JP2002174612A - Measurement method and device therefor - 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 imparting 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 measured substance 3 is adsorbed in a lipid thin film 12 (dissolved in the thin film), the resistance is reduced for imparting the ion conductivity to the lipid thin film 12, 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. By sensitizing an experimental animal with an antigen, it is possible to obtain antibodies corresponding to a large number of substances, and it is possible to generally construct a method for detecting the components and amounts of many kinds of substances. 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 detection 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. In addition, as an application of this method, there is a method in which a oxidoreductase is used as a label, and a change in the concentration of an oxidizing substance or a reducing substance, which is the result of an enzymatic reaction, is detected as an electrode potential or a current for titration (electrolysis). .

[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 types of 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. Alternatively, when the sensitivity is increased to increase the sensitivity, noise is picked up and the accuracy is deteriorated.

[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 detection of a substance in a living body, recognition and response of a neurotransmitter acetylcholine by an acetylcholine receptor at a nerve synapse is as follows.
Are 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 substance in 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. Thus, it can be said that this is an extremely rational detection method.

[0017]

However, the in vitro application technique of this method for detecting a substance in a living body is limited to extracting a receptor from the living body and reconstructing the receptor on a lipid bilayer. Due to the type of receptor, equivalent detection cannot be performed on many types 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 method having both rapidity and extracting an immune response as an electrical response.

[0019]

For this purpose, the measuring method of the present invention uses an immunization method using an ion channel marker as a label and a resistance detection method. That is, a substance that is soluble in lipid having high resistance (the inverse of resistance is the same concept as electrical conductivity) and that imparts ionic conductivity to lipid is bound to the substance to be measured as a label, and the label of the labeled substance is labeled. The part is adsorbed on the lipid thin film (dissolves in the thin film), and a 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.

The combined use of such a label and a resistance detection method has both the versatility of the immunoassay and the sensitivity, accuracy, simplicity, and rapidity found in the sensing of biological receptors.
An immunoelectrode method in which an immune reaction is extracted as an electrical response becomes possible. In addition, it becomes possible to provide the device.

[0021]

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, there is provided a method for measuring a change in electrical conductivity of a lipid thin film by adsorbing a substance to be measured, which is a labeled compound labeled with a fat-soluble ion-conductive substance, to a lipid thin film. The substance to be measured is measured by detecting the lipophilic ion-conducting labeled compound in an amount. It is possible to provide an immunoelectrode method that combines the versatility of an immunoassay with the sensitivity, accuracy, simplicity, and rapidity found in sensing of a receptor in a living body, and extracts an immune reaction as an electrical response.

According to a second aspect of the present invention, a substance to be measured, which is a labeled compound labeled with a fat-soluble ion-conductive substance, is adsorbed on a lipid thin film formed on the electrode surface, and the electrical conductivity of the lipid thin film is measured. The substance to be measured is measured by detecting the lipophilic ion-conductive label with the change. Versatility of immunization method,
It is possible to provide an immunoelectrode method that combines sensitivity, accuracy, simplicity, and rapidity found in sensing of receptors in a living body and extracts an immune reaction as an electrical response.

According to a third aspect of the present invention, there is provided an insolubilized antibody and a labeled antigen which is bound to the insolubilized antibody by an antigen-antibody reaction and which is labeled with a lipophilic ion-conductive substance. The labeled antigen released by the displacement reaction due to the contact is adsorbed on the lipid thin film formed on the electrode surface, and the lipid-soluble ion-conductive labeled compound is detected based on the change in the electrical conductivity of the lipid thin film to measure the antigen. It is possible to provide an immunoelectrode method that combines the versatility of an immunoassay with the sensitivity, accuracy, simplicity, and rapidity found in the detection of receptors in a living body, and extracts an immune reaction as an electrical response.

According to a fourth aspect of the present invention, there is provided an insolubilized antibody, a labeled antigen labeled with a lipophilic ion-conductive substance bound to the insolubilized antibody by an antigen-antibody reaction, and a pair of electrodes, a working electrode and a counter electrode. The labeled antigen, which has a lipid thin film formed on the extreme surface and is released by a displacement reaction due to contact with the antigen in the liquid to be measured, is adsorbed on the lipid thin film formed on the electrode surface, and the electrical conductivity of the lipid thin film changes. The amount of the fat-soluble ion-conductive labeled compound is detected to determine the antigen.
It is possible to provide an immunoelectrode device that combines the versatility of an immunoassay with the sensitivity, accuracy, simplicity, and quickness found in the detection of receptors in a living body, and extracts an immune reaction as an electrical response.

