以複合酵素系統配合碳漿電極開發電化學式總膽固醇生物感測器

Abstract

人體血液中總膽固醇的含量是判斷體內脂質代謝是否異常的指標，過高可能有冠狀動脈硬化、糖尿病或甲狀腺機能減退等徵兆；過低亦有貧血、憂鬱症或罹病率、死亡率高的可能。 本研究主要目的是開發一電化學式生物感測器用以檢測血清中總膽固醇。先以自製碳漿電極對各種電子傳遞媒介物以循環伏安法（cyclic voltammetry）及電流法（amperometry）來評估其效用及進行電極之電化學處理，實驗結果顯示自製之碳漿電極配合三種不同的電子傳遞媒介物皆可有效降低檢測 H2O2 之工作電壓，並選擇以K3Fe(CN)6 作為後續試驗。後續實驗以市售網版印刷技術製作之可拋棄式平版碳漿電極（screen-printed carbon paste electrodes），配合膽固醇氧化酶（ChOx）、膽固醇酯酶（ChE）及電子傳遞媒介物K3Fe(CN)6共同構成檢測系統。實驗中各酵素及電子傳遞媒介物以物理性吸附的方式固定於電極表面，並探討系統最適操作條件，分別選定操作電位為 0.2 V、pH 7.0 、7.5 mM K3Fe(CN)6、1.2 % Triton X-100、1.0 Unit ChOx及1.0 Unit ChE。於室溫下操作，可測得膽固醇線性範圍至333.3 mg/dl，R2＝0.995，而可測得膽固醇酯線性範圍至 100 mg/dl，R2＝0.951。此生物感測器所檢測的線性範圍可涵蓋血液中游離膽固醇之正常範圍。

Determination of serum cholesterol has diagnostic implications：an increase in its level is associated with coronary artery disease, nephrosis, diabetes, myxoedema, obstructive jaundice and hypothyroidism, whereas a decreased level of cholesterol is observed in cases of hypothyroidism, anaemia, malabsorption and wasting syndromes. It is desired to develop techniques which allow convenient and rapid determinations of cholesterol. The aim of this study was to develop disposable planar electrodes apply to cholesterol and cholesteryl ester detection. The feasibility of the fabricated electrodes was evaluated by electrochemical methods. The current responses were proportional to concentrations of electroactive substances, hydrogen peroxide. Three mediators：iron(II,III) oxide, thionin and potassium ferricyanide had been tested using cyclic voltammetry and amperometric method to detect hydrogen peroxide. Therefore, potassium ferricyanide was chosen as a mediator to use in planar electrode. To achieve a reagentless biosensor, potassium ferricyanide and cholesterol oxidase and cholesterol esterase were co-immobilized onto the electrode surface by adsorption. The optimal operation condition for total cholesterol detection was at 0.2 V potential in 0.02 M pH 7.0 phosphate buffer, 1.2% Triton X-100 and 7.5 mM potassium ferricyanide, 1.0 Unit of cholesterol oxidase and 1.0 Unit of cholesterol esterase. The sensor has a good linear response in the range 0~333.3 mg/dl for determination of cholesterol and 0~100 mg/dl for determination of cholesteryl ester. The range can include the normal value of serum free cholesterol.