CA1212146A - Sensor for components of a liquid mixture - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A sensor electrode to detect one or more compon-ents in a liquid mixture comprises an electrically conduc-tive material having at least an external surface, the com-bination of an enzyme catalytic for a reaction of the desi-red component, and a mediator comprising a ferrocene which transfers electrons from the enzyme to the electrode when such catalytic activity takes place. It can be used with bacterial glucose dehydrogenase or glucose oxidase as the enzyme and/or ferrocene or a ferrocene derivative as the mediator compound to give electrodes with improved linearity, speed of response and insensitivity to oxygen.

Description

1. Lyle his invention relates to equipment and methods for detecting the presence of, measuring the amount of, and/or monitoring the level of one or more selected components in a liquid mixture.

While use may be made of this invention in chemical industry, especially where complex mixtures are encountered (erg. in food chemistry or biochemical engineering) it is of particular value in biological investigation and control techniques. More particularly, it lends itself to animal or human medicine, and in particular to in viva measuring or monitoring of components in body fluids.

or convenience, the invention will be described with reference primarily to one such procedure, the lo determination of glucose in a diabetic human subject, by the use of equipment which, while usable on a specific or occasional basis also lends itself to temporary or permanent implantation. However, while the provision ox an plan table glucose sensor is a major object of the invention other and broader objects are not hereby excluded.

In viva glucose tensors have already been proposed. One proposal is based on direct oxidation of .~.

-- ~21~14~

2.
glucose at a catalytic platinum electrode (see Hormone and Metabolic Research, supplement Series Jo. 8, pup 10-12 (Betty suffers from -the drawback of being non-specific and of being easily poisoned by interfering substances. Another proposal, for a procedure more specific to glucose, involves the use of glucose oxidize on an oxygen electrode (Ad. ~xpOMed.Biol, 50 pup 189-197 (Betty is not very responsive to the high glucose concentrations. Other systems using glucose oxidize have been proposed but not fully investigated for in viva methods, see e.g. J. Solid-Phase ~iochem. 4 pup 253 - 262 (1979~.

The inventors Howe recently carried out in vitro studies of enzyme-catalysed reactions using a mediator in solution to transfer the electrons arising from the enzyme, during its action, directly to the electrode, as described in Biotechnology wetters 3 pup 187 - 192 (1981~.

It has now 'oxen realized that mediator compounds 20 can be associated with the sensor electrode structure thus rendering such electrodes available for use by in viva methods.

In one aspect the present invention consists in a sensor electrode for use in liquid mixtures ox 25 components for detecting the presence of, measuring the \

amount of, and/or monitoring the level of, one or more selected components capable of undergoing an enzyme-cataly-sod reaction, the electrode being composed of electrically conductive material and comprising, at least at an external surface thereof, the combination of an enzyme and a mediator comprising a foreseen which transfers electrons between the enzyme and the constructive material of the electrode when the enzyme is catalytically active to produce a current rep-resentative of said activity.
Preferably the electrode is designed to determine glucose in viva. The enzyme is therefore preferably a glut cove oxidize, or possibly a glucose dehydrogenase, for exam-pie a bacterial glucose dehydrogenase.
Glucose oxidize (~-D-glucose:oxygen oxidoreductase, of enzyme classification HO 1.1.3.4) is a well known type of enzyme. Bacterial glucose dehydrogenase is of more recent discovery, and is believed to be a quinoprotein with a polyp I cyclicquinone prosthetic group (PQQ). Reference is made to Dune et at TUBS, (Oct. 1981) 278 - 280 and Arch. ~icrobiol (1982) 131.27-31.

Use of such a bacterial glucose dehydrogenase in the present invention has certain advantages over the use of a glucose oxidize. The major advantage is that i-t can give an oxygen-insensitive glucose sensor, since the enzyme does not use oxygen as an electron acceptor. A suitable enzyme can be purified (as described in more detail below) either by conventional chromatographic techniques or by two-phase aqueous partition from a range of micro-organisms. A pro-- furred micro-organism is Acinetobacter calcoaceticus but various Gluconobacter species ego. Gluconobacter oxidant) or Pseudomonas species (e.g. Pseudomonas fluoresces, Pseudo-_ monks aeruginosa) can also be used.

~Z~Z~46 Mediator compounds which may be used in accordance with the invention all possess the electron-transfer prop-arty referred to above.

S The mediator compound is a foreseen or foreseen derivative.

