Disposable screen-printed enzyme sensor for simultaneous determination of starch and glucose

A disposable screen-printed biosensor with dual working electrodes was first established for simultaneous determination of starch and glucose. The electrochemical behavior of the sensor was assessed using cyclic voltammetry and chronoamperometry. The linear response ranges of the sensor were up to 0.4% (w/v) starch and 20 mmol glucose 1^–1. Real samples were analysed using the proposed method and the reference method and the correlation coefficient was 0.997.     

Screen-printed biosensors for glucose determination in grape juice

An approach to the glucose determination by amperometric biosensing in wine industry applications is presented. Integrated screen-printed biosensors based on horseradish peroxidase (HRP) and glucose oxidase (GOx) have been developed. The experimental design methodology has been used to find the optimum conditions of the experimental variables, in such a way that a chronoamperometric response specific for glucose was recorded. Under these conditions, repeatability and reproducibility of the modified electrodes have been analyzed. The detection limit for glucose has been calculated taking into account the probability of false positive (alpha) and negative (beta), reaching a medium value of 4.37+/-0.21 micromol dm-3 (alpha=beta=0.05, and a replicate n=4). The biosensor was applied to the determination of glucose in white wine samples.     

Some comparisons among the calming and pain-relieving effects of sucrose, glucose, fructose and lactose in infant rats

Ultrasonic vocalizations were recorded from 10-day-old albinorats while they were isolated from their dam and siblings. Eachrat received a 1.0% BW intra-oral infusion of sucrose, fructose,glucose or lactose at a concentration range of 0.22–0.66M for 3 min and their vocalizations were determined during theinfusion and for an additional 7 min. Sucrose, fructose andglucose all significantly reduced vocalizations to about 50%of baseline levels, whereas lactose, the milk sugar, was ineffective.Moreover, the dose–response function was flat for thethree effective sugars. In a second experiment, the effectsof these sugars on heat escape latency were measured. Sucrose,fructose and glucose each elevated the latency with which infantrats removed a paw from a 48°C surface; lactose did not.These findings of lactose ineffectiveness and the flat dose-responsefunction for the other three sugars exactly parallel those obtainedfor human newborns. Their implications are discussed.     

Amperometric biosensors/sequential injection analysis system for simultaneous determination of S- and R-captopril

Two amperometric biosensors based on L- and D-amino acid oxidase, respectively, are proposed for the simultaneous detection of S- and R-captopril in a sequential injection analysis system (SIA). The linear concentration ranges are: 0.4-1.6 micromol/l (S-captopril) and 120-950 nmol/l (R-captopril) with detection limits of 0.2 and 15 nmol/l, respectively. The biosensors/SIA system can be used reliably on-line in synthesis process control, for the simultaneous assay of S- and R-captopril with a frequency of 34 samples/h.     

Development of mediated amperometric biosensors for hypoxanthine, glucose and lactate: a new format

Electrochemical growth was used to form the organic conducting salt of tetrathiafulvalene (TTF) and tetracyanoquinodimethane (TCNQ) on platinum wires inserted in a glass capillary. Glucose oxidase, lactate oxidase and xanthine oxidase were deposited and crosslinked on the salt structure to produce mediated biosensors responsive to the corresponding analytes. Reliability, stability, interference, and the effect of oxygen on the electrode's response were studied. Among three common electroactive interfering substances tested, ascorbic acid was very active at the TTF-TCNQ structure and the highest response was exhibited by the enzyme-free electrode. Acetaminophen and uric acid displayed similar behaviour at a lower magnitude. The presence of oxygen significantly decreased the current responses of all electrodes.

The xanthine oxidase-bearing mediated electrodes were able to assay the hypoxanthine content of either the fish extract, fish homogenate or slurry of manually ground tissue, yielding results in good agreement with conventional enzymatic assays. The electrodes were stable more than 120 days and could be reused more than 30 times without losing their original activities.     

