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This application is related to U.S. Pat. Nos. 4,661,235 and 4,637,861, both
to Krull and Thompson.
TECHNICAL FIELD
The present invention relates to a lipid membrane-based device. More
specifically, this invention pertains to a protected, lipid membrane-based
device, to a lipid membrane-based gas sensor, and to the use of these
devices as chemoreceptive transducers for the analysis of specific
chemical test species.
BACKGROUND ART
An ordered lipid membrane useful as a chemoreceptive transducer in an
electrochemical cell is known, as illustrated by U.S. Pat. Nos. 4,661,235
and 4,637,861, both to Krull and Thompson. Such membranes are modified to
include a complexing agent for selectively interacting with a particular
analyte of interest. However, a drawback is that these membranes may also
interact non-selectively by adsorption/absorption of various species, with
resultant undesirable transmembrane current perturbation. Furthermore,
exposure of a membrane surface to a sample solution permits membrane
damage and unwanted solution convection effects.
Krull et al, Abstract 11-2, 67th Annual CIC Conference (June 1984) disclose
advances in Langmuir-Blodgett thin-film deposition technology for
providing substrate-stabilized, lipid membrane structures. This abstract
mentions techniques for such deposition, including schemes involving gel
protection.
Heckmann et al, Thin Solid Films, 99: 265 (1983) describe a hyperfiltration
membrane. It is an object of this work to produce an active layer on top
of a membrane for ion permeability control, thereby providing a decreased
electrolyte retention capacity with resultant increased water
permeability, compared to conventional membranes. The hyperfiltration
membrane is a cross-linked monolayer, prepared by cross-linking
surfactants having glucose hydrophilic head groups with epichlorohydrin.
To extend the selective permeability of the membrane into the range of
molecules of medium size, the incorporation of hydrophobic ionophores and
pore molecules into the membrane is proposed.
As illustrated by Thompson et al, Talanta, 30: 919 (1983), a gas sensor
cell that includes a Teflon.TM. semipermeable membrane and a bilayer lipid
membrane, modified to be selective for ammonium ion, is known. FIG. 5 of
this publication depicts calculated values for a hypothetical cell formed
by removal of the Teflon membrane, and replacement of the aqueous phase
with a hydrated gel-like layer.
To prevent membrane damage and undesirable solution convection effects,
there is a need for a protected, lipid membrane-based device useful as a
chemoreceptive transducer. The discovery of such a device would constitute
an even greater contribution to the art if it could also be used to
enhance selectivity by preventing interfering chemical species from
reaching the lipid membrane surface. Also needed is an improved lipid
membrane-based gas sensor. Such devices would beneficially make possible
improved processes for analysis.
DISCLOSURE OF THE INVENTION
It is accordingly an object of the present invention to provide a
protected, lipid membrane-based device useful as a chemoreceptive
transducer.
It is a further object of the present invention to provide a device of this
type that could be used to enhance selectivity by controlling the size of
the chemical species that reaches the lipid membrane surface.
It is an even further object to provide an improved lipid membrane-based
gas sensor.
It is an additional object to provide improved processes for quantitative
and qualitative analysis.
Additional objects, advantages and novel features of the present invention
are set forth in the description that follows, and in part will become
apparent to those skilled in the art upon examination of the following
description or may be learned by practice of the invention.
To achieve the foregoing objects and in accordance with the purpose of the
present invention, as embodied and broadly described herein, there is
provided a protected, lipid membrane-based device useful as a
chemoreceptive transducer for determining the concentration of a specified
chemical species. The device includes a porous, ion-permeable, hydratable,
membrane-protective layer, and an underlying lipid membrane, which
controls ion permeability.
The pores of the membrane-protective layer permit passage therethrough of
the specified chemical species, but block passage of a larger material
from which the lipid membrane is desirably shielded. The lipid membrane is
modified by the incorporation of a complexing agent for selectively
interacting with the specified chemical species to increase membrane ion
permeability.
In accordance with the present invention, there is also provided a process
for using the protected device to determine the concentration of the
chemical species in an aqueous electrolytic solution. The process includes
forming an electrochemical cell from the device and the aqueous solution.
There is then applied across the modified lipid membrane of the device an
electrical potential difference. The interaction of the chemical species
with the membrane-incorporated complexing agent can then produce an
analytical signal based upon an increase in membrane ion permeability. The
analytical signal is measured, and the concentration of the chemical
species is determined from the measured signal.
