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.     

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.     

In vitro testing of a simply constructed, highly stable glucose sensor suitable for implantation in diabetic patients

We have constructed and tested in vitro a potentially implantable, needle-type amperometric enzyme electrode which is suitable for continuous monitoring of glucose concentrations in diabetic patients. The major requirements of stability during operation and ease of manufacture have been met with a sensor design which involves a simple dip-coating procedure for applying to a platinum base electrode an inner membrane of glucose oxidase immobilised in polyhydroxyethyl methacrylate (pHEMA), and an outer membrane composed of a pHEMA/polyurethane mixture. Sensors were operated at 700 mV for detection of hydrogen peroxide. Calibration curves for the sensor were linear to at least 20 mM glucose and were unaffected by a reduction in PO2 from 20 to 5 kPa. During continuous operation in 5 mM buffered glucose solutions in vitro, sensors suffered no significant loss of response over periods of up to 60 h. Such electrodes are, therefore, useful for development as in vivo glucose sensors.     

Application of enzyme field-effect transistors for determination of glucose concentrations in blood serum

Glucose-sensitive enzyme field effect transistors (ENFETs) modified by an additional Nafion membrane have been developed and used for diluted blood samples analysis. The ENFET was used in the linear portion of the calibration curve up to 1.5 mM glucose in a model solution, which corresponds with up to 60 mM glucose in the undiluted samples (dilution 1:40). The high linearity of the Grans curve (factor of linearity is 1.03) obtained by the method of standard additions indicates the high precision of analysis. Glucose concentrations in different blood serum samples determined by ENFETs were compared with those measured by the commercial analyzer 'Eksan-G' and colorimetric method ('Diagluc' enzymatic kit), and good correlation between these methods was revealed. The high reproducibility and operational stability of the biosensor developed were demonstrated.     

Sensor and biosensor based on Prussian Blue modified gold and platinum screen printed electrodes

Gold (Au) and platinum (Pt) screen-printed electrodes were modified with Prussian Blue (PB) for the development of amperometric sensors selective for hydrogen peroxide detection. The sensors exhibited sensitivities towards H(2)O(2) equal to 2 A M(-1) cm(-2) for Au and 1 A M(-1) cm(-2) for Pt electrodes. The sensors were also employed as the basis for construction of glucose biosensors through further modification with crystallised glucose oxidase immobilised in a Nafion membrane. In order to improve the operational stability of the modified electrodes a buffer solution containing tetrabutylammonium toluene-4-sulfonate was used. The long-term performance of the sensors and biosensors were evaluated by continuous monitoring of hydrogen peroxide and glucose solutions (50 microM and 1 mM, respectively) in the flow-injection mode for 10 h.     

Implantable glucose sensors: Choosing the appropriate sensing strategy

The special requirements for implantable glucose sensors which differ from laboratory analysers and in vitro probes include continuous operation without drift, compatibility with in vivo body conditions, electrical and toxicological safety and patient acceptability. We have studied the effect of oxygen tension, operating temperature and pH, and the stability of various potentially implantable amperometric glucose sensors so as to aid the choice of the technologies most suitable for in vivo application.     

A method for evaluating in vivo the functional characteristics of glucose sensors

A simple system for evaluating ex vivo the functional characteristics of glucose sensors was set up. Normal rats implanted with carotid and jugular catheters were connected under free-moving conditions to an extracorporeal circuit. Blood was allowed to circulate in contact with an enzyme glucose electrode. Glucose or insulin was infused intravenously at different rates to produce glycaemic alterations appropriate for sensor checking. Comparison of the changes in signal output with the corresponding variations in plasma glucose enabled in vivo evaluation of the performances of the sensor, i.e. of the linearity and of the speed of its response to glucose. This method, suitable for small laboratory animals, could therefore be used for the preliminary evaluation of glucose sensors, under in vivo conditions.     

Intravascular glucose/lactate sensors prepared with nitric oxide releasing poly(lactide-co-glycolide)-based coatings for enhanced biocompatibility

