Immobilization of maltogenase onto polyurethane microparticles from poly(vinyl alcohol) and hexamethylene diisocyanate

A series of porous polyurethane (PU) microparticles from poly(vinyl alcohol) (PVA) and hexamethylene diisocyanate (HMDI) using different ratios of components were obtained by one step method. Molar compositions of PU microparticles were estimated by determination of nitrogen, isocyanate and hydroxyl groups. PU carriers which were synthesized using optimal initial molar ratios of PVA and HMDI were applied for immobilization of maltogenase (MG) from Bacillus stearothermophilus. Immobilized enzyme exhibited higher catalytic activity and enhanced temperature stability in comparison with the native MG. Maximal loading 7.78 mg/g wet carrier was reached when PU microparticles with initial molar ratio of PVA and HMDI = 1:3 was used as a carrier for immobilization. The high efficiency of immobilization (EI) was obtained using PU microparticles when initial molar ratio of HMDI and PVA was 1:1–1:10. High stability of MG immobilized onto PU microparticles during storage was demonstrated. Immobilized starch hydrolyzing enzyme was successfully tested in batch and column type reactors for hydrolysis of potato starch. MG immobilized onto PU enables easy separation from the reaction medium and reuse of the immobilized preparation over seven reaction cycles in bath operation and at least three cycles in column type reactor.     

Thin-film antimony-antimony-oxide enzyme electrode for penicillin determination

A potentiometric penicillinase electrode is reported in which the base pH transducer is a thin-film anti-mony-antimony-oxide electrode deposited by vacuum evaporation. Several enzyme immobilization procedures have been examined and a crosslinked protein film found to be the most appropriate to this type of sensor. The use of an adjacent antimony-antimony-oxide track as a pseudoreference electrode was successfully demonstrated. The overall response was shown to be independent of the stirring rate above 100 rpm, but the kinetics of the response were found to depend markedly on the stirring rate. The intrinsic linear response range was 3 x 10(-4)Mto 7 x 10(-3)M penicillin G. Linearizing transforms that extend the useful range were examined.     

Urea potentiometric biosensor based on modified electrodes with urease immobilized on polyethylenimine films

The development of a new electrochemical sensor consisting in a glass-sealed metal microelectrode coated by a polyethylenimine film is described. The use of polymers as the entrapping matrix for enzymes fulfils all the requirements expected for these materials without damaging the biological material. Since enzyme immobilization plays a fundamental role in the performance characteristics of enzymatic biosensors, we have tested four different protocols for enzyme immobilization to determine the most reliable one. Thus the characteristics of the potentiometric biosensors assembled were studied and compared and it appeared that the immobilization method leading to the most efficient biosensors was the one consisting in a physical adsorption followed by reticulation with dilute aqueous glutaraldehyde solutions. Indeed, the glutaraldehyde immobilized urease sensor provides many advantages, compared to the other types of sensors, since this type of urea biosensor exhibits short response times (15–30 s), sigmoidal responses for the urea concentration working range from 1×10^−2.5 to 1×10^−1.5 M and a lifetime of 4 weeks.     

Characterizing the conservation effect of clear coatings on photodegradation of wood

Photodiscoloration of clear-coated wood may be caused by the yellowing of both clear coating film and underlying wood, or either of them. Wood specimens covered with two types of free polyurethane films with/without light stabilizer were used to simulate the photodiscoloration of clear-coated wood. Percent UV transmission of aromatic polyurethane (PU) films decreased after irradiation, whereas aliphatic polyurethane (PUA) films significantly increased with irradiation time resulting in further photoyellowing of wood beneath the PUA film. A light reflection model was used to elucidate discoloration caused separately by the clear coating film and the underlying wood. After 24 days of light irradiation, clear coating and the underlying wood contributed respectively, 40.70% and 59.30% discoloration of PU-coated specimens, and the corresponding values for PUA-coated specimens were 5.15% and 94.85%. PU film with light stabilizer reduced lignin degradation and generation of carbonyl derivatives in the underlying wood.     

