Analysis system and analysis method

Abstract

The present invention addresses the problem of providing an analysis system which can specify a change in structure of a biomaterial without using a special device or carrying out a complicated pretreatment. An analysis system which addresses the above problem comprises: a signal generation unit for generating a plurality of signals based on an interaction between a biomaterial and two or more types of luminescence probes; a detection unit for detecting the plurality of signals from the signal generation unit; and an analysis unit for analyzing the plurality of signals detected by the detection unit and specifying a change in structure of the biomaterial, wherein the luminescence probes are each a compound having, in a molecule thereof, a binding part which binds to the biomaterial and a light emitting part which changes in light emission behavior depending on the structure of the biomaterial.

Description

Analysis system and analysis method

[0001] The present invention relates to an analysis system and an analysis method.

[0002] Biological substances such as proteins (e.g., antibodies, antigens, membrane proteins of extracellular vesicles, etc.) can be denatured and their structure changed by heat, stress, environmental changes, etc. If the structure of a biological substance changes, the function of the biological substance can be lost, and if the biological substance is used as a pharmaceutical, it can cause side effects.

[0003] For example, if the higher-order structure of an antibody's antigen-recognition site (variable region) changes, its affinity to the antigen decreases. Also, if the higher-order structure of its constant region changes, non-specific reactions may progress and side effects may occur.

[0004] Surface plasmon resonance (SPR) technology is known as a method for detecting such changes. This SPR technology measures the binding affinity between an antibody and its target, and infers changes in the variable region (Fab region). However, the dissociation constant (Kd) obtained by this technology varies widely. Furthermore, detailed analysis of changes in the higher-order structure of the entire antibody, particularly the constant region (Fc region), requires three-dimensional structural analysis techniques such as X-ray crystallography and cryo-electron microscopy (cryo-EM).

[0005] Furthermore, extracellular vesicles contain a variety of membrane proteins, and even if the binding affinity between a specific membrane protein and an antibody is confirmed, structural changes may occur in other membrane proteins. In particular, when using extracellular vesicles as pharmaceuticals, it is necessary to confirm changes in the entire membrane protein, not just specific membrane proteins. To track structural changes in these membrane proteins as a whole, a large-scale device such as the one described above is required.

[0006] X-ray crystallography and cryo-electron microscopy, which can perform detailed analysis of the above structural changes, are large and expensive instruments. They also require dedicated facilities and specific environmental conditions (such as a location with little vibration and strict temperature and humidity control). Furthermore, sample preparation requires technical techniques such as crystallization and rapid freezing. Therefore, obtaining good data is difficult for anyone other than an experienced technician. Meanwhile, nuclear magnetic resonance spectroscopy (NMR spectroscopy) utilizes the magnetic properties of atomic nuclei within proteins to obtain structural information such as interatomic distances and angles. This method can capture the dynamic properties of proteins and small structural changes. However, this method has limitations on the size of the sample.

[0007] On the other hand, a method has also been proposed for determining the type and amount of a protein using a probe having a cationic polymer capable of non-specifically interacting with the protein and a fluorophore bound to the polymer (for example, Patent Document 1).

[0008] Patent No. 6741259

[0009] However, the cationic polymer used in the method of Patent Document 1 mainly interacts with negatively charged amino acids such as aspartic acid and glutamic acid. Therefore, the cationic polymer cannot be expected to specifically interact with positively charged amino acids such as lysine and arginine, or hydrophobic or hydrophilic amino acids. Furthermore, the method of Patent Document 1 makes it difficult to accurately identify the arrangement of amino acids or minute structural changes in biological materials.

[0010] An object of the present invention is to provide an analytical system and analytical method that can identify structural changes in biological materials without using special equipment or performing complex pretreatments.

[0011] In order to achieve at least one of the above-mentioned objects, the following analysis system and analysis method are provided.

[0012] An analytical system embodying one aspect of the present invention is an analytical system for detecting structural changes in a biological substance, and includes a signal generating unit for generating a plurality of signals based on interactions between the biological substance and two or more types of luminescent probes, a detecting unit for detecting the plurality of signals from the signal generating unit, and an analyzing unit for analyzing the plurality of signals detected by the detecting unit and identifying structural changes in the biological substance, wherein each of the luminescent probes is a compound having, in its molecule, a binding unit that binds to the biological substance and a luminescent unit whose emission behavior changes depending on the structure of the biological substance.

[0013] An analytical method reflecting one aspect of the present invention is an analytical method for detecting a structural change in a biological substance, and includes the steps of: allowing the biological substance to interact with two or more types of luminescent probes; generating a plurality of signals from the two or more types of luminescent probes that have interacted with the biological substance; detecting the plurality of signals; and analyzing the detected plurality of signals to identify a structural change in the biological substance, wherein each of the luminescent probes is a compound having, in its molecule, a binding moiety that binds to the biological substance and a luminescent moiety whose emission behavior changes depending on the structure of the biological substance.

[0014] According to the analysis system and analysis method of one embodiment of the present invention, it is possible to identify structural changes in biological materials without using special equipment or performing complex pretreatments.

[0015] FIGS. 1A and 1B are schematic diagrams illustrating an analysis system and analysis method of the present invention. FIG. 2 is a flowchart of an analysis method according to one embodiment of the present invention. FIG. 3 is a schematic diagram of an analysis system according to one embodiment of the present invention. FIG. 4 shows the results of discriminant analysis performed on multiple samples in Example 1. FIG. 5 shows the results of discriminant analysis performed on multiple samples in Example 2. FIG. 6 shows the results of a comparison between blocking performance predicted by PLS regression and actual blocking performance for the samples in Example 2. FIG. 7 shows the results of discriminant analysis performed on multiple samples in Example 3. FIG. 8 shows the results of discriminant analysis performed on multiple samples in Example 4.

[0016] The present invention will be described in detail below based on embodiments, but the present invention is not limited to these embodiments.

[0017] The analytical system and analytical method using the same of this embodiment detect structural changes in biological materials. Conventional analytical systems and analytical methods for biological materials have evaluated biological materials by directly or indirectly binding a luminescent probe to a specific structure of the biological material (e.g., the amino acid sequence of a protein) to identify the position and amount of the specific structure. However, this method makes it difficult to evaluate the structure of parts to which the luminescent probe is not bound. In other words, conventional methods using luminescent probes have had difficulty in adequately detecting subtle structural changes in biological materials. In contrast, the analytical system and analytical method of this embodiment generate signals by allowing two or more types of luminescent probes to interact with various parts of the biological material. Furthermore, in this embodiment, these multiple signals are used to obtain not only information about the structure of the luminescent probes bound thereto, but also information about structural changes in the entire biological material.

[0018] The analysis system and analysis method of this embodiment will be described using an example in which one biological substance interacts with 88 types of luminescent probes to obtain multiple signals. FIG. 1A shows an image of 384 wells (88 types x N=4), each containing one of the 88 types of luminescent probes, each containing the same biological substance, after which the luminescent probes were lit. Note that in FIG. 1A, in order to make the color change easier to understand, not only an overall view (Merge) but also a view broken down into red, blue, and green is shown. Similarly, FIG. 1B shows an image of the luminescent probes lit up, except that the biological substance used was heated. FIG. 1B also shows not only an overall view (Merge) but also a view broken down into red, blue, and green. Comparing FIG. 1A and FIG. 1B reveals that the brightness and color of the light emitted by some of the luminescent probes (each well) change. In other words, the luminescent state of a luminescent probe bound to a biological material changes subtly depending on structural changes in the biological material, etc. In this embodiment, multiple signals emitted by such multiple luminescent probes are comprehensively analyzed, making it possible to identify subtle structural changes in the biological material. Note that, as used herein, "structural changes in a biological material" include changes in the higher-order structure of proteins and changes in membrane proteins of exosomes, etc. Furthermore, biological materials that can be analyzed using the analysis system and analysis method of this embodiment may include proteins, sugar chains, lipids, nucleic acids, peptides, amino acids, extracellular vesicles (including exosomes), substances containing cell membrane components, etc. Below, the analysis method will be described first, followed by the analysis system.

[0019] 1. Analysis Method The flow of the analysis method of this embodiment is shown in Figure 2. This analysis method includes the steps of: a step (interaction step) S101 of allowing a biological substance to interact with two or more types of luminescent probes; a step (signal generation step) S102 of generating a plurality of signals from the two or more types of luminescent probes that have interacted with the biological substance; a step (signal detection step) S103 of detecting the plurality of signals; and a step (analysis step) S104 of analyzing the detected plurality of signals and identifying a structural change in the biological substance. Note that the analysis method of this embodiment may further include steps other than these steps, as long as the purpose and effects of this embodiment are not impaired. For example, a step of predicting the performance of the biological substance by referring to a trained model may be further performed.

[0020] (Interaction step S101) In the interaction step S101, the biological substance is allowed to interact with two or more types of luminescent probes. The number of luminescent probes allowed to interact with the biological substance may be two or more types, and is selected appropriately depending on the type of biological substance to be analyzed, etc. If the number of types of luminescent probes is 50 or more types, more preferably 100 or more types, very detailed analysis can be performed.

[0021] Furthermore, in this embodiment, two or more types of luminescent probes may be allowed to interact with one molecule of a biological substance at a time, but it is preferable to allow one type of luminescent probe to interact with one molecule of a biological substance at a time. When the luminescent probes are allowed to interact with one type at a time, it becomes possible to individually detect signals from each luminescent probe in the signal detection step described below. This makes it easier to identify the extent to which the signal from each luminescent probe has changed when comparing with a reference or other results.

[0022] The method for causing an interaction between a biological substance and a luminescent probe is not particularly limited, and the biological substance and the luminescent probe may simply be mixed. A buffer solution or other compound may also be used as needed. A specific example of a method for causing an interaction between a biological substance and a luminescent probe is a method in which a luminescent probe is placed in each well of a microwell plate, and a liquid containing the biological substance is poured into the well to cause the two to interact. Alternatively, the biological substance may be placed first, and then the liquid containing the luminescent probe may be poured in later. Another example is a method in which a luminescent probe is immobilized on a microplate or microarray, and then the biological substance is introduced onto the microplate or microarray by a conventional method. In this case, the biological substance may also be immobilized first, and then the luminescent probe may be introduced later.

