JP2003287493A - Measuring apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve the throughput of screening in a measuring apparatus utilizing a total reflected light. <P>SOLUTION: The measuring apparatus comprises a reflection optical system 35 having a plurality of measuring units in a series relation with an optical beam 30. An optical beam 30 reflected on an interface of the wells 51 of a row Q1 by branching and introducing in parallel the beam 30 emitted from a light source 10 by an incident means 20 to an interface of a plurality of wells 51 aligned in a direction P of a row Q1 of a measuring plate 50 having the measuring units each having the well 51, a metal film 12 formed at a bottom of the wells and a dielectric block 52 projected under the wells arranged in a two-dimensional manner is reflected by a concave mirror 31 of the reflection optical system 35, introduced to the interface of the well 51 of a row Q2, and further the optical beam 30 reflected from the interface is reflected from the concave mirror 32, introduced to the interface of the wells 51 of a row Q3, and the optical beam reflected from the interface is detected by photodetecting means 27a to 73e. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a measuring apparatus utilizing generation of an evanescent wave due to totally reflected light, such as a surface plasmon measuring apparatus for analyzing characteristics of a substance 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 measuring devices have been proposed which analyze the characteristics of a substance to be measured 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).

The surface plasmon measuring device using the above system is basically a dielectric block formed in a prism shape, for example, and is contacted with a substance to be measured such as a liquid sample formed on one surface of the dielectric block. A metal film, a light source that generates a light beam, and an optical system that causes the light beam to enter the dielectric block at various angles so that total reflection conditions can be obtained at the interface between the dielectric block and the metal film. And a light detecting means for measuring the intensity of the light beam totally reflected at the interface to detect the state of surface plasmon resonance, that is, the state of attenuated total reflection.

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

In the surface plasmon measuring device having the above structure, when a light beam is incident on the metal film at a specific incident angle of a total reflection angle or more, an evanescent light having an electric field distribution in the substance to be measured in contact with the metal film. A wave is generated, and the surface plasmon is excited at the interface between the metal film and the substance to be measured by this 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. Note that 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.

If the wave number of the surface plasmon is known from the incident angle at which the attenuated total reflection (ATR) occurs, that is, the attenuated total reflection angle θsp, the dielectric constant of the substance to be measured can be obtained. That is, the wave number of the surface plasmon is Ksp, the angular frequency of the surface plasmon is ω, c is the speed of light in vacuum, and ε _m And ε _s Are the metals and the permittivity of the substance to be measured, respectively, the following relationships are established.

[0008]

[Equation 1] That is, by knowing the total reflection attenuation angle θsp, which is the incident angle at which the reflected light intensity decreases, the dielectric constant ε of the measured substance
It is possible to obtain a characteristic related to _s , that is, the refractive index.

Further, as a similar measuring apparatus 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 for contact with a sample solution. An optical waveguide layer, a light source for generating a light beam, and the light beam at various angles with respect to the dielectric block so that total reflection conditions can be obtained at the interface between the dielectric block and the cladding layer. The optical system is configured to be incident, and a light detection unit that measures the intensity of the light beam totally reflected at the interface and detects the excited state of the guided mode, that is, the attenuated total reflection state.

In the leaky 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 substance to be measured on the optical waveguide layer, the refractive index of the substance to be measured and the related measured substance to be measured can be obtained by knowing the specific incident angle at which attenuation of total reflection occurs. The properties of the substance can be analyzed.

As a measuring apparatus using total reflection such as a surface plasmon resonance measuring apparatus or a leaky mode measuring apparatus, light is made incident on an interface at an incident angle at which total reflection conditions are obtained, and an evanescent wave is generated by the light. When measuring the characteristics of the substance to be measured by measuring the change in the state of the light totally reflected at the interface, the device for measuring the specific incident angle that causes the above-mentioned attenuation of total reflection is used. A device that makes a light beam enter the interface and detects the degree of attenuation of total internal reflection for each angular wavelength, or makes the light beam enter the interface and divides a part of this light beam before it enters the interface. There are various types such as a device that interferes the generated light beam with the light beam reflected at the interface and measures the state of the interference.

[0012]

By the way, in the above-provided surface plasmon resonance measuring devices, leak mode sensors, and other measuring devices that utilize totally reflected light,
When measuring a large number of samples, there is a problem that the measurement takes a long time. In particular, in the case of performing several measurements at one time interval for one sample in order to detect a change associated with, for example, an antigen-antibody reaction or a chemical reaction,
If the measurement of one sample is not completed, the measurement of a new sample cannot be started, and it takes a very long time to measure the entire sample.

Therefore, in order to make it possible to measure a large number of samples in a short time, a plurality of light beams are made to respectively enter a plurality of dielectric blocks arranged in parallel, and each of the dielectric blocks is made to have a plurality of light beams. The reflected light beams reflected by the interface and propagating in mutually parallel optical paths are individually received by a plurality of light receiving means arranged in parallel corresponding to each dielectric block, and the state of total reflected light is obtained. A measuring device for detecting is also under consideration. With such a measuring device, it is possible to measure a plurality of samples at the same time, and thus it is possible to improve the throughput.

