JP2003202285A - Measuring plate of measuring device utilizing total reflection - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To efficiently perform measurement with high accuracy in a measuring device utilizing total reflection. <P>SOLUTION: This measuring plate is provided with a metallic film 14 formed on a bottom inner face of a well 12a of 8×6 among wells 12 arranged at pitches in accordance with predetermined standard in the shape of matrix of 8×12, on a surface of a plate base 11 having the outer shape same as the predetermined standard of a microtiter plate, and a dielectric block 16 is formed on a bottom outer face. The dielectric block 16 is composed of the thick projecting part formed on the bottom of the wall 12a, and has an incoming face 16a for allowing the predetermined optical beam to enter into an interface between the bottom face of the measuring well 12a and the metallic film 14, and an outgoing face 16b for allowing the optical beam total-reflected by the interface to outgo. <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 plate for holding a sample for measurement by a measuring apparatus utilizing total reflection such as a surface plasmon resonance measuring apparatus.

[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 for generating a beam and a dielectric block that directs the light beam to the dielectric block at the interface between the dielectric block and the metal film so that various incident angles including the surface plasmon resonance condition can be obtained. And an optical system for measuring the intensity of the light beam totally reflected at the interface to detect the state of surface plasmon resonance.

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 the state of convergent light or divergent light. In the former case, a light beam whose reflection angle changes with the deflection of the light beam can be detected by a small photodetector that moves synchronously with the deflection of the light beam, or by an area sensor extending along the direction of change of the reflection angle. Can be detected. 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 resonance measuring apparatus having the above structure, when a light beam is incident on the metal film at a specific incident angle θsp which is equal to or greater than the total reflection angle, an evanescent light having an electric field distribution in the sample 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 sample 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.

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 θsp at which the attenuated total reflection (ATR) occurs, the dielectric constant of the sample can be obtained. That is, the wave number of the surface plasmon is Ks
p, the angular frequency of the surface plasmon is ω, c is the speed of light in a vacuum, ε _m And ε _s Where is the metal and the permittivity of the sample, respectively, the following relationship is established.

[0009]

[Equation 1] Dielectric constant of sample ε _s If is known, the concentration of the specific substance in the sample can be known based on a predetermined calibration curve, etc., so that by finally knowing the incident angle θsp at which the reflected light intensity decreases,
The specific substance in the sample can be quantitatively analyzed.

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 sensors described on the page are also known. This leaky mode sensor is basically formed by, for example, a dielectric block formed in a prism shape, a clad layer formed on one surface of the dielectric block, and formed on the clad layer and brought into contact with a sample. An optical waveguide layer, a light source for generating a light beam, and the light beam are incident on the dielectric block at various angles so that total reflection conditions are obtained at the interface between the dielectric block and the cladding layer. It comprises an optical system and a light detecting means for measuring the intensity of the light beam totally reflected at the interface to detect the excited state of the guided mode, that is, the attenuated state of total reflection.

In the leak mode sensor 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.

As a measuring apparatus utilizing 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.

In a conventional surface plasmon resonance measuring apparatus or the like using the above system, it is practically necessary to replace the metal film in contact with the sample every measurement. Therefore, it is considered that the metal film is fixed to one surface of the dielectric block and a holding mechanism for holding the sample is integrally formed on the metal film to form a measuring chip (for example, the present applicant). See Japanese Patent Application No. 2000-016633). However, it has been recognized that it is difficult to increase the efficiency of the measurement process because such individual measuring chips are small and their handling properties are poor.

Further, the above-mentioned surface plasmon resonance measuring device and leak mode sensor are used in the field of drug discovery research, etc.
It may be used for random screening to find a specific substance that binds to a given sensing substance. In this case, a plurality of measurement chips having a sensing substance fixed on the thin film layer (a metal film in the case of a surface plasmon resonance measuring device, a clad layer and an optical waveguide layer in the case of a leaky mode measuring device) are prepared, Sample liquids containing the analyte are dropped on the sensing substance of each measurement chip, and the state of total reflection is measured. As described above, since it takes a long time to perform the measurement for a plurality of analytes, how to perform the efficient measurement is important.

[0015]

Therefore, if a plurality of measuring chips are connected in a row so that they can be handled integrally, a more efficient measurement can be performed as compared with the case where a plurality of measuring chips are handled individually. It is thought possible to do. In the case where a plurality of measuring chips are connected and can be handled integrally as described above, a peripheral device for handling the plurality of integrated measuring chips is required in order to further improve the handling performance.

However, it is not desirable to add a great deal of cost to newly design and develop a dedicated peripheral device for a plurality of integrated measurement chips.

Therefore, in order to solve the above problems, it is used for enzyme immunoassay in the field of biochemistry,
It is conceivable to use a measurement plate in which the measurement chips are arranged in a matrix of the same standard as the microtiter plate having wells of 96 holes, 384 holes, 1536 holes and the like. If it has such a standard, it is possible to effectively use existing equipment such as a dispensing device for dispensing the sample solution conventionally used for microtiter plates into each well. Handling ability can be improved without preparing various peripheral devices.

