JP2002277389A - Measuring method and measuring instrument using attenuated total reflectance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To reduce the influence of a bulk effect or the like generated by change in the refractive index of a solvent when a sample solution is supplied to a measuring unit, so as to enhance measuring precision for interaction between a sensing substance and the sample solution, in a measuring instrument using attenuated total reflectance by surface plasmon resonance or the like. SOLUTION: The sample solution 15 is supplied droppingwisely to the measuring unit 10 fixed with the sensing substance 14, a light beam 30 gets incident to bring a total reflectance condition in an interface 12a between a dielectric block 11 and a metal film 12, the beam 30 total-reflected in the interface 12a is detected by an optical sensor 40, and a total reflectance attenuation angle in the beam 30 is detected. A detection value detected just after the supply of the sample solution is stored as a correction value at first, then the correction value is subtracted from a detection value after the lapse of a prescribed time, and a change with the lapse of time in the total reflectance attenuation angle is found based on the corrected detection value to measure an interaction state between the sensing substance and the sample solution. The measuring precision is enhanced since the correction value reflecting mainly a noise such as the bulk effect is subtracted from the detection value.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a measuring method and a measuring apparatus utilizing attenuated total reflection, such as a surface plasmon measurement for analyzing a characteristic of a substance utilizing generation of a surface plasmon, and more particularly to a sensing substance. TECHNICAL FIELD The present invention relates to a measurement method and a measurement apparatus using attenuated total reflection for measuring an interaction state between a sample and a sample liquid.

[0002]

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

Conventionally, there have been proposed various surface plasmon measuring devices for analyzing characteristics of a substance to be measured by utilizing a phenomenon in which surface plasmons are excited by light waves.
Among them, a particularly well-known one uses a system called a Kretschmann configuration (see, for example, JP-A-6-167443).

A surface plasmon measuring apparatus using the above system basically has a dielectric block formed, for example, in the shape of a prism, and is formed on one surface of the dielectric block and brought into contact with a substance to be measured such as a liquid sample. A metal film, a light source for generating a light beam, and an optical system for causing the light beam to enter the dielectric block at various angles so as to obtain a total reflection condition at an interface between the dielectric block and the metal film. And an optical sensor for measuring the intensity of the light beam totally reflected at the interface to detect a surface plasmon resonance state, that is, a state of attenuated total reflection.

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

In the surface plasmon measuring apparatus having the above structure, when a light beam is incident on a metal film at a specific incident angle equal to or larger than the total reflection angle, an evanescent substance having an electric field distribution in a substance to be measured in contact with the metal film. A wave is generated, and surface plasmon is excited at the interface between the metal film and the substance to be measured by the evanescent wave. When the wave number 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 light is transferred to the surface plasmon. The intensity of the reflected light drops sharply. This decrease in light intensity is generally detected as a dark line by the optical sensor.

[0007] The above resonance occurs only when the incident beam is p-polarized. Therefore, it is necessary to set in advance so that the light beam is incident as p-polarized light.

[0008] incident angle The attenuated total reflection (ATR) occurs, that is, the wave number of the surface plasmon is determined from the total reflection attenuation angle theta _SP is known, the dielectric constant of a measured substance can be determined. That surface plasmon wave number K _SP of _the angular frequency of the surface plasmon omega, the speed of light in vacuum c, metal epsilon _m and epsilon _s respectively, when the dielectric constant of the measured substance, the following relationship.

[0009]

(Equation 1) That is, by knowing the attenuated total reflection angle θ _SP which is the incident angle at which the reflected light intensity decreases, the dielectric constant ε _{s of the} substance to be measured _, that is, the characteristics related to the refractive index can be obtained.

In this type of surface plasmon measuring apparatus, an array-like surface plasmon measuring apparatus, as disclosed in Japanese Patent Application Laid-Open No. H11-326194, is provided for the purpose of accurately measuring the total reflection attenuation angle θ _SP with a large dynamic range. It has been considered to use an optical sensor. In this optical sensor, a plurality of light receiving elements are arranged in a predetermined direction, and are arranged such that components of light beams totally reflected at various angles at the interface are received by different light receiving elements. It is.

In this case, differentiating means for differentiating a light detection signal output from each light receiving element of the array-shaped optical sensor with respect to the direction in which the light receiving element is provided is provided. In many cases, characteristics related to the refractive index of the substance to be measured are obtained based on

Further, as a similar measuring apparatus utilizing the attenuated total reflection (ATR), for example, "Spectroscopy", Vol. 47, No. 1 (1998), pp. 21-23 and 26-27.
A leak mode measuring device described on the page is also known. This leak mode measurement device basically has a dielectric block formed, for example, in the shape of a prism, a clad layer formed on one surface of the dielectric block, and formed on the clad layer and brought into contact with the sample liquid. 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 as to obtain a total reflection condition at an interface between the dielectric block and the cladding layer. An optical system for incidence is provided, and an optical sensor for measuring the intensity of the light beam totally reflected at the interface and detecting the excited state of the waveguide mode, that is, the attenuated state of total reflection, is provided.

[0013] In the leaky mode measuring device having the above configuration,
When the light beam is incident on the cladding layer through the dielectric block at an incident angle equal to or greater than the total reflection angle, only light having a specific wave number at a specific incident angle is transmitted through the cladding layer. The light propagates in a guided mode. When the waveguide mode is excited in this way, most of the incident light is taken into the optical waveguide layer, so that total reflection attenuation occurs in which the intensity of 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, knowing the specific incident angle at which the total reflection attenuation occurs, the refractive index of the substance to be measured and the related The properties of the substance can be analyzed.

In this leak mode measuring apparatus,
In order to detect the position of a dark line generated in reflected light due to attenuated total reflection, the above-mentioned array-shaped optical sensor can be used, and in addition to this, the above-described differentiating means is often applied.

The above-described surface plasmon measuring device and leak mode measuring device may be used in the field of drug discovery research and the like to study the interaction between a desired sensing substance and a sample liquid. For example, it is used for measuring an interaction such as a binding action between a sensing substance and a specific substance contained in a sample liquid and a dissociation action of a specific substance from a binding substance into a sample liquid.

When used for random screening for finding a specific substance that binds to a sensing substance, the thin film layer (a metal film in the case of a surface plasmon measuring device, a cladding layer and an optical waveguide in the case of a leakage mode measuring device). On the wave layer), a sensing substance is fixed as the substance to be measured, and a sample solution in which various analytes are dissolved in a solvent is added to the sensing substance. The angle of θ _SP is measured. If the analyte in the sample solution binds to the sensing substance, the binding changes the refractive index of the sensing substance with time. Therefore, the attenuated total reflection angle theta _SP was measured every predetermined time, and it is determined whether or not a change in the attenuated total reflection angle theta _SP occurs, measure a binding state between a test substance and a sensing substance Then, based on the result, it can be determined whether or not the subject is a specific substance that binds to the sensing substance. Examples of such a combination of the specific substance and the sensing substance include an antigen and an antibody, or an antibody and an antibody. Specifically, a rabbit anti-human IgG antibody can be immobilized on the measurement unit as a sensing substance, and a human IgG antibody can be used as the specific substance.

