JP2021081359A - Analysis method of intermolecular interaction and analyzer - Google Patents

Abstract

To provide an analysis method of an intermolecular interaction and an analyzer which can analyze the intermolecular interaction generated between molecules on the basis of a measurement result with reduced measurement error and high reliability.SOLUTION: An analysis method of an intermolecular interaction includes; a first step for making a sample solution containing nanoparticles (5) in each of which one or more first molecule (1) being an analysis object are coupled with one or more second molecules (2) being an internal standard contact with a baseboard (100) having a region to which a third molecule (3) is fixed, and a region to which a fourth molecule (4) is fixed; a second step for measuring the interaction of the first molecule (1) and the third molecule (3), and the interaction of the second molecule (2) and the fourth molecule (4); and a third step for analyzing a measurement result of the analysis object on the basis of a measurement result of the internal standard. An analyzer of the intermolecular interaction comprises the baseboard (100), a measurement part for measuring the intermolecular interaction, and an analysis part for analyzing the measurement result of the analysis object on the basis of the measurement result of the internal standard.SELECTED DRAWING: Figure 1

Description

The present invention relates to an analysis method and an analysis device for intermolecular interactions by sensing multiplexes.

In the living body, the interaction between various molecules is involved in biological functions such as metabolism, biological defense, and information transmission. For example, enzymes and substrates, antibodies and antigens, receptors and ligands, etc. form specific intermolecular interactions to perform the desired function. Generally, the interaction between molecules is analyzed as a reversible equilibrium reaction. The equilibrium reaction consists of a binding reaction in which molecules are bonded to each other to form a complex, and a dissociation reaction in which the complex is dissociated into individual molecules.

In recent years, analysis of intermolecular interactions has been actively carried out in various fields such as drug development, clinical diagnosis, and basic research. Kinetic analysis of intermolecular interactions has become important as a means of understanding biological functions in real time. In addition, the dissociation constant and the like obtained by kinetic analysis are becoming more useful as initial parameters when simulating biological functions. Drug discovery using kinetic analysis is also being performed, such as fast-acting drugs with a fast binding rate and long-acting drugs with a slow dissociation rate.

Conventionally, many examples of analysis of intermolecular interactions are related to an antigen-antibody reaction. ELISA (Enzyme Linked Immuno Sorbent Assay) using a fluorescent label and RIA (Radio Immuno Assay) using a radioactive label are often used for quantification of intermolecular interaction. Specific intermolecular interactions have been analyzed by binding fluorescent chromophores and radioisotopes to antigens and antibodies.

Further, as in Patent Document 1, surface plasmon resonance (SPR) analysis is also used for the analysis of intermolecular interactions. In SPR analysis, the analyte is flow-through added to the ligand immobilized on the sensor. Binding to the ligand is tested by passing a sample solution containing ananalite, and dissociation is tested by passing a buffer containing no ananalite. In the SPR analysis, the change in mass on the sensor is detected as a change in the resonance condition, so that the label to be analyzed is unnecessary.

Further, as in Patent Document 2, a magnetic label is also used for the analysis of the intermolecular interaction. Magnetic labels are expected as a highly accurate analysis method because they are not affected by optical noise or optical interference / shielding. In addition, the measuring device is relatively easy to miniaturize, and has the advantage that molecules can be guided by using a magnetic force.

Special Table 2006-527365 Japanese Unexamined Patent Publication No. 2017-049257

In analyzing the intermolecular interaction formed between desired molecules, it is required to accurately quantify the forming molecule forming the intermolecular interaction. In order to derive accurate dissociation constants, binding rate constants, dissociation rate constants, etc. that are close to the true values, highly reliable measurement results that accurately and quantitatively reflect actual intermolecular interactions are required. However, the conventional method using SPR analysis or the like has a problem in obtaining highly reliable measurement results.

The measurement results used for the analysis of intermolecular interactions require that the reaction process of bond / dissociation between molecules proceeds chemically correctly and is not affected by impurities. For example, when analyzing the intermolecular interaction of biomolecules, impurities that cause non-specific binding may be mixed in the sample solution containing the biomolecule of interest to be collected. When a large amount of impurities are mixed, there may be an influence on the intermolecular interaction and an influence on the measurement including false detection.

Therefore, in such a case, it is necessary to purify the sample solution in advance. However, since the purification operation requires labor and cost, there is also a demand for direct analysis without purification. When directly analyzing an unpurified sample solution, there is a problem that it is difficult to obtain highly reliable measurement results because it is affected by impurities and the like.

In addition, the measurement results used for the analysis of intermolecular interactions are required to have extremely small measurement errors. Usually, when deriving a kinetic constant, a plurality of measurements are performed by changing the concentration, and global fitting is performed on a plurality of measurement results. At this time, for each measurement in which the concentration is changed, an error on the measuring device, a concentration error with respect to the target concentration due to the adjustment of the concentration of the test molecule, a variation in the reaction environment at the time of measurement, a lot difference of the labeling reagent, and a reaction of the labeling reaction. Variations in rates, variations in labeling procedures, etc. may have an effect.

As a measure to minimize such measurement error, multiplex sensing that simultaneously measures a plurality of samples is also used. However, the analytical methods that can be multiplexed are limited in principle. Moreover, even if multiplex sensing is performed, concentration errors and labeling-related variations still pose problems.

As described in Patent Document 1, a response to be measured at equilibrium R _eq, when the initial concentration of the test molecules was C _o, since the association rate constant is expressed by K _{on = R} eq _/ C _{o When the accuracy of Co} as an input parameter is required, the amount of the test molecule to be measured and the reaction rate of the labeling reaction easily vary, so that the reliability of the measurement result is lowered. There's a problem.

On the other hand, in the conventional SPR analysis as described in Patent Document 1, the influence of non-specific binding by impurities is large, so that accurate measurement is difficult if the sample solution is not sufficiently purified. Further, since the SPR analysis uses optical technology, it is not possible to apply multiplex sensing, and it is difficult to reduce the measurement error between a plurality of measurements.

Further, in the conventional analysis method, when a measurement error occurs, it is difficult for the measurer to recognize it, and it is also difficult to evaluate ex post facto whether or not the measurement result is normal. When analyzing biomolecules, there is also a problem that the activity of intermolecular interactions of biomolecules deteriorates during storage or preparation. Since the reaction kinetic information of the molecule to be analyzed is often unknown, it is not easy to judge whether the measurement result is normal or abnormal.

Therefore, the present invention provides an analysis method and an analysis device for intermolecular interactions that can analyze intermolecular interactions formed between molecules based on measurement results with small measurement error and high reliability. With the goal.

That is, in order to solve the above-mentioned problems, the method for analyzing the intermolecular interaction according to the present invention is to use a sample solution containing nanoparticles in which the first molecule to be analyzed and the second molecule as an internal standard are bound to each other. The first step of contacting a substrate having a region in which a molecule is fixed and a region in which a fourth molecule is fixed, an intermolecular interaction between the first molecule and the third molecule, and the second molecule and the first molecule. The second step of measuring the intermolecular interaction with the four molecules and the measurement result of the intermolecular interaction between the first molecule and the third molecule to be analyzed are taken as an internal standard with the second molecule and the above. It includes a third step of analysis based on the measurement result of the intermolecular interaction with the fourth molecule.

Further, in the intermolecular interaction analyzer according to the present invention, the third molecule is fixed so that the sample solution containing the nanoparticles to which the first molecule to be analyzed and the second molecule as an internal standard are bound is brought into contact with each other. Measurement of the intermolecular interaction between the first molecule and the third molecule and the intermolecular interaction between the second molecule and the fourth molecule on a substrate having a region and a region in which the fourth molecule is fixed. The measurement result of the intermolecular interaction between the first molecule and the third molecule to be analyzed is measured by the measuring unit, and the intermolecular interaction between the second molecule and the fourth molecule, which is an internal standard, is measured. It includes an analysis unit that analyzes based on the results.

According to the present invention, it is possible to provide an analysis method and an analysis device for an intermolecular interaction capable of analyzing an intermolecular interaction formed between molecules based on a measurement result having a small measurement error and high reliability. it can.

It is a schematic diagram for demonstrating the principle of analysis of the intermolecular interaction. It is a figure which shows typically an example of the analysis apparatus of the intermolecular interaction of a magnetic measurement type. It is a figure which shows typically an example of the analysis apparatus of the intermolecular interaction of a fluorescence measurement type. It is a figure which shows an example of the measurement result of the intermolecular interaction. It is a figure which shows the fluctuation pattern of the measurement result of the intermolecular interaction. It is a figure which shows the measurement area of the sensor array used in Example 1. FIG. It is a figure which shows the measurement result of the intermolecular interaction of the analysis target in Example 1. FIG. It is a figure which shows the measurement result of the intermolecular interaction of the internal standard in Example 1. FIG. It is a figure which shows the fluorescence measurement result of the magnetic nanoparticle used in Example 1. FIG. It is a figure which shows typically the quantum dot particle used in Example 2. It is a figure which shows the reaction of the DNA polymerase used in Example 2. It is a figure which shows the measurement area of the array chip used in Example 2. It is a figure which shows the measurement result of the intermolecular interaction using the fluorescently labeled secondary antibody in Example 2. FIG. It is a figure which shows the measurement result of the intermolecular interaction using the quantum dot particle in Example 2. FIG. It is a figure which shows the result of having made the measurement result of the intermolecular interaction using the fluorescently labeled secondary antibody in Example 2 curve fitting.

Hereinafter, an analysis method and an analysis device for intermolecular interactions according to an embodiment of the present invention will be described with reference to the drawings. The same reference numerals are given to the configurations common to each of the following figures, and duplicate description will be omitted.

<Analysis method of intermolecular interaction>
The method for analyzing intermolecular interactions according to the present embodiment relates to a method for quantitatively analyzing intermolecular interactions formed between molecules. In this analysis method, the intermolecular interaction formed between the test molecule to be analyzed and the test pair molecule and the intermolecular interaction formed between the standard molecule functioning as an internal standard and the standard pair molecule are detected. , In substantially the same reaction environment, measure at substantially the same time, and analyze and process the measurement result of the analysis target based on the measurement result of the internal standard.

The intermolecular interaction formed by the test molecule to be analyzed may be a specific interaction or a non-specific interaction. The intermolecular interaction may be formed by any of electrostatic interaction, van der Waals force, hydrogen bond, hydrophobic bond, dipole interaction, a combination thereof and the like. The same applies to the intermolecular interactions formed by the standard molecules that serve as internal standards.

