JP2012215478A - Method of detection and detector - Google Patents

Abstract

PROBLEM TO BE SOLVED: To reduce errors due to fluctuation of incident angles of excitation light against a metal film and also improve detection sensitivity in a method of detection and a detector for optically measuring the quantity of a substance to be detected in a sample solution by utilizing a surface plasmon enhanced effect.SOLUTION: A sensor chip for measuring the quantity of a substance to be detected in a sample solution has a sensor part formed by laminating a metal film 16 on one surface of a dielectric plate 17, the sensor part being divided into five planar regions E1 to E5 of which inclination angles with respect to a predetermined reference plane are different from each other. A method for measuring the quantity of the substance to be detected in the sample solution includes: using the sensor chip and bonding a fluorescent label bonding substance of a quantity according to the quantity of the substance to be detected contained in the sample solution on the sensor part by bringing the sample solution into contact with the sensor part of the sensor chip; sequentially irradiating each of the planar regions of the sensor chip with excitation light; detecting the quantity of light generated in the sensor part according to irradiation of the excitation light for each planar region of the sensor chip; and measuring the quantity of the substance to be detected based on the detection result of a planar region in which a measured amount of light is the largest among the planar regions E1 to E5.

Description

  The present invention relates to a detection method and a detection apparatus for optically measuring the amount of a substance to be detected in a sample solution.

  Conventionally, a fluorescence detection method has been widely used as a highly sensitive and easy measurement method in biomeasurement and the like. In this fluorescence detection method, the presence of the detection target substance is detected by irradiating the sample, which is considered to contain a detection target substance that emits fluorescence when excited with light of a specific wavelength, with the excitation light of the specific wavelength. It is a method to confirm. In addition, when the detection target substance is not a fluorescent substance, this binding, that is, by detecting the fluorescence in the same manner as described above by contacting the sample with a substance that is labeled with a fluorescent dye and specifically binds to the detection target substance. The existence of a detection target substance is also widely confirmed.

  Further, in order to improve sensitivity in such a fluorescence detection method, a method using the surface plasmon enhancement effect is proposed in Patent Document 1 and the like. This is because the surface plasmon resonance is generated, so that the sensor part provided with the metal film in a predetermined region on the transparent support is opposite to the metal layer forming surface of the support with respect to the interface between the support and the metal film. S / N is obtained by making excitation light incident at a predetermined angle equal to or greater than the total reflection angle from the surface side of the substrate, generating surface plasmons on the metal layer by irradiation of the excitation light, and enhancing fluorescence by the electric field enhancement action. Is to improve.

  In addition, instead of detecting the fluorescence from the fluorescent label as in the fluorescence method shown above, the emitted light (SPCE: Surface Plasmon-Coupled Emission) generated by the fluorescence newly inducing a surface plasmon in the metal layer A method for detecting the above has also been proposed.

  The surface plasmon enhancement depends on the intensity and incident angle of the excitation light on the metal film. In particular, the surface plasmon enhancement shows a steep characteristic with respect to the incident angle of the excitation light on the metal film. The plasmon enhancement varies greatly, and as a result, the detected fluorescence intensity varies. That is, if there are variations in the installation accuracy of the sensor chip, the surface accuracy of the sensor portion of the sensor chip, and the incident angle accuracy of the excitation light, there is a problem that the measurement result is not stable and the measurement accuracy is lowered.

  In order to solve such a problem, Patent Document 2 proposes an apparatus capable of absorbing an angle error within a convergence angle by using convergent light having a plurality of angle components for excitation light. . Normally, the light emitted from the light source has a light intensity that changes depending on the position of the light beam (for example, the distance from the light beam center derived from the angle of divergence from the light source) (that is, there is an intensity distribution). Since there is a problem that the degree of the electric field enhancement effect by the surface plasmon changes due to the change in the angle at which the plasmon resonance is generated, in Patent Document 2, an optical system such as a diaphragm is further combined with the light at the center of the light beam. A device has also been devised to suppress variation in the intensity of the excitation light for each incident angle by using only a region having a high intensity.

JP-A-10-307141 JP 2009-204483 A

  However, the means described in Patent Document 2 can deal with errors caused by fluctuations in the incident angle of the excitation light with respect to the metal film, but only a small amount of light near the center of the light beam is emitted from the light source. As a result, the intensity of the excitation light is lowered as a whole and the detection sensitivity is lowered.

