An amperometric pyruvate biosensor based on pyruvate oxidase nanoparticles immobilized onto pencil graphite electrode

This present study was aimed to fabricate a sensitive and improved amperometric biosensor by the nanoparticles of pyruvate oxidase, which were prepared and immobilized covalently onto pencil graphite electrode. The biosensor showed ideal working within 5 s under defined conditions of pH 6.0 and incubation temperature of 30 °C at an applied voltage of -0.1 V. Under standard assay conditions, a linear response was obtained between pyruvate concentration ranging from 0.001 to 6000 μM and current (μA). A lower detection limit (0.58 μM) and an excellent correlation coefficient (R² = 0.999) with standard spectrophotometric assay was obtained for the present biosensor. Within and between batches of coefficients of variation were calculated and found to be 3.61 % and 3.33 %, respectively. The biosensor was put to continual use for over 210 days. The biosensor was employed for the measurement of pyruvate level in sera of normal healthy individuals and persons suffering from heart disease.     

Whole cell immobilized amperometric biosensor based on Saccharomyces cerevisiae for selective determination of vitamin B1 (thiamine)

A new amperometric whole cell biosensor based on Saccharomyces cerevisiae immobilized in gelatin was developed for selective determination of vitamin B1 (thiamine). The biosensor was constructed by using gelatin and crosslinking agent glutaraldehyde to immobilize S. cerevisiae cells on the Teflon membrane of dissolved oxygen (DO) probe used as the basic electrode system combined with a digital oxygen meter. The cells were induced by vitamin B1 in the culture medium, and the cells used it as a carbon source in the absence of glucose. So, when the vitamin B1 solution is injected into the whole cell biosensor system, an increase in respiration activity of the cells results from the metabolic activity and causes a decrease in the DO concentration of interval surface of DO probe related to vitamin B1 concentration. The response time of the biosensor is 3 min, and the optimal working conditions of the biosensor were carried out as pH 7.0, 50mM Tris-HCl, and 30 degrees C. A linear relationship was obtained between the DO concentration decrease and vitamin B1 concentration between 5.0 x 10(-3) and 10(-1) microM. In the application studies of the biosensor, sensitive determination of vitamin B1 in the vitamin tablets was investigated.     

A novel glucose biosensor based on the immobilization of glucose oxidase onto gold nanoparticles-modified Pb nanowires

A novel glucose biosensor was developed, based on the immobilization of glucose oxidase (GOD) with cross-linking in the matrix of bovine serum albumin (BSA) on a Pt electrode, which was modified with gold nanoparticles decorated Pb nanowires (GNPs-Pb NWs). Pb nanowires (Pb NWs) were synthesized by an l-cysteine-assisted self-assembly route, and then gold nanoparticles (GNPs) were attached onto the nanowire surface through –SH–Au specific interaction. The morphological characterization of GNPs-Pb NWs was examined by transmission electron microscopy (TEM). Cyclic voltammetry and chronoamperometry were used to study and to optimize the electrochemical performance of the resulting biosensor. The synergistic effect of Pb NWs and GNPs made the biosensor exhibit excellent electrocatalytic activity and good response performance to glucose. The effects of pH and applied potential on the amperometric response of the biosensor have been systemically studied. In pH 7.0, the biosensor showed the sensitivity of 135.5 μA mM⁻¹ cm⁻², the detection limit of 2 μM (S/N = 3), and the response time <5 s with a linear range of 5–2200 μM. Furthermore, the biosensor exhibits good reproducibility, long-term stability and relative good anti-interference.     

A biosensor based on the self-entrapment of glucose oxidase within biomimetic silica nanoparticles induced by a fusion enzyme

