Processes for preparing barrier layer films for use in enzyme electrodes and films made thereby

Methods for forming thin layer barrier layer films for use in enzyme containing laminated membranes and membranes formed thereby are disclosed. The barrier layers exhibit improved acetaminophen rejection and comprise a cellulose acetate/cellulose acetate butyrate blend. The thin layer barrier membranes are formed from a plural solvent containing solution and are cured at a critical temperature of about 102.degree.-114.degree. F., most preferably at about 106.degree. F.-114.degree. F. while traveling through a circulating hot air oven. Alternatively, the membranes can be cured at room temperature or in a stagnant oven at temperatures of from room temperature to about 175.degree. C. (350.degree. F.) for a period of from about 10 minutes to 1 hour.

FIELD OF THE INVENTION 
The invention pertains to improved methods for forming this barrier layer 
films that are useful as a component of an enzyme containing laminated 
membrane. 
BACKGROUND OF THE INVENTION 
Polarographic cell systems have met with wide acclaim particularly in the 
medical field, providing for detection and concentration measurement of 
many desired analytes. Enzymes are commonly used in such systems, 
especially in those situations wherein the analyte itself is not 
polarographically active but where a reaction product formed or reactant 
consumed by an enzymatic reaction with the analyte is polarographically 
active. 
For example, in medical applications, one common procedure is to measure 
glucose in the blood of a patient. Typically, blood samples are withdrawn 
from the patient for an in-line analysis for glucose concentration using a 
glucose oxidase electrode with a polarographic detector for detecting 
H.sub.2 O.sub.2 generated in accordance with the reaction: 
##STR1## 
The hydrogen peroxide generated by the reaction is measurable by a 
polarographic detector and, by appropriate calibration and calculation, 
glucose content in the sample can be accurately determined by the H.sub.2 
O.sub.2 formed in the reaction. 
The polarographic cell systems commonly used for these measurements include 
an enzyme containing laminated membrane that separates the analyte sample 
from the working electrode of the cell. These types of membranes are 
disclosed in U.S. Pat. Nos. 3,979,274 and 4,073,713 (Newman), both patents 
being hereby incorporated by reference herein. In such membranes, a thin 
innermost membrane referred to as a barrier layer composed of cellulose 
acetate, silicone rubber, or methyl methacrylate is located adjacent the 
working electrode of the polarographic cell. Glucose oxidase enzyme is 
interposed between this barrier layer and an outer polycarbonate support 
layer. The outer support layer is typically about 5 .mu.m in thickness and 
is in contact with the analyte containing sample. 
In a glucose analytical determination, glucose and oxygen permeate through 
the outer support layer and react in the presence of the enzyme. Hydrogen 
peroxide produced permeates through the inner barrier layer where it is 
polarographically detected. The support layer permits passage of glucose, 
oxygen and other molecules therethrough while not permitting passage of 
high molecular weight substances such as proteins, red blood cells and 
other macromolecules. 
The barrier layer permits access of hydrogen peroxide to the working 
electrode while blocking passage of substances having molecular weights on 
the order of about 250 and greater such as ascorbic acid and uric acid. 
One problem that has been encountered in polarographic systems as described 
above is that acetaminophen N-(4-hydroxy phenyl) acetamide! in the blood 
sample analyzed is itself polarographically detectable. This causes 
inaccurate readings when attempts are made to monitor glucose levels in 
whole or diluted blood samples. 
The problem of acetaminophen interference in glucose determination is 
especially grave because plasma glucose level is one of the factors used 
to assess the severity of acetaminophen poisoning. In a case of suspected 
acetaminophen poisoning, an episode of low plasma glucose is diagnostic of 
massive liver damage and indicates that heroic measures will be needed to 
save the life of the patient. However, a glucose membrane susceptible to 
acetaminophen interference will give an apparently elevated glucose 
reading, even when the actual plasma glucose is low, completely obscuring 
the diagnosis. 
Accordingly, there is a need in the art to provide a method for making 
improved films that are to be incorporated as the barrier layer in an 
enzyme containing laminated membrane. 
Practical considerations dictate that the barrier layer construction will 
inhibit acetaminophen migration therethrough while not substantially 
impeding migration of H.sub.2 O.sub.2 to the working electrode. In 
polarographic systems of the type described supra, it is desirable to 
permit as much of the H.sub.2 O.sub.2, produced via the enzymatic 
reaction, to reach the electrode to thereby result in an easily detectable 
current. Barrier membranes that inhibit acetaminophen migration at the 
expense of instrument sensitivity are of no significant advantage. 
It is therefore an object of the invention to provide a method for 
producing a barrier layer that will effectively inhibit acetaminophen 
migration to the working electrode while maintaining a desired instrument 
sensitivity level. 
Additionally, hydroxyurea, potassium iodide, and isoniazid are also 
therapeutically used agents that may be contained in blood samples. These 
also result in false glucose readings similar to those provided upon 
acetaminophen presence in the blood. Accordingly, it is an additional 
object of the invention to provide a barrier membrane that will also 
inhibit hydroxyurea, potassium iodide, and isoniazid migration to the 
electrode while permitting the desired electrically detectable species, 
for example, H.sub.2 O.sub.2 to pass therethrough substantially unimpeded 
to thereby result in an acceptable electrical sensitivity. 
PRIOR ART 
Cellulose acetate, silicone rubber or methyl methacrylate barrier film 
layers for incorporation into an enzyme containing laminated membrane are 
taught in the aforementioned Newman patents. Preferred barrier films 
taught in this patent are homogenous films composed of cellulose acetate 
made by a "water casting" technique. In accordance with this technique, 
the cellulose acetate is dissolved in cyclohexanone and the resulting 
solution is dropped into a quiescent pool of water. The cellulose acetate 
precipitates on the top of the water and is removed therefrom by a 
strippable carrier sheet such as polyethylene. Suitable cellulose acetate 
films having thicknesses less than 2 microns may be produced by this 
method. 
