JPH07234201A - Electrochemical measuring method and new p-phenylene diamine compound - Google Patents

Abstract

PURPOSE:To provide an electrochemical measuring method, which provides good measurement sensitivity, can perform stable measurement with good reproducibility even when a sample contains an interfering substance such as blood, and can be suitably used for disposable usage, and to provide a new p-phenylene diamine compound used for this method. CONSTITUTION:In an electrochemical measuring method using an oxidation- reduction enzyme, an oxidation-reduction enzyme, an electron transporting substance, and an electrode which can carry out electron transport, with the electron transporting substance are arranged in a measuring system, and for accomplishing the purpose, such compounds as p-phenylene diamine derivatives or their salts having one of more group selected from a hydroxyl group, a mercapto group, a carboxyl group, a phosphonoxy group, and a sulfo group, or p-phenylene diamine derivatives or their salts having an alkyl group, a hydrogen atom, and previously described groups are used for the electron transporting substance.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrochemical measuring method using an electron mediator and a novel p-phenylenediamine compound. Specifically, at least one oxidoreductase, an electrode, and a predetermined electron mediator (electron med
(iator), an electrochemical measurement method for measuring an analyte in a liquid sample, for example, an enzyme electrode method or a specific binding analysis method, and a novel p- that can be used for this electrochemical measurement method. It relates to a phenylenediamine compound.

[0002]

2. Description of the Related Art The enzyme electrode method is a method for selectively measuring a chemical substance by utilizing the molecular discrimination ability of an enzyme, generally using an apparatus in which an enzyme-immobilized membrane and an electrode are combined.
In the field of biochemical analysis method, a simple and rapid measurement method using an enzyme sensor or the like has already been disclosed. For example, Japanese Examined Patent Publication No. 2-59424 and JP-A-60-173.
The measurement methods disclosed in Japanese Patent Publication No. 46 and No. 60-17347 are exemplified. On the other hand, as a specific binding analysis method,
Many methods are known, including immunoassays that apply antigen-antibody reactions, receptor assays that use receptors, and nucleic acid probe assays that use hybridization of complementary nucleic acid sequences. It is widely used in a wide range of fields including the ones mentioned above.

On the other hand, attempts to apply the electrochemical sensor to a specific binding analysis method are being actively made. For example, JP-A-58-58467 discloses an enzyme (catalase).
Specific binding reaction on electrode surface using labeled antibody; Japanese Patent Laid-Open No. 2-179461 discloses competitive specific binding reaction on electrode surface using enzyme (glucose oxidase) labeled substance; Japanese Patent No. 17360 discloses a specific binding reaction using an oxidoreductase (glucose oxidase or the like) and an electron mediator (ferrocene derivative or the like); Further, as a technique similar to these, Japanese Patent Application Laid-Open Nos. 3-25360 and 60-
127450, 60-242361, 63-1
Nos. 39248 and 61-500706, U.S. Pat. No. 4,963,245, and U.S. Pat.
066,372, as well as Anal. Chem., V
ol.56,2355-2360 (1984) and Clin.Chem., Vol.31,1449-145.
2 (1985) and others have various disclosures.

Among the enzyme electrode method and the specific bond analysis method, in the amperometric enzyme electrode method and the specific bond analysis method using an electrochemical sensor, a biological redox reaction of an enzyme or the like is generated. Low molecular weight compounds that mediate electrode reactions, so-called electron mediators, are widely used. That is, the electron transfer substance is a compound that mediates transfer transfer of electrons between an enzymatic reaction and an electrode reaction in a measurement system using at least one oxidoreductase and an electrode capable of electron transfer in the system.

Such an electron transfer substance is "reduced / oxidized" by an enzyme reaction and "oxidized / reduced" by an electrode reaction. Circulation between these two reactions is carried out from the enzyme reaction to the electrode reaction. Alternatively, it mediates transfer transfer of electrons from the electrode reaction to the enzyme reaction. Therefore, such an electron transfer substance must be a compound that is efficiently oxidized or reduced by an enzymatic reaction; oxidized or reduced by an electrode reaction, and returned to a state where the enzymatic reaction can be used again; enzymatic reaction or electrode It is required that the compound oxidized or reduced by the reaction be stable; Furthermore, the electron mediator that mediates the transfer of electrons between the enzymatic reaction and the electrode reaction must be redox at a potential within the measurable range of the working electrode. The measurable range (potential window) of the electrode varies depending on the type of electrode and the solution conditions, but in the case of a carbon electrode, normally about -1.2 V to +1.0 V (vs.SCE) is preferable.

As these electron transfer substances, there are organometallic compounds such as ferrocene derivatives (UK Patent 813203).
No. 4), inorganic compounds such as potassium ferri (or ferro) cyanide, and p-phenylenediamine (PP).
A large number of organic compounds such as D) derivatives are known. For example, p-phenylenediamine (PPD) derivative N, N, N ', N'-tetramethyl-p-phenylenediamine (TMPD) is used to oxidize sulfite ion to sulfate ion. Catalyzing su
It is known to mediate electron transfer between lfite oxidase and the electrode (Anal. Chem., 1993,65,
242-246). Further, WO92 / 07263 discloses a benzene derivative having an amino group, an alkylamino group, a dialkylamino group, a morpholinyl group, and a piperidyl group at the 1- and 4-positions as an electron transfer substance whose reduced form is poorly soluble in water. It is disclosed.

On the other hand, PPD derivatives N, N, N ',
N'-tetrakis- (2'-hydroxyethyl) -p-
Phenylenediamine (THEPD) and N, N,
N ', N'-tetrakiscarboxymethyl-p-phenylenediamine (TCPD) has been conventionally used as a light-sensitive material / chelating agent (Photographic Science and
Engineering 1962, vol.6, No.1, p39; Nature 1964, vo
l.204, p180; Z.Chem.1964, vol.4, No.11, p436; Z.Che
m. 1964, vol.4, No.12, p463; An.Quin.Ser.B 1963, vol.
59, No7-8, p501; An.Quin.Ser.B 1965, vol.61, No.5, p71
7; An.Quin.Ser.B 1965, vol.61, No.5, p723; An.Quin.
Ser.B 1963, vol.59, No.7-8, p493; An.Quin.Ser.B 198
6, vol.82, No.2, p133; Research Disclosure 1981, vol.
212, p428). Further, THEPPD can be used as a color reagent for the peroxidase reaction, when the THEPD reacts in the presence of peroxidase, a naphthol derivative, and hydrogen peroxide, and the radical and the naphthol derivative combine to form a coloring substance. Was known (European Patent No. 0504663). However, T
It was not known that HEPD and TCPD act as electron mediators with the above properties.

By the way, in the measuring method using the electron mediator, since the main use of the conventional electron mediator is the enzyme electrode, as described in the above-mentioned WO92 / 07263, it is hardly soluble in water. Certain electron mediators are preferred. This is because in order to obtain a stable electrochemical signal when mediating the electron transfer between the redox enzyme immobilized or insolubilized on the electrode and the electrode, an electron mediator should be present near the electrode and This is because it is necessary to prevent the electron transfer substance from flowing out. When the electron transfer substance is a compound that is easily affected by interfering substances such as ascorbic acid, uric acid, hemoglobin, and oxygen in the sample, the electron transfer substance is allowed to exist in the electrode as a compound insoluble in water to prevent interference. It is necessary to avoid contact with substances. However, in this method, since the electron transfer substance is present in the electrode, the field of the reaction is limited, and only the enzyme capable of effectively contacting the electron transfer substance in the electrode can participate in the reaction, and the signal intensity is generally low. It had the drawback of becoming smaller.

On the other hand, recently, a disposable enzyme electrode analytical piece using a water-soluble electron transfer substance such as potassium ferricyanide has also been developed (see Japanese Patent Laid-Open No. 1-156658). Since the water-soluble electron transfer substance can also mediate electron transfer between the electrode and the enzyme at a position distant from the electrode to some extent, the number of effective enzyme molecules is increased and the characteristics of the enzyme electrode can be improved. In such a disposable enzyme electrode analytical strip, it is desirable that the components necessary for the operation of the electrode be incorporated into the analytical strip in advance and measurement can be performed only by adding a sample specimen. Therefore, it is desired that the electron transfer material has high water solubility, can be easily dried, and is stable in a dry state. Water-soluble ferrocene derivative,
Electron transfer substances of metal complexes such as potassium ferricyanide and osmium complex satisfy these conditions to some extent. However, they have a small speed of alternately transferring and transferring electrons with the enzyme, and there is a drawback that a high concentration of electron transfer substance is required to increase the speed. Further, since the water-soluble electron transfer substance reacts in the sample solution, it is easily affected by interfering substances such as ascorbic acid, uric acid, hemoglobin and oxygen in the sample. Therefore, the development of compounds that are less affected by interfering substances is desired.

Further, recently, by using an oxidoreductase as a labeling agent and developing it in a matrix together with a liquid sample, a specific binding reaction between an analyte and a specific binding substance in the liquid sample causes the labeling agent from the electrode portion. ME as a specific binding analysis method for forming a distance distribution and measuring a current value corresponding to the concentration of an analyte in a liquid sample, which is rate-controlled by diffusion of an electron carrier, and thereby measuring the concentration of the analyte.
DIA method (Mediator Diffusion-Controlled Immunoassa
y) is known (see JP-A-5-264552 (European Patent Publication No. 0 525 723 A2)). Also in such a measuring method, an electron transfer substance capable of mediating stable electron transfer between an enzyme and an electrode in a sample liquid such as blood or urine containing an interfering substance is desired.

[0011]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems of the prior art. An electrochemical measurement method using a water-soluble electron mediator, for example, an enzyme electrode method or For example, in a measurement method such as a specific binding analysis method such as the MEDIA method, even if the measurement sensitivity (responsiveness) is good and the sample contains an interfering substance such as blood or urine, it is always stable and reproducible. The electrochemical measurement method which can perform good measurement of the above, and can be suitably used for disposable applications, and a novel p which can be used as an electron transfer substance in this electrochemical measurement method.
-To provide a phenylenediamine compound.

[0012]

[Means for Solving the Problems] A measuring method using an electron transfer substance, more specifically, a measuring method for quantifying an analyte in a liquid sample using an electrode and at least one oxidoreductase, The following are typical examples of oxidoreductases used. Oxidase (oxidase) In the living body, oxygen is used as an electron acceptor and contains flavin coenzyme, metal atom, heme and the like. Examples: glucose oxidase, amino acid oxidase,
Lactate oxidase, urate oxidase, xanthine oxidase, ascorbate oxidase, etc. Hydroperoxidase In the living body, hydrogen peroxide serves as an acceptor. Examples: peroxidase, catalase, etc. Dehydrogenase (Dehydrogenase) In the living body, NAD (H) and NADP (H) are used as acceptors (or donors). Examples: alcohol dehydrogenase, lactate dehydrogenase, etc.

Among these oxidoreductases, glucose oxidase (GOD) is used as an enzyme electrode for measuring blood glucose level, and horseradish peroxidase (HRP) is used.
O) is used as a labeling enzyme for specific binding analysis methods such as DNA hybridization and immunoassay.
According to the experiments by the present inventors, glucose oxidase,
Horseradish peroxidase shows a good current response in buffer when hydroquinone (HQ) / benzoquinone (BQ) is used as an electron mediator, and is superior to conventional electron mediators such as ferrocene derivatives, inorganic ions, and metal complexes. Also had excellent properties. However, these electron mediators were interfered with by the presence of blood and urine, and stable responses with good reproducibility were not observed. Further, p-phenylenediamine (PPD), TMPD, N, N, N ', N'
-Tetraethyl-p-phenylenediamine (TEPD)
In the presence of blood, urine, and the like, similar interference was observed with PPD-based electron mediators that exhibit a good current response in a buffer solution.

