JP6026262B2 - Measuring apparatus and measuring method - Google Patents

Description

  The present invention relates to a measuring apparatus and a measuring method using surface plasmon resonance.

  Cited Document 1 discloses an analysis chip having a flow channel for use in a measuring apparatus using surface plasmon resonance. This analysis chip is characterized in that only a test substance bound to a surface where plasmon resonance occurs can be detected among the test substances in the specimen flowing in the flow path. A test area and a control area for detecting a test substance in the specimen are formed in the flow path of the analysis chip. The first antibody is immobilized on the test region, and the first antibody captures the antigen captured in the primary reaction by the second antibody labeled with the fluorescent substance, and reflects the amount of the antigen. Generate a signal (so-called sandwich method). On the other hand, a reference antibody is immobilized on the control region, and the reference antibody captures the second antibody labeled with a fluorescent substance regardless of the presence or absence of an antigen and generates a positive signal.

  FIG. 16 is a diagram for explaining the principle of the sandwich method and the competitive method, and FIG. 17 is a diagram for explaining a multilayer film model in the sandwich method and the competitive method. In these figures, the Au film 200, the first antibody 201, the reference antibody 202, the antigen label protein 203, the antigen 204, and the fluorescent particles 205 on which the capture antibody is immobilized. First, as shown in the sandwich method (1) of FIG. 16, in the sandwich method, the structure of the antigen recognition site differs between the first antibody 201 in the test region and the reference antibody 202 in the control region. It is the same immunoglobulin that is a complex. Further, in the total reflection attenuation and electric field enhancement strength of the analysis chip using surface plasmon resonance, the film made of molecules immobilized on the Au film 200 mainly composed of such protein complex is as shown in FIG. Can be modeled as a single-layer dielectric film on the Au film 200.

  Here, when the size of the antigen 204a to be detected is small enough to be ignored, the same substance is fixed to the test area and the control area as shown in the sandwich method (1) in FIG. Is so small that it can be ignored. Therefore, the optical constant (refractive index n × film thickness d) of the film modeled as a dielectric film is the same.

  Therefore, for example, even if there is a variation that causes the same change in the surface plasmon resonance of the test region and the surface plasmon resonance of the control region, such as the refractive index variation for each specimen, by normalizing with this positive signal, In principle, this can be canceled out and high-precision measurement can be performed.

  Further, cited document 2 discloses an apparatus having means for adjusting the position of excitation light as a fluorescence detection apparatus using the above-described analysis chip. This fluorescence detection device detects the fluorescence generated when the excitation light is irradiated while shifting the position with respect to the test area as a plurality of adjustment fluorescence signals, and adjusts the deviation of the irradiation position of the excitation light with respect to the test area based on them Thus, there is provided a means for preventing the deterioration of the analysis accuracy due to the solid difference of the analysis chip. Although there is no description in the cited document 2, even if the displacement of the irradiation position of the excitation light due to the solid difference of the analysis chip is adjusted by providing such means, if a plurality of devices are manufactured, the excitation light is produced by parts or assembly of the device. There are cases where the angle of the entire irradiation unit is deviated or the wavelength of the excitation light is deviated due to variations in the components of the excitation light source used.

  When the irradiation position is automatically adjusted by a device where the angle of the entire excitation light irradiation unit is different from that of the other device, the angle of total reflection in the analysis chip changes from that of the other device, and the surface plasmon resonance state changes. Is different from other devices. This phenomenon is the same for the excitation light wavelength.

  However, as in the above cited reference 1, these changes are positive when the excitation light is incident on the test region and the control region by a common incidence method in the sandwich method, and the optical constants of the films on the Au film are substantially equal. In principle, it can be canceled by normalizing with the signal.

JP 2012-122977 A JP 2011-215051 JP 2011-059003 A

  On the other hand, a competitive method is known as a method for detecting an antigen of a low molecular weight compound that cannot be assayed by the sandwich method. As an example of the competition method, in Cited Document 3, an antibody labeled with a fluorescent substance is prepared (in Cited Document 3, this is the first antibody), and the test substance on the substrate itself or the analyte A compound having a site similar to the test substance and having the same epitope as the test substance (for an antibody labeled with a fluorescent substance) is immobilized. When a sample and an antibody labeled with a fluorescent substance are mixed and contacted with a test substance or similar substance fixed on the substrate, the sample is labeled with a fluorescent substance when the sample does not contain the test substance. The antibody binds to the test substance immobilized on the test area on the substrate to generate a fluorescent signal in the test area. When the specimen contains a test substance, the antibody labeled with the fluorescent substance binds to the test substance in the specimen and does not bind to the test substance fixed to the test area on the substrate. Does not generate a fluorescence signal due to plasmon enhancement. Even in the case of the competitive method, the reference antibody can be immobilized on the control region. The functions of the control region and reference antibody are exactly the same as in the sandwich method.