Also in accordance with this invention, there is provided a lipid
membrane-based gas sensor. The sensor includes a gas-permeable, hydrated,
upper layer permeable to an inorganic ion-forming gas, which is attached
to an underlying lipid membrane. The lipid membrane includes a complexing
agent for selectively interacting with a specified inorganic ion formed by
the dissolution of the inorganic ion-forming gas in the hydrated, upper
layer, to increase permeability of the lipid membrane to the inorganic
ion.
Additionally in accordance with this invention, there is provided a process
for using the gas sensor to determine the concentration of the inorganic
ion in an aqueous electrolyte solution. The process includes applying an
electrical potential difference across the lipid membrane of the gas
sensor. As a result, the inorganic ion interacts with the lipid
membrane-incorporated complexing agent, to increase the permeability of
the lipid membrane to the inorganic ion, thereby producing an analytical
signal based upon the increased membrane ion permeability. The analytical
signal is measured, and the inorganic ion concentration is determined from
the measured signal.
BRIEF DESCRIPTION OF THE DRAWING
Reference is made to the accompanying drawing which forms a part of the
specification of the present invention.
FIG. 1 depicts an exemplary crosslinker useful in forming a lipid
membrane-based device in accordance with the present invention; and
FIG. 2 is a diagrammatic representation of a lipid membrane-based device in
accordance with the present invention.
BEST MODE PRESENTLY CONTEMPLATED FOR CARRYING OUT THE INVENTION
As explained earlier, the present invention is directed to a novel,
protected lipid membrane-based device useful as a chemoreceptive
transducer, and to a novel lipid membrane-based gas-sensor. Additionally,
this invention is directed to a process for using the protected device for
determining the concentration of a specified chemical species in an
aqueous electrolytic solution, and to a process for using the gas sensor
for determining the concentration of a specified inorganic ion-forming
gas.
Lipid membrane-based devices in accordance with the present invention
include an upper layer and, attached to the upper layer, an underlying,
perturbable lipid membrane. The lipid membrane controls ion permeability,
and may be a bilayer or monolayer.
Lipids forming the membrane may be natural or synthetic. Suitable lipids
include, but are not limited to, phospholipids such as phosphatidic acid,
phosphatidyl glycerol, phosphatidyl choline, phosphatidyl ethanolamine,
phosphatidyl serine and phosphatidyl inositol; and sphingolipids such as
sphingomyelins. Phosphatidyl serine may be advantageously used as a lipid
if biocompatibility is a consideration. The membrane could be formed by a
mixture of lipids.
The membrane-forming lipids typically include two long hydrophobic chains.
Any long chain useful in forming a natural or synthetic bilayer or
monolayer membrane is suitable. Generally, a chain will have a length of
from at least six carbon atoms up to and including about thirty,
preferably ten to twenty, carbon atoms. Illustrative long hydrophobic acyl
chains are caproyl, lauroyl, myristoyl, palmitoyl and stearoyl chains.
The lipid membrane is modified by the incorporation of a complexing agent
selective for a specified chemical species (stimulant). Interaction
between the complexing agent and the stimulant perturbs the ordered lipid
membrane. As a result, an analytical signal based upon an increase in
membrane ion permeability is produced.
An essential feature of lipid membrane-based devices according to the
present invention is that an upper layer is attached to the lipid
membrane. Attachment is preferably by physical bonding.
A suitable method of forming these devices is by the use of a crosslinker
that includes moieties that can form the upper layer and bond to the lipid
membrane. Thus, a very useful type of crosslinker includes a polymerizable
moiety that, upon polymerization, forms the upper layer, and a binding
site-providing moiety that is capable of bonding to the lipid membrane.
Useful crosslinkers of this type include polymerizable sugar monomers such
as 1,6-anhydro sugars, and long hydrophobic chains for physically bonding
to the hydrophobic membrane region. Sugar monomer polymerization
beneficially yields a polysaccharide mucous layer as the upper layer. For
purposes of this invention, by the term "mucous" is meant a physical
structure of polymeric chains which are randomly interwoven to form a mat.
Generally, a suitable long hydrophobic chain will have a length of from at
least six carbon atoms up to and including about thirty, preferably ten to
twenty carbon atoms. Exemplary long hydrophobic acyl chains are the
aforementioned caproyl, lauroyl, myristoyl, palmitoyl and stearoyl chains.