Further, for the purpose of producing a disposable type analytical piece, a solution of HQ, PPD, TMPD or TEPD is impregnated into cellulose filter paper or glass filter paper and dried on the electrode (freeze drying, reduced pressure drying, air drying, etc.). Alternatively, if it is placed on the electrode after drying and is configured to be dissolved by the sample at the time of measurement, the current response becomes small and only extremely poor characteristics are exhibited.

As a result of the detailed studies by the present inventors, the existence of the following problems as the main causes of these phenomena was estimated. The solubility of the oxidized or reduced compound of the compound as an electron transfer substance in water is low. The vapor pressure of the compound that becomes the electron carrier is high, and the stability after drying is poor. The stability of an oxidized (or radical state) or reduced compound of a compound serving as an electron transfer substance in a biological sample such as blood or urine is poor.

As a result of intensive studies to solve such problems, the present inventors have found that an oxidoreductase, an electron mediator capable of transferring electrons to and from this oxidoreductase, and this electron transfer. In a system having an electrode capable of electron transfer with a substance, by using a compound represented by the following formula [I] or [II] or a salt thereof as an electron transfer substance, it is suitable for various measuring methods of biological components. It has been found that it can be used. That is, as a result of intensive investigations by the present inventors, the compound of the formula [I] or the formula [II] or a salt thereof, in an electrochemical measurement such as an enzyme electrode method or a specific binding analysis method such as the MEDIA method, Acts as an excellent electron mediator and is highly soluble in water; easy to dry (freeze-dry, vacuum-dry, air-dry, etc.) and stable in the dry state: high electron transfer rate with enzyme;
The present invention was accomplished by discovering that it has excellent properties such as that it functions as an electron mediator that is less affected by interfering substances in specimen samples such as blood and urine.

The present invention provides a measurement method for measuring an analyte in a liquid sample using at least one oxidoreductase, wherein the measurement system includes a oxidoreductase, an electron transfer substance, and the electron transfer substance and an electron. Provided is a movable electrode, and a compound represented by the following formula [I] or [II] or a salt thereof is used as the electron transfer substance, which provides an electrochemical measurement method.

The formula [I] has the following structure.

[Chemical 6] (In the above formula [I], R ¹ , R ² , R ³ and R ⁴ each represent a linear or branched alkyl group having 1 to 4 carbon atoms which may have a substituent. In the above formula [I], at least one of R ¹ , R ² , R ³ and R ⁴ is at least one group selected from the group consisting of a hydroxyl group, a mercapto group, a carboxyl group, a phosphonooxy group and a sulfo group. And R ¹ , R
² , R ³ and R ⁴ may be the same or different from each other. )

In the above formula [I], R ¹ , R
^At least one of ² , R ³ and R ⁴ preferably has a hydroxyl group or a carboxyl group, and particularly preferably one or more of the compounds shown below.

[Chemical 7]

The formula [II] has the following structure.

[Chemical 8] (In the above formula [II], R ⁵ , R ⁶ , R ⁷ and R ⁸ are each a hydrogen atom or a carbon atom which may have a substituent.
4 to 4 represents an alkyl group having a straight chain or a branched chain. However, in the above formula [II], R ⁵ , R ⁶ , R ⁷ and R ⁸ may be the same or different, and at least one of them is a hydroxyl group, a mercapto group or a carboxyl group. , An alkyl group having at least one group selected from the group consisting of a phosphonooxy group and a sulfo group, at least one of which is a hydrogen atom. )

In the above formula [II], R ⁵ , R ⁶ , and R ⁷
It is preferable that at least one of R ⁸ and R ⁸ has a hydroxyl group, and the following compounds are exemplified.

[Chemical 9]

The analyte is a substrate, a cofactor, an activator or an inhibitor of the oxidoreductase, and the electrochemical reaction at the electrode is modulated by the enzymatic reaction that occurs depending on the amount of the substrate, cofactor, activator or inhibitor. It is preferably detected as a modulation of the dynamic signal.

Further, it is preferable to measure the analyte by a specific binding reaction using at least one specific binding substance for the analyte and using the oxidoreductase as a labeling substance.

Further, an example using the MEDIA method, that is, a matrix which is a region where a liquid sample expected to contain the analyte is developed, a sample introducing section for introducing the sample into the matrix, and A sample is introduced into the sample introduction part by using an apparatus having an electrode substantially in contact with a matrix, and an analyte in the sample, a signal in the first specific binding substance and the electrode part are introduced in the matrix. Reacting with a second specific binding substance labeled with an oxidoreductase capable of generating a generated electron mediator species; or, outside the matrix, with the analyte in the sample,
Reacting with the first specific binding substance and the second specific binding substance labeled with an oxidoreductase capable of generating an electron mediator species that emits a signal at the electrode part, and then introducing into the matrix; Complex of a second specific binding substance labeled with oxidoreductase and an analyte, complex of a second specific binding substance labeled with oxidoreductase, an analyte and a first specific binding substance , Or of the second specific binding substance labeled with an oxidoreductase that does not form a complex, the distribution of at least one molecular species is changed, and the change in distribution corresponding to the amount of the analyte in the sample is changed,
It is preferable to detect as the modulation of the signal at the electrode which is rate-controlled by the mass transfer of the electron mediator species generated by the redox enzyme.

Another example using the MEDIA method, that is, a matrix in which a liquid sample expected to contain the analyte is developed, and a sample introducing section for introducing the sample into the matrix , And a device having an electrode substantially in contact with the matrix, and either one of the specific binding substance for the analyte or the substance that competes with the analyte for the specific binding substance,
The sample is labeled with an oxidoreductase capable of generating an electron mediator species that generates a signal at the electrode, and a sample is introduced into the sample introduction part, and an analyte in the sample and a specific binding substance are introduced in the matrix. And competitively reacting a substance that competes with the analyte for the specific binding substance; or, outside the matrix, with the analyte in the sample,
A specific binding substance and a substance that competes with the analyte for the specific binding substance are competitively reacted and then introduced into the matrix; thereby, a complex of the specific binding substance and the analyte, As a complex of a binding substance and a substance that competes with the analyte for the specific binding substance, a specific binding substance that does not form a complex, or a specific binding substance that does not form a complex, Of the competing substances, the distribution of the molecular species labeled with at least one redox enzyme is changed, and the distribution change corresponding to the amount of the analyte in the sample is generated by the redox enzyme. It is preferable to detect as a modulation of the signal at the electrode that is rate-controlled by movement.

The electrochemical measuring method of the present invention and the novel p-phenylenediamine compound applicable to the electrochemical measuring method will be described in more detail below. As described above, the electrochemical measurement method of the present invention (hereinafter referred to as the measurement method) has at least one oxidoreductase, an electron transfer substance, and an electrode capable of electron transfer with the electron transfer substance in the measurement system. A compound represented by the following formula [I] or [II] or a salt thereof is used as the electron transfer substance.

[0027]

[Chemical 10]

In the formula [I], R ¹ , R ² , R ³ and R ⁴ each represent a linear or branched alkyl group having 1 to 4 carbon atoms which may have a substituent. Further, at least one of R ¹ , R ² , R ³ and R ⁴ has at least one group selected from the group consisting of a hydroxyl group, a mercapto group, a carboxyl group, a phosphonooxy group and a sulfo group. In the formula [II], R ⁵ , R ⁶ , R ⁷ and R ⁸ each represents a hydrogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms which may have a substituent. And at least one of R ⁵ , R ⁶ , R ⁷ and R ⁸ is a hydroxyl group, a mercapto group, a carboxyl group,
An alkyl group having at least one group selected from the group consisting of a phosphonooxy group and a sulfo group, at least one of which is a hydrogen atom.

As a result of intensive studies, the present inventors have for the first time found that the compound represented by the above formula [I] or formula [II] acts well as an electron transfer substance in electrochemical measurement. Furthermore, the compound represented by the above formula [I] or formula [II] is selected from the group consisting of a hydroxyl group, a mercapto group, a carboxyl group, a phosphonooxy group and a sulfo group for at least one of R ^{1 to} R ⁸ . When it has one or more groups, the resulting compound has a low vapor pressure and high solubility in water, so that it can be easily dried (freeze drying, reduced pressure drying, air drying, etc.) and is stable in a dry state, It was also found that when added, the compound immediately dissolved and showed a good response. That is, this compound can be suitably used for disposable purposes by impregnating and drying porous materials such as various filter papers; it is possible to reduce interference in specimen samples such as blood and urine; At the same time, it was found that the transfer reaction is not largely hindered and a good current response is exhibited. Therefore, according to the measuring method of the present invention in which the compound of the above formula [I] or [II] is used as an electron carrier, the measuring sensitivity (responsiveness)
It can be used favorably even for disposable applications, and even when the sample contains interfering substances such as blood and urine, stable and reproducible measurement should always be performed. Is possible.

In the compound of the above formula [I], R ¹ , R
^As R ² , R ³ and R ⁴ , all known groups satisfying the above-mentioned requirements can be used, and specifically, methyl group, ethyl group, propyl group, hydroxymethyl group, 1-hydroxyethyl group, 2- Hydroxyethyl group, 1,2-dihydroxyethyl group, 1-hydroxypropyl group, 2-hydroxypropyl group, 3-hydroxypropyl group, 2-
(1-hydroxy) propyl group, 2,3-dihydroxypropyl group, 2- (1,3-dihydroxy) propyl group, 3-hydroxy-2-methylpropyl group, mercaptomethyl group, carboxymethyl group, phosphonooxymethyl group A group and a sulfomethyl group are preferably exemplified. Among them, hydroxymethyl group, 1-hydroxyethyl group, 2
-Hydroxyethyl group, 1-hydroxypropyl group, 2
-Hydroxypropyl group, 3-hydroxypropyl group,
A 2- (1-hydroxy) propyl group, a 2,3-dihydroxypropyl group, a carboxymethyl group and the like are particularly preferably exemplified. Further, R ¹ , R ² , R ³ and R ⁴ may be the same or different from each other.

In the compound of the above formula [II], R ⁵ , R
All known groups satisfying the above requirements can be used as ⁶ , R ⁷ and R ⁸ , and specifically, a hydrogen atom, a methyl group, an ethyl group, a propyl group, a hydroxymethyl group, 1
-Hydroxyethyl group, 2-hydroxyethyl group, 1,
2-dihydroxyethyl group, 1-hydroxypropyl group, 2-hydroxypropyl group, 3-hydroxypropyl group, 2- (1-hydroxy) propyl group, 2,3-dihydroxypropyl group, 2- (1,3-dihydroxy group )
Propyl group, 3-hydroxy-2-methylpropyl group,
Suitable examples include a mercaptomethyl group, a carboxymethyl group, a phosphonooxymethyl group and a sulfomethyl group. Among them, hydrogen atom, hydroxymethyl group, 1-hydroxyethyl group, 2-hydroxyethyl group, 1-hydroxypropyl group, 2-hydroxypropyl group, 3-hydroxypropyl group, 2- (1-hydroxy) propyl group, 2 , 3-dihydroxypropyl group and carboxymethyl group are particularly preferably exemplified. In addition, R ⁵ , R ⁶ , R
⁷ and R ⁸ may be the same or different, and at least one is an alkyl group and at least one is a hydrogen atom.

In the measuring method of the present invention, specific examples of the compound represented by the formula [I] or the formula [II] include the following compounds (1) to (27).

[0033]

[Chemical 11]

[0034]

[Chemical 12]

[0035]

[Chemical 13]

In particular, among the N, N,
N ', N'-tetrakis- (2'-hydroxyethyl)
-P-phenylenediamine (THEPD), N, N, N ', N'-tetrakiscarboxymethyl-p-phenylenediamine (TCPD) shown in (2), N, N, N', shown in (3) N'-tetrakis- (2 ', 3'
-Dihydroxypropyl) -p-phenylenediamine (TDHPD), shown in (11) N-2'-hydroxyethyl-N, N ', N'-triethyl-p-phenylenediamine (HTEPD), (12). N,
N-bis- (2'-hydroxyethyl) -N, N'-diethyl-p-phenylenediamine (N, N-BHDP
D) and N, N'-bis- (2'-hydroxyethyl) -N, N'-diethyl-p-phenylenediamine (N, N'-BHDPD) shown in (13) are preferably used. . Further, N, N'-bis- shown in (21)
(2'-hydroxyethyl) -p-phenylenediamine (N, N'-BHPD), and N shown in (24)
2'-Hydroxypropyl-p-phenylenediamine (HPPD) is also preferably used.

