Improved selectivity and stability of glucose biosensor based on in situ electropolymerized polyaniline-polyacrylonitrile composite film

A new type of in situ electropolymerization method was used for electrochemical biosensor design. The biologic film was prepared by in situ electropolymerization of aniline into microporous polyacrylonitrile-coated platinum electrode in the presence of glucose oxidase. The novel glucose biosensor exhibited good selectivity, sensitivity and stability, which showed no apparent loss of activity after 100 consecutive measurements and intermittent usage for 100 days with storage in a phosphate buffer at 4 degrees C. Blood glucose determinations agreed well with standard hospital laboratory analysis. The construction and operational parameters of the biosensor were also optimized.     

Optimization of a polypyrrole glucose oxidase biosensor

An amperometric glucose biosensor was fabricated by the electrochemical polymerization of pyrrole onto a platinum electrode in the presence of the enzyme glucose oxidase in a KCl solution at a potential of + 0·65 V versus SCE. The enzyme was entrapped into the polypyrrole film during the electropolymerization process. Glucose responses were measured by potentio-statting the enzyme electrode at a potential of + 0·7 V versus SCE in order to oxidize the hydrogen generated by the oxidation of glucose by the enzyme in the presence of oxygen. Experiments were performed to determined the optimal conditions of the polypyrrole glucose oxidase film preparation (pyrrole and glucose oxidase concentrations in the plating solution) and the response to glucose from such electrodes was evaluated as a function of film thickness, pH and temperature. It was found that a concentration of 0·3 M pyrrole in the presence of 65 U/ml of glucose oxidase in 0·01 M KCl were the optimal parameters for the fabrication of the biosensor. The optimal response was obtained for a film thickness of 0·17 μm (75 mC/cm²) at pH 6 and at a temperature of 313 K. The temperature dependence of the amperometric response indicated an activation energy of 41 kJ/mole. The linearity of the enzyme electrode response ranged from 1·0 mM to 7·5 mM glucose and kinetic parameters determined for the optimized biosensors were 33·4 mM for the K_m and 7·2 μA for the I_max. It was demonstrated that the internal diffusion of hydrogen peroxide through the polypyrrole layer to the platinum surface was the main limiting factor controlling the magnitude of the response of the biosensor to glucose. The response was directly related to the enzyme loading in the polypyrrole film. The shelf life and the operational stability of the optimized biosensor exceed 500 days and 175 assays, respectively. The substrate specificity of the entrapped glucose oxidase was not altered by the immobilization procedure.     

Retention of enzyme by electropolymerized film: a new approach in developing a hypoxanthine biosensor

An amperometric biosensor for hypoxanthine was constructed by forming a layer of crosslinked xanthine oxidase on a platinum electrode, followed by electropolymerization of a submonolayer film of resorcinol and para-diaminobenzene. The fabricated electrodes were evaluated for speed of response, sensitivity, and reusability. Optimal performance was obtained with enzyme-based electrodes sparsely covered with film which was formed by electropolymerization in less than 6 min. The resulting electrodes exhibited linear response to hypoxanthine in the. range 5-300 muM with a response time of 2 min. Application of the biosensor in monitoring hypoxanthine content of fish extracts yielded results which agreed well with spectrophotometric assays using soluble xanthine oxidase. The biosensor was stable for 60 days when stored at 4 degrees C in phosphate buffer and it could be used continuously for 6 h with over 50 assays.     

Amperometric glucose biosensors based on Prussian Blue- and polyaniline-glucose oxidase modified electrodes.

The properties of glucose sensors fabricated by immobilization of glucose oxidase in a layer of electrochemically deposited polyaniline were investigated. Selective amperometric glucose sensors were prepared by immobilization of glucose oxidase on a Prussian Blue-modified platinum electrode in a layer of polyaniline during a one-step electropolymerization procedure from phosphate buffer. The influence of ascorbic acid and acetaminophen was completely eliminated due to impermeability of polyaniline to these substances.     

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.     

