JP2003270130A - Measuring instrument - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prevent a measured result from being dispersed by unevenness of a film thickness in a thin film layer, dust on the thin film layer and the like, in a measuring instrument such as a surface plasmon resonance measuring instrument. <P>SOLUTION: In this measuring instrument provided with a dielectric block 10, the thin film layer 12 formed on one face thereof to contact with a sample, a light source 14 for generating light beam 13, an incident optical system 15 for making the beam 13 get incident into the dielectric block 10 at an incident angle by which a total reflection condition is attained in an interface 10b between the dielectric block 10 and the thin film layer 12, and a photo detecting means 17 for detecting the light beam 13 total reflected in the interface 10b, means 32, 33 are provided to move the optical system 15 and the dielectric bock 10 relatively so as to change an incident position of the beams 13 in the interface 10b, and a plurality of data corresponding to intensities of the total- reflected beams, output respectively by the photo detecting means 17 when the incident positions of the beam 13 are brought into different conditions is processed statistically by a computing means 20, so as to acquire one representative data corresponding to the intensity. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a measuring device such as a surface plasmon resonance measuring device for quantitatively analyzing a substance in a sample by utilizing generation of surface plasmon.

[0002]

2. Description of the Related Art In a metal, free electrons oscillate collectively to generate compression waves called plasma waves. And, the quantized compression wave generated on the metal surface is
It is called surface plasmon.

Conventionally, various surface plasmon resonance measuring devices have been proposed for quantitatively analyzing a substance in a sample by utilizing the phenomenon that the surface plasmon is excited by a light wave. Among them, one that is particularly well known is one that uses a system called Kretschmann arrangement (see, for example, JP-A-6-167443).

A surface plasmon resonance measuring apparatus using the above system basically comprises, for example, a dielectric block formed in a prism shape, a metal film formed on one surface of the dielectric block and brought into contact with a sample, and a light beam. A light source that generates a beam, an incident optical system that causes the light beam to enter the dielectric block at an incident angle that allows total reflection conditions to be obtained at the interface between the dielectric block and the metal film, and total reflection at the interface. State of surface plasmon resonance by measuring the intensity of the light beam
That is, it is provided with a light detecting means for detecting the attenuated total reflection state.

As described above, in order to obtain various incident angles, a relatively thin light beam may be deflected to be incident on the interface, or a component which is incident on the light beam at various angles may be included. As described above, a relatively thick light beam may be incident on the interface in a convergent light state or a divergent light state. In the former case, a light beam whose reflection angle changes with the deflection of the light beam is detected by a small photodetector that moves in synchronization with the deflection of the light beam, or an area measuring device extending along the direction of change of the reflection angle. Can be detected by. On the other hand, in the latter case, each light beam reflected at various reflection angles can be detected by an area measuring device extending in a direction in which all the light beams can be received.

In the surface plasmon resonance measuring apparatus having the above structure, when a light beam is incident on the metal film at a specific incident angle θ _SP which is equal to or greater than the total reflection angle, an electric field distribution is generated in the sample in contact with the metal film. An evanescent wave is generated, and the surface plasmon is excited at the interface between the metal film and the sample by the evanescent wave. When the wave vector of the evanescent light is equal to the wave number of the surface plasmon and the wave number matching is established, both are in a resonance state and the energy of the light is transferred to the surface plasmon, so that at the interface between the dielectric block and the metal film. The intensity of the reflected light sharply decreases. This decrease in light intensity is generally detected as a dark line by the light detecting means.

The above resonance occurs only when the incident beam is p-polarized. Therefore, it is necessary to set in advance that the light beam is incident as p-polarized light.

When the wave number of the surface plasmon is known from the incident angle θ _{SP at} which this attenuated total reflection (ATR) occurs, the dielectric constant of the sample can be obtained. That is, the wave number of the surface plasmon is K
_{Let SP be} the angular frequency of the surface plasmons be ω, c be the speed of light in a vacuum, ε _m and ε _s be the metal, and the permittivity of the sample respectively, and the following relationships are established.

[0009]

[Equation 1] If the permittivity ε _s of the sample is known, the concentration of the specific substance in the sample can be known based on a predetermined calibration curve or the like. Therefore, by finally knowing the incident angle θ _{SP at} which the reflected light intensity decreases, the dielectric constant of the sample can be determined. A property related to the index, that is, the refractive index can be obtained.

Further, as a similar measuring device utilizing the attenuated total reflection (ATR), for example, "Spectroscopic Research" Vol. 47, No. 1 (1998), pages 21 to 23 and 26 to 27.
Leakage mode measuring devices described on the page are also known. This leak mode measuring device is basically a dielectric block formed, for example, in a prism shape, a clad layer formed on one surface of the dielectric block, and a clad layer formed on the clad layer and brought into contact with a sample. Optical waveguide layer, a light source for generating a light beam, and the light beam with respect to the dielectric block, a total reflection condition is obtained at an interface between the dielectric block and the cladding layer, and An optical system that is incident at various angles so that the attenuation of the total reflection due to the excitation of the guided mode may occur, and the intensity of the light beam totally reflected at the interface is measured to measure the excited state of the guided mode, that is, the attenuated total reflection state. And a light detecting means for detecting

In the leak mode measuring device having the above structure,
When a light beam is incident on the cladding layer through the dielectric block at an angle of incidence equal to or more than the total reflection angle, only light with a specific incident angle having a specific wave number is transmitted in the optical waveguide layer after passing through the cladding layer. It propagates in the guided mode. When the guided mode is excited in this manner, most of the incident light is taken into the optical waveguide layer, so that the total reflection attenuation occurs in which the intensity of the light totally reflected at the interface sharply decreases.
Since the wave number of the guided light depends on the refractive index of the sample on the optical waveguide layer, the refractive index of the sample and the related characteristics of the sample are analyzed by knowing the above-mentioned specific incident angle at which attenuation of total reflection occurs. be able to.

