CA1057180A - Hydrophobic polymeric membrane - Google Patents
Hydrophobic polymeric membraneInfo
- Publication number
- CA1057180A CA1057180A CA235,597A CA235597A CA1057180A CA 1057180 A CA1057180 A CA 1057180A CA 235597 A CA235597 A CA 235597A CA 1057180 A CA1057180 A CA 1057180A
- Authority
- CA
- Canada
- Prior art keywords
- reactive
- membrane
- compound
- protein
- electrode
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
- B01D67/0081—After-treatment of organic or inorganic membranes
- B01D67/0093—Chemical modification
- B01D67/00931—Chemical modification by introduction of specific groups after membrane formation, e.g. by grafting
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
- B01D67/0081—After-treatment of organic or inorganic membranes
- B01D67/0093—Chemical modification
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
- B01D71/06—Organic material
- B01D71/76—Macromolecular material not specifically provided for in a single one of groups B01D71/08 - B01D71/74
- B01D71/82—Macromolecular material not specifically provided for in a single one of groups B01D71/08 - B01D71/74 characterised by the presence of specified groups, e.g. introduced by chemical after-treatment
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/002—Electrode membranes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/002—Electrode membranes
- C12Q1/003—Functionalisation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/817—Enzyme or microbe electrode
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T428/00—Stock material or miscellaneous articles
- Y10T428/31504—Composite [nonstructural laminate]
- Y10T428/31725—Of polyamide
- Y10T428/31768—Natural source-type polyamide [e.g., casein, gelatin, etc.]
Abstract
A B S T R A C T
An organic membrane which is specifically coreactive to another compound or group, e.g. to a protein, comprises a hydrophobic organic poly-meric substrate having pendant therefrom essentially hydrocarbon chain, said chains carrying groups specifically reactive with a chemical compound, e.g.
protein reactive groups such as oxirane groups. Encapsulation of a highly conductive electrode, e.g. platinum in such a membrane yields an electrode which, together with a reference electrode enables one to meansure the pre-sence or concentration of a compound in a mixture by contacting the mixture with such an electrode system and detecting any resulting change in the electrical charge on the membrane.
An organic membrane which is specifically coreactive to another compound or group, e.g. to a protein, comprises a hydrophobic organic poly-meric substrate having pendant therefrom essentially hydrocarbon chain, said chains carrying groups specifically reactive with a chemical compound, e.g.
protein reactive groups such as oxirane groups. Encapsulation of a highly conductive electrode, e.g. platinum in such a membrane yields an electrode which, together with a reference electrode enables one to meansure the pre-sence or concentration of a compound in a mixture by contacting the mixture with such an electrode system and detecting any resulting change in the electrical charge on the membrane.
Description
~057~80 This invention relates to organic membranes and to the preparation and use thereof.
The specific reaction of a particular protein with another particular protein is well known, for example, the development within animals of a specific antibody to combat a particular antigen is well known.
This specificity of one protein for another has been utilised in affinity chromatography to separate a specific protein which was present in a particular solution.
The selective reaction between two compounds exists for many substances; thus proteins, including hormones and enzymes, and poly~ and mono-saccharides selectively react with specific compounds; and all such reactions are identified herein as an immunochemical reaction. Although for convenience we refer hereinafter primarily to protein and protein reactive groups, it will be understood that other appropriate combinations of specifically co-reacting compounds or groupings may be employed instead of the proteins and protein reactive groups.
Affinity chromatographic materials, e.g. column packing objects, are prepared from hydrophilic polymeric surfaces which, by their polar nature, have reactive sites to which a protein reactive compound can be attached. The hydeophilic nature of the substrate is not affected by the attachment of the protein.
A protein molecule such as an antibody or antigen in a buffer solution has a net electrical charge, the
The specific reaction of a particular protein with another particular protein is well known, for example, the development within animals of a specific antibody to combat a particular antigen is well known.
This specificity of one protein for another has been utilised in affinity chromatography to separate a specific protein which was present in a particular solution.
The selective reaction between two compounds exists for many substances; thus proteins, including hormones and enzymes, and poly~ and mono-saccharides selectively react with specific compounds; and all such reactions are identified herein as an immunochemical reaction. Although for convenience we refer hereinafter primarily to protein and protein reactive groups, it will be understood that other appropriate combinations of specifically co-reacting compounds or groupings may be employed instead of the proteins and protein reactive groups.
Affinity chromatographic materials, e.g. column packing objects, are prepared from hydrophilic polymeric surfaces which, by their polar nature, have reactive sites to which a protein reactive compound can be attached. The hydeophilic nature of the substrate is not affected by the attachment of the protein.
A protein molecule such as an antibody or antigen in a buffer solution has a net electrical charge, the
- 2 -~6357~80 magnitude and polarity of which depend on the iso-electric point of the protein and the composition of the buffer. This charge changes as a result of anti-body-antigen reaction. Although the net change in electrical charge is of a magnitude to be detected, immobilisation of a protein on a hydrophilic membrane is unsatisfactory because the ionic action of water on the polar groups of the hydrophilic substrate is so great that it masks the slight electrical potential change induced by the immunochemical reaction.
We have now discovered that if all antibody is covalently attached to the surface of a hydrophobic polymer which, in turn, is deposited on a conductor, then the surface charge of the polymer/solution interface will depend on the net charge of the immobilised antibody. When corresponding antigen is present in the solution and the binding site of the antibody has not been destroyed during immobilisation (attachment to the polymer), the immunochemical reaction will occur at the interface with resulting change of the surface charge. This change can be measured potentiometrically against a reference electrode immersed ln the same solution .
