JP2002174611A - Immuno-electrode sensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immuno-electrode method for realizing both of the versatility of immunological method and characteristics such as sensitivity, precision, easiness, and quickness seen in the sensing of the receptor of organism and taking out an immunoreaction as an electric response. SOLUTION: A substance soluble in lipid having high resistance and capable of applying ion conductivity to the lipid is used as a label and bound to a substance to be measured. When the labeled part of the labeled substance to be measured is adsorbed in a lipid thin film (dissolved in the thin film), the resistance is reduced for imparting the ion conductivity to the lipid thin film, and this reduction in the resistance (increase in electric conductivity) is detected to measure the substance to be measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrochemical analysis and measurement of a chemical substance.

[0002]

2. Description of the Related Art As a method for selectively detecting a specific substance, a method using an antibody is often used.

[0003] Antibodies are biological substances that perform specific recognition and binding. It is possible to obtain antibodies corresponding to a large number of substances by sensitizing an experimental animal with an antigen, and it is possible to construct a sensing method for a wide variety of substances in general. A reaction in which an antibody specifically recognizes an antigen and binds to form a complex is called an antigen-antibody reaction or an immune reaction. Many sensing methods have been developed utilizing this antigen-antibody reaction.

One of them is to use no labeling substance,
ABO blood type determination, which detects a complex resulting from an antigen-antibody reaction and performs visual detection, is an example that has been known for a long time. There are also other detection methods such as liquid chromatography in which the complex is subjected to molecular weight fractionation by gel filtration and analyzed.

[0005] However, many sensing methods in this field have been developed by using a label (marker) bound to an antibody or an antigen in combination with a label detecting means.

[0006] The radioimmunoassay uses a radioisotope as a label and detects radiation with a scintillation counter or radioautography.

[0007] In the fluorescent antibody method, a fluorescent substance is used as a label, and optical detection such as fluorescence spectroscopy and fluorescence microscopy is performed.

In the enzyme immunoassay, generally, peroxidase of mustard radish is used as a label, and detection is carried out by luminescence analysis utilizing the peroxidase reaction. As a variation of this method, it is possible to use oxidoreductase as a label and detect changes in the concentration of oxidizing or reducing substances resulting from the enzymatic reaction as electrode potentials or currents for amperometric (electrolysis). It is.

[0009] Colored substances such as pigments and pigments are detected by a photometric method or a visual judgment. Gold colloid or blue latex particles are often used as labels. These are simple methods that incorporate a labeled antibody on a substrate such as filter paper, separate the complex resulting from the antigen-antibody reaction with the movement of the test solution, and perform colorimetric detection on the complex as well. Is often used for immunochromatography.

In addition, there are many combinations of the label and the detection method of the label, such as using magnetite particles as a label and detecting magnetism.

However, these immunization methods have many problems.

[0012] The radioimmunoassay is a heavy burden on the management of radioactive substances and cannot be a simple measurement method.

The method using various optical detection methods has low sensitivity. Or, if you perform sensitization to increase the sensitivity,
Noise is picked up and the accuracy deteriorates.

[0014] Simple methods such as immunochromatography do not require complicated operations for the measurement, but are inevitably performed in binary or ternary determination with a threshold value due to problems of accuracy and sensitivity. .

On the other hand, as a typical example of a method for selectively detecting a specific substance in a living body, recognition and response of a neurotransmitter acetylcholine by an acetylcholine receptor at a nerve synapse is known.

The acetylcholine receptor penetrates the lipid bilayer and exists with the acetylcholine recognition site facing outward. The recognition and binding of acetylcholine of the acetylcholine receptor is specific, and has high accuracy and sensitivity. The acetylcholine receptor that recognizes and binds acetylcholine forms an ion channel that penetrates the lipid bilayer due to a conformational change. Normally, the inside and outside of the lipid bilayer are electrically polarized due to the bias of ions, but depolarization occurs due to the movement and mixing of ions through ion channels. This depolarized state moves as a pulse in a nerve cell and transmits information to a distant site in a living body (neural transmission).

As in this example, the detection of a living body has high accuracy and sensitivity, and the information processing is performed after the recognition information is converted into an electric signal by a simple and quick process of one step. This can be said to be an extremely rational detection method.

[0018]

However, the in vitro detection technique of the living body detection involves only extracting the receptor from the living body and reconstructing it on a lipid bilayer, and the type of receptor that the living body has is limited. Due to the restriction, it is not possible to perform the same sensing for many kinds of substances in general.

The present invention relates to the versatility of the immunoassay and the sensitivity, precision, and simplicity found in the sensing of receptors in living organisms.
It is an object of the present invention to provide an immunoelectrode sensor that has both rapidity and extracts an immune response as an electrical response.

[0020]

According to the present invention, an ion channel marker is used as a label, resistance detection is used in combination as a detection method for detecting a label, and a means for releasing an ion channel as a result of an antigen-antibody reaction is combined. The antigen, which is the test substance, is measured.

