Sonochemically fabricated microelectrode arrays for biosensors. Part III. AC impedimetric study of aerobic and anaerobic response of alcohol oxidase within polyaniline

A sonochemically fabricated alcohol oxidase enzyme micro-electrode array is reported. Sensors of this type were fabricated by first depositing an insulating polydiaminobenzene film on supporting gold electrodes. Sonication and subsequent ablation exposed discrete areas of the underlying conducting electrode, which collectively act as a microelectrode array. Electropolymerisation of aniline has been used to generate in situ polyaniline containing entrapped alcohol oxidase. The physical and electrochemical properties of these films were studied and reported within this paper. The final composites were shown to behave with microelectrode performance characteristics for the detection of aqueous ethanol concentrations.     

Moving known libraries to an addressable array: a site-selective hetero-Michael reaction

A two-step, Michael reaction-based strategy for site-selectively placing molecules by unique electrodes in an addressable microelectrode array has been developed. The strategy is compatible with the use of polypeptide nucleophiles and works with microelectrode arrays having either 1024 electrodes/cm (2) or 12,544 electrodes/cm (2). The chemistry should allow for the transfer of existing molecular libraries to microelectrode array devices for analysis.     

The modification of enzyme electrode properties with non-conducting electropolymerised films

Phenol and several phenol derivatives have been electropolymerised on to platinum anodes. The selective properties of the modified electrodes were dependent on the final polymeric structure. Although essentially non-conducting, dual phenolic films could be deposited on electrode surfaces. This was achieved by the initial deposition of a porous polymer formed from, phenol red, followed by the more diffusion limiting film of oxidised phenol. The resulting composite film showed the combined selective characteristics of the two component polymers. Attempts to enhance polymeric film selectivity via the incorporation of charged or neutral surfactants are outlined. The characteristics of glucose oxidase incorporated into (poly)phenol films are discussed.     

Controlled electrophoretic deposition of multifunctional nanomodules for bioelectrochemical applications

The design of an electrochemical glucose sensing device formed by the electrodeposition of multifunctional Au nanoparticles is reported here as a novel concept for an enhanced generic sensing platform. Initially gold nanoparticles (Au) were alternatively coated with a layer of positively charged redox polymer (ORP) and a negatively charged glucose oxidase (GOX) layer alternatively using layer-by-layer methodology to form multifunctional Au/ORP/GOX/ORP particles. The modification and stability of the Au nanoparticles was monitored by using UV-vis spectroscopy and zeta-potential measurements. The modified Au nanoparticles were electrophoretically deposited onto an electrode to produce an electrochemical glucose sensing device. A considerable influence of electrophoretic deposition time and potential was found on the sensing platform response. Preliminary responses to glucose addition showed an enhanced performance by applying an electrophoretic deposition potential of +1.2V vs. Ag/AgCl for 30min. The observed response in the case of microelectrode geometry was in the range of mAcm(2). This work also shows that the presence of a second outer ORP layer on the functionalised Au nanoparticles improved the response.     

Immobilization of glucose oxidase on thin-film gold electrodes produced by magnetron sputtering and their application in an electrochemical biosensor

An electrochemical biosensor is described consisting of a thin-layer gold film electrode prepared by cathodic sputtering using a poly(vinyl chloride) sheet as substrate, with voltammetric behaviour comparable to that of conventional polycrystalline gold electrodes, coated with the hydrolysed copolymer hydroxyethyl methacrylate-co-methyl methacrylate onto which glucose oxidase was immobilized. The mechanical properties of the plastic foil substrate permit easy construction of circular-shaped electrodes which were employed as working electrodes for batch injection analysis. The electrochemical biosensor fabrication is inexpensive and can be used as disposable enzyme sensor for the detection of hydrogen peroxide. The biosensor was tested for the determination of glucose in serum and a good correlation was obtained with the measurement using the electrochemical and the spectrophotometric methods.     

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.     

Sonochemically fabricated microelectrode arrays for biosensors offering widespread applicability: Part I

A novel and patented procedure is described for the sonochemical fabrication of a new class of microelectrode array based sensor with electrode element populations of up to 2 x 10(5) cm(-2). For some years it has been accepted that microelectrode arrays offer an attractive route for lowering minimum limits of detection and imparting stir (convectional mass transport) independence to sensor responses; despite this no commercial biosensors, to date, have employed microelectrode arrays, largely due to the cost of conventional fabrication routes that have not proved commercially viable for disposable devices. Biosensors formed by our sonochemical approach offer unrivalled sensitivity and impart stir independence to sensor responses. This format lends itself for mass fabrication due to the simplicity and inexpensiveness of the approach; in the first instance impedimetric and amperometric sensors are reported for glucose as model systems. Sensors already developed for ethanol, oxalate and a number of pesticide determinations will be reported in subsequent publications.     

