GB2218808A - Detecting device - Google Patents

Description

t DETECTING DEVICE This invention relates to detecting devices for example

for chemical entities such as.compounds and ions or for physical stimuli.

is Many devices exist which are able to measure a biologically significant entity such as an antigen in a biological fluid. Such devices include immunosensors, enzyme electrodes, electrochemical sensors, biocatalytic membrane electrodes, enzyme electrodes, peizoelectric crystal detectors, chemical sensors, optoelectronic devices, ion-sensitive electrodes, and devices using mass spectrometry and nuclear magnetic resonance. Schramm et al., 1987, "The Commercialization of Biosensors", Medical Device & Diagnostic Industry, Vol. 9, p. 52.

An immunosensor is a device which has a sensor associated with a population of antibodies or antigens that selectively bind the antibody or antigen to be measured. The antibody-antigen complex "cannot be directly measured by physiochemical means" and so a signal generator is required e.g., an antigen-enzyme conjugate. Schramm et al., Id. at 54. The conjugate converts a substrate for the enzyme to an electroactive product which is detected by an electrochemical detector. The amount of analyte present is determined by the detector's response.

McConnell, U.S. Patent 4,490,216, the disclosure of Which is to be regarded as bereby incorporated by reference, describes an elecLroanalytica.1 device having a solid electrically-sensitive layer with a lipid layer non-diffusively bound to it. Also included is a second lipid layer, formed on the 1-pi(k_ayer, having polar hydrophobic head groups posit-ioned at a distance from 22' 18 8 0 B 1 1 the lipid layer. These polar groups form a layer which acts as an electrostatic.layer, the polarity of which can be sensed by the solid electricallysensitive layer. Variations in the polarity of this layer are detected by the solid layer. The polar layer can be modified by binding a member of a ligand-receptor pair to one of the lipids within the layers, e.g., by binding dipalmitoyl phospholipid nitroxide (DPN). Such a modified device is useful for detecting the other member of this binding pair, if such binding results in a change in electrostatic field in the polar layer.

The present work may be seen in part an extension of the recognition that membrane receptor proteins act as is transduction elements which can convert a stimulus, such as contact with a specific molecule, into an electrical or chemical signal to elicit a response from the cell. A stimulus-triggered change in the three- dimensional structure of the receptor protein appears to be a universal step in this process. For example, binding of a chemical at a specific site on the receptor protein causes a change in configuration of the receptor which may change the permeability of the membrane to ions, and thus initiate an electrochemical transduction, or it may expose a site on the inside of the membrane, and thus &I activate an enzymatic process. Such conformational changes can also cause a change in distribution of charged side chains on the protein with respect to the membrane surface, and thus cause changes in the electrical potential at the membrane surface.

We have designed membrane sensors which make use of this property of entities, such as proteins held within membranes. These sensors can detect small movements (e.g., less than about 2 Angstroms) of charge i 1 A t 1 i is -)s occurring within the hydropho4ic portion of the membrane by measuring a local change in the electric field of the membrane. They can detect not only the movement of such charges, but also the dynamics of this movement. other changes, e.g., optical changes, can also be detected by the sensors.

Accordingly, the invention features a device for detecting a first entity including a support on whose surface there is non-diffusively bound a first hydrophobic layer, overlaid by a second hydrophobic layer to form a bilayer. Within the bilayer there is a second entity capable of reacting with the first entity to produce a local change in a detectable property of the bilayer structure.

In preferred embodiments, the first or second hydrophobic layers contain a plurality of molecules having aliphatic chains of at least six carbon atoms; the first entity is a chemical entity; the second entity is a protein (most preferably a membrane receptor protein) which is freely mobile within the bilayer; the detectable property is an electrical property or a physical property such as light; and the first and second entities are capable of reacting to form an affinity complex (e.g., a complex between a receptor protein and the protein or other entity for which it is specific, e.g., growth factors and their membrane-bound receptor proteins, e.g., interleukin-2 and the interleukin-2 receptor on lymphocytes; or neurotransmitters and neuroreceptors; or hormones and. hormone receptors; or an immune complex) in which the first and second entities interact non-covalently.

Our devices described in detail below provide mans for linking the detection capabilities of biological receptors with established signal processing capabilities of - 4 is solid-state microelectronic and optical devices. These bio-mimetic sensing assemblies offer exceptional measuring capabilities provided by the use of membrane receptor proteins, or similar molecules, in an almost natural environment. Thus, the response of these proteins to various stimuli can be measured, and this response related to the concentration of molecules in a solution which are reactive with these proteins. These membranes can also be constructed using a wide rnge of entities, including not only membrane receptor proteins, but also other proteins, e.g., antibodies, antigens, and enzymes, as well as modified polypeptides.

