Behavior of glucose oxidase immobilized in various electropolymerized thin films

Glucose oxidase was immobilized by electropolymerization into films of polyaniline, polyindole, polypyrrole, poly(o-phenylediamine), and polyaniline crosslinked with p-phenylenediamine. The kinetics and the behavior of the entrapped enzyme toward elevated temperature, organic solvent denaturation, and pH were investigated, along with the response of the films to electroactive species such as acetaminophen, ascorbate, cysteine, and uric acid. For most of the films, linearity to glucose extended from 7 to 10 mM. The poly(o-phenylenediamine)/glucose oxidase film gave the best signal/noise ratio and polypyrrole/glucose oxidase film gave the most reproducible current responses. No significant shift of the optimum reaction pH (5.5), except for polypyrrole (5.0), was observed after immobilization of glucose oxidase in the various films. Enzymatic activity decreased rapidly when pH was raised above 7.5. Thermodeactivation studies at 55 degrees , 60 degrees , and 65 degrees C have shown polypyrrole/and poly(o-phenylediamine)/glucose oxidase films to be the most resistant enzymatic films. Poly(o-phenylenediamine) films offered the best protection against glucose oxidase deactivation in hexane, chloroform, ether, THF, and acetonitrile when compared with the other electropolymerized films. All the enzymatic films exhibited permselection toward electroactive species. (c) 1996 John Wiley & Sons, Inc.     

Sonochemically fabricated microelectrode arrays for biosensors--part II. Modification with a polysiloxane coating

A polymer-modified sonochemically fabricated glucose oxidase microelectrode array with microelectrode population densities of up to 2.5 x 10(5) microelectrodes per square centimetres is reported. These microelectrode sensors were formed by first depositing an insulating film on commercial screen printed electrodes which was subsequently sonicated to form cavities of regular sizes in the film. Electropolymerisation of aniline at the microelectrode cavities formed polyaniline protrusions containing entrapped glucose oxidase. Chemical deposition of polysiloxane from dichlorodimethysilane was used to deposit a thin protective and diffusion mass transport controlling coating over the electrodes. The physical and electrochemical properties of these films were studied. The performance of the final glucose oxidase based microelectrode sensor array is reported.     

Glucose concentration determination by means of fluorescence emission spectra of soluble and insoluble glucose oxidase: some useful indications for optical fibre-based sensors

By using soluble and insoluble glucose oxidase, the changes in intrinsic emission fluorescence in the visible spectral region were studied as a function of glucose concentration. Insoluble glucose oxidase (GOD) was obtained by entrapment in a gelatine membrane or by covalent attachment on an agarose membrane grafted with hexamethylendiamine. The intensity of the fluorescence emission peak at 520 nm or the value of the integral fluorescence area from 480 to 580 nm were taken as physical parameters representative of the glucose concentration during the enzyme reaction. By using these parameters, linear calibration curves for glucose concentration were obtained. The extension of the calibration curve and the sensitivity of the adopted systems were found to be dependent on the enzyme state (free or immobilized) and on the immobilization method. In particular, it was found that the extent of the linear range of the calibration curves is increased of one order of magnitude when the glucose oxidase is immobilized, while the sensitivity of the measure is decreased of one order of magnitude by the immobilization process. Measures carried out by using the integral fluorescence area resulted more sensitive than those obtained with the peak size. Useful indications for the construction of optical fibre-based sensors were drawn from the reported results.     

Reproducible fabrication of miniaturized glucose sensors: preparation of sensing membranes for continuous monitoring.

The immobilization process of glucose oxidase(GOx) in the poly(1,3-diaminobenzene) (poly(1,3-DAB)) network was closely investigated in situ using an electrochemical quartz crystal microbalance(EQCM). GOx captured in approximately 50 nm thick poly-1,3-DAB layer causes a 514 Hz frequency increase, corresponding to 541 ng, and distributes mostly in the outer part of the polymer film. The presence of poly-L-lysine and glutaraldehyde during electropolymerization of poly(1,3-DAB) improves sensitivity by raising the amount of GOx immobilized. Adding a protective membrane on to the enzyme layer from poly(tetrafluoroethylene) (PTFE) dispersed in aqueous media lets the entire fabrication procedure finish perfectly without nonaqueous solvent. The finalized needle-type glucose sensors show competent functions in sensitivity, stability, biocompatibility, lifetime, interference and reproducibility.     

