JP2011002430A - Cortisol measurement method and cortisol sensor chip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cortisol measurement method for simply and accurately measuring a cortisol concentration by using a compact apparatus, and a cortisol sensor chip used for the measurement method.SOLUTION: The cortisol sensor chip 10 is provided with a pair of comb electrodes 12, 13. A cortisol antibody is immobilized in the comb electrode 12. The cortisol measurement method for measuring the cortisol concentration in a sample solution uses the cortisol sensor chip 10, and has a process for dropping the sample solution containing the cortisol and a solution containing an enzyme-labeled antigen labeled by a peroxidase enzyme on the comb electrodes 12, 13, and a process for dropping a solution containing an oxidation-reduction material oxidized by the peroxidase enzyme, applying an oxidation potential of the oxidation-reduction material to the comb electrode 12, and measuring an oxidation current generated by oxidizing the oxidation-reduction material on a surface of the comb electrode 12. The the cortisol sensor chip 10 is used for the measurement method.

Description

  The present invention relates to a cortisol measurement method and a cortisol sensor chip.

For analyzing and diagnosing physiological functions in living organisms, high-performance liquid chromatography (HPLC), liquid chromatography / mass spectrometry (HPLC), and the concentration of cortisol, an essential hormone present in blood and saliva ( LC / MS), enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay, and other general analytical methods can be applied.
Among these, HPLC, LC / MS, and ELISA are widely used in analytical institutions and medical institutions because they can perform highly sensitive analysis. However, these methods have a problem that the apparatus is large and a complicated measurement method is required.

On the other hand, the electrochemical immunoassay has a feature that the apparatus is small and the measurement technique is simple.
In an electrochemical immunoassay, a primary antibody against an analyte, an antibody labeled with an enzyme (hereinafter referred to as “secondary antibody”), a standard substance labeled with an enzyme (hereinafter referred to as “enzyme-labeled antigen”), an enzyme substrate. Etc. are used. At this time, if the enzyme substrate is a redox substance or the product of the enzyme reaction is a redox substance, the concentration of the measurement target substance can be measured by electrochemically detecting the redox reaction.

As a specific method of electrochemical immunoassay, the use of a competitive method or a sandwich method known as a biological component measurement method by ELISA has been attempted.
In the competitive method, the target antibody and enzyme-labeled antigen are added to the primary antibody immobilized on the electrode to cause a competitive antigen-antibody reaction, and further, an oxidation-reduction substance that is a substrate for the enzyme is added to the enzyme. This is a method for electrochemically detecting the product of the reaction.
Non-Patent Document 1 discloses a method for measuring cortisol using a competitive method and a capillary electrophoresis immunoassay.

In the sandwich method, the target substance is added to the primary antibody immobilized on the electrode, then the secondary antibody is added, the target substance is sandwiched between the two antibodies, and the enzyme substrate is added to the enzyme reaction. This is a method for electrochemically detecting the product produced by the method.
Non-Patent Document 2 shows a method for measuring 4-aminophenol using a sandwich method.

M. Jia, Z. He, W. Jin, J. Chromatogr. A, 2002, 966, 187-194 O. Niwa, Y. Xu, H. B. Halsall, W. R. Heineman, Anal Chem., 1993, 65, 1559-1563

  The electrochemical immunoassay is small in size and simple in measuring method, but generally has low sensitivity as compared with analysis by HPLC, LC / MS and ELISA. Therefore, even if the competition method is used as in Non-Patent Document 1, it is difficult to obtain sufficient sensitivity in the measurement of low concentration of cortisol. In particular, the concentration of cortisol in blood and saliva is as low as about 5 nM, and very high detection sensitivity is required to measure the concentration of cortisol.

In addition, the present inventors tried to apply the sandwich method as described in Non-Patent Document 2 to the measurement of cortisol. However, no change in detected current relative to cortisol could be observed. This is because in the sandwich method, two antibodies, a primary antibody and a secondary antibody, need to bind to the substance to be measured for detection, and in the case of a biological substance with a low molecular weight such as cortisol, an antibody that can bind It is thought that this is due to the limited number of
As described above, it is difficult to measure cortisol concentration in blood or saliva with high sensitivity by a simple measurement method using a small device.

  The present invention relates to a cortisol measurement method capable of measuring cortisol concentration with high sensitivity by an electrochemical immunoassay with a smaller apparatus and a simple measurement method compared with HPLC, LC / MS and ELISA, and a cortisol sensor chip used in the measurement method. For the purpose of provision.

