Immobilization of glucose oxidase and lactate dehydrogenase onto magnetic nanoparticles for bioprocess monitoring system

Glucose oxidase (GOD) and lactate dehydrogenase (LDH) were immobilized onto magnetic nanoparticles, viz. Fe₃O₄, via carbodiimide and glutaraldehyde. The immobilization efficiency was largely dependent upon the immobilization time and concentration of glutaraldehyde. The magnetic nanoparticles had a mean diameter of 9.3 nm and were superparamagnetic. The immobilization of GOD and LDH on the nanoparticles slightly decreased their saturation magnetization. However, the FT-IR spectra showed that GOD and LDH were immobilized onto the nanoparticles by different binding mechanisms, the reason for which was not well explained. The optimum pH values of the immobilized GOD and LDH were changed to 8 and 10, respectively. The free and immobilized enzyme kinetic parameters (K_m and V_max) were determined by Michaelis-Menten enzyme kinetics. The Km values for free and immobilized GOD were 0.168 and 0.324 mM, respectively, while those for free and immobilized LDH were 0.19 and 0.163 mM for NAD, and 2.976 and 4.785 mM for lactate, respectively. High operational stability was observed, with more than 80% of the initial enzyme activity being retained for the immobilized GOD up to 12 h and for the immobilized LDH up to 24 h. The immobilized GOD was applied to a sequential injection analysis system for the application of bioprocess monitoring.     

Enzyme sensor-FIA-system for on-line monitoring of glucose, lactate and glutamine in animal cell cultures.

Enzyme sensors for glucose, lactate and glutamine were connected via flow-injection analysis (FIA) devices to two different bioprocesses. They were used for on-line process control of perfused bioreactor systems containing mammalian cell lines producing a monoclonal antibody and recombinant interleukin-2. The biosensor system gives direct access to important process data which can be used as control parameters for long term cell cultivation systems.     

An autoclavable glucose biosensor for microbial fermentation monitoring and control

The design, construction, and characterization of a prototype-regenerable glucose biosensor based on the reversible immobilization of glucose oxidase (GOx) using cellulose binding domain (CBD) technology is described. GOx, chemically linked to CBD, is immobilized by binding to a cellulose matrix on the sensor-indicating electode. Enzyme immobilization can be reversed by perfusing the cellulose matrix with a suitable eluting solution. An autocavable sensor membrane system is employed which is shown to be practical for use in real microbial fermentations. The prototype glucose biosensor was used without failure or deterioration during fed-batch fermentations of Escherichia coli reaching a maximum cell density of 85 g (dry weight)/L. Medium glucose concentration based on sensor output correlated closely with off-line glucose analysis and was controlled manually at 0.44 +/- 0.2 g/L for 2 h based on glucose sensor output. The sensor enzyme component could be eluted and replaced without interrupting the fermentation. To our knowledge, no other in situ biosensor has been used for such an extended period of time in such a high-cell-density fermentation. (c) 1995 John Wiley & Sons, Inc.     

An effective automated glucose sensor for fermentation monitoring and control

An industrial glucose analyser was partnered to an automated injection system to evaluate glucose in the culture medium of a bioreactor. This sensor has been validated on continuous cultures ofSchizosaccharomyces pombe and continuous and fed-batch cultures ofSaccharomyces cerevisiae. In addition to the advantage of a more accurate process monitoring, the main interest of this sensor deals with the control of the substrate concentration to a prespecified reference signal. Several experiments have been carried out first to validate the sensor, then to control the process evolution.     

