利用奈米金球建構酪胺酸酶印刷碳電極生物感測片及此生物感測片的應用

Abstract

在農業發展上，使用了許多不同種類的殺蟲劑及除草劑，其中以有機磷及氨基甲酸鹽類的農藥最常被使用，這兩類的農藥殘留會與人體內的乙醯膽鹼酯脢（acetyl cholinesterase; AChE）作用，抑制此酵素的活性，阻礙神經傳導素乙醯膽素的水解，導致突觸的乙醯膽素過量，干擾神經脈衝的傳輸。除此之外穀胱甘肽（glutathione; GSH）為生物體內一重要的抗氧化物質，已有許多研究證實，生物體內GSH濃度的改變已成為某些病理學上的徵狀，例如：癌症、帕金森氏症、後天免疫不全症候群（AIDS）…等。本研究的目的為建構一可攜型酪胺酸酶網版印刷電極生物感測試片，並將其應用在蔬菜農藥殘留及血液中GSH含量的檢測。 此檢測系統係以酪胺酸酶為生物辨識元件，並藉由安培式訊號轉換器將網版印刷碳電極（screening-printed carbon electrode; SPCE）感測試片的訊號轉換。首先在試片上修飾戊二醛（glutaraldehyde; GA）後，利用奈米金球（gold nanoparticles; AuNPs）及介電子二茂鐵（ferrocendicarboxylic acid; FeDC）修飾電極表面，以提供電子在氧化還原酵素與電極間的電子傳遞，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。介電子可幫助生物分子的氧化還原反應且作為一個電子梭子，加速酵素及電極間的電子傳遞以提升訊號，接著將酪胺酸酶固定在SPCE感測試片上。在利用循環伏安法探討AuNPs及介電子在此系統的特性，結果顯示在AuNPs以及FeDC的加成作用下，其循環伏安法的還原波峰電流放大3.8倍。 此系統中使用鄰苯二酚（catechol）作為酪胺酸酶的受質，當鄰苯二酚與試片上酪胺酸酶作用氧化成醌（quinone）後，電極提供一低電壓使醌還原回鄰苯二酚。其中影響反應電流的訊號參數有：pH值、磷酸緩衝液濃度及操作電壓。此系統中的最適pH值為6.5及緩衝液濃度為50 mM的磷酸鹽緩衝液，當在-300 mV的電壓下進行反應，醌的最大反應電流可在一分鐘內獲得。此外在最適化的條件下，反應電流與鄰苯二酚濃度呈正相關且濃度線性範圍為2.5 □M到200 □M。 將酪胺酸酶生物感測試片應用在殺蟲劑檢測上，係量化抑制酪胺酸酶的作用進行數據分析。加保扶（carbofuran）抑制酪胺酸脢的活性，導致醌的產物減少，反應電流隨醌的濃度降低而降低。另外，藉由反應電流的抑制率可獲得降低電流值與加保扶濃度的關聯性。在最佳化條件下，使用酪胺酸酶生物感測試片量測加保扶，可測得的濃度線性範圍為0.02至0.1 ppm，偵測極限為0.02 ppm。 在於測量血液中GSH的濃度方面，利用血液中GSH抑制醌在酵素及電極間的循環，GSH與醌反應產生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濃度線性範圍為12.5至100 □M。本研究中，使用AuNPs及FeDC修飾酪胺酸酶生物感測試片，可成功的提供一個做為即時監測使用的安培檢測系統。此研究證實，利用AuNPs及FeDC修飾酪胺酸酶生物感測試片，具有相當潛力應用於檢測農藥加保伏的殘留，及血液中GSH的檢測。

Among the pesticides, organophosphate and carbamate compounds are widely used in agriculture. The mode of action of these pesticides is based on inhibition of the activity of acetylcholine esterase (AChE) enzyme in the hydrolysis of the neurotransmitter acetylcholine. The inhibition of the activity of AChE results in an excess of acetylcholine and effect the responsible for the transmission of nervous impulses. Additionally, reduced glutathione (GSH) is a major antioxidant in organisms. Alterations in GSH concentration have also been demonstrated to be a common feature of many pathological conditions including cancer, neurodegenerative diseases ( Alzheimer and Parkinson), and acquired immune deficiency syndrome (AIDS). In this study, a disposable tyrosinase-SPCE strip was fabricated for the detection of pesticide contamination in vegetable and GSH in blood. The system was based on tyrosinase as the biorecognition element and screen-printed carbon electrode (SPCE) as an amperometric transducer. Firstly, glutaraldehyde (GA) was immobilized on the SPCE strip, then, the strip were modified with colloidal gold nanoparticles (AuNPs) and mediator (ferrocendicarboxylic acid; FeDC). The AuNPs not only permit direct electron transfer between redox proteins 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 base electrodes, resulting in enhancing the sensing performance. Finally, tyrosinase was immobilized on the surface of SPCE strip. 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 3.8 folds. In the tyrosinase-SPCE strip, catechol was used as the substrate of enzyme. 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. Parameters affecting the response current such as pH, phosphate buffer concentration and working potential were investigated. The optimum pH and concentration of phosphate buffer were 6.5 and 50 mM, respectively. The tests were performed in the presence of the catechol substrate at -300 mV. The maximum response from the oxidation reaction of o-quinone was obtained within 1 minute. Under the optimized condition, the response was found to be linear and concentration-dependent in the range of 2.5 to 200 □M of catechol. Detection of pesticides with the tyrosinase-SPCE strip was based on the quantification of inhibitory effect on the tyrosinase. Tyrosinase activity was inhibited by carbofuran and the production of o-quinone was reduced. The response current decreased with the reduction of o-quinone. In addition, the correlation between decreased current and carbonfuran concentration was obtained by the inhibitory effect of response current. Under optimized condition, a linear range of the carbofuran detection by the tyrosinase-SPCE trip was from 0.02 to 0.1 ppm and with a detection limit of 0.02 ppm. This tyrosinase-SPCE strip also could be applied to determine the GSH in whole blood. The detection of GSH in blood 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 12.5 to 100 □M of GSH. In this system, modification of AuNPs and FeDC in the tyrosinase immobilized SPCE was successfully applied in an amperometric system with real-time monitoring of current response. We proved that the tyrosinase-SPCE strips modified with AuNPs and FeDC have potential for the forward applications in the detection of carbofuran in food, and also in the detection of blood GSH.