[學研計畫] 分子生物電子平台系統整合與應用( I )

Abstract

高通量篩選為藥物開發一個不可或缺的核心。我們提出”蛋白質電晶體酵素分析”(pro-Ten)的平台，結合單分子平台的敏感性及多樣性進一步增加高通量篩選的靈敏性並簡化藥物開發的流程。 酵素的抑制分子可為藥物開發的起點。檢測方式及高通量篩選往往是決定新藥開發成功的關鍵。不受基質濃度及目標物特性影響的極敏感一般化的檢驗方式有助於簡化藥物開發的流程。 我們建立了單分子蛋白質電晶體的及時單分子酵素活性檢測平台。以抗體為基礎的蛋白質電晶體(pro-T)是由抗體與鍵結在電極上的奈米金粒子建構而成。Pro-T對結構敏感的特性提供了從單分子層次監控酵素活性的機會。 Pro-T可以藉由在酵素上鍵結而成為酵素電晶體。鍵結beta-galactosidase使此pto-T具有偵測水解乳糖的功能(pro-Tgal)。Pro-Tgal具有監控酵素水解反應時的基質結合、結構改變、水解並釋放末端糖、釋出半乳糖以及回復酵素原來結構。Pro-Tgal藉由電容曲線的波型及強度及時辨別非基質、基質、以及抑制物的結合。相同的敏感性及專一性也適用在其他酵素。 預期達成的目標: 1. 建立高通量篩選平台以簡化藥物開發流程。將單分子偵測系統結合單分子螢光偵測方式。 2. 研究單分子酵素反應。 3. 將此技術應用於生物電子相關領域。此蛋白質電晶體可以進一步應用在單電池光電以及生物電路系統。

High throughput screening (HTS) is an integral part of drug discovery industry. Here we propose a pro-T-based enzyme assay (pro-Ten) platform that enables HTS with single molecule sensitivity and versatility thus will facilitate the process and enhance the sensitivity of drug discovery. Enzymes are the most attractive targets in human diseases. Inhibitory molecules may serve as the starting points for drug discovery. The leads obtained from HTS represent the starting points for medicinal chemistry. Assay development and HTS often determine the successful flow of drug development. An ultra-sensitive and universal assay independent of substrate concentration and target properties would be beneficial in these steps. A single-molecule and real-time enzyme activity measuring platform based on single molecule protein transistor is established in our lab. An antibody-based protein transistor (pro-T) is fabricated through the specific recognition between antibody and gold nanoparticles with gold nanoparticles attaching to electrodes. The structure-sensing ability of pro-T provides an opportunity to monitor enzymatic action at single molecular level. The pro-T can be transformed to an enzyme transistor by binding to an enzyme-conjugated secondary antibody. Binding of beta-galactosidase-conjugated secondary antibody enables pro-T to sense the enzymatic hydrolysis of lactose (pro-Tgal). Pto-Tgal is able to monitor the enzymatic hydrolysis of -gal includes substrate binding, structural change of enzyme, hydrolysis and release of the end sugar, release of galactose, and recover of native enzyme structure. Pro-Tgal instantaneously distinguishes non-substrate, substrate, and inhibitor by the shape and intensity of the conductivity. Incorporation of other enzymes, such as glucose oxidase, horse relish peroxidase, pepsin, also exhibited single molecule sensitivity and specificity. We propose to establish an HTS platform to accelerate the drug discovery efficiency. The specific aims of this proposal are: 1. To develop an HTS platform to facilitate drug discovery process. We will combine the single molecule detection of our system (detecting substrate binding and structural change of protein) with single molecule fluorescence detection (detection product conversion and distance between chromophores). Microfluidic and multichannel sampling system will be amended. 2. To investigate single molecule enzyme reaction. The advantage of pro-Ten is the nano-second timescale to observe enzymatic reaction in real time. The single molecule FRET system detects “snapshot” of reaction, thus is also called “single molecule ensemble experiment”. This will be the first opportunity to verify most enzymatic hypotheses based on Mechalis-Menten kinetics. Additionally, kinetics of protein folding will be investigated. 3. To explore possible applications in bioelectronics. Incorporating into HTS system is not the only option for application of pro-T. Single cell photovoltics and integrated bio-circuitry will be investigated. Additionally, sensors in non-conventional environment, such as air or vacuum, will be investigated. Pro-T is most suitable to apply for biological detection. X-ray crystallography will be performed to monoclonal antibody and gold nanoparticle conjugate. This is to explore the possibility of extracting extra information for the bio-nano interface from structural data. We expect to generate high quality patents attractive to pharmaceutical society. High impact publications in Single molecule enzyme kinetics, protein folding, structural biology will be generated.