Stabilisation of tyrosinase by reversed micelles for bioelectrocatalysis in dry organic media

The enzymatic and bioelectrocatalytic activity of tyrosinase from mushrooms was studied in a system of reversed micelles formed by Aerosol OT (AOT) in hexane. The optimal catechol oxidising activity of tyrosinase incorporated in reversed micelles was found at a hydration degree of w(0)=25. The catalytic activity was comparable with tyrosinase activity in aqueous media. When immobilized at an Au electrode, either directly or in reversed micelles, tyrosinase exhibited a similar efficiency of the bioelectrocatalytic reduction of O(2) mediated by catechol; however, a rapid decrease in the activity correlated with the destruction of reversed micelles and/or the removal of tyrosinase from the electrode surface. The system containing tyrosinase in reversed micelles with caoutchouk, spread on the surface of the Au electrode and successively covered with a Nafion membrane layer, was found to result in stable tyrosinase-modified electrodes, which were resistant to inactivation in dry acetonitrile. The proposed technique offers possibilities for further development of highly active and stable surfactant/enzyme-modified electrodes for measurements carried out in organic solvents.     

Disposable biosensor based on graphene oxide conjugated with tyrosinase assembled gold nanoparticles

A highly efficient enzyme-based screen printed electrode (SPE) was obtained by using covalent attachment between 1-pyrenebutanoic acid, succinimidyl ester (PASE) adsorbing on the graphene oxide (GO) sheets and amines of tyrosinase-protected gold nanoparticles (Tyr-Au). Herein, the bi-functional molecule PASE was assembled onto GO sheets. Subsequently, the Tyr-Au was immobilized on the PASE-GO sheets forming a biocompatible nanocomposite, which was further coated onto the working electrode surface of the SPE. The characterization of obtained nanocomposite and modified SPE surface was investigated by atomic force microscopy (AFM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Attributing to the synergistic effect of GO-Au integration and the good biocompatibility of the hybrid-material, the fabricated disposable biosensor (Tyr-Au/PASE-GO/SPE) exhibited a rapid amperometric response (less than 6s) with a high sensitivity and good storage stability for monitoring catechol. This method shows a good linearity in the range from 8.3×10(-8) to 2.3×10(-5) M for catechol with a squared correlation coefficient of 0.9980, a quantitation limit of 8.2×10(-8) M (S/N=10) and a detection limit of 2.4×10(-8) M (S/N=3). The Michaelis-Menten constant was measured to be 0.027 mM. This disposable tyrosinase biosensor could offer a great potential for rapid, cost-effective and on-field analysis of phenolic compounds.     

Analysis of thiols with tyrosinase-modified carbon paste electrodes based on blocking of substrate recycling

The enzyme, tyrosinase, was immobilized inside carbon paste electrodes (CPE) for the analysis of thiol-containing compounds such as the reduced form of glutathione (GSH) and L-cysteine. The measuring principle of this sensor is based on the blocking of the substrate recycling process between the enzyme and the electrode. The current response is monitored at -0.050 V versus Ag/AgCl. At this low potential, interferences from easily oxidizable species such as ascorbic acid and uric acid are minimized. The tyrosinase CPE is characterized both in steady state experiments and by flow injection analysis (FIA). GSH is used as the model thiol-containing compound for the study. The highest response for GSH was obtained around pH 6.5. A detection limit of 100 nM and 1 microM is achieved for GSH in steady state and in flow measurements, respectively. The analytical range for GSH is dependent on the concentration of the tyrosinase substrate (catechol). In steady state experiments, and at a lower substrate concentration (10 microM catechol), a linear range of 1-8 microM is found for GSH as compared with 5-30 microM at a higher substrate concentration of 20 microM catechol. Current response of the tyrosinase CPE is not affected by the oxidized form of GSH and L-cysteine (glutathione disulfide, GSSG, and L-cystine, respectively) and sulfur-containing compound such as methionine. The tyrosinase CPE can also detect coenzyme A, which makes it possible to construct biosensors based on enzymes producing or utilizing coenzyme A.     

