WO2018051426A1

WO2018051426A1 - Analysis device

Info

Publication number: WO2018051426A1
Application number: PCT/JP2016/077071
Authority: WO
Inventors: 加藤　宏一; 庄司　智広
Original assignee: 株式会社日立ハイテクノロジーズ
Priority date: 2016-09-14
Filing date: 2016-09-14
Publication date: 2018-03-22

Abstract

The present invention connects linear flow channels formed on a conventional flow cell so as to form an eddy-like flow channel on the flow cell. By rotating and horizontally driving the flow cell on a θX stage with respect to an object lens, it is possible to increase the length of the flow channel that can be scanned continuously. This configuration, thereby, enables reduction in both the number of interruptions of the scanning operation associated with movement to different flow channels and the number of autofocusing operations to be performed on the flow channels.

Description

Analysis equipment

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.

Conventionally, as a method for detecting the fluorescence signal, an area imaging method in which a fluorescence image is captured using a two-dimensional camera has been mainly used. In this method, an XY stage with a fixed flow cell is sequentially driven, and the flow cell tile positioned on the optical axis is imaged. The point here is that the flow cell needs to be stopped during imaging. For this reason, after the tile is moved by the XY stage, it is usually necessary to take a stage settling time of about several hundred milliseconds until the vibration in the apparatus is settled. In other words, the waiting time for vibration attenuation caused by the acceleration motion accompanying the step-and-repeat of the XY stage has been a problem.

On the other hand, in the time-delay integration method (hereinafter referred to as TDI), it is possible to perform imaging while moving the measurement object continuously without stopping. Since this is a constant speed motion without acceleration, it is not necessary to provide a settling time required for stopping the stage, which is necessary in the area imaging method. Accordingly, it is possible to omit the settling time of the stage, and as a result, TurnＴAround Time (hereinafter referred to as TAT) can be reduced and throughput can be improved. The TDI in the next generation sequence is described in detail in Patent Document 1. Illumina introduced HiSeq2000, which adopted TDI in 2010, and achieved 8.7 times the throughput compared to the conventional area imaging method, that is, 28.6 Gb / day.

Furthermore, Illumina announced HiSeq Ten X in 2014, the world's first fastest system that can analyze the human genome at a cost of less than $ 1000. However, even in HiSeq Ten X, 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. By adopting the 2-flow cell system, 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.

Further, the conventional flow cell has a rectangular shape, and a plurality of linear flow paths are arranged in the flow cell. On the other hand, 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.

US7329860 .WO2014 / 143010

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.

More specifically, it was necessary to interrupt the scan when moving to a different flow path, move to the next flow path, and then perform autofocus in the Z direction again. Due to the movement of the flow path and autofocus, there is a problem that the imaging time increases and TAT increases.

In order to solve the above problem, 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. By rotating and horizontally driving the flow cell with the θX 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. 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 θX 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.

As a first embodiment of the present invention, a sequencer using a flow cell having a spiral channel will be described below with reference to FIG.

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. Up to this point, 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. In order to achieve these, 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.

Next, the laser light that has passed through the line generator 108 is condensed on the pupil plane of the objective lens 114 through the second beam expander 109. 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. With the configuration of the illumination optical system described above, it is possible to efficiently illuminate a rectangular area of, for example, 2 mm × 1 μm, which is not a conventional circular shape, with a laser beam on the detection surface of the flow cell.

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. At this time, 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.

Next, the SBS (Sequencing By Synthesis) reaction, which is a representative base sequence analysis method in the next generation sequence, will be described. In the SBS reaction, 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.

More specifically, using the primer as a scaffold, 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. After the fluorescence measurement for determining the first base is completed, 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. Similarly, 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. Similarly, 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. As a result, 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 θ stage and the X stage. More specifically, TDI measurement can be performed. TDI measurement is one of readout methods in a CCD or CMOS camera that shoots while accumulating moving objects. In particular, 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.

In 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. On the other hand, 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.

In addition, 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. This is generally due to a decrease in quantum efficiency with increasing temperature, and it is desirable to perform fluorescence measurement at a low temperature in order to acquire an image with a higher S / N ratio. In addition, if the temperature is 25 ° C., it is possible to minimize the measurement error of the apparatus due to the thermal expansion of the apparatus components, and it is possible to avoid problems such as defocus due to the thermal expansion during imaging. It is.

