JP2003279478A - Measurement method, measurement device and measurement chip unit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To reduce a measurement error caused by individual difference of sensitivity of every measurement device from another to improve measurement accuracy, in a measurement device using an evanescent wave. <P>SOLUTION: This measurement device is provided with: a dielectric block 10; a laser light source 14 for generating a light beam 13; an optical system 15 for entering the light beam 13 into the interface 10b between the dielectric block 10 and a metal film 12 so as to obtain various incident angles; and a light detection means 17 for detecting the light beam 13 totally reflected by the interface 10b and changed into parallel light. Correction curve data are previously obtained by measuring total reflection elimination angles of a plurality of reference sample solutions each having a known refraction index so that the result of the measurement for a substance on the metal film 12 is corrected based on the correction curve data. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the total reflection of a light beam at the interface between a thin film layer in contact with a sample and a dielectric block to generate an evanescent wave, which causes changes in the intensity of the totally reflected light beam. The present invention relates to a measuring method, a measuring device, and a measuring chip unit that use an evanescent wave to measure a temperature and analyze a sample.

[0002]

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

Conventionally, various surface plasmon measuring devices have been proposed which analyze the characteristics of a substance to be measured by utilizing the phenomenon that the surface plasmon is excited by a light wave.
Among them, one that is particularly well known is one that uses a system called Kretschmann arrangement (see, for example, JP-A-6-167443).

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

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

In the surface plasmon measuring device having the above structure, when a light beam is incident on the metal film at a specific incident angle of a total reflection angle or more, an evanescent light having an electric field distribution in the substance to be measured in contact with the metal film. A wave is generated, and the surface plasmon is excited at the interface between the metal film and the substance to be measured by this evanescent wave. When the wave vector of the evanescent light is equal to the wave number of the surface plasmon and the wave number matching is established, both are in a resonance state and the energy of the light is transferred to the surface plasmon, so that at the interface between the dielectric block and the metal film. The intensity of the reflected light sharply decreases. This decrease in light intensity is generally detected as a dark line by the light detecting means. Note that the above resonance occurs only when the incident beam is p-polarized. Therefore, it is necessary to set in advance that the light beam is incident as p-polarized light.

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

[0008]

[Equation 1] That is, by knowing the attenuated total reflection angle θ _SP , which is the incident angle at which the intensity of the reflected light decreases, it is possible to obtain the characteristic relating to the dielectric constant ε _{s of the} substance to be measured, that is, the refractive index.

In this type of surface plasmon measuring device, as shown in Japanese Patent Laid-Open No. 11-326194, an array shape is provided for the purpose of measuring the attenuated total reflection angle θ _SP with high accuracy and a large dynamic range. It is considered to use the above-mentioned light detection means. The light detecting means comprises a plurality of light receiving elements arranged in a predetermined direction,
The components of the light beam totally reflected at various reflection angles on the interface are arranged so that different light receiving elements receive the components.

In that case, there is provided a differentiating means for differentiating the photodetection signal output by each light receiving element of the array of light detecting means with respect to the arrangement direction of the light receiving element, and the differentiating means outputs this differentiating means. A property related to the refractive index of the substance to be measured is often obtained based on the value.

Further, as a similar measuring device utilizing the attenuated total reflection (ATR), for example, "Spectroscopic Research" Vol. 47, No. 1 (1998), pages 21 to 23 and 26 to 27.
Leakage mode measuring devices described on the page are also known. This leak mode measuring device is basically a dielectric block formed, for example, in a prism shape, a clad layer formed on one surface of the dielectric block, and a clad layer formed on the clad layer for contact with a sample solution. An optical waveguide layer, a light source for generating a light beam, and the light beam at various angles with respect to the dielectric block so that total reflection conditions can be obtained at the interface between the dielectric block and the cladding layer. The optical system is configured to be incident, and a light detection unit that measures the intensity of the light beam totally reflected at the interface and detects the excited state of the guided mode, that is, the attenuated total reflection state.

