近年來，由於DNA晶片技術的成熟發展，導致蛋白質晶片應用於臨床醫學檢測疾病上的地位也越來越重要。然而在製作蛋白質晶片過程中，利用自組裝單分子層來固定蛋白質已經是目前蛋白質固定技術的主流之一。但是儘管不斷的努力，長碳鏈硫醇 (thiol) 的單分子層會因為氧化作用形成disulfide而導致沈澱在基材上產生結構障礙，使得蛋白質無法固定於基材上，而導致蛋白質晶片的靈敏度大幅下降。因此本研究希望利用短鏈自組裝單分子層4,4- DTBA (4,4- Dithiodibutyric acid, disulfide) 來做為蛋白質和基材之間的橋樑，直接消除結構障礙的問題，使得蛋白質能完全固定於基材表面，並同時利用protein A來達到降低非特定吸附所導致的靈敏度下降的問題。另外本研究還探討了多種溶液對蛋白質晶片的影響，實驗中清洗的過程，利用PBST(PBS + 5% Tween20)來降低NHS esters水解的問題。並探討不同比例 (3:1和5:2)EDC/NHS的混合和利用不同溶劑(DI water, DI water + 0.1M MES和alcohol)來溶解EDC對於蛋白質固定技術上的影響做更進一步的探討與了解。實驗中的定性主要是利用親疏水角量測儀(contact angle goniometry), 原子力顯微鏡(atomic force microscopy,AFM), 傅必略紅外線光譜儀(Fourier transform infrared spectroscopy, FTIR) 和橢偏儀(ellipsometry)。最後利用螢光protein A 作螢光強度分析並利用11- MUA (11-Mercapto-undecanoic acid, thiol) 作為實驗的對照組. 從實驗結果知道4,4- DTBA可以成功地用來作為蛋白質晶片的單分子層，PBST能有效低降低NHS esters的水解問題。最後酒精溶解EDC的.3:1混合比例的EDC/NHS在反應時間4小時能達到最佳的螢光強度。

Despite considerable efforts, the fabrication of protein chips using a self-assembled monolayer (SAM) with a long chain length remains a challenge, due to its steric hindrance and the formation of disulfides, which may generate multilayer and block the terminal carboxylate groups of the SAM, and thus reduce the sensitivity of the chips. To eliminate those problems, the feasibility of using a short-chain SAM, 4,4- Dithiodibutyric acid (4,4-DTBA, disulfide), as a monolayer for the protein chips based on a gold surface was studied. Additionally, the hydrolysis of NHS esters can reduce the fluorescent intensity of protein chips. Thus, the effects of the solvents used in the washing step, the various ratios of EDC/NHS and dissolving EDC in DI water, DI water + 0.1 M MES and alcohol were also investigated to solve this problem in this study. Experiments for characterizing 4,4- DTBA were performed by contact angle goniometry, atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR) and ellipsometry. Additionally, the fluorescent assay of 4,4- DTBA was characterized using protein A-FITC (fluorescein isothiocyanate). The results of 4,4- DTBA were compared with those of 11- Mercapto-undecanoic acid (11- MUA, thiol). The comparison results indicate that 4,4- DTBA can be adopted as a monolayer for the protein chips. The PBST (PBS+ 5% tween20) is more effective in reducing the hydrolysis of NHS esters than deionized water. Moreover, the 3:1 EDC/NHS ratio yields a higher fluorescent intensity than the 5:2 ratio. Finally, the solvent alcohol provides higher fluorescent intensity than other solvents with a 3:1 EDC/NHS ratio at the reaction time of 4 h with 160 mM 4,4- DTBA.

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