In order to measure the binding state between the subject and the sensing substance, it is not always necessary to detect the angle of the total reflection attenuation angle θ _SP itself. For example, it is also possible to add a sample liquid to a sensing substance, measure the angle change of the total reflection attenuation angle θ _SP thereafter, and measure the binding state based on the magnitude of the angle change. If the above-described array-shaped optical sensor and differentiating means are applied to a measuring device using attenuated total reflection, the amount of change in the differential value reflects the amount of change in the attenuated total reflection angle θ _SP . The binding state between the sensing substance and the analyte can be measured based on the magnitude of the change in the differential value (see Japanese Patent Application No. 2000-398309 by the present applicant).

The above-described random screening may be performed using a high-throughput screening apparatus in order to simultaneously screen a large number of subjects. In such a case, for example, by arranging a plurality of measurement units on a turntable and the like, and by rotating the turntable, the measurement units are sequentially arranged with respect to the measurement mechanism including the light source, the optical system, and the optical sensor.
It is moved to a predetermined position and the measurement of each measurement unit is performed intermittently.

[0019]

However, in the conventional measuring method and apparatus utilizing attenuated total reflection, a cup-shaped or petri-shaped plate in which a sensing substance is fixed on a thin film layer previously formed on the bottom surface. The sample liquid is supplied to the measurement unit of, and the above-described total reflection attenuation angle θ _SP
Is measured.

When the sample liquid is supplied to the measurement unit and the sensing substance and the subject are combined, the refractive index of the sensing substance changes, and the angle of the attenuated total reflection angle θ _SP changes. However, the angle change of the total reflection attenuation angle θ _SP immediately after supplying the sample liquid to the measurement unit does not strictly reflect only the change in the refractive index of the sensing substance, but the change in the refractive index of the sensing substance and the solvent. Reflects the total change in the refractive index due to the addition of. The change in the refractive index due to the addition of the solvent is called "bulk effect". This bulk effect changes depending on the type of the solvent in the sample liquid and the like. Therefore, due to the influence of the bulk effect, there is a possibility that the measurement accuracy of the binding state between the sensing substance and the analyte is reduced.

In many cases, a protective solution for protecting the sensing substance is previously stored in the measuring unit. Even in this case, when a solvent is added, the refractive index of the protective solution changes. Again, there was a risk that a bulk effect would occur.

In addition to the bulk effect, immediately after the sample solution is supplied to the measurement unit to which the sensing substance is fixed, the interaction with the interaction such as binding or dissociation due to an increase in the fluid pressure or the like. In some cases, different phenomena may occur, and the measurement accuracy of the interaction between the sensing substance and the sample liquid may be reduced due to the influence of the angle change of the attenuated total reflection angle θ _SP caused by these phenomena.

If one measuring unit is provided with one light detecting means, the change in the total reflection attenuation angle θ _SP is constantly monitored, and the change in the total reflection attenuation angle θ _SP before the sample is supplied is measured. In the case of continuous display, the angle change of the attenuated total reflection angle θ _SP caused by a phenomenon different from the bulk effect or the interaction as described above, that is, the interaction between the noise and the sensing substance and the sample liquid. the angular change of attenuated total reflection angle theta _{S P} caused by the action can be easily distinguished.

However, when performing the above-described random screening or the like, a high-throughput screening apparatus may be used in order to efficiently measure a large number of measurement units. Since the units are sequentially measured, the angle change of the total reflection attenuation angle θ _SP is intermittently detected. For example, if it is a high-throughput screening device using a turntable,
By rotating a turntable on which a plurality of measurement units are arranged, the measurement units are sequentially turned on by the light source,
The light detecting means including the optical system and the optical sensor is moved to a predetermined position, and the measurement of each measuring unit is intermittently performed.

For this reason, the state of the angle change of the total reflection attenuation angle θ _SP cannot be continuously detected, and the angle change of the total reflection attenuation angle θ _SP which is noise is caused by the interaction between the sensing substance and the subject. It is difficult to distinguish the change in the total reflection attenuation angle θ _{SP from} the change in angle, and there is a possibility that the measurement accuracy of the interaction between the sensing substance and the sample liquid may be reduced.

In view of the above circumstances, the present invention relates to a measuring method and apparatus using attenuated total reflection for sequentially measuring a plurality of measuring units, wherein the influence of noise generated immediately after the sample liquid is supplied to the measuring units is provided. It is an object of the present invention to provide a measurement method and an apparatus using attenuated total reflection, which can reduce measurement and improve the measurement accuracy of the interaction between a sensing substance and a sample liquid.

[0027]

SUMMARY OF THE INVENTION A measuring method using attenuated total reflection according to 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 one surface of the dielectric block, a sensing substance disposed on the surface of the thin film layer and interacting with a sample liquid, and the sensing substance A plurality of measurement units each including a sample liquid holding mechanism for holding the sample liquid on the surface of the plurality of measurement units, and applying the light beam to a dielectric block of one of the plurality of measurement units. An optical system that makes incident light at various angles so that total reflection conditions are obtained at the interface between the dielectric block and the thin film layer, and an optical sensor that detects the intensity of a light beam that is totally reflected at the interface. Means, a support for supporting the plurality of measurement units, and the light beam is sequentially incident on the dielectric blocks of the plurality of measurement units at the various incident angles. To allow detection by Sa, and the support is moved relative to said light detecting means,
In a measuring apparatus using attenuated total reflection provided with a moving means for arranging each measuring unit at a predetermined position with respect to the light detecting means, a supply of a sample liquid to and a supply of a sample liquid to a sample liquid holding mechanism of each measuring unit After the elapse of a predetermined time, the support is moved relative to the light detection means so that the measurement unit is disposed at the predetermined position with respect to the light detection means, and Storing the detection result detected by the light detection means immediately after the supply of the sample liquid to the sample liquid holding mechanism, and correcting the detection result detected after the lapse of the predetermined time based on the stored detection result. And measuring the change over time in the state of total reflection attenuation based on the corrected detection result.

The measuring method using attenuated total reflection according to the present invention can be particularly applied to the above-described surface plasmon measuring method. In this case, a light source for generating a light beam and a light source for the light beam are used. Transparent dielectric block,
A metal film formed on one surface of the dielectric block, a sensing substance arranged on the surface of the metal film to interact with the sample liquid, and a sample liquid holding the sample liquid on the surface of the sensing substance A plurality of measurement units each including a holding mechanism, and the light beam being applied to a dielectric block of one of the plurality of measurement units at an interface between the dielectric block and the metal film. It supports an optical system that makes incident light at various angles of incidence so as to obtain reflection conditions, an optical sensor that detects the intensity of a light beam that is totally reflected at the interface, and supports the plurality of measurement units. The support and the support so that the light beams are sequentially incident on the dielectric blocks of the plurality of measurement units at the various incident angles and can be detected by the optical sensor. A measuring unit using attenuated total reflection, comprising: a moving unit that moves relative to the light detecting unit and arranges each measuring unit at a predetermined position with respect to the light detecting unit. Immediately after the supply of the sample liquid to the sample liquid holding mechanism and at a predetermined time after the supply, the support is placed on the light detection means so that the measurement unit is disposed at the predetermined position with respect to the light detection means. Relative to the measuring unit, for each of the measurement units, storing the detection result detected by the light detecting means immediately after the supply of the sample liquid to the sample liquid holding mechanism, based on the stored detection result The detection result detected after the lapse of the predetermined time is corrected, and the change over time in the state of attenuated total reflection is measured based on the corrected detection result.

