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.     

Ethanol biosensors based on alcohol oxidase

The detection and quantification of ethanol with high sensitivity, selectivity and accuracy is required in many different areas. A variety of methods and strategies have been reported for the determination of this analyte including gas chromatography, liquid chromatography, refractometry and spectrophotometry, among other. The use of the enzyme alcohol oxidase (AOX) on the analysis of ethanol in complex samples allows a considerable enhancement in specificity. This paper reviews the state of the art on ethanol determination based on AOX sensors, using either electrochemical electrodes or immobilised enzyme reactors. Almost all AOX-based ethanol sensors developed so far are based on the monitoring of O2 consumption or H2O2 formation. This has been mostly achieved using amperometric electrodes set at appropriate potentials namely, -600 mV for O2 monitoring or +600 mV for H2O2 monitoring. Mediated and non-mediated bienzymatic systems have also been assembled using AOX coupled to horseradish peroxidase (HRP). Different types of electrodes have been proposed for the detection of ethanol, namely, membrane electrode, carbon paste electrodes, screen-printed electrodes and self-assembled monolayers. Another approach to work with this sensitive enzyme is to use high amounts of AOX in order to create an enzyme reservoir, a strategy which can be implemented using immobilised enzyme reactors. These reactors can be combined with a colorimetric detection in a flow-injection analysis system or with electrochemical transducers.     

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.     

An oxygen-rich fill-and-flow channel biosensor

An oxygen-rich fill-and-flow channel biosensor has been developed for the measurement of glucose in wine. Glucose oxidase (GOD), immobilised in carbon paste (CP), was located in a well adjacent to a downstream detector electrode. When the analyte solution flows, hydrogen peroxide produced in the enzyme reaction is swept down to the detector electrode. Mineral oil and Kel-F oil (poly(chlorotrifluorethylene)) were used to prepare an enzyme layer of GOD within a CP. The hydrophobicity of the CP confined the reaction between the enzyme and its substrate to the surface of the enzyme layer. The oxidation current of hydrogen peroxide was sensitive to the enzyme loading but insensitive to mass transport variations such as flow rate. This response was, therefore, limited by the kinetics of the reaction between the enzyme and the substrate. For Kel-F oil, which can support a high concentration of dissolved oxygen, good reproducibility and greater dynamic range was obtained and the response did not decrease after degassing for 40 min with argon. Analysis of wine samples showed good agreement with the values obtained by spectrophotometric enzyme assay.     

Graphite-Teflon composite bienzyme electrodes for the determination of L-lactate: application to food samples.

A bienzyme amperometric graphite-Teflon composite biosensor, in which lactate oxidase (LOD) and peroxidase, together with the mediator ferrocene, are incorporated into the electrode matrix, was developed for the determination of L-lactate in food samples such as wine and yogurt by using both batch- and flow-injection modes. This bienzyme electrode was fabricated by simple physical inclusion of the enzymes and the mediator in the bulk of the graphite-Teflon matrix. A Teflon content of 70%, an applied potential of 0.00 V, and a pH of 7.4 were employed as working conditions. The composite bioelectrode exhibited long-term operation because of the renewability of its surface by polishing. Reproducible amperometric responses were achieved with different electrodes fabricated from different composite matrices, and no significant loss of the enzyme activity occurred after 6 months of storage at 4 degrees C. Detection limits for L-lactate of 1.4 and 0.9 microM were obtained by batch amperometry in stirred solutions and flow-injection with amperometric detection, respectively. An interferences study with different substances which may be present in wine and yogurt together with L-lactic acid demonstrated very good selectivity for the determination of this analyte. The bienzyme composite electrode was applied to the determination of L-lactic acid in red wine and shaken yogurt, and the methods were validated by comparing these results with those obtained by applying a recommended reference method.     