Descripti_n of Invention A hydrophobic membrane having the capability of immobilising a protein has now been invented. The membrane comprises a substrate of a hydrophobic polymer capable of being swollen by solvent action 1~57~80 and in which a hydrocarbon, usually aliphatic, compound having an appropriately reactive group can be absorbed from a sol~ent system. The resulting hydrophobic membrane has pendant therefrom an essentially hydro-carbon chain which contains a protein reactive group, such as an oxirane group (epoxide) or other protein reactive group. The chain may have linkages other than carbon-carbon bonds, such as ether bonds and, for the purposes of this invention, will be considered an "essentially hydrocarbon chain".
Thus, according to one aspect of the invention we provide a specifically reactive membrane comprising a hydrophobic polymer having pendant therefrom essentially nydrocarbon chains, said chains carrying groups specific-ally reactive with a chemical compound. Preferably the membrane comprises hydrocarbon chains terminating in a group specifically reactive with a polypeptide.
Conveniently the membrane is provided with hydro-carbon'chains each having a reactive group capable of bonding to a specifically protein reactive grouping by contacting said reactive group with the protein reactive grouping whereby the said grouping becomes bound to the membrane via the chain. Convenient protein reactive grouping~ are antigens or antibodies.
The hydrophobic membrane having protein-immobilising ability is used to encapsulate a highly conductive electrode, for example platinum. A protein, e~g., an antibody, having a selectivity for reaction ~571~30 with a particular protein (antigen) is reacted at the protein-reactive site. Immersion of such a coated electrode in association with a reference electrode into an aqueous solution containing the antigen provides an electrically sensitive system capable of measuring the change in electrical charge of the solution-polymer interface caused by the capture of a particular protein (antigen) by the electrode with an - immunoreactive antibody. Alternatively, the antigen may be immobilised on the membrane to selectively capture the antibody.
Thus, according to a second aspect of the invention there is provided an electrode encapsulated within a sheath of specifically reactive membrane, According to a further aspect of the invention there is provided a method of detecting the presence, and optlmally the concentration,of a compound in a mixture containing the compound together with other molecules, e.g. solvent molecules, by contacting the mixture with an electrode encased within a membrane specifically reactive with the compound, and detecting any resulting change in the electrical charge on the membrane. Conveniently this may be accomplished by comparison wlth an appropriate reference electrode.
Process Description A hydrophobic polymeric membrane havlng selec-tive immunochemical ability(i.e. a specifically reactive membrane)is prepared by forming a membrane of a hydrophobic polymer. The polymer is preferably one which is capable of being swollen with an organic solvent, or mixture of solvents, particularly aliphatic solvents. Particularly useful polymers in the practice of this invention are those hydrophobic polymers which contain no pendant polar groups.
Typical polymers for this purpose include thermoplastic polymers such as polyvinyl chloride, polystyrene, poly-ethylene, polypropylene, silicone rubber, polyurethane, polycarbonate, polytetrafluoroethylene and the like.
Thermosetting polymers such as epoxy resins and cross-linked polyesters may also be used. Preferred polymers are those which may be coated upon an electrode by dip-casting or shrink-fitting.
The polymeric membrane is then treated with a solvent system capable of swelling the membrane for a period sufficient to result in swelling of the membrane at least to an extent that the hydrocarbon chain may be incorporated into the membrane in sufficient concentration, as subsequently described. The solvent system contains, besides an appropriate solvent, a hydrocarbon compound having a reactive site thereon, preferabIy at or near one end of the hydrocarbon chain.
The solvents used to swell the polymeric membrane are preferably those which may be readily removed by drying of the polymer. Thus, lower molecular weight solvents are generally preferred to higher molecular weight solvents.
As indicated hereinafter, it is preferred that the solvent is of a lower boiling point and more easily 1~57180 evaporated than the hydrocarbon compound having a reactive site thereon. A typical solvent mixture for PVC comprises petroleum ether of a 30 to 60C
boiling range and toluene. Other solvents or mixtures thereof may be employed for the swelling of PVC or other polymeric materials, but these will be known to the skilled man or they may be determined by simple tests.
A typical hydrocarbon compound having a reactive site thereon is n-decanol. Other compounds which are particularly useful in the invention are n-hexanol, n-decylamine, n-hexyl-amine, n-decanoic acid and like compounds having a labile hydrogen.
After the polymeric membrane has been soaked in the solvent system for a period suffici~ntly long to effect the required degree of swelling, the membrane is dried at an appropriate temperature to remove the solvent without removing substantial quantities of the hydrocarbon compound having the reactive site.
When petroleum ether, toluene and solvents of similar boiling point range are utilised a typical drying temperature is ahout 50 to 100C, conveniently 50 to 60 C, preferably under vacuum. Removal of the solvent gives a membrane having a low concentration of hydro-carbon chains pendant therefrom, each of said chains having a reactive group thereon, examples of which are hydroxyl, amine or carboxyl groups.
Selection of the solvent (and thereby its boiling point) and pressures to be employed in its removal will be made with regard to the nature and properties of membrane and of the hydrocarbon. The polymers used herein have known solvents for swelling same.
Following attachment of the hydrocarbon chain to the polymeric membrane a series of treatments may follow to provide a specif cally reactive membrane, i.e., a membrane having the ability to capture a specific compound, for example a protein, an enzyme, or a mono-or poly-saccharide, as in an immunochemical reaction.
It will be appreciated that the hydrocarbon chain may be provided with an appropriate specific reactive group before its introduction into the membrane (in which case it will of course be necessary to ensure that the group is free to display its reactivity -preferably by ensuring that it is not located less than about 4-6 carbon atoms from the membrane), or the group may be introduced into the chain after its attachment to the membrane. Specific reactive groups may be obtained by contacting the membrane with an appropriate material capable of reacting with the hydrocarbon chain to provide the necessary specific activity, or it may be necessary to employ a linking grouping to enable the reactive group on the chain to bond an appropriate specific reactive group. Such alternatives will be apparent to the skilled man and selection of the appropriate sequence will be made with regard to the -` ~057180 nature of the various materials and groups involved.
