JP2008122405A - Method of reaction analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve the problem wherein the dependence of the degree of change in the light quantity on the film thickness of a light-transmitting part of a sample support, its refractive index, incident angle of incident light, wavelengths, and light quantity, when a trace amount of small optical changes on the sample support are to be captured as the change in the light quantity has not been disclosed at all in the conventional reaction analysis apparatuses that utilize polarization. <P>SOLUTION: In a reaction analysis method, the light reflected from a first optical element for making polarized light fall incident on a part of a biological sample, and from a second optical element for reflecting light by rotating the plane of polarization of p-polarized light and s-polarized light of reflected light reflected at the part of the biological sample by nπ+π/2 (n: is an integer of 0, 1, ...) is made incident on a second sample and is quenched by an analyzer, and the biological sample is analyzed. The sample support has a light non-transmitting layer; a light-transmitting layer; and a reaction layer on the biological sample, and the layer thickness of the reaction layer on the biological sample changes, prior to and after reaction. Reflectivity of the s-polarized light at an angle of incidence to the sample support is 0.25 or smaller. By detecting the changes in the light quantity, by reversing of the positive and negative phase differences of the s-polarized light and the p-polarized light in the light-transmitting layer, the reaction results are acquired. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a reaction analysis method for detecting a change in film thickness of a directly facing sample in a reaction layer formed on a sample support as a change in light quantity.

  In general, the reaction analysis method using polarized light observes the change in the polarization state when light is reflected on the surface of the object, and knows the optical constant of the object itself, or the thickness and optical constant of the thin film attached to the surface. It is. This ellipsometry has been applied to the biophysics field, and is also applied to the measurement of the thickness of protein membranes used for antigen-antibody reactions, the measurement of protein adsorption membranes, and the measurement of plasma proteins at the solid-liquid interface. It has become so.

  For example, Non-Patent Document 1 discloses a technique for measuring the thickness of a protein used in the antigen-antibody reaction. Non-patent document 2 and non-patent document 3 disclose the technique relating to the measurement of the protein adsorption film. In addition, as a technique relating to measurement of plasma protein at the solid-liquid interface, a technique disclosed in Non-Patent Document 4 is known.

Here, the ellipsometry will be described. When a p-1 layer multilayer film is attached on a substrate having a refractive index m ₀ , the amplitude reflectance of reflected light when light is incident from a medium having a refractive index m _P thereon is expressed as amplitude reflectance = r _{P , 0} , when the thickness of the medium of m _P is d _P and a medium of m _{P + 1} is further superimposed thereon and light is incident from above, the reflected light of amplitude reflectance = r _{P + 1,0} is obtained. if,

It is represented by Here, the refractive index m _P is represented by a complex refractive index,

ρ _{P + 1, P} is the amplitude reflectivity at the interface between the p + 1 layer and the p layer, and r _{P, 0} is

Therefore, the final reflectance can be obtained by sequentially substituting values for the reflectance of the multilayer film in a hierarchical manner. The reflectance of the S-polarized light component and the P-polarized light component can be obtained by applying the amplitude reflectance of the S-polarized light and the amplitude reflectance of the P-polarized light to the terms ρ _{P + 1 and P} , respectively. For example,

S in the ellipsometry device is reflected by the sample, the polarization reflectance ratio of the P component is measured, the amplitude reflectance of the S polarized light A _SS, if the amplitude reflectance of the _P polarized light and A _SP,

The refractive index and the film thickness are obtained by applying tanφ (amplitude reflectance ratio) and Δ (phase difference) to the above formula.

  In addition, there are various methods for performing ellipsometry, but for example, the ellipsometer shown in FIG. 15 is used. This device is King's photoelectric ellipsometer using a Faraday cell, and shows the arrangement state of a measurement method with a fixed incident angle. In this device, the polarizer and the analyzer can be rotated with an accuracy equivalent to ± 0.002 ° by a stepping motor or the like, and the polarization state is modulated by the Faraday effect to easily find the extinction position. Yes.

