WO1992004438A1 - Electrochemical biosensor - Google Patents

Electrochemical biosensor Download PDF

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Publication number
WO1992004438A1
WO1992004438A1 PCT/DK1991/000248 DK9100248W WO9204438A1 WO 1992004438 A1 WO1992004438 A1 WO 1992004438A1 DK 9100248 W DK9100248 W DK 9100248W WO 9204438 A1 WO9204438 A1 WO 9204438A1
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WIPO (PCT)
Prior art keywords
layer
cellulose
membrane
substrate
sensor
Prior art date
Application number
PCT/DK1991/000248
Other languages
French (fr)
Inventor
Anne Rosengaard Eisenhardt
Anne-Marie Christensen
Original Assignee
Radiometer A/S
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Publication date
Application filed by Radiometer A/S filed Critical Radiometer A/S
Publication of WO1992004438A1 publication Critical patent/WO1992004438A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes

Definitions

  • the invention relates to an electrochemical biosensor of the type disclosed in the opening part of claim 1.
  • Electrochemical biosensors have attracted much atten ⁇ tion since a glucose sensor was disclosed for the first time by Clark et al. in "Annals of the New York Academy of Science” 102 (1962) 29-45.
  • Electrochemical biosensors are provided with an immobi ⁇ lized enzyme which is applied to decompose a biochemi ⁇ cal analyte or a substrate - usually by use of oxygen.
  • the concentration or activity of the substrate is measured by such biosensors by determining the consumed quantity of oxygen or the formed quantity of a reaction product from the reaction of the substrate with oxygen, for example hydrogen peroxide.
  • oxygen for example hydrogen peroxide.
  • An example of an electrochemical biosensor is an elec ⁇ trochemical glucose sensor in which the enzyme glucose oxidase is applied to decompose glucose according to the reaction scheme: glucose + 0 2 + H 2 0 grIuc ⁇ se ox ⁇ as %
  • gluconic acid + H_0 2 As seen from the reaction scheme it is possible to measure the concentration or the activity of glucose in a sample by determining either the consumed quantity of oxygen or the formed quantity of hydrogen peroxide.
  • the formed quantity of hydrogen peroxide can be measured polarographically by the oxidation reaction:
  • the H 2 0 2 oxidation current can be used as a measure of the glucose content.
  • Patent No. US 4759828 (Young) as well as by Shichiri M. et al. in "Wearable-type Artificial Endocrine Pancreas with Needle-type Glucose Sensor", Lancet 2. (1982) 1129- 1131 and by Abe H. et al. in the specification of US Patent No. US 4515584.
  • the unique response characteris ⁇ tics of these sensors have been accomplished as a ' result of the fact that a membrane, which limits the diffusion of glucose molecules, is provided between the enzyme and the sample, whose glucose content is to be determined.
  • Biosensors which similarly to the glucose sensor according to Shichiri or Abe comprise a polyurethane membrane exposed to the sample, have now proved to provide generally sensor-independent measuring results when measuring on aqueous samples, but have, in fact, proved to be unable to provide sensor-independent measuring results when the sample is whole blood.
  • the sensors are to a varying degree providing too low measurements on whole blood and on aqueous solutions, which follow whole blood measuring, irrespective of the fact that they were able to measure uniformly on aque- ous solutions prior to whole blood measuring.
  • the purpose of the invention is, therefore, to provide a biosensor which, to a larger extent than the biosen ⁇ sors already known, provides sensor-independent measur- ing results when measuring on whole blood.
  • the most preferred material for the protection layer consists of regenerated cellulose such as cellophane, but a number of cellulose plastic materials such as cellulose ethers or cellulose esters, for example cellulose acetate, cellulose butyrate, cellulose pro- pionate, compounds of esters thereof, and ethyl cellu- lose and other cellulose alkyl and aryl ethers are expected to be suitable as well.
  • cellulose plastic materials such as cellulose ethers or cellulose esters, for example cellulose acetate, cellulose butyrate, cellulose pro- pionate, compounds of esters thereof, and ethyl cellu- lose and other cellulose alkyl and aryl ethers are expected to be suitable as well.
  • Polyhydroxyethyl methacrylate is among other hydrophilic materials which are expected to be suitable as protection layer.
  • Biosensors are applied both for in vitro measuring and in vivo measuring, including extracorporeal measuring where the sample is directed from the patient to a measuring chamber and subsequently back to the patient.
  • a protection layer of cellophane is particularly suit- able for such in vivo measuring as the material is strong, stands disinfection, is non-toxic, and has good bio-compability.
  • the protection layer has a thickness rang- ing up to 100 ⁇ m, particularly 10-50 ⁇ m, and especially 12-25 ⁇ m. It is preferred to apply a protection layer being as thin as possible owing to i.a. the response time of the biosensors. In the biosensor according to the invention this may be achieved by providing the protection layer by chemical modification of the sur ⁇ face of the substrate-limiting layer turning away from the enzyme so that the surface by this chemical modifi ⁇ cation is provided with the hydrophilic properties according to the invention. In a preferred embodiment the protection layer and the substrate-limiting layer are lying closely together.
  • the preferred substrate- limiting layer comprises a polyurethane layer whose slightly adhesive surface is able to hold the cellulose layer.
  • the sensor membrane is constituted by a CuprophaneTM-glucose oxi- dase-Cuprophane membrane, and the main purpose of the Cuprophane membranes is to retain the enzyme glucose oxidase at an electrode surface.
  • an electrochemical glucose sensor with a cello ⁇ phane membrane is disclosed.
  • the glucose sensor is intended for measuring on blood, and the membrane serves to retain the enzyme glucose oxidase at the same side of the membrane as the anode and to keep the enzymes of blood, for example the H 2 0 2 -cleaving cata- lase, from the H 2 0 2 formed by the enzymatic glucose conversion.