  The present invention has been made in view of the above problems, and in a detection method and a detection apparatus for optically measuring the amount of a substance to be detected in a sample liquid using a surface plasmon enhancement effect, excitation light for a metal film It is an object of the present invention to provide a detection method and a detection apparatus that have a small error due to fluctuations in the incident angle and a high detection sensitivity.

  The detection method of the present invention includes a sensor unit formed by laminating a metal film on one surface of a dielectric plate, and the sensor unit is divided into a plurality of plane regions having different inclination angles with respect to a predetermined reference plane. Using the chip, the sample liquid containing the substance to be detected is brought into contact with the sensor part of the sensor chip, whereby an amount of the fluorescent label binding substance corresponding to the quantity of the substance to be detected contained in the sample liquid is placed on the sensor part. The sensor chip is irradiated with excitation light sequentially for each plane area of the sensor chip, and the amount of light generated in the sensor unit in response to the excitation light irradiation is detected for each plane area of the sensor chip. The amount of the substance to be detected is measured based on the detection result of the planar region in which the most amount of light is detected.

  In the detection method of the present invention, when the sensor chip includes a microchannel through which the sample liquid is circulated, and a plurality of sensor units having the same planar area configuration are provided in the microchannel, a plurality of sensors are provided. The amount of light is detected in each plane area of the reference sensor unit, and the amount of light is the largest among the plurality of plane areas of the reference sensor unit. The detected plane area is determined, and for the remaining sensor sections of the plurality of sensor sections, the amount of light is only in the plane area corresponding to the plane area where the most amount of light is detected in the reference sensor section. It is preferable to perform detection.

  The detection device of the present invention is a detection device used in the above-described detection method, and selectively irradiates excitation light for each planar area of a housing portion that houses a sensor chip and a sensor chip that is housed in the housing portion. Based on the excitation light irradiation optical system, the light detection means for detecting the amount of light generated in the sensor unit in response to the excitation light irradiation, and the amount of the detected substance based on the amount of light detected by the light detection means And measuring means for measuring.

  According to the detection method and the detection apparatus of the present invention, the sensor unit includes a sensor unit formed by laminating a metal film on one surface of the dielectric plate, and the sensor unit is provided in a plurality of plane regions having different inclination angles with respect to a predetermined reference plane. Using the divided sensor chip, the sample liquid containing the substance to be detected is brought into contact with the sensor part of the sensor chip, whereby an amount of fluorescent label binding corresponding to the quantity of the substance to be detected contained in the sample liquid A substance is bonded onto the sensor unit, and excitation light is sequentially irradiated to each planar region of the sensor chip, and the amount of light generated in the sensor unit is detected for each planar region of the sensor chip in response to irradiation of the excitation light. The amount of the substance to be detected is measured based on the detection result of the planar area in which the largest amount of light is detected among the plurality of planar areas.

  If a sensor chip that includes a sensor unit formed by laminating a metal film on one surface of a dielectric plate and is divided into a plurality of plane regions having different inclination angles with respect to a predetermined reference plane is used, parallel light Even when is used as excitation light, angle adjustment within the setting range of the inclination angle of the plurality of planar regions can be made unnecessary.

  In addition, since the excitation light is irradiated sequentially for each planar area of the sensor chip, all the excitation light emitted from the light source can be irradiated for each planar area, so that the excitation light intensity at the time of detection can be increased. Can be high.

  Therefore, it is possible to reduce errors due to fluctuations in the incident angle of the excitation light with respect to the metal film and to increase the detection sensitivity.

  In addition, when the sensor chip includes a micro flow channel through which the sample liquid is circulated and a plurality of sensor units having the same planar area configuration are provided in the micro flow channel, any one of the plurality of sensor units is provided. Using one sensor unit as a reference sensor unit, the amount of light is detected for each plane region of the reference sensor unit, and the plane region in which the most amount of light is detected among the plurality of plane regions of the reference sensor unit For the remaining sensor units of the plurality of sensor units, the amount of light is detected only in the plane region corresponding to the plane region where the most amount of light is detected in the reference sensor unit. By doing so, the total number of detections can be reduced, so that efficient measurement can be performed.

