Needle-type lactate biosensor

A needle-type lactate biosensor has been developed for continuous intravascular lactate monitoring. The sensor employs poly(1,3-phenylenediamine) as the inner layer on the platinum electrode in order to eliminate the interference from oxidizable physiological substances. Cross-linking with glutaraldehyde was used for enzyme immobilization. Dithiothreitol was used as the stabilizer of lactate oxidase. PVC (polyvinyl chloride) was chosen as the external diffusion control membrane. Sensor performance was evaluated in vitro and the sensor shows a sensitivity of 10-15 nA/mM, and a linear range from 1 mM to at least 15 mM lactate. Evaluation of the sensor response in blood plasma showed similar sensitivity and linear range as indicated by the calibration curves obtained in buffer solution. The sensor has a short response time of approximately 1 minute. The sensors were operated continuously for 7 days in phosphate buffer containing solution with a concentration at the physiological lactate level. No significant change in sensor sensitivity and its linear range has been observed. Sensors show a minimum change in its performance when stored in buffer at 4 degrees C for at least 9 months.     

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.     

Containment sensors for the determination of L-lactate and glucose

This paper reports some new results on enzyme based silicon containment sensors. For the first time an L-lactate sensor in containment technology is presented. Through optimization of the buffer system the stability of the lactate sensor was enhanced and the linear response of over 10 mM was achieved. The glucose sensor has also been optimized for a large linear measurement range exceeding 30 mM. A two-enzyme chip with glucose and lactate sensor elements which were integrated on one silicon chip is presented. The response behaviour of the two-enzyme chip was very similar to the single chip behaviour. No cross-talking effects could be observed. A fabrication process for mass-production is described.     

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.     

A new amperometric glucose microsensor: in vitro and short-term in vivo evaluation

For biosensor fabrication, it is important to optimize materials and methods in order to create predictable function in vitro and in vivo. For this reason, we designed a new glucose sensor ('revised protocol') that utilized an outer permselective membrane made of amphiphobic polyurethane which allows glucose passage through hydrophilic segments. An inner polyethersulfone membrane, stabilized with a trimethoxysilane, provided specificity. Before application of the inner membrane, it was necessary to etch the platinum electrode with a radio frequency oxygen plasma. The revised protocol sensors (n=185) were compared with sensors fabricated with an earlier ('original') protocol (n=204) which used an outer polyurethane without hydrophilic segments and a complex inner membrane of cellulose acetate and Nafion. The function of revised protocol sensors was more predictable in vitro as evidenced by a much lower variation of glucose sensitivity than the original protocol sensors. Revised and original protocol sensors were nearly linear up to a glucose concentration of 20 mM. In vitro interference from 0.1 mM acetaminophen was minimal in both groups of sensors and would be expected to represent about 2% of the total sensor response at normal glucose levels for revised protocol sensors. Prolonged testing of the revised protocol sensors for 11 days during immersion in buffer revealed stable sensitivities (day 1: 6.12+/-1.34 nA/mM; day 3: 6.33+/-1.40; day 8: 7.13+/-1.39; and day 11: 7.56+/-1.47; sensitivity for day 1 vs. each other day: not significant) and no critical loss of glucose oxidase activity. The response of the revised protocol sensors (n=7) to intraperitoneal glucose was tested in rats approximately one day after subcutaneous implantation and the sensors tracked glucose closely with a slight lag of 3-6 min.     

Biosensor system for continuous flow determination of enzyme activities. I. Determination of glucose oxidase and lactic dehydrogenase activities

A biosensor system for continuous flow determination of enzyme activity was developed and applied to the determination of glucose oxidase and lactic dehydrogenase activities. The glucose oxidase activity sensor was prepared from the combination of an oxygen electrode and a flow cell. Similarly, the lactic dehydrogenase activity sensor was prepared from the combination of a pyruvate oxidase membrane, an oxygen electrode, and a flow cell. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane, and glutaraldehyde. Glucose oxidase activity was determined from the oxygen consumed upon oxidation of glucose catalyzed by glucose oxidase. Lactic dehydrogenase activity was determined from the pyruvic acid formed upon dehydrogenation of lactic acid catalyzed by lactic dehydrogenase. The amount of pyruvic acid was determined from the oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. Calibration curves for activity of glucose oxidase and lactic dehydrogenase were linear up to 81 and 300 units, respectively. One assay could be completed within 15 min for both sensors and these were stable for more than 25 days at 5°C. The relative errors were ±4 and ±6% for glucose oxidase and lactic dehydrogenase sensors, respectively. These results suggest that the sensor system proposed is a simple, rapid, and economical method for the determination of enzyme activities.     