Further, the measuring method using attenuated total reflection according to the present invention can be particularly applied to the above-mentioned leak mode measuring method. In this case, a light source for generating a light beam and a light source for the light beam are used. Transparent dielectric block, a cladding layer formed on one surface of the dielectric block, an optical waveguide layer formed on the cladding layer, and disposed on the surface of the optical waveguide layer to interact with the sample liquid. , A plurality of measurement units each including a sample liquid holding mechanism for holding the sample liquid on the surface of the sensing substance, and one of the plurality of measurement units for transmitting the light beam. For the dielectric block, an optical system that is incident at various angles of incidence so that total reflection conditions are obtained at the interface between the dielectric block and the thin film layer, and the light is totally reflected at the interface A light detecting means comprising an optical sensor for detecting the intensity of the beam; a support for supporting the plurality of measuring units; and the light beams sequentially entering the various incident angles with respect to each dielectric block of the plurality of measuring units. In, the support is relatively moved with respect to the light detection means so that detection by the light sensor can be performed,
In a measuring apparatus using attenuated total reflection provided with a moving means for arranging each measuring unit at a predetermined position with respect to the light detecting means, a supply of a sample liquid to and a supply of a sample liquid to a sample liquid holding mechanism of each measuring unit After the elapse of a predetermined time, the support is moved relative to the light detection means so that the measurement unit is disposed at the predetermined position with respect to the light detection means, and Storing the detection result detected by the light detection means immediately after the supply of the sample liquid to the sample liquid holding mechanism, and correcting the detection result detected after the lapse of the predetermined time based on the stored detection result. And measuring the change over time in the state of total reflection attenuation based on the corrected detection result.

In the measuring apparatus utilizing attenuation of total reflection according to the present invention, a light source for generating a light beam, a dielectric block transparent to the light beam, a thin film layer formed on one surface of the dielectric block, A sensing substance arranged on the surface of the thin film layer and interacting with the sample liquid, and a plurality of measurement units including a sample liquid holding mechanism for holding the sample liquid on the surface of the sensing substance; The light beam is transmitted to one of the plurality of measurement units.
An optical system that enters the dielectric block of one measurement unit at various angles of incidence so as to obtain total reflection conditions at an interface between the dielectric block and the thin film layer, and a light beam that is totally reflected at the interface A light detecting means comprising an optical sensor for detecting the intensity of the light, a support for supporting the plurality of measuring units, and the light beam sequentially with respect to each of the dielectric blocks of the plurality of measuring units at the various incident angles. And a moving means for moving the support relative to the light detecting means, and disposing each measuring unit at a predetermined position with respect to the light detecting means, so that the light can be detected by the light sensor. In the measuring apparatus using attenuated total reflection provided with the measuring unit, the moving unit may be configured to measure the measuring unit immediately after the supply of the sample liquid to the sample liquid holding mechanism of each of the measuring units and at a predetermined time after the supply. The support is moved relatively to the light detecting means so that the sample is disposed at the predetermined position with respect to the light detecting means. Immediately after the supply of the sample liquid to the holding mechanism, the detection result detected by the light detection unit is stored, and based on the stored detection result, the detection result detected after the lapse of the predetermined time is corrected. Measuring means for measuring a change over time in the state of attenuated total reflection based on the detected result.

Further, the measuring apparatus using the total reflection attenuation according to the present invention can be particularly applied to the one configured as the above-mentioned surface plasmon measuring apparatus.
A light source for generating a light beam, a dielectric block transparent to the light beam, a metal film formed on one surface of the dielectric block, and disposed on the surface of the metal film to interact with the sample liquid. A plurality of measurement units including a generated sensing substance, and a sample liquid holding mechanism for holding the sample liquid on the surface of the sensing substance; and An optical system that enters the dielectric block at various angles of incidence so as to obtain the condition of total reflection at the interface between the dielectric block and the metal film, and the intensity of the light beam totally reflected at the interface is detected. Light detection means comprising an optical sensor
The support for supporting the plurality of measurement units, and the light beam is sequentially incident on each of the dielectric blocks of the plurality of measurement units at the various incident angles, so that the light sensor can detect the light beams. A moving means for moving a body relative to the light detecting means and disposing each measuring unit at a predetermined position with respect to the light detecting means; The support means is arranged such that the measurement unit is disposed at the predetermined position with respect to the light detection means immediately after the supply of the sample liquid to the sample liquid holding mechanism of each of the measurement units and at a predetermined time after the supply. The body is moved relatively to the light detection means, for each of the measurement units,
Immediately after the supply of the sample liquid to the sample liquid holding mechanism, the detection result detected by the light detection means is stored, and based on the stored detection result, the detection result detected after the lapse of the predetermined time is corrected, Measurement means for measuring a change over time in a state of attenuated total reflection by surface plasmon resonance based on the corrected detection result is provided.

In addition, the measuring apparatus using the total reflection attenuation according to the present invention can be particularly applied to the one configured as the above-described leak mode measuring apparatus. In this case, a light source for generating a light beam is used. A dielectric block transparent to the light beam, a clad layer formed on one surface of the dielectric block, an optical waveguide layer formed on the clad layer, and disposed on a surface of the optical waveguide layer. A plurality of measurement units each including a sensing substance which interacts with a sample liquid, and a sample liquid holding mechanism for holding the sample liquid on the surface of the sensing substance; and An optical system for making the dielectric block of one of the measurement units incident at various angles of incidence such that the total reflection condition is obtained at the interface between the dielectric block and the thin film layer; A light detecting means comprising an optical sensor for detecting the intensity of the light beam totally reflected at the interface; a support for supporting the plurality of measurement units; and the light for each dielectric block of the plurality of measurement units. The beam is incident at the various angles of incidence, and the support is moved relatively to the light detecting means so that detection by the light sensor can be performed, and each measuring unit is moved relative to the light detecting means. In a measuring apparatus using attenuated total reflection having a moving means disposed at a predetermined position, the moving means is provided immediately after supply of the sample liquid to the sample liquid holding mechanism of each of the measurement units and at a predetermined time after the supply, The support is moved relatively to the light detecting means so that the measuring unit is disposed at the predetermined position with respect to the light detecting means. Each time, the detection result detected by the light detection means is stored immediately after the supply of the sample liquid to the sample liquid holding mechanism, and the detection result detected after the predetermined time has elapsed based on the stored detection result. And a measuring means for measuring a change with time in the state of total reflection attenuation due to excitation of the waveguide mode in the optical waveguide layer based on the corrected detection result.

