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1.
An amperometric inhibitor biosensor for the determination of reduced glutathione (GSH) without any derivatization in some plants 总被引:8,自引:0,他引:8
In this study, an amperometric biosensor based on cucumber tissue homogenate was developed for the determination of glutathione. Cucumber (Cucumis sativus L.) tissue homogenate was used as the biological material. The cucumber tissue homogenate was cross-linked with gelatine using glutaraldehyde and fixed on a pretreated teflon membrane. The principle of the measurements was based on the determination of the decrease in the differentiation of oxygen level which had been caused by the inhibition of ascorbate oxidase in the biological material by glutathione. Determinations were carried out by standard curves which were obtained by the measurement of the decrease in the consumed oxygen level related to glutathione concentration. Optimization and characterization studies of the biosensor were carried out and a linearity in the gamma-L-glutamyl-L-cysteinyl-glycine (GSH) concentration range 0.1-2 microM was obtained when 600 microM ascorbic acid was used as a substrate. The repeatability experiments (n = 7) revealed that for 1.5 microM GSH, the average value (x), standard deviation (S.D.) and variation coefficient (C.V.) were 1.517 microM, 4.72 x 10(-5) 3.11%, respectively. The biosensor useful lifetime was at least 2 months. The results of some plant samples analyzed with the presented biosensor agreed well with the spectrophotometric method (Ellman's reagent) used as a reference. 相似文献
2.
A new amperometric microbial biosensor based on Saccharomyces cerevisiae NRRL-12632 cells, which had been induced for lysine oxidase enzyme and immobilized in gelatin by a cross-linking agent was developed for the sensitive determination of L-lysine amino acid. To construct the microbial biosensor S. cerevisiae cells were activated and cultured in a suitable culture medium. By using gelatine (8.43 mg cm(-2)) and glutaraldehyde (0.25%), cells obtained in the logarithmic phase of the growth curve at the end of a 14 h period were immobilized and fixed on a pretreated oxygen sensitive Teflon membrane of a dissolved oxygen probe. The assay procedure of the microbial biosensor is based on the determination of the differences of the respiration activity of the cells on the oxygenmeter in the absence and the presence of L-lysine. According to the end point measurement technique used in the experiments it was determined that the microbial biosensor response depended linearly on L-lysine concentrations between 1.0 and 10.0 microM with a 1 min response time. In optimization studies of the microbial biosensor, the most suitable microorganism quantities were found to be 0.97x10(5)CFU cm(-2). In addition phosphate buffer (pH 7.5; 50 mM) and 30 degrees C were obtained as the optimum working conditions. In characterization studies of the microbial biosensor some parameters such as substrate specificity, interference effects of some substances on the microbial biosensor responses, reproducibility of the biosensor and operational and storage stability were investigated. 相似文献
3.
An amperometric multienzyme biosensor for determination of triglycerides (TGs) was constructed by mounting three gelatin membrane-bound enzymes on a glassy carbon electrode (working electrode), then connecting it to electrometer along with an Ag/AgCl reference electrode and a Pt auxiliary electrode. Characterization and optimization of the multienzyme biosensor, which is prepared with glycerol kinase (GK) (E.C.2.7.1.30), glycerol-3-phosphate oxidase (GPO) (EC 1.1.3.21), and lipase (EC 3.1.1.3), were studied. In the optimization studies for the bioactive layer components of the prepared biosensor, the optimum amounts of gelatin, bovine serum albumin (BSA), and glutaraldehyde was calculated as 1 mg/cm2, 1 mg/cm2, and 2.5%, respectively. Optimum pH and temperature of the reaction of biosensor were determined as 7.0 and 40°C, respectively. Linear range of triolein for the biosensor was found from the calibration curve between several substrate concentration and Δ Current. After optimization and characterization of the biosensor, its operationability in triglycerides was also tested. 相似文献
4.