  • an electrochemical glucose sensor is disclosed with a multi-layer membrane in which one of the layers comprises a thin, dense layer that prevents low-molecular agents, which similarly to H 2 0 2 are oxid- able at the anode potential of glucose sensor, from getting into contact with the*working surface of the anode.
  • oxidable, low-molecular agents are uric acid, ascorbic acid, and various drugs, par- ticularly paracetamol.
  • the thin layer should be so thin that it is essential to use a less dense layer as carrier layer.
  • Cuprophane, cellulose acetate, or polymerized protein are suggested for this layer, and a hydrophobic material such as silicone rubber or a hydrophilic material such as cellulose acetate is suggested for the thin, dense layer.
  • a glucose sensor is disclosed with a multi-layer mem ⁇ brane in which the layer nearest the anode or the inner layer comprises a layer of silicone rubber, methyl methacrylate or cellulose acetate which cf. the above- mentioned prevents oxidable agents from getting into contact with the anode.
  • the layer nearest the sample or the outer layer constitutes a diffusion barrier, partly preventing passage of high-molecular agents and partly giving mechanical strength. Porous polycarbonate or methyl methacrylate is suggested.
  • An enzyme preparation is present between the inner layer and the outer layer.
  • a glucose sensor is disclosed with a multi-layer membrane consisting of an inner filter membrane, an enzyme membrane, and an outer asymmetri- cal, semipermeable membrane produced from cellulose acetate comprising a thin, semipermeable outer layer and a thick, porous inner layer.
  • the asymmetrical, semipermeable membrane is said to improve the stability of the sensor when measuring on whole blood compared to a sensor with a usual semipermeable outer membrane of reproduced cellulose or polycarbonate.
  • the linearity range of the sensor is not disclosed, only measurements on blood containing 0.2 mM glucose, that is a glucose content far below the clinically interesting values.
  • an amperometric sensor for example a glucose sensor with an outer cellophane membrane and a layer of a polymer containing chemically bound glucose oxidase.
  • the sensor is said to be suit ⁇ able for blood analysis, and the cellophane membrane is said to be permeable for the chemical compound which is determined by the sensor, but the specification con ⁇ tains no data for the sensor.
  • the present invention is exemplified by a glucose sensor only, it is considered equally useful in connection with other biosensors measuring the concen ⁇ tration or activity of a biochemical analyte or a substrate by means of an immobilized enzyme.
  • the invention is considered applicable in con ⁇ nection with biosensors for the following biochemical analytes, the respective immobilized enzymes being men ⁇ tioned right after the analyte: lactate/lactate oxi- dase, cholesterol/cholesterol oxidase, hypoxanthine/- hypoxanthine oxidase and pyruvate/pyruvate oxidase.
  • the working electrode is constituted by platinum anode with an exposed working surface onto which is provided a coating of cellulose acetate.
  • This coating prevents low-molecular agents such as for example ascorbic acid and paracetamol, which can be oxidized by the potential of the working electrode, from reaching the working electrode, being oxidated and consequently interfering with the substrate determina ⁇ tion.
  • a very thin layer having a thickness of for example 2-25, preferably 3-15 and particularly 5-10 ⁇ m has now proved sufficient to prevent the interfering agents from interfering the glucose determination.
  • the layer should be as thin as possible owing to the re ⁇ sponse time of the sensor which increases with increas ⁇ ing thickness of the cellulose acetate layer.
  • the substrate-limiting layer consists of a hydrophobic plastic layer, particularly a polyurethane layer.
  • the thickness of the hydrophobic plastic layer is pre ⁇ ferably 0.1-10 ⁇ m, particularly 0.2-5 ⁇ m and especially 0.5-3 ⁇ m.
  • the invention also relates to the type of sensor mem ⁇ brane stated in the opening part of claim 8 having the characteristic features stated in claims 8, 9, 10, 11, and 12.
  • FIG. 1 is a view of the sensor according to the inven ⁇ tion located in a measuring chamber
  • FIG. 2 is an exploded view of an embodiment of the sensor according to the invention.
  • FIG. 3 is a view of the sensor membrane in the sensor according to the invention.
  • the sensor 1 according to the invention shown in FIG. 1 is arranged in such way that the front 2 of the sensor constitutes one of the walls in a measuring chamber 3.
  • An inlet channel 4 opens into the measuring chamber 3, and an outlet channel 5 extends from the measuring chamber.
  • the measuring chamber 3 is not in contact with the environment when the sensor 1 is mounted.
  • the sensor 1 is located in a tubular part 6 which projects from a surface 7 of the block in which the measuring chamber 3 is provided.
  • the measuring chamber 3 is located in the surface 7 and encircled by the tubular part 6.
  • the block 8 with the measuring chamber 3 shown in FIG. 1 is the type which is applied in the blood gas ana ⁇ lyzer ABL500 manufactured and sold by RADIOMETER A/S, Copenhagen, Denmark.
  • the block contains several serial ⁇ ly connected measuring chambers with inlet channels and outlet channels of the same configuration as the ea- suring chamber 3, the inlet channel 4, and the outlet channel 5.
  • FIG. 2 shows an embodiment in further detail of the sensor 1 according to the invention.
  • the sensor con- sists of a base part 9, a jacket 10, a membrane 11, a membrane ring 12. Apart from the membrane 11 and the cellulose acetate coating mentioned below and the fact that the Pt-wire has a differing thickness, the sensor is constructed similarly to a sensor which is manu- factured and sold by RADIOMETER A/S, Copenhagen, Denmark under the designation E909.
  • the base part 9 comprises a working electrode 13 in the form of a 250 ⁇ m platinum wire 17 melted into a glass rod 16. Further, the base part comprises an Ag/AgCl reference electrode 14 in the form of an annular coat ⁇ ing on the glass rod 16. At the front of the glass rod 16 the platinum wire 17 is exposed, and a not shown coating of cellulose acetate is applied to this end of the glass rod 16 as further described below in Example 1. At the rear the sensor 1 is provided with a connec ⁇ tor 15 with electrically conductive connection to the working electrode 13 and the reference electrode 14. Further, the base part comprises a locking means 18 with a rim 19 that serves to maintain the jacket 10 on the base part 9 according to the snap lock principle. Finally, at the front 2 of the locking means 18 facing the sensor is provided a rubber seal 20 which seals the compartment 21 formed when the jacket 10 is mounted on the base part 9.