Canadian patent 1,307,826 discloses barrier membranes that may be composed 
of silicone rubber, methyl methacrylate or other porous and permeable 
material such as cellulose acetate butyrate or cellulose acetate. 
Preferably, the barrier layer comprises cellulose acetate. As reported in 
this patent, these inner membranes have thicknesses of from 2-10 mu and 
are prepared from an acetone/cyclohexanone solution spread onto the 
surface of a glass plate using a microfilm applicator. 
It is difficult or impossible to remove films from a glass plate if the 
film is cured entirely by air drying. Extracting the solvent(s) from the 
film by flooding with water permits removal of the film from the backing 
but also produces an asymmetric film with mainly coarse pores. The 
intrusion of water into the film results in a phase inversion process in 
which the film is solidified in the form of a gel thereby resulting in a 
heterogenous structure exhibiting inconsistent pore shape and non-uniform 
pore distribution throughout. 
Most combinations of resins and solvents cannot be water cast at all. Even 
those that can are erratic and unpredictable in the way they spread over 
the surface of the water. Since the film thickness and pore geometry 
depend very strongly on the way the casting dope spreads over the surface 
of the water, reproducible results cannot be expected. In particular, 
solutions in solvent other than cyclohexanone spread very poorly over the 
water. 
Thick CA/CAB films cured by drying with hot air give slow responses and low 
sensitivities to glucose. Their permeability to hydrogen peroxide is too 
low. 
Some acetaminophen interference problems were experienced in conjunction 
with the YSI Model 23A analyzer. In order to help overcome these problems, 
thick cellulose acetate/cellulose acetate butyrate (CA/CAB) membranes 
having average thicknesses of about 6 microns were used in laminated 
enzyme membrane structures of the type disclosed in the Newman patents. 
These thick CA/CAB membranes were made by a knife blade casting technique 
in which a doctor's blade was appropriately spaced over the casting 
solution which was in turn superimposed over the desired polymeric 
substrate. The casting solution was similar to that used in the instant 
invention (i.e. the CA/CAB polymers were cast in a solution comprising 
nitromethane and .gamma.-butyrolactone). Films cast from this solution 
were cured by baking at high temperature of about 150.degree. C. 
(300.degree. F.). 
These films, when incorporated as barrier layers in Newman-type membranes, 
served to provide satisfactory protection against false readings caused by 
acetaminophen presence, but they resulted in slow instrument response 
time. However, performance of these membranes on the Model 23A machines 
was acceptable. 
The shift toward quicker, fully automated systems, led to the introduction 
of the YSI Model 2300 series analyzers in 1989. These analyzers place much 
more emphasis on rapid measurement and include fully automatic, 
programmable measurement time intervals and measurement cell flush out 
timing cycles. The aforementioned thick, CA/CAB membranes proved 
unacceptable for use in conjunction with the 2300 series analyzers because 
they took too long to obtain plateau currents (i.e., measurement time was 
too long) and adequate membrane flushing could not be accomplished within 
the flushing time periods allotted by the machine for the sample 
measurement. 
Due to the poor performance of the thick CA/CAB membranes in the 2300 
series analyzers, the thin water cast CA films disclosed in the Newman 
patents were then used as the barrier layer component of the laminated 
membrane assembly in the 2300 systems. Although these barrier layers 
provided for adequate measurement time and could be adequately flushed 
within the allotted time period, they did not serve to reduce 
acetaminophen interference. 
It was not until the presently claimed methods and barrier layer films made 
thereby were discovered that an acetaminophen rejecting barrier layer 
could be successfully employed in a laminated membrane structure for our 
2300 series analyzers. Use of the CA/CAB barrier layers in accordance with 
the invention not only are effective in inhibiting acetaminophen 
interference but they also significantly provide rapid measurement time 
and they can be adequately flushed within the instrument's flushing cycle 
requirements. 
Accordingly, despite the prior art efforts, there remains a need for a 
method to produce thin barrier films on the order of 2 .mu.m and less, 
which films function to inhibit false polarographic detector readings 
caused by acetaminophen in the sample, without significantly decreasing 
instrument sensitivity. There is an additional need to provide methods for 
forming barrier films that will also serve to inhibit hydroxyurea, 
potassium chloride, and isoniazid interference. 
SUMMARY OF THE INVENTION 
These and other objects are met by the present methods and barrier films 
made thereby. 
The barrier film is a thin (i.e. 2 micron or less) cellulose 
acetate(CA)/cellulose acetate butyrate (CAB) film which is solvent cast 
from a plural component non-aqueous solvent system. The film may be 
produced using a conventional Mayer-rod coating machine. Curing 
temperatures of the casting solution are carefully controlled. 
At least two solvents are employed to dissolve the CA/CAB blend. A volatile 
solvent such as nitromethane, dimethylformamide, cyclohexanone, etc is 
used in combination with a liquid plasticizer. A casting solution 
containing CA, CAB, and the solvents is spread over a suitable carrier 
sheet to the desired thickness via conventional mayer-rod coating 
techniques using for example machinery purchased from Lamitec, Inc., 
Minneapolis, Minn. 
After the CA/CAB solution is applied to the carrier sheet in the desired 
thickness (i.e. a sufficient amount to result, after curing, in a 2 micron 
or less film) it is then forwarded to an oven for curing. Temperature of 
the curing step in the oven is strictly controlled to a range of about 
102.degree. F. to 114.degree. F. The cured film resides in the oven for 
about 0.5 to 1.5 or from about 0.5-10 minutes. The film is advanced to a 
take-up roll. 
In another embodiment of the invention, the desired non-aqueous solvent 
system including the CA/CAB is spread over a desired substrate such as PET 
in a thickness that will ultimately result in a cured film thickness of 
about 2 microns or less. The precursor solvent containing solution is then 
cured at a temperature of room temperature to about 350.degree. F. or 
less. 
The invention will be further described in conjunction with the attached 
drawings and following detailed description.

