JP2019052984A - Method for analysis - Google Patents

Abstract

To provide a method for analysis for generating such active oxygen species as hydrogen peroxide in an aqueous solution at room temperature without fixing gold nano-particles to a carrier.SOLUTION: The method includes: a first step of preparing gold nano-particles with first biological molecules on the surface and a solution containing second biological molecules, which specifically bind with the first biological molecules; and a second step of detecting active oxygen species generated in the solution.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an analysis method for quantitatively detecting a biomolecule such as an antigen.

  As a kind of immunoassay (immunoassay) using a specific binding reaction between an antigenic determinant of an antigen and an antibody and a color reaction by an enzyme labeled with the antibody or antigen, an ELISA method (Enzyme-Linked Immunosorbent Assay) is used. Enzyme-linked immunosorbent method) is known. In the ELISA method, a highly specific antigen-antibody reaction is used, and the color development based on the enzyme reaction is converted into a signal for measurement, so that it can be detected with high sensitivity and has excellent quantitativeness. Moreover, it is safer, cheaper and simpler than radioimmunoassay (radioimmunoassay, RIA) using a radioactive substance as a labeling substance. Therefore, the ELISA method is used for diagnosis of biological substances such as antibodies, influenza viruses, plasma proteins, cytokines, DNA, peptides, ligands; chemical substances such as residual agricultural chemicals and environmental hormones contained in foods; diabetes, cancer, etc. It is widely used for detection and quantification of various test substances such as blood glucose and diagnostic substances such as tumor markers.

  The ELISA method uses a colorimeter to spectroscopically measure a dye colored by an enzyme-labeled antibody or the like, but the spectroscopic measurement requires multiple devices such as a diffraction grating, an optical filter, and a high-sensitivity detector. There is a problem that the apparatus becomes large and expensive.

  Therefore, as a novel detection technique that can be applied to the ELISA method and can be substituted for the conventional spectroscopic measurement method, for example, an optical detection system based on a waveguide using a scanning light source (Patent Document 1), a disk-type analysis chip ( Patent Document 2), an optical waveguide type antibody chip (Patent Document 3), and the like have been proposed.

Special table 2012-525595 gazette JP 2012-215515 A JP 2008-224524 A

  As described above, the ELISA method is extremely useful as a means capable of quantitatively detecting and analyzing a trace amount of a test substance using an antigen-antibody reaction and a labeling enzyme. However, the measurement of the absorbance of the color-developing substance based on the enzyme reaction has problems such as being easily affected by the pH and temperature of the solvent and difficult to handle, and has a problem that the measurement time is long.

  The above problem is not limited to immunoassays such as ELISA, but a method for detecting a test substance such as glucose by measuring the absorbance of a chromogenic substance produced by an enzyme reaction (in the sense that an enzyme is used, in a broad sense enzyme assay) In the same manner).

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for quantitatively detecting a biomolecule such as an antigen, and a method capable of quantitatively detecting the biomolecule quickly and with high sensitivity. There is.

  The method of the present invention that has solved the above problem is a solution containing gold nanoparticles having a first biomolecule formed on the surface and a second biomolecule that specifically binds to the first biomolecule. And a second step of detecting active oxygen species generated in the solution.

  In the method of the present invention, it is preferable that the first biomolecule is an antibody and the second biomolecule is an antigen.

  In the method of the present invention, the antigen is preferably immobilized on a reaction vessel, and some gold nanoparticles are bound to the antigen, and some other gold nanoparticles are bound to the antigen. It is preferable to include a step of removing gold nanoparticles that are not bound and are not bound to the antigen between the first step and the second step.

  In the method of the present invention, the solution preferably contains a compound having an amino group and a hydroxy group, and the compound contains trishydroxymethylaminomethane, tri (hydroxymethyl) methylglycine, and tris (hydroxymethyl) methyl- It is preferably at least one selected from 3-aminopropanesulfonic acid.

  In the method of the present invention, the concentration of the compound with respect to the solution is preferably 1 mM or more, and the pH concentration of the solution is preferably 8 or more.

  In the method of the present invention, the gold nanoparticles preferably have a particle size of 1 nm or more.

  In the method of the present invention, the second step is preferably performed by an electrochemiluminescence method using luminol, and is preferably performed by a fluorescence method using peroxidase.

  In addition, the method for producing reactive oxygen species of the present invention that has solved the above-described problems is characterized in that gold nanoparticles are mixed with a compound having an amino group and a hydroxy group.

  According to the method of the present invention, active oxygen species such as hydrogen peroxide can be generated and detected in an aqueous solution at room temperature without immobilizing gold nanoparticles on a carrier. Can be detected automatically.

