CA1189007A - Multilayer enzyme electrode membrane - Google Patents
Multilayer enzyme electrode membraneInfo
- Publication number
- CA1189007A CA1189007A CA000414487A CA414487A CA1189007A CA 1189007 A CA1189007 A CA 1189007A CA 000414487 A CA000414487 A CA 000414487A CA 414487 A CA414487 A CA 414487A CA 1189007 A CA1189007 A CA 1189007A
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- CA
- Canada
- Prior art keywords
- layer
- polymer
- enzyme
- membrane
- cellulose acetate
- 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
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
Abstract
ABSTRACT OF THE DISCLOSURE
A multilayer membrane for electrochemical analysis is described formed of a first, relatively dense layer, and a plurality of relatively porous layers, which porous layers are separated from each other and in contact with a plurality of layers. The process of making the membrane by casting multiple layers of polymer compositions is also described.
A multilayer membrane for electrochemical analysis is described formed of a first, relatively dense layer, and a plurality of relatively porous layers, which porous layers are separated from each other and in contact with a plurality of layers. The process of making the membrane by casting multiple layers of polymer compositions is also described.
Description
~90~7 Docket No. MS-1207 MULTILAYER ENZYME ELECTRODE MEMBP~NE
The present invention relates to a multilayer membrane suitable for use with an electrochemical sensor and a method l of making said multilayer membrane. The membranes as described 1 herein are used in voltametric cells for electrochemical analysls commonly known as polarographic cells and are referred to 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 1~ the cells. A wide variety of assay techniques and sensors are available for the measurement oE various materi.als. Of particular interest to the medical fleld, is the measurement of small amounts o various substances contained in body fluids, such as blood, in body tissues, in foodstuffs, and the like.
5 Such sub~tances include glucose, urea, uric acid, triglycericles, 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 important, particularly, for maintaining normal blood glucose levels in a diabetic patient. Typically, blood samples are withdrawn from the patient for an on~line analysis for glucose concentrations using a glucose oxidase electrode with a polarographic detector for the generated hydrogen peroxide.
Customarily, 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 ~nzyme that has been immobilized.
Enzymes have been used in conjunction with polarographic cells in instances where the unknown substance 0~7 ~1 ¦ to be measured is not electrochemiczl:Ly active itself, but by ¦~ conversion or reaction of the enzyme with the unknown sample, a rea^tion product is obtained that may be measured; that is, l it is detectable by polarographic means. As stated above, the 1 most common problem of medical interest is the desire to measure glucose in the blood. The problem is that glucose is itself not electrochemically active. However, in the presence of the enzyme glucose oxidase the following reaction takes place:
glucose Glucose + 2 -3 gluconic acid + hydrogen peroxide (H202) oxidase The hydrogen peroxide that is generated by this reaction is measurable by a polarographic detector and therefore by appropriate calibration and calculations, it i5 possible to ;.
d~termine, Erom the amount of H202 liberated, what the glucose 15 ! conterlt was in the original specimen or sample.
Generally, a polarographic cell comprises an l electrically insulating receptacle, an indicator or sensing ¦¦ electrode electrically contacting a membrane and a reference ~ electrode which is electrically in contact with the membrane.
20~ By the expression "contacting" it is intended to include the i situation where the contact between membrane and electrode is obtained directly or through a layer of electrolyte. Cells of various designs are widely known and understood in the art. Of j,, particular interest for purposes of the invention is the cell 2S shown in Clemens et al, U.S. Patent No. 4,092,233.
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
The present invention relates to a multilayer membrane suitable for use with an electrochemical sensor and a method l of making said multilayer membrane. The membranes as described 1 herein are used in voltametric cells for electrochemical analysls commonly known as polarographic cells and are referred to 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 1~ the cells. A wide variety of assay techniques and sensors are available for the measurement oE various materi.als. Of particular interest to the medical fleld, is the measurement of small amounts o various substances contained in body fluids, such as blood, in body tissues, in foodstuffs, and the like.
5 Such sub~tances include glucose, urea, uric acid, triglycericles, 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 important, particularly, for maintaining normal blood glucose levels in a diabetic patient. Typically, blood samples are withdrawn from the patient for an on~line analysis for glucose concentrations using a glucose oxidase electrode with a polarographic detector for the generated hydrogen peroxide.
Customarily, 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 ~nzyme that has been immobilized.
Enzymes have been used in conjunction with polarographic cells in instances where the unknown substance 0~7 ~1 ¦ to be measured is not electrochemiczl:Ly active itself, but by ¦~ conversion or reaction of the enzyme with the unknown sample, a rea^tion product is obtained that may be measured; that is, l it is detectable by polarographic means. As stated above, the 1 most common problem of medical interest is the desire to measure glucose in the blood. The problem is that glucose is itself not electrochemically active. However, in the presence of the enzyme glucose oxidase the following reaction takes place:
glucose Glucose + 2 -3 gluconic acid + hydrogen peroxide (H202) oxidase The hydrogen peroxide that is generated by this reaction is measurable by a polarographic detector and therefore by appropriate calibration and calculations, it i5 possible to ;.
d~termine, Erom the amount of H202 liberated, what the glucose 15 ! conterlt was in the original specimen or sample.
Generally, a polarographic cell comprises an l electrically insulating receptacle, an indicator or sensing ¦¦ electrode electrically contacting a membrane and a reference ~ electrode which is electrically in contact with the membrane.
20~ By the expression "contacting" it is intended to include the i situation where the contact between membrane and electrode is obtained directly or through a layer of electrolyte. Cells of various designs are widely known and understood in the art. Of j,, particular interest for purposes of the invention is the cell 2S shown in Clemens et al, U.S. Patent No. 4,092,233.
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
- 2 -~' , is present. The free face of ~he second membrane provides the test surface to which the substra~e to be tested is applled.
This type of enzyme membrane is described in the Annals of t.he l New York Academy of Science, Vol. 102 (1962), pages 29-49. In ¦ that article, it was suggested ~hat a pH sensitive èlectrode could be used to detect gluconic acid produced by the reaction.
It was disclosed that the enz~ne in such a system could be trapped between two cellulose acetate membranes. Glucose difEuses 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 firs~ 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.
:l5 For example, this membrane is permeable to the reactants of the. ell~ymatic reacti.on but impermeable to the enzyme it~elf.
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 20 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, tetrafluoroe~hylene and the like.
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 weigh~
~ub~ances such as catalase and peroxidase do not, It is _ 3 _ I
l _ l . . ~ ~, . _., ~:~896 l ll disclosed that the enzymes may be held in a thin film directly between the platinum surface and the membrane by placing the enzyme Gn a porous film which has spaces large enough to hold l 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.
¦ Clark suggested a dual electrode system to overcome that problem.
¦ The compensating electrode, without an enzyme 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 di.f~iculties in the matching of the two cells.
It was ~hen proposed to have an enzyme elec~rode ~S which employs a thin filter membrane to prevent passage of Low molecular weight interfering materials, such as uric acid ancl ascorbic acid, while permitting hydrogen peroxide to pass therethrough with minimum hindrance. There exist membrane materials, such as silicone rubber and cellulose acetate, ~0 which permit passàge of hydrogen peroxide but which are effective barriers to inte~fering substances. 5ince this type of membrane must be placed between sensing electrode and 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 stlll 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.
30The art then turned to depositing the material in a thin layer on a porous substructure to provide the necessary _ 4 _ l l 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.
l In Newman, U.S. Patent No. 3,979,274" a laminated 5 ¦ two-ply membrane is described wherein an enzyme adhesive is used to bond the two-lies together. The membrane includes a support 1ayer which controls substrate diffusion and serves ~s a barrier to high molecular weight substances, an enzyme l preparation for reacting with the unknown and for bonding l0 ¦ the layers together, and an essentially homogeneous layer that serves as a barrier to interfering low molecular weight materials but perm:its hydrogen peroxide to pass through.
I{o~ever in this development, it is necessary to make a I sandwich consisting of two membranes with a layer of enzyme 15 ¦ between, the enzyme acting as the adhesive or binding agent.
l In this type o:E arrangement, the use o:E t:oo much enzyme could ¦ slow clo~n the ~ fusion o the diffusing species to an unacceptable amount. If a thinner layer of enzyme is used, l there is acceptable dif~usion, but the loading of enzyme may 20 ¦ not be adequate.
A still later de~elopment came in British Patent No. l,442,303 ~`Radiometer) wherein there was proposed a ¦ composite membrane which is an inhomogeneous membrane formed I as a unit~ The membrane has two different strata, one has 25 ¦ a thickness of less than -5 microns and the other is sufficiently I thick.to provide strength. The enzyme is bonded to a surface ¦ of th.e mem~rane~
¦ Other pr~or art has shown a number of disadvantages.
