Biosensors Based on Laccase for Detection of Commercially Reactive Dyes

The results obtained using Trametes versicolor laccase modified graphite electrodes for detection of eleven different commercially reactive dyes are presented for the first time herein. The increase in current upon injection of the analyzed substrate was shown to be approximated by Michaelis–Menten type dependence. The calculated kinetic constants were used to evaluate the applicability of the laccase modified graphite electrode for the detection of reactive dyes in textile effluents.     

Dioxygen reactivity of laccase

A study was carried out on the dioxygen reactivity of the laccases from Trametes villosa, Rhizoctonia solani, Myceliophthora thermophila, Scytalidium thermophilum, and Coprinus cinereus. At pH 5.5, these laccases showed an apparently constant K _m (about 20–50 μM) for O₂ with either 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) or methyl syringate as the reducing substrate, in contrast to the k _cat , which varied up to 100-fold. O₂ reactivity of the Trametes and Myceliophthora laccases was also studied at various pH and NaF concentrations. The apparent K _m of Trametes and Myceliophthora laccases varied only slightly when pH changed from 3.0 to 8.0 or when the laccases were inhibited by F⁻ at pH 5.5, although the apparent k _cat were more significantly affected by both factors. The dependence of the apparent K _m on the source of laccase, pH, and F⁻ inhibition suggested that the fungal laccases might have a conserved O₂ pocket and that the F⁻ or OH⁻ inhibition might affect the O₂ reduction step (k _cat ) more than the O₂ binding step (K _m ) under steady-state conditions.     

介孔碳掺杂氮材料-壳聚糖固定漆酶电极的直接电化学行为及化学传感性能

以壳聚糖和介孔碳氮材料共混所得复合物为固定漆酶的载体,将固酶复合物滴涂在裸玻碳电极表面并干燥后,得到固定漆酶基阴极.考察了此电极在不含底物的电解质溶液中的直接电化学行为,同时还研究了其对氧气还原反应的催化性能和电极的长期使用性、重现性和力学稳定性.在此基础上还考察了此电极作为氧气电化学传感器的性能.研究结果表明,介孔碳氮材料-壳聚糖固定漆酶修饰电极能在无任何电子中介体条件下,实现漆酶活性中心T1与电极之间的直接电子转移,而且能在较高的电位下实现氧气的电还原.此电极催化氧还原的起始电位约为860 mV,氧还原的半波电流密度约为78×10-6 A/cm2.这种漆酶基电极的重现性良好且具有优异的长期稳定性,但力学稳定性较差.此电极对氧的传感性能良好:检测限低达0.4 μmol/L,灵敏度高达(67.9×10-6A·L/mmol),具有良好的对氧亲和力(KM =764.0 μmol/L).     

Laccase

The present review was dedicated to laccase—the enzyme, belonging to the group of multinuclear copper, containing so called “blue” oxidases. The molecular structure, metals content, substrate specificity, and other physicochemical properties were described in this article. The authors considered the mechanism of enzymatic action and electrocatalytic oxygen reduction catalyzed by laccase in details. The data of laccase application in organic synthesis, biosensors, and immunoenzyme assay were presented.     

漆酶电极的研制及应用

本文报道二茂铁修饰电极为基底的漆酶电极的研制及应用，讨论了电极性能及响应机理，找出了实验最佳条件。并对环境污水中对苯二酚含量进行规定。一结果令人满意。     

4-巯基苯甲酸功能化纳米金粒子固定葡萄糖氧化酶/漆酶基燃料电池性能

以4-巯基苯甲酸修饰纳米金粒子作为固酶载体和导电基体构建了新型纳米结构固酶葡萄糖/O₂燃料电池,其制备简单,长期使用性能稳定。利用纳米金粒子通过表面修饰基团和酶分子活性中心附近疏水结合位之间的相互作用固定葡萄糖氧化酶(GO_x)和漆酶(Lac)分子,分别制备了固酶阳极-4-巯基苯甲酸功能化纳米金粒子固定葡萄糖氧化酶修饰金盘电极GO_x/4-MBA@GNP/Au和固酶阴极-4-巯基苯甲酸功能化纳米金粒子固定漆酶修饰金盘电极Lac/4-MBA@GNP/Au。电化学实验结果表明,两种电极在不引入任何外加电子中介的条件下,均可以实现酶活性中心-纳米金粒子之间的直接电子迁移,而且具有较快的催化反应能力(固酶阳极和阴极的转化速率分别为1.3和0.5 s^-1;催化葡萄糖氧化和氧气还原的起始电位分别为-0.23和0.76 V)。评估了固酶阳极和阴极组装成的纳米结构固酶葡萄糖/O₂燃料电池的能量输出性能。该燃料电池在没有Nafion薄膜和阳极无N₂气保护下,开路电压和最大输出能量密度分别可达0.56 V和760.0 μW/cm²,使用一周后输出能量密度仍然可以达到最初值的~88%。进一步测试结果显示,该燃料电池呈现出与游离漆酶类似的pH依赖关系和热稳定性,这些实验结果均暗示:影响整个酶燃料电池性能的关键在于漆酶基阴极催化氧还原的过程。此外,这种燃料电池的性能虽然受到共存干扰物抗坏血酸的影响,但在人类血清中测试结果显示其仍然具有较高的输出能量密度(132.0 μW/cm²,开路电压0.40 V)。本文研究结果给出了设计高性能葡萄糖/O₂燃料电池的新思路,同时也为研究固酶燃料电池的构效关系提供了实验依据和有价值的启示。     

