Nanoparticles for sensing and their manufacturing method

Abstract

To provide an artificial biosensor that is easy to manufacture and has excellent specificity, and a method for producing the same.SOLUTION: A nanoparticle for sensing includes a molecular imprinting polymer having a molecular imprinting space of a biological material. The molecular imprinting polymer includes a constitutional unit derived from a functional monomer having a functional group including an interacting group with the biological material and a signal substance bonding group that is different from the interacting group. The functional group is present on a surface of the molecular imprinting space. The nanoparticle for sensing is easy to produce and is useful as an artificial biosensor with an excellent specificity.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to sensing nanoparticles and a method for producing the same. [Background technology]

[0002] Biological substances such as proteins are involved in many life phenomena, including metabolism, signal transduction, immunity, and the formation of biological structures, and provide useful information for disease diagnosis, etc. To further enhance the utility of such biomolecules, research is being conducted into the application of materials that recognize biomolecules to biosensors.

[0003] Antibodies, which are used as receptors for recognizing biomolecules in biosensors, contribute greatly to the performance of biosensors due to their excellent specificity and affinity. However, because they are biomolecules, antibodies have inherent problems, such as vulnerability to external stimuli such as heat and pH, and high cost.

[0004] In light of the inherent challenges of antibodies, attempts have been made to apply molecular imprinting to artificially obtain antibody specificity and affinity. Molecular imprinting is an established technique for imprinting biomolecular templates within polymers. Specifically, a complex of the template biomolecule and a functional monomer is formed, copolymerized with a crosslinker, and then the template is removed to obtain a molecularly imprinted polymer (MIP) with a binding space (molecularly imprinted space) complementary to the template. Various artificial biosensor substrates have been reported, in which such MIP films are deposited on a substrate.

[0005] For example, Non-Patent Document 1 reports an artificial biosensor substrate that enables improved detection sensitivity of biomarkers bound to molecularly imprinted spaces. In this artificial biosensor substrate, the creation of molecularly imprinted spaces eliminates the need to use antibodies for initial binding, and the secondary labeled antibody is replaced with a biosensor that relies on capacitance, thereby improving the sensitivity of detecting and quantifying low concentrations of biomolecules in complex samples quickly, label-free, and with high sensitivity.

[0006] Furthermore, Non-Patent Document 2 reports an artificial biosensor substrate that improves the specificity and affinity of molecularly imprinted spaces and enables binding information to be read out by a change in fluorescence. In this artificial biosensor substrate, two types of functional monomers, each with a different reversible bond (a disulfide group and an oxyimino group), are covalently bonded to the template alpha-fetoprotein (AFP). After forming an MIP membrane by molecular imprinting, the reversible bond is cleaved and the AFP is removed, resulting in the localization of two types of functional groups (a thiol group and an oxyamino group) derived from the reversible bond within the molecularly imprinted space. Introducing a group that interacts with AFP into one of these two functional groups improves the specificity and affinity of AFP for the molecularly imprinted space, and introducing a fluorescent reporter molecule into the other allows the binding information of AFP in the molecularly imprinted space to be read out by a change in fluorescence. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Journal of Visualized Experiments, 132, e57208, doi:10.3791 / 57208 (2018) [Non-patent document 2] Angewandte Chemie International Edition., 55, 13023-13027(2016) Summary of the Invention [Problem to be solved by the invention]

[0008] As mentioned above, although research has been conducted to improve the performance of artificial biosensor substrates in which MIP films are formed on substrates, it must be said that there is still room for improvement.

[0009] The technology of Non-Patent Document 1 uses molecular imprinted spaces instead of antibodies, and aims to increase the detection sensitivity of recognized molecules. However, the recognition ability exerted by the molecular imprinted spaces is only based on the complementary shape of the target molecule surface, and its specificity and affinity are far from satisfactory.

[0010] The technology of Non-Patent Document 2 adds an interactive group with the target molecule within the molecular imprinted space, thereby improving the affinity between the molecular imprinted space and the target molecule not only by the shape of the space but also by utilizing the interaction with the interactive group. Furthermore, a molecular imprinting protocol for localizing the interactive group within the molecular imprinted space reduces nonspecific adsorption outside the space. However, these excellent performances require a special process for covalently bonding two types of functional monomers to the template in advance, which complicates the manufacturing process.

[0011] The present inventors have attempted to simplify the manufacturing method of the technology described in Non-Patent Document 2 by eliminating the above-mentioned special steps. Specifically, they attempted to use a functional monomer having a functional group (interactive group) that interacts non-covalently (by a non-covalent bond or a similar weak covalent bond) with the template biomolecule, thereby adsorbing the functional monomer to the template surface in a polymerization system and localizing the interactive group in the molecular imprint space. Furthermore, in this attempt, the functional monomer was designed to have both an interactive group and a signal substance binding group, with the aim of localizing a group (signal substance binding group) to introduce a fluorescent reporter molecule in the molecular imprint space. However, even with this manufacturing method, they faced the problem of poor specificity in the resulting artificial biosensor substrate.

[0012] Therefore, an object of the present invention is to provide an artificial biosensor that is easy to manufacture and has excellent specificity, and a method for manufacturing the same. [Means for solving the problem]

[0013] As a result of extensive research, the present inventors have found that by using a monomer having both an interacting group and a signal substance-binding group to form a particulate MIP, an artificial biosensor with excellent specificity can be obtained, despite the fact that this is a simple production method that does not require any special steps on the template.The present invention was completed based on this finding and through further research.

[0014] That is, the present invention provides the following aspects of the invention.

[0015] Item 1. A molecularly imprinted polymer having a molecularly imprinted space of a biological material, the molecularly imprinted polymer comprises a constituent unit derived from a functional monomer having a functional group including a group that interacts with the biological substance and a signal substance-binding group different from the interactive group, The functional group is present on the surface of the molecular imprinted space. Item 2. The sensing nanoparticle according to Item 1, wherein the functional group is represented by the following formula (1): [ka] [R 1 represents a group that interacts with the biological substance, and R 2 represents a linking group containing the signal substance-binding group, and L 1 represents a direct bond or a linking group.] Item 3. The sensing nanoparticle according to Item 1 or 2, wherein the interactive group is selected from the group consisting of a substituted or unsubstituted amino group, a substituted or unsubstituted aromatic group, an amidino group, a guanidino group, a carboxyl group, a sulfo group, a boronyl group, and a ligand of the biomolecule. Item 4. The sensing nanoparticle according to any one of Items 1 to 3, wherein the signal substance-binding group is selected from the group consisting of an amino group, a carboxyl group, a thiol group, a disulfide group-containing group, an aminooxy group, an aldehyde group, and a hydroxyl group. Item 5. The sensing nanoparticle according to any one of Items 1 to 4, wherein the functional group is represented by the following formula (11) or (12): [ka] [R 11 represents a carboxyl aryl group or a sulfoaryl group, L 11 represents an alkylene group having 1 to 4 carbon atoms.] [ka] [R 11 represents a carboxyl aryl group or a sulfoaryl group, L 12 represents a divalent linking group, and R 21 represents an amino group, a carboxyl group, a thiol group, a disulfide group-containing group, an aminooxy group, an aldehyde group, or a hydroxyl group; L 21 represents a divalent linking group. Item 6. The sensing nanoparticle according to any one of Items 1 to 5, which comprises a signal group bound to the signal substance-binding group. Item 7. The sensing nanoparticle according to any one of Items 1 to 6, wherein the molecularly imprinted polymer comprises a constitutional unit derived from N-substituted or unsubstituted (meth)acrylamide and / or a biocompatible monomer. Item 8. The sensing nanoparticle according to any one of Items 1 to 7, wherein the biological material is selected from the group consisting of a protein, a sugar chain, a lipid, and a composite molecule of two or more of these. Item 9. The sensing nanoparticle according to any one of Items 1 to 8, wherein the biological substance is albumin. Item 10. The sensing nanoparticle according to any one of Items 1 to 9, which is used for detecting a biological substance. Item 11. A sensing nanoparticle according to any one of Items 1 to 9, wherein the biological substance is a substance attached to or bound to a biological membrane of a membrane structure, a molecule corresponding to an exposed partial structure of the biological membrane, or a fragment of a specific biological molecule, and the sensing nanoparticle is used to detect the membrane structure or the specific biological molecule via capture of the biological molecule by the interactive group. Item 12. The sensing nanoparticle according to Item 11, wherein the membrane structure is a cell, a virus, an extracellular vesicle, or an organelle, and the specific biomolecule is an antibody. Item 13. The sensing nanoparticles according to any one of Items 1 to 12, wherein the biological material is a biological material derived from an animal or plant that falls under the haram category and is used for halal checks. Item 14. A sensing substrate comprising a substrate and the sensing nanoparticles according to any one of Items 1 to 13 immobilized on the substrate. Item 15. A sensing reagent comprising the sensing nanoparticles according to any one of Items 1 to 13. Item 16. A step 1 of synthesizing a molecularly imprinted polymer by polymerizing, using a biological substance as a template, a monomer component including a functional monomer having a functional group including a group that interacts with the biological substance and a signal substance-binding group different from the interactive group; Step 2: removing the template from the molecularly imprinted polymer; A method for producing nanoparticles for sensing, comprising: Item 17. A method for producing nanoparticles for sensing according to Item 16, comprising, after step 2, step 3 of binding a signal substance to the signal substance-binding group in the molecularly imprinted polymer. [Effects of the Invention]

[0016] According to the present invention, an artificial biosensor that is easy to manufacture and has excellent specificity, and a method for manufacturing the same are provided. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is a schematic diagram showing an example of the sensing nanoparticle of the present invention. [Figure 2] 10A and 10B are schematic diagrams illustrating other examples of the sensing nanoparticles of the present invention. [Figure 3] 1A and 1B are schematic diagrams illustrating examples of usage of the sensing nanoparticles of the present invention. [Figure 4] 1 is a schematic diagram showing step 1 in an example of the method for producing sensing nanoparticles of the present invention. [Figure 5] 1 is a schematic diagram showing steps 2 and 3 in an example of the method for producing sensing nanoparticles of the present invention. [Figure 6] 1 is a schematic diagram showing an example of a sensing substrate of the present invention. [Figure 7] 10A and 10B are schematic diagrams illustrating other examples of the sensing substrate of the present invention. [Figure 8] 7 shows the results of a protein adsorption test using Example 2 (corresponding to the sensing substrate in FIG. 7), obtained in Test Example 5. [Figure 9] 1 shows the results of a protein adsorption test using Comparative Example 2 (a control sensing substrate provided with an MIP film), obtained in Test Example 5. [Figure 10] 1 shows the correlation between the HSA concentration determined by the BCP method and the relative fluorescence intensity based on HSA binding measured using a sensing substrate, obtained in Test Example 6. [Figure 11]1 shows the correlation between the relative fluorescence intensity obtained in Test Example 7 and the PSA concentration for the sensing substrate on which fluorescent group-introduced MIP-NGs of Example 4 were immobilized. [Figure 12] 7 shows the results obtained in Test Example 7 of a protein adsorption test using a sensing substrate (corresponding to the sensing substrate in FIG. 7) on which fluorescent group-introduced MIP-NGs of Example 4 were immobilized. [Figure 13] 1 shows the results of a protein adsorption test using a sensing substrate on which fluorescent group-introduced NIP-NGs of Comparative Example 3 were immobilized, obtained in Test Example 7. [Figure 14] 7 shows the results of a PSA adsorption test in a PSA-contaminated beef extract sample using a sensing substrate (corresponding to the sensing substrate in FIG. 7) on which fluorescent group-introduced MIP-NGs of Example 4 were immobilized, obtained in Test Example 7. [Figure 15] This shows the results of a fluorescence response test in a beef extract sample mixed with a pork extract sample, obtained in Test Example 7, using a sensing substrate (corresponding to the sensing substrate in Figure 7) on which fluorescent group-introduced MIP-NGs from Example 4 were immobilized. [Figure 16] 1 shows the change in fluorescence spectrum (λex=647 nm) obtained in Test Example 8 when PSA was added to non-immobilized (free) particles of fluorescent group-introduced MIP-NGs of Example 4. [Figure 17] This is an experiment on the selectivity of non-fixed (free) particles of fluorescent group-introduced MIP-NGs of Example 4, obtained in Test Example 8, for PSA (sensing target protein) or transferrin (reference protein), and shows the change in relative fluorescence intensity at 667 nm when each protein was added at a specified concentration. [Figure 18] 1 shows the change in relative fluorescence intensity when an Fc domain fragment was added to the substrate for Fc domain sensing (and for IgG sensing via Fc domain capture) obtained in Test Example 9. [Figure 19] 1 shows the change in relative fluorescence intensity when an Fc domain fragment, complete IgG, or lysosomes was added to the substrate for Fc domain sensing (and for IgG sensing via Fc domain capture) obtained in Test Example 9. [Figure 20]1 shows the relationship between relative fluorescence intensity and exosome concentration using an exosome sensing substrate via HER2 capture, obtained in Test Example 10. [Figure 21] This shows the change in relative fluorescence intensity when exosomes derived from a HER2-overexpressing breast cancer cell line were added to the exosome sensing substrate via HER2 capture obtained in Test Example 10 and to a sensing substrate to which an anti-HSA affibody was introduced instead of an anti-HER2 affibody. DETAILED DESCRIPTION OF THE INVENTION

[0018] [1. Nanoparticles for sensing] The sensing nanoparticle of the present invention comprises a molecularly imprinted polymer having a molecularly imprinted space for a biological substance; the molecularly imprinted polymer comprises a constituent unit derived from a functional monomer having a functional group including a group that interacts with the biological substance and a signal substance-binding group different from the interactive group; and the functional group is present on the surface of the molecularly imprinted space. The sensing nanoparticle of the present invention will be described in detail below.

[0019] An example of a sensing nanoparticle of the present invention is shown in Fig. 1. As shown in Fig. 1, the sensing nanoparticle 10 includes a molecularly imprinted polymer 20 having a molecularly imprinted space 21 for a biological substance; the molecularly imprinted polymer 20 has an interactive group with the biological substance (R 1 ) and a signal substance binding group (in the figure, R 2 The sensing nanoparticle 10 includes a structural unit derived from a functional monomer having a functional group 22 containing a signal substance binding group (contained in a structure schematically represented by the following formula:), and the functional group 22 is present on the surface of the molecular imprinted space 21. The signal substance binding group of the sensing nanoparticle 10 is free, and by introducing a signal group into the signal substance binding group to form the sensing nanoparticle 10a, it can be used as a sensor for recognizing biological substances. The user can freely select and customize the signal group to be introduced into the signal substance binding group of the sensing nanoparticle 10.

[0020] Another example of the sensing nanoparticle of the present invention is schematically shown in Figure 2. In the sensing nanoparticle 10a shown in Figure 2, a signal group 31 has been previously introduced to the signal substance-binding group of the sensing nanoparticle 10 shown in Figure 1. Therefore, the sensing nanoparticle 10a itself can be used as a sensor for recognizing a biological substance.

[0021] Furthermore, an example of a usage mode of the sensing nanoparticle of the present invention is shown schematically in Fig. 3. Fig. 3 shows how the sensing nanoparticle 10a (sensor for recognizing a biological material) shown in Fig. 2 recognizes and captures a biological material 90 that is the sensing target.

[0022] [1-1. Molecularly imprinted polymers] [1-1-1. Shape] The molecularly imprinted polymer 20 is in the form of particles, and has molecular imprinted spaces (recesses) 21 for the biological material to be sensed (biological material 90 shown in FIG. 3). The number of imprinted spaces 21 provided in one molecularly imprinted polymer molecule 20 may be one as shown in the figure, or may be multiple (for example, two to three, preferably two). The molecular imprinted spaces 21 are formed by molecular imprinting using the biological material to be sensed as a template, and therefore have a shape complementary to the surface shape of the biological material.

