Method and apparatus for detecting hydrogen bond structure change of liquid

Abstract

To provide a method and an apparatus for detecting a hydrogen bond structure change of liquid that can detect a change in a hydrogen bond structure of liquid.SOLUTION: A method for detecting a hydrogen bond structure change of liquid includes: a step of placing hydrogen-bonding liquid 20 in a gap d between a reflector 10A and a coaxial probe 40; a measurement step of irradiating the hydrogen-bonding liquid 20 with microwaves from the coaxial probe 40 while sweeping the frequency, and obtaining a complex relative permittivity εr* from a reflected wave input to the coaxial probe 40; and a step of calculating the magnitude Δr of a change from the reference of the complex relative permittivity εr* during the frequency sweep in the measurement step. Here, the gap d is less than 2 mm.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a method for detecting a change in the hydrogen bond structure of a liquid, and also to an apparatus for detecting a change in the hydrogen bond structure of a liquid. [Background technology]

[0002] Techniques for measuring state changes of solutions and the like using microwaves are disclosed, for example, in Patent Documents 1 to 4. Patent Document 1 discloses adjusting the dielectric constant by microwave heating, but does not mention measuring the hydration state. Patent Document 2 discloses a medical instrument that supplies high-power microwaves to a probe and heats a living body in contact with the tip. Other known techniques include those disclosed in Patent Documents 3 and 4.

[0003] When a solvent containing water molecules is mixed with a solute molecule, hydration occurs due to interactions with the solute molecule and ions. In hydration, the movement and hydrogen bond structure of the water molecule group surrounding the solute molecule change from the state in pure water. It is known that this hydration makes the water molecule easier to move and makes the water molecule less mobile. In some cases, water molecules directly interact with the solute surface and change the surrounding hydrogen bond structure. In addition, in the hydrophobic cavities of proteins, water molecules cannot directly interact with the solute surface, so they form hydrogen bonds with each other to surround the solute molecule. In particular, in the latter case, it is known that hydrogen bonds are formed after a sufficient amount of time has passed after mixing the solute molecule with water. In this way, the hydrogen bond structures formed between the solute molecule dissolved in water and the water molecules are diverse, and it is thought that the hydrogen bond structures change over time. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 7137003 [Patent Document 2] JP 2008-142467 A [Patent Document 3] JP 2008-111816 A [Patent Document 4] International Publication No. 2013 / 052062 Summary of the Invention [Problem to be solved by the invention]

[0005] However, as described above, the hydrogen bond structure in a liquid in which water and a solute are mixed is complex and changes over time, so a method for detecting changes in the hydrogen bond structure between a solute and water molecules has not yet been developed. The present disclosure provides a method and device for detecting changes in the hydrogen bond structure of a liquid, which are capable of detecting changes in the hydrogen bond structure of the liquid. [Means for solving the problem]

[0006] The present inventors have conducted extensive research into a method and device for detecting a change in the hydrogen bond structure in a liquid, and as a result have succeeded in developing a method and device for detecting a change in the hydrogen bond structure in a liquid containing a solute.

[0007] That is, a method for detecting a change in a hydrogen-bonding structure in a liquid according to one aspect of the present disclosure includes the steps of providing a hydrogen-bonding liquid in a gap d between a reflector and a coaxial probe, irradiating the liquid with microwaves from the coaxial probe while sweeping the frequency, and detecting a complex relative dielectric constant ε from the reflected wave input to the coaxial probe. r * a measuring step for determining the complex relative dielectric constant ε r * and determining the magnitude Δr of the change from the reference, wherein the gap d is less than 2 mm.

[0008] The complex relative dielectric constant ε r * is the complex relative dielectric constant ε of a solution containing a solvent having properties correlated with those of the liquid and having a network structure. a * and the complex relative permittivity of the unloaded medium, ε v* and the scattering parameters (S parameters) of the reflected wave.

[0009] The microwaves are preferably swept within a frequency band including 3 GHz to 9 GHz.

[0010] The method for detecting a change in the hydrogen bond structure of a liquid preferably further includes the step of irradiating the liquid with electromagnetic waves having a frequency in the sub-terahertz band, thereby accelerating the change in the hydrogen bond structure of the liquid.

[0011] The solute contained in the hydrogen bonding liquid may be organic.

[0012] The hydrogen bonding liquid solvent can include water.

[0013] A detection device for detecting a change in a hydrogen bond structure in a liquid according to one aspect of the present disclosure includes a coaxial probe that irradiates a reflector with a microwave whose frequency is swept and receives a reflected wave of the microwave reflected by the reflector, and a complex relative dielectric constant ε r * and the complex relative dielectric constant ε obtained by the measurement unit during a microwave frequency sweep. r * and a calculation unit that calculates a magnitude Δr of the change from a reference, and a gap d between the reflector and the coaxial probe is set to less than 2 mm. Effect of the Invention

[0014] According to the method and device for detecting a change in the hydrogen bond structure of a liquid disclosed herein, a change in the hydrogen bond structure of the liquid can be detected. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a diagram showing an apparatus for detecting a change in the hydrogen bond structure of a liquid according to an embodiment. [Diagram 2]FIG. 2 is a diagram showing the relationship between the tip face of the coaxial probe and the surface of the reflector. [Diagram 3] FIG. 3 is a flow chart illustrating a method for detecting a change in a hydrogen bond structure. [Figure 4] FIG. 4 is a Nyquist plot of the complex relative permittivity εr*(ω) within a particular frequency range. [Diagram 5] FIG. 5 is a Nyquist plot of the theoretical complex dielectric constant (dotted line) and the actual complex dielectric constant (dashed line). [Figure 6] FIG. 6 is a Nyquist diagram illustrating the change in complex relative dielectric constant (Δr). [Figure 7] FIG. 7 is a graph of the frequency dependence of Δr. [Figure 8] FIG. 8 is a graph showing the relationship between each frequency and Δr at a certain time after the preparation of the egg white lysozyme solution. [Figure 9] FIG. 9 is a graph showing the relationship between each frequency and Δr at a certain time after preparation of the salmon sperm-derived DNA solution. [Figure 10A] FIG. 10A is a graph showing the frequency dependence of Δr when an egg-white lysozyme solution is irradiated with sub-terahertz waves. [Figure 10B] FIG. 10B is a graph showing the frequency dependence of Δr when an egg white lysozyme solution is heated. [Figure 10C] FIG. 10C is a graph showing the frequency dependence of Δr when no stimulus was applied to an egg white lysozyme solution. [Figure 10D] FIG. 10D is a graph showing the frequency dependence of Δr upon cooling of an egg white lysozyme solution. [Figure 11] FIG. 11 is a graph showing the relationship between time and temperature (° C.) when each stimulus was applied to an egg white lysozyme solution. [Figure 12A] FIG. 12A is a graph showing the frequency dependence of Δr when a polyethylene glycol (PEG) 6000 solution is irradiated with sub-terahertz waves. [Figure 12B]FIG. 12B is a graph showing the frequency dependence of Δr when a polyethylene glycol (PEG) 6000 solution is heated. [Figure 13] FIG. 13 is a table showing the experimental data of Examples 1-1 to 2-4. [Figure 14] FIG. 14 is a table showing the experimental data of Examples 3-0 to 3-4. [Figure 15] FIG. 15 is a table showing the experimental data of Examples 4-0 to 6-4. [Figure 16] FIG. 16 is a table showing the experimental data of Examples 7-0 to 8-3. [Figure 17] FIG. 17 is a diagram for explaining the relationship between proteins and hydration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Hereinafter, the embodiments will be described in detail with reference to the drawings. The same elements will be denoted by the same reference numerals, and duplicated explanations will be omitted.

