JP2018071995A - Cortisol concentration analysis chip and measuring method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an analysis chip for measuring cortisol concentration and a measuring method using the same which are not required to use the standard concentration solution at every measurement (for each analysis chip) and which are capable of suppressing the influence of individual differences of chips due to lot differences of an antibody as a used material and manufacturing process variations, to measure the cortisol concentration with sufficient reproducibility.SOLUTION: A cortisol concentration analysis chip for electrochemical measurement, includes a plurality of working electrodes in which at least one of the working electrodes is a measuring working electrode modified with a cortisol antibody while at least one other working electrode is a correcting working electrode modified with a cortisol antibody and coupled to the cortisol antibody until the cortisol becomes saturated. The cortisol concentration analysis chip is used to measure cortisol concentration from the difference between the measured values of the measuring working electrode and the correcting working electrode.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an analysis chip for measuring the concentration of cortisol and a method for measuring the concentration of cortisol using the same. More particularly, the present invention relates to an analysis chip for electrochemically measuring a cortisol concentration in a biological sample using an antigen-antibody reaction and a method for measuring the cortisol concentration using the same.

  Cortisol is one of corticosteroids, is contained in saliva, blood, urine, etc., and is known to change in concentration due to stress response, and is recognized as a typical stress index.

  General methods such as high performance liquid chromatography (HPLC), liquid chromatography / mass spectrometry (LC / MS), enzyme-linked immunosorbent assay (ELISA), and electrochemical analysis are methods for measuring cortisol concentration in biological samples. Analytical methods can be used. HPLC, LC / MS, and ELISA have high measurement sensitivity and are suitable for quantification, but there are problems that the measurement requires too much time, cost, and labor. As a method for solving this problem, electrochemical analysis is performed which is easy to measure and does not cost the apparatus.

  In electrochemical analysis, an electrochemical analysis chip having a working electrode modified with cortisol antibody via a self-assembled monolayer on the surface is immersed in a biological fluid sample such as blood or saliva. A method is generally used in which electrochemical analysis is performed before and after the antigen-antibody reaction with cortisol, and the cortisol concentration is measured from the difference between the output values. FIG. 3 is a plan view of an example of a standard conventional electrochemical analysis chip, and FIG. 4 is an explanatory view of a BB cross section thereof.

  In Patent Document 1, as a method for measuring cortisol concentration with high sensitivity, a measurement sample is provided on a sensor chip including a comb-shaped electrode on which a cortisol antibody is immobilized via a monomolecular film such as a self-assembled monolayer. An electrochemical immunoassay based on oxidation current measurement is disclosed, in which a solution containing an enzyme-labeled antigen in which cortisol is labeled with cortisol is added dropwise to cause competition.

JP 2011-002430 A

  However, in the conventional electrochemical analysis chip shown in FIG. 3 and FIG. 4 and the analysis chip using the comb-shaped electrode described in Patent Document 1, the electrode is modified with an antibody, the difference between antibody lots, the manufacturing process There is a problem in the reliability and reproducibility of the measured value because the output of the electrochemical analysis varies due to slight fluctuations. Therefore, when converting to the cortisol concentration, measure the cortisol-free standard solution in advance, then measure the biological sample to be measured with the same electrode, compare the measured values, and calculate the concentration from the displacement. In other words, it is necessary to perform electrochemical measurement of the standard solution and the biological sample to be measured using two analysis chips prepared at the same time, and to convert the difference into the concentration, which is troublesome and costly.

  Therefore, the present invention does not require measurement with a standard concentration solution for each measurement time (each analysis chip), and does not affect the effects of differences in lots of antibodies used as materials or individual differences in chips due to variations in manufacturing processes. An object of the present invention is to provide an analysis chip for measuring cortisol concentration and a measurement method using the same, which can suppress and measure cortisol concentration with good reproducibility.

  In order to achieve the above object, the present inventors use a chip in which a plurality of working electrodes (measuring working electrode 1 and correcting working electrode 2) are formed on an analysis chip 100 as shown in FIG. A standard curve obtained in advance from the difference between the measured value of the working electrode 1 for the measurement and the measured value of the working electrode 2 for the correction with respect to the test sample is used without measuring the standard concentration solution at each measurement time (each analysis chip). As a result, it was found that the concentration of cortisol in the sample was required, and the present invention was completed.

