JP2008064518A - Sample analyzer and channel clogging judgment method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technology for detecting clogging of a channel beforehand without using especially new sensors, in order to reduce a loss in analysis. <P>SOLUTION: A photodetector equipped with a device for detecting clogging of a channel, to put it concretely, an image sensor, is used. The clogging of the channel is detected by image processing. According to this invention, the clogging of the channel can be detected without adding a new sensor, and a loss of a sample or time of an analyzer can be prevented without increasing device cost. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a sample analyzer and a flow path clogging determination method, for example, a technique for detecting a state where a flow path into which a cartridge is inserted is blocked by clogging.

  For example, in a sample analyzer such as an SNP analyzer, a sample analysis is performed by inserting a cartridge holding a DNA chip or the like in a flow cell. In that case, if the solute of the reagent to be used is deposited, or if foreign substances block the thin flow path, the flow path becomes clogged and the sample is wasted or an incorrect result is output, which hinders analysis. Become. In particular, the sample is precious, and if the measurement fails, the sample may not be available anymore, so a means that can be used effectively is required. Therefore, it is considered that an apparatus for introducing a sample into a flow cell detects clogging in a narrow flow path and issues a warning to a user when clogging occurs.

  Regarding detection of clogging of the flow path, for example, Patent Document 1 discloses a method of detecting clogging of a nozzle by detecting a vibration by causing a discharge from a nozzle to collide with a diaphragm of a microphone. Patent Document 2 discloses a method for detecting clogging of a nozzle by pressure fluctuation of a pressure measuring device.

JP 2000-085149 A Japanese Patent Laid-Open No. 06-109745

  However, as in Patent Documents 1 and 2, incorporating a microphone diaphragm or pressure measuring device in a sample analyzer as an element for detecting clogging complicates the structure of the apparatus and increases the price.

  The present invention has been made in view of such a situation, and provides a technique for detecting blockage of a flow path in advance in order to reduce the loss of a sample in sample analysis and to realize an inexpensive sample analyzer. It is.

  In order to solve the above-mentioned problems, the sample analyzer according to the present invention includes a photodetector, and determines whether or not the channel is clogged based on the output of the photodetector. Specifically, an image sensor can be used as the photodetector.

  In other words, the sample analyzer according to the present invention is a sample analyzer that introduces a sample into a sample reaction section and analyzes the sample, and the flow path for introducing the sample into the sample reaction section, the sample, and a predetermined liquid On the basis of a detection result of the light detection unit, a light detection unit for detecting light from the sample reaction unit, a liquid supply mechanism for supplying liquid to the sample reaction unit via the flow path And a clogging determination unit that determines whether or not the road is clogged.

  Here, the clogging determination unit determines clogging of the flow path from the output image of the photodetector. More specifically, the photodetector includes a first image after performing an operation of emptying the sample reaction unit and a second image after performing an operation of filling the sample reaction unit with a liquid. The clogging determining unit acquires the clogging of the flow path by comparing the first image and the second image. As another aspect, the photodetector acquires an image after performing an operation of filling the sample reaction unit with a liquid, and the clogging determination unit is configured to fill the image and the sample reaction unit with a liquid. The clogging of the flow path may be determined by comparing with a reference image in a state in which it is in a closed state.

  The sample analyzer further includes a cleaning processing unit that automatically performs a cleaning process on the flow path when the clogging determination section determines that the flow path is clogged, or the clogging determination section includes When it is determined that the flow path is clogged, a means for notifying the user of the determination result and prompting the user to perform cleaning may be provided.

  A cartridge including a flow cell having a septum may be attached to the sample analyzer. In this case, the sample analyzer performs the insertion / removal operation of the needle by moving the cartridge interface section for mounting the cartridge, the needle that can be inserted into the septum, and the needle relative to the septum. A needle moving mechanism to be executed; a channel for introducing the sample into the flow cell; and a liquid feeding mechanism for feeding the sample and a predetermined liquid to the flow cell via the channel and the needle; A light detection unit that detects light from the flow cell, and a clogging determination unit that determines whether the flow path is clogged based on a detection result of the light detection unit.

  The channel clogging determination method according to the present invention is a channel clogging determination method for determining clogging of the channel in the sample analyzer described above, and detects light from the sample reaction unit by a light detection unit. And a step of determining by the determination unit whether or not the flow path is clogged based on a detection result of the light detection unit.

  In the determining step, the clogging determination unit determines clogging of the flow path from the output image of the photodetector. More specifically, in the step of detecting the light, the photodetector performed a first image after performing an operation of emptying the sample reaction unit and an operation of filling the sample reaction unit with a liquid. In the step of acquiring and determining a later second image, the clogging determination unit determines clogging of the flow path by comparing the first image and the second image. As another aspect, in the step of detecting the light, the photodetector acquires an image after performing an operation of filling the sample reaction unit with a liquid, and in the determination step, the clogging determination unit includes: The clogging of the flow path may be determined by comparing the image with a reference image in a state where the sample reaction part is filled with liquid.

  Further features of the present invention will become apparent from the best mode for carrying out the present invention and the accompanying drawings.

  ADVANTAGE OF THE INVENTION According to this invention, in order to reduce the loss of the sample in a sample analysis and implement | achieve an inexpensive sample analyzer, the technique which detects beforehand blockage of a flow path can be provided.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the present embodiment, a SNP analyzer is cited as an example of a sample analyzer, but the present invention is not limited to this, and the gist of the present invention can be applied to an apparatus that performs any sample analysis.