A biosensor based on pyruvate oxidase (POX) enzyme was developed for the investigation of the effect of thiamine (vitamin B(1)) molecule on the activity of the enzyme. The biosensor was prepared with a chemical covalent immobilization method on the dissolved oxygen (DO) probe by using gelatin and cross-linking agent, glutaraldehyde. POX catalyzes the degradation of pyruvate to acetylphosphate, CO(2) and H(2)O(2) in the presence of phosphate and oxygen. Thiamine is an activator for POX enzyme and determination method of the biosensor was based on this effect of thiamine on the activity of the enzyme. The biosensor responses showed increases in the presence of thiamine. Increases in the biosensor responses were related to thiamine concentration. Thiamine determination is based on the assay of the differences on the biosensor responses on the oxygenmeter in the absence and the presence of thiamine. The biosensor response depend linearly on thiamine concentration between 0.025 and 0.5 microM with 2 min response time. In the optimization studies of the biosensor the most suitable enzyme amount was found as 2.5 U cm(-2) and also phosphate buffer (pH 7.0; 50 mM) and 35 degrees C were obtained as the optimum working conditions. In the characterization studies of the biosensor some parameters such as activator and interference effects of some substances on the biosensor response and reproducibility were carried out. 相似文献
5.
Application of a biosensor for monitoring of ethanol 总被引:4,自引:0,他引:4
An alcohol biosensor for the measurement of ethanol has been developed. It comprises an alcohol oxidase/chitosan immobilized eggshell membrane and a commercial oxygen sensor. Ethanol determination is based on the depletion of dissolved oxygen content upon exposure to ethanol solution. The decrease in oxygen level was monitored and related to the ethanol concentration. The biosensor response depends linearly on ethanol concentration between 60 microM and 0.80 mM with a detection limit of 30 microM (S/N=3) and 1 min response time. In the optimization studies of the enzyme biosensor the most suitable enzyme and chitosan amounts were found to be 1.0 mg and 0.30% (w/v), respectively. The phosphate buffer (pH 7.4, 25 mM) and room temperature (20-25 degrees C) were chosen as the optimum working conditions. In the characterization studies of the ethanol biosensor some parameters such as interference effects, operational and storage stability were studied in detail. The biosensor was also tested with various wine samples. The results of this newly developed biosensor were comparable to the results obtained by a gas chromatographic method. 相似文献
6.
Polyaniline based catalase biosensor for the detection of hydrogen peroxide and azide 总被引:1,自引:0,他引:1
Ravindra P. Singh Da-Yeon Kang Byung-Keun Oh Jeong-Woo Choi 《Biotechnology and Bioprocess Engineering》2009,14(4):443-449
The CAT/PANi/ITO bioelectrode has been prepared as a catalase biosensor and shows response for monitoring not only of H2O2 but also azide. The sensor exhibited an excellent response to the H2O2 and azide. The linear range of H2O2 was 0.064∼1 mM and for azide 0.125∼4 mM, respectively. Catalase biosensor was based on the principle of the measurements
as the decrease in the differentiation of oxygen level, which has been caused by the inhibition of catalase in the bioactive
layer of the biosensor by azide. The repeatability experiments were done in triplicate. The logarithm response of the biosensor
to H2O2 (r2 = 0.99), as well as, for azide (r2 = 0.90) were reported, respectively. The bioelectrode was characterized by CV and AFM. The proposed biosensor would be applied
for the determination of H2O2 and azide in various biological samples. 相似文献
7.
Sagiroglu A Paluzar H Ozcan HM Okten S Sen B 《Preparative biochemistry & biotechnology》2011,41(4):321-336
Different branches of industry need to use phenolic compounds (PCs) in their production, so determination of PCs sensitively, accurately, rapidly, and economically is very important. For the sensitive determination of PCs, some biosensors based on pure polyphenol oxidase, plant tissue and microorganisms were developed before. But there has been no study to develop a microbial phenolic compounds biosensor based on Lactobacillus species, which contain polyphenol oxidase enzyme. In this study, we used different forms of Lactobacillus species as enzyme sources of biosensor and compared biosensor performances of these forms for determination of PCs. For this purpose, we used lyophilized Lactobacillus cells (containing L. bulgaricus, L. acidophilus, Streptococcus thermophilus), pure L. acidophilus, pure L. bulgaricus, and L. acidophilus- and L. bulgaricus adapted to catechol in Lactobacilli MRS Broth. The most suitable form was determined and optimization studies of the biosensor were carried out by using this form. For preparing the bioactive layer of the biosensor, the Lactobacillus cells were immobilized in gelatin by using glutaraldehyde. In the study, we used catechol as a substrate. Phenolic compound determination is based on the assay of the differences on the respiration activity of the cells on the oxygen meter in the absence and the presence of catechol. The microbial biosensor response depends directly on catechol concentration between 0.5 and 5.0 mM with 18 min response time. In the optimization studies of the microbial biosensor the most suitable microorganism amount was found to be 10 mg, and also phosphate buffer (pH 8.0; 50 mM) and 37.5 °C were obtained as the optimum working conditions. In the characterization studies of the microbial biosensor some parameters such as substrate specificity on the biosensor response and operational and storage stability were examine. Furthermore, the determination of PC levels in synthetic wastewater, industrial wastewater, and milk products was investigated by using the developed biosensor under optimum conditions. 相似文献
8.