[0012]

In the surface plasmon resonance measuring apparatus and the leaky mode measuring apparatus described above, a thin film layer (a metal film in the case of the surface plasmon resonance measuring apparatus, and a clad in the case of the leaky mode measuring apparatus). Layer and optical waveguide layer) are non-uniform in thickness, or
When a sensing substance that binds to a specific substance in a sample is immobilized on the thin film layer, the reaction characteristics of the sensing substance are non-uniform, or if dust is attached to the thin film layer, The measurement result will have different values depending on the incident position of the light beam at the interface with the dielectric block. Regarding the above sensing substances,
For example, detailed description is made in Japanese Patent Application No. 2001-113647 by the present applicant.

In order to prevent variations in the measurement results due to such circumstances, as shown in Japanese Patent Application No. 2000-149415, a light beam is formed so as not to focus at the interface between the thin film layer and the dielectric block. Is considered to be incident. That is, if the light beam is made to enter over a relatively wide range of the interface in this way, the measurement is performed under the condition that the film thickness of the thin film layer, the reaction characteristics of the sensing substance, etc. are averaged. It is possible to prevent variations in results.

However, if the light beam is made to enter over a wide range of the interface without being converged at the interface, the range of the light beam incident angle where attenuation of total reflection is recognized is widened and the measurement sensitivity is impaired. The problem arises.

Further, in order to prevent the variation in the above-mentioned measurement results, a plurality of measurement units each including a dielectric block and a thin film layer are prepared for one sample, and a plurality of measurement units are used for each sample by using these measurement units. It is also conceivable to perform one measurement and statistically process a plurality of obtained measurement data to obtain one representative data. However, in such a case, it is necessary to supply one sample to a plurality of measurement units or sequentially perform a measurement operation on a plurality of measurement units, so that it takes a long time to obtain the measurement result of one sample. The problem arises that it takes time.

The present invention has been made in view of the above circumstances, and it is possible to prevent variations in the measurement results due to nonuniformity of the film thickness of the thin film layer, the reaction characteristics of the sensing substance, and dust on the thin film layer. In addition, it is an object of the present invention to provide a measuring device capable of highly sensitive and efficient measurement.

[0017]

A first measuring device according to the present invention comprises a dielectric block as described above, a thin film layer formed on one surface of the dielectric block and brought into contact with a sample, and a light beam. And a light source for generating the light beam, an incident optical system for causing the light beam to be incident on the dielectric block at an incident angle at which total reflection conditions are obtained at the interface between the dielectric block and the thin film layer, and total light at the interface. In a measuring device comprising a light detecting means for measuring the intensity of the reflected light beam, the optical system and the dielectric block are relatively moved so that the incident position of the light beam on the interface changes. When the relative moving means and the incident position of the light beam are in different states, a plurality of kinds of data corresponding to the intensities output by the photodetecting means are statistically processed to correspond to the intensities. It is characterized in that the calculating means for obtaining one representative data is provided.

The second measuring device according to the present invention is particularly configured as the above-mentioned surface plasmon resonance measuring device, and is formed on the dielectric block and one surface of the dielectric block to contact the sample. A thin film layer made of a metal film, a light source for generating a light beam, and an incident angle at which the light beam is incident on the dielectric block at the interface between the dielectric block and the metal film. In a measuring device comprising an incident optical system for making incident light and a light detecting means for measuring the intensity of a light beam totally reflected at the interface, a relative moving means and a calculating means similar to those in the first measuring device are provided. It is characterized by being provided.

Further, the third measuring device according to the present invention is
Particularly, the device is configured as the above-mentioned leaky mode measuring device, and includes a dielectric block, a clad layer formed on one surface of the dielectric block, and an optical waveguide formed on the clad layer and brought into contact with a sample. A thin film layer including a layer, a light source that generates a light beam, and an incident light beam that is incident on the dielectric block at an incident angle such that total reflection conditions are obtained at the interface between the dielectric block and the clad layer. In a measuring device comprising an optical system and a light detecting means for measuring the intensity of the light beam totally reflected at the interface,
It is characterized in that the same relative movement means and calculation means as in the first measuring device are provided.

In the first to third measuring devices according to the present invention described above, the relative position of the light beam at the interface is obtained by moving the optical system and the dielectric block relatively. However, in order to obtain this state, a special structure may be adopted for the optical system. Hereinafter, the fourth to sixth embodiments of the present invention configured as described above
The measuring device will be described.

A fourth measuring apparatus according to the present invention comprises a dielectric block, a thin film layer formed on one surface of the dielectric block and brought into contact with a sample, a light source for generating a light beam, and the light beam. An incident optical system that is incident on the dielectric block at an angle of incidence at which total reflection conditions are obtained at the interface between the dielectric block and the thin film layer, and light for measuring the intensity of the light beam totally reflected at the interface. In a measuring device including a detecting means, the optical system is formed so that the light beam can be made incident on a plurality of different incident positions on the interface, and the optical system is the same as in the first measuring device. It is characterized in that arithmetic means is provided.

The fifth measuring apparatus according to the present invention is particularly configured as the above-mentioned surface plasmon resonance measuring apparatus, and is formed on the dielectric block and one surface of the dielectric block to contact the sample. A thin film layer made of a metal film, a light source for generating a light beam, and an incident angle at which the light beam is incident on the dielectric block at the interface between the dielectric block and the metal film. In a measuring device comprising an incident optical system for making incident light and a photodetecting means for measuring the intensity of a light beam totally reflected at the interface, the optical system comprises a plurality of different incidents of the light beam on the interface. It is characterized in that it is formed so that it can be incident on a position, and that the same calculation means as in the first measuring device is provided.

Further, the sixth measuring device according to the present invention is
Particularly, the device is configured as the above-mentioned leaky mode measuring device, and includes a dielectric block, a clad layer formed on one surface of the dielectric block, and an optical waveguide formed on the clad layer and brought into contact with a sample. A thin film layer including a layer, a light source that generates a light beam, and an incident light beam that is incident on the dielectric block at an incident angle such that total reflection conditions are obtained at the interface between the dielectric block and the clad layer. In a measuring device comprising an optical system and a light detecting means for measuring the intensity of the light beam totally reflected at the interface,
The optical system is formed so that the light beam can be made incident on a plurality of different incident positions on the interface, and the same calculation means as in the first measuring device is provided. It is a thing.

