JP2008111709A - Labelling agent and measuring method of target substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel biochemical substance labelling agent which enables a specific binding assay using an inexpensive and small-sized measuring instrument having high sensitivity, and a measuring method of a target substance using the labelling agent. <P>SOLUTION: A compound, which has a structure of reacting with the functional group in a sample compound to form a covalent bond, a structure responding to light irradiation to cleave and a structure liberated by cleavage to emit an electrochemical signal in its one molecule is used as a labelling agent and covalently bonded to a sample compound to be labelled. The electrochemical signal produced by light irradiation is detected and measured to detect and measure the target substance with which the sample compound interacts indirectly and specifically. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a labeling agent for a biochemical substance in a chemical assay, particularly a biochemical assay, more specifically a specific binding assay such as an immunoassay, and a biochemical substance labeled with the labeling agent. The present invention relates to a method for measuring the target substance used.

  Conventionally, chemical labeling with a radioactive substance or a fluorescent substance has been widely used as a method for labeling biochemical substances such as antibodies used in an assay method applying an antigen-antibody reaction such as Western blotting. However, when a radiolabeling agent is used, in order to detect and measure the target substance by the assay method, the experimenter has a technique for handling the radioactive substance in addition to the experimental technique for performing the assay experiment method. Will be required. In addition, the experiment method using a radioactive substance is harmful to the human body, and there is a problem that it requires a special experiment facility and experiment equipment for that purpose, so that it is expensive.

  In addition, chemical labeling using a fluorescent substance enables the target substance to be indirectly detected by detecting and measuring the fluorescence intensity. However, the equipment used for the detection and measurement of fluorescence intensity is of a certain size and weight at the present time, occupies a relatively large experiment space in the experiment facility and cannot be moved, so the place where the experiment is performed is limited There is a problem of being. In addition, a fluorescence intensity detection / measurement device is generally expensive, and there is a problem that all experimenters cannot easily use it. Furthermore, since the current fluorescence detection / measurement apparatus cannot detect a fluorescence intensity lower than a certain level, there is a problem that a low concentration target substance cannot be detected / measured.

  In recent years, a labeling agent using a luminescent substance such as luciferase capable of detecting a target substance at a low concentration has been developed (see Patent Document 1), but the luminescence reaction process takes time, and the target substance is measured over time. There is a problem of making it difficult. There is also a problem that the stability of the enzyme situation depends on the components of the reaction solution.

  As described above, in the biochemical assay method, the cost of the experiment including facilities and equipment, the convenience of the experiment, and the nature of the detection / measurement method are greatly affected by the labeling agent used. At present, no labeling agent that can detect all target substances at low concentrations, low cost, and downsizing of an experimental apparatus, or a biochemical assay using the labeling agent has not been developed.

Japanese National Patent Publication No. 10-504903

  In view of such circumstances, an object of the present invention is to provide a novel biochemical substance labeling agent that has a high sensitivity and enables a specific binding assay using an inexpensive and small measuring device, and the labeling agent. It is to provide a method for measuring a target substance.

  As a result of diligent research, the inventors of the present application have produced a structure that reacts with a functional group in a sample compound to covalently bond, a structure that cleaves in response to light irradiation, and is released by the cleavage to generate an electrochemical signal. The sample compound is indirectly specific by detecting and measuring the electrochemical signal generated by light-irradiation with a compound that has a structure to be bound in one molecule as a labeling agent. Experimentally confirmed that target substances that interact with each other can be detected and measured, and the present invention has been completed.

  That is, the present invention provides the following general formula [I]

(Wherein, X represents a structure that can be covalently bonded to another functional group, Y represents a structure that can be cleaved by light irradiation, Z represents a structure having electrochemically measurable redox activity, L ₁ and L ₂ may or may not be present; if present, L ₁ represents any spacer structure that covalently connects structures X and Y, and L ₂ represents structure Y Any spacer structure that connects Z and Z by a covalent bond)
The labeling agent which has a structure represented by this is provided. Furthermore, the present invention reacts a specific binding substance labeled with the labeling agent of the present invention with a target substance that specifically binds to the specific binding substance, and by specific binding of the specific binding substance and the target substance. After separation and purification of the formed complex, the labeled specific binding substance bound to the target substance is irradiated with light to cleave Y in the general formula [I], and thereby released in the general formula [I]. Provided is a method for measuring a target substance, comprising electrochemically measuring structure Z.

  According to the present invention, a labeling agent capable of detecting a target substance and measuring a concentration with high sensitivity is provided by a small and inexpensive measuring device. Thereby, the detection of the target substance and the concentration measurement assay can be performed using a detection / measurement device that is small enough to be carried without being limited to the experimental place.

  As described above, the labeling agent of the present invention comprises a structure X that can be covalently bound to a specific binding substance to be labeled, a structure Y that can be cleaved by light irradiation, and a structure Z that has an electrochemically measurable redox activity. And X, Y, and Z are covalently bonded directly or through a spacer structure to exert their respective functions.

In the general formula [I], the structure X represents a structure that can be bonded to another substance. As a preferred group of X, a structure having a functional group capable of reacting with a functional group in a specific binding substance to be commonly bonded can be exemplified. In this case, X may be any functional group as long as it is a functional group capable of reacting with the functional group in the specific binding substance. Since the specific binding substance has a specific binding property to the target substance, it is often a biological substance such as a protein or a nucleic acid. Therefore, a functional group often included in these substances, that is, an —NH ₂ group, A functional group that reacts with a —SH group, —COOH group, —OH group, —CHO group, —PO ₃ group, or the like to form a covalent bond is preferable. As examples of such functional groups, in addition to the succinimide group employed in the following examples, SCN-, ClO ₂ S-,

Maleimide group, ie

_{BrH 2 C-, ClOC -, -} NH 2, -NHNH 2, -CH 2 I, ( may or may not be the hydrochloride _{salt) -CH 2 ONH 2 (-HCl)} ,

Can be mentioned. The functional group (R ′) of the specific binding substance that reacts with these functional groups and the bond formed as a result of the reaction are shown in Table 1 below (when X is —NH ₂ , it is shown in Table 1). And X in the case where the functional group (R ′) in the specific binding substance is —NH ₂ can be bound as shown in Table 1).

Further, as X, CH ₃ CH (NH ₂ ) ═CH— can also be preferably employed. In this case, two molecules of the labeling agent react with the specific binding substance represented by R-CHO as follows.

  Preferred X is further represented by the general formula [II]

(However, Hal represents halogen),

And general formula [III]

(However, n represents an integer of 1 to 5)
The group represented by these can be mentioned. In the general formula [II], the halogen is preferably bromine.

  X represented by the above general formula [II] reacts with cytosine in the specific binding substance and binds as follows.

(However, in the formula, R represents an arbitrary group such as a nucleotide or a sugar moiety of a nucleic acid)

  Since this X can bind to the cytosine moiety in the specific binding substance, cytidine monophosphate, cytidine diphosphate, cytidine triphosphate, deoxycytidine monophosphate, deoxycytidine diphosphate and deoxycytidine triphosphate It can bind to nucleic acids such as DNA and RNA, including phosphate and cytosine.