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.     

Covalent enzyme immobilization by poly(ethylene glycol) diglycidyl ether (PEGDE) for microelectrode biosensor preparation

Poly(ethylene glycol) diglycidyl ether (PEGDE) is widely used as an additive for cross-linking polymers bearing amine, hydroxyl, or carboxyl groups. However, the idea of using PEGDE alone for immobilizing proteins on biosensors has never been thoroughly explored. We report the successful fabrication of microelectrode biosensors based on glucose oxidase, d-amino acid oxidase, and glutamate oxidase immobilized using PEGDE. We found that biosensors made with PEGDE exhibited high sensitivity and a response time on the order of seconds, which is sufficient for observing biological processes in vivo. The enzymatic activity on these biosensors was highly stable over several months when they were stored at 4 °C, and over at least 3d at 37 °C. Glucose microelectrode biosensors implanted in the central nervous system of anesthetized rats reliably monitored changes in brain glucose levels induced by sequential administration of insulin and glucose. PEGDE provides a simple, low cost, non-toxic alternative for the preparation of in vivo microelectrode biosensors.     

Bienzyme biosensors for glucose,ethanol and putrescine built on oxidase and sweet potato peroxidase

Amperometric biosensors for glucose, ethanol, and biogenic amines (putrescine) were constructed using oxidase/peroxidase bienzyme systems. The H(2)O(2) produced by the oxidase in reaction with its substrate is converted into a measurable signal via a novel peroxidase purified from sweet potato peels. All developed biosensors are based on redox hydrogels formed of oxidases (glucose oxidase, alcohol oxidase, or amine oxidase) and the newly purified sweet potato peroxidase (SPP) cross-linked to a redox polymer. The developed electrodes were characterized (sensitivity, stability, and performances in organic medium) and compared with similarly built ones using the 'classical' horseradish peroxidase (HRP). The SPP-based electrodes displayed higher sensitivity and better detection limit for putrescine than those using HRP and were also shown to retain their activity in organic phase much better than the HPR based ones. The importance of attractive or repulsive electrostatic interactions between the peroxidases and oxidases (determined by their isoelectric points) were found to play an important role in the sensitivity of the obtained sensors.     

L-lactate measures in brain tissue with ceramic-based multisite microelectrodes

A newly developed multisite array microelectrode for in vivo measurements of L-lactate is presented. The resulting microelectrode is composed of three functional layers. First, Nafion is used to repel interfering electroactive anions, such as ascorbate. Second, L-lactate oxidase immobilized onto the recording sites is used to convert L-lactate to hydrogen peroxide. The H2O2 produced is proportional to L-lactate concentrations and is quantified at the platinum recording sites. Third, a layer of polyurethane is coated over the L-lactate oxidase to adjust the linear range of the microelectrode to one that is compatible with in vivo measurements. This layer reduces the amount of L-lactate that diffuses to the enzyme while not significantly limiting oxygen diffusion. The resulting L-lactate microelectrodes were linear to 20 mM (R2 = 0.997 +/- 0.001) and beyond in some cases with detection limits of 0.078 +/- 0.013 mM (n = 12). The selectivity and response time of these electrodes make them suitable for in vivo measurements in brain tissue. Self-referencing recordings may be utilized to further improve the selectivity of the recordings. However this is not necessary for most applications in the brain, because the resting and stimulated levels of dopamine (DA), norepinephrine (NE), and other potentially interfering cations are two to three orders of magnitude lower than that of in vivo L-lactate, which is in the millimolar range. Preliminary in vivo measures of L-lactate in the brain of anesthetized rats support that the microelectrodes are capable of measuring rapid endogenous changes in vivo.     

Carbon nanotube/gold nanoparticles/polyethylenimine-functionalized ionic liquid thin film composites for glucose biosensing

A novel glucose biosensor based on immobilization of glucose oxidase (GOD) in thin films of polyethylenimine-functionalized ionic liquid (PFIL), containing a mixture of carbon nanotubes (CNT) and gold nanoparticles (AuNPs) and deposited on glassy carbon electrodes, was developed. Direct electrochemistry of glucose oxidase in the film was observed, with linear glucose response up to 12mM. The PFIL-stabilized gold nanoparticles had a diameter of 2.4+/-0.8nm and exhibited favorable stability (stored even over one month with invisible change in UV-vis spectroscopic measurements). In addition, CNT were also well dispersed in the PFIL matrix, then, the resulting CNT/AuNPs/PFIL composites film showed high electrocatalytic activity toward reduction of hydrogen peroxide and oxygen. Here, PFIL, due to its high ionic conductivity, good solubility to CNT, and stability to nanoparticles, played an important role in constructing stable CNT/AuNPs/PFIL/GOD composites. And good biocompatibility of PFIL also offered a friendly environment for the immobilization of biomolecules.     

