Compound detection sensor and compound concentration measurement method using the compound detection sensor

Abstract

To provide a measuring device and a measuring method capable of simply and qualitatively or quantitatively determining a compound by measuring absorbance of a solution containing the compound.SOLUTION: A compound detection sensor for detecting a compound by absorbance measurement includes an indicator solution containing a cucurbituril structure-containing compound and a dye, and an absorbance measurement device for measuring a change in absorbance of a solution before and after mixing a measurement object with the indicator solution.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a compound detection sensor using a compound having a cucurbituril structure and a method for measuring the concentration of a compound using this compound detection sensor. [Background technology]

[0002] Cucurbiturils have a unique structure that allows them to encapsulate molecules, and they have been studied in a variety of fields. Among these, a method for detecting amino acids by irradiating a solution containing cucurbituril and acridinium, a luminescent molecule, with light of a specific wavelength has been disclosed (Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Chinese Patent Application Publication No. 103472047 Summary of the Invention [Problem to be solved by the invention]

[0004] Although the above-mentioned measurement method using fluorescence has been known for some time, it has not been known as a sensor that can more simply qualitatively or quantitatively determine a compound by measuring absorbance without using fluorescence. The present inventors provide a compound detection sensor and a compound concentration measurement method that can qualitatively or quantitatively determine a measurement target using a cucurbituril structure-containing compound and a dye without using fluorescence. [Means for solving the problem]

[0005] That is, the present invention includes the following. [1] A compound detection sensor that detects a compound by absorbance measurement, an indicator solution containing a cucurbituril structure-containing compound and a dye; and an absorbance measuring device that measures changes in absorbance and absorption wavelength of the solution before and after mixing the object to be measured with the indicator solution. [2] The compound detection sensor according to [1], wherein the indicator solution contains a mixed solvent consisting of an organic solvent and water. [3] The compound detection sensor according to [2], wherein the organic solvent is present in an amount of 1.0% by weight or more and 50.0% by weight or less relative to the mixed solvent. [4] The compound detection sensor according to [3], wherein the pH of the indicator solution is 2.0 or more and 6.0 or less. [5] The compound detection sensor according to [1], wherein the dye is an azobenzene-based compound. [6] The compound detection sensor according to [1], wherein the object to be measured is an organic compound containing a hydrophobic structure, and the molecular weight or number average molecular weight thereof is in the range of 100 or more and 900 or less. [7] The compound detection sensor according to [1], wherein the object to be measured is a steroid compound. [8] A method for measuring the concentration of a compound using the compound detection sensor according to [1], preparing an indicator solution containing a cucurbituril structure-containing compound and a dye; mixing a substance to be measured having a known concentration into the indicator solution; creating a calibration curve from an absorbance curve for an indicator solution that does not contain the object to be measured and an absorbance curve for an indicator solution that contains the object to be measured; preparing a detection solution containing an unidentified or unknown concentration of a target substance; mixing the detection solution with the indicator solution to obtain an absorbance curve of the detection solution; and determining the concentration of an unidentified analyte or analyte contained in the detection solution from characteristics obtained by comparing the calibration curve with the detection solution absorbance curve. [9] The concentration measurement method according to [8], wherein the indicator solution is prepared for each of a plurality of types of dyes and for each concentration of the dyes.

[10] The concentration measurement method according to [8], wherein the indicator solutions are prepared for each of a plurality of types of compounds having a cucurbituril structure and for each concentration of the compounds having a cucurbituril structure.

[11] The concentration measurement method according to [8], wherein the indicator solution is prepared for each of a plurality of types of solvents and for each concentration of the solvents.

[12] The concentration measurement method according to [8], wherein the calibration curve is created for each of the objects to be measured. [Effects of the Invention]

[0006] Since measurement can be performed by absorbance without using fluorescence, no special equipment is required for fluorescence measurement, and, unlike fluorescence, stable qualitative or quantitative measurement with little change over time is possible. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a schematic diagram showing how a dye included in a compound having a cucurbituril structure is replaced with a substance to be measured. [Figure 2A] Figure 2A shows the change in the absorbance curve when the concentration of the dye (AzB-2-02) is fixed and the concentration of cucurbit[8]uril is changed without adding the object to be measured. [Figure 2B] Figure 2B shows an example of the color change of the solution when the concentration of each dye is fixed and the concentration of cucurbit[8]uril is changed without adding the object to be measured (photo). [Figure 3] FIG. 3 shows the change in the absorbance curve when the concentration of the cucurbit[8]uril and the concentration of the dye are fixed and the concentration of the object to be measured is changed. [Figure 4] FIG. 4 is a plot of absorbance at a given wavelength of the absorbance curve of FIG. [Figure 5] FIG. 5 shows calibration curves for cortisol and testosterone in the examples. [Figure 6] FIG. 6 is a diagram showing the difference in the change in absorbance of the measurement object when the type of dye is changed. [Figure 7] FIG. 7 shows the difference in the change in absorbance of the object to be measured when cucurbit[8]uril is replaced with cucurbit[7]uril. [Figure 8] FIG. 8 shows the results of linear discriminant analysis of the results for 15 steroid hormones. [Figure 9] FIG. 9 shows the results of FIG. 8 with the same characteristics circled. [Figure 10] FIG. 10 shows the results of FIG. 8, with the same characteristics circled. [Figure 11] FIG. 11 shows the results of FIG. 8, with the same characteristics circled. [Figure 12] FIG. 12 shows the results of linear discriminant analysis when the ratio of two steroid hormones was changed. [Figure 13] FIG. 13 shows the results of the concentration prediction of progesterone (H13). DETAILED DESCRIPTION OF THE INVENTION

