WO2018051426A1 - Dispositif d'analyse - Google Patents

Dispositif d'analyse Download PDF

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Publication number
WO2018051426A1
WO2018051426A1 PCT/JP2016/077071 JP2016077071W WO2018051426A1 WO 2018051426 A1 WO2018051426 A1 WO 2018051426A1 JP 2016077071 W JP2016077071 W JP 2016077071W WO 2018051426 A1 WO2018051426 A1 WO 2018051426A1
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Prior art keywords
flow cell
flow
reagent
flow path
tdi
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PCT/JP2016/077071
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English (en)
Japanese (ja)
Inventor
加藤　宏一
庄司　智広
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株式会社 日立ハイテクノロジーズ
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Priority to PCT/JP2016/077071 priority Critical patent/WO2018051426A1/fr
Publication of WO2018051426A1 publication Critical patent/WO2018051426A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the present invention relates to an analyzer. More specifically, the present invention relates to an analyzer that decodes a base sequence of a nucleic acid such as DNA or RNA at high speed.
  • Next-generation sequencing technology is a technology that has developed rapidly after the completion of the Human Genome Project and is currently replacing the Sanger method, which has been the mainstream until then.
  • the feature is that minute reaction fields are arranged on a substrate at a high density on a two-dimensional plane, and weak fluorescence signals emitted from the minute reaction fields are acquired in a massively parallel manner. More specifically, the chemical reaction in the next generation sequence proceeds on a large number of minute reaction fields fixed on the substrate surface of the flow cell. By measuring the weak fluorescence signals generated by the respective micro reaction fields as a result of the reaction in a massively parallel manner, it is possible to perform DNA base sequence analysis at high speed and at low cost.
  • an area imaging method in which a fluorescence image is captured using a two-dimensional camera has been mainly used.
  • an XY stage with a fixed flow cell is sequentially driven, and the flow cell tile positioned on the optical axis is imaged.
  • the flow cell needs to be stopped during imaging.
  • the waiting time for vibration attenuation caused by the acceleration motion accompanying the step-and-repeat of the XY stage has been a problem.
  • TDI time-delay integration method
  • TAT TurnTAround Time
  • the imaging time per cycle required for one flow cell is 7.2 minutes. Therefore, in the sequence, the imaging time is still longer and dominant over the chemistry time.
  • chemistry and imaging can be performed alternately. However, even if the chemistry and imaging are processed in parallel to reduce the throughput, the rate-limiting step of the throughput is still the imaging time.
  • Patent Document 2 discloses a method of reducing reagent consumption by arranging a circular flow path along the outer periphery of a circular flow cell, providing a reagent injection port directly under the flow cell, and rotating the flow cell successively. It is described in.
  • the conventional flow cell has a rectangular shape, and a plurality of linear flow paths are arranged in the flow cell. For this reason, even if a high-speed scan detection method such as TDI is adopted, there is a problem that a single flow path cannot be scanned all at once.
  • the linear flow paths that have been conventionally formed on the flow cell are connected to form a spiral flow path on the flow cell.
  • rotating and horizontally driving the flow cell with the ⁇ X stage with respect to the objective lens it is possible to increase the flow path length that can be continuously scanned. Thereby, it is possible to reduce the number of interruptions of the scanning operation and the number of autofocus operations for the flow paths accompanying movement to different flow paths.
  • the application of the present invention brings about the effect of shortening the imaging time and improving the throughput.
  • FIG. 3 is an explanatory diagram of a TDI sequence method using a flow cell having a spiral channel for the first embodiment.
  • FIG. 4 is an explanatory diagram of a flow cell having one to two spiral-shaped flow paths in the second embodiment.
  • FIG. 6 is an explanatory diagram of a flow cell having a spiral channel having more than two channels in Example 3.
  • FIG. 6 is an explanatory diagram of a flow cell having a spiral channel having more than two channels in Example 3.
  • FIG. 6 is an explanatory diagram of the configuration of a flow cell having a spiral flow path in the fourth embodiment.
  • FIG. 9 is an explanatory diagram of a method for installing a flow cell having a spiral flow path on a heat block in Example 5.
  • FIG. 9 is an explanatory diagram of a method for installing a flow cell having a spiral flow path on a heat block in Example 5.
  • FIG. 10 is an explanatory diagram of a reagent feeding method for a flow cell having a spiral channel in Example 6;
  • FIG. 10 is an explanatory diagram of a reagent feeding method for a flow cell having a spiral channel in Example 6;
  • FIG. 10 is an explanatory diagram of a reagent feeding method for a flow cell having a spiral channel in Example 6;
  • FIG. 10 is an explanatory diagram regarding the order of TDI scanning in a flow cell having a spiral channel for the seventh embodiment.