Thus, the use of a linking grouping for protein may involve the following sequences:
After the polymeric membrane is dried, it is immersed ln a solution containing a compound having a specific protein-reactive site and another reactive site reactive with the reactive group of the hydrocarbon chain now attached to the membrane. For example, if the reaction group of the hydrocarbon chain is a hydroxyl group, then a typical protein-reactive compound which may be employed is epichlorohydrin wherein an epoxide ether linking grouping is formed. Other hydrocarbon molecules may be introduced into the membrane, for example aliphatic amines, preferably primary and lS secondary amines, carboxyl containing aliphatic compounds and the like, and these may be reactive with epichloro-hydrin or with a bis-epoxide wherein a pair of oxirane rings is present for reaction. It is desirable that there is no likelihood of cross linking between a pair of pendant groups pending from the membrane surface.
A series of treatment suitable for an aliphatic alcohol pendant from a polymeric membrane is lllustrated as follows:
membrane / ~ OH + Cl-CH CH-CH
allphatic alcohol epichl8rohydrin / __~^--^^ O-CH2-C~-~H + HCl _ g _ Proteins having amino groups are capable of reacting with the oxirane group to attach to the membrane to provide a membrane with an immobilised protein capable of capturing a specific protein in an immuno-chemical type reaction, Other techniques for preparing an immobilised-protein membrane include:
(a) Thiophosgene or isocyanate coupling ~ /
membrane / ~______-NH2 + CSC1 / aliphatic amine phosgene /
/ ._~N=C=S + 2HCl _~ N=C=S + P--NH2 _~ /~N- -N-P
/ Protein (b) Azo coupling Cl 1 membrane / C=O
~NI2 + b / allphatic amine ~
/ paranitrobenzoyl chloride H ¦ ¦ 0 2 4 ~;~' / O NO2 sodium thiosulfate / ~~~~^~~~ N-~- 0 + 2 /
/ H ¦¦ 0 protein 0 N=NH
Another protein coupling system involves the formation of a diimide linkage between the hydrocarbon chain and a protein molecule; an aliphatic compound with a carboxyl group is reacted with carbodiimide, the reaction product of which is reacted with an appropriate protein.
After the protein-reactive linking compound (i.e.
compound containing an immobilising group) e.g. epi-chlorohydrin, is reacted with the labile hydrogen of the chain pending from the membrane surface to produce the protein linking grouping, it is preferably washed and placed in a solution containing the protein to be immobilised. A preferred reaction temperature is room temperature and it is generally preferred to allow one or two days for the reaction to proceed, The reaction is generally conducted in a slightly baslc medium.
After the protein i8 attached, it ls treated to wash off residue of unreacted materials and further reacted with a compound to neutralise any unreacted protein-reactive, i.e. immobilising, groups which remained after reaction with the protein molecule.
(Unreacted protein-reactive groups tend to be polar in ~057180 nature and are also undesirable in that they react nonspecifically with proteins and could give erroneous results in a protein detection device).
In conducting the process according to the instant invention, it is generally preferred to use a hydro-carbon compound which has a sufficient chain length to permit the reactive group to be somewhat remote from the surface of the membrane. Generally the hydrocarbon compound employed according to the invention has at least six carbon atoms in the main chain length. A
preferred length is one in which the main chain of the hydrocarbon compound contains about eight to twelve carbon atoms, and in any case is preferably selected so that the specific reactive group is distant from the membrane surface by at least 4, and preferably at least 6 or even 8 carbon atoms. If a protein-reactive linking compound which is reacted therewith is of significant chain length, so that the ultimate protein-reactive group is pendant from the surface by at least six carbon atoms, then the chain length of the hydrocarbon need not be as long.
Apparatus The novel membranes of the instant invention are particularly useful inasmuch as they can be utilised in devices for detecting the presence of a particular compound qualitatively and, preferably, quantitatively in a given mixture e.g. solution containing the compound.
-" 1057180 A sheath of the hydrophobic polymeric membrane con-taining a hydrocarbon chain with a reactive group is formed on the measuring electrode (the 'immunoelectrode') in a thickness of about 10 to 50 microns, with a thickness of about 20 to 40 microns being particularly preferred.
The membrane preferably does not exceed 100 ~, nor is less than 5 ~ in thickness. A protein or other appropriate compound of an immunochemical pair is immobilised in the membrane. The measuring electrode is used in conjunction with a reference electrode.
The two electrodes are immersed in a mixture, typically a solution which contains a protein or other compound of the type sought to be identified. The measuring electrode and the reference electrode are electrically lS connected to a meter sensitive to very slight changes in electrical potential. As the particular protein is captured by the measuring electrode, the electrical potential at the polymer-solution interface changes.
The slight change is detected by the meter, which has a high impedence in electrode circuitry, thus indicating the presence of the compound. By calibration, the meter may be used to determine quantitatively the amount of compound present in the solution.
The reference electrode may be of any of a wide range of convenient electrode materials many of which are known, although we have found it advantageous to employ as the reference electrode a second immunoelectrode, substantially identical with the measuring electrode ` `` 1057180 except that the specific reactive sites are blocked with a suitably reactive blocking agent so that the reference electrode is no longer responsive to the particular material being tested for. It does, however respond to non-selective adsorption of other molecules in the test solution as does the measuring elec-trode, so that the effect of non-selective adsorption, where it occurs, may be compensated for; measurement of the potential of an active measuring immuno-electrode against the potential of an identical (reference) immunoelectrode with blocked binding sites can effectively eliminate the effect of non-specific interactions.
