JP2014122888A - Method for quantifying adsorption of trace amount of water molecule to thin film by use of reflectometric interference spectroscopy, and measurement system for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means capable of analyzing a behavior of water molecules to a thin film made of various materials, from a viewpoint different from the prior arts.SOLUTION: There is provided an analyzing method relating to a behavior of gaseous water molecules with respect to a sample thin film including a step of measuring data (measurement step) relating to a film thickness of the sample thin film by RIfS (reflectometric interference spectroscopy) while continuously or discontinuously changing the humidity of the environment where the sample thin film formed on a surface of a measurement substrate is placed. A RIfS measurement system including humidity adjusting means for continuously changing the humidity of the environment where the sample thin film formed on a surface of a sensor chip is placed is also.provided.

Description

  The present invention relates to an analysis method applying RifS (Reflectometric Interference Spectroscopy) and a measurement system therefor.

  RIFS uses the principle that when a thin film with an optical thickness formed on the surface of a sensor chip is irradiated with light, the wavelength (bottom peak wavelength) at which the reflectance becomes lowest due to interference differs depending on the thickness. This is an analysis method. That is, by measuring the bottom peak wavelength for the sensor chip on which the sample thin film is formed, the difference (Δλ) from the measured bottom peak wavelength for the unmodified sensor chip is calculated, and from there, the sample thin film It can be converted into an optical path length (refractive index × thickness). For example, an intermolecular interaction (binding by antigen-antibody reaction) occurs between an analyte (antigen molecule, etc.) contained in a sample and a ligand (antibody, etc.) immobilized on the surface of the sensor chip, and the analyte When a light thin film is formed, Δλ changes according to the amount of formation (average thickness), so that it can be detected quantitatively in real time without labeling the analyte in the sample. it can. The basic principle of RIfS is described in Patent Document 1, Non-Patent Document 1, and the like.

As a means for analyzing water-related physical properties such as water absorption and swelling characteristics of various materials such as polymer materials, the following are known.
For example, the moisture content of a sample (value obtained by dividing the weight of water by the total weight of the sample containing water) is simply measured by measuring the weight before and after heating the sample containing water in an oven. More precisely, it can be measured by the Karl Fischer method, and various measuring devices are also sold. However, such a measurement method requires a relatively large amount of sample, and the amount of adsorption (moisture absorption) and the speed thereof that change according to the environmental humidity cannot be determined in real time.

  Patent Document 2 describes means using QCM (quartz crystal microbalance) or QCM-D which is an improved method thereof. That is, by using a sensor having a film-like sample formed on the surface and measuring the QCM or the like while changing the relative humidity of the minute space formed above the sample, the physical properties of the absorbed sample A method for analyzing (swellability) and an apparatus (module) for carrying out the method are described. Furthermore, Non-Patent Document 2 has an application example in which the thickness of a sample is measured while changing the relative humidity in several steps using the apparatus (module) sold by the applicant according to Patent Document 2. It is disclosed. However, in theory such as QCM, measurement cannot be performed if the measurement environment is not constant (if the measurement environment changes, the measurement baseline changes because the deviation from the Sauerbrey equation increases when water is adsorbed to the sample thin film). ), And the devices for these methods generally have a mechanical drive and therefore take a long time to measure. Therefore, as shown in the graph in Non-Patent Document 2, even when the relative humidity is changed, it is necessary to change it stepwise so that it is held at a constant value for a while after being raised or lowered. It is impossible to measure in real time while continuously changing the humidity. Moreover, since the above-mentioned error becomes larger as the thickness of the film increases, there is a disadvantage that the film thickness to be measured is very narrow.

  Non-Patent Document 3 discloses a device in which a precision electronic balance installed in a sealed space and a humidity control device are combined. In this device, it is possible to measure the moisture adsorption / desorption behavior on a solid material by changing the humidity condition in the sealed space and obtaining the water vapor adsorption isotherm by weighing with a precision electronic balance in the equilibrium state of each humidity. it can. However, the accuracy of the electronic balance is on the order of micrograms.

  According to the conventional technology as described above, in principle, it can be seen only as the difference between the measured amount before and after the adsorption / desorption of water, and the accuracy of the information obtained even when trying to analyze the characteristics of the sample related to the interaction with water. Was limited and could only grasp the behavior of water macroscopically (water as bulk).

Japanese Patent No. 3778673 International Publication No. WO2009 / 005452

Sandstrom et al, APPL.OPT., 24, 472, 1985 Application Note (QS 405-18-1) "Analysing vapor uptake & release with QCM-D" (http://www.q-sense.com/file/18-analyzing-humidity-effects-with-qcm-d- 1.pdf) Intrinsic-Compact and Economical Dynamic Vapor Sorption System (http: //www.thesorptionsolution/com/files/DVS%20Intrinsic%20Brochure.pdf)

  An object of the present invention is to provide a means that makes it possible to analyze the behavior of water molecules with respect to thin films made of various materials from an unconventional viewpoint.

  The inventors measured Δλ of the sample thin film formed on the surface of the sensor chip while continuously changing the humidity of the atmosphere in which the sensor chip was placed using an intermolecular interaction measuring apparatus based on RIfS. Surprisingly, however, it was possible to observe how Δλ (and the film thickness converted therefrom) changed at the sub-angstrom level in response to changes in humidity. Since the size of the water molecule is about 0.4 nm, if the film thickness is converted from Δλ to 0.02 nm, the water molecule adheres to about 1/20 of the surface area of the sensor chip (water molecule 1 on average) / 20 thin films are formed). That is, it was found that the behavior of gaseous water with respect to the sample thin film can be quantitatively observed at the molecular level, not as a bulk. Moreover, since the optical interface is viewed in RIfS, the baseline does not change even if the humidity (and temperature if necessary) is changed, and there is no mechanical drive, so the limit of the spectrometer (usually Up to 10 msec).

  The present invention completed based on such findings provides, in one aspect, an analysis method for the behavior of gaseous water molecules with respect to a thin film, and in another aspect, a measurement system for performing the analysis method. To do. That is, the present invention includes the following inventions.

  [1] Data on the film thickness of the sample thin film is obtained by RIFS (reflection interference spectroscopy) while continuously or discontinuously changing the humidity of the atmosphere in which the sample thin film formed on the surface of the measurement substrate is placed. A method for analyzing the behavior of gaseous water molecules with respect to a sample thin film, comprising a step of measuring (measuring step).

[2] The analysis method according to [1], wherein in the measurement step, 10 or more measurement steps are performed in which a humidity value interval is 0.1 to 10%.
[3] The analysis method according to [1] or [2], wherein in the measurement step, a change in humidity is repeated for 1 to 100 cycles.

[4] The analysis method according to any one of [1] to [3], wherein in the measurement step, the humidity is changed at a rate of 1 to 3600 seconds per 1%.
[5] The analysis method according to any one of [1] to [4], wherein in the measurement step, the initial humidity is increased and then the humidity is decreased.

[6] The analysis method according to any one of [1] to [5], including changing the temperature of the atmosphere or the temperature of the sample thin film in the measurement step.
[7] The analysis method according to any one of [1] to [6], wherein the measurement step is performed a plurality of times under conditions in which the humidity change rate of the atmosphere is different.

[8] The analysis method according to any one of [1] to [7], wherein the measurement step is performed a plurality of times under conditions in which the temperature of the atmosphere or the temperature of the sample thin film is different.
[9] A thin film in which the sample thin film is formed of a film-forming solid or liquid; a thin film formed of a solid, liquid, or gas that can be fixed to the surface of the measurement substrate; or a sealed space formed on the measurement substrate The analysis method according to any one of [1] to [8], which is a thin film formed of a substance that dissolves or floats inside.

[10] The analysis method according to any one of [1] to [9], wherein the thickness of the sample thin film is 1 nm to 100 μm.
[11] Based on the data obtained by the measurement step, the adsorption / desorption amount and adsorption / desorption rate of gaseous water molecules, the adsorption / desorption dynamics of gaseous water molecules, the saturation hydration amount of the sample thin film, the equilibrium time, the equilibrium membrane Thickness and equilibrium moisture content, wettability of minute area at molecular level, difference in environment (change in temperature and humidity), difference in hydration property of substance before and after stress, absorption / desorption of water molecule (humidification) And the step of analyzing at least one item selected from the group consisting of hysteresis, difference in hydration rate, and hydration structure, according to any one of [1] to [10] Analysis method.

[12] The analysis method according to [11], wherein the analysis of the equilibrium time, the equilibrium film thickness, or the equilibrium moisture content is performed based on a simulation curve or a calculation formula.
[13] Gaseous water molecules adsorbed and desorbed on the sample thin film calculated from the data on the film thickness of the sample thin film obtained by the measurement step for analysis of the adsorption / desorption amount or adsorption / desorption rate of the gaseous water molecules The analysis method according to [11], which is performed based on the volume or mass of

[14] The analysis method according to [11], wherein the hydration structure is analyzed based on an inflection point of a film thickness change amount-humidity curve.
[15] A RIfS measurement system comprising humidity adjusting means for continuously changing the humidity of the atmosphere in which the sample thin film formed on the surface of the measurement substrate is placed.

[16] The system according to [15], further comprising temperature adjusting means for changing the temperature of the atmosphere or the temperature of the sample thin film.
[17] The system according to claim 15 or 16, wherein a sealed space is formed around the sample thin film by overlapping a sealed space forming member having an opening through which gas flows in and out on the measurement substrate.

  The term “humidity” described in the specification includes both “relative humidity” and “absolute humidity”. If the temperature is constant, “changing the relative humidity continuously” and “changing the absolute humidity continuously” are common in that both change continuously. There is no problem in interpreting either “humidity” in the analysis method or measurement system. For example, as the humidity adjusting means in the present invention, a commercially available humidity adjusting unit or humidity adjusting device can be used. However, since the humidity is normally controlled by relative humidity, the relative humidity can be controlled accordingly. A humidity control pattern can be set. On the other hand, even if the relative humidity is the same, a higher amount of water molecules are present at higher temperatures (absolute humidity is higher), and conversely, even if there are water molecules of the same mass (even if the absolute humidity is the same), the temperature is higher. The relative humidity is lower. Therefore, when comparing the results measured at different temperatures based on a common standard, or when displaying the absolute amount of water molecules, it is more appropriate to measure or convert with absolute humidity.

  According to the present invention, since the change in the optical path length of the sample when the humidity is continuously changed can be measured in real time by RIfS, the behavior of adsorption and desorption of gaseous water molecules, The amount of adhesion (expressed in various terms such as retention, fixation, moisture absorption, moisture retention, and hydration) can be analyzed accurately. Such an analysis method of the present invention is not limited to the macroscopic physical properties expressed by words such as water absorption, water content, and swelling rate that could be measured by conventional methods, and has not been even conceived so far (of course). Analysis at the molecular level of water (not possible with conventional methods), for example, determination of a small amount of water molecules adhering to a sample, measurement of adsorption / desorption rate of water molecules, wettability at the molecular level, environmental differences This makes it possible to evaluate physical properties such as the difference in hydration properties of substances before and after stress. The present invention created by introducing the technical idea of performing measurement while continuously changing humidity into RIfS is a means capable of essentially analyzing the behavior of gaseous water molecules with respect to various substances. As groundbreaking.

