A specific enzyme electrode for l-glutamate-development and application

A biosensor for the specific determination of l-glutamate has been developed using l-glutamate oxidase in combination with a hydrogen peroxide indicating electrode. The biosensor response depends linearly on l-glutamate concentration between 0.001 and 1.0 mM. The measuring time is 2 min. The sensor is stable for more than 10 days during which more than 500 assays can be performed. The sensor has been applied to l-glutamate determination in liquid seasonings. Furthermore, transaminase activities have been determined by their catalytic l-glutamate production from alpha-ketoglutarate and l-alanine or l-aspartate. Also, the coimmobilization of glutaminase yielded a bienzyme electrode sensitive to l-glutamine.     

Electrochemical determination of gamma-glutamyl transpeptidase activity and its application to a miniaturized analysis system

A novel method to determine the activity of gamma-glutamyl transpeptidase (gamma-GTP) was developed. gamma-l-glutamyl-l-glutamate and glycyl-glycine were used as the substrates for gamma-GTP. l-glutamate produced by the enzymatic reaction was measured with an amperometric l-glutamate sensor. Following the mixing of the substrate solution and a sample solution, the current generated on the l-glutamate sensor continued to increase at a constant rate. The method was used to construct a miniaturized analysis system for the determination of gamma-GTP activity. The system consisted of the l-glutamate sensor formed on a glass substrate and a polydimethylsiloxane (PDMS) flow channel. Since the l-glutamate concentration in the solution increased as the solution was mobilized through the flow channel, a constant current increase was observed. The relation between the slope of the response curve and the activity of gamma-GTP was linear between 35 U l(-1) and 659 U l(-1). The rate analysis in the micro flow channel minimized the influence of interferents. The reproducibility of the output of the micro system was found to be good with a relative standard deviation (R.S.D.) of 5.6% at 659 U l(-1). The activities of gamma-GTP in human serum samples were also determined and compared with values obtained with a conventional spectroscopic method. The values obtained by the two methods were consistent with a correlation coefficient of 0.953.     

Development of an ultra high sensitive tissue biosensor for determination of swellfish poisoning, tetrodotoxin

A simple tissue biosensor for measuring Na⁺ channel blockers such as tetrodotoxin (TTX) and saxitoxin (STX) has been developed. The membrane of frog bladder has Na⁺ channels which control the passage of Na⁺. It is well known that TTX blocks Na⁺ channels. The tissue biosensor consists of a Na⁺ electrode integrated within a flow cell. The tip of the electrode was covered with frog bladder membrane sandwiched between two sheets of cellulose acetate membrane, and the electrode was set in a flow cell.

A solution of 8% NaCl was carried in the cell and the output of the electrode allowed to stabilize. TTX was injected into the sensor system and measured from the inhibition ratio of the sensor peak output. One assay took approximately 5 min. The lower limit of detection was 86 fg. The continuous determination of TTX was feasible for 250 h in the presence of 0·003% NaN₃. A Linear correlation was obtained between TTX activities of F-niphobles and F-parudale determined by the methods of TTX sensor and mouse assay.     

Amperometric biosensor for determining human salivary phosphate

An amperometric biosensor was constructed for analysis of human salivary phosphate without sample pretreatment. The biosensor was constructed by immobilizing pyruvate oxidase (PyOD) on a screen-printed electrode. The presence of phosphate in the sample causes the enzymatic generation of hydrogen peroxide (H(2)O(2)), which was monitored by a potentiostat and was in proportion to the concentration of human salivary phosphate. The sensor shows response within 2s after the addition of standard solution or sample and has a short recovery time (2 min). The time required for one measurement using this phosphate biosensor was 4 min, which was faster than the time required using a commercial phosphate testing kit (10 min). The sensor has a linear range from 7.5 to 625 microM phosphate with a detection limit of 3.6 microM. A total of 50 salivary samples were collected for the determination of phosphate. A good level of agreement (R(2)=0.9646) was found between a commercial phosphate testing kit and the phosphate sensor. This sensor maintained a high working stability (>85%) after 12h operation and required only a simple operation procedure. The amperometric biosensor using PyOD is a simple and accurate tool for rapid determinations of human salivary phosphate, and it explores the application of biosensors in oral and dental research and diagnosis.     

