CA1176145A - Enzyme electrode membrane - Google Patents
Enzyme electrode membraneInfo
- Publication number
- CA1176145A CA1176145A CA000413665A CA413665A CA1176145A CA 1176145 A CA1176145 A CA 1176145A CA 000413665 A CA000413665 A CA 000413665A CA 413665 A CA413665 A CA 413665A CA 1176145 A CA1176145 A CA 1176145A
- Authority
- CA
- Canada
- Prior art keywords
- membrane
- layer
- polymer
- cellulose acetate
- layers
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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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
- C12Q1/002—Electrode membranes
Abstract
ABSTRACT OF THE DISCLOSURE A membrane for electrochemical analysis is described comprising a first, relatively dense and thin layer and a second, relatively porous thick layer, which thick layer has dispersed therethrough the enzyme glucose oxidase. The process of making the membrane by casting two three layers of cellulose acetate compositions is also described.
Description
S Docket. I~o. 1~ 1205 E YI~IE ELECTRODE_M ~IBR~NE
¦ The present invention relates to a membrane ¦ suitable for use with an electrochemical sensor and a method of making said novel membrane. The membranes are used in ~oltametric cells for electrochemi.cal analysis con~only referred to as polarographic cells and mentioned as such hereinafter. These cells comprise an enzyme for converting a substance which is an unknown to be measured into a material ¦ which can be measured by way of an electrical signal from the I cells. A wide variety of assay techniques and sensors are available for the measurement of various materials. Of particular interest to the medical field, is the measurement of small amounts of various substances contained in body fluids, such as blood, in body tissues, in foodstuffs, and the likei Such substances include glucose, urea, uric acid, triglycerides, phospholipids, creatinine, amino acids, lactic acid, xanthine, chondroitin, etc. The development of a sensor for controlling or monitoring the glucose concentration . in blood or other body fluids is particularly important 2C especially for maintaining normal blood glucose levels in a diabetic patent. Typically, blood samples are withdrawn from the patient for an on-line analysis for glucose concentrati.ons using a glucose oxidase electrode with a polarographic detector for the generated hydrogen peroxide. Customarily, . 25 such detectors comprise an enzyme electrode for the determination of hydrogen peroxide with an anode, a cathode, an electrolyte, and a membrane of specific composition containing an enzyme that has been im~lobilized.
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Enzyr;les llave been used in C011jl.lllCtiOII W:itll pO].lrOgra.pll:iC
cells in instances where the unkllowll substance to he measured is not electrocllemically acti~e i~self, but by conversion or reaction of the enzyme Wit]l tlle unknown sample, a, rcact:ion product is obtained that may be measured; tliat i~s, i.t :is : , detectable by polarographic means. As stated abovc, t}-le most common problem of medical interest is the desi.re to measllrc glucose in the blood. In this measurement it is advalltageous to employ an enzyme to gain specificity. In the presence Or the enzyme glucose oxidase the following reaction takes place:
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. . .G.lucose + 2 glucose gluconic acid + hydrogen pero~i~le (ll2(~)2,) ¦ The hydrogen peroxide that is generated by this reclction is measurable by a-polarographic detector and tl-.erefore by .Ip~l^O-l priate calibration and calculations, it is possible to cletermi.ne, l from the amount of H2O2 liberated, what the glucose content was in the original specimen or sample.
,Generally, a polarographic cell comprises an electrically insulating receptacle, an indicator or sensing electrode electrically contacting a memhrane and a rererence ~0 electrode which is electri`cally in contact wit]l the membrane.
¦ By. the expression "contacting" it is intended to include the ¦ situation where the contact between membrane alld electro(le is ¦ obtained directly or througll a layer of electrolytc. Cells of ¦ various designs are widely known and understood in the art. An :5 ¦ especially suitable cell for purposes of the inventioll is l shown in Clemens et al, U.S. Patent No. 4,092,233.
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In the prior art, in the case of an enzyme membrane structure, it is known to arrange a second hydrophilic membrane at a distance.from the first mentioned membrane. In the space between the two membranes, a layer of concentrated enzyme is present. The free face of the second membrane provides the test surface to which the substrate to be .. tested is applied. This type of enzyme membrane is . described in the Annals of the New York Academy of Science, Vol. 102 (1962), pages 29-49. In that article, it was suggested that a pH sensitive electrode could be used to , detect gluconic acid produced by the reaction. It was disclosed that the enzyme in such a system could be trapped b~tween two cellulose acetate membranes. Glucose dif~uses through the membrane and is converted by the enzyme to gluconic acid which then diffuses both towards the pH
sensitive glass and back into the donor solution.
The first mentioned membrane facing the sensing electrode is made up of a material which can be penetrated by the substance to which the sensing electrode is sensitive.
. For example, this membrane is permeable to the reactants of the enzymatic reaction but impermeable to the enzyme . itself. It may be made of cuprophane but in the event that one of the reaction products is a gas at normal pressure and temperature and it is desired to measure via this gas, the membrane may consist of hydrophobic plastic impermeable to ions but slightly permeable to such gases as oxygen, carbon dioxide or ammonia. Numerous plastics having such properties are known including silicone rubber, tetrafluoroethylene and the like.
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In a later type of polarographic cell developed by Clark and described in U.S. Patent No. 3,539,455, the enzyme is placed on the electrode side of the membrane,and a platinum anode measures the hydrogen peroxide produced. Glucose, a low molecular weight species, passes through the membrane and reacts with the enzyme, but interfering high molecular weight substances such as catalase and peroxidase do not. It is disclosed that the enzymes may be held in a thin film directly !
between the platinum surface and the membrane by placing the enzyme on a porous film which has spaces large enough to hold enzyme molecules. However, cellophane membranes will not prevent low molecular weight interfering materials such as . uric acid or ascorbic acid from reaching the sensing electrode.
I Clark suggests a dual electrode system to overcome that problem. The compensating electrode, without an en~yme present, gives a signal for the interfering substances while the enzyme . electrode detects both the hydrogen peroxide and the interference. By calculation, the glucose level is determined.
. Such a dual sensor system, however, may encounter difficulties in the matching of the two cells.
It was then proposed to have an enzyme electrode which employs a thin filter membrane to prevent passage of low molecular weight interfering materials, such as uric acid and ascorbic acid, while permitting hydrogen peroxide to pass therethrough with minimum hindrance. There exist membrane materials, such as silicone rubber and cellulose acetate, which permit passage of hydrogen peroxide but which are effective barriers to interfering substances. Sin^e this type of membrane must be placed between the sensing electrode and . I
, . . .. ..
~ 76~15 . .1 some component of the sensing system, it follows that in order for measurement equilibrium to be as rapid as possible, the membrane must be as thin as possible while still retaining its selectivity. In the case of a hydrogen peroxide sensing probe, this membrane will need to be less than 2 microns thick.
A membrane of this thickness is difficult to use in practice because of its insufficient strength.
The art then turned to depositing the material in a thin layer on a porous substructure to provide the necessary strength while at the same time being of little hindrance to hydrogen peroxide passage, and the weak interference rejecting layer can be thin to enhance speed of response.
As described in Newman, U.S. Patent No. 3,979,274, in a laminated two-ply membrane, an enzyme adhesive is used to bond the two-plies together. The membrane includes a support layer which controls substrate diffusion and serves as a barrler to high molecular weight substances, an . enzyme preparation for reacting with the unknown and for bonding the layers together, and an essentially homogeneous layer that serves as a barrier to interfering low molecular weight materials but permits hydrogen peroxide to pass through. However ln this development, it is necessary to make a sandwich consisting of two membranes with a layer of enzyme between, the enzyme acting as the adhesive or bonding agent. In this type of arrangement, the use of too much enzyme could slow down the diffusion of the diffusing species to an unacceptable amount. If a thinner layer of enzyme is used, there is acceptable diffusion, but the loading of enzyme may not be adequate.