That is, the present invention
[1] Cortisol concentration analysis chip by electrochemical measurement including a plurality of working electrodes,
A measuring working electrode in which at least one working electrode is modified with a cortisol antibody;
Cortisol concentration analysis chip, wherein at least one other working electrode is modified with a cortisol antibody and cortisol is bound to the cortisol antibody until saturation, and [2] the above-mentioned [1] Supplying a biological sample to be measured on the working pole of the cortisol concentration analysis chip of
A step of allowing the antigen-antibody reaction between the cortisol antibody on the working electrode for measurement and the cortisol in the biological sample to proceed for a certain period of time;
The present invention relates to a method for measuring cortisol concentration, which includes a step of performing electrochemical analysis measurement for each of the measurement working electrode and the correction working electrode, and a step of obtaining the cortisol concentration in the biological sample from the difference between the obtained measurement values.

  According to the analysis chip for cortisol concentration of the present invention, the measurement working electrode 1 and the correction working electrode 2 are formed on the same chip, so that measurement with a standard concentration solution is required at every measurement time (each analysis chip). Therefore, it is possible to suppress the influence of the difference between the lots of the antibodies used as materials and the individual difference of the chip due to the variation in the manufacturing process, and to measure the cortisol concentration with reproducibility.

  In addition, according to the method for analyzing cortisol concentration of the present invention, using the above-mentioned analysis chip for cortisol concentration of the present invention, electrochemical measurement is performed for each of the measuring working electrode and the correcting working electrode, and the measured values obtained By obtaining the cortisol concentration in the biological sample from the difference between the samples, there is no need to measure with a standard concentration solution at each measurement time (each analysis chip), and there is a difference between lots of antibodies used as materials and variations in the manufacturing process. It is possible to measure the cortisol concentration with reproducibility by suppressing the influence of individual differences of chips.

It is a plane explanatory view of an example of the analysis chip of the present invention. It is explanatory drawing of the AA cross section of the analysis chip | tip of FIG. It is a plane explanatory view of an example of the conventional analysis chip. It is explanatory drawing of the BB cross section of the analysis chip | tip of FIG.

  The analysis chip 100 of the present invention will be described with reference to FIG. 1 and FIG. 2, but the present invention is not limited to these forms, and the members, arrangements, and the like described below are the gist of the present invention. Various modifications can be made within the range. In the drawings, the same reference numerals denote the same or the same parts.

  FIG. 1 is an example showing an embodiment of a planar shape when the analysis chip of the present invention is viewed from above, and a working electrode 1 for measurement and a working electrode 2 for correction are provided on the same surface on a substrate 5 as reference electrodes. A counter electrode 4 is provided so as to surround the measurement working electrode 1, the correction working electrode 2, and the reference electrode 3. The measuring working electrode 1 is connected to a measuring working electrode terminal 1a for connecting to a measuring device provided at the end of the substrate 5 by a conducting wire 1b. The working electrode 2 for correction, the reference electrode 3 and the counter electrode 4 are also connected to the respective terminals 2a, 3a and 4a for connection to the measuring device provided at the end of the substrate 5 by conducting wires 2b, 3b and 4b, respectively. Yes. FIG. 2 is an explanatory diagram of the AA cross section of FIG. 1, and a self-assembled monolayer 6 is formed on the surface of the working electrode 1 for measurement and the working electrode 2 for correction, and the cortisol antibody 7 is immobilized thereon. Has been. Further, cortisol 8 is bound in a saturated state to the cortisol antibody 7 fixed to the correction working electrode 2.

  The material of the substrate 5 is not particularly limited as long as it is an insulating material that does not affect the measurement of the target substance. For example, ceramics, glass, silicon, polyvinyl chloride, polypropylene, polystyrene, polycarbonate And thermoplastic resins such as acrylic resin, polybutylene terephthalate and polyethylene terephthalate (PET), thermosetting resins such as epoxy resins, and synthetic resin materials such as UV curable resins.