DEVELOPMENT OF A NEW BIOSENSOR FOR DETERMINATION OF CATALASE ACTIVITY

Catalase is one of the major antioxidant enzymes that catalyzes the hydrolysis of H₂O₂. The aim of this study was to suggest a new method for the assay of catalase activity. For this purpose, an amperometric biosensor based on glucose oxidase for determination of catalase activity was developed. Immobilization of glucose oxidase was made by a cross-linking method with glutaraldehyde on a Clark-type electrode (dissolved oxygen probe). Optimization and characterization properties of the biosensor were studied and determination of catalase activity in defined conditions was investigated in artificial serum solution. The results were compared with a reference method.     

Immobilization of glucose oxidase on thin-film gold electrodes produced by magnetron sputtering and their application in an electrochemical biosensor

An electrochemical biosensor is described consisting of a thin-layer gold film electrode prepared by cathodic sputtering using a poly(vinyl chloride) sheet as substrate, with voltammetric behaviour comparable to that of conventional polycrystalline gold electrodes, coated with the hydrolysed copolymer hydroxyethyl methacrylate-co-methyl methacrylate onto which glucose oxidase was immobilized. The mechanical properties of the plastic foil substrate permit easy construction of circular-shaped electrodes which were employed as working electrodes for batch injection analysis. The electrochemical biosensor fabrication is inexpensive and can be used as disposable enzyme sensor for the detection of hydrogen peroxide. The biosensor was tested for the determination of glucose in serum and a good correlation was obtained with the measurement using the electrochemical and the spectrophotometric methods.     

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.     

The use of differential measurements with a glucose biosensor for interference compensation during glucose determinations by flow injection analysis

A novel detection system for the determination of glucose in the presence of clinically important interferents, based on the use of dual sensors and flow-injection analysis (FIA), is described. The normalisation methodology involves measurement of the interference signal at a reference sensor; this signal can then be subtracted from the glucose sensor signal (post-run) to give a corrected measurement of the glucose concentration. The detection system consists of a thin layer cell with dual glassy carbon working electrodes. One electrode was surface modified to act asglucose biosensor by immobilisation of glucose oxidase (GOx) (from Aspergillus niger) with 1% glutaraldehyde and bovine serum albumin. The second electrode (glucose oxidase omitted) was utilised to measure the interference signal responding only to electroactive species present in the injected sample. A computer controlled multichannel potentiostat was used for potential application and current monitoring duties. The sensor responses were saved in ASCII format to facilitate post-run analysis in Microsoft Excel. Cyclic voltammetry (CV) was utilised to investigate the manner in which the interference signal contributed to the total signal obtained at the biosensor in the presence of glucose. The kinetic parameters I_max and the apparent Michaelis-Menten constant, K′_m, were calculated for the sensor operating under flow-injection conditions.     

Electrochemical detection of short sequences of hepatitis C 3a virus using a peptide nucleic acid-assembled gold electrode

Development of an electrochemical DNA biosensor, using a gold electrode modified with a self-assembled monolayer composed of a peptide nucleic acid (PNA) probe and 6-mercapto-1-hexanol, is described. The sensor relies on covalent attachment of the14-mer PNA probe related to the hepatitis C virus genotype 3a (pHCV3a) core/E1 region on the electrode. Covalently self-assembled PNA could selectively hybridize with a complementary sequence in solution to form double-stranded PNA-DNA on the surface. The increase of peak current of methylene blue (MB), upon hybridization of the self-assembled probe with the target DNA in the solution, was observed and used to detect the target DNA sequence. Some hybridization experiments with noncomplementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. Diagnostic performance of the biosensor is described and the detection limit was found to be 5.7 × 10⁻¹¹ M with a relative standard deviation of 1.4% in phosphate buffer solution, pH 7.0. This sensor exhibits high reproducibility and could be used for detection of the target DNA for seven times after the regeneration process.     