However, even with a measuring device capable of simultaneously measuring N samples in one measurement, the time required for one measurement is 10 minutes, and for example, the time required for measuring 10,000 samples is 1
It is 0000 × 10 / N (minute). It is possible to increase the number of specimens to be measured at the same time by increasing the number of light detecting means, but there is a limit because the apparatus is enlarged. That is, a conventional measuring device capable of simultaneously measuring N samples is N
Since the number of samples that can be measured at the same time cannot be made larger than the number of light detecting means, the number of specimens that can be simultaneously measured is limited, and thus there is a limit in shortening the time required for screening.

On the other hand, the number of hits of the sample in the screening is about 0.01 to 0.1%, and there is a case where there is no hit in one sample among N samples that can be measured at the same time. Therefore, in the case of a 98-well multititer plate-like measurement plate, there may be no hit in one measurement plate of 98 samples. Therefore, it is not necessary to spend time on such a measuring plate to perform highly accurate measurement.

In view of the above circumstances, it is an object of the present invention to provide a measuring device capable of performing simple screening at higher speed.

[0017]

The measuring device of the present invention comprises a light source for generating a light beam, a dielectric block transparent to the light beam, a thin film layer formed on the upper surface of the dielectric block, and A plurality of measurement units provided with a sample holding mechanism for holding a sample on the surface of the thin film layer, and the light beam to the dielectric block of the first measurement unit of the plurality of measurement units, A second to (2 + n) th measurement unit that reflects the light beam totally reflected at the incident optical system that makes the incident angle at which the total reflection condition is obtained at the interface between the dielectric block and the thin film layer, and the interface. To the dielectric block, and a reflective optical system that sequentially makes incident at an angle of incidence where total reflection conditions are obtained at the interface between the dielectric block and the thin film layer, and the interface of the (2 + n) th measurement unit. And it is characterized in by comprising a light detection means for measuring the intensity of the light beam totally reflected.

That is, in the measuring apparatus of the present invention, one light detecting means detects the light beam in which the state of the totally reflected light in the plurality of measuring units arranged in series with respect to the light beam is reflected. It is characterized by.

A plurality of light detecting means may be provided, and a light beam reflecting the state of the total reflected light in the plurality of measuring units may be detected for each light detecting means.

Here, n is a natural number (0, 1, 2, ...
・)

The measuring apparatus may be configured such that the thin film layer is made of a metal film and the measurement is performed by utilizing the effect of the surface plasmon resonance described above.

In the measuring device, the thin film layer is composed of a clad layer formed on the upper surface of the dielectric block and an optical waveguide layer formed on the clad layer. The measurement may be performed by utilizing the effect of the excitation of the wave mode.

Furthermore, in the measuring device according to the present invention, 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. The incident light is made incident at various incident angles where total reflection conditions are obtained at the interface, the intensity of the light beam totally reflected at the interface is measured at each position corresponding to each incident angle, and the position of the dark line generated by the attenuated total reflection is measured. Sample analysis may be carried out by detecting (angle), DVNoort, K.jo
hansen, C.-F.Mandenius, Porous Gold in Surface Plas
mon Resonance Measurement, EUROSENSORS XIII, 1999,
As described in pp.585-588, the light beams of multiple wavelengths are made incident at the incident angle at which the total reflection condition is obtained at the interface, and the intensity of the light beam totally reflected at the interface for each wavelength is measured. The sample analysis may be performed by measuring and detecting the degree of attenuation of total reflection for each wavelength.

Also, PINikitin, ANGrigorenko, AAB
eloglazov, MVValeiko, AISavchuk, OASavchuk, Sur
face Plasmon Resonance Interferometry for Micro-Ar
rayBiosensing, EUROSENSORS XIII, 1999, pp.235-238
The light beam is incident at an angle of incidence such that total internal reflection conditions are obtained at the interface, and a portion of the light beam is split before the light beam enters 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 measuring the intensity of the light beam after the interference.

In the case of a measuring device which detects the position (angle) of a dark line generated by attenuation of total reflection by injecting a light beam at various incident angles at which total reflection conditions are obtained, the reflection optical system is It can be configured by one or a plurality of optical elements having a function and a light converging function. Specifically, it may be provided with a concave surface and a mirror each having a light converging function and a reflecting function, or may be provided with a mirror as a reflecting element and a convex lens as a light converging element. . The concave surface having a light converging function and the mirror having a reflecting function may be configured to include a concave mirror having both functions at the same time.
Further, a mirror is provided as a reflective element having a reflective function,
The interface of the measurement unit may be concave so that the interface has a light converging function. The reflective optical system may be provided in a part of the measurement unit or may be provided separately from the measurement unit.

Further, in the case of a measuring device in which a light beam is made incident at a constant incident angle and the wavelength or phase of the light beam is used, the reflection optical system can be constituted by only a reflection element, and the light converging function is not provided. Not necessarily required. Also in this case, the reflection optical system may be provided in a part of the measurement unit or may be provided separately from the measurement unit.

Further, the dielectric block may be formed as one block having all of the incident surface and the exit surface of the light beam, and the surface on which the thin film layer is formed, or the light block. A portion having a beam incident surface and a beam emitting surface, and a portion having a surface on which the thin film layer is formed.
One of them may be joined via a refractive index matching means.

