Note: Descriptions are shown in the official language in which they were submitted.
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.1.
ORGANIC ION-SELECTIVE MEMBRANES
Background of the Invention
1. Field of the Invention
The present invention relates to ion-selective membranes and methods of their
preparation. Specifically it
relates to improved properties of polymer membranes that are formed without
the use of solvents.
2. Discussion of the Background
lon-selective membranes have uses in numerous applications, particularly in
biosensors and analytical
devices. Typically, such membranes are used to separate a test solution from a
reference solution, allowing
electrochemical measurements of the differences in ion concentration across
the membrane. Recent theoretical
advances have created prospects for a marked increase in the detection limits
of such devices. Chemical and
Engineering News, November 24, 1997, p. 13. However, presently available ion-
selective membranes impose
significant limitations on overall sensitivity. These membranes also have
other characteristics that seriously limit their
long-term stability in aqueous solutions.
A major use of ion-selective membranes is in the field of disposable
biosensors. Optimally, membranes made
for these devices would have a long shelf life, low detection limits, and
could be manufactured rapidly and efficiently in
large scale. However, because of the means by which conventional ion-selective
membranes are made, such
membranes have inherent limitations on shelf life, detection limits, and
efficiency of high volume manufacture.
The stability problems of available ion-selective membranes are due in large
measure to the tendency of such
membranes to swell in the presence of water. Swelling not only distorts the
structure of the membrane, but it also
changes membrane permeability, and it can cause components of the membrane to
leach out, further altering
membrane permeability and distorting the concentrations of ions or other
chemical entities in the area near the
membrane. This type of distortion affects both the detection limits and the
accuracy of electrochemical
measurements.
Physically or chemically unstable membranes further limit the potential for
development of implantable
biosensors. The use of implantable devices to detect physiological states,
such as, for example, blood glucose levels,
is not feasible if the membranes in such devices are subject to significant
distortion or degradation upon contact with
aqueous solutions. Accordingly, there is a great need for ion-selective
membranes that have long-term stability in
contact with aqueous solutions.
Ion-selective membranes of the prior art are manufactured by dissolving the
required components in a
common solvent and then casting the mixture into a suitable tool to form it
into the shape utilized in the electrode.
There are many general problems associated with this technology.
For example, solvent evaporation must occur under very tightly controlled
conditions. If the speed of
evaporation is too fast, a crust builds up and bubbles form beneath the crust.
The bubbles thus formed tend to isolate
the surface area of the membrane from the potential that generates the
transmitting function across the membrane.
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This, of course, causes diminished sensitivity of the membrane and in extreme
cases can result in membranes that are
entirely useless for electrode devices.
Another disadvantage is that the casting process must be repeated many times
to produce a reasonable
thickness of membrane that can be handled with tools. This repeated process
can increase the accumulation of
bubbles with each repeated step, thus disabling an increasingly larger area of
the membrane.
In many cases, finding a common solvent is difficult, if not impossible,
because not all of the membrane
components and additives that would be beneficial in combination are soluble
in the same solvent. This is particularly
relevant in automated production of precalibrated, disposable electrochemical
sensors, where components and
additives are required that are not typical for membranes used in laboratory
ion-selective sensors.
Given that the various desirable components in an ion-selective membrane are
not always soluble in a single
solvent, occasionally solvent mixtures are used to create the solution from
which the membranes are cast. However,
mixed solvents create additional problems in the evaporating step, especially
in the case of azeotropic mixtures having
high boiling points. Further, since the evaporation of solvents requires
energy, which is extracted from the vicinity of
the membrane, it can cause cooling of the membrane. This temperature reduction
can cause water vapor deposition,
1 S altering the membrane surface and inhibiting the adhesion of the next
layer of membrane material to the previously
deposited portion of the membrane. The traditional casting process also
requires precise measurement of the volume
of the solvent mixture and prevention of solvent evaporation from the casting
solution prior to the deposition of each
new layer, in order to avoid changes in viscosity and component concentrations
in the mixture.
Same modifications to the process of membrane manufacture have been developed
to address the probhams
associated with evaporation casting. In one such process, a glass ring is
adhered to a glass or metal plate. A
polymerlsolvent solution is then poured into the ring. The ring is covered by
a larger chamber and evaporation is
allowed to proceed at a relatively slow rate. After the solvent evaporates,
the membrane can be cut into discs with a
punch.
Since, in this procedure, the evaporation process is slower and more
controlled, it is not always necessary to
form the membrane in several layers. However, the advantage of one-step
casting of membranes in such a protocol is
diminished by the disadvantage of slow solvent evaporation, cumbersome setup,
and limits of the number of useful
membrane disks that can be recovered from each round of the process.
The membranes utilized in the automated production of sensors must be very
uniform within a production lot,
must also have a long shelf life, and must perform in a predictable manner.
Because of the numerous inherent
problems in forming electrode membranes by a solvent evaporation process,
manufacturers ate forced to produce
membranes in relatively small production lots. Making membranes in small lots
reduces the waste associated with a
failed process, but it also puts very restrictive limits on efficiency and
production capacity in the membrane formation
process.
Because of the great variability between different production lots in solvent-
based membrane formulations,
attempts at high volume production, such as in the manufacture of disposable
sensors, can be very inefficient,
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unproductive and costly. With al! of the above disadvantages, the casting of
solvent-based membranes for post-
manufacture integration into disposable biosensors is not very feasible.
