JP6788565B2 - Fluorescence detection device using surface plasmon resonance and its operation method - Google Patents

Description

The present invention relates to a fluorescence detection device using surface plasmon resonance and a method of operating the same.

A fluorescence detection device using a fluorescence method is known in biomeasurement and the like (see, for example, Patent Document 1). The fluorescence method is a method of irradiating a fluorescent label bound to a test substance with excitation light, detecting fluorescence generated from the fluorescent label, and analyzing the test substance. The fluorescence detection device described in Patent Document 1 enhances the fluorescence emitted by a fluorescent label by utilizing a surface plasmon resonance phenomenon to analyze a test substance. Such a fluorescence detection device is also called a measurement system using surface plasmon resonance excitation enhanced fluorescence spectroscopy (SPFS) (SPFS: Surface Plasmon field-enhanced Fluorescence Spectroscopy).

In such a fluorescence detection device, an analysis chip provided with a flow path through which a sample solution containing a test substance flows is used. The analysis chip is provided with a flow path through which the sample solution containing the test substance flows, a reaction region in which the antibody that specifically reacts with the test substance is fixed and the sample solution comes into contact with the reaction region, and a reaction region. It has a metal film to be formed and a main surface in contact with the back surface on the opposite side to the front surface having a reaction region in the metal film, and has a dielectric material transparent to excitation light. An injection port for injecting the sample solution is provided at one end of the flow path.

The excitation light is incident from the back side of the main surface through the dielectric. In order to enhance the fluorescence by utilizing the surface plasmon resonance, it is necessary to adjust the incident angle of the excitation light to the resonance angle at which the surface plasmon resonance occurs and the fluorescence is maximized. In Patent Document 1, an incident angle adjusting mechanism for adjusting the incident angle of the excitation light is provided, and the incident angle is changed by the incident angle adjusting mechanism to specify the resonance angle at which the fluorescence is maximized.

WO2012 / 108323

However, in the fluorescence detection device as described in Patent Document 1, even when the resonance angle is specified, if the specified resonance angle is not within the permissible range, some abnormality may occur. Conceivable. As a cause of the abnormality, for example, improper mounting of the analysis chip can be considered. When such an abnormality occurs, it may be determined that some abnormality has occurred by examining the measurement result after the measurement is completed. However, in such a case, re-examination is often required, and the user has requested a means to detect the abnormality at an early stage without looking at the measurement result.

An object of the present invention is to provide a fluorescence detection device using surface plasmon resonance and a method of operating the same, which can notify the user of improper mounting of an analysis chip at an early stage.

The fluorescence detection device using the surface plasmon resonance of the present invention has a flow path through which the sample solution containing the test substance flows and is arranged in the flow path, and an antibody that specifically reacts with the test substance is fixed to prepare the sample solution. It has a contact reaction region, a metal film provided with a reaction region, and a main surface in contact with the back surface opposite to the surface of the metal film where the reaction region is located, and has a dielectric material transparent to excitation light. Fluorescence using surface plasmon resonance, in which excitation light is incident from the back side of the main surface through a dielectric using an analysis chip and the fluorescence generated from the fluorescence label bonded to the test substance due to the incidence of the excitation light is detected. The detection device includes a light irradiation unit, a fluorescence detection unit, an incident angle adjusting mechanism, a resonance angle specifying unit, a determination unit, and a warning unit. The light irradiation unit irradiates the excitation light. The fluorescence detection unit detects fluorescence. The incident angle adjusting mechanism adjusts the incident angle of the excitation light with respect to the main surface. The resonance angle specifying unit changes the incident angle by the incident angle adjusting mechanism to specify the resonance angle at which the fluorescence detected by the fluorescence detecting unit is maximized. The determination unit determines whether or not the resonance angle specified by the resonance angle specifying unit exists within a preset allowable range. The warning unit warns of an abnormality when the determination unit determines that the resonance angle does not exist within the allowable range.

The abnormality is preferably an improper mounting of the analysis chip or an abnormality in the dilution ratio of the sample solution.

The resonance angle is preferably determined in a state where the sample solution is injected into the flow path.

It is preferable that the determination unit makes a determination based on the determination data prepared according to the type of the sample contained in the sample solution.

It is preferable that the resonance angle specifying unit acquires a fluorescence detection signal according to the amount of fluorescence detected by the fluorescence detecting unit, and determines whether or not the resonance angle is within an allowable range based on the maximum value of the fluorescence detection signal.

It is equipped with a reflected light detection unit that detects the reflected light of the excitation light reflected by the metal film, and the resonance angle identification unit resonates based on the reflected light detection signal according to the amount of reflected light detected by the reflected light detection unit. It is preferable to identify the corners.

The method of operating the fluorescence detection device using surface plasmon resonance is that the sample solution is arranged in the flow path through which the sample solution containing the test substance flows and the antibody that specifically reacts with the test substance is fixed. It has a contact reaction region, a metal film provided with a reaction region, and a main surface in contact with the back surface opposite to the surface of the metal film where the reaction region is located, and has a dielectric material transparent to excitation light. Fluorescence using surface plasmon resonance, in which excitation light is incident from the back side of the main surface through a dielectric using an analysis chip and the fluorescence generated from the fluorescence label bonded to the test substance due to the incidence of the excitation light is detected. The method of operating the detection device includes a light irradiation step, a fluorescence detection step, a resonance angle specifying step, a determination step, and a warning step. The light irradiation step irradiates the excitation light. The fluorescence detection step detects fluorescence. In the resonance angle specifying step, the resonance angle at which the fluorescence detected in the fluorescence detection step is maximized is specified while changing the incident angle of the excitation light with respect to the main surface. The determination step determines whether or not the resonance angle specified in the resonance angle specifying step exists within a preset allowable range. The warning step warns of an abnormality when it is determined in the determination step that the resonance angle does not exist within the permissible range.