[0023] Here, each luminescent probe may be a compound having a binding moiety that binds to a biological substance and a luminescent moiety whose luminescence behavior changes depending on the structure of the biological substance. The luminescent probe may be a substance that specifically binds to a specific structure of the biological substance, but is preferably a substance that non-specifically binds to the biological substance. In addition, the dissociation constant (Kd) between at least one of the two or more luminescent probes and the biological substance is 10 ６ It is preferable that the molecular weight is M or more. By using such a luminescent probe, the luminescent probe can easily bind to multiple structures of a biological substance. Therefore, it becomes easier to obtain more signals in the signal detection step described below. The specific structure of the luminescent probe will be described in detail later.

[0024] (Signal Generation Step S102) In the signal generation step S102, multiple signals are generated from two or more types of luminescent probes that have interacted with the biological material. The type of signal is not particularly limited as long as it is useful for analysis in the analysis step described below, but as described above, the luminescent probe has a light-emitting moiety whose emission behavior changes depending on the structure of the biological material. Therefore, in this embodiment, a method is preferred in which excitation light is irradiated onto each luminescent probe that has interacted with the biological material, causing each luminescent probe to generate light (signal). In this case, the wavelength of the irradiated excitation light and the irradiation method of the excitation light are appropriately selected depending on the signal detection method in the signal detection step, the type of signal used in the analysis, etc. Furthermore, light of a single wavelength may be irradiated only once, or may be irradiated multiple times as the excitation light. Furthermore, light of different wavelengths may be irradiated all at once, or multiple times as the excitation light.

[0025] (Signal detection step S103) In the signal detection step S103, two or more types of signals emitted from the two or more types of luminescent probes are detected. The signal detection method is not particularly limited and is selected appropriately depending on the type of signal. For example, when detecting light emitted by each luminescent probe, the luminance or wavelength may be detected as a signal. Furthermore, changes over time in the spectral distribution or chromaticity of the light emitted by the luminescent probe may be detected.

[0026] (Analysis step S104) In the analysis step S104, the multiple signals (analysis data) acquired in the signal detection step S103 are analyzed to identify structural changes in the biological material. For example, the structural changes in the biological material may be identified by comparing the analytical data with standard data (reference) acquired in advance by performing the interaction step S101, signal generation step S102, and signal detection step S103 for a standard biological material. Note that the standard data may be data relating to a biological material of known structure, and may be data relating to either before or after the structural change. In this step, the analytical data may be compared with multiple standard data.

[0027] When performing analysis using this method, the standard data and the analytical data may be simply compared, but for example, the comparison result between the standard data and the analytical data may be converted into a distance matrix and analyzed using a heat map (unweighted), or the distance matrix may be subjected to principal component analysis (also known as PCA, weighting that emphasizes anisotropy), or analysis by DL (weighting that emphasizes isotropy), etc.

[0028] On the other hand, the analysis data may be analyzed with reference to a trained model to identify structural changes in the biological material. When the analysis is performed with reference to a trained model, not only the structural changes may be detected but also the properties and performance of the biological material may be determined (predicted) from accumulated data, etc.

[0029] (Other Steps) As described above, the analysis method of the present embodiment may further include other steps in addition to the above steps. For example, the analysis method may further include a machine learning step of performing machine learning on arbitrary data to generate a trained model.

[0030] In the machine learning process, the above-described interaction process S101, signal generation process S102, signal detection process S103, etc. are performed on a large number of machine learning samples (biological substances). Then, multiple prediction models are constructed from the large number of signals acquired in the signal detection process S103. Then, by combining the results of the multiple prediction models, a trained model capable of predicting information about the biological substances (e.g., the presence or absence of structural changes, performance, properties, etc.) is created.

[0031] Machine learning can be either supervised learning or unsupervised learning. Supervised learning is a learning method that learns the "relationship between input and output" from training data with correct answer labels. Unsupervised learning is a learning method that learns the "structure of data groups" from training data without correct answer labels.

[0032] Machine learning may be reinforcement learning, deep learning, or deep reinforcement learning. Reinforcement learning is a learning method that learns an "optimal sequence of actions" through trial and error. Deep learning is a learning method that learns features contained in data in a stepwise and deeper manner (at a deeper level) from a large amount of data. Deep reinforcement learning is a learning method that combines reinforcement learning and deep learning.

[0033] Common analytical methods (algorithms) can be applied to machine learning, such as a predictive model constructed by an analytical method selected from linear regression (multiple regression analysis, partial least squares (PLS) regression, LASSO regression, Ridge regression, principal component regression (PCR)), random forest, decision tree, support vector machine (SVM), support vector regression (SVR), neural network, discriminant analysis, and the like.

[0034] (Regarding Luminescent Probes) The luminescent probes used in the analysis method of this embodiment may be any compound that has a binding moiety in the molecule that binds to a biological substance and a luminescent moiety whose luminescence behavior changes depending on the structure of the biological substance. The luminescent probe may have only the binding moiety and the luminescent moiety. Alternatively, the luminescent probe may further have a structure that does not contribute to binding to a biological substance or luminescence. However, it is preferable that the binding moiety and the luminescent moiety are located on the tip side of the luminescent probe, i.e., on the side that is more likely to come into contact with the biological substance.