As an alternative, the membranes may be formed in situ from a dissolved
membrane-forming mixture.
However, this requires expensive equipment capable of repeatedly dispensing
small amounts of high viscosity
solutions, subsequent protection against water deposition, controlled
evaporation of the solvent to minimize bubble
formation, long parking time in the equipment, and excessive delay in the
manufacturing process.
Indeed, the difficulties and inefficiencies of solvent-based ion-selective
membranes place severe limits on the
mass manufacture of disposable devices employing such membranes. This is the
case because of the variability of the
results, so that membranes formed for post-manufacture integration into
biosensor devices must be produced in small
lots to prevent waste of large lots of expensive materials. This is likewise
true because membranes formed in situ on
disposable medical devices create comparable inefficiencies in use of
equipment.
A problem, perhaps even more severe, with solvent-based ion-selective
membranes is the fact that such
membranes are inherently unstable when in contact with aqueous solutions. This
is because as the solvent leaves the
membrane structure by evaporation, it leaves behind pores, channels, and other
irregularities in the membrane
structure. These artifacts of solvent evaporation are readily attacked by
water, which causes rapid distortion of
membrane structure and electrochemical function.
Therefore, it has been necessary to develop elaborate means of preventing
water contact with prior art
membranes in electrode devices until the very moment that such devices are to
be used in a measurement. This is
because typical prior art membranes absorb significant amounts of water within
seconds at their first contact with an
aqueous solution. The absorption of water changes membrane geometry,
composition, and electrochemical function.
For example, as water moves through an electrode membrane, the situs of the
electrical potential is likewise in motion,
and additives and ions previously immobilized within the membrane can begin to
diffuse as well.
This phenomenon inhibits the formation of, and distorts the magnitude of, an
electrical potential across the
membrane, and can, within a short time, lead to a complete loss of potential
as the aqueous solution moves through
the membrane. This is because the infiltration of water into the membrane
causes the movement and diffusion of
previously immobilized active ingredients within the membrane, thus carrying
with them the ions responsible for
formation of the potential.
If these active ingredients move toward the outside of the membrane and
release ions at the membrane
surface, the released ions raise the local ion concentration outside the
membrane and interfere with the detection
limits of the electrode. If the active ingredients diffuse toward the
reference solution side of the membrane, the ions
previously immobilized within the membrane are depleted, and the ability to
establish a potential across the membrane
is reduced or destroyed. Because the electrochemical properties of the
membrane can change over the course of a
very few seconds. automated measurements of membrane potential must he done
extremely rapidly, and inherently
must involve approximations and extrapolations that are subject to significant
error.
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Accordingly, the disadvantages of solvent-based, ion-selective membranes
include both inefficiencies of
production and irregularities in performance. Such membranes have an
inherently short shelf life, particularly in the
presence of any form of humidity, and are thus not optimal for disposable
biosensors that may require long shelf life.
Of course such membranes are also wholly unsuitable for use in implantable
biosensor devices, both because of their
rapid physical and performance degradation in contact with aqueous solutions,
and because of the biocompatibility
problems commonly associated with implanted plastics. There is, therefore, a
great need for new approaches to
membrane manufacture and for improved ion-selective membranes having
significantly reduced rates of water
absorption, greatly simplified manufacturing requirements, and enhanced
biocompatibility. Disclosed herein are
membranes exhibiting highly desirable physical properties, as well as methods
of their manufacture that represent a
vast improvement over the methods disclosed in the prior art.
Summary of the Invention
The present invention provides a method of producing an ion-selective
membrane. In the method, membrane
components including a polymer and at least one additive are combined to form
a mixture without addition of a solvent.
A superior ion-selective membrane may be formed from this mixture. In this
aspect of the invention, the polymer may
be vinyl chloride. The polymer may also be polyvinyl chloride or it may be a
copolymer of vinyl chloride, such as, for
example vinyl acetate andlor vinyl alcohol. Also in this aspect of the
invention, the additive may be a plasticizer.
Suitable plasticizers may include, for example, one or more of the following:
aromatic ethers, aliphatic-aromatic ethers,
adipic acid esters, sebasic acid esters, phthalic acid esters, lauric acid
esters, glutaric acid esters, and phosphoric acid
esters. The additive may also be an ion-selective agent.
According to the method of the invention, the membrane components may include
more than one additive.
For example, the membrane components may include a plasticizer and an ion-
selective agent. The membrane
components further may include an additive such as, for example, plasticizer
modifiers, active ingredients, ion mobility
enhancers, heat stabilizers, light stabilizers, surface activity modifiers,
lipophilizers, and intermediary immobilizers.
In the method of the invention the combining step may include mixing the
components in a homogenizer. It
may also include heating the components. The forming step of the method may
include extruding the mixture onto a
device adapted for use with the membrane. The membrane may also be formed by
injection molding the mixture. In
the practice of this method, the invention also contemplates reacting a
biomolecule with the components of the
membrane to form a bond between the biomolecule and the membrane at the
membrane's surface. Biomolecules that
may be thus reacted include, for example, enzymes, receptors, hormones,
nucleic acids and antibodies.
The method of the invention may further include contacting a surface of the
membrane with an aqueous
material that may contain a biomolecule, to form a two-layer ion-selective
membrane having a solvent-free layer and an
aqueous layer. fn this aspect of the invention, the biomolecules may be, for
example, enzymes, receptors, hormones,
nucleic acids and antibodies.
