JP3209729B2 - Polarization measurement method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a polarization measurement method which enables a change in film thickness due to, for example, an antibody and an antigen on an immune support to be measured by ellipsometry.

[0002]

2. Description of the Related Art In the ellipsometry, the change in the state of polarization when light is reflected on the surface of an object is observed to determine the optical constant of the object itself, or the thickness and optical constant of a thin film attached to the surface. Is the way. Recently, this ellipsometry has been applied to the field of biophysics, such as measurement of changes in sample surface thickness based on biological binding reactions such as antigen-antibody reactions and complementary nucleic acid binding reactions. It has also been applied to the measurement of adsorption films, the study of plasma coagulation, and the like.

[0003] Techniques for measuring the thickness of a protein used in the antigen-antibody reaction include, for example, A. Rothen and C. Mat.
hot.Helvetica Chimica Aacta, Vol.54 (1971) Immunological Reactios Carried out at a Liquid-Sol
The technology disclosed in id Interface is known. The technology related to the measurement of the above protein adsorption film is UL
F JOENSSON, M.MALMQVIST, INGER ROENNBERG. Journal of
Colloid and Interface Science.Vol.103, No.2,1985
Adsorption of Immunoglobulin G, Protein A, and Fibr
onectin in the Submonolayer Region Evaluated by a
CombinedStudy of Ellipsometry and Radiotracer Tech
niqs and A.Rothen, C.Mathot.Surface Chemistry
of BiologicalSystems.1970 IMMUNOLOGICAL REACTIONS
CARRIED OUT AT A LIQUID-SOLID INTERFACE WITH THE H
The technology disclosed in ELP OF A WEAK ELECTRICCURRENT is known.

[0004] In addition, techniques relating to plasma coagulation include:
L.VROMAN AND ALADAMS. SURFACESCIENCE 16 (1969) FI
NDINGS WITH THE RECORDING ELLIPSOMETER SUGGESTING
RAPID EXCHANGE OF SPECIFIC PLASMA PROTEINS AT LIQU
The technology disclosed in ID / SOLID INTERFACES is known.

[0005] In ellipsometry, the refractive index

[0006]

(Equation 1)

On a substrate surface having a thickness d and a refractive index

[0008]

(Equation 2)

When there is an isotropic homogeneous thin film (see FIG. 9), when linearly polarized light is incident on the thin film at an incident angle φ, the amplitude reflectances of the P and S polarized components are respectively

[0010]

(Equation 3)

Is given by Where r _1P , r _2P , r _1s ,
r _2S is P, S in vacuum-film and film-substrate, respectively.
In the amplitude reflectance of the component, δ is a phase difference generated in the film.
This δ is

[0012]

(Equation 4)

[0013] Here, φ _f is the refraction angle in the film,

[0014]

(Equation 5)

The following holds.

At this time,

[0017]

(Equation 6)

Tanφ (amplitude reflectance ratio) and iΔ (phase difference) are measured by ellipsometry.

There are various methods for performing ellipsometry conventionally, and for example, an ellipsometer shown in FIG. 10 is used. This device uses Ki using Faraday cell.
ng of the photoelectric polarization analyzer, showing the arrangement of the measurement method with the fixed incident angle. This device allows the polarizer and analyzer to be rotated with a precision equivalent to ± 0.002 ° by a stepping motor or the like, and furthermore, modulates the polarization state by the Faraday effect and makes it easy to find the extinction position. ing. As described above, by obtaining the extinction condition using the ellipsometer shown in FIG. 10, it is possible to know the thickness and the optical constant of the thin film attached to the sample surface.

[0020]

However, in the conventional measuring method, as shown in FIG. 10, a Faraday cell must be provided, or a mechanism for accurately rotating both the polarizer and the analyzer must be provided. The disadvantage is that it is inferior in convenience, for example. When an optical system provided in a conventional apparatus is to be used for an immunoassay apparatus described in Japanese Patent Application No. 3-080124, which is a prior application, the complexity of the apparatus and the size of the optical system are increased. I will. As a result,
An optical system used in a conventional automatic analyzer that performs a plurality of different measurements greatly hinders the automation of the analyzer.

An object of the present invention is to provide a polarization measurement method which can easily carry out a plurality of different measurements in a small space when measuring a change in membrane pressure due to an antibody and an antigen on an immune support by an ellipsometry. It is an object.

