Amperometric Detection of Catechol and Catecholamines by Immobilized Laccase from DeniLite

DeniLite laccase immobilized Pt electrode was used for detection of catechol and catecholamines. The enzymatically oxidized substrates were measured amperometrically. The sensitivities are 210, 75, 60 and 45 nA/μM with the upper limits of linear ranges of 58, 40, 55 and 55 μM and the detection limits (S/N=3) of 0.07, 0.2, 0.3 and 0.4 μM for catechol, dopamine (DA), norepinephrine (NEPI) and epinephrine (EPI), respectively. The response time (t_90%) is about 2 seconds for each substrate and the long‐term stability is around 40–50 days with retaining 80% of initial activity. The very fast response and the remarkable long‐term stability are the principal advantages of this sensor. In case of catechol, the pH response of the sensor is mainly determined by enzyme's pH profile, however, in case of catecholamines, both enzyme's pH profile and reversibility of the substrate are operated and the optimal pHs for NEPI and EPI shift towards acidic range compared to that for DA. The presence of ascorbic acid (<50 μM) did not interfere with the measurement.     

伏安型植物组织生物微电极的研究——茄子组织碳糊微电极的研究

茄子组织生物微电极在儿茶酚测定中表现出了较高的生物催化活性,而对抗坏血酸测定则为稳态响应,这表明该电极能有效地消除抗坏血酸的干扰。该电极的灵敏度高,重现性好,对多巴胺测定的线性范围为5.8×10~(-6)～6.5×10~(-4)mol/L,检出下限为:2.9×10~(-6)mol/L。     

阻抑动力学光度法测定痕量邻苯二酚

在pH8．0的KH2PO4-Na2HPO4缓冲介质中，Mn(Ⅱ)催化高碘酸钾氧化二甲酚橙的褪色反应，邻苯二酚可灵敏地阻抑。研究了该指示反应的动力学参数及阻抑反应的机理，建立了动力学光度法测定痕量邻苯二酚的新方法。方法检出限为12．3ng／mL，线性范围在0．5～5．0μg／25mL和6．0～11．0μg／25mL。采用分馏法用于污水中邻苯二酚的测定，结果满意。     

Cyclic Voltammetric Study of the Oxidation of Catechols in the Presence of Cyanide Ion

Electrochemical oxidation of catechols has been studied in the presence of cyanide ion as nucleophile in aqueous solution, by means of cyclic voltammetry and controlled‐potential coulometry. The results indicate that the participation of catechols in the Michael reaction with cyanide ion to form the corresponding o‐dihydroxybenzonitrile. Based on an EC mechanism, the homogeneous rate constants were estimated by comparing the experimental cyclic voltammetric responses with the digital simulated results.     

伏安型植物组织生物微电极的研究：混合不同植物组织碳糊微电极的研究

由于香蕉组织中含有的氧化酶能够生物催化氧化多巴胺，去甲肾上腺素，Ｌ－多巴等，大大地提高了植物组织电极的灵敏度；又由于茄子组织中所含有的氧化酶在催化氧化儿茶酚的同时，能有效地抑制抗坏血酸在电极上的反应，将上述两种植物组织混合制成电极，使其更适于神经递质的测定，线性范围：２．４×１０＾－６－－９．８×１０＾－４ｍｏｌ／Ｌ；检出下限３．２×１０＾－７ｍｏｌ／Ｌ。     

正交试验选择组织电极的最佳分析条件

本文用正交试验法对花椰菜组织Ｌ－抗坏血酸传感器和香蕉组织儿茶酚类化合物传感器的分析条件进行了优化，通过与单因子分析方法及４种儿茶酚类化合物之间正交试验结果的比较，说明用正交试验选择组织传感器的最佳分析条件，在少量的试验次数内，得到较多的分析实验信息，是一种较理想的试验方法。     

Nucleophilic Addition of Thiaproline to Electrochemically Derived o‐Quinone,Application to the Sensitive Voltammetric Detection of Thiaproline