:
¦ Thus, the method of Koyama et al, Analytica Chemica l Acta, Vol. 116, pages 307-311 (1980), immobilizes glucose oxidase ! to a cellulose acetate membrane. This method is more time l consuming; it involves more steps and suffers from the 1 disadvantages that a monolayer of molecules would be the maximum possible enzyme load achievable.
Wingard et al, The Journal of Biomedical Materials ~e_earch, Vol. l3, pages 921-925 (1979) discloses a platinum l screen or wire for immobilization of the enzyme. This would 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. However, the approach of Wingard is also limited to a monolayer of enzyme and capable oE
sustaining high conversion rates of substrate diffusing through the open spaces in the platinum screen near the surface of the platinum wlre only.
In accordance with the present i.nventi.on, there is provided a multilayer membrane formed of a plurality of layers or strata of polymer and enzyme which function together to overcome problems that have occurred in the prior art. The multiple layer technique of the invention affords the opportunity to obtain the advantages of a distributed enzyme preparation through the utilization of more enzyme being present in the composite multiple layer structure without encountering the problems which have been observed in the use of single layer enzyme sandwich membranes of the past.
A significant advantage of the present inventi.on ¦ resides in the fact that because the enzyme appears in more l than one layer the overall distribution of the enzyme will appear to be homogeneous and thereby eliminate any inhQmogeneity which may be present in any individual enzyme layer.
In addition, any significant interference with analyte diffusion which may be caused by unusual concentrations o~ enzyme in individual layers will be balanced by the total l overall diffusion of ~he analyte through a multiplicity of enzyme containing layers where any discontinuities or high concentrations of enzyme will effectively be equalized.
The principles involved in the present invention may be illustrated with reference to the analysis of blood for glucose content. However, it should be noted that the ¦ present invention is applicable to use wi,h many different enzyme systems, polymer membrane formula~ions, and the resulting multiple layer membranes may be used for analysls o~ numerous 1uids for varl.ous components. The l.iqui.d portion of blood consists of proteins, lipids, and other substances.
Nonelectrolytes are present such as glucose, enz~nes such as catala~e, electrolytes such as ascorbic acid (vitamin C) ~Ind varlous metallic salts made up oE cations of sodium, potassium, magnesium, calcium, iron and copper, and anions such as chlorides, phosphates, bicarbonates, and carbonates.
The phosphates, carbonates and bicarbonates operate as buffers to maintain the pH of blood at a fixed level under normal conditions. If a sample of blood were placed on one side of a membrane in a cell and an aqueous solution of, for example, the . enzyme glucose oxidase and oxygen on the other side of the 25` membrane, certain low molecular weight materials will pass from the blood through the membrane to the glucose oxidase solution.
The high molecular weight materials such as the enzymes will not pass through the membrane. The rates of permeability of the various materials through the membrane are fixed because of the nature of the membrane. By selection of appropriate materials, the membranes may be designed to have a molecular cut off at any desired point; for example, at approximately 300. This means that materials of a molecular weight of greater than about 300 will not pass through.
Glucose, a low molecular weight material, will pass through SUC]I a membrane and react with the enzyme glucose oxidase in the presence of oxygen to form gluconolactone and hydrogen peroxide.
Gluconolactone in the presence of water will hydrolyze spontane-ously to form gluconic acid.
Gluconic acid and hydrogen peroxide, being relatively 1(~ low mol~clllar welght species compared to the enzyme glucose oxidase, will pass through such a membrane. Catalase and p~roxidases wllicll are large enzyme molecules capable of r~lpid:ly dcstroyillg 11202 and wh:ich are present in b.iochemical fl~ ls aro provcnted .Erom passing throu~h the membrane. The .lS ~or~going is given by way of illustration and it is to be understood that the present invention may be used with any suitable enzyme for analysis of various components in fluids.
According to the present invention, the multilayer membrane ma~ be utilized in a cell for electrochemical analysis comprising, in general, an electr-ically~ insulating recep~acle, an anode and a cathode as is shown in U.S. Patent No. 4,092,223.
The membrane of this invention may also be used in older ty~e devices such as those of Clark~ U.S. 3,53~,455, utili~ing a sensing electrode (anode), a reference electrode (cathode) in a space ln the receptacle which lS separated from the sensing electrode and adapted to hold an electrolyte. T~e membrane electrically contacts the electrodes; a path for an electrical current extends ~etween anode and cathode or between the reerence electrode and the sensing electrode and the membrane which is described herein.
.11 ~ 8 Mu1tilayer membranes of the invention are characterized by a plurality of layers or phases of polymer and a plurality of layers or phases containing enzyme. Since an intermingling or l diffusion of the layers is believed to occur, thc terms layers 5 ¦ and phases are used interchangeably to mean layers which may interact at their interfaces. Typically, the multilayer membrane will include at least one relatively high density polymer layer, ¦ a plurality of relatively lower density polymer layers and I cllzyme containing layers. I`he several polymer layers thus lO ¦ described may be separated by a layer of enzyme.
It is a characteristic feature of the present invention that the multilayer membrane include as one of the two outer mo~t Laycrs, a layer of relatively dense polymer. T}lis layer l ox outer stratum is formecl by cLisso1vlng a sultable polylllcr in a 15 ¦ solvent therefor and then casting the resulting solution on an apl~ropriate surface~ Illustratively, cellulose acetate in acetone mlly l)o Illelltione(l in this regard~ By dlssol~ln~ tlle polymer ln a solvellt alld casting a film thereof, there is obtained a layer or l stratum which is relatively dense compared to the other polymer 20 ¦ layers, which are cast from a dispersion of the polymer in a solvent/nonsolvent mixture. A skin comprised of dense polymer ¦ may be formed on the exposed surace of the less dense layer which ¦ functions to block the mi`g-ration to the sensing electrode of ¦ interfering substances such as uric acid, ascorbic acid3 and 25 ¦ large nongaseous ~olecules ànd similar substances and allows the ¦ passing of solvent and low molecular weight species, for example, enzymatic conversion products such as hydrogen peroxide.
¦ Although cellulose acetate is specifically mentioned l as the polymer used or preparation of the dense polymer layer, 30 ¦ a mem~rane exhibiting these properties can be made of other I materials as well 9 such as copolymers of cellulose acetate ¦ and the like which are known in the art for membrane formation.
, It has been determined that a reasonably short 1~ measuring time requires that the overall thickness of the multi- !
layer membrane should not exceed, preferably, about 70 microns although this can vary depending on the kind of measurement S to be c~rried out. It is to be understood that the several ¦ layers in the multiple layer membrane of the invention can l vary widely in thickness. Thus, the dense layer may be ! relatively thin compared with the less dense, more porous l polymer layer. They may also be of equal thickness. Generally~
1 the more dense layer will not be thicker than the less dense, more porous layer, although under certain conditions, the porous layer may be thinner than the dense layer.
The invention will be further understood with l reference to the drawings wherein: `
Figure 1 is a vertical partial section view of an oldcr type polarographic cell which may utilize the membrane o the invention, Figure 2 is a cross-section view, considerably -l enlarged and not to scale, of a multilayer membrane of the 1 invcntion, ¦ Figure 3, Figure 3a and Fi~ure 3b are cross-section ! views of three other embodiments of a multilayer membrane of the invention; and I Figure 4 is a cross-section view of another embodiment ¦ of a multilayer membrane of the invention.
In forming the multilayer membrane according to the invention, multilayer film forming equipment as is used in l `the photographic film industry can be used. The membrane ! consists of a multiplicity of phases which are not necessarily distinct but which when cast separately and independently of I;
il I
¦ each other are c]laracterized as se~er~-l relatively porous, ¦ phases in the mu:Ltilayer, and in the final mul-tila~er membrane a re:latively nonporo-ls, denser phase whi.ch in -the multilayer membrane faces the sample; e.g.~ -tile blood specimell. For 5 1 purposes of this description, this dense layer will be refer~ed ¦ to as the outer most layer of the membrane.