[0008] [About the compound detection sensor] The compound detection sensor of the present invention comprises an indicator solution containing a cucurbituril structure-containing compound and a dye, and an absorbance measuring device capable of measuring the change in absorbance of the solution before and after mixing the indicator solution with the analyte. In the compound detection sensor, the coexistence of the cucurbituril structure-containing compound and the dye results in the formation of a complex, such as the inclusion of the dye in the cucurbituril structure-containing compound, which changes the absorbance and absorption wavelength. Furthermore, when the analyte is added to the solution in which the dye is encapsulated within the cucurbituril structure-containing compound, the analyte is encapsulated in place of the encapsulated dye, which releases the dye from the complex, further changing the absorbance and absorption wavelength of the solution. This will be explained in detail using the schematic diagram in Figure 1. When dye 2 is mixed with inclusion compound 1 (in this invention, the cucurbituril structure-containing compound), dye 2 is encapsulated within inclusion compound 1, forming a complex, which changes the absorbance and absorption wavelength. In this state, if a target substance 3 (a compound to be measured) is further mixed, the target substance 3 is encapsulated in place of the dye 2, and the dye is released from the complex, causing the absorbance and absorption wavelength of the solution to change again.

[0009] (Cucurbituril structure-containing compound) The cucurbituril structure-containing compound used in the present invention is a compound containing the following structure (hereinafter referred to as a cucurbituril structure).

[0010] [ka]

[0011] In the formula, n is an integer from 5 to 10, and X independently represents a chalcogen atom selected from the group consisting of oxygen, sulfur, and selenium. A is a hydrogen atom, a group that can be substituted with a hydrogen atom (for example, A can be replaced with -OH (hydroxyl group) to improve solubility), or an organic group. Cucurbit[n]urils with n of 5, 6, 7, and 8 are particularly preferred, with n of 7 or 8 (CB[7] and CB[8]) being preferred, and n of 8 (CB[8]) being even more preferred. The rate of change in absorbance and absorption wavelength varies depending on the size of the encapsulation space of the cucurbituril ring of the cucurbituril structure-containing compound or the type of functional group of the cucurbituril structure-containing compound, and therefore can be determined depending on the object to be measured.

[0012] (dye) In the present invention, the dyes that can be used are not particularly limited, and any dye can be used. Dyes that have a strong interaction with cucurbituril structure-containing compounds, such as dyes that are easily included in cucurbituril structure-containing compounds, are particularly preferred, as are compounds that easily change their absorbance and absorption wavelength. Another advantage is that the interior of the cucurbituril ring of cucurbituril structure-containing compounds is hydrophobic, so hydrophobic dyes may be used. Here, hydrophobic dyes, i.e., hydrophobic organic compounds, refer to organic compounds that have hydrophobic groups at the terminals of the compound and exhibit hydrophobicity, such as organic compounds containing hydrophobic groups such as hydrocarbon groups. Furthermore, dyes that are easily soluble in organic solvents are preferred. For example, azobenzene-based compounds (compounds having an azobenzene skeleton or compounds in which one benzene ring in the azobenzene skeleton is replaced with pyridine) such as those shown in the following formula are preferred.

[0013] [ka] [ka] [ka] [ka]

[0014] In the above azobenzene-based compounds, R1 to R 10 are each independently a hydrogen atom, NH2, NHMe (Me represents a methyl group), NHPh (Ph represents a phenyl group), NMe2 (Me represents a methyl group), NEt2 (Et represents an ethyl group), OH, COOH, NO2, SO3Na, CH3, Cl, etc., and at least one of them is other than a hydrogen atom. Examples of dyes other than azobenzene-based compounds include pyridine-based compounds and naphthalene-based compounds.

[0015] The chemical formulas of specific dyes that can be used are shown below. [ka]

[0016] [ka]

[0017] [ka]

[0018] [ka]

[0019] [ka]

[0020] Other examples of dyes include the following: Azo dyes, anthraquinone dyes, phthalocyanine dyes, xanthene dyes, triarylmethane dyes, nitro dyes, indigoid dyes, metallocene dyes, acridine dyes, anthracene dyes, benzoquinone dyes, cyanine dyes, diarylmethane dyes, hemicyanine dyes, methine dyes, naphthol dyes, oxazine dyes, sulfonphthalein dyes Dyes, tetraphenylmethane dyes, dioxazine dyes, benzoin dyes, oxazine dyes, pyrrolidine dyes, thiophene dyes, pyryllium dyes, diazomethane dyes, isoxazole dyes, and isothiazole dyes.