Glucose biosensor based on platinum microparticles dispersed in nano-fibrous polyaniline

A sensitive and selective amperometric glucose biosensor based on platinum microparticles dispersed in nano-fibrous polyaniline (PANI) was investigated. Poly (m-phenylenediamine) (PMPD), which was employed as an anti-interferent barrier and a protective layer to platinum microparticles, was deposited onto platinum-modified PANI in the presence of glucose oxidase. The morphology of PANI, Pt/PANI and PMPD-GOD/Pt/PANI were investigated by scanning electron microscopy. The results show that PANI has a nano-fibrous morphology. The enzyme electrode exhibits excellent response performance to glucose with linear range from 2 x 10(-6) to 12 x 10(-3) M and fast response time within 7s. Due to the selective permeability of PMPD, the enzyme electrode also shows good anti-interference to uric acid and ascorbic acid. The Michaelis-Menten constant km and the maximum current density imax of the enzyme electrode were 9.34 x 10(-3) M and 917.43 microA cm(-2), respectively. Furthermore, this glucose biosensor also has good stability and reproducibility.     

The use of differential measurements with a glucose biosensor for interference compensation during glucose determinations by flow injection analysis

A novel detection system for the determination of glucose in the presence of clinically important interferents, based on the use of dual sensors and flow-injection analysis (FIA), is described. The normalisation methodology involves measurement of the interference signal at a reference sensor; this signal can then be subtracted from the glucose sensor signal (post-run) to give a corrected measurement of the glucose concentration. The detection system consists of a thin layer cell with dual glassy carbon working electrodes. One electrode was surface modified to act asglucose biosensor by immobilisation of glucose oxidase (GOx) (from Aspergillus niger) with 1% glutaraldehyde and bovine serum albumin. The second electrode (glucose oxidase omitted) was utilised to measure the interference signal responding only to electroactive species present in the injected sample. A computer controlled multichannel potentiostat was used for potential application and current monitoring duties. The sensor responses were saved in ASCII format to facilitate post-run analysis in Microsoft Excel. Cyclic voltammetry (CV) was utilised to investigate the manner in which the interference signal contributed to the total signal obtained at the biosensor in the presence of glucose. The kinetic parameters I_max and the apparent Michaelis-Menten constant, K′_m, were calculated for the sensor operating under flow-injection conditions.     

Highly selective amperometric glucose microdevice derived from diffusion layer gap electrode

Although most of enzyme catalytic reactions are specific, the amperometric detection of the enzymatic reaction products is largely nonselective. How to improve the detection selectivity of the enzyme-based electrochemical biosensors has to be considered in the sensor fabrication procedures. Herein, a highly selective amperometric glucose biosensor based on the concept of diffusion layer gap electrode pair which we previously proposed was designed. In this biosensor, a gold tube coated with a conductive layer of glucose oxidase/Nafion/graphite was used to create an interference-free region in its diffusion layer by electrochemically oxidizing the interfering electroactive species at proper potentials. A Pt probe electrode was located in this diffusion layer of the tube electrode to selectively detect hydrogen peroxide generated from the enzyme catalytic oxidation of glucose in the presence of oxygen in the solution. In practical performance of the microdevice, parameters influencing the interference-removing efficiency, including the tip-tube opening distance, the tube electrode potential and the electrolyzing time had been investigated systematically. Results showed that glucose detection free from interferents could be achieved at the electrolyzing time of 30s, the tip-tube opening distance of 3mm and the tube electrode potential of 0.4V. The electrochemical response showed linear dependence on the concentration of glucose in the range of 1 x 10(-5) to 4 x 10(-3) M (the correlation coefficient: 0.9936, without interferents; 0.9995, with interferents). In addition, the effectiveness of this device was confirmed by numerical simulation using a model system of a solution containing interferents. The simulated results showed good agreement with the experimental data.     