  • the jacket 10 which is made from a transparent plastic material, is tubular and slightly conical and has a projecting part 22 on the inner surface of its broad end adapted to interlock with the locking means 18.
  • the membrane 11 shown in further detail in FIG. 3 consists of a laminated membrane comprising a 14 ⁇ m protection layer 24 of cellophane, a 1 ⁇ m polyurethane layer 25, an approximately 1 ⁇ m glucose oxidase layer 26, and a 1 ⁇ m poly lrethane inner layer 27.
  • the mem ⁇ brane has a greater area than the small aperture in the jacket 10 and is consequently able to cover this aper ⁇ ture and be wrapped around the outer of the jacket 10 onto which it is pasted and further held by the mem ⁇ brane ring 12.
  • the protection layer 24 is turned outwards and the inner layer 27 against the aperture of the jacket 10.
  • the membrane 11 is prepared as described below in Example 1. Before mounting the jacket 10 on the base part 9, an electrolyte is introduced having the compound described further in Example 1. The electrolyte establishes the necessary electrical contact between the working elec- trode 13 and the reference electrode 14.
  • the small aperture in the jacket 10 i.e. the aperture covered by the mem ⁇ brane
  • the membrane has a thickness of approximately 17 ⁇ m.
  • the measuring chamber 3 contains approximately 5 ⁇ l sample and has a circular aperture with a maximum diameter of approxi ⁇ mately 3.5 mm at the surface 7.
  • the inlet channel 4 and the outlet channel 5 are tubular bores with a circular cross section of approximately 0.7 mm in diameter.
  • a polarization voltage of +0.625 is applied to the working electrode relatively to the reference electrode, and the measuring set-up is thermostated to 37°C.
  • the working electrode current is registered as a function of time on a not shown printer after transformation of the electrode current to a voltage signal.
  • the signal of the working electrode is collected every half second from 5 to 25 seconds after introduction of the sample into the measuring chamber 3.
  • the values for the signal of the working electrode are processed along with data from a previous calibration and printed as mM glucose. Calibration is performed on aqueous solutions contain- ing 0 mM and 10 mM glucose of the composition explained further under the paragraph Materials and Methods in Example 1.
  • a solution without glucose is applied; partly as rins ⁇ ing fluid in the measuring set-up and partly as cali- bration liquid.
  • This liquid has the following composi ⁇ tion:
  • Calibration Liguid_2_-_10 mM_GjLucose This liquid has the same composition as calibration liquid 1 except that it further contains glucose in a concentration of 10 mM.
  • This solution is applied onto a glass plate through a 0.1 mm slot in a metal box which slowly by the hand is moved across the glass plate. After some time of evapo- ration (approximately 10 minutes) the membrane is removed from the glass plate and washed in ion-e. changed water and put to dry. The thickness of this membrane is approximately 1 ⁇ m.
  • the enzyme solution has the following composition:
  • a solution of High Rejective Cellulose Acetate Membrane S-18914 in Nitromethane is prepared for the cellulose acetate coating on the front of the glass rod.
  • the exact composition of the solution is a trade secret belonging to the manufacturer.Yellow Springs Instru ⁇ ments, Ohio, USA.
  • the solution is applied to the front of the glass rod by a fine brush and dried on standing.
  • the layer thick ⁇ ness is 5-10 ⁇ m.
  • the provided membrane is dried on standing and subse- quently pasted on the jacket of FIG. 2 by LoctiteTM (Cyanoacrylate Adhesive 414) .
  • the two electrodes provide mutually corresponding measurements both when measuring on blood and when measuring on aqueous sample and that the measurements on the aqueous samples before and after the blood sample correspond properly.
  • the sensor has a linearity range of 0.5-30 mM glucose and satisfactory stability. Long-term experiments did not show any sign of deterioration in the measuring performance for membranes which had been stored dryly at room temperature for 9 weeks. Membranes used in the test set-up of FIG. 1 and kept in continually contact with calibration liquid 1 between measuring have proved to maintain the measuring performance after 4 weeks function at 37°C.
  • the two electrodes provide mutually corresponding measurements when measuring the first time on aqueous sample, but provide very different measurements on blood, particularly when measured the second time. It is noted that it is the same blood sample.
  • the measurements on aqueous sample are mutually corresponding prior to the blood measurements, but subsequently both too low and varying from the elec ⁇ trode a to electrode b.

Abstract

The biosensor comprises a working electrode, a reference electrode, and a membrane comprising a substrate-limiting layer and an enzyme layer. At the substrate-limiting layer is provided a protection layer having hydrophilic properties corresponding to the properties of regenerated cellulose or being more hydrophilic than regenerated cellulose. Particularly, the substrate-limiting layer is constituted by a polyurethane layer and the protection layer is constituted by a cellophane layer.

Description

ELECTROCHEMICAL BIOSENSOR
The invention relates to an electrochemical biosensor of the type disclosed in the opening part of claim 1.
Electrochemical biosensors have attracted much atten¬ tion since a glucose sensor was disclosed for the first time by Clark et al. in "Annals of the New York Academy of Science" 102 (1962) 29-45.
Electrochemical biosensors are provided with an immobi¬ lized enzyme which is applied to decompose a biochemi¬ cal analyte or a substrate - usually by use of oxygen. The concentration or activity of the substrate is measured by such biosensors by determining the consumed quantity of oxygen or the formed quantity of a reaction product from the reaction of the substrate with oxygen, for example hydrogen peroxide. In order to establish a linear relation between the signal of the biosensors and the substrate concentration it is a condition that an excess of oxygen is present at the enzyme, otherwise oxygen - and not the substrate - will become the limit¬ ing factor and that the substrate concentration at the enzyme is much smaller than the Km-value of the enzyme where Km is the Michaelis-Menten constant.