Electrochemical sensor based on photopolymeric membranes for determining urea

A new electrochemical enzymatic sensor based on the ion selective field effect transistors (ISFETs) and photocurable membrane was developed for the determination of urea. For the immobilization of urease on the gate surface of the ISFET a simple method, involving the use of liquid photocurable compositions on the basis of vinylpirollidone, oligouretanemetacrylate and oligocarbonatemetacrylate, was applied. The linearange of the response of the developed electrochemical sensor lies in the range 0.05-20 mM. The latter corresponds to the claims of the medical practice. The overall time of the analysis is 5-10 min. The effects of the buffer concentration and its pH as well as temperature and presence of ammonia ions in the measuring medium on the amplitude of the sensor response were estimated. The duration of sensor work is as shortest 40 days. The proposed sensor on the basis of the ISFET is promising for the express analysis of the level urea in blood, while the developed method of membrane preparation with entrapped enzyme can be combined with the integral technology of producing of the biosensors semiconductor transducers.     

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.     

Improving reuse cycles of Thermomyces lanuginosus lipase (NS-40116) by immobilization in flexible polyurethane

Enzyme immobilization using a low-cost support that allows increasing operational stability and reutilization arise as a great economic advantage for the industry. In this work, it was explored different methods of Thermomyces lanuginosus lipase (NS-40116) immobilization in flexible polyurethane foam (PU). PU polymer was synthesized using polyether and toluene diisocyanate as monomers. PU-NS-40116 immobilized was evaluated in terms of stability in a range of pH (7.0 and 9.0), temperature (24, 50 and 60?°C) for 24?h, and storage stability (room temperature and 4?°C) for 30?days. The results showed that after 30?days of storage immobilized enzyme kept 80% of initial enzyme activity. PU support before and after immobilization process was characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. Free and immobilized enzymes were compared in terms of hydrolysis of soybean oil. Immobilized enzyme by entrapment was evaluated in successive cycles of reuse showing catalytic activity above 50% even after 5 successive cycles of reuse, confirming the efficiency of immobilization process.     

Glucose sensor using a microfabricated electrode and electropolymerized bilayer films

A new type miniaturized glucose sensor with good selectivity and stable current response has been developed. The structure consists of a recessed rectangular microfabricated platinum electrode, inner layer of two electropolymerized nonconducting films, and outer bilayer of poly(tetrafluoroethylene) (Teflon) and polyurethane (PU) films. Glucose oxidase (GOx) is entrapped during the electropolymerization of a poly(m-phenylenediamine) (PMPD) film in an acetate buffer (AB) solution, on which a highly interference-resistive PMPD film is deposited in a phosphate buffered saline (PBS) solution. The second PMPD film causes no significant decrease in accessibility of glucose to GOx. The inner layer maintains less than 1% permeability to acetaminophen for 12 days. The fairly adhesive outer layer allows stable current response. Due to high permeability, the information about enzyme activity can be obtained without serious error in spite of outer layer intervening between enzymes and solution. The apparent Michaelis-Menten constant and the maximum steady-state current density were 24 mM and 80 microA cm(-2), respectively.     

Direct electrochemistry and electrocatalysis based on film of horseradish peroxidase intercalated into layered titanate nano-sheets

Intercalation of horseradish peroxidase (HRP) into layered titanate by assembling it with titanate nano-sheets (TNS) was firstly used for fabrication of enzyme electrode (HRP-TNS electrode). XRD result revealed that HRP-TNS film featured layered structure with HRP monolayer intercalated between the titanate layers. UV-vis spectra result indicated the intercalated HRP in TNS film well retained its native structure. The HRP-TNS film was uniform with porous structures which were confirmed by SEM. The immobilized HRP in the TNS film exhibited fast direct electron transfer and showed a good electrocatalytic performance to H2O2 with high sensitivity, wide linear range and low detection. The excellent electrochemical performance of the HRP-TNS electrode was attributed to biocompatibility of the titanate sheets, porous architectures of the HRP-TNS film which retained activity of HRP to large extent, avoided aggregation of HRP, provided better mass transport and allowed more HRP loading per unit area. Thus, the simple method described here provides a novel and effective platform for immobilization of enzyme in realizing direct electrochemistry and has a promising application in fabrication of the third-generation electrochemical biosensors.     

