Amperometric enzyme electrodes: Part II. Conducting salts as electrode materials for the oxidation of glucose oxidase

Results are reported for the direct oxidation of the enzyme glucose oxidase on 7 different one-dimensional conducting donor acceptor salts. Experiments conducted with the enzyme in bulk solution are shown to be in good agreement with theory. Three salts, made of the cations tetrathiafulvalinium (TTF⁺), N-methylphenazinium or quinolinium with the anion tetracyanoquinodimethanide (TCNQ^?) had the lowest background currents and were used to make membrane sensors for glucose. Analysis of the variation of current with glucose concentration identified the rate limiting processes as transport of gluycose through the membrane and electrochemical kinetics under unsaturated and saturated conditions respectively. The electrochemical rate constants for these three materials were all greater than 10^?2 cm s^?1. TTF⁺TCNQ^? is the material of choice and linear calibration plots were obtained for glucose concentrations between 50 μmol dm^?3 and 10 mmol dm^?3.     

Oxygen-independent glucose microsensor based on glucose oxidase

A glucose oxidase-based needle-type microsensor is described that is independent of the local oxygen concentration. The sensor consists of an enzyme-coated platinum microelectrode that is inserted in a glass capillary with a tip of 5–25 μm, and connected to the environment via an agar membrane. Oxygen is supplied to the enzyme coating from the shaft of the capillary. Depending on the electrode configuration, the sensor has a response time of 1–10 s, and the measuring range extends from 0.01 to 0.4 mmol l^?1 or from 0.4 to 10 mmol l^?1. The signal is not affected by stirring.     

Heavily fluorinated carbohydrates as enzyme substrates: oxidation of tetrafluorinated galactose by galactose oxidase

Galactose oxidase (GOase) was shown to oxidise several C2/C3 fluorinated galactose analogues. Interestingly, the enzyme was able to distinguish between the 2,3-tetrafluorinated galactose and its epimeric glucose analogue, and this represents the first reported biotransformation of a heavily fluorinated sugar.     

Laccase/glucose oxidase electrode for determination of glucose

The electrode involves a layer of co-immobilized glucose oxidase and laccase in a gelatin membrane placed over a modified oxygen electrode. Hexacyanoferrate(III) is added to the samples to oxidize reductive interferents such as ascorbic acid, and the hexacyanoferrate(II) formed is re-oxidized by a laccase-catalyzed reaction. Ascorbic acid is completely eliminated up to a concentration of 20 mM in the sample.     

Activated platinum electrodes as transducer for a glucose sensor using glucose oxidase in a photopolymer membrane

A planar platinum electrode was covered by a photopolymer membrane containing glucose oxidase (GOD) to construct an amperometric glucose sensor. The application of a photopolymer system in membrane formation gives the opportunity to manufacture cheap biosensors with good reproducibility by means of automated techniques and to miniaturise sensors using photolithography. The electrodes were pretreated mechanically and chemically resulting in a half-wave potential (E_1/2) of the H₂O₂ oxidation shifted towards more negative potentials. This shift allows the determination of glucose at a low working potential (300 mV vs. SCE) without addition of mediators. The important advantage of such applied potential decreasing lies in minimising the interference of oxidisable substances such as uric acid, bilirubin and paracetamol. The selectivity to ascorbic acid could also be proved without the application of additional protection layers. The glucose sensor developed has a high life-time, selectivity and sensitivity and a linear working concentration range from 0.05 up to 10 mmol/l of glucose. The sensor was used for the glucose determination in human serum samples with a very good correlation to a common photometric reference method. Received: 13 July 1996 / Revised: 11 September 1996 / Accepted: 14 September 1996     

Spherical polyelectrolyte brushes as bio-platforms to integrate platinum nanozyme and glucose oxidase for colorimetric detection of glucose

The integration of nanozyme and natural enzyme for cascade reactions has attracted great attention due to their huge potential applications in detection, biomedicine, and catalysis. Here the novel cascade bio-platforms were fabricated by using spherical poly[(2-methacryloyloxyethyl)trimethyl ammonium chloride] (PMOTA) brushes (SPB) as nanoreactors to prepare platinum nanoparticles in situ and as nanocarriers to immobilize glucose oxidase (GOX). The generated Pt nanoparticles possess high stability and peroxidase-like properties, which can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H₂O₂ to generate blue colored oxidized TMB (oxTMB). And the absorbed GOX can specifically catalyze the oxidation of glucose into gluconic acid and H₂O₂, while the produced H₂O₂ is subsequently catalyzed by the Pt nanozymes. Thus, the co-immobilized Pt nanozymes and GOX within SPB (SPB@Pt@GOX) acted as effective biosensors for colorimetric detection of glucose showing high selectivity and great feasibility. This work demonstrates a facile and general strategy to use SPB as bio-platforms to integrate nanozymes and natural enzymes for cascade reactions.     

