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

背景：许多生物传感器依赖于生物标志物与抗体或抗体模拟物如亲和分子、DARPins、纳米抗体和单链抗体结合具有高度的特异性。然而，结合是一种被动的事件，本身并不会导致容易检测到的信号，而是需要进行ELISA或开发阻抗生物传感器等过程。ELISA通常需要训练有素的技术人员和实验室环境，这使得它们不适合用于现场诊断或现场分析设备。目的：随着人们对更好、更可靠、更快速检测生物标志物的需求不断增加，本项目将设计合成酶开关，其中分析物与仿生抗体的结合直接破坏抑制物-酶的相互作用，导致可以直接用比色法或电化学法监测的化学反应。新颖性和及时性：以前报道过合成酶开关使用单一抗体仿制。然而，Jeuken和Walti实验室(与McFherson一起)最近开发了一种新的生物传感器结构，该结构依赖于两个亲和分子的同时结合。后者提供了灵敏度的增强，可与夹心ELISA法与直接ELISA法相媲美。因此，现在是时候将这一方法扩展到检测替代分析物，并测试这些合成酶开关在使用其他抗体构建物时是否也起作用，例如scFAB的纳米体。实验方法：DSLT可以获得高亲和力的纳米体和针对一些重要靶点的scFAB，用于检测例如立信或诊断埃博拉病毒。在这个博士项目中，我们将测试它们是否可以用于合成酶开关来开发敏感的生物传感器结构。

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.