[0017] FIG. 1 is a diagram showing an apparatus 100 for detecting a change in a hydrogen bond structure in a liquid according to an embodiment of the present invention.

[0018] The device 100 for detecting changes in hydrogen bond structure in a liquid includes a container 10 equipped with a reflector 10A, a sub-terahertz supply unit 30, a coaxial probe 40, a vector network analyzer 41, a temperature adjustment unit 50, a control device 60, and a display 70. The coaxial probe 40 emits microwaves and receives reflected microwaves. The frequency of the microwaves is swept. The control device 60 calculates the complex relative dielectric constant ε of the object to be measured from the reflected waves input to the coaxial probe 40. r * and the complex relative permittivity ε obtained by the measurement unit during the microwave frequency sweep. r * The present invention is also provided with a calculation unit for calculating the magnitude Δr of the change from the reference value.

[0019] The container 10 contains and holds the hydrogen-bonding liquid 20 therein. The container 10 has a recess for holding the hydrogen-bonding liquid 20, and the recess of the container 10 has a bottom surface (reflector 10A). The reflector 10A faces the tip surface 45 of the coaxial probe 40. The reflector 10A is irradiated with microwaves emitted from the tip surface 45 of the coaxial probe 40. The frequency of the microwaves is swept. The reflector 10A reflects the microwaves irradiated to the reflector 10A. The surface of the reflector 10A is flat.

[0020] An XYZ three-dimensional orthogonal coordinate system is set. The direction perpendicular to the surface of reflector 10A is defined as the Z-axis direction. The positive direction of the Z-axis is the direction pointing vertically upward. The direction perpendicular to the Z-axis direction is defined as the X-axis direction. The direction perpendicular to both the Z-axis and X-axis is defined as the Y-axis direction. The surface of reflector 10A is parallel to the XY plane.

[0021] The container 10 and the reflector 10A are made of silicone such as polydimethylsiloxane (PDMS). The shape and material of the reflector 10A are not particularly limited as long as they can reflect microwaves whose frequency is swept. A lid 11 can be placed on the upper open end of the container 10 as necessary.

[0022] The hydrogen-bonding liquid 20 is provided so as to fill the gap d between the surface position (Z0) of the reflector 10A of the container 10 and the coaxial probe tip surface position (Z1) of the tip surface 45 of the coaxial probe 40. The hydrogen-bonding liquid 20 may be any liquid containing water. Examples of the liquid containing water include a case where the solvent contains water and a case where a hydrate is dissolved as a solute.

[0023] The hydrogen-bonding liquid 20 is a solution containing a solvent and a solute. In the hydrogen-bonding liquid 20, hydrogen bonds are considered to be formed between solvent molecules, between solvent molecules and solute molecules, and between solute molecules and solute molecules, but from the viewpoint of detecting a change in the hydrogen bond structure, it is preferable that hydrogen bonds are formed between solvent molecules and between solvent molecules and between solute molecules.

[0024] Examples of the solvent for the hydrogen-bonding liquid 20 include water, ethanol, methanol, propanol, hexanol, octanol, ethylene glycol, glycerin, carboxylic acid, sulfonic acid, ammonia, amine, etc., and a solvent containing water is preferable from the viewpoint of containing a large amount of bulk water. The solvent for the liquid may be one or more selected from the group consisting of water, ethanol, methanol, propanol, hexanol, octanol, ethylene glycol, glycerin, carboxylic acid, sulfonic acid, ammonia, and amine listed above.

[0025] The solute of the hydrogen-bonding liquid 20 may be an organic substance, an inorganic substance, heavy water, a dispersion, etc. The solute may be one or more types selected from the group consisting of an organic substance, an inorganic substance, heavy water, and a dispersion, and preferably contains an organic substance.

[0026] Examples of organic substances include water-soluble polymers, amino acids, lipids, organic acids, etc., with water-soluble polymers being preferred.

[0027] The water-soluble polymer may be a water-soluble polymer of natural origin (produced in nature) or may be a water-soluble polymer obtained by artificial synthesis. Examples of the water-soluble polymer include proteins, deoxyribonucleic acid (DNA), polyethylene glycol, polypeptides, ribonucleic acid (RNA), polysaccharides, polyamines, polyacrylic acid, polyacrylamide, polyethylene oxide, polyvinyl alcohol, polyvinyl amide, etc., and proteins, deoxyribonucleic acid (DNA), and polyethylene glycol are preferred.

[0028] The protein may be a naturally occurring protein or an artificially synthesized protein such as a modified protein. The deoxyribonucleic acid (DNA) may be a naturally occurring DNA or an artificially synthesized DNA such as a chemically synthesized nucleic acid.

[0029] An example of the polyethylene glycol is polyethylene glycol (PEG6000) having an average molecular weight of 7300 to 9300 (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.).

[0030] Examples of inorganic substances include phosphoric acid, polyphosphoric acid, sodium chloride, calcium chloride, and water glass.

[0031] Examples of dispersions include inorganic fine powders such as zeolite, alumina, silica, magnesia, and titania; polymer fine powders such as crosslinked polymer gels and nanofibers; emulsion dispersoids such as latex, colloids, and micelles; and suspension dispersoids such as muddy water, slip, and paint.

[0032] The sub-terahertz supply unit 30 irradiates the hydrogen-bonding liquid 20 with sub-terahertz waves. 11 Hz~10 13 Terahertz waves are electromagnetic waves in the frequency range of 10 12 The sub-terahertz waves are electromagnetic waves in a frequency range lower than 1 THz (1 THz). Since the skeletal vibration and intermolecular vibration of proteins overlap with the main relaxation of water at a frequency of 0.1 THz, the sub-terahertz waves to be irradiated are preferably 0.1 THz or more and 0.3 THz or less.

[0033] The sub-terahertz supply unit 30 can also be controlled by the control device 60. The sub-terahertz supply unit 30 includes a preamplifier, an isolator, an attenuator, a klystron, a horn antenna, a plano-convex lens, and a bandpass filter along the propagation direction of the electromagnetic wave output from the oscillator. The oscillator generates an electromagnetic wave that serves as a transmission source. The preamplifier amplifies the electromagnetic wave. The isolator suppresses backward propagation of the electromagnetic wave. The attenuator attenuates the electromagnetic wave output from the isolator. The klystron amplifies the electromagnetic wave output from the attenuator. The horn antenna radiates the electromagnetic wave output from the klystron. The plano-convex lens converges the diffusion of the output electromagnetic wave of the horn antenna. The bandpass filter transmits electromagnetic waves in the sub-terahertz frequency band. The container 10 is made of a material that can transmit sub-terahertz waves.

[0034] The coaxial probe 40 receives microwaves transmitted from a vector network analyzer 41 (microwave transmission device) via a cable 42, a cable connector 43, and a probe connector 44. The coaxial probe 40 emits the received microwaves from a tip surface 45 of the coaxial probe 40. The longitudinal direction of the coaxial probe 40 is the Z-axis direction. The microwaves travel vertically downward, that is, along the negative direction of the Z-axis. The microwaves emitted from the tip surface 45 of the coaxial probe 40 pass through the hydrogen-bonding liquid 20 to be measured along the negative direction of the Z-axis and reach the reflector 10A. The microwaves that reach the reflector 10A are reflected by the reflector 10A, becoming reflected waves, which again pass through the hydrogen-bonding liquid 20 along the positive direction of the Z-axis and reach the tip surface 45 of the coaxial probe 40. The reflected wave that reaches the coaxial probe 40 is input to a vector network analyzer 41 (microwave receiving device) via a probe connector 44, a cable connector 43, and a cable 42 in this order.