The plurality of measuring units may be any one of a one-dimensional array, a two-dimensional array, and a three-dimensional array, and the reflection optical system may have a structure corresponding to the array.

The plurality of measuring units may be individually formed one by one, or may be one-dimensionally or two-dimensionally arranged and integrally formed. Further, the measurement unit may be provided with a sample supplying / discharging means for continuously supplying the sample onto the surface of the thin film layer and continuously discharging the supplied sample.

The intensity of the reflected light is measured by the light detecting means to confirm the state of the reflected light. The presence or absence of a hit in the plurality of measurement units that are the measurement targets is obtained by the change in the state of the reflected light with time. If it is confirmed that one of the multiple measurement units that was the target of measurement has hit, the light of the light beam is incident on the interface of each measurement unit to determine which sample of which measurement unit has hit. It may be specified by performing measurement using a conventional measuring device having a high measurement accuracy for measuring the reflected light from. The measuring device of the present invention may have the function of a conventional measuring device.

In particular, when the measuring device of the present invention is a measuring device which performs measurement based on the incident angle at which attenuation of total reflection occurs, it is assumed that the thin film layers of the plurality of measuring units have different thicknesses. , The refractive index of the dielectric block is different, the refractive index of the buffer dispensed to each measurement unit is different, or a plurality of the reflection optical system, which is arranged according to the number of measurement units. The dark lines generated by the plurality of measurement units may be separated so that the reflection elements have different angles.

[0032]

EFFECTS OF THE INVENTION The measuring apparatus of the present invention is provided with the reflection optical system, so that a plurality of measuring units are arranged in series with respect to one light beam, and the incidence and incidence are performed at the interfaces of the plurality of measuring units. Since the intensity of the light beam that has been repeatedly reflected is measured by the light detecting means, it is possible to detect the state of the totally reflected light in the plurality of measuring units with one light detecting means. Therefore, the throughput can be improved as compared with the conventional measuring device.

For example, in the case of a measuring device equipped with N light detecting means, conventionally, only N measuring units were measured at the same time, but as in the present invention, one light beam is measured. If a plurality of measuring units are arranged in series so that the light beam passing through the M measuring units is received by one light detecting means, N × M at the same time.
It is possible to perform measurements on individual specimens, and
The object can be measured at double speed. For example, in the case of screening for measuring the presence or absence of binding with a specific sensing substance for tens of thousands of analytes, the effect of shortening the time is remarkable.

[0034]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings. The sensor utilizing total internal reflection light of the embodiment of the present invention is a device for performing random screening to find a specific substance that binds to a desired sensing substance in the field of drug discovery research, and is used for high-speed simple screening. The light beams are made to enter the plurality of dielectric blocks in parallel, and the intensities of the light beams that have undergone the reflection and reflection to the plurality of measurement units arranged in series are measured by the light detection means, Is a surface plasmon sensor that uses surface plasmon resonance to simultaneously detect the attenuated total reflection state of the measurement unit. FIG. 1 is a plan view showing a schematic configuration of a surface plasmon sensor according to a first embodiment of the present invention, and FIG. 2 shows a side surface shape of this surface plasmon sensor.

In this surface plasmon sensor 1,
A measurement plate 50 having a plurality of wells (liquid reservoirs) 51 holding the sample 15 is provided, and the measurement of the plurality of samples 15 can be performed efficiently in a short time. The measuring plate 50 is made of, for example, a transparent resin, and has a plurality of wells 51 each having a shape obtained by cutting out a part of a cone on the upper surface as shown in an enlarged view in FIG. The membrane 12 and the sensing substance 14 are fixed in this order. Further, a dielectric block portion 52 protruding downward from each of the wells 51 is formed, and the measuring light beam 30 is made incident on the interface between the dielectric block portion 52 and the metal film 12. That is, one well 51, the metal film 12 on the bottom of the well and the dielectric block 52 constitute one measurement unit, and the measurement plate 50 is an assembly in which a plurality of measurement units are two-dimensionally arranged. Can be regarded as As shown in FIG. 2, the wells 51 are formed side by side in the horizontal direction (arrow P direction) and the vertical direction (arrow Q direction).
Is provided for measurement while being set on the measurement unit support base 60.

This surface plasmon sensor includes a laser light source 10 for emitting a light beam 30 and a light beam 30 emitted from the light source 10 on a plate 50 in the first row in the direction of arrow Q (hereinafter
The light reflected by the interface between the incident optical system 20 that makes parallel incidence on the bottom surface of a plurality (five in this example) of wells 51 arranged in the direction of arrow P in the Q1 column and the wells 51 in the Q1 column. Beam 30 in the second row in the direction of arrow Q in the same row (hereinafter,
A reflection optical system that makes the light beam 30 incident on the well 51 of the Q2 row) and further reflected on the interface of the well 51 of the Q2 row enters the well 51 of the third row (hereinafter referred to as the Q3 row) in the arrow Q direction. 35 and the light beam 30 reflected at the interface of wells 51 in Q3 row
The number of wells in the direction of arrow P is the same as that of the five light detecting means 27a to 27e.

The incident optical system 20 includes a collimator lens 21 for collimating a light beam 30 emitted from the laser light source 10 in a divergent light state, and half mirrors 22a to 22d for branching the light beam 30.
And a mirror 23, a cylindrical beam expander 24a-e for expanding the diameter of the branched light beam 30 only in the plane shown in FIG. 2, and a mirror 25a-for reflecting the light beam 30.
e, the light beam 30 reflected by the mirrors 25a-e is shown in FIG.
Cylindrical lens 26 that collects light only in the plane shown in
a to e.

The reflection optical system 35 is a concave mirror 3 fixed to a part of a measurement unit support base 60 that supports the measurement plate 50.
As shown in FIG. 3, the light beam 30 reflected at the interface of the well 51 in the Q1 row is reflected by the concave mirror 31, and is reflected at the interface of the well 51 in the Q2 row as shown in FIG. The light beam 30 reflected by the interface of the well 51 in the Q2 row is reflected by the concave mirror 32 and is similarly converged so as to be incident within the same incident angle range as that of the well in the Q3 row.
It is incident on the interface of 51. Due to the reflective optical system 35, the three wells 51 arranged in the Q direction are in series with the light beam 30.

Each of the light detecting means 27a to 27e is composed of a line sensor in which a large number of light receiving elements are arranged in one line, and the light receiving elements are arranged in the direction of arrow X in FIG. It is distributed.

A light beam 30 emitted from one laser light source 20 in a divergent state is collimated by a collimator lens 21 and then converted into a parallel beam by a half mirror 22a to 22d and a mirror 23.
The five branched light beams 30 are incident on the bottom surface of the well 51, that is, the interface between the dielectric block portion 52 and the metal film 12.

At this time, each of the branched light beams 30 is expanded in diameter only in the plane shown in FIG. 2 by the cylindrical beam expanders 24a to 24e, and is reflected by the mirrors 25a to 25e to change the traveling direction. After changing, the light is condensed only in the plane shown in FIG. 2 by the cylindrical lenses 26a to 26e. Thereby, each light beam 30 is applied to the interface between the dielectric block portion 52 and the metal film 12,
It is incident with various incident angle components. The laser light source 20 is arranged so that the linearly polarized light beam 30 enters the interface in the p-polarized state. In addition, in order to make the light beam 30 incident on the interface as p-polarized light, the polarization direction of the light beam 30 may be controlled by a wave plate.

Since the light beam 30 is condensed as described above, it will contain components that are incident on the interface at various incident angles θ. The incident angle θ is set to an angle equal to or larger than the total reflection angle. Therefore, the reflected light beam totally reflected at the interface
30 will include components that are totally reflected at various reflection angles. The incident optical system 20 may be configured so that the light beam 30 is incident on the interface in a defocused state without being condensed in a point shape. By doing so, the light beam 30 is totally reflected in a wider area on the interface,
The detection error of the attenuated total reflection state is averaged, and the measurement accuracy of the total reflection elimination angle can be improved.

The sample analysis by the surface plasmon sensor having the above structure will be described below.

As shown in FIG. 2, the light beam 30 emitted from the laser light source 10 is focused on the interface between the dielectric block 52 of the well 51 in the Q1 column and the metal film 12 through the incident optical system 20. The reflected light beam 30 that is converged on the interface and totally reflected at this interface is incident on the interface of the well 51 of the next Q2 row,
The light is reflected at the interface, and then is incident on the interface of the well 51 in the next Q3 row and reflected. The reflected light beam reflected by the interface of the wells 51 in the Q3 row is detected by the light detecting means 27a to 27e. The light detecting means 27a to 27e are photodiode arrays in which photodiodes, which are a plurality of light receiving elements, are arranged in a line. Therefore, different photodiodes receive the respective components of the reflected light beam 30 that are totally reflected at various reflection angles at the interface. Then, the light receiving means 27a to 27e output a signal indicating the intensity distribution of the reflected light beam 30 detected by each photodiode.

The component of the light beam 30 incident on the interface at the specific incident angle θ _SP excites surface plasmons at the interface between the metal film 12 and the liquid sample 15, so that the reflected light intensity sharply decreases for this light. . That is, the specific incident angle θ _SP is a total reflection elimination angle, and the reflected light intensity shows a minimum value at this angle θ _SP . The area where the reflected light intensity decreases is
Observed as a dark line in the reflected light beam 30. When the dielectric constant of the substance in contact with the metal film 12 on the bottom surface of the well, that is, the refractive index changes, the light intensity minimum region (dark line position) correspondingly changes.
Fluctuates horizontally.

In the case of this embodiment, since the light beam 30 repeats the incident reflection at the interfaces of the three wells 51, dark lines should be generated in the reflected lights at the respective interfaces.
Here, since the light beam is incident on each interface in the same incident angle range, the dark line positions are substantially the same before the sample is dropped, and the incident angle θ of the light beam 30 on the interface and the light detection means receive the light beam. The relationship with the light intensity I of the light beam shows a profile having a minimum value only in θ ₀ , as shown in FIG. After dropping the sample solution, the profile of the light beam is monitored and changes in the dark line are observed. After a lapse of a predetermined time, for example, as shown in FIG. 4B, when the minimal value appears separately in θ ₁ and θ ₂ , it means that the coincident dark line position is displaced. It is judged that the sample in any one of the two wells has bound (hit) to the sensing substance 14. On the other hand, it is judged that the sample in any of the wells has not been hit unless there is a dark line that does not cause a specific change without separation. Therefore, the group of wells judged to have no hit can be processed at a throughput three times faster than the conventional one. For the group of wells in which binding has occurred, that is, the group of hits, the change in the dark line position is measured using a conventional device for each well to identify in which well the hit has occurred. As described above, in the screening in the field of drug discovery research, since the number of hits is 0.01% to 0.1%, a plurality of wells are arranged in series with the light beam at the same time as in the present embodiment. By performing the measurement, the number of wells to be accurately measured can be significantly reduced, and as a result, the throughput can be improved and the speed can be increased.

In the above surface plasmon sensor, a leak mode sensor can be obtained by changing a part of the structure of the measuring unit. Hereinafter, an embodiment in which the leaky mode sensor is used as the measuring apparatus of the present invention will be described with reference to the drawings.

FIG. 5 is a diagram showing an example of a measuring unit of the leak mode sensor. In addition, in FIG.
Elements that are the same as the elements inside are given the same numbers, and descriptions thereof are omitted unless otherwise necessary.

The leaky mode sensor has, as a measuring unit, a measuring plate 50 'having a plurality of wells 51 similar to the above. However, as shown in FIG.
A clad layer 40 is formed on the bottom surface of 51 instead of the metal film,
Further, an optical waveguide layer 41 is formed on it. The other structure is the same as that of the surface plasmon sensor described above.

The measuring plate 50 'is made of, for example, synthetic resin or optical glass such as BK7. On the other hand, the clad layer 40 is formed in a thin film shape using a dielectric material having a lower refractive index than the plate 50 ′ 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 36.5 nm when it is formed of a gold thin film, and the optical waveguide layer 41 has a film thickness of, for example, PM.
When formed from MA, the thickness is about 700 nm.

In the leaky mode sensor having the above structure,
When the light beam 30 emitted from the laser light source 10 is made incident on the cladding layer 40 through the dielectric block portion 52 at an incident angle of not less than the total reflection angle, many components of the light beam 30 are generated by the dielectric block portion 52. Light having a specific wave number that is totally reflected at the interface with the cladding layer 40 but is transmitted through the cladding layer 40 and incident on the optical waveguide layer 41 at a specific incident angle is propagated through the optical waveguide layer 41 in a guided mode. become. When the guided mode is excited in this way, most of the incident light that has entered at the specific incident angle is taken into the optical waveguide layer 41, so that the intensity of the light that is incident on the interface at the specific incident angle and totally reflected is sharply reduced. Attenuation of total reflection occurs. Since the wave number of the guided light in the optical waveguide layer 41 depends on the refractive index of the liquid sample 15 on the optical waveguide layer 41, it is possible to know the change in the total reflection elimination angle, which is the specific incident angle where attenuation of total reflection occurs. The presence or absence of a specific substance can be obtained.

Also in the leak mode sensor, the measurement unit support 60 is provided with the reflection optical system 35, and a plurality of wells are provided.
The light beam 30 which is sequentially incident on the interface of 51 and is reflected by the interface
By measuring the intensity of, it is possible to obtain the presence or absence of hits in the plurality of wells 51 arranged in series,
Throughput can be improved.

In the above embodiment, the reflection optical system is provided on the measurement unit support, but as shown in the sectional view of the measurement unit and the reflection optical system in FIG. The measurement plate and the reflection optical system may be integrally configured. The measurement plate 70 shown in FIG. 6 is provided with a plurality of wells 71 that are two-dimensionally arranged in the arrow P and Q directions, like the measurement plate 50 shown in FIG. On the other hand, the dielectric block portion is not formed to project for each individual well,
A dielectric block portion 72 formed in the lower part of the wells aligned in the arrow Q direction and extending in the Q direction is provided. A part of the bottom surface of the dielectric block portion 72 is concave surfaces 73 and 74, and the concave surfaces 73 and 74 are mirror surfaces, and the reflection optical system functions as a concave mirror having a light converging function and a reflecting function. Make up 75.

The light beam 30 is incident from one surface 72a of the dielectric block portion 72 toward the interface of the well 71 in the Q1 row, and the light beam 30 reflected at the interface is formed on a part of the dielectric block portion 72. The light beam 30 reflected by the concave surface 73 is incident so as to converge on the interface of the well 71 in the Q2 row, and the light beam 30 reflected by the interface is further reflected by the concave surface 74 to form the interface of the well 71 in the Q3 row. Is incident so as to converge on the interface. The light beam 30 reflected at the interface of the wells 71 in the Q3 row is emitted from the other surface 72b of the dielectric block portion 72 and is received by the light detecting means.

FIG. 7 shows a sectional view of still another measuring unit and reflective optical system. A measurement plate 80, which is an assembly of measurement units shown in FIG. 7, is provided with a plurality of wells 81 arranged two-dimensionally in the arrow P direction and the Q direction, like the measurement plate 50 shown in FIG. A dielectric block portion 82 formed in the lower part of the wells aligned in the arrow Q direction and extending in the Q direction is provided. However, the bottom surface of the dielectric block portion 82 is a flat surface, and a mirror surface 84 is provided on a part thereof. Further, the main measurement plate 80 has a bottom surface 81a of the well 81.
Is a concave surface, that is, the dielectric block and the metal film
The interface with 12 is a concave surface, and the concave surface 81a and the mirror surface 82 constitute a reflective optical system including a concave surface having a light converging function and a reflecting function and a mirror. It should be noted that when the light beam is incident on the concave interface, the light beam is not condensed in a point shape on the interface but is incident in a defocused state so that the reflected light at the interface is converged on the mirror surface 82. To configure.

The light beam 30 is incident from one surface 82a of the dielectric block portion 82 toward the interface of the well 81 in the Q1 row, and the light beam 30 reflected at the interface is formed on a part of the bottom surface of the dielectric block portion 82. The light beam 30 reflected by the reflected mirror surface 84 is incident on the interface of the well 81 in the Q2 row, and the light beam 30 reflected by the interface is further reflected by the mirror surface 84 and incident on the interface of the well 81 in the Q3 row. The light beam 30 reflected at the interface of the well 81 of the Q3 row
Is emitted from the other surface 82b of the dielectric block portion 82 and is received by the light detecting means.

In the case of the measurement unit and the reflection optical system of FIGS. 6 and 7, the light beam 30 is incident on each interface in substantially the same angle range, and therefore, before the sample droplet is moved, the light beam 30 is incident on the interface of FIG.
As shown in (a), it is observed as one dark line, and then, with the passage of time, by observing a change in the profile such as the separation of the dark line as shown in FIG. Obtainable.

However, in the above case, it is not possible to specify which of the plurality of wells arranged in series hit, and then the hit well is determined by performing measurement for each well. Need to be identified.

FIG. 8 shows a sectional view of still another measuring unit and reflective optical system. The measurement plate 50, which is an assembly of measurement units in FIG. 8, is the same as the measurement plate 50 shown in FIG. On the other hand, reflective optics 95
3 is similar to FIG. 3 in that the measurement unit support base 60 is provided with a concave mirror, but the two concave mirrors 91 and 92 are arranged so that the inclinations thereof are different from each other.

The light beam 30 is incident on the interface of the wells 51 in the Q1 row at the maximum incident angle θ1, and the light beam 30 reflected by the interface is reflected by the concave mirror 91 and converges on the interface of the wells 51 in the Q2 row at the interface. Is incident. At this time, the maximum incident angle of the light beam 30 is θ2. Further, the light beam 30 reflected at the interface of the wells 51 in the Q2 row is incident on the concave mirror 92 so as to be converged at the interface of the wells 51 in the Q3 row. The maximum incident angle of the light beam 30 at this time is θ3 (θ1 <θ2 <θ
3). The light beam 30 reflected by the interface of the wells 51 in the Q3 row is received by the photodetection means. Thus, a concave mirror
When the light beam 30 is made to enter the interfaces in a slightly different angle range by 91 and 92, the light beam profile before dropping the sample is as shown in FIG.
Dark lines are observed at different positions x1, x2, x3 on the light detecting means. After the dropping of the sample, the position of the dark line deviates with the lapse of time, but the presence or absence of a hit can be obtained by changing the interval between the three dark lines. For example, the light beam profile before dropping the sample is as shown in FIG. 9A, and after a predetermined time has passed after dropping the sample, as shown in FIG.
Suppose the dark line spacing in a book changes. In this case, the change in position of the dark line moved from x2 to x2 ′ is larger than the changes in the other two dark lines, and it is possible that the sample in the well that generated the dark line represented by x2 (x2 ′) hits. Means big. By thus shifting the position of the dark line generated in each well on the photodetector, it is possible to simultaneously identify which well has hit. However, when performing measurement by arranging a larger number of wells in series, it may be difficult to specify the wells and the dark line positions even if the dark line positions are set to be different for each well. In that case, when it is determined that there is a hit due to a change in the light beam profile detected by the light detecting means, the measurement may be performed by a measuring device capable of measuring each well.

FIG. 10 shows a sectional view of still another measuring unit and reflective optical system. Measuring unit shown in FIG.
100 is formed in the shape of an individual chip. The measurement chip 100 as a measurement unit has, for example, an inverted truncated quadrangular pyramid shape formed of a transparent resin or the like, and a sample holding hole 113a having a circular cross section is formed on the upper part of the measurement chip 100 to form a sample holding portion 113. The sample holding hole 113a is configured.
A metal film 12 is deposited on the bottom surface of the. Further, the sensing substance 14 is further fixed on the metal film 12. The lower part of the sample holding part 113 of the measuring chip 100 is the dielectric block part.
111, and two facing surfaces of the four side surfaces are a light incident end surface 111a and a light emitting end surface 111b, respectively. The measurement chips 10 are fitted and fixed one by one in a plurality of chip holding holes 121 arranged in a two-dimensional arrangement in the P direction and the Q direction of the measurement unit support base 120. The thickness t1 of the metal film 12 formed on the bottom surface of the sample holding hole 113a of the plurality of measurement chips (here, three measurement chips) arranged in series (in the direction of arrow Q) with respect to the light beam 30. ~
t3 is different from each other. The reflective optical system 130 of the present embodiment is a mirror surface arranged below the measuring chip 100.
131 and two convex lenses 132 and 133 arranged on the mirror surface 131
It consists of and.

The light beam 30 is incident on the interface of the measurement chip 100 in the Q1 row, the light beam 30 reflected by the interface is reflected by the mirror surface 131, and the action of the convex lens 132 causes the interface of the measurement chip 100 in the Q2 row. The light beam 30 which is made to converge so as to be converged on and is further reflected on the interface is reflected on the mirror surface 131 again, and is made to be converged on the interface of the measuring chip 100 in the Q3 row by the action of the convex lens 133. The Q3 measuring chip 10
The light beam 30 reflected by the interface of 0 is received by the light detection means. Since the relationship between the incident angle of the light beam on the interface and the refractive index of the substance on the metal film 12 differs depending on the thickness of the metal film 12, three dark lines are present in the profile of the light beam received by the light detecting means. Observed with a shift. Before the sample is dropped, and for a predetermined time after the sample is dropped, the profile of the light beam is monitored,
The presence or absence of a hit can be obtained by changing the interval between the three dark lines. For example, if there is a noticeable change in the interval between the three dark lines, it is determined that the sample of any measurement chip has hit.

As a method of shifting the position of the dark line generated on each interface on the light detecting means, the inclination of the reflection optical means is changed and the thickness of the metal film is changed as described above. Various methods are conceivable, such as one in which the buffers of the sample liquid have different refractive indexes, and one in which the measuring unit is configured as a chip and which is provided with dielectric blocks having different refractive indexes.

In the above embodiment, the measurement units (wells) arranged one-dimensionally are connected in series to the light beam, but the measurement units arranged two-dimensionally and three-dimensionally are arranged with respect to the light beam. The reflective optical system may be configured to be in series with each other.

FIG. 11 is a plan view showing the outline of the surface plasmon sensor of the second embodiment of the measuring apparatus of the present invention. The surface plasmon sensor of this embodiment also includes the same measurement plate 50 as that shown in FIG. However, the measurement plate 50 is provided with only one set of each of the incident optical system 20 'and the photodetection means 27a, and the light beam 30 is transmitted to all the wells of the measurement plate 50 by the reflection optical system not shown.
It is configured to sequentially enter and reflect on the interface of 51. The light beam 30 passes through all the wells 51 in the order shown by the dotted arrow in the figure, and is detected by the light detecting means 27a. In this way, a plurality of measurement units arranged two-dimensionally are arranged in series with the light beam 30 by the reflection optical system, and the reflected light at the interface of more measurement units is made by one light detecting means. It is possible to detect the light beam including the above condition, and it is possible to obtain the presence or absence of a hit in the plurality of measurement units arranged in series with respect to the light beam.

The measuring apparatus of each of the above-described embodiments makes the light beam from the light source incident on the interface at various angles, measures the reflected light from the interface, and changes the incident angle, which is a dark line, to determine the total. The state of reflection attenuation is measured to obtain the binding state between the analyte and the sensing substance, but the incident angle of the light beam is set to a predetermined angle that satisfies the condition of total reflection at the interface, and light beams having various wavelengths are used. Alternatively, the wavelength of the incident or incident light beam may be changed, the reflected light from the interface may be measured, and the binding state between the analyte and the sensing substance may be obtained from the attenuated total reflection state for each wavelength.

Further, FIG. 12 shows a measuring apparatus utilizing the phase of light which is a surface plasmon sensor of the third embodiment of the measuring apparatus of the present invention, and will be described below. FIG. 12 is a schematic sectional view of a measuring device using the phase of light.

In the surface plasmon sensor according to the present embodiment, the measurement plate 140, which is an assembly of measurement units, is substantially the same as the measurement plate 80 shown in FIG. 7, but the bottom surface 141a of each well 141 is It has what is a plane. Further, similarly to the one used in the above-mentioned embodiment, the light beam is made to enter the five wells 141 arranged in the P direction in parallel. Also,
A reflection optical system in which a mirror surface 145 is formed on a part of the bottom surface of the dielectric block portion 142 of the measurement plate 140 is configured. In the present embodiment, the reflection optical system does not have a focusing function.

As shown in the side view of FIG. 12, the surface plasmon sensor according to this embodiment has a measuring plate 140.
Of the plurality of light sources 334a to 334e and the CCD 3 on the light beam incident surface 142a side and the light emitting surface 142b side of the dielectric block 142 of FIG.
60a-e are provided, and collimator lenses 350a-350a-e are provided between these light sources 334a-e and CCDs 360a-e.
e, an interference optical system, condenser lenses 355a to 355e, and apertures 356a to 356e.

The interference optical system includes the polarization filters 351a to 351a ...
e, half mirrors 352a-e, half mirrors 353a-e, and mirrors 354a-e.

Further, the CCDs 360a to 360e have a signal processing unit 361.
The signal processing unit 361 is connected to the display unit 362.