Further, the compound represented by the following formula can also be preferably used.

[Chemical 14]

The electron transfer material represented by the formula [I] or the formula [II] can be synthesized by various known methods. One example of the synthesis method, and p- ^{phenylenediamine, R 1, R 2, R} 3, R 4, R 5, R 6, R
^{An example is} a method in which a halide of a compound corresponding to ⁷ or R ⁸ is dissolved in a solvent such as alcohol and refluxed under heating to react, and the obtained target product is separated and purified by filtration or column chromatography. To be done. Also, R ¹
In the case of synthesizing a compound having a hydroxyl group at the 2-position carbon of R ⁸ to R ⁸ , p-phenylenediamine and an ethylene oxide derivative are dissolved in a solvent and reacted under reflux with heating to obtain the desired product. Examples of the method include separation and purification by filtration, column chromatography and the like.

In the measuring method of the present invention, a salt of the electron mediator represented by the formula [I] or the formula [II] can be preferably used. Specific examples thereof include hydrochloride, nitrate, sulfate, perchlorate, p-toluenesulfonate, and the like.
Further, when the electron carrier represented by the formula [I] or [II] has one or more groups selected from the group consisting of a carboxyl group, a phosphonooxy group and a sulfo group, its sodium salt, potassium salt and the like are also preferable. Used for. Among them, especially the THEPD hydrochloride, TCPD hydrochloride, TD
HPD hydrochloride and the like are preferably used.

The electron mediator represented by the formula [I] or [II] is redox-oxidized by an oxidoreductase or an electrode to be an electron carrier substance in an oxidized state or an electron carrier substance in a reduced state. When an electron transfer substance species in an oxidized state is generated by the redox reaction by a redox enzyme, the generated electron carrier substance in an oxidized state returns to a reduced state by an electrode reaction, and at that time, a reduction current is signaled at the electrode. Is detected (FIG. 1 (a)). On the contrary, when an electron transfer substance species in a reduced state is produced by the redox reaction by an oxidoreductase, the produced electron carrier substance in a reduced state returns to an oxidized state by an electrode reaction, and at that time, the electrode is oxidized. Current is detected as a signal (Fig. 1
(B)). That is, the measurement method of the present invention has the same effect in both FIG. 1 (a) and FIG. 1 (b) regardless of whether the reaction starts from an electron transfer substance species in an oxidized state or a reduced state. . Therefore, the electron mediator represented by the formula [I] or [II] includes an electron mediator species in which the electron mediator is in an oxidized state or an electron mediator species in which the electron mediator is in a reduced state.

In the measuring method of the present invention, the amount of the electron transfer substance represented by the formula [I] or the formula [II] used is not particularly limited, and it depends on the electrode, the oxidoreductase, and the substance to be measured. It may be appropriately determined according to the type and amount.

In the measuring method of the present invention, the electron transfer substance represented by the formula [I] or the formula [II] can be used in various states. For example, a method of directly dissolving and mixing in a measuring system, a method of drying or freeze-drying and dissolving at the time of measurement, a method of existing in an electrode paste, and a method of dissolving in a buffer solution and impregnating filter paper Examples thereof include a method of drying, drying, and dissolving with a sample solution or the like at the time of measurement. In any case, due to the excellent water solubility of this electron transfer material, the drying property and the stability after drying, the current response, and the low influence of interfering substances in the sample, It is possible to make chemical measurements.

The measuring method of the present invention is an electrochemical measurement using at least one redox enzyme. The oxidoreductase used in the present invention is not particularly limited, and various oxidoreductases are selected and used according to the kind of the target substance to be measured. Specifically, glucose oxidase, peroxidase such as horseradish peroxidase, galactose oxidase, amino acid oxidase, NAD (P) H oxidase, lactate oxidase,
Bilvic acid oxidase, alcohol oxidase, urate oxidase, cholesterol oxidase, choline oxidase, xanthine oxidase, nucleoside oxidase, ascorbate oxidase, diaphorase and the like are preferably used. Above all, glucose oxidase for measuring blood glucose level, horseradish peroxidase as a labeling enzyme used in the specific binding analysis method, and the like are preferably used. The amount of oxidoreductase used is not particularly limited, and may be appropriately determined depending on the type of electrode, electron transfer substance, type and amount of the measurement target, and the like.

Examples of the electrode used in the measuring method of the present invention include various electrodes usually used in electrochemical measurement such as enzyme electrode method and specific binding analysis method. Specifically, a glassy carbon electrode, a pyrolytic graphite electrode, a carbon paste electrode, a carbon electrode such as a carbon fiber electrode, a platinum electrode,
Examples thereof include conductive transparent electrodes such as gold electrodes and ITO electrodes, oxide electrodes such as TiO _2, and modified electrodes modified with a functional compound. In the measuring method of the present invention, an electrode pattern-printed on the substrate by screen printing or the like using a conductive carbon ink is particularly preferably used because of its wide applicability. In addition, there is no particular limitation on the shape (flat electrode, porous electrode, etc.) or size of the electrode, and the type and amount of the substance to be measured, the amount and characteristics of the measurement sample (such as viscosity), the measurement application and conditions, etc. It may be appropriately determined depending on the situation.

Basically, the measuring method of the present invention carried out in a system having such a redox enzyme, an electron transfer substance and an electrode is carried out by various known electrochemical methods such as an enzyme electrode method and a specific binding analysis method. It is suitable for practical measurement. Specifically, it can be used for an amperometric enzyme sensor, an immunosensor, a nucleic acid sensor, and the like. More specifically, measurement of blood glucose, cholesterol, uric acid, lactic acid, etc. using an enzyme sensor, human chorionic gonadotropin (hCG), luteinizing hormone (LH), human placental lactogen using a specific binding assay method. (HPL), measurement of various hormones such as estrogen, HBs antibody, HBs antigen, HBc antibody, HB
Measurement of various virus-related antigens / antibodies such as e-antigen, HCV antibody, HIV antibody, specific I corresponding to various allergens
Examples of use include measurement of gE antibody, measurement of tumor markers such as AFP and CEA, measurement of various blood proteins such as β ₂ m and CRP, drugs and their metabolites, measurement of virus- and tumor-related polynucleotide sequences Is preferably exemplified as.

In the measuring method of the present invention, various electrochemical measurements such as the well-known enzyme electrode method and specific binding analysis method can be carried out in the same manner as the ordinary measurement. The measuring method of the present invention makes use of the excellent water-solubility, drying property, drying stability and the like of the electron carrier represented by the formula [I] or [II] to make an electron carrier on a cellulose filter paper or a glass filter paper. Disposable measurement in which an electron transfer substance is impregnated and dried, and this electron transfer substance is dissolved by the sample solution at the time of measurement, diffused and moved in the matrix, and acts as a substance that transfers an electron that is a signal. The device is particularly preferably used.

FIG. 2 shows a disposable type specific binding reaction measuring apparatus utilizing the MEDIA method as an example for carrying out the measuring method of the present invention. A detailed description of the analysis method in this measuring device is described in Japanese Patent Application Laid-Open No. 5-264552.

The measuring apparatus shown in FIG. 2 includes an upper cover 10, a filter 12, a labeled antibody impregnated portion 14, an electron transfer substance impregnated portion 16, a communicating member 18, an electrode portion 20, a matrix 22, and an absorbing portion 24 from the top. And a lower substrate 26, and as shown in FIG. 3, each member is stacked and assembled in the above order. Further, the filter 12, the labeled antibody impregnated portion 14, the electron transfer substance impregnated portion 16 and the communication member 18 constitute a sample introduction portion 38. Incidentally, the measuring device utilizing the measuring method of the present invention is not limited to those having all of these members,
Various configurations described in the above-mentioned JP-A-5-264552 can also be used.

The upper cover 10 is made of various synthetic resins such as acrylic, polyvinyl chloride, glass, polystyrene, ABS resin and epoxy resin.
A sample introduction port 10a for injecting a sample is formed in the central portion. The filter 12 removes a solid substance (interfering substance) unnecessary for the measurement contained in the sample and allows the sample to be uniformly introduced, and is usually formed of a woven fabric, a non-woven fabric, or the like.

The labeled antibody impregnated portion 14 is made of glass fiber filter paper,
It is obtained by impregnating a cellulose fiber filter paper, a non-woven fabric or the like with an oxidoreductase-labeled antibody, which is one of the constituent elements of the present invention, and drying. That is, when the sample passes through the labeled antibody impregnated portion 14, the oxidoreductase labeled antibody is eluted and the analyte and the oxidoreductase labeled antibody are mixed or the reaction is started.

The electron transfer material impregnated portion 16 is formed by impregnating and drying glass fiber filter paper, cellulose fiber filter paper, non-woven fabric and the like with the electron transfer material which is a characteristic element of the present invention. When the sample passes through the electron transfer substance impregnated portion 16, the electron transfer substance is eluted and mixed with the sample. In addition,
In the measuring method of the present invention, since the compound represented by the formula [I] or the formula [II] is used as the electron carrier, the electron carrier has good drying property and dry stability,
Moreover, the rapid elution and mixing are as described above. In the apparatus of the illustrated example, as a preferred embodiment, a water impervious seal portion 16a is formed in the central portion of the upper surface of the electron transfer material impregnated portion 16. This seal portion 16a
By having the above, it is possible to change the flow of the sample in the vertical direction up to the labeled antibody-impregnated portion 14 to the horizontal direction, and it is possible to lengthen the flow time of the sample and secure a sufficient reaction time. At the same time, the reaction efficiency increases due to the mixing effect, and more accurate measurement can be performed. The material and method for forming the seal portion 16a are not particularly limited,
Various water-impermeable materials such as vinyl chloride and cellulose acetate may be attached to the central portion of the electron transfer substance impregnated portion 16 by using an adhesive such as an acrylic adhesive.

In the measuring device of the illustrated example, the labeled antibody impregnated portion 14 holding the oxidoreductase labeled antibody and the electron transfer substance impregnated portion 16 holding the electron transfer substance are separately formed. In addition to this configuration, the measuring device used in the invention may have a reagent impregnation unit that holds both the oxidoreductase-labeled antibody and the electron transfer substance. Further, in the measuring apparatus of the illustrated example, the electron transfer substance is held in the electron transfer substance impregnated portion 16. However, as described above, the electron transfer substance represented by the formula [I] or the formula [II] is good. Since it has excellent drying property and water solubility, other than this, for example, the matrix 22 and the absorbing portion 24 described later are used.
Good measurement is possible even when held in the same manner.

The communicating member 18 is the impregnated portion 16 of the electron transmitting substance.
The sample or the like that has passed through is introduced into the matrix 22 through a predetermined position of the electrode portion 20, and is formed of glass fiber filter paper, cellulose fiber filter paper, or the like. Such a communication member 18 is inserted and arranged in a through hole 30 of the electrode portion 20 described later.

The electrode section 20 has an electrode for measurement which is one of the constituent elements of the present invention, and in the apparatus of the illustrated example, as shown in FIGS. 2 and 4, an insulating substrate. A circular through hole 30 is formed in the substrate 28, and a reference electrode (counter electrode) 32 and its terminal 3 are formed around the through hole 30 on the upper surface of the substrate 28.
2a, a working electrode 34 as a detection electrode and its terminal 34a are formed around the through hole 30 on the lower surface of the substrate 28. At the time of measurement, the terminals of both electrodes are connected to an external measuring device. In addition, as shown by the hatched lines in FIG. 4, both the upper and lower surfaces of the substrate 28 of the electrode portion 20 are insulated from the contact portions of the reference electrode 32 and the working electrode 34 with the electron transfer material impregnated portion 16 and the matrix 22 described later. A layer 36 is formed.