According to a fifth aspect of the present invention, the fat-soluble ion-conductive substance according to the first to fourth aspects,
A fat-soluble substance that produces an inclusion compound with a water-soluble ion, its inclusion compound, a fat-soluble chelating agent, its chelating compound,
A measurement method and a measurement device, which are at least one of a liposoluble ion and a polymer or oligomer having a structure in which a polar group is oriented inward and a hydrophobic group is oriented outward,
Signs can be diversified.

The present invention according to claim 7 or 8 is a measuring method and a measuring apparatus in which a cationic or anionic dissociating group is introduced into the labeled compound according to any of claims 1 to 4, In addition to imparting water solubility to the labeled compound to avoid loss due to precipitation, the sensitivity, measurement range, response speed, and the like of the measurement can be controlled in combination with the invention of claims 9 and 12,
In addition, it contributes to the resistance of the lipid thin film and can increase the insulation resistance.

The present invention described in claim 9 or 10 is a measuring method and a measuring apparatus in which the lipid thin film according to claim 2 or 4 is a monomolecular film or a multilayer film. Can be provided.

The present invention according to claim 11 or 12 is:
A measurement method and a measurement device, wherein the lipid thin film of the invention according to claims 2 to 4 binds to a metal material of an electrode via a sulfur atom,
A method for easily forming a lipid thin film can be provided.

The present invention according to claim 13 or 14 provides:
The measurement in which the lipid thin film surface according to any one of claims 1 to 4 is covered with one or more of a hydrophobic group, a nonionic hydrophilic group, a cationic dissociating group, an anionic dissociating group, and a zwitterionic dissociating group. The present invention provides a method and a measurement device, which can provide a method for controlling the sensitivity, the measurement range, and the response speed of the measurement, and a method for increasing the insulation resistance.

The present invention according to claim 15 or 16 is:
A measuring method and a measuring apparatus using a monovalent antibody as the antibody according to the third and fourth aspects of the present invention, which can obtain a highly linear response to the concentration of an analyte.

The present invention according to claim 17 or 18 provides:
This is a measurement method using a monoclonal antibody as the antibody of the invention according to claims 3 and 4, and can provide a highly linear response to the concentration of a specific measured substance.

The present invention according to claim 19 or 20 is:
The method for measuring the change in electric conductivity according to the first to fourth aspects of the present invention is a measuring method in which an alternating current is applied or an alternating current is applied by applying a direct current bias, and a method of extracting an electric response to the outside can be provided.

[0033]

(Embodiment 1) A first embodiment of the present invention will be described with reference to FIG. In the figure, reference numeral 1 denotes a lipid thin film composed of a phospholipid bilayer, which is provided so as to partition between electrodes 6 and 7. Reference numeral 8 denotes an aqueous electrolyte solution that fills the space between the electrodes 6 and 7, which is a foreign substance in the present configuration example and corresponds to the liquid to be measured when viewed from the present measurement system. The electrolyte concentration is assumed to be a salt concentration on the order of body fluid or urine. The reference numeral 1 in the liquid to be measured is an object to be measured, which has already been bonded to the marker 2 outside the measurement system to form the labeled object 3. This marker is a fat-soluble ion-conducting substance, which is taken into the phospholipid bilayer,
It has the property of imparting ion conductivity.

The phospholipid bilayer, which is the lipid thin film of this example,
It has a high resistance of about 10 9 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 phospholipid bilayer. The analyte in the electrolyte aqueous solution diffuses in the electrolyte solution to reach the phospholipid bilayer.
At this time, as shown in FIG. 9, the marker portion is adsorbed and taken into the phospholipid bilayer, imparts conductivity to the phospholipid bilayer, and lowers the membrane resistance (increases electric conductivity). As described above, since the resistance of the entire measurement system is a film resistance,
Alternatively, the increase in electric conductivity is the contribution due to adsorption.

By the way, the relationship between the marker incorporated in the phospholipid bilayer and the marker, and the marker and the base phospholipid bilayer portion 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 phospholipid bilayer portion where the marker is not incorporated is set to approximately zero electric conductivity,
There is a proportional relationship between the number (amount) of the markers taken in and the electrical conductivity.

