An amperometric glucose biosensor based on glucose oxidase immobilized in electropolymerized poly(o-aminophenol) and carbon nanotubes composite film on a gold electrode.

An amperometric glucose biosensor is developed that is based on immobilization of glucose oxidase (GOD) in a composite film of poly(o-aminophenol) (POAP) and carbon nanotubes (CNT), which are electrochemically co-polymerized at a gold (Au) electrode. Because of the high surface per volume ratio and excellent electrical conductivity of CNT, the biosensor based on an Au/POAP/CNT/GOD electrode has lower detection limit (0.01 mM), larger maximum response current (0.24 mA cm(-2)) and higher sensitivity (11.4 mA M(-1) cm(-2)) than the values of the biosensor based on an Au/POAP/GOD electrode. Additionally, the biosensor shows fast response time, large response current, and good anti-interferent ability for ascorbic acid, uric acid and acetaminophen. Good reproducibility and stability of the biosensor are also observed.     

Electrochemical nitrate biosensor based on poly(pyrrole-viologen) film-nitrate reductase-clay composite

The immobilization of nitrate reductase (NR) was performed by entrapment in a laponite clay gel and cross-linking by glutaraldehyde. In presence of nitrate and methyl viologen, a catalytic current appeared at -0.60 V illustrating the enzymatic reduction of nitrate into nitrite via the reduced form of the freely diffusing methyl viologen. The electropolymerization of a water-soluble pyrrole viologen derivative within the interlamellar spaces and channels of the host clay matrix successfully carried out the electrical wiring of the entrapped NR. Rotating disk measurements led to the determination of kinetic constants, namely k(2)=10.7 s(-1) and K(M)=7 microM. These parameters reflect the efficiency of the electro-enzymatic reduction of nitrate and the substrate affinity for the immobilized enzyme.     

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.     

Construction of a l-lysine biosensor by immobilizing lysine oxidase on a gold-poly(o-phenylenediamine) electrode

The construction of a l-lysine biosensor on a Si-gold strip electrode (SGSE) is described in this study. The construction comprises (a) the formation of poly(o-phenylenediamine, o-PD) membrane on the electrode surface via electropolymerization and (b) the immobilization of lysine oxidase on the gold/poly(o-PD) electrode with glutaraldehyde. The behavior of the gold/poly(o-PD) electrode against H(2)O(2) and lysine, as well as the repeatability of the electropolymerization and the time stability of the polymer were studied. The study showed that the electropolymerization procedure is repeatable, and that the polymer is quite stable for at least 40 days. The biosensor showed a linear calibration curve in the range 0.01-1x10(-5) M (0.1-10 muM) lysine. The interfering effect of other amino acids on the biosensor performance was also studied and amperometric selectivity coefficients were calculated. The biosensor responded mainly against tyrosine and cysteine, while the response to phenylalanine, arginine, histidine and ornithine was very low. By changing the electropolymerization conditions, the effect of interferents was further reduced.     

Electroimmobilization of MAO into a Polypyrrole Film and Its Utilization for Amperometric Flow Detection of Antidepressant Drugs

The preparation of a biosensor based on the enzymatic immobilization in polypyrrole polymer for the detection of antidepressant drugs is described. The enzyme monoamine oxidase (MAO) was immobilized by electropolymerization of pyrrole around a platinum electrode, at a constant potential of +0.75 V (vs. Ag/AgCl) in such a way to obtain a membrane thickness, which was constant and equal to 100 mC/cm². The biosensor was obtained from a 0.1 M KCl saline solution containing pyrrole at a concentration equal to 0.4 M and 2.5 mU/mL of MAO. The biosensor was adapted to a continuous flow injection analysis system (FIA) with the amperometric detection of hydrogen peroxide produced by enzymatic reaction carried out at a potential of +0.7 V (vs. Ag/AgCl), pH 7.4 and temperature of 37 °C. In optimized flow conditions, the biosensor presented an analytical response for fluoxetine in the interval between 0.67 and 4.33 mM, with a detection limit of 0.10 mM. The analytical use of the biosensor developed was evaluated through analysis of commercial pharmaceutical products containing fluoxetine, available on the Portuguese market. The amperometric flow results obtained do not differ significantly from the values resulting from analysis of the same products by the method proposed by the US Pharmacopeia, with sampling rates of 20–25 samples/hour.     

