JP2004093478A - Element for electrochemical analysis of physiologically active substance, and analysis method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To perform functional analysis of a protein rapidly at a low cost and with high sensitivity useful for developing drugs or therapeutic methods or screening of environmental contaminant substances, etc. <P>SOLUTION: A nickel complex or a cobalt complex is fixed via a fixing reagent having an adsorbed site to a conductive substrate and a complex ligand onto the substrate. An element for electrochemical analysis of a physiologically active substance fixed and constituted via a receptor protein via a histidine tag bonded to the complex and specifically recognized with the receptor protein via the histidine tag bonded to the complex, is provided. The element for the electrochemical analysis via the physiologically active substance in which a fixed reagent is made of a bifunctional fixed agent, and a method of analyzing the physiologically active substance having a step of measuring current potential characteristics of a sample solution in the presence of an oxidation reducible marker with this element used as an operating electrode is provided. For example, as the physiologically active substance, it is used for determining an estrogen or a pseudo-estrogen substance. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention belongs to the technical field of protein function analysis applied to screening of environmental pollutants from new drugs or therapeutic methods, or a large amount of industrial chemicals and the like.
[0002]
[Prior art]
As all human genes are being identified by the Genome Project, functional analysis of proteins produced from genes has attracted attention as an important research topic in the post-genome. In vivo, various proteins maintain their homeostasis by interacting (transmitting information) with other proteins, low molecular weight compounds, or nucleic acid molecules such as DNA. Many diseases are associated with the disruption of this signaling mechanism, and in order to determine the cause of the disease and to search for disease-specific markers or new drugs, functional analysis at the protein level is necessary.
[0003]
Proteome analysis is one of the most common methods conventionally proposed for functional analysis of such proteins. This method combines two-dimensional gel electrophoresis and mass spectrometry.First, all proteins are developed by gel electrophoresis, each spot is cut out, digested with protease, and the molecular weight of the obtained peptide fragment is measured. This is a method of measuring with a time-of-flight mass spectrometer (TOF-MS) and determining the result of the gene based on the data of the gene bank and how it changes when a stimulus is applied. However, this method has disadvantages in that it is not fast, for example, it takes about one day for electrophoresis, plotting, and MS analysis, and the system is very expensive.
[0004]
There is also a method using surface plasmon resonance (SPR) as a method for analyzing the interaction between proteins. A typical example of such a system is a Biacore system, but in principle, it is difficult to detect the binding between a protein and a small molecule, and it is expensive.
[0005]
Furthermore, in recent years, unknown environmental pollutants that act on living organisms through various routes, including endocrine disruptors, have been revealed. At present, the development of powerful means for quantifying the dangers of these chemicals is awaited, but in order to evaluate the health risks of more than 100,000 types of industrial chemicals in circulation and to screen for environmental pollutants. Therefore, means that can analyze the interaction between a target protein and a chemical substance at high speed and with high sensitivity is required.
[0006]
Conventional methods for screening environmental pollutants include a method for evaluating the binding activity of these substances to a target protein (binding assay) and a method for observing gene expression caused by those chemicals (transcription activity assay). Method) has been used. However, these methods have drawbacks such as the necessity of special facilities for using radioisotopes and the fact that it takes about one day to one week for evaluation. In addition, particularly when the evaluation is performed using live cells in a transcription activity assay method, it is difficult to lack stability and quantitativeness.
[0007]
[Problems to be solved by the invention]
An object of the present invention is to provide a new technique which can be used for protein function analysis at a high speed, with high sensitivity and at low cost, which is useful for the development of drugs and therapeutic methods, screening for environmental pollutants, and the like.
[0008]
[Means for Solving the Problems]
As a result of repeated studies to achieve the above object, the present inventors have established a means for immobilizing an in vivo protein known as a receptor (receptor) on a solid surface while maintaining its function. Can be further improved to electrochemically detect structural changes caused by the binding or interaction between the receptor protein and the target substance (ligand: bioactive substance), and based on this characteristic, the quantification of the bioactive substance We have devised an analysis technology that can be used for screening and screening.