DETAILED DESCRIPTION OF THE EMBODIMENT 
With reference made to FIG. 1 of the drawings, there is shown an enlarged 
cross-section of an enzyme containing laminated membrane 28. Membrane 28 
is adapted for use in conjunction with commercially available analytical 
equipment such as the Models 1500, 2700 or 2300 stat analyzers available 
from The Yellow Springs Instrument Co., Inc. Yellow Springs, Ohio. 
Barrier layer 32 comprises an homogenous cellulose acetate/cellulose 
acetate butyrate polymer blend having a thickness of 2 microns or less, 
preferably 1-2 microns. Enzyme 34 is provided intermediate barrier layer 
32 and support layer 30. Enzyme 34 is typically cross-linked in situ 
between the layers 32,30 by use of glutaraldehyde although any one of a 
number of adhesives or cross-linking promoters may be used. Also, it 
should be mentioned that the enzyme itself may be used as the adhesive 
without any additional adhesive or cross-linking agent added. 
Support layer 30, as shown, is composed of a polycarbonate layer such as 
those that are commercially available from Nuclepore Filtration Products 
of Pleasanton, Calif. under the "Nucleopore" brand name. Other acceptable 
films may be purchased from Poretics, Inc. of Livermore, Calif. These 
films typically have thicknesses ranging from about 5 to 7 microns. 
As shown, support layer 30 comprises a single layer. It is to be understood 
however, that the support layer 30 may actually comprise a multi-layered 
structure with, for example, bovine serum albumin or other suitable 
adhesive interposed between layers to yield a composite structure. In this 
approach pore sizes and individual thicknesses of the layers can be 
controlled, for instance, to limit or promote migration of a given 
chemical species to the enzyme 34. 
It is to be appreciated that support layer 30 is positioned adjacent the 
analyte sample and that the barrier layer 32 is therefore adjacent a 
working electrode (typically platinum) in an electrolyte solution. An 
auxiliary electrode is also disposed in the electrolyte. Accordingly, a 
polarographic cell is provided in which the electrodes and electrolyte are 
separated from the analyte solution by the laminated membrane. 
As used throughout this disclosure, enzyme 34 will be described as glucose 
oxidase enzyme. The artisan of course will appreciate that depending on 
the particular desired analyte and reaction chosen, the enzyme may vary. 
For instance in analytical situations in which it is desired to monitor 
lactate levels in blood samples, lactate oxidase will be used as enzyme 
34. Other candidate analytes and corresponding oxidoreductase enzymes are 
noted as being exemplary: 
______________________________________ 
oxidoreductase 
analyte enzyme 
______________________________________ 
lactose galactose oxidase 
( invertase 
sucrose ( mutarotase 
( glucose oxidase 
alcohol alcohol oxidase 
galactose galactose oxidase 
______________________________________ 
Membrane 32 is composed of a blend of cellulose acetate/cellulose acetate 
butyrate cellulosic esters. The ratio (by weight) of cellulose acetate: 
cellulose acetate butyrate used to form barrier layer 32 varies widely 
over a range of 1.5-20:1. Based upon present indications, it is preferred 
to utilize a 4:1 (by weight) blend of cellulose acetate/cellulose acetate 
butyrate to cast the film used to form barrier layer 32 of membrane 28. 
The requisite ratio of cellulose acetate and cellulose acetate butyrate is 
dissolved in a two solvent non-aqueous system. The first solvent is a 
highly volatile organic solvent exhibiting a low boiling point. At 
present, nitromethane, dimethylformamide and cylcohexanone may be 
mentioned as being exemplary members of this class of highly volatile 
organic solvents. All of those have boiling points, under atmospheric 
conditions, of less than 200.degree. C. At present, it is preferred to use 
nitromethane as the highly volatile organic solvent. 
In addition to use of the volatile solvent, an organic liquid plasticizer 
is used as a second component of the casting solution. The CA/CAB blend is 
also soluble in the plasticizer. This plasticizer is characterized by 
having a boiling point of greater than about 200.degree. C. and must be 
capable of rendering the CA and CAB compatible (i.e. leading to the 
formation of a homogenous CA/CAB film). Exemplary organic liquid 
plasticizers include the phthalates, phosphates, lactones, esters of 
aliphatic dibasic acids, camphor, etc. Especially preferred are the 
lactones including .gamma.-butyrolactone and valerolactone. 
.gamma.-Butyrolactone is presently preferred. 
One of the surprising properties of butyrolactone and valerolactone is that 
they have high boiling points for such tiny molecules. 
Although Applicants do not wish to be bound to any particular theory of 
operation, it is thought that the highly volatile solvent leaves the 
casting solution quickly while the plasticizer leaves the solution much 
more slowly and ultimately defines the pores in the layer as it leaves. It 
is preferred that the platicizer have a boiling point of about 80.degree. 
F. higher than the volatile organic solvent. Since the highly volatile 
organic solvent will leave the solution first, the viscosity of the film 
increases rapidly enough so that it does not flow or sag appreciably after 
it is cast. The plasticizer helps to ensure that the cast film maintains 
its structural integrity with the pores in the film then being defined as 
it, the plasticizer solvent evaporates. 
The first and second solvents can be used in a wide range of addition to 
the cellulosic esters. The volume ratio of Volatile Organic 
Solvent:Plasticizer may for instance vary from about 0.5-1.5 
solvent:plasticizer with a ratio of about 1:1 presently preferred. 
The volatile organic solvent and plasticizer must be essentially free of 
high molecular weight impurities, because such impurities would become 
concentrated as the film dries and would exert an influence on the film 
out of proportion to their percentage in the starting solvent. 
The shape of the plasticizer molecule may also have an influence on pore 
geometry. Current wisdom is to the effect that linear molecules move 
through a film by "reptating" (i.e. a snake-like motion) which can allow 
the plasticizer to escape through a very irregular and tortuous pore. A 
substantially spherical molecule such as .gamma.-butyrolactone, on the 
other hand, has a definite diameter to escape. This suggests that more 
spherical plasticizer molecules will produce a better-defined pore as they 
depart from the film. 
