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

背景资料：许多生物传感器依赖于生物标志物以高特异性结合抗体或抗体模拟物（如亲和聚体、DARPin、纳米抗体和单链FAB（scFAB））的概念。然而，结合是一种被动事件，本身不会导致容易检测的信号，而是需要诸如ELISA或阻抗生物传感器的开发等过程。ELISA通常需要经过培训的技术人员和实验室环境，使其不适合即时诊断或“现场”分析设备。阻抗和类似的生物传感器装置（除了非常成功的葡萄糖生物传感器）遭受批次间变化，这迄今为止已经损害了这种生物传感器的商业开发。随着对更好、更可靠、更快的生物标志物检测的需求不断增加，该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了底物-酶的相互作用，导致化学反应，该化学反应可被比色或电化学直接监测。新奇和及时性：先前已经报道了使用单一抗体模拟物的合成酶开关。然而，Jeuken和Walti实验室（与麦克弗森一起）最近开发了一种新的生物传感器构建体，该构建体依赖于两个Affimers的同时结合。后者提供了灵敏度的增强，与夹心ELISA相对于直接ELISA测定的灵敏度相当。因此，现在是时候将这种方法用于检测替代分析物，并测试这些合成酶开关是否在使用其他抗体构建体（如scFAB.Experimental Approach的纳米抗体）时也起作用了。DALDESS已经获得了针对许多重要靶标的高亲和力纳米抗体和scFAB，用于检测例如Risin或诊断埃博拉病毒。在这个博士项目中，我们将测试这些是否可以用于合成酶开关，以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.