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

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

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

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

Amperometric Glucose Biosensor Based on Glucose Oxidase Encapsulated in Carbon Nanotube–Titania–Nafion Composite Film on Platinized Glassy Carbon Electrode

A highly sensitive and selective glucose biosensor has been developed based on immobilization of glucose oxidase within mesoporous carbon nanotube–titania–Nafion composite film coated on a platinized glassy carbon electrode. Synergistic electrocatalytic activity of carbon nanotubes and electrodeposited platinum nanoparticles on electrode surface resulted in an efficient reduction of hydrogen peroxide, allowing the sensitive and selective quantitation of glucose by the direct reduction of enzymatically‐liberated hydrogen peroxide at ?0.1 V versus Ag/AgCl (3 M NaCl) without a mediator. The present biosensor responded linearly to glucose in the wide concentration range from 5.0×10^?5 to 5.0×10^?3 M with a good sensitivity of 154 mA M^?1cm^?2. Due to the mesoporous nature of CNT–titania–Nafion composite film, the present biosensor exhibited very fast response time within 2 s. In addition, the present biosensor did not show any interference from large excess of ascorbic acid and uric acid.     

Detection of Galactose and Lactose by a Poly(Amphiphilic Pyrrole)-Galactose Oxidase Electrode

Abstract

The entrapment of galactose oxidase (GAO) on an electrode surface by coadsorption with a cationic amphiphilic pyrrole and electropolymerization of this pyrrole monomer is described. This simple and rapid procedure for biosensor construction provides very fast responsive and sensitive GAO-based sensors to galactose and lactose. The electrode response is based on the electrochemical detection of enzymically generated hydrogen peroxide. The stability, optimum pH and selectivity of the bioelectrode as well as the characteristics of the immobilized galactose oxidase have been determined. Poly(amphiphilic pyrrole) films have been electrogenerated on the surface of the bioelectrode and the effect of such additional coatings on the biosensor selectivity have also been examined.     

A promising dehydrogenase-based bioanode for a glucose biosensor and glucose/O2 biofuel cell

A new type of dehydrogenase-based amperometric glucose biosensor was constructed using glucose dehydrogenase (GDH) which was immobilized on the edge-plane pyrolytic graphite (EPPG) electrode modified with poly(phenosafranin)-functionalized single-walled carbon nanotubes (PPS-SWCNTs). The PPS-SWCNT-modified EPPG electrode was prepared by electropolymerization of phenosafranin on the EPPG electrode which had been previously coated with SWCNTs. The performance of the GDH/PPS-SWCNT/EPPG bioanode was evaluated using cyclic voltammetry and amperometry in the presence of glucose. The GDH/PPS-SWCNT/EPPG electrode possesses promising characteristics as a glucose sensor: a wide linear dynamic range of 50 to 700 μM, low detection limit of 0.3 μM, fast response time (1-2 s), high sensitivity (96.5 μA cm(-2) mM(-1)), and anti-interference and anti-fouling abilities. Moreover, the performance of the GDH/PPS-SWCNT/EPPG bioanode was tested in a glucose/O(2) biofuel cell. The maximum power density delivered by the assembled glucose/O(2) biofuel cell could reach 64.0 μW cm(-2) at a cell voltage of 0.3 V with 40 mM glucose.     

Biosensors based on immobilization of biomolecules by electrogenerated polymer films

The concept and potentialities of electrochemical procedures of biomolecule immobilization are described. The entrapment of biomolecules within electropolymerized films consists of the application of an appropriate potential to an electrode soaked in an aqueous solution containing monomer and biomolecules. This method of biosensor construction is compared with a two-step procedure based on the adsorption of an aqueous amphiphilic pyrrole monomer-biomolecule mixture on an electrode followed by the electropolymerization of the adsorbed monomers. Another approach is based on the electrogeneration of polymer films functionalized by specific groups allowing subsequently the attachment of biomolecules. The immobilization of biomolecules on these films by covalent binding or noncovalent interactions is described.     

Electrochemical characterization of biosensor based on nitrite reductase and methyl viologen co-immobilized glassy carbon electrode