According to the present invention, it is possible to notify the user at an early stage of improper mounting of the analysis chip.

It is an external view of the fluorescence detection apparatus of this invention. It is a block diagram of a fluorescence detection device. It is a schematic diagram which shows an example of the analysis chip used for a fluorescence detection apparatus. It is a schematic diagram which shows a mode that a sample is extracted from a sample container by a sample processing part using a nozzle tip. It is a schematic diagram which shows how the sample in a nozzle tip is injected into a reagent cell and agitated by a sample processing part. It is explanatory drawing which shows the state of the flow path in the state which the sample solution was injected. It is explanatory drawing which shows the positional relationship between a flow path and a measuring part, and the moving method of a measuring part. It is explanatory drawing explaining the outline of fluorescence detection using a measuring part. It is explanatory drawing of the incident angle of the excitation light and the plasmon intensification when the sample is serum. It is explanatory drawing of the incident angle of the excitation light and the plasmon intensification when the sample is plasma. It is explanatory drawing of the incident angle of the excitation light and the plasmon intensification when the sample is whole blood (HCT = 36%). It is explanatory drawing of the incident angle of the excitation light and the plasmon intensification when the sample is whole blood (HCT = 40%). It is explanatory drawing of the incident angle of the excitation light and the plasmon intensification when the sample is whole blood (HCT = 48%). It is explanatory drawing which shows the outline of the processing of abnormality determination of a resonance angle. It is explanatory drawing of the determination data. It is a flowchart which shows the procedure of a measurement process. It is explanatory drawing of the aspect which specifies the resonance angle based on the reflected light. It is explanatory drawing which shows the relationship between fluorescence and reflected light of excitation light. It is explanatory drawing of the judgment data in consideration of the dilution ratio. It is a graph which shows the measurement example of TSH concentration. It is a graph of the calibration curve obtained from the graph of FIG. It is a table which shows the appropriate range of TSH concentration. It is a table which shows the angle which the plasmon increasing strength shifts by 2.2%.

[First Embodiment]
The fluorescence detection device 100 shown in FIG. 1 is a fluorescence detection device 100 that utilizes surface plasmon resonance, and more specifically, it is a device used for analysis including an inspection of a biological substance by an antigen-antibody reaction or the like.

When the analysis is performed using the fluorescence detection device 100, a sample container CB containing the sample shown in FIG. 1, a nozzle tip NC used for extracting the sample and the reagent, a reagent cell and a microchannel are formed. The analysis chip 10 is set in the fluorescence detection device 100. The sample container CB, the nozzle tip NC, and the analysis tip 10 are all disposable items that are discarded once they are used. Then, the fluorescence detection device 100 injects the sample into the flow path 15 of the analysis chip 10 to perform quantitative or qualitative analysis on the test substance in the sample.

The sample is, for example, blood, more specifically serum, plasma, or whole blood. The sample may be other than blood, and may be urine, nostril fluid, saliva, stool, body cavity fluid, or the like. Examples of the test substance contained in the sample include nucleic acids, proteins, amino acids, sugars, lipids, modified molecules thereof, and complexes thereof. The complex may be, for example, a tumor marker, a signal transduction substance, a hormone, or the like.

As shown in FIG. 2, the fluorescence detection device 100 includes a mounting unit 101, a sample processing unit 20, a measuring unit 30, a control unit 40, and the like. The analysis chip 10 is mounted on the mounting portion 101. The sample processing unit 20 extracts a sample from the sample container CB (see FIG. 1) using the nozzle tip NC, and mixes the extracted sample with the reagent to generate a sample solution. Further, the sample processing unit 20 injects the generated sample solution into the analysis chip 10.

Specifically, the sample processing unit 20 includes a nozzle moving mechanism, a pump, and the like. The nozzle movement mechanism is a mechanism for moving the nozzle tip NC in the vertical direction and the horizontal direction. The pump is connected to the nozzle tip NC via a pipe to discharge and suck a liquid such as a sample.

The measuring unit 30 measures the concentration of the test substance in the sample by measuring the reaction status of the test substance in the sample on the analysis chip 10. The measuring unit 30 includes a light irradiation unit 31, an incident angle adjusting mechanism 33, a fluorescence detection unit 32, and the like.

The light irradiation unit 31 irradiates the analysis chip 10 with the excitation light L. The light irradiation unit 31 irradiates the excitation light L. The incident angle adjusting mechanism 33 adjusts the incident angle of the excitation light L to irradiate the analysis chip 10. The fluorescence detection unit 32 detects the fluorescence emitted by the fluorescence label excited by the excitation light L in the analysis chip 10 and outputs a fluorescence detection signal to the control unit 40. The fluorescence detection unit 32 includes a photodiode, a photomultiplier, a CCD (Charge Coupled Device) image sensor, a CMOS (Complementary Metal Oxide Semiconductor Image Sensor) image sensor, and the like.

The measuring unit moving mechanism 36 is a moving mechanism that moves the measuring unit 30. As will be described later, the analysis chip 10 is provided with a plurality of regions to be measured, and the measurement unit moving mechanism 36 analyzes the measurement unit 30 so that the plurality of regions of the analysis chip 10 can be measured. Move relative to chip 10.

The control unit 40 comprehensively controls each unit of the fluorescence detection device 100. An operation unit 51 and a display unit 52 are connected to the control unit 40. Further, the control unit 40 has a built-in timer for performing various timekeeping. The operation unit 51 is composed of buttons, cross keys, and the like, and inputs an operation instruction such as a measurement start instruction to the control unit 40. In addition, input of patient information related to the sample is also performed through the operation unit 51. The display unit 52 is composed of, for example, a liquid crystal panel or the like, and displays messages such as a measurement result, a status indicating an operating state, and a warning.

