Method for analyzing food component by self-assembled chemosensor

Abstract

[Problem] The present invention addresses the problem of providing a component analysis technique by which, by using a simpler configuration, a target substance, particularly a component contained in a foodstuff, can be efficiently detected through optical response. [Solution] It has been discovered that a multi-equilibrium self-assembled chemosensor system capable of patterning and discerning optical responses of respective target substances can be constructed by using a probe complex formed in a self-assembling manner via a non-covalent bond or a dynamic covalent bond by using three types of building blocks.

Description

Analysis method of food components using self-assembling chemosensor

[0001] The present invention relates to an analytical method for identifying the type and concentration of taste components in food using a self-assembling chemosensor, and to an analytical plate, reagent, and kit suitable for the method.

[0002] The flavor of food is determined by the type and concentration of flavor components with various chemical structures, and food freshness is closely related to structural changes caused by oxidation of flavor components. Also, considering the generation of allergens as food spoils, comprehensive food analysis targeting a wide variety of components is desirable.

[0003] Conventional techniques for analyzing taste components use large analytical instruments such as liquid chromatography / mass spectrometry (HPLC-MS). This method separates each type of taste component using a dedicated column, while detecting the mass of the specific component. However, the need for large analytical instruments makes it difficult to simultaneously analyze a wide variety of taste components without a separation procedure, and there are issues with the need for pretreatment of the actual sample and the lengthy measurement time due to complex procedures.

[0004] On the other hand, chemosensors are molecular-level chemical sensors that combine receptor and reporter units and exhibit optical response changes (color / fluorescence response) upon capture of target species (e.g., Patent Document 1). Chemosensors designed based on molecular recognition chemistry exhibit cross-response characteristics unique to artificial receptors, so a group of chemosensors arranged in an array (i.e., a chemosensor array) exhibits various optical response patterns depending on the type and concentration of target species. This optical response pattern can be considered a fingerprint pattern, and multivariate analysis can be used to simultaneously qualitatively analyze target species with diverse chemical structures and quantitatively determine their concentrations.

[0005] However, with conventional chemosensor arrays, a large number of chemosensors must be arranged to create one array, and furthermore, there was a problem in that only a small number of sensors among the large number of arranged chemosensors contribute to discrimination.

[0006] International Publication WO2018 / 169023

[0007] Therefore, an object of the present invention is to provide a component analysis method that can efficiently detect target substances, particularly components contained in food, by optical response with a simpler configuration.

[0008] The present inventors conducted extensive research to solve the above-mentioned problems and focused on the phenomenon of molecular self-assembly in order to design a chemosensor based on molecular recognition chemistry. In this invention, the building blocks of the chemosensor, a receptor compound and a reporter compound (dye compound), spontaneously form a probe complex (self-assembling chemosensor) through noncovalent or dynamic covalent bonds while exhibiting changes in optical behavior. When a target substance is added to the complex consisting of the receptor compound and the reporter compound, the optical response corresponding to the target substance changes further as the complex dissociates and a new complex is formed due to differences in the binding ability of the two compounds. The inventors discovered that by using multiple such probe complexes, it is possible to construct a multi-equilibrium self-assembling chemosensor system capable of patterning and distinguishing the optical responses of each target substance.

[0009] That is, in one aspect, the present invention provides: <1> a method for analyzing components in food, the method comprising: A) a step of preparing a probe solution containing a dye compound, a first receptor compound, and a second receptor compound, wherein the first receptor compound and the dye compound are linked to each other via a coordinate bond to form a first probe complex; and the second receptor compound and the dye compound are linked to each other via a dynamic covalent bond to form a second probe complex, and the first probe complex and / or the second probe complex exhibit an optical response due to a change in absorbance or fluorescence in the presence of a target substance; B) a step of adding a sample solution containing one or more target substances to the probe solution; C) a step of measuring the optical response accompanying the addition of the sample solution; and D) a step of identifying the type and / or concentration of the target substances contained in the sample solution based on the measurement result of the optical response; <2> The method according to <1> above, wherein the optical response occurs when the target substance binds to the first receptor compound, thereby breaking the coordinate bond, and / or when the target substance binds to either the second receptor compound or the dye compound, thereby breaking the dynamic covalent bond; <3> The method according to claim 1, wherein the change in absorbance or fluorescence is accompanied by a long-wavelength shift or a short-wavelength shift of the peak wavelength; <4> The method according to <1> above, wherein the dye compound is an aromatic compound having at least one OH group; <5> The method according to <1> above, wherein the dye compound is a compound having a catechol group; <6> The method according to <1> above, wherein the dye compound is selected from the following group: <7> The method according to <1> above, wherein the first receptor compound is a transition metal complex; <8> The method according to <1> above, wherein the first receptor compound is a transition metal complex having dipicolylamine as a ligand; <9> The method according to <1> above, wherein the second receptor compound is a phenylboronic acid derivative; <10> The method according to <1> above, wherein the target substance is a taste substance contained in food; <11> The method according to <1> above, wherein the target substance is selected from the group consisting of amino acids, nucleotide compounds, sugars, organic acids, kokumi components, and combinations thereof.

