"利用馬來醯亞胺官能化之聚(3,4-乙烯二氧噻吩)製備偵測鈣調蛋白之電化學適體型傳感器"

Abstract

適體(aptamer)為能夠與特定目標分子結合的人工核酸序列，可能由DNA、RNA、或多肽所組成，能夠經由體外選序的方式獲得，為了偵測樣品中的鈣調蛋白(calmodulin)濃度，我們開發了一種藉由固定特定胜肽序列於導電高分子表面來抓取樣品中鈣調蛋白的電化學適體傳感器。用核醣體體外選序的方式我們獲得了一段胜肽序列YWDKIKDFIGG，本次實驗將利用此胜肽序列作為鈣調蛋白的受體，並將此胜肽序列固定在馬來醯亞胺官能化之聚(3,4-乙烯二氧噻吩)(EDOT-MI)電極表面，進而製造出鈣調蛋白感測器。 首先，以電化學聚合的方式將含有馬來醯亞胺官能基之3,4-乙烯二氧噻吩聚合在金電極表面。為了在電極表面植上胜肽鏈，必須在該胜肽序列末端加上一半胱氨酸(cysteine)，藉此胜肽鏈能夠經由將半胱氨酸含硫一側與打斷環上碳-碳雙鍵之馬來醯亞胺官能基鍵結，使胜肽鏈固定於電極表面。為了使該胜肽序列更能實際應用在蛋白質感測器上，抗沾黏是其不可或缺的性質之一，因此在原有的鈣調蛋白受體胜肽上我們另外個別修飾上三種不同的胜肽序列，分別為SSSSC及帶有兩性離子性質的EKEKEKEC和EKEKEKEKEKEC。經由石英晶體微量天平(quartz crystal microbalance)來觀察牛血清蛋白和鈣調蛋白對各胜肽鏈的吸附現象後，我們發現帶有正電荷之離胺酸(lysine)與帶有負電荷之麩胺酸(glutamic acid)交錯結構之胜肽序列在抵抗牛血清蛋白吸附上展現出良好的性能，並同時維持抓取鈣調蛋白的能力，其中含有較長鏈兩性離子胜肽之序列(EKEKEKEKEKEKEC)展現出最佳的抗沾黏性能，同時它在電化學阻抗頻譜分析(EIS)中所增加之阻抗為所有胜肽序列最低。 最後，我們使用電化學阻抗頻譜分析來偵測樣品中的鈣調蛋白濃度，其偵測極限及偵測範圍分別為0.001 mg/mL和0.01 mg/mL ~ 10 mg/mL。藉由在導電高分子薄膜植上適體的方式，本實驗成功發展出一種於樣品中偵測鈣調蛋白濃度之電化學適體感測器。

In this study, a fast and sensitive electrochemical aptasenor based on electropolymerized EDOT-MI film was developed for calmodulin (CaM) detection. Poly(EDOT-MI) film was used as a platform for peptide probe immobilization, and peptide sequence YWDKIKDFIGG was obtained by in vitro ribosome display selection for CaM recognition. For the immobilization of peptide probe on electrode surface, cysteine was incorporated at the end of the peptide sequence. A maleimide-functionalized poly(3,4-ethylenedioxythiophene), poly(EDOT-MI), film was first electrogenerated onto the electrode surface. The cysteine labeled peptide probe was then anchored on the electrode through thiol-ene conjugation between the cysteine end and maleimide group. To achieve antifouling performance of the peptide sequence, it was modified with three different peptide linkers separately. The binding tests with bovine serum albumin (BSA) and CaM were monitored by a quartz crystal microbalance (QCM). A linker was composed of alternating positively charged lysine (K) and negatively charged glutamic acid (E), the EKEKEKEKEKEK, to provide excellent performance to resist BSA adsorption and remained highly CaM binding. It was also discovered that peptide probe with this linker increased less in the impedimetric detection. This aptasensor was applied for detecting CaM through electrochemical impedance spectroscopy with the detection limit 0.001 mg/L and linear range from 0.01 mg/L to 10 mg/L. From immobilizing the designed peptide onto the electrode surface, we successfully demonstrated an impedimetric aptasensor with antifouling property and selectivity. This work has been published in Analytical Chemistry. Most data reported in this study have been published in the paper.