In vivo voltammetric detection of rat brain lactate with carbon fiber microelectrodes coated with lactate oxidase.

To allow rat brain lactate measurement in vivo, a specific sensor based on a carbon fiber (phi = 30 microns) microelectrode coated with lactate oxidase was prepared. Combined with the differential normal pulse voltammetry measurement method, such a sensor, with a sensitivity of 9.15 +/- 0.91 mA.M-1.cm-2, provided a lactate linear response in concentrations ranging from 0.1 to 2.0 mM. The measurements performed appeared to be essentially insensitive to usual interference caused by the electroactive compounds present in the brain (ascorbic acid and peptides). In vivo detection performed in the cortex of the anesthetized rat led to the determination of a lactate concentration of 0.41 +/- 0.02 mM. Moreover, to validate the results obtained in vivo, an ex vivo determination of the lactate level was also performed in samples of brain tissue, plasma, and cerebrospinal fluid, using both voltammetry and a clinical analyzer with colorimetric-based detection. A good correlation was observed between the sets of data established by both methods.

Brain glucose: voltammetric determination in normal and hyperglycaemic rats using a glucose microsensor.

Pulsed voltammetry applied to glucose oxidase-coated carbon fibre electrodes (glucose sensor) was used for brain glucose determination in normal and streptozotocin-treated rats (experimental diabetes mellitus). Glucose levels increased in the frontal cortex of diabetic animals compared with the controls (+262%). Glucose levels were also increased in their CSF (+48%) and plasma (+64%), determined in ex vivo conditions. The validity of the glucose sensor determinations, as well as that of the experimental model of diabetes used, was checked using the Beckman glucose analyser and a radioimmunoassay for plasma insulin. Insulin, unlike glucose, was decreased in diabetic animals. The sensor described here ensures precise determinations and is suitable for use in experimental models where alterations in glucose metabolism occur.

In vivo brain glucose measurements: differential normal pulse voltammetry with enzyme-modified carbon fiber microelectrodes.

The enzyme glucose oxidase was immobilized on the surface of carbon fiber microelectrodes (CFMEs) either by cross-linking in glutaraldehyde vapor or by enzyme entrapment in electropolymerized films of m-phenylenediamine or resorcinol. The cross-linked enzymatic layer was, in the given conditions, covered with an additional membrane of Nafion or cellulose acetate. The prepared glucose sensors were tested using differential normal pulse voltammetry (DNPV, in which the scan comprises successive double pulses ("prepulse and pulse"), the prepulses are of increasing amplitude, and the current measured is the differential of the current existing between each prepulse and pulse). With properly chosen DNPV parameters, the response to glucose presented a peak at a potential of about 1 V versus an Ag/AgC1-reference, owing to the oxidation of enzymatically produced hydrogen peroxide. The calibration curves obtained (peak height/glucose concentration) were linear from 0.3-0.5 up to 1.5-6.5 mM and showed a sensitivity ranging from 1.4 up to 34.5 mA M-1 cm-2, depending on the sensor type. The DNPV response to glucose exhibited an essential insensitivity toward easily oxidizable interfering substances such as ascorbic acid and acetaminophen present at physiological concentrations. Peptides, the interfering species typical of the cerebral medium, were effectively retained by the above additional membranes. Concentration values of glucose in plasma and cerebrospinal fluid, determined in vitro from the DNPV peak height, agreed well with those measured by standard procedures. In the anesthetized rat, extracellular brain concentration of glucose was also monitored during administration of either insulin or glucagon. Under such pharmacological conditions, the changes observed in the peak height were in perfect agreement with the known effects induced by both substances.

Glucose-sensitive enzyme field effect transistor using potassium ferricyanide as an oxidizing substrate.

A glucose-sensitive field effect transistor was fabricated by immobilizing glucose oxidase on the gate of a pH-sensitive field effect transistor. Calibration curves of the biosensor were measured in phosphate and TRIS buffers in the presence of potassium ferricyanide. The use of the latter as an oxidizing substrate in the biocatalytic oxidation of glucose leads to an increase of the acidification rate of the solution inside the enzymatic layer, because three protons are now generated per one molecule of glucose instead of only one when the natural oxidizing cosubstrate, oxygen, is used. Depending on the concentration of ferricyanide we observe a 10-100 times increase of the biosensor response in concentrated buffer solutions and a substantial extension of its dynamic range. At sufficiently high concentrations of ferricyanide, the calibration curves in both buffers have a sigmoidal shape in linear coordinates with local pH changes on the surface of the field effect transistor reaching about two pH units in the saturation range. The resulting saturation of the curves at higher glucose concentrations is due to the inhibition of the activity of glucose oxidase at acidic pH by Cl- ions present in the solution. The proposed approach may be extended to allow the detection of a wide range of analytes using enzyme field effect transistors based on the enzymes for which reoxidation of the cofactor (coenzyme) leads to a liberation of H+ ions.