Analytical apparatus and analytical method

Abstract

We provide analytical equipment that enables highly accurate chemical analysis of various substances. [Solution] The analytical apparatus 100 of the present invention comprises a first substrate 101, a second substrate 102 arranged so that one side of the first substrate 101 faces the second substrate 102, an ultrasonic generator 103 connected to the first substrate 101 or the second substrate 102, a medium 104 that fills the space between one side 101a of the first substrate and one side 102a of the second substrate and propagates ultrasonic waves, a chain-like molecule 105 in the space filled with the medium 104 with one end 105a bonded to one side 101a of the first substrate, and microparticles 106 in the space filled with the medium 104 that are bonded to the other end 105b of the chain-like molecule and have sensitivity to ultrasonic waves S generated by the ultrasonic generator 103, wherein the binding force between the microparticles 106 and the target substance is adjusted so that the microparticles 106 are replaced by the supplied target substance and dissociated from the chain-like molecule 105.

Description

[Technical Field]

[0001] The present invention relates to an analytical apparatus and an analytical method. [Background technology]

[0002] Techniques for measuring specific target molecules using microparticles are known. For example, Patent Document 1 discloses a technique for measuring target molecules from changes in microparticle density based on the dissociation behavior of gold nanoparticle-modified microparticles from a substrate in a combined field of ultrasonic radiation force and gravity. However, in this technique, it is difficult to measure the changes in microparticle density with high accuracy because the chemical properties of the target molecule may change due to surface modification of the particles.

[0003] Non-patent document 1 discloses a technique for measuring target molecules without modification. This technique measures DNA based on the intermolecular interaction forces that occur when the target molecule's DNA dissociates from microparticles. The intermolecular interaction is adjusted by the number of bases that contribute to the binding of microparticles to DNA. However, since this technique is limited to DNA as the target molecule, there is a need for a technique that can measure the number of molecules in various target molecules other than DNA. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2022-172633 [Non-patent literature]

[0005] [Non-Patent Document 1] A. Miyagawa et al., Talanta 268 (2024) 125369 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] This invention has been made in view of the above circumstances, and aims to provide an analytical apparatus and analytical method that enable highly accurate chemical (quantitative) analysis of various substances. [Means for solving the problem]

[0007] To solve the above problems, the present invention employs the following means.

[0008] (1) An analytical apparatus according to one aspect of the present invention is an analytical apparatus for analyzing the intermolecular interaction forces of a target substance, comprising: a first substrate; a second substrate arranged such that one surface faces the first substrate; an ultrasonic generator connected to or integrated with the first substrate or the second substrate; a medium disposed between one surface of the first substrate and one surface of the second substrate for propagating ultrasonic waves; a chain-like molecule in the medium with one end bonded to one surface of the first substrate; and microparticles in the medium bonded to the other end of the chain-like molecule and having sensitivity to ultrasonic waves generated by the ultrasonic generator, wherein the bonding force between the microparticles and the target substance is adjusted such that the microparticles are replaced by the supplied target substance and dissociated from the chain-like molecule.

[0009] (2) In the analytical apparatus described in (1) above, the analytical apparatus for analyzing the concentration of the target substance may be adjusted such that the binding force between the microparticles and the chain-like molecules is weaker than the binding force between the target substance and the chain-like molecules.

[0010] (3) In the analytical apparatus described in (1) above, the analytical apparatus for analyzing the binding force of the target substance may be adjusted so that the binding force between the microparticles and the chain-like molecules is stronger than the binding force between the target substance and the chain-like molecules.

[0011] (4) In the analyzer according to any one of (1) to (3) above, the microparticles may be bound to the other end side of the chain-like molecule via a DNA molecule.

[0012] (5) In the analyzer according to any one of (1) to (4) above, the chain-like molecule may be an aptamer.

[0013] (6) In the analyzer according to any one of (1) to (5) above, the chain-like molecule may be an antibody or an antigen that undergoes an antigen-antibody reaction.

[0014] (7) In the analyzer according to any one of (1) to (6) above, the chain-like molecule may be a host molecule or a guest molecule that undergoes a host-guest reaction.

[0015] (8) In the analyzer according to any one of (1) to (7) above, it is preferable that the particle size of the microparticles is 5 μm or more and 50 μm or less.

[0016] (9) In the analyzer according to any one of (1) to (8) above, it is preferable that the distance between adjacent chain-like molecules is not less than the particle size of the microparticles.

