JP2002071556A - Surface analytical method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique by which a change in a sample in the vertical direction at about 0.1 to hundreds nm can be detected in a short time in a range of several cm square and which can measure the change even in a solution. SOLUTION: In the surface analytical method, reflected light from the sample is imaged by a two-dimensional photodetector regarding three or more wavelengths at an angle of incidence at which a surface plasmon resonance(SPR) is not generated, reflected-light images are measured, mean values of luminances in the images as a whole at the respective wavelengths are calculated while the maximum value is used as a reference, the means values at the respective wavelengths are standardized, background data with reference to the respective wavelengths is obtained, the reflected light from the sample regarding the three or more wavelengths at which the data is measured at an angle of incidence at which the SPR is generated is imaged by the photodetector, the luminances of respective pixels of a measured image are corrected by the data, the luminances of the respective corrected pixels at all the wavelengths are stored in the memory of a computer until a measurement at a required wavelength is finished, the corrected luminances at different wavelengths in the respective pixels of the image are approximated by a quadratic polynomial, the minimum wavelength of the luminances in the respective pixels of the image is calculated, and an SPR wavelength in the respective pixels of the reflected light image is found.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a surface analysis method using a two-dimensional surface plasmon resonance method.

[0002]

2. Description of the Related Art In recent years, in order to efficiently develop a compound having a new function, there has been a demand for a method for simultaneously evaluating many types of compounds prepared on a substrate. For example, in the development of an artificial antibody that artificially creates an antibody that binds and degrades a foreign substance (antigen) that has entered the body, the function of the artificial antibody is evaluated by the following method. First, a number of different types of artificial antibodies are prepared on a substrate, and a solution containing the antigen is evenly spread over the substrate to allow the antigen to bind to the artificial antibodies. At this time, the shape of the substrate surface changes in the vertical direction by several hundreds of pm (pm is one billionth of a meter) to several tens of nanometers (nm is one millionth of a meter) due to the binding of the antigen. By measuring the change, the function of the artificial antibody is evaluated.

[0003] Therefore, there is a need for a method capable of detecting a change in the shape of the substrate surface in the vertical direction in a short time and with high sensitivity within a range (usually several cm square) in which an artificial antibody is prepared on the substrate. Above all, the state where the solution is sprinkled on the substrate,
That is, the development of a method capable of performing such a measurement in a solution is desired.

[0004] As a conventional method of analyzing the surface shape,
A stylus-type roughness tester that measures by contacting a stylus with a sample, a scanning laser microscope using laser light interference, a light interference microscope using white light interference, a scanning electron microscope using an electron beam, An atomic force microscope using an atomic force between needles and a tunnel microscope using a change in tunnel current between a sample and a stylus are used.

The stylus type roughness meter is used in the atmosphere, has a measurement range of several cm square at the maximum, and has a vertical resolution of about 1 nm.
It is. Scanning laser microscopes and optical interference microscopes
Used in the atmosphere, the measurement range is up to several mm square, and the vertical resolution is about 0.1 nm. Scanning electron microscope is used in vacuum, the measuring range is up to several mm,
The vertical resolution is about 1 nm. Atomic force microscopes and tunneling microscopes are used in air or in solution,
The measurement range is up to several 100 μm square, and the vertical resolution is about 0.1 nm.

[0006] The measurement range by these methods is several mm square or less except for a stylus-type roughness meter, and cannot be used for the above-mentioned applications that normally require several cm square. Also, the maximum number
The stylus-type roughness meter having a measurement range of cm square has a disadvantage that it takes more than 30 minutes to scan the entire measurement range because mechanical scanning must be repeated. Atomic force microscopes and tunneling microscopes also require a mechanical stylus scan for measurement and take more than 10 minutes of measurement time. All other methods except atomic force microscopy and tunneling microscopy are used in air or vacuum and cannot be used in solution.

As described above, any of the conventional methods is unsuitable for use as a means for developing the above-mentioned compounds.

Therefore, about 0.1 n in a range of several cm square
It is necessary to develop a new method that can detect changes in the vertical direction from m to several hundreds of nm in a short time, and that can measure not only in air and vacuum but also in solution.
Conversely, if a technique that satisfies these requirements is developed, it will be useful not only as a means for compound development, but also as a technique that opens a new dimension in surface analysis.