  Thus, by obtaining the extinction conditions using the ellipsometer shown in FIG. 15, the thickness and optical constant of the thin film adhering to the sample surface can be known. However, such an apparatus has a drawback in that it is required to be equipped with a Faraday cell, or it is necessary to provide a mechanism for rotating both the polarizer and the analyzer with high accuracy. In addition, it has been difficult to accurately determine a minute optical change in the uppermost film in a multilayer structure.

  On the other hand, when the ellipsometer is used as an analyzer such as an antigen-antibody reaction, it is sufficient that the reactivity of the antigen-antibody reaction can be detected with higher sensitivity than measuring the refractive index and the film thickness. For this purpose, place the sample support before the reaction on the ellipsometer to quench the light, and then place the sample support after the reaction while fixing the position of the polarizer, analyzer, etc. And antigen-antibody reaction can be detected.

In applying this method to measure the change in film thickness due to the antibody on the immune support and the antigen by ellipsometry, the applicant of the present application has developed a ellipsometer with a small and simple optical system and the optical The structure of the sample support suitable for the system was proposed in Patent Document 1 and Patent Document 2.
JP-A-5-203564 JP-A-5-203565 A. Rothen and C. Mathot. Helvetica Chimica Acta, Vol. 54 (1971), Immunological Reactions Carried out at a Liquid-solid Interface ULF Joensson, M. Malmqvist, Inger Roenberg, Journal of Colloid and Interface Science, Vol. 103, No. 2 (1985), Adsorption of Immunoglobulin G, Protein A and Fibronectin in the Submonolayer Regions Evaluated by a Combined Study of Ellipsometry and Radiotracer Techniqs A. Rothen and C. Mathot. Surface Chemistry of Biologicalsystems (1970), IMMUNOLOGICAL REACTIONS CARRIED OUTAT A LIQIUID-SOLID INTERFACE WITHTHE HELP OF A WEAK ELECTRIC CURRENT L.Vroman and ALAdams, SURFACESCIENCE 16 (1969) FINDINGS WITH THERECORDING ELLIPSOMETER SUGGESTINGRAPID EXCHANGE OF SPECIFIC PLASMAPROTEINS AT LIQUID / SOLID INTERFACES

  However, in Patent Document 1 and Patent Document 2 described above, when a minute optical change on the sample support is regarded as a change in the amount of light using the proposed device or a conventional ellipsometer, the degree of the change in the amount of light. However, although it depends on the film thickness, refractive index, incident angle of incident light, wavelength, and light quantity of the light-transmitting portion of the sample support, nothing is described about them.

  Therefore, the present invention has an object to provide a reaction analysis method using polarized light that detects a change in light amount as a reaction result of a biological sample due to a minute change in film thickness on a multilayer structure formed by the biological sample. .

  In order to achieve the above object, the present invention provides a first optical element that causes polarized light to be incident on a part of a biological sample, and polarization planes of P-polarized light and S-polarized light reflected by the part of the biological sample. Is rotated by nπ + π / 2 (n: 0, 1, 2,... Integer) and reflected light from the second sample is incident on the second sample and quenched by the analyzer. A reaction analysis method for analyzing a biological sample, comprising a light-impermeable layer, a light-transmitting layer, and a reaction layer related to the biological sample, and a layer thickness of the reaction layer related to the biological sample before and after the reaction is Using a changing sample support, the change in the amount of light is detected on the basis of the phase difference between the S and P polarizations in the light-transmitting layer when the S-polarized reflectance at the incident angle to the sample support is 0.25 or less. Analysis of biological sample to obtain the reaction result Provide a method.