[0021] More specific dyes are listed below. Azo dyes include azobenzene, methyl red, orange II, Sudan III, Sudan IV, and oil red O. Xanthene dyes include fluorescein, rhodamine B, and eosin Y. Anthraquinone dyes include alizarin, quinoline blue, solvent red 19, and anthrapurin. Phthalocyanine dyes include phthalocyanine blue and phthalocyanine green. Metallocene dyes include ferrocene. Triarylmethane dyes include Crystal Violet, Malachite Green, and Brilliant Green. Nitro dyes include Dinitroaniline Orange and Para-nitroaniline. Indigoid dyes include Indigo and Indigo Carmine.

[0022] (Measurement object) One of the features of the present invention is that the object to be measured is placed inside a cucurbituril ring. Therefore, the object to be measured is not particularly limited as long as it is a compound that is easily included in a compound having a cucurbituril structure, but since the inside of the cucurbituril ring is hydrophobic, it is preferable that the object to be measured is also a hydrophobic compound. For example, biological components include lipids, hydrophobic vitamins, terpenoids, prostaglandins, and steroid compounds. Pharmaceutical and supplement ingredients include analgesics, antibiotics, tranquilizers, antidepressants, antihistamines, sedatives, antiepileptics, anticancer drugs, hydrophobic amino acids, hydrophobic peptides, and hormone preparations. Food ingredients include lipids, hydrophobic vitamins, carotenoids, flavonoids, polyphenols, and steroid compounds. Food additive ingredients include preservatives, antioxidants, flavorings, emulsifiers, fungicides, seasonings, thickeners, and emulsion stabilizers. Daily necessities and cosmetic ingredients include humectants, preservatives, UV absorbers, surfactants, fragrances, antioxidants, emollients, and film-forming agents. Pesticide ingredients include organophosphates, carbamates, pyrethroids, neonicotinoids, triazoles, and strobilurins.

[0023] Specific examples of each are as follows: Examples of lipids that constitute living organisms include triolein, dioleoylphosphatidylcholine, cholesterol, ceramide, sphingomyelin, 1-monooleoylglycerol, 1,2-dioleoylglycerol, lecithin, phosphatidylethanolamine, and phosphatidylserine. Hydrophobic vitamins include vitamin A, vitamin D, vitamin E, vitamin K, vitamin D2, vitamin D3, retinol, retinal, tocopherol, and menaquinone. Terpenoids include limonene, squalene, carotene, lycopene, geraniol, myrcene, phytol, nerol, and vitamin K2. Examples of prostaglandins include prostaglandin E2, prostaglandin D2, prostaglandin F2α, prostaglandin I2, thromboxane A2, leukotriene B4, and leukotriene D4. Steroid compounds include cortisol, aldosterone, testosterone, estradiol, progesterone, dehydroepiandrosterone, estrone, estriol, androstenedione, and pregnenolone. Other examples include tocotrienols, lecithin, and propolis.

[0024] Pain relievers available as medicines and supplements include ibuprofen, naproxen, diclofenac, and acetaminophen. Antibiotics include erythromycin, clarithromycin, ciprofloxacin, and rifampicin. Mood stabilizers include clozapine, risperidone, olanzapine, and quetiapine. Antidepressants include fluoxetine, paroxetine, sertraline, and imipramine. Antihistamines include loratadine, fexofenadine, cyproheptadine, and hydroxyzine. Sedative-hypnotic drugs include diazepam, lorazepam, alprazolam, and midazolam. Antiepileptic drugs include carbamazepine, valproic acid, lamotrigine, and levetiracetam. Anticancer drugs include tamoxifen, doxorubicin, imatinib, and paclitaxel. Hydrophobic amino acids include leucine, isoleucine, valine, phenylalanine, tryptophan, methionine, proline, and alanine. Hydrophobic peptides include glutathione, angiotensin II, endorphins, casein phosphopeptides, insulin, and glucagon. Examples of hormone preparations include testosterone, estradiol, progesterone, dexamethasone, epinephrine, norepinephrine, and the compounds used in the examples below.

[0025] Lipids contained in food include triglycerides, cholesterol, linoleic acid, oleic acid, palmitic acid, stearic acid, α-linolenic acid, DHA, and EPA. Hydrophobic vitamins include vitamin A, vitamin D, vitamin E, and vitamin K. Carotenoids include β-carotene, lutein, lycopene, and zeaxanthin. Flavonoids include quercetin, rutin, catechin, and naringin. Polyphenols include epigallocatechin gallate, resveratrol, and curcumin. Steroids in foods include ergosterol, brassicasterol, sitosterol, and campesterol.

[0026] Food additive preservatives include calcium propionate, potassium sorbate, and sodium benzoate. Antioxidants include butylhydroxyanisole, butylhydroxytoluene, and propyl gallate. Flavoring agents include vanillin, methyl amyl ketone, and ethyl vanillin. Emulsifiers include lecithin, monoglycerides, and diglycerides. Antifungal agents include natamycin, imidazolidinyl urea, and sorbic acid. Seasonings include sodium glutamate, sodium inosinate, and sodium guanylate. Thickeners include carrageenan, xanthan gum, and guar gum. Emulsion stabilizers include sodium caseinate, gum arabic, carob bean gum, polymethyl methacrylate, ethyl cellulose, and acrylates copolymers.