A biosensor based on mushroom tissue homogenate for detecting some phenolic compounds (PCs) and usage of the biosensor for quantifying certain substances that inhibit the polyphenol oxidase activity in mushroom (Agaricus bisporus) tissue homogenate is described. The mushroom tissue homogenate was immobilized to the top of a Clark-type oxygen electrode with gelatin and glutaraldehyde. Optimization of the experimental parameters was done by buffer system, pH, buffer concentration, and temperature. Besides, the detection range of eight phenolic compounds were obtained with the help of the calibration graphs. Thermal stability, storage stability, and repeatability of the biosensor were also investigated. A linear response was observed from 20 x 10(-3) to 200 x 10(-3) mM phenol. The biosensor retained approximately 74% of its original activity after 25 days of storage at 4 degrees C. In repeatability studies, variation coefficient (C.V.) and standard deviation (S.D.) were calculated as 2.44% and +/-0.002, respectively. Inhibition studies revealed that the proposed biosensor was applicable for monitoring benzoic acid and thiourea in soft drinks and fruit juices. 相似文献
9.
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. 相似文献
10.
Different branchs of industry need to use ethanol in their production and some progress and not only the industry also to determine ethanol sensitively, accurately, fast and economical is very important. For the sensitive determination of ethanol a new amperometric biosensor based on Candida tropicalis cells, which contains alcohol oxidase enzyme, immobilized in gelatin by using glutaraldehyde was developed. In the study, before the microbial biosensor construction C. tropicalis cells were activated and cultured in a culture medium. By using gelatine and glutaraldehyde (0.1%) C. tropicalis cells obtained in logarithmic phase were immobilized and fixed on a pretreated teflon membrane of a dissolved oxygen probe. Ethanol determination is based on the assay of the differences on the respiration activity of the cells on the oxygenmeter in the absence and the presence of ethanol. The microbial biosensor response was depend linearly on ethanol concentration between 0.5 and 7.5 mM with 2 min response time. In the optimization studies of the microbial biosensor the most suitable microorganism amount were found as 4.42 mg cm(-2) and also phosphate buffer (pH:7.5; 50 mM) and 30 degrees C were obtained as the optimum working conditions. In the characterization studies of the microbial biosensor some parameters such as substrate specificity, interference effects of some substances on the biosensor response, operational and storage stability were carried out. 相似文献
11.
beta-Galactosidase is an hydrolase enzyme that catalyzes the hydrolysis of beta-galactosides into monosaccharides. Substrates of different beta-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins. A novel aspect of the activity determination of beta-galactosidase was presented. A glucose oxidase biosensor based on Clark electrode was utilized in order to monitor beta-galactosidase. Immobilization of glucose oxidase was made by gelatin and glutaraldehyde as cross-linker. Several parameters such as glucose oxidase activity, gelatin amount, and glutaraldehyde percentage for cross-linking were optimized. The most important parameter, lactose concentration in working buffer was studied in detail. Optimum temperature, thermal stability, optimum pH, buffer system and its concentration effect on the biosensor system, repeatability, reproducibility, and storage and operational stabilities of the biosensor were identified. A linear detection range for beta-galactosidase was observed between 9.4 x 10(-5) and 3.2 x 10(-2)U/ml. Finally, beta-galactosidase activity in artificial intestinal juice was investigated by the biosensor and the results obtained were compared with a reference spectrophotometric method. 相似文献
12.
Electrochemically fabricated polypyrrole nanofiber-modified electrode as a new electrochemical DNA biosensor 总被引:4,自引:0,他引:4
A new biosensor employing immobilized DNA on a nano-structured conductive polymer fixed onto a platinum electrode is presented. Upon optimization of synthesis parameters, polypyrrole nanofibers, 30-90 nm in diameter, were synthesized in an aqueous media by the electropolymerization of pyrrole using normal pulse voltammetry (NPV). The nanofiber film was investigated by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Double-stranded DNA was physisorbed onto the PPy nanofiber films. Various parameters, including the pH and DNA concentration, were optimized. The DNA immobilized on the nanofiber films was characterized using differential pulse voltammetry (DPV) and Fourier-transform infrared (FTIR) spectroscopy. Using DPV to study the interaction of spermidine with DNA, a binding constant (K) value of 4.08 x 10(5)+/-0.05 M(-1) was obtained. For the determination of spermidine, the proposed method exhibited a good dynamic range, correlation coefficient (0.05-1.0 microM and 0.9983, respectively) and a low detection limit (0.02 microM), although Ca(2+) ions were found to electrostatically bind to DNA and weaken the spermidine-DNA interaction. 相似文献
13.