The working electrode 34, which is a detection electrode, is formed of the above-mentioned various materials. The reference electrode 32 is a silver / silver chloride electrode or the like. The substrate 28 is formed of various known insulating materials such as PET (polyethylene terephthalate), polyvinyl chloride, polyimide and polyester. The insulating layer 36 is formed of various known insulating ink materials such as acrylic resin and polyester. In addition, in the apparatus of the illustrated example, the working electrode 34, the reference electrode 32, and the insulating layer 36.
Both may be formed by a known film forming technique such as a thick film forming technique such as screen printing or a doctor knife, or a thin film forming technique such as sputtering or CVD.

The matrix 22 is a field for distributing an enzyme-labeled substance generated by the specific binding between the analyte in the liquid sample and the oxidoreductase-labeled antibody, and the distance distribution of this labeled substance from the working electrode 34 is It is measured as an electric current value through the aforementioned electron transfer substance. The matrix 22 is formed by, for example, insolubilizing and retaining and drying an antibody, an antigen, a nucleic acid or the like for a specific binding reaction on a porous film such as a membrane. When the sample flows into this matrix 22, a specific binding reaction between the insolubilized antibody and the analyte occurs, and the distribution of the enzyme-labeled product changes, the electron mediator species generated by the enzyme reaction diffuses and moves, and the working electrode. It is detected by 34 as an electrochemical modulation corresponding to the reaction. Also,
The surplus sample passes through the matrix 22 and is absorbed by the absorption section 24 described later.

As described above, the absorbing portion 24 is formed by the matrix 2
It absorbs the excess sample that has passed through 2, and is formed of, for example, a cellulose filter paper such as a chromatographic filter paper or a super absorbent polymer. In the apparatus of the illustrated example, the absorbing section 24 also functions as a substrate impregnating section for enzyme reaction,
The above-mentioned material holds an enzyme reaction substrate such as hydrogen peroxide-urea dried product. In the illustrated apparatus,
As a preferred embodiment, a watertight seal portion 24a is formed in the central portion of the upper surface of the absorbing portion 24. As with the above-mentioned seal portion 16a, by having the seal portion 24a,
The vertical flow of the sample can be changed to the horizontal direction, increasing the sample flow time, improving the efficiency of the specific binding reaction, and clarifying the distribution of the labeled substance in the matrix for more accurate measurement. Various measurements can be realized. The seal portion 24a is formed in the same manner as the seal portion 16a described above.

The measuring apparatus of the illustrated example is constructed by stacking these members on the lower substrate 26 in the order shown in FIGS. Like the upper cover 10, the lower substrate 26 is formed of various resin materials such as acrylic.

In such a measuring apparatus, the sample is injected from the sample introduction port 10a formed in the upper cover 10. The sample injected from the sample introduction port 10 a passes through the filter 12 to remove foreign matters and the like, and flows into the labeled antibody impregnated portion 14. In the labeled antibody impregnation section 14, the oxidoreductase-labeled antibody dried and held by the inflow of the sample is eluted and mixed with the sample, or the specific binding reaction between the analyte and the oxidoreductase-labeled antibody starts. To be done.

The sample is then impregnated with the electron transfer material 16
Flow into. As described above, since the electron transfer substance is dried and held in the electron transfer substance impregnated portion 16, the electron transfer substance is eluted and mixed with the sample by the inflow of the sample. In the apparatus of the illustrated example, since the seal part 16a is formed on the upper surface of the electron transfer substance impregnated part 16, the flow direction of the sample is changed from the vertical direction to the horizontal direction, and a sufficient reaction time can be secured. Is as described above.

The sample that has passed through the electron transfer material impregnated portion 16 passes through the communication member 18, passes through the through hole 30 of the electrode portion 20, and flows into the matrix 22. When the sample flows into the matrix 22, the distribution of the oxidoreductase-labeled body in the matrix 22 is determined by the specific binding between the analyte and the insolubilized antibody in the liquid sample, and the electrode reaction generated by the enzymatic reaction of the oxidoreductase-labeled body. Possible electron mediator species reach the working electrode 34 by diffusion. That is, the distribution of the analyte in the radial direction of the working electrode 34 is detected by the working electrode 34 as an electrochemical modulation corresponding to the enzymatic reaction. The excess sample passes through the matrix 22 and is absorbed by the absorbing section 24.

Therefore, according to the above-mentioned measuring device, as a result of the specific binding reaction between the analyte and the insolubilized antibody in the matrix 22, the enzyme generated by the specific binding reaction depending on the amount of the analyte contained in the sample. The distribution of the labeled body in the matrix 22 is determined, and the working electrode 3 is generated through the diffusion transfer of the electron mediator species generated by the enzyme labeled body.
Electrons are transferred and received at 4, and electron transfer due to electrode reaction is detected as electrochemical modulation. As a result, the distribution of the specific binding reaction in the radial direction of the matrix 22 can be measured to measure the analyte in the sample.

The electrochemical measurement method using the MEDIA method of the present invention and the novel p-phenylenediamine compound have been described above in detail, but the present invention is not limited to the above examples and deviates from the gist of the present invention. In the range that does not
Of course, various changes and improvements may be made.

[0064]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to specific examples of the present invention. The present invention is not limited to the examples below.

<Synthesis Example 1> (corresponding to the prior art) Synthesis of N, N, N ', N'-tetraethyl-p-phenylenediamine dihydrochloride (TEPD.2HCl) p-phenylenediamine (1 g), iodide Ethyl (1
0.7 g) and sodium carbonate (4.4 g) were dissolved in methanol (10 mL), and the mixture was refluxed under heating overnight. After cooling the reaction mixture to room temperature, the solvent was distilled off under reduced pressure, acetone was added to the residue, and the insoluble material was filtered off. The solvent was distilled off from the obtained filtrate under reduced pressure, and the residue was recrystallized with a mixed solution of ethyl acetate-ether to give 6.8 g of a quaternary ammonium salt.
Got

Then, 6.8 g of this quaternary ammonium salt
And 12.5 g of sodium thiosulfate pentahydrate were dissolved in 50 mL of dimethylformamide, and the mixture was refluxed under heating overnight. After cooling the reaction mixture to room temperature, distilled water was added and extraction was performed with toluene. The extracted layer was washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain 1.0 g of TEPD. . In addition, this TEPD has ethanol 10
mL was added, 5 mL of 4N hydrochloric acid / ethyl acetate was added dropwise under ice cooling, and the mixture was further stirred at 5 ° C. for 1 hour. The generated precipitate was filtered, and the obtained precipitate was washed with ethanol and ether to obtain 1.3 g of TEPD.2HCl.

From the results of ¹ HNMR, IR (infrared absorption analysis) and MS (mass spectrum) analysis shown below,
It was confirmed to be the desired TEP D.2HCl. ¹ HNMR: δ 1.2 (12H, t, J = 7Hz)
3.7 (8H, q, J = 7Hz) 7.9 (4H, S) IR (cm ^-1 ): 715, 840, 1020, 1276, ¹
516, 2420 MS: M ⁺ -2HCl ⁺ 1221

<Synthesis Example 2> (corresponding to the present invention) N, N, N ', N'-tetrakiscarboxymethyl-p
-Synthesis of phenylenediamine (TCPD) p-phenylenediamine (1 g), chloroacetic acid (3.5
g), sodium hydroxide (3.1 g) and potassium iodide (460 mg) were dissolved in distilled water (46 mL) and refluxed under heating for 1 hour. After cooling the reaction mixture to room temperature,
3.7 mL of concentrated hydrochloric acid was added dropwise under ice cooling, and the generated precipitate was filtered. The precipitate was recrystallized with a mixed solution of methanol-ethanol to obtain 700 mg of TCPD.

From the ¹ H NMR, IR and MS analysis results shown below, it was confirmed that the desired TCPD was obtained. ¹ HNMR: δ 3.9 (8H, S) 6.3 (4H,
S) 11.2 (4H, S) IR (cm ^-1 ): 700, 820, 1030, 1514,
1720, 2700, 3300 MS: M ⁺ +1 341

<Synthesis Example 3> (Corresponding to the present invention) N, N, N ', N'-tetrakis- (2'-hydroxyethyl) -p-phenylenediamine dihydrochloride (THEP
Synthesis of D.2HCl p-phenylenediamine (1 g), 2-chloroethanol (3.0 g), and sodium hydroxide (1.5 g)
Was dissolved in 6 mL of distilled water and refluxed under heating for 2 hours. After cooling the reaction mixture to room temperature, the solvent was distilled off under reduced pressure,
Ethanol was added to the residue and the insoluble material was filtered off. The solvent was distilled off from the obtained filtrate under reduced pressure, and the residue was chloroform-
Purification by silica gel column chromatography using a methanol mixed solution as an eluent gave 1.5 g of THEPD.

Next, 1.5 g of this THEPD was dissolved in 10 mL of ethanol, 5 mL of 4N hydrochloric acid / ethyl acetate was added dropwise to this solution under ice cooling, and the mixture was further stirred at 5 ° C. for 1 hour.
The generated precipitate was filtered and the obtained precipitate was washed with ethanol to obtain 1.1 g of THEPD.2HCl.

From the results of ¹ H NMR, IR and MS analysis shown below, it was confirmed that the desired compound was THEPD.2HCl. ¹ HNMR: δ 3.5 (16H, brS) 7.0
(4H, m) 7.1 (4H, S) IR (cm ⁻¹ ): 720,890,1020,1210,
1390, 1500, 2700, 3300 MS: M ⁺ -2HCl + 1 285

<Synthesis Example 4> (corresponding to the present invention) N, N, N ', N'-tetrakis- (2', 3'-dihydroxypropyl) -p-phenylenediamine dihydrochloride (TDHPD.2HCl) Synthesis p-phenylenediamine (1.0 g), 3-chloro-
1,2-Propanediol (4.6 g) and sodium hydroxide (1.6 g) were dissolved in 30 mL of distilled water and refluxed under heating for 2 hours. After cooling the reaction mixture to room temperature,
The solvent was distilled off under reduced pressure, ethanol was added to the residue, and the insoluble material was filtered off. The solvent was distilled off from the filtrate obtained under reduced pressure, and the residue was purified by silica gel column chromatography using chloroform-methanol as an eluent to obtain TDH.
1.1 g of PD was obtained.

Next, 1.1 g of this TDHPD was dissolved in 10 mL of ethanol, 5 mL of 4N hydrochloric acid / ethyl acetate was added dropwise to this solution under ice cooling, and the mixture was further stirred at 5 ° C. for 1 hour.
Then, the produced precipitate was filtered, and the obtained precipitate was washed with ethanol to obtain 820 mg of TDHPD · 2HCl.

From the analysis results by ¹ HNMR, IR and MS shown below, it was confirmed that the target was TDHPD · 2HCl. ¹ HNMR: δ 3.7 (20 H, m) 7.1 (4
H, S) IR (cm ^-1 ): 720,870,1040,1260,
1370, 1520, 3300 MS: M ⁺ -2HCl + 1 405.

<Synthesis Example 5> (Corresponding to the present invention) N-2'-hydroxyethyl-N, N ', N'-triethyl-p-phenylenediamine dihydrochloride (HTEPD.
2HCl) Synthesis of p-phenylenediamine (5 g) in methanol (1
Potassium carbonate (12.8 g) was added to (00 mL), and ethyl iodide (14.4 g) was added dropwise to this solution at room temperature over 10 minutes. The reaction solution was stirred at 50 ° C., and disappearance of the raw materials was confirmed by thin layer chromatography (chloroform: methanol = 10:
After confirmation in 1), concentration under reduced pressure was performed. Next, the concentrate was dissolved in n-hexane: ethyl acetate = 1: 1, the precipitated crystals were filtered off, and the filtrate was concentrated under reduced pressure. Then, the residue was purified by silica gel column chromatography (n-hexane: ethyl acetate = 1: 1) to obtain 1.5 g of N, N ′, N′-triethyl-p-phenylenediamine.