In addition to the solvent and plasticizer, described supra., a thinner or 
diluent may be added, as necessary, to accurately control the viscosity of 
the casting solution. For example, isopropanol, methyl ethyl ketone and 
ethyl acetate may be mentioned as exemplary. The thinner may be added in 
an amount by weight of about 0.5-1.5:1 based on the weight of plasticizer 
added. Presently, it is preferred to use isopropanol as the thinner, 
present in amount of 0.88 parts by weight isopropanol: parts by weight 
plasticizer. 
The cellulose esters are added to the highly volatile organic solvent and 
plasticizer in an amount sufficient to make 10-40 wt. % solutions of 
(cellulose esters): combined weight of cellulose esters+solvent and 
plasticizer). 
The cellulose acetate butyrate (CAB) that is used comprises a mixture of 
cellulose acetic acid esters and butyric acid esters. Commercially 
available CABs are graded according to butyryl content of 17, 27, 38, and 
a 50%. Presently preferred is a CAB product having 28-31% acetyl groups 
and about 16% butyryl. This product is available from Eastman Kodak. 
The cellulose acetate component should be of high molecular weight and good 
purity. Presently, this component is a film grade cellulose acetate that 
is commercially available from Eastman Kodak. 
Turning now to FIG. 2, there is shown a Mayer Rod coating machine of the 
type available from Lamitec, Inc. Machines of this type are preferred for 
use in the instant methods for preparing the cellulose acetate/cellulose 
acetate butyrate films. 
Feed roll 102 is provided with a wound spool of carrier film 124, 
preferable formed of PET. The PET is coated on the film forming side with 
a silicone release agent. The system is driven by take-up roll 122. 
Carrier film 124 is trained around tension adjustment rolls 104, 106 and 
108 and fed to and through the nip between resin transfer roller 200 and 
guide rollers 202, 204 and 206. In accordance with conventional 
techniques, the casting solution contained in bath 126 is applied to 
transfer roll 200 and then transferred onto the bottom surface of the 
carrier film. Mayer rods 110, 112 act as doctor blades and limit the 
coating depth to the desired thickness needed to provide a cured film of 2 
microns or less. The Mayer rods 110, 112 each contain worm flights thereon 
with the rotating worm flights serving to spread the coating over the 
carrier film to the desired thickness. 
In accordance with the presently preferred method, the thus coated carrier 
film is advanced forwardly by guide rollers 116, 118 through oven 128. In 
the oven, coated sheet 124 is heated to effect cure. Volatiles are vented 
via hood 130. Residence time of the coated carrier film 124 may vary from 
about 0.5 to 10 preferably around 4-5 minutes in the oven. All that is 
important is that the time should be sufficient to remove substantially 
all of the highly volatile organic solvent and plasticizer from the 
precursor casting solution. 
The coated carrier film 124 travels through the oven at a speed of about 15 
inches/minutes with the average residence time of the coating in the oven 
being about 4-6 minutes, preferably about 5 minutes. Hot air is circulated 
over the coated film from a series of five overhead air slits (not shown) 
formed in the top oven half with each slit having a dimension of about 
111/2".times.1/8". The linear speed of the air emanating from each of the 
slits is about 1,000 linear feet/min. Each slit thereby passes about 10 cu 
ft/min of warm air into the oven. Accordingly, based upon the preferred 
residence time of about 5 minutes, each slit would pass about 50 cu. 
ft./min. of warm air onto the coated film. Multiplied by the number of air 
slits (5), during the preferred residence time, about 250 cf of warm air 
is circulated throughout the oven. Based upon oven residence times of 
0.5-10 minutes, the coated film may be subjected to from about 25-500 
cubic feet of circulating warm air. 
In accordance with the preferred embodiment of the invention, it has been 
found critical to cure the film at a temperature of from 102.degree. F. to 
114.degree. F., most preferably from about 106.degree.-114.degree. F. When 
cast cellulose acetate/cellulose acetate butyrate films are prepared at 
lower temperatures on conveyor ovens of the type shown in FIG. 2, barrier 
layers 32 made therefrom, when employed in polarographic glucose 
determination cells of the type disclosed by Newman, exhibit unacceptable 
"false" glucose readings due to acetaminophen presence in the analyte 
sample. 
In the preferred method using the coating machine shown in FIG. 2 in those 
cases in which the casting solution is cured above the desired range, 
acetaminophen rejection is sufficient, but the membrane exhibits 
unacceptable H.sub.2 O.sub.2 current sensitivity. 
Although the process described above using the Mayer-rod coating machine 
shown in FIG. 2 is presently preferred for commercial practice, 
preliminary data suggest that other methods for curing the non-aqueous 
CA/CAB film solutions provide improvement not only in acetaminophen 
interference inhibition when the film is used as a barrier layer in Newman 
type laminated membranes, but also in hydroxyurea, potassium iodide, and 
isoniazid interference inhibition. Isoniazid is a commonly prescribed 
antibiotic that is useful in combating tuberculosis. Hydroxyurea is an 
antineoplastic agent that is especially useful in the treatment of 
carcinomas located on the head and neck regions. Potassium iodide, on the 
other hand, is a well-known expectorant treatment used to help relieve 
dyspnea. All of these compounds, when present in a diluted or undiluted 
blood sample can provide "false" glucose readings in polarographic 
instruments incorporating Newman-type glucose oxidase containing laminated 
membranes. 
In accordance with the alternate methods, the non-aqueous CA/CAB solvent 
solution described above is cured at a temperature of about room 
temperature to about 175.degree. C. (about 350.degree. F.) to ultimately 
result in a thin film of 2 micron or less in thickness. In those instances 
in which room temperature curing is desired, the solvent solution must be 
cured for a time period of about 40 minutes or greater. Otherwise, the 
insufficiently cured solution, when used as a barrier layer, will not show 
improved interference inhibition. 
As an alternative to room temperature curing, the substrate coated with 
solvent solution may be placed in a stationary, stagnant oven having air 
flow therethrough of less than about 25 cubic feet of air for a period of 
about 10 minutes-1 hour at a temperature of about 30.degree. C. 
