Wireless enzyme sensor system for real-time monitoring of blood glucose levels in fish

Periodic checks of fish health and the rapid detection of abnormalities are thus necessary at fish farms. Several studies indicate that blood glucose levels closely correlate to stress levels in fish and represent the state of respiratory or nutritional disturbance. We prepared a wireless enzyme sensor system to determine blood glucose levels in fish. It can be rapidly and conveniently monitored using the newly developed needle-type enzyme sensor, consisting of a Pt-Ir wire, Ag/AgCl paste, and glucose oxidase. To prevent the effects of interfering anionic species, such as uric acid and ascorbic acid, on the sensor response, the Pt-Ir electrode was coated with Nafion, and then glucose oxidase was immobilized on the coated electrode. The calibration curve of the glucose concentration was linear, from 0.18 to 144mg/dl, and the detection limit was 0.18mg/dl. The sensor was used to wirelessly monitor fish glucose levels. The sensor-calibrated glucose levels and actual blood glucose levels were in excellent agreement. The fluid of the inner sclera of the fish eyeball (EISF) was a suitable site for sensor implantation to obtain glucose sample. There was a close correlation between glucose concentrations in the EISF and those in the blood. Glucose concentrations in fish blood could be monitored in free-swimming fish in an aquarium for 3 days.     

Quantification of nisin in flow-injection immunoassay systems

A monoclonal-antibody-based, sequential competitive-flow-injection immunoassay system in expanded-bed mode has been developed for the determination of nisin. The system allows the determination of nisin in the presence of suspended particles without any significant interference, illustrating its potential for on-line monitoring of fermentation processes or the analysis of food matrices. The dose response range of the system when operated in expanded-bed mode was 6-90 microM. The detection limit under packed-bed conditions was 3 microM. The results correlated well with the results from conventional ELISA in the analysis of samples of processed cheese. When milk samples, fermentation samples and buffer were spiked with nisin, the mean recoveries were 86% for milk samples, 96% for fermentation samples and 98% for buffer solution.     

Studies on catabolite repression in solid state fermentation for biosynthesis of fungal amylases

Catabolite repression by glucose of the biosynthesis of alpha amylase and amyloglucosidase by Aspergillus niger CFTRI 1105 was studied in a solid state fermentation (SSF) and in submerged fermentation (SMF) systems and the results were compared. The addition of glucose did not enhance the production of alpha-amylase and amyloglucosidase in an earlier fermentation system. However, a drastic reduction in alpha-amylase production was observed in submerged fermentation by the addition of 5·0 mg ml⁻¹ glucose and of amyloglucosidase production by 10 mg ml⁻¹ glucose. Glucose concentrations above 50 mg ml⁻¹ completely suppressed the production of both enzymes in the initial hours. In contrast, in the SSF system the repression was negligible, even when the glucose level was raised to 150 mg g⁻¹ wheat bran for both alpha and amyloglucosidase synthesis.     

Potentiometric sensor for on line glucose determination

Summary An enzymatic biosensor, with a flow-through sensor for continuous monitoring of glucose coupled to a Flow Injection Analysis (FIA) sampling line has been used to determine glucose over the range of 5 to 120 g/l. without sample dilution. The system can analyse 10 samples per hour. Calibration and reproducibility analysis have shown a good linearity and excellent results as compared to a commercial glucose analyser.     

A Double-Needle Gold-Silver Electrodes Continuous Glucose Monitoring Device

ObjectivesDiabetes is a serious, long-term disease and the use of continuous glucose monitoring sensors can reduce reliance on other painful invasive blood testing methods such as the finger blood glucose test. According to our work, a low-cost continuous glucose sensor has been developed based on electrochemical measurement techniques.MaterialsThe sensor is based on a two needles system; a gold and a silver electrode are integrated into a circular shaped electronic printed circuit board (PCB). The sensing part is based on biological electrochemical measurements. Glucose oxidase (Gox) was used as the active sensing element and ferrocene (Fc) as a mediator. Simple and low-cost coating methods were used; these methods are self-assembled monolayers and deep coating. This will reduce the final cost of the sensor as no expensive technique was used. The electrical subsystem contains a low-noise and low-power trans-impedance front-end as well as a single-chip low-power Bluetooth microcontroller with a 12-bit Analog-to-Digital Converter (ADC).ResultsThe sensor was tested in various concentrations of glucose. As a result of initial in vitro experiments, detailed analytical performance metrics are presented. The device has consistently shown a sensitivity of 3.059 mV/(mg/dl) reading with a linear range of 0-400 mg/dl.ConclusionThe proposed study shows promising results for glucose detection. Thus, this type of sensor can be used for different analyzes targeting biological applications after further investigations and analysis.     