[0023] The particulate shape of the molecularly imprinted polymer 20 essentially contributes to the development of excellent specificity in the sensing nanoparticles 10a. A sensor substrate for recognizing biological substances, which is produced by forming a flat film of a molecularly imprinted polymer (MIP film) on a substrate with the same monomer composition as the molecularly imprinted polymer 20 and then introducing a signal group, cannot develop specificity as excellent as that of the sensing nanoparticles of the present invention. The specific mechanism by which such excellent specificity can be achieved as an effect unique to the present invention is not clear, but the following reason is one possible explanation. During membrane formation (polymerization) using a biological material as a template, the templates tend to be randomly present, resulting in the formation of non-uniform molecular imprinted spaces (with large variations in the opening diameters of the molecular imprinted spaces), which results in a large proportion of molecular imprinted spaces that may cause nonspecific adsorption (for example, spaces with opening diameters that are too small to exhibit sufficient selectivity) compared to all molecular imprinted spaces. On the other hand, during polymer particle formation (polymerization) using a biological material as a template, the biological material templates, which are relatively hydrophilic with respect to the polymer, tend to be present near the interface between the polymer and the polymerization reaction solution, which results in the formation of molecular imprinted spaces with high uniformity (with little variation in the opening diameters of the molecular imprinted spaces), which results in an extremely small proportion of molecular imprinted spaces that may cause nonspecific adsorption compared to all molecular imprinted spaces.

[0024] Furthermore, since the molecular imprinted polymer 20 is particulate, the shape of the sensor for recognizing specific biological substances is not limited to the shape of the substrate, and it can be formulated, for example, as a liquid sensing reagent, making it highly versatile.

[0025] The average particle size of the molecularly imprinted polymer 20 varies depending on the size and application of the biological material to be sensed, but examples thereof include a volume-average particle size (cumulative 50% value of the volume particle size distribution) measured by dynamic light scattering (DLS) of 5 to 500 nm, and a Z-average particle size measured by DLS of 5 to 500 nm. More specifically, when administered to a living body or used for in vitro imaging to observe the inside of cells, the average particle size of the molecularly imprinted polymer 20 is a volume-average particle size measured by dynamic light scattering (DLS) of 5 to 100 nm, and a Z-average particle size measured by DLS of 5 to 100 nm. When used without administering to a living body, the average particle size of the molecularly imprinted polymer 20 is a volume-average particle size measured by dynamic light scattering (DLS) of 5 to 500 nm, and a Z-average particle size measured by DLS of 5 to 500 nm. Preferably, the average particle size of the molecularly imprinted polymer 20 is 7 to 50 nm, 8 to 40 nm, 15 to 30 nm, or 20 to 25 nm in terms of volume average particle size, and 7 to 50 nm, 8 to 40 nm, 15 to 30 nm, or 20 to 25 nm in terms of Z average particle size.

[0026] [1-1-2. Functional groups arranged in the molecular imprint space] The molecularly imprinted polymer 20 has functional groups 22 present in the molecular imprinted spaces 21. The functional groups 22 contribute to the recognition of the biological substance to be sensed and the provision of signal information based on the recognition. In the present invention, the presence of functional groups 22 on the surface of the molecularly imprinted polymer 20 other than the molecular imprinted spaces 21 is permitted as long as the functional groups 22 are present in the molecular imprinted spaces 21. However, in the molecular imprinted spaces 21, the shape imprinted with the protein to be sensed and the presence of the functional groups 22 suppress nonspecific adsorption of molecules other than the biological substance to be sensed, thereby exhibiting excellent specificity.

[0027] [1-1-3. Structure of functional groups] As described above, the functional group 22 is a group that interacts with the biological substance to be sensed (R1 ) and a signal substance binding group (in the figure, R 2 Furthermore, the interacting group and the signal substance-binding group are designed to be different groups from each other.

[0028] [1-1-3-1. Functions of the interacting group and signal substance binding group] The interactive group is a group that can interact with the biological substance to be sensed and form a non-covalent bond. 1 ) interacts with the surface of the biological material 90 to be sensed. Non-covalent bonds include non-covalent bonds and covalent bonds that are weaker than non-covalent bonds. Examples of non-covalent bonds include hydrogen bonds, electrostatic interactions (ionic bonds), hydrophobic bonds, and van der Waals bonds. Examples of covalent bonds weaker than non-covalent bonds include covalent bonds that can be cleaved simply by changing the liquid properties without adding a cleavage reagent that exhibits strong oxidizing or reducing properties, such as an oxidizing agent or a reducing agent. Specifically, when the biological material is a glycoprotein, a covalent bond formed between the sugar group and the borate group of the glycoprotein is included. This facilitates the removal of the template biological material after the synthesis of the molecularly imprinted polymer particles in molecular imprinting.

[0029] Furthermore, the interactive groups are present at positions corresponding to the specific amino acid residues and / or specific hydrophobic regions of the biological substance on the surface of the imprinted space 21, which has a shape complementary to the surface shape of the protein. Therefore, the molecular imprinted polymer 20 exhibits excellent recognition ability for the biological substance because the molecular imprinted space 21 has a shape specific to the biological substance and the interactive groups present in the molecular imprinted space 21 are located at positions specific to the biological substance.

[0030] The signal substance binding group is a group that can bind to a signal substance. By binding the signal substance binding group to the signal substance, a signal group 31 is introduced, as in the sensing nanoparticle 10a. The signal group 31 undergoes an environmental change when the biological substance to be sensed binds non-covalently to the interactive group, and therefore causes a signal change before and after binding to the biological substance. In Figure 3, the signal group 31, which was exposed in Figure 2 (before recognizing the biological substance 90), is exposed when the biological substance 90 to be sensed binds to the interactive group (R 1 ) and thus the sensing nanoparticle 10a can read out the binding information of the biological substance to be sensed from the signal change.

[0031] The environmental change that the signal group 31 undergoes as a result of interaction with the biological substance to be sensed is not particularly limited as long as it results in a signal change due to the proximity of the biological substance to be sensed. Examples of such environmental changes include: (i) a polar group of the biological substance to be sensed approaches the vicinity of the signal group 31, causing electrons of the signal group 31 to be attracted to the polar group; (ii) a polar group of the biological substance to be sensed approaches the vicinity of the signal group 31, thereby eliminating water molecules that were present near the signal group 31, and thereby releasing electrons of the signal group 31 from being attracted by the nearby water molecules; (iii) a polar group of the biological substance to be sensed approaches the vicinity of the signal group 31, causing energy that was on the side of the signal group 31 to be transferred to the biological substance to be sensed to which it is bound; and (iv) a polar group of the biological substance to be sensed approaches the vicinity of the signal group 31, shielding the signal group 31, thereby preventing excitation energy from reaching the signal group 31.

[0032] [1-1-3-2. Positional relationship between interacting group and signal substance binding group] In the illustrated embodiment, the interacting group is located at the distal end of the functional group 22, and the signal substance-binding group is located at the proximal end of the functional group 22. However, the positional relationship between the interacting group and the signal substance-binding group in the functional group 22 is not limited thereto, as long as the biological substance to be sensed can be non-covalently bound to the interacting group and the signal group 31 undergoes an environmental change upon non-covalent binding. For example, the positional relationship between the interacting group and the signal substance-binding group may be reversed from that illustrated. However, from the viewpoint of obtaining higher recognition ability by making the non-covalent binding between the biological substance and the interacting group easier, and / or from the viewpoint of obtaining higher sensitivity of signal changes by more reliably changing the environment of the signal group 31 upon non-covalent binding between the biological substance and the interacting group, it is preferable that the interacting group be located at the distal end of the functional group 22, and the signal substance-binding group be located at the proximal end of the functional group 22, as illustrated.

[0033] [1-1-3-3. Specific examples of functional groups] (general formula) A preferred example of the functional group is represented by the following formula (1).

[0034] [ka]

[0035] In formula (1), R 1 represents an interacting group, and R 2 represents a linking group containing a signal substance-binding group, and L 1 represents a direct bond or a linking group.

[0036] (interaction group R 1 ) interaction group R 1 is designed depending on the surface state of the biological material to be sensed. For example, the interactive group R 1Examples of the interactive group R include a group that can form one or more bonds selected from the group consisting of a hydrogen bond, a bond due to electrostatic interaction (ionic bond), a hydrophobic bond, and a van der Waals bond through interaction with a specific structure that constitutes the biological substance. 1 Specific examples of the group include [1] a substituted or unsubstituted amino group, [2] a substituted or unsubstituted aromatic group, [3] an amidino group (-C(=NH)NH2), [4] a guanidino group (-NHC(=NH)NH2), [5] a carboxyl group, [6] a sulfo group, [7] a boronyl group (-B(OH)2), and / or [8] a ligand. The aromatic group in the [2] substituted or unsubstituted aromatic group includes

[21] a substituted or unsubstituted nitrogen-containing aromatic group and

[22] a substituted or unsubstituted non-nitrogen-containing aromatic group, as well as

[25] a substituted or unsubstituted aryl group (

[0251] a nitrogen-containing aryl group and

[0252] a non-nitrogen-containing aryl group) and

[26] a substituted or unsubstituted arylene group (

[0261] a nitrogen-containing arylene group and

[0262] a non-nitrogen-containing arylene group). The interacting group R 1 may be a group consisting of any one of these groups, or may be a group containing a combination of two or more selected from these groups.

[0037] If the biological material is a protein, the interacting group R 1 Specific examples of the group R include a group capable of forming a hydrogen bond and / or an electrostatic interaction through interaction with a specific amino acid residue constituting the protein, and a group capable of forming a hydrophobic bond with a specific hydrophobic region of the protein. 1 The following table shows combinations of specific groups selected as and the types of non-covalent bonds formed by their interaction.

[0038] [Table 1]

[0039] [1] Examples of the substituent in the substituted amino group include a linear or branched alkyl group having 1 to 8 carbon atoms, a substituted or unsubstituted aralkyl group, an alkenyl group having 4 to 6 carbon atoms constituting a cyclic secondary amino group, a substituted or unsubstituted aryl group, etc. Specific examples of the substituted or unsubstituted aryl group include the groups exemplified below in

[0251] substituted or unsubstituted nitrogen-containing aryl group and

[0252] substituted or unsubstituted non-nitrogen-containing aryl group. [1] Specific examples of the substituted or unsubstituted amino group include an amino group (unsubstituted); secondary amino groups such as an N-methylamino group, an N-ethylamino group, an Nn-propylamino group, an N-isopropylamino group, an Nn-butylamino group, an N-isobutylamino group, an N-tert-butylamino group, an N-benzylamino group, an N-phenylamino group, an N-mesylamino group, and an N-tosylamino group; secondary amino groups such as an N,N-dimethylamino group, an N,N-diethylamino group, an N,N-dibenzylamino group, an N-ethyl-N-methylamino group, an N,N-di-n-propylamino group, an N,N-diisopropylamino group, an N,N-diphenylamino group, an N-methyl-N-phenylamino group, an N-methyl-N-benzylamino group, and an N-mesyl-N-methylamino group; and cyclic secondary amino groups such as a piperidyl group and a pyrrolidyl group.

[0040] [2] Among the substituted or unsubstituted aromatic groups, the nitrogen-containing aryl group in the substituted or unsubstituted nitrogen-containing aryl group includes a nitrogen-containing aryl group having 2 to 12 carbon atoms. Examples of the substituent in the substituted nitrogen-containing aryl group include a linear or branched alkyl group having 1 to 8 carbon atoms.

[0251] Specific examples of the substituted or unsubstituted nitrogen-containing aryl group include a pyridyl group, a pyrimidyl group, a pyridazyl group, a pyrazyl group, an imidazolyl group, a triazolyl group, a methylpyridyl group (e.g., a 2-methylpyridyl group, a 3-methylpyridyl group, a 4-methylpyridyl group), a dimethylpyridyl group (e.g., a 2,6-dimethylpyridyl group), a methylethylpyridyl group (e.g., a 2-methyl-6-ethylpyridyl group), a methylimidazolyl group (e.g., a 1-methylpyridyl group), a methylimidazolyl group (e.g., a 1-methylpyridyl group), a methylpyridyl group (e.g., a 2-methyl-6-ethylpyridyl group), a methylimidazolyl group (e.g., a 1-methylpyridyl group), a methylpyridyl group (e.g., a 2-methyl-6-ethylpyridyl group), a methylpyridyl group (e.g., a 1-methyl ... imidazolyl group, etc.), dimethylimidazolyl group (1,2-dimethylimidazolyl group, etc.), ethylimidazolyl group (1-ethylimidazolyl group, etc.), propylimidazolyl group (1-n-propylimidazolyl group, etc.), butylimidazolyl group (1-n-butylimidazolyl group, etc.), pentylimidazolyl group (1-n-pentylimidazolyl group, etc.), hexylimidazolyl group (1-n-hexylimidazolyl group, etc.), and the like.

[0041] [2] Among the substituted or unsubstituted aromatic groups, the non-nitrogen-containing aryl group in the substituted or unsubstituted non-nitrogen-containing aryl group

[0252] includes a phenyl group, a naphthyl group (1-naphthyl group, 2-naphthyl group, etc.) having 6 to 16 carbon atoms, etc. Substituents in the substituted aryl group include a linear or branched alkyl group having 1 to 8 carbon atoms, a nitro group, a halogen group (fluoro group, chloro group, bromo group, etc.), etc.

[0252] Specific examples of substituted or unsubstituted aryl groups include phenyl, naphthyl, tolyl (o-tolyl, m-tolyl, p-tolyl, etc.), ethylphenyl (4-ethylphenyl, 3-ethylphenyl, 2-ethylphenyl, etc.), propylphenyl (4-n-propylphenyl, etc.), isopropylphenyl (4-isopropylphenyl, 2-isopropylphenyl, etc.), butylphenyl (4-n-butylphenyl, 4-isobutylphenyl, 4-t-butylphenyl, 3-t-butylphenyl, 2-t-butylphenyl, etc.), pentylphenyl (4-n-pentylphenyl, 4-isopentylphenyl, 2-neopentylphenyl, 4-t-pentylphenyl, etc.), hexylphenyl (4-n-hexylphenyl, etc.), 4-(2-ethylbutyl)phenyl, 4-n-heptylphenyl, 4-n-octylphenyl, etc. diphenyl group, 4-(2-ethylhexyl)phenyl group, 4-t-octylphenyl group, 4-ethyl-1-naphthyl group, 6-n-butyl-2-naphthyl group, dimethylphenyl group (2,4-dimethylphenyl group, 2,5-dimethylphenyl group, 3,4-dimethylphenyl group, 3,5-dimethylphenyl group, 2,6-dimethylphenyl group, etc.), trimethylphenyl group (2,3,5-trimethylphenyl group, 2,3,6-trimethylphenyl group, etc.), group, 3,4,5-trimethylphenyl group, etc.), diethylphenyl group (2,4-diethylphenyl group, 2,6-diethylphenyl group, etc.), 2,5-diisopropylphenyl group, 2,6-diisobutylphenyl group, di-t-butylphenyl group (2,4-di-t-butylphenyl group, 2,5-di-t-butylphenyl group, etc.), 4,6-di-t-butyl-2-methylphenyl group, 5-t-butyl-2-methylphenyl group, 4-t-butyl-2,6-dimethylphenyl group, fluorophenyl group (4-fluorophenyl group, 3-fluorophenyl group, 2-fluorophenyl group, etc.), chlorophenyl group (4-chlorophenyl group, 3-chlorophenyl group, 2-chlorophenyl group, etc.), bromophenyl group (4-bromophenyl group, 2-bromophenyl group, etc.), chloronaphthyl group (4-chloro-1-naphthyl group, 4-chloro-2-naphthyl group, etc.), 6-bromo-2-naphthyl group, dichlorophenyl group (2,3-dichlorophenyl group, 2,4-dichlorophenyl group, 2,5-dichlorophenyl group, 3,4-dichlorophenyl group, 3,5-dichlorophenyl group, etc.), Examples of such groups include a 2,5-dibromophenyl group, a 2,4,6-trichlorophenyl group, a dichloronaphthyl group (such as a 2,4-dichloro-1-naphthyl group or a 1,6-dichloro-2-naphthyl group), a chloromethylphenyl group (such as a 2-chloro-4-methylphenyl group, a 2-chloro-5-methylphenyl group, a 2-chloro-6-methylphenyl group, a 3-chloro-4-methylphenyl group, a 2-methyl-3-chlorophenyl group, a 2-methyl-4-chlorophenyl group, or a 3-methyl-4-chlorophenyl group), a trifluoromethylphenyl group (such as a 4-trifluoromethylphenyl group), and a nitrophenyl group (such as a 4-nitrophenyl group).