(86.degree. F.) to about 175.degree. C. (350.degree. F.). Stagnant oven 
temperatures on the order of 80.degree. C. (176.degree. F.) to about 
150.degree. C. (302.degree. F.) are preferred. The phrase stagnant oven 
means one in which the precursor CA/CAB solution is not moved or conveyed 
through the oven with the precursor maintained in a relatively stationary 
position in the oven during the curing step. 
The invention will be further described with reference to the following 
specific examples which are to be regarded solely as being illustrative, 
and not as restricting the scope of the invention. 
EXAMPLE 1 
(a) Cellulose Acetate Butyrate In Butyrolactone Solutions 
250 grams of cellulose acetate butyrate powder (Eastman Kodak) were mixed 
with 1000 g of butyrolactone in a 1 gallon teflon coated can. A heater 
blanket was placed around the can. A stir motor blade was placed into the 
can so that it did not touch the bottom. The can was then covered with 
aluminum foil and a split lid to accommodate the stirrer blade. The 
mixture was heated to 35.degree. C. and stirred for four days. 
(b) Cellulose Acetate In Butyrolactone 
The procedure set forth in (a) above was followed except that cellulose 
acetate in powder form (Eastman Chemical Products) was substituted for the 
cellulose acetate butyrate of (a) above. 
(c) 227.6 grams of cellulose acetate in butyrolactone produced in (b) above 
were placed in a stainless steel beaker using a large stainless steel 
spatula. 56.9 grams of cellulose acetate butyrate in butyrolactone 
produced in (a) above were added to this beaker. An additional amount of 
75.0 grams of butyrolactone was added with 340.5 grams of nitromethane 
then added. 
This resulting mixture was then stirred under a fume hood by hand until it 
appeared homogenous (ca. 10 minutes). 300 grams of 2-propanol were then 
added to the beaker while the mixture was continuously stirred. 
EXAMPLE 2 
Cellulose acetate/cellulose acetate butyrate solutions CA/CAB were prepared 
in accordance with Example 1. These solutions were each fed to the trough 
of a Mayer rod coating machine as shown in FIG. 1. All processing 
conditions utilized to produce membranes from the cellulose acetate 
butyrate solutions were maintained as constants except for the curing 
temperature in oven 128 (FIG. 2). The membrane curing temperatures are 
detailed in the Table. After these films had been made, they were used as 
a component of a laminated glucose oxidase containing membrane utilizing a 
polycarbonate membrane as a support layer. Glutaraldehyde was used to bind 
the enzyme interposed between the CA/CAB layer and the polycarbonate 
layer. 
The CA/CAB barrier layer was less than 2 .mu.m thick. 
Glucose measurements and "false" glucose measurements due to acetaminophen 
presence were recorded on a YSI 2300 STATPLUS.TM. analyzer run in normal 
mode to monitor glucose levels in analyte samples using the CA/CAB 
containing laminated membranes. In the normal mode, current measurements 
are made of current running through a circuit comprising a platinum 
electrode and an auxiliary electrode where the potential is maintained at 
+0.7 v. between the electrodes. Glucose containing samples are presented 
to the polarographic call (and enzyme containing laminated membrane 
adjacent the Pt electrode) diluted at a 20:1 volume ratio of buffer 
solution: analyte sample. When glucose is present in the analyte sample, 
the instrument measures the current resulting from the amount of H.sub.2 
O.sub.2 present at the working anode of the polarographic test system. 
Polarographic cells of the type used are generally shown in the 
aforementioned Newman U.S. patents. 
The following results were obtained under test runs designed to measure 
current for known samples containing 1.8 g/l glucose and those in which no 
glucose, but 100 mg/dl acetaminophen was present in the sample. In those 
instances in which no glucose, but acetaminophen, was tested, the results 
shown are given in terms of "apparent glucose readings". This means the 
current produced was compared to glucose calibration standards with the 
result then given in units (mg/dl) of glucose falsely detected present by 
the analyzer. 
TABLE 
______________________________________ 
Membrane Membrane Membrane 
1 2 3 Overall* 
______________________________________ 
I. Membrane - CA/CAB--cured at 106.degree. F. 
Instrument Sensitivity 
for 1.8 g/l glucose 
nanoamps 
(avg) 13.76 12.96 12.7 13.23 
(std dev) 1.07 0.32 1.1 0.75 
Apparent 
Glucose Reading 
Response To 100 mg/dl 
acetaminophen 
(given in mg/dl) 
(avg) 0 1.5 0 0.5 
(std dev) 0 0.51 0 0.8 
II. Membrane - CA/CAB--cured at 100.degree. F. 
Instrument Sensitivity 
for 1.8 g/l glucose 
nanoamps 
(avg) 19.4 17.86 15.56 17.6 
(std dev) 1.15 1.21 1.76 1.9 
Apparent 
Glucose Reading 
Response To 100 mg/dl 
acetaminophen 
(given in mg/dl) 
(avg) 2.79 2.43 3.11 2.75 
(std) 0.61 0.04 0.14 0.28 
III. Membrane - CA/CAB--cured at 115.degree. F. 
Instrument Sensitivity 
for 1.8 g/l glucose 
nanoamps 
(avg) 9.17 7.14 8.77 8.16 
(std dev) 1.34 0.71 0.61 0.95 
Apparent 
Glucose Reading 
Response To 100 mg/dl 
acetaminophen 
(given in mg/dl) 
(avg) 0 0 0 0 
(std dev) 0 0 0 0 
______________________________________ 
*N = 12 determinations per date point per membrane batch. 
DISCUSSION--EXAMPLE 2 
It is desirable that the instrument exhibit maximum current sensitivity for 
any given concentration of analyte. Accordingly, with only this 
characteristic considered, the membranes II would appear to the best since 
they average 17.6 nA. However, these membranes also gave the worst "false" 
glucose readings, averaging 2.75 mg/dl glucose readings when no glucose 
(but 100 mg/dl acetaminophen) was present in the sample. These "false" 
glucose readings are clearly unacceptable. 
