JPH0219767A - Membrane bio sensor - Google Patents

Abstract

PURPOSE: To make a membrane biosensor possible to respond to biologically active compounds of various kinds, e.g. hormone, nerve conduction elements, toxicants, etc., by designing the sensor based on a chemically sensitive receptor. CONSTITUTION: A device for detecting a first substance is provided with a supporting member 10 having a face, a first hydrophobic layer 14 coupled to the face not diffusingly, a second hydrophobic layer 16 overlapping with the first layer to form two layers, and a second substance reactive to the first substance and positioned between the two layers. The second substance reacts with the first substance when in the two layers thereby to generate a partial change of a detectable characteristic of the two-layer structure. The first or second hydrophobic layer includes a plurality of molecule having an aliphatic chain with at least 6 carbon atoms. The first substance is set as a chemical substance. The second substance is protein capable of freely moving in the two layers. The detectable characteristic is set as an electric or optical characteristic. The second substance reacts with the first substance thereby to form an affinitive complex without a covalent bond of the first and second substances.

Description

[Detailed description of the invention] Retired Emperor and Minister of Industry… The present invention relates to electronic devices containing selective membranes.

For example, it relates to a sensor that measures chemical entities such as compounds and ions.

There are many devices capable of measuring biologically significant entities such as antigens in biological fluids. This type of device includes immunosensors, enzyme electrodes, electrochemical sensors, biocatalytic membrane electrodes,
These include chemical sensors, optoelectronic devices, ion sensing electrodes, etc., and some devices use mass spectrometers and nuclear magnetic resonance meters. Schramm eta+, 1987 The C
Omercialization of Btose
nsorsMedical Device & Dia
Gnostic Industry, Vol, 9゜p
, 52.

An immunosensor is a device that has a sensor that combines a group of antibodies or antigens and selectively binds to the antibody or antigen to be measured. Antibody-antigen complexes cannot be measured directly by physiochemical means and require a single generator, such as an antibody-enzyme conjugate. The conjugate converts the enzyme's substrate into an electroactive product that can be detected by an electrochemical detector. The analyte is determined by the detector response.

US Pat. No. 4,490,216 describes an electroanalytical device that non-diffusively couples a lipid layer to a rigid electrosensing layer. A second lipid layer is placed on top of the lipid layer, and the polar hydrophobic heads are located away from the lipid layer. The layer formed by this polar group acts as an electrostatic layer.

Polarity is sensed by a rigid electrical sensing layer. The polar layer is changed to replace the group of ligand-receptor pairs with one lipid, 9 for example binding divarmitoyl phospholipid nitroxide (DPN). This modified device is effective in detecting other members of the bonding pair if this bonding causes a change in the electrostatic field within the polar layer.

Key points for skin development The present invention is based on the membrane receptor protein acting as a transduction element,
It is based on the recognition that stimulation caused by contact with specific molecules is converted into electrical or chemical signals, and a response from cells is manifested.

Stimulus-triggered changes in the ternary structure of receptor proteins are a common step in this process. For example, binding a compound to a specific side of a receptor protein causes a change in the shape of the receptor.
* changes its permeability to ions, initiates electrochemical transduction, exposes the inside of the membrane, and initiates enzymatic processes. This adaptive change causes a change in the distribution of the protein's charged side chains with respect to the membrane surface, resulting in a change in the potential at the membrane surface.

Membrane sensors according to the invention exploit the properties of entities such as proteins retained within membranes. This sensor is capable of detecting small movements, such as movements of less than 2 angstroms of the charge occurring within the hydrophobic portion of the membrane, by measuring local changes in the electric field of the membrane. Not only the movement of this charge but also the kinematics can be detected. Other changes, such as optical changes, can also be detected by the sensor of the invention.

A device for detecting a first entity according to the present invention includes a support member having a surface, a first hydrophobic layer non-diffusively bonded to one surface, and a second hydrophobic layer overlaid on the first layer to form two layers. sex layer and
a second entity positioned within the bilayer and reactive with the first entity, wherein the second entity reacts with the first entity when within the bilayer to cause a portion of the detectable property of the layer structure to react with the first entity; may cause physical changes.

According to a preferred embodiment of the invention, the first or second hydrophobic layer comprises a plurality of molecules with aliphatic chains having at least 6 carbon atoms. Let the first entity be a chemical entity. The second entity is a protein (preferably a membrane receptor protein) that can move freely within the two layers. The detectable property is an electrical property or an optical property. The second entity reacts with the first entity to form an affinity complex in which the first and second entities are non-covalently bonded (e.g., a complex between a receptor protein and an entity such as a specific white matter). Surface binding proteins 3 form interleukin 2 and interleukin 2 receptors of lymph cells, or neurotransmitter elements and neuroreceptors, or hormones and hormone receptors, or immune complexes).