  However, in the case of the above competition method, the test substance itself or the test substance-like substance (203 + 204c) is fixed to the test area as shown in the competition method of FIG. The substance is different from the immunoglobulin of the reference antibody 202, and as shown in the competition method of FIG. 17, the optical constant (refractive index n × film thickness d) when modeled as a dielectric film is also different.

  Even in the sandwich method, as shown in the sandwich method (2) in FIG. 17, when the size of the antigen 204b to be detected is so large that it cannot be ignored, the optical when modeled as a dielectric film is used. It may happen that the constants (refractive index n × film thickness d) are different.

  FIG. 13 is a graph showing the relationship between the excitation light incident angle and the detection signal in the surface plasmon enhanced fluorescence method. As shown in this graph, the optical constants of the dielectric film on the Au film are in the control region and the test region. If different, even if the fluorescence signal in the test area is normalized with the fluorescence signal in the control area, the standardized fluorescence signal is different when the excitation light wavelength and excitation light incident angle of the measurement apparatus differ from measurement apparatus to measurement apparatus due to manufacturing variations. It shifts from device to device. Explaining in detail with the example of FIG. 13, the value (dotted line in the figure) when the fluorescence signal in the test area is normalized with the fluorescence signal in the control area is ± 0 depending on the individual difference of the apparatus with reference to 75.5 °. If there is a difference in incident angle within a range of .5 °, an error of about ± 3% will occur.

  FIG. 14 is a graph showing the difference in individual apparatus depending on the type of test substance, and FIG. 15 is a graph showing the difference in optical constant of the dielectric film on the Au film depending on the type of test substance. The graphs shown are the values when the fluorescence signal in the test area of the reference chip to which the measurement target particles are fixed is normalized with the fluorescence signal in the control area for each of the analysis chips for T4 and TSH in three apparatuses. This is a result of relative comparison between the fluorescence signal in the test area of the analysis chip where the specimen was actually measured and the value obtained by normalizing the fluorescence signal in the control area.

  As shown in FIG. 15, the optical constants of the test area and the control area of T4 are different, and the optical constants of the test area and the control area of TSH are almost the same. When the relationship between the optical constants of the areas and the relationship between the control constants of different test substances and the optical constants of the test areas are different, the appearance of individual device differences differs depending on the test substances.

  In TSH, since the optical constants of the dielectric films in the test area and the control area are almost the same, the standardization as described above should not cause a difference in individual devices in principle. Since there was another error due to other than the optical constants of the body membrane (individual difference in the accuracy of installation of the analysis chip and the angle accuracy of the entire excitation light irradiation unit, etc.), there were some individual differences.

  In order to eliminate such problems of individual differences in the apparatus, it is conceivable that a calibration curve for the fluorescence signal and the test substance is created for each analysis chip dedicated to the apparatus, and attached as information such as a barcode when manufacturing the analysis chip. However, since samples and analysis chips are consumables and it is difficult to supply the same quality, it is difficult to grasp individual differences with high reproducibility, and it is not possible to create a calibration curve for each device. It is cumbersome and not realistic.

  The present invention has been made in view of the above problems, and in a measuring apparatus using surface plasmon resonance, a measuring apparatus capable of performing high-accuracy measurement by correcting individual apparatus differences that differ for each test substance and An object is to provide a measurement method.

  The measurement apparatus of the present invention includes a sensor region in which a substance that binds to or competes with a test substance in a specimen is fixed, and an identification information holding unit that holds identification information related to the type of the test substance, and the sensor area is a surface plasmon. A measurement device that uses an analysis chip configured to generate resonance and excite a fluorescent label, and stores information on individual device differences and / or correction factors calculated based on the difference depending on the test substance. Storage means, information acquisition means for acquiring identification information from the analysis chip, detection means for detecting a binding or competition state in the sensor area, device individual difference information extracted from the storage means in accordance with the identification information, and / or And an arithmetic means for correcting the individual device difference with respect to the result detected by the detecting means based on the correction coefficient.

