A catechol biosensor based on a gold nanoparticles encapsulated-dendrimer

Tyrosinase has been immobilized on a Au nanoparticles encapsulated-dendrimer bonded conducting polymer on a glassy carbon electrode for the estimation of catechol. The modified electrode was characterized by cyclic voltammetry and AFM techniques. The principle of catechol estimation was based on the reduction of biocatalytically liberated quinone species at +0.2 V versus Ag/AgCl (3 M KCl), with good stability, sensitivity, and featuring a low detection limit (about 0.002 μM) and wide linear range (0.005 μM-120 μM). The electrochemical redox peak of catechol on the GCE/PolyPATT/Den(AuNPs)/tyrosinase was also investigated. A response time of 7 s, reusability up to 5 cycles and a shelf life of more than 2 months under refrigerated conditions were reported. Various parameters influencing biosensor performance have been optimized including pH, temperature, and applied potential. The utility and application of this nanobiosensor was tested in a real water samples.     

Development and optimization of a solid composite tyrosinase biosensor for phenol detection in flow injection systems

Bulk-modified epoxy-graphite tyrosinase biosensors were fabricated by four different procedures. The influence of these fabrication procedures on the analytical performance of the enzyme electrode in an amperometric wall-jet flow cell has been studied. The bioprobe performance is assessed by cyclic voltammetry. Higher current densities and narrower peaks were obtained when the enzyme was introduced in the dry state into the epoxy-graphite material, instead of introducing it previously dissolved in the buffer. In the F1 system responses of 11.79 μA cm⁻² and 1.43 μA cm⁻² are then obtained for catechol and phenol respectively for 50 μL injections of 20 μM solutions. Moreover, if gold/palladium is introduced into the epoxy-graphite, a further increase in current is achieved resulting in 27.70μA cm⁻² and 4.90μA cm⁻²for catechol and phenol, respectively. This biosensor can operate in aqueous as well as in mixed aqueous-organic environments.     

Dopamine Electroanalysis Using Electrochemical Biosensors Prepared by a Sinusoidal Voltages Method

A biocomposite material of tyrosinase and poly(3,4‐ethylenedioxythiophene) was electrodeposited onto gold (Au) disk microelectrode arrays and Au interdigitated microband electrode arrays (IDEs) by using a combination of sinusoidal voltages and microgravimetric method to construct amperometric biosensors for dopamine (DA). The main advantage of this new strategy consists in a reliable enzyme immobilization and in additional information related to the electropolymerization process. The determination of DA was successfully achieved in pharmaceutical formulations. The best analytical performances, in terms of the lowest limit of detection of 6×10^?7 M DA and highest recovery and stability, were obtained for IDEs based biosensor.     

Electrode Reactions of Catechol at Tyrosinase‐Immobilized Latex Suspensions

Tyrosinase was immobilized on polystyrene latex particles in order to control amounts of the enzyme. The tyrosinase‐coated latex particles were composed of the core polystyrene and four successive coating layers: polystyrene sulfonate, polyallylamine, tyrosinase and polyallylamine again, built up by the layer‐by‐layer technique. They showed catalytic currents for the enzymatic oxidation of catechol to o‐quinone. The enzyme activity per particle was evaluated as 2.3×10^?7 units from UV absorption of o‐quinone. The relation between the catalytic current and the concentration of catechol leads to a Michaelis‐Menten type kinetic equation. The layer‐by‐layer method was found to have a deactivating effect on enzyme catalysis. In spite of this, the catechol oxidation current was larger than the current from free tyrosinase at a common value of enzyme units per volume. This is ascribed to strong adsorption of the latex particles on the electrode, leading to the enhancement of the local concentration of tyrosinase.     

Preparation and characterization of a new enzyme electrode based on solid paraffin and activated graphite particles

A new electrochemical biosensor was developed by incorporating an enzyme into a solid-paraffin-graphite-particle matrix. Tyrosinase served as model enzyme and the biosensor response was characterized with respect to its response to dopamine. The influence of different experimental parameters (tyrosinase loading, flow rate, oxygen dependence, pH, etc.) was investigated in order to optimize the biosensor performance. The electrode response was fast, reversible and linear in a large concentration domain (0.1 muM-1 mM). The enzyme-solid paraffin carbon paste electrode (CPE) showed markedly improved stability in flow injection analysis compared to the classical liquid paraffin-graphite-based biosensors. The biosensor allowed a sampling rate of 79 samples per hour, the repeatability of the injections was improved with respect to the classical CPE with a relative standard deviation of 2.2% (N = 63), and the detection limit for dopamine was 50 nM. The biosensor response to some phenol and catechol derivatives was also investigated.     

