JPH0658873A - Optical sensor, detection method using optical sensor, and formation of molecular recognizing film for optical - Google Patents

Abstract

PURPOSE:To perform the determination of specific molecule in a sample solution which shows a specific reaction against molecular recognizing substance, making use of surface plasmon resonance. CONSTITUTION:A prism 1 is provided with a metallic thin film 2, a molecular recognizing film 5, and a passage 6 in which a sample 6 to be analyzed, on one face thereof. An incident light 3 emitting from a while light source 8 is changed to a p-polarized light wave through a polarizer 10 and further into a parallel light through a colimeter lens 11. The light 3 enters the surface of the film 2 at a angle range where the light may be totally reflected, while changing an incident angle, and excites surface plasmon at a specific angle to decrease the intensity of a reflected light 4. When the refractive index, dielectric constant, etc., of the sample 6 vary due to the function of molecular recognizing substance fixed on the film 5, the incident angle (resonance angle) excited by the surface plasmon vary, and its change is detected so as to calculate the concentration, etc., of the specific substance in the sample 6.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention uses the surface plasmon resonance method, which is one of the optical analysis methods, for ion-sensing substances, enzymes, antigens, antibodies, and other molecular recognition functional membranes present in a sample solution. TECHNICAL FIELD The present invention relates to an optical sensor for quantifying or monitoring a specific molecule that specifically reacts with a fixed molecular recognition functional substance, a detection method using the optical sensor, and a method for forming a molecular recognition functional film used in the optical sensor. .

[0002]

2. Description of the Related Art The living body has the ability to recognize inorganic substances and organic substances with high selectivity. A biosensor is a device that quickly and easily detects a specimen in a sample solution by using a functional molecule having high selectivity, or a living body itself including cells and tissues as a substance selection functional site.

The function of the sensor is divided into a part for selectively recognizing and reacting a specific sample and a part for detecting changes in the sample's conductivity, heat generation, luminescence, etc. due to this reaction and converting it into a signal. Then you can think. Electrodes such as an oxygen electrode, a hydrogen peroxide electrode, an ion electrode, and a gas electrode are generally used in a portion that catches a reaction and converts it into a signal. Recently, a number of sensors utilizing light have been proposed, for example, in Japanese Patent Application Laid-Open No. 3-48139, for example, Japanese Patent Application Laid-Open Nos. 61-292045, 63-75542 and 63-63.
The surface plasmon resonance method described in Japanese Patent No. 271162 is one of them.

The surface plasmon resonance method occurs when a surface wave of a plasma wave in which the charge density oscillates collectively is irradiated with a light wave from the outside to cause resonance and coupling. To use this surface plasmon resonance method as a chemical sensor, Kreshman (Kretshma
n) Use a system called placement. As shown in FIG. 9, the structure is such that a metal thin film 2 is attached to one surface of the optical prism 1, and air as a sample to be analyzed is in contact with this metal thin film. The conditions for causing the above plasmon resonance depend on various structural parameters. For example, the refractive index n =
70 nm on the prism 1 of 1.817 (λ = 633 nm)
When a thick silver vapor deposition film 2 is formed and air is used as a sample,
It is observed that when the p-polarized light 3 is incident at about 42.9 degrees, surface plasmons are excited at the interface between the silver thin film 2 and the air layer, and as a result, the intensity of the reflected light 4 decreases. The intensity curve of the reflected light 4 with respect to the incident angle at this time is as shown in FIG.
The intensity decreases sharply at a certain incident angle. The angle (resonance angle) of the reflected light 4 at this time is the kind of metal of the metal thin film 2,
It has been confirmed that the film thickness, the refractive index (dielectric constant) of the sample to be measured, and the film thickness of the molecular recognition functional film change greatly, and in particular, the refractive index of the sample to be measured and the molecular recognition functional film. The change in the resonance angle due to the film thickness of is used for the chemical sensor.

