WO2014061408A1 - Method for measuring optical film thickness, system for measuring optical film thickness, and program for measuring optical film thickness - Google Patents

Abstract

The purpose of the present invention is to measure the optical film thickness of an interference film through reflectometric interference spectroscopy, and in the event that there is variation in the optical film thickness of an interference film, to measure the optical film thickness of the portion where there is a variation in the optical film thickness. The optical film thickness of an interference film in a laminate obtained by laminating an interference film on a substrate is measured through reflectometric interference spectroscopy, doing so while taking note of the tendency of the spectral distribution of spectral reflectivity (or the amount of variation thereof) to shift towards the long-wavelength end in the order curve b1->curve b2->curve b3->curve b4, and for the number of peaks thereof to increase in association with an increase in the optical film thickness of the interference film. For a number of different optical film thicknesses, the relationship between a predetermined characteristic value of the curve of spectral reflectivity (the amount of variation) for a laminate in which the optical film thickness of the interference film is known and the optical film thickness in question is created in advance; a predetermined characteristic value of the curve of spectral reflectivity (the amount of variation) in a laminated to be measured through reflectometric interference spectroscopy is plugged into the relationship; and the optical film thickness of the interference film to be measured is derived by identifying the optical film thickness that most closely approximates the characteristic value.

Description

Optical film thickness measurement method, optical film thickness measurement system, optical film thickness measurement program, etc.

The present invention relates to an optical film thickness measurement method, an optical film thickness measurement system, and an optical film thickness measurement program using reflection interference spectroscopy.

Conventionally, the measurement of bonds such as intermolecular interactions between biomolecules such as antigen-antibody reactions and intermolecular interactions between organic macromolecules is generally performed by using labels such as radioactive substances and phosphors. I have been. This labeling takes time, and in particular, labeling a protein may involve complicated methods or the property of the protein may change depending on the labeling. Therefore, in recent years, as a means for directly detecting a bond between a biomolecule and an organic polymer without using a label, an RIfS method (Reflectometric interference spectroscopy: reflection interference spectroscopy using an interference color change of an optical thin film). ) Has been proposed and already put into practical use. The basic principle of the RIfS method is mentioned in Patent Document 1, Non-Patent Document 1, and the like.

The RIfS method will be briefly described. In this method, as shown in FIG. 12, a substrate 102 provided with an optical thin film 104 is used. As shown in FIG. 12A, when the optical thin film 104 on the substrate 102 is irradiated with white light, the spectral intensity of the white light itself is represented by a solid line 106 as shown in a typical example of FIG. Is represented by a solid line 108. When the reflectance is obtained from each spectral intensity of the irradiated white light and the reflected light, a reflection spectrum 110 having a bottom peak (minimum portion in the spectrum curve) represented by a solid line is obtained as shown in FIG.

In detecting intermolecular interactions, a ligand 120 is provided on the optical thin film 104 as shown in FIG. 12b. When the ligand 120 is provided on the optical thin film 104, the optical thickness 112 at the site where the ligand 120 is provided changes, the optical path length changes, and the interference wavelength also changes due to the reflection interference effect. That is, the peak position of the spectral intensity distribution of the reflected light is shifted, and as a result, the reflected spectrum 110 is shifted to the reflected spectrum 122 (see the dotted line portion) as shown in FIG. In this state, when the sample solution is caused to flow on the optical thin film 104, the ligand 120 and the analyte 130 in the sample solution are bonded as shown in FIG. 12c. When the ligand 120 and the analyte 130 are bonded, the optical thickness 112 at the site where the analyte 130 is bonded further changes. The analyte 130 partially adheres to the ligand 120 to generate a heterogeneous layer. This macroscopic layer is macroscopically determined to have a predetermined optical thickness corresponding to the amount of the analyte 130 deposited. It is replaced with a homogeneous layer having a thickness. Accordingly, the optical thickness of the homogeneous layer through which incident light passes changes depending on the amount of the analyte 130 attached. As a result, as shown in FIG. 14, the reflection spectrum 122 is shifted to the reflection spectrum 132 (see the chain line). Then, by detecting the change in the bottom peak wavelength of the reflection spectrum 132 with respect to the bottom peak wavelength of the reflection spectrum 122 (the wavelength at which the reflectance becomes a minimum value), the presence or absence of the intermolecular interaction can be detected. Further, by detecting the amount of change in the bottom peak wavelength of the reflection spectrum 132 with respect to the bottom peak wavelength of the reflection spectrum 122, the progress of the intermolecular interaction can be detected.

When the transition of the change in the bottom peak wavelength is observed with time, the change in the bottom peak wavelength due to the ligand 120 can be confirmed at the time point 140, which is the first shoulder portion on the curve, as shown in FIG. At the time 142, which is the second shoulder portion above, a change in the bottom peak wavelength due to the binding between the ligand 120 and the analyte 130 can be confirmed.

Japanese Patent No. 3778673 Japanese Patent Application No. 2011-141348 Japanese Patent Application No. 2012-001095

However, according to the study by the present inventors, the method of observing the transition of the bottom peak wavelength over time has a limit in measurement accuracy due to the measurement principle, and the progress of intermolecular interaction is more accurately determined. When trying to catch it, it turned out that there was a problem that it could not be improved sufficiently. This is due to the following reasons.
In other words, intermolecular interactions between biomolecules such as antigen-antibody reactions, and intermolecular interactions between organic macromolecules, while microscopically repeating binding and detachment, the overall rate of binding Increases uniformly, that is, the progress of intermolecular interaction increases with time as shown in FIG. 16A.
However, the data that is actually output by the detection device that detects the spectral intensity of the reflected light repeats minute fluctuations, such as reflectance data 151 shown in FIG. 17, for example. An operation such as fitting the approximate curve 152 to the reflectance data 151 to calculate the approximate curve 152 and obtaining the minimum value of the approximate curve as the above-described bottom peak position is required. In addition, since the change in reflectance is small near the bottom peak, the method of determining the bottom peak position using the approximate curve as described above tends to cause the bottom peak position to be inaccurate in principle. This is disadvantageous when it is required to calculate the progress of the interaction with higher accuracy. As a result, the change amount Δλ of the bottom peak wavelength may change differently from the progress of the intermolecular interaction, as shown in FIG. 16B. In such a case, the change amount Δλ of the bottom peak wavelength. Is not suitable as a value for accurately determining the progress of intermolecular interaction.

In addition, the method of tracking the change amount Δλ of the bottom peak wavelength tracks only one point on the graph of the spectral characteristic of reflected light, does not capture the overall shift of the graph, and accurately changes There is a limit to catching it.
In addition, in order to perform the above-mentioned calculation for calculating the approximate curve, a high-performance calculation device or a complicated calculation is required, or the calculation cannot catch up in a case where the intermolecular interaction proceeds rapidly. There is also a concern.

Researching the background of the above circumstances, the present inventors have focused on the extreme value of the spectral characteristics of the detected light, and found that this extreme value shows a certain change tendency according to the thickness of the interference film. By specifying the relationship in advance, a method for measuring the optical path length of the interference film, that is, the optical film thickness of the interference film was found, and patent applications of Patent Documents 2 and 3 were filed. According to this method, it is possible to measure the optical film thickness as well as the film formed by the intermolecular interaction, and if there is a change in the optical film thickness over time like the intermolecular interaction, It is also possible to detect and measure the changed optical film thickness. That is, in the measurement of the intermolecular interaction, when the ligand and the analyte are bonded, the amount of change in the optical film thickness at the site where the analyte is bonded can be measured.
At the same time, the present inventors have determined that the entire curve is constant according to the thickness of the interference film and is not limited to the extreme value. The present inventors have found that it is possible to measure thickness and the like, and that there is a possibility that measurement can be performed even if the extreme value is out of the measurement range.

That is, the present invention is to measure the optical film thickness of the interference film by reflection interference spectroscopy using a predetermined feature value different from the wavelength giving the extreme value, if there is a change in the optical film thickness of the interference film, It is an object of the present invention to provide an optical film thickness measurement method, an optical film thickness measurement system, and an optical film thickness measurement program capable of measuring the changed optical film thickness.

The invention according to claim 1 for solving the above-described problems is an optical film thickness in which an optical film thickness of an interference film in a laminate in which one or two or more interference films are laminated on a substrate is measured by reflection interference spectroscopy. Measuring method,
In advance, a predetermined feature value different from the wavelength that gives the extreme value related to the spectral reflectance curve in the laminate having the known optical film thickness of the interference film is extracted, and the relationship between the feature value and the optical film thickness is determined. Create different optical film thicknesses,
By applying the predetermined feature value relating to the spectral reflectance curve in the laminate to be measured obtained by reflection interference spectroscopy to the relationship, by identifying the optical film thickness that approximates the feature value, It is an optical film thickness measuring method for obtaining an optical film thickness in the laminate to be measured.

