Indian Patents. 222754:SCREEN-PRINTABLE PASTE FOR PRODUCING A POROUS POLYMER MEMBRANE FOR A BIOSENSOR.

Title of Invention	SCREEN-PRINTABLE PASTE FOR PRODUCING A POROUS POLYMER MEMBRANE FOR A BIOSENSOR.
Abstract	The invention relates to a paste, which can undergo screen printing, for producing a porous polymer membrane. Said paste contains at least one polymer, one or more solvents for the polymer having a boiling point > 100°C, one or more non-solvents for the polymers (pore-forming agents) having a higher boiling point than that of the solvent (s), and contains a hydrophllic viscosity modifier.

Full Text	The present invention relaces to a screen-printable pasce for producing a porous polymer membrane which can be used in electrochemical sensors, especially in electrochemical biosensors, for integrated preparation of, m particular, whole blood samples. Biosensors are already in use in a large number of diagnostic methods, for example in the determination of the concentration of various factors in body fluids such as blood. The aim in this connection is to have sensors which require no elaborate processing of the (blood) sample but provide a rapid result simply by applying the body fluid to a test strip. This entails a specific biochemical reaction taking place, such as, for example, the enzymatic conversion of the component to be determined, which then brings about an electron transfer between different electrodes (working and reference electrodes), and this can be determined quantitatively. The disadvantage of most of the known electrochemical biosensors is that, on application of the blood to the region provided therefor on the test strip. the biochemical reaction which takes place is influenced by other constituents-present in the blood, especially the red blood corpuscles (erythrocytes). Thus, for example, when the value3 of the hematocrit (= volume of the erythrocytes as a proportion of the total amount of blood in vol.wt.%) are high, the value measured for glucose using conventional blood glucose sensors is lower than the actual value. This adverse affect arises from the face that the erythrocytes influence, through adsorption once the reaceive layor of the hinsensor. che diffusion of glucose into the latter and to the electrode and reduce the measured signal. To solve this problem, various membranes which are put on top of the enzyme layer, which is disposed on the electrodes, of the test strip in order to keep the erythrocytes away from this layer have been proposed. Thus, for example, US patent 5.658,444 describes an erythrocyre exclusion membrane for a sensor, which consists of a water-insoluble, hydrophobic polymer, of a water-soluble hydrophilic polymer and of an erythrocyte aggregating agent and is produced by spraying onto the surface of the test strip. One disadvantage of this membrane is that the membrane pore diameter varies as a function of the spraying distance and spray pressure. In addition, the spraying on of the membrane during production of the test strip means an additional operation which is different from the production of the remainder of the test strip and is therefore elaborate, which makes the production process complicated and thus costly. It is therefore an object of the present invention to provide a paste for producing a porous membrane which does not have the disadvantages mentioned since it can be applied during the biosensor production process by a method which first in with the remaining procedure and is therefore cost-effective, and provides a membrane of constant pore size. This object is achieved by a paste for a porous polymer membrane as claimed in claim 1. Advantageous developments are evident from claims 2 to 18. The invention is explained below by means of the figures, where Figure l shows diagrammatically che structure of a test scrip wach che membrane of che inversion, Figure 2 shows the rheological charactersatics of the paste of the invention. Figure 3a shows an electron micrograph of a. polymer membrane with inadequately developed pore structure, Figure 3b shows an electron micrograph of the polymer mambrane of che invention wich well developed pore structure, Figure 4 shows the results of measurement wich two biosensors, one of chem being provided with a membrane of the invention, comparing as the values of the hemacocrit increase, Figures 5a to 5d show the clinical performance on comparison of four blood glucose sensors. Figure 1 depicts the structure of a test strip with the polymer membrane of the., invention. An electrode arrangement 2 in the form of a carbon layer, which in turn is partly covered by an insulation 3, is located on a polyester support material 1. An enzyme and mediator layer 4 is disposed on che region of the electrode layer which is left free by che insulation. In the case of a blood glucose sensor, this layer comprises, for example, the enzyme glucose oxidase and che mediator Fe3T. The polymer membrane 5 of che invention is arranged above the enzyme and mediacor layer 4. The whole is covered by an adhesive layer 6 and a cover sheet 7. In che mass production of biosensors, the screen princing method is used For printing the various layers such as electrode, insulating and enzyme layers. The present invention provides a membrane which can be applied with the same technique. On the one hand, this has the advantage that the same screen printing device can be used for printing the membrane and thus throughout the sensor production process, which involves enormous economic advantages m mass produc- tion. On the other hand, it is possible to produce by the screen printing method reproducibly a membrane of uniform thickness and pore size, which is not ensured with the other methods such as spmcoating, dipping or spraying. For it to be possible to apply the paste used to produce the polymer membrane by screen printing, the solvent(s) present therein for the polymer must have a boiling point which is as high as possible (above 1OO°C) in order to avoid premature drying of the material in the printing machine. in. addition, the paste comprises a nonsolvent for the polymer, which acts as pore former and has a higher boiling point than the solvent(s) used. The paste must moreover have a suitable viscosity (30 000-50 000 cpi) in order to ensure uniform flow through the screen during the printing. The viscosity of the paste is preferably reduced on exposure to shear forces, as depicted in the rheological characteristics in Figure 2. The polymer preferably used in the paste of the inven- tion is cellulose acetate (50 kDa). It is preferably present in a proportion of about 8% by weight in the screen-printable paste. In addition, a farther polymer which may be present is cellulose nitrate in a proportion of up to 10% by weight. Solvents which can be used for the polymer are, for example. 