Polyphenol biosensor based on laccase immobilized onto silver nanoparticles/multiwalled carbon nanotube/polyaniline gold electrode

Laccase purified from Ganoderma sp. was immobilized covalently onto electrochemically deposited silver nanoparticles (AgNPs)/carboxylated multiwalled carbon nanotubes (cMWCNT)/polyaniline (PANI) layer on the surface of gold (Au) electrode. A polyphenol biosensor was fabricated using this enzyme electrode (laccase/AgNPs/cMWCNT/PANI/Au electrode) as the working electrode, Ag/AgCl as the reference electrode, and platinum (Pt) wire as the auxiliary electrode connected through a potentiostat. The biosensor showed optimal response at pH 5.5 (0.1 M acetate buffer) and 35 °C when operated at a scan rate of 50 mV s⁻¹. Linear range, response time, and detection limit were 0.1–500 μM, 6 s, and 0.1 μM, respectively. The sensor was employed for the determination of total phenolic content in tea, alcoholic beverages, and pharmaceutical formulations. The enzyme electrode was used 200 times over a period of 4 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors in that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective PANI microenvironment.     

Development of an amperometric sulfite biosensor based on SO(x)/PBNPs/PPY modified ITO electrode

A sulfite oxidase (SO(x)) (EC 1.8.3.1) purified from Syzygium cumini leaves was immobilized onto prussian blue nanoparticles/polypyrrole composite (PBNPs/PPY) electrodeposited onto the surface of indium tin oxide (ITO) electrode. An amperometric sulfite biosensor was fabricated using SO(x)/PBNPs/PPY/ITO electrode as working electrode, Ag/AgCl as standard and Pt wire as auxiliary electrode connected through a potentiostat. The working electrode was characterized by Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS) before and after immobilization of SO(x). The biosensor showed optimum response within 2s, when operated at 20mVs(-1) in 0.1M Tris-HCl buffer, pH 8.5 and at 35°C. Linear range and minimum detection limit were 0.5-1000μM and 0.12μM (S/N=3) respectively. There was good correlation (r=0.99) between red wine samples sulfite value by standard DTNB method and the present method. The sensor was evaluated with 97% recovery of added sulfite in red wine samples and 2.2% and 4.3% within and between batch coefficients of variation respectively. The sensor was employed for determination of sulfite level in red and white wine samples. The enzyme electrode was used 200 times over a period of 3 months when stored at 4°C.     

Self-assembled monolayer for low density lipoprotein detection

Heparin (HEP) has been covalently immobilized onto 4-aminothiophenol (ATP) self-assembled monolayer (SAM) deposited onto gold (Au)-coated glass plate for low density lipoprotein (LDL) detection. The HEP/ATP/Au and LDL/HEP/ATP/Au electrodes have been characterized using cyclic voltammetry (CV) and scanning electron microscopy (SEM). Surface plasmon resonance (SPR) measurements reveal that HEP/ATP/Au electrode is sensitive to detection of the LDL in the range 0.03 microM (10 mg/dl)-0.39 microM (130 mg/dl). The values of association and dissociation rate constants in the association phase calculated by kinetic analysis have been found to be k(a) = 9.67 x 10(1) M(-1) s(-1) and k(d) = 2.64 x 10(-4) s(-1).     

Immobilization of creatininase, creatinase and sarcosine oxidase on iron oxide nanoparticles/chitosan-g-polyaniline modified Pt electrode for detection of creatinine

Commercial enzymes, creatininase (CA) from Pseudomonas sp, creatinase (CI) from Pseudomonas sp, sarcosine oxidase (SO) from Bacillus sp were co-immobilized onto iron oxide nanoparticles/chitosan-graft-polyaniline (Fe(3)O(4)-NPs/CHIT-g-PANI) composite film electrodeposited on surface of Pt electrode through glutaraldehyde coupling. Transmission electron microscopy (TEM) was used for characterization of Fe(3)O(4)-NPs. A creatinine biosensor was fabricated using Enzymes/Fe(3)O(4)-NPs/CHIT-g-PANI/Pt electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode. The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopic and electrochemical impedance spectroscopy (EIS). The biosensor exhibited an optimum response within 2s at pH 7.5 and 30 °C, when polarized at 0.4V vs Ag/AgCl. The electrocatalytic response showed a linear dependence on creatinine concentration ranging from 1 to 800 μM. The sensitivity of the biosensor was 3.9 μA μM(-1) cm(-2), with a detection limit of 1 μM (S/N=3). Apparent Michaelis-Menton (K(m)) value for creatinine was 0.17 mM. The biosensor showed only 10% loss in its initial response after 120 uses over 200 days, when stored at 4 °C. The biosensor measured creatinine in the serum of apparently healthy persons which correlated well with a standard colorimetric method (r=0.99).     