[0042] Furthermore, examples of groups containing a combination of two or more selected from the above groups [1] to [7] include groups containing a combination of the monovalent groups of the groups [1] to [7], namely, [1] a substituted or unsubstituted amino group,

[0251] a nitrogen-containing aryl group,

[0252] a non-nitrogen-containing aryl group, [3] an amidino group, [4] a guanidino group, [5] a carboxyl group, [6] a sulfo group, and [7] a boronyl group, and

[26] a substituted or unsubstituted arylene group (

[0261] a nitrogen-containing arylene group,

[0262] a non-nitrogen-containing arylene group).

[0043]

[26] Among the substituted or unsubstituted arylene groups, the

[0261] nitrogen-containing arylene group is a group obtained by removing one hydrogen atom bonded to a carbon atom constituting an aromatic ring from the group listed above as the

[0251] substituted or unsubstituted nitrogen-containing aryl group.

[26] Among the substituted or unsubstituted arylene groups, the

[0262] non-nitrogen-containing arylene group is a group obtained by removing one hydrogen atom bonded to a carbon atom constituting an aromatic ring from the group listed above as the

[0252] substituted or unsubstituted nitrogen-containing aryl group.

[0044] Specific examples of groups containing a combination of two or more selected from the above groups [1] to [7] include an amidinoaryl group, which is a combination of an amidino group and a substituted or unsubstituted arylene group

[26] , a carboxyaryl group, which is a combination of a carboxyl group and a substituted or unsubstituted arylene group [5], a sulfoaryl group, which is a combination of a sulfo group and a substituted or unsubstituted arylene group

[26] , and a boronylaryl group, which is a combination of a boronyl group and a substituted or unsubstituted arylene group [7]. Among these groups, preferred groups are shown below.

[0045] [ka]

[0046] [8] The ligand is not particularly limited as long as it is a ligand corresponding to the biological substance to be sensed as the analyte. For example, when the biological substance is an antigen, examples include antibody mimetics (such as affibodies) and nanobodies, when the biological substance is a sugar chain, examples include lectins, when the biological substance is a phospholipid, examples include phospholipid-binding proteins (eventin 2, annexin A2, annexin V, Tim4), and when the biological substance is an enzyme, examples include enzyme substrates or enzyme inhibitors.

[0047] (Linking group R containing a signal substance binding group) 2 ) Linking group R containing a signal substance binding group 2 In the above, the signal substance-binding group is designed according to the binding group (binding group 32 in FIG. 65) possessed by the signal substance (signal substance 30 in FIG. 5 described later) from which the signal group 31 to be introduced is derived. Specific examples of the signal substance-binding group include an amino group (including a monovalent amino group and a divalent amino group), a carboxyl group, a thiol group, a disulfide group-containing group (monovalent group), an aminooxy group, an aldehyde group, and a hydroxyl group.

[0048] Linking group R 2 is an unbranched or branched divalent group.

[0049] Linking group R 2 is unbranched, the signal substance binding group may itself be linked to the linking group R 2 (i.e., the linking group R 2 (The signal substance-binding group is composed of the unbranched linking group R 2 That is, examples of the signal substance binding group include a divalent amino group represented by the following formula (2).

[0050] [ka]

[0051] In formula (2), R 3 represents hydrogen or a linear or branched alkyl group having 1 to 8 carbon atoms, and preferably represents hydrogen.

[0052] Linking group R 2 When R is branched, the signal substance binding group is a monovalent group present on the branch. 2 Examples of the group include a divalent group represented by the following formula (3).

[0053] [ka]

[0054] In formula (3), R 21represents an amino group (monovalent), a carboxyl group, a thiol group, a disulfide group-containing group (monovalent group), an aminooxy group, an aldehyde group, or a hydroxyl group, and preferably represents a thiol group or a disulfide group-containing group. The amino group (monovalent) is preferably an amino group (monovalent), such as the above-mentioned interacting group R 1 Examples of the substituted or unsubstituted amino group include the groups listed as [1].

[0055] The disulfide group-containing group (monovalent group) is represented by the following formula (4).

[0056] [ka]

[0057] In formula (4), R 211 represents a substituted or unsubstituted alkyl group. Examples of the alkyl group in the substituted or unsubstituted alkyl group include linear or branched alkyl groups having 1 to 8 carbon atoms. Examples of the substituent in the substituted alkyl group include an interacting group R 1 Those skilled in the art can appropriately select any functional group that does not interfere with the interaction between the disulfide bond and the biological substance serving as a template, does not interfere with the reaction between the disulfide bond and the signal substance, and ensures water solubility of the compound as a whole. Specifically, a hydroxyl group, an amino group (specifically, the above-mentioned interacting group R 1 Examples of the substituent in the substituted alkyl group include the groups listed as examples of [1] substituted or unsubstituted amino group), a carboxy group, an alkoxy group (for example, an alkoxy group having 1 to 4 carbon atoms, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1), a polyethylene glycol group (for example, an ethylene oxide addition number of 1 to 6), an alkyl group (for example, an alkyl group having 1 to 4 carbon atoms), an amide group, a sulfo group, a guanidino group, an amidino group, etc. The position of the substituent in the substituted alkyl group can be determined by the linking group R 2 Examples include the ends of the branches.

[0058] In formula (3), L 21represents a direct bond or a linking group. Examples of the linking group include an alkylene group, a carbonyl group, an ester bond (-COO- or -OCO-), an amide bond (-CONH- or -NHCO-), and a group consisting of any combination thereof, and preferably an alkylene group. Examples of the alkylene group include alkylene groups having 1 to 4 carbon atoms, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1 carbon atom.

[0059] (direct bond or linking group L 1 ) Direct bond or linking group L 1 is the interacting group R 1 and a linking group R containing a signal substance binding group 2 When the sensing nanoparticle 10a recognizes the biological substance 90 to be sensed, the linking group R 2 The signal group 31 is introduced into the signal substance binding group of the above, and the interacting group R of the biological substance is 1 In order to more effectively suppress interference with binding to L 1 is more preferably a linking group.

[0060] Examples of the linking group include an alkylene group, a carbonyl group, an ester bond (-COO- or -OCO-), an amide bond (-CONH- or -NHCO-), and any combination thereof. Examples of the alkylene group include alkylene groups having 1 to 4 carbon atoms.

[0061] (More specific examples) More specific structures of the functional group 22 include the following formulas (11) and (12).

[0062] [ka]

[0063] In equation (11), R 11 is an interacting group R of formula (1) 1and the divalent amino group (—NH—) corresponds to a linking group R 2 (which itself constitutes the signal substance binding group), and L 11 is a direct bond or a linking group L 1 is equivalent to

[0064] In equation (11), R 11 represents a carboxyaryl group or a sulfoaryl group, specifically, as described above, [5] a carboxyaryl group which is a combination of a carboxyl group and

[26] a substituted or unsubstituted arylene group, and [6] a sulfoaryl group which is a combination of a sulfo group and

[26] a substituted or unsubstituted arylene group. 11 represents an alkylene group having 1 to 4 carbon atoms. In the functional group represented by formula (11), the interacting group R 1 is R 11 The shorter the distance between the L and the divalent amino group (-NH-) that is the signal substance binding group, the more sensitive the signal change can be obtained when the sensing nanoparticle 10a recognizes the biological substance 90 that is the sensing target. 11 As the alkylene group, an alkylene group having preferably 1 to 3 carbon atoms, more preferably 1 to 2 carbon atoms, and even more preferably 1 carbon atom can be mentioned.

[0065] [ka]

[0066] In equation (12), R 11 is an interacting group R of formula (1) 1 and the divalent group (-CH(L 21 -R 21 )-) is a linking group R containing a signal substance binding group of formula (1) 2 is equivalent to L 12 is a direct bond or a linking group L 1 is equivalent to

[0067] In equation (12), R 11represents a carboxyaryl group or a sulfoaryl group, specifically, as described above as a carboxyaryl group which is a combination of [5] a carboxyl group and

[26] a substituted or unsubstituted arylene group, and a sulfoaryl group which is a combination of [6] a sulfo group and

[26] a substituted or unsubstituted arylene group. In addition, in formula (12), the divalent group (-CH(L 21 -R 21 )-) is as described above in formula (3). Furthermore, in formula (12), L 12 represents a divalent linking group, and examples of the divalent linking group include an alkylene group, a carbonyl group, an ester bond (-COO- or -OCO-), an amide bond (-CONH- or -NHCO-), and a group consisting of any combination thereof, and preferably an amide bond.

[0068] Further specific examples of the groups shown in the following formulae (11) and (12) are shown in the following formulae (11a), (11b), and (12a), (12b), respectively.

[0069] [ka]

[0070] These functional groups 22 may be present in one or more types, or may be present in one or more, per sensing nanoparticle. Furthermore, these functional groups 22 may be present in one or more types, or may be present in one or more molecular-printed spaces.

[0071] [1-1-4. Constituent Monomers of Molecularly Imprinted Polymers] The polymer constituting the molecularly imprinted polymer 20 contains at least a constituent unit derived from a functional monomer having a functional group 22. The polymer constituting the molecularly imprinted polymer 20 may contain, in addition to the constituent unit derived from the functional monomer, a constituent unit derived from N-substituted or unsubstituted acrylamide and / or a biocompatible monomer. Preferably, the polymer constituting the molecularly imprinted polymer 20 contains, in addition to the constituent unit derived from the functional monomer, a constituent unit derived from N-substituted or unsubstituted (meth)acrylamide and a biocompatible monomer. Note that the polymer constituting the molecularly imprinted polymer 20 may also contain a constituent unit derived from a monomer other than the functional monomer, the N-substituted or unsubstituted acrylamide, or the biocompatible monomer.

[0072] (functional monomer) The functional monomer having the functional group 22 is not particularly limited as long as it is a monomer containing a functional group and an ethylenically unsaturated group, and is, for example, represented by the following formula (5).

[0073] [ka]

[0074] In formula (5), X represents an ethylenically unsaturated group, L 5 represents a linking group, -R 2 -L 1 -R 1 represents a functional group. Examples of the ethylenically unsaturated group include an acrylic group and a methacrylic group. Examples of the linking group include an alkylene group, a carbonyl group, an ester bond (-COO- or -OCO-), an amide bond (-CONH- or -NHCO-), and a group consisting of any combination thereof, and preferably a group in which any number of alkylene groups and amide bonds are bonded. Examples of the functional group are as described in detail above in "1-1-3. Structure of Functional Group."

[0075] Specific examples of the functional monomer represented by formula (5) include functional monomers represented by the following formulae (51a), (52a), (51b), and (52b).

[0076] [ka]

[0077] (N-substituted or unsubstituted acrylamide) Regarding N-substituted or unsubstituted (meth)acrylamides, N-substituted (meth)acrylamides are represented by the following formula (6).

[0078] [ka]

[0079] In formula (6), R 61 represents hydrogen or a methyl group, preferably hydrogen, and R 62 and R 63 each independently represents hydrogen; a linear or branched alkyl group having 1 to 6 carbon atoms; a linear or branched hydroxyalkyl group having 1 to 6 carbon atoms; or a linear or branched aminoalkyl group having 1 to 6 carbon atoms, which may be the same or different (provided that R 62 and R 63 and R are hydrogen atoms. 62 and R 63 However, they may form a saturated 5- to 7-membered ring containing an oxygen atom together with the nitrogen atom that carries them.

[0080] Examples of linear or branched alkyl groups having 1 to 6 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl groups. Examples of linear or branched hydroxyalkyl groups having 1 to 6 carbon atoms include hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxy-n-propyl, 2-hydroxy-n-propyl, 3-hydroxy-n-propyl, 1-hydroxyisopropyl, 2-hydroxyisopropyl, 1-hydroxy-n-butyl, 1-hydroxy-n-pentyl, and 1-hydroxy-n-hexyl groups. Examples of the linear or branched aminoalkyl group having 1 to 6 carbon atoms include an aminomethyl group, a 1-aminoethyl group, a 2-aminoethyl group, a 1-amino-n-propyl group, a 2-amino-n-propyl group, a 3-amino-n-propyl group, a 1-aminoisopropyl group, a 2-aminoisopropyl group, a 1-amino-n-butyl group, a 1-amino-n-pentyl group, a 1-amino-n-hexyl group, etc. Examples of the saturated 5- to 7-membered ring containing an oxygen atom together with a nitrogen atom include a morpholine ring, etc.

[0081] Among these N-substituted or unsubstituted (meth)acrylamides, preferably N-substituted (meth)acrylamides are used, more preferably N-monosubstituted (meth)acrylamides are used, still more preferably N-alkyl(meth)acrylamides (wherein the alkyl group is the above-mentioned linear or branched alkyl group having 1 to 6 carbon atoms), still more preferably N-isopropyl(meth)acrylamide is used, and particularly preferably N-isopropylacrylamide is used.

[0082] (biocompatible monomer) Biocompatibility refers to the property of not inducing adhesion of biological materials. By including components derived from biocompatible monomers in the polymer constituting the molecularly imprinted polymer 20, nonspecific adsorption can be further suppressed. Preferred examples of biocompatible monomers include hydrophilic monomers, and more preferably zwitterionic monomers.

[0083] Zwitterionic monomers contain both an anionic group derived from an acidic functional group (e.g., phosphate group, sulfate group, and carboxyl group) and a cationic group derived from a basic functional group (e.g., primary amino group, secondary amino group, tertiary amino group, and quaternary ammonium group) in one molecule. Types of zwitterionic monomers include phosphobetaines, sulfobetaines, and carboxybetaines.

[0084] Examples of phosphobetaines include molecules having a phosphorylcholine group in the side chain, preferably 2-methacryloyloxyethyl phosphorylcholine (MPC). Examples of sulfobetaines include N,N-dimethyl-N-(3-sulfopropyl)-3'-methacryloylaminopropanaminium inner salt (SPB) and N,N-dimethyl-N-(4-sulfobutyl)-3'-methacryloylaminopropanaminium inner salt (SBB). Examples of carboxybetaines include N,N-dimethyl-N-(1-carboxymethyl)-2'-methacryloyloxyethanaminium inner salt (CMB) and N,N-dimethyl-N-(2-carboxyethyl)-2'-methacryloyloxyethanaminium inner salt (CEB).

[0085] Among these biocompatible monomers, phosphobetaine is preferred, molecules having a phosphorylcholine group in the side chain are more preferred, and 2-methacryloyloxyethyl phosphorylcholine (MPC) is even more preferred.

[0086] [1-2. Signal Groups] The signal group 31 is a signal group that interacts with the biological substance 90 to be sensed and the interaction group R 1 The signal group 31 functions as a signal that provides information on the binding between the biological substance 90 to be sensed and the interactive group R 1The signal may be changed by the change in the environment due to binding to the target molecule, and is not particularly limited thereto. The signal may be changed by a change in signal intensity or a change in spectrum (e.g., a peak shift).

[0087] Examples of the signal group 31 include a fluorescent group, a radioactive element-containing group, a magnetic group, etc. From the viewpoint of ease of detection, etc., the signal group 31 is preferably a fluorescent group. The fluorescent group is preferably a group of a non-biological substance, and examples thereof include fluorescent dyes such as fluorescein dyes, cyanine dyes such as indocyanine dyes, and rhodamine dyes. Examples of the radioactive element-containing group include: 18 Sugars, amino acids, nucleic acids, etc., labeled with radioactive isotopes such as F 19 Examples of the magnetic group include an MRI probe labeled with F. Examples of the magnetic group include a group having a magnetic substance such as ferrichrome.

