US-12618834-B2 - Measurement sample preparation method, analysis method, reagent, and reagent kit

US12618834B2US 12618834 B2US12618834 B2US 12618834B2US-12618834-B2

Abstract

Disclosed is a preparation method for preparing a measurement sample comprising an aggregate of metal nanoparticles having an analyte bound thereto, the preparation method comprising: contacting the analyte with a linker to bind the analyte to the linker; and contacting the linker that has been bound to the analyte with the metal nanoparticles to bind the linker to the metal nanoparticles.

Inventors

Yuki SHIMAOKA
Masaya Okada
Shigeki Iwanaga
Kazuki BANDO
Katsumasa Fujita
Yasunori Nawa
Satoshi Fujita

Assignees

SYSMEX CORPORATION
OSAKA UNIVERSITY
NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY

Dates

Publication Date: 20260505
Application Date: 20220304
Priority Date: 20210305

Claims (9)

1 . An analysis method for analyzing analytes in a sample using a spectroscopic analysis, the method comprising: preparing a measurement sample comprising, wherein the preparing comprises: contacting the analytes with linkers to bind the analytes to the linkers, whereby a plurality of complexes are formed, each complex having the analyte and the linker bound to the analyte; contacting the complexes with metal nanoparticles to bind the complexes to the metal nanoparticles, whereby a plurality of first aggregates are formed in a solution, each first aggregate having the metal nanoparticle and the complex bound to the metal nanoparticle; and adding an inorganic salt or an acid to the solution containing the plurality of the first aggregates, whereby a second aggregate is formed, the second aggregate consists of a plurality of the analytes, a plurality of the linkers and a plurality of the metal nanoparticles; obtaining an optical spectrum from the second aggregates as a measurement sample; and outputting information about a type of the analyte in the second aggregate on the basis of the obtained optical spectrum, wherein the analyte is at least one substance selected from the group consisting of a single amino acid, a nucleic acid, a catecholamine, a polyamine, an organic acid, an extracellular vesicle and a virus.
2 . The analysis method according to claim 1 , wherein the analyte has a functional group, and the linker has a reactive group capable of reacting with the functional group.
3 . The analysis method according to claim 2 , wherein the functional group is at least one selected from the group consisting of an amino group, a carboxyl group and a hydroxyl group.
4 . The analysis method according to claim 3 , wherein the functional group is an amino group and the reactive group is at least one selected from the group consisting of an N-hydroxysuccinimide ester group, an isothiocyanate group, an isocyanate group, an acyl azide group, a sulfonyl chloride group, an aldehyde group, an imide ester group, a fluorobenzene group, an epoxide group, a carbodiimide group, a carbonate group, and a fluorophenyl ester group.
5 . The analysis method according to claim 4 , wherein the reactive group is an N-hydroxysuccinimide ester group, and the linker is at least one component selected from the group consisting of dithiobis(succinimidyl propionate), dithiobis(succinimidyl undecanoate), dithiobis(succinimidyl octanoate) and dithiobis(succinimidyl hexanoate).
6 . The analysis method according to claim 1 , wherein the metal nanoparticle is a nanoparticle, the nanoparticle being at least one metal selected from the group consisting of gold, silver, platinum, copper and palladium.
7 . The analysis method according to claim 1 , wherein a particle diameter of the metal nanoparticle is equal or greater than 10 nm and equal or smaller than 150 nm.
8 . The analysis method according to claim 1 , wherein the sample is blood, serum, plasma, saliva or a body fluid, or is a solution of the analytes in which water or a buffer solution is used as a solvent.
9 . The analysis method according to claim 1 , wherein the spectroscopic analysis is a surface enhanced Raman scattering analysis.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from prior Japanese Patent Application No. 2021-035597, filed on Mar. 5, 2021, entitled “MEASUREMENT SAMPLE PREPARATION METHOD, ANALYSIS METHOD, REAGENT, AND REAGENT KIT”, and prior Japanese Patent Application No. 2021-035593, filed on Mar. 5, 2021, entitled “METHOD FOR ANALYZING TEST SUBSTANCE, ANALYZER, TRAINING METHOD, ANALYZER SYSTEM, AND ANALYSIS PROGRAM”, the entire contents of which are incorporated herein by reference. FIELD OF THE INVENTION The present invention relates to: a preparation method for preparing a measurement sample containing an aggregate of metal nanoparticles having an analyte bound thereto; an analysis method; a reagent; and a reagent kit. BACKGROUND US Patent Application Publication No. 2007/0155021 discloses a surface enhanced Raman scattering (SERS) active particle which comprises a metal-containing particle and a cationic coating provided on the metal-containing particle and carries a positive electric charge. US Patent Application Publication No. 2007/0155021 also discloses a SERS active particle which includes a metal-containing particle and a non-metal molecule, in which the metal-containing particle is derivatized with the non-metal molecule. US Patent Application Publication No. 2007/0155021 also discloses a method in which an analyte is contacted with SERS active particles to capture the analyte on the SERS active particles, the SERS active particles each having the analyte captured thereon are agglutinated, and a signal of a SERS spectrum is detected. The highly sensitive detection of an analyte has been required in various fields. For example, in a clinical test, it has been required to detect an amino acid in a protein with high sensitivity. In US Patent Application Publication No. 2007/0155021, detection sensitivity is improved by enhancing a signal of a SERS spectrum using a SERS active particle. However, further enhancement of the signal is still required. An object of the present invention is to provide: a measurement sample preparation method whereby the sensitivity of detection of an analyte can be improved; an analysis method; a reagent for preparing a measurement sample; and a reagent kit. SUMMARY OF THE INVENTION The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this summary. The present invention relates to a preparation method for preparing a measurement sample including an aggregate of metal nanoparticles bound to an analyte, the preparation method including: contacting the analyte with a linker to bind the analyte to the linker; and contacting the linker that has been bound to the analyte with the metal nanoparticles to bind the linker to the metal nanoparticles. It becomes possible to prepare a measurement sample whereby an analyte contained therein can be detected with high sensitivity. The present invention relates to an analysis method including: obtaining an optical spectrum from the measurement sample prepared by the preparation method; and outputting information about the analyte on the basis of the obtained optical spectrum. It becomes possible to detect the analyte with high sensitivity. The present invention relates to a reagent for use in the preparation of a measurement sample by the above-mentioned preparation method, the reagent including a linker that is not bound to a metal nanoparticle. The present invention relates to a reagent kit for use in the preparation of a measurement sample by the above-mentioned preparation method, the reagent kit including: the reagent; and metal nanoparticles that are packed separately from a linker. According to the present invention, the sensitivity of detection of an analyte can be improved. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing a measurement sample preparation method; FIG. 2 is a diagram showing an example of the configuration of an analysis device; FIG. 3A shows SERS spectra of measurement samples (Phe) which are prepared in accordance with the procedure of Examples. FIG. 3B shows SERS spectra of measurement samples which are prepared in accordance with the procedure of Examples except that no analyte is added. FIG. 3C shows SERS spectra of measurement samples which are prepared in accordance with the procedure of Examples except that DSP is not added. FIG. 3D shows SERS spectra of negative control samples in which neither an analyte nor DSP is added; FIG. 4 shows SERS spectra obtained using 20 types of amino acids as analytes. In FIG. 4, the symbol “a” indicates a spectrum obtained when an analyte is added, and the symbol “b” indicates a spectrum of a negative control which is obtained when the analyte is not added; FIG. 5 shows SERS spectrum obtained using dipeptides as analytes. In FIG. 5, the symbol “c” indicates a spectrum of a negative control which is obtained when the analyte is not added; FIG. 6A shows a SERS spectrum