用的潜力，
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