The orientationally controlled assembly of genetically modified enzymes in an amperometric biosensor

A novel, nitroreductase (NTR) containing a sequence of six cysteine amino acids, enabling strong thiolate bonds to form on a gold electrode surface without the loss of enzyme activity, was genetically engineered. The enzyme was directly immobilised at a gold electrode without the need for pre-treatment of the surface with a self-assembled monolayer or a conducting polymer. The ensemble was used to develop an amperometric biosensor for the detection of explosives containing nitroaromatic compounds. Preliminary results demonstrate detection levels down to 100 parts per trillion, signifying tremendous promise towards an in situ sensor for the detection of explosives.     

Biochemical and immunochemical characterization of the antigen-antibody reaction on a non-toxic biomimetic interface immobilized red blood cells of crucian carp and gold nanoparticles

A special protein assay system based on a highly hydrophilic, non-toxic and conductive biominetic interface has been demonstrated. To fabricate such assay system, red blood cells of crucian carp (RBC) was initially grown on a glassy carbon electrode surface (GCE) deposited nano-sized gold particles (GPs), a second gold nanoparticle layer (NG) was then absorbed on the RBC surface, and finally mammary cancer 15-3 antibody (anti-CA15-3) was attached on the functional RBC surface. A competitive immunoassay format was employed to detect CA15-3 with horseradish peroxidase (HRP)-labeled CA15-3 as tracer and hydrogen peroxide as enzyme substrate. When the immunosensor was incubated into a mixture solution containing HRP-labeled CA15-3 and CA15-3 sample for 1h at 37 degrees C, the amperometric response decreased with the increment of CA15-3 sample concentration. AFM images of the modified layer revealed a uniform distribution of protein and nanogold. In situ QCM and electrochemical measurements demonstrated that the wanted antibody-antigen reactions should occur with high specificity and selectivity. The specific immunoassay system can be developed further to yield sophisticated structures for other proteins.     

In vitro testing of a simply constructed, highly stable glucose sensor suitable for implantation in diabetic patients

We have constructed and tested in vitro a potentially implantable, needle-type amperometric enzyme electrode which is suitable for continuous monitoring of glucose concentrations in diabetic patients. The major requirements of stability during operation and ease of manufacture have been met with a sensor design which involves a simple dip-coating procedure for applying to a platinum base electrode an inner membrane of glucose oxidase immobilised in polyhydroxyethyl methacrylate (pHEMA), and an outer membrane composed of a pHEMA/polyurethane mixture. Sensors were operated at 700 mV for detection of hydrogen peroxide. Calibration curves for the sensor were linear to at least 20 mM glucose and were unaffected by a reduction in PO2 from 20 to 5 kPa. During continuous operation in 5 mM buffered glucose solutions in vitro, sensors suffered no significant loss of response over periods of up to 60 h. Such electrodes are, therefore, useful for development as in vivo glucose sensors.     

Measurement of protein using an electrochemical bi-enzyme sensor.

A screen-printed three-electrode amperometric biosensor for the rapid and quantitative measurement of single protein solutions is described. A membrane immobilised protease preparation of broad specificity was used to digest sample protein liberating free amino acids that were subsequently oxidised at a working electrode by immobilised L-amino acid oxidase (L-AAO). The enzymatically generated hydrogen peroxide was determined amperometrically. The fully optimised device required 30 mU L-AAO and 3.94 U protease and had a limit of detection of 170 microg ml(-1) and linearity of response up to 1 mg ml(-1) for Casilan 90 protein. The analytical performance of the device was comparable to that of a commercially available standard photometric protein test kit and required only a 10 microl volume of sample and a single dilution step. Unlike with photometry, the sensor is able to determine the protein content of turbid samples and hence should find widespread applications. The device was simple to use, low-cost and could be mass-produced, yielding results within 4 min of sample addition with acceptable assay repeatability.     