However, if the measurement chips are arranged on the plate of the same standard as the microtiter plate in a matrix according to the standard, the light beams entering and exiting at the time of measurement of the respective measurement chips are adjacent to each other. Since vignetting occurs due to the bottom of the chip, there is a problem that accurate measurement cannot be performed.

In view of the above circumstances, the present invention is a measuring plate which is used in the measurement of a measuring apparatus using total reflection such as a surface plasmon resonance measuring apparatus, which has an excellent handling property and enables accurate measurement. It is intended to provide.

[0020]

A measuring plate of a measuring apparatus utilizing total reflection according to the present invention has a position on the surface of a plate base having the same outer shape as a predetermined standard of a microtiter plate, according to the standard. A plurality of wells arranged in an array, and a predetermined light beam is incident on the back surface of the plate substrate only on the bottom outer surface of some of the plurality of wells so as to be totally reflected by the bottom surface of the well. A dielectric block having an entrance surface and an exit surface for emitting the light beam totally reflected on the bottom surface, and the wells provided with the dielectric block are arranged so as not to be adjacent to each other in at least one array direction. It is what

Here, the predetermined standard refers to, for example, JIS, ANSI, DIN, etc., 96, 384, and 1 with respect to the outer dimensions of the plate and the array of wells (number of rows and columns, etc.).
For example, the proposed SBS microplate standards of 536 wells (well) are the standards that are used or can be used as microtiter plates.

The dielectric block may be provided in wells in every one row or more or in every one column or more of the plurality of wells, or in the staggered wells of the plurality of wells. May be.

Another measuring plate of the present invention is one or more rows or one or more columns arranged in a matrix according to the standard on the surface of a plate substrate having the same outer shape as the predetermined standard of the microtiter plate. A plurality of wells are arranged at alternate positions, an outer surface of the bottom of each well of the plurality of wells is incident on a bottom surface of the well so that a predetermined light beam is incident on the bottom surface of the well, and the bottom surface. It is characterized by comprising a dielectric block having an emission surface for emitting the light beam totally reflected by.

Still another measuring plate according to the present invention has a plurality of staggered positions arranged in a matrix according to the standard on the surface of a plate substrate having the same outer shape as the predetermined standard of the microtiter plate. Wells are arranged, and an incident surface on which a predetermined light beam is incident on the bottom outer surface of each well of the plurality of wells so as to be totally reflected on the bottom surface of the well, and a light beam totally reflected on the bottom surface are emitted. It is characterized in that it is provided with a dielectric block having a light emitting surface.

In each of the measuring plates of the present invention, a thin film layer may be formed on the inner surface of the bottom of the well having the dielectric block on the outer surface of the bottom, and the thin film layer is a metal film. Yes, it may be used in a measuring device for measuring the state of attenuation of total reflection associated with surface plasmon resonance, or the thin film layer is composed of a cladding layer and an optical waveguide layer formed thereon. Yes, it may be used in a measuring device for measuring the state of attenuation of total reflection accompanying excitation of a guided mode in the optical waveguide layer.

As a measuring device utilizing total internal reflection, a light beam is made incident on the bottom surface of the well at various incident angles at which total reflection conditions can be obtained, and total reflection is made at the bottom surface at each position corresponding to each incident angle. DVNOort, K.johansen, a measurement device that performs sample analysis by measuring the intensity of the light beam and detecting the position (angle) of the dark line generated by attenuation of total internal reflection.
C.-F. Mandenius, Porous Gold in Surface Plasmon Res
onance Measurement, EUROSENSORS XIII, 1999, pp.585
-588, light beams of multiple wavelengths are incident on the bottom surface of the well at an incident angle at which total reflection conditions are obtained, and the intensity of the light beam totally reflected on the bottom surface is measured for each wavelength. , A device for analyzing samples by detecting the degree of total reflection attenuation for each wavelength, and PINikitin, ANGr
igorenko, AABeloglazov, MVValeiko, AISavchuk, O.
A. Savchuk, Surface Plasmon Resonance Interferometry
for Micro-Array Biosensing, EUROSENSORS XIII, 199
As described in 9, pp.235-238, the light beam is made incident on the bottom surface of the well at an incident angle at which total reflection conditions are obtained, and part of this light beam is made incident on the bottom surface. Before performing the splitting, the split light beam is caused to interfere with the light beam totally reflected on the bottom surface, and the intensity of the light beam after the interference is measured to perform sample analysis. There is a device that performs sample analysis by making the sample enter with an incident angle that allows the total reflection condition to be generated, exciting the sample with the evanescent light generated by the light beam to generate fluorescence, and detecting the fluorescence.