  • FIG. 10 is an explanatory diagram of a method for driving a flow cell having a spiral flow path by a ⁇ X stage in the eighth embodiment.
  • FIG. 10 is an explanatory diagram of a method for driving a flow cell having a spiral flow path by a ⁇ X stage in the eighth embodiment.
  • FIG. 10 is an explanatory diagram of a driving method when performing sequencing using a flow cell having a spiral flow path in the ninth embodiment.
  • FIG. 10 is an explanatory diagram of a method for applying the patterned array technology to a flow cell having a spiral flow path in the tenth embodiment.
  • FIG. 16 is an explanatory diagram of parameters when applying the TDI method to a flow cell having a spiral flow path in the eleventh embodiment.
  • FIG. 16 is an explanatory diagram of a drift of a minute reaction field that occurs when a flow cell having a spiral flow path is rotationally driven in Example 12.
  • FIG. 14 is an explanatory diagram of a TDI sequence method using two flow cells having a spiral channel in Example 13.
  • Laser light having a wavelength of 642 nm emitted from a diode laser 191 that is a light source is reflected by a mirror 105 through a laser line filter 103.
  • the laser light having a wavelength of 505 nm emitted from the other diode laser 192 is combined with the laser light having a wavelength of 642 nm via the laser line filter 104 and the dichroic mirror 106. Since the combined laser light is linearly polarized, it passes through the quarter-wave plate 123 in order to make it circularly polarized. Further, the laser light passes through the first beam expander 107, and the diameter of the light beam is enlarged.
  • the profile of the laser beam is circular, but in order to perform TDI scanning effectively, it is necessary to make this a rectangular shape.
  • the beam intensity distribution in the Z direction is preferably rectangular.
  • the laser light is passed through the line generator 108. More specifically, as the line generator 108, (1) a Powell lens which is an aspherical lens, (2) a cylindrical microlens array, or (3) a diffusion plate can be used.
  • the second beam expander 109 has a function of reducing the angle of view of the laser light formed into a rectangular shape, and has a function of efficiently irradiating a region within the diameter of the pupil plane of the objective lens 114.
  • the laser light is condensed by the objective lens 114 and illuminates a rectangular area.
  • a minute reaction field is fixed in the flow path of the flow cell 101.
  • the minute reaction field may be randomly arranged on the flow path, or may be regularly arranged by using a semiconductor lithography technique.
  • the objective lens 114 can be driven in the direction of the optical axis by the Z motor 115, thereby focusing on the minute reaction field.
  • Each micro reaction field fixed on the flow path includes a large number of one small fragment of DNA of a sample to be measured, and this is said to be “monoclonal” in terms of molecular biology. In other words, it can be said that each minute reaction field is an aggregate in which a fragment of DNA derived from a sample is amplified.
  • SBS Sequence By Synthesis
  • a primer serving as a reaction scaffold is bound to a micro reaction field immobilized on a substrate, and then 4 types of nucleotides (Alexa-488-dATP, Alexa-) labeled with different 4 types of fluorescent dyes are used.
  • 555-dTTP, Alexa-647-dCTP, Alexa-680-dGTP is a method of performing sequencing by incorporating only one base into a micro reaction field.
  • nucleotides corresponding to the complementary strand of the single-stranded DNA on the side of the microreaction field are sequentially taken up by the polymerase enzyme for one base in the 3 ′ end direction of the primer. After one base corresponding to each minute reaction field is incorporated, the floating fluorescent nucleotide is removed by washing, and then fluorescence measurement is performed. The reason why the extension of the second base does not occur is because a substance that inhibits the extension of the dye of the second base is bound to the fluorescent dye of the first base.
  • a reagent for cleaving the elongation inhibiting substance is injected into the flow cell, and after the completion of the cleavage, the SBS reaction of the second base is sequentially performed.
  • the incorporated fluorescent dye is irradiated with laser light and emits fluorescence.
  • the fluorescence is condensed by the objective lens 114, passes through the dichroic mirror 113, and passes through the 642 nm notch filter 161 and the 505 nm notch filter 162.
  • the dichroic mirror 116 reflects only the fluorescence wavelength band of Alexa-488, and transmits the wavelength bands of Alexa-555, Alexa-647, and Alexa-680, which are the other three fluorescent dyes.
  • the reflected fluorescence of Alexa-488 passes through the emission filter 119 and is collected by the tube lens 130 to form fluorescence images emitted from a large number of minute reaction fields on the sensor surface of the CMOS camera 134.