~21~6 A foreseen has, as its fundamental structure, an iron atom held "sandwiched" by dative bonds between two pentadienyl rings. It is an electroactive oryanome-tallic compound, acting as a pH-independent reversible one-electron donor. Various derivatives are available (e.g. with various substituents on the ring structure, possibly in polymer form) differing in redo potential, aqueous volubility and bonding constant to glucose oxidize or bacterial glucose dehydrogenase enzyme.
For instance, the redo potential of the parent compound is +422 my us THE. By introducing functional groups on to the ring system, Ego can be varied between ~300 and ~650 my. Moreover, the water-solubility of the carboxyl-substituted fierceness is greater than that of the parent compound. Further description will be found in Queen T., 1977, AS Symposium Series, 38, 154 Among specific mediator compounds of this type are foreseen itself, l,l'-ferrocene dicarboxylic acid, dim ethyl foreseen, and polyvinyl foreseen, e.g. of average Milwaukee-far weight of about 16000.

The electrically conductive material of the elect trove itself can be a metal, particularly silver, or carbon either as a preformed rod or as an electrode shape made up from a paste of carbon particles. Surface condition of the electrode is usually important If metal, the surface can be roughened where it contacts the active materials (enzyme and/or mediator). If solid carbon, the surface can be "ox-dosed" i.e. previously heat-treated in an oven with oxygen access.

212~6 Of the -two types of enzyme listed, the dehydrogen-aye is preferred Certain combinations of the above materials, and certain configurations of electrode, are preferable in pray-lice.

I

SLY

Optionally, enzyme immobilization materials, or polymeric electrode admixtures e.g. TEFLON*, or long-chain alkyd derivatives of mediators of increased molecular weight and thus decreased mobility, can be incorporated.

In a particularly valuable form of the invention, however, the electrode comprises a carbon core, a layer of foreseen or a foreseen derivative a-t a surface *TRADE MARK

thereof and a layer of glucose oxidize or glucose dehydrogenase at the surface of the foreseen layer.
the enzyme layer is preferably immobilized at the surface of the underlying mediator, retained in a self-sustaining gel layer thereupon and/or has a retention layer there over permeable to the glucose molecule.

The carbon core can itself be solid or a stiff paste of particles. normally, it will present a smooth surface for the foreseen or foreseen derivative, which may be adhered thereto in a umber of jays, for example, (a) or a monomeric foreseen or foreseen derivative, by deposition from a solution in a readily evaporatable liquid erg. an organic solvent such as Tulane.
(b) For a ferlocene polymeric derivative, deposition prom a readily evaporable organic solvent for the polymer such as chloroform. J. Polymer Sat. 1976, 14 2433 describes preparation ox a polyp errocene of average molecular weight about 16000 which can be deposited in this way.
I For a polymerisable ferrocene-type monomer, by electrochemicall~ induced polymerization in situ, e.g. by dissolving vinyl foreseen in an organic electrolyte containing tertiary bottle ammonium per chlorate in concentration about EM
and depositing at a potential of 700 my '10 .
vinyl foreseen radicals as a polymer in situ, (d) By covalent modification of the carbon electrode e.g. by carbo-diimide cross lining of the foreseen or foreseen derivative on to the carbon.

he enzyme to be coated on to the foreseen or foreseen derivative can be the glucose oxidize or the bacteria glucose dehydrogenase. the glucose oxidize can be immobilized to the underlying surface e.g. by the carbo-diimide material DCC (1-cyclohex~ (,2-morpholino ethyl) carbo-diimide metho-~-toluene sulphonate~ winch gives a thin strongly bound layer, a good linear response to lo glucose concentrations, and oxygen insensitivity (because of the competition from the ~errocene with oxygen for electrons trays-furred to the enzyme redo center from the substrate).
Using DCC immobilization of glucose oxidize on foreseen also extends the top end of the linear range of the sensor from about em to Ox Other methods of immobilization, or oilier forms of protection erg. incorporated into a self-supporting gelatin layer, are also possible.

he bacterial glucose dekydrogenase can also be immobilized at the mediator surface, but may be merely deposited from an evaporatable solution, or held in a gelatin layer, `' I 6 11 .
Optionally, but preferably when being used on live blood, a protective membrane surrounds both the enzyme and the mediator layers, permeable to water and glucose molecules. this can be a film of dialysis membrane resiliently held erg. by an elastic O-ring. It can however also with advantage be a layer of cellulose acetate, e.g. as formed by dipping the electrode into a cellulose acetate solution in acetone.

It will be apparent that while the invention has primary relevance to a sensor electrode, en-specially such an electrode specific for glucose, t also relates to the combination of such an electrode and temporary or permanent implantation means, e.g. a needle-like probe. Also, such an electrode, connected or connectable, with signal or control equipment, more especially with an insulin ad minis-traction means, constitutes an aspect of the invention.
Moreover, a method of monitoring a diabetic subject involving the use of a temporarily or permanently imp planted electrode as described above is also within the scope of the invention.

he electrodes according to the invention permit the manufacture of an improved macro-sensor for use in hospital analytical glucose-sensing in-struments of the existing type. the advantages compared . . .

'1 2 iLZ~L~a6 to Nemo instruments would be that the increased linear range together Truth very low oxygen sensitivity would allow omission of the dilution step invalid in blood analysis in current instruments. Moreover, as described in more detail below, the response times of such electrodes are short (24 - 36 seconds for 95% of steady state depending on complexity of solution The electrodes of the invention, on the macro-scale could be incorporated into simple, cheap electronic digital read-out instruments for doctors surgeries or diabetic home-testing kits.

Use of a smell version of the macro-sensor would be possible in a device which automatically takes a blood sample from the Ginger, brings it into contact with the sensor, amplifies the signal and gives a digital readout. Use of a micro-version of the sensor in a watch type device for monitoring glucose interstitial fluid in the skin could also be envisaged.
It would be worn on the wrist and would have a disk potable sensor cartridge in the back with one or more separate, fine, needle type sensors. Each would feed into the electronics which if several sensors were used would cross-refer the current inputs to ensure reliability.

Lo Connection of such devices to external insulin delivery systems could act as a feedback control loop for an insulin pump. Indeed, such a device could be housed in -the Connally used to feed insulin into -the body from a pump and again serve as a sensor for the feedback loop. Other uses such as a hypoglycemia alarm, or digital read-out monitor, are also possible.

The invention will be further described with rev-erroneous to the following Examples 1 to 3 and to the accompany-in drawings, in wish Figure 1 is a diagrammatic longitudinal cross-section through a glucose sensor electrode;
Figure 2 is a graph of the current sensed by the electrode of Figure I against glucose concentration;
Figure 3 is a diagrammatic longitudinal cross-section of the electrode of Figure 1 located within a hype-dermis needle;
Figure 4 is a diagrammatic longitudinal cross-section through a further glucose sensor electrode;
Figure 5 is a graph analogous to Figure 2 for the electrode of Figure 4; and Figure 6 is a graph analogous to Figure 2 for an electrode incorporating a glucose dehydrogenase.

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. t 14 ~212~
Example 1 Purification of Quinoprotein Glucose ~enydrogenase (GDH) from Acinetobacter calcoacetlcus (a) Growth of Organisms Strain ~CTC 7844 was grown on sodium succinate (20 glue) in batch culture at pi 8.5 and 20 I Cells were harvested after 20 hours Aye= 6.0) using a ~harples centrifuge, and stored frozen.
(b) Purification of Glucose Deh~drogenase the method it based on the method ox J A Dune et at (Arch Microbial, 1982 vise swooper) but with modifications as follows.
1. loo g. of cells were thawed, resuspended in 3 300 ml. of 56 my Tracy my Gleason and treated for 20 minutes at room temperature with 60 my. lyxozyme~

lo 2. Briton X-100 extracts were combined and treated with 0~01 mgml 1 of deoxyribomlclease I for 15 minutes at room temperature. The resulting suspension was then centrifuged at 48000 xg for 25 minutes at 4 C.
the supernatant from this centrifugation was then treated with ammonium sulfite. the yellow protein precipitating between 55 and 70% ammonium sulfite was resuspended in 36 my Russ my Gleason containing 1%
Briton X - 100 anddialysed against to buffer at 4C
for 5 hours.

25 3- Active fractions from the CM suffers C1-6B

15~
column were combined and concentrated using Millipore CX-30 immersible u3trafilters.
Example 2 Purification of ~uinoprotein Glucose Dehydxogenase from Acinetobacter calcoaceticus (alternative method) (a) Growth of Organisms the method ox Example 1 was repeated.
(by Purification of GDH
- the method is based on the partitioning of proteins between two liquid phases. the steps were:-1. Cells were thawed and resuspended at 3 ml/g wet weight in 50 my sodium phosphate, pi Zoo. whey were then prickled on ice and passed once through a Stansted pressure cell (made by Stansted fluid Power to Stansted, Essex, US) at 25000 psi. this provided the cell-free extract.

2. The cell-free extract was then mixed for 15 minutes at room temperature with 50% (ivy) polyethylene-glycol 'loo, 50% (w/v) sodium phosphate, pi 7.0 and distilled water in the proportions of 2:4:3:1 respectively. this mixture was centrifuged at 5000 rum for 5 minutes to break the emulsion.

I the lower layer was aspirated off and desalted immediately, by either diafiltration using an Am icon*
hollow-fibre ultrafiltration cartridge of 1000Q met cut off, or by passage through a Sephadex G50~(medium grade) gel filtration column.

16.
4. the resulting solution was concentrated using an Am icon Polo membrane in a nitrogen pressure cell.

Example 3 Interaction between foreseen and glucose oxidize DC cyclic voltmeter was used to investigate the homogeneous kinetics of the reaction between foreseen and the glucose oxidize enzyme under substrate excess conditions. A two compartment electromechemical cell of 1.0 ml volume fitted with a ~uggin capillary was used. the cell contained a 4.0 mm gold disc working electrode, a platinum gauze counter-electrode and a saturated calmly electrode as a reference A series of voltamogra~s for foreseen was recorded at scan rates of 1-1000 mVs~1 in 50 my potassium phosphate buffer, pi I the data showed that the mediator acted as a reversible, one-electron acceptor owe MY
SUE.

Addition of 50 my glucose has no discernible effect on the electrochemistry of the mediator (500~m).
Upon addition of glucose oxidize (lo m), however, an enhanced anodic current was observed in the ~oltamogran at oxidizing potentials with respect to the mediator. this 25 indicated catalytic regeneration of the reduced form of the mediator by glucose oxidize. Quantitative kinetic data was obtained for this reaction using an established I 3LZ14~
procedure (Nicholson, US and Skein, J., 1~64, Anal. Chum., 36, 707). The mediator gave a second order rate constant for the reaction between ferricinium ion and reduced glucose oxidize of K=104m is 1. This ability of the ferricinium ion to act as a rapid oxidant for glucose oxeyes facilitates the efficient coupling of the enzymic oxidation of glucose.

The procedure of Example 3 was repeated using 1,1l-foreseen dicarboxylic acid instead of foreseen. The value of Ho ' was determined to be +420 my, and the second order rate constant of the ferricinium ion and reduced glucose oxidize was again 104m lo 1, thus confirming the conclusions drawn from Example 3.

Glucose/Oxidase Dim ethyl Foreseen Mini electrode for in viva glucose sensing in skin A graphite rod 13 (Figure 1) with an oxidized sun-face, 30 mm long x 0.9 mm diameter is glued with epoxy resin into a nylon tube 14 25 mm long, 0.9 mm inside diameter, 1,3 mm outside diameter. The end 15 of the electrode is dipped into a solution of dim ethyl foreseen, (10 mg/ml) in -Tulane, and the solvent is then allowed to evaporate.
The end 15 of the electrode is placed into a soul-lion of water soluble DCC (25 mg/ml) in acetate buffer, pi 4.5 for 1 hour. It is then rinsed, in buffer only, for 5 minutes and thereafter placed in a solution of glucose ox-dBase (10 mg/ml~ in acetate buffer, pi 5.5, for I hours before again rinsing in buffer. The tip of -the electrode 15, with the layers of dim ethyl foreseen and immobilized enzyme is then dipped into a solution of cellulose acetate dozily-vied in acetone and formamide and put into ice water for several minutes, to give a protected and stable electrode.

lZlZ~6 This electrode was connected to a potentiosta-t, together with a suitable counter electrode and calmly rev-erroneous electrode and placed in a solution containing glucose.
The potential of the working electrode is kept at -~100 my to 300 my relative to the calmly electrode, i.e. as low as possible to avoid oxidation of potentially interfering sub-stances. A current is produced which is proportional to the glucose concentration. The time for 95% of response is less than 1 minute and the electrode gives a near linear response over the range 0-32 my glucose, as shown in Figure 2. Slot-loss of activity foreseen (due to slow loss of ferrociniumion) can be minimized by keeping the electrode at a potent trial between 0 and -100 my vs. a standard calmly electrode when not in use.

Figure 3 shows in section an electrode structure in which an electrode (references as in Figure 1) of much smaller size is held within a hypodermic needle 16 plugged at its point 17 but with side windows 18 for passage of blood or other body fluid. The small size of such an elect trove and its linear response over a large range of glucose concentrations makes it possible to use the electrode for in viva glucose determination on both severely diabetic and normal individuals.

EXAMPLE
Glucose Oxidase/Ferrocene - In vitro sensor A carbon rod 19 (Figure 4) Ultra carbon, grade Us, 6 mm x 15 mm) with a metal connector 20 secured in one end was sealed in glass tubing 21 (borosilicate, 6 mm I'd. x mm) with an epoxy resin (Araldite). (Not shown). The exposed surface at 22 was polished with emery paper and washed with distilled water. The entire rod was heated in an oven for 40 h at 200C to given an oxidized surface at 22.
1 1 of foreseen (20 mg/ml in ~oluene) was pipe-.

~2121~6 -ted on-to the oxidized surface and allowed -to dry completely.
The rod was then placed in 1 ml of water-soluble DCC (25 my/
ml in Old acetate buffer, pi 4.5) for I mix a-t room -them-portray. The rod was then washed in 0.2 M carbonate buffer, S pi 9.5 and placed in a glucose oxidize solution (Sigma type X, 12.5 mg/ml) for I hours at room temperature. It was finally washed with water with a pi 7 buffer containing 0.2 g/l glucose) and stored at 4C.

The characteristics of the above electrode were determined in a nitrogen-saturated buffer solution (0.2M
Nope, pi 7.3) and are shown in Figure 5. The curve is fin-ear from 2 to 25 my glucose and reaches saturation current at 100 my in glucose.
In separate tests with an air-saturated buffer a-t my glucose the current was measured as being a-t least 95%
of that produced in the nitrogen-saturated buffer.

Response time was also measured, being the time taken to achieve 95~ of maximum current for the given glut cove concentration. With the nitrogen-saturated buffer an electrode as described above had a response time of 24 sect onus at 2 my glucose and 60 seconds at 6 my glucose. With the same buffer, such an electrode modified by a cellulose acetate membrane coating prudes as in Example 5) gave response times of 36 seconds (2 my) and 72 seconds (6 my).
With blood this modified electrode gave response times of 36 seconds blood with a known 2 my glucose content) and 72 seconds blood at a known 6 my glucose content).

Electrodes above were stored in 20 my Nope, pi 7 for 4 weeks at 4C as a stability test and thereafter reexamined as above. The results were within 10% and usually within 5 of results with a freshly made electrode.

- 19 - .

.

12~ L46 Glucose Dehydrogenase/Ferrocene A stiff carbon paste was made up from 1.6 g of Derek activated charcoal and 2.5 ml of liquid paraffin. A
Pasteur pipette of 6 mm internal diameter was blocked 2 mm from its wide end by a silver disc to which a connecting wire was soldered. The space between the disc and the end of the pipette was filled with the carbon paste, and the surface of the paste was polished with paper until smooth and even.
A single 20 microlitre drop of a Tulane 25 soul-lion of foreseen (20 Mel was placed on the smooth surface and allow to spread and evaporate to leave a film of -the foreseen.
A further drop of 25 microlitres of bacterial glut cove dehydrogenase solution as obtained in Example 1, con-twining between 1 and 10 my. of protein per ml, was placed on this foreseen surface and allowed to spread.
A cover of dialysis membrane was secured over the so-coated end of the electrode by a ~ight-fitting O-ring.

EXAMPLE
Glucose Deh~_rogenanse/Ferrocene The procedure of Example 7 was repeated but using as electrode the same carbon paste packed into the space defined between the end of a length of nylon tubing and a stainless steel hypodermic needle shaft inserted therein terminating 2 mm. short of the tubing end, so as to define a small electrode body. The electrode was further fabricated using Only 5 microlitres of the foreseen solution and 1 microlitre ox the enzyme solution.

glucose Dehydrogenase/Ferrocene The procedure of Example 7 was repeated using as electrode a solid carbon rod (Ultra carbon grade Us 6 mm die meter) within a Pyrex* glass tube 3 cm long and 6 mm inter-net diameter and connected to a stainless steel hypodermic shaft, giving a construction similar -to that shown in Fig-use 4. The end of the carbon rod was polished smooth with emery cloth and aluminum oxide powder prior to the applique-lion of the foreseen solution.

Glucose Dehydrogenase/Ferrocene A gelation-entrapped glucose dehydrogenase was prepared by mixing at 37C, 25 my gelatin, 0.5 ml of the glucose dehydrogenase solution as described in Example 7 and 2.5 microlitres of TIMED. After complete dissolving of the gotten 200 microlitres of the solution was spread over an area of 2 cm2 and allowed to dry under a stream of cold air.
A disc of 0.25 cm2 area was then used instead of the drop of enzyme solution in Example 7.

Glucose Dehydrogenase/Ferrocene Example 10 was repeated using a disc of the gel of 1 mm2 area and applying it instead of the drops of enzyme solution in the construction of Example 8.

The results obtained from the electrodes described in Examples 7 to 11 are all similar, and show a very specie lie electrode of low oxygen sensitivity. By way of example, the electrode of Example 10 was calibrated and gave the results shown in Figure 6.
Devices such as shown in the Examples offer ad van-. .
`

Lo 6 taxes over most the of the enzyme-based sensors currently available. When compared to such sensors prior to dilution steps, the present electrode has an equal or faster response time, the ability to operate under anaerobic conditions, greater oxygen insensitivity (important in blood samples,.
where oxygen concentration is variable), extended linear range covering the complete physiological range and compare-bye specificity, stability and ease of manufacture.

Claims (21)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A sensor electrode for use in a liquid mixture of components, said electrode being responsive to the presence of at least one selected component capable of undergoing an enzyme-catalysed reaction, the electrode being composed of electrically conductive material and comprising, at least at an external surface thereof, the combination of an enzyme and a mediator comprising a ferrocene which transfers elec-trons beween the enzyme and the contactive material of the electrode when the enzyme is catalytically active to produce a current representative of said activity.

2. A sensor electrode as claimed in claim 1, in which the enzyme catalyses a reaction of glucose whereby there is provided a glucose sensor.

3. A sensor electrode as claimed in claim 2, in which the enzyme is a glucose oxidase.

4. A sensor electrode as claimed in claim 2, in which the enzyme is a bacterial glucose dehydrogenase.

5. A sensor electrode as claimed in claim 4, in which the glucose dehydrogenase is that separated from Acinetobacter calcoaceticus.

6. A sensor electrode as claimed in claim 1, 2 or 3, in which the mediator is selected from ferrocene, 1,1'-ferrocenedicarboxylic acid, dimethyl ferrocene, and polyvinyl ferrocene.

7. A sensor electrode as claimed in claim 1, 2 or 3, in which the electrode is made of a material selected from silver, carbon particle paste and solid carbon.

8. A sensor electrode for use in a liquid mixture including glucose, to be responsive to the presence of glucose therein, the electrode being composed of carbon, a layer of ferrocene at an external surface thereof as an electron-trans-ferring mediator compound, and an enzyme chosen from glucose oxidase and bacterial glucose dehydrogenase located upon the layer of mediator compound.

9. A sensor electrode as claimed in claim 8, in which the ferrocene is deposited on the surface from a readily evaportable organic solvent therefor.

10. A sensor electrode as claimed in claim 8, in which the ferrocene is in polymeric form and produced at the surface by polymerisation of the corresponding monomer.

11. A sensor electrode as claimed in claim 9, in which the ferrocene is bonded to the carbon electrode by carbo-diimide cross-linking.

12. A sensor electrode as claimed in claim 8, in which the enzyme is a glucose ozidase immobilized on the mediator by DCC.

13. A sensor electrode as claimed in claim 8, in which the enzyme is a bacterial glucose dehydrogenase deposi-ted on the mediator layer from an evaportable solution.

14. A sensor electrode as claimed in claim 8, in which the enzyme is a bacterial glucose dehydrogenase held in a gelatine layer at the surface of the mediator layer.

15. A sensor electrode as claimed in claim 8, having an outermost protective membrane permeable to water and glu-cose molecules.

16. A sensor electrode as claimed in claim 15, in which the protective membrane is a layer of cellulose acetate deposited from a solution thereof.

17. A sensor electrode as claimed in claim 1, in combination with implantation means suitable for implanta-tion in a human subject.

18. A sensor electrode combination as claimed in claim 17, in which the implantation means is a needle-like probe.

19. A sensor electrode combination as claimed in claim 18, in further combination with signal and control equipment.

20. A sensor electrode as claimed in claim 4 or 5, in which the mediator is chosen from ferrocene, 1,1'-ferrocenedicarboxylic acid, dimethyl ferrocene, and polyvinyl ferrocene.

21. A sensor electrode as claimed in claim 4 or 5, in which the electrode is made of a material chosen from silver, carbon particle paste and solid carbon.