Toward real-time continuous brain glucose and oxygen monitoring with a smart catheter

Oxygen and glucose biosensors have been designed, fabricated, characterized and optimized for real-time continuous monitoring on a new smart catheter for use in patients with traumatic brain injury (TBI). Oxygen sensors with three-electrode configuration were designed to achieve zero net oxygen consumption. Glucose sensors were based on the use of platinum nanoparticle-enhanced electrodes that were modified with polycation and glucose oxidase immobilized by chitosan matrix. An iridium oxide electrode was developed to work as a biocompatible reference electrode with enhanced durability and stability in the biological solutions. A study of the effect of temperature on oxygen sensor performance, and both temperature and oxygen effects on glucose sensor performance were accomplished to enhance their operative stability and provide useful information for in vivo applications. A new methodology for automatic correction of the temperature and oxygen dependence of biosensor outputs is demonstrated through programmed LabView™ software. In vitro experiments in both physiological and pathophysiological ranges (oxygen: 0–60 mmHg; glucose: 0.1–10 mM; temperature: 25–40 °C) with clinical samples of cerebrospinal fluid obtained from TBI patients have demonstrated stable measurements with enhanced accuracy, indicating the feasibility of the sensors for a real-time continuous in vivo monitoring.     

Real-time monitoring and control of glucose and lactate concentrations in a mammalian cell perfusion reactor

On-line monitoring and control of cell culture fermentation is important for optimal and consistent production of biologicals. In this work, glucose and lactate concentrations are monitored on-line using a commercially available analyzer (Model 2700, Yellow Springs Instruments, Yellow Springs, OH) during batch and perfusion hybridoma cell fermentation. Cell free samples from the reactor are obtained using a 0.45 mum hollow fiber filtering system placed in a circulation loop. The samples were analyzed at specified times and the data are collected on a computer. A process control strategy was developed to control the concentrations of glucose and lactate in a perfusion reactor where the feed rate is adjusted to maintain their concentrations at desired set points. Hybridoma cells (A10G10) were cultivated in a high density perfusion culture where cell density increased from 2 to 14 million cells/mL. During this period the control algorithm successfully adjusted the perfusion rate while maintaining constant glucose and lactate concentrations. Glucose consumption and lactate accumulation rates as well as net lactate yield on glucose were monitored continuously during perfusion culture. These metabolic rates were observed to be independent of cell concentration and were used for the estimation of viable cell density in the reactor. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 372-378, 1997.     

An automated monitoring system using on-line ultrafiltration and column liquid chromatography for Aspergillus niger fermentations

An automated system for the monitoring of fermentations of filamentous fungi is described. The system is based on the on-line combination of ultrafiltration, for the removal of cellular and macromolecular material from the fermentation broth. and column liquid chromatography for analysis of the filtrate. The performance of one hollow-fibre and two planar ultrafiltration modules is evaluated. The maximum sampling frequency as well as prevention from clogging by mycelium is strongly dependent on the construction of the module, best results being obtained with a planar membrane and a relative high flow rate (150 ml/min) of the broth through a single, wide-bore (3 mm) flow channel in the module. The method is used for the study of metabolic and regulatory processes of two different Aspergillus niger strains on multiple carbon sources. The selected system can be applied for at least 70 h without any negative effect on either the fermentation or the analytical system. Through an analysis frequency of once per hour detailed information regarding consumption and production of nine different compounds could be obtained.     

Quantitative on-line monitoring of hippocampus glucose and lactate metabolism in organotypic cultures using biosensor technology

Quantitative glucose and lactate metabolism was assessed in continuously perfused organotypic hippocampal slices under control conditions and during exposure to glutamate and drugs that interfere with aerobic and anaerobic metabolism. On-line detection was possible with a system based on slow perfusion rates, a half-open (medium/air interface) tissue chamber and a flow injection analytic system equipped with biosensors for glucose and lactate. Under basal conditions about 50% of consumed glucose was converted to lactate in hippocampal slice cultures. Using medium containing lactate (5 mm) instead of glucose (5 mm) significant lactate uptake was observed, but this uptake was less than the net uptake of lactate equivalents in glucose-containing medium. Glucose deprivation experiments suggested lactate efflux from glycogen stores. The effects of drugs compromising or stimulating energy metabolism, i.e. 2-deoxyglucose, 3-nitropropionic acid, alpha-cyano-4-hydroxycinnamate, l-glutamate, d-asparate, ouabain and monensin, were tested in this flow system. The data show that maintaining Na+ and K+ gradients consumed much of the energy but do not support the hypothesis that l-glutamate stimulates glycolysis in hippocampal slice cultures.     