1,4-dioxane (boiling point 102°C) and/or 4-hydroxymethylpentanone (boiling point 165-0C) . A. preferred composition comprises 0-20% by weight, more preferably 20% by weight, of 1,4-dioxar.e and 0-'70% by weight, more preferably 56% by weight, of 4-hydroxy- methylpentanone, it being possible alternatively to replace the 4-hydroxymethylpentanone by ethyl acetate or ethylene glycol diacetate. It has emerged that long-chain alcohols with a boiling pome of > 150aC are suitable as pore formers for the screen-printable membrane paste; preference is given to n-octanol. which has a boiling point of 196°C, and/or 2-methyl-2,4-pentanediol (MPD), which has a boiling point of 1970C. The paste is somewhat more tolerant to evaporation of dioxane or. use of 2-netyl-2,4-pentanediol (MPD) as pore former. Moreover the cellulose acetate remains in solution longer, which extends the time during -which the paste remains in a printable state. This extended "pot life" makes it possible to produce larger batches of constant quality. The pore former should be present in a proportion of 5-20% by weight, preferably 12-15% by weight. The viscosity modifiers used are, for example, hydrophilic silica xerogels or equivalent "fumed silicas", bentonite, clay, Natrosol or carbon black. They should be added in a proportion of from 1 to 10% by weight to the screen-printable paste. Preference is given to hydrophilic Cab-C-Sils (proprietary name for silica xerogels marketed by the Cabot organization). such as Cab-O-Sil M5, Cab-O-Sil H5, Cab-O-Sil LM50, Cab-O-Sil LM130, in a proportion of 4% by weight. It is also possible to add further additives such as Tween 20, Triton X, Silvet 7600 or 7280. lauryl sulfate (SDS), other detergents, and polyols such as glycerol. or hydrophilic polymers such as polyvinylpyrolidone (PVP) or vinylpyrolidone/vinyl acetate copolymers (Pvp/VA) co the paste of the invention. Addition of one or more of these additives is not obligatory for producing the membrane; however it has emerged chat chey may improve the wetting of the membrane and speed up the sensor response. Preference is given to the use of PVP/VA or PVP in a proportion of 0.1% by weight in the screen-printable paste. Moreover the addition of the additives Bioterge, poly- ethyl eneimine, BSA, dextran, dicyclohexyl phthalate, gelatin, sucrose and/or biuret may improve the separa- tion of erythrocytee and plasma. It is additionally possible to add enzyme, for example glucose oxidase, even to the cellulose acetate paste so that printing of the enzyme layer can be dispensed with in the biosensor production process. After application of a uniform layer of the printing paste to a suitable substrate, the membrane is formed during the drying process. There is formation of a porous layer and not of a continuous film, because the solvents used have a lower boiling point than the pore former; the solvents evaporate correspondingly quickly and the cellulose acetate polymer precipitates in the remaining film of the pore former. However, in connection with a biosensor, it is not permissible to use just a high temperature in the drying process, because the enzymes/proteins used are denatured if the temperatures are too high. The best results were achieved in connection with a biosensor for determining glucose in whole blood with a drying temperature of about 70°C. The boiling points of the solvents and pore formers used should be selected correspondingly. A crucial factor for the pore formation is che viscosity modifier used, which forms a gel together with the pore former in order to stabilize the polymer structure. With the substances used, the gel is produced through che interaction between the OK groups of the silica xerogel and the long-chain alcohol (e.g. octanol) . The amount and the distribution of the gel produced during the drying process eventually deter- mines the size and shape of the pores which develop. Without addition of a viscosity modifier there is formation of an emulsion from the solvent and the pore former, because the pore former is unable on its own to stabilize the polymer skeleton. The result is a white, smooth and unstructured film with entrapped pore former, which does not allow lateral liquid transport. By comparison, a clear film is obtained when no pore former is used in the paste. If the amounts of viscosity modifier used are too small ( inadequately developed pore structure, as shown in Figure 3a. Sir.ce the various suitable viscosity modifiers have different surface properties, the viscosity modifier can be selected depending on the required membrane or the required biosensor. For example, with high mechanical stress, e.g. with long printing times or on printing of very thin layers with a high squeegee pressure, the Cab-O-Sil H5 is "crushed". The surface then shows microscopically sharp fracture edges which may lead to lysis of che red blood cells. This is an unwanted property for a blood glucose sensor because the basic current of the sensor is increased thereby. On che other hand, this effect can be opti- mized, and the plasma from cells be utilized directly in the sensor for the electrochemical detection. One practical example would be the examination of hemoglobin in erychrocytes. In this case, che mediator of the biosensor, e.g. potassium hexacyancferrate(III) , reacts with the Fe(II) group of the hemoglobin, producing potassium hexacyanoferrate(II) which can be determined directly at the electrode of the biosensor. An enzyme like chat in the case of glucose determination is unnecessary in this case because the mediator reacts directly with the hemoglobin. It is possible in this way in practice to determine the value of che hematocrit for a patient with similar measuring equipment as in the monitoring of blood glucose, making the time-consuming use of capillary cubes and centrifuge unnecessary. Cab-O-Sil LM 150 consists of smaller particles than H5, which are therefore more stable and are not damaged by the mechanical stress during the printing process. This viscosity modifier is therefore most suitable for producing a membrane for blood glucose sensors. In accordance with the above statement, the difference in boiling points between solvent and pore former is, besides the stabilization of the polymer skeleton by the viscosity modifier, important for the formation of a suitable membrane. The difference should be about 30°C in this case, so chat there is formation in the drying process of a film which comprises a sufficiently high concentration of pore former in which the membrane polymer can precipicace. If the boiling point differen- ces are smaller the pore former starts to evaporate before a critical ratio between solvent and pore former is reached, which brings about the precipitation of the membrane polymer. After the screen-printable paste with che composition described previously has been printed, and the solvent has evaporated, there is formation through deposition of che cellulose esters of a membrane with an average pore size of from o.l to 2 µm, 1c being possible to influence the pore size by the amount of long-chain alcohol used. An electron micrograph of the membrane is shown in Figure 3b. Since erythrocytes have an average size of 8 to 10 µm, the membrane keeps them away from the enzyme layer, while the plasma can pass through unhindered. In addition, the membrane contributes no the mechanical stability of the enzyme layer and prevents the enzyme being detached from the electrode on application of the blood sample and then no longer being available for the electrochemical reaction. Figure 4 illustrates by means of a series of measure- ments the fact that at a constant: glucose concentration the test strip provided with a membrane of the inven- tion provides, in contrast to a test strip without membrane, constant results as the values of the hema- tocrit increase, whereas the response with the test strip without membrane decreases as the erythrccyte concentration increases. Because of the increased dif- fusion barrier between the enzyme layer and the blood sample, the response overall is somewhat reduced in the case of the sensor with membrane. The invention is illustrated by means of the following examples. Production of the printing paste: In accordance with the ratios of amounts indicated in the following examples, a mixture of the solvent (e.g. hydroxymethylpenanone, dioxane) and the pore former (e.g. octanol. MPD) is produced to ensure uniform distribution of the twc substances. In the next step, ail the additives (e.g. pvp/VA) are added and dissolved, if necessary with the aid of ultrasound. The membrane polymer (cellulose actate 50 KDa) is then mixed rapidly with the -previously produced solvent until a uniform suspension results. This suspension is rolled for 48 h in a closed container until a clear gel results, and ic is possible to add the viscosity modifier (e.g. Cab-O-Sil) to this. The