[0035] The vector network analyzer 41 receives a microwave generation signal from the control device 60 and generates microwaves. The vector network analyzer 41 can transmit a signal having information on parameters (S parameters, permittivity, magnetic permeability, etc.) obtained from the microwaves (reflected waves) received from the coaxial probe 40 to the control device 60. The S parameters related to reflection can be expressed as a ratio of the reflected wave intensity to the incident wave intensity. The control device 60 (measurement unit, calculation unit) can detect a hydrogen bond structure change in the liquid, that is, a change in the hydration state, based on the signal received from the vector network analyzer 41. The hydrogen bond structure change detected by the control device 60 (a graph of the frequency dependence of Δr described later) is displayed on the display 70.

[0036] The coaxial probe 40 irradiates the hydrogen-bonding liquid 20 and the reflector 10A with microwaves while sweeping the frequency. The microwaves are swept within a frequency band including 3 GHz to 9 GHz. Within this frequency band, changes in the hydrogen-bonding structure of water containing organic matter such as proteins are easily detected. The reason for this is that the peak value of Δr, P Δr This is because the specific phenomenon represented by appears at 3 GHz to 9 GHz when the gap d is less than 2 mm. The microwave may be swept within a frequency band including 200 MHz to 14 GHz. In this case, the frequency sweep range is wider, so the calculation accuracy of Δr is increased, and it is expected that the change in the hydrogen bond structure can be detected more sensitively in many cases. The microwave may be swept within a frequency band including 100 MHz to 40 GHz. In this case, the frequency sweep range is even wider, so the calculation accuracy of Δr is further increased, and the ε that appears at 16-22 GHz can be detected more precisely. r * From the measurement of the maximum value of the imaginary part of the equation, the relaxation time of water can be directly determined, and in many cases, it is expected that the change in the hydrogen bond structure can be detected sensitively.

[0037] The coaxial probe 40 is connected to a vector network analyzer 41 via a probe connector 44 , a cable connector 43 , and a cable 42 , and the vector network analyzer 41 is connected to a control device 60 .

[0038] The temperature adjustment unit 50 adjusts the temperature by heating or cooling the hydrogen-bonding liquid 20. The temperature adjustment unit 50 is provided so as to be in contact with the bottom surface of the container 10. The temperature adjustment unit 50 has an opening through which the sub-terahertz waves pass. The sub-terahertz waves emitted from the sub-terahertz supply unit 30 can pass through the opening of the temperature adjustment unit 50, and can easily reach the hydrogen-bonding liquid 20.

[0039] The temperature adjustment unit 50 does not need to be in contact with the container 10, and may be provided at any location in contact with the container 10, as long as it can heat or cool the hydrogen-bonding liquid 20 to a desired temperature. The location where the temperature adjustment unit 50 is provided may be, for example, the bottom or side surface of the exterior of the container 10, or the bottom or side surface of the interior of the container 10. Examples of the temperature adjustment unit 50 include a resistance heater, an infrared heater, a Peltier element, and a cooling pipe.

[0040] The control device 60 is a computer equipped with a central processing unit 61 (CPU) and a storage device 62, and the central processing unit 61 executes various control processes in accordance with a program 62A stored in the storage device 62. In the embodiment, the operations and calculations described are executed in accordance with the program 62A.

[0041] The control device 60 instructs the vector network analyzer 41 to generate microwaves and sweep their frequencies. As a result, the coaxial probe 40 receives the microwaves as reflected waves, and the vector network analyzer 41 outputs various parameters (S parameters, etc.) that correlate with the reflected waves. Signals of the various parameters (signals that correlate with the reflected waves) that have reached the coaxial probe 40 are input to the control device 60 (measurement unit). Based on the input signals, the control device 60 (measurement unit) calculates the complex relative dielectric constant ε of the measurement target for each microwave frequency. r * Calculate the S parameters and the complex relative permittivity ε r * The relationship between these is as shown in Equation 1 below.

[0042] When the microwave frequency is swept, the complex relative permittivity ε r * The control device 60 (calculation unit) changes the complex relative dielectric constant ε r * The control device 60 (calculation unit) calculates the magnitude of change Δr of the complex relative dielectric constant ε r * The control device 60 (calculation unit) calculates the difference Δr between the measured complex dielectric constant ε at each microwave frequency in the Nyquist diagram. The reference complex dielectric constant is a complex dielectric constant that forms a semicircle according to the Debye equation with a point on the horizontal axis as the center when expressed on a Nyquist diagram with the horizontal axis as the real part and the vertical axis as the imaginary part. r * The difference (Δr) in the complex relative dielectric constant of the reference semicircle is calculated.

[0043] The display unit 70 displays values ​​obtained in the control device 60 (measurement unit, calculation unit). An example of the display unit 70 is a display, but as long as it can display the obtained values, it may be a printer or the like, and the type is not particularly limited.

[0044] FIG. 2 is a diagram showing the relationship between the tip face of the coaxial probe and the surface of the reflector.

[0045] A gap d is provided between the reflector 10A and the tip surface 45 of the coaxial probe 40. The gap d is less than 2 mm. In FIG. 2, the gap d means the distance in the Z-axis direction between the container surface position Z0 and the coaxial probe tip surface position Z1. The microwave is swept within a frequency band including 3 GHz to 9 GHz. In a medium (for example, in a solution such as the hydrogen-bonding liquid 20), the electromagnetic wave is shortened to the −0.5 power of the dielectric constant. For example, the free space wavelength λ in pure water at 8 GHz is 4.5 mm. When the gap d is a distance equivalent to ¼ of the microwave wavelength λ, a specific example distance when the gap d is less than 2 mm is P Δr A unique phenomenon appears, as shown in the figure.

[0046] For example, the λ / 4 (mm) of microwaves with frequencies f = 8.2 GHz to 10.5 GHz for pure water is ε r (0)=79.8, ε r (∞)=4.8, τ(=1 / f c )=59.7(ps), we get the following.

[0047] When the frequency f is 8.2 GHz, λ / 4 (mm) = 1.1 (mm) (refractive index n = 8.3, wavelength λ = 4.5 (mm), real part of relative dielectric constant ε r '=65.9, relative permittivity imaginary part ε r ”=29.2).

[0048] When the frequency f is 9.2 GHz, λ / 4 (mm) = 1.0 (mm) (refractive index n = 8.1, wavelength λ = 4.0 (mm), real part of relative dielectric constant ε r '=62.4, relative permittivity imaginary part ε r ”=31.6).

[0049] When the frequency f is 10.5 GHz, λ / 4 (mm) = 0.9 (mm) (refractive index n = 7.9, wavelength λ = 3.6 (mm), real part of relative dielectric constant ε r '=58.6, relative permittivity imaginary part ε r ”=33.7).

[0050] In this technique, a unique phenomenon based on multiple reflections is used in the measurement technique when the gap d of the measurement sample is approximately 1 / 4 of the wavelength λ in the medium. When microwaves of at least 7 GHz to 9 GHz are irradiated onto a liquid, the multiple reflection phenomenon occurs. To utilize this phenomenon, the microwaves are swept within a frequency band including 3 GHz to 9 GHz. The microwave wavelength may reach 10 GHz. As the hydration (aging) of the sample progresses and the complex relative dielectric constant changes, a frequency that undergoes multiple reflections appears in the gap d, which is a fixed length, and the measurement ε that appears at that time r * The degree of hydration can be estimated by the change in the apparent dielectric constant.