Based on the above-mentioned principle, Nyla
Nder et al. formed a thin film of a silicone-glycol copolymer on the surface of a metal thin film and detected the change in the resonance absorption position with the absorption of halogenated hydrocarbon gas (“Sensors and Ac
tuators "7 (1982/83), 79). After this, NO ₂ gas,
Alcohol, anti-α-fetoprotein, anti-IgG, ions, etc. have been studied.

As a method of detecting surface plasmon resonance, (1) a method of injecting parallel monochromatic light at different angles for irradiation of excitation light, (2) a method of injecting monochromatic light having an angular distribution, (3) There is a method of injecting parallel white light, and (i) a method of changing the position and angle of the light detecting element for reflected light detection, and (ii) an angle distribution curve of reflectance using an array of light detecting elements. And (iii) a method of obtaining a wavelength spectrum curve of reflectance. Among these methods, the conventional method is (1)
Surface plasmon resonance was often detected by the combination of-(i), (2)-(ii), and (3)-(iii).

On the other hand, a protein sensor measures the amount of protein excreted in urine due to abnormal urinary tract in the human body, change in blood volume in the kidney, production of abnormal protein, etc. And (1) a method for measuring the amount of precipitate generated by the addition or heating of this reagent (2) a burette method for measuring the amount of amino groups in the protein by the amount of light absorption or luminescence (3) Low measured by the transmittance of
ry method (4) Method of measuring the rate of increase in the refractive index of urine with a refractometer (5) A dye binding method or the like in which a dye is specifically bound to a protein and the absorbance is measured. In addition, JP-A-2-27
A sensor using a crystal oscillator as described in Japanese Patent Publication No. 6966 is also proposed.

[0008]

In the combination of the excitation light irradiation method and the reflected light detection described above, in the case of (1)-(i), a mechanical mechanism for changing the positions of the excitation light source and the light receiving element is used. Since the driving means is required, the accuracy is poor and there is a problem in terms of price. On the other hand, in the case of (2)-(ii), the angle resolution function of the obtained reflectance angle distribution curve is
It depends on the number of elements of the arrayed optical sensor and the distance from the sample to be measured by the optical sensor. Therefore, if the distance is increased, the resolution is improved, but the light intensity is decreased. Further, in the case of (3)-(iii), there is a method in which light that is fixed and dispersed by using a diffraction grating or a prism is received by an array-shaped photodetector, but the wavelength resolution is different from that of the diffraction grating. Since it is determined by the number of elements of the optical sensor, there is a problem that the light intensity decreases when the resolution is increased.

Further, if the positional relationship between the excitation light source, the prism and the metal thin film is changed, the position where the reflected light reaches may shift or the incident angle and the reflection angle may not match. In such a case, the measurement of the angle distribution-reflectance curve of the reflected light becomes inaccurate. For example, when the method of irradiating monochromatic light having an angular distribution of the excitation light irradiation method (2) is adopted, even if the reflected light is at the same angle as the positional relationship is unstable, the light receiving element Since the position of incidence on the beam changes, the angle measurement value becomes inaccurate. Further, even when parallel light is incident as in (1) or (3) above, the reflected position is similarly displaced, and the detected reflected light intensity is different each time. In order to utilize the surface plasmon resonance, it is necessary to make light incident under the condition of total internal reflection, so that the incident angle is large, and if the thickness of the refractive index adjusting oil changes even a little, the above deviation occurs. In such a case, it becomes impossible to detect the surface plasmon resonance condition determined by the strict parameters.

Further, regarding the protein sensor, the measurement work is complicated in the method by adding each reagent described above,
There were problems such as the need to be skilled and the long measurement time.

[0011]

In order to solve the above-mentioned problems, an optical sensor according to the present invention comprises a molecular recognition functional film whose surface faces a solution flow path through which a sample to be analyzed and a back surface of the molecular recognition functional film. By providing a metal thin film provided, an excitation light source for injecting white (that is, light of all wavelengths) p-polarized parallel light from the metal thin film side under the condition of total reflection, and by reflecting the incident light on the surface of the metal thin film. The optical sensor includes a Fourier transform spectroscope that uses an interferometer that receives the reflected light that is generated. Such a Fourier transform spectroscopic analysis method is optimal for the surface plasmon resonance method that can obtain a reflectance curve that draws an extremely sharp curve as shown in FIG.

A transparent parallel plate and an optical prism are placed in this order on the excitation light source side of the metal film, and white p-polarized parallel light is transmitted through the optical prism and the transparent parallel plate to enter the metal film. In the case of (1), the gap between the optical prism and the transparent parallel plate is sandwiched by a gap adjusting member and filled with oil for adjusting the refractive index, or (2) the gap is formed on one surface of the optical prism facing the transparent parallel plate side. An adjustment groove or protrusion is formed and the space between the optical prism and the transparent parallel plate is filled with refractive index adjusting oil, or (3) a gap adjustment groove or protrusion is formed on the transparent parallel plate surface facing the optical prism side. It is possible to keep the positional relationship between the prism and the metal thin film constant by forming the above and filling the space between the optical prism and the transparent parallel plate with the refractive index adjusting oil.

Further, regarding the optical sensor for protein quantification, a molecular recognition functional film whose surface faces the solution flow path through which the sample to be analyzed flows, a metal thin film provided on the back surface of this molecular recognition functional film, and from this metal thin film side an excitation light source to be incident white p-polarized light and the collimated light, is provided with a photodetector for receiving the reflected light generated by reflection by the Nyushoku thin film surface, the molecular recognition layer is rational formula -SO ₃ The above problem can be solved by an optical sensor which is a functional film on which a compound containing a group represented by M (M is hydrogen or an alkali metal) is fixed.

In order to quantify protein using the above-mentioned optical sensor, white light emitted from an excitation light source is converted into white p-polarized / parallel light by a polarizer and a collimator lens to obtain a metal thin film and a molecular recognition function. The film and the sample solution flow path to be analyzed are incident on the metal thin film surface side formed in this order while gradually changing the incident angle under the condition of total reflection, and the reflected light generated by this is received by the light receiver. Since the protein in the sample solution to be analyzed is adsorbed on the molecular recognition function membrane on which the compound having the group represented by the above-mentioned rational formula —SO ₃ M (M is hydrogen or alkali metal) is immobilized, the molecule generated by this adsorption action The change in the permittivity of the recognition function film can be measured by utilizing the transition of the resonance angle due to the surface plasmon resonance in the reflected light, and from this, the protein concentration in the sample solution to be analyzed can be measured.

The above-mentioned molecular recognition functional film has the rational formula --SO ₃
It has a group represented by M (M is hydrogen or an alkali metal). When the compound that is a protein recognition functional substance is water-soluble, it is dissolved in pure water to form an aqueous phase, while it is selected from alcohols, thiols, carboxylic acids, quaternary ammonium salts or phosphates, and Forming a monomolecular film at the gas-liquid interface using at least one compound having a long-chain alkyl group (preferably having a carbon number of 15 to 24) as a film-forming component, and scooping the monomolecular film adsorbing a functional substance onto a substrate. Can be formed by.

When the compound which is a protein recognition functional substance is not dissolved in water, it is mixed with the above film-forming components to form these monomolecular films on pure water, and the monomolecular film containing the functional substance is formed. The molecular recognition functional film can be formed by scooping on the substrate.

The molecular recognition film can also be used as a cumulative film by repeating the method for forming a monomolecular film. Further, it is preferable to form a molecular recognition functional film using a mixture of octadecyl mercaptan and dioctadecyldimethylammonium bromide represented by the following (Chemical formula 1) as the film forming component.

[0018]

[Chemical 1]

[0019]

Function: The resonance angle resulting from the surface plasmon resonance changes in response to changes in the refractive index (dielectric constant) of the sample to be measured. In the present invention, this subtle fluctuation in the resonance angle is stably detected by a Fourier transform spectroscope using an interferometer. When the incident light intensity is weak, repeated measurement is performed, and the results can be integrated to obtain a spectral curve.

By keeping the positional relationship between the prism and the metal thin film constant and stabilizing the paths of incident light and reflected light, the positional relationship between the excitation light source, the light receiving element or the sample to be analyzed becomes constant. As a result, the surface plasmon resonance phenomenon can be detected with higher accuracy.