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A still later development came in Britisil Patent ~o. 1,442,303 (Radiometer) wherein there l~as ~roposed a compos;te membrane which is an inhomogeneous membrane formed as a Ullit.
The membrane has two different strata, one has a thickness of less than 5 microns and the other is sufficiently thick to provide strength. Ihe enzyme is bonded to a surace Gf thc membrane.
Other prior art has shown a number or disadvantages.
Thus, the method of Koyama et al, Anal~ a Chcmica ¦ Acta, Vol. 116, pages 307-311 (1980), immobilizes glucose ¦ oxidase to z cellulose acetate membrane. This method is more time consuming; it involves more steps and su:Efers from the disadvantages that a monolayer of molecules would be the maximum l possible enzyme load achievable.
¦ The invention described in the present applicationl however, allows much greater amounts of enzyme to be spacially ¦ distributed within the membranes such that much more enzyme is available for reaction with the substrate along the difusion I path of said substrate.
¦ Wingard et al, The Journal of Biomedical Materials Research, Vol. 13, pages 921-935 (1979) disclpses a platinum ¦ screen or wire for immobilization of the enzyme. This ~ould ¦ allow greater surface area to be utilized for binding than ¦ the method of Koyama et al and hence could employ greater ¦ numbers of enzyme molecules. Ilowever, the approach of ¦ Wingard is also limited to a monolayer of enzyme and I capable of sustaining high conversion rates of substrate .. I , ~ . .. ...
~ - ~ ~ " ,, ;, -~7 ~ ~ 5 diffusing through the open spaces in the platinum screen near the surface of the platlnum wire only. Hence, this prior art cannot achieve the theoretical conversion rates possible with an enzyrne spacially distributed throughout a membrane through which the substrate diffuses, as is ob~ainable by following this invention.
In accordance with the present invention, the need to prepare a discrete enzyme layer is eliminated by incorporating the enzyme directly into one portion of the membrane in a manner whereby the enzyme is homogeneously dispersed throughout that phase of the membrane and immobilized therein.
A number of advantages characterize the present invention including ease of preparation, the permanent attachment of two phases of the membrane with no chance of separation; i.e.,avoidance of lamlnation. Also the new membrane readily lends itself to a dip casting process whereby the membrane can be fixed directly to a miniature electrode.
In addition, a greater uniformity of enzyme - concentration may be achieved by the homogeneous distribution ' in a membrane than by sandwiching bulk enzyme between two layers.
The principles involved in the present invention may bè more fully understood with reference to the analysis of blood for glucose content. The liquid portion of blood consists of proteins, lipids, and other substances. Non-electrolytes are present such as glucose, enzymes such as catalase, electrolytes such as ascorbic acid (vitamin C) and various metallic salts made up of cations of sodium, potassium, 3Q magnesium, calcium, iron nd copper, and anions such as . . . 'I
_ . . . . , . _ w~-s chlorides, phosphates, bicarbonates, an~l carbonates. Ihe l~hos-¦ phates, carbonates and bicarboncltes operate as buffers to maintai ¦ the pH of blood at a fixecl level under normal conditions. If a I sample of blood were placed on one sicle of a Illcmbrane in a ce~]
¦ and an aqueous solution of the enzyme glucose oxidase and o~ger ¦ on the other side of the membrane, certain low molecular weigllt materials will pass from the blood throu~]i the melllbralle to tl~e ¦ glucose oxidase solution. The hig]~ molecular ~eig}lt materials I such as the enzymes will not pass throug]l the membrane. Ille ¦ rates of permeability of the various materials througll the membrcl]le ¦ are fix~d because of the nature of tne membrane. In this illVClltil)ll, ¦ the relatively thin phase has a molecular cut off of appro~ tely ¦ 300. This means that matcrials c)f a molecular weigllt or greater I than about 300 wi-ll not pass through.
¦ Glucose, a low molecular weight material, will pass through the membrane and react witll the enzyme glucose oxi~lase in the presence of oxygen to form gluconolactone and hyclroge peroxide. Gluconolactone in the presence of water will hydrolyze spontaneously to form gluconic acid.
¦ Gluconic acid and hydrogen peroxide, being relatively I 1DW molecular weight materials compared to the enzyme glucose ¦ oxidase, will pass through the membrane. Catalase and -¦ peroxidases which are large enzyme molecules capable of l rapidly destroying 1-l2O2 and which are present in biocl~emical ¦ fluids are prevented from passing through the membrane.
According to the present invention~ tlle mentbrane may ¦ be utilized in a cell for electrocllemical analysis comprising, ¦ in general, an elèctrically insulating receptacle, an ano~le allcl a cathode as is shown in U.S. Patent No. 4,092,223. Tlle membrane . ~1_ , . . . .. _, 7~5 of this in~rention may also be used in olcler type devices I utilizing a sensing electrode (anode), a reference electrode ¦ (cathode) in a space in the receptacle which is separated from ¦ the sensing electrode and adapted to hold an elcctrolyte.
¦ The membrane electrically contacts the electrodes; a path ~or ¦ an electrical current extends between anode and cathode or ¦ between the reference electrode an~ the sensing elcctro(lc a]l~l ¦ the membrane camprising the t~o component, integrated enzyme ¦ membrane which is described herein~
¦ One portion of the membrane of the invention has a ¦ relatively high density and is re]atively thin, and tlle other ¦ portion of the membrane has a relatlvely lo-~er density an~l a ¦ thicker cross-section. The portion of the membrane ~]liCll }laS
¦ the thicker cross-section has the enzyme incorporatcd and ¦ immobilized therein and distributed homogeneously thr~ugllout.
It is a characteristic feature of the present invention ¦ that the composite membrane is formed in two distinct steps ¦ and has different strata or portions paralIel to the surface ¦ of the membrane. If desired, however, anothcr iayer, witllout ZO ¦ enzyme, may be present between the first and second layers.
I The use of a second phase inversion layer (without enzyme) ¦ between the dense layer and the phase inversion enzyme layer ¦ appears to allow a better membrane to be manufactured by I providing more linear response characteristics. Tlle multilaye ¦ membrane blocks the migration to the sensing electrode of ¦ interfering substances such as uric acid, ascorbic acid, and large nongaseous molecules and similar substances and allo~s the passing of solvent and low molecular weight species, for l examplé, enzymatic conversion products such as hydrogen ~ peroxide.
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A membrane exhibiting these properties can be ma(lc of I- cellulose acetate as well as other materials such .lS copolymers ¦ of ceilulose acetate. -¦ It has been determi3led that a reasonably short measllring ¦ time requires that the thickness of the membrane should not ¦ exceed, preferably, about 70 microns althollgll this Ca~l vary ¦ depending on the kind of meas~lrement to be carried out. It ~ould ¦ be possib]e to achieve an acceptable short response time for an I equilibrium of diffusion, for example of hydrogen peroxidc by ¦ designing the membrane to be made up of the thinner, more dense ¦ layer of 2 to S microns and the thicker, less dense laycr Or ¦ about 65 microns.
¦ The weaknesses inherent in the prior art have been overcome ¦ by forming the composite membrane according to the novel method of the invention. It consists of two phases whicll are not ¦ necessarily distinct but which when cast separately and in~lepend-¦ ently of each other are characterized as: a highly porous, ¦ relatively thick phase which in the composite membrane faces the ¦ electrodes and a relatively nonporous, denser and thi]lner p]lasc I which in the comp-oslte membrane faces the sample; e.g., the blood I specimen. In the composite membrane, the porosities and thick-¦ nesses of the two membranes may become modified as they arc ¦ fused together. There is uniformly distributed throughout the I highly porous ph-ase, a particulate enzyme. This enzyme may, however, become distributed throughout the composite me]llbrane.