[0009]
Thus, according to the present invention, the nickel complex or the cobalt complex is immobilized on the conductive substrate via the immobilizing reagent containing the adsorption site for the conductive substrate and the ligand of the complex. A receptor protein is immobilized via a histidine tag bound to a complex, and is an element for electrochemical analysis of a physiologically active substance specifically recognized by the receptor protein, wherein the immobilized reagent is bifunctional. An element for electrochemical analysis of a physiologically active substance is provided, the element comprising a sex-immobilized reagent. In a preferred embodiment of the device for electrochemically analyzing a physiologically active substance according to the present invention, the conductive substrate is a gold electrode, the ligand of the nickel complex or the cobalt complex is nitrilotriacetic acid or iminodiacetic acid, and the conductive moiety The adsorption site for is a disulfide group. In a particularly preferred embodiment of the element for electrochemically analyzing a physiologically active substance of the present invention, the receptor protein comprises a nuclear receptor, and a preferred example of the nuclear receptor is an estrogen receptor.
[0010]
The present invention further provides an analytical method for quantifying or screening a physiologically active substance specifically recognized by a receptor protein. The analytical method of the present invention provides an electrochemical analysis of a physiologically active substance as described above. The present invention is characterized in that current-potential characteristics of a sample solution which may contain a physiologically active substance to be analyzed are measured in the presence of an oxidation-reduction marker using an element for working as a working electrode. In a preferred embodiment of the method for analyzing a physiologically active substance according to the present invention, the redox marker K₄Fe (CN)₆/ K₃Fe (CN)₆And the current-potential characteristics are measured by cyclic voltammetry. The analysis method of the present invention is suitable for quantifying a physiologically active substance, for example, an estrogen or a pseudoestrogenic substance.
[0011]
BEST MODE FOR CARRYING OUT THE INVENTION
The element for electrochemically analyzing a physiologically active substance of the present invention can be regarded as one kind of an electrochemical sensor constituted by immobilizing a protein structure on an electrode. As an electrochemical sensor in which a protein is immobilized on an electrode, an enzyme electrode represented by a glucose sensor has been well known. However, the conventional enzyme electrode is based on detecting an electrochemical change caused by the enzyme immobilized on the electrode decomposing the substrate, and differs from the principle of the present invention as described below. .
[0012]
With the exception of some enzymes, protein functions (such as binding of receptor proteins to ligands, protein-protein interactions, etc.) have no electrochemical activity. In the analysis of the physiologically active substance according to the present invention, the electrochemically active molecule (redox marker) added to the measurement solution is "permeated" to the protein (receptor) layer immobilized on the electrode surface. It is based on indirectly capturing changes in protein properties by using "easiness" as an index. That is, a change in the permeability of a marker substance due to a structural change of a receptor protein caused by binding or interaction of a ligand (bioactive substance) with the receptor protein is electrically measured.
[0013]
Another feature of the device for electrochemically analyzing a physiologically active substance of the present invention is that a protein (receptor protein) is immobilized on an electrode without impairing its function. Immobilization of a biomolecule on a solid surface in a controlled orientation is very important in developing a sensor with high selectivity. As a protein immobilization method, a carrier binding method for immobilization by covalent bonding, ionic bonding, biochemical affinity, and the like, a cross-linking method for cross-linking with a reagent such as glutaraldehyde, and the like are generally used. However, these methods have disadvantages in that amino acid residues on the protein are used for immobilization, so that the function of the protein cannot be completely maintained, and the orientation of the protein with respect to the solid surface cannot be controlled. In particular, when an immobilized protein is used as a recognition element on an electrode, these problems are fatal in terms of a detection mechanism. Therefore, in the present invention, a histidine tag capable of reversibly orienting a protein to a solid surface while retaining its function is used.
[0014]
The histidine tag is also called a His tag (His-tag), and as is well known, a small number (generally 5 to 10) of histidine residues added to the amino terminal or carboxyl terminal of a protein. Group. This histidine tag is known to strongly bind to a complex of nickel ions (nickel complex) or a complex of cobalt ions (cobalt complex).