New bioluminogenic substrates for monoamine oxidase assays

Novel bioluminogenic substrates were designed for probing monoamine oxidase (MAO) activity based on a simple and effective beta-elimination strategy. By modifying the amino group and the central core of luciferin derivatives, we have developed a series of substrates useful for assays of MAO A or B, or both. One of these substrates, exhibiting low Km values and high signal-to-background ratios with both isozymes, was shown to accurately measure the Ki values of known MAO inhibitors. This substrate is a key component in the development of a highly sensitive homogeneous MAO assay for high-throughput screening (HTS) of compounds in drug discovery and for monitoring MAO activity in complex biological systems. This design strategy should be applicable to fluorogenic MAO substrates and could broaden the structural requirements of substrates for other enzyme assays.     

Analytical use of immobilized glucose oxidase

With the aim of immobilizing glucose oxidase (GO) for routine determination of glucose, a covalent bond immobilization method on titanium (IV) chloride activated silica supports was used (1). Several parameters were studied in order to optimize the residual activity upon immobilization and during operation. The immobilized enzyme can be reutilized at 25°C for several h a day alternating with storage (4°C) for at least 3,300 h.     

Development of monolithic enzymatic reactors in glass microchips for the quantitative determination of enzyme substrates using the example of glucose determination via immobilized glucose oxidase

A one-step procedure for the immobilization of glucose oxidase in fused-silica capillaries and in glass microchips was developed based on enzyme entrapment in a polyacrylamide-based monolithic column. The inner capillary surface was silanized with gamma-methacryloxypropyltrimethoxysilane (gamma-MAPS) to allow covalent binding of the gel to the surface. The composition of the polymer was optimized to prevent the formation of bubbles, allow liquid transportation by electroosmotic flow and to maintain the enzymatic activity. These requirements resulted in the addition of polyethylene glycol and poly(acrylic acid) to the acrylamide mixture. The gel containing the enzyme was formed in situ in the capillaries, respectively, in one channel of the microchip. In the microchip, it was limited to the sample injection channel by accordingly controlled silanization of the inner capillary surface. Glucose was detected via the amperometric determination of hydrogen peroxide. A linear correlation between signals and glucose concentration was observed from 0.05 to 1.1 mM glucose with a correlation coefficient of 0.999. The enzymatic monolithic microreactor showed no loss of activity during 8 h of continuous use and during storage in the running buffer at 4 degrees C for about 2 months. Interferents, such as ascorbic acid, were separated from the analyte electrophoretically, so that glucose could be quantified in diluted juices.     

2-Methylpyridinium salts as 1,4-dinucleophiles. II. Westphal condensation with substituted pyridinium substrates

Condensation of α-methylpyridinium, quinolinium and isoquinolinium salts with 1,2-dicarbonyls in the presence of base, yielded quinolizinium derivatives. In an analogous process, α-benzyl derivatives produced 2,3-dihydroindolizin-2-ones by intramolecular cyclisation.     

Covalent attachment of glucose oxidase to an Au electrode modified with gold nanoparticles for use as glucose biosensor

A feasible method to fabricate glucose biosensor was developed by covalent attachment of glucose oxidase (GOx) to a gold nanoparticle monolayer modified Au electrode. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of ferrocyanide followed and confirmed the assemble process of biosensor, and indicated that the gold nanoparticles in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. CV performed in the presence of excess glucose and artificial redox mediator, ferrocenemethanol, allowed to quantify the surface concentration of electrically wired enzyme (Gamma(E)(0)) on the basis of kinetic models reported in literature. The Gamma(E)(0) on proposed electrode was high to 4.1 x 10(-12) mol.cm(-2), which was more than four times of that on electrode direct immobilization of enzyme by cystamine without intermediate layer of gold nanoparticles and 2.4 times of a saturated monolayer of GOx on electrode surface. The analytical performance of this biosensor was investigated by amperometry. The sensor provided a linear response to glucose over the concentration range of 2.0 x 10(-5)-5.7 x 10(-3) M with a sensitivity of 8.8 microA.mM(-1).cm(-2) and a detection limit of 8.2 microM. The apparent Michaelis-Menten constant (K(m)(app)) for the sensor was found to be 4.3 mM. In addition, the sensor has good reproducibility, and can remain stable over 30 days.     