In our devices in which the mobile entity within the bilayer is a protein, the device responds to stimuli that activate or change functional properties of the protein. Because the environment of the membrane mimics the environment in which the protein naturally functions, its maximal sensitivity is poised in the biological range of interest. The protein molecule, maintained in as near a natural environment as possible, retains its conformational dynamics and thus responds to its natural stimuli; i.e., the sensor will detect natural positive and negative stimuli, as well as mimics of these stimuli. The sensors are useful in medical diagnostics, pharmaceut-ica.L screening, chemical warfare and other toxic agent detection, and illegal drug-use surveillance, as well as in research into membrane receptor mechanics. In addition to their ability in detecting chemical entities such as proteins in solution, the devices can also be used to detect light, smells or odours and changes in pH and temperature.

Other features and advantages of the invention will be apparent from the following description of the

1% - 5 preferred embodiments thereof.

11 1 The drawings will first briefly be described.

is Fig. 1 is a diagrammatic representation of a membrane-mimetic assembly; Fig. 2 is a transverse section through an insulated gate field-effect transistor; and

Fig. 3 is a diagrammat.iq view,of dialysis apparatus useful in forming devices in accofrdance with this invention.

Referring to Fig. 1, surface-bound membrane structure 8, designed to detect a first entity, includes support 10 having surface 12 to which first hydrophobic layer 14 is non-diffusively (i.e., covalently) bonded. Second hydrophobic (lipid) layer 16, containing lipid molecules 17 having polar head-groups 18, is bound to layer 14 through hydrophobic interaction. Polar lipid head-groups 18 face outward, away from substrate 10. Incorporated into the bilayer are second entity (e.g., receptor protein) molecules 20, which are freely mobile in the bilayer. Membrane structure 8 is associated with standard auxiliary electronics to provide a detectable signal when the first entity being detected reacts with the second entity in the membrane. Each component will now be described in greater detail.

First Hydrophobic Layer The first hydrophobic layer provides a surface with which polar lipids can interact to form the bilayer structure resembling a natural membrane. This layer is formed by reacting the surface of the substrate with a solution that contains reactive long-chained hydrocarbon compounds capable of forming covalent bonds with the support e.g., octadecyltrichlorosilane, which can react I with hydroxyl groups on the surface of glass, as described in J. Sagiv., 1979, Isr. J. Chem., Vol. 18, p. 46. Other suitable hydrophobic layer- forming solutions are described in McConnell, supra.

Second H)rdrophobic Layer (Lipid Layer) The lipid layer is deposited on the surface simultaneously with the receptor protein, e.g., by a detergent dialysis procedure, described below. The lipid layer is held to the surface, and oriented as described above, by interaction with the first hydrophobic layer. The polar head-groups face outward from the surface,and the individual lipid molecules are free to diffuse in the plane of the membrane. Examples of lipids suitable in this layer are given in McConnell, supra. The lipids in both the first or second hydrophobic layers may be cross-linked or polymerized to reduce diffusion within the layer.

Second Entit The second entity is preferably a protein or protein-like compound, and most preferably a receptor protein. The second entity is restrained within the bilayer structure by its interaction with the hydrophobic interior of the bilayer.

Preferably, the second entity is a protein which is free to move within the bilayer, and free to undergo conformational rearrangements. Two general classes of proteins are associated with biological membranes peripheral proteins which bind to the hydrophilic surface of the membrane, and integral proteins which are inserted into the hydrophobic interior of the membrane. Preferred membrane structures of the invention utilize integral membrane proteins, particularly those having a polypeptide chain which crosses the juncture between the two membrane layers It 11 more than once. These "transmembrane" proteins are of particular importance in biological sensory processes, e.g., the sensing of light via the light-sensitive protein Rhodopsin.

As mentioned above, a primary attribute of our membrane structures is that they provide an environment about the protein closely resembling the natural membrane bilayer, so that the protein is maintained in an environment optimal for membrane protein function.

