JP2002310903A - Sensor utilizing attenuated total reflectance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To shorten the measuring time of a sensor utilizing the attenuated total reflectance caused by surface plasmon, etc., without lowering the measuring accuracy of the coupling between a sensing material and an object to be inspected. SOLUTION: A sample liquid 15 containing the object to be inspected is supplied to a measuring unit 10 in which the sensing material is fixed by means of a sample liquid supplying mechanism 70, and a light beam 30 is made incident on the unit 10 at various angles so that a total reflection condition may be obtained on the interface between a metallic film formed on the internal bottom face of the unit 10 and a dielectric block underlying the metallic film. In this state, the light beam 30 totally reflected by the interface is detected by means of a photodetector 40. A measuring means 61 finds the variation ΔI' corresponding to the change of the angle of attenuated total reflectance with time based on the detected value of the photodetector 40. A measuring control means 66 continues the measuring until the final discriminating time Tf (2 hours) elapses when the variation ΔI' is smaller than an upper-limit reference value Sp and larger than a lower-limit reference value Sn when initial discriminating time T1 (20 minutes) elapses. In the other case, the control means 66 terminates the measurement and performs discrimination. Consequently, the measuring time of this sensor can be shortened.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sensor using attenuated total reflection, such as a surface plasmon sensor for analyzing the characteristics of a substance utilizing the generation of surface plasmons. The present invention relates to a sensor using attenuated total reflection for measuring a state of a binding action with a specific substance contained therein.

[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, various surface plasmon sensors have been proposed for analyzing the characteristics of a substance to be measured by utilizing the phenomenon that 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).

[0004] A surface plasmon sensor using the above system basically includes, for example, a dielectric block formed in a prism shape and a metal formed on one surface of the dielectric block and brought into contact with a substance to be measured such as a liquid sample. A film, a light source that generates a light beam, and an optical system that causes the light beam to enter the dielectric block at various angles so as to obtain a total reflection condition at an interface between the dielectric block and the metal film. And light detecting means 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 total reflection attenuation.

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 sensor having the above-described structure, when a light beam is incident on a metal film at a specific angle of incidence equal to or greater than the total reflection angle, an evanescent wave having an electric field distribution is formed in a substance to be measured in contact with the metal film. Is generated, and surface plasmons are 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 light detection means.

[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 sensor, as shown in Japanese Patent Application Laid-Open No. H11-326194, an array-like surface plasmon sensor has been proposed for the purpose of accurately measuring the total reflection attenuation angle θ _SP with a large dynamic range. It has been considered to use light detection means. The light detecting means includes a plurality of light receiving elements arranged in a predetermined direction,
The components of the light beam totally reflected at the interface at various reflection angles are arranged so as to be received by different light receiving elements.

In this case, differentiating means for differentiating the light detection signal output from each light receiving element of the array-shaped light detecting means 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 the values.

Similar sensors utilizing attenuated total reflection (ATR) include, for example, “Spectroscopy”, Vol. 47, No. 1 (1998), pp. 21-23 and 26-27.
A leak mode sensor described on the page is also known. This leak mode sensor is basically formed of, 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 liquid. An optical waveguide layer, a light source for generating a light beam, and the light beam incident on the dielectric block at various angles so as to obtain a total reflection condition at an interface between the dielectric block and the cladding layer. And an optical system for measuring the intensity of the light beam totally reflected at the interface and detecting an excited state of the waveguide mode, that is, an attenuated total reflection state.

In the leakage mode sensor having the above configuration,
When a 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 of a specific incident angle having a specific wave number is transmitted to the optical waveguide layer after passing 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, and thus the total reflection attenuation occurs in which the intensity of the light totally reflected at the interface sharply decreases.
Since the wave number of the guided light depends on the refractive index of the substance to be measured on the optical waveguide layer, 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 leakage mode sensor,
In order to detect the position of the dark line generated in the reflected light due to the attenuated total reflection, the above-described array-like light detecting means can be used, and in addition to this, the above-described differentiating means is often applied.

Further, the surface plasmon sensor and the leak mode sensor described above are sometimes used for random screening for finding a specific substance binding to a desired sensing substance in the field of drug discovery research and the like. A sensing substance is immobilized as a substance to be measured on a layer (a metal film in the case of a surface plasmon sensor, and a cladding layer and an optical waveguide layer in the case of a leaky mode sensor). Is added, and the angle of the aforementioned total reflection attenuation angle θ _SP is measured every predetermined time. If the analyte in the sample solution binds to the sensing substance,
Due to this coupling, the refractive index of the sensing substance changes over 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. In particular,
Rabbit anti-human IgG antibody can be immobilized on a measurement unit as a sensing substance, and a human IgG antibody can be used as a specific substance.

[0016] In order to measure a binding state between a test substance and a sensing substance, it is not always necessary to detect the angle itself of an attenuated total reflection angle theta _SP. 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-like light detection means and differentiation means are applied to a sensor using attenuated total reflection, the change in the differential value with respect to the time when the sample liquid is added to the sensing substance is determined by the attenuated total reflection angle. because it reflects the angular change theta _SP, on the basis of changes in this differential value, No. by sensing state of bonding between materials and the object can be measured (the applicant 2000-39830
No. 9).