[0088] [1-3. Other structures] As described above in "1-1-2. Functional group arranged in molecular imprinted space," the present invention allows the functional group 22 to be present on the surface of the molecular imprinted polymer 20 other than the molecular imprinted space 21, as long as the functional group 22 is present in the molecular imprinted space 21. In the sensing nanoparticle of the present invention, when the functional group 22 is present on the surface of the molecular imprinted polymer 20 other than the molecular imprinted space 21, a capping modification group may be bonded to the functional group 22 present on the surface of the molecular imprinted polymer 20 other than the molecular imprinted space 21.

[0089] The capping modification group is a group introduced by a compound that inhibits the function of the functional monomer. Examples of compounds that inhibit the function of the functional monomer (capping compounds) include non-signal substances, such as compounds used to inhibit the adsorption of biological substances (e.g., compounds having an oligoethylene glycol chain) and compounds having a sugar chain structure. The bonding position of the capping modification group can be determined by the interaction group R of the functional group 22. 1and / or a signal substance binding group. Such a capping modification group shields the functional group 22 from the outside, thereby achieving an even greater effect of suppressing nonspecific binding and background signals.

[0090] [1-4. Biological substances to be sensed] The sensing target of the sensing nanoparticles of the present invention is biological material. In the present invention, biological material refers to a molecular species classified as a molecule that is a basic material constituting living organisms. Generally, biological material refers to a molecule that contains carbon and hydrogen, and further contains nitrogen, oxygen, phosphorus, and / or sulfur as constituent elements, and has biological function or biological significance. Specific examples of biological material include proteins (including peptides; the same applies hereinafter), sugar chains, lipids (e.g., phospholipids), composite molecules of two or more of these, and composite molecules containing proteins and cofactors (e.g., coenzymes and prosthetic groups, specifically organic molecules other than proteins and metal ions). In the present invention, biological material may be natural or artificial. For convenience, human serum albumin (HSA) is shown as the biological material 90 to be sensed in FIG. 3 , but this biological material 90 is intended to include any biological material that can be sensed in the present invention.

[0091] A preferred example of a biological material is a protein. In the following description, the term "protein" encompasses both normal proteins that do not contain sugars, lipids, or cofactors, and complex proteins that contain at least one of sugars, lipids, and cofactors (e.g., glycoproteins, lipoproteins, metalloproteins, etc.). Proteins may be natural or artificial. Natural proteins are proteins that have been produced as a result of natural selection. Artificial proteins include natural proteins in which part of the amino acid sequence has been artificially modified, and natural proteins in which modifying groups have been artificially introduced or removed.

[0092] Examples of biological material 90, in terms of their in vivo existence form, include free molecules secreted into body fluids (e.g., free proteins, free sugar chains, etc.), substances (e.g., membrane proteins, membrane sugar chains) attached to or bound to the biological membrane of membrane structures (e.g., cells, viruses, extracellular vesicles (e.g., exosomes), or organelles, etc.), and molecules corresponding to exposed partial structures of biological membranes (e.g., phospholipids such as phosphatidylserine, etc.). Biological material 90 may also be a fragment of a specific biological molecule. Examples of such fragments include molecules obtained by cutting out a specific region (e.g., a domain) from a specific biological molecule (e.g., an antibody), and molecules obtained by artificially producing only that region. Furthermore, examples of biological material 90, in terms of their function, include plasma proteins, biomarkers, domains, enzymes, hormones, etc. Examples of plasma proteins include albumin, γ-globulin, and fibrinogen. Biomarkers include kidney function markers, liver function markers, inflammatory markers, tumor markers, etc. More specifically, urinary albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), transferrin, ceruloplasmin, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (gamma-GTP), C-reactive protein (CRP), alpha-fetoprotein (AFP), various antigens (e.g., carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), exosome-specific antigens (CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, LAMP1, etc.), etc.) are included. In terms of the organism from which the biological material 90 is derived, examples include human-derived biological material and non-human-derived biological material. Non-human organisms are not particularly limited and include all living organisms, such as vertebrates such as fish, amphibians, reptiles, and mammals; invertebrates such as cnidarians and crustaceans; and plants such as seed plants, gymnosperms, and algae. Mammals are preferred, including mice, rats, monkeys, dogs, cats, cows, horses, pigs, hamsters, rabbits, and goats.Furthermore, biological materials 90 include biological materials derived from plants and animals that are considered haram from the viewpoint of Islamic law.

[0093] [1-5.Application] The sensing nanoparticles of the present invention can be used to detect the above-mentioned biological substances. When the biological substances are substances (e.g., membrane proteins, membrane sugar chains) attached to or bound to the biological membrane of membrane structures (e.g., cells, viruses, extracellular vesicles, organelles, etc.), molecules (e.g., phospholipids such as phosphatidylserine) corresponding to exposed partial structures of biological membranes, or fragments of specific biological molecules (e.g., specific regions such as domains), the sensing nanoparticles of the present invention can be used to detect, via the above-mentioned biological substances, membrane structures (e.g., cells, viruses, extracellular vesicles (e.g., exosomes), organelles, etc.) containing the biological substances or specific biological molecules (e.g., antibodies, etc.) having the fragments (e.g., specific regions such as domains) as partial structures.

[0094] More specific uses of the sensing nanoparticles of the present invention vary depending on the type of biological material, but for example, when the sensing nanoparticles of the present invention are designed for sensing biomarkers such as kidney function markers, liver function markers, inflammation markers, and tumor markers, examples of uses include diagnostic uses based on kidney function, liver function, the presence or degree of inflammation, the presence or degree of tumors, etc.

[0095] Furthermore, due to the excellent specificity of the sensing nanoparticles of the present invention, they can distinguish between the same type of biological material in different organisms. Therefore, a more specific use of the sensing nanoparticles of the present invention is in halal checks to confirm whether or not food contains haram biological materials of animal or plant origin (e.g., porcine serum albumin) when the biological material is a haram biological material of animal or plant origin.

[0096] [2. Manufacturing method of sensing nanoparticles] [2-1. Manufacturing method 1] The present invention also provides a method for producing the sensing nanoparticles of the present invention described above in "1. Sensing Nanoparticles." The method for producing the sensing nanoparticles of the present invention includes: Step 1: synthesizing a molecularly imprinted polymer by polymerizing, using a biological material as a template, a monomer component including a functional monomer having a functional group including a group that interacts with the biological material and a signal substance-binding group different from the interactive group; and Step 2: removing the template from the molecularly imprinted polymer. The method for producing the sensing nanoparticles of the present invention may also include, after Step 2, Step 3: binding a signal substance to the signal substance-binding group in the molecularly imprinted polymer. In the production method of the present invention, the monomer component in Step 1 may further include N-substituted or unsubstituted (meth)acrylamide and / or a biocompatible monomer.

[0097] In this manufacturing method 1, it is not necessary to perform surface modification by covalent bonding on the biological material serving as a template (for example, a treatment performed for the purpose of localizing an interactive group and a signal substance-binding group in the molecular imprint space) prior to step 1. Furthermore, this manufacturing method 1 can be applied to any of the sensing nanoparticles of the present invention described above in "1. Sensing nanoparticles," but is preferably applied when the interactive group is selected from the group consisting of a substituted or unsubstituted amino group, a substituted or unsubstituted aromatic group, an amidino group, a guanidino group, a carboxyl group, a sulfo group, and a boronyl group.

[0098] Figure 4 schematically shows step 1 in one example of the method for producing sensing nanoparticles of the present invention. Figure 5 also schematically shows steps 2 and 3 in one example of the method for producing sensing nanoparticles of the present invention. Hereinafter, the method for producing sensing nanoparticles of the present invention will be described in detail with reference to these figures.

[0099] [2-1-1. Process 1] In step 1, as shown in FIG. 4, a biological substance 90′ is used as a template to form an interactive group R 1 and the interacting group R 1A molecularly imprinted polymer is synthesized by polymerizing a monomer component including a functional monomer M22 having a functional group 22 containing a signal substance-binding group different from that of the functional monomer M22. In the example of Fig. 4, the monomer component includes an N-substituted or unsubstituted (meth)acrylamide M23 and a biocompatible monomer M24, along with the functional monomer M22.

[0100] The template biological material 90' is the same type of biological material as the biological material 90 to be sensed. For example, if the biological material 90 to be sensed is HSA, then HSA can also be used as the template biological material 90'. Therefore, examples of the template biological material 90' include those listed above in "1-4. Biological material to be sensed."

[0101] The concentration of the biological material 90' serving as a template in the polymerization reaction solution is not particularly limited, but may be, for example, 0.5 to 5 μmol / L, preferably 1 to 3 μmol / L, and more preferably 1.5 to 2.5 μmol / L.

[0102] The functional monomer M22 is as described above in "(Functional Monomer)" of "1-1-4. Constituent Monomers of Molecularly Imprinted Polymer."

[0103] The concentration of the functional monomer M22 in the polymerization reaction solution is not particularly limited, and may be, for example, 1 to 6 mmol / L, preferably 2 to 4 mmol / L, and more preferably 2.5 to 3.5 mmol / L. The amount of the functional monomer M22 used relative to the template biological material 90' is preferably 0.5 to 4 mmol, more preferably 1 to 2 mmol, and even more preferably 1.3 to 1.7 mmol per μmol of the template biological material 90'.

[0104] The N-substituted or unsubstituted (meth)acrylamide M23 is as described above in "(N-substituted or unsubstituted (meth)acrylamide)" in "1-1-4. Constituent Monomers of Molecularly Imprinted Polymer."

[0105] The concentration of N-substituted or unsubstituted (meth)acrylamide M23 in the polymerization reaction solution is not particularly limited, but may be, for example, 20 to 60 mmol / L, preferably 30 to 50 mmol / L, and more preferably 35 to 40 mmol / L. The amount of N-substituted or unsubstituted (meth)acrylamide M23 used relative to the template biological material 90' is preferably 5 to 40 mmol, more preferably 10 to 33 mmol, and even more preferably 15 to 20 mmol per μmol of the template biological material 90'. The amount of N-substituted or unsubstituted (meth)acrylamide M23 used relative to the functional monomer M22 is preferably 5 to 30 mol, more preferably 8 to 20 mol, and even more preferably 10 to 15 mol per mole of functional monomer M22.

[0106] The biocompatible monomer M24 is as described above in "(Biocompatible monomer)" of "1-1-4. Constituent monomers of molecularly imprinted polymer."

[0107] The concentration of the biocompatible monomer M24 in the polymerization reaction solution is not particularly limited, but may be, for example, 0.5 to 5 mmol / L, preferably 1 to 3.5 mmol / L, and more preferably 1.5 to 2.5 mmol / L. The amount of biocompatible monomer M24 used relative to the template biological material 90' is preferably 0.3 to 3.5 mmol, more preferably 0.5 to 2 mmol, and even more preferably 0.8 to 1.2 mmol per μmol of the template biological material 90'. The amount of biocompatible monomer M24 used relative to the functional monomer M22 is preferably 0.1 to 1.5 mol, more preferably 0.2 to 1 mol, and even more preferably 0.4 to 0.8 mol per mole of functional monomer M22.

[0108] The polymerization reaction liquid contains a crosslinking agent, an initiator, and a solvent as appropriate.

[0109] The crosslinking agent may be a compound in which two or more ethylenically unsaturated groups are linked by a linker group. Specific examples of the crosslinking agent include those represented by the following formula (7):

[0110] [ka]

[0111] In formula (7), W represents an ethylenically unsaturated group, which may be the same or different, and Z represents a linker group. Examples of the ethylenically unsaturated group include an acrylic group and a methacrylic group. Examples of the linker group include an alkylene group having 1 to 6 carbon atoms, preferably 2 to 6 carbon atoms, an amino group (-NH-), an ether group, a carbonyl group, an ester bond (-COO- or -OCO-), an amide bond ((-CONH- or -NHCO-)), a sulfoxide group (-SO-), a sulfonyl group (-SO2-), and groups formed by combining two or more of these groups. When two or more of the above groups are combined to form the linker group, the number of bonds is preferably 5 or less or 4 or less, and more preferably 3 or less or 2. More specific examples of the crosslinker agent include low-molecular-weight crosslinkers such as N,N'-methylenebisacrylamide and ethylene glycol dimethacrylate, and preferably N,N'-methylenebisacrylamide.

[0112] The concentration of the crosslinking agent in the polymerization reaction solution is not particularly limited, but may be, for example, 0.5 to 5 mmol / L, preferably 1 to 3.5 mmol / L, and more preferably 1.5 to 2.5 mmol / L.

[0113] Examples of the initiator include peroxides such as ammonium persulfate and potassium persulfate, and azo polymerization initiators such as azobisisobutyronitrile and 2,2'-azobis(2-methylpropionamidine) dihydrochloride, preferably azo polymerization initiators, and more preferably 2,2'-azobis(2-methylpropionamidine) dihydrochloride.

[0114] The concentration of the initiator in the polymerization reaction solution is not particularly limited, but may be, for example, 1 to 15 mmol / L, preferably 3 to 12 mmol / L, and more preferably 5 to 10 mmol / L.

[0115] The solvent used may be an aqueous solvent such as a buffer solution, preferably a phosphate buffer solution. The solvent preferably contains NaCl, with the NaCl concentration being, for example, 100 to 200 mM, preferably 120 to 160 mM. The pH of the solvent is appropriately selected so as not to denature the template 90', for example, 6 to 8, preferably 6.8 to 7.8, and more preferably 7.2 to 7.6.

[0116] In the polymerization reaction system of step 1, as shown in FIG. 4, the functional monomer M22 among the monomer components has an interacting group R 1 The functional monomer M22 interacts specifically with a predetermined site on the template 90' ​​via the functional group 22, forming a complex of the functional monomer M22 and the template 90'. When the polymerization reaction proceeds with such a complex formed, a molecular imprinted space (recess) complementary to the surface shape of the template 90' ​​is formed, and the functional group 22 can be present on the surface of the molecular imprinted space (recess). Furthermore, the template 90' ​​is likely to be present near the polymer interface, which allows the molecular imprinted space to be formed efficiently.

[0117] Specific polymerization methods for obtaining nanoparticles of molecularly imprinted polymers include emulsifier-free precipitation polymerization, dispersion polymerization, emulsion polymerization, and seed emulsion polymerization, with emulsifier-free precipitation polymerization being preferred.

[0118] In step 1, the polymerization reaction solution is preferably subjected to low-temperature conditions of 1 to 15°C, preferably 1 to 10°C, and more preferably 2 to 6°C before being subjected to temperature conditions for promoting the polymerization reaction, in order to sufficiently form a complex of functional monomer M22 and template 90'. The time for which the solution is subjected to low-temperature conditions is, for example, 20 to 30 hours. The temperature conditions for promoting the polymerization reaction are, for example, 40 to 76°C, preferably 50 to 74°C, and more preferably 60 to 72°C. The time for which the solution is subjected to temperature conditions for promoting the polymerization reaction is, for example, 10 to 15 hours. Even when the polymerization reaction is carried out under heated conditions, the shape of the template is maintained to an extent that it does not undergo denaturation that would cause a loss of the specificity of the molecular imprinted space, thereby effectively achieving the specificity of the molecular imprinted space.

[0119] In this way, the molecularly imprinted polymer 20 portion is synthesized, and a complex of the molecularly imprinted polymer 20 and the template 90' ​​is obtained. The complex of the molecularly imprinted polymer 20 and the template 90' ​​has an interaction group R 1 and template 90' ​​by non-covalent bonding (including non-covalent bonding and covalent bonding that is as weak as a non-covalent bonding).

[0120] [2-1-2. Process 2] In step 2, the template 90' ​​is removed from the complex of the molecularly imprinted polymer 20 and the template 90', as shown in Figure 5. In the complex of the molecularly imprinted polymer 20 and the template 90', the interactive group R 1 Since the bond between the template 90' ​​and the template 90' ​​is a weak non-covalent bond, the template 90' ​​can be easily dissociated and removed without adding a cleavage reagent that exhibits strong oxidizing or reducing action, such as an oxidizing agent or a reducing agent.

