Methods for analyzing organic molecules

Abstract

To provide a method for easily quantifying an organic molecule with high sensitivity.SOLUTION: According to one aspect of the present invention, an analysis method of an organic molecule is provided. The analysis method includes a first step, a second step and a third step, in that order. In the first step, a labeling material is brought into contact with an organic molecule. The second step includes at least one of a separation step and a concentration step. The separation step is a process of separating the organic molecule bound to the labeling material. The concentration process is a process of concentrating the organic molecules bound to the labeling material. In the third step, mass spectrometry with positional resolution is used to detect the labeling material, thereby performing mass spectrometry on the organic molecules labeled by the labeling material.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for analyzing organic molecules. [Background Art]

[0002] As means for analyzing organic molecules, nuclear magnetic resonance, gas chromatography, matrix-assisted laser desorption ionization and the like are employed (see Patent Document 1). Patent Document 1 describes a method for quantifying proteins contained in a test sample. [Prior Art Documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Unexamined Patent Publication No. 2014-102102 [Summary of the Invention] [Problem to be Solved by the Invention]

[0004] However, it is difficult for any of these methods to detect and quantify organic molecules with high sensitivity from a small amount of organic molecules. As another means, sensitivity can be increased by using a radioisotope or the like, but this cannot become a general analysis method in consideration of circumstances such as the burden on the operator and environmental handling.

[0005] In view of the above circumstances, the present invention provides a method that enables simple quantification of organic molecules with high sensitivity. [Means for Solving the Problem]

[0006] According to one aspect of the present invention, a method for analyzing organic molecules is provided. This analytical method comprises a first step, a second step, and a third step in that order. In the first step, a labeled substance is brought into contact with the organic molecules. The second step comprises at least one of a separation step and a concentration step. The separation step is a step of separating the organic molecules bound to the labeled substance. The concentration step is a step of concentrating the organic molecules bound to the labeled substance. In the third step, mass spectrometry of the organic molecules labeled with the labeled substance is performed by detecting the labeled substance using a mass spectrometry method with positional resolution.

[0007] According to this embodiment, a method can be provided that enables the quantification of organic molecules simply and with high sensitivity. [Brief explanation of the drawing]

[0008] [Figure 1] This diagram shows the basic structure of reagent devices used in immunochromatography. [Figure 2] This figure shows the results of the chromatographic development. [Figure 3] This is a graph showing the concentration information of the labeled substance. [Figure 4] This graph shows the results of elemental analysis performed simultaneously with the antibody being measured. [Modes for carrying out the invention]

[0009] The following describes the method for analyzing organic molecules according to the present invention. The various features shown in the embodiments below can be combined with each other.

[0010] Chromatography is a widely used method for qualitative and quantitative analysis of components contained in a sample. Chromatography employs two phases: a mobile phase and a stationary phase positioned in contact with the mobile phase. By flowing the sample through the mobile phase, the components can be separated by utilizing the differences in migration speed in the mobile phase and the varying adsorption forces between the different substances in the sample and the components in the stationary phase.

[0011] When the mobile phase is a gas, it is called gas chromatography, and when the mobile phase is a liquid, it is called liquid chromatography. Furthermore, depending on the shape of the equipment used, there are types such as column chromatography and thin-layer chromatography, and depending on the interaction used, there are types such as adsorption chromatography, ion exchange chromatography, and partition chromatography.

[0012] Immunochromatography is a type of chromatography that utilizes antigen-antibody reactions.

[0013] The following explanation will use immunochromatography as an example to describe the quantitative analysis of organic molecules.

[0014] In the following explanation, we will use the detection of antibodies to SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2), a type of virus classified in the genus Betacoronavirus of the family Coronaviridae, as an example.