Also, at the time of switching between chemistry and imaging, measurement was conventionally performed between two different and independent flow cells. Therefore, since the measurement surface is physically divided, it is necessary to focus the measurement surface again. However, in this embodiment, since imaging and chemistry are switched in different flow paths in the same flow cell, the measurement surface is physically continuous, so that the focus adjustment is easy and the time required for the focus is also long. There is an advantage that it can be shortened.

Furthermore, in the conventional method, it was necessary to increase the stroke of the XY stage in order to switch between the two flow cells. For this reason, the moving distance is large, and there is a time loss associated therewith. Further, since the stroke is large, the apparatus has to be large, and there is a problem that the apparatus cost increases. On the other hand, since only one flow cell is required in the present embodiment, the stroke of the θX stage can be reduced, the apparatus can be further downsized, and the apparatus cost can be reduced.

Next, the liquid delivery system of this sequencer will be described. 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. Note that, as the heat block 142 is cooled, the opposite surface of the Peltier element 144 is heated. In order to dissipate this heat, 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. 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. 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. By this operation, a plurality of reagents can be fed with one syringe pump 154. Further, if 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. In order to avoid this, it is necessary to always arrange the waste liquid tank 156 in the apparatus, and in order to detect this, a micro photo sensor 158 for monitoring the presence or absence of the waste liquid tank 156 is installed. For further safety, a liquid receiving tray 157 is installed under the waste liquid tank 156 in case the waste liquid leaks.

As a second embodiment of the present invention, a flow cell having a spiral channel shape will be described below with reference to FIG. FIG. 2a) 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. For this reason, continuous scanning, which is a feature of TDI, is frequently interrupted, and the advantages of TDI cannot be effectively utilized. As a result, there is a problem in that imaging time increases and TAT increases. To solve this problem, in 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.

1) Operability of the flow cell If the straight flow paths are simply connected in series, the flow length is extremely long and the operability is poor and the flow cell becomes unrealistic. Spiral-shaped channels are the most suitable for realizing two points of good operability while maintaining a long channel length.

2) Flow path occupancy ratio in the flow cell It is possible to make the flow path circular instead of spiral, but in this case, the start and end points of the flow path are combined on a circle, so the length of the flow path It cannot be longer than 2πr (where r is the radius of the flow cell). Moreover, when it makes it circular, the problem that the inside of the circular flow path formed in the flow cell cannot be used as a scanning range also arises. That is, there arises a problem that the flow passage occupation ratio in the flow cell decreases. In order to avoid this, a spiral channel was adopted.

Further, 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. Even when measuring multiple samples, it is possible to mix and handle these multiple samples as one sample by adding a barcode array specific to each sample in the pre-processing step for each sample. it can. Therefore, the influence on the measurement due to the small number of the

flow paths

225 and 226 in the present embodiment being one or two can be avoided by adding a barcode array to the sample in the preprocessing.

As a third embodiment of the present invention, 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.

In FIG. 3a), the flow cell has two flow paths. In FIG. 3b), the flow cell has three flow paths. In FIG. 3c), the flow cell has four flow paths. Furthermore, in FIG. 3d), the flow cell has five flow paths. These flow cells are useful when a plurality of samples are not desired to be mixed, and an optimum flow cell is adopted according to the application, and the sample processing time can be minimized by applying TDI.

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. As shown in FIG. 2 a), a typical Illumina flow cell has eight channels with a channel length of 60 mm. Therefore, the total flow path length per flow cell is 480 mm. Since the channel width is 2.5 mm, the channel area is 1200 mm ² . Since the size of the flow cell is 25 × 75mm ^² = 1875mm ^2, the channel occupancy stays at 64%.

On the other hand, in the flow cell shown in FIG. 3A) described in the present embodiment, 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%.

From the above comparison, the following performance improvement effects can be given in the measurement method using the spiral channel of this patent.

(1) While the length of one flow path in the flow cell is 60 mm in FIG. 2 a), it is 451 mm in FIG. Therefore, TDI scanning can be performed continuously without interruption, and TAT can be shortened.