[0017]

In the sensor using the total reflection attenuation provided conventionally, the detection is intermittently repeated and the total reflection attenuation angle θ _SP until a predetermined time elapses.
Is determined, and based on the result, it is determined whether or not the binding between the sensing substance and the analyte is present, that is, whether or not the analyte is a specific substance that binds to the sensing substance. The binding speed between the sensing substance and the analyte is various, and if the analyte has a high binding speed to the sensing substance, the presence or absence of the binding can be easily determined from the detection result after a short time has elapsed from the start of the measurement. For an analyte having a low binding rate to a sensing substance, it is desirable to make a determination based on a detection result after a long time has elapsed in order to accurately determine the presence or absence of binding to the sensing substance. On the other hand, in recent years, the types of subjects have increased, and it is desired to reduce the measurement time for each subject. However, when the measurement time is shortened uniformly, there is a possibility that an accurate determination cannot be made for an analyte having a low binding speed.

The present invention has been made in view of the above circumstances, and provides a sensor using attenuated total reflection which can reduce the measurement time without lowering the measurement accuracy of the binding state between a sensing substance and an analyte. With the goal.

[0019]

According to the present invention, there is provided a sensor utilizing attenuated total reflection, comprising: 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 binding to a specific substance in the sample liquid, and a sample holding the sample liquid on the sensing substance A measurement unit including a liquid holding mechanism, and the light beam is incident on the dielectric block 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. An optical system, a light detecting means for detecting the intensity of the light beam totally reflected at the interface, and a time-dependent change in the state of attenuated total reflection based on a plurality of detection results at intervals of the light detecting means. In a sensor using attenuated total reflection comprising a measuring means for measuring the amount of change and a measuring control means for controlling the operation of the measuring means, a final judgment time Tf and an initial judgment time T1
(Where T1 <Tf) and setting means for setting the reference range of the change amount to the measurement control means, wherein the measurement control means performs the measurement when the initial determination time T1 has elapsed from the start of measurement of the measurement unit. The variation measured by the means is compared with the reference range.If the variation is within the reference range, the measurement is terminated, and the measurement is continued when the final determination time Tf has elapsed from the start of the measurement. If the measurement has been performed, the measurement is controlled to end.

Further, the sensor using the attenuated total reflection according to the present invention can be particularly applied to the sensor configured as the above-mentioned surface plasmon sensor.
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 specific substance disposed on the surface of the metal film, and A sensing substance coupled to the sensing substance, and a measuring unit including a sample liquid holding mechanism for holding the sample liquid on the sensing substance, and the light beam is applied to the dielectric block with respect to the dielectric block and the metal. An optical system for incidence at various angles of incidence so as to obtain the condition of total reflection at the interface with the film, a light detecting means for detecting the intensity of the light beam totally reflected at the interface, and an interval between the light detecting means. Measuring means for measuring the amount of change reflecting the change over time in the state of total reflection attenuation due to surface plasmon resonance based on the detection results obtained a plurality of times, and measurement control means for controlling the operation of the measuring means. In sensor utilizing attenuated total reflection, final determination time Tf and the initial determination time T1 (where T1 <
Tf) and setting means for setting the reference range of the change amount to the measurement control means, wherein the measurement control means comprises:
At the elapse of the initial determination time T1 from the start of measurement of the measurement unit, the change amount measured by the measurement means is compared with the reference range, and if the change amount is within the reference range, the measurement ends. When the measurement is continued when the final determination time Tf has elapsed since the start of the measurement,
The measurement is controlled so as to end the measurement.

Further, the sensor utilizing the attenuated total reflection according to the present invention can be particularly applied to a sensor configured as the above-described leak mode sensor. In this case, a light source for generating a light beam and the light source A dielectric block transparent to the beam, a cladding layer formed on one surface of the dielectric block, an optical waveguide layer formed on the cladding layer, and a sample liquid disposed on the surface of the waveguide layer. A sensing substance that binds to a specific substance therein, and a measurement unit including a sample liquid holding mechanism that holds the sample liquid on the sensing substance; and An optical system for entering at various angles of incidence so as to obtain the condition of total reflection at the interface between the block and the cladding layer, and a light detecting means for detecting the intensity of the light beam totally reflected at the interface. Measuring means for measuring a change amount reflecting a temporal change in a state of total reflection attenuation due to excitation of a waveguide mode in the optical waveguide layer based on a plurality of detection results at intervals of the light detecting means. And a measurement control means for controlling the operation of the measurement means, using a total reflection attenuation, the final determination time Tf and the initial determination time T1
(Where T1 <Tf) and setting means for setting the reference range of the change amount to the measurement control means, wherein the measurement control means performs the measurement when the initial determination time T1 has elapsed from the start of measurement of the measurement unit. The variation measured by the means is compared with the reference range.If the variation is within the reference range, the measurement is terminated, and the measurement is continued when the final determination time Tf has elapsed from the start of the measurement. If the measurement has been performed, the measurement is controlled to end.