Schematic diagram of a fluorescence detection apparatus according to an embodiment of the present invention Block diagram of the fluorescence detection device The schematic diagram which shows an example of the sensor chip used for the said fluorescence detection apparatus FIG. 2 is a schematic diagram showing how a sample is extracted from a sample container using a nozzle tip by the sample processing means of FIG. FIG. 2 is a schematic diagram showing how the sample in the nozzle tip is injected and stirred into the reagent cell by the sample processing means of FIG. Schematic diagram showing an example of the light irradiation means and fluorescence detection means of FIG. Top view showing the configuration of the sensor portion of the sensor chip Sectional drawing for each planar area of the sensor section viewed from the direction of arrow VIII in FIG. The top view which shows the other structure of the sensor part of the said sensor chip XX sectional view in FIG.

  Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. 1 is a schematic diagram of a fluorescence detection device according to an embodiment of the present invention, FIG. 2 is a block diagram of the fluorescence detection device, FIG. 3 is a schematic diagram illustrating an example of a sensor chip used in the fluorescence detection device, and FIG. FIG. 5 is a schematic diagram showing how a sample is extracted from a sample container using a nozzle tip by the sample processing means in FIG. 2, and FIG. 5 is a diagram in which the sample in the nozzle chip is injected and stirred into the reagent cell by the sample processing means in FIG. 6 is a schematic diagram showing an example of the light irradiation means and the fluorescence detection means of FIG. 2, FIG. 7 is a top view showing the configuration of the sensor portion of the sensor chip, and FIG. 8 is a diagram in FIG. It is sectional drawing for every plane area | region of the sensor part seen from VIII arrow direction.

  This fluorescence detection device 1 is an immunological analysis device using surface plasmon resonance, and when performing analysis by the fluorescence detection device 1, a sample container CB containing the sample shown in FIG. 1 and a sample and a reagent are extracted. The nozzle chip NC used at the time, and the sensor chip 10 formed with the reagent cell and the micro flow path are loaded. The sample container CB, the nozzle tip NC, and the sensor chip 10 are all disposable items that are discarded once they are used. Then, the fluorescence detection apparatus 1 performs quantitative or qualitative analysis on the test substance in the sample while flowing the sample through the micro flow path 15 of the sensor chip 10.

  The fluorescence detection apparatus 1 includes a sample processing unit 20, a light irradiation unit 30, a fluorescence detection unit 40, a data analysis unit 50, and the like. The sample processing means 20 extracts a sample from the sample container CB containing the sample using the nozzle chip NC, and generates a sample solution obtained by mixing and stirring the extracted sample with a reagent.

  Here, FIG. 3 is a schematic diagram showing an example of the sensor chip 10. The sensor chip 10 has a structure in which an inlet 12, an outlet 13, sample cells 14 a and 14 b, and a flow path 15 are formed in a main body 11 made of a dielectric plate such as a light transmissive resin. The injection port 12 communicates with the discharge port 13 via the flow channel 15, and by applying a negative pressure from the discharge port 13, the specimen is injected from the injection port 12, flows into the flow channel 15, and is discharged from the discharge port 13. The The sample cells 14a and 14b are containers for storing a fluorescent reagent (second antibody) to be mixed with the specimen in the specimen container CB. Note that the openings of the sample cells 14a and 14b are sealed with a sealing member, and the sealing member is pierced when the specimen and the fluorescent reagent are mixed.

  Further, a test region TR as a measurement sensor unit for detecting a test substance in the specimen is formed in the flow path 15, and in the test region TR, downstream of the test region TR. A control region CR is formed as a correction sensor unit for acquiring information for correcting the measurement result.

  A first antibody is immobilized on the test region TR, and the labeled antibody is captured by a so-called sandwich method. In addition, a reference antibody is fixed to the control region CR, and the reference antibody captures the fluorescent substance when the sample solution flows on the control region CR. Two control regions CR are formed, and in order from the upstream side, a so-called negative control region CR for detecting non-specific adsorption and a so-called positive type for detecting a difference in reactivity due to a difference in specimen. The control region CR is formed.

  As shown in FIGS. 7 and 8, the test region TR and the two control regions CR are divided into five planar regions E1 to E5 in the traveling direction of the flow path 15. The five plane regions E1 to E5 have different inclination angles with respect to the horizontal direction (reference plane), and the plane region E1 is inclined by −2 ° with respect to the horizontal direction in the direction orthogonal to the traveling direction of the flow path 15. E2 is inclined by -1 ° in the direction orthogonal to the traveling direction of the flow path 15 with respect to the horizontal direction, the planar area E3 is parallel to the horizontal direction, and the planar area E4 is parallel to the horizontal direction. The plane region E5 is inclined 2 ° in the direction orthogonal to the traveling direction of the flow path 15 with respect to the horizontal direction. The configuration of the planar areas E1 to E5 of the test area TR and the two control areas CR is the same.

