Increasing amperometric biosensor sensitivity by length fractionated single-walled carbon nanotubes

In this work the sensitivity-increasing effect of single-walled carbon nanotubes (SWCNTs) in amperometric biosensors, depending on their average length distribution, was studied. For this purpose the SWCNTs were oxidatively shortened and subsequently length separated by size exclusion chromatography. Transmission electron micrographs of different fractions of SWCNTs were collected. Diaphorase "wired" to an osmium redox polymer was blended with the shortened SWCNTs of different lengths. Depending on the average length of the SWCNTs the sensitivity of the amperometric biosensor model system towards oxidation of 1,4-dihydronicotinamide adenine dinucleotide (NADH) was increased by a factor of five. The best performance was achieved with SWCNTs of medium length. The linear range for NADH detection was between 5muM and 7mM, the maximum sensitivity was 47nAmuM(-1)cm(-2), and the detection limit was 1muM. The biosensor exhibited excellent electrocatalytic properties. Even at relatively high NADH concentrations the oxidative current was limited by the diffusion rate of NADH.     

Supramolecular architecture based on the self-assembling of multiwall carbon nanotubes dispersed in polyhistidine and glucose oxidase: Characterization and analytical applications for glucose biosensing

We report for the first time the development of a sensitive and selective glucose biosensor based on the self-assembling of multiwall carbon nanotubes (MWCNTs) dispersed in polyhistidine (Polyhis) and glucose oxidase (GOx) on glassy carbon electrodes (GCE). The supramolecular architecture was characterized by SEM, FT-IR and electrochemical techniques. The optimum multistructure was obtained with five (MWCNT-Polyhis/GOx) bilayers and one layer of Nafion as anti-interferent barrier. The sensitivity at 0.700V was (1.94±0.03) mAM(-1) (r=0.9991), with a linear range between 0.25 and 5.00mM, a detection limit of 2.2μM and a quantification limit of 6.7μM with minimum interference from lactose (1.5%), maltose (5.7%), galactose (1.2%), ascorbic acid (1.0%), and uric acid (3.3%). The biocatalytic layer demonstrated to be highly reproducible since the R.S.D. for 10 successive amperometric calibrations using the same surface was 3.6%. The sensitivity of the biosensor after 15 day storage at 4°C remained at 90% of its original value. The combination of the excellent dispersing properties and polycationic nature of polyhistidine, the stability of the MWCNT-Polyhis dispersion, the electrocatalytic properties of MWCNTs, the biocatalytic specificity of GOx, and the permselective properties of Nafion have allowed building up a sensitive, selective, robust, reproducible and stable glucose amperometric biosensor for the quantification of glucose in milk samples.     

Highly ordered mesoporous carbons as electrode material for the construction of electrochemical dehydrogenase- and oxidase-based biosensors

In this work, the excellent catalytic activity of highly ordered mesoporous carbons (OMCs) to the electrooxidation of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H(2)O(2)) was described for the construction of electrochemical alcohol dehydrogenase (ADH) and glucose oxidase (GOD)-based biosensors. The high density of edge-plane-like defective sites and high specific surface area of OMCs could be responsible for the electrocatalytic behavior at OMCs modified glassy carbon electrode (OMCs/GE), which induced a substantial decrease in the overpotential of NADH and H(2)O(2) oxidation reaction compared to carbon nanotubes modified glassy carbon electrode (CNTs/GE). Such ability of OMCs permits effective low-potential amperometric biosensing of ethanol and glucose, respectively, at Nafion/ADH-OMCs/GE and Nafion/GOD-OMCs/GE. Especially, as an amperometric glucose biosensor, Nafion/GOD-OMCs/GE showed large determination range (500-15,000mumoll(-1)), high sensitivity (0.053nAmumol(-1)), fast (9+/-1s) and stable response (amperometric response retained 90% of the initial activity after 10h stirring of 2mmoll(-1) glucose solution) to glucose as well as the effective discrimination to the possible interferences, which may make it to readily satisfy the need for the routine clinical diagnosis of diabetes. By comparing the electrochemical performance of OMCs with that of CNTs as electrode material for the construction of ADH- and GOD-biosensors in this work, we reveal that OMCs could be a favorable and promising carbon electrode material for constructing other electrochemical dehydrogenase- and oxidase-based biosensors, which may have wide potential applications in biocatalysis, bioelectronics and biofuel cells.     

