JP2007047110A - Fluorescent material detection device and specimen analysis device including it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescent material detection device capable of performing highly accurate detection to various biochemical reaction cartridges in each proper condition. <P>SOLUTION: A reflection plate 33 on the surface of a light-transmissive DNA chip 12 is irradiated with excitation light from the back side, and reflected light by the reflection plate 33 is received by a photomultiplier 37, and the light intensity is measured. A light intensity measured value is compared with a light intensity reference value stored beforehand. The output intensity of a laser light generator 34 is adjusted corresponding to the comparison result. Then, excitation light is irradiated toward a DNA probe 32 of the DNA chip 12, and fluorescence from the excited fluorescent material is received by the photomultiplier 37. A specimen is analyzed based on its fluorescence detection pattern. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an apparatus for detecting a fluorescent substance in order to detect a sample of cells, microorganisms, chromosomes, nucleic acids, etc. in a specimen by using a biochemical reaction such as an antigen-antibody reaction or a nucleic acid hybridization reaction, The present invention relates to a sample analyzer including a fluorescent substance detection method and a sample analysis method.

  Sample analyzers for analyzing a sample such as blood include an immunological method using an antigen-antibody reaction and a method using nucleic acid hybridization. In an example of an immunological method, a protein such as an antibody or an antigen that specifically binds to a substance to be detected is used as a probe and immobilized on a solid phase surface such as a microparticle, a bead, or a glass plate, and a target in a specimen is detected. An antigen-antibody reaction with the detection substance is performed. In an example of a method using nucleic acid hybridization, a single-stranded nucleic acid is used as a probe and immobilized on a solid phase surface such as a fine particle, a bead, or a glass plate, and nucleic acid hybridization is performed with a target substance in a specimen. . These methods include labeled antibodies, labeled antigens, or labeled nucleic acids, which are labeled substances having a specific interaction carrying a labeling substance with high detection sensitivity, such as an enzyme, a fluorescent substance, or a luminescent substance. Etc. are used. Then, an antigen-antibody compound or a hybridized double-stranded nucleic acid is detected, and the presence or absence of a substance to be detected (target) is detected or quantified.

  As a development of these techniques, for example, Patent Document 1 discloses a so-called DNA array in which a large number of DNA (deoxyribonucleic acid) probes having different base sequences are arranged in an array on a substrate. .

  Non-Patent Document 1 discloses a method for preparing a protein array having the same structure as a DNA array by arranging many types of proteins on a membrane filter. As described above, by using a DNA array, a protein array, or the like, it has become possible to inspect a very large number of items at once.

  In addition, disposable biochemical reaction cartridges that allow biochemical reactions to be performed internally are proposed for the purpose of reducing sample contamination, improving reaction efficiency, miniaturizing equipment, and simplifying operations in various sample analyses. Has been. For example, Patent Document 2 discloses a configuration in which a plurality of chambers are disposed in a biochemical reaction cartridge including a DNA array. According to this biochemical reaction cartridge, a reaction such as extraction, amplification, or hybridization of DNA in a specimen can be performed internally by moving a solution to each chamber using a pressure difference.

  As a method for injecting a solution from the outside into such a biochemical reaction cartridge, there is a method using an external electric syringe pump or a vacuum pump. As a method for moving a solution inside a biochemical reaction cartridge, a method using gravity, capillary action or electrophoresis is known in addition to a pressure difference. Further, as micro pumps that can be disposed inside the biochemical reaction cartridge, Patent Document 2 includes a diaphragm pump, Patent Document 3 uses a heat generating element, and Patent Document 4 uses a piezoelectric element. Each is disclosed.

  There is a configuration in which an IC for storing information is provided on a DNA chip used as a biochemical reaction cartridge or as a part thereof, and DNA is identified using identification information stored in the IC. Specifically, Patent Document 5 discloses that information on the base sequence of a DNA chip and information on a sample are written in an information storage IC.

There is also a method of reading a fluorescent substance (fluorescent label) attached to a sample in order to detect the hybridization state of the sample on the DNA chip. Specifically, Patent Document 6 discloses a method for appropriately calibrating excitation light applied to a DNA chip so as to generate fluorescence in order to detect a target by reading a fluorescent substance.
US Pat. No. 5,445,934 Japanese National Patent Publication No. 11-509094 Japanese Patent No. 2832117 JP 2000-274375 A JP 2001-147231 A Special table 2002-538427 gazette Angelika Lueking and five others, “Protein Microarrays for Gene Expression and Antibody Screening”, Analytical Biochemistry Vol.270 (1), Academic Press (Academic Press), May 15, 1999, p.103-111

  A biochemical reaction cartridge that contains various solutions for supplying a biochemical reaction and is supplied with a specimen is preferably disposable from the viewpoint of prevention of secondary infection and contamination and ease of use. However, there is a problem that a biochemical reaction cartridge incorporating a micropump is expensive.

  Therefore, a disposable biochemical reaction cartridge is generally used, in which a solution is moved by the action of an external pump without incorporating a pump and a series of biochemical reactions can proceed without injecting the solution to the outside after sample injection. It's being used.

  Each DNA chip is for performing only a single biochemical reaction called hybridization. However, a biochemical reaction cartridge with a built-in DNA chip may continuously perform various biochemical reactions and finally perform a detection process. Depending on the biochemical reaction cartridge, for example, the material, thickness, translucency, and the like of the substrate of the built-in DNA chip may differ. As described above, even if fluorescence detection is performed by always irradiating excitation light to biochemical reaction cartridges having different materials, thicknesses, transmissivities, and the like under the same conditions, errors are large and high. The accuracy may not be detected.

  In addition, the fluorescent substance is generally easily deteriorated by excitation light, and when the excitation light is irradiated for a long time or with high intensity, the fluorescent substance may be deteriorated and the detection accuracy may be lowered.

  Accordingly, an object of the present invention is to provide a fluorescent substance detection apparatus capable of performing high-accuracy detection on various biochemical reaction cartridges under appropriate conditions, a sample analyzer including the same, a fluorescent substance detection method, and a sample analysis method. It is to provide.

  A feature of the present invention is a fluorescent substance detection apparatus for detecting a fluorescent substance on the surface of a substrate having translucency, an excitation light generating means for generating excitation light irradiated toward the back surface of the substrate, and a transparent substance. First light receiving means for receiving excitation light attenuated by passing through a substrate having optical properties, second light receiving means for receiving fluorescence emitted by a fluorescent material excited by excitation light, and first light receiving means And adjusting means for adjusting the output intensity of the excitation light in accordance with the measured light intensity value of the attenuated excitation light received.

  Another feature of the present invention is a fluorescent substance detection method for detecting a fluorescent substance on the surface of a light-transmitting substrate, a step of irradiating excitation light toward the back surface of the substrate, and a transmission through the substrate. A comparison of the step of receiving the excitation light attenuated by the light receiving means with the light intensity measurement value of the attenuated excitation light received by the light receiving means and a preset light intensity reference value In accordance with the step of adjusting the output intensity of the excitation light, the step of irradiating the back surface of the substrate with the excitation light whose output intensity has been adjusted, and the fluorescence emitted by the fluorescent material present at the position irradiated with the excitation light, And a step of detecting by the light receiving means.