[0017] (10) In the analyzer according to any one of (1) to (9) above, it is preferable that the first substrate and the second substrate have permeability to visible light.

[0018] (11) In the analyzer according to any one of (1) to (10) above, a recess is formed on one surface of the second substrate, and the space filled with the medium is the internal space of the recess.

[0019] (12) An analytical method according to one aspect of the present invention is an analytical method using the analytical apparatus described in (2) above, comprising the steps of: applying a voltage to the ultrasonic generator and generating an ultrasonic standing wave between the first substrate and the second substrate, and balancing the ultrasonic radiation force acting on the microparticles with other forces acting on the microparticles; supplying a reference first target substance into the medium and applying a predetermined voltage, measuring the ratio A at which the microparticles dissociate from the chain molecules multiple times by changing the concentration A of the supplied first target substance, and determining the relationship between the concentration A and the ratio A; and supplying a second target substance to be analyzed into the medium, measuring the ratio B at which the microparticles dissociate from the chain molecules, and analyzing the concentration B corresponding to the ratio B by referring to the relationship between the concentration A and the ratio A.

[0020] (13) An analytical method according to one aspect of the present invention is an analytical method using the analytical apparatus described in (3) above, comprising the steps of: applying a voltage to the ultrasonic generator and generating an ultrasonic standing wave between the first substrate and the second substrate, and balancing the ultrasonic radiation force acting on the microparticles with other forces acting on the microparticles; supplying a reference first target substance into the medium and applying a predetermined voltage, measuring the ratio C at which the microparticles dissociate from the chain molecules multiple times by changing the binding constant C of the microparticles to the chain molecules, and determining the relationship between the binding constant C and the ratio C; and supplying a second target substance to be analyzed into the medium, measuring the ratio D at which the microparticles dissociate from the chain molecules, and analyzing the binding constant D corresponding to the ratio D by referring to the relationship between the binding constant C and the ratio C.

[0021] (14) The analytical method according to either (12) or (13) further comprises the steps of: measuring a first voltage applied to the ultrasonic generator in order to dissociate all of the microparticles before supplying the first target substance; and measuring a second voltage applied to the ultrasonic generator in order to dissociate all of the microparticles after supplying the first target substance, wherein when measuring the ratio A, it is preferable that the voltage applied to the ultrasonic generator be a voltage intermediate between the first voltage and the second voltage. [Effects of the Invention]

[0022] According to the present invention, it is possible to provide an analytical apparatus and analytical method that enable highly accurate chemical (quantitative) analysis of various substances. [Brief explanation of the drawing]

[0023] [Figure 1] This figure schematically shows the configuration of the analytical apparatus according to the first embodiment of the present invention. [Figure 2] This is an enlarged cross-sectional view of the vicinity of one surface of the first substrate in the analytical apparatus of the same embodiment. [Figure 3] This figure illustrates the state in which an ultrasonic standing wave is generated between the first substrate and the second substrate in the analytical apparatus of the same embodiment. [Figure 4] This graph shows the voltage dependence of the dissociation rate of microparticles in the analytical apparatus of the same embodiment. [Figure 5] This is an enlarged perspective view of the vicinity of one surface of the first substrate when the target substance is supplied in the analytical apparatus of the same embodiment. [Figure 6] This graph shows the relationship between the concentration of the target substance and the dissociation rate of microparticles in the analytical apparatus of the same embodiment. [Figure 7] This graph shows the voltage dependence of the dissociation rate of microparticles obtained in Examples 1 to 10. [Figure 8]This graph shows the relationship between the concentration of the target substance and the dissociation rate of microparticles obtained in Examples 1 to 10. [Modes for carrying out the invention]

[0024] Hereinafter, an analytical apparatus and analytical method according to embodiments to which the present invention is applied will be described in detail with reference to the drawings. Note that, for convenience, the drawings used in the following description may show enlarged versions of characteristic parts to make the features easier to understand, and the dimensional ratios of each component may not be the same as in reality. Furthermore, the materials, dimensions, etc., exemplified in the following description are merely examples, and the present invention is not limited to them. It can be implemented with appropriate modifications without altering the essence of the invention.