[0015]
Thus, the present inventors have previously immobilized a nickel complex or a cobalt complex on a conductive substrate such as a gold electrode via an immobilization reagent, and further via a histidine tag bonded to the complex. A device for electrochemical analysis (electrochemical sensor) composed of immobilized receptor protein was devised, and the detection of endocrine disrupting substances such as estrogen and target DNA using the device was presented [The 79th Annual Meeting of the Chemical Society of Japan] Proceedings (2001), lecture numbers 3F404 and 3F407, etc.]. Although this electrochemical sensor provides a few techniques for realizing analysis for screening and quantification of endocrine disrupting substances and the like, its response sensitivity was not sufficient.
[0016]
Therefore, the present inventors have conducted intensive studies to solve this problem, and as a fixing reagent for fixing a nickel complex (or a cobalt complex) on a conductive substrate, a conventional monofunctional immobilization reagent has been used. It has been found that the sensitivity is significantly improved by using a bifunctional immobilizing reagent instead of the immobilizing reagent.
[0017]
The immobilization reagent has an adsorption site (for example, a disulfide group) on the conductive substrate and a ligand (for example, nitrilotriacetic acid) for forming a complex, and in addition to these, generally, Although a linker site is provided between the molecule and the adsorption site, a monofunctional immobilization reagent, that is, a system having a single ligand at only one end, has been used until now. (See Example 1 described later). When such a monofunctional immobilizing reagent is used, as shown in FIG. 1A, a single protein (a receptor protein to which a histidine tag is added) is immobilized on a conductive substrate (electrode). It is thought that it will only be converted.
[0018]
In contrast, the immobilization reagent used in the present invention is a bifunctional immobilization reagent, that is, one having ligands at both ends. Specifically, the bifunctional immobilizing reagent used in the present invention is a ligand in which an adsorption site (for example, a disulfide group) on a conductive substrate is disposed at the center, and a suitable linker site is disposed at both ends thereof. (For example, nitrilotriacetic acid) (see Example 1 described later). When such a bifunctional immobilization reagent is used, as shown in FIG. 1B, two proteins (a histidine-tagged receptor protein) are immobilized close to each other. Conceivable.
[0019]
The present invention examines the binding and interaction between various types of physiologically active substances that are specifically recognized by the receptor protein and the receptor protein immobilized on the conductive substrate, It is applied to the quantification and screening of an active substance, and is particularly suitable for immobilizing a protein that dimerizes upon binding or interacting with a physiologically active substance.
[0020]
For example, physiologically active substances such as steroid hormones (estrogens, androgens, etc.), thyroid hormones, fat-soluble vitamins (vitamin A, vitamin D) bind to nuclear receptors (intracellular receptors). Is known to dimerize. As the element for electrochemical analysis according to the present invention, an element in which these nuclear receptors (estrogen receptor, androgen receptor, thyroid receptor, vitamin A receptor, vitamin D receptor, etc.) are immobilized is used. If an attempt is made to analyze biologically active substances (estrogen, androgen, thyroid hormone, vitamin A, vitamin D, etc.) that are recognized as such, it is possible to quantify or screen each physiologically active substance with extremely high sensitivity. This is because, as described above, in the electrochemical analysis element of the present invention using a bifunctional immobilizing reagent, two receptor proteins are immobilized close to each other, so that the receptor protein and the physiologically active substance are immobilized. It is understood that the conformational change (dimerization) induced by the binding or interaction of can be more accurately simulated and detected.
[0021]
In the electrochemical analysis device according to the present invention, a ligand constituting a nickel complex or a cobalt complex binding a histidine tag is not particularly limited, but is generally well known in the art. As described above, nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) is preferable, and NTA is particularly preferable.
In general, a gold electrode is preferable as the conductive substrate constituting the electrochemical analysis element according to the present invention. In this case, as is well known, a sulfur-containing group such as a disulfide group is used as an adsorption site for a conductive substrate (gold electrode). The complex is immobilized on the gold electrode by bonding NTA (or IDA) which is a ligand of the nickel complex (or cobalt complex) via an appropriate linker site. When a conductive substrate other than the gold electrode is used, the means for attracting the conductive substrate is changed accordingly.
