Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-01-07
2002-08-13
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C204S400000, C204S403060, C422S082010, C422S082020, C422S082030, C435S287100, C436S071000, C436S528000, C436S806000
Reexamination Certificate
active
06432629
ABSTRACT:
The present invention relates to electrode membrane combinations for use in biosensors to detect analytes in a sample and to methods for the production of such electrode membrane combinations. The present invention also relates to methods for storing such electrode membrane combinations.
Biosensors based on ion channels or ionophores contained within lipid membranes that are deposited onto metal electrodes and where the ion channels are switched in the presence of analyte molecules have been described in International patent specification Nos WO 92/17788, WO 93/21528, WO 94/07593 and U.S. Pat. No. 5,204,239 (the disclosures of which are incorporated herein by reference). As is disclosed in these applications, ionophores such as gramicidin ion channels may be co-dispersed with amphiphilic molecules, thereby forming lipid membranes with altered properties in relation to the permeability of ions. There is also disclosure of various methods of gating these ion channels (for example, the lateral segregation mechanism disclosed in International Patent Application WO90/08783) such that in response to the binding of an analyte to a binding partner attached to the membrane, the conductivity of the membrane is altered. The applications also disclose methods of producing membranes with improved sensitivity using a surface amplifier effect, and improved stability and ion flux using chemisorbed arrays of amphiphilic molecules attached to an electrode surface. The applications further disclose means of producing lipid membranes incorporating ionophores on said chemisorbed amphiphilic molecules.
The present inventors have now determined improved means of producing electrode membrane combinations that result in sensor membranes with improved properties in terms of reproducibility, gating response towards an analyte, lateral segregation response, surface amplifier effect, stability in serum, plasma and blood, simplified production and the ability to store the membranes in a dry format (i.e. in the absence of any aqueous bath solution).
In the first aspect, the present invention consists in a method of producing a first layer electrode membrane comprising:
(1) Forming a solution containing Linker Lipid
A
(FIG.
1
), the disulfide of mercaptoacetic acid (MAAD) or similar molecule, such as EDS linker Gramicidin B (FIG.
2
), membrane spanning lipid C (MSL-C) (
FIG. 3
) and membrane spanning lipid D (MSL-D) (
FIG. 3
) or other linker molecules and ion channel or ionophore combinations as previously described;
(2) Contacting an electrode containing a clean gold surface with the solution, the disulfide containing components in the solution thus adsorbing onto the gold surface of the electrode;
(3) Rinsing the electrode with a suitable organic solvent; and
(4) Removing the excess organic solvent used for rinsing.
The nature of the membrane components are as follows:
Linker Lipid
A
comprising a benzyl disulfide attachment region, a hydrophilic region composed, in sequence, of tetraethylene glycol, succinic acid, tetraethylene glycol and succinic acid subgroups and an aliphatic chain;
The disulfide of mercaptoacetic acid (MAAD) or similar molecule, such as the disulfide of 2-mercaptoethanol (EDS).
Linker Gramicidin
B
is a linker molecule which comprises a benzyl disulfide attachment region, a hydrophilic region composed, in sequence, of tetraethylene glycol, succinic acid, tetraethylene glycol, succinic acid, and a hydrophobic region of gramicidin;
Membrane spanning lipid (MSL)
D
which comprises a benzyl disulfide attachment region, a hydrophilic region composed, in sequence, of tetraethylene glycol, succinic acid, tetraethylene glycol, succinic acid and a hydrophobic region of 1,1′dotriacontamethylenebis (2-3 RS,7R, 11-phytanyl) with an intermediate biphenyl region and a head group of phosphatidylcholine, hydroxyl, succinic acid, or PEG-400 COOH; and
Membrane spanning lipid
C
which comprises the same attachment and hydrophilic region as membrane spanning lipid
D
but differs in the head group which is a group consisting of (one to eight) 1,6-amino caproic acid and biotin.