finished printing paste is rolled for a further 24 h m order to ensure uniform distribution of the viscosity modifier. Example 1 Polymer(s): Cellulose acetate (Mw 30 000) 7.5% by weight Solvent: Ethylene glycol diacetate (b.p. 136°C} 65.5% by weight Pore former: n-Decanol (b.p. 23l°C) 25.0% by weight Viscosity modifier: Cab-O-Sil M5 2.0% by weight Example 2 Polymer(a): Cellulose acetate (Mw 50 000) 8.0% by weight Solvents: 1,4-Dioxane (b.p. 102°C) 35.0% by weight Ethyl acetate (b.p. 154CC) 35.0% by weight Pore former: n-Octanol (b.p. 196°C) 18.0% by weight Viscosity modifier: Cab-O-Sil M5 4.0% by weight, Example 3 Polymer(s): Cellulose acetate (Mw 50 000) 8.0% by weight. Solvents: 1,4-Dioxane ib.p. 102°C) 20.0% by weight 4-Kydroxymethylpencanone (b.p. 165°C) 56.0% by weight pore former: n-Octanol (b.p. 196°C) 12.0% by weight Viscos8ity modifier: Cab-O-Sil M5 4.0% by weight Additives: PVP/VA 0.1% by weight Example 4 Polymer(s): Cellulose acetate (Mw 50 000) 7.4% by weight Solvents: 1,4-Dioxane (b.p. 102°C) 18.5% by weight 4-Hydroxymechylpenranone (b.p. 165°C) 55.6% by weight Pore former: 2-Methyl-2,4-pentanediol 14.8% by weight Viscosity modifier: Cab-O-Sil M5 3.7% by weight Additives: PVP/VA 0.1% by weight Figure 5 shows the clinical performance of blood glucose sensors a) without polymer membrane b) with polymer membrane {composition of Example 2) c) wich polymer membrane (composition of Example 3) d) wich polymer membrane (composition of Example 4). In the comparative clinical investigations, the results of measurement with the various types of sensors were compared with the results of measurement by che reference method (YSI Model 23OC Stat Plus), and the percentage deviation was plotted against the values of che hemacocrit for the individual blood samples. The result in the ideal case is a measurement line hori- zontal to the x axis. The gradient of these measurement lines, which is shown in Table 1, provides information about che interference of che hemacocrit with the sensor system used. Table 1 The data unambiguously reveal the superior performance of the sensor system with the preferred membrane (com- position of Example 4). This improvement is achieved through the separation of whole blood and plasma directly in front of the electrode, because the Nernsc diffusion layer in front of the electrode can no longer be extended into the region with erythrocyces and therefore also can no longer be influenced by different values of che hematocric. The following comparative examples describe printing pastes in which there is no suitable accordance between the pore former, the solvents and the viscosity- modifier. Comparative Example 1 Polymer(3): Cellulose acetate (Mw 50 000) 8.0% by weight Solvent: Ethylene glycol diacetate (b.p. 186°C) 76.0% by weight Pore former: n-Octanol (b.p. 196°C) 12.0% by weight Viscosity modifier: Cab-O-Sil M5 (hydrophilic) 4.0% by weight Additives.- PVP/VA 0.1% by weight Comparative Exampl* 2 Polymer(s) .- Cellulose acetate (Mw 50 000)' 8.0% by weight Solvents: 1,4-Dioxane (b.p. 102°C) 20.0% by weight 4-Hydroxymethylpentanone (b.p. 165°C) 56.0% by weight Pore former; n-Octanol (b.p. X96°C) 12.0% by weight Viscosity modifier: Cab-O-Sil TS720 (hydrophobic) 4-0% by weight Additives: PVP/VA 0.1% by weight Comparative Example 3 Polymer(s) : Cellulose acetate propionate (Mw 75 Q00) 8.0% by weight Solvents: 1,4-Dioxane (b.p. 102°C) 20.0% by weight 4-Hydroxymethylpentanone (b.p. 165°C) 56.0% by weight Pore former: n-Octanol (b.p. 1S6°C) 12.0% by weight Viscosity modifier: Cab-O-Sil M5 (hydrophilic) 4.0% by weight Additives.- PVP/VA 0.1% by weight In Comparative Example l there is no formation of a porous membrane because the difference between the boiling points of the solvent (ethylene glycol diacetate) and pore former (n-octanol) used in che printing paste is too small. If, by contrast, n-decanol is used as pore former (as described in Example l) . a porous membrane is obtained after the drying process because the boiling point between the solvent and the pore former is sufficiently large. In Comparative Example 2 there is only inadequate gel formation between the pore former and the viscosity modifier, because of the use of hydrophobic Cab-O-Sil which is unable to react with the CH groups of the pore former, and thus there is inadequate stabilization of the polymer skeleton. This impedes the formation of a porous membrane. No porous membrane is formed in Comparative Example 3 either, where the solubility of the polymer' used (cellulose acetate propionate) in che pore former is coo high. • WE CLAIM: . i. A screen-printable paste for producing a 'porous polymer membrane, comprising at least one polymer, one or more solvents for the polymer with a boiling point of > 100°C, one or more nonsolvents (pore formers) for the polymer with a higher boiling point than the solvent(s) and a hydro- philic viscosity modifier. 2. A screen-printable paste as claimed in claim I, wherein the difference of the boil- ing points of solvent and pore former is at least 30°C. 3. A screen-print able paste as claimed in claim 1 or 2, wherein the paste comprises cellulose acetate as polymer. 4. A screen-printable paste as claimed m claim 3, wherein. . wherein the paste comprises 1.4-di- oxane and/or' 4-hydroxymethylpentanone and/or ethyl acetate as solvent. 5. A screen-printable paste as claimed in at least one of claims 1 to 4, wherein the paste comprises a long-chain alcohol as pore former. 6. A screen-printable paste as claimed in claim 5, wherein the paste comprises n-octanol and/or 2-methyl-2,4-pentanediol as pore former. 7. A screen-printable paste as claimed in claim 6, wherein n-octanol and/or 2-methyl- 2.4-pantanediol is present in a proportion of 5-20% by weight. 8. A screen-prlntable paste as claimed In at least one of the preceding claims, wherein the paste comprises hydrophillc silica xerogei as viscosity modifier. 9. A screen-printable paste as claimed in claim 8, wherein the silica xerogel Is present In a proportion of 1-10% by weight 10. A screen-printable paste as claimed in at least one of the preceding claims, wherein the paste additionally comprises vinylpyrolidone/vlnyl acetate copolymer (PVP/VA) and/or polyvinyl- pyrolidone (PVP). 11. A screen-printable paste as claimed in claim 10, wherein the PVP/VA Is present In a proportion of 0.1% by weight. 12. A screen-printable paste as claimed in at least one of the preceding claims, wherein the paste additionally comprises one or more enzymes. 13.A method for producing a screen-printable paste by producing a mixture of one or more solvents) for a polymer and one or more non-solvent(s) for a polymer (pore former), wherein said solvents) have a boiling point of > 100°C and said non-soivent(8) have a higher boiling point than said solvents); mixing in the polymer until a uniform suspension results; rolling the suspension until a clear gel results; adding a hydrophlllc viscosity modifier, and rolling the mixture until the viscosity modifier is uniformly distributed. 14. A porous polymer membrane produced from the screen-printabie paste as claimed in at least one of claims 1 to 12. 15. The membrane as claimed In claim 14, wherein said polymer membrane is introduced into a biosensor test strip. 16.The membrane as claimed in claim 15, wherein the biosensor is designed for measuring the blood glucose concentration. 17. The membrane as claimed In claim 15, wherein the biosensor Is designed for determining the value of the hematocrit. The invention relates to a paste, which can undergo screen printing, for producing a porous polymer membrane. Said paste contains at least one polymer, one or more solvents for the polymer having a boiling point > 100°C, one or more non-solvents for the polymers (pore-forming agents) having a higher boiling point than that of the solvent (s), and contains a hydrophllic viscosity modifier.

