Attachment of tyrosinase on mixed self-assembled monolayers for the construction of electrochemical biosensor

<正>A mixed self-assembled monolayers(SAMs) of thioctic acid(T-COOH) and thioctic acid amide(T-NH_2) were used to immobilize tyrosinase for fabricating biosensor.The results showed that the mixed SAMs prepared from solution at the ratio of 1:4 provided an excellent microenvironment for enzymatic reaction between tyrosinase and substrate.The biosensor exhibited a fast response and high sensitivity for sensing substrate.     

Electrically ‘wired’ tyrosinase enzyme inhibition electrode for the detection of respiratory poisons

The use of the solution redox species, [Os(bpy)₂Cl₂]^+/0, [Os(bpy)₂(MeIm)Cl]^2+/+ and [Fe(CN)₆]^4−/3−, where bpy is 2,2-bipyridine and MeIm is N-methylimidazole, as electron mediators in the enzymatic reduction of oxygen by tyrosinase is investigated. Co-immobilization of both enzyme and an osmium redox mediator in a hydrogel on glassy carbon electrodes results in a biosensor for the ‘reagentless’ addressing of enzyme activity, consuming only oxygen present in solution. Immobilized enzyme inhibition biosensors can thus be constructed for the detection of tyrosinase inhibitors, such as sodium azide, using this approach. The enzyme inhibition biosensor can detect levels of azide as low as 5 × 10⁻⁶ mol dm⁻³ in solution and may be useful in environmental monitoring applications and as an early warning poison sensor.     

Penicillamine determination using a tyrosinase micro-rotating biosensor

Tyrosinase [EC 1.14.18.1], immobilized on a rotating disk, catalyzed the oxidation of catechols to o-benzoquinone, whose back electrochemical reduction was detected on glassy carbon electrode surface at −150 mV versus Ag/AgCl/NaCl 3 M. Thus, when penicillamine (PA) was added to the solution, this thiol-containing compound participate in Michael type addition reactions with o-benzoquinone to form the corresponding thioquinone derivatives, decreasing the reduction current obtained proportionally to the increase of its concentration. This method could be used for sensitive determination of PA in drug and human synthetic serum samples. A linear range of 0.02–80 μM (r = 0.999) was obtained for amperometric determination of PA in buffered pH 7.0 solutions (0.1 M phosphate buffer). The biosensor has a reasonable reproducibility (R.S.D. < 4.0%) and a very stable amperometric response toward this compound (more than 1 month).     

Reagentless biosensor for phenolic compounds based on tyrosinase entrapped within gelatine film

A simple and new reagentless phenolic compound biosensor was constructed with tyrosinase immobilized in the gelatine matrix cross-linked with formaldehyde. The morphologies of gelatine and gelatine/tryosinase were characterized by SEM. The tyrosinase retains its bioactivity when being immobilized by the gelatine film. Phenolic compounds were determined by the direct reduction of biocatalytically liberated quinone at -0.1 V vs SCE. The process parameters for the fabrication of the enzyme electrode were studied. Optimization of the experimental parameters has been performed with regard to pH, operating potential, temperature and storage stability. This biosensor exhibits a fast amperometric response to phenolic compounds. The linear range for catechol, phenol, and p-Cresol determination was from 5×10⁻⁸ to 1.4×10⁻⁴ M, 5×10⁻⁸ to 7.1×10⁻⁵ M, and 1×10⁻⁷ to 3.6×10⁻⁵ M, with a detection limit of 2.1×10⁻⁸ M, 1.5×10⁻⁸ M, and 7.1×10⁻⁸ M, respectively. The enzyme electrode retained ca.77% of its activity after 7 days of storage at 4°C in a dry state. The proposed sensor presented good repeatability, evaluated in terms of relative standard deviation (R.S.D.=8.6%) for eight different biosensors and was applied for determination in water sample. The recovery for the sample was from 99.0% to 99.8%.     