An example of an electrochemical biosensor is an elec¬ trochemical glucose sensor in which the enzyme glucose oxidase is applied to decompose glucose according to the reaction scheme: glucose + 02 + H20 grIucσse ox^as%
gluconic acid + H_02 As seen from the reaction scheme it is possible to measure the concentration or the activity of glucose in a sample by determining either the consumed quantity of oxygen or the formed quantity of hydrogen peroxide. The formed quantity of hydrogen peroxide can be measured polarographically by the oxidation reaction:
Figure imgf000004_0001
On said condition that an excess of oxygen is present and that the substrate concentration at the enzyme is much smaller than the Km-value of the enzyme, the H202 oxidation current can be used as a measure of the glucose content.
A great deal of work has been involved in developing and providing biosensors which, when measuring directly on whole blood, provide a linear response in the entire clinically relevant concentration range for the bio¬ chemical analyte. Glucose sensors with a linear re¬ sponse in the entire clinically relevant glucose range (0.5-30 mM) are disclosed in the specification of US
Patent No. US 4759828 (Young) as well as by Shichiri M. et al. in "Wearable-type Artificial Endocrine Pancreas with Needle-type Glucose Sensor", Lancet 2. (1982) 1129- 1131 and by Abe H. et al. in the specification of US Patent No. US 4515584. The unique response characteris¬ tics of these sensors have been accomplished as a ' result of the fact that a membrane, which limits the diffusion of glucose molecules, is provided between the enzyme and the sample, whose glucose content is to be determined.
Biosensors, which similarly to the glucose sensor according to Shichiri or Abe comprise a polyurethane membrane exposed to the sample, have now proved to provide generally sensor-independent measuring results when measuring on aqueous samples, but have, in fact, proved to be unable to provide sensor-independent measuring results when the sample is whole blood. The sensors are to a varying degree providing too low measurements on whole blood and on aqueous solutions, which follow whole blood measuring, irrespective of the fact that they were able to measure uniformly on aque- ous solutions prior to whole blood measuring.
The purpose of the invention is, therefore, to provide a biosensor which, to a larger extent than the biosen¬ sors already known, provides sensor-independent measur- ing results when measuring on whole blood.
This is achieved by a biosensor which is characterized by the features stated in the characterizing part of claim 1.
The combination of a substrate-limiting layer and a protection layer comprising or consisting of cellulose or cellulose derivative material is hitherto unknown and as c"* eady mentioned this combination renders it possible to provide a biosensor being significantly improved compared to biosensors without said protection layer. Thus, with the protection layer the large sen¬ sor-to-sensor variation provided by the sensors without protection layer in connection with whole blood measur- ing is eliminated. This can also be seen from the comparison example below.
The most preferred material for the protection layer consists of regenerated cellulose such as cellophane, but a number of cellulose plastic materials such as cellulose ethers or cellulose esters, for example cellulose acetate, cellulose butyrate, cellulose pro- pionate, compounds of esters thereof, and ethyl cellu- lose and other cellulose alkyl and aryl ethers are expected to be suitable as well. Polyhydroxyethyl methacrylate is among other hydrophilic materials which are expected to be suitable as protection layer.
Biosensors are applied both for in vitro measuring and in vivo measuring, including extracorporeal measuring where the sample is directed from the patient to a measuring chamber and subsequently back to the patient. A protection layer of cellophane is particularly suit- able for such in vivo measuring as the material is strong, stands disinfection, is non-toxic, and has good bio-compability.
Preferably, the protection layer has a thickness rang- ing up to 100 μm, particularly 10-50 μm, and especially 12-25 μm. It is preferred to apply a protection layer being as thin as possible owing to i.a. the response time of the biosensors. In the biosensor according to the invention this may be achieved by providing the protection layer by chemical modification of the sur¬ face of the substrate-limiting layer turning away from the enzyme so that the surface by this chemical modifi¬ cation is provided with the hydrophilic properties according to the invention. In a preferred embodiment the protection layer and the substrate-limiting layer are lying closely together. The preferred substrate- limiting layer comprises a polyurethane layer whose slightly adhesive surface is able to hold the cellulose layer.
Application of a cellulose membrane in glucose sensors is in itself well-known, for example from the articles, patents, and patent applications mentioned below.
In the above-mentioned Clark 1962 article, the sensor membrane is constituted by a Cuprophane™-glucose oxi- dase-Cuprophane membrane, and the main purpose of the Cuprophane membranes is to retain the enzyme glucose oxidase at an electrode surface.
In the specification of US Patent No. US 3539455 (Clark) an electrochemical glucose sensor with a cello¬ phane membrane is disclosed. The glucose sensor is intended for measuring on blood, and the membrane serves to retain the enzyme glucose oxidase at the same side of the membrane as the anode and to keep the enzymes of blood, for example the H202-cleaving cata- lase, from the H202 formed by the enzymatic glucose conversion.
In the specification of British Patent No. GB 1442303 (Christiansen) an electrochemical glucose sensor is disclosed with a multi-layer membrane in which one of the layers comprises a thin, dense layer that prevents low-molecular agents, which similarly to H202 are oxid- able at the anode potential of glucose sensor, from getting into contact with the*working surface of the anode. Examples of such oxidable, low-molecular agents are uric acid, ascorbic acid, and various drugs, par- ticularly paracetamol. Out of consideration for the response time of the electrode, the thin layer should be so thin that it is essential to use a less dense layer as carrier layer. Cuprophane, cellulose acetate, or polymerized protein are suggested for this layer, and a hydrophobic material such as silicone rubber or a hydrophilic material such as cellulose acetate is suggested for the thin, dense layer.