It is a schematic diagram of the method concerning an embodiment of the invention. It is an antigen antibody reaction image figure concerning an embodiment of the invention. 1 is a conceptual diagram of a light emission principle and apparatus of an electrochemiluminescence method according to an embodiment of the present invention. It is a conceptual diagram of the light emission principle of the fluorescence method which concerns on embodiment of this invention. It is the figure which showed the time-dependent change of the electrochemiluminescence intensity accompanying the gold nanoparticle density | concentration which concerns on embodiment of this invention. It is the figure which showed the time-dependent change of the electrochemiluminescence intensity accompanying the Tris solvent density | concentration which concerns on embodiment of this invention. It is the figure which showed the time-dependent change of the electrochemiluminescence intensity by the difference in the solvent structure which concerns on embodiment of this invention. It is the figure which showed the time-dependent change of the electrochemiluminescence intensity | strength in the various solvent which concerns on embodiment of this invention. It is the figure which showed the time-dependent change of the electrochemiluminescence intensity | strength in the various solvent which concerns on embodiment of this invention. It is the figure which showed the electrochemiluminescence intensity by the pH concentration difference of the various solvent which concerns on embodiment of this invention. It is the figure which showed the electrochemiluminescence intensity by the particle size difference of the gold nanoparticle which concerns on embodiment of this invention. It is the figure which showed the electrochemiluminescence intensity | strength by the residual oxygen which concerns on embodiment of this invention. It is the figure which showed the electrochemiluminescence intensity before and behind nitrogen substitution which concerns on embodiment of this invention. It is the figure which showed the electrochemiluminescence intensity | strength by the surface state difference of the gold nanoparticle which concerns on embodiment of this invention. It is the figure which showed the fluorescence intensity at the time of using the fluorescence method which concerns on embodiment of this invention. It is the figure which showed the electrochemiluminescence intensity at the time of performing the antigen antibody reaction which concerns on embodiment of this invention.

  Based on the known catalytic activity of gold nanoparticles, the present inventors have developed a reaction for generating reactive oxygen species such as hydrogen peroxide in an aqueous solution at room temperature without fixing the gold nanoparticles to a support. As a result of various examinations of conditions, a method for generating reactive oxygen species with time in the aqueous solution was found. In addition, regarding this catalytic reaction, it has also been found that active oxygen species are expressed under room temperature conditions regardless of whether they are light or dark. Thereby, the reactive oxygen species generated in the solution containing the gold nanoparticle on which the first biomolecule is formed on the surface and the second biomolecule that specifically binds to the first biomolecule, It became possible to detect quantitatively according to the amount of the second biomolecule. According to the method of the present invention, it is possible to utilize the catalytic activity of gold nanoparticles instead of the conventional bioanalysis using a labeling material such as an enzyme or a fluorescent material. For example, fluorescence or luminescence is used. It can be used for highly sensitive analysis methods.

  The method of the present invention provides a first step of preparing a gold nanoparticle having a first biomolecule formed on a surface thereof, and a solution containing a second biomolecule that specifically binds to the first biomolecule. And a second step of detecting active oxygen species generated in the solution. The method of the present invention is based on a catalytic reaction in which the catalytic activity of gold nanoparticles does not appear due to a synergistic effect with the carrier as in the prior art, and generates active oxygen species such as hydrogen peroxide in an aqueous solution at room temperature. Is. Therefore, it does not require an advanced technique for generating stable nanoclusters as in the prior art. Specifically, it is not necessary to control the particle size of the gold nanoparticle alone to about 2 nm, and active oxygen species such as hydrogen peroxide can be easily generated in an aqueous solution at room temperature. Here, the “nanoparticle” described in the present specification is a substance obtained by making a substance into a nanometer order particle (1 nm or more and less than 1000 nm). In addition, “active oxygen species” described in the present specification is a general term for a compound in which oxygen molecules contained in the atmosphere are changed to a more reactive compound, and is generally a superoxide anion radical (commonly referred to as a superoxide radical). ), Hydroxyl radical, hydrogen peroxide, and singlet oxygen.

  The method for producing reactive oxygen species of the present invention is characterized in that a compound having an amino group and a hydroxy group is mixed with gold nanoparticles. According to the production method of the present invention, reactive oxygen species can be generated relatively easily by performing an operation of mixing a compound having an amino group and a hydroxy group into gold nanoparticles.

Note that the first biomolecule and the second biomolecule in the present invention are characterized by specific binding, and the site where the first biomolecule and the second biomolecule are bound is determined, so that it can be selectively used. Alternatively, it exhibits a particularly high affinity. Specific examples include an antigen-antibody reaction in which the first biomolecule is an antibody and the second biomolecule is an antigen. As another example, a ligand that specifically binds to a specific receptor (receptor) represented by a combination such as an enzyme protein and its substrate, a signal substance such as a hormone or neurotransmitter and its receptor, etc. Specific DNA-DNA, specific DNA-RNA, and the like. In the embodiment of the present specification, an antibody is used as the first biomolecule and an antigen is used as the second biomolecule as an example. However, the present invention is not limited to this.

  FIG. 1 shows a schematic diagram of the method of the present invention. In the first step of the present invention, a gold nanoparticle having an antibody as a first biomolecule formed on the surface and an antigen as a second biomolecule that specifically binds to the first biomolecule are included. Prepare the solution. Subsequently, in the second step, the first biomolecule and the second biomolecule are specifically bound by adding the solution to the gold nanoparticles prepared in the first step. In this specific binding, reactive oxygen species are generated in the solution, and this reactive oxygen species is detected in the second step.

