JP6497507B2 - Amino acid detection method using chiral gold nanorod tissue - Google Patents

Description

  The present invention relates to a measurement method for selectively measuring amino acids using AuNR (gold nanorod) -serum protein tissue.

  Amino acids are components of about 20% of the human body and play an important role in life activities. Furthermore, with the recent health orientation, interest in specific amino acids has increased remarkably, and the development of health foods containing these has been thriving. On the other hand, one of the interesting materials for supramolecular assembly research is gold nanorods with anisotropic rod-shaped metal particles. The rapid establishment of this synthesis method is expected to be applied as a building block material. We have succeeded in producing a supramolecular organization (AuNR-HSA) that exhibits a huge circular dichroism signal (CD signal) by interacting gold nanorods (AuNR) and human serum albumin (HSA). did. Therefore, with the aim of further application of this material, the purpose of this study was to selectively detect amino acids using AuNR-serum proteins.

  Patent Document 1 discloses a technique relating to a method for producing a chiral gold nanorod tissue using serum protein, a chiral gold nanorod tissue obtained by the production method, and a method for measuring a serum protein concentration using the chiral gold nanorod tissue. Have been described.

Japanese Patent Application No. 2014-030436

  As a result of further examination of the technique described in the above literature, it was found that when an amino acid was added to the prepared AuNR-serum protein, a CD spectrum change was observed only in the case of an amino acid having a specific functional group.

  The present invention is to provide a technique aimed at selectively measuring amino acids using AuNR (gold nanorod) -serum protein tissue.

  According to the present invention, a) preparing a cell infused with an aqueous solution containing a test body amino acid and a tissue composed of gold nanorods and serum protein, b) irradiating the cell with light, c) A step of detecting a circular dichroism spectrum of light transmitted through the cell, d) a calibration standard provided in advance for each kind of combination of the serum protein and the test amino acid, and step c). The amino acid concentration can be measured by the step of comparing the detection result and the step of e) calculating the test amino acid concentration from the comparison result obtained in step d).

  In addition, as the serum protein of the present invention, either human serum albumin or bovine serum albumin can be used.

  Moreover, the amino acid which this invention measures should just be an amino acid which has an acidic amino acid and a thiol group.

Further, the amino acid concentration of one or a plurality of combinations selected from aspartic acid (Asp), glutamic acid (Glu), and cysteine (Cys) can be measured.

