JP2009047507A - Detection method of target molecule in sample using molecule imprinting particulate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for constructing a sensing system that sufficiently puts the feature of a molecule imprinting particulate uniform in particle size and having a nanosize (average particle size is below 1 μm) to practical use. <P>SOLUTION: The target molecule in a sample is detected by detecting the interaction of a molecule imprinting particulate (A), which has a target molecule recognizing region constructed by a molecule imprinting method and has the average particle size of below 1 μm measured by a dynamic light scattering method, and a molecule accumulation (B) having a monomolecular layer composed of a target molecule or its derivative (B) and characterized in that the molecule imprinting particulate is bondable to the monomolecular layer. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for detecting a target molecule in a sample using molecularly imprinted fine particles, and more specifically, interaction between a molecularly imprinted fine particle and a molecular aggregate having a monomolecular layer of a target molecule or a derivative thereof. The present invention relates to a method for detecting a target molecule in a sample by detection of.

  A molecular imprint method (MI method) is known as one of artificial receptor synthesis methods capable of specifically recognizing a target molecule. The MI method is a method in which a recognition target molecule (target molecule) is used as a template and a binding site (target molecule recognition site) that is selective for the target molecule is artificially constructed in a material.

  A polymer synthesized using the MI method is called a molecular imprint polymer (MIP). MIP radicals a template molecule (target molecule or derivative thereof) and a functional monomer (a molecule having a site that interacts specifically with the template molecule and a polymerizable functional group such as a vinyl group) together with a crosslinking agent. It is constructed by polymerizing and removing the template molecule from within the polymer (see Non-Patent Document 1).

  As target molecules recognized by MIP, herbicides, drugs, insecticides, proteins and peptides, cholesterol, dyes, carbohydrates and the like have been reported, and MIP has been shown to be useful as a target molecule recognition technique. .

  Further, it has been reported that MIP can be applied as a sensor in combination with various detection means. Examples thereof include a quartz crystal chemical measurement device (see Non-Patent Document 2), a surface plasmon resonance measurement device (see Non-Patent Document 3), an electrode (see Non-Patent Document 4), and the like.

On the other hand, due to recent innovations in polymer fine particle synthesis technology, it is possible to control the particle size, shape, particle composition, and particle surface of polymer fine particles with a higher degree of precision, and MIP has also been attempted to make fine particles. (See Non-Patent Document 5).
Mikiharu Tsunoike, Takeshi Endo, “Radical Polymerization Handbook” (1999) NTS Kugimiya, A., Yoneyama, H., Takeuchi, T. Sialic Acid Imprinted Polymer-Coated Quartz Crystal Microbalance, Electroanalysis 2000, 12, 1322-1326. Matsunaga, T., Hishiya, T., Takeuchi, T., Surface Plasmon Resonance Sensor for Lysozyme Basedon Molecularly Imprinted Thin Films, Anal. Chim. Acta 2007, 591, 63-67. Shoji, R., Takeuchi, T., Kubo, I. Atrazine Sensor Based on Molecularly Imprinted PolymerModified Gold Electrode, Anal. Chem. 2003, 75, 4882-4886. Silvestri, D., Borrelli, C., Giusti, P., Cristallini, C., Ciardelli, G., Polymeric devices containing imprinted nanospheres: a novel approach to improve recognition inwater for clinical uses, Anal. Chim. Acta 2005, 542, 3-13.

  As described above, the advancement of polymer fine particle synthesis technology makes it possible to synthesize molecularly imprinted fine particles having a uniform particle size and having a uniform particle size (average particle size of less than 1 μm) easily and inexpensively. Such molecularly imprinted fine particles have an increased specific surface area compared to fine particles obtained by mechanically pulverizing conventional MIP polymers, so that they can effectively interact with target molecules. It is done. Therefore, by fully utilizing these characteristics, it is expected to construct a novel sensing system (detection system) using nano-sized molecular imprinted fine particles (average particle size is less than 1 μm). That is, the construction of a novel sensing system (detection system) using nano-sized molecularly imprinted fine particles (average particle size is less than 1 μm) is an issue in this research field.

  The present invention has been made to solve the above-described problems, and an object of the present invention is to provide a novel method for detecting a target molecule using molecular-imprinted fine particles of nanosize (average particle size is less than 1 μm). It is.

  As a result of intensive studies, the present inventor has accumulated the target molecule derivative on the surface of the surface plasmon resonance (SPR) sensor substrate, and sent the molecular imprinted fine particle, so that the molecular imprinted fine particle interacts with the target molecule derivative accumulated substrate. Thus, it has been found that the target molecule in the sample can be detected by utilizing the change in the SPR spectrum. Furthermore, the present inventor has shown that the fluorescent molecularly imprinted fine particles interact with the gold fine particles having the target molecule derivative accumulated on the surface, thereby absorbing light energy between the molecular imprinted fine particles and the target molecule derivative-integrated gold fine particles. And found that the target molecule in the sample can be detected by utilizing the release behavior, and the present invention has been completed.

That is, the target molecule detection method in a sample according to the present invention is a method for detecting a target molecule in a sample, and is characterized by detecting the following interaction between (A) and (B).
(A) Molecularly imprinted fine particles having a target molecule recognition site constructed by a molecular imprint method and having an average particle diameter of less than 1 μm measured by a dynamic light scattering method (B) A single molecule of a target molecule or a derivative thereof Molecular assembly having a layer and capable of binding molecularly imprinted fine particles to a monolayer

  In the method for detecting a target molecule in a sample according to the present invention, the molecular assembly preferably has a monomolecular layer formed on the surface of the substrate. In addition, the molecular aggregate preferably has a monomolecular layer formed on the surface of fine particles dispersible in a solvent.

  Further, the molecular assembly preferably absorbs or emits light energy. On the other hand, the molecularly imprinted fine particles preferably emit light energy when the molecular aggregate absorbs light energy, and absorb light energy when the molecular aggregate releases light energy. A more preferable combination is that the molecularly imprinted fine particles emit fluorescence, and the molecular aggregate absorbs the fluorescent energy emitted by the molecularly imprinted fine particles and quenches the fluorescence of the molecularly imprinted fine particles, or the fluorescent energy emitted by the molecularly imprinted fine particles. And the molecular aggregate itself emits further fluorescence. Conversely, the molecular aggregate emits fluorescence, and the molecularly imprinted fine particles absorb the fluorescent energy emitted by the molecular aggregate and quench the fluorescence of the molecular aggregate, or absorb the fluorescent energy emitted by the molecular aggregate and absorb the molecule. The imprint fine particles themselves may further emit fluorescence.

  The method for detecting a target molecule in a sample according to the present invention includes a sample contacting step in which a sample, the molecular imprinted fine particles, and the molecular aggregate are brought into contact with each other.

A target molecule detection kit in a sample according to the present invention is a reagent kit for detecting a target molecule in a sample, and includes the following (A) and (B).
(A) Molecularly imprinted fine particles having a target molecule recognition site constructed by a molecular imprint method and having an average particle diameter of less than 1 μm measured by a dynamic light scattering method (B) A single molecule of a target molecule or a derivative thereof Molecular assembly having a layer and capable of binding molecularly imprinted fine particles to a monolayer

  According to the present invention, there is provided a novel method for detecting a target molecule using nano-sized (average particle size is less than 1 μm) molecular imprinted fine particles and a molecular assembly having a monomolecular layer of the target molecule or its derivative. can do. The method for detecting a target molecule in a sample according to the present invention can be applied to various detection means, and has an effect that the application range is wide.

  First, the background to the completion of the present invention will be briefly described.

  In order to develop a biomimetic technique that artificially mimics the function of a biomolecule, the present inventor has conducted earnest research on sensing using a target molecule recognition structure constructed by a molecular imprint method. This time, the present inventors synthesized molecularly imprinted fine particles having bisphenol A (hereinafter referred to as “BPA”), which is known as an endocrine disrupting substance exhibiting a female hormone-like action, as target molecules. An attempt was made to detect the interaction with the target molecule BPA as a change in surface plasmon resonance (hereinafter referred to as “SPR”) spectrum.

  First, the present inventor produced a substrate in which BPA recognition molecular imprinted fine particles were self-assembled on the surface of an SPR glass substrate, set this as an SPR measurement substrate in an SPR measurement device, and fed a BPA solution to perform SPR. The change of the spectrum was observed. However, almost no change in the SPR spectrum was observed. Similarly, no change in the SPR spectrum was observed when a substance known to adsorb nonspecifically with BPA was used for the substrate. From these results, BPA is not adsorbed on BPA recognition molecule imprinted fine particles on the substrate surface, and the mass change (density change) on the substrate surface due to BPA adsorption is too small to be captured as a change in SPR spectrum. Became clear. This result is valid on the principle of SPR that the change in dielectric constant due to the density change on the substrate surface affects the resonance wavelength. Since the target BPA (Mw = 228) has a molecular weight that is too small, it is a target to be detected by SPR. It was considered inappropriate.