Nitrite reductase (NiR, nitric-oxide: ferricytochrome c oxidoreductase, EC 1.7.2.1) and methyl viologen (MV) were co-immobilized on glassy carbon electrode (GCE, d=3 mm) by polymer entrapment, and the electrode was tested as an electrochemical biosensor for amperometric determination of nitrite. The immobilization was performed by sequential loading and drying of a homogeneous mixture of poly(vinyl alcohol) (PVA), NiR and MV, followed by poly(allylamine hydrochloride) (PAH) solution, and finally hydrophilic polyurethane (HPU) dissolved in chloroform. The positively charged PAH layer could effectively keep immobilized cationic MV from diffusing through the membrane, holding mediator tightly near or on the electrode surface. The working principle of the biosensor was based on MV mediated electron transfer between electrode and immobilized NiR. The response time (t(90%)) of the biosensor was about 20 s and sensitivity was 11.8 nA/ microM (2.5 mU NiR) with linear response range of 1.5-260 microM (r(2)=0.996) and detection limit of 1.5 microM (S/N=3). Lineweaver-Burk plot showed that Michaelis-Menten constant (K(m,app)) was about 770 microM. The biosensor showed durable storage stability for 24 days (stored in ambient air at room temperature) retaining 80% of its initial activity, and showed satisfactory reproducibility (relative standard deviation (R.S.D.)=3.8%, n=9). Interference study showed that chlorate, chloride, sulfite, sulfate did not interfere with the nitrite determination, however, nitrate interfered with the determination with relative sensitivity of 38% (ratio of sensitivity for nitrate to that for nitrite). In addition to the full characterization of the biosensor, kinetic study was also conducted in solution and the homogeneous rate constant (k(2)) between NiR and MV were determined by chronoamperometry to be 5.8 x 10(5) M(-1) s(-1).     

Covalent attachment of glucose oxidase to an Au electrode modified with gold nanoparticles for use as glucose biosensor

A feasible method to fabricate glucose biosensor was developed by covalent attachment of glucose oxidase (GOx) to a gold nanoparticle monolayer modified Au electrode. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of ferrocyanide followed and confirmed the assemble process of biosensor, and indicated that the gold nanoparticles in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. CV performed in the presence of excess glucose and artificial redox mediator, ferrocenemethanol, allowed to quantify the surface concentration of electrically wired enzyme (Gamma(E)(0)) on the basis of kinetic models reported in literature. The Gamma(E)(0) on proposed electrode was high to 4.1 x 10(-12) mol.cm(-2), which was more than four times of that on electrode direct immobilization of enzyme by cystamine without intermediate layer of gold nanoparticles and 2.4 times of a saturated monolayer of GOx on electrode surface. The analytical performance of this biosensor was investigated by amperometry. The sensor provided a linear response to glucose over the concentration range of 2.0 x 10(-5)-5.7 x 10(-3) M with a sensitivity of 8.8 microA.mM(-1).cm(-2) and a detection limit of 8.2 microM. The apparent Michaelis-Menten constant (K(m)(app)) for the sensor was found to be 4.3 mM. In addition, the sensor has good reproducibility, and can remain stable over 30 days.     

Micro- to nanostructured poly(pyrrole-nitrilotriacetic acid) films via nanosphere templates: applications to 3D enzyme attachment by affinity interactions

We report the combination of latex nanosphere lithography with electropolymerization of N-substituted pyrrole monomer bearing a nitrilotriacetic acid (NTA) moiety for the template-assisted nanostructuration of poly(pyrrole-NTA) films and their application for biomolecule immobilization. The electrodes were modified by casting latex beads (100 or 900 nm in diameter) on their surface followed by electropolymerization of the pyrrole-NTA monomer and the subsequent chelation of Cu²⁺ ions. The dissolution of the nanobeads leads then to a nanostructured polymer film with increased surface. Thanks to the versatile affinity interactions between the (NTA)Cu²⁺ complex and histidine- or biotin-tagged proteins, both tyrosinase and glucose oxidase were immobilized on the modified electrode. Nanostructuration of the polypyrrole via nanosphere lithography (NSL) using 900- and 100-nm latex beads allows an increase in surface concentration of enzymes anchored on the functionalized polypyrrole electrode. The nanostructured enzyme electrodes were characterized by fluorescence microscopy, 3D laser scanning confocal microscopy, and scanning electron microscopy. Electrochemical studies demonstrate the increase in the amount of immobilized biomolecules and associated biosensor performances when achieving NSL compared to conventional polymer formation without bead template. In addition, the decrease in nanobead diameter from 900 to 100 nm provides an enhancement in biosensor performance. Between biosensors based on films polymerized without nanobeads and with 100-nm nanobeads, maximum current density values increase from 4 to 56 μA cm^?2 and from 7 to 45 μA cm^?2 for biosensors based on tyrosinase and glucose oxidase, respectively.     