<Overview of SNP analyzer>
FIG. 1 is an exploded perspective view (external view) of an SNP analyzer 101 using the DNA chip according to the present embodiment. The SNP analyzer 101 is mainly intended to detect DNA SNPs. SNP is an abbreviation for Single Nucleotide Polymorphism, which is called single nucleotide polymorphism, and refers to genes that differ by one nucleotide. This SNP is considered to be related to illness, and search for SNPs related to various illnesses has been vigorously conducted.

  The final output of the SNP analyzer 101 is the following three states. In the sample DNA, the SNP of interest exists in both alleles or does not exist (homo), and exists in only one allele (hetero). As a method for determining these, for example, a reagent that reacts with and binds to (hybridizes with) a sample having the SNP of interest is labeled with the first fluorescent dye, and a reagent that hybridizes with a sample that does not have it. The first is labeled with a second fluorescent dye having a different excitation wavelength and fluorescence wavelength. The both reagents are reacted with the sample DNA. Fluorescence from the first and second fluorescent dyes is detected, and a ratio between the detected intensities is obtained. Based on this ratio, the three states for the SNP of interest are determined.

  Here, a procedure for analyzing a sample which is a PCR product using the electric field control type chip will be described. There are two types of analysis procedures: “Amplicaon Down Format” and “Capture Down Format”.

  In the Amplicaon Down Format, first, a PCR product to be analyzed (Sample Oligos) whose part of the base sequence is unknown and biotinylated at one end is supplied onto a semiconductor chip, and a voltage is applied to a predetermined electrode on the semiconductor chip. Apply. Then, the sample oligos are attracted to the electrode and come into contact with the transmission layer structure on the surface of the semiconductor chip. Then, the biotin label of Sample Oligos reacts with the permeation layer structure (avidin-biotin reaction), and Sample Oligos is fixed to the permeation layer structure. The surface of the semiconductor chip is washed and the above-described steps are repeated using another Sample Oligos to form a desired oligonucleotide array made of Sample Oligos on the semiconductor chip. After forming an oligonucleotide array in which Sample Oligos are arranged in a matrix, an oligonucleotide (Reporter Oligos) fluorescently labeled at one end is supplied thereon. Reporter Oligos hybridizes with Sample Oligos having a complementary sequence. After cleaning the surface of the semiconductor chip, the semiconductor chip is irradiated with excitation light. As a result, fluorescence is generated from the Sample Oligos and the hybridized Reporter Oligos. By detecting and analyzing this fluorescent pattern, the base sequence of Sample Oligos can be analyzed.

  On the other hand, in Capture Down Format, first, an oligonucleotide having a known base sequence and biotinylated at one end (Capture Oligo) is supplied onto a semiconductor chip, and a voltage is applied to a predetermined electrode on the semiconductor chip. . Then, the Capture Oligo is attracted to the electrode and comes into contact with the transmission layer structure on the surface of the semiconductor chip. Then, the biotin label of Capture Oligo reacts with the transmission layer structure (avidin-biotin reaction), and Capture Oligo is fixed to the transmission layer structure. The surface of the semiconductor chip is washed, and the above-described steps are repeated using another Capture Oligo to form a desired oligonucleotide array made of Capture Oligo on the semiconductor chip. After the Oligonucleotide array in which Capture Oligo is arranged in a matrix is formed, a PCR product to be analyzed (Sample Oligos) whose partial base sequence is unknown is supplied thereon. Sample Oligos hybridizes with Capture Oligo having a complementary sequence. Thereby, Sample Oligos is fixed on a semiconductor chip via Capture Oligo. The surface of the semiconductor chip is washed, and an oligonucleotide (Reporter Oligos) that is fluorescently labeled at one end is supplied. Reporter Oligos hybridizes with Sample Oligos having a complementary sequence. After cleaning the surface of the semiconductor chip, the semiconductor chip is irradiated with excitation light. Thereby, fluorescence is generated from Reporter Oligos hybridized with Sample Oligos. By detecting and analyzing this fluorescent pattern, the base sequence of Sample Oligos can be analyzed.

  As shown in FIG. 1, the SNP analyzer 101 includes a top cover 102, a cover 103, an apparatus main body 104, and a front panel 105. The apparatus main body 104 of the SNP analyzer 101 is mainly composed of a dispensing unit (112 + 106, as will be described later), a flow path system unit 113 to 115, an interface unit 107, an optical system unit 108, and a power supply unit 109. Controlled by an external device (PC) (not shown). The SNP analyzer 101 is equipped with a cartridge equipped with a DNA chip and performs analysis. The cartridge contains a flow cell in which a DNA chip made of a semiconductor element is arranged. The DNA chip can arrange a maximum of 400 nucleic acid probes at predetermined positions to construct a desired probe array. It should be noted that the process from the attachment of the nucleic acid probe on the DNA chip to the measurement can be performed automatically, and there is no need for the operator to operate the apparatus for each sample. For example, analysis of 96 samples can be done in approximately 3 hours.

  The dispensing unit includes a sample handling station 106 capable of arranging and storing samples and reagents, and a robot arm 112 for transporting samples and reagents to a predetermined position. The sample handling station 106 can mount two sample trays 111 containing samples and reagents and four reagent bottles. Moreover, the reaction enzyme cooling part which can cool and store the reaction enzyme is provided. A robot arm 112 equipped with a probe tip capable of sucking and discharging the solution is used to dispense a predetermined sample or reagent and put it into the injection port.

