建構酪胺酸酶感測片以檢測酚類的汙染與農藥之殘留

Abstract

利用電化學結合生物感測試片之分析方法已廣泛應用於各種領域，包括環境、醫學、食品工業、農業等等，此乃由於電化學分析方法可提供簡便及低成本的檢測平台，因此其應用性與發展性日益受到重視。據此，本研究之目的在於建構一酪胺酸酶網版印刷碳電極之試片 (screen-printed carbon electrode; SPCE)，並期能應用於即時監控環境中的酚類化合物和農產品中的農藥殘留。 在本感測片中，電子的傳遞能力會影響偵測訊號結果，為了要提升SPCE試片之電子傳遞能力，本實驗運用介電子二茂鐵 (ferrocene- dicarboxylic acid, FeDC) 及奈米金球 (gold nanoparticles; AuNPs) 等物質參與偵測反應進行。FeDC在本感測片中作為電子梭子，協助生物分子進行氧化還原反應，加速酵素及電極表面之間的電子傳遞以提升電子傳遞訊號。AuNPs則作為導體，在氧化還原反應中加速酵素與電極表面之間的電子傳遞效率。本試驗中利用循環伏安法 (cyclic voltammetry; CV) 探討FeDC與AuNPs在感測片中的特性，實驗結果顯示在FeDC與AuNPs的加成作用下，其CV的氧化波峰電流放大了4.2倍。 在感測片中，酪胺酸酶 (tyrosinase) 可藉由脫氫作用將其受質磷苯二酚 (catechol) 氧化為醌分子 (o-quinone)，生成的醌分子為電化學反應中的活性物質，其可在低電位下再被還原成磷苯二酚。在農藥的檢測方面，其農藥分子可抑制酪胺酸酶的活性，使之無法與受質產生氧化還原反應，因此醌分子產物減少，導致測得之電流也隨之下降，並藉由電流下降之比例可推測農藥的含量。影響SPCE試片感測訊號之主要參數包含pH值、緩衝液之濃度及操作電壓等，而由一系列實驗結果顯示出在pH值5.0、60 mM磷酸鹽溶液及 -200 mV操作電壓下為最佳的偵測條件，而此在一分鐘內即可獲得最大的反應電流。 本研究中，酪胺酸酶生物感測試片用以偵測四種酚類化合物，其中包括磷苯二酚 (catechol)、酚 (phenol)、對甲酚 (ρ-cresol) 及鄰苯三酚 (pyrogallol)，另外也偵測三種農藥 (含有機磷與氨基甲酸鹽類) ，包括得滅菀 (aldicarb)、加保扶 (carbofuran) 及巴拉松 (parathion)。結果顯示上述物質的偵測極限分別為1.9 × 10−4 mM 磷苯二酚、3.7 × 10−4 mM酚、6.4 × 10−4 mM對甲酚、1.63 × 10−3 mM鄰苯三酚、5.3 × 10−5 mM得滅菀、4.5 × 10−5 mM加保扶及3.6 × 10−3 mM巴拉松。 我們利用FeDC與AuNPs於SPCE感測片中，除了能增加偵測反應之敏感度，也能夠放大反應訊號，因此本試片可成功作為一個安培量測系統，且具有相當潛力應用至環境中酚類化合物與蔬果農藥殘留之檢測。

The electrochemical devices have been used for a variety field to monitoring of water quality, medical, foods or agriculture quality in decades. Because of electrochemical analysis method can provide an easy and cost-effective platform, it is important to develop a biosensing strips and application in various filed. Accordingly, the purpose of this study is constructing a screen-printed carbon electrode (SPCE) strip that was prepared for rapid determination of phenolic compounds and pesticides residual. The electron transfer may affect the ability of detection signal results on SPCE strip. In order to enhance the capability of electron transfer, the 1, 1-ferrocene-dicarboxylic acid (FeDC) and gold nanoparticles (AuNPs) were involved the oxidation-reduction in this system. The FeDC as an electron shuttle that was accelerating electron transfer between the enzyme and electrode surface to enhance electron transfer signals, and assists redox biomolecule. AuNPs are used as a conductor that was accelerating electron transfer efficiency between enzyme and electrode surface in a redox reaction. It was explored the sensing feature of FeDC and AuNPs on SPCE strip by cyclic voltammogram (CV). The cathodic peak current of CV result was significantly enhanced 4.2-fold in presence of FeDC and AuNPs. In tyrosinase-SPCE strip, catechol was oxidized to quinone molecules via dehydrogenation by tyrosinase. The o-quinone is an electroactive species that can be reduced at a low potential and the reduction current can be measured in the process. Tyrosinase activity was inhibited by pesticides which cannot redox reaction with substrates, and the production of o-quinone was decreased. Therefore, the concentration of the pesticides can be calculated by the proportion of the current decline. It has main factors can be affected the SPCE strips, including pH, concentration of PBS and working potential. The optimum pH and concentration of PBS were 5.0 and 60 mM, respectively, the best condition of operating voltage at - 200 mV. The maximum response current of the oxidation reaction was obtained within one minute. In this study, tyrosinase biosensor can detect four kinds of phenolic compounds, including catechol, phenol, ρ-cresol and pyrogallol. It also detected for three pesticides, including aldicarb, carbofuran and parathion. The results show the limit of detection (LOD) was 1.9 × 10−4 mM catechol、3.7 × 10−4 mM phenol、6.4 × 10−4 mM ρ-cresol and 1.63 × 10−3 mM pyrogallol, and 5.3 × 10−5 mM aldicarb, 4.5 × 10−5 mM carbofuran and 3.6 × 10−3 mM parathion, respectively. We used both of FeDC and AuNPs on SPCE strip which can increase sensitivity and amplify signal. Moreover, this strip can be successfully used as an ampere measurement system, and tyrosinase-SPCE strip is a potential application to detect phenolic compounds and pesticides residual in environment and foods.