Batch III membranes, cured at 115.degree. F. provided excellent "false" 
glucose readings at the price of unacceptably low instrument sensitivity 
values (i.e. average 8.16 nA). 
Batch I membranes provided high sensitivity (i.e. 13.23 nA) while not 
providing any significant compromise in "false" glucose readings (i.e. 0.5 
mg/dl). 
Although Applicant is not bound to any particular theory of operation, it 
is thought that the Batch II membranes, cured at 100.degree. F. resulted 
in relatively large pore size formation in the membrane which increased 
the electrical sensitivity of the membrane to glucose while also allowing 
easy access of acetaminophen to the working electrode. Cured at the 
115.degree. F. temperature, the Batch III membranes appeared to form tight 
pores that unacceptably hindered instrument sensitivity. 
EXAMPLE 3 
Laminated membranes containing CA/CAB barrier layers made in accordance 
with the procedures set forth in Examples 1 and 2 were contrasted to 
laminated membranes containing CA only barrier layers made in accordance 
with water casting methods specified in the aforementioned Newman patents. 
Tests were conducted to assess propensity of the membranes in falsely 
measuring glucose in analyte sample containing acetaminophen. In 
accordance with the water casting method, the cellulose acetate used for 
the barrier layer was deposited in a cyclohexanone/water solution. 
Once again, after the requisite films had been made, they were fabricated 
into laminated membranes containing a polycarbonate layer as the support 
layer. Glucose oxidase was utilized as the enzyme and was adhered 
intermediate the CA or CA/CAB barrier layer and the polycarbonate support 
layer by the use of glutaraldehyde. 
These laminated membranes were tested using a YSI Model 2300 STATPLUS.TM. 
analyzer in accordance with the procedure set forth in Example 2. 
Results appear in the following Table. 
______________________________________ 
Membrane # 
Sensitivity 1 2 3 *Overall 
______________________________________ 
Batch A - CA/CAB containing laminated membrane. Tested 
on a model 2300 stat plus analyzer normal mode. 
Calibration value 1.8 g/l Glucose. 
N = 3 Determinations per data point per membrane. 
nA 14.1 15.1 12.1 
14 15 12.7 
14.4 16 12.5 
(avg) 14.16 15.36 12.43 13.98 
(std dev) 0.21 0.55 0.31 1.31 
Apparent glucose 
0 0 0 
response in mg/dl to 
0.01 0 0 
100 mg/dl 
acetaminophen 
0 0 0 
(avg) 0.003 0 0 0.001 
(std dev) 0.005 0 0 0.0003 
Batch B - CA (water casting technique) containing 
laminated membrane. Tested on 2300 stat plus analyzer 
normal mode. 
Calibration value 1.8 g/l glucose 
N = 3 Determinations per data point per membrane 
nA 21.6 17.9 21.72 
20.9 18.01 21.7 
22.9 18.28 22.4 
(avg) 21.8 18.06 21.94 20.6 
(std dev) 1.01 0.19 0.39 1.98 
Apparent glucose 
222 146 167 
response in mg/dl to 
225 152 168 
100 mg/dl 
acetaminophen 
226 155 167 
(avg) 224 151 167 180.88 
(std dev) 2 4 0.5 033.4 
______________________________________ 
*N = 9 Determinations per data point per membrane batch. 
DISCUSSION--EXAMPLE 3 
The laminated membrane containing a CA barrier layer produced by the water 
casting technique provides increased electrical sensitivity at the price 
of displaying high "false" glucose readings in the presence of 
acetaminophen. 
It is apparent that the present invention provides a thin film layer 
suitable for use as a barrier layer in an enzyme containing laminated 
membrane of the type disclosed in the Newman patents. Not only do these 
barrier layers minimize interference that may be caused by acetaminophen 
presence in the analyte solution, but it achieves this goal without 
significantly impairing membrane sensitivity. 
The CA/CAB barrier layers formed in accordance with the above when 
incorporated into a laminated membrane of the type shown in FIG. 1, 
provide an acceptable current level of from about 10 to 15 nanoamps for a 
1.8 g/l analyte solution (diluted at a 20:1 buffer: analyte solution 
ratio) in a polarographic cell of the type wherein the electrical 
potential is poised at 0.7 volts. Quite surprisingly, these membranes 
provide improved acetaminophen rejection. That is, in polarographic cells 
as described above, they provide "false" glucose readings of between about 
only 0-2.0 mg/dl in the presence of sample solutions of 100 mg/dl 
acetaminophen. Most preferably, they exhibit "false" glucose readings of 
about 0.5 mg/dl in these systems. 
The choice of volatile organic solvent and plasticizer used is not 
elementary. These components must: 
Level promptly when spread uncommonly thin; and 
Wet the web backing sufficiently to spread uniformly, without pinholes, 
fish eyes, orange peels or any other type of lumps or gaps in the 
continuity of the wet film. This is tougher than it might seem, because 
the web backing is chosen especially for its glossiness and slickness. 
Many solvents will bead up on such a web, rather than spread uniformly 
since we use an insoluble polyester film coated on one side with a solid 
silicone release layer. The fact that almost nothing sticks to the 
silicone allows us to peel off the cured CA/CAB film even though that film 
is very thin. Many commonly used solvents do not like to spread nicely on 
a silicone film. 
EXAMPLE 4 
In order to demonstrate the widespread versatility of the above disclosed 
alternative methods in producing barrier films that effectively inhibit 
acetaminophen, hydroxyurea, potassium iodide, and isoniazid interference, 
glucose measurement and false glucose measurements were recorded on the 
YSI 2300 STATPLUS.TM. analyzer as described in Example 2. In all cases, 
laminated membrane structures of the type specified in the aforementioned 
Newman patents were made to include thin (i.e. less than 2 micron) CA/CAB 
barrier films (made in accordance with the non aqueous solvent system 
described above in Example 1.) In each case, the enzyme used was glucose 
oxidase with the outer support layer composed of 5 micron thick 
polycarbonate having an average pore diameter of about 300 Angstrom units 
with a pore density of about 6.times.10.sup.8 pores/cm.sup.2. 
