Protein engineering of synthetic enzyme switches for the next generation of biosensors

点击翻译按钮获取中文摘要

Background: Many biosensors rely on the concept that biomarkers bind with high specificity to antibodies or antibody mimetics such as Affimers, DARPins, Nanobodies and single-chain FAB (scFAB). However, binding is a passive event and in itself does not lead to an easily detectable signal, instead requiring processes such as ELISAs or the development of impedimetric biosensors. ELISAs generally require trained technicians and a lab environment, making them unsuitable for point-of-care diagnostics or 'in-the-field' analytical devices. Impedimetric and similar biosensor devices (with the exception of the very successful glucose biosensor) suffer from batch-to-batch variation, which have so far impaired commercial exploitation of such biosensors.Objectives: As there is an ever-increasing demand for better, more reliable, and faster detection of biomarkers, this project will engineer synthetic enzyme switches, where binding of an analyte to a biomimetic antibody directly disrupts an inhibitor-enzyme interaction, resulting in a chemical reaction that can be directly monitored colorimetric or electrochemically.Novelty & Timeliness: Synthetic enzyme switches have previously been reported using a single antibody mimetics. However, the Jeuken and Walti labs (together with McPherson) have recently developed a novel biosensor construct that relies on the simultaneous binding of two Affimers. The latter provides an enhancement in sensitivity, comparable to that of a sandwich ELISA versus a direct ELISA assay. It is thus now timely to extend this methodology for the detection of alternative analytes and test if these synthetic enzyme switches also function when using other antibody constructs such as Nanobodies of scFAB.Experimental Approach: DSLT has access to high-affinity Nanobodies and scFAB against a number of important targets for the detection of, for instance, Risin or diagnosis of Ebola. In this PhD project, we will test whether these can be used in synthetic enzyme switches to develop sensitive biosensor constructs.