  The flow path system units 113 to 115 drive three syringe pumps, and can automatically carry the sample and sample introduced from the injection port, water stored in the water bottle, and the like to the DNA chip in the flow cell. In addition, the probe tip can be washed by the washing port, and the inside of the flow cell and the channel can be washed. Further, the waste liquid generated from the inside of the apparatus can be discharged to a waste liquid bottle arranged outside the apparatus.

  The interface unit 107 can detachably hold the cartridge, insert a needle into the cartridge, and connect the flow path through the needle. Thereby, a predetermined sample or reagent can be transported into the flow cell. In addition, the location of the nucleic acid probe can be controlled by electrical connection with the DNA chip. Furthermore, the temperature in the flow cell can be controlled by thermal connection with the DNA chip.

  The optical system unit 108 includes a light source that can excite a fluorescent reagent present on the DNA chip and a detector that can detect fluorescence generated from the fluorescent reagent, and can output an image on the DNA chip.

  The power supply unit 109 supplies driving power to each unit, and 100, 110, 120, 200, 220, 230, and 240 VAC can be used as the power supply voltage, the current value is 4 A or less, and the frequency is 50/60 Hz. It corresponds.

  An external device connection unit 110 is provided on the side of the SNP analyzer 101, and a personal computer (PC) and a barcode reader (not shown) can be connected to the SNP analyzer 101. For example, the PC has a role of operating the SNP analyzer 101 and displaying, analyzing, and storing measurement results. Further, for example, the SNP analyzer 101 and the PC are connected by Ethernet. It is also possible for one PC to operate up to four SNP analyzers 101 via the hub. The barcode reader facilitates management of samples, reagents, and cartridges to be measured by reading barcodes attached to sample trays and cartridges containing samples and transmitting them to a PC.

<Internal configuration of SNP analyzer>
FIG. 2 is a diagram illustrating a schematic internal configuration of the SNP analyzer 101 according to the present embodiment. Hereinafter, the mechanism and operation of the entire apparatus will be described with reference to FIG. 2 shows a state in which the cartridge 218 is inserted into the apparatus main body, and the apparatus main body and the flow cell 219 are connected by the needle 211.

  The flow path system units 113 to 115 include a probe tip 205 of the robot arm 112, a water bottle 201, a histidine bottle 202, a waste liquid bottle 203, a water pump 206, a histidine pump 207, a cartridge pump 208, a cleaning port 209, an injection port 210, and The needle 211 has a structure connected by a plurality of flow paths.

  The probe tip 205 is connected to a pipe for sucking and discharging a sample and a solution, and the tip of the probe tip 205 can be moved to a predetermined position by the robot arm 112. Then, a predetermined sample or reagent can be dispensed from a sample tray, a reagent bottle, and a reaction enzyme cooling unit arranged on the sample handling station, and can be put into an injection port. Here, the injection port 210 is a port tank that exists before the sample and the solution are sent to the flow cell 219 in the cartridge 218. The temperature of the injection port 210 can be controlled in the range of 40 ° C to 60 ° C. Spike noise occurs when the sample is introduced in a state where the introduced sample temperature is different from the temperature in the cartridge. However, spike noise can be reduced by adjusting the temperature from the injection port 210 before introducing the sample. In addition, the probe tip 205 can be cleaned at the cleaning port 209.

  The water pump 206 can send water from the water bottle 201 to the probe tip 205, perform suction and discharge of the sample and the sample by the probe tip 205, and send the solution to the waste liquid bottle 203.

  The histidine pump 207 also sends histidine from the histidine bottle 202 to the injection port 210, sends water from the water bottle 201 to the injection port 210, sends air to the injection port 210, and sends the solution in the syringe to the waste bottle 203. Can send.

  The cartridge pump 208 can also send solution and air from the injection port 210 to the flow cell 219 in the cartridge 218, and can send solution and air from the flow cell 219 in the cartridge 218 to the cartridge pump 208.

  The waste liquid generated in the apparatus can be discharged to a waste liquid bottle 203 outside the apparatus that is detachably mounted via the connection portion.

  By driving the water pump 206 and the cartridge pump 208, the reagent and the cleaning liquid introduced into the injection port can be conveyed into the flow cell 219 via the cartridge flow path. The cartridge flow path is cleaned with histidine by driving the histidine pump 207 and the cartridge pump 208. The flow path from the histidine pump 207 to the injection port 210 and the cartridge pump 208 has a structure capable of replacing the histidine solution with water. By washing the channel with water, the histidine solution is prevented from being deposited in the channel pipe.

  In addition, it is possible to put 1 L of water and histidine into the water bottle 201 and the histidine bottle 202, respectively. For this reason, it is not necessary for the user to add water or histidine from the pasting of 96 samples to the measurement.

  The interface unit 107 includes a needle 211 serving as a flow path connecting the flow cell 219 in the cartridge 218 and the apparatus main body, a cartridge cooling peltier 212, and a pogo pin 213. The cartridge cooling Peltier 212 contacts the cartridge and controls the temperature of the flow cell 219. The pogo pin 213 is connected to a terminal provided on the cartridge 218, and electrically connects the apparatus main body and the DNA chip. Since the number of pogo pins 213 is as small as 12, for example, connection to the cartridge 218 is facilitated.