Transmyocardial revascularization aggravates myocardial ischemia around the channels in the immediate phase

We examined whether transmyocardial revascularization (TMR) relieves myocardial ischemia by increasing regional perfusion via the transmural channels in acute canine experiments. Regional blood flow during transient coronary ligation (2 min) was compared before and 30 min after TMR, and at the third transient ischemia the mid-left ventricle (LV) was cut and immediately frozen along the short axis for the analysis of NADH fluorescence in the regions around the TMR channels. In low-resolution analysis (2-4 g tissue or 2-3 cm(2) area), regional perfusion was not significantly altered after TMR, and NADH fluorescence was observed throughout the ischemic region without significant spatial variation. High-resolution analysis (2.8 mg, 1 mm x 1 mm) revealed that the flow after TMR was lower, and NADH fluorescence was higher in the regions close to the channels (1-2 mm) than in the regions 3-4 mm away from them. Creating TMR channels did not improve the regional perfusion and rather aggravated the local ischemia in the vicinity of the channels in the immediate phase.     

Towards the real-time monitoring of glucose in tear fluid: holographic glucose sensors with reduced interference from lactate and pH

Glucose-selective holographic sensors were fabricated from unique tetrahedral 2-acrylamidophenylboronic acid (2-APB) incorporated with co-monomers poly(ethylene glycol) acrylate (PEG), (3-acrylamidopropyl)trimethylammonium chloride (ATMA) and [2-(acryloyloxy)ethyl]-trimethylammonium chloride (AETA) into thin hydrogel films which were transformed into volume holograms using a diffusion method coupled with holographic recording using a frequency-doubled Nd:YAG laser (532 nm). The results showed that the 2-APB-based holographic sensors contracted upon addition of glucose due to the formation of a 2:1 complex between the tetrahedral 2-APB and glucose. More significantly, the 2-APB-based holographic sensors had greatly reduced lactate dependence and a hugely reduced pH effect over the physiological range of pH. These features are vital for development of contact lens-based glucose sensor, where the pH variability is greater (pH 5.8-7.8) and the lactate concentration is substantially higher than in blood. Furthermore, the 2-APB-based holographic sensors also displayed fast response to glucose. The successful union of holograms and the tetrahedral 2-APB receptor for glucose detection in artificial tear fluid is also demonstrated. This new type of holographic sensors responding to glucose with features of minor pH effect and negligible interference from lactate is applicable to the detection of glucose concentrations in tear fluid for the management of diabetes.     

Micromachined sensor for lactate monitoring in saliva

A miniaturised sensor for continuous lactate measurement in saliva was developed and tested. The sensor was built using silicon microfabrication technologies. The size of the chip is 5.5 mmx6.4 mmx0.7 mm and features a working, a counter and an Iridium reference electrode. The chip has a cavity whose floor is perforated by fine pores. The cavity contains the enzyme lactate oxidase (LOD), which is immobilised in an agarose gel. Prior to the amperometric detection of the reaction product hydrogen peroxide at the working electrode, the analyte lactate has to pass the pores to reach the cavity with the lactate oxidase by diffusion. To test the silicon sensor, capillary blood and saliva samples were obtained during standardised ergometer tests. Salivary lactate concentrations were determined with the sensor and compared to photometrically derived data from a lab-automate. In addition the saliva data were compared to standard capillary blood lactate concentrations measured with a pocket photometer. Lactate concentration versus load graphs were plotted and compared visually showing very similar progressions. The novel approach enables a location independent, permanent real-time measurement of the lactate concentration during exercise.     