Serum glucose and insulin response in rats administered with sucrose or starch containing adenosine, inosine or cytosine

Blood glucose and insulin responses and gastric emptying were examined in rats intubated with sucrose or soluble starch that contained adenosine, inosine and cytosine. The increase in serum glucose and insulin levels in the rats following loading with sucrose (2.5 g/kg of body weight) or soluble starch (1.875 g/kg of body weight) was significantly reduced by the administration of adenosine, inosine and cytosine (0.0625-0.125 g/kg of body weight). The gastric emptying rates were only marginally affected by the nucleoside administration. The activities of sucrase, maltase, isomaltase and glucoamylase in a crude preparation from the small intestinal mucosa of rats were mildly inhibited by the nucleosides. The decrease in blood glucose and insulin levels may have been in response to a decrease in glucose absorption caused by the inhibiting effect of the nucleosides on the mucosal enzymes that digest sucrose, maltose, and malto- and isomalto-oligosaccharides.     

Electrodeposited nonconducting polytyramine for the development of glucose biosensors

Biosensors with the composition of carbon/Prussian blue/(glucose oxidase+glutaraldehyde+polytyramine) were constructed. Before tyramine monomers were electropolymerized, glucose oxidase and tyramine monomers were cross-linked with glutaraldehyde onto the surface of Prussian-blue-modified electrodes. The constructed biosensors produced highly reproducible and stable devices. The biosensors exhibited neglectable decrease in current response after 10 repeated uses or after 1 month of dry storage. The resultant biosensors had a linear range of 0.1-1 mM glucose and a detection limit of 0.05 mM. Since the following electrocatalytic process proceeds at a low electrode potential (ca. -0.3 V vs Ag/AgCl), ascorbate and uric acid do not produce observable interfering signal for the determination of glucose.     

Prospects for increasing starch and sucrose yields for bioethanol production

In the short term, the production of bioethanol as a liquid transport fuel is almost entirely dependent on starch and sugars from existing food crops. The sustainability of this industry would be enhanced by increases in the yield of starch/sugar per hectare without further inputs into the crops concerned. Efforts to achieve increased yields of starch over the last three decades, in particular via manipulation of the enzyme ADPglucose pyrophosphorylase, have met with limited success. Other approaches have included manipulation of carbon partitioning within storage organs in favour of starch synthesis, and attempts to manipulate source–sink relationships. Some of the most promising results so far have come from manipulations that increase the availability of ATP for starch synthesis. Future options for achieving increased starch contents could include manipulation of starch degradation in organs in which starch turnover is occurring, and introduction of starch synthesis into the cytosol. Sucrose accumulation is much less well understood than starch synthesis, but recent results from research on sugar cane suggest that total sugar content can be greatly increased by conversion of sucrose into a non-metabolizable isomer. A better understanding of carbohydrate storage and turnover in relation to carbon assimilation and plant growth is required, both for improvement of starch and sugar crops and for attempts to increase biomass production in second-generation biofuel crops.     

A polarographic method for the simultaneous determination of total glucosinolates and free glucose of cruciferous material

A rapid method for the simultaneous analysis of total free glucose and total glucosinolates in aqueous extracts of cruciferous material is described. The technique, which appears suitable for plant-breeding programs as it allows the processing of more than 100 samples per day, involves the polarographic determination of O2 uptake of free glucose by a system of double-coupled enzymes, such as myrosinase-glucose oxidase. The method has advantages over current methods, because it is very rapid (4 min per analysis), allows two determinations for each analysis, and appears to be very reproducible, accurate, and sensitive.     

Octanol-water partition coefficient of glucose, sucrose, and trehalose

The octanol-water partition coefficients (P) of glucose, sucrose, and trehalose were measured at temperatures between 5 and 20 degrees C using an enzymatic method. The measured log P is compared with calculated and experimental data previously reported. In the case of trehalose, the measured log P differs considerably from the theoretically estimated values and agrees with the expected value for a disaccharide. Some methods of assessing the partition coefficients are also analyzed and it is concluded that the atom/fragment contribution method overestimates the hydrophilicity of disaccharides and, probably in a larger extension, that of trisaccharides. The knowledge of P for these sugars is valuable both for basic or applied purposes, including food and biomolecules stabilization.     

