JP4341242B2 - Analytical method of binding analysis target substance that does not require labeling dye and analysis kit used therefor - Google Patents

Description

[0001]
[Technical field to which the invention belongs]
The present invention relates to a life science field that requires qualitative and quantitative analysis of biological components such as intermolecular interaction analysis and microchip technology.
[0002]
[Prior art]
Surface plasmon resonance (SPR) and QCM are mainly used for the analysis of the interaction between biomolecules. In addition, FRET is used in which biomolecules and molecules that bind complementarily are labeled with different fluorescent dyes.
[0003]
For microchip technologies such as DNA microarrays, biomolecules or molecules that bind complementarily are labeled with fluorescent dyes or gold nanoparticles, and the fluorescence of the dye or the absorbance or resonance Rayleigh scattering due to surface plasmon resonance of the gold nanoparticles is measured. A detection method is used.
[0004]
[Problems to be solved by the invention]
In SPR and QCM, it is necessary to immobilize molecules that bind complementarily on a solid phase such as a prism or a quartz crystal on which gold is vapor-deposited, and the immobilization yield and the stability of the immobilized molecules are a problem. It was. In addition, intermolecular interactions of biomolecules are originally analyzed in the same aqueous solution as in the living body, but are limited to intermolecular interactions with molecules immobilized on a solid phase.
[0005]
On the other hand, light detection methods using FRET, fluorescent dyes, and gold nanoparticle labels require complicated labeling operations such as linker molecule design, labeling reaction, and separation of unlabeled molecules.
[0006]
In addition, in these light detection methods, the wavelength region used for detection is at most a visible region, and thus it cannot be said to be sufficient to suppress the influence of impurities in the sample. Specifically, since the sample containing the substance to be analyzed is usually a cell or a body fluid, it may contain impurities in the living body that show absorption and fluorescence at wavelengths shorter than 650 nm, so accurate measurement is possible. There was something I couldn't do.
[0007]
In addition to the few fluorescent dyes available in the near-infrared region where there is little interference with impurities, these dyes were chemically unstable. Molecular design and synthesis were also difficult.
[0008]
Although gold nanoparticles are chemically stable, in order to have absorption or Rayleigh scattering at a certain wavelength, it is necessary to align the particle diameter and shape of the gold nanoparticles. A great deal of effort was required to synthesize such gold nanoparticles.
[0009]
In addition, in DNA microarrays, the use of multi-color labels using dyes of different wavelengths is required, but the half-value widths of the fluorescent peak and absorption peak due to the above-mentioned dyes and gold nanoparticles are as broad as several tens of nm. There was a problem that the label interfered optically.
[0010]
Further, contamination due to labeling and nonspecific adsorption are likely to occur, and it was difficult to detect at a high S / N ratio.
[0011]
[Means for Solving the Problems]
The present invention has been made in view of the above-mentioned problems, and without using a label such as a dye, the analysis of the intermolecular interaction of the analyte is performed in an aqueous solution, and detection of a plurality of analytes is performed. It is intended to be specific and quantitative.
[0012]
In order to achieve the above object, the analysis method of the present invention enhances the Raman active substance having a stoichiometric relationship with the substance to be analyzed by coexisting a substrate of surface-enhanced Raman scattering comprising a group of noble metal colloids. This is done by measuring surface-enhanced Raman scattering by an electric field. Thereby, since the wavelength-specific surface-enhanced Raman scattering of the Raman active substance is measured, the substance to be analyzed can be specifically and quantitatively performed without using any label.