  Therefore, the present inventor tried to produce an SPR measurement substrate in which a BPA derivative is integrated on the substrate surface, set the substrate in an SPR measurement device, and send a BPA recognition molecule imprinted fine particle solution. As a result, it was found that adsorption of molecularly imprinted fine particles on the substrate can be detected as a change in the SPR spectrum. Further, it was found that the change in the SPR spectrum when the BPA-recognized molecularly imprinted fine particles were added to a sample containing BPA and allowed to flow and then sent was different from the case where only the BPA-recognized molecularly imprinted fine particles were sent. As a result, a new target molecule detection method capable of detecting low molecular weight BPA as a change in the SPR spectrum was completed (see Example 2 in the subsequent stage).

  Furthermore, in a detection system using the above-described molecularly imprinted fine particles and a molecular assembly having a monomolecular layer of a target molecule or a derivative thereof, a method capable of detecting a target molecule without using an expensive device such as an SPR measurement device Researched to develop. This is because there are a limited number of facilities that have expensive devices such as SPR measurement devices, and not everyone can easily implement them.

Therefore, the present inventor has attempted to use fluorescent BPA-recognizing molecule-imprinted fine particles and gold fine particles in which BPA derivatives are accumulated on the surface. This is because gold fine particles are known to exhibit a quenching reaction when present in the vicinity of a fluorescent dye as a fluorescence quencher (see, for example, the following references: O. Yen-Yu, H. Michael). , J.Phys.Chem, 2006,110,2031.C.Fabio,
C. Giuseppe, B. Anna, C. Salvatore, J. Phys. Chem, 2006, 110, 16491. N. Kato, F. Caruso,
J.Phys.Chem, 2005,109,19604.H.Cheng, D.Silvester, G.Wang, G.Kalyuzhny,
A. Douglas, R. Murry, J. Phys. Chem, 2006, 110, 4637.). As a result, by adding the BPA-integrated gold fine particles to the fluorescent BPA recognition molecular imprinted fine particle solution, the BPA-integrated gold fine particles are adsorbed on the fluorescent molecularly imprinted fine particles, and the fluorescence of the fluorescent molecularly imprinted fine particles is quenched. I found it. As a result, in a detection system using molecular imprinted fine particles and a molecular assembly having a monomolecular layer of a target molecule or a derivative thereof, the target can be obtained by utilizing the release and absorption of light energy without using an expensive measuring instrument. It was shown that molecules can be detected (see Example 3 below).

  Hereinafter, the method for detecting a target molecule in a sample according to the present invention will be described in detail.

[Target molecule detection method]
A target molecule detection method in a sample according to the present invention (hereinafter referred to as “detection method according to the present invention”) is a method for detecting a target molecule in a sample, and is constructed by (A) a molecular imprint method. A molecular imprinted fine particle having a target molecule recognition site and having an average particle size of less than 1 μm measured by a dynamic light scattering method, and (B) a monomolecular layer of the target molecule or a derivative thereof, And detecting an interaction with the molecular aggregate to which the molecularly imprinted fine particles can bind.

  The molecularly imprinted fine particles used in the detection method according to the present invention may have a target molecule recognition site constructed by the molecular imprint method, and the average particle diameter measured by the dynamic light scattering method may be less than 1 μm. . Therefore, the material may be any material as long as it can construct a target molecule recognition site by applying the molecular imprint method and can form fine particles having an average particle diameter measured by the dynamic light scattering method of less than 1 μm. Specific examples include organic polymers, inorganic polymers, metal oxides, and the like. The “molecular imprint method” is a method of artificially constructing a binding site (target molecule recognition site) that is selective for the target molecule in a material using the molecule to be recognized (target molecule) as a template. means.

An organic polymer material can be obtained by using a target molecule or its derivative or similar compound as a template molecule in the radical polymerization reaction. A material made of an inorganic polymer can be obtained by using a target molecule or its derivative or similar compound as a template molecule in the sol-gel reaction. (Lee, SW,
Ichinose, I., Kunitake, T., Enantioselective binding of amino acid
Chemist. Lett. 2002, 678-679.) Metal oxides are used as template molecules for target molecules or their derivatives and similar compounds. Derived onto imprinted TiO2 ultrathin films. It can be obtained by using one type of liquid phase precipitation method (Feng, L., Liu, Y., Hu, J., Langmuir 2004, 20, 1786.). In these methods, a molecular recognition site that interacts complementarily with a template molecule during a polymerization reaction is constructed simultaneously with the synthesis of the polymer. More specifically, first, a functional monomer having both a functional group capable of binding to the target molecule or its derivative or similar compound and a polymerizable functional group is bound to the target molecule to form a target molecule / functional monomer complex. Let The bond for forming this complex may be a covalent bond or a non-covalent bond as long as it can be cleaved. Next, a crosslinking agent and a polymerization initiator are added to the target molecule / functional monomer complex, and a polymerization reaction is performed. Thereby, an organic polymer in which the shape of the template molecule and the arrangement of the interaction points are stored is obtained. Finally, the template molecule is cleaved and removed from the obtained polymer to obtain a polymer (molecular imprint polymer) having a molecular recognition site that interacts with the template molecule in a substrate-specific manner. For the method of synthesizing the molecularly imprinted polymer, for example, the reference “Komiyama, M., Takeuchi, T., Mukawa, T., Asanuma, H.“ Molecular
Refer to the description of “Imprinting”, WILEY-VCH, Weinheim, 2002.

  The method for obtaining molecularly imprinted fine particles is roughly classified into two methods: a method for obtaining fine particles by mechanical pulverization and a method for obtaining fine particles by growing nuclei. When mechanically pulverizing, known pulverizers such as a hammer pulverizer, an impact pulverizer, a roll pulverizer, and a jet airflow pulverizer can be used. The obtained pulverized product (fine particles) can be sieved as necessary to prepare a particle size.

Examples of the method for obtaining fine particles of molecularly imprinted polymer include precipitation polymerization method, dispersion polymerization method, emulsion polymerization method, seed emulsion polymerization method, etc. (reference: Mikiharu Tsunoike, Takeshi Endo, “Radical” Polymerization Handbook "(1999) NTS, G. Schmid Ed. Nanoparticles,
(See Wiley-VCH (2004)). The inventor has synthesized BPA-recognized molecularly imprinted polymer fine particles by a seed emulsion polymerization method using polystyrene particles as seeds (see Example 1).

  The molecularly imprinted fine particles used in the detection method according to the present invention have an average particle size measured by a dynamic light scattering method of less than 1 μm. Here, the “dynamic light scattering method” refers to fluctuations in the scattered light intensity depending on the Brownian motion of the particles detected when laser light is applied to the solution in which the particles are dispersed and changes in the scattered light are measured. This is a method for deriving the size (particle diameter) of particles based on the above. Particle size measuring devices based on the dynamic light scattering method are commercially available from various companies (Otsuka Electronics, Sysmex, Beckman Coulter, etc.) and suitable for measuring the average particle size of molecularly imprinted fine particles used in the detection method according to the present invention. Can be used.

  If the average particle size is less than 1 μm, the molecularly imprinted fine particles can be uniformly dispersed in a solvent (liquid dispersion medium). The lower limit of the average particle diameter is not particularly limited, and it is preferably as small as possible unless aggregation occurs. From the viewpoint of dispersibility and ease of handling, the average particle size of the molecularly imprinted fine particles is preferably 100 nm to 500 nm.

  The target molecule to be detected by the detection method according to the present invention is not particularly limited, and a wide range of molecules from low molecular compounds such as drugs to high molecular compounds such as proteins can be used as target molecules. Preferable target molecules include biomolecules. The biomolecule may be a molecule that exists in an organism. By targeting a biomolecule, it is possible to provide a method for detecting a target molecule that can be used for diagnosis of disease, clinical examination, basic research of biology, and the like.

  The sample to which the detection method according to the present invention is applied may be any sample that can contain a target molecule. The form of the sample is not particularly limited, and examples thereof include liquids, solids, granules, powders, fluids, and tissue sections. When targeting biomolecules, animal and plant biological components can be suitably used. Examples of samples derived from animals including humans include body fluids such as blood, tissue fluid, lymph fluid, cerebrospinal fluid, pus, mucus, nasal discharge, sputum, urine, feces, ascites, skin, lung, kidney, mucous membrane, various organs, Examples thereof include a washing solution after washing tissue such as bone, nasal cavity, bronchi, skin, various organs, bone, and the like, and dialysis drainage.

  The molecular assembly used in the detection method according to the present invention may be any one having a monomolecular layer of a target molecule or a derivative thereof and capable of binding the molecularly imprinted fine particles (A) to the monomolecular layer. . The monomolecular layer of the target molecule or derivative thereof is preferably formed on the surface of the substrate or the surface of fine particles that can be dispersed in a solvent. The fine particles dispersible in the substrate and the solvent will be described later. This monomolecular layer may be formed on the entire surface of the molecular assembly or may be formed on a part of the surface. “Monolayer of target molecule or its derivative” means that the target molecule or its derivative is immobilized by creating a monomolecular layer on the substrate surface or fine particle surface like a self-assembled film or Langmuir-Blodgett film. Means that

  In the detection method according to the present invention, the interaction between the above-described molecularly imprinted fine particles and a molecular aggregate having a monomolecular layer of a target molecule or a derivative thereof (hereinafter referred to as “molecular aggregate”) is a molecularly imprinted fine particle. The target molecule recognition site of the molecule specifically recognizes the target molecule or its derivative on the surface of the molecular aggregate, and the molecular imprinted microparticle and the molecular aggregate are in close proximity so that they can interact physically and chemically. Means.

