JP2018185220A - Analyzer and analysis method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an analyzer with which it is possible to identify multiple kinds of antigen to be detected for exosome simply in a short time.SOLUTION: An analyzer 1 comprises a laser light source 2, a photodetector 13, fluorescence detectors 14, 15, and a detection signal generation circuit 20. The laser light source 2 irradiates, with a laser light L1, a disk 100 for sample analysis in which is captured an exosome 200 having an antigen 203 having fluorescent particulates 120 coupled therewith or an antigen 204 having fluorescent particulates 130 coupled therewith. The photodetector 13 detects the laser beam reflected by the disk 100 for sample analysis as a detection light L2. The fluorescence detector 14 detects a fluorescence FL1 of the fluorescent particulates 120. The fluorescence detector 15 detects a fluorescence FL2 of the fluorescent particulates 130. The detection signal generation circuit 20 generates a gate signal GS on the basis of the detection light L2 and generates fluorescence detection signals FS1, FS2 from the fluorescence FL1, FL2 on the basis of the gate signal GS.SELECTED DRAWING: Figure 7

Description

  The present invention relates to an analyzer and an analysis method for analyzing biological materials such as antigens and antibodies.

  An immunoassay is known in which a specific antigen associated with a disease is detected and analyzed as a biomarker to quantitatively analyze the effect of disease detection and treatment.

  In Patent Document 1, an antibody fixed on a sample analysis disk is bound to an antigen in a sample, the antigen is labeled with fine particles having an antibody, and laser light emitted from an optical pickup is scanned, An analyzer for detecting fine particles trapped on a sample analysis disk is described. The analyzer described in Patent Document 1 uses an optical disk device for specimen detection.

Japanese Patent Laying-Open No. 2015-127691

  In recent years, minute membrane vesicles called exosomes are expected as new biomarkers. In general, exosomes contain a plurality of different types of antigens. Among them, the antigen to be detected is a protein present on the surface of the lipid bilayer membrane, such as a transmembrane protein, an adhesion molecule, a membrane transport protein, a membrane fusion protein, a glycoprotein, and the like. Hereinafter, a protein to be detected is referred to as a detection target antigen.

  If a plurality of types of detection target antigens can be distinguished from the exosome, exosome can be selectively detected. Therefore, it can be expected that the disease specificity of detection can be increased and the accuracy and accuracy of diagnosis can be improved.

  However, conventionally, in order to discriminate a plurality of types of antigens to be detected from exosomes, it is necessary to carry out a plurality of measurements using fine particles on which different antibodies are immobilized. For this reason, the measurement time becomes long and it is difficult to make a quick diagnosis.

  An object of the present invention is to provide an analysis apparatus and an analysis method that can identify a plurality of types of detection target antigens with respect to exosomes in a simpler manner and in a shorter time than before.

  The present invention relates to a laser for irradiating a sample analysis disk on which a exosome having a first antigen to which first fluorescent particles are bound or a second antigen to which second fluorescent particles are bound is captured. A light source, a photodetector that detects laser light reflected by the sample analysis disk as detection light, and first fluorescence emitted from the first fluorescent fine particles when the laser light is irradiated are detected. A first fluorescence detection unit that detects the second fluorescence emitted from the second fluorescence fine particle when irradiated with the laser light, and a gate based on the detection light. A detection signal for generating a signal, generating a first fluorescence detection signal from the first fluorescence based on the gate signal, and generating a second fluorescence detection signal from the second fluorescence based on the gate signal Living Providing an analysis device, characterized in that it comprises a circuit.

  The present invention irradiates a sample analysis disk on which an exosome having a first antigen to which a first fluorescent fine particle is bound or a second antigen to which a second fluorescent fine particle is bound is captured with a laser beam, The laser light reflected by the sample analysis disk is detected as detection light, and the first fluorescent light emitted from the first fluorescent fine particles or the laser light is irradiated when the laser light is irradiated. And detecting a second fluorescence emitted from the second fluorescent fine particles, generating a gate signal based on the detection light, and detecting the first fluorescence from the first fluorescence based on the gate signal. Provided is an analysis method characterized by generating a signal and generating a second fluorescence detection signal from the second fluorescence based on the gate signal.

  According to the analysis apparatus and the analysis method of the present invention, it is possible to identify a plurality of types of detection target antigens with respect to exosomes in a simpler and shorter time than in the past.

It is sectional drawing of the track area | region of the disk for sample analysis. 6 is a flowchart for explaining a method of capturing exosomes and a plurality of types of fluorescent fine particles on a sample analysis disk. It is a typical sectional view of an exosome. It is sectional drawing which shows typically the state by which the exosome is trapped on the track | truck area | region of the disk for sample analysis. It is a typical sectional view of fluorescent fine particles. It is sectional drawing which shows typically the state by which the fluorescent fine particle is trapped on the track area | region of the sample analysis disk. It is a block diagram which shows the analyzer of 1st Embodiment. It is a time chart for demonstrating operation | movement of the detection signal generation circuit in the analyzer of 1st Embodiment. It is a figure which shows the spectrum of the background noise of an Example and a comparative example. It is a block diagram which shows the analyzer of 2nd Embodiment. It is a time chart for demonstrating operation | movement of the detection signal generation circuit in the analyzer of 2nd Embodiment. It is a block diagram which shows the analyzer of 3rd Embodiment. It is a block diagram which shows the analyzer of 4th Embodiment.