  ADVANTAGE OF THE INVENTION According to this invention, the reaction analysis method which detects the change of the light quantity by the change of the minute film thickness on the multilayer film structure formed with the biological sample as a reaction result of a biological sample can be provided.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
First, the elliptically polarized light incident on the sample

Expressed in, after being reflected by the reference sample, the amplitude A _f of S components by quenching _operation, it is assumed that the amplitude of the P component is linearly polarized light of A _f tan [phi _r, the amplitude reflectance of the two components A _rs, A _rp , if the phase difference and Δ _r,

Therefore,

Then, the amplitude reflectance of the second measurement sample A _{SS, A SP,} elliptically polarized light after reflected on the measurement sample and the phase difference and delta _S is as follows.

Therefore, the reflected light amount _{I S} to be detected,

It becomes.

a. Assuming an optimum device setting, A _f ² sin ² φ _r in equation (1) is an amount related to the device setting.

From A _ps ² + A _op ² = 1, A ₀ ² is eliminated, and the optimum condition is obtained by differentiation.

Substituting into equation (1) and introducing the value into equations (4), (5), and (8), FIGS. 3, 4, and 5 were obtained. The conditions used when determining the reflectance of the multilayer film are shown below. Therefore,

Incident wavelength 6700Å SiO ₂ film thickness 4100Å at an incident angle of 70 ° in any case, SiO ₂ film thickness 4000Å at an incident angle of 65 °, SiO ₂ film thickness 3900Å at an incident angle of 60 °, SiO ₂ film thickness 3700Å at an incident angle of 50 ° It can be seen that the maximum amount of reflected light is given in the vicinity. In addition, FIG. 6, FIG. 7, FIG. 8, and FIG. 9 show the S-polarized light reflectance, the P-polarized light reflectance, the polarized light reflectance ratio, and the phase difference in the vicinity of these film thicknesses. From these results, it can be seen that the P-polarized reflectance hardly changes, but the S-polarized reflectance becomes the smallest at the SiO ₂ film thickness that gives the maximum amount of reflected light.

  Therefore, at that time, the polarization reflectance ratio | Ap / As | shows the maximum value. On the other hand, it can be seen that the phase difference changes greatly in the vicinity of the film thickness. That is, it can be seen that the maximum amount of reflected light can be obtained by setting the sample and the apparatus so that the S-polarized reflectance is the smallest, the polarized reflectance ratio is the largest, and the phase difference changes greatly. Further, it can be seen that when the optimum apparatus setting is performed, the reflected light amount is about twice as much as that obtained when the λ / 4 plate is set to 45 °. Both the analytical method and the results calculated using the Poincare sphere yield the same result.

Theoretically, the minimum thickness of the S-polarized reflectance is optimum, but when the SiO ₂ film is actually formed on the Si wafer, the thickness is 5 to 10% of the target value. Have variations. Therefore, as shown in FIG. 16, for example, a 4000 mm film is formed, and the sensor chip is manufactured so that the reflectance is minimized. There was no. Accordingly, the S-polarized reflectance is theoretically the minimum, but in practice, the S-polarized reflectance is preferably 0.25 or less.

Next, an experiment for confirming the calculation result in the first embodiment was performed. In the experiment, an LB film capable of easily forming a reproducible thin film having an arbitrary film thickness was used as a simulation sample. As shown in FIG. 10, the LB film is sequentially laminated stepwise on the SiO ₂ film having a predetermined thickness, and after quenching only at the SiO ₂ film, the amount of reflected light at the position where the LB films having various thicknesses are accumulated. Was measured and compared with the calculation results. Since the experimental apparatus used was a λ / 4 plate set at a position of 45 °, the formula (5) or (8) can be used as the calculation formula, but this time formula (5) was used.
a. In order to make the calculation calculation result for the confirmation experiment correspond to the output of the apparatus, the output of the light source and the photoelectric conversion efficiency of the light receiving element were added as follows.

This equation is the light amount when assuming the device setting that gives the maximum light amount.
b. When the λ / 4 plate is set to 45 degrees, this setting is most often applied to a conventional reaction analyzer using polarized light (hereinafter referred to as a polarization analyzer).