Then, N, N ', N'-triethyl-p
-1.5 g of phenylenediamine was added to n-butanol (20
2-chloroethanol (4.4 g) was added dropwise to the solution containing sodium hydroxide (2.2 g) at room temperature over 5 minutes, and the mixture was refluxed for 1 hour. After confirming the disappearance of the raw materials by thin layer chromatography (n-hexane: ethyl acetate = 1: 1), the reaction solution was cooled to room temperature and the precipitated crystals were separated by filtration. After that, the residue was purified by silica gel column chromatography (n-hexane: ethyl acetate = 1: 1) to obtain HTEPD. Next, HTEPD was dissolved in 15 mL of ethanol, 4N hydrochloric acid / ethyl acetate (2.4 mL) was added dropwise under ice-cooling, and the generated crystals were washed with ethanol to obtain 1.0 g of HTEPD.2HCl.

From the analysis results by ¹ HNMR, IR and MS shown below, it was confirmed that the target was HTPD.2HCl. ¹ HNMR: δ 1.1 (9H, t, J = 7Hz)
3.5 (10H, m) 7.4 (4H, m) IR (cm ^-1 ): 700, 830, 1060, 1370,
1450, 1500, 2350, 2900, 3350 MS: M ⁺ -2HCl + 1 237.

<Synthesis Example 6> (corresponding to the present invention) N, N-bis- (2'-hydroxyethyl) -N ',
Synthesis of N′-diethyl-p-phenylenediamine dihydrochloride (N, N-BHDPD · 2HCl) N, N-diethyl-p-phenylenediamine (5 g) in n-butanol solution (30 mL) was added with sodium hydroxide. 3.7 g was added, and further 2-chloroethanol (7.3
g) was added dropwise at room temperature in 10 minutes. The solution was refluxed for 4 hours, and after disappearance of the raw materials was confirmed by thin layer chromatography (chloroform: methanol = 10: 1), the solution was cooled to room temperature. The precipitated crystals were filtered off, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform: methanol = 10: 1) to obtain N, N-BHD.
I got PD.

Next, N, N-BHDPD was dissolved in ethanol (20 mL), 4N hydrochloric acid / ethyl acetate (2.74 mL) was added dropwise under ice cooling, and the resulting precipitated crystals were washed with ethanol to obtain N, N-BHDPD. 1.22 g of N-BHDPD.2HCl
Obtained.

From the analysis results by ¹ HNMR, IR and MS shown below, the desired N, N-BHDPD
It was confirmed to be HCl. ¹ HNMR: δ 1.0 (6H, t, J = 7Hz)
3.5 (12H, m) 6.7 (2H, d, J = 9Hz) 7.5 (2H, d,
J = 9 Hz) IR (cm ^-1 ): 700, 830, 1050, 1380,
1450, 1500, 2480, 2900, 3300 MS: M ⁺ -2HCl + 1 253

<Synthesis Example 7> (corresponding to the present invention) Synthesis of N, N'-bis- (2'-hydroxyethyl) -p-phenylenediamine (N, N'-BHPD) p-phenylenediamine (5 g) Ethanol solution (1
Sodium hydroxide (4.4 g) was added to (00 mL) and the mixture was heated to 55 ° C. Further, 8.9 g of 2-chloroethanol was added dropwise to this solution in 10 minutes. After refluxing overnight, the disappearance of the raw materials was confirmed by thin layer chromatography (chloroform: methanol = 10: 1), and then the mixture was cooled to room temperature. The precipitated crystals were separated by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform: methanol = 10: 1) to obtain 5.0 g of N, N′-BHPD.

<Synthesis Example 8> (corresponding to the present invention) N, N'-bis- (2'-hydroxyethyl) -N,
Synthesis of N'-diethyl-p-phenylenediamine dihydrochloride (N, N'-BHDPD.2HCl) N, N'-BHPD 4.5 g ethanol solution (100
(mL) to potassium carbonate 8.9 g and ethyl iodide 9.5.
g was added and refluxed overnight. After confirming the disappearance of the raw materials by thin layer chromatography (chloroform: methanol = 5: 1), the mixture was cooled to room temperature. The precipitated crystals were separated by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate 100%) to give N, N'-B.
233 mg of HDPD was obtained. Then, N, N'-BHD
After PD was dissolved in 5 mL of ethanol, 10% hydrogen chloride / methanol was added dropwise under ice cooling, and the generated crystals were separated by filtration.
266 mg of N, N′-BHDPD · 2HCl was obtained.

From the results of ¹ H NMR, IR and MS analysis shown below, the desired N, N'-BHDPD
It was confirmed to be 2HCl. ¹ HNMR: δ 1.1 (6H, t, J = 7Hz)
3.5 (12H, m) 7.4 (4H, s) IR (cm ^-1 ): 670, 830, 1070, 1500,
2500, 2900, 3350 MS: M ⁺ -2HCl + 1 253

<Synthesis Example 9> (Corresponding to the present invention) Synthesis of each substitution product on N atom by reaction of p-phenylenediamine and propylene oxide p-phenylenediamine (5 g) in methanol solution (1
Sodium hydroxide (1.3 g) was added to (00 mL), and propylene oxide (1.1 g) was added dropwise. 1 at 30 ° C
After stirring the mixture overnight, it was concentrated under reduced pressure. Then, chloroform was added to the concentrate to obtain 7 g of chloroform extract.
Then, using acetonitrile: 50 mM ammonium acetate = 15.6: 84.4 as an eluent, an ODS column (YMC-Pack ODS, 20 mmφ × 250 m).
m, manufactured by YMC), HPLC of chloroform extract
The collection was done. At this time, the flow rate should be 15 mL / min and 2
The detection wavelength was 54 nm. By this fractionation, 1, 2, and 3 substituents on the p-phenylenediamine N atom were separated. From the results of ¹ H NMR and MS analysis shown below, the product was confirmed to be the desired product. The number of substitutions was determined by mass spectrum. 1-Substituted ¹ HNMR: δ 1.1 (3H, d, J =
6 Hz) 2.8 (2H, m) 3.8 (1H, m)
4.9 (3H, brs) 6.4 (4H, m) MS: 167 (M + 1) 2-substituted compound MS: 225 (M + 1) 3-substituted compound MS: 283 (M + 1) Also, from the results of analysis by ¹ HNMR and MS. It was confirmed that the 1-substituted product was N-2'-hydroxypropyl-p-phenylenediamine (HPPD).

Experimental Example 1 p-phenylenediamine (PPD), N, N, N ',
N'-tetramethyl-p-phenylenediamine (TMP
D), TEPD, TCPD, THEPD and TDHP
Vapor pressure measurement of D PPD (manufactured by Wako Pure Chemical Industries, Ltd.), TMPD (manufactured by Tokyo Chemical Industry Co., Ltd.), and TEPD and T obtained during the above-mentioned Synthesis Examples 1 to 4.
Vapor pressure was measured for CPD, THEPD and TDHPD. Solid sample at constant temperature (PPD, TMPD, T
A carrier gas (nitrogen gas) is caused to flow so as to come into contact with EPD, TCPD, THEPD and TDHPD, and the vapor of the sample is saturated to measure the vapor density (evaporation amount / volume),
The vapor pressure of each compound was calculated by assuming that the vapor follows the ideal gas law (gas flow method). The evaporation rate (weight reduction rate) of the sample was measured using an electric balance, and the carrier gas flow rate was measured using a flow meter to determine the vapor density of the sample. The following equation was used to obtain the vapor pressure from the vapor density. P = (k / v) Vπ / M In the above formula, P: vapor pressure [mmHg], k: evaporation rate [mg /
min], v: carrier gas flow rate [mL / min], k / v: vapor density [mg / mL], M: molecular weight of sample, π: system pressure [mmHg],
V: The molar volume [L / mol] of the carrier gas.

FIG. 5 shows TEPD and T at each temperature.
The vapor pressure curve of HEPD is shown as an example. Also, 25
The vapor pressure of each compound at ° C is shown in Fig. 24. As shown in FIGS. 5 and 24, TCPD, THEPD and TDHP are compared to PPD, TMPD and TEPD.
The vapor pressure of D is low. Specifically, TPD at 25 ° C,
The vapor pressures of TCPD, THEPD and TDHPD are 4.2 × 10 ⁻⁴ mmHg, 8.7 × 10 ⁻⁹ mmHg and 1.3, respectively.
TCP with x10 ^-6 mmHg and 1.0 x10 ^-9 mmHg
The vapor pressures of D, THEPD and TDHPD are about 1/50000 and 1/3, respectively, compared to the vapor pressure of TEPD.
00, about 1 / 420,000. From this result, TC
PD, THEPD and TDHPD are PPD and TMPD
It can be seen that the composition is superior in drying property (freeze-drying, reduced-pressure drying, air-drying, etc.) and stability of the dried product, as compared with TPD.

Experimental Example 2 TEPD.2HCl, TCPD, THEPD.2HC
1, TDHPD.2HCl, HTEPD.2HCl,
N, N-BHDPD ・ 2HCl, N, N'-BHDPD
Cyclic voltammetry (CV) measurement of each substitution product on the N atom (hereinafter, 2HCl is omitted) generated by the reaction of 2HCl and p-phenylenediamine with propylene oxide. , TCPD, T
HEPD, TDHPD, HTEPD, N, N-BHDP
D, N, N'-BHDPD and each substitution product on the N atom produced by the reaction of p-phenylenediamine with propylene oxide were dissolved in 0.1 M phosphate buffer containing 0.1 M NaCl at pH 6.0. , 5.0 × 10 ⁻⁴ M solution was prepared, and their CVs were measured. Glassy carbon electrode (MF2012, manufactured by BIOANALYTICAL SYSTEMS) as a working electrode, Ag / AgCl electrode (MF2020, manufactured by BIOANALYTICAL SYSTEMS) as a reference electrode, platinum wire (made by BIOANALYTICAL SYSTEMS) as a counter electrode
Respectively, and these are used for potentiostat HA-
It was connected to 150 (manufactured by Hokuto Denko). Furthermore, a function generator HB-104 for setting the electrode potential
(Manufactured by Hokuto Denko) and a recorder were connected. The recorder was connected to a computer through the GPIB line to perform measurement and data processing. CV measurement is initial potential -50
0 mV (vs Ag / AgCl, the same applies below) to reversal potential +800 mV,
Alternatively, the initial potential was set to -200 mV to the reversal potential +300 mV, and the potential was swept at a rate of 50 mV / sec in each case.

The results of -500 mV to +800 mV of TEPD are shown in FIG. 6, the results of -200 mV to +300 mV are shown in FIG. 7, and similarly, the results of TCPD are shown in FIG. 8 and FIG.
The results of PD are shown in FIGS. 10 and 11, and the results of TDHPD are shown in FIGS. 12 and 13. Also, HTE
The results of PD are shown in FIG. 25 and FIG.
27 and 28 show the results of D, N, N'-BHDP.
29 and 30 show the results of D, and FIGS. 31 and 32 show the results of the monosubstituted product (HPPD) on the N atom produced by the reaction of p-phenylenediamine and propylene oxide.
FIG. 33 and FIG. 34 show the results for the same 2-substituted product, and FIGS. 35 and 36 show the results for the same 3-substituted product.

CVs of FIGS. 8 to 13 and 25 to 36
As shown in, TCP used in the measurement method of the present invention
D, THEPD, TDHPD, HTEPD, N, N-B
HDPD, N, N'-BHDPD and N produced by the reaction of p-phenylenediamine with propylene oxide
It is suggested that each substitution product on the atom is also a substance capable of being redox-reduced on the electrode similarly to the above-mentioned TEPD (FIGS. 6 and 7), and is suitable as an electron transfer substance in the electrochemical measurement method. It turns out that it is available. Furthermore, FIG. 9, FIG. 11, FIG. 13, FIG. 26, FIG. 28, FIG.
As shown in FIG. 2, FIG. 34, and FIG. 36, it is considered that according to these substances, the reversible redox reaction is carried out in the potential range of −200 mV to +300 mV.

