利用表面電漿共振影像儀探討核酸共軛之蛋白質晶片最佳化研究

Optimization of DNA-Conjugate Protein Chip Studies by Surface Plasmon Resonance Image

黄莉雅(Li-Ya Huang)

指導教授 ： 陳文逸

國立中央大學/工學院/化學工程與材料工程學系/碩士(2011年)

摘要

近年來，蛋白質生物感測器廣泛的應用於環境監測、生物檢測或疾病診斷方面，但是因為蛋白質的不穩定性和不易保存，可能會隨著使用次數的增加而失去活性，進而影響檢測的準確性。為了改善這些問題，本研究加入了穩定性較高的去氧核醣核酸(deoxyribonucleic, DNA)作為生物感測器的元件之一，並使用表面電漿共振影像儀(surface plasmon resonance image, SPRi)觀測晶片表面蛋白質吸附情形來做最佳化研究之探討。
利用乙烯乙二醇(ethylene glycol, EG)可抵抗蛋白質吸附的特性，製作寡聚乙烯乙二醇(oligo ethylene glycol-thiol, OEG-thiol)與終端為羧基的硫醇類EG分子(COOH-thiol)組成的混合自組裝單分子層膜(mixed SAMs)，接著將單股DNA修飾於mixed SAMs表面，再利用雜交的方式固定上修飾有互補股DNA的抗體，形成DNA共軛蛋白質的蛋白質晶片。藉由調控晶片上單股DNA的比例與鹼基數的多寡，減少蛋白質非專一性的吸附；調整再生溶劑的濃度或種類，使晶片能有良好的再現性；再改變緩衝溶液的酸鹼值，增加目標物的吸附，以降低偵測極限。研究發現：單股DNA佔表面2 %以及鹼基數為20個時，晶片表面的抗非專一性吸附效果及專一性結合能力都有很好的表面，1 N氫氧化鈉溶液可使晶片達92 %左右的再現性。這些結果證實最佳化研究的確可使蛋白質晶片擁有良好的精確性，且此種檢測方式可用於各種檢測上，可說具有相當的發展潛力。

關鍵字

生物感測器 ； 最佳化 ； DNA蛋白質共軛物

並列摘要

Protein-based biosensors are widely used for disease diagnoses, environmental monitoring, drug screening and etc... . However, the fabrication, stability, and accuracy of protein-based biosensors are usually affected by the decreasing biological activity of the immobilized protein. With the knowledge of DNA-based biosensors, in this study we utilized the ssDNA-protein conjugates to immobilize onto the complementary ssDNA-modified self-assembled monolayers (SAMs) through the help of the surface plasmon resonance image (SPRi) measurement. Thereby a DNA-based biosensor can be transformed into protein-based biosensor.
The specificity and sensitivity of DNA-conjugate anti-HSA with its target were measured to demonstrate the feasibility of this platform. The mixed SAMs composed of oligo(ethylene glycol) (OEG) terminated thiols and carboxylic-functionalized OEG (COOH-OEG)termiated thiols, and DNA length were examined to obtain the optimized detection efficiency. Moreover, the regeneration conditions of protein-based biosensor were also discussed. The results revealed that the DNA length with 20 based pairs immobilized onto OEG:COOH-OEG = 50:1 mixed SAMs owns the high sensitivity and low non-specific binding. It could be also found that the using of 1N NaOH is able to dehybridize the immobilized protein-ssDNA, and the regeneration efficiency of protein-ssDNA biochip is about 90 %. Consequently, the optimized stability of protein biosensors seems to be feasible under the DNA mediation; and our approach provides important information on high sensitive protein-based biosensors.

並列關鍵字

DNA-conjugate antibody ； optimization ； biosensor

參考文獻

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