This type of crosslinker advantageously further includes a glycerophosphate
moiety, to stabilize the sugar moiety. For example, the lipid system may
contain a phosphatidyl moiety as well as acyl chains. One of the acyl
chains may carry a 1,6-anhydro sugar group as a terminal group.
Interaction of the phosphatidyl moiety with this terminal group of the
underlying lipid membrane is then primarily responsible for the physical
bonding of the glycophospholipid to the membrane.
An illustrative crosslinker is
2-capramido-1,6-anhydro-2-deoxy-.beta.-D-glucopyranose. Other crosslinkers
can be prepared by reacting
2-amino-1,6-anhydro-2-deoxy-.beta.-D-glucopyranose with a carboxylic acid
having the desired hydrophobic chain length. Thus, myristic acid can be
selected for reaction when a fourteen carbon acyl chain is desired.
A commercially available starting material for making
2-amino-1,6-anhydro-2-deoxy-.beta.-D-glucopyranose, is levoglucosan.
Conversion of the 2-hydroxyl group of levoglucosan to a 2-amino group may
be achieved by tosylating the 2-hydroxyl and 4-hydroxyl groups, forming an
epoxide from the 3-hydroxyl and tosylated 4-hydroxyl groups, opening the
epoxide with benzyl alcohol to protect the 4-hydroxyl group with a benzyl
moiety, forming an epoxide from the 3-hydroxyl group and the tosylated
2-hydroxyl group, opening the epoxide with ammonia, and restoring the
4-hydroxyl group. An alternative method for making this
2-amino-1,6-anhydro-2-deoxy glucose starts with an N-protected
2-amino-2-deoxy glucose, which is commercially available, and building up
the 1,6-anhydro system. Tosylation of the C.sub.6 -primary hydroxyl,
followed by acetylation of the remaining three hydroxyls (on C.sub.1,
C.sub.3 and C.sub.4) yields the N-protected 6-tosyl-1,3,4-triacetoxy
glucose which, on treatment with base, gives the desired
2-amino-1,6-anhydro-2-deoxy glucose after removal of the protecting group.
Another exemplary crosslinker is the glycophospholipid shown in FIG. 1.
This crosslinker is a derivatized phosphatidyl choline containing a
1,6-anhydro-.beta.-D-glucopyranose as the polymerizable sugar monomer, and
a lauroyl moiety as the long hydrophobic chain. Preparation of the
glycophospholipid shown in FIG. 1 involves treating
2-amino-1,6-anhydro-2-deoxy glucose with nonanedioic acid to introduce the
C.sub.9 -chain with a terminal carboxyl moiety, which on reaction with
lysolauroyl lecithin yields the product shown in FIG. 1. Various
modifications thereof can be obtained by substituting other straight chain
dicarboxylic acids for the nonanedioic acid in the above procedure.
In a further type of crosslinker, the lipid membrane-bonding moiety
provides for covalent bonding to the lipid matrix. A covalent-bonding
crosslinker could, for instance, be identical to a long hydrophobic
chain-containing crosslinker, except that the long chain terminates in a
hydroxyl-reactive groups such as a carboxyl group. This modification
permits covalent bond formation between the crosslinker long chain and a
glycerol hydroxyl group. For example, phosphatidyl glycerol can be
tritylated to protect the C.sub.3 -primary hydroxyl group, then treated
with N-(.omega.-carboxy alkanoyl)-1,6-anhydro glucosamine to introduce the
sugar moiety onto the C.sub.2 -position of the lipid, followed by
detritylation and treatment with alkanedioic acid.
The length of a crosslinker hydrophobic chain should be selected based upon
the lipid membrane hydrophobic chain length. Desirably, a crosslinker
chain length should not exceed the membrane chain length and should
therefore occupy one chain volume or less.
In forming lipid membrane-based devices according to the present invention
using a crosslinker, a low ratio of crosslinker to the membrane-forming
lipids is generally employed. By a low ratio is meant a range of typically
from about 1:5 to about 1:100, advantageously about 1:10, parts of the
crosslinker to the membrane-forming lipids. When each crosslinker has only
one chain available for associating into the lipid membrane, this density
will provide an association of crosslinker chains into the membrane
ranging from about 1 chain per 10 lipids to 1 chain per 1000 lipids. This
density provides sufficient association to enable the upper layer to be
securely attached to the lipid membrane. An association of about 1 chain
per 20 lipids is especially suitable.