In the fourth to sixth measuring devices,
There are various methods for analyzing the sample by measuring the intensity of the light beam totally reflected at the interface by the light detecting means,
For example, the light beam is made incident on the interface at various incident angles under which total reflection conditions are obtained, and the intensity of the light beam totally reflected at this interface is measured at each position corresponding to each incident angle,
The sample analysis can be performed by detecting the position (angle) of the dark line generated by the attenuation of the total reflection. Also, D.
V.Noort, K.johansen, C.-F.Mandenius, PorousGold in S
urface Plasmon Resonance Measurement. EUROSENSORS
As described in XIII, 1999, pp.585-588, light beams of a plurality of wavelengths are made incident on the interface at an incident angle at which total reflection conditions are obtained, and the light beams totally reflected at this interface are The sample analysis may be performed by measuring the intensity for each wavelength and detecting the degree of attenuation of total reflection for each wavelength.

Furthermore, PINikitin, ANGrigorenko,
AABeloglazov, MVValeiko, AISavchuk, OASavchu
k, Surface Plasmon Resonance Interferometry for Mi
cro-Array Biosensing. EUROSENSORS XIII, 1999, pp.2
As described in 35-238, the light beam is incident on the interface at an angle of incidence such that total internal reflection conditions are obtained, and a portion of this light beam is split before it is incident on the interface. The sample analysis may be performed by causing the divided light beam to interfere with the light beam totally reflected at the interface and detecting the intensity of the light beam after the interference.

More specifically, as the optical system in the fourth to sixth measuring devices, more specifically, one light beam is branched into a plurality of light beams, and the light beams are respectively made to enter a plurality of different beams on the interface. It is possible to use one that is incident on a position, or one that deflects one light beam and makes it incident on a plurality of different incident positions on the interface.

On the other hand, more specifically, as the calculating means in the first to sixth measuring devices, more specifically, median values of a plurality of types of data corresponding to the intensity of the light beam are obtained and used as the one representative data. What can be used can be used.

As this calculating means, the average value of the data included in the upper and lower predetermined widths is calculated from the median values of the plural kinds of data corresponding to the intensity, and the average value is used as the one representative data. A thing can also be used.

Further, as the calculating means, an average value of data excluding the maximum value and / or the minimum value of a plurality of kinds of data corresponding to the intensity is obtained and used as the one representative data. A thing can also be used.

[0030]

INDUSTRIAL APPLICABILITY The measuring device according to the present invention is such that the light beam is incident on the interface between the dielectric block and the thin film layer (the metal film in the case of the surface plasmon resonance measuring device and the clad layer in the case of the leaky mode measuring device). When a position is changed and a plurality of incident positions of the light beam are in different states, a plurality of kinds of data corresponding to the intensities of the light beams totally reflected at the interface are statistically processed, and one representative corresponding to the intensity is obtained. Since it is configured to obtain data, this representative data excludes fluctuations due to the film thickness of the thin film layer and the reaction characteristics of the sensing substance, and fluctuations caused by disturbance such as dust. obtain. Therefore, according to this measuring device, even if the film thickness of the thin film layer or the reaction characteristics of the sensing substance are non-uniform, or dust is present on the thin film layer, it is possible to prevent variations in the measurement results due to them. Becomes

In addition, since the measuring device of the present invention does not need to make the light beam incident so that it is not focused at the interface between the thin film layer and the dielectric block, the range of the light beam incident angle at which attenuation of total reflection is recognized is limited. It does not become wider, so that it becomes possible to perform measurement with high sensitivity.

Moreover, the measuring device of the present invention is basically
Since it is possible to prevent variations in measurement results by using only one measurement unit consisting of a dielectric block and a thin film layer for one sample, it is possible to use a plurality of measurement units for measurement of one sample. By comparison, the time required for sample supply and measurement operation can be shortened and the measurement can be performed efficiently.

[0033]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

FIG. 1 shows a side view of a measuring apparatus according to the first embodiment of the present invention, and FIG. 2 shows a plan view of an optical system portion thereof. The apparatus of the present embodiment is the surface plasmon resonance measuring apparatus described above as an example, and this apparatus has, for example, a dielectric block 10 having a shape in which a part of an approximately quadrangular pyramid is cut off, and the dielectric block 10. It has a metal film 12 formed on one surface (upper surface in the drawing) and made of, for example, gold, silver, copper, aluminum or the like.

The dielectric block 10 is, for example, a dielectric block.
Reference numeral 10 is made of, for example, transparent synthetic resin or optical glass such as BK7, and the periphery of the portion where the metal film 12 is formed is raised, and the raised portion 10a is a sample holder for storing the liquid sample 11. Function as. In this example, the sensing substance 30 is fixed on the metal film 12, and the sensing substance 30 will be described later.

The dielectric block 10 constitutes a disposable measuring chip (measuring unit) together with the metal film 12,
For example, a plurality of moving tables 32 attached to the turntable 31
They are fitted and fixed one by one in the chip holding holes 32a. The movable table 32 is movably attached to the turntable 31 in a direction perpendicular to the plane of FIG. 1, that is, in the vertical direction in FIG. 2, and is moved in this direction by driving the actuator 33.

After the dielectric block 10 is fixed to the turntable 31 as described above, the turntable 31 is intermittently rotated by a fixed angle and the liquid sample 11 is stopped with respect to the dielectric block 10 stopped at a predetermined position. Is dropped and the liquid sample 11 is held in the sample holding portion 10a. Thereafter, when the turntable 31 is further rotated by a certain angle, the dielectric block 10 is sent to the measurement position shown in FIG. 1 and stopped there.

In the surface plasmon resonance measuring apparatus of this example, in addition to the dielectric block 10, one light beam 13
A laser light source 14 composed of a semiconductor laser or the like for generating
The optical system 15 which passes the light beam 13 through the dielectric block 10 and makes it incident on the interface 10b between the dielectric block 10 and the metal film 12 so as to obtain various incident angles, and the interface 10
The cylindrical lens 16 for collimating the light beam 13 totally reflected by b only in the plane of the display of FIG. 1, the light detecting means 17 for detecting this collimated light beam 13, and the light detecting means 17 A driver 19 connected thereto, a signal processing unit 20 including a computer system, and a display unit 21 connected to the signal processing unit 20 are provided.

