WO2023082063A1 - 液路系统、基因测序仪和试剂回收方法 - Google Patents

液路系统、基因测序仪和试剂回收方法 Download PDF

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Publication number
WO2023082063A1
WO2023082063A1 PCT/CN2021/129640 CN2021129640W WO2023082063A1 WO 2023082063 A1 WO2023082063 A1 WO 2023082063A1 CN 2021129640 W CN2021129640 W CN 2021129640W WO 2023082063 A1 WO2023082063 A1 WO 2023082063A1
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Prior art keywords
reagent
flow
port
channel
power unit
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PCT/CN2021/129640
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English (en)
French (fr)
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陆灏
牛子华
李松霖
邢楚填
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深圳华大智造科技股份有限公司
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Priority to PCT/CN2021/129640 priority Critical patent/WO2023082063A1/zh
Publication of WO2023082063A1 publication Critical patent/WO2023082063A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • 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

Definitions

  • the application relates to a liquid path system, a gene sequencer and a reagent recovery method.
  • Various detection instruments used for clinical diagnosis and life science research usually have a set of fluid systems to realize the transportation of different solutions between multiple regions.
  • samples containing detection targets cells, DNA fragments, etc.
  • biochemical reagents that react with the samples various buffers, cleaning solutions, etc. are generally transported from containers such as sample tubes and reagent tanks to one or more reaction areas.
  • the detection area after the reaction or detection is completed, it is transferred to the waste liquid area.
  • concentration and uniformity of reagents entering the reaction zone determine the efficiency of biochemical reactions, and the former is closely related to the overall design of the fluid system.
  • samples and biochemical reagents are often precious and costly, which means that the amount of reagents used in the transportation process must be sufficiently small.
  • the fluid systems of high-end detection instruments mostly adopt the form of pressure drive, and the above-mentioned reagent storage area, reaction area or detection area, waste liquid area and other areas are fluidly connected by high-precision, low-volume pump valve components and pipelines .
  • biochemical reactions are often carried out step by step, different reagents need to pass through the reaction area in sequence, which involves the substitution of reagents.
  • a closed pipe or channel also known as Poiseuille flow
  • the fluid viscosity makes the flow velocity near the wall smaller, and the flow velocity farther away from the wall is higher, and the overall velocity is in a parabolic distribution.
  • r is a number related to many variables, such as pipe geometry, viscosity ratio and density ratio of reagents a and b, flow rate, etc. Both experiments and numerical simulations show that for a straight pipe with a circular cross-section, r is usually between 4 and 5; for a channel whose height is much smaller than its length and width (for example, its length and width are 100 times its height), r is 1.5 ⁇ 2 range. These two basic shapes are very common in fluid systems of medical testing instruments.
  • DNA fragments to be tested are usually immobilized on a flow cell.
  • the typical height of the flow cell channel is generally only 50-100 Microns are much smaller than the length and width (the length and width make up the detection plane) on the order of millimeters or centimeters.
  • the flow cell is fluidly connected with its upstream and downstream reagent tanks, waste liquid tanks, pump valve elements, etc., usually using standard circular pipes that are easy to process.
  • the volume of the final reagent b used is generally between 2V 1 and 2V 1 +5V 2 . It can be seen that, due to the characteristics of Poiseuille flow, the amount of reagent used not only depends on the inner volume V 1 of the detection zone, but also highly correlates with the inner volume V 2 of the pipeline upstream of the detection zone. Especially for some fluid system designs where the detection area is smaller than the volume of the upstream pipeline (ie, V 1 ⁇ V 2 ), a large amount of reagents is required to ensure the reagent concentration and reaction efficiency in the detection area.
  • V 2 or V 2 /V 1 In order to reduce the amount of reagents used, an obvious solution is to reduce the volume or relative volume of the upstream pipeline (ie reduce V 2 or V 2 /V 1 ), which can be achieved by reducing the cross-sectional area or length of the pipeline.
  • V 2 or V 2 /V 1 the volume or relative volume of the upstream pipeline
  • the application provides a liquid system, a gene sequencer and a reagent recovery method, so as to avoid cross-contamination caused by the recovery of the reagents in the previous step into the flow cell.
  • the present application provides a liquid circuit system, which includes at least two reagent storage containers, a flow cell, a flow splitting module, and a fluid power unit, and the at least two reagent storage containers are used to store at least two different reagent.
  • the flow cell is used to accommodate samples, and the flow cell is connected with at least two reagent storage containers.
  • the splitting module includes a splitting structure and at least two splitting channels.
  • the splitting structure has a confluence port in fluid communication with the flow cell and at least two splitting ports corresponding to the at least two splitting channels.
  • the fluid power unit is connected to the splitting module, the fluid power unit is selectively communicated with one of the at least two splitting channels, and the fluid power unit is configured to drive the reagent to flow forward from the reagent storage container toward the splitting module, and The fluid power unit is further configured to drive reverse flow of reagent from the splitter module towards the reagent storage container.
  • At least two distribution channels and at least two reagent storage containers are provided in one-to-one correspondence.
  • the at least two distribution channels include a first distribution channel and a second distribution channel
  • the distribution structure includes a three-way pipe
  • the three-way pipe includes a confluence port in fluid communication with the flow cell, a second flow port in communication with the first distribution channel A split port and a second split port communicated with the second split channel.
  • the flow distribution structure further includes an on-off control valve, and the on-off control valve is arranged on the first flow distribution channel and/or the second flow distribution channel.
  • the at least two flow distribution channels include a first flow distribution channel and a second flow distribution channel
  • the flow distribution structure includes a first reversing valve
  • the first reversing valve has a first port, a second port and a third port
  • the second port One port forms a confluence port
  • the second port forms a first diversion port communicated with the first diversion channel
  • the third port forms a second diversion port communicated with the second diversion channel
  • the first reversing valve operates to control the first port and the second diversion channel.
  • the second or third port is connected.
  • the fluid power unit includes a syringe pump, the syringe pump includes a first power port and a second power port, the first power port is fluidly connected to the first split channel, and the second power port is fluidly connected to the second split channel.
  • the liquid circuit system further includes a waste liquid pool
  • the syringe pump further includes a third power port, and the third power port communicates with the waste liquid pool.
  • the fluid power unit includes a syringe pump and a second reversing valve
  • the syringe pump includes a first power port
  • the second reversing valve has a first port, a second port and a third port
  • the first port and the second port The two ports are respectively connected to the first diversion channel and the second diversion channel
  • the third port is connected to the first power port of the injection pump
  • the second reversing valve operates to control the third port to communicate with the first port or the second port.
  • the liquid circuit system further includes a waste liquid pool
  • the syringe pump further includes a second power port
  • the second power port of the syringe pump communicates with the waste liquid pool
  • the fluid power unit includes a first peristaltic pump and a second peristaltic pump
  • the hydraulic system further includes a waste liquid pool
  • the first split channel and the second split channel are both connected to the waste liquid pool
  • the first peristaltic pump is set On the first distribution channel
  • the second peristaltic pump is arranged on the second distribution channel.
  • the liquid circuit system further includes a reagent selection element, the reagent selection element includes a common hole and at least two branch holes, the at least two branch holes are correspondingly connected to at least two reagent storage containers, the common hole is connected to the flow cell, The common hole optionally communicates with one of the at least two branch holes.
  • the fluid power unit includes a syringe pump
  • the fluid circuit system further includes a waste liquid pool, a reagent selection element, the first split channel and the second split channel are both connected to the waste liquid pool
  • the syringe pump includes a power port
  • the reagent selection element includes a common hole and a plurality of branch holes
  • the common hole can be selectively communicated with one of the plurality of branch holes
  • the plurality of branch holes include at least two reagent branch holes correspondingly communicated with at least two reagent storage containers and connected with the flow cell
  • the branch hole of the connected flow cell, the power port of the syringe pump is connected with the common hole.
  • the fluid circuit system further includes a buffer storage container for storing the buffer, the buffer storage container is connected to the flow cell, and the fluid power unit is configured to drive the buffer from the buffer storage container to the flow distribution module Forward flow.
  • the fluid power unit is configured to drive the reagent to flow reversely from the splitting module toward the reagent storage container and back into the pipeline connected to the outlet end of the reagent storage container.
  • the present application provides a gene sequencer, which includes a sequencing slide and the above-mentioned fluid system, and the flow cell is arranged on the sequencing slide.
  • the present application provides a reagent recovery method based on the above liquid circuit system, at least two different reagents include a first reagent and a second reagent, and at least two split channels include a first split channel and a second split channel,
  • This reagent recovery method comprises the steps:
  • controlling the fluid power unit to communicate with the first split channel and driving the first reagent through the flow cell and the split structure into the first split channel of the at least two split channels, where the first reagent undergoes a first reaction with the sample in the flow cell;
  • the reagent recovery method further includes, after the first reaction, controlling the action of the fluid power unit to drive the buffer solution through the flow cell and the split structure and into the first split channel for cleaning.
  • the fluid power unit is controlled to drive the second reagent through the flow cell and the split structure, the second reagent undergoes a second reaction with the sample in the flow cell, and the fluid power unit is controlled to drive the second reagent after the second reaction
  • the flow back toward the reagent storage container includes: controlling the fluid power unit to communicate with the second flow channel and driving the second reagent through the flow cell and the flow structure into the second flow channel of the at least two flow channels, and in the second reaction Afterwards, the fluid power unit is controlled to communicate with the second distribution channel and drive the second reagent to flow back toward the reagent storage container.
  • the fluid power unit is controlled to drive the second reagent through the flow cell and the split structure, the second reagent undergoes a second reaction with the sample in the flow cell, and the fluid power unit is controlled to drive the second reagent after the second reaction
  • the flow back toward the reagent storage container includes: controlling the fluid power unit to communicate with the first flow channel and driving the second reagent through the flow cell and the flow structure into the first flow channel, and after the second reaction, controlling the fluid power unit to communicate with the first flow channel
  • the second distribution channel communicates with and drives the second reagent to flow back toward the reagent storage container.
  • the reagent recovery method further includes, after the first reaction, controlling the fluid power unit to communicate with the first split channel and driving the first reagent to flow back toward the reagent storage container so that the recovered first reagent flows back to the storage container.
  • the outlet end of the reagent storage container of the first reagent is connected to the pipeline.
  • controlling the fluid power unit to drive the second reagent to flow back toward the reagent storage container includes: controlling the fluid power unit to drive the second reagent to flow back toward the reagent storage container so that the recovered second reagent flows back into the reagent storage container.
  • controlling the fluid power unit to drive the second reagent to flow back toward the reagent storage container so that the recovered second reagent flows back into the reagent storage container.
  • the liquid circuit system is provided with a shunt module, which includes a shunt structure and at least two shunt channels, so that the reagents that need to be recovered can enter different shunt channels from the reagents that were reacted in the previous step In order to avoid the cross-contamination of reagents from the previous step being recycled into the flow cell.
  • a shunt module which includes a shunt structure and at least two shunt channels, so that the reagents that need to be recovered can enter different shunt channels from the reagents that were reacted in the previous step In order to avoid the cross-contamination of reagents from the previous step being recycled into the flow cell.
  • FIG. 1 is a schematic structural diagram of a fluid circuit system in the prior art when a first reagent flows in a forward direction.