Further, in the optical sensor for protein quantification, the occurrence of surface plasmon resonance is sensitively affected by changes in the film thickness and the dielectric constant of the substance existing in the immediate vicinity of the metal thin film. That is, when the protein is adsorbed on the molecular recognition functional film adjacent to the metal thin film, the dielectric constant increases,
The resonance angle, which is the angle at which surface plasmon resonance occurs, becomes large. Therefore, it is possible to quantify the protein by measuring the change in this angle.

[0022]

Embodiments of the present invention will be described below with reference to the accompanying drawings. Here, FIG. 1 is a schematic diagram showing an example of an optical sensor according to the present invention, and FIG. 2 is a schematic diagram showing an example of a Fourier transform spectroscope including an interferometer according to the present invention. 3 is a partial schematic view showing another example of the optical sensor of the present invention, and FIG. 4 is a perspective view showing the gap adjusting member according to the present invention.
FIG. 5 is a perspective view showing a transparent parallel plate and an optical prism having a gap adjusting groove according to the present invention. Further, FIG. 6 is a schematic diagram showing an example of a single-tank Langmuir trough for forming the molecular recognition functional film for protein quantification according to the present invention, and FIG. 7 is an example of resonance angle change by the optical sensor for protein quantification according to the present invention. FIG. 8 is a resonance angle-bovine serum albumin concentration diagram derived from the results of FIG. 7.

In FIG. 1, an optical prism 1 having a high refractive index
The metal thin film 2, the molecular recognition functional film 5, and the flow path 7 through which the sample 6 to be analyzed flows are arranged in this order on one surface. Incident light 3 emitted from the white light source 8 can be adjusted in incident angle to the optical prism 1 by the mirrors 9a and 9b. A lens, an optical fiber, or the like may be used instead of the mirrors 9a and 9b. Next, the white light is converted into a p-polarized light wave by the polarizer 10. This is because surface plasmons couple only with p-polarized light waves. This light is further collimated by the collimator lens 11. Polarizer 1
The arrangement of 0 and the collimator lens 11 may be reversed.

The metal thin film 2 is conveniently formed on one surface of the high refractive index prism 1. Further, a flat plate in which the metal thin film 2 and the molecular recognition functional film 5 are laminated is used as one of the transparent substrates,
If the high refractive index prism 1 is brought into contact with the high refractive index prism 1 through the refractive index adjusting oil, there is an advantage that the molecular recognition function film 5 can be easily replaced, and this configuration is also preferable. As a method for forming the metal thin film 2, there are known vacuum deposition method, sputtering method and the like.
The metal may be of any type, but for example, because the molecular recognition function film 5 is formed adjacently, the hydrophilicity or hydrophobicity of the metal surface,
It is necessary to consider the ease with which an oxide film is formed, etc., and also to consider the adhesion to the base material, etc. Examples of such metal include gold and silver.

Known enzymes, antibodies, etc. can be used as the molecular recognition functional substance in the molecular recognition functional film 5, and by exchanging these, an optical sensor capable of measuring many kinds of substances can be formed. it can.

The white light converted into p-polarized light and parallel light by the above operation is incident on the optical prism 1. The incident light 3 is incident on the surface of the metal thin film 2 in an angle range where the incident angle is changed, and the surface plasmon is excited at a specific angle to reduce the intensity of the reflected light 4 at that time. . Here, when the refractive index, the dielectric constant, etc. of the sample 6 to be analyzed are changed by the action of the molecular recognition functional substance fixed to the molecular recognition functional film 5, the incident angle (resonance angle) at which the surface plasmon is excited Changes, it is possible to detect the change and calculate the concentration of the specific substance in the sample 6 to be analyzed.

The emitted reflected light 4 is incident on a Fourier transform spectroscope (FT spectroscope) 12 through mirrors 9c and 9d, and a change in the outgoing angle of the reflected light 4 corresponding to the change in the incident angle is detected. . It is necessary that the angles of the mirrors 9c and 9d can be freely adjusted, and a lens or an optical fiber can be used instead of the mirror. In particular, when an optical fiber is used instead of the mirrors 9a, 9b, 9c and 9d, the load on the rotary drive portion can be reduced.

The holographic FT spectroscopic device 12 shown in FIG. 2 has an interferometer 20 of an optical system, the light adjusted here is detected by a multi-channel light receiving element 21, and an interferogram is obtained by a signal processing device 22. This is a device that is created and obtains a spectrum from this interferogram by a Fourier transform operation. The reflected light 4 is incident on the interferometer 20, is collimated by a collimator lens 23, and is split into two light beams by a beam splitter 26 provided with equi-tilt mirrors 24a and 24b and a semi-transmissive mirror 25. Is generated and enters the light receiving element 21 through the concave mirror 27, the reflecting mirror 28 and the cylindrical lens 29.

The interferometer 20 has the following features. That is, since the optical system is structurally fixed, there is no driving part, and it is compact, has high resolution, and has high intensity, and the multi-channel light receiving element 21 is adopted for the detection part, so that the measurement can be completed instantly. The measured values are stable and the reproducibility is high. Further, this interferometer 20 exhibits its performance especially when measuring a bright line, a narrow band spectrum, and a spectrum group.

According to the optical sensor of this embodiment, the change in the absorption peak of the reflected light 4 due to the change in the surface plasmon resonance condition can be detected quickly and accurately.

FIG. 3 shows an optical prism 1 and a transparent parallel plate 30.
It shows an embodiment of an optical sensor in which the gap between and is adjusted to be constant. Incident light 3 that is white light that is p-polarized and parallel light
Passes through the optical prism 1 and the refractive index adjusting oil 31 and the transparent parallel plate 30, and is totally reflected by the surface of the metal thin film 2 to become reflected light 4, which is incident on the FT spectroscope 12.

Here, the refractive index adjusting oil 31 is
It is sandwiched between the optical prism 1 and the transparent parallel plate 30 together with the gap adjusting member 32a or 32b shown in FIGS. 4 (a) and 4 (b), and is kept at a constant thickness by these gap adjusting members. The gap adjusting member 32a is made of a polytetrafluoroethylene sheet having a film thickness of 1 μm, and the gap adjusting member 32b is made of a metal aluminum thin plate having a thickness of 1.2 μm. By using the gap adjusting member 32 in this way, the thickness of the refractive index adjusting oil 31 can be kept constant, so that the arrival position of the reflected light 4 can be stabilized. Further, unlike the case where only the refractive index adjusting oil 31 is used, once the arrival position of the reflected light 4 is adjusted, it is not necessary to perform the adjustment again. Further, since the member does not block the position of the light passage, the transparency does not deteriorate.

FIG. 5 shows another example of a mechanism for keeping the thickness of the refractive index adjusting oil 31 constant without using the gap adjusting member 32. In FIG. 3A, a gap adjusting groove 33a is formed on the upper surface of the transparent parallel plate 30. It is possible to keep the thickness of the oil 31 constant by dropping the refractive index adjusting oil 31 into this groove and combining it with the optical prism 1. Further, FIG. 2B shows that the gap adjusting groove 3 is formed on one surface of the optical prism 1.
3b is an example in which the refractive index adjusting oil 31 is dropped into this groove and combined with the transparent parallel plate 30, so that the thickness of the oil 31 can be kept constant.
Further, instead of these gap adjusting grooves 33, a plurality of protrusions are provided on the surface of the optical prism 1 or the transparent parallel plate 30, and the refractive index adjusting oil 31 is dropped between them to keep the thickness of the oil 31 constant. You can also That is, these gap adjusting grooves or protrusions keep the thickness of the oil 31 constant,
The arrival position of the reflected light 4 can be stabilized.

The gap adjusting member 32 and the gap adjusting groove 3 described above.
When the gap between the optical prism 1 and the transparent parallel plate 30 is kept constant by 3 or the protrusions for adjusting the gap, it is possible not to use the refractive index adjusting oil 31.