[0015] Figure 1 shows the basic structure of a reagent device used in immunochromatography. The test specimen 1, which is a reagent device used in immunochromatography, is generally composed of the components shown in Figure 1. As shown in Figure 1, the test specimen 1 has a support substrate 10 and, arranged on the support substrate 10, a sample supply unit 11, a reagent unit 12, a development layer 13, a capture unit 14, a control unit 15, and an absorption unit 16. When the development direction in which the test sample develops the development layer 13 is taken as the reference, the reagent unit 12 is located upstream of the development layer 13, and the absorption unit 16 is located downstream of the development layer 13.

[0016] The support substrate 10 is a strip-shaped (flat) component called a backing sheet. The support substrate 10 supports the various components that make up the test piece 1. The main material used for the support substrate 10 is resin. For example, polystyrene, polypropylene, polycarbonate, polyester, cellulose acetate, polyethylene terephthalate, etc., are commonly used.

[0017] The sample supply unit 11 is strip-shaped, and is configured to absorb a dropped test sample and send the absorbed test sample to the development layer 13. The sample supply unit 11 is arranged such that an edge thereof is in contact with the reagent unit 12, and supplies the dropped test sample to the reagent unit 12. The sample supply unit 11 is not particularly limited as long as it has liquid permeability, and is formed of, for example, a porous material, a fibrous material, or the like.

[0018] The reagent unit 12 is impregnated with a SARS-coronavirus-2 antigen and a labeled antibody. The antigen and the labeled antibody specifically bind to an antibody contained in the test sample, and are supplied to the development layer 13 in a state where a labeled antigen-antibody complex is formed. In the reagent unit 12, the antigen and the labeled antibody are releasably supported (held) on a porous carrier called a conjugate pad. The constituent material of the reagent unit 12 is not particularly limited as long as it is liquid-permeable, and for example, similarly to the sample supply unit 11, the reagent unit 12 is formed using a porous material or a fibrous material.

[0019] Here, the labeled antibody is an antibody (secondary antibody) labeled by binding to a labeling substance. Examples of the secondary antibody include IgG antibodies, IgA antibodies, IgM antibodies, IgE antibodies, and IgD antibodies.

[0020] Examples of the labeling substance include biotin, enzymes, fluorescent dyes, metal nanoparticles, and the like, but nanoparticles containing a considerable number of atoms are preferable. Known particles such as latex particles can be employed as the nanoparticles, but metal nanoparticles are preferred. With this configuration, a plurality of signals can be generated for one organic molecule through a simple labeling operation. Further, examples of metal nanoparticles include gold nanoparticles, silver nanoparticles, and the like, but it is preferable to use gold nanoparticles. One gold nanoparticle has 10 7 gold atoms on the order of, whereby, compared to directly detecting organic molecules, 10 7A doubled signal can be obtained. In the present specification, commercially available metal nanoparticles or metal nanoparticles produced by a known method shall be used. In addition, the average particle diameter of the metal nanoparticles is typically 10 nm or more and 150 nm or less, preferably 20 nm or more and 40 nm or less. In the present specification, the "average particle diameter" refers to the value of D50 (median diameter; particle diameter corresponding to a cumulative 50% frequency distribution) measured by a laser diffraction particle size analyzer.

[0021] Furthermore, gold nanoparticles exist as a colloid in a fluid. When an antibody is labeled with gold nanoparticles, the labeling is performed by mixing the antibody and gold colloid in a buffer solution. As a result, gold colloid adheres to the antibody, the antibody is labeled, and a labeled antibody can be obtained.

[0022] In addition, as a method for forming the reagent section 12 by supporting the labeled antibody on a porous carrier, for example, a method including preparing an aqueous solution containing the labeled antibody, impregnating the porous carrier with this aqueous solution, and then drying the porous carrier can be mentioned. By such a method, the labeled antibody can be uniformly supported on the porous carrier.

[0023] The developing layer 13 is a mobile phase that is a section for chromatographically developing a test sample by capillary action. Hereinafter, "chromatographic development" refers to developing a sample with the developing layer 13. The developing layer 13 has a strip shape, and is arranged such that its upstream edge in the longitudinal direction is in contact with the reagent section 12, and its downstream edge is in contact with the absorbing section 16. The developing layer 13 is composed of, for example, a porous carrier, and may be composed of a membrane formed of a cellulose derivative such as nitrocellulose or cellulose acetate, a glass filter, filter paper, or the like.

