GB2259771A - Electrochemical throughflow enzyme biosensor - Google Patents

Abstract

The sensor comprises a flow chamber containing an immobilised enzyme electrode and an auxiliary electrode and, upstream of the flow chamber, an electrochemical preconditioning cell which nullifies interfering ions. The enzyme electrode may respond to glucose, lactate, glutamate or choline and the preconditioning cell is preferably a long thin platinum flow tube set at 1.5V which pre-oxidises species such as ascorbic acid before they can reach the flow chamber containing the detector. The flow chamber may also comprise a reference electrode and the reference electrode may be operated potentiostatically.

Description

IMPROVED ELECTROCHEMICAL ENZYME BIOSENSOR This invention relates to an electrochemical enzyme biosensor.

In vivo microdialysis is a powerful tool, but when it is associated with high pressure liquid chromatography, its possibilities are limited by the large number of samples which are collected for subsequent off-line analysis. One way to circumvent this problem is to couple microdialysis to on-line enzyme fluorimetric analysis, which provides information on a substrate of interest rapidly and with a good time resolution. A better alternative in some cases may be an enzyme electrode in a biosensor.

Thus, for example, glutamic acid (GLU) is now known to be the principal excitatory neurotransmitter in the mammalian brain, and an excessive GLU release into the synaptic space may play a major role in selective neuronal injury following various insults to the nervous system. There is therefore now a major need for accurate and detailed real-time information on GLU changes in extracellular fluid (ECF) in vivo.

Although GLU can be detected by amperometric response on an enzyme electrode in vitro, in vivo the ECF contains various species which are liable to interfere with that response. This is merely an example of a general diagnostic problem with enzyme electrodes. Therefore the invention provides an assay method which is liable to interference by ions, characterised by electrochemically nullifying such ions beforehand, e.g. by changing the oxidation number of the ion from a harmful to a harmless value, by discharging the ion altogether or by causing it to react yielding a harmless product.

According to the present invention, an enzyme electrochemical biodetector for fluid comprises an enzyme electrode in a flow chamber containing also an auxiliary electrode and preferably also a reference electrode, characterised in that upstream of the flow chamber a preconditioning electrochemical cell is provided for subjecting the fluid to oxidation or reduction before the fluid enters the flow chamber; the preconditioning cell is preferably poised at a fixed voltage, e.g. exceeding l.OV between its two electrodes, which for example can be a tube (e.g.

platinum) containing the flow and an axial counter electrode (e.g. stainless steel). The preconditioning is to eliminate chemical components in the fluid which would have generated an interfering signal at the enzyme electrode.

The tube may be elongated, that is having an aspect ratio (e.g. if circular a length:internal diameter ratio) of at least 20, preferably at least 100.

The enzyme electrode is preferably operated amperometrically i.e. potentiostatically.

The invention will now be described by way of example.

EXAMPLE 1 A microflow amperometric enzyme cell was made from two facing blocks (25 x 25 x 13mm), defining a 1 p1 cavity between them, one block accommodating an inlet tube and an outlet tube and the other a 3-electrode system. The working electrode was a 1.5-mm disk of platinised carbon paper (PCP) fitted into a small well, glued to a copper connector with carbon-powder-loaded epoxy resin. The remarkable properties of PCP make it especially suitable for preparing oxidase-based biosensors with minimal oxygen-dependency. The reference (Ag/AgCl) and auxiliary electrodes were inserted into two other wells. Glucose oxidase was immobilised on the PCP by absorption.

Electrochemical measurements and validation in vitro: A constant potential (0.6V) was applied to the working electrode by a potentiostat. The background current was allowed to fall to a steady and low level before starting the experiment. In vitro studies were carried out in degassed 7mM phosphate buffer saline (PBS) containing 145mM NaCl or artificial cerebral spinal fluid (CSF, composition in mmol/l: NaCl 125, KC1 2.5, Mac12 1.18, CaC12 1.26), flowing at 1 pl/min directly from the syringe pump or through a dialysis probe. Glucose was either included in the perfusing medium, or added as small aliquots of a concentrated glucose solution to the stirred medium bathing the probe.