In the specification of US Patent No. 4073713 (Newman) a glucose sensor is disclosed with a multi-layer mem¬ brane in which the layer nearest the anode or the inner layer comprises a layer of silicone rubber, methyl methacrylate or cellulose acetate which cf. the above- mentioned prevents oxidable agents from getting into contact with the anode. The layer nearest the sample or the outer layer constitutes a diffusion barrier, partly preventing passage of high-molecular agents and partly giving mechanical strength. Porous polycarbonate or methyl methacrylate is suggested. An enzyme preparation is present between the inner layer and the outer layer.
In the specification of US Patent No. 4172770 (Semersky) a system is disclosed where a single-layer or double-layer membrane of the cellulose material Spectrapor™ separates a reaction chamber and an elec¬ trochemical sensor. This membrane in combination with a controlled flow rate of sample past the membrane pre¬ vents interfering species from getting into contact with the active electrode surface of the sensor.
In the specification of European Patent Application No. EP 25110 (Suzuki) a glucose sensor is disclosed with a multi-layer membrane consisting of an inner filter membrane, an enzyme membrane, and an outer asymmetri- cal, semipermeable membrane produced from cellulose acetate comprising a thin, semipermeable outer layer and a thick, porous inner layer. The asymmetrical, semipermeable membrane is said to improve the stability of the sensor when measuring on whole blood compared to a sensor with a usual semipermeable outer membrane of reproduced cellulose or polycarbonate. The linearity range of the sensor is not disclosed, only measurements on blood containing 0.2 mM glucose, that is a glucose content far below the clinically interesting values.
In the specification of US Patent No. US 3948745 (Guilbault) an amperometric sensor is disclosed, for example a glucose sensor with an outer cellophane membrane and a layer of a polymer containing chemically bound glucose oxidase. The sensor is said to be suit¬ able for blood analysis, and the cellophane membrane is said to be permeable for the chemical compound which is determined by the sensor, but the specification con¬ tains no data for the sensor.
Finally, in the specification of US Patent No. US 4005002 (Racine) a sensor for glucose or lactate is disclosed with a cellophane membrane facing the sample. Measuring is performed on a diluted biological fluid which is said to be necessary owing to the lifetime of the sensor. The sensor does not measure on reoxidation of H202 but the reoxidation of a reduced acceptor.
None of the reference cited above suggest that it would be desirable oi -bvious to apply an outer, hydro¬ philic protection layer in a glucose sensor of the type having a membrane comprising .a substrate-limiting layer and having due to the presence of this layer a line¬ arity range relevant for clinical applications. Several electrochemical biosensors are available. Especially potentiometrical, polarographical, conducto- metrical, redox- ediator-based, and FET-based sensors, and among the polarographical ones are both sensors basing the substrate determination on determination of 02 and sensors basing the substrate determination on determination of H202. Gronow M. et al. has further described the various types of electrochemical biosen¬ sors in "Biosensors", Royal Society of Chemistry Spe- cial Publication, Molecular Biology and Biotechnology 54 (1985) 295-324. The contents of this reference is considered part of the present specification, and the claims of the present application are to cover all the various types of electrochemical biosensors.
Although the present invention is exemplified by a glucose sensor only, it is considered equally useful in connection with other biosensors measuring the concen¬ tration or activity of a biochemical analyte or a substrate by means of an immobilized enzyme. Particu¬ larly, the invention is considered applicable in con¬ nection with biosensors for the following biochemical analytes, the respective immobilized enzymes being men¬ tioned right after the analyte: lactate/lactate oxi- dase, cholesterol/cholesterol oxidase, hypoxanthine/- hypoxanthine oxidase and pyruvate/pyruvate oxidase.
In a preferred embodiment of the sensor according to the invention the working electrode is constituted by platinum anode with an exposed working surface onto which is provided a coating of cellulose acetate. This coating prevents low-molecular agents such as for example ascorbic acid and paracetamol, which can be oxidized by the potential of the working electrode, from reaching the working electrode, being oxidated and consequently interfering with the substrate determina¬ tion. A very thin layer having a thickness of for example 2-25, preferably 3-15 and particularly 5-10 μm has now proved sufficient to prevent the interfering agents from interfering the glucose determination. The layer should be as thin as possible owing to the re¬ sponse time of the sensor which increases with increas¬ ing thickness of the cellulose acetate layer.
In a further preferred embodiment of the invention the substrate-limiting layer consists of a hydrophobic plastic layer, particularly a polyurethane layer. The thickness of the hydrophobic plastic layer is pre¬ ferably 0.1-10 μm, particularly 0.2-5 μm and especially 0.5-3 μm.
The invention also relates to the type of sensor mem¬ brane stated in the opening part of claim 8 having the characteristic features stated in claims 8, 9, 10, 11, and 12.
The invention will now be further explained in con¬ nection with the drawing where
FIG. 1 is a view of the sensor according to the inven¬ tion located in a measuring chamber;
FIG. 2 is an exploded view of an embodiment of the sensor according to the invention, and
FIG. 3 is a view of the sensor membrane in the sensor according to the invention.
In the figures of the drawing same parts are provided with identical reference numerals. The sensor 1 according to the invention shown in FIG. 1 is arranged in such way that the front 2 of the sensor constitutes one of the walls in a measuring chamber 3. An inlet channel 4 opens into the measuring chamber 3, and an outlet channel 5 extends from the measuring chamber. Besides, the measuring chamber 3 is not in contact with the environment when the sensor 1 is mounted. The sensor 1 is located in a tubular part 6 which projects from a surface 7 of the block in which the measuring chamber 3 is provided. The measuring chamber 3 is located in the surface 7 and encircled by the tubular part 6.
The block 8 with the measuring chamber 3 shown in FIG. 1 is the type which is applied in the blood gas ana¬ lyzer ABL500 manufactured and sold by RADIOMETER A/S, Copenhagen, Denmark. The block contains several serial¬ ly connected measuring chambers with inlet channels and outlet channels of the same configuration as the ea- suring chamber 3, the inlet channel 4, and the outlet channel 5.