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 concentration of the analyte) in the electrolyte solution. Accordingly, the electric conductivity at the equilibrium of the measurement 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, so that the concentration of the substance to be measured can be calculated from this relationship.

The above is the measurement using the proportional region of the electrical conductivity response, and it is easy to use the proportional region. However, the measurement of the concentration of the substance to be measured is possible even if it is not in the proportional region, provided that a one-to-one relationship is established between the concentration of the substance to be measured and the response. Except for the proportional region, a calibration curve can be separately prepared and determined as a correspondence between the response on the calibration curve and the concentration of the substance to be measured.

The increase in electrical conductivity due to the adsorption of one marker to the phospholipid bilayer is about 10 −9 Siemens, which is a high-precision resistance measurement for detecting one molecule of the analyte. Is possible, and the sensitivity is extremely high.

As described above, the measuring method of the present invention essentially measures the change in resistance (electric conductivity) to obtain the concentration of the substance to be measured. The distinction from other methods of measuring electrode potential is 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. Therefore, as for the electric conductivity or resistance in the present invention, any measurement method or unit system may be used as long as the change in resistance of the film is observed.

It is also possible to provide a measuring device equipped with the method of the present invention.

(Embodiment 2) A second embodiment of the present invention will be described with reference to FIG. In the figure, reference numeral 12 denotes a lipid monolayer formed of molecules of lipid 11 formed on the working electrode 13 and is a lipid thin film. Reference numeral 8 denotes an aqueous electrolyte solution that fills the space between the electrodes 13 and 14, which is a foreign substance in the present configuration example and corresponds to the liquid to be measured, as viewed from the measurement system. The electrolyte concentration is assumed to be a salt concentration on the order of body fluid or urine. The reference numeral 1 in the liquid to be measured is an object to be measured, which has already been bonded to the marker 2 outside the measurement system to form the labeled object 3.
This marker is a fat-soluble ion-conductive substance, and has a property of being incorporated into twelve lipid monolayers to impart ion conductivity.

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 cm 2. 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 analyte to be labeled in the electrolyte aqueous solution diffuses in the electrolyte solution to reach the lipid monolayer. At this time, as shown in FIG. 9, the marker portion is adsorbed and taken into the lipid monolayer, imparts conductivity to the lipid monolayer, and lowers the membrane resistance (increases 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.

By the way, the relationship between the marker incorporated into the lipid monolayer and the marker, and the marker and the base lipid monolayer portion 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 electric conductivity is approximately zero for the phospholipid bilayer portion where the marker is not incorporated, and the number of the incorporated markers and the electric 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 amount of parallel adsorption and the initial rate of adsorption are proportional to the marker concentration in the electrolyte solution (which is also the concentration of the analyte). Therefore, the electric conductivity at the equilibrium of the measurement or the rising slope of the electric conductivity at the initial stage of contact with the liquid is proportional to the concentration of the substance to be measured.

The above is the measurement using the proportional region of the electrical conductivity response, and it is easy to use the proportional region. However, the measurement of the concentration of the substance to be measured is possible even if it is not in the proportional region, provided that a one-to-one relationship is established between the concentration of the substance to be measured and the response. Except for the proportional region, a calibration curve can be separately prepared and determined as a correspondence between the response on the calibration curve and the concentration of the substance to be measured.

In addition, 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, and has extremely high 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). However, if a response to an external power supply (not shown in FIG. 2) is considered, the current value under a constant voltage application state is synonymous with the electric conductivity, so that the current may be observed. Therefore, the electrical conductivity or resistance referred to in the present invention may be any measurement method or unit system as long as the change in resistance of the film is observed.

It is also possible to provide a measuring device equipped with the method of the present invention.

(Embodiment 3) A third embodiment of the present invention will be described with reference to FIGS. FIG. 3 is a diagram showing an example of the overall configuration of the sensor according to the present invention. In FIG. Reference numeral 22 denotes an insolubilized antibody layer in which the inside of the cylindrical counter electrode 14 is filled with granular insolubilized antibody. Reference numeral 21 denotes an inlet of the liquid to be measured. The liquid to be measured, which is an aqueous electrolyte solution containing the antigen, passes through the insolubilized antibody layer 22 by capillary action, reaches the tip of the working electrode 13, and comes into contact with the working electrode 13.