  The optical system unit 108 includes a CCD camera 214, an EM filter 215, an EX filter 216, and an LED 217. The LED 217 is filtered with a wavelength characteristic by the EM filter 215 and can irradiate the phosphor on the DNA chip in the flow cell 219 with light. Then, the fluorescence generated from the phosphor is filtered with the wavelength characteristic by the EX filter 216, and the image of the flow cell 219 is acquired by the CCD camera 214. Since the LED 217 is used as the light source and the life of the light source is sufficiently longer than that of a conventional laser, light source replacement work during the life of the apparatus can be avoided, and maintenance is facilitated.

<About the interface unit>
FIG. 3 is an exploded configuration diagram of the Cartridge processor 301 constituting the interface unit 107. The interface between the cartridge 218 and the SNP analyzer 101 will be described with reference to FIG.

  In FIG. 3, a cartridge holder 302 is for holding the cartridge 218 in the apparatus. The needle block 304 inserts the needle 211 into the cartridge 218 to form a flow path connection. The slide base 305 is electrically and thermally connected to and disconnected from the cartridge 218 by the pogo pin 213 and the cartridge cooling peltier 212. A needle block 304 is attached to the needle base 306, receives power from the actuator 307, inserts and removes the needle, and drives the slide base 305 via the shaft 308 and two types of springs 309 and 310. The mechanical stopper 311 keeps the position of electrical and thermal contact constant by contacting the slide base 305 and restricting movement when the actuator 307 is driven. The photo interrupter 312 detects that the light shielding plate attached to the needle base 306 has come to a predetermined position, and determines an initial position when the apparatus is driven.

<About cartridge configuration>
FIG. 4 is a diagram showing a more detailed configuration of the cartridge. The function of the cartridge will be described with reference to FIG. 4A is an overall view of the cartridge, and FIG. 4B is an enlarged plan view and a cross-sectional view of the chip portion.

  The cartridge 218 includes a DNA chip 402, a flow cell 219 that is disposed below the chip and serves as a flow path for sending a sample to the chip, and a septum that seals the flow cell at the portion where the sample at the end of the flow cell is introduced (partition part: realizing a seal structure) 404, which is an appearance of the cartridge and is constituted by a housing 405 that holds a chip, a flow cell, a septum, and the like. The cartridge 218 can be attached to and detached from the sample analyzer, and different cartridges can be attached to the apparatus for each detection purpose and each measurement object to perform inspection.

  The DNA chip 402 has DNA probes arranged in an array. DNA as a sample is fed onto the chip with a fluorescent substance, hybridized with a DNA probe, and fluorescence detection is performed to determine SNPs of the DNA. In this embodiment, the DNA probe has 400 sites, and the DNA is electrically attracted and pasted when the probe is formed.

  The flow cell 219 introduces and holds the sample on the DNA chip 402 by storing the sample in the cartridge 218. In this embodiment, the flow cell size is about 7 mm in length and about 1.5 mm in width. The septum 404 seals at the needle insertion ports at both ends of the cartridge flow cell 219 to prevent sample leakage. In this embodiment, a septum made of silicon and having a thickness of about 1.6 mm is used. The housing 405 holds and accommodates the DNA chip 402, the flow cell 219, the septum 404, and the like. Since electricity and heat are used in various reactions, a flame retardant is used for the housing 405.

<About cartridge insertion / fixing mechanism>
FIG. 5 shows a mechanism for inserting and fixing the cartridge 218 in the apparatus. FIG. 5A shows the positional relationship between the cartridge, the cartridge presser, and the switch (microswitch), and FIG. 5B shows the fixing method using the three pins of the cartridge and the apparatus-side long hole.

  The cartridge retainer 302 has a shape that leads to an appropriate position when the cartridge 218 is inserted. A micro switch 503 is attached to the tip of the cartridge presser 302 to detect the insertion of the cartridge 218. The positioning of the cartridge 218 is determined by three pins 505 on the cartridge side and three long holes 506 on the apparatus side.

  The cartridge retainer 302 is in point contact with a cartridge 218 having a curved outer shape, thereby reducing contact resistance and realizing smooth insertion. When the micro switch 503 attached to the deepest portion of the cartridge presser 302 is pressed by the cartridge 218 to detect insertion, the cartridge presser 302 is pressed only at the tip of the cartridge when the cartridge 218 is in the correct position. It has a shape like this. Therefore, the risk of erroneous detection that may occur when the cartridge is inserted is avoided.

  The positioning of the cartridge 218 is realized by three pins 505 having a spherical tip attached to the cartridge and three elongated holes 506 provided on the apparatus side. When viewed in a cross-sectional view, the spherical surface of each pin tip and the inclined surface of the elongated hole are in contact with each other to determine the position state. The DNA chip of the cartridge is positioned at the center of the three pins 505, and the detector optical system measurement center is positioned at the center of the three long holes 506. With this configuration, even if the machining position of the pin or hole is slightly deviated from the design value, the deviation of the center position of the center of the 3 pins and the center of the 3 long holes is small. It can be suppressed to the limit.

<Needle block and needle base>
FIG. 6 is an assembly diagram of the needle block and the needle base. A method for adjusting the insertion position of the needle will be described with reference to FIG.

  The needle base system 601 includes a needle block 602, a positioning pin 603, an N adjustment base 604, a fixing screw 605, and a needle base 606. The needle block 602 is attached to the N adjustment base 604 with reference to the positioning pin 603. The N adjustment base 604 is attached to the needle base 606 with a fixing screw 605 through two holes 607. Since the two holes 607 have a likelihood of 2 mm with respect to the fixing screw 605, position adjustment is possible. By adjusting the position of the N adjustment base 604, the position of the needle block and needle attached thereto is adjusted. When replacing the needle, only the needle block 602 is removed from the N adjustment base 604, the needle is replaced, and attachment is performed based on the positioning pin 603, so readjustment is unnecessary.