[0051] The gap d may be 0.1 mm or more, 0.5 mm or more, or 0.8 mm or more. The microwaves emitted from the coaxial probe 40 are reflected by the reflector 10A constituting the bottom surface of the container, and are also reflected by the tip surface 45 of the coaxial probe 40, causing multiple reflections between them. From this viewpoint, the gap d is preferably less than 2 mm, more preferably 1.5 mm or less, and particularly preferably 1.2 mm or less.

[0052] Next, a method for detecting a change in the hydrogen bond structure of a liquid will be described.

[0053] The method for detecting a change in a hydrogen bond structure in a liquid according to the present disclosure includes the following three steps. Basic information and preferred examples of each component are the same as those of the above-mentioned device for detecting a change in a hydrogen bond structure. (1) A step of providing a hydrogen-bonding liquid in the gap d between the reflector and the coaxial probe. (2) A microwave is irradiated onto the liquid while sweeping the frequency from a coaxial probe, and the complex dielectric constant ε is calculated from the reflected wave input to the coaxial probe. r * Measurement process to find (3) Complex relative permittivity ε during frequency sweep in the measurement process r * A step of determining the magnitude of change Δr from the reference

[0054] In the step (1), a hydrogen-bonding liquid 20 is provided in a gap d between a reflector 10A of a container 10 and a tip surface 45 of a coaxial probe 40.

[0055] 3 is a flow chart illustrating a method for detecting a change in a hydrogen bond structure. Hereinafter, (2) and (3) will be explained based on FIG. 3.

[0056] (2) The process includes a step S1 of irradiating microwaves, a step S2 of receiving microwaves, and a step S3 of measuring a complex relative dielectric constant εr * The method includes a step S3 of measuring:

[0057] In step S1, based on an input from the control device 60, the vector network analyzer 41 generates microwaves while sweeping the frequency, and the coaxial probe 40 irradiates the frequency-swept microwaves onto the reflector 10A.

[0058] In step S2, the microwaves reflected by the reflector 10A reach the coaxial probe 40, and are input as a signal via the vector network analyzer 41 to the measurement section (control device 60).

[0059] In step S3, the measurement unit (controller 60) records and measures the S parameters based on the signal input from the vector network analyzer 41, and further calculates the complex relative dielectric constant ε r * Calculate.

[0060] In step (3), the complex relative dielectric constant ε r * The method includes step S4 of calculating the magnitude Δr of the change from the reference, and step S5 of displaying Δr.

[0061] In step S4, the calculation unit (control device 60) calculates the complex relative dielectric constant ε r * The value of and the magnitude of change Δr from the reference complex relative dielectric constant are calculated.

[0062] In step S5, the calculation unit (control device 60) causes the display device 70 to display the calculated Δr.

[0063] This embodiment will now be described in more detail.

[0064] The data from the hydrogen bond structural change detection device of the present disclosure may be calibrated by the following method. In the desired frequency range, the vector network analyzer 41 is calibrated at the cable connector 43 using initial data measured at the cable end face of the cable connector 43. The calibration method for the cable connector 43 may be mechanical, electrical, manual, or automatic. Calibration may be performed in three ways: open (O), short (S), and load (L), and the order may be arbitrary. · Connect the coaxial probe 40 to the cable connector 43 via the probe connector 44. Perform movement calibration (offset calibration) of the end face according to the length of the coaxial probe 40. This is not essential if the vector network analyzer 41 does not have this calculation function.

[0065] Next, the measurement of the reference value will be described.

[0066] The detection method of the present disclosure is r * The following four S-parameters are measured to determine the following:

[0067] S s is a scattering parameter consisting of real and imaginary parts. The reference material may be any material that provides uniform electrical conduction, such as soft conductive materials such as copper foil, tin foil, conductive resin, and liquid metal. S s The measurement is performed by pressing a conductor against the tip surface 45. s The conductive material used was copper foil.

[0068] S ois a scattering parameter consisting of a real part and an imaginary part, and is measured with the tip surface 45 open. o was measured with the tip surface 45 open.

[0069] S a is a scattering parameter consisting of real and imaginary parts. The reference material is pure water or the measurement target (immature sample) immediately after dissolving a solute. S a The measurement is performed with the tip surface 45 immersed in the hydrogen-bonding liquid 20 contained in the container 10. a was based on pure water.

[0070] S m is a scattering parameter consisting of a real part and an imaginary part. The reference substance is a liquid sample to be evaluated. The measurement is performed with the tip surface 45 immersed in the hydrogen-bonding liquid 20 contained in the container 10. In this embodiment, S m In Examples 1 and 3 to 6, a liquid sample containing egg white lysozyme was used as a reference substance, and in Example 2, a liquid sample containing salmon sperm-derived DNA was used as a reference substance.

[0071] Next, the calculation of the complex relative dielectric constant of the liquid to be evaluated will be described.

[0072] The obtained S s , S o , S a , S m From the scattering parameters, the complex relative dielectric constant ε of the liquid to be evaluated is calculated according to the following formula (1): r * where ε a * is the complex dielectric constant of pure water, ε v * is the complex dielectric constant of the unloaded medium (complex dielectric constant expressed as 1-j0, with the real part being 1 and the imaginary part being 0). This calculation method is the same as the general calculation method used in the reflection probe method, but when the device shown in the figure is used, it does not match the true complex dielectric constant value because it is not a semi-infinite reflection condition.

[0073]

number

[0074] Complex relative permittivity ε r * is the complex relative dielectric constant ε of a solution containing a solvent having properties correlated with those of a liquid and having a network structure. a * and the complex relative permittivity of the unloaded medium, ε v * and the scattering parameters (S parameters) of the reflected wave.

[0075] Next, the principle of the complex dielectric constant becoming a circular arc will be described.

[0076] Complex relative permittivity ε of a substance r * (ω) is a function of angular frequency ω, a complex number with the real part ε r '(ω), the imaginary part is ε r When f is expressed as “ω”, the relationship given by the following equation (2) holds. Here, ω is the angular frequency obtained by multiplying frequency f by 2π, and j is the imaginary unit.

[0077]

number

[0078] The relative dielectric constant of common dielectric liquids follows the Debye relaxation equation (Equation 3).

[0079]

number

[0080] Rewriting these equations, we obtain (Equation 4) and (Equation 5). Here, τ is the relaxation time, ε r (∞) is the relative permittivity at a sufficiently large frequency, ε r (0) is the relative dielectric constant in a static electric field.

[0081]

number

[0082]

number

[0083] The response diagram using (Equation 4) and (Equation 5) with the real part on the horizontal axis and the imaginary part on the vertical axis is called a Nyquist diagram. When a dielectric liquid has one relaxation time τ, the complex relative permittivity draws a regular semicircle on the Nyquist diagram (Figure 4). Hereinafter, this regular semicircle will be called the theoretical semicircle.

[0084] When the length of the sample is semi-infinite, the actual measurement values ​​obtained by the reflection probe method conform to the theoretical semicircle. However, due to the limitations of the measurement device specifications, the measurement range of frequencies is finite, and the obtained trajectory is only a part of the theoretical semicircle. For example, in the range of 200 MHz to 14 GHz, the measured values ​​of water (solid line and dashed dotted line) show a part of a circular arc compared to the theoretical semicircle of water (dashed line) (Figure 5).

[0085] Next, the principle of calculating the arc from the measured value of the complex relative dielectric constant will be described.

[0086] When the actual measured values ​​sufficiently retain the shape of a circular arc, the center and radius of the theoretical semicircle can be found from the measured values. Specifically, a provisional theoretical semicircle is created by setting the initial values ​​of the center coordinates and radius of the theoretical semicircle, and the distance r from the center coordinates of the provisional theoretical semicircle to each measured value is calculated. s and the difference between radius r, Δr s Calculate Δr s The center coordinates and radius of the theoretical semicircle are searched for so that the sum of the squares of is minimized. This task is called optimization.

[0087] The theoretical semicircle obtained as a result of optimization is called the measurement semicircle. From the center coordinate and radius of the measurement semicircle, ε r (∞), ε as the right intercept r (0) is obtained, where ε r (∞), ε r (0) is a calculated value and is not necessarily an actual measured value at an angular frequency of ∞ or 0.

[0088] Next, a method for calculating a circular arc in this embodiment will be described.

[0089] Actual liquid water has various network structures due to hydrogen bonds and has multiple relaxation times τ (Popovet et al., Phys.Chem.Chem.Phys., 2016, 18,13941, Shiraga et al., Phys.Chem.Chem.Phys., 2018, 20, 26200). The relaxation time τ is mainly due to the interaction between water molecules (specifically hydrogen bonds), and the relaxation frequency f c is in the region around 20 GHz. This relaxation time τ is main In liquid water, there are no interactions other than between water molecules, and τ main The portion of water in which this can be considered the main relaxation phenomenon is called bulk water.

[0090] When a solute that undergoes hydration enters bulk water, hydration occurs on the solute surface. The hydration structure consists of hydration water that directly interacts with the surface of the solute (first hydration sphere) and hydration water that interacts with the water in the first hydration sphere (second hydration sphere). The network structures of the first hydration sphere, second hydration sphere, and bulk water are different from each other. When a hydrophilic substance is selected as the solute, the first hydration sphere has a strong interaction with the solute surface (specifically, ion-dipole interaction), which causes binding of the water molecules, lengthening the relaxation time τ and decreasing f c Generally, this relaxation time τ occurs in the range of 0.1 GHz to 0.01 GHz. slow In addition, the water in the second hydration sphere has limited interaction with the water in the first hydration sphere, which is strongly bound to the solute, and the binding becomes weaker than that of bulk water (relaxation time τ becomes shorter), and f c occurs in the region around 600 GHz. This relaxation time τ is fast Let us assume that.

[0091] After mixing of the solutes, in the short time period, τ slow In addition, τ fast In some cases, a water structure with a relaxation time of τ may occur (e.g., when the solute is egg white lysozyme).main The theoretical semicircle, which assumes only τ fast , τ slow Due to the mixture of , it is no longer a regular semicircle. fast If a hydration structure with ε occurs, the left intercept of the measurement semicircle is r (∞) moves slightly to the right of the left intercept of the theoretical semicircle. Similarly, τ slow If a hydration structure with τ occurs, the right intercept of the measured semicircle will shift slightly to the left of the right intercept of the theoretical semicircle. fast The relaxation frequency of is around 600 GHz and τ main The relaxation frequency is very high for τ slow The relaxation frequency of this occurs in the range of 0.1 GHz to 0.01 GHz, which is very low. τmain for τ fast and τ slow The proportion of is very small. Therefore, main When the occupancy rate of is large, the measured value can be approximated as roughly following a semicircle. fast In order to directly measure the change in the complex dielectric constant, a special device is required, and it is not easy.

[0092] Next, a general reflection probe method will be described.

[0093] Generally, when measuring the dielectric constant of a liquid using the reflection probe method, if the object being measured is not thick enough, the electromagnetic waves will reach the bottom surface of the object being measured, and will be reflected due to impedance mismatch at the bottom surface, resulting in a failure to give a correct measurement signal. Therefore, when measuring the dielectric constant of a liquid using the reflection probe method, the object being measured must have a sufficient thickness. Generally, the penetration depth is an index of the depth to which electromagnetic waves penetrate, and its calculation method is expressed as the length at which a certain amount of attenuation is reached. Here, the length at which the power in a material is attenuated to 1 / e of its original value due to penetration is called L 1 / e Here, e represents the Napier's constant (e = 2.718···). If the thickness of the object to be measured is L 1 / eIn this case, 1 / e, or 37% of the power remains at the end surface after transmission. If the reflected wave generated at this surface reaches the measurement probe again and mixes with the reflected signal, a maximum of 14% of the power will be disturbed and a correct measurement signal will not be obtained. 1 / e Since the length of the fiber is 10.8 mm at 3 GHz, the power at the end face after transmission at 3 GHz is 1 / e 3 That is, L is 5% or less. 1 / e If the thickness is 33 mm or more, which is three times the length of the reflected wave, the power of the reflected wave mixed into the reflected signal is 1 / e 6 In other words, the value is 0.3% or less, and interference is avoided. Therefore, when performing precise measurement of the complex relative permittivity of water in this frequency range, it is preferable that the object to be measured has a thickness of 33 mm or more.

[0094] Next, a reflection probe method according to an embodiment will be described.

[0095] As mentioned above, the true complex dielectric constant evaluation by the reflection probe method must be performed on a sample with a sufficient penetration depth of the electromagnetic waves. fast , τ main , τ slow Therefore, in the present invention, instead of measuring and evaluating the true complex relative dielectric constant, we have developed a method to measure the complex relative dielectric constant sensitively under special conditions. fast We provide a method to evaluate the amount of hydration water having a hydrogen-bonded structure characterized by the above-mentioned, using an index equivalent to the complex dielectric constant in the microwave band.

[0096] Specifically, by appropriately thinning the sample thickness, a singular point is provided where the microwave reflected from the container surface position Z0 has the opposite phase at the tip surface position Z1 of the coaxial probe that introduces the microwave. main The change in the arc caused by the change in the dielectric constant is measured sensitively. The singular point here is the value based on the complex relative dielectric constant mentioned above, which is observed at a frequency where the gap d between Z0 and Z1 is close to 1 / 4 of the wavelength of the microwave passing through the measurement object, and is calculated using the same method.

[0097] Next, the Δr method for calculating the singular point will be described.

[0098] Here is a specific method for calculating the singular point. After calculating the measurement semicircle, when a line segment is applied from the center of the semicircle to the measurement value, the difference between the semicircle radius r and the distance of the line segment is Δr (Figure 6). Here, Δr is a function of frequency. If the horizontal axis is the measurement frequency and the vertical axis is Δr, the maximum value P of Δr in a specific frequency band is Δr In other words, the standard P Δr-s After the state change, P Δr-t (For example, P in Fig. 7 Δr-t1 , P Δr-t2 etc.) Δr-t / P Δr-s By measuring the S-parameters of the reflected wave input to the coaxial probe, the measurement unit of the control device can measure the complex relative permittivity ε r * The calculation unit calculates the complex relative dielectric constant ε r * The magnitude of change Δr from the reference (e.g. Δr = 0) is calculated. Δr is monitored while sweeping the frequency, and the peak value of Δr is the reference value P Δr-s is the peak value of the measurement target, P Δr-t When the value of the input signal changes to the value of the input signal, the calculation unit calculates the ratio of this change (P Δr-t / P Δr-s If the calculation result is equal to or greater than a threshold value (e.g., 1.06), it is determined that the hydrogen bond structure has changed so that hydration proceeds (determination A), and if the calculation result is less than the threshold value (e.g., 1.06), it is determined that the hydrogen bond structure has not changed substantially so that hydration proceeds (determination B). The control device can display this determination result on a display.

[0099] ε obtained by optimization through microwave measurement r (∞) is τ fast The value increases as the relative amount of hydration water with τ increases, and thus it is an index for evaluating the hydration structure. mainBy measuring the change in the complex relative permittivity of bulk water with main The left intercept of the theoretical semicircle derived from r (∞) (similarly, the right intercept of the theoretical semicircle, ε r (0) change), τ fast (or τ slow ) can be inferred from the change in the relative amount of hydration water.

[0100] Here, P Δr The increase in is correlated with the decrease in the real part of the dielectric constant between 0.1 GHz and 10 GHz and the increase in the imaginary part below 3 GHz. Δr , and the ratio to a specific reference value, P Δr-t / P Δr-s The measurement of τ fast (or τ slow This method can sensitively measure the quantitative change of hydration water having a complex dielectric constant (Δr) in correlation with the complex dielectric constant. This evaluation method proposed in the present invention is called the Δr method.

[0101] The relationship between the Δr method and hydration is specifically shown. Egg white lysozyme is mixed with water, and the P Δr The response of the samples was compared. Figure 8 shows the relationship between frequency and Δr under each condition. The response of the samples was compared. Δr This is because the amount of secondary water of hydration decreased with the progress of hydration, i.e., maturation, and the amount of τ fast The relative amount of hydration water with τ main ε r (∞) has decreased, and therefore τ main The measurement semicircle derived from shifted to the left, and as a result, Δr This results in an increase in the P value of the target sample compared to the reference sample. Δr An increase in the amount of hydration indicates the progress of hydration reactions in complex molecules such as proteins, i.e., the degree of maturation.

[0102] To confirm the progress of hydration, a sample of egg white lysozyme mixed with water was irradiated with sub-terahertz waves. 11 Hz or higher, 10 13 It refers to electromagnetic waves in the frequency range below τ fast It selectively interacts with the molecular motion associated with the P Δr In comparison with the room temperature sample, which required 24 hours for the P to increase three-fold, the P Δr In contrast, the P Δr This suggests that selective stimulation of the motion of water molecules with short relaxation times (i.e., water in the second hydration sphere, where the hydrogen-bond network structure is disrupted or lost) probably promotes rearrangement of the hydrogen-bond network in the first hydration sphere, thereby increasing the rate of the slow hydration reaction.

[0103] The above results were obtained by combining sub-THz wave irradiation from a klystron light source with complex permittivity measurement. This light source can generate electromagnetic waves in the frequency range of 95±0.25GHz, generating a square wave with a repetition rate of 10kHz and a pulse width of 0.8μs. Scattering parameters were measured at a frequency range of 100MHz-14GHz and an intensity of 32μW using a vector network analyzer (VNA) (ZVB14, Rohde & Schwarz). The permittivity was calculated using the average value of five measurements and the measured value of a calibration performed with a similar equipment configuration. The measurement time per frequency during the frequency sweep (the time to apply the external electromagnetic field generated by the VNA to the sample) was estimated to be (approximately 5μs) according to the manufacturer's instructions. The application time of (5μs) is close to the pulse width of the 0.1THz incident wave (approximately 1μs).

[0104] Here, if interference occurs between the 0.1 THz wave and the electromagnetic field generated by the VNA during irradiation, a large noise should occur in the dielectric spectrum obtained in each measurement. However, since no such noise was detected in five measurements per sample, the influence of interference between the 0.1 THz wave generated by the klystron and the external field generated by the VNA for measurement can be ignored. As the open-ended coaxial probe in this measurement, a SUS304 stainless steel coaxial line (PT292M2024486, KMCO (Kawashima Seisakusho Co., Ltd.)) with Z0 = 50 Ω and outer diameter of 2.2 mm was used. Similarly, measurements were performed using the same coaxial probe as the VNA (Agilent (Keysight Technologies), N5230A; 300 kHz-20 GHz), and it was confirmed that the measurement results were independent of the VNA equipment and coaxial transmission line used. When the frequency range was expanded to evaluate the signal PΔr generated in the sample cell, measurements were performed using a VNA (Anritsu Corporation, MS46131A; 20 MHz-43.5 GHz) combined with a coaxial probe (Keysight Technologies, Inc., N1501A performance probe of the dielectric probe kit).

[0105] (Experimental Example 1) Evaluation of hydrogen bond structure changes in lysozyme solution Pure water was used as the calibration standard. In the apparatus shown in FIG. 1, 30 mg of egg white lysozyme powder (Sigma-Aldrich Co., Cat. No. 62971) was mixed with 1 mL of pure water (resistivity = 18.2 MΩ·cm, total organic carbon < 5 ppb), and 0.8 mL was introduced into a poly-dimethylsiloxane (PDMS) container with a diameter of 20 mm. After introduction, the silicone lid 11 was placed and left to stand for 1 hour until the set temperature of 24°C became stable. This was used as the reference sample shortly after mixing, and the measurement of the reference sample was used as the standard for the difference. Next, a sample that had been mixed at the same ratio for 24 hours was left to stand until it reached the same temperature and measured. As a result, as shown in FIG. 8 and FIG. 13, the P of the sample that had been mixed for 24 hours was 1.0 μg / L. ΔrThe response of increased to 2.87 times the standard (Examples 1-1 to 1-4). FIG. 8 shows the frequency dependence of Δr after 1 hour has elapsed since mixing 10 mg / mL solute with pure water (Example 1-1), the frequency dependence of Δr after 1 hour has elapsed since mixing 30 mg / mL solute with pure water (Example 1-2), the frequency dependence of Δr after 1 hour has elapsed since mixing 70 mg / mL solute with pure water (Example 1-3), and the frequency dependence of Δr after 24 hours has elapsed since mixing 30 mg / mL solute with pure water (Example 1-4). As a standard for the difference in Experimental Example 1, the P Δr-t / P Δr-s The value was set to 1.0.

[0106] (Experimental Example 2) Evaluation of hydrogen bond structure changes in DNA solution Pure water was used as the calibration standard. In the device shown in FIG. 1, 100 mg of salmon sperm-derived DNA powder (manufactured by Fujifilm Wako Pure Chemical Industries, Cat. No. 049-31383) was mixed with 1 mL of pure water, and 0.8 mL was introduced into the PDMS container. The container was then covered with the lid 11 and left to stand for 1 hour until the set temperature of 24°C stabilized. This was used as the standard sample shortly after mixing, and the measurement of the standard sample was used as the standard for the difference. Next, a sample that had been mixed at the same ratio for 24 hours was left to stand until it reached the same temperature and measured. As a result, as shown in FIG. 9 and FIG. 13, P Δr The response of increased 4.10 times the standard (Examples 2-1 to 2-4). FIG. 9 shows the frequency dependence of Δr after 1 hour has elapsed since mixing 10 mg / mL solute with pure water (Example 2-1), the frequency dependence of Δr after 1 hour has elapsed since mixing 20 mg / mL solute with pure water (Example 2-2), the frequency dependence of Δr after 1 hour has elapsed since mixing 30 mg / mL solute with pure water (Example 2-3), and the frequency dependence of Δr after 24 hours has elapsed since mixing 20 mg / mL solute with pure water (Example 2-4). As a standard for the difference in Experimental Example 2, the P Δr-t / P Δr-s The value was set to 1.0.

[0107] (Experimental Example 3) Observation of promotion of hydrogen bond structural change by sub-terahertz wave irradiation In the device shown in Fig. 1, egg white lysozyme powder (100 mg) was mixed with pure water (1 mL), and 0.8 mL was introduced into the PDMS container. The lid 11 was then placed on the container in the same manner, and the measured value was taken after 18 minutes had elapsed for stabilization to the set temperature of 24°C. This value was used as the calibration standard. To observe the effect, the difference was taken as the standard for the measurement when sub-terahertz waves were irradiated. Sub-terahertz waves (0.1 THz, 16 mW / cm) were irradiated from the bottom of the PDMS container for 10 minutes, from 20 to 30 minutes after the sample was introduced into the container. 2 ) was irradiated, the temperature rose by 4°C during irradiation, and P Δr The response increased to 1.43 times the standard 30 minutes after the end of irradiation (60 minutes after mixing of the sample) (FIGS. 10A, 11, and 14). (Examples 3-0 to 3-4) Note that FIG. 10A shows the frequency dependence of Δr 18 minutes after mixing of the sample (Example 3-0), the frequency dependence of Δr 28 minutes after mixing of the sample (Example 3-1), the frequency dependence of Δr 40 minutes after mixing of the sample (Example 3-2), the frequency dependence of Δr 50 minutes after mixing of the sample (Example 3-3), and the frequency dependence of Δr 60 minutes after mixing of the sample (Example 3-4). The value of Example 3-0 was used as the calibration standard for Experimental Example 3. The P of Example 3-1 was used as the standard for the difference in Experimental Example 3. Δr-t / P Δr-s The value was set to 1.0.