In another aspect of the invention is provided a solvent-free ion-selective
membrane made of a polymer and at
least one additive, wherein the membrane is formed without a solvent. The
polymer of the membrane may be, for
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example, vinyl chloride, polyvinyl chloride, or a copolymer of vinyl chloride,
such as vinyl acetate andlor vinyl alcohol.
The additive may be a plasticizes. Useful plasticizers include, for example,
aromatic ethers, aliphatic-aromatic ethers.
adipic acid esters, sebasic acid esters, phthalic acid esters, lauric acid
esters, glutaric acid esters. and phosphoric acid
esters. The additive also may be an ion-setecttve agent. Membrane components
may also include mere than one
additive, such as a plasticizes and an ion-selective agent. The membrane
components further may include one or more
additives such as, for example, plasticizes modifiers, active ingredients, ion
mobility enhancers, heat stabilizers, light
stabilizers, surface activity modifiers, lipophilizers, and intermediary
immobilizers.
The ion-selective membranes of this aspect of the invention may have a curing
mass loss less than 10%, or in
some embodiments less than 1 %. The membranes may have a water absorption
index less than 0.5%, or in some
embodiments less than 0.1 %. In some embodiments of the invention, a
biomolecule may be bound to a surface of the
membrane. Useful biomolecules may include, for example, enzymes, receptors,
hormones, nucleic acids and antibodies.
The membranes may also have a layer of aqueous material in contact with a
surface of the membrane, and the
aqueous material may contain a biomolecule.
In another aspect of the invention, there is provided an improved biosensor
device including an electrode,
1 S wherein the electrode has an ion-selective membrane. In this aspect of the
invention, the improvement is a solvent-free
ion-selective membrane made up of a polymer and at least one additive, wherein
the improved membrane is formed
without a solvent.
Detailed Descriotion of the Preferred Embodiment
The membranes of the present invention are formed without the use of
extraneous solvents. An extraneous
solvent is a solvent that is added to increase the solubility of the
components, which solvent is later removed as part
of the formation of the membrane. Because the membranes of the invention do
not employ extraneous solvents, they
do not require evaporation steps. And since these membranes are formed without
evaporation of a solvent, they
exhibit structural and physical properties that are distinct from prior art
membranes. Among the most important of
those properties is a greatly reduced degree and rate of water absorption.
Because the membranes of the present
invention do not readily absorb water, they are not subject to the rapid
distortions in structure, composition, and
electrochemical properties that have plagued membranes of the prior art.
Rather, they are highly stable and exhibit
much more reproducible electrochemical properties over a much longer period of
time in contact with aqueous
solutions. "Aqueous solutions" as discussed herein may include solutions with
any appreciable water content,
including, for example, beverages, pharmaceutical solutions or suspensions,
culture or fermenter media, blood, plasma,
urine, cerebrospinal fluid, mucous secretions, and the like.
In addition to their stability in contact with aqueous solutions, these
membranes also have a very long shelf
life, and devices employing these membranes do not require specialized
packaging or other measures intended to
protect the membranes from moisture or humidity. Further, membranes of the
present invention are capable of lower
limits of ion detection, more rapid response, more accurate and reproducible
measurements over time, and greater
mechanical strength, such as would be beneficial for use under positive
pressure or vacuum. Additionally, because of
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their greatly reduced water absorption, the membranes of the present invention
are much mare effective in
immobilizing membrane components. That is, important additives in the
membrane, as discussed below, do not tend to
leach out of the membrane, as is commonly the case with membranes of the prior
art.
The membranes of the present invention are made without solvents; they are
instead made by combining a
polymer, a plasticizer, and other optional ingredients, which are mixed
mechanically to form a mixture for membrane
formation. For example, mechanical mixing may employ a high speed blender or
homogenizer, which is welt known in
the plastics industry. Components may be added individually or in groups into
the base polymer. Such components
can be plasticizers, heat stabilizers, UV absorbers, gamma ray stabilizers,
anti static agents, ion-selective agents and
other ligands, conductive agents, waxes, hydrophobic agents, flow reduction
agents, and the like. Without the
problems associated with solvent removal, membranes of the invention can be
formed to virtually any desired
thickness, eliminating the need to form membranes in a series of thin-layer
depositions with controlled evaporation
steps in between.
The solvent-free mixture of membrane components can be pre-granulated or it
can be directly fed into an
extruder or injection molding machine. With extrusion, a ribbon of nascent
membrane material can be fed easily into
high speed production equipment. In this manner, membranes with new
characteristics or mixtures of characteristics
may be produced, many of which mixtures were heretofore impossible with
previously known ion-selective membrane
technology. The thickness of the membrane may be controlled with tooting or
extrusion dies. The membrane thus
produced may be rolled in quantities for prolonged production shifts of days
or weeks. Since there are no solvents
involved, the membranes display many superior properties over prior art
membranes discussed above. The per-unit
production cost is very low, and high volume automated production is
relatively simple.