According to the second aspect of the present invention, the optical film thickness of the molecules adsorbed on the interference film is reduced by the intermolecular interaction performed on the interference film of the laminate in which one or more interference films are laminated on the substrate. An optical film thickness measuring method for measuring by reflection interference spectroscopy,
In advance,
Spectral reflectance in the first laminate in which the first interference film having a known optical thickness is laminated;
Spectral reflectance in a second laminate in which a second interference film having a known optical thickness is laminated on the interference film of the first laminate, and
A predetermined feature value that is different from a wavelength that gives an extreme value related to a curve of the amount of change in the spectral reflectance of the second laminate relative to the spectral reflectance of the first laminate is extracted, and the feature value and the optical properties of the second interference film are extracted. The relationship with the film thickness is created in advance for the optical film thickness of the different second interference film,
Spectral reflectance before the start of intermolecular interaction of the laminate to be measured is obtained by reflection interference spectroscopy, and spectral reflectance after initiation of intermolecular interaction of the laminate to be measured by reflection interference spectroscopy By applying the predetermined feature value related to the curve of the change amount before the start to the relationship and identifying the optical film thickness of the second interference film that approximates the feature value, This is an optical film thickness measurement method for obtaining an optical film thickness of molecules adsorbed on an interference film by intermolecular interaction in the laminate.

According to a third aspect of the present invention, when there are a plurality of the predetermined feature values in a predetermined wavelength band, the relationship is created for the plurality of feature values, and the measurement object is measured from the laminate to be measured by reflection interference spectroscopy. The optical film thickness measuring method according to claim 1 or 2, wherein the identification is performed by obtaining each feature value.

The invention according to claim 4 is the optical film thickness measuring method according to any one of claims 1 to 3, wherein the predetermined characteristic value is a wavelength value.

The invention according to claim 5 is characterized in that the predetermined characteristic value is a wavelength value of a point on the curve having a spectral reflectance separated from the extreme value of the curve by a predetermined amount. 3 is an optical film thickness measuring method according to any one of 3 above.

The invention according to claim 6 is the optical film thickness measurement according to any one of claims 1 to 3, wherein the predetermined feature value is a wavelength value of an inflection point of the curve. Is the method.

The invention according to claim 7 is characterized in that the predetermined feature value is a wavelength value of a point on the curve having a spectral reflectance separated from the inflection point of the curve by a predetermined amount. Item 4. The optical film thickness measurement method according to any one of Items 3 above.

The invention according to claim 8 is the optical film thickness measuring method according to any one of claims 1 to 7, wherein the relation is created by a function.

The invention according to claim 9 is the optical film thickness measuring method according to claim 8, wherein the function is a quadratic function, and the quadratic function is created for each feature value.

According to a tenth aspect of the present invention, the identification is performed by specifying an optical film thickness that approximates the feature value most closely, and an optical film thickness to be obtained is estimated from the optical film thickness that is most approximated. Item 10. The optical film thickness measurement method according to any one of Items 9 above.

Invention of Claim 11 irradiates the said laminated body of a measuring object with a white light by reflection interference spectroscopy, and obtains the said spectral reflectance of the reflected light from the said laminated body among Claim 1-10 It is an optical film thickness measuring method as described in any one.

The invention according to claim 12 is the optical film thickness measuring method according to any one of claims 1 to 11, wherein the interference film is measured with a liquid layer covering the interference film.

The invention according to claim 13, by dividing the optical film thickness of the molecule obtained by the optical film thickness measurement method according to claim 2 by the refractive index of the molecule, for a molecule having a known refractive index. This is a film thickness measurement method for obtaining a film thickness.

In the invention described in claim 14, for a molecule whose optical size in a specific direction is known, the optical film thickness of the molecule obtained by the film thickness measuring method according to claim 2 and the optical size in the specific direction are calculated. This is a molecular direction estimation method for estimating the direction of molecules adsorbed on the interference film by intermolecular interaction with respect to the interference film by comparison.

The invention according to claim 15 compares the film thickness of the molecule determined by the film thickness measurement method according to claim 13 and the size in the specific direction for molecules whose size in the specific direction is known. This is a molecular direction estimation method for estimating the direction of molecules adsorbed on an interference film by intermolecular interaction with respect to the interference film.

The invention according to claim 16 is obtained by the optical film thickness measurement method according to claim 2 for each different time point when environmental variables such as temperature, pressure, pH, and salt concentration of the measurement environment where the molecule is placed have changed. This is an optical film thickness measurement method that records the environmental variable in association with the optical film thickness of the molecule.

The invention according to claim 17 is a molecule obtained by the film thickness measurement method according to claim 13 at each of different time points when environmental variables such as temperature, pressure, pH, and salt concentration of the measurement environment where the molecule is placed are changed. The film thickness measurement method records the environmental variable in association with the film thickness.

The invention according to claim 18 is an optical film thickness measurement system for measuring an optical film thickness of an interference film in a laminate in which one or more interference films are stacked on a substrate by reflection interference spectroscopy.
A light source;
Irradiating means for irradiating the laminate with light from the light source;
A light receiving means for receiving reflected light from the laminate;
Spectral detection means for detecting spectral characteristics of reflected light received by the light receiving means;
In advance, a predetermined feature value different from the wavelength that gives the extreme value related to the spectral reflectance curve in the laminate having the known optical film thickness of the interference film is extracted, and the relationship between the feature value and the optical film thickness is determined. Storage means for storing data configured by creating different optical film thicknesses;
Based on the spectral characteristics detected by the spectral detection means, for the spectral reflectance in the laminate to be measured, the predetermined feature value relating to the spectral reflectance curve is specified, and the data stored in the storage means And calculating means for determining the optical film thickness in the laminate to be measured by identifying the optical film thickness that approximates the feature value, and
Is an optical film thickness measurement system.

According to the nineteenth aspect of the present invention, the optical film thickness of the molecules adsorbed on the interference film is reduced by the intermolecular interaction performed on the interference film of the laminate in which one or more interference films are laminated on the substrate. An optical film thickness measurement system for measuring by reflection interference spectroscopy,
A light source;
Irradiating means for irradiating the laminate with light from the light source;
A light receiving means for receiving reflected light from the laminate;
Spectral detection means for detecting spectral characteristics of reflected light received by the light receiving means;
The spectral reflectance in the first laminated body in which the first interference film having a known optical film thickness is laminated in advance, and the second interference film in which the second optical film having a known optical film thickness is laminated on the interference film of the first laminated body. The spectral reflectance of the two laminated body is obtained, and a predetermined feature value different from the wavelength that gives the extreme value relating to the curve of the amount of change in the spectral reflectance of the second laminated body with respect to the spectral reflectance of the first laminated body is extracted. Storage means for storing data constituted by creating in advance the relationship between the characteristic value and the optical film thickness of the second interference film with respect to the optical film thickness of the different second interference film;
Spectral reflection after the start of the intermolecular interaction of the laminate of the measurement object with respect to the spectral reflectance before the start of the intermolecular interaction of the laminate of the measurement object based on the spectral characteristics detected by the spectroscopic detection means By specifying the predetermined feature value relating to the curve of the rate change amount, applying it to the data stored in the storage means, and identifying the optical film thickness of the second interference film that approximates the feature value, An arithmetic means for obtaining an optical film thickness of molecules adsorbed on the interference film by intermolecular interaction in the laminate to be measured;
Is an optical film thickness measurement system.

In the invention according to claim 20, when there are a plurality of the predetermined feature values in a predetermined wavelength band, the relationship is created for the plurality of feature values, and the calculation means calculates each of these extreme values. The optical film thickness measurement system according to claim 18 or 19, wherein the identification is performed.

The invention according to claim 21 is the optical film thickness measurement system according to any one of claims 18 to 20, wherein the predetermined feature value is a wavelength value.

According to a twenty-second aspect of the present invention, the predetermined feature value is a wavelength value of a point on the curve having a spectral reflectance separated from the extreme value of the curve by a predetermined amount. The optical film thickness measurement system according to any one of 20.

23. The optical film thickness measurement according to claim 18, wherein the predetermined characteristic value is a wavelength value of an inflection point of the curve. System.

According to a twenty-fourth aspect of the present invention, the predetermined characteristic value is a wavelength value of a point on the curve having a spectral reflectance separated from the inflection point of the curve by a predetermined amount. Item 21. The optical film thickness measurement system according to any one of Items 20 above.

The invention according to claim 25 is the optical film thickness measurement system according to any one of claims 18 to 24, wherein the light source is a light source that emits white light.

According to a twenty-sixth aspect of the present invention, a member that forms a flow path on the interference film and a liquid that contains molecules that are connected to the flow path and interact with molecules provided on the interference film are supplied to the flow path. It is an optical film thickness measuring system as described in any one of Claims 18-25 provided with the liquid feeding means to flow.