¦ Since an intermingling or diffusion of the layers is believed ¦ to occur, the terms layers and phases are used interchangeably ¦ to mean layers W]liC]l may interact at their interaces.
¦ The indlvidual properties of the phases formillg the conipositc .
¦ membrane, if cast separately should be as follows: the relativelv nonporous phase should if cast by itself and tested have a molccu-lar weight cut off of approximately 300; the highly porous phase _... IL. . . . . ~ _ ~ ' . ~ .. , . .~ . .
~ 7~ 5 if cast by itself and tested should -freely pass the substrate for ¦ the enzyme (at the surface adjacent to the surface onto which it ¦ has been cast) and yet exclude macromolecules such as large ¦ proteins.
S ¦ In order to achieve the desired properties for detection ¦ of analytej the membrane of the invention is fabricated iJI a two ¦ stage process. First, an ultra thin ce]lulose acetate mellll)ralle I is cast or spread on a suitable surface which does not interact ¦ with or bond to the membrane. Replesentative surfaces to provi~le 10 ¦ a support for the cast film are glass and some ~lastics sucll as polyethylene. The film is cast with conventional equipment ¦ whereby it is possible to control the thickness of the resulting film. After being~spread on the surface, the cast Eilm is dried.
This thin film serves as the relatively nonporous thin phase.
15 ¦ The thickness of this phase generally ranges from about 1 to 10 ¦ microns, preferably from 2 to 5 microns.
¦ A thicker phase inversion type of cellulose acetate mem-I brane containing the enzyme is then cast directly on top of the ¦ ultra thin membrane. Since both casting solutions are of the 20 ¦ same polymer base, and preferably use the same solvent, there is ¦ a diffusion zone of the two at the interface or boundary and no clear distinction can be made between the two phases. Indeed, the order of casting may also be reversed, although it is ~referre l to c^ast the thin film first. The films may be allowed to clry uncle 25 1 ambient conditions, or a heating device may be utilized. T]le first film need not be absolutely dry when the second film is cast on it; i.e., the first film may be tacky to the touch. It is believed that a skin forms on the top surface of the tllick film ater drying. .
30 ¦ The solution of the cellulose acetate for the formation of l tlle thin, more dense membra]le component is formecl by clissoll~ing I a cellulose acetate polymer in an inert or~anic solvellt SUCll as . ................ ,.,, L ~ ' ' ' - ' -,W,~
¦ ketones. Typical are acetone, cyclohe~anone, rnetl~ylethylketone ~ and the like. Mixtures of miscible solvents may also be used.
¦ Concentration of the polymer in solvent may vary, as from 1 to 5 ¦ preferably 2 to 3%. Tlle film is cast with any suitc3~1e filnl 5 ¦ applicator such as will produce a final product film thickness ¦ of 1-10 microns, preferably 2-5 microns in tllickness.
The phase inversion member; tllat is the relatively porolls ¦ thicker portion of the composite membrane of this inverltion, is ¦ prepared by forming a cellulose acetate polymer in solutiorl in an 10 ¦ inert organic solvent such as a ketone. ~ nonsolvent or non-solvent mixture for the cellulose acetate SUC]I as an etha]-ol all(l water mixture is then mixed with the cellulosc acetatc solvcllt critical and others may be used. Lower alcohols mixe~ Wit]l i~ate3 15 ¦ are usually preferred for this purpose. An aqueous enzyme solution is included as part of the nonsolvent phase. Ihe enzyllle, glucose oxidase, is usually employed in an aqueous solution con-taining from 500 to 5000 units of the enzyme per cc of water, ¦ although this can vary as will be apparent to those skilled in 20 ¦ the art. Typical electrochemical sensors which can be em.ployed ~¦ .with the membrane of this invention include the BIOST~TOR glucose electrode of Miles Laboratories, Inc. See U.S. Patent No. 4,092,233.
l The overall thickness of the membrane of thc inventio]l 25 ¦ can vary from about 40 to about 100 microns, but is preferably approximately 70 microns. The thinner, more dense layer ranges ¦ from about 1 to 10 microns, preferably 2 to about 5 microns and the thicker, less dense range from about 40-80 microns. Somc l variation in these values is permissible within the contemplation .
of this invention. The preferred membrane is about 70 microns in thickness, with one layer about 2 microns an(l another laycr about 65 microns in thickness.
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. ~l The following drawings illustrate the invention in ~urther detail and the invention will be more fullv understood by reference-to these drawings wherein:' Figure 1 is a vertical section view (partial) of a conventional polarographic cell utilizing the membrane of the present invention, and Figure 2 is an enlarged view of a cross-section of the membrane of the present invention.
Referring to Figure 1, there is sho~n a polarographic cell assembly which includes a receptacle in the form of an electrically insulating container 10 made of a plastic or glass material or any other suitable material and which may be of any cross-sectional area and shape, but is preferably cylindrical. This is covered by an electrically insulating cap 11. Positioned within the receptacle is an electrically insulating member rod, or cylindrical column 12, which contains in it àn electrical conductor 13. This conductor is connected to an active or exposed element 14 whic'n may be platinum, gold, silver, - graphite or the like.
- A lead is attached to the electrode which passes through the rod or column and through the cap to be'connected with a ~. ~. voltage source 15.
The lower end of the receptacle is provided with a support means 16 such as a ring or retainer 'and the membrane 17 in accordance with the present invention is supported over the end of the supporting receptacle nearest the central electrode and spaced a capillary distance from the active face of the electrode. The membrane can be held in posit~on with any suitable means, for example, by an 0-ring .. I
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fitting into a circular groove or other convenient means in the receptacle. A current measuring instrument (not shown) is connected in series with the cell.
Typically, the receptacle is provided with a vent 18 to permit gases to escape if pressure inside the rece~tacle rises to a sufficiently high degree.
An annular space is provided between the central rod and the receptacle walls and receives a reference electrode 19 which may be for example, silver chloride coated silver wire. The space 20 inbetween is at least partially and preferably campletely filled with a liquid mixture of electrolyte which may be introduced into the chamber through an aperture.
In polarographic measurements, two electrodes are commonly used, one of which is polarized and does not allow current to flow until depolarized by the substance being measured. In the cell structure shown in Figure 1, electrode 19 is the cathode and is polarized and ~requently referred to as the reference electrode. The other electrode, electrode 14 as shown in Figure 1, functions as an anode and is not polarized in the presence of the substance being measured and therefore will not restrict the flow of relatively large current and is frequently referred to as the sensor electrode. The electrodes shown in Figure 1 are in an electrically insulating relation and the electrolyte material which occupies the chamber provides a conductive patn between the two electrodes. Typical electrolytes include sodium or potassium chloride, buffers including carbonates, phosphates, . bicarbonates, acetates, alkali or rare earth salts or other organic buffers or mixtures thereof may be used. The solvent .
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¦ for such an electrolyte may be water, glycols, glycerine an~
¦ mixtures thereof as is well known in the art.
¦ Figure 2 shows a membrane in cross-sectional detail. The ¦ nonhomogeneous membrane haS a tllin, dense layer 2] an~ a tllick, ¦ less dense or porous layer 22 ~hich laycrs togctllcr rorm tllc I composite structure. The enzyme shown symbolically by ~ots is ¦ dispersed uniformly in the thick portion or strata oL tlle ¦ membrane. ~owever, some of the enzyme may difruse into the thin ¦ layer during preparation of the membrane before tlle solvent for ¦ the cellulose acetate has evaporated. ~lembrane surface 24 is in ¦ electrical contact ~ith tlle electrode. The membranc comprises ¦ the nonhomogeneous combination of the two layers ancl tlle enzymc, I the outer free surface of which 23 represents the test surface ¦ which is to be brought into contact with the solution to bc 15 I analyzed.