  According to the present invention, a fluorescent substance can be detected with high accuracy by using excitation light having an appropriate light intensity even when using various substrates.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  In the following embodiments, a form using a biochemical reaction cartridge will be described in detail. However, the present invention is not limited to this, and can also be used as a so-called DNA chip detection device. In that case, depending on the optical system used, there may be a form in which no reflecting means is provided.

[First Embodiment]
FIG. 1 schematically shows a sample analyzer according to the first embodiment of the present invention. The processing apparatus of this sample analyzer has a table 13 on which the biochemical reaction cartridge 1 that is a reaction field in the present embodiment is placed. On the table 13, an electromagnet 14, Peltier elements 15 and 16, and a first communication unit 26 are arranged, and these are connected to a control unit 17 that controls the entire processing apparatus. The electromagnet 14 applies electromagnetic force to the biochemical reaction cartridge 1. The Peltier elements 15 and 16 are for controlling the temperature of the biochemical reaction cartridge 1. The first communication unit 26 transmits power and transmits / receives signals to / from the IC chip 25 in order to store data of the processing apparatus in the IC chip 25 in the biochemical reaction cartridge 1 described later. .

  On both sides of the table 13, there are electric syringe pumps 18, 19 and pump blocks 22, which are outlets for discharging or sucking air by these pumps 18, 19, each having ten pump nozzles 20, 21. 23 are arranged. A plurality of electric switching valves (not shown) are arranged between the electric syringe pumps 18 and 19 and the pump nozzles 20 and 21. An electric switching valve (not shown) and pumps 18 and 19 are connected to the control unit 17.

  The control unit 17 is connected to an input unit 24 for input by an inspector. The controller 17 controls the connection and disconnection of the electric syringe pumps 18 and 19 by independently opening and closing the pump nozzles 20 and 21 one by one, or opening and closing all the pump nozzles 20 and 21 simultaneously. It can be performed. Further, the controller 17 causes the biochemical reaction in the biochemical reaction cartridge 1 to be executed by appropriately operating the electromagnet 14 and the Peltier elements 15 and 16.

  A detection area 28 is provided outside the table 13 and the pump blocks 22 and 23. This detection area 28 is provided for detecting the hybridization state of the DNA chip 12 of the biochemical reaction cartridge 1, that is, for detecting a fluorescent substance.

  In the detection area 28, an XY stage 29, a fluorescence detection unit 30, and a second communication unit 31 are provided, and these are also connected to the control unit 17. The XY stage 29 is for moving the biochemical reaction cartridge 1 in the X direction (main scanning) and moving in the Y direction (sub scanning). Each time one main scan is performed, the sub-scan is performed in a direction (in the present embodiment, a direction orthogonal) intersecting the main scan direction by the scan width of the main scan. Although the detailed configuration of the fluorescence detection unit 30 will be described later, it is mainly configured by an optical device, and can measure the intensity of fluorescence generated by exciting the phosphor. Moreover, the fluorescence detection unit 30 can also adjust the intensity | strength of excitation light suitably. The second communication unit 31 transmits power and transmits / receives signals to / from the IC chip 25 in the biochemical reaction cartridge 1.

  Further, a cartridge transport unit 27 that transports the biochemical reaction cartridge 1 from the table 13 to the detection area 28 in accordance with a command from the control unit 17 is provided and connected to the control unit 17.

  Next, the configuration of the fluorescence detection unit 30 which is one of the main features of the present invention will be described with reference to FIG. The fluorescence detection unit 30 mainly includes a laser light generator 34, a splitter 35, an objective lens 36, a photomultiplier tube 37, a lens 38, and a filter 39.

  The laser light generator 34 is a laser light source for generating fluorescence by irradiating laser light (excitation light) to excite a fluorescent substance in a specimen located on the DNA chip 12 of the biochemical reaction cartridge 1. The output intensity can be adjusted. The splitter 35 bends the laser light from the laser light generator 34 by 90 degrees toward the DNA chip 12 and transmits reflected light and fluorescence from the DNA chip 12. The objective lens 36 opposes the DNA chip 12 and is adjusted in position by the control unit 17 and a vertical mechanism (not shown) to focus the laser beam on the DNA chip 12. The lens 38 collects the reflected light or fluorescence from the DNA chip 12 that has passed through the splitter 35 on the photomultiplier tube 37. The photomultiplier tube 37 receives light collected by the lens 38 and detects the incident light. The filter 39 can be moved between the optical path from the DNA chip 12 to the photomultiplier tube 37 and the outside of the optical path by the control unit 17 and a moving mechanism (not shown). When this filter 39 is located in the optical path, it transmits the fluorescence from the DNA chip 12 but blocks the reflected light from the DNA chip 12.

  Due to such a configuration, parallel light (laser light) incident on the objective lens 36 from the laser light generator 34 via the splitter 35 is focused on the DNA chip 12 by the objective lens 36. Then, the fluorescence emitted from the fluorescent material excited by the laser light and the reflected light from the later-described reflecting plate 33 provided on the DNA chip 12 are converted into parallel light by the objective lens 36 and then transmitted through the splitter 35. . The parallel light transmitted through the splitter is left as it is when the filter 39 is outside the optical path, and only the fluorescence is transmitted when the filter 39 is located on the optical path, and the photomultiplier tube is passed through the lens 38. 37 is incident. The photomultiplier tube 37 detects incident light and measures its light intensity.

  Next, the biochemical reaction cartridge 1 serving as a reaction field in this processing apparatus will be described in detail.

  The body (housing) of the biochemical reaction cartridge 1 is a transparent or translucent synthetic material such as polymethyl methacrylate (PMMA), acrylonitrile-butadiene-styrene (ABS) copolymer, polystyrene, polycarbonate, polyester, polyvinyl chloride, etc. It is made of resin.

  As shown in FIG. 3, a specimen inlet 2 a for injecting a specimen such as blood using a syringe or the like is provided at the top of the biochemical reaction cartridge 1 and sealed with a rubber cap 2. The side surface of the biochemical reaction cartridge 1 is provided with a plurality of nozzle inlets 3a to 3j for inserting and pressurizing or depressurizing the nozzle to move the solution inside, and sealed by the rubber cap 3. Has been. Both sides of the cartridge 1 have the same configuration.

  The biochemical reaction cartridge 1 is provided with an IC chip 25. The IC chip 25 includes a nonvolatile storage unit and a communication unit for receiving power from outside and transmitting / receiving signals. Identification information (ID) for identifying the biochemical reaction cartridge 1 is written in the storage unit of the IC chip 25. Furthermore, it is preferable that the storage unit is also written with the type of biochemical reaction cartridge 1, the procedure of the processing step, the analysis information for analyzing the result of the biochemical reaction, and the like.

  In general, the processing apparatus or an apparatus attached thereto stores a procedure of processing steps for each type of biochemical reaction cartridge 1. As described above, when the type of the biochemical reaction cartridge 1 is written in the IC chip 25, the type of the biochemical reaction cartridge 1 is read to select a procedure of a processing step suitable for the type, and processing is performed in accordance with the procedure. However, when the type of the biochemical reaction cartridge 1 is a type that is not stored in the processing apparatus or the apparatus attached thereto, the procedure of the processing process written in the IC chip 25 is read and the procedure is read. Follow the process. The data written as the procedure of the processing step may include a temperature condition that is applied to cause a biochemical reaction.