  X is above

In the case of, it reacts with guanine in the specific binding substance and binds as follows.

(However, in the formula, R represents an arbitrary group such as a nucleotide or a sugar moiety of a nucleic acid)

  Since this X can bind to the guanine moiety in the specific binding substance, guanosine monophosphate, guanosine diphosphate, guanosine triphosphate, deoxyguanosine monophosphate, deoxyguanosine diphosphate and deoxyguanosine triphosphate. It can bind to nucleic acids such as DNA and RNA containing phosphate and guanine.

  When X is a group represented by the above general formula [III], it reacts with the phosphate ester moiety in the specific binding substance and binds as follows (however, in the following example, n is 2) Show).

  In this reaction formula, the specific binding substance is deoxyadenosine monophosphate (that is, the base in the nucleotide is adenine and the sugar is deoxyribose), but X is bound to the phosphate ester moiety. Thus, the base may be a base other than adenine such as cytosine, guanine, thymine or uracil, and the sugar may be deoxyribose or ribose. In addition, the number of phosphate esters is one in the above reaction formula, but X is bonded to the phosphate ester at the end, so the number of condensed phosphate esters is any one of 1 to 3. There may be. Therefore, X represented by the above general formula [III] can bind to all kinds of nucleoside monophosphates, nucleoside diphosphates and nucleoside triphosphates. Furthermore, since a free phosphate ester is present at the end of the nucleic acid, X represented by the above general formula [III] can be bound to a nucleic acid such as DNA or RNA.

  The structure Y is not particularly limited as long as it is a chemical structure that can be covalently bonded to the structure X and can be cleaved by light irradiation to release the structure Z, and a known chemical structure should be used. Can do. A preferred example is a 2-nitrobenzyl structure (see Examples below).

  The structure Z is not particularly limited as long as it is a chemical structure that can be liberated from the structure Y by light irradiation and has a redox activity capable of electrochemically measuring the liberated state. Can be used. Preferred examples include ferrocene (see Examples below), osmium complex / ruthenium complex, and the like.

  In the case of ferrocene, which is an example of the above-described structure Z, the structure is a coordinate bond between iron ion (II) and two molecules of cyclopentadiene, and a cyclopentadienyl ring that is a carbon plane of two molecules of a regular pentagon structure. Has a sandwich structure in which the iron ion (II) is centered in parallel, and in a free state, a reversible redox reaction is caused between the iron ion and the complex carbon skeleton by resonance of a shared electron pair. Can do. Since such a reversible oxidation-reduction reaction generates a voltage due to the donation of electrons, the target substance specifically bound to the specific binding substance labeled with the labeling agent of the present invention is measured by measuring the voltage. It can be detected indirectly, and the concentration of the target substance can be indirectly measured by comparing with a voltage value when a known concentration is measured in advance.

In the general formula [I], L ₁ and L ₂ are optional spacer portion. The labeling agent of the present invention binds to a specific binding substance by X, and irradiates the labeled specific binding substance bound to the target substance with light to cleave Y in the general formula [I], thereby releasing it. L ₁ located between X and Y and L ₂ located between Y and Z exist because the principle of electrochemically measuring the structure Z in the general formula [I] is present. Even if it exists, it may take an arbitrary structure. Examples include an alkylene group having about 1 to 20 carbon atoms, but the skeletal carbon may be substituted with nitrogen, oxygen or the like, and may contain a carbonyl group, a carboxyl group, an amide group, a hydroxyl group or the like. Good. Preferred examples include an alkylene group (preferably having 2 to 10 carbon atoms), an alkylene group having an amide bond or an ester bond for bonding with another structure at one or both ends, and an amino bond / peptide bond. Etc. can be illustrated.

The synthesis of the compound which is the labeling agent of the present invention may be a method in which the known chemical structures X, Y and Z are covalently bonded with or without the known spacers L ₁ and L _2. It is not particularly limited, and those skilled in the art can synthesize it according to common sense of organic chemistry. In the following examples, specific synthesis methods will be described, but the present invention is not limited to the examples.

  The labeling of the specific binding substance using the labeling agent of the present invention can be performed by a technique well known in the field of synthetic organic chemistry via the structure X and the reactive group of the specific binding substance. The reactive group of the structure X and the specific binding substance and the binding structure formed are as described in Table 1 and above.

  The results of organic chemical synthesis of the labeling agent of the present invention can be verified by methods well known in the field of organic synthetic chemistry. The method is not particularly limited as long as it can verify the results of organic synthesis, but examples include electron ionization, chemical ionization, electrolytic desorption, fast atom bombardment, matrix-assisted laser desorption / ionization (MALDI) And mass spectrometry such as electrospray ionization. According to these methods, the result of the synthesis reaction can be grasped by analyzing the change in the mass spectrum representing the detection intensity of the analytical sample with respect to the mass electrification ratio before and after the organic synthesis reaction.

  In addition, as described above, the present invention reacts the specific binding substance labeled with the labeling agent represented by the general formula [I] with a target substance that specifically binds to the specific binding substance, and the specific binding After separation of the complex formed by the specific binding between the target substance and the target substance, the labeled specific binding substance bound to the target substance is irradiated with light to cleave Y in the general formula [I], thereby releasing it. There is also provided a method for measuring a target substance, which comprises electrochemically measuring the structure Z in the general formula [I]. Each step of the measurement method will be described below. In the present invention, “measurement” includes both quantification and detection.

When a specific binding substance is labeled with the labeling agent of the present invention, the specific binding substance may be a substance that can covalently bind to the structure X and can specifically bind to the target substance. For example, when the target substance is a protein, sugar chain, etc., specific binding substances such as antibody proteins and proteins that specifically interact with the target substance to form a complex are listed. Can do. When the target substance is a nucleic acid or the like, it has complementarity with the target substance such as a protein or oligonucleotide that recognizes the base sequence of the target substance and specifically binds to form a nucleic acid-protein complex. Specific binding substances can be mentioned. Preferred examples include antigens, antibodies and antigen-binding fragments thereof (Fab, F (ab ′) ₂ etc.), nucleic acids, DNA-binding transcription factors, receptors and their ligands, and DNA sequences represented by MutS protein. Examples include proteins having a mismatch detection function, chromatin structural proteins such as histones, and RNA.

  The reaction for labeling the specific binding substance with the labeling agent of the present invention is carried out by a known technique depending on the X of the labeling agent and the type of functional group to be bound to X in the specific binding substance. Based on appropriate conditions. The following examples also specifically describe the conditions for the binding reaction between the preferred labeling agent and the specific binding substance.