On the other hand, a surface plasmon resonance method (SPR method) is used as a method for analyzing a certain point on a sample surface. According to this method, when a sample on a metal thin film is irradiated with light at a certain incident angle via a prism, a single sharp attenuation occurs in the reflectance spectrum (surface plasmon resonance: S
PR). It is possible to detect minute changes in properties such as the thickness, refractive index, and dielectric constant of a sample on a metal thin film from the wavelength and the incident angle of light at which SPR occurs. At present, an attempt has been made to perform SPR measurement in two dimensions to use it for surface analysis in a certain range (two-dimensional SPR method).

For example, Okamoto et al. Mechanically move a sample together with a prism in the X and Y directions, and SP
The angle of incidence causing R was determined (Optics Commun. 1992, 93, 2
65). However, in this method, since it is necessary to mechanically move the sample, it takes 30 minutes or more to scan the entire range, which is a serious problem as a practical method.

Have developed a method for measuring the intensity distribution of reflected light using a CCD camera (Nature).
1988, 332, 615). As the thickness or refractive index of the sample increases, the wavelength at which the reflected light intensity is minimized (ie, the surface plasmon resonance wavelength) shifts to longer wavelengths. The wavelength is set immediately before or immediately after the position where SPR occurs only with the metal thin film, and the change in reflected light intensity is measured. Since the reflectance changes abruptly in the portion where the sample is present, it is possible to display an image of the sample surface. This method is an excellent method in that the surface shape of the sample can be detected at one time.

However, according to this method, when the same sample is measured, the reflectivity increases or decreases depending on the measurement wavelength, so that the quantitativeness is poor. In addition, the detectable range in the vertical direction is limited to several nanometers, and there is a drawback that a further change cannot be measured.

[0013]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a two-dimensional surface plasmon resonance surface analysis method capable of quantifying a vertical change of about 0.1 nm to several hundreds nm in a short time in a range of several cm square. The main purpose is.

A further object of the present invention is to provide a surface analysis method using a two-dimensional surface plasmon resonance method that can be measured not only in air or vacuum but also in a solution.

[0015]

SUMMARY OF THE INVENTION The present inventor has determined the wavelength at which the luminance of each pixel becomes minimum in an image obtained by a two-dimensional photodetector, thereby obtaining a problem which the conventional two-dimensional SPR method has. We focused on being able to solve the points at once. As a result of intensive research, the present inventors have recorded and collected images of reflected light at several different wavelengths in the wavelength range where SPR occurs, calculated the surface plasmon resonance wavelength at each pixel of the image,
The inventors have found that a surface analysis method using a two-dimensional surface plasmon resonance method for reconstructing an image can achieve the above object, and have completed the present invention.

That is, the present invention provides a surface analysis method comprising the following techniques; A sample prepared on a metal thin film provided on a transparent substrate is analyzed, and the sample is irradiated with light from the substrate side through a prism, an image of the reflected light is measured, and a minute change in the property of the sample is reflected. In the surface analysis method using the two-dimensional surface plasmon resonance method that detects the change in intensity, 1) two-dimensional light detection of reflected light from the sample at three or more wavelengths at an incident angle at which surface plasmon resonance (SPR) does not occur The image of the reflected light is measured by taking an image with a device, the average value of the luminance in the entire image is calculated for each wavelength, and the average value at each wavelength is calculated based on the average value of the largest luminance among the calculated average values. By normalization, background data for each wavelength is obtained. 2) For three or more wavelengths at the incident angle where SPR occurs, reflected light from the sample is imaged with a two-dimensional photodetector. Measure the image of the reflected light more, correct the luminance of each pixel of the image measured for each wavelength with background data, and correct each pixel after correction at all wavelengths until the end of the measurement at the required wavelength. The luminance is stored in the memory of the computer. 3) By approximating the corrected luminance: y of each pixel at different wavelengths: x by the following second-order polynomial, the wavelength at which the luminance becomes minimum at each pixel (surface plasmon resonance wavelength)
A surface analysis method characterized by determining

[0017]

(Equation 3)

2. Assuming that the film thickness of the sample is infinite, the dielectric constant of the transparent substrate is assumed to be constant even when the wavelength changes,
Using known data representing the relationship between the wavelength of light and the dielectric constant of the metal thin film and the following relational expression, converting the surface plasmon resonance wavelength at each pixel of the image into the dielectric constant or refractive index of the sample. 2. The surface analysis method according to 1.