Experimental Example 3 TEPD, TCPD, THEPD, TDHPD, HTE
PD, N, N-BHDPD, N, N'-BHPD, N,
Measurement of electron transfer activity of N'-BHDPD and each substitution product on N atom produced by reaction of propylene oxide with p-phenylenediamine 1 Glucose oxidase (GOD, manufactured by Behringer Mannheim) 0.1M NaCl containing pH 6.0.
Dissolved in 1M phosphate buffer, 0M and 4.0 × 10 ^-4
A solution of M was prepared. Further, glucose (manufactured by Junsei Chemical Co., Ltd.) was dissolved in the same buffer to prepare a 1.5 M solution. Furthermore, TEP D, TCP synthesized in Synthesis Examples 1 to 9
D, THEPD, TDHPD, HTEPD, N, N-B
HDPD, N, N'-BHPD, N, N'-BHDPD
And each compound of each substitution product on the N atom produced by the reaction of p-phenylenediamine and propylene oxide was dissolved in the same buffer solution to prepare a 5.0 × 10 ⁻³ M solution. Immediately before measurement, measure the pH of each of these solutions at the following volume ratios.
Mixing in 6.0 M 0.1 M phosphate buffer containing 0.1 M NaCl to prepare solutions represented by composition A and composition B shown below, and in the presence (composition A) or absence of GOD (composition A). The current value for composition B) was measured continuously.

Composition A ────────────────────────────────── GOD 4.0 × 10 ⁻⁴ M 100 μL gluco -S 1.5M 1.0mL compound solution 5.0 × 10 ^-3 M 1.0mL 0.1M NaCl-containing 0.1M phosphate buffer pH 6.0 7.9mL ──────────── ─────────────────────── Composition B ───────────────────────── ───────── GOD 0M 100 μL Glucose 1.5M 1.0mL Compound solution 5.0 × 10 ⁻³ M 1.0mL 0.1M NaCl-containing 0.1M phosphate buffer pH 6.0 7 .9mL ──────────────────────────────────

The measurement was carried out by the same device as in the above-mentioned Experimental Example 2, and a potential of +300 mV (vs Ag / AgCl) was applied to the working electrode during the measurement, in the presence of GOD (composition A) and in the absence of GOD (composition). The current value of B) was measured for 10 minutes after applying the potential.

FIG. 14 shows the result of the TEPD measurement (GOD-glucose-TEPD), and the result of the TCPD measurement (GOD-GOD-glucose-TEPD).
-Glucose-TCPD) is shown in FIG. 15, and the measurement result of THEPD (GOD-glucose-THEDP) is shown in FIG.
In addition, the measurement results of TDHPD (GOD-glucose-TD
HPD) is shown in FIG. 17, respectively. In addition, HTEPD
37 shows the measurement result of N, N-BHDPD, and FIG. 38 shows the measurement result of N, N'-BHDPD.
FIG. 40 shows the measurement results of the monosubstituted product (HPPD) on the N atom produced by the reaction of p-phenylenediamine and propylene oxide, and FIG.
Similarly, FIG. 42 shows the measurement results of the 3-substituted product.

As shown in FIG. 14 to FIG. 17 and FIG. 37 to FIG. 42, in any measurement by the substances, when GOD is contained (composition A), compared with the case where GOD is not contained (composition B). Since the oxidation current value increases, T of the present invention example
CPD, THEPD, TDHPD, HTEPD, N, N
-BHDPD, N, N'-BHDPD, and each substitution product on the N atom formed by the reaction of p-phenylenediamine and propylene oxide, are similar to the conventional TEPD, and reversible electron transfer between the GOD and the electrode. It is clear that it has a function as a substance. In addition, N, N'-BH
PD also had a function as a reversible electron transfer substance between the GOD and the electrode.

Experimental Example 4 TEPD, TCPD, THEPD, TDHPD, HTE
PD, N, N-BHDPD, N, N'-BHPD, N,
Measurement of electron transfer activity of N'-BHDPD and each substitution product on N atom produced by reaction of propylene oxide with p-phenylenediamine 2 Horseradish peroxidase (HRPO, manufactured by Toyobo Co., Ltd.)
Was dissolved in 0.1 M phosphate buffer containing 0.1 M NaCl at pH 6.0 to prepare solutions of 0 M and 5.0 × 10 ⁻⁷ M. Further, hydrogen peroxide (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in the same buffer solution to prepare a 1.0 × 10 ^-1 M solution.
Furthermore, the TEPD, TCPD synthesized in Synthesis Examples 1 to 9,
THEPD, TDHPD, HTEPD, N, N-BHD
4. PD, N, N'-BHPD, N, N'-BHDPD and each compound of each substitution product on the N atom produced by the reaction of p-phenylenediamine and propylene oxide were dissolved in the same buffer solution, and A 0 × 10 ⁻³ M solution was prepared.
Immediately before measurement, each of these solutions was adjusted to pH 6.0 with the following quantitative ratio.
In 0.1 M phosphate buffer containing 0.1 M NaCl to prepare solutions represented by the following composition A and composition B, in the presence (composition A) or absence (composition B) of HRPO. The current value at was continuously measured.

────────────────────────────────── HRPO 5.0 × 10 ^-7 M 100 μL hydrogen peroxide 1.0 × 10 ^-1 M 100 μL Compound solution 5.0 × 10 ^-3 M 1.0 mL 0.1 M NaCl-containing 0.1 M phosphate buffer pH 6.0 8.8 mL ─────────── ──────────────────────── Composition B ──────────────────────── ────────── HRPO 0M 100 μL Hydrogen peroxide 1.0 × 10 ⁻¹ M 100 μL Compound solution 5.0 × 10 ⁻³ M 1.0 mL 0.1M NaCl-containing 0.1M phosphate buffer pH 6.0 8.8mL ──────────────────────────────────

The measurement was carried out by the same device as in Experimental Example 2 described above. At the time of measurement, TEPD, THEPD and TDHP were used.
In the case of D, the potential of -80 mV (vs Ag / AgCl and so on)
TCPD, HTEPD, N, N-BHDPD, N, N '
In the case of each substitution product on the N atom produced by the reaction of -BHDPD and p-phenylenediamine with propylene oxide, a potential of -150 mV was applied to the working electrode, and H
The current values in the presence of RPO (composition A) and in the absence of HRPO (composition B) were measured for 10 minutes or 5 minutes after applying the potential.

FIG. 18 shows the measurement result of TEPD (HRPO-hydrogen peroxide-TEPD), and the measurement result of TCPD (HR
PO-hydrogen peroxide-TCPD) is shown in FIG.
20 shows the measurement result (HRPO-hydrogen peroxide-THEPD), and the measurement result of TDHPD (HRPO-hydrogen peroxide-TDHPD) is shown in FIG. Also, HT
The EPD measurement result is shown in FIG. 43, the N, N-BHDPD measurement result is shown in FIG. 44, the N, N′-BHDPD measurement result is shown in FIG. Monosubstituted on atom (HPP
FIG. 46 shows the measurement result of D), FIG. 47 shows the measurement result of the same 2-substituted product, and FIG. 48 shows the measurement result of the same 3-substituted product.

As shown in FIG. 18 to FIG. 21 and FIG. 43 to FIG. 48, in the case where any of the substances contains HRPO (composition A), the reduction current value is higher than that in the case where no substance is contained (composition B). Is increasing, and TCPD and THE of the example of the present invention are increasing.
PD, TDHPD, HTEPD, N, N-BHDPD,
N, N′-BHDPD and each substitution product on the N atom produced by the reaction of p-phenylenediamine and propylene oxide are the same as those of the conventional TEPD, as a reversible electron transfer material between the HRPO and the electrode. It is clear that it has a function. In addition, N, N'-BHPD is also HRP
It had a function as a reversible electron transfer substance between O and the electrode.

<Example> The specific binding analyzer shown in FIGS. 2 and 3 was prepared and used to measure the hCG concentration in a sample. First, the following members were produced in order to produce a specific binding analyzer.

Preparation of Conjugate of Anti-hCG Antibody and Horseradish Peroxidase (Labeled Antibody) Mouse monoclonal antibody HM81 recognizing hCG (manufactured by Mochida Pharmaceutical Co., Ltd.) was added to 100 mM sodium chloride-1 mM EDTA.
-60 mM triethanolamine buffer (pH 8.0 T
EA buffer) to a concentration of 8.3 mg / mL,
It was thoroughly dialyzed against a TEA buffer solution that had been purged with nitrogen gas. To 1.1 mL of this antibody solution, 61 μL of 50 mM 2-iminothiolane hydrochloride (manufactured by Pierce) solution prepared in TEA buffer was added, and after stirring, under a nitrogen gas atmosphere, at 4 ° C. for 1.5 hours. I let it stand. After that, 100 with nitrogen gas replacement
Sufficiently dialyzed with mM sodium chloride-1 mM EDTA-100 mM phosphate buffer (pH 7.0 EDTA-PB), S
An anti-hCG antibody HM81 into which an H group was introduced was obtained.

On the other hand, 100 mM phosphate buffer (pH 7.
3) 500 μL of horseradish peroxidase (HRPO, Toyobo Co., Ltd.) solution prepared to a concentration of 20 mg / mL in 0)
50 mM sulfo SMCC with slow stirring at 0 ° C
(Pierce) 500 μL was added and reacted for 20 minutes. After the reaction, the unreacted sulfo SMC was passed through a Sephadex G-25 (Pharmacia) column (2.6φ × 15 cm) equilibrated with EDTA-PB purged with nitrogen gas.
C was removed, and the mixture was concentrated using a concentrator CENTRIPREP-10 (manufactured by Amicon) to obtain maleimidated HRPO. The concentration of the obtained maleimidated HRPO was determined from the absorbance at 403 nm.

1.25 × 10 ^-8 mol or 1.56 ×
To 10 ^-8 mol of maleimidized HRPO solution, each 3
Double molar amount or 1/3 times molar amount of SH group-introduced anti-hCG
The antibody HM81 was added and mixed, and then reacted at 4 ° C. for 12 hours under a nitrogen gas atmosphere. Then, 50 μL of 50 mM cysteamine solution was added to each, and the mixture was placed in a nitrogen gas atmosphere at 4 ° C.
At room temperature for 60 minutes and then replaced with nitrogen gas
ULTROGEL AcA34 (IBF equilibrated with TA-PB
Gel filtration chromatography was performed using a Biotechnics column. Absorbance measurements at 280 nm and 403 nm were carried out on the eluate fractions of gel filtration chromatography to obtain free enzyme-free HM81 and HRPO.
Fractions of the conjugate with and were collected and concentrated. The concentration of the obtained concentrate (hereinafter referred to as HRPO-HM81) is confirmed by electrophoresis with a Phast system (Pharmacia), and then the amount of antibody and enzyme contained is determined from the absorbance and the enzyme activity, which will be described later. It was used as an oxidoreductase-labeled antibody in the measurement.

Horseradish peroxidase labeled anti-h
Preparation of Dried Product Impregnated with CG Antibody (HRPO-HM81) HRPO-HM81 prepared above was prepared using 5% normal rabbit serum (NRS) -10% saccharose / pH 6.0 phosphate buffer containing 0.1 M NaCl. The solution was diluted with a solution to prepare a solution of peroxidase activity of 0.63 U / mL. Then
140 μL of the above solution was spotted on a circular filter paper having a diameter of 12 mm prepared by punching from a glass fiber filter paper (GA100, manufactured by Advantech Toyo Co., Ltd.) and freeze-dried to prepare a dried product of HRPO-HM81 to be the labeled antibody-impregnated part 14. did.