[0108] (Experimental Example 4) In a similar comparison to Example 3, a sample was used within 1 hour after mixing, and the temperature history was set to the same as in Example 3 without irradiating with sub-terahertz waves. Δr The response of Δr decreased to 0.8 times the standard 60 minutes after the sample was introduced (FIG. 10B, FIG. 11, FIG. 15) (Examples 4-0 to 4-4). FIG. 10B shows the frequency dependence of Δr 18 minutes after sample mixing (Example 4-0), the frequency dependence of Δr 28 minutes after sample mixing (Example 4-1), the frequency dependence of Δr 40 minutes after sample mixing (Example 4-2), the frequency dependence of Δr 50 minutes after sample mixing (Example 4-3), and the frequency dependence of Δr 60 minutes after sample mixing (Example 4-4). The value of Example 4-0 was used as the calibration standard for Experimental Example 4. The P of Example 4-1 was used as the standard for the difference in Experimental Example 4. Δr-t / P Δr-s The value was set to 1.0.

[0109] (Experimental Example 5) In a similar comparison to Example 3, a sample was used within 1 hour after mixing, and the temperature was set to a constant value without irradiating sub-terahertz waves or heating. Δr The response increased to 1.28 times the reference value 60 minutes after sample introduction (FIG. 10C, FIG. 11, FIG. 15). (Examples 5-0 to 5-4) Note that FIG. 10C shows the frequency dependence of Δr 18 minutes after sample mixing (Example 5-0), the frequency dependence of Δr 28 minutes after sample mixing (Example 5-1), the frequency dependence of Δr 40 minutes after sample mixing (Example 5-2), the frequency dependence of Δr 50 minutes after sample mixing (Example 5-3), and the frequency dependence of Δr 60 minutes after sample mixing (Example 5-4). The value of Example 5-0 was used as the calibration reference for Experimental Example 5. The P of Example 5-1 was used as the reference for the difference in Experimental Example 5. Δr-t / P Δr-s The value was set to 1.0.

[0110] (Experimental Example 6) In a similar comparison to Example 3, a sample was used within 1 hour after mixing, and was not irradiated with sub-terahertz waves. The sample was cooled to a temperature 4°C lower for 10 minutes, from 20 to 30 minutes after being introduced into the container. Δr No response of PΔr was observed (FIG. 10D, FIG. 11, FIG. 15). (Examples 6-0 to 6-4) FIG. 10D shows the frequency dependence of Δr 18 minutes after sample mixing (Example 6-0), the frequency dependence of Δr 28 minutes after sample mixing (Example 6-1), the frequency dependence of Δr 40 minutes after sample mixing (Example 6-2), the frequency dependence of Δr 50 minutes after sample mixing (Example 6-3), and the frequency dependence of Δr 60 minutes after sample mixing (Example 6-4). The value of Example 6-0 was used as the calibration standard for Experimental Example 6. Since no response of PΔr was observed in Experimental Example 6, the value of P in Examples 6-0 to 6-4 was used as the calibration standard for Experimental Example 6. Δr-t / P Δr-s The notation has been changed to -.

[0111] (Experimental Example 7) In a similar comparison to Example 3, polyethylene glycol (PEG) 6000 powder was mixed with pure water (1 mL) at a weight ratio of 4% on the day before the measurement, and 0.8 mL of a sample within one day after mixing was introduced into a PDMS container and then irradiated with sub-terahertz waves. ΔrThe response increased to 1.28 times the standard 30 minutes after the end of irradiation (60 minutes after sample introduction) (FIG. 12A, FIG. 16). (Examples 7-0 to 7-4) Note that FIG. 12A shows the frequency dependence of Δr 18 minutes after sample introduction (Example 7-0), the frequency dependence of Δr 28 minutes after sample introduction (Example 7-1), the frequency dependence of Δr 40 minutes after sample introduction (Example 7-2), the frequency dependence of Δr 50 minutes after sample introduction (Example 7-3), and the frequency dependence of Δr 60 minutes after sample introduction (Example 7-4). The value of Example 7-0 was used as the calibration standard for Experimental Example 7. As the standard for the difference in Experimental Example 7, the value of PΔr-t / PΔr-s of Example 7-1 was set to 1.0.

[0112] (Experimental Example 8) In a similar comparison to Example 3, PEG6000 powder was mixed with pure water (1 mL) at a weight ratio of 4% on the day before the measurement, and 0.8 mL of a sample was introduced into a PDMS container within one day of mixing. After that, the sample was not irradiated with sub-terahertz waves, was not heated, and was set at a constant temperature. As a result, for 50 minutes after the sample was introduced, Δr No response was observed (FIG. 12B, FIG. 16). (Examples 8-0 to 8-3) Note that FIG. 12B shows the frequency dependence of Δr 18 minutes after sample introduction (Example 8-0), the frequency dependence of Δr 28 minutes after sample introduction (Example 8-1), the frequency dependence of Δr 40 minutes after sample introduction (Example 8-2), and the frequency dependence of Δr 50 minutes after sample introduction (Example 8-3). The value of Example 8-0 was used as the calibration standard for Experimental Example 8. In Experimental Example 8, P Δr Since no response was observed, P in Examples 8-0 to 8-3 Δr-t / P Δr-s The notation has been changed to -.

[0113] As the above experimental results show, it was possible to measure changes in the hydration state of protein (egg white lysozyme), deoxyribonucleic acid (DNA) (DNA derived from salmon sperm), and polyethylene glycol (PEG6000). The cause of the changes is thought to be a change in the amount of hydrogen bonds formed between solvent molecules, which is caused by a change in the hydrogen bond structure between solvent molecules and solute molecules, so it is believed that this detection method can also be applied to other water-soluble polymers. The experimental results show that the hydration state of water-soluble polymers can be detected using this form of detection method (or detection device).

[0114] That is, the method for detecting a change in the hydrogen bond structure of a liquid includes the steps of: placing a solution containing a water-soluble polymer and water in the gap d between the reflector and the coaxial probe, the gap d being less than 2 mm; irradiating the solution with microwaves from the coaxial probe while sweeping the frequency, and detecting the reflected waves input to the coaxial probe. By performing the above-mentioned calculations on the detected reflected waves, the change in the hydration state can be detected and determined.

[0115] A method for detecting a change in hydrogen bond structure in a liquid, comprising the steps of: placing a solution containing nucleic acid in a gap d between a reflector and a coaxial probe, the gap d being less than 2 mm; irradiating the solution with microwaves while sweeping the frequency from the coaxial probe, and detecting the reflected wave input to the coaxial probe.

[0116] A method for detecting a change in hydrogen bond structure in a liquid, comprising the steps of: placing a solution containing a protein in a gap d between a reflector and a coaxial probe, the gap d being less than 2 mm; irradiating the solution with microwaves while sweeping the frequency from the coaxial probe, and detecting the reflected wave input to the coaxial probe.