  Next, the method of the present invention will be described in detail with reference to an image diagram of an antigen-antibody reaction, taking as an example the case where the first biomolecule is an antibody and the second biomolecule is an antigen. Fig.2 (a)-FIG.2 (f) are the antigen antibody reaction image diagrams in this invention. 2A to 2E illustrate the first step of the present invention, and FIG. 2F illustrates the second step of the present invention.

  As shown in FIG. 2A, after binding the antibody 2 to the magnetic particles 1 in a reaction vessel (not shown), the solution containing the antigen 3 is put in the reaction vessel as shown in FIG. 2B. In addition, an antigen-antibody reaction between the antibody 2 formed on the surface of the magnetic particle 1 and the antigen 3 occurs, and the antigen 3 is bound to the antibody 2. Next, as shown in FIG. 2C, the magnetic particles 1 to which the antibody 2 and the antigen 3 are bound are collected by a magnet 4 at a predetermined location and washed. By doing so, only the antigen 3 that binds to the antibody 2 is left, and the antigen 3 (unreacted antigen 3 that does not bind to the antibody 2) attached to the surface of the magnetic particle 1 or the like can be removed. Next, as shown in FIG. 2 (d), gold nanoparticles 5 on which antibody 2 is formed on the surface are added. By doing so, an antigen-antibody reaction occurs between the antibody 2 formed on the surface of the gold nanoparticle 5 and the antigen 3 bound to the antibody 2 formed on the surface of the magnetic particle 1. The antibody 2 formed on the surface is bound to the antigen 3. Next, as shown in FIG. 2 (e), the magnetic particles 1, the antigens 2, and the gold nanoparticles 5 are collected at predetermined locations by the magnet 4 and washed. By doing so, only the gold nanoparticles 5 that specifically bind to the antigen 3 that binds to the antibody 2 of the magnetic particle 1 are left, and the excess gold nanoparticles 5 that are not bound to the antigen 3 are removed. can do. Next, as shown in FIG. 2 (f), the surface of the gold nanoparticle 5 becomes electron-rich by adding a solution 6 that imparts electron donating properties and hydrophilicity to the gold nanoparticle 5. The active oxygen species hydrogen peroxide 7 is generated by the catalytic action of the gold nanoparticles 5. When luminol 8, which is a luminescent substance, is added to hydrogen peroxide 7, luminol 8 enters an excited state, and a light emission phenomenon occurs when the excited state returns to the ground state. In the present invention, based on this luminescence phenomenon, luminol 8 is dropped on the printed electrode 9 to determine the change in the electrochemiluminescence intensity accompanying the change in the antigen concentration, whereby the antigen-antibody reaction using the catalytic activity of the gold nanoparticles 5 is obtained. The amount of antigen required for quantification can be quantified.

  In the embodiment of the present invention, the antigen 3 is preferably fixed to the reaction vessel. That is, in the present invention, some of the gold nanoparticles 5 are bound to the antigen 3 fixed to the container, and the other part of the gold nanoparticles 5 are not bound to the antigen 3, Between the step and the second step, a step of removing the gold nanoparticles 5 not bound to the antigen 3 may be included. By configuring in this way, excess gold nanoparticles 5 that do not bind to the antigen 3 are removed to the outside of the container, leaving only the gold nanoparticles 5 that bind to the antigen 3 in the container, thereby causing an actual antigen-antibody reaction. The amount of antigen 3 can be quantified. Specifically, the step of removing the gold nanoparticles 5 not bound to the antigen 3 is performed by washing the gold nanoparticles 5 floating in the solution 6 with washing water and discharging them to the outside of the reaction vessel. That's fine.

  In the embodiment of the present invention, two types of antibodies 2 may exist. That is, in the present invention, an antibody for capturing antigen 3 (hereinafter referred to as antibody 2A) is present in advance, antigen 3 is added to antibody 2A, and antibody 2A and antigen are reacted by antigen-antibody reaction. Then, an antibody different from antibody 2A (hereinafter referred to as antibody 2B) may be added, and reacted at a site different from the antigen-antibody reaction. In this way, a sandwich structure of antibody 2A-antigen 3-antibody 2B is formed, and antigen 3 can be detected using two types of antibodies 2A and 2B. Therefore, one type of antibody 2 is used. Thus, the specificity is higher than the detection method and the detection sensitivity can be further improved.

  In the embodiment of the present invention, the antibody 2 is bound to the magnetic particle 1 and the antibody 2 is bound to the surface of the gold nanoparticle 5 in parallel. By adding the antigen 3 and adding the antigen 3, the antigen-antibody reaction of the antibody 2 and the antigen 3 bound to the magnetic particle 1 and the antibody 2 and the antigen 3 bound to the gold nanoparticle 5 may be simultaneously generated. By doing so, the amount of surplus antigen 3 not involved in the antigen-antibody reaction can be suppressed, and the unreacted antigen 3 that adheres to the surface of the magnetic particles 1 and does not bind to the antibody 2 or antigen 3 It is possible to reduce the excess gold nanoparticles 5 that are not bonded to.