[0010] In another aspect, the present invention relates to an analysis plate, an analysis reagent, and an analysis kit suitable for the above-mentioned analysis method, specifically: <12> An analysis plate for analyzing components in food, the analysis plate having a plurality of sensing regions for detecting target substances on a substrate, the plurality of sensing regions forming a pattern with a predetermined distance from each other, the plurality of sensing regions containing probe reagents which may be the same or different, the probe reagent containing a dye compound, a first receptor compound, and a second receptor compound, the first receptor compound and the dye compound being linked to each other via a coordinate bond to form a first probe complex; and the second receptor compound and the dye compound being linked to each other via a dynamic covalent bond to form a second probe complex, the analysis plate being used in a method for identifying the type and / or concentration of the target substances contained in a sample solution from a change in absorbance or fluorescence accompanying the addition of the sample solution containing one or more of the target substances; <13> The analysis plate according to <12> above, wherein each of the plurality of sensing regions is separated by a hydrophobic material applied to the substrate; <14> A reagent for analyzing a component in food, comprising a dye compound, a first receptor compound, and a second receptor compound, wherein the first receptor compound and the dye compound are linked to each other via a coordinate bond to form a first probe complex; and the second receptor compound and the dye compound are linked to each other via a dynamic covalent bond to form a second probe complex, the reagent being used in a method for identifying the type and / or concentration of the target substance contained in a sample solution containing one or more target substances from a change in absorbance or fluorescence accompanying the addition of the sample solution; <15> A kit for detecting a target substance that is a component in food, comprising a solution containing a dye compound, a first receptor compound, and a second receptor compound stored therein, wherein the first receptor compound and the dye compound are linked to each other via a coordinate bond to form a first probe complex;and the kit, in which the second receptor compound and the dye compound are linked to each other via a dynamic covalent bond to form a second probe complex, is used in a method for identifying the type and / or concentration of the target substance contained in a sample solution based on a change in absorbance or fluorescence caused by the addition of the sample solution containing one or more target substances.

[0011] According to the present invention, by using a reagent containing multiple self-assembling chemosensors (probe complexes), it is possible to perform colorimetric / fluorescent detection of target substances, such as flavor components in food, thereby enabling the type and concentration of unknown components in a sample solution to be identified easily and with high accuracy. Because the self-assembling chemosensors are composed of non-covalent or dynamic bonds, it is possible to identify multiple target substances using reagents of the same composition by utilizing equilibrium bias based on differences in binding ability with the target substances. The self-assembling chemosensors can be constructed by mixing commercially available reagents without requiring new synthesis, which is advantageous in terms of simplicity and low cost.

[0012] Furthermore, in this invention, the detection response is imaged using a CCD camera or the like, and the image is then subjected to an automatic image analysis algorithm and pattern recognition method, enabling on-site qualitative / quantitative analysis through a simplified sensing process. This invention can be applied to the analysis of not only flavor components in food, but also biomarkers, environmental pollutants, etc.

