發展新型生物奈米感測技術於蛋白質之研究：C-反應蛋白之結構和構型分析與分子間交互作用

為發展一C反應蛋白感測晶片作為心血管粥狀硬化症與冠狀動脈疾病的臨床檢測，本研究使用原子力顯微術與雙偏極化干涉術進行C反應蛋白表面微結構的量測，並定量五聚體蛋白三維結構與x光結晶學之結果比較。另外，利用此C反應蛋白感測晶片配合雙偏極化干涉術分析，經由表面厚度、單層密度與表面濃度等結構參數，分析C反應蛋白於不同pH值條件的構形變化，結果顯示在中性環境pH值6.0到7.0為五聚體的構形；酸性環境pH值4.0下，C反應蛋白分子薄膜解離為單體的CRP；在pH值2.0時則發現部份的單體的CRP會產生聚集作用；而在鹼性環境pH值8.0和10.0下，則只存在單體的C反應蛋白結構。此發現將有助於C反應蛋白生物性功能與構形的研究。另一方面，了解抗體-抗原間的交互作用可作為生物檢測的依據，本研究藉由多單體的C反應蛋白與單株C反應蛋白抗體間的系統，計算其中蛋白動力學與結合常數的化學量論，並驗證小分子化學（鈣離子）與C反應蛋白分子薄膜的結合試驗。最後，開發一新型的電子轉移生物結合分子OT-3C，提供電化學生物晶片的應用，研究成果更發現此化學分子可將銦錫氧化物基材的導電性提高數倍之上，大大增加C反應蛋白感測晶片的發展性。

關鍵字

雙偏極化干涉術；原子力顯微術；導電性原子力顯微術； C反應蛋白；蛋白構形變化；抗體-抗原試驗；蛋白動力學；化學量論

並列摘要

In order to develop the C-reactive protein (CRP) sensor chips for clinical detection of atherosclerosis and coronary heart disease, we used an atomic force microscope (AFM) and a dual polarization interferometric (DPI) biosensor to probe the surface ultrastructure and to measure the dimensions of CRP. The quantitative measurements of the dimensions of the protein were basically corresponds to that previously obtained from the structure of CRP determined by X-ray crystallography. Moreover, the DPI sensor was used to investigate the structure and conformational changes by structural parameters (thickness, layer density, surface mass loading concentration) under different physiological condition. This current work revealed that the conformations of CRP monolayer in the acidic region were existed two forms include of the monomeric CRP at pH 4.0 and a “partial” subunits’ aggregation at pH 2.0. On the other hands, only compact monomeric form was observed in the pH 8.0 and 10.0 alkaline region. These informations were useful to discover the biological role in the function of homopentamer CRP. For the purposed of biosensing, understanding the interaction kinetics between a ‘homopolyvalent’ antigen (Ag) and a monoclonal antibody (Ab). A model system, which uses a monoclonal Ab against a homopentameric Ag, CRP, was presented with principle and experiments for the study of the interactions between an Ab and an Ag that had multiple identical epitopes. This allowed evaluation of the dissociation constant (KD) and of the binding stoichiometry. In the final parts, the novel electron transfer type biolinker called 5"-formyl-5-carboxylic acid-2,2',5',2'-terthiophene, OT-3C, was designed into an EC sensor chip development. The aldehyde (-CHO) and carboxyl (-COOH) head groups could provide a higher potential for biomolecules immobilization and modification of hydroxyl ITO surfaces, which conductivity increased several folds.

並列關鍵字

DPI ； AFM ； cAFM ； CRP ； conformational change ； Ab–Ag assay ； kinetic ； stoichimetry

參考文獻

Buckwalter, J.A., Martin, J., Mankin, H.J., 2000. Synovial joint degeneration and the syndrome of osteoarthritis. Instr Course Lect 49, 481-489.

Agrawal, A., Shrive, A.K., Greenhough, T.J., Volanakis, J.E., 2001. Topology and structure of the C1q-binding site on C-reactive protein. J. Immunol. 166, 3998-4004.

Ahluwalia, A., Rossi, D.D., Schirone, A., 1992. Antigen recognition properties of antibody monolayers.Thin Solid Films 210 (2), 726-729.

Armstrong, J., Salacinski, H.J., Mu, Q., Seifalian, A.M., Peel, L., Freeman, N., Holt, C.M., Lu, J.R., 2004. Interfacial adsorption of fibrinogen and its inhibition by RGD peptide: a combined physical study. J. Phys.: Condens. Matter 16, S2483-S2491.

Arwin, H., 2000. Ellipsometry on thin organic layers of biological interest: characterization and applications. Thin Solid Films 377/378, 48-56.

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發展新型生物奈米感測技術於蛋白質之研究：C-反應蛋白之結構和構型分析與分子間交互作用

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