Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
2000-03-13
2001-01-23
Tung, T. (Department: 1743)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C204S403060, C204S412000, C427S002130
Reexamination Certificate
active
06177000
ABSTRACT:
This invention relates to a biosensor, and in particular to a biosensor of the type which operates by detecting or measuring the transport of ions across a lipid membrane.
Biosensors based on the use of a gated channel protein spanning a bilayer membrane are of considerable interest. Each individual binding event can give rise to the passage of as many as 10
9
individual ions through the channel during a practical measurement interval. Also, the operation of distinct channels is essentially independent and the currents through them combine linearly. These two factors inspire the hope of a general principle for sensing biomolecules which displays both excellent sensitivity and high dynamic range.
In order to achieve high dynamic range it is necessary to choose channel proteins which open in the presence of the target biomolecule. This is the case for a number of naturally-occurring channel proteins, typically neurotransmitter receptors in nerve cells. In a more generally applicable approach, robust ungated channels, particularly the gramicidins, are bound chemically to antibody molecules in such a way as normally to obstruct the channel, and to unblock it when an antigen binds.
The electric charge transported through these channel proteins consists physically of solvated ions. In order to allow further processing, the ions must be exchanged for the flow of electrons through a wire at electrodes located at both the front and rear of the bilayer. In one known approach, the bilayer is located immediately adjacent to the rear noble-metal electrode. It is not clear where the ions flow to. If they discharge at the electrode, the associated chemical changes will inevitably lead to degradation.
In another known approach, there is electrolyte behind the bilayer contained in a gel. The bilayer is fabricated by a standard technique across an aperture adjacent to the gel. The gel provides some physical support for the bilayer, so that it is able to withstand quite vigorous agitation of the test liquid. However the bilayer cannot be dehydrated and must be formed immediately prior to the measurement in the aqueous medium to be monitored. It is directly exposed to the medium and cannot withstand contact with a solid.
There has now been devised a novel form of biosensor based on measurement of ion transport across a lipid membrane which overcomes or substantially mitigates the disadvantages of known forms of such biosensor.
According to a first aspect of the invention, a biosensor comprises a lipid membrane containing gated ion channels sensitive to the presence or otherwise of an analyte molecule in a sample applied, in use, to a first side of said lipid membrane, the lipid membrane being disposed between a pair of electrodes, wherein a first layer of a porous gel is applied to the first side of the lipid membrane.
The biosensor according to the invention is advantageous primarily in that the first layer of porous gel applied to the lipid membrane protects the membrane from dehydration and physical damage caused by mechanical contact, yet still permits molecules contained within the sample access to the lipid membrane. Because the membrane is not destroyed by drying of the biosensor, the biosensor can be packaged and stored in the dry state, for rehydration immediately prior to use.
Preferably, a second layer of gel is also applied to the second side of the lipid membrane, to further protect the membrane and to provide the necessary separation from the adjacent electrode and to accommodate a reservoir of ions required by that electrode.
The gel is preferably a biocompatible and porous gel, most preferably a hydrogel. Suitable gel materials include agarose, dextran, carrageenan, alginic acid, starch, cellulose, or derivatives of these such as eg carboxymethyl derivatives, or a water-swellable organic polymer such as eg polyvinyl alcohol, polyacrylic acid, polyacrylamide or polyethylene glycol. A particularly preferred gel material is agarose. Other gel materials considered particularly suitable include polyacrylaimide gels.
The thickness of particularly the first layer of gel is preferably such as to permit diffusion of biomolecules of approximately 1 kD to occur in reasonably short time periods, eg less than 5 minutes, more preferably less than 2 minutes. The first and second layers of gel preferably have thicknesses of less than 5 mm, eg 0.1 to 2 mm, most preferably approximately about 1 mm.
The lipid membrane is preferably a bilayer of amphiphilic molecules, most preferably one or more phospholipids, eg phosphatidylcholines and/or phosphatidylethanolamines. The lipids may have hydrocarbon tails with chain lengths of C
12
-C
22
, most preferably C
12
-C
18
. A particularly preferred phospholipid is dioleylphosphatidylcholine. Other membrane forming molecules which may be employed include amphiphilic polymers, eg hydrophobic polymer chains with hydrophilic side groups. One example of such a polymer is a polysiloxane with phosphatidylcholine side groups.
Suitable molecules defining the gated ion channels are incorporated into the lipid membrane, eg membrane-bound proteins.
Preferably a perforated sheet of an inert and impermeable material is interposed between the lipid membrane and the second gel layer. A suitable such material is polytetrafluoroethylene. The sheet is preferably thin, eg less than about 100 &mgr;m in thickness, more preferably about 10 &mgr;m in thickness. The sheet is preferably formed with one or more perforations of diameter 10-200 &mgr;m, more preferably about 50-100 &mgr;m. The sheet of material permits the flow of current between the electrodes only in the region of the perforation(s) in the sheet.
The lipid membrane may be formed by dissolving the membrane-forming lipid and the molecules defining the gated ion channels in a solvent and applying the solution so formed to the second gel layer (or to the perforated sheet of inert material abutting the second gel layer). Any suitable solvent may be used, provided that it is substantially immiscible with water. Polar solvents, capable of initiating hydrogen bonds, are preferred since their use provides a strong driving force for complete coverage of the solution over the surface. A particularly preferred solvent is chloroform. The solution preferably has a concentration of 0.01 to 5% w/v, more preferably less than 1% w/v, eg about 0.2% w/v.
However, the method of forming the lipid membrane described above may not always be suitable. For example, some ligand-gated channel proteins may be denatured by chloroform. One alternative method for the formation of the lipid membrane which may be suitable in such cases involves forming an inverted micellar solution or emulsion containing the membrane forming lipid and molecules defining the gated ion channels, the micellar solution or emulsion having a hydrocarbon continuous phase. The hydrocarbon is preferably an alkane, most preferably hexane.
One functional ligand-gated channel protein for which the alternative method described in the immediately preceding paragraph may be applicable is the nicotinic acetycholine receptor (nAChR—see G Puu et al,
Biosens. Bioelectron
. 10 (1995), 463). This neuroreceptor, with many slight variations, is found in most animals with nervous systems, and a very rich source of supply is available in the electric organ of the common marbled ray
Torpedo marmorata
. A crude extract formed by homogenizing the electric organ of the ray and centrifuging in a CsC1 gradient to isolate the membrane-bound fraction has a continuous phase which is essentially aqueous. By reducing the proportion of water it is possible to invert the emusion and to prepare from it an inverse emulsion with a hydrocarbon continuous phase having the characteristics required for formation of the lipid membrane.
The electrodes are preferably noble metal electrodes of generally conventional form. Most preferably the electrodes are silver/silver chloride electrodes formed on sheets of a suitable substrate such as mica. Preferably the electrode on the first side of the lipid membrane is f
Coventry University
Noguerola Alex
Oppenheimer Wolff & Donnelly LLP
Tung T.
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