A disposable biosensor for urea determination in blood based on an ammonium-sensitive transducer

A potentiometric urea-sensitive biosensor using a NH4(+)-sensitive disposable electrode in double matrix membrane (DMM) technology as transducer is described. The ion-sensitive polymer matrix membrane was formed in the presence of an additional electrochemical inert filter paper matrix to improve the reproducibility in sensor production. The electrodes were prepared from one-side silver-coated filter paper, which is encapsulated for insulation by a heat-sealing film. A defined volume of the NH4(+)-sensitive polymer matrix membrane cocktail was deposited on this filter paper. To obtain the urea-biosensor a layer of urease was cast onto the ion-sensitive membrane. Poly (carbamoylsulfonate) hydrogel, produced from a hydrophilic polyurethane prepolymer blocked with bisulfite, served as immobilisation material. The disposable urea sensitive electrode was combined with a disposable Ag/AgCl reference electrode to obtain the disposable urea biosensor. The sensor responded rapidly and in a stable manner to changes in urea concentrations between 7.2 x 10(-5) and 2.1 x 10(-2)mol/l. The detection limit was 2 x 10(-5) mol/l urea and the slope in the linear range 52 mV/decade. By taking into consideration the influence of the interfering K(+)- and Na(+)-ions the sensor can be used for the determination of urea in human blood and serum samples (diluted or undiluted). A good correlation was found with the data obtained by the spectrophotometric routine method.     

Immobilization of glucose oxidase on polymer membranes treated by low-temperature plasma

Low-temperature plasma was employed for activation of polymer membranes as a carrier for enzyme immobilization. Glucose oxidase was immobilized on polypropylene (PP), polyvinylidene fluoride (PVDF), or polytetrafluoroethylene (PTFE) membrane surfaces treated by nitrogen or ammonia gas plasma using glutaraldehyde as a linking agent. Enzyme activity was evaluated by the response of glucose sensor composed of the immobilized enzyme membrane and a dissolved oxygen electrode. The sensor response was found to depend on the kind of carrier membrane and to become maximum at suitable conditions of plasma treatment.     

Aspergillus niger inulinase immobilized in polyurethane foam and treated in pressurized LPG: A potential catalyst for enzymatic synthesis of fructooligosaccharides

Inulinase from Aspergillus niger was immobilized in polyurethane foam (PU). Immobilized catalyst was treated in pressurized liquefied petroleum gas (LPG) system. This biocatalyst was used in the fructooligosaccharide production using sucrose as substrate in aqueous system. The main objective of this study was to evaluate the reaction yield and productivity by using polyurethane foam as a low-cost support for enzyme immobilization in an alternative processes for fructooligosaccharide production in pressurized LPG system with potential for industrial application. The total FOS concentration obtained were 31% as a result of sucrose concentration reduction, and formation of FOS long chain (GF3 and GF4) from kestose (GF2). FOS concentrations of 5%, 22%, and 3% were obtained for GF2, GF3, and GF4, respectively. The methodology suggested in this research work, enzyme immobilization in a low-cost support, and treatment in LPG, showed potential technology for fructooligosaccharide synthesis.     