Therefore, if this is substituted into equation (2),

Therefore,

c. Analysis using the Poincare sphere Referring to FIG. 1, the state of polarization is described in sequence. (1) The light polarized by P (polarizer) is installed at an angle of 45 ° with respect to the incident surface. After passing through the four plates, the light is located on a great circle containing 45 ° linearly polarized light (L ₀ ).

(2) When a reference sample is set and extinction adjustment is performed, the light after reflection of the reference sample becomes linearly polarized light (R). The amplitude reflectances of both components at this time are A _rs and A _rp, and the phase difference is Δ _r .

At this time, the analyzer is located at the opposite point A of R.
(3) The reflected light when the measurement sample is measured with the arrangement fixed is S, the amplitude reflectances of both components are A _ss , A _sp, and the phase difference is Δ _s .

At this time, the relative light quantity I observed at the position A is c, where the length of the arc RS is c.

Paying attention to the triangular DSR on the spherical surface,

Cosine theorem of spherical triangle,

Than,

Therefore,

When the incident light quantity is 1, the total reflected light quantity I ₀ is

Accordingly, the amount of reflected light observed at the position of the analyzer A is expressed by the following equation.

Also, equations (5) and (8) should be equivalent. Here, as a reference sample and a measurement sample, a multilayer structure including a silicon oxide film (SiO ₂ ) 2, a silane film 3, and an antigen-antibody reaction layer 4 on a semiconductor substrate 1 as shown in FIG. 2 was assumed. The amount of reflected light was calculated by using the above-mentioned equation, using SiO ₂ having a certain thickness as a reference sample, and using a sample whose thickness increased by 200 mm as a measurement sample. This was sequentially shifted by 200 mm to obtain a change in the amount of reflected light with respect to the thickness of SiO ₂ as a reference sample and a measurement sample.

The polarization reflectance and the phase difference used for the calculation were obtained from the reflectance formula of the multilayer film described in the prior art. The output (P) is reduced to ½ after I ₀ (LD output) passes through the polarizing prism, and becomes incident light on the sample. The reflected light (I _S ) is an electric signal based on the photoelectric conversion efficiency (S) of the light receiving element. It is converted into that is amplified by the amplifier (amplification factor 10 ⁶ times). Therefore,

The calculation conditions are shown below.

The results are shown in FIG.
b. Four types of SiO ₂ film thickness Si wafers shown in the preparation and measurement of the samples were cut into a size of 10 × 76 mm, and LB films were accumulated in steps of 10 mm intervals. The LB film material and cumulative conditions are shown in the table below.

The produced sample was set in the apparatus as described above, and the amount of reflected light was measured while automatically moving by 10 mm. In the measurement, first, the polarizer (P) and the analyzer (A) are rotated little by little in the portion of the SiO ₂ film where the LB film is not accumulated to obtain the extinction position. Next, while the positions of P and A were fixed, the portion where the LB film was accumulated was moved to the measurement point, and the amount of reflected light at each film thickness was measured.

The measurement results are shown in FIG. Even on a single Si wafer, the SiO ₂ film thickness varies by several tens of millimeters, and a 10 mm square photodiode is used for the light receiving element. However, since the actual light receiving spot is about 1 mmφ, the linearity of the element deteriorates. The calculated results and the measured values do not completely coincide with each other because the extinction ratio of the Glan-Thompson prism and the transmittance of the λ / 4 plate are assumed to be ideal. However, in both cases, the result of calculation with the SiO ₂ film 4000 mm can be obtained with the maximum amount of reflected light, and the logarithm of the amount of reflected light and the thickness of the LB film has a linear relationship as can be seen from the correlation coefficient. Can be seen to reflect actual measurements.