[0022]

In order to solve the above-mentioned problems, a polarization measurement method according to one aspect of the present invention includes a step of movably disposing first and second sample surfaces required for measurement, A step of entering and reflecting light for measurement in a state where the first sample surface is positioned at the measurement position, and the step of: reflecting the light reflected from the first sample surface while maintaining the polarization direction at the time of reflection. Making the light incident and reflected on a reference surface for comparison positioned at a position different from the measurement position of the first sample surface, and a second sample surface for measurement different from the first sample surface. Positioning the light at the measurement position in the same plane as the first sample surface, and maintaining the polarization direction at the time of reflection of the light reflected at the second sample surface with the second sample surface. Is a reference for comparison placed at different locations Relative scan plane, a step of incident and reflected, and light reflected by the said reference surface for said first light and said second specimen surface reflected the record full Aren <br/> scan plane for the sample surface Is detected.

It is preferable that the optical path incident on and reflected from the reference surface is oriented so that the optical path incident on and reflected from the first and second sample surfaces returns in parallel. Further, the first and second
It is preferable that the sample surfaces have different concentrations of substances for binding reaction reacted with each other.

[0024]

[0025]

Embodiments of the present invention will be described below. The polarization analyzer suitable for performing the polarization measurement method of the present invention has a feature in an optical system, and thus the optical system will be described in detail.

The above-mentioned ellipsometer has an optical system as shown in FIG. This optical system includes a polarizing beam splitter 1 also serving as a polarizer and an analyzer, a 波長 wavelength plate 5, and a non-polarizing prism 7. Reference numeral 3 denotes a sample support (sample portion) suitable for the optical system, and the sample portion is movably supported on a stage (not shown).

When the light emitted from a light source (not shown) and incident on the polarization beam splitter 1 is unpolarized, the light source is fixed and only the polarization beam splitter 1 rotates, and when the light is polarized, the light source and the polarization beam splitter 1 are rotated. 1 is adapted to rotate integrally. The quarter-wave plate 5 has p
Alternatively, it is fixed on the front surface of the non-polarizing prism 7 so that the azimuth becomes 45 ° with respect to the s direction.

FIG. 2 shows a configuration of the non-polarizing prism 7 and a light path in the non-polarizing prism 7. As shown in FIG. 2, the non-polarizing prism 7 has a shape such that light incident from point A is reflected four times in the prism. In this case, the p-polarized light incident from the point A is reflected on the reflection surface a to become s-polarized light, the polarization components are kept as they are on the next reflection surfaces b and c, and become p-polarized light on the next reflection surface d. From the point B. That is, the polarization state is p-s along the path of the reflecting surface abcd.
−sp. Similarly, the s-polarized light incident from point A is
In the path of the reflecting surface abcd, the path becomes spps in order, and the phase difference between the two is preserved. As described above, the non-polarizing prism 7 is configured to emit the light incident at the point A from the position B different from the point A without changing the polarization characteristics.

Next, the entire light path of the optical system will be described.

The linearly polarized light that has passed through the polarizing beam splitter 1 is reflected by the sample unit 3 and becomes elliptically polarized light.
The light enters the four-wavelength plate 5. In this case, the polarization angle of the linearly polarized light passing through the polarization beam splitter 1 is φ, and the light reflected by the sample unit 3 is elliptically polarized light having a phase difference δ. FIG. 3 shows the relationship between the polarization angle and the phase difference at this time for the electric field vector component.

Here, the change of the polarization angle at each point is illustrated. When the reflected light from the sample unit 3 is incident on the quarter-wave plate 5 at an angle of 45 ° (the angle formed by the main section of the quarter-wave plate and the vibration plane of the electric field vector), the polarization angle becomes as shown in FIG. As shown by φ, it becomes φ + π / 4. At this time, the phase changes to δ + π / 2. Next, the light passes through the non-polarizing prism 7 and again becomes 1 /
The light that has passed through the four-wavelength plate 5 becomes elliptically polarized light as shown in FIG. At this time, the polarization angle is φ + π / 2, and the phase changes to δ + π. When this elliptically polarized light is reflected again by the sample section 3, it is converted into linearly polarized light. At this time, the polarization angle changes to π / 2−φ, and the phase difference changes to π.

