Chemistry: electrical and wave energy – Apparatus – Electrolytic
Reexamination Certificate
2001-01-16
2004-05-25
Noguerda, Alex (Department: 1753)
Chemistry: electrical and wave energy
Apparatus
Electrolytic
C204S403050
Reexamination Certificate
active
06740213
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a biosensor comprising an electrode and membrane in which the conductance of the membrane changes in response to the presence of an analyte. In particular, the invention relates to the use of composite layer membranes incorporating a supporting layer comprising regions of low ionic or electronic mobility with regions of high ionic or electronic mobility.
International patent application WO 90/08783, the disclosure of which is incorporated herein by reference, discloses a membrane in which the conductance of the membrane is dependent on the presence or absence of an analyte. The membrane comprises a closely packed array of amphiphilic molecules and a plurality of ionophores comprising first and second half membrane spanning monomers. Ionophores within the top layer monomer are capable of lateral diffusion within the membrane. The membrane also includes receptor molecules and the binding of the analyte to the receptor molecules causes a change in the relationship between the first and second half membrane spanning monomers thereby altering the conductivity of the membrane. This type of gating mechanism is referred to as “lateral segregation”.
In the lateral segregation gating mechanism described in WO 90/08783 it is essential that the membrane include dimeric ion channels with at least one of the monomers being capable of diffusion within the membrane so that the relationship between the two monomers may be altered. The present inventors have developed a biosensor in which gating is achieved by allowing ion channels to diffuse in a lipid membrane between zones of the electrode with differing states of polarisation, conductivity, ion reservoir capacity, or redox potential. On binding of an analyte, the ion channels are locked into one of these regions. With this arrangement any ion channel, membrane spanning or otherwise, can be used.
The provision of the hydrophilic tethers has permitted the construction of a robust membrane but the elimination of that element would further increase the stability of the membrane. This is significant because the membranes themselves are only one or two molecules in thickness. Further, the membranes in use should be able to contact blood and other biological materials that have interfering substances which tend to disturb the integrity of the membrane and its ionic reservoir.
The use of molecular wires in connection with macromolecular devices has been reviewed in J. -M. Lehn, Perspectives in Supramolecular Chemistry—From Molecular Recognition towards Molecular Information Processing and Self-Organisation, (Agnew, Chem. Int. Ed. Engl. 29, 1990, 1304-1319). The molecular wire is a connector permitting electron flow between the different elements of a molecular electronic system. An example is based on caroviologens, long, conjugated, polyolefinic chains bearing pyridinium groups at each end. These have been incorporated into dihexadecyl phosphate vesicles but were not successful as conductors in that environment. Small quantities of zwitterionic caroviologens have been incorporated into phospholipid vesicles and accelerated the rate of reduction of an internal oxidant making it probable that electron conduction occurred. It has also been suggested that one could make polarised molecular wires which would have rectifying properties from conjugated polyolefinic chains, bearing an electron-acceptor group at one end and a donor on the other end.
Highly conducting molecular crystals prepared from porphyrin and phthalocyanine complexes have been prepared as derivatives of the metalloporphyrine skeleton (Hoffman B A and Ibers J A. Porphyrinic Molecular Metals, Acc. Chem. Res. 16, 1983, 15-21).
Molecular wires formed of bispyridium polyenes have been synthesised and incorporated into the bilayer membrane of sodium dihexadecyl phosphate vesicles so that they have formed membrane-spanning electron channels. These molecular wires were not adapted to provide conduction between a membrane and its supporting electrode but had the pyridinium sites close to the negatively charged outer and inner surfaces of the vesicles and the polyene chain crossed the lipidic interior of the membrane. (Arrhenius T S, Blanchard-Desce, M, Dvolaitzky, M, Lehn J-M and Maithete, J. Molecular devices: Caroviologens as an approach to molecular wires—synthesis and incorporation into vesicle membranes, Proc. Natl Acad. Sci. USA 83, 1986, 5355-5359).
Conducting organic materials similar to and including tetrabenzoporphyrine has been reported in the literature and its electrical properties noted. (Hanack M and Zipplies T. Synthesis and Properties of Doped &mgr; Oxo (tetrabenzoporphyrinato) germanium(IV). J. Am. Chem. Soc. 107, 1985, 6127-6129).
SUMMARY OF THE INVENTION
In a first aspect the present invention consists in a biosensor comprising an electrode and a membrane, the biosensor including at least two zones each zone differing from each other zone in a property; the membrane including a plurality of ionophores, at least a proportion of the ionophores being capable of lateral diffusion within the membrane, a plurality of first binding partner molecules attached to membrane elements positioned within a first zone such that the first binding partner molecules are prevented from diffusing laterally into a second zone, second binding partner molecules attached to the ionophores, the rate of lateral diffusion within the membrane of the first binding partner molecules and second binding partner molecules being different.
In a preferred embodiment of the invention there is provided an intermediate region between at least portions of the membrane and the electrode, the intermediate region functioning as a reservoir or as a source or sink for ions.
In a further preferred embodiment of the invention the at least two zones of the biosensor are due to differing zones in the electrode, the membrane, the intermediate region or combinations thereof.
In yet another preferred embodiment the property is selected from the group consisting of chemical, polarisation, admittance, ionic reservoir capacity or redox potential.
In a preferred forms of the invention, the electrode comprises a silicon silver composite or a silicon gold composite. Similarly, the electrode may comprise a pattern of silver or gold islands deposited onto silicon oxide or a pattern of silicon oxide islands deposited onto gold or silver. Indeed, a number of possible arrangements will readily occur to those skilled in this area. These include gold/aluminium, silver/gold, silicon rubber/gold, rubber/silver, titanium/gold and niobium/gold, or patterned lipid monolayers attached to the gold so that the lipid regions provide electronic/ionic insulation and the lipid free regions provide an ion reservoir. The essential. criterion is that the electrode comprises zones of differing states of polarisation, conductivity, ionic reservoir capacity or redox potential.
In the situation where the at least two zones of the biosensor comprise a pattern of islands, the islands are preferably arranged to be insulated from each other so that they may be measured independently, or electrically interconnected for simultaneous measurement of all gating sites. In International patent application No PCT/AU89/00352 arrangements for independent measurements are disclosed. This disclosure of PCT/AU89/00352 is hereby incorporated by reference.
In yet a further preferred embodiment of the present invention, the first and second zones comprise two interleaved but separated comb electrodes at different potentials. Preferably, the separation between adjacent teeth of the respective combs and the width of each tooth is less than one micron. The total number of teeth on each electrode is preferably approximately 500.
The first and second binding partner molecules may be attached to the membrane elements and ionophores by any of the techniques disclosed in Australian patent No 617687 or PCT/AU93/00509, the disclosures of which are incorporated by reference. The first binding molecules are attached to membrane
Crossley Maxwell John
Martin Alastair Scott
Osman Peter Damien John
Pace Ronald John
Australian Membrane and Biotechnology Research Institute
Gottlieb Rackman & Reisman P.C.
Noguerda Alex
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