GB2197486A - Enzyme electrode sensor - Google Patents

Description

1 GB2197486A 1 9

SPECIFICATION

Sensor This invention relates to a sensor of the enzyme-electrode type, to a step in preparing the 5 sensor for use to an analytical method using the sensor.

Enzyme electrodes are increasingly used. in medical and other laboratories particularly for the determination of materials such as glucose and urea in specimens of blood and other physiolog ical fluids. Such electrodes are described in many publications notably an article by Clark and Lyons (Annals of the New York Academy of Science, 102, 29-45, 1962) and US Patents 3539455 and 3979274 to Clark and Newman respectively. Enzyme electrodes are generally used to determine materials which themselves are not electrochemically active but which in the presence of suitable enzymes take part in reactions which produce species which can be readily detected by the electrodes. In enzyme electrodes the enzymes are frequently located within polymeric materials in close proximity to the underlying electrode. Enzyme electrodes proposed 15 more recently include those of our co-pending European Patent Application 86303907.9 and our co-pending UK Patent Application 85228374 and 8529300.

The determination of glucose can be taken as an example of the determination of a material by an enzyme electrode. In the presence of the enzyme glucose oxidase the following reaction occurs-.- glucose Glucose+02 -Gluconic acid+H202 oxidase The hydrogen peroxide produced in this reaction passes through the first layer of a membrane such as that of US Patent 3979274 and can be determined using the electrode. Since the hydrogen peroxide produced is dependent upon the glucose present in a specimen, the glucose concentration can be determined using a suitably calibrated sensor.

Continuous in vivo monitoring of blood metabolites such as glucose can provide the clinician 30 with immediate data on a patient's condition and hence can enable more effective treatment to be provided. If continuous in vivo monitoring of glucose were possible for an extended period of time, the construction to a portable, closed-loop insulin delivery system for diabetics would be possible. All the components for such a system have been made except for a long term enzyme-electrode sensor for use in vivo. To date the most effective system of this type to be 35 reported is that developed by M. Shichiri et al (Lancet, 1982, (8308), 1129-31; Chem. Abs., 1984, 100, 144957g and Chem. Abs., 1985, 102. 2004956) who achieved successful in vivo terms monitoring of pancreatectomised dogs over 3 day periods and shorter- term successful monitoring of forearm tissue glucose in human volunteers.

We have concluded that producers of an in vivo sensor should aim for an instrument having a 40 combination of the following properties:

1. Long term stability; - 2. Reproducible (preferably linear) response to a substrate over the clinically important range, i.e. 0-40 mM; 3. Selectivity, i.e. freedom from interference by other chemical species; 4. Independence of electrode performance from variations in in sample oxygen tension (cosubstrate for glucose oxidase); 5. A fast response time, preferably less then 2 minutes; 6. Biocompatibility giving minimal trauma, thrombus formation and tissue necrosis; 56 7. Small size; 8. Independence from stirring and sample viscosity changes; 9. Robustness; and 10. Sterilisability. Additionally, the electrode response should remain substantially unaffected by exposure to an essentially hostile biological environment. 55 According to the present invention we provide a sensor of the enzyme-electrode type for the 55 determination of an analyte, said analyte being convertable in the presence of an enzyme into a species which can be detected by the sensor, which comprises an electrode covered by a membrane permeable to liquids and solutes which can be contacted by a specimen containing the analyte, said membrane comprising a layer containing one or more enzymes and an outer- most layer of material positioned between the enzyme-containing layer and the specimen characterised in that the outermost layer has been treated with a protei n-denatu ring reagent.

Further according to the present invention we provide a step in preparing for use, a sensor of the enzyme-electrode type for the determination of an analyte convertable in the presence of an enzyme into a species which can be detected by the sensor, the sensor comprising an electrode covered by a membrane permeable to liquids and solutes and having a layer containing one or 2 GB2197486A 2 more enzymes and an outermost layer of material positioned between the enzyme-containing layer and a specimen containing the analyte which step is characterised in that during it the outermost layer is treated with a protein-denaturing reagent.

Further according to the invention we provide a method for determining a analyte in a specimen which comprises contacting the specimen with an electrode of a sensor of the enzyme-electrode type, which electrode is covered by a membrane permeable to liquids and solutes and comprising a layer comprising one or more enzymes, in the presence of which the analyte is conveirtable into a species detectable by the sensor, and an outermost layer of material positioned between the enzyme-containing layer and the specimen, and measuring the response of the sensor to the species, characterised in that the outermost layer has been 10 treated with a protein-denaturing reagent.

