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.     

Toward continuous glucose monitoring with planar modified biosensors and microdialysis. Study of temperature, oxygen dependence and in vivo experiment

Glucose biosensors based on the use of planar screen-printed electrodes modified with an electrochemical mediator and with glucose oxidase have been optimised for their application in the continuous glucose monitoring in diabetic patients. A full study of their operative stability and temperature dependence has been accomplished, thus giving useful information for in vivo applications. The effect of dissolved oxygen concentration in the working solution was also studied in order to evaluate its effect on the linearity of the sensors. Glucose monitoring performed with serum samples was performed to evaluate the effect of matrix components on operative stability and demonstrated an efficient behaviour for 72 h of continuous monitoring. Finally, these studies led to a sensor capable of detecting glucose at concentrations as low as 0.04 mM and with a good linearity up to 2.0 mM (at 37 degrees C) with an operative stability of ca. 72 h, thus demonstrating the possible application of these sensors for continuous glucose monitoring in conjunction with a microdialysis probe. Moreover, preliminary in vivo experiments for ca. 20 h have demonstrated the feasibility of this system.     

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.     

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.     

Glucose biosensor prepared by glucose oxidase encapsulated sol-gel and carbon-nanotube-modified basal plane pyrolytic graphite electrode

A new glucose biosensor has been fabricated by immobilizing glucose oxidase into a sol-gel composite at the surface of a basal plane pyrolytic graphite (bppg) electrode modified with multiwall carbon nanotube. First, the bppg electrode is subjected to abrasive immobilization of carbon nanotubes by gently rubbing the electrode surface on a filter paper supporting the carbon nanotubes. Second, the electrode surface is covered with a thin film of a sol-gel composite containing encapsulated glucose oxidase. The carbon nanotubes offer excellent electrocatalytic activity toward reduction and oxidation of hydrogen peroxide liberated in the enzymatic reaction between glucose oxidase and glucose, enabling sensitive determination of glucose. The amperometric detection of glucose is carried out at 0.3 V (vs saturated calomel electrode) in 0.05 M phosphate buffer solution (pH 7.4) with linear response range of 0.2-20 mM glucose, sensitivity of 196 nA/mM, and detection limit of 50 microM (S/N=3). The response time of the electrode is < 5s when it is stored dried at 4 degrees C, the sensor showed almost no change in the analytical performance after operation for 3 weeks. The present carbon nanotube sol-gel biocomposite glucose oxidase sensor showed excellent properties for the sensitive determination of glucose with good reproducibility, remarkable stability, and rapid response and in comparison to bulk modified composite biosensors the amounts of enzyme and carbon nanotube needed for electrode fabrication are dramatically decreased.     

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.     

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.     

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.     

Interference elimination of an amperometric glucose biosensor using poly(hydroxyethyl methacrylate) membrane

A permselective membrane fabricated from photo‐cross‐linked poly(hydroxyethyl methacrylate) (pHEMA) was studied as a potential selective membrane that can eliminate electrochemical interferences commonly faced by a hydrogen peroxide‐based biosensor. The quantitative selection of the permselective membrane was based on the permeabilities of hydrogen peroxide and acetaminophen (AC). AC was used as a model of the interfering substance due to its neutral nature. pHEMA membrane with the cross‐linking ratio of 0.043 was found to achieve a selectivity of hydrogen peroxide over AC of 10, while maintaining an acceptable degree of hydrogen peroxide response. A two‐layer glucose biosensor model consisting of glucose oxidase entrapped within a freeze‐thawed poly(vinyl alcohol) matrix and the cross‐linked pHEMA membrane was challenged with AC, ascorbic acid and uric acid. 0.2 mM AC and 0.2 mM ascorbic acid were completely eliminated. However, 0.2 mM uric acid could not be completely eliminated and still gave a bias of approximately 6.6% relative to 5 mM glucose. The results showed that cross‐linked pHEMA was quite promising as an interference eliminating inner membrane.     