  Examples of specific binding reaction conditions are described below for each of the specific combinations described above (see Table 1) of X and the functional group (R ′) in the specific binding substance that binds to X. However, these are merely examples, and these functional groups can be bonded to each other under other reaction conditions, and it will be apparent to those skilled in the art that the bonding reaction conditions are not limited to the following. is there. In addition, with respect to X that is not described here, a person skilled in the art can easily perform a binding reaction according to chemical common sense. In the description of the binding conditions between the labeling agent of the present invention and the specific binding substance, “sample” means a specific binding substance.

When X is SCN- and R 'is -NH ₂
Add 0.2 ml (ethanol: water: triethylamine = 2: 2: 1 v / v) to a sample of 2-3 mg and dry under reduced pressure, then 0.5 ml (ethanol: water: triethylamine: probe = 7: 7: 1: Add 1 v / v) and react at room temperature for 20 minutes. After evaporating the solvent under reduced pressure, the solvent is dissolved in the eluent to give a sample solution (see BA Bidlingmeyer, et al, J. Chromatogr., 336, 93 (1984)).

When X is ClO ₂ S— and R ′ is —NH ₂
Add 500 μL of 1M NaHCO ₃ aqueous solution and 200 μL of 1 mg / mL probe / acetone solution to 2-3 mg sample and heat at 60 ° C. for 30 minutes. Acetone is distilled off, and then dissolved in an eluent to obtain a sample solution (see Meffin, PJ, et al, J. Pharm. Sci., 66, 583 (1977)).

X is

で、Ｒ’が−ＮＨ₂の場合

And R ′ is —NH ₂

  Dissolve 1.5 mg sample in 5.0 mL THF, add 50 mg probe and heat at 60 ° C. for 30 min. After THF is distilled off, it is dissolved in the eluent to obtain a sample solution (see Jupill, T.H., Am. Lab., 8 (5), 85-92 (1976)).

X is

で、Ｒ’が−ＳＨの場合

And R 'is -SH

  Dissolve 2-3 mg of sample in 500 μL of water, add 200 μL of 1 mg / mL probe / acetonitrile solution, and heat at 60 ° C. for 30 minutes. After distilling off the solvent, dissolve in the eluent to obtain a sample solution (see Nakashima K., et al, Talanta., 32, 167 (1985)).

X is BrH ₂ C-or the IH ₂ C-a, when R 'is -COOH
Dissolve 2-3 mg of sample in 500 μL of acetone, add 200 μL of 1 mg / mL probe / acetone solution and 31 mg of K2CO, and heat at 60 ° C. for 30 minutes. After the solvent is distilled off, the sample is dissolved in the eluent to obtain a sample solution (see Dunges, W., Anal. Chem., 49, 442 (1977)).

When X is ClOC- and R 'is -OH
Dissolve 2-3 mg of sample in 0.5 ml of pyridine, add 0.2 mL of 1 mg / mL probe / pyridine solution, and heat at 40 ° C for 1 hour. After distilling off the solvent, it is dissolved in an eluent to obtain a sample solution (see Suzuki, A., et al., J. Biochem., 82, 1185 (1977)).

X is

で、Ｒ’が−ＣＨＯの場合

And R ′ is —CHO

  Add 2-3 mg of sample to 1.0 ml of methanol, 0.1 ml of acetic acid, 0.2 mL of 1 mg / mL probe / methanol solution, and heat at 40 ° C. for 30 minutes. After distilling off the solvent, the sample solution is dissolved in the eluent.

X is

で、Ｒ’が−ＮＨ₂の場合

And R ′ is —NH ₂

Add 1.5 mL of a 10 mg / mL probe / ethanol solution to 0.1 mL of the sample ethanol solution (10 ⁻⁴ to 10 ⁻³ M), and stir at room temperature for 5 to 10 minutes. After evaporating the solvent under reduced pressure, the solvent is dissolved in the eluent to prepare a sample solution (see Roth, M., Anal. Chem., 43, 880 (1971)).

X is is R 'with -CH ₂ ONH ₂ · HCl

の場合

in the case of

  Heat 1-5 mg sample, 2 drops of triethylamine, 50 mg of probe at 50 ° C. for 30 minutes. After distilling off the solvent under reduced pressure, the sample is dissolved in the eluent to prepare a sample solution (see Jupille, T.H., Am. Lab., 8 (5), 85-92 (1976)).

When X is —NHNH ₂ and R ′ is —CHO
After dissolving 1 to 5 mg of sample in 0.5 ml of water, a solution in which the probe was dissolved in a 30% HclO ₄ aqueous solution at a rate of 20 mg / mL was added and stirred at room temperature for 10 minutes. After evaporating the solvent under reduced pressure, the sample is dissolved in the eluent to prepare a sample solution (see Newberg, C., et al., Anal. Chim. Acta, 7, 238 (1952)).

X is

で、Ｒ’が

And R 'is

の場合

in the case of

  Dissolve 0.1-5.0 μg of sample in 50 μL of phosphate buffer solution (pH 7.0), add 200 μL of 1 mg / mL probe / phosphate buffer solution (pH 7.0), and stir at room temperature for 5 minutes. After evaporating the solvent under reduced pressure, the solvent is dissolved in the eluent to obtain a sample solution.

X is

で、Ｒ’が

And R 'is

の場合

in the case of

  Dissolve 0.1-5.0 μg of sample in 50 μL of phosphate buffer solution (pH 7.0), add 200 μL of 1 mg / mL probe / phosphate buffer solution (pH 7.0), and stir at room temperature for 5 minutes. After evaporating the solvent under reduced pressure, the solvent is dissolved in the eluent to obtain a sample solution.

X is —CH ₂ CH ₂ NH ₂ and R ′ is

の場合

in the case of

  After dissolving 0.1-5.0 μg of sample in 50 μL of phosphate buffer solution (pH 7.0), 200 μL of 1 mg / mL probe / phosphate buffer solution (pH 7.0) and 1-ethyl-3- (3- Add (dimethylaminopropyl) carbodiimide and stir at room temperature for 30 minutes. After evaporating the solvent under reduced pressure, the solvent is dissolved in the eluent to obtain a sample solution.

  The result of the organic synthesis reaction in which the specific binding substance is labeled with the labeling agent of the present invention as shown above can also be verified by a method well known in the field of synthetic organic chemistry. The method is not particularly limited as long as it can verify the results of organic synthesis, but examples include electron ionization, chemical ionization, electrolytic desorption, fast atom bombardment, matrix-assisted laser desorption / ionization (MALDI) And mass spectrometry such as electrospray ionization. According to these methods, the result of the synthesis reaction can be grasped by analyzing the change in the mass spectrum representing the detection intensity of the analytical sample with respect to the mass electrification ratio before and after the organic synthesis reaction.