(1) A method using a substrate on which a monomolecular layer of a target molecule or a derivative thereof is formed on the surface (first embodiment)
In one embodiment of the detection method according to the present invention, the molecular aggregate is preferably a substrate (hereinafter, referred to as “molecular integrated substrate”) on which a monomolecular layer of a target molecule or a derivative thereof is formed. The substrate may be any substrate as long as a monomolecular layer of the target molecule or derivative thereof can be formed on the surface. Examples of suitable substrates include metal substrates, glass substrates, silicon substrates, optical waveguide substrates, and the like. Moreover, it is preferable to select the material of the substrate as appropriate according to the detection means used for detecting the interaction between the molecularly imprinted fine particles and the target molecule on the substrate surface or a derivative thereof. For example, when applied to the surface plasmon resonance (SPR) method, a gold substrate for SPR is preferably used, and when applied to the localized plasmon resonance (LSPR) method, an optical waveguide substrate is preferably used. When applied to a sensor, it is preferable to use a quartz crystal as a sensing chip, and when applied to detection by an electrochemical method, it is preferable to use a metal substrate (for example, a gold substrate) serving as an electrode. When applied to detection by light emission or color development, it is preferable to use a glass, quartz substrate, optical fiber, or optical waveguide substrate.

  As a method for forming a monolayer of a target molecule on the surface of a substrate, for example, the present inventor has used a self-assembled monolayer (SAM: Self-Assembled) of BPA into which a thiol group is introduced using a gold-thiol bond. Monolayer) is formed to produce a molecular integrated substrate of a BPA derivative to which BPA imprinted fine particles can bind (see Example 2 below).

  FIG. 1 is a schematic diagram showing a state where molecularly imprinted fine particles are adsorbed on a molecular integrated substrate. As shown in FIG. 1, in this embodiment, when the target molecule or its derivative on the surface of the molecular integrated substrate and the molecularly imprinted fine particle come into contact with each other, the target molecule recognition site of the molecularly imprinted fine particle changes the target molecule or its derivative. Specifically recognized, the molecularly imprinted fine particles are adsorbed on the molecular integrated substrate by the interaction between the two. By detecting this interaction, the target molecule in the sample can be detected. As a means for detecting the interaction between the molecularly imprinted fine particles and the target molecule on the surface of the molecular integrated substrate or its derivative, surface plasmon resonance (SPR) method, localized plasmon resonance (LSPR) method, quartz crystal sensor, electrochemical Known methods such as a method, a light emitting method, a color developing method, and an optical waveguide spectroscopy can be suitably applied. In any case, contrary to the normal case, the molecular imprint fine particles having the target molecule recognition site for capturing the target molecule are not immobilized. In the case of a flow type detection system, the substrate is dispersed and flowed in a carrier solution, and a substrate in which a target molecule or a derivative thereof forms a monomolecular layer and is accumulated on the surface is used. This point is a feature of the detection method according to the present invention, and it is possible to detect even a low molecular target molecule having a very small interaction with the molecularly imprinted fine particles.

  When detecting a target molecule in a sample, the strength of the interaction when the molecular imprinted fine particle and the molecular integration substrate are brought into contact with each other, the sample that may contain the target molecule, the molecular imprinted fine particle, and the molecular integration. The strength of the interaction when the three parties are brought into contact with the substrate is compared. That is, if a target molecule is present in the sample when the three are brought into contact with each other, the target molecule in this sample competes with the target molecule or its derivative accumulated on the substrate surface, and the target molecule of the molecularly imprinted microparticle Adsorb to the recognition site. As a result, since the interaction between the molecularly imprinted fine particles and the molecular integrated substrate is smaller than when the two are brought into contact with each other, it can be seen that the target molecule is present in the sample. At this time, it is possible to quantify the target molecule in the sample by creating a calibration curve.

  When contacting the sample, the molecularly imprinted fine particle, and the molecular integrated substrate, the molecularly imprinted fine particle may be brought into contact with the sample before contacting the molecularly imprinted fine particle and the molecular integrated substrate. After contacting the molecular integrated substrate, the molecularly imprinted fine particles may be contacted with the sample, or the three may be simultaneously contacted. It is preferable to select an optimal order according to the target molecule to be detected, the detection means used, and the like.

(2) Method using a fine particle having a monomolecular layer of a target molecule or its derivative formed on the surface (second embodiment)
In another embodiment of the detection method according to the present invention, the molecular aggregate is a fine particle (hereinafter referred to as “molecular integrated fine particle”) having a monomolecular layer of a target molecule or derivative thereof formed on the surface and dispersible in a solvent. It is preferable. Dispersible in a solvent means a state in which a uniform dispersion state is maintained in a solvent (liquid dispersion medium) and aggregation or precipitation does not occur. Accordingly, a suitable size of the fine particles is selected depending on conditions such as a substance constituting the fine particles, a target molecule, and a solvent to be used. Usually, the average particle diameter is selected in the range of several tens nm to several hundreds nm.

  The fine particles are not particularly limited as long as a monomolecular layer of a target molecule or a derivative thereof can be formed on the surface thereof. Suitable fine particles include organic polymer fine particles (polystyrene, polymethyl methacrylate, etc.), inorganic polymer fine particles (silica, alumina, etc.) and metal fine particles (gold, silver, platinum, titanium, etc.).

  In the case of organic polymer fine particles or inorganic polymer fine particles, fine particles with a target molecule or derivative monomolecular layer formed on the surface are introduced into the polymer surface layer by introducing a functional group capable of chemically bonding to the target molecule or derivative thereof in advance. It can be obtained by binding a target molecule or a derivative thereof. In the case of metal fine particles, it can be obtained by introducing a thiol group into the target molecule or derivative thereof to form a self-assembled film on the surface of the metal fine particles. The inventor has synthesized gold fine particles having a BPA monomolecular layer formed on the surface from BPA into which a chloroauric acid solution and a thiol group have been introduced (see Example 3 in the subsequent stage).

  In this embodiment, it is preferable that the molecular integrated fine particles absorb or emit light energy. “Absorb light energy” means that the molecule absorbs light energy and goes from the ground state to the excited state, and “releases light energy” means when the molecule returns from the excited state to the ground state. It means that energy is released as light.

  When using molecularly imprinted fine particles that emit light energy, the use of molecularly integrated fine particles that absorb the light energy emitted by molecularly imprinted fine particles eliminates the quenching phenomenon that occurs when molecularly imprinted fine particles interact with molecularly integrated fine particles. Sensing can be used. In addition, when using molecularly imprinted fine particles that emit light energy, it is possible to absorb the light energy emitted from the molecularly imprinted fine particles, and when the absorbed light energy becomes excited and returns to the ground state, Occurs when molecularly imprinted fine particles interact with molecularly integrated fine particles by using molecularly integrated fine particles that generate resonance energy transfer of light that emits new light on a longer wavelength side that is different from the wavelength of light emitted by imprinted fine particles Sensing using the resonance energy transfer phenomenon of light becomes possible.

  Contrary to the above, in the case of using molecularly integrated fine particles that emit light energy and molecularly imprinted fine particles that absorb light energy emitted by the molecularly integrated fine particles, the same sensing is possible, and molecules that emit light energy can be obtained. It is possible to absorb the light energy emitted by the integrated fine particles and molecular integrated fine particles, and when the absorbed light energy enters the excited state and returns to the ground state, the wavelength is different from the wavelength of the light emitted by the molecular integrated particles. The same sensing is also possible when using molecularly imprinted fine particles that generate resonance energy transfer of light that emits new light.

  The substance that absorbs light energy is not specified as a specific compound, and any compound having an absorption maximum in the vicinity of the emission wavelength of the light emitting substance to be used may be used. In the case of utilizing the resonance energy transfer phenomenon of light, any compound may be used as long as it is a compound having an absorption maximum near the emission wavelength of the light-emitting substance to be used and generates a new light emission phenomenon using the absorbed light energy. Examples of the substance that emits light energy include fluorescent substances such as pyrene, fluorescein, rhodamine, coumarin, dansyl, Cy3 and Cy5, and inorganic light-emitting substances called quantum dots such as CdSe fine particles.

  Therefore, molecularly integrated fine particles that absorb light energy are doped with a compound having an absorption maximum in the vicinity of the emission wavelength of the light-emitting substance used during the fine particle synthesis, or a functional group is introduced on the fine particle surface to introduce covalent bonds or non- It can be obtained by chemically bonding with a covalent bond. Similarly, molecularly integrated microparticles that emit light energy can be doped with a material selected from the above-mentioned substances as appropriate during the synthesis of the microparticles, or introduced with functional groups on the surface of the microparticles to chemistry by covalent bonding or noncovalent bonding. Can be obtained by combining them.

  In addition, molecularly imprinted fine particles that absorb light energy can be doped or copolymerized by mixing a compound having an absorption maximum near the emission wavelength of the luminescent material used in the synthesis of the molecularly imprinted fine particles. It can be obtained by introducing a functional group and chemically bonding it with a covalent bond or a non-covalent bond. Similarly, the molecularly imprinted fine particles that emit light energy are mixed with a material appropriately selected from the above-listed materials during the synthesis of the molecularly imprinted fine particles, copolymerized, or functional groups are introduced on the surface of the fine particles. It can be obtained by chemically bonding with a covalent bond or a non-covalent bond.