Amperometric glucose sensor based on coimmobilization of glucose oxidase and Poly(p-phenylenediamine) at a platinum microdisk electrode

A miniaturized glucose biosensor in which glucose oxidase (GOD) and poly(p-phenylenediamine) (poly-PPD) were coimmobilized at the surface of a platinum microdisk electrode was developed and used successfully for amperometric determination of glucose. The performance of sensors prepared at different monomer concentrations and polymerization potentials with different media was investigated in detail. It was found that similarly to poly(o-phenylenediamine) (poly-OPD), (poly-PPD) noticeably eliminated the electrochemical interference of ascorbic acid, uric acid, and l-cysteine. The amperometric response of glucose with the biosensor under optimal conditions exhibited a linear relationship in the range of 5.0 x 10(-5) to 3.0 x 10(-3) M with correlation coefficient 0.9995. According to the Michaelis-Menten equation, the apparent Michaelis constant for glucose and the maximum steady-state current density of the poly-PPD/GOD-modified microelectrode were 3.94 mM and 607.5 microA cm(-2), respectively. The current density of the sensor responding to glucose in the linear range can reach 160 microA cm(-2) mM(-1), which is far greater than that obtained using poly-OPD and poly(phenol) film. In addition, the stability of the sensor was examined over a 2-month period.     

Glucose sensors based on electrodeposition of molecularly imprinted polymeric micelles: a novel strategy for MIP sensors

A voltammetric glucose sensor was prepared from novel molecularly imprinted polymeric micelles (MIPMs) through direct electrodeposition. The MIPMs, which were photo-crosslinkable and nano-scaled with high specific surface area, were prepared via macromolecule self-assembly of an amphiphilic photo-crosslinkable copolymer, combined with a molecular imprinting technique using glucose as the template molecule. A MIP film was formed in situ on the electrode surface by electrodeposition of the MIPMs, while photo-crosslinking led to a robust film which showed good solvent resistant to dissolution. With these features, the resulting sensor showed good response and selectivity towards glucose. In particular, the linear response of this glucose sensor ranged from 0.2 mM to 8 mM and its comparatively higher detection limit, about 10 mM, indicated numerous effective recognition sites among the polymer matrix due to the large specific surface area of MIPM. In addition, this MIP sensor also showed good stability and reversibility. The contribution of this work lies in not only the invention of a new type of glucose MIP sensor with good performance, but also the creation of a novel strategy to develop advanced MIP sensors for a wide range of templates in viewing of the versatility of the amphiphilic copolymers and the ease of control and applicability of the electrodeposition process.     

Amperometric determination of total assimilable sugars in fermentation broths with use of immobilized whole cells

A microbial sensor consisting of immobilized living whole cells of Brevibacterium lactofermentum and an oxygen electrode was prepared for continuous determination of total assimilable sugars (glucose, fructose and sucrose) in a fermentation broth for glutamic acid production. Total assimilable sugars were evaluated from oxygen consumption by the immobilized microorganisms. When a sample solution containing glucose was applied to the sensor system, increased consumption of oxygen by the microorganisms caused a decrease in the dissolved oxygen around the Teflon membrane of the oxygen electrode and the current of the electrode decreased markedly with time until steady state was reached. The response time was ≈ 10 min by the steady state method and 1 min by the pulse method. A linear relationship was found between the decrease in current and the concentration of glucose (<1 mM), fructose (<1 mM) and sucrose (<0.8 mM). The ratio of the sensitivity of the microbial sensor to glucose, fructose and sucrose was 1.00:0.80:0.92. The decrease in current was reproducible to within 2% of the relative standard deviation when a sample solution containing glucose (0.8 mM) was employed for experiments. The selectivity of the microbial sensor for assimilable sugars was satisfactory for use in the fermentation process. The additivity of the response of the microbial sensor for glucose, fructose and sucrose was examined. The difference between the observed and calculated values was within 8%. The microbial sensor was applied to a fermentation broth for glutamic acid production. Total assimilable sugars can be determined by the microbial sensor which can be used for more than 10 days and 960 assays.     