  • the dichroic mirror 117 reflects only the fluorescence derived from Alexa 555, and the reflected fluorescence of Alexa 555 passes through the emission filter 120 and the tube lens 131 and is collected on the CMOS camera 135 to form an image.
  • the dichroic mirror 118 reflects the fluorescence derived from Alexa 647 and transmits the fluorescence derived from Alexa 680, and forms images on the CMOS cameras 136 and 137 through the emission filters 121 and 122 and the tube lenses 132 and 133, respectively.
  • four types of fluorescence that is, four types of bases can be identified by one fluorescence measurement.
  • the flow cell 101 having a spiral channel is installed in the heat block 102.
  • the flow cell 101 can continuously drive the spiral flow path directly under the objective lens 114 by simultaneously driving the ⁇ stage and the X stage.
  • TDI measurement can be performed.
  • TDI measurement is one of readout methods in a CCD or CMOS camera that shoots while accumulating moving objects.
  • the CCD camera performs vertical transfer in units of one line when reading charges. If the timing of this transfer and the timing of movement of the target image incident on the CCD surface are matched, exposure can be repeated by the number of vertical stages of the CCD.
  • This method is called TDI, and is a measurement method capable of imaging a moving object at high speed and with high sensitivity.
  • the flow cell 101 two spiral channels are formed. This is because imaging and chemistry are alternately performed in one cycle of the SBS reaction. What is characteristic in this embodiment is that two flow paths are prepared for two imaging steps in one cycle. In other words, one imaging process is completed within one flow path.
  • the conventional flow cell having a plurality of linear flow paths has the following problems. That is, it is the movement time of the XY stage that accompanies movement between a plurality of flow paths during the imaging process, and the time that accompanies autofocus operation that occurs at the start of measurement in different flow paths. These times were one of the causes of TAT delay. In this embodiment, since one imaging process is completed within one flow path, it is possible to avoid the operation time described above. Therefore, the imaging time can be greatly shortened.
  • the heat block controls the temperature of the flow cell 101 to 60 ° C. That is, while the SBS chemistry reaction is performed at 60 ° C., the imaging process is performed at 60 ° C. at the same time. As a result, the time required for heating and cooling the heat block can be omitted, and TAT can be shortened. Moreover, this temperature control temperature is not limited to 60 degreeC, 50 degreeC, 40 degreeC, and 30 degreeC may be sufficient. Furthermore, the room temperature of 25 ° C. is most desirable. It is known that the fluorescence intensity of a fluorescent dye becomes weaker as it is exposed to a higher temperature.
  • Reagent for primer hybridization Reagent for primer hybridization, extension reagent containing 4 types of fluorescent nucleotides and polymerase, cleavage reagent for dissociating protecting group of fluorescent nucleotide, unnecessary reaction of reactive group after cleaving protecting group
  • a cap reagent, a cleaning reagent, an imaging reagent for fluorescence observation, and the like are arranged and injected in advance in the reagent cartridge 140.
  • the reagent cartridge 140 is installed in the reagent rack 141 and cooled to 4 ° C.
  • the Peltier element 144 cools the heat block 142 installed in the reagent cartridge 140, and the fan 146 blows air in the reagent rack 141 to the fins 145.
  • the cooled air circulates in the reagent rack 141 and indirectly cools the plurality of reagents installed in the reagent cartridge 140 to 4 ° C.
  • the opposite surface of the Peltier element 144 is heated.
  • a fin 148 and a fan 143 are installed on the Peltier element 144. Thereby, the fan 143 can exhaust heat outside the apparatus and cool the Peltier element 144.
  • a sipper tube is inserted into each reagent well held by the reagent cartridge 140. Reagents are aspirated from the tips of these sipper tubes.
  • the sipper tube is connected to the switching valve 147.
  • the switching valve 147 can be connected to an arbitrary flow path to select an arbitrary reagent.
  • the reagent selected by the switching valve 147 is sent through the flow path 150 to the flow cell 101 that holds the minute reaction field.
  • a syringe pump 154 serving as a power source for sucking the reagent is disposed downstream of the flow cell 101.
  • a two-way valve 152 is disposed upstream of the syringe pump 154 and a two-way valve 155 is disposed downstream.
  • the two-way valve 152 When aspirating the reagent, the two-way valve 152 is controlled to connect the flow cell 101 and the syringe pump 154, and the two-way valve 155 is closed to drive the syringe pump 154.
  • the two-way valve 152 When discarding the reagent, the two-way valve 152 is closed, the two-way valve 155 is opened, the syringe pump 154 is driven, and the reagent is sent to the waste liquid tank 156.
  • a plurality of reagents can be fed with one syringe pump 154.