In the multilayer membrane, the porosities and thickllesses o~ the various components or stra-ta may become l mQd:i.Eied as they are :fused together. In general, there is 10 ¦ a diffusion zone at the boundary of two adjacent strata. The relatively dense layer is formed o-E polymer as is the more porous, less dense :Layer. In between the individual polymer str~lt~l tllere ;is clisposed a layer con-taill:ing en~ymes. ~dcl;it:ion-l a.Lly, there may be clistr:ihuted uni:Eorlllly tllroughout tlle rel.ati.vcly .15 ¦ porous polymer phase, a particula-te d:ispetsed enzyme or a protcctlve:ly ellcaps-llated ellzyllle. This etlzyme nlay, however, be ~I;isl:~ril)~ltQcl t;lll~o~lgllout the~ mu:ltilclye~ mc!ml)l~c~ . r[~lle :inclividlla.l.
rol~e.r~.ies of the phases or :layers formi.ng the m~lltilayer l membrane, lf cast separately should be as follows: the ¦ relatively nonporous dense polymer phase should if cast by itself and tested have a molecular weight cut off at a ¦ specific poi`nt; e.g., approximately 300; the highly porous ¦ pol~ner ~hase if cast ~y~ i`tself and tested should freely pass ¦ t~e substrate for th.e enzyme (at the surface adjacent to the 25 ¦ surface onto which ~t ~as been cast~ and yet exclude l macromolecules such as large proteins.
.¦ In order to achieve desired properties for detection of anal~te) the membrane of the invention is fabricated in a l multi~stage process. For example, the relatively dense membrane, 30 ¦ or first component, is formed by casting or spreading a solution 8~ ~ ~7 of an appropriate polymer, such as cellulose acetate in a solvent therefor; e.g., acetone, on a suitable surface which does no-t interact with or bond to the membrane. Representative surfaces to provide a support for the cast film are glass and some plastics such as polyethylene. The film is cast with conventional film forming equipment, well known in the art, whereby it is possible to control the thickness of the resulting film. After being spread on the surface, the cast film may be dried. This thin film serves as the relatively nonporous phase and may also be thin, relative to the more porous phasè. The thickness of -this phase generally ranges :Erom 2 to 5 microns, although the dimension of the film is not critical for purposes of the inventi.on.
A plurality of relatively more porous strata are prepared by casting a film by dissolving a suitable polymer in ~ sol~en~ m:lx~ure therefore. The solvent mixture used to form the more porous layer includes a solvent for the polymer and a nonsolvent for the polymer. The solvent mixture used is such as to be capable of phase inversion to form pores.
A phase inversion type of polymer layer can be forn;ed in this way and may be then cast directly on top of the relatively more dense layer. Since both casting solutions may be of the same polymer base, and preferably use the same solvent, there may be formed a diffusi.on zone of the two at the interface or boundary and no clear distinction can be made between the two phases. Indeed, the order oE
I casting may also be reversed, although it is preferred to cast the more dense film first. The first film need not be l 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 l skin forms on the top of the film after drying.
1~1L1~9007 I The solution of the polymer Such as cellulose acetate ¦¦ for the formation of the first component, or more dense layer is formed by dissolving a cellulose acetate polymer in ¦ an inert organic solvent such as a ketone. Typical ketone 1 solvents are acetone, cyclohexanone, methylethylketone and the like. Mixtures of miscible solvents may also be used.
Concentrations of the polymer in solvent may vary, as from 1 to 5%l preerably 2 to 3%. The film is cast with any suitable l ilm applicator suçh as will produce a final product film thickness of 1-10 microns, preferably 2-5 microns in l thickness.
! The phase invers;.on member or second type of layer present; tha~ is the relatively porous port:ion of the m~lltilayer membrane of this inventi.on, is prepared by ~orming ~5 a cellulose acetate, or other suitable polymer solution in an :L~ o~ganic solvent such as a ketone, A nonsolvent or nonsolvent mixture for the polymer is then mixed with the polymer' l solvent solution~ The particular polymer such as cellulose ¦ acetone and the particular nonsolvent; e.g.,ethanol and water, 20 1 is not critical a~d others may be used. ~en using cellulose acetate as the polymer, lower alcohols mixed wlth water are l usually preferred for this purpose.
¦ According to the invention, a plurality of less ~ dense polymer layers are provided in the multi].ayer membrane 25 1 each of which layers is separated from the next adjacent layer by an intermediate layer of enzyme. The enzyme layer may be formed by ~queous solutions or suspensions of enzyme which may contain additives to facilitate spreading~ Generally, the l outer most or top layer is the relatively dense` layer which 30 1 has coated on one side or surface thereof a layer of less 118911i0~
dense or porous polymer layer. m en, the next layer may be an enzyme layer followed by another layer of porous polymer followed by another layer of enzyme, etc.
~s described herein, the layer technique aEfords the opportunity to obtain the advantages of a distributed enzyme preparation through the utilization of more enzyme in the overall membrane structure and thereby obtain a higher substrate ¦ conversion rates. This may be accomplished without suffering l from the previously acknowledged drawbacks of prior art I layering of the enzyme. These drawbacks include any inhomogeneity;
i.e., gaps or spaces in some areas an.d aggregates of enzyme in ot;hers, and interference with the transport of analyte species ¦ by blockage of di:Efusion in areas of aggregated enzy~e. With l the repitition of enzyme layers in the multilayer membrane of lS ¦ khe invention, if any inhomogeneous distribution of enzyme ppear~ on the microscopic level, in any one layer of enzyme, tll:L will on ~he average equalize or distribute itself in a homogeneous way at the macroscopic level. As a result of the I multiple layers of enzyme, there will be achieved an essentially equal amount of enzyme per unit area of membrane which considered from the plane parallel to the major axis of the membrane surface.
Any interference with analyte dlffusion in regions of enzyme concentration in any one layer of enzyme can be minimized by using a multiplicity of thinner layers of enzyme.
At the same time, larger amounts of enzyme may be incorporated in the total membrane, than is the case if one thicker, discrete enzyme layer were utilized.
As will be seen from the drawings, the number and I thicknesses of the individual layers may be varied for desired 30 ¦ transport and system response characteristics~ e.g., see Figure 3. Moreover, it should be apparent that a variety il oE enz~mes ma~ be used in one multilayer membrane.
~ lso, various film thickness and combinations of phases of dense and less dense membranes may be utilized to obtain a range of film porosity properties not easily obtainable by using one or two layers alone, see Figure 4.
In certain situations, enzymes may be adversely affected by the solvents with which they come into contact.
Hence, particularly in those instances where enzyme sensitivity is a problem, the present invention offers an advantage.
Greater protection may be afforded to the enzyme from damage by the solvent (e.g., acetone) for the membrane forming polymer (e.g., cellulose acetate) than in the case where the enzyme may be in contact with the solvent. This is because adj~cent layers may be formed in a cascading sequence such that prior layers are completely or partially soldified before subsequen~ layers are applied.
To further ensure protection of solvent sensitive cn~ymes, the enzyme may be microencap~3ulated according to the teachin~s of copending Canadian patent application No.~13,673 ~lled October 18, 1982 and entitled "Enzyme Electrode Membrane Wherein Enzyme is Protectively Encapsulated", especially if additional protection is desired from solvents in adjacent layers.
Typical electrochemical sensors which can by employed with the membrane of this invention include the BIOSTATOR
glucose electrode of Miles Laboratories, Inc. See ~.S.
Patent No. 4,092,233.
The overall thickness of the membrane of the invention can vary from about 40 to about 100 microns, but is preferably approximately 70 microns. The more dense layer ranges from 2 to about 5 microns and the less dense individual layer range ~rom about 2 to 20 microns. Variation in these values i.s permissible within the contemplation of this ir~vention as the l precise thickness of the layers can range widely as will be ¦ seen by reference -to the drawings. The preferred membrane is ¦ about 70 microns in thickness.
R~ferring to Figure 1, there is shown 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 i.9 covered by an electrically insulating ci~p 11. Positioned within the receptacle i.s an electrically l insulating member rod, or cylindrical column 12, which contains in it an electrical conductor 1~. Th:is conductor is connected to nn activ~ or exposed elemen~ 14 which may be platinum, p~olcl, silv~r, graphite or the like. I
A lead is attached to the eLectrode which passes through the rod or column and through the cap to be connected with a D. C. voltage source 15.
The lower end of the rèceptacle 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 position with any suitable means, for example, by an 0-ring fitting into a circular groove or other convenient means in the receptacle. A current measuring instrument (not shown) 30 1 is connected in series with the cell.
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1.
_ I . .
~ ~ ob~
Typically, the receptacle is provided with a vent 18 to permit g~ses to escape if pressure inside the receptacle rises to a sufficiently high degree.
An annular space is provided be~ween the central rod and the receptacle walls and receives a reference electrode 1~ which may be for example, silver chloride coated silver wire. The space 20 inbetween is at leas~ partially and preferably compleLely filled with a liquid mixture of ¦ electrolyte which may be introduced into the chamber through I an aperture.