Automated HPLC monitoring of glucose,glutamine, lactate and alanine on suspended mammalian cell reactors

Summary An isocratic HPLC method has been developed for the single step analysis of glucose, lactate, alanine and glutamine (without prederivatization) in mammalian cell culture media. The method has been successfully connected and automated on a stirred tank reactor to monitor these components during hybridoma culture.     

Early pre-diabetic state alters adaptation of myocardial glucose metabolism during ischemia in rats

Pre-diabetic subjects with high insulin secretory capacity have double risk of cardiovascular disease compared with subjects who do not develop insulin-resistance. It is well established that the ability of the myocardium to increase its glycolytic ATP production plays a crucial role in determining cell survival under conditions of ischemia. Up to now, whether the pre-diabetic state reduces the tolerance of the heart to ischemia by affecting its ability to increase its energy production through glycolysis remains unknown. The aim of the present study was to assess whether insulin resistance affects the ability of the myocardium to increase glycolysis under ischemic conditions. Male Wistar rats were fed for 8 weeks a fructose-enriched (33%) diet to induce a pre-diabetic state. Hearts were isolated and subjected to ex-vivo low-flow (2%) ischemia for 30 min. The fructose diet increased sarcolemmal GLUT4 localisation in myocardial cells under basal conditions compared with controls. This effect was not accompanied by increased glucose utilisation. Ischemia induced the translocation of GLUT4 to the plasma membrane in controls but did not significantly modify the distribution of these transporters in pre-diabetic hearts. Glycolytic flux under ischemic conditions was significantly lower in fructose-fed rat hearts compared with controls. The reduction of glycolytic flux during ischemia in fructose-fed rat hearts was not due to metabolic inhibition downstream hexokinase II since no cardiac accumulation of glucose-6-phosphate was detected. In conclusion, our results suggest that the pre-diabetic state reduces the tolerance of the myocardium to ischemia by decreasing glycolytic flux adaptation.     

An ultrafiltration assay for lysyl oxidase

A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware.     

溶液中木质素和葡萄糖的超滤分离

以木质素和葡萄糖的混合溶液为木质纤维素水解液模型,采用截留相对分子质量为5 000的卷式聚醚砜膜对葡萄糖和木质素进行全回流模式的分离,探讨了木质素和葡萄糖浓度、操作压力、错流速率对通量、木质素和葡萄糖截留率的影响。结果表明:在实验条件范围内,通量随葡萄糖浓度和木质素浓度的增加而降低,并随操作压力、错流速率的增加而增加。木质素截留率不受任何条件的影响,基本稳定在97%。葡萄糖截留率随木质素浓度的增加而增加,并随错流速率的增加而减小。在0.8 g/L的木质素质量浓度条件下,当错流速率从0.12 m/s增加到0.17 m/s时,葡萄糖截留率从14%减小到7.3%。由此可见,在混合溶液的超滤过程中,通过合理选择错流速率,能够改善木质素和葡萄糖的分离。     

Presence of lactate dehydrogenase and lactate racemase in Megasphaera elsdenii grown on glucose or lactate.

Activity of D-lactate dehydrogenase (D-LDH) was shown not only in cell extracts from Megasphaera elsdenii grown on DL-lactate, but also in cell extracts from glucose-grown cells, although glucose-grown cells contained approximately half as much D-LDH as DL-lactate-grown cells. This indicates that the D-LDH of M. elsdenii is a constitutive enzyme. However, lactate racemase (LR) activity was present in DL-lactate-grown cells, but was not detected in glucose-grown cells, suggesting that LR is induced by lactate. Acetate, propionate, and butyrate were produced similarly from both D- and L-lactate, indicating that LR can be induced by both D- and L-lactate. These results suggest that the primary reason for the inability of M. elsdenii to produce propionate from glucose is that cells fermenting glucose do not synthesize LR, which is induced by lactate.