In the reaction for forming a specific bond between the specific binding substance labeled with the labeling agent of the present invention and the target substance, the target substance specifically binds to the labeled specific binding substance. There is no particular limitation as long as it can be used. For example, when the specific binding substance is an antibody or an antigen-binding fragment of the antibody such as Fab or F (ab ′) ₂ , an antigen having antigen-antibody reaction activity with the specific binding substance as a target substance Can be mentioned. On the other hand, the case where the specific binding substance is an antigen and the target substance is an antibody or an antigen-binding fragment of the antibody such as Fab or F (ab ′) ₂ can also be mentioned. Moreover, when the specific binding substance is a protein, the target substance can include a protein having a binding property due to specific interaction with the specific binding substance, or a peptide fragment thereof, a nucleic acid, and the like. Further, when the specific binding substance is a nucleic acid, the target substance specifically binds to a nucleic acid molecule such as DNA or RNA having a base sequence complementary to the specific binding substance, or the specific binding substance. Examples include proteins and peptides.

The reaction with the target substance that specifically binds to the specific binding substance labeled with the labeling agent of the present invention is performed when the specific binding substance is an antibody or an antibody such as Fab or F (ab ′) ₂ . In the case where the target substance is an antigen when it is an antigen-binding fragment and vice versa, an antigen-antibody reaction may be mentioned. it can.

  The reaction with the target substance that specifically binds to the specific binding substance labeled with the labeling agent of the present invention is based on the specific interaction between the specific binding substance and the target binding substance. In the case of a protein having a binding property, a peptide fragment thereof, a nucleic acid or the like, a specific interaction (for example, a reaction between a receptor and its ligand, a transcription factor protein, and the like) occurring between the specific binding substance and a target substance , Reaction with a nucleic acid site that specifically binds to the protein, reaction with a MutS protein and a mismatched site of a double-stranded nucleic acid, etc.). be able to.

  The reaction with the target substance that specifically binds to the specific binding substance labeled with the labeling agent of the present invention is carried out in such a manner that the specific binding substance is a nucleic acid and the target substance is a complementary base sequence to the specific binding substance. In the case of a nucleic acid molecule such as DNA or RNA having a specific bond, specific binding by hybridization can be mentioned. In this case, a specific reaction can be caused under well-known conditions.

Further, the influence of the labeling on the specific binding ability between the specific binding substance labeled with the labeling agent of the present invention and the target substance can be measured by a method for quantitatively measuring various specific binding ability. . Methods for quantitatively measuring these various specific binding capacities are techniques well known among those skilled in the art. For example, the specific binding substance is an antibody or an antibody such as Fab or F (ab ′) ₂ . An antigen-binding fragment, and when a specific binding is formed by an antigen-antibody reaction with a target substance that is an antigen, or in the opposite combination, or the specific binding substance is a protein or peptide, When a complex is formed by specific interaction with a target substance that is a protein, peptide, or nucleic acid molecule, or in the opposite combination, the surface plasmon resonance (SPR) method is used as one measurement method. Specificity, kinetics and affinity of specific binding can be measured. An example of a measuring apparatus using the principle of the SPR method is Biacore 2000 manufactured by BIACORE. In addition, for example, when the specific binding substance is a nucleic acid and forms a specific bond by hybridization with a target substance having a complementary base sequence with the specific binding substance, the cyber Measure the specific binding ability by incorporating a fluorescent substance such as green into the complex by intercalation and measuring the temperature at which the two hybridized nucleic acids dissociate using a real-time PCR analyzer Can do. Other known measurement methods can also be used.

  Next, the complex formed by the specific binding between the specific binding substance and the target substance is separated (so-called B / F separation). Usually, since the target substance is immobilized on a solid phase, washing with a buffer, centrifugation (such as when beads are used as a solid phase), magnetic separation (such as when magnetic beads are used as a solid phase), etc. B / F separation can be performed by Even when the target substance is not immobilized on a solid phase, B / F separation can be performed by well-known techniques such as a separation method based on a difference in fluorescence intensity and a separation method based on molecular weight (electrophoresis, gel filtration chromatography, etc.).

Next, the labeled specific binding substance bound to the target substance is irradiated with light to cleave Y in the general formula [I]. A method of cleaving Y by irradiating the label specific binding substance bound to the target substance by light irradiation can also be easily performed by a known method. Examples of the light to be used include ultraviolet rays (preferably wavelengths of 250 nm to 400 nm), visible rays (400 nm to 480 nm), and the like. Irradiation conditions can be appropriately set according to the type of Y. In the case of the 2-nitrobenzyl group used in the following examples, ultraviolet rays having a wavelength of about 300 nm to 350 nm, preferably about 365 nm, 1 mW / cm ^{2 to} 10,000 mW / cm ² , preferably about 3500 to 5000 mW / cm ² are used. Y can be easily cleaved by light irradiation.

  The measurement of the electrochemical signal generated by the reversible oxidation-reduction reaction of Z does not require a special device, and can be easily measured by a well-known technique in the electrochemical field. Preferable examples include an apparatus capable of measuring a current change amount generated with respect to a constant voltage by an electrochemical measurement apparatus including a reference electrode, a working electrode, and a counter electrode. Specifically, examples of the measurement apparatus include the following examples. it can. Such as a potentiostat.

  According to the method described above, the specific binding substance labeled with the labeling agent of the present invention can be used to detect the target substance and indirectly measure the concentration of the target substance. An example of target concentration measurement is given below.

  As described above, the reaction between the specific binding substance labeled according to the present invention and the target substance that specifically binds to the specific binding substance includes the antibody protein labeled according to the present invention or the antigen binding of the antibody. An antigen-antibody reaction can be considered in which a specific fragment is a specific binding substance and a target substance is an antigen. As a biochemical assay method using an antigen-antibody reaction, a target substance can be detected and measured by an immunoprecipitation method, Western blotting method, protein chip analysis method, ELISA method, ELISPOT method or the like. In this case, if a sample containing the target substance at a known concentration can be obtained, a calibration curve representing the relationship between the target substance concentration and the voltage generated when the labeling agent is irradiated with light is prepared. The concentration of the target substance contained can be quantified indirectly.

  In addition, for example, when the target substance is secreted by a cell in response to a stimulus, or when the target substance is a substance generated by an enzymatic reaction in a reaction system to be analyzed, the target substance concentration changes over time. In such a case, it is possible to quantify the concentration of the target substance over time using the property that the voltage changes instantaneously when the labeling agent is irradiated with light.

  Furthermore, by performing an assay using the same principle as the assay method such as chromatin immunoprecipitation, an antibody labeled with a labeling agent consisting of structures X, Y and Z is used to further specifically target the target substance. It is also possible to detect and measure non-peptide molecules such as nucleic acid molecules to bind electrochemically.

  As described above, in the reaction between the specific binding substance labeled according to the present invention and the target substance that specifically binds to the specific binding substance, the protein or peptide labeled according to the present invention is used as the specific binding substance. And a specific interaction with the partner of the protein interaction as a target substance is considered. Examples of biochemical assays utilizing specific interactions by proteins include in vitro and in vivo assays such as pull-down assays and two-hybrid methods.