Auxiliar Electronics Fig. 2 shows the structure of a conventional insulated gate field-effect transistor (IGFET) 30 which can be operationally associated with membrane structure is 8 using known, standard techniques. Briefly, IGFET has a base 32 formed of p-type silicon. This is treated to form n-type silicon in a drain region 34, and a source region 36. Metal contacts for the source and drain regions are applied, followed by an insulator 39, and a metal layer to form a gate electrode 38 on top of the insulator 39.. In such devices the potential applied to gate electrode 38 attracts or repels charge carriers from the silicon surface of the device. By regulating the population of charge carriers at the surface, the gate potential controls the flow of current between source 36 to drain 34. This current flow is a sensitive measure of the potential field at the device's surface.

The device essentially responds to the potential-dependent accumulation or depletion of carriers at the insulated silicon surface.

To measure the charge movements accompanying membrane protein functions, the conventional IGFET structure.of Fig. 2 is modified by replacing metal gate electrode 38 with surface-bound is membrane assembly 8 (Fig. 1). In this system, the membrane acts as the gate area of the IGFET. The device is encapsulated with an appropriate polymer (e.g. epoxy) in a manner which leaves the gate region with surface-bound membrane exposed to the solution containing the analyte. The insulator isolates the rest of the device including the source and drain electrodes. When used in this manner a reference electrode, in the solution being measured, must be added to provide electrical contact with the solution.

The substrate (support) is chosen to respond to some property of the bilipid layer which changes upon interaction with a stimulus. For example, an electronically responding substrate such as a field effect transistor senses charge movements induced by the interaction of the receptor protein with a stimulus; and an optically responding substrate senses changes in the optical properties of the film upon interaction with the stimulus. One such substrate is described in Hafeman et, al., US Patent 4591550, the disclosure of which is to be regarded as liereby incorporated by reference. Others include such substrLtes as optical fibres. The surface of the substrate is generally modified to provide chemically reactive groups nee ed for coupling with the hydrophobic layer of membrane structure B. Suitable substrates are composed of glass, platinum oxide, silicon dioxide, silicon nitrate, or coated silicon, or any other surface which can be derivatized with lonq-changed hydrocarbons. The surface may be planar or non-planar, e.g., beads as well as flat surfaces can be used. Other suitable substrates are described in McConnell, supra. Manufacture

The membrane structures are formed directly onto solid substrate surfaces using a modified detergent dialysis technique. Surfaces with exposed hydroxyl and other reactive groups are modified by reaction with a variety of organosilanes to form the first layer of the bilayer. For example, the substrate is made hydrophobic by linking long-chained fatty acids to surface groups, such as is achieved by reaction with octadecyltrichlorosilane. The second layer of the bilayer is deposited by detergent dialysis with simultaneous incorporation of protein. The protein and outer leaflet of the bilayer are held to the substrate surface through hydrophobic interactions with the covalently attached inner alkyl group leaflet of the bilayer.

In detergent dialysis, the detergent is slowly removed from solubilized lipid/protein/detergent solutions, and the lipids and proteins recombine to form vesicular bilayer structures. If appropriate choices of detergent and other conditions are made, functional membrane vesicles can be formed. The detergent used must be non-denaturing to the protein and it must have a relatively high critical micelle concentration, to facilitate its removal by dialysis. Octyl glucoside and deoxycholate are most preferable. If a suitable hydrophobic surface is present during the dialysis then some of the lipid and protein will be driven to reform with that surface instead of forming free-floating vesicles. Structures created by such techniques have compositions and electrical characteristics consistent with the formation of bilayer membranes on the substrate surfaces.

The photoreceptor protein rhodops - in was incorporated into membrane structures as follows. Retinal rod outer segment disks were isolated by the method of Smith et al., 1982, Meth. Enzymol., Vol. 81, is p. 57, solubilized with the detergent octylglucoside (OG), and placed into chamber "C" of a flow dialysis apparatus, shown in Fig. 3, such that the solubilized membrane solution was in contact with planar support 10, which previously had been alkylated by treatment with octAdecyltrichlorosilane. Dialysis was performed, using a dialysis membrane A, against a linear concentration gradient (shown in Fig. 3) decreasing from 50 mM to 0 mM octylglucoside over 5 hours, followed by dialysis against detergent-free buffer for 18 hours. The amount of lipid and protein in the suspension was in excess of that required to create a monolayer on the surface and thus substantial amounts of vesicles were formed. Planar substrate 10 was freed of these vesicles by dipping it into buffer solutions.

If glass beads, rather than planar surfaces, are coated, they are separated from vesicles by centrifugation in 50% sucrose, which is sufficiently dense to float the vesicles, but not the membrane-coated glass beads.