[0024] In addition, in the middle of the developing layer 13, a capture section 14 and a control section 15 arranged downstream of the capture section 14 are disposed.

[0025] The capture unit 14 is a stationary phase on which a capture antibody (primary antibody) that specifically binds to the antigen-antibody complex formed in the reagent unit 12 is immobilized. As shown in Figure 1, the capture unit 14 is formed in a line (band) shape perpendicular to the longitudinal direction of the development layer 13. Hereinafter, the line formed in the capture unit 14 will be referred to as the "detection line". When a test sample is supplied from the reagent unit 12, the primary antibody immobilized in the capture unit 14 captures the antigen contained in the test sample. At this time, since an antigen-antibody complex has been formed from the antigen supplied to the development layer 13, when the antigen is captured in the capture unit 14, the labeled antibody is also captured. When the labeled antibody is captured in the capture unit 14, the detection line becomes colored, a band is formed, and a signal can be detected. Note that if the sample does not contain an antigen, the labeled antibody is not captured in the capture unit 14, and no band is formed.

[0026] Furthermore, capture antibodies (primary antibodies), like secondary antibodies, are not particularly limited as long as they specifically recognize the antigen-antibody complex, but examples include IgG antibodies, IgA antibodies, IgM antibodies, IgE antibodies, and IgD antibodies.

[0027] Furthermore, as a method for immobilizing the capture antibody on the development layer 13, if a nitrocellulose membrane is used as the porous support of the development layer 13, the capture portion 14 can be formed by dropping an aqueous solution containing the capture antibody in a line, followed by drying and washing.

[0028] The control section 15 is a stationary phase on which an anti-antibody (anti-labeled antibody) that captures the labeled antibody is immobilized. The control section 15 is located on the opposite side of the capture section 14 from the reagent section 12. The control section 15 is formed in a linear shape perpendicular to the longitudinal direction of the development layer 13.

[0029] Secondary antibodies that do not form an antigen-antibody complex pass through the capture unit 14 and are captured in the control unit 15. If a reaction between the immobilized anti-antibody and the labeled antibody is confirmed in the control unit 15, it indicates that the labeled antibody contained in the test sample supplied to the development layer 13 has spread from the upstream side of the development layer 13 to the control unit 15 located downstream.

[0030] Antibodies are not particularly limited as long as they have the function of capturing labeled antibodies, but examples include IgG antibodies, IgA antibodies, IgM antibodies, IgE antibodies, and IgD antibodies.

[0031] The absorbent section 16 is shaped like a strip, and its upstream edge in the longitudinal direction is positioned to contact the unfolding layer 13. It absorbs (absorbs water from) the inspection sample that has been unfolded by the unfolding layer 13 from the downstream end of the unfolding layer 13. As a result, the absorbent section 16 has the function of assisting capillary action in the developing layer 13, allowing for smoother chromatographic development in the developing layer 13. The absorbent section 16 is not particularly limited as long as it is permeable to liquid, but for example, it can be formed from a porous material, a fibrous material, etc., similar to the sample supply section 11.

[0032] Inductively coupled plasma mass spectrometry (ICP-MS) is a mass spectrometry method that uses plasma as an ion source and is used for quantitative analysis of electronic materials and other substances. In ICP-MS, high-frequency radio waves are used to ionize argon gas, generating a high-temperature plasma. This plasma ionizes the atoms in the sample, and then the concentration is measured in a vacuum to perform mass analysis.

[0033] ICP-MS can be combined with other techniques, such as liquid chromatography-inductively coupled plasma mass spectrometry (IC-ICP-MS) and laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS). Laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS) is a technique that combines laser ablation (LA) technology with ICP-MS. In LA-ICP-MS, a pulsed laser is irradiated onto the sample to atomize it into particles, which are then introduced into the ICP-MS for analysis.

