Characterization and Potential Applications of Immobilized Glucose Oxidase and Polyphenol Oxidase

Pyrrole functionalized polystyrene (PStPy) was copolymerized with pyrrole to obtain a conducting copolymer, P(PStPy‐co‐Py) which is used as the immobilization matrix. Glucose oxidase and polyphenol oxidase enzymes were immobilized via the entrapment method by electrochemical polymerization. Enzyme electrodes were prepared by electrolysis at a constant potential using sodium dodecyl sulfate (SDS) as the supporting electrolyte during the copolymerization of PStPy with pyrrole. Maximum reaction rates (V_max) and enzyme affinities (Michaelis‐Menten constants, K_m) were determined for the enzyme entrapped both in polypyrrole (PPy) and P(PStPy‐co‐Py) matrices. Optimizations of enzyme electrodes were done by examining the effects of temperature and pH on enzymes' activities along with the shelf life and operational stability investigations. Glucose oxidase enzyme electrodes were used for human serum analysis and glucose determination in two brands of orange juices. Polyphenol oxidase enzyme electrodes were used for the determination of phenolics in red wines of Turkey.     

Amperometric detection of phenolic compounds by polypyrrole-based composite carbon paste electrodes

This contribution describes new composite carbon paste electrodes (CPEs) for the determination of phenolic compounds. The composite CPEs were prepared by in situ generation of polypyrrole (PPy) within a paste containing the enzyme polyphenol oxidase (PPO). The best paste composition (enzyme/pyrrole monomer/carbon particles/Nujol) was determined for a model enzyme, glucose oxidase (GOx) according to the enzymatic activity of the resulting electrodes and to the enzyme leakage from the paste during storage in phosphate buffer. The in situ electrogenerated PPy improves the enzyme immobilisation within the paste since practically no enzyme was lost in solution after 72 h of immersion. Moreover, the enzyme activity remains particularly stable under storage since the biocomposite structure conserves 80% of its activity after 1 month of storage. Following the optimisation of the paste composition, PPO-based carbon paste biosensors were prepared and presented excellent analytical properties toward catechol detection with a sensitivity of 4.7 A M(-1) cm(-2) and a response time lower than 20 s. The resulting biosensors were applied to the determination of polyphenolic compounds (e.g., epicatechin and ferulic acid).     

Immobilization of invertase and glucose oxidase in poly 2-methylbutyl-2-(3-thienyl) acetate/polypyrrole matrices

Immobilization of invertase and glucose oxidase in conducting polypyrrole and copolymers of poly 2-methylbutyl-2-(3-thienyl) acetate with pyrrole were achieved via electrochemical method. Sodium dodecyl sulphate was found to be the most suitable supporting electrolyte. Maximum reaction rate, Michaelis-Menten constant and optimum temperatures were determined for native and immobilized enzymes. Storage and operational stabilities of enzyme electrodes were also investigated.     

Immobilization of invertase in conducting copolymers of 3-methylthienyl methacrylate

Immobilization of invertase in conducting copolymer matrices of 3-methylthienyl methacrylate with pyrrole and thiophene was achieved by constant potential electrolysis using sodium dodecyl sulfate (SDS) as the supporting electrolyte. Polythiophene (PTh) was also used in entrapment process for comparison. Kinetic parameters, Michaelis-Menten constant, K(m), and the maximum reaction rate, V(max), were investigated. Operational stability and temperature optimization of the enzyme electrodes were also examined.     

Immobilization of Invertase in Copolymer of 2,5-Di(thiophen-2-yl)-1-p-Tolyl-1H-Pyrrole with Pyrrole

Immobilization of invertase in conducting copolymer matrix of 2,5-di(thiophen-2-yl)-1-p-tolyl-1H-pyrrole with pyrrole (poly(DDTP-co-Py)) was achieved via electrochemical polymerization. Kinetic parameters, Michaelis-Menten constant, K_m and the maximum reaction rate, V_max were investigated. Operational stability and temperature optimization of the enzyme electrodes were also examined.