  FIG. 7 is a detailed view of the needle and the needle block. A method for attaching the needle to the needle block will be described with reference to FIG. FIG. 7A is a detailed view of the needle and the needle block, FIG. 7B is a detailed view of the needle guide, and FIG. 7C is a detailed view of the needle block.

  The needle system 701 includes a needle 211, a needle guide 703, a needle block 602, a needle seal 705, and a needle holding screw 706.

  The needle 211 is inserted into the flow cell 219 of the cartridge 218 via a septum to create a flow path with the cartridge. In this embodiment, the tip of the needle is cut at 45 °, and the cut end is deburred to prevent clogging of the septum.

  The needle guide 707 has a protrusion 708 in a part of a cylindrical shape, and is bonded to the needle 211. The needle block 602 has a cylindrical hole 710 that receives the needle guide, and a recess 711 that receives the protrusion of the needle guide on the bottom surface of the cylindrical hole. The protrusion 708 of the needle guide enters the depression 711 of the needle block, so that the cut end of the needle 211 bonded to the needle guide 703 is oriented. In the present embodiment, the cut end cut at 45 ° is attached to the two needles so as to face outward. In this manner, a needle moving mechanism that moves the needle relative to the septum and executes the needle insertion / removal operation is realized.

  The concentricity between the needle 211 and the needle block 602 can be increased by incorporating the cylindrical portion of the needle guide 703 into the cylindrical hole of the needle block 602. Therefore, position reproducibility by needle replacement can be realized, and maintainability is improved. Further, by arranging two cylindrical holes of the needle block 602 in which the needle guide 703 is accommodated in parallel, it is possible to arrange the two needles incorporated in the holes in a state where the needle directions are aligned in parallel. Therefore, by adjusting the position of one needle block 602, the positions of the two needles 211 can be determined, and compared with the case of adjusting the position of each needle, simple and highly accurate position adjustment is possible. realizable. When the two needles 211 are attached to the needle block 602, if the lengths are not uniform, it is difficult to insert the cartridge into the cartridge and to feed liquid. In this method, if the positional relationship at the time of adhesion of the needle 211 and the needle guide 703, the dimensions of the needle guide 703, and the guide receiving hole dimensions of the needle block 602 are accurate, the cylindrical side surface of the needle guide 703 is the cylindrical hole of the needle block 602. Along the side surface, the needle bottom surface of the needle guide 703 is pressed by the needle holding screw 706 so as to come into contact with the bottom surface of the cylindrical hole of the needle block 602. Therefore, it is possible to align the lengths of the two needles without adjustment. .

  The needle seal 705 is provided at a connection portion between the needle guide 703 and the needle block 602, and prevents the sample from flowing out during liquid feeding. In the present embodiment, the needle seal 705 is made of fluororesin, and the needle seal 705 is brought into close contact with each of the needle guide 703 and the needle block 602 by being pushed by the needle holding screw 706 to elastically deform the needle seal 705. Thus, the sealing function is achieved.

<About processing of SNP analyzer>
FIG. 8 is a diagram illustrating a processing flow of the SNP analysis apparatus 101. First, the SNP analyzer 101 is powered on and initialized (S801). Confirm the water capacity in the water bottle and the histidine capacity in the histidine bottle.

  Next, as a sample preparation (S802), the barcode reader reads each of the low salt buffer, high salt buffer, NaOH, microtiter plate containing the sample, and the barcode of the cartridge, and the LED lights each time. , Install each one correctly in its place.

  The cartridge is inserted into the apparatus (S803), and the flow paths are connected (S804). Specifically, the needle system 701 slides and the needle 211 is inserted into the flow cell 219 of the cartridge 218 via a septum.

  Here, clogging detection is performed in order to confirm that the flow paths are securely connected and that there is no clogging due to foreign matter (S805). The method will be described later.

  Next, sample DNA is affixed (S806) as follows. The robot 112 moves the probe tip 205 to the cleaning port and operates the water pump 206 to clean the probe tip 205. After moving the probe tip 205 to the target sample position, the robot 112 sucks the sample from the probe tip 205 by the water pump 206 that has been operated. Thereafter, the robot 112 is moved to the PI detection position, and calibration is confirmed. The robot 112 moves the probe tip 205 to the injection port, and then discharges the sample from the probe tip 205 by the water pump 206. Subsequently, the cartridge pump 208 is operated, and the sample is moved to the cartridge flow cell 219. Further, a current of approximately 0.2 mA is applied to a target position in the cartridge Active Chip for 60 seconds. The histidine pump 207 is activated and histidine is sent to the injection port 210. The cartridge pump 208 is activated and histidine is moved to the cartridge flow cell 219 for cleaning the cartridge flow cell. Then, the cartridge pump 208 is operated to move histidine to the waste liquid bottle 203.

  In addition, introduction of the reporter DNA (S807) is performed as follows. The robot 112 moves the probe tip 205 to the cleaning port 209 and cleans the probe tip 205 with the water pump 206 that has been operated. Then, the robot 112 moves the probe tip 205 to the High Salt Buffer position, and then sucks the High Salt Buffer from the probe tip 205 by the water pump 206. After moving the robot 112 to the PI detection position, the calibration is confirmed. After moving the probe tip 205 to the injection port 210, the robot 112 discharges a high salt buffer from the probe tip 205 by the water pump 206. Subsequently, the cartridge pump 208 is operated, and the high salt buffer is moved to the cartridge flow cell 219. Further, after the cartridge pump 208 is operated and the high salt buffer is moved to the cartridge flow cell 219, the cartridge pump 208 is operated and the high salt buffer is moved to the waste bottle 203.