In addition, in this specification, a specific interaction means an interaction in which the affinity of the molecule forming the interaction is remarkably high as compared with the case of other general molecules. Further, in the case of a specific bond or the like, as in the case of a specific interaction, it means a bond in which the affinity of the molecule forming the bond is remarkably high as compared with the case of other general molecules.

Any molecule can be used as the test molecule to be analyzed. The test molecule may be any of synthetic organic low molecule, synthetic organic polymer, natural organic low molecule, natural organic polymer, inorganic low molecule, inorganic polymer, organic metal molecule, organic-inorganic composite molecule and the like.

The type, molecular structure, molecular weight, and the like of the test pair molecule for which the intermolecular interaction with the test molecule is analyzed are not particularly limited. The test pair molecule may be any of synthetic organic low molecule, synthetic organic polymer, natural organic low molecule, natural organic polymer, inorganic low molecule, inorganic polymer, organic metal molecule, organic-inorganic composite molecule and the like. Further, the test pair molecule may be a bulk-like inorganic compound, metal, biological tissue, biological organ, biological membrane or the like.

As the standard molecule that serves as an internal standard and the standard pair molecule for which the intermolecular interaction with the standard molecule is measured, a combination in which the intermolecular interaction is known is used. That is, the standard molecule is a molecule that forms a specific intermolecular interaction with a predetermined standard pair molecule, and a molecule whose measurement result of the intermolecular interaction is known, or a predetermined standard pair molecule. For a specific binding reaction, a molecule is used in which kinetic constants such as dissociation constant, binding rate constant, and dissociation rate constant are known, and the most probable values of these constants are available.

In the present specification, the measurement result of the intermolecular interaction means a quantitative result over time in which the amount of the forming molecule forming the intermolecular interaction is associated with the measurement time. The measurement result of the intermolecular interaction may be expressed as a relationship between the measurement time and other physical quantities having a correlation with these, in addition to the amount and concentration of the molecule. Further, it may be represented by a kinetic constant such as a dissociation constant, a binding rate constant, and a dissociation rate constant of an equilibrium reaction.

Further, in the present specification, the most probable value means a value evaluated to be substantially closest to the true value from the result of an appropriate number of measurements. Generally, the most probable value is obtained by regression analysis such as the least squares method from the measurement results of a large number of measurements. The most probable value may be either a test value obtained by measuring the intermolecular interaction performed in advance, a literature value, or the like. For example, a kinetic constant obtained by a conventional method such as SPR analysis can be used as the most probable value. The most preferable value is a value obtained under the condition that the influence of impurities and the measurement error are extremely small.

The test molecule to be analyzed and the test-to-molecule for which the intermolecular interaction with the test molecule is analyzed have the number of types of molecules involved in the intermolecular interaction, the ratio of the number of molecules involved in the intermolecular interaction, and the like. , There are no particular restrictions. However, from the viewpoint of analyzing the measurement result as a primary reaction, a combination in which the test molecule and the test pair molecule interact in a ratio of 1: 1 is preferable. The same applies to the standard molecule that serves as an internal standard and the standard pair molecule for which the intermolecular interaction between the standard molecules is analyzed.

Specific examples of test molecules, test pair molecules, standard molecules, standard pair molecules, etc. include proteins, peptides, amino acids, DNA, RNA, these polynucleotides, nucleic acids such as these oligonucleotides, polysaccharides, oligosaccharides, etc. Examples thereof include saccharides such as monosaccharides, lipids such as fatty acids, simple lipids and phospholipids, glycolipids, lipoproteins, glycoproteins and the like, and derivatives thereof. Specific examples include, in particular, enzymes, antibodies, receptors and ligands.

Examples of the enzyme include oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase and the like. The form of the enzyme may be any of apoenzyme, holoenzyme, cofactor, enzyme subunit and the like.

Examples of the antibody include a monoclonal antibody and a polyclonal antibody. Examples of the type of antibody include IgA, IgD, IgE, IgG, IgM and the like. The form of the antibody may be any of a complete antibody molecule, Fab, Fc, Fab', F (ab') ₂ , ScFv and the like.

Examples of the receptor include a cell membrane receptor, a nuclear receptor and the like. Examples of the type of receptor include G protein-coupled receptor, ion channel type receptor, enzyme-linked receptor and the like. The form of the receptor may be either a complete receptor molecule, a receptor subunit, or the like.

As the ligand, an agonist, an antagonist, or the like can be used in addition to the true ligand for a specific receptor. Examples of the type of ligand include hormones, cytokines, lymphokines, neurotransmitters, endocrine disruptors, toxins and the like.

Specific examples of intermolecular interactions include interactions by hybridization between nucleic acids, interactions between proteins or peptides, and interactions between proteins or peptides and nucleic acids. Specific examples include the interaction between an enzyme and a substrate, the interaction between an antigen and an antibody, and the interaction between a receptor and a ligand. The analysis results of these intermolecular interactions are useful for kinetic evaluation in fields such as drug development and clinical diagnosis.

The analysis method according to the present embodiment uses multiplex sensing for measurement. In the analysis method according to the present embodiment, the first step of bringing the sample solution containing nanoparticles into contact with the array substrate, and the measurement of the intermolecular interaction of the analysis target and the intermolecular interaction of the internal standard that occur in the sample solution. It includes two steps and a third step of analyzing the measurement result of the analysis target based on the measurement result of the internal standard.

FIG. 1 is a schematic diagram for explaining the principle of analysis of intermolecular interactions.
As shown in FIG. 1, in the first step, the sample liquid 200 containing the nanoparticles 5 to which the analyte is bound is brought into contact with the array substrate 100 on which the ligand is fixed. The sample liquid 200 is a dispersion liquid in which nanoparticles 5 are dispersed. The nanoparticles 5 have a function as a carrier of analite and a function as a label for quantification. The array substrate 100 is a substrate in which measurement regions (6, 7) for measuring intermolecular interactions are arrayed.

As the nanoparticles 5, fine particles to which the test analyzer (first molecule) 1 to be analyzed and the standard analyzer (second molecule) 2 as an internal standard are bound are used. The test analyzer 1 is a molecule for measuring the intermolecular interaction between the test molecule and the test pair molecule. A standard analyzer is a molecule for measuring the intermolecular interaction between a standard molecule and a standard pair molecule.

The array substrate 100 has a measurement region 6 in which the test ligand (third molecule) 3 to be analyzed is fixed, and a measurement region 7 in which the standard ligand (fourth molecule) 4 as an internal standard is fixed. ing. The test ligand is a molecule for measuring the intermolecular interaction between the test molecule and the test pair molecule. A standard ligand is a molecule for measuring the intermolecular interaction between a standard molecule and a standard pair molecule.

The test molecule to be analyzed may be added to the array substrate 100 as the test analyzer 1 or may be immobilized on the array substrate 100 as the test ligand 3. On the other hand, the test pair molecule is preliminarily immobilized as the test ligand 3 on the array substrate 100 to which the test molecule is added, or is added as the test analyzer 1 to the array substrate 100 on which the test molecule is preliminarily immobilized.

Similarly, the standard molecule that serves as an internal standard may be added to the array substrate 100 as the standard analyze 2 or may be immobilized on the array substrate 100 as the standard ligand 4. On the other hand, the standard pair molecule is preliminarily fixed as the standard ligand 4 to the array substrate 100 to which the standard molecule is added, or is added as the standard analyze 2 to the array substrate 100 to which the standard molecule is preliminarily fixed.

As shown in FIG. 1, when the sample solution 200 is brought into contact with the array substrate 100 in the first step, the measurement region 6 in which the test ligand 3 is immobilized and the measurement region 7 in which the standard ligand 4 is immobilized are nano-sized. Immerse in the sample solution 200 containing the particles 5. Since the test-analyte 1 and the standard-analyte 2 are bound to the nanoparticles 5, the test-ligand 3 can come into contact with the test-analyte 1 and the standard ligand 4 can come into contact with the standard-analyte 2. Become.

When the test analyzer 1 and the test ligand 3 can form an intermolecular interaction, a complex 8 is formed in the measurement region 6 for analysis. As a result, the nanoparticles 5 to which the test Annalite 1 is bound are in a state of being fixed in the measurement region 6. In the second step, the nanoparticles 5 forming the complex 8 existing on the measurement region 6 are individually quantified. That is, the molecules forming the intermolecular interaction are quantified over time for each measurement region for the analysis target, and the measurement result of the intermolecular interaction to be analyzed can be obtained.

On the other hand, since the standard analyze 2 and the standard ligand 4 can form an intermolecular interaction, a complex 9 is formed in the measurement region 7 for the internal standard. As a result, the nanoparticles 5 to which the standard Analite 2 is bound are fixed in the measurement region 7. In the second step, the nanoparticles 5 forming the complex 9 existing on the measurement region 7 are individually quantified. That is, the molecules forming the intermolecular interaction are quantified over time for each measurement region for the internal standard, and the measurement result of the intermolecular interaction of the internal standard is obtained.

Thus, in the second step, both the intermolecular interaction between the test molecule and the test pair molecule and the intermolecular interaction between the standard molecule and the standard pair molecule are both achieved by sensing the multiplex using the array substrate. , Measured at substantially the same time under the same equipment conditions.

In general, intermolecular interactions are affected by the concentration, size, three-dimensional structure, etc. of molecules. Therefore, in order to accurately analyze the intermolecular interaction between desired molecules, it is necessary to perform a plurality of measurements with different molecular concentrations. On the other hand, according to multiplex sensing using an array substrate, the variation in device conditions and measurement conditions for each measurement is reduced, so the measurement error for each concentration or between the analysis target and the internal standard can be reduced. it can.

In the second step, both the intermolecular interaction between the test molecule and the test pair molecule and the intermolecular interaction between the standard molecule and the standard pair molecule are measured for the same sample solution in contact with the same array substrate. Will be done. That is, when performing multiplex sensing using an array substrate, the analysis target and the internal standard are tested in substantially the same reaction environment on the same substrate.

In the conventional multiplex sensing, the test group for testing the analysis target and the control group for testing the control are divided into a plurality of wells, a test time, etc., and measurement is performed in individual sample solutions. Is common. On the other hand, when measurement is performed with the same sample solution in contact with the same array substrate, in addition to the variation in the device conditions and measurement conditions for each measurement, the variation in the reaction conditions is reduced. The measurement error between and can be further reduced.