Immobilized invertase reveals maximum activity at 50°C and; pH 8 and pH 4 for two copolymer matrices. Although the same two monomers are utilized for the copolymer synthesis, the way the copolymer is produced results in quite different responses in terms of enzyme activity, optimum pH and kinetic parameters. Excellent operational stability of the enzyme electrodes enables their repetitive use in the determination of invert sugar.     

Simultaneous Determination of Epinephrine and Dopamine by Using Candida tropicalis Yeast Cells Immobilized in a Carbon Paste Electrode Modified with Single Wall Carbon Nanotube

A new electrochemical microbial biosensor system based on Candida tropicalis was developed for the fast detecting of dopamine and epinephrine. Candida tropicalis was immobilized in a carbon paste electrode (CPE) with single wall carbon nanotube (SWCNT). Immobilized cells were used as a origin of the polyphenol oxidase (PPO) to develop voltammetric epinephrine and dopamine biosensor. Voltammetric determination of phenolic compounds such as epinephrine and dopamine a simple technique which is available. Direct oxidation of phenols can be used, but the oxidation potentials of this compounds are similar and they can not be detected distinctively. Another possibility is the use of biosensors based on the polyphenol oxidase (tyrosinase) enzyme that oxidizes the phenolic compounds into their related quinones. By this way, phenolic compounds are epinephrine and dopamine which were used in this study as well detected at different potentials. In this study differential pulse voltammetry and amperometry techniques were used for the determination of dopamine and epinephrine. The effect of varying the amounts of SWCNT and the response of microorganism to epinephrine was investigated to find the optimum composition of the sensor. The effects of pH and temperature were also examined. Increases in biosensor responses obtained by amperometric measurements were linearly related to dopamine concentrations between 0.025 and 0.25 mM and epinephrine concentrations between 0.01 and 0.1 mM. Limits of detection of the biosensor for dopamine and epinephrine were calculated to be 0.008 and 0.0023 mM, respectively. Finally, proposed system was applied to epinephrine and dopamine analysis in pharmaceutical drugs and synthetic serum and the results were compared with LC MS MS method.     

New Amperometric Cholesterol Biosensors Using Poly(ethyleneoxide) Conducting Polymers

Accumulation of cholesterol in human blood can cause several health problems such as heart disease, coronary artery disease, arteriosclerosis, hypertension, cerebral thrombosis, etc. Therefore, simple and fast cholesterol determination in blood is clinically important. In this study, two types of amperometric cholesterol biosensors were designed by physically entrapping cholesterol oxidase in conducting polymers; thiophene capped poly(ethyleneoxide)/polypyrrole (PEO-co-PPy) and 3-methylthienyl methacrylate-co-p-vinyl benzyloxy poly(ethyleneoxide)/polypyrrole (CP-co-PPy). PEO-co-PPy and CP-co-PPy were synthesized electrochemically and cholesterol oxidase was immobilized by entrapment during electropolymerization. The amperometric responses of the enzyme electrodes were measured by monitoring oxidation current of H₂O₂ at +0.7 V in the absence of a mediator. Kinetic parameters, such as Km and Imax, operational and storage stabilities, effects of pH and temperature were determined for both entrapment supports. Km values were found as 1.47 and 5.16 mM for PEO-co-PPy and CP-co-PPy enzyme electrodes, respectively. By using these Km values, it can be observed that ChOx immobilized in PEO-co-PPy shows higher affinity towards the substrate.     

Strong π-delocalization and substitution effect on electronic properties of dithienylpyrrole-containing bipyridine ligands and corresponding ruthenium complexes

The first dithienylpyrrole (DTP)-based bipyridine ligands has been prepared and coordinated with ruthenium to give the corresponding homoleptic complexes. Bipyridine was bound at pyrrole (DTP(1)) or thiophene (DTP(2)) ring. A strong bathochromic effect was obtained by switching from pyrrole to thiophene for ligands and complexes. Interestingly the DTP(2) series offered a wide absorption window from UV to visible domain with an almost constant absorbance. These effects are due to a larger extent of delocalization as supported by DFT calculations and photophysical measurements.     

