利用電化學固定鹼性磷酸酶在戊二醛交聯聚苯胺的石英晶體微天平上並探討TAU蛋白之去磷酸化作用

Immobilization of ALP on the PANI-GA Modified Working Electrode by Electrochemical and that Dephosphorylates Tau Protein

蔡欣宜(Hsin-Yi Tsai)

指導教授 ： 陳榮治

國立交通大學/資訊學院/生醫工程研究所/碩士(2018年)

摘要

阿茲海默症為失智症之一，主要2大病因為大腦皮質周圍的β−amyloid42堆積形成斑塊以及tau protein過磷酸化造成神經糾結。本研究主要是探討過磷酸化的Tau protein是否能用修飾過後的電極有效的去磷酸化。首先利用即時觀測設備電化學石英晶體微天平(EQCM)作為反應器，以Polyaniline膜為載體形成導電之半導體，並以Glutaraldehyde作為交聯劑連接生物酵素-鹼性磷酸酶，由定電位技術透過電流-電位及頻率響應觀察膜上修飾程度，再經由加入磷酸化受質觀測其吸光度而判別鹼性磷酸酶之活性程度。以酵素修飾後的電極進而使用在過度磷酸化的Tau protein，去磷酸化以膜上吸附及釋出之重量變化加以驗證。實驗結果由-0.8V之定電位製作的Polyaniline膜可以得到較好的導電性，且可以快速沉積在此膜，接合劑Glutaraldehyde拍攝SEM圖觀察到整齊排列，EDS中碳量增多，推測接上帶有為烴鏈的有機化合物，而後使用修飾後的官能基固定鹼性磷酸酶，再用ρ-Nitrophenyl phosphate (pNPP) 作為鹼性磷酸酶受質，EILSA reader在波長405nm測得吸光度得知酵素有活性。利用GSK-3B與ATP磷酸化tau蛋白，將帶有磷酸跟的蛋白先沉積在聚苯胺膜上再用不同濃度的鹼性磷酸酶去除蛋白質上的磷酸跟，經實驗發現在123Unit Alkaline phosphatase有最佳穩定的構型及去磷酸作用。此研究對於阿茲海默症的病人有治療上的創新發展性。

關鍵字

電化學 ； 磷酸化 ； 去磷酸化 ； TAU蛋白 ； 石英晶體微天平 ； 鹼性磷酸酵素 ； 聚苯胺 ； GSK-3β

並列摘要

Alzheimer’s disease is the most common cause of dementia. There are two major causes of disease. One is the accumulation of beta-amyloid 42 around the cerebral cortex to form plaques, the other is tau protein hyperphosphorylation triggers tau aggregation and nerve fibers entangled, leading to neuronal death. Consequently, the study will investigate whether Tau protein can be dephosphorylated by the method of interaction with human phosphatase. Firstly, we use the electrochemical quartz crystal microbalance (EQCM) to be the sensor that investigate the electrical conductivity of polyaniline polymer film. Polyaniline polymer films immobilized on the gold surface was optimized to observe the current-potential and frequency responses by Potentiostatic technique. The EQCM system is used to cross-linking with glutaraldehyde and immobilize ALP on the polymer film. We use Absorbance principle to measure ALP activity. Finally, we use QCM adsorption principle to measure the weight differ of Tau protein dephosphorylation. As a result of three parts of the experiments, the conductivity of polyaniline film can be triggered by a cathodic potential of -0.8 V could be better and deposited rapidly. The results of SEM pictures indicate PANI-GA modified working electrode to be drawn up in orderly ranks. Compared with before and after of the carbone atom is significantly increased by the EDS content. Then the functional group of GA can immobilize ALP using the covalent bond. ALP catalyzes the hydrolysis of ρ-Nitrophenyl phosphate (pNPP) to ρ-Nitrophenol. PNPP is colorless but ρ-Nitrophenol has a strong absorbance at 405 nm. The rate of increased absorbance at 405 nm is proportional to the enzyme activity.
We want to demonstrate dephosphorylates Tau protein by ALP and we make another experiments. Add the tau protein on the membrane and its phosphorylation by the protein kinase GSK-3β and ATP. The experiments are monitored by frequency responses. And using different concentration of ALP interacts with phosphorylation tau protein to look at whether the phosphate can be removed from the membrane. Finally, ALP concentration is 123Unit on the polymer film which is the best structure and dephosphorylation. This device will be a sensor for the treatment of Alzheimer's patients in the further.

並列關鍵字

Electrochemical quartz crystal microbalance ； Tau protein ； Dephosphorylation ； Polyaniline ； GSK-3β protein kinase ； glutaraldehyde ； Alkaline phosphatase ； ρ-Nitrophenyl phosphate

參考文獻

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