  • the waste liquid tank 156 is not provided, the waste liquid spills into the apparatus cabinet, which may cause problems such as electric shock, rust of the apparatus, and generation of a strange odor.
  • a micro photo sensor 158 for monitoring the presence or absence of the waste liquid tank 156 is installed.
  • a liquid receiving tray 157 is installed under the waste liquid tank 156 in case the waste liquid leaks.
  • a flow cell having a spiral channel shape is a typical flow cell used in next generation sequencers.
  • the flow cell 201 has a rectangular shape, and eight linear flow paths 202 are arranged in the flow cell 201.
  • the channel 202 has a reagent inlet 203 and an outlet 204. Since the flow cell 201 has a plurality of flow paths, even if a high-speed scan detection method such as TDI is adopted, the scan is temporarily interrupted when moving to a different flow path, and after moving to a new flow path, autofocus in the Z direction is performed again. Had to do.
  • FIG. 2 b all the flow paths in FIG. 2 a) are connected to form one spiral-shaped flow path 211.
  • the channel 211 has a reagent inlet 212 and a outlet 213. The reason why the spiral channel is used is the following two points.
  • FIG. 2c shows a flow cell in which two spiral flow paths 225 and 226 are formed in one flow cell.
  • the channel 225 has a reagent inlet 220 and outlet 223, and the channel 226 has a reagent inlet 221 and outlet 224, respectively.
  • This flow cell is a flow cell for performing a chemistry process and an imaging process isothermally. While the TDI scanning is performed on the flow path 225, the chemistry process can be performed in the flow path 226. Since the two flow paths 225 and 226 are respectively integrated, the TDI scanning operation can be continuously performed, and the imaging time can be shortened.
  • a flow cell having two or more spiral channel shapes will be described below with reference to FIGS. 3A and 3B.
  • This embodiment is particularly useful when it is highly necessary to perform measurement in an independent flow path without mixing a plurality of different samples, and it is desired to realize a short TAT. In other words, it is useful when it is desired to perform high-precision measurement at high speed while avoiding contamination between samples as much as possible.
  • the flow cell has two flow paths.
  • the flow cell has three flow paths.
  • the flow cell has four flow paths.
  • the flow cell has five flow paths.
  • FIG. 3e shows a comparison between a conventional flow cell having a linear flow path and a flow cell having a spiral flow path described in this patent.
  • the flow cell shown in FIG. 3A two flow paths having a flow path length of 451 mm are formed. Therefore, the total flow path length per flow cell is 901 mm. Since the channel width is 2.5 mm, the channel area is 2253 mm 2 . The radius of the flow cell is 31.5 mm, and the flow path occupancy exceeds 72%.
  • the flow cell is composed of three members: a substrate 401 having a circular shape, a spacer 410, and a cover glass 413.
  • the flow cell is manufactured by bonding these members.
  • Examples of the material of the substrate 401 include glass, quartz, silicon, titanium, and sapphire.
  • a cover glass 413 provided with reagent inlets 404 and 405 and reagent outlets 402 and 403 on the substrate 401 is light-transmitting, and transmits visible light of 400 to 800 nm with high transmittance.
  • Examples of the material of the cover glass 413 include glass, quartz, and sapphire.
  • the spacer 410 is generally manufactured from a material such as PDMS.
  • the thickness of the spacer 410 is 30 to 100 ⁇ m, and more specifically 50 ⁇ m is desirable.
  • two flow paths 411 and 412 are formed.
  • the channel 411 has an inlet 405 and an outlet 402
  • the channel 412 has an inlet 404 and an outlet 403.
  • the flow paths 411 and 412 have a spiral shape, and the planar area of the disk-shaped flow cell can be efficiently utilized by adopting a shape in which the two flow paths are intricately arranged. Further, by adopting a spiral channel shape as compared with the conventional linear channel, the channel length per channel can be increased.
  • minute reaction fields are arranged in a random shape or a lattice shape on the upper and lower surfaces of the channels 411 and 412.
  • the minute reaction field may be formed through a DNA amplification reaction in the flow cell, or may be adjusted outside the flow cell.
  • a so-called bridge PCR may be used, or a method of adjusting outside the flow cell like a DNA nanoball may be used.
  • the flow cell 507 is vacuum chucked on the surface of the heat block 501 by sucking the suction hole 506 with a vacuum pump while being pressed against the guides 510 and 511 of the heat block 501.
  • the stress applied to the flow cell 507 by the vacuum chuck method can be reduced, and as a result, the distortion of the flow cell 507 can be reduced.
  • the flatness of the ⁇ X stage is ⁇ 30 ⁇ m. Further, by using the guides 510 and 511, the flow cell 507 can be fixed to the heat block 501 with high positional accuracy.