On the high density end of the range (1 chain per 10 lipids), fluidity and
packing parameters would be greatly affected, and the lipids forming the
membrane, would have relatively less mobility. As a result, membrane ion
permeability would be relatively less, and the degree of perturbability
that a stimulated complexing agent could induce in a lipid matrix would be
reduced. Membrane structural stability would be relatively greater on this
end of the range.
On the other hand, on the low density end of the range (1 chain per 1000
lipids), the upper layer would be only loosely bound to the lipid
membrane, and would therefore be more easily physically displaced. Also,
the structural stability of the membrane would be relatively less.
Another useful type of physical bonding requires a very high energy for
detaching the upper layer from the lipid membrane. Electrostatic
complexation and chemisorption exemplify this type of bonding.
To form the upper layer from polymerizable sugar monomers, UV irradiation
may be advantageously employed. A wavelength of abut 254 nm is suitable.
The result, with crosslinkers having long hydrophobic chains which have
associated with the lipid membrane, is an upper layer that includes long
hydrophobic chains embedded in the lipid membrane by a hydrophobic effect.
FIG. 2 diagrammatically depicts a mucous layer 10 derived from a
glycophospholipid attached by hydrophpbic bonding to an ordered, bilayer
lipid membrane 12. The mucous layer long chains 14 are shown incorporated
into a hydrophobic region 16 of membrane 12. Membrane 12 comprises lipids
having polar, hydrophilic head groups 18 and hydrophobic tails 20. The
head groups form separate aqueous phase regions 22, 24, which are bordered
for illustrative purpose by dotted lines.
If desired, polymerizable sugar monomers lacking lipid membrane-bonding
groups (interlinkers), may be added prior to polymerization. During upper
layer formation, the interlinkers react with one another to form long
polymeric chains, interlinking the upper layer-forming moieties, and
building the upper layer thickness. Monomeric interlinkers are suitably
used in a ratio ranging from about 1:5 to about 1:100 parts of the upper
layer-forming moieties to the monomeric interlinkers. An illustrative
interlinker is 2-acetamido-1,6-anhydro-2-deoxy-.beta.-D-glucopyranose.
Typically, irradiation with UV light (about 254 nm wavelength), for about 5
to 30 minutes at an intensity of about 10 to 100 milliwatts/cm.sup.2, may
be employed. The length of time and the intensity of irradiation, in
conjunction with the concentration of the crosslinker and interlinker,
control the degree of density and the thickness of the mucous layer.
Polysaccharides have large dipole moments. Therefore, when the upper layer
is a polysaccharide, this characteristic may be used to control membrane
dipolar potential. Moreover, the polysaccharide layer can be employed for
controlling lipid packing and mobility, and hence the ion energy barrier
across the membrane for improvement of the signal-to-noise ratio.
An essential feature of the protected lipid membrane-based device of the
present invention is a porous, membrane-protective layer as the upper
layer. On the one hand, the pores in the layer are large enough to allow a
stimulant to pass through so that it reaches the underlying lipid
membrane. However, on the other hand, the average size of the pores is
chosen to exclude contaminants or interfering chemical species of larger
size than the stimulant from passage through the protective, upper layer.
Of particular concern are organic compounds of a molecular weight of about
1000 or greater. These compounds often threaten membrane destruction, or
the possibility of interacting with the complexing agent. Hence, the
average pore size is typically chosen to exclude these organic compounds.
As can therefore be understood, the upper layer functions in this device as
a barrier layer by screening out the larger-sized chemical species.
Furthermore, an additional benefit is provided: selectivity of the
complexing agent is enhanced.
A further essential feature of the membrane-protective layer is that it is
ion-permeable. Thus, the underlying lipid membrane controls ion
permeability of the device. Furthermore, the membrane-protective layer
must be hydratable so that ion conduction in the layer is high.
Rate-limiting ion conduction must be by the lipid membrane, not by the
upper layer.
When the characteristics of porosity, ion-permeability and hydratability
are considered, it can be understood that the membrane-protective layer of
the protected device is suitably a mucous layer. Advantageously, the
membrane-protective layer is a polysaccharide mucous layer. Monomeric
sugars for forming a polysaccharide mucous layer are well known in the
biochemistry art, and include derivatives of
1,6-anhydro-.beta.-D-glucopyranose.
