Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is organic
Reexamination Certificate
1995-10-10
2003-06-03
Swartz, Rodney P (Department: 1645)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is organic
C204S403060, C204S415000, C204S418000, C205S778000, C205S793000, C435S007100, C435S817000
Reexamination Certificate
active
06573109
ABSTRACT:
The present invention relates to membranes for use in detecting the presence of an analyte.
BACKGROUND OF THE INVENTION
In International patent application No W090/08783, it is disclosed how a biosensor of high sensitivity and specificity may be constructed based on a lateral segregation principle incorporating ionophores in a supported bilayer membrane. The preferred embodiment of the invention described in this application included gramicidin as the ionophore, which is known to form a conducting channel only when two monomers, one in each of the two bilayer leaflets, align themselves appropriately to form a bilayer spanning dimer. The monomers in one monolayer (called the “bottom” monolayer) are restrained from lateral mobility by chemical crosslinking in that monolayer, or by attachment through suitable linking groups to an underlying substrate, or by some other means. The monomers in the other (called “top”) monolayer are free to diffuse laterally within that monolayer and form conducting channels by alignment with the bottom layer monomers. The top layer monomers have receptor moieties attached, which are accessible to the analyte in the solution phase above the membrane. These receptors may be any of the general types previously described, such as polyclonal or monoclonal antibodies, antibody fragments including at least one Fab fragment, antigens, etc. Another class (called “complementary”) of receptor moieties are also attached at the membrane surface. This second class of receptor moieties is restrained from lateral mobility by attachment through to the bottom (immobilised) layer. Detection of analyte occurs when an analyte molecule is bound, at complementary sites on itself, to two receptors of both the mobile and immobilised class. This restrains the gramicidin monomer attached to one receptor from aligning itself with a monomer in the bottom layer, so causing a lowering of membrane electrical conduction which constitutes the detection event.
Such biosensors typically possess comparable surface concentrations of channel attached and immobilised receptor moieties. As such, it is necessary to ensure that all immobilised and mobile receptors are respectively of the same type, as analyte induced cross-linking between mobile channel attached receptors will typically not lead to efficient gating. In addition, the detection sensitivity of such a device in a convenient time (approximately 100 seconds) is set by the known diffusion rate constant, K
on
(approximately 10
8
M
−1
s
1
) for binding from solution under physiological conditions. In order that a significant (approximately 50%) fraction of detection sites be occupied (here channels to be gated), the analyte concentration, c, must satisfy,
c
>1/(
K
on
X
100) (1)
This general requirement limits any detection device, operating under the above requirements, without some additional means of detection amplification and sets an analyte detection concentration limit of approximately 10
−10
M.
DESCRIPTION OF THE INVENTION
The present inventors have found that an improvement in sensitivity of membranes for use in detecting the presence of an analyte can be obtained by increasing the ratio of fixed receptor molecules to mobile receptor molecules above a ratio of 1:1.
Accordingly, the present invention consists in a membrane for use in detecting the presence of an analyte, the membrane comprising an array of closely packed self-assembling amphiphilic molecules and a plurality of first and second receptor molecules, the first receptor molecules being reactive with one site on the analyte and second receptor molecules being reactive with another site on the analyte, the first receptor molecules being prevented from lateral diffusion within the membrane whilst the second receptor molecules are free to diffuse laterally within the membrane, the membrane being characterized in that the ratio of first receptor molecules to second receptor molecules is 10:1 or greater.
In a preferred embodiment of the present invention the ratio of first receptor molecules to second receptor molecules is in the range 10:1 to 10
5
:1 and is preferably about 1,000:1.
In yet a further preferred embodiment of the present invention the first and second receptor molecules bind to different epitopes on the analyte.
In a preferred embodiment of the present invention a membrane is a bilayer and includes a plurality of ionophores comprising first half membrane spanning monomers provided in one layer and second half membrane spanning monomers provided in the other layer, the first half membrane spanning monomers being prevented from lateral diffusion within the membrane whilst the second half membrane spanning monomers are free to diffuse laterally within the membrane, the second receptor molecules being bound to the second half membrane spanning monomers such that the binding of the analyte to the first and second receptor molecules causes a change in the conductance of the membrane.
The first and second half membrane spanning monomers may be any such molecules known in the art, however, it is presently preferred that the first and second half membrane spanning monomers are gramicidin or one of its derivatives.
In a further preferred embodiment of the present invention the membrane includes membrane spanning lipids. It is further preferred that the first receptor molecules are attached to the membrane spanning lipids.
The present inventors have also developed a novel method of increasing the number of first receptor molecules by using a loose polymer network attached to the membrane. Accordingly, in another embodiment of the present invention linear polymer chains of radius of gyration of approximately 100 to 300 Å are attached to the surface of the membrane, the first receptor molecules being attached to the linear polymer chains.
The linear polymer chains are preferably attached to the membrane at one or two points through suitably functionalised lipids in the top layer. These may be membrane spanning lipids.
In a preferred embodiment of the present invention the radius of gyration of the linear polymer chains is approximately 200 Å. All antibodies then attached to the polymer chain will be within approximately 500 Å of the surface of the membrane.
It is preferred that the ratio of linear polymer chains to lipids in the membrane is approximately 1:10
4
. This should give “loose contact” packing of the polymer on the surface of the membrane thereby allowing free diffusion of the first half membrane spanning monomers.
The polymer chains are preferably condensed polyethylene glycol.
The radius of gyration, S
o
is given by
S
0
2
=⅓&agr;
2
l
2
n for a chain containing tetrahedral bonds of length, l, n is the number of links and &agr;
2
a constant characteristic or the polymer. For PEO (polyethylene oxide) (—CH
2
—CH
2
—O—)
m
1 is the average CH
2
—CH
2
or CH
2
—0 bond length, ~1.5 Å, and &agr;
2
~2.
n=3 m for PEO (ie ~3 times no. of monomer units).
If S
o
~200 Å, then n~25,000 (mw~400,000).
The mean mass fraction of polymer in the 500 Å thick layer is
∼
4
×
10
5
6
×
10
23
×
(
5
×
10
-
6
)
3
∼
0.005
,
i.e. <1%.
This should still permit reasonably easy lateral movement of antibody/ion channel complexes on the surface.
The readily available form of PEO is PEG, poly-ethyleneglycol. OH—CH
2
—CH
2
—(CH
2
—CH
2
—O) mCH
2
—CH
2
—OH. This has hydroxyl groups at each chain end. So a chain with n~25,000 and ~10 functional attachment points for membrane anchoring or antibody binding) might be formed by condensing shorter chained PEG (n~2,500) with a suitably bifunctional (e.g. dicarboxylic acid) molecule containing also a side chain (e.g. hydrazide) for antibody/lipid attachment.
It is envisaged that a common attachment chemistry be used for the antibodies and membrane attachment lipids (e.g. hydrazide linkage to aldehydes). The polymer may be attached first to the membrane surface (by adding it as a ~1% solution in saline) and the unreacted exces
Cornell Bruce A.
Pace Ronald J.
Australian Membrane and Biotechnology Research Institute
Nixon & Vanderhye
Swartz Rodney P
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