[0025] [First Embodiment] <Analyzer> Figure 1 is a schematic diagram showing the configuration of an analytical apparatus 100 according to the first embodiment of the present invention. The analytical apparatus 100 is an analytical apparatus that chemically (quantitatively) analyzes a target substance to be analyzed. The target substance is not particularly limited and can be, for example, amino acids such as arginine, metal ions, small organic molecules, proteins, sugars, cells, etc. The analytical apparatus 100 mainly comprises a first substrate 101, a second substrate 102, an ultrasonic generator 103, a medium 104, chain molecules 105, and microparticles 106. The first substrate 101 and the second substrate 102 are arranged so that one surface of each other faces each other, and the medium 104, chain molecules 105, and microparticles 106 are arranged between one surface 101a of the first substrate and one surface 102a of the second substrate. The ultrasonic generator 103 is arranged to be connected to or integrated with the first substrate 101 or the second substrate 102 so that the generated ultrasonic waves can be applied to the second substrate 102.

[0026] The first substrate 101 is made of a material that is transparent to electromagnetic waves (such as visible light) because it is necessary to transmit a predetermined electromagnetic wave (such as visible light) when observing a target material. Examples of such materials include glass, polystyrene, and acrylic, with quartz glass being particularly preferred. From the viewpoint of transmitting electromagnetic waves, it is preferable that both one surface 101a and the other surface 101b opposite to it of the first substrate are substantially flat.

[0027] The second substrate 102 is made of a material that is transparent to ultrasonic waves, as it is necessary to transmit the ultrasonic waves supplied from the ultrasonic generator 103 when generating an ultrasonic radiation force between it and the first substrate 101. Examples of such materials include glass, polystyrene, and acrylic. It is preferable that the other surface 102b of the second substrate, opposite to one surface 102a, is substantially flat.

[0028] The ultrasonic generator 103 is a device that generates ultrasonic waves and includes a function generator 107, a power amplifier 108, an oscilloscope 109, a transducer 110, etc. The function generator 107 generates a predetermined electrical signal, the power amplifier 108 amplifies this electrical signal, the amplified electrical signal is confirmed by the oscilloscope 109, and the transducer 110 converts it into ultrasonic waves.

[0029] Figure 1 illustrates a configuration in which the transducer 110 of the ultrasonic generator 103 is in contact with (directly connected to) the other surface 102b of the second substrate, or is indirectly connected by having a fixing member such as an adhesive, a buffer material that attenuates or amplifies the amplitude, etc. in between. Alternatively, the first substrate 101 or the second substrate 102 may be made of a substrate such as a piezoelectric material that has an ultrasonic generating function, and the substrate may be integrated with the ultrasonic generator 103.

[0030] The ultrasonic generator 103 in Figure 1 generates ultrasonic waves in a predetermined area between the first substrate 101 and the second substrate 102 via the second substrate 102. In the area where ultrasonic waves are generated, one surface of the first substrate 101 and the second substrate 102 are spaced apart, and the distance between the surfaces is designed so that the generated ultrasonic waves become standing waves.

[0031] The medium 104 fills the space between one surface 101a of the first substrate and one surface 102a of the second substrate. The medium 104 is a substance that propagates ultrasonic waves, and examples of materials for the medium 104 include water, ethanol, and glycerol. The material of the medium 104 is selected considering the distance between the surfaces of the first substrate 101 and the second substrate 102 so that the ultrasonic waves become standing waves.

[0032] Figure 1 illustrates a case where a recess 102c is formed on one surface 102a of the second substrate, and the portion of surface 102a other than the recess 102c is in contact (joined, bonded) with one surface 101a of the first substrate 101. In this case, the medium 104 fills only the internal space of the recess 102c. The first substrate 101 and the second substrate 102 may be integrated.

[0033] The chain molecule 105 is formed by linking multiple atoms in a space filled with the medium 104 so as to extend in approximately one direction. Figure 2 is an enlarged cross-sectional view of region R1 near one surface 101a of the first substrate in the analytical apparatus 100 of Figure 1. One end 105a of the chain molecule is bonded (chemically bonded) to one surface 101a of the first substrate. The length of the chain molecule 105 is not particularly limited, but it is preferable that the other end 105b of the chain molecule is far enough away from one surface 101a that it is not affected by intermolecular interaction forces of the first substrate 101. Furthermore, it is preferable that the length of the chain molecule 105 is such that it does not become entangled with other adjacent chain molecules 105. As the chain molecule 105, for example, one consisting of 20 to 60 bases is preferred, and the length of such a chain molecule 105 is about 6.8 nm to 20.4 nm.

[0034] The microparticle 106 is bound to the other end 105b of the chain molecule by intermolecular interactions in the space filled with the medium 104. Here, the microparticle 106 is bound to the other end 105b of the chain molecule via the DNA molecule 113. The strength of the bond can be adjusted by changing the number of bases that contribute to the binding.