Biosensor for continuos glucose monitoring

A potentially implantable glucose biosensor for continuous monitoring of glucose levels in diabetic patients has been developed. The glucose biosensor is based on an amperometric oxygen electrode and glucose oxidase immobilized on carbon powder held in a form of a liquid suspension. The enzyme material can be replaced (the sensor recharged) without sensor disassembly. Recharging of the biosensor is achieved by injecting fresh immobilized enzyme into the sensor using a septum. Diffusion membranes made of silastic latex-rubber coatings over a microporous polycarbonate membrane are used. Calibration curves of the amperometric signal show linearity over a wide range of glucose concentrations-up to 500 mg/dL (28 mM), covering hypoglycemic, normoglycemic, and hyperglycemic conditions. Preliminary in vitro studies of the biosensor show stable performance during several recharge cycles (of 14 days each) over a period of 4 months. (c) 1994 John Wiley & Sons, Inc.     

Improved specificity of reagentless amperometric PQQ-sGDH glucose biosensors by using indirectly heated electrodes

Indirectly heated electrodes operating in a non-isothermal mode have been used as transducers for reagentless glucose biosensors. Pyrroloquinoline quinone-dependent soluble glucose dehydrogenase (PQQ-sGDH) was entrapped on the electrode surface within a redox hydrogel layer. Localized polymer film precipitation was invoked by electrochemically modulating the pH-value in the diffusion zone in front of the electrode. The resulting decrease in solubility of an anodic electrodeposition paint (EDP) functionalized with Osmium complexes leads to precipitation of the redox hydrogel concomitantly entrapping the enzyme. The resulting sensor architecture enables a fast electron transfer between enzyme and electrode surface. The glucose sensor was operated at pre-defined temperatures using a multiple current-pulse mode allowing reproducible indirect heating of the sensor. The sensor characteristics such as the apparent Michaelis constants K(M)(app) and maximum currents I(max)(app) were determined at different temperatures for the main substrate glucose as well as a potential interfering co-substrate maltose. The limit of detection increased with higher temperatures for both substrates (0.020 mM for glucose, and 0.023 mM for maltose at 48 degrees C). The substrate specificity of PQQ-sGDH is highly temperature dependent. Therefore, a mathematical model based on a multiple linear regression approach could be applied to discriminate between the current response for glucose and maltose. This allowed accurate determination of glucose in a concentration range of 0-0.1mM in the presence of unknown maltose concentrations ranging from 0 to 0.04 mM.     

Enzyme electrodes based on sono-gel containing ferrocenyl compounds

An amperometric-mediated glucose sensor has been developed by employing a silica sono-gel carbon composite electrode (SCC). The chosen mediators, ferrocene (Fc) and 1,2-diferrocenylethane (1), have been immobilized in the sono-gel composite matrix. The complex 1 has been employed for the first time as an electron transfer mediator for signal transduction from the active centre of the enzyme to the electrode conductive surface. After the optimisation of the construction procedure the best operative conditions for the analytical performance of the biosensor have been investigated in terms of pH, temperature and applied potential. Cyclic voltammetric and amperometric measurements have been used to study the response of both the glucose sensors, which exhibit a fast response and good reproducibility. The sensitivity to glucose is quite similar (6.7+/-0.1 microA/mM versus 5.3+/-0.1 microA/mM) when either Fc or 1 are used as mediators as are the detection limit ca. 1.0 mM (S/N=3) and the range of linear response (up to 13.0 mM). However, the dynamic range for glucose determination results wider when using 1 (up to 25.0 mM). The apparent Michaelis-Menten constants, calculated from the reciprocal plot under steady state conditions, are 27.7 and 31.6 mM for SCC-Fc/GOx and SCC-1/GOx electrodes, respectively, in agreement with a slightly higher electrocatalytic efficiency for the mediator 1.     