  Hereinafter, each component according to the embodiment of the present invention will be described.

  The gold nanoparticles 5 can be prepared by a known citrate reduction method. The particle size of the gold nanoparticles 5 is preferably 200 nm or less, more preferably 150 nm or less, still more preferably 100 nm or less, more preferably 1 nm or more, more preferably 2 nm or more, and even more preferably 5 nm or more. The catalytic action on the surface of the gold nanoparticle 5 can be enhanced, and the amount of hydrogen peroxide 7 generated from the surface of the gold nanoparticle 5 can be increased.

  The magnetic particles 1 may have a structure in which a superparamagnetic layer is introduced into polymer core particles having a uniform particle size. By using a structure having such a structure, B / F separation by a magnetic field [a binding type forming an antigen-antibody complex (B: Bound) and a free type not forming an antigen-antibody complex (F: Free) ) And the need for easy redispersion after removing the magnetic field, the residual magnetism can be eliminated.

  As the solution 6, a solution capable of generating active oxygen species from the surface of the gold nanoparticle 5 is used. Specifically, it is preferable to use a compound containing a compound having an amino group and a hydroxy group. By including an amino group and a hydroxy group, as a result, a reaction field for expressing a catalytic activity to generate reactive oxygen species from oxygen (oxygen is bonded, stabilization to an intermediate state, and a product of reactive oxygen species It is surmised that it is possible to provide a place where conversion to

Examples of the solvent of the solution 6 that can be used in the method of the present invention are shown in the following [Chemical Formula 1] to [Chemical Formula 14]. Trishydroxymethylaminomethane, tri (hydroxymethyl) methylglycine, and tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid, glycine, glycinamide, 2-amino include compounds having an amino group and a hydroxy group 2-methyl-1-propanol, 2-amino-2methyl-1,3-propanediol, 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid, bis-trismethane, piperazine-N, N-bis (2-ethanesulfonic acid), 4- (2-hydroxyethyl) -1-piperazinepropanesulfonic acid, bis-trispropane, triethanolamine, 2- [1,3-dihydroxy-2- (hydroxymethyl) propane- 2-Amino] ethanesulfonic acid is preferred. In particular, selected from trishydroxymethylaminomethane (hereinafter referred to as Tris), tri (hydroxymethyl) methylglycine (hereinafter referred to as Tricine), and tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (hereinafter referred to as TAPS) More preferably, at least one of the above is used.

  The concentration of the compound with respect to the solution 6 is preferably 1 mM or more, more preferably 5 mM or more, further preferably 10 mM or more, and further preferably 50 mM or more. If the said density | concentration is 1 mM or more, the generation amount of the reactive oxygen species with time passage can be increased.

  The pH concentration of the solution 6 is preferably 8 or more, more preferably 10 or more, and further preferably 12 or more. The amount of active oxygen species generated can be increased as the pH concentration of the solution increases.

  In the second step, as a method for detecting the hydrogen peroxide 7 generated in the solution 6, an electrochemiluminescence method using luminol or a fluorescence method using peroxidase is preferable.

  FIG. 3 shows a light emission principle of the electrochemiluminescence method and a conceptual diagram of the apparatus. FIG. 3A is an overall view, and FIG. 3B is an enlarged view of a dotted line portion in FIG. The electrochemiluminescence method is an optical amplifier (photophotograph) in which light emitted when a luminol oxidized by applying a voltage chemically reacts with active oxygen species such as hydrogen peroxide and returns to the ground state from an excited state is connected to a computer. This is a method of measuring with a multiplier tube. In addition to high sensitivity and practicality, this method has an advantage that measurement can be performed with a small amount of sample by using a printed electrode as an electrode.

FIG. 4 shows the light emission principle of the fluorescence method. In the fluorescence method, Amplite (substrate) is enzymatically reacted with peroxidase (enzyme) in the presence of hydrogen peroxide, and the Amplite after this enzymatic reaction.
This fluorescence value is measured by utilizing the fact that 590 nm fluorescence is emitted when ^TM Red is irradiated with 540 nm light. The fluorescence value shows how much hydrogen peroxide is contained in the substance.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited only to the following examples, and may be implemented with modifications within a range that can be adapted to the purpose described above and below. All of these are possible within the scope of the present invention.

(Preparation of gold nanoparticles)
50 mL of 40 mM citric acid was heated to 70 ° C., chloroauric acid prepared so that the aqueous solution was 0.2 mM was added, and stirred vigorously for 10 minutes to prepare a red-purple gold colloid. Thereafter, centrifugation and washing with distilled water were repeated three times, and gold nanoparticles were produced by firing in a heating furnace. Gold nanoparticles other than those produced by the citric acid reduction method are commercially available 15 nm (manufactured by SIGMA-ALDRICH), commercially available 5 nm, 15 nm, 30 nm, 50 nm, 80 nm, and 100 nm (all manufactured by Tanaka Kikinzoku). Was used.

(Preparation of trishydroxymethylaminomethane solution)
The trishydroxymethylaminomethane (Tris) solution was prepared with distilled water and hydrochloric acid (in some cases, sodium hydroxide) so as to have a predetermined pH. Table 1 shows the configuration of 1 L of Tris solution having a concentration of 1M.

(Method for producing luminol)
Room temperature solid luminol (manufactured by Wako) (17.716 mg) was dissolved in 0.1 M NaOH to prepare a concentration of 10 mM luminol. Thereafter, 1 mL was dispensed and stored frozen at −20 ° C. At the time of confirming electrochemiluminescence, the frozen 10 mM luminol was thawed, diluted with 200 mM Tris buffer or 200 mL borate buffer, adjusted to 0.2 mM luminol, and used as a luminescent reagent.

(Measurement by electrochemiluminescence method)
As the applied voltage, a method called linear sweep voltammetry (LSV) in which the voltage is linearly increased from 0 to 700 mV by 50 mV is used. At the time of electrochemiluminescence measurement, the pH was adjusted to 8 and the luminescence was counted with a photodetector every 500 msec.

(Measurement by fluorescence method)
According to the instructions of the fluorescence reagent kit, 20 U / ml-HRP stock solution was mixed with 200 μL, Amplified red stack solution was mixed with 50 μL, and assay buffer was mixed with 4.75 mL to prepare a fluorescence reagent. To a 96-well plate reader, 25 μL of 56 mg / L gold nanoparticles (manufactured by SIGMA-ALDRICH) and 25 μL each of 1 mM, 10 mM Tris, Tricine, TAPS and PBS were added. Thereafter, 50 μL of a fluorescent reagent (Amplite ^™ Fluorogen Hydrogen Peroxide Assay Kit ^* Red Fluorescene ^* , − (−), manufactured by ABD) is added to the above sample, and measurement is performed with a fluorometer. Measurement conditions are to measure 590 nm light by applying light of 540 nm. Three samples were prepared for one condition and measured.

(Preparation method and measurement / analysis method of sample for basic experiment)
After adding 500 μL of the gold nanoparticle dispersion to 500 μL of a solvent such as Tris, the system waited for several minutes to generate active oxygen species from the gold nanoparticles. Then, 100 μL of luminol having a concentration of 0.2 mM was added to 100 μL of this gold nanoparticle sample, and 20 μL was dropped on the electrode, and electrochemiluminescence measurement was performed. Each sample was measured three times to obtain an ECL (Electrochemiluminescence) graph, and the peak value of the obtained graph was plotted and analyzed.

(Preparation method of antigen-antibody reaction sample)
(A) Immobilization of antibody on the surface of gold nanoparticles In a dispersion of gold nanoparticles (particle size: 15 nm) having a concentration of 40 mg / L, IgA antibody (Human IgA antibody, Goat polyantigen, Affinity Purified, manufactured by BET) with a concentration of 1 mg / mL ) 100 μL was added and allowed to stand for 1 day. After centrifugation, the plate was washed 3 times with 200 mM PBS (pH 7.4). Then, the IgA antibody was fixed by concentrating to 1.5 mL and storing at 4 ° C. The centrifugation conditions are 15000 rpm and 30 minutes.
(B) Antibody fixation of magnetic particles To 250 μL of a dispersion of 20 mg / mL magnetic particles (particle size 180 nm ± 30 nm, NHS beads: manufactured by Tamagawa Seiki), 250 μL of 1 mg / mL IgA antibody and 200 mM PBS (pH 7) .4) 250 μL was added and allowed to stand for 1 day. After centrifugation, the plate was washed 3 times with 200 mM PBS (pH 7.4) and concentrated to 1.5 mL. The centrifugation conditions are 12000 rpm and 10 minutes. Subsequently, 200 μL of 1M aminoethanol was added and allowed to stand overnight, then centrifuged, washed 3 times with 200 mM PBS (pH 7.4), diluted to 3 mL and stored at 4 ° C. to fix the IgA antibody. did. The centrifugation conditions are 12000 rpm and 10 minutes.

  Hereinafter, Experimental Examples 1 to 9 are basic experimental examples for confirming the catalytic mechanism of gold nanoparticles, and Examples 1 to 2 are examples based on an antigen-antibody reaction.

(Experimental example 1)
(Effect on electrochemiluminescence intensity due to gold nanoparticle concentration)
500 μL of 200 mM Tris (pH 8) was added to 500 μL each of the gold nanoparticle dispersions having concentrations of 0.56 mg / L, 5.6 mg / L, and 56 mg / L, and after 0 minutes from the time Tris was added, After 30 minutes, 100 μL of the gold nanoparticle sample after 30 minutes was mixed with 100 μL of a concentration of 0.2 mM luminol, and electrochemiluminescence measurement was performed. As the gold nanoparticles, 15 nm gold nanoparticles (manufactured by SIGMA-ALDRICH) were used.

  In FIG. 5, the time-dependent change of the electrochemiluminescence intensity | strength of each gold nanoparticle is shown. As shown in FIG. 5, it was confirmed that the electrochemiluminescence intensity increased as the concentration of the gold nanoparticles increased.

(Experimental example 2)
(Influence on electrochemiluminescence intensity associated with Tris solvent concentration)
After adding 500 μL of 0.01 mM-Tris, 0.1 mM-Tris, 1 mM-Tris, 10 mM-Tris to 500 μL of a gold nanoparticle dispersion at a concentration of 56 mg / L (all Tris uses pH 8), Tris was added. From the added time, 100 μL of the gold nanoparticle sample after 0 minutes, 30 minutes, and 60 minutes was mixed with 100 μL of a concentration of 0.2 mM luminol, and electrochemiluminescence measurement was performed. As the gold nanoparticles, 15 nm gold nanoparticles (manufactured by SIGMA-ALDRICH) were used.

FIG. 6 shows the change with time of the electrochemiluminescence intensity at each Tris solvent concentration. As shown in FIG. 6, it was found that the electrochemiluminescence intensity tends to increase as the Tris solvent concentration increases.

(Experimental example 3)
(Effects of gold nanoparticles on the electrochemiluminescence intensity associated with solvent species 1)
Using two types of solvents having a structure similar to Tris, whether or not reactive oxygen species are generated from gold nanoparticles was examined. As the solvent having a structure similar to Tris, 1,1,1-trisethane having only a hydroxy group and 2-amino-2methylpropane having only an amino group were used. [Chemical 15] shows the solvent structure diagram of 1,1,1-trisethane, and [Chemical 16] shows the solvent structure of 2-amino-2methylpropane. As the gold nanoparticles, 15 nm gold nanoparticles (manufactured by SIGMA-ALDRICH) were used. First, 1 mM Tris, 1 mM 1,1,1-trisethane, 1 mM 2-amino-2methylpropane were added to 500 μL of a dispersion of gold nanoparticles having a concentration of 56 mg / L, and then Tris and the like were added. The gold nanoparticle samples 100 μL after 0 minutes, 15 minutes, and 30 minutes from the time were mixed with 100 μL of 0.2 mM luminol concentration, and electrochemiluminescence measurement was performed. As a comparative experiment, 1 mM 1,1,1-trisethane and 1 mM 2-amino-2-methyl were used to confirm the effect of using a solvent in which a compound having an amino group and a compound having a hydroxy group were mixed. Electrochemiluminescence measurement was performed on the solvent mixed with propane by the same method as described above.

  In FIG. 7, the time-dependent change of the electrochemiluminescence intensity in each solvent is shown. As shown in FIG. 7, only the Tris solvent having both amino group and hydroxy group structures showed an increase in electrochemiluminescence intensity, and the amino group and hydroxy group structures contributed to the generation of reactive oxygen species. It was suggested. In addition, an increase in electrochemiluminescence intensity was not confirmed in a solvent containing a compound having only one of the amino group and the hydroxy group or a solvent obtained by simply mixing the two.

(Experimental example 4)
(Effect of gold nanoparticle on electrochemiluminescence intensity associated with solvent species 2)
In order to further investigate that the structure of amino group and hydroxy group is related to the generation of reactive oxygen species from gold nanoparticles, Tris, Tricine, TAPS having the above structure, and PBS (Phosphate not having the above structure) buffered saline), borate, and KCl. These six types are generally used as buffer solutions. In addition, 15 nm gold nanoparticles (manufactured by SIGMA-ALDRICH) were used as the gold nanoparticles.

  First, after adding 500 μL of 100 mM Tris, Tricine, TAPS, PBS, borate, and KCl to 500 μL of a dispersion of gold nanoparticles having a concentration of 56 mg / L, 0 minutes after adding a solvent such as Tris, After minutes, 20 minutes and 30 minutes, 100 μL of the gold nanoparticle sample was mixed with 100 μL of 0.2 mM luminol, and electrochemiluminescence measurement was performed.

  FIG. 8 shows the change over time of the electrochemiluminescence intensity in each solvent. As shown in FIG. 8, only Tris, Tricine, and TAPS having both amino group and hydroxy group structures were confirmed to increase in electrochemiluminescence intensity with time, and the structures of amino group and hydroxy group were reactive oxygen species. It was suggested that it contributed to the generation.

(Experimental example 5)
(Effect of gold nanoparticle on electrochemiluminescence intensity associated with solvent species 3)
In order to further investigate that the structure of amino groups and hydroxy groups is related to the generation of reactive oxygen species from gold nanoparticles, seven types of solvents having a structure similar to Tris were examined. These 7 types include 2-amino-2-methyl-1-propanol (hereinafter referred to as AMP) of [Chemical Formula 6], 2-amino-2methyl-1,3-propanediol (hereinafter referred to as AMPD) of [Chemical Formula 7]. ), [Chemical Formula 13] triethanolamine (hereinafter referred to as TEA), [Chemical Formula 11] bis-trismethane, [Chemical Formula 12] bis-trispropane, [Chemical Formula 8] 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (hereinafter referred to as HEPES), [Chemical Formula 10] and 4- (2-hydroxyethyl) -1-piperazinepropanesulfonic acid (hereinafter referred to as HEPPS).