Electrochemical nucleic acid hybridization biosensor based on poly(L-Aspartic acid)-modified electrode for the detection of short oligonucleotide sequences related to hepatitis C virus 1a

Abstract

This work describes, for the first time, the fabrication of poly(L-aspartic acid) (PAA) film modified pencil graphite electrode (PGE) for the detection of hepatitis C Virus 1a (HCV1a). The presence of PAA on the electrode surface can provide free carboxyl groups for covalent binding of biomolecules. The PGE surface was first coated with PAA via electropolymerization of the L-aspartic acid, and avidin was subsequently attached to the PAA modified electrode by covalent attachment. Biotinylated HCV1a probes were immobilized on avidin/PAA/PGE via avidin-biotin interaction. The morphology of PAA/PGE was examined using a scanning electron microscope. The hybridization events were monitored with square wave voltammetry using Meldola’s blue (MDB). Compared to non-complementary oligonucleotide sequences, when hybridization was carried out between the probe and its synthetic targets or the synthetic polymerase chain reaction analog of HCV1a, the highest MDB signal was observed. The linear range of the biosensor was 12.5 to 100?nM and limit of detection was calculated as 8.7?nM. The biosensor exhibited favorable stability over relatively long-term storage. All these results suggest that PAA-modified electrode can be used to nucleic acid biosensor application and electropolymerization of L-aspartic acid can be considered as a good candidate for the immobilization of biomolecules.     

A nano-Ni based ultrasensitive nonenzymatic electrochemical sensor for glucose: Enhancing sensitivity through a nanowire array strategy

Highly ordered Ni nanowire arrays (NiNWAs) were synthesized for the first time using a template-directed electropolymerization strategy with a nanopore polycarbonate (PC) membrane template, and their morphological characterization were examined by scanning electron microscopy (SEM) and transmission electron microscope (TEM). A NiNWAs based electrode shows very high electrochemical activity for electrocatalytic oxidation of glucose in alkaline medium, which has been utilized as the basis of the fabrication of a nonenzymatic biosensor for electrochemical detection of glucose. The biosensor can be applied to the quantification of glucose with a linear range covering from 5.0 × 10⁻⁷ to 7.0 × 10⁻³ M, a high sensitivity of 1043 μA mM⁻¹ cm⁻², and a low detection limit of 1 × 10⁻⁷ M. The experiment results also showed that the sensor exhibits good reproducibility and long-term stability, as well as high selectivity with no interference from other oxidable species.     

Bienzymatic glucose biosensor based on co-immobilization of peroxidase and glucose oxidase on a carbon nanotubes electrode

A bienzymatic glucose biosensor was proposed for selective and sensitive detection of glucose. This mediatorless biosensor was made by simultaneous immobilization of glucose oxidase (GOD) and horseradish peroxidase (HRP) in an electropolymerized pyrrole (PPy) film on a single-wall carbon nanotubes (SWNT) coated electrode. The amperometric detection of glucose was assayed by potentiostating the bienzymatic electrode at -0.1 versus Ag/AgCl to reduce the enzymatically produced H(2)O(2) with minimal interference from the coexisting electroactive compounds. The single-wall carbon nanotubes, sandwiched between the enzyme loading polypyrrole (PPy) layer and the conducting substrate (gold electrode), could efficiently promote the direct electron transfer of HRP. Operational characteristics of the bienzymatic sensor, in terms of linear range, detection limit, sensitivity, selectivity and stability, were presented in detail.     

Fabrication of glucose oxidase/polypyrrole biosensor by galvanostatic method in various pH aqueous solutions

The pH effect of pyrrole electropolymerization in the presence of glucose oxidase (GODx) on the performance and characteristic of galvanostatically fabricated glucose oxidase/polypyrrole (Ppy) biosensor is reported. Preparing the GODx/Ppy biosensors in 0.1 M KCl saline solution with various pH containing 0.05 M pyrrole monomer and 0.5 mg/ml GODx at 382 microA/cm2 current density for 100 mC/cm2 film thickness, both the galvanostatic responses and characteristics of these resulted biosensors were obtained. The results revealed that the galvanostatic glucose biosensor fabricated at neutral pH condition exhibited much higher sensitivity than those fabricated at lower or higher pH conditions, and had a good linearity form zero to 10 mM glucose with the sensitivity of 7 nA/mM. Finally, the long-term stability and the kinetic parameters, Michaelis constant and maximum current, of this biosensor were also reported.     