An example of forming a molecular recognition functional film for protein quantification according to the present invention will be shown. In FIG. 6, a single tank type Langmuir trough 40 is provided with a tank (trough) 41 for forming a monomolecular film. The tank 41 contains a solution of the protein recognition function substance dissolved in pure water. Examples of this protein recognition functional substance include the compounds shown in (Chemical Formula 2) to (Chemical Formula 18).

[0036]

[Chemical 2]

[Chemical 3]

[Chemical 4]

[Chemical 5]

[Chemical 6]

[Chemical 7]

[Chemical 8]

[Chemical 9]

[Chemical 10]

[Chemical 11]

[Chemical 12]

[Chemical 13]

[Chemical 14]

[Chemical 15]

[Chemical 16]

[Chemical 17]

[Chemical 18]

On the surface of this aqueous solution, a film-forming component dissolved in an organic solvent is dropped to form a monomolecular film. This organic solvent volatilizes and only the component of the monomolecular film is developed as a gas film. The monomolecular film thus formed is compressed by a barrier (not shown) and compressed to a desired surface pressure to form a condensed film. When two or more compounds are used as film-forming components, the mixing ratio can be freely changed to obtain a monomolecular film having desired physical properties. Examples of film-forming components include octadecyl mercaptan, dioctadecyldimethylammonium bromide, octadecyl alcohol, dihexadecylphosphoric acid, and the like. The water-soluble molecule recognition functional substance is adsorbed on the monolayer.

On the other hand, as the base material for forming the molecular recognition functional film, the optical prism 1 having the metal thin film 2 formed on the surface thereof is used.
Alternatively, it is convenient to use the transparent parallel plate 30. When the optical prism 1 is used, a metal thin film 2 is formed by a vacuum deposition method or the like, the metal surface is made hydrophilic by oxygen plasma irradiation, etc., and immersed in a single tank type Langmuir trough 40 to adsorb the molecular recognition functional substance. One layer of the above-mentioned monomolecular film (Langmuir-Blodgett film; hereinafter abbreviated as LB film) is formed. When the transparent parallel plate 30 is used as the substrate, this surface can be modified with, for example, a silane coupling agent, and then immersed as shown in FIG. 6 to form an LB film. When the LB film is to be a cumulative layer, the transparent parallel plate 30 may be moved up and down a required number of times as indicated by the arrow in the figure. Further, the unnecessary film formed on the back surface can be easily removed by the solvent or the like in which the film forming components are dissolved.

Another example of the method of forming the LB film is a method of forming by a polyion complex.

The protein quantification molecular recognition functional film 5 formed as described above can be used as an optical sensor for protein quantification with the configuration shown in FIG. 1 or 3. However, the optical sensor for protein quantification of the present invention does not necessarily need to use the FT spectroscopic device 12, and a known light receiver and arithmetic device can be used.

The optical sensor for protein quantification of the present invention comprises:
It is considered that changes in film thickness and dielectric constant caused by irreversible adsorption of protein to the molecular recognition function film 5 are detected by surface plasmon resonance. The adsorptivity of proteins depends on the density and the state of sulfo groups existing in the molecular recognition function film 5. Therefore, the degree of adsorption changes depending on the acidity or basicity of the sample 6 to be analyzed, or when hydrogen of the sulfo group is replaced with an alkali metal. Although the adsorbed protein is irreversible as described above, it can be released by washing with wash water prepared to have an acidity or basicity that is significantly different from the acidity or basicity of the sample 6 to be analyzed. You can

The optical sensor for protein quantification of the present invention will be described in more detail. That is, a chromium (Cr) film having a film thickness of about 5 nm and a gold (Au) film having a film thickness of 50.8 nm were formed in this order on the rectangular prism 1 having a refractive index nd = 1.5143 by a vacuum vapor deposition method. This surface is irradiated with oxygen plasma (MARCH
Using PM-600 type plasma ashing device manufactured by the same company. Oxygen flow rate 1
L at which Acid Red 112 of (Chemical Formula 2) was fixed here by making it hydrophilic by treating with 0 ml / min, input power 100 W for 30 seconds).
One layer of B film was formed.