Amperometric biosensor based on tyrosinase immobilized in hydrotalcite-like compounds film for the determination of polyphenols

A tyrosinase (Tyr) biosensor has been constructed by immobilizing tyrosinase on the surface of Mg–Al–CO₃ hydrotalcite-like compound film (HTLc) modified glassy carbon electrode (GCE) for the determination of polyphenols. The negatively charged tyrosinase was adsorbed firmly on the surface of a positively charged HTLc/GCE by electrostatic interactions and retained its activity to a great degree. The modified electrode was characterized by cyclic voltammetry and AC impedance spectra. Polyphenols were determined by a direct reduction of biocatalytically generated quinone species. The different parameters, including pH, temperature, and enzyme loading were investigated and optimized. Under the optimum conditions, Tyr/HTLc electrode gave a linear response range of 3–300, 0.888–444, and 0.066–396 μM with a detection limit (S/N = 3) of 0.1, 0.05, and 0.003 μM for catechol, caffeic acid, and quercetin, respectively. In addition, the repeatability and stability of the enzyme electrode were estimated. Total polyphenol contents of real samples were also determined to study the potential applicability of the Tyr/HTLc/GCE biosensor.     

Enzyme immobilisation on electroactive nanostructured membranes (ENM): optimised architectures for biosensing

Electroactive nanostructured membranes have been produced by the layer-by-layer (LbL) technique, and used to make electrochemical enzyme biosensors for glucose by modification with cobalt hexacyanoferrate redox mediator and immobilisation of glucose oxidase enzyme. Indium tin oxide (ITO) glass electrodes were modified with up to three bilayers of polyamidoamine (PAMAM) dendrimers containing gold nanoparticles and poly(vinylsulfonate) (PVS). The gold nanoparticles were covered with cobalt hexacyanoferrate that functioned as a redox mediator, allowing the modified electrode to be used to detect H₂O₂, the product of the oxidase enzymatic reaction, at 0.0 V vs. SCE. Enzyme was then immobilised by cross-linking with glutaraldehyde. Several parameters for optimisation of the glucose biosensor were investigated, including the number of deposited bilayers, the enzyme immobilisation protocol and the concentrations of immobilised enzyme and of the protein that was crosslinked with PAMAM. The latter was used to provide glucose oxidase with a friendly environment, in order to preserve its bioactivity. The optimised biosensor, with three bilayers, has high sensitivity and operational stability, with a detection limit of 6.1 μM and an apparent Michaelis–Menten constant of 0.20 mM. It showed good selectivity against interferents and is suitable for glucose measurements in natural samples.     

L-Dopa Synthesis on Conducting Polymers

With regards to the synthesis of L-Dopa (l-3,4-dihydroxy phenylalanine) two types of biosensors were designed by immobilizing tyrosinase on conducting polymers; polypyrrole (PPy) and poly(3,4-ethylenedioxythiophene) (PEDOT). PPy and PEDOT were synthesized electrochemically and tyrosinase immobilized by entrapment during electropolymerization. The kinetic parameters of the designed biosensors, maximum reaction rate of the enzyme (V_max) and Michaelis Menten constant (K_m) were determined. V_max were found as 0.013 for PPy matrix and 0.041 μ mol/min.electrode for PEDOT matrix. K_m values were determined as 3.7 and 5.2mM for PPy and PEDOT matrices respectively. Calibration curves for enzyme activity vs. substrate concentration were drawn for the range of 0.8 to 2.5 mM L-Tyrosine. Optimum temperature and pH, operational and shelf life stabilities of immobilized enzyme were also examined.     

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.     

A comparison of amperometric screen-printed, carbon electrodes and their application to the analysis of phenolic compounds present in beers

In this paper a comparison between three commercially-available, screen-printable graphite inks for the construction of phenolic biosensors is made. The enzyme tyrosinase was immobilised within a polymer matrix and the substrate catechol was used to characterise the bio-electroanalytical response of each electrode. Biosensors fabricated from Gwent graphite inks exhibited the greatest sensitivity (5740 mA mol cm(-2)) compared to Dupont and Acheson graphite-based inks. This difference in sensitivity was attributed to a combination of a larger electroactive surface area, and thus a greater number of immobilised enzyme molecules. However, the dynamic range was considerably smaller (0.025-14 muM) indicating that the enzyme molecules were easily accessible to the substrate catechol. The surface properties of the biosensors were characterised using ac impedance, which indicated that the presence of the polymer on the electrode surface not only increased the charge-transfer kinetics of the three biosensors, but also increased the surface roughness of biosensors fabricated from Gwent inks. On the basis of these results Gwent graphite-based inks were used for analysis of phenolic compounds in lager beers by flow-injection analysis. The biosensor displayed favourable response characteristics, but cannot differentiate between the various phenolic compounds present in the samples. Nevertheless, the biosensor maybe suitable for indicating the phenolic status of beer or brew samples compared to time-consuming traditional methods, e.g. colorimetric or chromatographic methods.     