羧基化石墨烯/半胱胺修饰金电极对多巴胺的电化学行为研究

为了基于羧基化石墨烯/半胱胺修饰金电极建立更为先进的多巴胺生物传感器，以定量检测儿茶酚胺类神经递质多巴胺，利用自组装技术将半胱胺修饰于金电极上，再利用1-乙基-\[3-二甲基氨基丙基\]碳酰二亚胺盐酸化物/N-羟基琥珀酰亚胺（EDC/NHS）交联剂将羧基化石墨烯固定在修饰后的金电极上制成多巴胺电化学传感器。先对修饰电极进行表征以检验其灵敏度，再利用循环伏安法研究该电极在多巴胺溶液中的电化学行为，包括检测条件的优化和传感器性能的测定。修饰电极表征结果表明，羧基化石墨烯/半胱胺修饰金电极提高了电极传递电子的能力，具有较高的灵敏度。经单因素实验得出，最佳检测条件为利用pH 6.00的0.30 mol·L-1磷酸盐缓冲溶液（PBS）配制多巴胺溶液，扫描速率设定为200 mV·s-1。在最佳检测条件下，制备的多巴胺电化学传感器电流的大小随着多巴胺浓度的增大而增大，在1.0×10-3~3.5×10-3 mol·L-1范围内呈现良好的线性关系，线性回归方程为I=8.120 6C+7.017，相关系数R2为0.999 5。且该传感器精密度好，稳定性强，具有一定的抗干扰能力。研究结果为药物盐酸多巴胺注射液中多巴胺含量测定提供了支撑。     

Amperometric cholesterol biosensor based on in situ reconstituted cholesterol oxidase on an immobilized monolayer of flavin adenine dinucleotide cofactor

A new amperometric biosensor for determining cholesterol based on deflavination of the enzyme cholesterol oxidase (ChOx) and subsequent reconstitution of the apo-protein with a complexed flavin adenine dinucleotide (FAD) monolayer is described. The charge transfer mediator pyrroquinoline quinone (PQQ) was covalently bound to a cystamine self-assembled monolayer (SAM) on an Au electrode. Boronic acid (BA) was then bound to PQQ using the carbodiimide procedure, and the BA ligand was complexed to the FAD molecules on which the apo-ChOx was subsequently reconstituted. The effective release of the FAD from the enzyme and the successful reconstitution were verified using molecular fluorescence and cyclic voltammetry. The optimal orientation of FAD toward the PQQ mediator and the distances between FAD and PQQ and between PQQ and electrode enhance the charge transfer, very high sensitivity (about 2,500 nAmM(-1)cm(-2)) being obtained for cholesterol determination. The biosensor is selective toward electroactive interferents (ascorbic acid and uric acid) and was tested in reference serum samples, demonstrating excellent accuracy (relative errors below 3% in all cases). The biosensor activity can be successfully regenerated in a simple process by successive reconstitution with batches of recently prepared apo-ChOx on the same immobilized Au/SAM-PQQ-BA-FAD monolayer (it was tested five times); the lifetime of the biosensor is about 45-60 days.     

Amperometric sulfide detection using Coprinus cinereus peroxidase immobilized on screen printed electrode in an enzyme inhibition based biosensor

In the present work, an amperometric inhibition biosensor for the determination of sulfide has been fabricated by immobilizing Coprinus cinereus peroxidase (CIP) on the surface of screen printed electrode (SPE). Chitosan/acrylamide was applied for immobilization of peroxidase on the working electrode. The amperometric measurement was performed at an applied potential of -150 mV versus Ag/AgCl with a scan rate of 100 mV in the presence of hydroquinone as electron mediator and 0.1M phosphate buffer solution of pH 6.5. The variables influencing the performance of sensor including the amount of substrate, mediator concentration and electrolyte pH were optimized. The determination of sulfide can be achieved in a linear range of 1.09-16.3 μM with a detection limit of 0.3 μM. Developed sensor showed quicker response to sulfide compared to the previous developed sulfide biosensors. Common anions and cations in environmental water did not interfere with sulfide detection by the developed biosensor. Cyanide interference on the enzyme inhibition caused 43.25% error in the calibration assay which is less than the amounts reported by previous studies. Because of high sensitivity and the low-cost of SPE, this inhibition biosensor can be successfully used for analysis of environmental water samples.     