The sample solution 200 used for the analysis of the intermolecular interaction includes a dispersion medium such as water and additives such as a pH buffer, an inorganic salt, a surfactant, and a reducing agent, in addition to the test analyze 1 and the standard analyze 2. Can be included. In addition, the sample solution may contain other substances involved in the intermolecular interaction and other substances not involved in the intermolecular interaction, depending on the purpose of the analysis. The sample liquid 200 may be a liquid derived from a living body such as a culture liquid, an interstitial fluid, blood, serum, or the like. The sample liquid 200 may or may not have been separated / purified.

The test announcer 1 and the standard analyzer 2 may be fixed to the nanoparticles 5 by chemisorption by covalent bond or the like, or by physical adsorption by intermolecular force or the like. However, from the viewpoint of reducing the variation in the immobilization reaction for immobilizing the nanoparticles 5 and the dropout after immobilization, the test analysts 1 and the standard analysts 2 are preferably immobilized by covalent bonds. In addition, the test announcer 1 and the standard announcer 2 may be fixed to the nanoparticles 5 via a linker.

A plurality of measurement regions 6 to which the test ligand 3 is fixed and a plurality of measurement regions 7 to which the standard ligand 4 is fixed are provided on the array substrate 100, respectively. The plurality of measurement regions can be provided independently of each other at positions on the array substrate 100 where they can come into contact with the same sample liquid. The plurality of measurement regions can be arranged one-dimensionally or two-dimensionally. When a plurality of measurement regions are provided, a plurality of measurements with different molecular concentrations such as a dilution series can be measured on one substrate at substantially the same time under the same device conditions.

The test ligand 3 may be immobilized on the array substrate 100 by chemical adsorption or physical adsorption. Further, the standard ligand 4 may be fixed on the array substrate 100 by chemical adsorption or physical adsorption. In addition, other substances that do not significantly inhibit intermolecular interaction, such as blocking agents and carriers for ligands, are fixed in the measurement region where the test ligand is fixed and the measurement region where the standard ligand is fixed. May be good.

The array substrate 100 has a measurement region 6 in which the test ligand 3 is fixed, a measurement region 7 in which the standard ligand 4 is fixed, a region in which the ligand is not fixed, and a measurement region for performing a control experiment. You may be. The measurement region for performing the control experiment may or may not have the ligand fixed. In the measurement area for performing the control experiment, for example, the interaction with the substrate itself, the interaction with the blocking agent treated with the substrate, and the like can be measured.

In the array substrate 100, the method of contacting the sample liquid may be a batch type or a flow type. The batch type array substrate may be provided with, for example, a well, a dish portion, or the like in order to hold the sample liquid 200 on the substrate. The flow type array substrate may include, for example, a channel, a flow cell, or the like in order to allow the sample liquid 200 to flow on the substrate.

The shape of the nanoparticles 5 is not particularly limited. Specific examples of the shape of the nanoparticles 5 include a spherical shape, an elliptical spherical shape, a block shape, a plate shape, a columnar shape, an annular shape, a needle shape, a tubular shape, a fibrous shape, and an indefinite shape. The nanoparticles 5 may have a solid structure, a hollow structure, a core-shell structure, or the like. The nanoparticles 5 may be formed of any of an organic material, an inorganic material, an organic-inorganic composite material, and the like, but are preferably formed of a material that is chemically stable in the sample solution. When an aqueous sample solution is used, the nanoparticles 5 preferably have hydrophilicity on the surface from the viewpoint of preventing aggregation of the particles.

The particle size of the nanoparticles 5 is preferably equal to or smaller than the size of the analite. The particle size of the nanoparticles 5 is, for example, 1 to 500 nm, preferably 2 to 300 nm, more preferably 5 to 200 nm, and even more preferably 10 to 100 nm. The larger the nanoparticles 5 are at 1 nm or more, the easier it is to handle the particles and bind the analysts. Further, as the nanoparticles 5 are smaller at 500 nm or less, the steric hindrance to the intermolecular interaction and the kinetic influence of the diffusion process tend to be smaller.

As the nanoparticles 5, a magnetic material, a magnetically labeled material, a fluorescent material, a fluorescently labeled material, or the like can be used. When a magnetic substance or a magnetic labeling substance is used, the quantification of molecules forming intermolecular interactions can be performed using a magnetic measurement type device. Further, when a fluorescent substance or a fluorescent labeling substance is used, the quantification of molecules forming intermolecular interactions can be performed using a fluorescence measurement type device.

<Analyzer for intermolecular interaction>
Here, a magnetic measurement type intermolecular interaction analysis device and a fluorescence measurement type intermolecular interaction analysis device will be described with reference to the drawings.

FIG. 2 is a diagram schematically showing an example of a magnetic measurement type intermolecular interaction analysis device.
As shown in FIG. 2, the magnetic measurement type intermolecular interaction analysis device (magnetic measurement type device) 10 includes a sensor array 11, a circuit board 12, a movable support device 13, a digitizer 14, and a data analysis device. It includes 15, a magnetic field generator 16, and an analysis control device (analysis unit) 40. The liquid tank 17 is housed in the magnetic field generator 16 at the time of measurement. The test sample liquid 18 is prepared in the liquid tank 17.

The magnetic measurement type device 10 is a device capable of measuring the magnetism in the test sample liquid 18 by the sensor array 11. In the magnetic measurement type device 10, the magnetism of the measurement target in the test sample liquid 18 can be measured while applying an external magnetic field to the test sample liquid 18 in which the measurement target exists by the magnetic field generator 16.

The sensor array 11 is a device in which magnetic sensors for measuring magnetism to be measured are arrayed on a substrate. The sensor array 11 includes a plurality of arrayed magnetic sensors, and has a magnetic sensor for analysis (first magnetic sensor) as a measurement region in which a test ligand (third molecule) is fixed, and is standard. As the measurement region to which the ligand (fourth molecule) is fixed, the array substrate 100 having an internal standard magnetic sensor (second magnetic sensor) is used.

The test ligand may be fixed to the surface of the magnetic sensor to be analyzed by chemical adsorption or physical adsorption. Further, the standard ligand may be fixed to the surface of the internal standard magnetic sensor by chemisorption or physical adsorption. In addition, other substances that do not significantly inhibit intermolecular interaction, such as blocking agents and carriers for ligands, are fixed in the measurement region where the test ligand is fixed and the measurement region where the standard ligand is fixed. May be good. Further, the test ligand and the standard ligand may be fixed to the surface of the substrate instead of the surface of the magnetic sensor.

A plurality of magnetic sensors for analysis and an internal standard magnetic sensor are provided on the sensor array 11, respectively. The plurality of magnetic sensors can be provided independently of each other so as to correspond to the measurement area to be arrayed. The plurality of magnetic sensors can be arranged one-dimensionally or two-dimensionally. The magnetic sensor can be provided, for example, with an area of a measurement region of, for example, 100 μm ^{2 to} 10 cm ² , 10 to 500 μm square, or the like.

The sensor array 11 may include a control magnetic sensor for measuring a control experiment in addition to the analysis target magnetic sensor and the internal standard magnetic sensor. The control magnetic sensor does not have to have a ligand immobilized on the surface. When a control magnetic sensor is used, for example, the interaction with the substrate itself, the interaction with the blocking agent used for processing the substrate, and the like can be measured.

The magnetic sensor for analysis, the magnetic sensor for internal standard, and the magnetic sensor for control are provided at positions on the substrate where they can come into contact with the same liquid. The magnetic sensor for analysis, the magnetic sensor for internal standard, and the magnetic sensor for control are magnetic sensors of the same type, and can measure magnetic fields in substantially the same conditions for each measurement region on the sensor array 11. As long as it is possible, an appropriate type can be used. The magnetic sensor can be provided on the sensor array 11 in a disk shape, a flat plate shape, or the like.

As the magnetic sensor, an MR sensor using a magneto resistance effect (MR) is preferable. According to the MR sensor, the magnetic sensor can be easily miniaturized. Further, according to the MR sensor, since the detection distance is appropriate and the temperature dependence is low, accurate measurement can be performed.

As the MR sensor, a GMR sensor using a giant magnetoresistive effect (GMR) or a TMR sensor using a tunnel magnetoresistive effect is preferable. According to the GMR sensor and the TMR sensor, weak magnetism is detected with high sensitivity, so that it is possible to quantify the measurement target having magnetism with high accuracy.

MR sensors include, for example, antiferromagnetic layers such as IrMn and PtMn, ferromagnetic layers such as CoFe, CoFeB, CoPt, FePt and GdFe, non-magnetic layers such as Cu, and insulating layers such as MgO and resists. , A spin valve using Cu, Pd or the like as a capping and Ta, Cu, Au, Ag, Pt, Al or the like as an electrode can be used.

In FIG. 2, the sensor array 11 is incorporated on the circuit board 12. The circuit board 12 includes a processing circuit such as an amplifier. The circuit board 12 receives the measurement signal measured by each magnetic sensor on the magnetic sensor array 11 and performs signal processing such as amplification for each magnetic sensor.

The movable support device 13 movably supports the circuit board 12. The movable support device 13 can move the circuit board 12 up and down relative to each other, and can move the sensor array 11 back and forth into the liquid tank 17. According to the movable support device 13, since the sensor array 11 can be immersed in the test sample liquid 18 prepared in the liquid tank 17, the magnetic measurement of the measurement target in the test sample liquid 18 can be uniformly started. it can.

The digitizer 14 digitizes the measurement signal signal-processed by the circuit board 12. The digitized measurement signal is input to the data analysis device 15. The data analysis device 15 quantifies the measurement target based on a measurement signal corresponding to the magnetic strength of the measurement target, for example, a measurement signal converted into the magnitude of electrical resistance by the magnetoresistive effect.

The magnetic field generator 16 is a device that generates a uniform magnetic field, and applies the generated magnetic field to the measurement target in the test sample liquid 18. According to the magnetic field generator 16, the measurement target can be magnetized by applying an external magnetic field to perform measurement. Further, according to the magnetic field generator 16, in the case of a magnetic measurement object, relaxation of the magnetic field due to orientation is prevented.