Interference elimination of an amperometric glucose biosensor using poly(hydroxyethyl methacrylate) membrane

A permselective membrane fabricated from photo‐cross‐linked poly(hydroxyethyl methacrylate) (pHEMA) was studied as a potential selective membrane that can eliminate electrochemical interferences commonly faced by a hydrogen peroxide‐based biosensor. The quantitative selection of the permselective membrane was based on the permeabilities of hydrogen peroxide and acetaminophen (AC). AC was used as a model of the interfering substance due to its neutral nature. pHEMA membrane with the cross‐linking ratio of 0.043 was found to achieve a selectivity of hydrogen peroxide over AC of 10, while maintaining an acceptable degree of hydrogen peroxide response. A two‐layer glucose biosensor model consisting of glucose oxidase entrapped within a freeze‐thawed poly(vinyl alcohol) matrix and the cross‐linked pHEMA membrane was challenged with AC, ascorbic acid and uric acid. 0.2 mM AC and 0.2 mM ascorbic acid were completely eliminated. However, 0.2 mM uric acid could not be completely eliminated and still gave a bias of approximately 6.6% relative to 5 mM glucose. The results showed that cross‐linked pHEMA was quite promising as an interference eliminating inner membrane.     

Bi-enzyme L-arginine-selective amperometric biosensor based on ammonium-sensing polyaniline-modified electrode

A novel L-arginine-selective amperometric bi-enzyme biosensor based on recombinant human arginase I isolated from the gene-engineered strain of methylotrophic yeast Hansenula polymorpha and commercial urease is described. The biosensing layer was placed onto a polyaniline-Nafion composite platinum electrode and covered with a calcium alginate gel. The developed sensor revealed a good selectivity to L-arginine. The sensitivity of the biosensor was 110 ± 1.3 nA/(mM mm(2)) with the apparent Michaelis-Menten constant (K(M)(app)) derived from an L-arginine (L-Arg) calibration curve of 1.27 ± 0.29 mM. A linear concentration range was observed from 0.07 to 0.6mM, a limit of detection being 0.038 mM and a response time - 10s. The developed biosensor demonstrated good storage stability. A laboratory prototype of the proposed amperometric biosensor was applied to the samples of three commercial pharmaceuticals ("Tivortin", "Cytrarginine", "Aminoplazmal 10% E") for L-Arg testing. The obtained L-Arg-content values correlated well with those declared by producers.     

Amperometric biosensor for ethanol analysis in wines and grape must during wine fermentation

The amperometric biosensor for ethanol determination based on alcohol oxidase immobilised by the method of electrochemical polymerization has been developed. The industrial screen-printed platinum electrodes were used as transducers for creation of amperometric alcohol biosensor. Optimal conditions for electrochemical deposition of an active membrane with alcohol oxidase has been determined. Biosensors are characterised by good reproducibility and operational stability with minimal detection limit of ethanol 8 x 10(-5) M. The good correlation of results for ethanol detection in wine and during wine fermentation by using the developed amperometric biosensor with the data obtained by the standard methods was shown (r = 0.995).     

Development of amperometric α-ketoglutarate biosensor based on ruthenium-rhodium modified carbon fiber enzyme microelectrode

A rapid and highly sensitive miniaturized amperometric biosensor for the detection of α-ketoglutarate (α-KG) based on a carbon fiber electrode (CFE) is presented. The biosensor is constructed by immobilizing the enzyme, glutamate dehydrogenase (GLUD) on the surface of single carbon fiber modified by co-deposition of ruthenium (Ru) and rhodium (Rh) nanoparticles. SEM and EDX shed useful insights into the morphology and composition of the modified microelectrode. The mixed Ru/Rh coating offers a greatly enhanced electrocatalytic activity towards the detection of β-nicotinamide adenine dinucleotide (NADH), with a substantial decrease in overpotential of ～ 400 mV compared to the unmodified CFE. It also imparts higher stability with minimal surface fouling, common to NADH oxidation. Further modification with the enzyme, GLUD leads to effective amperometric biosensing of α-KG through monitoring of the NADH consumption. A very rapid response to dynamic changes in the α-KG concentrations is observed with a response time of 6s. The current response is linear between 100 and 600 μM with a sensitivity of 42 μAM(-1) and a detection limit of 20 μM. This proof of concept study indicates that the GLUD-Ru/Rh-CFE biosensor holds great promise for real-time electrochemical measurements of α-KG.     