[0027] Moisturizing agents contained in daily necessities or cosmetics include glyceryl stearate, cetyl alcohol, stearic acid, squalane, and isopropyl myristate. Preservatives include parabens, phenoxyethanol, methylchloroisothiazolinone, and methylisothiazolinone. UV absorbers include oxybenzone, avobenzone, homosalate, and octocrylene. Surfactants include sodium lauryl sulfate, sodium laureth sulfate, cocamidopropyl betaine, and polysorbate 80. Fragrances include linalool, citronellol, geraniol, and limonene. Antioxidants include vitamin E, vitamin C derivatives, butylhydroxytoluene, and butylhydroxyanisole. Emollients include shea butter, cocoa butter, jojoba oil, and mineral oil. Film formers include polyvinyl alcohol, polymethyl methacrylate, ethyl cellulose, and acrylate copolymers.

[0028] Organophosphate pesticides include parathion, malathion, chlorpyrifos, dimethoate, and fenitrothion. Carbamate pesticides include carbaryl, methomyl, carbofuran, and propoxur. Examples of pyrethroid pesticides include cypermethrin, deltamethrin, permethrin, and baifenthrin. Neonicotinoid pesticides include imidacloprid, clothianidin, acetamiprid, and thiamethoxam. Examples of triazole pesticides include tebuconazole, propiconazole, and myclobutanil. Strobilurin pesticides include azoxystrobin, pyraclostrobin, and trifloxystrobin. Other examples of pesticides include glyphosate, paraquat, dinotefuran, spinosad, abamectin, and fenbuconazole.

[0029] Among the compounds listed, steroid compounds are components that make up the human body, and are also used in medicines and supplements, and are found in food, so there is a demand for easy measurement of them. Therefore, it is preferable to measure cortisol, testosterone, corticosterone, cholic acid, dehydroepiandrosterone, estrone, estradiol, β-estradiol 17-acetate, β-estradiol 3-benzoate, estriol, ethinylestradiol, megestrol acetate, nandrolone, pancuronium bromide, prednisolone, progesterone, spironolactone, cholesterol, cholecalciferol / dehydrocholesterol, drospirenone, vecuronium bromide, etc.

[0030] The ease with which the substance to be measured is included in the cucurbituril structure-containing compound depends on the size of the part of the molecule that is easily included. When CB[7] and CB[8] are used, the molecular weight or number average molecular weight of the substance to be measured is preferably 100 to 900, more preferably 200 to 600, and even more preferably 250 to 400.

[0031] Since the object to be measured needs to be included in place of the dye that is included in the compound having a cucurbituril structure, the relationship between the object to be measured and the dye is preferably such that the binding constant of the object to be measured to the compound having a cucurbituril structure is higher than the binding constant of the dye to the compound having a cucurbituril structure.

[0032] (concentration) Regarding the concentrations used in the measurement, the molar ratio of the dye to the compound having a cucurbituril structure is not particularly limited, but it is preferable that 1 / 3 < (molar concentration of dye / molar concentration of compound having a cucurbituril structure) < 3.0, and it is more preferable that the molar concentration of the dye is equal to the molar concentration of the compound having a cucurbituril structure. Furthermore, the molar concentration of the object to be measured is preferably greater than the molar concentrations of the dye and the compound having a cucurbituril structure, because a higher molar concentration of the object to be measured than the molar concentrations of the dye and the cucurbituril facilitates substitution of the included compound.

[0033] (organic solvent) The organic solvent used as the solvent for the dye and analyte solution need not be particularly limited as long as it can dissolve the cucurbituril, analyte, and dye so that the absorbance of the indicator solution can be measured. However, an organic solvent that produces a large color difference between a solution containing both the dye and the cucurbituril structure-containing compound and a solution containing only the dye is preferred. Usable organic solvents include ethanol, dimethyl sulfoxide, 2-propanol, 1-methyl-2-pyrrolidone, ethylene glycol, γ-butyrolactone, tetraethylene glycol dimethyl ether, and dimethyl sulfoxide (DMSO).

[0034] (Water and pH) The indicator solution used in the present invention preferably uses water as the solvent, and therefore the mixed solvent used in the present invention is preferably a mixture of water and an organic solvent. The ratio of water to organic solvent is not particularly limited, but the color of the dye may change depending on the ratio. A preferred ratio, for example, by volume, is organic solvent:water = 1:1 to 0.1:9.9, more preferably 2:8 to 0.5:9.5, and even more preferably 2:8 to 1:9. The organic solvent may be 1.0 wt. % or more and 50.0 wt. % or less of the organic solvent / water mixture. Note that, depending on the type of dye used, it may be sensitive to pH. For example, the degree of color change of azo dyes varies depending on pH. Therefore, it is preferable to select a pH that produces a large color change depending on the dye. For example, when an azo dye is used, it is preferable to use it in an acidic state with a pH of about 2.0 to 6.0. The pH adjuster is not particularly limited, but examples thereof include hydrochloric acid, citric acid, nitric acid, sulfuric acid, sodium hydroxide, and aqueous ammonia.