  In the present embodiment, it is preferable to use molecularly imprinted fine particles that emit fluorescence and molecular integrated fine particles that absorb fluorescence energy emitted from the molecularly imprinted fine particles and quench the fluorescence of the molecularly imprinted fine particles. Thereby, the interaction between the molecularly imprinted fine particles and the molecular integrated fine particles can be detected using the fluorescence intensity as an index.

  Alternatively, molecularly imprinted fine particles that emit fluorescence and molecularly integrated fine particles that absorb fluorescence energy emitted from the molecularly imprinted fine particles and generate a fluorescence resonance energy transfer phenomenon in which they themselves emit fluorescence may be used. Similarly, the interaction between the molecularly imprinted fine particles and the molecular integrated fine particles can be detected using the fluorescence intensity based on the fluorescence resonance energy transfer phenomenon as an index.

  The molecularly imprinted fine particles emitting fluorescence can be obtained by adding a fluorescent monomer when synthesizing the molecularly imprinted fine particles of the organic polymer material. As the fluorescent monomer, for example, pyrene, fluorescein, rhodamine, coumarin, dansyl, Cy3, Cy5 and the like can be used.

Molecularly integrated fine particles that absorb and extinguish fluorescence energy are the same as described above using metals (gold, silver, platinum, titanium, etc.) and organic compounds (organic compounds having an absorption wavelength at the fluorescence wavelength of the fluorescent substance). Can be obtained by the method. In addition, molecular integrated fine particles that absorb fluorescence energy and emit fluorescence further can be obtained in the same manner as described above using a fluorescent substance that can be excited at a wavelength near the emission wavelength of the fluorescent substance that emits light first. .

  Conversely, it is also possible to use molecularly integrated fine particles that emit fluorescence and molecularly imprinted fine particles that absorb the fluorescence energy emitted from the molecular integrated body and quench the fluorescence of the molecularly integrated fine particles. Similarly, it is also possible to use molecularly integrated fine particles that emit fluorescence and molecularly imprinted fine particles that absorb fluorescence energy emitted from the molecular integrated body and emit fluorescence further. These molecular integrated fine particles and molecular imprinted fine particles can also be obtained by the same method as described above.

  In the case where the interaction between the molecularly imprinted fine particles and the molecular integrated fine particles is detected using the fluorescence intensity as an index, the particle diameters of the fluorescent molecularly imprinted fine particles and the molecular integrated fine particles are preferably not more than the excitation wavelength of the fluorescence. This is because the fine particles in the dispersion are not visible even when irradiated with excitation light.

  In the following, the target molecule is detected using the fluorescence intensity as an index by the interaction between the molecularly imprinted fine particles that emit fluorescence and the molecularly integrated fine particles that absorb the fluorescence energy emitted from the molecularly imprinted fine particles and quench the fluorescence of the molecularly imprinted fine particles. A detection method will be described.

  FIG. 2 is a schematic diagram showing an interaction between molecularly imprinted fine particles (fluorescent molecularly imprinted fine particles) that emit fluorescence and molecularly integrated gold fine particles. As shown in FIG. 2, when only fluorescent molecularly imprinted fine particles are present in the system, strong fluorescent emission is observed by excitation. When molecularly integrated gold microparticles with a monomolecular layer of a target molecule derivative formed on the surface of gold microparticles that absorb fluorescence energy are added, the molecularly integrated gold microparticles adsorb to the target molecule recognition site on the surface of the fluorescent molecularly imprinted microparticle. And since this acts as a quencher, a quenching reaction proceeds. Furthermore, it is considered that fluorescence is restored when a target molecule, a target compound derivative, or a compound that inhibits the interaction between particles is added.

  When detecting a target molecule in a sample, the fluorescence intensity when the two molecules of the fluorescent molecule imprinted fine particle and the molecular integrated fine particle acting as a quencher are brought into contact with each other, the sample that may contain the target molecule, and the fluorescence A comparison is made of the fluorescence intensity when the three molecules of the molecular imprinted fine particles and the molecular integrated fine particles (quenching agent) are brought into contact with each other. That is, if a target molecule is present in the sample when the three are brought into contact with each other, the target molecule in the sample competes with the target molecule on the surface of the molecule-integrated fine particle or its derivative to target the fluorescent molecularly imprinted fine particle. Adsorbs to the molecular recognition site. As a result, compared with the case where the two are brought into contact with each other, the interaction between the fluorescent molecularly imprinted fine particles and the molecular integrated fine particles is reduced, so the degree of quenching is also reduced. That is, the fluorescence intensity increases and it can be seen that the target molecule is present in the sample. At this time, it is possible to quantify the target molecule in the sample by creating a calibration curve.

  When the sample, the molecularly imprinted fine particle, and the molecularly integrated fine particle are brought into contact with each other, the molecularly imprinted fine particle may be brought into contact with the sample before contacting the molecularly imprinted fine particle and the molecularly integrated fine particle. Then, the molecular imprinted fine particles may be brought into contact with the sample, or the three members may be brought into contact at the same time. It is preferable to select an optimal order according to the target molecule to be detected.

  In this embodiment, the liquid phase detection system using the molecularly imprinted fine particles that emit fluorescence and the molecular integrated fine particles that act as a quencher has been described. However, the molecularly imprinted fine particles that emit fluorescence are immobilized on the substrate. Thus, a detection system using the fluorescence intensity on the substrate as an index can also be used.

  Further, the present invention can also be carried out using a molecular integrated substrate instead of the molecular integrated fine particles. Specifically, for example, the fluorescence intensity on the substrate when only the molecularly imprinted fine particles are dropped on the molecular integrated substrate, and the sample that may contain the target molecule and the molecularly imprinted fine particles on the substrate when the molecularly integrated substrate is dropped. It is possible to detect the target molecule in the sample by comparing the fluorescence intensity of the sample.

[Target molecule detection kit]
The target molecule detection kit in the sample according to the present invention may be any one provided with the following (A) and (B).
(A) Molecularly imprinted fine particles having a target molecule recognition site constructed by a molecular imprint method and having an average particle diameter of less than 1 μm measured by a dynamic light scattering method (B) A single molecule of a target molecule or a derivative thereof There is no particular limitation on the structure of the specific kit other than the molecular assembly in which the molecular imprinted fine particles can be bound to the monomolecular layer, and necessary reagents and instruments are appropriately selected. The kit may be configured. By using the kit according to the present invention, the detection method according to the present invention can be carried out simply and quickly.

  (B) is a substrate on which a monomolecular layer of a target molecule or a derivative thereof is formed on a surface (molecular integrated substrate), or a fine particle (a monomolecular layer of a target molecule or a derivative thereof formed on the surface and dispersible in a solvent) Molecularly integrated fine particles) are preferable, and fine particles (molecular integrated fine particles) in which a monomolecular layer of a target molecule or a derivative thereof is formed on the surface and can be dispersed in a solvent are more preferable.

  As a preferred combination of (A) and (B), as described in the second embodiment, (A) molecular integrated fine particles that absorb light energy and (B) molecular imprinted fine particles that emit light energy. Or a combination of (A) molecular integrated fine particles that emit light energy and (B) molecular imprinted fine particles that absorb light energy.

  More specifically, (1) a combination of molecularly imprinted fine particles that emit fluorescence and molecularly integrated fine particles that absorb fluorescence energy emitted from the molecularly imprinted fine particles and quench the fluorescence of the molecularly imprinted fine particles; (2) Combination of molecularly imprinted fine particles that emit fluorescence and molecular integrated fine particles that absorb fluorescence energy emitted by the molecularly imprinted fine particles and emit themselves further, (3) molecular integrated fine particles that emit fluorescence and the molecular integrated In combination with molecularly imprinted fine particles that absorb the fluorescence energy emitted by the body and quench the fluorescence of the molecularly integrated fine particles, (4) the molecular integrated fine particles emitting fluorescence and the fluorescent energy emitted by the molecular integrated body absorb themselves Furthermore, a combination with molecularly imprinted fine particles that emit fluorescence is mentioned. Since these details have been described in the second embodiment, description thereof will be omitted here.

  Moreover, this kit can be used according to the implementation procedure of the detection method which concerns on the above-mentioned this invention.

  As used herein, a “kit” is intended to be a package with a container (eg, bottle, plate, tube, dish, etc.) containing a specific material. Preferably, an instruction manual for using the material is provided. The instructions for use may be written or printed on paper or other media, or may be affixed to electronic media such as magnetic tape, computer readable disk or tape, CD-ROM, and the like.