Full Text

The present invention relaces to a screen-printable
pasce for producing a porous polymer membrane which can
be used in electrochemical sensors, especially in
electrochemical biosensors, for integrated preparation
of, m particular, whole blood samples.
Biosensors are already in use in a large number of
diagnostic methods, for example in the determination of
the concentration of various factors in body fluids
such as blood. The aim in this connection is to have
sensors which require no elaborate processing of the
(blood) sample but provide a rapid result simply by
applying the body fluid to a test strip. This entails a
specific biochemical reaction taking place, such as,
for example, the enzymatic conversion of the component
to be determined, which then brings about an electron
transfer between different electrodes (working and
reference electrodes), and this can be determined
quantitatively.
The disadvantage of most of the known electrochemical
biosensors is that, on application of the blood to the
region provided therefor on the test strip. the
biochemical reaction which takes place is influenced by
other constituents-present in the blood, especially the
red blood corpuscles (erythrocytes). Thus, for example,
when the value3 of the hematocrit (= volume of the
erythrocytes as a proportion of the total amount of
blood in vol.wt.%) are high, the value measured for
glucose using conventional blood glucose sensors is
lower than the actual value. This adverse affect arises
from the face that the erythrocytes influence, through
adsorption once the reaceive layor of the hinsensor.
che diffusion of glucose into the latter and to the
electrode and reduce the measured signal.
To solve this problem, various membranes which are put
on top of the enzyme layer, which is disposed on the
electrodes, of the test strip in order to keep the
erythrocytes away from this layer have been proposed.
Thus, for example, US patent 5.658,444 describes an
erythrocyre exclusion membrane for a sensor, which
consists of a water-insoluble, hydrophobic polymer, of
a water-soluble hydrophilic polymer and of an
erythrocyte aggregating agent and is produced by
spraying onto the surface of the test strip.
One disadvantage of this membrane is that the membrane
pore diameter varies as a function of the spraying
distance and spray pressure. In addition, the spraying
on of the membrane during production of the test strip
means an additional operation which is different from
the production of the remainder of the test strip and
is therefore elaborate, which makes the production
process complicated and thus costly.
It is therefore an object of the present invention to
provide a paste for producing a porous membrane which
does not have the disadvantages mentioned since it can
be applied during the biosensor production process by a
method which first in with the remaining procedure and
is therefore cost-effective, and provides a membrane of
constant pore size.
This object is achieved by a paste for a porous polymer
membrane as claimed in claim 1. Advantageous
developments are evident from claims 2 to 18.
The invention is explained below by means of the
figures, where
Figure l shows diagrammatically che structure of a test
scrip wach che membrane of che inversion,
Figure 2 shows the rheological charactersatics of the
paste of the invention.
Figure 3a shows an electron micrograph of a. polymer
membrane with inadequately developed pore structure,
Figure 3b shows an electron micrograph of the polymer
mambrane of che invention wich well developed pore
structure,
Figure 4 shows the results of measurement wich two
biosensors, one of chem being provided with a membrane
of the invention, comparing as the values of the
hemacocrit increase,
Figures 5a to 5d show the clinical performance on
comparison of four blood glucose sensors.
Figure 1 depicts the structure of a test strip with the
polymer membrane of the., invention. An electrode
arrangement 2 in the form of a carbon layer, which in
turn is partly covered by an insulation 3, is located
on a polyester support material 1. An enzyme and
mediator layer 4 is disposed on che region of the
electrode layer which is left free by che insulation.
In the case of a blood glucose sensor, this layer
comprises, for example, the enzyme glucose oxidase and
che mediator Fe3T. The polymer membrane 5 of che
invention is arranged above the enzyme and mediacor
layer 4. The whole is covered by an adhesive layer 6
and a cover sheet 7.
In che mass production of biosensors, the screen
princing method is used For printing the various layers
such as electrode, insulating and enzyme layers. The
present invention provides a membrane which can be
applied with the same technique. On the one hand, this
has the advantage that the same screen printing device
can be used for printing the membrane and thus
throughout the sensor production process, which
involves enormous economic advantages m mass produc-
tion. On the other hand, it is possible to produce by
the screen printing method reproducibly a membrane of
uniform thickness and pore size, which is not ensured
with the other methods such as spmcoating, dipping or
spraying.
For it to be possible to apply the paste used to
produce the polymer membrane by screen printing, the
solvent(s) present therein for the polymer must have a
boiling point which is as high as possible (above
1OO°C) in order to avoid premature drying of the
material in the printing machine. in. addition, the
paste comprises a nonsolvent for the polymer, which
acts as pore former and has a higher boiling point than
the solvent(s) used.
The paste must moreover have a suitable viscosity
(30 000-50 000 cpi) in order to ensure uniform flow
through the screen during the printing. The viscosity