[0013]
In the present invention, a laser in the near-infrared region can be used as a light source, and both excitation light and Raman scattered light are near-infrared light. It becomes optical analysis. Since Raman scattering is specific to the structure of the Raman active molecule, specific detection is possible. Furthermore, Raman scattering has significantly higher wavelength resolution than fluorescence and absorption, and can easily identify a plurality of molecules having different structures.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
The present invention includes an analysis target substance, an analysis target substance binding molecule, at least one Raman active molecule that competes with the analysis target substance and binds to the analysis target substance binding molecule, and surface-enhanced Raman scattering. A system comprising a substrate, wherein the binding force of the Raman-active molecule to the analyte-binding molecule is weaker than that of the analyte, the analyte-binding molecule and the Raman activity An analyte is added to the complex to which the molecule is bound, and the Raman-active molecule is released by displacement of the analyte, then captured by a substrate of surface-enhanced Raman scattering, and irradiated with excitation light in this state. This is performed by measuring the surface-enhanced Raman scattering light of the Raman active molecule. As a result, the analysis of the intermolecular interaction between the analyte and the analyte-binding molecule and the specific and quantitative measurement of the analyte are achieved.
[0015]
The present invention also provides an analysis target substance, an analysis target substance binding molecule, at least one Raman active molecule that competes with the analysis target substance binding molecule and binds to the analysis target substance, and surface-enhanced Raman scattering. A system comprising a substrate, wherein the binding force of the Raman-active molecule to the analyte is weaker than that of the analyte-binding molecule, wherein the analyte and the Raman-active molecule are bound to each other The analyte-binding molecule is added to the complex, and the Raman-active molecule is liberated by displacement of the analyte-binding molecule, and then captured by a substrate of surface-enhanced Raman scattering. In this state, excitation light is irradiated, This is performed by measuring the surface-enhanced Raman scattering light of the generated Raman active molecule. As a result, the analysis of the intermolecular interaction between the analyte and the analyte-binding molecule and the specific and quantitative measurement of the analyte are achieved.
[0016]
The present invention also provides an analysis target substance, an analysis target substance binding molecule, at least one Raman active molecule that competes with the analysis target substance and binds to the analysis target substance binding molecule, and surface enhanced Raman scattering. A binding substance of the Raman-active molecule to the analyte binding molecule is stronger than the analyte, wherein the binding property of the analyte and the analyte is The Raman-active molecule is generated when the Raman-active molecule is added to the complex to which the molecule is bound, and the Raman-active molecule is bound by substitution of the analyte, and the excitation light is irradiated in this state, and the analyte is not present. This is done by measuring the difference between the surface enhanced Raman scattered light of the molecules. As a result, the analysis of the intermolecular interaction between the analyte and the analyte-binding molecule and the specific and quantitative measurement of the analyte are achieved.
[0017]
The present invention also provides an analysis target substance, an analysis target substance binding molecule, at least one Raman active molecule that binds to a complex in which the analysis target substance and the analysis target substance binding molecule are bound, and a surface. A system composed of a substrate for enhanced Raman scattering, wherein the Raman active molecule is bound to the complex in which the analyte and the analyte binding molecule are bound, and in this state, excitation light is irradiated, and the analyte is This is done by measuring the difference between the Raman-active molecules generated when they are not present and the surface-enhanced Raman scattered light. As a result, the analysis of the intermolecular interaction between the analyte and the analyte-binding molecule and the specific and quantitative measurement of the analyte are achieved.
[0018]
The substance to be analyzed can be selected from the group consisting of oligonucleotides, enzymes, and antibodies, but is not limited thereto. As the analyte binding molecule, a molecule that can complementarily or specifically bind to the analyte can be selected. An oligonucleotide or a peptide can be contained in at least a part of the Raman active molecule. The Raman-active molecule particularly preferably contains a nitrogen-containing heterocyclic compound as a part of the molecule.
[0019]
Examples of such a combination of an analysis target substance, an analysis target substance substance binding molecule, and a Raman active molecule are not particularly limited, and examples thereof include an oligonucleotide complementary to each other and a primer having adenine at the end.
[0020]
Surface-enhanced Raman scattering substrates include rough electrode metal surfaces, colloids of metal particles, films with metal particles deposited in islands, metal particles encapsulated in a glass matrix by the sol-gel method, and polymers. Inclusive metal particulates have been reported so far. Among these substrates, a colloid of noble metal nanoparticles in an aqueous solution is considered to be most convenient in practice. The reasons are as follows: 1) Fine particles can be synthesized by a liquid phase method, and handling is simple. 2) Applicable to continuous flow analysis systems. 3) The particle size and shape can be controlled. 4) The surface area can be easily defined. 5) The form can be changed for theoretical analysis. Such advantages are pointed out.