The LB film was formed as follows. That is, Acid Red 112 is included in the single tank Langmuir trough 40.
An aqueous solution of 25 mg / l dissolved in pure water was added. As a film forming component, octadecyl mercaptan (C
₁₈ H ₃₇ SH, manufactured by Tokyo Chemical Industry Co., Ltd. and dioctadecyldimethylammonium bromide ( ₂ (C ₁₈ H ₃₇ ) N _{2 2}
CH ₃ Br, manufactured by Tokyo Chemical Industry Co., Ltd., 1 mmol / l each
The mixture was mixed at a concentration of (molar ratio 1: 1), and this was dissolved in chloroform ( _CHC13 , Dojindo Laboratories, for ultraviolet spectroscopic analysis) and used. The above film-forming components were spread on the Acid Red 112 aqueous solution, left for 1 hour, and then compressed at a speed of 14 cm ² / min to form an LB film at a surface pressure of 5 mN / m.

Using the optical prism 1 formed by integrating the sulfo group-containing molecule recognition function film 5 and the metal thin film 2 formed as described above, the surface plasmon resonance was detected by the apparatus shown in FIG. Analyte sample 6 has a concentration of 2
Bovine serum albumin aqueous solutions of 0, 50, 100 and 200 mg / l were used. As a result, as shown in FIG. 7, with respect to the resonance angle of pure water used as a standard, it was observed that the resonance angle in the case of using the bovine serum albumin aqueous solution increased as the albumin concentration increased. .

FIG. 8 shows a resonance angle-albumin concentration line drawn based on the change in the resonance angle. In the figure, since the resonance angle and the albumin concentration have a very high correlation, the albumin concentration can be accurately, stably and easily measured from the inclination of the figure by using the optical sensor of the present invention. .

As a comparative example, the above system was used to measure the resonance angle using a glucose aqueous solution having the same concentration instead of the bovine serum albumin aqueous solution. The difference between the resonance angle of pure water and the aqueous solutions having different concentrations were measured. It was found that there was almost no difference between the two, and therefore it was not suitable for measurement of the glucose aqueous solution concentration.

[0047]

As described above, according to the present invention, it is possible to provide an optical sensor that can perform quantitative analysis of a sample to be analyzed quickly and accurately. In addition, the gap adjusting member, gap adjusting groove or protrusion can keep the thickness of the refractive index adjusting oil constant and stabilize the arrival position of the reflected light. The reflected light can be satisfactorily observed in terms of both sex.

Furthermore, by using the optical sensor of the present invention as a protein sensor, it is possible to easily obtain stable and reproducible results as compared with the conventional method.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing an example of an optical sensor of the present invention.

FIG. 2 is a schematic diagram showing an example of a Fourier transform spectroscope including an interferometer according to the present invention.

FIG. 3 is a partial schematic view showing another example of the optical sensor of the present invention.

FIG. 4 is a perspective view showing a gap adjusting member according to the present invention.

FIG. 5 is a perspective view showing a transparent parallel plate and an optical prism in which a gap adjusting groove is formed.

FIG. 6 is a schematic view showing an example of a single tank type Langmuir trough for forming a molecular recognition functional film for protein quantification according to the present invention.

FIG. 7 is a graph showing an example of resonance angle change by the optical sensor for protein quantification according to the present invention.

FIG. 8: Resonance angle-bovine serum albumin concentration diagram derived from the results of FIG.

FIG. 9 is a schematic diagram showing the Creshman arrangement.

FIG. 10 is an intensity curve of reflected light with respect to an incident angle for explaining surface plasmon resonance.

[Explanation of symbols]

1 ... Optical fiber, 2 ... Molecular recognition functional film, 3 ... Excitation light,
4 ... Fluorescence, 5 ... Detector, 6 ... Core part, 7 ... High refractive index prism, 8 ... Metal thin film, 9 ... Molecular recognition functional film, 10 ... Flow path, 11 ... Excitation light generator, 12 ... Photodetector , 13 ... Transparent substrate, 14 ... Refractive index adjusting oil, 20 ... Interferometer, 21 ...
Multi-channel light receiving element, 22 ... Signal processing device, 30 ... Transparent parallel plate, 31 ... Refractive index adjusting oil, 32a, 32
b ... Gap adjusting member, 33a, 33b ... Gap adjusting groove, 4
0 ... Single tank Langmuth rough.