As explained earlier, sugar monomers can be polymerized to form a mucous
layer by UV irradiation. By, for instance, controlling the time and
intensity of the irradiation, the degree of cross-linking and the mucous
layer pore size may be regulated. Accordingly, a relatively smaller pore
size can be produced by a relatively longer time and/or intensity of
irradiation; whereas, a relatively larger pore size results from a
relatively shorter time of irradiation.
Control of the pore size also depends upon the density of polymerizable
sugar monomer at the layer surface. Thus, a relatively higher monomer
density yields a relatively smaller pore size, and a relatively lower
monomer density results in a relatively larger pore size.
A complexing agent useful in the protected lipid membrane-based device may
be selective for an inorganic ion, or may be a receptor selective for an
organic compound.
A useful complexing agent selective for an inorganic ion includes a
polypeptide, such as an antibiotic polypeptide. Illustrative antibiotic
polypeptides are known in this art and include gramicidin A, valinomycin
and nonactin.
The receptor could be, for example, chemically bound in the lipid membrane,
and may be a product of nature or a synthetic organic compound. Exemplary
receptor-organic compound pairs, all of which are known in this art, are
as follows: antibody-antigen, hormone receptor-hormone, enzyme-substrate,
enzyme inhibitor-enzyme, and lectin-polysaccharide. An advantageous
glycoreceptor is concanavalin A, which is useful for dextran analysis.
An essential feature of a lipid membrane-based gas sensor in accordance
with the present invention is a gas-permeable layer as the upper layer.
This layer must be hydrated for the gas sensor to function. Therefore, the
gas-permeable layer is chosen so that it may be hydrated, and
advantageously so that the head groups of the underlying lipid membrane
can assist in maintaining water of hydration after removal of the sensor
from a bulk aqueous environment.
A mucous layer is easily hydrated since it is quite polar, and can easily
form hydrogen bonds with water due to a high density of hydroxyl groups.
Furthermore, a mucous structure is porous, thereby enabling water to be
retained in the cavities, as in a sponge. Therefore, the gas-permeable
layer may suitably be a mucous layer. An advantageous gas-permeable layer
is a polysaccharide, mucous layer. Monomeric sugars for forming a
polysaccharide, mucous layer are well known in the biochemistry art, and
include derivatives of 1,6-anhydro-.beta.-D-glucopyranose.
The gas sensor may require a replacement of water to offset evaporation and
thereby maintain hydration. This may be achieved by a reservoir of water,
which would provide replacement of water as needed, by, for instance,
capillary action.
In the gas sensor, the lipid membrane-modifying complexing agent is
selective for a specified inorganic ion formed by dissolution of a gas,
such as ammonia, in an aqueous portion of the hydrated, upper layer. The
upper layer is permeable to the ion-forming gas. Interaction between the
complexing agent and the inorganic ion increases permeability of the lipid
membrane to the inorganic ion, which results in the production of an
analytical signal.
Suitable complexing agents useful in the gas sensor include a polypeptide,
such as an antibiotic polypeptide. Illustrative antibiotic polypeptides
are known in this art, and include gramicidin A, valinomycin and nonactin.
A procedure for making lipid membrane-based devices in accordance with the
present invention will now be described.
An ordered, bilayer lipid membrane is produced from the membrane-forming
lipids, using a Langmuir-Blodgett thin-film trough. Afterwards, the lipid
structure is modified with a selected complexing agent and added to an
aqueous solution.
Crosslinkers, including sugar monomers and long hydrophobic chains, are
added to the aqueous solution. The crosslinker chains are spontaneously
incorporated into the lipid matrix. Spontaneous incorporation may be
assisted by stirring, and slight heating above room temperature may be
employed. Chain incorporation is permitted to proceed until a significant
density of the crosslinker, typically on the order of about 1 chain per 10
to 30 lipids, is physically bonded to the lipid membrane.
At this point, sugar monomer interlinkers may be added to the mixture.
Mucous layer formation is then catalyzed using UV irradiation. In the
mucous layer formation, the interlinkers react with one another to form
long polymeric chains. As interchain polymerization occurs, an upper
polymeric layer physically bonded to the lipid membrane through the
hydrophobic interaction between the hydrophobic chains and hydrophobic
lipid membrane chains, is formed in situ.
Preferably, a device in accordance with the present invention includes a
lipid membrane-stabilizing support. The support may be an ion-conductive
support or, as described in U.S. Pat. No. 4,637,861, an electrically
conductive, solid substrate.