  • Fig. 2 is a schematic structural view of the first reagent of the liquid circuit system in the prior art during reverse recovery.
  • Fig. 3 is a schematic structural diagram of a liquid circuit system in some embodiments of the present application.
  • FIG. 4 is a flow chart of a reagent recovery method based on an embodiment of the liquid circuit system shown in FIG. 3 .
  • Fig. 5 is a step diagram of a reagent recovery method in some embodiments of the present application.
  • Fig. 6 is a step diagram of a reagent recovery method according to another embodiment of the present application.
  • Fig. 7 is a flowchart of a reagent recovery method in some embodiments of the present application.
  • FIG. 8 is a schematic structural diagram of the liquid circuit system according to the first embodiment of the present application.
  • Fig. 9 is a diagram showing the change of the concentration of the reagent in the reagent storage container with the number of cycles when the liquid circuit system shown in Fig. 8 is used for reagent recovery.
  • FIG. 10 is a schematic structural diagram of a fluid circuit system according to a second embodiment of the present application.
  • FIG. 11 is a schematic structural diagram of a liquid circuit system according to a third embodiment of the present application.
  • FIG. 12 is a schematic structural diagram of a fluid circuit system according to a fourth embodiment of the present application.
  • FIG. 13 is a schematic structural diagram of a fluid circuit system according to a fifth embodiment of the present application.
  • FIG. 14 is a schematic structural diagram of a fluid circuit system according to a sixth embodiment of the present application.
  • spatially relative terms may be used here, such as “on !, “over !, “on the surface of !, “above”, etc., to describe The spatial positional relationship between one device or feature shown and other devices or features. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, devices described as “above” or “above” other devices or configurations would then be oriented “beneath” or “above” the other devices or configurations. under other devices or configurations”. Thus, the exemplary term “above” can encompass both an orientation of “above” and “beneath”. The device may be positioned in other different ways and the spatially relative descriptions used herein interpreted accordingly.
  • the fluid circuit system of the related art includes a flow cell C1, a first pipeline L1 and a second pipeline L2, the first pipeline L1 is connected to the first end of the flow cell C1, and the second pipeline L2 is connected to the The second end of the flow cell C1.
  • the arrow FD shows the flow direction.
  • the sample in each reaction cycle, the sample first undergoes a first reaction with the first reagent, and then undergoes a second reaction with the second reagent, and then repeats the above reaction cycle for many times. Then when two reaction cycles occur, the second reagent in the first reaction cycle reacts first, and the first reagent in the second reaction cycle reacts second.
  • Figure 1 shows a schematic diagram of the first reagent R1 flowing forward from the first pipeline L1 to the second pipeline L2 in a certain reaction cycle.
  • Figure 1 shows that when the first reagent R1 flows forward from the first pipeline L1 When reaching the second pipeline L2, buffer S and the second reagent R2 of the previous reaction cycle still remain in the second pipeline L2.
  • Figure 2 shows that the first reagent R1 flows back from the second pipeline L2 back to the second pipeline.
  • a schematic diagram of a pipeline L1 that is to say, FIG. 2 shows a schematic diagram of recovering the first reagent R1.
  • the interface between the reagents is not a straight line but a more complex curve. It can be seen from Figure 1 that when the first reagent R1 flows into the flow cell C1 from left to right, the second pipeline L2 contains the buffer S replaced by the first reagent R1 and the second reagent replaced earlier R2, at this time, the interfaces between the first reagent R1 and the buffer S, and between the buffer S and the second reagent R2 are all parabolic; the buffer S is used as a partition between the first reagent R1 and the second reagent R2 .
  • the free bases with fluorescent groups enter the flow cell with the synthesis reagent as the medium, and under the action of the polymerase, complementarily pair with the single-strand DNA to be tested immobilized on the surface of the flow cell.
  • the optical system visualizes the fluorophore, which identifies the base of the current cycle.
  • the cleaving reagent enters the flow cell to cleave the fluorophore, ending the current cycle.
  • the first reagent R1 is a synthesis reagent
  • the second reagent R2 is an excision reagent in the previous cycle.
  • the existing recovery schemes are to directly recover the recovered reagents in the reagent storage containers. Since the reagent inevitably mixes with the liquid previously occupying the flow cell and its first pipeline in the process of entering the flow cell in the forward direction, its concentration after being reversely recovered will inevitably decrease, so the concentration of the reagent is reused after multiple recovery In this way, the effective concentration of the reagents cannot be guaranteed when they react in the flow cell. Also, when these recovered reagents enter the storage pool or reservoir, they further dilute the never-used portion of the reagent in the reservoir. For a reaction system in which the concentration is positively correlated with the reaction efficiency, the diluted reagent may significantly reduce the reaction efficiency in the flow cell.
  • the decrease of reagent concentration is positively correlated with the recovery times. For example, if the concentration of the recovered reagent is reduced by 10% each time, the concentration will be reduced by 34% after four cycles. Therefore, the higher the recovery ratio, the lower the overall reaction efficiency within the flow cell. Therefore, how to ensure the concentration of reagents involved in biochemical reactions is also a problem to be solved.
  • this application proposes a technical solution for setting a shunt channel at the second end of the flow cell C1 (the downstream end when the reagent is flowing forward), and the reagents that need to be recovered Separate the reagents from the previous step to avoid cross-contamination.
  • the liquid circuit system of the embodiment of the present application includes at least two reagent storage containers (not shown in the figure), a flow cell C1, a flow splitting module and a fluid power unit (not shown in the figure), wherein at least two The reagent storage containers are used to separately store at least two different reagents.
  • the flow cell C1 is used to accommodate samples, and the flow cell C1 is connected with at least two reagent storage containers.
  • the distribution module includes a distribution structure C2 and at least two distribution channels.
  • the distribution structure C2 has a confluence port in fluid communication with the flow cell C1 and at least two distribution ports corresponding to at least two distribution channels.
  • the fluid power unit is connected to the flow splitting module.
  • the fluid power unit is selectively communicated with one of the at least two flow distribution channels, and the fluid power unit is configured to drive the reagent to flow forward from the reagent storage container toward the flow distribution module. And the fluid power unit is further configured to drive the reverse flow of the reagent from the distribution module towards the reagent storage container.
  • the liquid circuit system includes at least two reagent storage containers (not shown in the figure), a flow cell C1, a first pipeline L1 connected to the first end of the flow cell C1, and a second end connected to the flow cell C1
  • the second pipeline L2 the distribution structure C2 and at least two distribution channels.
  • at least two distribution channels include a first distribution channel L3 and a second distribution channel L4 .
  • first pipeline L1 is used for fluid connection with the reagent storage container, and components for realizing reagent selection, such as switching valves, may also be arranged between the first pipeline L1 and the reagent storage container.
  • first distribution channel L3 and the second distribution channel L4 can also be fluidly connected with other functional modules, such as a valve for controlling the flow path and a fluid power unit for driving fluid.
  • the shunt structure C2 can be a simple three-way element (such as a T-shaped three-way, a Y-shaped three-way), or a control element such as a three-way solenoid valve, or a combination of the former two.
  • the shunt structure C2 can be divided into more than two branches, and each branch can be divided into more branches through one or more shunt modules.
  • S1 switch the reagent selection element located upstream of the first pipeline L1 to be in fluid connection with the reagent storage container containing the first reagent 101 (the reagent selection element and the reagent storage container are not shown in FIG. 4 ), and make the fluid power unit It communicates with the first distribution channel L3, so that the first reagent 101 is driven by the fluid power unit to successively replace the buffer solution 102 in the first pipeline L1, the flow cell C1, the second pipeline L2 and the distribution structure C2, and finally along the first A split channel L3 flows out, and at this moment, the parabolic interface between the first reagent 101 and the buffer solution 102 is located in the first split channel L3.
  • the fluid power unit reversely drives the second reagent 103 to leave the second distribution channel L4, the distribution structure C2, the second pipeline L2 and the flow cell C1 in order to realize the recovery of the second reagent 103.
  • S1 to S6 describe the recovery strategy of a single reagent (ie, the second reagent 103 ) in the two-step reaction system.
  • a single reagent ie, the second reagent 103
  • the first reagent 101 and the second reagent 103 By sending the first reagent 101 and the second reagent 103 to the first split channel L3 and the second split channel L4 respectively, and setting a buffer between the first reagent 101 and the second reagent 103, when the second reagent 103 is recovered , the first reagent 101 will not flow back into the flow cell so as to avoid the recurrence of the first reaction.
  • the recovery method shown in Figure 4 shows the recovery process for the second reagent 103, but in another possible reagent recovery method, the first reagent 101 can also be recovered after the first reaction be recycled afterwards.
  • the fluid power unit reversely drives the first reagent 101 to leave the first distribution channel L3 , the distribution structure C2 , the second pipeline L2 and the flow cell C1 in order to realize the recovery of the first reagent 101 .
  • the second reagent 103 in the last cyclic reaction flows into the second split channel L4
  • the second reagent 103 will not flow back into the flow cell C1 to avoid cross-contamination.
  • the liquid circuit system of the present application allows the first reagent and the second reagent to enter different flow channels through the split module, so that when a certain reagent is recovered, the other reagent will not flow back into the flow cell, that is, It is said that the liquid circuit system of the present application can recover both the first reagent and the second reagent, not only the second reagent can be recovered.
  • the fluid power unit in order to realize that the first reagent 101 and the second reagent 103 respectively enter into different distribution channels, it is necessary for the fluid power unit to be able to select between the first distribution channel and the second distribution channel, so as to ensure that the While the liquid is flowing, the liquid in the other channel remains still. That is, the fluid power unit is configured to selectively communicate with one of the at least two flow distribution channels. Moreover, in the above embodiments, the reagent needs to flow forward into the flow cell to react, and when it needs to be recovered, the reagent needs to flow backward from the splitting module, and the above-mentioned two-way flow of the reagent is realized by the fluid power unit.
  • the fluid power unit is configured to drive a forward flow of reagent from the reagent storage container towards the splitter module. And the fluid power unit is further configured to drive the reverse flow of the reagent from the distribution module towards the reagent storage container.
  • the fluid power unit may include a power device capable of both forward driving and reverse driving.
  • the fluid power unit may also include a forward power device capable of forward drive and a reverse power device capable of reverse drive.
  • the split module includes a split structure and at least two split channels, so that the reagents that need to be recovered can enter into different channels from the reagents that were reacted in the previous step.
  • the split channel to avoid cross-contamination caused by the reagents of the previous step being recycled into the flow cell.
  • the embodiment of the present application also provides a reagent recovery method.
  • This reagent recovery method comprises the steps:
  • the fluid circuit system further includes a buffer storage container for storing the buffer, the buffer storage container is connected to the flow cell, and the fluid power unit is configured to drive the buffer from the buffer storage container to the flow distribution module Forward flow.
  • the reagent recovery method further includes, after the first reaction, controlling the action of the fluid power unit to drive the buffer solution through the flow cell and the split structure and into the first split channel for cleaning. Then after cleaning, the control fluid power unit communicates with the second split channel and drives the second reagent through the flow cell and the split structure into the second split channel of the at least two split channels, and after the second reaction, the control fluid The power unit communicates with the second distribution channel and drives the second reagent to flow back toward the reagent storage container. That is to say, in this embodiment, a buffer washing process is set between the first reaction and the second reaction, and the first reagent and the second reagent enter into two different distribution channels respectively.