  FIG. 4 shows a plan sectional view of the biochemical reaction cartridge 1. As described above, ten nozzle inlets 3a to 3j are provided on one side surface, and ten nozzle inlets 3k to 3t are provided on the opposite side surface. Most of the nozzle inlets 3a to 3t communicate with chambers 5a to 5t, which are places where solutions are stored or where reactions occur, via air flow paths 4a to 4t through which the respective air flows. That is, the nozzle inlets 3a to 3j communicate with the chambers 5a to 5j through the flow paths 4a to 4j. The opposite nozzle inlets 3k, 3l, 3m, 3o, 3r, 3t communicate with the chambers 5k, 5l, 5m, 5o, 5r, 5t via the flow paths 4k, 4l, 4m, 4o, 4r, 4t, respectively. ing. However, in this embodiment, the nozzle inlets 3n, 3p, 3q, 3s are not communicated with the chamber and are reserved, and are not used.

  The specimen inlet 2a communicates with the chamber 7, and the chamber 7 communicates with the chambers 5a, 5b, 5c, 5k through the flow paths 6a, 6b, 6c, 6k, and communicates with the chamber 8 through the flow path 10. is doing. The chamber 8 communicates with the chambers 5 g and 5 o through the flow paths 6 g and 6 o and also communicates with the chamber 9 through the flow path 11. The chamber 9 communicates with the chambers 5h, 5i, 5j, 5r, and 5t via the flow paths 6h, 6i, 6j, 6r, and 6t. The flow path 10 communicates with the chambers 5d, 5e, 5f, 5l, and 5m via the flow paths 6d, 6e, 6f, 61, and 6m.

A square hole is formed in the bottom surface of the chamber 9, and the DNA chip 12 shown in FIGS. 2 and 5 is attached to the square hole with the probe surface facing upward. The DNA chip 12 is obtained by arranging several tens to several hundreds of thousands of different DNA probes 32 on a solid surface of a glass plate having a size of about 1 square inch (about 645 mm ² ). In the present embodiment, a number of genes can be examined at a time by using this DNA chip 12 to cause a hybridization reaction with DNA in the specimen. These DNA probes 32 are regularly arranged in a matrix, and the addresses (positions indicated by how many rows and what columns) of each DNA probe 32 can be easily taken out as information. Examples of genes to be examined include infectious disease viruses, bacteria, disease-related genes, and individual gene polymorphisms. Further, the DNA chip 12 is provided with a reflecting plate 33 juxtaposed with a region where the DNA probes 32 are arranged in a matrix. The reflecting plate 33 reflects excitation light (laser light shown in FIG. 2) from the back surface of the DNA chip 12.

  Here, an example of the setting of the chambers 5a to 5t for processing the specimen in the present embodiment is shown. The chamber 5a contains a first hemolytic agent containing EDTA (ethylenediaminetetraacetic acid) that destroys the cell wall, and the chamber 5b contains a second hemolytic agent containing a protein denaturant such as a surfactant. . The chamber 5c contains silica-coated magnetic particles on which DNA is adsorbed. The chambers 5l and 5m contain first and second extraction detergents used for DNA purification during DNA extraction, respectively.

  The chamber 5d contains an eluate composed of a low-concentration salt buffer that elutes DNA from magnetic particles. The chamber 5g contains a mixture of chemicals necessary for PCR (polymerase chain reaction), ie, Cy3-dUTP (fluorescent label manufactured by Amersham Biosciences) containing primer, polymerase, dNTP solution, buffer, and fluorescent agent. Contained. In the chambers 5h and 5j, a cleaning agent containing a surfactant for cleaning the sample DNA with the fluorescent substance (fluorescent label) that has not been hybridized and the fluorescent substance (fluorescent label) is accommodated. The chamber 5 i contains alcohol for drying the inside of the chamber 9 including the DNA chip 12.

  The chamber 5e is a chamber for collecting dust other than blood DNA. The chamber 5f is a chamber for storing waste liquids of the first and second extracted cleaning agents from the chambers 5l and 5m. The chamber 5r is a chamber for storing waste liquids of the first and second cleaning agents. The chambers 5k, 5o, and 5t are blank chambers provided to prevent the solution from flowing into the nozzle inlet.

  In the present embodiment, a liquid specimen such as blood is injected into the biochemical reaction cartridge 1 and set in the processing apparatus (see FIG. 1). Then, DNA and the like are extracted and amplified inside the biochemical reaction cartridge 1, and hybridization is performed between the amplified sample DNA and the DNA probe 32 of the DNA chip 12 inside the biochemical reaction cartridge 1. To do. On the other hand, the sample DNA with the fluorescent substance (fluorescent label) that has not been hybridized and the fluorescent substance (fluorescent label) can be washed.

  A method for generating a biochemical reaction of a specimen in the biochemical reaction cartridge 1 in this embodiment using such a processing apparatus and the biochemical reaction cartridge 1 will be specifically described.

  First, an examiner passes a rubber cap 2 that closes the sample inlet 2a of the biochemical reaction cartridge 1 through a needle (not shown) of a syringe containing blood as a sample, and samples the blood in the syringe. Injection into the chamber 7 from the inlet 2a. Thereafter, the examiner places the biochemical reaction cartridge 1 on the table 13. Then, the inspector operates the lever (not shown) to move the pump blocks 22 and 23 in the direction of the arrow in FIG. Then, the pump nozzles 20 and 21 are inserted through the rubber cap 3 into the nozzle inlets 3 a to 3 t on both sides of the biochemical reaction cartridge 1.

  When the inspector inputs an instruction to start an experiment from the input unit 24, the process starts. FIG. 6 is a flowchart for explaining the procedure of biochemical reaction and subsequent treatment.

  First, in step S <b> 1, the control unit 17 controls the pump nozzles 20 and 21 to open only the nozzle inlets 3 a and 3 k, ejects air from the electric syringe pump 18, and sucks air from the electric syringe pump 19. Thereby, the first hemolytic agent in the chamber 5a is poured into the chamber 7 containing blood. As described above, the pump nozzle 20 is inserted into the nozzle inlet 3a and air is ejected to pressurize, and the pump nozzle 21 is inserted into the nozzle inlet 3k to suck and reduce the pressure of the air in the chamber 5a. The hemolytic agent flows into the chamber 7 containing the blood. This is shown in FIG. 7, which is a cross-sectional view through the chambers 5a, 7, 5k.

  In the present embodiment, by shifting the timing of air supply and suction, it is possible to control the pressurization and decompression of each chamber to smoothly flow the solution through the cartridge 1. Furthermore, it is possible to flow the solution more smoothly by performing fine control such as linearly increasing the suction of air by the electric syringe pump 19 from the start of the air from the pump 18. For example, depending on the viscosity of the hemolytic agent and the resistance of the flow path, in step S1, the suction of air from the electric syringe pump 19 is started and the ejection of air from the electric syringe pump 18 is started for 10 to 200 milliseconds. Control to start later. According to this, the solution does not jump out at the beginning of the flowing solution, and the solution flows smoothly. The same applies to the movement of the solution in the following steps.