[Example 1: Synthesis and evaluation of bisphenol A-recognized molecular imprinted fine particles]
Bisphenol A (BPA) recognizing molecular imprinted fine particles were synthesized and evaluated. All reagents used in this example were purchased commercially. Moreover, the following was used as an analytical instrument. That is, high performance liquid chromatography (HPLC) was used for evaluation of the synthesized molecularly imprinted fine particles. For HPLC, a personal computer was connected to an instrument (GILSON) consisting of an auto sample injector (AUTOINJECTOR 234), a pump (322 pump), and a UV detector (UV / VIS-152), and Unipoint V. 3.00 was used. A centrifuge SRX-201 manufactured by Tommy Seiko Co., Ltd. was used for washing the polymer. A dynamic light scattering photometer (DL-6500) manufactured by Otsuka Electronics Co., Ltd. was used for measurement of particle size distribution and average particle size. The centrifugal evaporator used was CVE-2000 type manufactured by Tokyo Science Instruments Co., Ltd. Eppendorf Thermomixer Comfort was used for the incubation. As a scanning analytical electron microscope (SEM), JSM-5610LVS manufactured by JEOL Ltd. (JEOL) was used.

(1-1) Synthesis of BPA-recognizing molecule-imprinted fine particles Styrene particles for use as seeds (seed particles) were synthesized. The styrene monomer was obtained by removing the polymerization inhibitor with an inhibitor remover. A 500 ml separable flask was charged with a monomer and distilled water according to the recipe of Table 1, and heated to a polymerization temperature of 80 ° C. in a nitrogen atmosphere while stirring. Initiator V-50 was dissolved in 5 ml distilled water, and the aqueous solution was added to the heated separable flask. Under nitrogen atmosphere, stirring polymerization was carried out at 80 rpm using a mechanical stirrer at 80 ° C. for 12 hours. After completion of the polymerization, centrifugal washing was performed three times to remove unreacted monomers and initiators, and the emulsion solid content concentration was adjusted to 0.74%. The polymerization rate was 100%, and the average particle size measured by a dynamic light scattering photometer (DLS) was 381 nm.

  Using the seed obtained above, BPA-recognized molecular imprinted fine particles were synthesized. 30 g of a polystyrene seed solution (0.997 wt%) was weighed into a 50 ml vial, a predetermined amount of monomer was added according to the recipe in Table 2, and the mixture was stirred at room temperature for 6 hours to swell. After confirming disappearance of the monomer phase, the mixture was heated to a polymerization temperature of 80 ° C. in a nitrogen atmosphere, and an aqueous solution in which initiator V-50 was dissolved in 5 ml distilled water was added to the heated vial. Under a nitrogen atmosphere, stirring polymerization was performed at 80 ° C. and a stirrer at 200 rpm for 12 hours. After completion of the polymerization, centrifugal washing was performed 3 times to remove unreacted monomers and initiators. The polymerization rate was 88.5%, and the average particle size by DLS was 438.5 nm. Blank fine particles (“BNP” in Table 2) were also synthesized without using the BPA dimethacrylate monomer under the same conditions as the BPA-recognized molecular imprinted fine particles (“INP” in Table 2).

  In order to remove BPA from the BPA-recognized molecularly imprinted fine particles, a hydrolysis reaction was performed. 80 ml of the obtained molecular imprinted fine particle solid concentration of 0.7 wt% was added to a 100 ml eggplant flask, and sodium hydroxide was added to adjust to 1M. It was stirred for 24 hours under reflux for hydrolysis. After 24 hours, stirring was stopped and the mixture was cooled to room temperature, and then the supernatant was removed by centrifugation. An aqueous HCl solution (1M) was added thereto, and ultrasonic irradiation was performed for 10 minutes, and the solvent was removed by centrifugation. Washing with an acid was repeated three times, and finally, washing was performed twice with distilled water and stored as an emulsion.

(1-2) Evaluation of BPA-recognized molecularly imprinted fine particles A recombination experiment by HPLC was performed as an evaluation of the molecular recognition ability of the obtained polymer (BPA-recognized molecularly imprinted fine particles). 5 mg of the synthesized BPA recognition molecule imprinted fine particles are weighed into a 1.5 ml vial, and there are four types (BPA, bisphenol B (BPB), 1-naphthol, 17β-estradiol (FIG. 3) containing the target molecule BPA. 1.5 ml each of the sample solution (see 3) each and 1.1 mM dry toluene solution) was added, and incubation was performed with a thermomixer at 25 ° C. for 24 hours. Thereafter, the solution was filtered with a syringe filter (0.2 μm), and 1 ml of the filtrate was collected with a micropipette. 100 μl of an internal standard sample (bis (4-hydroxyphenyl) methane 6.28 mM toluene solution) was added thereto, and then the solvent was distilled off with a centrifugal evaporator. After distilling off, it was redissolved in acetonitrile, and its concentration was quantified using HPLC, and the binding amount to BPA-recognized molecular imprinted fine particles was calculated (HPLC measurement conditions; eluent: MeCN / H ₂ O = 4/6, Flow rate: 0.8 ml / min, injection volume: 10 μl, detection wavelength: 278 nm, column: SUPELCO LC-8-DB).

(1-3) Results FIG. 4 shows an SEM image of the synthesized BPA recognition molecule imprinted fine particles. From FIG. 4, it was confirmed that particles having a relatively monodispersed particle diameter of about 450 nm were formed.

  FIG. 5 shows the result of calculating the amount of each sample adsorbed on the synthesized BPA-recognizing molecule-imprinted fine particles. As is apparent from FIG. 5, the adsorption amount of BPA as a target molecule is 13.8 μmol per 1 g of polymer, 1-naphthol (1.42 μmol / g polymer), 17β-estradiol (1.03 μmol / g polymer). The amount of adsorption was larger than that. Although 1-naphthol has many differences in structure and functional group from BPA, the adsorption of 17β-estradiol was suppressed even though it was the same diol compound. Moreover, the adsorption amount of BPB having almost the same structure was 11.0 μmol / g polymer, and the same degree of adsorption as BPA as the target molecule was observed. From this result, it was difficult to identify BPB having almost the same structure as BPA, but it was shown that a BPA-specific recognition site was constructed in the molecularly imprinted fine particles synthesized this time.

[Example 2: SPR sensing using BPA-SAM substrate and BPA-recognized molecular imprinted fine particles]
SPR sensing was attempted using a BPA-SAM substrate and BPA recognition molecule imprinted fine particles. All reagents used in this example were purchased commercially. Moreover, the following was used as an analytical instrument. That is, silica gel (Wakogel C-300HG) manufactured by Wako Pure Chemical Industries was used for silica gel chromatography used for derivative synthesis. The NMR spectrum was measured using a JEOL Co., Ltd. NMR measurement apparatus (JNM-LA300). ESI-MS (API2000) and MALDI-TOF-MS (Voyager-2000 [accelating voltage 20 kV]) manufactured by Applied Biosystems Co., Ltd. were used for mass spectrometry. DIGILAB EXCULIBUR SERIES FT 3000 was used for IR spectrum measurement. For the evaluation of the molecularly imprinted fine particles, a plasmon optical waveguide spectrometer (S-SPR6000) manufactured by System Instruments Co., Ltd. was used. As a scanning analytical electron microscope (SEM), JSM-5610LVS manufactured by JEOL Ltd. (JEOL) was used.

(2-1) Production of BPA-SAM substrate In order to form BPA-SAM (Self-Assembled Monolayer) as a means for integrating BPA on the SPR substrate, first, a thiol group of BPA Introduced.

(i) Synthesis of 4,4-Bis- (4-hydroxy-phenyl) -Pentanoic acid (11-mercapto-undecyl) -amide (see the following reaction formula (1))
Diphenolic acid (3.43 g), K ₂ CO ₃ (4.15 g), and Ac ₂ O (4.51 ml) were placed in a 200 mL two-necked eggplant flask, acetonitrile (150 ml) was added, and the mixture was stirred at 60 ° C. for 12 hours. The reaction was stopped after confirming that there were no two raw materials by TLC, and the solvent was distilled off with an evaporator. After extraction with H ₂ O: CH ₂ Cl ₂ , purification was performed by silica gel column chromatography (CH ₂ Cl ₂ : MeOH = 20: 1) to obtain a white powder. ^The target compound 1 was confirmed by ¹ H-NMR and MALDI-TOF-MS (amount 3.05 g, yield 68.7%).

(ii) Synthesis of alkylthiol derivative 2 (see the following reaction formula (2))
Extraction of 11-amino-1-undecanethiol hydrochloride (NaHCO ₃ aqueous solution: CH ₂ Cl ₂ ) was performed, and dehydrochlorination treatment was performed. 11-amino-1-undecanethiol (53.8 mg) and compound 1 (98 mg) were dissolved in 20 ml CH ₂ Cl ₂ . WSC (75.1 mg), NHS (45.7 mg), and DIEA (138.4 μl) were added thereto, and the mixture was stirred at room temperature for 24 hours. Three spots were confirmed by TLC. Therefore, extraction was performed (AcOEt: 4% NaHCO ₃ aqueous solution, 10% citric acid aqueous solution, saturated saline). Then, purification was performed twice with silica gel column chromatography with CH ₂ Cl ₂ : MeOH = 100: 1 and CH ₂ Cl ₂ : MeOH = 100: 3, and 3 spots were separated. ¹ confirmed ¹ H-NMR and MALDI-TOF-MS of target product 2 (yield 25 mg, yield 17%).