  First, a 5 nm gold nanoparticle (Tanaka Kikinzoku) concentration 0.04 mg / mL and a solution (100 mM, pH 12) were mixed at a ratio of 1: 1, and after 0 minutes, 10 minutes, and 20 minutes, luminol (200 μM, The mixture was diluted with a borate buffer solution of pH 9 at a ratio of 1: 1, and the electrochemiluminescence intensity was measured.

  FIG. 9 shows the change over time of the electrochemiluminescence intensity in each solvent. As shown in FIG. 9, amplification of electrochemiluminescence accompanying changes with time was confirmed in all of the above seven solvents having both amino group and hydroxy group structures.

(Experimental example 6)
(Effect on electrochemiluminescence intensity due to difference in solvent pH)
The pH dependence of the Tris solvent with respect to the amount of reactive oxygen species produced from the gold nanoparticles was investigated. As the gold nanoparticles, 30 nm gold nanoparticles (manufactured by Tanaka Kikinzoku) were used.

  First, 500 μL of 200 mM concentration of pH 6, pH 7, pH 8, pH 9, pH 10, pH 11, and pH 12 was added to 500 μL of a gold nanoparticle dispersion having a concentration of 40 mg / L, and 10 minutes after the time of adding the Tris solvent. 100 μL of the gold nanoparticle sample was mixed with 100 μL of 0.2 mM luminol (diluted with a borate buffer at pH 8), and electrochemiluminescence measurement was performed.

  FIG. 10 shows the value of the electrochemiluminescence intensity at the pH of each solvent. As shown in FIG. 9, it was confirmed that the value of the electrochemiluminescence intensity increases as the pH concentration of the solvent increases.

(Experimental example 7)
(Influence on electrochemiluminescence intensity associated with the particle size of gold nanoparticles)
The relationship between the particle size of gold nanoparticles and the emission intensity was investigated. In addition, 5 nm, 15 nm, 30 nm, 50 nm, 80 nm, and 100 nm (made by Tanaka Kikinzoku) were used for the gold nanoparticles.

  First, 500 μL of a 200 mM concentration of Tris (pH 8) was added to 500 μL of each gold nanoparticle dispersion having a concentration of 40 mg / L, and then 100 μL of a gold nanoparticle sample 30 minutes after the solvent was added. The mixture was mixed with 100 μL of 2 mM luminol, and electrochemiluminescence measurement was performed.

  FIG. 11 shows the value of electrochemiluminescence intensity in each gold nanoparticle. As shown in FIG. 11, it was confirmed that the electrochemiluminescence intensity tends to increase as the particle size of the gold nanoparticles decreases.

(Experimental example 8)
(Effect of dissolved oxygen on electrochemiluminescence intensity)
In order to investigate the effect of dissolved oxygen on the generation of reactive oxygen species from gold nanoparticles, measurements were performed while nitrogen substitution was performed. In addition, 15 nm (made by Tanaka Kikinzoku) was used for the gold nanoparticles.

  First, 500 μL of 100 mM Tris (pH 8) was added to 500 μL of a gold nanoparticle dispersion having a concentration of 40 mg / L, and two samples were prepared. Thereafter, 100 μL of the gold nanoparticle sample after 20 minutes and 30 minutes was mixed with 100 μL of 0.2 mM luminol, and electrochemiluminescence measurement was performed.

  FIG. 12 shows the change over time in the electrochemiluminescence intensity associated with the nitrogen substitution, and FIG. 13 shows the change over time in the electrochemiluminescence intensity before and after the nitrogen substitution. As shown in FIG. 12, when nitrogen substitution was not performed, an increase in electrochemiluminescence intensity was observed over time. On the other hand, when nitrogen substitution was performed, an increase in electrochemiluminescence intensity was not confirmed. Further, as shown in FIG. 13, when the nitrogen substitution was stopped at 30 points and further time was taken, an increase in electrochemiluminescence intensity was confirmed. From the above results, it was suggested that dissolved oxygen is required for the generation of reactive oxygen species from gold nanoparticles.

(Experimental example 9)
(Effect of electrochemiluminescence intensity by the surface state of gold nanoparticles)
By blocking the surface of the gold nanoparticle with BSA (Bovine Serum Albumin: bovine serum albumin), the influence of the gold nanoparticle surface state on the electrochemiluminescence intensity was examined. Gold nanoparticles prepared by a citrate reduction method were used.

  First, BSA (manufactured by SIGMA-ALDRICH, molecular weight to 6600) was added to 10 mL of a gold nanoparticle dispersion having a concentration of 1 mg / mL so that the concentration would be 0 mg / L, 0.66 mg / L, 66 mg / L, 6600 mg / mL. In addition, the surface of the gold nanoparticles was blocked with BSA for 1 hour at 4 ° C. Thereafter, centrifugation and washing were repeated three times to produce BSA-blocked gold nanoparticles. After adding 500 μL of 200 mM Tris (pH 8) to 500 μL of the prepared BSA-blocking gold nanoparticles, 100 μL of the gold nanoparticle sample after 0 minutes, 15 minutes, and 30 minutes after adding the solvent such as Tris is 0 .2 mM-Luminol 100 μL was mixed with and electrochemiluminescence measurement was performed.