(2) By reducing the number of channels from eight to two, the channel system can be simplified and the dead volume of the reagent can be reduced. Therefore, the sequence cost can be reduced.

(3) By forming the spiral flow path, the flow path occupancy with respect to the entire area of the flow cell can be improved.

As a fourth embodiment of the present invention, the structure of a flow cell will be described below with reference to FIG.

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.

Generally, the spacer 410 is generally manufactured from a material such as PDMS. The thickness of the spacer 410 is 30 to 100 μm, and more specifically 50 μm is desirable. By sandwiching the spacer 410 between the cover glass 413 and the substrate 401, two flow paths 411 and 412 are formed. Here, the channel 411 has an inlet 405 and an outlet 402, and 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. Therefore, it is possible to continuously perform TDI scanning with the increased flow path length. As a result, it is possible to prevent TDI scan operation interruption, movement between channels, and increase in TAT associated with autofocus operation after switching channels, which were problems with the conventional TDI method in a linear channel. .

Also, 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.

As a fifth embodiment of the present invention, a method of fixing a flow cell having a spiral flow path shape to a heat block will be described below with reference to FIG.

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. Compared with the method of mechanically clamping the outer periphery of the flow cell 507, 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 θX stage is ± 30 μm. Further, by using the guides 510 and 511, the flow cell 507 can be fixed to the heat block 501 with high positional accuracy. Although not shown, 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 θX 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 θX stage in the θ 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.

As a sixth embodiment of the present invention, a mechanism and a method for feeding a reagent in a flow cell having a spiral channel shape will be described below with reference to FIGS.

In FIG. 6 a), a method of supplying a reagent to the flow channel 615 in the flow cell 605 having two flow channels 615 and 616 will be described. 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 θX stage. On the other hand, 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 θX stage. Therefore, distortion and twist are not accumulated in the flow path 612 as the θX 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. By operating the multi-way valve 618, a predetermined reagent can be selectively supplied to the two flow paths 615 and 616 of the flow cell 605. In addition, the multi-way valve 618 is installed on the θX stage, and rotates with the θX stage.

As the θX 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. First, 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 θX stage. In order to reduce the amount of reagent, it is important to shorten the piping length from the reagent arranged in the reagent rack to the flow cell channel as much as possible. Therefore, it is important to use linear tubes 601 and 612 as much as possible to supply the reagent to the flow cell 605, and to use a spiral tube that does not cause any problems even if the flow path length becomes long as the flow path of the used reagent. is there.

Similarly, FIG. 6b) describes a method of supplying the reagent to the flow path 616. Note that a chemistry reaction is performed on the channel 616 while the reagent is supplied to the channel 616. On the other hand, 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. In the conventional DNA sequence, the TAT of the sequence becomes longer due to the time required for the imaging process compared to the chemistry process. However, TAT can be reduced by using this method.

In order to selectively supply the reagent to the flow path 616, 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 θX stage. By using the rotary joint, multi-way valve and spiral tube described above, it becomes possible to smoothly supply a reagent to the flow path in the flow cell. .

In addition, a mechanism for heating before injecting the reagent into the flow cell will be described with reference to FIG. In this embodiment, both imaging and chemistry are performed at 60 ° C. However, in 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. In order to avoid this possibility, 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.

Next, as a seventh embodiment of the present invention, a TDI scanning method in a flow cell having a spiral channel will be described below with reference to FIG.

Also in this embodiment, the flow cell has two flow paths, and the flow path width is 2.5 mm. One flow path is divided into two vertically long scan areas called swaths. That is, the width of swath is 2.5 mm / 2 = 1.25 mm, and the length of swath in the vertical direction is the same as that of the flow path. 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. In FIG. 7, for the sake of explanation, the spiral channel is simplified to a semicircular channel. FIG. 7a) shows the scan order within one of the two channels. Moreover, the solid line part in FIG. 7b) shows the scanning order of another flow path. By repeating the imaging process and the chemistry process alternately using these two flow paths, the TAT can be shortened. Further, as described above, each flow path has two swath regions. A specific TDI scan order will be described below.