In the measuring method and measuring apparatus utilizing the above-mentioned various types of attenuated total reflection, it is preferable that the time immediately after the supply is within a period of 60 seconds after the supply of the sample liquid to the measuring unit. It may be until 30 seconds elapse or until 10 seconds elapse after supply.
The predetermined time is preferably at least five times the elapsed time from when the sample liquid was supplied to the sample liquid holding mechanism of the measurement unit to immediately after the supply.

The light detecting means detects when each measuring unit is arranged at a predetermined position with respect to the light detecting means.

The “interaction” includes protein-protein interaction, DNA-protein interaction,
It includes sugar-protein interaction, protein-peptide interaction, lipid-protein interaction, chemical substance interaction, and the like.

[0036]

According to the measuring method and the measuring apparatus utilizing the attenuated total reflection according to the present invention, the attenuated total reflection is utilized by sequentially arranging a plurality of measuring units supported by a support at predetermined positions with respect to the light detecting means. In the measuring apparatus described above, first, for each measuring unit, immediately after the sample liquid of the sample liquid holding mechanism is supplied, detection is performed by the light detecting means, and the detection result is stored. Immediately after the supply of the sample liquid, the interaction between the sensing substance and the sample liquid has not yet progressed. On the other hand, when the sample liquid is supplied, the noise effect due to the bulk effect and the liquid pressure has occurred. The detection result immediately after can be regarded as a detection result mainly reflecting the noise phenomenon.

For this reason, based on the detection results immediately after the supply of the sample liquid, the detection results detected a predetermined time after the supply of the sample liquid to the measuring unit are corrected, and the total reflection is performed based on the corrected detection results. By measuring the change over time in the state of attenuation, the influence of noise generated immediately after the sample liquid is supplied to the measurement unit can be reduced, and the accuracy of measurement of the interaction between the sensing substance and the sample liquid can be improved.

If the time immediately after the supply is set to be 60 seconds after the supply of the sample liquid, the interaction between the sensing substance and the sample liquid has not progressed so much in 60 seconds. The result can be regarded as a detection result that mainly reflects the noise phenomenon, and the measurement accuracy of the interaction between the sensing substance and the sample liquid can be improved when the subsequent time-dependent change is measured. There is sufficient time to dispose the measurement unit at a predetermined position with respect to the light detecting means, so that there is no need to change the configuration of the moving means or the like.

Further, if the time immediately after the supply is set to be 30 seconds after the supply, the progress of the interaction between the sensing substance and the sample liquid is further reduced, so that the measurement of the interaction between the sensing substance and the sample liquid is performed. Accuracy can be further improved. In addition, if the time immediately after the supply is set until 10 seconds elapse after the supply, the interaction between the sensing substance and the sample liquid hardly progresses. It can be further improved.

Further, if the predetermined time is set to be five times or more the elapsed time from the time when the sample liquid is supplied to immediately after the supply, the error that occurs when measuring the change over time is small, and the reliability of the measurement result is low. Can be improved.

[0041]

Embodiments of the present invention will be described below in detail with reference to the drawings. FIG. 1 shows an overall shape of a surface plasmon measuring device according to a first embodiment of the present invention, and FIG. 2 shows a side shape of a main part of the device. In this surface plasmon measurement device, the amount of change in the total reflection attenuation angle θ _SP due to surface plasmon resonance is measured, and it is determined whether or not the sensing substance and the analyte are bonded, that is, whether or not the analyte is a specific substance. ing.

As shown in FIG. 1, the surface plasmon measuring apparatus includes a plurality of measuring units 10, a turntable 20 supporting the plurality of measuring units 10, and a support body for rotating the turntable 20 intermittently. Means 21, a laser light source 31 such as a semiconductor laser for generating a measurement light beam (laser beam) 30, a condenser lens 32 constituting an incident optical system, an optical sensor 40, the laser light source 31, and a support. While controlling the driving of the moving means 21, the optical sensor
It has a controller 60 that receives the output signal S of 40 and performs processing described below, and a sample liquid supply mechanism 70.

As shown in FIG. 2, the measuring unit 10 has a dielectric block 11 having, for example, a substantially quadrangular pyramid shape, and is formed on one surface (the upper surface in the figure) of the dielectric block 11.
For example, it has a metal film 12 made of gold, silver, copper, aluminum or the like.

The dielectric block 11 is made of, for example, a transparent resin, and has a raised portion around the portion where the metal film 12 is formed. The raised portion 13 serves as a sample liquid holding mechanism for storing the sample liquid 15. Function. In this example, the metal film
A sensing substance 14 is fixed on the top 12, and this sensing substance 14 will be described later.

On the turntable 20, a plurality (11 in this example) of through-holes 22 for fitting and holding the measurement unit 10 are provided.
Are provided at equal angular intervals on the circumference of the turntable 20 around the rotation shaft 23. The measurement unit 10 is held in the turntable 20 in a replaceable state. The support moving means 21 is composed of a stepping motor or the like,
The turntable 20 is intermittently rotated by an angle equal to the arrangement angle of the measurement unit 10.

The condenser lens 32 has a light beam as shown in FIG.
30 is condensed and passed through the dielectric block 11 in a convergent light state, and is incident on the interface 12a between the dielectric block 11 and the metal film 12 so that various incident angles can be obtained. The range of the incident angle is a range including an angle range in which the condition of total reflection of the light beam 30 at the interface 12a is obtained and surface plasmon resonance can occur.

The light beam 30 enters the interface 12a as p-polarized light. In order to do so, the laser light source 31 may be disposed in advance so that the polarization direction thereof becomes a predetermined direction. Alternatively, the direction of polarization of the light beam 30 may be controlled by a wave plate or a polarizing plate.

The optical sensor 40 is a photodiode array in which a large number of photodiodes are arranged in one row, and the photodiodes are arranged so that the photodiodes are arranged in the direction indicated by the arrow X in FIG.

On the other hand, the controller 60 is
Receives an address signal A indicating the rotation stop position, and outputs a drive signal D for operating the support moving means 21 based on a predetermined sequence. In the present embodiment, within 60 seconds after the sample liquid is supplied dropwise to the measurement unit 10, the measurement unit 10
The above sequence is created so that the optical sensor 40 is moved to a predetermined position, the first detection is performed, and the change over time is measured at intervals of 5 minutes after the supply of the sample liquid 15. Thereafter, detection is sequentially performed at predetermined time intervals. Further, the controller 60 includes a measuring unit 61 that receives the output signal S of the optical sensor 40, and a display unit 62 that receives an output from the measuring unit 61.

As shown in FIG. 3, the measuring means 61 includes a differential amplifier array 63 connected to the optical sensor 40 and a driver 64
And a signal processing unit 65 composed of a computer system or the like and performing signal processing including correction of measured values and the like.

As shown in the figure, the driver 64 samples and holds the outputs of the differential amplifiers 63a, 63b, 63c... Of the differential amplifier array 63.
b, 52c..., these sample and hold circuits 52a, 52
b, 52c,.
The A / D converter 54 that digitizes the output of the multiplexer 53 and inputs it to the signal processing unit 65, the driving circuit 55 that drives the multiplexer 53 and the sample and hold circuits 52a, 52b, 52c,. The control circuit 56 controls the operation of the drive circuit 55 based on the instruction.