The optical system 15 has cylindrical lenses 15a and 15b for diverging, collimating, and converging the light beam 13 emitted from the laser light source 14 in the state of thin parallel light only within the plane shown in FIG. , 15c.

FIG. 3 is a block diagram showing the electrical construction of this surface plasmon resonance measuring apparatus. Light detection means 17
Is a plurality of photodiodes 17a, 17b, 17c ...
It is a photodiode array that is arranged in rows. These photodiodes 17a, 17b, 17c ... Are connected to the multiplexer 18 of the driver 19 via signal lines H, respectively.

Here, the signal line H is grounded via a resistor R. Although only one signal line H is grounded in FIGS. 1 and 3, all signal lines H are similarly grounded.

The driver 19 is a multiplexer 18
Differential amplifier 22 connected to the two output lines of, an amplifier 23 that amplifies the output of the differential amplifier 22, and an A / D converter that digitizes the output of the amplifier 23 and inputs it to the signal processing unit 20. 24, a drive circuit 25 that inputs the clock CLK and the address signal SA to the multiplexer 18 to drive the multiplexer 18, and a controller 26 that controls the operation of the drive circuit 25 based on an instruction from the signal processing unit 20. is doing.

The light beam 13 emitted from the laser light source 14 in the state of thin parallel light advances in the state of parallel light as it is in the plane shown in FIG. On the other hand, in the plane shown in FIG. 1, this light beam 13 is diverged by the cylindrical lens 15a, is then collimated by the cylindrical lens 15b, and is then condensed by the cylindrical lens 15c. Is incident on the interface 10b between the metal film 12 and the metal film 12 so as to converge there. Therefore, the light beam 13 includes components that are incident on the interface 10b at various incident angles θ. It should be noted that this incident angle θ is set to an angle that is equal to or greater than the total reflection angle and that excites surface plasmons. Therefore, the light beam 13 is totally reflected at the interface 10b, and the reflected light beam 13 contains components that are reflected at various reflection angles.

The light beam 13 is incident on the interface 10b as p-polarized light. In order to do so, the laser light source 14 may be arranged in advance so that the polarization direction thereof becomes a predetermined direction. In addition, the polarization direction of the light beam 13 may be controlled by a wave plate or a polarizing plate.

The light beam 13 which is totally reflected on the interface 10b and then collimated by the cylindrical lens 16 in the plane of the display of FIG. 1 is detected by the light detecting means 17. In the photodetection means 17 which is a photodiode array, the parallel arrangement direction of the photodiodes 17a, 17b, 17c ... Is substantially perpendicular to the traveling direction of the collimated light beam 13 in the plane of FIG. It is arranged in a different direction. Therefore, the light beam 13 totally reflected at various reflection angles at the interface 10b is
Each component of the photodiodes 17a, 17
b, 17c ... will receive light.

The photodiodes 17a, 17b, 17c ...
Each output of ... Is input to the multiplexer 18. The multiplexer 18, which is controlled by the drive circuit 25, sequentially selects the outputs of two photodiodes adjacent to each other and inputs them to the differential amplifier 22. Differential amplifier 22
Calculates the difference between the outputs of the two photodiodes sequentially input in this way. Therefore, the differential signal Sd sequentially output from the differential amplifier 22 is a plurality of photodiodes 17a,
It can be considered that the photodetection signals output by 17b, 17c ... Are differentiated with respect to their parallel arrangement direction.

The differential signal Sd sequentially output from the differential amplifier 22.
Is amplified by the amplifier 23 and then input to the A / D converter 24. The A / D converter 24 digitizes the difference signal Sd and inputs it to the signal processing unit 20.

FIG. 4 shows the light beam 13 totally reflected at the interface 10b.
The relationship between the light intensity for each incident angle θ and the output of the differential amplifier 22 will be described. Here, the interface 10b of the light beam 13
It is assumed that the relationship between the incident angle .theta. And the light intensity I is as shown in the graph of FIG.

Light incident on the interface 10b at a specific incident angle θ _SP excites surface plasmons at the interface between the metal film 12 and the sample 11, so that the reflected light intensity I of this light sharply decreases. That is, θ _SP is the total reflection elimination angle, and the reflected light intensity I takes the minimum value at this angle θ _SP . This decrease in the reflected light intensity I is observed as a dark line in the reflected light, as indicated by D in FIG.

Further, FIG. 4B shows the photodiode 17
a, 17b, 17c ... are shown in a line, and as described above, these photodiodes 17a, 17b, 17c are shown.
The position in the parallel direction of ...... uniquely corresponds to the incident angle θ.

Then, the photodiodes 17a, 17b, 17c
……, the parallel installation position, that is, the incident angle θ and the differential amplifier
The relationship between the output Sd of 22 and the differential value I ′ of the reflected light intensity I is as shown in FIG. It should be noted that, in the same figure, for the sake of simplicity of explanation, the differential amplifier 22 is shown as being connected to each of two photodiodes adjacent to each other, but in reality, the common amplifier 1 operates due to the operation of the multiplexer 18. 2 connected to two differential amplifiers 22
Two photodiodes are selected.

Based on the value of the differential value I'indicated by the output of the A / D converter 24, the signal processing section 20 causes the differential value I'which is the closest to the differential value I '= 0 corresponding to the total reflection elimination angle θ _SP. A combination of photodiodes for which "'is obtained (photodiodes 17d and 17e in the example of FIG. 4) is selected, and its differential value I'is displayed on the display means 21. Depending on the case, there may be a combination of photodiodes for which the differential value I ′ = 0 is obtained, and in that case, the differential value I ′ is naturally obtained.
= 0 is displayed.

Thereafter, every time a predetermined time elapses, a differential value I'for the outputs of the selected photodiodes 17d and 17e is obtained, and the value is displayed on the display means 21. This differential value I ′ is a value in which the curve shown in FIG. 4 (1) moves in the left-right direction due to a change in the dielectric constant of the substance in contact with the metal film 12 (see FIG. 1) of the measurement chip, that is, the refractive index. When it changes with, it goes up and down accordingly. Therefore, by continuously measuring the differential value I ′ with the passage of time, it is possible to investigate the change in the refractive index of the substance in contact with the metal film 12, that is, the change in the characteristic.

In particular, in this example, the sensing substance 30 that binds to the specific substance in the liquid sample 11 is fixed to the metal film 12, and the refractive index of the sensing substance 30 changes according to the binding state, By continuously measuring the differential value I ′, it is possible to check the state of the change in the binding state. That is, in this case, both the liquid sample 11 and the sensing substance 30 are samples to be analyzed. Examples of such a combination of the specific substance and the sensing substance 30 include an antigen and an antibody.

As is apparent from the above description, in the present embodiment, the plurality of photodiodes 17a, 17b, as the light detecting means 17,
Since 17c ... are arranged in a row in a photodiode array, even if the curve shown in FIG. Dark line detection is possible. That is, the use of such an array-shaped light detecting means 17 can secure a large dynamic range of measurement.

Further, in this example, since the signal lines H connected to the photodiodes 17a, 17b, 17c, ... Of the light detecting means 17 are grounded, charges are accumulated in the multiplexer 18 when the reflected light intensity I is measured. Then, the charge is prevented from riding on the output of the photodiodes 17a, 17b, 17c ... At the next measurement. Therefore, generation of so-called charge noise due to this accumulated charge can be prevented and a high S / N photodetection signal can be obtained, and the accuracy of sample analysis can be improved.

In order to investigate the change in the binding state between the specific substance in the liquid sample 11 and the sensing substance 30 with the passage of time, the differential value I '
In addition to obtaining and displaying, the difference ΔI ′ between the first measured differential value I ′ (0) and the differential value I ′ (t) measured after a predetermined time has elapsed.
May be displayed in search of.

Basically, the binding state between the specific substance in the liquid sample 11 and the sensing substance 30 can be examined by performing the above-mentioned operation, but as described above, the reaction characteristics of the sensing substance 30. If the thickness of the metal film 12 or the metal film 12 is not uniform within the surface of the metal film 12, or if dust is attached on the metal film 12 or the sensing substance 30, the above-mentioned bonding state is determined according to the state. There will be variations in the measurement results. Hereinafter, how to prevent the occurrence of such a problem will be described.

In this case, unlike the above-described operation, the movable table 32 is intermittently moved in the vertical direction in FIG. 2 by the drive of the actuator 33 during one measurement, and N times including the first and last stop. (N is a plurality) The moving table 32 is stopped.
Then, in each stop state, the light beam 13 is irradiated to the interface 10b in the same manner as above, and the light beam 13 is totally reflected at the interface 10b.
The intensity of 13 is detected by the photodiodes 17a, 17b, 17c .... That is, in this case, the intensity of the totally reflected light beam 13 is detected at N different light beam incident positions on the interface 10b.

Therefore, N differential values I'are input to the signal processing unit 20 from the A / D converter 24 for each of the nine positions shown in FIG. 4 (the positions of circled numbers 1 to 9). It will be. Then, the signal processing unit 20 sets the N for each position.
The median value of each of the differential values I ′ is calculated and used as the differential value I ′ that represents the position. In this way, one differential value I'obtained as representative data from the N differential values I'is used in the same manner as in the case described above, and a plurality of such differential values I '(example shown in FIG. If so, the binding state between the specific substance in the liquid sample 11 and the sensing substance 30 is examined based on 9).

The actuator 33 is driven by the overall control unit.
Controlled by the control unit 34, a predetermined timing signal is input from the overall control unit 34 to the signal processing unit 20 every time the moving table 32 is stopped N times. Then, the signal processing unit 20 takes in the differential value I ′ from the A / D converter 24 each time this timing signal is input, and obtains N differential values I ′ at each of the above positions.

As described above, one differential value I ′ obtained as representative data from the N differential values I ′ relating to different light beam incident positions on the interface 10b is the film thickness of the metal film 12 or the sensing substance. It is possible to eliminate the difference in the reaction characteristics of 30 specifically, and further, to eliminate the fluctuation caused by disturbance such as dust on the sensing substance 30. Therefore, according to this measuring device, the film thickness of the metal film 12 and the sensing substance 30
Even if the reaction characteristics of are non-uniform or dust is present on the sensing substance 30, it is possible to prevent variations in the measurement results due to them.

Further, in this measuring apparatus, since the light beam 13 is made incident so as to be focused at the interface 10b, the range of the light beam incident angle at which attenuation of total reflection is recognized does not become wide, and therefore the sensitivity is high. It becomes possible to measure.

Moreover, this measuring apparatus can basically prevent the variation of the measurement result by using only one measuring chip composed of the dielectric block 10 and the metal film 12 for one sample 11. Therefore, as compared with the case where the measurement for one sample 11 is performed using a plurality of measurement chips, the time required for the sample supply and the measurement operation can be shortened and the measurement can be performed efficiently.

In the above embodiment, the signal processing unit 20 as the calculating means is configured to obtain the median value of the N differential values I'and use it as the representative data. Instead, the average value of the data included in the upper and lower predetermined widths is calculated from the median value of the N differential values I ′,
You may make it a representative data. Moreover,
Of the N differential values I ′, the average value of the differential values I ′ excluding the maximum value and / or the minimum value thereof may be obtained and used as the representative data.

Next, a second embodiment of the present invention will be described. FIG. 5 shows a side view of the measuring device according to the second embodiment of the present invention. In FIG. 5, elements that are the same as the elements in FIG. 1 are given the same numbers, and descriptions thereof are omitted unless necessary.

This measuring apparatus is the leaky mode measuring apparatus described above, and is configured to use the dielectric block 10 formed into a measuring chip also in this example. A clad layer 40 is formed on one surface (upper surface in the figure) of the dielectric block 10, and an optical waveguide layer 41 is further formed thereon.