The following Examples illustrate the invention. In the drawings;
Figure 1 shows graphically the dependence of the potential of a con-canavalin A immunoelectrode on the concentration of yeast mannan precipitated;
Figure 2 illustrates the concentration dependence for mannose at pH
6; and Figure 3 illùstrates the concentration dependence for 7S gamma globu-lin at 25C.
Example l A protein-immobilised membrane was formed on a platimum electrode by dip casting the electrode in polyvinyl chloride solution. The coating was dried.
A coating of about 25 microns in thickness was formed.
The polyvinyl chlorido coatcd electrode was inlmersed in a solvent sol-ution for three hours at room temperature. The solvent was 4.5 parts by volume or petroleum ether and 4.5 parts by volume of toluene and contained 1 part by volume of the hydrocarbon n-decanol. The electrode was then placed in a vacuum oven at 50C for a period of about 16 hours.
The dry electrode was immcrsed in a solution containing 10% epich-lorohydrin in l molar sodium hydroxide solution for 2 hours at about 60C. The ~.
' 1057~80 electrode was then washed with distilled water and placed in a 0.5 molar sodium bicarbonate solution containing the protein concanavalin A at room temper-ature for a period of about 48 hours.
The measuring immunoelectrode was then removed from the solution, washed with 0.5 molar solution bicarbonate buffer, and with 0.1 molar potassium hydrogen phthalate buffer at a pH of 3.0 containing 1 molar sodium chloride, then with distilled water, then with 0.1 molar solution of tris (hydroxyl methyl) aminomethane buffer having a pH of 7.8 containing 0.5 molar solution ethanolamine. (The ethanolamine is employed to block any unreacted epoxy groups).
The measuring electrode was then electrically connected through a sensitive meter to a reference electrode. It is generally desired to have a large amount of impedence in the connecting system between the two electrodes. Concanavalin A is known to precipitate selectively certain branch polysaccharides containing non-reducing alpha-D-hexapyranosyl or beta-D-fructofuranosyl end groups. Yeast mannan belongs to thls group and is known to be most reactive. The response of concanavalin A immunoelectrode to varying concentrations of yeast mannan at pH 6.0 and pH 3.5 is shown in Fig 1. The potential of the system was shown to alter with the concentration of yeast mannan solution. No change of potential, however, was observed when a solution of agar was added to the measured 1057~80 - solution. Agar is a polysaccharide which does not react with concanavalin A. The lack of change of electrical characteristics of the system when agar was added indicates that the concanavalin A selectivity was retained despite its immobilisation of the hydro-phobic membrane.
Example 2 Another example of the immunoelectrode specificity is illustrated in Eig 3.
An immunoelectrode was prepared as in Example 1.
Rabbit anti-human 7S gamma-globulin was reacted with the pendant epoxide group. The immunoelectrode was then immersed in a solution of human 7S gamma-globulin at pH 5.00 wherein the concentration was changed. The change in concentration was detected electrically.
The immunochemical reaction between rabbit anti-human 7S gamma-globulin and human 7S gamma-globulin is known not to occur at pH 7.8. When the immunoelectrode containing immobilised rabbit anti-human 7S gamma-globulin was immersed in a solution of human 7S gamma-globulin, no electrical response was noted.
The immunochemical reactlons rema~ned even though one compound was immobilised on a hydrophobic polymeric membrane.
The preferred electrode material is platinum, although any other suitable conducting material, either metal, e.g. copper or silver, or non-metal, e.g. carbon, may be employed. The electrode may be of any convenient ~057180 configuration, e.g. rod, wire or sheet. It is desirable that areas of the electrode not covered or ensheathed by the membrane should be insulated, and this is conveniently accomplished by enclosing other-wise exposed portions of the electrode and, where appropriate, its associated leads, within an appropriate insulating materiaL for example with platinum we prefer to employ a glass insulant, although other materials, e.g. PTFE may be employed.
What we claim is:-
We have now discovered that if all antibody is covalently attached to the surface of a hydrophobic polymer which, in turn, is deposited on a conductor, then the surface charge of the polymer/solution interface will depend on the net charge of the immobilised antibody. When corresponding antigen is present in the solution and the binding site of the antibody has not been destroyed during immobilisation (attachment to the polymer), the immunochemical reaction will occur at the interface with resulting change of the surface charge. This change can be measured potentiometrically against a reference electrode immersed ln the same solution .
Descripti_n of Invention A hydrophobic membrane having the capability of immobilising a protein has now been invented. The membrane comprises a substrate of a hydrophobic polymer capable of being swollen by solvent action 1~57~80 and in which a hydrocarbon, usually aliphatic, compound having an appropriately reactive group can be absorbed from a sol~ent system. The resulting hydrophobic membrane has pendant therefrom an essentially hydro-carbon chain which contains a protein reactive group, such as an oxirane group (epoxide) or other protein reactive group. The chain may have linkages other than carbon-carbon bonds, such as ether bonds and, for the purposes of this invention, will be considered an "essentially hydrocarbon chain".
Thus, according to one aspect of the invention we provide a specifically reactive membrane comprising a hydrophobic polymer having pendant therefrom essentially nydrocarbon chains, said chains carrying groups specific-ally reactive with a chemical compound. Preferably the membrane comprises hydrocarbon chains terminating in a group specifically reactive with a polypeptide.
Conveniently the membrane is provided with hydro-carbon'chains each having a reactive group capable of bonding to a specifically protein reactive grouping by contacting said reactive group with the protein reactive grouping whereby the said grouping becomes bound to the membrane via the chain. Convenient protein reactive grouping~ are antigens or antibodies.