Comparison of different methods of glucose oxidase immobilization

Glucose oxidase was immobilized by covalent bond to two basic types of sorbents—glycidylmethacrylate copolymers and bead cellulose. These two types of carries were chemically modified, if needed, by the employing various procedures and subsequently used in the immobilization of native and oxidized glucose oxidase. The samples thus obtained were compared with those of immobilized glucose oxidase bound onto some common carriers. Samples which possessed not only a high absolute activity but also adequate mechanical and flow properties were characterized in greater detail with respect to the immobilization efficiency and kinetic properties of bound glucose oxidase.     

Second generation biosensors.

Enzyme-membrane electrodes using glucose oxidase in combination with peroxide detection dominate in the field of laboratory analyzers for diluted samples. Using the same indication principle, extremely fast responding glucose sensors have been fabricated by covering thin metal electrodes with a porous enzyme layer. In the second generation auxiliary enzymes and/or co-reactants are coimmobilized with the analyte converting enzyme in order to improve the analytical quality and to simplify the performance. Following this line oxidizable interferences are suppressed by using a glucose oxidase/peroxidase complex which communicates with the electrode at a low working potential. Furthermore, fluctuations of pH or buffer capacity are ineffective when using a glucose oxidase/peroxidase layer covered fluoride FET in the potentiometric glucose determination. Enzymatic recycling of the analyte and/or accumulation of intermediates increase the sensitivity by several orders of magnitude. Inclusion of NAD bound to PEG in the glucose dehydrogenase layer allows a reagentless glucose measurement.     

Intravascular glucose/lactate sensors prepared with nitric oxide releasing poly(lactide-co-glycolide)-based coatings for enhanced biocompatibility

Intravenous amperometric needle-type enzymatic glucose/lactate sensors intended for continuous monitoring are prepared with a novel nitric oxide (NO) releasing layer to improve device hemocompatibility. To create an underlying NO release coating, the sensors with immobilized enzymes (either glucose oxidase or lactate oxidase) are prepared with a thin layer of poly(lactide-co-glycolide) (PLGA) loaded with lipophilic diazeniumdiolate species that slowly release NO via a proton driven reaction. An outer thin layer (ca. 30 μm) of PurSil (polyurethane/dimethylsiloxane copolymer) limits the flux of glucose and lactate to the inner layer of enzyme, to provide the desired linear amperometric response. A 30 μm coating of PLGA containing 33 wt% of the appropriate NO donor (N-diazeniumdiolated dibutylhexanediamine, DBHD/N?O?) can release NO at a physiologically relevant rate > 1 × 10?1?mol min?1 cm?2 for at least 7 days without influencing the analytical performance of the glucose/lactate sensors. In vitro, the sensors exhibit relatively stable amperometric response over a one-week period with high selectivity over interferences (e.g., ascorbic acid) required for blood monitoring applications. Glucose sensors implanted in the veins of rabbits for 8h exhibit significantly enhanced hemocompatibility for the NO release sensors vs. corresponding controls (without NO release in same animals), with greatly reduced thrombus formation on their surfaces. Further, the analytical performance of the NO release glucose sensors are superior to controls placed in the veins of the same animals, with a greater accuracy in measuring blood glucose levels as evaluated using a Clarke error grid type analysis.     

Miniaturized glucose biosensor modified with a nitric oxide-releasing xerogel microarray

An enzyme-based glucose biosensor modified to release nitric oxide (NO) via a xerogel microarray is reported. The biosensor design is as follows: (1) glucose oxidase (GOx) is immobilized in a methyltrimethoxysilane (MTMOS) xerogel layer; (2) a blended polyurethane/hydrophilic polyurethane coating prevents enzyme leaching and imparts selectivity for glucose; and (3) micropatterned xerogel lines (5 microm wide) separated by distances of 5 or 20 microm provide NO-release capability. This configuration allows for increased glucose sensitivity relative to sensors modified with NO-releasing xerogel films since significant portions of the sensor surface remain unmodified. Glucose diffusion to the GOx layer is thus less inhibited. The micropatterned NO-releasing biosensors generate sufficient NO levels to reduce both Pseudomonas aeruginosa and platelet adhesion without significantly compromising the enzymatic activity of GOx. The glucose response, linearity and stability of the NO-releasing micropatterned sensors are reported.     