[0035] (Absorbance measuring device) The absorbance measuring device used in the present invention is not particularly limited as long as it is an absorbance measuring device that measures changes in the absorbance and absorption wavelength of a solution before and after mixing a measurement target with an indicator solution, and examples include ultraviolet-visible spectrophotometers such as the UV-2600 and UV-2700 manufactured by Shimadzu Corporation. Furthermore, since changes in absorbance and absorption wavelength result in changes in color, a method may be used in which the color of the solution is recorded by imaging using an image sensor such as a CMOS sensor. Alternatively, if a light source is located behind the solution from the image sensor, the color of the transmitted light can be recorded by imaging using the image sensor. As mentioned above, in this specification, the image sensor is also considered to be included in the absorbance measuring device.

[0036] (Absorbance measurement) In the present invention, by comparing data from solutions under different conditions, it becomes possible to qualitatively (discriminately) identify and quantify the substance to be measured. Regarding the qualitative characterization of the object to be measured, if the characteristics of the object to be measured are reflected in the absorbance and absorption wavelength, in the case of a single substance, quantification is possible with just one measurement. As an example, the absorbance and absorption wavelength when using CB[7] may differ from those when using CB[8], and the difference in absorbance may allow the object to be identified. Regarding the quantification of a measurement target, if the measurement target is a single target and the target is already known, quantification can be achieved by performing only one measurement. When the object to be measured is a mixture, it is necessary to distinguish and quantify the substances by measuring multiple types, and it is required that the absorbance and absorption wavelength also change accordingly. The following describes a quantification method when the object to be measured (one type) is already known.

[0037] One aspect of the present invention is a method for measuring the concentration of a compound using a compound detection sensor, comprising: preparing an indicator solution containing a cucurbituril structure-containing compound and a dye; mixing a substance to be measured having a known concentration into the indicator solution; creating a calibration curve from an absorbance curve for an indicator solution that does not contain the object to be measured and an absorbance curve for an indicator solution that contains the object to be measured; preparing a detection solution containing an unidentified or unknown concentration of a target substance; mixing the detection solution with the indicator solution to obtain an absorbance curve of the detection solution; and determining the concentration of the unidentified analyte or analyte contained in the detection solution from characteristics obtained by comparing the calibration curve with the detection solution absorbance curve. The following explains each situation separately.

[0038] (1) Preparation of indicator solution First, the type of cucurbituril structure-containing compound and dye to be used for measurement are determined, and then the cucurbituril structure-containing compound and dye are added to an organic solvent such as DMSO. It is preferable to obtain multiple indicator solutions by varying the concentrations of the dye and cucurbituril structure-containing compound.

[0039] (2) Determination of the concentration of cucurbituril structure-containing compounds and dyes Next, the concentrations of the cucurbituril structure-containing compound and the dye are determined. The absorbance and absorption wavelength of the multiple indicator solutions prepared in (1) are measured to obtain an absorbance curve. From the manner in which the concentrations of the cucurbituril structure-containing compound and the dye change, the molar concentrations of the cucurbituril structure-containing compound and the dye are identified, and the absorption wavelength with the largest change is also identified. In the example, the absorbance measurement in Figure 2 corresponds to this step. For multifaceted measurements that also include identification of the substance to be measured, it is desirable to prepare an indicator solution using multiple types of cucurbituril structure-containing compounds and multiple types of dyes. Alternatively, indicator solutions may be prepared by fixing the concentrations of the cucurbituril structure-containing compound and the dye, and varying the type of solvent or mixing ratio.

[0040] (3) Measurement of absorbance of the solution containing the target substance Next, a solution containing a specific concentration of a cucurbituril structure-containing compound and a dye is prepared, and multiple concentrations of the target substance are added. The absorbance, absorbance, and absorption wavelength of each solution are measured. As the target substance replaces the included dye and the amount of non-included dye increases, an absorbance curve is obtained, showing an increase or decrease in absorbance at a specific wavenumber. While the replacement is typically performed at room temperature, the temperature and pressure may be adjusted as necessary. By plotting the absorbance at a specific absorption wavelength for each concentration, absorbance data corresponding to the target substance concentration can be obtained (referred to herein as a calibration curve, which corresponds to a theoretical curve). In the examples, the measurement results shown in Figures 3 and 4 correspond to the results of this step. It is preferable to prepare a calibration curve in advance for each target substance. This not only allows for the determination of concentration, but also allows for the identification of unidentified compounds.

[0041] (4) Measurement of the object to be measured Finally, the object to be measured is added to a solution containing a cucurbituril structure-containing compound and a dye at the same specific concentration as the solution used in (3), and the absorbance and absorption wavelength are measured and compared with the absorbance and absorption wavelength of the base, thereby enabling the identification or concentration of the object to be measured.

[0042] Although absorbance can be measured in any of the ultraviolet, visible, and infrared regions, it is preferable to measure it in the visible region, for example, from 400 nm to 600 nm, because the concentration of a pigment can be intuitively predicted visually.

[0043] It is also preferable to prepare a calibration curve using multiple dyes, compounds having a cucurbituril structure, and solvents to be used for identification or concentration measurement. By preparing a calibration curve in a matrix using multiple dyes, compounds having a cucurbituril structure, and solvents, it becomes possible not only to specify the concentration but also to identify unidentified compounds. [Example]

[0044] The present invention will be specifically described below using examples. However, the examples are merely specific examples of the present invention, and the present invention is not limited to the examples.