(iii) Synthesis of BPA-thiol derivative 3 (see the following reaction formula (3))
10 mg of Compound 2 was dissolved in 6 ml of THF, 1 ml of NaOH aqueous solution (0.25 M) was added, and the mixture was stirred at room temperature for 6 hours. After confirming that the spot of the raw material disappeared and a highly polar spot appeared by TLC, the reaction was stopped, and after neutralization, the solvent was distilled off with an evaporator. Thereafter, extraction (AcOEt: 10% aqueous citric acid solution) was performed, and Compound 3 was confirmed by ¹ H-NMR and MALDI-TOF-MS. In addition, ¹ H-NMR was confirmed for the compound 3 obtained using Kiriyama wax and washed with Hx and CH ₂ Cl ₂ (yield: 8 mg, yield: 93%).

(iv) Synthesis of BPA-SAM film using BPA-thiol derivative 3 In order to use as a SPR measurement substrate, a Piranha solution (concentrated sulfuric acid: 30% hydrogen peroxide solution = 3: 1) is used on a gold thin film on a highly refractive glass substrate. Was dropped and allowed to stand for 5 minutes, and then washed with distilled water. Thereto, the above BPA-thiol derivative 3 solution (1 mM ethanol solution) was dropped, covered with a cover glass, and allowed to stand overnight in a nitrogen atmosphere. Thereafter, the substrate was washed with EtOH and dried. Moreover, in order to confirm formation of a BPA-SAM film, what produced the SAM film in the same procedure on another gold substrate was observed with the high sensitive reflection infrared spectrometer (IR-RAS).

(2-2) Synthesis of BPA recognition molecular imprinted fine particles Styrene particles for use as seeds were synthesized in the same manner as in Example 1 except that the rotation of the mechanical stirrer was changed to 200 rpm. The emulsion solid content concentration after centrifugal washing was adjusted to 1.44%. The polymerization rate was 100%, and the average particle size by DLS was 270 nm (dw / dn = 1.14).

  BPA recognition molecular imprinted fine particles were synthesized using the obtained seed. 50 g of a polystyrene seed particle solution (1.44 wt%) was weighed into a 100 ml vial, a predetermined amount of monomer was added according to the recipe in Table 3, and the mixture was allowed to swell by stirring at room temperature for 6 hours. After confirming disappearance of the monomer phase, the mixture was heated to a polymerization temperature of 80 ° C. in a nitrogen atmosphere, and an aqueous solution in which initiator V-50 was dissolved in 5 ml distilled water was added to the heated vial. Under a nitrogen atmosphere, stirring polymerization was performed at 80 ° C. and a stirrer at 200 rpm for 12 hours. After completion of the polymerization, centrifugal washing was performed 3 times to remove unreacted monomers and initiators. The polymerization rate was 91% and the average particle size by DLS was 292.9 nm (dw / dn = 1.11). Blank fine particles (“BNP” in Table 3) were also synthesized without using the BPA dimethacrylate monomer under the same conditions as the BPA recognition molecule imprinted fine particles (“INP” in Table 3).

  In order to remove BPA from the BPA-recognized molecularly imprinted fine particles, hydrolysis was performed in the same manner as in Example 1, and washing with acid and washing with distilled water were performed. Furthermore, in this example, in order to perform SPR measurement using a chloroform solvent, substitution with chloroform was performed after substitution with methanol. In subsequent experiments, “INP” is a molecular imprinted fine particle that has undergone template molecule (BPA) excision processing, “RNP” is a molecular imprinted fine particle that has not been excised from a template molecule (BPA), template Fine particles synthesized without adding molecules (BPA) are denoted as “BNP”.

(2-3) Sensing of interaction between BPA-SAM substrate and BPA-recognized molecular imprinted fine particles by SPR BPA-SAM substrate is set on stage, cell and flow path are installed, INP solution, RNP solution as analyte And a BNP solution (0 to 3 wt% aqueous solution) were fed, and the SPR spectrum was measured 5 minutes after reaching equilibrium. Moreover, the value of the shift to the long wavelength side from the SPR spectrum when fine particles are not included (0 wt%) was Δλ [nm], and the adsorption isotherm between each fine particle and the BPA-SAM substrate was graphed and evaluated.

  Further, as a competition experiment, BPA as a target molecule was added to the INP solution to adsorb BPA, and then the solution was sent to the flow path to measure the spectral change. Specifically, a predetermined amount of BPA was added to an INP 3 wt% solution to adjust to 50 mM. The solution was incubated at 25 ° C. for 12 hours and then fed, and the time course of the SPR spectrum was observed. As a comparison, an experiment was similarly performed on an INP solution to which BPA was not added.

Next, for the purpose of dissociation of INP from the BPA-SAM substrate, the target molecule BPA and BPA analog 4,4'-diaminodiphenylmethane (DADPM, see formula (4) below), hydrogen bonding with polar solvent Methanol was weakened to observe the change. First, an INP solution (3 wt% chloroform solution) was allowed to flow for 5 minutes to adsorb INP, and the change with time was followed. Chloroform (CHCl ₃ ), which is a solvent, was further fed for 5 minutes, and the time course of INP dissociation due to concentration equilibrium was followed. Finally, BPA solution (50 mM), DADPM (25 mM), or methanol was fed as an inhibitor for 5 minutes, respectively, and the time course of INP dissociation was followed. About methanol, it measured by returning to chloroform which is a solvent after methanol feeding.
The SPR measurement conditions are as follows.
Incident angle 28.1 °
Detection angle 28.6 °
Integration time 10ms
Addition average 5 times smoothing 5 points P polarized light incident flow rate 20μl / min

(2-4) Results
(i) Identification of molecularly imprinted fine particles by SEM SEM images of each synthesized molecularly imprinted fine particle are shown in FIGS. 6 is an SEM image of INP, FIG. 7 is an SEM image of RNP, and FIG. 8 is an SEM image of BNP. As is apparent from FIGS. 6 to 8, each of the obtained molecularly imprinted fine particles is a polymer (INP: dw / dn = 1.1, RNP: dw / dn = 1.11). From this, it can be assumed that each surface area is the same, and the change of the SPR spectrum is considered to reflect the binding behavior.

(ii) Identification of BPA-SAM substrate by IR FIG. 9 shows an IR-RAS spectrum of the BPA-SAM substrate. From FIG. 9, it was confirmed that the BPA-SAM film was formed on the substrate as follows.
3500-3700 cm ⁻¹ : Phenol OH monosubstituted amide NH stretching vibration around 1600 cm ⁻¹ : aromatic C═C and C—H stretching vibration around 1700 cm ⁻¹ : monosubstituted amide C═O stretching vibration 1200 to 1350 cm ⁻¹ : Twist and roll vibration of C10 long chain alkyl

(iii) SPR measurement using BPA-SAM substrate and BPA-recognized molecularly imprinted fine particles FIG. 10 is an adsorption isothermal plotting wavelength shift values (Δλnm) at respective concentrations of three kinds of fine particles (INP, RNP, BNP). A line is shown. As is clear from FIG. 10, even when any of the fine particles was fed, a shift toward the long wavelength side of the SPR spectrum was observed as the concentration increased. Among the three types of fine particles, the shift value of INP was The largest was confirmed. From this result, it was shown that the difference in interaction (adsorption) between the BPA-SAM substrate and each fine particle can be sensed by the wavelength shift amount of the SPR spectrum.

(iv) Competitive Experiment in the Presence of BPA As a competitive experiment, BPA as a target molecule was added to the INP solution to adsorb BPA to INP, and then the solution was sent to the flow path, and 0 minutes to 5 minutes. Spectral changes were measured over time. As a control, an INP solution to which BPA was not added was sent to the channel, and the change in spectrum was measured in the same manner.

  FIG. 11 shows an adsorption isotherm in which the wavelength shift value (Δλnm) at each time from the start of liquid feeding is plotted. As is clear from FIG. 11, the behavior of reaching the equilibrium after 1 minute is the same both when BPA is added and when it is not added, but the amount of change is overwhelmingly when BPA is added. It was small. This result indicates that the interaction between INP and the BPA-SAM substrate is inhibited because BPA is pre-adsorbed to the BPA recognition site on the surface of INP by incubating in the presence of BPA and INP. it was thought. Therefore, it was shown that the interaction (adsorption) between INP and the BPA-SAM substrate was changed by competition of BPA, and BPA in the sample could be detected by detecting this change.

(v) Dissociation of INP from BPA-SAM substrate Fig. 12 shows the change in SPR spectrum when the solvent (chloroform) was fed after the INP solution was fed to the BPA-SAM substrate, and finally BPA was fed. showed that. As shown in FIG. 12, first, when the INP solution was fed for 5 minutes, INP was adsorbed on the BPA-SAM substrate, and the SPR spectrum was greatly shifted to the longer wavelength side. Next, when chloroform as a solvent was fed, the concentration equilibrium was lost, and the INP that was weakly bound on the substrate was slightly dissociated, so the SPR spectrum shifted to the short wavelength side. However, since it was not possible to completely dissociate INP, further dissociation was attempted using BPA as the target molecule, and no change in the SPR spectrum was observed even when a solution with a BPA saturation concentration of 50 mM was fed. .

  FIG. 13 shows the change in the SPR spectrum when the INP solution was sent to the BPA-SAM substrate, the solvent (chloroform) was sent, and finally DADPM was sent. As shown in the above formula (4), DADPM is a compound having an amino group that is considered to form a hydrogen bond strongly with a carboxylic acid residue present at the BPA recognition site on the surface of INP, and having a BPA-like skeleton. is there. As shown in FIG. 13, when the DADPM solution was fed, the SPR spectrum was shifted to a shorter wavelength side than when chloroform was fed, and further dissociation of INP was observed.