It is noted that plasticizers are capable of solubilizing polymers in many
cases. Accordingly, in some
publications (e.g. Suzuki, et al., Anal. Chem. 1989, 61:382-3841 plasticizers
themselves have been misdesignated as
solvents. More correctly, and for the purposes of this application, a
distinction is drawn between a true solvent and a
plasticizer in membrane production. A true (extraneous) solvent is used to
dissolve the components of the membrane
and is then, either in whole or in part, removed from the membrane as it
cures. The removal of the solvent generally is
accomplished by means of evaporation, the disadvantages of which have been
discussed above. Examples of true
solvents that commonly have been used in solvent-based polymeric membranes of
the prior art include cyclohexanone,
methylene chloride, propylene carbonate. tetrahydrofuran, toluene, methanol,
and water. The membranes of the
present invention are thus made without addition of these or any other
extraneous solvents, and have the benefit of
being highly resistant to degradation caused by contact with water or
solutions containing water.
In contrast to true solvents, a plasticizer may also be used to dissolve
membrane components, but it remains
part of the membrane and does not require any evaporation steps, nor does it
produce the structural artifacts of
evaporation. To further draw this distinction, a solvent-based membrane
solution that is cast to form a membrane will
lose significant mass during the process of solvent evaporation. However, a
solvent-free mixture used to form a
membrane will not lose significant mass during the manufacturing process. Of
course, much more important than
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issues of loss of mass are the resultant disadvantages of solvent evaporation,
such as structural distortions and
membrane porosity, water deposition on the curing membrane, elaborate control
of evaporation parameters andlor the
long delays and equipment downtime associated with curing by means of
evaporation.
Membranes of the invention, being solvent-free, permit greatly increased rates
of production, require
significantly less control aver environmental variables such as humidity,
avoid the expense and environmental
regulation difficulties associated with using and removing large amounts of
organic solvents, and ultimately produce
membranes having a degree of water absorption that is a smalt fraction of the
water absorption of membranes of the
prior art. Water absorption by membranes of the present invention, compared
with ion-selective membranes of the
prior art, thus may be reduced by a factor of 5,10, 10D, or more.
The final density of a cured membrane provides a distinction between the
solvent-based ion-selective
membranes of the prior art and the solvent-free ion-selective membranes of the
present invention- As discussed at
length above, solvent evaporation creates artifacts in the membrane, causing
undesirable irregularities of structure and
membrane porosity. In contrast, solvent-free membranes do not display
artifacts of evaporation, and are essentially
non-porous.
In the filtration membrane art, bulk porosity is defined as the "dead space"
within a membrane, and is
calculated by weighing a membrane sample of known volume, and comparing its
density with the overall density of the
base polymer and any additives that may be present. For filtration membranes,
high bulk porosity is a desirable
condition. In contrast, ion-selective membranes would ideally approach zero
bulk porosity--they are ideally non-porous.
The difference between the ideal and the actual can be expressed as the
Membrane Density Ratio IMDR). Solvent-
based ion-selective membranes having bubbles or pores may have an MDR of BD%
or less, while solvent-free
membranes generally have an MDR greater than 85%, preferably greater than 95%,
and most preferably greater than
99%.
The membranes of the present invention may contain a polymer, typically
polyvinylchloride IPVC) or a PVC
copolymer; plasticizers; plasticizer modifiers; active ingredients; ion
mobility enhancers; heat stabilizers; light
stabilizers; surface activity modifiers;, lipophilizers; intermediary
immobilizers; andlor other components. PVC polymers
or copolymers include low, medium, high and ultra-high molecular weight PVC.
Also useful are copolymers of vinyl
chloride such as vinyl chloridelvinyl acetate, vinyl chloridelvinyl alcohol.
and vinyl chloride/vinyl acetatefvinyl alcohol.
Another suitable polymer is carboxylated polyvinyl chloride.
Depending on the formulation, any of several known plasticizers may be used.
Examples of appropriate
plasticizers are aromatic ethers; aliphatic-aromatic ethers; esters of adipic
acid, sebasic acid, phosphoric acid, glutaric
acid, phthalic acid, andlor lauric acid; and long chain aliphatic alcohols.
Non-limiting examples of plasticizer modifiers
are halogenated paraffins such as chloroparaffins, long chain aliphatic
alcohols, substituted nitrobenzenes, and
acetophenones. Active ingredients that may be used in membranes of the present
invention include, among others,
antibiotics, liquid ion exchangers, neutral carriers, substituted amines,
organo-ammonium salts, crown ethers,
hormones, enzymes, antigens, antibodies, DNA binding factors, nucleic acids,
and the like. Ion mobility enhancers
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include, for example, salts of stearic acid, long chain alcohols, and waxes
including but not limited to paraffin waxes.
Examples of useful heat stabilizers are salts of stearic acid, organo-metallic
compounds, and chlorine receptors- Light
stabilizers can include both UV absorbers and light-to-heat converters.
Examples of surface activity modifiers are
cellulose triacetate, polyacrylamide, organo-ammonium salts, and the like.
While the possible active ingredients are tar too many to list exhaustively,
several particularly useful ion-
selective agents are listed in Table 1. It will be recognized by those of
skill in the art that other ion-selective agents,
as well as other types of active ingredients, can be selected and employed in
the membranes of the invention, to
optimize membrane function based on the particular desired use and properties
of the membrane.