FIG. 1 is a schematic diagram showing an embodiment of a measurement system according to the present invention (a first embodiment of a measurement environment adjusting mechanism). FIG. 2 is a schematic view showing one embodiment of the measurement system of the present invention (second embodiment of the measurement environment adjusting mechanism). FIG. 3 is a graph showing various humidity change patterns. 4 shows a sensor chip No. of Example. It is a graph showing the measurement result (Δλ, relative humidity) of 1 (bare substrate). 5 shows the sensor chip No. of Example. 2 is a graph showing measurement results (Δλ, relative humidity) of 2 (OPTOOL DSX). 6 shows the sensor chip No. of Example. 3 is a graph showing measurement results (Δλ, relative humidity) of 3 (Fressera R). 7 shows the sensor chip No. of Example. 4 is a graph showing measurement results (Δλ, relative humidity) of 4 (Triethoxy-1H, 1H, 2H, 2H-tridecafluoro-n-octylsilane). 8 shows a sensor chip No. of Example. 5 is a graph showing measurement results (Δλ, relative humidity) of 5 (Octadecyl-trimethoxysilane). 9 shows a sensor chip No. of Example. 6 is a graph showing measurement results (Δλ, relative humidity) of 6 (Hydroxymethyl triethoxysilane). 10 shows a sensor chip No. of Example. 7 is a graph showing measurement results (Δλ, relative humidity) of 7 (N- (3-Triethoxysilylpropyl) gluconeamide). 11 shows the sensor chip No. of Example. 8 is a graph showing measurement results (Δλ, relative humidity) of 8 (CMD-L-05B1). 12 shows the sensor chip No. of Example. 9 is a graph showing measurement results (Δλ, relative humidity) of 9 (CMD-D40-021205). FIG. 13 shows a sensor chip No. of Example. 10 is a graph showing a measurement result (Δλ, relative humidity) of 10 (CMD500-06F1). 14 shows a sensor chip No. of Example. 11 is a graph showing measurement results (Δλ, relative humidity) of 11 (BSA). 15 shows a sensor chip No. of Example. It is a graph showing the measurement result ((DELTA) (lambda), relative humidity) of 12 (neutravidin). 16 shows a sensor chip No. of Example. 13 is a graph showing measurement results (Δλ, relative humidity) of 13 (cell membrane fraction). 17 shows a sensor chip No. of Example. 14 is a graph showing a measurement result (Δλ, relative humidity) of 14 (DOPC). 18 shows a sensor chip No. of Example. 15 is a graph showing a measurement result (Δλ, relative humidity) of 15 (PS1). 19 shows a sensor chip No. of Example. 16 is a graph showing a measurement result (Δλ, relative humidity) of 16 (PS2). 20 shows a sensor chip No. of Example. 17 is a graph showing measurement results (Δλ, relative humidity) of 17 (PS3). 21 shows a sensor chip No. of Example. 18 is a graph showing a measurement result (Δλ, relative humidity) of 18 (PMMA1). 22 shows the sensor chip No. of Example. 19 is a graph showing measurement results (Δλ, relative humidity) of 19 (PMMA2). 23 shows a sensor chip No. of Example. It is a graph showing the measurement result (Δλ, relative humidity) of 20 (PMMA3). 24 shows a sensor chip No. of Example. It is a graph showing the measurement result ((DELTA) (lambda), relative humidity) of 21 (PA). 25 shows a sensor chip No. of Example. 22 is a graph showing measurement results (Δλ, relative humidity) of 22 (Hydran CP7610). 26 shows a sensor chip No. of Example. It is a graph showing the measurement result (Δλ, relative humidity) of 23 (SiO ₂ ). 27 shows a sensor chip No. of Example. It is the graph which contrasted the measurement result before and behind 12 hours about 12 (neutravidin). 28 shows a sensor chip No. of Example. 15 is a graph comparing the measurement results (Δλ, relative humidity) of 15, 16, 17 (PS1-3: difference in coating concentration). 29 shows a sensor chip No. of Example. 18 is a graph comparing the measurement results (Δλ, relative humidity) of 18, 19, 20 (PMMA1 to 3: difference in coating concentration). 30 shows a sensor chip No. of Example. 20 (PMMA3) is a graph comparing the measurement results (λ, temperature vs. temperature) when the temperature is continuously changed while maintaining the relative humidity in three stages (from the top relative humidity is 50%, 30 %, 10%). 31 shows a sensor chip No. of Example. 23 is a graph comparing measurement results (Δλ, relative humidity) when the humidity is continuously changed while maintaining the temperature in two stages for 23 (SiO ₂ ). FIG. 32 is an image view of a sealed space forming member (gas flow path) used in Example Group 2. FIG. 33 is a graph showing an adsorption / desorption curve relating to Example 8. FIG. 34 is a plot showing the water adsorption / desorption dynamics of Example 8 (2). FIG. 35 is a graph showing a drying curve and a wetting curve for Example 8 (3). FIG. 36 is a graph showing changes in film thickness in Example 9. Three graphs in the left column each show a change in film thickness with respect to the humidity control pattern, and three graphs in the right column show a change in film thickness at the time of water adsorption and desorption, respectively. FIG. 37 is a graph showing a change in film thickness in which an auxiliary line for obtaining the inflection point of sample (c) is drawn in Example 9. FIG. 38 is a graph used in the simulation of the equilibrium humidity control time (left) and the simulation of the equilibrium moisture content (right) in Example 9. FIG. 39 is a graph showing changes in film thickness of a thin film composed of normal NeutrAvidin (upper) and a thin film composed of modified NeutrAvidin (lower) in Example 10. FIG. 40 is a graph showing changes in film thickness of PS (polystyrene) (upper stage) and PMMA (polymethyl methacrylate) (lower stage) in Example 11. The two graphs in the left column each show the change in film thickness with respect to the humidity control pattern, and the two graphs in the right column show the film thickness change during water adsorption and desorption, respectively. FIG. 41 is a graph showing changes in film thickness of CMD (carboxymethyl dextran) (upper stage) and dextran (lower stage) in Example 12. The two graphs in the left column each show the change in film thickness with respect to the humidity control pattern, and the two graphs in the right column show the film thickness change during water adsorption and desorption, respectively. FIG. 42 is a plot showing the water adsorption / desorption dynamics of CMD (carboxymethyldextran) (upper) and dextran (lower) in Example 12. FIG. 43 is a graph (upper stage) showing the change in the film thickness of Fressela R and a plot (lower stage) showing the water adsorption / desorption dynamics in Example 13 (1) (temperature difference between the humidity control temperature and the sample surface temperature is 5 ° C.). ). FIG. 44 is a graph (upper stage) showing the change in the film thickness of Fresseler R and a plot (lower stage) showing the water adsorption / desorption dynamics in Example 13 (2) (temperature difference between the humidity control temperature and the sample surface temperature is 10 ° C.). ).

-Measurement system-
In one aspect, the present invention provides a measurement system suitable for carrying out the analysis method of the present invention relating to the behavior of water molecules with respect to a sample thin film. The measurement system of the present invention is based on RIfS, in particular reflective RIfS. Examples of embodiments of the measurement system of the present invention are shown in FIG. 1 and FIG. 2, but the embodiments of the present invention are not limited to those shown in these drawings, and are modified within a range that does not impede the effects of the present invention. Embodiments are also included.

  The RIfS measurement system 1 is basically configured by an RIfS apparatus 10, a sensor chip 21 used by being set in the RIfS apparatus 10, a control apparatus 50, and a system control means including a connection means 60 between the control apparatus 50 and each element. Is done. The RIfS measurement system 1 of the present invention further includes measurement environment adjusting means for continuously or discontinuously changing the temperature and humidity of the atmosphere in which the sensor chip 21 is placed, and preferably the sensor chip 21 (sample thin film 21c). In addition, a sealed space forming member 23 that forms a sealed space around the sensor chip and a sensor chip temperature adjusting means for directly adjusting the temperature of the sensor chip 21 (sample thin film 21c) are also included.

(RIfS device)
The RIfS apparatus 10 accommodates a RIfS measurement mechanism, a measurement environment adjustment mechanism, a sensor chip 21 and a sensor chip set part (stage) and a chip cover for setting a gas flow path forming member 23 used as necessary. A part of the housing is provided with a shading cover that can be opened and closed in part.

RIfS measurement mechanism The RIfS measurement mechanism includes a white light source 11, a spectroscope 12, and a measurement probe 13. When the white light source 11 is turned on, the white light is irradiated from the probe 13 directed to the measurement region A through the optical fiber 13a, and the reflected light from the measurement region A is guided to the spectroscope 12 through the optical fiber 13b. . Arrows in the figure represent incident light and reflected light. The range in which the white light irradiated from the probe 13 reaches corresponds to the measurement region A, and the area of the measurement region A (hereinafter sometimes referred to as “measurement area”) is, for example, 0.126 mm ² (with a diameter of 0.4 mm). Yen).

  The white light source 11 includes a halogen lamp and a casing that stores the halogen lamp. The housing is provided with a connection port for connecting the first optical fiber 13a, and is arranged so that the end face of the optical fiber 13a connected to the connection port faces the halogen lamp.

  The spectroscope 12 detects the intensity for each wavelength of the light received by the light receiving unit (CCD or the like), and outputs it to the control device 50 as the spectral intensity. The housing is provided with a connection port for connecting the second optical fiber 13b, and is arranged so that the end face of the optical fiber 13b connected to the connection port and the light receiving portion face each other.

  The measurement probe 13 is an optical fiber 13a as a first light transmission path for guiding white light from the white light source 11 to the measurement region A, and reflected light in the measurement region A of the white light irradiated from the optical fiber 13a. And an optical fiber 13b as a second light transmission path for guiding to the spectroscope 12. Each of the optical fibers 13a and 13b has a structure in which fine fibers are bundled. One end of the optical fiber 13 a is connected to the connection port of the white light source 11, and one end of the optical fiber 13 b is connected to the connection port that receives light from the spectrometer 12. The other end of each of the optical fibers 13a and 13b is combined with the measurement probe 13 so that each of the fine fibers forms one bundle. The fine fibers constituting the optical fiber 13a are distributed in the center of the measurement probe 13, and the fine fibers constituting the optical fiber 13b are distributed around the bundle of the fine fibers of the optical fiber 13a.

  In the present embodiment, reflected light from the sensor chip 21 is received by the spectroscope 12 (reflective type RIfS). It is also possible to arrange the spectroscope 12 so as to irradiate the sensor chip 21 with light and to receive the light transmitted through the sensor chip 21 and to detect the spectral intensity of the transmitted light.

(Sensor chip / measurement board)
The sensor chip 21 is generally rectangular, and includes a substrate 21a, an optical thin film 21b formed thereon, and a sample thin film 21c formed on the optical thin film 21b in the present invention. A part of the sample thin film 21c becomes a measurement region A where the reflectance is measured by irradiation with white light.

  In addition, in the conventional general RIfS, in order to form a flow path capable of feeding various liquids, a flow cell serving as a frame material is loaded on a sensor chip and used (a combination of a sensor chip and a flow cell). Measurement member). In the present invention, the sample thin film is not placed in a liquid, but is measured in a state where it is placed in a gas and brought into contact with gaseous water molecules. Therefore, it is not necessary to use such a flow cell. Instead, in one of the preferred embodiments of the present invention, a sealed space forming member for sealing around the sample thin film formed on the surface of the sensor chip and for introducing and discharging a gas whose humidity and / or temperature is adjusted. In some cases, a member similar to a conventional flow cell and superposed on a sensor chip may be used.

  In this specification, a sensor chip in which the sample thin film 21c is formed on the surface is referred to as an “analysis target sensor chip”. On the other hand, a sensor chip having a minimum configuration as a sensor chip for RIfS, typically, only the substrate 21a and the optical thin film 21b is referred to as an “unmodified sensor chip”. In addition, such unmodified sensor chip and a state in which pretreatment (for example, treatment with a silane coupling agent) for forming a sample thin film is performed on the unmodified sensor chip (a state immediately before forming the sample thin film) This is referred to as a “reference sensor chip” for comparing the measurement data of the sensor chip to be analyzed.