  Furthermore, in the reaction of the specific binding substance labeled according to the present invention and the target substance that specifically binds to the specific binding substance, for example, a nucleic acid molecule such as a DNA or RNA fragment labeled according to the present invention Specific binding by hybridization is conceivable using a specific binding substance as a target substance and a nucleic acid having a complementary base sequence to the specific binding substance as a target substance. As a biochemical assay method using specific binding by such hybridization, it becomes possible to detect and measure a target substance by Southern blotting, Northern blotting, DNA microarray method, and the like. In addition to the above method, the target substance can be detected and the target substance concentration can be measured by hybridizing with the target substance in the analysis sample as a probe. In this case, if a sample containing the target substance at a known concentration can be obtained, a calibration curve representing the relationship between the target substance concentration and the voltage generated when the labeling agent is irradiated with light is prepared. The concentration of the target substance contained can be quantified indirectly.

  In addition, for example, when the target substance is a nucleic acid molecule transcribed by a cell in response to a stimulus, or when the target substance is a substance generated by an enzymatic reaction in a reaction system to be analyzed, such as PCR amplification, or a virus The nucleic acid concentration of the target substance changes over time, such as when analyzing changes in the nucleic acid concentration of the target substance into cells by infection or gene transfer experiments, or when analyzing the transcriptional activity of the introduced gene. In this case, it is possible to quantify the concentration of the target substance over time using the characteristic that voltage changes instantaneously by light irradiation to the labeling agent.

  In addition, when the target substance is a peptide exhibiting the ability to bind to a nucleic acid molecule, the nucleic acid labeled with the labeling agent of the present invention is used as a specific binding substance, and the target substance is specifically bound to the target substance. Substances can be detected and measured. Examples of peptides exhibiting the ability to bind to nucleic acid molecules include DNA-binding transcription factors, receptors and their ligands, proteins with DNA sequence mismatch detection functions typified by MutS protein, and chromatin typified by histones. Examples include structural proteins.

  On the other hand, a peptide showing binding ability to a nucleic acid molecule is labeled as a specific binding substance with the labeling agent of the present invention, and a nucleic acid molecule having a base sequence that specifically binds to the specific binding substance is detected as a target substance. It is also possible to do. Examples of peptides exhibiting the ability to bind to nucleic acid molecules include DNA-binding transcription factors, receptors and their ligands, proteins with DNA sequence mismatch detection functions typified by MutS protein, and chromatin typified by histones. Examples include structural proteins.

  As specifically described in the following examples, the present inventor applied an anti-prostate specific antigen antibody labeled with the labeling agent of the present invention to an immunoassay, and as a result of detailed research and development, the prostate using an antigen-antibody reaction was used. We have succeeded in developing a test method for specific antigens. The present invention employs the measurement principle of detecting an electrochemical signal emitted from a labeling agent by light irradiation, and has high sensitivity to a target substance. Therefore, the method for examining prostate-specific antigen, which is an early warning index for cancer, using the labeling agent of the present invention enables detection of a target substance from a small amount of sample. In addition, since the above inspection method uses a simple measurement principle, it is considered to have very high applicability to the development of protein / microchip analysis methods, and the present invention provides a more comprehensive high throughput. It can be said that it provides the technical basis for the analysis method.

  Further, when the MutS protein labeled with the labeling agent of the present invention is used as a specific binding substance, the inventors of the present invention have a mismatch site on the K-ras gene sequence that is the specific binding substance and the target substance. After confirming the formation of a complex by specific binding at the nano level with an atomic force microscope, the labeling agent of the present invention is cleaved by light irradiation and the target is detected by detecting the electrochemical signal generated from Z. We have succeeded in detecting and quantitatively measuring substances. The theoretical value of the detection limit was calculated that the target substance concentration was on the order of femtomole. That is, by reacting a MutS protein labeled with the labeling agent of the present invention with a double-stranded nucleic acid, the MutS protein is specifically bound to a mismatch site in the double-stranded nucleic acid, and an unbound MutS protein is After removal, the structure Z is liberated by light irradiation, and the liberated structure Z is measured by electrochemical measurement using its redox activity to specifically detect mismatches in the double-stranded nucleic acid. be able to. The method of detecting and quantitatively measuring a DNA mismatch site using the labeling agent of the present invention combines high accuracy using specific binding and very high detection sensitivity using detection of an electrochemical signal. is there.

  Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited to the following examples.

Design and Synthesis of Labeling Agent A compound represented by the following general formula [VIII] having N-hydroxysuccinimide (NHS) group as structure X, 2-nitrobenzyl group as structure Y, and ferrocene group as structure Z Synthesized as one specific example. As the spacer structure, a structure represented by the following [A] in L ₁ and a structure represented by the following [B] in L ₂ were used. The following compound is a labeling agent having redox activity, and in the following, the compound name will be referred to as KOX for convenience.

  The KOX compound was synthesized according to the synthesis scheme shown in the following reaction formulas [IX]-[XI]. Specific reaction conditions are shown below. First, 1 shown below is reacted in chlorobenzene at 50 degrees for 4 hours. Then, it reacts in hydrochloric acid to obtain 3, and in the process of 4-6, Boc is reacted to attach a protecting group. Then, ferrocene which is an electrochemically active group is added to obtain 8. Finally, attaching the NHS group gives the final product 13.

  N-hydroxysuccinimide, which is a specific example of the structure X of the KOX compound, is a site that specifically binds the α-amino group of the protein and the ε-amino group of the lysine side chain, and KOX is a specific binding substance. It can be combined with a protein which is one specific example.

  The 2-nitrobenzyl group, which is a specific example of the structure Y of the KOX compound represented by the above chemical formula 2, is a site that undergoes photocleavage when irradiated with ultraviolet rays. The nitrobenzyl group is a chemical structure frequently used as a photodeprotection group for caged compounds. A caged compound refers to a molecule in which a physiologically active molecule is protected with a photodegradable protecting group and temporarily loses its activity, and has the function of instantly appearing the original physiologically active molecule by light irradiation. Therefore, the timing and part at which the function of the signal molecule is expressed can be controlled by the timing and part of the light irradiation, and the amount of expression can be adjusted in principle by the amount of irradiation. It is a chemical structure that enables real-time control of spatiotemporal dynamics.

UV irradiation experiment of KOX When 2-nitrobenzyl group, which is a specific example of structure Y, is irradiated with ultraviolet light, photocleavage occurs as shown in the following reaction formula.

  Therefore, when KOX is irradiated with ultraviolet light, KOX undergoes photocleavage as shown in the following reaction formula [XII].

  Specifically, the UV irradiation experiment of KOX was performed by the following method. In a dark box, an ultraviolet laser (wavelength, 350 nm) was irradiated to a solution adjusted to 5 mM KOX in a vial to perform an irradiation experiment.

  When KOX was irradiated with ultraviolet rays according to the above method, the following results were obtained. As a result, it was confirmed by the analysis result of the MALDI-MS spectrum that the ferrocene group and the NHS group were separated.