FIG. 4 is a diagram for explaining the internal configuration of FIG. 3 together with the movement of the liquid to be measured. Reference numeral 31 denotes an antigen in the liquid to be measured 8 which corresponds to the object to be measured in this example. The liquid to be measured enters the insolubilized antibody layer 22 by capillary action. Here, the insolubilized antibody substrate 33
An antibody 32 corresponding to the antigen 31 is bonded to the surface of the sample to form an insolubilized antibody 34. Further, a labeled antigen 3 obtained by modifying the same antigen as the antigen to be measured with a marker is subjected to an antigen-antibody reaction. Previously bound to the insolubilized antibody. The marker is a fat-soluble ion-conducting substance.

The antigen and the antigen portion of the labeled antigen are the same substance, and have the same binding power to the insolubilized antibody. Therefore, the antigen 31 that has flowed into the insolubilized antibody layer undergoes a substitution reaction with the labeled antigen 3 bound to the insolubilized antibody, and the labeled antigen is released. The released labeled antigen reaches the working electrode 13 with the movement of the liquid to be measured, and the working electrode 13 and the counter electrode 14 are connected via the liquid to be measured.

Here, reference numeral 12 denotes a lipid monolayer composed of molecules of lipid 11 formed on the working electrode 13 and is a lipid thin film. Reference numeral 8 denotes an aqueous electrolyte solution that fills the space between the electrodes 13 and 14, and in the present configuration example, is a measured liquid that has passed through the insolubilized antibody layer. The electrolyte concentration is assumed to be a salt concentration on the order of 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 cm 2. 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 labeled antigen 3 in the aqueous electrolyte solution diffuses in the electrolyte solution to reach the lipid monolayer. At this time,
As shown at 9 in FIG. 4, the marker portion is adsorbed and taken into the lipid monolayer, imparts conductivity to the lipid monolayer, and lowers the membrane resistance (increases electrical conductivity). As described above, since the resistance of the entire measurement system is a film resistance, an increase in resistance or an increase in electrical conductivity is a contribution due to adsorption.

The relationship between the marker incorporated in the lipid monolayer and the marker, and the marker and the base lipid monolayer constitute a parallel connection circuit of the 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 labeled antigen concentration) in the electrolyte solution. Therefore, the electrical conductivity at the equilibrium of the measurement or the rising slope of the electrical conductivity at the initial contact with the liquid is proportional to the concentration of the substance to be measured. Therefore, the concentration of the labeled antigen may be calculated from this relationship.

The concentration of the free labeled antigen reaching the working electrode is determined by the probability of the substitution reaction between the antigen in the insolubilized antibody layer and the labeled antigen bound to the insolubilized antibody. Since the substitution probability is constant while the substitution rate is low, the concentration of the released labeled antigen is proportional to the concentration of the antigen in the test solution. Therefore, over the entire process, the concentration of the antigen in the liquid to be measured is proportional to the electric conductivity at the equilibrium of the measurement, or the rising slope of the electric conductivity at the initial contact with the liquid. What is necessary is just to obtain the antigen concentration of.

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.

The marker detection sensitivity is extremely high as described in the above two examples. In the present embodiment, this is multiplied by the substitution rate between the labeled antigen on the insolubilized antibody and the antigen to be measured. This replacement rate is mainly
It is determined by the magnitude of the space velocity in the insolubilized antibody layer. If the space velocity is low, the sensitivity is high, and if the space velocity is high, the sensitivity is low and can be controlled.

As described above, the measuring method and measuring apparatus of the present invention essentially determine the concentration of a substance to be measured by observing a change in resistance (electric conductivity). However, if it is considered that the response is to an external power supply (not shown in FIG. 3), the current value in a state where a constant voltage is applied 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 4 An example of the fourth marker of the present invention will be described with reference to the chemical structural formula shown in FIG. 5 showing an example of the structure of a labeled analyte or labeled antigen. In the first and second embodiments of the present invention, the structure shown in FIG. 5 is synthesized externally and brought into a measurement system. In the third embodiment of the present invention, the structure is used as a labeled antigen bound to an insolubilized antibody by an antigen-antibody reaction.