[0121] Here, unlike the manufacturing method of the present invention, when a functional monomer having a reversible bond (a covalent bond such as a disulfide group or an oxyimino group) is introduced into the template in advance, nanoparticles are formed by molecular imprinting polymerization, and then the reversible bond is cleaved with a cleavage reagent to remove the template, and functional groups derived from the reversible bond (such as thiol groups or oxyamino groups that function as signal substance-binding groups and / or interactive groups) are localized within the molecularly imprinted space, the template cannot be easily removed even with the cleavage reagent because of the covalent bond between the template and the molecularly imprinted polymer. In this case, to more reliably remove the template, it is necessary not only to introduce the functional monomer into the template in advance, but also to support such a template on a carrier such as silica, and then remove the entire carrier when removing the template after molecular imprinting polymerization. On the other hand, the manufacturing method of the present invention is also superior in terms of ease of manufacturing, since it does not require a step of introducing a functional monomer into the template 90' ​​prior to molecular imprinting polymerization, nor a step of supporting the template 90' ​​on a carrier, and it is also easy to remove the template 90' ​​from the complex of the molecularly imprinted polymer 20 and the template 90'.

[0122] Removal of template 90' ​​results in the release of the interacting group R 1 The binding mode between the template 90' ​​and the template 90' ​​can be appropriately determined by those skilled in the art. Preferably, the reaction solution after step 1 is subjected to chromatography (preferably anion exchange chromatography), and the mobile phase used in this step is also used as an eluent for the template molecule, thereby removing the template 90'. The removed template 90' ​​is separated from the molecularly imprinted polymer 20 by chromatography, and the molecularly imprinted polymer 20 is purified.

[0123] The mobile phase is preferably a Tris-HCl buffer solution. The mobile phase preferably contains NaCl, and the NaCl concentration may be the same as that in the solvent used in step 1, for example, 100 to 200 mM, preferably 120 to 160 mM. The pH of the mobile phase may be the same as that in the solvent used in step 1, for example, 6 to 8, preferably 6.8 to 7.8, more preferably 7.2 to 7.6.

[0124] In this manner, the sensing nanoparticles 10 are synthesized.

[0125] [2-1-3. Process 3] In step 3, as shown in FIG. 5, a signal substance 30 is bound to the signal substance-binding group of the molecularly imprinted polymer 20.

[0126] The signal substance 30 includes a signal group 31 and a binding group 32. The signal group 31 is as described above in "1-2. Signal Group." The binding group 32 is a group capable of covalently bonding to the signal substance-binding group, and specifically, can be appropriately determined by those skilled in the art depending on the type of signal substance-binding group (amino group (including monovalent amino group and divalent amino group), carboxyl group, thiol group, disulfide group-containing group (monovalent group), aminooxy group, aldehyde group, or hydroxyl group).

[0127] The specific conditions for introducing the signal substance 30 into the molecularly imprinted polymer 20 can be appropriately determined by a person skilled in the art based on the type of binding group 32 and the type of signal substance binding group, so that the two can chemically react to form a covalent bond.

[0128] In this manner, the sensing nanoparticles 10a are synthesized.

[0129] [2-1-4. Other processes] When obtaining sensing nanoparticles having the capping modification group described above in "1-3. Other Structures," the following steps can be performed on the synthesized sensing nanoparticles 10. In this case, it is preferable to introduce a signal group after introducing the capping modification group.

[0130] First, a very small amount of biological material (the same type of biological material as the biological material 90 to be sensed and the biological material 90' to be used as a template) is added to the sensing nanoparticle 10, and the molecular imprinted space 21 is blocked and protected with the biological material (protection step). Next, a compound (capping compound) that inhibits the function of the functional monomer is reacted to block the interaction group R of the functional group 22 present on the surface of the molecular imprinted polymer 20 other than the molecular imprinted space 21. 1 A capping modification group is bound to the signal substance-binding group (capping modification step). Compounds that inhibit the function of the functional monomer (capping compounds) include non-signal substances, such as compounds commonly used to inhibit the adsorption of biological substances (e.g., compounds having an oligoethylene glycol chain) and compounds having a sugar chain structure. Next, the bound biological substance is removed to regenerate the molecular imprinted space 21 (deprotection step).

[0131] [2-2. Manufacturing method 2] The method for producing the sensing nanoparticles of the present invention described in "1. Sensing Nanoparticles" above is not limited to the method described in "2-1. Production Method 1" above. The method for producing the sensing nanoparticles of the present invention can include the following steps: Step 11: preparing a molecularly imprinted template in which the surface of a biological material is modified with a polymerizable functional group via a reversible linking group; Step 12: synthesizing a molecularly imprinted polymer on a portion of the surface of the template using the polymerizable functional group as a substrate; Step 13: cleaving the reversible linking group to remove the template from the molecularly imprinted polymer; and Step 14: introducing, via the reversible linking group, a molecule having a functional group containing a group for binding an interactive group with the biological material and a signal substance-binding group. Furthermore, Production Method 2 can include, in addition to steps 11 to 14 above, the following steps: binding a molecule that provides the interactive group with the biological material to the group for binding the interactive group with the biological material; and binding a signal substance to the signal substance-binding group.

[0132] This manufacturing method 2 can be applied to any of the sensing nanoparticles of the present invention described above in "1. Sensing nanoparticles," but is preferably applied when the interactive group is a ligand for the biomolecule.

[0133] [3. Sensing substrate and sensing reagent] Examples of applications of the sensing nanoparticles of the present invention described above in "1. Sensing Nanoparticles" include sensing substrates and sensing reagents.

[0134] [3-1.Sensing board] A sensing substrate includes a substrate and sensing nanoparticles fixed to the substrate. FIG. 6 schematically shows an example of a sensing substrate. FIG. 7 schematically shows another example of a sensing substrate. The sensing substrate 1 shown in FIG. 6 includes a substrate 40 and sensing nanoparticles 10 fixed to the substrate 40. The sensing substrate 1a shown in FIG. 7 includes a substrate 40 and sensing nanoparticles 10a fixed to the substrate 40.

[0135] The substrate 40 may be made of a material selected from the group consisting of metal, glass, and resin. Examples of metals include gold, silver, copper, aluminum, tungsten, and molybdenum. Examples of resins include polymethacrylate, polyacrylate, polystyrene, ABS (acrylonitrile-butadiene-styrene copolymer), polycarbonate, polyester, polyethylene, polypropylene, nylon, polyurethane, silicone resin, fluororesin, methylpentene resin, phenol resin, melamine resin, epoxy resin, and vinyl chloride resin.

[0136] The substrate 40 may be formed by combining a plurality of materials selected from the above-mentioned materials. For example, the substrate 40 may be formed by providing a metal film on the surface of glass or resin.

[0137] The substrate 40 can be one that is compatible with the sensing means of the signal group 31. For example, the substrate 40 can be a surface plasmon (SPR) substrate, a surface enhanced Raman scattering (SERS) substrate, or the like.

[0138] The sensing substrate 1 fixed to the substrate 40 is as described above in "1. Sensing Nanoparticles" as the sensing nanoparticles 10. In the illustrated embodiment, for convenience, the sensing nanoparticles 10 are oriented in a uniform direction, but the sensing nanoparticles 10 may be oriented randomly.

[0139] The sensing substrate 1 shown in FIG. 6 is obtained by fixing the sensing nanoparticles 10 described above in "1. Sensing nanoparticles" to the surface of a substrate 40.

[0140] The method for immobilizing the sensing nanoparticles 10 on the surface of the substrate 40 is not particularly limited, and those skilled in the art can appropriately determine a general method for immobilizing polymer nanoparticles on a substrate surface. Such a method can be performed, for example, by a molecular film formation step of forming a molecular film having binding functional groups on the surface of the substrate 40, and a step of binding the sensing nanoparticles 10 to the binding functional groups on the surface of the formed molecular film. Note that examples of the functional groups on the surface of the sensing nanoparticles 10 that bind to the binding functional groups on the surface of the molecular film include interactive groups and / or signal substance binding groups derived from functional monomers remaining on the surface, and / or functional groups derived from initiators remaining at the ends of the polymers that constitute the sensing nanoparticles 10.

[0141] In the illustrated embodiment, a molecular film having carboxyl groups on the surface of the substrate 40 as binding functional groups is formed, and an amide bond is formed between the carboxyl groups and amino groups on the surface of the sensing nanoparticles 10, thereby binding the sensing nanoparticles 10.

[0142] 7 can be obtained by introducing a signal group into the above-mentioned sensing substrate 1. The method for introducing the signal group is as described above in "2-3. Step 3." The sensing substrate 1a shown in FIG. 7 can also be obtained by using sensing nanoparticles 10a instead of sensing nanoparticles 10 as the sensing nanoparticles fixed to the substrate 40, in the same manner as the manufacturing method of the above-mentioned sensing substrate 1.

[0143] The sensing substrate can be used to detect the biological substances described in items 1-4 above. When the biological substance is a substance (e.g., a membrane protein or a membrane sugar chain) attached to or bound to the biological membrane of a membrane structure (e.g., a cell, a virus, an extracellular vesicle, or an organelle), a molecule (e.g., a phospholipid such as phosphatidylserine) corresponding to an exposed partial structure of the biological membrane, or a fragment of a specific biological molecule (e.g., a specific region such as a domain), the sensing substrate can be used to detect, via the biological substance, a membrane structure (e.g., a cell, a virus, an extracellular vesicle (e.g., an exosome), or an organelle) containing the biological substance or a specific biological molecule (e.g., an antibody) having the fragment (e.g., a specific region such as a domain) as a partial structure. More specific applications vary depending on the type of biological substance to be sensed. For example, sensing substrates designed for sensing biomarkers such as kidney function markers, liver function markers, inflammation markers, and tumor markers can be used for diagnostic purposes based on kidney function, liver function, the presence or degree of inflammation, the presence or degree of tumors, etc. Furthermore, a more specific use of the sensing substrate of the present invention is a halal check to confirm whether or not food contains haram-derived biological substances derived from animals and plants (for example, porcine serum albumin) when the biological substances are haram-derived biological substances derived from animals and plants.

[0144] [3-2. Sensing Reagents] The sensing reagent includes sensing nanoparticles. The sensing nanoparticles in the sensing reagent are not immobilized on a bulk substrate such as a substrate.

[0145] The sensing reagent is formulated in a liquid or solid form (e.g., powder). The sensing reagent contains other pharmaceutically acceptable components. As the other components, solids and / or liquids that do not affect the stability and recognition ability of the sensing reagent are appropriately selected, and examples thereof include water, salts such as NaCl, buffers, stabilizers, antioxidants, preservatives, pH adjusters, surfactants, excipients, binders, etc.

[0146] The sensing reagent can be obtained by formulating the sensing nanoparticles 10 or sensing nanoparticles 10a and the other components described above based on a conventional formulation method.

[0147] Examples of uses of sensing reagents include the same uses as those of the sensing substrates described above. Other examples of uses of sensing reagents include in vitro imaging and in vivo imaging, which take advantage of the feature that sensing particles are not fixed to a substrate. One example of in vitro imaging is a method in which sensing nanoparticles 10a are prepared to sense intracellular or surface biological substances, and then added to the cells, thereby imaging the dynamics of intracellular or surface biological substances 90 using a signal detection means such as a fluorescence microscope. One example of in vivo imaging is a method in which sensing nanoparticles 10a are prepared to sense intracellular or surface biological substances, and then administered to the circulatory system, e.g., blood, of an animal such as a laboratory animal, and then imaging the dynamics of the biological substances 90 using a signal detection means. [Example]

[0148] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0149] [Test Example 1: Synthesis of nanoparticles for sensing - 1] In this test example, human serum albumin (HSA) sensing nanoparticles (hereinafter also referred to as "MIP-NGs") were synthesized, which correspond to sensing nanoparticle 10 in Figure 1. For comparison, nanoparticles (hereinafter also referred to as "NIP-NGs") were also synthesized in the same manner except that molecular imprinting was not performed.

[0150] 1-1. Synthesis of molecularly imprinted polymer (Step 1) A mixture of the components shown in the table below (prepolymer solution) was prepared in a 50 mL Schlenk flask and incubated at 4°C for 24 hours to allow sufficient interaction between the functional monomer and HAS, followed by emulsifier-free precipitation polymerization at 70°C for 12 hours under a nitrogen atmosphere. For comparison, NIP-NGs were subjected to emulsifier-free precipitation polymerization under the same conditions, except that the prepolymer solution did not contain HSA.

[0151] [Table 2]

[0152] [ka]

[0153] The functional monomer shown in formula 51a has a benzene ring, a carboxyl group, and a secondary amino group. In MIP-NGs, the benzene ring is thought to interact with the hydrophobic pocket of HSA, and the carboxyl group is thought to interact with positively charged sites such as lysine residues in HSA. Furthermore, the secondary amino group is used to introduce fluorescent molecules in the post-imprinting modification (step 3) described below.

[0154] 1-2. Removal of template (step 2) The particles were purified using anion exchange chromatography as follows: In this step, the template was removed from the molecularly imprinted polymer by utilizing the strong interaction between the anion exchange chromatography support and HSA.

[0155] The solution after the polymerization reaction was subjected to ultrafiltration (25°C, 7500 × g, 20 min) three times using an Amicon Ultra-4, 10 kDa dialysis tube to remove unreacted monomers and to solvent replacement from PBS to 10 mM Tris-HCl buffer, pH 7.4, containing 140 mM NaCl, to obtain a crude sample.

[0156] 1.0 g of DEAE-Sephadex, a chromatographic packing material for anion exchange chromatography, was added to 35 mL of Tris-HCl buffer (pH 7.4) containing 140 mM NaCl and allowed to swell for 24 hours. The mixture was then packed into a polypropylene Big LibraTube. (R) The column was filled to a height of approximately 5.5 cm. 1 mL of the crude sample was added, and the eluate was fractionated into 1.5 mL aliquots using 10 mM Tris-HCl buffer (pH 7.4) containing 140 mM NaCl as the mobile phase. The fluorescence derived from the functional monomer (functional group) (400 nm at an excitation wavelength of 280 nm) and the fluorescence derived from HSA (340 nm at an excitation wavelength of 280 nm) of the fractionated fractions were measured using a Hitachi High-Technologies F-2500 to confirm the removal of HSA. The second to seventh fractions were all mixed, and the solvent was replaced with PBS using a 10 kDa dialysis tube. The mixture was then concentrated to 5 mL. This yielded a purified sample solution of MIP-NGs or NIP-NGs.

[0157] 1-3. Particle size measurement The particle sizes of the obtained MIP-NGs and NIP-NGs were evaluated by DLS measurement (ZETASIZER NANO-ZS MAL500735, manufactured by Malvern) as follows.

[0158] 500 μL of the purified sample solution was added to a 1.5 mL microtube and dried overnight at 90°C. The solids concentration was calculated from the masses before and after drying using the following formula (where w0 is the weight (g) of the 1.5 mL microtube, w1 is the weight (g) before drying, and w2 is the weight (g) after drying). The results are shown in the table below.

[0159]

number

[0160] [Table 3]

[0161] As shown in Table 3, the presence of nanometer-order particles was confirmed for MIP-NGs and NIP-NGs.

[0162] [Test Example 2: Preparation of sensing substrate-1] In this test example, MIP-NGs (Example 1) and NIP-NGs (Comparative Example 1) obtained in Test Example 1 were immobilized on a substrate as follows. Specifically, the MIP-NGs obtained in Test Example 1 were immobilized on a substrate to prepare a human serum albumin (HSA) sensing substrate corresponding to sensing substrate 1 in Figure 6. NIP-NGs was also immobilized on the substrate in the same manner.

[0163] 2-1. Fixing particles to the substrate An SPR gold substrate (GE Healthcare SIA Kit Au) was washed with ethanol and pure water, then immersed in a 1 mM ethanol solution of 11-Mercaptoundecanoic acid and incubated for 24 hours at 25°C. After the reaction, the substrate was washed with ethanol and pure water and dried with nitrogen gas.