The hydrophobic membrane having protein-immobilising ability is used to encapsulate a highly conductive electrode, for example platinum. A protein, e~g., an antibody, having a selectivity for reaction ~571~30 with a particular protein (antigen) is reacted at the protein-reactive site. Immersion of such a coated electrode in association with a reference electrode into an aqueous solution containing the antigen provides an electrically sensitive system capable of measuring the change in electrical charge of the solution-polymer interface caused by the capture of a particular protein (antigen) by the electrode with an - immunoreactive antibody. Alternatively, the antigen may be immobilised on the membrane to selectively capture the antibody.
Thus, according to a second aspect of the invention there is provided an electrode encapsulated within a sheath of specifically reactive membrane, According to a further aspect of the invention there is provided a method of detecting the presence, and optlmally the concentration,of a compound in a mixture containing the compound together with other molecules, e.g. solvent molecules, by contacting the mixture with an electrode encased within a membrane specifically reactive with the compound, and detecting any resulting change in the electrical charge on the membrane. Conveniently this may be accomplished by comparison wlth an appropriate reference electrode.
Process Description A hydrophobic polymeric membrane havlng selec-tive immunochemical ability(i.e. a specifically reactive membrane)is prepared by forming a membrane of a hydrophobic polymer. The polymer is preferably one which is capable of being swollen with an organic solvent, or mixture of solvents, particularly aliphatic solvents. Particularly useful polymers in the practice of this invention are those hydrophobic polymers which contain no pendant polar groups.
Typical polymers for this purpose include thermoplastic polymers such as polyvinyl chloride, polystyrene, poly-ethylene, polypropylene, silicone rubber, polyurethane, polycarbonate, polytetrafluoroethylene and the like.
Thermosetting polymers such as epoxy resins and cross-linked polyesters may also be used. Preferred polymers are those which may be coated upon an electrode by dip-casting or shrink-fitting.
The polymeric membrane is then treated with a solvent system capable of swelling the membrane for a period sufficient to result in swelling of the membrane at least to an extent that the hydrocarbon chain may be incorporated into the membrane in sufficient concentration, as subsequently described. The solvent system contains, besides an appropriate solvent, a hydrocarbon compound having a reactive site thereon, preferabIy at or near one end of the hydrocarbon chain.
The solvents used to swell the polymeric membrane are preferably those which may be readily removed by drying of the polymer. Thus, lower molecular weight solvents are generally preferred to higher molecular weight solvents.
As indicated hereinafter, it is preferred that the solvent is of a lower boiling point and more easily 1~57180 evaporated than the hydrocarbon compound having a reactive site thereon. A typical solvent mixture for PVC comprises petroleum ether of a 30 to 60C
boiling range and toluene. Other solvents or mixtures thereof may be employed for the swelling of PVC or other polymeric materials, but these will be known to the skilled man or they may be determined by simple tests.
A typical hydrocarbon compound having a reactive site thereon is n-decanol. Other compounds which are particularly useful in the invention are n-hexanol, n-decylamine, n-hexyl-amine, n-decanoic acid and like compounds having a labile hydrogen.
After the polymeric membrane has been soaked in the solvent system for a period suffici~ntly long to effect the required degree of swelling, the membrane is dried at an appropriate temperature to remove the solvent without removing substantial quantities of the hydrocarbon compound having the reactive site.
When petroleum ether, toluene and solvents of similar boiling point range are utilised a typical drying temperature is ahout 50 to 100C, conveniently 50 to 60 C, preferably under vacuum. Removal of the solvent gives a membrane having a low concentration of hydro-carbon chains pendant therefrom, each of said chains having a reactive group thereon, examples of which are hydroxyl, amine or carboxyl groups.
Selection of the solvent (and thereby its boiling point) and pressures to be employed in its removal will be made with regard to the nature and properties of membrane and of the hydrocarbon. The polymers used herein have known solvents for swelling same.
Following attachment of the hydrocarbon chain to the polymeric membrane a series of treatments may follow to provide a specif cally reactive membrane, i.e., a membrane having the ability to capture a specific compound, for example a protein, an enzyme, or a mono-or poly-saccharide, as in an immunochemical reaction.
It will be appreciated that the hydrocarbon chain may be provided with an appropriate specific reactive group before its introduction into the membrane (in which case it will of course be necessary to ensure that the group is free to display its reactivity -preferably by ensuring that it is not located less than about 4-6 carbon atoms from the membrane), or the group may be introduced into the chain after its attachment to the membrane. Specific reactive groups may be obtained by contacting the membrane with an appropriate material capable of reacting with the hydrocarbon chain to provide the necessary specific activity, or it may be necessary to employ a linking grouping to enable the reactive group on the chain to bond an appropriate specific reactive group. Such alternatives will be apparent to the skilled man and selection of the appropriate sequence will be made with regard to the -` ~057180 nature of the various materials and groups involved.
Thus, the use of a linking grouping for protein may involve the following sequences:
After the polymeric membrane is dried, it is immersed ln a solution containing a compound having a specific protein-reactive site and another reactive site reactive with the reactive group of the hydrocarbon chain now attached to the membrane. For example, if the reaction group of the hydrocarbon chain is a hydroxyl group, then a typical protein-reactive compound which may be employed is epichlorohydrin wherein an epoxide ether linking grouping is formed. Other hydrocarbon molecules may be introduced into the membrane, for example aliphatic amines, preferably primary and lS secondary amines, carboxyl containing aliphatic compounds and the like, and these may be reactive with epichloro-hydrin or with a bis-epoxide wherein a pair of oxirane rings is present for reaction. It is desirable that there is no likelihood of cross linking between a pair of pendant groups pending from the membrane surface.