An exemplary ion-conductive support is a hydrogel, for instance, a
polyacrylamide hydrogel. An ordered lipid membrane may be deposited onto
this type of support by Langmuir-Blodgett thin film deposition.
Alternatively, the support could be an electrically conductive, solid
substrate, the surface of which has been modified to provide reactive
binding sites. Surface modification to provide binding sites can be
accomplished through conventional chemical means, such as oxidation or
nitridation. Oxidation and hydration yield hydroxyl binding sites, and
nitridation gives nitrogen-containing binding sites.
An ordered lipid membrane may be anchored to binding sites on the support
surface through long chains originating in the membrane-forming lipids.
Anchoring may be by covalent bonding. One particularly useful technique
for covalent bonding involves the reaction of the support surface reactive
sites with a bridging species, such as aminopropyltriethoxysilane,
followed by reaction of silane terminal amino groups with terminal
carboxyl groups of the lipid long chains.
Illustrative electrically conductive, solid substrates include, but are not
limited to, a conductive metal such as silver, platinum and gold;
electrolytic glassy carbon; and amorphous silver chloride. Each of these
exemplary electrically conductive, solid substrates is amenable to surface
modification to form reactive binding sites.
After the organized lipid assembly has been stabilized on a support and
modified by a complexing agent, the stabilized membrane may then be added
to an aqueous solution. Crosslinkers, including sugar monomers and long
hydrophobic chains, may then be introduced into the aqueous solution, and
after sufficient association of the crosslinker chains into the lipid
matrix has occurred, a device in accordance with the present invention may
be produced by sugar monomer polymerization.
The devices of the present invention are useful for determining the
concentration of a stimulant in an aqueous electrolyte solution. When an
ion conductive support is used, ion current may be measured. On the other
hand, if an electrically conductive, solid substrate is used as the
support, the change in internal capacitance of the lipid membrane may be
measured.
To determine the concentration of a stimulant, a lipid membrane-based
device in accordance with the present invention is used, in combination
with a reference electrode, an electrometer or a capacitance bridge
measurement device, a power supply, and an electrolyte. The concentration
of the stimulant is determined as follows: Several known concentrations of
the stimulant are used to prepare a calibration curve. Then the same
electrical parameter, for example, ion current or capacitance, is measured
for an unknown concentration of the stimulant, and the concentration is
determined by comparison with the calibration curve.
In the Examples that follow and throughout this description and the claims
set forth below, all percentages are by weight/weight, and all procedures
are carried out at ambient temperature and pressure, unless otherwise
specified.
EXAMPLE 1
An ordered, bilayer membrane assembly prepared from egg-derived
phosphatidyl choline is physically bonded to a polyacrylamide support by
Langmuir-Blodgett thin-film deposition. Afterwards, a 50 .ANG..times.50
.ANG. cross-sectional area density of lecitin known as concanavalin A, is
adsorbed onto the lipid membrane surface by hydrophilic effects at a
density not exceeding more than 50% of the membrane surface.
The lecitin-modified, supported lipid assembly is placed into an aqueous
solution. There is then added to the aqueous solution
2-capramido-1,6-anhydro-2-deoxy-.beta.D-glucopyranose as a crosslinker.
Each molecule of this crosslinker has one hydrophobic chain available for
associating with the lipid membrane. The amount of crosslinker added
provides a density of 1 part of crosslinker to 10 parts of the
membrane-forming lipids.
The mixture is gently stirred and heated slightly above ambient temperature
to assist spontaneous incorporation of the hydrophobic chains into the
lipid matrix. Once a density of 1 chain per 20 lipids is bound into the
lipid matrix, 2-acetamido-1,6-anhydro-2-deoxy-.beta.-D-glucopyranose is
added as an interlinker, in a ratio of 10 parts of the interlinker to 1
part of the crosslinker.
The mixture is then irradiated with UV light (254 nm) for 15 minutes at an
intensity of 40 milliwatt to effect a ring-opening polymerization of the
sugar monomers to form a mucous upper layer. As a result, there is
produced a lipid membrane-based device in accordance with the present
invention, having a polyacrylamide support, a complexing agent-modified,
ordered lipid membrane physically bonded to the polyacrylamide support,
and a polysaccharide mucous layer physically bonded to the lipid membrane.