of the paste is preferably reduced on exposure to shear
forces, as depicted in the rheological characteristics
in Figure 2.
The polymer preferably used in the paste of the inven-
tion is cellulose acetate (50 kDa). It is preferably
present in a proportion of about 8% by weight in the
screen-printable paste. In addition, a farther polymer
which may be present is cellulose nitrate in a
proportion of up to 10% by weight.
Solvents which can be used for the polymer are, for
example. 1,4-dioxane (boiling point 102°C) and/or
4-hydroxymethylpentanone (boiling point 165-0C) . A.
preferred composition comprises 0-20% by weight, more
preferably 20% by weight, of 1,4-dioxar.e and 0-'70% by
weight, more preferably 56% by weight, of 4-hydroxy-
methylpentanone, it being possible alternatively to
replace the 4-hydroxymethylpentanone by ethyl acetate
or ethylene glycol diacetate.
It has emerged that long-chain alcohols with a boiling
pome of > 150aC are suitable as pore formers for the
screen-printable membrane paste; preference is given to
n-octanol. which has a boiling point of 196°C, and/or
2-methyl-2,4-pentanediol (MPD), which has a boiling
point of 1970C.
The paste is somewhat more tolerant to evaporation of
dioxane or. use of 2-netyl-2,4-pentanediol (MPD) as pore
former. Moreover the cellulose acetate remains in
solution longer, which extends the time during -which
the paste remains in a printable state. This extended
"pot life" makes it possible to produce larger batches
of constant quality.
The pore former should be present in a proportion of
5-20% by weight, preferably 12-15% by weight.
The viscosity modifiers used are, for example,
hydrophilic silica xerogels or equivalent "fumed
silicas", bentonite, clay, Natrosol or carbon black.
They should be added in a proportion of from 1 to 10%
by weight to the screen-printable paste. Preference is
given to hydrophilic Cab-C-Sils (proprietary name for
silica xerogels marketed by the Cabot organization).
such as Cab-O-Sil M5, Cab-O-Sil H5, Cab-O-Sil LM50,
Cab-O-Sil LM130, in a proportion of 4% by weight.
It is also possible to add further additives such as
Tween 20, Triton X, Silvet 7600 or 7280. lauryl sulfate
(SDS), other detergents, and polyols such as glycerol.
or hydrophilic polymers such as polyvinylpyrolidone
(PVP) or vinylpyrolidone/vinyl acetate copolymers
(Pvp/VA) co the paste of the invention.
Addition of one or more of these additives is not
obligatory for producing the membrane; however it has
emerged chat chey may improve the wetting of the
membrane and speed up the sensor response. Preference
is given to the use of PVP/VA or PVP in a proportion of
0.1% by weight in the screen-printable paste.
Moreover the addition of the additives Bioterge, poly-
ethyl eneimine, BSA, dextran, dicyclohexyl phthalate,
gelatin, sucrose and/or biuret may improve the separa-
tion of erythrocytee and plasma.
It is additionally possible to add enzyme, for example
glucose oxidase, even to the cellulose acetate paste so
that printing of the enzyme layer can be dispensed with
in the biosensor production process.
After application of a uniform layer of the printing
paste to a suitable substrate, the membrane is formed
during the drying process. There is formation of a
porous layer and not of a continuous film, because the
solvents used have a lower boiling point than the pore
former; the solvents evaporate correspondingly quickly
and the cellulose acetate polymer precipitates in the
remaining film of the pore former.
However, in connection with a biosensor, it is not
permissible to use just a high temperature in the
drying process, because the enzymes/proteins used are
denatured if the temperatures are too high. The best
results were achieved in connection with a biosensor
for determining glucose in whole blood with a drying
temperature of about 70°C. The boiling points of the
solvents and pore formers used should be selected
correspondingly.
A crucial factor for the pore formation is che
viscosity modifier used, which forms a gel together
with the pore former in order to stabilize the polymer
structure. With the substances used, the gel is
produced through che interaction between the OK groups
of the silica xerogel and the long-chain alcohol (e.g.
octanol) . The amount and the distribution of the gel
produced during the drying process eventually deter-
mines the size and shape of the pores which develop.
Without addition of a viscosity modifier there is
formation of an emulsion from the solvent and the pore
former, because the pore former is unable on its own to
stabilize the polymer skeleton. The result is a white,
smooth and unstructured film with entrapped pore
former, which does not allow lateral liquid transport.
By comparison, a clear film is obtained when no pore
former is used in the paste.
If the amounts of viscosity modifier used are too small
( inadequately developed pore structure, as shown in
Figure 3a.
Sir.ce the various suitable viscosity modifiers have
different surface properties, the viscosity modifier
can be selected depending on the required membrane or
the required biosensor. For example, with high
mechanical stress, e.g. with long printing times or on
printing of very thin layers with a high squeegee
pressure, the Cab-O-Sil H5 is "crushed". The surface
then shows microscopically sharp fracture edges which
may lead to lysis of che red blood cells.
This is an unwanted property for a blood glucose sensor
because the basic current of the sensor is increased
thereby. On che other hand, this effect can be opti-
mized, and the plasma from cells be utilized directly
in the sensor for the electrochemical detection. One
practical example would be the examination of
hemoglobin in erychrocytes. In this case, che mediator
