CA1067457A - Membrane for enzyme electrodes - Google Patents

Abstract

Abstract of the Disclosure A laminated membrane for use with an enzyme electrode without the need for use of a compensating electrode. The laminated membrane comprises a layer of essentially homogeneous material such as cellulose acetate or silicone rubber which will prevent passage of even low molecular weight interfering materials, an adhesive layer containing the enzyme (with or without other materials that may be blended with it), and a layer of support film which will also prevent the passage of high molecular weight interfering materials.

Description

~0674~7 Background of th.e ~nvention .
This invention relates to an improved membrane for an enzyme electrode, and more particularly to a laminated membrane wherein the enzyme (with or without other ~aterials blended with it) is actually the adhesive between the lamina or is included in or bonded to the adhesive between the lamina, Polarographiccell ~ystems have become quite . ~ .
popular in the medical field for measurement of various sub-stances. In addition, enzymes have been used in conjunction with polarographic cells, especially in instances where the : unknown substance to be measured is not polarographically active~
: but a material produced or consumed by an enzymatic reaction ~.
with that unknown is detectable. For example, it is known that glucose is not polarographically active but that the following reaction takes place in the presence of tbe enzyme glucose oxidase:
Glucose GlucoSe ~ 2 ~ Gluconic acid ~H22 Oxidase The existence of this reaction is signiflcant in enabling polarographic measurement of glucose.
Thus, in an article by Clark and Lyons in the Annals of the ~ew York Academy of Science, 102, 29-45 (1962), it was suggested that a pH sensitive electrode could be used to detect the gluconic acid produced by the reaction. It ~as disclosed that the enzyme in such ~ syste~ could be trapped between cuprophane membranes. The glucose diPfuses through the membrane and is converted by the enzyme to gluconic acid, which then diEfuses both toward the p~l sensitive glass and back into the donor solution.
Alternatively, it was suggested that by using a hydrophobic membrane, a dialysis membrane, glucose oxidase, and a P~2 electrode, a system could be arranged that is sensitive to glucose by virtue of the fact that oxygen is consumed from the flowing glucose solution in proportion to its glucose content.
Later, Clark obtained a patent on an improvement in such a system. In U.S. patent 3,539,455, it is stated that the system disclosed therein "differs in simplicity, reliability and in function from the cell disclosed in 'Annals of the ~ew York Academy of Sciences~". Rather than measuring the pH change or the oxygen consumption, the Clark patent discloses using a p~lati umanode to measure the hydrogen peroxide produced. In the polarographic cell described in that patent, the enzyme is placed on the anode side of a Cellophane membrane. The lo~ molecular weight glucose passes through the membrane and reacts with the enzyme, but interfering high molecular weight catalase and peroxidase materials 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. The use of polymeric gels to stabilize the *Registered Trade Mark. - 3 -^ Docket 6265 1067~57 enzyme is also disclosed. ' Since the cellophane membrane will not prevent low molecular wei~ht interfering materials such as uric acid or ascorbic acid from reaching the~anode, Clark suggests a dual elec-trode system. The compen'sating electrod0, wi~hout an enzyme present, gives a signal for the interfering ' substances while the enzyme electrode detects both the hydroyen peroxide and the interference. By subtracting the reading of the compensating electrode'from that of the glucose electrode, the amount of hydrogen peroxide production,'~and thus, the glucose level is determined. Still, such a' dual sensor system may encoun~er difficulties in the matching of the ~wo cells.
Under the; circumstances, then, it would be '15 desirable to have an enzyme electrode which employs a' thin filter membrane to prevent passage of even low molecular weight interfering materials,~such as uri~c acid and ascorbic acid, while permitting hydrogen'peroxide to' pass therethrough with minimum hindrance. There exist 20 ' membrane materials, such as~silicone rubber:and cellulose acetate, which permit passage of hydrogen peroxide but which are-effective barriers to interfering substances.
Since this type of membrane must be placed between the - anode and some component o the sensing system, it follows that in order for measurement equilibrium to be as rapid as possible, the membrane must be as thin as possibl '' ' " ' ~.