  • the direction of backflow includes: controlling the fluid power unit to communicate with the first split channel and driving the second reagent to enter the first split channel through the flow cell and the split structure, and after the second reaction, controlling the fluid power unit to communicate with the second split channel communicate and drive the second reagent to flow back in the direction of the reagent storage container.
  • the second reagent directly replaces the first reagent in the flow cell and finally flows out along the first split channel.
  • the interface between the first reagent and the second reagent is located in the first split channel, and then The recovery of the second reagent is switched to the second split channel.
  • At least two distribution channels and at least two reagent storage containers are provided in one-to-one correspondence. That is, each reagent enters a different flow channel, thereby preventing other reagents from flowing back into the flow cell during recovery.
  • the flow distribution module of the liquid circuit system includes two flow distribution channels. If a cyclic reaction involving two reagents is involved, one reagent is controlled to flow into the first split channel, and the other reagent is controlled to flow into the second split channel, that is to say, it can be recovered according to the recovery strategy shown in Figure 6 . In the case of a cyclic multi-step reaction involving multiple reagents, the reagents in each step need to enter into a split channel different from the reagents used in the previous step to prevent the reagents used in the previous step from flowing back into the flow cell and cause disordered reactions.
  • the fluid power unit is configured to drive the reagent to flow reversely from the splitting module toward the reagent storage container and back into the pipeline connected to the outlet end of the reagent storage container.
  • controlling the fluid power unit to drive the second reagent to flow back in the direction of the reagent storage container includes: controlling the fluid power unit to drive the second reagent to flow back in the direction of the reagent storage container so that the recovered second reagent flows back to the direction of the reagent storage container.
  • controlling the fluid power unit to drive the second reagent to flow back in the direction of the reagent storage container so that the recovered second reagent flows back to the direction of the reagent storage container.
  • the recovered second reagent only flows back into the pipeline connected to the outlet end of the reagent storage container, and will not return to the reagent storage container, so that the reagent in the reagent storage container can be guaranteed not to be diluted.
  • the recovered reagent can be prevented from returning to the reagent storage container by adjusting the recovery ratio. For example, by controlling the volume of recovered reagents.
  • the liquid circuit system includes a first storage container 111 , a second storage container 112 , a third storage container 113 , a reagent selection element C3 , a first pipeline L1 , a flow cell C1 and a second pipeline L2 .
  • the first storage container 111 is used to store the first reagent 101
  • the second storage container 112 is used to store the buffer solution 102
  • the third storage container 113 is used to store the second reagent 103 .
  • Fig. 7A shows the recovery process of the current cycle
  • Fig. 7B shows the second reaction of the next cycle. As shown in FIG.
  • a certain volume of the second reagent 103 is recovered to the upstream of the flow cell C3, including a portion 1031 whose concentration is diluted due to direct contact with the buffer solution 102 and a portion whose concentration is hardly diluted.
  • the diluted part 1031 since the diluted part 1031 is downstream of the undiluted part 1032, as shown in Figure 7B, when the second reagent 103 is used in the next cycle, the diluted part 1031 must first participate in the first pipeline L1 and Reagent replacement in flow cell C1, combined with a faster flow rate, when the diluted part 1031 passes through the flow cell, only a weak second reaction (almost negligible) will be triggered, and the second reagent 103 continues to flow downstream , the portion 1032 whose concentration is almost undiluted enters the flow cell, at which point the flow stops, and this portion fully reacts with the sample in the flow cell.
  • the technical solution of the application realizes efficient reagent recovery and reuse through the design of the liquid system and recovery logic, which can greatly reduce the consumption of reagents in closed pipes and channels, thereby greatly reducing the cost of reagent consumables in medical testing instruments. Meanwhile, the present application mainly has the following two advantages:
  • the reagent After the reagent is recovered, it will not cause obvious dilution of the overall concentration of the reagent in the storage container. At the same time, the low-concentration part of the recovered reagent is mainly used to replace another liquid in the flow cell and its upstream public pipeline, rather than to participate in biochemical reactions. The biochemical reaction is still carried out at a higher reagent concentration, which ensures the efficiency of the reaction.
  • At least two flow distribution channels include a first flow distribution channel 206 and a second flow distribution channel 207
  • the flow distribution structure includes a three-way pipe 202
  • the three-way pipe 202 includes a confluence port in fluid communication with the flow cell
  • the first distribution port communicates with the first distribution channel 206 and the second distribution port communicates with the second distribution channel 207 .
  • the liquid circuit system of the gene sequencer of the first embodiment shown in FIG. 8 is taken as an example for specific description.
  • the liquid circuit system includes a storage container 208, a reagent selection element 203, a first pipeline 204, a sequencing slide 201, a second pipeline 205, a tee 202, a first shunt channel 206, and a second shunt channel 207 , a syringe pump 209 , a third pipeline 210 and a waste liquid pool 211 .
  • the storage container 208 includes a first storage container for storing the synthesis reagent 221, a second storage container for storing the scanning reagent 222, a third storage container for storing the ablation reagent 223, and a fourth storage container for storing the buffer solution 224. storage container.
  • the sequencing slide 201 has a flow cell.
  • the reagent selection element 203 is used to selectively fluidly communicate the flow cell with one of the storage containers 208 so that a corresponding reagent can enter the flow cell.
  • the reagent selection element 203 includes a common hole and at least two branch holes, the at least two branch holes are correspondingly connected to at least two reagent storage containers, the common hole is connected to the flow cell through the first pipeline 204, and the common hole is optionally connected to at least two One of the branch holes is connected.
  • the reagent selection element 203 is a reagent selection valve.
  • the three-way pipe 202 forms a flow distribution structure.
  • the tee pipe 202 includes a confluence port in fluid communication with the flow cell, a first diversion port in communication with the first diversion channel 206 , and a second diversion port in communication with the second diversion channel 207 .
  • the three-way pipe 202 is a T-shaped three-way pipe, and in other embodiments, it may also be a Y-shaped three-way component.
  • the flow distribution structure may also include multi-way pipes, such as four-way pipes, to realize more flow distribution channels.
  • the syringe pump 209 forms a fluid power unit.
  • the syringe pump 209 has three power ports, the first power port is fluidly connected to the first flow channel 206, the second power port is fluidly connected to the second flow channel 207, and the third power port is fluidly connected to the waste liquid pool 211 through the third pipeline 210. connect.
  • the syringe pump 209 can optionally provide a driving force to one of the three power ports, and the syringe pump 209 can provide a forward driving force to make the reagent flow forward, and can also provide a reverse driving force to make the reagent flow in a reverse direction.
  • the fluid power unit may also include two independently arranged syringe pumps, one syringe pump is used to provide a positive driving force for the reagent, and the other syringe pump is used to provide a reverse driving force for the reagent.
  • the solid line arrows indicate the flow direction of the reagent when it flows forward
  • the dotted line arrows indicate the flow direction of the reagent when it is reversely recovered.
  • the DNA fragments to be tested are immobilized on the surface of the flow cell of the sequencing slide, and the entire sequencing process is a cyclic "synthesis-detection-excision" system as described above.
  • a buffer is used as a partition between reagents between two steps. It should be noted that the volume of the flow cell in this example is about 4 ⁇ L, which is a typical small volume flow cell.
  • the volume of the first pipeline 204 is 4 ⁇ L
  • the total volume of the reagent selection element 203 and the pipeline between it and the storage container 208 is about 30 ⁇ L
  • the volume of the second pipeline 205 is 10 ⁇ L
  • the first split channel 206 and the second split channel The volumes of the channels 207 are all above 100 ⁇ L.
  • a complete reaction cycle includes the following steps:
  • the reagent selection element 203 is switched to be fluidly connected to the first storage container, the syringe pump 209 is switched to be fluidly connected to the first shunt channel 206, and 40 ⁇ L of synthetic reagent 221 is extracted to be sequenced at a high flow rate (such as 2000 ⁇ L/min, the same below). Sheet 201.
  • the synthetic reagent 221 replaces the buffer 224 originally in the first pipeline 204 and the flow cell;
  • the syringe pump 209 pushes 30 ⁇ L of the synthetic reagent 221 upstream along the first split channel 206 (see the dotted arrow in FIG. 8 ), and the diluted part of the synthetic reagent 221 flows back to the reagent selection element 203 and the storage container 208 between, but not into the storage container 208;
  • the reagent selection element 203 is switched to be in fluid connection with the fourth storage container, and the syringe pump 209 remains in fluid connection with the first shunt channel 206, and draws 50 ⁇ L of buffer solution 224 through the sequencing slide 201, replacing the fluid in the first pipeline 204 and the flow cell. Residual synthetic reagents 221;
  • the reagent selection element 203 is switched to be fluidly connected to the second storage container, the syringe pump 209 remains fluidly connected to the first shunt channel 206, and draws 40 ⁇ L of the scanning reagent 222 through the sequencing slide 201.
  • the optical system (not shown in the figure) ) detecting the sequencing slide 201;
  • the reagent selection element 203 is switched to fluidly connect with the fourth storage container, and the syringe pump 209 pumps 50 ⁇ L of buffer solution 224 through the sequencing slide 201 to replace the scanning reagent 222 in the first pipeline 204 and the flow cell;
  • the reagent selection element 203 is switched to be fluidly connected to the third storage container, the syringe pump 209 is switched to be fluidly connected to the second shunt channel 207, and 40 ⁇ L of the cutting reagent 223 is drawn through the sequencing slide 201;
  • the reagent selection element 203 is switched to be in fluid connection with the fourth storage container, the syringe pump 209 remains in fluid connection with the second shunt channel 207, and draws 50 ⁇ L of buffer solution 224 through the sequencing slide 201 to replace the first pipeline 204 and the flow cell.
  • the syringe pump 209 is switched to be in fluid connection with the third pipeline 210 and the waste liquid pool 211, and the liquid in the syringe pump is emptied.
  • Table 1 compares the sequencing quality indicators of no recovery and recovery of 75%, both of which are at the same level in terms of read length (Total Reads) and quality value (Q30).
  • the problem of cross-contamination in the flow cell caused by the reflux of the removal reagent 223 caused by reagent recovery was successfully solved.
  • the concentration of the residual cutting reagent in the recovered flow cell the residual concentration is 0.7% when the tee pipe 202 and the shunt channel are not introduced, and the residual concentration after the introduction is negligible.
  • the technical solution of the present application is very important for gene sequencing because a very small amount of excision reagent can cause reaction disorder and sequencing errors.
  • the reagents of each step can enter into different flow channels from the reagents of the previous step, but the following situations may also occur, when a reagent is When recovering, the reagent before two or more steps may be in the same shunt channel as the reagent to be recovered, so it may flow back. At this time, the above situation can be avoided by increasing the buffer volume between the reagents .
  • the key is that when the reagent in this step enters the same flow channel as the reagent in the previous step Whether it will cause out-of-order reactions later. For example, in the embodiment shown in FIG.
  • the reaction sequence is synthesis-scanning-cutting, since the scanning reagent 222 flows back into the flow cell and will not cause disordered reactions, and The reflow of the ablation reagent 223 into the flow cell will cause a disordered reaction, so in the above embodiment, the synthesis reagent 221 and the scanning reagent 222 both flow into the first distribution channel 206, while the ablation reagent 223 flows into the second distribution channel 207 inside.