[0022]
In the method for analyzing a physiologically active substance according to the present invention, the electrochemical analysis element configured as described above is used as a working electrode, and in the presence of an oxidation-reduction marker, a current is applied to a sample solution containing the target physiologically active substance. This is performed by measuring a potential characteristic. The redox marker to be used can be used as long as it is water-soluble and does not adversely affect the receptor protein or its ligand.₄Fe (CN)₆/ K₃Fe (CN)₆(Hereinafter, in this specification, this system is referred to as [Fe (CN)₆]^{3 − / 4 −}May be written). Other examples of the redox marker to be used include ferrocene and hexaamine ruthenium (III) complex. Changes in the current-potential characteristics are generally based on cyclic voltammetry (CV), but other electrochemical measurement methods such as differential pulse voltammetry can also be used.
In the present invention, a receptor protein used for immobilization on an electrode (conductive substrate) is generally a nickel complex obtained by obtaining a histidine-tagged protein by a gene recombination technique well known in the art. (Or cobalt complex) affinity purified by a column.
[0023]
【Example】
Examples are shown below to further clarify the features of the present invention, but the present invention is not limited by these examples.
Example 1: Preparation of estrogen receptor-immobilized electrode
An electrode having an estrogen receptor immobilized thereon was produced as an electrochemical analysis element according to the present invention.
An important role in immobilizing proteins is the development of reagents that connect proteins to solid surfaces. As described above, the present inventor newly designed and synthesized a bifunctional immobilizing reagent instead of the conventional monofunctional immobilizing reagent [(5) in FIG. 2]. The newly designed and synthesized bifunctional immobilization reagent has the structural formula shown in (4) of FIG. 2, in which nitrilotriacetic acid (NTA: ligand) is linked to both ends of a disulfide group via linker sites. It is configured. The synthesis procedure is as follows (see FIG. 2). 140 mg of Nα ', Nα-bis (carboxymethyl) -L-lysine hydrate (1) was dissolved in 3 ml of DMF containing 1035 mg of diisopropylethylamine (DIEA). After purging with nitrogen for 10 minutes, trimethylsilyl chloride {[(CH₃)₃While adding 870 mg of [SiCl] little by little, the reaction was carried out at room temperature under a nitrogen atmosphere for 2 hours to protect the carboxyl group. To the obtained compound (2), 1.5 ml of DMF in which half equivalent of 3,3′-dithiodipropionsandi (N-succinimidyl) [DTPS, (3)] is dissolved is added, and the mixture is stirred at room temperature under a nitrogen atmosphere for 2 days. did. After adding 7 ml of an aqueous tartaric acid solution to the reaction solution and sufficiently shaking, 15 ml of dichloromethane was added thereto to extract an organic phase. The extraction operation was performed twice. After the solvent was concentrated under reduced pressure, it was redissolved in 1.5 ml of dichloromethane. Purification is performed by preparative TLC (thin layer chromatography) and is performed at 250 MHz.¹The product (4) was confirmed using 1 H-NMR.
[0024]
Gold disk electrode as conductive substrate (electrode area 0.08cm²) Is sufficiently polished with an alumina abrasive, and then 0.1 M MH₂SO₄Electropolishing was carried out using a / 10 mM KCl aqueous solution. The above-mentioned bifunctional nitrilotriacetic acid (NTA) derivative (indicated by (4) in FIG. 2), which is a bifunctional immobilization reagent newly designed and synthesized by the present inventors, is dissolved in chloroform and polished gold electrode Was immersed in 50 μl of this solution at 4 ° C. for 3 hours. Thereafter, the electrode was washed with chloroform and dried with nitrogen gas. This is again 0.1M NiSO₄It was immersed in 30 µl of the solution for 10 minutes to form a Ni (II) -NTA complex. This electrode is immersed in an aqueous solution of human estrogen receptor α fused with histidine residues (His-tag) at the gene level at 5 ° C. for 10 minutes at 4 ° C. -HCl (pH 7.4)) to give an estrogen receptor-immobilized electrode.