The invention according to claim 27 comprises the optical film thickness measurement system according to claim 19, and the molecular film thickness obtained by the optical film thickness measurement system according to claim 19 is calculated for a molecule having a known refractive index. It is a film thickness measuring system further comprising a film thickness calculating means for obtaining the film thickness of the molecule by dividing by the refractive index of the molecule.

The invention according to claim 28 comprises the optical film thickness measurement system according to claim 19, and the molecule obtained by the film thickness measurement system according to claim 19 is obtained for a molecule whose optical magnitude in a specific direction is known. Molecular direction estimation further comprising molecular direction estimation means for estimating the direction of molecules adsorbed on the interference film by intermolecular interaction with respect to the interference film by comparing the optical film thickness with the optical size in the specific direction. System.

The invention according to claim 29 is provided with the film thickness measurement system according to claim 27, and the molecular film thickness obtained by the film thickness measurement system according to claim 27 is determined for a molecule whose size in a specific direction is known. The molecular direction estimation system further comprising molecular direction estimation means for estimating the direction of molecules adsorbed on the interference film by intermolecular interaction with respect to the interference film by comparing with the size of the specific direction.

The invention according to claim 30 includes the optical film thickness of the molecule determined by the calculation means at different time points when environmental variables such as temperature, pressure, pH, and salt concentration of the measurement environment in which the molecule is placed are changed. The optical film thickness measurement system according to claim 19, further comprising recording means for recording environmental variables in association with each other.

The invention according to claim 31 includes the thickness of the molecule determined by the thickness calculation means at each different time when environmental variables such as temperature, pressure, pH, and salt concentration of the measurement environment where the molecule is placed are changed. 28. The film thickness measurement system according to claim 27, further comprising recording means for recording the environment variable in association with each other.

According to a thirty-second aspect of the present invention, there is provided an optical film thickness for causing a computer to execute a process of measuring an optical film thickness of an interference film in a laminate in which one or two or more interference films are stacked on a substrate by reflection interference spectroscopy. A measurement program,
In advance, a predetermined feature value different from the wavelength that gives the extreme value related to the spectral reflectance curve in the laminate having the known optical film thickness of the interference film is extracted, and the relationship between the feature value and the optical film thickness is determined. A process of reading out data configured by creating different optical film thicknesses;
Identifying the predetermined feature value relating to a spectral reflectance curve in the laminate to be measured obtained by reflection interference spectroscopy, and applying it to the data to identify an optical film thickness that approximates the feature value By the arithmetic processing to obtain the optical film thickness in the laminate to be measured,
Is an optical film thickness measurement program that causes the computer to perform

According to a thirty-third aspect of the present invention, there is provided an optical film thickness of molecules adsorbed on the interference film by intermolecular interaction performed on the interference film of the laminate in which one or more interference films are laminated on the substrate. An optical film thickness measurement program for causing a computer to execute processing to be measured by reflection interference spectroscopy,
In advance,
Spectral reflectance in the first laminate in which the first interference film having a known optical thickness is laminated;
Spectral reflectance in a second laminate in which a second interference film having a known optical thickness is laminated on the interference film of the first laminate, and
A predetermined feature value that is different from a wavelength that gives an extreme value related to a curve of the amount of change in the spectral reflectance of the second laminate relative to the spectral reflectance of the first laminate is extracted, and the feature value and the optical properties of the second interference film are extracted. A process of reading out data configured by preparing in advance the relationship between the film thickness and the optical film thickness of different second interference films;
Spectral reflectance before the start of intermolecular interaction of the laminate to be measured is obtained by reflection interference spectroscopy, and spectral reflectance after initiation of intermolecular interaction of the laminate to be measured by reflection interference spectroscopy The measurement is performed by identifying the predetermined feature value related to the curve of the change amount before the start, and applying the data to identify the optical film thickness of the second interference film that approximates the feature value. A calculation process for obtaining the optical film thickness of the molecules adsorbed on the interference film by the intermolecular interaction in the target laminate;
Is an optical film thickness measurement program that causes the computer to perform

The invention according to claim 34 is provided with the optical film thickness measurement program according to claim 33, and for molecules having a known refractive index, the molecules obtained by the computer based on the optical film thickness measurement program according to claim 33. It is a film thickness measurement program for causing the computer to further execute a film thickness calculation process for obtaining the film thickness of the molecule by dividing the optical film thickness by the refractive index of the molecule.

The invention according to claim 35 is provided with the optical film thickness measurement program according to claim 33, and the molecule having a known optical size in a specific direction is based on the optical film thickness measurement program according to claim 33. Molecular direction estimation processing for estimating the direction of molecules adsorbed on the interference film by intermolecular interaction by comparing the optical film thickness of the molecule obtained by the computer and the optical size in the specific direction. Is a molecular direction estimation program for causing the computer to execute.

A thirty-sixth aspect of the invention includes the film thickness measurement program according to the thirty-fourth aspect, and the computer obtains a molecule whose size in a specific direction is known based on the thickness measurement program according to the thirty-fourth aspect. By comparing the film thickness of the molecule and the size of the specific direction, the computer further executes a molecular direction estimation process for estimating the direction of the molecule adsorbed on the interference film by the intermolecular interaction with respect to the interference film. Is a molecular orientation estimation program.

The invention according to claim 37 is the optical film thickness of the molecule obtained by the computer by the arithmetic processing at different time points when environmental variables such as temperature, pressure, pH, and salt concentration of the measurement environment where the molecule is placed have changed. In addition, the optical film thickness measurement program according to claim 33, further causing the computer to execute a recording process in which the environment variable is associated and recorded.

The invention according to claim 38 is the molecular film obtained by the computer by the film thickness calculation process at different time points when environmental variables such as temperature, pressure, pH, and salt concentration of the measurement environment where the molecule is placed have changed. 35. The film thickness measurement program according to claim 34, further causing the computer to execute a recording process for recording the environmental variable in association with the thickness.

According to the present invention, the optical film thickness of the interference film can be measured. In particular, according to the inventions according to claims 2, 19 and 33, there is a change in the optical film thickness of the interference film due to intermolecular interaction. For example, the optical film thickness corresponding to the change can be measured.

It is a schematic diagram which shows schematic structure of the measurement system of the intermolecular interaction which concerns on one Embodiment of this invention. It is a perspective view showing a schematic structure of a measuring member concerning one embodiment of the present invention. It is a schematic diagram which shows the mode of a measurement of the measuring system of intermolecular interaction. It is a circuit block diagram of the measurement system of intermolecular interaction. It is sectional drawing which represented typically the mode of the coupling | bonding of the ligand and analyte which concern on one Embodiment of this invention. New drawing from here It is a typical model figure for demonstrating the principle of a reflection interference spectroscopy. . It is each curve which shows the variation | change_quantity of the spectral reflectance at the time of making an interference film increase from a certain reference | standard film thickness. It is a graph which shows the relationship between the sample film thickness d obtained by simulation, and the extreme value position wavelength of the spectral reflectance R. FIG. 9 is a graph created from the graph of FIG. 8 by converting the horizontal axis to optical path length (optical film thickness) nd and the vertical axis to 1000 / λ. It is a graph which shows the relationship between the sample film thickness d obtained by simulation, and the extreme position wavelength of spectral reflectance change amount (DELTA) R. 11 is a graph created from the graph of FIG. 10 by converting the horizontal axis to optical path length (optical film thickness) nd and the vertical axis to 1000 / λ. It is a schematic diagram for demonstrating the outline of a RIfS system. It is a graph of an example which shows the rough relationship between a wavelength and spectral intensity. It is a graph of an example which shows the rough relationship between a wavelength and a reflectance. It is a graph of an example which shows the rough transition of the change of a bottom peak wavelength. It is a graph which shows the time change of the progress degree of intermolecular interaction. It is a graph which shows an example of the time change of variation | change_quantity (DELTA) (lambda) of the bottom peak wavelength in intermolecular interaction. It is a graph which shows an example of the detection data of a spectral reflectance, and its approximated curve. In the schematic diagram for explaining the estimation of the molecular direction, a single molecule is shown. It is a schematic diagram for explaining the estimation of the molecular direction, and shows molecules adsorbed on the interference film. It is a schematic diagram for explaining estimation of the molecular direction, and shows molecules adsorbed on the interference film in a different direction from FIG. 18B.

Hereinafter, an embodiment of the present invention will be described with reference to the drawings. The following is one embodiment of the present invention and does not limit the present invention.

First, with reference to FIG. 1 to FIG. 5, an outline of the intermolecular interaction measurement system 1 which is an embodiment for carrying out the optical film thickness measurement system of the present invention will be described. The intermolecular interaction measurement system 1 executes the optical film thickness measurement method of the present invention, and the computer included in the system configuration executes a program reflecting the optical film thickness measurement program of the present invention. It is remembered as possible. In the following, the case of measuring the intermolecular interaction will be described as an example, but the application of the present invention is not limited to the measurement of the intermolecular interaction. In addition, the intermolecular interaction measurement system 1 is usually configured to output various measurement values, but only the measurement of the optical film thickness of the interference film according to the implementation of the present invention will be described below.