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. Next, in order to move from the end point 702 to the start point 703 of the second swath, the θX 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. After moving to the second swath start point 703, this time, 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. Next, the TDI scan is similarly performed on the minute reaction field fixed on the top surface of the flow cell.

More specifically, 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. Next, 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. Further, by the step-and-repeat driving, 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 TDI scan speed is 16.6 mm / s as described in the eleventh embodiment. Therefore, the imaging time required for one flow path is 451 mm / swath × 4 swath ÷ 16.6 mm / s = 109 s.
Thus, it is possible to scan one flow path in less than 2 minutes. Since Illumina's SBS chemistry currently requires 2 minutes per cycle, the imaging time can be made shorter than the chemistry time by employing this method. That is, it means that the imaging time that is a bottleneck in TAT can be shortened to a time that does not become the rate-limiting factor of TAT.

The throughput for one cycle in one channel is estimated as follows. The area of the scan area is 451 mm / swath × 4 swath × 2.5 mm = 4507 mm ² / lane
It becomes. Assuming that the minute reaction fields are fixed every 1 μm, the number of minute reaction fields in the region is 4507 mm ² ÷ (1 μm) ² = 4.5G. Since this can be completed in 2 minutes, the throughput is as follows.

4.5 Gb / 2 minutes = 3.2 Tb / day This is higher than the 0.6 Tb / day throughput of HiSeqXTEN, which boasts the highest throughput in 2016, with 10 current HiSeq units connected.

While imaging is being performed from the start point 701 to the end point 708 in one of the two channels in the flow cell, or at the same time as the imaging is completed, the other channel in the flow cell has a chemistry per cycle. Has been completed. In order to continue imaging in this flow path, in FIG. 7 b), the θX stage is moved horizontally from the end point 708 to the start point 711. Next, after continuously performing a TDI scan in the forward direction from the start point 711 to the end point 712, the θX stage is moved horizontally from the end point 712 to the start point 713 in a step-and-repeat manner. After the TDI scan is continuously performed in the reverse direction from the start point 713 to the end point 714, 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. Similarly, a TDI scan is performed in the forward direction from the start point 715 to the end point 716. The θX 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.

It is possible to perform sequencing by repeating the operations per cycle described above. 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.

By applying this embodiment, it is possible to continuously and continuously perform TDI scanning by connecting a number of conventional channels and forming a long channel. Further, 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.

Next, as an eighth embodiment of the present invention, a θX stage control method for a disc-shaped flow cell having a spiral flow path will be described below with reference to FIG.

In this example, consider the case where the minute reaction field is randomly fixed on the flow cell channel surface. In this case, 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.

In this embodiment, in order to keep the TDI scan speed at an arbitrary point on the flow path constant, a control method is adopted in which the angular speed ω [radians / s] of the θX stage is made variable. In order to explain more specifically, the following description will be given using two different points on the spiral channel. For these two points on the spiral flow path, the distance from the center of the circular flow cell and the angular velocity are r _1, r ₂ , ω ₁ , and ω ₂ , respectively. In that case, the scanning speed at each point (that is, the speed in the flow path direction) v ₁ , ₂ is v ₁ = ω ₁ × r ₁
v ₂ = ω ₂ × r ₂
Can be written. In this embodiment, control is performed to keep the scan speed at an arbitrary point on the flow path constant. In that case, v ₁ = v ₂ = v _const
ω ₁ × r ₁ = ω ₂ × r ₂
Can be written. When r ₁ > r ₂ , the angular velocity is ω ₁ <ω ₂ . In other words, in order to make the scanning speed constant, the angular velocity is increased as the TDI scanning is closer to the center of the spiral flow path, and the θX stage is controlled so as to decrease the angular velocity when performing TDI scanning in a region away from the center. . Specific examples of this control method are shown in FIGS. 8a) and 8). Since t = 20 s and r = 5 mm are small, it is necessary to increase the angular velocity to ω = 72 [deg / s]. On the other hand, since r = 29 mm at t = 70 s, it is necessary to reduce the angular velocity ω to 12.7 [deg / s]. As seen in FIG. 8c), the distance r from the center position of the disc-shaped flow cell and the angular velocity ω are in an inversely proportional relationship. In other words, if the distance r from the center position and the angular velocity ω are applied, the scanning velocity v is constant. By performing a TDI scan of the spiral flow path in accordance with this relationship, fluorescence measurement from a micro reaction field can be performed in the center part and the outer peripheral part of the disk-like flow cell similarly. In particular, 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 ω with the transition from the inner periphery to the outer periphery.