[0164] Using an SPR molecular interaction analyzer (GE Healthcare Biacore 3000), a 1:1 mixed solution of 0.1 M N-hydroxysuccinimide (NHS) and 0.4 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was injected at a flow rate of 20 μL / min for 7 minutes to form a molecular film, and then the purified sample (466 μg / mL) obtained in step 2 of Test Example 1 was injected at a flow rate of 20 μL / min for 7 minutes to immobilize the particles to the substrate. In this immobilization, the NHS ester on the molecular film surface reacts with the functional groups on the particle surface (presumably amino groups derived from functional monomers and / or amidino groups derived from initiators remaining at the ends of the polymers that make up the particles), thereby immobilizing the particles to the substrate. Subsequently, 1 M 2-aminoethanol (pH 8.5) was injected at a flow rate of 20 μL / min for 7 minutes to block unreacted NHS ester on the molecular membrane surface.

[0165] 2-2. Protein adsorption test Adsorption experiments were performed by injecting human serum albumin (HSA; Mw: 66.5 kDa, pI: 4.6), cytochrome C (Cyt c; Mw: 11.7 kDa, pI: 10.3), or transferrin (Trf; 80 kDa, pI: 6.1) dissolved in PBS (10 mM, pH 7.4, containing 140 mM NaCl) onto the particles immobilized on the substrate under the following conditions.

[0166] [Table 4]

[0167] The binding constant was calculated from the obtained adsorption isotherm by curve fitting. The curve fitting was performed using DeltaGraph 5.4.5v (manufactured by Nippon Pola Digital Co., Ltd.) based on the following formula (where K is the binding constant, G is the protein concentration, D is the maximum change in SPR signal intensity, and H is the independent variable). The results are shown in the table below.

[0168]

number

[0169] [Table 5]

[0170] [Table 6]

[0171] As shown in Table 5, the binding constant of MIP-NGs is significantly larger than that of NIP-NGs, confirming that it exhibits high affinity for HSA due to the molecular imprinting effect. Furthermore, as shown in Table 6, the binding constant of HSA is significantly larger than that of the three proteins, suggesting that MIP-NGs has a specific binding space for HSA.

[0172] Test Example 3: Preparation of a comparative sensing substrate with an MIP film For the comparative test in Test Example 5 described below, a flat MIP film was formed on a substrate using the same monomer as that used in Test Example 1, to prepare an HSA sensing substrate for comparison.

[0173] 3-1. Preparation of polymerizable functional group-immobilized substrate A gold-coated glass substrate (5 x 10 mm) was washed with ethanol and pure water and then treated with UV-O3 for 30 minutes. The treated substrate was immersed in a 1 mM ethanol solution of 11-mercapto-1-undecanol and incubated at 25°C for 24 hours. The substrate was then washed with ethanol and pure water and dried with nitrogen gas. The resulting substrate was immersed in a CHCl solution containing 10 mM methacryloyl chloride and 10 mM triethylamine and incubated at 25°C for 24 hours. The substrate was then washed with ethanol and pure water and dried with nitrogen gas. This resulted in a substrate with immobilized polymerizable functional groups.

[0174] 3-2. Synthesis of MIP membrane A prepolymer solution was prepared by mixing the components shown in the table below. The polymerizable functional group-immobilized substrate was covered with a silicone rubber sheet (thickness: 0.5 mm) with holes of 3 mm radius drilled therein. The prepolymer solution was dropped onto the exposed part of the gold substrate surface within the holes, and a cover glass was placed on top. This was placed in a photoreactor (KeyChem-Lumino, YMC Co., Ltd.) and a photopolymerization reaction was carried out (λ em =365 nm, 4°C, 10 minutes), thereby obtaining an MIP membrane (Comparative Example 2).

[0175] [Table 7]

[0176] [ka]

[0177] 3-3. Template removal The substrate provided with the MIP film (Comparative Example 2) was washed with pure water, and then immersed in a 1 M NaCl aqueous solution and a 0.5 wt % SDS aqueous solution (for 2 hours each) to remove HSA.

[0178] 3-4. Fluorescence introduction After washing, 10 μL of a 50 μg / mL ATTO647N NHS solution (in 10 mM PBS containing 0.5% DMSO) was dropped onto the MIP membrane of the substrate, and the substrate was left to stand at room temperature for 1 hour. Thereafter, the substrate was washed with pure water.

[0179] This resulted in the production of a comparative HSA sensing MIP membrane substrate. This comparative HSA sensing MIP membrane substrate has molecular imprinted spaces (recesses) for HSA on the surface of an MIP membrane provided on the substrate, and functional groups and fluorescent groups derived from functional monomers are disposed at least within the molecular imprinted spaces (recesses). The fluorescent groups increase in fluorescence in response to environmental changes when the imprinted spaces (recesses) adsorb the sensing target.

[0180] [Test Example 4: Synthesis of sensing nanoparticles-2] In this test example, HSA sensing nanoparticles corresponding to the sensing nanoparticles 10a in FIG. 2 were synthesized.

[0181] Introduction of signal substance (fluorescent substance) (Step 3) To 1 mL of the purified sample (466 μg / mL) of MIP-NGs (Example 1) obtained in step 2 of Test Example 1, 5 μL of a 0.5 wt % DMSO solution containing 10 mg / mL of a fluorescent substance (ATTO647N NHS, excitation wavelength 646 nm, emission wavelength 664 nm) in 10 mM PBS was added, and the mixture was incubated at 25° C. for 2 hours. The MIP-NGs were then ultrafiltered (25° C., 7500 × g, 20 minutes, 3 times) using an Amicon Ultra-4 10 kDa, and washed with PBS to obtain MIP-NGs with fluorescent groups introduced therein.

[0182] [Test Example 5: Preparation of sensing substrate-2] In this test example, an HSA sensing substrate corresponding to the sensing substrate 1a in FIG. 7 was fabricated.

[0183] 5-1. Immobilization of fluorescent group-introduced MIP-NGs on a substrate A gold-coated glass substrate (5 mm × 10 mm) was washed with ethanol and pure water, then immersed in a 1 mM ethanol solution of 11-Mercaptoundecanoic acid and incubated for 24 hours at 25°C. After the reaction, the substrate was washed with ethanol and pure water and dried with nitrogen gas.

[0184] 100 μL of an aqueous solution containing 0.2 M EDC and 50 mM NHS was added dropwise and incubated at room temperature for 1 hour. After washing with pure water, 100 μL of the fluorescent group-introduced MIP-NGs (47 μg / mL) obtained in Test Example 4 was added dropwise and allowed to react at room temperature for 2 hours for immobilization. After washing with pure water, 100 μL of 1 M 2-aminoethanol (pH 8.5) was added dropwise and allowed to react for 30 minutes for blocking. This resulted in an HSA sensing substrate with fluorescent group-introduced MIP-NGs immobilized on the substrate.

[0185] 5-2. Protein adsorption test-1 The obtained fluorescent group-introduced MIP-NGs-immobilized HSA sensing substrate was subjected to a protein adsorption test in the same manner as in Test Example 2. The results are shown in the table below.

[0186] [Table 8]

[0187] As shown in Table 8, the binding constant of HSA was further improved by introducing a fluorescent group. This is presumably because the introduced fluorescent group not only did not inhibit the binding of the sensing target HSA to the interactive group in the molecular imprint space, but also because the introduced fluorescent group was hydrophobic, an interaction between the hydrophobic region of HSA and the hydrophobic region of HSA was also observed in addition to the interaction between the benzene ring in the interactive group and the hydrophobic region of HSA.

[0188] 5-3. Protein adsorption test-2 A protein adsorption test based on the selectivity factor was performed on the obtained HSA sensing substrate with immobilized fluorescent group-introduced MIP-NGs and a control HSA sensing substrate provided with the MIP membrane obtained in Test Example 3. The characteristics of each of the compared HSA sensing substrates are shown in the table below. [Table 9]

[0189] Human serum albumin (HSA; Mw: 66.5 kDa, pI: 4.6), cytochrome C (Cyt c; Mw: 11.7 kDa, pI: 10.3), or transferrin (Trf; 80 kDa, pI: 6.1) dissolved in PBS (10 mM, pH 7.4, containing 140 mM NaCl) was incubated on the sensing substrate at 25°C for 10 minutes, and measurements were performed using a fluorescence microscope equipped with an automatic SIC dispenser under the following conditions.

[0190] (Fluorescence microscope with SIC automatic dispensing device, measurement conditions) Fluorescence microscope Filter: Cy5 Objective lens: x5 Exposure time: 0.1 seconds Light output: 12% Light source: mercury lamp SIC automatic dispensing device sequence 1. Chip Attachment 2. Sample aspiration (150 μL) 3. Incubation (25°C, 10 minutes) 4. Measurement position keep (measurement port) *Steps 2 to 4 were repeated for each protein.

[0191] The selectivity factor was calculated by dividing the rate of change in fluorescence intensity before and after the addition of each protein to the sensing substrate by the rate of change in fluorescence intensity of HSA, the sensing target. Specifically, the selectivity factor is expressed by the following formula: In the formula, F0 represents the fluorescence intensity on the substrate surface in the above buffer solution, and F sample represents the fluorescence intensity on the substrate surface for each protein-containing sample, and F HSA represents the fluorescence intensity on the substrate surface in a sample containing HSA, which is the sensing target.

[0192]

number

[0193] The results for the HSA sensing substrate (Example 2) with fluorescent group-introduced MIP-NGs (particles) immobilized thereon are shown in Figure 8, and the results for the comparative HSA sensing MIP membrane substrate (Comparative Example 2) with the MIP membrane obtained in Test Example 3 provided thereon are shown in Figure 9.

[0194] A selectivity factor value of less than 1 indicates the ability to recognize HSA. As shown in Figures 8 and 9, all sensing substrates were found to have the ability to recognize HSA. However, as shown in Figure 9, the control HSA sensing substrate (Comparative Example 2) provided with an MIP membrane was observed to exhibit large fluorescence changes not only for the sensing target HSA but also for Cyt c and Trf, which are not sensing targets, demonstrating poor specificity. In contrast, as shown in Figure 8, the HSA sensing substrate (Example 2) in which particulate MIPs were immobilized on the substrate was found to have high specificity for the sensing target HSA.

[0195] [Test Example 6: Measurement of HSA in serum samples] In this test example, an HSA sensing substrate (Example 2) corresponding to the sensing substrate 1a in FIG. 7, which was prepared in Test Example 5, was used to measure HSA in a serum sample.

[0196] Eight human serum samples approved by the Kobe University Graduate School of Medicine Medical Ethics Committee under registration number 180128 were used. First, the HSA concentration in each sample was confirmed by measuring it using bromocresol purple (BCP method). The confirmed HSA concentrations are shown in the table below.

[0197] [Table 10]

[0198] Next, each sample was diluted 1000 times with a buffer solution (PBS (10 mM, pH 7.4, containing 140 mM NaCl)), and the relative fluorescence intensity was determined in the same manner as in Protein Adsorption Test-2 of Test Example 5 using an HSA sensing substrate corresponding to sensing substrate 1a in Figure 7 prepared in Test Example 5.

[0199] The correlation between the obtained relative fluorescence intensity and the concentration obtained by the BCP method is shown in Figure 10. The relative fluorescence intensity was expressed as F-F0 / F0 (F0 represents the fluorescence intensity on the substrate surface in the above buffer solution, and F represents the fluorescence intensity on the substrate surface in the sample). As shown in Figure 10, a high correlation was observed between the HSA concentration measured by the BCP method and the relative fluorescence intensity based on HSA binding measured with the HSA sensing substrate. This demonstrated that the HSA sensing substrate can detect and quantify HSA even in human serum.

[0200] [Test Example 7: Halal check using PSA sensing substrate] In this test example, a PSA sensing substrate was prepared by immobilizing particles with porcine serum albumin (PSA) as the sensing target onto a substrate, and a halal check was performed using this substrate, based on the fact that PSA is used as a marker for the presence of pig-derived ingredients in halal food.

[0201] 7-1. Synthesis of nanoparticles for sensing - 3 First, PSA sensing nanoparticles corresponding to sensing nanoparticle 10 in Fig. 1 were synthesized. Specifically, MIP-NGs were synthesized in the same manner as in Test Example 1 (Example 3) using a mixture (prepolymer solution) of the components shown in the table below, except that PSA was used as the template protein.

[0202] [Table 11]

[0203] 7-2. Measurement of particle size The particle sizes of the obtained MIP-NGs and NIP-NGs were measured in the same manner as in Test Example 1, and the presence of particles on the order of nanometers was confirmed.

[0204] [Table 12]

[0205] 7-3. Synthesis of nanoparticles for sensing - 4 Next, PSA sensing nanoparticles were synthesized that correspond to the sensing nanoparticle 10a in Fig. 2. Specifically, the fluorescent substance ATTO647N NHS was introduced into the MIP-NGs of Example 3 in the same manner as in Test Example 4, thereby obtaining fluorescent group-introduced MIP-NGs (Example 4).

[0206] 7-4.Sensing board-3 Next, the fluorescent group-introduced MIP-NGs of Example 4 were immobilized on a substrate as follows.

[0207] A gold-coated glass SPR substrate (5 mm x 10 mm) was washed with ethanol and pure water, then treated with UV-O3 cleaner for 20 minutes. It was then immersed in a 1 mM ethanol solution of 11-amino-1-undecaneoctanethiol hydrochloride and incubated at 25°C for 24 hours. After the reaction, the substrate was washed with ethanol and pure water and dried with nitrogen gas. This resulted in a substrate with a molecular film formed on its surface. Furthermore, an aqueous solution (100 μL) of EDC (0.4 M) and NHS (0.1 M) was dripped onto the molecular film on the substrate and allowed to stand for 1 hour. 100 μL (300 μg / mL) of the fluorescent group-introduced MIP-NGs from Example 3 was then dripped onto the substrate and allowed to react for 2 hours, thereby immobilizing the particles to the substrate. During this immobilization, the amino groups on the molecular film surface reacted with the functional groups (carboxyl groups derived from functional monomers) on the particle surface, immobilizing the particles to the substrate. 100 μL of 10 mM sulfo-NHS acetate aqueous solution was added dropwise and reacted for 30 minutes to block the amino groups on the surface.

[0208] 7-5. Measurement of PSA in a sample A fluorescent substance was also introduced into the NIP-NGs of Comparative Example 1 in the same manner as above, yielding fluorescently-introduced NIP-NGs (Comparative Example 3). The fluorescent group-introduced NIP-NGs of Comparative Example 3 were similarly immobilized on a substrate to prepare a comparative sensing substrate. Using a PSA sensing substrate on which the fluorescent group-introduced MIP-NGs of Example 4 were immobilized and a sensing substrate on which the fluorescent group-introduced NIP-NGs of Comparative Example 3 were immobilized, the PSA concentration contained in samples with PSA concentrations of 0 to 20 nM in a buffer solution (PBS (10 mM, pH 7.4, containing 140 mM NaCl)) was measured in the same manner as in Test Example 6. The detection limit was 12 ng / mL.

[0209] Figure 11 shows the correlation between the relative fluorescence intensity and PSA concentration obtained for the PSA sensing substrate on which fluorescent group-introduced MIP-NGs of Example 4 were immobilized. The relative fluorescence intensity was the same as in Test Example 6. As shown in Figure 11, a high correlation was observed between the PSA concentration and the relative fluorescence intensity based on PSA binding measured on the PSA sensing substrate. This indicates that by changing the template for the imprinted recesses, it is possible to detect and quantify not only human serum albumin (HSA) but also porcine serum albumin (PSA). On the other hand, similar fluorescence measurements were performed on the sensing substrate on which fluorescent group-introduced NIP-NGs of Comparative Example 3 were immobilized, but almost no change in fluorescence was observed. Furthermore, the apparent binding constants calculated from the obtained fluorescence change rates are shown in the table below.

[0210] [Table 13]

[0211] As shown in Table 13, the binding constant of MIP-NGs (Example 4) was significantly larger than that of NIP-NGs (Comparative Example 3), confirming that it exhibited high affinity for PSA due to the molecular imprinting effect.