Biosensor system for continuous flow determination of enzyme activities. I. Determination of glucose oxidase and lactic dehydrogenase activities

A biosensor system for continuous flow determination of enzyme activity was developed and applied to the determination of glucose oxidase and lactic dehydrogenase activities. The glucose oxidase activity sensor was prepared from the combination of an oxygen electrode and a flow cell. Similarly, the lactic dehydrogenase activity sensor was prepared from the combination of a pyruvate oxidase membrane, an oxygen electrode, and a flow cell. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane, and glutaraldehyde. Glucose oxidase activity was determined from the oxygen consumed upon oxidation of glucose catalyzed by glucose oxidase. Lactic dehydrogenase activity was determined from the pyruvic acid formed upon dehydrogenation of lactic acid catalyzed by lactic dehydrogenase. The amount of pyruvic acid was determined from the oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. Calibration curves for activity of glucose oxidase and lactic dehydrogenase were linear up to 81 and 300 units, respectively. One assay could be completed within 15 min for both sensors and these were stable for more than 25 days at 5°C. The relative errors were ±4 and ±6% for glucose oxidase and lactic dehydrogenase sensors, respectively. These results suggest that the sensor system proposed is a simple, rapid, and economical method for the determination of enzyme activities.     

A Study on the Comparative Stability of Insoluble Complexes of Glucose Oxidase Obtained with Concanavalin A and Specific Polyclonal Antibodies

Summary The formation of insoluble complexes of glycoenzymes with lectins and antibodies is one of the simplest methods of enzyme immobilization. Insoluble complexes of glucose oxidase were simply obtained by mixing the enzyme with concanavalin A or a specific polyclonal antibodies solution. The concanavalin A and immunocomplexes of glucose oxidase retained more than 80% of the original enzyme activity. Expression of very high enzyme activity in insoluble complexes suggested that these aggregates were quite porous and easily accessible to substrates. Insoluble complexes of glucose oxidase showed very high stability against denaturation induced by pH, temperature, urea and water-miscible organic solvents. Complexes of glucose oxidase obtained with concanavalin A and glycosyl-specific antiglucose oxidase polyclonal antibodies were quite comparable in stability while complexes prepared using polyclonal antibodies raised against the native glucose oxidase were slightly less stable. The complexes of glucose oxidase obtained with glycosyl-specific antiglucose oxidase polyclonal antibodies showed very high stability against inactivation mediated by exposure to water-miscible organic solvents. Insoluble complexes of glucose oxidase were cross-linked with glutaraldehyde to maintain their integrity in the presence of substrates. The cross-linking of complexes resulted in a slight decrease in enzyme activity but showed a pronounced enhancement in stability against various forms of denaturation.     

Immobilization of biocatalysts with bombyx mori silk fibroin by several kinds of physical treatment and its application to glucose sensors

Biocatalyst-immobilized Bombyx mori silk fibroin membrane was prepared. The insolubilization of the water-soluble membranes was performed by physical treatments only, i.e. stretching, compressing and standing under high humidity and methanol-immersion treatment, without any use ofcovalently binding reagent. All physical treatments performed were effective for the purpose of the immobilization of the enzymes in the membranes. The structural characterization of the glucose oxidase (GOD) immobilized membrane was performed in detail. The permeability of the substrate depends on the crystalline structure, i.e. the fraction of Silk I and Silk II of the membrane. The activity yield of the immobilized GOD was more than 80% of the value of free enzyme when 0–002% of the enzyme was entrapped in the membrane, but it decreased with increasing the concentration of the GOD in the membrane. This seems to result from diffusion limitation of the substrate. The pH and thermal stabilities of the immobilized enzyme were much improved, and were essentially independent of the methods of the immobilization. Development of the GOD or microorganism, Pseudomonas fluorescens immobilized silk fibroin membranes as glucose sensors are described.     