A flow injection system, comprising a biosensor based on a screen-printed carbon electrode containing Meldola’s Blue-Reinecke salt coated with glucose dehydrogenase, for the measurement of glucose

A biosensor for the measurement of glucose in serum has been developed, based on a screen-printed carbon electrode modified with Meldola’s Blue-Reinecke salt, coated with the enzyme glucose dehydrogenase (from Bacillus sp.), and nicotinamide adenine dinucleotide coenzyme (NAD⁺). A cellulose acetate layer was deposited on top of the device to act as a permselective membrane. The biosensor was incorporated into a commercially available, thin-layer, amperometric flow cell operated at a potential of only +0.05 V versus Ag/AgCl. The mobile phase consisted of 0.2 M phosphate buffer (pH 7.0) containing 0.1 M potassium chloride solution, and a flow rate of 0.8 ml min⁻¹ was used throughout the investigation. The biosensor response was linear over the range of 0.075-30 mM glucose, with the former representing the detection limit. The precision of the system was determined by carrying out 20 repeat injections of a 5-mM glucose standard, and the calculated coefficient of variation was 3.9%. It was demonstrated that this biosensor system could be applied to the direct measurement of glucose in serum without pretreatment. Therefore, this would allow high-throughput analysis, at low cost, for this clinically important analyte.     

Purification and biochemical characterization of simplified eukaryotic nitrate reductase expressed in Pichia pastoris

NAD(P)H:nitrate reductase (NaR, EC 1.7.1.1-3) is a useful enzyme in biotechnological applications, but it is very complex in structure and contains three cofactors-flavin adenine dinucleotide, heme-Fe, and molybdenum-molybdopterin (Mo-MPT). A simplified nitrate reductase (S-NaR1) consisting of Mo-MPT-binding site and nitrate-reducing active site was engineered from yeast Pichia angusta NaR cDNA (YNaR1). S-NaR1 was cytosolically expressed in high-density fermenter culture of methylotrophic yeast Pichia pastoris. Total amount of S-NaR1 protein produced was approximately 0.5 g per 10 L fermenter run, and methanol phase productivity was 5 microg protein/g wet cell weight/h. Gene copy number in genomic DNA of different clones showed direct correlation with the expression level. S-NaR1 was purified to homogeneity in one step by immobilized metal affinity chromatography (IMAC) and total amount of purified protein per run of fermentation was approximately 180 mg. Polypeptide size was approximately 55 kDa from electrophoretic analysis, and S-NaR1 was mainly homo-tetrameric in its active form, as shown by gel filtration. S-NaR1 accepted electrons efficiently from reduced bromphenol blue (kcat = 2081 s(-1)) and less so from reduced methyl viologen (kcat = 159 s(-1)). The nitrate KM for S-NaR1 was 30 +/- 3 microM, which is very similar to YNaR1. S-NaR1 is capable of specific nitrate reduction, and direct electric current, as shown by catalytic nitrate reduction using protein film cyclic voltammetry, can drive this reaction. Thus, S-NaR1 is an ideal form of this enzyme for commercial applications, such as an enzymatic nitrate biosensor formulated with S-NaR1 interfaced to an electrode system.     