[0212] 7-6. Specificity for PSA Samples were prepared by dissolving porcine serum albumin (PSA), human serum albumin (HSA), bovine serum albumin (BSA), lysozyme (LyZ), or transferrin (Trf) at a concentration of 40 nM in a buffer solution (PBS (10 mM, pH 7.4, containing 140 mM NaCl)). These samples were measured in the same manner as in Test Example 6, using a sensing substrate on which fluorescent group-introduced MIP-NGs of Example 4 were immobilized or a sensing substrate on which fluorescent group-introduced NIP-NGs of Comparative Example 3 were immobilized. The selectivity factor is the ratio of the fluorescence change in the sample to the fluorescence change in 40 nM PSA, and is expressed by the following formula: In the formula, F0 represents the fluorescence intensity on the substrate surface in the buffer solution, and F samplerepresents the fluorescence intensity on the substrate surface at the sample, and F PSA represents the fluorescence intensity on the substrate surface at 40 nM PSA.

[0213]

number

[0214] The results for the sensing substrate on which fluorescent group-introduced MIP-NGs of Example 4 were immobilized are shown in Figure 12, and the results for the sensing substrate on which fluorescent group-introduced NIP-NGs of Comparative Example 3 were immobilized are shown in Figure 13.

[0215] A selectivity factor value of less than 1 indicates the ability to recognize PSA. As shown in Figures 12 and 13, both sensing substrates were found to have the ability to recognize PSA. However, as shown in Figure 13, the sensing substrate on which the fluorescent group-introduced NIP-NGs of Comparative Example 3 were immobilized showed a large change in fluorescence not only for the sensing target PSA but also for proteins that were not the sensing target, indicating poor specificity. In contrast, as shown in Figure 12, the sensing substrate on which the fluorescent group-introduced MIP-NGs of Example 4 were immobilized was found to have high specificity for the sensing target PSA.

[0216] 7-7. Halal Check-1 One gram of minced beef was added to 4 mL of phosphate buffer (pH 7.4), and the beef was homogenized at 10,000 rpm for 1 minute using a homogenizer. The resulting supernatant was centrifuged (16,000 × g, 30 minutes, 4°C), filtered three times through a 0.2 μm filter, and diluted 500-fold with phosphate buffer (pH 7.4) to obtain a beef extract sample.

[0217] PSA was added at various concentrations to the resulting beef extract samples to prepare PSA-contaminated beef extract samples. Separately, PSA samples were prepared by adding PSA at similar concentrations to phosphate buffer (pH 7.4). The changes in fluorescence of the PSA-contaminated beef extract samples and the PSA samples were measured using a sensing substrate immobilized with fluorescent group-introduced MIP-NGs (Example 4), and the changes in relative fluorescence intensity were calculated as described above. The results are shown in Figure 14.

[0218] As shown in Figure 14, it was confirmed that the sensing substrate on which the fluorescent group-introduced MIP-NGs of Example 4 was immobilized could quantify PSA mixed in a beef extract sample (in 500x beef extract) in the same way as PSA in phosphate buffer (in PBS buffer).

[0219] 7-8. Halal Check-2 A beef extract sample contaminated with a pork extract sample was prepared. First, a 500-fold diluted beef extract sample was obtained in the same manner as above. A 500-fold diluted pork extract sample was also obtained in the same manner, except that pork was used instead of beef. The beef extract sample and the pork extract sample contamination ratio were mixed so that the beef extract sample content and the pork extract sample contamination ratio were as shown in the table below, to obtain a beef extract sample contaminated with a pork extract sample. The ratios shown in the table indicate the amounts equivalent to the protein content (by weight) in the extract sample.

[0220] [Table 14]

[0221] For the beef extract samples contaminated with these pork extract samples, changes in fluorescence were measured using a sensing substrate to which the fluorescent group-modified MIP-NGs of Example 4 were immobilized, and the relative fluorescence intensity was calculated in the same manner as described above. The results are shown in Figure 15. In Figure 15, the horizontal axis represents the contamination rate of pork extract samples, and the vertical axis represents the relative fluorescence intensity.

[0222] As is clear from Figure 15, almost no change in fluorescence was observed in the negative control, which was 100% beef extract sample without any pork extract sample contamination, as was the case with PBS buffer. In contrast, in the beef extract sample contaminated with pork extract sample, PSA contained in the pork extract sample was detected. In particular, since PSA was detected even when the pork extract sample contamination rate was 0.1 wt%, it was confirmed that the sensing substrate on which the fluorescent group-introduced MIP-NGs of Example 4 were immobilized is suitable for highly sensitive halal checks.

[0223] [Test Example 8: Immune response using PSA sensing particles (non-immobilized)] In this test example, particles with porcine serum albumin (PSA) as the sensing target were used in a free state without being immobilized on a substrate, and immune responses to PSA and transferrin were tested.

[0224] Particles (fluorescent group-introduced MIP-NGs; Example 4) for sensing PSA were prepared by the same method as in Test Example 7, "Synthesis of sensing nanoparticles - 3" and "Synthesis of sensing nanoparticles - 4."

[0225] Protein solutions (PSA or transferrin in 10 mM phosphate buffer (pH 7.4) containing 140 mM NaCl) were added to 80 μg / mL of fluorescently labeled MIP-NGs to achieve final protein concentrations of 5, 10, 50, 100, 500, or 1000 μg / mL (final solution volume: 500 μL) and incubated at 25°C for 15 min. The fluorescence spectra of the solutions were then measured using a spectrophotometer (F-2500, Hitachi High-Technologies) (excitation wavelengths: λex: 647 nm, λem: 658-690 nm). The results are shown in Figure 16. The relative fluorescence intensity at a fluorescence wavelength of 667 nm was plotted against the protein concentrations of PSA and transferrin (PSA: 5, 10, 50, 100, 500, and 1000 μg / mL; transferrin: 10, 50, 100, 500, and 1000 μg / mL). The results are shown in Figure 17.

[0226] As shown in Figure 16, when a PSA solution was added to the fluorescent group-introduced MIP-NGs of Example 4 in a free state, an increase in fluorescence intensity was observed as the concentration increased. A similar phenomenon was observed when the fluorescent group-introduced MIP-NGs of Example 4 were immobilized on a substrate (Test Example 7), suggesting that the increase in fluorescence intensity shown in Figure 16 is a change in fluorescence intensity associated with PSA binding to the fluorescent group-introduced MIP-NGs of Example 4. Furthermore, as shown in Figure 17, a greater change in fluorescence intensity was observed when PSA was added compared to the change in fluorescence when the reference protein, transferrin, was added. This demonstrates that the fluorescent group-introduced MIP-NGs of Example 4, even in a free state not immobilized on a substrate, can selectively adsorb PSA and transmit binding information as a fluorescent signal.

[0227] [Example 9: Synthesis of sensing nanoparticles-5] In this test example, nanoparticles for sensing Fc domains (and for sensing IgG via capturing Fc domains) corresponding to the sensing nanoparticles 10a in FIG. 2 were synthesized.

[0228] 9-1.Synthesis of functional monomers The functional monomer shown in formula 51b was designed and synthesized according to the following scheme. Note that the functional monomer shown in formula 51b has a boronylaryl group and a divalent amino group. In MIP-NGs, the boronylaryl group interacts with the glycan of the IgG Fc domain, and the secondary amino group is used to introduce fluorescent molecules in post-imprinting modification.

[0229] [ka]

[0230] Ethylenediamine monomethacrylamide hydrochloride (ca. 1.57 mmol), 4-formylphenylboronic acid (215 mg, 1.5 mmol), and triethylamine (210 μL, 1.5 mmol) were dissolved in methanol (10 mL) and stirred at room temperature for 0.5 h. The reaction was monitored by TLC (MeOH → UV @ 254 nm and ninhydrin). Based on the appearance of a spot stained with UV and ninhydrin (Rf: 0.55, purple), it was determined that a Schiff base had formed. NaBH4 (120 mg, 9.0 mmol) was added to the mixture. The mixture was further stirred at room temperature overnight (8 h). Upon confirmation of the desired product spot (Rf: 0.25, yellow) by TLC (MeOH → UV @ 254 nm and ninhydrin), the reaction was terminated, and the solvent was removed under reduced pressure on a rotary evaporator. The residue was purified using an autocolumn (silica: universal premium, MeOH / DCM=70 / 30→100 / 0, GR) to obtain the target product. Yield: 175 mg (0.668 mmol, 42.5%) 1 H-NMR (500 MHz, D2O): δ=7.62 (d, 2H, phenyl), 7.30 (d, 2H, phenyl), 5.69 (s, 1H, vinyl), 5.44 (s, 1H, vinyl), 3.97 (s, 2H, N-CH2-), 3.47 (t, 2H, -CH2-), 2.99 (br, 2H, -CH2-), 1.89 (s, 3H, -Me)

[0231] 9-2. Preparation of Fc domain An antibody solution was prepared by dissolving 10 mg of IgG in 10 mM phosphate buffer (pH 7.4, 140 mM NaCl) (5 mL). An enzyme solution was prepared by dissolving 0.02 M L-cysteine, 0.002 M EDTA, and 0.1 mg / mL papain in 10 mM phosphate buffer (pH 7.4, 140 mM NaCl). Equal amounts of the antibody solution and enzyme solution were mixed and reacted at 37°C for 24 hours. The reaction solution was then eluted with 20 mM phosphate buffer (pH 7.0) using a 10 kDa ultrafiltration membrane (7500 g, 20 min × 3). Unreacted IgG was then separated using a 100 kDa ultrafiltration membrane (Amicon Ultra) (7500 g, 20 min × 3). The resulting solution (1 mL) was then passed through a Protein A column (Hitrap TM Purification was performed using Protein A HP (Cytiva). 20 mM phosphate buffer (pH 7.0) was used as the binding buffer, and 0.1 M citrate buffer (pH 3.0) was used as the elution buffer. Purification was performed using a syringe (flow rate: approximately 1 mL / min). After purification, the solvent was replaced with 10 mM carbonate buffer (pH 9.2) using a 10 kDa ultrafiltration membrane (7500 g, 20 min × 3). Purification of the Fc domain was confirmed by SDS-PAGE.

[0232] 9-3.Synthesis of nanoparticles for sensing - 5 A mixture of the components shown in the table below (prepolymer solution) was subjected to emulsifier-free precipitation polymerization at 50°C for 12 hours to synthesize a polymer nanogel (Step 1). To facilitate particle purification, a labeled monomer was also used as the monomer to be incorporated into the prepolymer solution. The polymerized solution (2 mL) was eluted with 10 mM phosphate buffer (pH 7.4, 140 mM NaCl) using an ultrafiltration membrane (10 kDa, 7500 × g, 20 min × 3). The resulting solution (2 mL) was passed through size exclusion chromatography (Sephadex G-100) to remove unreacted monomer. The resulting solution (1 mL) was mixed with 40 mg / mL SDS aqueous solution (1 mL) and incubated for 5 minutes. The mixture was then loaded onto an anion exchange resin (DEAE-Sephadex, 10 cm, 1.5 cm id) and eluted with 10 mM Tris-HCl buffer (pH 7.4, 140 mM NaCl) (Step 2). Purification was then performed by passing the mixture through a desalting column (PD-10) (eluent: 10 mM phosphate buffer (pH 7.4, 140 mM NaCl) to remove the SDS.

[0233] [Table 15]

[0234] The particle sizes of the obtained MIP-NGs (Example 5) and NIP-NGs (Comparative Example 4) were measured in the same manner as in Test Example 1, confirming the presence of nanometer-order particles. Specifically, the Z-average particle size was 21 nm for MIP-NGs (Example 5) and 18 nm for NIP-NGs (Comparative Example 4). The Z-potential was 19 mV for MIP-NGs (Example 5) and 2.9 mV for NIP-NGs (Comparative Example 4), indicating a positive charge. This charge is thought to be derived from the secondary amino group of the functional monomer (and the polymerization initiator). These findings suggest that particles incorporating functional monomer moieties have been synthesized. Furthermore, fluorescence measurements (λ) of the particle solutions before and after purification showed that the Z-average particle size (λ) of the particles was 19 mV for MIP-NGs (Example 5) and 2.9 mV for NIP-NGs (Comparative Example 4). exFrom the results (fluorescence at 340 nm, 280 nm), it was confirmed that the tryptophan-derived fluorescence in the Fc region, which was observed before purification, disappeared, and therefore most of the Fc domain fragments were successfully removed by washing.

[0235] Furthermore, 5 μL of ATTO647N NHS (10 mg / mL) DMSO solution was added to the purified MIP-NGs (0.5 mg / mL, 1 mL) and incubated at 25°C for 2 hours (Step 3). The reaction solution was ultrafiltered (10 kDa, 7500 × g, 20 min × 3) to remove unreacted fluorescent molecules, yielding fluorescent group-introduced MIP-NGs (Example 5). Fluorescence measurements (excitation wavelength: 647 nm) before and after the fluorescent molecule (ATTO647N) incorporation procedure revealed a fluorescence peak around 670 nm that was not observed before incorporation, confirming the incorporation of fluorescence. The same procedure was performed on NIP-NGs to obtain fluorescent group-introduced NIP-NGs (Comparative Example 4).

[0236] 9-4.Sensing board-4 A gold-sputtered glass substrate (4.3 × 9.8 mm) was washed with pure water and EtOH and then subjected to UV-O3 cleaning (20 min). After the cleaning, the substrate was immersed in a 1 mM EtOH solution of 11-mercaptoundecanoic acid to introduce carboxyl groups onto the surface via self-assembled monolayer formation (25°C, 24 h). After rinsing with EtOH, the substrate was immersed in an aqueous solution containing 0.2 M EDC and 0.05 M NHS to activate the surface carboxyl groups (25°C, 1 h). After the reaction, 50 μL of a solution (0.5 mg / mL in PBS) of fluorescently group-introduced MIP-NGs (Example 5) or fluorescently group-introduced NIP-NGs (Comparative Example 4) was added to the substrate, and nanoparticles were immobilized by amine coupling (25°C, 1 h). Subsequently, unreacted active esters were inactivated by dropwise addition of 50 μL of a 1 M aminoethanol aqueous solution (25°C, 0.5 h). Finally, protein-free blocking buffer (50 μL) was added dropwise to block the surface (25°C, 0.5 hours). The fluorescence intensity on the surface was measured before and after immobilization, and an increase in fluorescence intensity was observed on the substrate after immobilization, confirming the immobilization of the particles.

[0237] 9-5.Fluorescence measurement The fluorescent detection ability of the protein on the fabricated sensing substrate was examined using a fluorescence microscope equipped with an automatic liquid handling robot. The measurement sequence conditions were as follows: Fluorescence microscope Filter: Cy5 Objective: x5 Exposure time: 0.1 seconds Light intensity: 12% Light source: Mercury lamp ROI: 15 locations in 3x5 grid at the center of the board Automatic dispensing device sequence 1. Chip Attachment 2. Aspirate 150 μL of sample 3. React for 5 minutes (25℃) 4. Measurement position: Keep 2 → 4 repeated

[0238] Figure 18 shows the change in relative fluorescence intensity when the template protein, an Fc domain fragment (0-1600 nM), was added to the prepared sensing substrate. Compared to the substrate on which NIP-NGs (Comparative Example 4) was immobilized, the substrate on which MIP-NGs (Example 5) was immobilized showed a larger change in fluorescence. This suggests that a binding space for the Fc domain fragment in MIP-NGs (Example 5) was formed by molecular imprinting, and that a functional monomer was incorporated into this space and fluorescently labeled by post-imprinting modification, thereby converting the binding of the Fc domain fragment into a fluorescent signal. In addition, the apparent binding constant (K d ) is 9.8 × 10 -9 It was calculated as M.

