Development of an electrochemical biosensor of total cholesterol by the combination of a multienzymatic system and a carbon paste electrode

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.目錄------------------------------------------------------Ⅰ 圖目錄----------------------------------------------------Ⅳ 表目錄----------------------------------------------------Ⅶ 中文摘要--------------------------------------------------Ⅷ 英文摘要--------------------------------------------------Ⅸ 第一章 緒論 1.0 實驗背景------------------------------------ 1 1.1 生物感測器---------------------------------- 3 1.1.1 生物感測器之構成元件------------------------ 3 1.1.2 生物感測器之種類---------------------------- 4 1.1.2.1 電化學型生物感測器-------------------------- 4 1.1.2.2 光學型生物感測器---------------------------- 7 1.1.2.3 熱學型生物感測器---------------------------- 8 1.1.2.4 聲學型生物感測器---------------------------- 8 1.1.3 優良生物感測器需具備之要件------------------ 9 1.1.3.1 辨識元固定技術------------------------------10 1.1.3.2 干擾排除之技術------------------------------12 1.1.4 電子傳遞媒介物------------------------------12 1.1.5 碳漿電極------------------------------------13 1.1.6 網版印刷技術--------------------------------15 1.2 膽固醇與膽固醇酯----------------------------16 1.2.1 膽固醇之生合成------------------------------16 1.2.2 膽固醇與膽固醇酯之結構----------------------19 1.2.3 膽固醇在體內的運輸與代謝--------------------20 1.2.4 膽固醇與血管硬化及心臟病之關係--------------22 1.2.5 膽固醇氧化酶與膽固醇酯酶簡介----------------22 1.2.5.1 膽固醇氧化酶--------------------------------22 1.2.5.2 膽固醇酯酶----------------------------------22 1.2.6 膽固醇分析法的發展--------------------------23 1.3 實驗原理------------------------------------25 1.4 實驗目的------------------------------------25 1.5 實驗架構------------------------------------25 第二章 材料與方法 2.0 實驗藥品與器材------------------------------26 2.1 碳漿電極之製作方法--------------------------26 2.2 Thionin 修飾之碳漿電極----------------------28 2.3 Fe3O¬4 修飾之碳漿電極-------------------------28 2.4 酵素之固定化方法----------------------------29 2.5 電化學實驗方法------------------------------30 2.6 感測器操作之方法----------------------------31 2.7 以呈色法進行膽固醇氧化酶活性之分析----------32 第三章 結果與討論 3.1 自製碳漿電極--------------------------------34 3.1.1 空白碳漿電極--------------------------------34 3.1.2 Thionin 修飾之碳漿電極----------------------35 3.1.3 Fe3O¬4 修飾之碳漿電極-------------------------39 3.1.4 空白碳漿電極對K3Fe(CN)6之探討---------------44 3.2 平版碳漿電極--------------------------------49 3.2.1 平版碳漿電極之前處裡------------------------49 3.2.2 基礎性質之探討------------------------------51 3.2.3 檢測游離膽固醇------------------------------53 3.2.3.1 循環伏安法----------------------------------53 3.2.3.2 最適 K3Fe(CN)6-------------------------------57 3.2.3.3 最適膽固醇氧化酶添加量----------------------57 3.2.3.4 最適操作 pH---------------------------------57 3.2.3.5 最適 Triton X-100 添加量--------------------60 3.2.3.6 以最適化條件檢測游離膽固醇¬------------------60 3.2.4 檢測膽固醇酯--------------------------------64 3.2.5 血液中干擾物質探討--------------------------68 3.3 酵素呈色法檢測游離膽固醇--------------------68 第四章 結論 4.1 結論----------------------------------------72 4.2 未來展望------------------------------------72 參考文獻 ¬----------------------------------------------------7