FIG. 14 shows the value of the electrochemiluminescence intensity depending on the BSA concentration. As shown in FIG.
It was confirmed that the increase in the electrochemiluminescence intensity value was smaller as the concentration of was increased, and that the electrochemiluminescence intensity value decreased when the surface of the gold nanoparticle was covered. This result suggested that active oxygen species were generated on the surface of the gold nanoparticles.

(Experimental example 10)
(Detection of reactive oxygen species by fluorescence method using peroxidase)
As a method for detecting reactive oxygen species other than the electrochemiluminescence method, it was confirmed using a fluorescence method. FIG. 15 shows the value of fluorescence intensity associated with the generation of active oxygen species from gold nanoparticles in each solvent. As shown in FIG. 15, in the mixed solution of Tris, Tricine, TAPS and gold nanoparticles, an increase in the fluorescence intensity value was confirmed, which was the same as the result by the electrochemiluminescence method using luminol. From this result, it was found that a fluorescence method using peroxidase was useful for detecting reactive oxygen species as well as an electrochemiluminescence method using luminol.

Example 1
(Detection of reactive oxygen species of gold nanoparticles in antigen-antibody reaction)
Antibody-fixed magnetic particles (NHS beads, manufactured by Tamagawa Seiki) 90 μL of antigen (Secretary Immunoglobulin A, Human Costruum, manufactured by ART) at concentrations of 0 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL 10 μL each of 10 μg / mL was added and incubated at room temperature for 1 hour. Next, the magnetic particles were collected with a magnet and washed twice with 200 mM PBS (pH 7.4), and then 200 μL of gold nanoparticles after antibody fixation was added. Subsequently, magnetic particles, antigens, and gold nanoparticles were collected with a magnet and washed twice with 200 mM PBS (pH 7.4), and then 200 μL of 200 mM Tris (pH 12) was added and left for 15 minutes. Next, 100 μL of luminol was added to 100 μL of the sample after adding Tris, 20 μL of which was dropped onto the electrode, and electrochemiluminescence measurement was performed.

  FIG. 16 shows the value of electrochemiluminescence intensity at each IgA antigen concentration. As shown in FIG. 16, it was confirmed that the electrochemiluminescence intensity could be detected to the level of 1 ng / mL IgA antigen concentration in the antigen-antibody reaction. This result suggests the usefulness of application to a biosensor used for antigen-antibody reaction.

(Example 2)
(Light emission measurement with EM-CCD camera)
About the measurement of electrochemiluminescence intensity, it measured using the EM-CCD camera instead of the optical amplifier (photomultiplier tube), and examined the influence by the difference in a measuring method. In the same manner as in Example 1, the luminescence from the sample after the antigen-antibody reaction with the antigen having a concentration of 10 μg / mL was measured with an EM-CCD camera. As a result, light emission was confirmed only in the portion where the gold nanoparticles were present. From this result, it was confirmed that the luminescence measurement by the EM-CCD camera was also effective, and it was suggested that this technique can be applied to inspection techniques such as biological imaging.

DESCRIPTION OF SYMBOLS 1 Magnetic particle 2 Antibody 3 Antigen 4 Magnet 5 Gold nanoparticle 6 Solution 7 Hydrogen peroxide 8 Luminol 9 Printed electrode

Claims (12)

A first step of preparing a gold nanoparticle having a first biomolecule formed on a surface thereof, and a solution containing a second biomolecule that specifically binds to the first biomolecule;
And a second step of detecting active oxygen species generated in the solution.   The method of claim 1, wherein the first biomolecule is an antibody and the second biomolecule is an antigen.   The method according to claim 2, wherein the antigen is immobilized in a reaction vessel. Some gold nanoparticles are bound to the antigen, and some other gold nanoparticles are not bound to the antigen,
4. The method according to claim 2 or 3, comprising a step of removing gold nanoparticles not bound to the antigen between the first step and the second step.   The method according to claim 1, wherein the solution contains a compound having an amino group and a hydroxy group.   6. The compound according to claim 1, wherein the compound is at least one selected from trishydroxymethylaminomethane, tri (hydroxymethyl) methylglycine, and tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid. The method described.   The method according to claim 5, wherein the concentration of the compound with respect to the solution is 1 mM or more.   The method according to claim 1, wherein the pH concentration of the solution is 8 or more.   The method according to claim 1, wherein the gold nanoparticles have a particle size of 1 nm or more.   The method according to claim 1, wherein the second step is performed by an electrochemiluminescence method using luminol.   The method according to claim 1, wherein the second step is performed by a fluorescence method using peroxidase. A method for producing reactive oxygen species, comprising mixing gold nanoparticles with a compound having an amino group and a hydroxy group.