DE3736910A1 - SENSOR - Google Patents

Abstract

A sensor of the enzyme- electrode type for the 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 sensor comprises a membrane having an inner layer 9 containing one or more enzymes and an outer layer 10 treated with a protein denaturing reagent. The membrane may be coated on the sensor which comprises a stainless steel tube containing an insulated platinum wire. The outer layer is preferably polyurethane of effective porosity of 5% or less and the protein-denaturing agent may be an inorganic acid or base e.g. HCl or KOH. <IMAGE>

Description

The present invention relates to a sensor from Enzyme electrode type, a process step for application of the sensor and one using the sensor he following analytical procedure.

Enzyme electrodes are increasingly used in medical and other laboratories, especially for loading materials such as glucose and urea Blood and other physiological fluid samples keiten. Such electrodes are used in numerous Described publications, especially in an article by Clark and Lyons (Annals of the New York Academy of Science, 102, 29-45, 1962) or in the U.S. patents 35 39 455 and 39 79 274 (owners Clark and Newman). Enzyme electrodes are usually used to determine Materials used that are not electrochemical themselves are active, but in the presence of suitable enzymes participate in reactions that produce species, which can be easily detected by the electrodes nen. In enzyme electrodes, the enzymes are often inside of polymeric materials in close proximity to underneath the electrode. In youth Enzyme electrodes currently proposed include those as they go back to the same applicant European patent application 86 303 907.9 and in the UK Patent applications 85 22 834 and 85 29 300 are described.

The determination of glucose can serve as an example of that Determination of a material by an enzyme electrode serve. In the presence of the enzyme glucose oxidase takes place the following reaction takes place:

The hydrogen peroxide formed in this reaction penetrates through the first layer of a membrane, e.g. one those of US Pat. No. 3,979,274 and can be found at Use of the electrode can be determined. Because that generated Hydrogen peroxide from that present in a sample Glucose depends, the glucose concentration under Ver using a suitably calibrated sensor will.

Continuous control of blood metabolism in vivo Like glucose, the clinician can provide immediate data care about the condition of a patient and thus enable more effective treatment to be used. If continuous in vivo monitoring of glucose would be possible for a longer period of time the construction of a portable insulin supply closed circuit systems for diabetics be possible. All components for such a Systems have already been created with the exception of one Long-term enzyme electrode sensor for use in vivo. The most effective system of this type is currently over that was reported, that by M. Shichiri et al (Lancet, 1982, 2 (8308), 1129-31; Chem. Abs., 1984, 100, 144957g and Chem. Abs., 1985, 102, 20 04 956) ent was wrapped up in successful tissue in vivo control of pancreas-resected dogs 3 days and the successful monitoring of Forearm tissue glucose in itself for shorter periods volunteered people succeeded.

It was assumed that manufacturers of one in vivo sensor strive for an instrument that is a station wagon nation has the following characteristics:

• 1. Long-term stability;
• 2. reproducible (preferably linear) response onto a substrate over the clinically important area rich, i.e. 0-40 mM;  
• 3. Selectivity, i.e. Freedom from disturbing legs flow through other chemical substances;
• 4. Independence of the electrode behavior from changes in the oxygen pressure in the sample (cosubstrate for Glucose oxidase);
• 5. rapid response time, preferably less than 2 min;
• 6. Biocompatibility that is minimal to trauma, Thrombus formation and tissue necrosis leads;
• 7. small size;
• 8. Independence from stirring and viscosity changes in the sample;
• 9. robustness; and
• 10. Sterilizability.

In addition, the electrode responsiveness should be in the we remain significantly unaffected by exposure to the the same as a practically hostile biological reverse exercise.

According to the invention, a sensor is of the enzyme electrode type for determining an analytical substance or an analyte, which converts to a species in the presence of an enzyme bar that can be detected by the sensor created, the sensor having an electrode, the one with liquids and solutes permeable membrane is covered by a Sample containing analyte can be contacted, and where the membrane is a layer containing one or more Contains enzymes, and one between the enzyme-containing Layer and the sample outer material layer has, the sensor being characterized in that that the outer layer with a protein-denaturing Reagent has been treated.  

According to the invention, a method step for Use of an enzyme electrode type sensor Determination of an analyte in the presence of an enzyme can be converted into a species by the sensor can be detected, indicated by the sensor has an electrode which through a membrane is drawn for liquids and solutes is permeable and a layer containing one or more Contains enzymes, and one between the enzyme-containing Layer and a sample containing the analyte Liche outer material layer, the Ver stage is characterized in that during performing the outer layer with a protein denaturing reagent is treated.