[0035] Here, the binding moiety is not particularly limited as long as it has a structure that can bind to a biological substance. However, from the viewpoint of binding to a biological substance, it preferably contains a nucleic acid structure. When the luminescent probe contains a nucleic acid structure, it can be physically or chemically bound to a protein, or to a peptide or amino acid that constitutes a protein. In this specification, the term "nucleic acid structure" includes not only structures derived from DNA or RNA, but also structures derived from phosphorothioate oligodeoxynucleotides, 2'-O-(2-methoxy)ethyl-modified nucleic acids, siRNA, crosslinked nucleic acids, peptide nucleic acids, aTNA, SNA, GNA, LNA, and morpholino antisense nucleic acids.

[0036] Furthermore, the type of luminescence emitted by the light-emitting moiety is not particularly limited as long as its luminescence behavior changes upon interaction with a biological substance. The light-emitting moiety may emit only one type of light in response to a single excitation light. However, it is preferable that the light-emitting moiety exhibit two or more types of luminescence selected from the group consisting of fluorescence, phosphorescence, excimer luminescence, exciplex luminescence, thermally activated delayed fluorescence, excited-state intramolecular proton luminescence, triplet triplet annihilation luminescence, twisted intramolecular charge transfer luminescence, and aggregation-induced luminescence in response to a single excitation light. When the light-emitting moiety exhibits such two or more types of luminescence, more information can be obtained in the signal detection step described above. For example, when a luminescent probe exhibiting two or more types of luminescence is bound to a biological substance, the structure and electronic state of the luminescent moiety change, resulting in complex luminescence behavior different from that of the luminescent probe alone. For example, when a luminescent probe exhibiting three different types of luminescence (fluorescence, phosphorescence, and excimer luminescence) in response to a single excitation light is allowed to interact with a biological substance, the two types of luminescence interact with each other. The processes that produce fluorescence, phosphorescence, and excimer luminescence change, and the wavelength and lifetime of each light change. Therefore, depending on the structure of the biological material, a complex and large amount of data can be obtained that combines these lights. This complex and large amount of data makes it possible to understand the structure and state of the biological material in great detail.

[0037] Specific examples of luminescent probes include a structure having a binding moiety consisting of a nucleic acid structure and at least two chromophores or luminophores (luminescent moieties) bound to the backbone of the nucleic acid structure. More specifically, the present invention includes molecules having a backbone having one or more structural units containing a pentose- or hexose-derived sugar structure and a phosphate ester bond bound to the sugar structure, and one or more chromophores or luminophores bound to the sugar structure.

[0038] In this molecule, it is preferable that 50% or more of the sugar structures to which the luminescent moiety is bound are in the β form. Generally, DNA has a structure in which bases are bound to a backbone (deoxyribose) containing phosphate ester bonds and structures derived from deoxyribose. Furthermore, in natural DNA, all deoxyribose in the backbone is in the β form. Therefore, if 50% or more of the sugar structures to which the luminescent moiety is bound are in the β form, it can be said that the structure is highly similar to a biological substance. When mixed with a biological substance, such a luminescent probe is less likely to cause steric hindrance, and can penetrate into the interior or conform to the shape of the biological substance. Therefore, more detailed analysis of the biological substance is possible. Whether the sugar structure is in the β form or the α form can be confirmed by NMR analysis, X-ray crystal structure analysis, or the like. Luminescent probes having this structure are described in detail below.

[0039] The main chain of the binding portion of the luminescent probe may have one or more structural units containing a sugar structure derived from a pentose or hexose and a phosphate ester bond bound to the sugar structure. The main chain may contain only one of the structural units, or may contain multiple structural units. That is, the main chain may have one of the sugar structures and one of the phosphate ester bonds bound to the sugar structure, or may have a structure containing the sugar structure and phosphate ester bonds alternately. Usually, both ends of the main chain of the luminescent probe are sugar structures, so that the number of sugar structures is one more than the number of phosphate ester bonds. When the main chain contains multiple structural units, the multiple structural units may be the same or different from each other.

[0040] The number of the structural units contained in the main chain of the binding moiety is selected appropriately depending on the type of biological substance, etc., but is preferably 2 or more and 6 or less. As the amount of the structural units increases, the binding moiety becomes more likely to specifically bind to the biological substance. However, it is preferable that the luminescent probe has appropriate (not excessive) specificity for the biological substance, and the number of the structural units is preferably 6 or less. By using multiple probes that are not excessively specific or have excessive binding strength, it becomes easier to detect various structural changes compared to probes that react only to a specific three-dimensional structure.

[0041] The main chain of the bond may partially contain a structure other than the structural unit containing the pentose- or hexose-derived sugar structure and the phosphate ester bond, as long as the purpose and effect of this embodiment are not impaired. The structures at both ends of the main chain are not particularly limited and may be various structures, such as an OH group or an alkoxy group.

[0042] Examples of the pentose include ribose, deoxyribose, and xylose. Specific examples of hexose include allose, glucose, mannose, etc. Among these, a sugar structure derived from ribose or deoxyribose is particularly preferred, since the main chain of the luminescent probe has a structure similar to that of DNA or RNA, which facilitates interaction with biological substances.

[0043] When the structural unit contains a structure derived from ribose or deoxyribose, the phosphate ester bond is preferably bonded to the 3-carbon and 5-carbon of the ribose or deoxyribose. Furthermore, the luminescent moiety described below is preferably bonded to the 1-position of the ribose or deoxyribose. In other words, the luminescent probe preferably contains a structure represented by the following general formula (1a) or (1b): In the general formulae (1a) and (1b), Y represents a light-emitting moiety described below.