We constructed a fusion protein (GOx-R5) consisting of R5 (a polypeptide component of silaffin) and glucose oxidase (GOx) that was expressed in Pichia pastoris. Silaffin proteins are responsible for the formation of a silica-based cell matrix of diatoms, and synthetic variants of the R5 protein can perform silicification in vitro[1]. GOx secreted by P. pastoris was self-immobilized (biosilicification) in a pH 5 citric buffer using 0.1 M tetramethoxysilane as a silica source. This self-entrapment property of GOx-R5 was used to immobilize GOx on a graphite rod electrode. An electric cell designed as a biosensor was prepared to monitor the glucose concentrations. The electric cell consisted of an Ag/AgCl reference electrode, a platinum counter electrode, and a working electrode modified with poly(neutral red) (PNR)/GOx/Nafion. Glucose oxidase was immobilized by fused protein on poly(neutral red) and covered by Nafion to protect diffusion to the solution. The morphology of the resulting composite PNR/GOx/Nafion material was analyzed by scanning electron microscopy (SEM). This amperometric transducer was characterized electrochemically using cyclic voltammetry and amperometry in the presence of glucose. An image produced by scanning electron microscopy supported the formation of a PNR/GOx complex and the current was increased to 1.58 μA cm⁻¹ by adding 1 mM glucose at an applied potential of −0.5 V. The current was detected by way of PNR-reduced hydrogen peroxide, a product of the glucose oxidation by GOx. The detection limit was 0.67 mM (S/N = 3). The biosensor containing the graphite rod/PNR/GOx/Nafion detected glucose at various concentrations in mixed samples, which contained interfering molecules. In this study, we report the first expression of R5 fused to glucose oxidase in eukaryotic cells and demonstrate an application of self-entrapped GOx to a glucose biosensor.     

Effects of thiamine and pyridoxine on respiratory activity in Saccharomyces carlsbergensis strain 4228

Studies were made to elucidate the relationship among the thiamine-induced growth inhibition, decrease in cellular vitamin B₆ content and respiratory deficiency in Saccharomyces carlsbergensis strain 4228 [Nakamura et al., Biochem. Biophys. Res. Commun. 59, 771–776 (1974)]. Addition of pyridoxine to the thiamine-added culture at the beginning or in the course of cultivation brought about appearance of cytochrome spectra and the increase in the activity of heme-containing enzymes and in respiratory activity (Q _{O 2}). The effects of pyridoxine occurred prior to the restoration of growth. Pyridoxine was effective even in the presence of high levels of glucose in the growth medium (not less than 3%). On the basis of these results, the mechanism of the effects of thiamine and pyridoxine was discussed.     

Microglial activation is a major contributor to neurologic dysfunction in thiamine deficiency

In Wernicke’s encephalopathy and thiamine deficiency (TD), the cause of this brain disorder, development of inflammation is an important aspect of the disease process. How this pathological mechanism relates to the neurologic impairment associated with TD, however, remains unclear. A key feature of the inflammatory process is the activation of microglia. In the present study, we evaluated the role of microglial activation in the pathophysiology of TD by examining the relationship between levels of CD11b/c and CD68, two proteins associated with microglial activation, and neurological dysfunction under conditions of TD. Rats with TD showed large increases in expression of both CD11b/c and CD68 in the vulnerable thalamus and inferior colliculus, with no change in mRNA levels in the relatively non-vulnerable frontal cortex. These alterations in CD11b/c and CD68 expression were reflected in dramatic upregulation of both proteins by immunoblotting and immunohistochemical methods. Co-treatment of rats with TD and the anti-inflammatory drug minocycline prevented microglial activation, and onset of neurological changes, including loss of righting reflex, was delayed by approximately 39 h, compared to animals with TD alone. In addition, co-treatment of rats with TD and N-acetylcysteine prevented the increase in CD11b/c and CD68, but did not alter the onset of neurological impairment. These results suggest that microglial activation plays a role in the development of neurological impairment in TD and possibly Wernicke’s encephalopathy, and that while development of oxidative stress may be involved in microglial activation, the basis of this neurologic dysfunction is likely to be multifactorial in nature.     