The present invention provides a means of combining the detectability of bioreceptors with the known signal processing possibilities of So'J sodostate microelectronic and optical devices. The biomimetic sensing assembly according to the invention gains significant measurability through the use of molecules such as membrane receptor proteins in their near-natural environment.

The response of a protein to various stimuli can be measured, and this response is related to the concentration of molecules in solution that react with the protein. A wide variety of entities can be used to construct membranes, including membrane-receptor proteins as well as other proteins such as antibodies, antigens, enzymes, modified polypeptides, and the like.

In the device according to the invention, the internally moving entity is a protein, and the device responds to stimuli that actuate or change the functional properties of the protein. The membrane environment mimics the environment in which proteins naturally function, and maximum sensitivity is commensurate with their biological range. Protein molecules closely approximate their natural environment, maintain adaptive dynamics, and respond to natural stimuli. That is, the sensor detects natural positive and negative stimuli as well as simulated stimuli. The sensor of the present invention is effective for medical diagnosis, drug screening, detection of toxic substances such as chemical warfare, testing for illegal drug use, and research on membrane receptor mechanisms. Additionally, this device can be used to detect chemical entities such as proteins in solution.

It can detect light, odor, pH, and temperature changes.

DETAILED DESCRIPTION OF THE INVENTION Examples and drawings illustrating the present invention will be described.

FIG. 1 is a diagrammatic representation of a membrane-mimetic assembly in which a five-sided membrane structure 8 is designed for the detection of a first entity and does not diffuse the first hydrophobic FW 14 onto the surface 12 of the support member IO. For example, as a covalent bond. The second hydrophobic (lipid) layer 16 contains lipid molecules I7 with polar head groups 18 and is bound to the layer 14 by hydrophobic interactions. Polar lipid head group 1
8 faces outward with respect to the substrate 10. The second layer combined with both layers
The entity molecule 20 is, for example, a receptor protein and is freely movable within both layers.

The membrane structure 8 is combined with standard auxiliary electronics to produce a detectable signal when the first entity to be detected reacts with the second entity within the membrane. Each part will be explained in detail below.

The first hydrophobic layer will be explained.

In the first hydrophobic layer, polar lipids interact to form a surface, forming a bilayer structure similar to natural membranes.

A solution containing a reactive long chain carbohydrate, such as octadoethyltrichlorosilane, capable of forming covalent bonds with the support member is reacted on the surface of the substrate to form this layer, and on the surface of the glass is reacted with hydroxyl groups to form J, Sag. iv+
1979+ I sr, J, Chem, + V
It is described in ol-18+ p46. Other suitable hydrophobic layering solutions are found in the literature such as McConne L 1.

The second hydrophobic N (lipid layer) will be explained.

The lipid layer is deposited on the surface simultaneously with the receptor protein using, for example, the surfactant dialysis technique described below.

The lipid layer is held on the surface and interacts with the first hydrophobic layer to maintain said position. The polar head group faces outward from the surface,
Individual lipid molecules diffuse freely in the plane of the membrane. Examples of suitable lipids for this purpose are found in McConnal et al. Lipids within the first and second layers may cross-link or polymerize to reduce diffusion within the layers.

The second entity will be explained.

The second entity is a protein or protein-like compound, preferably a receptor protein. The second entity interacts with the hydrophobes within both layers and becomes constrained within the bilayer structure.

In a preferred example, the second entity is a protein that moves freely within both layers and undergoes conformational rearrangement. 21 [1] Assemble the proteins on the general paper into the biofilm, 9. Assemble the peripheral proteins on the hydrophilic side of the membrane, and insert the main proteins into the hydrophobic interior of the membrane. A preferred membrane structure of the present invention uses a major membrane protein, particularly a protein having a polypeptide chain that crosses the glabellar junction of both membranes two or more times.

This intermembrane protein is particularly important for biological sensing processes 5, such as the sensing of light by the light-sensing protein rhodopsin.

As mentioned above, 9 the key properties of the membrane structure of the present invention create an environment surrounding proteins that closely resembles the bilayers of natural membranes, and proteins are retained within an environment suitable for membrane protein function.

Describe the auxiliary electronic circuit.