[0239] The protein selectivity of the sensing substrate was tested using the Fc domain fragment and complete IgG and lysozyme (Lyz) as reference proteins. Figure 19 shows the fluorescence response upon addition of each reference protein (100 nM), with the relative fluorescence intensity change upon addition of the Fc domain fragment (100 nM) set to 1. MIP-NGs (Example 5) showed the largest fluorescence change upon addition of the Fc domain fragment. Furthermore, complete IgG, which contains the Fc domain fragment as a partial structure, also responded at approximately 70%, suggesting that it is capable of recognizing the Fc portion of IgG. This demonstrates that MIP-NGs (Example 5), which were prepared using the Fc domain fragment as a template, can also detect IgG via Fc domain capture. On the other hand, in NIP-NGs (Comparative Example 4), both complete IgG and Lysozyme showed greater fluorescence responses than the Fc domain fragment. This is presumably due to nonspecific binding to the functional monomer residues randomly present on the surface of NIP-NGs (Comparative Example 4) and the fluorescent molecules introduced thereto. The pI of whole IgG is approximately 8.5, the Lyz is 11.2, and it is positively charged in a neutral solution. This suggests that electrostatic interactions with the boronyl aryl group of the functional monomer and hydrogen bonding with the amide bond site contribute to binding.

[0240] [Test Example 10] In this test example, a particle having a molecularly imprinted recess for sensing HER2 (biological substance), and a functional group containing an anti-HER2 affibody (a group that interacts with biological substances) that interacts with HER2 and the signal substance ATTO647N in the molecularly imprinted recess, was produced as a nanoparticle for sensing exosomes (exosomes expressed by SK-BR-3 cells, a HER2-overexpressing breast cancer cell line) via HER2 capture.

[0241] 10-1. Template modification (step 11) Template HSA modified with a methacryloyl group (polymerizable functional group) via a disulfide bond (reversible linking group) was prepared as follows.

[0242] 10-1-1.Reagents Albumin from Human Serum (HSA) (Wako Pure Chemical Industries, Ltd.) Compound (5) synthesized by the following method:

[0243] [ka]

[0244] 3-mercaptopropionic acid (0.900 g, 8.50 mmol) was dissolved in 20 mL of ethanol, and 1.6 mL of acetic acid was added. 2,2-dipyridyl disulfide (3.75 g, 17 mmol) dissolved in 30 mL of ethanol was added dropwise with stirring at room temperature. The mixture was allowed to react at 25 °C for 3 hours to obtain compound (1).

[0245] Purified compound (1) (1.43 g, 3.97 mmol) was dissolved in 15 mL of ethanol, and then 2-(Boc-amino)ethanethiol (1.69 mL, 9.99 mmol, 2 eq) dissolved in 20 mL of ethanol was added. The reaction was allowed to proceed at room temperature for 3 hours to yield compound (2).

[0246] 4N HCl dioxane (6.60 mL, 25.5 mmol, 5 eq) dissolved in 10 mL of CHCl was added dropwise to purified compound (2) (1.43 g, 5.10 mmol) dissolved in 5 mL of CHCl under ice-cooling and stirring. The mixture was then allowed to react overnight at room temperature to obtain compound (3).

[0247] Purified compound (3) (531.6 mg, 2.45 mmol) was dispersed in 5 mL of CHCl, and DIEA (1.28 mL, 7.35 mmol, 3 eq) was added. N-succinimidyl methacrylate (670 mg, 3.68 mmol, 1.5 eq) dissolved in 3.5 mL of CHCl was added dropwise under a nitrogen atmosphere, and the mixture was allowed to react at room temperature overnight to obtain compound (4).

[0248] The purified compound (4) (290 mg, 1.16 mmol, 1.1 eq), sulfo-NHS (230 mg, 1.1 mmol), and DCC (304.5 mg, 1.54 mmol, 1.4 eq) were dissolved in 5 mL of DMA and reacted at room temperature for 24 hours. After the reaction, the solution was cooled to 4°C, and the precipitate was removed by filtration. The filtrate was then evaporated under reduced pressure to remove the DMA. The resulting white solid was dissolved in AcOEt, and hexane was added to precipitate again, yielding compound (5). 1 H-NMR chart 7 (500MHz,CD3OD):δ=8.1 (t,1H, -CO-NH-C-), 5.7-5.3 (s,2H, CH3-C=CH2), 4.0-3.8 (d,1H,-CO-CH-SO3Na), 3.4-3.3 (m, 2H,NaSO3-CH-CH2-), 3.3-2.7 (m,8H, -NH-CH2-CH2-SS-CH2-CH2-), 1.85 (s, 3H, CH3-C=CH2))

[0249] 10-1-2. Experimental procedure HSA (100 mg, 2.23 μmol) in 2 mL of 10 mM phosphate buffer (pH 7.4) was mixed with compound (5) (20 mg, 44.6 μmol, 20 eq) in 1 mL of 10 mM phosphate buffer (pH 7.4) and incubated overnight at 4 °C. The mixture was then washed three times with an Amicon Ultra-4 10 kDa column (4 °C, 7500 G, 20 min) to obtain template HSA modified with a methacryloyl group (polymerizable functional group) via a disulfide bond (reversible linking group).

[0250] 10-2. Synthesis of molecularly imprinted polymer (step 12) Molecularly imprinted polymer particles were synthesized as follows.

[0251] Specifically, molecularly imprinted polymer particles MIP-NGs were synthesized by emulsifier-free precipitation polymerization in a 50 mL Schlenk flask under a nitrogen atmosphere at 70°C for 16 hours according to the polymerization recipe in the table below. [Table 16]

[0252] 10-3. Removal of template (step 13) The template was removed from the molecularly imprinted polymer.

[0253] The PBS solution containing the complex of molecularly imprinted polymer particles MIP-NGs and template HSA was subjected to ultrafiltration (25°C, 7500 g, 20 min, 3 times) using Amicon Ultra-4 (10 kDa) dialysis tubing to replace the solvent with pure water. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to the complex to a concentration of 20 mM and reacted overnight at 25°C. This reduced and cleaved the disulfide bond (reversible linking group) between the template HSA and MIP-NGs, exposing the thiol group.

[0254] The eluate was then sieved using Sephadex G-100, a size-exclusion chromatography column, and ultrafiltration (25°C, 7500 g, 20 min, 3 times) using Amicon Ultra-4 10 kDa dialysis tubing to remove TCEP and exchange the solvent with 140 mM NaCl, 10 mM Tris-HCl buffer, pH 7.4. The template HSA was then removed by anion exchange chromatography. This yielded molecularly imprinted polymer particles, MIP-NGs.

[0255] 10-4. Fixing particles to a substrate The resulting molecularly imprinted polymer particles, MIP-NGs, were immobilized on a substrate as follows.

[0256] After washing the SPR gold substrate with pure water and ethanol, it was immersed in a 1 mM ethanol solution of 11-mercapto-nectanoic acid and incubated overnight at 25°C. After the reaction, the substrate was washed with ethanol and pure water and dried with nitrogen gas. A PBS solution of 0.1 M N-hydroxysuccinimide (NHS) and 0.4 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was injected into the substrate using a Biacore3000 and incubated for 10 minutes at 25°C. The resulting substrate (with the surface modified with carboxylic acid active ester groups as binding groups) was then injected with a 400 μg / mL MIP-NGs solution and incubated for 10 minutes at 25°C to obtain a substrate with immobilized MIP-NGs.

[0257] 10-5.Synthesis of functional molecules According to the scheme below, a functional molecule (compound (3) below) having a functional group including a group for binding an interactive group with a biological substance and a signal substance binding group, which will be used in item 10-6 described later, was synthesized.

[0258] [ka]

[0259] Tris(2-aminoethyl)amine (1800 μL, 12 mmol) was dissolved in dioxane (4 mL) and stirred under ice cooling. To this was slowly added dropwise 440 mg (2.0 mmol) of BocO in dioxane (10 mL), followed by stirring overnight (0°C → room temperature). After the reaction, the solvent was removed under reduced pressure, and the residue was dissolved in Milli-Q water and extracted with CHCl (15 mL) (×4). The organic layer was dried over NaSO, and the solvent was removed under reduced pressure using a rotary evaporator. The mixture was then dried in vacuo to obtain the target compound (1) as a clear oil. (Yield: 364 mg, 74%) 1 H-NMR (300 MHz, CDCl3) δ = 5.29 (br, 1H, carbamide), 3.18 (t, 2H, -CH2-NH-Boc), 2.75 (t, 4H, H2N-CH2-), 2.53 (m, 6H, -CH2-), 1.45 (s, 9H, Boc)

[0260] 323 mg (1.5 mmol) of (3-pyridyldithio)propionic acid and 330 mg (1.6 mmol) of DCC were dissolved in CHCl (10 mL) and stirred under ice cooling. 130 mg (0.53 mmol) of compound (1) was added and stirred for an additional 1 hour. After the reaction, the precipitate was removed by filtration, and the solvent was evaporated under reduced pressure using a rotary evaporator. The residue was purified by silica gel chromatography (EA / Hx = 45 / 55 → 100 / 0, EA / MeOH = 99 / 1 → 80 / 20) to obtain compound (2). (Yield: 230 mg, 68%) 1H-NMR (300 MHz, CDCl3) δ= 8.44 (d, 2H, pyridyl), 7.70-7.60 (m, 4H, pyridyl), 7.18 (br, 2H, amide), 7.12-7.07 (m, 2H, pyridyl), 5.01 (br, 1H, carbamide), 3.34-3.29 (m, 4H, H2N-CH2-), 3.14-3.07 (m, 6H, -CH2-NH-Boc, -CH2-), 2.68 (t, 4H, -CH2-), 2.61-2.51 (m, 6H, -CH2-), 1.43 (s, 9H, Boc)

[0261] 230 mg (0.36 mmol) of compound (2) was dissolved in 5 mL of CHCl, and 0.5 mL of 4 N HCl in dioxane was added and stirred under ice cooling. After stirring overnight (from 0 °C to room temperature), an excess amount of diethyl ether was added, and the precipitate was separated by decantation. The resulting solid was washed with diethyl ether and dried in vacuo to obtain the target product (compound (3)). (Yield: 255 mg, quant.) 1 H-NMR (300 MHz, D2O) δ= 8.51 (d, 2H, pyridyl), 8.14(t, 2H, pyridyl), 8.01 (d, 2H, pyridyl), 7.56 (m, 2H, pyridyl), 3.67-3.3.56 (m, 6H, HN-CH2-), 3.43 (t, 6H, -CH2-N), 3.09 (t, 4H, -CH2-), 2.74 (t, 4H, -CH2-)

[0262] 10-6. Introduction of functional groups (step 14) The functional molecules were introduced into the binding groups of the molecular imprint recesses via disulfide bonds (reversible linking groups) as follows.

[0263] The substrate on which the molecularly imprinted polymer particles MIP-NGs obtained in 10-4 above were fixed was immersed in a 900 μg / mL PBS solution of the functional molecule (compound (3)) obtained in 10-5 above, and the reaction was allowed to proceed at room temperature for 2 hours, thereby converting the pyridyl disulfide group of the functional molecule and the thiol group of the molecularly imprinted polymer into a disulfide bond (reversible linking group).

[0264] 10-7. Introduction of affibodies and fluorescent substances Anti-Her2 affibody molecules (molecules that provide groups that interact with biological substances) and fluorescent substances (signal substances) were introduced onto the substrate obtained in 10-6 above as follows.

[0265] Anti-Her2 Affibody (R) Ten microliters of a 1 mg / mL solution of Molecule (anti-Her2 affibody molecule, Abcam) (10 mM PB, 137 mM NaCl, 0.05 vol% Tween 20 (pH 7.4); PBST) was added to 40 μL of a 20 mM DTT solution (50 mM PB, 137 mM NaCl (pH 7.5)). The mixture was incubated at 25°C for 2 hours, then subjected to ultrafiltration (4°C, 14,000 × g, 15 minutes) three times using an Amicon Ultra-0.5 (MWCO: 10 kDa) and purified with PBST. This yielded a molecule that conferred anti-Her2 affibody.

[0266] A 10 μg / mL PBS solution of anti-Her2 affibody molecules (molecules that provide interactive groups with biological substances) was reacted with the substrate obtained in step 10-6 (room temperature, 2 hours) to introduce anti-Her2 affibody groups (groups for binding interactive groups with biological substances) to the pyridyl disulfide groups (groups for binding interactive groups with biological substances). The remaining pyridyl disulfide groups were then capped by reacting with 100 μM 2-mercaptoethanol solution. The substrate was then reacted with Atto647N-NHS solution (room temperature, 2 hours) to introduce Atto647N (signal group) to the amino groups (signal substance binding groups). This resulted in a sensing substrate with molecularly imprinted recesses using HER2 as a template, and particles bearing functional groups containing anti-HER2 affibody molecules that interact with HER2 and the signal substance ATTO647N were immobilized in the molecularly imprinted recesses.

[0267] 10-8. Analysis of target substances by fluorescence measurement Using the sensing substrate obtained in 10-7 above, an exosome capture test was performed using a fluorescence microscope equipped with an automatic SIC dispenser.

[0268] 10-8-1. Experimental Procedure Exosomes expressed by SK-BR-3 cells were diluted in PBS (10 mM phosphate, 140 mM NaCl, pH 7.4) at 0–1.0 × 10 -13 An exosome solution was prepared by dissolving exosomes at a concentration of M. The prepared exosome solution was added dropwise to the analytical sensor for the detection target obtained in 10-7 above.

[0269] The measurement conditions for the fluorescence microscope were as follows: measurement points were three points on the substrate, filter was Cy5, objective lens was 5x, exposure time was 0.1 seconds, light intensity was 100%, and light source was a mercury lamp.

[0270] 10-8-2.Results The relative fluorescence intensity was calculated, and the relationship between the relative fluorescence intensity and the exosome concentration was examined. The results are shown in Figure 20.

[0271] As shown in Figure 20, it was confirmed that the degree of quenching increased depending on the exosome concentration in the sensing substrate obtained in 10-7 above. In other words, it demonstrated the ability to recognize exosomes expressed by SK-BR-3 cells. In addition, the dissociation constant K d As a result, the dissociation constant K d is 1.0 x 10 -17 (M).

[0272] Another sensing substrate was prepared in the same manner as above, except that an anti-HSA affibody was used instead of the anti-HER2 affibody. The sensing substrate with the anti-HER2 affibody and another sensing substrate with the anti-HSA affibody were subjected to the same assay. The exosomes (5 × 10) expressed by SK-BR-3 cells, a HER2-overexpressing breast cancer cell line, were then analyzed. -17 The change in relative fluorescence intensity when M) was added is shown in FIG.

[0273] As shown in Figure 21, exosomes expressed by SK-BR-3 cells were specifically detected by the exosome-sensing substrate, which was loaded with anti-HER2 affibody and mediated HER2 capture. [Explanation of symbols]

[0274] 1, 1a...Sensing board 10, 10a…Sensing nanoparticles 20...Molecularly imprinted polymer 21...Molecular imprint space R 1 ...interaction group R 2 ...Linking group containing a signal substance binding group 22...Functional group F22...functional monomer F23...Acrylamide F24...Biocompatible monomer 30...Signaling substances 31...Signal group 40...Substrate 90...Biological material (sensing target) 90'...Biomaterial (template)

Claims

1. a molecularly imprinted polymer having a molecularly imprinted space of a biological material; the molecularly imprinted polymer comprises a constituent unit derived from a functional monomer having a functional group including a group that interacts with the biological substance and a signal substance-binding group different from the interactive group, The functional group is present on the surface of the molecular imprinted space.

2. 2. The sensing nanoparticle according to claim 1, wherein the functional group is represented by the following formula (1): 【Chemistry 1】 [R 1 represents a group that interacts with the biological substance, and R 2 represents a linking group containing the signal substance-binding group, and L 1 represents a direct bond or a linking group.

3. 3. The sensing nanoparticle according to claim 1, wherein the interactive group is selected from the group consisting of a substituted or unsubstituted amino group, a substituted or unsubstituted aromatic group, an amidino group, a guanidino group, a carboxyl group, a sulfo group, a boronyl group, and a ligand of the biological substance.

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