Intravenous amperometric needle-type enzymatic glucose/lactate sensors intended for continuous monitoring are prepared with a novel nitric oxide (NO) releasing layer to improve device hemocompatibility. To create an underlying NO release coating, the sensors with immobilized enzymes (either glucose oxidase or lactate oxidase) are prepared with a thin layer of poly(lactide-co-glycolide) (PLGA) loaded with lipophilic diazeniumdiolate species that slowly release NO via a proton driven reaction. An outer thin layer (ca. 30 μm) of PurSil (polyurethane/dimethylsiloxane copolymer) limits the flux of glucose and lactate to the inner layer of enzyme, to provide the desired linear amperometric response. A 30 μm coating of PLGA containing 33 wt% of the appropriate NO donor (N-diazeniumdiolated dibutylhexanediamine, DBHD/N?O?) can release NO at a physiologically relevant rate > 1 × 10?1?mol min?1 cm?2 for at least 7 days without influencing the analytical performance of the glucose/lactate sensors. In vitro, the sensors exhibit relatively stable amperometric response over a one-week period with high selectivity over interferences (e.g., ascorbic acid) required for blood monitoring applications. Glucose sensors implanted in the veins of rabbits for 8h exhibit significantly enhanced hemocompatibility for the NO release sensors vs. corresponding controls (without NO release in same animals), with greatly reduced thrombus formation on their surfaces. Further, the analytical performance of the NO release glucose sensors are superior to controls placed in the veins of the same animals, with a greater accuracy in measuring blood glucose levels as evaluated using a Clarke error grid type analysis.     

In vivo glucose monitoring: the clinical reality and the promise

Glucose monitoring is an essential component of modern diabetes management. Three in vivo glucose sensors are now available for clinical use: a subcutaneously implanted amperometric enzyme electrode, a reverse iontophoresis system and a microdialysis-based device. Improvements in glucose-sensing technology continue to be sought, e.g. wired enzyme technology, viscometric affinity sensing and totally implanted glucose sensors. Non-invasive glucose sensing is the ultimate goal of glucose monitoring, but the most investigated approach, near-infrared (NIR) spectroscopy, is presently too imprecise for clinical application. Fluorescence-based glucose sensing offers several advantages and we are investigating strategies which include NIR-based fluorescence resonance energy transfer using concanavalin A/dextran; changes in the intrinsic fluorescence of hexokinase encapsulated in sol-gel; and non-invasive glucose monitoring of cells by measuring glucose-related changes in NADP(H).     

Miniaturized glucose biosensor modified with a nitric oxide-releasing xerogel microarray

An enzyme-based glucose biosensor modified to release nitric oxide (NO) via a xerogel microarray is reported. The biosensor design is as follows: (1) glucose oxidase (GOx) is immobilized in a methyltrimethoxysilane (MTMOS) xerogel layer; (2) a blended polyurethane/hydrophilic polyurethane coating prevents enzyme leaching and imparts selectivity for glucose; and (3) micropatterned xerogel lines (5 microm wide) separated by distances of 5 or 20 microm provide NO-release capability. This configuration allows for increased glucose sensitivity relative to sensors modified with NO-releasing xerogel films since significant portions of the sensor surface remain unmodified. Glucose diffusion to the GOx layer is thus less inhibited. The micropatterned NO-releasing biosensors generate sufficient NO levels to reduce both Pseudomonas aeruginosa and platelet adhesion without significantly compromising the enzymatic activity of GOx. The glucose response, linearity and stability of the NO-releasing micropatterned sensors are reported.     

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.     

辣椒碱对3T3-L1 前脂肪细胞葡萄糖摄取的影响

目的:探讨辣椒碱对3T3-L1前脂肪细胞葡萄糖摄取的影响。方法:不同浓度的辣椒碱作用于3T3-L1前脂肪细胞,采用MTT测定细胞活性,GLU Test试剂盒法测定葡萄糖摄取,Western Blot法检测葡萄糖转运蛋白1(GLUT-1)表达的变化。结果:25μM辣椒碱作用72 h和50μM、100μM辣椒碱作用48 h、72 h,可显著抑制3T3-L1细胞增殖,6.25、12.5、25μM辣椒碱作用可显著促进3T3-L1细胞的葡萄糖摄入,Western Blot结果显示辣椒碱能够显著增加GLUT1蛋白表达量,差异均具有统计学意义(P0.05)。结论:低剂量辣椒碱具有降糖作用,其作用机制可能与增加GLUT-1蛋白表达有关。     

An ultrafiltration catheter for monitoring of venous lactate and glucose around myocardial ischemia

Early detection of myocardial ischemia is of major importance in critical-care medicine. Changes of lactate or glucose levels in the cardial venous efflux may be useful parameters. We succeeded in integrating an ultrafiltration membrane in a cardiac catheter for continuous sampling. The ultrafiltrate was analyzed outside the body, resulting in a lag-time of about 24 min. Biosensors in a flow-injection analysis system were used for minute by minute sample analyses. The coronary sinus of pigs was catheterized to monitor the effects of 5, 15 or 45 min ischemia by coronary artery obstruction or myocardial stress by dobutamine infusion. A total of 27 h was monitored. The intravascular response time was 1.33+/-0.61 min (10-90%). Linear regression in vivo of blood and ultrafiltrate samples was 0.977 for lactate and 0.994 for glucose. Lactate levels rose 0.38+/-0.10 mM above baseline within 5 min after ischemia. Reperfusion was clearly marked by a promptly peaking lactate release (maximum 9.27 mM). Myocardial stress by dobutamine increased glucose but not lactate levels. Once, a wall effect was noted at the catheter tip. In vivo semi-continuous myocardial monitoring of absolute lactate and glucose concentrations was thus achieved by an ultrafiltration catheter. Ischemia and reperfusion can be detected very early by a lactate level rise. Further, development of the ultrafiltration catheter will be focused on the diagnostic potential of lactate monitoring for patients.     