Enzyme Deposition by Polydimethylsiloxane Stamping for Biosensor Fabrication

High‐performance biosensors were fabricated by efficiently transferring enzyme onto Pt electrode surfaces using a polydimethylsiloxane (PDMS) stamp. Polypyrrole and Nafion were coated first on the electrode surface to act as permselective films for exclusion of both anionic and cationic electrooxidizable interfering compounds. A chitosan film then was electrochemically deposited to serve as an adhesive layer for enzyme immobilization. Glucose oxidase (GOx) was selected as a model enzyme for construction of a glucose biosensor, and a mixture of GOx and bovine serum albumin was stamped onto the chitosan‐coated surface and subsequently crosslinked using glutaraldehyde vapor. For the optimized fabrication process, the biosensor exhibited excellent performance characteristics including a linear range up to 2 mM with sensitivity of 29.4±1.3 μA mM⁻¹ cm⁻² and detection limit of 4.3±1.7 μM (S/N=3) as well as a rapid response time of ∼2 s. In comparison to those previously described, this glucose biosensor exhibits an excellent combination of high sensitivity, low detection limit, rapid response time, and good selectivity. Thus, these results support the use of PDMS stamping as an effective enzyme deposition method for electroenzymatic biosensor fabrication, which may prove especially useful for the deposition of enzyme at selected sites on microelectrode array microprobes of the kind used for neuroscience research in vivo .     

Comparison of biosensors based on gold and nanocomposite electrodes for monitoring of malic acid in wine

Amperometric biosensors based on a gold planar electrode and on two types of nanocomposite electrodes consisting of multi-walled carbon nanotubes for the determination of L-malic acid designed for wine-makers were developed. The biosensors designed for wine-makers were constructed by immobilization of L-malate dehydrogenase and diaphorase within chitosan layers on the surface of the electrodes. The coenzyme NAD+ and the electrochemical mediator ferricyanide were present in the measuring solution. The current resulting from re-oxidation of produced ferrocyanide was measured at a working potential of +300 mV against an Ag/AgCl reference electrode. The biosensor based on a gold electrode showed linearity over the range 10–520 μM with a detection limit of 5.41 μM. Calibration curves for biosensors utilizing nanocomposites were obtained both with the linear range of 10 to 610 μM. The detection limits were 1.57 and 1.77 μM, respectively. The biosensors showed satisfactory operational stability (no loss of sensitivity after 30 consecutive measurements) and storage stability (90% of the initial sensitivity after one year of storage at room temperature). The results obtained from measurements of wine samples were in a good correlation with the standard HPLC method. Satisfactory biosensor sensitivity, specificity and stability allowed their successful commercialization.     

Pure Organic Phase Phenol Biosensor Based on Tyrosinase Entrapped in a Vapor Deposited Titania Sol‐Gel Membrane

A novel amperometric biosensor was constructed for the determination of phenols in pure organic phase. This biosensor was fabricated by immobilizing tyrosinase in a titania sol‐gel membrane which was obtained with a vapor deposition method. This method was facile and avoided the calcination step needed in conventional titania sol‐gel process. The titania sol‐gel membrane could effectively retain the essential water layer around the enzyme molecule needed for maintaining its activity in organic phase. The experimental parameters such as solvent and operating potential were optimized. At ?100 mV this biosensor showed a good amperometric response to phenols in pure chloroform without any mediator and rehydration of the enzyme. For catechol determination the sensor exhibited a fast response of less than 5 seconds. The sensitivity of different phenols was as follows: catechol > phenol > p‐cresol. Additionally, the apparent Michaelis‐Menten constants of the encapsulated tyrosinase to catechol, phenol and p‐cresol were found to be 0.15±0.003, 0.17±0.008 and 0.21±0.004 mM, respectively. The biosensor had also good reproducibility and stability. This work provided a promising platform for the construction of pure organic phase biosensors and the determination of substrates with poor water solubility.     

Diazirine‐functionalized Nanostructured Platform for Enzymes Photografting and Electrochemical Biosensing

A simple technique for the construction of a versatile diazirine‐functionalized nanostructured platform for enzymes photografting and electrochemical biosensing was proposed in this work. The feasibility of the approach was proved by photo crosslinking of an enzyme, tyrosinase, to diazirine‐activated aminated carbon nanotubes coated glassy carbon electrode. The analytical performances of the realized biosensor were evaluated employing catechol as analyte. Then the sensor based on the diazirine‐functionalized nanostructured platform with photografted tyrosinase was applied together with the high resolution technique Differential Alternative Pulses Voltammetry for dopamine determination in the linear concentration range of 5–25 μmol L^?1 in the presence of interfering agents as uric acid up to its 100‐fold excess.     