Nano polyurethane-assisted ultrasensitive biodetection of H(2)O(2) over immobilized microperoxidase-11

Nanostructured polyurethane (PU) synthesized by an emulsion polymerization with narrow size distribution was employed for the first time directly as a novel matrix for enzyme immobilization to develop sensitively amperometric biosensors. When Microperoxidase-11 (MP-11) was selected as a model protein, the resulting hydrogen peroxide (H(2)O(2)) biosensor exhibited improved sensitivity of 29.6μAmM(-1)cm(-2) with quite good response time of (1.3±0.4)s and remarkable limit of detection as low as 10pM (S/N 3) over existing protocols. A linear calibration curve for hydrogen peroxide was obtained up to 1.3μM under the optimized conditions with a relative low calculated Michaelis-Menten constant (K(M)(app)) (1.87±0.05)μM, which indicated the enhanced enzymatic affinity of MP-11 to H(2)O(2) via PU. The possible interferents had negligible effect on the response current and time of the prepared biosensor. Results suggest that the PU nanoparticles (PU-NPs) with good biocompatibility and sufficient interfacial adhesion hold promise as an attractive support material for construction of ultrasensitive amperometric biosensor.     

Urea potentiometric enzymatic biosensor based on charged biopolymers and electrodeposited polyaniline

A potentiometric biosensor based on urease was developed for the quantitative determination of urea concentration in aqueous solutions for biomedical applications. The urease was either physisorbed onto an electrodeposited polyaniline film (PANI), or immobilized on a layer-by-layer film (LbL) assembled over the PANI film, that was obtained by the alternate deposition of charged polysaccharides (carboxymethylpullulan (CMP) and chitosan (CHI)). In the latter case, the urease (Urs) enzyme was either physically adsorbed or covalently grafted to the LbL film using carbodiimide coupling reaction. Potentiometric responses of the enzymatic biosensors were measured as a function of the urea concentration in aqueous solutions (from 10(-6) to 10(-1) mol L(-1) urea). Very high sensitivity and short response time were observed for the present biosensor. Moreover, a stability study showed a higher stability over time for the potentiometric response of the sensor with the enzyme-grafted LbL film, testifying for the protective nature of the polysaccharide coating and the interest of covalent grafting.     

Co-immobilization of glucose oxidase and hexokinase on silicate hybrid sol-gel membrane for glucose and ATP detections

The co-immobilization of glucose oxidase (GOD) and hexokinase/glucose-6-phosphate dehydrogenase (HEX) in the silica hybrid sol-gel film for development of amperometric biosensors was investigated. The silica hybrid film fabricated by hydrolysis of the mixture of tetraethyl orthosilicate and 3-(trimethoxysiyl)propyl methacrylate possessed a three-dimension vesicle structure and good uniformity and conformability, and was ready for enzyme immobilization. The electrochemical and spectroscopic measurements showed that the silica hybrid sol-gel provided excellent matrice for the enzyme immobilization and that the immobilized enzyme retained its bioactivity effectively. The immobilized GOD could catalyze the oxidation of glucose, which could be used to determine glucose at +1.0 V without help of any mediator. The competition between GOD and HEX for the substrate glucose involving ATP as a co-substrate led to a decrease of the glucose response, which allowed us to develop an ATP sensor with a good stability. The fabricated silica hybrid sol-gel matrice offered a stage for further study of immobilization and electrochemistry of proteins.     

Potentiometric sensor for dipicolinic acid

A potentiometric chemosensor for selective determination of dipicolinic acid (2,6-pyridinedicarboxylic acid, DPA) was developed based on the surface imprinting technique coupled with a nanoscale transducer: an indium tin oxide (ITO)-coated glass plate. The sensor fabrication conditions, optimal recognition condition, as well as selectivity, sensitivity, and stability of the DPA sensor have been investigated. The DPA sensor could recognize DPA from 3,5-pyridinedicarboxylic acid. Potentiometric measurements demonstrated selective detection of DPA in a concentration range of 1.5 x 10(-6) to 0.0194 M. The response time of DPA sensor for 4 x 10(-4) M DPA was 25 s. The potentiometric response of the DPA sensor to DPA is at 90% of its initial magnitude after 550 times measurement. The viability of such a modified ITO electrode in the presence of other inorganic, organic, and biological materials was probed.     