According to such a magnetic measuring device 10, the measurement target existing in the measurement range of the magnetic sensor on the sensor array 11 can be quantified by the magnetic strength. When the sample liquid 200 containing the nanoparticles to which the test analyzer (first molecule) and the standard analyzer (second molecule) are bound is prepared as the test sample liquid 18 in the liquid tank 17, the test sample liquid 18 is prepared. By immersing the sensor array 11 in the sample, the test of the intermolecular interaction for each measurement region is uniformly started.

Then, the intermolecular interaction to be analyzed is measured based on the magnetic strength of the nanoparticles measured by the sensor for analysis on the sensor array 11. The intermolecular interaction of the internal standard is measured based on the magnetic strength of the nanoparticles measured by the internal standard sensor on the sensor array 11. Therefore, from the quantification result of only the molecule associated with the magnetic label, the measurement result of the intermolecular interaction by the sensing of multiplex can be obtained.

As the magnetic nanoparticle, for example, particles such as paramagnetic material, ferromagnetic material, and ferrimagnetic material, superparamagnetic particles, and particles in which a ferromagnetic material and an antiferromagnetic material are combined can be used. it can. Further, as the nanoparticles of the magnetically labeled body, inorganic particles, organic particles, etc. labeled with a magnetic material such as a paramagnetic material, a ferromagnetic material, and a ferrimagnetic material, and inorganic particles, organic particles, etc. in which the magnetic material is composited are used. Can be used. Magnetic nanoparticles and magnetically labeled nanoparticles are prepared so that their magnetizations are substantially uniform. Specific examples of the magnetic material include hematite, magnetite, magnetite, iron, cobalt, nickel and the like.

According to the method for measuring a magnetic label, unlike the conventional SPR analysis or the like, a measurement result that is not significantly affected by impurities or the like can be obtained from the quantification result of only the molecule associated with the magnetic label. Since the false detection of the magnetic label can be reduced depending on the test conditions, the measurement error of the intermolecular interaction can be reduced. Further, according to the method of measuring a magnetic label, it is possible to perform measurement accurately and stably as compared with a fluorescent label which may cause light interference, fading, quenching and the like. When analyzing a sample collected from a living body, the influence of optical interfering substances such as red blood cell components can be avoided.

FIG. 3 is a diagram schematically showing an example of an analysis device for an intermolecular interaction of a fluorescence measurement type.
As shown in FIG. 3, the fluorescence measurement type intermolecular interaction analyzer (fluorescence measurement type device) 20 includes an array plate 21, a flow path forming member 22, a temperature control device 23, and a test sample solution channel 24. , The liquid feed unit 25, the liquid feed port valve 26, the excitation light source 31, the condenser lens 32, the dichroic mirror 33, the objective lens 34, the optical filter 35, the imaging lens 36, and the image sensor 37. And an analysis control device (analysis unit) 40.

The fluorescence measurement type device 20 is a device capable of measuring the fluorescence emitted from the measurement target by photoexciting the measurement target on the array plate 21. In the fluorescence measurement type device 20, the measurement target is fed onto the array plate 21 in a flow manner through the test sample liquid channel 24, the measurement target bound on the array plate 21 is irradiated with excitation light, and the measurement target changes from the excited state to the ground state. The fluorescence emitted when returning to can be measured.

The array plate 21 is a substrate in which measurement regions having transparency of excitation light emitted from the measurement target and fluorescence emitted from the measurement target are arrayed. The array plate 21 has a measurement region in which a test ligand (third molecule) is fixed and a measurement region in which a standard ligand (fourth molecule) is fixed, and has light transmission of excitation light and fluorescence. Array substrate 100 is used. The conditions for fixing the test ligand and the standard ligand and the conditions for the measurement region are the same as those for the array substrate 100 described above.

The flow path forming member 22 covers the flat plate-shaped temperature control device 23, and forms a tubular flow path for allowing the test sample liquid to flow on the temperature control device 23. The array plate 21 is placed flat in the flow path formed on the temperature control device 23. The temperature control device 23 is a device for controlling the temperature of the reaction field of the intermolecular interaction, and adjusts the temperature of the array plate 21 or the like so that the intermolecular interaction is tested at a predetermined temperature.

The flow path formed on the temperature control device 23 extends to both outer sides of the temperature control device 23 and forms the test sample liquid channel 24. The test sample liquid channel 24 is a flow path for flowing the test sample liquid through the array plate 21. One end of the test sample liquid channel 24 communicates with the liquid feeding unit 25. The liquid feed port of the liquid feed unit 25 is provided with a liquid feed port valve 26 which can open and close the test sample liquid channel 24 and can adjust the flow velocity of the test sample liquid. The other end of the test sample liquid channel 24 communicates with a recovery tank or the like (not shown) for collecting the test sample liquid.

As shown in FIG. 3, the excitation light source 31, the objective lens 34, and the image sensor 37 constitute a fluorescence measuring device together with other optical system devices. The excitation light source 31 is a device that generates excitation light for photoexciting the measurement target. A condenser lens 32 and a dichroic mirror 33 are installed in the optical path of the excitation light emitted from the excitation light source 31. The excitation light emitted from the excitation light source 31 reaches the dichroic mirror 33 after being focused by the condenser lens 32.

The dichroic mirror 33 reflects the light on the short wavelength side including the excitation light and transmits the light on the long wavelength side not including the excitation light. The excitation light emitted from the excitation light source 31 is reflected by the dichroic mirror 33 and sent to the objective lens 34. On the other hand, unnecessary stray light emitted from the excitation light source 31 is removed by passing through the dichroic mirror 33.

The objective lens 34 is installed so as to face the array plate 21 placed flat in the test sample liquid channel 24. The excitation light reflected by the dichroic mirror 33 is irradiated to the measurement region on the array plate 21 by the objective lens 34. The measurement target on the array plate 21 is excited by the excitation light, and fluorescence is emitted when returning from the excited state to the ground state. The fluorescence emitted by the measurement target is observed by the objective lens 34 and reaches the dichroic mirror 33.

The fluorescence emitted by the measurement target passes through the dichroic mirror 33 and reaches the optical filter 35. The optical filter 35 absorbs light in an unnecessary wavelength region and transmits fluorescence in a predetermined wavelength region emitted by the measurement target. The fluorescence transmitted through the optical filter 35 is detected by the image sensor 37 after being focused by the imaging lens 36.

The image sensor 37 is provided for measuring the fluorescence intensity of each measurement region on the array plate 21. The image sensor 37 images the measurement area on the array plate 21 and quantifies the measurement target based on the measurement signal of the fluorescence intensity of each pixel in which fluorescence is detected. As the image sensor 37, for example, a two-dimensional image sensor such as a CCD (Charge-Coupled Device) image sensor or a CMOS (Complementary Metal Oxide Semiconductor) image sensor can be used.

According to such a fluorescence measuring device 20, the measurement target existing in the measurement region on the sensor array 11 can be quantified by the intensity of fluorescence. When the sample solution 200 containing the nanoparticles containing the test-analyte (first molecule) and the standard-analyte (second molecule) is flown onto the array plate 21 by the liquid feeding unit 25, the test ligand (third molecule) is passed. ) And the standard ligand (4th molecule) are immobilized on the array plate 21, and the intermolecular interaction for each measurement region is tested.

Then, the intermolecular interaction to be analyzed and the intermolecular interaction of the internal standard are measured as the fluorescence intensity due to the nanoparticles trapped on the array plate 21. Therefore, from the quantification result of only the molecule associated with the fluorescent label, the measurement result of the intermolecular interaction by the sensing of multiplex can be obtained.

As the nanoparticles of the phosphor, for example, particles of oxides, sulfides, nitrides, fluorides, rare earths and the like, and particles of quantum dots can be used. Further, as the nanoparticles of the fluorescently labeled substance, inorganic particles, organic particles or the like labeled with a fluorescent substance, a fluorescent dye or the like can be used. Specific examples of quantum dot materials include ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, InP, InAs, InSb, InN, GaP, GaAs, GaSb, GaN, SnS, SnSe, SnTe, PbS, PbSe, PbTe and the like. Can be mentioned. Specific examples of the fluorescent dye include fluorescein dye, luciferin dye, coumarin dye, rhodamine dye, acridine dye, perylene dye and the like.

According to the method for measuring a fluorescent label, unlike the conventional SPR analysis or the like, a measurement result that is not significantly affected by impurities or the like can be obtained from the quantification result of only the molecule associated with the fluorescent label. Since erroneous detection of fluorescent labels due to light interference, fading, quenching, etc. can be reduced depending on the test conditions, the measurement error of intermolecular interaction can be reduced. Further, according to the method of measuring the fluorescent label, the detection distance can be easily secured as compared with the magnetic label, so that the degree of freedom in the device configuration is increased.

The analysis control device 40 is a device that analyzes the measurement result of the intermolecular interaction. The analysis control device 40 is, for example, a calculation device (calculation unit) such as a CPU (Central Processing Unit), a storage device such as a ROM (Read Only Memory), a RAM (Random Access Memory), a hard disk, or an SSD (Solid State Drive). It is composed of a (storage unit), a display device (display unit), an input means, and the like.

The data analysis device 15 of the magnetic measurement device 10, the image sensor 37 of the fluorescence measurement device 20, and the input means are connected to the analysis control device 40 via the input interface. The input means includes a keyboard, a mouse, a touch pad, and the like. Further, a display device is connected to the analysis control device 40 via an output interface. The display device is composed of a liquid crystal display, a plasma display, an organic EL display, and the like.

FIG. 4 is a diagram showing an example of the measurement result of the intermolecular interaction.
As shown in FIG. 4, the measurement results of the intermolecular interaction between the test ligand (first molecule) and the test analyzer (third molecule), and the standard ligand (second molecule) and the standard analyze (fourth molecule) The measurement result of the intermolecular interaction with the molecule can be obtained as a sensorgram showing the relationship between the response measured using the nanoparticles as a measurement control and the reaction time.

In FIG. 4, _{the section from time T 1} to time T ₂ shows the coupled phase. If the ligand and the analyte to form intermolecular interactions, when starting the contact of the ligand in the sample solution containing an analyte at time T _1, the ligand and the analyte to form a complex, on the measurement region The amount of nanoparticles increases. The response measured with nanoparticles as the measurement control _{gradually rises from the baseline (R 0} ) and reaches a certain value (R _m).