Monitoring and control of enzymic sucrose hydrolysis using on-line biosensors

Summary Previously reported flow microcalorimeter devices for enzymic reaction heat measurement, enzyme thermistors, have here been extended with systems for on-line sample treatment. Glucose analysis was performed by intermittent flow injections of 50 l samples through such an enzyme thermistor device containing immobilized glucose oxidase and catalase. Sucroce analysis was performed by allowing diluted samples to continuously pass through an additional enzyme thermistor containing immobilized invertase. The reaction heats were recorded as temperature changes in the order of 10–50 m°C for concentrations of 0.05–0.30 M glucose or sucrose present in the original non-diluted samples.The performance of this system was investigated by its ability to follow concentration changes obtained from a gradient mixer. The system was applied to monitoring and controlling the hydrolysis of sucrose to glucose and fructose in a plug-flow reactor with immobilized invertase. The reactor was continuously fed by a flow of scurose of up to 0.3 M (100 g/l). Glucose and remaining sucrose were monitored in the effluent of the column. By using flow rate controlled feed pumps for sucrose and diluent the influent concentration of sucrose was varied while the overall flow rate remained constant.On-line control of the effluent concentration of lucose and sucrose was achieved by a proportional and integral regulator implemented on a microcomuter. Preset concentration of glucose in the effluent could be maintained over an extended period of time espite changes in the overall capacity of the invertase reactor. Long delay times in the sensor system and the enzyme column made it necessary to carefully tune the control parameters. Changes of set-point value and temperature disturbances were used to verify accuracy of controlling performance.     

Novel planar glucose biosensors for continuous monitoring use

Novel planar glucose biosensors to be used for continuous monitoring have been developed. The electrodes are produced with the "screen printing" technique, and present a high degree of reproducibility together with a low cost and the possibility of mass production. Prior to enzyme immobilisation, electrodes are chemically modified with ferric hexacyanoferrate (Prussian Blue). This allows the detection of the hydrogen peroxide produced by the enzymatic reaction catalysed by GOD, at low applied potential (ca. 0.0 V versus Ag/AgCl), highly limiting any electrochemical interferences. The layer of Prussian Blue (PB) showed a high stability at the working conditions (pH 7.4) and also after 1 year of storage dry at RT, no loss of activity was observed. The assembled glucose biosensors, showed high sensitivity towards glucose together with a long-term operational and storage stability. In a continuous flow system, with all the analytical parameters optimised, the glucose biosensors detected glucose concentration as low as 0.025 mM with a linear range up to 1.0mM. These probes were also tested over 50-60 h in a continuous flow mode to evaluate their operational stability. A 0.5 mM concentration of glucose was continuously fluxed into a biosensor wall-jet cell and the current due to the hydrogen peroxide reduction was continuously monitored. After 50-60 h, the drift of the signal observed was around 30%. Because of their high stability, these sensors suggest the possibility of using such biosensors, in conjunction with a microdialysis probe, for a continuous monitoring of glucose for clinical purposes.     

Electrochemiluminescent biosensors array for the concomitant detection of choline, glucose, glutamate, lactate, lysine and urate

A multifunctional bio-sensing chip was designed based on the electrochemiluminescent (ECL) detection of enzymatically produced hydrogen peroxide. Six different oxidases specific for choline, glucose, glutamate, lactate, lysine and urate were non-covalently immobilised on imidodiacetic acid chelating beads (glucose oxidase only) or on diethylaminoethyl (DEAE) anion exchanger beads, and spotted on the surface of a glassy carbon foil (25 mm(2) square), entrapped in PVA-SbQ photopolymer. The chip measurement was achieved by applying during 3 min a +850 mV potential between the glassy carbon electrode and a platinum pseudo-reference, while capturing a numeric image of the multifunctional bio-sensing chip with a CCD camera. The use of luminol supporting beads (DEAE-Sepharose) included in the sensing layer was shown to enable the achievement of spatially well defined signals, and to solve the hydrogen peroxide parasite signal which appeared between contiguous spots using luminol free in solution. The detection limits of the different biosensor were found to be 1 microM for glutamate, lysine and uric acid, 20 microM for glucose and 2 microM for choline and lactate. The detection ranges were 1-25 microM (uric acid), 1 microM-0.5 mM (glutamate and lysine), 20 microM-2 mM (glucose) and 2 microM-0.2 mM (choline and lactate). The ECL chip was used for the detection of glucose, lactate and uric acid in human serum matrix. Good correlations between measured and expected values were found without the need of internal calibration of the sample, demonstrating the potentiality of the ECL multifunctional bio-sensing chip.     