The mechanism of electrochemical behavior of catechol in the presence of thiaproline is investigated by cyclic voltammetry, controlled‐potential coulometry and spectrophotometric tracing of the reaction coordinate. The results indicate that thiaproline participate in with an ECEC mechanism in a nucleophilic (Michael) addition to o‐quinone. Effect of pH of buffer solution on reaction pathway is studied and showed that addition of thiaproline to the o‐quinone is performed only in solutions with pHs higher than 5. These results indicate that the addition of thiaproline is occurred first from amine functional group. In the second step, the addition of carboxylate group of thiaproline to C‐5 of catechol results the final product with a lactone ring in its structure. Observation of two isosbestic point in absorption spectrum during the progress of the electrolysis together with the FT‐IR results for final product can be presented as evidence for two step addition of thiaproline to catechol. Final product, due to the electron donor property of thiaproline, more easily oxidized respect to the former catechol and as a result, a new redox couple is obtained for this compound in lower potentials. The easier anodic oxidation of addition product (relative to catechol) caused to an increase in anodic current for catechol, which is proportional to the thiaproline concentration. The differential pulse voltammetry (DPV) is applied as a sensitive voltammetric method for the detection of thiaproline. A linear range from 5×10^?8 to 5×10^?6 M with a detection limit of 1×10^?8 M is resulted for thiaproline. With respect to the reversibility of the electrochemical reactions in the mechanism, and also more facile oxidation of the addition products, the square‐wave voltammetry is presented as a method with considerably more sensitivity for the detection of sub‐micromolar amounts of thiaproline. The advantageous properties of the voltammetric method for thiaproline detection lie in its excellent catalytic activity, sensitivity and simplicity.     

Chromatographic molecular recognition for catechol-related compounds using thiacalix[4]arene as an effective selector

Thiacalix[4]arene (5,11,17,23-tetra-tert-butyl-25,26,27,28-tetrahydroxy-2,8,14,20-tetrathiacalix[4]arene) is an amphiphilic molecule comprising four p-tert-butylphenol-like groups ortho-linked by single sulfur atoms. This molecule has a high electron density area owing to the close proximity of the hydroxyl groups and sulfur atoms. We studied the applicability of this interesting compound as a selector for high-performance liquid chromatography (HPLC) thereby presumably exploiting this feature. Firstly, uniformly sized polymer particles were prepared by using a multi-step swelling and polymerization method with ethylene glycol dimethacrylate (EDMA) as a cross-linker. Methacrylic acid (MAA) was introduced onto the surface of the resulting polymer particles through a new modification method. Thiacalix[4]arene was chemically bonded through the MAA group by using 1,4-dibromobutane as a spacer to reduce steric hindrance around the MAA and the polymer particle itself. The performance of the prepared polymer-based thiacalix[4]arene-modified stationary phase was evaluated with HPLC. Specific chromatographic retention behavior was observed for catechol relative to positional isomers of xylene, cresol, and benzene-diol. Catecholamine and catechol showed specific chromatographic retention behavior.     

Laccase Biosensor on Monolayer‐Modified Gold Electrode

Laccase enzymes from two different sources, namely, tree laccase from Rhus vernicifera and fungal laccase from Coriolus hirsutus were used for the development of biosensor for catechol. Laccase was immobilized onto the amine terminated thiol monolayers on gold surface by glutaraldehyde coupling. From the different thiol monolayers investigated, cystamine was found to be optimal with respect to sensitivity, stability, reproducibility, and other electrochemical properties of the enzyme electrode. Linear calibration in the range between 1 and 400 μM for catechol was obtained for fungal laccase covalently coupled on the electrode surface. The kinetic parameters determined using the Lineweaver‐Burk and Eadie‐Hofstee plots were K_m=0.65 mM and V_max=24.5 μA for fungal laccase compared to K_m=5.4 mM and V_max=6.6 μA for tree laccase on cystamine monolayer. The electrode showed good stability for 1 month without loosing appreciable activity when stored dry in a refrigerator at ?20 °C.     

Kinetic studies of dicopper complexes in catechol oxidase model reaction by using an approximationless evaluating method

The oxidation of 3,5-di-tert-butylcatechol to 3,5-di-tert-butyl-o-benzoquinone catalyzed by dinuclear copper(II) complexes {[Cu₂(L¹)(CF₃SO₃)₂(H₂O)₄]-(CF₃SO₃)₂ (1) and [Cu₂(L²O)](CF₃SO₃)₂ (2)| has been investigated in methanol saturated with O₂ at ambient temperature. Detailed kinetic studies were carried out and for the treatment the fitting software ZiTa was applied. On the basis of the results in the kinetic studies a possible mechanism of the catalytic reaction is proposed.This revised version was published online in December 2005 with corrections to the Cover Date.