[0019]

(Equation 4)

3. The magnitude of the shift of the resonance wavelength obtained from the difference between the lower limit value of the measurement wavelength range and the surface plasmon resonance wavelength or the dielectric constant or refractive index of the sample described in 2 above is represented on a two-dimensional visual scale or a three-dimensional graph. 3. The surface analysis method as described in 1 or 2 above, wherein an image representing the properties of the sample surface is obtained by the conversion. 4. 1 to above
3. A method for evaluating the function of an antibody, measuring the concentration of an antigen, evaluating an interaction between DNAs, evaluating an interaction between proteins, or evaluating a DNA-protein interaction, using the surface analysis method described in any of 3 above.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention analyzes a sample prepared on a metal thin film provided on a transparent substrate, irradiates the sample with light from the substrate side through a prism, and measures an image of the reflected light. In surface analysis using two-dimensional surface plasmon resonance, which detects minute changes in sample properties as changes in the intensity of reflected light, 1) For wavelengths of 3 or more at incident angles at which surface plasmon resonance (SPR) does not occur An image of the reflected light is measured by imaging the reflected light from the sample with a two-dimensional photodetector, and an average value of the luminance in the entire image is calculated for each wavelength, and the highest luminance value among the calculated average values is calculated. By normalizing the average value at each wavelength on the basis of the average value, background data for each wavelength is obtained. 2) Reflected light from the sample at two or more wavelengths at an incident angle at which SPR occurs. The image of the reflected light is measured by imaging with a two-dimensional photodetector, the brightness of each pixel of the image measured for each wavelength is corrected with background data, and all are measured until the measurement at the required wavelength is completed. 3) The luminance of each pixel after correction at the wavelength of is stored in the memory of the computer. 3) The luminance at each pixel at different wavelengths: x is approximated by the following quadratic polynomial: y Is determined, that is, a surface plasmon resonance wavelength (hereinafter sometimes simply referred to as “resonance wavelength”).

[0022]

(Equation 5)

As a sample to be subjected to the present invention, a metal thin film is provided on a transparent substrate such as a glass plate or a quartz plate, and a sample to be analyzed is manufactured on the metal thin film. Alternatively, a prism can be used as a transparent substrate. That is,
A metal thin film is provided on the prism surface, and a sample to be analyzed is manufactured on the metal thin film. The method for producing the sample is not particularly limited as long as the sample is fixed on the metal thin film. The thickness of the transparent substrate is not particularly limited, but is usually about 0.2 to 3 mm,
It is preferably about 0.5 to 2 mm.

The kind of the metal of the metal thin film is not particularly limited, and examples thereof include gold and silver. Examples of the metal thin film include a metal thin film deposited on a transparent substrate (including a prism) and a metal thin film manufactured by extending a metal.

Although the thickness of the metal thin film is not particularly limited,
It is usually about 30 to 80 nm, preferably about 40 to 60 nm.

As a two-dimensional photodetector, for example, a CCD
Examples include a camera and an imaging tube. Since the resolution of the image is determined to some extent by the photodetector used, it may be appropriately selected as needed. For example, a CCD camera has about 200 to 1500 vertical pixels and about 250 to 2000 horizontal pixels.

The light source used is not particularly limited, and examples thereof include a halogen lamp and a xenon lamp.

When measuring a sample in a vacuum, for example, a sample and a prism are set in a vacuum container, a pair of transparent windows made of a glass plate, a quartz plate, or the like are provided in the vacuum container, and a vacuum is passed through the window. An example is a method of irradiating a prism and a sample in a container with light, and detecting light reflected from the sample through the prism through another window. Alternatively, a method in which one transparent window is provided in a vacuum container and this is used as a substrate can be exemplified. In this method, a prism is provided so as to be in contact with the outside air side of a window, and a sample is manufactured on the vacuum side of the window.
The sample formed on the vacuum side of the window is irradiated with light through the prism, and the reflected light can be detected.

When a sample in a solution is measured, a plate made of resin or the like is mounted on the sample side of the substrate via a spacer or an O-ring, and a gap formed between the substrate and the plate is filled with an arbitrary solution. By making two through holes in the plate and connecting a tube or the like to the through holes, the measurement can be performed while circulating the solution from the outside.

In the present invention, first, background data for correcting a difference in image brightness caused by wavelength characteristics such as light source brightness at at least three or more different wavelengths and diffraction efficiency of a monochromator is determined. I do.