  The robot 112 moves the probe tip 205 to the cleaning port, and at that time, operates the water pump 206 to clean the probe tip 205. After the robot 112 moves the probe chip 205 to the position of the DNA (reporter DNA) with the fluorescent substance, the water pump 206 is operated, and the robot 112 sucks the reporter DNA from the probe chip 205. After moving the robot 112 to the PI detection position, the calibration is confirmed. After the robot 112 moves the probe chip 205 to the injection port 210, the water pump 206 is operated, and the robot 112 discharges the reporter DNA from the probe chip 205. The cartridge pump 208 is operated to move the reporter DNA to the cartridge flow cell 219. After the cartridge pump 208 is activated and the reporter DNA is moved to the cartridge flow cell 219, it is maintained for about 60 seconds. Thereafter, the cartridge pump 208 is operated again, and the reporter DNA is moved to the waste liquid bottle 203.

  The robot 112 moves the probe tip 205 to the cleaning port 209 and cleans the probe tip 205 using the activated water pump 206. The robot 112 moves the probe tip 205 to the high salt buffer position, and then sucks the high salt buffer from the probe tip 205 using the water pump 206. After moving the robot 112 to the PI detection position, the calibration is confirmed. After moving the probe tip 205 to the injection port 210, the robot 112 discharges the high salt buffer from the probe tip 205 using the water pump 206. Thereafter, the cartridge pump 208 is operated, and the high salt buffer is moved to the cartridge flow cell 219. Further, the cartridge pump 208 is operated to move the high salt buffer to the waste liquid bottle 203. After raising the temperature of the cartridge flow cell 219 to the set temperature, it is maintained for about 60 seconds.

  The nonspecific adsorption reporter DNA is washed (S808) as follows. The robot 112 moves the probe tip 205 to the cleaning port 209 and cleans the probe tip 205 using the water pump 206. The robot 112 moves the probe tip 205 to the Low Salt Buffer position, and then sucks the Low Salt Buffer from the probe tip 205 with the water pump 206. After moving the robot 112 to the PI detection position, the calibration is confirmed. After moving the probe tip 205 to the injection port 210, the robot 112 discharges the low salt buffer from the probe tip 205 using the water pump 206. Thereafter, the temperature of the injection port 209 is raised to the set temperature and maintained for about 60 seconds. Subsequently, the cartridge pump 208 is operated, and the Low Salt Buffer is moved to the cartridge flow cell 219. Then, the cartridge pump 208 is operated, and the Low Salt Buffer is moved to the waste liquid bottle 203.

  After moving the probe tip 205 to the cleaning port 209, the robot 112 cleans the probe tip 205 using the water pump 206. After moving the probe tip 205 to the Low Salt Buffer position, the robot 112 sucks the Low Salt Buffer from the probe tip 205 using the water pump 206. After moving the robot 112 to the PI detection position, the calibration is confirmed. After moving the probe tip 205 to the injection port 209, the robot 112 discharges the low salt buffer from the probe tip 205 by the water pump 206. The cartridge pump 208 is operated, and the Low Salt Buffer is moved to the cartridge flow cell 219. The temperature of the cartridge flow cell 219 is set to a set temperature.

  Subsequently, an image is acquired by the CCD camera (S809). Here, clogging is detected in order to confirm whether or not the flow path is clogged at the time of image acquisition (S810). As will be described later, the clogging detection is performed by continuously obtaining CCD images in a state where the liquid in the flow cell is emptied and a state where the flow cell is filled with the liquid, and performing image processing. Therefore, in the process described from S806 to S808, the process in which the liquid in the flow cell is emptied and the state in which the flow cell is filled with the liquid may continue. Further, even if the above steps are not repeated, a state in which the liquid in the flow cell is intentionally emptied and a state in which the flow cell is filled with the liquid may be continuously created.

  Thereafter, the cartridge pump 208 is operated, and the Low Salt Buffer is moved to the cartridge flow cell 219. The cartridge pump 208 is operated again, and the Low Salt Buffer is moved to the waste bottle 203. After the apparatus termination process (S807), the apparatus is turned off.

<Detection of clogged flow path>
Detection of clogging of the flow path is performed according to the following procedure. FIG. 9 is a flowchart for explaining the flow channel clogging detection operation.

  First, an operation is performed so that the interior of the flow cell 219 becomes empty (S901), and an image is acquired (S902). At this time, the focus is set at a position where the inside of the flow cell 219 is filled with the liquid. The image can be easily acquired by using an image sensor, but the image may be synthesized by processing by scanning irradiation light like a laser scanning microscope.

  Next, the flow cell 219 is operated to fill the flow cell 219 with the liquid feeding mechanism of the SNP analyzer 101 (S903), and an image is acquired (S904).

If the flow path is clogged at this time, the following two states are assumed.
(1) The flow path is clogged and the flow cell is not filled with liquid. (2) The flow path is clogged and the flow cell is filled with liquid.

In the case of (1), the images acquired in S902 and S904 are both “empty” images that are not filled with liquid.
In the case of (2), the images acquired in S902 and S904 are both “full” images that are filled with liquid.

