Wiring Laccase on Covalently Modified Graphene: Carbon Nanotube Assemblies for the Direct Bio‐electrocatalytic Reduction of Oxygen

Reduced graphene oxide (RGO) was covalently functionalized by the in situ generation and reduction of anthraquinone diazonium salt. Deposition on multi‐wall carbon nanotube (MWCNT) electrodes prevents the aggregation of RGO nanosheets and allows the stable deposition of modified graphene, accompanied with excellent electron transfer properties. Laccases were immobilized on the nanostructured electrode by the interaction between the anthraquinone moiety and the laccase hydrophobic pocket located near the T1 copper center. The MWCNT/f‐RGO electrode exhibits efficient bioelectrocatalytic oxygen reduction, with current densities of up to 0.9 mA cm^?2.     

Laccase Containing Sol-Gel Based Optical Biosensors

Abstract

Laccase-containing sol-gels were synthesized by hydrolysis and condensation-polymerization of tetramethylorthosilicates. Two types of laccase containing sol-gel based optical biosensors were designed and tested. The first type is based on 2–8 mm thick monolith sol-gel blocks. It was observed that laccase-containing sol-gel blocks accumulate high molecular weight products of oxidation of the laccase substrate - 2,6-dimethoxyphenol. This leads to at least ten fold increase of optical bioassay sensitivity in comparison with the homogeneous phase. The response time of such biosensors is 3–24 hours. The second type of biosensor is based on laccase-containing 0.3 mm diameter solgel particles in a flow cell and includes an optical fiber measuring system. This sensor is characterized by a fast response time in comparison with the first type. The latter biosensor also possesses a homogeneous phase assay sensitivity and yields a linear calibration curve.

     

壳聚糖-g-N-羧甲基-2-硫代-4,5-2H咪唑啉酮-多壁碳纳米管复合物固定漆酶基化学传感器的性能

通过壳聚糖-g-N-羧甲基-2-硫代-4,5-2H咪唑啉酮(CTS-g-N-CSIDZ)非共价功能化多壁碳纳米管(MWCNTs)的方式制备固定漆酶载体,该复合物载体主要通过物理吸附和漆酶活性中心与载体上配体之间的配位作用来固定漆酶,较大程度地保持了游离漆酶活性位原始构象.将固定了漆酶的复合物附着在裸玻碳电极上便构筑了复合物固定漆酶修饰玻碳电极.在以分光光度法测定了这种复合物载体对漆酶的担载量、固定漆酶比活力、稳定性、重复使用性及其催化2,6-二甲氧基苯酚(DMP)氧化动力学参数的基础上,还对基于此种复合物固定漆酶修饰玻碳电极作为化学传感器(以DMP作为底物)的性能进行了研究.结果表明,该复合物具有较高的固酶担载量(81.7 mg/g)和固定漆酶比活力(1.33 U/mg);而作为电化学传感器的复合物固定漆酶修饰玻碳电极对底物DMP具有较高的亲和力(对DMP的米氏常数KM是0.0918 mmol/L),较高的灵敏度( 3680 mA· L/mol),较低的检测限(3.3×10-4 mmol/L),较高的响应选择性,良好的重现性、重复使用性和长期稳定性.这种漆酶基电极有望用作电流型特定结构的酚类传感器.     

Nanobiomolecular Multiprotein Clusters on Electrodes for the Formation of a Switchable Cascadic Reaction Scheme

A supramolecular multicomponent protein architecture on electrodes is developed that allows the establishment of bidirectional electron transfer cascades based on interprotein electron exchange. The architecture is formed by embedding two different enzymes (laccase and cellobiose dehydrogenase) and a redox protein (cytochrome c) by means of carboxy‐modified silica nanoparticles in a multiple layer format. The construct is designed as a switchable dual analyte detection device allowing the measurement of lactose and oxygen, respectively. As the switching force we apply the electrode potential, which ensures control of the redox state of cytochrome c. The two signal chains are operating in a non‐separated matrix and are not disturbed by the other biocatalyst.     