Highly sensitive amperometric biosensors for phenols based on polyaniline-ionic liquid-carbon nanofiber composite

A novel polyaniline-ionic liquid-carbon nanofiber (PANI-IL-CNF) composite was greenly prepared by in situ one-step electropolymerization of aniline in the presence of IL and CNF for fabrication of amperometric biosensors. The scanning electron micrographs confirmed that the PANI uniformly grew along with the structure of CNF and the PANI-IL-CNF composite film showed a fibrillar morphology with the diameter of around 95 nm. A phenol biosensor was constructed by immobilizing tyrosinase on the surface of the composite modified glassy carbon electrode via the cross-linking step with glutaraldehyde. The biosensor exhibited a wide linear response to catechol ranging from 4.0 x 10(-10) to 2.1 x 10(-6)M with a high sensitivity of 296+/-4 AM(-1)cm(-2), a limit of detection down to 0.1 nM at the signal to noise ratio of 3 and applied potential of -0.05 V. According to the Arrhenius equation, the activation energy for enzymatic reaction was calculated to be 38.8 kJmol(-1) using catechol as the substrate. The apparent Michaelis-Menten constants of the enzyme electrode were estimated to be 1.44, 1.33, 1.16, 0.65 microM for catechol, p-cresol, phenol, m-cresol, respectively. The functionalization of CNF with PANI in IL provided good biocompatible platform for biosensing and biocatalysis.     

A mediator-free phenol biosensor based on immobilizing tyrosinase to ZnO nanoparticles

A mediator-free phenol biosensor was developed. The low-isoelectric point tyrosinase was adsorbed on the surface of high-isoelectric point ZnO nanoparticles (nano-ZnO) facilitated by the electrostatic interactions and then immobilized on the glassy carbon electrode via the film forming by chitosan. It was found that the nano-ZnO matrix provided an advantageous microenvironment in terms of its favorable isoelectric point for tyrosinase loading and the immobilized tyrosinase retaining its activity to a large extent. Moreover, there is no need to use any other electron mediators. Phenolic compounds were determined by the direct reduction of biocatalytically generated quinone species at -200mV (vs. saturated calomel electrode). The parameters of the fabrication process and the various experimental variables for the enzyme electrode were optimized. The resulting biosensor can reach 95% of steady-state current within 10s, and the sensitivity was as high as 182microAmmol(-1)L. The linear range for phenol determination was from 1.5x10(-7) to 6.5x10(-5)molL(-1) with a detection limit of 5.0x 10(-8)molL(-1) obtained at a signal/noise ratio of 3. In addition, the apparent Michaelis-Menten constant (K(m)(app)) and the stability of the enzyme electrode were estimated. The performance of the developed biosensor was compared with that of biosensors based on other immobilization matrices.     

Direct electrochemistry of horseradish peroxidase immobilized on a colloid/cysteamine-modified gold electrode

Direct electron transfer of immobilized horseradish peroxidase on gold colloid and its application as a biosensor were investigated by using electrochemical methods. The Au colloids were associated with a cysteamine monolayer on the gold electrode surface. A pair of redox peaks attributed to the direct redox reaction of horseradish peroxidase (HRP) were observed at the HRP/Au colloid/cysteamine-modified electrode in 0.1 M phosphate buffer (pH 7.0). The surface coverage of HRP immobilized on Au colloid was about 7.6 x 10(-10) mol/cm(2). The sensor displayed an excellent electrocatalytic response to the reduction of H(2)O(2) without the aid of an electron mediator. The calibration range of H(2)O(2) was 1. 4 microM to 9.2 mM with good linear relation from 1.4 microM to 2.8 mM. A detection limit of 0.58 microM was estimated at a signal-to-noise ratio of 3. The sensor showed good reproducibility for the determination of H(2)O(2). The variation coefficients were 3. 1 and 3.9% (n = 10) at 46 microM and 2.8 mM H(2)O(2), respectively. The response showed a Michaelis-Menten behavior at higher H(2)O(2) concentrations. The K(app)(M) value for the H(2)O(2) sensor was found to be 2.3 mM.     

Glucose biosensor based on electrodeposition of platinum nanoparticles onto carbon nanotubes and immobilizing enzyme with chitosan-SiO(2) sol-gel

A novel amperometric biosensor, based on electrodeposition of platinum nanoparticles onto multi-walled carbon nanotube (MWNTs) and immobilizing enzyme with chitosan-SiO(2) sol-gel, is presented in this article. MWNTs were cast on the glass carbon (GC) substrate directly. An extra Nafion coating was used to eliminate common interferents such as acetaminophen and ascorbic acids. The morphologies and electrochemical performance of the modified electrodes have been investigated by scanning electron microscopy (SEM) and amperometric methods, respectively. The synergistic action of Pt and MWNTs and the biocompatibility of chitosan-SiO(2) sol-gel made the biosensor have excellent electrocatalytic activity and high stability. The resulting biosensor exhibits good response performance to glucose with a wide linear range from 1 microM to 23 mM and a low detection limit 1 microM. The biosensor also shows a short response time (within 5s), and a high sensitivity (58.9 microAm M(-1)cm(-2)). In addition, effects of pH value, applied potential, rotating rate, electrode construction and electroactive interferents on the amperometric response of the sensor were investigated and discussed in detail.     