An immersible manometric sensor for measurement of humidity and enzyme mediated changes in dissolved gas

An immersible manometric sensor was made by covering the gaseous cavity of a pressure transducer with a 1 microm controlled pore membrane. Transfer of gas across the membrane allowed the pressure transducer to record changes in humidity or dissolved gas when immersed in solution. By immersing the sensor in distilled water, atmospheric humidity could be estimated by the deficit of atmospheric vapor pressure from saturation. In another application of the sensor, CO(2) was monitored continuously. This was not possible in previous closed-reactor type manometric sensors, and may allow the new technology to be used in applications requiring continuous monitoring of a process or stream. By coupling the sensor with enzymes liberating or consuming dissolved gas, different chemicals could be estimated. Urea was estimated by first hydrolyzing it with urease and then measuring the resulting CO(2) gas in solution. Glucose was measured through its enzymatic oxidation by glucose oxidase. The sensitivity to urea over the range 0-2.5 mM was about 1.02 kPa/mM, and the standard error was 0.086 mM. Due to the lower solubility of oxygen, the sensitivity to glucose in a range from 0 to 10 microM was over 100 kPa/mM, with a standard error of only 0.76 microM. This sensitivity was not possible in closed-reactor type manometric sensors due to constraints of dimensioning the head space gas volume for reproducibility and effective mass transfer. The 90% rise times for the sensor ranged from about 1-60 min for the different applications. The dynamic characteristics of the device may be improved by using a membrane with greater porosity, higher rigidity and lower thickness, and by reducing the dimensions of the cavity volume in the sensor through integrated microfabrication of the membrane onto the transducer.     

Studies on gluconeogenesis and ketogenesis in isolated hepatocytes from alloxan diabetic rats.

Gluconeogenesis and ketogenesis were studied in isolated hepatocytes obtained from normal and alloxan diabetic rats. Insulin treatment maintained near-normal blood glucose levels and caused an increase in glycogen deposition. The third day after insulin withdrawal the rats displayed a diabetic syndrome marked by progressive hyperglycemia and glycogen depletion. Net glucose production in liver cells isolated from alloxan diabetic rats progressively increased with time up to 72 hr after the last in vivo insulin injection. Maximal glucose production was observed at 72 hr with 10 mM alanine, lactate, pyruvate, or fructose. Glucose production decreased at 96 hr. The same pattern was observed with the incorporation of labeled bicarbonate into glucose. Ketogenesis in liver cells and hepatic lipid content also peaked at 72 hr.     

A novel continuous subcutaneous lactate monitoring system

A novel continuous lactate monitoring system has been developed modifying the GlucoDay portable medical device (A. Menarini Diagnostics), already present in the European market, and used to continuously measure glucose levels. Lactate oxidase based biosensors have been developed immobilising the enzyme on nylon net and placing it on a Pt electrode. The biosensor was connected to the portable device provided with a micro-pump and coupled to a microdialysis system. It is capable to record subcutaneous lactate every 3 min. In vitro analytical results confirmed that the sensors respond linearly in the interval of concentration between 0.1 and 10 mmol/L, covering the whole physiological range. During prolonged monitoring periods, the response of the biosensors remained stable, showing a limited drift of 8%, within 60 h. Stability tests are still on route. However, preliminary results have shown a shelf life of about 10 months. In vivo experiments performed on healthy rabbits have demonstrated the good accuracy and reproducibility of the system. A correlation coefficient equal to 0.9547 (N=80) was found, which represents a good correlation between the GlucoDay and the laboratory reference analyser. A 16 h in vivo monitoring on a healthy volunteer has been also performed.     