The sample liquid supply mechanism 70 is composed of a pipette 71 for sucking and holding a predetermined amount of the sample liquid and a means 72 for moving the pipette 71, and a sample liquid container 73 set at a predetermined position. Then, the sample liquid is sucked and held by the pipette 71, and the sample liquid is dropped and supplied into the sample liquid holding frame 13 of the measurement unit 10 at a predetermined stop position on the turntable 20.

The operation of the surface plasmon measuring device having the above-described configuration for measuring the amount of change in the total reflection attenuation angle θ _SP due to surface plasmon resonance will be described. First, before performing an actual measurement, a plurality of types of sample liquids containing different analytes are prepared. Also, the same number of measurement units 10 as the types of the sample liquid are prepared. Normally, the sensing substance fixed to the measurement unit 10 differs depending on the measurer. For this reason, the measurer purchases a measurement unit to which the sensing substance 14 is not fixed (hereinafter referred to as a measurement cup) and fixes the sensing substance 14 to the measurement cup. For example, when the sensing substance 14 is streptavidin, which is a kind of protein, first, 10 μg / mL of streptavidin is added to the measuring cup.
After pooling for 5 minutes, washing with PBS (phosphate buffer solution) is performed, and then ethanolamine is pooled for 5 minutes to perform blocking. After washing with PBS, a 1% BSA (Bovine Serum Albumin: bovine serum albumin) solution as a non-specific adsorbent is pooled for 10 minutes, washed again with PBS, and finally PBS for protecting the sensing substance is added. Thus, a measurement unit 10 to which streptavicin as a sensing substance is fixed is created. These measuring units 10, for example, are arranged in a 96-well cassette. Thereafter, each measuring unit 10 is moved by a measuring unit moving mechanism (not shown).
Are sequentially arranged in the through holes 22 on the turntable 20.

The sample liquid supply mechanism 70 sequentially suctions and holds the sample liquid from the sample liquid container 73 to the pipette 71, and drops the sample liquid into the sample liquid holding frame 13 of the measurement unit 10. As the sample liquid, for example, a liquid containing 0.1% BSA / PBS liquid (PBS liquid containing 0.1% BSA) as a solvent and biotinylated insulin as a subject is prepared.

When the sample liquid is dropped and supplied to the measurement unit 10 by the sample liquid supply mechanism 70, the turntable 20 is stopped after rotating several times, and the measurement unit 10 It is in a state of resting at the measurement position where the beam 30 enters (the position of the measurement unit 10 on the right side in FIG. 2). In this state, the laser light source 31 is driven by a command from the controller 60, and the light beam 30 emitted from the laser light source 31 is incident on the interface 12a between the dielectric block 11 and the metal film 12 while being converged as described above. I do. The light beam 30 totally reflected at the interface 12a is detected by the optical sensor 40. Note that the above operation is performed according to a sequence set in the controller 60 in advance, and within 60 seconds after the sample liquid is dropped into the measurement unit 10, the measurement unit 10 is moved to the measurement position and measurement is performed. .

The optical sensor 40 in this embodiment is a photodiode array in which a plurality of photodiodes 40a, 40b, 40c... Are arranged in a line, and the light beam 30 travels within the plane shown in FIG. The direction in which the photodiodes are disposed is substantially perpendicular to the direction. Therefore, different components of the light beam 30 totally reflected at the interface 12a at various reflection angles are received by the different photodiodes 40a, 40b, 40c,.

The above photodiodes 40a, 40b, 40c ...
… Each output of the differential amplifier 63 of the differential amplifier array 63
a, 63b, 63c... At this time, outputs of two photodiodes adjacent to each other are input to a common differential amplifier. Therefore, each differential amplifier 63a, 63b,
The output of 63c is a plurality of photodiodes 40a, 40
It can be considered that the light detection signals output from b, 40c,... are differentiated with respect to their disposition directions.

The outputs of the differential amplifiers 63a, 63b, 63c... Are respectively sampled and held by circuits 52a, 52b, 52c.
.. Are sampled and held at a predetermined timing and input to the multiplexer 53. The multiplexer 53 is provided for each of the sampled and held differential amplifiers 63a, 63b, 63c,.
Are input to the A / D converter 54 in a predetermined order. The A / D converter 54 digitizes these outputs and inputs them to the signal processing unit 65.

FIG. 4 shows a light beam 30 totally reflected at the interface 12a.
Light intensity for each incident angle θ and differential amplifiers 63a, 63b, 63c
This explains the relationship with the output of... Here, it is assumed that the relationship between the incident angle θ of the light beam 30 to the interface 12a and the light intensity I is as shown in the graph of FIG.

The light incident on the interface 12a at a specific incident angle θ _SP excites surface plasmons at the interface between the metal film 12 and the sensing substance 14, so that the intensity I of the reflected light sharply decreases. That is, θ _SP is the total reflection attenuation angle, and the reflected light intensity I takes a minimum value at this angle θ _SP . This decrease in the reflected light intensity I is observed as a dark line in the reflected light, as indicated by D in FIG.

FIG. 4B shows a photodiode 40.
a, 40b, 40c... indicate the directions in which the photodiodes 40a, 40b, 40c are arranged as described above.
.. Correspond to the incident angle θ uniquely.

Then, the photodiodes 40a, 40b, 40c
…… the direction of arrangement, that is, the incident angle θ, and the differential amplifier
The relationship between 63a, 63b, 63c... And the output I ′ (differential value of the reflected light intensity I) is as shown in FIG.

Before measuring the change with time, first, for each measurement unit 10, a process of selecting a differential amplifier to be used and a process of setting a correction value I'r of the differential value I 'are performed. The signal processing unit 65 performs differential amplifiers 63a, 63b, 63c,... Based on the value of the differential value I ′ input from the A / D converter 54.
From the local maximum value of the change in reflected light intensity I, i.e. selects a differential amplifier closest differential value I'min on the differential value I '= 0 corresponding to the attenuated total reflection angle theta _SP is obtained. In the example of FIG. 4, the differential amplifier 63e is used.

The signal processing unit 65 stores the differential value I'min output from the differential amplifier 63e as a correction value I'r corresponding to the measurement unit in a storage unit not shown, and displays a zero point on the display unit 62. To be displayed. That is, at the time of the first measurement, the displayed value is 0 regardless of the magnitude of the differential value I ′ output from the differential amplifier 63e.

When the first measurement of the measurement unit 10 is completed, the turntable 20 is intermittently rotated by the support moving means 21. Thereafter, after the sample liquid 15 is supplied to the measurement unit 10, the turntable 20 is rotated. Every time the minutes elapse, the measurement unit 10 is moved to the detection position and the detection is performed. Signal processing section
In 65, each time the measurement unit is measured, a change amount ΔI ′ which is a value obtained by subtracting the correction value I′r corresponding to the measurement unit from the differential value I ′ output from the selected differential amplifier 63e is calculated. The calculated value is displayed on the display unit 62. Similar processing is performed on each measurement unit 10.