[0045] Example 1 Preparation and measurement of cortisol-containing solutions (Preparation of indicator solution) A 30% aqueous solution of hydrochloric acid (9.5 M) was diluted with ultrapure water to prepare Solution 1 so that the concentration of hydrochloric acid was 0.57 mM. Cucurbit[8]uril was dissolved in Solution 1 so that the concentration was 100 μM to prepare Solution 2. Next, 4-hydroxy-4'-dimethylaminoazobenzene was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 μM to prepare solution 3. Solutions 1, 2, and 3 were mixed to prepare a measurement solution (indicator solution). At this time, solution 3 was mixed so that its volume ratio relative to the total measurement mixture solution was 1 / 10. The ratios of Solution 1 and Solution 2 were adjusted to 0, 0.5, 1, 3, 7, 10, 20, 30, 40, 50, 60, and 70 μM for each sample. The 4-hydroxy-4'-dimethylaminoazobenzene concentration was 10 μM, and the DMSO content was 10% by volume.

[0046] DMSO was added to Solution 1 at a volume ratio of 1 / 10 to prepare a reference solution.

[0047] Measurements using the UV-visible spectrophotometer utilize a double-beam measurement method, measuring the reference and sample together to correct for fluctuations in the light source, enabling highly accurate measurements with a stable baseline for absorbance and absorption wavelength. The specific measurement method is as follows:

[0048] Two 3 mL reference solutions were placed in screw-capped quartz cells with a 1 cm optical path length. These reference solutions were placed on both the reference and sample sides of an ultraviolet-visible spectrophotometer (Shimadzu UV-2600) and baseline measurements were performed. Next, 3 mL of the measurement solution was placed in a measurement container, which was then placed on the sample side of a UV-Visible spectrophotometer, and the absorbance and absorption wavelength were measured. The UV-Visible spectrophotometer used was equipped with a mechanism that could adjust the sample temperature to 25°C. When changing the solution in the quartz cell during measurements with the UV-Visible spectrophotometer, it was thoroughly washed and dried before use. The measurement results are shown in Figure 2A. The values ​​listed next to the figure in Figure 2A are the concentrations of cucurbit[8]uril. From these results, it is predicted that at 20 μM, approximately 90 vol% of the dye is encapsulated in cucurbit[n]uril. Figure 2B also shows the appearance of the container containing the obtained sample. Figure 2B also shows the appearance when other dyes are used.

[0049] (Test using the object to be measured) Indicator solution 1 was prepared by mixing solutions 1, 2, and 3 so that the cucurbit[8]uril concentration was 20 μM. The concentration of 4-hydroxy-4'-dimethylaminoazobenzene was 10 μM, and the DMSO concentration was 10% by volume. Next, cortisol was dissolved in DMSO to a concentration of 50 mM to prepare cortisol-containing solution 50. Cortisol-containing solution 50 was also diluted with DMSO to a cortisol concentration of 10 mM to prepare cortisol-containing solution 10. Cortisol-containing solution 10 was also diluted with DMSO to a cortisol concentration of 2 mM to prepare cortisol-containing solution 2. Baseline absorbance measurements were performed using a UV-Vis spectrophotometer in the same manner as above, except that the liquid volume was 2.8 mL. Next, 2.8 mL of indicator solution 1 was placed in a screw-cap quartz cell with a 1 cm optical path length, a stir bar was added, and the cell was placed next to the sample. Absorbance and absorption wavelength measurements were then performed. Next, cortisol-containing solution was added to the quartz cell, according to the amount of cortisol-containing solution added (as shown in Table 1). The solution was stirred for 5 minutes using a magnetic stirrer, and absorbance and absorption wavelength measurements were performed using a UV-Vis spectrophotometer. Repeating this process allowed us to obtain the absorbance and absorption wavelength of each solution as the cortisol concentration increased. The results are shown in Figures 3 and 4. Figure 4 is a graph plotting absorbance values ​​at a specific wavelength against concentration. It was found that when cortisol was added to a solution containing a dye and cucurbit[n]uril, the absorbance and absorption wavelength gradually approached those of the original dye solution (Figure 3). Figure 4 shows the plot of the change in absorbance at 517 nm. It was found that the inclusion of cortisol in cucurbit[n]uril caused a change in absorbance. Furthermore, because the decrease in absorbance with an increase in concentration was monotonically decreasing, it was possible to create a calibration curve based on the absorbance and concentration values. This indicated that even when measuring a solution containing cortisol of unknown concentration, it was possible to derive the concentration from the absorbance value.

[0050] [Table 1]

[0051] Example 2 Preparation and measurement of testosterone-containing solution A solution was prepared and measured in the same manner as in Example 1, except that testosterone was used instead of cortisol, and the absorbance and absorption wavelength of the solution were obtained. The results are shown in Figures 3 and 4. Since the decrease in absorbance with an increase in testosterone concentration is also a monotonic decrease, it was found that even when a solution containing testosterone of unknown concentration is measured, it is possible to estimate the concentration from the absorbance value. In Figure 4, the horizontal and vertical axes are aligned for comparison. It can be seen that testosterone undergoes a more rapid change in absorbance than cortisol. This means that the binding constant of testosterone to cucurbit[n]uril is expected to be larger than that of cortisol.