The control unit 40 controls the sample processing unit 20 in accordance with the measurement start instruction from the operation unit 51 to inject the sample solution into the analysis chip 10. Then, the measuring unit moving mechanism 36 and the measuring unit 30 are operated to perform measurement. In the measurement, the control unit 40 performs data analysis based on the fluorescence detection signal acquired from the fluorescence detection unit 32, and outputs the measurement result including the analysis result to the display unit 52.

FIG. 3 is a schematic view showing an example of the analysis chip 10. The analysis chip 10 has a structure in which an injection port 12, a discharge port 13, sample cells 14A and 14B, and a flow path 15 are formed in a main body 11 formed of a dielectric material such as a light-transmitting resin. The injection port 12 communicates with the discharge port 13 via the flow path 15. The nozzle tip NC is inserted into the injection port 12. The sample processing unit 20 injects the sample solution into the flow path 15 via the nozzle tip NC. The sample cells 14A and 14B are containers for containing the fluorescent reagent to be mixed with the sample in the sample container CB. The fluorescent reagent undergoes pretreatment such as adsorbing to a protein in the sample to dissociate the target, for example, for pH adjustment. The openings of the sample cells 14A and 14B are sealed by a sealing member, and the sealing member is perforated when the sample and the fluorescent reagent are mixed.

Further, a reaction region 16 for detecting the test substance in the sample is provided in the flow path 15. A test region TR and a control region CR are formed in the reaction region 16. In the flow path 15, when the side where the injection port 12 is located is the upstream side of the reaction region 16, the control region CR is provided on the downstream side of the test region TR.

The first antibody is immobilized on the test region TR, and the antibody labeled by the so-called sandwich method is captured. Further, the reference antibody is fixed in the control region CR, and the reference antibody captures the fluorescent substance by flowing the sample solution on the control region CR. Two control regions CR are formed, a so-called negative type control region CR for detecting non-specific adsorption and a so-called positive control region CR for detecting a difference in reactivity due to a sample difference. Is formed.

Then, when the measurement start is instructed, the sample processing unit 20 sucks the sample from the sample container CB using the nozzle tip NC as shown in FIG. After that, the sample processing unit 20 punches 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 also performed for the sample cell 14B. The reagent is a second antibody B2 labeled with a fluorescent label F. The second antibody B2 specifically binds to the test substance (antigen) A present in the sample. Therefore, by mixing and stirring the sample and the reagent, the second antibody B2 and the fluorescent label F are attached to the surface of the test substance (antigen) A by binding between the second antibody B2 and the test substance (antigen) A. A modified sample solution is produced.

Then, as shown in FIG. 6, the sample processing unit 20 inserts the nozzle tip NC containing the sample solution SL into the injection port 12. Then, by operating the pump 22 to perform a discharge operation of discharging the sample solution SL from the nozzle tip NC, the sample solution SL in the nozzle tip NC is injected into the flow path 15. As described above, the atmosphere may be sucked from the discharge port 13 in synchronization with the discharge operation from the injection port 12. In this way, the sample solution SL can be injected more smoothly into the flow path 15. Further, after the sample solution SL is injected into the flow path 15, the pump 22 repeats discharge and suction to reciprocate the sample solution SL in the first direction and the second direction. By reciprocating the sample solution SL, specific binding between the test substance and the antibody can be efficiently performed even when the amount of the sample is small.

Although the case where the sample processing unit 20 supplies the sample solution SL in which the sample and the reagent are mixed into the flow path 15 is illustrated, the sample processing unit 20 is filled with the reagent in advance in the flow path 15. May allow only the sample to flow in through the inlet 12.

FIG. 7 is an explanatory diagram showing a test region TR and a control region CR of the analysis chip 10 and a state in which the measurement unit 30 moves with respect to the analysis chip 10. The test region TR and the two control regions CR are arranged along the flow direction (X direction) of the sample solution SL in the flow path 15. The main body 11 is provided with a prism 11A having an incident surface on which the excitation light L is incident, corresponding to the test region TR and the two control regions CR.

In the measuring unit 30, the light irradiation unit 31 is arranged at a position facing the incident surface of the prism 11A of the analysis chip 10 when the analysis chip 10 is mounted on the mounting unit 101. On the other hand, the fluorescence detection unit 32 is arranged above the flow path 15 of the analysis chip 10 at a position facing the test region TR and the control region CR, and is arranged at a position where fluorescence from each region TR and CR can be detected. ing.

The measuring unit moving mechanism 36 linearly moves the light irradiation unit 31 and the fluorescence detecting unit 32 along the flow direction (X direction) of the flow path 15, that is, the arrangement direction of the test region TR and the control region CR. As a result, the measuring unit 30 can selectively move to a position facing each of the test region TR and the two control regions CR, and measure the reaction status of each region.

FIG. 8 is an explanatory view of the relationship between the reaction region 16 of the analysis chip 10 and the light irradiation unit 31 and the fluorescence detection unit 32 as viewed from the X direction. Although the test region TR will be described in FIG. 8, the same applies to the control region CR. The main body 11 of the analysis chip 10 has a dielectric plate 17. In the dielectric plate 17, the front surface 17A constitutes the bottom surface of the flow path 15, and the prism 11A is provided on the back surface 17B. A metal film 18 constituting the test region TR and the control region CR is formed. The material of the metal film 18 is gold in this example. The dielectric plate 17 and the prism 11A are integrally molded, and the prism 11A is also a dielectric.

