King's College London, Pharmaceutical Science Division, Analytical Sciences Research Group, School of Biomedical & Health Sciences, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom.
Talanta. 2010 Apr 15;81(1-2):68-75. doi: 10.1016/j.talanta.2009.11.036. Epub 2009 Nov 18.
Fluorescent antibody protein (IgG) was attached to the surface of an integrated optical glass waveguide chip via specific binding to a covalently attached hapten and used as a substrate for the measurement of protease activities. Exposure of the optical chip to proteases resulted in digestion of the bound fluorescent antibody molecules and proportional decrease in the detectable fluorescence resulting from loss of fluorescence from the evanescent field. The bound fluorescent antibody protein was used as a unique universal protease substrate in which the combined biological activity and fluorescence signal were the basis of measurement. The action of proteases was monitored in real-time mode where the gradual decrease in evanescent fluorescence was recorded. The chip was regenerated by complete digestion of the antibody substrate by excess pepsin and recharged by incubation with a fresh sample of the labelled antibody. The biosensor was used to detect activity of several proteases including a bacterial protease preparation, Pronase E. The linear range of measurable Pronase E activity was from 0.03 to 2 units/mL. A measurement cycle took 40 min for samples with high protease concentration (>or=0.5 units/mL), when the concentration of the protease was less measurement times up to 100 min were required. The method demonstrates the principle of a new mode of real-time biosensing of proteases. The modular integrated optical glass waveguide biosensor system used in this study is compact and controlled by a laptop computer and could easily be miniaturised and utilized as a true probe device for detecting proteases with potential applications in a wide range of areas including research, clinical diagnostics, biotechnology processing and food and detergent manufacturing industries.
荧光抗体蛋白（IgG）通过与共价连接的半抗原的特异性结合附着在集成光学玻璃波导芯片的表面上，并用作测量蛋白酶活性的底物。将光学芯片暴露于蛋白酶会导致结合的荧光抗体分子被消化，并且由于消失场的荧光损失而导致可检测荧光的比例降低。结合的荧光抗体蛋白被用作独特的通用蛋白酶底物，其中组合的生物活性和荧光信号是测量的基础。以实时模式监测蛋白酶的作用，其中记录逐渐减少的消逝荧光。通过过量胃蛋白酶完全消化抗体底物并通过孵育标记的抗体的新鲜样品来再生芯片。该生物传感器用于检测几种蛋白酶的活性，包括细菌蛋白酶制剂 Pronase E。可测量 Pronase E 活性的线性范围为 0.03 至 2 单位/毫升。对于高蛋白酶浓度（>或= 0.5 单位/毫升）的样品，测量周期为 40 分钟，而当蛋白酶浓度较低时，测量时间最多可达 100 分钟。该方法展示了蛋白酶实时生物传感的新模式的原理。本研究中使用的模块化集成光学玻璃波导生物传感器系统紧凑，由笔记本电脑控制，很容易微型化并用作用于检测蛋白酶的真正探头设备，其潜在应用范围广泛，包括研究、临床诊断、生物技术加工以及食品和洗涤剂制造业。