Table 1. Examples of ion-selective agents
Ion or Selective Agents)
other
Analyte
ammonium nonactin
calcium calcimycin
(N,N,N',N'-tetracyclohexyl-3-oxapentanediamide)
(-1-(R,R)-N,N'-fbis(11-(ethoxycarbonyllundecyl)]-N,N'-4,5-tetramethyl-3,6-
dioxaoctane-diamide
N,N-dicyclohexyl-N',N'-dioctadecyt-3-oxapentanediamide
bis[4-(1,1,3,3-tetramethylbutyllphenyllphosphate
calcium salt
carbonate heptyl4-trifluoroacetytbenzoate
1-(dodecylsulfonyl)-4-triftuoroacetytbenzene
N-dodecyl-4-trifluoroacetylacetanilide
4-butyl-a,a,a-trifluoroacetophenone
chloride tridodecylmethylammonium chloride
5-10-15-20-tetraphenyl-21H,23H-porphyrin manganesehll)
chloride
4,5-dimethyl-3,6-dioctyloxy-1,2-phenylene-bis (mercury-trifluoroacetate)
hydrogen tridodecylamine
4-nonadecylpyridine
octadecyl isonicotinate
lithium N,N,N',N'-tetraisobutyl-cis-cyclohexane-1,2-dicarboxamide
N,N-dicyclohexyl-N',N'-disisobutyl-cis-cyclohaxane-1,2-dicarboxamide
5-butyl-5-ethyl-N,N,N',N'-tetracyclohexyl-3,7-dioxaazelaic
diamide
12-crown-4;1,4,7,10-tetraoxacyclododecane
6,6-dibenzyl-14-crown-4
6-[2-(diethylphosphonooxy)-ethyl]-6-dodecyl-14-crown-4
N,N,N',N',N",N"-hexacyclohexyl-4,4',4"-propylidyne-tris-(3-oxabutyramide)
magnesium N,N'-diheptyl-N,N'-dimethyl-1,4-butanediamide
N,N"-octamethylen-bis(N'-heptyl-N'-methyl-methylmalonamide
N.N"-octamethytene-bis(N'-heptyl-N'-methylmalonamide)
N,N'N"-tris[3-Iheptylmethytamino)-3-oxopropionyt]-8,8'-iminodioctylamine
potassium valinomycin
bis[(benzo-15-crown-5)-4'-ylmethyl]pimetate
2-dodecyl-2-methyl-1,3-propanediyl-bis [N-(5'-vitro(benzo-15-crown-5)-4'-
yllcarbamate]
sodium (N,N',N"-triheptyl-N,N',N"-trimethyl-4,4',4"-propylidyne-tris(3-
oxabutyramide)
N,N'-dibenzyl-N,N'-Biphenyl-1,2-phenylendioxydiacetamide
N,N,N',N'-tetracyclohexyl-1,2-phenylendioxydiacetamide
4-octadecanoyloxymethyl-N,N,N',N'-tetracyclohexyl-1.2-phenylendioxydiacetamide
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Ion or Selective Agents)
other
Analyte
sodium bis(112-crown-)methyl] dodecylmethylmalonate
(coot.)
4-tert-butylcalix(4]arene-tetraacetic acid tetraethylester
decyl monensin
urea urease enzymelammonium ion
glucose glucose oxidaseloxygen
The membrane components are mixed by combining the polymer, the plasticizer,
and any other optional
additives. In some formulations, a piasticizer may also function as an active
ingredient, or an active ingredient may
function as a piasticizer. Thus, the invention contemplates solvent-free
membranes made from as few as two
components, as well as membranes made from a combination of numerous
components.
Mixing may be facilitated with a high speed blender or by heat gelation and
diffusion. Mixing may occur at
ambient temperature, or an alternative temperature may be selected based on
the particular combination and
properties of the components. For example, where antibodies or other proteins
are among the additives to the
membrane, elevated temperatures may cause problems of protein denaturation,
and would therefore dictate limits on
the temperature that could be used in forming the membrane. Other
considerations in selecting membrane formation
temperature include the desired working viscosity of the membrane mixture,
temperature tolerances of other
components of disposable devices onto which the membranes may be formed, and
the like.
The membranes may be formed by extrusion and stamping, including in situ
extrusion directly onto an
electrode device. The membranes may also be formed by injection molding or
capillary extrusion, either in situ onto a
device, or in a separate fabrication step. Additional means of membrane
formation include, for example, vacuum
forming, vacuum molding, compression molding, blow molding, and calendaring.
Membranes may be formed to virtually
any useful thickness.
The invention also contemplates solvent-free membranes as described herein
with aqueous-active molecules
adhered or bound to their surface. For example, catalysts, including enzymes,
may function at the surface of the
membrane to modify a chemical species which is not directly detectable into a
product which can be detected with the
electrode of the device. In other embodiments of the invention, other
molecules, structures, or complexes, may
function at the membrane surface to enhance or modify the function of the
membrane or the electrode device as a
whole. Advantageous molecules, structures, or complexes include, for example,
hormones, antibodies, antigens,
nucleic acids, receptors, binding proteins, pharmaceutical preparations,
crystalline substances, and the like.
For example, an enzyme, such as urease, may be bound or attached to the
surface of a solvent-free
membrane. This modification allows a membrane electrode that is capable of
detecting an ionic species such as
ammonium to also indirectly detect a non-ionic species such as urea. That is,
as urea molecules in a test salution come
into contact with the urease enzyme immobilized at the surface of the solvent-
free membrane, the enzyme catalyzes
the breakdown of urea to produce ammonium ions, which are detected by the
electrode.