In the dielectric plate 17, the front surface 17A corresponds to the main surface of the metal film 18 in contact with the back surface opposite to the front surface on which the test region TR corresponding to the reaction region is provided.

The light irradiation unit 31 causes the excitation light L to enter the surface 17A from the back side of the front surface 17A of the dielectric plate 17 in contact with the back surface of the metal film 18 via the prism 11A. The incident angle θ of the optical axis with respect to the surface 17A is an angle equal to or higher than the critical angle that satisfies the total reflection condition. As a result, the excitation light L is applied to the back surface of the metal film 18 in the test region TR and the control region CR. The light irradiation unit 31 is composed of, for example, an LD (Laser Diode) which is a light source that emits excitation light L, a reflection mirror that reflects the excitation light L, and the like. The reflection mirror can be rotationally moved, and the light irradiation unit 31 can change the incident angle θ of the excitation light L by rotationally moving the reflection mirror. The incident angle adjusting mechanism 33 adjusts the incident angle of the excitation light L without changing the irradiation position of the excitation light L on the back surface of the metal film 18 by rotating and moving the reflection mirror by using a lens in combination.

When the excitation light L is incident on the back surface of the metal film 18 at a specific incident angle equal to or higher than the critical angle by the light irradiation unit 31, the evanescent wave Ew exudes onto the metal film 18, and the metal is generated by the evanescent wave Ew. Surface plasmons are excited on the surface of the film 18. This surface plasmon creates an electric field distribution on the surface of the metal film 18, and an electric field enhancement region is formed. Then, the fluorescent label F bound to the first antibody B1 fixed on the metal film 18 is excited by the evanescent wave Ew to generate enhanced fluorescent Lf. The fluorescence detection unit 32 receives the enhanced fluorescence Lf and outputs a fluorescence detection signal according to the amount of light of the received fluorescence Lf.

Here, a specific incident angle θ at which surface plasmon resonance occurs and the enhanced fluorescence Lf is maximized is called a resonance angle. The resonance angle changes depending on the type of the sample solution SL that comes into contact with the surface of the metal film 18. More specifically, the resonance angle changes according to the refractive index of the sample solution SL. Since the resonance angle changes for each type of the sample solution SL, the incident angle θ of the excitation light L is adjusted by the incident angle adjusting mechanism 33.

9 to 13 are graphs showing the relationship between the plasmon increasing intensity of fluorescent Lf and the incident angle θ of various samples of serum, plasma, and whole blood. The profiles of FIGS. 9 to 13 are examples in the case where the wavelength of the excitation light L is 658 nm, the thickness of the metal film 18 is 36 nm, the material of the metal film 18 is gold, and the material of the prism 11A is PMMA (polymethylcrylic). .. Here, the plasmon increasing intensity is an index indicating how many times the amount of light of the fluorescent Lf after the enhancement is multiplied by the reference value, using the amount of light of the fluorescence Lf when there is no enhancement as a reference value. The plasmon enhancement has a proportional relationship with the amount of light of the fluorescence Lf detected by the fluorescence detection unit 32, and even if the vertical axis is the amount of light of the fluorescence Lf in FIGS. 9 to 13, the relationship with the incident angle θ is the same profile. It becomes.

11 to 13 are graphs of whole blood, but each graph has a different value of hematocrit (HCT), which is the ratio of blood cells in blood. The average value of HCT varies from person to person, for example, it differs between men and women. Even in the same whole blood, the resonance angle changes depending on the HCT value.

In each graph, in addition to the curve of plasmon intensification of fluorescence Lf shown by the solid line, the curve showing the relationship between the reflectance of the reflected light RL of the excitation light L reflected by the metal film 18 and the incident angle θ is shown by a broken line. ing. Since the excitation light L consumes energy for plasmon enhancement, the reflected light RL of the excitation light L is greatly attenuated near the resonance angle, and the reflectance shows a minimum value, contrary to the plasmon enhancement of the fluorescence Lf.

In each of the graphs of FIGS. 9 to 13, the incident angle θ at which the plasmon intensification of the fluorescence Lf shows a peak value and becomes maximum is specified as a resonance angle. In each graph, the resonance angle is indicated by a broken straight line extending in the vertical direction. The resonance angle of the serum shown in FIG. 9 is 72.4 °, and the resonance angle of the plasma shown in FIG. 10 is 73.6 °. The resonance angle of whole blood (HCT = 36%) shown in FIG. 11 is 76.86 °, and the resonance angle of whole blood (HCT = 40%) shown in FIG. 12 is 79.2 °, which is shown in FIG. The resonance angle of whole blood (HCT = 80.82 °) is 80.82 °.

By adjusting the incident angle, the resonance angle at which the plasmon increasing intensity of the fluorescence Lf shows the maximum value is specified as shown in the graphs of FIGS. 9 to 13.

In FIG. 8, the control unit 40 has a resonance angle specifying unit 41, a determination unit 42, and a memory 43. The control unit 40 controls the incident angle adjusting mechanism 33 to change the incident angle of the excitation light L irradiated by the light irradiation unit 31 to specify the resonance angle at which the fluorescence Lf detected by the fluorescence detection unit 32 is maximized. To do. The determination unit 42 determines whether or not the resonance angle specified by the resonance angle specifying unit 41 exists within a preset allowable range. Judgment data 61 for use in determination is stored in the memory 43. The control unit 40 functions as a warning unit that warns of an abnormality when the determination unit 42 determines that the resonance angle does not exist within the allowable range.