大尺寸SiO2大孔材料固定化漆酶

以表面固定Cu2+的改性大尺寸SiO2大孔材料作为载体,考察了时间、pH和给酶量对漆酶固定化效果的影响,并对固定化漆酶的活性和稳定性进行了研究。结果表明:5 h时吸附达到平衡,pH为4.5、漆酶与载体比例为5 mg·g-1时固定化效果最好,酶活回收率可达到100.4%;固定化漆酶的最适pH和最适温度较游离漆酶的均有升高且范围变宽,固定化后,漆酶的pH稳定性和热稳定性都得到显著提高;固定化漆酶的K m值略高于游离漆酶的;固定化漆酶具有良好的操作稳定性,与底物反应反复操作10批次后剩余酶活为72.7%。     

基于分子模拟和光谱法分析漆酶与甲酚的相互作用

该文运用计算模拟与光谱法研究了甲酚与漆酶的相互作用。首先,计算模拟表明漆酶与3种甲酚同分异构体都能发生相互作用,分子对接研究结果表明漆酶与甲酚同分异构体能以氢键和疏水作用力相结合,且结合位点相似。通过分子动力学模拟比较漆酶结合甲酚前后残基的柔性差异,验证了分子对接结合位点的可靠性。其次,选取计算模拟中结果较好的间甲酚,利用光谱法探究间甲酚与漆酶的荧光猝灭机制及结合前后漆酶二级结构的变化。荧光猝灭实验证实漆酶与间甲酚间是形成非荧光复合物的静态猝灭,与分子对接结果一致。红外光谱研究结果表明,漆酶与间甲酚结合后二级结构发生变化,其内部的β-转角和β-反向平行结构向β-折叠、无规则卷曲和α-螺旋结构转化,这与分子动力学模拟结果相呼应。该研究为利用漆酶转化环境中甲酚污染物提供了理论基础与数据支持。     

A Laccase/Chitosan‐Lambda‐Carrageenan Based Voltammetric Biosensor for Phenolic Compound Detection

In this contribution, a new concept of voltammetric catechol biosensor, based on the encapsulation of laccase (LAC) in a chitosan+lambda‐carrageenan (CHIT+CAR) polyelectrolyte complex (PEC) employing a simple coacervation process is presented. Chitosan (CHIT) was prepared from α‐chitin extracted from shrimp shells and lambda‐carrageenan (CAR) was extracted from red algae, both polysaccharides and PEC being characterized using FTIR spectrometry and electrochemistry. Cyclic voltammetry was utilized to determine the analytical features of the laccase (LAC) biosensor for catechol detection. The linear range was from 10^?20 M to 10^?14 M with a sensitivity of 1.55 mA/p[catechol] and a limit of detection of 3×10^?21 M.The laccase biosensor exhibits good repeatability (RSD 2.38 %) and stability (four weeks). The developed biosensor was tested by applying it to the evaluation of the total polyphenolic content in natural oil samples.     

Sensitive and renewable picloram immunosensor based on paramagnetic immobilisation

Picloram (4-amino-3,5,6-trichloro-2-pyridincarboxylic acid) is one of the chlorinated pesticides. It is widely used for control of wood plants, wheat, barley and wide range of broadleaf weeds as a plant growth regulator. An immunosensor was developed for detection of picloram concentration in compost extracts and river water. The laccase-picloram was prepared. The magnetic core-shell (Fe₃O₄-SiO₂) nanoparticles were modified with anti-picloram-IgG and attached to the surface of carbon paste electrode (CPE) with the aid of paramagnetism. Following competitive immunoreaction with picloram and the picloram-laccase to form immunocomplex, electrochemical measurement was carried out. After immunoassay, the electrode was immersed in glycin-hydrochloric acid buffer or polished with diamond paper for regeneration. The linear range for picloram detection was 1?×?10^–4–10?µg?mL^–1 with the correlation coefficient of 0.9936, and the detection limit is 1?×?10^–4?µg?mL^–1. The laccase labelled on the picloram for competitive immunoassay showed good activity, and the current response was strong and stable in electrochemical detection. The current reached 95% of the steady-state current within about 100?s. The proposed immunosensor exhibited good precision, sensitivity, selectivity, reusability, and storage stability.     

The Thermophilic CotA Laccase from Bacillus subtilis: Bioelectrocatalytic Evaluation of O2 Reduction in the Direct and Mediated Electron Transfer Regime

The thermophilic bacterial laccase CotA from Bacillus subtilis adsorbed on graphite electrodes enables monitoring of the temperature dependent direct biocatalytic O₂ reduction. Its entrapment in two different Os‐complex modified redox hydrogels is the basis for mediated bioelectroreduction of O₂. Besides the temperature and pH dependence of the bioelectrocatalytic response chloride and fluoride inhibition studies demonstrate that the Os‐complexes are bound to the T1 copper centre of the enzyme. The interaction between CotA and the Os‐complex modified polymers prevents inhibition by chloride ions.