  When the start of analysis is instructed, the sample processing means 20 aspirates the sample from the sample container CB using the nozzle tip NC as shown in FIG. Thereafter, the sample processing means 20 punctures the seal member of the sample cell 14a as shown in FIG. 5, mixes and stirs the sample with the reagent in the sample cell 14a, and then sucks the sample solution again using the nozzle tip NC. . This operation is similarly performed for the sample cell 14b. Then, a sample solution is generated in which the second antibody B2, which is a second binding substance specifically binding in the reagent, to the test substance (antigen) A present in the sample is modified on the surface. Then, the sample processing means 20 installs the nozzle chip NC containing the sample solution on the injection port 12, and the sample solution in the nozzle chip NC flows into the flow path 15 by the negative pressure from the discharge port 13.

  In addition, although the case where the sample processing unit 20 supplies the sample solution in which the sample and the reagent are mixed into the flow path 15 is illustrated, the reagent is filled in the flow path 15 in advance, and the sample processing means 20 Only the specimen may be allowed to flow from the inlet 12.

  FIG. 6 is a schematic diagram showing an example of the light irradiation means 30 and the fluorescence detection means 40. In FIG. 6, the description will be given focusing on the test region TR, but the excitation light L is similarly applied to the control region CR. The light irradiating means 30 in FIG. 2 selects the excitation light L, which is parallel light from the back side of the sensor chip 10, at one of the plane regions E1 to E5 of the test region TR through the prism at an incident angle that is a total reflection condition. Is intended to be irradiated. The fluorescence detection means 40 is composed of, for example, a CCD, a CMOS, or the like, and detects the amount of fluorescence Lf generated in the test area TR or the control area CR.

  Then, the excitation light L is incident on the interface between the dielectric plate 17 and the metal film 16 at a specific incident angle equal to or greater than the total reflection angle by the light irradiation means 30, thereby entering the sample S on the metal film 16. The evanescent wave Ew oozes out, and surface plasmons are excited in the metal film 16 by the evanescent wave Ew. This surface plasmon causes an electric field distribution on the surface of the metal film 16 to form an electric field enhancement region. Then, the fluorescent labeling substance F bound to the first antibody B1 fixed on the metal film 16 is excited by the evanescent wave Ew to generate enhanced fluorescence Lf.

  In detection using plasmon enhancement, metal quenching may occur and the sensitivity may be lowered. For example, if a quenching prevention layer made of a silica layer, a polystyrene layer, or the like is provided on the metal layer 16, Such a problem can be solved. In addition, as for the fluorescent labeling substance F, for example, as a quenching preventive substance such as a fluorescent dye encapsulated in polystyrene particles or silica particles, or a gold colloid surface coated with polystyrene, the problem of metal quenching is solved. be able to.

  The data analysis means 50 in FIG. 2 analyzes the test substance based on the signal FS based on the light quantity of the fluorescence Lf detected by the fluorescence detection means 40.

  Here, the analysis processing in this embodiment will be described in detail.

  In the present embodiment, first, for each of the planar regions E1 to E5 in the positive control region CR, as shown in FIG. 7, the irradiation position of the excitation light L is moved along the traveling direction of the flow path 15, The excitation light L is sequentially irradiated as described above, and the amount of light generated at that time is detected. And the plane area with the highest detected light quantity is specified among the plane areas E1 to E5. Then, the measurement result in the control region CR is acquired based on the detection result of the planar region having the highest detected light amount.

  In addition, the test region TR and the negative control region CR were irradiated with the excitation light L only in the same planar region as the planar region having the highest detected light amount in the detection in the positive control region CR, and occurred at that time. Detect the amount of light. For example, when the detected light amount in the plane area E2 is the highest in the positive type control area CR, the test area TR and the negative type control area CR are irradiated with the excitation light L only in the plane area E2 and generated at that time. Detect the amount of light.

  The analysis result obtained as described above is output from the information output means 4 comprising a monitor, a printer or the like.