[0013] FIG. 1 is a schematic diagram showing the process of forming the first and second probe complexes in the method of the present invention. FIG. 2 shows the change in absorption spectrum when glycine is added to the probe solution prepared in Example 1. FIG. 3 shows the change in absorption spectrum when guanylic acid is added to the probe solution prepared in Example 1. FIG. 4 shows the change in absorption spectrum when guanylic acid is added to the probe solution (ARS:3-NPBA:DPA.Zn ２＋ The change in absorption spectrum when fructose was added to the probe solution (ARS:3-NPBA:DPA.Zn) (the molar ratio of the two solutions was 1:100:12.5). ２＋The changes in absorption spectrum when fructose was added to the probe solution (ARS:3-NPBA:DPA.Zn) at a molar ratio of 1:100:37.5. Figure 6 shows the changes in absorption spectrum when glutathione was added to the probe solution prepared in Example 1. Figure 7 shows the changes in absorption spectrum when glutathione was added to the probe solution (ARS:3-NPBA:DPA.Zn) ２＋ The changes in absorption spectrum when γ-Glu-Val-Gly was added to a probe solution (ARS:3-NPBA:DPA.Zn) with a molar ratio of 1:100:12.5. ２＋ The graph shows the change in absorption spectrum when γ-Glu-Val-Gly was added to a sample solution (a molar ratio of 1:100:37.5). FIG. 9 shows an LDA plot (results of qualitative analysis) classified by taste components contained in the sample solution. FIG. 10 shows an LDA plot (results of semi-quantitative analysis) classified by taste components contained in the sample solution. FIG. 11 shows a graph showing the predicted concentration of each component in an unknown sample (SVM calibration). FIG. 12 shows the change in absorption spectrum when γ-Glu-Val-Gly was added to a sample solution (a molar ratio of 1:100:37.5) containing ARS:3-NPBA:DPA.Zn. ２＋ 13 is a graph showing the response pattern (fingerprint pattern) based on the change in absorption spectrum of the two-component mixed sample solution obtained for probe solution 2 (ARS:3-NPBA:DPA.Zn ２＋ FIG. 14 is an LDA plot (results of semi-quantitative analysis) for a two-component mixed sample solution. FIG. 15 is a graph showing a response pattern (fingerprint pattern) based on changes in the absorption spectrum of a three-component mixed sample solution. FIG. 16 is an LDA plot (results of semi-quantitative analysis) for a three-component mixed sample solution (in the presence of 1 mM fructose). FIG. 17 is an LDA plot (results of semi-quantitative analysis) for a three-component mixed sample solution (in the presence of 10 mM fructose). FIG. 18 is an LDA plot (results of semi-quantitative analysis) for an actual sample (umami seasoning). FIG. 19 is an LDA plot (results of semi-quantitative analysis) for an actual sample (umami seasoning) displayed from a different angle.

[0014] The following describes embodiments of the present invention. The scope of the present invention is not limited to these descriptions, and other than the following examples, the present invention can be implemented with appropriate modifications within the scope that does not depart from the spirit of the present invention.

[0015] 1. Detection Method of the Present Invention As described above, the method of the present invention is a method for analyzing ingredients in food, and includes the following steps A) to D): A) preparing a probe solution containing a dye compound, a first receptor compound, and a second receptor compound; B) adding a sample solution containing one or more target substances to the probe solution; C) measuring the optical response accompanying the addition of the sample solution; and D) identifying the type and / or concentration of the target substances contained in the sample solution based on the measurement results of the optical response.

[0016] 1-1. Step A) Step A) is a step of preparing a probe solution, which is an analytical reagent used to detect a target substance.

[0017] Here, the dye compound, the first receptor compound, and the second receptor compound are each building blocks that constitute a self-assembling chemosensor molecule, and they self-assemble to form a complex in solution. Specifically, the first receptor compound and the dye compound self-assemble to form a first probe complex by linking to each other via a coordinate bond. The second receptor compound and the dye compound self-assemble to form a second probe complex by linking to each other via a dynamic covalent bond. Therefore, two types of self-assembling chemosensor molecules, the first probe complex and the second probe complex, exist in the probe solution. Here, the term "dynamic covalent bond" refers to a bond form that is a covalent bond but undergoes reversible dissociation-binding in response to a specific external stimulus such as temperature, light, or an inhibitor.

[0018] Because the coordination bonds and dynamic covalent bonds used in the self-assembly of these probe complexes are both reversible chemical bonds, differences in binding ability lead to equilibrium bias among the probe complexes. Typically, when the three building blocks coexist, the first probe complex formed by the coordination bond is preferentially formed over the second probe complex formed via the dynamic covalent bond (Figure 1).

[0019] The first probe complex and / or the second probe complex exhibit an optical response due to a change in absorbance or fluorescence in the presence of a target substance. When a target substance is present, it binds to one of the building blocks forming these probe complexes, thereby dissolving the coordinate bond or dynamic covalent bond that constitutes the probe complex, resulting in a change in absorbance (color change) / fluorescence. From another perspective, when the probe complex is dissolved by the target substance in this way, the chemical equilibrium between the probe complexes further changes. As described below, the type and concentration of components contained in an unknown sample can be identified from the optical response pattern associated with this shift in multiple equilibria.

