DK170103B1 - Electrochemical biosensor and biosensor membrane - Google Patents

Description

in DK 170103 B1

ELECTROCHEMICAL BIOSENSOR AND BIOSENSOR MEMBRANE

The invention relates to an electrochemical biosensor of the kind mentioned in the preamble of claim 1.

Electrochemical biosensors have been the subject of much attention since a glucose sensor was first described by Clark et al. in "Annals of the New York 10 Academy of Science" 102 (1962) 29-45.

Electrochemical biosensors include an immobilized enzyme that is used to degrade a biochemical analyte or substrate - most often when consuming oxygen.

The concentration or activity of the substrate is measured with such biosensors by determining the amount of oxygen consumed or the amount of reaction product produced from the substrate's reaction with oxygen, for example hydrogen peroxide. A prerequisite for achieving a linear relationship between the signal of the biosensor and the substrate concentration is partly that there is excess oxygen present at the enzyme, since oxygen - and not the substrate - otherwise - becomes the limiting factor and partly that the substrate concentration at the enzyme is substantially less than the Km value of the enzyme, where Km is the Michae lisMenten constant.

An example of electrochemical biosensor is an electrochemical glucose sensor in which the enzyme glucose oxidase is used to break down glucose according to the reaction scheme: glucose + 02 + gluconic acid + H202 DK 170103 B1 2

As shown in the reaction scheme, it is possible to measure the concentration or activity of glucose in a sample by determining either the amount of oxygen consumed or the amount of hydrogen peroxide formed. The amount of hydrogen peroxide formed can be measured polarographically by the oxidation reaction: H 2 O 2 -> 2 H + + O 2 + 2e * 10 Under the aforementioned assumption that excess oxygen is present and that the substrate concentration at the enzyme is substantially less than the enzyme Km value, The H 2 O 2 oxidation stream is used as an expression of the glucose content.

15

There has been much development work on providing biosensors which, when measured directly on whole blood, have a linear response over the entire clinically relevant concentration range of the biochemical analyte. Glucose sensors with linear response over the entire clinically relevant glucose range (0.5-30 mM) are now described, partly in the description of US Patent No. US 4759828 (Young) and partly by Shichiri M. et al. in "We-arable-type Artificial Endocrine Pancreas with Needle-25 Type Glucose Sensor", Lancet 2 (1982) 1129-1131 and by Abe H. et al. in the specification to US Patent No. US 4515584. The special response properties of these sensors have been obtained because a membrane is provided between the enzyme and the sample whose glucose content is desired to be determined which limits the diffusion of glucose molecules.

It has now been found that biosensors which, similar to the glucose sensor of Shichiri or Abe, have a polyurethane membrane exposed to the sample, show quite a large number of sensor independent measurement results when measured on aqueous samples, but not is capable of providing sensor independent measurement results when the sample is whole blood. The sensors to varying degrees give too low measurements on whole blood and also on aqueous solutions that follow a whole blood measurement, even though they were able to measure similarly on aqueous solutions before being used for whole blood measurement.

The object of the invention is therefore to provide a biosensor which, to a greater extent than the known biosensors, gives sensor independent measurement results in whole blood measurements.

This is achieved with a biosensor which is peculiar to the characterizing part of claim 1.

The combination of substrate-limiting layers and a protective layer comprising or consisting of a cellulose or cellulose derivative material is novel and allows, as mentioned, to provide a biosensor which is substantially improved over biosensors without this protective layer. Thus, with the protective layer, the large sensor to sensor variation eliminated by the sensors without protective layers in connection with whole blood measurements is eliminated. This will also be apparent from the comparative example below.

The most preferred material for the protective layer 30 comprises regenerated cellulose such as cellophane, but a variety of cellulosic plastic materials such as cellulose ethers or cellulose esters, for example, cellulose acetate, cellulose butyrate, cellulose propionate, mixed esters thereof and ethyl cellulose, and . Among other DK 170103 B1 4 hydrophilic materials which are expected to be suitable as protective layers may be mentioned polyhydroxyethyl methacrylate.