  • the orientation of the flow cell 507 can be specified by providing an orientation flat and its guide on the circumference in order to align the flow cell 507. Thereby, the positions of the reagent inlet and the reagent outlet on the heat block 501 and the flow cell 507 can be accurately aligned.
  • the heat block 501 is fixed on the ⁇ X stage, and it is possible to continuously position the measurement visual field of the spiral channel 509 with respect to the optical axis fixed in the vertical direction through the objective lens 508. . More specifically, it is possible to continuously perform scanning by the TDI operation in the flow channel regions from the reagent inlets 503 and 502 to the reagent outlets 504 and 505 in the spiral flow channels 509 and 510, respectively. Become. By simultaneously driving the ⁇ X stage in the ⁇ rotation direction and the X horizontal direction along the inside of the spiral channel, TDI scanning can be continuously performed without interrupting the spiral channel. Focusing during a TDI scan of the flow path is achieved by driving the objective lens 508 with a Z motor 512.
  • the reagent is connected to the rotary joint 602 via the tubes 601 and 612.
  • the rotary joint 602 is composed of two parts.
  • the lower part of the rotary joint 602 is connected to the flow path 612 from the reagent cartridge and does not follow the rotational movement of the ⁇ X stage.
  • the upper portion of the rotary joint 602 can freely rotate with respect to the lower portion thereof. More specifically, the upper part of the rotary joint 602 can follow the rotational movement of the ⁇ X stage. Therefore, distortion and twist are not accumulated in the flow path 612 as the ⁇ X stage rotates.
  • Tubes 603 and 617 are connected to the ports 604 and 610 in the multi-way valve 618 from the upper part of the rotary joint 602, respectively.
  • Multi-way valve 618 can be opened and closed independently for ports 608, 609, 604, and 610.
  • a predetermined reagent can be selectively supplied to the two flow paths 615 and 616 of the flow cell 605.
  • the multi-way valve 618 is installed on the ⁇ X stage, and rotates with the ⁇ X stage.
  • the tube 612 As the ⁇ X stage rotates, the multi-way valve 618 and the flow paths 615 and 616 rotate in the same manner, but the tube 612 is connected to the multi-way valve 618 and the flow paths 615 and 616 via the lower part of the rotary joint 602. Therefore, the tube 612 does not generate distortion such as torsion, and can stably supply the reagent to the flow cell 605.
  • a method for feeding the reagent to the spiral channel 615 will be described more specifically with reference to FIG.
  • the ports 604 and 608 are opened, the ports 609 and 610 are closed, and a negative pressure is generated by a syringe pump below the flow path to suck the reagent into the flow path 615.
  • the reagent reaches the reagent inlet 606 in the flow path via the tube 631. In the direction of the arrow, the reagent advances in the flow path along the spiral flow path.
  • the reagent that has reached the reagent outlet 607 is connected to the port 608 through the tube 632.
  • the reagent to be discharged is connected to the spiral tube 613 from the port 608 and further discharged to the waste liquid tank through the tube 614.
  • the reason why the spiral tube 613 is used here is to absorb and relieve the twist associated with the rotational movement of the ⁇ X stage.
  • FIG. 6b describes a method of supplying the reagent to the flow path 616.
  • a chemistry reaction is performed on the channel 616 while the reagent is supplied to the channel 616.
  • an imaging operation can be performed in parallel on the flow path 615.
  • the time required for the chemistry reaction and the time required for the imaging operation are the same as 3 minutes. Therefore, by alternately nesting chemistry and imaging, it becomes possible to always perform imaging during sequencing.
  • the TAT of the sequence becomes longer due to the time required for the imaging process compared to the chemistry process.
  • TAT can be reduced by using this method.
  • the ports 604 and 608 of the multi-way valve 618 are closed and the ports 609 and 610 are opened.
  • the reagent reaches the reagent inlet 621 of the flow path 616 through the tube 633.
  • the reagent travels inside the spiral channel 616 in the flow cell and reaches the reagent outlet 622. Thereafter, it reaches the port 609 through the tube 634, and further passes through the spiral tube 613 and the tube 614 and is discharged to the waste liquid tank.
  • a flow cell having a spiral flow path rotates by a ⁇ X stage.
  • both imaging and chemistry are performed at 60 ° C.
  • the reagent cooler the reagent is cooled at 4 ° C., and the temperature inside the tube is about 30 ° C. until the reagent reaches the flow cell. If a reagent near 30 ° C. is directly injected into the flow cell, the temperature environment in which imaging and chemistry are already performed at 60 ° C. may be disturbed.