  Here, in the present invention, as shown in FIG. 1, not only the case where the sample thin film 21c is formed on the upper layer of the unmodified sensor chip (the optical thin film 21b on the outermost surface) but also the substrate 21a not covered with the optical thin film 21b. Even in the case where the sample thin film 21c is directly formed on the upper layer of the film itself (therefore, it is difficult to call the sensor chip), the measurement based on RIfS and the analysis of the sample thin film can be performed. In the present specification, the present invention is described using the term “(analysis object) sensor chip” composed of the substrate 21a, the optical thin film 21b, and the sample thin film 21c based on the former which is a typical embodiment. However, it is possible to interpret the invention by appropriately reading the term as “(analysis target) substrate” composed of the substrate 21a and the sample thin film 21c. Similarly, “unmodified sensor chip” and “reference sensor chip” can be read as “unmodified substrate” and “reference substrate”. The substrate 21a on which the sample thin film 21c is to be applied, which may or may not be laminated with the optical thin film 21b, is collectively referred to as a “measurement substrate”.

-Substrate and optical thin film The substrate 21a and the optical thin film 21b are formed of a material having a refractive index and a thickness such that the minimum wavelength of reflectance observed when irradiated with white light is within an appropriate range.

In the conventional general sensor chip, the substrate 21a is preferably a substrate made of Si (silicon, silicon wafer). On the other hand, in the embodiment of the present invention, when the sample thin film 21c is formed on the upper layer of the substrate 21a, the substrate 21a is a substrate other than Si, for example, SiO ₂ (quartz glass) or other glass, polystyrene, polymethyl, or the like. It may be a substrate made of a plastic such as methacrylate, polycarbonate, or cycloolefin polymer.

When the substrate 21a is made of Si, the optical thin film 21b can be a thin film made of SiN, SiO ₂ , TiO ₂ , Ti ₂ O ₅ or the like, but a thin film made of SiN (silicon nitride) is preferable. The refractive index of SiN is about 2.0 to 2.5 in the wavelength range of about 400 to 800 nm in the visible light region. By setting the film thickness of SiN to about 45 to 90 nm, the minimum reflectance wavelength is about 400 nm to It can be adjusted to a range of 800 nm. The optical thin film 21b can be laminated on the upper surface of the substrate 21a by vapor deposition.

Sample thin film The sample thin film 21c is not particularly limited as long as it is formed of an appropriate material and has properties such as a film thickness and a refractive index to which RIfS can be applied.

  The film thickness of the sample thin film can be, for example, in the range of 1 nm to 100 μm, preferably 10 nm to 10 μm, more preferably 10 nm to 1 μm. The sample thin film 21c may be formed on the entire surface of one side of the sensor chip 21 or may be formed on a part thereof.

  Examples of the sample thin film 21c include a thin film formed from a film-forming solid or liquid; a thin film formed from a solid, liquid or gas that can be fixed to the surface of the sensor chip; or a sealed space formed on the sensor chip. Examples include a thin film formed from a substance that dissolves or floats inside.

Among these, “film-forming solids” include hydrophobic polymers (polystyrene, polymethyl methacrylate, etc.), hydrophilic polymers (polymethyl methacrylate hydrophilized by plasma treatment, etc.), water-soluble polymers, biological Materials (proteins, phospholipids, nucleic acids, sugar chains, cells, cell membrane fractions, skin, biologically derived secretory components, etc.), surface treatment agents (fluorinated water repellent treatment agents, hydrophilic surface modifiers, etc.), paints ( Ink, paint, etc.) and functional materials, but also inorganic compounds. A thin film (layer) itself made of a substance capable of adsorbing water molecules such as SiO ₂ , Si, SiN, ZnO, TiO ₂ (which changes the optical path length) may be used as the sample thin film. “Solids that can be fixed to the surface of the sensor chip” include fine particles (colloidal silica, pigments, toners, etc.) and monomolecular compounds (silane coupling agent-forming films, LB film-forming films, vapor depositions, etc.). May be.

The method for forming the sample thin film 21c is not particularly limited, and a known method similar to that used for manufacturing a sensor chip for RIfS can be used.
For example, when forming a thin film made of a synthetic or natural polymer material, use a coating technique such as dip coating, spin coating, or spray coating after preparing a solution with an appropriate viscosity using a solvent as necessary. Can be applied to the surface of an unmodified sensor chip. In addition, the surface of the unmodified sensor chip is treated with a silane coupling agent or an amine coupling agent to introduce reactive functional groups (amino group, carboxyl group, etc.) and reacted with the functional group of the polymer material. Thus, a thin film made of the polymer material may be formed. Instead of such a reaction between functional groups, a thin film made of a polymer material can be formed by intermolecular interaction or electrostatic adsorption. Alternatively, a thin film can be formed by introducing a polymerizable monomer by light, heat or the like onto the surface of the unmodified sensor chip and graft polymerizing it to produce a polymer material from the monomer. In addition, film forming techniques such as a casting method, chemical vapor deposition (CVD), and physical vapor deposition (PVD) can also be used.

  When the sample thin film 21c is made of a biological material such as protein or nucleic acid, the biological material is captured by a specific reaction such as an antigen-antibody reaction or DNA hybridization, or a non-specific reaction. A film can be formed by previously immobilizing a substance that can be formed on the surface of the sensor chip and bringing the aqueous solution of the biological substance into contact with the surface of the sensor chip. At this time, in the step of immobilizing the ligand and the step of capturing the analyte, the flow cell is placed on the sensor chip in the state before the step is performed to form a sealed channel, and the solution containing the material is fed there. It is possible to do so. Furthermore, biomaterials (collected tissues or artificial products) such as bones and skin can be fixed to the surface of the sensor chip.

-Reagent supply means The measurement system 1 may be provided with a reagent supply means for adding a small amount of reagent to the sample thin film, if necessary. In this case, the RIfS measurement apparatus 1 includes a reagent supply mechanism by spraying with a vibrator or electrostatic spraying, for example. The control device 50 storing a control program related to the reagent supply mechanism is connected so as to be communicable with each member (liquid transporting means, etc.) constituting the mechanism. In the embodiment using the sealed space forming member 23, since the reagent supply port 23d is provided in the member, the reagent supply mechanism is installed at a position where the reagent can be sprayed from the reagent supply port 23d.

(System control means)
The system control means includes a control device 50, a connection means 60 that enables communication of electrical signals, data, and the like between the control device 50 and each element constituting the measurement system 1, and a program stored in the control device 50. (Software). In addition to the white light source 11 and the spectroscope 12 that are basic elements in RIfS, the above-described elements that are the targets of the system control means include measurement environment adjustment mechanisms that are required in the present invention, such as those constituting the system control means. The temperature / humidity adjusting unit 110 and the temperature / humidity sensor 120 are included, and when the RIfS measuring device includes a sensor chip temperature controller and a reagent supply mechanism, those components are also included.

  The control device 50 receives an input of an instruction related to system operation or data processing from an operator, transmits an execution command for operating the system component (a microcomputer included in the system) according to the instruction, a system configuration Appropriate interfaces are provided for receiving measurement data from elements, operating status and reception of the system, and displaying processed data.

  The control device 50 also includes a calculation means for processing data received from each element constituting the measurement system 1, a program (software) for performing the processing, and a recording medium for storing various data. For example, the calculation of the displacement amount (Δλ) of the bottom peak wavelength based on the spectral intensity data received from the spectroscope 12 and the integration of the temperature / humidity data received from the temperature / humidity sensor 120 corresponding to the value are performed by a predetermined program. Can be processed.

  As the control device 50 having the above functions, a personal computer is generally used. In the analysis system of the present invention, it is preferable that processes such as each measurement step, start of process, repetition, and end are (semi-) automatically performed by a program stored in the control device 50.

(Measurement environment adjustment means)
The measurement environment adjusting means is a means for adjusting the humidity and / or temperature in the sealed space 23 to a desired state according to the purpose of analysis. The RIfS measurement system of the present invention includes at least humidity adjustment means, and preferably further includes temperature adjustment means, but it is desirable to include measurement environment adjustment means in which these two are integrated.

  As a first embodiment of the measurement environment adjustment mechanism, it is constituted by a temperature / humidity adjustment unit 110, a temperature / humidity sensor 120, and a casing 190 that houses them and forms a sealed space 23a, as shown in FIG. Measurement environment adjusting mechanism 100 to be used. The control device 50 storing a control program related to the measurement environment adjustment mechanism 100 is connected to the temperature / humidity adjustment unit 110 and the temperature / humidity sensor 120 so as to be controllable.

  In the present embodiment, the RIfS apparatus 10 is installed in the housing 190 of the measurement environment adjustment mechanism 100. In this case, the gas is kept in and out so that the temperature and humidity in the housing 190 can be regarded as the temperature and humidity of the atmosphere in which the sensor chip 21 set in the RIfS apparatus 10 is placed.

  As such a measurement environment adjustment mechanism 100 of the first embodiment, a temperature / humidity adjustment unit 110, a humidity sensor 120, and a housing 190 for housing them are integrated integrally, such as a thermo-hygrostat. An apparatus may be used. Examples of commercially available products include humidity control chambers ARL-0680-J, ARL-1100-J, ARS-0680-J, ARS-1100-J, PFL-3K, PFL-3KH, PFU-3K , SH-221, SH-241, SH-246, SH-661 (manufactured by Espec).

  The measurement environment adjustment mechanism 100, particularly the integrated apparatus as described above, measures the humidity in the apparatus by the temperature / humidity sensor 120, and the humidity set by the humidity / dehumidification by the temperature / humidity adjustment unit 110 is measured. It is preferable that the humidity control can be executed, and that the program is controlled by an electronic circuit (microcomputer) or system control means so that the humidity can be automatically increased, decreased, or maintained in an arbitrary pattern. An input means for storing the program in an electronic circuit, a temperature / humidity data recording / outputting means, a display means for confirming various types of information may be provided in the measurement environment adjusting mechanism 100 itself as a control panel, The control device 50 connected to the measurement environment adjusting mechanism 100 may have the function.

  As a second embodiment of the measurement environment adjustment mechanism, there is a measurement environment adjustment mechanism 100 configured by a temperature / humidity adjustment unit 110, a temperature / humidity sensor 120, and an exhaust mechanism 130 arranged as shown in FIG. In the present embodiment, the temperature generated from the temperature / humidity adjusting unit 110 after the sealed space 23a is formed around the sample thin film 21c (particularly, the measurement region A) of the sensor chip 21 by the sealed space forming member 23 similar to the flow cell. A gas whose humidity is adjusted is introduced into the sealed space 23a and supplied to the sample thin film.

  Moreover, the control apparatus 50 which memorize | stored the control program (measurement environment control program) concerning the measurement environment adjustment mechanism 100 is connected so that it can control with each element of these measurement environment adjustment mechanisms. In such a measurement environment adjusting means, the humidity and / or temperature in the sealed space 23a is measured in real time by the temperature / humidity sensor 120, and the measurement value is fed back to the control device 50 and stored in the control device 50. Compare with the value. Based on the result, by appropriately operating the temperature / humidity adjusting unit 110 and the exhaust mechanism 130, the sealed space 23a can be controlled to have the set humidity and / or temperature.

  It is preferable that the measurement environment control program can automatically increase, decrease or maintain the humidity and / or temperature of the sealed space 23a in an arbitrary pattern. A PC generally used as the control device 50 includes an input unit for the measurement environment control program, a recording / output unit for temperature / humidity data, a display unit for confirming various information, and the like. The measurement environment control program may be, for example, the following embodiments: (1) Read a preset schedule of “time” and “set temperature / humidity”, and (2) temperature / humidity adjustment unit and sensor chip The temperature / humidity sensor installed in the vicinity of the measurement area is input by converting the temperature / humidity by A / D conversion, and (3) calculating the difference between the measured temperature / humidity and the set temperature / humidity. Drive the adjustment unit to control the temperature and humidity of the wet and dry gas. The calculation of the degree of heating / cooling and the degree of humidification / dehumidification (%) can be, for example, a PID calculation method suitable for the sampling method (separation value) and can be tuned by three PID control parameters (Kp, Ki, Kd). preferable.

  The temperature / humidity adjusting unit 110 and the exhaust mechanism 130 are respectively connected to an opening 23b (inlet) and an opening 23c (exhaust) provided at both ends of the sealed space 23a. It is connected via the path 140b. The gas flow paths 140a and 140b preferably include a moisture trap mechanism 150 for removing condensed moisture.