  FIG. 14 shows the change in the SPR spectrum when the INP solution was sent to the BPA-SAM substrate, the solvent (chloroform) was sent, and finally methanol was sent. As shown in FIG. 14, when methanol as a polar solvent was fed, the long wavelength shift value (Δλ [nm]) was almost “0”, and INP was almost completely dissociated from the BPA-SAM substrate. It has been shown.

  As described above, the adsorption of INP on the BPA-SAM substrate could be dissociated by the amine and the polar solvent, suggesting that the interaction between INP and the SAM substrate is a hydrogen bond-based specific bond. .

[Example 3: Fluorescence quenching sensing using BPA-recognized molecular imprinted fine particles]
As described above, it is known that gold fine particles exhibit a quenching reaction when present in the vicinity of a fluorescent dye as a fluorescence quencher. Therefore, fluorescence quenching sensing is performed using BPA-integrated gold fine particles in which the target molecule BPA is integrated on the surface of the gold fine particles, and fluorescent molecule imprinted fine particles (fluorescent BPA recognition molecular imprinted fine particles) having BPA as the target molecule. (See FIG. 2).

  All reagents used in this example were purchased commercially. Moreover, the following was used as an analytical instrument. That is, silica gel (Wakogel C-300HG) manufactured by Wako Pure Chemical Industries was used for silica gel chromatography used for derivative synthesis. The NMR spectrum was measured using a JEOL Co., Ltd. NMR measurement apparatus (JNM-LA300). ESI-MS (API2000) and MALDI-TOF-MS (Voyager-2000 [accelating voltage 20 kV]) manufactured by Applied Biosystems Co., Ltd. were used for mass spectrometry. A centrifuge SRX-201 manufactured by Tommy Seiko Co., Ltd. was used for washing the polymer. A dynamic light scattering photometer (DL-6500) manufactured by Otsuka Electronics Co., Ltd. was used for measurement of particle size distribution and average particle size. The transmission electron microscope (TEM) used was a Hitachi transmission electron microscope H-7500. Ultraviolet / visible spectrophotometric analysis uses an ultraviolet / visible spectrophotometer (JASCO V-560), system control and data processing use special software Spectra Manager (JASCO), and a 1 cm square quartz cell is used for measurement. Using. Hitachi F-2500 was used for the fluorescence spectrum measurement, and a 1 cm square quartz cell was used for the measurement.

(3-1) Synthesis of fluorescent BPA-recognizing molecule imprinted fine particles
(i) Synthesis of pyrene monomer (see the following reaction formula (5))
In a 50 mL eggplant flask, 1-pyrenemethylamine 500 mg (2 mmol) was dissolved in 10 ml of dry dichloromethane (CH ₂ Cl ₂ ). Under nitrogen atmosphere, methachloroyl chloride (377 mg, 4 mmol) and TEA (0.36 ml, 4 mmol) were added. Stir for 16 hours. Extraction (CH ₂ Cl ₂ : NaHCO ₃ aqueous solution, HCl aqueous solution, NaCl aqueous solution) and purification by silica gel column chromatography (CH ₂ Cl ₂ ) gave a white target product. ^It was confirmed by ¹ H-NMR and MALDI-TOF-MS (yield 300 mg, yield 50%).

(ii) Synthesis of fluorescent BPA-recognizing molecule imprinted fine particles Using the obtained pyrene monomer and the styrene seed with a particle size of 270 nm (DLS measurement) synthesized in Example 2, the synthesis of fluorescent BPA-recognized molecular imprinted fine particles was performed. went. 10 g of a polystyrene seed solution (1.4 wt%) was weighed into a 25 ml vial, a predetermined amount of monomer was added according to the recipe in Table 4, and the mixture was stirred for 6 hours at room temperature to swell. For the pyrene-containing molecular imprinted fine particles, a small amount of acetone was added to dissolve the pyrene monomer for the solubility of the monomer. After confirming disappearance of the monomer layer, the mixture was heated to a polymerization temperature of 80 ° C. in a nitrogen atmosphere, and an aqueous solution in which initiator V-50 was dissolved in 5 ml distilled water was added to the heated vial. Under a nitrogen atmosphere, stirring polymerization was performed at 80 ° C. and a stirrer 200 rpm for 24 hours. After completion of the polymerization, centrifugal washing was performed 3 times to remove unreacted monomers and initiators. The polymerization rate of the pyrene-containing molecularly imprinted fine particles was 95%.

  In order to remove BPA from the fluorescent BPA-recognizing molecule-imprinted fine particles, hydrolysis was performed in the same manner as in Example 1, and washing with acid and distilled water was performed. Furthermore, in this example, in order to perform fluorescence measurement with a methanol / toluene = 7/13 solution, it was washed once with methanol and then replaced with a methanol / toluene = 7/13 solution.

(3-2) Synthesis of BPA-integrated gold fine particles Gold particles were synthesized using the BPA-thiol derivative synthesized in Example 2 as a protective agent in order to accumulate BPA as a target molecule on the gold fine particle surface (reference: M. Brust, M. Walker, D. Bethell, D. Schiffrin, R. Whyman, Chem. Commun., 1994, 801.
M. Brust, J. Fink, D. Bethell, D. Schiffrin, C. Kiely, Chem. Commun., 1995, 1655.).

  In advance, a silicon coat of an instrument used for synthesis was performed. 2% dimethyldichlorosilane (solvent: toluene) was prepared, poured into a 10 ml vial, and the solution was spread while rotating the container. The solution was returned, and the solution was evaporated as it was, rinsed with distilled water and dried.

  As shown in Table 5, 2 ml of BPA-thiol derivative solution (5.8 mM in MeOH) and 2 ml of chloroauric acid solution (5.8 mM methanol solution) were placed in a vial, and 80 μl of acetic acid was added. While stirring this solution with a stirrer, a 0.4 M sodium borohydride aqueous solution was slowly added dropwise in 100 μl portions in 5 portions. Since the color of the solution changed to reddish purple instantly, stirring was stopped after 30 minutes. This reaction solution was applied to an evaporator and the solvent was distilled off. Ether was then added, and the resulting precipitate was filtered using a Kiriyama funnel, and further washed in the order of ethyl acetate and distilled water. The obtained gold fine particles were redissolved with a methanol / toluene = 7/13 solution (yield: 4.3 mg).

(3-3) Fluorescence quenching sensing using fluorescent BPA-recognized molecular imprinted fine particles and BPA-integrated gold fine particles When only fluorescent BPA-recognized molecularly imprinted fine particles (fluorescent INP) are present in the system, fluorescence emission is saturated. A titration experiment was conducted to examine the concentration. Specifically, 3 ml of a methanol / toluene = 7/13 solution is added to a 1 cm square quartz cell, and 5 μl of a BPA fluorescent molecule imprinted fine particle solution (1.2 wt% methanol / toluene = 7/13) is added dropwise thereto. The concentration at which fluorescence was saturated and quenching due to concentration quenching was confirmed. The same procedure was used to measure fluorescent BPA-recognized molecularly imprinted fine particles (fluorescent RNP) that had not been subjected to template molecule cutting-out.

Next, in order to detect the fluorescence quenching reaction, a titration experiment of BPA-integrated gold fine particles in a fluorescent INP solution or a fluorescent RNP solution was performed. In the fluorescence spectrum measurement, both the concentration of fluorescent INP or fluorescent RNP was adjusted to 2.4 × 10 ⁻³ wt% (methanol / toluene = 7/13), and 3 ml of each was added to a 1 cm square quartz cell. Thereto, 10 μl of 3 mg / ml solution of BPA-integrated gold fine particles (methanol / toluene = 7/13) was dropped and stirred for 1 minute, and then the fluorescence spectrum was measured and the quenching was observed. The concentration of fluorescent INP was corrected with the addition of BPA-integrated gold fine particles.

  Inhibitor solution that inhibits adsorption of BPA-integrated gold fine particles to fluorescent INP in fluorescent INP solution or fluorescent RNP solution that has been quenched by adding BPA-integrated gold fine particles to measure fluorescence recovery from the quenched state A titration experiment in which Add 3 ml of fluorescent INP solution or fluorescent RNP solution 3.2 × 10 −3 wt% (methanol / toluene = 7/13) to a 1 cm square quartz cell and add BPA-integrated gold fine particle solution to 0.02 mg / ml to quench the state. Formed. A BPA solution, which is a target molecule, or an acetic acid solution, which is a carboxylic acid, was added thereto as an inhibitor and stirred for 1 minute, and then fluorescence measurement was performed. A BPA solution having a concentration of 400 mM (methanol / toluene = 7/13) and an acetic acid solution having a concentration of 100 mM (methanol / toluene = 7/13) were used for titration. The concentration of fluorescent INP was corrected with the addition of the acetic acid solution.