Paraquat and menadione exposure of rainbow trout (Oncorhynchus mykiss)--studies of effects on the pentose-phosphate shunt and thiamine levels in liver and kidney

Possible xenobiotic interactions with thiamine were studied in salmonid fish, by repeatedly injecting two model substances, paraquat and menadione, into juvenile rainbow trout (Oncorhynchus mykiss). These two substances were chosen because of their well-known ability to redox-cycle and cause depletion of NADPH in several biological systems. Depletion of NADPH increases metabolism through the pentose-phosphate shunt and may thereby increase the need for thiamine diphosphate by heightened transketolase activity. A special food was produced with lower thiamine content than commercial food, usually enriched with thiamine, which could mask an effect on the thiamine level. After 9 weeks of exposure, glucose-6-phosphate dehydrogenase, transketolase, glutathione reductase and ethoxyresorufin O-deethylase were analysed in liver and kidney cellular sub-fractions as well as analysis of total thiamine concentrations in liver, kidney and muscle. The results showed that paraquat caused a large increase in hepatic glutathione reductase activity and induced hepatic glucose-6-phosphate dehydrogenase activity, i.e., the rate-limiting enzyme in the oxidative part of the pentose-phosphate shunt. Despite this paraquat exposure did not affect transketolase activity and total thiamine concentration.     

A biocatalyst for pyruvate preparation from -lactate: lactate oxidase in a Pseudomonas sp.

For the purpose of producing pyruvate from -lactate by enzymatic methods, four microorganism strains that produce lactate oxidase (LOD) were screened and isolated from many soil samples. Among them, strain SM-6, which showed high potential for pyruvate production, was chosen for further research. Physiological studies and 16S rDNA relationship reveal that SM-6 belongs to Pseudomonas putida. The optimized pH and temperature of the enzyme-catalyzed reaction were pH 7.2, and 39 °C, respectively. Low-concentration EDTA (1 mM) could improve the stability of pyruvate and conversion ratio of lactate oxidase. V_max and K_m value for -lactate were 2.46 μmol/(min mg) protein and 9.53 mM, respectively. On preparation scale, cell-free extract from SM-6, containing 300 mg/l of crude enzyme (4037 U/ml lactate oxidase), could convert 66% of 116 mM of -lactate into 76.6 mM pyruvate in 18 h, and 82% of substrate was transformed after 48 h, giving 95.0 mM (10.5 mg/ml) of pyruvate. The ratio of product to biocatalyst was 34.8:1 (g/g).     

A nanocomposite/crude extract enzyme-based xanthine biosensor

A novel amperometric biosensor for xanthine was developed based on covalent immobilization of crude xanthine oxidase (XOD) extracted from bovine milk onto a hybrid nanocomposite film via glutaraldehyde. Toward the preparation of the film, a stable colloids solution of core–shell Fe₃O₄/polyaniline nanoparticles (PANI/Fe₃O₄ NPs) was dispersed in solution containing chitosan (CHT) and H₂PtCl₆ and electrodeposited over the surface of a carbon paste electrode (CPE) in one step. Scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectrophotometry, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were used for characterization of the electrode surface. The developed biosensor (XOD/CHT/Pt NPs/PANI/Fe₃O₄/CPE) was employed for determination of xanthine based on amperometric detection of hydrogen peroxide (H₂O₂) reduction at –0.35 V (vs. Ag/AgCl). The biosensor exhibited a fast response time to xanthine within 8 s and a linear working concentration range from 0.2 to 36.0 μM (R² = 0.997) with a detection limit of 0.1 μM (signal/noise [S/N] = 3). The sensitivity of the biosensor was 13.58 μA μM⁻¹ cm⁻². The apparent Michaelis–Menten (K_m) value for xanthine was found to be 4.7 μM. The fabricated biosensor was successfully applied for measurement of fish and chicken meat freshness, which was in agreement with the standard method at the 95% confidence level.     

Amperometric glucose biosensor based on adsorption of glucose oxidase at platinum nanoparticle-modified carbon nanotube electrode

A new amperometric biosensor, based on adsorption of glucose oxidase (GOD) at the platinum nanoparticle-modified carbon nanotube (CNT) electrode, is presented in this article. CNTs were grown directly on the graphite substrate. The resulting GOD/Pt/CNT electrode was covered by a thin layer of Nafion to avoid the loss of GOD in determination and to improve the anti-interferent ability. The morphologies and electrochemical performance of the CNT, Pt/CNT, and Nafion/GOD/Pt/CNT electrodes have been investigated by scanning electron microscopy, cyclic voltammetry, and amperometric methods. The excellent electrocatalytic activity and special three-dimensional structure of the enzyme electrode result in good characteristics such as a large determination range (0.1-13.5mM), a short response time (within 5s), a large current density (1.176 mA cm(-2)), and high sensitivity (91mA M(-1)cm(-2)) and stability (73.5% remains after 22 days). In addition, effects of pH value, applied potential, electrode construction, and electroactive interferents on the amperometric response of the sensor were investigated and discussed. The reproducibility and applicability to whole blood analysis of the enzyme electrode were also evaluated.     