Lifespan of subcutaneous glucose sensors and their performances during dynamic glycaemia changes in rats

Performances of a glucose sensor have been investigated during dynamic variations of plasma glucose levels. Subcutaneous glucose concentrations measured by the sensors were calculated by a one-point calibration, performed in basal conditions. A first group of sensors were chronically implanted in the subcutaneous tissue of normal rats. The animals were submitted to glucagon and insulin injection, in order to induce rapid modifications of their glycaemia. This test was repeated at different days after implantation in order to investigate the lifespan and the performance of the sensors. All the sensors were working 1 or 2 days after implantation, and 70% adequately responded to glycaemia variations at day 3 or 4. The quality of the sensors' performance remained constant as a function of the time. With a second group of sensors, we demonstrated that an efficient sterilization procedure did not alter the sensors' characteristics. At the day of implantation, the sterilized sensors' performance, during dynamic variations of plasma glucose levels, was closely similar to that of the non-sterilized sensors. The animals bearing the sterilized devices were rendered diabetic by steptozotocin (STZ) injection. Once the rats had developed a severe hyperglycaemia (1–3 days after STZ), they were injected with intravenous insulin. The subcutaneously implanted glucose sensors correctly followed the decline in plasma glucose levels. We therefore conclude that our sensor could represent a useful tool for short-term continuous blood monitoring.     

Development of biosensors for the simultaneous determination of sucrose and glucose, lactose and glucose, and starch and glucose

Sensors for the simultaneous determinations of sucrose and glucose, lactose and glucose, and starch and glucose were prepared by a combination of the enzyme system shown below and an oxygen electrode: The mechanism for separating the substrates with the proposed sensors is based on the time lag arising from reaction and diffusion. Invertase, beta-galactosidase, amyloglucosidase, mutarotase, and glucose oxidase were covalently immobilized on triacetyl cellulose membranes containing 1,8-diamino-4-aminomethyloctane. A glucose oxidase membrane, mutarotase membrane, three sheets of triacetyl cellulose membranes, and invertase, or beta-galactosidase or amyloglucosidase membrane were placed in that order on the tip of the oxygen electrode. Calibration curves for sucrose, lactose, and starch were linear up to 40 mM, 60-180 mM, and 10%, respectively. The simultaneous determination of sucrose and glucose, lactose and glucose, and starch and glucose was possible when the amount of glucose coexised was in the range of 2-16% sucrose, 2.8-8.3% lactose, or 0.1-1% starch. The relative errors were +/-4% for sucrose and +/-3% for lactose in 100 assays. The starch sensor was reused only five times. Each enzyme membrane was fairly stable for more than 10 days.     

Extended-gate FET-based enzyme sensor with ferrocenyl-alkanethiol modified gold sensing electrode

We developed a field-effect transistor (FET)-based enzyme sensor that detects an enzyme-catalyzed redox-reaction event as an interfacial potential change on an 11-ferrocenyl-1-undecanethiol (11-FUT) modified gold electrode. While the sensitivity of ion-sensitive FET (ISFET)-based enzyme sensors that detect an enzyme-catalyzed reaction as a local pH change are strongly affected by the buffer conditions such as pH and buffer capacity, the sensitivity of the proposed FET-based enzyme sensor is not affected by them in principle. The FET-based enzyme sensor consists of a detection part, which is an extended-gate FET sensor with an 11-FUT immobilized gold electrode, and an enzyme reaction part. The FET sensor detected the redox reaction of hexacyanoferrate ions, which are standard redox reagents of an enzymatic assay in blood tests, as a change in the interfacial potential of the 11-FUT modified gold electrode in accordance with the Nernstian response at a slope of 59 mV/decade at 25 degrees C. Also, the FET sensor had a dynamic range of more than five orders and showed no sensitivity to pH. A FET-based enzyme sensor for measuring cholesterol level was constructed by adding an enzyme reaction part, which contained cholesterol dehydrogenase and hexacyanoferrate (II)/(III) ions, on the 11-FUT modified gold electrode. Since the sensitivity of the FET sensor based on potentiometric detection was independent of the sample volume, the sample volume was easily reduced to 2.5 microL while maintaining the sensitivity. The FET-based enzyme sensor successfully detected a serum cholesterol level from 33 to 233 mg/dL at the Nernstian slope of 57 mV/decade.     

Modeling of spherical fluorescent glucose microsensor systems: design of enzymatic smart tattoos

A two-substrate mathematical model of microspherical optical enzymatic glucose sensors is presented. The sensors are based on the well-known oxidation of glucose by glucose oxidase, and are constructed by the encapsulation of glucose oxidase within hydrogel microspheres coated with ultrathin polyelectrolyte multilayer films. In order to measure glucose via changes in oxygen concentration, a fluorescent oxygen indicator is co-encapsulated with the enzyme. The model was used to predict the temporal and spatial distributions of glucose and oxygen within the sphere for step increases in bulk glucose concentration. In addition, the model was used to observe the effect of varying sensor parameters, namely sphere size, film thickness, enzyme concentration, and mass transport of substrate and co-substrate within the sphere and film coatings, on the response of the sensors. A major finding was that the application of {PSS/PAH} films as thin as 12 nm can drastically improve the sensor performance over uncoated sensors based on calcium alginate microspheres. The model is proposed as an important tool for a priori design of these complex sensor structures.     

