The present invention relates to a dialysis elec¬ trode or biosensor device suitable for use in medicine and the food, drink, pharmaceutical and environmental monitor¬ ing industries and other industries where it is desirable to analyse or monitor the presence of chemical substances.

Dialysis is a general sampling technique which can be used in conjunction with an appropriate analytical technique, such as electrochemical analysis, to analyse chemical substances recovered by the dialysis. Microdialy- sis is a technique for sampling in vi vo body fluids, for example, as described in an article by Urban Ungerstedt entitled "Microdialysis - A New Bioanalytical Sampling Technique" published in Current Separations, Volume 7, No. 2 (1986) by Bioanalytical Systems, Inc. It may be used in the electrochemical analysis of chemical substances ex¬ tracted from the brain and various other organs of the human or animal body. With this technique, a chemical substance diffuses into a microdialysis probe, which may be the size of a syringe needle, implanted in tissue and the concentration of the substance is determined outside the body. Particularly, the introduction of a microdialysis probe into the brain makes it possible closely to monitor the chemical events of the extracellular space, wherein chemical transmission involved in neuro ^' transmission of information takes place.

The problems with the dialysis technique are three¬ fold. Firstly, the dialysate is diluted into a flowing stream. Therefore, in order to have a sufficient quantity to measure, typically, a sample must be collected over ten to twenty minutes. This leads to the second problem that the technique has poor time resolution. Thirdly, the continuous removal by dialysis disturbs the concentration of the perfusing species in the region of the probe. With

a view to resolving the problem of slow response time, it has been proposed to use an on-line electrode for the analysis but even so there is a delay whilst the solution flows slowly from the probe to the electrode in the elec- -rochemical analysing equipment.

Fast response times of less than half a second may be obtained from implanted enzyme electrodes. However, the general problem wit such implanted electrodes, in which an enzymic reaction is monitored by sensing changes in elec¬ trical parameters, is that the enzyme becomes inactive. It is also not easy to supply a cofactor, such as, NADH. is difficult to sustain the enzymatic reaction for periods Furthermore, toxicity may be a barrier to use in clinical practice.

It is an object of the present invention to allevi¬ ate the problems experienced with hitherto known dialysis and electrode techniques of sampling and to provide a dialysis electrode device which produces rapid measurements of variations in the concentrations of chemical substances and, more particularly, the in vivo concentrations of chemical substances, such as, the important neurotransmit- ter, glutamate, occurring in the extracellular space in a human or animal brain.

The present invention consists in a dialysis elec¬ trode device comprising a hollow probe having an internal chamber and a dialysis membrane forming a wall of the chamber, a working electrode mounted in the chamber, con¬ duit means for supplying electrolyte to the chamber and removing electrolyte therefrom, and a reference electrode arranged to be electrically coupled to the working elec¬ trode by the electrolyte.

Conventional dialysis requires the continuous flow of perfusing solution as, for example, in the case of carrying out dialysis with a probe implanted in the brain or any other organ of a human or animal body, and relative-

ly large amounts of chemical substances may be removed. With the present invention, the dialysis electrode device may simply be primed intermittently with a flow of electro¬ lyte solution and electrical measurements of a particular dialysate are made with the probe remaining in situ in the organ. Because measurements are made when there is no flow through the device, disturbance of the organ tissue is minimised. Also, when the device is not in use all enzyme may be removed to prevent any depletion.

Conventional platinum electrodes may be used or conducting organic salts or other mediators, such as, TTFTCNQ or NMPTCNQ, can be electrochemically plated onto the working electrode. A target molecule, such as, gluta- mate diffuses through the dialysis membrane. Instead of being carried away by the flowing solution, it is immedi¬ ately consumed by the in situ enzyme electrode. Using a dialysis pump connected to the conduit means, the solution around the electrode can readily be changed. This means that the electrode can be supplied with fresh enzyme and cofactor, as well as different enzyme solutions, thereby enabling the performance of control and test measurements. It can also be replated with the same or a different media¬ tor.

Accordingly, the present invention alleviates the problem of short lifetime hitherto experienced with the enzyme electrodes of known electrochemical biosensors. Furthermore, it enables good time resolution and sensitivi¬ ty to be achieved and the control of the in vivo environ¬ ment of the working electrode. The device may also be made biocompatible.

Electrochemical enzyme electrodes of prior electro¬ chemical biosensors can only measure one chemical substance because a preselected enzyme is immobilised on the elec¬ trode. The present invention enables different enzyme loaded liquids to be used as electrolyte solutions to

detect different chemical substances. For example, the invention can be used to detect glutamate or acetylcholine in neurology applications, glutamate or ascorbate in the food industry and glucose in clinical diagnoses in vitro . In a modification of the invention, the device may be filled with dry powder enzyme as an electrolyte for emer¬ gency purposes.