  That is, only when the flow path is not clogged, the images acquired in S902 and S904 are different images when they are empty and when they are satisfied.

  An example of the acquired image is shown in FIG. 10 (a) and 10 (c) show a state where the flow cell is empty, and FIGS. 10 (b) and 10 (d) show a case where the flow cell is filled with liquid. The images in the state where the flow cell is empty and filled with the liquid are clearly different, and it can be easily confirmed by visual observation that the flow path is not clogged. In the case of clogging, for example, FIGS. 10A and 10C are acquired, or FIGS. 10B and 10D are acquired, and the images are not different from each other.

  In the image acquisition part of the determination flow shown in FIG. 9, a reference image for comparison may be stored in advance in a storage device part (not shown) of the sample introduction apparatus and compared with the image. Specifically, an image in a state where the flow path is filled with the liquid is stored in advance (this is called F1), and the newly acquired liquid and the liquid for actually checking the clogging of the flow path are stored. The image processing described below is performed on the filled image (referred to as F2). Both should be almost the same, but if the flow path is clogged, it will be an empty image, or bubbles will be mixed in from the flow path, and a difference will occur between F1 and F2 to detect this Can do.

  In addition, automatic determination can be performed in an apparatus that has advanced automation. First, the signal output of each pixel with the flow cell emptied is represented by e (i, j). Here, i and j are image coordinates. The signal output of each pixel in a state where the flow cell is filled with the liquid is represented by f (i, j).

  In order to automatically determine clogging of the flow path, Σ | e (i, j) −f (i, j) | is calculated. This calculation may be performed on all pixels of the acquired image, or may be performed on a part of the screen or partially on a plurality of locations. This sum is small when two images are similar, and large when two images are different. Therefore, automatic determination is possible by comparison with a preset threshold value. In the calculation, the difference may be emphasized by calculating the square sum of the differences instead of the sum of the absolute values of the differences.

  Alternatively, for the signal output of each pixel of the two images divided by the average value of the output of each image (standardization), the sum of the ratios of the normalized pixels of the two images is obtained and You may compare with a threshold value. If the images are similar, the normalized ratio of each pixel approaches 1, and the sum total approaches the total number of images. If the sum is far from the number of pixels, it is determined that the two images are different, and it is determined that there is no blockage in the flow path. If necessary, calculation can be performed by taking a ratio without standardization, so that measurement conditions and the state of the apparatus (such as intensity change of the excitation light source) can also be detected and used accordingly.

After the above calculation of the difference or ratio, the average value between the pixels of the difference or ratio may be used instead of the sum.
Next, FIG. 11 shows an example in which the sum of absolute values of differences is calculated for all pixels. Hereinafter, the calculation was made assuming that the flow cell was empty and filled with liquid, and that FIG. 10a was empty, FIG. 10b was full, FIG. 10c was empty, and FIG. If the above procedure is followed, the empty → full line is in a normal state without clogging of the flow path, and the empty → empty state and the full → full state are abnormal states in which the flow path is clogged.

  In FIG. 11, the calculation result shows a large value in the normal case in both the absolute value sum of differences and the normalized sum of ratios. In this imaging condition, when the sum of absolute values of differences is about 4.00E + 09, in the case of the sum of standardized ratios, the threshold is set to about 410000, and the flow path is clogged. Separation is possible when it is not clogged.

  If an abnormality in the apparatus such as a clogged channel is detected, the control unit in the SNP analyzer (sample analyzer) may leave an abnormality detection log in the apparatus log. Then, if necessary, the operator is notified on the screen that an abnormality has been detected. Further, if necessary, the operator may be prompted to confirm by selecting a confirmation button.

  Subsequently, the SNP analyzer 101 cleans the flow path automatically or after obtaining confirmation from the operator. Specifically, the operation of filling the liquid in the flow cell 219 is performed once or a plurality of times. By this operation, foreign substances and deposits are washed away and the normal state is restored. After cleaning, clogging detection processing is performed again to confirm the cleaning effect. If the operation does not return to normal even after one or more repetitions, the operator may be prompted to clean the corresponding part using a technique, or may be asked to request maintenance. Further, cleaning and maintenance may be promoted without performing the cleaning process.

<Summary>
In the sample analyzer (SNP analyzer 101) according to the present embodiment, a predetermined liquid such as a sample and water is sent to the flow cell via a flow path for introduction into the flow cell (sample reaction unit), and light detection is performed. The flow cell image is acquired by the unit (CCD etc.), and it is determined whether or not the flow path is clogged by the image. Therefore, the clogging determination can be performed by adding a simple configuration, and the cost of the apparatus can be reduced. Can do.

  In addition, the image after the operation of emptying the flow cell by the photodetector and the image after the operation of filling the flow cell with the liquid are acquired, and the clogging of the flow path is performed by comparing these images. judge. Therefore, the clogging of the flow path can be detected easily and easily as compared with the conventional case. Note that an image after the operation of filling the flow cell with the liquid (or an image after the operation of emptying the flow cell) is acquired, and the state in which the image and the flow cell are filled with the liquid (or the flow cell You may make it determine clogging of a flow path by comparing with the reference image obtained by imaging (the state made empty).

  Further, when it is determined that the flow path is clogged, the flow path cleaning process is automatically performed, so that the sample can always be analyzed in a normal state. If it is determined that the flow path is clogged, the same effect can be obtained by notifying the user of the determination result and prompting the user to perform cleaning.