[0034] The steps in this invention will be described in order. The analytical method in this invention includes a first step, a second step, and a third step, in that order.

[0035] <First step> In the first step, the labeling substance is brought into contact with the antibody to be measured, which is an organic molecule. Specifically, for example, a blood sample containing the antibody to be measured is dropped onto a reagent section 12 impregnated with an antigen and a labeled antibody containing the labeling substance, bringing the antigen, the labeled antibody, and the organic molecule into contact. This forms an antigen-antibody complex between the antigen, the antibody to be measured, and the labeled antibody, and the antibody to be measured binds to the labeling substance via the antigen. However, the method is not limited to this; the blood sample containing the antibody to be measured may also be mixed with a reagent containing the antigen and the labeled antibody to bind the antibody to the labeling substance. The following explanation will use the case where the labeling substance is a metal nanoparticle as an example, but is not limited to this. The labeling substance may be any nanoparticle other than metal.

[0036] <Second step> The second step includes at least one of a separation step and a concentration step. For example, the second step may combine multiple steps by chromatographically developing a test sample containing an organic substance that has been in contact with a labeled substance. The separation step is a step of separating organic molecules bound to the labeled substance. Specifically, for example, by chromatographically developing a blood sample dropped into the reagent section 12, the antigen-antibody complex formed in the first step is separated by capillary action from interfering molecules such as other components in the blood, free labeled substances not bound to the antibody to be measured, and components pre-contained in each component constituting the test piece 1. In this specification, "organic molecules bound to a labeled substance" includes not only cases where the labeled substance and the organic molecule are directly bound, but also cases where the substance bound to the labeled substance binds to an organic substance to form a complex.

[0037] The concentration step is a process of concentrating organic molecules bound to the labeled substance. Specifically, for example, the antigen-antibody complex formed in the first step is specifically captured in the capture unit 14 and concentrated on the detection line.

[0038] By incorporating these processes, it is possible not only to further increase the signal obtained from the labeled substance, but also to reduce background noise and dramatically reduce the risk of detecting non-organic molecules as if they were the organic molecules in question. In addition, this increases the signal-to-noise ratio and dramatically improves quantitative accuracy.

[0039] Here, after careful consideration, the inventors concluded that in order to increase the analytical sensitivity of organic molecules, as a means of generating multiple signals for a single organic molecule, fine particles containing a considerable number of atoms, such as metal nanoparticles, could be attached to or bound to the organic molecule as a labeling substance. By detecting the labeling substance rather than the organic molecule, the apparent signal could be dramatically increased. However, in reality, due to losses in the mass spectrometry process, the signal obtained from a single gold nanoparticle is only about 100 to 1000 signals. In contrast, as described above, by performing the second step, the labeling substance can be separated or concentrated, ensuring sufficient reliability and quantitative accuracy.

[0040] Furthermore, the second step may further include a dispersion step. The dispersion step is a step of dispersing organic molecules bound to the labeling substance. Specifically, antigen-antibody complexes, interfering molecules, free labeling substances, and components contained in each component constituting test piece 1 are dispersed and held in a nitrocellulose thin film. The nitrocellulose thin film functions as a controlled matrix, not only separating and stably holding the antigen-antibody complexes from other substances, but also enabling the quantification of these substances in the third step. Such quantification does not affect the quantification of organic molecules and can be performed simultaneously with the quantification of organic molecules. This makes it possible to calculate the total sample volume of the test sample in reverse, eliminating the need for highly accurate control of the sample volume.

[0041] <Third step> In the third step, the labeled substance is detected by a mass spectrometry method with positional resolution, thereby performing mass spectrometry of the organic molecules labeled with the labeled substance. Specifically, in the third step, the labeled substance is detected from the membrane support used in chromatographic development by a mass spectrometry method with positional resolution, thereby performing mass spectrometry of the organic molecules labeled with the labeled substance. With this configuration, the apparent signal can be dramatically increased by detecting the labeled substance rather than the organic molecules.