In the sensor utilizing the various types of total reflection attenuation, if an upper limit reference value is set as the lower limit value of the reference range, the measurement control means sets the above-mentioned initial determination time T1 to the above-mentioned value. When the amount of change is equal to or more than the upper limit reference value, it may be determined that a specific substance that binds to the sensing substance is present in the measurement unit.

If the lower limit reference value is set as the upper limit value of the reference range, the measurement control means
If the change amount is equal to or less than the lower limit reference value after the elapse of the initial determination time T1, it may be determined that there is no specific substance that binds to the sensing substance in the measurement unit.

The reference range includes a range in which the lower limit is the upper limit reference value and a range in which the upper limit is the lower limit reference value (however, lower limit reference value <upper limit reference value). Further, when the final reference value is to be set in the measurement control means, the measurement control means, when the initial determination time T1 has elapsed, the change amount measured by the measurement means is not less than the upper limit reference value. In some cases, it is determined that a specific substance that binds to the sensing substance is present in the measurement unit, and when the amount of change is equal to or less than the lower reference value, the measurement unit is coupled to the sensing substance. It is determined that the specified substance does not exist, and if the amount of change is smaller than the upper reference value and larger than the lower reference value, the measurement is continued and the final When the time Tf has elapsed, the measurement is terminated, and at the end, the variation measured by the measuring means is compared with the final reference value.If the variation is larger than the final reference value, the measurement is completed. The unit may determine that a specific substance that binds to the sensing substance is present in the unit.

Further, the reference range includes a range in which the lower limit value is the upper limit reference value and a range in which the upper limit value is the lower limit reference value (however, lower limit reference value <upper limit reference value). However, if the final reference value is to be set to the measurement control means, the measurement control means,
If the amount of change measured by the measuring means is equal to or more than the upper reference value when the initial determination time T1 has elapsed, it is determined that a specific substance that binds to the sensing substance is present in the measurement unit. If the amount of change is equal to or less than the lower limit reference value, it is determined that there is no specific substance that binds to the sensing substance in the measurement unit, and the amount of change is smaller than the upper limit reference value, and If it is larger than the lower reference value, the measurement is continued, and each time the measurement is performed by the measuring means, the change amount is compared with the final reference value, and if the change amount is larger than the final reference value, It is determined that the specific substance that binds to the sensing substance is present in the measurement unit, and the measurement is terminated, and the measurement is continued when the final determination time Tf has elapsed from the start of the measurement. If so, control may be performed to end the measurement.

In the above-described sensors utilizing the total reflection attenuation, the initial determination time T1 is equal to the final determination time Tf.
Is preferably equal to or less than 1/2 times of

Further, in the above-mentioned sensors utilizing various types of attenuated total reflection, a plurality of measuring units are provided, a support for detachably holding the plurality of measuring units, and a dielectric member of each of the plurality of measuring units. The support is moved relative to the optical system and the light detecting means so that the various incident angles are obtained sequentially with respect to the body block, and each measurement unit is moved relative to the optical system and the light detecting means. It may be provided with a moving means arranged at a predetermined position and a means for removing the measurement unit having completed the measurement from the support.

In the above-mentioned various measuring methods and sensors, “start of measurement” means a point in time when the sample liquid is supplied onto the sensing substance, and “change amount” means a time from the start of measurement to the measurement time. The change reflects the time-dependent change in the state of total reflection attenuation during the measurement, and can be obtained, for example, as the difference between the measurement value reflecting the state of total reflection attenuation during measurement and the measurement value at the start of measurement. In the case where it is difficult to measure at the start of measurement or when there is a large possibility that the measured value at the start of measurement contains an error, the measured values before and after the start of the measurement, or corrected by mathematical processing The measured value or the like may be used as the measured value at the start of the measurement.

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

[0030]

According to the sensor utilizing attenuated total reflection according to the present invention, the amount of change measured by the measuring means is compared with the reference range when the initial determination time T1 has elapsed from the start of measurement by the measuring unit. If the amount of change is within the reference range, the measurement is terminated, so that it is not necessary to continue the measurement for all the measurement units uniformly until the final determination time Tf, and the measurement time can be reduced. In addition, at the time when the initial determination time T1 has elapsed, measurement can be continuously performed for the measurement unit whose change amount is not within the reference range, so that the identification unit that binds to the sensing substance in the sample liquid after the initial determination time T1 has elapsed. When it is difficult to determine whether or not a substance is present, the measurement by the measurement unit can be continued, so that the measurement time can be shortened without lowering the measurement accuracy.

An upper limit reference value is set as a lower limit value of the reference range. If the amount of change after the lapse of the initial determination time T1 is equal to or greater than the upper limit reference value, a specific substance that binds to the sensing substance exists in the measurement unit. When the initial determination time T1 has elapsed, it can be determined that the specific substance is present, and the measurement of the measurement unit that does not need to continue the measurement any more can be terminated.