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. Together, they form a residue of an estradiol glucuronide conjugate. The estradiol glucuronic acid conjugate is a urinary excretion type of ovarian hormone and is an antigen or an analyte when urinary hormone is measured using urine as a liquid to be measured. 43 is an aminobenzyl-15-crown-5 residue, which is a fat-soluble ion-conductive label (marker);
This is an example. 44 and 45 are residues of a cross-linking agent connecting the antigen and the marker. The raw material of 44 is mercaptosuccinic anhydride, and the raw material of 45 is succinimidylpropionic maleimide which is a bivalent cross-linking agent. The marker portion 43 is a kind of a crown compound, and has oxygen with 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 of this example
In the crown-5 structure, sodium ions are mainly included. On the one hand, the crown compound directs the hydrocarbon around the outside, which is hydrophobic and easily soluble in oil. Due to this property, the crown compound moves from the aqueous solution and dissolves (adsorbs) in the lipid thin film.
And take the ions into the lipid thin film. 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. . If the lipid film becomes thicker,
The reaction is carried out by electrophoresis of the conjugated compound ions having conjugated ions in the lipid thin film. This is 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-conducting substances, such as valinomycin, which are natural and antibiotics, are known as fat-soluble ion-conducting 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. All of these compounds can be used as a lipophilic ion-conductive marker if they introduce a modifying group that can be used for crosslinking, such as the amino group of aminobenzyl-5-crown-5.

A porphyrin compound which has not been modified with a hydrophilic group 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.

The 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.

(Example 5) An example of the fifth dissociating group of the present invention will be described with reference to the chemical structural formula of FIG. 5 showing an example of the structure of a labeled analyte or labeled antigen. In the figure, reference numeral 46 denotes a carboxyl group. Carboxyl groups are anionic dissociating groups. In general, not only carboxyl groups but also ionic substances have high affinity for water and high water solubility. In this structural example, a carboxyl group is introduced to increase the water solubility of the labeled antigen. The aminobenzyl-15-crown-5 residue of the marker 43 in the figure is a fat-soluble substance and is hardly soluble in water.
Not limited to this, the marker used in the present invention is hardly soluble in water because it must be incorporated into a lipid thin film. In addition, the antigen of this example is an estradiol glucuronic acid conjugate. Among them, the estradiol portion has a structure in which a small hydrophilic group hydroxyl group is bonded to a large hydrophobic steroid skeleton, and this portion is also hardly soluble in water. Therefore, it is difficult to deposit and reach the lipid thin film on the working electrode without taking measures to increase the water solubility.

In this example, a ring-opening reaction of an acid anhydride portion of mercaptosuccinic anhydride is used to introduce a carboxyl group. This reaction also serves as a crosslinking reaction with the hydroxyl group of the glucuronic acid residue. The thiohydryl group of mercaptosuccinic anhydride also serves as a target for a cross-linking reaction with the adjacent succinimidylpropionic maleimide. However, it is not necessary to consolidate such a number of functions into one cross-linking agent, and any method of introducing a carboxyl group may be used for the purpose of enhancing water solubility. The dissociating group to be introduced may be another anionic dissociating group such as a sulfone group, or may be a cationic dissociating group such as a primary to quaternary amine.

On the other hand, introduction of a dissociation group is a major factor that changes the adsorptivity to the lipid thin film in relation to the modifying group on the surface of the lipid thin film. This will be described later.

Example 6 An example of the sixth lipid thin film of the present invention will be described with reference to FIG. 6 showing the structure of the lipid thin film. Reference numeral 51 in FIG. 6 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, the dielectric breakdown occurs through the hydrocarbon layer having no marker for the excessive application, but the insulation resistance is advantageously higher when the hydrocarbon chain length is longer.

The terminal modifying group exhibits 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.

Phospholipids such as phosphatidylcholine and phosphatidylethanolamine can be prepared by applying a solution of the organic solvent to the pores in the gas phase and then evaporating the solvent to form a double layer called a phospholipid double layer. A lipid overlayer is formed. This embodiment has been described above.

(Embodiment 7) An embodiment of the seventh lipid thin film of the present invention will be described with reference to FIG. Numeral 53 in FIG. 6 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 a 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 8) An eighth embodiment of the present invention will be described with reference to FIGS. 6 and 5. 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 labeled antigen (labeled analyte) already shown in FIG.
It has 46 carboxyl groups and one carboxyl group among 42 gluconic acid residues. These carboxyl groups are anionic dissociating groups. Therefore, the labeled antigen shown in FIG. 5 forms an ion pair with the amino group on the surface of the lipid thin film shown in FIG. 6 to be adsorbed, and the labeled antigen is concentrated on the surface of the lipid thin film. From this concentrated state, the marker portion 43, which is a fat-soluble ion-conductive substance, is further incorporated into the 54 hydrocarbon layers. 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 a cationic dissociating group such as a primary to quaternary amine is introduced into a labeled antigen, 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 labeling antigen and the dissociating group on the surface of the lipid thin film have the same sign, it becomes difficult for the labeling antigen to approach the surface of the lipid thin film, so that the equilibrium adsorption amount of the marker portion and the adsorption speed are low. Become.