[0027]

The measuring plate of the measuring apparatus utilizing total reflection of the present invention has the same outer shape as the predetermined standard of the microtiter plate and a plurality of wells arranged at the positions according to the standard. Therefore, it is not necessary to newly design and develop the peripheral devices, since the peripheral devices such as the sample liquid dispenser which have been conventionally developed for the microtiter plate can be used. The handling becomes easier compared to the conventional case where individual measuring chips are handled, and by using the existing peripheral devices, the handling property can be further improved.

Further, of the plurality of wells arranged at the positions according to the predetermined standard, only the outer surface of the bottom of some of the wells is
A well is provided with a dielectric block having an incident surface on which a predetermined light beam is incident on the bottom surface of the well and an emission surface on which the light beam totally reflected by the bottom surface is emitted, and the wells including the dielectric block are adjacent to each other in at least one array direction. Since the light beam is arranged so as not to enter, the light beam can enter and exit from the well side not provided with the dielectric block, and the light beam is eclipsed by the dielectric block of the adjacent well when the light beam enters and exits. Measurement can be performed accurately.

Further, since a plurality of samples can be held simultaneously on one plate, it is possible to carry out measurement on a large number of samples more efficiently.

Further, another measuring plate of the present invention is arranged on the surface of a plate substrate having the same outer shape as the predetermined standard of the microtiter plate, at a position of every other row arranged in a matrix according to the standard. Since a plurality of wells provided with the dielectric block are arranged, the light beam incident and the light beam emitted from the dielectric block are not eclipsed as in the case described above, and the measurement can be performed accurately. It is possible to use peripheral devices such as a sample liquid dispenser that have been conventionally developed for microtiter plates, and it is possible to improve handling properties.

Still another measuring plate of the present invention is a dielectric plate, which is arranged in a zigzag position in a matrix according to the standard on the surface of a plate substrate having the same outer shape as the predetermined standard of the microtiter plate. Since a plurality of wells including blocks are arranged, as in the case described above, the light beam entering and exiting the dielectric block can be accurately measured without vignetting and the conventional micro Peripheral devices such as a sample liquid dispenser developed for the titer plate can be used, and the handling property can be improved.

In each measurement plate, a thin film layer is formed on the inner surface of the bottom of the well having the dielectric block, and if a metal film is formed as the thin film layer, the state of attenuation of total reflection due to surface plasmon resonance is obtained. It can be used in a measuring device for measuring, and if a thin film layer consisting of a cladding layer and an optical waveguide layer formed thereon is formed, all of the light accompanying the excitation of the waveguide mode in the optical waveguide layer can be formed. It can be used in a measuring device for measuring the state of return loss. In any of the apparatuses, the measurement plate of the present invention can be used to efficiently measure a large number of samples.

[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 is a perspective view of a measuring plate according to the first embodiment of the present invention. As shown in FIG. 1A, the plate 10 for measurement has a pitch of 96 wells 12 according to a predetermined standard on the surface of a plate base 11 having the same outer shape as the predetermined standard of a microtiter plate. (9 mm) and arranged in a matrix of 8 rows and 12 columns (8 × 12). The wells 12 have a circular cross-sectional shape whose diameter gradually decreases downward from the upper surface of the base body 11. Of the wells 12, a dielectric block 16 is formed on the outer surface of the bottom of every other well 12a. There is. That is, the wells 12a provided with the dielectric blocks 16 are not adjacent to each other in the column direction (at least not adjacent to each other in one arrangement direction), and only the wells 12a of 8 × 6 out of the wells 12 of 8 × 12 are dielectric blocks. 16 are equipped. As shown in the figure, the dielectric block 16 is formed by forming the bottom of the well 12a in a thick and convex shape. Hereinafter, the well provided with the dielectric block 16 will be referred to as a measurement well 12a. The dielectric block 16 has an incident surface 16a on which a predetermined light beam is incident on the bottom surface 15 of the measuring well 12a and an emission surface on which the light beam totally reflected on the bottom surface 15 is emitted.
16b, which has an entrance surface 16a and an exit surface
16b is provided on the side of the well 12b that does not include the dielectric block 16 among the adjacent wells.

The dielectric block 16 may be individually formed in each measuring well 12a as shown in FIG. 1B, or a plurality of dielectric blocks 16 arranged in a line as shown in FIG. 1C. It may be formed continuously over the individual measurement wells 12a.

Since the present measurement plate 10 has the same outer dimensions and wells 12 as the standard of the microtiter plate, as a means for supplying the liquid sample 20 to the measurement well 12a, a portion as shown in FIG. An injector 50 can be used. The dispenser 50 comprises eight dispenser nozzles 51 supported by a support member 52 at a predetermined pitch, and is used in a conventional dispenser such as an elisa using a microtiter plate. It is a machine. As described above, if the main measurement plate 10 is used, it is possible to use a peripheral device such as a pipetting machine conventionally used for a microtiter plate, and it is possible to improve the handling property.

If the metal film 14 is formed on the inner surface of the bottom of the measuring well 12a having the dielectric block 16, the measuring plate 10 'used in the surface plasmon sensor (see FIG. 3).
If the cladding layer 41 and the optical waveguide layer 42 are formed in this order on the inner surface of the bottom, the measurement plate 10 ″ (see FIG. 5) used in the leaky mode sensor can be obtained.

FIG. 3 is a schematic view of a surface plasmon sensor using a measuring plate 10 'having a metal film 14 provided on the inner surface of the bottom of the measuring well of the measuring plate 10 shown in FIG. A surface plasmon sensor, which is one form of a measuring device using total reflection, is a laser light source 31 such as a semiconductor laser that generates a light beam (laser beam) 30 for measurement.
, A condenser lens 32 that constitutes the incident optical system, and a photodetector 40.
And a signal processing unit 60 for receiving the output signal S of the light detecting means 40 and performing a process described later, and a moving means (not shown) for relatively moving the measuring system and the measuring plate 10 '. Be prepared.

In FIG. 3, a partially enlarged side section of the measuring plate 10 'is shown. As described above, among the wells 12 arranged in the column direction, every other well 12a (pitch P
= 18 mm), a dielectric block 16 is formed on the outer surface of the bottom, and a metal film 14 is formed on the inner surface of the bottom of the well 12a having the dielectric block 16.

The condenser lens 32, as shown in FIG.
30 is condensed and passed through the dielectric block 16 in a converged light state, and is incident on the interface 15 (well bottom surface) between the dielectric block 16 and the metal film 14 so as to obtain various incident angles. The range of this incident angle is the interface between the metal film 14 and the dielectric block 16.
At 15, a total reflection condition of the light beam 30 is obtained, and a range including an angular range where surface plasmon resonance can occur is set.

The light beam 30 is incident on the interface 15 as p-polarized light. In order to do so
It suffices to dispose 31 so that its polarization direction is a predetermined direction. In addition, the polarization direction of the light beam 30 may be controlled by a wave plate or a polarizing plate.

The photodetector 40 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 so that they are arranged in the direction of arrow X in FIG. .

The moving means two-dimensionally moves the measuring system or the measuring plate 10 relatively in the direction perpendicular to the paper surface and in the left-right direction on the paper surface so that the measuring well 12a is located at a predetermined position with respect to the measuring system. It is what makes me.

The sample analysis by the surface plasmon sensor having the above-mentioned structure will be described below. The sample to be measured is supplied to the measurement well 12a by the dispenser 50 shown in FIG.

Measuring well 12a holding sample 20
However, in a state where the dielectric block 16 is installed at the measurement position where the light beam 30 is incident by the moving means, the laser light source 31 is driven, and the light beam 30 emitted from the laser light source 31 is converged as described above. Then, it is incident on the interface 15. This interface
The light beam 30 totally reflected at 15 is detected by the photodetector 40.

Since the light beam 30 is incident on the dielectric block 16 in a convergent light state as described above, it includes components that are incident on the interface 15 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 light beam 30 is totally reflected at the interface 15, and this reflected light beam
30 will include components that reflect at various reflection angles.

When the light beam 30 is totally reflected in this way,
An evanescent wave seeps out from the interface 15 toward the metal film 14 side.
When the light beam 30 is incident on the interface 15 at a certain incident angle θsp, this evanescent wave is generated by the metal film.
Since it resonates with the surface plasmons excited on the surface of 14, the reflected light intensity I of this light sharply decreases. Figure 4
3 schematically shows the relationship between the incident angle θ and the reflected light intensity I when the phenomenon of attenuation of total reflection occurs.

Therefore, the amount of light detected for each light receiving element is checked from the light amount detection signal S output from the photodetector 40, and the incident angle (decay angle for total reflection) θsp is obtained based on the position of the light receiving element where the dark line is detected. Based on the relationship curve between the reflected light intensity I and the incident angle θ which is obtained in advance, the specific substance in the sample can be quantitatively analyzed. The signal processing unit 60 quantitatively analyzes the specific substance in the sample 20 based on the above principle, and outputs the analysis result to a display unit (not shown).

As shown in FIG. 3, at the bottom of the well 12b on the incident surface 16a side and the emission surface 16b side of the dielectric block 16 of the measuring well 12a, the dielectric block as shown by the dotted line in the figure.
If 16 ′ is provided, the measurement accuracy decreases because the light beam 30 that enters or exits the dielectric block 16 of the measurement well 12 a suffers from vignetting, but in the measurement plate 10 Since no dielectric block is provided at the bottom of the non-measurement well 12b, vignetting does not occur and the measurement accuracy does not deteriorate.

Then, the measuring plate 10 is moved by moving means.
Alternatively, the measurement systems are moved relative to each other so that the measurement well 12a to be used for the measurement next is at a predetermined position with respect to the measurement system. In this way, the measurement wells 12a can be used for measurement one after another as the measurement plate 10 moves. This allows the surface plasmon sensor to measure a large number of samples 20 in a short time.

A sensing substance that binds to a specific substance is previously fixed on the metal film 14, and the sample solution 20 containing the analyte is dropped on the sensing substance, and the total reflection attenuation angle θsp due to surface plasmon resonance is set. It is also possible to determine whether or not the sensing substance is bound to the analyte, that is, whether the analyte is a specific substance, by measuring the amount of change in angle.

That is, in this case, the refractive index of the sensing substance changes according to the binding state of the specific substance and the sensing substance, and the characteristic curve of FIG. The antigen-antibody reaction can be detected according to the attenuation angle θsp. In this case, both the sample 20 and the sensing substance are samples to be analyzed.

FIG. 5 uses total reflection using a measurement plate 10 ″ in which a cladding layer and an optical waveguide layer are provided in this order on the bottom inner surface of the measurement well of the measurement plate 10 shown in FIG. FIG. 6 is a schematic view of a leaky mode sensor which is another form of the measuring device described above. Note that, in this figure, elements equivalent to those shown in FIG. 3 are shown with the same numbers as in FIG.

In the measurement plate 10 ″, the cladding layer 41
Is formed into a thin film using a dielectric material having a lower refractive index than the dielectric block 16 or a metal such as gold. Also optical waveguide layer
42 is a dielectric having a higher refractive index than the cladding layer 41, for example, P
It is formed into a thin film using MMA. Clad layer 41
Has a thickness of 36.5 nm when formed from a gold thin film,
The film thickness of the optical waveguide layer 42 is, for example, about 700 nm when it is formed from PMMA.

In the leaky mode sensor shown in FIG. 5,
The measurement is performed with the measurement well 12a holding the sample 20 installed at a predetermined position. When the light beam 30 emitted from the laser light source 31 is incident on the cladding layer 41 through the dielectric block 16 at an angle of incidence equal to or more than the total reflection angle, the light beam 30 causes the interface between the dielectric block 16 and the cladding layer 41 ( Light having a specific wave number, which is totally reflected by the bottom surface 15 of the well but is transmitted through the cladding layer 41 and is incident on the optical waveguide layer 42 at a specific incident angle, propagates through the optical waveguide layer 42 in a guided mode. When the guided mode is excited in this manner, most of the incident light is taken into the optical waveguide layer 42, so that the total reflection attenuation occurs in which the intensity of the light totally reflected at the interface 15 sharply decreases.

The wave number of the guided light in the optical waveguide layer 42 depends on the refractive index of the sample 20 on the optical waveguide layer 42. Therefore, by knowing the specific incident angle at which attenuation of total reflection occurs, the sample can be obtained.
The refractive index of 20 and its associated properties of the sample 20 can be analyzed. The signal processing unit 60 quantitatively analyzes the specific substance in the sample 20 based on the above principle, and the analysis result is displayed on the display unit (not shown).

Then, the measuring plate 1 is moved by moving means.
0 ″ or the measurement systems are moved relative to each other so that the measurement well 12a to be used for the measurement next is at a predetermined position with respect to the measurement system. In this way, the measurement well 12a is
With the movement of the measurement plate 10 ″, it can be used for measurement one after another. In this way, the main measurement plate 10 ''
By using, even in this leaky mode sensor, it is possible to obtain the same effect as that obtained when the measuring plate 10 ′ is used in the above-mentioned surface plasmon sensor.

The above-mentioned measuring plate 10 has the same standard structure as the 96-well microtiter plate, but it is also possible to use a measuring plate having the same standard as the 384-well and 1536-well microtiter plates. For example, in a substrate having 384 wells of 16 rows and 24 columns (16 × 24) and a cell pitch of 4.5 mm, a thin film layer is formed on the inner surface of the bottom of every other row of wells (wells of 8 rows and 24 columns), and a dielectric block is formed on the outer surface. It may be provided. 38
In the case of a 4-well measurement plate, the thickness of the dielectric block is preferably 3.5 mm or less.

Further, wells arranged in rows and columns arranged in rows and columns according to the standard may be used as measurement wells, and wells arranged in multiple rows or columns may be used as measurement wells. As shown in the figure, of the wells 72 arranged in a matrix on the base 71, a dielectric block is provided only in the wells 72a in the staggered position, which are shaded in the figure, and the measurement wells 72a are non-measurement wells. The measurement plates 70 may be arranged in a zigzag pattern alternately formed with 72b.

The arrangement of the measuring wells in the measuring plate of the present invention may be any arrangement as long as the measuring wells are not adjacent to each other in at least one arrangement direction. In this way, the measurement wells are not arranged adjacent to each other in one predetermined array direction, that is, the dielectric blocks may exist adjacent to the light beam entrance surface and the light beam exit surface side of the dielectric block of each measurement well. With no arrangement, the state of total reflection can be measured without causing eclipse of the light beam.

7 to 9 show still another form of the measuring plate of the present invention. 7 and 8 are top views of the measurement plate, and FIG. 9 is a side sectional view of the plate shown in FIG.

The measuring plate 80 shown in FIG. 7 is arranged on every other row in a matrix arrangement according to the standard for 96 wells on the surface of the plate substrate 81 having the same outer shape as the predetermined standard of the microtiter plate. The wells 82 are arranged only at the positions, and a dielectric block is provided on the outer surface of the bottom of each well 82. That is, of the 96 wells of the measurement plate 10 in FIG. 1, only the measurement wells are formed and the non-measurement wells are not formed. The point 83 in the figure is the location where the well originally exists in the standard for 96 wells.

The measuring plate 90 shown in FIGS. 8 and 9 is arranged in a matrix on the surface of the plate base 91 having the same outer shape as the predetermined standard of the microtiter plate according to the standard for 384 wells. The wells 92 are arranged only at the staggered positions, and the dielectric block 96 is provided on the outer surface of the bottom of each well 92. That is, this is a form in which only the measurement wells are formed in a staggered arrangement, and the non-measurement wells are not formed. The point 93 in FIG. 8 indicates the location where a well originally exists in the standard for 384 wells. As shown in FIG. 9, the light beam
Vignetting does not occur when 30 enters the entrance surface 96a of the dielectric block 96 and exits from the exit surface 96b of the dielectric block 96, so that the measurement accuracy does not deteriorate.

As described above, even in the measurement plate constructed by thinning out the wells from the matrix arrangement according to the standard as shown in FIGS. 7 and 8, the wells are not thinned out and the measurement wells and non-measurement wells are not thinned out. Measuring plate with wells
The same effect as in the case of 10 can be obtained.

The wells of the above measuring plates are
Although the cross-section has a circular shape whose diameter gradually decreases downward from the upper surface of the substrate, the shape of the well is not limited to this. Examples of well shapes are shown in FIGS.
In each figure, (a) is a side sectional view of the well, and (b) is a view of the well as seen from the back surface of the substrate.

Well 1 of the measuring plate 100 shown in FIG.
02 has a circular cross section whose diameter gradually decreases downward from the upper surface of the substrate 101, but has a portion in the lower portion of the well where the reduction rate of the diameter increases. Thin film layer on the bottom of the well
94 is formed, the bottom of the well is formed thickly so as to be convex with respect to the back surface of the substrate 101, and the dielectric block 10 is formed.
6 are configured. The dielectric block 106, that is, the convex portion on the back surface of the substrate 101 has a shape extending in the well-arrangement direction on the outer surface of the bottom of the well adjacent to the depth of the drawing in FIG.

Well 1 of measurement plate 110 shown in FIG.
In the case of 12, the opening of the well 112 is quadrangular and has an inverted truncated square prism shape. A thin film layer 94 is formed on the bottom surface of the well, and the dielectric block 116 is formed by forming the bottom of the well thickly so as to be convex toward the back surface of the substrate 111. Dielectric block 116, that is, substrate 1
The convex portion on the back surface of 11 has a shape extending in the direction in which the wells are arranged on the outer surface of the bottom of the well adjacent to the depth of the paper in FIG.

Well 1 of the measuring plate 120 shown in FIG.
22 is a well lower part of the well 112 shown in FIG.
Further, it has a portion where the reduction rate of the cross-sectional area becomes large. Also in this case, similarly, the thin film layer 94 is formed on the bottom surface of the well, and the dielectric block 126 is formed on the outer surface of the well bottom portion.

In each measuring plate, a thin film layer
As 94, a metal film may be formed when used in a surface plasmon sensor, and a clad layer and an optical waveguide layer may be formed when used in a leaky mode sensor.

The above-described measuring device using total reflection provided with the measuring plate of the present invention makes the light beam from the light source incident on the interface (well bottom surface) at various angles, and then from the interface. The reflected light is measured to measure the state of attenuation of total reflection from the incident angle that becomes a dark line for sample analysis, or the binding state between the analyte and the sensing substance is obtained. With a predetermined angle that satisfies the condition of total reflection, light beams having various wavelengths are made incident, or the wavelength of the light beam to be made incident is changed,
The reflected light from the interface may be measured, and the sample analysis may be performed based on the attenuated total reflection state for each wavelength, or the binding state between the analyte and the sensing substance may be obtained.

Further, the measuring plate 10 'shown in FIG.
A side view of a measuring device of another form using total reflection using is shown in FIG. 13 and briefly described.

Here, a sensing substance 18 that binds to a specific substance is further provided on the metal film 14 provided on the inner surface of the bottom of the measuring well 12a of the measuring plate 10 '.

As shown in the side view of FIG. 13, the surface plasmon sar of this embodiment has the above-described measurement plate 1
0 ', and the dielectric block 16 of the measuring plate 10'
On the light beam incident surface 16a side and the emission surface 16b side of
A light source 334 and a CCD 360 are provided, and these light sources 33
A collimator lens 350, an interference optical system, a condenser lens 355, and an aperture 356 are arranged between 4 and the CCD 360.

The interference optical system is composed of a polarization filter 351, a half mirror 352, a half mirror 353 and a mirror 354.