[0052] [Creating a calibration curve] Calibration curves were prepared for cortisol and testosterone under the same conditions as in Examples 1 and 2 (dye concentration: 10 μM, solvent concentration: 0.57 mM (HCl) and 10 vol% DMSO; cucurbit[n]uril was CB[8]; however, the dye used was AzB-1-06). (The curves in Figure 5 correspond to the calibration curves.) Next, solutions containing the same dye and solvent as above were prepared, and cortisol and testosterone were added to prepare 20 μM and 100 μM cortisol solutions, and 6.0 μM and 20 μM testosterone solutions. The absorbance was measured. Based on the absorbance results and the calibration curve, the concentrations were estimated to be 23 μM and 96 μM for cortisol, and 6.3 μM and 20 μM for testosterone, respectively. Although there was some error, it was found that both cortisol and testosterone could be quantified.

[0053] Figure 6 shows the results when the dye concentration (10 μM) and solvent concentration (0.57 mM (HCl) and 10 vol% DMSO) were the same as those used to create the calibration curve, but the type of dye was changed (the type of dye is shown in Figure 6). The substances to be measured were cortisol (Figure 6(a)) and testosterone (Figure 6(b)), and the concentrations were the same as when creating the calibration curve.

[0054] By using multiple indicator solutions, multiple absorbance curves can be obtained, and Figure 6 shows a graph in which the absorbance value at a specific wavelength is plotted against the concentration on the horizontal axis. It was found that the absorbance value and the degree of change also vary depending on the dye. From this, a comprehensive analysis of the absorbance changes makes it possible to estimate the concentration and identify the substance.

[0055] Figure 7 shows the difference in absorbance when using CB[7] and CB[8] under the same conditions as when creating the calibration curve (dye concentration: 10 μM, solvent concentration: 0.57 mM (HCl) and 10 vol% DMSO, except that the dye used was AzB-2-02). The concentrations of CB[7] and CB[8] used were the same. The analytes to be measured were cortisol (Figure 7(a)) and testosterone (Figure 7(b)), and their concentrations were the same as when creating the calibration curve. Figure 7 shows that using multiple indicator solutions can generate multiple absorbance curves. From this, complex analysis of the absorbance changes makes it possible to estimate the concentration and identify the substance. A comparison of CB[7] and CB[8] is shown in Figure 7. Because the absorbance differs depending on the analyte, it was found that the substance can be identified from these differences.

[0056] Example 3 An experiment was conducted to determine whether various steroid hormones can be detected simultaneously. In this example, colorimetry was performed in combination with a pattern recognition technique to confirm whether steroid hormones can be simultaneously and quantitatively identified. To achieve colorimetric detection, 4-(N,N'-dimethylamino)-azobenzene derivatives (those in which X = -NH2 in the formula (1) below, indicator 1), those in which X = -OH in the formula (2) below, indicator 2) and 4-phenylazopyridine derivatives (indicator 3, formula (3) below) were used as dyes (hereinafter referred to as indicators).

[0057] [ka] [ka]

[0058] The conversion of indicators 1 and 2 to azonium structures results in dramatic color changes, and indicator 3 exhibits unique optical properties due to the presence of a pyridine unit as a proton acceptor.

[0059] The steroid hormones used were the following 15 types, and the detection ability of the sensor of the present invention (combination of indicator 1 and CB[8], combination of indicator 2 and CB[8], and combination of indicator 3 and CB[8]) was evaluated for these. The indicators were all 10 μM, CB[8] was 20 μM, and the steroid hormones were 30 μM. In the figure, "Control" does not contain steroid hormones. Other than these conditions, the experiment was carried out under the same conditions as in Example 1.

[0060] [ka]

[0061] The steroid hormones of interest are classified based on their structural geometry, including, for example, structures with a cyclohexan-2-one structure in the A ring and a methyl group at the 10-position, cationic skeletons, cyclohexanol, and phenol structures.

[0062] A linear discriminant analysis was performed on the results for these steroid hormones. Information on the absorbance and absorption wavelength before and after the addition of each hormone was obtained through 20 repeated measurements. The results are shown in Figure 8 (see Figures 9 to 11 for circles showing similar characteristics). Note that F in the figure indicates the contribution rate. In this experiment, the absorbance and absorption wavelength corresponding to the number of sensors x number of target species x repeated characteristics were used as input data. Because this input data is highly dimensional, classification was performed by compressing the dimensions to two or three using linear discriminant analysis. F1-F3 in the figure represent the contribution rates when all data was dimensionally compressed.

[0063] The group of circled compounds in Figure 9 (the cluster to the right of Factor 1 (F1)) was classified as compounds with a cyclohex-2-en-1-one structure in the A ring and a methyl group at carbon 10 (i.e., testosterone (H2), progesterone (H13), and drospirenone (H14)) and compounds with a cationic skeleton (i.e., pancuronium bromide (H11) and vecuronium bromide (H15)). The group of compounds circled in Figure 10 (the cluster in the middle of Factor 1 (F1)), namely cortisol (H1) and corticosterone (H3), exhibited low binding affinity due to their hydrophilic groups. Furthermore, in Figure 11, the circled compounds (cluster located to the left of F1) contained cyclohexanol structures (i.e., cholic acid (H4) and dehydroepiandrosterone (H5)) and phenolic structures (i.e., estrone (H6), 17β-estradiol (H7), 17α-ethynylestradiol (H8), and prednisolone (H12)). Thus, in this example, the compounds could be classified based on the differences in the A-ring structure, and the cluster distribution was mainly based on the order of binding affinity. It is noteworthy that the cluster distribution was almost entirely dependent on the order of binding affinity.