The invention has two further advantages. Firstly, by flowing a known concentration of target analyte over the working electrode and making test measurements, the device can be calibrated so that it measures absolute, as opposed to relative, concentrations of analyte. This is impossible with conventional implanted enzyme electrodes or with conventional dialysis. Secondly, electrical current trans¬ mitted by interfering substances, such as, ascorbate in the case of brain microdialysis, can be mitigated or blocked by surrounding the working electrode with a suitable second enzyme. For example, in the case of microdialysis of the brain, interference from ascorbate may be blocked by using ascorbate oxidase.

Measurements of the analyte concentration may be produced by means of a potentiostat connected to the work¬ ing and reference electrodes of the dialysis electrode device. With this potentiometric method of measurement, the potential difference between the two electrodes is measured, when no current flows. As an alternative to the potentiometric method, current flow measurement is pre¬ ferred as this results in reduced errors. In this regard, the dialysis electrode device, preferably , includes an auxiliary or counter electrode arranged to be electrically coupled to the working electrode by the electrolyte. With this preferred method of measurement, the electrical cur¬ rent, caused by oxidation of the enzyme, is measured as it flows between the working and counter electrodes, the reference electrode being used to retain the working elec-

trode at a predetermined potential .

The dialysis electrode device may include a second working electrode which may be useful for compensating for spurious electrical signals , as well as measuring interfer¬ ence from electroactive molecules .

Conveniently, the probe comprises a semi-permeable dialysis membrane formed as a needle-like hollow shank , closed at its distal end . Such a configuration facilitates introduction of the probe into the matter to be tested . The shank may be made of suitable size for microdialysis procedures , f or example , not greater than 250 μm f or neurophysiological work . The length of the shank can be selected so that the probe alone , and no other parts of the device , is implanted in the test matter, thereby minimising any damage caused by introduction of the probe . The pas¬ sage in the hollow shank constitutes the internal chamber and the conduit means may comprise an inlet conduit pro¬ jecting into the passage to a position adjacent the distal end of the shank and an outlet conduit connected to the proximal end of the shank . At its proximal end, the hollow shank may be connected to an auxiliary chamber to which the outlet conduit is connected . The auxiliary chamber may also house the reference electrode and/or counter elec¬ trode . The second working electrode , if provided , may be spirally wound about the outside of the shank .

In order that the present invention may be more readily understood , reference will now be made to the accompanying drawings , in which : -

Figure 1 schematically illustrates an axial section through a microdialysis electrode device constructed in accordance with the invention; igure 2 is an axial section similar to Figure 1 and illustrating modifications ;

Figure 3 is a diagram illustrating the reaction scheme when the electrode device of Figure 1 is used to

measure glutamate; and

Figure 4 is a schematic illustration of a potentio¬ metric measuring circuit which may be used with the inven¬ tion.

Referring to Figure 1 of the drawings, the device comprises a shank 1 of needle-like configuration and formed from a hollow, semi-permeable, dialysis, fibre membrane. The shank 1 is closed at its distal end 2, which may be pointed, by an epoxy resin plug 3. The passage 4 within the hollow dialysis fibre shank 1 constitutes in internal chamber and, at its proximal end, the shank is connected to a tubular plastics body part 5, for example, made from polyethylene tubing, which defines an auxiliary chamber 6 communicating with the internal chamber 4 of the hollow shank. The shank and body 1,5 are arranged with their axes mutually parallel and with the shank disposed eccentrically with respect to the body. The body is sealed to the proxi¬ mal end 8 of the shank by means of a suitable sealing material 9, such as an epoxy sealing material, and is encased in a tubular housing 20 made from a stiffening material, such as stainless steel or glass tubing.

Projecting axially through the tubular body 5 and into the chamber 4 of the hollow shank is an inlet conduit or cannula 10 for supplying electrolyte solution to the chamber. The outlet end 11 of the inlet conduit 10 termi¬ nates at a position adjacent the distal end 2 of the shank, and spaced a small distance from the plug 3. Juxtaposed the inlet conduit 10 is a dialysis or first working elec¬ trode 12 which is insulated save for a length 13 thereof disposed within the dialysis fibre shank 1. A counter electrode 16 and a reference electrode 17 are also mounted in the auxiliary chamber 6.