The present invention employs the following configuration in order to solve the above problems.
[1] A method for measuring the concentration of cortisol in a sample solution using a cortisol sensor chip having a pair of comb-shaped electrodes and having a cortisol antibody immobilized on at least one of the comb-shaped electrodes, At the same position on the comb-shaped electrode, a sample solution containing cortisol as a measurement target substance and a labeled antigen-containing solution containing an enzyme-labeled antigen obtained by labeling peroxidase enzyme with cortisol are dropped, and the cortisol and the enzyme-labeled antigen are added. A competitive reaction step of competitively binding to the cortisol antibody, and a substrate-containing solution containing a redox substance that is oxidized by the peroxidase enzyme are further dropped at the position on the pair of comb-shaped electrodes, and the cortisol antibody Applying the oxidation potential of the redox substance to the immobilized comb electrode and applying the oxidation potential Cortisol measurement method having a current measuring step of measuring the oxidation current resulting from oxidation of the redox substance in the interdigital electrode surface.
[2] The cortisol antibody is immobilized only on one comb electrode of the pair of comb electrodes, the oxidation potential of the redox substance is applied to the comb electrode on which the cortisol antibody is immobilized, and the other The method for measuring cortisol according to [1], wherein the reduction potential of the redox substance is applied to a comb electrode.
[3] The cortisol measurement method according to [1] or [2], wherein the amount of the redox substance to be dropped is an amount that is completely oxidized in one minute by the peroxidase enzyme of the enzyme-labeled antigen to be dropped.
[4] When the enzyme activity of the peroxidase enzyme is P (units / mg), the molecular weight of the peroxidase enzyme is Q, the number of moles of the redox substance is X, and the number of moles of the enzyme-labeled antigen is Y, X < Cortisol measuring method as described in said [3] satisfy | filling ^Px (Qx10 < ^-3 >) xY.
[5] A Cortisol sensor chip for use in the cortisol measurement method according to [1], wherein the Cortisol sensor chip comprises a pair of comb-shaped electrodes, and a Cortisol antibody is immobilized on at least one of the comb-shaped electrodes.
[6] The Cortisol sensor chip used for the cortisol measurement method according to [2], wherein the chip comprises a pair of comb-shaped electrodes, and the cortisol antibody is immobilized only on one of the comb-shaped electrodes. ] Cortisol sensor chip of description.

The cortisol measurement method of the present invention can measure cortisol concentration with high sensitivity by an electrochemical immunoassay with a smaller apparatus and a simple measurement method compared with HPLC, LC / MS, and ELISA.
Moreover, this invention provides the cortisol sensor chip used for the said cortisol measuring method.

It is the top view which showed an example of embodiment of the cortisol sensor chip of this invention. It is the conceptual diagram which showed the cross section which fix | immobilized the cortisol antibody to the comb-shaped electrode of FIG. It is the conceptual diagram which showed an example of embodiment of the apparatus which has a cortisol sensor chip of this invention.

[Cortisol sensor chip]
The cortisol sensor chip of the present invention is a sensor chip used in the cortisol measurement method for measuring the cortisol concentration of the present invention. Hereinafter, an example of an embodiment of the cortisol sensor chip of the present invention will be shown and described in detail.
As shown in FIG. 1, a cortisol sensor chip 10 (hereinafter referred to as “sensor chip 10”) of this embodiment includes a substrate 11, a pair of comb-shaped electrodes 12 and 13 formed on the substrate 11, and a reference. It has an electrode 14 and a counter electrode 15.

The board | substrate 11 will not be specifically limited if it does not have a bad influence on the measurement of the cortisol density | concentration by the sensor chip 10, For example, a glass substrate is mentioned.
The shape of the substrate 11 is rectangular. However, the shape of the substrate 11 is not particularly limited as long as each electrode can be formed on the substrate 11 and a cortisol concentration can be measured by dropping a sample solution or the like onto the electrodes. The size of the substrate 11 is not particularly limited as long as the cortisol concentration can be measured.

The comb electrodes 12 and 13 are comb-shaped electrodes corresponding to each other.
As the comb-shaped electrodes 12 and 13, known electrodes can be used, and examples thereof include electrodes made of copper, aluminum, gold, silver, silver chloride, platinum, chromium, nickel, iron, and carbon.