A lipid-based method for the preparation of a piezoelectric DNA biosensor

A piezoelectric DNA biosensor was prepared by immobilizing DNA probes on a quartz crystal microbalance (QCM) using a lipid-based method. A QCM electrode was coated with a hybrid bilayer membrane composed of an octadecanethiol monolayer and a lipid monolayer containing biotinylated lipids to establish biotin groups on the electrode surface. A DNA biosensor was prepared by sequentially immobilizing avidin and the biotinylated probe. The DNA biosensor was stable throughout repeated surface regeneration and showed higher sensitivity than that prepared by the conventional chemical method using diimide. We also optimized the surface regeneration conditions and flow rate for flow injection analysis.     

A flow injection system, comprising a biosensor based on a screen-printed carbon electrode containing Meldola’s Blue-Reinecke salt coated with glucose dehydrogenase, for the measurement of glucose

A biosensor for the measurement of glucose in serum has been developed, based on a screen-printed carbon electrode modified with Meldola’s Blue-Reinecke salt, coated with the enzyme glucose dehydrogenase (from Bacillus sp.), and nicotinamide adenine dinucleotide coenzyme (NAD⁺). A cellulose acetate layer was deposited on top of the device to act as a permselective membrane. The biosensor was incorporated into a commercially available, thin-layer, amperometric flow cell operated at a potential of only +0.05 V versus Ag/AgCl. The mobile phase consisted of 0.2 M phosphate buffer (pH 7.0) containing 0.1 M potassium chloride solution, and a flow rate of 0.8 ml min⁻¹ was used throughout the investigation. The biosensor response was linear over the range of 0.075-30 mM glucose, with the former representing the detection limit. The precision of the system was determined by carrying out 20 repeat injections of a 5-mM glucose standard, and the calculated coefficient of variation was 3.9%. It was demonstrated that this biosensor system could be applied to the direct measurement of glucose in serum without pretreatment. Therefore, this would allow high-throughput analysis, at low cost, for this clinically important analyte.     

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.     

Polyphenol biosensor based on laccase immobilized onto silver nanoparticles/multiwalled carbon nanotube/polyaniline gold electrode

Laccase purified from Ganoderma sp. was immobilized covalently onto electrochemically deposited silver nanoparticles (AgNPs)/carboxylated multiwalled carbon nanotubes (cMWCNT)/polyaniline (PANI) layer on the surface of gold (Au) electrode. A polyphenol biosensor was fabricated using this enzyme electrode (laccase/AgNPs/cMWCNT/PANI/Au electrode) as the working electrode, Ag/AgCl as the reference electrode, and platinum (Pt) wire as the auxiliary electrode connected through a potentiostat. The biosensor showed optimal response at pH 5.5 (0.1 M acetate buffer) and 35 °C when operated at a scan rate of 50 mV s⁻¹. Linear range, response time, and detection limit were 0.1–500 μM, 6 s, and 0.1 μM, respectively. The sensor was employed for the determination of total phenolic content in tea, alcoholic beverages, and pharmaceutical formulations. The enzyme electrode was used 200 times over a period of 4 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors in that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective PANI microenvironment.     

Regulation of alanine dehydrogenase in Bacillus (licheniformis)

Cell extracts of Bacillus licheniformis were found to contain nicotinamide adenine dinucleotide (NAD)-dependent l-alanine dehydrogenase (ADH) (l-alanine: NAD oxidoreductase, EC 1.4.1.1). High specific activities (3.5 to 6.0 IU/mg of protein) were found in extracts of cells throughout growth cycles only when l-alanine served as the primary source of carbon or carbon and nitrogen. Specific activities were minimal (0.02 to 0.04 IU/mg of protein) during growth on glucose, but increased at least sevenfold during the first 5 h of postlogarithmic-phase metabolism. Addition of 10 mM glucose to cultures during logarithmic-phase growth on l-alanine resulted in a rapid decrease in enzyme activity. Addition of 20 mM l-alanine to cells near the completion of log-phase growth on glucose resulted in a 20-fold increase in ADH specific activity during less than one cell generation. Extracts of postlogarithmic-phase cells cultured on glucose, malate, l-glutamate, or Casamino Acids contained intermediate levels of ADH activity. The enzyme was partially purified from crude extracts of B. licheniformis, and apparent kinetic constants were estimated. A role for ADH in the catabolism of l-alanine to pyruvate during vegetative growth on l-alanine and during sporulation of cells cultured on glucose is proposed on the basis of these experimental results.     