Further, in FIG. 4, _{the section from time T 2} to time T ₃ shows a dissociated phase. Discontinue contact with the ligand of a sample solution containing analyte, when starting the contact to the ligand of the buffer that does not contain the analyte at time T _2, to dissociate the analyte from the complex, nanoparticle on the measurement region The amount of The response measured with nanoparticles as the measurement control begins to descend from _{a certain value (R m} ) at the end of the bound phase and usually asymptotics above _{baseline (R 0).} Contacting the cleaning solution to eliminate intermolecular interactions at time T _3, the response is returned to the base line (R _0).

From such a relationship between the response and the reaction time, the intermolecular interaction can be analyzed kinetically. In the second step, the array substrate on which the ligand is immobilized is blocked, if necessary, and then the array substrate is washed before measurement is started. In the second step, only the bound phase may be measured using a sample solution containing an analyte, or the dissociated phase may be measured by substituting the bound phase with a buffer containing no ananalite.

FIG. 5 is a diagram showing a variation pattern of the measurement result of the intermolecular interaction.
As shown in FIG. 5, when the intermolecular interaction of the analysis target or the internal standard is measured, the step of preparing the ligand and the analysis, the step of measuring the intermolecular interaction, and the binding / dissociation of the ligand and the analysis are performed. The normal line 110 is obtained when the reaction process of No. 1 proceeds normally with few errors and variations. The normal line 110 is a line _{in which the response at a certain time (T n} ) gradually _{approaches a value (R 1} ) close to the maximum value obtained in the saturated state.

On the other hand, _{the response at a certain time (Tn} ) becomes low, and an abnormal line 120 that asymptotes to a _{value (R 2} ) significantly lower than the maximum value obtained in the saturated state may be obtained. In such an abnormal line 120, each step does not proceed normally, an error on the measuring device, a concentration error with respect to the target concentration due to the adjustment of the concentration of the test molecule, a variation in the reaction environment at the time of measurement, and a lot of labeling reagent. It indicates the possibility that a difference, a variation in the reaction rate of the labeling reaction, a variation in the procedure at the time of labeling, etc. occurred.

Therefore, in the third step, the measurement result of the intermolecular interaction between the test analyzer (first molecule) and the test ligand (third molecule), which is the analysis target obtained in the second step, is obtained in the second step. The analysis is performed based on the measurement results of the intermolecular interaction between the standard analyze (second molecule) and the standard ligand (fourth molecule), which are the internal standards obtained. The analysis of the measurement result of the intermolecular interaction is performed by comparing the measurement result data with each other.

The measurement result of the internal standard can be compared with the normal measurement result obtained in advance to evaluate the reliability of the data. The reliability of the data can be evaluated by comparing the measurement results of the analysis target tested in the reaction environment substantially the same as the internal standard with the measurement results of the internal standard in which the reliability of the data was evaluated. Therefore, it is possible to perform processing such as normality or abnormality test and correction on the measurement result of the analysis target whose intermolecular interaction is unknown.

In the third step, the measurement results obtained in the second step are fitted or linearized to a predetermined reaction model such as a Langmuir adsorption model to obtain kinetic constants such as a dissociation constant, a binding rate constant, and a dissociation rate constant. , The reliability of kinetic constant measurements can be evaluated. When the ligand and the analyte interact at a ratio close to 1: 1, the kinetic constant can be obtained by regarding the binding reaction between the ligand and the analyte as a primary reaction.

In the third step, first, the measurement result of the intermolecular interaction between the standard analyst and the standard ligand obtained in the second step as the internal standard, and the standard analyst and the standard ligand as the internal standard obtained in advance are used. Compare with the measurement result of the intermolecular interaction of. For internal standards, it is possible to obtain normal measurement results in advance by preliminary analysis or another analysis method. For example, the measurement can be repeated a plurality of times, and the measurement results of a large number of measurements can be regression-analyzed to obtain the most probable value. The measurement result of the internal standard obtained in the second step can be compared with such the most probable value.

In the magnetic measurement type device 10 and the fluorescence measurement type device 20, the analysis of the intermolecular interaction is performed by the analysis control device 40. The analysis of intermolecular interactions may be performed by hardware or software. The program for executing the analysis is stored in a storage medium such as a ROM, a hard disk, an SSD, a magnetic disk, or an optical disk, and is read into the memory when the analysis is executed. The measurement result of the internal standard obtained in advance by a preliminary analysis or another analysis method, for example, the most probable value, is stored in the storage device in advance.

In the third step, the third step is based on the similarity between the measurement result of the internal standard intermolecular interaction obtained in the second step and the most probable value of the measurement result of the internal standard intermolecular interaction obtained in advance. It is possible to test the normality or abnormality of the measurement result of the intermolecular interaction to be analyzed obtained in two steps. For example, when the mathematical distance between the measurement result obtained in the second step and the measurement result obtained in advance is closer than the threshold value, it can be determined that there is no abnormality in the measurement and the measurement error is small. On the other hand, when the mathematical distance is farther than the threshold value, it can be determined that there is an abnormality in the measurement and a large measurement error has occurred.

In the magnetic measurement type device 10 and the fluorescence measurement type device 20, the arithmetic device of the analysis control device 40 is the measurement result of the intermolecular interaction of the internal standard obtained in the second step and the intermolecular of the internal standard obtained in advance. When the absolute value of the difference in the mathematical distance between the measurement result obtained in the second step and the most probable value exceeds a predetermined value by comparing with the most probable value of the measurement result of the interaction, the intermolecular interaction to be analyzed It is possible to determine that the measurement result is abnormal. On the other hand, when the absolute value of the difference in mathematical distance is not more than a predetermined value, it can be determined that the measurement result of the intermolecular interaction to be analyzed is normal.

When the measurement result of the intermolecular interaction to be analyzed is determined to be abnormal or normal, the arithmetic unit of the analysis control device 40 can display the determination result on the display device. For example, the degree of similarity is that the measurement result is normal, the velocity constant based on the normal measurement result, or the measurement result is abnormal, the velocity constant based on the abnormal measurement result, etc. It can be displayed in numbers, symbols, languages, etc., together with numerical values representing.

Alternatively, in the third step, the similarity between the measurement result of the internal standard intermolecular interaction obtained in the second step and the most probable value of the measurement result of the internal standard intermolecular interaction obtained in advance is used. , The measurement result of the intermolecular interaction to be analyzed obtained in the second step can be corrected. For example, the kinetic constant based on the measurement result obtained in the second step, the response value at a predetermined time, the time change rate of the response, the maximum value, the average value, etc. are compared with the corresponding most probable values, and each other is compared. It is possible to obtain a correction coefficient that reflects the similarity of the above and correct the measurement result of the analysis target to a value close to the true value.

In the magnetic measurement type device 10 and the fluorescence measurement type device 20, the arithmetic device of the analysis control device 40 uses the measurement result of the internal standard intermolecular interaction obtained in the second step and the intermolecular standard internal standard obtained in advance. The measurement obtained in the second step is performed when the absolute value of the difference in the mathematical distance between the measurement result obtained in the second step and the most probable value exceeds a predetermined value by comparing with the most probable value of the measurement result of the interaction. The measurement result of the intermolecular interaction to be analyzed can be corrected by the correction coefficient obtained from the result and the most probable value.

The correction coefficient can be obtained, for example, by dividing the measurement result of the internal standard intermolecular interaction obtained in the second step by the most probable value of the measurement result of the internal standard intermolecular interaction obtained in advance. it can. For example, when a kinetic constant based on the measurement result obtained in the second step is divided by the most probable values such as a test value of the kinetic constant and a literature value, a calculated value reflecting the degree of similarity with each other is obtained. The measurement result to be analyzed can be corrected by using the reciprocal of the calculated value obtained in this way as a correction coefficient (multiplication factor).

According to the above analysis method and analysis device for intermolecular interactions, nanoparticles in which multiple types of analysts are bound are used, so even when a single analysis sample is used as an analysis target, multiple types of intermolecular interactions are used. The interaction is measured. Therefore, it is possible to multiplex the measurement without individually measuring the sample solution to be analyzed and the sample solution of the internal standard. That is, the intermolecular interaction to be analyzed and the intermolecular interaction of the internal standard can be measured with the same sample solution in contact with the same array substrate at substantially the same time under the same equipment conditions. .. Since it is possible to accurately compare the measurement result of the intermolecular interaction of the analysis target with the measurement result of the intermolecular interaction of the internal standard, the measurement result of the internal standard is used as an index for judging the validity of the data to be analyzed. The reliability of the measurement result can be evaluated. Therefore, the intermolecular interaction formed between the molecules can be analyzed based on the measurement result with a small measurement error and high reliability. For example, it is possible to eliminate the influence of noise due to an external magnetic field when a magnetic label is used, and light interference, fading, quenching, etc. when a fluorescent label is used.

Although the present invention has been described above, the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the spirit of the present invention. For example, the present invention is not necessarily limited to those having all the configurations included in the above-described embodiment. Replacing part of the configuration of one embodiment with another, adding part of the configuration of one embodiment to another, or omitting part of the configuration of one embodiment. Can be done.

For example, in the above-mentioned method and apparatus for analyzing intermolecular interactions, magnetic labels and fluorescent labels are used. However, in the method and apparatus for analyzing the intermolecular interaction, other labeling methods such as scattered light labeling such as colloidal gold, phosphorescence labeling, and radioactive labeling may be used instead of the magnetic labeling and the fluorescent labeling.

Further, in the above-mentioned method and apparatus for analyzing intermolecular interactions, tests and corrections are performed as analysis of intermolecular interactions in the third step. However, as long as the measurement result of the intermolecular interaction between the test ligand and the test analyst and the measurement result of the intermolecular interaction between the standard ligand and the standard analyzer are compared, the intermolecular interaction in the third step As an analysis, appropriate processing can be performed, for example, construction of a database that rejects abnormal measurement results.

Further, in FIGS. 2 and 3, a batch type magnetic measurement type device 10 and a flow type fluorescence measurement type device 20 are shown. However, for the measurement of the intermolecular interaction, a magnetic measurement type device 10 having a flow type device configuration or a fluorescence measurement type device 20 having a batch type device configuration can also be used. The configuration of the measuring device and the configuration of the analysis device are not particularly limited as long as they do not deviate from the gist of the present invention.

Hereinafter, the present invention will be specifically described with reference to examples, but the technical scope of the present invention is not limited thereto.