Resonance phenomenon of the ATP motor as an ultrasensitive biosensor

We designed a rotary biosensor as a damping effector, with the rotation of the F₀F₁-ATPase driven by Adenosine Triphosphate (ATP) synthesis being indicated by the fluorescence intensity and a damping effect force being induced by the binding of an RNA molecule to its probe on the rotary biosensor. We found that the damping effect could contribute to the resonance phenomenon and energy transfer process of our rotary biosensor in the liquid phase. This result indicates that the ability of the rotary motor to operate in the vibration harmonic mode depends on the environmental conditions and mechanism in that a few molecules of the rotary biosensor could induce all of the sensor molecules to fluoresce together. These findings contribute to the theory study of the ATPase motor and future development of biosensors for ultrasensitive detection.     

Statistical optimization of medium for the production of pyruvate oxidase by the recombinant Escherichia coli

Pyruvate oxidase (PyOD) is a very useful enzyme for clinical diagnostic applications and environmental monitor. Optimization of the fermentation medium for maximization of PyOD constitutively, production by Escherichia coli DH5α/pSMLPyOD was carried out. Response surface methodology (RSM) was used to optimize the medium constituents. A 2^6–2 fractional factorial design (first order model) was carried out to identify the significant effect of medium components towards PyOD production. Statistical analysis of results shows that yeast extract, ammonium sulfate and composite phosphate were significant factors on PyOD production. The optimized values of these three factors were obtained by RSM based on the result of a 2³ central composite rotatable design. Under these proposed optimized medium, the model predicted a PyOD activity of 610 U/L and via experimental rechecking the model, an activity of 670 U/L was attained.     

Crystallographic study on the interaction of L-lactate oxidase with pyruvate at 1.9 Angstrom resolution

L-Lactate oxidase (LOX) from Aerococcus viridans catalyzes the oxidation of L-lactate to pyruvate by the molecular oxygen and belongs to a large family of 2-hydroxy acid-dependent flavoenzymes. To investigate the interaction of LOX with pyruvate in structural details and understand the chemical mechanism of flavin-dependent L-lactate dehydrogenation, the LOX-pyruvate complex was crystallized and the crystal structure of the complex has been solved at a resolution of 1.90 Angstrom. One pyruvate molecule bound to the active site and located near N5 position of FMN for subunits, A, B, and D in the asymmetric unit, were identified. The pyruvate molecule is stabilized by the interaction of its carboxylate group with the side-chain atoms of Tyr40, Arg181, His265, and Arg268, and of its keto-oxygen atom with the side-chain atoms of Tyr146, Tyr215, and His265. The alpha-carbon of pyruvate is found to be 3.13 Angstrom from the N5 atom of FMN at an angle of 105.4 degrees from the flavin N5-N10 axis.     

Dipicolinato complexes of cobalt(II), copper(II) and zinc(II) with thiamine dications

Cobalt(II), copper(II) and zinc(II) dipicolinato complexes having thiamine dication with compositions [HT][CoL₂]·5H₂O (1), [HT][CuL₂]·5H₂O (2) and [HT][ZnL₂]·5H₂O (3) (L = dipicolinato anion, T = thiamine cation) are synthesized, characterized by X-ray diffraction and other spectroscopic techniques. The thiamine part in these complexes exists as divalent cations. The second protonation of thiamine takes place at the less crowded nitrogen atom of the pyrimidine ring. These complexes are stabilized by electrostatic and hydrogen-bonding interactions of -N⁺-H and -COO⁻ groups along with crystallized water molecules. The complexes are stable in solution as determined by ¹H NMR and visible study. The complex 1 has two medicinal components which can be easily separated by controlling pH in aqueous medium. It gets decomposed on treatment with sodium hydroxide at pH > 8 to form neutral complex {[Na₂(μ-H₂O)₃(H₂O)₃][CoL₂]·H₂O}₂.     