EXAMPLE 2
Following the procedure of Example 1 except that the irradiation step is
appropriately modified, a device in accordance with the prsent invention
is prepared with an upper layer having a pore size that will permit a
compound having a molecular weight of about 800 to pass through, but that
blocks passage of an organic compound having a molecular weight of about
1000 or more. Using this device, an Ag/AgCl reference electrode, an
electrometer, a DC power supply, and 0.1M KCl at pH 7 as an electrolyte, a
liquid electrochemical cell is prepared. The cell is employed using
several known concentrations of a dextran having an average molecular
weight of about 800, to prepare a calibration curve. Then an aqueous
sample containing an unknown concentration of the dextran is introduced
into the electrochemical cell, the conductivity change is measured, and
the concentration is determined by comparison of the conductivity change
with the calibration curve.
EXAMPLE 3
The procedure of Example 1 is followed except that the lipid membrane is
doped with nonactin, which is maintained at a solution concentration of
10.sup.-5 M. The resulting gas sensor is used with a constant ionic
strength buffer (0.1M LiCl) directly in the gas phase to prepare a
calibration curve based upon several known concentrations of ammonia gas.
Then an unknown concentration of ammonia gas is analyzed using the gas
sensor, and the concentration is determined by comparison with the
calibration curve.
EXAMPLE 4
An ordered monolayer of the glycophospholipid (FIG. 1) is prepared by the
Langmuir-Blodgett thin-film deposition technique on a polyacrylamide or
metal-metaloxide surface. The glycophospholipid is prepared by treatment
of 2-amino-1,6-anhydro-2-deoxy glucose with nonanedioic acid, followed by
reaction of the resultant addition product with lysolauroyl lecithin, as
described, supra. The anhydro sugar moiety of the glycophospholipid, which
is laid on the surface as a monolayer, is polymerized by treatment with an
etherial solution of borontrifluoride-etherate. Alternatively,
polymerization can be effected by exposure to U.V. light of suitable
wavelength, say 254 nm. This procedure affords an ordered lipid membrane
covalently bonded to a polysaccharide umbrella.
If desired, a 2-acrylamido-1,6-anhydro-deoxy glucose can be utilized as a
cross-linking agent in the above-described polymerization procedure to
obtain the polysacharide.
The lipid membrane obtained by this procedure is then treated with
concanavalin A, followed by cross-linking and U.V. irradiation all as
described in Example 1, above, to obtain a lipid membrane-based device in
accordance with the present invention.
EXAMPLE 5
A silicon wafer containing an oxide layer of about 1000-1200 .ANG.
thickness is refluxed in chloroform for 2 to 3 hours to clean the surface
from any adhering hydrocarbons and greasy materials. Thereafter, the
chloroform is decanted, and the wafer is dried under vacuum. The dried
wafer is then silanized by refluxing it for three hours under a nitrogen
blanket with a solution of (3-aminopropyl)triethoxy silane in toluene in
the presence of a catalytic amount of triethyl amine. The wafer is taken
from the liquid, washed several times with chloroform and acetone, and is
then dried in vacuum. The thus silanized wafer is treated with
glycophospholipid (formula as shown in FIG. 1, except that it carries a
--COOH group in place of the terminal --CH.sub.3 group at the acyl chain)
in a stirred chloroform solution in the presence of catalytic amounts of
dimethylaminopyridine and dicyclohexylcarbodiimide under a nitrogen
atmosphere at room temperature for 48 hours. The wafer is thereafter
recovered from the reaction mixture, washed several times with chloroform,
dried under vacuum and stored under nitrogen.
The preparation of the glycophospholipid from phosphatidyl glycerol is as
described, supra.
The phosphoglycolipid covalently bound to the silicon surface obtained as
described above can be polymerized by exposing the wafer to U.V. light
(for example at 254 nm wavelength) or by treatment with
borontrifluoride-etherate in anhydrous ether solution. An anhydro-sugar
cross-linker may be added during this process, if desired. Further, the
procedure described in this Example 5 can be used in sensor applications
as described in Examples 2 and 3, above.
The above examples are illustrative of the present invention. It is to be
understood that these examples are not in any way to be interpreted as
limiting the scope of the invention. Rather, it is intended that the scope
of the invention be defined by the claims set forth below. It is
contemplated that the invention as hereinafter claimed, will be subject to
various modifications, which modifications are within the scope thereof.
INDUSTRIAL APPLICABILITY
The devices of the present invention are useful for the quantitative and
qualitative analysis of a specific chemical species, including certain
inorganic ion-forming gases such as ammonia.
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