It is a conceptual diagram for demonstrating the mechanism in which a gold nanorod forms a side-by-side organization body in the aqueous solution containing a gold nanorod and serum protein. (Top) Absorption spectrum of AuNR-HSA structure, (Bottom) CD spectrum of AuNR-HSA structure (Upper) Change in absorption spectrum due to addition of 0 to 150 μM Lys to AuNR-HSA tissue (Lower) Change in CD spectrum due to addition of 0 to 150 μM Lys to AuNR-HSA tissue (Top) Change in absorption spectrum with addition of 0 to 150 μM Arg to AuNR-HSA tissue, (Bottom) Change in CD spectrum with addition of 0 to 150 μM Arg to AuNR-HSA tissue (Upper) Change in absorption spectrum due to addition of 0 to 150 μM His to AuNR-HSA tissue, (Lower) Change in CD spectrum due to addition of 0 to 150 μM His to AuNR-HSA tissue (Upper) Change in absorption spectrum due to addition of 0 to 150 μM Asp to AuNR-HSA tissue, (Lower) Change in CD spectrum due to addition of 0 to 150 μM Asp to AuNR-HSA tissue (Upper) Change in absorption spectrum due to addition of 0 to 150 μM Glu to AuNR-HSA tissue, (Lower) Change in CD spectrum due to addition of 0 to 150 μM Glu to AuNR-HSA tissue (Upper) Change in absorption spectrum due to addition of 0 to 150 μM Ser to AuNR-HSA tissue, (Lower) Change in CD spectrum due to addition of 0 to 150 μM Ser to AuNR-HSA tissue (Upper) Change in absorption spectrum with addition of 0 to 150 μM Asn to AuNR-HSA tissue, (Lower) Change in CD spectrum with addition of 0 to 150 μM Asn to AuNR-HSA tissue (Upper) Change in absorption spectrum due to addition of 0 to 150 μM Gln to AuNR-HSA tissue, (Lower) Change in CD spectrum due to addition of 0 to 150 μM Gln to AuNR-HSA tissue (Top) Change in absorption spectrum due to addition of 0 to 150 μM Ala to AuNR-HSA tissue, (Bottom) Change in CD spectrum due to addition of 0 to 150 μM Ala to AuNR-HSA tissue (Upper) Change in absorption spectrum with addition of 0 to 150 μM Phe to AuNR-HSA tissue, (Lower) Change in CD spectrum with addition of 0 to 150 μM Phe to AuNR-HSA tissue (Upper) Change in absorption spectrum with addition of 0-100 μM Try to AuNR-HSA tissue, (Lower) Change in CD spectrum with addition of 0-100 μM Try to AuNR-HSA tissue (Upper) Change in absorption spectrum due to addition of 0 to 150 μM Trp to AuNR-HSA tissue, (Lower) Change in CD spectrum due to addition of 0 to 150 μM Try to AuNR-HSA tissue (Upper) Change in absorption spectrum with addition of 0 to 150 μM Met to AuNR-HSA tissue, (Lower) Change in CD spectrum with addition of 0 to 150 μM Met to AuNR-HSA tissue (Top) Change in absorption spectrum with addition of 0-100 μM Cys to AuNR-HSA tissue, (Bottom) Change in CD spectrum with addition of 0-100 μM L-Cys to AuNR-HSA tissue (Upper) Change in absorption spectrum with addition of 0 to 150 μM Pro to AuNR-HSA tissue Figure 32 Change in absorption spectrum with addition of 0 to 150 μM Pro to AuNR-HSA tissue (Upper) Change in absorption spectrum due to addition of 0 to 150 μM Cys-Cys to AuNR-HSA tissue (Lower) Change in CD spectrum due to addition of 0 to 150 μM Cys-Cys to AuNR-HSA tissue (Top) Concentration change of absorption spectrum (at 745 nm) with addition of amino acid to AuNR-HSA tissue (Bottom) Concentration change of CD spectrum (at 701 nm) with addition of amino acid to AuNR-HSA tissue (Upper) Absorption spectrum of AuNR-BSA structure (Lower) Absorption spectrum of AuNR-BSA structure (Upper) Change in absorption spectrum due to addition of 0 to 150 μM Asp to AuNR-BSA structure (Lower) Change in CD spectrum due to addition of 0 to 150 μM Asp to AuNR-BSA structure (Upper) Change in absorption spectrum due to addition of 0 to 150 μM Glu to AuNR-BSA tissue (Lower) Change in CD spectrum due to addition of 0 to 150 μM Asp to AuNR-BSA (Upper) Change in absorption spectrum due to addition of 0 to 150 μM Cys to AuNR-BSA structure (Lower) Change in CD spectrum due to addition of 0 to 150 μM Cys to AuNR-BSA structure (Top) Concentration change of absorption spectrum (at 674 nm) with addition of amino acid to AuNR-BSA tissue (Bottom) Concentration change of CD spectrum (at 638 nm) with addition of amino acid to AuNR-BSA tissue (Top) Change of absorption spectrum with time change of AuNR-BSA structure, (Bottom) Change of CD spectrum with time change of AuNR-BSA structure

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In this specification, “A to B” means A or more and B or less.

<Chiral gold nanorod structure>
FIG. 1 is a conceptual diagram for explaining a mechanism in which gold nanorods form side-by-side tissue bodies in an aqueous solution containing gold nanorods and serum proteins. As shown in FIG. 1, the chiral gold nanorod structure is a structure having circular dichroism composed of a plurality of gold nanorods. In this tissue body, a layered gold nanorod array is formed and has a chiral orientation due to serum proteins. The shape of the gold nanorod used in the present embodiment may be a rod-like anisotropic particle, and may be any of a cylindrical shape, a triangular prism shape, a quadrangular prism shape, a dog bone shape, and the like.

  The method for producing gold nanorods used in the present embodiment is not particularly limited. For example, a high yield gold nanorod synthesis method described in Tapan K. Sau and Catherine J. Murphy. Langmiur 2004, 20, 6414—CTAB protected gold It can be manufactured using a method for synthesizing gold nanorods with nanoparticles as the core.

  The aspect ratio of the gold nanorod used in the present embodiment is preferably 1.5 to 10, and more preferably 2.0 to 5.0. This is because a chiral gold nanorod structure having good circular dichroism can be formed if the aspect ratio is within these ranges.

  The volume average minor axis diameter of the gold nanorods used in the present embodiment is preferably 2 nm to 200 nm, and more preferably 17 nm to 27 nm. This is because if this volume average minor axis diameter is within these ranges, a chiral gold nanorod structure having good circular dichroism can be formed.

  The volume average major axis diameter of the gold nanorods used in the present embodiment is preferably 5 nm to 500 nm, and more preferably 46 nm to 66 nm. This is because if this volume average major axis diameter is within this range, a chiral gold nanorod structure having good circular dichroism can be formed.

  In the chiral gold nanorod textured body of the present embodiment, it is preferable that a plurality of gold nanorods are arranged in parallel so that the long axes are in contact with each other. And it is preferable that adjacent gold nanorods are arrange | positioned in the state twisted mutually.

  The chiral gold nanorod structure of the present embodiment includes two or more gold nanorods. If the number of gold nanorods is 2 or more, good circular dichroism is exhibited.

  The chiral gold nanorod tissue of the present embodiment is preferably configured in an aqueous solution containing gold nanorods and serum protein. As described above, the presence of the gold nanorod and the serum protein in the aqueous solution can stably maintain the chiral gold nanorod structure having good circular dichroism. However, once the chiral gold nanorod structure is formed, the surrounding aqueous solution, protein, and other organic substances may be removed to form only the chiral gold nanorod structure. It has been confirmed that even when it is naturally dried, a layered gold nanorod array is formed and the structure in which the serum protein is in a chiral orientation is maintained.

  When the chiral gold nanorod tissue of the present embodiment is constituted in an aqueous solution containing gold nanorods and serum protein, the serum protein is preferably albumin or globulin in mammalian or avian serum. When experiments were conducted using various proteins, BSA (Bovine Serum Albumin), HSA (Human Serum Albumin), and γ-globulin (bovine serum albumin), which are albumins or globulins in the serum of mammals or birds, were tested. This is because it has been demonstrated that chiral gold nanorod structures having good circular dichroism can be formed when γ-globulin) is used. In addition, examples of albumin or globulin in mammalian or avian serum that can be used in the present embodiment include ovalbumin, lactalbumin, chicken egg albumin, and the like.

  When the chiral gold nanorod tissue of the present embodiment is constituted in an aqueous solution containing gold nanorods and serum protein, the concentration of the gold nanorods in the aqueous solution is preferably 0.010 nM to 0.500 nM, More preferably, it is from .050 nM to 0.100 nM. This is because a concentration within this range can stably maintain a chiral gold nanorod structure having good circular dichroism.

<Method for measuring amino acid concentration>
The method for measuring the amino acid concentration of the present embodiment includes: a) preparing a cell infused with a tissue composed of gold nanorods and serum protein and an aqueous solution containing a test amino acid; and b) irradiating the cell with light. C) a step of detecting a circular dichroism spectrum of the light transmitted through the cell; d) a calibration standard provided in advance for each kind of combination of the serum protein and the test amino acid; and step c. And b) a step of comparing the detection result obtained in step e), and e) a step of calculating the test amino acid concentration from the comparison result obtained in step d).

  According to this method, a CD signal is confirmed at a gold nanorod absorption region of about 500-1000 nm in an aqueous solution containing a side-by-side tissue composed of gold nanorods and serum proteins and a test amino acid. It will be. Therefore, if the circular dichroism spectrum of the aqueous solution containing the tissue and the test amino acid is measured and compared with a calibration standard provided in advance, the type and concentration of the amino acid contained in the aqueous solution are measured. be able to. The calibration standard can be used as a calibration curve, for example, within a range in which a CD spectrum and amino acid concentration are plotted and a linear relationship is obtained. Moreover, since the inclination of a straight line changes with amino acids, it can be used for qualitative judgment.

  In step a) of the measurement method of this embodiment, when preparing a cell into which an aqueous solution containing gold nanorods and serum protein is prepared, various materials, various concentrations, and various conditions can be appropriately adjusted.

  As the measurement cell used in the present embodiment, a known cell generally used for measurement of a circular dichroism spectrum (CD spectrum) can be used. For example, quartz attached to a CD spectrum measurement apparatus used for measurement is used. A cell may be used. Further, the size of the measurement cell is not particularly limited, but generally a cell having a detection cell length (optical path length) of 1 mm is often used.

  In step b) of the measurement method of this embodiment, the above cell is irradiated with light. As a light source used in this case, a known light source generally used for measuring a circular dichroism spectrum (CD spectrum) can be used. For example, an optical wavelength attached to a CD spectrum measuring apparatus used for measurement. A light source capable of covering wavelengths from 190 nm to 1100 nm in the ultraviolet region to the visible / near infrared region can be suitably used. This is because by preparing an aqueous solution containing gold nanorods and serum proteins in step a) of this embodiment, a CD signal can be confirmed in the gold nanorod absorption region of about 500 to 1000 nm as shown in the examples described later. It is.

  In step c) of the measurement method of this embodiment, a circular dichroism spectrum of the light transmitted through the cell is detected. As a detector of the circular dichroism spectrum used at this time, for example, a photomultiplier tube or a silicon photodiode attached to a CD spectrum measuring apparatus used for measurement may be used. As a wavelength detectable by this detector, it is preferable that wavelengths from the ultraviolet region to the visible / near infrared region with a wavelength of 300 nm to 1100 nm can be covered. This is because by preparing an aqueous solution containing gold nanorods and serum proteins in step a) of this embodiment, a CD signal can be confirmed in the gold nanorod absorption region of about 500 to 1000 nm as shown in the examples described later. It is.

  In step d) of the measurement method of the present embodiment, the calibration standard provided in advance for each type of test serum protein is compared with the detection result obtained in step c). At this time, a sample in which the concentration is shaken in a predetermined range for each type of amino acid is prepared, and the above steps a) to c) are performed in advance, for each type of combination of the above serum protein and test amino acid. It is preferable to prepare a calibration standard. As a result, for example, BSA (Bovine Serum Albumin), HSA (Human Serum Albumin), gamma-globulin (γ-globulin), ovalbumin, which are albumins or globulins in mammalian or avian serum (Ovalbumin), lactalbumin (Lactalbumin), and chicken egg albumin (Albumin, from chicken egg) and amino acids lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu), serine (Ser), Asparagine (Asn), Glutamine (Gln), Alanine (Ala), Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp), Methionine (Met), Cysteine (Cys), Proline (Pro), Cystine ( Calibration standards can be created for each combination of Cys-Cys).

In step e) of the measurement method of this embodiment, the test amino acid concentration is calculated from the comparison result obtained in step d). In this case, if the above calibration standard is used, lysine (Lys), arginine (Arg), histidine (His), uppartic acid (Asp), glutamic acid (Glu), serine (Ser), asparagine (Asn) can be applied to it. ), Glutamine (Gln), alanine (Ala), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), methionine (Met), cysteine (Cys), proline (Pro), cystine (Cys-Cys) Can be calculated. Particularly preferred amino acids are acidic amino acids and amino acids having a thiol group, and aspartic acid (Asp), glutamic acid (Glu), and cysteine (Cys) can be preferably measured.

  Even a mixture of a plurality of amino acids can be qualitatively determined from the difference in the degree of change in the CD spectrum.

  As mentioned above, although embodiment of this invention was described, these are illustrations of this invention and various structures other than the above are also employable.

  For example, in the above embodiment, the cell and the detector have been described as parts of the CD spectrum measuring device. However, a general CD spectrum measuring device is provided with a sample chamber having a cell holder for storing the cell. It has been.

  A general CD spectrum measuring apparatus includes a spectroscope. In many cases, a prism, a diffraction grating, or the like is used as a dispersive element of a spectroscope. When a diffraction grating is used as the dispersive element of the spectroscope, the spectroscope is configured by, for example, an entrance slit, an exit slit, a diffraction grating, and a mirror associated therewith.

  A general CD spectrum measuring apparatus also includes a polarizer that linearly polarizes light split into monochromatic light by a spectroscope. As this polarizer, an absorption polarizer, a crystal polarizer, a reflective polarizer, or the like is used. Further, a general CD spectrum measuring apparatus includes a phase modulator that converts light linearly polarized by a polarizer into circularly polarized light. As this phase modulator, for example, a photoelastic modulator (PEM) composed of a transparent and high-quality optical crystal such as quartz and a piezoelectric actuator can be used.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these.

<Preparation of sample>
(1) Preparation method of gold nanorod (AuNR) solution [0.32 nM] (1-1) Preparation of nuclear solution (at 25 ° C.)
Put a 0.01 M chloroauric acid aqueous solution (0.25 mL, 2.5 μM) and a 0.1 M CTAB (hexadecyltrimethylammonium bromide) aqueous solution (7.5 mL, 0.75 mM) in a 14 mL sample bottle at room temperature. An ice-cooled 0.01 M aqueous sodium borohydride solution (0.6 mL, 6 μM) was added all at once with vigorous stirring, and the mixture was vigorously stirred for 2 min. It kept warm until it used in a 25 degreeC thermostat. The final concentration of the preparation conditions for this nuclear solution is shown below.

Final concentration [Chloroauric acid] = 0.3 mM
[CTAB] = 0.09M
[Sodium borohydride] = 0.72 mM

(1-2) Preparation of gold nanorod (AuNR) solution [0.32 nM] (at 25 ° C.)
In a sample bottle containing 0.1 M CTAB aqueous solution (4.75 mL, 0.475 mM), 0.01 M basic gold acid aqueous solution (0.2 mL, 2 μM), 0.01 M silver nitrate aqueous solution (0.03 mL, 0.3 μM) And slowly mixed. The color was light brown. When 0.1 mM aqueous ascorbic acid solution (0.032 mL, 3.2 μM) was added and mixed gently, the solution became colorless. When 20 μL of the nucleus solution (3 hours after preparation) was added thereto, the color changed. In addition, the final concentration of the preparation conditions of this gold nanorod solution and the literature information referred to are shown below.

Final concentration [Chloroauric acid] = 0.4 mM
[CTAB] = 0.09M
[Silver nitrate] = 0.06mM
[Ascorbic acid] = 0.64 mM
* Tapan K. Sau and Catherine J. Murphy. Langmiur 2004, 20, 6414
High yield gold nanorod synthesis method-Prepared based on a gold nanorod synthesis method using CTAB reduced gold nanoparticles as a core.

(1-3) Preparation method of gold nanorod (AuNR) concentrated solution [1.6 nM] 0.32 nM AuNR-0.1 M CTAB sol. C. 60 mL was centrifuged at 12,000 rpm for 30 min, and the supernatant was removed. W. To 30 mL. Further, the mixture was centrifuged at 12,000 rpm for 30 min, and 18 mL of the supernatant was removed and redispersed (concentrated 5 times, 1.6 nM). Excess CTAB was removed by filtering this with a stationary glass filter for 2 hours in a refrigerator (2 ° C.).
Using the above sample, an aqueous solution containing gold nanorods and protein was prepared under the conditions shown below, and amino acids were added for experiments.

<Conditions and results of each experiment>
(2) Preparation of AuNR-HSA tissue AuNR concentrated stock solution (AuNR = 1.6 nM) 80 μL acetonitrile (MeCN) 400 μL, 0.01 M CTABaq 1510 μL, 0.21 mM HSA (human serum albumin) 10 μL Were mixed at room temperature, and absorption spectrum measurement and CD spectrum measurement were performed.

Measurement conditions Solvent = 20% MeCNaq.
Temperature = 25 ° C
[AuNR] = 0.064 nM
[CTAB] = 7.7 mM
[HSA] = 0.5 μM

FIG. 2 shows the absorption spectrum and CD spectrum of the AuNR-HSA tissue.

(2-1) Addition of amino acid to AuNR-HSA tissue solution Addition of 21 mM amino acid 0-15 μL to 2000 μL of AuNR-HSA tissue solution 10 min, followed by absorption spectrum measurement and CD spectrum measurement.
The amino acids used in this experiment were lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu), serine (Ser), asparagine (Asn), glutamine (Gln), alanine. (Ala), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), methionine (Met), cysteine (Cys), proline (Pro), and cystine (Cys-Cys).

Absorption spectrum measurement conditions: wavelength 300 nm to 1100 nm
CD spectrum measurement conditions: slit width 300 μM
Response 0.125 sec
Sensitivity standard
Data acquisition interval 1 nm
Scanning speed 500 nm / min
Integration count 1

Amino acid measurement conditions Solvent = 20% MeCNaq.
Temperature = 25 ° C
[AuNR] = 0.064 nM
[CTAB] = 7.7 mM
[HSA] = 1 μM
[Amino acid concentration] = 0 to 150 μM

FIGS. 3 to 18 show changes in absorption spectrum and CD spectrum with addition of 0 to 150 μM amino acids to AuNR-HSA tissue.

  Further, the measurement results of each amino acid are summarized, and FIG. 19 shows the change in the concentration of the absorption spectrum (at 745 nm) and the change in the concentration of the CD spectrum (at 701 nm) accompanying addition of the amino acid to the AuNR-HSA tissue. As a result, almost no change was confirmed in the absorption spectrum, whereas in the CD spectrum, the change was confirmed only with Asp, Glu, and Cys.

Asp and Glu have a carboxyl group in the side chain, and Cys has a thiol group.
Therefore, it was suggested that the carboxyl group and the thiol group act electrostatically or non-covalently on the AuNR-HSA tissue. This technique enables selective amino acid detection.

(3) Preparation of AuNR-BSA tissue AuNR concentrated stock solution (AuNR = 1.6 nM) MeCN 400 μL, 0.01 M CTAB (hexadecyltrimethylammonium bromide) aq 1510 μL, 0.21 mM BSA (cow) Serum albumin (10 μL) was mixed at room temperature, and absorption spectrum measurement and CD spectrum measurement were performed.

Measurement conditions Solvent = 20% MeCNaq.
Temperature = 25 ° C
[AuNR] = 0.064 nM
[CTAB] = 7.7 mM
[BSA] = 0.5 μM

FIG. 20 shows the absorption spectrum and CD spectrum of the AuNR-BSA tissue.

(3-1) Addition of Amino Acid to AuNR-BSA Tissue Solution Solution Absorption spectrum measurement and CD spectrum measurement were carried out after adding 21 mM amino acid 0-15 μL to AuNR-BSA tissue solution 2000 μL for 10 min.
The amino acids used in this experiment are aspartic acid (Asp), glutamic acid (Glu), and cysteine (Cys).

Absorption spectrum measurement conditions: wavelength 300 nm to 1100 nm
CD spectrum measurement conditions: slit width 300 μM
Response 0.125 sec
Sensitivity standard
Data acquisition interval 1 nm
Scanning speed 500 nm / min
Integration count 1

Amino acid measurement conditions Solvent = 20% MeCNaq.
Temperature = 25 ° C
[AuNR] = 0.064 nM
[CTAB] = 7.7 mM
[BSA] = 0.5 μM
[Amino acid concentration] = 0 to 150 μM

FIGS. 21 to 23 show changes in absorption spectrum and CD spectrum with addition of 0 to 150 μM Asp to AuNR-BSA tissue.

Furthermore, the measurement results of each amino acid are summarized, and FIG. 24 shows the concentration change and absorption spectrum (at) of the absorption spectrum (at 674 nm) accompanying addition of amino acid to the AuNR-BSA tissue.
638 nm) concentration change.

  In addition, FIG. 25 shows changes in absorption spectrum and CD spectrum with time change of AuNR-BSA tissue.

  In the AuNR-BSA tissue as well as in the AuNR-HSA tissue, changes in the CD spectrum due to the addition of Asp, Glu, and Cys were confirmed. However, since the stability of the AuNR-BSA tissue is low, the CD spectrum changes with time.

  In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

Claims (4)

A method for measuring amino acid concentration,
a) preparing a cell infused with an aqueous solution containing a tissue body composed of gold nanorods and serum proteins and a test amino acid;
b) irradiating the cell with light;
c) detecting a circular dichroism spectrum of the light transmitted through the cell;
d) a step of comparing a calibration standard provided in advance for each type of combination of the serum protein and the test amino acid with the detection result obtained in step c);
e) calculating the test amino acid concentration from the comparison result obtained in step d);
A method for measuring an amino acid concentration, comprising:   The method for measuring amino acid concentration according to claim 1, wherein the serum protein is either human serum albumin or bovine serum albumin. The test amino acid, method of measuring amino acid concentration according to any one of claims 1 or 2, characterized in that an amino acid having an acidic amino acid and a thiol group. The test amino acid, aspartic acid (Asp), glutamic acid (Glu), according to any one of claims 1 to 3, characterized in that one selected from cysteine (Cys), or a plurality of combinations Method for measuring amino acid concentration.