[0035] The microparticles 106 are sensitive to ultrasound generated by the ultrasound generator 103 and are particles that are attracted to the nodes of the standing waves of ultrasound by the ultrasound radiation force. The particle size of the microparticles 106 is on the order of micrometers, preferably 5 μm or more and 50 μm or less, and more preferably 10 μm or more and 30 μm or less.

[0036] The binding force between the microparticles 106 and the chain molecules 105 is adjusted so that the microparticles 106 dissociate by replacing the target substance supplied into the medium 104. In this embodiment, assuming an analytical device for analyzing the concentration of the target substance, the binding force between the microparticles 106 and the chain molecules 105 is adjusted to be weaker than the binding force between the target substance and the chain molecules 105.

[0037] The materials for the microparticles 106 and chain molecules 105 are not particularly limited, as long as the combination of materials results in a binding force (intermolecular interaction force) between the microparticles 106 and chain molecules 105 that is weaker than the binding force between the target substance being analyzed and the chain molecules 105. For example, when the target substance is arginine, the chain molecules 105 can be anything that specifically interacts (binds) to the target substance, such as an aptamer, an antibody or antigen that undergoes an antigen-antibody reaction, a host molecule or guest molecule that undergoes a host-guest reaction, or a protein that undergoes a vidin-biotin interaction. As the microparticles 106, polystyrene particles, acrylic particles, silica particles, etc., can be used. When the chain molecules 105 are aptamers, the aptamers have a DNA sequence that can interact with the target substance. When the chain-like molecule 105 is used as an antibody or antigen for antigen-antibody reaction, examples of antibodies include biomolecules such as cedar pollen antibody Cry j1, dust mite allergen antibody Der p1, and cat allergen antibody Fel d1, while examples of antigens include biomolecules such as cedar pollen antigen SBP, dust mite antigen, and cat antigen.

[0038] Figure 3 is an enlarged view of the region R2 near a single microparticle 106 in the analytical apparatus 100 of Figure 1, with an ultrasonic standing wave S generated between the first substrate 101 and the second substrate 102. It is preferable that the standing wave S is generated such that it contains only one node. The microparticle 106 is subjected to ultrasonic radiation force F ac As a result, it is pulled toward the position of node S1 of the standing wave. As shown in Figure 3, the ultrasonic radiation force F when the microparticle 106 is on the side (below) the first substrate 101 closer to node S1 of the standing wave. ac This pulls the microparticle 106 toward the second substrate 102. The ultrasonic radiation force F when the microparticle 106 is on the side (above) the second substrate 102 relative to the standing wave node S1. ac This pulls the microparticle 106 toward the first substrate 101.

[0039] For microparticles 106, the ultrasonic radiation force F ac In addition, there is a sinking force F generated by the gravitational field. sed, the intermolecular force F due to the intermolecular interaction with the binding partner acts. The ultrasonic radiation force F bind , and the sedimentation force F ac are represented by the following equations (1) and (2), respectively. sed

[0040]

Equation

[0041]

Equation

[0042] The definitions of the symbols in the above equations (1) and (2) are as follows. r is the radius of the microparticle. k is the wave number of the ultrasonic wave (2π / λ). λ is the wavelength of the ultrasonic wave. α is a device-dependent parameter. V is the voltage applied to the ultrasonic generator (transducer). ρ * is the density of the microparticle. ρ is the density of the medium. γ * is the compressibility of the microparticle. γ is the compressibility of the medium. z is the distance from the node of the standing wave of the ultrasonic wave. g is the acceleration due to gravity.

[0043] When the ultrasonic radiation force F ac , the sedimentation force F sed , and the intermolecular interaction force F bind are in equilibrium, their resultant force becomes zero, and the following equation (3) holds. F ac + F sed + F bind = 0 (3)

[0044] The voltage V that satisfies the above equation (3) is the upper limit voltage for maintaining the binding between the chain molecule 105 and the microparticle 106, and the intermolecular interaction force F​bind It is a function of this. If the voltage applied to the ultrasonic generator 103 is increased above this upper limit voltage, the balance of the three forces is disrupted, and the microparticles 106 dissociate from the chain molecules 105.

[0045] The dissociation state of the microparticles 106 can be observed using a microscope 111 positioned opposite the other surface 101b of the first substrate. The microscope 111 may be connected to a computer 112 or the like for analyzing the observation results.

[0046] Figure 4 is a graph showing the relationship between the voltage applied to the ultrasonic generator 103 and the dissociation rate of the microparticles 106. Before supplying the target substance T, and when the bond between all microparticles 106 and chain molecules 105 is maintained, the microparticles 106 are subjected to a strong intermolecular interaction force F from the chain molecules 105. bind It is subjected to a high voltage (first voltage) V in order to dissociate it from the chain molecule 105. H It is necessary to apply a voltage. The relationship between the applied voltage and the dissociation rate in this case is represented by the solid curve on the right side of Figure 4.

[0047] Figure 5 is an enlarged perspective view of the vicinity of one surface 101a of the first substrate when the target substance T is supplied into the medium 104. The supplied target substance T is assumed to be able to bind more strongly (preferentially) to the chain molecules 105 than to the microparticles 106. In this case, the microparticles 106 are dissociated from the chain molecules 105, and the target substance is newly bound to the chain molecules 105.

[0048] The ratio of microparticles 106 that are dissociated is determined by the number (concentration) of target substances supplied. If the number of target substances supplied is equal to or greater than the number of microparticles 106 bound to the chain molecules 105, all microparticles 106 will be dissociated. If the number of target substances supplied is less than the number of microparticles 106 bound to the chain molecules 105, some microparticles 106 will not be dissociated and will maintain their bond with the chain molecules 105.

[0049] The dissociated microparticles 106 either float near node S1 of the ultrasonic standing wave or fall and physically adhere to one surface 101a of the first substrate. Therefore, it is preferable that the distance between adjacent chain molecules 105 is greater than or equal to the particle size of the microparticles 106. Microparticles 106A that float near node S1 are bound only by the ultrasonic radiation force and settling force because there are no intermolecular interaction forces. Microparticles 106B that adhere to one surface 101a are bound almost entirely by the ultrasonic radiation force and settling force because there are no intermolecular interaction forces and the adhesion force (adsorption force) to one surface 101a is extremely weak compared to the intermolecular interaction forces.

[0050] Therefore, in this case, the microparticles 106A and 106B have a lower voltage (second voltage) V compared to when they are bound to the chain molecule 105. L The microparticles can be released from their bound state, and the relationship between the applied voltage and the dissociation rate is represented by the solid curve on the left side of Figure 4. The magnitude of the dissociation rate changes depending on the number of target substances supplied. When some of the microparticles 106 bound to the chain molecule 105 dissociate while the rest maintain their bond, the relationship between the applied voltage and the dissociation rate is represented by the dashed curve in Figure 4. For example, when the number of target substances supplied is half the number of microparticles bound to the chain molecule, the voltage V L The dissociation rate of microparticles is 50%. It is preferable to evaluate the dissociation rate under conditions where the influence of applied voltage fluctuations is minimal, and the applied voltage should be set to voltage V L and voltage V H The voltage V between (preferably in the middle) M It is preferable to do so when that is the case.

[0051] Figure 6 is a graph showing the relationship between the concentration of the target substance and the dissociation rate of microparticles. The horizontal axis of the graph represents the logarithmic scale of the target substance concentration, and the vertical axis represents the dissociation rate of microparticles. As shown in Figure 6, there is a linear relationship between the logarithmic scale of the target substance concentration and the dissociation rate of microparticles. Therefore, for the target substance being analyzed, the concentration corresponding to the measured dissociation rate can be easily estimated by referring to this relationship.

[0052] <Analysis method> The analytical method using the analytical apparatus 100 described above mainly comprises a first step of balancing the forces acting on the microparticles 106, a second step of determining the relationship between the concentration A of the reference target substance and the dissociation rate A of the microparticles 106, and a third step of analyzing the concentration B of the target substance to be analyzed using this relationship.

[0053] (first step) An ultrasonic standing wave S is generated between the first substrate 101 and the second substrate 102 using an ultrasonic generator 103, and an ultrasonic radiation force is applied to the microparticles 106. The voltage applied to the ultrasonic generator 103 is adjusted so that the ultrasonic radiation force balances other forces acting on the microparticles 106 (sedimentation force, intermolecular interaction force).

[0054] (Second process) With the forces acting on the microparticles 106 balanced, a reference first target substance T1 is supplied to the medium 104 at a predetermined concentration, and a predetermined voltage is applied to the ultrasonic generator. The applied voltage is between the first voltage and the second voltage, that is, the voltage at which the ratio of microparticle dissociation from the chain molecules 105 (dissociation rate) A becomes approximately constant, and preferably, it is a voltage midway between the first voltage and the second voltage. At this time, the dissociation rate A is measured multiple times by changing the concentration A of the supplied first target substance T1. From the measurement results, the relationship (relational formula) between the concentration A (logarithm of A) and the dissociation rate A is determined, as shown in Figure 6.

[0055] (Third step) A second target substance T2 of unknown concentration is supplied to the medium 104, and the ratio (dissociation rate) B at which microparticles 106 dissociate from chain molecules 105 is measured. The concentration B corresponding to the dissociation rate B is analyzed by referring to the relationship between concentration A and dissociation rate A obtained in the second step.

[0056] In order to measure the dissociation rate A under conditions where the influence of fluctuations in the applied voltage is minimal, it is preferable to further include the following two steps: First, before supplying the first target material for reference, apply a first voltage (V in Figure 4) to the ultrasonic generator 103 to dissociate all the microparticles 106. H This is a step in which the voltage (corresponding to) is measured. Another step is to apply a second voltage (V in Figure 4) to the ultrasonic generator 103 in order to dissociate all the microparticles 106 after supplying the first target material. L This is a process for measuring (corresponding to). From the viewpoint of improving measurement accuracy, when measuring the dissociation rate A, the voltage applied to the ultrasonic generator 103 is set to a voltage midway between the first voltage and the second voltage (V in Figure 4). M It is preferable to use the following: (corresponding to)

[0057] [Second Embodiment] <Analyzer> The analytical apparatus according to the second embodiment of the present invention is an analytical apparatus for analyzing the binding force of a target substance. In this analytical apparatus, the binding force between microparticles and chain molecules is adjusted to be stronger than the binding force between the target substance and chain molecules. The other configurations are the same as those of the analytical apparatus of the first embodiment.

[0058] <Analysis method> The analytical method using the analytical apparatus of this embodiment mainly comprises a first step of balancing the forces acting on the microparticles, a second step of determining the relationship between the binding constant C of a reference first target material and the dissociation rate C of the microparticles, and a third step of analyzing the binding constant of the target material to be analyzed using this relationship.

[0059] (first step) Similar to the first embodiment, when a voltage is applied to the ultrasonic generator and ultrasonic standing waves are generated between the first substrate and the second substrate, the ultrasonic radiation force acting on the microparticles is balanced with other forces acting on the microparticles.

[0060] (Second process) With the forces acting on the microparticles balanced, a reference first target substance is supplied into the medium, and a predetermined voltage is applied. At this time, the ratio C of microparticles dissociating from the chain molecule is measured multiple times while varying the binding constant C of the microparticles to the chain molecule, and the relationship between the binding constant C and the ratio C is determined. The binding constant C can be adjusted by the composition of the DNA intervening in the binding with the chain molecule, specifically by the number of bases that make up the DNA.

[0061] (Third step) When the second target substance to be analyzed is supplied into the medium, the ratio D at which microparticles dissociate from the chain molecule is measured. The binding constant D corresponding to the ratio D is analyzed by referring to the relationship between the binding constant C obtained in the second step and the ratio C. Even if the binding constant of the target substance (first target substance, second target substance) is smaller than the binding constant of the microparticles, the microparticles can be dissociated and replaced with the target substance by increasing the amount of target substance supplied. For example, even if the microparticles are strongly fixed to the chain molecule with DNA composed of about 20 bases, this replacement can be achieved by supplying a large amount of target substance containing DNA composed of 19 bases or less.

[0062] As described above, the analysis using the analytical apparatus and analytical method of the above embodiments (first embodiment, second embodiment) makes it possible to perform trace measurements of the concentration and bonding constant of a target substance by utilizing the fact that the intermolecular interaction force acting on microparticles changes in proportion to the concentration and bonding constant of the target substance supplied to the medium.

[0063] When the target substance is supplied, the binding partners of the microparticles are replaced from chain molecules with intermolecular interactions to glass substrates or the like. Since the microparticles are no longer subjected to intermolecular interaction forces, they become more susceptible to dissociation. The higher the concentration of the supplied target substance, the more the binding partners are replaced and the number of microparticles that are not subjected to intermolecular interaction forces increases, thus increasing the dissociation rate of the microparticles. Furthermore, as the amount of supplied target substance increases and the overall binding constant of the target substance increases, the number of microparticles that are not subjected to intermolecular interaction forces increases, thus increasing the dissociation rate of the microparticles. In this way, by confirming the dissociation behavior of microparticles based on the concentration of the target substance and the binding constant, it becomes possible to perform unmodified trace measurements and measure changes in the density of the target substance with high accuracy.