In the above experiment, the fluorescence measurement was performed under the following conditions.
Exiting wavelength 347nm
Temperature 25 ℃,
Photomal voltage 400V

(3-4) Results
(i) Identification of fluorescent BPA-recognizing molecule imprinted fine particles and BPA-integrated gold fine particles FIG. 15 shows an SEM image of fluorescent BPA-recognized molecular imprinted fine particles containing a pyrene monomer. As is clear from FIG. 15, it was confirmed that the particles were fine particles having an average particle diameter of 368 nm that maintained a clean shape.

  FIG. 16 shows the absorption spectrum of the BPA-integrated gold fine particle solution measured with a visible ultraviolet absorption photometer. As shown in FIG. 16, plasmon-derived absorption of gold particles was observed near 520 nm, and BPA-derived absorption was observed near 279 nm. FIG. 17 shows a TEM image of the BPA-integrated gold fine particles. As shown in FIG. 17, it was revealed that particles having a particle diameter of about 30 nm were formed, although the sizes varied.

(ii) Dependence of fluorescence intensity on fluorescent BPA recognition molecule imprinted fine particles FIG. 18 and FIG. 19 show the relationship between the concentration of fluorescence INP and fluorescence intensity, and the relationship between the concentration of fluorescence RNP and fluorescence intensity, respectively. As is clear from FIGS. 18 and 19, the fluorescence intensity reached saturation at around 0.02 wt% for both fluorescence INP and fluorescence RNP, and it was confirmed that concentration quenching was caused when the concentration was further increased. . Based on this result, in subsequent experiments, a low concentration (2 to 3 × 10 ⁻³ wt%) at which concentration quenching does not occur was used as the concentration of fluorescent INP (fluorescent RNP).

(iii) Fluorescence quenching based on the interaction between fluorescent BPA recognition molecule imprinted fine particles and BPA-integrated gold fine particles. FIG. 20 shows the fluorescence intensity when the BPA-integrated gold fine particle solution is added to the fluorescent INP solution and the fluorescent RNP solution, respectively. The rate of change is shown. The change rate of the fluorescence intensity was indicated by the ratio (F / F ₀ ) of the fluorescence intensity (F) after addition of the BPA-integrated gold fine particles to the fluorescence intensity (F ₀ ) before addition of the BPA-integrated gold fine particles at a wavelength of 397 nm. As shown in FIG. 20, it was confirmed that the degree of quenching of the fluorescent INP solution was greater than the degree of quenching of the fluorescent RNP. From this result, it has been clarified that the fluorescence is efficiently quenched by the adsorption of the BPA-integrated gold fine particles to the BPA recognition site formed on the surface of the fluorescent INP.

(iv) Fluorescence recovery by BPA and polar solvent FIG. 21 shows changes in the fluorescence spectrum when the BPA solution was added dropwise to the fluorescence INP solution that was quenched by adding the accumulated gold fine particles. In the figure, the fluorescence spectrum near the wavelength of 397 nm is shown enlarged in the upper right. BPA was added dropwise until the BPA concentration in the cell reached 20 mM. As is clear from FIG. 21, no large spectral change was observed. This result was consistent with the result of Example 2.

  FIG. 22 shows the recovery rate of the fluorescence intensity when the acetic acid solution is dropped into the fluorescent INP solution and the fluorescent RNP solution which have been quenched by adding the accumulated gold fine particles. The recovery rate of the fluorescence intensity is 100% of the fluorescence intensity (wavelength 397 nm) of the fluorescent INP solution and the fluorescent RNP solution before the addition of the accumulated gold fine particles, and the fluorescence intensity when the fluorescence is quenched after the addition of the accumulated gold fine particles is 0%. Calculated. As apparent from FIG. 22, the fluorescence recovery of the fluorescent INP was observed by the addition of acetic acid, and the fluorescence recovery rate was 15.5%. On the other hand, almost no recovery of fluorescence was observed with the fluorescent RNP, and the fluorescence recovery rate was 2%. This result is consistent with the result of Example 2, and the dissociation between the fluorescent INP and the accumulated gold fine particles was promoted by the carboxylic acid, so that the interaction between the fluorescent INP and the accumulated gold fine particles is specific due to hydrogen bonding. It was suggested that this is a natural bond.

  The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  The present invention can be used in fields such as public health, environmental health, and medical care. It can also be used in the reagent industry.

It is a schematic diagram which shows the state which the molecular imprint fine particle has adsorb | sucked to the molecular integration | stacking board | substrate. It is a schematic diagram which shows the interaction of fluorescent molecularly imprinted fine particles and molecular integrated gold fine particles. It is a figure which shows the chemical structural formula of four types of compounds used for evaluation of BPA recognition molecule | numerator imprint fine particle. It is a SEM image of the synthetic | combination BPA recognition molecule | numerator imprint fine particle. It is a figure which shows the result of having calculated the adsorption amount of each sample to the synthetic | combination BPA recognition molecule | numerator imprint fine particle. It is a SEM image of INP. It is a SEM image of RNP. It is a SEM image of BNP. It is a figure which shows the IR-RAS spectrum of a BPA-SAM board | substrate. It is a figure which shows the adsorption isotherm which plotted the shift value ((DELTA) (lambda) nm) of the wavelength in each density | concentration of three types of microparticles | fine-particles (INP, RNP, BNP). It is a figure which shows the adsorption isotherm which plotted the shift value ((DELTA) (lambda) nm) of the wavelength in each time from the liquid feeding start. It is a figure which shows the change of a SPR spectrum when a solvent (chloroform) is sent after sending an INP solution to a BPA-SAM board | substrate, and BPA is sent last. It is a figure which shows the change of a SPR spectrum when a solvent (chloroform) is sent after sending an INP solution to a BPA-SAM board | substrate, and DADPM is sent last. It is a figure which shows the change of a SPR spectrum when a solvent (chloroform) is sent after sending an INP solution to a BPA-SAM board | substrate, and methanol is finally sent. It is a SEM image of fluorescent BPA recognition molecule imprint microparticles containing a pyrene monomer. It is a figure which shows the absorption spectrum of the BPA integration | stacking gold fine particle solution measured with the visible ultraviolet absorption photometer. It is a TEM image of BPA accumulation gold fine particles. It is a figure which shows the relationship between the density | concentration of fluorescence INP, and fluorescence intensity. It is a figure which shows the density | concentration of fluorescence RNP, and the relationship of fluorescence intensity. It is a figure which shows the change rate of a fluorescence intensity when a BPA accumulation | aggregation gold fine particle solution is added to the fluorescence INP solution and the fluorescence RNP solution, respectively. It is a figure which shows the change of a fluorescence spectrum when a BPA solution is dripped at the fluorescence INP solution which added the accumulation | aggregation gold microparticles and is in the quenching state. It is a figure which shows the recovery | restoration rate of the fluorescence intensity when an acetic acid solution is dripped at the fluorescence INP solution and fluorescence RNP solution which are in the quenching state by adding the accumulation | aggregation | fine-particle fine particle, respectively.

Claims (9)

A method for detecting a target molecule in a sample, the method comprising detecting a target molecule in a sample, wherein the following interaction between (A) and (B) is detected.
(A) Molecularly imprinted fine particles having a target molecule recognition site constructed by a molecular imprint method and having an average particle diameter of less than 1 μm measured by a dynamic light scattering method (B) A single molecule of a target molecule or a derivative thereof Molecular assembly having a layer and capable of binding the molecularly imprinted fine particles to the monomolecular layer   The method for detecting a target molecule in a sample according to claim 1, wherein the monomolecular layer is formed on a surface of a substrate of the molecular assembly.   The method for detecting a target molecule in a sample according to claim 1, wherein the molecular aggregate has the monomolecular layer formed on the surface of fine particles dispersible in a solvent.   The method of detecting a target molecule in a sample according to any one of claims 1 to 3, wherein the molecular assembly absorbs or emits light energy.   5. The molecular imprinted fine particles emit light energy when the molecular aggregate absorbs light energy, and absorb light energy when the molecular aggregate emits light energy. 2. A method for detecting a target molecule in a sample according to 1.   The molecularly imprinted fine particles emit fluorescence, and the molecular aggregate absorbs the fluorescent energy emitted by the molecularly imprinted fine particles and quenches the fluorescence of the molecularly imprinted fine particles, or the fluorescent energy emitted by the molecularly imprinted fine particles. 6. The method for detecting a target molecule in a sample according to claim 5, wherein the molecular aggregate itself further emits fluorescence upon absorption.   The molecular aggregate emits fluorescence, and the molecular imprinted fine particles absorb fluorescence energy emitted from the molecular aggregate to quench the fluorescence of the molecular aggregate, or absorb fluorescence energy emitted from the molecular aggregate. The method for detecting a target molecule in a sample according to claim 5, wherein the molecularly imprinted fine particles themselves further emit fluorescence.   The method for detecting a target molecule according to any one of claims 1 to 7, further comprising a sample contacting step in which a sample, the molecularly imprinted fine particles, and the molecular aggregate are brought into contact with each other. A reagent kit for detecting a target molecule in a sample, comprising the following (A) and (B):
(A) Molecularly imprinted fine particles having a target molecule recognition site constructed by a molecular imprint method and having an average particle diameter of less than 1 μm measured by a dynamic light scattering method (B) A single molecule of a target molecule or a derivative thereof Molecular assembly having a layer and capable of binding the molecularly imprinted fine particles to the monomolecular layer

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