¦ In polarographic measurements, two electrodes a.re commonly used, one o:E which is polarized and does not allow current to flow until depolarized by the substance l being measured. In the cell stîucture shown in Figure 1, elec~rocle 1~ is the cathode and is polariæed and frequently ~.er~e~l to as the refercncc 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 oE relatively 2Q 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 path between the two electrodes. Typical electrolytes include sodium ~5 or po~assium chloride, buffers including carbonates, phosphates, bicarbonates, acetates, alkali or rare earth salts or other organic buffers or mixtures thereof may be used. The solvent for such an electrolyte may be water, glycols, glycerine and mixtures thereof as is well known in the art.
!1 17 -I
I
I
I:igure 2 shows a multilayeI membrane in cross-sectional detail. A dense layer 21 and a plura]ity of less dense or porous layer 22 comprise the strata which less dense layers l are separated by a layer of enzyme 23. The enzyme layer 23 is 5 ¦ shown in Figure 2 as being of the same thickness as the dense l layer 21 and the porous layer 2~. However9 as shown in Figu-re 3, ¦ these layers may be different. They may also be different che1nically in addition to being different in physical dimensions.
l Sc\mc oE the enzyme may di~fuse into the adjacent layers during 10 ¦ preparation of the membrane if the solvent for the cellulose acetate has not yet fully evaporated. Membrane surface 24 is i~ electrical contact Wit}l the electrode. The outer free surface o`~ laY~1 21 repres~nts the test surface which is to be brought l :;lltO contact with the so:lution to be analyzed.
IS ¦ As shown in Figure 4, the multilayer membrane may in~:lu~I~ severa:l layers of the dense strata as well as several .L~y~rs o~ ~IIzyme 23 and porous layer 22.
'l'I~e mem~rane o the invent.ion may be produced by l initially castlng the relatively dense polymer layer, stratum 20 ¦ or film onto a suitable surface and permitting it to at least partially dry. This first layer is referred to as the first compollent of the multilayer membrane. If this dense layer is omitted, the measurements may be more subject to nonlinearity I due to oxygen depletion which is, in turn, caused by an increased 25 ¦ ` flux of glucose molecules transported through the membrane and reacting with enzyme.
The porous phase inversion type polymer layer which lS referTed to as the second component may be cast directly l on top of the thin layer. It may be possible to first cast 3Q ¦ the porous portion of the membrane and then cast the dense portion directly on top of It. The phase inversion member or more porous portion of the membrane composite may be formed by providing a solution of the polymer in an inert organic ~ 7 ."
solvent such as acetone. Ths ~olution is then mixed with anonsolvent for the polymer. Suitable nonsolvents include I¦ alcohol and ~ater mixtures. In the case of cellulose acetate, ¦l ethanol and water is a suitable nonsolvent.
¦ The third component of the multilayer membrane ¦l comprlses the enzyme containing layer. Typically, this is onned o an aqueous solution or suspension of the enzyme, which may further contain additives to facilitate spreading.
As a fourth component another phase inversion porous 1 polymer layer may be utilized.
A Eifth layer of enzyme containing substance is then al~o utilized. The multilayer membrane of the invention thus contains at least one irst component; i.e. a relatively l dense polymer layer and at least two phase inversion type ¦ polymer layers; that is, a porous layer o the second type.
¦¦ In ~ddi.tion, the membrane contains at. least two layers o enzyme material. Preferably, the memb~ane o:E the invention contains at least 3 or 4 enzyme layers and 3 or 4 porous polymer layers.
In preparing the membranes of the invention, the 2~ sequence of forming layers may be carried out according to the process steps stated above and thereby produce a membrane as shown, or example, in Figures 2 or ~. Or, the sequence may be varied to produce a structure such as shown in Figure 4.
i ¦ Typically~ the mixing of enzyme should take place at 25 ! low temperatures; i.e., 0C or below 0C. The time of mixing is also to be minimized in order to avoid inactivating the enzyme.
The following specific example illustrates how the l invention may be carried out but should not be considered as limiting thereof in any way.
l - 19- ' i 9~g~7 ¦ EXAMPLE
~n a clean glass plate, spread a 3% ceilulose acetate ! in acetone solution with 2 mil film applicator to prepare 1~ the dense film portion.
Prepare the phase inversion cellulose acetate casting solution by mixing 1.5 cc of e~hanol with 5 cc of a 10%
¦ cellulose acetate in acetone solution. This is then placed in a salt water ice bath and stirring of the solution is continued.
l The second solution which will form the porous layer 10 1 is then spread on top of the first membrane withan 18 mil applicator. The spread film is then permitted to dry Eor ~everal hours at room temperature.
The enzyme layer can be cast on top of the porous layer at any convenient time such as when the surface oE tlle E~lm is tacky to the touch, or partially dry.
The enzyme preparation may simply be a mixture o:E
the appropriate enzyme such as glucose oxidase in water. For example, 1.0 cc of an aqueous glucose oxidase solution may I be used. This`solution contains 2,000 to 3,000 units of the 20 ~ glucose oxidase per cc of solution. Of course, other materials ¦ such as a binder or cross-linking agent like glutaraldehyde may be included in the enzyme preparation. Likewise, the proportion of enzyme to water in the preparation is immaterial as long l as a rlowable paste or solution is formed which may be easily 25 1 coated or deposited. Sufficient enzyme is incorporated into l the solutlon to prepare an adequate reactive amount for ¦ measurement.
The forPgoing procedure of casting the polymer films l and enzyme layers is then repeated until the multilayer ¦ mèmbrane including at least 1 dense layer, and at least two porous and enzyme layers areobtained. Preferably, the product l l contains at least 3 or 4 layers of enzyme and at least 3 or 4 layers of porous film.
The multilayer membrane of the yresent invention is a sel:E-supporting film of a total thickness which may range from about 50 to 100 microns, preferably about 70 microns. Tlle multilayer membrane may ~e shaped to any particular configuration or size or may be cut or dimensioned in any par~icular way to .~it receptacles for polarograp}lic cells or elec-trodes of any sllitable dimension. It may, in par-ticularJ be fastened to an O-ring for use in an electrode such as described in U.S. ~atent No. 4,092,233.
To fasten the membrane to a rubbery O-ring o:E an ~ ropr:i~tc s:ize, a gluing operation may be employed. The mcmbralle may also be cast clirectly OlltO an electrode surace.
.1~ In addition to cellulose acetate, other polynlers capable I
o: bein~ clissolved in solvents and undergoing phase inversion w.ith l tllo (Icklit.ioll o:~ a weak solvcnt or nonso:lvent would be~ potential momb.rano materia:ls. Such polymers include cellulose nitrate, ethylcellulose and other cellulose derivatives. In addition, polycarbonate is a suitable alternative if methylene chloride is employed as a solvent instead of acetone or other ketones.
As a substitute or alternative ~or the lower alcohols present in th'e phase inversion mixture formamide can be used.
Furth.er variations and modifications of the invention as ~Yi~ e apparent to those skilled in the art after reading t~.e foregoing are intended to be encompassed by the claims that are ap~ended hereto.
~ .
This type of enzyme membrane is described in the Annals of t.he l New York Academy of Science, Vol. 102 (1962), pages 29-49. In ¦ that article, it was suggested ~hat a pH sensitive èlectrode could be used to detect gluconic acid produced by the reaction.
It was disclosed that the enz~ne in such a system could be trapped between two cellulose acetate membranes. Glucose difEuses 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 firs~ 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.
:l5 For example, this membrane is permeable to the reactants of the. ell~ymatic reacti.on but impermeable to the enzyme it~elf.
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 20 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, tetrafluoroe~hylene and the like.
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 weigh~
~ub~ances such as catalase and peroxidase do not, It is _ 3 _ I
l _ l . . ~ ~, . _., ~:~896 l ll disclosed that the enzymes may be held in a thin film directly between the platinum surface and the membrane by placing the enzyme Gn a porous film which has spaces large enough to hold l 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.
¦ Clark suggested a dual electrode system to overcome that problem.
¦ The compensating electrode, without an enzyme 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 di.f~iculties in the matching of the two cells.
It was ~hen proposed to have an enzyme elec~rode ~S which employs a thin filter membrane to prevent passage of Low molecular weight interfering materials, such as uric acid ancl ascorbic acid, while permitting hydrogen peroxide to pass therethrough with minimum hindrance. There exist membrane materials, such as silicone rubber and cellulose acetate, ~0 which permit passàge of hydrogen peroxide but which are effective barriers to inte~fering substances. 5ince this type of membrane must be placed between sensing electrode and 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 stlll 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.