FIG. 2 shows an embodiment in further detail of the sensor 1 according to the invention. The sensor con- sists of a base part 9, a jacket 10, a membrane 11, a membrane ring 12. Apart from the membrane 11 and the cellulose acetate coating mentioned below and the fact that the Pt-wire has a differing thickness, the sensor is constructed similarly to a sensor which is manu- factured and sold by RADIOMETER A/S, Copenhagen, Denmark under the designation E909.
The base part 9 comprises a working electrode 13 in the form of a 250 μm platinum wire 17 melted into a glass rod 16. Further, the base part comprises an Ag/AgCl reference electrode 14 in the form of an annular coat¬ ing on the glass rod 16. At the front of the glass rod 16 the platinum wire 17 is exposed, and a not shown coating of cellulose acetate is applied to this end of the glass rod 16 as further described below in Example 1. At the rear the sensor 1 is provided with a connec¬ tor 15 with electrically conductive connection to the working electrode 13 and the reference electrode 14. Further, the base part comprises a locking means 18 with a rim 19 that serves to maintain the jacket 10 on the base part 9 according to the snap lock principle. Finally, at the front 2 of the locking means 18 facing the sensor is provided a rubber seal 20 which seals the compartment 21 formed when the jacket 10 is mounted on the base part 9.
The jacket 10, which is made from a transparent plastic material, is tubular and slightly conical and has a projecting part 22 on the inner surface of its broad end adapted to interlock with the locking means 18.
The membrane 11 shown in further detail in FIG. 3 consists of a laminated membrane comprising a 14 μm protection layer 24 of cellophane, a 1 μm polyurethane layer 25, an approximately 1 μm glucose oxidase layer 26, and a 1 μm poly lrethane inner layer 27. The mem¬ brane has a greater area than the small aperture in the jacket 10 and is consequently able to cover this aper¬ ture and be wrapped around the outer of the jacket 10 onto which it is pasted and further held by the mem¬ brane ring 12. When the membrane is correctly mounted on the jacket 10, the protection layer 24 is turned outwards and the inner layer 27 against the aperture of the jacket 10. The membrane 11 is prepared as described below in Example 1. Before mounting the jacket 10 on the base part 9, an electrolyte is introduced having the compound described further in Example 1. The electrolyte establishes the necessary electrical contact between the working elec- trode 13 and the reference electrode 14.
Finally, in order to illustrate the size of the sensor according to the invention and the related measuring set-up it can be mentioned that the small aperture in the jacket 10, i.e. the aperture covered by the mem¬ brane, has a diameter of approximately 7 mm and that the membrane has a thickness of approximately 17 μm. Furthermore, it can be mentioned that the measuring chamber 3 contains approximately 5 μl sample and has a circular aperture with a maximum diameter of approxi¬ mately 3.5 mm at the surface 7. The inlet channel 4 and the outlet channel 5 are tubular bores with a circular cross section of approximately 0.7 mm in diameter.
The test results mentioned below have been achieved by the embodiment shown in FIG. 2 of the sensor according to the invention located in the measuring chamber of FIG. 1.
In the measuring situation a polarization voltage of +0.625 is applied to the working electrode relatively to the reference electrode, and the measuring set-up is thermostated to 37°C. After a sample has been intro¬ duced into the measuring chamber 3 and brought into contact with the sensor 1, the working electrode current is registered as a function of time on a not shown printer after transformation of the electrode current to a voltage signal. Alternatively, the signal of the working electrode is collected every half second from 5 to 25 seconds after introduction of the sample into the measuring chamber 3. The values for the signal of the working electrode are processed along with data from a previous calibration and printed as mM glucose. Calibration is performed on aqueous solutions contain- ing 0 mM and 10 mM glucose of the composition explained further under the paragraph Materials and Methods in Example 1.
The invention is further illustrated by the examples below.
Example 1
Materials and Methods
starting Materials
High Rejective Cellulose Acetate Membrane; S18914 from
Yellow Springs Instruments, Ohio, USA Nitromethane; Aldrich Chemie, Stenhei , West Germany
Cellulose acetate/butyrate; Eastman 4623 from Eastman
Kodak Co. , Rochester, USA
Cellulose acetate; Eastman 394-60 from Eastman Kodak
Co. , Rochester , USA Polyurethane; Desmopan™ 786 from Bayer AG, Dormagen,
West Germany
Tetrahydrofuran; E. Merck, Darmstadt, West Germany
N,N-Dimethyl formamide; E. Merck, Darmstadt, West
Germany Glucose oxidase; G-7141 from Sigma, St. Louis, USA
Glutaric aldehyde, G-6257 from Sigma, St. Louis, USA
Na-EDTA, Titriplex™ III, Art._ 8418 from E. Merck,
Darmstadt, West Germany
Na-benzoate; M 6290 from Bie and Bentsen A/S, Rødovre, Denmark NaH2P04, H20 z.A.; Art. 6346 from E. Merck, Darmstadt, West Germany
Na2HP04, 2H20 z.A.; Art. 6580 from E. Merck, Darmstadt, West Germany NaCl z.A. ACS, ISO; Art. 6404 from E. Merck, Darmstadt, West Germany
Kathori™ 886 MW; Rohm and Haas Co., Philadelphia, USA Glucose; D(+)-Glucose, wasserfrei Art. 8337, from E. Merck, Darmstadt, West Germany Cellophane; Dialysierschlauch 44110 Visking 20/32 from Serva Feinbiochemica, Heidelberg, West Germany
The starting materials mentioned above are applied in the production of the electrolyte, calibration liquids, and membranes stated below.
Calibration Liguid_l_-_0_mM Glucose
A solution without glucose is applied; partly as rins¬ ing fluid in the measuring set-up and partly as cali- bration liquid. This liquid has the following composi¬ tion:
0.56 g Na-EDTA
0.92 g Na-benzoate 1.52 g NaH2P04, 2H20
6.90 g Na2HP04, 2H20
2.72 g NaCl
60 μl Kathon™ 886 MW Ion-exchanged water to 1 1
Calibration Liguid_2_-_10 mM_GjLucose This liquid has the same composition as calibration liquid 1 except that it further contains glucose in a concentration of 10 mM. Electrolyte
As electrolyte is applied the same liquid as calibra¬ tion liquid 1.
Po3.yurethane_Membrane
First is produced a solution of the composition:
1.9 g Polyurethane 41.0 ml Tetrahydrofuran 9.0 ml N,N-Dimethyl formamide
This solution is applied onto a glass plate through a 0.1 mm slot in a metal box which slowly by the hand is moved across the glass plate. After some time of evapo- ration (approximately 10 minutes) the membrane is removed from the glass plate and washed in ion-e. changed water and put to dry. The thickness of this membrane is approximately 1 μm.
Enz.yine_Soluti.on
The enzyme solution has the following composition:
Glucose oxidase Calibration liquid 1
Figure imgf000017_0001
Glutaric aldehyde
Ce3.lu.lgse Acetate Coating
A solution of High Rejective Cellulose Acetate Membrane S-18914 in Nitromethane is prepared for the cellulose acetate coating on the front of the glass rod. The exact composition of the solution is a trade secret belonging to the manufacturer.Yellow Springs Instru¬ ments, Ohio, USA. The solution is applied to the front of the glass rod by a fine brush and dried on standing. The layer thick¬ ness is 5-10 μm.
Prgductign_of Sensgr_Membrane
From the Cellophane Dialysierschlauch described under the paragraph "Starting Materials" is cut out a piece of approximately 2x2 cm. This piece, the thickness of which is 14 μm, is put together with a piece of the above-mentioned polyurethane membrane of corresponding size. 2-3 μl enzyme solution is applied to a second piece of polyurethane membrane of corresponding size as the first one. The first membrane part consisting of a cellophane layer and a polyurethane layer and the second membrane part consisting of a polyurethane layer with enzyme solution are pressed together, thus provid¬ ing a membrane consisting of a cellophane layer, a polyurethane layer, an enzyme layer, and a polyurethane layer in this order. The polyurethane layer in the second membrane part may be replaced by a second inner layer, for example micro-porous polypropylene of the type Celgard™ 3401.
The provided membrane is dried on standing and subse- quently pasted on the jacket of FIG. 2 by Loctite™ (Cyanoacrylate Adhesive 414) .
Results
Two sensors of the type shown in FIG. 2, and wherein the sensor membrane was provided as mentioned above, were located in two similar, adjacent measuring cham¬ bers in connection with the measuring set-up of FIG. 1. Measuring was performed on a solution consisting of calibration liquid 1 with 5 mM glucose added (5 mM glucose) and blood. The results stated in table 1 were obtai
Figure imgf000019_0001
It is seen that the two electrodes provide mutually corresponding measurements both when measuring on blood and when measuring on aqueous sample and that the measurements on the aqueous samples before and after the blood sample correspond properly.
The sensor has a linearity range of 0.5-30 mM glucose and satisfactory stability. Long-term experiments did not show any sign of deterioration in the measuring performance for membranes which had been stored dryly at room temperature for 9 weeks. Membranes used in the test set-up of FIG. 1 and kept in continually contact with calibration liquid 1 between measuring have proved to maintain the measuring performance after 4 weeks function at 37°C.
Comparison Example
Two sensors of the type showed in FIG. 2, and wherein the sensor membrane was provided as mentioned above, yet the cellulose layer in the sensor membrane was left out, were located in the measuring set-up of FIG. 1. Measuring was performed on 5 mM glucose and blood, respectively. The results stated in table 2 were ob¬ tained. Table 2
Figure imgf000020_0001
It is seen that the two electrodes provide mutually corresponding measurements when measuring the first time on aqueous sample, but provide very different measurements on blood, particularly when measured the second time. It is noted that it is the same blood sample. The measurements on aqueous sample are mutually corresponding prior to the blood measurements, but subsequently both too low and varying from the elec¬ trode a to electrode b.

Claims

C L A I M S
1. Electrochemical biosensor comprising a working electrode, a reference electrode, and a membrane comprising a substrate-limiting layer and an en¬ zyme layer c h a r a c t e r i z e d in that at the substrate-limiting layer is provided a protection layer having hydrophilic properties corresponding to the hydrophilic properties of regenerated cellulose or being more hydrophilic than regenerated cellulose.
2. Electrochemical biosensor according to claim 1, c h a r a c t e r i z e d in that the protection layer comprises or consists of a cellulose material or a cellulose derivative material.
3. Electrochemical biosensor according to claims 2-3, c h a r a c t e r i z e d in that the protection layer comprises a layer of regenerated cellulose or cellulose plastic such as cellulos-: -ather or cellulose ester.
4. Electrochemical biosensor according to claims 1-3, c h a r a c t e r i z e d in that the protection layer has a thickness of up to 100 μm, preferably 10-50 μm and particularly 12-25 μm.
5. Electrochemical biosensor according to claims 1-4, c h a r a c t e r i z e d in that the working electrode is constituted by a platinum anode and that the platinum anode has an exposed working surface on which a coating of cellulose acetate is provided.
6. Electrochemical biosensor according to claim 5, c h a r a c t e r i z e d in that the cellulose acetate coating is in direct contact with the platinum anode and has a thick¬ ness of 2-25 μm, preferably 3-15 μm and particu¬ larly 5-10 μm.
7. Electrochemical biosensor according to claims 1-6, c h a r a c t e r i z e d in that the substrate-limiting layer consists of a hydrophobic plastic layer, preferably a polyure- thane layer.
8. Biosensor membrane comprising a substrate-limiting layer, an enzyme layer, and an inner layer c h a r a c t e r i z e d in that at the substrate-limiting layer is provided a protection layer having hydrophilic properties corresponding to the hydrophilic properties of regenerated cellulose or being more hydrophilic than regenerated cellulose.
9. Biosensor membrane according to claim 8, c h a r a c t e r i z e d in that the protection layer comprises or consists of a cellulose material or a cellulose derivative material.
10. Biosensor membrane according to claims 8-9, c h a r a c t e r i z e d in that the protection layer comprises a layer of regenerated cellulose or a cellulose plastic such as a cellulose ether or a cellulose ester.
11. Biosensor membrane according to claims 8-10, c h a r a c t e r i z e d in that the protection layer has a thickness of up to 100 μm, preferably 10-50 μm an particularly 12-25 μm.
12. Biosensor membrane according to claims 8-11, c h a r a c t e r i z e d in that the substrate-limiting layer consists of a hydrophobic plastic layer, preferably a polyure- thane layer.
PCT/DK1991/000248 1990-08-31 1991-08-27 Electrochemical biosensor WO1992004438A1 (en)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997004954A1 (en) * 1995-08-01 1997-02-13 Ysi Incorporated Improved laminated membrane structure for polarographic measurement and methods of making said structures
US5611900A (en) * 1995-07-20 1997-03-18 Michigan State University Microbiosensor used in-situ
US5766839A (en) * 1994-06-17 1998-06-16 Ysi Incorporated Processes for preparing barrier layer films for use in enzyme electrodes and films made thereby
WO2000003222A2 (en) * 1998-07-09 2000-01-20 Geun Sig Cha Method for fabricating biosensors using hydrophilic polyurethane
US6020052A (en) * 1996-07-30 2000-02-01 Ysi Incorporated Laminated membrane structure for polarographic measurement and methods of making said structures
US6099804A (en) * 1996-03-29 2000-08-08 Radiometer Medical A/S Sensor and membrane for a sensor
WO2006122554A2 (en) 2005-05-17 2006-11-23 Radiometer Medical Aps Enzyme sensor with a cover membrane layer covered by a hydrophilic polymer
EP2261647A1 (en) * 2001-05-31 2010-12-15 Instrumentation Laboratory Company Cross-linked enzyme matrix and uses thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0079502A1 (en) * 1981-11-05 1983-05-25 Miles Laboratories, Inc. Multilayer enzyme electrode membrane, method of making same and polarographic cell structure
EP0216577A2 (en) * 1985-09-16 1987-04-01 Imperial Chemical Industries Plc Sensor
US4759828A (en) * 1987-04-09 1988-07-26 Nova Biomedical Corporation Glucose electrode and method of determining glucose
DE3822911A1 (en) * 1987-07-17 1989-01-26 Daikin Ind Ltd BIOSENSOR
DE3824258A1 (en) * 1987-07-23 1989-02-16 Akad Wissenschaften Ddr MODIFIED ENZYME MEMBRANE FOR ENZYME ELECTRODES WITH HIGH SELECTIVITY AND METHOD FOR THEIR USE
EP0327018A2 (en) * 1988-02-05 1989-08-09 Nova Biomedical Corporation Enzyme Electrode

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0079502A1 (en) * 1981-11-05 1983-05-25 Miles Laboratories, Inc. Multilayer enzyme electrode membrane, method of making same and polarographic cell structure
EP0216577A2 (en) * 1985-09-16 1987-04-01 Imperial Chemical Industries Plc Sensor
US4759828A (en) * 1987-04-09 1988-07-26 Nova Biomedical Corporation Glucose electrode and method of determining glucose
DE3822911A1 (en) * 1987-07-17 1989-01-26 Daikin Ind Ltd BIOSENSOR
DE3824258A1 (en) * 1987-07-23 1989-02-16 Akad Wissenschaften Ddr MODIFIED ENZYME MEMBRANE FOR ENZYME ELECTRODES WITH HIGH SELECTIVITY AND METHOD FOR THEIR USE
EP0327018A2 (en) * 1988-02-05 1989-08-09 Nova Biomedical Corporation Enzyme Electrode

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5766839A (en) * 1994-06-17 1998-06-16 Ysi Incorporated Processes for preparing barrier layer films for use in enzyme electrodes and films made thereby
US5611900A (en) * 1995-07-20 1997-03-18 Michigan State University Microbiosensor used in-situ
WO1997004954A1 (en) * 1995-08-01 1997-02-13 Ysi Incorporated Improved laminated membrane structure for polarographic measurement and methods of making said structures
US6099804A (en) * 1996-03-29 2000-08-08 Radiometer Medical A/S Sensor and membrane for a sensor
US6020052A (en) * 1996-07-30 2000-02-01 Ysi Incorporated Laminated membrane structure for polarographic measurement and methods of making said structures
WO2000003222A2 (en) * 1998-07-09 2000-01-20 Geun Sig Cha Method for fabricating biosensors using hydrophilic polyurethane
WO2000003222A3 (en) * 1998-07-09 2000-03-30 Geun Sig Cha Method for fabricating biosensors using hydrophilic polyurethane
US6509148B2 (en) 1998-07-09 2003-01-21 I-Sens, Inc. Method for fabricating biosensors using hydrophilic polyurethane
EP2261647A1 (en) * 2001-05-31 2010-12-15 Instrumentation Laboratory Company Cross-linked enzyme matrix and uses thereof
US8426192B2 (en) 2001-05-31 2013-04-23 Instrumentation Laboratory Company Composite membrane containing a cross-linked enzyme matrix for a biosensor
US9388503B2 (en) 2001-05-31 2016-07-12 Instrumentation Laboratory Company Cross-linked enzyme matrix and uses thereof
WO2006122554A2 (en) 2005-05-17 2006-11-23 Radiometer Medical Aps Enzyme sensor with a cover membrane layer covered by a hydrophilic polymer

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DK209990A (en) 1992-03-01

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