A novel glucose biosensor using bi-enzyme incorporated with peptide nanotubes

A novel amperometric glucose biosensor was developed using the bio-inspired peptide nanotube (PNT) as an encapsulation template for enzymes. Horseradish peroxidase (HRP) was encapsulated by the PNT and glucose oxidase (GO(x)) was co-immobilized with the PNT on a gold nanoparticle (AuNP)-modified electrode. A binary SAM of 3-mercaptopropionic acid (MPA) and 1-tetradecanethiol (TDT) was formed on the surface of the electrode to immobilize the PNT and GO(x). The resulting electrode appeared to provide the enzymes with a biocompatible nanoenvironment as it sustained the enhanced enzyme activity for an extended time and promoted possible direct electron transfer through the PNT to the electrode. Performance of the biosensor was evaluated in terms of its detection limit, sensitivity, pH, response time, selectivity, reproducibility, and stability in a lab setting. In addition the sensor was tested for real samples. The composite of AuNP-SAM-PNT/HRP-GO(x) to fabricate a sensor electrode in this study exhibited a linear response with glucose in the concentration range of 0.5-2.4mM with a R(2)-value of 0.994. A maximum sensitivity of 0.3mAM(-1)and reproducibility (RSD) of 1.95% were demonstrated. The PNT-encapsulated enzyme showed its retention of >85% of the initial current response after one month of storage.     

A commercial whole blood glucose biosensor with a low sensitivity to hematocrit based on an impregnated porous carbon electrode

Erythrocytes (red blood cells) are a major source of response variation in biosensor electrodes expected to operate in whole blood. Such a blood-to-plasma difference (hematocrit effect) must be minimized for those sensors directed towards the hospital market where wide variations in hematocrit can be seen. Typically, many current glucose sensors demonstrate a decreasing response to the analyte in the presence of increasing hematocrit levels. A sensor electrode for glucose is described which displays a reduced sensitivity to changes in hematocrit. The working electrode comprises a base porous conducting carbon layer, which is impregnated with a mixture including glucose oxidase and a ferrocene redox mediator. The base carbon layer has a void volume of 50%, an average pore diameter of less than 0.1 microm and a thickness of about 20 microm. The interior void volume of the base carbon layer is filled entirely with a substantial proportion of the impregnating mixture such that very little remains on the exterior. The resulting impregnated porous electrode excludes erythrocytes and is consequently capable of operating acceptably in venous, capillary, arterial and neonatal blood over a wide hematocrit range of 20-70%.     

Covalent conjugation of avidin with dye-doped silica nanopaticles and preparation of high density avidin nanoparticles as photostable bioprobes

A sensitive, selective and stable amperometric glucose biosensor employing novel PtPd bimetallic nanoparticles decorated on multi-walled carbon nanotubes (PtPd-MWCNTs) was investigated. PtPd-MWCNTs were prepared by a modified Watanabe method, and characterized by XRD and TEM. The biosensor was constructed by immobilizing the PtPd-MWCNTs catalysts in a Nafion film on a glassy carbon electrode. An inner Na?on film coating was used to eliminate common interferents such as uric acid, ascorbic acid and fructose. Finally, a highly porous surface with an orderly three-dimensional network enzyme layer (CS-GA-GOx) was fabricated by electrodeposition. The resulting biosensor exhibited a good response to glucose with a wide linear range (0.062-14.07 mM) and a low detection limit 0.031 mM. The biosensor also showed a short response time (within 5 s), and a high sensitivity (112 μA mM(-1)cm(-2)). The Michaelis-Menten constant (K(m)) was determined as 3.3 mM. In addition, the biosensor exhibited high reproducibility, good storage stability and satisfactory anti-interference ability. The applicability of the biosensor to actual serum sample analysis was also evaluated.     