FIG. 5 shows a light path from the light emitted from the light source to passing through the polarizing beam splitter 1 and returning to the polarizing beam splitter 1 again via the sample section 3 and the non-polarizing prism 7. This is easily shown. Light passing through the polarizing beam splitter 1 is applied to one side 3 of the sample section 3.
The light is reflected by a and enters the point A of the non-polarizing prism 7. A
The light incident at the point is repeatedly reflected four times in the prism as described above, is emitted from the point B, is reflected by the other side surface 3b of the sample unit 3, and is incident on the polarization beam splitter 1 again. The sample section 3 is arranged such that light from the polarizing beam splitter 1 side and light from the non-polarizing prism 7 side are incident on one side 3a and the other side 3b of the sample section 3, respectively. The one side surface 3a and the other side surface 3b are symmetric about the central axis M of the sample section 3. The output light from the polarization beam splitter 1 is detected by the photoelectric detector 10 or
Alternatively, a change in the amount of light can be visually observed.

As described above, the reflected light from the sample section 3
By making the light incident on the 波長 wavelength plate 5 at an angle of 45 °,
The extinction condition can be obtained in the polarization beam splitter 1. Here, the reflected light from the sample part 3 is incident on the quarter-wave plate 5 at an angle of 45 ° (the angle formed by the main section of the quarter-wave plate and the vibration plane of the electric field vector) so as to prevent the reflected light.
It is assumed that the wave plate 5 has been arranged. Then, FIG.
The changes in the polarization angles shown in FIGS.
(A) and (B). That is, the light that has passed through the quarter-wave plate 5 again has a polarization angle like K in FIG. 6B, and thus cannot be extinguished in the polarization beam splitter 1.

Next, the structure of the immune support suitable for the above-described optical system and the method for measuring the same will be described.

A silicon oxide film having a thickness of 1000 to 1900 angstroms (hereinafter referred to as A) is formed on a silicon single crystal (having 100 planes).
Sample 3 was prepared by silane treatment and immobilization of the antibody by the method presented in No. 4. FIG. 7 shows the structure of the sample section 3. 7, reference numeral 20 denotes a Si single crystal substrate, reference numeral 21 denotes an SiO ₂ film, reference numeral 22 denotes a silane coupling agent, and reference numeral 23 denotes an antibody.

FIG. 8A is a plan view of the sample section 3. The sample section 3 is arranged as shown in FIG. One side surface 3a of the sample portion is a surface 25 on which only the antibody is immobilized as a reference, and the other side surface 3b is divided into three regions 26 to 28. Reference numeral 26 indicates a portion where the same concentration of the antigen is reacted with the same antibody as the surface 25, and reference numerals 27 and 28 indicate portions where the actual sample was sensitized.

The method of immunoassay using the above-mentioned sample section is described below. (A). The sample section 3 shown in FIG. 8A is arranged as shown in FIG. (B). To the portions indicated by reference numerals 26, 27, and 28 in the sample section 3, the required amounts of the control solution and the specimen having the lowest concentration are respectively dropped. The reaction time is set to the optimal time depending on the measurement item. (C). After the completion of the reaction, the portions indicated by reference numerals 26, 27 and 28 are washed with distilled water, and the surface moisture is blown off with N ₂ gas or the like. (D). When the light source to be used is non-polarized light, the polarization beam splitter 1 is rotated, and the polarization angle of extinction is obtained by the photoelectric detector 10. At this time, it should be noted that the sample unit 3 is positioned so that only the portions 25 and 26 of the sample unit are irradiated with the light from the polarizing beam splitter 1 and the light from the non-polarizing prism 7. That is. (E). The operation performed in (d) corresponds to zero adjustment. (F). Next, the position of the sample unit 3 is shifted so that only the portions 25 and 27 of the sample unit 3 are irradiated with the light from the polarizing beam splitter 1 and the light from the non-polarizing prism 7, and the output of the photoelectric detector 10 is changed. Read. The same operation is performed on the portions 25 and 28 of the sample section 3. (G). From the calibration curve prepared in advance, the concentration of the sample dropped on 27 and 28 of the sample section 3 can be detected.

The structure of the immune support is shown in FIG.
It is also possible to configure as shown in FIG. Reference numeral 40 denotes a portion for sensitizing the specimen, and reference numeral 31 denotes a surface on which only the antibody is immobilized. One side 3a of the sample portion is divided into five regions 32 to 36 except for the surface 31 on which only the antibody is immobilized. The number of divided regions is arbitrary, and is five in this embodiment. Each of the regions 32 to 36 is a site where the same antibody is immobilized and then sensitized with five kinds of control solutions having known and different antigen concentrations.