[0044] On the other hand, the main chain of the linking portion is not limited to a structural unit containing a sugar structure derived from pentose or hexose and a phosphate ester bond. Representative examples of other structural units include peptide nucleic acid structural units such as those shown below. Peptide nucleic acids have no charge and are free from electrostatic repulsion, allowing them to form stronger complexes with biological materials. Furthermore, they are resistant to enzymes such as nucleases and proteases, making them suitable for use in cells. Furthermore, they can be synthesized on a relatively large scale. However, due to their nonionic structure, they may aggregate in water, reducing their solubility. Therefore, when the binding moiety of a luminescent probe has a structure derived from peptide nucleic acids, it is preferable to appropriately select the type of base linked to the signal generating moiety and the solvent.

[0045] On the other hand, the light-emitting portion (chromophore or luminophore) may have a structure that emits a predetermined type of light by itself in response to a single excitation light, or that emits a predetermined light by the action of multiple chromophores or luminophores. In this specification, the term "chromophore" refers to a structure that absorbs light with a wavelength of 300 nm or more, and the term "luminophore" refers to a structure that absorbs light with a wavelength of 300 nm or more and emits light.

[0046] The number of chromophores or luminophores possessed by each luminescent probe may be only one, as long as the luminescent probe is capable of exhibiting multiple types of luminescence. However, from the viewpoint that the luminescent probe is likely to exhibit multiple types of luminescence, two or more are preferred, and three to six are even more preferred. When the luminescent probe has multiple chromophores or luminophores, the types may be only one type, or two or more types. Usually, one chromophore or luminophore is bound to one sugar structure at the binding moiety. Therefore, when the luminescent probe has two or more chromophores or luminophores, it is preferable that the sugar structures in the main chain of the binding moiety are also two or more. In other words, it is preferable that the number of chromophores or luminophores in the luminescent probe is the same as or less than the number of sugar structures (or peptide structures) in the main chain of the above-mentioned signal generating moiety.

[0047] In addition, when the number of chromophores or luminophores in the luminescent probe is less than the number of sugar structures (or peptide structures) in the binding portion, some sugar structures will not have chromophores or luminophores bound to them. The sugar structures to which no chromophores or luminophores are bound may not have other atomic groups bound to them, and may have natural nucleic acid bases bound to them. In this specification, natural nucleic acid bases refer to adenine, guanine, cytosine, thymine, and uracil. However, the total number of natural nucleic acid bases bound to the main chain is preferably 50% or less, more preferably 25% or less, of the total number of structural units constituting the binding portion of the luminescent probe. When the number of natural nucleic acid bases is 50% or less, the association between luminescent probes is suppressed, and the binding between biological substances and luminescent probes is more likely to be dominant.

[0048] Here, examples of chromophores or luminophores that emit fluorescence include structures derived from fluorescein, rhodamine, boron dipyrromethene, etc. Examples of chromophores or luminophores that emit phosphorescence include structures derived from iridium complexes, platinum complexes, etc. Examples of chromophores or luminophores that emit excimer emission include structures derived from pyrene, anthracene, perylene, etc. Examples of chromophores or luminophores that emit exciplex emission include structures derived from pyrene-dimethylaniline, etc. Examples of chromophores or luminophores that emit thermally activated delayed fluorescence include structures derived from 4CzIPN, DABNA, etc. Examples of chromophores or luminophores that emit excited-state intramolecular proton emission include structures derived from hydroxyphenylbenzoxazole, etc. Examples of chromophores or luminophores that emit triplet triplet annihilation emission include structures derived from 9,10-diphenylanthracene, rubrene, etc. Examples of chromophores or lumophores that emit twisted intramolecular charge transfer luminescence include structures derived from diaminoanthracene, diaminonaphthalene, etc. Examples of chromophores or lumophores that emit aggregate organic luminescence include structures derived from tetraphenylethene, hexaphenylsilole, etc.

[0049] Furthermore, luminescent compounds used as luminescent materials or hosts, electron transport materials, hole transport materials, or luminescent materials in organic electroluminescence (EL) can also be suitably used as the chromophore or luminophore material. Specific examples of such luminescent compounds include those described in "State-of-the-art Organic EL" (CMC Publishing), Organic EL Material Technology (CMC Publishing), All About Organic EL (Nippon Jitsugyo Publishing), and Diverse Dye Materials Pioneering the Future (Kagaku Dojinsha). Furthermore, the luminescent probe may further include a structure that exerts various functions as a site for controlling the interaction between the binding moiety and the biological substance.

[0050] In this embodiment, it is preferable that the luminescent probe contains, as a chromophore or luminophore, at least one structure selected from a structure that emits fluorescence, a structure that emits excimer emission, and a structure that emits exciplex emission. It is particularly preferable that the luminescent probe contains at least a structure that emits fluorescence. When the luminescent probe emits fluorescence, this has the advantage of being easy to analyze using various measuring devices.

[0051] Furthermore, the luminescent probe preferably exhibits multiple types of luminescence when irradiated with light of a wavelength of 300 to 400 nm. If the luminescent probe exhibits multiple types of luminescence when irradiated with light of the wavelength, a special light source is not required when analyzing biological materials, and the biological materials are less likely to be damaged.