Study of xanthine oxidase immobilized electrode based on modified graphite

Xanthine oxidase (E. C. 1.2.3.2) was immobilized by adsorption on electrochemically modified graphite plate to obtain an enzyme electrode. The current of the enzyme electrode in substrate (xanthine) solutions was found to be a result of the electrooxidation of H2O2 generated in the enzyme layer. The linearity of the amperometric signal was up to a substrate concentration of 65 microM at 0.6 V (vs. Ag/AgCl). The response time was 2 minutes. The enzyme electrode preserves 80% of its initial activity after a three-week storage in air at room temperature.     

Development and performance of an enzyme-linked amperometric immunosensor for the detection of Staphylococcus aureus in foods

An amperometric enzyme-linked immunosensor was developed to detect and quantify levels of Staphylococcus aureus electrically in pure cultures and in foods. The assay was a modification of a 'sandwich' ELISA for the protein A of Staph. aureus, employing catalase-labelled anti-protein A antibody. On addition of hydrogen peroxide to the assay system the catalase released O2 which was monitored using an amperometric oxygen electrode. The rate of current increase was proportional to the antigen concentration (protein A or Staph. aureus). Protein A was detected reliably at 0.1 ng/ml representing a 20-fold increase in sensitivity over the conventional ELISA that used horseradish peroxidase. Pure cultures of Staph. aureus were detected at 10(-3)-10(-4) cfu/ml with the amperometric electrode (cf greater than 10(5)/ml for conventional ELISA). The same level of sensitivity was achieved for inoculated food samples. Low levels of contamination (1 cfu/g) of Staph. aureus were detected after incubation at 37 degrees C for 18 h, and the immunosensor could from the basis of a test for screening and identification of protein A-bearing Staph. aureus in 24 h, although natural variations in protein A content between different strains could make the system unreliable in accurate quantification of cell numbers.     

Polyvinylferrocenium modified Pt electrode for anaerobic glucose monitoring

An amperometric enzyme electrode for the determination of glucose under anaerobic solution conditions was developed by immobilizing glucose oxidase and then by adsorbing ferrocene in polyvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of ferrocene that the reduced flavin adenine dinucleotide centers of glucose oxidase was measured at a constant potential. The response characteristics of the enzyme electrode were investigated. The effects of the thickness of the polymeric film, the amount of the enzyme immobilized, the amount of the mediator, the glucose concentration, the applied potential, operating pH and temperature on the response of the enzyme electrode were studied. The response time and the optimum pH were found to be 30-40 s and pH 7.4 at 25 degrees C, respectively. The linear response was observed up to 5.0 mM glucose concentration that the produced detectable current was 0.0075 mM glucose concentration. The activation energy (E(a)) of immobilized enzyme reaction was calculated to be 41.3 kJ mol(-1) from the Arrhenius plot. The apparent Michaelis-Menten constant (K(Mapp)) was found to be 6.05 mM glucose according to the Lineweaver-Burk graph of the Michaelis-Menten equation under the optimum conditions. The interference signal due to the most common electrochemical interfering species was also evaluated.     

Investigation of concentration profiles inside operating biocatalytic sensors with scanning electrochemical microscopy (SECM)

Scanning electrochemical microscopy (SECM) with amperometric or potentiometric measuring tips was used to investigate biocatalytic reactions inside the enzyme layer of a biosensor during its operation. The well known glucose oxidase catalyzed oxidation of glucose has been selected for the studies. Local, instantaneous concentration of dissolved oxygen and hydrogen peroxide was studied observing the amperometric current while miniaturized potentiometric tip served for local pH measurements. Liquid enzyme layer immobilized with Cellophane membrane or cross linked polyacrilamide gel membrane containing entrapped enzyme served for biocatalytic media in the SECM imaging. Local maximum of H(2)O(2) and minimum of O(2) profiles were found at approximately 200 microm far from the substrate/enzyme layer boundary. From the experimental findings guidelines to design well functioning biocatalytic sensors could be concluded. The concentration profiles obtained with SECM techniques were compared with the results of simple model calculations carried out with the method of finite changes. Most of earlier made SECM studies dealing with enzyme reactions imaged the electrolyte being in contact with the immobilized enzyme. The data in our investigation, however, were collected inside the working catalytic layer.     