A rapid and easy procedure of biosensor fabrication by micro-encapsulation of enzyme in hydrophilic synthetic latex films. Application to the amperometric determination of glucose

Novel enzyme electrodes based on synthetic hydrophilic latex matrices are described for the detection of glucose. Glucose oxidase was immobilised through micro-encapsulation, by the simple adsorption of enzyme–latex suspensions on the surface of a platinum electrode. Two latex films functionalised by a hydroxy or a gluconamide group were used. The response of these biosensors to glucose additions was measured by potentiostating the modified electrodes at 0.6 V/SCE in order to oxidise the hydrogen peroxide generated by the enzymatic oxidation of glucose in the presence of dioxygen. The response of such electrodes was evaluated as a function of film thickness and temperature. The sensitivity for a two-layer latex-based biosensor was found to be 38.78 mA M⁻¹ cm⁻² with a response time of 3–5 s. Moreover, a marked improvement of the thermal stability of the biosensor was observed. Only at temperatures higher than 65°C the enzyme started to be denatured and being inactive.     

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.     

Biotinylated alginate immobilization matrix in the construction of an amperometric biosensor: application for the determination of glucose

The use of biotinylated alginate as an immobilization matrix of enzymes on the surface of the amperometric transducer is described herein. The model used is that of the well-established glucose detection. Several types of immobilization protocols were tested. In the exception of one protocol, biotin labeled glucose oxidase was shown to first require conjugation with avidin, before its immobilization onto a biotin-alginate gel matrix. The response of the biosensors to incremental additions of glucose, was measured by potentiostating the modified electrodes at 0.6 V/SCE. The permeability of the modified electrodes was thereafter measured by using rotating disk electrode (RDE) voltammetry with ferrocenemonocarboxylic acid as the electroactive probe.     

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.     

Gold nanoparticles-enhanced bisphenol A electrochemical biosensor based on tyrosinase immobilized onto self-assembled monolayers-modified gold electrode

A novel electrochemical sensor based on the immobilization of tyrosinase(tyr) onto gold nanoparticles(nano-Au) and thioctic acid amide(T-NH2) self-assembled monolayers(SAMs)-modified gold electrode has been developed for the determination of bisphenol A(BPA).It was found that the nano-Au could significantly enhance the electrochemical response of tyr/nano-Au/T-NH2/Au electrode to BPA,and the enhancement effect of nano-Au on the current response was also related to the enzyme.The results indicated that the biosensor could be used as a detector for BPA determination with a linear range from3.99 ×10-7mol/L to 2.34 ×10-4mol/L and a detection limit of 1.33×10-7mol/L.In addition,this biosensor showed good reproducibility.     

A phenol biosensor based on immobilizing tyrosinase to modified core-shell magnetic nanoparticles supported at a carbon paste electrode

A phenol biosensor was developed based on the immobilization of tyrosinase on the surface of modified magnetic MgFe₂O₄ nanoparticles. The tyrosinase was first covalently immobilized to core-shell (MgFe₂O₄-SiO₂) magnetic nanoparticles, which were modified with amino group on its surface. The resulting magnetic bio-nanoparticles were attached to the surface of carbon paste electrode (CPE) with the help of a permanent magnet. The immobilization matrix provided a good microenvironment for the retaining of the bioactivity of tyrosinase. Phenol was determined by the direct reduction of biocatalytically generated quinone species at −150 mV versus SCE. The resulting phenol biosensor could reach 95% of steady-state current within 20 s and exhibited a high sensitivity of 54.2 μA/mM, which resulted from the high tyrosinase loading of the immobilization matrix. The linear range for phenol determination was from 1 × 10⁻⁶ to 2.5 × 10⁻⁴ M with a detection limit of 6.0 × 10⁻⁷ M obtained at a signal-to-noise ratio of 3. The stability and the application of the biosensor were also evaluated.     