Insulin and glucose-dependent regulation of the glucose transport system in the rat L6 skeletal muscle cell line

Differentiated rat L6 skeletal muscle cell cultures maintained in glucose-deficient medium containing 25 mM xylose displayed a rapid, reversible, time- and concentration-dependent 3-5-fold increase in glucose transport activity. Glucose deprivation in the continuous presence of insulin (24 h) resulted in an overall 9-10-fold stimulation of glucose transport activity. In contrast, acute (30 min) and chronic (24 h) insulin treatment of L6 cells maintained in high glucose (25 mM)-containing medium resulted in a 1.5- and 4-fold induction of glucose transport activity, respectively. Acute glucose deprivation and/or insulin treatment had no significant effect on the total amount of glucose transporter protein, whereas the long-term insulin- and glucose-dependent regulation of glucose transport activity directly correlated with an increase in the cellular expression of the glucose transporter protein. In situ hybridization of the L6 cells demonstrated a 3-, 4-, and 6-fold increase in glucose transporter mRNA induced by glucose deprivation, insulin, and glucose deprivation plus insulin treatments, respectively. Similarly, Northern blot analysis of total RNA isolated from glucose-deprived, insulin, and glucose-deprived plus insulin-treated cells resulted in a 4-, 3-, and 9-fold induction of glucose transporter mRNA, respectively. The continuous presence of insulin in the medium, either in the presence or absence of glucose, resulted in a transient alteration of the glucose transporter mRNA. The relative amount of the glucose transporter mRNA was maximally increased at 6-12 h which subsequently returned to the basal steady-state level within 48 h. These data demonstrate a role for insulin and glucose in the overall regulation of glucose transporter gene expression which may account for the alteration of glucose transporter activity of muscle tissue observed in pathophysiological states such as type II diabetes mellitus.     

Real-time in situ monitoring of freely suspended and immobilized cell cultures based on mid-infrared spectroscopic measurements.

Glucose and lactate profiles in Chinese hamster ovary cell cultures were accurately monitored in real time and in situ during three bioreactor batch cultures lasting 11,15, and 15 days performed within a 60-day period. Monitoring was accomplished using in situ-collected mid-infrared spectra analyzed with a priori one-time established partial least-squares regression models. The robustness of the technique was demonstrated by application of these models without modification after 2.3 years. Neither recalibration nor instrument maintenance was required during the 2.3-year period, except for the daily filling of liquid nitrogen for detector cooling during operation. The lactate calibration model yielded accurate absolute concentration estimations during each of the batch cultures with standard errors of estimate from 1 to 3 mM. The a priori-established glucose calibration model yielded concentration estimations with an off-set, which was constant throughout a culture. Adjustment of the off-set before inoculation resulted in accurate concentration estimations with Standard errors of estimate of approximately 1 mM for each of the bioreactor cultures. Sensitivity in detecting differences of 0.5 mM and selectivity against variation of one metabolite while the other was kept constant was demonstrated during standard additions of either glucose or lactate. The sensor system proved to be reliable, simple, accurate, sterile, and capable of long-term automatic operation and is considered to be mature enough to be routinely applied for in situ (on-line) cell culture monitoring.     

Effects of sugar concentration on recombinant human alpha(1)-antitrypsin production by genetically engineered rice cell

Productivity of recombinant human alpha(1)-antitrypsin (rAAT) with a genetically engineered rice cell using an inducible promoter has been studied by batch-wise and continuous production. A simple model explained the effect of proteases released from the disrupted cells on the rAAT degradation. Glucose concentration in the medium significantly affected the rAAT productivity in the continuous production, because the rAAT was induced by sugar depletion. When the fresh medium containing 5mM glucose was supplied to the continuous bioreactor, induction time was long and the productivity was low, indicating that the glucose concentration in the cells was high enough as to repress the promoter. When the glucose concentration in the fresh medium was reduced to 0.5mM, total amount of rAAT produced in 70h cultivation reached 6.7-7.6mg/g-dry cell, which was two times larger than the control medium without glucose.     

Continuous optical in-line glucose monitoring and control in CHO cultures contributes to enhanced metabolic efficiency while maintaining darbepoetin alfa product quality

Great efforts are directed towards improving productivity, consistency and quality of biopharmaceutical processes and products. One particular area is the development of new sensors for continuous monitoring of critical bioprocess parameters by using online or in-line monitoring systems. Recently, we developed a glucose biosensor applicable in single-use, in-line and long-term glucose monitoring in mammalian cell bioreactors. Now, we integrated this sensor in an automated glucose monitoring and feeding system capable of maintaining stable glucose levels, even at very low concentrations. We compared this fed-batch feedback system at both low (< 1 mM) and high (40 mM) glucose levels with traditional batch culture methods, focusing on glycosylation and glycation of the recombinant protein darbepoetin alfa (DPO) produced by a CHO cell line. We evaluated cell growth, metabolite and product concentration under different glucose feeding strategies and show that continuous feeding, even at low glucose levels, has no harmful effects on DPO quantity and quality. We conclude that our system is capable of tight glucose level control throughout extended bioprocesses and has the potential to improve performance where constant maintenance of glucose levels is critical.