  • a cylindrical heating mechanism 651 is installed in the tube 612. As a result, the reagent immediately before being injected into the flow cell is heated to 60 ° C. in advance, so that imaging and chemistry reaction in the flow cell can be stably performed at 60 ° C.
  • the flow cell has two flow paths, and the flow path width is 2.5 mm.
  • the flow path has two surfaces, an upper surface and a lower surface, and a minute reaction field is fixed to each of the surfaces. Therefore, it is possible to increase the number of minute reaction fields that can be detected by scanning the upper and lower surfaces.
  • FIG. 7 shows the scanning order in a flow cell having two spiral channels.
  • the spiral channel is simplified to a semicircular channel.
  • FIG. 7a) shows the scan order within one of the two channels.
  • the solid line part in FIG. 7b) shows the scanning order of another flow path.
  • the TDI scan starting from the start point 701 reaches the end point 702 of the first swath. From the start point 701 to the end point 702, it is possible to perform a TDI scan in the forward direction without continuous interruption.
  • the ⁇ X stage is driven in the X direction by a step-and-repeat method. Since the movement from the end point 702 to the start point 703 moves in a direction perpendicular to the advancing direction of the TDI line scan, it is not necessary to perform the TDI scan.
  • a TDI scan is performed in the direction opposite to the traveling direction of the first swath. This continues until the second swat termination point 704.
  • the operation so far is the TDI scan of the minute reaction field fixed on the bottom surface of the flow cell.
  • the TDI scan is similarly performed on the minute reaction field fixed on the top surface of the flow cell.
  • the Z stage holding the objective lens is driven to move the focus from the bottom end point 704 to the top start point 705.
  • the TDI scan is continuously performed in the forward direction from the start point 705 to the end point 706 of the second swath without interruption.
  • the flow cell moves from the terminal point 706 on the upper surface of the flow cell to the start point 707, and similarly, the TDI scan is continuously performed in the reverse direction from the start points 707 to 708.
  • the time from the start to completion of this imaging time is as follows.
  • the length of the swath is 451 mm, and a TDI scan for 4 swaths is performed for one flow path.
  • the throughput for one cycle in one channel is estimated as follows.
  • the minute reaction fields are fixed every 1 ⁇ m
  • the ⁇ X stage is moved horizontally from the end point 708 to the start point 711.
  • the ⁇ X stage is moved horizontally from the end point 712 to the start point 713 in a step-and-repeat manner.
  • the focus is moved from the end point 714 to the start point 715. Thereby, the lower surface can be scanned from the upper surface in the flow cell channel.
  • a TDI scan is performed in the forward direction from the start point 715 to the end point 716.
  • the ⁇ X stage is translated in the X direction from the end point 716 to the start point 717, and the TDI scan is again performed in the reverse direction from the start point 717 to the end point 718. From the start point 701 to the end point 711 is a TDI scan operation per cycle.
  • FIG. 7c) shows the imaging operation entering the second cycle, and FIG. 7c) is basically the same as FIG. 7a). Further progress of the cycle is achieved by repeating FIG. 7b) ⁇ FIG. 7c) ⁇ FIG. 7b) ⁇ FIG. 7c) up to a predetermined number of cycles.
  • the spiral channel can be formed so as to fill the substrate surface, and this shape is useful from the viewpoint of the manufacturing cost of the flow cell.
  • the minute reaction field is randomly fixed on the flow cell channel surface.
  • the interval between the minute reaction fields is not controlled, which means that the average distance of the reaction fields does not depend on the panels in the flow cell and is constant. For this reason, it is desirable that the TDI scanning speed be constant at any location of the flow cell.
  • a control method in which the angular speed ⁇ [radians / s] of the ⁇ X stage is made variable.
  • the distance from the center of the circular flow cell and the angular velocity are r 1, r 2 , ⁇ 1 , and ⁇ 2 , respectively.
  • the scanning speed at each point that is, the speed in the flow path direction
  • control is performed to keep the scan speed at an arbitrary point on the flow path constant.
  • the angular velocity is ⁇ 1 ⁇ 2 .
  • the angular velocity is increased as the TDI scanning is closer to the center of the spiral flow path, and the ⁇ X stage is controlled so as to decrease the angular velocity when performing TDI scanning in a region away from the center.
  • FIGS. 8a) and 8 Specific examples of this control method are shown in FIGS. 8a) and 8).
  • this method is an effective measurement method when a minute reaction field is fixed on a substrate randomly, that is, not regularly. This is because when the minute reaction field is fixed at random, the distance between the minute reaction fields is constant in both the inner and outer circumferences and is not regular. Therefore, as the TDI scan is moved to the outer periphery, the average distance of the physical minute reaction field does not change. For the reasons described above, it is necessary to keep the scanning speed constant at the outer periphery. For this reason, as described in the present embodiment, it is necessary to control to gradually reduce the angular velocity ⁇ with the transition from the inner periphery to the outer periphery.