Further, the CCD 360 is connected to the signal processing unit 361, and 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.

The light source 334 is driven and the light beam 330 is emitted in a divergent state. The light beam 330 is collimated by the collimator lens 350 and enters the polarization filter 351. The light beam 330 transmitted through the polarization filter 351 and incident on the interface 15 as p-polarized light is transmitted by the half mirror 3
A part of the light beam is split as a reference light beam 330R by 52, and the remaining light beam 330S transmitted through the half mirror 352.
Is incident on the interface 15. Light beam 330 totally reflected at interface 15
Reference light beam 330 reflected by S and mirror 354
R enters the half mirror 353 and is combined. The combined light beam 330 'is condensed by the condenser lens 355, passes through the aperture 356, and is detected by the CCD 360.
At this time, the light beam 330 'detected by the CCD 360 generates interference fringes according to the state of interference between the light beam 330S and the reference light beam 330R.

Here, the sensing substance 18 fixed on the surface of the metal film 14 binds to a specific substance in the sample 20. Examples of such a combination of the specific substance and the sensing substance 18 include an antigen and an antibody. In that case, measure continuously after dispensing 20 samples and use CCD36
The presence or absence of an antigen-antibody reaction can be detected by detecting the change in the interference fringes detected by 0. That is, in this case, when the refractive index of the sensing substance 18 changes according to the binding state between the specific substance and the sensing substance 18, the light beam 330S and the reference light beam 330R totally reflected at the interface 15 are combined by the half mirror 353. When the interference occurs, the state of interference changes, so that the antigen-antibody reaction can be detected according to the change in the interference fringe. In this case, both the sample 20 and the sensing substance 18 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.

Then, the measuring plate 10 'or the measuring system is moved relative to each other by a moving means (not shown) so that the measuring well 12a to be used for the next measurement will be at a predetermined position with respect to the measuring system. . In this way, the measurement wells 12a can be used for measurement one after another as the measurement plate 10 'moves.

As described above, by using the main measurement plate 10 ′, the light beam 33 that enters or exits the dielectric block 16 of the measurement well 12 a.
Vignetting does not occur in 0S, there is no decrease in measurement accuracy,
Measurement can be performed efficiently.

Further, FIG. 14 is a side view of a measuring apparatus of another form using total reflection using the measuring plate of the present invention.
, And briefly explained.

The measuring device of which side view is shown in FIG.
The measurement plate 10 shown in FIG. 2 is provided, and the light beam is incident on the bottom surface 15 of the measurement well 12a under the condition of total reflection, and is injected into the well by the evanescent wave generated on the sample side of the well bottom surface 15 at the time of the total reflection. The sample is analyzed by exciting the sample 420 and detecting the fluorescence 435 emitted from the sample 420. This measuring device is used, for example, in a fluorescent immunoassay method (fluorescent immunoassay) for measuring an antigen-antibody reaction using a fluorescent substance as a label.

Further, the present measuring apparatus includes an excitation optical system including a laser light source 431 such as a semiconductor laser for generating a light beam 430 and a condenser lens 32, and a sample side when the light beam 430 is totally reflected on the bottom surface 15 of the well. Condensing lens 436 that collects fluorescence 435 generated from the sample excited by the evanescent wave that exudes and fluorescence spectroscope 437 that receives the fluorescence 435.
And a signal processing unit 438 connected to the fluorescence spectroscope 437.

The sample analysis by the measuring device having the above-mentioned structure will be described below. The sample 420 to be measured is supplied to the measurement well 12a by the pipetting machine 50 shown in FIG. 2 as in the case of the above-described other measuring device.

Measurement well 12a holding sample 420
However, the laser beam source 431 is driven in a state where the dielectric block 16 is installed at the measurement position where the light beam 430 is incident by the moving means, and the light beam 430 emitted therefrom is converged by the lens 432 and wells. It is incident on the bottom surface 15 under the condition of total reflection.

When the light beam 430 is totally reflected in this way, an evanescent wave seeps out from the well bottom surface 15 to the well side. Then, this evanescent wave causes the sample 420
Are excited, and then the sample 420 fluoresces when returning to the ground state.

The fluorescence 435 emitted from the sample 420 is condensed by the condenser lens 436 and detected by the fluorescence spectroscope 437. The signal processing unit 438 analyzes the properties of the sample by detecting the fluorescence emitted from the sample, and outputs the analysis result to a display unit (not shown).

Then, the measuring plate 10 or the measuring system is moved relative to each other by a moving means (not shown),
Next, the measurement well 12a to be used for measurement is set at a predetermined position with respect to the measurement system. In this way measuring well 12
a can be used for measurement one after another as the measurement plate 10 moves.