Bird nest-like zinc oxide nanostructures for sensitive electrochemical glucose biosensor

In this research, a novel bird nest-like zinc oxide (BN-ZnO) nanostructures were prepared by a simple solvothermal method. A sensitive electrochemical glucose biosensor was for the first time developed based on the immobilization of glucose oxidase (GOx) on nanostructured BN-ZnO modified electrode. The BN-ZnO nanostructure and the resultant biosensor were characterized by scanning electron microscope, X-ray diffraction spectroscopy, Fourier transform infrared spectroscopy, and electrochemical impedance spectroscopy. BN-ZnO nanostructures have large specific surface area and can load large amounts of GOx molecules. Meanwhile, BN-ZnO provides an excellent microenvironment to retain the native bioactivity of enzymes and to promote direct electron transfer between GOx and electrode surface. The proposed biosensor shows a wide linear range of 0.005–1.6 mmol/L, high sensitivity of 15.6 mA L mol⁻¹ cm⁻² with a low detection limit of 0.004 mmol/L. The resulting biosensor also shows excellent selectivity, acceptable stability and reproducibility, and can be successfully applied in the detection of glucose in human serum samples at −0.37 V.     

Investigation of a Biosensor Based on Phenylalanine Dehydrogenase Immobilized on a Polymer‐Blend Film for Phenylketonuria Diagnosis

We investigated a L ‐phenylalanine (L ‐phe) biosensor, functionalized through enzyme immobilization on a polymer‐blend film. The electron mediator 3,4‐dihydroxybenzaldehyde (3,4‐DHB) was employed at the electrode surface to improve direct oxidation of NADH to NAD⁺ and no additional reagents is required to be added to the sample solution. The bioactivated electrode was coated with a semi‐permeable cellulose acetate membrane in order to prevent dissolution of biofunctionalized polymer‐blend film. This constructed enzyme electrode is the first selective biosensor for phenylketonuria (PKU) detection. The sensitivity of the enzyme electrode was determined as 12.014 mA/M cm². The Michaelis–Menten and current responses as well as sensitivity of the electrode showed improved values than those of previous works. This selective biosensor presented an excellent electroanalytical response for L ‐phe, with a high steady‐state current being obtained after 20 s. The sensitivity of our biodevice is quite sufficient for the purpose of PKU detection because the reference range of clinical concern for L ‐phenylalanine concentration is C_{L ‐phe}>0.5 mM. This surface‐bioactivated enzyme electrode retained more than 80 % of its electrocatalytic activity after 16 days.     

Mass transport controlled oxygen reduction at anthraquinone modified 3D-CNT electrodes with immobilized Trametes hirsuta laccase

Carbon nanotubes covalently modified with anthraquinone were used as an electrode for the immobilization of Trametes hirsuta laccase. The adsorbed laccase is capable of oxygen reduction at a mass transport controlled rate (up to 3.5 mA cm(-2)) in the absence of a soluble mediator. The storage and operational stability of the electrode are excellent.     

Amperometric biosensor based on glucose dehydrogenase and plasma-polymerized thin films.

A novel design is described for an amperometric biosensor based on NAD(P)-dependent glucose dehydrogenase (GDH) combined with a plasma-polymerized thin film (PPF). The GDH is sandwiched between several nanometer thick acetonitrile PPFs on a sputtered gold electrode (PPF/GDH/PPF/Au). The lower PPF layer plays the role as an interface between enzyme and electrode because it is extremely thin, adheres well to the substrate (electrode), has a flat surface and a highly-crosslinked network structure, and is hydrophilic in nature. The upper PPF layer (overcoating) was directly deposited on immobilized GDH. The optimized amperometric biosensor characteristics covered 2.5-26 mM glucose concentration at +0.6 V of applied potential; the least-squares slope was 320 nA mM(-1) cm(-2) and the correlation coefficient was 0.990. Unlike conventional wet-chemical processes that are incompatible with mass production techniques, this dry-chemistry procedure has great potential for enabling high-throughput production of bioelectronic devices.     

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).     

Effects of fabrication parameters on the enzyme loading and sensor response of enzyme-carrying conductive polymer electrodes.

Hydrogen peroxide sensors where horseradish peroxidase (HRP) is incorporated into pyrrole/3-alkylsulfonate pyrrole copolymer films deposited on an SnO2 electrode (HRP/Py-PS electrode) were investigated with regard to the effects of the fabrication parameters (electropolymerization charge, deposition current density, and electrodeposition solution pH) on the amount of surface-immobilized enzyme and the sensor response. The amount of incorporated enzyme was determined with a method recently developed by ourselves. The results suggest that the amount of entrapped enzyme increases almost linearly with the total charge passed, and strongly depends on the polymer film growth rate and the electropolymerization pH. These findings open up a way to control the amount of enzyme and the resultant response of the biosensor by modifying the preparation conditions.