  The ferrocene group which is a specific example of the structure Z of the KOX compound is a site having redox activity. Ferrocene is a compound in which an iron ion (II) and a cyclopentadiene bimolecule are coordinated, and is a typical organic compound that causes a reversible redox reaction. Therefore, the voltage generated when the reversible redox reaction is accompanied by charge transfer can be electrochemically measured. The reversible redox reaction of the ferrocene group and the resonance state of the shared electron pair are shown in the following reaction formula [XIII].

Labeling of rabbit-derived anti-mouse IgG protein In the following Examples, rabbit-derived anti-mouse IgG protein was labeled with KOX as a specific binding substance. In addition, a confirmation experiment of KOX labeling was performed by a MALDI mass spectrometry experiment.

The experimental reagents and equipment used for labeling are as follows.
<Reagent>
1M Tris-HCl pH 7.5 (Wako Pure Chemical Industries)
ADP (Wako Pure Chemical Industries)
DTT (MP Biomedicals)
Glycerol (Wako Pure Chemical Industries)
HEPES
KCl (Wako Pure Chemical Industries)
MgCl ₂ (Wako Pure Chemical Industries)
NaOH (Wako Pure Chemical)
PMSF (Wako Pure Chemical Industries)
Surfactant P-20 (BIACORE)
Rabbit-derived anti-mouse IgG (CHEMICON)
<Solvent>
DMSO (Wako Pure Chemical Industries)
<Purified separation (centrifugal concentration method)>
Amicon-Ultra 4 (MILLIPORE)

The labeling of the rabbit-derived anti-mouse IgG protein followed the following protocol. First, 200 μg of anti-mouse IgG was taken, and PBS pH 7.6 to 10 mM HEPES pH 7.9 (50 mM KCl, 5 mM MgCl ₂ , 0.2 mM PMSF) in which anti-mouse IgG was dissolved at 4 ° C. and 4000 rpm using Amicon-Ultra 4 , 1 mM DTT, 10% Glycerol). Secondly, a KOX 100 μg solution dissolved in 4 μL of DMSO was added to 200 μg / 200 μL of anti-mouse IgG, and at the same time, 4 μL of DMSO was added to 200 μg / 200 μL of anti-mouse IgG was also prepared as a blank. Thirdly, the cells were incubated at room temperature under two conditions of 2 hours or 4 hours, and concentrated and washed 3 times at 4 ° C. and 4000 rpm using Amicon-Ultra 4. Fourth, the reaction was stopped by removing excess KOX. Finally, washing was performed using 20 mM Tris-HCl pH 7.6 (150 mM KCl, 5 mM MgCl ₂ , 1 mM DTT, 1 mM ADP, 0.005% Surfactant P-20) to obtain an anti-mouse IgG-KOX complex.

The experimental reagents and equipment used in the mass spectrometry experiment for labeling confirmation are as follows.
<Reagent>
SA (TCI)
TFA (Wako Pure Chemical Industries)
<Solvent>
MeCN (Wako Pure Chemical Industries)
<Measurement equipment>
Mass spectrometer (MALDI-TOF MS)

  The mass spectrometry experiment for confirming the KOX labeling was performed according to the following protocol. The ionization method of the mass spectrometer was the MALDI method, and the mass spectrometer was an ultraflex of TOF BURKER DALTONICS. In the above-mentioned labeling of rabbit-derived anti-mouse IgG protein, KOX label for one molecule of anti-mouse IgG protein using MALDI-TOF MS for those incubated at room temperature for 2 hours or 4 hours The number of chemical molecules was confirmed. The matrix used was a saturated solution of SA dissolved in MeCN: water = 1: 1, 0.1% TFA mixed solution. The mass spectrum obtained under each condition is shown in FIG.

  From FIG. 1, the spectrum was shifted to the right before and after the labeling of the redox tag probe: KOX to the antibody. Thus, it was confirmed that KOX was also labeled on the anti-mouse IgG.

  Fig. 2 is an enlargement of the vicinity of the spectrum peak in Fig. 1. Based on the peak shift obtained from Fig. 2, the number of KOX molecules bound per molecule of anti-mouse IgG is calculated based on the fact that the molecular weight of KOX is 633. Can be sought. The results of the number of labels under each condition are shown in Table 2 below.

  From Table 2, it was confirmed that 1 to 2 and 6 to 8 molecules of KOX were labeled per antibody under the labeling conditions of room temperature (r.t.) for 2 hours and 4 hours, respectively. It was also confirmed that the number of labels increased as the labeling time increased. The reaction mechanism of the labeling is shown in the following chemical formula 7.

KOX labeling of anti-prostate specific antigen (PSA) antibody (Detection-PSA)
Since it was confirmed that KOX was labeled on the IgG antibody, the PSA antibody selected as a substance actually having a specific binding property to the target substance was labeled. In addition, a confirmation experiment of KOX labeling was performed by a MALDI mass spectrometry experiment.

The experimental reagents and equipment used for labeling are as follows.
<Reagent>
1M Tris-HCl pH 7.5 (Wako Pure Chemical Industries)
ADP (Wako Pure Chemical Industries)
DTT (MP Biomedicals)
Glycerol (Wako Pure Chemical Industries)
HEPES
KCl (Wako Pure Chemical Industries)
MgCl ₂ (Wako Pure Chemical Industries)
NaOH (Wako Pure Chemical)
PMSF (Wako Pure Chemical Industries)
Surfactant P-20 (BIACORE)
Anti-PSA monoclonal antibody (Detection-PSA) (BIODEESIGN, catalog: M86506M)
<Solvent>
DMSO (Wako Pure Chemical Industries)
<Purified separation (centrifugal concentration method)>
Amicon-Ultra 4 (MILLIPORE)

The labeling of Detection-PSA followed the following protocol. First, 200 μg of Detection-PSA is taken, and PBS pH 7.4 to 10 mM HEPES pH 7.9 (50 mM KCl, 5 mM MgCl ₂ , 0.2 mM PMSF) in which Detection-PSA is dissolved at 4000 rpm at 4 ° C. using Amicon-Ultra 4 , 1 mM DTT, 10% Glycerol). Secondly, a KOX 100 μg solution dissolved in DMSO 4 μL was added to Detection-PSA 200 μg / 200 μL, and at the same time, Detection-PSA 200 μg / 200 μL containing DMSO 4 μL was also prepared as a blank. Thirdly, the cells were incubated at room temperature under two conditions of 2 hours and 4 hours, and concentrated and washed 3 times at 4 ° C. and 4000 rpm using Amicon-Ultra 4. Fourth, the reaction was stopped by removing excess KOX. Finally, washing was performed using 20 mM Tris-HCl pH 7.6 (150 mM KCl, 5 mM MgCl ₂ , 1 mM DTT, 1 mM ADP, 0.005% Surfactant P-20) to obtain a Detection-PSA-KOX complex.

