電流式酵素電極檢測於新式交流電化學量測和生物燃料電池先期開發

Enzymatic electrode based AC mode electrochemical technique for glucose detection and biofuel cell electrode development

賴威任(Wen-Ren Lai)

指導教授 ： 李世光

國立臺灣大學/工學院/工程科學及海洋工程學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.01245

摘要

以研究團隊以往所提出之新式電化學檢測方法為基礎，本研究提出創新基礎模型，並據以指出輸入弦波電壓為供電模式的可能優勢，進而比較其與傳統直流電壓供電相較的優勢效果。研究過程中，並自行開發血糖酵素電極檢測試片配方，所得實驗資料證實其對於血球容積比40%的全血，在葡萄糖濃度50至500 mg/dL，在反應訊號對濃度的關係上，有高線性度和低於5%的變異係數(CV)值表現。依此血糖酵素電極檢測試片，和另行調配不同濃度之葡萄糖標準液作為主要待測樣本，來做為本研究新式電化學檢測方法驗證的測試平台。本文所提創新方法，不同於前人供電量測觸發方式，可於當標準液滴入血糖酵素檢測試片時，利用其自我產生之微小電流於瞬間馬上觸發供電量測動作，而得一酵素回溶過程之訊號，依此判斷出一真正最大有效訊號擷取時間。在此一新式供電模式下，血糖酵素檢測試片將有更大的反應電流表現，因此和濃度電流反應關係也將因此有著更高的解析度。
本文並對新式供電模式調整其振幅和頻率來找尋一最佳的新式供電模式參數，且至葡萄糖高濃度600mg/dL下得到和傳統供電模式相比，較高的高線性度表現，整體量測範圍寬達50~600mg/dL。除此之外，本文並依所推導完成的理論模型，提出如何依據輸入的弦波電壓頻率來選取對反應電流積分的最佳特定時間區間，以能避開因交流供電模式在高頻供電下將形成電雙層電容效應，進而影響與放大非法拉第電流訊號的不良效應。利用此一創新的以積分為基礎的信號處理方法所取得的實驗資料，在和快速傅立葉轉換於頻譜大小上的DC項大小互相對照下，證明了交流頻率的引入有助於在反應電流DC項中之表現，因此乃能於理論及實驗兩方面盡皆證實交流信號的引將可對血糖酵素檢測試片一類的電化學反應和濃度的靈敏度產生一增加效應。在經過數百種類型的實驗分析後，本文擇定交流頻率50Hz、10mV振幅的測試條件，在本文所用的血糖酵素電極檢測試片配方條件下，具有最佳反應，分析反應開始後之10秒至20秒訊號，本文所提創新檢測方式和傳統供電模式相比，可增進約40%的訊號靈敏度。考量目前全球各大公司所擁有的血糖儀專利對我國血糖儀公司的箝制，本文所提新式電化學檢測技術，或可為國內血糖儀廠商帶來另一重點發展技術的方向。
本文同時嘗試進行生物燃料電池的先期開發，希望藉由酵素的轉換來將化學能轉為電能，由於研究願景所考量的未來主要應用為植入式生物體內裝置之電力來源，因此研究過程中以生物體內之葡萄糖和氧氣作為陽極、陰極之反應燃料，同時使用開發血糖偵測儀之酵素電極試片為出發點，採用葡萄糖氧化酶(Glucose oxidase, EC 1.1.3.4)和膽紅素氧化酶(Bbilirubin oxidase, EC 1.3.3.5)來進行催化反應進行，再以電聚合方式在金膜電極上，進行導電高分子pyrrole的聚合反應，將酵素和中間物分子包埋其中以做為酵素固定方式。在循環伏安法電聚合下，嘗試以單獨使用葡萄糖氧化酶作為pyrrole包埋之分子，而陰極採用膽紅素和赤血鹽為中間物包埋其中，藉由控制其掃引圈數來增加pyrrole聚合沉積，並且在此系統下以10mV/s的電壓變化速率最為適合pyrrole沉積，在200mg/dL的葡萄糖標準液中、負載50k歐姆下最大可得一約0.15V之初始瞬間電壓輸出，整體系統於操作過程中可輸出的穩定輸出功率約為0.242μW/cm2