A series of treatment suitable for an aliphatic alcohol pendant from a polymeric membrane is lllustrated as follows:
membrane / ~ OH + Cl-CH CH-CH
allphatic alcohol epichl8rohydrin / __~^--^^ O-CH2-C~-~H + HCl _ g _ Proteins having amino groups are capable of reacting with the oxirane group to attach to the membrane to provide a membrane with an immobilised protein capable of capturing a specific protein in an immuno-chemical type reaction, Other techniques for preparing an immobilised-protein membrane include:
(a) Thiophosgene or isocyanate coupling ~ /
membrane / ~______-NH2 + CSC1 / aliphatic amine phosgene /
/ ._~N=C=S + 2HCl _~ N=C=S + P--NH2 _~ /~N- -N-P
/ Protein (b) Azo coupling Cl 1 membrane / C=O
~NI2 + b / allphatic amine ~
/ paranitrobenzoyl chloride H ¦ ¦ 0 2 4 ~;~' / O NO2 sodium thiosulfate / ~~~~^~~~ N-~- 0 + 2 /
/ H ¦¦ 0 protein 0 N=NH
Another protein coupling system involves the formation of a diimide linkage between the hydrocarbon chain and a protein molecule; an aliphatic compound with a carboxyl group is reacted with carbodiimide, the reaction product of which is reacted with an appropriate protein.
After the protein-reactive linking compound (i.e.
compound containing an immobilising group) e.g. epi-chlorohydrin, is reacted with the labile hydrogen of the chain pending from the membrane surface to produce the protein linking grouping, it is preferably washed and placed in a solution containing the protein to be immobilised. A preferred reaction temperature is room temperature and it is generally preferred to allow one or two days for the reaction to proceed, The reaction is generally conducted in a slightly baslc medium.
After the protein i8 attached, it ls treated to wash off residue of unreacted materials and further reacted with a compound to neutralise any unreacted protein-reactive, i.e. immobilising, groups which remained after reaction with the protein molecule.
(Unreacted protein-reactive groups tend to be polar in ~057180 nature and are also undesirable in that they react nonspecifically with proteins and could give erroneous results in a protein detection device).
In conducting the process according to the instant invention, it is generally preferred to use a hydro-carbon compound which has a sufficient chain length to permit the reactive group to be somewhat remote from the surface of the membrane. Generally the hydrocarbon compound employed according to the invention has at least six carbon atoms in the main chain length. A
preferred length is one in which the main chain of the hydrocarbon compound contains about eight to twelve carbon atoms, and in any case is preferably selected so that the specific reactive group is distant from the membrane surface by at least 4, and preferably at least 6 or even 8 carbon atoms. If a protein-reactive linking compound which is reacted therewith is of significant chain length, so that the ultimate protein-reactive group is pendant from the surface by at least six carbon atoms, then the chain length of the hydrocarbon need not be as long.
Apparatus The novel membranes of the instant invention are particularly useful inasmuch as they can be utilised in devices for detecting the presence of a particular compound qualitatively and, preferably, quantitatively in a given mixture e.g. solution containing the compound.
-" 1057180 A sheath of the hydrophobic polymeric membrane con-taining a hydrocarbon chain with a reactive group is formed on the measuring electrode (the 'immunoelectrode') in a thickness of about 10 to 50 microns, with a thickness of about 20 to 40 microns being particularly preferred.
The membrane preferably does not exceed 100 ~, nor is less than 5 ~ in thickness. A protein or other appropriate compound of an immunochemical pair is immobilised in the membrane. The measuring electrode is used in conjunction with a reference electrode.
The two electrodes are immersed in a mixture, typically a solution which contains a protein or other compound of the type sought to be identified. The measuring electrode and the reference electrode are electrically lS connected to a meter sensitive to very slight changes in electrical potential. As the particular protein is captured by the measuring electrode, the electrical potential at the polymer-solution interface changes.
The slight change is detected by the meter, which has a high impedence in electrode circuitry, thus indicating the presence of the compound. By calibration, the meter may be used to determine quantitatively the amount of compound present in the solution.
The reference electrode may be of any of a wide range of convenient electrode materials many of which are known, although we have found it advantageous to employ as the reference electrode a second immunoelectrode, substantially identical with the measuring electrode ` `` 1057180 except that the specific reactive sites are blocked with a suitably reactive blocking agent so that the reference electrode is no longer responsive to the particular material being tested for. It does, however respond to non-selective adsorption of other molecules in the test solution as does the measuring elec-trode, so that the effect of non-selective adsorption, where it occurs, may be compensated for; measurement of the potential of an active measuring immuno-electrode against the potential of an identical (reference) immunoelectrode with blocked binding sites can effectively eliminate the effect of non-specific interactions.
The following Examples illustrate the invention. In the drawings;
Figure 1 shows graphically the dependence of the potential of a con-canavalin A immunoelectrode on the concentration of yeast mannan precipitated;
Figure 2 illustrates the concentration dependence for mannose at pH
6; and Figure 3 illùstrates the concentration dependence for 7S gamma globu-lin at 25C.
Example l A protein-immobilised membrane was formed on a platimum electrode by dip casting the electrode in polyvinyl chloride solution. The coating was dried.
A coating of about 25 microns in thickness was formed.
The polyvinyl chlorido coatcd electrode was inlmersed in a solvent sol-ution for three hours at room temperature. The solvent was 4.5 parts by volume or petroleum ether and 4.5 parts by volume of toluene and contained 1 part by volume of the hydrocarbon n-decanol. The electrode was then placed in a vacuum oven at 50C for a period of about 16 hours.