Electrolyte solution supplied to the chamber 4 in the dialysis fibre shank 1, via the inlet conduit 10, flows from the ©utlet end 11 thereof, through the chamber 4 and

about the working electrode 12, and is removed from the device via the proximal end 8 of the shank, the auxiliary chamber 6 and an outlet conduit or cannula 18 projecting from the opposite end of the tubular body 5 to the. shank. The electrodes 12, 16, 17 and conduits 10,18 are secured in position at this opposite end of the tubular body 5 by a suitable epoxy sealing material 19, such as cyano acrylate sealing material.

In a modified embodiment illustrated in Figure 2, like reference numerals indicate similar parts to those of Figure 1. In this modified embodiment, the distal end of the uninsulated length 13 of the working electrode 12 is embedded in the epoxy plug 3 and the electrode serves as an internal reinforcing strut for the shank 1. Furthermore, a second working electrode 14 extends through the tubular body 5 and the sealing material 9 and is spirally wound about the exterior of the shank 1. The insulation of the electrode 14 is removed from the length 15 of the electrode wound about the shank.

In a typical dialysis electrode device as illus¬ trated in Figure 1 or 2, the dialysis membrane is made of cellulose. An upper length of the membrane of approximate¬ ly 5 mm is coated with the epoxy resin sealing material 9 leaving a probe length of approximately 4mm. The working electrode 12 may, for example, be Teflon coated platinum wire, with approximately 4mm of Teflon being removed from the distal end of the electrode to expose the length 13 of platinum wire. The reference electrode may be an Ag/AgCl electrode made from 75 μm Teflon coated silver wire, whilst the counter electrode 16 may be a silver electrode. The conduits 10,18 may be vitreous silica conduits.- In one embodiment suitable for microdialysis procedures, the tubular body 5 has an external diameter of approximately lmm, the tubular casing 20 has an external diameter of 1.5mm, the hollow dialysis fibre shank 1 has an external

diameter of 220 μm, the inlet conduit 10 has an external diameter of 140 μ , the first working electrode 12 is insulated 50 μm platinum wire and the second working elec¬ trode 14 is insulated 25 μm platinum wire.

Schematically illustrated in Figure 1 is the electrical circuitry via which electrical energy is sup¬ plied to the electrodes 12,16,17 and the electrical current in the working electrode 12 is detected in order to provide a measurement of the quantity of analyte diffused through the dialysis fibre shank 1 into the electrolyte contained in the chamber . The reference and counter electrodes 17,16 are interconnected via an amplifier 21 having its inputs connected to the reference electrode and ground and its output connected to the counter electrode. This ar¬ rangement maintains the counter electrode 16 at a fixed potential with respect to the reference electrode 17 and ground. The working electrode 12 is connected to one input of an amplifier 22 having its other input connected to a suitable electrical source for applying a potential differ¬ ence between the input and ground and, hence, across the electrodes. The output terminal 23 of the amplifier 22 is connected to a suitable device (not shown) for detecting the current flow through the working electrode and provid¬ ing a measurement of the quantity of analyte diffused into the chamber 4. The variable resistor 24 connected ^' across the amplifier provides for gain control.

In the embodiment of Figure 2 , the electrical potential is applied to the second working electrode 14 and the current flow in this electrode is measured by circuitry 22' ,23',2 ' similar to the circuitry 22,23,24 connected to the first working electrode 12.

-Alternatively to the current measuring systems shown in Figures 1 and 2 , analyte may be measured by a potentiometric system as schematically illustrated in Figure 4. In this event, the counter electrode 16 is not re-

quired and the reference electrode 17 is interconnected with the working electrode by the electrical circuitry. Hence, the working electrode 12 is connected to the posi¬ tive input of a first amplifier 25 having its negative input connected to the output of this first amplifier. The output of the first amplifier 25 is also connected to the negative input of a second amplifier 26, the positive input of which is connected to the output of a third amplifier 27 having its positive input connected to the reference elec¬ trode 17. The negative input of the third amplifier 27 is connected to its output. Changes in potential are detected at the output terminal 28 of the second amplifier 26 in order to produce an electrical signal representative of the quantity of analyte diffused through the dialysis fibre shank 1.

In carrying out a microdialysis procedure in the brain, for example, the shank 1 is introduced into the required position in the brain and its length can be prede¬ termined so that the shank, alone, and not the wider tubu¬ lar body part 5, is implanted thereby minimising, any damage caused. The electrodes 12,14,16,17 are connected_to elec¬ trical measuring circuitry, as described above, all poten¬ tials being reported with respect to the reference elec¬ trode 17. The inlet and outlet conduits 10,18 are connect¬ ed to a suitable microdialysis pump capable of pumping electrolyte solution into the probe at a flow rate of, for example, 0.5mm^ per in. The device is intermittently primed with an enzyme electrolyte solution, with the added advantage that fresh enzyme can be introduced when required or, as described below, mixtures of enzymes can be used, and electrical measurements are taken in situ of the con¬ centration of a chemical substance diffusing into the shank chamber 4 through the dialysis fibre membrane 1. The latter may be designed to allow or prevent flow of prede¬ termined chemical substances.