Flavin-protein interaction in bound glucose oxidase

Glucose oxidase was bound to Sepharose, Sephadex, gelatin, and dextran, yielding immobilized soluble and insoluble derivatives of the enzyme. The soluble preparations possessed higher enzymic activity than the analogous insoluble ones. The reversible dissociation process of the bound enzyme into apoenzyme and flavin adenine dinucleotide (FAD) was studied with the soluble and insoluble glucose oxidase in relation to enzymic activity and conformational changes as measured by circular dichroism and fluorescence methods. Bound apoenzyme was found to be more stable than the apoenzyme obtained from the unmodified glucose oxidase. The binding constant of FAD in bound glucose oxidase (K_diss≈10^-8M) calculated from fluorescent studies was lower than that of FAD in the native enzyme (K_diss10^-10M). The circular dichroism measurements indicated that dextran-bound glucose oxidase has a conformation similar to that of the native enzyme.     

Electrodeposited glucose oxidase/anionic clay for glucose biosensors design

An amperometric glucose biosensor was developed using an anionic clay matrix (layered double hydroxide (LDH), Ni/Al-NO₃) for the immobilization of glucose oxidase (GOx). The biofilm was prepared by electrodeposition of the clay and GOx and subsequent cross-linking with glutaraldeyde. The Pt surface modified with the Ni/Al-NO₃ shows a much reduced noise, giving rise to a better signal to noise ratio for the currents relative to H₂O₂ oxidation, and a linear range for H₂O₂ determination wider than the one observed for bare Pt electrodes. Under the optimised operative conditions, the performances of the biosensor have been evaluated by measuring the steady-state currents (at +0.45 V versus SCE) to increasing concentrations of glucose in “air saturated” 0.1 M phosphate buffer (pH 7.0). Both batch and flow injection modes were explored. The response to glucose was linear up to 8.0 and 12.0 mM, and the sensitivities were 7.7 ± 0.1 and 19.1 ± 0.2 mA M⁻¹ cm⁻², respectively. The current response of the biosensors does not significantly change for 15 consecutive days in batch and for 10 days in flow, at least, if stored at 4 °C in phosphate buffer, when not in use. The effects of interferants and applicability to fruit juices and soft drinks analysis of the biosensor were also investigated.     

Reversible denaturation behavior of immobilized glucose oxidase

Glucose oxidase (GOD) was immobilized by using glutaraldehyde crosslinking and various stabilizing agents such as BSA, gelatin, lysozyme, and polyethylenimine (PEI). Studies on the denaturation of the soluble as well as immobilized GOD were carried out for 1 h at various concentrations of guanidine hydrochloride (GdmCl) in 50 mM phosphate buffer, pH 6.0 at 25±1°C. The soluble enzyme required a GdmCl concentration of 5M for total activity loss, whereas for GOD immobilized with BSA, gelatin, lysozyme, and heat-inactivated lysozyme, the corresponding GdmCl concentration required was 8 M. GOD immobilized with PEI, however, was more stable and retained 25% activity when denatured for 1 h using 8 M GdmCl. However, after undergoing denaturation for 1 h, GOD immobilized with lysozyme regained 72% original activity within 20 min of renaturation, while GOD immobilized with BSA, PEI, gelatin, and heat-inactivated lysozyme regained only 39, 21, 20, and 25% of activity, respectively. After five cycles of repeated denaturation and renaturation with 8 M GdmCl, GOD immobilized with lysozyme retained 70% of the original activity. Refolding ability of lysozyme, glutaraldehyde crosslinkages between lysozyme and GOD, together with ionic interactions between them, appear to play an important role in the denaturation-renaturation behavior of the immobilized enzyme.     

Microfabricated glucose biosensor with glucose oxidase entrapped in sol-gel matrix

A microfabricated glucose biosensor based on an amperometeric hydrogen peroxide electrode has been developed. A sol-gel layer with 5 A pore size and 2 mum thickness was used as the glucose oxidase entrapping matrix. The sol-gel matrix formed over the silicon-based sensor has good mechanical and chemical stability, and the ability to entrap a large amount of enzyme. The miniaturized electrode sensing system is composed of platinum as both working and counter electrodes and silver as a reference electrode. Nafion(R) coating was applied as the interference limiting layer. A series of technologies, such as standard photolithography, electron beam evaporation and image reverse lift-off were utilized for mass production allowing 143 electrodes to be produced at the same time. The effect of oxidable interferences was <10% of the background value of the sensor response. Calibration tests of a series of individual sensors manufactured from the same silicon wafer and dip coated in the same conditions, showed a highly reproducible response characteristics (linear range up to 500 mg dl(-1) and mean sensitivity of 0.54+/-0.14 nA mg(-1) dl(-1) (n=10)).