Since the comb-shaped electrodes 12 and 13 are electrodes in which a large number of parallel electrodes are connected in parallel, the current density can be increased while the apparatus is kept small. For this reason, the current value obtained is larger than when using a simple parallel electrode, and the sensitivity can be increased.
The width d ₁ and the interval d ₂ of the parallel electrode portions of the comb-shaped electrode 12 are preferably as small as possible because a large current can be obtained, but 0.1 to 10 μm in that they can be manufactured by a conventional photolithography technique. It is preferable that
The comb electrode 13 may have a shape corresponding to the comb electrode 12 and preferably has the same width and interval.

As shown in FIG. 2, a cortisol antibody 23 is immobilized on the comb electrode 12 through a monomolecular film 21 and a cross-linking molecule 22. The cortisol antibody 23 may be immobilized on both of the pair of comb-shaped electrodes 12 and 13, but is preferably immobilized on only one as in this embodiment.
The monomolecular film 21 is a monomolecular film that is self-assembled on the electrode surface by adsorbing a disulfide compound. Specifically, it consists of 10-carboxydisulfide (10-CDD), 7-carboxyheptyl disulfide (7-CHD), 5-carboxypentyl disulfide (5-CPD), 4,4′-dithiobutanoic acid (DDA) and the like. A monomolecular film may be mentioned.

The cortisol antibody 23 can be immobilized on the comb-shaped electrode 12 via the monomolecular film 21 and the cross-linking molecule 22 by activating the monomolecular film 21 with a cross-linking reagent and reacting the cortisol antibody.
For example, a monomolecular film 21 made of 10-CDD is formed on the comb-shaped electrode 12, 2,4-dinitrophenylamine which is a cross-linking molecule 22 is bound, and then the cortisol antibody 23 is reacted to form a comb-shaped Cortisol antibody 23 can be immobilized on electrode 12.
Further, a monomolecular film 21 made of 10-CDD is formed on the comb-shaped electrode 12, and N-hydroxysuccinimide and a benzophenone derivative which are cross-linked molecules 22 are bonded, and then the cortisol antibody 23 is reacted by light irradiation. Thus, the cortisol antibody 23 can be immobilized on the comb electrode 12.
However, the immobilization of the cortisol antibody on the comb-shaped electrode 12 is not limited to the above-described method, and a known immobilization method can be appropriately used.

  The cortisol antibody 23 is a polyclonal or monoclonal antibody that specifically recognizes cortisol. As the cortisol antibody 23, one produced by a known method may be used, or a commercially available one may be used.

The formation method of the comb-shaped electrodes 12 and 13, the reference electrode 14, and the counter electrode 15 is not particularly limited, and a known method can be used. For example, when forming an electrode made of gold, the following method can be used.
First, a portion that becomes a groove of each electrode is drawn on the substrate 11 by electric beam (EB) drawing, and a negative resist is applied so that the groove portion remains by development. Then, gold is deposited and each electrode is formed by lift-off. Next, the electrodes having a desired shape can be formed by adjusting the electrodes into a desired shape by UV photolithography (positive resist) and etching.

The reference electrode 14 is not particularly limited, and examples thereof include a silver / silver chloride electrode. A portion 14a forming a circuit in the reference electrode 14 may be formed of a metal such as gold.
Moreover, the counter electrode 15 is not specifically limited, For example, a platinum electrode and a carbon electrode are mentioned.
The shape of the reference electrode 14 and the counter electrode 15 is not specifically limited, For example, a flat electrode is mentioned.

As an apparatus for measuring the concentration of cortisol using the sensor chip 10, for example, as shown in FIG. A device in which the comb electrode 13 and the counter electrode 15 are connected to the ammeter 40 can be used. In this apparatus, a desired potential can be applied to the comb-shaped electrode 12 and the comb-shaped electrode 13 with the reference electrode 14 as a reference by the power supply 30. Further, the current generated in the comb electrode 12 and the comb electrode 13 can be measured by the ammeter 40.
Examples of an apparatus that can be used for cortisol concentration measurement using the sensor chip 10 include a commercially available electrochemical analyzer (trade name: ALS600C, BIAS Co., Ltd.).