A lower limit reference value is set as an upper limit value of the reference range, and if the amount of change when the initial determination time T1 elapses is equal to or less than the lower limit reference value, the specific substance that binds to the measurement unit with the sensing substance is measured. In the case where it is determined that no specific substance exists, it is possible to determine that the specific substance does not exist at the time when the initial determination time T1 has elapsed, and the measurement unit that does not need to continue the measurement any more is measured. Can be terminated.

The reference range includes a range in which the lower limit is the upper limit reference value and a range in which the upper limit is the lower limit reference value (however, lower limit reference value <upper limit reference value). If the amount of change is equal to or greater than the upper limit reference value at the time when elapses, it is determined that the specific substance that binds to the sensing substance is present in the measurement unit, and the measurement of the measurement unit is terminated. Is less than or equal to the lower reference value, it is determined that the specific substance that binds to the sensing substance is not present in the measurement unit, and the measurement of the measurement unit is terminated. If it is larger than the reference value, the measurement is performed until the final judgment time Tf elapses, and if the amount of change at the elapse of the final judgment time Tf is larger than the final reference value, the measurement unit is combined with the sensing substance. For those that determine that a constant substance is present, at the time when the initial determination time T1 has elapsed, the measurement of the measurement unit that can determine the presence or absence of the specific substance is terminated, and the measurement unit that is difficult to determine Since only the measurement is continued until the final determination time Tf elapses, the measurement time can be further reduced without lowering the measurement accuracy.

Further, the reference range includes a range in which the lower limit value is the upper limit reference value and a range in which the upper limit value is the lower limit reference value (however, lower limit reference value <upper limit reference value). If the amount of change is equal to or greater than the upper limit reference value at the time when elapses, it is determined that the specific substance that binds to the sensing substance is present in the measurement unit, and the measurement of the measurement unit is terminated. Is less than or equal to the lower reference value, it is determined that the specific substance that binds to the sensing substance is not present in the measurement unit, and the measurement of the measurement unit is terminated. If the change is larger than the reference value, the measurement is continued, and the change amount at that time is compared with the final reference value for each measurement. Sensing In the case where the determination is made that there is no specific substance that binds to the substance and the measurement is terminated, if the determination is possible at the time when the initial determination time T1 has elapsed, the measurement of the measurement unit is terminated and the determination is performed. If it is difficult to continue the measurement, and even before the final judgment time Tf has elapsed, the measurement is terminated when the amount of change becomes larger than the final reference value. And the measurement time can be further reduced.

Note that the initial judgment time T1 is equal to the final judgment time Tf.
If it is not more than 1/2 times, the measurement time can be efficiently shortened.

Further, among the sensors utilizing attenuated total reflection of the present invention, a plurality of measurement units are supported on a support,
The support was moved relatively to the optical system and the light detecting means so that each measuring unit could be sequentially arranged at a predetermined position with respect to the measuring optical system and the light detecting means, and the measurement was completed. In the apparatus provided with means for removing the measurement unit from the support, the measurement units for which determination has been completed among the measurement units can be sequentially removed from the support, and new measurement units can be successively used for measurement. It is possible to efficiently perform the measurement for the measurement unit.

[0037]

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 driving mechanism for intermittently rotating the turntable 20. Means 21, a laser light source 31 such as a semiconductor laser for generating a measuring light beam (laser beam) 30, a condenser lens 32 constituting an incident optical system, a photodetector 40, the laser light source 31 and a support Controlling the driving of the body driving means 21 and the photodetector
40, a controller 60 for performing the processing described below in response to the output signal S, a sample liquid supply mechanism 70 for supplying a sample liquid, setting means 80 for setting a predetermined set value to the controller 60, and a turntable Access mechanism to get in and out of 20
81.

As shown in FIG. 2, the measuring unit 10 is formed on 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 measuring 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 driving means 21 is constituted by 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 photodetector 40 is a photodiode array in which a large number of photodiodes are arranged in a 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 driving means 21 based on a predetermined sequence. Further, the controller 60 receives the output signal S of the photodetector 40, calculates a measurement value reflecting the angle change amount of the total reflection attenuation angle θ _SP , and a display receiving the output from the measurement means 61. A measurement control unit that determines a measurement value when a predetermined time has elapsed based on the setting value set by the unit 62 and the setting unit 80
66 and. Further, it is connected to the sample liquid supply means 70 and the access mechanism 81, and controls its operation as needed.

As shown in FIG. 3, the measuring means 61 includes a differential amplifier array 63 connected to the photodetector 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, the driver 64 samples and holds the outputs of the respective 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 setting unit 80 includes a keyboard and the like.
By the manual input operation of the measurer, the final determination time Tf (120 minutes in the present embodiment), the initial determination time T1 (20 minutes in the present embodiment), and the upper limit reference value Sp as the reference range of the change amount. The lower reference value Sn and the final reference value Sf are set in the measurement controller 66 of the controller 60.