The dielectric block 10 is made of synthetic resin or B, for example.
It is formed using an optical glass such as K7. On the other hand, the clad layer 40 is formed in a thin film shape using a dielectric having a lower refractive index than the dielectric block 10 or a metal such as gold. The optical waveguide layer 41 is a dielectric material having a higher refractive index than the cladding layer 40,
For example, PMMA is also used to form a thin film. The clad layer 40 has a film thickness of, for example, 36.5 nm when formed from a gold thin film, and the optical waveguide layer 41 has a film thickness of, for example, PMMA.
When formed from, the thickness is about 700 nm.

In the leak mode measuring device having the above structure,
Dielectric block the light beam 13 emitted from the laser light source 14.
When the light beam 13 is incident on the cladding layer 40 through 10 at an angle of total reflection or more, the light beam 13 is totally reflected at the interface 10c between the dielectric block 10 and the cladding layer 40.
Light having a specific wave number that has passed through the optical waveguide layer 41 and is incident on the optical waveguide layer 41 at a specific incident angle propagates through the optical waveguide layer 41 in the guided mode. When the guided mode is excited in this manner, most of the incident light is taken into the optical waveguide layer 41, so that the total reflection attenuation occurs in which the intensity of the light totally reflected at the interface 10c sharply decreases.

The wave number of the guided light in the optical waveguide layer 41 depends on the refractive index of the sample 11 on the optical waveguide layer 41. Therefore, by knowing the specific incident angle at which attenuation of total reflection occurs,
It is possible to analyze the refractive index of 11 and the characteristics of the sample 11 related thereto. Then, the characteristics of the sample 11 can be analyzed based on the reflected light intensity I in the vicinity of the specific incident angle and the differential value I ′ thereof.

Also in the second embodiment, the interface 10
Statistical processing is performed on a plurality of differential values I ′ obtained under different incident positions of the light beam 13 in c to obtain a differential value I ′ as one representative data, and sample analysis is performed based on the differential value I ′. As a result, the same effect as that in the first embodiment can be obtained.

In order to change the incident position of the light beam at the interface between the dielectric block and the thin film layer, the above first
The mechanism adopted in the second embodiment, that is, the mechanism for moving the dielectric block 10 with respect to the light beam 13 is not limited to this, and another mechanism can be used. Hereinafter, third to sixth embodiments of the present invention using such another mechanism will be described. The devices of these embodiments are configured as a surface plasmon resonance measuring device, but the mechanism used in each embodiment is also applicable to the leaky mode measuring device.

FIG. 6 shows a planar shape of the optical system portion of the surface plasmon resonance measuring apparatus according to the third embodiment of the present invention. In FIG. 6 as well, elements similar to those in FIG. 2 are denoted by the same reference numerals, and description thereof will be omitted unless otherwise necessary (the same applies hereinafter).

In this embodiment, a mirror is provided between the cylindrical lens 15b and the cylindrical lens 15c.
51 and 52 are provided so that the optical path of the light beam 13 is bent twice at a right angle in a plan view, and the optical path of the light beam 13 emitted from the dielectric block 10 is also reflected twice by the mirrors 53 and 54. It is bent at a right angle. The mirrors 52 and 53 are mounted on a movable base 55 which is movable in the vertical direction in the figure, and the movable base 55 is moved in this direction by driving an actuator 33.

As is apparent from the above description, in this embodiment, the light beam 13 is blocked by the cylindrical lenses 15a, 15b, 15c and the mirrors 51, 52 as a dielectric block.
An optical system for making the light incident on 10 is configured.

In this structure, when the dielectric block 10 is kept stationary at a predetermined measuring position during measurement, and the actuator 33 is driven to move the moving table 55 in the above-mentioned direction, the dielectric block 10 and the metal film are moved. The incident position of the light beam 13 at the interface with the metal film 12 (not shown; see the metal film 12 in FIG. 1) can be changed.

In order to dispose the dielectric block 10 at the predetermined measurement position, it may be arranged automatically by using, for example, the turntable 31 shown in FIG. 1 or by manual operation. You may arrange.

Next, FIG. 7 shows a planar shape of the optical system portion of the surface plasmon resonance measuring apparatus according to the fourth embodiment of the present invention. In the present embodiment, an AOD (acousto-optical light deflector) 60 is provided between the cylindrical lens 15b and the cylindrical lens 15c, and between the cylindrical lens 15c and the dielectric block 10, as shown in FIG. A cylindrical lens 61 having a refractive power is arranged only in the plane shown, and the light beam 13 is deflected in the direction of arrow Q in the figure by driving the AOD 60.

In this structure, when the dielectric block 10 is kept stationary at a predetermined measurement position during measurement and the AOD 60 is driven to deflect the light beam 13, the dielectric block 10
The incident position of the light beam 13 at the interface between 10 and the metal film (not shown; see the metal film 12 in FIG. 1) can be changed.

In the case of the present embodiment, when the light beam 13 is deflected as described above, the light detecting means 17 for the light beam 13 is generated.
The incident position on the plane will change within the plane shown. Therefore, in this example, in order to cope with the change of the incident position, as the light detecting means 62, a light receiving element is arranged also in the direction in which the incident position changes (vertical direction in the drawing), for example, a two-dimensional CCD. Etc. are used as the light detecting means.

Next, FIG. 8 shows a planar shape of an optical system portion of the surface plasmon resonance measuring apparatus according to the fifth embodiment of the present invention. The surface plasmon resonance measuring apparatus of the present embodiment is different from the apparatus shown in FIG. 7 in that instead of the cylindrical lens 16, a condensing lens 65 made of a circular lens having a refractive power in the plane shown in FIG. 8 is used. , Again 2
The difference is that the light detecting means 62 including a one-dimensional direction is used instead of the light detecting means 62 such as a dimensional CCD.

In this structure, the light beam 13 emitted from the dielectric block 10 is condensed by the condenser lens 65 even in a plan view, so that it is always guided to the thin light receiving portion of the light detecting means 17 regardless of its deflected state. He will then converge.