[Method for measuring cortisol]
Hereinafter, a cortisol concentration measuring method using the sensor chip 10 described above will be described as an example of an embodiment of the cortisol measuring method of the present invention.
In the cortisol measurement method of the present embodiment, a sample solution containing cortisol as a measurement target substance and a labeled antigen-containing solution containing an enzyme-labeled antigen obtained by labeling peroxidase enzyme on cortisol are dropped at the same position on the comb electrodes 12 and 13. And a substrate-containing solution containing a redox substance that is oxidized by the peroxidase enzyme is further dropped at the position on the comb-shaped electrodes 12 and 13 to the comb-shaped electrode 12 on which the cortisol antibody 23 is immobilized. A current measuring step of applying an oxidation potential of the oxidation-reduction substance and measuring an oxidation current generated by oxidation of the oxidation-reduction substance on the surface of the comb electrode to which the oxidation potential is applied.

In the competitive reaction step, the sample solution and the labeled antigen-containing solution are dropped at the same position. Thereby, the cortisol in the sample solution and the enzyme-labeled antigen in the labeled antigen-containing solution cause an antigen-antibody reaction competitively with the cortisol antibody 23 immobilized on the comb electrode 12. Therefore, the higher the concentration of cortisol in the sample solution, the smaller the amount of enzyme-labeled antigen that binds to the cortisol antibody 23.
The sample solution and the labeled antigen-containing solution may be dropped separately, or may be dropped after mixing in advance, as long as cortisol in the sample solution can compete with the enzyme-labeled antigen in the labeled antigen-containing solution.

  The peroxidase enzyme of the enzyme-labeled antigen is an enzyme that oxidizes the redox substance in the substrate-containing solution, and can be appropriately selected in consideration of the combination with the redox substance. An example is horseradish peroxidase (HRP).

  When Ma is the maximum value of the molar amount of cortisol to be measured, the molar amount Mb of the enzyme-labeled antigen to be dropped is preferably 0.5Ma <Mb <2Ma, and more preferably Ma = Mb. . Further, the molar amount Mc of the cortisol antibody 23 immobilized on the comb electrode 12 is preferably 0.5 Mb <Mc ≦ Mb, and more preferably Mb = Mc. By satisfying these conditions, the cortisol concentration in the sample solution and the enzyme-labeled antigen are made to compete with each other, so that the cortisol concentration can be easily measured with high sensitivity even with a small apparatus.

  In the competitive reaction step, it is preferable that the sample solution and the labeled antigen-containing solution are dropped and then allowed to stand for about 60 minutes in order to sufficiently react cortisol with the enzyme-labeled antigen.

  In the cortisol measurement method of the present embodiment, since the competition method is applied in this way, the sensitivity is higher than the method employing the sandwich method. That is, in the sandwich method, measurement cannot be performed unless both the primary antibody and the secondary antibody are bound to cortisol, which is a substance to be measured. On the other hand, in the present invention, detection is possible simply by binding one antibody (cortisol antibody 23) to the substance to be measured, and therefore the concentration of a substance having a low molecular weight such as cortisol can be measured. Become.

In the current measurement step, a substrate-containing solution containing a redox substance that is oxidized by the peroxidase enzyme of the enzyme-labeled antigen is dropped at the position where the sample solution and the labeled antigen-containing solution are dropped.
The redox material is a substrate for the peroxidase enzyme of the enzyme-labeled antigen. Specifically, in the case of HRP, examples include tetramethylbenzidine (TMB) and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS).

  It is preferable that the amount of the redox substance to be dropped is an amount that is completely oxidized in one minute by the peroxidase enzyme of the enzyme-labeled antigen that is dropped in the competitive reaction step. As a result, when the cortisol concentration in the sample solution is zero and all the enzyme-labeled antigen is bound to the cortisol antibody 23, all redox substances are oxidized by the peroxidase enzyme of the enzyme-labeled antigen. Therefore, oxidation of the redox substance does not occur on the surface of the comb electrode to which an oxidation potential is applied, and the measured oxidation current becomes zero.

Specifically, the relationship between the number of moles (X) of the redox substance to be added and the number of moles (Y) of the enzyme-labeled antigen bound to the cortisol antibody is preferably X <aY. a is a constant obtained from the peroxidase enzyme to be used. When the enzyme activity of the enzyme is P (units / mg, units = μmol) and the molecular weight is Q (g / mol), a = P × Q × 10 ^{− 3} .
If X is set to a value smaller than aY, the amount of the enzyme-labeled antigen is such that when all of them bind to the cortisol antibody 23, the peroxidase enzyme of the enzyme-labeled antigen can oxidize all the redox substances. When the cortisol concentration in the sample solution is zero, only the enzyme-labeled antigen binds to the cortisol antibody 23 and the surface of the type electrode 12 is covered with the peroxidase enzyme, so that all the redox substances are oxidized by the enzyme, In the comb electrode to which a potential is applied, no oxidation occurs and no oxidation current is detected. Therefore, the concentration range that can be measured is widened, and the concentration can be measured with high sensitivity even when cortisol is at a low concentration.
For example, since the enzyme activity P of HRP is 50 units / mg and the molecular weight Q is 44,000 g / mol, a = 2200. Therefore, the redox material may be dropped so that the number of moles X satisfies X <2200 × Y.