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Likewise, a receptor complex with ATPase activity may be immobilized at the
surface of a solvent-free
membrane sensitive to phosphate ions, to indirectly measure presence of the
receptor's ligand. As the ligand binds the
receptor, phosphate ions are released from ATP and these ions are detected by
the electrode- This type of indirect
detection of analytes assisted by soluble biomolecules held at or near the
surface of a solvent-free membrane has
broad applicability that will be appreciated by those of skill in the art.
There are numerous ways to form membranes having biomolecules or other
desirable molecules or structures
at their surface. In one embodiment of the invention, a solvent-free membrane
is formed as disclosed herein.
Subsequent to membrane formation, a selected molecule or structure is
chemically linked to the membrane via active
moieties within the polymer or co-polymer of the membrane. For example, an
amine group may be reacted to a chlorine
group on a polyvinyl chloride membrane in a coupling reaction driven by silver
ions as a catalyst.
In this embodiment, covalent bonds are formed between the biomolecule and the
polymer of which the
membrane is formed. In another embodiment, one or more selected biemolecules
are solubilized in water, and then a
water soluble polymerizing agent is added to form a solution. The solution is
contacted in a thin layer with a pre-
formed solvent-free membrane according to the invention. Upon or after contact
between the aqueous solution and the
solvent-free membrane, the aqueous layer polymerizes and forms a thin layer
bound to the solvent-free membrane,
which thin layer immobilizes the biomolecules or other structures it contains.
In another embodiment of the invention, two separate membranes are formed. The
first is a solvent-free
membrane as described extensively herein. The second is a water-based membrane
containing one or more water
soluble active ingredients, such as biomolecules or other complexes or
structures. The thickness of the water-based
membrane would typically be 1 to 100 microns, although a membrane layer of any
useful thickness may be applied to
the solvent-free membrane. When the water-based membrane comes into contact
with the solvent-free membrane it
approximates and immobilizes the biomolecuies or other complexes or structures
at or near the surface of the solvent-
free membrane.
In many of these embodiments, the end result is a membrane having the
desirable water-excluding properties
of the solvent-free membranes of the invention, while also having immobilized
thereon molecules that may require
water to perform their desired function. Accordingly, the invention is not
limited merely to solvent-free membranes
that may contain one or more active ingredients, but also encompasses solvent-
free membranes that may bear one or
more desirable water soluble molecules on their surface- The water soluble
molecules thus immobilized may function
as enzymatic catalysts to convert a non-measurable species into a measurable
species- They may also perform other
useful functions, such as concentration of desirable particles, exclusion of
undesirable particles, initiation of
biochemical pathways leading to a desirable andlor measurable product, and the
like.
Examples
Representative solvent-free membranes were prepared for comparison with prior
art solvent-based
membranes. Samples of membranes selective for calcium, potassium, and ammonium
were tested.
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Example 1. Calcium-selective solvent-free membrane
A solvent-free membrane, selective for calcium ions, was prepared according to
the following formulation:
PVC, ultra high molecular weight, 100.00 parts; 2-nitrephenyl octyl ether,
250.00 parts; (-1-(R,R)-N,N'-Ibis(11-
ethoxycarbonyl)undecyl)-N,N'-4,5-tetramethyl-3,6-dioxaoctanediamide (calcium
ion selective ligand), 7.50 parts:
S sodium tetraphenylborate, 1.50 parts: calcium stearate, 1.00 parts. All
components were mixed to homogeneity and
the membrane was formed to a thickness of about 1-3 mils (25-75 microns).
After 3 h at 75-105°C, the membrane
was fully solidified and functionally selective for calcium ions. Membranes of
this formulation were then compared
with conventional calcium-selective membranes. The results of such comparisons
are provided and discussed below.
Comparative Example 1. Conventional calcium-selective membrane
A conventional formulation was used to prepare a solvent-based calcium-
selective membrane for comparison
to the calcium-selective membrane of the invention. The membrane was made
using: N,N,N',N'-tetracyclo-3-
oxapentanediamide, 10 mg; 2-nitrophenyl-octyl ether, 655 mg; potassium
tetrakis(4-chlorophenyl)borate, 6 mg; PVC,
high molecular weight, 328 mg. All components were dissolved in 8.0 ml (11,256
mgt tetrahydrofuran. Thus, the
total mass of the formulation, prior to evaporation of the solvent, was 12,255
mg--about 8% solids and about 92%
solvent. The membranes were cast and cured using conventional techniques, and
the resulting membranes were
functionally selective for calcium ions.
It is possible to prepare this type of conventional membrane using more or
less solvent than the amount used
in this comparative example, within a limited range. The optimal amount of
solvent to be used is based on the desired
viscosity of the solution, which is governed by the membrane manufacturing
parameters. Solutions prepared with too
little solvent are typically too viscous to dispense and do not properly
settle into uniform, flat layers. Solutions
containing excessive amounts of solvent are much less viscous, but require
longer evaporation times and generally also
require more dispensing and curing steps to achieve a specified membrane
thickness.
The viscosity of the solution used in this comparative example, and the other
comparative examples herein,
was about equal to the viscosity of solutions that would be used in typical
manufacturing processes of these
conventional membranes. Thus, measures of mass or volume lost during curing of
the membranes of the comparative
examples are representative of the mass or volume loss in conventional,
solvent-based, ion-selective membrane
manufacture.