The differential value I ′ is determined by the fact that the dielectric constant, that is, the refractive index of the substance in contact with the metal film 12 (see FIG. 1) of the measurement chip changes, and the curve shown in FIG. When it changes in the form of doing, it goes up and down accordingly. Therefore, by continuously measuring the variation ΔI ′ of the differential value I ′ with the passage of time, the change in the refractive index of the sensing substance 14 in contact with the metal film 12 can be checked.

The analyte in the sample solution 15 is a sensing substance
If the substance binds to 14, the refractive index of the sensing substance 14 changes in accordance with the state of their binding. Therefore, by continuously measuring the above-mentioned change ΔI ′, the binding state of the subject and the sensing substance 14 is measured. Based on the measurement result, it can be determined whether or not the analyte is a specific substance that binds to the sensing substance.

That is, as shown in FIG.
If the value of changes, it can be determined that the refractive index of the sensing substance 14 has changed, that is, the analyte contained in the sample liquid 15,
It can be determined that the substance binds to the sensing substance 14.
If the value of the differential value I 'does not change, it can be determined that the subject is not a substance that binds to the sensing substance.

In the present embodiment, the sample liquid 15 is supplied to the measurement unit 10 from the differential value I ′ instead of the differential value I ′ itself.
Is measured and displayed as the amount of change ΔI ′ obtained by subtracting the correction value I′r detected immediately after the supply of. Immediately after the supply of the sample liquid, the interaction between the sensing substance and the sample liquid has not progressed yet, while the noise effect due to the bulk effect and the liquid pressure has occurred. It can be considered that the detection result reflects the phenomenon. Also,
It is considered that the differential value I ′ detected thereafter reflects the noise phenomenon and the progress of the interaction between the sensing substance and the sample liquid. Therefore, it can be considered that the change amount ΔI ′ obtained by subtracting the correction value I′r from the differential value I ′ mainly reflects the progress of the interaction between the sensing substance and the sample liquid. Therefore, if the angle change of the total reflection attenuation angle θ _SP , that is, the change over time of the state of total reflection attenuation, is measured based on these ΔI ′, the influence of noise generated immediately after the sample liquid is supplied to the measurement unit is reduced. And
The measurement accuracy of the binding action between the sensing substance and the analyte, that is, the interaction between the sensing substance and the sample liquid can be improved.

Further, if the detection of the correction value I′r is performed at a point of time 60 seconds after the supply of the sample liquid to the measuring unit 10,
At 0 seconds, the interaction between the sensing substance and the sample liquid has not progressed so much, so that it is possible to improve the measurement accuracy of the interaction between the sensing substance and the sample liquid at the time of measuring the change with time thereafter. There is sufficient time to arrange the measuring unit to which the liquid has been supplied at a predetermined position with respect to the light detecting means, so that it is not necessary to change the configuration of the moving means used in the conventional measuring apparatus.

Since the detection of the correction value I′r was performed at the point of time 60 seconds after the supply of the sample liquid to the measuring unit 10, strictly speaking, when the analyte binds to the sensing substance, The correction value I′r also includes the angle change of the attenuated total reflection angle θ _SP due to the coupling action between the sensing substance and the subject. However, since the first measurement of the change with time was performed at the time when 5 minutes had elapsed after the sample liquid 15 was supplied to the measurement unit 10, the detected value after the lapse of 5 minutes includes the correction value I′r
Since the angle change of the total reflection attenuation angle θ _SP caused by the binding action between the sensing substance and the subject included in the above is sufficiently large, a sufficient measurement accuracy can be obtained.

If there is a possibility that the speed of the interaction between the sensing substance and the sample solution may be high, it is desirable to shorten the elapsed time from the supply of the sample solution to the detection of the correction value I′r. For example, immediately after the supply, 30
If the time until the elapse of seconds is set, the progress of the interaction between the sensing substance and the sample liquid is small, so that the measurement accuracy of the interaction between the sensing substance and the sample liquid can be further improved. In addition, if the time immediately after the supply is set until 10 seconds elapse after the supply, the interaction between the sensing substance and the sample liquid hardly progresses. It can be further improved.

When it takes more than 60 seconds for the noise phenomenon immediately after the sample liquid is supplied to the measuring unit 10 to be completed, the correction value I′r is detected after the noise phenomenon is completed. It is desirable to do. That is, the most desirable timing for detecting the correction value I′r may be appropriately determined in consideration of the progress speed of the interaction between the sensing substance and the sample liquid and the timing when the noise phenomenon ends.

Further, a plurality of measurement units 10 are arranged on a turntable 20, and by rotating this turntable 20, each measurement unit 10 can be sequentially arranged at a predetermined position with respect to the condenser lens 32 and the optical sensor 40. With such a configuration, the differential values in the plurality of measurement units 10 can be measured one after another with the rotation of the turntable 20, and the measurement for a large number of measurement units 10 can be performed in a short time. .

In each of the embodiments described above, the rotating turntable 20 is used as a support for supporting the measuring unit 10. However, the shape and the moving method of the support are not limited to this. . For example, a support that supports a plurality of measurement units is configured to reciprocate linearly, and with the movement, the plurality of measurement units are sequentially set on a light detection unit including a light source 31, a condenser lens 32, and an optical sensor 40. You may do it.

If a plurality of the light detecting means are provided, the measuring units are sequentially set on the light detecting means in accordance with the movement of the support, so that measurement can be performed in each measuring section. Alternatively, only one such measurement unit is provided, the support is moved in one direction, the measurement unit is set on the light detection means, and after the measurement is performed, the support is moved in the opposite direction, and the measurement unit is moved. May be set in the light detecting means again to perform the measurement.

The method of moving the support in the normal and reverse directions can be applied to the case where the turntable 20 described above is used. When this turntable 20 is used, a plurality of light detecting means are provided, and the turntable 20 is provided.
It is also possible to configure so that one measurement unit performs measurements a plurality of times during one rotation.

Further, as a modified example of the first embodiment, it is conceivable to fix the support, move the light detecting means, and measure each measuring unit 10 sequentially. Also in this case, it is possible to perform measurement on a large number of measurement units in a short time.

As described above, the dielectric block 11,
The metal film 12 and the sample holding frame are not limited to the measurement unit 10 in which the metal film 12 and the sample holding frame 13 are integrally formed.
It is also possible to apply a measurement unit in which the dielectric block 13 is integrated and is exchangeably formed with the dielectric block 11.

Next, a second embodiment of the present invention will be described with reference to FIGS.

Since the overall configuration of the second embodiment is substantially the same as that of the first embodiment, only the numbers of the different components in FIG. 1 are added in the figure. Also, in FIG. 6, the same elements as those in FIG. 2 are denoted by the same reference numerals, and description thereof will be omitted unless otherwise required.

The sensor using the attenuated total reflection according to the second embodiment is the leak mode measuring device described above.
It is configured to use the measurement unit 90. One surface of the dielectric block 11 of this measurement unit 90 (the upper surface in the figure)
, A cladding layer 91 is formed thereon, and an optical waveguide layer 92 is further formed thereon.