As shown in FIG. 1, the intermolecular interaction measurement system 1 includes a measurement device 80 that includes a measurement member 10 that holds a sample to be measured, a measurement mechanism including a light source and a spectroscope, which will be described later, The control arithmetic device 50 is a computer connected to the device 80, and a display 91 and an input / output device 92 connected to the control arithmetic device 50.
In the measurement system 1, the control arithmetic device 50 includes a control unit for a measurement mechanism such as a light source and a spectroscope built in the measurement device 80, a calculation unit for detection information, and an input / output unit for inputting / outputting control commands and detection information. Functions as (interface).

The measuring device 80 includes a lower housing 82 and an upper housing 81 that is rotatably attached to the lower housing 82. The lower housing 82 is provided with a table 83 for holding the measuring member 10. Inside the upper housing 81, a connection portion 84 having an injection port 85 and a suction port 87 for connecting the measurement member 10 and circulating the sample and a detection window 86 is provided. As will be described later, a white light source 20, a spectroscope 30, and optical fibers 40 and 41 (see FIG. 3) are provided in the upper housing 81, and light is emitted from the detection window 86 and from the detection window 86. It is configured to receive incident light. When performing measurement, first, the upper casing 81 is rotated upward to open the table 83 on the lower casing 82, and the measuring member 10 is set on the table 83. Thereafter, the upper casing 81 is rotated downward and closed, whereby the injection port 85 and the suction port 87 are connected to the measurement member 10, and the detection window 86 faces the measurement member 10 to complete the measurement preparation.

As shown in FIG. 2, the measurement member 10 includes a sensor chip 12 provided with an optical thin film and a flow cell 14 that forms a flow path together with the sensor chip 12. The sensor chip 12 has a silicon substrate 12a. A SiN film 12b (silicon nitride) is deposited on the silicon substrate 12a. The SiN film 12b is an example of an optical thin film.
The flow cell 14 is a transparent member made of silicone rubber. A groove 14 a is formed in the flow cell 14. When the flow cell 14 is brought into close contact with the sensor chip 12, a sealed flow path 14b is formed as shown in FIG. Both ends of the groove 14a are exposed from the surface of the flow cell 14, and one end functions as a sample solution inlet 14c and the other end functions as an outlet 14d. A ligand 16 is previously bonded to the bottom of the groove 14a (see FIG. 3).

In the measurement member 10, the flow cell 14 can be replaced with the sensor chip 12, and the flow cell 14 can be used in a disposable manner. The surface of the sensor chip 12 may be modified with a silane coupling agent or the like. In this case, the flow cell 14 can be easily replaced.

As described above, after the measurement member 10 is set, the upper casing 81 is rotated downward and closed, so that the detection window 86 faces the flow cell 14 as shown in FIG. The optical fiber 40 is installed above the path 14b. Here, the white light source 20 is connected to one end of the optical fiber 40. For example, a halogen light source is used as the white light source 20. Further, a pseudo white light source having a plurality of spectral intensity peaks in the visible light wavelength region, such as an LED light source, may be used. The other end of the optical fiber 40 faces the detection window 86. The spectroscope 30 is connected to one end of the optical fiber 41, and the other end faces the detection window 86. When the white light source 20 is turned on, the light is applied to the sealed flow path 14 b through the optical fiber 40, and the reflected light is detected by the spectrometer 30 through the optical fiber 41. The white light source 20 and the spectroscope 30 are connected to a control arithmetic device 50, and the control arithmetic device 50 controls the operation of these modules.
In addition, the control arithmetic device 50 executes data stored in a storage device 503 (see FIG. 4) described later, and stores data representing the spectral characteristics of reflected light through the interface at a predetermined timing linked to detection operation control. While obtaining an input, it functions as a calculation means for calculating the value of the optical film thickness based on the input data.

FIG. 4 is a schematic circuit block diagram of the measurement system 1. As shown in FIG. 4, the control arithmetic device 50 includes a CPU 500, a ROM 501, a RAM 502, a storage device 503 such as a hard disk, a communication device 504, a reader / writer 505 of a storage medium such as a memory card, and each part and display of the measurement device 80. An interface 506 is provided for exchanging signals with the input device. A program for measuring the optical film thickness in the intermolecular interaction and other programs for measurement are stored in the storage device 503, and the CPU 500 is controlled by this program to execute various operations.

Subsequently, the operation and measurement method of the measurement system 1 until spectral characteristics are input to the control arithmetic device 50 will be described.

As shown in FIG. 3, the sample solution 60 containing the analyte 62 is circulated from the inlet 14c to the outlet 14d through the sealed channel 14b. At this time, the control arithmetic device 50 controls the liquid feeding device 35 (see FIG. 4) for feeding the sample solution 60. The analyte 62 is a substance that specifically binds to the ligand 16 and is a target molecule to be detected. As the analyte 62, for example, biomolecules such as proteins, nucleic acids, lipids, and sugars, and foreign substances that bind to biomolecules such as drug substances and endocrine confusion chemical substances are used.
The control arithmetic device 50 turns on the white light source 20 at a timing before the sample solution 60 flows into the measurement member 10, and the reflected light of the optical thin film (SiN film 12 b) before the start of the intermolecular interaction from the spectroscope 30. An input of spectral characteristic data including data indicating the intensity of light is obtained.

The control arithmetic device 50 turns on the white light source 20 while the sample solution 60 is circulating in the closed flow path 14b. The white light passes through the flow cell 14 and is irradiated to the sensor chip 12, and the reflected light is detected by the spectroscope 30. The spectral characteristic of the reflected light detected by the spectroscope 30 is transmitted to the control arithmetic device 50.

In this case, as shown in FIG. 5, when the analyte 62 in the sample solution 60 binds to the ligand 16, the optical thickness, that is, the optical film thickness 70 changes, and the reflected light characteristics (for example, by the spectroscope 30). The wavelength at which the detected intensity is the smallest) changes. The control arithmetic device 50 obtains from the spectroscope 30 input of spectral characteristic data including data indicating the intensity of reflected light during or after the progress of the intermolecular interaction.

As described above, spectral characteristic data necessary for measuring the optical film thickness of the interference film is input to the control arithmetic unit 50.

As can be seen from the configuration, function, and operation of the measurement system 1 described above, the sensor chip 12 is a laminate in which one or more interference films (SiN film 12b) are laminated on a substrate (silicon substrate 12a). The measurement system 1 can measure the optical film thickness (such as the optical film thickness 70) of the interference film in the laminate by reflection interference spectroscopy.
The optical fiber 40 receives irradiation light that irradiates light from the light source (white light source 20) to the laminate (sensor chip 12), and the optical fiber 41 receives reflected light from the laminate (sensor chip 12). The spectroscope 30 corresponds to a spectroscopic detector that detects the spectral characteristics of the reflected light.
The RAM 502 functions as a data storage unit that is referred to when the control arithmetic device 50 calculates the optical film thickness. The data can be read from any of a storage device 503, a server connected via the communication device 504, an external storage device connected via the interface 506, a memory card read by the reader / writer 505, and the like. Good. In addition, a hardware configuration in which another computer that has received spectral characteristic data necessary for measuring the optical film thickness of the interference film via the communication device 504 performs calculations for measuring the optical film thickness of the interference film. Does not matter. In this case, the other computer is a computer in which the optical film thickness measurement system of the present invention is incorporated and the optical film thickness measurement program of the present invention is installed.
The flow cell 14 corresponds to a member that forms a flow path on the interference film. As described above, the flow cell 14 is connected to the flow path formed by the flow cell 14 in the measuring device 80 and is provided on the interference film. A liquid feeding means is configured to flow a liquid containing molecules that interact with the molecules through the flow path.

Next, two measurement principles 1 and 2 of the optical film thickness will be described.
[Measurement principle 1]
First, the measurement principle 1 will be described as follows.
(Create reference data)
When performing measurement based on this measurement principle, the data to be referred to when calculating the optical film thickness is obtained by extracting in advance the predetermined characteristic value related to the spectral reflectance curve in the laminate with the known optical film thickness of the interference film. This is data configured by creating a relationship between the characteristic value and the optical film thickness for different optical film thicknesses. This will be described in detail below.