[Method of capturing exosomes and fluorescent fine particles]
An example of a method for capturing exosomes and a plurality of types of fluorescent fine particles on a sample analysis disk will be described with reference to FIGS. FIG. 1 shows a cross section of a sample analysis disk 100.

  As shown in FIG. 1, a track region 103 in which convex portions 101 and concave portions 102 are alternately arranged in the radial direction is formed on the surface of the sample analysis disk 100. The convex portion 101 and the concave portion 102 are formed in a spiral shape from the inner peripheral portion toward the outer peripheral portion. The sample analysis disc 100 has a disk shape that is the same as or similar to an optical disc such as a Blu-ray disc (BD), DVD, or compact disc (CD).

  The sample analysis disk 100 is made of, for example, a resin material such as polycarbonate resin or cycloolefin polymer generally used for optical disks. Note that the sample analysis disk 100 is not limited to the optical disk described above, and an optical disk conforming to another form or a predetermined standard may be used.

  The convex portion 101 in the track region 103 of the sample analysis disc 100 corresponds to a land of the optical disc, and the concave portion 102 corresponds to a groove. The track pitch corresponding to the pitch in the radial direction of the recesses 102 is, for example, 320 nm.

  As shown in FIG. 3, the exosome 200 is covered with a lipid bilayer membrane 201. In the lipid bilayer membrane 201, a plurality of types of membrane proteins such as a transmembrane protein, a membrane surface protein, and a membrane-bound protein are present as surface molecules. The type and number of these membrane proteins, or the position in the lipid bilayer membrane 201 vary depending on the type of exosome and also varies from individual to individual. Exosomes are recognized by antigen-antibody reaction using these surface molecules as antigens.

  In addition to CD9, CD81, CD63, HER2, CD147, EpCAM, Caveolin-1, etc., the presence of molecules such as various proteins has been reported in many papers as surface molecules that are antigens. In FIG. 3, three types of proteins (antigens to be detected) for identifying exosomes are designated as antigen 203 (first antigen), antigen 204 (second antigen), and antigen 202 (third antigen). It is shown. Antigen 202 is, for example, a CD9 protein present in many exosomes. Antigen 203 and antigen 204 are HER2 protein and CD147 protein that are expected to be associated with diseases.

  The average particle diameter of the exosome 200 is about 100 nm. The average particle diameter of the exosome 200 means an average value of values obtained by measuring the particle diameter of the exosome 200 by an arbitrary measurement method. As the measurement method, for example, a wet measurement method in which the exosome 200 is observed in a solution state using a nanoparticle tracking method, or a dry measurement method in which the exosome 200 is observed while maintaining the form of the exosome 200 with a transmission electron microscope. Etc.

  An example of an exosome detection procedure will be described with reference to the flowchart of FIG. 2 and FIG. In step S1 of the flowchart shown in FIG. 2, the operator mounts a cartridge on the track area 103 of the sample analysis disk 100 to form a well. Specifically, a plurality of wells are formed by the plurality of through holes of the cartridge and the track region 103 of the sample analysis disk 100. The well functions as a container for storing a solution such as a sample solution and a buffer solution.

  In step S2, the operator injects a buffer containing an antibody 111 (capture antibody) for capturing exosomes into the well. The buffer is incubated at the appropriate temperature for the appropriate time. For example, a sufficient amount of antibody 111 can be fixed on the track region 103 of the sample analysis disk 100 by reacting overnight at room temperature.

  The operator causes the well into which the buffer solution containing the antibody 111 has been injected to incubate at an appropriate temperature for an appropriate time. The buffer solution is preferably a carbonate buffer solution or the like. As a result, as shown in FIG. 4A, the antibody 111 is fixed on the track region 103 of the sample analysis disk 100 constituting the bottom surface of the well. Antibody 111 is an anti-CD9 antibody that specifically binds to the CD9 protein. The operator drains the buffer containing the remaining antibody 111 not fixed from the well, and the well is washed with another buffer containing no antibody. The antibody 111 that is not fixed to the track region 103 is removed by this washing.

  In step S <b> 3, the operator performs a blocking process on the track region 103 in order to prevent nonspecific adsorption of exosomes on which the antigens 202, 203, and 204 do not exist on the track region 103. Specifically, the operator injects skim milk diluted with a buffer solution such as a phosphate buffer solution into the well and shakes the well for an appropriate time. The skim milk covers the surface of the sample analysis disk 100 or well, or blocks the site where the antibody protein immobilized on the surface of the sample analysis disk 100 or well is adsorbed, thereby preventing further protein adsorption. This prevents nonspecific adsorption of exosomes or microparticles. For this reason, skim milk is suitable for a blocking process. In addition, the substance used for the blocking treatment is not limited to skim milk as long as it has the same effect.

  The operator drains the buffer containing skim milk from the well and rinses the well with another buffer. As a buffer used for washing, skim milk may or may not be contained. It is also possible to omit the cleaning. As shown in FIG. 4A, a block layer 112 is formed on the track area 103 of the sample analysis disk 100.