  • the main purpose is to make the ablation reagent 223 enter into a shunt channel different from that of the scanning reagent 222 .
  • the synthetic reagents in this example are recovered after the reaction, and will not be recovered into the storage container 208; when the next cycle is repeated, the diluted part mainly participates in the replacement of reagents, and the concentration of reagents participating in the reaction in the flow cell maintained at a high level.
  • This advantage is evidenced by the curve of synthesis reagent concentration in the flow cell as a function of cycle number shown in Figure 9. It can be seen from the figure that the concentration of the synthetic reagent is always maintained at an effective relative concentration above 98.7% within 100 cycles, fully illustrating a major advantage of the technical solution of the present application.
  • the diversion module further includes an on-off control valve.
  • the on-off control valve is disposed on the first distribution channel 206 and/or the second distribution channel 207 . Setting on-off control valves on the first split channel 206 and/or the second split channel 207 can strengthen the physical isolation, so that when the reagent is recovered, the reagent in the previous step is completely isolated in another split channel.
  • the fluid circuit system of the gene sequencer of the second embodiment shown in FIG. 10 is taken as an example for specific description.
  • the change of the second embodiment is that an on-off control valve, specifically a two-position two-way solenoid valve 212 is added in the second flow channel 207 .
  • the function of the solenoid valve is to strengthen the physical separation, so that the ablation reagent is completely left in the second distribution channel 207 during the process of recovering the synthesis reagent.
  • the three-way pipe 202 and the first split channel 206 and the second split channel 207 all form passages, so when the synthesis reagent is recovered along the first split channel 206, the second split channel 207 Due to the unequal flow resistance of the first split channel 206 and the second split channel 207, air bubbles in the pipeline, etc., the ablation reagent in the tube may still partly flow back into the flow cell.
  • the solenoid valve 212 is open in the energized state, and is open in the de-energized state.
  • the steps of reagent recovery are slightly different from the first embodiment, specifically:
  • the reagent selection element 203 is switched to be fluidly connected to the first storage container, the solenoid valve 212 is switched to the power-off state, the syringe pump 209 is switched to be fluidly connected to the first shunt channel 206, and 40 ⁇ L of synthetic reagent 221 is extracted at a high flow rate (such as 2000 ⁇ L /min, the same below) through the sequencing slide 201.
  • the synthetic reagent 221 replaces the buffer 224 originally in the first pipeline 204 and the flow cell;
  • the reagent selection element 203 is switched to be fluidly connected to the third reagent storage container, the solenoid valve 212 is switched to the energized state, the syringe pump 209 is switched to be fluidly connected to the second shunt channel 207, and 40 ⁇ L of the ablation reagent 223 is drawn through the sequencing slide 201;
  • the on-off control valve can also be arranged in the first distribution channel 206 .
  • on-off control valves are respectively set in the first distribution channel and the second distribution channel.
  • the on-off control valve can be a solenoid valve, or other components that can realize the on-off control of the pipeline.
  • the at least two split channels include a first split channel 206 and a second split channel 207 .
  • the diversion structure includes a first reversing valve.
  • the first reversing valve has a first port, a second port, and a third port.
  • the first port forms a confluence port
  • the second port forms a first diversion port communicated with the first diversion channel 206
  • the third port forms a junction with the second diversion channel 206.
  • the channel 207 communicates with the second diverter port, and the first reversing valve operates to control the communication between the first port and the second port or the third port.
  • liquid circuit system of the gene sequencer of the third embodiment shown in FIG. 11 is taken as an example for specific description.
  • This embodiment is an improved design of the first embodiment. It can be seen from FIG. 11 that the only change in this example compared to the first embodiment is that the three-way pipe is replaced with a reversing valve, specifically a two-position three-way solenoid valve 213 . The normally open end of the solenoid valve is in fluid connection with the first distribution channel 206 , and the normally closed end is in fluid connection with the second distribution channel 207 . Like the second embodiment, this embodiment has better physical separation than the first embodiment.
  • the reagent selection element 203 is switched to be fluidly connected to the first storage container, the solenoid valve 213 remains in a power-off state, the syringe pump 209 is switched to be fluidly connected to the first shunt channel 206, and 40 ⁇ L of synthetic reagent 221 is extracted at a high flow rate (such as 2000 ⁇ L/ min, the same below) through the sequencing slide 201.
  • the synthetic reagent 221 replaces the buffer 224 originally in the first pipeline 204 and the flow cell;
  • the reagent selection element 203 is switched to be fluidly connected to the third storage container, the solenoid valve 213 is switched to the energized state, the syringe pump 209 is switched to be fluidly connected to the second shunt channel 207, and 40 ⁇ L of the ablation reagent 223 is drawn through the sequencing slide 201;
  • the fluid power unit includes a syringe pump 209, the syringe pump 209 includes a first power port and a second power port, the first power port is fluidly connected to the first split channel, and the second power port is fluidly connected to the second split channel connect.
  • the syringe pump 209 includes a first power port and a second power port, the first power port is fluidly connected with the first split channel 206 , and the second power port The port is in fluid connection with the second split channel 207.
  • the liquid system further includes a waste liquid pool 211
  • the syringe pump 209 also includes a third power port, which communicates with the waste liquid pool 211 .
  • the fluid power unit includes a syringe pump 214 and a second reversing valve
  • the syringe pump includes a first power port
  • the second reversing valve has a first port, a second port and a third port
  • the first port and the second port are respectively connected to the first diversion channel and the second diversion channel
  • the third port is connected to the first power port of the syringe pump 214
  • the second reversing valve acts to control the connection between the third port and the first port or The second port is connected.
  • the fluid circuit system of the gene sequencer of the fourth embodiment shown in FIG. 12 is taken as an example for specific description. It can be seen from FIG. 12 that the change of this embodiment compared with the first embodiment is that a reversing valve, specifically a two-position three-way solenoid valve 213 is added at the rear end of the three-way pipe 202 .
  • the normally open end of the solenoid valve is in fluid connection with the second flow channel 207
  • the normally closed end is in fluid connection with the first flow channel 206 .
  • the syringe pump 214 in this example has only two optional power ports, one end is in fluid connection with the solenoid valve 213 , and the other end is in fluid connection with the third pipeline 210 and the waste liquid pool 211 .
  • the reagent selection element 203 is switched to fluidly connect with the first storage container, the solenoid valve 213 is switched to the energized state, and the syringe pump 214 pumps 40 ⁇ L of synthetic reagent 221 through the sequencing slide 201 at a high flow rate (such as 2000 ⁇ L/min, the same below).
  • the synthetic reagent 221 replaces the buffer 224 originally in the first pipeline 204 and the flow cell;
  • the syringe pump 214 pushes 30 ⁇ L of the reagent upstream along the first split channel 206 (see the dotted arrow in FIG. 12 ), and the diluted part of the synthetic reagent 221 flows back to between the reagent selection element 203 and the reagent storage container 208 pipeline, but will not enter the reagent storage container 208;
  • the reagent selection element 203 is switched to fluidly connect with the fourth storage container, and the syringe pump 214 pumps 50 ⁇ L of buffer solution 224 through the sequencing slide 201 to replace the remaining synthesis reagent 221 in the first pipeline 204 and the flow cell;
  • the reagent selection element 203 is switched to be fluidly connected to the second storage container, and the syringe pump 214 pumps 40 ⁇ L of the scanning reagent 222 through the sequencing slide 201, and the optical system (not shown in the figure) detects the sequencing slide 201 after completion;
  • the reagent selection element 203 is switched to be in fluid connection with the fourth storage container, and the syringe pump 214 draws 50 ⁇ L of buffer solution 224 through the sequencing slide 201 to replace the scanning reagent 222 in the first pipeline 204 and the flow cell;
  • the reagent selection element 203 is switched to be in fluid connection with the ablation reagent 223, the solenoid valve 213 is switched to a power-off state, and the syringe pump 214 pumps 40 ⁇ L of the ablation reagent 223 through the sequencing slide 201 and into the second shunt channel 207;
  • the reagent selection element 203 is switched to be in fluid connection with the fourth storage container, and the syringe pump 214 draws 50 ⁇ L of buffer solution 224 through the sequencing slide 201 to replace the first pipeline 204 and the excision reagent 223 in the flow cell;
  • the syringe pump 214 is switched to be in fluid connection with the third pipeline 210 and the waste liquid storage container 211 to empty the liquid in the syringe.
  • the liquid circuit system further includes a waste liquid pool 211
  • the syringe pump 214 further includes a second power port
  • the second power port of the syringe pump 214 communicates with the waste liquid pool.
  • the syringe pump 214 includes two power ports, one power port is connected to the reversing valve (two-position three-way solenoid valve 213 ), and the other power port is connected to the waste liquid pool 211 through the third pipeline 210 .
  • the fluid power unit includes a first peristaltic pump 216 and a second peristaltic pump 217 .
  • the liquid circuit system also includes a waste liquid pool 211, the first split channel 206 and the second split channel 207 are both in communication with the waste liquid pool 211, the first peristaltic pump 216 is arranged on the first split channel 206, and the second peristaltic pump 217 is arranged on the on the second distribution channel 207 .
  • Fig. 13 shows the fluid circuit system of the gene sequencer of the fifth embodiment.
  • this embodiment is a modified design of the third embodiment, and the main change is that the syringe pump is replaced by two peristaltic pumps 216 and 217 .
  • the normally open end of the solenoid valve 213 is fluidly connected to the first peristaltic pump 216 through the first split channel 206
  • the normally closed end is fluidly connected to the second peristaltic pump 217 through the second split channel 207 .
  • the reagent selection element 203 is switched to be in fluid connection with the first storage container, the solenoid valve 213 remains powered off, and the syringe pump 217 pumps 40 ⁇ L of synthetic reagent 221 through the sequencing slide 201 at a high flow rate (such as 2000 ⁇ L/min, the same below).
  • the synthetic reagent 221 replaces the buffer 224 originally in the first pipeline 204 and the flow cell;
  • the first peristaltic pump 216 pushes 30 ⁇ L of the reagent upstream along the first split channel 206 (see the dotted arrow in FIG. 14 ), and the diluted part of the synthetic reagent 221 flows back to the reagent selection element 203 and the reagent storage container 208 between, but will not enter the reagent storage container 208;
  • the reagent selection element 203 is switched to be in fluid connection with the fourth storage container, and the second peristaltic pump 217 draws 50 ⁇ L of buffer solution 224 through the sequencing slide 201 to replace the remaining synthesis reagent 221 in the first pipeline 204 and the flow cell;
  • the reagent selection element 203 is switched to be fluidly connected to the second storage container, and the second peristaltic pump 217 pumps 40 ⁇ L of the scanning reagent 222 through the sequencing slide 201. After completion, the optical system (not shown in the figure) detects the sequencing slide 201;
  • the reagent selection element 203 is switched to fluidly connect with the fourth storage container, and the second peristaltic pump 217 pumps 50 ⁇ L of buffer solution 224 through the sequencing slide 201 to replace the scanning reagent 222 in the first pipeline 204 and the flow cell;
  • the reagent selection element 203 is switched to be in fluid connection with the third storage container, and the solenoid valve 213 is switched to the energized state.