Development of an amperometric biosensor for the determination of phenolic compounds in reversed micelles

The possibilities of amperometric enzyme electrodes in reversed micellar systems for the determination of phenol, 4-chloro-3-methylphenol and 2,4-dimethylphenol are illustrated. The used enzymatic reaction consisted of the oxidation of the phenolic compounds by oxygen, catalysed by tyrosinase. The reduction of the liberated quinones was amperometrically detected. The concentration of the components of the reversed micelles, as well as the potential applied to the tyrosinase electrode have been optimized. The stability of the enzyme electrode with time was also evaluated. The effect of the analyte solubility in water upon the analytical performance of the electrode was explored. Advantages of amperometric biosensors in reversed micelles are shown with respect to aqueous media and organic phase enzyme electrodes.     

Immobilization of CoII-(N,N′-bis(salicylidene)-2-aminobenzylamine) on Poly(pyrrole-co-o-anisidine)/Chitosan Composite Films: Application to Electrocatalytic Oxidation of Catechol

In this study, poly (pyrrole-co-o-anisidine)/chitosan composite (Cs) films were prepared by cyclic voltammetry technique on platinum electrode using different pyrrole and o-anisidine mole ratios. Immobilization process was accomplished in Co^II-(N,N′-bis(salicylidene)-2-aminobenzylamine)(CoL) dissolved 0.15 M acetonitrile-LiClO₄ solution by cyclic voltammetry technique at 0.2–2.0 V potential range. Three electrode methods were applied in all electrochemical studies. After immobilization process, the characterizations of the electro catalytic surfaces (Cs−CoL−Pt) were carried out by cyclic voltammetry and SEM images. The SEM images clearly indicated that the [CoL] complex is immobilized onto composite films. The electrocatalytic activity of the modified electrodes on the catechol was investigated using buffer solutions of different pH values. The results of catalytic studies revealed that, pH=10 buffer solution was the optimal solution and 1 : 1 Cs−CoL−Pt electrode was the best electrode for catechol oxidation. In square wave voltammetry measurements using this electrode, two linear working ranges were determined. The linear response ranges for catechol determination were found as 3.0 μM–6.0 μM and 16 μM–80 μM for the first and the second linear working ranges, respectively, with 1.1 μM detection limit.     

A glassy carbon electrode modified with graphene and tyrosinase immobilized on platinum nanoparticles for sensing organophosphorus pesticides

An amperometric biosensor is described for the detection of organophosphorus pesticides. It is based on the enzyme tyrosinase immobilized on platinum nanoparticles and the use of a glassy carbon electrode modified with graphene. Tyrosinase was immobilized on the electrode surface via electrostatic interaction between a monolayer of cysteamine and the enzyme. In the presence of catechol as a substrate, the pesticides chlorpyrifos, profenofos and malathion can be determined as a result of their inhibition of the enzyme which catalyzes the oxidation of catechol to o-quinone. Platinum nanoparticles and graphene effectively enhance the efficiency of the electrochemical reduction of o-quinone, thus improving sensitivity. Under optimum experimental conditions, the inhibition effect of the pesticides investigated is proportional to their concentrations in the lower ppb-range. The detection limits are 0.2, 0.8 and 3?ppb for chlorpyrifos, profenofos and malathion, respectively. The biosensor displays good repeatability and acceptable stability.

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.     

Characterization and Optimization of Interdigitated Ultramicroelectrode Arrays as Electrochemical Biosensor Transducers

Interdigitated ultramicroelectrode arrays (IDUAs) were fabricated on glass wafers and investigated to obtain optimal oxidation and reduction reactions of potassium ferro/ferrihexacyanide, Fe^2+/3+(CN)₆, when using a 2‐electrode set up. These electrodes will be used as transducers in portable microfluidic‐based biosensors in the future for the detection in an aqueous, biocompatible matrix. IDUAs were designed to maximize the signal‐to‐noise ratio (S/N) investigating electrode height, gap size, finger width, and material. Interesting differences in the electrode materials gold and platinum were found, which were due to the oxidization of platinum and gold during the IDUA fabrication process. It resulted in gold IDUAs being by far superior in respect to signal‐to‐noise ratio and overall signal magnitude to those made of platinum. The effects of gap size, electrode width and number of electrode fingers were as expected. Optimal electrode heights were in the range of 70 nm–140 nm, much larger and smaller electrodes had lower signal‐to‐noise ratios due to overall reduced signal or increased background. The optimized IDUA was made out of gold, had 400 fingers with a finger width of 2.7 μm, a finger height between 70 nm and 140 nm and a gap size of 0.9–1 μm. A detection limit of as low as 0.1 μM ferro/ferrihexacyanide measured in a simple 2‐electrode set up was obtained with a signal‐to‐noise ratio of 9.7.     

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.