The experimental reagents and equipment used in the mass spectrometry experiment for labeling confirmation are as follows.
<Reagent>
SA; sinapinic acid (TCI)
TFA; trifluor acetic acid
Protein calibration standard II (BURKER DALTONICS)
<Solvent>
MeCN (Wako Pure Chemical Industries)
EtOH (Wako Pure Chemical Industries)
<Measurement equipment>
Mass spectrometer (MALDI-TOF MS)

The mass spectrometry experiment for confirming the KOX labeling was performed according to the following protocol. The ionization method of the mass spectrometer was the MALDI method, and the mass spectrometer was an ultraflex of TOF BURKER DALTONICS. The number of labels was confirmed using MALDI-TOF MS for those incubated at room temperature for 2 hours and 4 hours. In the saturated solution of sinapinic acid (SA) dissolved in MeCN: water = 1: 1, 0.1% TFA mixed solution used for detection of anti-mouse IgG, the matrix had a ratio of the intensity of the monovalent peak to the divalent peak. Because it was small, I examined the matrix,
SA1: SA EtOH saturation
SA2: SA TA saturation (TA 2: 1 = 0.1% TFA water: MeCN)
The SA was saturated with two types of solvents. As a specific procedure for preparing a mixed crystal, first take 1 μL of SA1 with a pipette, place it on the target plate to make a thin film of SA, then mix the sample to be measured and 0.5 μL of SA2 in equal amounts on the thin film. It was then dried and measured. During the measurement, Protein calibration standard II was used as an external standard for calibration.

  As a result of the calibration, the obtained spectrum peak value of calibration with a known molecular weight was corrected as an external standard, and actual measurement was performed. The spectrum obtained as an actual measurement result is shown in FIGS.

  FIG. 5 is an enlarged view of the vicinity of the spectrum in FIGS. 3 and 4, which confirmed a peak shift before and after labeling. Table 3 shows the results under each labeling condition based on the peak of the spectrum obtained.

  From Table 3, it was confirmed that the KOX 2.4 molecule was labeled against the PSA 1 antibody at a labeling time of 2 hours, and the KOX 3.3 molecule was labeled against the PSA 1 antibody at a labeling time of 4 hours. In addition, the longer the labeling time, the greater the number of KOX labels. As the number of KOX labels increases, the amount of ferrocene attached per PSA antibody increases, so that it is possible to detect PSA with high sensitivity even with a small amount of PSA antigen.

Confirmation experiment of detection-PSA antibody antigen binding by SPR method
In order to confirm whether Detection-PSA labeled with KOX retains the ability to cause an antigen-antibody reaction even in the state of Detection-PSA-KOX complex, The binding amount of the labeled Detection-PSA antibody was measured.

The reagents and equipment used in the confirmation experiment of the antigen-binding ability of Detection-PSA antibody by the SPR method are as follows.
<Reagent>
1M Tris-HCl pH 7.5 (Wako Pure Chemical Industries)
ADP (Wako Pure Chemical Industries)
DTT (Wako Pure Chemical Industries)
KCl (Wako Pure Chemical)
MgCl ₂ (Wako Pure Chemical Industries)
Surfactant P-20 (BIACORE)
Sensor Chip SA (BIACORE)
<Running buffer>
20mM Tris-HCl pH7.6 (including 150mM KCl, 5mM MgCl2, 1mM DTT, 1mM ADP, 0.005% Surfactant P-20)
<Measurement equipment>
Biacore 2000 (BIACORE)

  The experiment was performed according to the following protocol, and for the measurement, Detection-PSA antibody labeled based on the protocol shown in [0058] was used. First, biotinylated Capture-PSA antibody was flowed and immobilized on a streptavidin-modified SA sensor chip. Second, PSA antigen was flowed to cause an antigen-antibody reaction. Finally, the KOX-labeled Detection-PSA antibody with different labeling conditions was flowed to further cause an antigen-antibody reaction, and the binding amount of the Detection-PSA antibody was calculated.

FIG. 6 shows a sensorgram obtained from the experiment by the SPR method, and Table 4 shows the result of calculating the amount of KOX-labeled Detection-PSA antibody bound to the PSA antigen from the sensorgram. The RU value was taken at 1000 seconds. At this time, the antibody binding amount was determined from 1000 RU corresponding to a mass change of 1 ng / mm ² of protein on the sensor chip surface.

  As shown in FIG. 6 and Table 4, when comparing the labeled Detection-PSA antibody with the unlabeled Detection-PSA antibody, the unlabeled Detection-PSA antibody binds better to the PSA antigen. I understand. Compared to the unlabeled Detection-PSA antibody, the 4h-labeled antibody has an activity maintenance rate of 44.6%. From the value of RU value 568.0, it is considered that the KOX-labeled Detection-PSA antibody has sufficient ability as an antibody, and there is no problem in the detection and concentration measurement of the target substance. Therefore, it was confirmed that the detection-PSA antibody maintained its activity as an antibody even after being labeled with KOX.

Electrochemical detection of PSA antigen using KOX labeled Detection-PSA antibody
A confirmation experiment was performed to actually detect the PSA antigen as the target substance by irradiating the KOX-labeled Detection-PSA antibody with ultraviolet light.

The reagents and equipment used in the PSA antigen detection confirmation experiment are as follows.
<Reagent>
1 M Tris-HCl pH 7.5 (Wako Pure Chemical Industries)
ADP (Wako Pure Chemical Industries)
BSA (Wako Pure Chemical Industries)
EDTA
MagnaBindTM Streptavidin Beads (PIERCE)
MgCl ₂ (Wako Pure Chemical Industries)
NaCl (Wako Pure Chemical Industries)
PBS (NIPPON GENE)
Triton-X100 (Wako Pure Chemical Industries)
Biotin labeling Kit (Dojindo)
<Ultraviolet light source>
LIGHTNINGCURE L8868 (Hamamatsu Photonics)
<Electrode>
Reference electrode: Ag | AgCl electrode
Working electrode: Glass-carbon electrode, diameter 1.0 mm
Counter electrode: Platinum electrode (manufactured by BAS)

Capture-PSA was biotinylated to obtain Capture-PSA at a concentration of 188 μg / Tris-HCl pH 7.8 200 μL. The lamp of the UV light source is a mercury xenon lamp, and the UV irradiation intensity is 3500 mW / cm ^{2 on} average at 365 nm. The experiment was conducted at an intensity of 50%. The PSA antigen detection experiment was performed according to the following protocol. First, 50 μL (1 mg) of streptavidin magnetic beads was placed in a 1.5 mL tube, allowed to stand for 1 minute on a magnetic stand, and then the supernatant was removed. Second, after washing the beads 3 times with 20 mM Tris-HCl pH 7.8 (1.0 M NaCl, 1 mM EDTA, 0.02% Triton-X100), add 5 μg / 100 μL of biotinylated Capture-PSA and incubate for 30 minutes at room temperature. After standing for 1 minute on a magnetic stand, the supernatant was removed and washed twice with 10 mM Tris-HCl pH 8.0 (1 mM EDTA, 1 M NaCl, 0.1% Triton X-100). Thirdly, in order to prevent non-specific adsorption to the surface of the beads on which Capture-PSA was immobilized, blocking was performed with 2% BSA / PBS buffer at 4 ° C. overnight. Fourth, 200 μL of PSA antigen was added and allowed to stand at 37 ° C. for 2 hours. After removing the supernatant, the plate was washed twice with 10 mM Tris-HCl pH 8.0 (1 mM EDTA, 1 M NaCl, 0.1% Triton X-100). Fifth, 200 μg / 100 μL of Detection-PSA-KOX complex was allowed to act and incubated at 37 ° C. for 1.5 h. Sixth, after 1 hour, excess KOX was removed with a magnetic stand. Washing was performed 5 times using 20 mM Tris-HCl pH 7.6 (150 mM KCl, 5 mM MgCl ₂ , 1 mM DTT, 1 mM ADP, 0.005% Surfactant P-20). Finally, after UV irradiation for 180 seconds, photocleavage occurred, and the released ferrocene site was measured by CV.