A composite amperometric tyrosinase biosensor for the determination of the additive propyl gallate in foodstuffs

The performance of a graphite-Teflon composite amperometric tyrosinase biosensor for the determination of the food additive propyl gallate (PG) in different types of foodstuffs is reported. The enzyme reaction involves the catalytic oxidation of PG to the corresponding o-quinone, and the electrochemical reduction of this o-quinone was employed to monitor the enzyme reaction. Depending on the nature of the food sample analysed and on the presence of other phenolic antioxidants in these samples, aqueous buffer solutions or predominantly nonaqueous acetonitrile-Tris buffer mixtures were employed as working media. Experimental conditions such as the aqueous solution percentage in the predominantly nonaqueous medium, pH, and the potential to be applied were optimised. Control charts constructed showed a useful lifetime for the biosensor of 40 days when working in phosphate buffer of pH 6.5, and of 50 days in 80:20 acetonitrile-Tris buffer (pH 7.4) mixture. The limits of detection obtained for PG in these media were 9.0×10⁻⁷ and 1.1×10⁻⁶ mol L⁻¹, respectively. The composite bioelectrode also performed well in the flow-injection mode. PG was determined in dehydrated broth bars using the phosphate buffer solution of pH 6.5 as working medium. However, PG was determined in spiked olive oil in the working medium formed by the 80:20 acetonitrile-Tris buffer mixture, because a liquid-liquid extraction step was carried out. Comparison of the results with those obtained by applying reference methods showed that no significant differences existed at a significance level of 0.05.     

A novel amperometric inhibition biosensor based on HRP and gold sononanoparticles immobilised onto Sonogel-Carbon electrode for the determination of sulphides

A novel inhibition biosensor used for the detection of sulphides (Na₂S) has been developed. The biosensor is based on the immobilisation of horseradish peroxidase (HRP) on the Sonogel-Carbon (SNGC) electrode using glutaraldehyde, Poly(4-vinylpyridine) and gold sononanoparticles (AuSNPs). The Poly(4-vinylpyridine) was used due to its high affinity for sulphide anions, while the presence of gold sononanoparticles enhances the electron transfer reaction and improves the analytical performance of the biosensor. The amperometric measurements were performed at an applied potential of ?0.15 V vs. Ag/AgCl in 50 mM sodium acetate buffer solution pH = 6.0. The apparent kinetic parameters (K_mapp, V_max) of immobilised HRP were calculated in the absence of inhibitor (sulphide) using caffeic acid as substrate. Under the optimal experimental conditions, the determination of sulphide can be achieved in a dynamic range of 0.4–2.8 µM with a low limit of detection of 0.15 µM. The electrochemical impedance spectroscopy (EIS) was also used to characterise the interactions of substrate and inhibitor with the enzyme-modified electrode. The developed biosensor exhibited high sensitivity, selectivity and stability, and can be successfully applied to the detection of sulphide in water.     

Eggplant-based bioamperometric sensor for the detection of catechol

Polyphenol oxidases from eggplant have a high catalytic activity for the aerobic oxidation of catechol to o-quinone with selectivity over other phenolic substrates. An amperometric biosensor can therefore be constructed by incorporating selected portions of eggplant tissue in a carbon paste electrode. The proposed biosensor provides a selective response for catechol in the micromolar range, with a very fast response time and a useful lifetime of at least 3 weeks.     

Bienzymatic amperometric biosensor for glucose based on polypyrrole/ceramic carbon as electrode material

A novel amperometric biosensor utilizing two enzymes, glucose oxidase (GOD) and horseradish peroxidase (HRP), was developed for the cathodic detection of glucose. The glucose biosensor was constructed by electrochemical formation of a polypyrrole (PPy) membrane in the presence of GOD on the surface of a HRP-modified sol-gel derived-mediated ceramic carbon electrode. Ferrocenecarboxylic acid (FCA) was used as mediator to transfer electron between enzyme and electrode. In the hetero-bilayer configuration of electrode, all enzymes were well immobilized in electrode matrices and showed favorable enzymatic activities. The amperometric detection of glucose was carried out at +0.16 V (versus saturated calomel reference electrode (SCE)) in 0.1 M phosphate buffer solution (pH 6.9) with a linear response range between 8.0×10⁻⁵ and 1.3×10⁻³ M glucose. The biosensor showed a good suppression of interference in the amperometric detection.     