Next, a third embodiment will be described. The device configuration is different from that described above, and the devices proposed in Patent Document 1 and Patent Document 2 were also examined. In this apparatus, as shown in FIG. 13, the polarization plane (X and Y) of the light reflected by the reference sample is rotated by π / 2 using a λ / 4 plate or prism and is incident on the measurement sample. The calculation formula is as follows as in the first embodiment.

A _rs : S-polarized reflectance of reference sample A _rp : P-polarized reflectance of reference sample A _SS : S-polarized reflectance of measurement sample A _sp : P-polarized reflectance of measurement sample Δ _S : S and P-polarized light of reference sample The value obtained by subtracting the phase difference between the S and P polarizations of the measurement sample from the phase difference of φ: the inclination of the incident linearly polarized light with respect to the S axis of the reference sample. As can be seen from this equation, φ is π / 4, that is 45 The value is maximized when the device is set in degrees.

Calculation was performed using the polarization reflectance and phase difference used in the first embodiment. The result is shown in FIG. This Similar to the results obtained in the first embodiment, SiO ₂ film thickness 4100Å at an incident angle of 70 °, SiO ₂ film thickness 4000Å at an incident angle of 65 °, SiO ₂ film thickness 3900Å at an incident angle of 60 °, the angle of incidence It can be seen that the maximum amount of reflected light is given in the vicinity of 3700 mm of SiO ₂ film thickness at 50 °.

The S-polarized light reflectance, P-polarized light reflectance, polarized light reflectance ratio, and phase difference in the vicinity of these film thicknesses are shown in FIGS. From these results, it can be seen that the P-polarized reflectance hardly changes, but the S-polarized reflectance becomes the smallest at the SiO ₂ film thickness that gives the maximum amount of reflected light. Therefore, at that time, the polarization reflectance ratio | Ap / As | shows the maximum value. On the other hand, it can be seen that the phase difference changes greatly in the vicinity of the film thickness. That is, it can be seen that the maximum amount of reflected light can be obtained by setting the sample and the apparatus so that the S-polarized reflectance is the smallest, the polarized reflectance ratio is the largest, and the phase difference changes greatly.

In the described embodiment, the reference sample and the measurement sample are a silicon substrate as a light-impermeable reflection substrate, a SiO ₂ film as a light-transmissive transparent thin film, a silane film, and a protein film for causing an antigen-antibody reaction. In addition, although a sample obtained by accumulating an LB film on a SiO ₂ film was used as a simulation sample, it is not limited to these.

The material applicable to the calculation of the embodiment of the present invention is, for example, a metal such as gold, silver, and aluminum as a light impermeable reflective substrate, or a semiconductor such as silicon, instead of SiO ₂ as a light transmissive transparent thin film. A single layer or composite film of aluminum oxide, titanium oxide, tantalum oxide or the like, a single layer or composite film of an organic thin film such as titanate or thiol instead of a silane film, and a protein film for causing an antigen-antibody reaction as a reaction layer If a receptor such as an antigen, an antibody, or a receptor is used alone or a mixture with dextran, cellulose, or the like, it can be used for specific detection of an antigen, an antibody or the like in a test solution such as liquid or urine.

  Moreover, although these samples comprise the multilayer film, it is also possible to abbreviate | omit organic thin films, such as a silane film, by using a mixture with functional dextran, a cellulose, etc.

It is also possible to make the SiO ₂ film thinner or omitted by increasing the thickness of the silane film that has amino, carboxyl, vinyl, epoxy, thiol, aryl, etc. at the end. It is.

When the thickness of the SiO ₂ film was changed from 1100 mm to 4000 mm based on the calculation result according to the present invention and measurement was performed at an incident angle of 60 degrees, a measurement sensitivity of about 10 times could be obtained.

  In the sample support and its support method in the ellipsometer having the above-described configuration, an optimum measurement condition is obtained by simulation, and an LB film is formed on the sample support by the LB (Langmuir-Blodgett) method. Create and confirm. The simulation is first performed in a standard ellipsometer arrangement. The standard arrangement of the ellipsometer was as follows.