Next, FIG. 9 shows a planar shape of the optical system portion of the surface plasmon resonance measuring apparatus according to the sixth embodiment of the present invention. In the surface plasmon resonance measuring apparatus of the present embodiment, the light beam 13 emitted from the cylindrical lens 15b is branched into three systems by the half mirrors 70, 71 and the mirror 72 so as to proceed in parallel with each other. Therefore, the light beams 13 of these three systems are simultaneously incident on mutually different positions at the interface between the dielectric block 10 and the metal film (not shown; see the metal film 12 in FIG. 1).

In this embodiment, a plurality of light beams 13 which are totally reflected at different positions on the interface are simultaneously emitted. Therefore, in the present example, in order to correspond to the plurality of light beams 13, as the light detection means 62, a light receiving element is arranged also in the arrangement direction of the plurality of light beams 13 (vertical direction in the drawing). For example, a light detecting means such as a two-dimensional CCD is used.

Next, the side surface shape of the surface plasmon resonance measuring apparatus according to the seventh embodiment of the present invention will be shown. In the surface plasmon resonance measuring apparatus of this embodiment, as in the sixth embodiment, the light beam is simultaneously incident on three different positions at the interface 10b between the dielectric block 10 and the metal film 12. Is configured to let.

That is, here, three blocks of optical fibers 334a, 334b and 334c and three CCDs 360, 360b and 360c are arranged in such a manner that the block 2 holding the dielectric block 10 is sandwiched from the left and right respectively. A collimator lens 350a-c, an interference optical system, a condenser lens 355a-c, and an aperture 356a-c are disposed between the optical fibers 334a-c and the CCD 360a-c. The interference optical system includes polarization filters 351a to 351a ...
c, half mirrors 352a-c, half mirrors 353a-c, and mirrors 354a-c. CCD3 above
60a to 60c are connected to the signal processing unit 361, and the signal processing unit 3
Reference numeral 61 is connected to the display unit 362.

The measurement of the sample by the surface plasmon resonance measuring apparatus of this embodiment will be described below. Here, one system from the optical fiber 334a to the CCD 360a will be described as an example, but the measurement is similarly performed in other systems.

The rear end (not shown) of the optical fiber 334a is
It is optically connected to a light source similar to the laser light source 14 shown in FIG. 1 and the like, and a light beam 330 is emitted from the tip thereof in a divergent state. This light beam 330 is used by the collimator lens 3
The light is collimated by 50a and enters the polarization filter 351a. A linearly polarized light beam 33 that passes through the polarization filter 351a and is incident on the interface 10b as p-polarized light 33
In the case of 0, a part is split as a reference light beam 330R by the half mirror 352a, and the remaining light beam 330S transmitted through the half mirror 352a enters the interface 10b.

The light beam 330 totally reflected at the interface 10b
The reference light beam 330R reflected by S and the mirror 354a enters the half mirror 353a and is combined therewith. The combined light beam 330 'is condensed by the condenser lens 355a, passes through the aperture 356a, and is detected by the CCD 360a. At this time, the light beam 330 'is changed to the light beam 330S.
Interference fringes are generated according to the state of interference between the reference light beam 330R and the reference light beam 330R, and the interference fringes are detected by the CCD 360a. Here, the sensing substance 30 fixed on the surface of the metal film 12 is in the sample 11. It binds to a specific substance. Examples of such a combination of the specific substance and the sensing substance 30 include an antigen and an antibody. In that case, the presence or absence of the antigen-antibody reaction can be detected by detecting the change in the interference fringes detected by the CCD 360a. That is, in this case, when the refractive index of the sensing substance 30 changes in accordance with the binding state between the specific substance and the sensing substance 30, the light beam 330 totally reflected at the interface 10b.
S and reference light beam 330R are half mirrors 353
Since the state of interference changes when combined by a,
The antigen-antibody reaction can be detected according to the change in the interference fringe. In this case, both the sample 11 and the sensing substance 30 are samples to be analyzed.

The signal processing unit 361 detects the presence or absence of the above reaction based on the above principle, and the result is displayed on the display unit 362. At this time, the output signals of the remaining two CCDs 360b and 360c in addition to the CCD 360a are also processed by the signal processing unit.
Input to 361, the signal processing unit 361 obtains representative data of those signals, for example, an average value, and detects the presence or absence of the reaction based on the representative data.

In the present embodiment, by detecting the presence or absence of the above reaction based on the above representative data, the film thickness of the metal film 12 and the reaction characteristics of the sensing substance 30 are different, Can eliminate the influence of disturbance such as dust on the sensing substance 30 and accurately detect the presence or absence of the above reaction.

[Brief description of drawings]

FIG. 1 is a partially cutaway side view showing a surface plasmon resonance measuring apparatus according to a first embodiment of the present invention.

FIG. 2 is a plan view showing an optical system portion of the surface plasmon resonance measuring apparatus.

FIG. 3 is a block diagram showing an electrical configuration of the surface plasmon resonance measuring apparatus.

FIG. 4 is a schematic diagram showing a relationship between a light beam incident angle and a detected light intensity, and a relationship between a light beam incident angle and a differential value of a light intensity detection signal in the surface plasmon resonance measuring apparatus.

FIG. 5 is a partially cutaway side view showing a leaky mode measuring device according to a second embodiment of the present invention.

FIG. 6 is a plan view showing an optical system portion of a surface plasmon resonance measuring apparatus according to a third embodiment of the present invention.

FIG. 7 is a plan view showing an optical system portion of a surface plasmon resonance measuring apparatus according to a fourth embodiment of the present invention.

FIG. 8 is a plan view showing an optical system portion of a surface plasmon resonance measuring apparatus according to a fifth embodiment of the present invention.

FIG. 9 is a plan view showing an optical system portion of a surface plasmon resonance measuring apparatus according to a sixth embodiment of the present invention.

FIG. 10 is a side view showing a surface plasmon resonance measuring apparatus according to a seventh embodiment of the present invention.

