利用體外核醣體展示技術發展之多肽探針電化學感測器

Abstract

本實驗設計為利用由核醣體展示技術篩選出對鈣調蛋白 (Calmodium, CaM) 具有專一性的多肽來發展電化學偵測系統，希望能藉由系統建立將此偵測技術推廣至其他具疾病診斷因子，例如人體C反應蛋白 (C-reactive protein, CRP) ，流感病毒等。為了在電極上修飾此具有專一性之多肽，在多肽序列尾端加入帶有硫醇(-SH)官能基的胺基酸，並在電極上以電聚合方式形成帶有maleimide官能基的高分子薄膜，利用maleimide和thiol 之間形成穩定共價鍵，同時多肽本身由於部分胺基酸帶正負電近似兩性離子材料，具有良好親水特性能在表面形成的水層，達到防止非特定吸附的效果。首先利用石英晶體天平驗證電極上成功修飾多肽並探討多肽修飾後對不同蛋白質非特定吸附的效果，同時利用電化學阻抗的方式量測電極修飾前後的阻抗變化，更進一步利用此方法量測不同濃度目標蛋白接上電極後阻抗的改變以建立濃度偵測曲線並找出線性區間。而未來希望將此偵測模板推廣至其他目標，建立一個穩定且具多樣性的偵測基板。因此我們同時利用核醣體展示技術從人工設計的隨機DNA序列庫中去篩選對人體C反應蛋白有專一性的多肽序列，希望將篩選出的多肽利用的同樣的修飾方式，建立人體C反應蛋白的偵測器。

This work is to develop an electrochemical biosensor immobilized with a selected peptide sequence. We demonstrated the electrochemical biosensor system using a selected peptide probe for Calmodium (CaM) detection and this peptide sequence (YWDKIKDFIGG) was obtained from in vitro ribosome display selection. In order to immobilized this peptide probe on the electrode surface, an amino acid containing thiol group was used as the end of this peptide sequence. A maleimide-functionalized poly(3,4-ethylenedioxythiophene), poly(EODT-MI), film was coated on the electrode surface and then immobilization of the peptide probe was achieved through thiol-ene conjugation. The charged amino acid in the peptide probe also provided an antifouling effect to non-specific protein binding. We used a quartz crystal microbalance to demonstrate the bioconjugation of peptide probe and evaluated the antifouling effect of this immobilized peptide probe against proteins. In electrochemical impedance analysis, the increase of charge transfer resistance after peptide immobilization and protein binding was also measured and quantitatively analyzed. The linear detection range for CaM is from100 ngL-1 to 10 mgL-1. Based on our results, this platform provides good sensitivity and a low detection limit. We plan to further apply this electrochemical biosensing system to other proteins, such as C-reactive proteins (CRPs).