Development of a D-alanine sensor for the monitoring of a fermentation using the improved selectivity by the combination of D-amino acid oxidase and pyruvate oxidase

A D-alanine (D-Ala) sensor for the monitoring of a fermentation process was developed using flow injection analysis (FIA). The FIA system consisted of a D-amino acid oxidase (D-AAOx) reactor, a Pyruvate oxidase (PyOx) electrode and a contrast electrode in the flow cell, and through the oxidation of D-amino acids in the D-AAOx reactor, pyruvic acid was formed only from D-Ala. The pyruvic acid was further oxidized with PyOx via the D-AAOx reaction. The amount of oxygen consumed in the PyOx reaction was proportional to the amount of D-Ala. It was possible to continuously repeat the assay up to 60 times at pH 6.8 and a flow rate of 0.18-ml min(-1). A linear relationship was obtained in the range of 0.1-1 mM D-Ala with a correlation coefficient of 0.987 and the detection limit was 0.05 mM. The relative standard deviation (R.S.D.) was 4.9% (n=5) for 0.5 mM D-Ala. The D-Ala content in some fish sauces was also determined using the proposed sensor system. The results obtained indicated a linear relationship between the amounts of D-Ala determined by the proposed sensor system and the conventional method. From the results, even if the substrate specificity of the enzyme (D-AAOx) was low, it was evident that the concentration of the original material (D-Ala) could be determined specifically when the first reaction product was changed by the second reaction (PyOx).     

Offline glucose biomonitoring in yeast culture by polyamidoamine/cysteamine-modified gold electrodes

This article deals with the use of pyranose oxidase (PyOx) and glucose oxidase (GOx) enzymes in amperometric biosensor design and their application in monitoring fermentation processes with the combination of flow injection analysis (FIA). The amperometric studies were carried out at -0.7 V by following the oxygen consumption due to the enzymatic reactions for both batch and FIA modes. Optimization studies (enzyme amounts and pH) and analytical parameters such as linearity, repeatability, effect of interference, storage, and operational stabilities have been studied. Under optimized conditions, for the PyOx-based biosensor, linear graph was obtained from 0.025 to 0.5 mM glucose in phosphate buffer (50 mM) at pH 7.0 with the equation of y = 3.358x + 0.028 and R(2) = 0.998. Linearity was found to be 0.01-1.0 mM in citrate buffer (50 mM and pH 4.0) with the equation of y = 1.539x + 0.181 and R(2) = 0.992 for the GOx biosensor. Finally, these biosensor configurations were further evaluated in a conventional flow injection system. Results from batch experiments provide a guide to design sensitive, stable, and interference-free biosensors for FIA mode. Biosensor stability, dynamic range, and repeatability were also studied in FIA conditions, and the applicability for the determination of glucose in fermentation medium could be successfully demonstrated. The FIA-combined glucose biosensor was used for the offline monitoring of yeast fermentation. The obtained results correlated well with HPLC measurements.     