[Explanation of symbols]

10 Dielectric block 10a Dielectric block sample holder 10b Interface between dielectric block and metal film 10c Interface between dielectric block and cladding layer 11 samples 12 Metal film 13 light beam 13P fluorescence image 13D diffused image 14 Laser light source 15 Optical system 15a, 15b, 15c, 16 Cylindrical lens 17 Light detection means (photodiode array) 17a, 17b, 17c ... Photodiodes 18 multiplexer 19 driver 20 Signal processing unit (calculation means) 21 Display means 22 Differential amplifier 23 amplifier 24 A / D converter 25 Drive circuit 26 Controller 30 Sensing substances 31 turntable 32 mobile platform 33 Actuator 34 Overall control unit 40 clad layer 41 Optical waveguide layer 51, 52, 53, 54 mirror 55 Mobile platform 60 AOD (acousto-optic light deflector) 61 Cylindrical lens 62 Light detection means 65 Condensing lens 70,71 Half mirror 72 mirror 330 light beam 334a-c Optical fiber 350a-c Collimator lens 351a-c Polarizing filter 352a-c Half mirror 353a-c Half mirror 354a-c Mirrors 355a-c Condensing lens 356a-c aperture 360a-c CCD 361 Signal processor

Claims (11)

[Claims] 1. A dielectric block, a thin film layer formed on one surface of the dielectric block and brought into contact with a sample, a light source for generating a light beam, and the light beam with respect to the dielectric block. Measurement comprising: an incident optical system that makes an incident angle at which the total reflection condition is obtained at the interface between the dielectric block and the thin film layer; and a light detection unit that measures the intensity of the light beam totally reflected at the interface. In the device, means for relatively moving the optical system and the dielectric block so that the incident position of the light beam at the interface changes, and the light detection when the incident positions of the light beam are different from each other. And a calculation unit that statistically processes a plurality of types of data corresponding to the intensity output by the unit to obtain one representative data corresponding to the intensity. Place 2. A dielectric block, a thin film layer made of a metal film formed on one surface of the dielectric block and contacting a sample, a light source for generating a light beam, and the dielectric block On the other hand, an incident optical system that makes an incident angle at which the total reflection condition is obtained at the interface between the dielectric block and the metal film, and a light detection unit that measures the intensity of the light beam totally reflected at the interface are provided. In the measuring apparatus, the means for relatively moving the optical system and the dielectric block so that the incident position of the light beam on the interface changes, and respectively when the incident position of the light beam is different from each other. Arithmetic means for statistically processing a plurality of types of data corresponding to the intensities output from the light detecting means to obtain one representative data corresponding to the intensities. Measuring device. 3. A thin film layer comprising a dielectric block, a clad layer formed on one surface of the dielectric block, and an optical waveguide layer formed on the clad layer and brought into contact with a sample, and a light beam A light source to be generated, an incident optical system that causes the light beam to be incident on the dielectric block at an incident angle at which total reflection conditions are obtained at the interface between the dielectric block and the cladding layer, and total reflection is performed at the interface. In a measuring device comprising a light detecting means for measuring the intensity of a light beam, a means for relatively moving the optical system and the dielectric block so that the incident position of the light beam at the interface changes, When the incident positions of the light beams are in different states, a plurality of types of data corresponding to the intensities, which are output by the light detecting means, are statistically processed to obtain one data corresponding to the intensities. And a calculation means for obtaining the representative data of the measuring device. 4. A dielectric block, a thin film layer formed on one surface of the dielectric block and brought into contact with a sample, a light source for generating a light beam, and the light beam with respect to the dielectric block. Measurement comprising: an incident optical system that makes an incident angle at which the total reflection condition is obtained at the interface between the dielectric block and the thin film layer; and a light detection unit that measures the intensity of the light beam totally reflected at the interface. In the device, the optical system is formed so that the light beam can be made incident on a plurality of different incident positions on the interface, and the light detection means is respectively provided when the incident positions of the light beam are different. A measuring device provided with an arithmetic means for statistically processing a plurality of types of data corresponding to the intensity to be output and obtaining one representative data corresponding to the intensity. 5. A dielectric block, a thin film layer made of a metal film formed on one surface of the dielectric block and brought into contact with a sample, a light source for generating a light beam, and the dielectric block On the other hand, an incident optical system that makes an incident angle at which the total reflection condition is obtained at the interface between the dielectric block and the metal film, and a light detection unit that measures the intensity of the light beam totally reflected at the interface are provided. In the measuring device, the optical system is formed so that the light beam can be made incident on a plurality of different incident positions on the interface, and the light beams are respectively generated when the incident positions of the light beam are different. A measuring device, comprising: a calculation unit that statistically processes a plurality of types of data corresponding to the intensities output by the detection unit to obtain one representative data corresponding to the intensities. 6. A thin film layer comprising a dielectric block, a clad layer formed on one surface of the dielectric block, and an optical waveguide layer formed on the clad layer and brought into contact with a sample, and a light beam A light source to be generated, an incident optical system that causes the light beam to be incident on the dielectric block at an incident angle at which total reflection conditions are obtained at the interface between the dielectric block and the cladding layer, and total reflection is performed at the interface. In a measuring device comprising a light detecting means for measuring the intensity of a light beam, the optical system is formed so that the light beam can be made incident on a plurality of different incident positions on the interface, and the light beam When the incident positions of are different from each other, the plurality of kinds of data corresponding to the intensities output by the photodetector are statistically processed to obtain one representative data corresponding to the intensities. A measuring device provided with a calculating means for obtaining. 7. The optical system splits one light beam into a plurality of light beams, and makes these light beams respectively enter a plurality of different incident positions on the interface. Item 7. A measuring device according to any one of items 4 to 6. 8. The optical system according to claim 4, wherein the optical system deflects one light beam and makes the light beam incident on a plurality of different incident positions on the interface.
The measuring device according to the item. 9. The calculating means obtains median values of a plurality of types of data corresponding to the intensity, and calculates the median values.
9. The measuring device according to claim 1, wherein the measuring device is one representative data. 10. The arithmetic means obtains an average value of data included in a predetermined upper and lower width from median values of a plurality of kinds of data corresponding to the intensity, and sets it as the one representative data. The measuring device according to any one of claims 1 to 8, wherein the measuring device comprises: 11. The calculating means obtains an average value of data excluding a maximum value and / or a minimum value of a plurality of kinds of data corresponding to the intensity, and sets it as the one representative data. The measuring device according to any one of claims 1 to 8, wherein the measuring device comprises: