Amperometric selective biosensing of dimethyl- and diethyldithiocarbamates based on inhibition processes in a medium of reversed micelles

An amperometric tyrosinase electrode has been used for biosensing of dimethyl- and diethyldithiocarbamates based on the inhibition effects of these substances on the catalytic activity of the enzyme. A working medium consisting of reversed micelles, and phenol as the substrate has been used. The tyrosinase electrode was constructed by direct adsorption of the enzyme on the surface of a graphite-disk electrode. Reversible inhibition processes are shown to be involved for ziram, diram and zinc diethyldithiocarbamate. Following a simple regeneration of the enzyme electrode, an acceptable reproducibility for the measurements of the inhibition response was obtained. Experimental variables, such as temperature, phenol concentration and the presence of chloroform, affecting the inhibition processes, were optimized. The type of enzyme inactivation for each inhibitor tested was studied, and the inhibition constants were calculated. Detection limits of 0.074, 1.3 and 1.7 μmol l⁻¹ were achieved for ziram, diram and zinc diethyldithiocarbamate, respectively. Other carbamates belonging to families different from dimethyl- and diethyldithiocarbamates showed no amperometric response at the tyrosinase electrode, except for pyrimidine-derivative carbamates. The developed analytical methodology was applied to determine ziram in spiked apple samples.     

Screen-printed multienzyme arrays for use in amperometric batch and flow systems

Screen-printing technology for electrode fabrication enables construction of amperometric devices suitable for combination of several enzyme electrodes. To develop a biosensor array for characterisation of wastewaters, tyrosinase and horseradish peroxidase (HRP) or cholinesterase-modified electrodes were combined on the same array. The behaviour of the tyrosinase-modified electrode in the presence of hydrogen peroxide (required co-substrate for the HRP-modified electrode) and acetylthiocholine chloride (required co-substrate for cholinesterase) was studied. Performance of bi-enzyme biosensor arrays in the batch mode and in the flow-injection system are discussed.     

Detection of phenolic compounds in flow systems based on tyrosinase-modified reticulated vitreous carbon electrodes

The fabrication and performance of a reticulated vitreous carbon (RVC)-based tyrosinase flow-through electrode, in which the enzyme was covalently immobilized, is reported. The bioelectrode was tested as an amperometric detector for phenolic compounds. Variables affecting the construction of the enzyme flow-through electrode such as the RVC chemical pretreatment procedure, the enzyme immobilization method in the RVC matrix, the enzyme loading and the pH value of the buffer solution used, were optimized by flow-injection with amperometric detection. A good immobilization of the enzyme in the RVC matrix, in spite of the hydrodynamic conditions, was found. The same tyrosinase-RVC electrode could be used with no significant loss of the amperometric response for around 20 days, and reproducible responses could be achieved with different electrodes constructed in the same manner. Moreover, the operational stability of the bioelectrode was tested under continuous monitorization conditions. Calibration plots by flow injection with amperometric detection at -0.20 V were obtained for phenol, 2,4-dimethylphenol; 3-chlorophenol; 4-chlorophenol; 4-chloro-3-methylphenol and 2-aminophenol, with detection limits ranging from 2 mug l(-1) (4-chloro-3-methylphenol) to 2 mg l(-1).     

Silicone-grease-based immobilisation method for the preparation of enzyme electrodes

An approach to the construction of amperometric biosensors based on the incorporation of an enzyme in silicone grease and using the grease to fill micropores on a graphite surface is described. The enzyme-grease electrode concept, illustrated with the enzyme tyrosinase, offers a very simple, rapid and inexpensive approach to the fabrication of enzyme electrodes. The tyrosinase electrode responds very rapidly to dynamic changes in the concentration of phenolic compounds. A response time (t95%) as low as 5 s has been determined. With flow injection, 120 samples per hour can be processed with a relative standard deviation of 2.4%. The electrode remains active for about 12 d. The detection limit for dopamine is 6 x 10(-6) M. This method of biosensor construction should be applicable to other enzyme-substrate systems.     

Design of a composite amperometric enzyme electrode for the control of the benzoic acid content in food

A graphite–Teflon–tyrosinase composite biosensor for the determination of benzoic acid in foodstuffs is reported. The biosensor functioning is based on the inhibition effect of benzoic acid on the biocatalytic activity of the enzyme in a reversed micelle working medium formed with ethyl acetate as the continuous phase, a 0.05 mol l⁻¹ phosphate buffer solution of pH 7.4 (5%) as the aqueous dispersed phase, and 0.10 mol l⁻¹ dioctyl sulfosuccinate (AOT) as the emulsifying agent. A potential value of −0.10 V, and a constant enzyme-substrate (phenol) concentration of 2.0×10⁻⁴ mol l⁻¹ were selected to carry out the amperometric inhibition measurements. The tyrosinase inhibition process by benzoic acid is reversible and of the competitive type, with an apparent inhibition constant of 0.016 mmol l⁻¹. The composite bioelectrodes allow the regeneration of the electrode surface by polishing and exhibit long-term operation and stability. A limit of detection of 9.0×10⁻⁷ mol l⁻¹ benzoic acid was obtained. An interference study from other substances which can be found in foodstuffs together with benzoic acid was performed. Taking advantage of the capabilities of reversed micelles as universal solubilization media, the composite tyrosinase electrode was used for the determination of benzoic acid in two different kind of samples: mayonnaise sauce, which is a highly hydrophobic matrix, and Cola soft drinks, a hydrophilic matrix for which practically no sample treatment is necessary.     

Organophosphorus and carbamate pesticide analysis using an inhibition tyrosinase organic phase enzyme sensor; comparison by butyrylcholinesterase+choline oxidase opee and application to natural waters

Recent research performed in our laboratory (using a butyrylcholinesterase + choline oxidase enzyme electrode) suggested the validity of the biosensor approach using enzyme inhibition OPEEs (i.e. enzyme electrodes working in organic phase) in the case of organophosphorus and carbamate pesticides, which are poorly soluble in aqueous solutions. Since these pesticides are generally much more soluble in chloroform than in water, the present research aimed at analysing this class of pesticides using a tyrosinase inhibition OPEE operating in water-saturated chloroform medium. The tyrosinase biosensor was assembled using an oxygen amperometric transducer coupled to the tyrosinase enzyme, immobilized in kappa-carrageenan gel. Lastly a detailed comparison between the inhibition monoenzymatic tyrosinase and inhibition bienzymatic (butyrylcholinesterase + choline oxidase) OPEEs was performed and discussed in this work.     

Detection of zeptomolar concentrations of alkaline phosphatase based on a tyrosinase and horse-radish peroxidase bienzyme biosensor

A bienzyme biosensor based on tyrosinase and horse-radish peroxidase is described in a flow injection analysis and cyclic voltammetry for measurement of phenol. Tyrosinase and horse-radish peroxidase were immobilized on the surface of a glassy carbon electrode by bovine serum albumin and glutaric dialdehyde. Phenol was oxidized by tyrosinase and horse-radish peroxidase via catechol to o-quinone in the presence of oxygen and hydrogen peroxide. The o-quinone was reduced to produce catechol (the substrate recycling) on the electrode surface. The enhanced sensitivity of the bienzyme electrode to phenol was observed in the flow injection system comparing with tyrosinase and horse-radish peroxidase monoenzyme electrodes. The mechanisms for enhanced amperometric response to phenol of bienzyme electrode were discussed. The biosensor was used to detect alkaline phosphatase (ALP). A detection limit of 1.4×10⁻¹⁵ M ALP (140 zmol/100 μl) was obtained after 1 h incubation with phenyl phosphate.     

电聚吡咯固定化酪氨酸酶电极的制备及性能

在应用恒电位法电化学聚合吡咯的同时 ,将酪氨酸酶固定在导电聚吡咯膜内 ,制成一种灵敏、稳定的酪氨酸电极 .讨论了溶液 pH值和聚合电位对酶固定化的影响 ,对酶分子嵌入吡咯膜前后的SEM图和CV曲线进行了分析、比较 .该电极对甲苯酚响应的线性范围为 5 .0× 10 -8～ 1.0× 10 -6mol/L ,最适 pH值为 6 .6 ,酶反应表观上遵循Michaelis_Menten动力学 ,表观米氏常数为 2 .2× 10 -5mol/L .     

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.     

Biosensing of Aromatic Amines in Reversed Micelles with Self‐Generation of Hydrogen Peroxide at Glucose Oxidase‐Peroxidase Bienzyme Electrodes

The amperometric biosensing of aromatic amines using a composite glucose oxidase (GOD)‐peroxidase (HRP) biosensor in reversed micelles is reported. Rigid composite pellets of graphite and Teflon, in which GOD and HRP were coimmobilized by simple physical inclusion, were employed for the biosensor design. This design allows the in situ generation of the H₂O₂ needed for the enzyme reaction with the aromatic amines, thus preventing the negative effect that the presence of a high H₂O₂ concentration in solution has on HRP activity. The H₂O₂ in situ generation is performed by oxidation of glucose catalyzed by GOD. The effect of the composition of the reversed micelles, i.e., the nature of the organic solvent used as the continuous phase, the nature and concentration of the surfactant used as emulsifying agent, the aqueous 0.05 mol L^?1 phosphate buffer percentage used as the dispersed phase, and the glucose concentration in the aqueous phase, on the biosensor response was evaluated. Reversed micelles formed with ethyl acetate, a 5% of phosphate buffer (pH 7.0) containing 3.0×10^?3 mol L^?1 glucose, and 0.1 mol L^?1 AOT (sodium dioctylsulfosuccinate), were selected as working medium. Well‐defined and reproducible amperometric signals at 0.00 V were obtained for p‐phenylenediamine, 2‐aminophenol, o‐phenylenediamine, m‐phenylenediamine, 1‐naphthylamine, o‐toluidine and aniline. The useful lifetime of one single biosensor was of 60 days. The trend in sensitivity observed for the aromatic amines is discussed considering the effect of their structure on the stabilization of the radicals formed in the enzyme reaction which are electrochemically reduced. The behavior of the composite bienzyme electrode was also evaluated in a FI (flow injection) system using reversed micelles as the carrier. The suitability of the composite bienzyme electrode for the analysis of real samples was demonstrated by determining aniline in spiked carrots.     

Conducting polymer based amperometric enzyme electrodes

The construction and the properties of conducting-polymer based amperometric enzyme electrodes are reviewed. The main aim is to focus on the properties of conducting polymer films which are important for the construction of amperometric enzyme electrodes. Additionally, the review is focused on electron-transfer pathways between conducting-polymer integrated immobilized enzyme molecules and the modified electrode using free-diffusing redox mediators as well as direct electron transfer via the conducting-polymer wires. Possible future applications using microstructured conducting-polymer films will be discussed.     

A Reagentless Tyrosinase Biosensor Based on 1,6‐Hexanedithiol and Nano‐Au Self‐Assembled Monolayers

An amperometric tyrosinase biosensor was developed via a simple and effective immobilization method using the self‐assembled monolayers (SAMs) technique. The organic monolayer film was first formed by the spontaneous assembly of thiolor sulfur compound (1,6‐hexanedithiol, HDT) from solution onto gold electrode. When these thiol‐rich surfaces were exposed to Au colloid, the sulfurs form strong bonds to gold nanoparticles, anchoring the clusters to the electrode substrate. After the assembly of gold nanoparticles layer, a new nano‐Au surface was obtained. Thus, the tyrosinase could be immobilized onto the electrode. The tyrosinase retained its activity well in such an immobilization matrix. The various experimental variables for the enzyme electrode were optimized. The resulting biosensor can reach 95% of steady‐state current within 10 s, and the trend in the sensitivity of different phenolic compounds was as follows: catechol>phenol>p‐cresol. In addition, the apparent Michaelis–Menten constant (K and the stability of the enzyme electrode were estimated.     

Integrated, electrically contacted NAD(P)+-dependent enzyme-carbon nanotube electrodes for biosensors and biofuel cell applications

Integrated, electrically contacted beta-nicotinamide adenine dinucleotide- (NAD(+)) or beta-nicotinamide adenine dinucleotide phosphate- (NADP(+)) dependent enzyme electrodes were prepared on single-walled carbon nanotube (SWCNT) supports. The SWCNTs were functionalized with Nile Blue (1), and the cofactors NADP(+) and NAD(+) were linked to 1 through a phenyl boronic acid ligand. The affinity complexes of glucose dehydrogenase (GDH) with the NADP(+) cofactor or alcohol dehydrogenase (AlcDH) with the NAD(+) cofactor were crosslinked with glutaric dialdehyde and the biomolecule-functionalized SWCNT materials were deposited on glassy carbon electrodes. The integrated enzyme electrodes revealed bioelectrocatalytic activities, and they acted as amperometric electrodes for the analysis of glucose or ethanol. The bioelectrocatalytic response of the systems originated from the biocatalyzed oxidation of the respective substrates by the enzyme with the concomitant generation of NAD(P)H cofactors. The electrocatalytically mediated oxidation of NAD(P)H by 1 led to amperometric responses in the system. Similarly, an electrically contacted bilirubin oxidase (BOD)-SWCNT electrode was prepared by the deposition of BOD onto the SWCNTs and the subsequent crosslinking of the BOD units using glutaric dialdehyde. The BOD-SWCNT electrode revealed bioelectrocatalytic functions for the reduction of O(2) to H(2)O. The different electrically contacted SWCNT-based enzyme electrodes were used to construct biofuel cell elements. The electrically contacted GDH-SWCNT electrode was used as the anode for the oxidation of the glucose fuel in conjunction with the BOD-SWCNT electrode in the presence of O(2), which acted as an oxidizer in the system. The power output of the cell was 23 muW cm(-2). Similarly, the AlcDH-SWCNT electrode was used as the anode for the oxidation of ethanol, which was acting as the fuel, with the BOD-SWCNT electrode as the cathode for the reduction of O(2). The power output of the system was 48 microW cm(-2).     

Amperometric sensors based on tyrosinase-modified screen-printed arrays

This paper describes the design, development and characteristics of a tyrosinase (polyphenol oxidase) modified amperometric screen-printed biosensor array, with the enzyme cross-linked in a redox-hydrogel namely the PVI₁₃-dmeOs polymer. Two types of Au-screen-printed four-channel electrode arrays, differing in design and insulating layer, were compared and investigated. Au-, graphite-coated-Au- and Carbopack C-coated-Au-surfaces, serving as the basis for tyrosinase immobilisation, were investigated and the performances of the different arrays were evaluated and compared in terms of their electrocatalytic characteristics, as well as operational- and storage stability using catechol as model substrate. It was found that the Carbopack C-coated array was the best choice for tyrosinase immobilisation procedure mainly due to a higher mechanical stability of the deposited enzyme layer, combined with good sensitivity and stability for up to 6 months of use. In the batch mode the biosensors responded linearly to catechol up to 30 μM with limits of detection from 0.14 μM. Parameters from cyclic voltammograms indicated that the reversibility of the direct electrochemical reaction for catechol on the three types of electrode surfaces (no tyrosinase modification) was not the limiting factor for the construction and performance of tyrosinase biosensors.     

Analytical performance characteristics of thin and thick film amperometric microcells

The analytical performance of amperometric microcells with different electrode geometries is compared for enzyme activity measurements. The microcells were fabricated with thin film photolithography or thick film screen-printing in four different designs. The cells made with the thin film process used flexible substrate with microelectrode array or a circular, disk-shaped working electrode. The screen-printed working electrodes had semicircle or disk shape on ceramic chips. Putrescine oxidase (PUO) activity measurement was used as a model. The determination of PUO activity is important in the clinical diagnosis of premature rupture of the amniotic membrane. An electropolymerized m-phenylenediamine size-exclusion layer was used to eliminate common interferences. The size exclusion layer revealed also to be advantageous in protecting the electrodes from fouling by putrescine (enzyme substrate). The electrode fouling of bare electrodes was insignificant for screen-printed electrodes, but very severe for electroplated platinum working electrodes. The microelectrode array electrodes demonstrated smaller RSD and higher normalized sensitivities for hydrogen peroxide and PUO activity. All the other electrodes were demonstrating comparable analytical performances.     

Analytical performance characteristics of thin and thick film amperometric microcells

The analytical performance of amperometric microcells with different electrode geometries is compared for enzyme activity measurements. The microcells were fabricated with thin film photolithography or thick film screen-printing in four different designs. The cells made with the thin film process used flexible substrate with microelectrode array or a circular, disk-shaped working electrode. The screen-printed working electrodes had semicircle or disk shape on ceramic chips. Putrescine oxidase (PUO) activity measurement was used as a model. The determination of PUO activity is important in the clinical diagnosis of premature rupture of the amniotic membrane. An electropolymerized m-phenylenediamine size-exclusion layer was used to eliminate common interferences. The size exclusion layer revealed also to be advantageous in protecting the electrodes from fouling by putrescine (enzyme substrate). The electrode fouling of bare electrodes was insignificant for screen-printed electrodes, but very severe for electroplated platinum working electrodes. The microelectrode array electrodes demonstrated smaller RSD and higher normalized sensitivities for hydrogen peroxide and PUO activity. All the other electrodes were demonstrating comparable analytical performances.     

Electropolymerized network of polyamidoamine dendron-coated gold nanoparticles as novel nanostructured electrode surface for biosensor construction

Polyfuntionalized gold nanoparticles were prepared by using 2-mercaptoethanesulfonic acid, p-aminothiophenol and cysteamine core polyamidoamine G-4 dendron as capping ligands. The nanoparticles were electropolymerized on a Au electrode surface through the formation of a bisaniline-cross-linked network. The enzyme tyrosinase was further crosslinked on this nanostructured matrix. The enzyme electrode, poised at -100 mV, was used for the amperometric quantification of cathecol. The biosensor showed a linear response from 50 nM to 10 μM cathecol, with a low detection limit of 20 nM and a sensitivity of 1.94 A M(-1) cm(2). The electrode retained 96% and 67% of its initial activity after 16 and 30 days of storage at 4 °C under dry conditions.     

Direct electron transfer enhancement of covalently bound tyrosinase to glassy carbon via Woodward's reagent K

This work describes the reaction mechanism for chemical modification of tyrosinase by Woodward's Reagent K and its covalent attachment to a glassy carbon electrode. The spectrophotometric studies revealed that the modification does not cause a significant structural change to tyrosinase. The direct electrochemistry of modified enzyme was achieved after immobilization on an oxidatively activated glassy carbon electrode. The enzyme film exhibited a pair of well-defined quasi-revesible voltammetric peaks corresponding to the Cu (II)/Cu (I) redox couple located in the active site of tyrosinase. The formal potential of immobilized enzyme was measured to be 90mV (vs. Ag/AgCl) in phosphate buffer solution at pH 7.0. The charge-transfer coefficient and apparent heterogeneous electron transfer rate constant were estimated to be 0.5 and 0.9±0.06s(-1), respectively. Finally, the electrochemical behavior of the immobilized enzyme in the presence of caffeic acid and L-3,4-dihydroxyphenylalanine as substrates was investigated. The amperometric study of biosensor toward L-3,4-dihydroxyphenylalanine resulted a linear response in the concentration range from 1.66×10(-6) to 8.5×10(-5)M with detection limit of 9.0×10(-5)M and sensitivity of 135mAμM(-1)cm(-2).     

A comparative study of immobilization methods of a tyrosinase enzyme on electrodes and their application to the detection of dichlorvos organophosphorus insecticide

The use of several designs of amperometric enzymatic biosensors based on the immobilized tyrosinase enzyme (Tyr) for determining dichlorvos organophosphate pesticide are described. The biosensors are based on the reversible inhibition of the enzyme and the chronocoulometric measurement of the charge due to the charge-transfer mediator 1,2-naphthoquinone-4-sulfonate (NQS). Tyr becomes active when reducing the quinone form of the mediator molecule (NQS) to the reactive o-diol form substrate of Tyr (H₂NQS) at the working electrode, thus permitting modulation of the catalytic activity of the enzyme and measurement of the inhibition produced by the pesticide. The full activity of the enzyme reversibly recovers after removal of the pesticide and re-oxidation of H₂NQS.Tyr was immobilized onto electrodes using different procedures: (i) entrapment within electropolymerized conducting and non-conducting polymers, (ii) covalent attachment to self-assembled monolayers (SAM), (iii) cross-linking with glutaraldehyde (and nafion covering) and (iv) dispersion within carbon-paste electrodes. The mediator was co-immobilized onto the working electrode next to the enzyme and reagentless biosensors were subsequently constructed. In the SAM design (ii) NQS was added to the solution. The analytical properties of the different biosensors based on the competitive inhibition produced by dichlorvos were then compared. A detection limit of about 0.06 μM was obtained for dichlorvos with entrapment of NQS and Tyr within electropolymerized poly(o-phenylenediamine) polymer (oPPD), which was the design that proved to have the best analytical performance.     

Amperometric measuring cell for the determination of putrescine oxidase activity

A microfabricated, flat form, amperometric microcell (microchip) is used in a simple, two-electrode arrangement for putrescine oxidase enzyme activity determinations. The cell contains a platinum microdisk working electrode and an Ag/AgCl reference electrode covered by a porous, hydrophilic membrane. An electrochemically-prepared size-exclusion layer is applied on the working electrode surface, to avoid the effect of electroactive interferences in the sample. The hydrophilic membrane, resting on the bottom of the cell, is soaked with a small volume of buffered substrate solution and a few mul enzyme containing sample solution is dispensed over the electrodes. During the enzyme activity measurement a catalytic reaction takes place in the membrane-supported liquid film over the working electrode surface. The hydrogen peroxide produced in the reaction is detected amperometrically. The amperometric current-time curves are used for evaluation. In our work putrescine was used as a substrate to determine the unknown putrescine oxidase enzyme activity of the sample. Elevated diamine oxidase enzyme activity in the vaginal milieu can indicate premature rupture of the amniotic membrane. Results with membrane discs, containing all the necessary chemicals in solid or lyophilized form, are very encouraging with respect of a single use, 'reagentless' biosensor for home care.