FIG. 6 is a schematic model diagram for explaining the principle of reflection interference spectroscopy. As shown in FIG. 6, the laminated body is composed of a substrate a1 and an interference film a2 having a film thickness d laminated thereon, and the liquid layer a3 covers the interference film a2. The liquid layer a3, the interference film a2, and the substrate a1 have refractive indexes n1, n2, and n3, respectively. When light a4 having a wavelength λ0 as shown in FIG. 6 is incident thereon, reflected light a5 reflected from the surface of the interference film a2 and reflected light a6 passing through the interference film a2 and reflected from the interface with the substrate a1. Interfere as shown by part a7. At this time, the optical path of the reflected light a6 is longer than the reflected light a5 by 2n2d. When the optical path difference 2n2d causes a half-wavelength shift between the reflected light a5 and the reflected light a6, it interferes so as to weaken the reflection intensity most, as shown by a7. As a result, the reflection intensity varies depending on the wavelength of the light to be irradiated. By irradiating light in a wide wavelength band such as white light, a spectral distribution in which the reflection intensity changes depending on the wavelength can be obtained.
Therefore, since the spectral distribution of the reflected light is different depending on the optical film thickness n2d of the interference film a2, the optical film thickness n2d of the interference film a2 can be specified by analyzing the spectral characteristics of the reflected light. This is the basic principle of the present invention using reflection interference spectroscopy. The following description will clarify how the reflected light is analyzed to determine the optical film thickness.
The silicon substrate 12a described above corresponds to the substrate a1, and the SiN film 12b corresponds to the interference film a2. As shown in FIG. 5, when the ligand 16 is placed on the SiN film 12b and when the analyte 62 is further bonded to the ligand 16, the portion corresponding to the optical film thickness 70 added to the SiN film 12b is the interference film. It corresponds to a2.

The curve of the spectral reflectance and the amount of change due to reflection interference changes as if it moves in the longer wavelength direction as the thickness of the interference film increases.
An example is shown in FIG. The spectral reflectance change amount curves b1, b2, b3, and b4 shown in FIG. 7 are obtained from the curve b2, the curve b2, the curve b3, and the curve b4 having a thicker interference film than the curve b1. It is. The spectral reflectance change amount ΔR is a change amount based on the spectral reflectance in an interference film having a certain film thickness. As shown in FIG. 7, the peak position moves to the long wavelength side in the order of curve b1, curve b2, curve b3, and curve b4.
Moreover, not only the peak moves, but also changes so that the number of peaks increases.
From these graphs, it can be seen that as the film thickness increases, the peak of the spectral reflectance change amount curve increases with the shift toward the longer wavelength side. As the film thickness increases, one peak (top peak) giving the maximum value moves to the long wavelength side. Further, as the film thickness increases, the first peak moves further to the longer wavelength side, and a peak (bottom peak) giving a minimum value appears on the shorter wavelength side, and also moves to the longer wavelength side together with the first top peak. . Furthermore, the movement toward the longer wavelength side and the increase in the number of peaks proceed.
Note that the vertical axis in FIG. 7 represents the spectral reflectance change amount ΔR with respect to the spectral reflectance R, but the observed peak shift and increase change appear in the same way for both R and ΔR. .

This change characteristic depends on the optical film thickness of the interference film. Therefore, the physical properties of the interference film are extracted by focusing on the wavelength that gives the extreme value from the spectral reflectance curve and the change amount curve, and instead extracting the feature by paying attention to a predetermined feature value that is different from the wavelength that gives the extreme value. Regardless, it is possible to extract the optical properties depending on the optical film thickness of the interference film. And it can expand | deploy to the measurement of the optical film thickness of the interference film | membrane of a different physical property.
That is, since the movement of the spectral reflectance (or the spectral reflectance change amount) is unique, the characteristic of this curve may be extracted. In other words, any element that characterizes a curve may be used. The feature here is to characterize a curve corresponding to a certain optical film thickness from a curve corresponding to another optical film thickness.
For example, as shown in FIG. 7, extreme values (maximum value P, minimum value B), inflection point E of the curve, and points G1, G2 on the curve where the spectral reflectance is separated from the maximum value P by a predetermined amount F. The points J1 and J2 on the curve where the spectral reflectance is separated by a predetermined amount H from the minimum value B, and the points M1 and M2 on the curve where the spectral reflectance is separated by a predetermined amount K from the inflection point E appear.
In addition to the wavelength that gives the extreme value (the wavelength value of the maximum value P, the wavelength value of the minimum value B), the characteristic values include the wavelength value of the inflection point E, the value of the slope of the curve at the inflection point E, the point Wavelength value of G1, wavelength value of point G2, wavelength value of point J1, wavelength value of point J2, wavelength value of point M1, wavelength value of point M2, difference between wavelength value of point G1 and wavelength value of point G2, point The difference between the wavelength value of J1 and the wavelength value of the point J2 and the difference of the wavelength value of the point M1 and the wavelength value of the point M2 can be mentioned, and these can be used alone or in combination.
By tracking these feature values as the film thickness changes, a reference data set can be created.

Here, a wavelength that gives an extreme value as a feature value is taken as an example.
Curves of spectral reflectance R are calculated for samples having different film thicknesses by simulation, the wavelength λ at the extreme position is obtained from the curve, and the relationship between the sample film thickness d and the extreme position wavelength λ is shown in the graph of FIG. . This is obtained by examining the substrate a1 as a silicon substrate and changing the SiN film as the interference film a2 thereon in the range of 66.5 nm to 2000 nm. In the graph of FIG. 8, the solid line indicates the wavelength at the maximum value position with respect to the sample film thickness, and the broken line indicates the wavelength at the minimum value position with respect to the sample film thickness.
The sample film thickness d on the horizontal axis of the graph of FIG. 8 is converted to the optical path length (optical film thickness) nd by multiplying the refractive index n of the interference film, and the wavelength λ on the vertical axis is converted to 1000 / λ. The resulting graph is shown in FIG.
In the graph of FIG. 9, a curve indicating the wavelength at the minimum value position appears on the far left. This is a curve B1. When going to the right from the curve B1, a curve indicating the wavelength at the local maximum position appears. This is a curve P1. When going further to the right from the curve P1, a curve indicating the wavelength at the minimum value position and a curve indicating the wavelength at the maximum value position alternately appear. These are designated as curves B2, P2, B3, P3,... B24, P24 in this order. Each of the curves B1 to P24 is approximated by a quadratic function, and the approximate quadratic function is expressed by a mathematical expression. When the expression form is y = ax2 + bx + c, the coefficients a and b and the constant c corresponding to each of the curve B1 to the curve P24 are shown in Table 1 below.

 

The above function data refers to data that is referred to when calculating the optical film thickness according to the present measurement principle, that is, the wavelength λ that gives the extreme value of the spectral reflectance in a laminate in which the optical film thickness of the interference film is known in advance. It is data configured by creating a relationship with the optical film thickness for different optical film thicknesses.
Such function data is stored so that it can be used by a computer (control arithmetic unit 50) for calculating the optical film thickness.

(Optical film thickness identification calculation)
In the measurement system 1, based on the spectral characteristic data input from the spectroscope 30 by the control arithmetic device 50, first, for the spectral reflectance in the laminate to be measured, the wavelength that gives the extreme value of the spectral reflectance is determined. Identify. The extreme value is specified including the maximum value and the minimum value.
In the graph of FIG. 9, when a line parallel to the vertical axis y is drawn at an arbitrary value of the optical film thickness (horizontal axis x), it intersects one of the curves B1 to P24. The y coordinate of the intersection is a value of 1000 / λ giving an extreme value to the spectral reflectance curve at the optical film thickness. If the intersection intersects the curve B1-24, the extreme value is a minimum value, and the curve P1-24 If they intersect, the extreme value is a local maximum.
Using this in a reverse calculation, the control arithmetic unit 50 converts the wavelength λ giving the extreme value of the spectral reflectance obtained from the object to be measured into 1000 / λ to obtain the y value, and distinguishes between the maximum value and the minimum value. In consideration of the number of y values including, the magnitude relationship of wavelengths giving extreme values, etc., and further considering errors, while narrowing down to possible fitting patterns, input to the above quadratic function, the common solution x, that is, the optical film thickness nd value is identified. The control arithmetic unit 50 estimates and outputs the optical film thickness of the interference film in the measurement target laminate from the identified optical film thickness nd value.
In actual measurement, there is almost no case where the solutions x completely coincide with each other. Therefore, the identification is performed by specifying those that approximate each other in the narrowest range. Then, for example, the average value of the distributed solution x is output as an estimated measurement value. In addition, the reliability of the measurement is ensured to be constant by providing a threshold value of the approximation that may be output as a measurement value and the approximation that is output as a measurement error. The degree of approximation may also be output.
Possible fitting patterns are SP1, SP2,... Of the maximum value of the spectral reflectance curve obtained from the measurement object from the short wavelength side to the long wavelength direction, and the minimum values are SB1, SB2,. , SP1, SP2... Are sequentially applied to the expressions P1, P2..., SP1, SP2... Are sequentially applied to the expressions P2, P3. A pattern in which SB2... Is sequentially applied to the expressions B1, B2,..., A pattern in which SB1, SB2.
On the other hand, since the pattern in which SP1 is applied to the formula of B1 is erroneously distinguished from the maximum value and the minimum value, the pattern in which SP1 is applied to the formula of P2 and SP2 is applied to the formula of P1 is related to the magnitude of the wavelength giving the extreme value. Is incorrect and should not be included in possible fitting patterns.
When SB2 is specified between SP1 and SP2, when calculating by applying SP1, SP2... To the expressions P2, P3... In order, there is a pattern for applying SB2 to the expression B2. Since the magnitude relationship between the wavelengths giving the extreme values is incorrect, it is appropriate that they are not included in the possible fitting patterns.