More specifically, in adjusting the incident angle, the control unit 40 performs the process as shown in FIG. When the excitation light L is changed by the incident angle adjusting mechanism 33, a fluorescence detection signal corresponding to each incident angle is input from the fluorescence detection unit 32 to the resonance angle specifying unit 41. The resonance angle specifying unit 41 generates an actual measurement profile based on the fluorescence detection signal corresponding to each incident angle. This actually measured profile is similar to the profile showing the relationship between the plasmon intensification and the incident angle θ as shown in FIGS. 9 to 13. By generating such an actual measurement profile, the resonance angle at which the plasmon intensification is maximized and the actual measurement value of the maximum value are specified. The data of such an actual measurement profile is input to the determination unit 42.

When specifying the resonance angle, for example, if the mounting posture of the analysis chip 10 deviates significantly from the proper posture, the peak value does not exist in the actually measured profile, and the resonance angle may not be specified. In this case, the resonance angle specifying unit 41 outputs to that effect to the determination unit 42.

On the other hand, the determination data 61 as shown in FIG. 15 is stored in the memory 43. The determination data 61 is theoretical profile data showing the relationship between the plasmon intensification and the incident angle θ shown in FIGS. 9 to 13. This profile data includes data on the resonance angle and the maximum value shown in FIGS. 9 to 13. The profile data 1 is serum data, and the profile data 2 is plasma data. Profile data 31 to 32 are whole blood data. Further, the determination data 61 includes data indicating a preset allowable range RG as an allowable range in which the resonance angle exists. As for the data of the allowable range RG, the allowable ranges RG1, RG2, and RG31 to 33 are stored according to each profile data.

Sample type information indicating the type of sample (serum, plasma, etc.) is input from the operation unit 51 to the determination unit 42. The determination unit 42 accesses the memory 43 and reads out profile data and allowable range data corresponding to the sample type from the determination data 61 based on the sample type information. For example, when the sample type information is serum, the determination unit 42 reads out the serum profile data and the allowable range data from the determination data 61. Then, the determination unit 42 collates the actually measured profile with the profile data, which is the determination data 61 prepared according to the type of the sample, and the actually measured resonance angle exists within a preset allowable range. Determine whether or not to do so.

When the analysis chip 10 is mounted on the mounting portion 101 in an appropriate posture, the measured resonance angle should match the theoretical profile data. However, if improper mounting occurs, the measured resonance angle may deviate from the theoretical profile data. When the measurement is performed in such a state, the reliability of the measurement result cannot be ensured.

Therefore, the determination unit 42 outputs a determination result of abnormality when the measured resonance angle does not exist within the preset allowable range, and outputs a determination result of normal when the measured resonance angle exists within the allowable range. .. When the determination result is abnormal, the control unit 40 outputs the determination result to the display unit 52 to warn that the determination result is abnormal. The warning may be given by voice.

Further, as described above, the resonance angle may not be specified when the mounting posture of the analysis chip 10 deviates significantly from the proper posture. In that case, the resonance angle specifying unit 41 inputs that fact to the determination unit 42. Also in this case, the determination unit 42 outputs a determination result of abnormality, and the control unit 40 warns that the abnormality is present.

Hereinafter, the operation of the above configuration will be described with reference to the flowchart shown in FIG. When performing analysis using the fluorescence detection device 100, first, step (S) 1001 is executed, and the analysis chip 10 and the sample container CB containing the sample are set in the fluorescence detection device 100. The analysis chip 10 is mounted on the mounting portion 101.

Next, sample information such as patient information is input through the operation unit 51, and the control unit 40 receives the input sample information (S1002). The sample information also includes sample type information indicating the type of sample. The control unit 40 waits for the input of the measurement start instruction from the operation unit 51 (S1003). When the measurement start instruction is input (Y in S1003), the control unit 40 starts the operation of the sample processing unit 20. As shown in FIG. 4, the sample processing unit 20 sucks the sample from the sample container CB with the nozzle tip NC. Then, as shown in FIG. 5, the sucked sample is injected into the sample cell 14A from the nozzle tip NC, and the fluorescent reagent containing the second antibody B2 and the fluorescent label F and the sample solution SL are mixed to prepare the sample solution. Generate SL.

Then, the sample processing unit 20 inserts the tip of the nozzle tip NC into the injection port 12 of the analysis tip 10 and injects the generated sample solution SL into the flow path 15 (S1004). The sample processing unit 20 reciprocates the sample solution SL in the flow path 15 to bring the reaction region 16 sample solution SL into contact. By reciprocating the sample solution SL with respect to the reaction region 16 in this way, the reaction amount of the test substance (antigen) A and the first antibody B1 can be increased with a small amount of the sample solution SL.

After completing the reciprocating operation of the sample solution SL, the control unit 40 starts adjusting the incident angle of the excitation light L of the light irradiation unit 31 (S1005). The control unit 40 adjusts the incident angle θ of the excitation light L of the light irradiation unit 31 to a preset reference angle. The reference angle is determined according to, for example, the type of sample. Then, the control unit 40 changes the incident angle θ in the plus direction and the minus direction within a predetermined range with reference to the reference angle in a state where the excitation light L is irradiated from the light irradiation unit 31 (S1006). During this time, the fluorescence detection unit 32 detects the fluorescence Lf according to the reaction amount. The fluorescence detection unit 32 outputs a fluorescence detection signal according to the amount of light of the detected fluorescence Lf to the control unit 40.

In the control unit 40, the resonance angle specifying unit 41 identifies the resonance angle at which the plasmon increasing intensity is maximized by the procedure shown in FIG. 15 based on the fluorescence detection signal detected by the fluorescence detecting unit 32 (S1007). If the resonance angle cannot be specified (N in S1007), the resonance angle specifying unit 41 outputs to that effect to the determination unit 42, and the control unit 40 executes an error process for warning that the abnormality is abnormal (S1009).