In the preferred embodiment, the inner surface 24 ~ ich is ¦ an electrical contact with the electrode is about 65 microns in ¦ thickness and the opposite layer in contact with tlle sample to ¦ be analyzed is about 2 microns. The overall thickness of tlle 20 ¦ membrane lS preferably about 70 microns.
The membrane of the invention may be pro~uced by first ¦ casting an ultra thin, relatively dense cellulose acetate mem-¦ brane onto a suitable surface and permitting it to dry. If the l thin layer is omitted, the measurements may be morc subject to 25 I nonlinearity due`to oxygen depletion which is, in turn, caused by an increased flux of glucose molecules transported through the membrane and reacting witll enzyme. Then the tllicker p1lase inversion type cellulose acetate membrane ~llic}l is relatively .
I porous may be cast directly on top of the thin membrane. It may 30 ¦ be possible to first cast the thick portion Or tl~e melllbranc an(l l then cast the thin portion directly on top of it.
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The phase inversion member or more porous portion of ¦ the membrane composite is formed by providing a solution of cellulose acetate in an organic inert solvent such as acetone.
The solution is thell mixed with a nonsolvent for tlle cellulose acetate. Suitable nonsolvents include ethanol ~nd l~ater mixtures.
It is also desirable that-the a~ueous ellzyme solutioll be introduced as a part of the nonsolvent phase.
The following specific example iilustrates ho~ the invention may be carried out bu-t should not be considercd as limiting thereof in any way.
EXAMPLE
On a clean glass plate, spread a 3~ cellulose acetate in acetone solution with 2 mil film applicator to prepare the first film portion.
Prepare the phase inversion cellulose acetate casting solu-tion by mixing l.5 cc of ethanol with 5 cc of a 10~i cellulose acetate in acetone solution. This is then placed in a salt water ice bath and stirring of the solution is continued. In ¦ 0.1 cc increments, a total of 1.0 cc of an aqueous glucose ¦ oxidase solution is then added to the solution. This solution I contains 2,0C0 to 3,000 units of the glucose oxidase per cc of ¦ solution. This is mixed for 10 to 15 minutes. The ]lliXing iS
¦ then stopped and the material is allowed to deaerate for 25 ¦ 5 minutes.
The second membrane solution is then spread Oll t~p of the first membrane Wit]l a 18 mil a~plicator. Ille spread film is .
then permitted to dry for several hours at room temperature.
The membrane is then ready for use.
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The enzyme preparation may simply be a mixture of the appropriate enzyme such as glucose oxidase in water. O~ course, other materials such as a binder or cross-linking a~ent liKe glutaraldehyde may be included in the enzyllle preparcltion. Iike-¦ wise, the proportion of enzyme to water in tlle preparation is¦ immaterial as long as a flowable paste or so:lution is formed l~hicl~
¦ may be coated or pressed easily into the solution. Su~ficicll~
¦ enzyme is incorporated into the solution to prepare an ade~uate ¦ reactive amount for measurement.
I The membrane composite of the present inve]1tion is a self-supporting film of a total thickness whicl1 may ran~e rrom about 50 to l00 microns, preferably abollt 70 microns. ~he composite membrane may be shaped to any particular configuration l or size or may be cut or dimensioned in any particular way to lS I fit receptacles for polarographic cells or electrodes o~ any suitable dimension. It may, in particu~ar, be fastened to an O-ring for use in an electrode such as described in U.S. Patent No. 4,092,233.
To fasten the membrane to a rubbery O-ring of an appro7l)riate size, a glucing operation may be employed. The membrane may also be cast directly onto an electrode surface.
In addition to cellulose acetate, other polymers capable of being dissolved in solvents and undergoing phase in~ersion witl~
thé addition of a weak solvent or nonsolvent would be potential membrane materials. Such polymers include cellulose nitrate, ethylcellulose and other cellulose derivatives. In adclition, polycarbonate is a suitable alternative if methylene c!1loride is employed as a solvent instead of acetone or other ketones. .
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~ As a substitute or alternative for the lower alcohols ¦ present in the phase inversion mixture formamide can be used.
Further variations and modifications of the invention as will be apparent to those skilled in the art af~er reading the foregoing are intended to be encompassed by thc claims tllat . are appended hereto.
¦ The present invention relates to a membrane ¦ suitable for use with an electrochemical sensor and a method of making said novel membrane. The membranes are used in ~oltametric cells for electrochemi.cal analysis con~only referred to as polarographic cells and mentioned as such hereinafter. These cells comprise an enzyme for converting a substance which is an unknown to be measured into a material ¦ which can be measured by way of an electrical signal from the I cells. A wide variety of assay techniques and sensors are available for the measurement of various materials. Of particular interest to the medical field, is the measurement of small amounts of various substances contained in body fluids, such as blood, in body tissues, in foodstuffs, and the likei Such substances include glucose, urea, uric acid, triglycerides, phospholipids, creatinine, amino acids, lactic acid, xanthine, chondroitin, etc. The development of a sensor for controlling or monitoring the glucose concentration . in blood or other body fluids is particularly important 2C especially for maintaining normal blood glucose levels in a diabetic patent. Typically, blood samples are withdrawn from the patient for an on-line analysis for glucose concentrati.ons using a glucose oxidase electrode with a polarographic detector for the generated hydrogen peroxide. Customarily, . 25 such detectors comprise an enzyme electrode for the determination of hydrogen peroxide with an anode, a cathode, an electrolyte, and a membrane of specific composition containing an enzyme that has been im~lobilized.
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Enzyr;les llave been used in C011jl.lllCtiOII W:itll pO].lrOgra.pll:iC
cells in instances where the unkllowll substance to he measured is not electrocllemically acti~e i~self, but by conversion or reaction of the enzyme Wit]l tlle unknown sample, a, rcact:ion product is obtained that may be measured; tliat i~s, i.t :is : , detectable by polarographic means. As stated abovc, t}-le most common problem of medical interest is the desi.re to measllrc glucose in the blood. In this measurement it is advalltageous to employ an enzyme to gain specificity. In the presence Or the enzyme glucose oxidase the following reaction takes place:
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. . .G.lucose + 2 glucose gluconic acid + hydrogen pero~i~le (ll2(~)2,) ¦ The hydrogen peroxide that is generated by this reclction is measurable by a-polarographic detector and tl-.erefore by .Ip~l^O-l priate calibration and calculations, it is possible to cletermi.ne, l from the amount of H2O2 liberated, what the glucose content was in the original specimen or sample.
,Generally, a polarographic cell comprises an electrically insulating receptacle, an indicator or sensing electrode electrically contacting a memhrane and a rererence ~0 electrode which is electri`cally in contact wit]l the membrane.
¦ By. the expression "contacting" it is intended to include the ¦ situation where the contact between membrane alld electro(le is ¦ obtained directly or througll a layer of electrolytc. Cells of ¦ various designs are widely known and understood in the art. An :5 ¦ especially suitable cell for purposes of the inventioll is l shown in Clemens et al, U.S. Patent No. 4,092,233.
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In the prior art, in the case of an enzyme membrane structure, it is known to arrange a second hydrophilic membrane at a distance.from the first mentioned membrane. In the space between the two membranes, a layer of concentrated enzyme is present. The free face of the second membrane provides the test surface to which the substrate to be .. tested is applied. This type of enzyme membrane is . described in the Annals of the New York Academy of Science, Vol. 102 (1962), pages 29-49. In that article, it was suggested that a pH sensitive electrode could be used to , detect gluconic acid produced by the reaction. It was disclosed that the enzyme in such a system could be trapped b~tween two cellulose acetate membranes. Glucose dif~uses through the membrane and is converted by the enzyme to gluconic acid which then diffuses both towards the pH
sensitive glass and back into the donor solution.