Generally, in the analysis of intermolecular interactions formed between molecules, a primary reaction in which a ligand and an analyst react in a ratio of 1: 1 is preferably used. In general pharmacokinetic analysis using a living body, antigen-antibody reaction and the like are often analyzed. However, commonly used complete antibodies have two Fab regions in which variable regions are present. Therefore, different kinetic constants are observed depending on the number of Fab regions involved in the antigen-antibody reaction.

In this way, when the ratio of the number of molecules involved in the intermolecular interaction is not constant, one of the ligand and the analyst is greatly excessive and the other is used in order to perform a kinetic analysis as a pseudo-first-order reaction. The measurement is performed diluted. In the following examples, similar to the publication (Richard S. Gaster et al., Nature Nanotechnology, 2011, 6 (5), p.314-320), nanoparticles that act as an analyto-bound label are diluted. The intermolecular interaction was analyzed.

<Example 1>
In Example 1, the intermolecular interaction between the antigen and the antibody was analyzed using magnetic nanoparticles. As the magnetic nanoparticles, a MACS labeling kit (manufactured by Miltenyi Biotec) was used. An anti-biotin antibody was used as the standard analyst. As a test analyst, an anti-CEA antibody, which is an antibody against CEA (CarcinoEmbryonic Antigen) known as a colorectal cancer marker, was used.

In 100 μL of a solution in which 2 mg of magnetic nanoparticles having an active reactive group introduced in N-Hydroxysuccinimide (NHS) dispersed, 10 μL of a solution of 10 mg / mL (pH 8.5) of an anti-biotin antibody and 10 μL of anti-biotin antibody. 10 μL of a solution of CEA antibody was added, and the reaction was carried out at 4 ° C. for 24 hours. Then, a reaction stop solution (Stop Solution) was added to the reaction solution, and the mixture was allowed to stand for 10 minutes to stop the reaction.

Subsequently, the reaction solution was passed through a magnetic column to which a magnet was attached to remove the remaining unreacted antibody. Then, the solution in the magnetic column is replaced with an elute buffer in the magnetized state, the magnet is removed from the magnetic column, and the magnetic nanoparticles (MNP-1) to which the anti-biotin antibody and the anti-CEA antibody are bound are obtained. A solution was obtained.

Further, a solution of magnetic nanoparticles (MNP-2) was obtained in the same manner as the solution of MNP-1 except that the solution of the antibody was added and left at room temperature for 1 hour before the reaction was carried out day and night.

In addition, the ligand was immobilized on the sensor array provided with the magnetic sensor by the following procedure. As a standard ligand, biotinylated bovine serum albumin (BSA) (biotin-BSA) was used. As a test ligand, a gene recombinant of CEA (recombinant CEA) was used.

1. PBS solution of biotin-BSA (0.01% BSA) adjusted to 10 μg / mL, 1 μg / mL, 0.1 μg / mL and 0.01 μg / mL, respectively, in different measurement regions on the sensor array. 5 nL was applied and dried for 1 hour to immobilize biotin-BSA. It can be said that the preferable condition is that the number density of the ligand with respect to the measurement region is smaller than the number density of the nanoparticles bound to the measurement region.

Also, PBS solutions of recombinant CEA (0.01% BSA) adjusted to 10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL, respectively, in different measurement regions on the sensor array. 1.5 nL was applied and dried for 1 hour to immobilize recombinant CEA.

In addition, a solution of BSA adjusted to 1 mg / mL, a solution of anti-mouse antibody adjusted to 1 mg / mL, and BSA-PEG (polyethylene glycolified) adjusted to 1 mg / mL in a measurement region different from the ligand on the sensor array. BSA) solutions were applied, dried and immobilized. BSA is a control sample for baseline correction. Anti-mouse antibody is a positive control capable of binding to both anti-biotin antibody and anti-CEA antibody. BSA-PEG is a negative control that does not bind to both anti-biotin and anti-CEA antibodies.

FIG. 6 is a diagram showing a measurement area of the sensor array used in the first embodiment.
In FIG. 6, the rectangular area indicates the surface of the sensor array, and the round area indicates the measurement area in which the magnetic sensor is arranged. The ligand, control sample, positive control, and negative control are immobilized on the magnetic sensor in a round area.

A1 to A4 in FIG. 6 are biotin-BSA-immobilized regions adjusted to 10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL, respectively. B1 to B5 are regions in which recombinant CEA adjusted to 10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL is immobilized. The shaded area is the region where the control sample BSA is immobilized. The PC is a region in which an anti-mouse antibody, which is a positive control, is immobilized. NC is a region where BSA-PEG, which is a negative control, is immobilized.

The sensor array was mounted on a magnetic measurement device as shown in FIG. 2 after immobilizing a ligand or the like in the sequence shown in FIG. First, the sensor array was immersed in a 5% BSA blocking treatment solution for 15 minutes to block the surface of the sensor array. Then, the sensor array was brought into contact with a sample solution containing magnetic nanoparticles to which the analyte was bound, and the binding phase of the intermolecular interaction was tested. A solution of MNP-1 and a solution of MNP-2 were brought into contact with each other for 30 minutes in the measurement area of the sensor array, and the magnetism during this period was measured over time.

Subsequently, the sample solution in contact with the sensor array was replaced with a phosphate buffer solution (pH 7.4, 0.05% Tween 20) to test the dissociated phase of the intermolecular interaction. A phosphate buffer solution containing no MNP-1 or MNP-2 was brought into contact with the measurement area of the sensor array, and the magnetism during this period was measured over time.

FIG. 7A is a diagram showing the measurement results of the intermolecular interaction to be analyzed in Example 1. FIG. 7B is a diagram showing the measurement results of the intermolecular interaction of the internal standard in Example 1.
In FIGS. 7A and 7B, the vertical axis represents the concentration (ppm) of magnetic nanoparticles converted from the magnetic strength measured in the measurement region of the sensor array, and the horizontal axis represents the measurement time (min) of the bound phase. .. FIG. 7A shows the measurement results of the intermolecular interaction between the recombinant CEA, which is the test ligand, and the anti-CEA antibody, which is the test analyzer. FIG. 7B shows the standard ligand, biotin-BSA, and the standard analyzer, anti-CEA antibody. It is a measurement result of the intermolecular interaction with a biotin antibody.

As shown in FIGS. 7A and 7B, the increase in concentration was measured with the passage of reaction time in both the analysis target and the internal standard. However, in both the measurement result of the analysis target and the measurement result of the internal standard, the concentration of the solution of MNP-2 was lower than that of the solution of MNP-1. It can be said that in the solution of MNP-2, the NHS for immobilizing the analog was inactivated at room temperature, so that the reaction rate of the labeling reaction varied.

Assuming that the intermolecular interaction in Example 1 follows the Langmuir adsorption model (see Patent Document 2), the concentration of the analyte that forms the intermolecular interaction is y, and the maximum of the analysts that can form the intermolecular interaction. the concentration _{n max,} the association rate constant _{K on,} the initial concentration of the analyte _{C o,} when the reaction time was t, the following formula (I) is satisfied.
y = n _max × [1-exp (−K _on・_Co・ t)] ・ ・ ・ (I)

Further, the fitting curve for the equation (I) is approximately expressed by the following equation (II) _{when the response at equilibrium is R eq.}
y = n _max × [1-exp (−R _eq · (t−t ₀ ))] ・ ・ ・ (II)

Therefore, the measurement result of intermolecular interactions in Example 1 to obtain association rate constant K _on approximated by the formula (II). The results are shown in Table 1. The literature values in the table are values indicated by the manufacturer of the anti-biotin antibody.

As shown in Table 1, when the result of MNP-1 and the result of MNP-2 are compared, the binding rate constant obtained from the measurement result of the analysis target and the binding rate constant obtained from the measurement result of the internal standard are obtained. It can be seen that they are equally proportional to each other. Since the binding rate constant obtained from the measurement results of the internal standard of MNP-1 is close to the literature value, the labeling reaction and measurement are correct even for the analysis target tested in the reaction environment substantially the same as the internal standard. It can be said that it is highly likely that it was done. It can be said that the binding rate constant of MNP-1 obtained from such measurement results is close to the true value.

On the other hand, the internal standard binding rate constant of MNP-1 deviates from the literature value. In the solution of MNP-2, it is presumed that the reaction rate of the labeling reaction decreased because the NHS for immobilizing the analyze was inactivated at room temperature. Therefore, in order to confirm the reactivity of the magnetic nanoparticles when left at room temperature, evaluation by fluorescence measurement was also performed.

FIG. 8 is a diagram showing the fluorescence measurement results of the magnetic nanoparticles used in Example 1.
FIG. 8 shows the measurement results of the fluorescence intensity when each of MNP-1 and MNP-2 is fluorescently labeled via the NHS remaining on the magnetic nanoparticles. The spots in the figure are fluorescent images of MNP-1 and MNP-2 that are fluorescently labeled after being fixed on a slide glass.

Each of the MNP-1 solution and the MNP-2 solution was diluted to 1 nM, and 1 μL of each of these diluted solutions was dropped onto a slide glass “APS coat” (manufactured by Matsunami Glass Ind. Co., Ltd.) coated with aminosilane. Then, it was dried for 1 hour and fixed. Then, biotin-labeled R-phycoerythrin "P811" (manufactured by Thermo Fisher Scientific) was diluted 1000-fold with PBS, and the reaction solution was reacted with each of MNP-1 and MNP-2 for 30 minutes for fluorescence. After labeling, the fluorescence image was observed and the fluorescence intensity was measured.

As shown in FIG. 8, not only in the magnetic measurement but also in the fluorescence measurement, the result that the response of MNP-2 was lowered as compared with MNP-1 was obtained. The ratio of the fluorescence intensity of MNP-1 to the fluorescence intensity of MNP-2 is equivalent to the ratio of the internal standard binding rate constant of MNP-1 to the internal standard binding rate constant of MNP-2 (see Table 1). It has become. Since a _{K on = R eq / C o} , kinetic constants determined from the measurement results, it can be said that it is possible to correct the normal numerical correction factor corresponding to the ratio (86%). When calculated at + 86%, the result is small, and when calculated at -86%, the result is large like normal MNP-1.

Therefore, it can be said that the validity of the internal standard can be confirmed by utilizing the sensing characteristics of the multiplex by using nanoparticles in which a plurality of types of analysts are bound. Even when a single analysis sample is used as the analysis target, multiple types of intermolecular interactions will be measured, and by confirming the validity of the internal standard, the reliability of the measurement results of the analysis target will be measured. Has been proven to be able to be guaranteed.