Responses to dietary starch or sucrose on protein and glucose kinetics in goats fed a high-concentrate diet

Abstract

Responses of whole body protein synthesis (WBPS) and glucose irreversible loss rate (ILR) were compared between dietary starch and sucrose in four male goats. Diets were fed at 1.2 times maintenance requirements of ME and CP with 30% of the ME as starch, starch plus sucrose or sucrose, twice daily. The diets consisted of 33, 32, 11 and 24% of alfalfa hay, corn, soybean meal and the carbohydrates, respectively. The WBPS and glucose ILR during 5 – 7 h after feeding were determined by an isotope dilution method of [²H₅]phenylalanine, [²H₂]tyrosine, [²H₄]tyrosine and [¹³C₆]glucose. Sucrose elevated ammonia nitrogen and lowered acetate concentrations in the rumen, but did not differ from starch in nitrogen retention. Glucose ILR and WBPS were similar between the carbohydrates. It was concluded that dietary sucrose would have effects similar to starch on WBPS and glucose kinetics in the absorptive state in goats fed a high-concentrate diet.     

Polypyrrole nanotube array sensor for enhanced adsorption of glucose oxidase in glucose biosensors

A novel amperometric biosensor based on polypyrrole (PPy) nanotube array deposited on a Pt plated nano-porous alumina substrate and its performances are described. Glucose oxidase (GOx) enzyme was selected as the model enzyme in this study. Commercially available nano-porous alumina discs were used to fabricate electrodes in order to study the feasibility of enzyme entrapment by physical adsorption. A PPy/PF6- film comprising of nanotube array was synthesized using a solution containing 0.05 M Pyrrole and 0.1 M NaPF6 at a current density of 0.3 mA/cm2 for 90 s. The immobilization was done by physical adsorption of 5 microL of GOx (from a stock solution of 2 mg/mL of 210 U/mg) on each electrode. A sensitivity of 7.4 mA cm(-2) M(-1) was observed with PPy nanotube array where the maximum tube diameter was 100 nm. A linear range of 500 microM-13 mM and a response time of about 3 s were observed with a nanotube array where the maximum tube diameter was 200 nm. The synthesized nanotube arrays were characterized by galvanostatic electrochemical technique. Calculated value of apparent Michaelis-Menten constant (Km) was 7.01 mM. The use of nano-porous template electrodes leads to an efficient enzyme loading and provides an increased surface area for sensing the reaction. These factors contribute to increase the characteristic performances of the novel biosensor.     

Possible structure and active site residues of starch,glycogen, and sucrose synthases

A group of enzymes that include muscle glycogen phosphorylase and sugar transferases involved in, for example, the glucosylation of DNA and the synthesis of peptidoglycan are known to possess the same basic three-dimensional fold. Here the possibility is examined that other monosaccharide transferases, those that catalyze synthesis of starch, glycogen, and the disaccharide sucrose, resemble the phosphorylase-type enzymes in structure. In particular, a clear relationship is shown, for the first time, between mammalian glycogen synthases and the phosphorylase structural group of proteins. Domain architecture and secondary structure are discussed, and the possible role of several conserved amino acids at the active site is explored.     

Responses to dietary starch or sucrose on protein and glucose kinetics in goats fed a high-concentrate diet

Responses of whole body protein synthesis (WBPS) and glucose irreversible loss rate (ILR) were compared between dietary starch and sucrose in four male goats. Diets were fed at 1.2 times maintenance requirements of ME and CP with 30% of the ME as starch, starch plus sucrose or sucrose, twice daily. The diets consisted of 33, 32, 11 and 24% of alfalfa hay, corn, soybean meal and the carbohydrates, respectively. The WBPS and glucose ILR during 5-7 h after feeding were determined by an isotope dilution method of [2H5]phenylalanine, [2H2]tyrosine, [2H4]tyrosine and [13C6]glucose. Sucrose elevated ammonia nitrogen and lowered acetate concentrations in the rumen, but did not differ from starch in nitrogen retention. Glucose ILR and WBPS were similar between the carbohydrates. It was concluded that dietary sucrose would have effects similar to starch on WBPS and glucose kinetics in the absorptive state in goats fed a high-concentrate diet.