生物高感度偵測技術快速鑑別人類血紅素結合蛋白之表現型

Abstract

人體血紅素結合蛋白(Human Haptoglobin, Hp)可以被劃分為三種型態，分別是 Hp 1-1, 2-1, 及 2-2。 Hp 1-1 具有雙單元 (α1β)2 構型, Hp 2-2 為不同單元數之環形聚合 的混合物 (α2β)n ，以及 Hp 2-1 由不同長度的線性聚合 (α1.2.β)n 所組成。就如同 血型一樣，每個人只會有其中一種型態，目前報導指出，許多疾病與 Hp 的表現型以及 在血液中的Hp 濃度有關聯，例如腎衰竭、狹心症、以及冠心病等。然而，目前要從血 清中以傳統生化分析方式分辨出Hp 表現型與血清中濃度，必需精準的操作與耗費漫長 的實驗時間，例如ELISA 以及RIA 等方法。所以，本實驗致力於發展生物電化學感測器 來作抗原(Hp)抗體的親和附著分析，作一快速，操作簡便之生物感測器。分析步驟如下。 先將抗體固定在電極的金表面上，再將含有待測的血紅素結合蛋白樣品放入反應槽，測 量電極表面的氧化還原電流與阻抗。藉由循環伏安法與電化學阻抗頻譜(electrochemical impedance spectroscopy,EIS)分析，我們可以得知是否有血清結合珠蛋白的存在。而利用 Randles’等效電路，更可以將流到電極電子的等效串電阻(electron transfer resistance ,Rct) 予以量化，並藉此有效的對血紅素結蛋白進行表現性之分型與濃度量化。

Haptoglobin (Hp) is an acute phase protein, similar to blood type, Hp have been classified into three phenotypes: Hp 1-1, 2-1, and 2-2. Briefly, Hp 1-1 represents the dimeric (α1β)2 conformation, Hp 2-2 are mixed cyclic complexes of (α2β)n, and Hp 2-1 are heterogeneous linear complexes (α1β)2(α2β)n. Hp phenotypes have been reported to be associated with diseases, such as myocardial infarction, kidney failure, and coronary artery diseases in diabetics. Meanwhile, it is a challenge to determine the phenotypes and concentrations of human haptoglobin in serum. In this study, a homemade portable, high sensitivily bio-electrochemical analyzer (BEA) is design and developed. The developed BEA can rapidly identify the phenotype and quantitate the concentration of Hp. After immobilizing antibodies onto the gold electrode, the interfacial electron transfer rate on the electrode surface with and without antigens is measured by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Furthermore, Randles’ equivalent circuit is applied to evaluate the electron transfer resistance (Rct) of bio-molecular interactions. The heterogeneous of Hp and concentrations can be determined easily by this analysis.