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

In this leak mode measuring device as well,
In order to detect the position of the dark line generated in the reflected light due to the attenuation of the total reflection, the above-mentioned array-shaped light detecting means can be used, and in addition to that, the differentiating means is often applied.

The surface plasmon measuring device and the leak mode measuring device described above are sometimes used in random screening to find a specific substance that binds to a desired sensing substance in the field of drug discovery research and the like. A sensing substance is fixed as the substance to be measured on the thin film layer (a metal film in the case of a surface plasmon measuring device, a clad layer and an optical waveguide layer in the case of a leaky mode measuring device), and various sensing substances are fixed on the sensing substance. A sample solution in which a test object is dissolved in a solvent is added, and the angle of the above-described attenuated total reflection angle θ _SP is measured every predetermined time.

If the analyte in the sample solution binds to the sensing substance, this binding causes the refractive index of the sensing substance to change 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 analyte 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 surface of the thin film layer as a sensing substance, and a human IgG antibody can be used as the specific substance.

It should be noted that the angle itself of the attenuated total reflection angle θ _SP does not necessarily have to be detected in order to measure the binding state between the analyte and the sensing substance. For example, it is also possible to add a sample solution to the sensing substance, measure the angle change amount of the total reflection attenuation angle θ _SP after that, and measure the binding state based on the magnitude of the angle change amount. In the case of applying the above-mentioned array-shaped light detecting means and the differentiating means to the measuring apparatus using the attenuated total reflection, the variation amount of the differential value reflects the angular variation amount of the attenuated total reflection angle θ _SP . Therefore, the binding state between the sensing substance and the analyte can be measured based on the amount of change in the differential value. (Patent application 20 by the applicant
No. 00-398309) In such a measurement method and apparatus using attenuation of total reflection, a cup-shaped or petri dish-shaped measurement chip in which a sensing substance is fixed on a thin film layer formed in advance on the bottom surface is used as a solvent. A sample liquid consisting of the subject is dropped and supplied, and the above-described attenuated total reflection angle θ
The angle change amount of _SP is measured.

The applicant of the present invention makes it possible to measure a large number of samples in a short time by sequentially measuring a plurality of measuring chips mounted on a turntable or the like. Device as Japanese Patent Laid-Open No. 2001-3
Proposed by No. 30560.

The applicant of the present invention filed Japanese Patent Application No. 2001-397.
No. 411 also proposes a measuring device using attenuated total reflection that performs measurement using a measuring chip provided with a plurality of sample liquid holding portions. By using the measuring device having such a configuration, it is possible to simultaneously measure many samples without moving the measuring chip.

[0019]

When a sample solution is supplied to the above-mentioned measurement chip and the sensing substance and the analyte are bound to each other,
The refractive index of the sensing material changes and the total reflection attenuation angle θ _SP
Angle changes. Therefore, it is determined whether or not the analyte binds to the sensing substance by determining the amount of change in the attenuated total reflection angle θ _SP from the start of the measurement at the time when a predetermined time has elapsed from the start of the measurement. In addition, the binding state between the analyte and the sensing substance can be analyzed. However, strictly speaking, the amount of change in the attenuated total reflection angle θ _SP detected by the measurement device does not accurately reflect the change in the refractive index due to the binding between the sensing substance and the analyte, and the measurement sensitivity of the measurement device Some errors may occur due to individual differences.

In view of the above circumstances, it is an object of the present invention to provide a measuring method and apparatus capable of improving the measurement accuracy by reducing the measurement error caused by the individual difference in the measurement sensitivity of the measurement apparatus. .

[0021]

A measuring method according to the present invention comprises a dielectric block, a thin film layer formed on one surface of the dielectric block, and a sample holding mechanism for holding a sample on the surface of the thin film layer. And a light source for generating a light beam, and an incident optical system for making the light beam incident on the dielectric block at an angle of incidence at which the total reflection condition is obtained at the interface between the dielectric block and the thin film layer, In a measuring method for analyzing a sample by a measuring device comprising a light detecting means for measuring the intensity of a light beam totally reflected at the interface, a measurement is performed on a plurality of types of reference samples whose refractive indexes are known, Is measured, and the measurement result of the measurement on the sample is calibrated based on the measurement result of the measurement on the reference sample.