Generally, a method for labeling a protein such as an antibody is roughly classified into an amino type in which a labeled compound is bound to an amino group and a thiol type in which a labeled compound is bound to a thiol group. The amino type is suitable for binding low molecular weight labeled compounds, and the thiol type is suitable for binding high molecular weight labeled compounds. However, since the labeled compound itself is often unstable, the reaction rate of the labeling reaction tends to vary easily due to slight differences in the reaction environment or the like.

Further, when measurement is performed using a protein as an analyst, it is necessary to adjust the concentration of the analytical test to be tested by measuring ultraviolet absorption or the like. However, when contaminants are mixed in the sample solution containing the analyte, the apparent concentration of the analyte appears at a higher concentration than the target concentration, so that a concentration error with respect to the target concentration tends to occur.

In addition, when a protein is used as an analyzer for measurement, it is necessary to reduce the disulfide bond using dithiothreitol (DTT) or β-mercaptoethanol when binding the labeled compound to the thiol group. However, recrosslinking of disulfide bonds is likely to occur in air or the like. In such a case, the reaction rate of the labeling reaction becomes low, and the apparent concentration of the analyst appears at a concentration lower than the target concentration, so that a concentration error with respect to the target concentration tends to occur.

In addition, when a protein is used as an analyst for measurement, an active group is often introduced by NHS or the like when the labeled compound is bound to the molecular chain. However, the active group used for binding is easily inactivated depending on the reaction environment and storage environment, and the apparent concentration of the analyte appears at a concentration lower than the target concentration, so that a concentration error with respect to the target concentration tends to occur. ..

Therefore, when measuring intermolecular interactions involving proteins, it is desirable to reduce variations in reaction conditions and reaction rates of labeling reactions as much as possible. Since it is not easy to confirm the variation in the reaction rate of the labeling reaction and it becomes a large factor of the measurement error, it can be said that the test based on the measurement result of the internal standard as shown in Example 1 is indispensable. Further, when the kinetic constant of the internal standard is known, the analysis target can be corrected based on the gap between the internal standard and the true value regardless of the measurement error, so that more accurate analysis can be performed. It can be said that it will be possible.

<Example 2>
In Example 2, the intermolecular interaction between protein and nucleic acid was analyzed using fluorescent nanoparticles. Fluorescent nanoparticles include a fluorescently labeled secondary antibody of the protein labeling kit "Alexa Fluor 633" (manufactured by Thermo Fisher Scientific) and a protein labeling kit "Qdot 705 ITK Amino (PEG) Quantum Dots" (manufactured by Thermo Fisher). Quantum dot particles were used. Biotin was used as the standard analyst. DNA polymerase was used as the test analyst.

0.5 mL of PBS solution of 2 mg / mL DNA polymerase and _{50 μL of Alexa kit solution (pH 8.3, in NaHCO 3} ) were mixed and reacted at room temperature for 1 hour. Then, the reaction solution was transferred to a filtration tube "Centricon" (MW: 10000 type, manufactured by Merckmillipore), centrifuged, and washed with PBS 3 to 5 times to remove unreacted fluorescently labeled secondary antibody. Then, the filter medium was collected with PBS to obtain a solution of DNA polymerase (Alexa-POL) to which the fluorescently labeled secondary antibody was bound.

Further, to a 10 mg / mL Qdot solution (pH 8.3, in 50 mM boret buffer) containing 2 nmol quantum dot particles, a PBS solution of DNA polymerase and 10 μL of the same amount of NHS esterified biotin (biotin-NHS) were added. The reaction was carried out at 37 ° C. for 1 hour. Then, the reaction solution was dropped onto a filtration tube "Amicon Ultra" (MW: 100 kDa type, manufactured by Merckmillipore), and washing with 1 × PBS (pH 7.4) was repeated 3 to 5 times to obtain unreacted quantum dot particles. The DNA polymerase was removed. Then, the filter medium was recovered with PBS to obtain a solution of quantum dot particles (Qdot-POL) to which biotin and DNA polymerase were bound.

FIG. 9 is a diagram schematically showing the quantum dot particles used in Example 2.
In FIG. 9, reference numeral 60 is a quantum dot particle, reference numeral 61 is a core portion, reference numeral 62 is a shell portion, reference numeral 63 is a coating, reference numeral 64 is biotin, and reference numeral 65 is a DNA polymerase.

As shown in FIG. 9, the quantum dot particles (Qdot-POL) 60 are core-shell type quantum dot particles, and have a property of not fading even when continuously irradiated with excitation light. Biotin 64 and DNA polymerase 65 are covalently bonded to the coating 63 formed of the resin.

On the other hand, the ligand was immobilized on the microchannel type array chip by the following procedure. An anti-biotin antibody was used as the standard ligand. As the test ligand, four types of DNA aptamer candidates having different base lengths in the range of 20 to 40 bp were used.

A 10 mg / mL salmon sperm DNA solution (500 to 1000 bp, manufactured by Gene Mark) diluted 100-fold with PBS is mixed with each of the four types of DNA aptama candidates, and these mixed solutions are microchannel type. It was applied to different measurement areas on the array chip. Then, each DNA aptamer candidate was immobilized by irradiating with ultraviolet rays having a wavelength of 254 nm for 5 seconds.

When the DNA is 20 bp, the length is about 6.8 nm, which is equivalent to the size of the antibody. Using large-sized salmon sperm DNA as a carrier allows the ligand to react substantially 1: 1.

FIG. 10 is a diagram showing the reaction of the DNA polymerase used in Example 2.
In FIG. 10, reference numeral 71 is a DNA polymerase, reference numeral 72 is a single difference DNA, reference numeral 73 is a DNA prima (aptamer), and reference numeral 74 is a complementary strand DNA. The upper figure is the dissociated DNA polymerase and the leading strand DNA, the lower right figure is the bound DNA polymerase and the leading strand DNA, and the lower left figure is the synthesized double-stranded DNA.

As shown in the upper and lower right of FIG. 10, a DNA primer 73 having a complementary sequence specifically binds to the single-strand DNA 72 dissociated from the double strand on a specific sequence. The DNA polymerase 71 can specifically bind to the conjugate of the single-difference DNA 72 and the DNA primer 73 formed in this way.

As shown in the lower left of FIG. 10, in the case of the leading strand, the DNA polymerase 71 bound to the conjugate extended the daughter strand from the 3'end of the DNA primer 73, and the single-difference DNA 72 and the complementary strand DNA 74 were associated with each other. It forms double-stranded DNA. The DNA polymerase 71 then dissociates from the double-stranded DNA.

DNA polymerase repeats such a cycle of binding and dissociation to replicate DNA. When DNA polymerase is used as an analyzer, the intermolecular interaction between a protein and a nucleic acid can be analyzed using a DNA aptamer candidate that has been mixed with a carrier to form a replication initiation conjugate as a ligand. In general, when screening for DNA polymerase, those with fast binding and dissociation to DNA are preferred.

FIG. 11 is a diagram showing a measurement area of the array chip used in the second embodiment.
In FIG. 11, the white area indicates the channel of the microchannel type array chip, and the round area indicates the measurement area in the channel. The ligand and control sample are immobilized on the surface of a round region within the channel. The microchannel array chip is made of Polydimethylsiloxane (PDMS) and is molded from a non-fluorescent material.

1 to 4 in FIG. 11 are regions in which four types of DNA aptamer candidates 1 to 4, which are test ligands, are immobilized. The net line is the region where the anti-biotin antibody, which is a standard ligand, is immobilized. R is the area of the blocking-treated substrate for the control experiment.

The microchannel type array chip was placed in a CCD imaging fluorescence microscope after immobilizing a ligand or the like in the sequence shown in FIG. First, a blocking treatment liquid of 5% BSA was flowed through a microchannel type array chip at 0.1 μL / sec, and the inner surface of the channel was blocked. Then, a solution of Analite was flowed through this channel to test the binding phase of the intermolecular interaction. Each channel was flushed with 0.1 nM Alexa-POL PBS solution and 0.1 nM Qdot-POL PBS solution at 0.5 μL / sec and contacted for 30 minutes, during which fluorescence was fluoresced over time. It was measured.

Subsequently, the liquid flowing through the channel was changed to PBS and the dissociated phase of the intermolecular interaction was tested. PBS containing no Alexa-POL or Qdot-POL was flowed through the channel at 0.5 μL / sec, and the fluorescence during this period was measured over time.

FIG. 12A is a diagram showing the measurement results of the intermolecular interaction using the fluorescently labeled secondary antibody in Example 2. FIG. 12B is a diagram showing the measurement results of the intermolecular interaction using the quantum dot particles in Example 2.
In FIGS. 12A and 12B, the vertical axis represents the fluorescence intensity measured in the measurement region of the microchannel type array chip, and the horizontal axis represents the measurement time (sec) of the bound phase. FIG. 12A shows the measurement results of one of four types of DNA aptamer candidates (DNA aptamer candidate 1) and DNA polymerase (Alexa-POL) to which a fluorescently labeled secondary antibody was bound. FIG. 12B shows four types of DNA. It is a measurement result of one of the aptamer candidates (DNA aptamer candidate 1) and a quantum dot particle (Qdot-POL) in which biotin and DNA polymerase are bound.

In addition, FIG. 12A and FIG. 12B show the measurement result (◯: continuous irradiation) when the measurement region is continuously irradiated with the excitation light, and the measurement region is intermittently irradiated with the excitation light every 15 sec. The measurement result (Δ: 15s interval) is shown. This is because a general fluorescent dye is used for Alexa-POL, and fluorescence fading, quenching, etc. are expected.

As shown in FIG. 12A, the DNA polymerase (Alexa-POL) to which the fluorescently labeled secondary antibody is bound is fluorescent when the excitation light is intermittently irradiated as compared with the case where the excitation light is continuously irradiated. The strength has increased. When the excitation light was continuously irradiated, the fluorescence intensity attenuated with the lapse of the measurement time. Continuous irradiation of excitation light may have an effect on fluorescence fading, quenching, etc., or an effect on intermolecular interaction.

On the other hand, as shown in FIG. 12B, with respect to the quantum dot particles (Qdot-POL) in which biotin and DNA polymerase are bound, there are cases where the excitation light is continuously irradiated and cases where the excitation light is intermittently irradiated. No difference in fluorescence intensity was observed. When quantum dots are used, it can be said that measurement errors due to optical causes are unlikely to occur.