In the current measurement step, the power supply 30 applies an oxidation potential of the redox substance to the comb-shaped electrode 12 on which the cortisol antibody 23 is immobilized. Thereby, when cortisol in the sample solution binds to the cortisol antibody 23 and the binding amount of the enzyme-labeled antigen to the cortisol antibody 23 decreases, the oxidation-reduction substance is applied on the surface of the comb electrode to which an oxidation potential is applied accordingly. Oxidized, and the ammeter 40 detects the oxidation current.
The oxidation potential is a potential sufficient to oxidize the redox substance on the electrode surface and does not adversely affect the measurement of the oxidation current due to electrode deterioration or reaction of other substances. I just need it. For example, when the redox substance is TMB and the redox potential is 200 mV, it is preferably set to 400 to 600 mV.

In the current measurement step, when the concentration of cortisol in the sample solution is high, the number of cortisol that binds to the cortisol antibody 23 increases, and the number of enzyme-labeled antigens that bind to the cortisol antibody 23 decreases accordingly. Therefore, the number of redox substances oxidized by the peroxidase enzyme of the enzyme-labeled antigen decreases, while the number of redox substances oxidized by the comb-type electrode to which an oxidation potential is applied increases. Therefore, the larger the cortisol in the sample solution, the larger the oxidation current can be obtained.
On the other hand, when the concentration of cortisol in the sample solution is low, the number of cortisol that binds to the cortisol antibody decreases, and accordingly, the number of enzyme-labeled antigens that bind to the cortisol antibody increases. Therefore, the number of redox substances that are oxidized by the peroxidase enzyme of the enzyme-labeled antigen increases, and the number of redox substances that are oxidized by the comb electrode to which an oxidation potential is applied decreases. Therefore, the smaller the cortisol in the sample solution, the smaller the oxidation current obtained.
Thus, in the cortisol measurement of this embodiment, the oxidation current depending on the cortisol concentration in the sample solution is detected. Therefore, highly sensitive measurement of cortisol concentration is possible by using a method such as preparing a calibration curve in advance using a solution containing cortisol at a known concentration.

  In the cortisol measurement method of this embodiment, the comb electrodes 12 and 13 are applied to the competitive method of electrochemical immunoassay. Since the comb-shaped electrodes 12 and 13 are electrodes having a shape in which a large number of parallel electrodes are connected in parallel, the current density can be increased. Therefore, compared with a simple parallel electrode, the current value obtained while keeping the device small can be increased, and the sensitivity can be improved.

Moreover, in the cortisol measurement method of this embodiment, the cortisol antibody 23 is fixed only to the comb electrode 12. In the cortisol measurement method of the present invention, the cortisol antibody 23 is immobilized only on one of the comb-type electrodes 12, and the oxidation potential of the redox substance is applied to the comb-type electrode 12, and the reduction potential of the redox substance is applied to the comb-type electrode 13. Each is preferably applied.
As a result, the redox material oxidized at the comb electrode 12 to which the oxidation potential is applied reaches the comb electrode 13 to which the reduction potential is applied by diffusion, and is reduced at the comb electrode 13. The redox material reduced by the comb electrode 13 reaches the comb electrode 12 again by diffusion and is oxidized. Therefore, it becomes possible to cause a redox reaction repeatedly by performing measurement in the above-described form, and it is possible to measure cortisol concentration with higher sensitivity.

  The reduction potential applied to the comb-shaped electrode 13 is a potential sufficient to reduce the redox material and does not adversely affect the measurement of the oxidation current due to electrode deterioration or reaction of other materials. Any range is acceptable. For example, when the redox substance is TMB and the redox potential is 200 mV, it is preferably 0 to −200 mV.