  The humidity adjustment unit 110 is a unit for generating a gas having a desired humidity and / or temperature and supplying the gas to the sealed space 23a, and includes a wet gas generation mechanism, a dry gas generation mechanism, a dry / humid gas supply control unit, and It is preferable to construct by combining a temperature control mechanism for dry and wet gas.

  The humidification method that can be used in the wet gas generation mechanism is a dew point control type (a bubble is generated in water and humidity is generated at the dew point of the ambient temperature) a steam type (water is boiled by heating with a heater, and its steam ), Vaporization type (water is contained in a filter, and water is vaporized and released by applying air to the filter), ultrasonic type (water is made into very fine particles by ultrasonic vibration) And a hybrid type (the vaporization type is the base, but warm air heated by a heater is blown to promote vaporization and release).

  On the other hand, the dehumidification method that can be used for the dry gas generation mechanism is a compressor type (cooling air with a compressor, liquefying and recovering moisture in the air, and releasing dried air by collecting moisture) , Desiccant type (using a moisture absorbent called desiccant to adsorb moisture in the air to dehumidify), hybrid type (combined compressor type and desiccant type), etc.

  The dry / humid gas supply control unit is a mechanism for supplying the wet gas generated from the wet gas generation mechanism and the dry gas generated from the dry gas generation mechanism to the sealed space 23a in an appropriate amount at an appropriate timing. A control device 50 storing a control program related to the dry / wet gas supply control unit is connected so as to be communicable with each component of the dry / wet gas supply control unit.

  The temperature control mechanism for the wet and dry gas is the same as the temperature control mechanism for the sensor chip described later, and can be an appropriately modified embodiment as necessary. For example, the temperature of the wet and dry gas is controlled by a temperature controller, a temperature control unit (for example, a Peltier element and a cooling fan) connected to the temperature controller and in contact with each of the wet gas generation mechanism and the dry gas generation mechanism, and a temperature sensor. Regulatory mechanisms can be constructed.

  As the humidity sensor 120, various temperature / humidity meters (wet and humidity meters) using a general humidity measuring method such as a hair type, a polymer resistance type, a polymer capacitance type, an aluminum oxide capacitance type, an Asman ventilation type, etc. are used. be able to.

  The exhaust mechanism 130 discharges the unnecessary gas after being supplied from the temperature / humidity adjustment unit 110 to the outside of the RIfS measurement apparatus 10. The exhaust mechanism 130 includes a fan and, if necessary, a mechanism for collecting moisture in the gas. The exhaust capacity of the exhaust mechanism 130 can be appropriately set in consideration of the volume of the sealed space 23a and the like, but can be adjusted, for example, in the range of 0.1 to 10 mL / min.

  As a third embodiment of the measurement environment adjustment mechanism, a structure suitable for a sensor chip set part (stage) for setting a sensor chip and a chip cover for covering the periphery of the sensor chip 21 installed inside the RIfS apparatus 10 with a flow cell. Or by opening / closing the sensor chip when attaching / detaching the sensor chip to / from the RIfS apparatus 10 and providing an appropriate structure for the light-shielding cover that blocks light from the outside during RIFS measurement, the sealed space 23a (chamber) is formed. In the above, the gas whose temperature and humidity are generated from the temperature and humidity adjustment unit 110 may be introduced into the sealed space 23a (chamber).

  Examples of external humidity control devices (humidity generator, dew point meter, humidity calibrator, temperature / humidity control device, etc.) that can be used in the second and third embodiments of the measurement environment adjustment mechanism described above are as follows: Humilab (Toyo Technica), SRG-1R (Shinei Technology), KTC-Z02A-S (Kotohira Industry), GenRH-A, GenRH-T (Surface Measurement Systems Ltd.), OVG-4, OHG-4 (Owlstone) co. ltd).

  The range of humidity and / or temperature adjusted by the measurement environment adjusting mechanism 100 is not particularly limited, and can be set as appropriate according to the purpose of analysis. It is preferable that the temperature can be arbitrarily set within a range of 100% and a temperature within a range of 10 to 60 ° C. Moreover, the temperature and humidity adjustment unit 110 is 1 to 3600 seconds per 1%, for example, 15 to 150 seconds per 1% of humidity so that the humidity and / or temperature can be adjusted in an arbitrary pattern according to the purpose of analysis. In the case of temperature, those capable of continuously changing humidity and / or temperature at a rate of 1 to 3600 seconds per 1 ° C., for example, at a rate of 10 to 120 seconds are preferable.

  Furthermore, the pressure in the sealed space 23a can be reduced or increased according to the purpose of analysis (for example, to accelerate or control the movement of water molecules). Therefore, the control device 50 (control program) adjusts the balance between the gas supply amount by the temperature / humidity adjustment unit 110 and the gas discharge amount by the exhaust mechanism 130 to set the pressure in the sealed space 23a to a desired value. It may have a function.

  In addition, not only air but also various gases according to the measurement purpose such as nitrogen gas and hydrogen gas can be used as the gas with adjusted humidity and / or temperature supplied to the sealed space 23a. For this purpose, the measurement environment adjustment mechanism is further provided with a mechanism for supplying a gas other than air, such as nitrogen gas or hydrogen gas, to the temperature / humidity adjustment unit 110, for example, a mechanism for generating such gas by a chemical reaction, or a cylinder (gas ) To a tank (liquid) to introduce those gases. The wet gas generation mechanism and the dry gas generation mechanism can prepare a gas having a predetermined humidity and / or temperature by using the gas previously enclosed by replacing with air, and supply the gas to the sealed space 23a.

(Temperature adjustment means for sensor chip)
The measurement system 1 may include sensor chip temperature adjusting means for heating and cooling the sensor chip 21 (sample thin film 21c) itself more directly as necessary.

  In this case, the RIfS measurement apparatus 1 includes, for example, a temperature adjustment mechanism 200 for a sensor chip that includes a temperature controller 210, a temperature adjustment unit 220 connected to the temperature controller 210, and a temperature sensor 230 that is in contact with the sensor chip 21. Is provided. The control device 50 storing the control program related to the sensor chip temperature adjustment mechanism 200 is connected so as to be communicable with the temperature controller 210. The temperature of the sensor chip 21 controlled by the temperature adjusting means for sensor chip can be regarded as the temperature of the sample thin film 21c (particularly the measurement area A) or the reagent application area.

  The temperature range adjusted by the temperature adjusting means for sensor chip is not particularly limited, and can be set as appropriate according to the purpose of analysis. For example, it may be possible to cope with a temperature rise of 100 to 200 ° C. so that the sample thin film can be analyzed in a high temperature range. Conversely, it may be one that can cope with a temperature drop of -50 to -20 ° C so as to easily cause a temperature difference with dry and humid air.

  As long as the temperature can be adjusted according to the purpose, the temperature adjusting means for the sensor chip may not necessarily be able to continuously change the temperature, but the temperature can be adjusted in an arbitrary pattern. For example, a material capable of continuously changing the temperature at a rate of 10 to 3600 seconds per 1 ° C. is preferable.

  The temperature controller 210 measures the temperature of the sensor chip 21 with the temperature sensor 230 and performs temperature control so that the sensor chip 21 becomes a set temperature by heating or cooling by the temperature adjustment unit 220.

  It is preferable that the temperature controller 210 be program-controlled by an electronic circuit (microcomputer) so that heating, cooling or temperature maintenance by the temperature adjustment unit 220 can be automatically performed in an arbitrary pattern. An input means for storing the program in the electronic circuit, a temperature data recording / outputting means, a display means for confirming various types of information may be provided in the temperature controller 210 itself as a control panel or the like. The control device 50 connected to the device 210 may perform the function.

  The temperature adjustment unit 220 can be configured by combining a member having a heating function and a cooling function, such as a Peltier element and a cooling fan, so as to produce a desired temperature. As the temperature sensor 230, for example, a thermistor can be used. The temperature control unit 220 and the temperature sensor 230 may be selected corresponding to the temperature in the range according to the purpose of analysis.

-Analysis method-
The analysis method relating to the behavior of water molecules with respect to the sample thin film according to the present invention is performed by increasing and / or decreasing the humidity and / or temperature of the atmosphere in which the sensor chip to be analyzed is continuously or discontinuously. Including a step (RIfS measurement step) of acquiring data relating to the film thickness of the sample thin film.

  Note that the analysis method of the present invention as described below is preferably carried out using the measurement system of the present invention as described above, but means for carrying out the analysis method of the present invention is not limited thereto. However, the present invention is not limited, and the analysis method of the present invention is included in the scope of the present invention even when it is performed by other means.

(Measurement process)
In the measurement process, the temperature and humidity of the atmosphere in which the sensor chip (analysis target sensor chip) having the sample thin film formed on the surface is continuously or discontinuously changed (increased and / or lowered) by RIfS. Data on the thickness of the sample thin film is measured. That is, in the measurement process, the measurement step is repeated a plurality of times at predetermined time intervals in synchronization with continuous or discontinuous changes in temperature and humidity. If the RIfS measuring apparatus is used, it is possible to measure at intervals of 1 second or less (for example, 700 milliseconds).

  When the humidity is continuously changed, the interval between the humidity values in the two adjacent measurement steps can be appropriately adjusted so as to be relatively small, but is usually 0.1 to 10%, preferably Is 0.1 to 2%. Although the number of measurement steps included in one measurement process can be adjusted as appropriate, it is usually 10 times or more, preferably 20 times or more. When the humidity is automatically changed by the humidity adjustment means, the system is controlled so that the measurement step is automatically performed while adjusting the timing so that the humidity interval between measurements is within the above range. It is preferable to do.

  On the other hand, when the humidity is discontinuously changed, measurement is performed after the humidity is kept constant for a predetermined time (for example, a time sufficient for the adsorption / desorption of water molecules to the sample thin film to reach equilibrium), and thereafter, at certain intervals. (Usually larger than the interval for the continuous change described above), and the measurement is repeated after the humidity is kept constant for a predetermined time again. Also in this case, the interval between the humidity values in two adjacent measurement steps and the number of measurement steps included in one measurement step can be adjusted as appropriate.

  Moreover, you may perform the measurement process which changes humidity several times in different temperature as needed. For example, one measurement target sensor chip is measured at a first temperature while changing the humidity in a predetermined pattern (first measurement step). After the step is finished, the temperature is changed (increased or lowered), and the second Measure again with the same humidity pattern at the same temperature (second measurement step), change the temperature again after the end of the measurement step, and measure again at the third temperature with the same humidity pattern (third measurement step) The measurement process may be repeated. In such an embodiment, the temperature is basically fixed constant during one measurement process in which the measurement is performed while continuously changing the humidity in a certain pattern. Conversely, the measurement process for changing the temperature may be performed a plurality of times at different humidity.

  On the other hand, both temperature and humidity can be changed within one measurement process. For example, it is possible to measure with a pattern that first increases the humidity to a predetermined value, then increases the temperature while maintaining the humidity, and finally decreases the humidity to the initial value while maintaining the temperature. It is.

  Humidity adjustment (humidification or dehumidification) and temperature adjustment (heating or cooling) are performed by the temperature / humidity adjustment means and the sensor chip temperature adjustment means as described above so that the humidity and temperature can be adjusted at any time. Is preferred.

・ Humidity control pattern The humidity control pattern (plot when the horizontal axis represents time and the vertical axis represents humidity or temperature) continuously or discontinuously provides data on the film thickness of the sample thin film at any temperature and humidity range. It is only necessary to continuously or discontinuously change the numerical value of the temperature and humidity so as to be acquired, and various patterns can be applied depending on the purpose of analysis.

  The change in temperature and humidity may consist of only one of rising or lowering, or may include both. Further, the cycle for changing the humidity within a predetermined range may be included once to plural times (for example, 2 to 100 times). The above cycle may be a pattern in which the humidity or temperature is first increased and then the humidity is decreased, or a pattern in which the humidity is first decreased and then increased.