Erol Akyilmaz Gizem Oyman Ezgi Cınar Gorkem Odabas 《Preparative biochemistry & biotechnology》2017,47(1):86-93
A new biosensor based on catalase enzyme immobilized on electrochemically constructed polyaniline (PANI) film modified with glutaraldehyde has been developed for the determination of hydrogen peroxide (H2O2) in milk samples. Assembly processes of polyaniline and immobilization of the enzyme were monitored with the help of electrochemical impedance spectroscopy. Amperometric measurements have been performed at cathodic peak (?0.3?V vs. Ag/AgCI) which was attributed to reduction of PANI. Hydrogen peroxide was determined by using amperometric method at ?0.3?V. The biosensor responses were correlated linearly with the hydrogen peroxide concentrations between 5.0?×?10?6 and 1.0?×?10?4?M by amperometric method. Detection limit of the biosensor is 2.18?×?10?6?M for H2O2. In the optimization studies of the biosensor, some parameters such as optimum pH, temperature, concentration of aniline, amount of enzyme, and number of scans during electropolymerization were investigated. 相似文献
14.
Basu AK Chattopadhyay P Choudhury UR Chakraborty R 《Indian journal of experimental biology》2005,43(7):646-653
A hypoxanthine (Hx) biosensor based on immobilized xanthine oxidase (XO) as the bio-component was developed and studied for the rapid analysis of fish (sweet water and marine) and goat meat samples. The biosensor was standardized for the determination of Hx in the range of 0.05 to 2 mM. Crosslinking with glutaraldehyde in presence of BSA as a spacer molecule was used for the method of immobilization. One layer of gelatin (10%) was applied over the immobilized enzyme layer to reduce the leaching out of enzyme from the membrane (cellulose acetate) matrix. The optimum pH of the immobilized system was determined to be 8.5 at 25 degrees C instead 7.0-7.2 for free enzyme system. Km and Vmax values were determined for the immobilized system. The developed sensor was applied to determine the amount of Hx present in fish and meat over a period of time. The stability of the enzyme immobilized membrane was also tested over a period of 30 days. 相似文献
15.
Shumyantseva V Deluca G Bulko T Carrara S Nicolini C Usanov SA Archakov A 《Biosensors & bioelectronics》2004,19(9):971-976
A screen-printed enzyme electrode based on flavocytochrome P450scc (RfP450scc) for amperometric determination of cholesterol has been developed. A one-step method for RfP450scc immobilization in the presence of glutaraldehyde or by entrapment of enzyme within a hydrogel of agarose is discussed. The sensitivity of the biosensor based on immobilization procedures of flavocytochrome P450scc by glutaric aldehyde is 13.8 nA microM(-1) and the detection limit is 300 microM with a coefficient of linearity 0.98 for cholesterol in the presence of sodium cholate as detergent. The detection limits and the sensitivity of the agarose-based electrode are 155 microM and 6.9 nA microM(-1) with a linearity coefficient of 0.99. For both types of electrodes, the amperometric response to cholesterol in the presence of detergent was rather quick (1.5-2 min). 相似文献
16.
《Process Biochemistry》2014,49(9):1393-1401
In this study, a microbial biosensor for hydrogen sulfide (H2S) detection based on Thiobacillus thioparus immobilized in a gelatin matrix was developed. The T. thioparus was immobilized via either surface adsorption on the gelatin matrix or entrapment in the matrix. The bacterial and gelatin concentration in the support were then varied in order to optimize the sensor response time and detection limit for both methods. The optimization was conducted by a statistical analysis of the measured biosensor response with various bacterial and polymer concentrations. According to our experiments with both immobilization methods, the optimized conditions for the entrapment method were found to be a gelatin concentration of 1% and an optical density of 82. For the surface adsorption method, 0.6% gelatin and an optical density of 88 were selected as the optimal conditions. A statistical model was developed based on the extent of the biosensor response in both methods of immobilization. The maximum change in the potential of the solution was 23.16 mV for the entrapment method and 34.34 mV for the surface absorption method. The response times for the entrapment and adsorption methods were 160 s and 105 s, respectively. The adsorption method is more advantageous for the development of a gas biosensor due to its shorter response time. 相似文献
17.