On-line monitoring of glucose and/or lactate in a fermentation process using an expanded micro-bed flow injection analyser

A novel flow injection biosensor system for monitoring fermentation processes has been developed using an expanded micro bed as the enzyme reactor. An expanded bed reactor is capable of handling a mobile phase containing suspended matter like cells and cell debris. Thus, while the analyte is free to interact with the adsorbent, the suspended particulate matter passes through unhindered. With the use of a scaled down expanded bed in the flow injection analysis (FIA) system, it was possible to analyse samples directly from a fermentor without the pretreatment otherwise required to extract the analyte or remove the suspended cells. This technique, therefore, provides a means to determine the true concentrations of the metabolites in a fermentor, with more ease than possible with other techniques.Glucose oxidase immobilised on STREAMLINE was used to measure glucose concentration in a suspension of dead yeast cells. There was no interference from the cell particles even at high cell densities such as 15 gm dry weight per litre. The assay time was about 6 min. Accuracy and reproducibility of the system was found to be good. In another scheme, lactate oxidase was covalently coupled to STREAMLINE for expanded bed operation. With the on-line expanded micro bed FIA it was possible to follow the fermentation with Lactobacillus casei.     

An automated flow injection analysis system for on-line monitoring of glucose and L-lactate during lactic acid fermentation in a recycle bioreactor

The study concerns on-line sequential analysis of glucose and L-lactate during lactic acid fermentation using a flow injection analysis (FIA) system. Enzyme electrodes containing immobilized glucose oxidase and L-lactate oxidase were used with an amperometric detection system. A 12-bit data acquisition card with 16 analog input channels and 8 digital output channels was used. The software for data acquisition was developed using Visual C++, and was devised for sampling every hour for sequential analyses of lactate and glucose. The detection range was found to be 2–100 g l^–1 for glucose and 1–60 g l^–1 for L-lactate using the biosensors. This FIA system was used for monitoring glucose utilization and L-lactate production by immobilized cells of Lactobacillus casei subsp. rhamnosus during a lactic acid fermentation process in a recycle batch reactor. After 13 h of fermentation, complete sugar utilization and maximal L-lactate production was observed. A good agreement was observed between analysis data obtained using the biosensors and data from standard analyses of reducing sugar and L-lactate. The biosensors exhibited excellent stability during continuous operation for at least 45 days.     

Micro-analytical GO/HRP bioreactor for glucose determination and bioprocess monitoring

A bi-enzymatic micro-analytical bioreactor integrated in a FIA system for glucose measurements is described. Its robustness and small dimensions (working volume of about 70 microl containing approximately 1.2 mg GO and 0.26 mg HRP) make it easy to operate. The column is based on immobilisation of glucose oxidase (GO) and horseradish peroxidase (HRP) on alkylamine controlled pore glass (CPG) beads. The column has excellent shelf life (no significant loss of activity after 1 year if kept at 4 degrees C), and a very high operational stability that was demonstrated through extensive usage for glucose determinations over 1 year period during which the column retained almost all of its activity. More importantly, this operational stability allows glucose monitoring in the culture media without a decay of signal over the experiment time and consequently no signal correction or re-calibration is needed. This high operational stability was also confirmed by continuous glucose conversion with 30% activity loss after converting quantity of glucose equivalent to 21600 FIA injections of 20 microl with 1.7 mM glucose. Such good performance is a result of an optimised immobilisation method and moreover of the implementation of in situ enzyme stabilisation strategy which consisted on promoting the instantaneous H2O2 consumption produced by the GO. This strategy has the additional advantage of allowing concomitant assay of the H2O2 based on the HAP catalysed co-oxidation of phenol-4-sulphonic acid (PSA) in the presence of 4-aminoantipyrine (4-AAP). The glucose measurements are reproducible with high precision against the standard HPLC method. Linear range and sensitivity depend on sample injection volume; the upper limit is about 1.1 g/l. Lower detection limit is 10mg/l. The column performance has been validated for E. coli and S. cerevisiae fermentation monitoring, and glucose measurements in an animal cell culture (rat Langerhans islets).     

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.     

Analysis of micromolar concentrations of glucose by an interference free flow injection based biosensor

A fast, sensitive, interference-free, single enzyme single reagent glucose biosensor, operated in flow injection analysis (FIA) mode, was developed. The method used involved formation of colored complex of titanium sulfate reagent with the peroxide generated by glucose oxidase immobilized in a packed bed reactor. The color developed was detected spectrophotometrically in a flow cuvette. The system could measure down to 0.5 mg glucose l^–1 and the response was reproducible and linear in the range 1 mg l^–1 to 100 mg l^–1. The analysis time for a 500 l sample was 35 s and was free of interference from a number of substances tested. Analysis results using an off-line batch kit were observed to be in agreement with the developed system for determination of glucose in blood plasma samples.     