[0021]
The precious metal colloid is preferably used in a state where it aggregates due to the addition of salt or the like and forms a group in water.
[0022]
However, the state of aggregated noble metal colloid is usually unstable in an aqueous solution.
[0023]
In the present invention, it is desirable that the group of noble metal colloids constituting the surface-enhanced Raman scattering substrate are held by the dispersible fine particles. Thereby, the stability of the noble metal colloids forming a group can be improved, and surface-enhanced Raman scattering can be easily used (PCT / JP01 / 01854). Here, a swellable layered silicate such as synthetic smectite can be used as the dispersible fine particles.
[0024]
Several such swellable layered silicates are commercially available, for example, trade name Lucentite SWN or Lucentite SWF (synthetic hectorite) or ME (fluorine mica), trade name Smecton SA (synthetic saponite) or trade name Examples thereof include Kunipia F (purified montmorillonite), trade name Tixopy W (synthetic hectorite), trade name Laponite XLG or Laponite RD or Laponite XLS (synthetic hectorite), trade name Multigel (bentonite), and swellable synthetic fluorinated mica.
[0025]
In the present invention, the surface-enhanced Raman scattering of the Raman-active substance having a stoichiometric relationship with the intermolecular interaction is specifically measured by the enhanced electric field of the noble metal colloids that form a group in water. Can be analyzed.
[0026]
The present invention also has a great feature in that it enables high-sensitivity analysis by Raman spectroscopy in the near infrared region. Analysis by Raman spectroscopy in the near-infrared region eliminates the influence of impurities in the matrix containing the analyte and improves specificity and selectivity in the detection of the analyte. It is possible to obtain an extremely practical effect.
[0027]
These Raman-active molecules and analyte-binding molecules constitute a useful analysis kit together with a surface-enhanced Raman scattering substrate.
[0028]
【Example】
The present invention will be specifically described below with reference to examples.
[Example 1]
FIG. 1 is a schematic diagram showing an embodiment of the present invention.
[0029]
[Figure 1]
[0030]
In the above figure, the analysis target substance A, the analysis target substance binding molecule B, at least one Raman active molecule C that competes with the analysis target substance and binds to the analysis target substance binding molecule, and the surface A system comprising a substrate D for enhanced Raman scattering, wherein the binding force of the Raman active molecule C to the analyte binding molecule B is weaker than that of the analyte A. An analyte A is added to a complex BC in which the substance-binding molecule B and the Raman-active molecule C are bound, and the Raman-active molecule C is liberated by substitution of the analyte A, and then the substrate D for surface-enhanced Raman scattering. In this state, the surface is irradiated with excitation light, and the generated surface-enhanced Raman scattering light of the Raman active molecule C is measured.
[0031]
[Example 2]
FIG. 2 shows the specific surface-enhanced Raman scattering spectrum of an aqueous solution of adenine due to the enhanced electric field of a group of noble metal colloids retained by smectite. The adenine concentrations of a, b, c, and d in FIG. 2 are 62.5 μM, 20 μM, 6.25 μM, and 0 μM, respectively. As a Raman spectroscope, FT-Raman Magna manufactured by Thermo Nicolet was used and irradiated with a 1064 nm near-infrared laser and accumulated 128 times. A calibration curve at 735 cm −1 is shown in FIG. In FIG. 3, a is surface-enhanced Raman scattering, and b is normal Raman scattering. Adenine could be quantified by near infrared light.