Further, the measuring device according to the present invention comprises a dielectric chip, a thin film layer formed on one surface of the dielectric block, and a sample holding mechanism for holding a sample on the surface of the thin film layer. A light source that generates a light beam, an incident optical system that causes the light beam to enter the dielectric block at an incident angle at which total reflection conditions are obtained at the interface between the dielectric block and the thin film layer, and at the interface. In a measuring device comprising a light detecting means for measuring the intensity of the reflected light beam, the measurement result measured using a plurality of types of reference samples of which the refractive index is known as the reference measurement result, the reference measurement result A calibration means for calibrating the measurement result of the measurement based on the sample is provided.

As the above-mentioned measuring device, the above-mentioned surface plasmon measuring device using a metal film as the above-mentioned thin film layer, a clad layer formed on one surface of the dielectric block,
There is the above-mentioned leaky mode measuring device which uses a layer composed of an optical waveguide layer formed on the clad layer as the thin film layer.

In the measuring apparatus according to the present invention, there are various methods for measuring the intensity of the light beam totally reflected at the interface by the light detecting means to analyze the sample. For example, the light beam is totally reflected at the interface. The intensity of the light beam totally reflected by the interface is measured at each position corresponding to each incident angle, and the position (angle) of the dark line generated by the attenuated total reflection is measured. Sample analysis may be carried out by detecting, DVNoort, K.johansen, C.-F.Ma.
ndenius, Porous Gold in Surface Plasmon Resonance
As described in Measurement, EUROSENSORS XIII, 1999, pp.585-588, light beams with a plurality of wavelengths are made incident at an incident angle at which total reflection conditions are obtained at the interface, and total wavelength is obtained at the interface for each wavelength. The sample analysis may be performed by measuring the intensity of the reflected light beam and detecting the degree of attenuation of total reflection for each wavelength.

In the measuring device according to the present invention, the refractive index of two kinds of samples is obtained by mixing two kinds of samples whose refractive indexes are already known and changing the mixing ratio of these two kinds of samples. It is desirable to provide a reference sample generating means for generating reference samples having a plurality of refractive indices between.

The measurement using the reference sample is not limited to the measurement of a liquid reference sample, and a solid material having a known refractive index (dielectric constant) is fixed (deposited) on the thin film layer of the measuring chip. The measurement may be performed using the measured chip (calibration jig).

As such a measurement chip (calibration jig), a plurality of measurement chips are arranged and integrated,
A measurement chip unit in which solid materials having different known refractive indexes are fixed on the thin film layers of the plurality of measurement chips is preferable. In this measurement chip unit, the dielectric blocks of the plurality of measurement chips forming the measurement chip unit may be integrally formed.

Further, the light detecting means is arranged in parallel in a predetermined direction, and a plurality of light receiving elements for receiving the light beams totally reflected at the interface, and light detection signals output by the plurality of light receiving elements are arranged in parallel to the light receiving elements. Differentiating means that differentiates with respect to the installation direction and outputs a differential value, and the initial value of this differential value is subtracted from the differential value existing near the point where the change in the photodetection signal in the installation direction of the light receiving elements changes from decrease to increase. However, it is desirable to use a measuring means for measuring the change with time of the differential value obtained by subtracting the initial value.

Here, "the above-mentioned differential value existing in the vicinity of the point where the change of the photodetection signal changes from decrease to increase" means the differential value which is the closest to the point where the change of the photodetection signal changes from decrease to increase. Although preferable, the present invention is not limited to this, and it is sufficient that it exists in the vicinity of the point where the change of the photodetection signal changes from decrease to increase. Also, the above "initial value of differential value"
As for, the value of the differential value existing near the point where the change of the photodetection signal changes from decrease to increase, which is measured at the beginning of the measurement, may be used as it is, or the calculation such as feedback may be performed at the start of the measurement. Alternatively, the value may be set as an initial value by performing a process to obtain a value that shifts the measured value at the start of measurement to near 0. Further, when performing the “subtraction”, a subtracter or the like may be used to subtract the initial value of the differential value from the differential value existing in the vicinity of the point where the change of the photodetection signal changes from decrease to increase. Alternatively, the subtraction may be performed by obtaining the difference between the two using a differential circuit or the like.

[0030]

EFFECT OF THE INVENTION In the measuring method and the measuring apparatus using the evanescent wave of the present invention, the measurement result of the state of attenuated total reflection measured by using a plurality of types of reference sample liquids having known refractive indices is referred to. As a result, by calibrating the measurement result of the measurement on the sample based on the reference measurement result, it is possible to reduce the measurement error caused by the individual difference in the measurement sensitivity of the measurement device, and thus it is possible to improve the measurement accuracy. .

[0031]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings. A measuring device according to an embodiment of the present invention is a surface plasmon measuring device capable of simultaneously analyzing a plurality of samples by making light beams enter a plurality of dielectric blocks in parallel, and FIG. It is a top view which shows the schematic structure of the surface plasmon measuring apparatus of this Embodiment, and FIG. 2 shows the side surface shape of this surface plasmon measuring apparatus.

The surface plasmon measuring device 101 comprises a plurality of surface plasmon measuring units 101 having the same structure.
A, 101B, 101C ...

The configuration of each measuring unit will be described by omitting the symbols A, B, C ... Representing the individual elements. Each measuring unit comprises a measuring tip 9 and a light beam 1.
A laser light source 14 which is a light source for generating 3 and an incident optical system 1 for making the light beam 13 incident on the measuring chip 9.
5, a collimator lens 16 that collimates the light beam 13 reflected by the measuring chip 9 and emits the collimator lens 16 toward the photodetector 17, and a light intensity of the light beam 13 emitted from the collimator lens 16 A photodetector 17 for detecting
It includes a differential amplifier array 18 connected to the photodetector 17, a driver 19 connected to the differential amplifier array 18, and a signal processing unit 20 connected to the driver 19 such as a computer system. The signal processing unit 20
The storage unit (not shown) for storing the reference measurement result data used in the calibration operation described later is built in, and also functions as a calibration unit.

The measuring chip 9 has a shape in which a part including the apex angle at which the four ridges of the quadrangular pyramid are gathered is cut out, and the bottom surface of the quadrangular pyramid is formed with a recess 10c functioning as a sample holding mechanism for storing the sample liquid 11. Dielectric block 10 of
It is composed of a metal film 12 which is a thin film layer made of, for example, gold, silver, copper, aluminum or the like, which is formed on the bottom surface of the concave portion 10c of the dielectric block 10. This dielectric block 10
Can be formed of, for example, a transparent resin or the like. The sensing medium 30 described below may be provided on the metal film 12. In addition, the dielectric block 10 of the measurement chip 9
As shown in FIG. 3, may be integrally formed with the dielectric block of the measurement chip of the plurality of surface plasmon measurement units adjacent to each other.

The incident optical system 15 has a collimator lens 15a for collimating the light beam 13 emitted from the laser light source 14, and a condenser for converging the collimated light beam 13 toward the interface 10b. It is composed of a lens 15b.

Since the light beam 13 is condensed by the condensing lens 15b as described above, it contains components that are incident on the interface 10b at various incident angles θ. The incident angle θ is set to an angle equal to or larger than the total reflection angle. Therefore, the light beam 13 totally reflected at the interface 10b contains components totally reflected at various reflection angles. The incident optical system 15 directs the light beam 13 into the interface 10b.
The light may be incident in a defocused state without being condensed in a point shape. By doing so, the interface 10b
Since the light beam 13 is totally reflected in the wider area above, the detection error in the attenuated total reflection state is averaged, and the measurement accuracy of the total reflection elimination angle can be improved.