[0042] The mass spectrometry method used in the third step is not limited to any analytical means that has spatial resolution, but examples include quadrupole type, field sweep type, time-of-flight type, etc. Preferably, the mass spectrometry method is inductively coupled plasma mass spectrometry, and in particular, laser ablation inductively coupled plasma mass spectrometry is preferred. This makes it possible to perform localized ultratrace elemental analysis only at the laser irradiation site, and to simultaneously quantify multiple elements while having a spatial resolution of 10 μm or less. This means that multiple labeling materials can be used simultaneously, that is, multiple target organic molecules can be analyzed at the same time. Also, since mass separation is performed, even if it is desired to label with the same element, isotopes can be used, and the chemical properties can be completely preserved, but they can be detected as different labeling materials at the detection stage. Having high spatial resolution is synonymous with spatial concentration. For this reason, even with 1 nm gold nanoparticles (an aggregate of about 1,000 gold atoms), a signal of tens of thousands of cps can be obtained. In other words, by attaching one gold nanoparticle to each protein molecule, that protein molecule can be reliably detected. By rapidly moving the laser irradiation position and recording the resulting mass spectrum along with positional information—a technique known as elemental mapping—the labeled substance, which has been spatially dispersed, can be detected and quantified very efficiently and quickly. Furthermore, even when the same labeled substance is attached to two or more analytes, by making the spatial dispersion location-dependent and performing elemental mapping, the two or more analytes can be accurately distinguished. In the third step of spatially dispersing the organic molecules containing the labeled substance, it is possible to choose whether to concentrate them in a very narrow area or disperse them over a wide area, depending on the purpose and concentration.

[0043] The analytical methods for organic molecules described above offer the following additional advantages.

[0044] Traditionally, labeling materials have been preferred for their high coloration and diverse coloration, and labeling materials possessing the quantum dot effect, which allows for the free manipulation of emission color, have been used. While labeling materials with the quantum dot effect can simultaneously detect multiple target molecules, the degree of freedom in their color diversity is limited to approximately ±10-20 nm, which is the resolution of optical filters and monochromators used in optical detectors. Therefore, in principle, the simultaneous analysis of 12 to 25 substances is limited. Even with the quantum dot effect, detection still relies on optical methods, making it difficult to significantly improve detection sensitivity compared to conventional labeling methods.

[0045] In contrast, the present invention, in principle, allows for the simultaneous analysis of a number of substances equivalent to the product of the number of elemental types and isotopes with high sensitivity, significantly improving the simultaneous analysis capability compared to labeling methods using the quantum dot effect.

[0046] Furthermore, when attempting to detect a single organic molecule, one method involves ionizing that single organic molecule and then performing mass spectrometry. However, simply executing this method requires measures to prevent the organic molecule from decomposing during the ionization process, measures to accurately separate very large molecular weight molecules by mass, and measures to reliably detect a single ion, making it extremely difficult. In contrast, the present invention allows for the simple and highly sensitive quantification of organic molecules.

[0047] Furthermore, it has been essential that labeling substances do not ionize, i.e., remain in a dissolved state. This is because ionization increases fluidity, making it difficult to detect as a color difference; in other words, it worsens contrast and consequently reduces detection sensitivity. Additionally, it becomes difficult to distinguish between the color development of the original fine particles and the color development of the ions, leading to the disadvantage of exhibiting a false positive reaction.

[0048] However, with the detection method according to the present invention, the mass spectra obtained for particles and ions are decisively different, so interference with the analysis due to the ionization of the labeling material can be minimized. For this reason, ionizable materials can be used as labeling materials, and it is expected that the diversity of usable labeling materials will greatly expand. Furthermore, they may be provided in the following embodiments.

[0049] (1) A method for analyzing organic molecules, comprising a first step, a second step, and a third step in that order, wherein the first step involves contacting the organic molecules with a labeled substance, the second step comprises at least one of a separation step and a concentration step, the separation step being a step of separating the organic molecules bound to the labeled substance, the concentration step being a step of concentrating the organic molecules bound to the labeled substance, and the third step being a method for performing mass spectrometry of the organic molecules labeled with the labeled substance by detecting the labeled substance using a mass spectrometry method having positional resolution.