  Also, in the sample analysis process, the clogging of the flow path is determined at both timings before the process of attaching the sample to DNA and immediately after the analysis process. If the correct determination result is obtained at both timings, the sample analysis process is performed. It will be possible to judge that it is reliable.

It is a disassembled perspective view of the SNP analyzer which uses a DNA chip. It is a figure which shows schematic structure inside a SNP analyzer. It is a figure which shows the structure of the Cartridge processor in an interface unit. It is a figure which shows the detailed structure of a cartridge. It is a figure for demonstrating the mechanism which inserts and fixes a cartridge in an apparatus. It is an assembly drawing of a needle block and a needle base. It is a figure which shows the detailed structure of a needle and a needle block. It is a flowchart for demonstrating the processing operation in a SNP analyzer. It is a flowchart for demonstrating the processing operation of clogging detection. It is a figure which shows the example of the clogging detection using an actual image. It is a calculation result of clogging detection using an actual image.

Claims (18)

A sample analyzer for analyzing a sample introduced into a sample reaction unit,
A flow path for introducing the sample into the sample reaction section;
A liquid feeding mechanism for feeding the sample and a predetermined liquid to the sample reaction section via the flow path;
A light detection unit for detecting light from the sample reaction unit;
A clogging determination unit that determines whether the flow path is clogged based on a detection result of the light detection unit,
A sample analyzer comprising:   The sample analysis apparatus according to claim 1, wherein the clogging determination unit determines clogging of the flow path from an output image of the photodetector. The photodetector acquires a first image after performing an operation of emptying the sample reaction unit, and a second image after performing an operation of filling the sample reaction unit with a liquid,
The sample analyzer according to claim 2, wherein the clogging determination unit determines clogging of the flow path by comparing the first image and the second image. The photodetector acquires an image after performing an operation of filling the sample reaction part with a liquid,
The sample analysis according to claim 2, wherein the clogging determination unit determines clogging of the flow path by comparing the image with a reference image in a state where the sample reaction unit is filled with a liquid. apparatus.   The sample analysis according to claim 1, further comprising: a cleaning processing unit that automatically performs a cleaning process on the flow path when the clogging determination unit determines that the flow path is clogged. apparatus.   Furthermore, when the said clogging determination part determines with the said flow path being clogged, it has a means to notify a user of a determination result and to encourage the said user to wash | clean. Sample analyzer. A sample analyzer for mounting a cartridge including a flow cell having a septum and introducing a sample into the flow cell for analysis,
A cartridge interface unit for mounting the cartridge;
A needle insertable into the septum;
A needle moving mechanism for moving the needle relative to the septum and performing an insertion / extraction operation of the needle;
A flow path for introducing the sample into the flow cell;
A liquid feeding mechanism for feeding the sample and a predetermined liquid to the flow cell via the flow path and the needle;
A light detection unit for detecting light from the flow cell;
A clogging determination unit that determines whether the flow path is clogged based on a detection result of the light detection unit,
A sample analyzer comprising:   The sample analysis apparatus according to claim 7, wherein the clogging determination unit determines clogging of the flow path from an output image of the photodetector. The photodetector acquires a first image after performing an operation of emptying the sample reaction unit, and a second image after performing an operation of filling the sample reaction unit with a liquid,
The sample analyzer according to claim 8, wherein the clogging determination unit determines clogging of the flow path by comparing the first image and the second image. The photodetector acquires an image after performing an operation of filling the sample reaction part with a liquid,
The sample analysis according to claim 8, wherein the clogging determination unit determines clogging of the flow path by comparing the image with a reference image in a state where the sample reaction unit is filled with a liquid. apparatus.   The sample analysis according to claim 7, further comprising a cleaning processing unit that automatically performs a cleaning process on the flow path when the clogging determination unit determines that the flow path is clogged. apparatus.   Furthermore, when the said clogging determination part determines with the said flow path being clogged, it has a means to notify a user of a determination result and to encourage the said user to wash | clean. Sample analyzer. A flow path for introducing a sample into the sample reaction section; and a liquid feed mechanism for feeding the sample and a predetermined liquid to the sample reaction section via the flow path, and the sample reaction A channel clogging determination method for determining clogging of the channel in a sample analyzer that analyzes the sample at a part,
Detecting light from the sample reaction unit by a light detection unit;
A step of determining whether or not the flow path is clogged based on a detection result of the light detection unit by a determination unit;
A channel clogging determination method comprising:   The channel clogging determination method according to claim 13, wherein, in the determining step, the clogging determination unit determines clogging of the channel from an output image of the photodetector. In the step of detecting light, the photodetector detects a first image after performing an operation of emptying the sample reaction unit and a second image after performing an operation of filling the sample reaction unit with a liquid. Get images and
The channel clogging determination according to claim 14, wherein, in the determining step, the clogging determination unit determines whether the channel is clogged by comparing the first image and the second image. Method. In the step of detecting the light, the photodetector acquires an image after performing an operation of filling the sample reaction part with a liquid,
The clogging determination unit determines the clogging of the flow path by comparing the image with a reference image in a state where the sample reaction unit is filled with a liquid in the determining step. 14. The method for determining clogging of the flow path according to 14.   Furthermore, when the said clogging determination part determines with the said flow path being clogged, the said sample analyzer comprises the process of performing the washing process of the said flow path automatically. Flow path clogging judgment method.   Furthermore, when the clogging determination unit determines that the flow path is clogged, the sample analyzer includes a step of notifying a user of a determination result and urging the user to perform cleaning. Item 14. The method for determining clogging of a flow path according to item 13.

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