  The temperature / humidity change rate (the humidity or temperature gradient with respect to time) can be adjusted as appropriate according to the purpose of the analysis, and may be constant from the start to the end, or once in the middle as needed. It may be changed multiple times. It may be a pattern that does not substantially include the time during which the temperature and humidity are constant (no increase or decrease) from the start to the end, and if necessary, any time when the temperature and humidity are constant Alternatively, it may be a pattern that is included a plurality of times (stepped pattern in which the time during which the humidity or temperature changes and the time during which the humidity or temperature changes is regularly repeated). For example, when a thin film whose change in film thickness cannot be observed if the change in humidity is too fast is to be analyzed, an appropriate analysis may be performed by changing the humidity or temperature in a stepwise manner.

  The rate of increase and / or decrease in temperature and humidity can be adjusted as appropriate according to the purpose of analysis, but for humidity, for example, a rate of 1 to 3600 seconds per 1%, for temperature, for example, 1 per 1 ° C. The speed can be up to 3600 seconds. For example, the adsorption rate or desorption rate of water molecules can be obtained from the measurement result based on the humidity rate at this time (the time taken when the humidity is changed within a predetermined range).

  Furthermore, as shown in FIG. 3, a pattern in which the initial humidity is lowered and the humidity is increased from that (normal humidity pattern) is generally used. However, in the present invention, the initial humidity is increased. Then, the pattern may be changed so as to decrease the humidity (reverse humidity control pattern), or a pattern in which the reverse humidity control pattern and the normal humidity control pattern are mixed (normal / reverse humidity control pattern). The same applies to the temperature. The reverse humidity pattern and the normal / inverse humidity pattern are useful when, for example, a highly hydrophilic material (such as a biomaterial) that may be denatured when dried is used for the test thin film.

  The temperature / humidity change as described above may be a change between a plurality of patterns as well as a change in one pattern. For example, when the humidity is changed at the first speed in the first pattern and the humidity is changed at the second speed in the second pattern, the humidity change rate is calculated from the comparison of the measurement results of those patterns. It is possible to analyze the difference in the behavior of the analysis sample thin film and water molecules due to the difference.

-Reagent supply When a reagent is added to a sample thin film and the influence of the reaction is analyzed, an operation for adding the reagent at an appropriate timing may be performed. For example, after measuring a predetermined temperature / humidity change for a certain sample thin film (before addition of the reagent), an operation of spraying the reagent into the sealed space according to the above-described embodiment is performed. Next, the sample thin film (after addition of the reagent) can be reacted, and a measurement relating to a predetermined change in temperature and humidity can be performed.

-Data relating to film thickness "Data relating to the film thickness of a sample thin film" is generally based on the optical path length in RIfS, and the acquisition method is as follows.

  First, with respect to the sensor chip to be measured, the spectral intensity data of reflected light obtained by the spectrometer in each measurement step of the measurement process is compared with the spectral intensity data of white light as a reference, and the reflectance for each wavelength (= reflected light) Intensity / white light intensity). The spectral intensity data of the white light may be measured and stored in advance when the apparatus is assembled and adjusted, or may be acquired at every measurement by an appropriate means (for example, using a reference reflector).

  Subsequently, a reflection spectrum is created by plotting the wavelength on the horizontal axis and the calculated reflectance on the vertical axis, and the wavelength at which the reflectance is minimized (bottom wavelength) and the wavelength at which the reflectance is maximized (peak wavelength) are determined. The waveform of the reflection spectrum has an irregular shape that repeats unevenness as the film thickness increases, and it may be difficult to specify the bottom wavelength and peak wavelength, For example, by approximating the reflection spectrum with a high-order function using a known method, the solution (extreme value) can be obtained from a high-order polynomial, and this can be used as an extreme value.

-Thickness calculation method The data on the optical path length of the sample thin film is based on the reflection spectrum (wavelength and light intensity) obtained by the spectroscope, for example, as described below (a) bottom peak method (Δλ etc.), ( b) The film thickness d, the refractive index n, or the optical path length (= film thickness d × refractive index n) can be converted by the cOPL method, (c) Fourier analysis method, or the like. Regardless of which calculation method is used, this measurement data is the sum of the optical path lengths of the sample thin film and water at each point in time. Essentially, the difference between the two states is not water, but water and thin film. This amount can be derived in real time as an absolute value in a non-destructive manner (as a film thickness or a converted weight). The method of calculating the film thickness is not particularly limited, and an appropriate one can be used according to the required accuracy. For example, when using a simulation, the accuracy should be improved or the measured value corrected. Is possible.

  Note that water molecules in the atmosphere that are not retained in the sample thin film are not measured by the change in the optical path length because they are outside the optical interface, and only the water molecules retained on the surface or inside the sample thin film are in the optical path. It can be measured and quantified by changes in length. Optically, the refractive index during humidification can be determined by “Edren's empirical formula”, but in the measurement system of the present invention, the difference in refractive index between dry air and wet air is so small that it can be ignored.

(A) Bottom peak method The bottom peak method is a method generally used in conventional RIfS, and the film thickness d equivalent value of the thin film to be measured is simply calculated from the value of Δλ by a predetermined conversion formula. It is also possible. However, it is not easy to obtain the absolute value of the film thickness for the film thickness exceeding the periodicity.

  Here, in the bottom peak method, the conversion method of Δλ differs depending on the value of d. When λ is in the range of 400 to 800 nm, the measured value of Δλ itself can be used for calculating d only when d is about 100 nm or less.

  When d is about 100 nm or less, it is not necessary to consider the periodicity in the relationship between d and Δλ. At this time, a first-order approximation of Δλ / d = an + b is established (the values of a and b vary depending on the measurement conditions). Therefore, Δλ / d = an + b is obtained from a plot of n and Δλ / d by measuring several points of Δλ for a sample whose d and n are known in advance under a predetermined measurement condition or by simulating it. The regression equation represented by is acquired. Then, Δλ measured under the same measurement conditions is applied to a conversion formula of d = Δλ / (an + b), in other words, 1 / (an + b) is multiplied by Δλ as a “film thickness conversion factor” to calculate d. can do. However, such a calculation method of d can be applied when n is known and can be regarded as not changing even if water is adsorbed to the sample thin film. Conversely, n = (Δλ / d−b) / a = Δλ / da−b / a is obtained from the approximate expression, and therefore n can be calculated by applying Δλ measured to this conversion expression. it can. However, such a calculation method of n can be applied when d is known and it can be considered that it does not change even if water is adsorbed to the sample thin film. Alternatively, by establishing a relational expression between Δλ and the optical path length dn and applying Δλ measured to the expression, it is possible to calculate dn that integrally reflects changes in d and n. The sample may be analyzed.

  On the other hand, when d is about 100 nm or more, it is necessary to consider the periodicity in the relationship between d and Δλ. Therefore, in order to obtain separately information (for example, based on the approximate film thickness estimated from the formation conditions of the sample thin film) as to what period the measured λ corresponds to, thereby reflecting the correct film thickness It is necessary to determine Δλ to be added to (addition Δλ value). Then, using the correction value obtained by adding the added Δλ value to the measured Δλ, d and n are individually or based on the conversion formula created in the same manner as in the case where d is about 100 nm or less. It can be calculated as the optical path length dn.

  The boundary of periodicity is determined as follows. That is, (1) determine the period, (2) calculate the film thickness conversion coefficient (coefficient × refractive index−adjustment term) based on the periodicity and the refractive index n, and then calculate the value of Δλ by the film thickness conversion coefficient. (3) In the second and subsequent cycles, the value obtained by adding the film thickness addition value is used as the film thickness value. The variation amount of the film thickness is similarly calculated using the film thickness conversion coefficient. In order to see the film thickness value and the amount of change of the substance itself, it is necessary to subtract the λ value of the reference (for example, the sensor chip before modification) in advance, but if the period is not the same, the effect on the measured value is small. There is no need.

  In the present invention, as a result of one optical simulation, when the refractive index of water is 1.33, when there is no periodicity (0 period), d = Δλ / 1.5, when there is periodicity (multi-period) ) Can use a conversion formula of d = Δλ / 0.86.

(B) cOPL method (film thickness calculation program manufactured by Konica Minolta)
The cOPL method (converted optical length) is a method for calculating the optical path length of a sample thin film based on the positions (number and bottom and peak wavelength information) of extreme values appearing in the reflectance curve. In this method, a change in the optical path length, that is, the wavelength shift relationship of the extremum position of the reflectance curve in which the film thickness is changed for each refractive index is simulated in advance, and this is mathematically processed to obtain a template. Create. When the measured wavelength of the extreme value position of the reflectance curve is illuminated on the template, the optical path length (L) of the sensor chip to be analyzed is approximately obtained. If the optical path length (L ′) of the reference sensor chip is subtracted from L, the optical path length (ΔL) of the sample thin film itself can be obtained. Since ΔL is the optical path length of the sample thin film (= refractive index n × film thickness d) itself, the thickness d can be directly calculated by calculating the value based on the refractive index n of the sample thin film or the refractive index of water 1.33. Can be calculated. By conducting a detailed analysis of the optical path length, the refractive index n and the film thickness d can be separated in principle. When the cOPL method is used, it has been confirmed that the measurement accuracy (resolution) of the water film thickness is, for example, about 0.05 nm from the molecular signal / noise level in the case of a high-precision spectrometer. Unlike the above-mentioned Δλ, the cOPL method can calculate the absolute film thickness even if the periodicity is exceeded. In addition, since a thick film (approximately 100 nm or more) has a plurality of bottoms or peaks (referred to as extreme values), it is more resistant to noise than the bottom peak method in which only one peak can be traced, and the measurement accuracy is in principle high accuracy. . In the calculation of the amount of water adsorbed and desorbed, for example, the film thickness of an arbitrary dry state is simply calculated as the film thickness of the sample thin film, and from there the water evaporates or the water increases due to humidification. It can be easily calculated by calculating the film thickness as water molecules. In addition, the film thickness of the thin film and the film thickness of the water can be calculated independently in real time by analyzing the refractive index from the optical path length at a predetermined humidity or the optical path length that changes with humidity control by the least square method. Is also possible.

(C) Fourier analysis method Similar to the cOPL method, a waveform of a reflectance curve with respect to a change in optical path length is simulated in advance, and a template is created by mathematically processing it. Then, the waveform of the actually measured reflectance curve is Fourier analyzed, and the optical path length can be obtained with reference to the template. By conducting a detailed analysis of the optical path length, the refractive index n and the film thickness d can be separated in principle.

-Analysis content In the present invention, based on the data obtained by the measurement process as described above, the behavior of water molecules with respect to the sample thin film, various physical properties of the sample thin film, and other various items can be analyzed. The analysis content in the present invention is not particularly limited. For example, the following items can be analyzed. The analysis for each item can be performed based on the film thickness, or can be performed based on the volume or mass that can be calculated from the film thickness.

  (A) By changing the humidity, uptake of gaseous water molecules into the sample thin film is observed as a change in film thickness. In relatively thin films, hydration in the molecular state proceeds on the surface, and in relatively thick films, such hydration on the surface of the film is combined with the uptake of water molecules inside the film, that is, the swelling of the film. It becomes a thing. By measuring the film thickness under high humidity, the film thickness in which water molecules are saturated (wet film thickness) or the saturation hydration amount can be obtained. By measuring the film thickness after deaeration in a low humidity, for example, in vacuum, a film thickness without water molecules (dry film thickness) can be obtained. If no change in thickness is observed, it can be seen that the sample adsorbs little or no water molecules. In addition, since the RIfS measuring apparatus can measure the film thickness at an extremely short time interval (1 second or less), the adsorption / desorption dynamics of gaseous water molecules can be calculated by calculating the difference in the film thickness change amount in each measurement. You can create plots to represent.