30The art then turned to depositing the material in a thin layer on a porous substructure to provide the necessary _ 4 _ l l 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.
l In Newman, U.S. Patent No. 3,979,274" a laminated 5 ¦ two-ply membrane is described wherein an enzyme adhesive is used to bond the two-lies together. The membrane includes a support 1ayer which controls substrate diffusion and serves ~s a barrier to high molecular weight substances, an enzyme l preparation for reacting with the unknown and for bonding l0 ¦ the layers together, and an essentially homogeneous layer that serves as a barrier to interfering low molecular weight materials but perm:its hydrogen peroxide to pass through.
I{o~ever in this development, it is necessary to make a I sandwich consisting of two membranes with a layer of enzyme 15 ¦ between, the enzyme acting as the adhesive or binding agent.
l In this type o:E arrangement, the use o:E t:oo much enzyme could ¦ slow clo~n the ~ fusion o the diffusing species to an unacceptable amount. If a thinner layer of enzyme is used, l there is acceptable dif~usion, but the loading of enzyme may 20 ¦ not be adequate.
A still later de~elopment came in British Patent No. l,442,303 ~`Radiometer) wherein there was proposed a ¦ composite membrane which is an inhomogeneous membrane formed I as a unit~ The membrane has two different strata, one has 25 ¦ a thickness of less than -5 microns and the other is sufficiently I thick.to provide strength. The enzyme is bonded to a surface ¦ of th.e mem~rane~
¦ Other pr~or art has shown a number of disadvantages.
:
¦ Thus, the method of Koyama et al, Analytica Chemica l Acta, Vol. 116, pages 307-311 (1980), immobilizes glucose oxidase ! to a cellulose acetate membrane. This method is more time l consuming; it involves more steps and suffers from the 1 disadvantages that a monolayer of molecules would be the maximum possible enzyme load achievable.
Wingard et al, The Journal of Biomedical Materials ~e_earch, Vol. l3, pages 921-925 (1979) discloses a platinum l screen or wire for immobilization of the enzyme. This would 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. However, the approach of Wingard is also limited to a monolayer of enzyme and capable oE
sustaining high conversion rates of substrate diffusing through the open spaces in the platinum screen near the surface of the platinum wlre only.
In accordance with the present i.nventi.on, there is provided a multilayer membrane formed of a plurality of layers or strata of polymer and enzyme which function together to overcome problems that have occurred in the prior art. The multiple layer technique of the invention affords the opportunity to obtain the advantages of a distributed enzyme preparation through the utilization of more enzyme being present in the composite multiple layer structure without encountering the problems which have been observed in the use of single layer enzyme sandwich membranes of the past.
A significant advantage of the present inventi.on ¦ resides in the fact that because the enzyme appears in more l than one layer the overall distribution of the enzyme will appear to be homogeneous and thereby eliminate any inhQmogeneity which may be present in any individual enzyme layer.
In addition, any significant interference with analyte diffusion which may be caused by unusual concentrations o~ enzyme in individual layers will be balanced by the total l overall diffusion of ~he analyte through a multiplicity of enzyme containing layers where any discontinuities or high concentrations of enzyme will effectively be equalized.
The principles involved in the present invention may be illustrated with reference to the analysis of blood for glucose content. However, it should be noted that the ¦ present invention is applicable to use wi,h many different enzyme systems, polymer membrane formula~ions, and the resulting multiple layer membranes may be used for analysls o~ numerous 1uids for varl.ous components. The l.iqui.d portion of blood consists of proteins, lipids, and other substances.
Nonelectrolytes are present such as glucose, enz~nes such as catala~e, electrolytes such as ascorbic acid (vitamin C) ~Ind varlous metallic salts made up oE cations of sodium, potassium, magnesium, calcium, iron and copper, and anions such as chlorides, phosphates, bicarbonates, and carbonates.
The phosphates, carbonates and bicarbonates operate as buffers to maintain the pH of blood at a fixed level under normal conditions. If a sample of blood were placed on one side of a membrane in a cell and an aqueous solution of, for example, the . enzyme glucose oxidase and oxygen on the other side of the 25` membrane, certain low molecular weight materials will pass from the blood through the membrane to the glucose oxidase solution.
The high molecular weight materials such as the enzymes will not pass through the membrane. The rates of permeability of the various materials through the membrane are fixed because of the nature of the membrane. By selection of appropriate materials, the membranes may be designed to have a molecular cut off at any desired point; for example, at approximately 300. This means that materials of a molecular weight of greater than about 300 will not pass through.
Glucose, a low molecular weight material, will pass through SUC]I a membrane and react with the enzyme glucose oxidase in the presence of oxygen to form gluconolactone and hydrogen peroxide.
Gluconolactone in the presence of water will hydrolyze spontane-ously to form gluconic acid.
Gluconic acid and hydrogen peroxide, being relatively 1(~ low mol~clllar welght species compared to the enzyme glucose oxidase, will pass through such a membrane. Catalase and p~roxidases wllicll are large enzyme molecules capable of r~lpid:ly dcstroyillg 11202 and wh:ich are present in b.iochemical fl~ ls aro provcnted .Erom passing throu~h the membrane. The .lS ~or~going is given by way of illustration and it is to be understood that the present invention may be used with any suitable enzyme for analysis of various components in fluids.
According to the present invention, the multilayer membrane ma~ be utilized in a cell for electrochemical analysis comprising, in general, an electr-ically~ insulating recep~acle, an anode and a cathode as is shown in U.S. Patent No. 4,092,223.
The membrane of this invention may also be used in older ty~e devices such as those of Clark~ U.S. 3,53~,455, utili~ing a sensing electrode (anode), a reference electrode (cathode) in a space ln the receptacle which lS separated from the sensing electrode and adapted to hold an electrolyte. T~e membrane electrically contacts the electrodes; a path for an electrical current extends ~etween anode and cathode or between the reerence electrode and the sensing electrode and the membrane which is described herein.
.11 ~ 8 Mu1tilayer membranes of the invention are characterized by a plurality of layers or phases of polymer and a plurality of layers or phases containing enzyme. Since an intermingling or l diffusion of the layers is believed to occur, thc terms layers 5 ¦ and phases are used interchangeably to mean layers which may interact at their interfaces. Typically, the multilayer membrane will include at least one relatively high density polymer layer, ¦ a plurality of relatively lower density polymer layers and I cllzyme containing layers. I`he several polymer layers thus lO ¦ described may be separated by a layer of enzyme.
It is a characteristic feature of the present invention that the multilayer membrane include as one of the two outer mo~t Laycrs, a layer of relatively dense polymer. T}lis layer l ox outer stratum is formecl by cLisso1vlng a sultable polylllcr in a 15 ¦ solvent therefor and then casting the resulting solution on an apl~ropriate surface~ Illustratively, cellulose acetate in acetone mlly l)o Illelltione(l in this regard~ By dlssol~ln~ tlle polymer ln a solvellt alld casting a film thereof, there is obtained a layer or l stratum which is relatively dense compared to the other polymer 20 ¦ layers, which are cast from a dispersion of the polymer in a solvent/nonsolvent mixture. A skin comprised of dense polymer ¦ may be formed on the exposed surace of the less dense layer which ¦ functions to block the mi`g-ration to the sensing electrode of ¦ interfering substances such as uric acid, ascorbic acid3 and 25 ¦ large nongaseous ~olecules ànd similar substances and allows the ¦ passing of solvent and low molecular weight species, for example, enzymatic conversion products such as hydrogen peroxide.
¦ Although cellulose acetate is specifically mentioned l as the polymer used or preparation of the dense polymer layer, 30 ¦ a mem~rane exhibiting these properties can be made of other I materials as well 9 such as copolymers of cellulose acetate ¦ and the like which are known in the art for membrane formation.
, It has been determined that a reasonably short 1~ measuring time requires that the overall thickness of the multi- !
layer membrane should not exceed, preferably, about 70 microns although this can vary depending on the kind of measurement S to be c~rried out. It is to be understood that the several ¦ layers in the multiple layer membrane of the invention can l vary widely in thickness. Thus, the dense layer may be ! relatively thin compared with the less dense, more porous l polymer layer. They may also be of equal thickness. Generally~
1 the more dense layer will not be thicker than the less dense, more porous layer, although under certain conditions, the porous layer may be thinner than the dense layer.
The invention will be further understood with l reference to the drawings wherein: `
Figure 1 is a vertical partial section view of an oldcr type polarographic cell which may utilize the membrane o the invention, Figure 2 is a cross-section view, considerably -l enlarged and not to scale, of a multilayer membrane of the 1 invcntion, ¦ Figure 3, Figure 3a and Fi~ure 3b are cross-section ! views of three other embodiments of a multilayer membrane of the invention; and I Figure 4 is a cross-section view of another embodiment ¦ of a multilayer membrane of the invention.