The resulting substrates have properties which are consistent with the formation of membrane-like structures on the alkylated surfaces The rhodopsincontent on 37 pm diameter glass beads is 145+28 pg rhodopsin retained per gram of beads. This corresponds to one rhodopsin (Rh) molecule per 2600 Angstroms 2 of surface, which is comparable to the 2500 Angstroms 2 to 3600 Angstroms 2 estimated for the natural disk membrane. The lipid content of these beads is calculated as 83+15 mol phosphate (P)/mol Rh. Again this valu.e is very close to that usually reported for the native disk membrane (50 to 100 mol P/mol Rh). These results indicate that these surface-bound structures have the composition expected for a 9 i i 1 - 11 is membrane-mimetic bilayer.

When the above assembly is formed on the oxidized surface of a planar platinum electrode, and the electrical properties determined by cyclic voltammetry, the measured resistance is 10 7 ohm-cm 2, and the capacitance 0.2 - 0.5 uF/cm 2. These results are close to typical values for natural membranes (resistance, 10 3 _ 10 6 ohm-cm 2; and capacitance, 1 uF/cm 2 Use The membrane assemblies can be used to detect any entity which is reactive with the protein or other entity within the bilayer. For example, the protein in the bilayer can be the human interleukin-2 (IL-2) receptor (described, e.g., in Leonard et al., 1982), Nature (Lond.), Vol. 300, pp. 267-69) and the device used to detect or measure IL-2 in biological fluids such as serum to provide information on the immune status of a human patient.

The invention is also applicable to the detection or measurement of entities reactive with any other membrane receptors and transduction elements including visual, olfactory, neurotransmitter, and hormone receptors. The combination of biological with electronic and optical systems provides a new class of sensors whose specificity is determined by choosing an appropriate membrane receptor protein.

Sensors based upon chemically sensitive receptors can be designed to respond to a variety of biologically active compounds including hormones, neurotransmitters, and toxins. These devices respond both to the natural stimulus and to compounds which inhibit interaction between the receptor and the stimulus. Such chemically sensitive devices have widespread applications including toxic agent detection and assay, drug surveillance, medical diagnostics, and pharmaceutial screening.

Other embodiments are feasible.

For example, the membrane structure can be constructed by known techniques such as those of Langmuir-Blodgett, rather than detergent dialysis.

r, g - 13

Claims (13)

1. Claims

   A device for detecting a first entity, comprising: a support having a surface, a first hydrophobic layer non- diffusively bound to'said surface, a second hydrophobic layer overlying said first layer to form a bilayer, and a second entity, reactive with said first entity, positioned within said bilayer, said second entity, while within said bilayer, being capable of reacting with said first entity to produce a local change in a detectable property of said bilayer structure.

   is
2. 2. The device of claim 1, wherein said first or said second hydrophobic layer comprises a plurality of molecules having aliphatic chains of at least six carbon atoms.
3. 3. The device of claim 1, wherein said first entity is a chemical entity.
4. 4. The device of claim 1, wherein said first entity is a physical stimulus.
5. 5. The device of claim 1, wherein said second entity is a protein which is freely mobile within said bilayer.
6. 6. The device of claim 1, wherein said detectable property is an electrical property.

   is 14 -
7. 7. The device of claim 1, wherein said detectable property is an optical property.
8. 8. The device of claim 5, wherein said protein is a receptor protein.
9. 9. The device of claim 1, wherein said first and second hydrophobic layers comprise a polymerized or cross-linked region.
10. 10. The device of claim 1, wherein said second entity is capable of reacting with said first entity to form an affinity complex in which said first and second entities are bound non-covalently.
11. 11, A method for manufacturing the device of claim 1, comprising the steps of:

    a) forming said first hydrophobic layer on said solid substrate, b) mixing said second entity with a hydrophobic liquid and a detergent to form a liquid mixture, mixture, and c) contacting said substrate with said d) dialysing said mixture to remove said detergent from said mixture.
12. 12. For manufacturing the device of Claim 1, a method substantially as hereinbefore described with reference to the accompanying drawings.
13. 13. A device for detecting a first entity substantially as hereinbefore described with reference to and as shown in the accompanying drawings.

    Published 1989 atThe PatentOffice, Stau House, 66/71 High Holborn, London WCIR4TP- Further copies maybe obtainedfrom The Patentoffice. Sales Branch, St Mary Cray, Orpington, Kent BR5 3RD. Printed by Multiplex techniques ltd, St Mazy Cray, Kent, Con. 1/87 1 1