關鍵字

血糖檢測 ； 電化學檢測法 ； 生物燃料電池

並列摘要

Taking the research results on electrochemistry based detection methods developed in Nano-BioMEMS team of National Taiwan University, this dissertation proposed a fundamental theoretical model for the previously reported detection method and used this model to point out the potential advantages of using sine wave voltage as the primary power source. In order to prove the underlying advantage with respect to traditional electrochemical method, we developed a glucose enzymatic strip as the testing platform. The experimental data obtained indicated that higher linearity and lower than 5% coefficient of variation with respect to the glucose concentration rangking between 50 to 500 mg/dL can be obtained for 40% hematocrit whole blood test. Using different glucose concentrations standard solutions as the test samples, we compared the testing data obtained from our new voltage applied method and that of the traditional method. To determine right current sampling time, instant trigger voltage method was adopted, i.e., the response current created by dropping the glucose sample on strip was used to start the measurement process. By modulating the frequency and the amplitude of the applied sine wave voltage, a higher resolution and stronger response current can be obtained between measured voltage and the glucose concentration. Even at the high glucose concentration of 600 mg/dL, the new method achievees higher linearity when compared to traditional electrochemical method. To eliminate the non-Faraday current and to increase the signal-to-noise ratio this thesis proposed a new signal processing algorithm using the newly developed theoretical modeling. This approach recognized that non-Faraday current was induced by the double layer capacity effect created by applying the sine wave voltage and can thus be eliminated by proper signal processing algorithm. More specifically, it was identified that non-Faraday current can be integrated to zero if the integration time is set to be the integer times of the applied sine voltage period. The experimental data obtained clearly verified that this newly proposed integration signal processing algorithm and the newly proposed sine wave driving voltage method led to better singla to noise ratio and higher linearity when comparing the FFT spectrum of the signal at DC and at the applied driving voltage signal frequency. This research proves that the frequency of sine wave voltage can enhance the DC term of response current and this current is directly proportional to the sample glucose concentration and has higher sensitivity with respect to the glucose concentration. After several hundred measurements at the testing condition of 50Hz and 10mV amplitude sine wave voltage, it was found that this new approach can have better than 40% sensitivity when compared to the respone current obtained from the traditional method.
This thesis also attempted to develop biofuel cells, which can use biocatalysts to convert chemical energy to electrical energy. As the long-term vision of this biofuel is to be used as the power source for embedded biosensors, glucose was applied as the substrate for the oxidation processes at the anode and oxygen was uesd as the substrates for the reduction processes at the cathode. Taking the enzymatic strip of glucose sensor as the starting point, we used glucose oxidase (EC 1.1.3.4) and bilirubim oxidase(EC 1.3.3.5) to catalyze the processes on the anode and the cathode. The enzyme and mediator were entrapped by the polypyrrole on the Au electrode through electropolymerization. The Cyclic Voltammetry was used to polymerize the pyrrole, which control the scan cycles and the scan rate to determine the deposit of polypyrrole. For final testing of this newly attempted biofuel cell, the GOx-polypyrrole electrode and the BOD- -polypyrrole electrode were integrated and it was identified that for glucose concentration 200mg/dL, 0.15V maximum voltage and 0.242μW/cm2 output power can be obtained when connecting to a 50kΩ load.

並列關鍵字

glucose ； biofuel cell ； electrochemical

參考文獻

李明峰 (2003). "比咯高分子於微型生醫感測元件之表面改質與細胞生理應用之探討." 臺灣大學醫學工程學研究所碩士論文.

鄭弘美, 溫., 周慧玲,李婉瑜,蔡晨瑩 (2005). "利用健保申報資料探討糖尿病住院醫療照護." 內科學誌 16: 121-128.

Allen J. Bard (1966). electroanalyrical chemistry 1.

Anthony P.F. Turner, I. K., and George S. Wilson (1987). Biosensors : fundamentals and applications

Delahay, T. B. a. P. (1955). Z. Electrochem. 59: 792.