Conducting graft copolymers of pyrrole and thiophene with random copolymers of methyl methacrylate and 3-methylthienyl methacrylate

Random copolymers of 3-methyl thienylmethacrylate and methyl methacrylate were synthesized via free radical polymerization. Electro-copolymerizations of random copolymers with thiophene and/or pyrrole were carried out in acetonitrile-tetrabutylammonium tetrafluoroborate (TBAFB), water-p-toluene sulfonic acid (PTSA) solvent-electrolyte couples. Oxidative polymerization of thiophene functionalized random copolymer was also achieved by constant current electrolysis and chemical polymerization. The characterizations were done by conductivity measurements, cyclic voltammetry (CV), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), thermal gravimetry analysis (TGA), scanning electron microscopy (SEM).     

Quinoprotein glucose dehydrogenase modified thick-film electrodes for the amperometric detection of phenolic compounds in flow injection analysis

The use of thick-film electrodes as basic transducers for highly sensitive amperometric biosensors using PQQ (pyrroloquinoline quinone) dependent glucose dehydrogenase (GDH) with short response times is described. The enzyme is embedded in a polyurethane matrix on top of a platinum based thick film electrode and its ability to reduce oxidized phenolic compounds is exploited. The electrochemical amplification is based on the oxidation of the analyte on the surface of the electrode followed by its enzymatic reduction. Different parameters of the glucose dehydrogenase electrode system using dopamine as a model analyte were optimized, e.g., membrane thickness, pH value, buffer system, flow rate and storage conditions. Using optimized parameters the sensitivity and detection limits for various phenolic compounds were evaluated. The comparison of electrodes from the identical as well as from different batches shows the ability to produce a number of well reproducible sensors showing remarkably small differences with respect to parameters as sensitivity, response times and measuring range.     

Functionalized conducting polymers for chemical sensors – an in situ ESR/UV‐Vis‐NIR voltammetric study

The redox and optical properties of various well defined polymer and copolymer films containing pyrrole or thiophene units were studied. The in situ ESR/UV‐Vis‐NIR spectroelectrochemistry was applied to investigate polymers and copolymers deposited both electrochemically or by a special chemical procedure using adhesion promoter onto the optically transparent indium‐tin‐oxide (ITO) electrodes. The spectroelectrochemical responses of chemically and electrochemically prepared polythiophenes on ITO were compared and the electronic structures of both polymers found to be similar. In situ ESR/UV‐Vis‐NIR voltammetric studies on electrochemically prepared copolymers containing pyrrole units and various N,N′‐ethylene‐bis(salicylidenimine) (salen) transition metal complexes indicate the presence of both polysalen and polypyrrole redox active centers in the copolymer.     

Quinoprotein glucose dehydrogenase modified thick-film electrodes for the amperometric detection of phenolic compounds in flow injection analysis

The use of thick-film electrodes as basic transducers for highly sensitive amperometric biosensors using PQQ (pyrroloquinoline quinone) dependent glucose dehydrogenase (GDH) with short response times is described. The enzyme is embedded in a polyurethane matrix on top of a platinum based thick film electrode and its ability to reduce oxidized phenolic compounds is exploited. The electrochemical amplification is based on the oxidation of the analyte on the surface of the electrode followed by its enzymatic reduction. Different parameters of the glucose dehydrogenase electrode system using dopamine as a model analyte were optimized, e.g., membrane thickness, pH value, buffer system, flow rate and storage conditions. Using optimized parameters the sensitivity and detection limits for various phenolic compounds were evaluated. The comparison of electrodes from the identical as well as from different batches shows the ability to produce a number of well reproducible sensors showing remarkably small differences with respect to parameters as sensitivity, response times, and measuring range. Received: 15 August 2000 / Revised: 17 October 2000 / Accepted: 24 October 2000     

Poly(pyrrole) versus poly(3,4-ethylenedioxythiophene): amperometric cholesterol biosensor matrices

Two conducting polymers, poly(pyrrole) (PPy) and poly(3,4-ethylenedioxythiophene) (PEDOT) were used as immobilization matrices for cholesterol oxidase (ChOx). The amperometric responses of the enzyme electrodes were measured by monitoring oxidation current of H₂O₂ at +0.7 V in the absence of a mediator. Kinetic parameters, such as K _m and I _max, operational and storage stabilities, effects of pH and temperature were determined for both entrapment supports. K _m values are found as 7.9 and 1.3 mM for PPy and PEDOT enzyme electrodes, respectively; it can be interpreted that ChOx immobilized in PEDOT shows higher affinity towards the substrate.     