An amperometric biosensor based on laccase immobilized onto MnO2NPs/cMWCNT/PANI modified Au electrode

A method is described for construction of an amperometric biosensor for detection of phenolic compounds based on covalent immobilization of laccase (Lac) onto manganese dioxide nanoparticles (MnO(2)NPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/PANI composite electrodeposited onto a gold (Au) electrode through N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. The modified electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response at pH 5.5 (0.1M sodium acetate buffer) and 35°C, when operated at 0.3 V vs. Ag/AgCl. Linear range, response time, detection limit were 0.1-10 μM (lower concentration range) and 10-500 μM (higher concentration range), 4s and 0.04 μM, respectively. Biosensor measured total phenolic content in tea leaves extract. The enzyme electrode was used 150 times over a period of 5 months.     

Bienzymatic amperometric biosensor for choline based on mediator thionine in situ electropolymerized within a carbon paste electrode

An amperometric enzyme biosensor for the determination of choline utilizing two enzymes, choline oxidase (CHOD) and horseradish peroxidase (HRP), is described. The biosensor consisted of CHOD cross-linked onto a HRP-immobilized carbon paste electrode. The biosensor was prepared by in situ electropolymerization of poly(thionine) within a carbon paste containing the enzyme HRP and thionine monomer and then CHOD was immobilized by using chitosan film through cross-linking with glutaraldehyde. The in situ electrogenerated poly(thionine) displays excellent electron transform efficiency between the enzyme HRP and the electrode surface, and the polymer enables improvement in enzyme immobilization within the paste. Several parameters such as the amount of thionine and enzyme, the applied potential, the pH, etc. have been studied. Amperometric detection of choline was realized at an applied potential of -0.2V vs saturated calomel electrode in 1/15M phosphate buffer solution (pH 7.4) with a linear response range between 5.0 x 10(-6) and 6.0 x 10(-4)M choline and a response time of 15s. When applied to the analysis of phosphatidylcholine in serum samples, a 0.997 correlation was obtained between the biosensor results and those obtained by a hospital method.     

Electrochemical biosensor for catechol using agarose-guar gum entrapped tyrosinase

An electrochemical biosensor using tyrosinase was constructed for the determination of catechol. The enzyme was extracted from a plant source Amorphophallus companulatus and entrapped in agarose-guar gum composite biopolymer matrix. Catechol was determined by direct reduction of biocatalytically liberated quinone species at -0.1 V versus Ag/AgCl (3M KCl). The response was found to be linear and concentration dependent in the range of 6 x 10(-5) to 8 x 10(-4)M with a lower detection limit of 6 microM. It has reusability up to 20 cycles and a shelf life of more than 2 months when stored at 4 degrees C.     

An electrochemical biosensor for fructosyl valine for glycosylated hemoglobin detection based on core-shell magnetic bionanoparticles modified gold electrode

A high-performance amperometric fructosyl valine (FV) biosensor was developed, based on immobilization of fructosyl amino-acid oxidase (FAO) on core-shell magnetic bionanoparticles modified gold electrode. Chitosan was used to introduce amino groups onto the surface of core-shell magnetic bionanoparticles (MNPs). With FAO as an enzyme model, a new fructosyl valine biosensor was fabricated. The biosensor showed optimum response, when operated at 50 mVs(-1) in 0.1M potassium phosphate buffer, pH 7.5 and 35°C. The biosensor exhibited excellent sensitivity [the detection limit is down to 0.1mM for FV], fast response time (less than 4s), wide linear range (from 0 to 2mM). Analytical recovery of added FV was 95.00-98.50%. Within batch and between batch coefficients of variation were <2.58% and <5.63%, respectively. The enzyme electrode was used 250 times over 3 months, when stored at 4°C.     