The access mechanism 81 is composed of a holding portion 82 for holding the measuring unit 10 and a means 83 for moving the holding portion 82, and is also a removing means of the present invention.

Hereinafter, the operation of the surface plasmon measuring apparatus 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) solution as a non-specific adsorption inhibitor is pooled for 10 minutes, washed again with PBS, and finally PBS that protects the sensing substance is added. Then, a measurement unit 10 to which streptavicin as a sensing substance is fixed is created. These measurement units 10 are arranged, for example, in a 96-well cassette. Thereafter, the measuring units 10 are sequentially arranged in the through holes 22 on the turntable 20 by the access mechanism 81.

After the turntable 20 has been rotated a number of times and then stopped, the measuring unit 10 determines the measuring position where the light beam 30 is incident on the dielectric block 11 (the position of the measuring 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 interface 12a between the dielectric block 11 and the metal film 12 is kept in a state where the light beam 30 emitted therefrom converges as described above.
Incident on. The light beam 30 totally reflected at the interface 12a is
It is detected by the photodetector 40.

The photodetector 40 in this embodiment is a photodiode array in which a plurality of photodiodes 40a, 40b, 40c,... Are arranged in one row. The photodiodes are arranged so that the direction in which the photodiodes are arranged is substantially perpendicular to the traveling 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 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 supplied to sample-and-hold 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.

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 reflected light intensity I of this 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 shown by oblique lines 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.

In order to obtain a measurement value reflecting the amount of change in the total reflection attenuation angle θ _SP , first, each measurement unit 10
For each time, the selection process of the differential amplifier to be used and the differential value I ′
Of the correction value I′r is performed. Signal processing unit 65
Is the maximum value of the change in the reflected light intensity I, that is, the total reflection attenuation, from among the differential amplifiers 63a, 63b, 63c... Based on the value of the differential value I 'input from the A / D converter 54. Angle θ
_The differential value I'mi closest to the differential value I '= 0 corresponding to _SP
Select the differential amplifier for which n is obtained. In the example of FIG. 4, the differential amplifier 63e is used.

In the signal processing section 65, the differential value I'min output from the differential amplifier 63e is stored in a storage section (not shown) as a correction value I'r which is a measured value corresponding to the start of measurement by the measuring unit 10. At the same time, the zero point is displayed on the display unit 62.

Next, after the turntable 20 has been rotated a number of times, the measuring unit 10 stops at the position where the sample liquid is supplied. The sample liquid supply mechanism 70 sequentially suctions and holds the sample liquid from the sample liquid container 73 to the pipette 71, and supplies the sample liquid dropwise into the sample liquid holding frame 13 of the measurement unit 10. As a sample solution, for example, a 0.1% BSA / PBS solution (0.1% B
A solution containing biotinylated insulin as a subject in a PBS solution containing SA) 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 again
After being rotated by the support driving means 21, the measuring unit 10 is stopped at the measuring position at substantially predetermined time intervals thereafter, and the measurement is performed. The above measurement is performed intermittently until the initial determination time T1 elapses after the sample liquid is supplied to the measurement unit 10. In the signal processing unit 65, every time the same measurement unit is measured, the change amount ΔI ′ is a value obtained by subtracting the correction value I′r from the differential value I ′ output from the selected differential amplifier 63e.
Is calculated and displayed on the display unit 62. A graph showing the relationship between the elapsed time and the variation ΔI ′ as shown in FIG. 5A is displayed on the display unit 62 for each measurement unit, that is, 11 graphs are simultaneously displayed.

That is, at the time of the first measurement, the differential amplifier
Regardless of the magnitude of the differential value I 'output by 63e, the displayed value is 0. Thereafter, every time the measurement unit stops at the measurement position and the measurement is performed, the differential value is measured first, that is, the amount of change in the differential value from before the sample liquid is supplied to the measurement unit until the time of measurement. ΔI ′ is displayed on the display 62
Will be displayed on the graph. In the present embodiment, the measured value before the sample liquid is supplied to the measuring unit 10 is used as the measured value at the start of the measurement.

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.

When the initial determination time T1 (20 minutes) has elapsed since the sample liquid was supplied to the measurement unit 10, the measurement control means 66 determines the change amount ΔI 'calculated by the measurement means 61 and the upper limit reference value. Sp and the lower reference value Sn are compared.

As shown by the broken line in FIG. 5B, when the variation ΔI ′ is equal to or more than the upper limit reference value Sp, a specific substance that binds to the sensing substance is present in the measurement unit 10. Is determined, the determination result is displayed on the display unit 62,
The measurement by the measurement unit 10 ends. Also, the change amount Δ
If I ′ is equal to or less than the lower reference value Sn, it is determined that there is no specific substance that binds to the sensing substance in the measurement unit 10, and the measurement is also terminated.

As shown by the solid line in FIG. 5B, when the variation ΔI ′ is equal to or less than the upper limit reference value Sp and equal to or greater than the lower limit reference value Sn, the measurement of the measurement unit 10 is performed by the sample liquid supply. It is repeated at predetermined time intervals from the time until the final determination time Tf elapses.