  Further, while the air supply is easily controlled using the electric syringe pumps 18 and 19, only the nozzle inlets 3 a and 3 o are opened, and the electric syringe pumps 18 and 19 alternately repeat the ejection and suction of air. In this way, the solution in the chamber 7 is caused to flow through the flow path 10 and then returned to the agitation to repeat the stirring. Alternatively, stirring is performed while bubbles are generated by continuously ejecting air from the electric syringe pump 19.

  When the first hemolytic agent flow and stirring step (step S1) is performed in this way, a signal is transmitted from the first communication unit 26 to the communication unit (not shown) of the IC chip 25. . This signal represents the completion of the first hemolytic fluid flow and stirring process, various conditions of the process, and the end time, and is stored in a storage unit (not shown) of the IC chip 25. .

  Next, in step S2, only the nozzle inlets 3b and 3k are opened, and the second hemolytic agent in the chamber 5b is poured into the chamber 7 on the same principle as in step S1. And the same stirring as step S1 is performed.

  When the second hemolytic agent flow and stirring step (step S2) is performed as described above, a signal is transmitted from the first communication unit 26 to the communication unit (not shown) of the IC chip 25. This signal indicates the completion of the second hemolytic agent flow and stirring process, various conditions of the process, and the end time, and is stored in a storage unit (not shown) of the IC chip 25. .

  In step S3, only the nozzle inlets 3c and 3k are opened, and the magnetic particles in the chamber 5c are poured into the chamber 7 on the same principle as in steps S1 and S2. And the same stirring as step S1 is performed. By this step S3, the DNA obtained by the cell lysis in steps S1 and S2 adheres to the magnetic particles.

  After the magnetic particle flow and stirring step (step S3) is performed in this way, a signal is transmitted from the first communication unit 26 toward the communication unit (not shown) of the IC chip 25. This signal represents the completion of the flow and stirring steps of the magnetic particles, various conditions of the steps, and the end time, and is stored in a storage unit (not shown) of the IC chip 25.

  In step S4, the electromagnet 14 is turned on, only the nozzle inlets 3e and 3k are opened, air is ejected from the electric syringe pump 19, air is sucked from the electric syringe pump 18, and the solution in the chamber 7 is discharged into the chamber 5e. Move to. During this movement, the magnetic particles and DNA are captured at a position above the electromagnet 14 in the flow channel 10. Note that the efficiency of DNA capture is improved by alternately repeating the suction and ejection of air by the electric syringe pumps 18 and 19 to reciprocate the solution between the chambers 7 and 5e twice. If the number of reciprocations is further increased, the capture efficiency can be further increased. However, extra processing time is required.

  As described above, in steps S1 to S4, the DNA in a fluidized state is captured very efficiently using magnetic particles on the small flow path 10 having a width of about 1 to 2 mm and a height of about 0.2 to 1 mm. To do. It should be noted that if the capture target substance is not DNA but RNA or protein, efficient capture can be performed similarly.

  When the magnetic particle and DNA capturing step (step S4) is thus performed, a signal is transmitted from the first communication unit 26 toward the communication unit (not shown) of the IC chip 25. This signal represents the completion of the magnetic particle and DNA capturing process, various conditions of the process, and the end time, and is stored in a storage unit (not shown) of the IC chip 25.

  Next, in step S5, the electromagnet 14 is turned off, and only the nozzle inlets 3f and 3l are opened. Then, air is ejected from the electric syringe pump 19 and air is sucked from the electric syringe pump 18 to move the first extraction cleaning liquid in the chamber 5l to the chamber 5f. At this time, the magnetic particles and DNA captured in step S4 move together with the extraction cleaning liquid to perform cleaning. Further, the electromagnet 14 is turned on, and the magnetic particles, DNA, and extraction cleaning liquid are reciprocated twice between the chamber 5l and the chamber 5f by the same principle as in step S4. Thus, the washed magnetic particles and DNA are collected at a position above the electromagnet 14 in the flow path 10 and the first extraction washing liquid is returned to the chamber 5l.

  As described above, after performing the step of capturing the magnetic particles and DNA (step S5) after washing with the first extraction washing liquid, the first communication unit 26 sends the communication unit (not shown) of the IC chip 25 to the communication unit. Send a signal to. This signal represents the completion of the process of capturing the magnetic particles and DNA after washing with the first extraction washing liquid, various conditions of the process, and the end time. (Not shown).

  In step S6, only the nozzle inlets 3f and 3m are opened, and the same process as in step S5 is performed. That is, the second extraction washing liquid in the chamber 5m, the magnetic particles and the DNA are moved, and the magnetic particles and the DNA are further washed and then recovered at a position above the electromagnet 14, and the second extraction washing liquid is used as the second extraction washing liquid. Return to chamber 5m.

  As described above, after performing the step of capturing the magnetic particles and DNA (step S6) after washing with the second extraction washing liquid, the first communication unit 26 sends the communication unit (not shown) of the IC chip 25 to the communication unit. Send a signal to. This signal represents the completion of the process of capturing the magnetic particles and DNA after washing with the second extraction washing liquid, various conditions of the process, and the end time. (Not shown).

  Subsequently, with the electromagnet 14 turned on in step S7, only the nozzle inlets 3d and 3o are opened, air is ejected from the electric syringe pump 18, and air is sucked from the electric syringe pump 19. Thereby, the eluate in the chamber 5 d is moved to the chamber 8. Due to the action of the eluate, the magnetic particles and DNA are separated, and only the DNA moves to the chamber 8 together with the eluate, and the magnetic particles remain in the flow path 10.

  In this way, when the step of separating the magnetic particles and DNA by flowing the eluate (step S7) is performed, a signal is sent from the first communication unit 26 to the communication unit (not shown) of the IC chip 25. Send. This signal indicates the completion of the step of separating the magnetic particles and DNA by flowing the eluate, the various conditions of the step, and the end time, and is stored in a storage unit (not shown) of the IC chip 25. Remembered.

  In step S7, DNA extraction and purification are performed. In this embodiment, the chambers 5l and 5m for storing the extracted cleaning liquid and the chamber 5f for storing the waste liquid after the cleaning are prepared, so that DNA can be extracted and purified in the cartridge 1.

  Next, in step S8, only the nozzle inlets 3g and 3o are opened, air is ejected from the electric syringe pump 18, and air is sucked from the electric syringe pump 19. Thereby, the PCR agent (for example, a mixed solution of primer, polymerase, dNTP solution, buffer, fluorescent label, etc.) in the chamber 5 g is poured into the chamber 8. Further, only the nozzle inlets 3g and 3t are opened, and the ejection and suction of air by the electric syringe pumps 18 and 19 are alternately repeated, the solution in the chamber 8 is allowed to flow through the flow path 11, and then the operation of returning to it is repeated for stirring. Do. Then, after controlling the Peltier element 15 and holding the solution in the chamber 8 at a temperature of 96 ° C. for 10 minutes, a cycle of 96 ° C. · 10 seconds, 55 ° C. · 10 seconds, 72 ° C. · 1 minute is repeated 30 times. PCR is performed to amplify the eluted DNA.