Sensitive flow-injection method with peroxyoxalate chemiluminescence detection combined with preparative high-performance liquid chromatography for determination of choline-containing phospholipids in human serum

A sensitive and rapid flow-injection analysis (FIA) of total choline-containing phospholipids (PLs) and a selective FIA method for the class assay of choline-containing PLs combined with preparative HPLC were described. The FIA method is based on peroxyoxaxalate chemiluminescence (PO-CL) detection of hydrogen peroxide enzymatically formed from choline-containing PL. The linear standard curves were obtained up to 1 nmol/20-μl injection (r>0.999) with the detection limits of 1.3–1.6 pmol at a signal-to-noise ratio of 2. The total amounts of choline-containing PLs in human serum were ranged from 1.63 to 3.19 mg/ml. The HPLC separation of choline-containing PLs was achieved with an aminopropyl-modified silica gel column using a mixture of acetonitrile-methanol-10 mM ammonium phosphate buffer pH 5.8 as eluent. The eluate corresponding to each choline-containing PL was collected, evaporated, dissolved in 0.1% Triton X-100 aqueous solution, and then injected into FIA system. The FIA method combined with preparative HPLC was applied to the assay of human serum.     

Kalman filter based glucose control at small set points during fed-batch cultivation of Saccharomyces cerevisiae

A glucose control system is presented, which is able to control cultivations of Saccharomyces cerevisiae even at low glucose concentrations. Glucose concentrations are determined using a special flow injection analysis (FIA) system, which does not require a sampling module. An extended Kalman filter is employed for smoothing the glucose measurements as well as for the prediction of glucose and biomass concentration, the maximum specific growth rate, and the volume of the culture broth. The predicted values are utilized for feedforward/feedback control of the glucose concentration at set points of 0.08 and 0.05 g/L. The controller established well-defined conditions over several hours up to biomass concentrations of 13.5 and 20.7 g/L, respectively. The specific glucose uptake rates at both set points were 1.04 and 0.68 g/g/h, respectively. It is demonstrated that during fed-batch cultivation an overall pure oxidative metabolism of glucose is maintained at the lower set point and a specific ethanol production rate of 0.18 g/g/h at the higher set point.     

Theanine production by coupled fermentation with energy transfer using gamma-glutamylmethylamide synthetase of Methylovorus mays No. 9

Gamma-glutamylmetylamide synthetase (GMAS) of Methylovorus mays No. 9, produced by Eschericia coli AD494 (DE3) harboring pET21aGM, formed theanine from glutamic acid and ethylamine with coupling of the reaction with sugar fermentation of baker's yeast cells as an ATP-regeneration system. Theanine formation was stimulated by the addition of Mn(2+) to the reaction mixture, whereas Mg(2+) was less effective. Increases to a certain level in the concentrations of GMAS and the substrates in the mixture were effective in increasing theanine formation, but high concentrations of ethylamine (900 mM or more) inhibited yeast sugar fermentation, and eventually decreased theanine formation. The inhibitory effect of ethylamine was restored by increasing the concentration of potassium phosphate buffer in the mixture. Approximately 600 mM (110 mg/ml) theanine was formed in 48 h in an improved reaction mixture containing 600 mM sodium glutamate, 600 mM ethylamine.HCl, 300 mM glucose, 200 mM potassium phosphate buffer (pH 7.0), 30 mM MgCl(2), 5 mM MnCl(2), 5 mM AMP, 30 units/ml of GMAS, and 40 mg/ml of yeast cells. The yield of theanine was 100% on the substrates (glutamic acid and ethylamine) and also on the energy source (glucose consumed).     

