奈米力學建構之感測器應用於生物分子辨識之研究

Abstract

生物分子之辨識(biomolecular recognition)是生命體之自然特性，存在於核酸雜交反應、蛋白質與蛋白質交互作用、核酸與蛋白質交互作用、脂質與蛋白質交互作用、酵素與基質反應、細胞與配體鍵結等過程中，探究生物分子間辨識之特性有助於促進新的藥物之研發、生物技術方法之提升、新的治療診斷技術之開發。本論文研究已成功地將抗原與抗體間分子辨識之特性轉換至微型懸臂梁之奈米力學響應，透過晶片表面之化學修飾與生物分子固定化技術，以及感測元件之微製造技術，由生物膜引入奈米力學之換能機制以建立了一個可即時、免螢光、可定量檢測之生物感測器平台，應用於生物分子辨識之分析與生物醫學之診斷上。 本論文研究，係利用奈米力學建構之生物感測器，首先成功地應用於疾病標記C反應蛋白(C-reactive protein)之即時檢測，此微型生物感測晶片與系統可定量檢測C反應蛋白於具臨床意義之濃度範圍(1-500 µg/mL)，並且實驗之結果具有高度重複性(repeatability, 7%)，以使得本系統可潛在成為急性發炎(inflammatory)或心血管疾病(cardiovascular disease)之臨床診斷技術。再者，電場操控於抗原－抗體複合物之分離以使感測表面再生(sensing surface regeneration)之技術已首次成功地引入於奈米力學建構之生物檢測系統中，本研究係施加低頻之交替電場於晶片感測面上以取代傳統感測面暴露於極酸環境(pH 2.5)的方法，以使本系統達成可重複檢測且微型化之目的。同時，電場操控於感測表面再生之技術相較於傳統方法不但可以減低蛋白質活性之喪失，也能提供相對高的感測表面再生效率，而且將使整個檢測系統能進一步微小化，並可潛在地解決目前生物感測器轉為植入式晶片之關鍵瓶頸問題。 運用純熟之半導體工業技術，本研究更進一步成功地將無線傳輸CMOS元件整合於奈米力學建構之生物感測器中，並用以C反應蛋白之遠端即時地定量檢測，整個生物檢測系統預期將可微小化至CD隨身聽的大小，達成可攜式的目的，而可望應用於災區，如颶風與海嘯，或疫區之就地診斷，並由於微懸臂梁感測器與無線CMOS元件於材料與製程上具有高度相容性，故將可預期使整個系統整合於單一晶片上(system-on-chip)，而達到更進一步地微小化。 總結，本研究中奈米力學建構之生物感測器已展現其廣泛之可應用性，相容於微電子技術之高度整合性，以及未來將可望朝向無線感測網絡(wireless sensor networks)之發展性。

Biomolecular recognition is a natural characteristic in DNA hybridization, protein-protein interaction, DNA-protein interaction, lipid-protein interaction, enzyme-substrate reaction, and cell-ligand binding. Exploring the biomolecular recognition event facilitates the discovery of new drugs, biotechnological methods, and materials for therapeutics and diagnostics. This work has shown antibody-antigen recognition into a direct nanomechanical response of microfabricated cantilever beams. With the bottom-up technology in chemical surface treatment and biomolecular functionality, and the top-down microfabrication on physical devices, the biofilm-induced nanomechanical transduction establishes a biosensor platform in real-time, label-free, and quantitative analysis on biomolecular recognition and thus diagnostics. First of all, this nanomechanics-based biosensor demonstrates the real-time and in-vitro quantitative detection of disease-related C-reactive protein (CRP). A wide range of clinically relevant CRP concentrations from 1 to 500 µg/mL have been successfully measured with the repeatability of within 7 %, making this biosensor a potential diagnostic technique for inflammatory events or cardiovascular disease. In addition to successful biosensing, sensing surface regeneration is a washing process prior to re-use of biosensors in dissociation of antigen molecules out of anchored antibody. To cater to portability and miniaturization of biosensors in point-of-care applications, the physically electrical regeneration in replacement of highly acidic washing with no additional, sizable containers has been proven on separation of antibody-antigen complexes for nanomechanics-based biosensors. The advantageous feature of the electrical technique over the conventional treatment is evident in long-standing protein activity, providing a relatively high efficiency in dissociation, and miniaturization in entire bioassay system. Leveraging the mature semiconductor industry technology, this work successfully integrates the microcantilever biosensor with a wireless CMOS device for C-reactive protein detection and electrical regeneration. The entire bioassay system is expected in miniaturization to a size as small as a commercial CD player, and thus to be portable. Therefore, the portable biosensor system allows to be applied for on-site diagnosis to a remote area beyond hospital, for instances, in cases of hurricane and Tsunami. Owing to high compatibility of the microcantilever and standard-CMOS wireless device in terms of process and materials, the System-On-Chip (SOC)-based biosensor is highly expected and potentially miniaturized to a grain size for future implanted health monitoring. In summary, the nanomechanics-based immunoassay biosensor exhibits its broad applicability, effectiveness, high microelectronics integration, promising wireless sensor networks as well.