Wutipraditkul N Boonkomrat S Buaboocha T 《Bioscience, biotechnology, and biochemistry》2011,75(10):1900-1906
Catalase is the major H(2)O(2)-scavenging enzyme in all aerobic organisms. From the cDNA sequences of three rice (Oryza sativa L.) genes that encode for predicted catalases (OsCatA, OsCatB, and OsCatC), complete ORFs were subcloned into pET21a and expressed as (His)(6)-tagged proteins in Escherichia coli. The recombinant (His)(6)-polypeptides were enriched to apparent homogeneity and characterized. With H(2)O(2) as substrate, the highest catalase k(cat) value (20±1.71×10(-3) min(-1)) was found in recombinant OsCatB. The optimum temperatures for catalase activity were 30 °C for OsCatA and OsCatC and 25 °C for OsCatB, while the pH optima were 8.0, 7.5, and 7.0 for OsCatA, OsCatB, and OsCatC respectively. All the catalases were inhibited by sodium azide, β-mercaptoethanol, and potassium cyanide, but only weakly by 3-amino-1,2,4-triazole. The various catalases exhibited different catalase activities in the presence of different salts at different concentrations, OsCatC showing higher salt inhibitory effects than the two other OsCats. 相似文献
18.
Zhang Z Xia S Leonard D Jaffrezic-Renault N Zhang J Bessueille F Goepfert Y Wang X Chen L Zhu Z Zhao J Almeida MG Silveira CM 《Biosensors & bioelectronics》2009,24(6):1574-1579
A conductometric biosensor for nitrite detection was developed using cytochrome c nitrite reductase (ccNiR) extracted from Desulfovibrio desulfuricans ATCC 27774 cells immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin, methyl viologen (MV), Nafion, and glycerol. The configuration parameters for this biosensor, including the enzyme concentration, ccNiR/BSA ratio, MV concentration, and Nafion concentration, were optimized. Various experimental parameters, such as sodium dithionite added, working buffer solution, and temperature, were investigated with regard to their effect on the conductance response of the biosensor to nitrite. Under the optimum conditions at room temperature (about 25 degrees C), the conductometric biosensor showed a fast response to nitrite (about 10s) with a linear range of 0.2-120 microM, a sensitivity of 0.194 microS/microM [NO(2)(-)], and a detection limit of 0.05 microM. The biosensor also showed satisfactory reproducibility (relative standard deviation of 6%, n=5). The apparent Michaelis-Menten constant (K(M,app)) was 338 microM. When stored in potassium phosphate buffer (100mM, pH 7.6) at 4 degrees C, the biosensor showed good stability over 1 month. No obvious interference from other ionic species familiar in natural waters was detected. The application experiments show that the biosensor is suitable for use in real water samples. 相似文献
19.
In this work, a multiwalled carbon nanotube (MWCNT)–Nafion–cysteamine (CA) modified tyrosinase biosensor brings a new and original perspective to biosensor technology intended for the development of dopamine determination. Dopamine measurements were done at 0.2 V with the amperometric method by the developed biosensor system. In addition, in this study dopamine determination was carried out by using the differential pulse voltammetry method between potentials of 0.4 and −0.15 V. In the optimization studies of the biosensor, some parameters such as optimal pH, optimal temperature, optimal enzyme amount, and effect of MWCNT concentration were investigated. Afterward, in the characterization studies, some parameters such as linearity and reproducibility were determined. In the reproducibility experiment, an average value of 1.026 μM, a standard deviation of ±0.03975, and a coefficient of variation of 3.8% were determined for a 1-μM dopamine concentration (n = 15). Determination of dopamine was carried out in drug samples by the developed biosensor. 相似文献
20.
《Biocatalysis and Biotransformation》2013,31(4):130-140
AbstractA biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; an ion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensor response and showed that 30 μL of cell-free extract containing 7.47 mg protein mL?1, 2 μL of glutaraldehyde (5%, v/v) and 10 μL of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation of membranes in urea. The biosensor exhibited a linear response in the range of 4.0–10.0 μM urea, a detection limit of 2.0 μM for urea, a response time of 20 s, a sensitivity of 58.245 % per μM urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition. 相似文献