  Thus, after performing the amplification process (step S8) of the eluted DNA, a signal is transmitted from the first communication unit 26 to the communication unit (not shown) of the IC chip 25. This signal represents the completion of the amplification process of the eluted DNA, various conditions of the process, and the end time, and is stored in a storage unit (not shown) of the IC chip 25.

  In step S 9, only the nozzle inlets 3 g and 3 t are opened, air is ejected from the electric syringe pump 18, air is sucked from the electric syringe pump 19, and the solution in the chamber 8 is moved to the chamber 9. Further, the Peltier element 16 is controlled to hold the solution in the chamber 9 at 45 ° C. for 2 hours to perform hybridization. At this time, the ejection and suction of air by the electric syringe pumps 18 and 19 are alternately repeated, the solution in the chamber 9 is moved to the flow path 6t, and the subsequent return operation is repeated to perform the hybridization while stirring. .

  In this way, when the hybridization step (step S9) is performed, a signal is transmitted from the first communication unit 26 toward the communication unit (not shown) of the IC chip 25. This signal represents completion of the hybridization process, various conditions of the process, and end time, and is stored in a storage unit (not shown) of the IC chip 25.

  Next, in step S10, while maintaining the temperature at 45 ° C., only the nozzle inlets 3h and 3r are opened, air is ejected from the electric syringe pump 18, and air is sucked from the electric syringe pump 19. Accordingly, the solution in the chamber 9 is moved to the chamber 5r, and the first cleaning liquid in the chamber 5h is poured into the chamber 5r through the chamber 9. Thus, the first cleaning liquid in the chamber 5h is inserted by inserting the pump nozzle 20 into the nozzle inlet 3h and ejecting air to pressurize it, and inserting the pump nozzle 21 into the nozzle inlet 3r and sucking air to reduce the pressure. Flows into the chamber 5r through the chamber 9. This is shown in FIG. 8, which is a cross-sectional view through the chambers 5h, 9, 5r. By alternately repeating the suction and ejection of the electric syringe pumps 18 and 19, this solution is reciprocated twice between the chambers 5h, 9, and 5r, and finally returned to the chamber 5h. In this way, the fluorescently labeled sample DNA and the fluorescent label that have not been hybridized are washed.

  In this way, when the cleaning process using the first cleaning liquid (step S10) is performed, a signal is transmitted from the first communication unit 26 to the communication unit (not shown) of the IC chip 25. This signal represents the completion of the first cleaning process, various conditions of the process, and the end time, and is stored in the storage unit (not shown) of the IC chip 25.

  Further, in step S11, while maintaining the temperature at 45 ° C., only the nozzle inlets 3j and 3r are opened, and the same process as in step S10 is performed. That is, the second washing solution in the chamber 5j is poured into the chamber 5r through the chamber 9 to further wash the DNA, and finally the solution is returned to the chamber 5j.

  Thus, when the cleaning process using the second cleaning liquid (step S11) is performed, a signal is transmitted from the first communication unit 26 to the communication unit (not shown) of the IC chip 25. This signal represents the completion of the cleaning process with the second cleaning liquid, various conditions of the process, and the end time, and is stored in a storage unit (not shown) of the IC chip 25.

  In this embodiment, the chambers 5h and 5j for storing the first and second cleaning liquids and the chamber 5r for storing the waste liquid after the cleaning are prepared, so that the DNA chip is provided in the biochemical reaction cartridge 1 as described above. It is possible to perform 12 washings.

  In step S12, only the nozzle inlets 3i and 3r are opened, air is ejected from the electric syringe pump 18, air is sucked from the electric syringe pump 19, and the alcohol in the chamber 5i is moved through the chamber 9 to the chamber 5r. . Thereafter, only the nozzle inlets 3i and 3t are opened, air is ejected from the electric syringe pump 18, and air is sucked from the electric syringe pump 19 to dry the inside of the chamber 9.

  When the alcohol flow and drying step (step S12) is performed in this way, a signal is transmitted from the first communication unit 26 toward the communication unit (not shown) of the IC chip 25. This signal represents the completion of the alcohol flow and drying process, various conditions of the process, and the end time, and is stored in a storage unit (not shown) of the IC chip 25.

  Through the steps S1 to S12 described above, the biochemical reaction (for example, DNA hybridization) of the specimen can be performed in the biochemical reaction cartridge 1.

  In the present embodiment, the nozzle inlets 3a to 3t are concentrated on the two surfaces of the cartridge 1, that is, both side portions. Therefore, the shape and arrangement of the electric syringe pumps 18 and 19, the electric switching valve, the pump blocks 22 and 23 incorporating the pump nozzle, and the like can be simplified. Furthermore, the pump nozzles 20 and 21 can be inserted by a simple operation of simultaneously sandwiching the biochemical reaction cartridge 1 by the pump blocks 22 and 23 while securing necessary chambers and flow paths. Accordingly, the configuration of the pump blocks 22 and 23 can be simplified. Further, by arranging the nozzle inlets 3a to 3t so as to be all linearly arranged at the same height, the heights of the flow paths 4a to 4t connected to the nozzle inlets 3a to 3t are all the same, and the flow path 4a. Production of ˜4t is facilitated.

  Further, in the processing apparatus shown in FIG. 1, the pump blocks 22 and 23 can be configured to be n times longer so that n biochemical reaction cartridges 1 can be used simultaneously. In that case, n biochemical reaction cartridges 1 can be arranged in series, and necessary steps can be simultaneously performed on each of the n biochemical reaction cartridges 1. Therefore, the biochemical reaction can be performed simultaneously in a large number of biochemical reaction cartridges, though the configuration is extremely simple.

  After the processing steps described above (steps S1 to S12), in step S13, the inspector operates a lever (not shown) to move the pump blocks 22 and 23 away from the biochemical reaction cartridge 1. Accordingly, the pump nozzles 20 and 21 are detached from the nozzle inlets 3 a to 3 t of the biochemical reaction cartridge 1. In step S14, the biochemical reaction cartridge 1 is transported from the table 13 to the detection area 28 using the cartridge transport unit 27 (see FIG. 1). In this detection area 28, the biochemical reaction of the biochemical reaction cartridge 1 is measured and analyzed.

  In step S <b> 15, the hybridized DNA captured by the DNA chip 12 is detected by the fluorescence detection unit 30. The detection result is transmitted from the second communication unit 31 to the communication unit (not shown) of the IC chip 25 and stored in the storage unit (not shown) of the IC chip 25. In this way, the transition (including the passage of time) and various conditions of each step in steps S1 to S12 and the detection result of the DNA chip 12 that has passed through each step are stored in the IC chip 25 of the biochemical reaction cartridge 1. The details of this fluorescence detection step (step S15) will be described later.

  Then, in step S16, the control unit 17 analyzes the fluorescence detected pattern. When the biochemical reaction cartridge 1 is of a type that is not stored in the processing apparatus in advance, the analysis information written in the IC chip 25 is read in analyzing the detection pattern, and based on the analysis information. Analyze.