[0020] In a preferred embodiment, the dye compound, which is one of the building blocks, is an aromatic compound having at least one OH group. More preferably, the dye compound is a compound having a catechol group. As such a compound having a catechol group, a derivative in which an OH group is introduced into the phenyl group of an anthraquinone skeleton, a xanthene skeleton, a triphenylmethane skeleton, or a coumarin skeleton to form a catechol group can be used.

[0021] Specific examples of dye compounds having an anthraquinone skeleton, a xanthene skeleton, or a triphenylmethane skeleton include the following compounds.

[0022] These compounds are, from left to right, Alizarin Red S (ARS), Bromopyrogallol Red (BPR), Pyrogallol Red (PR), Pyrocatechol Violet (PV), and m-carboxyphenylfluorone (m-CPF).

[0023] Furthermore, specific examples of the first compound having a coumarin skeleton include 4-methylesculetin (ML: left) and esculetin (right) having the following structures.

[0024] The first receptor compound is capable of binding to the dye compound through a coordinate bond, and is preferably a transition metal complex. A preferred example of such a transition metal complex is a transition metal complex having dipicolylamine (DPA) as a ligand. The transition metal forming such a complex may be an element of Groups 3 to 12 of the periodic table, but is preferably an element of Group 12, and more preferably zinc (II) ion (Zn ２＋ The first receptor compound has the property of easily binding to amino acids among the target substances.

[0025] The second receptor compound can be bound to the dye compound through a dynamic covalent bond, and is preferably a phenylboronic acid derivative. These compounds form dynamic covalent bonds with two OH groups of the catechol group in the dye compound. Typically, the phenylboronic acid derivative is a phenylboronic acid derivative having a nitro group, and specific examples include 3-nitrophenylboronic acid (3-NPBA) and its esters.

[0026] In a preferred embodiment, the first probe complex is formed from a compound having a catechol group and a dipicolylamine zinc(II) complex; and the second probe complex is formed from a compound having a catechol group and 3-nitrophenylboronic acid (3-NPBA). However, the present invention is not limited to this combination.

[0027] 1-2. Step B) Next, step B) is a step of adding a sample solution containing one or more target substances to the probe solution prepared in step A).

[0028] The target substances of the present invention are components in foods, and although the scope can be expanded to any substance depending on the type of probe reagent, they are typically taste substances contained in foods. In this specification, the term "taste substance" refers to a substance that provides tastes such as kokumi (rich flavor) in addition to the five basic tastes of sweetness, saltiness, sourness, bitterness, and umami.

[0029] More specifically, the target substance, which is a taste component, is one or more selected from the group consisting of amino acids, nucleotide compounds, sugars, organic acids, kokumi components, and combinations thereof.

[0030] The amino acids to be targeted may be any amino acid, including aromatic amino acids and branched-chain amino acids, and may be either D- or L-isomers. Specific examples of preferred amino acids include, but are not limited to, taurine, glutamine, glutamic acid, proline, glycine, alanine, arginine, methionine, pyroglutamic acid, histidine, salts thereof, and protein hydrolysates.

[0031] Examples of nucleotide compounds (nucleic acids) that serve as target substances include inosinic acid, guanylic acid, and xanthylic acid.

[0032] The sugars that can be used as target substances can be any of monosaccharides, disaccharides, and polysaccharides, and examples thereof include, but are not limited to, the following: D-fructose (Fru), D-glucose (Glc), D-arabinose (Ara), L-rhamnose (Rha), L-ribose (Rib), L-fucose (Fuc), D-xylose (Xyl), N-acetyl-D-glucosamine (Ace), D-galactose (Gal), lactose (Lac), maltose (Mal), and sucrose (Suc).

[0033] Examples of organic acids that can be targeted include lactic acid, citric acid, and acetic acid.

[0034] Examples of target kokumi components include glutathione (γ-Glu-Cys-Gly), γ-glutamyl tripeptides such as γ-Glu-Val-Gly, and γ-glutamyl dipeptides such as γ-Glu-Met and γ-Glu-Thr.

[0035] When adding the sample solution to the probe solution, means known in the art, such as a micropipette or a syringe, can be used.

[0036] 1-3. Step C) Step C) is a step of measuring the optical response accompanying the addition of a sample solution. The change in absorbance (color change) or fluorescence of the probe solution to which the sample solution has been added is observed as the optical response.

[0037] As described above, the optical response is caused by the addition of a target substance, which dissolves the self-assembly of the first probe complex or the second probe complex, thereby shifting their chemical equilibrium. More specifically, the optical response is caused by the target substance binding to the first receptor compound, thereby dissolving the coordinate bond, and / or by the target substance binding to either the second receptor compound or the dye compound, thereby dissolving the dynamic covalent bond.