The light beam 13 is incident on the interface 10b as p-polarized light. In order to do so, the laser light source 14 may be arranged in advance so that the polarization direction thereof is the above predetermined direction. In addition, the light beam 13 is applied to the interface 10
To make p-polarized light incident on b, use a wave plate 1
The direction of the polarized light of No. 3 may be controlled.

Further, the surface plasmon measuring device 101 is
Signal processing units 20A, 20B, 20C of each measurement unit ...
It has one display means 21 connected to.

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

As shown in FIG. 2, the light beam 13 emitted from the laser light source 14 is converged on the interface 10b between the dielectric block 10 and the metal film 12 through the incident optical system 15.

It converges on the interface 10b, and this interface 10b
The light beam 13 totally reflected by the collimator lens 1
It is detected by the photodetector 17 through 6. The photodetector 17 is a photodiode 17 which is a plurality of light receiving elements.
is a photodiode array in which a, 17b, 17c ... Are arranged side by side in a row, and the parallel arrangement of the photodiodes is performed through the collimator lens 16 such that the parallel arrangement direction of the photodiodes is substantially parallel to the paper surface of FIG. Incident light beam 1
It is arranged so as to be substantially orthogonal to the propagation direction of 3. Therefore, the different photodiodes 17a, 17b, 17c, ... Receive the respective components of the light beam 13 totally reflected at various reflection angles at the interface 10b. Then, the photodetector 17 outputs a signal indicating the intensity distribution of the light beam 13 detected by the photodiodes 17a, 17b, 17c ...

The component of the light beam 13 incident on the interface 10b at the specific incident angle θ _SP is the metal film 12 and the metal film 1.
Since surface plasmons are excited at the interface with the substance in contact with 2, the reflected light intensity sharply decreases for this light. That the specific incident angle theta _{S P} is total reflection eliminated angle, the reflected light intensity at the angle theta _SP indicates a minimum value. The area where the reflected light intensity decreases is observed as a dark line in the light beam 13 totally reflected at the interface 10b, as shown by D in FIG.

Next, the processing of the signal indicating the intensity distribution of the light beam 13 output from the photodetector 17 will be described in detail.

FIG. 4 is a block diagram showing the electrical construction of this surface plasmon measuring device. As shown in the figure, the driver 19 is provided for each differential amplifier 18 of the differential amplifier array 18.
The sample-hold circuits 22a, 22b, 22c, which sample and hold the outputs of a, 18b, 18c ..., the multiplexer 23 to which the outputs of these sample-hold circuits 22a, 22b, 22c. A / D converter 24 which is converted to be input to the signal processing unit 20, a driving circuit 25 which drives the multiplexer 23 and the sample and hold circuits 22a, 22b, 22c ..., And a driving circuit 25 based on an instruction from the signal processing unit 20. It is composed of a controller 26 for controlling the operation of.

The photodiodes 17a, 17b, 1
.. of the differential amplifier array 18 are input to the differential amplifiers 18a, 18b, 18c. At this time, the outputs of two photodiodes adjacent to each other are input to a common differential amplifier. Therefore, each differential amplifier 18
The outputs of a, 18b, 18c ... Are the photodetection signals output by the plurality of photodiodes 17a, 17b, 17c.
It can be considered that they are differentiated with respect to their juxtaposition direction.

The outputs of the differential amplifiers 18a, 18b, 18c ...
.. are sample-held at a predetermined timing by b, 22c, ... And inputted to the multiplexer 23. The multiplexer 23 uses the sample-and-hold differential amplifiers 18
Outputs a, 18b, 18c, ...
Input to the / D converter 24. The A / D converter 24 digitizes these outputs and inputs them to the signal processing unit 20.