  Here, the principle of fluorescence detection and analysis in this embodiment will be described. For example, when the biochemical reaction cartridge 1 and the DNA chip 12 are for detecting an infectious disease, several infectious diseases can be detected at a time depending on how the DNA probe 32 is set. Taking the DNA chip 12 in which the DNA probes 32 are arranged in a 5 × 5 matrix as an example, patterns when infectious diseases A to E are detected alone are shown in FIGS. 9 (a) to 9 (e). It can be set to be 5 ways. 9A to 9E, the black circles indicate a state in which the target is captured by hybridization with the DNA probe 32, and the fluorescent substance (fluorescent label) attached to the target emits fluorescence. . On the other hand, a white circle indicates a state in which the target is not captured without being hybridized with the DNA probe 32 and there is no fluorescent label.

  In the example shown in FIGS. 9A to 9E, all the DNA probes 32 included in the same column are set to hybridize with DNA existing when infected with the same infection. Each column is set so as to correspond to a different infectious disease DNA. Therefore, when the detection result of the pattern of FIG. 9A is obtained, infection with the infectious disease A is recognized, and similarly, the detection result of the pattern of FIGS. 9B to 9E is obtained. In the case of infection, infectious diseases B to E are respectively recognized. The infection determination method based on such a detection pattern is included in the analysis information stored in advance in the control unit 17.

  As described above, in order to accurately perform the target sample analysis based on the sequence pattern of the DNA probe 32 to which the fluorescent substance is attached, it is important to accurately detect the fluorescence in step S15. In particular, it is necessary to perform accurate detection each time corresponding to various types of biochemical reaction cartridges 1 and DNA chips 12.

  Therefore, the fluorescence detection process of this embodiment will be described in detail with reference to the flowchart shown in FIG.

  First, in step S101, the filter 39 is removed from the optical path from the DNA chip 12 to the photomultiplier tube 37. Then, the XY stage 29 (see FIG. 1) is operated to move the biochemical reaction cartridge 1 so that the laser light (excitation light) from the laser light generator 34 enters the reflector 33 of the DNA chip. Accordingly, in step S102, the laser light generator 34 irradiates the reflecting plate 33 with excitation light, and in step S103, the photomultiplier tube 37 starts measuring the light intensity of the reflected light from the reflecting plate 33. In this state, in step S104, the position where the light intensity measurement value by the photomultiplier tube 37 is maximized is searched while moving the objective lens 36 up and down. Then, the objective lens 36 is fixed at a position where the light intensity measurement value is maximized. Then, in step S105, the maximum measured light intensity value is compared with a prestored light intensity reference value to calculate the ratio. The light intensity reference value is the intensity of reflected light experimentally obtained in advance using a reference substrate and reference excitation light.

  Then, in step S106, the output intensity of the laser light generator 34 is adjusted based on the ratio obtained in step S105. That is, when the light intensity measurement value is larger than the light intensity reference value, the output intensity is weakened. When the light intensity measurement value is smaller than the light intensity reference value, the output intensity is increased, and the light intensity measurement value is Match the light intensity reference value as much as possible. An adjusting means (not shown) may adjust the output intensity of the laser light generator 34, but the laser light generator 34 or the control unit 17 may act as an adjusting means for adjusting the output intensity.

  When the preparatory work is performed in the above steps S101 to S106, the filter 39 is moved on the optical path from the DNA chip 12 to the photomultiplier tube 37 in step S107. In step S108, the XY stage 29 is operated so that the laser beam (excitation light) from the laser beam generator 34 is incident on the detection start position of the DNA probe 32 group of the DNA chip 12. Move. Therefore, in step S109, the laser light generator 34 irradiates the region for one line of the DNA chip 12 with excitation light while the XY stage 29 is main-scanned in the X direction. If the fluorescent substance exists in the DNA probe 32 at the position irradiated with the excitation light, that is, if the DNA to which the fluorescent substance (fluorescent label) is attached is hybridized, the fluorescent substance generates fluorescence. This fluorescence passes through the filter 39 and is detected by the photomultiplier tube 37. Thus, the fluorescence light intensity for one line is measured. From this measurement result, the presence or absence of infection with infectious disease A can be known. In addition, since light other than fluorescence, such as reflected light, is blocked by the filter 39 and does not enter the photomultiplier tube 37, detection noise hardly occurs.

  In this embodiment, since the output intensity of the excitation light is adjusted in step S105, the detection error is small even if the fluorescence light intensity is measured based on the analysis information stored in advance.

  However, if it is difficult in step S105 to match the light intensity measurement value and the light intensity reference value, a correction formula for conversion to the light intensity reference value is obtained. In step S110, it is possible to detect fluorescence with high accuracy using the analysis information stored in advance and the correction formula. For example, when the light intensity measurement value becomes larger than the light intensity reference value even after the adjustment in step S105, correction is performed to reduce the measurement value by 1/2 of the difference. On the contrary, if the light intensity measurement value becomes smaller than the light intensity reference value even after the adjustment in step S105, a correction is added to the measurement value by 1/2 of the difference.

  When the fluorescence detection for one line is completed in this way, the XY stage 29 is operated in step S111, and the biochemical reaction cartridge 1 is sub-scanned in the Y direction by one line.

  In step S112, the main scanning and fluorescence detection (steps S109 to S110) and the sub-scanning (step S111) are alternately repeated until it is confirmed that the fluorescence detection of the final line of the matrix-like DNA probe 32 is completed. When it is confirmed in step S112 that the fluorescence detection of the final line of the matrix-like DNA probe 32 is completed, the XY stage 29 is operated in step S113 to move the biochemical reaction cartridge 1 to the initial position.

  As described above, in the present embodiment, when the fluorescent substance is detected using various types of biochemical reaction cartridges 1 such as the material, thickness, and refractive index of the substrate of the DNA chip 12 are different, The light intensity measurement can be adjusted to match the light intensity reference value.

  In the configuration as shown in FIG. 2, the reflection plate 33 that reflects the excitation light is arranged on the front side of the substrate of the DNA chip 12, and the excitation light is irradiated from the back side of the substrate. is there. Therefore, in this embodiment, the attenuation rate at the substrate is grasped in advance in steps S102 to S105, and the output intensity of the excitation light is adjusted in step S106 so that the fluorescence is excited at a constant intensity based on that. To do. Furthermore, in step S110, the fluorescence intensity obtained by the adjusted excitation light is further corrected to obtain a final detection result.

  Thus, even when measuring the biochemical reaction cartridge 1 having a different substrate of the DNA chip 12 incorporated, the measurement can be performed under substantially the same fluorescent substance excitation conditions and fluorescence detection conditions. Therefore, the measurement result is stable and can be easily compared with the analysis information (for example, pass / fail judgment data) obtained in advance. In addition, since analysis can be easily performed using analysis information (for example, pass / fail judgment data) obtained in advance, the processing speed is increased, and the cause of errors such as data conversion is reduced, and the analysis is quickly performed. There is an effect that an accurate result can be obtained. As a result, the sample can always be analyzed with high accuracy.

[Second Embodiment]
Next, a second embodiment of the present invention will be described in detail.

  Although not shown, the reflector of the present embodiment is provided not in the DNA chip 12 but in the processing apparatus main body to which the biochemical reaction cartridge 1 is attached. The reflector touches the storage position that does not interfere with the irradiation of the excitation light from the laser light generator 34 to the DNA chip 12 to detect fluorescence and the substrate surface, and is irradiated from the laser light generator 34. It is possible to move between reflection positions where the excitation light is incident. Therefore, in steps S102 to S106, a series of steps of adjusting the output intensity by irradiating the excitation light from the laser light generator 34 and measuring the intensity of the reflected light with the reflector disposed at the reflection position. Do. Thereafter, the reflector is moved from the reflection position to the storage position before the fluorescent material is detected by performing main scanning and sub-scanning in steps S109 to S112.

  According to this embodiment, since it is not necessary to provide a reflector on the minute DNA chip 12, the manufacturing process is simplified. Since the configuration other than the reflector is the same as that of the first embodiment, the description thereof is omitted. And although description is not repeated, in this embodiment, the effect similar to 1st Embodiment can be acquired by performing the process similar to 1st Embodiment (FIG. 6, 10).

[Third Embodiment]
Next, a third embodiment of the present invention will be described in detail.

  In the present embodiment, as shown in FIG. 11, in the detection region of the DNA chip 12, a reflector having a length equal to or longer than the length of the matrix region on the side of the region where the DNA probes 32 are arranged in a matrix. 40 is arranged. In particular, this configuration is such that, before the main scanning for one line for actually detecting fluorescence, that is, after the sub-scanning is performed once, the excitation light is periodically radiated on the reflection plate 40 to reflect the reflected light. Suitable for measuring intensity and adjusting output intensity.

  A specific configuration in this case is shown in FIG. That is, in addition to the laser beam generator 34, the splitter 35, the objective lens 36, the lens 38, and the photomultiplier tube 37 similar to those of the first embodiment, the light receiving element (photodiode) 42, the dichroic mirror 41, and the like. The lens 43 is provided. The photodiode 42 is for detecting the light intensity of the reflected light from the DNA chip 12. The dichroic mirror 41 bends the reflected light from the DNA chip 90 by 90 degrees and reflects the reflected light toward the photodiode 42, while transmitting the fluorescence having a wavelength different from that of the reflected light as it is. The lens 43 condenses the reflected light reflected by being bent 90 degrees by the dichroic mirror 41 on the photodiode 42.

  As in the first embodiment, if the filter 39 is configured to move on the optical path from the DNA chip 12 to the photomultiplier tube 37 and outside the optical path, the filter 39 is moved very frequently. This must be done and is cumbersome and time consuming. Therefore, in the present embodiment, the filter 39 is not used, and as described above, in addition to the photomultiplier tube 37 for fluorescence detection, a light receiving means for receiving reflected light and obtaining data for adjusting the output intensity of the excitation light ( Photodiode) 42 is separately provided. Therefore, a dichroic mirror 41 is further added.

  Next, the fluorescence detection process in this embodiment will be described with reference to the flowchart shown in FIG.

  First, the biochemical reaction in the biochemical reaction cartridge 1 is performed in steps S1 to S14 (see FIG. 6) as in the first embodiment. Thereafter, in step S121, the XY stage 29 is operated so that the laser light (excitation light) from the laser light generator 34 is adjacent to the detection start position of the DNA probe 32 group of the DNA chip 12 on the reflector 40. The biochemical reaction cartridge 1 is moved so as to be incident on.

  In step S122, the laser light generator 34 irradiates the reflecting plate 40 with excitation light, and in step S123, the intensity of the reflected light from the reflecting plate 40 bent by 90 degrees by the dichroic mirror 41 is expressed as a photodiode. 42 starts measurement. In this state, in step S124, a position where the light intensity measurement value by the photodiode 42 is maximized is searched for while moving the objective lens 36 up and down. Then, the objective lens 36 is fixed at a position where the light intensity measurement value is maximized. Then, in step S125, the maximum measured light intensity value is compared with a previously stored light intensity reference value to calculate the ratio. Then, in step S126, the output intensity of the laser light generator 34 is adjusted based on the ratio obtained in step S125. That is, when the light intensity measurement value is larger than the light intensity reference value, the output intensity is weakened. When the light intensity measurement value is smaller than the light intensity reference value, the output intensity is increased, and the light intensity measurement value is Match the light intensity reference value as much as possible.

  Subsequently, in step S127, laser light (excitation light) is irradiated from the laser light generator 34 to the region of one line of the DNA chip 12 while the XY stage 29 is main-scanned in the X direction. If the fluorescent substance exists in the DNA probe 32 at the position irradiated with the excitation light, that is, if the DNA to which the fluorescent substance (fluorescent label) is attached is hybridized, the fluorescent substance generates fluorescence. This fluorescence passes through the dichroic mirror 40 and is detected by the photomultiplier tube 37. Thus, the light intensity of one line of fluorescence is measured, and when the measurement is completed, the biochemical reaction cartridge 1 is moved to a position where the excitation light is incident on the reflection plate 40.

  In step S125, when it is difficult to match the light intensity measurement value and the light intensity reference value, a correction formula for conversion to the light intensity reference value is obtained. In step S128, the light intensity of the fluorescence can be accurately obtained by using the analysis information stored in advance and the correction formula. For example, when the light intensity measurement value becomes larger than the light intensity reference value even after the adjustment in step S105, correction is performed to reduce the measurement value by 1/2 of the difference. On the contrary, when the light intensity measurement value becomes smaller than the light intensity reference value even after the adjustment in step S125, a correction is added to the measurement value by 1/2 of the difference.

  When the fluorescence detection for one line is completed in this way, the XY stage 29 is operated in step S129, and the biochemical reaction cartridge 1 is sub-scanned in the Y direction by one line.

  In step S130, the main scanning and fluorescence detection (steps S109 to S110) and the sub scanning (step S111) are alternately repeated until it is confirmed that the fluorescence detection of the final line of the matrix-like DNA probe 32 is completed. . However, in this embodiment, before performing main scanning and fluorescence detection (steps S109 to S110), processing for adjusting the output intensity of excitation light (steps S122 to S126) is always performed. As a result, even if any change in processing conditions occurs during the analysis of one DNA chip 12, highly accurate fluorescence detection can be performed in response to each change.

  When it is confirmed in step S130 that the fluorescence detection of the final line of the matrix-like DNA probe 32 is completed, the XY stage 29 is operated in step S131 to move the biochemical reaction cartridge 1 to the initial position.

  In the present embodiment, as described above, the output intensity adjustment of the excitation light is always performed in the actual fluorescence detection operation for each line. As a result, even when any change in processing conditions occurs during the analysis of one DNA chip 12, it is possible to detect fluorescence with high accuracy in response to each change. Moreover, although description is not repeated, the effect similar to 1st Embodiment can be acquired also in this embodiment.

  Further, immediately after the laser light generator 34 is turned on, a situation occurs in which the excitation light is not stable. Even in that case, according to the present embodiment, the power of the laser light generator 34 is measured in order to measure the light intensity of the reflected light from the reflector 40 and adjust the output intensity of the excitation light based on the measured light intensity before each main scanning. Immediately after being turned on, it is possible to detect the fluorescence with the same high accuracy as in the state where the excitation light is stable, without having to wait. As a result, the power of the laser generator 34 is not kept on, and even if it is a short time, even if the excitation light is unnecessary, even if the power is turned off, the work efficiency is not lowered, and the power can be saved. There is also an effect of contributing.

It is a block diagram which shows the structure of the sample analyzer of the 1st Embodiment of this invention. It is a schematic side view which shows the fluorescent substance detection apparatus of the sample analyzer shown in FIG. It is a perspective view of the biochemical reaction cartridge of this invention. FIG. 4 is a plan sectional view of the biochemical reaction cartridge shown in FIG. 3. FIG. 4 is a plan view of the DNA chip of the biochemical reaction cartridge shown in FIG. 3. It is a flowchart which shows the process process in the sample analyzer shown in FIG. FIG. 5 is a longitudinal sectional view passing through a part of the chamber of the biochemical reaction cartridge shown in FIGS. FIG. 5 is a longitudinal sectional view through another chamber of the biochemical reaction cartridge shown in FIGS. It is a schematic diagram which shows the example of the fluorescent substance detection pattern of a DNA chip. It is a flowchart which shows the fluorescent substance detection process of the process shown in FIG. It is a top view of the DNA chip of the 2nd Embodiment of this invention. It is a schematic side view which shows the fluorescent substance detection apparatus of the 2nd Embodiment of this invention. It is a flowchart which shows the fluorescent substance detection process of the 2nd Embodiment of this invention.

Explanation of symbols

1 Biochemical Reaction Cartridge 12 DNA Chip 17 Controller 25 IC Chip 30 Fluorescence Detection Unit 33 Reflector 34 Laser Light Generator 37 Photomultiplier Tube 40 Reflector 42 Photodiode

Claims (17)

A fluorescent substance detection apparatus for detecting a fluorescent substance on the surface of a substrate having translucency,
Excitation light generating means for generating excitation light irradiated toward the back surface of the substrate;
A first light receiving means for receiving the excitation light attenuated by transmitting through the light-transmitting substrate; a second light receiving means for receiving the fluorescence emitted by the fluorescent material excited by the excitation light;
Adjusting means for adjusting the output intensity of the excitation light according to the light intensity measurement value of the attenuated excitation light received by the first light receiving means;
A fluorescent substance detection device comprising: Reflecting means is provided on the surface side of the substrate,
The fluorescent substance detection apparatus according to claim 1, wherein the reflecting means is arranged so that the transmitted excitation light is reflected toward a back surface side of the substrate.   The fluorescent substance detection apparatus according to claim 2, wherein the first and second light receiving means are a single light receiving means. A main scan that scans the substrate in a fixed direction relative to the excitation light generating means so that the excitation light crosses at least a part of the substrate, and the substrate intersects the main scanning direction. In the direction, the sub-scan that is moved relative to the excitation light generating means is alternately repeated a plurality of times to irradiate the entire detection region of the substrate with the excitation light,
In the detection area, an area where the fluorescent substance may exist and the reflection means are juxtaposed,
Prior to each of the main scans that are performed a plurality of times, the excitation light generating means irradiates the excitation light to the reflection means, and the light receiving means receives the reflected light to obtain a light intensity measurement value of the reflected light. The fluorescent substance detection device according to any one of claims 2 to 3, wherein an output intensity of the excitation light is adjusted on the basis of the excitation light.   The fluorescent substance detection apparatus according to claim 2, wherein the reflection unit is provided integrally with the substrate on a surface of the substrate.   The fluorescent substance detection apparatus according to claim 2, wherein the reflection unit is disposed at a position adjacent to the surface of the substrate.   The fluorescent substance detection apparatus according to claim 1, wherein a plurality of DNA probes capable of capturing DNA to which a fluorescent substance is attached are provided in the detection area of the substrate. The fluorescent substance detection device according to any one of claims 1 to 7,
Control for analyzing the specimen by knowing the presence / absence and / or biochemical reaction state of the specimen supplied to the fluorescent substance detection apparatus based on the detection result of the fluorescent substance by the fluorescent substance detection apparatus And a sample analyzer.   The sample analyzer according to claim 8, wherein the controller is capable of controlling an operation of the fluorescent substance detection device.   The sample analyzer according to claim 9, further comprising a processing device in which a biochemical reaction cartridge including the substrate to which the sample is supplied is set to cause a biochemical reaction of the sample. It is a board | substrate for using for the apparatus of any one of Claims 1-10,
A probe binding substrate comprising a substrate having translucency, a probe for capturing a target substance bound to the substrate surface, and reflecting means provided on the substrate surface. In the fluorescent substance detection method for detecting the fluorescent substance on the surface of the substrate having translucency,
Irradiating excitation light toward the back surface of the substrate;
Receiving the excitation light attenuated by passing through the substrate by a light receiving means;
Comparing the light intensity measurement value of the attenuated excitation light received by the light receiving means with a preset light intensity reference value, and adjusting the output intensity of the excitation light according to the comparison result When,
Irradiating the back surface of the substrate with the excitation light whose output intensity is adjusted;
And a step of detecting, by the light receiving means, fluorescence emitted from the fluorescent material present at a position irradiated with the excitation light. In the fluorescent substance detection method for detecting the fluorescent substance on the surface of the substrate having translucency,
Irradiating excitation light toward the back surface of the substrate having reflecting means on the surface;
Receiving the reflected light of the excitation light reflected by the reflecting means by a light receiving means;
Comparing the light intensity measurement value of the reflected light received by the light receiving means and a preset light intensity reference value, and adjusting the output intensity of the excitation light according to the comparison result;
Irradiating the back surface of the substrate with the excitation light whose output intensity is adjusted;
And a step of detecting, by the light receiving means, fluorescence emitted from the fluorescent material present at a position irradiated with the excitation light.   The fluorescent substance detection method according to claim 13, wherein the reflected light and the fluorescence are received by a single light receiving means.   The fluorescent substance detection method according to claim 13, wherein the reflected light is received by the reflected light receiving means of the light receiving means, and the fluorescence is received by the fluorescent light receiving means of the light receiving means. A main scan that scans the substrate in a fixed direction relative to the excitation light generating means so that the excitation light crosses at least a part of the substrate, and the substrate intersects the main scanning direction. Irradiating the entire region to be detected of the substrate with the excitation light by alternately repeating a plurality of sub-scans that move relative to the excitation light generation means in the direction,
Prior to each of the main scans that are performed a plurality of times, the excitation light generating means irradiates the excitation light to the reflection means, and the light receiving means receives the reflected light to obtain a light intensity measurement value of the reflected light. The fluorescent substance detection method according to any one of claims 13 to 15, wherein an output intensity of the excitation light is adjusted on the basis of the excitation light.   The knowledge of the presence / absence and / or biochemical reaction state of a specimen based on each step of the fluorescent substance detection method according to any one of claims 12 to 16 and the detection result of the fluorescence. A sample analysis method comprising the step of analyzing the sample.

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