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Doc~e~ 6265 10674S7 .. .
while still retaining its selectivity. In the case oL a hydrogen peroxide sensing probe, this membrane will need to be less than 2 microns thick. A membrane of this thickness is difficult, if not impossible to use in prac~i~e because of its insufficient strength~
Some support is needed. Depositing ~he material in a thin layer on a porous substructure will be in some respects satisfactory. The porous substructure will provide the necessary strength while at the same time being of little . .
hindrance to hydrogen peroxide passage, and the weak inter-ference rejecting layer can be thin to enhance speed of response. It remains that this-laminated membrane be .. . . . .
combined with a polarographic electrode and appropriate enzyme in such a fashion-that the completed sensor responds satis-factorily to the desired non-polarographic substrate. In-a common configuration with a typical membrane, the enzyme is placed between the anode and membrane as disclosed in the Clark patent. With the laminated membrane just described, the enzyme in this configuration would be as effectively shielded as the anode. Therefore the in~erference rejection must be li~ited to molecules the same size or larger than the substrate of the enzyme. Membrane materials that would rejèct smaller interferences would also prevent t~e substrate from reaching the enzyme.
Alternatively, the enzyme may be placed on the side of this laminated membrane away from the anode. In this case it may-be captured by a third outer membrane layer which is permeable to the substrate but impermeable to .

_5_ `" 1()67457 the enzyme. In this con~igurat~on, the substrate is not unnecessarily hindered from reaction with the enæyme, and good interEerence rejection is possible since the filter layer need pass only the resultant polarographic substance, i.e., hydrogen peroxide. The polarographic æubstance, however, is now produced two layers away from the sensing anode, being separated from it by the thin interference-rejecting layer and the porous substructure, and speed of response is limited by the reservoir effect of this spacing.
As a further alternative, the enzyme may be placed within the porous substructure and captured b~ an outer membrane, but this configuration also has the limita-tions on speed imposed by the multiple layers, and specifically by the thickness of the porous substructure~
for now the enzyme is dispersed in this thick layer and is less accessible to its substrate.
As a still further alternative, the enzyme may be placed within and bonded to the porous substructure so that the third outer membrane may be eliminated. Thus, the polarographic substance, hydrogen peroxide, is produced close to the anode and the enzyme is readily accessible to its substrate. This approach, however, requires very sophisticated enzyme immobilization techniques, and presents difficulties in the control of the diffusion of the sub-strate which determines the range of linearity of the electrode.
The above enumerated alternatives are all discussed in Swiss patent No. 569,973, published November 28, 1975, Docket 626S 106~45~

Another problem with such a membrane is that iE
it is too thin it will not have sufficient strength;
whereas, if it i5 too thick, then the all-important speed o~ measurement is lengthened beyond that tolerable.
That is, for measurement of the unknown in any one sample, time is consumed while the~ reaction takes place and the potentiometer equilibrates and records the amount of H202 produced. Then, be~ore another sample can be tested 'the potentiometer must go back down to the null point. As is apparent, when a large number of samples are to be analyzed, any reduction in this time period is quite significant.
Accordingly, the need exists for an enzyme electrode membrane which will prevent passage of both high and low molecular weigh-t interfering chemicals,,and does not re~uire an in~rdinant amount o~ time for sample . .
measurement.
Summary of the Invention In accordance with the present invention there is provided a laminated, two-ply membrane wherein the enzyme itself is used to bond the two plys together or wherein the aahesive layer bonding the two plys together includes the enzyme. The membrane includes (1) a support layer which controls substrate diffusion and serves as a barrier to high molecular weight substances, (2) an enzyme ' preparation for reacting with the unknown, and (3) an essentially homogeneous layer which serves as a barrier to ' ' '' . ' ~'.

Docket 6265 1~67457 interfering low molecular weight materials, but permits hydrogen peroxide to pass through. All of this can be achieved in a total membrane thickness prefera~ly less than around 10 microns, although somewhat thicker membranes, up to around 25 microns, are con~emplated;
As such, the laminated membrane of the present invention is capable of equilibrating speeds as low as 10 seconds when used with a polarographic cell such as that disclosed in Clark patent No. 3,539,455.~
When used with an enzyme electrode, the layer nearest the anode is a silicone rubber, methyl methacrylate, cellulose acetate or other material which will prevent passage of interfering chemicals such as ascorbic acid and uric acid. This layer may be less than two microns thick and~preferably has a thickness of between a. s and 1.0 microns. The layer nearest the sample is a diffusion barrier which prevents passage of high molecular w~ight substances while at the same time providing the tensile strength to hold the shape of the membrane and maintain 2Q intimate contact with the electrode. This material is .
preferably a porous po]ycarbonate, but may be of other types such as metal mesh. It has preferred thickness of of less than 20 microns, more preferably of between 1 - and lQ microns, and most preferred of between 5 and 7 microns.
The adhesive bonding these two layers together may be an enz~me preparation, i.e., glucose oxidase, ylactose oxidase, uricase, etc., which may be mixed with, .

, ~t ``` 1~674~i7 for instance, gluteraldehyde. In that case, the gluteral-dehyde may act as the adhesive bonding the t:wo layers to-gether and the enzyme is merely included therein. Other adhesives could also be used as well. An example is bovine syrum albumin. Likewise, the adhesive may be applied to the inner face of either outer membrane layer and the enzyme joined to it. In either event the enzyme-adhesive is placed in a thin uniform layer from an aqueous paste or solution onto an essentially homogeneous film which is supported on a carrier sheet. A self-sustaining support film is then brought into contact with the enzyme-adhesive preparation on the substrate to form a laminate. The laminate is then dried to adhesiveIy set the enzyme-adhesive preparation and securely bond the layers together. The membrane may be used in this form after the carrier is removed. Alternatively, for easy application onto a polarographic cell, an appropriately sized O-ring may be glued onto the support layer surface, individual laminated membranes punched out of the sheet, and the carrier layer removed from each.
Accordingly, it is an object of the present invention to provide an improved laminated membrane for use in an enzyme electrode.
Thu$,the invention in one aspect provides a laminated mPmbrane having a total thickness of less than 25 microns for use in a polarographic cell consisting essentially of a first layer of essentially homogeneous material selected from the group consisting of silicone rubber, methyl methacrylate and cellulose acetate and having a thickness of less than two microns, a second layer of a support material which permits passage of low molecular weight substances but excludes high molecular weight substances, said support material having a thickness of ~_ g _ ~:' !.

between 1 and 20 microns, and an adhesive layer positioned between and bonding said first and second layers, including at least an enzyme in said adhesive layer.
In a further aspect the invention provides a polarographic cell structure for use in polarographic analysis of an unknown comprising an electrically insulating support means, and electrode means mounted in said support means, said electrode means including means defining an active exposed working face, wherein the improvement comprises: a laminated membrane of a total thickness of less than 25 microns positioned between said working face and said unknown, the laminated membrane having the characteristics recited above.
In a still further aspect there is provided a process for the production of a laminated membrane for use in a polarographic cell comprising the steps of: (a) depositing a thin film of essentially homogeneous material selected from the group consisting of silicone rubber, methyl methacrylate and cellulose acetate on a smooth surface, (b) picking up said ~ deposited film with a strippable carrier sheet, (c) placing an aqueous enzyme-adhesive preparation on said essentially homogeneous film, (d) bringing a self-sustaining porous support sheet into contact with the coated substrate to form a laminate, (e) drying said laminate to adhesively set the enzyme, and (f) stripping said strippable carrier sheet from said laminated membrane.
Other objects and advantages of the invention will be apparent from the following description, the accompanying drawing and the appended claims.

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1~)67457 Bri`e`f'D'es'c'r`i'p'tio'n of the Drawin~
Fig. 1 is a view partly in section and partly in elevation of a polarographic cell having in place the lami-nated membrane of the present invention, and Fig. 2 is an enlarged view of the lower central portion of the polarographic cell of Fig. 1 and showing in more detail the laminated membrane of the present invention, Brief Description of the P'refe'rred E~bodi'ment Referring to Fig. 1, there is shown a cell assembly 10~ which includes an electrically in:ulatlng support body 12 of plastic or glass ~hich is preferably cylindrical and which is covered by an electrically insulating cap 14. Positioned within'the cylindrical body 12 is an electrically insulating member or rod 15 of plastic or glass which supports a plati-num electrode 16, the latter including an active or exposed face 17, and a conductor 18 attached to the electrode 16 and which passes through the rod 15 and through the cap 14.
The lower end of the support body 12 i5 provided ~with an annular ring or retainer 19, and a laminated mem-brane 20 in accordance with the present invention is supported over the end of the supporting body nearest the electrode 16 '~
and spaced a capillary distance from the active face 17. The ; membrane is held in position on the supporting body by an 0-;ring 21 or the like.
An annular sp:ce 25 is provided between the rod 15 :nd the supporting body 12 and receives a reference electrode 30 which may for example be silver c~loride coated silver wire.

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`` 1~67457 The space 25 is at least partly and prefera`bly completely filled with a liquid mixture of electrolyte which contacts both electrodes 30 and 16 and which may be ~ntroduced into the chamber through an aperture 31 provided beneath the cap 14.
In polarographic measurements two electrode~ are commonly used, one of which is polarized and does not allow current to flow until depolarized by a substance being mea-sured. In the cell structure shown in Fig. 1, electrode 30 is the cathode and is polarized and frequently referred to as the reference electrode, The other electrode, electrode 16 as shown in Fig. 1, functions as an anode and i5 not polarized in the presence of the sub6tances being measured and therefore will not restrict the flow of relatively large current and is frequently referred to as the senscr electrode~
The electrodes as shown in Fig. l are in electrically insu~at-ing relation, and the electrolyte material which occupies the chamber 25 provides an electrical path between the two elec-trodes. Typical electrolytes include sodium or potassium chloride buffers including carbonate, phosphate, bi:carbonate, acetates, or alkali or rare earth salts, or other organic buffers or mixtures thereof. The solvent for such electro-lyte may be water, glycols, glycerine, and mixtures thereof.
A more detailed description of the enzyme electrode itself, exclusive of the laminated membrane of the present invention, is found in Clark patent ~o. 3,539,455.

1~67457 Fig. 2 shows me~brane 20 m~re fully and will be referred to primarily in the description of that membrane.
~ayer 32, as sho~n, is that ad~acent the active face 17 of anode 16. That layer is the essentially homogeneous slli-cone, methyl methacrylate or cellulose acetate material.
~ayer 34 is the outer layer which w-lll be in contact with the sample to be analyzed. In the preferred embodiment, this is a .03 micron pore size perforated polycarbonate film having a thickness of 5 microns, a nitrogen flow rate of 25 ml/min/cm at 10 psi, and having 6 x 108 holes/cm . Such films~are available from Nuclepore Filtration Products of Pleasanton, California. ~hen an approximately 5-7 micron thick support film is used, the overall thickness of the laminated membrane is le5s than 10 microns as is preferred, Typical thicknesses would be 5 microns for layer 34, one micron for layer 32, and one for layer 36, or a total of 7 microns thickness. Layer 36 ig the adhesive-enzyme material bond~ng layers 32 and 34 together.
The laminated membrane 20 is preferably produced by first placing the essentially homogenous layer ON a strippable carrier sheet. In the case of cellulose acetate, this is done by depositing the cellulose acetate in a solvent (cyclo-hexanone, for example) solution onto water. A film forms which can be picked up by a strippable carrier sheet such as polyethylene. A similar process can be used for silicones and other essentially homogenous materials such as methyl methacrylate. As mentioned, the preferred thickness for the essentially homogenous layer i5 in the range of .5 to l.0 microns.

"~ ~0~7457 The enzyme-adhesive preparation may be simply a mixture of an appropriate enzyme such as glucose oxidase, glactose oxidase, etc. in water. In another embodiment other materials such as a binder or a cross-linking agent like gluteraldehyde may be included In the enzyme preparation.
In that case the gluteraIdehyde may be said to be the adhesire~
Likewise an adhesive may be applied to the inner face of either of the outer layers and the enzyme joined to it.
All that is required is that the adhesive material be non-interferîng with the screening of the outer layers~
Likewise, the proportion of enzyme to water in the prepara-tion is immaterial as long as flowable paste or solution is formed which may be coated or pressed easily into a thin uniform layer, and sufficîent enzyme îs incorporated to provide an adequate reactive amount for measurement.
After placing the aqueous enzyme-adhesive solution or paste onto the essentially homogeneous layer, a self-sustaining support sheet of diffusion barrier material 34, preferably a porous polycarbonate, is brought into contact with the enzyme-adhesive preparation on the cellulose acetate layer to form a laminate. The laminate is then dried by allowing it to sit in air at room temperature~for a half-hour or more. Additionally, to condition the laminate for transit and storage it may be baked at 45C
for approximately half-an-hour. When the carrier sheet is removed the laminated membranes are ready for installation onto a polarographic cell.

Docket 6265 1~6745~

However, if pxe~erred, the laminating procedure may be followed by ~luing onto the support layer 34 a rubbery O-ring 21 of an appropriate size ~or fitting into the retainer 19 on the polarographic cell 10 (see Fiy. 1).
S Laminated membranes 20 ready for use may then be punched out around the O-rings. Of caurse, the;support layer is stripped off the face of the essentially homogeneous layer in.this case,.too.
As ready for use and in use, the laminated 10 . :: membrane 20 need not be kept moist.since the~bond~between layers 32 and 34 will. withstand the di~ferentlal expansion c.aused by drying. That is, drying of the laminate will not cause cracking or other destruation o~ the interference rejecting.layer. ~ :
Most significantly, because the laminated membrane ~: may be less than 10 microns thickness, less than thlrty seconds (and even in some cases as few as ten secondsj ~ ~ : is taken for a polarographic analysis.~ During that short -~ period of time the unknown and oxygen.diffuse through layer 34, react with the enzyme in layer 36, and then the hydrogen peroxide formed diffuses through layer 32 to contact . the active face 17 af the anode 16. The potentiometer -: then equilibrates in the measurement of the amount of hydrogen peroxide. This quick measurement time is extremely : 25 important to laboratories and hospitals where numerous analyses must be made each day.
While the article and method herein described constitute preerred embodimen~s of the invention, it is to . -14-- ' ~ Docket 6265 10~7457 be understood that the invention i5 not limi~ed to this precise article and method, and that changes may be made therein without depàrting form the scope of the invention.

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Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. A polarographic cell structure for use in polarographic analysis of an unknown comprising an electrically insulating support means, and electrode means mounted in said support means, said electrode means including means defining an active exposed working face, wherein the improvement comprises:
a laminated membrane of a total thickness of less than 25 microns positioned between said working face and said unknown, said membrane consisting essentially of a first layer of an essentially homogeneous material selected from the group consisting of silicone rubber, methyl methacrylate, and cellulose acetate and having a thickness of less than two microns, a second layer of a support material which permits passage of low molecular weight substances but excludes high molecular weight substances, said support material having a thickness of between 1 and 20 microns, and an adhesive layer positioned between and bonding said first and second layers, including at least an enzyme in said adhesive layer.
2. A polarographic cell structure as set forth in claim 1 wherein the essentially homogeneous material is cellulose acetate having a thickness of .5 to 1.0 micron.
3. A polarographic cell structure as set forth in claim 2 wherein said enzyme is selected from the group consisting of glucose oxidase, glactose oxidase and uricase.

A polarographic cell structure as set forth in claim 3 wherein said second layer is a porous poly-carbonate film having a thickness of approximately 5-7 microns and the overall thickness of said membrane is less than 10 microns.

A laminated membrane haying a total thickness of less than 25 microns for use in a polarographic cell consisting essentially of a first layer of essentially homogeneous material selected form the group consisting of silicone rubber, methyl methacrylate and cellulose acetate and having a thickness of less than two microns, a second layer of a support material which permits passage of low molecular weight substances but excludes high molecular weight substances, said support material having a thickness of between 1 and 20 microns, and an adhesive layer positioned between and bonding said first and second layers, including at least an enzyme in said adhesive layer.

A laminated membrane as set forth in claim 5 wherein the essentially homogeneous material is cellulose acetate having a thickness of .5 to 1.0 micron.

7. A laminated membrane as set forth in claim 6 wherein said second layer is a porous polycarbonate film having a thickness of approximately 5-7 microns, and the overall thickness of said membrane is less than 10 microns.
8. A laminated membrane as set forth in claim 7 further including a O-ring adhesively bonded to said second layer for purposes of attaching the membrane to a polaro-graphic cell.
9. A laminated membrane as set forth in claim 7 wherein said enzyme is selected from the group consisting of uricase, glactose oxidase, and glucose oxidase.
10. A process for the production of a laminated membrane for use in a polarographic cell comprising the steps of:
(a) depositing a thin film of essentially homogeneous material selected from the group consisting of silicone rubber, methyl methacrylate and cellulose acetate on a smooth surface, (b) picking up said deposited film with a strippable carrier sheet, (c) placing an aqueous enzyme-adhesive preparation on said essentially homogeneous film, (d) bringing a self-sustaining porous support sheet into contact with the coated substrate to form a laminate, (e) drying said laminate to adhesively set the enzyme, and (f) stripping said strippable carrier sheet from said laminated membrane.
11. A process for producing a laminated membrane as set forth in claim 10 further including the steps of gluing an O-ring onto said self-sustaining porous support sheet and then punching out the laminated membrane around said O-ring prior to stripping of the strippable carrier sheet from said laminated membrane.
12. A process as set forth in claim 11 wherein said exzyme is selected from the group consisting of glucose, oxidase, glactose oxidase and uricase.