  By setting it as such an aspect, even when parallel light is used as the excitation light L, the angle error within the inclination angle range of the planar regions E1 to E5 can be absorbed. Further, since the excitation light L is sequentially irradiated for each of the planar regions E1 to E5, all the excitation light L emitted from the light source can be irradiated for each of the planar regions E1 to E5. The intensity of the excitation light L can be increased. Therefore, it is possible to reduce errors due to fluctuations in the incident angle of the excitation light with respect to the metal film 16 and to increase the detection sensitivity. In addition, since the excitation light L can be parallel light, the optical system can have a simple configuration without requiring a special optical system as described in Patent Document 2 above, so that the cost can be reduced. Can be kept low.

  As mentioned above, although preferable embodiment of this invention was described, this invention is not limited to the said embodiment.

  For example, the test region TR and the two control regions CR may be divided into five planar regions E1 to E5 in a direction orthogonal to the traveling direction of the flow path 15, as shown in FIGS. Here, the five plane regions E1 to E5 have different inclination angles with respect to the horizontal direction (reference plane), and the plane region E1 is inclined by −2 ° in the direction perpendicular to the traveling direction of the flow path 15 with respect to the horizontal direction. The plane region E2 is inclined by -1 ° in the direction orthogonal to the traveling direction of the flow path 15 with respect to the horizontal direction, the plane region E3 is parallel to the horizontal direction, and the plane region E4 is parallel to the horizontal direction. The plane region E5 is inclined by 2 ° in the direction orthogonal to the traveling direction of the flow path 15 with respect to the horizontal direction. The configuration of the planar areas E1 to E5 of the test area TR and the two control areas CR is the same. In this case, for each of the planar regions E1 to E5, as shown in FIG. 10, the excitation light L is sequentially irradiated by moving the irradiation position of the excitation light L along the direction orthogonal to the traveling direction of the flow path 15, The amount of light generated at that time is detected.

  Moreover, about the structure of a sensor part, the number divided | segmented into each plane area | region is not restricted to five, It is good also as a several other number. Further, the division mode may be divided in a two-dimensional manner instead of a one-dimensional manner as described above. Further, the inclination angle of each planar region is not limited to the above.

  Furthermore, it goes without saying that various improvements and modifications other than those described above may be made without departing from the scope of the present invention.

DESCRIPTION OF SYMBOLS 1 Fluorescence detection apparatus 10 Sensor chip 20 Sample processing means 30 Light irradiation means 40 Fluorescence detection means 50 Data analysis means CR Control area FS Fluorescence signal L Excitation light LF Fluorescence TR Test area

Claims (3)

Using a sensor chip that includes a sensor unit formed by laminating a metal film on one surface of a dielectric plate, and the sensor unit is divided into a plurality of plane regions having different inclination angles with respect to a predetermined reference plane,
By contacting a sample liquid containing a substance to be detected on the sensor part of the sensor chip, an amount of the fluorescent label binding substance corresponding to the quantity of the substance to be detected contained in the sample liquid is placed on the sensor part. Combined
Irradiate excitation light sequentially for each planar area of the sensor chip,
Detecting the amount of light generated in the sensor unit according to the irradiation of the excitation light for each planar region of the sensor chip;
A detection method, comprising: measuring an amount of the substance to be detected based on a detection result of a planar area in which the largest amount of light is detected among a plurality of planar areas. When the sensor chip includes a microchannel through which the sample liquid is circulated, and a plurality of sensor units having the same planar region configuration are provided in the microchannel,
Using one of the plurality of sensor units as a reference sensor unit, the amount of the light is detected for each planar region of the reference sensor unit,
Determining the plane area in which the most amount of light is detected among the plurality of plane areas of the reference sensor unit;
For the remaining sensor units of the plurality of sensor units, the amount of light is detected only in the plane region corresponding to the plane region in which the most amount of light is detected in the reference sensor unit. The detection method according to claim 1. A detection device used in the detection method according to claim 1 or 2,
An accommodating portion for accommodating the sensor chip;
An excitation light irradiation optical system that selectively emits excitation light for each planar region of the sensor chip housed in the housing portion;
Light detecting means for detecting the amount of light generated in the sensor unit in response to irradiation of the excitation light;
A detection apparatus comprising: a measurement unit that measures the amount of the substance to be detected based on the amount of light detected by the light detection unit.