  The shape of the substrate 5 can be a rectangle, a circle, an ellipse, or the like, but a rectangle is preferable from the viewpoint of manufacturing in the form of a lattice.

  As the working electrode for the measuring working electrode 1 and the working working electrode 2, known electrode materials can be used, for example, from copper, aluminum, gold, silver, silver chloride, platinum, chromium, nickel, iron, carbon An appropriate material can be selected depending on the type of modification to the working electrode. For example, when the working electrode is modified via a thiol group as described later, the working electrode can be selected from the group consisting of gold, platinum, silver, and copper. Further, the measuring working electrode 1 and the correcting working electrode 2 can be formed of the same material.

  A conducting wire 1b extending from the measuring working electrode 1 formed on the substrate 5 and a subsequent measuring working electrode terminal 1a extend from the measuring working electrode 1 to the end of the substrate, for example, as shown in FIG. The working electrode 1 can be formed of the same material. Similarly, for example, as shown in FIG. 1, a conducting wire 2b extending from the correction working electrode 2 and a subsequent correction working electrode terminal 2a extend from the correction working electrode 2 to the end of the substrate. 2 can be formed of the same material as 2.

  Cortisol antibody 7 is fixed to both the working electrode 1 for measurement and the working electrode 2 for correction. As a method for immobilizing the cortisol antibody 7, various methods known in the field of biosensors can be used, and examples thereof include a method using a self-assembled monolayer (SAM). In SAM, a functional group of an organic molecule, such as a thiol group, is chemically adsorbed on the substrate surface by being immersed in a solution containing the organic molecule or placed in a vapor containing the organic molecule, and spontaneously formed on the substrate surface. It is a high density and highly oriented monomolecular film. Specifically, SAMs composed of alkyl thiols and disulfide compounds, SAMs with thiols as fixed groups and oriented polyethylene glycol (PEG), SAMs with benzene rings, and alkyl thiols with different carbon chain lengths are mixed. SAM and the like. These SAM constituent molecules preferably have a functional group for imparting characteristics to the SAM surface separately from the functional group that is chemisorbed on the substrate, and the cortisol antibody is immobilized using this functional group. Can do. In order to perform selective SAM modification on the measurement working electrode 1 and the correction working electrode 2, the other electrode portions are masked, and only the measurement working electrode 1 and the correction working electrode 2 are formed with the SAM. Alternatively, after the SAM formation process is performed on the entire electrode portion, a potential is applied to electrodes other than the measurement working electrode 1 and the correction working electrode 2 to reduce and desorb the SAM.

  The cortisol antibody 7 is a polyclonal antibody or a monoclonal antibody that can specifically recognize cortisol, and can be produced by a known method, but a commercially available antibody can also be used.

  Cortisol antibody 7 immobilized on the corrective working electrode 2 is bound to cortisol until saturated. This is because a solution containing cortisol is selectively dropped onto the corrective working electrode 2 and left for a sufficient time to complete the antigen-antibody reaction between the cortisol antibody on the corrective working electrode 2 and the cortisol in the solution. do it. Unreacted cortisol is removed.

  The shapes of the measurement working electrode 1 and the correction working electrode 2 are not particularly limited, but preferably have the same size and shape. From the viewpoint of miniaturization of the sensor chip, a flat plate or a thin film is preferable, and film formation / pattern formation can be performed by a sputtering method, a vacuum evaporation method, a plating method, various printing methods, and the like. Further, the measuring working electrode 1 and the correcting working electrode 2 may be comb-shaped electrodes.

  A known electrode material can be used for the reference electrode 3 and the counter electrode 4, and examples thereof include materials made of copper, aluminum, gold, silver, silver chloride, platinum, chromium, nickel, iron, and carbon. The shapes of the reference electrode 3 and the counter electrode 4 are not particularly limited, but are preferably flat or thin from the viewpoint of miniaturization of the sensor chip, and include sputtering, vacuum deposition, plating, It is possible to form a film and pattern by the printing method.

  The conductive wire 3b extending from the reference electrode 3 formed on the substrate 5 and the subsequent reference electrode terminal 3a extend from the reference electrode 3 to the end of the substrate, and can be formed of the same material as that of the reference electrode 3. Similarly, the conductive wire 4b extending from the counter electrode 4 and the subsequent counter electrode terminal 4a can be formed of the same material as the counter electrode 4.

