Biosensor for detection of small molecule analytes

Details Biosensor for detection of small molecule analytes Biosensor for detection of small molecule analytes

1998-03-13

2001-11-13

Mertz, Prema (Department: 1646)

Chemistry: analytical and immunological testing

Involving an insoluble carrier for immobilizing immunochemicals

C436S527000, C436S532000, C435S007200

Reexamination Certificate

active

06316273

ABSTRACT:

The present invention relates generally to mechanisms for modifying the electrical conductivity of ionophore containing membrane based biosensors in response to the concentration of small analytes or haptens in a sample.
Biomembranes have been constructed from a double layer of closely packed amphiphilic lipid molecules. The molecules of these bilayers exhibit the random motions characteristic of the liquid phase, in which the hydrocarbon tails of the lipid molecules have sufficient mobility to provide a soft, flexible, viscid surface. The molecules can also diffuse in two dimensions freely within their own monolayer so that two neighbouring lipids in the same monolayer exchange places with each other in a time interval several orders of magnitude less than the time for lipid molecules in opposite monolayers to exchange places.
These membrane may incorporate a class of molecules, called ionophores, which facilitate the transport of ions across these membranes. Ion channels are a particular form of ionophore, which as the term implies, are channels through which ions may pass through membranes. A favoured ionophore is gramicidin A which forms aqueous channels in the membrane. Australian Patent Specification No. 40123/85 discloses the use of membranes including ionophores in biosensors. Further examples of gated ionophores are found in Australian Patent Specification No. 21279/88, which discloses receptor molecules conjugated with a support that is remote from the receptor site. The support may be a lipid head group, a hydrocarbon chain, a cross-linkable molecule or a membrane protein or membrane polypeptide. The inner level of the membrane may be adjacent to a solid surface with groups reactive with the solid surface, and spaced from the surface to provide a reservoir region as disclosed in International Patent Specification No. 92/17788 (the disclosure of which is incorporated herein by reference).
Biosensors based on ion channels or ionophores contained within lipid membranes tethered to or deposited onto metal electrodes are disclosed in Australian Patent Specification Nos. 50334/90 and 40787/89. Those references disclose a membrane bilayer in which each layer has incorporated therein ionophores and in which the conductance of the membrane is dependent upon the presence or absence of an analyte. The disclosure of Australian Patent Specification No. 50334/94 (incorporated herein by reference) describes various ionophore gating mechanisms to modify the conductivity of the membrane in response to the presence of an analyte. In each of those gating mechanisms an inner layer of the membrane (the layer closer to the solid electrode surface, if any) contains immobilised or tethered half membrane spanning ion channels while an outer layer contains more mobile half membrane spanning ion channels. One method for immobilising the ion channels or the inner layer is to employ a polymerizable lipid layer and then cross-link the molecules of the inner monolayer and the ionophore. The conductivity of the membrane is altered by the extent to which opposing half membrane spanning ion channels align to establish a membrane spanning channel for ion transmission across the membrane.
Other biosensors based on ion channels or ionophores contained within membranes are described in International Patent Specification Nos.
WO92/17788, WO93/21528, WO94/07593 and U.S. Pat. No. 5,204,239 (the disclosures of which are incorporated herein by reference). These applications also disclose methods of producing membranes with improved sensitivity using a surface amplifier effect, stability and ion flux using chemisorbed arrays of amphiphilic molecules attached to an electrode surface and means of producing lipid membranes incorporating ionophores on said chemisorbed amphiphilic molecules.
The present inventors have now developed a modified biosensor for use in the detection of small analytes. The modified biosensor can, however, also be used with larger size analytes.
Accordingly in the first aspect the present invention consists in a biosensor for use in detecting analytes, the biosensor comprising a membrane and an electrode and a reservoir defined there between, the membrane having an inner layer proximate the electrode and an outer layer remote from the electrode comprising a closely packed array of amphiphilic molecules, a plurality of ionophores, and a plurality of membrane spanning lipids, the ionophores comprising first and second half membrane spanning monomers, the first half membrane spanning monomers being provided in the inner layer and being prevented from lateral diffusion within the membrane and the second half membrane spanning monomers being provided in the outer layer and being free to diffuse laterally within the membrane, the second half membrane spanning monomers having attached thereto a first receptor which is reactive with the analyte, wherein a carrier to which is attached a plurality of the analyte is reversibly bound to the first receptor via the analyte.
In a preferred embodiment of the first aspect of the present invention, a second receptor which is also reactive with the analyte is provided on the membrane spanning lipids which are prevented from lateral diffusion in the membrane.
The first and second receptors may be the same or different.
The carrier is preferably bound, reversibly, to two or more first receptors or two or more first and second receptors, such that the carrier forms a “bridge” between the membrane members to which the receptors are attached. In this way, a portion of the second half membrane spanning monomers may be caused to locate into a position out of registration with the first half membrane spanning monomers, thereby reducing the conductivity of the membrane.
Preferably, the carrier is substantially larger than the analyte, for example 2-50 times the molecular weight of the analyte.
In a second aspect the present invention consists in a biosensor for use in detecting analytes, the biosensor comprising a membrane and an electrode and a reservoir defined there between, the membrane having an inner layer proximate the electrode and an outer layer remote from the electrode comprising a closely packed array of amphiphilic molecules, a plurality of ionophores, and a plurality of membrane spanning lipids, the ionophores comprising first and second half membrane spanning monomers, the first half membrane spanning monomers being provided in the inner layer and being prevented from lateral diffusion within the membrane and the second half membrane spanning monomers being provided in the outer layer and being free to diffuse laterally within the membrane, the second half membrane spanning monomers having attached through a carrier and/or linker group at least one analyte, wherein a receptor which is reactive with the analyte is reversibly bound to the second half membrane spanning monomers via the analyte and said carrier and/or linker group.
In a preferred embodiment the receptor has two or more analyte binding sites thereby allowing the receptor to form a bridge between two or more second half membrane spanning monomers. In this way, a portion of the second half membrane spanning monomers may be caused to aggregate, out of registration with the first half membrane spanning monomers, thereby reducing the conductivity of the membrane.
Preferably, the analyte binding sites on the receptor are separated by less than 80% of the distance between the first half membrane spanning monomers. Also, preferably, the second half membrane spanning monomer have attached through a carrier and/or linker group a plurality of the analyte.
In a further preferred embodiment of the present invention, the first receptors or, in the case of a biosensor according to the second aspect, the analyte or carrier is attached to the second half membrane spanning monomers via linker groups. Similarly, it is preferred that any second receptors are attached to the membrane spanning lipids via linker groups. Examples of suitable linker groups include protein(s) and polymers. Preferred linker groups

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