That is, a soluble labeled antibody labeled with a fat-soluble ion-conductive substance, an immobilized antigen corresponding to the labeled antibody, a pair of electrodes, a working electrode and a counter electrode, and a lipid thin film formed on the surface of the working electrode. The complex of the antigen and the labeled antibody generated by contacting the labeled antibody with the antigen in the liquid to be measured is adsorbed to the immobilized antigen, and the unreacted labeled antibody is adsorbed to the lipid thin film formed on the electrode surface. Then, a decrease in resistance (an increase in electrical conductivity) caused by the labeling portion imparting ionic conductivity to the lipid thin film is detected, and the antigen as the measurement object is measured.

By using such a label in combination with the detection of the label, the versatility of the immunoassay and the sensitivity, accuracy, simplicity, and rapidity found in the sensing of a receptor in a living body are combined, and the immune response is converted into an electrical response. It becomes possible to take out an immunoelectrode sensor.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION According to the present invention, there is provided a soluble labeled antibody labeled with a fat-soluble ion-conductive substance, an immobilized antigen corresponding to the labeled antibody, and a pair of electrodes, a working electrode and a counter electrode. And a lipid thin film formed on the surface of the working electrode, and a complex of the antigen and the labeled antibody generated by contacting the labeled antibody with the antigen in the liquid to be measured is adsorbed to the immobilized antigen, and unreacted labeling is performed. Absorb the antibody to the lipid thin film formed on the electrode surface,
The lipid-soluble ion-conductive label is detected based on the change in the electrical conductivity of the lipid thin film, and the antigen in the liquid to be measured is detected.

An immunoelectrode sensor which has the versatility of the immunization method and the sensitivity, accuracy, simplicity, and rapidity found in the sensing of receptors in a living body and extracts an immune reaction as an electric response is provided.

According to a second aspect of the present invention, there is provided the fat-soluble ion-conductive substance according to the first aspect of the invention, wherein the fat-soluble ion-conductive substance forms a conjugate compound with a water-soluble ion;
A fat-soluble chelating agent, a chelate compound thereof, a fat-soluble ion, a polymer or oligomer having a structure in which a polar group is oriented inward and a hydrophobic group is oriented outward, and the immunoelectrode sensor is at least one of: More types of signs.

According to a third aspect of the present invention, there is provided a measuring method in which the lipid thin film according to the first aspect of the present invention is a monomolecular film or a multilayer film, and can stably maintain high sensitivity.

According to a fourth aspect of the present invention, there is provided an immunoelectrode sensor in which the lipid thin film according to the first aspect of the present invention is bonded to a metal material of an electrode via a sulfur atom. Can be.

According to a fifth aspect of the present invention, the surface of the lipid thin film according to the first aspect has a hydrophobic group, a nonionic hydrophilic group,
An immunoelectrode sensor covered with at least one of a cationic dissociative group, an anionic dissociative group, and a zwitterionic dissociative group, and can control measurement sensitivity, a measurement range, and a response speed. , Insulation resistance can be improved.

According to a sixth aspect of the present invention, there is provided an immunoelectrode sensor using a monovalent antibody as a soluble labeled antibody labeled with the fat-soluble ion-conductive substance according to the first aspect of the present invention. , A response with good linearity can be obtained.

The present invention according to claim 7 is an immunoelectrode sensor using a monocolonal antibody as a soluble labeled antibody labeled with the fat-soluble ion-conductive substance according to claim 1 of the present invention, and has a blank value for measurement. Can be kept low.

An eighth aspect of the present invention is the immunoelectrode sensor according to the first aspect of the present invention, wherein the electric conductivity measurement is an AC marking or an AC marking applied with a DC bias.
The electrical response can be taken out.

According to a ninth aspect of the present invention, a pH buffer is incorporated in the immunoelectrode sensor according to the first aspect of the present invention. Can be.

[0033]

(Embodiment 1) The first embodiment of the present invention is shown in FIGS.
It is explained based on. FIG. 1 is a diagram showing an example of the overall configuration of a sensor according to the present invention. In FIG. Reference numeral 6 denotes a labeled antibody layer formed by coating and filling a labeled antibody on cellulose powder. Reference numeral 3 denotes an immobilized antigen layer in which a cylindrical immobilized antigen is filled inside the cylindrical counter electrode 1. Reference numeral 4 denotes an inlet of the liquid to be measured, from which the liquid to be measured, which is an aqueous electrolyte solution containing the antigen, enters the labeled antibody layer, passes through the immobilized antigen layer 3 by capillary action, and reaches the tip of the working electrode 2; Contact with working electrode.

FIG. 2 is a diagram for explaining the internal structure of FIG. 1 together with the movement of the liquid to be measured. Reference numeral 11 denotes an antigen in the liquid to be measured 12, which corresponds to the object to be measured in this example. The liquid to be measured 12 enters the labeled antigen layer 6 by capillary action. Here, a labeled antibody 17 in which a marker is bound to an antibody corresponding to the antigen 11 that is the measurement object is applied. This marker 16 is a fat-soluble ion-conductive substance. This labeled antibody 17 is
2 to be free. Antigen 1 contained in the liquid to be measured
1 binds to a labeled antibody by an antigen-antibody reaction to form an antigen-labeled antibody complex 18.