According to the invention, a method for determination is also provided of an analyte in a sample in which the Sample with an electrode of a sensor of the enzyme elec trode type, which electrode by one for liquid and solute permeable membrane coated and is a layer that contains one or more enzymes the presence of the analyte in a by the sensor detectable species is convertible, contains, as well as a between the enzyme-containing layer and the sample Liche outer layer of material in contact brings and the response of the sensor to the species is measured, the method being characterized is that the outer layer with a protein denaturie reagent has been treated.

The electrode can be of any shape and e.g. the Type to be assigned, which can be obtained by dropping a sample is clocked. However, it is preferably rod-shaped and has a (possibly beveled) tip, which is covered by the membrane. With fiction  According to analytical methods, the membrane is coated Tip of such an electrode inserted into the sample. In the further course of the description of the Invention appropriate sensor as a rod-shaped or "needle" electrode designated.

In its simplest form, the membrane consists of the invention appropriate sensor from the enzyme-containing layer and a another layer of material, which is preferably around a layer with limited permeability like them in pending European patent application no. 86 30 011.6 is described. This one more Layer is the outer layer in this simple form the membrane and it will contact directly through the sample tiert in the inventive method for determination of an analyte.

However, it is preferred that the membrane be laminated Membrane is of the type for which those described in U.S. Patent Scripture 39 79 274 described membrane is an example poses. Such a membrane has a first or inner layer of material between the enzyme-containing Layer and the electrode is arranged, the enzymhal layer and a second layer of material on the other side of the enzyme-containing layer, where it the second layer is preferably one layer with limited permeability In the following description, the one explained sensor according to the invention a laminated membrane with a first and second layers, where the layer, which is preferably the porous material with limited Permeability has to do with the second layer.

It deserves to be emphasized that the membranes in the sensor according to the invention more than two materials  layers in addition to the enzyme-containing layer can hold. For example, the second shift is not necessary maneuverably the outermost layer of the membrane. It can one or more additional layers of material, i.e. a third, fourth, etc. layers exist between the two th layer or the layer with limited permeability act and the rehearsal. Often, however, is the second layer the outer layer and its outer surface is contacted by the sample.

As a rule, this is used in the second layer Material is a polymer material, but there are others suitable materials can be used. For example, the second Layer of glass or metal with laser cut pores.

The second material layer is expediently made of a material with an effective porosity of not formed greater than 2%. Very low effective poro sities are preferred, e.g. in the range of 0.001 or 0.005 to 0.5%.

The second layer acts in the sensor according to the invention Membrane as a diffusion barrier and also prevent changes or limits the passage of connections with high molecular weight. Suitable porous materials for the second layer comprise porous polycarbonates, Polyurethanes and modified cellulose, in particular Cellulose nitrate, cellulose acetate and regenerated Cellu loose. The second layer is preferably made of one Polyurethane formed. Polyurethanes can affect the enzyme layer either as polymer precursor compounds or as a preformed polymer to form the second Layer can be applied.  

The sensor according to the invention can be a removable membrane or it can be a disposable sensor with a act adherent membrane. To form more suitable Electrodes used for the sensors contain inert metals and / or carbon.

If the sensor has a layered membrane in the U.S. Patent 3,979,274 includes the type described, is the first layer between the enzyme layer and the electrode is arranged, expediently Polymethyl methacrylate, polyurethane, cellulose acetate or another permeable material that forms the Passage of electroactive interfering compounds such as Ascorbic acid and tyrosine, restricted or prevented. It is particularly preferred that the first layer be made of a sulfonated or unsulfonated polyaryl ketone or in particular from a sulfonated or non-sul formulated polyarylsulfone, as in the UK patent application No. 85 29 300 is described, is formed. The usage of sulfonated polyarylsulfones for the first layer is particularly useful if the sensor is a stab shaped electrode. With rod-shaped electrodes the certain difficulties observed in education of three separate layers at the end of the bar and often the first layer and enzyme combined layer. The use of a sulfonated polyaryl sulfons for the first layer reduces the difficulty speed of coating the rod tip with three layers. It should be noted that if a rod tip with the layer components of a membrane is to be coated, the first and second layers expediently made of Ma materials are formed in different Solvents are soluble. Conveniently, the first layer a thickness in the range of 0.2 to 1.0 microns.

The enzyme present in the sensor according to the invention can  housed in the membrane in any suitable manner be. It is in a laminated membrane preferably between the first and second layers made of porous material and the enzyme-containing layer bil the link between these two. In this Situation and more generally the enzyme is preferred immobilized by mixing with a material that the occurrence of networking. A very suitable net material for this purpose is glutaraldehyde; Proteins such as albumin and other materials are used preferably also incorporated. To achieve faster it will facilitate stable readings from the sensor preferred that the enzyme-containing layer be thin, i.e. has a thickness of not greater than 5 microns.