The measurement of the sample in the surface plasmon sensor of this embodiment will be described below. It should be noted that, of the wells 141 of the measurement plate 140, the well 141 of one row in the P direction aligned with the light source 34a and the CCD 360a will be described as an example, but the same is true for other wells. Done

The light source 334a is driven and the light beam 330 is emitted in a divergent state. The light beam 330 is collimated by the collimator lens 350a and enters the polarization filter 351a. A part of the light beam 330 transmitted through the polarization filter 351a and incident on the interface 141a as p-polarized light is referred to by the half mirror 352a.
The remaining light beam 330S split as R and transmitted through the half mirror 352a enters the interface 141a of the well 141 in the Q1 column. The light beam 330S totally reflected at the interface 141a has a mirror surface.
The light beam 330S reflected by 145 and incident on the interface 141a of the well 141 in the Q2 row is reflected by the mirror surface 145 again and is incident on the interface of the well 141 in the Q3 row. Light beam 330 reflected at the interface of the Q3 wells
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 ′ detected by the CCD 360a is the light beam 330S and the reference light beam 33.
Interference fringes are generated according to the state of interference with 0R.

When the refractive index of the sensing substance 14 changes according to the binding state between the specific substance and the sensing substance, the interference state between the light beam totally reflected at the interface and the reference light beam changes, which causes a change in the interference fringes. The presence or absence of binding can be detected accordingly. In the present embodiment, since the plurality of wells 141 are arranged in series with respect to the light beam 330, if the analyte in any one of the plurality of wells 141 arranged in series binds to the sensing substance. , Changes in interference fringes are detected.

The signal processing section 361 detects the presence or absence of the above reaction based on the above principle, and the result is displayed on the display section 362. This measurement shows that multiple wells 14
If a hit is found in the sample in any one of the wells 1, the well that has hit in the measuring device capable of measuring each well is identified.

The above measurement operation is similarly performed in parallel for the wells 141 in the other four rows in the P direction.

The signal processing unit 361 includes five CCDs 360a.
~ E may be provided with dedicated ones, or one shared with 5 CCDs 360a ~ e
It is also possible to provide only one and sequentially process the light amount detection signals S output from the CCDs 360a to e.

Needless to say, the structure of this surface plasmon sensor can be applied to the leaky mode sensor as described above.

[Brief description of drawings]

FIG. 1 is a plan view of a surface plasmon sensor according to an embodiment of the present invention.

FIG. 2 is a side sectional view of the surface plasmon sensor shown in FIG.

FIG. 3 is an enlarged view of a measurement plate and a reflection optical system of the surface plasmon sensor of FIG.

FIG. 4 is a diagram showing changes in dark line position.

FIG. 5 is a partial side sectional view of a leaky mode sensor according to an embodiment of the present invention.

FIG. 6 is a side sectional view showing another measuring unit and a reflective optical system in the measuring apparatus of the present invention.

FIG. 7 is a side sectional view showing another measuring unit and a reflective optical system in the measuring apparatus of the present invention.

FIG. 8 is a side sectional view showing another measuring unit and a reflective optical system in the measuring apparatus of the present invention.

9 is a diagram showing changes in the dark line position when the measuring device including the measuring unit and the reflective optical system shown in FIG. 8 is used.

FIG. 10 is a side sectional view showing another measuring unit and a reflective optical system in the measuring apparatus of the present invention.

FIG. 11 is a side view of a surface plasmon sensor according to a second embodiment of the present invention.

FIG. 12 is a side view of a surface plasmon sensor according to a third embodiment of the present invention.

[Explanation of symbols]

10 light source 12 Metal film 14 Sensing substances 20 Incident optical system 27a-e Light detection means 30 light beams 31, 32 concave mirror 35 Reflective optics 50 Measuring plate 51 well 52 Dielectric block 60 Measuring unit support

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 2G057 AA02 AB04 AB07 AC01 BA01                       BB01 BB06                 2G059 AA01 BB04 BB12 CC16 DD13                       DD16 EE02 EE05 EE09 GG01                       GG04 JJ11 JJ13 JJ14 JJ19                       JJ22 KK04 MM01 MM03 MM09                       MM11 PP04

Claims (5)

[Claims] 1. A light source for generating a light beam, a dielectric block transparent to the light beam, a thin film layer formed on the upper surface of the dielectric block, and a sample held on the surface of the thin film layer. A plurality of measurement units provided with a sample holding mechanism, and the light beam to the dielectric block of the first measurement unit of the plurality of measurement units at an interface between the dielectric block and the thin film layer. An incident optical system that makes an incident angle at which a total reflection condition is obtained, and a light beam that is totally reflected at the interface is reflected to the interfaces of the second to (2 + n) th measurement units sequentially at the interface. A reflection optical system which is incident at an angle of incidence where a total reflection condition is obtained; and a light detection unit which measures the intensity of the light beam totally reflected at the interface of the (2 + n) th measurement unit. Do Constant apparatus. 2. The measuring device according to claim 1, wherein the reflective optical system includes a concave surface and a mirror. 3. The measuring device according to claim 1, wherein the reflective optical system includes a mirror and a convex lens. 4. The measuring device according to claim 1, wherein the plurality of measuring units are one-dimensionally or two-dimensionally arranged and integrally formed. 5. The thin film layer of each of the plurality of measurement units has a different thickness.
The measuring device according to claim 1.