Removal of interfering electroactive compounds was achieved by the use of a pre-oxidising cell (20mm length of platinum tube, O.lmm internal diameter) poised at 1.5V versus a stainless steel counter electrode, using a battery as voltage source. Its efficacy to oxidise endogenous ECF electroactive compounds was tested in vitro with a solution of PBS containing ascorbic acid (AA), dopamine (DA), dihydroxyphenyl acetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA), with concentrations much higher than basal or post-mortem ECF levels (200, 1, 1, 1, 0.1 and 1 pM, respectively). This solution was perfused through the device at lpl/min, and 5-minute samples collected at its outlet for HPLC analysis.

The biosensor response was found to be linear in the range 0-3mM glucose (approximately 200 nA/mM glucose) at lpl/min flow rate, with a detection limit of around 511M. The T95 response time to a step change in glucose concentration showed a variation of 0.5 to 1% over 5 hours, and this drift corresponded to a slight drop in the background current. These characteristics, which were not markedly altered even by repeated use over a month, clearly demonstrated the suitability of the system for measuring glucose on-line at slow flow rates. The PBS flowing through the cell at 1 pl/min produced a current of 250nA (cf.

200nA for lmM glucose). HPLC analysis showed that a potential of 1.4V applied to the platinum pre-oxidising cell decreased DA, DOPAC, HVA, 5-HT and 5-HIAA by 98, 91.5, 99.5, 99.5 and 99.6% respectively.

An in vivo recording of ECF glucose showed the glucose concentration in striatal dialysate was 1.13 + 0.06mM (mean +/- SEM; n = 6) during the control period. During the stabilisation-control period the pre-oxidising cell reduced the measured current by 11.6 i 1.9%.

In comparison to on-line enzyme fluorimetry, this biosensor may offer the advantage of providing information rapidly and with a better time resolution. The design of the prototype described herein may be suitable for on-line analysis of other substrates which can react with an enzyme which can be immobilized on an electrode able to detect such reaction, for example lactate, glutamate (see Example 2) and choline. Moreover, the electrochemical pre-treatment method could also be used in conjunction with a non-enzyme sensor or other type of assay procedure in which components of the analyte-containing fluid cause interferences (electrochemical or otherwise).

EXAMPLE 2 The amperometric response to glutamate was tested using a cell utilising glutamate oxidase instead of glucose oxidase but otherwise identical to that of Example 1. L-glutamate oxidase (GLO) from Streptomyces sp. X-119-6 was immobilised by absorption (Example 2A) or carbodiimide covalent coupling (Example 2B).

This enzyme catalyses the following reaction: Glutamate + 02 + H20 = 2-Ketoglutarate + H202 + NH3 The enzyme electrode responded rapidly to changes in glutamate concentration when tested in pH7 buffer solutions containing glutamate.

It was then tested using a glutamate-containing solution of artificial CSF, but also using the electrochemical pre-treatment procedure as described in Example 1. The background signal from oxidation of interferents was almost totally eliminated by the pre-treatment, whereas the signal from glutamate was unaffected.

Claims (9)

1. An electrochemical enzyme biosensor for fluid, comprising an immobilised-enzyme electrode in a flow chamber containing also an auxiliary electrode, characterised In that upstream of the flow chamber a preconditioning electrochemical cell is provided for pre-oxidising or pre-reducing chemical components in the fluid (which produce an interfering electrochemical signal) before the fluid enters the flow chamber.

2. An electrochemical enzyme biosensor according to Claim 1, wherein the preconditioning cell is poised at a fixed voltage.

3. An electrochemical enzyme biosensor according to Claim 2, wherein said voltage exceeds l.0V between the two electrodes of the preconditioning cell.

4. An electrochemical enzyme biosensor according to any preceding claim, wherein one electrode of the preconditioning cell has the form of a tube containing the flow.

5. An electrochemical enzyme biosensor according to Claim 4, wherein the tube has an aspect ratio of at least 20.

6. An electrochemical enzyme biosensor according to any preceding claim, wherein the enzyme electrode is operated potentiostatically.

7. An electrochemical enzyme biosensor according to any preceding claim, wherein the flow chamber contains also a reference electrode.

8. An electrochemical enzyme biosensor according to any preceding claim, wherein the enzyme reacts with glucose, lactate, glutamate or choline.

9. An assay method which is liable to interference by ions, characterised by electrochemically nullifying such ions beforehand.