The enzyme to be used in the sensor according to the invention depends on the analyte whose concentration is to be determined should. If the analyte is glucose, then is the enzyme e.g. Glucose oxidase. Other enzymes that may include uricase and lactate oxidase Determination of uric acid or lactic acid. Enzyme systems with two or more enzymes may also be present.

For the protein-denaturing reagent that is used for treatment the outermost membrane layer (usually the second Layer) is used, it can be any suitable Act reagent. Inorganic acids and bases are used preferred, with hydrochloric acid, sodium and potassium hydroxide, especially in a concentration in the range of 0.01 up to 0.5 M, are particularly suitable. The treatment area conditions that are preferred depend on the enzyme and the protein matrix in which it is located. At rod-shaped electrodes will tip into the reagent submerged for a few seconds as a simple kind and ways of effecting treatment.  

The application of the method according to the invention results the advantage of expanding the concentration range, over which a concentration curve against the sensor speakability is linear. Stretches with usual methods the linearity is usually only up to approximately a concentration of 3 mmol per l for glucose. At Use of the method according to the invention Linearity increased and the range can go up to Glucose concentrations of 50 mmol per liter and even higher extend. The range thus covers the concentrations on glucose expected in blood samples where by making it possible to lower blood glucose levels more easily to determine. This can be a considerable advantage in situations where there are large amounts of provisions regularly and with minimal trial preparations (namely Dilution) must be made.

Potential applications for sensors according to the invention with rod-shaped electrodes are the following:

• (1) Medical application:
  Multiple in vivo analysis of metabolic products. Artificial closed circuit system of the pancrea type. Intensive in vivo care monitoring (e.g. patients in diabetic coma or certain genetic disorders in babies, where blood glucose can quickly drop to zero with possible fatal consequences). Simple flow cell system with integral needle electrode (pre-sterilized) for use outside the body. Dental research tool for monitoring sugars and the like in the mouth area.
• (2) Veterinary applications:
  (Mainly as for 1). Possible monitoring of animal hormone levels if these are accompanied by changes in the usual metabolic concentrations.
• (3) Food applications:
  Monitoring of food production due to (e.g. jams, drinks and the like). Production of alcoholic beverages (beers, wines and the like). Food quality inspection for meat materials, vegetables, fruits and the like. Microbial surveillance.
• (4) Agricultural applications:
  Harvest maturity, quality and the like (as for 3). Silage production. Milk production.
• (5) Fermentation applications:
  (As for 3). Continuous monitoring of feeds for single-cell protein production.
• (6) Contamination tests:
  Monitoring river water for runoff, sewerage and the like.

The invention is illustrated by the drawings and the following Example explained in more detail.

In the drawings, FIGS. 1A and 1B, two forms of bar-shaped electrodes, which were prepared as described in the case of play and tested. The electrode of FIG. 1 (A) is a 0.5 mm electrode and the electrode of FIG. 1 (B) is a 0.25 mm electrode. Figure 1 (C) shows the membrane used to coat the tips of the electrodes.

The electrodes consist of tubes 1 made of stainless steel, which act as reference and counter electrodes and are embedded in the platinum wires 2 . Existing insulating materials include a glass envelope 3 and epoxy resin 4 in Fig. 1 (A) and a "TEFLON" (registered trademark) sleeve 5 and epoxy resin 6 in Fig. 1 (B). The electrical connections are soldered to the ends of the electrodes and the electrode tips 7 are polished using emery paper. The polished tips are then coated with the membrane layers shown in Fig. 1 (C). These consist of a first layer of a sulfonated poly aryl ether sulfone (PES) 8 , a layer 9 containing glucose oxidase and a second layer of polyurethane 10 . The entire membrane was treated by immersion in hydrochloric acid.

The polyurethane layer is made by one or more fold immersion coating applied to the enzyme layer brought and the solvent will evaporate in air calmly. It dealt with the polyurethanes used either precursor types (the polymerization in situ results in a layer insoluble in solvents) SC771, SC762, Trixane H 35 (Baxenden Chemical Co. Ltd.) or around dissolved Estane 5701F 1 polyurethane rubber, 7507F 1, 57111F 1 (B.F. Goodrich Co., Belgium). In all In some cases the linearity can be expanded. Either the precursor type as well as the rubber polymer coatings (which as separate prefabricated sheets from 0.2 to 1 µm thickness can be applied) result in similar Linearity, but have become long for the Estane products more responses found. The polyurethane he stretches down the linear area of the electrode setting the diffusion of glucose to the enzyme layer. It is believed that all polyurethanes used act as microporous diffusion-limiting barriers, because complete layers are impermeable to glucose. The electrode is subjected to an acid treatment to further increase linearity. He will is sufficient by immersing the electrode tip in 0.1 M HCl for 5 to 50 s, then washing and he Immerse again in stirred buffer solution until one stable baseline is achieved. Without one under laid membrane can linearity by acid treatment tion from 7 to 70 mM, although after  several hours this electrode linearity stabilized about 35 mM.