  • the second peristaltic pump 217 pumps 40 ⁇ L of the ablation reagent 223 through the sequencing slide 201 and into the second shunt channel 207 ;
  • the reagent selection element 203 is switched to be in fluid connection with the buffer 224, and the first peristaltic pump 216 pumps 50 ⁇ L of the buffer 224 through the sequencing slide 201 to replace the excision reagent 223 in the first pipeline 204 and the flow cell.
  • the fluid power unit includes a syringe pump 214 .
  • the liquid circuit system also includes a waste liquid pool 211, a reagent selection element 203, the first shunt channel 206 and the second shunt channel 207 are in communication with the waste liquid pool 211, the syringe pump 214 includes a power port, and the reagent selection element 203 includes a common hole and multiple a plurality of branch holes, the common hole can be selectively communicated with one of the plurality of branch holes, and the plurality of branch holes include at least two reagent branch holes correspondingly communicated with at least two reagent storage containers and a flow cell branch hole communicated with the flow cell , the power port of the syringe pump 214 is connected to the common hole.
  • Fig. 14 shows the fluid circuit system of the gene sequencer of the sixth embodiment.
  • This hydraulic system is a modified embodiment of the third embodiment shown in FIG. 11 .
  • the main change is that the syringe pump is put in front of the reagent selection element, so that the positive reagent flow becomes a positive pressure driving mode.
  • the syringe pump 214 is in fluid connection with the common hole of the reagent selection element 203 through the pipeline 215
  • the common pipeline 204 is in fluid connection with a branch hole of the reagent selection element 203 instead.
  • the flow distribution channels 206 and 207 of the solenoid valve 213 are both in fluid connection with the waste liquid pool 211 .
  • the steps of reagent recovery are basically the same as those in the third embodiment, except that each time the liquid is pumped in the forward direction, the syringe pump 214 first draws the reagent from the common hole of the reagent selection element 203 into the pipeline 215, and the reagent selection element 203 then Switch to fluid connection with the first pipeline 204 , and finally the syringe pump 214 pushes the reagent to the first pipeline 204 .
  • the syringe pump 214 When the reagent needs to be recovered, the syringe pump 214 first extracts the used reagent from the first pipeline 204 into the pipeline 215, and then the reagent selection element 203 is switched to the hole corresponding to the reagent, and finally the syringe pump 214 pushes the reagent to the reagent hole. recovered reagents.
  • the embodiment of the present application also provides a gene sequencer, which includes a sequencing slide and the above-mentioned fluid system, and the flow cell is arranged on the sequencing slide.

Abstract

一种液路系统、基因测序仪和试剂回收方法。液路系统包括至少两个试剂储存容器、流动池、分流模块和流体动力单元。流动池与至少两个试剂储存容器连接。分流模块包括分流结构和至少两个分流通道。流体动力单元与分流模块连接，流体动力单元可选择地与至少两个分流通道中的一个分流通道连通，且流体动力单元被配置为驱动试剂从试剂储存容器朝分流模块的方向正向流动，且流体动力单元还被配置为驱动试剂从分流模块朝试剂储存容器的方向反向流动。

Description

液路系统、基因测序仪和试剂回收方法 技术领域
本申请涉及一种液路系统、基因测序仪和试剂回收方法。
背景技术
用于临床诊断和生命科学研究的各类检测仪器(如基因测序仪、流式细胞仪、高压液相色谱仪等)通常具有一套流体系统来实现不同溶液在多个区域间的输运。例如，含有检测目标(细胞、DNA片段等)的样本、与样本反应的生化试剂、各类缓冲液、清洗液等一般从样本管、试剂槽等容器中分别被输送到一个或多个反应区或检测区，待反应或检测完成后再转移至废液区中。试剂进入到反应区时的浓度高低与均匀性决定了生化反应的效率，而前者又与流体系统的整体设计紧密关联。此外，样本及生化试剂通常珍贵且成本高昂，这意味着在输运过程中所使用的试剂量必须足够少。
目前，高端检测仪器的流体系统多采用压力驱动的形式，以高精度、低内体积的泵阀元件和管道将上述的试剂存储区、反应区或检测区、废液区等区域流体地连接起来。由于生化反应往往是分步进行的，不同试剂需依次地经过反应区，这又涉及到试剂间的替代问题。对于封闭管道或槽道的流动(又称泊肃叶流动)，流体粘性使得靠近壁面处流速较小，远离壁面处流速较大，整体速度呈抛物线分布，因此当一段管道中的一种试剂被后一种试剂替代或冲洗时，靠近壁面的区域往往较难以替代，导致最终需使用数倍于该管道体积的试剂量才能把管道冲洗干净。
具体来说，若要体积为V且内部充满某种试剂a的管道或流动池完全充满另一种试剂b，试剂b的所需用量至少应为rV，此处r＞1定义为替代比。r是一个与众多变量相关的数，如管道几何、试剂a和b的粘度比及密度比、流速等。实验与数值仿真共同表明，对于一段圆形截面的直管道，r通常在4～5之间；对于一个高度远小于长宽的槽道(如长宽为高的100倍)，r则在1.5～2的范围。这两种基础形状在医疗检测仪器的流体系统十分常见，例如在基因测序仪中，待测DNA片段通常被固定在一个流动池上。为了尽可能地缩小流动池的内体积从而减少所需的试剂用量，同时又保证待测DNA片段尽量多地在一个检测平面上铺展，该流动池槽道典型的高度尺寸一般仅为50～100微米，远小于在毫米或厘米量级的长度和宽度(长和宽组成检测平面)。另外，流动池与其上下游的试剂槽、废液槽、泵阀元件等流体地连接，通常使用易加 工的标准圆形管道。现在假设有体积为V 1的流动池，其上游与一段内体积为V 2的圆管平滑地连通，流动池和圆管内均充满了试剂a。为了计算方便，将流动池的r值固定为2，圆管的r值固定为5。此时，在圆管的入口端注入试剂b，则需要5V 2的试剂b才能将圆管中的试剂a完全替代，另外需要2V 1的试剂b才能进一步地将流动池中地试剂a完全替代。考虑到上游圆管的替代过程中，有部分试剂b已经进入了流动池中，最终使用的试剂b体积一般在2V 1和2V 1+5V 2之间。可以看出，泊肃叶流动的特性使试剂用量不仅取决于检测区的内体积V 1，还与检测区上游的管道内体积V 2高度相关。尤其是对于一些检测区小于上游管道体积(即V 1<V 2)的流体系统设计，需要使用大量的试剂才能保证检测区的试剂浓度及反应效率。为了降低试剂用量，一种显而易见的解决方案是减小上游管道的体积或相对体积(即减小V 2或V 2/V 1)，这可以通过缩小管道截面积或管道长度来实现。但是，管道截面积的减小不可避免地会导致整体压力降的上升，从而增大流体系统的负载；管道长度则会受到仪器内部物理空间的约束而存在一个下限。总之，V 2或V 2/V 1无法降低至零。
除此以外，另一种可以有效降低试剂用量的技术方案是试剂回收。如何在进行试剂回收时防止其他试剂被回收到流动池内发生交叉污染是需要解决的问题。
发明内容
本申请提供一种液路系统、基因测序仪和试剂回收方法，以避免前一步反应的试剂被回收到流动池内而发生交叉污染。
第一方面，本申请提供一种液路系统，该液路系统包括至少两个试剂储存容器、流动池、分流模块和流体动力单元，至少两个试剂储存容器用于分别储存至少两种不同的试剂。流动池用于容纳样本，且流动池与至少两个试剂储存容器连接。分流模块包括分流结构和至少两个分流通道，分流结构具有与流动池流体连通的合流口以及与至少两个分流通道对应设置的至少两个分流口。流体动力单元与分流模块连接，流体动力单元可选择地与至少两个分流通道中的一个分流通道连通，且流体动力单元被配置为驱动试剂从试剂储存容器朝分流模块的方向正向流动，且流体动力单元还被配置为驱动试剂从分流模块朝试剂储存容器的方向反向流动。