[0117] A method for detecting changes in hydrogen bond structure in a liquid, comprising the steps of: placing a solution containing a synthetic polymer in a gap d between a reflector and a coaxial probe, the gap d being less than 2 mm; irradiating the solution with microwaves while sweeping the frequency from the coaxial probe, and detecting the reflected wave input to the coaxial probe. In particular, it has been confirmed that changes in hydrogen bond structure can be detected in polyethylene glycol.

[0118] FIG. 17 is a diagram showing the relationship between proteins and hydration.

[0119] In the state before irradiation with sub-terahertz waves (Figure 17(A)), there are shown bulk water WAT1 with a network, hydrated water WAT2, water with a destroyed network (shorter relaxation time than bulk water) WAT3, water that strongly interacts with the surface charge (longer relaxation time than bulk water) WAT4, and a space CAVITY5 where water cannot easily enter due to thermal motion due to the strong ion-dipole-ion interaction on the surface. Hydrated water WAT2 consists of water with a destroyed network (shorter relaxation time than bulk water) WAT3, and water that strongly interacts with the surface charge (longer relaxation time than bulk water). The positive charges on the amino acid side chains are indicated by +. Here, WAT1 is the aforementioned τ main , WAT3 is τ fast , WAT4 is τ slow has a relaxation time of

[0120] In the state after irradiation with sub-terahertz waves (Figure 17(B)), hydrated water WAT6, in which the total number of hydrogen bonds has increased, including hydrophobic hydration, and water that has entered the hydrophobic interior (relaxation time longer than bulk water) WAT7 are shown. Here, WAT6 and WAT7 are the aforementioned τ slow has a relaxation time of

[0121] Proteins are biological materials that perform mechanical and chemical work. The collective interaction (hydration) between protein molecules and the many water molecules that surround them is complex. Proteins are polymers made up of approximately 20 types of amino acids linked together. Depending on the type of amino acid contained, the surface of a protein can have areas that like water (hydrophilic) and areas that dislike water (hydrophobic). Immediately after mixing water with dried protein, the bond (hydrogen bond) network between water molecules breaks down in the water. After that, a stable hydration structure is formed by removing the strong bonds with electric charges and remodeling the network. When sub-terahertz waves are irradiated, WAT3, which has a short relaxation time, is activated, shortening the time required for this change.

[0122] Hydration of liquids progresses when sub-terahertz waves are irradiated. According to the above-mentioned detection method, the Δr method can be used to detect minute changes in the dielectric constant remaining in the sample after irradiation. In the above-mentioned technique, a type of Fabry-Perot resonator was introduced into the coaxial probe reflection method in order to evaluate the signal derived from the multiple internal reflection of the incident electromagnetic wave. The change in the hydrogen bond structure (change in hydration state) was consistent with the interpretation from the change in the methyl group signal intensity on the lysozyme side measured using a nuclear magnetic resonance (NMR) spectroscopy device. It was found that the state change due to irradiation was localized around the hydrophobic cavity of the protein. It can be seen that water also enters the hydrophobic cavity due to irradiation with sub-terahertz waves. When the change in hydration state was measured by combining microwave dielectric constant measurement, terahertz spectroscopy, and NMR spectroscopy under irradiation with sub-terahertz waves, it was found that the hydration structure containing water, in which the initial network was collapsed, changed to a hydration structure in which the network was strengthened, including in highly hydrophobic regions, due to irradiation of sub-terahertz waves on a lysozyme aqueous solution.

[0123] According to the above-mentioned detection method, it is considered that the change in the state of the hydrogen bond structure can be detected not only for a solution containing an organic substance such as a protein in water as the liquid to be measured, but also for various types of solutions. By placing a measurement probe that emits microwaves so as to penetrate a dish-shaped container, placing water in the lower layer and oil in the upper layer, and placing a reflector on top of them, and appropriately selecting the oil film, water film, and probe-reflector distance, the change in state can be detected. Alternatively, liquids containing inorganic solids in water, alcohol for aging, etc. are also considered as the measurement target. For example, other organic substances, phosphoric acid, etc. are also considered as the measurement target.

[0124] As described above, it was confirmed that the hydration change of proteins, which usually takes a long time, is significantly accelerated by irradiation with sub-terahertz waves. By irradiating with sub-terahertz waves, it is possible to control the hydration phenomenon, which is essential for the expression of protein structure and function. This technology can also be used for activating enzyme reactions, preserving food and beverages, aging, and research on protein abnormalities. Data analysis using this evaluation method is useful for examining the mechanism of aging for foods, especially beverages, especially alcoholic beverages, which are particularly preferred for their tastes as they are aged over a long period of time. It is also possible to attach data from this evaluation method to products, and it is thought that products with this data attached will increase the interest of buyers. For medical applications, it is expected that functional expression will change by aging the hydration of dried and solidified medications. It can also be used to detect the hydration state in various situations where protein functions are actively utilized, such as in biopharmaceuticals such as antibodies, enzyme reactions in polymerase chain reaction (PCR) for detecting the new coronavirus, and other situations in which protein functions are actively utilized. [Explanation of symbols]

[0125] 10...container, 10A...reflector, 11...lid, 20...hydrogen-bonding liquid, 30...sub-terahertz supply unit, 40...coaxial probe, 41...vector network analyzer, 42...cable, 43...cable connector, 44...probe connector, 45...tip surface, 50...temperature adjustment unit, 60...control unit, 61...central processing unit (CPU), 62...storage device, 62A...program, 70...display, 100...device for detecting changes in hydrogen-bonding structure of liquid, Z0...container surface position, Z1...coaxial probe tip surface position, d...gap.

Claims

1. Providing a hydrogen-bonding liquid in a gap d between the reflector and the coaxial probe; The liquid is irradiated with microwaves while sweeping the frequency from the coaxial probe, and the complex relative dielectric constant ε is calculated from the reflected wave input to the coaxial probe. ｒ ＊ A measuring step for determining The complex relative dielectric constant ε ｒ ＊ determining a magnitude Δr of change from a reference; Including, The gap d is less than 2 mm. A method for detecting changes in hydrogen bond structure in liquids.

2. The complex relative dielectric constant ε ｒ ＊ teeth, The complex relative dielectric constant ε of a solution containing a solvent having properties correlated with those of the liquid and having a network structure ａ ＊ and, Complex relative permittivity ε of unloaded medium ｖ ＊ and, Scattering parameters of the reflected wave; It is calculated using The method for detecting a change in the hydrogen bond structure of a liquid according to claim 1.

3. The microwaves are swept within a frequency band including 3 GHz to 9 GHz; The method for detecting a change in the hydrogen bond structure of a liquid according to claim 1.

4. The method further includes a step of irradiating the liquid with electromagnetic waves having a frequency in the sub-terahertz band. The method for detecting a change in the hydrogen bond structure of a liquid according to claim 1.

5. The solute of the liquid is organic. The method for detecting a change in the hydrogen bond structure of a liquid according to claim 1.

6. The liquid solvent comprises water. The method for detecting a change in the hydrogen bond structure of a liquid according to claim 1.

7. a coaxial probe that irradiates a reflector with a microwave having a swept frequency and receives a reflected wave of the microwave reflected by the reflector; The complex relative dielectric constant ε ｒ ＊ A measurement unit for determining The complex relative dielectric constant ε obtained by the measuring unit during a microwave frequency sweep. ｒ ＊ A calculation unit for calculating a magnitude Δr of change from a reference value of Equipped with A gap d between the reflector and the coaxial probe is set to less than 2 mm. A device for detecting changes in hydrogen bond structure in liquids.

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