The final determination time Tf (120
Minute), the measurement control unit 66 compares the change amount ΔI ′ measured by the measurement unit with the final reference value Sf. If the change amount ΔI ′ is larger than the final reference value Sf, the measurement unit 10 It is determined that there is a specific substance that binds to the sensing substance, and when the variation ΔI ′ is smaller than the final reference value Sf, the specific substance that binds to the sensing substance does not exist in the measurement unit 10. And terminate the measurement.

When the measurement is completed after the elapse of the initial judgment time T1 or the elapse of the final judgment time Tf, the access mechanism is stopped.
The control unit 60 controls the measurement unit 10 that has completed the measurement by the measurement control
And a new measurement unit 10 to which the sample liquid is not supplied is placed in the through hole 22. After the detection of the correction value, the sample liquid supply mechanism 70 supplies a sample liquid containing a different type of subject under the control of the controller 60, and the same operation as that described above is repeated. The entry / exit mechanism
81, when removing the measurement unit 10 for which measurement has been completed, based on the determination result in the measurement control means 66, the measurement unit in which the analyte bound to the sensing substance is present and the measurement unit in which the analyte is not present can be easily determined. So that they are stored in different cassettes.

As is clear from the above description, in the present embodiment, when the initial determination time T1 has elapsed, the change amount ΔI ′
Is greater than or equal to the upper reference value Sp, it is determined that the analyte in the sample liquid is a specific substance that binds to the sensing substance, and the measurement of the measurement unit is terminated, and the change ΔI ′ is smaller than the lower reference value Sn. If it is less than or equal to, the analyte is determined not to be a specific substance that binds to the sensing substance and the measurement of the measurement unit is terminated, and the change amount ΔI ′ is equal to or less than the upper reference value Sp and equal to or greater than the lower reference value Sn. In some cases, the final judgment time Tf
Is measured until the elapsed time, and the change amount ΔI ′ at that time is measured.
Is greater than the final reference value Sf, the analyte is a specific substance that binds to the sensing substance, and if the variation ΔI ′ is less than or equal to the final reference value Sf, the analyte binds to the sensing substance. Since it is determined that the substance is not a specific substance, if the determination is possible at the time when the initial determination time T1 has elapsed, the measurement of the measurement unit 10 can be terminated, and the measurement time can be reduced. If the determination is difficult, the measurement may be continued until the final determination time Tf elapses, so that the measurement accuracy of the binding state between the analyte and the sensing substance is not reduced.

In the present embodiment, a plurality of measurement units 10 are arranged on a turntable 20, and the turntable 20 is moved to sequentially connect each measurement unit 10 to the condenser lens 32 and the photodetector 40. On the other hand, since it is configured to be able to be arranged at a predetermined position, the variation ΔI ′ in the plurality of measurement units 10 can be used for measurement one after another with the above-mentioned movement, and measurement for a large number of measurement units 10 can be performed. It can be performed in parallel.

The measurement units 10 whose measurement has been completed are sequentially taken out of the turntable 20, and a new measurement unit is obtained.
Since the measurement can be performed by arranging the 10 on the turntable 20 and supplying the next sample liquid (analyte), the determination of many analytes can be performed in a short time.

In the present embodiment, with respect to the measurement unit 10 which has been difficult to determine at the time when the initial determination time T1 has elapsed, all the measurement units which have been difficult to determine until the final determination time Tf has elapsed. The measurement of the unit was continued, but as a modified example, when the initial judgment time T1 elapses, the amount of change Δ
When I ′ is equal to or less than the upper reference value Sp and equal to or greater than the lower reference value Sn, the measurement is continued, and the change Δ
I ′ is compared with the final reference value Sf, and if the variation ΔI ′ is larger than the final reference value Sf, it is determined that the specific substance that binds to the sensing substance does not exist in the measurement unit, and the measurement is terminated. Things are also possible. In this case, a larger number of measurement units are required before the final judgment time Tf elapses.
Since 10 measurements are completed, the measurement time can be further reduced. The reference value to be compared for each measurement may be set to a different value for each elapsed time.

Further, in each of the embodiments described above, the rotating turntable 20 is used as a support for supporting the measuring unit 10, but 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 the plurality of measurement units are sequentially moved to a light measurement unit including a light source 31, a condenser lens 32, and a photodetector 40 with the movement. It may be set.

If a plurality of the light measuring units are provided,
The measuring unit is sequentially set in the light measuring unit with the movement of the support, and the measurement can be performed in each light measuring unit. Alternatively, only one such measurement unit is provided, the support is moved in one direction, the measurement unit is set on the optical measurement unit, measurement is performed, and then the support is moved in the opposite direction, and the measurement unit is moved. May be set in the optical measurement unit again to perform measurement.

The method of moving the support in the forward and reverse directions is applicable to the case where the turntable 20 described above is used. When the turntable 20 is used, it is also possible to provide a plurality of light measuring units so as to measure one measurement unit a plurality of times while the turntable 20 makes one rotation.

