Methylphenazonium-Modified Enzyme Sensor Based on Polymer Thick Films for Subnanomolar Detection of Phenols

Abstract

A methylphenazonium-zeolite-modified enzyme sensor based on a planar, screen-printed, two-electrode arrangement is described for the subnanomolar detection of phenols. Using tyrosinase (EC 1.14.18.1), a novel polyurethane hydrogel was applied for immobilization and stabilization of the enzyme, which forms a self-adhering layer on the active surface of the strip sensor. Performance and characteristics of the sensor were evaluated with regard to response time, detection limit, selectivity, and dependences on temperature and pH as well as operating and storage stabilities. The sensor shows marked sensitivity to eight phenolic compounds usually present in industrial waste waters. The detection limit for phenol was obtained with 0.25 nM. Comparing sensor responses with DIN and EPA standard methods for the chemical analyses of both synthetic and real sample matrices, the results indicate the feasibility of the strip sensor for sensitive determination of phenols in field analysis.Phenolic compounds are important but toxic starting materials in a broad range of chemical manufacturing processes. Especially in coal conversion industry, phenolic residues are considered an acute enviromental problem. Often soil and surface waters of areas around former production and processing plants are contaminated by phenolic compounds with risk to ground water resources. Therefore, screening, monitoring, and control of these pollutants are of great importance.Photometric analyses by DIN and EPA standard methods12 are now commonly used for the determination of phenols. These procedures usually require sample pretreatment by filtration and distillation. Consequently, a simple and fast alternative method for the determination of phenols is desirable. For this purpose, biosensors based on phenol-oxidizing oxygenases appear promising; tyrosinase (EC 1.14.18.1) and as laccase (EC 1.10.3.2) are suitable receptor compounds, although the catalyzed reactions are different.* 123 Basic studies of physicochemical properties and catalyzed pathways of tyrosinase have shown that the tetrameric enzyme, + Umweltforschungszentrum Leipzig-Halle. * Institut ftir Chemo-und Biosensorik. § Bundesforschungsanstalt fur Landwirtschaft. (1) Deutsche Einheitsverfahren zur Wasser-, Abwasser-, und Schlammuntersuchung Band III; Summarische Wirkungs und Stoffkenngrdssen (Gruppe H); DIN 38409-H16; Bestimmung des Phenolindex; VCH: Weinheim, Germany; Jun 1984.(2) Methods for chemical analysis of water and wastes; EPA manual 600/4-79-020, Method 420.1:

Biossensores amperométricos para determinação de compostos fenólicos em amostras de interesse ambiental

“…Supõe-se que este efeito de amplificação é responsável pelos baixos limites de detecção reportados para fenol e o-difenol 34,35,63,66,71,75 . Os mediadores ou modificações de superfície de maior sucesso para tal propósito são o tetracianoquinodimetano (TCNQ) 73 e metilfenazônio 23 . Eletrodos enzimáticos a base de tirosinase têm sido utilizado para a determinação seletiva de fenóis em matrizes ambientais 1,2,6,8,65,66,69,[76][77][78] .…”

“…Os biossensores revelam grandes perspectivas quanto a sua utilização no monitoramento "on-line" de efluentes (e outras matrizes de interesse ambiental), possibilitando uma rápida adaptação nos processos de tratamento. A incorporação de moléculas com atividade biológica em metodologias analíticas tem aumentado sensivelmente nos últimos anos, obtendo sucesso nos mais variados procedimentos analíticos, principalmente nos que visam a área de controle ambiental 5,[14][15][16][17][18][19][20][21][23][24][25][26][27][28][29][30][31][32][33][34][35][36] . Biossensores representam uma ferramenta promissora para suplementar as técnicas existentes, devido as suas características únicas, tais como: seletividade; relativo baixo custo de construção e estocagem; potencial para miniaturização; facilidade de automação e construção de equipamentos simples e portáteis para um monitoramento "on site" rápido.…”

Sensing Characteristics of Tyrosinase Immobilized and Tyrosinase, Laccase Co-immobilized Platinum Electrodes

“…2,[21][22][23] This provides the additional advantages of the enzymatic/ electrochemical recycling of the substrate, giving rise to an amplification of the signal (Figure 1). We have reported the detection of di-phenols such as PPD, PAP (p-aminophenol), 24,25 catechol, and catecholamines 26 with DeniLite TM laccase covalently immobilized platinum electrode, in which substrate recycling principle was employed and the obtained amplification factors were about 10−30.…”

“…This sensitivity order of tyrosinase sensor for the three substrates was also reported by other authors. 2,50,51 The sensitivities of the sensor for phenol and p-cresol are 122 and 139 nA/µM, respectively. These values are comparable to that of Deng et al reported (43 nA/µM or 127 nA/ µM for phenol, φ 3 mm glassy carbon electrode), 21,52 Wang et al reported (80 nA/µM for phenol, electrode size unknown), 53 and Freire et al reported (100 nA/µM for pcresol, 20 carbon fibers, φ 8 mm for each), 28 etc.…”

“…It was reported that tyrosinase can oxidize p-aminophenol which is a structural analogue of PPD, and the relative sensitivity of tyrosinase sensor for this substrate was 25% of that for catechol. 2 Yaropolov et al reported that the relative responses of laccase electrode and bi-enzyme (tyrosinase and laccase) electrode for catechol were 87 and 590, respectively. 20 In that case, the largely increased response after additional immobilization of tyrosinase with laccase originated from the relatively high response of tyrosinase for catechol (relative response of 700).…”