EXAMPLE

Electrodes were labeled in isotonic phosphate buffer (pH 7.4 ). The electrode behavior is highly dependent on the membrane construction and the pretreatment used and can therefore be tailored to the intended use. The linear electrode range for glucose can be expanded without an underlying membrane, as shown in Fig. 2, with a response time of 20 to 60 s in stirred solution. The linearity was extended to <30 mM by incorporating the underlying PES layer. The response times in a non-stirred solution range from 5 to 240 s (typically 30 s). The response time in completely undiluted, unmixed blood is typically T _95% 10 to 90 s. Figure 3 shows a response curve to glucose addition; for a linear electrode up to 30 mM ( T _95% 15 s, resolution of +/- 0.2 mM) the current value remained unchanged at 40 mM for <4 h (no underlying membrane in this case). With an underlying membrane made of PES, the electrodes showed negligible interference from other biological species (ascorbic acid, glutathione, uric acid and the like). The electrodes were largely independent of stirring effects.

In complete, undiluted, undisturbed blood, agreement with a spectrophotometric routine assay was good (see FIG. 4), y = 1.006 x +0.096, r = 0.996, n = 26. However, the response time for this electrode increased from 30 s in buffer to 60 to 90 s in blood. In diluted stirred blood (1 in 3 in isotonic buffer pH 7.4) an agreement of y = 0.971 x +0.024, r = 0.987, n = 28 was obtained. In both diluted and undiluted, complete blood, responsiveness to standard solutions decreased 1-4% after the first exposure to blood, but the standard responsiveness remained unchanged thereafter. Intermittent measurements were taken in complete undiluted blood over a period of 10 days without significant change in the electrode characteristics (storage in a dry state overnight). In diluted blood, the electrode behavior was unaffected after 200 blood measurements over 8 weeks (storage in isotonic buffer at ambient temperature overnight).

• Legends for FIGS. 2 to 4: FIG. 2: Electrode current dependence on the glucose concentration (stirred buffer solution).
  Fig. 3: Electrode current / time response curve against the addition of glucose for a sensor of extended linearity.
  Fig. 4: Whole blood correspondence with a spectrophotometric routine assay (y = 1.006 x +0.096, r = 0.996, n = 26).

Claims (10)

1. Sensor of the enzyme electrode type for determining an analyte which can be converted in the presence of an enzyme into a species which can be detected by the sensor, the sensor having an electrode which is coated with a membrane permeable to liquids and solutes which can be contacted by a sample containing the analyte, and wherein the membrane has a layer which contains one or more enzymes and an outer material layer located between the enzyme-containing layer and the sample, characterized in that the outer layer with a protein-denaturing reagent has been treated. 2. Sensor according to claim 1, wherein the membrane first or inner layer of material between the enzyme-containing layer and the electrode is arranged, and a second layer of material on the other side of the enzyme-containing layer. 3. Sensor according to claim 1 or 2, wherein the outer or second layer of a porous material with be restricted permeability, which is an effective porosity of not more than 5%. 4. Sensor according to claim 3, wherein the outer or second layer an effective porosity of no greater than 2%. 5. Sensor according to claim 4, wherein the outer or second layer effective porosity in the range of 0.001 to 0.5%.   6. Sensor according to claim 5, wherein the outer or second layer has an effective porosity in the range of 0.005 to 0.5%. 7. Sensor according to one of the preceding claims, in which the outer or second layer of polyurethane is formed. 8. Sensor according to one of the preceding claims, in which the enzyme-containing layer does not have a thickness has more than 5 µm. 9. Process stage for the application of a sensor from Enzyme electrode type for determining an analyte that convertible to a species in the presence of an enzyme which can be detected by the sensor wherein the sensor has an electrode which with a membrane permeable to liquids and solutes is coated, the one layer, which one or more Contains enzymes, and one between the enzyme-containing Layer and a sample containing the analyte Liche outer material layer, the Ver stage is characterized in that during They carry out the outer layer with a protein denaturing reagent is treated. 10. Method for determining an analyte in a Sample in which the sample with an electrode of a Enzyme electrode type sensors, which electrode with is covered with a membrane that is suitable for liquids and solutes is permeable and a layer that is a or several enzymes, in the presence of which the analyte in a species detectable by the sensor can be transferred is contains, as well as one between the enzyme-containing Layer and the sample outer material layer  has contacted and the response of the sensor is measured on the species, characterized in that that the outer layer is denatured with a protein the reagent has been treated.