探討導電高分子在生物轉換器和免疫感測器上應用

Abstract

本論文主要探討導電高分子在生物轉換器和免疫分析法上的應用。本研究嘗試應用導電高分子電聚合固定氧化脢在電極表面上，並作為氧化脢之脢輔基電子傳導的媒介，以取代氧分子產生過氧化氫的過程。本論文前階段主要以葡萄糖氧化脢作為模型，在嘗試應用導電高分子Polypyrrole取代氧氣產生過氧化氫的研究中，並未能直接作為葡萄糖氧化脢的脢輔基電子傳導的媒介。而測試山葵過氧脢，利用導電高分子Polypyrrole可直接作為此酵素脢輔基Heme的電子傳遞媒介。本論文第二個探討是應用導電高分子，開發一新穎免疫分析之電化學免疫感測器。利用導電高分子電聚合方式固定抗體製作免疫電極和檢體中抗原以及架接好電活性物之抗原，作競爭型結合反應後，以提供電壓方式氧化電極表面之電活性物，偵測淨電流變化得到檢體中抗原含量。本研究以人血清蛋白HSA作為抗原，維他命C和Ferrocene作為電活性物。而以HSA-VitC（1.149 mg / 40ml）和HSA（1.149~5.744 mg）競爭型AntiHSA電極結合反應後，偵測淨電流變化(6.02±30.6~111.0±36.8 nA)。(linear regression r2 = 0.913) 本研究已成功開發出一種新穎電流式免疫分析方法(Direct Amperometric Immuno Assay ,DAIA)可應用在電化學免疫感測器上，期望未來能應用在不同抗原和抗體，進一步落實於臨床診斷之應用分析。

In this research , we have focused on the application of conductive polymer in biotechnology . First of all , we have examined the possibility of applying conductive polymer to transfer electrons to the prosthetic group of enzyme active site in redox reactions when dealing with immobilized oxidases on electrode surface . The results of our observation when using glucose oxidase as model oxidative enzymes were not successful . It’s postulated that the active sites of glucose oxidase examined may bury deep inside of the enzyme molecules . The conductive polymer encapsulated enzymes only tangle the surface of the enzymes and not accessible to the prosthetic group inside the non-conductive protein environment housing the prosthetic group for direct electron conduction .Our second approach is to explore the possibility of a novel immunoassay protocol involing conjugating electroactive species using conductive polymer . It is expected that a electroactive apecies can be either oxidized or reduced on electrode surface when it is brought to close proximity of electrode by antigen-antibody immune response . The electroactive species is polarized on electrode surface and the change of current can be observed , in the presence of polarized potential of the electroactive species. We conjugate a large molecular antigen such as protein to ascorbic acid or ferrocene and brought these conjugate to electrode surface by their antibodies . A current change(6.02±30.6~111.0±36.8 nA) were repetitively observed and were concentration dependent HSA-VitC（1.149 mg / 40ml）and HSA（1.149 ~ 5.744 mg）when using VitC conjugate . (linear regression r2=0.913) We successfully set up this model . This new finding opens an avenue which leads to the era of “Current Immuno Assay “ , CIA , and shall greatly facilitate the biosensing of both large and small organic molecules in post genome clinical research .