PET substrates were coated with the CA/CAB non aqueous solvent system of 
Example 1 by using an apparatus similar to that shown in FIG. 2. Instead 
of passage through the oven 128 depicted in that figure, the thus coated 
substrates were subjected to either room temperature or stagnant oven 
curing under the time and temperature conditions noted in the following 
table. 
In each case, false or apparent glucose readings were obtained for test 
samples containing the specified amounts of potassium iodide, hydroxyurea, 
isoniazid and acetaminophen acting as interferants. In this regard, a base 
line efficiency was established (referred to as the average relative 
composite interference) using CA/CAB membranes cured in accordance with 
the curing apparatus schematically shown in FIG. 2 and described in 
Example 2. Average relative composite interference values less than 1 
indicate improved interference inhibition in comparison to CA/CAB barrier 
membranes made in accordance with Example 2 and cured at 106.degree. F. 
for 5 minutes using the oven and conveyor system schematically depicted in 
FIG. 2. Additionally, the laminated membranes were also tested in 
conjunction with samples including the specified amounts of H.sub.2 
O.sub.2 (a polarographically or electrically detectable species) and with 
glucose containing samples. These latter mentioned tests are used to 
ensure that use of the CA/CAB containing barrier layers in Newman type 
laminated membranes resulted in acceptable electrical sensitivity levels 
produced at the electrode. 
Results form these tests are shown in the following table: 
TABLE 
__________________________________________________________________________ 
All Membranes were Cured external of the Lamitec machine 
(FIG. 2) for the temp (degrees C)/time(min) indicated in a stagnant oven 
Average Average Reading (g/L apparent glucose) 
Plateau Currents (nA) 
Potassium 
Hydroxy- Average 
Average 
Cure/Tem 
H2O2 Glucose 
Iodide 
urea Isoniazid 
Acetaminophen 
Composite 
RelComp 
Time 3 mg % 
1.8 g/L 
1 mM 1 mM 1 mM 1 mM Interferen 
Interference 
__________________________________________________________________________ 
RT-24HrB 9.069803 
0.008093 
0.11308 
0.015533 
0.007233 
0.143939 
0.835086 
RT-36Hrs 8.071013 
0.001812 
0.117244 
0.014503 
0.007782 
0.14134 
0.820011 
30/10 
7.3525 
14.22568 
0.001162 
0.067824 
0.012685 
0.005616 
0.087287 
0.638023 
30/30 
5.764167 
11.33932 
0.000374 
0.068306 
0.008994 
0.002644 
0.080317 
0.587077 
40/10 
6.949167 
13.6792 
0.00101 
0.056803 
0.01176 
0.005717 
0.075289 
0.550324 
40/30 
7.295 
14.31057 
0.000968 
0.068794 
0.016754 
0.008726 
0.095242 
0.696167 
50/10 
6.99 13.39114 
0.000111 
0.064446 
0.011268 
0.006522 
0.082348 
0.803731 
50/30 
7.681667 
14.2703 
0.000231 
0.059615 
0.010796 
0.00533 
0.075972 
0.741502 
60/10 
7.635 
14.00697 
0.000581 
0.054093 
0.011969 
0.006732 
0.073375 
0.71616 
60/30 
6.559167 
11.1267 
0.001245 
0.058875 
0.009543 
0.003815 
0.073478 
0.717165 
70/10 
6.32 12.1533 
0.001148 
0.058768 
0.011783 
0.006497 
0.078196 
0.763208 
70/30 
7.628333 
13.98557 
0.000152 
0.06366 
0.01024 
0.005553 
0.079605 
0.776964 
80/10 
6.248333 
10.24943 
0.001287 
0.054268 
0.007131 
0.001952 
0.064638 
0.630877 
80/30 
6.571667 
11.49716 
0.000291 
0.053736 
0.007657 
0.002065 
0.063748 
0.622197 
90/10 
5.994167 
10.61057 
0.000194 
0.055517 
0.006446 
0.002103 
0.064259 
0.627178 
90/30 
6.34 10.80379 
0.000212 
0.051308 
0.006369 
0.002997 
0.060887 
0.594268 
100/10 
6.265833 
12.07818 
0.000332 
0.047462 
0.006971 
0.002418 
0.057182 
0.558114 
100/30 
5.640417 
9.96875 
0.000332 
0.046172 
0.004 
0.001039 
0.051543 
0.503068 
115/10 
6.2625 
11.96989 
0.001107 
0.05719 
0.010228 
0.003778 
0.072302 
0.52849 
115/30 
5.734167 
11.20432 
0.000346 
0.057038 
0.00744 
0.001876 
0.066699 
0.487536 
130/10 
8.085833 
15.42966 
0.000457 
0.042277 
0.004606 
0.001511 
0.048851 
0.357072 
130/30 
6.8475 
13.17761 
0.000457 
0.0403 
0.0032 
0.000403 
0.04436 
0.324244 
150/10 
8.753333 
16.92 
0.000235 
0.027201 
0.003794 
0.001372 
0.032602 
0.238306 
150/30 
6.021667 
11.45886 
0 0.027822 
0.002183 
0.000504 
0.030508 
0.222998 
175/10 
5.084167 
7.855795 
5.53E-05 
0.024222 
0.000491 
0.00073 
0.025499 
0.297913 
175/30 
5.099044 
8.596502 
3.55E-05 
0.023887 
0.0006 
0.000572 
0.025094 
0.293187 
__________________________________________________________________________ 
"Average Composite Interference" is the sum of the average apparent 
glucose readings in g/L obtained for each of the four interferences. 
"Average Rel Comp Interference" is the "Average Interference Composite" 
obtained for any given membrane type devided by the "Average Composite 
Interference" obtained on the set of 106F/5 membranes (control) which wer 
made and evaluated at the same time as the indicated experimental 
membranes At least four membranes of each type were evaluated. 