Biosensors are used for both in vitro and in vivo measurements, including extracorporeal measurements, where the sample is passed from the patient to a measuring chamber and then back to the patient. A cellophane protective layer is particularly suitable for such in vivo measurements as the material is strong, resistant to disinfection, is non-toxic and has good biocompatibility.

The protective layer preferably has a thickness of up to 100 µL, in particular 10-50 µια and especially 12-25 µια. It is preferred to use, among other things, the response time of the biosensor to the thinnest possible protective layer.

Optionally, in the biosensor of the invention, this can be achieved by providing the protective layer by chemically modifying the substrate-limiting layer away from the enzyme-facing surface, so that the surface of this chemical modification imparts the hydrophilic properties of the invention. In a preferred embodiment, the protective layer and the substrate-limiting layer are close together. The preferred substrate-limiting layer is a polyurethane layer whose lightly adherent surface is capable of retaining the cellulose layer.

Use of a cellulose membrane in glucose sensors is well known in the art, for example, from the following 30 articles, patents and patent applications.

In the Clark 1962 article, cf. above, the sensor membrane is a Cuprophane "glucose oxidase-Cuprophane membrane, and the primary purpose of the Cuprophane membranes is to hold the enzyme glucose oxidase at an electrode surface.

In the specification to US Patent No. US 3539455 (Clark) an electrochemical glucose sensor is disclosed which includes a cellophane membrane. The glucose sensor is intended for measurement on blood, and the membrane serves to keep enzyme glucose oxidase on the same side of the membrane as the anode and to keep the enzymes of the blood, for example, the H 2 O 2 cleavage catalase, from the H2 O 2 formed by the enzymatic glucose reaction.

In the disclosure of English Patent GB 1442303 (Christiansen), an electrochemical glucose sensor having a multilayer membrane, wherein one layer is a thin, dense layer which prevents low molecular weight, which, like H202, is oxidizable by the anode potential of the glucose sensor, come into contact with the anode's working surface.

Examples of such oxidizable low molecular weight substances are uric acid, ascorbic acid and various drugs, especially paracetamol. For the sake of electrode response time, it is necessary that the thin layer be so thin that a less dense layer may be used as a carrier layer. For this layer, among others, Cuprophane, cellulose acetate or polymerized protein is proposed, and for the thin, dense layer a hydrophobic material such as silicone rubber or a hydrophilic material such as cellulose acetate is proposed.

In U.S. Patent No. 4073713 (Newman), U.S. Pat. contact with the anode. The layer closest to the sample or outer layer is a diffusion barrier which partly prevents the passage of high molecular weight substances and partly gives mechanical strength. Porous polycarbonate DK 170103 B1 6 or methyl methacrylate is proposed. An enzyme preparation exists between the inner layer and the outer layer. »In the US Patent Specification No. 4172770 (Semersky) 5, a system is disclosed wherein a single layer or double layer membrane of the Spectrapor ™ cellulosic material separates a reaction chamber and an electrochemical sensor.

This membrane in combination with a controlled flow rate of sample past the membrane prevents interfering species from contacting 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 multilayer membrane consisting of an inner filter membrane, an enzyme membrane and an outer asymmetric semipermeable membrane made of cellulose acetate comprising a thin semi-permeable outer layer and a thick porous inner layer. The asymmetric semipermeable membrane is stated to improve the stability of the sensor when measured on whole blood relative to a sensor with a traditional semipermeable outer membrane of reproduced cellulose or polycarbonate. The linearity range of the glucose sensor is not discussed, and only measurements on blood containing 0.2 25 mM glucose have been described, that is, a glucose content well below the clinically interesting values.

U.S. Patent No. 4,948,745 to Guilbault discloses an amperometric sensor, for example, a glucose sensor having an outer cellophane membrane and a layer of a polymer containing chemically bound glucose oxidase. The sensor is indicated as suitable for blood analysis and cell.

The lofan membrane is stated to be permeable to the chemical compound determined by the sensor, but the description contains no data for the sensor.