The free-labeled antibody 24 thus unreacted with the antigen-labeled antibody complex 18 enters the next immobilized antigen layer. Here, the antigen 1 is placed on the surface of the immobilized substrate 13.
The same antigen 14 as in Example 1 binds to the immobilized antigen 15
Is formed. The free labeled antibody 24 binds to the immobilized antigen 15 by an antigen-antibody reaction. The antibody part of one antigen-labeled antibody complex 18 has already bound to the antigen and cannot bind to the immobilized antigen 15. Therefore, only the free labeled antigen 24 is selectively removed from the liquid to be measured.

The remaining antigen-labeled antibody complex reaches the working electrode 19 as the liquid to be measured moves, and the working electrode 19 and the counter electrode 20 are connected via the liquid to be measured.

Here, 21 is a lipid monolayer composed of lipid molecules 22 formed on 19 working electrodes, and is a lipid thin film. Reference numeral 12 denotes an aqueous electrolyte solution that fills the space between the electrodes 19 and 20, and in the present configuration example, is a liquid to be measured that has passed through the immobilized antigen layer. The electrolyte concentration is assumed to be a salt concentration of a body fluid or urine.

The lipid monolayer, which is the lipid thin film of this example, has a high resistance of about 10 8 ohms under an area of 1 square centimeter. This is significantly higher than the ionic conduction resistance (usually expressed by conductivity) of the electrolyte and the resistance of the electrode surface, and the resistance of the entire measurement system is approximately the resistance of the lipid monolayer. The complex 18 of the labeled antibody and the antigen in the electrolyte aqueous solution diffuses in the electrolyte solution to reach the lipid monolayer. At this time, the marker portion is adsorbed and taken into the lipid monolayer, as shown at 23 in FIG. 2, and imparts conductivity to the lipid monolayer to lower the membrane resistance (increase the electrical conductivity). As described above, since the resistance of the entire measurement system is the film resistance, the decrease in the resistance or the increase in the electrical conductivity is the contribution due to the adsorption.

When the lipid monolayer is regarded as an electric circuit placed between the working electrode and the counter electrode, the relationship between the marker incorporated in the lipid monolayer and the marker, and the relationship between the marker and the base lipid monolayer is considered. Is a parallel connection circuit of resistors. In the calculation process in this case, if the electric conductivity is used, the sum of the electric conductivities of the respective portions becomes the electric conductivity of all the films, so that the calculation process is easy. That is, the electrical conductivity is approximately zero for the lipid monomolecular film portion where the marker is not incorporated, and the number of the incorporated markers and the electrical conductivity have a proportional relationship.

On the other hand, the phenomenon of incorporation of a marker is a general adsorption phenomenon, and the relationship between the amount of adsorbed marker and the time draws a saturation curve. At this time, the equilibrium adsorption amount and the initial rate of adsorption are proportional to the marker concentration (which is also the complex concentration) in the electrolyte solution. Accordingly, since the electric conductivity at the time of measurement equilibrium or the rising slope of the electric conductivity at the initial contact with the liquid is proportional to the concentration of the substance to be measured, the concentration of the complex between the labeled antibody and the antigen can be calculated from this relationship.

Since the concentration of the antigen-labeled antibody complex reaching the working electrode is the same as the concentration of the antigen initially in the liquid to be measured, as described above, the electric conductivity at equilibrium of the measurement or the electric conductivity Since the rising slope at the initial stage of liquid contact is directly proportional to the antigen concentration in the liquid to be measured, the concentration of the antigen to be measured can be calculated from this relationship.

The above is the measurement using the proportional area, and it is easy to use the proportional area. However, even in the non-proportional region, the antigen concentration can be measured if a one-to-one relationship is established between the antigen concentration and the response. Except for the proportional region, a calibration curve can be separately prepared and determined as the correspondence between the response on the calibration curve and the antigen concentration.

Further, marker 1 for lipid monolayer
The increase in electrical conductivity due to the adsorption of individual pieces is 10 minus 8
It is about the power of Siemens, which is capable of detecting one molecule of an object by high-precision resistance measurement, has extremely high marker detection sensitivity, and has extremely high measurement sensitivity.

As described above, the measuring method of the present invention essentially determines the concentration of a substance to be measured by observing a change in resistance (electric conductivity), and uses another measuring method for measuring the current of electrolysis. And other measurement methods for measuring the electrode potential are clear. However, if a response to an external power supply (not shown in FIG. 1) is considered, the current value under a constant voltage applied state is synonymous with the electric conductivity, so that the current may be observed. Accordingly, the electrical conductivity or resistance referred to in the present invention may be any measurement method or display unit system as long as the change in resistance of the film is observed.

Example 2 An example of the second marker of the present invention will be described with reference to FIG. 3 showing an example of the structure of a labeled antibody.
In FIG. 3, 31 is an antibody corresponding to the antigen of the substance to be measured, and 32 is an amino-benzyl-15-crown-5 residue, which is an example of a fat-soluble ion-conductive label (marker). 33 is a residue of a cross-linking agent connecting the antibody and the marker, and the raw material is succinimidylpropionic maleimide. This cross-linking agent is a bivalent reagent that reacts with the sulfohydryl group contained in the antibody 31 and the amino group of the marker 32 at two positions. The 32 marker portion is a kind of crown compound, and has oxygen having high electronegativity with molecules directed inward and having a defined space inside. For this reason, this space is adapted to the size of the space and can easily take in electrically positive chemical species. 15-crown of this example
In the five structure, sodium ions are mainly included.