[0038] In a preferred embodiment, the change in absorbance or fluorescence in the optical response is accompanied by a long- or short-wavelength shift of the peak wavelength in the ultraviolet-visible absorption (UV) spectrum or fluorescence spectrum, thereby enabling more accurate identification of the target substance based on the optical response pattern.

[0039] The measurement of the optical response in step C) can be carried out using a UV spectrum measuring device or a fluorescence spectrum measuring device known in the art. In a preferred embodiment, the probe solution or an area containing the probe solution can be imaged by an imaging means to obtain an image showing the optical response due to a change in fluorescence or absorbance (color change) caused by the target substance. This allows the optical response caused by the target substance to be visually recognized and rapidly observed.

[0040] Such imaging means may be any imaging means capable of visualizing the fluorescence change or color change of the probe reagent as a two-dimensional image, and any device known in the art may be used. Typically, an imaging device such as a CCD camera or a scanner device such as a flatbed scanner may be used. This has the great advantage that the analysis in step D) described below can be performed using an imaging image obtained by a simple operation without using a conventional plate reader, large-scale spectrophotometer, fluorometer, or the like.

[0041] 1-4. Step D) Next, step D) is a step of identifying the type and / or concentration of the target substance contained in the sample solution based on the results of measuring the optical response.

[0042] Typically, linear discriminant analysis (LDA) is used to analyze the optical response patterns of the color changes or fluorescence changes obtained for various target substances, and the type and concentration of the target substance contained in a sample solution containing an unknown concentration of the target substance can be identified and predicted.

[0043] In a preferred embodiment, the image obtained in step C) can be analyzed using image pattern recognition such as a support vector machine (SVM), which is a machine learning model algorithm.

[0044] For example, when using SVM analysis, changes in fluorescence and the like accompanying the reaction between a probe reagent and a target substance are calculated in advance for each substance, and this is used as statistical training data. Then, from the images obtained as a result of measuring a sample solution containing multiple target substances at unknown concentrations, it is possible to determine what types of components are contained in the sample solution and further determine and predict their concentrations, which is called multi-component simultaneous quantitative analysis.

[0045] The analytical method of the present invention can be performed on any liquid sample containing a desired target substance, including liquid industrial products, pharmaceuticals, beverages, animal body fluids, etc. Non-limiting examples include soft drinks, vegetable juices, and sake.

[0046] The method of the present invention described above can be suitably used, for example, for quality control of food products or for monitoring the manufacturing process of the food products, including, but not limited to, controlling the fermentation process of fermented foods and monitoring precision fermentation.

[0047] 2. Analysis Plate, Analysis Reagent, and Analysis Kit The analysis method of the present invention described above is preferably performed using an analysis plate having multiple sensing regions that hold a probe solution (probe reagent) containing the first and second probe complexes. Use of such an analysis plate enables simultaneous analysis of multiple components.

[0048] More specifically, in one aspect, the present invention provides an analytical plate for analyzing components in food, which has a plurality of sensing areas on a substrate for detecting target substances, the plurality of sensing areas forming a pattern with a predetermined spacing from each other, and the plurality of sensing areas each containing probe reagents which may be the same or different.

[0049] Here, the probe reagent corresponds to the above-mentioned probe solution, and the details thereof have already been explained.

[0050] The base material of the analysis plate may be polyethylene, acrylic resin, glass, paper, or the like.

[0051] The sensing regions in the analysis plate may be arranged in a separate arrangement to detect multiple substances contained in a sample solution, i.e., they may form a pattern with a predetermined distance between them. In some cases, the predetermined distance is preferably a fixed distance. Therefore, each of the sensing regions is separated by an outer frame made of a hydrophobic (water-repellent) material applied to the substrate. The hydrophobic material is preferably an inert material that acts as an outer frame to separate each sensing region and prevent the probe reagents from mixing when applied to the individual sensing regions, and that does not interact with the probe reagents or target substances in the sample solution through chemical reactions or other interactions. Examples of such hydrophobic materials include waxy substances such as natural wax (e.g., paraffin) and synthetic wax (e.g., polyethylene wax), as well as fluororesins. When using a wax printer for patterning, the hydrophobic material is wax ink.

[0052] The analytical plate having such a patterned arrangement of sensing regions can be used as a chemical sensor array. Specifically, the plurality of sensing regions can form an array consisting of 96 to 1536 sensing regions, and typically can be a 96 array, a 384 array, or a 1536 array.