The measurement of the antigen concentration in the portion 40 where the sample has been sensitized is performed by the following procedure. First, zero adjustment is performed using the surface 31 on which only the antibody on one side 3a of the sample section 3 is immobilized and the surface 31 on which only the antibody on the other side 3b is immobilized. Next, while shifting the sample portion 3 in the M-axis direction, the region 32 and the portion 40 where the sample was sensitized,
, And the portion 40 where the sample was sensitized, and subsequently the region 34 and the portion 40 where the sample was sensitized.

Since the antigen concentration of each of the regions 32 to 36 is known, the regions 32 to 36 serve as a calibration curve. For example, if the output becomes zero when photometry is performed using the region 34, the antigen concentration in the portion 40 where the sample is sensitized is equal to the antigen concentration in the region 34. The use of such an immune support structure eliminates the need for a calibration curve. Of course, one side 3a of the sample part
Is further divided into a larger number of regions, it is possible to detect a more accurate density.

[0041]

As described above, in the polarization measurement method of the present invention, the second sample surface is positioned at the same measurement position in the same plane as the first sample surface, Since the measurement optical path for each reference of the second sample surface is incident and reflected at a position near the measurement position of the sample surface, a plurality of different polarization measurements can be easily performed in a small space. In particular, it is preferable that the optical system can be arranged in a small space by directing the incident light path and the reflected light path to the reference surface so that the reflected light path returns so that the incident light path and the reflected light path to the sample surface become parallel. In addition, since the first and second sample surfaces are caused to react with substances for binding reaction having different concentrations from each other, there is an advantage that calibration curve data can be obtained.

[Brief description of the drawings]

FIG. 1 is a diagram showing an outline of an optical system in a polarization analyzer used for a polarization measurement method of the present invention.

FIG. 2 is a diagram showing a detailed configuration of a non-polarizing prism in the optical system of FIG.

FIG. 3 is a diagram illustrating a relationship between a polarization angle and a phase difference of light reflected by a sample unit with respect to an electric field vector component in the optical system of FIG. 1;

FIG. 4 includes (A) and (B), and in the optical system of FIG. 1, the quarter-wave plate has an angle of 45 ° (a main cross section of the quarter-wave plate and a vibration plane of an electric field vector). (Angle), the polarization angle when the reflected light from the sample part is incident, and the polarization angle of the light that has passed through the non-polarizing prism and passed through the quarter-wave plate again.

FIG. 5 is a diagram schematically showing a light path in the optical system of FIG. 1;

FIGS. 6A and 6B respectively correspond to FIG. 4 and show an angle of 45 ° to a quarter-wave plate in the optical system of FIG. 1;
FIG. 9 is a diagram showing a state in which reflected light from a sample unit does not enter in °.

FIG. 7 is a sectional view of a sample support used in the optical system of FIG. 1;

8A and 8B are plan views showing a configuration of a sample support suitable for the optical system of FIG. 1, wherein FIG. 8A is a diagram showing a first embodiment, and FIG. 8B is a diagram showing a second embodiment.

FIG. 9: When there is an isotropic homogeneous thin film on a substrate,
It is a figure which shows the path | route of the light when linearly polarized light enters this.

FIG. 10 is a diagram showing a configuration of a conventional polarization analyzer.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Polarization beam splitter, 3 ... Sample part, 5 ... 1/4 wavelength plate, 7 ... Non-polarization prism, 10 ... Photoelectric detector.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-5-203564 (JP, A) JP-A-5-203565 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01B 11/02 G01N 21/00-21/01 G01N 21/17-21/61 G01N 33/543

Claims (3)

(57) [Claims] 1. A step of movably arranging first and second sample surfaces required for measurement, and a step of entering and reflecting light for measurement while the first sample surface is positioned at a measurement position. The light reflected from the first sample surface is reflected while maintaining the polarization direction at the time of reflection, with respect to a reference surface for comparison positioned at a position different from the measurement position on the first sample surface. Incident and reflecting; positioning a second sample surface for measurement different from the first sample surface at the measurement position in the same plane as the first sample surface; A step of causing the light reflected from the sample surface to enter and reflect on a reference surface for comparison arranged at a different position near the second sample surface while maintaining the polarization direction at the time of the reflection, the LES off for the first sample surface Polarization measurement method is characterized in that so as to detect the light reflected by the said reference surface for the light reflected by the Reference face the second sample surface. 2. The optical system according to claim 1, wherein an optical path incident on and reflected from the reference surface is directed back to be parallel to an optical path incident on and reflected from the reference surface. The polarization measurement method according to claim 1. 3. The polarization measurement method according to claim 1, wherein the first and second sample surfaces are obtained by reacting different concentrations of substances for a binding reaction with each other.