The dielectric block 11 is made of, for example, synthetic resin or B
It is formed using an optical glass such as K7. On the other hand, the cladding layer 91 is formed in a thin film shape using a dielectric material having a lower refractive index than the dielectric block 11 or a metal such as gold. Further, the optical waveguide layer 92 is a dielectric having a higher refractive index than the cladding layer 91,
For example, this is also formed into a thin film using PMMA. The thickness of the cladding layer 91 is, for example, 36.5 nm when it is formed from a gold thin film, and the thickness of the optical waveguide layer 92 is, for example, PMMA.
Is formed to about 700 nm.

In the leakage mode sensor having the above configuration,
Light beam 30 emitted from laser light source 31 is a dielectric block
When the light beam 30 is incident on the cladding layer 91 at an incident angle equal to or larger than the total reflection angle through the interface 11, the light beam 30 is totally reflected at the interface 91 a between the dielectric block 11 and the cladding layer 91.
The light of a specific wave number that has passed through the optical waveguide layer 92 and entered the optical waveguide layer 92 at a specific incident angle propagates through the optical waveguide layer 92 in a guided mode. When the waveguide mode is excited in this way, most of the incident light is taken into the optical waveguide layer 92, and thus the total reflection attenuation occurs in which the intensity of the light totally reflected at the interface 91a sharply decreases.

Since the wave number of the guided light in the optical waveguide layer 92 depends on the refractive index of the sensing substance 14 on the optical waveguide layer 92, knowing the specific incident angle at which the total reflection attenuation occurs gives the sensing substance 14 Can be measured. Further, each measurement unit is based on a differential value I ′ which is a difference between detection values of adjacent photodiodes of the optical sensor 40.
At 90, the change over time of the differential value, that is, the change over time in the state of attenuated total reflection, can be measured to measure the binding state between the subject and the sensing substance 14.

Also in the present embodiment, the measuring means 61 stores the correction value I′r, and thereafter measures a change ΔI ′ obtained by subtracting the correction value I′r from the detected differential value I ′.
Since the information is displayed, the influence of the bulk effect and the like can be reduced and the binding state between the sensing substance 14 and the analyte can be accurately measured as in the first embodiment.

Further, with respect to other effects, the same effects as in the first embodiment can be obtained.

[Brief description of the drawings]

FIG. 1 is an overall view of a surface plasmon measuring device according to a first embodiment of the present invention.

FIG. 2 is a partially broken side view showing a main part of the surface plasmon measuring device of FIG.

FIG. 3 is a block diagram of measuring means used in the surface plasmon measuring device.

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

FIG. 5 is a graph showing an example of a measurement result by the surface plasmon resonance measurement method of the present invention.

FIG. 6 is a partially cutaway side view showing a main part of a leakage mode measuring apparatus according to a second embodiment of the present invention.

[Explanation of symbols]

 10, 90 Measuring unit 11 Dielectric block 12 Metal film 12a Interface between dielectric block and metal film 13 Sample liquid holding frame 14 Sensing substance 15 Sample liquid 20 Turntable 21 Support moving means 30 Light beam 31 Laser light source 32 Focusing Lens 40 Optical sensor 60 Controller 61 Measurement means 62 Display section 70 Sample liquid supply mechanism 91 Cladding layer 91a Interface between dielectric block and cladding layer 92 Optical waveguide layer

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G057 AB04 AB09 AC01 BA01 BC07 DA03 DB01 HA04 2G058 AA02 CA04 CB04 EA02 EA11 ED03 GA02 2G059 AA01 BB04 DD12 DD13 EE02 FF03 FF04 GG01 GG04 JJ11 KK10 MM10

Claims (14)