[0052] However, when an LED or organic EL element is used as the excitation light source, excitation in the visible light range is advantageous. Therefore, when using such a light source, the absorption wavelength of the luminescent probe is preferably 400 to 700 nm.

[0053] The molecular weight of the luminescent probe is appropriately selected depending on the type of binding moiety and luminescent moiety possessed by the luminescent probe, and is usually preferably from 500 to 10,000, more preferably from 500 to 4,000. When the molecular weight of the luminescent probe is 10,000 or less, the specificity for a biological substance is appropriately low, making it possible to cause the luminescent probe to react nonspecifically with multiple sites on the biological substance.

[0054] The method for producing the luminescent probe is selected appropriately depending on the structure of the binding site in the luminescent probe. For example, a luminescent probe having the above-mentioned sugar structure can be produced by the following method. A monomer is prepared in which the above-mentioned chromophore or luminophore and a phosphate ester are bound to a pentose or hexose. The monomer can be synthesized by polymerizing the desired sequence using the phosphoramidite method with a DNA / RNA synthesizer or the like. According to this method, multiple types of monomers with different types of chromophores or luminophores are prepared, and the desired number of monomers can be bound by changing the sequence order. In other words, a wide variety of luminescent probes can be synthesized from multiple types of monomers with different types of chromophores or luminophores. By changing the type of monomer used and the number of monomers bound, a very large number of luminescent probes can be synthesized.

[0055] 2. Analysis System A schematic diagram showing the configuration of an analysis system for carrying out the above-described analysis method is shown in Figure 3. However, the configuration of the analysis system is not limited to this configuration. The analysis system 100 shown in Figure 3 includes a signal generation unit 11 for generating multiple signals based on the interaction between a biological material and two or more types of luminescent probes, a detection unit 12 for detecting the multiple signals from the signal generation unit 11, and an analysis unit 13 for analyzing the multiple signals detected by the detection unit 12 and identifying structural changes in the biological material. The analysis system 100 may include other components depending on its application. Each component will be described below.

[0056] (Signal Generation Unit) The signal generation unit 11 is configured to generate a signal based on the interaction between a biological substance and two or more types of luminescent probes. The structure of the signal generation unit 11 is selected appropriately depending on the type of signal to be generated. The signal generation unit 11 of this embodiment irradiates light onto the luminescent probes, causing them to emit light. The signal generation unit 11 has a light source 111, a storage unit 112 for storing the luminescent probes and biological substance, and an optical system 114 for guiding the light from the light source 111 to the storage unit 112 (or to the luminescent probes stored in it).

[0057] The light source 111 is not particularly limited as long as it is a means capable of irradiating light of a desired wavelength for a desired period of time onto a luminescent probe that has interacted with a biological substance. Examples of preferred light sources include picosecond diode lasers, tunable lasers, supercontinuum light sources, and LED light sources. These light sources 111 can irradiate light of a predetermined wavelength onto the luminescent probe for only a short period of time. In consideration of the signal-to-noise ratio (SN) in the detection unit 12, it is preferable to select a light source that can be quenched before the luminescent probe emits light.

[0058] The storage section 112 is not particularly limited as long as it has a structure capable of storing the above-mentioned luminescent probes and biological substances. Examples of the storage section 112 include a microwell plate, a microplate, a microarray, etc. Note that these storage sections 112 may be pre-stored with luminescent probes. Alternatively, they may be pre-stored with biological substances.

[0059] The optical system 114 is not particularly limited as long as it is capable of guiding light from the light source 111 to the storage section 112 that stores the luminescent probes and biological substances, and is capable of guiding light emitted by the luminescent probes to the detection section 12. The optical system 114 may have, for example, an excitation light filter (not shown) for cutting out light of unnecessary wavelengths emitted from the light source 111. The optical system 114 may also have a dichroic mirror (not shown) that reflects light from the light source 111 to the storage section 112 while transmitting light emitted by the luminescent probes. The optical system 114 may also have an optical filter or the like that cuts out light of unnecessary wavelengths from the light that has passed through the dichroic mirror.

[0060] (Detection Unit) The detection unit 12 is not particularly limited as long as it is a means capable of acquiring each of the multiple signals (here, multiple lights) emitted by the multiple luminescent probes. It is appropriately selected according to the type of signal to be acquired. When the signal is light, as in this embodiment, it may be a known camera or the like. It may also be, for example, a CCD camera, CMOS camera, or the like that captures images intermittently or continuously.

[0061] (Analysis Unit) The analysis unit 13 may be any means capable of analyzing a plurality of signals acquired by the above-described detection unit 12. For example, the analysis unit 13 may read separately acquired standard data and compare the reference standard data with a plurality of signals (analysis data) acquired by the detection unit 12 to analyze structural changes in biological materials. The analysis unit 13 may also read a trained model from an external storage device (not shown) or internal storage means (not shown) and perform a comparison operation between the trained model and the analysis data.

[0062] A general-purpose computer equipped with storage means such as a hard disk drive (HDD), a solid-state drive (SSD), or a read-only memory (ROM) for storing programs, data, etc., and a central processing unit (CPU) for executing programs, performing calculations, etc., can be used as the analysis unit 13. The computer may further have input means such as a keyboard and a mouse, and output means such as a monitor and a printer.