One-step construction of biosensor based on chitosan-ionic liquid-horseradish peroxidase biocomposite formed by electrodeposition

A simple and controllable electrodeposition approach was established for one-step construction of hydrogen peroxide (H(2)O(2)) biosensors by in situ formation of chitosan-ionic liquid-horseradish peroxidase (CS-IL-HRP) biocomposite film on electrode surface. A highly porous surface with orderly three-dimensional network was revealed by scanning electron microscopy (SEM) investigation. The biocomposite provided improved conductivity and biocompatible microenvironment. The developed biosensor exhibited a fast amperometric response for the determination of H(2)O(2) and 95% of the steady-state current was obtained within 2s. The linear response of the developed biosensor for the determination of H(2)O(2) ranged from 6.0x10(-7) to 1.6x10(-4)M with a detection limit of 1.5x10(-7)M. Performance of the biosensor was evaluated with respect to possible interferences and a good selectivity was revealed. The fabricated biosensor exhibited high reproducibility and long-time storage stability. The ease of the one-step non-manual technique and the promising feature of biocomposite could serve as a versatile platform for the fabrication of electrochemical biosensors.     

One-step method embedding superoxide dismutase and gold nanoparticles in silica sol-gel network in the presence of cysteine for construction of third-generation biosensor

A new and one-step method has been developed for the fabrication of superoxide dismutase (SOD) based biosensor. This method was used to form a silica sol-gel (SG) thin film and to immobilize SOD and gold nanoparticles (GNPs) in silica SG network for the fabrication of biosensor. The immobilized superoxide dismutase realized direct electron transfer between the enzyme and electrode surface, and the rate constants of the electrochemical process (ks) of SOD was markedly enhanced by GNPs. The electrochemical performance and influencing factors of the resulting biosensor were studied in detail. The resulting biosensor exhibited fast amperometric response to superoxide anion. The calibration range of superoxide anion was from 0.05 to 0.4 micromol L(-1). The proposed method exhibited the benefits of the advantages of self-assembly, nanoparticles and SG techniques. The fabrication of the SOD-modified electrode was easy and simple. The biosensor exhibited high sensitivity and long-term stability.     

Development of a carbon nanotube paste electrode osmium polymer-mediated biosensor for determination of glucose in alcoholic beverages

This paper describes a new amperometric biosensor for glucose monitoring. The biosensor is based on the activity of glucose dehydrogenase (GDH) and diaphorase (DI) co-immobilized with NAD(+) into a carbon nanotube paste (CNTP) electrode modified with an osmium functionalized polymer. This mediator was demonstrated to shuttle the electron transfer between the immobilized diaphorase and the CNTP electrode, thus, showing a good electrocatalytic activity towards NADH oxidation at potentials around +0.2V versus Ag|AgCl, where interfering reactions are less prone to occur. The biosensor exhibits a detection limit of 10 micromol L(-1), linearity up to 8 x 10(-4) mol L(-1), a sensitivity of 13.4 microA cm(-2)mmol(-1)L(-1), a good reproducibility (R.S.D. 2.1%, n=6) and a stability of about 1 week when stored dry at 4 degrees C. Finally, the proposed biosensor was applied for the determination of glucose in different samples of sweet wine and validated with a commercial spectrophotometric enzymatic kit.     

A porous poly(acrylonitrile-co-acrylic acid) film-based glucose biosensor constructed by electrochemical entrapment

A novel glucose biosensor was constructed by electrochemical entrapment of glucose oxidase (GOD) into porous poly(acrylonitrile-co-acrylic acid), which was synthesized via radical polymerization of acrylonitrile and acrylic acid. The obtained biosensor showed a better stability and higher sensitivity than the biosensor prepared by simple physical adsorption. Effects of some experimental variables such as immobilization time, enzyme concentration, pH, applied potential, and temperature on the amperometric response of the sensor were investigated. The biosensor exhibited a rapid response to glucose (< 30s) with a linear range of 5 x 10(-6) to 3 x 10(-3)M and a sensitivity of 6.82 mAM(-1)cm(-2). The apparent Michaelis-Menten constant (K(M)(app)) was 7.3mM.     