[0053] The size of each sensing region can be set appropriately depending on the volume of the sample solution to be measured, the number of arrays, or the resolution of an imaging means if used in step C), but for example, each sensing region can be independently a rectangle with a long side of 1 to 20 mm or a circle with a diameter of 1 to 20 mm.It is preferable that each sensing region has the same size.

[0054] Since each of the plurality of sensing regions holds a probe reagent, qualitative / quantitative detection of a target substance can be performed based on changes in fluorescence and absorbance obtained after the addition of a sample reagent. Note that, in this specification, the term "detection" is interpreted in the broadest sense to include measurements for various purposes, such as quantitative and qualitative measurements.

[0055] The multiple sensing regions may all be coated with the same probe reagent, or in some cases, two or more groups of sensing regions may be configured, one group coated with the same probe reagent and the other group coated with a different probe reagent. These can be adjusted as appropriate depending on the number of target substances contained in the sample solution, the purpose of detection, etc. The probe reagent can be applied to the sensing regions using an inkjet printer or the like.

[0056] In another aspect, the present invention relates to the above-mentioned probe reagent, i.e., an analytical reagent for analyzing ingredients in food, which comprises a dye compound, a first receptor compound, and a second receptor compound, thereby forming first and second probe complexes by self-assembly. The details of these dye compound, first receptor compound, and second receptor compound have been described above.

[0057] In yet another aspect, the present invention relates to a kit for detecting a target substance that is a component of food. The kit contains a solution containing a dye compound, a first receptor compound, and a second receptor compound. Details of the dye compound, the first receptor compound, and the second receptor compound have been described above.

[0058] Here, the dye compound, the first receptor compound, and the second receptor compound may be stored in separate containers or may be stored together in the same container. Furthermore, the dye compound, the first receptor compound, and the second receptor compound may be stored in the kit in the form of a solution in which they are dissolved, or may be stored as a powder sample.

[0059] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0060] Example 1 Preparation of Probe Solution A probe solution containing three building blocks of the present invention, namely, a dye compound, a first receptor compound, and a second receptor compound, was prepared. The concentrations of each were as follows:

[0061] The UV spectrum of the obtained probe solution was measured, and it was confirmed that an absorption peak was present around 530 nm.

[0062] Example 2 Identification and Analysis of Taste Components Using the probe solution prepared in Example 1, analysis of sample solutions containing various components was carried out.

[0063] The components to be measured were the following 5 groups and 17 types.

[0064] Here, the concentrations of all the components to be measured were 100 mM or less.

[0065] The changes in absorption spectra when glycine, guanylic acid, fructose, glutathione, and γ-Glu-Val-Gly were added to the probe solution are shown in Figures 2 to 8. As a result, different spectral changes were observed for each, and it was confirmed that the absorption intensity around 530 nm changed in all cases. Furthermore, a short-wavelength shift of the peak wavelength was observed for glycine, guanylic acid, glutathione, and γ-Glu-Val-Gly. On the other hand, when fructose was added, a long-wavelength shift of the peak wavelength was observed.

[0066] Furthermore, for γ-Glu-Val-Gly and fructose, the first receptor compound, DPA·Zn ２＋ The same measurement was carried out by changing the concentration of ARS:3-NPBA:DPA.Zn ２＋ Two probe solutions were used, with the molar ratios of DPA / Zn at 1:100:12.5 and 1:100:37.5. As shown in Figures 4 and 5, in the case of fructose, a long wavelength shift was observed with the addition of fructose. ２＋ A larger spectral change was observed when the ratio was 12.5, indicating that the optical response of fructose was due to a competitive reaction with the ARS·3-NPBA complex.

[0067] On the other hand, as shown in Figures 7 and 8, the addition of γ-Glu-Val-Gly, which is a kokumi component, resulted in a shift to longer wavelengths. ２＋ A larger spectral change was observed when the ratio was 37.5, suggesting that the optical response of γ-Glu-Val-Gly was similar to that of ARS, DPA, and Zn. ２＋ It was found that this was due to a competitive reaction with the complex.

[0068] The obtained changes in absorption spectrum were used to carry out qualitative analysis (species discrimination) and semi-quantitative analysis.

[0069] Qualitative analysis: The results (20 repeated measurements) of the absorption spectrum changes (color changes) obtained under fixed conditions of the concentration of the component to be measured were input into SYSTAT software manufactured by HULINKS, and qualitative analysis was performed using LDA (Linear Discriminant Analysis), a type of cluster analysis. The results are shown in Figure 9. As a result, an LDA plot classified by target substance contained in the sample solution was obtained, and the detection of each component could be identified from the color change.