The background data is obtained by measuring the reflected light image by imaging the reflected light from the sample at at least three or more wavelengths at an incident angle at which surface plasmon resonance (SPR) does not occur with a two-dimensional photodetector. For each wavelength, calculate the average value of the luminance in the entire image, and reference the average value of the largest luminance among the calculated average values (for example, 1.
It is determined for each wavelength by normalizing the average value at each wavelength as 0). Here, the average value of the luminance in the entire image is calculated by summing the luminance in each pixel and dividing the total by the number of pixels. The number of pixels is not particularly limited, and can be appropriately set according to the size of the sample, the required accuracy, and the like.

The incident angle at which SPR does not occur means an incident angle at which SPR does not occur at all wavelengths at which background data is measured. The range of the incident angle at which SPR does not occur depends on the type of the medium to be measured (for example, whether it is performed in air or in a solution), the type of the substrate, and the type of the metal thin film. Can be set. The incident angle at which SPR does not occur is, for example,
Estimation can be performed using computer simulation prior to measurement, and the determination can be made by actually performing measurement and confirming that SPR does not occur. For example, when quartz is used for the substrate in air and silver is used for the metal thin film, the angle of incidence used for background measurement is usually about 44 ° or less, preferably about 40 to 44 °. When glass is used and gold is used for the metal thin film, it is usually about 42 ° or less, preferably about 39 to 42 °.

The angle at which SPR does not occur depends on the type of medium to be measured (for example, in air or in solution), the type of substrate, and the type of metal thin film. If the type of the measurement medium, the type of the substrate, and the type of the metal thin film are the same, it is not necessary to reset the incident angle at which the SPR does not occur in the background measurement every time the measurement is performed.

The wavelength characteristics of the measurement system are determined mainly by the light source and the monochromator, then by the type of the substrate, the type and the thickness of the metal thin film, and hardly depend on the measurement medium. Therefore, when the light source, the monochromator, the type of the substrate, the type of the metal thin film and its thickness, etc. change, it is necessary to measure the background data again, but when using the same combination of measurement systems, Even if the background data is not measured again, the measurement can be repeatedly performed using the background data measured and recorded in the past.

Considering that repeated measurements are made on a plurality of samples using the same background data, it is desirable to measure the background data over as many wavelengths as possible over as wide a range as possible. However,
The wavelength range that can be measured is limited by the light source used, the diffraction grating of the monochromator, and the like. The wavelength range that can be measured is usually about 400 to 1100 nm. The background measurement is usually performed at a wavelength of 5 to 10 nm, preferably 1 to 5 nm. The measured background data is saved as a file on the computer's hard disk, etc., and read into the computer's memory as needed.
It can be used to correct measurement data.

Next, at three or more wavelengths at an incident angle at which SPR occurs, the reflected light from the sample is imaged by a two-dimensional photodetector to measure the image of the reflected light, and the image measured for each wavelength is measured. The luminance of each pixel is corrected with the background data, and the corrected luminance of each pixel at all wavelengths is stored in the memory of the computer until the measurement at the required wavelength is completed.

As described above, the incident angle at which the SPR occurs depends on the type of the measurement medium, the type of the substrate, and the type of the metal thin film. For example, estimation is performed by computer simulation prior to measurement, and the measurement is actually performed to confirm. At this time, it is preferable to set the incident angle so that the surface plasmon resonance wavelength of the portion where the sample is not present on the metal thin film is located near the lower limit of the wavelength range. Normally, the surface plasmon resonance wavelength shifts to the shorter wavelength side as the incident angle increases. The incident angle at which SPR occurs depends on the type of measurement medium, substrate, and metal thin film.Therefore, if the measurement medium, substrate, and metal thin film are the same, the incident angle must be changed each time the sample is changed. There is no need to reset the angle, and subsequent measurements can be made using the same angle of incidence. Furthermore, when the same type of measurement medium, the same type of substrate, and the same type of metal thin film are used, the measurement can be performed at the incident angle used in the past. The incident angle used for measurement is, for example, when using silver for the metal thin film using quartz for the substrate in the air, usually about 46 to 52 °, preferably
When BK7 glass is used for the substrate in the air and gold is used for the metal thin film, the angle is usually about 44 to 50 °, preferably about 45 to 49 °.