Amperometric glucose biosensor based on polymerized ionic liquid microparticles

A glucose amperometric biosensor based on the immobilization of glucose oxidase (GOx) in microparticles prepared by polymerization of the ionic liquid 1-vinyl-3-ethyl-imidazolium bromide (ViEtIm⁺Br⁻) using the concentrated emulsion polymerization method has been developed. The polymerization of the emulsion dispersed phase, in which the enzyme was dissolved together with the ionic liquid monomer, provides poly(ViEtIm⁺Br⁻) microparticles with entrapped GOx. An anion-exchange reaction was carried out for synthesizing new microparticles of poly(ViEtIm⁺(CF₃SO₂)₂N⁻) and poly(ViEtIm⁺BF₄⁻). The enzyme immobilization method was optimized for biosensor applications and the following optimal values were determined: pH 4.0 for the synthesis medium, 1.23 M monomer concentration and 3.2% (w/w) cross-linking content. The performance of the biosensor as a function of some analytical parameters such as pH and temperature of the measuring medium, and enzymatic load of the microparticles was also investigated. The effect of the substances which are present in serum samples such as uric and ascorbic acid was eliminated by using a thin Nafion layer covering the electrode surface. The biosensor thus prepared can be employed in aqueous and in non-aqueous media with satisfactory results for glucose determination in human serum samples. The useful lifetime of this biosensor was 150 days.     

Development of a disposable ethanol biosensor based on a chemically modified screen-printed electrode coated with alcohol oxidase for the analysis of beer

A disposable amperometric biosensor for the measurement of ethanol has been developed. It comprises a screen-printed carbon electrode doped with 5% cobalt phthalocyanine (CoPC-SPCE), and coated with alcohol oxidase; a permselective membrane on the surface acts as a barrier to interferents. The measurement of ethanol is based on the signal produced by H2O2, the product of the enzymatic reaction. Optimisation studies were performed using amperometry in stirred solution and the magnitude of the signal was found to be dependent on pH, enzyme loading, type of membrane and applied potential. The same technique was used to evaluate the biosensor for the determination of ethanol in samples. The results obtained compared well with the manufacturers specifications. In order to test the possibility of using the devices in the field, chronoamperometry was also used to determine ethanol in the same beer samples. The precision and recovery data again indicated that the biosensor should give reliable results under the conditions described.     

Sensitive amperometric biosensor for the determination of biogenic and synthetic amines using pea seedlings amine oxidase: a novel approach for enzyme immobilisation

We prepared a new inorganic sorbent based on modified triazine (2-[4,6-bis (aminoethylamine)-1,3,5-triazine]-Silasorb; BAT-Silasorb) which binds pea seedlings amine oxidase (PSAO) very tightly without loss of its catalytic activity. This unique feature as well as the wide substrate specificity of PSAO was successfully utilised in the construction of an amperometric biosensor based on a carbon paste electrode for the fast and sensitive detection of various amines at a formal potential 0 mV versus Ag/AgCl reference electrode. The reaction layer of the biosensor is created by the direct immobilisation of PSAO at the electrode surface via affinity carrier BAT-Silasorb. Used arrangement facilitates a simple restoration of the inactive biosensor. An amperometric signal results from horseradish peroxidase catalysed reduction of H2O2, a secondary product of the oxidative deamination of amines, catalysed by PSAO. The sensor was used for the basic characterisation of 55 biogenic and synthetic amines, from numerous mono-, di- and polyamines to various hydroxy-, thio-, benzyl- and aromatic derivatives in order to establish its suitability as a postcolumn detector. Its high sensitivity to putrescine 20.0 +/- 0.64 mA l-1 per mol (636.9 +/- 2.03 mA l-1 per mol per cm2), a limit of detection of 10 nmol l-1 (determined with respect to a signal-to-noise ratio 3:1), a linear range of current response to 0.01-100 mumol l-1 concentration of substrate and good reproducibility all indicate that the sensor could be applied to future industrial and clinical analyses.     

Design of an amperometric biosensor using polypyrrole-microgel composites containing glucose oxidase