The electrode can be of any shape and for instance be of the type which is contacted by drops of a specimen. However preferably it is rod-shaped having a tip (optionally tapered) which is covered by the membrane. In the analytical method of the invention the membrane-covered tip of such an electrode is inserted into the specimen. Throughout the remainder of this specifica- 15 tion the sensor of the invention will be described in terms of a rod- shaped or---needle electrode.

In its most simple form the membrane in the sensor of the invention consists of the enzyme containing layer and another layer of material preferably a layer of restricted permeability as described in our European Patent Application No. 86307011.6. The other layer is the outer layer 20 in this simple form of membrane and is contacted directly by the specimen in the method of the invention for determining an analyte.

However, it is preferred that the membrane is a laminated membrane of the type of which that disclosed in US Patent 3979274 is an example. Such a membrane comprises a first or inner layer of material positioned between the enzyme-containing layer and the electrode, the enzyme- 25 containing layer and a second layer of material on the other side of the enzyme-containing layer which second layer is preferably a layer having restricted permeability.

Hereafter in this specification the sensor of the invention which is described will contain a laminated membrane having first and second layers, the layer preferably comprising the porous material having restricted permeability being the second layer.

It should be understood that the membranes in the sensor of the invention can contain more than two layers of material in addition to the enzyme-containing layer. For instance the second layer is not necessarily the outermost layer of the membrane. There may be a further layer or layers of material, i.e. third, fourth etc. layers, between the second layer or layer of restricted permeability and the specimen. Often however the second layer will be the outer layer and its 35 outer face will be contacted by the specimen.

Generally the material used in the second layer will be a polymeric material but other suitable materials may be used. Thus the second layer may be formed from a glass or a metal having pores cut by lasers.

Suitably the second layer of material is formed from material having an effective porosity not 40 greater than 2%. Very low effective porosities are preferred for instance in the range 0.001% or 0.005% to 0.5%.

In the sensor of the invention the second layer of the membrane acts as a diffusion barrier and also prevents or restricts the passage of compounds of high molecular weight. Suitable porous materials for the second layer include porous polycarbonates, polyurethanes, and modi- 45 fied cellulose particularly cellulose nitrate, cellulose acetate and regenerated cellulose. Prefeably the second layer is formed from a polyurethane. Polyurethanes can be coated onto the enzyme layer either as polyer precursors or as pre-formed polymer to form the second layer.

The sensor of the invention may have a detachable membrane or it may be a disposable sensor with an adherent membrane. Materials used in the formation of suitable electrodes for the 50 sensors include inert metals and/or carbon.

When the sensor incorporates a laminated membrane of the type disclosed in US Patent 3979274 the first layer which is to be located between the enzyme layer and the electrode is suitably formed from polymethyl methacrylate, polyurethane, cellulose acetate or another permea ble material which will restrict or prevent passage of electroactive interfering compounds such as 55 ascorbic acid and tyrosine. It is particularly preferred that the first layer is formed from a sulphonated or unsulphonated polyaryiketone or, especially a sulphonated or unsulphonated poly aryisulphone as described in our UK Patent Application No. 8529300. The use of sulphonated polya ryisu 1 phones for the first layer has a particular benefit when the sensor has a rod-shaped electrode. With rod-shaped electrodes some difficulty is experienced in getting three discrete 60 layers onto the end of the rod and often the first layer and the enzyme layer have been combined. The use of a sulphonated polyaryisu [phone for the first layer reduces the difficulty of coating the rod lip with three layers. It should be noted that when coating a rod tip with the component layers of a membrane, the first and second layers are suitably formed from materials soluble in different solvents. Suitably the first layer has a thickness in the range 0.2 microns to 65 3 GB2197486A 3 1.0 microns.

The enzyme present in the sensor of the invention may be located in the membrane in any suitable manner. Preferably in a laminated membrane it is present between the first and second layers of porous material and the enzyme-containing layer forms the bond between them. In this situation, and also generally, the enzyme is preferably immobilised by mixing with a material which causes crosslinking to occur. A very suitable material for this purpose is glutaraldehyde; proteins such as albumin and other materials are preferably also included. In order to facilitate the obtaining of rapid stable readings from the sensor it is preferred that the enzyme-containing layer is thin, i.e. not greater than 5 microns thick.

The enzyme to be used in the sensor of the invention will depend upon the analyte whose 10 concentration is to be determined. If the analyte is glucose then the enzyme will be for example glucose oxidase. Other enzymes which may be present include uricase and lactate oxidase for determination of uric acid and lactic acid respectively. Enzyme systems comprising two or more enzymes may also be present.