Fabrication of ultra-thin polypyrrole-glucose oxidase film from supporting electrolyte-free monomer solution for potentiometric biosensing of glucose

A simple electropolymerisation process is described for the fabrication of an ultra-thin ( approximately 55 nm) polypyrrole (PPy)-glucose oxidase (GOD) film in a supporting electrolyte-free monomer solution for potentiometric biosensing of glucose. The optimum conditions for growing the ultra-thin film include 0.1 M pyrrole, 55-110 U/ml GOD, an applied current density of 0.05 mA/cm(2) and an electrical charge of 25 mC/cm(2). Long-term storage of the biosensor in acetate buffer improved the sensitivity of the biosensor by a factor of approximately two. The biosensor can also be used repeatedly for over 2 months with little or no loss in sensitivity. The interference effect of ascorbic acid was successfully reduced by inclusion of an outer PPy-Cl layer.     

Development of a biosensor for endocrine disrupting compounds based on tyrosinase entrapped within a poly(thionine) film

Preparation of semiconducting films by electropolymerisation of a monomer which is itself a redox mediator is an attractive and simple method for biosensor fabrication. A polymeric film of the redox dye thionine (phenothiazine) enables the stable immobilisation of polyphenol oxidase (tyrosinase) while acting as mediator for the enzymatic process. The immobilisation method is based on an inner crosslinked tyrosinase layer which contains thionine with an electropolymerised film of poly(thionine) on top. This method gave the most stable redox couple for poly(thionine) and exhibited the greatest response stability. The sensor was tested using a range of synthetic oestrogens and phenolic compounds, which are suspected endocrine disruptors/oestrogen mimics. The device responded well to all compounds tested with limits of detection ranging from 1 to 23 microM (based on three times S/N ratio). The tyrosinase/poly(thionine) electrode response to phenol was 3 orders of magnitude greater than the unmediated response in the absence of poly(thionine).     

A new type of electrochemical immunosensor based on ion-selective field effect transistors

A new type of an electrochemical sensor based on the use of ion-selective field effect transistors (ISFETs) and conjugates of horse radish peroxidase with specific monoclonal antibodies was developed for the express determination of myoglobine in a solution. For this purpose a simple method of covalent immobilization of myoglobine on the surface of ISFET gate was worked out, an appropriate biochemical approach which allowed potentiometrical registration of the peroxidase activity was used, and an immune chemical analysis was accomplished in competitive way. It was shown that the sensitivity of the analysis with the help of the electrochemical immune sensor corresponds to the demands of medical practice to reveal early stages of myocardial infarction. This sensitivity was significantly higher then that which can be obtained by the traditional ELISA-method. Moreover, overall time of the analysis by the immune sensor was almost one order shorter than this by the ELISA-method. It is concluded that the proposed electrochemical immune sensor based on the ISFETs was very perspective for the express analysis of the level of different types of antigens and antibodies.     

Simultaneous enzymatic/electrochemical determination of glucose and L-glutamine in hybridoma media by flow-injection analysis

A split-stream flow-injection analysis system is described for simultaneous determination of glucose and L-glutamine in serum-free hybridoma bioprocesses media. Amperometric measurement of glucose is based on anodic oxidation of hydrogen peroxide produced by immobilized glucose oxidase within a triple layer membrane of an integrated flow-through glucose-selective biosensor. Determination of L-glutamine is based on quantitating ammonium ions produced in a flow-through enzymes reactor containing immobilized glutaminase enzyme, and subsequent downstream potentiometric detection of these ions by a nonacting-based ion-selective polymer membrane electrode. Endogenous potassium and ammonium ion interference in the L-glutamine determination are eliminated by using a novel in-line tubular cation-exchange membrane unit to exchange these interferent species for cations undetectable by the membrane electrode. The first generation split-steam flow-injections system can assay 12 samples/h using direct injections of 50 muL of media samples, with linear responses to glucose in the range of 0.03 to 30mM, and log-linear response to L-glutamine from 0.1 to 10 mM. (c) 1993 Wiley & Sons, Inc.