  In step S4, the operator injects a sample solution containing the exosome 200 into the well. The operator causes the well into which the sample solution containing the exosome 200 has been injected to be incubated at an appropriate temperature for an appropriate time, for example, 37 degrees for 2 hours. Thereby, the antigen 202 of the exosome 200 and the antibody 111 fixed on the track region 103 of the sample analysis disk 100 are specifically bound by the antigen-antibody reaction.

  The operator drains the sample solution from the well, and the well is washed with a buffer solution. The exosomes that are not bound to the antibody 111 and are dispersed in the sample solution, and the exosomes that are attached to the wells due to non-specific adsorption that is not an antigen-antibody reaction are removed by this washing.

  As shown in FIG. 4B, the exosome 200 is captured on the track area 103 of the sample analysis disk 100. Since the CD9 protein that is the antigen 202 is present in most exosomes, most of the exosomes 200 can be captured on the track region 103. For easy understanding, the exosome 200 shown in FIG. 14B and the like is schematically shown by simplifying the exosome 200 shown in FIG. In addition, although there are various exosome particle sizes, in FIG. 4B and FIGS. 6A and 6B described later, the particle sizes of the exosomes 200 are all the same.

  In step S5, the operator injects a buffer containing fluorescent fine particles 120 (first fluorescent fine particles) to be labeled into the well. As shown in FIG. 5A, an antibody 121 (first detection antibody) that specifically binds to a specific protein of the exosome 200 by an antigen-antibody reaction is formed on the surface of the fluorescent fine particle 120. The size of the fluorescent fine particles 120 is about 200 nm.

  Antibody 121 is an anti-HER2 antibody that specifically binds to HER2 protein. The fluorescent fine particle 120 emits fluorescence FL1 (first fluorescence) having a wavelength λ1 (first wavelength) by exciting a fluorescent substance contained in the fluorescent fine particle 120 when irradiated with laser light from the outside.

  The operator causes the well into which the buffer solution containing the fluorescent fine particles 120 has been injected to incubate at an appropriate temperature for an appropriate time. As a result, as shown in FIG. 6A, the antibody 121 of the fluorescent fine particle 120 and the exosome 210 having the antigen 203 among the exosomes 200 captured on the track region 103 of the sample analysis disk 100 are antigens. It binds specifically by antibody reaction. The fluorescent fine particles 120 are captured on the track region 103 in a state of being coupled to the exosome 210.

  The operator drains the buffer from the well, and the well is washed with another buffer and dried. The fluorescent fine particles 120 that are not bonded to the exosome 200 and are dispersed in the buffer solution are removed by washing.

  In step S6, the operator injects a buffer containing fluorescent fine particles 130 (second fluorescent fine particles) to be labeled into the well. As shown in FIG. 5B, an antibody 131 (second detection antibody) that specifically binds to a specific protein of the exosome 200 by an antigen-antibody reaction is formed on the surface of the fluorescent fine particle 130. The size of the fluorescent fine particles 130 is about 200 nm.

  Antibody 131 is an anti-CD147 antibody that specifically binds to CD147 protein. When the fluorescent fine particles 130 are irradiated with laser light from the outside, the fluorescent substance contained in the fluorescent fine particles 130 is excited, and emits fluorescence FL2 (second fluorescence) having a wavelength λ2 (second wavelength).

  The operator causes the well into which the buffer solution containing the fluorescent fine particles 130 has been injected to incubate at an appropriate temperature for an appropriate time. As a result, as shown in FIG. 6B, the antibody 131 of the fluorescent fine particle 130 and the exosome 220 having the antigen 204 among the exosomes 200 captured on the track region 103 of the sample analysis disk 100 are antigens. It binds specifically by antibody reaction. The fluorescent fine particles 130 are captured on the track region 103 in a state of being coupled to the exosome 220.

  It is also possible to capture the plurality of fluorescent particles 120 and 130 on the track region 103 at a time using a buffer solution including a plurality of fluorescent particles, for example, a buffer solution including the fluorescent particles 120 and 130. However, when a plurality of fluorescent fine particles 120 and 130 are captured on the track region 103 at a time, a protein (for example, CD147 protein) and fluorescent fine particles having a large amount of the exosome 200 having two types of proteins having different abundances. 130 specifically binds.

  Therefore, there is a possibility that the probability that a protein having a small amount (for example, HER2 protein) and the fluorescent fine particles 120 are specifically bound to each other is reduced. The decrease in the binding probability becomes a factor that deteriorates the detection accuracy of a protein having a small abundance.

  Therefore, it is preferable to select an antibody having a smaller abundance than the antibody 131 as the antibody 121. For example, when detecting CD147 protein and HER2 protein as targets, the antibody 121 may be an anti-HER2 antibody that specifically binds to a HER2 protein that is known to be more likely to be less abundant than CD147 protein. desirable.

  As described above, it is possible to detect almost the entire amount of exosomes having a protein with a small abundance by specifically binding fluorescent particles having different fluorescence wavelengths in order from a protein with a small abundance. For exosomes with abundant protein, a slight deficiency may occur with respect to the total amount, but reproducibility can be ensured by always using the same procedure.

  An analyzer that detects a plurality of fluorescent fine particles 120 and 130 captured on the track region 103 of the sample analysis disk 100 will be described. By detecting exosomes with a plurality of detection antibodies that specifically bind to a plurality of antigens that are expected to be related to the disease, there is a possibility that the state of the disease can be grasped with higher accuracy.

