Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
2000-07-28
2003-03-25
Warden, Sr., Robert J. (Department: 1749)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C205S777500, C204S403010, C422S082020, C436S524000, C436S525000, C436S806000, C427S002110, C427S058000
Reexamination Certificate
active
06537441
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to biosensors which include or are fabricated using optically sensitive moieties.
BACKGROUND
Biosensors have been constructed comprising biomembranes which are 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 hydrogen tails of the lipid molecules have sufficient mobility to provide a soft, flexible, viscid surface. The molecules can also diffuse sideways freely within their own monolayer so that two neighbouring lipids in the same monolayer exchange places with each other about once every microsecond, while the lipid molecules in opposite monolayers exchange places on the average of one a year.
These membranes 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. Examples of such biosensors are disclosed in the following International Patent Applications, the disclosures of which are incorporated herein by cross reference:
PCT/AU88/00273, PCT/AU89/00352, PCT/AU90/00025,
PCT/AU92/00132, PCT/AU93/00509, PCT/AU93/00620,
PCT/AU94/00202, PCT/AU95/00763, PCT/AU96/00304,
PCT/AU96/00368, PCT/AU96/00369 and PCT/AU98/00482.
The first of these references 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.
The inner level of the membrane may be adjacent a solid surface with groups reactive with the solid surface, and spaced from the surface to provide a reservoir region as disclosed in U.S. Pat. No. 5,401,378.
Biosensors based on ion channels or ionophores contained within lipid membranes tethered to or deposited onto metal electrodes are disclosed in Australian Patent 623,747 and U.S. Pat. No. 5,234,566. 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 Austalian Patent 623,747 (incorporated herein by reference) describes various ionophore gating mechanisms termed local disruption gating, extended disruption gating, vertical disruption gating, and extended displacement 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 which an outer layer contains more mobile half membrane spanning ion, channels. One method for immobilising the ion channels of the inner layer is to employ a polymerisable 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 (dimer) for ion transmission across the membrane.
In local disruption gating receptor molecules are linked to mobile ionophores in the outer layer that are aligned with tethered or immobilised ionophores in the inner layer. The introduction of an analyte particle that binds to two adjacent receptors in the outer layer causes the disruption of the orderly alignment of the membrane spanning ionophore. In the case of local disruption gating a loss of conductivity occurs due to the deformation of the ionophores of the outer layer caused by the bonding of the analyte with the adjacent receptors.
The mechanism of extended disruption gating is similar, except that the displacement of the mobile ionophore is greater. In extended disruption gating the binding of pairs of receptors to the same analyte particle cause the outer layer ionophores to move completely out of alignment with the inner layer ionophores.
The mechanism of vertical disruption gating is also similar. In that case the presence of the analyte particle bound to two receptor molecules causes a separation of the two layers that disrupts the continuity of the ion channel across the membrane.
The mechanism of extended displacement gating utilises two different receptors that bind to each other and are linked receptively to a half membrane ionophore and a membrane molecule. The binding of these two receptor molecules to each other displaces the ionophore and disrupts conductivity. The analyte competes with the second receptor for the binding site on the first receptor. The presence of the analyte breaks the bond between the two receptors and allows the half membrane ionophores to realign (dimerize) and provide an ion conductive path. Each of these mechanisms has in common that the binding of the analyte to the receptor molecule causes a change in the relationship between two half membrane spanning monomers such that the flow of ions across the membrane via the ionophores is allowed or prevented.
In a number of sensing applications it is beneficial to incorporate within the one detection cell a positive or negative control to add to the utility of the biosensor. As will be recognised the fabrication of a biosensor having discrete areas of membrane which act as either a test area or control area can be very complex. The present inventors have developed methods by which such biosensors may be fabricated in a less complex manner using optically sensitive moieties.
In a number of sensing applications it is beneficial to incorporate within the one detection cell a positive or negative control to add to the utility of the biosensor. As will be recognised the fabrication of a biosensor having discrete areas of membrane which act as either a test area or control area can be very complex. The present inventors have developed methods by which such biosensors may be fabricated in a less complex manner using optically sensitive moieties.
SUMMARY
Accordingly in a first aspect the present invention consists in a method of fabricating a biosensor in which there is at least one discrete test and at least one discrete control zone, the method comprising the following steps:
(i) providing a conductive substrate;
(ii) forming a membrane including membrane spanning lipids and ion channels comprising first and second half membrane spanning monomers such that the membrane is tethered to the conductive substrate such that a functioning reservoir exists between the membrane and the conductive substrate;
(iii) linking a ligand reactive with an analyte of interest to the ion channel and linking a ligand reactive with an analyte of interest to the membrane spanning lipid components of the tethered membrane via photocleavable linkers; and
(iv) exposing the membrane to a focused light source to cleave the photocleavable linkers thereby releasing the ligands from the ion channel and membrane spanning lipid components in discrete areas of the membrane.
In a preferred embodiment the method further includes the following step:
(v) binding control ligands to the ion channels and membrane spanning lipid components after the ligands have been removed in step (iv).
In a further preferred embodiment the membrane is rinsed between steps (iv) and (v).
In a second aspect the present invention consists in a method of fabricating a biosensor in which there is at least one discrete test and at least one discrete control zone, the method comprising the following steps:
(i) providing a conductive substrate;
(ii) forming a membrane including membrane spanning lipids and ion channels comprising first and second half membrane spanning monomers such that the membrane is tethered to the conductive substrate such that a functioning reservoir exists between the membrane and the conductive substrate;
(iii) providing on the ion channels and membrane spannin
Burns Christopher J.
King Lionel G.
Martin Alistair S.
Osman Peter D.
Australian Membrane and Biotechnology Research
Nixon & Vanderhye
Olsen Kaj K.
Warden, Sr. Robert J.
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