Amperometric tyrosinase biosensor based on Fe3O4 nanoparticles-chitosan nanocomposite

A novel tyrosinase biosensor based on Fe(3)O(4) nanoparticles-chitosan nanocomposite has been developed for the detection of phenolic compounds. The large surface area of Fe(3)O(4) nanoparticles and the porous morphology of chitosan led to a high loading of enzyme and the entrapped enzyme could retain its bioactivity. The tyrosinase-Fe(3)O(4) nanoparticle-chitosan bionanocomposite film was characterized with atomic force microscopy and AC impedance spectra. The prepared biosensor was used to determine phenolic compounds by amperometric detection of the biocatalytically liberated quinone at -0.2V vs. saturated calomel electrode (SCE). The different parameters, including working potential, pH of supporting electrolyte and temperature that governs the analytical performance of the biosensor have been studied in detail and optimized. The biosensor was applied to detect catechol with a linear range of 8.3 x 10(-8) to 7.0 x 10(-5)mol L(-1), and the detection limit of 2.5 x 10(-8)mol L(-1). The tyrosinase biosensor exhibits good repeatability and stability. Such new tyrosinase biosensor shows great promise for rapid, simple, and cost-effective analysis of phenolic contaminants in environmental samples. The proposed strategy can be extended for the development of other enzyme-based biosensors.     

Glucose biosensor based on the room-temperature phosphorescence of TiO2/SiO2 nanocomposite

The direct immobilization of glucose oxidase (GOD) on TiO₂/SiO₂ nanocomposite and its application as glucose biosensor were investigated. The room-temperature phosphorescence of TiO₂/SiO₂ nanocomposite can be quenched by hydrogen peroxide (H₂O₂). The detection of glucose may be accomplished by monitoring the formation of hydrogen peroxide which generated in the oxidation process of glucose with the catalysis of GOD. To our surprise, by using a 96-hole polyporous plate accessory of fluorescence spectrophotometer, the biosensor exhibits excellent linear response to glucose concentrations ranging from 1.0 × 10⁻⁹ to 1.0 × 10⁻² M with a detection limit of 1.2 × 10⁻¹⁰ M. The TiO₂/SiO₂ nanocomposite can be used as both supporting material and signal transducer. The phosphorescence intensity and color of the biosensor change obviously and even could be observed with naked eyes by continuous addition of glucose. Based on the room-temperature phosphorescence of TiO₂/SiO₂ nanocomposite, a new method of solid substrate-room-temperature phosphorimetry (SS-RTP) for glucose determination is proposed. A glucose biosensor was fabricated with wide determination concentration range, low detection limit, high sensitivity, and fast response time. And the biosensor has been successfully applied to the determination of glucose in human blood serum. The coacervation of GOD enzyme and its interaction with TiO₂/SiO₂ nanocomposite enlarge the surface area and enhance the chemical stability of GOD. The nice biocompatibility, large surface area, good chemical stability and nontoxicity of the TiO₂/SiO₂ nanocomposite have made this material suitable for functioning as biosensor.     

Application of vitamin B(12)-targeting site on Lactobacillus helveticus B-1 to vitamin B(12) assay by chemiluminescence method

Lactobacillus helveticus B-1 is assumed to have a vitamin B(12)-targeting (or B(12)-binding) site on the cells, since the binding reaction of vitamin B(12) with L. helveticus B-1 cells proceeded instantly and quantitatively. This reaction is specific to complete B(12) compounds, cobalamins, and can be used for a vitamin B(12) assay method by chemiluminescence. The calibration graph was linear from 0.1 to 10.0 ng/mL. The B(12) contents in oyster and sardine were 75.9 and 39.4 microg/100g, respectively. These values were very close to those obtained using a chemilumi-ADVIA Centaur immunoassay system with intrinsic factor and to those obtained by microbiological assays.     