  The electrode terminals (1a, 2a, 3a, 4a) are preferably provided at the ends of the substrate so that they can be easily connected to measuring instruments. The corresponding electrodes (1, 2, 3, 4) and conductors (1b, 2b) 3b, 4b).

  Examples of the biological sample include body fluid, blood, saliva and the like, and saliva is preferable.

  The measurement target of the analysis chip 100 is cortisol. For example, if cortisol can be easily measured as a stress index, it is preferable to provide a simple measurement method for stress check accompanying the recent mandatory stress check.

  The biological sample is applied to the part so as to simultaneously cover the surface of the working electrode 1 for measurement and the surface of the working electrode 2 for correction. Specifically, both application by dripping and application by immersion can be used.

  The cortisol concentration measurement method of the present invention is characterized by using the above-described cortisol concentration analysis chip of the present invention, (1) a step of supplying a biological sample to be measured on the working electrode, and (2) static measurement for a fixed time. And a step of causing an antigen-antibody reaction between the cortisol antibody on the measuring working electrode 1 and cortisol of the biological sample, and (3) a step of performing electrochemical analysis measurement on the measuring working electrode 1 and the correcting working electrode 2 respectively. And (4) a step of obtaining a cortisol concentration in the biological sample from the difference between the obtained measurement values.

  In step (1), the biological sample to be measured is supplied onto the working electrode by dropping the biological sample on the surface of the working electrode or immersing the working electrode in the biological sample. The supply region includes the entire region of the measurement working electrode 1 and the correction working electrode 2, the reference electrode 3 and the counter electrode 4, and may include a part of each conductive wire (1b, 2b, 3b, 4b). It is preferable that a biological sample does not contact each electrode terminal (1a, 2a, 3a, 4a).

  The standing time in the step (2) is set to a time sufficient for completing the antigen-antibody reaction between the cortisol antibody on the working electrode and the cortisol in the biological sample to be measured.

  For the electrochemical analysis measurement in the step (3), a known method such as an impedance method or a cyclic voltammetry method can be used.

  The cortisol concentration in the step (4) is obtained from the difference between the measured values of the measuring working electrode 1 and the correcting working electrode 2, and at this time, a calibration curve based on this difference prepared in advance is used. The calibration curve based on the difference is prepared by alternately performing electrochemical analysis of the working electrode 1 for measurement and the working electrode 2 for correction using a plurality of biological samples whose cortisol concentrations are known in advance, and the difference between the measured values and cortisol. It can be made from the concentration. By using a calibration curve based on such a difference, the cortisol concentration can be obtained without comparison with the standard concentration solution of cortisol for each measurement (each analysis chip). Therefore, the cortisol concentration can be measured with high reliability and reproducibility using only the analysis chip, the measuring device, and the biological sample to be measured, and it is very simple and can be applied to point-of-care.

DESCRIPTION OF SYMBOLS 100 Analytical chip 1 Working electrode for measurement 2 Working electrode for correction 3 Reference electrode 4 Counter electrode 1a Working electrode terminal for measurement 2a Working electrode terminal for correction 3a Reference electrode terminal 4a Counter electrode terminal 5 Substrate 6 Self-assembled monolayer 7 Cortisol antibody 8 Cortisol

Claims (2)

A chip for analyzing cortisol concentration by electrochemical measurement including a plurality of working electrodes,
A measuring working electrode in which at least one working electrode is modified with a cortisol antibody;
A cortisol concentration analysis chip, which is a correcting working electrode in which at least one other working electrode is modified with a cortisol antibody and cortisol is bound to the cortisol antibody until saturation. Supplying a biological sample to be measured onto the working electrode of the cortisol concentration analysis chip according to claim 1;
A step of allowing the antigen-antibody reaction between the cortisol antibody on the working electrode for measurement and the cortisol in the biological sample to proceed for a certain period of time;
A method for measuring cortisol concentration, comprising a step of performing electrochemical analysis measurement for each of the measurement working electrode and the correction working electrode, and a step of obtaining a cortisol concentration in a biological sample from the difference between the obtained measurement values.

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