[0064] Example 4 Next, we conducted an experiment to determine whether quantitative analysis was possible using a mixture of the sex hormones testosterone (H2) and 17β-estradiol (H7). The total concentration of the two steroid hormones was 25 μM, the indicator was 10 μM, and CB[8] was 20 μM. The dyes used were indicators 1 to 3 from Example 3. Other conditions were the same as those in Example 1. The results are shown in Figure 12. Note that, as in Figures 8 to 11, F in the figure indicates the contribution ratio. In this experiment, the absorbance and absorption wavelength corresponding to the number of sensors × number of target species × repetition characteristics were used as input data. Because this input data was highly dimensional, classification was performed by compressing the dimensions to two or three dimensions using linear discriminant analysis. F1–F3 in the figure represent the contribution ratios when all data was compressed. Figure 12, which shows the canonical score plot of LDA (linear discriminant analysis), shows a distribution of molar ratios (H2:H7) of 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100, indicating that discrimination of the two sex hormones was achieved with a 100% accurate classification rate.

[0065] Example 5 This example further demonstrated the detection of progesterone (H13) in diluted human saliva samples. The goal was to predict the concentration of progesterone (H13) in human saliva samples. Regression analysis was performed using a support vector machine (SVM), which allows for the construction of a linear calibration curve from data showing a nonlinear sensor response. The concentrations of the salivary marker (progesterone (H13)) used were those shown in Figure 13, with the indicator at 10 μM and CB[8] at 20 μM. Other aspects were the same as in Example 1. As a result, the predicted data (diamonds) were distributed on the calibration curve by SVM (Figure 13). The RMSEC (root mean square error of the calibration curve) and RMSEP (root mean square error of prediction) were low, indicating that progesterone (H13) could be accurately predicted. The sensor consisting of indicators 1-3 and CB[8] showed a clear color change based on the substitution mode upon addition of the target steroid hormone. [Industrial Applicability]

[0066] The compound detection sensor and the method for measuring the concentration of a compound using this compound detection sensor can easily qualitatively or quantitatively determine the compound by measuring the absorbance of a solution containing the compound to be measured, and are therefore industrially applicable. [Explanation of symbols]

[0067] 1. Inclusion compounds (compounds containing cucurbituril structure) 2 Dye 3. Measurement object

Claims

1. A compound detection sensor that detects a compound by absorbance measurement, an indicator solution containing a cucurbituril structure-containing compound and a dye; and an absorbance measuring device that measures changes in absorbance and absorption wavelength of the solution before and after mixing the object to be measured with the indicator solution.

2. 2. The compound detection sensor according to claim 1, wherein the indicator solution comprises a mixed solvent consisting of an organic solvent and water.

3. 3. The compound detection sensor according to claim 2, wherein the organic solvent is present in an amount of 1.0% by weight or more and 50.0% by weight or less with respect to the mixed solvent.

4. 4. The compound detection sensor according to claim 3, wherein the pH of the indicator solution is 2.0 or more and 6.0 or less.

5. 2. The compound detection sensor according to claim 1, wherein the dye is an azobenzene-based compound.

6. 2. The compound detection sensor according to claim 1, wherein the measurement object is an organic compound containing a hydrophobic structure, and the molecular weight or number average molecular weight thereof is in the range of 100 or more and 900 or less.

7. 2. The compound detection sensor according to claim 1, wherein the measurement object is a steroid compound.

8. A method for measuring a concentration of a compound using the compound detection sensor according to claim 1, preparing an indicator solution containing a cucurbituril structure-containing compound and a dye; mixing a substance to be measured having a known concentration into the indicator solution; creating a calibration curve from an absorbance curve for an indicator solution that does not contain the object to be measured and an absorbance curve for an indicator solution that contains the object to be measured; preparing a detection solution containing an unidentified or unknown concentration of a target substance; mixing the detection solution with the indicator solution to obtain an absorbance curve of the detection solution; and determining the concentration of an unidentified analyte or analyte contained in the detection solution from characteristics obtained by comparing the calibration curve with the detection solution absorbance curve.

9. 9. The concentration measurement method according to claim 8, wherein the indicator solutions are prepared for each of a plurality of types of dyes and for each concentration of the dyes.

10. The concentration measurement method according to claim 8 , wherein the indicator solutions are prepared for each of a plurality of types of compounds having a cucurbituril structure and for each concentration of the compounds having a cucurbituril structure.

11. The concentration measurement method according to claim 8 , wherein the indicator solutions are prepared for a plurality of types of solvents and for a plurality of concentrations of the solvents.

12. The concentration measurement method according to claim 8 , wherein the calibration curve is created for each of the measurement objects.

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