On the other hand, the crown compound directs the hydrocarbon so as to surround the outside, and this portion is hydrophobic and easily soluble in oil. Due to such a property, the crown compound moves from the aqueous solution, is dissolved (adsorbed) into the lipid thin film and is taken in, and brings ions into the lipid thin film to impart conductivity. When the lipid thin film is thin and is about the molecular size of the crown compound, the movement of ions in this ion conduction is performed by direct passage of ions by the space inside the crown compound becoming a through hole of the lipid thin film. . When the lipid thin film becomes thicker, the whole of the conjugate compound ions that have bound the ions are performed by electrophoresis in the lipid thin film. This can be considered as ion conduction by fat-soluble ions. Therefore, the increase in conductivity per adsorption marker is greater when the lipid thin film is thinner, and the sensitivity is high.

Many kinds of fat-soluble ion-conductive substances, such as valinomycin, which are natural and antibiotics, are known as fat-soluble ion-conductive substances which bind ions similarly to crown compounds.
These are called neutral ion carriers or ionophores, and are practically applied to ion electrodes and the like. These compounds are aminobenzyl-15-
If a modifying group that can be used for crosslinking, such as the amino group of crown-5, is introduced, it can be used as a lipophilic ion-conductive marker.

A porphyrin compound having no hydrophilic group modification is a fat-soluble substance. It is a compound having a space inside like a crown compound, but is different from a crown in that amines are lined inward. The porphyrin compound incorporates a metal cation therein to form a metal chelate which forms a coordination bond with an amine. This metal chelate is taken into the lipid thin film and imparts ionic conductivity to the lipid thin film in the same manner as the above-mentioned conjugated compound. In addition, the amines arranged toward the inside of the porphyrin compound are cationic dissociation groups, and the porphyrin compound itself is a fat-soluble ionic substance. For this reason, the ionic conductivity by fat-soluble ions is given to a lipid thin film similarly to the above-mentioned inclusion ion. The porphyrin compound can be used as a fat-soluble ion-conductive marker if a modifying group that can be used for crosslinking is introduced.

Phthalocyanine is a substance having a structure similar to that of porphyrin and giving the same ionic conductivity as porphyrin. In addition, amines having a long-chain alkyl group are substances that impart ionic conductivity to lipid thin films as fat-soluble ions. In addition, many hydrophobic chelating agents are commercially available as a substance that forms a metal chelate and adsorbs on a lipid thin film to exhibit ion conductivity. Any of these can be used as a lipophilic ion-conductive marker if a modifying group that can be used for crosslinking is introduced.

A polymer or oligomer in which L-alanine, which is an amino acid, is a peptide bond (amide bond) has a helical structure due to hydrogen bonding between polar groups of the peptide main chain, and has a helical main chain polar group. Are oriented inward, and a three-dimensional structure is surrounded by hydrocarbon side chains which are hydrophobic groups. As a result, a cylindrical structure is formed in which the periphery is surrounded by a hydrophobic group around the hydrophilic portion. This tubular substance is fat-soluble due to the hydrophobic group covering the outside, is taken into the lipid thin film, and the hydrophilic portion functions as a through-hole for ions, giving ionic conductivity to the lipid thin film. At this time, the ionic species involved in ionic conduction are mainly hydrogen ions which are small in size and easy to move. A cross-linking reaction for producing a labeled antigen using the polymer or oligomer can be performed on the N-terminal amino group.

As described above, according to the present embodiment, a fat-soluble ion-conductive substance is converted into a fat-soluble substance that forms a conjugate compound with a water-soluble ion, the conjugate compound, a liposoluble chelating agent,
By using at least one of the chelate compound, the fat-soluble ion, and a polymer or oligomer having a structure in which a polar group is oriented inward and a hydrophobic group is oriented outward, the type of label can be increased. .

Example 3 An example of the third lipid thin film of the present invention will be described with reference to FIG. 4 showing the structure of the lipid thin film. Reference numeral 51 in FIG. 4 denotes a metal copper electrode. 52 is a lipid molecule, which forms a monolayer covering the copper surface,
A working electrode is formed in combination with the metal copper electrode. The raw material for lipid molecules in this example is 6-amino-mercaptohexane. The lipid molecule and copper bind via the sulfur atom 53. 55 is an amino group which is a terminal modifying group. Numeral 54 denotes a hydrocarbon chain, and this layer is a hydrophobic substance and is a main constituent of the lipid film with high resistance. The monomolecular film of this example has the ultimate thinness as a lipid thin film and has a uniform thickness. As described above, the thinner the lipid film, the higher the sensitivity. In addition, since uniform sensitivity can be obtained at any part, stable high sensitivity can be obtained. The effect of the hydrocarbon chain length is such that the longer the chain length, the lower the sensitivity.
The effect is small up to about 8. On the other hand, when a voltage is applied at the time of measurement, an ion is penetrated through a hydrocarbon layer without a marker for excessive application, and dielectric breakdown occurs. However, the insulation resistance is advantageously higher when the hydrocarbon chain length is longer.

The terminal modifying group exhibits a characteristic behavior depending on the type of the modifying group. This will be described later.