(B) From the measurement result when the humidity is continuously changed, the amount of water molecules retained in each sample at each humidity, the amount of adsorption (plus amount of variation) or the amount of desorption expressed as the amount of variation in the amount retained ( Negative change amount) can be measured quantitatively. By multiplying the film thickness of water converted from the measurement results by the measurement area (for example, 0.126 mm ² ), the volume of water molecules in the region can be obtained, and if the volume is multiplied by the specific gravity of water (ie, 1), When the mass of the molecule is obtained and the mass is divided by the mass number of water 18, the substance amount (mol) of the water molecule is obtained. As shown in the examples described later, the resolution of the film thickness of water is, for example, 0.05 nm. In this case, the resolution of the mass of water molecules is 6.3 pg. When the thickness of the sample thin film is relatively large, it is possible to obtain a volume-based moisture content (vol%) or the like by dividing the film thickness of water by the film thickness of the sample thin film. On the other hand, if the specific gravity of the sample thin film is known and the mass of the sample thin film can be obtained from the film thickness × measured area × specific gravity, the water content on the mass basis (wt %) Can be obtained.

  (C) The film thickness effect (for example, the degree of water penetration into the membrane) by comparing the values of water molecule retention, adsorption amount, desorption amount, etc. with respect to a certain sample thin film. ) Can be observed.

  (D) The behavior of water molecules due to the difference in humidity change rate can be observed by comparing values such as the retention amount, adsorption amount, and desorption amount of water molecules at several different humidity change rates for a certain sample thin film. For example, when the humidity change rate is fast (when the humidity is changed by a certain amount in a short time), when the humidity change rate is slower than the retention amount of water molecules at the upper limit of humidity (a constant amount of humidity over a long period of time) If the retention amount of water molecules at the upper limit of humidity at the time of change is higher, the latter retention amount is closer to the saturation amount of the true water molecule (closer to the equilibrium hydration rate), while the former is saturated ( It can be considered that the equilibrium hydration rate has not been reached. In addition, the time until the water molecule retention reaches equilibrium (equilibrium time), the film thickness (equilibrium film thickness) when water adsorption / desorption is balanced at a certain humidity, or the moisture content based on the film thickness ( It is possible to predict the equilibrium water content using a simulation curve or a calculation formula. For example, the simulation curve and calculation formula for the equilibration time are as follows: humidity adjustment time (time required from one end of the humidity range to the other end) on the horizontal axis, and average film thickness difference (film thickness at a certain humidity adjustment time) on the vertical axis It is obtained by plotting the measurement result by taking the average value of the difference. The humidity control time at which the average film thickness difference becomes 0 in the simulation curve or calculation formula can be predicted as the humidity control time during which continuous hydration proceeds most stably. On the other hand, when the measurement results are plotted with the humidity adjustment time on the horizontal axis and the film thickness or film thickness standard on the vertical axis, the film thickness or film thickness reference moisture content at the predicted equilibrium time is The moisture content can be predicted.

  (E) By dividing the amount of adsorption or desorption of water molecules by the time taken to change the humidity at that time, the adsorption rate and desorption rate of water molecules on the sample can be determined. For example, by comparing the adsorption speed of water molecules with the humidity at that time, water adsorption is slow because structured water is formed, or water adsorption is fast because free water is adsorbed. We can guess whether there is. That is, it is possible to further analyze (analyze) the hydration structure from the difference in the adsorption rate and desorption rate (hydration rate) of water molecules. For example, in a graph with the vertical axis representing the amount of change in film thickness and the horizontal axis representing humidity, the slope of the straight line (or tangent to the curve) represents the adsorption / desorption rate, but the hydration structure changes greatly at the inflection point. it seems to do. Such analysis can be performed by measuring in real time using RIfS.

  (F) When a cycle by humidification and dehumidification is performed, if the initial film thickness before humidification is restored under low humidity after dehumidification, it can be evaluated that the balance between hydration and dehydration is balanced. On the other hand, in the case of a highly hydratable material, water molecules are easy to enter, and the film thickness after dehumidification is larger than the initial film thickness. In the case of a highly hydrophobic material, the water that has entered is easy to spread out, or because there is a barrier against hydration such as wettability, the film thickness after dehumidification is smaller than the initial film thickness. These behaviors indicate that the slope of the plot when dehumidifying / the slope when humidifying is 1 (the same slope when humidifying and dehumidifying) or less than 1 (the slope when dehumidifying is smaller than the slope when humidifying), It is replaced by whether it is larger than 1 (the inclination during dehumidification is larger than the inclination during humidification).

  (G) The difference in hydration behavior at different temperatures can be observed by comparing the values of the retention amount, adsorption amount, desorption amount, etc. of water molecules at several different temperatures for a certain sample thin film. Furthermore, by adjusting the temperature and humidity of the gas by the measurement environment adjustment mechanism and the temperature of the sample thin film by the temperature adjustment mechanism for the sensor chip, the temperature and humidity are changed in a coordinated manner, and the gas from high temperature and high humidity to low temperature and low humidity. The antifogging property can be evaluated by observing the state of adsorption or dew condensation of the water molecules.

  (H) Hydration / desorption of water molecules, that is, changes in humidity (humidification and dehumidification) are repeated multiple times (multi-cycle), so that hysteresis of the sample thin film, deterioration status, change in water molecule retention due to repetition, etc. Can be evaluated.

(I) It is possible to evaluate both the thermal responsiveness and hydration property (environmental resistance and environmental adaptability) of a sample by changing the combination of humidity and temperature at three or more points.
(J) When a biomaterial (bone, (artificial) skin, etc.) is used as a sample, moisture retention and the like can be quantitatively evaluated from measured values such as the amount of water molecules retained, the amount of adsorption, and the amount of separation. For example, the effect of lotion can be estimated.

  (K) When a polymer material (natural, synthetic) is used as a sample, the water retention ability and the like can be quantitatively evaluated from measured values such as the retention amount, adsorption amount, and desorption amount of water molecules. For example, the water passage capacity of a battery separator can be measured.

  (L) With respect to the dried material, from the data obtained while increasing the humidity, the behavior of hydration (the amount of water molecules for sufficiently hydrating the substance) can be known. On the other hand, for a sample holding water molecules such as protein, the humidity is first kept high, and the behavior associated with drying can be known from the data obtained while reducing the humidity. For example, if the protein is easily denatured when dried, and the hydrated protein does not hydrate smoothly when the humidity is increased, it can be assumed that hysteresis due to drying occurs.

  (m) It is known that as the humidity increases, the behavior in water approaches, and the critical humidity differs depending on the hydrophilicity of the material. For example, it is possible to quantify the amount of water molecules required for the protein to gather in water (salt) and take a structure in the living body.

  (N) When a drug or the like is used as a sample, it is possible to estimate the penetrability of the drug into the living body (molecular level wettability) from the data obtained while increasing the humidity. Conventionally, wettability that has been evaluated using the contact angle as an index is considered not to have a quantitative relationship with hydration at the molecular level, but according to the present invention, hydration can be quantified at the molecular level. .

  (O) It is possible to evaluate the ease of water molecule uptake by fixing the model cell membrane and performing measurement while changing the humidity. As the model cell membrane, for example, a cell membrane fraction collected from a living body or a synthesized phospholipid bilayer can be used. The latter contains membrane proteins such as aquaporins that can selectively pass water molecules in the phospholipid bilayer, so the water uptake rate of aquaporins, and further elucidation of the mechanism of aquaporins, judgment of normality / abnormality, various types of aquaporins It may be possible to predict the risk of disease. In the latter case, it is possible to quantify the evaluation of the blending ratio of the different phospholipids by the moisture adhering amount.

  (P) The water repellency and hydrophilicity of various thin films can be quantitatively evaluated at the molecular level. Further, the initial level of durability can be determined by measuring the amount of water molecules adsorbed when a surface treatment agent or paint is applied to the substrate.

  (Q) The porosity can be measured from the amount of moisture adsorbed on the thin film (layer) formed of the porous material. Since water has a specific gravity of 1, it can be easily converted to mass. In addition, the followability of water molecule adsorption and desorption with respect to humidity is also known.

-Example group 1: Examples 1-7-
In Examples 1 to 7, a reflection type RIfS intermolecular interaction measurement device “MI-Affinity” (registered trademark, Konica Minolta Technology Center Co., Ltd.) is used as the RIfS device, and an unmodified sensor chip (bare substrate). As described above, a sensor chip (Si, thickness 1 mm / SiN, thickness 66 nm) dedicated to “MI-Affinity” was used. As the temperature and humidity adjusting device, a constant temperature bath PDR-3KT manufactured by ESPEC and a dedicated humidity control device were used, and the RIfS device was installed in the constant temperature bath. The humidity control program is to hold 15% (RH: Relative Humidity) for 15 minutes at 25 ° C, then humidify from 15% (RH) to 60% (RH) in 30 minutes, and 60% (RH ) For 15 minutes, then dehumidification from 60% (RH) to 15% (RH) was performed for 30 minutes, and finally, 15% (RH) was maintained for 15 minutes. As a film thickness calculation program, Δλ (bottom wavelength) of the bottom peak method was measured, and the film thickness was converted by a conversion formula.

(Production of sensor chip to be analyzed)
Using the sample, film forming method and substrate shown in Table 1, the sensor chip No. 1-23 were made.

(A) Application by spin coater (analysis target sensor chip No. 2, 3, 14-20, 22)
On the unmodified sensor chip, various materials were applied at 3000 rpm with a spin coater according to the conditions shown in Table 2, and dried by heating to produce an analysis target sensor chip.

(B) Treatment with silane coupling agent (sensor chip No. 4 to 7 to be analyzed)
100 μL of each of the drugs shown in Table 3 was added to 10 mL of 95% ethanol aqueous solution and stirred at room temperature for 1 hour. An unmodified sensor chip was immersed in the obtained silane coupling agent for 1 hour at room temperature to react. The sensor chip was lifted, washed with ethanol and ultrapure water in sequence, dried by nitrogen blowing, and dehydrated and condensed at 80 ° C. for 1 hour with a dry heat machine. 4-7 were produced.

(C) Coupling fixation using an amino group substrate (analysis target sensor chips Nos. 8 to 11 and 21)
(C-1: Preparation of aminated sensor chip)
100 μL of 3-Aminopropyltrimethoxysilane was added to 10 mL of 95% ethanol aqueous solution and stirred at room temperature for 1 hour. In the obtained silane coupling agent, an unmodified sensor chip was immersed at room temperature for 1 hour to react with the silane coupling agent. The sensor chip was pulled up, washed sequentially with ethanol and ultrapure water, dried by nitrogen blowing, and dehydrated and condensed at 80 ° C. for 1 hour with a dry heat machine to prepare an aminated sensor chip (amino group substrate).

(C-2: Fixation to aminated sensor chip)
An aqueous solution containing NHS (N-hydroxysuccinimide) and WSC (water-soluble carbodiimide) at concentrations of 50 mM and 200 mM, respectively, was prepared using 25 mM MES buffer (pH 5.0). Each reagent shown in Table 4 was added to the obtained aqueous solution so as to have a concentration of 10 mM (concentration: 5 wt%). After stirring for 30 minutes, the amination sensor chip was immersed for 20 minutes at room temperature with stirring. Thereafter, the sensor chip was pulled up and washed with ultrapure water. In order to hydrolyze the active ester not used for chemical bonding, the sensor chip after the reaction was immersed in a 1/10 normal aqueous sodium hydroxide solution for 10 minutes. After neutralizing with dilute hydrochloric acid, washing with ultrapure water and drying were performed to obtain the target sensor chip for analysis.

(D) Adhering in the flow path (analysis target sensor chip No. 12, 13)
(D-1: Neutravidin)
A dedicated flow cell made of PDMS was set in MI-Affinity, and 100 μg / ml of NeutraAvidin was injected from a 100 μl injector while PBS buffer was running. After 10 minutes of feeding at 20 μl / min., Nitrogen gas was sprayed to cut off the water attached to the surface and immediately used for measurement.