Micromachined sensor for lactate monitoring in saliva

A miniaturised sensor for continuous lactate measurement in saliva was developed and tested. The sensor was built using silicon microfabrication technologies. The size of the chip is 5.5 mmx6.4 mmx0.7 mm and features a working, a counter and an Iridium reference electrode. The chip has a cavity whose floor is perforated by fine pores. The cavity contains the enzyme lactate oxidase (LOD), which is immobilised in an agarose gel. Prior to the amperometric detection of the reaction product hydrogen peroxide at the working electrode, the analyte lactate has to pass the pores to reach the cavity with the lactate oxidase by diffusion. To test the silicon sensor, capillary blood and saliva samples were obtained during standardised ergometer tests. Salivary lactate concentrations were determined with the sensor and compared to photometrically derived data from a lab-automate. In addition the saliva data were compared to standard capillary blood lactate concentrations measured with a pocket photometer. Lactate concentration versus load graphs were plotted and compared visually showing very similar progressions. The novel approach enables a location independent, permanent real-time measurement of the lactate concentration during exercise.     

Development and study of an amperometric biosensor for the in vitro measurement of low concentration of putrescine in blood

An amperometric biosensor was developed for the in vitro determination of putrescine in blood samples because elevated level of putrescine in blood can be a diagnostic indicator of certain kinds of cancer. The electrochemical transducer consisted of a flat form, three electrode amperometric micro-cell fabricated with thin film photolithography on flexible Kapton substrate. An immobilized putrescine oxidase (PUO) layer provided the biocatalytic oxidation of the putrescine, while the generated hydrogen peroxide was detected on the platinum-working electrode. An electropolymerized poly(m-phenylenediamine) (pPDA) size-exclusion layer was used to protect the working electrode from fouling and to prevent signal generation by common electroactive interferents present in blood. The preparation of the biocatalytic enzyme- and outer protective layers was optimized for improved sensitivity and response time. A detection limit of 50 nM was achieved in pH-adjusted whole blood samples, which is below pathological levels.     

Amperometric enzyme electrode for organic peroxides determination prepared from horseradish peroxidase immobilized in poly(vinylferrocenium) film

Organic peroxides, t-butyl hydroperoxide, 2-butanone peroxide, cumene hydroperoxide and t-butyl peracetate, were determined by an amperometric enzyme electrode. The enzyme electrode was prepared through electrostatic immobilization of horseradish peroxidase (HRP) in a polyvinylferrocenium (PVF) film. A PVF(+)ClO(4)(-) film was coated on a Pt foil at +0.70 V by electrooxidation of polyvinylferrocene in methylene chloride with 0.1 M tetrabutylammonium perchlorate (TBAP). The enzyme modified electrode PVF(+)HRP(-) was prepared by anion-exchange in a solution of HRP(-) in 0.05 M phosphate buffer at pH 8.5. FTIR spectroscopy was used to identify PVF, PVF(+)ClO(4)(-), and PVF(+)HRP(-). The immobilized amount of the enzyme in the film was determined by UV spectroscopy. The effects of the polymeric film thickness, bulk enzyme concentration used in the immobilization treatment and the temperature on the performance of enzyme electrode were investigated. The inhibitory effect of oxygen was also examined. Linearities, lower detection limits, active life times and sensitivities of the electrode were determined for each peroxide.     

Periodically interrupted amperometry. A way of improving analytical performance of membrane coated electrodes

Amperometry is a powerful voltammetric measuring method. Its application is specially advantageous when used in combination with a separation step or with some other sample treatment method providing selectivity. The selectivity is often achieved by coating the amperometric working electrode surface with a membrane of special character. Size exclusion membrane, immobilized enzyme containing reaction layer, protecting dialysis membrane, perm selective ion exchange film etc can be mentioned here. In conventional amperometry the measuring potential is continuously applied, therefore in case of membrane coated electrodes the electrode process depletes the diffusion layer. In this work the performance of a new periodically interrupted amperometric (PIA) measuring program has been investigated in case of glucose enzyme sensor. The measuring program allowing time for reloading the diffusion layer provided higher current and therefore improved sensitivity and lower limit of detection.     