In carrying out a microdialysis procedure in the brain, two different techniques for measuring glutamate may be utilised. Firstly, a TTFTCNQ modified working electrode 12 may be used or, secondly, a platinum electrode. Utilis¬ ing the TTFTCNQ electrode as a mediator has the advantage or working at a low potential, reducing interference from some electroactive species, although ascorbate, the main interferent, is oxidized very effectively. With the second technique, oxidation of hydrogen peroxide has proved to be more reliable for measuring glutamate, resulting in a glutamate sensor with a 100% activity over a three day period. With a TTFTCNQ electrode 12, the device is fully active for only one day.

With the peroxide system (see Figure 3), the background electrolyte may be Krebs Ringer buffer which consists of the following mmol dm~^ concentrations, that is, Na Cl 136, KC1 2.54, KH ₂ P0 ₄ 1.18, NaHC0 ₃ 16, CaCl ₂ 1.18, MgSC^ 1.18 and glucose 10. The pH was adjusted to 7.4. All chemicals were of AnalaR quality. To measure glutamate, the enzyme glutamate oxidase is added to the buffer solution. A typical activity is 0.4 U mm" . The electrical current from oxidising the product of the enzyme reaction, hydrogen peroxide, is measured at a potential of 650 mV. Current voltage curves show that this potential gives a limiting current.

The disadvantage of the peroxide system is the high potential used for oxidation of the hydrogen peroxide. At this potential, there is considerable oxidation of electro- active interferents,such as, ascorbate, uric acid and HVA. In order to reduce interference from ascorbate, the working electrode 13 may be coated with Nafion or other suitable polymers. Tests also show that ascorbate can be removed with ascorbate oxidase and that the other interferents are either at a very low concentration or that their levels are stable enough so as not to interfere with the measurement

-li¬

ef glutamate .

With the dialys is electrode device o f Figure 2 having the second working electrode 14 and the probe cham¬ bers 4 , 6 containing both glutamate and ascorbate oxidases , the first working electrode 12 , an internal platinum elec¬ trode , measures enzymatically generated peroxide and , hence , measures glutamate , and the second working electrode 14 , an external platinum electrode , measures ascorbate but not glutamate . The device can be calibrated beforehand in vi tro in the concentration range of interest f or the in vi vo measurements so that it can then be used to produce measurements which indicate to a neurophysio logist how either or both glutamate and ascorbate vary with time .

The peroxide system , i s about 100 to 1000 times more sensitive that the TTFTCNQ technique . Tests in which the device is utilised as a glutamate sensor have shown real time measurement of glutamate changes during a re¬ sponse to a behavioural stimulus . This type of work has hitherto been impos s ible because there was no suitable method for measuring glutamate changes over short periods . Previously , glutamate had to be measured by one of three techniques :

( 1 ) derivatization with an absorbing marker fol¬ lowed by HPLC separation .

( 2 ) Araino acid analysis or

( 3 ) Enzymatic assay using glutamate dehydrogenase , measuring the NADH produced.

These prior methods rely on removal of samples from the biological tissue, which not only dilutes the glutamate but also requires long sampling intervals in order for sufficient glutamate to be collected .

The sensitivity of the three prior techniques (about n ) is lower than that produced by the device ac¬ cording to the present invention ( about 100 nM) , but this is unlikely to be a significant problem as physiological

changes in glutamate are of the order of 1-2 μM. In any case, the temporal resolution of the present device, that is 1 sec. compared to 20 min., outweighs any sensitivity advantage.

The main advantage of a dialysis electrode device as described above is the ability to control the local environment of the implanted electrode. Firstly, by using different enzymes one can select the analyte of choice in the same animal for the same probe. Secondly, ascorbate interference is removable so that, for the first time, sufficient sensitivity and time resolution is achieved and real time variations in glutamate levels can be measured. Thirdly, change in glutamate levels in response to a mild behavioural stimulus can be measured. As a result, neuro- chemical changes may be linked with behavioural studies. By suitable choices of enzymes, the electrode device may be used to measure real time changes in concen¬ tration of a wide variety of metabolites. For instance, good in vitro calibration plots have been obtained for glucose using glucose oxidase and for acetylchol.ine using acetyl choline esterase followed by choline oxidase. This provides the prospects of measuring metabolites such as these in vivo in the practice of clinical medicine.

Whilst particular embodiments have been described, it will be understood that modifications may be made with¬ out departing from the scope of the invention as defined by the appended claims.