In a preferred embodiment of the present invention the ratio of Linker Lipid
A
to the disulfide of mercaptoacetic acid (MAAD) or 2-mercaptoethoethanol (EDS) is 5:1 to 1:2, more preferably is 2:1.
It is further preferred that in order to improve the stability of the membrane, the amount of MSL-D in the first layer is as high as can be allowed and still maintain reasonable gramicidin conduction. The ratio of (Linker Lipid
A
+MAAD or EDS) to MSL-D is therefore preferably between 10:1 to 100:1.
In a further preferred embodiment, the amount of MSL-C is such that in the final sensor membrane an effective surface amplification on addition of analyte occurs, while still making it possible to suppress the lateral segregation induced gating on addition of the streptavidin, avidin or other similar biotin-binding protein. It should be noted that if the amount of MSL-C in the final sensor membrane is too large, then the excess protein that is bound to the MSL-C on addition of the streptavidin, avidin or similar biotin-binding protein will restrict the mobility of the gramicidin/receptor couple thereby reducing the gating response. In cases where the analyte molecule has multiple identical epitopes, MSL-C may capture the analyte molecules in preference to gramicidin/receptor couple, reducing the biosensor response.
It is therefore preferred that the ratio of (Linker Lipid
A
+MAAD or EDS) to membrane spanning lipid
C
is between 20,000:1 and 100:1.
It is further preferred that the ratio of (Linker Lipid
A
+MAAD or EDS) to MSL-C is 20,000:1.
As is known in the art, gramicidin exists in a monomer/dimer equilibrium in a bilayer membrane. In order for the gramicidin lateral segregation switch to function effectively, the ratio of monomer to dimer must be controlled. It is preferred that a proportion of the gramicidin ion channels exist as freely diffusing monomers in the outer membrane layer. The ratio of monomers to dimers can be controlled, amongst other methods, by changing the concentration of gramicidin in the first and second half of the membrane.
It is therefore preferred that the ratio of (Linker Lipid
A
+MAAD or EDS) to linker Gramicidin B is 10,000:1.
It is further preferred that the ratio of (Linker Lipid
A
+MAAD or EDS) to linker Gramicidin B is between 20,000:1 and 100,000:1 in those cases where it is necessary to minimise the amount of background leakage due to the adsorbed linker Gramicidin B.
It is preferred that the gold electrode consists of a freshly evaporated or sputtered gold electrode. It is further preferred that the gold electrode surface be freshly cleaned using a plasma etching process or an ion beam milling process.
It is preferred that the solvent for the adsorbing solution (step (1) and for the rinsing step (4) is ethanol.
In a second aspect, the present invention consists in a method of producing a monolayer electrode membrane comprising:
(1) Forming a solution containing the disulfide of mercaptoacetic acid (MAAD) or similar molecule (e.g. 2-mercaptoethanol (EDS)), membrane spanning lipid C(MSL-C) and/or membrane spanning lipid D (MSL-D) and, optionally, Linker Lipid
A
, linker Gramicidin B or other linker molecules or ion channel or ionophore combinations;
(2) Contacting an electrode containing a clean gold surface with the solution, the disulfide containing components in a solution thus adsorbing onto the gold surface of the electrode;
(3) Rinsing the electrode with a suitable organic solvent; and
(4) Removing the excess organic solvent used for rinsing,
wherein the solution in step (1) contains more than a molar % of 50% of a membrane spanning lipid.
More preferably, the solution in step (1) contains more than a molar % or 70% of a membrane spanning lipid, 29% MAAD or EDS and 1% other membrane spanning lipids.
The preferred features and embodiments discussed above in regard to the method of the first aspect of the invention, may be equally applicable to the method of
Braach-Maksvytia Vijoleta Lucija
Cornell Bruce
King Lionel George
Pace Ronald John
Raguse Burkhard
Australian Membrane and Biotechnology Research Institute
Chin Christopher L.
Gottlieb Rackman & Reisman P.C.
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