建構修飾聚砒硌及奈米碳管之酪胺酸酶生物感測試片與其應用

Abstract

此研究之目的在於架構一可攜式應用於即時監控環境中酚類化合物、食物中農藥殘留及健康指標榖胱胺肽(glutathione; GSH)之酪胺酸酶網版印刷碳電極試片(screening printed carbon electrode; SPCE)。我們製備此可攜帶之拋棄型安培式生物感測試片並將其應用於快速檢測酚類化合物、農藥及榖胱胺肽。由於此生物感測系統所感測的為電子傳遞訊號，為提升此網版印刷碳電極試片之電子傳遞能力，我們於其上電聚合聚砒硌(polypyrrole; PPy)與奈米碳管(carbon nanotubes; CNTs)複合材料，再將酪胺酸酶以物理性吸附固定於其上做為生物辨識元件。由於聚砒硌具共軛π鍵之特性且與酸化之奈米碳管摻雜，此複合材料具良好之電子傳遞能力並可增加電極表面積，在利用循環伏安法探討聚砒硌與奈米碳管複合材料於此系統之特性，結果顯示修飾有聚砒硌或聚砒硌與奈米碳管複合材料之電極試片，在循環伏安法的還原波峰電流各別放大了3.9及4.8倍。 酪胺酸酶為一多酚氧化酶，可藉脫氫作用將酚類化合物氧化為醌 (o-quinone)，生成的醌為電化學活性物質可在低電位下被還原，而此還原電流即為我們偵測的目標。於農藥的檢測上，因酪胺酸酶活性可被農藥所抑制，所產生的醌也相對減少，導致還原電流降低，故在此偵測系統中農藥的濃度，可由相對減少之反應電流數據量化得知。而在偵測榖胱胺肽這方面，則利用榖胱胺肽可和醌反應形成榖胱胺肽-醌(glutathione-quinone)複合物，導致還原電流降低，我們則可測定其電流抑制率估算榖胱胺肽之濃度。其中，影響此電極試片感測訊號之參數有pH值、緩衝液濃度及操作電壓已最適化。此系統中最適之pH值為6.5及緩衝溶液濃度為50 mM之磷酸鹽緩衝溶液，-400 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。 在此研究中，我們使用此酪胺酸酶生物感測試片偵測四種酚類化合物:酚(phenol)、鄰苯二酚(catechol) 、對甲酚(p-cresol) 、鄰苯三酚(pyrogallol)，三種農藥(含有機磷及氨基甲酸鹽類):加保扶(carbofuran)、得滅莞(aldicarb)、馬拉松(malathion) 及榖胱胺肽。所量測得之偵測極限為酚 50 □M、鄰苯二酚 1 □M、對甲酚 2.5 □M、鄰苯三酚 10 □M、加保扶 20 ppb、得滅莞 20 ppb、馬拉松 20 ppb 及榖胱胺肽 6.25□□M。除此之外，此研究也針對各種化合物進行偵測範圍的量測。研究證實，此修飾有砒硌及奈米碳管之酪胺酸酶網版印刷碳電極試片，具有相當潛力應用於環境中酚類化合物之監控、檢測農藥的殘留，及健康指標榖胱胺肽的檢測。

The purpose of this study is to fabricate the tyrosinase-SPCE strips possessing the potential to detect phenolic compounds for environment monitoring, pesticides for food safety and glutathione (GSH) for health care. Disposable amperometric biosensing strips were prepared for rapid determination of phenolic compounds, pesticides and GSH. Tyrosinase was immobilized on the surface of screening printed carbon electrode (SPCE) strips, which were modified by electrochemical polymerization of polypyrrole (PPy) and carbon nanotubes (CNTs). In the presence of PPy and PPy/CNTs modificaton, the cathodic peak current of the cyclic voltammogram of SPCE strips was significantly enhanced 3.9- and 4.8- fold, respectively. Tyrosinase is a polyphenol oxidase that catalyzes the oxidation of phenolic compounds via dehydrogenation to o-quinones. The generated o-quinones are electoactive species that can be reduced at a low potential and the reduction current can be measured. In pesticides detection, tyrosinase activity inhibited by pesticides and the production of o-quinone was reduced. Thus, the concentration of the pesticides can be calculatedly obtained from the reduction of response current in the detection system. In GSH detection, GSH reacted with o-quinone and then produced glutathione-quinone complex. The complex reduced the quantity of o-quinone and made the reduction current decreasing. We obtained the inhibition percentage in the detection system to evaluate the concentration of GSH. The factors affecting the biosensing response of the SPCE strips such as pH, concentration of phosphate buffer and working potential were investigation. The optimum pH and concentration of phosphate buffer were 6.5 and 50mM, respectively. The tests were performed in presence of catechol substrate at -400 mV. The maximum response from the oxidation reaction of o-quinone was obtained within 1 minute. Four phenolic compounds (phenol, catechol, p-cresol and pyrogallol), three pesticides (organophosphate: malathion and carbamate: aldicarb, carbofuran) and GSH were detected by the SPCE strips. The detection limit for phenol (50 □M), catechol (1□M), p-cresol (2.5 □M), pyrogallol (10 □M), aldicarb (20 ppb), malathion (20 ppb), carbofuran (20 ppb), and GSH (6.25 □M) was determined. Moreover, the detection range for each compound determined in this study was also evaluated. In conclusion, the SPCE strips modified with PPy and CNTs are potential for the forward applications in the detection of phenolic compounds to environment monitoring, pesticides in food to provide food safety and also GSH for body health.