Preparation of Dried Body Impregnated with THEPD (Corresponding to the Example of the Present Invention) The THEPD synthesized in the above Synthesis Example 3 was dissolved in a phosphate buffer containing 0.1 M NaCl at pH 6.0 to obtain 5.0.
A solution of mM was prepared. Then, glass fiber filter paper (GA1
00, manufactured by Advantech Toyo Co., Ltd.), 140 μL of the above solution was spotted on a circular filter paper having a diameter of 12 mm, which was produced by punching, and freeze-dried to obtain a THEPD dried body to be the electron transfer substance impregnated portion 16 corresponding to the present invention. It was made.

Preparation of Dried Body Impregnated with TEPD (Corresponding to Comparative Example) The TEPD synthesized in Synthesis Example 1 described above was mixed with 1 M of pH 6.0.
It was dissolved in a phosphate buffer containing NaCl to prepare a 5.0 mM solution. Next, 140 μL of the above solution was spotted on a circular filter paper having a diameter of 12 mm prepared by punching from a glass fiber filter paper (GA100, manufactured by Advantech Toyo Co., Ltd.), and freeze-dried to form an electron transfer substance impregnated portion 16 TEPD.
The dried body of was produced.

Preparation of Dry Body Impregnated with Hydrogen Peroxide Hydrogen peroxide (manufactured by Wako Pure Chemical Industries, Ltd.) and urea (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved in distilled water to prepare 0.5 M hydrogen peroxide.
A 0.5 M urea solution was prepared. Next, 120 μL of the above solution was spotted on a circular filter paper having a diameter of 12 mm, which was punched from a chromatographic filter paper (17 Chr, manufactured by Whatman), and freeze-dried to form an absorption part 24.
A dried form of urea was prepared.

Preparation of Anti-hCG Antibody-Insoluble Porous Cellulose Mixed Ester Membrane Pore size 8. in a beaker containing 200 mL of 1.0% (W / V) PBS solution of bovine gamma globulin (product code G7516, manufactured by Sigma). 200 sheets of circular cellulose acetate / cellulose nitrate mixed ester porous membrane (catalog number SCWP01300, manufactured by Nippon Millipore Industrial Co., Ltd.) having a diameter of 0 μm and a diameter of 13 mm were put and heated at 60 ° C. for 2 hours while slowly stirring. After removing the supernatant liquid and removing the residual liquid by suction, a sufficient amount of 0.076M phosphate buffered saline pH 6.4 (PBS) was added as a washing liquid and well stirred, and the contents liquid was removed again for washing. It was The PBS was washed twice more, and then the washing with distilled water was repeated 7 times.
After cleaning, 1.0% glutaraldehyde aqueous solution 200
mL was added, and the mixture was reacted at 25 ° C. for 3 hours with slow stirring. After the reaction, washing was performed 10 times with distilled water, and the porous membranes were arranged one by one on a glass plate and dried.

Mouse monoclonal antibody HM21 (manufactured by Mochida Pharmaceutical Co., Ltd.) recognizing hCG was dissolved in 0.05M sodium hydrogen carbonate-0.05M sodium chloride aqueous solution to adjust the concentration to 1.0 mg / mL. 25 μL of this solution was infiltrated from the central part per porous membrane (13 mmφ) dried on a glass plate. After reacting at room temperature for 1 hour, the porous membrane was covered with 0.2% bovine serum albumin (BSA) / PB.
Transfer to 200 mL of S solution and shake at 4 ° C for 2 with stirring.
Blocking reaction was carried out for a day. After that, 0.1% Twee
The membrane was washed 3 times with an n20 / PBS solution, further 7 times with PBS, and dried to obtain an anti-hCG antibody (HM21) insolubilized porous cellulose mixed ester membrane serving as the matrix 22.

Fabrication of Detection Part (Electrode) 20 As shown in FIG. 4, conductive carbon ink (400-CT, 400-CT, (Made by Asahi Kaken)
Screen printed with. Further, a reference electrode 32 was screen-printed with a conductive silver ink (LS411N, manufactured by Asahi Kaken Co., Ltd.) on the upper layer of the circular portion at the center of the front side. Further, the insulating layer 36 on the back side and the front side of the electrode is covered with an insulating ink (XB-101
Screen-printed with G Fujikura Kasei Co., Ltd.). Next, the center of the circular portion was punched to form a hole having a diameter of 3 mm to form the through hole 30, and the annular electrodes were left on the front surface and the back surface of the through hole 30. It was also confirmed that the electrodes on the front side and the back side were electrically independent from each other after the opening.
Next, an electrolysis reaction (+1.0 V vs Ag / AgCl) was performed for 10 minutes in a 0.1 M sodium chloride aqueous solution on the portion where the conductive silver ink was screen-printed to form a silver chloride layer on the surface. Silver / in the exposed ring
The side on which the silver chloride layers were overlaid was used as the reference electrode 32 (and the counter electrode), and the side containing only carbon was used as the working electrode 34 for detection.

[Production of Device for Inventive Example] Using each member produced as described above, the measurement device shown in FIGS. 2 and 3 for carrying out the measurement method of the present invention utilizing the MEDIA method was produced. did. That is, the absorbing part 24 prepared above is placed on an acrylic lower substrate 26, and a seal having a diameter of 6 mm prepared by punching from a mending tape (manufactured by Sumitomo 3M Ltd.) is attached to the center of the upper surface to form a seal part 24a. did. On the upper part of this, the matrix 22 produced above was laminated with the center position aligned with the seal part 24a of the absorbing part 24. Next, the center of the matrix 22 and the through holes 3 are formed on the matrix 22 with the reference electrode 32 as the upper surface.
Matching 0 with each other, the electrode part 20 prepared above was overlaid, and a circular filter paper with a diameter of 3 mm prepared by punching from a glass fiber filter paper (GA55, manufactured by Advantech Toyo Co., Ltd.) was fitted into the through hole 30 to establish a communication member. It was set to 18. next,
The electron transfer material impregnated portion 16 obtained by impregnating and drying the THEPD produced above is provided with the center of the through hole 30 of the electrode portion 20.
Stacked to coincide with the center of. A seal having a diameter of 6 mm, which was produced by punching from a mending tape, was attached to the center of the upper surface of the electron transfer substance impregnated portion 16 to form a seal portion 16a. On top of that, the labeled antibody impregnated part 14 prepared above was stacked, and a circular member having a diameter of 12 mm prepared by punching from a surfactant-treated Ertus (catalog number A05070, manufactured by Asahi Kasei) was stacked. And made the filter 12. An upper cover 10 having a sample introduction port 10a having a diameter of 6 mm, which is made of an acrylic plate having a thickness of 5 mm, is laminated thereon so that the center of the sample introduction port 10a coincides with the center of the through hole 30 of the electrode part 20. And h
A specific binding analyzer for measuring CG concentration was prepared. In addition,
In this device, the distance between the lower surface of the upper plate and the upper surface of the lower support was maintained at 3400 μm.

[Preparation of Device for Comparative Example] The electron transfer material impregnated section 16 prepared by impregnating and drying the TEPD prepared above is used without using the electron transfer material impregnated section 16 prepared by impregnating and drying the THEPD prepared above. Except for the above, a specific binding analyzer for measuring hCG concentration was prepared in exactly the same manner as in the above-mentioned invention example.

[Measurement of hCG in whole blood] The specific binding analyzers for measuring hCG concentration of the above-mentioned invention examples and comparative examples prepared as described above were measured in the same manner as in Experimental Example 2 described above, such as potentiostat. Connected. On the other hand, by adding hCG to heparin-added healthy male blood, hCG 0IU /
L whole blood (A) and hCG 1000 IU / L whole blood (B) were prepared. 260 μL of each of the solution (A) and the solution (B) was introduced into the sample introduction part 38 through the sample introduction port 10a of the upper cover 10 for each of the specific binding analyzers prepared in the invention example and the comparative example. . After 30 seconds from the time of spotting the sample, the working electrode was set to a potential of −150 mV with respect to the counter electrode / reference electrode, and the current value was recorded. The result of the invention example is shown in FIG. 22, and the result of the comparative example is shown in FIG. 23.

As shown in FIG. 22, in the specific binding analyzer according to the invention example in which the THEPD dried material is introduced as the electron carrier impregnating portion 16 for holding the electron carrier, hC is used.
The current value increased depending on the G concentration. In contrast,
In the specific binding analyzer of the comparative example in which the dried TEPD was introduced as the electron transfer material impregnated portion 16, the current value did not increase even if the hCG concentration was increased, as shown in FIG. This is, as shown in the above-mentioned Experimental Example 1, T
Since EPD has poor drying property and drying stability, and is easily affected by interfering substances in the sample, and has poor stability in the sample, EPD can act as a good electron transfer substance during measurement. It is thought that it was because there was not.

From these results, it was clarified that the hCG concentration in the sample can be easily and quickly quantified by the apparatus using the electron transfer substance of the present invention, and the effect of the present invention is clarified.

[0117]

INDUSTRIAL APPLICABILITY As described in detail above, according to the electrochemical measuring method and the novel p-phenylenediamine compound of the present invention, it is possible to measure in the enzyme electrode method or the specific binding analysis method such as MEDIA method. It has good measurement sensitivity (responsiveness) and can be used favorably even for disposable applications. Moreover, even if the sample contains interfering substances such as blood and urine, it is always stable and reproducible. A good measurement of can be performed.

[Brief description of drawings]

FIG. 1A and FIG. 1B are schematic diagrams for explaining the action of an electron mediator, respectively.

FIG. 2 is a schematic exploded perspective view showing an example of a (specific binding) analyzer for carrying out the electrochemical measurement method of the present invention.

FIG. 3 is a view conceptually showing a cross section of an assembled state of the (specific binding) analysis device shown in FIG.

FIG. 4 is a schematic plan view of an electrode unit of the (specific binding) analyzer shown in FIG.

FIG. 5 is a graph showing vapor pressures of TEPD and THEPD.

FIG. 6 is a graph showing cyclic voltammetry (potential scanning range −500 mV to +800 mV) of TEPD.

FIG. 7 is a graph showing cyclic voltammetry (potential scanning range −200 mV to +300 mV) of TEPD.

FIG. 8 is a graph showing cyclic voltammetry (potential scanning range −500 mV to +800 mV) of TCPD.

FIG. 9 is a graph showing cyclic voltammetry (potential scanning range −200 mV to +300 mV) of TCPD.

FIG. 10 is a graph showing cyclic voltammetry of THEPD (potential scanning range −500 mV to +800 mV).

FIG. 11 is a graph showing cyclic voltammetry of THEPD (potential scanning range −200 mV to +300 mV).

FIG. 12 is a graph showing cyclic voltammetry (potential scanning range −500 mV to +800 mV) of TDHPD.

FIG. 13 is a graph showing cyclic voltammetry (potential scanning range −200 mV to +300 mV) of TDHPD.

FIG. 14 is a graph showing the electron transfer activity of TEPD.

FIG. 15 is a graph showing electron transfer activity of TCPD.

FIG. 16 is a graph showing the electron transfer activity of THEPD.

FIG. 17 is a graph showing the electron transfer activity of TDHPD.

FIG. 18 is a graph showing another example of the electron transfer activity of TEPD.

FIG. 19 is a graph showing another example of the electron transfer activity of TCPD.

FIG. 20 is a graph showing another example of the electron transfer activity of THEPD.

FIG. 21 is a graph showing another example of the electron transfer activity of TDHPD.

FIG. 22 is a graph showing a measurement result according to an example using the electron carrier of the present invention.

FIG. 23 is a graph showing a measurement result by a conventional measuring method using an electron transfer substance.

FIG. 24: PPD, TMPD, TEPD, TCPD, T
It is a graph which shows the vapor pressure in 25 degreeC of HEPD and TDHPD.

FIG. 25 is a graph showing cyclic voltammetry (potential scanning range −500 mV to +800 mV) of HTPD.