As a modification of the first embodiment, as shown in FIG. 6, a stage 25 on which the measuring units 10 can be arranged in a grid is fixed, and a laser light source 31 and a condenser lens are arranged.
The light measuring head 35 composed of 32 and the photodetector 40 is connected to X
It is also conceivable that the measurement unit 10 is moved along a linear guide composed of the axis linear guide 36 and the Y-axis linear guide 37 to sequentially measure the measurement units 10. Also, in this case,
A sample liquid supply mechanism 77 including a pipette 75 and an XYZ stage 76 for moving the pipette 75 is used in place of the sample liquid supply mechanism 70, and a holding unit that holds the measurement unit 10 is used instead of the access mechanism 81. With the use of the access mechanism 87 including the 85 and the XYZ stage 86 for moving the holding unit 85, even while one measurement unit 10 is being measured, the other measurement unit 10 can be taken in and out, Supply can be performed, and measurement of a large number of measurement units can be performed in a shorter time. In addition, if the apparatus includes a plurality of optical measurement heads 35, the measurement can be performed more efficiently.

As described above, the dielectric block 11,
Not only the measurement unit 10 in which the metal film 12 and the sample liquid holding frame 13 are integrally formed, but also the measurement unit in which the metal film 12 and the sample holding frame 13 are integrated and exchangeably formed with respect to the dielectric block 11 Units can also be applied.

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

Since the overall configuration of the second embodiment is almost the same as that of the first embodiment, only the numbers of the different components in FIG. 1 are added in the figure. In FIG. 7, 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,
This is also formed in a thin film shape using, for example, 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.
From 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, the sensing substance 14 Can be measured. Further, each measurement unit is determined based on a differential value I ′ which is a difference between detection values of adjacent photodiodes of the photodetector 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 this embodiment, when the change amount ΔI ′ is equal to or larger than the upper limit reference value Sp or the change amount ΔI ′ is equal to or smaller than the lower limit reference value Sn at the time when the initial determination time T1 has elapsed. In this case, the determination is made, the measurement of the measurement unit is terminated, and if the variation ΔI ′ is equal to or less than the upper reference value Sp and equal to or greater than the lower reference value Sn, the measurement is performed until the final determination time Tf elapses. Since the determination is performed after the continuation of the measurement, the measurement of the measurement unit 10 capable of making the determination can be terminated when the initial determination time T1 has elapsed, and the measurement time can be reduced. If the determination is difficult, the measurement may be continued until the final determination time Tf elapses.
The accuracy of determining whether the subject is a specific substance that binds to the sensing substance is not reduced. Further, modifications similar to those of the first embodiment are also possible, and the same effects as those of 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 a photodetector, and a relationship between an incident angle of a light beam 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 modification of the first embodiment of the present invention.

FIG. 7 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 driving means 25 Stage 30 Light beam 31 Laser light source 32 Condensing lens 35 Optical measuring head 40 Optical detector 60 Controller 61 Measurement means 62 Display 66 Measurement control means 70, 77 Sample liquid supply mechanism 80 Setting means 81, 87 Access mechanism 91 Cladding layer 91a Interface 92 Optical waveguide layer

 ──────────────────────────────────────────────────続 き Continuing on the front page F term (reference) 2G057 AA02 AB04 AB07 AC01 BA01 BA03 BB01 BB06 HA04 2G058 AA01 AA10 CB04 CC08 CF02 CF12 CF21 EA02 EA12 GA02 2G059 AA01 BB04 BB12 CC16 DD12 EE02 EE05 FF04 JJ01 KK04 MM05 PP04

Claims (9)