of the biosensor, e.g. potassium hexacyancferrate(III) ,
reacts with the Fe(II) group of the hemoglobin,
producing potassium hexacyanoferrate(II) which can be
determined directly at the electrode of the biosensor.
An enzyme like chat in the case of glucose
determination is unnecessary in this case because the
mediator reacts directly with the hemoglobin. It is
possible in this way in practice to determine the value
of che hematocrit for a patient with similar measuring
equipment as in the monitoring of blood glucose, making
the time-consuming use of capillary cubes and
centrifuge unnecessary.
Cab-O-Sil LM 150 consists of smaller particles than H5,
which are therefore more stable and are not damaged by
the mechanical stress during the printing process. This
viscosity modifier is therefore most suitable for
producing a membrane for blood glucose sensors.
In accordance with the above statement, the difference
in boiling points between solvent and pore former is,
besides the stabilization of the polymer skeleton by
the viscosity modifier, important for the formation of
a suitable membrane. The difference should be about
30°C in this case, so chat there is formation in the
drying process of a film which comprises a sufficiently
high concentration of pore former in which the membrane
polymer can precipicace. If the boiling point differen-
ces are smaller the pore former starts to evaporate
before a critical ratio between solvent and pore former
is reached, which brings about the precipitation of the
membrane polymer.
After the screen-printable paste with che composition
described previously has been printed, and the solvent
has evaporated, there is formation through deposition
of che cellulose esters of a membrane with an average
pore size of from o.l to 2 µm, 1c being possible to
influence the pore size by the amount of long-chain
alcohol used. An electron micrograph of the membrane is
shown in Figure 3b. Since erythrocytes have an average
size of 8 to 10 µm, the membrane keeps them away from
the enzyme layer, while the plasma can pass through
unhindered. In addition, the membrane contributes no
the mechanical stability of the enzyme layer and
prevents the enzyme being detached from the electrode
on application of the blood sample and then no longer
being available for the electrochemical reaction.
Figure 4 illustrates by means of a series of measure-
ments the fact that at a constant: glucose concentration
the test strip provided with a membrane of the inven-
tion provides, in contrast to a test strip without
membrane, constant results as the values of the hema-
tocrit increase, whereas the response with the test
strip without membrane decreases as the erythrccyte
concentration increases. Because of the increased dif-
fusion barrier between the enzyme layer and the blood
sample, the response overall is somewhat reduced in the
case of the sensor with membrane.
The invention is illustrated by means of the following
examples.
Production of the printing paste:
In accordance with the ratios of amounts indicated in
the following examples, a mixture of the solvent (e.g.
hydroxymethylpenanone, dioxane) and the pore former
(e.g. octanol. MPD) is produced to ensure uniform
distribution of the twc substances. In the next step,
ail the additives (e.g. pvp/VA) are added and
dissolved, if necessary with the aid of ultrasound. The
membrane polymer (cellulose actate 50 KDa) is then
mixed rapidly with the -previously produced solvent
until a uniform suspension results. This suspension is
rolled for 48 h in a closed container until a clear gel
results, and ic is possible to add the viscosity
modifier (e.g. Cab-O-Sil) to this. The finished
printing paste is rolled for a further 24 h m order to
ensure uniform distribution of the viscosity modifier.
Example 1
Polymer(s):
Cellulose acetate (Mw 30 000) 7.5% by weight
Solvent:
Ethylene glycol diacetate (b.p. 136°C} 65.5% by weight
Pore former:
n-Decanol (b.p. 23l°C) 25.0% by weight
Viscosity modifier:
Cab-O-Sil M5 2.0% by weight
Example 2
Polymer(a):
Cellulose acetate (Mw 50 000) 8.0% by weight
Solvents:
1,4-Dioxane (b.p. 102°C) 35.0% by weight
Ethyl acetate (b.p. 154CC) 35.0% by weight
Pore former:
n-Octanol (b.p. 196°C) 18.0% by weight
Viscosity modifier:
Cab-O-Sil M5 4.0% by weight,
Example 3
Polymer(s):
Cellulose acetate (Mw 50 000) 8.0% by weight.
Solvents:
1,4-Dioxane ib.p. 102°C) 20.0% by weight
4-Kydroxymethylpencanone (b.p. 165°C) 56.0% by weight
pore former:
n-Octanol (b.p. 196°C) 12.0% by weight
Viscos8ity modifier:
Cab-O-Sil M5 4.0% by weight
Additives:
PVP/VA 0.1% by weight
Example 4
Polymer(s):
Cellulose acetate (Mw 50 000) 7.4% by weight
Solvents:
1,4-Dioxane (b.p. 102°C) 18.5% by weight
4-Hydroxymechylpenranone (b.p. 165°C) 55.6% by weight
Pore former:
2-Methyl-2,4-pentanediol 14.8% by weight
Viscosity modifier:
Cab-O-Sil M5 3.7% by weight
Additives:
PVP/VA 0.1% by weight
Figure 5 shows the clinical performance of blood
glucose sensors
a) without polymer membrane
b) with polymer membrane {composition of Example 2)
c) wich polymer membrane (composition of Example 3)
d) wich polymer membrane (composition of Example 4).
In the comparative clinical investigations, the results
of measurement with the various types of sensors were
compared with the results of measurement by che
reference method (YSI Model 23OC Stat Plus), and the
percentage deviation was plotted against the values of
che hemacocrit for the individual blood samples. The
result in the ideal case is a measurement line hori-
zontal to the x axis. The gradient of these measurement
lines, which is shown in Table 1, provides information
about che interference of che hemacocrit with the
sensor system used.
Table 1
The data unambiguously reveal the superior performance
of the sensor system with the preferred membrane (com-
