以場效電晶體為平台,建立一套新穎的偵測方法應用於中性小分子分析物之檢測

Abstract

高偵測靈敏度之場效電晶體於生物感測器之應用，主要是利用待測物 (蛋白質、DNA、抗體等) 吸附於通道表面時，待測物本身之帶電特性影響表面電位造成元件傳導性質之改變。然而針對部分不帶電荷之重要分子 (如:類固醇賀爾蒙)而言，如何設計一新穎的偵測系統並有效應用場效電晶體生物感測器偵測該類型待測物將是本研究之重點。本研究整合了蛋白質基因工程、奈米元件製作、電子工程與表面化學修飾等跨領技術，發展而成一套新穎的類固醇偵測平台。以成熟的分子生物技術為基礎，成功地取得人工重組酵素 (△5-3-酮基類固醇異構酵素；Art_KSI)，並以化學鍵結方法在不影響酵素功能的前提下，將帶電荷之探針分子 mA51；1, 5-EDANS 之化學衍生物)修飾在酵素上 (Art_KSI/mA51)。再經由酵素固定化的方法將此辨識類固醇的受體固定化於奈米元件表面。當類固醇待測物 (19-norandrostendione; 19-NA) 加入感測系統時，19-NA將與mA51競爭並趨使帶電荷之探針脫離活性區進而暴露於奈米線表面，此時便可利用場效電晶體的特性間接針對不帶電待測物做精準、快速的分析 (偵測極限可達10-15莫耳濃度)。同時也利用螢光與超快雷射的理論基礎進一步去證明酵素與小分子間的作用關係。此外，為避免複雜的生物檢測系統設計，本實驗亦成功以Open–Sandwich Enzyme-Linked Immunoassay (OS-ELISA) 並結合場效電晶體為技術平台來偵測另一不帶電荷分子 (Bisphenol A)。

The novel steroid-sensing model was constructed by intramolecular binding of a ligand through an alkyl linker [(-CH2)3] covalently bonded to a residue in the proximity of the active site of the protein. A genetically engineered △5-3-ketosteroid isomerase (KSI) was designed to conjugate uniquely with this ligand at its Cys-86 by the formation of disulfide bond. The steady-state protein-ligand binding, mediated by hydrophobic interactions, was confirmed by fluorescence spectroscopy and the identification of fluorophore-labeled peptides sequence using tandem mass spectrometry. The comparison of steady-state fluorescence spectra of various fluorophore-labeled proteins revealed that the emission characteristics varied with the change in environmental factors. Furthermore, the evaluation of fluorescence anisotropy decay of the fluorophore suggested the existence of the intramolecular protein-ligand binding interaction. The time-resolved fluorescence anisotropy studies of the different protein-ligand complexes yielded various values of anisotropy decay representing the degrees of freedom of the fluorophore in accordance to its location, inside or outside the steroid binding domain. When 19-norandrostendione was added to this protein-ligand system, a competitive binding between the ligand and the steroid was observed to confirm the feasibility of the design on steroid detection by engineered KSI. This protein-ligand system is utilized to overcome the intrinsic limitation of SiNW-FETs, because the general detection of SiNW-FETs is only feasible for charged analytes (such as protein, DNA, antibody, virus etc.). This engineered KSI protein was designed to function as the steroid acceptor, which was chemically modified by a carbon chain linked 1, 5-EDANS moiety, and further immobilized on the surface of silicon nanowire. In the presence of steroid, the negatively charged 1, 5-EDANS moiety, which presumably accommodates in the steroid-binding site, is expeled and exposes to the nanowire surface. The electrical response yielding from 1, 5-EDANS moiety is measured and the concentration is calculated accordingly. The sensitivity of this novel nano-bio-device can reach femto-molar level. This is the very first successful demonstration that a SiNW-FETs can detect non-charged analytes. Further medical applications can be evolved from this nano-biosensing system or same sort. At the same time, we also successfully demonstrate this novel noncharged analyte biosensing model on Ultra Thin Body Field Effect Transistors (UTB-FETs). On the other hand, a novel immunobioassay system (Open–Sandwich Enzyme-Linked Immunoassay; OS-ELISA) has been utilized to avoid this complex bio-and chem-design of non-charged analytes detection platform. In case, non-charged Bisphenol A has been successfully used to demonstrate the possibility of the application-based OS-ELISA on SiNW-FETs.