Graphite-Teflon composite bienzyme amperometric biosensors for monitoring of alcohols

Composite graphite-Teflon electrodes, in which the enzymes alcohol oxidase (AOD) and horseradish peroxidase (HRP), as well as the mediator ferrocene, are incorporated into the electrode matrix, are reported for the reliable monitoring of alcohols in food and beverages. The bienzyme electrodes are constructed by simple physical inclusion of the enzymes and the mediator in the bulk of graphite-70% Teflon rigid cylindrical pellets. The composite biosensors are robust and reusable because of the renewability of the electrode surface by polishing. Reproducible amperometric responses at 0.00 V were obtained with different electrodes constructed from the same pellet and from different pellets. No significant loss of the enzymes activities was found after at least 3 months of storage at 0 degrees C. The detection limits obtained by amperometry in stirred solutions can be advantageously compared with those achieved with commercial sensors for alcohols. The bienzyme electrodes are suitable to be used under flow-injection conditions, as well as for amperometric detection in HPLC. The bioelectrodes were employed for the determination of ethanol in beers, wines and liquors, using both batch- and flow-injection modes, and for the determination of methanol in wines and liquors by HPLC with amperometric detection. Only a dilution of the beverages was needed as sample treatment in all cases.     

Electrocatalytic four-electron reduction of oxygen at the cytochrome c3-adsorbed electrode

The electrocatalytic activity of cytochrome c3 for the reduction of molecular oxygen was characterized from the studies of the adsorption of cytochrome c3 and the co-adsorption of cytochrome cs with cytochrome c on the mercury electrode by the a.c. polarographic technique. The adsorption of cytochrome c3 on the mercury electrode is irreversible and is diffusion-controlled. The maximum amount of cytochrome c3 absorbed was 0.92 . 10(-11) mol . cm-2 at -0.90 V. The amount of cytochrome c3 in the mixed adsorbed layer with cytochrome c was determined from the differential capacitance measurement. It was shown that the fractional coverage of cytochrome c3 can be estimated from its bulk concentration and the diffusion coefficient (1.05 . 10(-6) cm2 . s-1). Cytochrome c3 catalyzes the electrochemical reduction of molecular oxygen from the two-electron pathways via hydrogen peroxide to the four-electron pathway at the mercury electrode in neutral phosphate buffer solution. The catalytic activity varies with the bulk concentration of cytochrome c3. The highest catalytic activity for the oxygen reduction (no hydrogen peroxide formation) is attained when one-half of the mercury electrode surface is covered by cytochrome c3. The addition of cytochrome c or bovine serum albumin to the cytochrome c3 solution inhibits the catalytic activity of cytochrome c3. The reversible polarographic behavior of cytochrome c3 through the mixed adsorbed layer of cytochrome c3 and cytochrome c was also investigated.     

Biocompatible glucose sensor prepared by modifying protein and vinylferrocene monomer composite membrane

This paper proposes a very simple procedure for preparing a biocompatible sensor based on a protein (bovine serum albumin, BSA), enzyme and vinylferrocene (VF) composite membrane modified electrode. The membrane was prepared simply by first casting vinylferrocene and then coating it with BSA and glucose oxidase immobilised with glutaraldehyde. The sensor response was independent of dissolved oxygen concentration from 3 to 10 ppm and showed good stability for serum sample measurement, unlike the commonly used BSA/enzyme modified electrode. The sensor response was almost unchanged over the measurement time (>10 h) whereas the responses of a BSA and glucose oxidase modified platinum electrode and an osmium-polyvinylpyridine wired horseradish peroxidase modified electrode (Ohara et al., 1993) fell to 68% of their initial value in a serum sample containing 10mM glucose.