position of Example 4). This improvement is achieved
through the separation of whole blood and plasma
directly in front of the electrode, because the Nernsc
diffusion layer in front of the electrode can no longer
be extended into the region with erythrocyces and
therefore also can no longer be influenced by different
values of che hematocric.
The following comparative examples describe printing
pastes in which there is no suitable accordance between
the pore former, the solvents and the viscosity-
modifier.
Comparative Example 1
Polymer(3):
Cellulose acetate (Mw 50 000) 8.0% by weight
Solvent:
Ethylene glycol diacetate (b.p. 186°C) 76.0% by weight
Pore former:
n-Octanol (b.p. 196°C) 12.0% by weight
Viscosity modifier:
Cab-O-Sil M5 (hydrophilic) 4.0% by weight
Additives.-
PVP/VA 0.1% by weight
Comparative Exampl* 2
Polymer(s) .-
Cellulose acetate (Mw 50 000)' 8.0% by weight
Solvents:
1,4-Dioxane (b.p. 102°C) 20.0% by weight
4-Hydroxymethylpentanone (b.p. 165°C) 56.0% by weight
Pore former;
n-Octanol (b.p. X96°C) 12.0% by weight
Viscosity modifier:
Cab-O-Sil TS720 (hydrophobic) 4-0% by weight
Additives:
PVP/VA 0.1% by weight
Comparative Example 3
Polymer(s) :
Cellulose acetate propionate
(Mw 75 Q00) 8.0% by weight
Solvents:
1,4-Dioxane (b.p. 102°C) 20.0% by weight
4-Hydroxymethylpentanone (b.p. 165°C) 56.0% by weight
Pore former:
n-Octanol (b.p. 1S6°C) 12.0% by weight
Viscosity modifier:
Cab-O-Sil M5 (hydrophilic) 4.0% by weight
Additives.-
PVP/VA 0.1% by weight
In Comparative Example l there is no formation of a
porous membrane because the difference between the
boiling points of the solvent (ethylene glycol
diacetate) and pore former (n-octanol) used in che
printing paste is too small. If, by contrast, n-decanol
is used as pore former (as described in Example l) . a
porous membrane is obtained after the drying process
because the boiling point between the solvent and the
pore former is sufficiently large.
In Comparative Example 2 there is only inadequate gel
formation between the pore former and the viscosity
modifier, because of the use of hydrophobic Cab-O-Sil
which is unable to react with the CH groups of the pore
former, and thus there is inadequate stabilization of
the polymer skeleton. This impedes the formation of a
porous membrane.
No porous membrane is formed in Comparative Example 3
either, where the solubility of the polymer' used
(cellulose acetate propionate) in che pore former is
coo high.
• WE CLAIM: .
i. A screen-printable paste for producing a 'porous
polymer membrane, comprising at least one polymer,
one or more solvents for the polymer with a
boiling point of > 100°C, one or more nonsolvents
(pore formers) for the polymer with a higher
boiling point than the solvent(s) and a hydro-
philic viscosity modifier.
2. A screen-printable paste as claimed in claim I,
wherein the difference of the boil-
ing points of solvent and pore former is at least
30°C.
3. A screen-print able paste as claimed in claim 1 or
2, wherein the paste comprises
cellulose acetate as polymer.
4. A screen-printable paste as claimed m claim 3,
wherein. .
wherein the paste comprises 1.4-di-
oxane and/or' 4-hydroxymethylpentanone and/or ethyl
acetate as solvent.
5. A screen-printable paste as claimed in at least
one of claims 1 to 4, wherein the
paste comprises a long-chain alcohol as pore
former.
6. A screen-printable paste as claimed in claim 5,
wherein the paste comprises
n-octanol and/or 2-methyl-2,4-pentanediol as pore
former.
7. A screen-printable paste as claimed in claim 6,
wherein n-octanol and/or 2-methyl-
2.4-pantanediol is present in a proportion of
5-20% by weight.
8. A screen-prlntable paste as claimed In at least one of the
preceding claims, wherein the paste comprises hydrophillc silica
xerogei as viscosity modifier.
9. A screen-printable paste as claimed in claim 8, wherein the silica
xerogel Is present In a proportion of 1-10% by weight
10. A screen-printable paste as claimed in at least one of the
preceding claims, wherein the paste additionally comprises
vinylpyrolidone/vlnyl acetate copolymer (PVP/VA) and/or polyvinyl-
pyrolidone (PVP).
11. A screen-printable paste as claimed in claim 10, wherein the
PVP/VA Is present In a proportion of 0.1% by weight.
12. A screen-printable paste as claimed in at least one of the
preceding claims, wherein the paste additionally comprises one or
more enzymes.
13.A method for producing a screen-printable paste by
producing a mixture of one or more solvents) for a polymer and one
or more non-solvent(s) for a polymer (pore former), wherein said
solvents) have a boiling point of > 100°C and said non-soivent(8)
have a higher boiling point than said solvents);
mixing in the polymer until a uniform suspension results;
rolling the suspension until a clear gel results;
adding a hydrophlllc viscosity modifier, and
rolling the mixture until the viscosity modifier is uniformly distributed.
14. A porous polymer membrane produced from the screen-printabie
paste as claimed in at least one of claims 1 to 12.
15. The membrane as claimed In claim 14, wherein said polymer
membrane is introduced into a biosensor test strip.
16.The membrane as claimed in claim 15, wherein the biosensor is
designed for measuring the blood glucose concentration.
17. The membrane as claimed In claim 15, wherein the biosensor Is
designed for determining the value of the hematocrit.
The invention relates to a paste, which can undergo screen printing,
for producing a porous polymer membrane. Said paste contains at
least one polymer, one or more solvents for the polymer having a
boiling point > 100°C, one or more non-solvents for the polymers
(pore-forming agents) having a higher boiling point than that of the
solvent (s), and contains a hydrophllic viscosity modifier.