(D-2: cell membrane fraction)
A dedicated flow cell made of PDMS was set in MI-Affinity, and 100 μg / ml of membrane fraction was injected from a 100 μl injector while PBS buffer was running. The pump was stopped while the membrane fraction was being fed at 20 μl / min. And allowed to stand for 1 hour to be fixed to the substrate. Nitrogen gas was jetted to drain the water on the surface and used immediately for measurement.

(Quantification of water molecules)
RIfS measurement was performed while continuously changing the humidity according to the humidity control program, and the film thickness of water molecules and the like were calculated based on the measured value. The results are shown in Table 5. The film thickness of the water molecule was determined by dividing the difference between Δλ of 60% (RH) and 15% (RH) by a conversion factor based on the refractive index of water (with or without periodicity). Since it is a real-time measurement, the amount of water molecules at any humidity% can be determined. It is also useful to divide and compare the obtained water molecule film thickness by the material film thickness. In addition, the behavior due to the difference between humidification and dehumidification is also important in analyzing the interaction between the material and water.

Moreover, the graph of a measurement result when performing RIfS measurement, changing humidity continuously according to the said humidity control program is shown to FIGS. Hereinafter, the graph of these measurement results was considered.

[Example 1] No. 1-7 (Bare substrate, wettability of ultrathin film, hydration, hydrophilicity, hydrophobicity, water repellency evaluation)
As a reference, water molecule adsorption of a bare substrate (No. 1: FIG. 4) was measured. Since it is not a dew condensation condition, water molecules should not be adsorbed originally, but as the relative humidity increased, an increase in Δλ, which seems to be the adsorption of water molecules, was observed. There is a possibility that SiN of the sensor chip is oxidized and partially becomes SiO ₂ . It was confirmed that water molecule adsorption observed on the bare substrate was suppressed by performing water-repellent fluorine coating (No. 2: FIG. 5, No. 4: FIG. 7). It can also be seen that the thick No. 2 has a higher water repellency effect than the No. 4 thin film at the molecular level. No. 5 (FIG. 8) having a hydrophobic alkyl group also suppressed water molecule adsorption. This result suggests that the method of the present invention enables not only hydration properties but also molecular level hydrophobicity and water repellency evaluation. On the other hand, the hydrophilic surface modifier No. 3 (FIG. 6) absorbs 20 to 40 times as much water as the hydrophobic material. The hydrophilized surface modifiers No. 6 (FIG. 9) and No. 7 (FIG. 10) have more water molecule adsorption than the hydrophobic surface modifier, but not as much as No. 3. As described above, even in a film with almost no thickness and almost no swelling due to water, the effect of moisture can be measured with high precision, and hydration occurs at the molecular level on the film surface, which could not be measured with conventional equipment. Is considered to be quantified.

[Example 2] No. 8-14 (Evaluation of hydration and hygroscopicity of biological substances)
No. 8 (Fig. 11), No. 9 (Fig. 12) and No. 10 (Fig. 13) CMDs are relatively highly hydratable, and No. 9 with a low degree of substitution (carboxyl group introduction amount) is water. It was confirmed that the Japanese ability was the lowest. BSA No. 11 (FIG. 14) has a higher hydration amount than CMD, but the behavior differs greatly between humidification and dehumidification, suggesting that hydration during humidification hardly occurs. Since BSA was a sample left in the atmosphere for a long time, it is thought that alteration occurred. The neutravidin of No. 12 (Fig. 15) and the cell membrane fraction of No. 13 (Fig. 16) have a very high hydration amount. ing. This suggests that the rate of water absorption into the membrane is superior to the release rate due to the high hydration properties of the material. The phospholipid membrane of No. 14 (FIG. 17) showed a very large hydrophilicity relative to the thinness of the membrane, suggesting that a phospholipid bilayer was formed.

[Example 3] No. 12 (Drying evaluation of neutravidin)
The neutral avidin (No. 12) used in Example 2 was left in the atmosphere for 12 hours, and then the water adsorptivity was measured again (humidification 2, dehumidification 2). As is clear from FIG. 27, the humidification 2 after being left is dry, so the hydration rate is slow, but in the dehumidification 2 cycle, it is the same as before being left as it is, and it is the original under high humidity. It is thought that it returned to. Such measurement shows that drying evaluation of various materials is possible.

[Example 4] No. 15-22 (hydrophobic PS, 2% moisture content PMMA, difference in hydration behavior)
With polystyrene (No. 15: FIG. 18, No. 16: FIG. 19, No. 17: FIG. 20), the smaller the film thickness, the greater the change due to hydration. In particular, No. 15 PS1 was such that water molecules were less likely to be adsorbed to the membrane during humidification, and water molecules were likely to be generated during dehumidification. On the other hand, polymethyl methacrylate (No. 18: FIG. 21, No. 19: FIG. 22, No. 20: FIG. 23), which has higher hydrophilicity, resulted in greater water molecule adsorption at higher film thicknesses. . Water-soluble acrylic acid (No. 21: Fig. 24) and "Hydran CP7610" (No. 22: Fig. 25), which has a cationic surface and strong hydration, are both highly hydratable, but long under drying. Since it was kept for a long time, water molecules did not easily enter during humidification, and it was likely to come out during dehumidification. If multiple humidification cycles are performed, the relationship between humidification and dehumidification is considered to be gradually reversed.

[Example 5] (Evaluation of difference in film thickness of polymer)
Looking at the behavior of the polymer film thickness difference, it can be seen that the adsorption ratio of water molecules to the film thickness (water film thickness / sample film thickness,%) is higher for both polystyrene and polymethyl methacrylate (Table 5). . This suggests that the efficiency of hydration decreases as the film thickness increases. As shown in FIGS. 28 and 29, in polystyrene, the absolute amount of hydration is higher as the film thickness is thinner, whereas in polymethyl methacrylate, the reverse is true, and the hydrophobicity of the polymer It is thought that it originates in the grade of.

[Example 6] (Combination of PMMA thermal response and hydration determination)
For polymethyl methacrylate (PMMA3: No. 20), a humidity control program was set up to maintain the same temperature at 10%, 30%, and 50% for 30 minutes each while changing the humidity. In synchronism with these humidity of 10%, 30%, and 50%, a thermal response program was run to raise the substrate from 10 ° C to 40 ° C in 15 seconds per 1 ° C. The results are shown in FIG. By measuring at a humidity of 10%, the effect of only heat on the substance can be measured, and by referring to the measurements of 30% and 50% for a humidity of 10%, the influence of humidity can be quantitatively separated. It can be seen that this is an excellent measurement method that can separate the factors of heat and moisture in a single measurement.

[Example 7] (Evaluation of SiO ₂ , temperature difference, and estimation of SiO 2 content of bare substrate)
For SiO ₂ (No. 23), only humidification was performed. As can be seen from the results shown in FIG. 26, by increasing the temperature, the absolute moisture content (absolute humidity) is high even if the relative humidity is the same. This sample can be estimated from becoming SiO ₂ 100%, when compared with partially bare substrate the absolute value became SiO ₂ of No.1 SiO ₂ content of about 50%.

-Example group 2: Examples 8-12-
In Examples 8 to 12, a reflection-type RIfS intermolecular interaction measurement device “MI-Affinity” (registered trademark, Konica Minolta Technology Center Co., Ltd.) is used as the RIfS device, and an unmodified sensor chip (bare substrate). As described above, a sensor chip (Si, thickness 1 mm / SiN, thickness 66 nm) dedicated to “MI-Affinity” was used. As a small temperature / humidity adjusting device, “GenRH-A” (manufactured by Surface Measurement Systems, UK) was used. A gas flow path was constructed by superimposing a member having the shape shown in FIG. 32 on the sensor chip to be analyzed manufactured as described below, and the gas flow path was installed in the sensor section of “MI-Affinity”. The apparatus and “MI-Affinity” were connected to the gas flow path, and the humidity control gas generated from the apparatus was supplied to the sample thin film in the measurement region. Program for continuous PID control of humidification and dehumidification using SpecView (manufactured by SpecView Corporation, USA), SCADA (Supervisory Control And Data Acquisition) package software as control software for automatically performing humidity control in real time It was created. The hygrometer used for PID control was the data of the hygrometer built in GenRH-A. As the film thickness calculation software, cOPL (manufactured by Konica Minolta) was used to create a two-layer model (thin film sample in the lower layer and water molecules in the upper layer), and the absolute film thickness was calculated.

(Production of sensor chip to be analyzed)
A coating solution containing the samples shown in Table 6 was prepared and applied to the surface of the unmodified sensor chip using a spin coater under conditions of slope (SL) 5 seconds and 1000 rpm-60 seconds, and a sample thin film consisting of each sample Sensor chip No. with the surface formed on the surface. 24-29 were produced. A plurality of these analysis target sensor chips were prepared. Analysis target sensor chip No. 27 and no. No. 28 produced two types having different film thicknesses.

[Example 8]
Analysis target sensor chip No. RIfS was measured using No. 25 (CMD: coated with carboxymethyl dextran as a sample). The temperature control of MI-Affinity was set to 25 ° C. The humidity is controlled at a flow rate of 100 ml / min., As shown in Fig. 33, 10 minutes at a low humidity of 10RH%, and continuously to a high humidity of 80RH% at a humidity change rate of 300RH% / hr. , Hold for 10 minutes under high humidity of 80RH%, then bring it continuously to low humidity of 10RH% at a humidity change rate of 300RH% / hr. And hold for 10 minutes under low humidity of 10RH% Is.

(1) When the refractive index of the CMD film was 1.45, the film thickness in the most dry state was calculated to be 343.55 nm. When the humidity was changed from 10RH% to 80RH%, the increase in film thickness (water adsorption film thickness) due to the amount of water adsorption was 51.16nm. It can be quantitatively calculated that the moisture content on the basis of the film thickness in the wet state of 80RH% is 12.96%. On the other hand, by multiplying the film thickness by the area of the measurement region (cross-sectional area of the optical fiber, 0.126 mm ² ), the film thickness can be converted into volume and further into mass. From the water adsorption film thickness, it can be seen that 6.43 ng of water was adsorbed in the measurement region. Moreover, since the specific gravity of CMD is 1.05, it turns out that the wet film thickness is 52.08 ng in terms of weight, and the moisture content on the weight basis is 12.34%. According to the present invention, it is understood that the adsorption / desorption phenomenon of water can be quantified in a practical system in which an arbitrary set humidity changes in a short time using a very small amount of sample.

(2) Water adsorption / desorption dynamics (see FIG. 34) can be expressed by measuring the film thickness change amount (d) at a time interval of about 1 second or less and calculating the difference (Δd). For example, if a very small amount of water at the molecular level is recalculated with the amount of change in a short time, a dynamic equilibrium analysis of water that cannot be analyzed by ordinary measurement is possible. In Δd, plus indicates the amount of adsorption and minus indicates the amount of desorption, and the initial adsorption is balanced at 10RH%, but the phenomenon that the amount of adsorption gradually increases as humidity increases can be quantified. I understand.

(3) Prediction of equilibrium state under set conditions (10RH%, 100ml / min.) Since the humidity control rate does not form a completely stable wet state within the range of 10-80RH%, 10RH% and 80RH By creating an arbitrary simulation curve from the graph of time and film thickness in% (see Table 8 and FIG. 35) and obtaining a calculation formula, it is possible to obtain an optimum humidity control condition. Further, by using this calculation formula, it is possible to quantify the water adsorption amount in a stable dry or wet state of the sample thin film. Although the water adsorption film thickness in this sample was 51.16 nm, it can be predicted to be 67.75 nm when measured under stable humidity control conditions.