For comparison, an estrogen receptor-immobilized electrode was prepared using the monofunctional immobilizing reagent shown as (5) in FIG. The receptor-immobilized electrode using this monofunctional immobilizing reagent is the same as the above-mentioned bifunctional immobilizing reagent except that the compounds (2) and (3) are reacted in the same amount in the synthesis step of the immobilizing reagent shown in FIG. It is prepared through the same steps as in the case of using a sex-fixing reagent.
[0025]
Example 2: Sensing of endocrine disrupting substance (1)
Next, in order to confirm whether the receptor immobilized on the gold electrode has a normal ligand binding ability, a natural female hormone 17β-estradiol is allowed to act on the hER immobilized electrode (electrochemical analysis element), The electrochemical response at that time was observed. That is, the hER-immobilized electrode prepared using the bifunctional immobilizing reagent according to Example 1 was used as a working electrode, and a 1 nM to 100 M 17β-estradiol-containing sample solution [10 mM Tris buffer, pH 7.4, [ KCl] = 10 mM, [Fe (CN)₆]^{4 − / 3 −}= 5 mM (redox marker), 25 ° C], and the current-voltage characteristics were measured by cyclic voltammetry (CV).
FIG. 3 shows the measurement results. For comparison, FIG. 4 shows the results of CV measurement similarly performed on an hER-immobilized electrode prepared using a monofunctional immobilizing reagent.
[0026]
As shown in FIG. 3, in the case of the hER-immobilized electrode prepared using the bifunctional immobilizing reagent, the peak current value was greatly reduced according to the added concentration. In contrast, the electrode immobilized with NTA alone (unmodified hER) did not respond to the same concentration of 17β-estradiol at all, so the decrease in the peak current value was due to the difference between the receptor and ligand on the electrode surface. It has been suggested that this is due to a specific interaction. And 10^-10Good electrochemical response has been obtained at concentrations as low as M, and its detection sensitivity is comparable to competition assays using RI (radioisotope) labeled ligands. On the other hand, as shown in FIG. 4, in the case of the hER-immobilized electrode prepared from the monofunctional immobilizing reagent, even when 100 μM of 17β-estradiol was added, the electrode response was extremely slight, and the bifunctional It can be seen that the response sensitivity was significantly improved when the immobilized reagent was used.
[0027]
In the sensing system according to the present invention, the marker ion has a detection mechanism of “easiness of permeation” of the marker ion to the protein layer formed on the electrode surface, and a decrease in peak current value upon addition of the ligand is immobilized on the electrode. It is understood that the hER changes its physical properties by binding to its ligand, and as a result, the properties of the entire protein layer affect marker ion permeability.
It is known that ER changes its three-dimensional structure by binding to a ligand to form a homodimer (homodimer). Have shown that the ligand-binding domain of hER is composed of 12 α-helices (H) and two β-sheets (S), and has a three-layered firm α-helix sandwich structure. It was also revealed that the ligand binding pocket is located below the central layer of the three-layer structure and is composed of H3, ΔH6, ΔH8, a loop, ΔH11, ΔH12, and an S1 / S2 hairpin. When a ligand molecule is caught in this binding pocket, the conformation changes significantly to become a homodimer, and the ER is activated. Interestingly, Witowska et al. Report that when ER is activated by binding to 17β-estradiol, basic amino acid residues are reduced and acidic amino acid residues are increased on the protein surface. If hER immobilized on the electrode surface binds to the ligand and takes a similar activation mechanism, the protein layer on the electrode will be negatively charged. On the other hand, the redox marker ([Fe (CN)₆]^{3- / 4-}) Is also anionic, and the permeability of marker ions decreases due to electrostatic repulsion when the protein layer gradually takes on a negative charge in accordance with the ligand addition concentration. As a result, it is inferred that the number of marker ions that reach the vicinity of the electrode surface after passing through the protein layer relatively decreased, and the peak current value based on the redox reaction also decreased.