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Patent Number

222754

Indian Patent Application Number

IN/PCT/2002/00798/KOL

PG Journal Number

34/2008

Publication Date

22-Aug-2008

Grant Date

21-Aug-2008

Date of Filing

13-Jun-2002

Name of Patentee

LIFESCAN SCOTLAND LIMITED

Applicant Address

COLLINS HOUSEE,RUTLAND SQUAE, EDINBURGH EH 12AA GREAT BRITAIN

Inventors:

#	Inventor's Name	Inventor's Address
1	STAINE,MATTHIAS	66 CROWN DRIVE, INVERNESS IV2 3QG GREAT BRITAIN
2	VON TIEDEMANN,BIRGIT	4 B ARGYLE TERRACE INVERNESS IV2 3HN GREAT BRITAIN
3	RODERS, JAMIE	30 STRATHERRICK RD. LOCHARDIL, INVERNESS IV2 4LL
4	MACGREGOR,LUCY	92 MILLER STREET, INSHES PARK, INVERNESS IV2 3DL GREAT BRITAIN
5	MCALEER, JERRY	52 NOBELS CLOSE, GROVE (OXON) OX12 ONR, GREAT BRITAIN
6	MCNEILAGE, ALAN	31 FIRTH VIEW ROAD, INVERNESS IV3 8LZ GREAT BRITAIN

PCT International Classification Number

B01D 71/10

PCT International Application Number

PCT/EP01/12073

PCT International Filing date

2001-10-18

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA