JP2018132350A - Method for detecting hetero cyclic amine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for easily detecting hetero cyclic amines (HCA).SOLUTION: A test piece, a colloid liquid of noble metal nanoparticles, and an aqueous solution of inorganic salts aggregating colloid are brought into contact with each other so that a self-assembly of the noble metal nanoparticles including the test piece is formed, a measurement sample liquid is prepared and is subjected to a Raman spectrometer so that a surface enhanced Raman scattering spectrum is measured, and hetero cyclic amines are detected in the test piece.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a simple detection technique for harmful substances in food, and more particularly to a simple detection method for heterocyclic amines (HCAs) suspected of causing cancer.

  When meat is heated, for example, PhIP (2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine: 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine), Trp-P-1 (3-Amino-1,4-dimethyl-5H-pyrido [4,3-b] indole: 3-amino-1,4-dimethyl-5H-pyrido [4,3-b] indole) , MeIQx (2-Amino-3,8-dimethylimidazo [4,5-f] quinoxaline: 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline), MeIQ (2-Amino-3,4 -dimethylimidazo [4,5-f] quinoline: 2-amino-3,4-dimethylimidazo [4,5-f] quinoline), A-α-C (2-Amino-9H-pyrido [2,3-b) ] indole: 2-amino-9H-pyrido [2,3-b] indole), Glu-P-1 (2-Amino-6-methyldipyrido [1,2-a: 3 ', 2'-d] imidazole: 2-amino-6-methyldipyrido [1,2-a: 3 ', 2'-d] imidazole), Glu-P-2 (2-Aminodipyrido [1,2-a: 3', 2'-d] imidazole) : 2-aminodipyrido [1,2-a: 3 ', 2'-d] imidazole), MeA-α-C (2-Amino-3-methyl-9H-pyrido [2,3-b] indole: 2- Amino-3-methyl-9H-pyrido [2,3-b] indole) Trp-P-2 (3-Amino-1-methyl-5H-pyrido [4,3-b] indole: 3-amino-1-methyl-5H-pyrido [4,3-b] indole), IQ (2 -Amino-3-methylimidazo [4,5-f] quinoline: 2-amino-3-methylimidazo [4,5-f] quinoline) and other heterocyclic amines (HCAs) are known to be produced .

  These HCAs are suspected to be mutagenic and cancer-inducing. It is thought that there is a threshold of HCA concentration in the cancer-inducing action of HCA. There is a need for a method for simply detecting whether the HCA contained in a food is below a threshold or above a threshold. As described in Non-Patent Document 1, mass spectrometry is mainly used for high-sensitivity and rapid detection of HCA. On the other hand, Raman spectroscopy in which a sample is irradiated with a laser to measure a Raman scattering spectrum peculiar to HCA is an excellent simple detection method for heterocyclic compounds including HCA. However, since the concentration that can be detected by Raman spectroscopy is usually a percentage (%) level, higher sensitivity is required.

  As a technique for enhancing the sensitivity of Raman spectroscopy, surface-enhanced Raman scattering (SERS) using an enhanced electric field of a noble metal nanostructure having appropriate localized plasmon resonance is well known (Patent Document 1, Non-Patent Document 1). Reference 2). In fact, an example of SERS in which a heterocyclic compound such as pyridine, which is an HCA analog in a broad sense, is sensitized using a silver nanofilm having nano-level roughness has been reported (Non-patent Document 3). As a method for measuring substances with SERS, Patent Document 1, Patent Document 2, Non-Patent Document 3, and Non-Patent Document 4 have been proposed, but as far as the inventors know, SERS of HCA including MeIQx has yet to be demonstrated. It wasn't been done. At least, a method that can easily obtain HERS SERS has not yet been developed.

Japanese Patent No. 4772273 US Pat. No. 8,699,019

Formation of heterocyclic amine adducts by heating food and its control by cooking method, Hiroyuki Kataoka, Urakami Foundation Research Report, 16, 130 (2006) Spectroscopy of surface plasmons II, surface-enhanced Raman scattering, Tamtake Ito, spectroscopic study, 64 (2), 1-16 (2015) D. L. Jeanmaire and R. P. Van Duyne, J. Electroanal. Chem., 84, 1-20 (1977) Ultrasensitive analysis of gold nanoparticle self-assembly by surface-enhanced Raman scattering, Takao Fukuoka, Yasumori Mori, Oreoscience, 14 (1), 5-10 (2014)

  An object of this invention is to provide the simple detection method and detection kit of heterocyclic amine (HCA).

  Surface Enhanced Raman Scattering (SERS) is a phenomenon in which Raman scattering of Raman active molecules in the vicinity of nanostructures is significantly enhanced by localized plasmons generated by collective oscillation of electrons in noble metal nanostructures. It is. The present inventors have found that trace amounts of heterocyclic amines (HCA) can be easily and rapidly detected using noble metal nanoparticle self-assembly and surface-enhanced Raman scattering, and have completed the present invention. .

The present invention provides a simple method for detecting the following heterocyclic amines.
[1] A specimen, a colloid liquid of noble metal nanoparticles, and an aqueous solution of an inorganic salt that aggregates the colloid are brought into contact to form a self-assembly of noble metal nanoparticles including the specimen, and a measurement sample liquid is prepared. Prepared,
Simple measurement of heterocyclic amines by measuring the surface-enhanced Raman scattering spectrum by applying the measurement sample solution to a Raman spectrometer, and detecting the cyclic amines in the specimen by the expression of specific Raman shift of the heterocyclic amines Detection method.
[2] The simple detection method according to [1], wherein the noble metal nanoparticles are gold nanoparticles or silver nanoparticles.
[3] The heterocyclic amine is PhIP (2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine: 2-amino-1-methyl-6-phenylimidazo [4,5- b] pyridine), Trp-P-1 (3-Amino-1,4-dimethyl-5H-pyrido [4,3-b] indole: 3-amino-1,4-dimethyl-5H-pyrido [4,3 -b] indole), MeIQx (2-Amino-3,8-dimethylimidazo [4,5-f] quinoxaline: 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline), MeIQ (2- Amino-3,4-dimethylimidazo [4,5-f] quinoline: 2-amino-3,4-dimethylimidazo [4,5-f] quinoline), A-α-C (2-Amino-9H-pyrido [ 2,3-b] indole: 2-amino-9H-pyrido [2,3-b] indole), Glu-P-1 (2-Amino-6-methyldipyrido [1,2-a: 3 ', 2' -d] imidazole: 2-amino-6-methyldipyrido [1,2-a: 3 ', 2'-d] imidazole), Glu-P-2 (2-Aminodipyrido [1,2-a: 3', 2 '-d] imidazole: 2-aminodipyrido [1,2-a: 3', 2'-d] imidazole), MeA-α-C (2-Amino-3-methyl-9H-pyrido [2,3-b ] indole: 2-amino-3-methyl-9H-pyrido [2,3- b] indole), Trp-P-2 (3-Amino-1-methyl-5H-pyrido [4,3-b] indole: 3-amino-1-methyl-5H-pyrido [4,3-b] indole ), IQ (2-Amino-3-methylimidazo [4,5-f] quinoline: 2-amino-3-methylimidazo [4,5-f] quinoline), [1] or [2] Simple detection method as described.

  According to the simple detection method of the present invention, regardless of the concentration of the sample HCA, an appropriate aggregate dispersion (measurement sample) in which the respective absorbances at the laser wavelength of the Raman spectrometer and the Raman scattering wavelength of the HCA are sufficiently increased. Therefore, by irradiating a laser having a wavelength suitable for the dispersion, the SERS of HCA in the subject can be easily obtained, and quantitative analysis can be performed easily and rapidly.

In the examples, a SERS spectrum (solid line) and a background spectrum of MeIQx acquired by irradiating a laser of 785 nm (8 mW) with a 20 × objective lens for 1 second using a colloid of gold nanoparticles and a microscopic Raman spectrometer (Example) (Dotted line). FIG. 3 is an absorption spectrum of a dispersion of gold nanoparticle aggregates obtained by mixing a solution containing a sample HCA and a colloidal solution of gold nanoparticles in an example and adding a salt to cause aggregation.

Hereinafter, the present invention will be described in detail, but the present invention is not limited thereto.
The present invention is an HCA detection method for simple analysis of HCA by measuring a measurement sample solution in which a specimen is dispersed in a noble metal nanoparticle self-assembly by a surface enhanced Raman scattering (SERS) method.

  The present invention comprises mixing an analyte, a colloidal solution of noble metal nanoparticles such as gold nanoparticles or silver nanoparticles, and a salt solution for aggregating gold nanoparticles or silver nanoparticles in this order, A dispersion of an aggregate of gold nanoparticles or silver nanoparticles adsorbing HCA molecules in a specimen is prepared, measured directly with a Raman spectrometer, and identified by a specific peak of HCA. The detection method of the present invention realizes high-sensitivity detection of HCA by SERS easily and quickly.

The subject can be prepared by a normal method. For example, a solution that may contain HCA can be prepared from meat by chloroform extraction.
Colloids of noble metal nanoparticles such as gold nanoparticles or silver nanoparticles can be synthesized by known methods. The conditions for aggregating the colloid vary depending on the concentration of the colloid and the surface charge of the nanoparticles, but it is sufficient to add a salt at a concentration that gives sufficient ionic strength for rapid aggregation, which is obvious from Schulze-Hardy's law. . The kind of the salt is not particularly limited as long as it is a substance having a salting-out effect, but an aqueous solution of an inorganic salt such as sodium chloride, potassium chloride, calcium chloride, sodium nitrate, potassium nitrate, or calcium nitrate can be suitably used.

As the noble metal nanoparticles, particularly gold nanoparticles and silver nanoparticles can be preferably used.
The conditions for aggregating the colloid of the noble metal nanoparticles differ depending on the concentration of the colloid and the surface charge of the nanoparticles, but a salt having a concentration that gives an ionic strength sufficient to cause rapid aggregation may be added. The kind of the salt is not particularly limited, and an aqueous solution of an inorganic salt such as sodium chloride, potassium chloride, calcium chloride, sodium nitrate, potassium nitrate, or calcium nitrate can be suitably used. In many cases of gold nanoparticles obtained by reducing 0.2 mM to 1.5 mM chloroauric acid, sodium chloride may be added to a concentration of 30 mM to 100 mM, but is not limited thereto. .

  When the colloid is aggregated, the progress of aggregation of the gold nanoparticles or silver nanoparticles adsorbed with the sample HCA molecules can be adjusted by adjusting either or both of the mixing operation and the aggregation time. Adjustment of mixing operation can be performed by the presence or absence of stirring or the adjustment of stirring speed. As the stirring method, (1) a method of allowing a container containing aggregates to be covered and tumbling the container to flow a solution containing aggregates, and (2) shaking the container containing aggregates to remove the aggregates. (3) A method in which a stirrer is placed in a container containing aggregates, and the stirrer is rotated by a magnetic stirrer. (4) A centrifugal force is applied to the container in which aggregates are added to form aggregates. (5) A method of applying pressure to a solution containing aggregates to cause the solution containing aggregates to flow; (6) Pipetting that sucks up the solution containing aggregates with a pipette and immediately discharges the solution. It can be preferably selected from a method of flowing a solution containing aggregates by operation, and a method of (7) applying a pressure to a container containing aggregates and deforming the container to flow a solution containing aggregates. To these Not a constant. Stirring can be carried out intermittently or continuously, but it is preferable to carry out stirring for a short time intermittently to obtain a coagulated state suitable for SERS measurement. In addition, the aggregation time can be adjusted by adjusting the time required from the addition of salt to the colloidal solution containing the sample HCA to the measurement by the Raman spectrometer. The shorter the time it takes to measure with a Raman spectrometer after aggregating and preparing a measurement sample solution, the more preferable. Alternatively, both the mixing operation and the aggregation time may be adjusted. The progress of aggregation can be adjusted by adjusting either the mixing operation or the aggregation time, or both. Once an aggregate dispersion is obtained, it is measured with a Raman spectrometer. The measurement by the Raman spectroscope is desirably completed before precipitation occurs in the aggregate dispersion.

  From the knowledge of Non-Patent Document 2, in the absorption spectrum of the measurement sample solution, in the absorption spectrum of the measurement sample solution, the wavelength of the irradiation laser and the wavelength of Raman scattering generated thereby are preferable so that the aggregate of gold nanoparticles or silver nanoparticles is preferable. It is desirable to adjust so that absorption is sufficiently strong. At this time, when comparing the measured values of a plurality of samples, it is more desirable to adjust either the mixing operation or the aggregation time, or both so that the absorption becomes comparable.

  The Raman scattering wavelength X [nm] can be calculated from the peak Raman shift Y [1 / cm] appearing in the SCA spectrum of HCA and the wavelength L [nm] of the laser used by the following equation.

X = 1 / (1 / L−Y / 10000000)
Therefore, in the absorption spectrum of the measurement sample solution, attention can be paid to the absorption intensity at the wavelength L of the laser and the wavelength X of Raman scattering, and it can be predicted whether it is preferable for developing SERS.

  HCA expresses a Raman shift of a specific peak in Raman spectroscopic analysis. In the present invention, by performing surface enhanced Raman scattering (SERS) analysis using noble metal nanoparticles (colloid), the above characteristic Raman shift is enhanced to detect and identify a trace amount of chemical agent. be able to. In particular, using a Raman analyzer that is suitable for on-the-spot analysis of ultra-small portable devices, the self-assembly formed by adding an inorganic salt such as sodium chloride to a specimen mixed with precious metal nanoparticles (colloid) is analyzed. Therefore, it is possible to perform simple and quick on-site analysis with high sensitivity.

Hereinafter, the present invention will be specifically described with reference to examples.
All the equipment used was washed with aqua regia, and the operation was performed in a laboratory equipped with a clean bench. 50 ml of 0.01% tetrachloroauric acid (HAuCl ₄ ) aqueous solution is heated to boiling, and 0.5 ml of 1% trisodium citrate (Na ₃ (C ₃ H ₅ O (COO) ₃ )) aqueous solution is added. Then, tetrachloroauric acid was reduced to synthesize a colloid of gold nanoparticles having a particle diameter of approximately 40 nm. The colloid of gold nanoparticles was red.

  To 54 μL of this gold nanoparticle colloid, add a methanol solution of MeIQx to a predetermined concentration (60 nM, 100 nM, 500 nM), add an aqueous sodium chloride solution, and agglomerate by repeatedly pipetting with a 1 mL micropipette. An aggregate dispersion containing MeIQx was prepared. At this time, a measurement sample solution was prepared so that the total amount of the dispersion was 60 μL. The operation for preparing the measurement sample did not require as much as 30 seconds. In this way, a measurement sample solution, which is a dispersion of aggregates containing 60 nM, 100 nM, and 500 nM MeIQx, was prepared. The dispersion of gold nanoparticle aggregates (measurement sample solution) was reddish purple regardless of the MeIQx concentration. Further, in order to examine the size of the background, a blank solution was prepared by adding ultrapure water instead of the methanol solution of MeIQx.

  Using a Raman Raman spectrometer RAM300 / 785 manufactured by Lambda Vision equipped with a 20 × objective lens, the measurement sample solution and the blank solution were irradiated with a 785 nm (8 mW) laser for 1 second, and a Raman spectrum was obtained.

  The result is shown in FIG. As can be seen from FIG. 1, characteristic and clear SERS can be obtained from a dispersion containing MeIQx in a measurement time of 1 second, and SERS spectra having different intensities according to MeIQx concentrations of 60 nM, 100 nM, and 500 nM are obtained. Recognize. Therefore, it can be seen that the MeIQx concentration can be quantitatively analyzed from the intensity of Raman spectroscopy by the simple detection method of the present invention.

  Further, the absorption spectrum of the measurement sample solution was measured and summarized in FIG. In FIG. 2, “before aggregation X1 / 2” is an absorption spectrum of a sample obtained by diluting gold nanoparticles before aggregation twice. A dispersion of gold nanoparticle aggregates (measurement sample liquid) obtained by mixing a solution containing the sample HCA and a colloidal solution of gold nanoparticles and aggregating them by adding salt has a strong absorption between 700 nm and 800 nm. I understood it. It was found that the absorption spectra of the dispersions containing MeIQx concentrations of 100 nM and 500 nM were almost the same.

  From these results, it can be seen that a dispersion of gold nanoparticle aggregates expressing SERS can be obtained by the method of the present invention regardless of the MeIQx concentration, and SERS having a strength corresponding to the MeIQx concentration can be obtained in a short time. It was.

  Heterocyclic amines suspected of being cancer-inducing can be detected easily and quickly with high sensitivity using SERS.

Claims (3)

A test sample solution is prepared by bringing a specimen into contact with a colloid liquid of noble metal nanoparticles and an aqueous solution of inorganic salts that aggregate the colloid to form a self-assembly of noble metal nanoparticles containing the specimen.
Simple measurement of heterocyclic amines by measuring the surface-enhanced Raman scattering spectrum by applying the measurement sample solution to a Raman spectrometer, and detecting the cyclic amines in the specimen by the expression of specific Raman shift of the heterocyclic amines Detection method. The simple detection method according to claim 1, wherein the noble metal nanoparticles are gold nanoparticles or silver nanoparticles. The heterocyclic amines include PhIP (2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine: 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine. ), Trp-P-1 (3-Amino-1,4-dimethyl-5H-pyrido [4,3-b] indole: 3-amino-1,4-dimethyl-5H-pyrido [4,3-b] Indole), MeIQx (2-Amino-3,8-dimethylimidazo [4,5-f] quinoxaline: 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline), MeIQ (2-Amino-3 , 4-dimethylimidazo [4,5-f] quinoline: 2-amino-3,4-dimethylimidazo [4,5-f] quinoline), A-α-C (2-Amino-9H-pyrido [2,3 -b] indole: 2-amino-9H-pyrido [2,3-b] indole), Glu-P-1 (2-Amino-6-methyldipyrido [1,2-a: 3 ', 2'-d] imidazole: 2-amino-6-methyldipyrido [1,2-a: 3 ', 2'-d] imidazole), Glu-P-2 (2-Aminodipyrido [1,2-a: 3', 2'-d) ] imidazole: 2-aminodipyrido [1,2-a: 3 ′, 2′-d] imidazole), MeA-α-C (2-Amino-3-methyl-9H-pyrido [2,3-b] indole: 2-Amino-3-methyl-9H-pyrido [2,3-b] in ), Trp-P-2 (3-Amino-1-methyl-5H-pyrido [4,3-b] indole: 3-amino-1-methyl-5H-pyrido [4,3-b] indole) A simple according to claim 1 or 2, selected from IQ (2-Amino-3-methylimidazo [4,5-f] quinoline: 2-amino-3-methylimidazo [4,5-f] quinoline) Detection method.

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