  Light source-> P-> C-> S-> AP: Polarizer, C: Compensator, S: Sample, A: Two analyzer samples are prepared. The amount of light is measured with a sample. Therefore, the first reference sample corresponds to the sample before the reaction, and the second sample corresponds to the sample after the reaction. In addition, it is assumed that the apparatus condition is an ideal state.

Next, in order to confirm the result by experiment, an LB film was sequentially laminated in a step shape on a SiO ₂ film having a predetermined thickness, and after quenching only at the SiO ₂ film, LB films having various thicknesses were accumulated. Measure the amount of reflected light at the position. Further, this simulation is applied to the devices proposed in Patent Document 1 and Patent Document 2.

    As described above in detail, according to the present invention, when measuring a minute change in film thickness on a multilayer structure formed by an antibody on an immune support and an antigen as a change in light amount by ellipsometry, It is possible to provide a sample support method in an ellipsometer capable of obtaining the optimum measurement conditions.

It is a figure which shows the state of the polarization by the setting of the sample for demonstrating the sample support method of this invention. It is a figure which shows the structural example of the measurement sample used for description of a present Example. Is a diagram showing the reflection quantity of light with respect to _SiO 2 _-200Å change when the device settings were optimized. is a diagram showing the reflection quantity of light with respect to _SiO 2 _-200Å change when the lambda / 4 plate to 45 °. is a diagram showing the reflection quantity of light with respect to _SiO 2 _-200Å change from the Poincare sphere when the lambda / 4 plate to 45 °. In a present Example, it is a figure which shows S polarization | polarized-light reflectance in the film thickness vicinity, and S component reflectance). In a present Example, it is a figure which shows the P polarized light reflectance (P component reflectance) in the film thickness vicinity. In a present Example, it is a figure which shows the polarization | polarized-light reflectance ratio in the film thickness vicinity. In a present Example, it is a figure which shows the phase difference in the film thickness vicinity. It is a figure which shows the structural example of the measurement sample for measuring reflected light quantity. It is a figure which shows the calculation result (reflectance calculated value) based on the measurement performed using the measurement sample shown in FIG. It is a figure which shows the measurement result (reflectance actual value) based on the measurement performed using the measurement sample shown in FIG. It is a figure which shows the state of the reflected light which injects into a measurement sample in the apparatus structural example shown 2nd. It is a figure which shows the reflectance computed in the apparatus structural example shown in the 2nd using the polarization | polarized-light reflectance and phase difference which were used in 1st Example. It is a figure which shows the schematic structure of the conventional ellipsometer. In the S component reflectance in the present embodiment, and shows a characteristic of the reflectivity for SiO ₂ film thickness.

Explanation of symbols

1 ... semiconductor substrate, 2 ... silicon oxide film _(SiO 2), 3 ... silane film, 4 ... antigen-antibody reaction layer.

Claims (2)

A first optical element that makes polarized light incident on a part of a biological sample, and a polarization plane of P-polarized light and S-polarized light reflected by the part of the biological sample are expressed as nπ + π / 2 (n: 0, 1, 2,... Integer) a reaction analysis method for analyzing the biological sample by rotating the reflected second optical element and the reflected light from the second sample and quenching by the analyzer. And
Using a sample support having a light-impermeable layer, a light-transmitting layer, and a reaction layer related to the biological sample, wherein the layer thickness of the reaction layer related to the biological sample changes before and after the reaction
The reaction result is obtained by detecting the change in the light amount based on the phase difference between the S and P polarizations in the light transmissive layer, and the S polarization reflectance at the incident angle to the sample support is 0.25 or less. A method for analyzing a reaction of a biological sample. In the reaction analysis method,
The reaction analysis method according to claim 1, wherein the polarized light is incident on the biological sample within a range of 50 ° to 70 ° with respect to the S axis of the biological sample.

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