In forming the multilayer membrane according to the invention, multilayer film forming equipment as is used in l `the photographic film industry can be used. The membrane ! consists of a multiplicity of phases which are not necessarily distinct but which when cast separately and independently of I;
il I
¦ each other are c]laracterized as se~er~-l relatively porous, ¦ phases in the mu:Ltilayer, and in the final mul-tila~er membrane a re:latively nonporo-ls, denser phase whi.ch in -the multilayer membrane faces the sample; e.g.~ -tile blood specimell. For 5 1 purposes of this description, this dense layer will be refer~ed ¦ to as the outer most layer of the membrane.
In the multilayer membrane, the porosities and thickllesses o~ the various components or stra-ta may become l mQd:i.Eied as they are :fused together. In general, there is 10 ¦ a diffusion zone at the boundary of two adjacent strata. The relatively dense layer is formed o-E polymer as is the more porous, less dense :Layer. In between the individual polymer str~lt~l tllere ;is clisposed a layer con-taill:ing en~ymes. ~dcl;it:ion-l a.Lly, there may be clistr:ihuted uni:Eorlllly tllroughout tlle rel.ati.vcly .15 ¦ porous polymer phase, a particula-te d:ispetsed enzyme or a protcctlve:ly ellcaps-llated ellzyllle. This etlzyme nlay, however, be ~I;isl:~ril)~ltQcl t;lll~o~lgllout the~ mu:ltilclye~ mc!ml)l~c~ . r[~lle :inclividlla.l.
rol~e.r~.ies of the phases or :layers formi.ng the m~lltilayer l membrane, lf cast separately should be as follows: the ¦ relatively nonporous dense polymer phase should if cast by itself and tested have a molecular weight cut off at a ¦ specific poi`nt; e.g., approximately 300; the highly porous ¦ pol~ner ~hase if cast ~y~ i`tself and tested should freely pass ¦ t~e substrate for th.e enzyme (at the surface adjacent to the 25 ¦ surface onto which ~t ~as been cast~ and yet exclude l macromolecules such as large proteins.
.¦ In order to achieve desired properties for detection of anal~te) the membrane of the invention is fabricated in a l multi~stage process. For example, the relatively dense membrane, 30 ¦ or first component, is formed by casting or spreading a solution 8~ ~ ~7 of an appropriate polymer, such as cellulose acetate in a solvent therefor; e.g., acetone, on a suitable surface which does no-t interact with or bond to the membrane. Representative surfaces to provide a support for the cast film are glass and some plastics such as polyethylene. The film is cast with conventional film forming equipment, well known in the art, whereby it is possible to control the thickness of the resulting film. After being spread on the surface, the cast film may be dried. This thin film serves as the relatively nonporous phase and may also be thin, relative to the more porous phasè. The thickness of -this phase generally ranges :Erom 2 to 5 microns, although the dimension of the film is not critical for purposes of the inventi.on.
A plurality of relatively more porous strata are prepared by casting a film by dissolving a suitable polymer in ~ sol~en~ m:lx~ure therefore. The solvent mixture used to form the more porous layer includes a solvent for the polymer and a nonsolvent for the polymer. The solvent mixture used is such as to be capable of phase inversion to form pores.
A phase inversion type of polymer layer can be forn;ed in this way and may be then cast directly on top of the relatively more dense layer. Since both casting solutions may be of the same polymer base, and preferably use the same solvent, there may be formed a diffusi.on zone of the two at the interface or boundary and no clear distinction can be made between the two phases. Indeed, the order oE
I casting may also be reversed, although it is preferred to cast the more dense film first. The first film need not be l 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 l skin forms on the top of the film after drying.
1~1L1~9007 I The solution of the polymer Such as cellulose acetate ¦¦ for the formation of the first component, or more dense layer is formed by dissolving a cellulose acetate polymer in ¦ an inert organic solvent such as a ketone. Typical ketone 1 solvents are acetone, cyclohexanone, methylethylketone and the like. Mixtures of miscible solvents may also be used.
Concentrations of the polymer in solvent may vary, as from 1 to 5%l preerably 2 to 3%. The film is cast with any suitable l ilm applicator suçh as will produce a final product film thickness of 1-10 microns, preferably 2-5 microns in l thickness.
! The phase invers;.on member or second type of layer present; tha~ is the relatively porous port:ion of the m~lltilayer membrane of this inventi.on, is prepared by ~orming ~5 a cellulose acetate, or other suitable polymer solution in an :L~ o~ganic solvent such as a ketone, A nonsolvent or nonsolvent mixture for the polymer is then mixed with the polymer' l solvent solution~ The particular polymer such as cellulose ¦ acetone and the particular nonsolvent; e.g.,ethanol and water, 20 1 is not critical a~d others may be used. ~en using cellulose acetate as the polymer, lower alcohols mixed wlth water are l usually preferred for this purpose.
¦ According to the invention, a plurality of less ~ dense polymer layers are provided in the multi].ayer membrane 25 1 each of which layers is separated from the next adjacent layer by an intermediate layer of enzyme. The enzyme layer may be formed by ~queous solutions or suspensions of enzyme which may contain additives to facilitate spreading~ Generally, the l outer most or top layer is the relatively dense` layer which 30 1 has coated on one side or surface thereof a layer of less 118911i0~
dense or porous polymer layer. m en, the next layer may be an enzyme layer followed by another layer of porous polymer followed by another layer of enzyme, etc.
~s described herein, the layer technique aEfords the opportunity to obtain the advantages of a distributed enzyme preparation through the utilization of more enzyme in the overall membrane structure and thereby obtain a higher substrate ¦ conversion rates. This may be accomplished without suffering l from the previously acknowledged drawbacks of prior art I layering of the enzyme. These drawbacks include any inhomogeneity;
i.e., gaps or spaces in some areas an.d aggregates of enzyme in ot;hers, and interference with the transport of analyte species ¦ by blockage of di:Efusion in areas of aggregated enzy~e. With l the repitition of enzyme layers in the multilayer membrane of lS ¦ khe invention, if any inhomogeneous distribution of enzyme ppear~ on the microscopic level, in any one layer of enzyme, tll:L will on ~he average equalize or distribute itself in a homogeneous way at the macroscopic level. As a result of the I multiple layers of enzyme, there will be achieved an essentially equal amount of enzyme per unit area of membrane which considered from the plane parallel to the major axis of the membrane surface.
Any interference with analyte dlffusion in regions of enzyme concentration in any one layer of enzyme can be minimized by using a multiplicity of thinner layers of enzyme.
At the same time, larger amounts of enzyme may be incorporated in the total membrane, than is the case if one thicker, discrete enzyme layer were utilized.
As will be seen from the drawings, the number and I thicknesses of the individual layers may be varied for desired 30 ¦ transport and system response characteristics~ e.g., see Figure 3. Moreover, it should be apparent that a variety il oE enz~mes ma~ be used in one multilayer membrane.
~ lso, various film thickness and combinations of phases of dense and less dense membranes may be utilized to obtain a range of film porosity properties not easily obtainable by using one or two layers alone, see Figure 4.
In certain situations, enzymes may be adversely affected by the solvents with which they come into contact.
Hence, particularly in those instances where enzyme sensitivity is a problem, the present invention offers an advantage.
Greater protection may be afforded to the enzyme from damage by the solvent (e.g., acetone) for the membrane forming polymer (e.g., cellulose acetate) than in the case where the enzyme may be in contact with the solvent. This is because adj~cent layers may be formed in a cascading sequence such that prior layers are completely or partially soldified before subsequen~ layers are applied.
To further ensure protection of solvent sensitive cn~ymes, the enzyme may be microencap~3ulated according to the teachin~s of copending Canadian patent application No.~13,673 ~lled October 18, 1982 and entitled "Enzyme Electrode Membrane Wherein Enzyme is Protectively Encapsulated", especially if additional protection is desired from solvents in adjacent layers.
Typical electrochemical sensors which can by employed with the membrane of this invention include the BIOSTATOR
glucose electrode of Miles Laboratories, Inc. See ~.S.
Patent No. 4,092,233.
The overall thickness of the membrane of the invention can vary from about 40 to about 100 microns, but is preferably approximately 70 microns. The more dense layer ranges from 2 to about 5 microns and the less dense individual layer range ~rom about 2 to 20 microns. Variation in these values i.s permissible within the contemplation of this ir~vention as the l precise thickness of the layers can range widely as will be ¦ seen by reference -to the drawings. The preferred membrane is ¦ about 70 microns in thickness.