Conducting graft copolymers of poly(3‐methylthienyl methacrylate) with pyrrole and thiophene

A thiophene‐functionalized methacrylate monomer (3‐methylthienyl methacrylate) was synthesized via the esterification of 3‐thiophene methanol with methacryloyl chloride. The methacrylate monomer was polymerized by free‐radical polymerization in the presence of azobisisobutyronitrile as the initiator. Graft copolymers of poly(3‐methylthienyl methacrylate) (PMTM2) and polypyrrole and of PMTM2 and polythiophene were synthesized by constant‐potential electrolyses. p‐Toluene sulfonic acid, sodium dodecyl sulfate, and tetrabutylammonium tetrafluoroborate were used as the supporting electrolytes. PMTM2‐coated platinum electrodes were used as anodes in the polymerization of pyrrole and thiophene. Moreover, the oxidative polymerization of poly(3‐methylthienyl methacrylate) (PMTM1) was studied with FeCl₃ as the oxidant. The self‐polymerization of PMTM1 was also investigated by galvanostatic electrolysis both in dichloromethane and in propylene carbonate. The structures of PMTM1 and PMTM2 were investigated by several spectroscopic and thermal methods. The grafting process was elucidated with conductivity measurements, Fourier transform infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis, and scanning electron microscopy studies. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 4131–4140, 2002     

Synthesis and Characterization of Conducting Copolymers of Bisphenol A-Diglycidyl Ether with Thiophene Side-Groups and Pyrrole

Copolymers of bisphenol A-diglycidyl ether with thiophene side-groups and pyrrole were synthesized by electrochemical polymerization. Bisphenol A-diglycidyl ether with thiophene side-groups (DGEBATh) was obtained from the reaction between bisphenol A-diglycidyl ether (DGEBA) and thiophene-3-acetic acid. The syntheses of copolymers of DGEBATh and pyrrole were achieved electrochemically using three different supporting electrolytes, p-toluene sulfonic acid (PTSA), sodium dodecyl sulfate (SDS) and tetrabutylammonium tetrafluoroborate (TBAFB). Characterizations of DGEBATh and copolymers were performed by combination of techniques including cyclic voltammetry, scanning electron microscopy, gel permeation chromatography, differential scanning calorimetry, ¹H-NMR and FT-IR. The conductivities were measured by the four-probe technique.     

Amperometric tyrosinase based biosensor using an electrogenerated polythiophene film as an entrapment support

An amperometric enzyme sensor using tyrosinase, also called polyphenol oxidase (PPO), was constructed for determination of phenolic compounds and herbicides. The enzyme was entrapped in a conducting polymer, poly 3,4-ethylenedioxythiophene (PEDT), electrochemically generated on a glassy carbon electrode. Several experimental parameters in the electropolymerisation process and working conditions were determined to optimise biosensor performances. Mono-phenol and di-phenol were tested in oxygenated solutions, by amperometric measurements at −200 mV (vs. SCE) in a batch system. The limit of detection of these molecules ranges from 5 to 500 nM. Detection of herbicides was obtained from the inhibition of tyrosinase electrode responses. The limit of detection for atrazine and diuron was 1 and 0.5 mg l⁻¹ respectively. These data suggest that PEDT film is a promising PPO immobilisation method.     