[0028]
The excellent response characteristics as shown in FIG. 3 are the results achieved by using a bifunctional immobilization reagent. The device of the present invention is for electrochemically detecting the structural change of a protein induced by ligand binding and the subsequent protein / protein interaction, so that the interacting protein is located nearby. Is an important condition. In particular, in the case of a biosensor in which a protein is immobilized on a solid surface, a means for bringing the protein closer to the vicinity is required because the mobility is limited. The bifunctional immobilization reagent used in the present invention can freely adjust the distance between the proteins to be immobilized by performing molecular design. Therefore, a protein immobilized sensor such as the detection system of the present invention. Very effective as a protein immobilization method
[0029]
Example 3: Sensing of endocrine disrupting substance (2)
As shown in Example 2, it was revealed that the method of the present invention using a human estrogen receptor-immobilized electrode enables the evaluation of protein binding to a ligand. Then, this electrode was next applied to risk assessment of industrial chemicals and the like whose pseudo estrogenic activity is currently concerned. Using hER-immobilized electrodes made from bifunctional immobilization reagents as described in Example 1, synthetic estrogens (diethylstilbestrol), and industrial chemicals (bisphenol A, nonylphenol, dibutyl phthalate) and natural Each substance of androgen (testosterone) was allowed to act, and the response was measured by cyclic voltammetry under the same conditions as in Example 2. The measurement was performed by preparing stock solutions of 100 mM (100% DMSO solution), 1 mM (50% DMSO aqueous solution), and 100 μM (0.5% DMSO), and dropping these into the measurement solution while stirring. In addition, the same measurement was carried out using a DMSO solution having the same concentration and not containing the substance to be measured, and used as a control.
[0030]
As a result, it became clear that not only human estrogen (17β-estradiol) and synthetic estrogen (diethystilbestrol) but also all three chemical substances of bisphenol A, nonylphenol and dibutylphthalate respond to the present detection system. . Therefore, as shown in FIG. 5, the relationship between the amount of decrease in peak current (Δip) and the concentration when the test substance was added was plotted as 10^-8From M to 10^-4An almost linear response was obtained up to M, indicating that the present system is also useful for quantification of these substances. The endocrine disrupting activity of these substances has already been confirmed by several risk assessment methods, and the results confirm this. In contrast, under the measurement conditions, the hER-immobilized electrode did not show a significant response to DMSO used as a control.
[0031]
These results indicate that the analytical element (biosensor) of the present invention, which uses the human estrogen receptor as a recognition element, exhibits a good electrochemical response not only to natural female hormones but also to endocrine disrupting substances. it is obvious. These substances, like 17β-estradiol, are thought to bind to the ligand binding pocket composed of H3, ΔH6, ΔH8 and a loop, ΔH11, ΔH12 and S1 / S2 hairpin, and induce a similar conformational change. . In estradiol, the hydroxyl group of the steroid A ring is strongly hydrogen-bonded (pincers structure) between the carboxyl group of Glu353 (H3) and the guanidium group of Arg394, and is further hydrogen-bonded to one molecule of water. It has also been clarified that a hydrogen bond between the 17β-hydroxyl group of the D ring and His524 (H11) and a hydrophobic interaction with a hydrophobic site of the ligand binding domain surrounding the A, B, and D rings are formed. However, many endocrine disruptors may also be stabilized by a similar bond using a hydrophobic backbone and phenolic hydroxyl groups. At this time, as in the case of Example 2, it is considered that the physical properties of the protein layer composed of hER formed on the electrode surface changed, and this caused a change in the peak current value.
[0032]
【The invention's effect】
As is apparent from the above detailed description, according to the present invention, an electrode (electrochemical analysis element) having a receptor protein immobilized on a solid surface such as gold while retaining its function is obtained. An analytical system that can electrochemically detect structural changes caused by the binding or interaction between a receptor protein and its ligand, a biologically active substance, using the element to quantify or screen the biologically active substance. Is provided. The analytical system of the present invention, in which a receptor protein is immobilized using a bifunctional immobilization reagent, specifically detects the binding or interaction between a receptor protein that dimerizes upon binding or interaction with a ligand and the ligand. Suitable to do.