On the other hand, when the resonance angle can be specified (N in S1007), the data of the measured profile is input to the determination unit 42. As shown in FIG. 15, the determination unit 42 reads out the profile data and the permissible range according to the sample type from the determination data 61, and collates the data with the actually measured profile. Then, the determination unit 42 determines whether or not the actually measured resonance angle exists within a preset allowable range. If the analysis chip 10 is improperly mounted, the measured resonance angle will be out of the permissible range. In that case (N in S1008), the determination unit 42 outputs a determination result of abnormality. When the determination result is abnormal, the control unit 40 warns through the display unit 52 that the determination result is abnormal (S1009). In this case, the process ends without measuring.

On the other hand, when the measured resonance angle is within the permissible range (Y in S1008), the determination unit 42 outputs a determination result of normality. When the control unit 40 receives the determination result that it is normal, the control unit 40 ends the incident angle adjustment (S1010).

After the incident angle adjustment (S1010) is completed, the measurement is performed (S1011). The light irradiation unit 31 irradiates the excitation light L at a specified resonance angle for a predetermined time, during which the fluorescence detection unit 32 detects fluorescence. Such fluorescence detection is performed for the test region TR and the control region CR while moving the measuring unit 30.

The control unit 40 analyzes the fluorescence detection signal detected by the fluorescence detection unit 32 in each of the test region TR and the control region CR. Then, the reaction amount of the test substance (antigen) A and the second antibody B2 is calculated, the concentration of the test substance (antigen) A in the sample is measured, and this is output as the measurement result (S1012). The measurement result is displayed on the display unit 52 and recorded as data in the memory. Further, the measurement result may be printed out.

As described above, in this example, the incident angle of the excitation light L is adjusted to specify the resonance angle, and if the specified resonance angle does not exist within the permissible range, an abnormality is warned. It is possible to notify the user of an abnormality caused by improper mounting of the device at an early stage. As a result, unreliable measurement results can be eliminated at an early stage, which reduces the time and effort required for re-examination.

Further, the abnormality determination is performed in a state where the sample solution SL is injected into the flow path 15. Therefore, it is possible to perform an appropriate abnormality determination according to the type of the sample.

[Second Embodiment]
The second embodiment shown in FIGS. 17 and 18 is an example in which the resonance angle is specified based on the reflected light RL. Since other configurations and processing procedures are the same as those in the first embodiment, the same components are designated by the same reference numerals and the description thereof will be omitted.

As shown in FIG. 17, the second embodiment is provided with an optical sensor 66 that receives the reflected light RL reflected by the excitation light L on the back surface of the metal film 18. The optical sensor 66 is, for example, a CCD image sensor or a CMOS image sensor. The optical sensor 66 corresponds to a reflected light detection unit. The optical sensor 66 outputs a reflected light detection signal corresponding to the amount of reflected light RL to the control unit 40.

The resonance angle specifying unit 41 acquires the reflected light detection signal and generates an actual measurement profile based on the reflected light detection signal. As shown in FIGS. 9 to 13, the reflected light RL is greatly attenuated when surface plasmon resonance occurs, contrary to the fluorescence Lf. Further, as shown in FIG. 18, there is a correlation between the minimum value shown by the profile of the reflected light RL and the maximum value of the fluorescence Lf. Therefore, the incident angle θ at which the reflected light RL shows the minimum value is converted into the incident angle θ at which the fluorescence Lf shows the maximum value. As a result, the resonance angle at which the plasmon strengthening intensity is maximized can be obtained. Other configurations and processing procedures are the same as those in the first embodiment, and description thereof will be omitted.

[Modification 1]
In each of the above embodiments, the example in which the sample is used without being diluted has been described, but when serum or plasma is used as the sample, dilution may be performed. There are two possible modes of dilution, manual dilution and automatic dilution. The manual dilution is an embodiment in which the user manually dilutes the sample before mounting the analysis chip 10 on the fluorescence analyzer 100, and the manually diluted sample is used. The automatic dilution is an embodiment in which, when the fluorescence analyzer 100 has a dilution function, the fluorescence analyzer 100 dilutes the sample according to the specified dilution ratio after the analysis chip 10 is attached.

When the sample is diluted for measurement, it is advisable to prepare profile data according to the dilution ratio as shown in FIG. As shown in FIG. 19, the resonance angle changes depending on the dilution ratio. For example, in the case of serum, the resonance angle changes to 72.04 ° without dilution, while the resonance angle changes to 71.70 ° when the dilution ratio is 10 times. Further, in the case of serum, the resonance angle is 73.60 ° without dilution, whereas the resonance angle changes to 71.68 ° when the dilution ratio is 10 times. In this way, if the resonance angle changes, it also affects the determination of whether or not the resonance angle exists within the allowable range. Therefore, it is advisable to prepare profile data for each dilution ratio.

In this case, profile data is selected based on the dilution factor entered by the user. In the case of automatic dilution, the dilution ratio is input to perform the dilution process. In the case of manual dilution, when the user inputs the sample type information from the operation unit 51, the dilution ratio is also input in addition to the sample type. The dilution factor thus entered is used to select profile data. In the example of FIG. 19, there is no change in the resonance angle when the dilution ratio is 10 times and 100 times for both serum and plasma. In this case, the profile data is not prepared separately. You may.

In addition, when the sample is diluted and used in this way, it is considered abnormal even if the dilution ratio of the sample solution SL is not appropriate by determining whether the specified resonance angle exists within the preset allowable range. It becomes possible to detect. This is because even if the analysis chip 10 is properly mounted and there is no mounting defect, if the dilution ratio of the sample solution SL is not appropriate, the resonance angle is considered to be out of the allowable range. The cause of the case where the dilution ratio becomes inappropriate is considered to be, for example, a user's dilution error in the case of manual dilution, and a defect in the dilution function of the device in the case of automatic dilution.