在一些实施例中，至少两个分流通道和至少两个试剂储存容器一一对应设置。
在一些实施例中，至少两个分流通道包括第一分流通道和第二分流通道，分流结构包括三通管，三通管包括与流动池流体连通的合流口、与第一分流通道连通的第一 分流口以及与第二分流通道连通的第二分流口。
在一些实施例中，分流结构还包括通断控制阀，通断控制阀设置在第一分流通道和/或第二分流通道上。
在一些实施例中，至少两个分流通道包括第一分流通道和第二分流通道，分流结构包括第一换向阀，第一换向阀具有第一口、第二口和第三口，第一口形成合流口，第二口形成与第一分流通道连通的第一分流口，第三口形成与第二分流通道连通的第二分流口，第一换向阀动作以控制第一口与第二口或第三口连通。
在一些实施例中，流体动力单元包括注射泵，注射泵包括第一动力口和第二动力口，第一动力口与第一分流通道流体连接，第二动力口与第二分流通道流体连接。
在一些实施例中，液路系统还包括废液池，注射泵还包括第三动力口，第三动力口与废液池连通。
在一些实施例中，流体动力单元包括注射泵和第二换向阀，注射泵包括第一动力口，第二换向阀具有第一口、第二口和第三口，第一口和第二口分别和第一分流通道和第二分流通道连接，第三口与注射泵的第一动力口连接，第二换向阀动作以控制第三口与第一口或第二口连通。
在一些实施例中，液路系统还包括废液池，注射泵还包括第二动力口，注射泵的第二动力口与废液池连通。
在一些实施例中，流体动力单元包括第一蠕动泵和第二蠕动泵，液路系统还包括废液池，第一分流通道和第二分流通道均与废液池连通，第一蠕动泵设置在第一分流通道上，第二蠕动泵设置在第二分流通道上。
在一些实施例中，液路系统还包括试剂选择元件，试剂选择元件包括公共孔和至少两个分支孔，至少两个分支孔与至少两个试剂储存容器对应连接，公共孔与流动池连接，公共孔可选择地与至少两个分支孔中的一个连通。
在一些实施例中，流体动力单元包括注射泵，液路系统还包括废液池、试剂选择元件，第一分流通道和第二分流通道均与废液池连通，注射泵包括动力口，试剂选择元件包括公共孔和多个分支孔，公共孔可选择地与多个分支孔中的一个连通，多个分支孔包括与至少两个试剂储存容器对应连通的至少两个试剂分支孔以及与流动池连通的流动池分支孔，注射泵的动力口与公共孔连接。
在一些实施例中，液路系统还包括用于储存缓冲液的缓冲液储存容器，缓冲液储存容器与流动池连接，流体动力单元被配置为驱动缓冲液从缓冲液储存容器向分流模 块的方向正向流动。
在一些实施例中，流体动力单元被配置为驱动试剂从分流模块朝试剂储存容器的方向反向流动并回流到与试剂储存容器的出口端连接的管路内。
第二方面，本申请提供一种基因测序仪，包括测序载片和上述液路系统，流动池设置在测序载片上。
第三方面，本申请提供一种基于上述液路系统的试剂回收方法，至少两种不同的试剂包括第一试剂和第二试剂，至少两个分流通道包括第一分流通道和第二分流通道，该试剂回收方法包括如下步骤：
控制流体动力单元与第一分流通道连通并驱动第一试剂通过流动池和分流结构进入到至少两个分流通道中的第一分流通道中，第一试剂在流动池内与样本发生第一反应；和
控制流体动力单元动作以驱动第二试剂通过流动池和分流结构，第二试剂在流动池内与样本发生第二反应，并在第二反应后控制流体动力单元驱动第二试剂向试剂储存容器的方向回流。
在一些实施例中，试剂回收方法还包括在第一反应后，控制流体动力单元动作以驱动缓冲液通过流动池和分流结构并进入到第一分流通道中以清洗。
在一些实施例中，控制流体动力单元动作以驱动第二试剂通过流动池和分流结构，第二试剂在流动池内与样本发生第二反应，并在第二反应后控制流体动力单元驱动第二试剂向试剂储存容器的方向回流包括：控制流体动力单元与第二分流通道连通并驱动第二试剂通过流动池和分流结构进入到至少两个分流通道中的第二分流通道中，并在第二反应后，控制流体动力单元与第二分流通道连通并驱动第二试剂向试剂储存容器的方向回流。
在一些实施例中，控制流体动力单元动作以驱动第二试剂通过流动池和分流结构，第二试剂在流动池内与样本发生第二反应，并在第二反应后控制流体动力单元驱动第二试剂向试剂储存容器的方向回流包括：控制流体动力单元与第一分流通道连通并驱动第二试剂通过流动池和分流结构进入到第一分流通道中，并在第二反应后，控制流体动力单元与第二分流通道连通并驱动第二试剂向试剂储存容器的方向回流。
在一些实施例中，试剂回收方法还包括在第一反应后，控制流体动力单元与第一分流通道连通并驱动第一试剂向试剂储存容器的方向回流以使得回收的第一试剂回流到与储存第一试剂的试剂储存容器的出口端连接的管路内。
在一些实施例中，控制流体动力单元驱动第二试剂向试剂储存容器的方向回流包括：控制流体动力单元驱动第二试剂向试剂储存容器的方向回流以使得回收的第二试剂回流到与试剂储存容器的出口端连接的管路内。
基于本申请的各方面，液路系统通过设置分流模块，该分流模块包括分流结构和至少两个分流通道，这样可使得需要回收的试剂进入到与前一步反应的试剂进入的不同的分流通道内，以避免前一步反应的试剂被回收到流动池内而发生交叉污染。
通过以下参照附图对本申请的示例性实施例的详细描述，本申请的其它特征及其优点将会变得清楚。
附图说明
此处所说明的附图用来提供对本申请的进一步理解，构成本申请的一部分，本申请的示意性实施例及其说明用于解释本申请，并不构成对本申请的不当限定。在附图中：
图1为现有技术的液路系统的第一试剂正向流动时的结构示意图。
图2为现有技术的液路系统的第一试剂在反向回收时的结构示意图。
图3为本申请一些实施例的液路系统的结构示意图。
图4为基于图3所示的液路系统的一个实施例的试剂回收方法的流程图。
图5为本申请一些实施例的试剂回收方法的步骤图。
图6为本申请另一些实施例的试剂回收方法的步骤图。
图7为本申请一些实施例的试剂回收方法的流程图。
图8为本申请第一实施例的液路系统的结构示意图。
图9为采用图8所示的液路系统进行试剂回收时试剂储存容器内的试剂浓度随循环次数的变化图。
图10为本申请第二实施例的液路系统的结构示意图。
图11为本申请第三实施例的液路系统的结构示意图。
图12为本申请第四实施例的液路系统的结构示意图。
图13为本申请第五实施例的液路系统的结构示意图。
图14为本申请第六实施例的液路系统的结构示意图。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。以下对至少一个示例性实施例的描述实际上仅仅是说明性的，决不作为对本申请及其应用或使用的任何限制。基于本申请中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
除非另外具体说明，否则在这些实施例中阐述的部件和步骤的相对布置、数字表达式和数值不限制本申请的范围。同时，应当明白，为了便于描述，附图中所示出的各个部分的尺寸并不是按照实际的比例关系绘制的。对于相关领域普通技术人员已知的技术、方法和设备可能不作详细讨论，但在适当情况下，技术、方法和设备应当被视为授权说明书的一部分。在这里示出和讨论的所有示例中，任何具体值应被解释为仅仅是示例性的，而不是作为限制。因此，示例性实施例的其它示例可以具有不同的值。应注意到：相似的标号和字母在下面的附图中表示类似项，因此，一旦某一项在一个附图中被定义，则在随后的附图中不需要对其进行进一步讨论。
为了便于描述，在这里可以使用空间相对术语，如“在……之上”、“在……上方”、“在……上表面”、“上面的”等，用来描述如在图中所示的一个器件或特征与其他器件或特征的空间位置关系。应当理解的是，空间相对术语旨在包含除了器件在图中所描述的方位之外的在使用或操作中的不同方位。例如，如果附图中的器件被倒置，则描述为“在其他器件或构造上方”或“在其他器件或构造之上”的器件之后将被定位为“在其他器件或构造下方”或“在其他器件或构造之下”。因而，示例性术语“在……上方”可以包括“在……上方”和“在……下方”两种方位。该器件也可以其他不同方式定位，并且对这里所使用的空间相对描述作出相应解释。
如图1和图2所示，相关技术的液路系统包括流动池C1、第一管道L1和第二管道L2，第一管道L1连接在流动池C1的第一端，第二管道L2连接在流动池C1的第二端。其中，箭头FD示出的是流动方向。在图1和图2示出的液路系统中，在每一个反应循环中，样本先与第一试剂发生第一反应，再与第二试剂发生第二反应，然后多次重复以上反应循环。那么当两个反应循环发生时，第一个反应循环中的第二试剂先发生反应，而第二个反应循环中的第一试剂后发生反应。图1示出的是某一个反应循环的第一试剂R1从第一管道L1正向流动到第二管道L2的示意图，如图1所示，当第一试剂R1从第一管道L1正向流动到第二管道L2时，第二管道L2中还残留有缓冲液S和上一个反应循环的第二试剂R2，图2示出的是第一试剂R1从第二管道L2反向流回到第一管道L1的示意图，也就是说图2示出的是对第一试剂R1进行回 收的示意图。
由于泊肃叶流动特性，即封闭管道或槽道内流速的抛物线式分布，试剂之间的交界面并非直线，而是更加复杂的曲线。从图1可以看到，当第一试剂R1从左往右正向流入流动池C1后，第二管道L2中是被第一试剂R1替代的缓冲液S和更早之前被替代的第二试剂R2，此时第一试剂R1和缓冲液S之间，以及缓冲液S和第二试剂R2之间的交界面均为抛物线；缓冲液S在第一试剂R1和第二试剂R2之间作为隔断。这样当部分第一试剂R1被从右往左反向回收后(见图2)，由于流动池C1中部的流速最快，少量的第二试剂R2可能沿流动池中部回流到流动池C1中。在一些需按照某种顺序进行的反应体系中，这很可能会造成负面影响。例如，基因测序通常包含对DNA单链片段上每个碱基周期性的“合成-检测-切除”过程。在合成阶段，带有荧光基团的游离碱基以合成试剂为介质进入到流动池中，在聚合酶作用下，与固定在流动池表面的待测DNA单链互补配对。随后的检测阶段中，光学系统对荧光基团进行显影，从而识别出当前周期的碱基。最后，切除试剂进入流动池，对荧光基团进行切除，结束当前循环。在图1和图2中，假设第一试剂R1是合成试剂，第二试剂R2是上一个循环中的切除试剂。当第一试剂R1进入流动池C1(图1)，即开始了一个新的循环的合成反应，此时按照“合成-检测-切除”的顺序，接下来应进行光学显影。但由于试剂回收使部分切除试剂回流到流动池中(图2)，进而把部分已经结合的荧光基团与碱基分离，这种在光学显影之前意外发生的切除反应使整个流动池的测序过程出现了不同步，导致了测序错误的产生。因此在回收试剂时，如何避免前一步反应的试剂回流到流动池内而产生交叉污染是亟待解决的技术问题。
另外，现有的回收方案均是将回收的试剂直接回收在试剂储存容器中。由于试剂在正向进入流动池的过程中不可避免地与此前占据流动池及其第一管道的液体混合，因此其被反向回收后的浓度必然会下降，因此试剂浓度在多次回收复用中被反复稀释，这样就无法保证试剂在流动池内反应时的有效浓度。而且，当这些回收的试剂进入到贮藏池或储器中时，会进一步稀释储器中从未使用的那部分试剂。对于浓度与反应效率成正相关的反应体系，稀释后的试剂可能会使流动池中的反应效率显著下降。进一步地，试剂浓度的下降是与回收次数正相关的。举例来说，若每次回收试剂浓度降低10％，则四次循环后浓度将降低34％。因此，回收比越高，流动池内的整体反应效率越低。因此如何保证参与生化反应的试剂的浓度也是需要解决的问题。
为了避免前一步反应的试剂回流到流动池而产生交叉污染，本申请提出了在流动池C1的第二端(试剂正向流动时的下游端)设置分流通道的技术方案，将需要回收 的试剂和前一步反应的试剂分流，进而避免交叉污染。