Further, when the surface of the lipid thin film is covered with a hydrophilic group having a large nonionic polarity such as a hydroxyl group or a formamide group, and when the surface is a methyl group as a hydrophobic group,
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.

Also, like the above-mentioned phospholipids such as phosphatidylcholine and phosphatidylethanolamine, the surface of the lipid thin film has 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 and the response speed of the antigen, and contributes to the resistance of the lipid thin film to increase the insulation resistance. be able to.

(Example 9) An example of the ninth antibody of the present invention will be described based on the constitutional example of the insolubilized antibody in FIG. 7 and the preparation example of the monovalent antibody in FIG. Numeral 63 in FIG. 7 denotes an antibody corresponding to the antigen of the analyte. Reference numeral 61 denotes polystyrene beads selected as the insolubilizing substrate in this example. Polystyrene has a molecular structure in which a side chain of a phenyl group is bonded to a main chain of a hydrocarbon. When a bromine molecule is allowed to act on the polystyrene, a brominated polystyrene in which the hydrogen of the phenyl group is replaced with a bromine atom is obtained. This brominated polystyrene is active, and can react with a protein such as an antibody to form a crosslink via a phenyl group. The insolubilized antibody layer shown at 22 in FIG. 3 is obtained by filling the thus obtained insolubilized antibody in a cylindrical counter electrode. In addition to the exemplified methods, there are many types of insolubilization methods, and various types of shapes other than beads are known. The present invention does not limit these.

On the other hand, the antibody is a tetramer in which four protein chains, two long H chains and two short L chains, are connected by a disulfide bond, as shown in FIG. 8A. FIG. 8a
Is a schematic representation of the primary structure, in which the parallel chain portion in which the H and L chains are linked by disulfide bonds takes a more three-dimensional higher-order structure to perform specific recognition and binding of the antigen-antibody reaction. . Thus, an antibody is necessarily divalent and can bind to two antigens.

By the way, these divalents should be equivalent in terms of their symmetry and have the same binding power to the antigen, but when either one binds to the antigen, the other binding site is already bound to the antigen. Disturbed by the antigens present (steric hindrance)
It becomes difficult to combine. That is, the first antigen and the second antigen have different binding forces. In Example 3, the displacement reaction between the labeled antigen on the insolubilized antibody and the antigen to be measured was described. However, as long as a bivalent antibody is used, if the displacement probability is equal between the first and second, No. Therefore, the response of the electric conductivity has poor linearity. This is improved by the use of monovalent antibodies.

As shown in the example of FIG. 8, the monovalent antibody is acted on by a protease such as porcine pepsin to remove a part of the H chain which is not involved in the antigen-antibody reaction (FIG. 8b). It can be obtained by opening a disulfide bond by the action of a reducing agent such as ethylamine (FIG. 8c). In addition, a sulfohydryl group that is not involved in the antigen-antibody reaction is exposed in the monovalent antibody thus formed. A method for obtaining an insolubilized antibody by cross-linking this sulfohydryl group with a divalent cross-linking agent is as follows. Called the hinge method,
It is also useful in the present invention as an insolubilization method without a decrease in antibody titer.

(Example 10) An example of the tenth monoclonal antibody of the present invention will be described.

By sensitizing laboratory 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 polycolonal 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 monocolonal antibody.

A polycolonal antibody is a mixture of a number of antibodies that recognize and bind to distinct recognition positions within the same antigen, so that the antigen-binding power differs between the antibodies.
Further, the composition is not constant, which varies with each production. In Example 3, the substitution reaction between the labeled antigen and the antigen to be measured on the insolubilized antibody was described. However, as long as a polycolonal antibody is used, the substitution probability is not sharp and constant, and the reproducibility is poor. The response of the electrical conductivity is poor in linearity with respect to the antigen concentration of the analyte. In contrast, a monocolonal antibody is a homogeneous antibody,
Excellent reproducibility and linearity of electrical conductivity response.