Imaging of electrochemical enzyme sensor on gold electrode using surface plasmon resonance

Three types of imaging, namely layer structure, electrochemical reaction, and enzyme sensor response, were achieved by applying surface plasmon resonance (SPR) measurement to an electrochemical biosensor. We constructed glucose oxidase based mediator type sensors on a gold electrode by spotting the mediator that contained horseradish peroxidase and spin coating the glucose oxidase film. The layer structure of the sensor was imaged by means of angle scanning SPR measurement. The single sensor spot (about 1 mm in diameter) consisted of about 100 x 100 pixels and its spatial structure was imaged. The multilayer structure of the enzyme sensor had a complex reflectance-incident angle curve and this required us to choose a suitable incident angle for mapping the redox state. We chose an incident angle that provided the most significant reflection intensity difference by using data obtained from two angle scanning SPR measurements at different electrode potentials. At this incident angle, we controlled the electrochemical states of the spotted mediator in cyclic voltammetry and imaged the degree to which the charged site density changed. Finally, we mapped the enzymatic activity around the mediator spot by the enzymatic reoxidation of pre-reduced mediator in the presence of glucose.     

High-yield method for immobilization of enzymes

Two types of polyethylenimine-coated glass microbeads (13–44 μm) were synthesized and used for the immobilization of glucose oxidase from Aspergillus niger and catalase from A. niger and beef liver. The two types of beads were distinguishable by differences in their surface topography. Immobilizations were performed by adsorption followed by treatment with glutaraldehyde. The immobilized-enzyme activities per unit support of all of the enzymes tested were compared with and found to be superior to the immobilized activities attainable on aminopropyl-activated glass microbeads. When enzyme was present in less than saturating amounts, the coated beads were able to remove 100% of the glucose oxidase activity initially present in the immobilization solution, with 78–87% of that activity expressed on the support surface. Bound glucose oxidase was more stable to thermal inactivation than native enzyme.     

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.     

Comparative study of hydrogel-immobilized l-glutamate oxidases for a novel thick-film biosensor and its application in food samples

Novel, thick-film biosensors have been developed for the determination of l-glutamate in foodstuffs. The sensors were prepared by immobilization of l-glutamate oxidase by using polycarbamylsulfonate-hydrogel on a thick-film sensor. l-Glutamate oxidases obtained from Streptomyces sp. with different degree of purification were compared with their characteristic response to l-glutamate at different conditions and for their specificity, inhibition, and storage properties. These sensors were applied to determine monosodium glutamate in soy sauce samples and show good correlation with colorimetric method.     

The immobilization of enzymes on nylon structures and their use in automated analysis

1. Glucose oxidase (EC 1.1.3.4) and urease (EC 3.5.1.5) were covalently attached through glutaraldehyde to low-molecular-weight nylon powder. 2. Immobilized derivatives of glucose oxidase and urease were prepared by cross-linking the respective enzymes within the matrix of a nylon membrane. 3. An improved process is described for the immobilization of glucose oxidase and urease on the inside surface of partially hydrolysed nylon tube. 4. Automated analytical procedures are described for the determination of glucose with each of the three immobilized glucose oxidase derivatives and for the determination of urea with each of the three immobilized urease derivatives. 5. The efficiencies of the three immobilized enzyme structures as reagents for the automated determination of their substrates were compared.     

Glucose microbiosensor based on alumina sol-gel matrix/electropolymerized composite membrane

A procedure is described that provides co-immobilization of enzyme and bovine serum albumin (BSA) within an alumina sol-gel matrix and a polyphenol layer permselective for endogenous electroactive species. BSA has first been employed for the immobilization of glucose oxidase (GOx) on a Pt electrode in a sol-gel to produce a uniform, thin and compact film with enhanced enzyme activity. Electropolymerization of phenol was then employed to form an anti-interference and protective polyphenol film within the enzyme layer. In addition, a stability-reinforcing membrane derived from (3-aminopropyl)-trimethoxysilane was constructed by electrochemically-assisted crosslinking. This hybrid film outside the enzyme layer contributed both to the improved stability and to permselectivity. The resulting glucose sensor was characterized by a short response time (<10 s), high sensitivity (10.4 nA/mM mm(2)), low interference from endogenous electroactive species, and a working lifetime of at least 60 days.     

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.     

Simultaneously immobilized glucose oxidase and catalase in controlled-pore titania

The immobilization of glucose oxidase and catalase by adsorption within the pores of controlled-pore titania has yielded a remarkably stable enzyme system. Catalase apparently acts as both a stabilizer and an activator for glucose oxidase within the pores of this material. Hydrogen peroxide concentrations and flow rates have a marked effect upon the apparent activity of the immobilized enzyme system. The carrier parameters were varied to obtain optimum loading and stability information.     

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.