参考图3，本申请实施例的液路系统包括至少两个试剂储存容器(图中未示出)、流动池C1、分流模块和流体动力单元(图中未示出)，其中，至少两个试剂储存容器用于分别储存至少两种不同的试剂。流动池C1用于容纳样本，且流动池C1与至少两个试剂储存容器连接。分流模块包括分流结构C2和至少两个分流通道。分流结构C2具有与流动池C1流体连通的合流口以及与至少两个分流通道对应设置的至少两个分流口。流体动力单元与分流模块连接。流体动力单元可选择地与至少两个分流通道中的一个分流通道连通，且流体动力单元被配置为驱动试剂从试剂储存容器朝分流模块的方向正向流动。且流体动力单元还被配置为驱动试剂从分流模块朝试剂储存容器的方向反向流动。
如图3所示，液路系统包括至少两个试剂储存容器(图中未示出)、流动池C1、与流动池C1第一端相连的第一管道L1、与流动池C1第二端相连的第二管道L2、分流结构C2和至少两个分流通道。具体地，图3中示出至少两个分流通道包括第一分流通道L3和第二分流通道L4。
需要说明的是，第一管道L1用于与试剂储存容器流体连接，第一管道L1与试剂储存容器之间还可以设置实现试剂选择的元件，如切换阀等。同样，第一分流通道L3和第二分流通道L4亦可继续与其他功能模块流体地连接，如实现流路控制的阀、驱动流体的流体动力单元。分流结构C2可以是简单的三通元件(如T型三通、Y型三通)，也可以是三通电磁阀等控制元件，还可以是前两者的组合。另外，根据实际应用的需要，分流结构C2可分流出两个以上的支路，每个支路又可以再经过一个或多个分流模块分成更多的支路，这些向下延伸的旁路设计均属于本申请保护的范围。
基于图3的液路系统设计，可以建立一套防止流动池C1内交叉污染的试剂回收逻辑，如图4所示。这里为了便于理解，只涉及到两步反应，即第一反应和第二反应；同时需要说明的是，图4中只对第二反应中使用的试剂进行回收，以简化讨论，但这并不代表第一反应中使用的试剂无法被回收。结合图4，整个回收逻辑主要包含以下步骤：
S1：切换位于第一管道L1上游的试剂选择元件，使其与装有第一试剂101的试剂储存容器流体连接(试剂选择元件与试剂储存容器在图4中未标示)，并使流体动力单元与第一分流通道L3连通，进而使得第一试剂101在流体动力单元的驱动下依次替代第一管道L1、流动池C1、第二管道L2及分流结构C2内的缓冲液102，并最终沿第一分流通道L3流出，此时第一试剂101与缓冲液102的抛物线交界面位于第 一分流通道L3内。
S2：S1结束后，流动池C1内的第一试剂101随即与固定在流动池C1上的样本发生第一反应。
S3：第一反应完成后，切换试剂选择元件，使第一管道L1与装有缓冲液102的缓冲液储存容器流体连接，缓冲液102在流体动力单元的驱动下依次清洗第一管道L1、流动池C1、第二管道L2及分流结构C2内的第一试剂101，并最终沿第一分流通道L3流出，此时缓冲液102与第一试剂101的交界面位于第一分流通道L3内，保证第一试剂101完全离开分流结构C2。
S4：切换试剂选择元件，使第一管道L1与装有第二试剂103的试剂储存容器流体连接，并使流体动力单元与第二分流通道L4连通，进而使得第二试剂103在流体动力单元的驱动下依次替代第一管道L1、流动池C1、第二管道L2及分流结构C2内的缓冲液102，并最终沿第二分流通道L4流出，此时第二试剂103与缓冲液102的抛物线交界面位于第二分流通道L4内，且分流结构C2中的第二试剂103不会与第一分流通道L3中的第一试剂101混合。
S5：S4结束后，流动池C1内的第二试剂103随即与固定在流动池C1上的样本发生第二反应。
S6：第二反应完成后，流体动力单元反向驱动第二试剂103依次离开第二分流通道L4、分流结构C2、第二管道L2和流动池C1，实现对第二试剂103的回收。
S1至S6描述了单一试剂(即第二试剂103)在两步反应体系中的回收策略。通过将第一试剂101和第二试剂103分别输送至第一分流通道L3和第二分流通道L4，并在第一试剂101和第二试剂103间设置缓冲液，在第二试剂103被回收时，第一试剂101不会回流至流动池中进而避免第一反应的再次发生。
在此需要说明的是，图4示出的回收方法示出的是对第二试剂103的回收流程，但是在另一种可能的试剂回收方法中，第一试剂101也可以在第一反应结束后被回收。例如，在第一反应后，流体动力单元反向驱动第一试剂101依次离开第一分流通道L3、分流结构C2、第二管道L2和流动池C1，实现对第一试剂101的回收。对于循环反应来说，正是由于在上一个循环反应中的第二试剂103是流动到第二分流通道L4内的，因此对第一试剂101回收时，第二试剂103不会回流到流动池C1内进而避免发生交叉污染。综上可知，本申请的液路系统通过分流模块使得第一试剂和第二试剂进入到不同的分流通道内，使得在对某一个试剂回收时，另一个试剂不会回流到流动池内，也就是说本申请的液路系统对第一试剂和第二试剂均能回收，并不是仅能对第二 试剂进行回收。
在以上实施例中，为了实现第一试剂101和第二试剂103分别进入到不同的分流通道内，需要流体动力单元可以在第一分流通道和第二分流通道中选择，从而保证一个通道中的液体流动时，另一个通道中的液体保持静止。也就是说，流体动力单元被配置为可选择地与至少两个分流通道中的一个分流通道连通。并且，在以上实施例中，试剂需要正向流动至流动池内以发生反应，在需要回收时，试剂还需要从分流模块反向流动，试剂的上述双向流动是由流体动力单元来实现的。流体动力单元被配置为驱动试剂从试剂储存容器朝分流模块的方向正向流动。且流体动力单元还被配置为驱动试剂从分流模块朝试剂储存容器的方向反向流动。具体地，流体动力单元可以包括既能够实现正向驱动又能够实现反向驱动的动力装置。流体动力单元也可以包括能够实现正向驱动的正向动力装置和能够实现反向驱动的反向动力装置。
综上可知，在本申请实施例的技术方案中，通过设置分流模块，该分流模块包括分流结构和至少两个分流通道，这样可使得需要回收的试剂进入到与前一步反应的试剂进入的不同的分流通道内，以避免前一步反应的试剂被回收到流动池内而发生交叉污染。
参考图5，本申请实施例还提供一种试剂回收方法。该试剂回收方法包括如下步骤：
S101:控制流体动力单元与第一分流通道L3连通并驱动第一试剂101通过流动池C1和分流结构C2进入到至少两个分流通道中的第一分流通道L3中，第一试剂101在流动池C1内与样本发生第一反应；和
S102:控制流体动力单元动作以驱动第二试剂103通过流动池C1和分流结构C2，第二试剂103在流动池C1内与样本发生第二反应，并在第二反应后控制流体动力单元驱动第二试剂103向试剂储存容器的方向回流。
在一些实施例中，液路系统还包括用于储存缓冲液的缓冲液储存容器，缓冲液储存容器与流动池连接，流体动力单元被配置为驱动缓冲液从缓冲液储存容器向分流模块的方向正向流动。
在一些实施例中，试剂回收方法还包括在第一反应后，控制流体动力单元动作以驱动缓冲液通过流动池和分流结构并进入到第一分流通道中以清洗。然后在清洗后，控制流体动力单元与第二分流通道连通并驱动第二试剂通过流动池和分流结构进入到至少两个分流通道中的第二分流通道中，并在第二反应后，控制流体动力单元与第二分流通道连通并驱动所述第二试剂向试剂储存容器的方向回流。也就是说，在本实 施例中，在第一反应和第二反应之间设置缓冲液的清洗过程，并且使得第一试剂和第二试剂分别进入到两个不同的分流通道中。
在另一些实施例中，在第一反应和第二反应之间可以不设置缓冲液的清洗过程。控制流体动力单元动作以驱动第二试剂通过流动池和分流结构，第二试剂在流动池内与样本发生第二反应，并在第二反应后控制流体动力单元驱动第二试剂向所述试剂储存容器的方向回流包括：控制流体动力单元与第一分流通道连通并驱动第二试剂通过流动池和分流结构进入到第一分流通道中，并在第二反应后，控制流体动力单元与第二分流通道连通并驱动第二试剂向试剂储存容器的方向回流。也就是说在第一反应后，第二试剂直接替代流动池中的第一试剂并最终沿第一分流通道流出，此时第一试剂与第二试剂的交界面位于第一分流通道内，之后对第二试剂的回收切换至第二分流通道内进行。
在一些实施例中，至少两个分流通道和至少两个试剂储存容器一一对应设置。也就是说，每一种试剂均进入到不同的分流通道，进而避免在回收时其他试剂回流到流动池内。
在另一些实施例中，为了减小液路系统的体积并简化其结构，液路系统的分流模块包括两个分流通道。若是涉及两种试剂的循环反应，则控制一种试剂流到第一分流通道内，控制另一种试剂流到第二分流通道内，也就是说可按照如图6所示的回收策略来回收。若是涉及多种试剂的循环多步反应，则需要将每一步的试剂进入到与上一步反应所用的试剂不同的分流通道中，防止上一步所用的试剂回流到流动池中而导致乱序反应。
在一种可能出现的情况中，当一个试剂被回收时，不仅前一步的试剂有可能回流至流动池中，两步或更多步之前的试剂也有可能回流。此时可以通过增加试剂间的缓冲液体积，或降低回收率，或在分流模块后增设更多的试剂通道来解决，例如上述实施例中提到的至少两个分流通道和至少两个试剂储存容器一一对应设置，这样就使得每个试剂都进入到对应的一个分流通道内，进而避免交叉污染。
针对如何在对试剂进行回收的情况下保证参与生化反应的试剂的浓度这一问题，本申请的发明人也进行了深入研究。在一些实施例中，流体动力单元被配置为驱动试剂从分流模块朝试剂储存容器的方向反向流动并回流到与试剂储存容器的出口端连接的管路内。例如在回收第二试剂时，控制流体动力单元驱动第二试剂向试剂储存容器的方向回流包括：控制流体动力单元驱动第二试剂向试剂储存容器的方向回流以使得回收的第二试剂回流到与试剂储存容器的出口端连接的管路内。也就是说回收的第 二试剂仅回流到与试剂储存容器的出口端连接的管路内，而不会回到试剂储存容器中，这样可以保证试剂储存容器中的试剂不被稀释。
具体地，可以通过调节回收比例，使回收的试剂不会回到试剂储存容器内。例如，通过控制回收试剂的体积。
当然，若有需要，回收的试剂部分或全部回到试剂储存容器中也是允许的，只要保证试剂储存容器中被稀释的试剂满足再次被使用的要求即可。
参考图7，该液路系统包括第一储存容器111、第二储存容器112、第三储存容器113、试剂选择元件C3、第一管道L1、流动池C1和第二管道L2。其中第一储存容器111用于储存第一试剂101，第二储存容器112用于储存缓冲液102，第三储存容器113用于储存第二试剂103。其中图7A示出的是当前循环的回收过程，图7B示出的是下一个循环的第二反应。如图7A所示，在回收结束后，一定体积的第二试剂103被回收到流动池C3的上游，其中包括与缓冲液102直接接触导致浓度被稀释的部分1031和浓度几乎未被稀释的部分1032，由于被稀释的部分1031处在未被稀释的部分1032的下游，那么如图7B所示，在下一个循环使用第二试剂103时，被稀释的部分1031必定先参与第一管路L1和流动池C1中的试剂替代，配合较快的流动速度，被稀释的部分1031经过流动池时，仅会引发微弱的第二反应(几乎可以忽略不计)，随着第二试剂103继续向下游流动，浓度几乎未被稀释的部分1032进入流动池，此时流动停止，该部分与流动池内的样品进行充分的反应。
本申请技术方案通过液路系统设计与回收逻辑实现了高效的试剂回收重复利用，可以大大降低在封闭管道和槽道中消耗的试剂用量，从而大幅降低医疗检测仪器中的试剂耗材成本。与此同时，本申请主要具有以下两个优点：
1)通过设置分流模块，将需要回收的试剂和前一个试剂分流，进而解决了试剂回收后流动池内交叉污染的问题，使基因测序仪等精密仪器中复杂的循环式有序反应体系不会因为试剂回收而混乱，保证了生化反应的质量。对于流动池体积相对上下游管道较小的情况，试剂回收极其容易造成交叉污染，本方案使得在小流动池中实现高回收率成为了可能。