The prptein-denaturing reagent which is used to treat the outermost membrane layer (usually 15 the second layer) can be any suitable reagent. Inorganic acids and bases are preferred with hydrochloric acid sodium and potassium hydroxide, particularly at a concentration in the range 0.01 M to 0.5 M, being especially suitable. The treatment conditions which are preferred will depend upon the enzyme and the protein matrix in which it is placed. With rod-shaped elec- trodes the tip is dipped into the reagent for a few seconds as a simple way of effecting the treatment.

Use of the method of the invention gives the advantage of an increase in the concentration range over which a graph of concentration against sensor response is linear. With conventional methods linearity generally extended only up to approximately a concentration of 3 m mol per litre for glucose. Using the method of the invention linearity is increased and the range can extend to glucose concentrations of 50 m mol per litre and even higher. Thus the range covers the concentration of glucose which can be anticipated in blood samples thereby enabling blood glucose levels to be determined more readily. This could be a considerable advantage in situations where large numbers of determinations must be made regularly and with minimal sample preparation (viz dilution).

Potential uses for sensors of the invention having rod-shaped electrodes are as follows:- (1) Medical:

Multiple in vivo analysis of metaolites. Artificial pancreas type closedloop system. In vivo intensive care monitoring (e.g. patients in diabetic coma or some genetic disorders in babies, 35 where the blood glucose can rapidly drop to zero with possible fatal results). Simple flow cell system with integral needle electrode (pre-sterilised), for use extracorporeally, Dental research tool for monitoring sugars etc. in the mouth.

(2) Veterinary uses:

(Mainly as for 1). Possible monitoring of animals hormoneal levels where accmpanied by changes in common metabolite levels.

(3) Food uses:

Monitoring of food production lines (e.g. jams, drinks etc.). Alcoholic beverage (beers, wines 45 etc.). Food quality probe for meats, vegetables, fruit etc. Microbial monitoring.

(4) agricultural:

Crop ripeness, quality etc. (as for 3). Silage production. Milk production.

(5) Fermentation:

(as for 3). Continuous monitoring of feedstocks for single cell protein production.

(6) Pollution:

Monitoring of river water for effluent, sewage etc.

The invention is illustrated by the drawings and the following Example:In the drawings Figs. 1A and 113 show two forms of rod-shaped electrodes which were prepared and tested as described in the Examples. The electrode of Fig. l(A) is a 0.5 mm electrode and the electrode of Fig. 1(13) is a 0.25 mm electrode. Fig. l(C) shows the membrane- used to coat the tips of the electrodes.

55, The electrodes are formed from stainless steel tubes 1 which act as reference and counter electrodes and into which platinum wires 2 are embedded. Insulating materials present comprise a glass sleeve 3 and epoxy resin 4 in Fig. 1(A) and a 'TEFLON' (Registered Trade Mark) sleeve and epoxy resin 6 in Fig. l(B). The electrical connections are soldered onto the ends of the electrodes and the electrode tips 7 are polished using emery paper. The polished tips are then 65 4 GB2197486A 4 coated with the membrane layers shown in Fig. 1 (C). These are a first layer of a sulphonated polyarylether sulphone (PES) 8, a layer comprising glucose oxidase 9 and a second layer of polyurethane 10. The complete membrane was treated by dipping in hydrochloric acid.

The polyurethane layer is applied by single or multiple dip-coating onto the enzyme layer, and the solvent is allowed to evaporate off in air. The polyurethanes used have been either precursor type (polymerising in situ giving a solvent insoluble layer) SC771, SC762, Trixane H35 (Baxenden Chemical Co Ltd) or dissolved polyurethane rubbers Estane 5701F1, 5707F1, 5711 1F1 (B. F. Goodrich Co, Belgium). In all cases the linearity can be extended. Both the precursor type and the rubber polymer coats (which can be applied as descrete precast sheets 0.2-1 am thick) give similar linearity, but slower responses are found for the Estanes. The polyurethane extends the 10 linear range of the electrode by reducing the diffusion of glucose to the enzymic layer. It is believed that all the polyurethanes used operate as microporous diffusion limiting barriers, since complete layers are impermeable to glucose. The electrode is given acid treatment to extend linearity further. This is achieved by dipping the electrode tip in 0.1 M HCI for 5-50 s then washing and re-immersing in stirred buffer until a stable baseline is achieved. Without an, underlying membrane the linearity can be extended by acid treatment from 7 to 70 mM although after several hours this electrode linearity stabilised at about 35 mM.