Dynamic response of the expression of hxt1, hxt5 and hxt7 transport proteins in Saccharomyces cerevisiae to perturbations in the extracellular glucose concentration

Glucose transport in Saccharomyces cerevisiae relies on a multi-factorial uptake system. The modulation of its efficiency depends on the differential expression of various sets of hexose transport-related proteins whose glucose affinity differs considerably. The expression of three different glucose transport proteins (HXT1, HXT5 and HXT6/7 with low-, intermediate- and high-affinity, respectively) was monitored as a result of modified extracellular glucose concentrations. Cultivation at glucose-limited (continuous) conditions was instantly replaced by a batch (and thus, non-limited) mode. Further, to mimic concentration gradients in large-scale production bioreactors, multiple and rapid transient glucose pulses were applied to chemostat cultivation. Antibodies against the HXT-proteins were used to monitor the proteins' expression levels prior to and after perturbing the external glucose concentrations. HXT5 and HXT6/7 were either expressed during the starvation-like steady-state phases in the chemostat cultivations, whereas HXT1 could not be detected at all. HXT1, however, is subsequently expressed during the excess of glucose in the batch mode, while the HXT5 and HXT6/7 transporters were at least found to decline. These findings coincide well with the transporters' affinity profiles. As a result of repeated and rapid transient glucose pulses during continuous fermentation, especially HXT6/7 pointed out to alter the protein expression pattern.     

An expression system matures: a highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha

An efficient process was developed for the low-cost production of phytases using Hansenula polymorpha. Glucose or glucose syrups, previously reported as repressive substrates, were used as main carbon sources during fermentation. Glucose was even the most productive substrate for high-level production of phytases. Compared with the process using glycerol, the standard carbon source used for this process until now, the use of glucose led to a reduction of more than 80% in the raw materials costs. In addition, exceptionally high concentrations of active enzyme (up to 13.5 g/L) were obtained in the medium, with phytase representing over 97% of the total accumulated protein. These levels greatly exceed those reported so far for any yeast-based expression system. Very efficient downstream processing procedures were developed with product recovery yields over 90%. Both the fermentation and downstream processing were successfully tested in pilot scale up to 2000 L. As a result, H. polymorpha can be used as a highly competitive system for low-cost phytase production.     

On-line monitoring of monoclonal antibody formation in high density perfusion culture using FIA

An automated flow injection system for on-line analysis of proteins in real fermentation fluids was developed by combining the principles of stopped-flow, merging zones flow injection analysis (FIA) with antigen-antibody reactions. IgG in the sample reacted with its corresponding antibody (a-IgG) in the reagent solution. Formation of insoluble immunocomplexes resulted in an increase of the turbidity which was determined photometrically. This system was used to monitor monoclonal antibody production in high cell density perfusion culture of hybridoma cells. Perfusion was performed with a newly developed static filtration unit equipped with hydrophilic microporous tubular membranes. Different sampling devices were tested to obtain a cell-free sample stream for on-line product anlysis of high molecular weight (e.g., monoclonal antibodies) and low molecular weight (e.g., glucose, lactate) medium components. In fermentation fluids a good correlation (coefficient: 0.996) between the FIA method and an ELISA test was demonstrated. In a high density perfusion cultivation process mAb formation was succesfully monitored on-line over a period of 400 h using a reliable sampling system. Glucose and lactate were measured over the same period of time using a commercially available automatic analyser based on immobilized enzyme technology.Abbreviations TIA Turbidimetric immunoassay - mAb Monoclonal Antibody     

A flow injection analysis system for fermentation monitoring and control

An automated analysis system for on-line fermentation monitoring is presented. The modular system consists of an in-line sterilizeable crossflow microfilter, a selection valve that allows injection of sample or standards, a degassing unit, a dilution module, and a FIA manifold with a spectrophotometric UV/VIS detector. In the dilution module samples are conditioned and diluted depending upon concentration of analyte and the working range of the analyzer. Methods for the monitoring of glucose, ethanol, ammonia and phosphate are described. Results from the monitoring of glucose and their use in fermentation control are presented. The maximal analysis frequency is 30 samples per hour including the dilution of 1 : 200. Detection limits are 5 mg/L for ethanol and glucose, 1 mg/L for phosphate and 50 mg/L for ammonia.     

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.