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Description  |
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BACKGROUND OF THE INVENTION
This invention relates to an improved membrane for an enzyme electrode, and
more particularly to a laminated membrane wherein the enzyme itself (with
or without other materials blended with it) is used as the adhesive
between the lamina.
Polarographic cell systems have become quite popular in the medical field
for measurement of various substances. 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 the enzyme glucose oxidase:
##EQU1##
The existence of this reaction is significant in enabling polarographic
measurement of glucose.
Thus, in an article by Clark and Lyons in the Annals of the New 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 was disclosed that the enzyme in such a system could be
trapped between Cuprophane membranes. The glucose diffuses through the
membrane and is converted by the enzyme to gluconic acid, which then
diffuses both toward the pH 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 pO.sub.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.
Pat. No. 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 New York Academy of Sciences`". Rather than
measuring the pH change or the oxygen consumption, the Clark patent
discloses using a platinum anode 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 low 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 enzyme is also disclosed.
Since the cellophane membrane will not prevent low molecular weight
interfering materials such as uric acid or ascorbic acid from reaching the
anode, Clark suggests a dual electrode system. 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 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 encounter difficulties in the matching of the two cells.
Under the circumstances, then, it would be desirable to have an enzyme
electrode which employs a thin filter membrane to prevent passage of even
low molecular weight interfering materials, such as uric acid and ascorbic
acid, while permitting hydrogen peroxide to pass therethrough with minimum
hindrance. There exist membrane materials, such as silicone rubber and
cellulose acetate, which permit passage of hydrogen peroxide but which are
effective barriers to interfering substances. Since this type of membrane
must be placed between the anode 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 still retaining
its selectivity. In the case of a hydrogen peroxide sensing probe, this
membrane will need to be less than 2 microns thick. A membrane of this
thickness is difficult, if not impossible to use in practice because of
its insufficient strength.
Some support is needed. Depositing the 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
interference 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 satisfactorily 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 interference
rejection must be limited to molecules the same size or larger than the
substrate of the enzyme. Membrane materials that would reject smaller
interferences would also prevent the 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 the enzyme. In this configuration, the substrate is not unnecessarily
hindered from reaction with the enzyme, and good interference rejection is
possible since the filter layer need pass only the resultant polarographic
substance, i.e., hydrogen peroxide. The polarographic substance, 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 by an outer membrane, but this configuration
also has the limitations 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 substrate which determines the range of linearity of the
electrode.
Another problem with such a membrane is that if it is too thin it will not
have sufficient strength; whereas, if it is too thick, then the
all-important speed of 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 H.sub.2 O.sub.2 produced. Then, before 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 weight interfering
chemicals, and does not require an inordinant amount of time for sample
measurement.
SUMMARY OF THE INVENTION
In accordance with the present invention there is provided a laminated,
two-ply membrane wherein an enzyme adhesive is used to bond the two plys
together. 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
for bonding the layers together, and (3) an essentially homogeneous layer
which serves as a barrier to interfering low molecular weight materials,
but permits hydrogen peroxide to pass through. All of this can be achieved
in a total membrane thickness preferably less than around 10 microns,
although somewhat thicker membranes, up to around 25 microns, are
contemplated. 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 U.S. Pat. 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 2 microns thick and preferably
has a thickness of between 0.5 and 1.0 microns. The layer nearest the
sample is a diffusion barrier which prevents passage of high molecular
weight substances while at the same time providing the tensile strength to
hold the shape of the membrane and maintain intimate contact with the
electrode. This material is preferably a porous polycarbonate, but may be
of other types such as metal mesh. It has a preferred thickness of less
than 20 microns, more preferably of between 1 and 10 microns, and most
preferred of between 5 and 7 microns.
The adhesive bonding these two layers together is an enzyme preparation,
i.e., glucose oxidase, glactose oxidase, uricase, etc., which may be mixed
with, for instance, gluteraldehyde. It 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 preparation on the substrate to
form a laminate. The laminate is then dried to adhesively set the enzyme
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.
Other objects and advantages of the invention will be apparent from the
following description, the accompanying drawing and the appended claims.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a view partly in section and partly in elevation of a
polarographic cell having in place the laminated 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 PREFERRED EMBODIMENT
Referring to FIG. 1, there is shown a cell assembly which includes an
electrically insulating support body 12 of plastic or glass which is
preferably cylindrical and which is convered 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 platinum
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 is provided with an annular ring or
retainer 19, and a laminated membrane 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 O-ring 21 or the
like.
An annular space 25 is provided between the rod 15 and the supporting body
12 and receives a reference electrode 30 which may for example be silver
chloride coated silver wire. The space 25 is at least partly and
preferably completely filled with a liquid mixture of electrolyte which
contacts both electrodes 30 and 16 and which may be introduced into the
chamber through an aperture 31 provided beneath the cap 14.
In polarographic measurements two electrodes are commonly used, one of
which is polarized and does not allow current to flow until depolarized by
a substance being measured. 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 is not polarized in the presence of the
substances being measured and therefore will not restrict the flow of
relatively large current and is frequently referred to as the sensor
electrode. The electrodes as shown in FIG. 1 are in electrically
insulating relation, and the electrolyte material which occupies the
chamber 25 provides an electrical path between the two electrodes. Typical
electrolytes include sodium or potassium chloride buffers including
carbonate, phosphate, bicarbonate, acetates, or alkali or rare earth
salts, or other organic buffers or mixtures thereof. The solvent for such
electrolyte 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 U.S.
Pat. No. 3,539,455, which is hereby incorporated by reference.
FIG. 2 shows membrane 20 more fully and will be referred to primarily in
the description of that membrane. Layer 32, as shown, is that adjacent the
active face 17 of anode 16. That layer is the essentially homogeneous
silicone, methyl methacrylate or cellulose acetate material. Layer 34 is
the outer layer which will be in contact with the sample to be analyzed.
In the preferred embodiment, this is a 0.03 micron pore size perforated
polycarbonate film having a thickness of 5 microns, a nitrogen flow rate
of 25 ml/min/cm.sup.2 at 10 psi, and having 6 .times. 10.sup.8
holes/cm.sup.2. Such films are available from Nuclepore Filtration
Products of Pleasanton, Calif. When an approximately 5-7 micron thick
support film is used, the overall thickness of the laminated membrane is
less than 10 microns as is preferred. Typical thicknesses would be 5
microns for layer 34, 1 micron for layer 32, and one micron for layer 36,
or a total of 7 microns thickness. Layer 36 is the adhesive enzyme
material bonding layers 32 and 34 together.
The laminated membrane 20 is preferably produced by first placing the
essentially homogeneous layer on a strippable carrier sheet. In the case
of cellulose acetate, this is done by depositing the cellulose acetate in
a solvent (cyclohexanone, 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
homogeneous materials such as methyl methacrylate. As mentioned, the
preferred thickness for the essentially homogeneous layer is in the range
of 0.5 to 1.0 microns.
The enzyme preparation may be simply a mixture of an appropriate enzyme
such as glucose oxidase, glactose oxidase, etc. in water. Of course, other
materials such as a binder or a cross-linking agent like gluteraldehyde
may be included in the enzyme preparation. Likewise, the proportion of
enzyme to water in the preparation is immaterial as long as flowable paste
or solution is formed which may be coated or pressed easily into a thin
uniform layer, and sufficient enzyme is incorporated to provide an
adequate reactive amount for measurement.
After placing the aqueous enzyme 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 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 45.degree.C for
approximately half-an-hour. When the carrier sheet is removed the
laminated membranes are ready for installation onto a polarographic cell.
However, if preferred, the laminating procedure may be followed by gluing
onto the support layer 34 a rubbery O-ring 21 of an appropriate size for
fitting into the retainer 19 on the polarographic cell 10 (see FIG. 1).
Laminated membranes 20 ready for use may then be punched out around the
O-rings. Of course, 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 membrane 20 need not be kept
moist since the bond between layers 32 and 34 will withstand the
differential expansion caused by drying. That is, drying of the laminate
will not cause cracking or other destruction of the interference rejecting
layer.
Most significantly, because the laminated membrane may be less than 10
microns in thickness, less than 30 seconds (and even in some cases as few
as 10 seconds) 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 of the anode 16. The
potentiometer then equilibrates in the measurement of the amount of
hydrogen peroxide. This quick measurement time is extremely important to
laboratories and hospitals where numerous analyses must be made each day.
While the article and method herein described constitute preferred
embodiments of the invention, it is to be understood that the invention is
not limited to this precise article and method, and that changes may be
made therein without departing from the scope of the invention.
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