  In the measurement apparatus of the present invention, it is preferable that the individual apparatus difference information and / or the correction coefficient is acquired using a reusable reference analysis chip in which a liquid having a known refractive index is held on the sensor region. .

  In this case, the reference analysis chip is preferably one in which a liquid having a known refractive index is held on the sensor region of the analysis chip capable of measuring the test substance. The “analysis chip capable of measuring a test substance” means an analysis chip for performing a normal test on the test substance or an analysis chip having a configuration equivalent to this analysis chip.

  In addition, the analysis chip has a first sensor region in which a first substance that binds or competes with a test substance in a sample is fixed, and a second substance that binds to a labeled substance that binds or competes with the test substance. A second sensor region, and the computing means calculates the third signal by normalizing the first signal acquired in the first sensor region with the second signal acquired in the second sensor region, It is preferable to correct individual device differences for the three signals.

  The measurement method of the present invention includes a sensor region in which a substance that binds to or competes with a test substance in a specimen is fixed, and an identification information carrier that carries identification information related to the type of the test substance, and the sensor area is a surface plasmon. A measurement method that uses an analysis chip configured to generate resonance and excite a fluorescent label, and stores information on individual device differences that differ depending on the test substance and / or correction coefficients calculated based on the information. In addition, the identification information is obtained from the analysis chip, the state of binding or competition in the sensor area is detected, and detected by the detection means based on the individual device difference information and / or the correction coefficient extracted according to the identification information. It is characterized in that the individual device difference is corrected for the result.

  In the measurement method of the present invention, the device individual difference information and / or the correction coefficient are preferably acquired using a reusable reference analysis chip in which a liquid having a known refractive index is held on the sensor region. .

  In this case, the reference analysis chip is preferably one in which a liquid having a known refractive index is held on the sensor region of the analysis chip capable of measuring the test substance. The “analysis chip capable of measuring a test substance” means an analysis chip for performing a normal test on the test substance or an analysis chip having a configuration equivalent to this analysis chip.

  In addition, the analysis chip has a first sensor region in which a first substance that binds or competes with a test substance in a sample is fixed, and a second substance that binds to a labeled substance that binds or competes with the test substance. Two sensor regions, the first signal acquired in the first sensor region is normalized with the second signal acquired in the second sensor region to calculate a third signal, and the third signal It is preferable to correct the individual device difference.

  According to the measuring apparatus and the measuring method of the present invention, the correction coefficient of the apparatus individual difference according to the type of the test substance based on the identification information is stored in advance, the identification information is obtained from the analysis chip, and the identification information is Since the individual device difference is corrected with respect to the result detected by the detection means based on the extracted individual device difference information and / or the correction coefficient, the individual device difference that is different for each test substance is corrected. It becomes possible to measure the accuracy.

  Further, if the device individual difference information and / or correction coefficient is acquired using a reusable reference analysis chip in which a liquid having a known refractive index is held on the sensor region, the same reference analysis chip is used. Since individual device differences between a plurality of devices are acquired by using, it is possible to acquire accurate device individual difference information with high reproducibility without being influenced by individual differences between specimens and analysis chips.

  In this case, if the reference analysis chip is a liquid having a known refractive index held on the sensor area of the analysis chip capable of measuring the test substance, accurate apparatus individual difference information corresponding to the actual measurement can be obtained. Since it can be acquired, it becomes possible to correct the apparatus individual difference more accurately.

  In addition, the analysis chip has a first sensor region in which a first substance that binds or competes with a test substance in a sample is fixed, and a second substance that binds to a labeled substance that binds or competes with the test substance. Two sensor regions, the first signal acquired in the first sensor region is normalized with the second signal acquired in the second sensor region to calculate a third signal, and the third signal By correcting the individual device difference, not only the individual device difference but also the refractive index variation for each specimen can be corrected, so that a more accurate result can be obtained.

