利用電化學沉積法將NTA修飾於金表面以建立固定化目標分子之平台及其應用

Abstract

石英晶體微天平(Quartz Crystal Microbalance, QCM)可以將石英晶體表面的質量變化轉換成頻率變化，可以做為生物感測器(Biosensor)的偵測元件，其優點為靈敏度高，偵測極限達納米克(nano gram)，而且可以即時地偵測表面分子的吸附或脫離，藉此即時地偵測生物分子的分離或結合。NTA(Nitrilotriacetic acid)常做為純化蛋白質所使用之分子，其結構有一個三級胺及三個羧基，具四個配位，可與二價過渡金屬敖合而形成錯合物，再藉由多個Histidine的胜肽(His-tag)與蛋白質形成NTA-M2+-Histag-protein錯合物，因此廣泛的被應用於蛋白質純化、蛋白質固定化及生物感測器中。本研究合成三種不同長度之苯胺-NTA衍生物，並將其與NaNO2反應後形成重氮鹽，再利用電化學還原使其與金形成共價鍵而修飾至QCM晶片表面，並以氧化還原電位圖、表面電阻抗及接觸角分析鏈長對於表面物理性質的影響，並發現這三個分子中，鏈長最長的NTA修飾的晶片與具有His-tag的分子結合能力最佳。最後藉由修飾NTA之QCM晶片做為固定具有His-tag分子(胜肽、蛋白質…等)之平台，並以QCM即時地觀察這些分子的結合或分離，以做為生物感測器並可以計算這些生物分子間的解離常數(Dissociation constant, Kd)。

Quartz crystal microbalance (QCM) can convert mass change on the quartz surface to frequency change, so QCM can be used as transducer in biosensor. The adventage of QCM is high sensitivity, which is up to nano gram, and real-time detection of adsorption and dissociation of molecules on quartz surface. Nitrilotriacetic acid (NTA),which consist of one tertiary amine and three carboxylic acid, used to be apply in protein purification and NTA can also form four coordinate bond with transition metal ion, generating NTA-M2+- His-tag-Protein complex by coordination with two histidine in His-tag, an oligomer which usually compose of six to ten histidine. Therefore, NTA widely apply in protein purification, protein immobilization and biosensor. This research synthesized three different length aniline-NTA derivatives and covert those aniline-NTA derivatives into diazonium salts by reaction with NaNO2 under acid condition, and then modify those diazonium salts on gold surface, which was coated on QCM chip, by electrodeposition. We investigate surface physical properties by analysis surface oxidation-reduction voltammetry, surface impedence and contact angle and discover that His-tag-protein binding most on the chip by QCM when modify longest aniline-NTA of the three derivatives. Finally, Using QCM chip, which was modified the longest NTA, as His-tag-biomolecule, such as peptide, protein…etc, immobilization platform to real-time observe association or dissociation reaction of biomolecules and calculate dissociation constant (Kd) of those biochemical reaction.