Example 2. Potassium-selective solvent-free membrane
A solvent-free membrane, selective for potassium ions, was prepared according
to the following formulation:
PVC, ultra high molecular weight, 100-00 parts; dioctyl adipate, 150.00 parts;
valinomycin (potassium ion selective
ligand), 2.85 parts; sodium tetraphenyibarate. 2.00 parts; stearic acid, 1.00
parts. All components were mixed to
homogeneity and the membrane was formed to a thickness of about 1-3 mils (25-
75 microns). After 6 h at 85-1 i 5°C,
the membrane was fully solidified and functionally selective for potassium
ions. Membranes of this formulation were
then compared with conventional potassium-selective membranes. The results of
such comparisons are provided and
discussed below.
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Comparative Example 2. Conventional potassium-selective membrane
A conventional formulation was used to prepare a solvent-based potassium-
selective membrane for
comparison to the potassium-selective membrane of the invention. The membrane
was made using: valinomycin, 10
mg; bisll-butylpentyll decane-1,10-diyl diglutarate, 650 mg; potassium
tetrakis(4-chlorophenyllborate; 5 mg; PVC, high
molecular weight, 330 mg. All components were dissolved in 8.0 ml (11,256 mg)
tetrahydrofuran. Thus, the total
mass of the formulation, prior to evaporation of the solvent, was 12,251 mg--
about 8% solids and about 92% solvent.
The membranes were cast and cured using conventional techniques, and the
resulting membranes were functionally
selective for potassium ions.
Examule 3. Ammonium-selective solvent-free membrane
A solvent-free membrane, selective for ammonium ions, was prepared according
to the following formulation:
PVC, ultra high molecular weight, 100.00 parts; bis(ethylhexyl) adipate,
250.00 parts; nonactin (ammonium ion
selective ligandh 1.50 parts; ammonium tetraphenyl borate, 0.36 parts; stearic
acid. 1.00 parts. All components were
mixed to homogeneity and the membrane was formed to a thickness of about 1-3
mils (25-75 microns). After 6 h at
85-105°C, the membrane was fully solidified and functionally selective
for Ammonium ions. Membranes of this
formulation were then compared with conventional ammonium-selective membranes.
The results of such comparisons
are provided and discussed below.
Comaarative Example 3. Conventional ammonium-selective membrane
A conventional formulation was used to prepare a solvent-based ammonium-
selective membrane for
comparison to the ammonium-selective membrane of the invention. The membrane
was made using: nonactin, 10 mg;
bis(butylpentylladipate, 668 mg; PVC, high molecular weight, 322 mg. All
components were dissolved in 8.0 ml
(11,256 mg) tetrahydrofuran. Thus, the total mass of the formulation, prior to
evaporation of the solvent, was 12,256
mg--about 8% solids and about 92% solvent. The membranes were cast and cured
using conventional techniques, and
the resulting membranes were functionally selective for ammonium ions.
Example 4. Curing Mass Loss
The membranes of the invention do not lose significant mass during curing (or
solidification), because they do
not contain a solvent. In contrast, prior art ion-selective membranes, since
they are formed using solvents, lose
substantial mass as the solvent evaporates. Curing Mass Loss (CML) is a
numerical expression of the percent loss of
mass of the membrane solution during curing, and thus provides a meaningful
and measurable distinction between the
membranes of the invention and those of the prior art.
CML can be calculated based on the relative concentrations of the components
in the solvent-based mixture
by subtracting the percentage concentration of the solvent from 100. This of
course assumes complete solvent
removal. CML also may be determined empirically by weighing a given quantity
of solvent-based membrane solution
before casting. and later weighing the fully cured membrane produced
therefrom. Table 2 provides calculated CML for
prior art membranes and for solvent-free membranes of the invention.
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Theoretical CML of the membranes of the present invention is 0%, and measured
CML is generally less than
10%, preferably less than 1 %, and most preferably less than 0.1 %.
Table 2. Curing Mass Loss (CML)
Membrane CMt (%1
Solvent-free calcium-selective membrane < 0.1
Conventional calcium-selective membrane ' 92
Solvent-free potassium selective membrane < 0.1
Conventional potassium-selective membrane " 92
Solvent-free ammonium selective membrane < 0.1
Conventional ammonium-selective membrane ' 92
Similar to mass loss is volume loss of a membrane as it solidifies. Membranes
of the present invention
exhibit little, if any, change in volume after solidification. In contrast,
the membranes of the comparative examples
exhibited volume loss of 91.6% (potassium-sensitive membrane) and 89.3%
(ammonium-sensitive membranel. Volume
loss was not measured for the conventional calcium-sensitive membrane.
Example 5. Water Absorption Index
Membranes of the present invention differ significantly from prior art organic
ion-selective membranes
because of their greatly reduced tendency to absorb water. The functional
importance of this difference has been
discussed above. A useful measure of this difference is the water absorption
index (WAIL. WAI is determined by
placing a sample of membrane having a known mass into distilled water for 24h
at room temperature. After 24h, the
sample is re-weighed and the mass gain, if any, is expressed as a percentage
of the original mass of the membrane.
Different membranes then may be compared based on the WAI of each.
While WAI is an arbitrarily selected measure of water absorption, it is a
simple, universal test that is clearly
proportional to and indicative of differences in water affinities of different
membranes. Depending on the intended
uses of particular organic ion-selective membranes, other measures similar to
WAI, as defined above, may also be
useful.
