建構與運用穀胱胺肽生物感測試片

Abstract

穀胱胺肽（glutathione; GSH）是生物體內一重要的抗氧化物質。它不只可以直接清除自由基，還可以增強其他重要抗氧化劑的能力，例如：維生素C和維生素E。現今有許多研究顯示，GSH濃度的改變和癌症、阿茲海默症、帕金森氏症、糖尿病、黃斑病變、後天免疫不全症候群（AIDS）等疾病有相關。然而，傳統上偵測GSH的方法有許多限制，例如檢測時間較長、有些樣品需要做前處理及缺乏足夠的靈敏度等。本研究的目的為建構一可攜帶且一次性使用的酪胺酸酶（tyrosinase）網版印刷碳電極（screening-printed carbon electrode; SPCE）生物感測試片，並將此試片應用在細胞和動物檢體中GSH濃度的檢測。 此GSH檢測系統以酪胺酸酶做為生物辨識元件，並藉著安培式訊號轉換器將SPCE感測試片的訊號轉換。我們先將試片的工作電極與參考電極上修飾氨水（ammonium hydroxide; NH4OH），再修飾奈米金球（colloidal gold nanoparticles; AuNPs）與介電子二茂鐵（ferrocendicarboxylic acid; FeDC）於工作電極和參考電極表面。AuNPs的功用為提供電子在氧化還原酵素與電極間的傳遞，及提供生物分子（酵素）在固定化時一個穩定的電極表面，以維持生物分子的活性。介電子FeDC可幫助生物分子的氧化還原反應並做為一個電子梭子，加速酵素及電極間的電子傳遞以提升訊號。最後將酪胺酸酶固定在SPCE感測試片上，再利用循環伏安法探討AuNPs及介電子在此生物感測系統的特性，經結果可以得知在AuNPs以及FeDC的加成作用下，其循還伏安法的還原波峰電流放大5.2倍。 在此系統中使用鄰苯二酚（catechol）為酪胺酸酶的受質。當鄰苯二酚與試片上的酪胺酸酶氧化生成醌（ｏ-quinone）後，電極提供一低電壓使醌還原成鄰苯二酚。其中影響此反應的參數包括pH值、緩衝液濃度及操作電壓。此系統之最佳化條件為pH值為6.5、緩衝液濃度為50 mＭ與-200 mV的電壓。當在-200 mV的電壓下進行反應時，可在40秒內獲得最大的反應電流。此外，在最佳化條件下，反應電流與鄰苯二酚濃度呈正相關且濃度線性範圍為0到1000 uM。 此系統再現性測試方面，在血清中加入不同濃度的GSH，用此生物感測系統檢測可以得到極佳的再現性。此外，長時間穩定性測試方面，將修飾好的SPCE長時間保存在4°C下，可以維持84%至少六周的活性，由結果顯示，此酪胺酸酶修飾之生物感測試片可以長時間穩定的保存在4°C下且不會失去活性。 將酪胺酸酶修飾之生物感測試片應用在細胞與動物的GSH濃度檢測上，可以成功地檢測動物血液、血清及器官中和細胞中的GSH濃度。利用GSH抑制醌在酵素及電極間的循環，GSH和醌生成GSH-醌 (glutathione-quinone) 複合物，導致還原電流降低。使用酪胺酸酶生物感測試片測得的GSH線性濃度範圍為32.5到500 uM。 此酪胺酸酶修飾之電化學生物感測系統可以成功的做為一個即時檢測的安培量測系統。此研究證實，利用AuNPs和FeDC修飾酪胺酸酶生物感測試片具有相當的潛力於臨床GSH的樣品檢測。