The wavelength range that can be measured is mainly determined by the type of the metal thin film. For example, when gold is used, it is usually about 500 nm or more, and when silver is used, it is usually about 450 nm.
That is all. In the actual measurement, if the wavelength range is set to be wide, the range of the property of the sample that can be measured becomes large, but the measurement takes time correspondingly. The number of measurement wavelengths is
It can be appropriately selected according to the wavelength range that can be measured, the required accuracy, and the like. The measurement is usually performed every 5 to 50 nm, preferably every 5 to 20 nm.

Next, the background data at the required wavelength is read from the memory, and the luminance of each pixel of the image measured using the read background data is corrected. The correction is performed by multiplying the luminance of each pixel of the image by the background value at each wavelength. The corrected data is stored in the memory of the computer at least until the measurement at the predetermined wavelength is completed.

Thereafter, the corrected luminance y at each pixel of the image at three or more different wavelengths: x is approximated by the following quadratic polynomial, thereby obtaining the wavelength at which the luminance at each pixel of the image becomes the minimum, that is, Find the surface plasmon resonance wavelength.

[0041]

(Equation 6)

The surface plasmon resonance wavelength at each pixel of the image thus obtained is basic information for expressing the state of the sample surface two-dimensionally. The surface plasmon resonance wavelength always shifts to the longer wavelength side as the thickness or dielectric constant or refractive index of the sample increases.

The surface plasmon resonance wavelength obtained as described above has a good correlation with properties such as the thickness, refractive index and dielectric constant of the sample. Therefore, if necessary, the obtained resonance wavelength can be converted into parameters relating to the properties of the sample. For example, assuming that the thickness of the sample is infinite, the dielectric constant of the transparent substrate is assumed to be constant even if the wavelength changes, and known data representing the relationship between the wavelength of light and the dielectric constant of the metal thin film are used. Using the following relational expression, the resonance plasmon resonance wavelength at each pixel of the image can be converted into a physical property value such as a dielectric constant or a refractive index of the sample. As the permittivity of the transparent substrate, for example, a known value at a certain wavelength can be used.

[0044]

Equation 7

Although there is no relational expression directly linking the surface plasmon resonance wavelength and the film thickness of the sample at present, a plurality of samples whose film thicknesses are known are measured in advance and a calibration curve is created to obtain an unknown sample. The film thickness can be determined.

Further, parameters relating to the properties of the sample, such as the dielectric constant or refractive index of the sample, which can be calculated using the above-mentioned relational expression, or the magnitude of the shift of the resonance wavelength are represented by two-dimensional visual values such as luminance, color, and contour lines. By converting to a scale or a three-dimensional graph, the properties of the sample surface can be visually displayed. Here, the magnitude of the shift of the resonance wavelength is the difference between the lower limit of the wavelength and the resonance wavelength in the measured wavelength range.

The present invention can sufficiently detect a change in the vertical direction of about 0.05 to 0.2 nm, and thus, for example, evaluates the function of an antibody with a slight change in height in the vertical direction due to the binding of an antigen; Measurement of concentration of drugs, drugs, physiological substances, etc .; evaluation of DNA-protein interaction; evaluation of protein-protein interaction;
It can be suitably used for evaluation of protein interaction and the like.

According to the present invention, properties such as film thickness, refractive index, and dielectric constant of an analysis sample can be quantified. For example, the refractive index or permittivity can be determined using a mathematical relationship with the resonance wavelength measured according to the present invention. In addition, the thickness of an unknown sample can be quantified by measuring a plurality of samples whose thicknesses are known in advance and creating a calibration curve.

[0049]

According to the method of the present invention, not only the physical properties such as the refractive index and the dielectric constant can be directly obtained, but also the film thickness can be indirectly measured.

According to the method of the present invention, as compared with the conventional method of simply measuring and displaying the intensity of reflected light, the range of properties that can be measured is wide, and the quantification is significantly improved.

According to the method of the present invention, it is possible to correct a difference in brightness of an image due to the wavelength characteristic of the measurement system, and to determine a large number of reflected light images measured at different wavelengths through determination of a resonance wavelength. Information can be aggregated, so the accuracy of the obtained image is remarkably improved, especially for the vertical measurement, which has the accuracy of quantitatively measuring a small change of about 0.05 to 0.2 nm .