FIG. 5 illustrates the relationship between the light intensity of the light beam 13 totally reflected at the interface 10b at each incident angle θ on the interface 10b and the outputs of the differential amplifiers 18a, 18b, 18c. is there. Here, it is assumed that the relationship between the incident angle θ of the light beam 13 on the interface 10b and the light intensity I of the reflected light beam 13 is as shown in the graph of FIG.

Further, FIG. 5B shows the photodiode 1
7a, 17b, 17c, ... Are shown in parallel, and as described above, these photodiodes 17a, 17b are provided.
The positions of b, 17c, ... Arranged in parallel are uniquely corresponding to the incident angle θ.

Then, the photodiodes 17a, 17b,
The relation between the positions of the 17c in parallel, that is, the incident angle θ, and the output I ′ (differential value of the reflected light intensity I) of the differential amplifiers 18a, 18b, 18c, ... Is as shown in FIG. It will be

The signal processing unit 20 outputs from the A / D converter 24.
Based on the input differential value I ′, the differential amplifier 18
From a, 18b, 18c ...
Has and total reflection elimination angle θ_SPThe differential value I '=
An output that is closest to 0 is obtained (example in Fig. 5 (3))
Then it becomes a differential amplifier 18e) and a negative value as a differential value
And the total reflection elimination angle θ_SPDifferential value I'corresponding to
Output that is closest to = 0 (see (3) in FIG.
Select the difference amplifier 18d in the example), and select the difference between them.
The total reflection elimination angle θ based on the differential value output by the dynamic amplifier
_SPTo calculate. In some cases, the differential value I ′ = 0
There may be a differential amplifier that outputs
Then the total reflection elimination angle θ based on the differential amplifier_SPTo
calculate. After that, every time a predetermined time elapses, the same as above
Repeated operation, total reflection elimination angle θ _SPTo calculate the measurement
The angle change amount from the beginning is calculated and displayed on the display means 21.

As described above, when the dielectric constant of the substance in contact with the metal film 12 of the measuring chip, that is, the refractive index changes,
Since the total reflection canceling angle θ _SP also changes accordingly, the change in the refractive index of the substance in contact with the metal film 12 is examined by continuously measuring the amount of change in the total reflection canceling angle θ _SP with the passage of time. be able to.

When the sensing medium 30 that binds to a specific substance in the sample liquid 11 is fixed on the metal film 12, the refractive index of the sensing medium 30 changes depending on the binding state of the sample liquid 11 and the sensing medium 30. Changes, it is possible to check the state of change in the binding state by continuously measuring the differential value I ′. So in this case,
Both the sample liquid 11 and the sensing medium 30 serve as a sample to be analyzed. Examples of such a combination of the specific substance and the sensing medium 30 include an antigen and an antibody.

By the way, strictly speaking, the angle change of the total reflection elimination angle θ _SP measured by the above measuring apparatus does not accurately reflect the change in the refractive index of the substance in contact with the metal film 12 of the measuring chip. However, some errors may occur due to individual differences in measurement sensitivity of the measuring device.

Therefore, in the measuring device of the present invention,
The total reflection elimination angle θ _SP is measured in advance for a plurality of reference sample liquids having known refractive indices, and the data of the reference measurement results obtained by measuring these reference sample liquids is used as a calibration curve data as shown in FIG. It is stored in a storage unit (not shown) in the processing unit 20.

Such a reference sample liquid is prepared by automatically blending a plurality of these two types of sample liquids with different blending ratios, based on two types of sample liquids having known refractive indices, to obtain a plurality of refraction samples. By using a reference sample generating means (not shown) that generates a reference sample solution at a specific rate, efficient and accurate generation can be achieved.

In this embodiment, the refractive index is 1.33 to 1.
Although the measurement is performed in 0.01 steps up to 40, such a reference sample solution is prepared by preparing distilled water having a refractive index of 1.33 and a standard refractive solution having a refractive index of 1.40. The refractive index of 1.
Multiple reference sample solutions of 33 to 1.40 can be generated.

When the reference sample solution is measured, the metal film 12 is not fixed to the sensing medium 30.
The reference sample solution is dropped directly onto the top surface for measurement.