A carboxylic acid having a long-chain hydrocarbon (this is called a higher fatty acid) or a primary to quaternary amine having a long-chain hydrocarbon is added to a monomolecular film having a methyl group as a terminal modifying group. When acted in water, long-chain hydrocarbons are oriented toward a monomolecular layer, and carboxyl groups or amine groups are oriented toward water, so that a double-layer lipid membrane can be easily obtained. This bilayer lipid thin film is uniform, similar to a monolayer, has the second highest sensitivity next to a monolayer, and has a higher insulation resistance than a monolayer.

A multilayer film such as a bimolecular film can also be formed by forming an LB film (Langmuir-Blodgett film) on the electrode.

As described above, according to the present embodiment, high sensitivity can be stably maintained by using a lipid thin film as a monomolecular film or a multilayer film.

Example 4 An example of the fourth lipid thin film of the present invention will be described with reference to FIG. Numeral 53 in FIG. 4 denotes a sulfur atom, which was present as a thiohydryl group in 6-amino-mercaptohexane as a raw material. Thiohydryl groups act on many metals, such as silver, copper, iron, and tungsten, and readily bind. The bond is the bond with the electrode metal via the sulfur atom indicated by 53 in FIG. As described above, the hydrocarbon having a thiohydryl group can easily form a lipid monolayer on the metal electrode. The monolayer thus formed is called a self-assembled monolayer. The present invention is an application of this, and a homogeneous and highly sensitive working electrode can be easily produced.

A long-chain hydrocarbon having a triazithiol group in which two thiohydryl groups are introduced into a triazine ring is known as one that produces a similar self-assembled monolayer. Also in this case, the thiohydryl group of the tridithiol forms a bond with the metal, and a working electrode having a uniform and highly sensitive lipid thin film can be obtained as in the case of the bond of only a simple thiohydryl group.

(Embodiment 5) An embodiment of the fifth lipid thin film of the present invention will be described with reference to FIGS. 4 and 3. 55 in FIG.
Is an amino group that is a terminal modifying group of a lipid monolayer.
The amino group is a cationic dissociating group, and forms an ion pair by adsorbing an anion in an electrolyte solution (measurement liquid). On the other hand, the antibody 31 in the labeled antibody shown in FIG. 3 is a protein and is an amphoteric electrolyte having a plurality of cations (mainly amino groups) and anions (mainly carboxyl groups). Whole antibodies range from cations in excess of anions to anions in excess of cations. The pH at which the charge of the dissociated anion and the charge of the cation of the amphoteric electrolyte are equal is called the isoelectric point, and is a value that indicates the balance between the cation and the anion, and is one of the properties that distinguish proteins such as antibodies. I have. When the pH of the electrolyte in which the antibody is dissolved is higher than the isoelectric point, the antibody behaves as an anion as a whole, and when the pH of the electrolyte is lower than the isoelectric point, it behaves as a cation.

Therefore, the labeled antibody using an antibody having an isoelectric point smaller than that of the electrolyte solution is adsorbed and adsorbed on the amino group on the surface of the lipid thin film in FIG. 4, and the labeled antibody is concentrated on the surface of the lipid thin film. Become. From this concentrated state, the marker portion 33, which is a fat-soluble ion-conductive substance, is further taken into the 54 hydrocarbon layer. For this reason, the equilibrium adsorption amount of the marker portion is large, and the adsorption speed is high. As the equilibrium adsorption amount increases, the measurable marker measurement range increases. In addition, the response speed of the measurement increases as the adsorption speed increases.

The same occurs when the isoelectric point of the labeled antibody is higher than the pH of the electrolyte solution and the surface of the lipid thin film is covered with an anionic dissociating group such as a carboxyl group. That is, when the dissociated groups on the surface of the lipid thin film and the labeled antigen have opposite signs, the equilibrium adsorption amount and the adsorption speed increase.

When the labeled antigen and the dissociating group on the surface of the lipid thin film have the same sign, the labeled antigen becomes difficult to approach the surface of the lipid thin film, so that the equilibrium adsorption amount of the marker portion and the adsorption speed are reduced. Become.

The case where the surface of the lipid thin film is covered with a hydrophilic group having a large nonionic polarity such as a hydroxyl group and a formamide group and the case where the surface of the lipid thin film is a methyl group of a hydrophobic group are as follows:
Although there is no significant difference in the equilibrium adsorption amount, the adsorption speed is faster when the hydrophilic group is used. When the lipid surface is covered with a hydrophilic group, hydrogen bonds are formed with water molecules in the liquid to be measured, and the hydrogen bonds between water molecules at the lipid thin film interface are reduced accordingly. For this reason, compared to the case of a hydrophobic group that does not form a hydrogen bond with a water molecule,
This is because the interfacial tension decreases and the marker can break into the interface with a small force and penetrate into the hydrophobic layer of the hydrocarbon of the lipid thin film.

Further, like the above-mentioned phospholipids such as phosphatidylcholine and phosphatidylethanolamine, the surface of the lipid thin film has a zwitterionic electrolyte having both a cationic dissociating group of an amine and an anionic dissociating group such as a phosphate ester. When it is covered with, the surface structure of the lipid thin film in which an anion layer such as a phosphate ester and a cation layer of an amine are layered on the surface of the lipid thin film is formed. The cation layer rejects cations and the anion layer rejects anions. For this reason, cations can pass through this multilayer structure,
It also blocks the passage of anions, thus increasing the resistance of the lipid film and making it more resistant to breakdown, which occurs as ions move through the lipid film.