According to the present invention, it is possible to greatly reduce image disturbance due to unevenness of the irradiation light intensity distribution and contamination of the prism. According to the present invention, two-dimensional measurement of properties such as sample thickness, refractive index, and dielectric constant can be performed.
Accurate and quick evaluation of a thin film sample on a metal thin film becomes possible. Further, since the distribution state of these properties can be displayed as an image, visual evaluation becomes possible.

According to the method of the present invention, since the time required for one measurement is short, it is possible to analyze the change over time in the properties of the sample.

[0054]

EXAMPLES The present invention will be described more specifically with reference to examples and comparative examples, but the present invention is not limited to the following examples. Example 1 and Comparative Example 1 FIG. 1 shows an outline of an apparatus used for measuring two-dimensional SPR in Example 1 and Comparative Example 1. FIG. 2 is a schematic diagram showing the vicinity of the sample.

A halogen lamp was used as a light source, and the light from the light source was separated by a monochromator. The monochromatic light obtained from the monochromator was converted into parallel light using a lens and a pinhole, and was applied to a sample to be analyzed through a 60 ° prism at a fixed incident angle. The sample was irradiated with light from the opposite side (the metal thin film side) through a prism. The measuring device was set so that the reflected light from the sample passed through the polarizer (analyzer) and then formed an image on the sensor surface of the CCD camera using the lens and the pinhole.

As shown in FIG. 3, when the angle between the irradiation system and the sample system is adjusted using the biaxial rotary stage so as to be 40 °, the incident angle becomes 4 due to the refraction by the prism.
6.5 °. Both the prism and the substrate were made of quartz. A silver thin film (thickness: 50 nm) was deposited on the quartz substrate.

As a measurement sample, lactoglobulin (LG), which is a kind of protein, was used.
%, 0.01%, 0.05% and 0.1%) aqueous solutions were prepared. A droplet (about 0.26 μl) of each concentration of the aqueous solution was placed on the silver thin film on the substrate, and a spot having a diameter of about 1 mm was formed, dried, and the measurement sample was fixed on the substrate. Concentration 0.002%, 0.01%, 0.05% and 0.1%
The films prepared by drying the LG aqueous solution of
It is believed to have thicknesses of 2.6 nm, 13 nm and 26 nm. The sample thus prepared was brought into close contact with the bottom surface of the prism using water.

First, as Comparative Example 1, the above-mentioned sample was evaluated by a conventional method. FIG. 4 shows the incident angle: 46.5 °, p-polarized light (4
8 shows a CCD camera image of reflected light measured at 80 nm and 520 nm). Comparing the two images, the slight difference in the measured wavelengths reverses the brightness of the images. At 480 nm, SPR occurs only in the silver thin film, and the reflectance of the portion where the protein film exists is high. Also, the difference in brightness between samples with different film thicknesses made using solutions of different concentrations is
rare.

On the other hand, at 520 nm, SPR occurs in the portion where the protein film exists, and the reflectance is low. There is a difference in brightness between films made from aqueous solutions of different concentrations.

As described above, in the conventional method, since the reflectance increases or decreases depending on the measurement wavelength, it is difficult to find a correlation between the brightness or darkness and the film thickness. According to the conventional method, it is possible to display an image of a thin film at a nanometer level, and it is possible to determine whether or not a film exists, but it is not enough to discriminate a difference in film thickness.

Next, as Example 1, the same sample as Comparative Example 1 was evaluated by using the surface analysis method of the present invention (an embodiment in which the dielectric constant of the sample is converted into luminance and displayed).

First, the background was measured. Background measurements are taken at an angle of incidence where SPR does not occur.
The measurement was performed at an interval of 5 nm in a wavelength range of 400 to 700 nm using 4 °. The incident angle at which SPR does not occur was estimated using a computer simulation prior to the measurement, and actual measurement was performed to confirm that SPR did not occur at all measurement wavelengths.

The average value of the luminance of the entire image at each wavelength was calculated by summing the luminance of each of 486 × 640 pixels at each wavelength and dividing by the total number of pixels 311040. The largest value among the calculated average values is 1.0,
The average value of the luminance at each wavelength was normalized.

Next, an image of the reflected light was measured by taking an image of the reflected light from the sample with a CCD camera at an interval of 10 nm in a wavelength range of 450 to 670 nm, using 46.5 ° as the incident angle at which SPR occurs.

The correction was performed by multiplying the luminance of each pixel by the background value of the corresponding wavelength. This operation was performed for each wavelength. The corrected luminance of each pixel at all wavelengths was stored in a computer memory.