Based on this calibration curve data, sample liquid 1
1 (and the sensing medium 30) is calibrated to reduce the influence of individual differences in the measurement sensitivity of the measuring device and to measure the refractive index of the substance in contact with the metal film 12. Can be improved.

The measurement for obtaining the calibration curve data may be performed using a measurement chip unit (calibration jig) as shown in FIG. This measuring chip unit 5
The reference numeral 0 is integrally formed with the dielectric blocks of the measurement chips of the plurality of surface plasmon measurement units adjacent to each other, and the solid materials 53a and 53b of different known refractive indexes are provided on the plurality of thin film layers 52. , 53c ... Are fixed. The calibration curve data can be efficiently obtained even by using this measurement chip unit 50.

Further, as described in Japanese Patent Application No. 2001-382817, first, the initial value of the differential value to be measured is obtained, and the differential value output from the differential amplifier 18 (differentiating means) for each measurement of the differential value. If the initial value of the differential value is subtracted from the value, the differential value from which this initial value is subtracted does not include the variation in absolute value according to the positional relationship between the photodiode 17 and the dark line, and the differential value from the start of measurement. The value reflects only the change with time. Therefore, the value after this subtraction is smaller in absolute value than the normal differential value and can be amplified with a sufficiently high amplification factor, so the change in differential value over time can be measured with high sensitivity and sample analysis can be performed accurately. Therefore, it is possible to perform more accurate measurement.

The surface plasmon measuring device described above can be made into a leaky mode measuring device by changing a part of the configuration. FIG. 8 shows the surface plasmon measuring device 1 described above.
It is a side view of a measurement unit of a leaky mode measuring device configured by changing a part of 01. In FIG. 8, elements that are the same as the elements in FIG. 2 are given the same numbers, and descriptions thereof are omitted unless necessary.

This leaky mode measuring device is also configured to use the measuring chip 9 similarly to the above-mentioned surface plasmon measuring device. A clad layer 40 is formed on the bottom surface of the recess 10c formed on the upper surface of the measurement chip 9, and an optical waveguide layer 41 is further formed on the clad layer 40. The clad layer 40 and the optical waveguide layer 41 form a thin film layer.

The dielectric block 10 is formed of, for example, synthetic resin or optical glass such as BK7. On the other hand, the clad layer 40 is formed into a thin film using a dielectric material having a lower refractive index than the dielectric block 10 or a metal such as gold. The optical waveguide layer 41 is also formed in a thin film using a dielectric material having a higher refractive index than the cladding layer 40, for example, PMMA. The film thickness of the clad layer 40 is, for example, 36.5 nm when it is formed of a gold thin film, and the film thickness of the optical waveguide layer 41 is approximately 700 nm when it is formed of PMMA.

In the leak mode measuring device having the above structure,
When the light beam 13 emitted from the laser light source 14 is incident on the cladding layer 40 through the dielectric block 10 at an angle of incidence equal to or more than the total reflection angle, many components of the light beam 13 are included in the dielectric block 10 and the cladding layer. Interface 10 with 40
Light having a specific wave number, which is totally reflected by b but is transmitted through the cladding layer 40 and is incident on the optical waveguide layer 41 at a specific incident angle, is propagated in the optical waveguide layer 41 in a waveguide mode. When the guided mode is excited in this way, most of the incident light that has entered at the specific incident angle is taken into the optical waveguide layer 41, so that the intensity of the light that is incident on the interface 10b at the specific incident angle and totally reflected is sharp. Decreasing total internal reflection attenuation occurs.

Since the wave number of the guided light in the optical waveguide layer 41 depends on the refractive index of the sample liquid 11 on the optical waveguide layer 41,
By knowing the total reflection elimination angle which is the above-mentioned specific incident angle at which the total reflection attenuation occurs, the refractive index of the sample liquid 11 and the characteristics of the sample liquid 11 related thereto can be analyzed, and the same as in the above-described embodiment. The effect can be obtained.