As shown in the above embodiment, the abnormality determination is performed in a state where the sample solution SL is injected into the flow path 15. Therefore, it is possible to determine the abnormality of the dilution ratio of the sample solution SL.

[Modification 2]
Further, in each of the above embodiments, the abnormality determination is performed only by the resonance angle, but the abnormality determination may be performed in consideration of the maximum value in addition to the resonance angle.

"Specific example of allowable range"
With reference to FIGS. 20 to 23, a method of obtaining the allowable range RG of the resonance angle and a specific example of numerical values will be described. The examples shown in FIGS. 20 to 23 are examples of how to obtain the permissible range RG and numerical values when measuring the concentration of TSH (Thyroid Stimulating Hormone) as a test item in a blood test.

First, as shown in FIG. 20, measurement examples of samples having TSH concentrations of 0.25 ng / mL, 0.45 ng / mL, and 0.74 ng / mL, respectively, are prepared. The TSH measurement example shows the relationship between the measurement time and the signal value of the fluorescence detection signal (hereinafter, referred to as the fluorescence signal value). The higher the TSH concentration and the longer the measurement time, the higher the fluorescence signal value (digit).

FIG. 21 is a graph obtained by obtaining the slope of each graph of the TSH concentration measurement example of FIG. 20 as a rate signal (digit / minute) which is a fluorescence signal value per unit time, and plotting the obtained rate signal for each concentration. Is. The graph of FIG. 21 is used when a rate signal is grasped from the fluorescence detection signal output by the fluorescence detection unit 32 at the time of actual measurement and the TSH concentration is calculated based on the grasped rate signal, and is a calibration curve. Is called.

As is clear from the graph of the calibration curve shown in FIG. 21, the larger the rate signal, the higher the TSH concentration, and the higher the TSH concentration, the steeper the slope of the calibration curve. This means that the higher the TSH concentration, the greater the influence of a slight change in the fluorescence signal value on the TSH concentration. The fluorescence signal value changes greatly with a change in the incident angle θ of the excitation light L. After all, when the deviation of the incident angle θ with respect to the theoretical resonance angle is the same, the higher the TSH concentration, the larger the error of the TSH concentration will appear. Therefore, it is necessary to narrow the permissible range RG in the region where the TSH concentration is high.

FIG. 22 is a table showing evaluation criteria for evaluating the TSH concentration as a measurement result. In FIG. 22, the range of the reference value recognized as the appropriate range of the TSH concentration is 0.44 ng / mL to 4.95 ng / mL, and if it is out of the range, an increase or decrease in the TSH concentration is observed, which is inappropriate. Is evaluated as.

As described above, from the above consideration of the calibration curve shown in FIG. 21, the higher the TSH concentration, the greater the influence of the deviation of the incident angle θ on the error of the measurement result. Therefore, in the appropriate range of TSH concentration, when the deviation of the incident angle θ is the same, the allowable range RG is obtained based on the case of the upper limit value of 4.95 ng / mL, which is the worst case where the error is the largest.

For example, set the permissible range for the error of TSH concentration to ± 5%, and for the case of the upper limit value of 4.95 ng / mL, set the permissible range of the error of the rate signal that causes the error of TSH concentration according to the predetermined calculation formula. When calculated, for example, a range of ± 2.2% can be obtained. The error of the rate signal corresponds to the error of plasmon augmentation. Then, the permissible range of the error of ± 2.2% is converted into the permissible range RG of the deviation of the incident angle θ.

FIG. 23 shows the result of measuring the deviation angle of the incident angle θ when the plasmon increasing intensity deviates by ± 2.2%. In FIG. 23, the most severe value is −0.46 ° in the case of serum. From this result, the permissible range RG is set in the range of ± 0.46 °.

For example, if the difference between the resonance angle identified by actual measurement and the theoretical resonance angle read from the profile data is within the range of ± 0.46 °, the determination unit 42 determines that it is normal and is out of the range. In the case of, it is judged as abnormal. More specifically, when the sample is serum, the theoretical resonance angle is 72.04 ° (see FIG. 15). In this case, if the resonance angle specified by actual measurement is in the range of 71.58 ° to 72.50 °, it is determined to be normal, and if it is outside that range, it is determined to be abnormal.

In this example, the most severe value among the plurality of types of samples is set as the permissible range RG of all the samples, but of course, as in the permissible range RG1, RG2 ... shown in FIG. The allowable range RG value may be set for each sample.

The examples of FIGS. 20 to 23 are examples of how to obtain the permissible range, and may be obtained by other methods. Further, the examples of FIGS. 20 to 23 are examples of measuring the TSH concentration, and when measuring the values of other test items or measuring a sample other than blood, an appropriate permissible range is provided. Of course, it can change.

Although the preferred embodiments of the present invention have been described above, the present invention is not limited to the above embodiments, and various configurations such as combinations of the respective embodiments and the respective modifications are provided as long as the gist of the present invention is not deviated. Of course, it can be adopted.

In each of the above embodiments, for example, the hardware structure of the processing unit that executes the processing of the electric signal such as the control unit 40 is various processors as shown below.

Various processors include a CPU, a programmable logic device (PLD), a dedicated electric circuit, and the like. As is well known, a CPU is a general-purpose processor that executes software (program) and functions as various processing units. PLD is a processor such as FPGA (Field Programmable Gate Array) whose circuit configuration can be changed after manufacturing. A dedicated electric circuit is a processor having a circuit configuration specially designed for executing a specific process such as an ASIC (Application Specific Integrated Circuit).