The dry electrode was immcrsed in a solution containing 10% epich-lorohydrin in l molar sodium hydroxide solution for 2 hours at about 60C. The ~.
' 1057~80 electrode was then washed with distilled water and placed in a 0.5 molar sodium bicarbonate solution containing the protein concanavalin A at room temper-ature for a period of about 48 hours.
The measuring immunoelectrode was then removed from the solution, washed with 0.5 molar solution bicarbonate buffer, and with 0.1 molar potassium hydrogen phthalate buffer at a pH of 3.0 containing 1 molar sodium chloride, then with distilled water, then with 0.1 molar solution of tris (hydroxyl methyl) aminomethane buffer having a pH of 7.8 containing 0.5 molar solution ethanolamine. (The ethanolamine is employed to block any unreacted epoxy groups).
The measuring electrode was then electrically connected through a sensitive meter to a reference electrode. It is generally desired to have a large amount of impedence in the connecting system between the two electrodes. Concanavalin A is known to precipitate selectively certain branch polysaccharides containing non-reducing alpha-D-hexapyranosyl or beta-D-fructofuranosyl end groups. Yeast mannan belongs to thls group and is known to be most reactive. The response of concanavalin A immunoelectrode to varying concentrations of yeast mannan at pH 6.0 and pH 3.5 is shown in Fig 1. The potential of the system was shown to alter with the concentration of yeast mannan solution. No change of potential, however, was observed when a solution of agar was added to the measured 1057~80 - solution. Agar is a polysaccharide which does not react with concanavalin A. The lack of change of electrical characteristics of the system when agar was added indicates that the concanavalin A selectivity was retained despite its immobilisation of the hydro-phobic membrane.
Example 2 Another example of the immunoelectrode specificity is illustrated in Eig 3.
An immunoelectrode was prepared as in Example 1.
Rabbit anti-human 7S gamma-globulin was reacted with the pendant epoxide group. The immunoelectrode was then immersed in a solution of human 7S gamma-globulin at pH 5.00 wherein the concentration was changed. The change in concentration was detected electrically.
The immunochemical reaction between rabbit anti-human 7S gamma-globulin and human 7S gamma-globulin is known not to occur at pH 7.8. When the immunoelectrode containing immobilised rabbit anti-human 7S gamma-globulin was immersed in a solution of human 7S gamma-globulin, no electrical response was noted.
The immunochemical reactlons rema~ned even though one compound was immobilised on a hydrophobic polymeric membrane.
The preferred electrode material is platinum, although any other suitable conducting material, either metal, e.g. copper or silver, or non-metal, e.g. carbon, may be employed. The electrode may be of any convenient ~057180 configuration, e.g. rod, wire or sheet. It is desirable that areas of the electrode not covered or ensheathed by the membrane should be insulated, and this is conveniently accomplished by enclosing other-wise exposed portions of the electrode and, where appropriate, its associated leads, within an appropriate insulating materiaL for example with platinum we prefer to employ a glass insulant, although other materials, e.g. PTFE may be employed.
What we claim is:-
Claims (2)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A hydrophobic membrane having one member of an immunochemically re-active pair immobilized thereon comprising:
a. a hydrophobic polymeric substrate swellable by an organic sol-vent, b. hydrocarbon chains of at least 6 carbon atoms in length parti-ally absorbed into the surface of said polymeric substrate, said hydrocarbon chains having a reactive site reactive with one compound of an immunochemi-cally reactive pair attached to its non-absorbed portion, and c. one member of an immunochemically reactive pair reacted with the reactive site of said hydrocarbon chain.
2. The membrane of claim 1 wherein the reactive sites of said hydrocar-bon chains unreacted with one compound of an immunochemically reactive pair contain a reaction-blocking molecule.
3. The membrane of claim 1 wherein said reactive site of the hydro-carbon chain is an oxirane group.
4. The membrane of claim 1 wherein said organic polymeric substrate is swellable by aliphatic solvents.
5. The membrane of claim 1 wherein the said hydrocarbon chain is the reaction product of n-decanol and epichlorohydrin.
6. A hydrophobic membrane of claim 1 wherein the compound of an immuno-chemically reactive pair is a protein.
7. The hydrophobic membrane of claim 1 wherein the compound of an im-munochemically reactive pair is an enzyme.
8. The hydrophobic membrane of claim 1 wherein the compound of an im-munochemically reactive pair is a monosaccharide.
9. The hydrophobic membrane of claim 1 wherein the compound of an im-munochemically reactive pair is a polysaccharide.
10. A quantitative device measuring electric potential difference for detecting presence of compounds reactive with an immobilized compound of an immunochemically reactive pair by utilization of an immunochemically chemical reaction comprising:
a. a reference electrode;
b. a reactive electrode comprising:
1. an electrical metal conductor;
2. a sheath of hydrophobic polymeric membrane comprising:
a. a hydrophobic polymeric substrate swellable by an organic solvent, b. hydrocarbon chains of at least 6 carbon atoms in length partially absorbed into the surface of said polymeric substrate, said hydro-carbon chains having a reactive site reactive with one compound of an immuno-chemically reactive pair attached to its non-absorbed portion, c. one member of an immunochemically reactive pair reacted with the reactive site of said hydrocarbon chain, and c. electrical conductor means connecting said reference electrode and measuring electrode through a meter sensitive to very slight changes in electrical potential.