Amperometric glucose biosensor based on multilayer films via layer-by-layer self-assembly of multi-wall carbon nanotubes, gold nanoparticles and glucose oxidase on the Pt electrode

A novel amperometric glucose biosensor based on the nine layers of multilayer films composed of multi-wall carbon nanotubes (MWCNTs), gold nanoparticles (GNp) and glucose oxidase (GOD) was developed for the specific detection of glucose. MWCNTs were chemically modified with the H₂SO₄–HNO₃ pretreatment to introduce carboxyl groups which were used to interact with the amino groups of poly(allylamine) (PAA) and cysteamine via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide cross-linking reaction, respectively. A cleaned Pt electrode was immersed in PAA, MWCNTs, cysteamine and GNp, respectively, followed by the adsorption of GOD, assembling the one layer of multilayer films on the surface of Pt electrode (GOD/GNp/MWCNTs/Pt electrode). Repeating the above process could assemble different layers of multilayer films on the Pt electrode. PBS washing was applied at the end of each assembly deposition for dissociating the weak adsorption. Film assembling and characterization were studied by transmission electron microscopy and quartz crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The marked electrocatalytic activity of Pt electrode based on multilayer films toward H₂O₂ produced during GOD enzymatic reactions with glucose permitted effective low-potential amperometric measurement of glucose. Taking the sensitivity and selectivity into consideration, the applied potential of 0.35 V versus Ag/AgCl was chosen for the oxidation detection of H₂O₂ in this work. Among the resulting glucose biosensors, the biosensor based on nine layers of multilayer films was best. It showed a wide linear range of 0.1–10 mM glucose, with a remarkable sensitivity of 2.527 μA/mM, a detection limit of 6.7 μM estimated at a signal-to-noise ratio of 3 and fast response time (within 7 s). Moreover, it exhibited good reproducibility, long-term stability and the negligible interferences of ascorbic acid, uric acid and acetaminophen. The study can provide a feasible approach on developing new kinds of oxidase-based amperometric biosensors, and can be used as an illustration for constructing various hybrid structures.     

A kinetic study on iron stimulation of the xanthine oxidase dependent oxidation of ascorbate

Xanthine oxidase reduces molecular oxygen to H₂O₂ and superoxide radicals during its catalytic action on xanthine, hypoxanthine or acetaldehyde. Ascorbate is catalytically oxidized by the superoxide radicals generated, when present in the reaction solution (Nishikimi 1975). The present study shows that iron ions markedly stimulate the enzyme dependent ascorbate oxidation, by acting as a red/ox-cycling intermediate between the oxidase and ascorbate. An apparent K_m-value of 10.8 M characterized the iron stimulatory effect on the reaction at pH 6.0. Reduced transition-state metals can be oxidized by H₂O₂ through a Fenton-type reaction. Catalase was found to reduce the effect of iron on the enzyme dependent ascorbate oxidation, strongly suggesting that H₂O₂, produced during catalysis, is involved in the oxidation of ferrous ions.     

微嗜酸寡养单胞菌中磷酸吡哆胺氧化酶基因pnpox生物学功能研究北大核心

【目的】研究微嗜酸寡养单胞菌(Stenotrophomonas acidaminiphila)CW117中磷酸吡哆胺氧化酶基因pnpox(phosphopyridoxamine oxidase,pnpox)在维生素B6(VB6)合成中的贡献及对黄曲霉毒素B1(aflatoxin B1,AFB1)的降解活性。【方法】采用基因插入突变方式,对菌株CW117中磷酸吡哆胺氧化酶基因pnpox进行突变,得到突变菌株。通过高效液相色谱法(high performance liquid chromatography,HPLC)检测突变株对AFB1的降解活性,以及突变株中吡哆醇和吡哆醛的合成情况,确定基因pnpox在寡养单胞菌体内VB6合成中的贡献和黄曲霉毒素降解代谢作用。【结果】成功构建了磷酸吡哆胺氧化酶基因突变子pnpox::pK19mobΩ2HMB,突变子吡哆醛的合成量较野生型菌株显著减少,吡哆醇合成量与野生型菌株无显著性差异;同时,突变子与野生型株CW117对AFB1的降解活性未发现显著性差异。【结论】菌株CW117中磷酸吡哆胺氧化酶在吡哆醛合成的过程中起着重要作用,该基因突变会导致VB6的严重缺乏,影响寡养单胞菌正常生长,但该基因对CW117降解黄曲霉毒素无显著性贡献。