[Analyzer of First Embodiment]
The analyzer according to the first embodiment will be described with reference to FIG. The analysis apparatus 1 of the first embodiment includes a laser light source 2, a collimator lens 3, beam splitters 4 and 5, an objective lens 6, condenser lenses 7a, 7b, 8, and 9, a wavelength separation prism 10, Band pass filters 11 and 12, a photodetector 13, fluorescence detectors 14 and 15, a detection signal generation circuit 20, and a measurement unit 16 are provided. The detection signal generation circuit 20 includes a particle detection circuit 21, a gate signal generation circuit 22, and a fluorescence detection circuit 23. The measurement unit 16 may be configured to be provided in an external device.

  The laser light source 2 emits laser light L1 having a wavelength of, for example, 405 nm. The collimator lens 3 makes the laser light L1 parallel light. The beam splitter 4 (first beam splitter) transmits the laser light L1. The objective lens 6 focuses the laser beam L1 on the track region 103 of the sample analysis disk 100, specifically on the recess 102 of the track region 103.

  The laser light L1 is reflected by the track region 103 and enters the beam splitter 4 through the objective lens 6 as detection light L2. The beam splitter 4 reflects the detection light L2 toward the beam splitter 5 (second beam splitter). The beam splitter 5 transmits the detection light L2. The condensing lenses 7a and 7b collect the detection light L2 toward the photodetector 13. The photodetector 13 detects the light amount LV (detection light amount) of the detection light L 2 and outputs it to the detection signal generation circuit 20.

  When the fluorescent fine particles 120 are captured in the recesses 102 of the track region 103, the fluorescent fine particles 120 are excited by irradiating the fluorescent fine particles 120 with the laser light L1, and the fluorescent fine particles 120 have a wavelength. Fluorescence FL1 of λ1 is emitted. The fluorescence FL1 enters the beam splitter 4 via the objective lens 6. The beam splitter 4 reflects the fluorescence FL1 toward the beam splitter 5.

  The beam splitter 5 reflects the fluorescent light FL1 toward the wavelength separation prism 10. The wavelength separation prism 10 transmits the fluorescent light FL1. The band-pass filter 11 (first band-pass filter) is a fluorescent filter that has a wavelength band (first wavelength band) that transmits the fluorescent light FL1 and blocks light having a wavelength other than the wavelength band. The fluorescence FL1 is very susceptible to background noise because the light amount and the light intensity are very small compared to the detection light L2. The bandpass filter 11 can reduce the influence of background noise.

  The condensing lens 8 condenses the fluorescence FL1 toward the fluorescence detector 14 (first fluorescence detection unit). The fluorescence detector 14 detects the light amount FV1 (first fluorescence light amount) of the fluorescence FL1 and outputs it to the detection signal generation circuit 20.

  When the fluorescent fine particles 130 are captured in the concave portion 102 of the track region 103, the fluorescent fine particles 130 are excited by irradiating the fluorescent fine particles 130 with the laser light L1, and the fluorescent fine particles 130 have a wavelength. Fluorescence FL2 of λ2 is emitted. The fluorescence FL2 enters the beam splitter 4 via the objective lens 6. The beam splitter 4 reflects the fluorescence FL 2 toward the beam splitter 5.

  The beam splitter 5 reflects the fluorescence FL2 toward the wavelength separation prism 10. The wavelength separation prism 10 reflects the fluorescence FL2 toward the bandpass filter 12 (second bandpass filter). The bandpass filter 12 is a fluorescent filter that has a wavelength band (second wavelength band) that transmits the fluorescent light FL2 and blocks light of wavelengths other than the wavelength band. The fluorescent light FL2 is very susceptible to background noise because the light amount and the light intensity are very small compared to the detection light L2. The bandpass filter 12 can reduce the influence of background noise.

  The condensing lens 9 condenses the fluorescence FL2 toward the fluorescence detector 15 (second fluorescence detection unit). The fluorescence detector 15 detects the amount of light FV2 (second amount of fluorescence) of the fluorescence FL2 and outputs it to the detection signal generation circuit 20.

  The operation of the detection signal generation circuit 20 will be described with reference to the time charts shown in FIGS. The light amount LV of the detection light L2 is input from the photodetector 13 to the fine particle detection circuit 21 of the detection signal generation circuit 20. The laser beam L1 is scanned along the recess 102 of the track area 103. When the laser light L1 is scanned on the fluorescent fine particles 120 and 130, the light quantity LV of the detection light L2 changes. For example, in FIGS. 8A and 8B, when the laser light L1 is scanned over the fluorescent fine particles 120 and 130, the light quantity LV of the detection light L2 decreases.

  The fine particle detection circuit 21 generates a fine particle detection signal BS that is a pulse signal that becomes a high level during a period in which the light amount LV exceeds the threshold value, and outputs it to the measurement unit 16 and the gate signal generation circuit 22. The fine particle detection circuit 21 may detect the time point t1 when the light amount LV exceeds the threshold value and output the detected time point t1 to the gate signal generation circuit 22.

  The gate signal generation circuit 22 generates a gate signal GS that rises after the period Ta from the time t1 when the particle detection signal BS rises and further falls after the period Tb, and outputs the gate signal GS to the fluorescence detection circuit 23. The period Tb corresponds to the pulse width of the gate signal GS. The period Ta, the period Tb, and the pulse width Tbs of the particle detection signal BS preferably have a relationship of (Ta + Tb) ≦ Tbs. Note that Ta = 0 may be used.

  The fluorescence detection circuit 23 receives the light amount FV1 of the fluorescence FL1 from the fluorescence detector 14, receives the light amount FV2 of the fluorescence FL2 from the fluorescence detector 15, and receives the gate signal GS from the gate signal generation circuit 22. The fluorescence detection circuit 23 extracts the light amount FV1 during the period when the gate signal GS is at a high level, generates a fluorescence detection signal FS1 (first fluorescence detection signal), and outputs the fluorescence detection signal FS1 to the measurement unit 16. Further, the fluorescence detection circuit 23 extracts the light amount FV2 during the period when the gate signal GS is at a high level, generates a fluorescence detection signal FS2 (second fluorescence detection signal), and outputs the fluorescence detection signal FS2 to the measurement unit 16.

  The measuring unit 16 counts the input fine particle detection signal BS and measures the total number of fine particles captured on the track region 103. The measuring unit 16 compares the input fluorescence detection signal FS1 with a threshold value, and counts the fluorescence detection signal FS1 exceeding the threshold value, thereby measuring the number of fluorescent fine particles 120 captured on the track region 103. . Further, the measurement unit 16 compares the input fluorescence detection signal FS2 with a threshold value and counts the fluorescence detection signal FS2 exceeding the threshold value, thereby determining the number of fluorescent fine particles 130 captured on the track region 103. measure.

  By analyzing the ratio of the fluorescent fine particles 120 and the fluorescent fine particles 130 to the total number of fine particles captured on the track region 103, or the ratio of the fluorescent fine particles 120 and the fluorescent fine particles 130, a plurality of types of detection for the exosomes. The antigen of interest can be identified.

  FIG. 9 shows a spectrum of background noise when fluorescent microparticles bound to exosomes are detected by the analyzer 1 at a reproduction linear velocity of 4.917 m / s using the sample analysis disk 100 having a track pitch of 320 nm. Yes. FIG. 9 shows a background noise spectrum when the analysis apparatus 1 detects the gate signal GS as an example, and a fluorescence detection signal FS detected continuously without using the gate signal GS as a comparative example. It shows the spectrum of background noise. When fluorescent particles are actually detected, a particle detection signal having a wide frequency band is added to the background noise, so that comparison is difficult.

  Here, the noise level of the sample analysis disk 100 in which no fluorescent fine particles are present is measured using a gate pulse generated based on the existence ratio of the fluorescent fine particles under stable measurement conditions in which aggregation of the fluorescent fine particles is difficult to occur. Under stable measurement conditions, the abundance ratio of the fluorescent fine particles does not exceed 20%. In this case, the noise level can be reduced to 1/5.

  Therefore, according to the analyzer 1, a plurality of types of detection target antigens can be identified with respect to exosomes in a simpler manner and in a shorter time than before. Moreover, according to the analyzer 1, the gate signal GS is generated based on the light amount of the detection light L2, and the fluorescence detection signals FS1 and FS2 during the period when the gate signal GS is at a high level are extracted, thereby affecting the influence of background noise. Can be reduced. Thereby, the erroneous detection in the period when the fluorescence detection signals FS1 and FS2 are not detected can be suppressed.

[Analyzer of Second Embodiment]
The analyzer according to the second embodiment will be described with reference to FIG. In addition, in order to make description easy to understand, the same reference numerals are given to the same components as those of the analyzer 1 of the first embodiment.

  The analyzer 31 of the second embodiment includes a laser light source 2, a collimator lens 3, beam splitters 4 and 5, an objective lens 6, condenser lenses 7a, 7b, 8, and 9, a wavelength separation prism 10, Band pass filters 11 and 12, a photodetector 13, fluorescence detectors 14 and 15, a detection signal generation circuit 40, and a measurement unit 16 are provided.

  The detection signal generation circuit 40 includes a particle detection circuit 21, a gate signal generation circuit 22, a fluorescence detection circuit 23, and a laser light source control circuit 41. That is, the analyzer 31 of the second embodiment is different from the analyzer 1 of the first embodiment in that the detection signal generation circuit 40 further includes a laser light source control circuit 41. The configuration is the same. Therefore, differences from the analyzer 1 of the first embodiment will be described.

  The operation of the detection signal generation circuit 40 will be described with reference to the time charts shown in FIGS. (A) and (b) in FIG. 11 correspond to (a) and (b) in FIG. In addition, in order to make the explanation easy to understand, the same reference numerals are given to the same signals as those in FIGS. 8A and 8B.

  The light amount LV of the detection light L2 is input from the photodetector 13 to the fine particle detection circuit 21. The laser beam L1 is scanned along the recess 102 of the track area 103. When the laser light L1 is scanned on the fluorescent fine particles 120 and 130, the light quantity LV of the detection light L2 changes. For example, in FIGS. 11A and 11B, when the laser light L1 is scanned over the fluorescent fine particles 120 and 130, the light quantity LV of the detection light L2 decreases.

  The fine particle detection circuit 21 generates a fine particle detection signal BS that is a pulse signal that becomes a high level during a period in which the light amount LV exceeds the threshold value, and outputs it to the measurement unit 16 and the gate signal generation circuit 22. The fine particle detection circuit 21 may detect the time point t1 when the light amount LV exceeds the threshold value and output the detected time point t1 to the gate signal generation circuit 22.

  The gate signal generation circuit 22 generates a gate signal GS that rises after a period Ta from the time t1 when the particle detection signal BS rises and further falls after a period Tb, and outputs the gate signal GS to the fluorescence detection circuit 23 and the laser light source control circuit 41.

  The laser light source control circuit 41 outputs a laser drive signal CS for adjusting the light quantity LB1 of the laser light L1 to the laser light source 2 during the period when the gate signal GS is at a high level. Based on the laser drive signal CS, the laser light source 2 increases the light quantity LV1 of the laser light L1 during the period when the gate signal GS is at a high level. Thereby, the light amount FV11 of the fluorescent light FL1 emitted from the fluorescent fine particles 120 and the light amount FV12 of the fluorescent light FL2 emitted from the fluorescent fine particles 130 are increased. Therefore, the SN ratio of the light amounts FV11 and FV12 can be increased.

  The fluorescence detection circuit 23 receives the light amount FV11 of the fluorescence FL1 from the fluorescence detector 14, the light amount FV12 of the fluorescence FL2 from the fluorescence detector 15, and the gate signal GS from the gate signal generation circuit 22. The fluorescence detection circuit 23 extracts the light amount FV11 during the period when the gate signal GS is at a high level, generates the fluorescence detection signal FS11, and outputs the fluorescence detection signal FS11 to the measurement unit 16. In addition, the fluorescence detection circuit 23 extracts the light amount FV12 during the period when the gate signal GS is at a high level, generates the fluorescence detection signal FS12, and outputs the fluorescence detection signal FS12 to the measurement unit 16.

  The measuring unit 16 counts the input fine particle detection signal BS and measures the total number of fine particles captured on the track region 103. The measuring unit 16 compares the input fluorescence detection signal FS11 with a threshold value, and counts the fluorescence detection signal FS11 exceeding the threshold value, thereby measuring the number of fluorescent fine particles 120 captured on the track region 103. . Further, the measurement unit 16 compares the input fluorescence detection signal FS12 with a threshold value, and counts the fluorescence detection signal FS12 exceeding the threshold value, thereby determining the number of fluorescent fine particles 130 captured on the track region 103. measure.

  By analyzing the ratio of the fluorescent fine particles 120 and the fluorescent fine particles 130 to the total number of fine particles captured on the track region 103, or the ratio of the fluorescent fine particles 120 and the fluorescent fine particles 130, a plurality of types of detection for the exosomes. The antigen of interest can be identified.

  Therefore, according to the analyzer 31, a plurality of types of detection target antigens can be identified with respect to exosomes in a simpler manner and in a shorter time than before. Further, according to the analysis device 31, the gate signal GS is generated based on the light amount of the detection light L2, and the fluorescence detection signals FS11 and FS12 are extracted during a period in which the gate signal GS is at a high level, thereby affecting the influence of background noise. Can be reduced. Thereby, the erroneous detection in the period when the fluorescence detection signals FS11 and FS12 are not detected can be suppressed.

  Further, according to the analyzer 31, the SN ratio of the fluorescence detection signals FS1 and FS2 is increased by increasing the light quantity LV1 of the laser light L1 during the period when the gate signal GS is at a high level. , 130 fluorescence detection accuracy can be improved.

  Although the amount of fluorescent light can be increased by always setting the output of the laser beam L1 to a high level, the drive current of the laser beam L1 increases and the temperature of the laser chip constituting the laser light source 2 rises. The life of the laser chip is reduced. According to the analyzer 31, the presence of the fluorescent fine particles can be detected and the output of the laser light L1 can be increased, so that the time for increasing the output of the laser light L1 for fluorescence detection can be limited. Thereby, it is possible to suppress the heat generation of the laser chip, and it is possible to prevent the life of the laser chip from being reduced.

  The analyzer 31 performs servo control such as tracking control and focus control based on the detection light L2 detected by the photodetector 13. If the light quantity LV1 of the laser beam L1 increases during the period when the gate signal GS is at a high level, the light reception level of the photodetector 13 may be saturated. When the light receiving level of the photodetector 13 is saturated, there are cases where desired servo control cannot be executed.

  Therefore, it is preferable to control each circuit so that the detection light L2 is not detected or the state immediately before the light quantity LV1 of the laser light L1 increases during the period in which the gate signal GS is at a high level. . The period during which the gate signal GS is at a high level is very short compared to the period during which servo control is required, and therefore the influence on the servo control is small.

[Analyzer of Third Embodiment]
The analyzer according to the third embodiment will be described with reference to FIG. In addition, in order to make description easy to understand, the same reference numerals are given to the same components as those of the analyzer 1 of the first embodiment.

  The analyzer 51 of the third embodiment includes a laser light source 2, a collimator lens 3, beam splitters 4 and 5, an objective lens 6, condenser lenses 7a, 7b, and 8, a bandpass filter 52, and a diffraction grating. 53, a photodetector 13, a fluorescence detector 54, a detection signal generation circuit 20, and a measurement unit 16.

  The diffraction grating 53 separates the fluorescence FL1 and the fluorescence FL2. The fluorescence detector 54 has a plurality of fluorescence detection units 55 and 56. The fluorescence detection unit 55 detects fluorescence FL1, and the fluorescence detection unit 56 detects fluorescence FL2. The fluorescence detection unit 55 corresponds to the fluorescence detector 14, and the fluorescence detection unit 56 corresponds to the fluorescence detector 15.

  That is, the analysis device 51 of the third embodiment is different from the analysis device 1 of the first embodiment in that the diffraction grating 53 is provided and the fluorescence detector 54 has a plurality of fluorescence detection units 55 and 56. Is different. Specifically, in the analyzer 1, the fluorescence FL 1 and the fluorescence FL 2 are separated by the wavelength separation prism 10, whereas in the analyzer 51, they are separated by the diffraction grating 53. Therefore, in the analyzer 51, the wavelength separation prism 10 and the condenser lens 9 can be omitted from the configuration.

  In addition, the analyzer 1 includes a plurality of fluorescence detectors 14 and 15, whereas in the analyzer 51, the fluorescence detector 54 includes a plurality of fluorescence detectors 55 and 56. Therefore, a plurality of fluorescence FL1, FL2 can be detected by a single fluorescence detector 54. The band-pass filter 52 is a fluorescent filter that has a wavelength band that transmits the fluorescences FL1 and FL2 and blocks light having a wavelength other than the wavelength band.

  The fluorescent light FL1 and FL2 are easily affected by background noise because the light amount and the light intensity are very small compared to the detection light L2. The bandpass filter 52 can reduce the influence of background noise. Therefore, the analyzer 51 can be a single bandpass filter 52. Except for the above differences, the configuration of the analyzer 1 is the same.

  The laser light source 2 emits laser light L1. The collimator lens 3 makes the laser light L1 parallel light. The beam splitter 4 transmits the laser light L1. The objective lens 6 focuses the laser beam L1 on the track region 103 of the sample analysis disk 100, specifically on the recess 102 of the track region 103.

  The laser light L1 is reflected by the track region 103 and enters the beam splitter 4 through the objective lens 6 as detection light L2. The beam splitter 4 reflects the detection light L2 toward the beam splitter 5. The beam splitter 5 transmits the detection light L2. The condensing lenses 7a and 7b collect the detection light L2 toward the photodetector 13. The photodetector 13 detects the light quantity LV of the detection light L2 and outputs it to the detection signal generation circuit 20.

  When the fluorescent fine particles 120 are captured in the recesses 102 of the track region 103, the fluorescent fine particles 120 are excited by irradiating the fluorescent fine particles 120 with the laser light L1, and the fluorescent fine particles 120 are fluorescent. FL1 emits light. The fluorescence FL1 enters the beam splitter 4 via the objective lens 6. The beam splitter 4 reflects the fluorescence FL1 toward the beam splitter 5.

  The beam splitter 5 reflects the fluorescence FL <b> 1 toward the band pass filter 52. The band pass filter 52 transmits the fluorescence FL1. The diffraction grating 53 diffracts the fluorescence FL1 so that the fluorescence FL1 is irradiated to the fluorescence detection unit 55 of the fluorescence detector 54. The condensing lens 8 condenses the fluorescence FL1 toward the fluorescence detection unit 55. The fluorescence detector 54 detects the amount of light FV1 of the fluorescence FL1 by the fluorescence detection unit 55 and outputs it to the detection signal generation circuit 20.

  When the fluorescent fine particles 130 are captured in the recesses 102 of the track region 103, the fluorescent fine particles 130 are excited by irradiating the fluorescent fine particles 130 with the laser light L1, and the fluorescent fine particles 130 are excited. FL2 is emitted. The fluorescence FL2 enters the beam splitter 4 via the objective lens 6. The beam splitter 4 reflects the fluorescence FL 2 toward the beam splitter 5.

  The beam splitter 5 reflects the fluorescent light FL <b> 2 toward the band pass filter 52. The band pass filter 52 transmits the fluorescence FL2. The diffraction grating 53 diffracts the fluorescence FL2 so that the fluorescence FL2 is irradiated to the fluorescence detection unit 56 of the fluorescence detector 54. The condensing lens 8 condenses the fluorescence FL2 toward the fluorescence detection unit 56. The fluorescence detector 54 detects the light amount FV2 of the fluorescence FL2 by the fluorescence detection unit 56 and outputs it to the detection signal generation circuit 20.

  The configurations and operations of the detection signal generation circuit 20 and the measurement unit 16 of the analyzer 51 are the same as the configurations and operations of the detection signal generation circuit 20 and the measurement unit 16 of the analysis device 1.

  Therefore, according to the analyzer 51, it is possible to identify a plurality of types of detection target antigens with respect to exosomes in a simpler manner and in a shorter time than before. Further, according to the analysis device 51, the gate signal GS is generated based on the light amount of the detection light L2, and the fluorescence detection signals FS1 and FS2 in a period in which the gate signal GS is at a high level are extracted. Can be reduced. Thereby, the erroneous detection in the period when the fluorescence detection signals FS1 and FS2 are not detected can be suppressed.

[Analyzer of Fourth Embodiment]
The analyzer of 4th Embodiment is demonstrated using FIG. In addition, in order to make explanation easy to understand, the same reference numerals are given to the same components as the analyzers 1, 31, 51 of the first to third embodiments.

  The analysis device 61 of the fourth embodiment includes a laser light source 2, a collimator lens 3, beam splitters 4 and 5, an objective lens 6, condenser lenses 7a, 7b, and 8, a bandpass filter 52, a diffraction grating. 53, a photodetector 13, a fluorescence detector 54, a detection signal generation circuit 40, and a measurement unit 16.

  The analysis device 61 of the fourth embodiment is different from the analysis device 51 of the third embodiment in that it includes a detection signal generation circuit 40 of the analysis device 31 of the second embodiment, and other configurations and operations. Are the same. The configuration and operation of the detection signal generation circuit 40 of the analysis device 61 of the fourth embodiment are the same as those of the detection signal generation circuit 40 of the analysis device 31 of the second embodiment.

  Therefore, according to the analyzer 61, the gate signal GS is generated based on the light amount of the detection light L2, and the fluorescence detection signals FS11 and FS12 are extracted during a period in which the gate signal GS is at a high level. Can be reduced. Thereby, the erroneous detection in the period when the fluorescence detection signals FS11 and FS12 are not detected can be suppressed.

  Further, according to the analyzer 61, the SN ratio of the fluorescence detection signals FS1 and FS2 is increased by increasing the light quantity LV1 of the laser light L1 during the period when the gate signal GS is at the high level. , 130 fluorescence detection accuracy can be improved.

  Note that the analyzer 61 performs servo control such as tracking control and focus control based on the detection light L2 detected by the photodetector 13. If the light quantity LV1 of the laser beam L1 increases during the period when the gate signal GS is at a high level, the light reception level of the photodetector 13 may be saturated. When the light receiving level of the photodetector 13 is saturated, there are cases where desired servo control cannot be executed.

  Therefore, it is preferable to control each circuit so that the detection light L2 is not detected or the state immediately before the light quantity LV1 of the laser light L1 increases during the period in which the gate signal GS is at a high level. . The period during which the gate signal GS is at a high level is very short compared to the period during which servo control is required, and therefore the influence on the servo control is small.

  The analysis apparatuses 1, 31, 51, 61 and the analysis method of the first to fourth embodiments described above may be applied to analysis of viruses, protein complexes, protein aggregates, and the like other than exosomes.

  The present invention is not limited to the embodiment described above, and various modifications can be made without departing from the spirit of the present invention.

  For example, three or more types of fluorescent fine particles may be captured on the sample analysis disk 100. By specifically binding fluorescent microparticles having different fluorescence wavelengths in order from proteins with abundant abundance, almost all exosomes having proteins with abundant abundance can be detected. Finally, proteins present in many exosomes and fluorescent fine particles may be bound.

  In the analysis devices 31 and 61, the laser light source control circuit 41 may increase the light amount LV1 of the laser light L1 in a period shorter than the above period within the period in which the gate signal GS is at a high level. .

DESCRIPTION OF SYMBOLS 1 Analyzer 2 Laser light source 13 Photodetector 14 Fluorescence detector (1st fluorescence detection part)
15 Fluorescence detector (second fluorescence detector)
20 Detection signal generation circuit 100 Sample analysis disk 120 Fluorescent fine particles (first fluorescent fine particles)
130 Fluorescent fine particles (second fluorescent fine particles)
200 Exosome 203 antigen (first antigen)
204 antigen (second antigen)
L1 laser light L2 detection light FL1 fluorescence (first fluorescence)
FL2 fluorescence (second fluorescence)
GS gate signal FS1 fluorescence detection signal (first fluorescence detection signal)
FS2 fluorescence detection signal (second fluorescence detection signal)

Claims (3)

A laser light source for irradiating a sample analysis disk in which an exosome having a first antigen to which a first fluorescent fine particle is bound or a second antigen to which a second fluorescent fine particle is bound is captured;
A photodetector for detecting, as detection light, laser light reflected by the sample analysis disk;
A first fluorescence detection unit that detects first fluorescence emitted from the first fluorescent fine particles when irradiated with the laser beam;
A second fluorescence detection unit for detecting second fluorescence emitted from the second fluorescent fine particles by irradiation with the laser light;
A gate signal is generated based on the detection light, a first fluorescence detection signal is generated from the first fluorescence based on the gate signal, and a second fluorescence is generated from the second fluorescence based on the gate signal. A detection signal generation circuit for generating a detection signal;
An analysis apparatus comprising:   The analyzer according to claim 1, wherein the detection signal generation circuit includes a laser light source control circuit that adjusts a light amount of the laser light based on the gate signal. Irradiating the sample analysis disk on which the exosome having the first antigen to which the first fluorescent fine particles are bound or the second antigen to which the second fluorescent fine particles are bound is captured with a laser beam;
Detecting the laser beam reflected by the sample analysis disk as detection light,
First fluorescence emitted from the first fluorescent fine particles when irradiated with the laser light, or second fluorescence emitted from the second fluorescent fine particles when irradiated with the laser light. Detect
Generating a gate signal based on the detection light;
An analysis method comprising: generating a first fluorescence detection signal from the first fluorescence based on the gate signal; and generating a second fluorescence detection signal from the second fluorescence based on the gate signal. .

Images