Claims (11)

[Claims] 1. A molecular recognition functional film, the surface of which faces a solution flow path through which a sample to be analyzed flows, a metal thin film provided on the back surface of the molecular recognition functional film, and white p-polarized / parallel light from the metal thin film side. And a Fourier transform spectroscope using an interferometer that receives reflected light generated by reflecting incident light on the surface of the metal thin film. 2. A transparent parallel plate for causing white p-polarized parallel light from the excitation light source to enter the metal thin film and an optical prism are arranged in this order on the back surface side of the metal thin film. The optical sensor according to claim 1. 3. The optical sensor according to claim 2, wherein a gap adjusting member is sandwiched between the optical prism and the transparent parallel plate, and a refractive index adjusting oil is filled. 4. A gap adjusting groove or protrusion is formed on one surface of the optical prism facing the transparent parallel plate side, and a refractive index adjusting oil is filled between the optical prism and the transparent parallel plate. The optical sensor according to claim 2, wherein: 5. A transparent parallel plate surface facing the optical prism is provided with a gap adjusting groove or a protrusion, and a refractive index adjusting oil is filled between the optical prism and the transparent parallel plate. The optical sensor according to claim 2, wherein: 6. A molecular recognition functional film, the surface of which faces a solution flow path through which the sample to be analyzed, a metal thin film provided on the back surface of this molecular recognition functional film, and white p-polarized / parallel light from the metal thin film side. Is provided, and a light receiver for receiving reflected light generated by reflecting incident light on the surface of the metal thin film, and the molecular recognition functional film is a rational formula-
An optical sensor, wherein a compound having a group represented by SO ₃ M (M is hydrogen or an alkali metal) is immobilized. 7. The white light emitted from the excitation light source is converted into white p-polarized / parallel light by a polarizer and a collimator lens, and a metal thin film, a molecular recognition functional film and a sample solution channel to be analyzed are formed in this order. It is incident on the metal thin film surface side while varying the incident angle under the condition of total reflection, and the reflected light is received by the light receiver to have a group represented by the rational formula —SO ₃ M (M is hydrogen or an alkali metal). The change in the dielectric constant of the molecular recognition functional film caused by the adsorption action of the protein contained in the sample solution to be analyzed to the molecular recognition functional film on which the compound is immobilized is measured by the change in the resonance angle due to the surface plasmon resonance in the reflected light. A method for detecting a protein concentration in a sample solution to be analyzed by using an optical sensor for protein, the method comprising: 8. A water-soluble compound having a group represented by the rational formula —SO ₃ M (M is hydrogen or an alkali metal), which is a protein recognition functional substance, is dissolved in pure water to form an aqueous phase, and alcohols, At least one film-forming component selected from thiols, carboxylic acids, quaternary ammonium salts or phosphates and having a long-chain alkyl group forms a monomolecular film at the gas-liquid interface and adsorbs the recognition functional substance. A method for forming a molecular recognition functional film for use in an optical sensor for protein, which comprises scooping a monomolecular film onto a substrate. 9. A water-insoluble compound which is a protein recognition functional substance and has a group represented by the rational formula —SO ₃ M (M is hydrogen or an alkali metal), and alcohols, thiols, carboxylic acids, and quaternary ammonium. At least one selected from salts or phosphates and having a long-chain alkyl group
An optical sensor for proteins, characterized in that these monomolecular films are formed on pure water by mixing with various film forming components and the monomolecular film containing the recognition function substance is scooped onto a substrate. A method for forming a molecular recognition functional film to be used. 10. The forming method according to claim 9, wherein the monomolecular film forming method is repeated to form a cumulative film. 11. The method according to claim 8, wherein the film forming component is a mixture of octadecyl mercaptan and dioctadecyl dimethyl ammonium bromide.