Schematic configuration diagram of a fluorescence detection apparatus which is an embodiment of the measurement apparatus of the present invention The block diagram which shows preferable embodiment of the said fluorescence detection apparatus The schematic diagram which shows an example of the analysis chip used for the fluorescence detection apparatus of FIG. 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. A graph showing how quantitative or qualitative analysis is performed by the rate method in the data analysis means of FIG. Schematic diagram showing an example of the irradiation position adjusting means of FIG. The schematic diagram which shows a mode that the irradiation position of excitation light moves to the arrow Y direction by the irradiation position adjustment means of FIG. Graph showing an example of the shift amount of the irradiation position and the intensity of the fluorescent signal for adjustment Schematic diagram showing an example of the scanning drive means of FIG. FIG. 8 is a schematic diagram showing how the irradiation position of excitation light moves in the direction of arrow X by the scanning drive means of FIG. Graph showing the relationship between excitation light incident angle and detection signal in surface plasmon enhanced fluorescence Graph showing differences in individual device differences depending on the type of test substance Graph showing the difference in the optical constants of the dielectric film on the Au film depending on the type of test substance Diagram for explaining the principle of sandwich method and competitive method Diagram for explaining multilayer film model in sandwich method and competitive method

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. FIG. 1 is a schematic configuration diagram of a fluorescence detection apparatus 1 which is an embodiment of the measurement apparatus of the present invention. For example, the immunological analysis apparatus uses surface plasmon resonance. When the analysis is performed by the fluorescence detection device 1, the sample container CB containing the sample shown in FIG. 1, the nozzle chip NC used for extracting the sample and the reagent, the reagent cell, and the micro flow channel are formed. Chip 10 is loaded. Note that the sample container CB, the nozzle tip NC, and the analysis chip 10 are all disposable items that are discarded once they are used. Then, the fluorescence detection apparatus 1 performs a quantitative or qualitative analysis on the test substance in the sample while flowing the sample through the microchannel 15 of the analysis 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, an information acquisition unit 90, a storage unit 100, 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 analysis chip 10. The analysis chip 10 corresponds to the sandwich method, and has a structure in which an inlet 12, an outlet 13, sample cells 14a and 14b, and a flow path 15 are formed in a main body 11 made of a light-transmitting resin. ing. The injection port 12 formed on the upper surface of the main body 11 communicates with the discharge port 13 via the flow path 15, and the sample is injected into the injection port by applying negative pressure from the discharge port 13 formed on the upper surface of the main body 11. 12 is injected into the flow path 15 and discharged from the discharge port 13. 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 (first sensor region) TR for detecting a test substance in the specimen and two control regions CR provided on the upstream side and the downstream side of the test region TR are formed in the flow path 15. ing. A first antibody (first substance) is fixed on the test region TR, and the labeled antibody is captured by a so-called sandwich method. In addition, a reference antibody (second substance) 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. A so-called negative control region CR for detecting non-specific adsorption is arranged upstream of the test region TR, and a so-called positive control region (second sensor region) for detecting a difference in reactivity due to a difference in specimen. ) CR is arranged downstream of the test area TR.

  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.

  Furthermore, on the lower surface of the main body 11 of the analysis chip 10, a barcode (not shown) is printed as an identification information carrier that carries identification information relating to the type of the test substance. The barcode may be a one-dimensional barcode or a two-dimensional barcode.

  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 irradiates the dielectric plate 17 and the metal film 16 in the test region TR through the prism with the excitation light L from the back surface side of the analysis chip 10 at an incident angle that is a total reflection condition. . The fluorescence detection means 40 is made of, for example, a photodiode, CCD, CMOS, etc., and detects fluorescence generated from the test region TR by the irradiation of the excitation light L of the light irradiation means 30 as a fluorescence signal FS.

  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 bound fluorescent labeling substance F is excited by the evanescent wave Ew and generates enhanced fluorescence.

  The data analysis means 50 in FIG. 2 analyzes the test substance based on the temporal change of the fluorescence signal FS detected by the fluorescence detection means 40. Specifically, since the fluorescence intensity changes depending on the amount of the fluorescent labeling substance F bound thereto, the fluorescence intensity changes with time as shown in FIG. The data analysis means 50 acquires a plurality of fluorescent signals FS at a predetermined sampling period (for example, a period of 5 seconds) in a predetermined period (for example, 5 minutes), and analyzes the change rate of the fluorescence intensity over time, thereby analyzing the test within the sample. Perform quantitative analysis of substances (rate method). The analysis result is output from the information output means 4 comprising a monitor, a printer or the like.