The cortisol measurement method of the present invention described above uses a competition method using a sensor chip to which a pair of comb electrodes are applied. Therefore, even for cortisol, which is a small molecule for applying the sandwich method, it is possible to easily measure a highly sensitive concentration with a small apparatus.
The cortisol measurement method of the present invention is not limited to the method using the sensor chip 10 and the device 1. For example, a sensor chip in which a cortisol antibody is immobilized on both electrodes of a pair of comb electrodes may be used. In this case, the oxidation potential may be applied to both of the comb electrodes, or may be applied to only one of them.

Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples. However, the present invention is not limited by the following description.
In this example, cortisol was detected using an electrochemical immunoassay using a cortisol sensor chip.
[Example 1]
The sensor chip 10 illustrated in FIG. 1 was created. On the glass substrate having a vertical D ₁ of 12 mm and a horizontal D ₂ of 20 mm, which is the substrate 11, a portion to be a groove of each electrode is drawn by drawing an electric beam (EB), a negative resist is applied, and a groove portion is left by development. Each electrode is formed by metal vapor deposition and lift-off, and further, comb electrodes 12 and 13, a reference electrode 14, and a counter electrode 15 are provided by adjusting these electrodes to a desired shape by UV photolithography (positive resist) and etching. A sensor chip 10 was obtained. The comb electrodes 12 and 13 have a width d ₁ of 10 μm and a distance d ₂ of 5 μm. Each electrode was formed of silver / silver chloride for the reference electrode 14 and gold for the other electrodes.

On the comb electrode 12, as illustrated in FIG. 2, a monomolecular film 21 and a cross-linking molecule 22 are used as a Carboxydecyl disulfide SAM reagent, and 1 μg / mL cortisol antibody 23 (trade name: ab1949, manufactured by abcam) 10 μL was immobilized.
Each electrode of the obtained sensor chip 10 was connected to a commercially available electrochemical analyzer (trade name: ALS600C, BIAS Co., Ltd.) using four electric cables and used for measurement.
Cortisol labeled with horseradish peroxidase (HRP) was used as the enzyme-labeled antigen. Tetramethylbenzine (TMB) was used as the redox material.

(Competitive reaction process)
A sample solution (10 μL) having a cortisol concentration of 0 nM and a labeled antigen-containing solution (10 μL) containing 100 ng / mL of HRP-labeled cortisol are dropped on the comb-type electrode 12 and the comb-type electrode 13 on which the cortisol antibody 23 is immobilized. Let stand for hours.

(Current measurement process)
The amount of TMB used as a redox substance was determined as follows.
In the relational expression X <P × (Q × 10 ⁻³ ) × Y between the number of moles (X) of the redox substance and the number of moles (Y) of the enzyme-labeled antigen that binds to the cortisol antibody, the enzyme activity P of HRP is 50 units. The molecular weight Q of HRP is 44,000 g / mol. The dropping volume of the substrate-containing solution containing TMB was 10 μL. At this time, a = 2200, Y = 2.3 × 10 ⁻¹⁴ mol, and X <5.0 × 10 ⁻¹¹ . In addition, the amount of TMB necessary for the measurement of the sample solution having a contisol concentration of 10 nM is X> 10 ⁻¹⁴ mol when the volume of the substrate-containing solution to be dropped is 10 μL.
Therefore, the concentration of TMB in the substrate-containing solution to be dropped was set to 1 μM.

Further, 10 μL of a substrate-containing solution (1 μM) containing TMB is dropped at the position where the sample solution and the labeled antigen-containing solution on the comb-shaped electrode 12 and the comb-shaped electrode 13 are dropped, and the comb-shaped electrode 12 with respect to the reference electrode 14 is dropped. A potential of 400 mV was applied, and the oxidation current obtained with the comb electrode 12 was measured. As a result, the current value was 0A.
This is because cortisol is not present in the sample solution, so that the enzyme-labeled antigen is all bound to the cortisol antibody 23 immobilized on the comb-shaped electrode 12, and the amount of TMB in the dripping substrate-containing solution This is because all the TMB was oxidized by the peroxidase enzyme of the enzyme-labeled antigen because X was smaller than aY, and no oxidation occurred on the comb-type electrode 12.

Further, the cortisol concentration of the sample solution was changed in the range from 0 nM to 100 nM, and the oxidation current was measured for each sample solution in the same manner as described above.
As a result, the oxidation current measured as the cortisol concentration of the sample solution increased, and the oxidation current measured at 100 nM was 20 nA. Further, the detection limit of cortisol in the sample solution by measuring the oxidation current was 10 nM.
As described above, in Example 1, it was confirmed that a low concentration of cortisol can be detected with high sensitivity by simple measurement using diffusion of the redox substance using a small apparatus.