  FIG. 7 shows the results of measuring 150 μL of the supernatant in a microcell before and after UV irradiation and measuring with CV. Since the ferrocene peak appeared before and after photocleavage, the PSA antigen was detected electrochemically. I can say that. A response of about 0.8 μA was obtained at a PSA antigen concentration of 590 nM (17.7 μg / ml). Since CV can measure up to pA, it can theoretically be detected up to 59 fM (1.77 pg / ml).

Verification of correlation between target substance concentration and electrochemical signal intensity According to the protocol described above, the PSA antigen concentration of the target substance was set to a level different from 0, 88.5, and 885 ng / mL, and electrochemical detection was performed. The result is shown in FIG.

  As is clear from FIG. 8, with the increase of the PSA antigen concentration, a clear increase in peak current was observed at the oxidation potential of ferrocene: 596 mV vs. Ag | AgCl. From this, it can be said that the PSA antigen could be electrochemically detected indirectly using a redox activity labeling agent. In addition, since there is a clear correlation between an increase in PSA antigen concentration and an increase in peak current, the target substance concentration can be electrochemically detected by a well-known technique such as combined use with a calibration curve using the redox active labeling agent of the present invention. Can be quantified indirectly.

KOX labeling of MutS protein and mass spectrometry of KOX labeled MutS protein KUT labeling of MutS protein was carried out by the following method.
MutS protein was labeled by KOX labeling by allowing the MutS protein and KOX to coexist in a vial in a PBS buffer solution and reacting at room temperature.

  Next, mass spectrometry of the KOX-labeled MutS protein was performed by the following method to confirm KOX binding to the Muts protein. This was carried out by confirming a mass spectrum shift of about two molecules of KOX in the MALDI-MS spectrum.

Verification of enzyme activity of MutS-KOX complex The effect of MutS protein on the enzyme activity of MutS protein was verified by the following method. The enzyme activity was measured using SPR (Surface Plasmon Resonance Method). The DNA was immobilized on a substrate in a state of mismatch hybridization of DNA, and MutS-KOX was flowed to the substrate by a flow. As a result, an increase response of the SPR signal could be obtained, and the interaction could be confirmed.

Mutation between MutS-KOX complex and DNA The interaction between MutS-KOX complex and target DNA was verified by the specific method described below. The interaction was verified using SPR (Surface Plasmon Resonance Method). The DNA was immobilized on a substrate in a state of mismatch hybridization of DNA, and MutS-KOX was flowed to the substrate by a flow. As a result, an increase response of the SPR signal could be obtained, and the interaction could be confirmed.

Mismatch DNA Detection Experiment Using MutS-KOX Complex Mismatch DNA detection experiment using MutS-KOX complex was performed using the following method.
The solution containing the MutS-KOX complex interacted in the vial was irradiated with UV, and only the amount interacting with the target DNA was opened to selectively separate only the reaction amount of the target DNA. Thereafter, the supernatant of the reaction solution was transferred to a vial in which an electrochemical electrode was inserted, and the potential response was confirmed by ferrocene oxidation potential by applying a potential to detect mismatched DNA.

It is a figure which shows the mass spectrometry result for confirming the coupling | bonding of mouse | mouth IgG protein and a KOX labeling agent. It is the figure which expanded the peak vicinity of the mass spectrum of FIG. It is a figure which shows the mass-spectrometry result for confirming the coupling | bonding of Detection-PSA and a KOX label | marker under incubation conditions at room temperature for 2 hours. It is a figure which shows the mass-spectrometry result for confirming the coupling | bonding of Detection-PSA and a KOX label | marker under incubation conditions of room temperature and 4 hours. FIG. 5 is an enlarged view of the vicinity of the peak of the mass spectrum of FIGS. 3 and 4. An SPR analysis sensorgram of the binding between KOX-labeled Detection-PSA and PSA-specific antigen is shown. The graph which shows the electrochemical detection of the electric current produced by reaction of KOX labeled Detection-PSA and a PSA specific antigen is shown. It is a graph which shows the correlation about the increase in the electric current produced by the reaction of the increase in a PSA specific antigen concentration, and the reaction of KOX labeling Detection-PSA.

Claims (10)

The following general formula [I]

(Wherein, X represents a structure that can be covalently bonded to another functional group, Y represents a structure that can be cleaved by light irradiation, Z represents a structure having electrochemically measurable redox activity, L ₁ and L ₂ may or may not be present; if present, L ₁ represents any spacer structure that covalently connects structures X and Y, and L ₂ represents structure Y Any spacer structure that connects Z and Z by a covalent bond)
A labeling agent having a structure represented by:   2. The labeling agent according to claim 1, wherein, in the general formula [I], Z is selected from the group consisting of ferrocene, osmium complex, and ruthenium complex.   3. The labeling agent according to claim 2, wherein Z in the general formula [I] is ferrocene.   The labeling agent according to any one of claims 1 to 3, wherein, in the general formula [I], Y is selected from the group consisting of a 2-nitrobenzyl structure.   The labeling agent according to claim 4, wherein Y in the general formula [I] has a 2-nitrobenzyl structure.   The labeling agent according to any one of claims 1 to 5, wherein, in the general formula [I], X is selected from the group consisting of a succinimide structure and a maleimide structure.   The labeling agent according to claim 6, wherein X in the general formula [I] has a succinimide structure.   The specific binding substance labeled with the labeling agent according to any one of claims 1 to 7 is reacted with a target substance that specifically binds to the specific binding substance, and the specific binding substance and the target substance are reacted. After separation of the complex formed by specific binding, the labeled specific binding substance bound to the target substance is irradiated with light to cleave Y in the general formula [I], thereby releasing the general formula [I ] The measuring method of a target substance including measuring the structure Z in [inside]] electrochemically.   The method according to claim 8, wherein the specific binding substance is selected from the group consisting of an antigen, an antibody and an antigen-binding fragment thereof, a MutS protein, a receptor and its ligand, a nucleic acid, and an RNA. The method according to claim 9, wherein the specific binding substance is selected from the group consisting of an antigen, an antibody and an antigen-binding fragment thereof, and a MutS protein.

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