The measuring apparatus using each of the above-mentioned total reflections is provided with one measuring system and moving means for moving the plate two-dimensionally with respect to the measuring system. For example, it is provided with eight measuring systems and moving means for moving the plates relative to the measuring systems only in a direction perpendicular to the arrangement direction of the measuring systems. Simultaneously perform measurements on eight measuring wells that are lined up, and move the measuring system or plate in a direction perpendicular to the direction in which the measuring systems are arranged by the moving means so that the measuring wells in each row are sequentially measured. Good.

[Brief description of drawings]

FIG. 1 is a perspective view showing a measuring plate according to a first embodiment of the present invention.

FIG. 2 is a front view showing a dispensing device for dispensing a sample liquid on a measurement plate.

FIG. 3 is a side view of a surface plasmon sensor using the measurement plate shown in FIG.

FIG. 4 is a graph showing a schematic relationship between an incident angle of a light beam on a surface plasmon sensor and a light intensity detected by a photodetector.

FIG. 5 is a side view of a leaky mode sensor using the measurement plate of the present invention.

FIG. 6 is a top view showing another embodiment of the measuring plate of the present invention.

FIG. 7 is a top view showing another embodiment of the measuring plate of the present invention.

FIG. 8 is a top view showing another embodiment of the measuring plate of the present invention.

9 is a partially enlarged cross-sectional view of the measurement plate shown in FIG.

FIG. 10 is a view for explaining the well shape of the measurement plate of the present invention.

FIG. 11 is a view for explaining the well shape of the measurement plate of the present invention.

FIG. 12 is a view for explaining the well shape of the measurement plate of the present invention.

FIG. 13 is a side view of another measuring apparatus using total reflection using the measuring plate shown in FIG.

FIG. 14 is a side view of a measuring device using still another total reflection using the measuring plate shown in FIG.

[Explanation of symbols]

10 Measuring plate 11 Base 12 well 12a Measuring well 12b Non-measurement well 14 Metal film 16 Dielectric block 30 light beams 31 light source 32 condenser lens 40 photo detector 41 Cladding layer 42 Optical waveguide layer 50 dispenser 60 Signal processing unit

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 2G043 AA01 BA16 CA03 DA06 EA01                       EA15 GA07 GB01 GB05 GB16                       HA01 HA02 HA05 HA07 KA09                       LA03                 2G057 AA02 AA04 AB04 AB07 AC01                       BA01 BA03 BB06                 2G059 AA01 AA05 BB04 BB12 CC16                       DD13 EE02 EE05 EE09 FF08                       FF09 FF11 GG02 GG04 JJ11                       JJ13 JJ17 JJ19 JJ20 JJ22                       KK04 PP04

Claims (8)

[Claims] 1. A microtiter plate is provided with a plurality of wells arranged at positions according to the standard on the surface of a plate base having the same outer shape as the predetermined standard, and the plurality of wells on the back surface of the plate base are provided. A dielectric having an incident surface on which a predetermined light beam is incident so as to be totally reflected on the bottom surface of the well and an emission surface for emitting the light beam totally reflected on the bottom surface only on the outer surface of the bottom of some of the wells. A measuring plate of a measuring device using total reflection, comprising a body block, and wells having the dielectric block are arranged so as not to be adjacent to each other in at least one arrangement direction. 2. The measuring apparatus utilizing total internal reflection according to claim 1, wherein the dielectric block is provided in wells in every one row or more or in every one column or more of the plurality of wells. Measuring plate. 3. The measuring plate of a measuring device using total internal reflection according to claim 1, wherein the dielectric block is provided in wells at staggered positions among the plurality of wells. . 4. A plurality of wells are provided on a surface of a plate substrate having the same outer shape as a predetermined standard of a microtiter plate at a position of one row or more or one column or more arranged in a matrix according to the standard. The wells of the plurality of wells are disposed on the outer surface of the bottom of each well so that a predetermined light beam is incident on the bottom surface of the well so that the light beam is totally reflected by the well surface. A measuring plate of a measuring device utilizing total internal reflection, comprising a dielectric block having a surface. 5. A plurality of wells are arranged at staggered positions in a matrix according to the standard on the surface of a plate substrate having the same outer shape as the predetermined standard of the microtiter plate. A dielectric block having an incident surface on a bottom outer surface of each well for allowing a predetermined light beam to be totally reflected on the bottom surface of the well and an emission surface for emitting the light beam totally reflected on the bottom surface. A measuring plate for a measuring device using total reflection, which is characterized by being provided. 6. The total internal reflection according to claim 1, wherein a thin film layer is formed on the inner surface of the bottom of the well having the dielectric block on the outer surface of the bottom. Plate for the measured measuring device. 7. The measuring device utilizing total internal reflection according to claim 6, wherein the thin film layer is a metal film and is used in a measuring device for measuring a state of attenuation of total internal reflection due to surface plasmon resonance. Measuring plate. 8. A measurement for measuring a state of attenuation of total reflection due to excitation of a guided mode in the optical waveguide layer, wherein the thin film layer comprises a cladding layer and an optical waveguide layer formed thereon. The measuring plate of a measuring device utilizing total internal reflection according to claim 6, which is used in the device.