EXAMPLE

Electrodes have been characterised in isotonic phosphate buffer (pH 7.4). The electrode per- 20 formance is highly dependent on the membrane construction and the pretreatment used, and can thus be tailored to the intended use. The electrode linear range to glucose can be extended without an underlying membrane as shown in Fig. 2 with a response time of 20s to 60s in stirred solution. With the incorporation of the PES underlying layer, linearity has been extended to >30 mM. Response times in unstirred solution range from 5-240s (typically 30s). Response 25 time in whole undiluted unstirred blood is typically T,,, 10-90s. Fig. 3 illustrates a response curve to glucose addition; for an electrode linear to 30 mM (T151 15s, resolution of +/- 0.2 mM), the current level remained unchanged at 40 mM for >4 hours (no underlying membrane in this case). With an underlying membrane of PES, the electrodes showed neglible interference from other biological species (ascorbic acid, glutathione, uric acid, etc. ). Electrodes were largely 30 independent of stirring effects.

In whole, undiluted, unstirred blood the correlation with a routine spectrophotometric assay was good (see Fig. 4), y= 1.006 x +0.096, n=26. However, the response time for this electrode increased from 30s in buffer to 60-90s in blood. In diluted stirred blood (1 to 3 in isotonic buffer pH 7.4) a correlation of y=0.971 x +0.024, r=0.987, n=28, was obtained. 35 Both in diluted and undiluted whole blood the response to standard solutions decreased by 1-4% after the first blood exposure but thereafter the standard response remained unaltered. In whole undiluted blood intermittant measurements have been made over a 10 day period with no significant change in the electrode characteristics (storage in dry state overnight). In diluted blood, the electrode performance appeared unimpaired after 200 blood measurements over 8 40 weeks (storage in isotonic buffer at ambient temperature overnight).

Legends for Figs. 2 to 4 Fig. 2: electrode current dependence on glucose concentration (stirred buffer solution).

Fig. 3: electrode current/time response curve to glucose additions for a sensor of extended 45 linearity.

Fig. 4: Whole blood correlation with a routine spectrophotomeric assay (y=1.006 X +0.096, r=0.996, n=26).

Claims (10)

1. A sensor of the enzyme-electrode type for the determination of an analyte, said analyte being convenable in the presence of an enzyme into a species which can be detected by the sensor, which comprises an electrode covered by a membrane permeable to liquids and solutes which can be contacted by a specimen containing the analyte, said membrane comprising a layer containing one or more enzymes and an outermost layer of material positioned between the enzyme-containing layer and the specimen characterised in that the outermost layer has been treated with a proteindenaturing reagent.

2. A sensor as claimed in claim 1 wherein the membrane comprises a first or inner layer of material positioned between the enzyme-containing layer and the electrode, and a second layer of material on the other side of the enzyme-containing layer.

3. A sensor as claimed in claim 1 or claim 2 wherein the outermost or second layer is formed from a porous material of restricted permeability having an effective porosity no greater than 5%.

4. A sensor as claimed in claim 3 wherein the outermost or second layer has an effective porosity not greater than 2%.

' GB2197486A

5 5. A sensor as claimed in claim 5 wherein the outermost or second layer has an effective porosity in the range 0.001 to 0.5%.

6. A sensor as claimed in claim 5 wherein the outermost or second layer has an effective porosity in the range 0.005 to 0.5%.

7. A sensor as claimed in any one of the preceding claims wherein the outermost or second 5 layer is formed from polyurethane.

8. A sensor as claimed in any one of the preceding claims wherein the enzyme-containing layer is not greater than 5 microns thick.

9. A step in preparing for use, a sensor of the enzyme-electrode type for the determination of an analyte convertable in the presence of an enzyme into a species which can be detected by 10 the sensor, the sensor comprising an electrode covered by a membrane permeable to liquids and solutes and having a layer containing one or more enzymes and an outermost layer of material positioned between the enzyme-containing layer and a specimen containing the analyte which step is characterised in that during it the outermost layer is treated with a protein-denaturing reagent.

10. A method for determining an analyte in a specimen which comprises contacting the specimen with an electrode of a sensor of the enzyme-electrode type, which electrode is covered by a membrane permeable to liquids and solutes and comprising a layer comprising one or more enzymes in the presence of which the analyte is convenable into a species detectable by the sensor, and an outermost later of material positioned between the enzyme-containing 20 layer and the specimen, and measuring the response of the sensor to the species, characterised in that the outermost layer has been treated with a protein-denaturing reagent.

Published 1988 at The Patent Office, State House, 66/71 High Holborn, London WC1R 4TP. Further copies may be obtained from The Patent Office, Sales Branch, St Mary Cray, Orpington, Kent BR5 3RD. Printed by Burgess & Son (Abingdon) Ltd. Con. 1/87.