Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1999-03-08
2002-07-09
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S071000, C436S528000, C436S806000, C435S287100, C435S289100, C435S291400, C435S817000, C422S082010, C422S082030, C427S002110, C427S002130, C427S058000, C427S337000, C427S338000, C204S296000, C204S418000, C204S400000, C204S403060, C204S415000, C204S416000
Reexamination Certificate
active
06417009
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel molecules which may be advantageously incorporated in membrane based biosensors.
BACKGROUND OF THE INVENTION
Previous patents such as WO 92/17788, U.S. Pat. No. 5,204,239 and WO 93/21528 (the disclosures of which are incorporated herein by reference) have described how functional biosensor bilayer or monolayer lipid membranes may be formed on a metal substrate such that a functioning ionic reservoir is formed between the metal surface and the lipid membrane. The inner leaflet of the membrane, or in the case of the monolayer membrane the whole membrane is typically assembled using molecules that comprise within the same molecule a hydrophobic group linked to a hydrophilic group onto which is attached an attachment group such as a disulfide or thiol group capable of attaching the molecule to an electrode. Furthermore, it has been disclosed in WO 94/07593 (the disclosure of which is also included herein by reference) that in order to provide improved reservoir characteristics and fluidity characteristics of the membrane a small spacer molecule, such as the disulfide of mercaptoacetic acid, should be incorporated between the reservoir molecules that had been adsorbed onto the metal surface.
The present inventors have now determined that if the functionality of the small spacer molecule is covalently incorporated into the reservoir molecules described previously, such that a single molecule is formed, then improvements in stability and reproducibility of the membrane formation, as well as improved ionophore conduction can be achieved. Additionally the manufacture of the membrane is simplified as fewer components are required.
SUMMARY OF THE INVENTION
Accordingly, in a first aspect, the present invention consists in a linker lipid for use in attaching a membrane including a plurality of ionophores to an electrode and providing a space between the membrane and the electrode in which the membrane is either in part or totally made up of the linker lipid, the linker lipid comprising within the same molecule a hydrophobic region capable of spanning the membrane, an attachment group used to attach the molecule to an electrode surface, a hydrophilic region intermediate said hydrophobic region and the attachment group, and a polar head group region attached to the hydrophobic region at a site remote from the hydrophilic region wherein said attachment group has a cross sectional area that is at least two times the cross sectional area of the hydrophilic region.
It is preferred that the head group, hydrophobic region, and hydrophilic region are as described previously in WO 92/17788 and WO 94/07593. The linker lipid in this case may be wholly synthetic or derived from naturally occurring membrane spanning lipids or archaebacterial lipids.
The hydrophilic region of the linker lipid is preferably a long chain hydrophilic compound. The hydrophilic region of the linker lipid may be composed of oligo/poly ethers, oligo/poly peptides, oligo/poly amides, oligo/poly amines, oligo/poly esters, oligo/poly saccharides, polyols, multiple charged groups (positive and/or negative), electroactive species or combinations thereof. The main requirement of the hydrophilic region of the linker lipid is that it allows the diffusion of ions through the ionophores provided in the membrane. This is achieved by the placement of suitable ion and/or water binding sites along or within the length of the long chain that makes up the reservoir region.
In a preferred embodiment of the invention the hydrophilic region consists of an oligoethylene oxide group. The oligoethylene oxide group may consist of four to twenty ethylene oxide units.
In a further preferred embodiment the hydrophilic region consists of a subunit of tetraethylene glycol attached to succinic acid. This tetraethylene glycol/succinic acid subunit may be repeated 1-4 times.
In a further preferred embodiment the hydrophilic region is formed by group transfer or anionic polymerisation of suitable monomers.
In a further preferred embodiment the hydrophilic region consists of mercaptoethanol, succinic acid, 1,4-diesterified 1,2,3,4-butanetetraol and succinic acid subunits. The succinic acid/1,4-diesterified 1,2,3,4-butanetetraol may be repeated 1-4 times.
In yet another embodiment the hydrophilic region may consist of an oligopropylene glycol of between 1 to 20 propylene glycol units in length. It is further preferred that the hydrophilic region consists oligopropylene glycols of between 2 and 8 propylene glycol units that are functionalised at each end with an N-alkyl amine functionality and that may be joined together via acid units forming tertiary amides.
It is further preferred that the hydrophilic region consists of oligoethylene glycols of between 2 and 10 ethylene glycol units that are functionalised at each end with an N-alkyl amine functionality and that may be joined together via acid units forming tertiary amides.
In a preferred embodiment of the present invention the head group of the linker lipid comprises a receptor reactive with an analyte or a group capable of attaching to a protein receptor.
In a preferred embodiment, the head group comprises a biotin or biotin derivative capable of complexing streptavidin, avidin or one of the common biotin binding proteins.
In a further preferred embodiment the biotin group is linked to the linker lipid via 1 to 8 aminocaproyl groups.
In a further preferred embodiment two biotin groups are attached to the linker lipid such that both biotin groups are capable of complexing a single avidin or streptavidin molecule so as to increase the overall complexing ability and strength of the linker lipid to the avidin or streptavidin.
In a further preferred embodiment of the present invention the hydrophobic region of the membrane spanning lipid comprises a hydrocarbon backbone of between 20-60 angstroms in length with sites of attachment at either end of the hydrocarbon backbone to which are attached at least two hydrocarbon side chains such as phytanyl chains.
In a further preferred embodiment of the present invention the hydrophobic region of the membrane spanning lipid comprises a hydrocarbon backbone of between 20-60 angstroms in length with sites of attachment at either end of the hydrocarbon backbone to which are attached at one end zero or one hydrocarbon sidechain and at least two to four hydrocarbon sidechains at the other end.
The hydrocarbon backbone may comprise a straight methylene chain hydrocarbon, or a hydrocarbon chain optionally substituted with additional groups selected from alkyl, aryl, ether and amine groups, or may comprise two shorter hydrocarbon chains that have been joined via ether, amine, or biphenyl ether groups. Those skilled in the art will appreciate that other functionalities that can link two hydrocarbon chains may also be employed.
It is preferred that the means by which the hydrocarbon chains are attached to the hydrocarbon backbone is via a polyhydroxylated hydrocarbon containing from 3 to 20 hydroxyl groups.
It is further preferred that the means by which the hydrocarbon sidechains are attached to the hydrocarbon backbone is via glycerol, glutamic acid, erythritol, threitol or pentaerythritol groups.
It is preferred that the length of the hydrocarbon sidechains are approximately half the total length of the hydrocarbon backbone.
It is further preferred that the hydrocarbon sidechains are phytanyl chains.
It is further preferred that the hydrocarbon sidechains are mono- or per-methylated hydrocarbon chains or a hydrocarbon chain optionally substituted with additional groups selected from alkyl, aryl, ether and amine groups.
It is preferred that for the case of the electrode material being a gold, platinum, palladium, silver or other coinage metal substrate or combination thereof, the attachment region includes sulfur containing groups such as thiols, disulfides, sulfides thiocyanates. However as previously described, other groups such as organosilanes that form strong attachment to a variety of conductive substrates may
Burns Christopher John
Field Leslie David
Raguse Burkhard
Ridley Damon Donald
Australian Membrane and Biotechnology Institute
Chin Christopher L.
Nixon & Vanderhye
Padmanabhan Kartic
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