Rechargeable glucose electrodes for long-term implantation

A unique enzyme electrode was designed using glucose oxidase immobilized on fine graphite powder. The graphite-enzyme is in a fluid state which enables recharging of the system when the enzyme activity decreases, therefore allowing the system to have a long lifetime in a diabetic patient. The sensor was tested using glucose concentration in the range of 20–300 mg d⁻¹. The electrode construction includes a hydrophobic membrane and a platinum electrode for detection of hydrogen peroxide. The signal output is large with minimal noise when tested in buffer. The sensor has been performing at ambient temperature for 4 months when stored overnight at 4° C.     

A fast estimation of biochemical oxygen demand using microbial sensors

Summary Microbial amperometric sensors for biochemical oxygen demand (BOD) determination using Bacillus subtilis or Trichosporon cutaneum cells immobilized in polyvinylalcohol have been developed. These sensors allow BOD measurements with very short response times (15–30s), a level of precision of ±5% and an operation stability of 30 days. A linear range was obtained for a B. subtilis-based sensor up to 20 mg/l BOD and for a T. cutaneum-based sensor up to 100 mg/l BOD using a glucose/glutamic acid standard.     

Carbon post-microarrays for glucose sensors

A novel design and fabrication method of glucose sensors based on high aspect ratio carbon post-microarrays is reported in this paper. Apart from the fact that carbon has a wide electrochemical stability window, a major advantage of using carbon post-microarrays as working electrodes for an amperometric glucose sensor is the large reactive surface per unit footprint substrate area, improving sensitivity of the glucose sensor. The carbon post-microarrays were fabricated by carbon-microelectromechanical systems (C-MEMS) technology. Immobilization of enzyme onto the carbon post-electrodes was carried out through co-deposition of glucose oxidase (GOx) and electrochemically polymerized polypyrrole (PPy). Sensing performance of the glucose sensors with different post-heights and various post-densities was tested and compared. The carbon post-glucose sensors show a linear range from 0.5 mM to 20 mM and a response time of about 20 s, which are comparable to the simulation result. Sensitivity per unit footprint substrate area as large as 2.02 mA/(mM cm²) is achieved with the 140 μm high (aspect ratio around 5:1) carbon post-samples, which is two times the sensitivity per unit footprint substrate area of the flat carbon films. This result is consistent with the hypothesis that the number of reaction sites scales with the reactive surface area of the sensor. Numerical simulation based on enzymatic reaction and glucose diffusion kinetics gives the optimum geometric design rules for the carbon post-glucose sensor. Glucose sensors with even higher sensitivity can be achieved utilizing higher carbon post-microarrays when technology evolution will permit it.     

Occurence of adverse events due to continuous glucose monitoring

Aims: Continuous glucose monitoring (CGM) using transcutaneous sensors is becoming a sophisticated method to control and regulate glucose metabolism. The transcutaneous sensor of the CGM system (CGMStrade mark Medtronic Minimed, Northridge, CA, USA) is chosen to measure glucose concentration in interstitial fluid up to three days after insertion even though its function remains stable for a longer period. The question arises, which factors really limit the period of sensor insertion without unnecessary risk. The aim of this study was to assess any adverse events occurring in the course of 9 days after the sensor insertion. Methods: In a group of 22 healthy volunteers aged 21.8+/-1.30 y (mean +/- SE) a total of 26 sensors was inserted subcutaneously in gluteal or lumbar region for 9 days. Before insertion the site was sprayed with an antiseptic (Cutasept F, Bode Chemie, Hamburg, Germany). Local adverse reactions and disturbances in general condition were examined. Results: In the course of 184 sensor-days, there were only minor local adverse events: hypersensitivity, itching, pain, redness, burning, subcutaneous hemorrhage. Additionally, sleep disturbances, attention deficits, problems related to the CGMS monitor, to adhesive tape and/or sensor were found. None of these resulted in sensor withdrawal. In 12 volunteers (55 %) no complications were observed. The sensor function measured according to electrical signals (ISIG) failed (always on day 1-2) in 4 cases (16 %). Conclusions: The present FDA approved 3-day insertion period for Medtronic transcutaneous sensor does not seem to limit its use and appears to be worth a careful revision.     