[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 sensing substance that binds to a specific substance in the liquid, and a measurement unit including a sample liquid holding mechanism that holds the sample liquid on the sensing substance; and An optical system for entering at various angles of incidence so as to obtain a total reflection condition at an interface between the body block and the thin film layer; a light detection unit for detecting the intensity of a light beam totally reflected at the interface; A measuring means for measuring an amount of change reflecting a temporal change in the state of total reflection attenuation based on a plurality of detection results at intervals of the means, and a measuring control means for controlling the operation of the measuring means. Reflection The sensor using the decay, further comprising a setting means for setting a final determination time Tf, an initial determination time T1 (where T1 <Tf) and a reference range of the change amount to the measurement control means, wherein the measurement control means At the elapse of the initial determination time T1 from the start of measurement by the measurement unit, the change amount measured by the measuring means is compared with the reference range, and when the change amount is within the reference range, the measurement is terminated. A sensor using attenuated total reflection, wherein if the measurement is continued when the final determination time Tf elapses from the start of the measurement, the measurement is controlled to end. 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 sensing substance that binds to a specific substance in the liquid, and a measurement unit including a sample liquid holding mechanism that holds the sample liquid on the sensing substance; and An optical system for entering at various angles of incidence so as to obtain a total reflection condition at an interface between the body block and the metal film; a light detection unit for detecting the intensity of a light beam totally reflected at the interface; A measuring means for measuring a change amount reflecting a temporal change of a state of attenuated total reflection by surface plasmon resonance based on a plurality of detection results at intervals of the means, and a measuring system for controlling the operation of the measuring means. And a setting means for setting a final judgment time Tf, an initial judgment time T1 (where T1 <Tf) and a reference range of the change amount to the measurement control means. The measurement control means compares the variation measured by the measurement means with the reference range when the initial determination time T1 elapses from the start of measurement of the measurement unit, and the variation is within the reference range. In some cases, the measurement is terminated, and when the final determination time Tf has elapsed from the start of the measurement, if the measurement is continued, control is performed so as to terminate the measurement. Sensor using. 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 Disposed on the surface of the optical waveguide layer, a sensing substance that binds to a specific substance in the sample liquid, and a measurement unit including a sample liquid holding mechanism that holds the sample liquid on the sensing substance, An optical system for causing the light beam to enter the dielectric block at various angles of incidence so as to obtain a total reflection condition at an interface between the dielectric block and the cladding layer; and a light totally reflected at the interface. A light detecting means for detecting the intensity of the beam, and a time-dependent change in a state of attenuated total reflection due to excitation of a waveguide mode in the optical waveguide layer based on a plurality of detection results at intervals of the light detecting means. Anti A sensor using total reflection attenuation provided with a measuring means for measuring the amount of change and a measuring control means for controlling the operation of the measuring means, wherein a final judgment time Tf and an initial judgment time T1 (where T1 <Tf) and The measurement control unit further includes a setting unit that sets a reference range of the change amount to the measurement control unit. And comparing the reference range, the measurement is terminated when the amount of change is within the reference range, and when the final determination time Tf has elapsed since the start of the measurement, when the measurement is continued, the A sensor using attenuated total reflection, wherein the sensor controls the measurement to be completed. 4. The measurement unit, wherein the lower limit value of the reference range is an upper limit reference value, and the measurement control unit is configured to, when the change amount is equal to or more than the upper limit reference value when the initial determination time T1 has elapsed. 4. The sensor using attenuated total reflection according to claim 1, wherein it is determined that a specific substance that binds to the sensing substance is present. 5. The measurement unit, wherein an upper limit value of the reference range is a lower limit reference value, and the measurement control unit is configured to determine that the change amount is equal to or less than the lower limit reference value when the initial determination time T1 has elapsed. 4. The sensor using attenuated total reflection according to claim 1, wherein it is determined that there is no specific substance that binds to the sensing substance. 5. 6. The reference range includes a range whose lower limit is an upper limit reference value and a range whose upper limit is a lower limit reference value (however, lower limit reference value <upper limit reference value). And further setting a final reference value to the measurement control means.When the measurement control means has passed the initial determination time T1, the change measured by the measurement means is equal to or greater than the upper limit reference value. It is determined that a specific substance that binds to the sensing substance is present in the measurement unit, and when the amount of change is equal to or less than the lower reference value, a specific substance that binds to the sensing substance to the measurement unit is determined. When it is determined that the substance does not exist, and the amount of change is smaller than the upper reference value and larger than the lower reference value, the measurement is continued, and the final determination time Tf from the start of measurement is obtained. At the time of passing, the measurement is terminated, and at the end, the variation measured by the measuring means is compared with the final reference value, and when the variation is larger than the final reference value, the measurement unit The sensor using attenuated total reflection according to any one of claims 1 to 3, wherein it is determined that a specific substance that binds to the sensing substance is present. 7. The reference range comprises a range whose lower limit is an upper limit reference value and a range whose upper limit is a lower limit reference value (however, lower limit reference value <upper limit reference value). And further setting a final reference value to the measurement control means.When the measurement control means has passed the initial determination time T1, the change measured by the measurement means is equal to or greater than the upper limit reference value. It is determined that a specific substance that binds to the sensing substance is present in the measurement unit, and when the amount of change is equal to or less than the lower reference value, a specific substance that binds to the sensing substance to the measurement unit When it is determined that the substance does not exist, and the amount of change is smaller than the upper limit reference value and larger than the lower limit reference value, the measurement is continued, and every time the measurement is performed by the measuring means, Comparing the amount of change with the final reference value, and if the amount of change is greater than the final reference value, determines that a specific substance that binds to the sensing substance is present in the measurement unit and performs measurement. 4. The method according to claim 1, wherein when the measurement is continued when the final determination time Tf has elapsed from the start of the measurement, the measurement is controlled so as to end the measurement. Sensor using total reflection attenuation described. 8. The initial judgment time T1 is equal to the final judgment time.
The sensor using attenuated total reflection according to any one of claims 1 to 7, wherein the sensor is equal to or less than 1/2 of Tf. 9. A plurality of said measurement units, a support for detachably holding said plurality of measurement units, and said various incident angles are sequentially obtained for each dielectric block of said plurality of measurement units. Moving means for moving the support relative to the optical system and the light detecting means, and disposing each measuring unit at a predetermined position with respect to the optical system and the light detecting means; The sensor using attenuated total reflection according to any one of claims 1 to 8, further comprising means for removing a measurement unit from the support.