R~ferring to Figure 1, there is shown 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 i.9 covered by an electrically insulating ci~p 11. Positioned within the receptacle i.s an electrically l insulating member rod, or cylindrical column 12, which contains in it an electrical conductor 1~. Th:is conductor is connected to nn activ~ or exposed elemen~ 14 which may be platinum, p~olcl, silv~r, graphite or the like. I
A lead is attached to the eLectrode which passes through the rod or column and through the cap to be connected with a D. C. voltage source 15.
The lower end of the rèceptacle 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 position with any suitable means, for example, by an 0-ring fitting into a circular groove or other convenient means in the receptacle. A current measuring instrument (not shown) 30 1 is connected in series with the cell.
!
1.
_ I . .
~ ~ ob~
Typically, the receptacle is provided with a vent 18 to permit g~ses to escape if pressure inside the receptacle rises to a sufficiently high degree.
An annular space is provided be~ween the central rod and the receptacle walls and receives a reference electrode 1~ which may be for example, silver chloride coated silver wire. The space 20 inbetween is at leas~ partially and preferably compleLely filled with a liquid mixture of ¦ electrolyte which may be introduced into the chamber through I an aperture.
¦ In polarographic measurements, two electrodes a.re commonly used, one o:E which is polarized and does not allow current to flow until depolarized by the substance l being measured. In the cell stîucture shown in Figure 1, elec~rocle 1~ is the cathode and is polariæed and frequently ~.er~e~l to as the refercncc 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 oE relatively 2Q 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 path between the two electrodes. Typical electrolytes include sodium ~5 or po~assium chloride, buffers including carbonates, phosphates, bicarbonates, acetates, alkali or rare earth salts or other organic buffers or mixtures thereof may be used. The solvent for such an electrolyte may be water, glycols, glycerine and mixtures thereof as is well known in the art.
!1 17 -I
I
I
I:igure 2 shows a multilayeI membrane in cross-sectional detail. A dense layer 21 and a plura]ity of less dense or porous layer 22 comprise the strata which less dense layers l are separated by a layer of enzyme 23. The enzyme layer 23 is 5 ¦ shown in Figure 2 as being of the same thickness as the dense l layer 21 and the porous layer 2~. However9 as shown in Figu-re 3, ¦ these layers may be different. They may also be different che1nically in addition to being different in physical dimensions.
l Sc\mc oE the enzyme may di~fuse into the adjacent layers during 10 ¦ preparation of the membrane if the solvent for the cellulose acetate has not yet fully evaporated. Membrane surface 24 is i~ electrical contact Wit}l the electrode. The outer free surface o`~ laY~1 21 repres~nts the test surface which is to be brought l :;lltO contact with the so:lution to be analyzed.
IS ¦ As shown in Figure 4, the multilayer membrane may in~:lu~I~ severa:l layers of the dense strata as well as several .L~y~rs o~ ~IIzyme 23 and porous layer 22.
'l'I~e mem~rane o the invent.ion may be produced by l initially castlng the relatively dense polymer layer, stratum 20 ¦ or film onto a suitable surface and permitting it to at least partially dry. This first layer is referred to as the first compollent of the multilayer membrane. If this dense layer is omitted, the measurements may be more subject to nonlinearity I due to oxygen depletion which is, in turn, caused by an increased 25 ¦ ` flux of glucose molecules transported through the membrane and reacting with enzyme.
The porous phase inversion type polymer layer which lS referTed to as the second component may be cast directly l on top of the thin layer. It may be possible to first cast 3Q ¦ the porous portion of the membrane and then cast the dense portion directly on top of It. The phase inversion member or more porous portion of the membrane composite may be formed by providing a solution of the polymer in an inert organic ~ 7 ."
solvent such as acetone. Ths ~olution is then mixed with anonsolvent for the polymer. Suitable nonsolvents include I¦ alcohol and ~ater mixtures. In the case of cellulose acetate, ¦l ethanol and water is a suitable nonsolvent.
¦ The third component of the multilayer membrane ¦l comprlses the enzyme containing layer. Typically, this is onned o an aqueous solution or suspension of the enzyme, which may further contain additives to facilitate spreading.
As a fourth component another phase inversion porous 1 polymer layer may be utilized.
A Eifth layer of enzyme containing substance is then al~o utilized. The multilayer membrane of the invention thus contains at least one irst component; i.e. a relatively l dense polymer layer and at least two phase inversion type ¦ polymer layers; that is, a porous layer o the second type.
¦¦ In ~ddi.tion, the membrane contains at. least two layers o enzyme material. Preferably, the memb~ane o:E the invention contains at least 3 or 4 enzyme layers and 3 or 4 porous polymer layers.
In preparing the membranes of the invention, the 2~ sequence of forming layers may be carried out according to the process steps stated above and thereby produce a membrane as shown, or example, in Figures 2 or ~. Or, the sequence may be varied to produce a structure such as shown in Figure 4.
i ¦ Typically~ the mixing of enzyme should take place at 25 ! low temperatures; i.e., 0C or below 0C. The time of mixing is also to be minimized in order to avoid inactivating the enzyme.
The following specific example illustrates how the l invention may be carried out but should not be considered as limiting thereof in any way.
l - 19- ' i 9~g~7 ¦ EXAMPLE
~n a clean glass plate, spread a 3% ceilulose acetate ! in acetone solution with 2 mil film applicator to prepare 1~ the dense film portion.
Prepare the phase inversion cellulose acetate casting solution by mixing 1.5 cc of e~hanol with 5 cc of a 10%
¦ cellulose acetate in acetone solution. This is then placed in a salt water ice bath and stirring of the solution is continued.
l The second solution which will form the porous layer 10 1 is then spread on top of the first membrane withan 18 mil applicator. The spread film is then permitted to dry Eor ~everal hours at room temperature.
The enzyme layer can be cast on top of the porous layer at any convenient time such as when the surface oE tlle E~lm is tacky to the touch, or partially dry.
The enzyme preparation may simply be a mixture o:E
the appropriate enzyme such as glucose oxidase in water. For example, 1.0 cc of an aqueous glucose oxidase solution may I be used. This`solution contains 2,000 to 3,000 units of the 20 ~ glucose oxidase per cc of solution. Of course, other materials ¦ such as a binder or cross-linking agent like glutaraldehyde may be included in the enzyme preparation. Likewise, the proportion of enzyme to water in the preparation is immaterial as long l as a rlowable paste or solution is formed which may be easily 25 1 coated or deposited. Sufficient enzyme is incorporated into l the solutlon to prepare an adequate reactive amount for ¦ measurement.
The forPgoing procedure of casting the polymer films l and enzyme layers is then repeated until the multilayer ¦ mèmbrane including at least 1 dense layer, and at least two porous and enzyme layers areobtained. Preferably, the product l l contains at least 3 or 4 layers of enzyme and at least 3 or 4 layers of porous film.
The multilayer membrane of the yresent invention is a sel:E-supporting film of a total thickness which may range from about 50 to 100 microns, preferably about 70 microns. Tlle multilayer membrane may ~e shaped to any particular configuration or size or may be cut or dimensioned in any par~icular way to .~it receptacles for polarograp}lic cells or elec-trodes of any sllitable dimension. It may, in par-ticularJ be fastened to an O-ring for use in an electrode such as described in U.S. ~atent No. 4,092,233.
To fasten the membrane to a rubbery O-ring o:E an ~ ropr:i~tc s:ize, a gluing operation may be employed. The mcmbralle may also be cast clirectly OlltO an electrode surace.
.1~ In addition to cellulose acetate, other polynlers capable I
o: bein~ clissolved in solvents and undergoing phase inversion w.ith l tllo (Icklit.ioll o:~ a weak solvcnt or nonso:lvent would be~ potential momb.rano materia:ls. Such polymers include cellulose nitrate, ethylcellulose and other cellulose derivatives. In addition, polycarbonate is a suitable alternative if methylene chloride is employed as a solvent instead of acetone or other ketones.
As a substitute or alternative ~or the lower alcohols present in th'e phase inversion mixture formamide can be used.
Furth.er variations and modifications of the invention as ~Yi~ e apparent to those skilled in the art after reading t~.e foregoing are intended to be encompassed by the claims that are ap~ended hereto.
~ .