Time dependent study to evaluate the efficacy of zinc on hepatic marker enzymes and elemental profile in serum and liver of protein deficient rats

This study was designed to determine the time dependent protective effects of zinc sulfate on the serum and liver marker enzymes along with elemental profile in protein deficient Sprauge Dawley (S.D.) female rats. Zinc sulfate in the dose of 227 mg/l in drinking water was administrated to normal control as well as protein deficient rats for a total duration of 8 weeks. The effects of different treatments were studied on enzymes like alkaline phosphatase (ALP), aspartate aminotransferases (AST) and alanine aminotransferases (ALT) in rat serum at different time intervals of 1, 2, 4 and 8 weeks and in the rat liver at the end of study. The status of different essential elements in liver was also studied. The serum ALP activity got significantly depressed when estimated at the intervals of 4 and 8 weeks. Activity of serum ALT was significantly increased after 4 weeks interval in protein deficient rats and the increasing trend continued upto 8 weeks of protein deficiency. On the other hand, activity of AST showed a significant increase just after 2 weeks and activity continued to be increased up to 8 weeks. Moreover activities of all the hepato marker enzymes showed a significant increase in liver of protein deficient rats. Interestingly, supplementation of Zn to protein deficient rats helped in regulating the altered activities of ALP, AST and ALT both in serum and liver. However, zinc treatment alone to normal rats did not indicate any significant change in the activities of all the enzymes in liver as well serum except at the interval of 2 weeks where a marginal increase in the activity of AST was seen. It has also been observed that concentrations of zinc, copper, iron and selenium were found to be decreased significantly in protein deficient animals. However, the levels of these elements came back to within normal limits when zinc was administrated to protein deficient rats. Published online December 2004     

Amperometric phenol biosensor based on laponite clay-chitosan nanocomposite matrix

A novel strategy to fabricate an amperometric biosensor for phenol determination based on chitosan/laponite nanocomposite matrix was described. The composite film was used to immobilize PPO on the surface of a glassy carbon electrode. Chitosan was utilized to improve the analytical performance of the pure clay-modified bioelectrode. The biosensor exhibited a series of properties: good affinity to its substrate (the apparent Michaelis-Menten constant for the sensor was found to be 0.16 mM), high sensitivity (674 mA M(-1)cm(-2) for catechol) and remarkable long-term stability in storage (it retains 88% of the original activity after 60 days). In addition, optimization of the biosensor construction as well as effects of experimental variables such as pH, operating potential and temperature on the amperometric response of the sensor were discussed.     

Electrophoretic transfer protein zymography

A mixture of commercial creatinine amidohydrolase (CA), creatine amidinohydrolase (CI), and sarcosine oxidase (SO) was coimmobilized covalently via N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry onto carboxylated multiwalled carbon nanotube (c-MWCNT)/polyaniline (PANI) nanocomposite film electrodeposited over the surface of a platinum (Pt) electrode. A creatinine biosensor was fabricated using enzyme/c-MWCNT/PANI/Pt as working electrode, Ag/AgCl as reference electrode, and Pt wire as auxiliary electrode connected through potentiostat. The enzyme electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and electrochemical impedance spectroscopy (EIS). The biosensor detected creatinine levels as low as 0.1 μM, estimated at a signal-to-noise ratio of 3, within 5 s at pH 7.5 and 35 °C. The optimized biosensor showed a linear response range of 10 to 750 μM creatinine with sensitivity of 40 μA/mM/cm². The fabricated biosensor was successfully employed for determination of creatinine in human serum. The biosensor showed only 15% loss in its initial response after 180 days when stored at 4 °C.