Figure 2 shows a typical insulated gate field effect transistor (IG).
FET) 30 is shown and operatively coupled to membrane structure 8 using known standard techniques. Let me explain it simply.

IGFE 730 has a base 32 made of p-type silicon. This is processed to form n-type silicon in the drain section 34 and source section 36. The source and drain portions are coated with metal contacts, then an insulator 39 is attached, and a metal layer is coated on top of the insulator 39 to form a gate electrode 38. The gate charge is adjusted to the source 36 by adjusting the density of charge carriers on the surface.
and the drain 34. This current provides the precision with which the electric field is sensed in the plane of the device. The device essentially responds to charge-dependent accumulation or dissipation of carriers in an insulating silicon surface.

In order to measure the charge movement associated with membrane protein function according to the present invention, the conventional IGFET structure shown in FIG. 2 is modified to replace the metal gate electrode 38 with the surface-filled gold film assembly 8 shown in FIG. In this system, the membrane acts as the gate portion of the IGFET. This device is encapsulated in a polymer such as epoxy, and the gate portion of the nine-sided membrane is exposed to a solution containing an analytical agent. Insulating material isolates other parts of the device including the source and drain electrodes. During use, it is necessary to add a reference electrode within the measurement solution to make electrical contact with the solution.

The substrate or support member may be selected to respond to certain properties of both thoracic layers that change upon interaction with a stimulus, e.g.
Electrically responsive substrates, such as field effect transistors, sense charge movement induced by the interaction of receptor proteins and stimuli. A photoresponsive substrate senses changes in the optical properties of the film upon interaction with a stimulus. An example of this type of substrate is described in US Pat. No. 4,591,550. Another example of a substrate is an optical fiber. Typically, the substrate surface is modified to provide the chemically active groups necessary to bond to the hydrophobic layer of the membrane structure 8. Suitable substrates are surfaces capable of introducing long chain hydrocarbons such as glass, platinum oxide, silicon dioxide, silicon nitrate, coated silicon, and the like. The surface can be flat or non-flat, and both bead and flat surfaces can be used. Other suitable substrates are Mc
It is described in the literature by Connall.

Manufacturing will be explained.

The membrane structure of the present invention is formed directly on the substrate surface using a modified surfactant dialysis technique. The surface with reactive groups such as exposed hydroxyls is reacted with various organosilanes to form the first layer of the bilayer. For example, the substrate can be made hydrophobic by attaching long chain fatty acids to the face groups and reacted with, for example, octadoethyltrichlorosilane. The second layer of the bilayer is simultaneously deposited with surfactant dialysis in combination with the protein, and the other leaflet of the bilayer is covalently bound to the substrate surface by hydrophobic interactions to the internal alkyl group leaflets of the bilayer. do.

In surfactant dialysis, the surfactant is gradually removed from a solution of lipids, proteins, and surfactants.

Lipids and proteins recombine to form the bilayer structure of the vesicle0
The surfactant is non-denaturing to proteins.

It has a relatively high critical micelle concentration, making it easy to remove by dialysis. Octyl glucoside and deoxychlorate are most preferred. Once the required hydrophobic surface is formed during dialysis, the required amount of lipids and proteins are driven to improve the surface and eliminate the free floating mass.

The structures created by this technique are consistent in composition and electrical properties with the formation of a bilayer film on the substrate surface.

The photoreceptor protein rhodopsin is assembled into a membrane structure as follows. Smlth et al, +1982. Meth, E.
nzymol,.

Vol, 81. 57, the retinal rod outer segment disc was removed and solubilized with the surfactant octyl glucoside (OG) and placed in chamber C of a flow dialysis machine, as shown in Figure 3, and solubilized. The membrane solution is brought into contact with the planar support member 10, which has previously been treated with octadoethyltrichlorosilane to alkylate it. Dialysis was performed using 11llA, and octyl glucoside was adjusted from 50mM to OmM over 5 hours using the linear concentration gradient shown in Figure 3.
Dialysis for 3 hours. If the amount of lipid and protein in the suspension is greater than or equal to that required to form a monolayer on the surface, a large amount of small amounts will be formed. The vesicles may be removed by dipping the planar carrier into a buffer solution.