The first mentioned membrane facing the sensing electrode is made up of a material which can be penetrated by the substance to which the sensing electrode is sensitive.
. For example, this membrane is permeable to the reactants of the enzymatic reaction but impermeable to the enzyme . itself. It may be made of cuprophane but in the event that one of the reaction products is a gas at normal pressure and temperature and it is desired to measure via this gas, the membrane may consist of hydrophobic plastic impermeable to ions but slightly permeable to such gases as oxygen, carbon dioxide or ammonia. Numerous plastics having such properties are known including silicone rubber, tetrafluoroethylene and the like.
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In a later type of polarographic cell developed by Clark and described in U.S. Patent No. 3,539,455, the enzyme is placed on the electrode side of the membrane,and a platinum anode measures the hydrogen peroxide produced. Glucose, a low molecular weight species, passes through the membrane and reacts with the enzyme, but interfering high molecular weight substances such as catalase and peroxidase do not. It is disclosed that the enzymes may be held in a thin film directly !
between the platinum surface and the membrane by placing the enzyme on a porous film which has spaces large enough to hold enzyme molecules. However, cellophane membranes will not prevent low molecular weight interfering materials such as . uric acid or ascorbic acid from reaching the sensing electrode.
I Clark suggests a dual electrode system to overcome that problem. The compensating electrode, without an en~yme present, gives a signal for the interfering substances while the enzyme . electrode detects both the hydrogen peroxide and the interference. By calculation, the glucose level is determined.
. Such a dual sensor system, however, may encounter difficulties in the matching of the two cells.
It was then proposed to have an enzyme electrode which employs a thin filter membrane to prevent passage of low molecular weight interfering materials, such as uric acid and ascorbic acid, while permitting hydrogen peroxide to pass therethrough with minimum hindrance. There exist membrane materials, such as silicone rubber and cellulose acetate, which permit passage of hydrogen peroxide but which are effective barriers to interfering substances. Sin^e this type of membrane must be placed between the sensing electrode and . I
, . . .. ..
~ 76~15 . .1 some component of the sensing system, it follows that in order for measurement equilibrium to be as rapid as possible, the membrane must be as thin as possible while still retaining its selectivity. In the case of a hydrogen peroxide sensing probe, this membrane will need to be less than 2 microns thick.
A membrane of this thickness is difficult to use in practice because of its insufficient strength.
The art then turned to depositing the material in a thin layer on a porous substructure to provide the necessary strength while at the same time being of little hindrance to hydrogen peroxide passage, and the weak interference rejecting layer can be thin to enhance speed of response.
As described in Newman, U.S. Patent No. 3,979,274, in a laminated two-ply membrane, an enzyme adhesive is used to bond the two-plies together. The membrane includes a support layer which controls substrate diffusion and serves as a barrler to high molecular weight substances, an . enzyme preparation for reacting with the unknown and for bonding the layers together, and an essentially homogeneous layer that serves as a barrier to interfering low molecular weight materials but permits hydrogen peroxide to pass through. However ln this development, it is necessary to make a sandwich consisting of two membranes with a layer of enzyme between, the enzyme acting as the adhesive or bonding agent. In this type of arrangement, the use of too much enzyme could slow down the diffusion of the diffusing species to an unacceptable amount. If a thinner layer of enzyme is used, there is acceptable diffusion, but the loading of enzyme may not be adequate.
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A still later development came in Britisil Patent ~o. 1,442,303 (Radiometer) wherein there l~as ~roposed a compos;te membrane which is an inhomogeneous membrane formed as a Ullit.
The membrane has two different strata, one has a thickness of less than 5 microns and the other is sufficiently thick to provide strength. Ihe enzyme is bonded to a surace Gf thc membrane.
Other prior art has shown a number or disadvantages.
Thus, the method of Koyama et al, Anal~ a Chcmica ¦ Acta, Vol. 116, pages 307-311 (1980), immobilizes glucose ¦ oxidase to z cellulose acetate membrane. This method is more time consuming; it involves more steps and su:Efers from the disadvantages that a monolayer of molecules would be the maximum l possible enzyme load achievable.
¦ The invention described in the present applicationl however, allows much greater amounts of enzyme to be spacially ¦ distributed within the membranes such that much more enzyme is available for reaction with the substrate along the difusion I path of said substrate.
¦ Wingard et al, The Journal of Biomedical Materials Research, Vol. 13, pages 921-935 (1979) disclpses a platinum ¦ screen or wire for immobilization of the enzyme. This ~ould ¦ allow greater surface area to be utilized for binding than ¦ the method of Koyama et al and hence could employ greater ¦ numbers of enzyme molecules. Ilowever, the approach of ¦ Wingard is also limited to a monolayer of enzyme and I capable of sustaining high conversion rates of substrate .. I , ~ . .. ...
~ - ~ ~ " ,, ;, -~7 ~ ~ 5 diffusing through the open spaces in the platinum screen near the surface of the platlnum wire only. Hence, this prior art cannot achieve the theoretical conversion rates possible with an enzyrne spacially distributed throughout a membrane through which the substrate diffuses, as is ob~ainable by following this invention.
In accordance with the present invention, the need to prepare a discrete enzyme layer is eliminated by incorporating the enzyme directly into one portion of the membrane in a manner whereby the enzyme is homogeneously dispersed throughout that phase of the membrane and immobilized therein.
A number of advantages characterize the present invention including ease of preparation, the permanent attachment of two phases of the membrane with no chance of separation; i.e.,avoidance of lamlnation. Also the new membrane readily lends itself to a dip casting process whereby the membrane can be fixed directly to a miniature electrode.
In addition, a greater uniformity of enzyme - concentration may be achieved by the homogeneous distribution ' in a membrane than by sandwiching bulk enzyme between two layers.
The principles involved in the present invention may bè more fully understood with reference to the analysis of blood for glucose content. The liquid portion of blood consists of proteins, lipids, and other substances. Non-electrolytes are present such as glucose, enzymes such as catalase, electrolytes such as ascorbic acid (vitamin C) and various metallic salts made up of cations of sodium, potassium, 3Q magnesium, calcium, iron nd copper, and anions such as . . . 'I
_ . . . . , . _ w~-s chlorides, phosphates, bicarbonates, an~l carbonates. Ihe l~hos-¦ phates, carbonates and bicarboncltes operate as buffers to maintai ¦ the pH of blood at a fixecl level under normal conditions. If a I sample of blood were placed on one sicle of a Illcmbrane in a ce~]
¦ and an aqueous solution of the enzyme glucose oxidase and o~ger ¦ on the other side of the membrane, certain low molecular weigllt materials will pass from the blood throu~]i the melllbralle to tl~e ¦ glucose oxidase solution. The hig]~ molecular ~eig}lt materials I such as the enzymes will not pass throug]l the membrane. Ille ¦ rates of permeability of the various materials througll the membrcl]le ¦ are fix~d because of the nature of tne membrane. In this illVClltil)ll, ¦ the relatively thin phase has a molecular cut off of appro~ tely ¦ 300. This means that matcrials c)f a molecular weigllt or greater I than about 300 wi-ll not pass through.
¦ Glucose, a low molecular weight material, will pass through the membrane and react witll the enzyme glucose oxi~lase in the presence of oxygen to form gluconolactone and hyclroge peroxide. Gluconolactone in the presence of water will hydrolyze spontaneously to form gluconic acid.
¦ Gluconic acid and hydrogen peroxide, being relatively I 1DW molecular weight materials compared to the enzyme glucose ¦ oxidase, will pass through the membrane. Catalase and -¦ peroxidases which are large enzyme molecules capable of l rapidly destroying 1-l2O2 and which are present in biocl~emical ¦ fluids are prevented from passing through the membrane.
According to the present invention~ tlle mentbrane may ¦ be utilized in a cell for electrocllemical analysis comprising, ¦ in general, an elèctrically insulating receptacle, an ano~le allcl a cathode as is shown in U.S. Patent No. 4,092,223. Tlle membrane . ~1_ , . . . .. _, 7~5 of this in~rention may also be used in olcler type devices I utilizing a sensing electrode (anode), a reference electrode ¦ (cathode) in a space in the receptacle which is separated from ¦ the sensing electrode and adapted to hold an elcctrolyte.
¦ The membrane electrically contacts the electrodes; a path ~or ¦ an electrical current extends between anode and cathode or ¦ between the reference electrode an~ the sensing elcctro(lc a]l~l ¦ the membrane camprising the t~o component, integrated enzyme ¦ membrane which is described herein~
¦ One portion of the membrane of the invention has a ¦ relatively high density and is re]atively thin, and tlle other ¦ portion of the membrane has a relatlvely lo-~er density an~l a ¦ thicker cross-section. The portion of the membrane ~]liCll }laS
¦ the thicker cross-section has the enzyme incorporatcd and ¦ immobilized therein and distributed homogeneously thr~ugllout.
It is a characteristic feature of the present invention ¦ that the composite membrane is formed in two distinct steps ¦ and has different strata or portions paralIel to the surface ¦ of the membrane. If desired, however, anothcr iayer, witllout ZO ¦ enzyme, may be present between the first and second layers.
I The use of a second phase inversion layer (without enzyme) ¦ between the dense layer and the phase inversion enzyme layer ¦ appears to allow a better membrane to be manufactured by I providing more linear response characteristics. Tlle multilaye ¦ membrane blocks the migration to the sensing electrode of ¦ interfering substances such as uric acid, ascorbic acid, and large nongaseous molecules and similar substances and allo~s the passing of solvent and low molecular weight species, for l examplé, enzymatic conversion products such as hydrogen ~ peroxide.
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A membrane exhibiting these properties can be ma(lc of I- cellulose acetate as well as other materials such .lS copolymers ¦ of ceilulose acetate. -¦ It has been determi3led that a reasonably short measllring ¦ time requires that the thickness of the membrane should not ¦ exceed, preferably, about 70 microns althollgll this Ca~l vary ¦ depending on the kind of meas~lrement to be carried out. It ~ould ¦ be possib]e to achieve an acceptable short response time for an I equilibrium of diffusion, for example of hydrogen peroxidc by ¦ designing the membrane to be made up of the thinner, more dense ¦ layer of 2 to S microns and the thicker, less dense laycr Or ¦ about 65 microns.
¦ The weaknesses inherent in the prior art have been overcome ¦ by forming the composite membrane according to the novel method of the invention. It consists of two phases whicll are not ¦ necessarily distinct but which when cast separately and in~lepend-¦ ently of each other are characterized as: a highly porous, ¦ relatively thick phase which in the composite membrane faces the ¦ electrodes and a relatively nonporous, denser and thi]lner p]lasc I which in the comp-oslte membrane faces the sample; e.g., the blood I specimen. In the composite membrane, the porosities and thick-¦ nesses of the two membranes may become modified as they arc ¦ fused together. There is uniformly distributed throughout the I highly porous ph-ase, a particulate enzyme. This enzyme may, however, become distributed throughout the composite me]llbrane.
¦ Since an intermingling or diffusion of the layers is believed ¦ to occur, the terms layers and phases are used interchangeably ¦ to mean layers W]liC]l may interact at their interaces.
¦ The indlvidual properties of the phases formillg the conipositc .
¦ membrane, if cast separately should be as follows: the relativelv nonporous phase should if cast by itself and tested have a molccu-lar weight cut off of approximately 300; the highly porous phase _... IL. . . . . ~ _ ~ ' . ~ .. , . .~ . .
~ 7~ 5 if cast by itself and tested should -freely pass the substrate for ¦ the enzyme (at the surface adjacent to the surface onto which it ¦ has been cast) and yet exclude macromolecules such as large ¦ proteins.
S ¦ In order to achieve the desired properties for detection ¦ of analytej the membrane of the invention is fabricated iJI a two ¦ stage process. First, an ultra thin ce]lulose acetate mellll)ralle I is cast or spread on a suitable surface which does not interact ¦ with or bond to the membrane. Replesentative surfaces to provi~le 10 ¦ a support for the cast film are glass and some ~lastics sucll as polyethylene. The film is cast with conventional equipment ¦ whereby it is possible to control the thickness of the resulting film. After being~spread on the surface, the cast Eilm is dried.
This thin film serves as the relatively nonporous thin phase.
15 ¦ The thickness of this phase generally ranges from about 1 to 10 ¦ microns, preferably from 2 to 5 microns.
¦ A thicker phase inversion type of cellulose acetate mem-I brane containing the enzyme is then cast directly on top of the ¦ ultra thin membrane. Since both casting solutions are of the 20 ¦ same polymer base, and preferably use the same solvent, there is ¦ a diffusion zone of the two at the interface or boundary and no clear distinction can be made between the two phases. Indeed, the order of casting may also be reversed, although it is ~referre l to c^ast the thin film first. The films may be allowed to clry uncle 25 1 ambient conditions, or a heating device may be utilized. T]le first film need not be absolutely dry when the second film is cast on it; i.e., the first film may be tacky to the touch. It is believed that a skin forms on the top surface of the tllick film ater drying. .
30 ¦ The solution of the cellulose acetate for the formation of l tlle thin, more dense membra]le component is formecl by clissoll~ing I a cellulose acetate polymer in an inert or~anic solvellt SUCll as . ................ ,.,, L ~ ' ' ' - ' -,W,~
¦ ketones. Typical are acetone, cyclohe~anone, rnetl~ylethylketone ~ and the like. Mixtures of miscible solvents may also be used.
¦ Concentration of the polymer in solvent may vary, as from 1 to 5 ¦ preferably 2 to 3%. Tlle film is cast with any suitc3~1e filnl 5 ¦ applicator such as will produce a final product film thickness ¦ of 1-10 microns, preferably 2-5 microns in tllickness.
The phase inversion member; tllat is the relatively porolls ¦ thicker portion of the composite membrane of this inverltion, is ¦ prepared by forming a cellulose acetate polymer in solutiorl in an 10 ¦ inert organic solvent such as a ketone. ~ nonsolvent or non-solvent mixture for the cellulose acetate SUC]I as an etha]-ol all(l water mixture is then mixed with the cellulosc acetatc solvcllt critical and others may be used. Lower alcohols mixe~ Wit]l i~ate3 15 ¦ are usually preferred for this purpose. An aqueous enzyme solution is included as part of the nonsolvent phase. Ihe enzyllle, glucose oxidase, is usually employed in an aqueous solution con-taining from 500 to 5000 units of the enzyme per cc of water, ¦ although this can vary as will be apparent to those skilled in 20 ¦ the art. Typical electrochemical sensors which can be em.ployed ~¦ .with the membrane of this invention include the BIOST~TOR glucose electrode of Miles Laboratories, Inc. See U.S. Patent No. 4,092,233.
l The overall thickness of the membrane of thc inventio]l 25 ¦ can vary from about 40 to about 100 microns, but is preferably approximately 70 microns. The thinner, more dense layer ranges ¦ from about 1 to 10 microns, preferably 2 to about 5 microns and the thicker, less dense range from about 40-80 microns. Somc l variation in these values is permissible within the contemplation .
of this invention. The preferred membrane is about 70 microns in thickness, with one layer about 2 microns an(l another laycr about 65 microns in thickness.
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. ~l The following drawings illustrate the invention in ~urther detail and the invention will be more fullv understood by reference-to these drawings wherein:' Figure 1 is a vertical section view (partial) of a conventional polarographic cell utilizing the membrane of the present invention, and Figure 2 is an enlarged view of a cross-section of the membrane of the present invention.
Referring to Figure 1, there is sho~n a polarographic cell assembly which includes a receptacle in the form of an electrically insulating container 10 made of a plastic or glass material or any other suitable material and which may be of any cross-sectional area and shape, but is preferably cylindrical. This is covered by an electrically insulating cap 11. Positioned within the receptacle is an electrically insulating member rod, or cylindrical column 12, which contains in it àn electrical conductor 13. This conductor is connected to an active or exposed element 14 whic'n may be platinum, gold, silver, - graphite or the like.
- A lead is attached to the electrode which passes through the rod or column and through the cap to be'connected with a ~. ~. voltage source 15.
The lower end of the receptacle is provided with a support means 16 such as a ring or retainer 'and the membrane 17 in accordance with the present invention is supported over the end of the supporting receptacle nearest the central electrode and spaced a capillary distance from the active face of the electrode. The membrane can be held in posit~on with any suitable means, for example, by an 0-ring .. I
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fitting into a circular groove or other convenient means in the receptacle. A current measuring instrument (not shown) is connected in series with the cell.
Typically, the receptacle is provided with a vent 18 to permit gases to escape if pressure inside the rece~tacle rises to a sufficiently high degree.
An annular space is provided between the central rod and the receptacle walls and receives a reference electrode 19 which may be for example, silver chloride coated silver wire. The space 20 inbetween is at least partially and preferably campletely filled with a liquid mixture of electrolyte which may be introduced into the chamber through an aperture.
In polarographic measurements, two electrodes are commonly used, one of which is polarized and does not allow current to flow until depolarized by the substance being measured. In the cell structure shown in Figure 1, electrode 19 is the cathode and is polarized and ~requently referred to as the reference electrode. The other electrode, electrode 14 as shown in Figure 1, functions as an anode and is not polarized in the presence of the substance being measured and therefore will not restrict the flow of relatively large current and is frequently referred to as the sensor electrode. The electrodes shown in Figure 1 are in an electrically insulating relation and the electrolyte material which occupies the chamber provides a conductive patn between the two electrodes. Typical electrolytes include sodium or potassium chloride, buffers including carbonates, phosphates, . bicarbonates, acetates, alkali or rare earth salts or other organic buffers or mixtures thereof may be used. The solvent .
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¦ for such an electrolyte may be water, glycols, glycerine an~
¦ mixtures thereof as is well known in the art.
¦ Figure 2 shows a membrane in cross-sectional detail. The ¦ nonhomogeneous membrane haS a tllin, dense layer 2] an~ a tllick, ¦ less dense or porous layer 22 ~hich laycrs togctllcr rorm tllc I composite structure. The enzyme shown symbolically by ~ots is ¦ dispersed uniformly in the thick portion or strata oL tlle ¦ membrane. ~owever, some of the enzyme may difruse into the thin ¦ layer during preparation of the membrane before tlle solvent for ¦ the cellulose acetate has evaporated. ~lembrane surface 24 is in ¦ electrical contact ~ith tlle electrode. The membranc comprises ¦ the nonhomogeneous combination of the two layers ancl tlle enzymc, I the outer free surface of which 23 represents the test surface ¦ which is to be brought into contact with the solution to bc 15 I analyzed.
In the preferred embodiment, the inner surface 24 ~ ich is ¦ an electrical contact with the electrode is about 65 microns in ¦ thickness and the opposite layer in contact with tlle sample to ¦ be analyzed is about 2 microns. The overall thickness of tlle 20 ¦ membrane lS preferably about 70 microns.
The membrane of the invention may be pro~uced by first ¦ casting an ultra thin, relatively dense cellulose acetate mem-¦ brane onto a suitable surface and permitting it to dry. If the l thin layer is omitted, the measurements may be morc subject to 25 I nonlinearity due`to oxygen depletion which is, in turn, caused by an increased flux of glucose molecules transported through the membrane and reacting witll enzyme. Then the tllicker p1lase inversion type cellulose acetate membrane ~llic}l is relatively .
I porous may be cast directly on top of the thin membrane. It may 30 ¦ be possible to first cast the thick portion Or tl~e melllbranc an(l l then cast the thin portion directly on top of it.
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The phase inversion member or more porous portion of ¦ the membrane composite is formed by providing a solution of cellulose acetate in an organic inert solvent such as acetone.
The solution is thell mixed with a nonsolvent for tlle cellulose acetate. Suitable nonsolvents include ethanol ~nd l~ater mixtures.
It is also desirable that-the a~ueous ellzyme solutioll be introduced as a part of the nonsolvent phase.
The following specific example iilustrates ho~ the invention may be carried out bu-t should not be considercd as limiting thereof in any way.
EXAMPLE
On a clean glass plate, spread a 3~ cellulose acetate in acetone solution with 2 mil film applicator to prepare the first film portion.
Prepare the phase inversion cellulose acetate casting solu-tion by mixing l.5 cc of ethanol with 5 cc of a 10~i cellulose acetate in acetone solution. This is then placed in a salt water ice bath and stirring of the solution is continued. In ¦ 0.1 cc increments, a total of 1.0 cc of an aqueous glucose ¦ oxidase solution is then added to the solution. This solution I contains 2,0C0 to 3,000 units of the glucose oxidase per cc of ¦ solution. This is mixed for 10 to 15 minutes. The ]lliXing iS
¦ then stopped and the material is allowed to deaerate for 25 ¦ 5 minutes.
The second membrane solution is then spread Oll t~p of the first membrane Wit]l a 18 mil a~plicator. Ille spread film is .
then permitted to dry for several hours at room temperature.
The membrane is then ready for use.
..
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The enzyme preparation may simply be a mixture of the appropriate enzyme such as glucose oxidase in water. O~ course, other materials such as a binder or cross-linking a~ent liKe glutaraldehyde may be included in the enzyllle preparcltion. Iike-¦ wise, the proportion of enzyme to water in tlle preparation is¦ immaterial as long as a flowable paste or so:lution is formed l~hicl~
¦ may be coated or pressed easily into the solution. Su~ficicll~
¦ enzyme is incorporated into the solution to prepare an ade~uate ¦ reactive amount for measurement.
I The membrane composite of the present inve]1tion is a self-supporting film of a total thickness whicl1 may ran~e rrom about 50 to l00 microns, preferably abollt 70 microns. ~he composite membrane may be shaped to any particular configuration l or size or may be cut or dimensioned in any particular way to lS I fit receptacles for polarographic cells or electrodes o~ any suitable dimension. It may, in particu~ar, be fastened to an O-ring for use in an electrode such as described in U.S. Patent No. 4,092,233.
To fasten the membrane to a rubbery O-ring of an appro7l)riate size, a glucing operation may be employed. The membrane may also be cast directly onto an electrode surface.
In addition to cellulose acetate, other polymers capable of being dissolved in solvents and undergoing phase in~ersion witl~
thé addition of a weak solvent or nonsolvent would be potential membrane materials. Such polymers include cellulose nitrate, ethylcellulose and other cellulose derivatives. In adclition, polycarbonate is a suitable alternative if methylene c!1loride is employed as a solvent instead of acetone or other ketones. .
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~ As a substitute or alternative for the lower alcohols ¦ present in the phase inversion mixture formamide can be used.
Further variations and modifications of the invention as will be apparent to those skilled in the art af~er reading the foregoing are intended to be encompassed by thc claims tllat . are appended hereto.
Claims (15)
1. A method of making a 40 to 100 micron conti-guous multilayer membrane suitable for use with an electro-chemical sensor in the measurement of an unknown which comprises:
providing a first polymer dissolved in an inert organic solvent and casting said polymer in solution onto an inert support surface which is unreactive with said polymer and does not form a bond to said polymer, permitting said solution to form a 1 to 10 micron dense relatively nonporous film and thereby obtain a first layer, providing a second polymer dissolved in an inert organic solvent mixing said second polymer dissolved in sol-vent with a nonsolvent for said polymer and with glucose oxidase to obtain a dispersion and thereafter casting said dispersion onto said first layer, and thereafter permitting said second polymer to dry to form a second 40 to 80 micron highly porous layer less dense than the first layer, thereby forming said contiguous multilayer membrane, said layers of the membrane being fused together such that no clear distinction can be made between the layers at the boundary and the boundary between the layers is a diffusion zone.
providing a first polymer dissolved in an inert organic solvent and casting said polymer in solution onto an inert support surface which is unreactive with said polymer and does not form a bond to said polymer, permitting said solution to form a 1 to 10 micron dense relatively nonporous film and thereby obtain a first layer, providing a second polymer dissolved in an inert organic solvent mixing said second polymer dissolved in sol-vent with a nonsolvent for said polymer and with glucose oxidase to obtain a dispersion and thereafter casting said dispersion onto said first layer, and thereafter permitting said second polymer to dry to form a second 40 to 80 micron highly porous layer less dense than the first layer, thereby forming said contiguous multilayer membrane, said layers of the membrane being fused together such that no clear distinction can be made between the layers at the boundary and the boundary between the layers is a diffusion zone.
2. A method of making a 40 to 100 micron conti-guous multilayer membrane suitable for use with an electo-chemical sensor in the measurement of an unknown which com-prises:
providing a first polymer dissolved in an inert organic solvent by mixing said first polymer in solution with a nonsolvent for said polymer and with glucose oxidase to obtain a dispersion, casting said dispersion onto an inert support surface which is unreactive with said polymer and does not form a bond to said polymer, permitting said dispersion to form a 40 to 80 micron highly porous film and thereby obtain a first layer, providing a second polymer dissolved in an inert organic solvent, casting said second polymer onto said first layer and permitting said second polymer to dry to form a second 1 to 10 micron relatively nonporous layer more dense than the first layer, thereby forming said contiguous multilayer polymer membrane, said layers of the membrane being fused together such that no clear distinction can be made between the layers at the boundary and the boundary between the layers is a diffusion zone.
providing a first polymer dissolved in an inert organic solvent by mixing said first polymer in solution with a nonsolvent for said polymer and with glucose oxidase to obtain a dispersion, casting said dispersion onto an inert support surface which is unreactive with said polymer and does not form a bond to said polymer, permitting said dispersion to form a 40 to 80 micron highly porous film and thereby obtain a first layer, providing a second polymer dissolved in an inert organic solvent, casting said second polymer onto said first layer and permitting said second polymer to dry to form a second 1 to 10 micron relatively nonporous layer more dense than the first layer, thereby forming said contiguous multilayer polymer membrane, said layers of the membrane being fused together such that no clear distinction can be made between the layers at the boundary and the boundary between the layers is a diffusion zone.
3. The method of claim 1 wherein the inert organic solvent reacted with said first and second polymer is the same.
4. The method of claim 2 wherein the inert organic solvent reacted with said first and second polymer is the same.
5. The method of claim 3 wherein the inert organic solvent is a ketone.
6. The method of claim 3 wherein the inert organic solvent is acetone.
7. The method of claims 1 or 2 wherein the glu-cose oxidase is present in a mixture of water and ethanol.
8. The method of claim 1 wherein said first layer is formed from a 3% cellulose acetate in acetone and said first layer is about 2 microns in thickness.
9. The method of claim 1 wherein said second layer is formed from 10% cellulose acetate solution in acetone which is mixed with a solution containing ethanol and glucose oxidase in water or buffer.
10. The method of claim 2 wherein said first layer is formed from 10% cellulose acetate in acetone solu-tion which is mixed with a solution containing ethanol and glucose oxidase in water or buffer.
11. The method of claim 2 wherein said second layer is formed from a 3% cellulose acetate solution in acetone and said second layer is about 2 microns in thick-ness.
12. The method as defined in claims 1 or 2 wherein in forming the layer containing glucose oxidase, cellulose acetate solution is mixed with glucose oxidase by mixing 1.5 cc of ethanol with 5 cc of a 10% cellulose acetate solution in acetone in a salt water ice bath with continuous stirring, the 1 cc of an aqueous glucose oxidase solution containing 2,000 to 3,000 units per cc, is added incremental-ly with continuing mixing for 10 to 15 minutes to the cel-lulose acetate solution to form a dispersion and deaerating for 5 minutes.
13. In a polarographic cell structure for use in electrochemical analysis of an unknown comprising an elec-trically insulating receptacle, an electrode means mounted in said receptacle, and a membrane means, the improvement which comprises utilizing the membrane defined by claims 1 or 2.
14. A membrane having a total thickness of about 40 to about 100 microns for use in a polarographic cell for the electrochemical analysis of an unknown comprising a first 1 to 10 micron dense relatively nonporous layer of essentially homogeneous cellulose acetate and a second 40 to 80 micron highly porous less dense layer of cellulose acetate separately fused to said first layer, said second layer containing glucose oxidase dispersed throughout, wherein the boundary between the two layers is a diffusion zone and no clear distinction can be made between the layers at the boundary.
15. A membrane for use in a polarographic cell for the electrochemical analysis of an unknown and which is made by the method of claims 1 or 2.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/318,626 US4415666A (en) | 1981-11-05 | 1981-11-05 | Enzyme electrode membrane |
| US318,626 | 1981-11-05 |
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| Publication Number | Publication Date |
|---|---|
| CA1176145A true CA1176145A (en) | 1984-10-16 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000413665A Expired CA1176145A (en) | 1981-11-05 | 1982-10-18 | Enzyme electrode membrane |
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| US (1) | US4415666A (en) |
| EP (1) | EP0080601B1 (en) |
| JP (1) | JPS5886450A (en) |
| AU (1) | AU540000B2 (en) |
| CA (1) | CA1176145A (en) |
| DE (1) | DE3276341D1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| US3539455A (en) * | 1965-10-08 | 1970-11-10 | Leland C Clark Jr | Membrane polarographic electrode system and method with electrochemical compensation |
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| GB1442303A (en) * | 1972-09-08 | 1976-07-14 | Radiometer As | Cell for electro-chemical analysis |
| US3947325A (en) * | 1974-04-11 | 1976-03-30 | Snamprogetti S.P.A. | Preparation of high permeability cellulose fibers containing enzymes |
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| JPS6029475B2 (en) * | 1978-09-29 | 1985-07-10 | 株式会社日立製作所 | Immobilized enzyme membrane and its manufacturing method |
| JPS57127841A (en) * | 1981-02-02 | 1982-08-09 | Mitsubishi Rayon Co Ltd | Biological electrochemical sensor and manufacture thereof |
-
1981
- 1981-11-05 US US06/318,626 patent/US4415666A/en not_active Expired - Lifetime
-
1982
- 1982-10-13 AU AU89315/82A patent/AU540000B2/en not_active Ceased
- 1982-10-18 CA CA000413665A patent/CA1176145A/en not_active Expired
- 1982-10-27 EP EP82109922A patent/EP0080601B1/en not_active Expired
- 1982-10-27 DE DE8282109922T patent/DE3276341D1/en not_active Expired
- 1982-11-04 JP JP57192654A patent/JPS5886450A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| AU8931582A (en) | 1983-05-26 |
| EP0080601A1 (en) | 1983-06-08 |
| US4415666A (en) | 1983-11-15 |
| AU540000B2 (en) | 1984-10-25 |
| DE3276341D1 (en) | 1987-06-19 |
| EP0080601B1 (en) | 1987-05-13 |
| JPS5886450A (en) | 1983-05-24 |
| JPH0210902B2 (en) | 1990-03-12 |
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