[0070] Semi-quantitative analysis: Similarly, the results (measured 10 times) of the absorption spectrum changes (color changes) obtained when the concentration of the component to be measured was changed were entered into SYSTAT software manufactured by HULINKS, and semi-quantitative analysis was performed using LDA (linear discriminant analysis), a type of cluster analysis. The results are shown in Figure 10. As a result, an LDA plot (qualitative analysis) was obtained in Figure 9, which distinguished the type of target substance present at a specific concentration in the sample solution, and an LDA plot (semi-quantitative analysis) was obtained, which distinguished target species present at different concentrations, and the detection of each component could be identified from the color change.

[0071] Furthermore, quantitative analysis was performed using support vector machine (SVM) regression analysis with the optical response behavior obtained above as training data. As a result, the concentrations of various components, including sugars, amino acids, nucleic acids (nucleotide compounds), organic acids, and kokumi components, in a sample of unknown concentration (solvent: 100 mM HEPES buffer at pH 7.4) were predicted with high accuracy (Figure 11). In each figure, RMSEC is the calibration value of the root-mean-square error (RMSE), and RMSEP is the predicted value of RMSE. The closer the observed value and the predicted value, the smaller the value, indicating higher accuracy of the concentration prediction.

[0072] Example 3. Semi-quantitative analysis in a mixed solution (3-1) Two-component mixed system (five taste components) As in Example 1, the dye compound (ARS) of the present invention, the first receptor compound (DPA·Zn ２＋ Using a probe solution containing three building blocks, i.e., the first building block (3-NPBA) and the second receptor compound (3-NPBA), semi-quantitative analysis was performed on mixed sample solutions containing various combinations of two of the five taste components.

[0073] The following two types of probe solutions were used: <Probe solution 1> ARS:3-NPBA:DPA·Zn2+ = 1:100:12.5 (mol / mol) <Probe solution 2> ARS:3-NPBA:DPA·Zn2+ = 1:100:37.5 (mol / mol)

[0074] The combination of taste components in the mixed sample solution used is as follows:

[0075] As in Example 2, the results (measurements repeated 10 times) of the absorption spectrum changes (color changes) obtained when the concentration of each taste component in the mixed sample solution was changed were input into SYSTAT software manufactured by HULINKS, and semi-quantitative analysis was performed using LDA (linear discriminant analysis), a type of cluster analysis. As a result, different response patterns (fingerprint patterns) were obtained for the five types of taste components and their mixing conditions.

[0076] The response patterns obtained for probe solutions 1 and 2 are shown in Figures 12 and 13, respectively (in the figures, A ０ A: absorbance before adding the taste component, A: absorbance after adding the taste component. The same applies below.) As a result, it was revealed that the mode of change in absorption spectrum was significantly different depending on the composition ratio of the probe solution.

[0077] Furthermore, based on the obtained changes in absorption spectrum, semi-quantitative analysis was performed using an LDA plot in the same manner as in Figure 10. The results are shown in Figure 14. These results demonstrate that semi-quantitative analysis of taste components present at different concentrations in a mixed sample solution is possible.

[0078] (3-2) Three-component mixed system Next, a similar evaluation was performed on a three-component mixed system sample solution containing guanylate, glycine, and fructose. The probe solutions used were the above-mentioned probe solutions 1 and 2.

[0079] The response patterns (fingerprint patterns) obtained from the changes in absorption spectrum when the concentrations of each taste component in the mixed sample solution were changed are shown in Figure 15. As a result, it was found that even in the case of a three-component mixed sample solution, different response patterns were obtained depending on the solution conditions.

[0080] Furthermore, based on the observed changes in absorption spectra, semi-quantitative analysis was performed using LDA plots similar to that shown in Figure 10. The results showed that the tendency of the cluster distribution changed compared to the two-component system of guanylic acid and glycine. LDA plots for the presence of 1 mM and 10 mM fructose are shown in Figures 16 and 17, respectively. These results demonstrate that the self-assembling chemosensor system of the present invention can visualize the flavor balance composed of multiple taste components.

[0081] Example 4. Application to real samples Next, measurements were carried out on real seasoning samples using the self-assembling chemosensor system of the present invention.