[Example 9]
Analysis target sensor chip No. The RifS was measured using No. 26 (applied with Fresella R, which is a superhydrophilic material, as a sample). This thin film sample is held for 10 minutes at a low humidity of 10RH%, then continuously changed to a high humidity of 80RH% at a humidity change rate of 300RH% / hr. And held at a high humidity of 80RH% for 10 minutes. After that, it was continuously brought to a low humidity of 10 RH% at a humidity change rate of 300 RH% / hr., And further kept under a low humidity of 10 RH% for 10 minutes (a). Using the same thin film sample, measurement was performed in exactly the same manner except that all continuous humidity control rates were changed to 100 RH% / hr. In the humidity control pattern (b). Using the same thin film sample, measurement was performed in exactly the same manner except that all continuous humidity control rates were changed to 50 RH% / hr. In the humidity control pattern (c). The time required for measurement at 10-80 RH% is (a) 14 minutes, (b) 42 minutes, and (c) 84 minutes. The results are shown in Table 9.

Comparing (a), (b), and (c) where the humidity control time was changed (see FIG. 36), when the humidity control time was short, the moisture content was large and the adsorption / desorption curve was asymmetric (asymmetry) (A)>(b)> (c)), it can be seen that the water intake proceeds in an unstable state. On the other hand, in the long-term humidity control (c), it can be seen that the symmetry is improved and water is taken up in a stable state.

Further, when the intersection of the inflection points is obtained from the calculation formula for the sample (c), it can be seen that the humidity is 63 RH% (see FIG. 37).
By changing the humidity control time, the time to reach the equilibrium of water absorption / desorption can be determined from the values of adsorption / desorption. For example, by calculating the average adsorbed film thickness and the average desorbed film thickness, taking the difference between them, and determining the time at which the difference becomes 0, the humidity control speed at which continuous hydration reaches the maximum stability can be determined. . Here, as shown in Table 10 and FIG. 38, the approximate expression of the samples of (a), (b), and (c) was derived, and the time to become 0 was found to be 107 minutes. In the sample, if the change of 10-80RH% is performed in 107 minutes, the most stable hydration condition can be estimated. It is also possible to calculate that the film thickness reference moisture content when the equilibrium is reached at that time is 7.44%.

  Table 10 summarizes the adsorption rate when the humidity control rate was changed during water adsorption. It can be seen that water is difficult to adsorb at low humidity, and water is likely to adsorb at high humidity. This indicates that water adsorption is slow due to the formation of structured water, for example, because the interaction between water and substances is strong under low humidity. On the other hand, under high humidity, other water adsorption, for example, it is suggested that the water has low interaction like free water.

[Example 10]
The two unmodified sensor chips were immersed in a 1 mg / ml NeutrAvidin-PBS solution for 10 seconds and then washed with water to prepare a sensor chip to be analyzed in which NeutrAvidin was adsorbed nonspecifically on the surface. One sheet was a normal sample, which was held at 80 RH% for 10 minutes to prevent denaturation due to drying after washing, and the humidity was changed from 80 RH% to 10 RH%. Furthermore, after holding at 80 RH% for 10 minutes, the humidity was changed from 10 RH% to 80 RH%, and was held at 80 RH% for 10 minutes. The other sheet was a denatured sample, water was evaporated at 70 ° C., kept at the same temperature for 3 hours, then immersed in water again for 1 hour, and then the humidity measurement was performed. The results are shown in Table 11 and FIG. In the normal sample, a smooth hydration change was observed, but in the denatured sample that had been stored dry, there was a region where the hydration did not go smoothly. As a result, a remarkable hysteresis was observed. In addition, the denatured sample has a significantly higher moisture content than the normal sample, and the three-dimensional structure of the protein is dispersed, suggesting that more hydrated regions have appeared on the surface than the normal sample.

[Example 11]
Analysis target sensor chip No. 27 (coated with polymethyl methacrylate (PMMA), which is a hydrophobic synthetic polymer as a sample) and sensor chip No. RifS was measured using 28 (also coated with polystyrene (PS) as a sample). Regarding these hydrophobic synthetic polymers, there is data that it takes half a year to obtain a stable water-containing state at atmospheric pressure, but in this example, the water content immediately after adjustment was measured. The results are shown in Table 12 and FIG. According to the result of this example, the moisture content of PS was 0.1% or less on both the film thickness basis and the mass basis, and the amount of hydration was overwhelmingly small. From this result, the degree of hydrophobicity of PS became clear. On the other hand, with PMMA, which is considered to have a mass-based moisture content of around 1%, the amount of hydration per film thickness is overwhelmingly larger than that of PS, and the hydrophilicity between hydrophobic polymers is determined by the film-based moisture content. Can be quantified with high accuracy.

[Example 12]
Analysis target chip No. 24 (with dextran applied as a sample) and no. The RIfS was measured using 25 (coated with CMD as a sample). Dextran and CMD are hydratable materials (polysaccharides) with the same basic structure. For hydratable materials, an irreversible change can often occur by going through the dry state in the initial stage.Therefore, a reverse humidity control pattern was devised to adjust the humidity from a high humidity state to a low humidity state. To see if the Japanese structure is restored, the humidity control pattern changes from 80RH% to 10RH% at a rate of 300RH% / hr. From 80RH% to 10RH%, 10RH% for 10 minutes, from 10RH% to 80RH% A humidity control pattern was devised at a rate of 300RH% / hr. The results are shown in Table 13 and FIG. The film thickness reference moisture content was not so different as the basic structure may have the same sugar chain structure. In CMD, hydration proceeds quickly by adjusting the humidity from 10RH% to 80RH% even after the dry state, whereas in dextran, after passing through the dry state, the film thickness due to hydration does not return, It may have caused irreversible film degradation. It can be seen that by using such a humidity control pattern, it is possible to measure the hydration restoration property and the hydration speed.

In addition, when comparing the dynamics shown in FIG. 42, the CMD is proceeding with adsorption / desorption similar in shape due to desorption and adsorption of water, whereas dextran causes a decrease in the film thickness of water desorption at various frequencies. Distribution of film thickness difference is very large. On the other hand, the distribution of the difference in film thickness of the subsequent water adsorption is very narrow and orderly, indicating that water is adsorbed.

[Example 13]
Analysis target sensor chip No. The RifS was measured using No. 26 (applied with Fresella R, which is a superhydrophilic material, as a sample). Super hydrophilic and anti-dyeing materials are attracting attention as surface treatment agents. The phenomenon of condensation and devitrification at a minute level when the ambient temperature and humidity change rapidly, such as when breathing indoors and outdoors, is a major issue. However, scientific measurement methods have not been established. Regarding anti-fogging properties, the temperature-humidity relationship was evaluated from various aspects by using the program temperature control function of MI-Affinity because it was noticeably caused by the change from high temperature and high humidity to low temperature and low humidity. The difference between the humidity control temperature and the sample surface temperature was measured at 5 ° C and 10 ° C.

(1) Humidity adjustment temperature 30 ℃, sample surface temperature 25 ℃ (temperature difference 5 ℃)
In a measurement system in which the temperature of the enclosed space (humidity control gas) is adjusted to 30 ° C and the temperature of the sample surface (sensor chip) is adjusted to 25 ° C, the humidity control is performed in the order of 10RH%, 80RH%, and 10RH%. went. The results are shown in FIG. In FRESERA®, there was a point of condensation (clouding point) near a humidity of 80 RH% at a temperature difference of 5 ° C, and it was possible to observe the appearance of water drops out of the measurement range (large water drops cannot be measured).

(2) Humidity adjustment temperature 30 ℃, sample surface temperature 20 ℃ (temperature difference 10 ℃)
In a measurement system in which the temperature of the enclosed space (humidity control gas) is adjusted to 30 ° C and the temperature of the sample surface (sensor chip) is adjusted to 20 ° C, the humidity control is performed in the order of 10RH%, 80RH%, and 10RH%. went. The results are shown in FIG. Up to 60% humidity, normal water adsorption was observed, but after that, measurement was impossible due to condensation. It seems that condensation is apparently occurring at lower humidity than when the temperature difference is 5 ° C.

DESCRIPTION OF SYMBOLS 1 Measurement system 10 RifS measuring apparatus 11 White light source 12 Spectrometer 13 Measurement probe 13a First optical fiber 13b Second optical fiber 21 Sensor chip 21a Substrate (Si)
21b Optical thin film (SiN)
21c Sample thin film (sample)
23 Sealed space forming member 23a (broken line) Sealed space 23b Opening (gas inlet)
23c Opening (gas outlet)
23d Opening (reagent supply port)
A measurement area 50 control device 60 connection means 100 measurement environment adjustment mechanism 110 temperature / humidity adjustment unit 120 temperature / humidity sensor 130 exhaust mechanism 140a first gas flow path 140b second gas flow path 150 moisture trap mechanism 190 housing (humidity control) box)
200 Sensor Chip Temperature Control Mechanism 210 Temperature Controller 220 Temperature Control Unit 230 Temperature Sensor 300 Reagent Supply Mechanism

Claims (17)

  A step of measuring data relating to the film thickness of the sample thin film by RIFS (reflection interference spectroscopy) while continuously or discontinuously changing the humidity of the atmosphere in which the sample thin film formed on the surface of the measurement substrate is placed (A measuring process), The analysis method regarding the behavior of the gaseous water molecule with respect to a sample thin film characterized by the above-mentioned.   2. The analysis method according to claim 1, wherein in the measurement step, 10 or more measurement steps in which an interval between humidity values is 0.1 to 10% are performed.   The analysis method according to claim 1 or 2, wherein in the measurement step, the change in humidity is repeated for 1 to 100 cycles.   The analysis method according to any one of claims 1 to 3, wherein in the measurement step, the humidity is changed at a rate of 1 to 3600 seconds per 1%.   The analysis method according to any one of claims 1 to 4, wherein, in the measurement step, the initial humidity is increased and changed so as to decrease the humidity.   The analysis method according to any one of claims 1 to 5, wherein in the measurement step, the temperature of the atmosphere or the temperature of the sample thin film is changed.   The analysis method according to any one of claims 1 to 6, wherein the measurement step is performed a plurality of times under conditions in which the humidity change rate of the atmosphere is different.   The analysis method according to any one of claims 1 to 7, wherein the measurement step is performed a plurality of times under conditions in which the temperature of the atmosphere or the temperature of the sample thin film is different.   The sample thin film is a thin film formed of a film-forming solid or liquid; a thin film formed of a solid, liquid or gas that can be fixed to the surface of the measurement substrate; or dissolved in a sealed space formed on the measurement substrate. Or the analysis method as described in any one of Claims 1-8 which is a thin film formed with the substance which floats.   The analysis method according to claim 1, wherein the sample thin film has a thickness of 1 nm to 100 μm.   Based on the data obtained by the measurement process, the adsorption / desorption amount and adsorption / desorption rate of gaseous water molecules, the adsorption / desorption dynamics of gaseous water molecules, the saturation hydration amount of the sample thin film, the equilibrium time, the equilibrium film thickness and the equilibrium For moisture content, wettability of minute area at molecular level, difference in environment (change in temperature and humidity), difference in hydration property of substance before and after stress, adsorption / desorption of water molecule (humidification and dehumidification) The analysis method according to any one of claims 1 to 10, comprising a step of analyzing at least one item selected from the group consisting of hysteresis derived, a difference in hydration rate, and a hydration structure.   The analysis method according to claim 11, wherein the analysis of the equilibrium time, the equilibrium film thickness, or the equilibrium moisture content is performed based on a simulation curve or a calculation formula.   Analyzing the adsorption / desorption amount or adsorption / desorption rate of the gaseous water molecules, the volume of gaseous water molecules adsorbed / desorbed on the sample thin film calculated from the data on the film thickness of the sample thin film obtained by the measurement step, or The analysis method of Claim 11 performed based on mass.   The analysis method according to claim 11, wherein the hydration structure is analyzed based on an inflection point of a film thickness change amount-humidity curve.   An RIfS measurement system comprising humidity adjusting means for continuously changing the humidity of an atmosphere in which a sample thin film formed on the surface of a measurement substrate is placed.   The system according to claim 15, further comprising temperature adjusting means for changing a temperature of the atmosphere or a temperature of the sample thin film.   The system according to claim 15 or 16, wherein a sealed space is formed around the sample thin film by overlapping a sealed space forming member having an opening through which gas flows in and out on the measurement substrate.