Enzymatic rotating biosensor for ciprofloxacin determination

The high sensitivity that can be attained using an enzymatic system and mediated by catechol has been verified by on-line interfacing of a rotating biosensor and continuous flow/stopped-flow/continuous-flow processing. Horseradish peroxidase, HRP [EC 1.11.1.7], immobilized on a rotating disk, in the presence of hydrogen peroxide, catalyzed the oxidation of catechol, whose back electrochemical reduction was detected on a glassy carbon electrode surface at −200 mV. Thus, when ciprofloxacin (CF) was added to the solution, this piperazine-containing compound participate in Michael addition reactions with catechol to form the corresponding piperazine-quinone derivatives, decreasing the peak current obtained, in proportion with the increase of its concentration. The highest response for CF was obtained around pH 7. This method could be used to determine CF concentration in the range of 0.02-65 μM (r = 0.999). The determination of CF concentration was possible with a detection limit of 0.4 nM, in the processing of as many as 25 samples per hour. Application of this analysis to different pharmaceutical samples containing CF supports the utility of the HRP-rotating biosensor.     

Biosensor for determination of hydrogen peroxide based on catalase activity of human erythrocytes

A hydrogen peroxide biosensor based on human erythrocytes is described. Erythrocytes are retained on the surface of an oxygen electrode by a semipermeable membrane. The response is based on the catalase activity of the erythrocytes. The sensitivity of 10^?4 mol 1^?1 and linearity from 1.5×10^?4 to 5×10^?3 mol^?1 are comparable to those of analogous enzyme biosensors for hydrogen peroxide determination. The greatest advantages of this biosensor are its easy preparation and a lifetime of 2 months together with good reproducibility (relative standard deviation <5%) and selectivity; only ascorbic acid appeared to interfere with the measurements.     

Electrochemical determination of allopurinol based on its interaction with xanthine oxidase

Allopurinol was determined in the range 0.20–50 μM utilizing xanthine oxidase immobilized onto a carbon paste electrode. Alternative assay methods relying on either inhibition of enzymatic activity or direct formation of a product are evaluated. Regeneration of xanthine oxidase activity under various conditions is also surveyed.     

Development of an amperometric biosensor based on covalent immobilization of tyrosinase on a boron-doped diamond electrode

An amperometric enzyme electrode based on direct covalent immobilization of tyrosinase on a boron-doped diamond (BDD) electrode has been developed for the detection of phenolic compounds. Combined chemical and electrochemical modifications of the BDD film with 4-nitrobenzenediazonium tetrafluoroborate, an aminophenyl-modified BDD (AP–BDD) surface was produced, and then the tyrosinase was covalently immobilized on the BDD surface via carbodiimide coupling. The response dependences of the enzyme electrode (Tyr–AP–BDD electrode) on pH of solution, applied potential, oxygen level and phenolic compounds diffusion were studied. The Tyr–AP–BDD electrode shows a linear response range of 1–200, 1–200 and 1–250 μM and sensitivity of 232.5, 636.7 and 385.8 mA M⁻¹ cm⁻² for phenol, p-cresol and 4-chlorophenol, respectively. 90 percent of the enzyme activity of the Tyr–AP–BDD electrode is retained for 5 weeks storing in 0.1 M PBS (pH 6.5) at 4 °C.     

A quercetin-modified biosensor for amperometric determination of uric acid in the presence of ascorbic acid

The present work reports a quercetin-modified wax-impregnated graphite electrode (Qu/WGE) prepared through an electrochemical oxidation procedure in quercetin-containing phosphate buffer solution (PBS), for the purpose of detecting uric acid (UA) in the presence of ascorbic acid (AA). During modification quercetin was oxidized to the corresponding quinonic structure, and in the blank buffer solution the electrodeposited film exhibits a voltammetric response anticipated for the surface-immobilized quercetin. Retarding effect of the film towards the reaction of anionic species was found; therefore the pH of sample solutions was selected to ensure the analyte in molecular form. At suitable pHs the Qu/WGE shows excellent electrocatalytic effect towards the oxidation of both AA and UA, and separates the voltammetric signal of UA from AA by about 280 mV, allowing simultaneous detection of these two species. A linear relation between the peak current and concentration was obtained for UA in the range of 1-50 μM in the presence of 0.5 mM AA, with a detection limit 1.0 μM (S/N = 3). This sensor was stable, reproducible and outstanding for long-term use.     

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.