[0082] Ten types of seasonings with different sensory qualities were used to prepare sample solutions for measurement according to the following procedure. The seasonings stored frozen were dissolved in a thermoshaker at 50°C for 10 minutes to prepare a stock solution. The stock solution was diluted 100-fold with HEPES buffer (pH 7.4), and the resulting solution was centrifuged (12,000 rpm, 4°C, 5 minutes). The supernatant was collected and filtered through a 0.5 μm filter.

[0083] The absorption spectrum of the sample solution was measured (measured 10 times) using the same method as in Example 2. The obtained changes in the absorption spectrum (380-800 nm) were subjected to semi-quantitative analysis using LDA (linear discriminant analysis). The LDA plots are shown in Figures 18 and 19. As a result, it was possible to distinguish between the sample group (samples 1-9) that showed a sensory flavor-enhancing effect and sample 10, which showed no enhancing effect.

Claims

1. A method for analyzing ingredients in food, comprising: A) preparing a probe solution containing a dye compound, a first receptor compound, and a second receptor compound, wherein the first receptor compound and the dye compound are linked to each other via a coordinate bond to form a first probe complex; and the second receptor compound and the dye compound are linked to each other via a dynamic covalent bond to form a second probe complex, and the first probe complex and / or the second probe complex exhibit an optical response due to a change in absorbance or fluorescence in the presence of the target substance; B) adding a sample solution containing one or more target substances to the probe solution; C) measuring the optical response accompanying the addition of the sample solution; and D) identifying the type and / or concentration of the target substance contained in the sample solution based on the measurement result of the optical response.

2. The method of claim 1, wherein the optical response is generated by the target substance binding to the first receptor compound, thereby breaking the coordinate bond, and / or by the target substance binding to either the second receptor compound or the dye compound, thereby breaking the dynamic covalent bond.

3. The method according to claim 1, wherein the change in absorbance or fluorescence is accompanied by a longer or shorter wavelength shift of the peak wavelength.

4. The method of claim 1, wherein the dye compound is an aromatic compound having at least one OH group.

5. The method according to claim 1, wherein the dye compound is a compound having a catechol group.

6. The method of claim 1, wherein the dye compound is selected from the group consisting of:

7. The method of claim 1, wherein said first receptor compound is a transition metal complex.

8. The method of claim 1, wherein the first receptor compound is a transition metal complex having a dipicolylamine ligand.

9. The method of claim 1, wherein said second receptor compound is a phenylboronic acid derivative.

10. The method according to claim 1, wherein the target substance is a taste substance contained in food.

11. The method of claim 1, wherein the target substance is selected from the group consisting of amino acids, nucleotide compounds, sugars, organic acids, kokumi components, and combinations thereof.

12. An analytical plate for analyzing ingredients in food, comprising a plurality of sensing regions for detecting target substances on a substrate, the plurality of sensing regions forming a pattern spaced apart from one another by a predetermined distance, the plurality of sensing regions each containing a probe reagent which may be the same or different, the probe reagent comprising a dye compound, a first receptor compound and a second receptor compound, the first receptor compound and the dye compound being linked to each other via a coordinate bond to form a first probe complex, and the second receptor compound and the dye compound being linked to each other via a dynamic covalent bond to form a second probe complex, the analytical plate being used in a method for identifying the type and / or concentration of the target substances contained in a sample solution from a change in absorbance or fluorescence accompanying the addition of a sample solution containing one or more of the target substances.

13. The microtiter plate of claim 12, wherein individual regions in said plurality of sensing regions are defined by a hydrophobic material coated on said substrate.

14. A reagent for analyzing ingredients in food, comprising a dye compound, a first receptor compound, and a second receptor compound, wherein the first receptor compound and the dye compound are capable of linking to each other via a coordinate bond to form a first probe complex; and the second receptor compound and the dye compound are capable of linking to each other via a dynamic covalent bond to form a second probe complex, said reagent being used in a method for identifying the type and / or concentration of one or more target substances contained in a sample solution from a change in absorbance or fluorescence accompanying the addition of the sample solution containing the target substances.

15. A kit for detecting a target substance that is an ingredient in food, the kit storing a solution containing a dye compound, a first receptor compound, and a second receptor compound, the first receptor compound and the dye compound being linked to each other via a coordinate bond to form a first probe complex; and the second receptor compound and the dye compound being linked to each other via a dynamic covalent bond to form a second probe complex, the kit being used in a method for identifying the type and / or concentration of the target substance contained in a sample solution based on a change in absorbance or fluorescence accompanying the addition of a sample solution containing one or more target substances.

16. The method according to claims 1 to 11 for quality control or monitoring of the production process of said food.

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