An amperometric biosensor based on laccase immobilized onto Fe(3)O(4)NPs/cMWCNT/PANI/Au electrode for determination of phenolic content in tea leaves extract

A method is described for the construction of an amperometric biosensor for detection of phenolic compounds based on covalent immobilization of laccase onto iron oxide nanoparticles (Fe(3)O(4)NPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite electrodeposited onto a gold (Au) electrode. The modified electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 3s at pH 6.0 (0.1M sodium acetate buffer) and 35°C, when operated at 0.3V vs. Ag/AgCl. Linear range, detection limit were 0.1-10μM (lower concentration range) and 10-500μM (higher concentration range), and 0.03μM respectively. The sensor measured total phenolic content in tea leaves extract. The enzyme electrode lost 25% of its initial activity after its 150 uses over a period of 4 months, when stored at 4°C.     

Development of a biosensor for endocrine disrupting compounds based on tyrosinase entrapped within a poly(thionine) film

Preparation of semiconducting films by electropolymerisation of a monomer which is itself a redox mediator is an attractive and simple method for biosensor fabrication. A polymeric film of the redox dye thionine (phenothiazine) enables the stable immobilisation of polyphenol oxidase (tyrosinase) while acting as mediator for the enzymatic process. The immobilisation method is based on an inner crosslinked tyrosinase layer which contains thionine with an electropolymerised film of poly(thionine) on top. This method gave the most stable redox couple for poly(thionine) and exhibited the greatest response stability. The sensor was tested using a range of synthetic oestrogens and phenolic compounds, which are suspected endocrine disruptors/oestrogen mimics. The device responded well to all compounds tested with limits of detection ranging from 1 to 23 microM (based on three times S/N ratio). The tyrosinase/poly(thionine) electrode response to phenol was 3 orders of magnitude greater than the unmediated response in the absence of poly(thionine).     

Hydrogen peroxide sensitive amperometric biosensor based on horseradish peroxidase entrapped in a polypyrrole electrode

The enzyme horseradish peroxidase (HRP) has been entrapped in situ by electropolymerization of pyrrole onto a platinum electrode. The latter was previously coated by a polypyrrole layer for better adhesion of the biocatalyst film and in order to avoid the enzyme folding onto the Pt electrode. The biosensor allowed the determination of hydrogen peroxide in the concentration range comprised between 4.9 x 10(-7) and 6.3 x 10(-4) M. The biosensor retained more than 90% of its original activity after 35 days of use.     

Au-nanoparticles as an electrochemical sensing platform for aptamer-thrombin interaction

A novel electrochemical method for the detection of bioaffinity interactions based on a gold-nanoparticles sensing platform and on the usage of stripping voltammetry technique was developed. The oxidation of gold surface (resulted in gold oxide formation) upon polarization served as a basis for analytical response. As a model, thrombin-thrombin binding aptamer couple was chosen. The aptamer was immobilized on a screen-printed electrode modified with gold-nanoparticles by avidin-biotin technology. Cathodic peak area was found proportional to thrombin quantity specifically adsorbed onto electrode surface. Sigmoid calibration curve as is typical for immunoassay was obtained, with thrombin detection limit of 10(-9)M. Linear range corresponds from 10(-8) to 10(-5)M thrombin concentration or 2 x 10(-14) to 2 x 10(-11)mol/electrode (R=0.996). Binding of thrombin to an aptamer has also been detected using the ferricyanide/ferrocyanide redox couple as electrochemical indicator.     

Amperometric tyrosinase biosensor based on Fe3O4 nanoparticles-chitosan nanocomposite

A novel tyrosinase biosensor based on Fe(3)O(4) nanoparticles-chitosan nanocomposite has been developed for the detection of phenolic compounds. The large surface area of Fe(3)O(4) nanoparticles and the porous morphology of chitosan led to a high loading of enzyme and the entrapped enzyme could retain its bioactivity. The tyrosinase-Fe(3)O(4) nanoparticle-chitosan bionanocomposite film was characterized with atomic force microscopy and AC impedance spectra. The prepared biosensor was used to determine phenolic compounds by amperometric detection of the biocatalytically liberated quinone at -0.2V vs. saturated calomel electrode (SCE). The different parameters, including working potential, pH of supporting electrolyte and temperature that governs the analytical performance of the biosensor have been studied in detail and optimized. The biosensor was applied to detect catechol with a linear range of 8.3 x 10(-8) to 7.0 x 10(-5)mol L(-1), and the detection limit of 2.5 x 10(-8)mol L(-1). The tyrosinase biosensor exhibits good repeatability and stability. Such new tyrosinase biosensor shows great promise for rapid, simple, and cost-effective analysis of phenolic contaminants in environmental samples. The proposed strategy can be extended for the development of other enzyme-based biosensors.