(Embodiment 11) An eleventh embodiment of the present invention for measuring electric conductivity will be described with reference to FIGS. Reference numerals 6 and 7 in FIG. 1 denote a pair of electrodes provided so as to sandwich the lipid thin film. In FIG. 2, the electrodes are also arranged so as to sandwich the lipid thin film, but the lipid thin film is closely attached to one of the electrodes to form the working electrode 13.

To measure the resistance, a potential difference is applied to both sides of the lipid thin film. This can be performed through a pair of electrodes from an external power supply (not shown), and in the configuration of the example of FIG. 1, it can be performed by providing a difference in the chemical potential of the electrolyte solution on both sides of the lipid thin film.

When an external power supply is used, the resistance is determined from the response of the current to the voltage. Also, instead of one of the resistors of the Whiston bridge circuit,
Using a circuit composed of a lipid thin film and having the structure shown in FIG. 1 or FIG. When a chemical potential difference is used in place of an external power supply, the amount of movement of ions passing through the lipid thin film per time is electrochemically analyzed as a change in the difference in monopolar potential to obtain a current, The resistance can be obtained as a current per chemical potential difference. At this time, the pair of electrodes is used to pick up the monopolar potential of the electrolyte solution on each side of the lipid thin film.

However, when the external power supply is a DC voltage application, the chemical potential changes according to the movement of the ions, and a reverse potential is generated, and the potential of the external power supply cancels out with time in a short time. Therefore, it is difficult to measure the resistance change 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, an external power supply applies an AC voltage to a pair of electrodes sandwiching the lipid thin film to determine the resistance. 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 electric current in a certain direction with respect to the dissociation group of the labeled antigen described in Example 5 is obtained. Electrophoretic driving force is applied. As a result, the labeled antigen having an anionic dissociating group 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. . The DC bias voltage at which electrolysis does not occur, especially when there is no oxidizing substance or reducing substance, is the sum of the maximum AC voltage and the hydroxyl ion of water,
It is less than 0.83 volts, which is the theoretical potential for dissociation into hydrogen ions. In addition, unless electrolysis occurs, continuous electrophoresis cannot be performed at this bias voltage. Therefore, the method is to increase the sensitivity only to a trace amount of labeled antigen.

Further, even when the labeled antigen has a cationic dissociating group and the working electrode is a cathode, the marker is taken into the lipid thin film, so that the adsorption of the marker to the lipid thin film is accelerated and the response becomes faster. Further, the amount of equilibrium adsorption of the marker to the lipid thin film is increased, and the sensitivity is increased and the same effect is obtained.

[0096]

As described above, according to the present invention, a substance which is soluble in a lipid having high resistance and imparts ion conductivity to a lipid is bound to a substance to be measured as a label, and the label of the substance to be labeled is labeled. The part is adsorbed on the lipid thin film (dissolves 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 a label in combination with the resistance detection method, the immunity that combines the versatility of the immunoassay with the sensitivity, accuracy, simplicity, and rapidity found in the sensing of biological receptors, and extracts the immune response as an electrical response The electrode method and the device become possible.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of a first embodiment of the present invention.

FIG. 2 is a configuration diagram of a second embodiment of the present invention.

FIG. 3 is an overall configuration diagram of a third embodiment of the present invention.

FIG. 4 is a configuration diagram of a third embodiment of the present invention.

FIG. 5 is a structural diagram of a labeled analyte and a labeled antigen of Examples 5 and 8 of the present invention.

FIG. 6 is a structural diagram of a lipid thin film of Example 7 of the present invention.

FIG. 7 is a structural diagram of an insolubilized antibody of Example 9 of the present invention.

FIG. 8 is a diagram showing a process of preparing a monovalent antibody in Example 9 of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Measurement object 2 Marker 3 Labeling measurement object or labeled antigen 5 Lipid thin film 12 Lipid thin film 13 Working electrode 14 Counter electrode 31 Antigen 32 Antibody 33 Insolubilized base material 34 Insolubilized antibody 41 Estradiol residue 42 Glucuronic acid residue 43 Aminobenzyl-15 -Crown-5 residue 44 Crosslinker residue 45 Crosslinker residue 51 Electrode 52 Lipid molecule 53 Sulfur atom 54 Hydrocarbon chain 55 Amino group 61 Polystyrene bead 62 Phenyl group 63 Antibody 71 H chain 72 L chain