[0032]
[Example 3]
In the schematic diagram of FIG. 1, oligoadenine (20 base pairs) was measured using oligoadenine (10 base pairs) as the analyte, oligothymine (20 base pairs) as the analyte binding molecule, and adenine as the Raman active molecule. . FIG. 3 a is a surface-enhanced Raman spectrum of a mixed solution of 1 μM oligothymine (20 base pairs) and 20 μM adenine. FIG. 3b is a surface enhanced Raman spectrum of a mixed solution obtained by adding 1 μM oligoadenine (10 base pairs) to a mixed solution of oligothymine (20 base pairs) 1 μM and adenine 20 μM. FIG. 3C is a surface enhanced Raman spectrum of oligothymine (20 base pairs) 5 μM. It was found that the surface-enhanced Raman intensity of adenine weakens when bound to oligothymine, and the surface-enhanced Raman of oligothymine hardly appears. When oligoadenine was added to the complex of oligothymine and adenine, adenine was liberated and the surface-enhanced Raman intensity of adenine was increased. Therefore, it was suggested that the method of the present invention enables the analysis of oligoadenine and the analysis of the interaction between oligoadenine and oligothymine.
[0033]
【The invention's effect】
According to the present invention, it is possible to analyze an intermolecular interaction of an analysis target substance in an aqueous solution without using a label such as a dye, and to detect a plurality of analysis target substances specifically and quantitatively. it can. Moreover, the measurement by near-infrared light can be used easily.
[Brief description of the drawings]
FIG. 1 shows a schematic diagram of an embodiment of the invention.
FIG. 2 shows surface enhanced Raman scattering of adenine. FIG. 3 shows a calibration curve of adenine.
FIG. 4 shows the response of oligoadenine when the analyte is oligoadenine, the analyte binding molecule is oligothymine, and the Raman active molecule is adenine.

Claims (8)

A system comprising an analyte, an analyte-binding molecule, at least one Raman-active molecule that competes with the analyte and binds to the analyte-binding molecule, and a surface-enhanced Raman scattering substrate In the system characterized in that the binding force of the Raman active molecule to the analyte binding molecule is weaker than that of the analyte, the analyte binding molecule and the Raman active molecule are bound to each other. An analyte substance is added to the complex, and the Raman active molecule is liberated by displacement of the analyte substance. Then, the Raman active molecule is captured by a surface-enhanced Raman scattering substrate, and irradiated with excitation light in this state. A method for analyzing intermolecular interactions and substances, characterized by measuring surface-enhanced Raman scattering light. A system comprising an analyte, an analyte-binding molecule, at least one Raman-active molecule that competes with the analyte-binding molecule and binds to the analyte, and a surface-enhanced Raman scattering substrate. In the system characterized in that the binding force of the Raman active molecule to the analyte is weaker than that of the analyte binding molecule, analysis is performed on a complex in which the analyte and Raman active molecule are bound. The target substance-binding molecule is added, the Raman-active molecule is liberated by displacement of the target substance-binding molecule, and then captured by a surface-enhanced Raman scattering substrate. In this state, the excitation light is irradiated and the generated Raman activity A method for analyzing an intermolecular interaction and a substance, which comprises measuring surface-enhanced Raman scattering light of the molecule. The intermolecular interaction and the substance according to claim 1 or 2, wherein at least one of the analyte and the analyte binding molecule is selected from the group consisting of an oligonucleotide, an enzyme, and an antibody. Analysis method. The method for analyzing an intermolecular interaction and a substance according to claim 1 or 2, wherein at least a part of the Raman active molecule contains an oligonucleotide or a peptide. 3. The molecular interaction and substance analysis method according to claim 1 or 2, wherein the analysis target substance and the analysis target substance binding molecule are each composed of complementary oligonucleotides. 3. The method for analyzing intermolecular interactions and substances according to claim 1 or 2, wherein the substrate for surface-enhanced Raman scattering is a group of noble metal colloids. The molecular interaction and substance analysis according to claim 1 or 2, wherein the group of noble metal colloids are held by dispersible fine particles made of a swellable layered silicate such as synthetic smectite. Method. The method for analyzing an intermolecular interaction and a substance according to claim 1 or 2, wherein the binding reaction proceeds in a solution.

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