环氧基修饰周期性介孔有机氧化硅对漆酶的固定化作用

以三嵌段共聚物P123为模板剂, 在酸性条件下通过1,2-三乙氧基硅基乙烷(BTESE)和3-含氧缩水甘油基-丙基-三甲氧基硅烷(GPTMS)共水解缩聚合成环氧基修饰的周期性介孔氧化硅(PMOs), 以修饰后的PMOs为载体对漆酶进行固定化, 研究了环氧基修饰对固定化酶稳定性的影响. 通过XRD、TEM、固态NMR和N₂吸附等手段表征材料的修饰效果、孔结构以及漆酶的固定化. 结果表明, 修饰后的材料保持良好的孔结构, 环氧基的修饰有利于提高固定化酶的活力, 基于环氧基修饰PMOs的固定化酶具有较高稳定性.     

Development of Electrochemical Biosensor Based on Nanostructured Carbon Materials for Paracetamol Determination

This work describes the development of a biosensor for paracetamol (PAR) determination based on a glassy carbon electrode (GCE) modified with multiwalled carbon nanotubes (MWCNT) and laccase enzyme (LAC), which was immobilized by means of covalent crosslinking using glutaraldehyde. Voltammetric investigations were carried out by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and square wave voltammetry (SWV). The biosensor was characterized by Scanning Electron Microscope (SEM) and Fourier Transform Infrared Spectroscopy (FT‐IR). The results showed that the use of MWCNT/LAC composite increased the sensor sensitivity, compared to bare glassy carbon electrode. Factors affecting the voltammetric signals such as pH, ionic strength, scan rate and interferents were assessed. Linear range, limit of detection (LOD) and limit of quantitation (LOQ) obtained were 10–320 μmol L^?1, 7 μmol L^?1 and 10 μmol L^{? 1}, respectively. The developed biosensor was successfully applied to PAR determination in urine and pharmaceutical formulations samples, with recovery varying from 99.96 to 106.20 % in urine samples and a relative standard deviation less than 1.04 % for PAR determination in pharmaceutical formulations. Therefore, the MWCNT‐LAC/GCE exhibits excellent sensitivity and can be used to PAR determination as a viable alternative in clinical analyzes and quality control of pharmaceutical formulations, through a simple, fast and inexpensive methodology.     

High performance liquid chromatography coupled to an optical fiber detector coated with laccase for screening catecholamines in plasma and urine

An analytical method based on separation by high performance liquid chromatography (HPLC) and detection by optical fiber (OF) coated with an enzyme (laccase), has been developed for separation and quantification of catecholamines, namely epinephrine, dopamine and norepinephrine. The application of OF as a detector in this analytical system relies on the variation of the reflected optical power detected when the catecholamines eluted from the HPLC column act as the substrate of the laccase immobilized on a tip of a single-mode OF. The developed method shows a high linearity in a range between 5 and 125 pg/mL and detection limits of 3.5, 2.9 and 3.3 pg/mL for epinephrine, dopamine and norepinephrine, respectively. The analytical performance of the proposed method was compared with a classical analytical method, namely high performance liquid chromatography-electrochemical detector (HPLC-ED) regarding catecholamines detection, showing great analytical advantages such as low cost of equipment. Additionally, the proposed method was applied to catecholamines determination in actual samples of plasma and human urine.     

Electrochemical Studies of Intramolecular Electron Transfer in Laccase from Trametes versicolor

This paper describes electrochemical behavior of laccase from the fungus Trametes versicolor. The issues related to discrimination of the redox potentials corresponding to copper centers T1 and T2/T3 in the active site and possible mechanism of intramolecular electron transfer have been discussed. The electron‐transfer rate constant for laccase immobilized on carbon electrode is 3.4 s^?1. The bioelectrocatalytic activity of the enzyme was studied in the presence of 1,4‐hydroquinone (HQ). The kinetics of HQ oxidation is very fast (K_M=3.8 μM). However, the catalytic activity of laccase in the presence of high concentration of HQ decreases drastically. It is suggested that the T2/T3 copper center is able to accept electrons from HQ molecules directly via intramolecular channel.