[0063] 1. Preparation of Luminescent Probes Eighty-eight types of luminescent probes were prepared, as represented by the chemical formulas below. The 88 types of luminescent probes have a structure in which 13 types of groups (luminophores or chromophores, or other groups) shown on the right side are bonded to the R position of the structure on the left side of the following chemical formula. The 88 types are composed of combinations of groups represented by R.

[0064] 2. Example 1 2-1. Preparation of Biological Materials Bovine serum albumin (BSA, manufactured by Nacalai Tesque, product code: 01281-84) was mixed with 1x PBS to prepare a BSA solution with a BSA concentration of 1% by mass. Then, the following four types of samples, totaling 25 samples, were prepared from this BSA solution: (i) BSA solution stored in a refrigerator for 14 hours: 10 samples (ii) BSA solution heated at 50°C for 10 minutes: 5 samples (iii) BSA solution heated at 70°C for 10 minutes: 5 samples (iv) BSA solution heated at 90°C for 10 minutes: 5 samples

[0065] 2-2. Interaction Step A 96-well microwell plate with wells having an opening diameter of 7 mm arranged in 12 columns and 8 rows at 9 mm intervals was prepared for the number of samples (25). Luminescent probes 1 to 88 were individually placed in the wells of each 96-well microwell plate at 10 μl using an automatic dispenser (NichiMart CUBE, manufactured by NICHIRYO). Similarly, luminescent probes 1 to 88 were individually placed in the wells of all the microwell plates. 10 μl of sample was placed in each well containing the luminescent probe of each microwell plate, and the luminescent probe and BSA in the sample were allowed to interact. One microwell plate was used for one sample.

[0066] 2-3. Signal generation step and signal detection step The microwell plate was irradiated with excitation light (wavelength 365 nm) (signal generation step). The fluorescence spectrum at this time was photographed with a camera, and RGB information for each microwell plate was obtained (signal detection step). The same procedure was performed for all microwell plates.

[0067] 2-4. Analysis Step Principal component analysis was performed on the RGB data of each microwell plate obtained in the signal detection step. As a result, as shown in Figure 4, in the two-dimensional space plotting principal components 1 and 2, it was possible to separate (i) the BSA solution stored in a refrigerator, (ii) the BSA solution heated at 50°C for 10 minutes, (iii) the BSA solution heated at 70°C for 10 minutes, and (iv) the BSA solution heated at 90°C for 10 minutes.

[0068] 3. Example 2 3-1. Preparation of Biological Materials Seven types of bovine serum albumin were prepared, and each was mixed with 1x PBS to prepare seven types of BSA solutions (undenatured) so that the BSA concentration was 1% by mass. Furthermore, a portion of each BSA solution was heated at 90°C for 1 minute to prepare seven types of thermally denatured BSA solutions. One of the undenatured BSA solutions served as a reference sample. Six undenatured samples were combined 1:1 (15 samples in total), seven denatured samples were combined 1:1 (21 samples in total), and six undenatured samples were combined 1:1 with seven denatured samples (42 samples in total). Six samples each comprised one undenatured sample and seven denatured samples were also prepared, for a total of 91 samples.

[0069] 3-2. Interaction step, signal generation step, signal detection step, and analysis step As in Example 1 above, each sample (91 types) was mixed with 88 types of luminescent probes on a microwell plate. Then, excitation light was irradiated onto each microwell plate, and RGB information was obtained. When the obtained RGB information was subjected to principal component analysis, it was possible to separate each type, as shown in Figure 5.

[0070] 3-3. Verification of the Blocking Effect of Samples 150 μl of each of the 91 samples was placed in six wells (N=6) of a white microwell plate. A reference sample (unmodified) was also placed in the microwell plate. A PCR seal was attached to the microwell plate and stored at 4°C. The plate was then left to stand at room temperature for 20 minutes, and the sample was aspirated from the wells using a suction pump. Each well was then washed three times with 150 μl of 1x PBS. After washing, 50 μl of a solution containing HRP-labeled streptavidin was added to each well. The wells were then kept at 37°C for 2.5 hours. The plate was then returned to room temperature, and the HRP-labeled streptavidin was aspirated using a suction pump. Each well was then washed three times with 150 μl of 1x PBS. Furthermore, 100 μl of SuperSignal (Thermo Fisher Scientific) A+B adjusted product was added to each well as a substrate and stirred for 20 seconds using a stirrer. The luminescence intensity of each well was then measured using a microplate reader. Analysis using the plate reader was performed using the average value of N=4, excluding the maximum and minimum values ​​out of N=6. Evaluation was also performed using the relative value (signal ratio) to the reference sample. The smaller the signal ratio, the higher the blocking performance.

[0071] Meanwhile, PLS regression was performed using the RGB information obtained from the above samples as explanatory variables and each signal ratio as correct data. The results of this PLS regression (predicted blocking performance) and the actual blocking performance (correct answer) are shown in Figure 6. As shown in Figure 6, there was a good correlation between these results, suggesting that blocking function can be predicted from RGB information.