The corrected luminance obtained as described above:
By approximating the correlation between y and the measured wavelength: x by the following second-order polynomial, the wavelength (-b / 2a) at which the luminance becomes minimum for each pixel of the image was obtained.

[0067]

(Equation 8)

Assuming that the film thickness of the sample is infinite and the dielectric constant of the transparent substrate: ε _g is a constant value, known data representing the relationship between the wavelength of light and the dielectric constant of the metal thin film (ED Palik, edited by Han
The surface plasmon resonance wavelength at each pixel was converted into the dielectric constant of the sample using the following relational expression (obtained from a dbook of optical constants of solids).

[0069]

[Equation 9]

Next, the obtained dielectric constant of each pixel is set to 25.
The luminance was converted to six gradations (0 to 255). The transformation subtracts 0.96, a value close to the dielectric constant of air (1.0), from the obtained dielectric constant,
The difference is a value close to the maximum value of the permittivity corresponding to each pixel 1.07
Is normalized by dividing by 0.11 the difference of
Performed by multiplying by 5. In this way, the distribution of the dielectric constant when the film thickness of the sample was assumed to be infinite was reconstructed as an image (FIG. 5).

As shown in FIG. 5, the difference in the film thickness depending on the concentration of the solution used for manufacturing the sample is well displayed. By measuring at multiple wavelengths and determining the resonance wavelength at each pixel of the image, the SP with different resonance wavelengths depending on the difference in film thickness
It is thought that the information obtained using R was successfully aggregated. Compared with the conventional method of simply displaying the reflected light intensity at a single wavelength (Comparative Example 1), the information amount is significantly increased,
The quantification was significantly improved. Example 2 and Comparative Example 2 The same apparatus as in Example 1 was used.

Four types of proteins (hemoglobin (HG) and papain (P) were formed on a silver thin film (thickness: 71 nm) provided on a transparent substrate.
A), Ovalbumin (OA) or Albumin (A)) 0.01%
A measurement sample was prepared by placing a droplet of the aqueous solution, adsorbing the protein, drying and washing with water. When produced in this manner, the protein is considered to be a monomolecular film. Assuming that the molecule has a spherical shape, the film thicknesses of HG, PA, OA and A are expected to be 6.0 nm, 4.2 nm, 5.1 nm and 5.9 nm, respectively. The prepared sample was brought into close contact with the bottom surface of the prism using immersion oil for quartz.

First, as Comparative Example 2, the above-mentioned sample was evaluated using the conventional method. Fig. 6 shows the angle of incidence: 46.5 °, p-polarized light
4 shows a CCD camera image of reflected light measured at (480 nm and 510 nm). The result is the same as that of Comparative Example 1 (FIG. 4). In the conventional method, it is possible to display an image of a thin film at the nanometer level, but the reflectivity increases or decreases depending on the measurement wavelength. However, it is difficult to determine whether a thin film is present.

Next, as Example 2, the same sample as Comparative Example 1 was evaluated using the surface analysis method of the present invention (an embodiment in which the dielectric constant of the sample is converted into luminance and displayed). The analysis was performed as in Example 1. The same values as in Example 1 were used for ε _g and ε ′ (λ). However, the measurement was performed at 450 to 580 nm by 10
Performed at nm intervals. FIG. 7 shows the results.

As shown in FIG. 7, the difference in the thickness of the monomolecular film depending on the type of protein is well displayed. However, the thickness of HG is expected to be much larger than the molecular diameter. This is considered to be due to multi-molecule adsorption despite the above-mentioned treatment. Compared with the conventional method of simply displaying the reflected light intensity at a single wavelength, the amount of information has been significantly increased, and the quantitativeness has been significantly improved. Example 3 In Example 3, an attempt was made to measure a sample in water. Except for the vicinity of the sample, the same measurement device as in Example 1 was used. However, the angle of incidence during measurement is 7
3.6 °, incident angle at background measurement is 42 °
And

FIG. 8 is a schematic view showing the vicinity of the sample used in the third embodiment. A 6 mm thick acrylic resin plate was fixed to the sample side of the substrate via a 1 mm thick silicone resin spacer.
Two through holes were provided in the resin plate, and water was circulated from outside. Both the prism and the substrate were made of BK7 glass, and a gold thin film (thickness: 50 nm) was further deposited on the substrate.