[Brief description of drawings]

FIG. 1 is a plan view showing a schematic configuration of a surface plasmon measuring device according to an embodiment of the present invention.

FIG. 2 is a diagram showing a side surface shape of a surface plasmon measuring device.

FIG. 3 is a schematic configuration diagram of a measurement chip.

FIG. 4 is a block diagram showing an electrical configuration of a surface plasmon measuring device.

FIG. 5 is a diagram showing a relationship between an incident angle of a light beam on an interface and an output of a differential amplifier.

FIG. 6 is an explanatory diagram of reference data.

FIG. 7 is a diagram showing an example of a measurement chip.

FIG. 8 is a diagram showing an example of a leaky mode measuring device.

[Explanation of symbols]

9 Measuring Chip 10 Dielectric Block 13 Light Beam 14 Laser Light Source 15 Incident Optical System 16 Collimator Lens 17 Photodetector 18 Differential Amplifier Array 19 Driver 20 Signal Processing Unit 21 Display Means 50 Measuring Chip Unit 101 Surface Plasmon Measuring Devices 101A, 101B , 101C ... Surface plasmon measurement unit

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 2G057 AA02 AB04 AB07 AC01 BA01                       BB01 BB06                 2G059 AA01 BB04 BB12 CC16 DD13                       EE02 EE05 EE09 GG01 GG04                       JJ11 JJ19 JJ22 KK04 MM01                       MM03 MM09 MM11 MM14 PP04

Claims (5)

[Claims] 1. A measuring chip comprising a dielectric block, a thin film layer formed on one surface of the dielectric block, and a sample holding mechanism for holding a sample on the surface of the thin film layer, and a light beam is generated. A light source, an incident optical system that causes the light beam to be incident on the dielectric block at an incident angle such that total reflection conditions are obtained at an interface between the dielectric block and the thin film layer, and light totally reflected at the interface In a measuring method for analyzing the sample by a measuring device comprising a light detecting means for measuring the intensity of the beam, a plurality of types of reference samples having a known refractive index are measured, and the sample is measured, A measurement method comprising calibrating the measurement result of the measurement on the sample based on the measurement result of the measurement on the reference sample. 2. A measuring chip comprising a dielectric block, a thin film layer formed on one surface of the dielectric block, and a sample holding mechanism for holding a sample on the surface of the thin film layer, and a light beam is generated. A light source, an incident optical system that causes the light beam to be incident on the dielectric block at an incident angle such that total reflection conditions are obtained at an interface between the dielectric block and the thin film layer, and light totally reflected at the interface In a measuring device comprising a light detecting means for measuring the intensity of the beam, a measurement result measured using a plurality of types of reference samples of which the refractive index is known as a reference measurement result, based on the reference measurement result A measuring device comprising a calibrating means for calibrating the measurement result of the measurement on the sample. 3. Based on two kinds of samples having known refractive indexes, plural kinds of the two kinds of samples are mixed at different mixing ratios to obtain a plurality of kinds of refractive indexes between the two kinds of samples. The measuring apparatus according to claim 2, further comprising a reference sample generating unit that generates the reference sample having a refractive index. 4. The plurality of light detecting elements, which are arranged in parallel in a predetermined direction, receive the light beam totally reflected at the interface, and the light detecting signals output from the plurality of light receiving elements. Differentiating means for differentiating with respect to the juxtaposed direction of the elements and outputting a differential value, and the differentiating from the differentiated value existing in the vicinity of the point where the change of the light detection signal in the juxtaposed direction of the light receiving elements turns from decrease to increase. 4. The measuring device according to claim 2, further comprising a measuring unit that subtracts the initial value of the value and measures the change over time of the differential value obtained by subtracting the initial value. 5. A plurality of measuring chips used in the measuring apparatus according to claim 2 are arranged side by side and integrated, and solid materials having different known refractive indexes are provided on respective thin film layers of the plurality of measuring chips. A measuring chip unit characterized by being fixed.