DK 170103 B1 7

Finally, US Patent No. 4005002 (Racine) discloses a glucose or lactate sensor with a cellophane membrane facing the sample. A diluted biological fluid is measured, which is stated to be necessary for the life of the sensor. The sensor does not measure reoxidation of H2O2, but reoxidation of a reduced acceptor.

None of the above-mentioned literature points to the desirability or proximity of using an outer hydrophilic protective layer in a glucose sensor of the type having a membrane comprising a substrate-limiting layer and which, due to the presence of this layer, has a linearity range relevant for clinical use.

There are several types of electrochemical biosensors.

Particular mention may be made of potentiometric, polarographic, conductometric, redox mediator-based and FET-based sensors, and among the polarographic are also sensors which base the substrate determination on a determination of 02 and sensors which base the substrate determination on a determination of H 2 O 2. Gronow M. et al. has given a detailed description of the various 25 types of electrochemical biosensors in "Biosensors", Royal Society of Chemistry Special Publication, Molecular Biology and Biotechnology £ 4 (1985) 295-324.

Although the invention is exemplified here only by a glucose sensor, it is considered equally useful in connection with other biosensors which measure the concentration or activity of a biochemical analyte or substrate by an immobilized enzyme. In particular, the invention is considered useful in connection with biosensors for the following biochemical analytes, the respective immobilized enzymes being mentioned immediately after the analyte: lactate / lactate oxidase * cholesterol / cholesterol oxidase, hypoxanthine / hypoxanthine oxidase / pyruvate oxidase .

In a preferred embodiment of the sensor according to the invention, the working electrode is a platinum anode with an exposed working surface on which a coating of cellulose acetate is provided. This coating prevents low molecular weight substances such as, for example, ascorbic acid and paracetamol, which are oxidizable by the potential of the working electrode, reach the working electrode, and thus interfere with the substrate determination. It has been found that a quite thin layer of, for example, 2-25, preferably 3-15, and especially 5-10 μιη in thickness is sufficient to prevent the interfering substances from interfering with the glucose determination.

The layer is desired as thin as possible for the response time of the sensor, which increases with increasing thickness of the cellulose acetate layer.

In a further preferred embodiment of the invention, the substrate-limiting layer is a hydrophobic plastic layer, in particular a polyurethane layer. The thickness of the hydrophobic plastic layers is preferably 0.1-10 μιη, in particular 0.2-5 μιη and most preferably 0.5-3 μη.

The invention also relates to a sensor membrane of the type set forth in the preamble of claim 7 and having the properties set forth in claims 7, 8, 9 and 10.

The invention will now be explained in more detail in connection with the drawing, in which DK 170103 B1 9 FIG. 1 is a view of the sensor of the invention located in a measuring chamber; FIG. 2 is an exploded view of one embodiment of the sensor of the invention; and FIG. 3 is a view of the sensor membrane of the sensor of the invention.

10 In the drawings, the same reference numbers are used in the various figures to denote the same parts.

The one shown in FIG. 1, sensor 1 according to the invention is arranged such that the front face 2 of the sensor forms one wall of a measuring chamber 3. A supply channel 4 opens into the measuring chamber 3 and a delivery channel 5 exits the measuring chamber. In addition, the measuring chamber 3 does not communicate with the surroundings when the sensor 1 is mounted.

The sensor 1 is mounted in a tubular portion 6 which exits 20 from a surface 7 of the block in which the measuring chamber 3 is provided. The measuring chamber 3 lies in the surface 7 and is surrounded by the tubular part 6.

The one shown in FIG. 1, block 8 with the measuring chamber 3 is of the type used in the blood gas analyzer ABL500 manufactured and sold by RADIOMETER A / S, Copenhagen,

Denmark. The block contains several serially connected measuring chambers with supply channels and delivery channels in the same configuration as the measuring chamber 3, supply channel 4 and delivery channel 5.