As described above, the surface group of the lipid thin film relates to the equilibrium adsorption amount and the adsorption speed, can control the measurement range of the antigen, the response speed, and is involved in the resistance of the lipid thin film.
The insulation resistance can be increased.

(Example 6) An example of the sixth antibody of the present invention will be described with reference to the structural example of the labeled antibody of FIG. 3, the preparation example of the monovalent antibody of FIG. 6, and the structural example of FIG. explain. Reference numeral 31 in FIG. 3 denotes an antibody corresponding to the antigen of the test object. The antibody is a tetramer in which four protein chains, two long H chains 71 and two short L chains 72, are connected by a disulfide bond, as shown in FIG. 6a. FIG. 6a schematically shows the primary structure, in which the parallel chain portion in which the H chain and the L chain are linked by disulfide bonds has a more three-dimensional higher-order structure, and has a specific recognition binding in the antigen-antibody reaction. I do. Therefore,
Antibodies are necessarily divalent and can bind to two antigens.

However, since the antibody is bivalent, one antigen binds to the labeled antibody, and a monovalent complex of the antigen-labeled antibody is formed, the other of which preserves the binding ability. This monovalent complex has a binding ability to the immobilized antigen, is bound to the immobilized antigen, is removed, and does not reach the working electrode. Therefore, it does not respond to the electrical conductivity measurement and does not start the measurement.

This problem is solved by using a monovalent antibody. If a labeled antibody using a monovalent antibody binds to one antigen, it will not be able to bind to the immobilized antigen, so it will reach the working electrode without being removed by the immobilized antigen,
Starts electrical conductivity measurement.

As shown in the example of FIG. 6, the monovalent antibody is subjected to the action of a protease such as porcine pepsin to excise a part of the H chain which is not involved in the antigen-antibody reaction (FIG. 6b). It can be obtained by opening a disulfide bond by the action of a reducing agent such as ethylamine (FIG. 6c). Further, the monovalent antibody thus produced exposes a sulfohydryl group not involved in the antigen-antibody reaction, and a method for cross-linking this sulfohydryl group with a divalent crosslinker to obtain a labeled antibody is as follows. Called the hinge method,
It is also useful in the present invention as a labeling method without a decrease in antibody titer.

Example 7 An example of the seventh monoclonal antibody of the present invention will be described.

By sensitizing experimental animals such as rats, mice, rabbits, goats, and cows with antigens, various antibodies corresponding to the antigens are produced in plasma. In the body of a laboratory animal, the number of immune cells that produce antibodies is one antibody per cell, and a large number of immune cells produce different antibodies against the same antigen, thereby increasing antibody diversity. Such antibodies are called polyclonal antibodies. In contrast,
Only one immune cell is removed and immortalized (or immature) by cell fusion with myeloma cells, etc.
Make seed antibodies. This antibody is called a monoclonal antibody.

A polyclonal antibody is a mixture of multiple antibodies that recognize and bind to distinct recognition sites within the same antigen. Among many antibodies, there are antibodies having a recognition position at a binding portion between the antigen and the immobilized substrate of the immobilized antigen. Free labeled antibodies made with such antibodies cannot be removed with the immobilized antigen. Therefore, this labeled antibody reaches the working electrode and is adsorbed on the lipid thin film, thereby increasing the electric conductivity. That is, when a polyclonal antibody is used, the blank value of the electric conductivity measurement increases.

On the other hand, since the monoclonal antibody is a uniform antibody, the blank value in the electric conductivity measurement does not increase.

(Embodiment 8) An eighth embodiment of the present invention for measuring electric conductivity will be described with reference to FIGS. In FIG. 2, the electrodes are provided so as to sandwich the lipid thin film. The lipid thin film is closely attached to one of the electrodes to form the working electrode 19.

To measure the resistance, a potential difference is applied to both sides of the lipid thin film. This is shown in FIG. 1 from an external power supply (not shown).
The measurement is performed through a pair of electrodes via the electrode terminal 5 and the resistance is obtained from the response of the current to the voltage. Instead of one of the resistors of the Wheatstone bridge circuit, the electrode terminal 5
Fig. 1 consisting of electrodes, electrolyte aqueous solution, and lipid thin film
Can be obtained as a ratio with a known resistance from the equilibrium condition of the bridge circuit.

However, if the external power supply is a DC voltage applied, the chemical potential changes and a reverse potential is generated in accordance with the movement of the ions, and the potential of the external power supply cancels out over time within a short time. Therefore, it is difficult to measure the change in resistance of the lipid thin film. The same applies to the case where the chemical potential difference is used as a power source, because the chemical potential difference decreases with time as a result of the movement of ions.

As a countermeasure, the resistance is obtained by applying an AC voltage to a pair of electrodes sandwiching the lipid thin film from an external power supply. This is because the alternating current allows the resistance measurement to be performed without changing the chemical potential. Since the term in this case is based on alternating current, resistance is impedance and electric conductivity is read as admittance.