Next, as a ninth embodiment of the present invention, a θX stage control method for a disc-shaped flow cell in a plurality of chemist recycling will be described below with reference to FIG.

FIG. 8 illustrates the TDI scan operation corresponding to one cycle of the sequence reaction for the spiral channel, whereas FIG. 9a) illustrates the TDI scan operation for two cycles of the sequence reaction in one spiral channel. It is carried out. As described in the seventh embodiment, TDI scans are continuously performed from the inner periphery to the outer periphery to the inner periphery on the lower surface of the flow cell from t = 0 s to t = 120 s. As described above, the two swaths in the flow path are changed when the TDI scan direction is changed from the outer periphery to the inner periphery. In addition, after the focus is moved from t = 130 s to t = 240 s to the upper surface of the flow cell, a TDI scan is performed for the inner circumference → the outer circumference → the inner circumference. From t = 240 s to t = 480 s, the time of the chemistry reaction after the end of imaging. It becomes possible to proceed with imaging in the second cycle after t> 480 s.

Next, 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. In order to clarify that the two flow paths are alternately switched, the value of the position r when the TDI scan is performed in the second spiral flow path is negative. Like FIG. 9 a), there are two swaths for one flow path, and TDI scan is performed on these two surfaces, the lower surface and the upper surface. Compared with FIG. 9a), it is a significant difference that the imaging of the first channel is completed and then the second channel is immediately followed to continue the TDI scanning. In FIG. 9a), since the camera cannot be driven with respect to the chemistry reaction time, the duty ratio, which is an index indicating the operation rate of the camera, is 50%. In contrast, in FIG. Since imaging of one channel can be continued, the duty ratio can be improved. In particular, when the chemistry time and the imaging time can be made equal, the duty ratio can be improved to nearly 100%.

Similarly to FIG. 8c), in FIG. 9c), the distance | r | from the center position of the disc-shaped flow cell and the angular velocity ω are in an inversely proportional relationship.

Next, as a tenth embodiment of the present invention, a patterned array that regularly arranges minute reaction fields in a spiral channel or a circular channel and improves throughput will be described below with reference to FIGS. 10a) and b). explain.

As described in the eighth and ninth embodiments, when the minute reaction field is irregularly fixed on the flow path surface, the scan speed v is made constant by decreasing the angular velocity ω along with the transition from the inner periphery to the outer periphery. There is a need. However, by using a semiconductor lithography technique, 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). Can be fixed to. Here, in general, in the case of a straight flow path, it is natural that the minute reaction fields are fixed at equal intervals at the same pitch in the scanning direction in any part of the flow cell. On the other hand, a method for regularly fixing a minute reaction field to a spiral channel will be described below.

First, referring to FIG. 10 a), a method for performing TDI scanning with a constant rotation speed ω will be described. In 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. By creating a mask using a semiconductor lithography technique and forming a DNA adsorption site such as aminosilane on the silicon surface, it becomes possible to selectively fix the DNA on the channel surface. Here, although there are three flow paths with X = 5 mm, 10 mm, and 15 mm, the pitches of the minute reaction fields in the scanning direction are δd ₁ , δd ₂ , and δd ₃ , respectively. In FIG. 10a), δd ₁ ≠ δd ₂ ≠ δd ₃ and more specifically, δd ₁ = ω × 5 mm, δd ₂ = ω × 10 mm, and δd ₃ = ω × 15 mm. Here, since the angular velocity ω is constant, the relationship is δd ₁ : δd ₂ : δd ₃ = 1: 2: 3. If patterning is performed using photolithography while maintaining this relationship, TDI scanning can be performed at a constant rotational speed ω in a spiral channel or a circular channel. The control method of the θX stage in TDI scanning is simplified, and the risk of occurrence of measurement errors can be reduced. In addition, in the direction perpendicular to the TDI scan direction and the XY plane, 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.