[0072] 4. Example 3 4-1. Preparation of Biological Materials Anti-CD9 antibody (MBL MEX001-3 clone: ​​A-100) was mixed with 1x PBS to adjust the anti-CD9 antibody concentration to 10 μg / ml, thereby obtaining an antibody solution. Seven types of samples were prepared from this antibody solution using the following procedure. Four samples of the same type were prepared: (i) Stored at -80°C for 3 days (ii) Stored at -80°C for 10 days (iii) Stored at -80°C for 20 days (iv) Heated at 50°C for 10 minutes (v) Heated at 70°C for 10 minutes (vi) Heated at 90°C for 10 minutes

[0073] 4-2. Interaction step, signal generation step, signal detection step, and analysis step As in Example 1 above, each sample (7 types x 4 samples) was mixed with 88 types of luminescent probes on a microwell plate. Then, excitation light was irradiated onto each microwell plate to obtain RGB information. When the obtained RGB information was subjected to principal component analysis, it was possible to separate each type, as shown in Figure 7.

[0074] 5. Example 4 5-1. Preparation of Biological Substances Exosomes (LNCaP) were mixed with 1x PBS and the exosome concentration was adjusted to 1 μg / ml to obtain an exosome solution. Eight types of four samples were prepared from the exosome solution using the following procedure: (i) Store at -80°C for 3 days; (ii) Store at -80°C for 10 days; (iii) Store at -80°C for 20 days; (iv) Heat at 50°C for 10 minutes; (v) Heat at 70°C for 10 minutes; (vi) Heat at 90°C for 10 minutes; (vii) Repeated freezing and thawing 10 times.

[0075] 5-2. Interaction Step, Signal Generation Step, Signal Detection Step, and Analysis Step As in Example 1 above, each sample (8 types x 4 samples) was mixed with 88 types of luminescent probes on a microwell plate. Then, excitation light was irradiated onto each microwell plate to obtain RGB information. Principal component analysis of the obtained RGB information allowed for separation by type, as shown in Figure 8. More specifically, it was confirmed that exosome solutions stored frozen, exosome solutions in which membrane proteins had undergone thermal denaturation due to heating, and exosomes damaged by repeated freezing and thawing could be classified and distinguished.

[0076] This application claims priority from Japanese Patent Application No. 2024-108879, filed July 5, 2024. The contents of the specification and drawings of that application are incorporated herein by reference in their entirety.

[0077] The above-described analytical system and analytical method make it possible to identify structural changes in biological materials without using special equipment or performing complex pretreatments, and are therefore extremely useful in fields such as medicine.

[0078] REFERENCE SIGNS LIST 11 signal generating unit 12 detection unit 13 analysis unit 111 light source 112 storage unit 114 optical system 100 analysis system

Claims

1. An analytical system for detecting structural changes in a biological substance, comprising: a signal generation unit for generating a plurality of signals based on interactions between the biological substance and two or more types of luminescent probes; a detection unit for detecting the plurality of signals from the signal generation unit; and an analysis unit for analyzing the plurality of signals detected by the detection unit and identifying structural changes in the biological substance, wherein each of the luminescent probes is a compound having a binding part in its molecule that binds to the biological substance and a luminescent part whose emission behavior changes depending on the structure of the biological substance.

2. The analytical system according to claim 1, wherein the binding moiety binds non-specifically to the biological material.

3. The analytical system according to claim 1, wherein the binding moiety comprises a nucleic acid structure.

4. The analysis system according to claim 1, wherein the light-emitting portion exhibits luminescence selected from the group consisting of fluorescence, phosphorescence, excimer luminescence, exciplex luminescence, thermally activated delayed fluorescence, excited-state intramolecular proton luminescence, triplet triplet annihilation luminescence, twisted intramolecular charge transfer luminescence, and aggregation-induced luminescence in response to a single excitation light.

5. The analysis system according to claim 1, wherein the structural change in the biological material is a change in the higher-order structure of a protein.

6. The dissociation constant between at least one of the luminescent probes and the biological substance is 10 ６ The analysis system according to claim 1 , wherein the number of the plurality ...

7. The analysis system according to claim 1, wherein the signal generating unit includes the luminescent probe.

8. The analysis system according to claim 7, wherein the signal generating section includes a container that contains the luminescent probe.

9. The analytical system according to claim 8, wherein the container is any one selected from the group consisting of a microwell plate, a microplate, and a microarray.

10. The analysis system according to claim 1, wherein the signal generating unit includes a light source for exciting the light emitting unit of the luminescent probe, and the detecting unit detects the luminance and / or chromaticity of the light.

11. The analysis system according to claim 1, wherein the analysis unit further predicts the function of the biological material by referring to a trained model.

12. An analytical method for detecting structural changes in a biological substance, comprising the steps of: allowing the biological substance to interact with two or more types of luminescent probes; generating a plurality of signals from the two or more types of luminescent probes that have interacted with the biological substance; detecting the plurality of signals; and analyzing the detected plurality of signals to identify structural changes in the biological substance, wherein each of the luminescent probes is a compound having a binding moiety in its molecule that binds to the biological substance and a luminescent moiety whose emission behavior changes depending on the structure of the biological substance.

13. The analytical method according to claim 12, wherein the step of generating a signal is a step of irradiating the two or more types of luminescent probes with excitation light, and the step of detecting the signal is a step of detecting the luminance and / or chromaticity of the light.

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