The measurement sample was prepared as follows.
First, a gold thin film on a substrate is treated with mercaptoundecenoic acid, and then a polymer, polyallylamine (PAA)
Was placed on the treated gold thin film, dried and washed with water. The prepared sample was brought into close contact with the bottom surface of the prism using immersion oil for BK7 glass.

The measurement was performed in the same manner as in Example 1. Measurements were taken at 600 nm to 670 nm at 10 nm intervals.
FIG. 9 shows an image showing the distribution of the magnitude of the shift of the resonance wavelength.

It is known that PAA is naturally adsorbed on a negatively charged solid in an aqueous solution with a thickness of about 0.5 nm. According to the analysis method of the present invention, it can be seen that the film thickness is considerably large especially at the outer peripheral portion of the film. Thus, it was demonstrated that measurement was possible even in water, and an image corresponding to the film thickness of the sample was obtained.

[Brief description of the drawings]

FIG. 1 is a diagram showing an outline of an apparatus used for two-dimensional SPR measurement in Example 1 and the like.

FIG. 2 is a schematic diagram showing the vicinity of a sample in the measuring device shown in FIG.

FIG. 3 is a schematic view of the vicinity of a sample of the measuring device according to the first embodiment.

FIG. 4 is an image obtained by measuring a protein film using a conventional method in Comparative Example 1.

FIG. 5 shows the result of measurement of a protein membrane using the measurement method of the present invention in Example 1.

FIG. 6 is an image obtained by measuring a protein monolayer by a conventional method in Comparative Example 2.

FIG. 7 is an image obtained by measuring a protein monolayer by the method according to the present invention in Example 2.

FIG. 8 is a schematic view of the vicinity of a sample of a measuring device according to a third embodiment.

FIG. 9 shows the result of measurement of a polymer film using the measurement method of the present invention in Example 3.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G059 AA05 BB12 CC16 DD13 EE02 EE05 FF01 GG04 GG10 HH01 HH02 HH06 JJ05 JJ11 JJ12 JJ19 KK04 MM03 MM05 MM10 MM12 PP04

Claims (4)

[Claims] An object to be analyzed is a sample prepared on a metal thin film provided on a transparent substrate. The sample is irradiated with light from the substrate side through a prism, an image of the reflected light is measured, and the properties of the sample are measured. Surface analysis using two-dimensional surface plasmon resonance, which detects a significant change as a change in reflected light intensity. 1) Reflected light from a sample at three or more wavelengths at an incident angle at which surface plasmon resonance (SPR) does not occur. The image of the reflected light is measured by imaging with a two-dimensional photodetector, the average value of the luminance in the entire image is calculated for each wavelength, and the average value of the largest luminance among the calculated average values is used as a reference. By normalizing the average value at the wavelength, background data for each wavelength is obtained. 2) For three or more wavelengths at the incident angle at which SPR occurs, the reflected light from the sample is taken with a two-dimensional photodetector. The image of the reflected light is measured by imaging, and the luminance of each pixel of the image measured for each wavelength is corrected with the background data, and after the correction at all wavelengths is completed until the measurement at the required wavelength is completed. The luminance of each pixel is stored in a computer memory. 3) The corrected luminance: y of each pixel at a different wavelength: x is approximated by the following second-order polynomial to obtain a wavelength ( (Surface plasmon resonance wavelength)
A surface analysis method characterized by determining (Equation 1) 2. Description of the Related Art Assuming that the thickness of a sample is infinite, the dielectric constant of a transparent substrate is assumed to be constant even when the wavelength changes, and the relationship between the wavelength of light and the dielectric constant of a metal thin film is known. The surface analysis method according to claim 1, wherein the surface plasmon resonance wavelength at each pixel of the image is converted into a dielectric constant or a refractive index of the sample using the following data and the following relational expression. (Equation 2) 3. A two-dimensional visual measure of the magnitude of the shift of the resonance wavelength obtained from the difference between the lower limit of the measurement wavelength range and the surface plasmon resonance wavelength or the dielectric constant or refractive index of the sample according to claim 2. 3. The surface analysis method according to claim 1, wherein an image representing properties of the sample surface is obtained by converting the data into a three-dimensional graph. 4. Use of the surface analysis method according to claim 1 to evaluate the function of an antibody, measure the concentration of an antigen,
Of protein-protein interactions, protein-protein interactions or
A method for evaluating DNA-protein interaction.