As described above, the measurement system 1 measures the optical film thickness of the interference film a2 according to the measurement principle 1. The measurement system 1 is based on the measurement principle 1, regardless of whether the interference film a2 includes a layer formed by intermolecular interaction or not, by using reflection interference spectroscopy. Can be measured.

[Measurement principle 2]
Next, the measurement principle 2 will be described as follows.
(Create reference data)
When measurement is performed based on this measurement principle, the data to be referred to when calculating the optical film thickness includes the spectral reflectance in the first laminated body in which the first interference film having a known optical film thickness is previously laminated, and the first Spectral reflectance in the second laminate in which a second interference film having a known optical film thickness is laminated on the interference film of the laminate, and spectral reflection in the second laminate relative to the spectral reflectance in the first laminate By extracting a predetermined feature value related to the curve of the rate change amount and creating a relationship between the feature value and the optical film thickness of the second interference film in advance for different optical film thicknesses of the second interference film It is data that is composed. This will be described in detail below.

This measurement principle also presupposes the matters with reference to FIGS. 6 and 7 in the above measurement principle 1. As shown in FIG. 7, the curve of the change amount ΔR of the spectral reflectance with respect to the wavelength shows the change in peak movement and increase as the interference film becomes thicker as described above.

Here, a wavelength that gives an extreme value as a feature value is taken as an example.
The curve of the spectral reflectance change amount ΔR is calculated for samples having different film thicknesses by simulation, the wavelength λ at the extreme position is obtained in the curve, and the relationship between the sample film thickness d and the extreme position wavelength is shown in the graph of FIG. Described. In this method, the substrate a1 is a silicon substrate, and the first interference film laminated thereon is a 66.5 nm SiN film. The second interference film laminated on the first interference film is BK7, and the second interference film (BK7) is examined by changing in the range of 0 nm to 1000 nm. It is a change amount of the spectral reflectance in the second laminated body with the second interference film with respect to the spectral reflectance in the first laminated body composed of a1 and the first interference film. The sample film thickness d on the horizontal axis in the graph of FIG. 10 is for the second interference film (BK7). Note that BK7 is a virtual sample for simulation in which properties such as a refractive index are specified, corresponds to a typical kind of glass, and has a refractive index close to that of a biomaterial or a polymer material.
In the graph of FIG. 10, the solid line indicates the wavelength at the maximum value position with respect to the sample film thickness, and the broken line indicates the wavelength at the minimum value position with respect to the sample film thickness.
FIG. 10 is a graph created by converting the sample film thickness d on the horizontal axis to the optical path length (optical film thickness) nd by multiplying by the refractive index n, and converting the wavelength λ on the vertical axis to 1000 / λ. It is shown in FIG.
In the graph of FIG. 11, a curve indicating the wavelength at the local maximum position appears on the leftmost side. This is a curve P1. When going to the right from the curve P1, a curve showing the wavelength of the minimum value position appears. This is a curve B1. When going further to the right from the curve B1, a curve indicating the wavelength at the maximum value position and a curve indicating the wavelength at the minimum value position appear alternately. These are sequentially designated as curves P2, B2, P3, B3... B7, P8. Each of the curves P1 to P8 is approximated by a quadratic function, and the approximate quadratic function is expressed by a mathematical expression. When the expression form is y = ax2 + bx + c, the coefficients a and b and the constant c corresponding to each of the curves P1 to P8 are shown in Table 2 below.

 

The above function data refers to data that is referred to when calculating the optical film thickness according to the present measurement principle, that is, the spectral reflectance in the first laminated body in which the first interference film having a known optical film thickness is previously laminated, Spectral reflectance in the second laminate in which a second interference film having a known optical thickness is laminated on the interference film of the first laminate, and in the second laminate relative to the spectral reflectance in the first laminate This is data configured by creating in advance the relationship between the wavelength that gives the extreme value of the change amount of the spectral reflectance and the optical film thickness of the second interference film for the different optical film thicknesses of the second interference film.
Such function data is stored so that it can be used by a computer (control arithmetic unit 50) for calculating the optical film thickness.

(Optical film thickness identification calculation)
In the measurement system 1, first, before the start of the intermolecular interaction, the control arithmetic device 50 receives the input from the spectroscope 30 before the start of the intermolecular interaction. Spectral reflectance is obtained.
After the start of the intermolecular interaction, the control arithmetic unit 50 obtains the spectral reflectance after the start of the intermolecular interaction in the measurement target laminate based on the spectral characteristic data input from the spectroscope 30.
When the spectral reflectance after the start is obtained, the control arithmetic device 50 compares the spectral reflectance before the start of the intermolecular interaction of the laminate to be measured with respect to the spectral reflectance after the start of the intermolecular interaction of the laminate to be measured. The wavelength giving the extreme value of the change amount ΔR of the spectral reflectance is specified. The extreme value is specified including the maximum value and the minimum value.
In the graph of FIG. 11, when a line parallel to the vertical axis y is drawn at an arbitrary value of the optical film thickness (horizontal axis x), it intersects one of the curves P1, B1,. The y coordinate of the intersection is a value of 1000 / λ that gives an extreme value to the spectral reflectance change amount curve at the optical film thickness. If the intersection intersects the curves P1-P8, the extreme value is the maximum value, and the curve B1- If it crosses B7, the extreme value is a minimum value.
Using this in a reverse calculation, the control arithmetic device 50 obtains the y value by converting the wavelength λ giving the extreme value of the change amount of the spectral reflectance obtained from the measurement object into 1000 / λ, and the maximum value and the minimum value. In consideration of the number of y values, including the difference in wavelength, the magnitude relationship of wavelengths giving extreme values, etc., and further considering errors, input to the above quadratic function while narrowing down to possible fitting patterns, common X, that is, the optical film thickness nd value is identified. Thereafter, similarly to the measurement principle 1, the control arithmetic device 50 estimates and outputs the optical film thickness of the interference film in the laminate to be measured from the identified optical film thickness nd value.

As described above, the measurement system 1 can measure the optical film thickness increased by the intermolecular interaction of the interference film a2 according to the measurement principle 2. That is, the measurement system 1 can measure the optical film thickness corresponding to the analyte 62 bound to the ligand 16 as shown in FIG.
Due to the progress of the intermolecular interaction, the ligand 16 to which the analyte 62 is bound gradually increases. Even if the analyte 62 is combined even partly, there is a change in the wavelength of reflection interference, so that a curve of the spectral reflectance change amount can be obtained even in the initial stage of the intermolecular interaction. At this time, since the optical film thickness of the portion where the analyte 62 is bonded is constant regardless of the progress of the intermolecular interaction, the wavelength that gives the extreme value of the spectral reflectance change amount even at the initial stage of the intermolecular interaction is set. Can be identified. That is, the extreme value of the change amount of the spectral reflectance specified at the initial stage of the intermolecular interaction is not different from that specified thereafter. Therefore, the measurement system 1 can calculate and output the measured value of the optical film thickness from the stage where the wavelength that gives the extreme value of the spectral reflectance change amount can be specified first, and can notify the user at an early stage.
When the sample solution 60 includes a plurality of types of molecules, each molecule has a unique size. Even if molecules of different sizes are included, if the present invention is applied and the optical film thickness that changes due to the intermolecular interaction of the target molecule (analyte 62) is known, the known optical film thickness Compare the measured value with the measured optical film thickness value. If they match, it can be determined that there was an intermolecular interaction of the target molecule, and if it does not match, it can be determined that there was no intermolecular interaction of the target molecule. The presence or absence of intermolecular interaction of the target molecule can be detected.

(Film thickness measurement)
In the above embodiment, the optical film thickness corresponding to the analyte 62 bound to the ligand 16 is measured, but the film thickness can be obtained for molecules having a known refractive index. When the film thickness measurement system is configured to function as a film thickness calculation unit that measures the film thickness, the control arithmetic device 50 stores the refractive index of the molecule (analyte 62) in advance in the RAM 502 and obtains it as described above. The film thickness of the molecule is determined by dividing the optical film thickness of the molecule by the refractive index of the molecule.