Next, a method of performing a TDI scan with the rotation speed ω being variable will be described with reference to FIG. In FIG. 10a), 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. In other words, the rotation speed ω 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. In FIG. 10a), there is a problem that the pitch of the minute reaction field is increased with the shift to the outer peripheral portion, and as a result, the number of minute reaction fields (= density) per unit area is reduced. By making the reaction field pitch constant and the angular velocity ω variable, the number of micro reaction field measurements per unit time in the TDI scan can be made constant. By adopting this method, while enjoying the benefits of the TDI scan method with less interruptions in the spiral channel, the fixed density of the micro reaction field associated with the rotational motion is made constant, resulting in improved throughput. It becomes possible to bring

Next, as an eleventh embodiment of the present invention, the parameters of the sensor and stage driving operations specifically used for TDI scanning when TDI scanning is executed will be described with reference to FIG.

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 μm, and the number of TDI stages is 128. FIG. 11 shows the relationship between the sensor surface of the TDI image sensor and the measurement target surface. The side surface of the flow cell meter is projected at a total magnification of 36 times with respect to this sensor surface (the objective lens uses 20 times and the tube lens enlarges 1.8 times).
The pixel size on the flow cell measurement surface is 12 μm ÷ 36 = 0.33 μm. Similarly, the sensor surface size 1101 projected onto the flow cell measurement surface is 1387 μm × 43 μm. Since the line rate is 50 kHz, the scan speed 1103 on the flow cell measurement surface is 0.33 μm × 50 kHz = 16.6 mm / s. The passage time of one line with respect to the measurement object is 1/50 kHz = 20 μs, and therefore the exposure time measured by integrating 128 lines is 20 μs × 128 lines = 2.56 ms. As a matter of course, the distance that the bright spot having the size of 1 pixel crosses the sensor surface in the TDI scan is 0.33 μm / pixel × 128 pixels≈43 μm.

Next, as a twelfth embodiment of the present invention, 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.

In the TDI scan in the conventional flow cell, the minute reaction field moves linearly by linear drive by the XY stage. On the other hand, in this patent, the rotational drive by the θX stage is adopted, so that the minute reaction field performs a circular motion or a spiral motion. Since the scan direction of the TDI scan sensor is usually a linear direction, when the minute reaction field moves linearly, the scan direction of the TDI scan sensor coincides with the movement direction of the minute reaction field. However, when 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.

Specifically, the drift amount will be described below using numerical values based on the TDI sensor employed in the eleventh embodiment. In FIG. 12a) the case where the rotational speed ω is constant will be described. The distance traveled by the minute reaction field during exposure was determined by the physical size of the TDI sensor and the overall magnification of the optical system, and was 43 μm in Example 11. Now, let us consider the motion of a micro reaction field when the diameter of a flow cell having a spiral channel is 65 mm, and the distances r ₁ = 5 mm, r ₂ = 10 mm, and r ₃ = 30 mm from the center of the flow cell. The angle θ becomes 1.4 × 10 ⁻³ radians when the moving distance of the minute reaction field at r ₃ = 30 mm becomes 43 μm. Now, since the rotation speed ω is constant, the angle θ during a constant exposure time is constant. Under this condition, the drift amount of the minute reaction field during the exposure time is δx = r−r × cos θ.
Can be calculated. More specifically, the drift amounts of the minute reaction field at distances r ₁ = 5 mm, r ₂ = 10 mm, and r ₃ = 30 mm from the circle center of the flow cell are 0.05 μm, 0.01 μm, and 0.03 μm, respectively. . This is an amount of 1/10 or less for a pixel size of 0.33 μm on the surface of the flow cell. Therefore, this is an amount that causes no problem in the fluorescence measurement in the TDI scanning in the rotational motion, and the fluorescence measurement can be performed without any problem by rotationally driving the flow cell having the spiral flow path. When the rotational speed ω 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.