Claims (15)
1. A method of making a contiguous multilayer membrane of about 40 to about 100 microns in overall thickness suit-able for use with an electochemical sensor in the measure-ment of unknown which comprises:
Providing as a first layer, a polymer dissolved in an inert organic solvent and casting said polymer in solution into 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 film and thereby obtaining a first relatively nonporous dense poly-mer layer of about 2 to about 5 microns in thickness;
providing as a second layer, a composition comprising a polymer dissolved in an inert organic solvent, mixing said polymer dissolved in solvent with a nonsolvent for said polymer to obtain a dispersion and thereafter casting said dispersion onto said first layer and thereafter permitting said second layer to dry to form a porous polymer layer of about 2 to about 20 microns in thickness and less dense and more porous than the first layer;
providing a third layer comprising an enzyme solu-tion or suspension and depositing said third layer onto the exposed surface of the second layer;
providing as a fourth layer, a composition comprising a polymer dissolved in an inert organic solvent, mixing said polymer dissolved in solvent with a nonsolvent for said polymer to obtain a dispersion and thereafter casing said dispersion onto the ex-posed surface of the third layer to provide a fourth porous polymer layer of about 2 to about 20 microns in thickness and less dense and more porous than the first layer; and thereby forming said contiguous multilayer membrane.
Providing as a first layer, a polymer dissolved in an inert organic solvent and casting said polymer in solution into 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 film and thereby obtaining a first relatively nonporous dense poly-mer layer of about 2 to about 5 microns in thickness;
providing as a second layer, a composition comprising a polymer dissolved in an inert organic solvent, mixing said polymer dissolved in solvent with a nonsolvent for said polymer to obtain a dispersion and thereafter casting said dispersion onto said first layer and thereafter permitting said second layer to dry to form a porous polymer layer of about 2 to about 20 microns in thickness and less dense and more porous than the first layer;
providing a third layer comprising an enzyme solu-tion or suspension and depositing said third layer onto the exposed surface of the second layer;
providing as a fourth layer, a composition comprising a polymer dissolved in an inert organic solvent, mixing said polymer dissolved in solvent with a nonsolvent for said polymer to obtain a dispersion and thereafter casing said dispersion onto the ex-posed surface of the third layer to provide a fourth porous polymer layer of about 2 to about 20 microns in thickness and less dense and more porous than the first layer; and thereby forming said contiguous multilayer membrane.
2. The method of claim 1 wherein each of said layers is deposited one or more times in sequence to obtain a multilayer membrane.
3. The method of claim 1 wherein the polymer is cellulose acetate or a copolymer of cellulose acetate.
4. The method of claim 1 wherein the inert organic solvent is a ketone.
5. The method of claim 1 wherein said second layer further includes an enzyme homogenously dispersed therein.
6. The method of claim 1 wherein said second layer further contains an enzyme protectively encapsulated therein.
7. The method of claim 1 wherein said second layer is a phase inversion layer containing pores formed by dissolving said polymer in a solvent mixture, including solvent and a nonsolvent for said polymer.
8. The method of claim 1 wherein the enzyme is glucose or oxidase.
9. A method of making a contiguous multilayer mem-brane of about 40 to about 100 microns in overall thickness suitable for use with an electrochemical sensor in the measurement of an unknown which comprises:
a) providing a first cellulose acetate polymer dis-solved in an inert organic solvent;
b) casting said first polymer in solution onto an inert support surface which is unreative with said cel-lulose acetate and does not form a bond to said cellulose acetate;
c) permitting said first polymer in solution to form a film and thereby obtain a first relatively nonporous dense layer of cellulose acetate of about 2 to about 5 microns in thickness;
d) providing a second cellulose acetate polymer dissolved in an inert organic solvent mixing said second polymer with a nonsolvent for said polymer to form a dispersion thereof;
e) casting said second cellulose acetate polymer dis-persion onto said first layer of cellulose acetate and permitting said cellulose acetate polymer to form a porous second cellulose acetate layer of about 2 to about 20 microns in thickness and less dense and more porous than the first layer;
f) providing an aqueous solution or suspension of an enzyme and depositing said enzyme in a layer onto the surface of said second polymer; and g) repeating (d), (e) and (f) at least once to deposit at least one additional said second cellulose acetate layer and at least one additional enzyme layer:
thereby forming said continuous multilayer cellulose acetate polymer membrane.
a) providing a first cellulose acetate polymer dis-solved in an inert organic solvent;
b) casting said first polymer in solution onto an inert support surface which is unreative with said cel-lulose acetate and does not form a bond to said cellulose acetate;
c) permitting said first polymer in solution to form a film and thereby obtain a first relatively nonporous dense layer of cellulose acetate of about 2 to about 5 microns in thickness;
d) providing a second cellulose acetate polymer dissolved in an inert organic solvent mixing said second polymer with a nonsolvent for said polymer to form a dispersion thereof;
e) casting said second cellulose acetate polymer dis-persion onto said first layer of cellulose acetate and permitting said cellulose acetate polymer to form a porous second cellulose acetate layer of about 2 to about 20 microns in thickness and less dense and more porous than the first layer;
f) providing an aqueous solution or suspension of an enzyme and depositing said enzyme in a layer onto the surface of said second polymer; and g) repeating (d), (e) and (f) at least once to deposit at least one additional said second cellulose acetate layer and at least one additional enzyme layer:
thereby forming said continuous multilayer cellulose acetate polymer membrane.
10. The method of claim 9 wherein the inert organic solvent used with said first and second cellulose acetate polymer is the same.
11. The method of claim 10 wherein the inert organic solvent is a ketone.
12. The method of claim 10 wherein the inert organic solvent is acetone.
13. The method of claim 9 wherein the enzyme is glucose oxidase and is present in a mixture of water and ethanol.
14. In a polargraphic cell structure for use in elec-trochemical analysis of an unknown comprising an elec-trically insulating receptacle an electrode means, mounted in said receptacle and a membrane means, the improve-ment which comprises utilizing the multilayer membrane produced by the method of claim 1 or 9.
15. A multilayer membrane produced by the method of claim 1 or 9.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US318,627 | 1981-11-05 | ||
| US06/318,627 US4418148A (en) | 1981-11-05 | 1981-11-05 | Multilayer enzyme electrode membrane |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1189007A true CA1189007A (en) | 1985-06-18 |
Family
ID=23238951
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000414487A Expired CA1189007A (en) | 1981-11-05 | 1982-10-29 | Multilayer enzyme electrode membrane |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US4418148A (en) |
| EP (1) | EP0079502B1 (en) |
| JP (1) | JPS5892853A (en) |
| AU (1) | AU538211B2 (en) |
| CA (1) | CA1189007A (en) |
| DE (1) | DE3275408D1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3539455A (en) * | 1965-10-08 | 1970-11-10 | Leland C Clark Jr | Membrane polarographic electrode system and method with electrochemical compensation |
| IL32406A (en) * | 1968-06-26 | 1973-01-30 | Snam Progetti | Enzyme preparations comprising a solution or dispersion of enzyme occluded in filaments of cellulose esters or synthetic polymers |
| US3947325A (en) * | 1974-04-11 | 1976-03-30 | Snamprogetti S.P.A. | Preparation of high permeability cellulose fibers containing enzymes |
| US4004980A (en) * | 1975-03-25 | 1977-01-25 | Purdue Research Foundation | Enzyme entrappment with cellulose acetate formulations |
| US3979274A (en) * | 1975-09-24 | 1976-09-07 | The Yellow Springs Instrument Company, Inc. | Membrane for enzyme electrodes |
| US4092233A (en) * | 1975-12-18 | 1978-05-30 | Miles Laboratories, Inc. | Membrane apparatus |
| US4063017A (en) * | 1976-04-22 | 1977-12-13 | Purdue Research Foundation | Porous cellulose beads and the immobilization of enzymes therewith |
| DE3010119A1 (en) * | 1979-04-25 | 1980-11-06 | Akad Wissenschaften Ddr | ENZYME ELECTRODE FOR DETERMINING COFACTORS |
| US4356074A (en) * | 1980-08-25 | 1982-10-26 | The Yellow Springs Instrument Company, Inc. | Substrate specific galactose oxidase enzyme electrodes |
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1981
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1982
- 1982-10-28 DE DE8282109958T patent/DE3275408D1/en not_active Expired
- 1982-10-28 EP EP82109958A patent/EP0079502B1/en not_active Expired
- 1982-10-29 CA CA000414487A patent/CA1189007A/en not_active Expired
- 1982-11-01 AU AU90054/82A patent/AU538211B2/en not_active Ceased
- 1982-11-04 JP JP57192655A patent/JPS5892853A/en active Granted
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| DE3275408D1 (en) | 1987-03-12 |
| EP0079502B1 (en) | 1987-02-04 |
| JPH0210903B2 (en) | 1990-03-12 |
| EP0079502A1 (en) | 1983-05-25 |
| AU538211B2 (en) | 1984-08-02 |
| US4418148A (en) | 1983-11-29 |
| AU9005482A (en) | 1983-05-26 |
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