  Here, the fluorescence detection apparatus 1 has a function of adjusting the irradiation position RR of the excitation light L in accordance with individual differences of the fluorescence detection apparatus 1 before performing the above-described analysis of the test substance. More specifically, the angle of the entire light irradiation means 30 may be shifted due to component accuracy or assembly accuracy for each individual fluorescence detection device 1. In such a case, the test region TR or The irradiation area of the excitation light L with respect to the control region CR is different. Since the electric field enhancement field (evanescent wave) described above changes in intensity according to the irradiation amount of the excitation light, the intensity of the electric field enhancement field and the accompanying fluorescence amount differ depending on the irradiation area. As a result, there is a problem that the fluorescence intensity varies depending on individual differences of the fluorescence detection device 1 and the analysis accuracy is deteriorated. Therefore, the fluorescence detection device 1 has a function of automatically adjusting the irradiation position of the excitation light L according to individual device differences using an adjustment chip at the time of manufacture.

  As this adjustment chip, an adjustment chip having the same material and shape as the main body 11 of the analysis chip 10 is used. In the adjustment chip, the region including the portion corresponding to the sensor region of the analysis chip 10 is made into a glass shape by blasting or white paint is applied, and the incident excitation light is scattered and the incident light position can be known. ing.

  As shown in FIG. 2, the fluorescence detection apparatus 1 includes an irradiation position adjusting unit 60, an irradiation position control unit 70, and a scanning driving unit 80. The irradiation position adjusting unit 60 adjusts the irradiation position RR of the excitation light L with respect to the test region TR by the light irradiation unit 30. FIG. 8 is a schematic diagram showing an example of the light irradiation means 30 and the irradiation position adjustment means 60. In FIG. 8, the light irradiation means 30 includes a light source 31, an optical system 32 including a concave lens 32a and a convex lens 32b, and a diaphragm 33. Then, the excitation light L emitted from the light source 31 is condensed by the optical system 32 and is incident on the prism via the diaphragm 34. The irradiation position adjusting means 60 irradiates the excitation light L as shown in FIG. 9 by moving the optical system 32 in a direction substantially perpendicular to the optical axis (direction of arrow α) and substantially parallel to the incident surface of the excitation light L. The position RR is adjusted in a direction (arrow Y direction) that is substantially parallel to the surface of the test region TR and substantially parallel to the incident surface of the excitation light L. In addition, although the optical system 32 has illustrated about the case where the excitation light L is condensed, you may make it parallel light and a spreading | diffusion light.

  The irradiation position control means 70 in FIG. 2 drives the irradiation position adjustment means 60 to irradiate the excitation light L while shifting the irradiation position RR with respect to the test region TR. Based on the adjustment fluorescence signal AFS, the irradiation position RR of the excitation light L with respect to the test region TR when the test substance is analyzed is determined. Specifically, the irradiation position control means 70 controls the irradiation position adjustment means 60 so that the irradiation position RR of the excitation light L is scanned within a 1.5 mm scanning range while being shifted by 0.1 mm, for example, in the arrow Y direction. To do. Then, as shown in FIG. 10, the fluorescence detection means 40 detects a plurality of adjustment fluorescence signals AFS for different irradiation positions RR. In FIG. 10, the shift amount 0 indicates a preset initial position.

  Then, the irradiation position control means 70 detects the largest adjustment fluorescence signal AFSmax among the plurality of adjustment fluorescence signals AFS. Then, the irradiation position control means 70 determines that the irradiation position RR from which the maximum adjustment fluorescence signal AFSmax has been acquired is the irradiation position RR optimal for analysis, and the irradiation position so that the above analysis is performed at the irradiation position RR. The adjusting means 60 is controlled. Alternatively, the irradiation position control unit 70 may control the irradiation position adjustment unit 60 to perform the above analysis at the irradiation position RR at the center of the scanning range that exceeds 50% of the largest adjustment fluorescent signal AFSmax. By performing the adjustment operation of the irradiation position RR at the time of manufacturing the fluorescence detection device 1, it is possible to eliminate the angle shift of the entire light irradiation means 30 for each individual fluorescence detection device 1.

  Incidentally, as described above, the analysis chip 10 is provided with two control regions CR in addition to the test region TR, and fluorescence detection is also performed on the control region CR. Irradiation of the excitation light L to the test region TR and the control region CR is performed by causing the light irradiation means 30 to scan. FIG. 11 is a schematic diagram showing an example of the scanning drive unit 80. The scanning drive unit 80 moves the light irradiation unit 30 along the flow path formation direction (arrow X direction), and reciprocates between the test region TR and the control region CR, for example, at a cycle of 5 seconds. .