FIG. 26 is a graph showing cyclic voltammetry (potential scanning range −200 mV to +300 mV) of HTPD.

FIG. 27: Cyclic voltammetry of N, N-BHDPD (potential scanning range −500 mV to +800 mV)
It is a graph which shows.

FIG. 28 shows cyclic voltammetry of N, N-BHDPD (potential scanning range −200 mV to +300 mV).
It is a graph which shows.

FIG. 29 is a cyclic voltammetry of N, N′-BHDPD (potential scanning range −500 mV to +800 m).
It is a graph which shows V).

FIG. 30 shows cyclic voltammetry of N, N′-BHDPD (potential scanning range −200 mV to +300 m).
It is a graph which shows V).

FIG. 31 is a monosubstituted product on the N atom (HPP produced by the reaction of p-phenylenediamine and propylene oxide).
D) cyclic voltammetry (potential scanning range-
It is a graph which shows 500 mV- + 800 mV).

FIG. 32 is a monosubstituted product on the N atom (HPP produced by the reaction of p-phenylenediamine and propylene oxide).
D) cyclic voltammetry (potential scanning range-
It is a graph which shows 200 mV- + 300 mV).

FIG. 33 shows cyclic voltammetry (potential scanning range −500 mV to +) of a disubstituted product on an N atom produced by a reaction of p-phenylenediamine and propylene oxide.
It is a graph which shows 800 mV).

FIG. 34: Cyclic voltammetry (potential scanning range -200 mV to +) of a di-substituted product on the N atom produced by the reaction of p-phenylenediamine and propylene oxide.
It is a graph which shows 300 mV).

FIG. 35 is a cyclic voltammetry (potential scanning range −500 mV to +) of a tri-substituted product on the N atom produced by the reaction of p-phenylenediamine and propylene oxide.
It is a graph which shows 800 mV).

FIG. 36 is a cyclic voltammetry (potential scanning range −200 mV to +) of a tri-substituted product on the N atom produced by the reaction of p-phenylenediamine and propylene oxide.
It is a graph which shows 300 mV).

FIG. 37 is a graph showing electron transfer activity of HTPD.

FIG. 38 is a graph showing the electron transfer activity of N, N-BHDPD.

FIG. 39 is a graph showing the electron transfer activity of N, N′-BHDPD.

FIG. 40 is a monosubstituted product on the N atom (HPP produced by the reaction of p-phenylenediamine and propylene oxide).
It is a graph which shows the electron transfer activity of D).

FIG. 41 is a graph showing the electron transfer activity of the di-substituted product on the N atom produced by the reaction of p-phenylenediamine and propylene oxide.

FIG. 42 is a graph showing the electron transfer activity of the tri-substituted product on the N atom produced by the reaction of p-phenylenediamine and propylene oxide.

FIG. 43 is a graph showing another example of the electron transfer activity of HTPD.

FIG. 44 is a graph showing another example of the electron transfer activity of N, N-BHDPD.

FIG. 45 is a graph showing another example of the electron transfer activity of N, N′-BHDPD.

FIG. 46 is a monosubstituted product on the N atom (HPP produced by the reaction of p-phenylenediamine and propylene oxide).
It is a graph which shows another example of the electron transfer activity of D).

FIG. 47 is a graph showing another example of the electron transfer activity of the disubstituted product on the N atom produced by the reaction of p-phenylenediamine and propylene oxide.

FIG. 48 is a graph showing another example of the electron transfer activity of the tri-substituted product on the N atom produced by the reaction of p-phenylenediamine and propylene oxide.

[Explanation of symbols]

 10 Upper Cover 10a Sample Inlet 12 Filter 14 Label Impregnated Part 16 Electron Transfer Material Impregnated Part 16a, 24a Seal Part 18 Communication Member 20 Electrode Part 22 Matrix 24 Seal Part 26 Lower Substrate 28 Substrate 30 Through Hole 32 Reference Electrode 34 Working Electrode 36 Insulation part 38 Sample introduction part

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location G01N 27/416 33/543 593

Claims (15)

[Claims] 1. A method for measuring an analyte in a liquid sample using at least one oxidoreductase, which comprises a oxidoreductase, an electron transfer material, and an electron transfer capable of transferring the electron transfer material to the measurement system. A different electrode is provided, and a compound represented by the following formula [I] or a salt thereof is used as the electron transfer substance, an electrochemical measuring method. [Chemical 1] (In the above formula [I], R ¹ , R ² , R ³ and R ⁴ each represent a linear or branched alkyl group having 1 to 4 carbon atoms which may have a substituent. In the above formula [I], at least one of R ¹ , R ² , R ³ and R ⁴ is at least one group selected from the group consisting of a hydroxyl group, a mercapto group, a carboxyl group, a phosphonooxy group and a sulfo group. And R ¹ , R
² , R ³ and R ⁴ may be the same or different from each other. ) 2. The electrochemical measuring method according to claim 1, wherein in the formula [I], at least one of R ¹ , R ² , R ³ and R ⁴ has a hydroxyl group or a carboxyl group. 3. The electrochemical measuring method according to claim 1, wherein the formula [I] is at least one selected from the following compounds and salts thereof. [Chemical 2] 4. The analyte is a substrate, a cofactor, an activator or an inhibitor of the oxidoreductase, and the electrochemical reaction at the electrode is modulated by the enzymatic reaction that occurs depending on the amount of the substrate, cofactor, activator or inhibitor. The electrochemical measuring method according to any one of claims 1 to 3, which is detected as modulation of a dynamic signal. 5. The analyte is measured by a specific binding reaction using at least one specific binding substance for the analyte and the oxidoreductase as a labeling substance. Electrochemical measuring method. 6. A matrix, which is a region in which a liquid sample expected to contain the analyte is spread, a sample introducing section for introducing the sample into the matrix, and an electrode substantially in contact with the matrix. A sample is introduced into the sample introduction part, and an analyte in the sample and a first sample are introduced in the matrix.
Of the specific binding substance and a second specific binding substance labeled with an oxidoreductase capable of generating an electron mediator species that generates a signal at the electrode portion; or, outside the matrix, the analyte in the sample Reacting the substance with a first specific binding substance and a second specific binding substance labeled with an oxidoreductase capable of generating an electron mediator species that emits a signal at the electrode part, and then introducing the substance into the matrix; A complex of the second specific binding substance labeled with the oxidoreductase and the analyte, the second specific binding substance labeled with the oxidoreductase, the analyte and the first specific binding substance. Before changing the distribution of at least one molecular species of the second specific binding substance labeled with the complex or the complex-free oxidoreductase, which corresponds to the amount of the analyte in the sample. The electrochemical measuring method according to claim 5, wherein the change in distribution is detected as modulation of a signal at an electrode that is rate-controlled by mass transfer of an electron mediator species generated by an oxidoreductase. 7. A matrix, which is a region in which a liquid sample expected to contain the analyte is developed, a sample introducing section for introducing the sample into the matrix, and an electrode substantially in contact with the matrix. Oxidation capable of generating an electron mediator species that generates a signal at the electrode, using either a device having a specific binding substance or a substance that specifically competes with the analyte for the specific binding substance. Labeled with a reductase, a sample is introduced into the sample introduction part, and in the matrix, an analyte in the sample, a specific binding substance and a substance that competes with the analyte for the specific binding substance Or reacts with the analyte in the sample and the specific binding substance and the substance that competes with the analyte for the specific binding substance outside the matrix. After reacting competitively, it is introduced into the matrix; thereby, a complex of the specific binding substance and the analyte, a complex of the specific binding substance and a substance that competes with the analyte for the specific binding substance Body, a specific binding substance that does not form a complex, or a substance that competes with the analyte for a specific binding substance that does not form a complex, the molecular species labeled with at least one oxidoreductase The distribution is changed, and the distribution change corresponding to the amount of the analyte in the sample is detected as modulation of a signal at an electrode that is rate-controlled by mass transfer of an electron mediator species produced by an oxidoreductase. Electrochemical measurement method. 8. A compound represented by the following chemical formula or a salt thereof. [Chemical 3] 9. A compound represented by the following chemical formula or a salt thereof. [Chemical 4] 10. A measuring method for measuring an analyte in a liquid sample using at least one redox enzyme,
A redox enzyme, an electron mediator, and an electrode capable of electron transfer with the electron mediator are arranged in the measurement system, and a compound represented by the following formula [II] or a salt thereof is used as the electron mediator. Electrochemical measuring method. [Chemical 5] (In the above [II], R ⁵ , R ⁶ , R ⁷ and R ⁸ are each a hydrogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms which may have a substituent. In the above formula [II], R ⁵ , R ⁶ , R ⁷ and R ⁸ may be the same or different, but at least one of them is a hydroxyl group, a mercapto group, a carboxyl group, a phosphonooxy group and a sulfo group. An alkyl group having at least one group selected from the group consisting of groups, at least one of which is a hydrogen atom.) 11. The electrochemical measuring method according to claim 10, wherein in the formula [II], at least one of R ⁵ , R ⁶ , R ⁷ and R ⁸ has a hydroxyl group. 12. The analyte is a substrate, a cofactor, an activator or an inhibitor of the oxidoreductase, and the electrochemical reaction at the electrode is modulated by the enzymatic reaction that occurs depending on the amount of the substrate, cofactor, activator or inhibitor. 10. Detecting as modulation of a dynamic signal.
1. The electrochemical measurement method according to any one of 1. 13. The analyte according to claim 10, wherein the analyte is measured by a specific binding reaction using at least one specific binding substance for the analyte and the oxidoreductase as a labeling substance. Electrochemical measuring method. 14. A matrix, which is a region in which a liquid sample expected to contain the analyte is developed, a sample introducing section for introducing the sample into the matrix, and an electrode substantially in contact with the matrix. A sample is introduced into the sample introduction part, and an analyte in the sample and a first sample are introduced in the matrix.
Of the specific binding substance and a second specific binding substance labeled with an oxidoreductase capable of generating an electron mediator species that generates a signal at the electrode portion; or, outside the matrix, the analyte in the sample Reacting the substance with a first specific binding substance and a second specific binding substance labeled with an oxidoreductase capable of generating an electron mediator species that emits a signal at the electrode part, and then introducing the substance into the matrix; A complex of the second specific binding substance labeled with the oxidoreductase and the analyte, the second specific binding substance labeled with the oxidoreductase, the analyte and the first specific binding substance. Before changing the distribution of at least one molecular species of the second specific binding substance labeled with the complex or the complex-free oxidoreductase, which corresponds to the amount of the analyte in the sample. The electrochemical measuring method according to claim 13, wherein the change in distribution is detected as modulation of a signal at an electrode that is rate-controlled by mass transfer of an electron mediator species generated by an oxidoreductase. 15. A matrix, which is a region in which a liquid sample expected to contain the analyte is developed, a sample introducing section for introducing the sample into the matrix, and an electrode substantially in contact with the matrix. Oxidation capable of generating an electron mediator species that generates a signal at the electrode, using either a device having a specific binding substance or a substance that specifically competes with the analyte for the specific binding substance. Labeled with a reductase, a sample is introduced into the sample introduction part, and in the matrix, an analyte in the sample, a specific binding substance and a substance that competes with the analyte for the specific binding substance Or reacts with the analyte in the sample outside the matrix with a specific binding substance and a substance that competes with the analyte for the specific binding substance. Are reacted with each other competitively and then introduced into the matrix, whereby a complex of the specific binding substance and the analyte, a specific binding substance and a substance that competes with the analyte for the specific binding substance A molecular species labeled with at least one oxidoreductase of a complex, a specific binding substance that does not form a complex, or a substance that competes with an analyte for a specific binding substance that does not form a complex 14. The method according to claim 13, wherein the distribution change is detected, and the change in distribution corresponding to the amount of the analyte in the sample is detected as modulation of the signal at the electrode that is rate-controlled by the mass transfer of the electron mediator species produced by the redox enzyme. Electrochemical measuring method.