A novel wireless glucose sensor employing direct electron transfer principle based enzyme fuel cell

In this paper we present a novel wireless glucose biosensing system employing direct electron transfer principle based enzyme fuel cell. Using the glucose dehydrogenase complex, which is composed of a catalytic subunit containing FAD, the cytochrome c subunit that harbors heme c as the electron transfer subunit, and chaperone-like subunit, a direct electron transfer-type glucose enzyme fuel cell was constructed. The enzyme glucose fuel cell generated electric power, and the open-circuit voltage showed glucose concentration dependence, which suggests potential applications for this glucose-sensing system. We constructed a miniaturized "all-in-one" glucose enzyme fuel cell, which represents a compartmentless fuel that is based on the direct electron transfer principle. This involved the combination of a wireless transmitter system and a simple and miniaturized continuous glucose monitoring system, which operated continuously for about 3 days with stable response. This is the first demonstration of an enzyme-based direct electron transfer-type enzyme fuel cell and fuel cell-type glucose sensor which can be utilized as a subcutaneously implantable system for continuous glucose monitoring.     

Fabrication of a sensing module using micromachined biosensors

Micromachining is a powerful tool in constructing micro biosensors and micro systems which incorporate them. A sensing module for blood components was fabricated using the technology. The analytes include glucose, urea, uric acid, creatine, and creatinine. Transducers used to construct the corresponding sensors were a Severinghaus-type carbon dioxide electrode for the urea sensor and a Clark-type oxygen electrode for the other analytes. In these electrodes, detecting electrode patterns were formed on a glass substrate by photolithography and the micro container for the internal electrolyte solution was formed on a silicon substrate by anisotropic etching. A through-hole was formed in the sensitive area, where a silicone gas-permeable membrane was formed and an enzyme was immobilized. The sensors were characterized in terms of pH and temperature dependence and calibration curves along with detection limits. Furthermore, the sensors were incorporated in an acrylate flow cell. Simultaneous operation of these sensors was successfully conducted and distinct and stable responses were observed for respective sensors.     

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.     

Miniaturized multi-enzyme biosensors integrated with pH sensors on flexible polymer carriers for in vivo applications

An electrochemical glucose sensor has been integrated, together with a pH sensor, on a flexible polyimide substrate for in vivo applications. The glucose sensor is based on the measurement of H₂O₂ produced by the membrane-entrapped enzyme glucose oxidase (GOD). To minimize electrochemical interference, an electrode configuration was designed to perform differential measurements. The solid-state pH sensor employs a PVC-based neutral carrier membrane. The enzymes GOD and catalase were immobilized into two layers of photolithographically patterned hydrogels. The intended use of this device is the short-term monitoring of glucose and pH in intensive care units and operating theatres, especially for neurosurgical applications. The developed immobilization technique can also be used to create integrated multi-sensor chips for clinical analysers. The glucose and pH sensor exhibited excellent performance during tests in buffer solutions, serum and whole blood.     

A flow-through amperometric sensor based on dialysis tubing and free enzyme reactors.

A generic flow-through amperometric microenzyme sensor is described, which is based on semi-permeable dialysis tubing carrying the sample to be analyzed. This tubing (300 microm OD) is led through a small cavity, containing the working and reference electrode. By filling this cavity with a few microl of an appropriate enzyme solution, an amperometric enzyme sensor results. As the dialysis tubing is impermeable for large molecular species such as enzymes, this approach does not require any immobilization chemistry, and as a consequence the enzyme is present in its natural free form. Based on this principle, amperometric sensors for lactate, glucose, and glutamate were formed by filling cavities, precision machined in Perspex, with buffered solutions containing respectively, lactate-, glucose-, and glutamate-oxidase. All sensors showed a large linear range (0-35 mM for glucose, 0-3 mM for lactate, and 0-5 mM for glutamate) covering the complete physiological range. The lower detection limit was in the order of 15-50 microM. Applicability in flow injection analysis systems is demonstrated.     

Direct glucose determination in blood using a reagentless optical biosensor

This paper demonstrates that glucose determination in blood can be done directly (without sample pretreatment) using a reagentless reversible biosensor based on the intrinsic spectroscopic properties of peroxidase (HRP). The biosensor, prepared by HRP and glucose oxidase entrapment in a polyacrylamide gel matrix, works in continuous mode, presents a linear response range from 1.5 × 10⁻⁶ up to 5.5 × 10⁻⁵ M and can be used for at least 750 measurements; in the best conditions (0.1 M pH 6 phosphate buffer, HRP and GOx amounts in the polymersation mixture for the sensor film preparation 0.0165 and 0.0010 g, respectively) the minimum samples rate is 30 h⁻¹. For glucose determination, blood is simply diluted in water (until haemolysis is completed) and fed into the sensor without a cleaning step between samples; the blood absorption is corrected in a simple way by working at a proper reference wavelength. The biosensor signals have been mathematically modeled in order to facilitate the design of sensors based on the same idea for other biochemical compounds.