Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Patent
1995-05-17
1997-01-07
Housel, James C.
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
436525, 436528, 436532, 436806, 435 72, 435 75, 422 8202, 21050027, G01N 33543
Patent
active
055916473
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a membrane incorporating ionophores the conductance of which may be gated.
BACKGROUND OF THE INVENTION
The concept of membranes incorporating ionophores, the conductance of which are gated and the use of such membrane in biosensors is disclosed in International patent application Nos WO89/01159, WO90/08783, PCT/AU89/00352, PCT/AU92/00132 and PCT/AU93/00509. The disclosure of each of these documents is incorporated herein by reference.
The present invention stems from an observation made by the present inventor during the work conducted and disclosed in International application No WO90/08783. In this earlier application it was shown that the conduction of biotinylated gramicidin ion channels in a lipid membrane is greatly reduced by the binding of streptavidin by the biotins attached to the gramicidin, and that the changing conduction in this system is directly related to the quantity of streptavidin bound to the membrane ion channels. From this observation the present inventor has developed a general mechanism for analyte detection using a membrane incorporating ionophores.
DESCRIPTION OF THE INVENTION
Accordingly, in a first aspect the present invention consists in a membrane for use in the detection of an analyte, the membrane comprising a closely packed array of self-assembling amphiphilic molecules, a plurality of ionophores and a first and a second ligand attached to an end of the ionophore adjacent the surface of the membrane, characterised in that the binding of the first ligand to its binding partner prevents the flow of ions across the membrane via the ionophores and in that binding of the second ligand to its binding partner prevents the binding of the first ligand to its binding partner.
In a preferred embodiment of the present invention the first ligand is biotin, the binding partner of which is streptavidin.
In a further preferred embodiment of the present invention the ionophores are gramicidin or gramicidin analogues.
Typically, the second ligand is the analyte which the membrane is to be used for detecting. It is, however, possible that the second ligand may vary from ionophore to ionophore. In this manner the membrane could be used to detect the presence of more than one analyte.
BRIEF DESCRIPTION OF THE DRAWINGS
In order that the nature of the present invention may be more clearly understood the operation of the membrane of the present invention will be described with reference to the following schematic representations of the operation of the gating mechanism, in which
FIG. 1 is a schematic representation of a modified gramicidin ion channel with two ligands attached to the channel;
FIG. 2 is a schematic representation of the effect of adding anti-analyte antibody to the arrangement shown in FIG. 1;
FIG. 3 is a schematic representation of the use of the system shown in FIG. 1;
FIG. 4 is a graph of final/initial impedance against concentration of 2,4-dinitroanaline; and
FIG. 5 is a representation of gramicidin B.
DETAILED DESCRIPTION OF THE INVENTION
FIG. 1 shows a modified gramicidin ion channel (10) with two ligands (12 and 14) attached to the C terminus of the channel (10). One ligand (12) is biotin and is capable of binding streptavidin (16) so as to reduce the gramicidin (10) conductance. The second ligand (14), connected in close proximity to the biotin ligand (12), is the analyte of interest, an epitopic portion of the analyte, a structural analogue of either of the above or any ligand capable of competitively binding with an antibody raised against the analyte. In the absence of any species binding to the second ligand (14), binding of streptavidin (16) to the biotin ligand (12) effects a gating of the channel (10).
FIG. 2 demonstrates the effect of adding an anti-analyte antibody (18) to the system described above. Attachment of the antibody (18) to the second ligand (14), while not of itself effecting the conductance of channel (10), sterically precludes binding of streptavidin (16) to the biotin ligand (12). As many channels (10) as
REFERENCES:
patent: 5168057 (1992-12-01), Oh et al.
patent: 5436170 (1995-07-01), Cornell et al.
Australian Membrane and Biotechnology Research Institute
Housel James C.
Murthy Prasad A.
The University of Sydney
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