a. a hydrophobic polymeric substrate swellable by an organic solvent, b. hydrocarbon chains of at least 6 carbon atoms in length partially absorbed into the surface of said polymeric substrate, said hydro-carbon chains having a reactive site reactive with one compound of an immuno-chemically reactive pair attached to its non-absorbed portion, c. one member of an immunochemically reactive pair reacted with the reactive site of said hydrocarbon chain, and c. electrical conductor means connecting said reference electrode and measuring electrode through a meter sensitive to very slight changes in electrical potential.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US05/506,464 US3966580A (en) | 1974-09-16 | 1974-09-16 | Novel protein-immobilizing hydrophobic polymeric membrane, process for producing same and apparatus employing same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1057180A true CA1057180A (en) | 1979-06-26 |
Family
ID=24014697
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA235,597A Expired CA1057180A (en) | 1974-09-16 | 1975-09-16 | Hydrophobic polymeric membrane |
Country Status (15)
| Country | Link |
|---|---|
| US (1) | US3966580A (en) |
| JP (1) | JPS5928257B2 (en) |
| AU (1) | AU501914B2 (en) |
| BE (1) | BE833473A (en) |
| CA (1) | CA1057180A (en) |
| CH (1) | CH629596A5 (en) |
| DE (2) | DE2560557C2 (en) |
| FR (1) | FR2284632A1 (en) |
| GB (1) | GB1527772A (en) |
| IT (1) | IT1042602B (en) |
| MX (1) | MX3075E (en) |
| NL (1) | NL181735C (en) |
| NZ (1) | NZ178701A (en) |
| SE (1) | SE415211B (en) |
| ZA (1) | ZA755903B (en) |
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| EP1991110B1 (en) | 2006-03-09 | 2018-11-07 | DexCom, Inc. | Systems and methods for processing analyte sensor data |
| US20080071158A1 (en) | 2006-06-07 | 2008-03-20 | Abbott Diabetes Care, Inc. | Analyte monitoring system and method |
| US20200037874A1 (en) | 2007-05-18 | 2020-02-06 | Dexcom, Inc. | Analyte sensors having a signal-to-noise ratio substantially unaffected by non-constant noise |
| DK3575796T3 (en) | 2011-04-15 | 2021-01-18 | Dexcom Inc | ADVANCED ANALYZE SENSOR CALIBRATION AND ERROR DETECTION |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3574062A (en) * | 1967-04-20 | 1971-04-06 | Beckman Instruments Inc | Method of immobilizing enzymes and product |
| CH453269A4 (en) * | 1968-03-29 | 1973-01-31 | ||
| US3843446A (en) * | 1971-04-20 | 1974-10-22 | Research Corp | Preparation of enzymatically active membranes |
| AT317424B (en) * | 1971-06-09 | 1974-08-26 | Boehringer Mannheim Gmbh | Process for the production of carrier-bound proteins |
| DE2247163A1 (en) * | 1972-09-26 | 1974-03-28 | Merck Patent Gmbh | CARRIER MATRIX FOR FIXING BIOLOGICALLY ACTIVE SUBSTANCES AND THE PROCESS FOR THEIR PRODUCTION |
| US3843443A (en) * | 1973-03-30 | 1974-10-22 | J Fishman | Polypeptide materials bound to fluorocarbon polymers |
-
1974
- 1974-09-16 US US05/506,464 patent/US3966580A/en not_active Expired - Lifetime
-
1975
- 1975-09-16 CA CA235,597A patent/CA1057180A/en not_active Expired
- 1975-09-16 NZ NZ178701A patent/NZ178701A/en unknown
- 1975-09-16 AU AU84861/75A patent/AU501914B2/en not_active Expired
- 1975-09-16 SE SE7510337A patent/SE415211B/en not_active IP Right Cessation
- 1975-09-16 NL NLAANVRAGE7510868,A patent/NL181735C/en not_active IP Right Cessation
- 1975-09-16 GB GB38037/75A patent/GB1527772A/en not_active Expired
- 1975-09-16 DE DE2560557A patent/DE2560557C2/de not_active Expired
- 1975-09-16 JP JP50112008A patent/JPS5928257B2/en not_active Expired
- 1975-09-16 CH CH1196275A patent/CH629596A5/en not_active IP Right Cessation
- 1975-09-16 BE BE160090A patent/BE833473A/en not_active IP Right Cessation
- 1975-09-16 DE DE2541308A patent/DE2541308C2/en not_active Expired
- 1975-09-16 FR FR7528422A patent/FR2284632A1/en active Granted
- 1975-09-16 IT IT27297/75A patent/IT1042602B/en active
- 1975-09-16 ZA ZA00755903A patent/ZA755903B/en unknown
- 1975-09-17 MX MX10018675U patent/MX3075E/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| DE2541308C2 (en) | 1984-12-20 |
| AU8486175A (en) | 1977-03-24 |
| AU501914B2 (en) | 1979-07-05 |
| FR2284632B1 (en) | 1979-06-22 |
| JPS5928257B2 (en) | 1984-07-11 |
| DE2560557C2 (en) | 1988-09-15 |
| CH629596A5 (en) | 1982-04-30 |
| BE833473A (en) | 1976-03-16 |
| SE415211B (en) | 1980-09-15 |
| FR2284632A1 (en) | 1976-04-09 |
| MX3075E (en) | 1980-03-18 |
| NL181735C (en) | 1987-10-16 |
| SE7510337L (en) | 1976-03-17 |
| JPS5185794A (en) | 1976-07-27 |
| NL7510868A (en) | 1976-03-18 |
| DE2541308A1 (en) | 1976-07-29 |
| IT1042602B (en) | 1980-01-30 |
| NL181735B (en) | 1987-05-18 |
| NZ178701A (en) | 1978-03-06 |
| GB1527772A (en) | 1978-10-11 |
| ZA755903B (en) | 1976-08-25 |
| US3966580A (en) | 1976-06-29 |
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