On the other hand, in such impedance measurement, when a DC bias voltage that does not cause electrolysis is applied to the alternating current of the external power supply, the dissociation groups of the labeled antibody, which is an amphoteric electrolyte ion, described in Example 5 are removed. A driving force for electrophoresis in a certain direction is applied. As a result, when the isoelectric point of the labeled antibody is lower than the pH of the electrolyte solution, the labeled antibody moves to the anode side. When the anode is the working electrode, the marker is incorporated into the lipid thin film, so that the adsorption of the marker to the lipid thin film is accelerated and the response is accelerated, and the equilibrium adsorption amount of the marker to the lipid thin film is increased, thereby increasing the sensitivity. . Conversely, when the cathode is the working electrode, the marker moves away from the lipid thin film, so that the adsorption of the marker to the lipid thin film is inhibited and the response becomes slow, and the equilibrium adsorption amount of the marker to the lipid thin film decreases. , The sensitivity decreases.

Also, the isoelectric point of the labeled antibody is determined by the pH of the electrolyte solution.
If it is larger, the labeled antibody moves to the cathode side.
When the cathode is the working electrode, the marker is incorporated into the lipid thin film, so that the adsorption of the marker to the lipid thin film is accelerated and the response is accelerated, and the equilibrium adsorption amount of the marker to the lipid thin film is increased, thereby increasing the sensitivity. . Conversely, when the anode is the working electrode, the marker moves away from the lipid thin film, which inhibits the adsorption of the marker to the lipid thin film, slows down the response, and reduces the equilibrium adsorption amount of the marker to the lipid thin film. , The sensitivity decreases.

The DC bias voltage at which electrolysis does not occur, particularly when no oxidizing substance or reducing substance is present, is the sum of the maximum AC voltage and the water bias to hydroxyl ions and hydrogen ions. It is less than the theoretical potential of dissociation of 0.83 volts. In addition, unless electrolysis occurs, continuous electrophoresis cannot be performed at this bias voltage. Therefore, a method for increasing or decreasing the sensitivity only to a trace amount of labeled antigen is used.

(Embodiment 9) An embodiment of the ninth pH buffer according to the present invention will be described with reference to FIG. In the figure, reference numeral 1 denotes a cylindrical counter electrode, and reference numeral 2 denotes a working electrode having a tip inserted into the counter electrode 1.
Reference numeral 3 denotes an immobilized antigen layer in which a granular immobilized antigen is filled in a cylindrical counter electrode 1, and 6 denotes a labeled antibody layer. 81 is constant pH
PH buffer layer consisting of carboxymethyl cellulose, a weakly acidic ion exchanger equilibrated to the value, 4 is the inlet of the liquid to be measured, and the liquid to be measured, which is an aqueous electrolyte solution containing the antigen, has a capillary effect. Into the pH buffer layer by pH
Is adjusted, it passes through the immobilized antigen layer 3 by capillary action, reaches the tip of the working electrode 2, and comes into contact with the working electrode.

The carboxymethylcellulose ion exchanger is obtained by introducing a large number of carboxyl groups into fibrous cellulose. A carboxyl group is a weakly acidic dissociating group that changes its dissociation and non-dissociation by adsorbing and desorbing alkaline substances such as sodium ions in accordance with the pH of surrounding water, and thus has a pH buffering action. Furthermore, since a large number of carboxyl groups interfere with each other in the carboxymethylcellulose ion exchanger, a pH buffering effect is exerted.
The area is extensive. The operation of immersing the ion exchanger at a certain pH and performing adsorption equilibrium with alkali is called equilibration.When equilibrated carboxymethylcellulose is transferred to another environment, the surrounding environment is brought to the equilibrated pH. Adjust the pH
Exhibits a buffering action.

Therefore, in the structure of FIG. 7, the liquid to be measured which has entered the pH buffer layer 81 is adjusted to an equilibrated pH by the action of the pH buffer, and then enters the next labeled antibody layer 6 to form the antigen-labeled antibody complex. A body is formed, and further enters the immobilized antigen layer to remove excess labeled antibody, and the remaining antigen-labeled antibody complex reaches the working electrode. The effect of the pH adjustment here is shown in Examples 5 and 8.
As described above, the character of the labeled antibody as an ion is determined in relation to the isoelectric point of the labeled antibody. This means that when controlling the measurement sensitivity and measurement range in relation to the surface modification group of the lipid thin film or the DC bias voltage, the variation in the measured value due to the variation in the pH of the solution to be measured is suppressed. This has the effect of ensuring measurement accuracy.

Further, in this embodiment, the dissociating group is not limited to a carboxyl group, and an anionic weak dissociating group such as a phosphate ester can be used in addition to the carboxyl group. Also,
The substrate of the ion exchanger does not need to be cellulose, and may be polystyrene, fluorocarbon, or the like. Further, the shape does not necessarily have to be fibrous, but may be beads or a porous body.