[0064] The analysis in the above embodiment can be performed for various target substances by selecting a combination of aptamer base sequence and target molecule. By selecting a combination of microparticles and chain molecules that are easily dissociated when the target substance is supplied, the concentrations of various substances can be measured. [Examples]

[0065] The effects of the present invention will be made clearer by the following examples. However, the present invention is not limited to the following examples and can be modified as appropriate without altering its essence.

[0066] (Examples 1-10) Trace measurements of arginine concentration were performed using the analytical apparatus described above. Glass substrates were used as the first and second substrates, and one side of each was placed in contact with the other, as shown in Figure 1. Water was filled as the medium into a recess formed on one side of the second substrate. An aptamer having 28 bases was used as the chain molecule. Polystyrene particles with a particle size of 15.1 ± 0.4 μm were used as the microparticles. The polystyrene particles were bonded to the aptamer by a 5-base interaction. The resonance frequency of the ultrasound generated between the first and second substrates was set to 1 MHz. The concentrations of arginine supplied to the medium are shown in Table 1.

[0067] [Table 1]

[0068] In an ultrasonic generator, the applied voltage was swept, and the voltage dependence of the dissociation rate of polystyrene particles was measured. Figure 7 is a graph showing the results. The horizontal axis of the graph represents the applied voltage, and the vertical axis represents the dissociation rate of polystyrene particles. The plots are arranged in order from closest to the horizontal axis of the graph: Examples 1, 2, ..., 12.

[0069] In Examples 1 and 2, the voltage at which all polystyrene particles dissociated was the highest. In Examples 1 and 2, no arginine was supplied, and all polystyrene particles were bound to the aptamer and subjected to strong intermolecular interaction forces. Therefore, it is clear that in order to dissociate the polystyrene particles, it is necessary to apply a voltage that generates a high ultrasonic radiation force that overcomes the intermolecular interaction forces.

[0070] In Examples 11 and 12, the voltage at which all polystyrene particles dissociated was the lowest. In Examples 11 and 12, arginine was supplied at a high concentration and bound to all aptamers, causing all polystyrene particles to dissociate from the aptamers and be adsorbed onto the glass substrate, etc. Therefore, since no intermolecular interaction forces act on the polystyrene particles, it can be shown that polystyrene particles can be dissociated even with a weak ultrasonic radiation force, and the applied voltage can be lowered.

[0071] In Examples 3-10, dissociation of polystyrene particles begins at a low voltage, similar to Examples 11 and 12, but the dissociation rate is less than 100%. This means that the concentration of supplied arginine is low, and arginine has not reached all aptamers. Polystyrene particles dissociate from aptamers to which arginine is bound, resulting in the same state as Example 10, but polystyrene particles remain bound to aptamers to which arginine is not bound, resulting in the same state as Example 1. The higher the concentration of supplied arginine, the more arginine binds to the aptamers, and the more polystyrene particles are released from their binding to the aptamers, thus increasing the dissociation rate of polystyrene particles.

[0072] Figure 8 is a graph showing the relationship between the arginine concentration and the dissociation rate of polystyrene particles in Examples 1 to 12. The horizontal axis of the graph represents the logarithmic scale of the arginine concentration C, and the vertical axis represents the dissociation rate of polystyrene particles. The dissociation rate used is the value in the voltage region where the plots are flat in Figure 7.

[0073] There is a linear relationship between the logarithm of the arginine concentration C and the dissociation rate of polystyrene particles. By referring to this relationship, the concentration corresponding to the measured dissociation rate of an unknown target substance can be easily estimated. [Explanation of symbols]

[0074] 100...Analyzer 101...First board 101a...One side of the first substrate 101b...Other side of the first substrate 102...Second board 102a...One side of the second substrate 102b...Other side of the second substrate 102c···recess 103. Ultrasonic generator 104... Medium 105...chain molecule 105a... one end of a chain-like molecule 105b...the other end of a chain-like molecule 10⁶, 10⁶A, 10⁶B... microparticles 107...Function Generator 108... Power amplifier 109...Oscilloscope 110...Transducer 111... Microscope 112... Computer 113...DNA molecule F ac ...ultrasonic radiation F sed ...Sedimentation force F bind ...Intermolecular interaction force R1, R2...area S... Ultrasonic standing waves S1... Node of a standing wave T, T1... Target substance

Claims

1. An analytical instrument for chemically analyzing a target substance, First circuit board and A second substrate is arranged so that one side of it faces the first substrate, An ultrasonic generating device connected to or integrated with the first substrate or the second substrate, A medium for propagating ultrasonic waves is placed between one surface of the first substrate and one surface of the second substrate, In the medium, a chain-like molecule with one end bonded to one surface of the first substrate, The medium comprises microparticles that are bound to the other end of the chain-like molecule and are sensitive to ultrasonic waves generated by the ultrasonic generator, An analytical apparatus characterized in that the binding force between the microparticles and the target substance is adjusted so that the microparticles are replaced by the supplied target substance and dissociated from the chain-like molecules.

2. An analytical device for analyzing the concentration of the target substance, The analytical apparatus according to claim 1, characterized in that the binding force between the microparticles and the chain-like molecules is adjusted to be weaker than the binding force between the target substance and the chain-like molecules.

3. An analytical device for analyzing the binding force of the target substance, The analytical apparatus according to claim 1, characterized in that the binding force between the microparticles and the chain-like molecules is adjusted to be stronger than the binding force between the target substance and the chain-like molecules.

4. The analytical apparatus according to any one of claims 1 to 3, characterized in that the microparticles are bound to the other end of the chain-like molecule via DNA molecules.

5. The analytical apparatus according to any one of claims 1 to 3, characterized in that the chain-like molecule is an aptamer.

6. The analytical apparatus according to any one of claims 1 to 3, characterized in that the chain-like molecule is an antibody or antigen that undergoes an antigen-antibody reaction.

7. The analytical apparatus according to any one of claims 1 to 3, characterized in that the chain-like molecule is a host molecule or a guest molecule that undergoes a host-guest reaction.

8. The analytical apparatus according to any one of claims 1 to 3, characterized in that the particle size of the microparticles is 5 μm or more and 50 μm or less.

9. The analytical apparatus according to any one of claims 1 to 3, characterized in that the distance between adjacent chain molecules is greater than or equal to the particle size of the microparticles.

10. The analytical apparatus according to any one of claims 1 to 3, characterized in that the first substrate and the second substrate are transparent to visible light.

11. The analytical apparatus according to any one of claims 1 to 3, characterized in that a recess is formed on one surface of the second substrate, and the space in which the medium is filled is the internal space of the recess.

12. An analytical method using the analytical apparatus described in claim 2, A step of applying a voltage to the ultrasonic generator and generating an ultrasonic standing wave between the first substrate and the second substrate, thereby balancing the ultrasonic radiation force acting on the microparticles with other forces acting on the microparticles, A step of supplying a reference first target substance into the medium and applying a predetermined voltage, measuring the ratio A at which the microparticles dissociate from the chain molecules multiple times while varying the concentration A of the supplied first target substance, and determining the relationship between the concentration A and the ratio A, The analytical method is characterized by comprising the steps of: measuring the ratio B at which microparticles dissociate from the chain molecules when a second target substance to be analyzed is supplied to the medium; and analyzing the concentration B corresponding to the ratio B by referring to the relationship between the concentration A and the ratio A.

13. An analytical method using the analytical apparatus described in claim 3, A step of applying a voltage to the ultrasonic generator and generating an ultrasonic standing wave between the first substrate and the second substrate, thereby balancing the ultrasonic radiation force acting on the microparticles with other forces acting on the microparticles, A step of supplying a first target material for reference into the medium and applying a predetermined voltage, measuring the ratio C at which the microparticles dissociate from the chain molecules multiple times while changing the binding constant C of the microparticles to the chain molecules, and determining the relationship between the binding constant C and the ratio C, The analytical method is characterized by comprising the steps of: measuring the ratio D at which microparticles dissociate from the chain molecules when a second target substance to be analyzed is supplied to the medium, and analyzing the binding constant D corresponding to the ratio D by referring to the relationship between the binding constant C and the ratio C.

14. Before supplying the first target material, a step of measuring a first voltage applied to the ultrasonic generator in order to dissociate all of the microparticles, The method further includes the step of measuring a second voltage applied to the ultrasonic generator in order to dissociate all of the microparticles after supplying the first target material, The analysis method according to either 12 or 13, characterized in that when measuring the ratio A, the voltage applied to the ultrasonic generator is set to a voltage intermediate between the first voltage and the second voltage.

Images