  • FIG. 8 illustrates the TDI scan operation corresponding to one cycle of the sequence reaction for the spiral channel
  • FIG. 9a illustrates the TDI scan operation for two cycles of the sequence reaction in one spiral channel. It is carried out.
  • the two swaths in the flow path are changed when the TDI scan direction is changed from the outer periphery to the inner periphery.
  • FIG. 9b) is a graph showing the time change of the position r from the center of the flow cell when imaging and chemistry are alternately repeated for two flow paths in the disc-shaped flow cell. More specifically, the operation corresponding to FIGS. 7 a) and b) in the seventh embodiment is described.
  • the value of the position r when the TDI scan is performed in the second spiral flow path is negative.
  • the duty ratio which is an index indicating the operation rate of the camera, is 50%.
  • the duty ratio can be improved.
  • the duty ratio can be improved to nearly 100%.
  • the scan speed v is made constant by decreasing the angular velocity ⁇ along with the transition from the inner periphery to the outer periphery.
  • a minute reaction field with a size of about 0.5 to 1 micron can be regularly formed at a desired interval (0.5 to 1 micron, or any value from 1 to 5 microns).
  • the minute reaction fields are fixed at equal intervals at the same pitch in the scanning direction in any part of the flow cell.
  • a method for regularly fixing a minute reaction field to a spiral channel will be described below.
  • FIG. 10a a method for performing TDI scanning with a constant rotation speed ⁇ will be described.
  • FIG. 10a there are three arc-shaped channels, which actually form one channel as a spiral channel.
  • Each channel width is 1.1 mm, and a disk-like minute reaction field having a pitch of 1 micron and a diameter of 0.5 microns is fixed within the width.
  • the pitches of the minute reaction fields in the scanning direction are ⁇ d 1 , ⁇ d 2 , and ⁇ d 3 , respectively.
  • ⁇ d 1 ⁇ ⁇ 5 mm
  • ⁇ d 2 ⁇ ⁇ 10 mm
  • ⁇ d 3 ⁇ ⁇ 15 mm.
  • TDI scanning can be performed at a constant rotational speed ⁇ in a spiral channel or a circular channel.
  • the control method of the ⁇ X stage in TDI scanning is simplified, and the risk of occurrence of measurement errors can be reduced.
  • minute reaction fields are regularly arranged on the upper and lower surfaces of the channel surface at equal intervals of 1 micron pitch as described above.
  • the pitch of the minute reaction field in the scanning direction is increased according to the distance of the center radius r, but in FIG. 10b), the pitch is made the same.
  • the rotation speed ⁇ is variable as described in the eighth and ninth embodiments, and the scanning direction speed v is constant. Since the scan speed v is constant and the fixed pitch of the minute reaction field is constant at any center radius r, the number of minute reaction fields per unit time in the TDI scan can be made constant.
  • the TDI sensor 1102 specifically employed here is Hamamatsu Photonics S10202-08-01. This is a back-illuminated CCD image sensor, the number of effective pixels (H) ⁇ (V) is 4160 ⁇ 128 pixels, and the line rate is 50 kHz. The pixel size is 12 ⁇ 12 ⁇ m, and the number of TDI stages is 128.
  • FIG. 11 is used for the drift in the direction perpendicular to the TDI scan direction, that is, the radial direction, which occurs when TDI scanning is applied to spiral and circular flow paths. Will be described below.
  • the minute reaction field moves linearly by linear drive by the XY stage.
  • the rotational drive by the ⁇ X stage is adopted, so that the minute reaction field performs a circular motion or a spiral motion.
  • the scan direction of the TDI scan sensor is usually a linear direction
  • the scan direction of the TDI scan sensor coincides with the movement direction of the minute reaction field.
  • the minute field reaction field is not a linear motion but a circular motion or a spiral motion accompanied by a rotational motion
  • a minute reaction field drift occurs in a direction perpendicular to the sensor integration direction. This drift amount will be described below.
  • the drift amount will be described below using numerical values based on the TDI sensor employed in the eleventh embodiment.
  • FIG. 12a the case where the rotational speed ⁇ is constant will be described.
  • the rotation speed ⁇ is constant, the angle ⁇ during a constant exposure time is constant.
  • the rotational speed ⁇ is constant, it is desirable to increase the pitch of the minute reaction field with the transition from the inner periphery to the outer periphery of the flow cell as described in FIG. By increasing the pitch of the minute reaction field according to the radius, it becomes possible to cope with the amount of movement necessary for efficiently detecting the minute reaction field.