Further, it is not always necessary to use an insoluble pH buffer such as an ion exchanger. A dry composition of a water-soluble buffer such as a phosphate buffer or a trishydroxymethylaminomethane hydrochloride may be used as a pH buffer layer. The same effect can be obtained by disposing (applying) this in the liquid to be measured. As described in the present embodiment, when the pH is adjusted using an ion exchanger, if the antigen to be measured is an ionic substance having the opposite sign to the dissociating group of the ion exchanger, the antigen is adsorbed to the ion exchanger. Removed and cannot be measured. In such a case,
It is preferred to use a soluble pH buffer composition.

Example 10 An example of the immobilized antigen of the present invention will be described with reference to FIG. 5 showing the chemical structure of the immobilized antigen. In FIG. 5, reference numeral 41 denotes a residue of estradiol, which is a kind of estrogen, and reference numeral 42 denotes a glucuronic acid residue, which together form a residue of an estradiol glucuronic acid conjugate. Estradiol glucuronic acid conjugate is an excretory form of ovarian hormone in urine, and is an antigen or an analyte when urine is measured using urine as a test solution. 43 is a polyvinyl aniline as an immobilization base material, and 44 is an aniline residue thereof. 45 and 46 are residues of a cross-linking agent connecting the antigen and the marker. The raw material 46 is mercaptosuccinic anhydride, which reacts with the hydroxyl group of glucuronic acid. The raw material of 45 is succinimidyl propionic maleimide which is a divalent cross-linking agent, and reacts with the thiohydryl group of mercaptosuccinic anhydride of 46 and the amino group of polyvinyl aniline to form a cross-link.

The immobilized antigen of this example forms a complex with a labeled antibody corresponding to the estradiol glucuronic acid conjugate by an antigen-antibody reaction. The measurement method of Example 1 uses this reaction to remove excess labeled antibody.

[0088]

As described above, according to the present invention, a substance which is soluble in lipid having high resistance and imparts ionic conductivity to lipid is bound to a substance to be measured as a label, and the labeled substance is labeled. The part is adsorbed on the lipid thin film (dissolved in the thin film), and the decrease in resistance (increase in electrical conductivity) caused by imparting ionic conductivity to the lipid thin film is detected, and the substance to be measured is measured. By using such labeling and label detection together, immunity that combines the versatility of the immunoassay with the sensitivity, precision, simplicity, and rapidity found in the sensing of biological receptors and extracts the immune response as an electrical response An electrode sensor becomes possible.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating an entire configuration according to a first embodiment of the present invention.

FIG. 2 is a diagram illustrating an internal configuration according to a first embodiment of the present invention.

FIG. 3 is a diagram showing the structure of a labeled antibody in Example 2 of the present invention.

FIG. 4 is a diagram showing a configuration of a lipid thin film in Examples 3, 4, and 5 of the present invention.

FIG. 5 is a view showing the structure of an immobilized antigen in Example 10 of the present invention.

FIG. 6 is a diagram showing a process of preparing a monovalent antibody in Example 6 of the present invention.

FIG. 7 is a diagram showing the implementation of a pH buffer in Example 9 of the present invention.

[Explanation of symbols]

 Reference Signs List 11 antigen 15 immobilized antigen 16 marker 17 labeled antibody 18 antigen-labeled antibody complex 19 working electrode 21 lipid monolayer 24 free labeled antibody 54 sulfur atom

Claims (9)

[Claims] 1. A soluble labeled antibody labeled with a fat-soluble ion-conductive substance, an immobilized antigen corresponding to the labeled antibody, a pair of electrodes, a working electrode and a counter electrode, and a lipid thin film formed on the surface of the working electrode. Having a complex of an antigen and a labeled antibody generated by contacting the labeled antibody and an antigen in a liquid to be measured adsorbed to the immobilized antigen, thereby forming the unreacted labeled antibody on the electrode surface. An immunoelectrode sensor that adsorbs on a lipid thin film and detects a lipid-soluble ion-conductive label based on the amount of change in the electrical conductivity of the lipid thin film to detect an antigen in a liquid to be measured. 2. A fat-soluble substance which forms a conjugated compound with a water-soluble ion, a conjugated compound thereof, a liposoluble chelating agent, a chelate compound thereof, a liposoluble ion, wherein a polar group is oriented inward and a hydrophobic group is directed outward. 2. The immunoelectrode sensor according to claim 1, wherein at least one of the polymer or oligomer having a structure in which is oriented is a fat-soluble ion-conductive substance. 3. The immunoelectrode sensor according to claim 1, wherein the lipid thin film is a monomolecular film or a multilayer film. 4. The immunoelectrode sensor according to claim 1, wherein the lipid thin film is bonded to the metal material of the electrode via a sulfur atom. 5. The lipid thin film surface is covered with at least one of a hydrophobic group, a nonionic hydrophilic group, a cationic dissociating group, an anionic dissociating group, and a zwitterionic dissociating group. Immunoelectrode sensor. 6. The immunoelectrode sensor according to claim 1, wherein the soluble labeled antibody labeled with a fat-soluble ion-conductive substance is a monovalent antibody. 7. The immunoelectrode sensor according to claim 1, wherein the soluble labeled antibody labeled with a fat-soluble ion conductive substance is a monocolonal antibody. 8. The immunoelectrode sensor according to claim 1, wherein the measurement of the change in the electric conductivity is performed by applying an alternating current or applying a direct current bias. 9. The immunoelectrode sensor according to claim 1, further comprising a pH buffer.