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Masako Tamaki 1006 Kazuma Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. F-term (reference) 2G046 AA34 DD01 DD02 EB09 FA03 FA04 FA07 FA09

Claims (20)

[Claims] 1. A substance to be measured labeled with a fat-soluble ion-conductive substance is adsorbed on a lipid thin film, and the compound labeled with the fat-soluble ion conductivity is detected based on a change in electric conductivity of the lipid thin film. Measurement method of the substance to be measured. 2. The method according to claim 1, wherein the substance to be measured is adsorbed on a lipid thin film formed on the electrode surface. 3. An insolubilized antibody, and a labeled antigen labeled with a fat-soluble ion-conductive substance bound to the insolubilized antibody by an antigen-antibody reaction, and the antigen and the labeled antigen are contacted with the antigen in the liquid to be measured. A method for measuring an antigen in a liquid to be measured, in which a labeled antigen released by a displacement reaction is adsorbed to a lipid thin film formed on an electrode surface, and a lipid-soluble ion-conductive labeled antigen is detected based on a change in electrical conductivity of the lipid thin film. 4. An insolubilized antibody, a labeled antigen labeled with a fat-soluble ion-conductive substance bound to the insolubilized antibody by an antigen-antibody reaction, a pair of electrodes, a working electrode and a counter electrode, and a lipid formed on the surface of the working electrode It has a thin film, and by contacting the antigen in the liquid to be measured, the labeled antigen released by the substitution reaction between the antigen and the labeled antigen is adsorbed on the lipid thin film formed on the electrode surface, and the change in the electrical conductivity of the lipid thin film An apparatus for measuring an antigen in a liquid to be measured, which detects a lipid-soluble ion-conductive labeled antigen. 5. 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. The method according to any one of claims 1 to 3, wherein at least one of a polymer or an oligomer having a structure in which is oriented is a fat-soluble ion-conductive substance. 6. A fat-soluble substance which forms a conjugated compound with a water-soluble ion, a conjugated compound thereof, a lipophilic chelating agent, a chelate compound thereof, a liposoluble ion, wherein a polar group is oriented inward and a hydrophobic group is directed outward. 5. The measuring device according to claim 4, wherein at least one of the polymer or oligomer having a structure in which is oriented is a fat-soluble ion-conductive substance. 7. The measuring method according to claim 1, wherein a cationic or anionic dissociating group is introduced into the labeled compound labeled with a fat-soluble ion-conductive substance. 8. The measuring apparatus according to claim 4, wherein a cationic or anionic dissociating group is introduced into the labeled compound labeled with a fat-soluble ion-conductive substance. 9. The method according to claim 1, wherein the lipid thin film is a monomolecular film or a multilayer film. 10. The measuring device according to claim 4, wherein the lipid thin film is a monomolecular film or a multilayer film. 11. The method according to claim 1, wherein a cationic or anionic dissociating group is introduced into the labeled compound labeled with a fat-soluble ion-conductive substance. 12. The measuring apparatus according to claim 4, wherein a cationic or anionic dissociating group is introduced into the labeled compound labeled with a fat-soluble ion-conductive substance. 13. The lipid thin film according to claim 1, wherein at least one of a hydrophobic group, a nonionic hydrophilic group, a cationic dissociating group, an anionic dissociating group, and a zwitterionic dissociating group is used as a lipid thin film surface. The measurement method according to any one of the preceding claims. 14. The measuring apparatus according to claim 4, wherein at least one of a hydrophobic group, a nonionic hydrophilic group, a cationic dissociating group, an anionic dissociating group, and a zwitterionic dissociating group is used as a lipid thin film surface. . 15. The insolubilized antibody is a monovalent antibody.
The measurement method described. 16. The insolubilized antibody is a monovalent antibody.
The measuring device as described. 17. The method according to claim 3, wherein the insolubilized antibody is a monoclonal antibody. 18. The measuring device according to claim 4, wherein the insolubilized antibody is a monoclonal antibody. 19. The method according to claim 1, wherein the measurement of the amount of change in the electric conductivity is an AC application or an AC application with a DC bias applied.
The measurement method according to any one of Items 3 to 3. 20. The method according to claim 4, wherein the measurement of the change in the electric conductivity is performed by applying an alternating current or applying an alternating current to which a direct current bias is applied.
The measuring device as described.