Nanocrystalline diamond modified gold electrode for glucose biosensing

Boron-doped diamond has drawn much attention in electrochemical sensors. However there are few reports on non-doped diamond because of its weak conductivity. Here, we reported a glucose biosensor based on electrochemical pretreatment of non-doped nanocrystalline diamond (N-NCD) modified gold electrode for the selective detection of glucose. N-NCD was coated on gold electrode and glucose oxidase (GOx) was immobilized onto the surfaces of N-NCD by forming amide linkages between enzyme amine residues and carboxylic acid groups on N-NCD. The anodic pretreatment of N-NCD modified electrode not only promoted the electron transfer rate in the N-NCD thin film, but also resulted in a dramatic improvement in the reduction of the dissolved oxygen. This performance could be used to detect glucose at negative potential through monitoring the current change of oxygen reduction. The biosensor effectively performs a selective electrochemical analysis of glucose in the presence of common interferents, such as ascorbic acid (AA), acetaminophen (AP) and uric acid (UA). A wide linear calibration range from 10 microM to 15 mM and a low detection limit of 5 microM were achieved for the detection of glucose.     

Simultaneous monitoring of glucose and lactate by an interference and cross-talk free dual electrode amperometric biosensor based on electropolymerized thin films.

An interference and cross-talk free dual electrode amperometric biosensor integrated with a microdialysis sampling system is described, for simultaneous monitoring of glucose and lactate by flow injection analysis. The biosensor is based on a conventional thin layer flow-through cell equipped with a Pt dual electrode (parallel configuration). Each Pt disk was modified by a composite bilayer consisting of an electrosynthesised overoxidized polypyrrole (PPYox) anti-interference membrane covered by an enzyme entrapping gel, obtained by glutaraldehyde co-crosslinking of glucose oxidase or lactate oxidase with bovine serum albumin. The advantages of covalent immobilization techniques were coupled with the excellent interference-rejection capabilities of PPYox. Ascorbate, cysteine, urate and paracetamol produced lactate or glucose bias in the low micromolar range; their responses were, however, completely suppressed when the sample was injected through the microdialysis unit. Under these operational conditions the flow injection responses for glucose and lactate were linear up to 100 and 20 mM with typical sensitivities of 9.9 (+/- 0.1) and 7.2 (+/- 0.1) nA/mM. respectively. The shelf-lifetime of the biosensor was at least 2 months. The potential of the described biosensor was demonstrated by the simultaneous determination of lactate and glucose in untreated tomato juice samples; results were in good agreement with those of a reference method.     

Potentiometric glucose determination in human serum samples with glucose oxidase biosensor based on iodide electrode

Glucose potentiometric biosensor was prepared by immobilizing glucose oxidase on iodide-selective electrode. The hydrogen peroxide formed after the oxidation of glucose catalysed by glucose oxidase (GOD) was oxidized by sodium molybdate (SMo) at iodide electrode in the presence of dichlorometane. The glucose concentration was calculated from the decrease of iodide concentration determined by iodide-selective sensor. The sensitivity of glucose biosensor towards iodide ions and glucose was in the concentration ranges of 1.0 × 10^?1–1.0 × 10^?6 M and 1.0 × 10^?2?1.0 × 10^?4 M, respectively. The characterization of proposed glucose biosensor and glucose assay in human serum were also investigated.     

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 noninterference polypyrrole glucose biosensor

In order to eliminate the interference of impurities, such as ascorbic acid, a noninterference polypyrrole glucose biosensor was constructed with a four-electrode cell consisting of a polypyrrole film electrode, a polypyrrole-glucose oxidase electrode, a counter electrode and a reference electrode. The pure catalytic current of glucose oxidase (GOD) can be obtained from the difference between response currents of two working electrodes with and without GOD. The effects of potential, pH and temperature on analytical performance of the glucose biosensor were discussed. The optimum pH and apparent activation energy of enzyme-catalyzed reaction are 5.5 and 25 kJ mol(-1), respectively. The response current of the biosensor increases linearly with the increasing glucose concentration from 0.005 to 20.0 mmol dm(-3). The results show the glucose biosensor with under 2% of relative deviation has good ability of anti-interference. The glucose biosensor was also characterized with FT-IR and UV-vis spectra.