The properties of the formed substrate are consistent with the formation of a film-like structure on the alkylated surface. The rhodopsin content of a glass bead with a diameter of 37 μ- is 145 i. 28 μg of rhodopsin is
Contains per gram. This corresponds to 1 rhodopsin (Rh) molecule per 2600 faces, which is the expected 2500-. Equivalent to 3600K. The lipid content of the beads is 83 soil 15 moI phosphate (P)/mol
It is calculated as Rh. This value is close to the reported 50-50-1 O0P/mol Rb for natural disk membranes. From this result, the composition of the surface-bonded structure of the present invention becomes a required film pseudo-bilayer.

forming the above assembly on the oxidized surface of a planar platinum electrode;
When electrical properties are measured by a periodic voltammeter, the /1III constant resistance is 10 ohm cni and the capacitance is 0.2-0.5 uF/c%. This result is the standard value of natural film, resistance 10'-10'ohmc
nl, capacitance IuF/aII! Approximate to

Explain its use.

The membrane assembly according to the present invention detects entities that react with proteins;
or can be used to detect other entities within the bilayer. For example, the protein within the bilayer is human interleukin 2 (IL-2
) receptor, Leonard et al., 198
2Nature (Lond), Vol, 300.
It is described in pp. 267 = 69.

The device is used to measure IL-2 in biological fluids such as serum to obtain information regarding the patient's immune status.

The present invention may be used to detect or measure entities responsive to other membrane receptors and transduction elements 9 such as visual olfactory neurotransmitter elements, hormonal receptors, and the like.

The combination of biological and electronic and optical systems provides a novel sensor, defined by specialization by selecting the appropriate membrane receptor protein.

Chemically sensitive receptor-based sensors can be designed to respond to a variety of biologically active compounds such as hormones, neurotransmitters, toxins, and the like. The device responds to both natural stimuli and compounds that inhibit the interaction of the receptor with the stimulus. This chemically sensitive device has wide applications including toxin detection and analysis, drug research, medical diagnostics, and pharmaceutical screening.

In another embodiment, for example, the membrane structure of the present invention is not subjected to surfactant dialysis, but using known techniques such as LangmuirBlo.
It can also be configured using the dgett technique.

[Brief explanation of the drawing]

FIG. 1 is a diagrammatic representation of a membrane cell assembly, FIG. 2 is a cross-sectional view of an insulated gate field effect transistor, and FIG. 3 is a diagram of a dialysis device suitable for forming the device of the present invention. 818. Membrane structure 100. Supporting members! 4.16. ．． ．．
Hydrophobic layer 171. Lipid molecules 206. Receptor protein molecule 309. Insulated gate field effect transistor 320. Base 341. Drain 369. Source 381. Gate electrode 391. Engraving of insulator drawing (no change in content) (4 others: Procedural amendment 1゜Display of case 1999 Patent Application No. 120209 2, Name of invention Membrane biosensor 3゜Relationship with the case Tokuko 1 Applicant address Name: Easy & Tsubo G Incorporated 4, Agent address: 12-1 Otemachi 2-chome, Chiyoda-ku, Tokyo 206-ku 5, Shin-Otemachi Building, Applicant subject to amendment Application form stating the representative of

Claims (1)

[Claims] 1. A device for detecting a first entity, comprising: a support member having a surface; a first hydrophobic layer bonded to the surface in a non-diffusion manner; a second hydrophobic layer forming a bilayer; and a second entity positioned within the bilayer and reactive to the first entity, the second entity being reactive to the first entity when within the bilayer. A membrane biosensor characterized in that it is capable of causing a partial change in the detectable properties of a layered structure upon reaction with an entity of 1. 2. The sensor of claim 1, wherein the first or second hydrophobic layer comprises a plurality of molecules having aliphatic chains having at least 6 carbon atoms. 3. The sensor according to claim 1, wherein the first entity is a chemical entity. 4. The sensor according to claim 1, wherein the first entity is a physical stimulus. 5. The sensor according to claim 1, wherein the second entity is a protein that can move freely within the two layers. 6. The sensor according to claim 1, wherein the detectable characteristic is an electrical characteristic. 7. The sensor according to claim 1, wherein the detectable characteristic is an optical characteristic. 8. The sensor according to claim 1, wherein the protein is a receptor protein. 9. The sensor according to claim 5, wherein the first and second hydrophobic layers have a polymerized or cross-linked portion. 10. The second entity reacts with the first entity to form the first and second entities.
The sensor of claim 1, wherein the entities form a non-covalently bonded affinity complex. 11. A method of manufacturing a membrane biosensor, comprising: a) forming a first hydrophobic layer on a rigid substrate; and b) mixing a second entity with a hydrophobic liquid and a detergent to form a mixture. A method for producing a membrane biosensor, comprising: c) contacting a mixed solution with a substrate; and d) dialyzing the mixed solution to remove a detergent from the mixed solution.