  Here, due to individual differences of the fluorescence detection device 1, there may be a positional shift not only in the Y direction but also in the X direction. For this reason, the irradiation position control means 70 also adjusts the irradiation position RR using the scanning drive means 80 also in the X direction. Specifically, as shown in FIG. 12, the irradiation position control means 70 drives the scanning drive means 80 to irradiate the test region TR with the excitation light L while shifting the irradiation position RR. Then, the fluorescence detection means 40 detects a plurality of adjustment fluorescence signals AFS for each irradiation position RR. The irradiation position control means 70 determines the irradiation position of the excitation light L in the X direction using the same method as the irradiation position determination method in the arrow Y direction described above.

  In the X direction, the scanning drive unit 80 is used to adjust the irradiation position RR, and the Y direction is adjusted using the irradiation position adjusting unit 60. However, the X direction is also applied to the irradiation position adjusting unit. You may make it adjust using 60. At this time, the irradiation position adjusting means 60 adjusts the irradiation position RR by moving the optical system 32 in the arrow α direction and the direction orthogonal thereto. In addition, although the case where the irradiation position RR is adjusted for the test region TR is illustrated in FIG. 8, the irradiation position RR is similarly adjusted for the control region CR. Furthermore, although the case where the light irradiation means 30 is scanned in the direction of the arrow X is illustrated, the fluorescence detection means 40 may be moved in synchronization with the movement of the light irradiation means 30, or the test region TR and the control. A light detection unit may be provided for each region CR.

Next, a preferred embodiment of the fluorescence detection method of the present invention will be described.
As a preliminary stage, first, for each type of test substance expected to be used in the fluorescence detection apparatus 1, a test area TR in which the test substance is fixed and a control area CR in which the test substance is not fixed are provided. A reference analysis chip is prepared in which a liquid or solid having a known refractive index not containing a labeling substance is held on the region TR and the control region CR.

  Information on the ratio of the surface plasmon resonance amounts of the test region TR and the control region CR is acquired by measuring the reference analysis chip for each type of test substance in a predetermined reference fluorescence detection device.

  Next, by measuring the reference analysis chip for each type of test substance in the fluorescence detection device 1, information on the ratio of the surface plasmon resonance amounts of the test region TR and the control region CR is obtained, and this fluorescence detection device 1 2 is acquired and stored as a correction coefficient in the storage means 100 shown in FIG. 2.

  At the time of actual measurement, the fluorescence detection device 1 is first loaded with the analysis chip 10, the sample container CB, and the nozzle chip NB. Then, the barcode printed on the lower surface of the analysis chip 10 is read by the information acquisition means 90 (specifically, a barcode reader), and identification information regarding the type of the test substance on the analysis chip 10 is acquired.

  Next, a sample solution in which the sample and the reagent are mixed is generated by the sample processing means 20, and this sample solution is flowed down to the flow path of the analysis chip 10, and has a strength necessary for adjustment to the test region TR and the control region CR. Wait for an appropriate time for fluorescence to occur.

  Next, irradiation with excitation light L for quantitative analysis of the test substance is performed. At this time, the light irradiation means 30 scans in the direction of the arrow X, so that the test region TR and the control region CR are irradiated with the excitation light L. Then, as shown in FIG. 7, the above-described acquisition of the fluorescence signal FS is performed, for example, at a predetermined sampling period in a predetermined period.

  Thereafter, quantitative analysis is performed by the rate method in the data analysis means (detection means, calculation means) 50, and the measurement result in the test area (first sensor area) TR is the first signal, positive control area (second sensor area). The measurement result in CR is detected as the second signal. Next, the third signal is calculated by normalizing the first signal with the second signal. As the normalization process here, any technique such as dividing the first signal by the second signal may be used. Further, the correction coefficient extracted from the storage unit 100 according to the identification information acquired in the initial measurement stage is calculated as the third signal to correct the individual difference of the fluorescence detection device 1 and obtain the final result. As the correction processing here, any method such as multiplying the third signal by a correction coefficient may be used. The analysis result finally obtained is printed out or displayed on the screen.

  By setting it as such an aspect, it becomes possible to correct | amend the apparatus individual difference which changes for every test substance, and to perform a highly accurate measurement.