A new material consisting of a water-dispersed complex of polypyrrole-polystyrensulfonate (PPy) embedded in polyacrylamide (PA) has been prepared and tested as enzyme immobilizing system for its use in amperometric biosensors. Glucose oxidase (GOx) and the water-dispersed polypyrrole complex were entrapped within polyacrylamide microgels by polymerization of acrylamide in the dispersed phase of concentrated emulsions containing GOx and PPy. Polymerization of the dispersed phase provides microparticles whose size lies between 3.5 and 7 microm. The aim of incorporating polypyrrole into the polyacrylamide microparticles was to facilitate the direct transfer of the electrons released in the enzymatic reaction from the catalytic site to the platinum electrode surface. The conductivity of the microparticles was measured by a four-point probe method and confirmed by the successful anaerobic detection of glucose by the biosensor. Thus, the polyacrylamide-polypyrrole (PAPPy) microparticles combine the conductivity of polypyrrole and the pore size control of polyacrylamide. The effects of the polyacrylamide-polypyrrole ratio and cross-linking on the biosensor response have been investigated, as well as the influence of analytical parameters such as pH and enzymatic loading. The PAPPy biosensor is free of interferences arising from ascorbic and uric acids, which allows its use for quantitative analysis in human blood serum.     

Palladium hexacyanoferrate hydrogel as a novel and simple enzyme immobilization matrix for amperometric biosensors

An amperometric glucose biosensor with glucose oxidase (GOx) immobilized into palladium hexacyanoferrate (PdHCF) hydrogel has been prepared and evaluated. The sensor was based on a two-layer configuration with biocatalytic and electrocatalytic layers separately deposited onto the electrode. To reduce the overpotential for reduction of hydrogen peroxide liberated in the enzyme catalyzed oxidation of glucose, an inner thin layer of nickel hexacyanoferrate (NiHCF) electrodeposited onto the surface of graphite electrode was used as an electrocatalyst. As an outer layer, the hydrogel of palladium hexacyanoferrate with entrapped glucose oxidase was used. Under optimal operating conditions (pH 5.0 and E = -0.075 V versus calomel (3.0 M KCl) reference electrode), sensor showed high sensitivity to glucose (0.3-1.0 microA/mM) and a response time of less than 30s. The linear response to glucose was obtained in the concentration range between 0.05 and 1.0 mM in batch analysis mode and 0-7.0 mM in FIA. During the 32 days testing period, no significant decrease in the sensor sensitivity was observed. The sensor was applied for the determination of glucose concentration in fruit juice and yoghurt drink, and the results obtained showed good correlation with results obtained by reference spectrophotometric enzyme method.     

Biosensor for the enantioselective analysis of S-perindopril

Because S-perindopril enantiomer is the eutomer which is responsible for the angiotensin-converting enzyme inhibition activity, it is necessary to develop a reliable method for its assay from its distomer, the R-enantiomer. For this purpose, an amperometric biosensor was developed based on L-amino acid oxidase. The working range of the described biosensor was 20pmol/L-10 micromol/L on the 7.0-7.4 pH range, with a detection limit of 2pmol/L. The low enantioselectivity for R-perindopril, as compared with S-enantiomer, was demonstrated by both mixed solutions and separate solutions methods (amperometric selectivity coefficient is 1.0 x 10(-4)). The biosensor was also selective towards D-proline and polyvinylpyrrolidone. The amperometric biosensor can be used for enantioselective analysis of S-perindopril in raw material, with an RSD < 1%. The life time (t95%) of the biosensor is three weeks.     

A probe for NADH and H2O2 amperometric detection at low applied potential for oxidase and dehydrogenase based biosensor applications

Modified screen-printed electrodes for amperometric detection of H(2)O(2) and nicotinamide adenine dinucleotide (NADH) at low applied potential are presented in this paper. The sensors are obtained by modifying the working electrode surface with Prussian Blue, a well known electrochemical mediator for H(2)O(2) reduction. The coupling of this sensor with phenazine methosulfate (PMS) in the working solution gives the possibility of measuring both NAD(P)H and H(2)O(2). PMS reacts with NADH producing PMSH, which in the presence of oxygen, gives an equimolar amount of H(2)O(2). This allows the measurement of both analytes with similar sensitivity (357 mA mol(-1)L cm(-2) for H(2)O(2) and 336 mA mol(-1)L cm(-2) for NADH) and LOD (5x10(-7)mol L(-1) for H(2)O(2) and NADH) and opens the possibility of a whole series of biosensor applications. In this paper, results obtained with a variety of dehydrogenase enzymes (alcohol, malic, lactate, glucose, glycerol and glutamate) for the detection of enzymatic substrates or enzymatic activity are presented demonstrating the suitability of the proposed method for future biosensor applications.