(Molecular direction estimation)
Furthermore, if the size or optical size of a specific direction of a molecule is known, the direction of the molecule relative to the interference film can be estimated.
As shown in FIG. 18A, a molecule having a size d1 in one direction and a size d2 in a different direction may be adsorbed on the interference film with the size d1 as the stacking direction as shown in FIG. 18B. As shown in 18C, when there is a possibility that the size d2 is adsorbed to the interference film as the stacking direction, the control arithmetic unit 50 functions as a molecular direction estimation unit and constitutes a molecular direction estimation system. And the size d2 stored in the RAM 502 in advance, and the molecules adsorbed on the interference film by the intermolecular interaction by comparing the film thicknesses of the molecules obtained as described above with the sizes d1 and d2, respectively. Is estimated with respect to the interference film.
When the size d1 and the size d2 are optical sizes, that is, values obtained by multiplying the actual size by the refractive index, the control arithmetic unit 50 stores and holds these optical sizes in the RAM 502 in advance. Then, the direction of the molecules adsorbed on the interference film by the intermolecular interaction with respect to the interference film is estimated by comparing the optical film thicknesses of the molecules obtained as described above with these optical sizes.
Even when the size or optical size in only one direction is known and stored in the RAM 502, it can be estimated whether or not the suction is performed in that direction.

(Record of environmental change characteristics)
Changes in environmental variables such as temperature, pressure, pH, and salt concentration in the measurement environment where the molecules are placed may cause the molecules to expand and contract, and the change characteristics differ depending on the molecule.
By providing the measurement system 1 with the measurement environment control device or the measurement environment detection device, it is possible to record the change characteristic of the molecular size due to the environmental change.
For example, the measurement environment control device includes a device that controls one or more of environmental variables such as temperature, pressure, pH, and salt concentration of the measurement environment. The detection unit of the measurement environment control device detects the value of the controlled environment variable.
The control arithmetic unit 50 gives an instruction to the measurement environment control device based on the program to change the environment variable, and calculates the optical film thickness or film thickness of the molecule obtained as described above for each different time point when the environment variable changes. It is stored in the RAM 502 in association with the environment variable at the same time.
Further, when there is no need to provide the environmental change cause from the measurement system 1 such as when the cause of the environmental change is on the measurement target side, for example, as the measurement environment detection device, the environment such as the temperature, pressure, pH, salt concentration, etc. of the measurement environment One that detects any one or more of the variables is provided.
The control arithmetic unit 50 stores in the RAM 502 the optical film thickness or film thickness of the molecules obtained as described above for each different time point when the environmental variable changes in association with the environmental variable detected by the measurement environment detection device at the same time. To do.
In either case, the measurement system 1 further draws a graph based on the recorded information and displays it on the display 91.

Note that the above program can be updated to the latest one through the communication device 504 connected to a public line such as the Internet through a LAN or the like.
Further, as the above reference data, (1) created for each of interference films having different properties (refractive index, etc.), (2) created for each of cases where light sources of different wavelength bands are applied, (3) molecules Created for each of different ligands and analytes when creating an interference film formed by the intermolecular interaction, (4) Created for each of the different conditions depending on the combination of (1), (2) and (3) In view of the above, it is preferable to create a plurality of types of reference data. This is to improve the measurement accuracy.

INDUSTRIAL APPLICATION This invention can be utilized for measuring the optical thickness of the film | membrane which grows by the coupling | bonding of the intermolecular interaction of biomolecules, such as an antigen antibody reaction, and the intermolecular interaction of organic polymers. .

1 Measurement System 10 Measurement Member 12 Sensor Chip (Laminate)
12a Silicon substrate 12b SiN film 14 Flow cell 14a Groove 14b Sealed flow path 14c Inlet 14d Outlet 16 Ligand 20 White light source 30 Spectroscope 40, 41 Optical fiber 50 Control arithmetic device 60 Sample solution 62 Analyte 80 Measuring device R Spectral reflectance ΔR Amount of change in spectral reflectance a1 Substrate a2 Interference film a3 Liquid layer

Claims (38)