  The preferred embodiments of the present invention have been described above. However, the present invention is not limited to the above-described embodiments. For example, the assay method of the present invention detects the presence / absence of a substance to be detected and detects the target. It is intended to be interpreted as the broadest concept, including measurement (ie, quantification) of the amount of a substance, and so on, and includes any known immunological measurement method without limitation, and is described in the above embodiment. In addition to the sandwich method, various methods such as a competition method are included.

  In addition, although the embodiment has been described with respect to the rate method in which the fluorescence signal changes with time depending on the reaction, the analysis chip based on the endpoint method that measures the steady fluorescence signal after stopping the reaction by washing is also adjusted by the same adjustment. It is possible to prevent degradation of analysis accuracy.

  In addition, the identification information carrier that carries identification information relating to the type of test substance is not limited to a barcode, and an IC chip or the like that can read information from the outside may be attached to the analysis chip.

  Further, the method for measuring the first sensor region and the second sensor region is not limited to the so-called surface plasmon enhanced fluorescence method as in the above embodiment, but is based on the surface plasmon resonance state of each sensor region. May be detected.

  In addition to the above, it goes without saying that various improvements and modifications may be made without departing from the scope of the present invention.

DESCRIPTION OF SYMBOLS 1 Fluorescence detection apparatus 10 Analysis chip 20 Sample processing means 30 Light irradiation means 40 Fluorescence detection means 50 Irradiation position adjustment means 50 Data analysis means 60 Irradiation position adjustment means 70 Irradiation position control means 80 Scan driving means 90 Information acquisition means 100 Storage means AFS Fluorescence signal CR for adjustment Control area FS Fluorescence signal L Excitation light RR Irradiation position TR Test area

Claims (8)

A sensor region in which a substance that binds to or competes with a test substance in a sample is fixed; and an identification information carrier that carries identification information related to the type of the test substance. The sensor area causes surface plasmon resonance to generate fluorescence. A measuring device using an analysis chip configured to excite a sex label,
Storage means for storing device individual difference information which varies depending on the test substance and / or a correction coefficient calculated based on the information,
Information acquisition means for acquiring the identification information from the analysis chip;
Detecting means for detecting a state of binding or competition in the sensor region;
Computation means for correcting device individual differences with respect to the result detected by the detection means based on the device individual difference information and / or the correction coefficient extracted from the storage means according to the identification information. A measuring device.   The apparatus individual difference information and / or the correction coefficient are acquired using a reusable reference analysis chip in which a liquid having a known refractive index is held on the sensor region. 1. The measuring device according to 1.   3. The measuring apparatus according to claim 2, wherein the reference analysis chip is a liquid having a known refractive index held on the sensor region of the analysis chip capable of measuring a test substance. The analysis chip fixes a first sensor region to which a first substance that binds or competes with a test substance in a sample, and a second sensor that fixes a second substance that binds to a labeled substance that binds or competes with the test substance. Sensor area,
The calculation means normalizes the first signal acquired in the first sensor region with the second signal acquired in the second sensor region to calculate a third signal, and an individual device for the third signal The measuring apparatus according to claim 1, wherein the difference is corrected. A sensor region in which a substance that binds to or competes with a test substance in a sample is fixed; and an identification information carrier that carries identification information related to the type of the test substance. The sensor area causes surface plasmon resonance to generate fluorescence. A measurement method using an analysis chip configured to excite a sex label,
Store the device individual difference information and / or the correction coefficient calculated based on it depending on the test substance,
Obtaining the identification information from the analysis chip;
Detecting binding or competition conditions in the sensor region;
A device individual difference is corrected with respect to a detection result of a binding or competition state in the sensor region based on the device individual difference information and / or the correction coefficient extracted according to the identification information. Method.   The apparatus individual difference information and / or the correction coefficient are acquired using a reusable reference analysis chip in which a liquid having a known refractive index is held on the sensor region. 5. The measuring method according to 5.   The measurement method according to claim 6, wherein the reference analysis chip is a liquid having a known refractive index held on the sensor region of the analysis chip capable of measuring a test substance. The analysis chip fixes a first sensor region to which a first substance that binds or competes with a test substance in a sample, and a second sensor that fixes a second substance that binds to a labeled substance that binds or competes with the test substance. Sensor area,
Normalizing the first signal acquired in the first sensor region with the second signal acquired in the second sensor region to calculate a third signal, and correcting individual differences with respect to the third signal The measurement method according to claim 5, wherein:

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