Preventing inhibition of tyrosinase with modified electrodes

Wines, especially red wines, contain numerous biologically active compounds, the most important of which are polyphenols, whose nutritional importance is attributed to their antioxidant power. Because of this, the detection of the amount of phenolic compounds in red wines becomes extremely important. However, using free enzyme in the determination of phenolic compounds in wines cannot reflect the actual values since there are also naturally found inhibitors in red wines. In this study, benzoic acid, cinnamic acid, and sorbic acid were utilized to understand the behavior of immobilized polyphenol oxidase in the conducting polymer matrices toward inhibition. Cinnamic acid was found to be the most powerful inhibitor for both free and immobilized enzyme in copolymer matrix of poly(terephthalic acid bis-(2-thiophen-3-yl-ethyl) ester) (PTATE) with polypyrrole (PPy). In the case of immobilized enzyme in PPy matrix, it was observed that sorbic acid is a stronger inhibitor than cinnamic acid. The inhibitory effects of these inhibitors on PPO were compared with respect to both the structural differences of inhibitors and conducting polymer matrices.     

Block copolymers of thiophene-capped poly(methyl methacrylate) with pyrrole

Poly(methyl methacrylate) with a thiophene end group having narrow polydispersity was prepared by the Atom Transfer Radical Polymerization (ATRP) technique. Subsequently, electrically conducting block copolymers of thiophene-capped poly(methyl methacrylate) with pyrrole were synthesized by using p-toluene sulfonic acid and sodium dodecyl sulfate as the supporting electrolytes via constant potential electrolysis. Characterization of the block copolymers were performed by CV, FTIR, SEM, TGA, and DSC analyses. Electrical conductivities were evaluated by the four-probe technique. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 4218–4225, 1999     

Laccase Biosensor Based on N‐Succinimidyl‐3‐Thiopropionate‐Functionalized Gold Electrodes

A laccase biosensor, in which the enzyme was immobilized on N‐succinimidyl‐3‐thiopropionate (NSTP)‐modified gold electrodes, is reported. Two different approaches for the preparation of N‐succinimidyl‐terminated monolayers were evaluated: a) activation of a preformed 3‐mercaptopropionic acid (MPA) SAM by reaction with 1‐(3‐dimethylaminopropyl)‐ 3‐ethylcarbodiimide (EDC) and N‐hydroxysulfosuccinimide (NHS); b) assembling of dithiobisuccinimidyl propionate (DTSP). NSTP‐modified electrodes were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Biosensors prepared by covalent binding of the enzyme and by cross‐linking with glutaraldehyde atop NSTP‐modified electrodes were compared in terms of sensitivity and operational range for caffeic acid. A much better analytical performance was found using the latter approach. Variables affecting the amperometric detection (enzyme loading, pH and applied potential) were optimized. The operational stability and characteristics of functioning of the laccase biosensor in terms of repeatability of the amperometric measurements, reproducibility with different biosensors and useful lifetime, were evaluated. The kinetic parameters of the enzyme reactions and the analytical characteristics of the corresponding calibration plots were calculated for eight phenolic compounds. Limits of detection of 0.07 μM, 0.05 μM and 0.09 μM were obtained for caffeic acid, catechol and 3,4‐dihydroxyphenylacetic acid (DOPAC), respectively. The practical usefulness of the developed biosensor was evaluated by estimating the “pool” of phenolic compounds in olive oil mill wastewaters (OMW).     

Improvement of poly(amphiphilic pyrrole) enzyme electrodes via the incorporation of synthetic laponite-clay-nanoparticles

The electropolymerization of an enzyme-amphiphilic pyrrole ammonium-laponite nanoparticles mixture preadsorbed on the electrode surface provides the simultaneous immobilization of the enzyme and the hydrophilic laponite-clay-nanoparticles in a functionalized polypyrrole film. The presence of incorporated laponite particles within the electrogenerated polymer induces a strong improvement of the analytical performances (I_max and sensitivity) of amperometric biosensors based on polyphenol oxidase. These beneficial effects have been attributed to a marked enhancement of the apparent specific activity of the immobilized enzyme (from 0.21 to 0.85% of the specific activity of the free enzyme), the permeability of the host polymer being unchanged. This strategy of biosensor performance improvement was tested with cholesterol oxidase as an enzyme model. The presence of laponite additive in the poly(amphiphilic pyrrole) host matrix induces a similar enhancement of sensitivity and I_max for cholesterol biosensing as well as a large improvement of the storage stability of the polypyrrole-cholesterol oxidase electrode.