FIG. 2 shows in more detail an embodiment of the sensor 1 according to the invention. The sensor consists of a base member 9, a jacket 10, a membrane 11, a membrane ring 35 12. Except for the membrane 11 and the cellulose acetate coating mentioned below, and the Pt thread has a different thickness, the sensor is built up quite identically with a sensor manufactured and marketed under the designation E909 from RADIOMETER A / S, Copenhagen, Denmark, 5 mark.

The base member 9 comprises a working electrode 13 in the form of a 250 μτη platinum wire 17 fused to a glass rod 16.

The base member also comprises an Ag / AgCl reference electrode 14 in the form of an annular coating on the glass rod 16. At the front of the glass rod 16, the platinum wire 17 is exposed, and on this end of the glass rod 16 is applied a non-shown cellulose acetate coating which described below in Example 1. At its rear end, the sensor 1 has a contact portion 5 with electrically conductive connection to the working electrode 13 and the reference electrode 14. The base member further has a fixing member 18 with a flange 19 which serves to hold the cover 10 on the base member 9 after snap lock pin. Finally, at the side of the sensor face 2 of the fixing member 18, there is a rubber gasket 20 which seals the space 21 formed when the casing 10 is mounted on the base member 9.

The sheath 10, which is made of a transparent plastic material, is tubular and slightly tapered and has on its inner surface at its wide end projections 22 which are adapted to engage the fixing means 18.

30 '

The diaphragm 11, which is shown in more detail in FIG. 3, is a laminated membrane comprising a 14 μτα. protective layer 24 of cellophane, a 1 μιη polyurethane layer 25, an approx. 1 μια glucose oxidase layer 26 and a 1 μη polyurethane inner layer 35 27. The membrane has a larger extent than the small opening in the casing 10, and can thus cover this opening and fold up around the outside of the casing 10 where it is glued and further is retained by the membrane ring 12.

When the diaphragm is properly mounted on the casing 10, the protective layer 24 faces outward and the inner layer 27 faces the luminance of the casing 10. The diaphragm 11 is prepared as described below in Example 1.

Prior to mounting the casing 10 on the base portion 9, an electrolyte 10 of the composition described in Example 1 is charged. The electrolyte creates the necessary electrical contact between the working electrode 13 and the reference electrode 14.

Finally, to illustrate the order of magnitude of the sensor of the invention and the associated measurement array, it can be mentioned that the small aperture in the casing 10, i.e. the membrane-covered aperture, has a diameter of approx. 7 mm, and the membrane has a thickness 20 of approx. 17 yum. Furthermore, it can be mentioned that the measuring chamber 3 contains approx. 5 µl sample and has a circular aperture with a maximum diameter of approx. 3.5 mm at the surface 7. The supply duct 4 'and the discharge duct 5 are tubular bores having a circular cross-section with a diameter 25 of approx. 0.7 mm.

The test results discussed below are obtained with the one in FIG. 2 shows the embodiment of the sensor according to the invention arranged in the embodiment of FIG. 1.

In the measurement situation, a polarization voltage of +0.625 V is applied to the working electrode relative to the reference electrode and the measurement array is thermostated to 37 ° C. After a sample has been inserted into the measurement chamber 3 and brought into contact with the sensor 1, the course of the working electrode current is not recorded on a printer not shown, with time after the electrode current has been converted into a voltage signal. Alternatively, the working electrode signal is collected every 5 seconds from 5 to 25 seconds for 5 after the measuring chamber 3 is filled with sample. These values for the working electrode signal are processed together with data from a previous calibration and printed as mM glucose. Calibrate on aqueous solutions containing 0 mM and 10 mM glucose, respectively, with the 10 in Example 1 of the Materials and Methods described in the composition.

The invention is further illustrated by the following examples.