Enzyme entrapped polypyrrole modified electrode for flow-injection determination of glucose

Immobilization of glucose oxidase in electropolymerized polypyrrole film on the surface of a platinum wire electrode, provides a convenient sensor for flow-injection glucose determination. An upper limit of linear response for 100 microliters injected sample volume was estimated as 20 mM, whereas a 500 microliters injected sample volume gave an estimated detection limit of 0.5 mM. A simple electrode preparation procedure allows quick electrode renewal before each series of measurements.     

A contact lens with embedded sensor for monitoring tear glucose level

We report the design, construction, and testing of a contact lens with an integrated amperometric glucose sensor, proposing the possibility of in situ human health monitoring simply by wearing a contact lens. The glucose sensor was constructed by creating microstructures on a polymer substrate, which was subsequently shaped into a contact lens. Titania sol-gel film was applied to immobilize glucose oxidase, and Nafion? was used to decrease several potential interferences (ascorbic acid, lactate, and urea) present in the tear film. The sensor exhibits a fast response (20s), a high sensitivity (240 μA cm(-2) mM(-1)) and a good reproducibility after testing a number of sensors. It shows good linearity for the typical range of glucose concentrations in the tear film (0.1-0.6 mM), and acceptable accuracy in the presence of interfering agents. The sensor can attain a minimum detection of less than 0.01 mM glucose.     

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.     

Improvement of the performance of H2O2 oxidation at low working potential by incorporating TTF-TCNQ into a platinum wire electrode for glucose determination

A micro-biosensor was constructed by incorporating the organic conducting salt tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ) into a platinized platinum (Pt) wire and further covering with the electrochemical polymerical heteropolypyrrole film, in which glucose oxidase (GOx) was entrapped. The enzyme electrode can sensitively determine glucose at a low working potential, mainly based on the oxidation of H2O2. The incorporated TTF-TCNQ can significantly improve the oxidation of H2O2 on the electrode, although a part of the TTF-TCNQ functions as a mediator. Compared with the same electrode prepared without TTF-TCNQ incorporated, the TTF-TCNQ modified electrode had better performance characteristics at a working potential of 200 mV (versus SCE). The response time to 90% of the steady value was shortened from about 40 s to less than 10 s, the lower limit of the linear response was greatly extended from about 1.6 mM to 10 microM, the linear range was shifted from 1.6-10.0 to 0.01-5 mM and the sensitivity was increased from about 1 to 1.5 microA/mM. The electrode was quite stable. For continuous operation, the electrode could work for about 5 weeks and only lost 60% of its original sensitivity. Stored at 4 degrees C for intermittent determinations, the electrode kept 80% sensitivity for over 6 months. Due to covering the electrode with a non-conductive heteropolypyrrole film, ascorbate, urate and 4-acetamidophenol caused only negligible current response at an applied potential of 200 mV.     

Glucose enzyme electrode using cytochrome b(562) as an electron mediator

We demonstrate the construction of glucose sensors employing pyrroloquinoline quinone (PQQ) glucose dehydrogenase (PQQGDH) from Acinetobacter calcoaceticus and glucose oxidase (GOD) from Aspergillus nigar coupled with Escherichia coli soluble cytochrome b(562) (cyt b(562)) as electron acceptor. PQQGDH and GOD do not show direct electrochemical recycling of the prosthetic group at the electrode surface leading to a corresponding current signal. We constructed PQQGDH and GOD electrodes co-immobilized with 100-fold molar excess of cyt b(562) and investigated the electrochemical properties without synthetic electron mediators. PQQGDH/cyt b(562) and GOD/cyt b(562) electrodes both responded well to glucose whereas no current increase was observed from the electrode immobilizing enzyme alone. The detection limits for the PQQGDH/cyt b(562) and GOD/cyt b(562) electrodes were 0.1 and 0.8 mM, respectively, and their linearity extended to over 2 and 9 mM, respectively. These results demonstrate that a sensor system can be constructed without a synthetic electron mediator by using a natural electron acceptor. Furthermore, we have demonstrated the potential application of cyt b(562) in direct electron transfer type sensor systems with oxidoreductases whose quaternary structure do not contain any electron transfer subunit.