Labeless AC impedimetric antibody-based sensors with pgml(-1) sensitivities for point-of-care biomedical applications

This paper describes the development and characterisation of labeless immunosensors for (a) the cardiac drug digoxin and (b) bovine serum albumin (BSA). Commercial screen-printed carbon electrodes were used as the basis for the sensors. Two methods were used to immobilise antibodies at the electrode surface. Aniline was electropolymerised onto these electrodes to form a thin planar film of conductive polyaniline; the polyaniline film was then utilised as a substrate to immobilise biotinylated anti-digoxin using a classical avidin-biotin affinity approach. As an alternative approach, poly(1,2-diaminobenzene) was electrodeposited onto the carbon electrodes and this modified surface was then sonochemically ablated to form an array of micropores. A second electropolymerisation step was then used to co-deposit conductive polyaniline along with antibodies for BSA within these pores to produce a microarray of polyaniline protrusions with diameters of several mum, containing entrapped anti-BSA. The resulting antibody grafted electrodes were interrogated using an AC impedance protocol before and following exposure to digoxin or BSA solutions, along with control samples containing a non-specific IgG antibody. The impedance characteristics of both types of electrode were changed by increasing concentrations of antigen up to a saturation level. Calibration curves were obtained by subtraction of the non-specific response from the specific response, thereby eliminating the effects of non-specific adsorption of antigen. Both the use of microelectrode arrays and affinity binding protocols showed large enhancements in sensitivity over planar electrodes containing entrapped antibodies and gave similar sensitivities to our other published work using affinity-based planar electrodes. Detection limits were in the order of 0.1ngml(-1) for digoxin and 1.5ngml(-1) for BSA.     

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.     

Protein modified- and membrane electrodes: strategies for the development of biomolecular sensors

Different procedures used for constructing protein/enzyme-modified electrodes are examined, in particular adsorption, covalent attachment and film deposition. The performances of such modified electrodes with electroactive proteins or enzymes attached to their active surface are examined, especially in the case of c-type cytochromes, hydrogenases and glucose oxidase. Another strategy presented in this review consists of the use of membrane electrodes with an electroactive protein imprisoned between a dialysis membrane and the electrode surface. The versatility and other advantages of such a procedure are underlined. Applications of membrane electrodes to the bioremediation of soils and effluents and as models for investigating interactions between proteins and soils are described.     

Amperometric glucose sensor based on coimmobilization of glucose oxidase and Poly(p-phenylenediamine) at a platinum microdisk electrode

A miniaturized glucose biosensor in which glucose oxidase (GOD) and poly(p-phenylenediamine) (poly-PPD) were coimmobilized at the surface of a platinum microdisk electrode was developed and used successfully for amperometric determination of glucose. The performance of sensors prepared at different monomer concentrations and polymerization potentials with different media was investigated in detail. It was found that similarly to poly(o-phenylenediamine) (poly-OPD), (poly-PPD) noticeably eliminated the electrochemical interference of ascorbic acid, uric acid, and l-cysteine. The amperometric response of glucose with the biosensor under optimal conditions exhibited a linear relationship in the range of 5.0 x 10(-5) to 3.0 x 10(-3) M with correlation coefficient 0.9995. According to the Michaelis-Menten equation, the apparent Michaelis constant for glucose and the maximum steady-state current density of the poly-PPD/GOD-modified microelectrode were 3.94 mM and 607.5 microA cm(-2), respectively. The current density of the sensor responding to glucose in the linear range can reach 160 microA cm(-2) mM(-1), which is far greater than that obtained using poly-OPD and poly(phenol) film. In addition, the stability of the sensor was examined over a 2-month period.     