15

Example 1

Materials and Methods 2 0 Starting materials

High Rejective Cellulose Acetate Membrane; S-18914 from Yellow Springs Instruments, Ohio, USA Nitromethane; Aldrich Chemie, Stenheim, 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, 30 West Germany?

tetrahydrofuran; E. Merck, Darmstadt, West Germany Ν, Ν-Dimethylformamide; E. Merck, Darmstadt, West Germany Glucose oxidase; G-7141 from Sigma, St. Louis, USA Glutaraldehyde, G-6257 from Sigma, St. Louis, USA

DK 170103 B1 13

Post-EDTA, Titriplex ™ III, Art. 8418 from E. Merck, Darmstadt, West Germany

Sodium benzoate; M 6290 from Bie and Bentsen A / S, Rødovre,

Denmark 5 NaH 2 PO 4, HjO Z.A.; Nature. 6346 from E. Merck, Darmstadt, West Germany

Na 2 HPO 4, 2H 2 O e.g. Nature. 6580 from E. Merck, Darmstadt, West Germany

NaCl z.A. ACS, ISO; Nature. 6404 from E. Merck, Darmstadt, 10 West Germany

Kathon ™ 886 MW; Rohm and Haas Co., Philadelphia, USA Glucose; D (+) - glucose, washer-free Art. 8337, from E.

Merck, Darmstadt, West Germany

Cellophane; Dialysierschlauch 44110 Whisking 20/32 from 15 Serva Feinbiochemica, Heidelberg, West Germany

The above starting materials are used in the preparation of electrolyte, calibration fluids and membranes described hereinafter.

20

Calibration fluid 1-0 mM_ glucose A solution without glucose content is used partly as a cleaning fluid in the measuring system and partly as a calibration fluid. This liquid has the following composition: 0.56 g Na-EDTA 0.92 g Na-benzoate 1.52 g NaH2 PO4, 2H20 6.90 g Na2HPO4, 2H20 30 2.72 g NaCl 60 μΐ Kathon ™ 886 MW ion-exchanged water 1 1 DK 170103 B1 14

Calibration Fluid 2 - 10 µM Glucose This fluid has the same composition as Calibration Fluid 1 except that it further contains glucose at a concentration of 10 mM.

5 5

electrolyte

The same fluid as calibration fluid 1 is used as the electrolyte.

Polyurethane Membrane First a solution of the composition is prepared: 1.9 g Polyurethane 41.0 ml Tetrahydrofuran 15 9.0 ml Ν, Ν-Dimethylformamide

This solution is dosed on a glass plate through a 0.1 mm slit in a metal block which is slowly passed by hand over the glass plate. After some evaporation time 20 (about 10 minutes), the membrane is removed from the glass plate and washed out in ion-exchanged water and hung to dry. The thickness. of the membrane thus prepared is approx. 1 ^ m.

Enzyme solution 25 The enzyme solution has the composition: 1 mg Glucose oxidase 100 μΐ Calibration fluid 1 8 μΐ Glutaraldehyde 30

Celluloseacetatbelægning

For the cellulose acetate coating on the glass face, a solution of High Rejective Cellulose Acetate Membrane S-18914 in Nitromethane is prepared. The near-35 more composition of the solution is a manufacturing secret of the manufacturer Yellow Springs Instruments, Ohio, USA.

The solution is applied to the front surface of the glass rod with a 5 fine brush and wipe on standing. The layer thickness is 5-10 μχη.

Sensor membrane fabrication

Of the Cellophane 10 Dialysierschlauch mentioned in the "Starting materials" section, a piece of approx. 2x2 cm.

This piece, the thickness of which is 14 μιη, is added with a piece of the above-described polyurethane membrane of similar size. 2-3 μΐ of enzyme solution is applied to the second piece of polyurethane membrane of the same size as the first. The first membrane portion consisting of cellophane layer and polyurethane layer and the second membrane portion consisting of polyurethane layer with enzyme solution are compressed, thereby forming a membrane consisting of a cellophane layer, a polyurethane layer, an enzyme layer and a polyurethane layer in the order.

The polyurethane layer in the second membrane portion can be replaced, if desired, by another inner layer, for example, Celgard ™ 3401 microporous polypropylene.

The membrane thus prepared is dried on standing and then glued to the one of FIG. 2 with Loctite ™ (Cyanoacrylate Adhesive 414).

Results 30 2 sensors of the one shown in FIG. 2, in which the sensor membrane was made as described above, was mounted in two similar adjacent measuring chambers in the one in connection with FIG. 1. A solution consisting of calibration fluid 1 containing 5 in 1 glucose (5 mM glucose) and blood was measured. The results shown in Table 1 were obtained.

Table 1 $ 5

Sensor Sensor

Sample # 1 # 2

5 mM glucose 5.07 mM 5.12 mM

Blood 3.96 mM 3.94 mM

5 5 mM glucose 5.29 mM 5.25 mM

5 mM glucose 5.10 mM 5.13 mM

It can be seen that the two electrodes provide mutually consistent measurements both in blood and in aqueous sample, and that the measurements on the aqueous samples before and after the blood test match nicely.

The sensor has a linearity range of 0.5-30 mM glucose and good stability properties. Long-term experiments showed 20 no deterioration in the measurement properties of membranes that have been stored dry at room temperature for 2 1/2 months. Membranes used in the embodiment of FIG. 1, and between the measurements have always been in contact with calibration fluid 1, 25 has been found to maintain the measurement properties after 4 weeks of operation at 37 ° C.

Comparative Example 2 sensors of the one shown in FIG. 2, in which the sensor membrane was made as described above, however, excluding the cellulose layer in the sensor membrane, was mounted in it in connection with FIG. 1. 5 mM glucose and blood were measured, respectively. The results shown in Table 2 were obtained.

35 DK 170103 B1 17

Table 2

Sensor Sensor

Prove a b

5 5 mM glucose 5.15 mM 5.19 mM

Blood 4.93 mM 4.16 mM

Blood 3.98 mM 1.99 mM

5 mM glucose 4.55 mM 2.17 mM

5 mM glucose 4.70 mM 2.10 mM

10

It is seen that the two electrodes provide mutually consistent measurements at the first measurement on aqueous sample, but give very different measurements on blood, especially the second time. It is noted that this is the same blood sample. The measurements on the aqueous sample are mutually consistent before the blood measurements, but then both too low and different from electrode a to electrode b.

Claims (8)

An electrochemical biosensor comprising a working electrode, a reference electrode and a membrane V 5 comprising a substrate-limiting layer and an enzyme layer characterized in that a substrate-limiting layer is provided with the sample-facing protective layer which comprises or consists of a cellulose material. or a cellulose derivative material. Electrochemical biosensor according to claim 1, characterized in that the protective layer comprises a layer of regenerated cellulose or a cellulosic plastic such as a cellulose ether or a cellulose ester. Electrochemical biosensor according to claims 1-2, characterized in that the protective layer has a thickness of up to 100 μιη, preferably 10-50 μιη and especially 12-25 μιη. An electrochemical biosensor according to claims 1-3, characterized in that the working electrode is a platinum anode and that the platinum anode has an exposed working surface on which a coating of cellulose acetate is provided. 30 x 2 Electrochemical biosensor according to claim 4, characterized in that the cellulose acetate coating is in direct contact with the platinum anode and has a thickness of 2-25 μιη, 35 preferably 3-15 μιη and in particular 5-10 μιη. DK 170103 B1 19 Electrochemical biosensor according to claims 1-5, characterized in that the substrate-limiting layer is a hydrophobic plastic layer, preferably a polyurethane layer. 5 A biosensor membrane comprising a substrate-limiting layer, an enzyme layer and an inner layer, characterized in that the substrate-limiting layer provides a sample-facing protective layer comprising or consisting of a cellulose material or a cellulose derivative material. A biosensor membrane according to claim 7, 15, characterized in that the protective layer comprises a layer of regenerated cellulose or a cellulosic plastic such as a cellulose ether or a cellulose ester. Biosensor membrane according to claims 7-8, characterized in that the protective layer has a thickness of up to 100 µm, preferably 10-50 µηι and especially 12-25 µιη. Biosensor membrane according to claims 7-9, characterized in that the substrate-limiting layer is a hydrophobic plastic layer, preferably a polyurethane layer.