[Claims] 1. A light source for generating a light beam, a dielectric block transparent to the light beam, a thin film layer formed on one surface of the dielectric block, and a sample disposed on the surface of the thin film layer, A plurality of measurement units each including a sensing substance that interacts with a liquid, and a sample liquid holding mechanism that holds the sample liquid on the surface of the sensing substance; and An optical system for entering the dielectric block of one measurement unit at various angles of incidence so as to obtain a total reflection condition at an interface between the dielectric block and the thin film layer, and light totally reflected at the interface. Light detection means comprising an optical sensor for detecting the intensity of the beam; a support for supporting the plurality of measurement units; and a dielectric block for each of the plurality of measurement units. The support is moved relatively to the light detecting means so that the beam is incident at the various incident angles and the light sensor can perform detection, and each measuring unit is moved relative to the light detecting means. In a measuring apparatus using attenuated total reflection provided with a moving means arranged at a predetermined position, the measuring unit is configured to be connected to the sample liquid holding mechanism of each of the measuring units immediately after the supply of the sample liquid and at a predetermined time after the supply. The support is moved relatively to the light detecting means so as to be disposed at the predetermined position with respect to the light detecting means, and for each of the measuring units, the sample liquid is transferred to the sample liquid holding mechanism. Immediately after the supply, the detection result detected by the light detection means is stored, and based on the stored detection result, the detection result detected after the lapse of the predetermined time is corrected, and based on the corrected detection result. Measurement method using attenuated total reflection, characterized in that measuring the time course of condition of the attenuated total reflection. 2. A light source for generating a light beam, a dielectric block transparent to the light beam, a metal film formed on one surface of the dielectric block, and a sample disposed on the surface of the metal film, A plurality of measurement units each including a sensing substance that interacts with a liquid, and a sample liquid holding mechanism that holds the sample liquid on the surface of the sensing substance; and An optical system that enters the dielectric block of one measurement unit at various angles of incidence so as to obtain a total reflection condition at an interface between the dielectric block and the metal film, and light that is totally reflected at the interface. Light detection means comprising an optical sensor for detecting the intensity of the beam; a support for supporting the plurality of measurement units; and a dielectric block for each of the plurality of measurement units. The support is moved relatively to the light detecting means so that the beam is incident at the various incident angles and the light sensor can perform detection, and each measuring unit is moved relative to the light detecting means. In a measuring apparatus using attenuated total reflection provided with a moving means arranged at a predetermined position, the measuring unit is configured to be connected to the sample liquid holding mechanism of each of the measuring units immediately after the supply of the sample liquid and at a predetermined time after the supply. The support is moved relatively to the light detecting means so as to be disposed at the predetermined position with respect to the light detecting means, and for each of the measuring units, the sample liquid is transferred to the sample liquid holding mechanism. Immediately after the supply, the detection result detected by the light detection means is stored, and based on the stored detection result, the detection result detected after the lapse of the predetermined time is corrected, and based on the corrected detection result. Measurement method using attenuated total reflection, characterized by measuring the total reflection change with time of the attenuation state by surface plasmon resonance. 3. A light source for generating a light beam, a dielectric block transparent to the light beam, a cladding layer formed on one surface of the dielectric block, and an optical waveguide layer formed on the cladding layer A plurality of measurement units including a sensing substance arranged on the surface of the optical waveguide layer and interacting with the sample liquid, and a sample liquid holding mechanism for holding the sample liquid on the surface of the sensing substance. And transmitting the light beam to a dielectric block of one of the plurality of measurement units at various angles of incidence such that a total reflection condition is obtained at an interface between the dielectric block and the thin film layer. An optical system that makes the light incident on the optical interface; a light detection unit that includes an optical sensor that detects the intensity of the light beam that is totally reflected at the interface; For each dielectric block of the fixed unit, the light beam is sequentially incident at the various incident angles, and the support is relatively moved with respect to the light detection means so that detection by the light sensor can be performed. In a measuring apparatus using attenuated total reflection, comprising: a moving unit for arranging each measuring unit at a predetermined position with respect to the light detecting unit; After a lapse of a predetermined time, the support is moved relative to the light detection means so that the measurement unit is disposed at the predetermined position with respect to the light detection means. And storing the detection result detected by the light detection unit immediately after the supply of the sample liquid to the sample liquid holding mechanism, and detecting the detection result after the lapse of the predetermined time based on the stored detection result. Measurement using the total reflection attenuation, wherein the measurement result is corrected based on the corrected detection result, and the time-dependent change in the state of total reflection attenuation due to excitation of the waveguide mode in the optical waveguide layer is measured based on the corrected detection result. Method. 4. The measuring method using attenuated total reflection according to claim 1, wherein the time immediately after the supply is a time until 60 seconds elapse after the supply. 5. The measuring method using attenuation of total reflection according to claim 4, wherein the time immediately after the supply is a time until 30 seconds elapse after the supply. 6. The measuring method using attenuated total reflection according to claim 5, wherein the time immediately after the supply is until 10 seconds elapse after the supply. 7. The apparatus according to claim 1, wherein the predetermined time is at least five times an elapsed time from the time when the sample liquid is supplied to the sample liquid holding mechanism of the measuring unit to immediately after the supply. A measurement method using attenuated total reflection according to any one of the preceding claims. 8. A light source for generating a light beam, a dielectric block transparent to the light beam, a thin film layer formed on one surface of the dielectric block, a sample disposed on the surface of the thin film layer, A plurality of measurement units each including a sensing substance that interacts with a liquid, and a sample liquid holding mechanism that holds the sample liquid on the surface of the sensing substance; and An optical system for entering the dielectric block of one measurement unit at various angles of incidence so as to obtain a total reflection condition at an interface between the dielectric block and the thin film layer, and light totally reflected at the interface. Light detection means comprising an optical sensor for detecting the intensity of the beam; a support for supporting the plurality of measurement units; and a dielectric block for each of the plurality of measurement units. The support is moved relatively to the light detecting means so that the beam is incident at the various incident angles and the light sensor can perform detection, and each measuring unit is moved relative to the light detecting means. In a measuring apparatus using attenuated total reflection provided with a moving means disposed at a predetermined position, the moving means, immediately after the supply of the sample liquid to the sample liquid holding mechanism of each of the measurement units and at a predetermined time after the supply,
The support is moved relatively to the light detection means so that the measurement unit is disposed at the predetermined position with respect to the light detection means, and the sample is provided for each of the measurement units. Immediately after the supply of the sample liquid to the liquid holding mechanism, the detection result detected by the light detection means is stored, and based on the stored detection result, the detection result detected after the predetermined time has elapsed is corrected, and the correction is performed. A measuring device using attenuated total reflection, comprising: a measuring means for measuring a change over time in a state of attenuated total reflection based on the detected result. 9. A light source for generating a light beam, a dielectric block transparent to the light beam, a metal film formed on one surface of the dielectric block, a sample disposed on the surface of the metal film, A plurality of measurement units each including a sensing substance that interacts with a liquid, and a sample liquid holding mechanism that holds the sample liquid on the surface of the sensing substance; and An optical system that enters the dielectric block of one measurement unit at various angles of incidence so as to obtain a total reflection condition at an interface between the dielectric block and the metal film, and light that is totally reflected at the interface. A light detecting means comprising an optical sensor for detecting the intensity of the beam; a support for supporting the plurality of measurement units; and the light for each dielectric block of the plurality of measurement units. The support is moved relatively to the light detecting means so that the beam is incident at the various incident angles and the light sensor can perform detection, and each measuring unit is moved relative to the light detecting means. In a measuring apparatus using attenuated total reflection having a moving means disposed at a predetermined position, the moving means is provided immediately after the supply of the sample liquid to the sample liquid holding mechanism of each of the measurement units and at a predetermined time after the supply,
The support is moved relatively to the light detection means so that the measurement unit is disposed at the predetermined position with respect to the light detection means, and the sample is provided for each of the measurement units. Immediately after the supply of the sample liquid to the liquid holding mechanism, the detection result detected by the light detection means is stored, and based on the stored detection result, the detection result detected after the predetermined time has elapsed is corrected. A measuring apparatus using attenuated total reflection, comprising: a measuring means for measuring a change over time in a state of attenuated total reflection due to surface plasmon resonance based on the detected result. 10. A light source for generating a light beam, a dielectric block transparent to the light beam, a cladding layer formed on one surface of the dielectric block, and an optical waveguide layer formed on the cladding layer A plurality of measurement units including a sensing substance arranged on the surface of the optical waveguide layer and interacting with the sample liquid, and a sample liquid holding mechanism for holding the sample liquid on the surface of the sensing substance. And transmitting the light beam to a dielectric block of one of the plurality of measurement units at various angles of incidence such that a total reflection condition is obtained at an interface between the dielectric block and the thin film layer. An optical system for causing the light to be incident on the optical interface; a light detection unit including an optical sensor for detecting the intensity of the light beam totally reflected at the interface; a support supporting the plurality of measurement units; The light beam is sequentially incident on the various dielectric blocks of the measurement unit at the various incident angles, so that the detection can be performed by the optical sensor, by moving the support relative to the light detection means, A measuring device using total reflection attenuation, comprising: a moving unit that arranges each measuring unit at a predetermined position with respect to the light detecting unit; Immediately after the supply and at the elapse of a predetermined time after the supply,
The support is moved relatively to the light detection means so that the measurement unit is disposed at the predetermined position with respect to the light detection means, and the sample is provided for each of the measurement units. Immediately after the supply of the sample liquid to the liquid holding mechanism, the detection result detected by the light detection means is stored, and based on the stored detection result, the detection result detected after the predetermined time has elapsed is corrected, and the correction is performed. A measuring device for measuring a change over time in a state of attenuated total reflection due to excitation of a waveguide mode in the optical waveguide layer based on the detected result. 11. The apparatus according to claim 8, wherein the time immediately after the supply is a time until 60 seconds elapse after the supply.
0. A measuring apparatus utilizing the total reflection attenuation according to any one of the above. 12. The measuring apparatus using attenuated total reflection according to claim 11, wherein the time immediately after the supply is a time until 30 seconds elapse after the supply. 13. The measuring apparatus using attenuated total reflection according to claim 12, wherein the time immediately after the supply is a time until 10 seconds elapse after the supply. 14. The apparatus according to claim 8, wherein the predetermined time is at least five times the elapsed time from the time when the sample liquid is supplied to the sample liquid holding mechanism of the measurement unit to immediately after the supply. A measuring device using the attenuated total reflection according to any one of the preceding claims.