Next, the case where the rotational speed ω is variable will be described with reference to FIG. When the rotational speed ω 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. By applying 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. Since the rotational speed w can be made variable, the amount of movement of the micro reaction field at the distances r ₁ = 5 mm, r ₂ = 10 mm, r ₃ = 30 mm from the circle center of the flow cell can be unified to 43 μm. In this case, the angles θ at r ₁ = 5 mm, r ₂ = 10 mm, and r ₃ = 30 mm are 8.6 × 10 ⁻³ radians, 4.3 × 10 ⁻³ radians, and 1.4 × 10 ⁻³ radians, respectively. Become. Similar to the description in FIG. 12a), the drift amount of the minute reaction field during the exposure time can be calculated. These are 0.18 μm, 0.09 μm, and 0.03 μm, respectively, which are sufficiently smaller than the size of 0.33 μm / 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.

Next, as a thirteenth embodiment of the present invention, an apparatus in which two θX stages are mounted on one apparatus will be described below with reference to FIG.

In the present embodiment, the apparatus of the first embodiment has one θX stage mounted thereon, whereas two

θX stages

102 and 161 are mounted. In addition, 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. In this embodiment, 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 θX 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.

191 and 192 Diode lasers 103 and 104 Laser line filter 105 Mirrors 106, 113, 116, 117, 118 Dichroic mirror 123 1/4 wavelength plate 107 First beam expander 109 Second beam expander 108 Line generators 114 and 508 Objective lens 115, 512 Z motor 119, 120, 121, 122 Emission filter 130, 131, 132, 133 Tube lens 134, 135, 136, 137 CMOS camera 161 Notch filter for 642nm 162 Notch filter for 505nm 101, 161, 507, 605 Uzumaki Flow cell 201 having a flow path 102 Flow cells 102, 142, 161, 501 Heat block 140 Reagent cartridge 141 Medicine rack 144 Peltier element 145, 148 Fin 143, 146 Fan 147 Switching valve 150, 202, 225, 226, 411, 412, 509, 510, 612, 615, 616 Channel 154 Syringe pump 152, 155 Two-way valve 156 Waste liquid Tank 157 Liquid receiving tray 158 Micro photosensor 203, 212, 220, 221, 404, 405, 502, 503, 606, 621 Inlet 204, 213, 223, 224, 402, 403, 504, 505, 607, 622 Exit 401 Substrate 410 Spacer 413 Cover glass 510, 511 Guide 506 Suction hole 602 Rotary joint 601, 603, 612, 614, 617, 631, 632, 633, 634 Tube 604, 608, 609, 610 Port 613 Spiral tube 618 Multi-way valve 651 Heating mechanism 701, 703, 705, 707, 711, 713, 715, 717 Start point 702, 704, 706, 708, 712, 714, 716, 718 End point 1101 Projected on the flow cell measurement surface Sensor surface size 1103 Scan speed 1102 TDI sensor

Claims

A flow cell having light permeability and having a curved channel inside;
Means for holding the flow cell relative to the optical axis and rotating and translating the flow cell continuously and simultaneously;
Means for selectively irradiating the flow cell with a specific wavelength;
Means for continuously detecting a signal generated from a micro reaction field fixed to a curved flow path in the flow cell;
Means for feeding a reaction reagent to the flow cell;
Means for controlling the temperature of the flow cell;
The analyzer characterized by having.
In claim 1,
An analyzer characterized in that the means for continuously rotating and translating the flow cell simultaneously is a θX stage.
In claim 1,
An analyzer characterized in that the means for continuously detecting the signal performs detection using a Time Delay Integration (in Japanese) method.
In claim 2,
In the θX stage, according to the distance from the rotation center of the flow cell,
An analyzer characterized by being able to arbitrarily control the rotation speed and translation speed.
In claim 1,
The analyzer for feeding a reaction reagent to the flow cell comprises a rotary joint, a switching valve, and a spiral tube.
In claim 1,
An analytical apparatus characterized in that minute reaction fields fixed on the flow cell are regularly arranged.
In claim 1,
Regular arrangement of micro reaction fields in the flow cell
An analyzer characterized by being arranged at equal intervals without depending on the distance from the rotation center of the flow cell.
In claim 1,
Regular arrangement of micro reaction fields in the flow cell
An analyzer that changes regularly according to a distance from a rotation center of the flow cell.
In claim 1,
The flow cell has a plurality of curved channels,
Perform optical measurement in any one of a plurality of curved channels,
An analyzer characterized by advancing a chemical reaction through other curved channels.
In claim 1,
An analyzer having two or more means for holding the flow cell.