For example, an absorption index using blood plasma as the aqueous solution,
at body temperature, for a one
week duration, would fairly indicate the degree to which membranes of distinct
formulations would distort during one
week in an implanted biosensor electrode. Tests of membranes intended to be
selective for a particular ion also may
be conducted using a solution containing the ion and, in many cases, other
additives such as chelators of undesirable
ions, pH buffers, andlor solution preservatives. While WAI parameters provide
a simple, universal basis for comparison,
other solutions more specific for the membranes in question may be used in
comparison tests to accelerate water
absorption, allowing more rapid detection of differences between membranes.
See Example 7.
Membranes of the present invention absorb water much less than organic ion-
selective membranes of the
prior art, and are therefore much more stable in contact with aqueous
solutions, as shown below in Table 3. Typically,
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membranes of the invention have a WAI of less than 0.5%, preferably less than
0.1 %, and most preferably less than
0.05%.
Table 3. Water Absorption index (WAI)
Membrane WAI (%)
Solvent-free calcium-selective membrane < 0.1
Conventional calcium-selective membrane 0.69
Solvent-free potassium selective membrane < 0.2
Conventional potassium-selective membrane 0.95
Solvent-free ammonium selective membrane < 0.1
Conventional ammonium-selective membrane 0.72
S To clarify the interpretation of the results presented in Table 3, if 1000
mg of dry membrane were used as
starting material, the membranes of the invention would weigh between 1000 and
1002 mg after 24h in water, while
a conventional potassium-selective membrane would weigh 1009.5 mg after 24h in
water.
Example 6. Water Mass Loss
Another benefit of the membranes of the invention is that, since they do not
absorb water to an appreciable
degree, they likewise are not subject to loss of membrane components due to
the leaching effects caused by water
absorption andlor permeability. In contrast, conventional solvent-based ion-
selective membranes actually lose mass,
presumably due to the leaching out of internal membrane components during 24h
contact with water.
As a subsequent procedure to the WAI test described in Example 5, membranes
that had been soaked in
water for 24h and surface dried where then air dried for 24h to remove
residual water. The dried membranes were
then re-weighed, and the mass of each soaked and dried membrane was compared
with its original mass before
exposure to water. As shown in Table 4, conventional membranes were shown to
have lost mass, while the
membranes of the invention exhibited no significant change in mass.
Table 4. Water Mass Loss (WML)
Membrane WML (%)
Solvent-free calcium-selective membrane < 0.1
Conventional calcium-selective membrane 0.45
Solvent-free potassium selective membrane < 0.2
Conventional potassium-selective membrane 0.81
Solvent-free ammonium selective membrane < 0.1
Conventional ammonium-selective membrane 0.54
To clarify the interpretation of the results presented in Table 4, if 1000 mg
of dry membrane were used as
starting material, the membranes of the invention would weigh between 1000 and
998 mg after 24h in water followed
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by 24h of air drying, while a conventional potassium-selective membrane would
weigh 993.9 mg after 24h in water
followed by 24h of air drying.
Example 7. Ion Solution Absorption
Perhaps even more meaningful than the WAI, as a measure of the distortion of
prior art membranes by the
solutions they measure, is the membranes' absorption of solutions containing
the target ions of those membranes.
That is, a conventional calcium-selective membrane exhibits a greater mass
gain when in contact with a calcium
solution than when in contact with deionized water.
This is presumed to be due to the affinity of the ions in solution with the
ion-selective agents in the
membrane. As a membrane absorbs water, it is believed that ions also enter the
membrane and interact with the
active ingredients of the membrane. Internal ion accumulation can then lead to
greater water accumulation as well.
The result is that the membranes absorb the solution and become structurally
distorted and functionally impaired. In
contrast, the membranes of the examples do not exhibit significant ion
solution absorption. In fact, the solvent-free
membranes of the invention show no measurable difference in fluid absorption
whether in contact with ion solutions or
deionized water.
Ion solution absorption was determined by placing membrane samples in
solutions containing 4 m eq of the
ion for which the membrane is selective. Membranes were left in the ion
solution for 24h at room temperature, then
were blotted dry and weighed. Prior art membranes exhibited significantly
increased mass, while mass increases in
the membranes of the invention were at or near detection limits.
Table 5. Ion Solution Absorption (ISA)
Membrane ISA 1%)
Solvent-free calcium-selective membrane < 0.1
Conventional calcium-selective membrane 2.37
Solvent-free potassium selective membrane < 0.2
Conventional potassium-selective membrane 1.$3
Solvent-free ammonium selective membrane < 0.1
Conventional ammonium-selective membrane 1.16
Example 8. Ion Solution Retention
After the ion solution absorption experiment of Example 7, the membranes were
air dried for 24h and re-
weighed. Conventional membranes retained mass from the ion solution even after
24 of drying, while membranes of
the invention showed no significant change in mass.
Table 6. Ion Solution Retention (ISR)
Membrane ISR (%1
Solvent-free calcium-selective membrane < 0.1
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Membrane ISR (%I
Conventional calcium-selective membrane 0.39
Solvent-free potassium selective membrane < 0.2
Conventional potassium-selective membrane 0.47
Solvent-free ammonium selective membrane < 0.1
Conventional ammonium-selective membraneI 0.47
The foregoing examples are provided to demonstrate the significant advances in
the ion-selective membrane
art achieved by the present invention, and are merely representative of some
embodiments of the invention. The true
scope of the present invention is to be measured only by the following claims.