EXAMPLE 5 
An additional series of tests were performed on the CA/CAB membranes made 
with the non aqueous solvent combination detailed in Example 1. Here, 
similar to example 4, substrates coated with the precursor solvent 
combination were not transported thru an oven such as that shown by 
reference number 128 in FIG. 2. Instead, they were subjected to room 
temperature (about 22.degree. C. 72.degree. F.) curing for the specified 
times. Again, similar to the other examples, the so produced CA/CAB film 
samples were incorporated into glucose oxidase containing Newman type 
laminated membranes as described above and were tested on the 2300 
STATPLUS.TM. analyzer also described above. 
Results are shown in the following table. 
TABLE 3 
__________________________________________________________________________ 
Glucose Membrane Performance (42629) Vs. Time of CA/CAB cure at Room 
Temperature 
Unless otherwise noted all membranes were cured external of the Lamitec 
machine (FIG. 2) 
at room temperature for the Time (Mins.) indicated. 
Average Average Reading (g/L apparent glucose) 
Plateau Currents (nA) 
Potassium 
Hydroxy Average 
Average 
Cure 
H2O2 Glucose 
Iodide 
urea Isoniazid 
Acetaminophen 
Composit 
RelComp 
Time 
3 mg % 
1.8 g/L 
1 mM 1 mM 1 mM 1 mM Interferen 
Interfenren 
__________________________________________________________________________ 
106F/5.degree. 
6.488 
11.13 
0.001088 
0.091984 
0.014988 
0.002482 
0.11052 
1 
0 0.04 0.1675 
0.015583 
0.216743 
0.032142 
0 0.264447 
2.392759 
10 3.398333 
3.543333 
0.086158 
0.434861 
0.182618 
0.094742 
0.788378 
7.223859 
20 2.688 
2.1525 
0.397588 
1.472191 
0.839198 
0.4641 3.173077 
28.71049 
40 4.547143 
5.584286 
0.002439 
0.146236 
0.020846 
0.001454 
0.170975 
1.54701 
80 7.168 
11.09 
0.001565 
0.071476 
0.009035 
0.000462 
0.082539 
0.746823 
120 6.4075 
10.23167 
0.002169 
0.07413 
0.007966 
0.006388 
0.090632 
0.820051 
__________________________________________________________________________ 
"Lamitec process made and cured on machine at indicated temp (degrees F) 
at a web speed of 15 in/min (approx 5 min residence time) 
"Average Composite Interference" is the sum of the average apparent 
glucose readings in g/L obtained for each of the four interferences. 
"Average RelComp Interference" is the "Average Interference Composite" 
obtained for any given membrane type devided by the "Average Composite 
Interference" obtained on the set of 106F/5 membranes (control) which wer 
made and evaluated at the same time 
At least four membranes of each type were evaluated 
DISCUSSION EXAMPLES 4 AND 5 
It is apparent that a variety of curing temperatures may be used to cure 
the CA/CAB film with the resulting films showing effective interference 
inhibition. When cured at room temperature, effective interference 
inhibition commences at cure times on the order of about 40 minutes and 
greater. In contrast, when cured in a stagnant oven, effective 
interference inhibition may be achieved for example after 10 or 30 
minutes. In the stagnant oven, interference inhibition increases with an 
increase in curing temperature. 
Attention is now directed to the graphical representation shown in FIG. 4. 
Here, the relative composite interference is shown as the y-axis with the 
curing temperature attained in the stagnant oven shown along the x-axis. 
Both 10 minute and 30 minute curing time plots are provided. Markedly 
increased inhibition performance is shown when curing temperatures in the 
oven are 80.degree. C. (176.degree. F.) and greater. Within this range, 
curing at temperatures from 80.degree. C. (176.degree. F.) to about 
150.degree. C. (302.degree. F.) is particularly preferred. 
FIGS. 5 and 6 demonstrate that electrical sensitivity (in terms of 
nanoamperes sensed at the polarographic electrode) is not significantly 
altered for the tests shown graphically in FIG. 4. This is important since 
increased interference inhibition at the expense of diminished electrical 
sensitivity would be of little commercial value. Importantly, in the 
tested 1.8 g/L glucose containing solutions, current above 10 nanoamps was 
detected for all curing times less than about 150.degree. C. (302.degree. 
F.). Above this temperature, electrical sensitivity begins to diminish at 
cure temperatures of greater than about 150.degree. C. in the stagnant 
oven. 
The room temperature curing tests however indicated that curing times on 
the order of 40 minutes and greater should be used in order to provide 
increased interference inhibition. This is shown graphically in FIG. 7 of 
the drawings wherein with cure times of less than 40 minutes curing the 
average relative composite interference values were greater than the 
baseline (i.e. greater than 1). 
It is therefore apparent that the CA/CAB non aqueous solvent solutions 
detailed in Example 1 can be used to make a barrier film component for use 
in enzyme containing laminated membranes to inhibt interference or false 
readings caused by hydroxyurea, isoniazid, and potassium iodide. In 
conjunction with the schematic shown in FIG. 3, barrier layer 32 has been 
prepared and cured as per either the conveyor oven (FIG. 2) or stagnant 
oven--room temperature methods described above. When the outer support 
layer 30 is contacted by an aqueous sample 200 containing one or more of 
the interferants expressed above, the electrically detectable species 202, 
such as H.sub.2 O.sub.2, is allowed to pass through the barrier layer 32 
substantially unimpeded to provide the desired electrical measurement in 
conjunction with electrode 204. However, the interfering species is 
inhibited form such passage. 
Although the current methods of producing film and films produced thereby 
are directed toward use of such films in enzyme containing laminate 
membrane structures, membranes produced in accordance with this disclosure 
can also be utilized in non-enzyme containing structures and methods. For 
example, these membranes can be used to detect and determine H.sub.2 
O.sub.2, hydroxylamine, hydrazine and some other important compounds which 
are electrochemically active without any mediating enzyme. Specificity is 
highly desirable in such non-enzymatic membranes, just as in an enzyme 
membrane, and the improved barrier films and methods of producing same 
disclosed herein provide significant increase in specificity without any 
serious loss of speed or sensitivity.