Glutathione (GSH) is a major antioxidant in organisms. It not only detoxifies reactive oxygen species directly, but also enhances the functional ability of other crucial antioxidants, such as Vitamins C and E. Alteration in GSH concentration has been implicated in a number of pathological conditions, including cancer, Alzheimer’s, Parkinson’s diseases, diabetes, macular degeneration, acquired immune deficiency syndrome (AIDS) and HIV disease. However, GSH detection has always proven to be difficult. Most of the methods are time consuming, needing derivatization and lack sufficient sensitivity. In this study, the disposable and tyrosinase-modified electrochemical test strips were fabricated for the detections of GSH level in vitro and in vivo. The system was based on tyrosinase as biorecognition element and screen-printed carbon electrode (SPCE) as an amperometric transducer. The bilateral modification of the electrodes was utilized because the method had higher current compared to the modified unilateral electrode. At first, the working and reference electrodes of the SPCE strips were immobilized by ammonium hydroxide (NH4OH). The strips were modified with colloidal gold nanoparticles (AuNPs) and mediator (ferrocendicarboxylic acid; FeDC). The AuNPs not only permit electron transfer between redox chemicals directly and bulk electrode materials, but also provide a stable surface for the immobilized biomolecules retaining their biological activities. The immobilized mediator promotes the redox reactions of biomolecules and shuttles the electrons more efficiently between tyrosinase and electrodes, resulting in enhancing the sensing performance. Finally, tyrosinase was immobilized on the surface of working electrode of SPCE strips. The electrochemical performance of the modified SPCE strips was characterized by cyclic voltammetry. In the presence of AuNPs and FeDC, the cathodic peak current of the cyclic voltammogram was significantly enhanced by 5.2 folds. In the tyrosinase-SPCE strips, catechol was used as the substrate of tyrosinase. The oxidation reaction from catechol to o-quinone catalyzed by the immobilizing tyrosinase. This enzymatically generated o-quinone could be reduced electrochemically back to catechol by providing with a low potential and the reduction current was measured to determine the quantity of substrate. Factors affecting the response current, including pH, phosphate buffer concentration and working potential were investigated. The optimum pH was 6.5 and the optimum concentration of phosphate buffer was 50 mM. The tests were performed in the presence of the catechol substrate at -200 mV. The maximum response from the oxidation reaction of o-quinone was obtained within 40 sec. Under the optimized condition, the response was found to be linear and concentration-dependent in the range to 0 to 1,000 uM of catechol. The detection mechanism of GSH in the above-samples was accomplished by suppressing the recycling process which occurs between tyrosinase and electrode of biostrip. The GSH reacted with o-quinone and glutathione-quinone complex was produced. The production of glutathione-quinone complex reduced the quantity of o-quinone and made the reduction current decreasing. The linear response of the tyrosinase-SPCE strip was ranging from 32.5 to 500 uM of GSH. For testing the system stability and recovery for GSH determination in biological samples, the plasma samples were then spiked with different concentrations of GSH and the recovery values were calculated. For all the concentrations tested (200, 400, 600, 800, and 1000 uM of GSH), good recovery values were obtained. The results demonstrate that the thiol–disulfide exchange reaction is not influenced by the presence of a complex matrix. Both the time and completion of the reaction are similar of those obtained in standard solutions. GSH was tested in the concentration range which could be found in normal and pathologic conditions, with no substantial differences in the recovery values. In long-term storage stability of the modified-SPCE strips, the prepared tyrosinase SPCE strips were stored at 4°C; it retained 84% of its initial response at 6th week. It means that the developed tyrosinase-SPCE strips were stable for several weeks if they are stored at 4°C. The tyrosinase-SPCE strips were applied in biomedical experiments. In the experiments, the mice were challenged with doxorubicin and administrated with N-acetylcysteine or astaxanthin. After 14 days of treatment, the mice were scarified and the blood and tissues, including heart, lung and liver, were sampled for the detection of GSH levels. The results show that doxorubicin treatment could reduce the blood and visceral GSH level and N-acetylcysteine and astaxanthin could compensate this physiological GSH depletion. In conclusion, a disposable amperometric biosensor, the tyrosinase and AuNPs modified SPCE strips were fabricated. The strips were successfully utilized to real-time detect the GSH level in biological samples indicating that the strips are potential of forward applications in GSH detection for clinical diagnosis.