One processing unit may be composed of one of these various processors, or may be composed of a combination of two or more processors of the same type or different types (for example, a plurality of FPGAs or a combination of a CPU and an FPGA). May be done. Further, a plurality of processing units may be configured by one processor. As an example of configuring a plurality of processing units with one processor, first, there is a form in which one processor is configured by a combination of one or more CPUs and software, and this processor functions as a plurality of processing units. .. Secondly, as typified by System On Chip (SoC) and the like, there is a form in which a processor that realizes the functions of the entire system including a plurality of processing units with one IC chip is used. As described above, the various processing units are configured by using one or more of the above-mentioned various processors as a hardware structure.

Further, the hardware structure of these various processors is, more specifically, an electric circuit (circuitry) in which circuit elements such as semiconductor elements are combined.

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

11 Main body 11A Prism 12 Injection port 13 Outlet 14A, 14B Sample cell 15 Flow path 15A, 15B Cross-sectional area change part 16 Reaction area 17 Dielectric plate 17A Front side surface 17B Back side surface 18 Metal film 20 Specimen processing part 26 Piping 30 Measuring unit 31 Light irradiation unit 32 Fluorescence detection unit 33 Incident angle adjustment mechanism 36 Measuring unit movement mechanism 40 Control unit 41 Resonance angle identification unit 42 Judgment unit 43 Memory 51 Operation unit 52 Display unit 61 Judgment data 66 Optical sensor 100 Fluorescence detection device 101 Mounting part A Test substance (antigen)
B1 1st antibody B2 2nd antibody CB Specimen container CR Control area Ew Evanescent wave F Fluorescent label L Excitation light Lf Fluorescent NC Nozzle tip RL Reflected light RG Tolerance range SL Specimen solution TR Test area θ Incident angle

Claims (7)

The reaction region is provided with a flow path through which the sample solution containing the test substance flows, a reaction region arranged in the flow path, where an antibody that specifically reacts with the test substance is fixed, and the sample solution comes into contact with the sample solution. The dielectric material is used by using an analysis chip having a main surface in contact with the back surface opposite to the surface surface of the reaction region in the metal film and a dielectric material transparent to excitation light. In a fluorescence detection device using surface plasmon resonance, in which the excitation light is incident from the back side of the main surface through the surface to detect fluorescence generated from a fluorescent label bonded to the test substance due to the incident of the excitation light. ,
The light irradiation unit that irradiates the excitation light and
The fluorescence detection unit that detects the fluorescence and
An incident angle adjusting mechanism that adjusts the incident angle of the excitation light with respect to the main surface,
A resonance angle specifying unit that changes the incident angle with the incident angle adjusting mechanism to specify a resonance angle that maximizes the fluorescence detected by the fluorescence detecting unit.
A determination unit that determines whether or not the resonance angle specified by the resonance angle specifying unit exists within a preset allowable range.
A warning unit that warns of an abnormality when the determination unit determines that the resonance angle does not exist within the allowable range.
Provided,
The sample solution is a liquid obtained by diluting a sample containing the test substance at a predetermined dilution ratio.
The determination unit makes the determination using the allowable range according to the dilution ratio.
The abnormality includes the abnormality of the dilution ratio.
Fluorescence detection device using surface plasmon resonance. The abnormalities, fluorescence detection device using surface plasmon resonance according to claim 1 including the poor mounting of the analysis chip. The fluorescence detection device using surface plasmon resonance according to claim 1 or 2, wherein the determination of the resonance angle is performed in a state where the sample solution is injected into the flow path. Fluorescence detection using surface plasmon resonance according to any one of claims 1 to 3, wherein the determination unit makes a determination based on determination data prepared according to the type of the sample contained in the sample solution. apparatus. The resonance angle specifying unit acquires a fluorescence detection signal corresponding to the amount of fluorescence of the fluorescence detected by the fluorescence detection unit, and determines whether or not the resonance angle is within the allowable range based on the maximum value of the fluorescence detection signal. The fluorescence detection device using the surface plasmon resonance according to any one of claims 1 to 4. It is provided with a reflected light detection unit that detects the reflected light of the excitation light reflected by the metal film.
The surface according to any one of claims 1 to 4, wherein the resonance angle specifying unit specifies the resonance angle based on a reflected light detection signal corresponding to the amount of reflected light detected by the reflected light detecting unit. Fluorescence detection device using plasmon resonance. The reaction region is provided with a flow path through which the sample solution containing the test substance flows, a reaction region arranged in the flow path, where an antibody that specifically reacts with the test substance is fixed, and the sample solution comes into contact with the sample solution. The dielectric material is used by using an analysis chip having a main surface in contact with the back surface opposite to the surface surface of the reaction region in the metal film and a dielectric material transparent to excitation light. A fluorescence detection device using surface plasmon resonance that causes the excitation light to be incident from the back side of the main surface through the surface to detect fluorescence generated from a fluorescent label bonded to the test substance due to the incident of the excitation light. In the operating method
The light irradiation step of irradiating the excitation light and
The fluorescence detection step for detecting the fluorescence and
A resonance angle specifying step for specifying a resonance angle at which the fluorescence is maximized, which is detected in the fluorescence detection step, while changing the incident angle of the excitation light with respect to the main surface.
A determination step for determining whether or not the resonance angle specified in the resonance angle specifying step exists within a preset allowable range, and a determination step.
In the determination step, a warning step for warning an abnormality when it is determined that the resonance angle does not exist within the allowable range is defined as a warning step.
Provided,
The sample solution is a liquid obtained by diluting a sample containing the test substance at a predetermined dilution ratio.
In the determination step, the determination is made using the allowable range according to the dilution ratio.
The abnormality includes the abnormality of the dilution ratio.
A method of operating a fluorescence detector using surface plasmon resonance.

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