Simultaneous detection of the release of glutamate and nitric oxide from adherently growing cells using an array of glutamate and nitric oxide selective electrodes

The simultaneous detection of nitric oxide and glutamate using an array of individually addressable electrodes, in which the individual electrodes in the array were suitably modified with a highly sensitive nitric oxide sensing chemistry or a glutamate oxidase/redox hydrogel-based glutamate biosensor is presented. In a sequence of modification steps one of the electrodes was covered first with a positively charged Ni porphyrin entrapped into a negatively charged electrodeposition paint followed by the manual modification of the second working electrode by a bienzyme sensor architecture based on crosslinked redox hydrogels with entrapped peroxidase and glutamate oxidase. Adherently growing C6-glioma cells were grown on membrane inserts and placed in close distance to the modified sensor surfaces. The current responses recorded at each electrode after stimulation of glutamate and NO release by means of K+ and bradykinin clearly demonstrate the ability of the individual electrode in the array to detect the analyte towards which its sensitivity and selectivity was targeted without interference from the neighbouring electrode or other analytes present in the test mixture.     

Modification of carbon nanotubes with redox hydrogel: improvement of amperometric sensing sensitivity for redox enzymes

This study demonstrated that redox hydrogel-modified carbon nanotube (CNT) electrodes can be developed as an amperometric sensor that are sensitive, specific and fast and do not require auxiliary enzymes. A redox polymer, poly(vinylimidazole) complexed with Os(4,4'-dimethylbpy)(2)Cl (PVI-dmeOs) was electrodeposited on Ta-supported multi-walled CNTs. The resulted PVI-dmeOs thin film did not change the surface morphology of the CNTs, but turned the CNT surface from hydrophobic to hydrophilic, as studied by scanning electron microscopy (SEM) and static water contact angle measurements. Cyclic voltammetry measurements in a Fe(CN)(6)(3-) solution and electrochemical impedance measurements in an equimolar Fe(CN)(6)(3-/4-) solution demonstrated that the PVI-dmeOs hydrogel thin film was electronic conductive with a resistance of about 15Omega. The PVI-dmeOs/CNT electrodes sensed rapidly, sensitively and specifically to model redox enzymes (glucose oxidase (GOD) and lactate oxidase (LOD)) in amperometric experiments in electrolyte solutions containing the substrates of the measured redox enzymes. Both the CNT substrate and the thin PVI-dmeOs film enhanced the sensing sensitivities. Exploration of the mechanisms suggests that the PVI-dmeOs film may enhance the sensing sensitivities by wiring the enzyme molecules through the redox centers tethered on the mobile redox polymer backbones to the CNT electrodes.     

Fabrication of microband glucose biosensors using a screen-printing water-based carbon ink and their application in serum analysis

Microband glucose biosensors were fabricated by screen-printing a water-based carbon ink formulation containing cobalt phthalocyanine redox mediator and glucose oxidase (GOD) enzyme, then insulating and sectioning through the thick (20mum) film to expose a 3mm-long working electrode edge. The performance of these biosensors for glucose analysis was investigated at 25 degrees C. Voltammetry in glucose-containing buffer solutions established that an operating potential of +0.4V vs. Ag/AgCl was suitable for analysis under both stirring and quiescent conditions. The influence of pH on biosensor performance was established and an operational pH of 8.0 was selected. Steady-state responses were obtained under quiescent conditions, suggesting a mixed mechanism predominated by radial diffusion, indicative of microelectrode behaviour. Calibration studies obtained with these biosensors showed steady-state currents that were linearly dependent on glucose concentration from the limit of detection (0.27mM) up to 2.0mM, with a precision for replicate biosensors of 6.2-10.7%. When applied to the determination of glucose in human serum, the concentration compared favourably to that determined by a spectroscopic method. These results have demonstrated a simple means of fabricating biosensors for glucose measurement and determination in situations where low-current real-time monitoring under quiescent conditions would be desirable.     

Characterization of the biochemical behavior of glucose oxidase entrapped in a polypyrrole film

This article reports the characterization of the biochemical behavior of glucose oxidase entrapped in polypyrrole. The immobilization of glucose oxidase in a polypyrrole film was performed by entrapment during the electropolymerization of pyrrole at a platinum electrode poised at 0.65 V vs. SCE in aqueous solution in a one-compartment electrochemical cell. Thin films of polypyrrole (0.11 mum) were obtained and the entrapped enzyme obeyed Michaelis kinetics, indicating no diffusional constraints of the substrate. Our results indicate that the entrapped glucose oxidase is more resistant to denaturation conditions such as alkaline pH and temperature (50 and 60 degrees C) than the soluble form of the enzyme. The autoinactivation constant for the entrapped enzyme was also determined in presence of 0.25M of glucose and was 6.19 x 10(-4) min(-1), i.e., corresponding to a half-life value of 20 h. The results reported here show clearly that polypyrrole matrix has a strong stabilizing effect on the stucture and on the activity of glucose oxidase.