FIG. 13 is a diagram showing the result of curve fitting the measurement result of the intermolecular interaction using the fluorescently labeled secondary antibody in Example 2.
In FIG. 13, the vertical axis represents the fluorescence intensity measured in the measurement region of the microchannel type array chip, and the horizontal axis represents the measurement time (sec) of the binding phase. The plot of ● is the measurement result (actual value) of one of the four types of DNA aptamer candidates (DNA aptamer candidate 1) and the DNA polymerase (Alexa-POL) to which the fluorescently labeled secondary antibody is bound, and the plot of 〇. Is a fitting curve approximately obtained based on the Langmuir adsorption model.

As shown in FIG. 13, in the first half of the measurement before the measurement time: around 750 sec, the fitting curve according to the Langmuir adsorption model was well applied to the actual value. However, in the latter half of the measurement after the measurement time: around 750 sec, the fitting curve deviated from the actual value. Result of obtaining a binding rate constant _{K on} the DNA aptamer candidates 1 from the fitting curve _became K on = 1.4E + 07.

In the actual value shown in FIG. 13, the fluorescence intensity decreases with the lapse of the measurement time. From this result, it is inferred that some factors contrary to Langmuir's adsorption model are working. On the other hand, in the case of quantum dot particles (Qdot-POL), the fitting curve according to Langmuir's adsorption model fits well with respect to the actual value, and the factor of measurement error is reduced.

The measurement results of the intermolecular interaction in Example 2 were globally fitted with a plurality of measurement results for each of the DNA aptamer candidates 1 to 4, and the binding rate constant _Kon was obtained by approximating with the equation (II). The results are shown in Table 2. The test values in the table are values obtained by conventional SPR analysis.

As shown in Table 2, the coupling rate constants obtained from the measurement results of the internal standard are close to the test values. Therefore, the labeling reaction and the intermolecular interaction of the DNA aptamer candidates 1 to 4 to be analyzed have been correctly measured, and the DNA polymerase and the DNA aptamer have been measured in the same manner as the intermolecular interaction between the biotin and the anti-biotin antibody. It can be said that the intermolecular interaction with Candidates 1 to 4 was also properly tested. It can be said that the binding rate constants of the DNA aptamer candidates 1 to 4 obtained from such measurement results are all likely to be close to the true values.

1 Test Anna Reed (1st molecule)
2 Standard Analytes (2nd molecule)
3 Test ligand (third molecule)
4 Standard ligand (4th molecule)
5 Nanoparticles 6 Measurement area (area)
7 Measurement area (area)
8 Complex 9 Complex 10 Magnetic measurement device (analysis device)
11 Sensor array (magnetic sensor)
12 Circuit board 13 Movable support device 14 Digitizer 15 Data analysis device 16 Magnetic field generator 17 Liquid tank 18 Test sample liquid 20 Fluorescence measurement type device (analysis device)
21 Array plate 22 Flow path forming member 23 Temperature control device 24 Test sample liquid channel 25 Liquid feeding unit 26 Liquid feeding port valve 31 Excitation light source 32 Condensing lens 33 Dichroic mirror 34 Objective lens 35 Optical filter 36 Imaging lens 37 Image sensor 40 Analysis control device (analysis unit, calculation unit, storage unit, display unit)
100 array board (board)
200 sample solution

Claims (17)

A sample solution containing nanoparticles containing the first molecule to be analyzed and the second molecule as an internal standard is brought into contact with a substrate having a region in which the third molecule is fixed and a region in which the fourth molecule is fixed. The first step and
The second step of measuring the intermolecular interaction between the first molecule and the third molecule and the intermolecular interaction between the second molecule and the fourth molecule, and
The measurement result of the intermolecular interaction between the first molecule and the third molecule to be analyzed is analyzed based on the measurement result of the intermolecular interaction between the second molecule and the fourth molecule, which is an internal standard. A method for analyzing intermolecular interactions including the third step. The method for analyzing an intermolecular interaction according to claim 1.
In the third step, the measurement result of the intermolecular interaction between the second molecule and the fourth molecule, which is an internal standard, and the previously obtained intermolecular interaction between the second molecule and the fourth molecule. The normality or abnormality of the measurement result of the intermolecular interaction between the first molecule and the third molecule to be analyzed is compared with the most probable value and based on the similarity between the measurement result and the most probable value. A method for analyzing molecular interactions that test sex. The method for analyzing an intermolecular interaction according to claim 1.
In the third step, the measurement result of the intermolecular interaction between the second molecule and the fourth molecule, which is an internal standard, and the previously obtained intermolecular interaction between the second molecule and the fourth molecule. Intermolecular that compares the most probable value and corrects the measurement result of the intermolecular interaction between the first molecule and the third molecule to be analyzed based on the similarity between the measurement result and the most probable value. How to analyze the interaction. The method for analyzing an intermolecular interaction according to claim 3.
In the third step, the measurement result of the intermolecular interaction between the second molecule and the fourth molecule, which is an internal standard, and the previously obtained intermolecular interaction between the second molecule and the fourth molecule. When the difference between the most probable value and the most probable value exceeds a predetermined value, the measurement result of the intermolecular interaction between the first molecule and the third molecule to be analyzed is determined by the measurement result and the correction coefficient obtained from the most probable value. A method for analyzing molecular interactions to be corrected. The method for analyzing an intermolecular interaction according to claim 1.
The nanoparticles are magnetic or magnetically labeled bodies.
The substrate is a sensor array having a first magnetic sensor to which the third molecule is fixed and a second magnetic sensor to which the fourth molecule is fixed.
The first magnetic sensor and the second magnetic sensor are provided on the substrate at positions where they can come into contact with the same liquid, and can measure a magnetic field under the same conditions.
The intermolecular interaction between the first molecule and the third molecule is measured based on the magnetic strength of the nanoparticles measured by the first magnetic sensor.
The intermolecular interaction between the second molecule and the fourth molecule is a method for analyzing an intermolecular interaction measured based on the magnetic strength of the nanoparticles measured by the second magnetic sensor. The method for analyzing an intermolecular interaction according to claim 5.
The first magnetic sensor and the second magnetic sensor are GMR sensors or TMR sensors, which are methods for analyzing intermolecular interactions. The method for analyzing an intermolecular interaction according to claim 1.
The nanoparticles are fluorescent or fluorescently labeled, and are
The intermolecular interaction between the first molecule and the third molecule and the intermolecular interaction between the second molecule and the fourth molecule are measured based on the intensity of fluorescence emitted from the nanoparticles. A method for analyzing intermolecular interactions. The method for analyzing an intermolecular interaction according to claim 1.
The intermolecular interaction between the first molecule and the third molecule to be analyzed includes the interaction between nucleic acids by hybridization, the interaction between proteins or peptides, the interaction between proteins or peptides and nucleic acids, and enzymes. A method for analyzing an intermolecular interaction that is one of a substrate interaction, an antigen-antigen interaction, and a receptor-ligand interaction. A substrate having a region in which a third molecule is fixed and a region in which a fourth molecule is fixed, to which a sample solution containing nanoparticles containing a first molecule to be analyzed and a second molecule as an internal standard is bound is brought into contact with each other. When,
A measuring unit for measuring the intermolecular interaction between the first molecule and the third molecule and the intermolecular interaction between the second molecule and the fourth molecule.
The measurement result of the intermolecular interaction between the first molecule and the third molecule to be analyzed is analyzed based on the measurement result of the intermolecular interaction between the second molecule and the fourth molecule, which is an internal standard. An analysis device for intermolecular interactions, which comprises an analysis unit. The intermolecular interaction analyzer according to claim 9.
An intermolecular interaction analysis device including a storage unit that stores the most probable value of the intermolecular interaction between the second molecule and the fourth molecule obtained in advance. The intermolecular interaction analysis apparatus according to claim 10.
Comparison between the measurement result of the intermolecular interaction between the second molecule and the fourth molecule, which is an internal standard, and the most probable value of the intermolecular interaction between the second molecule and the fourth molecule obtained in advance. Then, based on the similarity between the measurement result and the most probable value, a calculation unit that tests the normality or anomaly of the measurement result of the intermolecular interaction between the first molecule and the third molecule to be analyzed. An intermolecular interaction analyzer comprising. The intermolecular interaction analysis apparatus according to claim 11.
Difference between the measurement result of the intermolecular interaction between the second molecule and the fourth molecule, which is an internal standard, and the most probable value of the intermolecular interaction between the second molecule and the fourth molecule obtained in advance. Is an intermolecular interaction analyzer that determines that the measurement result is abnormal and displays the determination result on the display unit when the value exceeds a predetermined value. The intermolecular interaction analysis apparatus according to claim 10.
Comparing the measurement result of the intermolecular interaction between the second molecule and the fourth molecule, which is an internal standard, with the most probable value of the intermolecular interaction between the second molecule and the fourth molecule obtained in advance. Then, an intermolecular interaction including a calculation unit for correcting the measurement result of the intermolecular interaction between the first molecule and the third molecule to be analyzed based on the similarity between the measurement result and the most probable value. Analytical device. The intermolecular interaction analysis apparatus according to claim 13.
Difference between the measurement result of the intermolecular interaction between the second molecule and the fourth molecule, which is an internal standard, and the most probable value of the intermolecular interaction between the second molecule and the fourth molecule obtained in advance. When exceeds a predetermined value, the intermolecular interaction that corrects the measurement result of the intermolecular interaction between the first molecule and the third molecule to be analyzed by the measurement result and the correction coefficient obtained from the most probable value. Analytical device. The intermolecular interaction analyzer according to claim 9.
The substrate is a sensor array having a first magnetic sensor to which the third molecule is fixed and a second magnetic sensor to which the fourth molecule is fixed.
The first magnetic sensor and the second magnetic sensor are provided on the substrate at positions where they can come into contact with the same liquid, and are an intermolecular interaction analyzer capable of detecting a magnetic field under the same conditions. The intermolecular interaction analysis apparatus according to claim 15.
The first magnetic sensor and the second magnetic sensor are GMR sensors or TMR sensors, which are intermolecular interaction analyzers. The intermolecular interaction analyzer according to claim 9.
The measuring unit is an intermolecular interaction analysis device that is an image sensor capable of detecting fluorescence under the same conditions.

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