[Example 2]
A potential of 400 mV with respect to the reference electrode is applied to the comb-shaped electrode 12 to which the cortisol antibody 23 is immobilized, and a potential of 0 mV to the other comb-shaped electrode 13 to which the cortisol antibody is not immobilized. The current obtained by the comb-shaped electrode 12 was measured in the same manner as in Example 1 except that. That is, in Example 2, the measurement using the redox cycle in which the oxidation-reduction reaction is balanced was performed.
As a result, as the cortisol concentration increases, the oxidation current obtained in the comb-type electrode 12 increases. When the cortisol concentration is 0 nM and 100 nM, the currents are 0 nA and 100 nA, respectively, and the sensitivity is 5 compared to Example 1. Doubled. That is, the detection limit of cortisol concentration was 2 nM.
The cortisol concentration in saliva is as low as about 5 nM in normal times. From this result, it was confirmed that the cortisol concentration in the living body can be measured with extremely high sensitivity by the method of the present invention.

[Example 3]
The oxidation current was measured in the same manner as in Example 1 except that 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was used as the redox substance instead of TMB.
As a result, as the cortisol concentration increased, the current obtained from the comb electrode 12 increased. When the cortisol concentration was 0 nM and 100 nM, the current was 0 nA and 20 nA, respectively.
Thus, it was confirmed that the cortisol concentration in the living body can be measured with extremely high sensitivity even when a redox substance other than TMB is used.

  The cortisol concentration measurement method of the present invention can measure the concentration of a low-molecular substance such as cortisol in a living body with a high sensitivity by a simple method using a small device. It can also be used to analyze and diagnose physiological functions of living bodies at home.

  DESCRIPTION OF SYMBOLS 10 Cortisol sensor chip 11 Substrate 12 Comb electrode 13 Comb electrode 14 Reference electrode 15 Counter electrode 21 Monomolecular film 22 Cross-linked molecule 23 Cortisol antibody

Claims (6)

A method for measuring the concentration of cortisol in a sample solution using a cortisol sensor chip comprising a pair of comb-shaped electrodes and having a cortisol antibody immobilized on at least one of the comb-shaped electrodes,
A sample solution containing cortisol, which is a measurement target substance, and a labeled antigen-containing solution containing an enzyme-labeled antigen obtained by labeling cortisol with a peroxidase enzyme are dropped at the same position on the pair of comb-shaped electrodes, and the cortisol and the enzyme label A competitive reaction step of competitively binding an antigen to the cortisol antibody;
A substrate-containing solution containing a redox substance that is oxidized by the peroxidase enzyme is further dropped at the position on the pair of comb-type electrodes, and the oxidation potential of the redox substance is applied to the comb-type electrode on which the cortisol antibody is immobilized. A current measuring step of measuring an oxidation current generated by oxidation of the redox substance on the surface of the comb electrode to which the oxidation potential is applied;
A method for measuring cortisol.   The cortisol antibody is immobilized only on one comb-shaped electrode of the pair of comb-shaped electrodes, the oxidation potential of the redox substance is applied to the comb-shaped electrode on which the cortisol antibody is immobilized, and the other comb-shaped electrode The method for measuring cortisol according to claim 1, wherein a reduction potential of the redox substance is applied to an electrode.   The method for measuring cortisol according to claim 1 or 2, wherein the amount of the redox substance to be dropped is an amount that is oxidized in one minute by the peroxidase enzyme of the enzyme-labeled antigen to be dropped. When the enzyme activity of the peroxidase enzyme is P (units / mg), the molecular weight of the peroxidase enzyme is Q, the number of moles of the redox substance is X, and the number of moles of the enzyme-labeled antigen is Y, X <P × ( The method for measuring cortisol according to claim 3, wherein Q × 10 ⁻³ ) × Y is satisfied. A cortisol sensor chip used in the cortisol measurement method according to claim 1,
A cortisol sensor chip comprising a pair of comb-shaped electrodes and having a cortisol antibody immobilized on at least one of the comb-shaped electrodes. A cortisol sensor chip used for the cortisol measurement method according to claim 2,
The cortisol sensor chip according to claim 5, comprising a pair of comb-shaped electrodes, wherein a cortisol antibody is immobilized only on one of the comb-shaped electrodes.

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