1. An optical film thickness measuring method for measuring an optical film thickness of an interference film in a laminate in which one or more interference films are laminated on a substrate by reflection interference spectroscopy,
   In advance, a predetermined feature value different from the wavelength that gives the extreme value related to the spectral reflectance curve in the laminate having the known optical film thickness of the interference film is extracted, and the relationship between the feature value and the optical film thickness is determined. Create different optical film thicknesses,
   By applying the predetermined feature value relating to the spectral reflectance curve in the laminate to be measured obtained by reflection interference spectroscopy to the relationship, by identifying the optical film thickness that approximates the feature value, An optical film thickness measurement method for obtaining an optical film thickness in the laminate to be measured.
2. An optical system for measuring the optical film thickness of molecules adsorbed on an interference film by reflection interference spectroscopy by intermolecular interaction performed on the interference film of a laminate in which one or more interference films are laminated on a substrate. A film thickness measuring method,
   In advance,
   Spectral reflectance in the first laminate in which the first interference film having a known optical thickness is laminated;
   Spectral reflectance in a second laminate in which a second interference film having a known optical thickness is laminated on the interference film of the first laminate, and
   A predetermined feature value that is different from a wavelength that gives an extreme value related to a curve of the amount of change in the spectral reflectance of the second laminate relative to the spectral reflectance of the first laminate is extracted, and the feature value and the optical properties of the second interference film are extracted. The relationship with the film thickness is created in advance for the optical film thickness of the different second interference film,
   Spectral reflectance before the start of intermolecular interaction of the laminate to be measured is obtained by reflection interference spectroscopy, and spectral reflectance after initiation of intermolecular interaction of the laminate to be measured by reflection interference spectroscopy By applying the predetermined feature value related to the curve of the change amount before the start to the relationship and identifying the optical film thickness of the second interference film that approximates the feature value, An optical film thickness measurement method for obtaining an optical film thickness of molecules adsorbed on an interference film by intermolecular interaction in the laminate.
3. When there are a plurality of the predetermined feature values in a predetermined wavelength band, the relationship is created for the plurality of feature values, and each of the feature values is obtained from the laminate to be measured by reflection interference spectroscopy. The optical film thickness measuring method according to claim 1 or 2, wherein identification is performed.
4. The optical film thickness measuring method according to claim 1, wherein the predetermined characteristic value is a wavelength value.
5. The predetermined characteristic value is a wavelength value of a point on the curve having a spectral reflectance separated from the extreme value of the curve by a predetermined amount. The optical film thickness measuring method.
6. The optical film thickness measuring method according to any one of claims 1 to 3, wherein the predetermined characteristic value is a wavelength value at an inflection point of the curve.
7. The predetermined characteristic value is a wavelength value of a point on the curve whose spectral reflectance is separated from the inflection point of the curve by a predetermined amount. The optical film thickness measuring method as described.
8. The optical film thickness measuring method according to claim 1, wherein the relationship is created as a function.
9. The optical film thickness measuring method according to claim 8, wherein the function is a quadratic function, and the quadratic function is created for each feature value.
10. The identification is performed by specifying an optical film thickness that approximates the feature value most closely, and the optical film thickness to be obtained is estimated from the optical film thickness that is most approximated. The optical film thickness measuring method as described.
11. The optical film according to any one of claims 1 to 10, wherein the spectral reflectance of reflected light from the laminate is obtained by irradiating the laminate to be measured with white light by reflection interference spectroscopy. Thickness measurement method.
12. The optical film thickness measuring method according to any one of claims 1 to 11, wherein measurement is performed in a state where a liquid layer covers the interference film.
13. A film thickness measuring method for determining a film thickness of a molecule having a known refractive index by dividing the optical film thickness of the molecule determined by the optical film thickness measuring method according to claim 2 by the refractive index of the molecule. .
14. By comparing the optical film thickness of the molecule determined by the film thickness measurement method according to claim 2 with the optical size in the specific direction for molecules having a known optical size in the specific direction, A molecular direction estimation method for estimating a direction of molecules adsorbed on an interference film by an action relative to the interference film.
15. The molecule thickness determined by the film thickness measurement method according to claim 13 and the size in the specific direction are compared for molecules having a known size in a specific direction, thereby causing intermolecular interaction on the interference film. Molecular direction estimation method for estimating the direction of molecules adsorbed on the interference film.
16. For each different point in time when environmental variables such as temperature, pressure, pH, and salt concentration of the measurement environment in which the molecule is placed are changed, along with the optical film thickness of the molecule determined by the optical film thickness measurement method according to claim 2, An optical film thickness measurement method that records environmental variables in association with each other.
17. The environmental variables together with the film thicknesses of the molecules determined by the film thickness measurement method according to claim 13 at different points in time when the environmental variables such as temperature, pressure, pH, and salt concentration of the measurement environment where the molecules are placed have changed. Is a film thickness measurement method for recording in association with each other.
18. An optical film thickness measurement system for measuring an optical film thickness of an interference film in a laminate in which one or more interference films are laminated on a substrate by reflection interference spectroscopy,
    A light source;
    Irradiating means for irradiating the laminate with light from the light source;
    A light receiving means for receiving reflected light from the laminate;
    Spectral detection means for detecting spectral characteristics of reflected light received by the light receiving means;
    In advance, a predetermined feature value different from the wavelength that gives the extreme value related to the spectral reflectance curve in the laminate having the known optical film thickness of the interference film is extracted, and the relationship between the feature value and the optical film thickness is determined. Storage means for storing data configured by creating different optical film thicknesses;
    Based on the spectral characteristics detected by the spectral detection means, for the spectral reflectance in the laminate to be measured, the predetermined feature value relating to the spectral reflectance curve is specified, and the data stored in the storage means And calculating means for determining the optical film thickness in the laminate to be measured by identifying the optical film thickness that approximates the feature value, and
    An optical film thickness measurement system.
19. An optical system for measuring the optical film thickness of molecules adsorbed on an interference film by reflection interference spectroscopy by intermolecular interaction performed on the interference film of a laminate in which one or more interference films are laminated on a substrate. A film thickness measuring system,
    A light source;
    Irradiating means for irradiating the laminate with light from the light source;
    A light receiving means for receiving reflected light from the laminate;
    Spectral detection means for detecting spectral characteristics of reflected light received by the light receiving means;
    The spectral reflectance in the first laminated body in which the first interference film having a known optical film thickness is laminated in advance, and the second interference film in which the second optical film having a known optical film thickness is laminated on the interference film of the first laminated body. The spectral reflectance of the two laminated body is obtained, and a predetermined feature value different from the wavelength that gives the extreme value relating to the curve of the amount of change in the spectral reflectance of the second laminated body with respect to the spectral reflectance of the first laminated body is extracted. Storage means for storing data constituted by creating in advance the relationship between the characteristic value and the optical film thickness of the second interference film with respect to the optical film thickness of the different second interference film;
    Spectral reflection after the start of the intermolecular interaction of the laminate of the measurement object with respect to the spectral reflectance before the start of the intermolecular interaction of the laminate of the measurement object based on the spectral characteristics detected by the spectroscopic detection means By specifying the predetermined feature value relating to the curve of the rate change amount, applying it to the data stored in the storage means, and identifying the optical film thickness of the second interference film that approximates the feature value, An arithmetic means for obtaining an optical film thickness of molecules adsorbed on the interference film by intermolecular interaction in the laminate to be measured;
    An optical film thickness measurement system.
20. When there are a plurality of the predetermined feature values in a predetermined wavelength band, the relationship is created for the plurality of feature values, and the calculation means obtains each of these extreme values and performs the identification. The optical film thickness measurement system according to 18 or 19.
21. The optical film thickness measurement system according to any one of claims 18 to 20, wherein the predetermined feature value is a wavelength value.
22. The predetermined characteristic value is a wavelength value of a point on the curve in which a spectral reflectance is separated from the extreme value of the curve by a predetermined amount. Optical film thickness measurement system.
23. 21. The optical film thickness measurement system according to claim 18, wherein the predetermined feature value is a wavelength value of an inflection point of the curve.
24. The predetermined characteristic value is a wavelength value of a point on the curve that is separated from the inflection point of the curve by a predetermined amount in spectral reflectance, according to any one of claims 18 to 20. The optical film thickness measurement system described.
25. The optical film thickness measurement system according to any one of claims 18 to 24, wherein the light source is a light source that emits white light.
26. A member that forms a flow path on the interference film, and a liquid feeding means that flows a liquid containing molecules that interact with the molecules connected to the flow path and provided on the interference film to the flow path. The optical film thickness measurement system according to any one of 18 to 25.
27. The molecule having the optical film thickness measurement system according to claim 19 having a known refractive index, the molecular film thickness obtained by the optical film thickness measurement system according to claim 19 is divided by the refractive index of the molecule. Thus, a film thickness measurement system further comprising a film thickness calculation means for determining the film thickness of the molecule.
28. An optical film thickness measurement system according to claim 19, the molecular optical film thickness obtained by the film thickness measurement system according to claim 19, and the specific direction, for a molecule whose optical magnitude in a specific direction is known. A molecular direction estimation system further comprising molecular direction estimation means for estimating the direction of molecules adsorbed on the interference film by intermolecular interaction with respect to the interference film by comparing with the optical size of.
29. For a molecule having the film thickness measurement system according to claim 27 and having a known size in a specific direction, the film thickness of the molecule determined by the film thickness measurement system according to claim 27 and the size in the specific direction are calculated. A molecular direction estimation system further comprising molecular direction estimation means for estimating the direction of molecules adsorbed on the interference film by intermolecular interaction with respect to the interference film by comparison.
30. For each time point when the environmental variables such as temperature, pressure, pH, and salt concentration of the measurement environment where the molecules are placed have changed, record the environmental variables in association with the optical film thicknesses of the molecules obtained by the calculation means. The optical film thickness measurement system according to claim 19, further comprising a recording unit.
31. At different points in time when environmental variables such as temperature, pressure, pH, and salt concentration of the measurement environment where the molecules are placed have changed, the environmental variables are recorded in association with the film thicknesses of the molecules obtained by the film thickness calculation means. 28. The film thickness measurement system according to claim 27, further comprising a recording unit that performs the recording.
32. An optical film thickness measurement program for causing a computer to execute a process of measuring the optical film thickness of an interference film in a laminate in which one or two or more interference films are laminated on a substrate by reflection interference spectroscopy,
    In advance, a predetermined feature value different from the wavelength that gives the extreme value related to the spectral reflectance curve in the laminate having the known optical film thickness of the interference film is extracted, and the relationship between the feature value and the optical film thickness is determined. A process of reading out data configured by creating different optical film thicknesses;
    Identifying the predetermined feature value relating to a spectral reflectance curve in the laminate to be measured obtained by reflection interference spectroscopy, and applying it to the data to identify an optical film thickness that approximates the feature value By the arithmetic processing to obtain the optical film thickness in the laminate to be measured,
    An optical film thickness measurement program for causing the computer to execute
33. A process of measuring the optical film thickness of molecules adsorbed on the interference film by reflection interference spectroscopy by intermolecular interaction performed on the interference film of the laminate in which one or two or more interference films are laminated on the substrate An optical film thickness measurement program for causing a computer to execute
    In advance,
    Spectral reflectance in the first laminate in which the first interference film having a known optical thickness is laminated;
    Spectral reflectance in a second laminate in which a second interference film having a known optical thickness is laminated on the interference film of the first laminate, and
    A predetermined feature value that is different from a wavelength that gives an extreme value related to a curve of the amount of change in the spectral reflectance of the second laminate relative to the spectral reflectance of the first laminate is extracted, and the feature value and the optical properties of the second interference film are extracted. A process of reading out data configured by preparing in advance the relationship between the film thickness and the optical film thickness of different second interference films;
    Spectral reflectance before the start of intermolecular interaction of the laminate to be measured is obtained by reflection interference spectroscopy, and spectral reflectance after initiation of intermolecular interaction of the laminate to be measured by reflection interference spectroscopy The measurement is performed by identifying the predetermined feature value related to the curve of the change amount before the start, and applying the data to identify the optical film thickness of the second interference film that approximates the feature value. A calculation process for obtaining the optical film thickness of the molecules adsorbed on the interference film by the intermolecular interaction in the target laminate;
    An optical film thickness measurement program for causing the computer to execute
34. 35. An optical film thickness measurement program according to claim 33, and a molecule having a known refractive index, the optical film thickness of the molecule determined by the computer based on the optical film thickness measurement program according to claim 33. A film thickness measurement program for causing the computer to further execute a film thickness calculation process for determining the film thickness of the molecule by dividing by a ratio.
35. 34. An optical film of a molecule obtained by the computer based on the optical film thickness measurement program according to claim 33, comprising the optical film thickness measurement program according to claim 33, and having a known optical magnitude in a specific direction. To cause the computer to further execute a molecular direction estimation process for estimating the direction of molecules adsorbed on the interference film by intermolecular interaction with respect to the interference film by comparing the thickness with the optical size in the specific direction. Molecular orientation estimation program.
36. 35. A molecule thickness measurement program according to claim 34, the molecule thickness determined by the computer based on the film thickness measurement program according to claim 34 for the molecule whose magnitude in a specific direction is known, and the identification A molecular direction estimation program for causing the computer to further execute a molecular direction estimation process for estimating a direction of a molecule adsorbed on an interference film by an intermolecular interaction with respect to the interference film by comparing the magnitude of the direction.
37. For each time point when environmental variables such as temperature, pressure, pH, and salt concentration of the measurement environment where the molecule is placed changed, the computer associates the environmental variable with the optical film thickness of the molecule obtained by the calculation process. 34. The optical film thickness measurement program according to claim 33, further causing the computer to execute a recording process for recording.
38. Corresponding to the environmental variables along with the molecular thickness obtained by the computer through the film thickness calculation process at different points in time when environmental variables such as temperature, pressure, pH, and salt concentration of the measurement environment where the molecules are placed have changed. 35. The film thickness measurement program according to claim 34, further causing the computer to execute a recording process of attaching and recording.

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