[0050] According to this embodiment, high molecular weight organic molecules such as proteins can be easily quantified with high sensitivity.

[0051] (2) The analytical method described in (1) above, wherein the second step further comprises a dispersion step, the dispersion step being a step of dispersing the organic molecule bound to the labeling substance.

[0052] This configuration makes it possible to reduce the effects of decomposition, contamination, and dispersion without affecting the quantitative determination of organic molecules.

[0053] (3) The analytical method described in (1) or (2) above, wherein in the second step, the test sample containing the organic molecule that has been in contact with the labeling substance is subjected to chromatographic development, which also serves as a plurality of steps included in the second step, and in the third step, the organic molecule labeled with the labeling substance is subjected to mass spectrometry by detecting the labeling substance from the membrane carrier used in the chromatographic development using a mass spectrometry method with positional resolution.

[0054] In this configuration, quantitative measurement of antibodies becomes possible using immunochromatography.

[0055] Like this

[0056] (4) An analytical method according to any one of (1) to (3) above, wherein the labeled substance is a nanoparticle.

[0057] This configuration makes it possible to eliminate false positives caused by pigments.

[0058] (5) An analytical method in which, in any one of (1) to (4) above, the mass spectrometry method is inductively coupled plasma mass spectrometry.

[0059] (6) The analytical method described in (5) above, wherein the inductively coupled plasma mass spectrometry is laser ablation inductively coupled plasma mass spectrometry. Of course, this is not always the case.

[0060] As previously described, various embodiments of the present invention have been explained, but these are merely examples and do not limit the scope of the invention in any way. These novel embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their variations are included in the scope and spirit of the invention, as well as in the claims of the invention and its equivalents. [Examples]

[0061] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In these examples, blood 25 days after vaccination with the SARS-CoV-2 virus was used as the test sample. Gold nanoparticles were used as the labeling substance. Antibodies of the SARS-CoV-2 virus in human blood were used as the organic molecule to be quantified in the test. In the second step, the organic molecule containing the labeling substance was separated, concentrated, and spatially dispersed using an immunochromatographic method with SARS-CoV-2 virus antigen.

[0062] In this experimental system, interfering molecules include proteins in the blood, matrix substances include metallic substances such as iron and zinc in the blood, sodium chloride in the immunochromatographic developing solution, and nitrocellulose is used as a controlled matrix.

[0063] Figure 2 shows the results of chromatographic development. As shown in Figure 2, the detection line of the capture unit 14 was colored and a band was formed, confirming that a large number of gold nanoparticles, which are the labeling substance, had accumulated in the capture unit 14. This confirmed that the test sample contained antibodies against the SARS-CoV-2 virus. In this way, the presence or absence of antibodies against the SARS-CoV-2 virus can be determined by utilizing the fact that metal nanoparticles can be optically identified as coloration.

[0064] Figure 3 is a graph of the concentration information of the labeled substance. In the third step, when elemental mapping analysis is performed using LA-ICP-MS with the nitrocellulose paper matrix, concentration information of the labeled substance, including positional information, can be obtained, as shown in Figure 3. The elemental concentration for IgG can be calculated by multiplying the height of the detected signal by the amount corresponding to the positional relationship, i.e., from the area of ​​the graph.

[0065] In this embodiment, the elemental concentration related to positional IgG can be calculated by multiplying the height of the detected signal by the amount corresponding to the positional relationship, i.e., from the area of ​​the graph. Based on the height of the detected signal, the detection sensitivity is estimated to be at least 100 times greater than that of visual inspection.

[0066] The positional resolution of a single laser shot is approximately 10 μm, and it is impossible to distinguish lines of this width by visual inspection using coloring. Therefore, as shown in Figure 3, the lines are dispersed over a horizontal area of ​​1.5 mm. However, using LA-ICP-MS, quantitative analysis is possible even at 10 μm, and all of the dispersed areas over a horizontal area of ​​15 mm can be integrated, resulting in a signal that is equivalent to obtaining 150 times greater signal strength.

[0067] Furthermore, in this experiment, vertical integration was not performed as shown in Figure 3. Since this width is 3 mm, it was confirmed that integrating the entire width would yield a signal 300 times stronger. Combining all of these factors, a sensitivity 4.5 million times higher than that of visual inspection was achieved.

[0068] Figure 4 is a graph showing the results of elemental analysis performed simultaneously with the labeled substance. As shown in Figure 4, it is also possible to simultaneously quantify metallic substances such as iron, copper, and zinc present as matrix material in the blood. Isotope ratios can also be determined as needed. Furthermore, it is possible to distinguish between ionized substances and particles such as nanoparticles in the test sample, and to detect and quantify them.

[0069] Conventionally, precise control of the sample volume (amount of test sample) is necessary to enable quantitative determination of the substance to be measured using immunochromatography, but it has been difficult to accurately control the amount of test sample dropped onto test piece 1. In contrast, according to the present invention, it is possible to quantify substances other than the substance to be measured contained in the test sample simultaneously with the quantification of the substance to be measured. A typical example is as follows: That is, the amount of iron contained in human blood is usually determined almost uniquely, but in this embodiment, as shown in Figure 4, since the iron concentration information has been obtained, the amount of blood (sample volume) dropped onto test piece 1 can be calculated in reverse. In this way, by combining the second and third steps, it becomes possible to accurately calculate the amount of test sample used for analysis. Furthermore, this makes it possible to quantify the concentration of antibodies in the blood.

[0070] One major problem with immunochromatography is the occurrence of false positives, known as ghost lines. While there are various possible causes, it is generally thought to be due to the specific detachment of the labeling substance due to abnormally strong antigen-antibody binding, leaving the chromophore behind. In the results for Day 0-20 shown in Figure 3, a faint line is observed at the IgG site visually, which corresponds to a ghost line. In other words, it would normally result in a false positive. However, analysis using the present invention showed that no gold was detected at the IgG site, and the result remained negative. This demonstrates that the present invention can effectively eliminate false positives.

[0071] Furthermore, the analysis time required by this invention is less than 1 second when analyzing a 15 mm dispersion interval with a resolution of 10 μm using a 60 kHz laser. Since the ELISA method, which allows for semi-quantification, takes about 3 hours for analysis, this demonstrates that the present invention is also superior in terms of rapid analysis.

[0072] Based on these results, organic molecules can be quantified easily and with high sensitivity. [Explanation of symbols]

[0073] 1: Test specimen 10: Support substrate 11: Sample supply unit 12: Reagent Department 13: Deployment layer 14: Supplementary section 15: Control Unit 16: Absorbent part

Claims

1. A method for analyzing organic molecules, The process includes the first step, the second step, and the third step in this order. In the first step described above, the labeled substance is brought into contact with the organic molecule. The second step described above includes a separation step and a concentration step, The separation step is a step of separating the organic molecule bound to the labeling substance, The aforementioned concentration step is a step of concentrating the organic molecule bound to the labeled substance, In the third step, the labeled substance is detected by a mass spectrometry method having positional resolution, thereby performing mass spectrometry on the organic molecules labeled with the labeled substance. Here, in the second step, the test sample containing the organic molecule that has been in contact with the labeled substance is subjected to chromatographic development, which also serves as one of the steps included in the second step. In the third step, by using laser ablation inductively coupled plasma mass spectrometry as the mass spectrometry method, the labeled substance is detected from the membrane carrier used in the chromatographic development, and the mass spectrometry of the organic molecules labeled with the labeled substance is performed. Analysis method.

2. In the analytical method described in claim 1, The second step further includes a dispersion step, The aforementioned dispersion step is a step of dispersing the organic molecule bound to the labeling substance. Analysis method.

3. In the analytical method described in claim 1, The labeling substance is a nanoparticle. Analysis method.

Images