  • the rotational speed ⁇ is variable as described in FIG. B) of Example 11
  • the pitch between the minute reaction fields can be made constant without depending on the radius r from the center.
  • the resolution in the optical system as this pitch, it is possible to increase the number of minute reaction fields that can be fixed for a flow cell having a certain area and improve the throughput.
  • the drift amount of the minute reaction field during the exposure time can be calculated. These are 0.18 ⁇ m, 0.09 ⁇ m, and 0.03 ⁇ m, respectively, which are sufficiently smaller than the size of 0.33 ⁇ m / pixel on the object plane of the TDI scan sensor. Therefore, it is possible to measure fluorescence without problems by rotationally driving a flow cell having a spiral channel.
  • the apparatus of the first embodiment has one ⁇ X stage mounted thereon, whereas two ⁇ X stages 102 and 161 are mounted.
  • the flow chip described in the first embodiment has two spiral channels, but in the present embodiment, one flow chip has a single spiral channel.
  • the sequence reaction can proceed at 25 ° C. for imaging and 60 ° C. for chemistry. That is, independent temperature control is possible for imaging and chemistry. Therefore, the imaging process and the chemistry process are performed independently for each of the two ⁇ X stages.
  • a single flow path is formed in one flow cell, and TDI scanning can be continuously continued for this flow path during imaging. Since the movement time to another flow path and the time required for focusing at the time of movement can be skipped, the TAT can be shortened.

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Abstract

La présente invention concerne le raccordement de canaux d'écoulement linéaires formés sur une cellule d'écoulement classique de façon à former un canal d'écoulement de type à tourbillon sur la cellule d'écoulement. Par rotation et entraînement horizontal de la cellule d'écoulement sur un étage θX par rapport à une lentille d'objectif, il est possible d'augmenter la longueur du canal d'écoulement qui peut être balayé en continu. Cette configuration permet ainsi de réduire à la fois le nombre d'interruptions de l'opération de balayage associée au mouvement vers différents canaux d'écoulement et le nombre d'opérations d'autofocalisation à effectuer sur les canaux d'écoulement.
PCT/JP2016/077071 2016-09-14 2016-09-14 Dispositif d'analyse WO2018051426A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009520985A (ja) * 2005-12-21 2009-05-28 ジェ・チャーン・ユ バイオメモリディスク及びバイオメモリディスクドライブ装置及びそれを用いた分析方法
US20100041562A1 (en) * 2004-12-10 2010-02-18 Simon Fraser University Microfluidic microarray assemblies and methods of manufacturing and using
JP2010172270A (ja) * 2009-01-29 2010-08-12 Soka Univ マイクロ反応容器及びマイクロ反応容器を用いたポリメラーゼ連鎖反応方法
JP2010284101A (ja) * 2009-06-11 2010-12-24 Hitachi High-Technologies Corp 反応容器，並列処理装置、及びシーケンサ
JP2012172981A (ja) * 2011-02-17 2012-09-10 Jikei Univ 特異的反応検出キット、特異的反応検出装置、及び特異的反応検出方法
WO2014034275A1 (fr) * 2012-08-30 2014-03-06 株式会社 日立ハイテクノロジーズ Dispositif d'analyse d'acides nucléiques
JP2015090458A (ja) * 2013-11-07 2015-05-11 株式会社日立ハイテクノロジーズ 分析装置

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100041562A1 (en) * 2004-12-10 2010-02-18 Simon Fraser University Microfluidic microarray assemblies and methods of manufacturing and using
JP2009520985A (ja) * 2005-12-21 2009-05-28 ジェ・チャーン・ユ バイオメモリディスク及びバイオメモリディスクドライブ装置及びそれを用いた分析方法
JP2010172270A (ja) * 2009-01-29 2010-08-12 Soka Univ マイクロ反応容器及びマイクロ反応容器を用いたポリメラーゼ連鎖反応方法
JP2010284101A (ja) * 2009-06-11 2010-12-24 Hitachi High-Technologies Corp 反応容器，並列処理装置、及びシーケンサ
JP2012172981A (ja) * 2011-02-17 2012-09-10 Jikei Univ 特異的反応検出キット、特異的反応検出装置、及び特異的反応検出方法
WO2014034275A1 (fr) * 2012-08-30 2014-03-06 株式会社 日立ハイテクノロジーズ Dispositif d'analyse d'acides nucléiques
JP2015090458A (ja) * 2013-11-07 2015-05-11 株式会社日立ハイテクノロジーズ 分析装置

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