[0033]
Thus, the analysis system of the present invention distinguishes and screens harmful substances (endocrine disrupting substances, environmental pollutants, etc.) corresponding to physiologically active substances that interact with or bind to specific receptor proteins from among a large amount of chemical substances. , And further, can be used to quantify them. For example, as described in the Examples, the bioaffinity sensor of the present invention in which an estrogen receptor is immobilized as a receptor protein is not only a natural female hormone, 17β-estradiol, but also an industrially suspected estrogen-like agonist. It shows good electrochemical response to the product.
Furthermore, the analysis system of the present invention can be used to identify or select a bioactive substance that binds or interacts with a specific receptor protein as an agonist or antagonist when developing a new drug or therapeutic method. it can.
[0034]
The analysis system of the present invention that utilizes an electrochemical reaction can perform extremely rapid detection as compared with the above-described conventional techniques, and can often detect within a few minutes. Further, the analysis system of the present invention is extremely excellent in sensitivity. For example, the detection sensitivity in the analysis using the hER-immobilized electrode shown in the Examples is comparable to the competitive assay using an RI-labeled ligand, which is currently the most sensitive method (detection lower limit: 10).^-10M).
Furthermore, since the analysis system of the present invention can be constructed using general-purpose products, it is advantageous in terms of cost, and can be easily made into a microchip or miniaturized as necessary.
[Brief description of the drawings]
FIG. 1 schematically shows the structure of a receptor protein-immobilized electrode (electrochemical analysis element) produced according to the present invention.
FIG. 2 schematically shows a synthesis step of a preferred example of an immobilization reagent used in the present invention.
FIG. 3 shows the cyclic voltammetric response of 17β-estradiol measured using a human estrogen receptor immobilized electrode made from a bifunctional immobilized reagent according to the present invention.
FIG. 4 shows the cyclic voltammetric response of 17β-estradiol measured using a human estrogen receptor immobilized electrode made from a monofunctional immobilized reagent for comparison.
FIG. 5 shows the results of cyclic voltammetry measurement of various endocrine disrupting substances performed using the human estrogen receptor-immobilized electrode according to the present invention.

Claims (8)

A nickel complex or a cobalt complex is immobilized on a conductive substrate via an immobilizing reagent having an adsorption site for the conductive substrate and a complex ligand, and further, a receptor is imposed via a histidine tag bonded to the complex. An element for electrochemical analysis of a physiologically active substance which is constituted by immobilizing a protein and specifically recognized by the receptor protein, wherein the immobilization reagent comprises a bifunctional immobilization reagent. For electrochemical analysis of physiologically active substances. 2. The physiology according to claim 1, wherein the conductive substrate is a gold electrode, the ligand of the nickel complex or the cobalt complex is nitrilotriacetic acid or iminodiacetic acid, and the adsorption site on the gold electrode is a disulfide group. Element for electrochemical analysis of active substances. The element for electrochemical analysis of a physiologically active substance according to claim 1 or 2, wherein the receptor protein is a nuclear receptor. The element for electrochemical analysis of a physiologically active substance according to claim 3, wherein the nuclear receptor is an estrogen receptor. An analytical method for quantifying or screening a physiologically active substance specifically recognized by a receptor protein, wherein the element for electrochemical analysis of a physiologically active substance according to any one of claims 1 to 4 is used as a working electrode, A method comprising measuring current-potential characteristics of a sample solution that may contain an analyte protein in the presence of a redox marker. Method for analyzing biologically active substance according to claim 5, wherein the use of _{K 4 Fe (CN) 6 /} K 3 Fe (CN) 6 as a redox marker. 7. The method for analyzing a physiologically active substance according to claim 5, wherein the current-potential characteristics are measured by cyclic voltammetry. The physiologically active substance analysis according to any one of claims 5 to 7, wherein an estrogen or a pseudoestrogenic substance is quantified as the physiologically active substance using the electrochemical analysis element according to claim 4. Method.

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