2)试剂回收后不会造成储存容器内试剂整体浓度的明显稀释。同时，回收的试剂中低浓度的部分主要用于替代流动池及其上游公共管道中的另一种液体，而非用于参与生化反应。生化反应仍旧在一个较高的试剂浓度下进行，保证了反应的效率。
参考图8，在一些实施例中，至少两个分流通道包括第一分流通道206和第二分 流通道207，分流结构包括三通管202，三通管202包括与流动池流体连通的合流口、与第一分流通道206连通的第一分流口以及与第二分流通道207连通的第二分流口。
以图8所示第一实施例的基因测序仪的液路系统为例来具体说明。如图8所示，该液路系统包括储存容器208、试剂选择元件203、第一管道204、测序载片201、第二管道205、三通管202、第一分流通道206、第二分流通道207、注射泵209、第三管道210和废液池211。
其中储存容器208包括用于储存合成试剂221的第一储存容器、用于储存扫描试剂222的第二储存容器、用于储存切除试剂223的第三储存容器以及用于储存缓冲液224的第四储存容器。测序载片201具有流动池。
试剂选择元件203用于使流动池可选择地与储存容器208中的一个储存容器流体连通，进而可使得相应的试剂进入流动池。试剂选择元件203包括公共孔和至少两个分支孔，至少两个分支孔与至少两个试剂储存容器对应连接，公共孔通过第一管道204与流动池连接，公共孔可选择地与至少两个分支孔中的一个连通。具体地，试剂选择元件203为试剂选择阀。
三通管202形成分流结构。该三通管202包括与流动池流体连通的合流口、与第一分流通道206连通的第一分流口以及与第二分流通道207连通的第二分流口。具体地，该三通管202为T形三通管，在其他实施例中，也可以是Y形等三通部件。
在其他实施例中，分流结构也可以包括多通管，例如四通管以实现更多分流通道。
注射泵209形成流体动力单元。注射泵209具有三个动力口，第一动力口与第一分流通道206流体连接，第二动力口与第二分流通道207流体连接，第三动力口通过第三管道210与废液池211流体连接。注射泵209可选择地给三个动力口中的一个动力口提供驱动力，且注射泵209可提供使试剂正向流动的正向驱动力，也可以提供使试剂反向流动的反向驱动力。当然，在其他实施例中，流体动力单元也可以包括两个独立设置的注射泵，一个注射泵用于给试剂提供正向驱动力，另一个注射泵用于给试剂提供反向驱动力。
下面对图8所示的第一实施例的液路系统的工作过程进行详细说明。图中，实线箭头表示试剂在正向流动时的流动方向，虚线箭头表示试剂在反向回收时的流动方向。在测序载片的流动池表面固定有待测的DNA片段，整个测序过程为如前所述的循环式“合成-检测-切除”体系。为了简化描述，每个步骤只涉及一种试剂，两个步骤间使用缓冲液作为试剂间的隔断。需要额外说明的是，本例中的流动池体积约为4 μL，属于典型的小体积流动池。与之相比，第一管道204体积为4μL，试剂选择元件203及其与储存容器208之间管道的总体积约为30μL，第二管道205体积为10μL，第一分流通道206和第二分流通道207体积均为100μL以上。测序开始前，储存容器208中的各试剂均预先灌注各自与试剂选择元件203之间的管道内，因此在测序过程中，试剂替代主要发生在第一管道204和测序载片201的流动池中。
在本例中，一个完整的反应循环包括以下步骤：
1)试剂选择元件203切换至与第一储存容器流体连接，注射泵209切换至与第一分流通道206流体连接，抽取40μL合成试剂221以高流速(如2000μL/min，下同)经过测序载片201。在此步中，合成试剂221替代原本在第一管道204和流动池中的缓冲液224；
2)流动池中进行持续一段时间的合成反应；
3)注射泵209将30μL的合成试剂221沿第一分流通道206反向推送至上游(见图8中虚线箭头)，合成试剂221中浓度被稀释的部分回流至试剂选择元件203与储存容器208之间的管道，但不会进入到储存容器208中；
4)试剂选择元件203切换至与第四储存容器流体连接，注射泵209保持与第一分流通道206流体连接，并抽取50μL缓冲液224经过测序载片201，替代第一管道204和流动池中残余的合成试剂221；
5)试剂选择元件203切换至与第二储存容器流体连接，注射泵209保持与第一分流通道206流体连接，并抽取40μL扫描试剂222经过测序载片201，完成后光学系统(图中未标示)对测序载片201进行检测；
6)试剂选择元件203切换至与第四储存容器流体连接，注射泵209抽取50μL缓冲液224经过测序载片201，替代第一管道204和流动池中的扫描试剂222；
7)试剂选择元件203切换至与第三储存容器流体连接，注射泵209切换至与第二分流通道207流体连接，抽取40μL切除试剂223经过测序载片201；
8)流动池中进行持续一段时间的切除反应；
9)试剂选择元件203切换至与第四储存容器流体连接，注射泵209保持与第二分流通道207流体连接，并抽取50μL缓冲液224经过测序载片201，替代第一管道204和流动池中的切除试剂223；
10)注射泵209切换至与第三管道210和废液池211流体连接，将注射泵中的液体排空。
在上述步骤中，合成试剂221先被抽取40μL，再被回收30μL，因此回收率为 30/40＝75％，净使用量为40-30＝10μL，相对于流动池体积(4μL)的替代比r值为10/4＝2.5。若不进行试剂回收，替代比则为40/4＝10。可见，替代比在引入本申请的回收策略后降低为原来的1/4。
从测序质量看，高比例的试剂回收未对测序的结果有明显影响。表1对比了无回收和回收75％的测序质量指标，两者在读长(Total Reads)和质量值(Q30)上都处于同一水平。
表1无回收和回收75％策略下的测序结果对比
Figure PCTCN2021129640-appb-000001
本例在流动池出口端引入T形的三通管202及第一分流通道206和第二分流通道207后，成功解决了试剂回收带来的切除试剂223回流造成流动池内交叉污染的问题。通过对回收后流动池中残留切除试剂浓度的测量，未引入三通管202及分流通道时残留浓度为0.7％，引入后残留浓度可忽略不计。由于极微量的切除试剂即可造成反应乱序和测序错误，本申请的技术方案对基因测序十分重要。
在图8示出的实施例中，合成试剂221进入的是第一分流通道206，切除试剂223进入的是第二分流通道207，因此在对合成试剂221回收时，避免了切除试剂223回流到流动池内发生交叉污染的问题。当然，当有需要时，我们也可以对切除试剂223进行回收，同样可避免合成试剂221回流到流动池内发生交叉污染的问题。另外，当涉及多种试剂的循环多步反应时，在一些实施例中，可以使每一步的试剂都进入到与上一步试剂不同的分流通道内，但是也可能发生以下情况，当一个试剂被回收时，两步或更多步之前的试剂可能会与该要回收的试剂在同一个分流通道内，那么可能会回流，此时可以通过增加试剂之间的缓冲液体积来避免以上情况的发生。在另一些实施例中，可以根据试剂的种类来决定每一步的试剂是否要进入到与上一步试剂不同的分流通道内，关键在于当该步的试剂进入到与上一步试剂相同的分流通道内后是否会造成乱序反应。例如在图8示出的实施例中，涉及到合成试剂221、扫描试剂222和切除试剂223，反应顺序是合成-扫描-切除，由于扫描试剂222回流到流动池内不会造成乱序反应，而切除试剂223回流到流动池内会造成乱序反应，因此在上述实施例中，合成试剂221和扫描试剂222均是流入到第一分流通道206内，而切除试剂223是流 入到第二分流通道207内的。主要是为了使得切除试剂223进入到与扫描试剂222不同的分流通道内。
本例中的合成试剂在反应后才被回收，且不会被回收至储存容器208中；在下一次循环复用时，浓度被稀释的部分主要参与试剂替代，在流动池中参与反应的试剂浓度维持在较高水平。图9所示的流动池中合成试剂浓度随循环数变化的曲线可以证明这一优点。从图中可见，合成试剂浓度在100个循环内始终保持在98.7％以上的有效相对浓度，充分说明了本申请技术方案的一大优点。
参考图10，在一些实施例中，分流模块还包括通断控制阀。通断控制阀设置在第一分流通道206和/或第二分流通道207上。在第一分流通道206和/或第二分流通道207上设置通断控制阀，可加强物理隔断，使得在回收试剂时，上一步试剂被完全隔离在另一个分流通道中。
具体地，以图10所示第二实施例的基因测序仪的液路系统为例来具体说明。
与第一实施例相比，第二实施例的改变是在第二分流通道207中增设了一个通断控制阀，具体地为两位两通电磁阀212。该电磁阀的作用是加强物理隔断，使得在回收合成试剂的过程中，切除试剂完全被留在第二分流通道207中。由于在图8所示实施例的设计中，三通管202和第一分流通道206、第二分流通道207均形成通路，因此在合成试剂沿第一分流通道206回收时，第二分流通道207中的切除试剂因第一分流通道206和第二分流通道207流阻不等、管道中有气泡等原因，仍然有可能部分回流至流动池中。本实施例在引入电磁阀212后，即可解决此问题。电磁阀212在通电状态下为通路，在断电状态下为断路。
在本实施例中，试剂回收的步骤与第一实施例稍有不同，具体为：
1)试剂选择元件203切换至与第一储存容器流体连接，电磁阀212切换至断电状态，注射泵209切换至与第一分流通道206流体连接，抽取40μL合成试剂221以高流速(如2000μL/min，下同)经过测序载片201。在此步中，合成试剂221替代原本在第一管道204和流动池中的缓冲液224；
2)～6)同第一实施例；
7)试剂选择元件203切换至与第三试剂储存容器流体连接，电磁阀212切换至通电状态，注射泵209切换至与第二分流通道207流体连接，抽取40μL切除试剂223经过测序载片201；
8)～10)同第一实施例。
在其他一些附图未示出的实施例中，通断控制阀也可以设置在第一分流通道206 中。或者第一分流通道和第二分流通道中分别设置通断控制阀。通断控制阀可以是电磁阀，也可以是可以实现控制管路通断的其他元件。
参考图11，在一些实施例中，至少两个分流通道包括第一分流通道206和第二分流通道207。分流结构包括第一换向阀。第一换向阀具有第一口、第二口和第三口，第一口形成合流口，第二口形成与第一分流通道206连通的第一分流口，第三口形成与第二分流通道207连通的第二分流口，第一换向阀动作以控制第一口与第二口或第三口连通。
具体地，以图11所示第三实施例的基因测序仪的液路系统为例来具体说明。
本实施例为第一实施例的一个改进设计。从图11可见，本例相对第一实施例的唯一改变是将三通管更换为一个换向阀，具体为两位三通电磁阀213。该电磁阀的常开端与第一分流通道206流体连接，常闭端与第二分流通道207流体连接。和第二实施例一样，本例相比第一实施例具有更好的物理隔断。
在本例中，试剂回收的步骤与第一实施例稍有不同，具体为：
1)试剂选择元件203切换至与第一储存容器流体连接，电磁阀213保持断电状态，注射泵209切换至与第一分流通道206流体连接，抽取40μL合成试剂221以高流速(如2000μL/min，下同)经过测序载片201。在此步中，合成试剂221替代原本在第一管道204和流动池中的缓冲液224；
2)～6)同第一实施例；
7)试剂选择元件203切换至与第三储存容器流体连接，电磁阀213切换至通电状态，注射泵209切换至与第二分流通道207流体连接，抽取40μL切除试剂223经过测序载片201；
8)～10)同第一实施例。
在一些实施例中，流体动力单元包括注射泵209，注射泵209包括第一动力口和第二动力口，第一动力口与第一分流通道流体连接，第二动力口与第二分流通道流体连接。
具体地，参考图9、图10和图11示出的三个实施例，注射泵209包括第一动力口和第二动力口，第一动力口与第一分流通道206流体连接，第二动力口与第二分流通道207流体连接。
参考图9、图10和图11示出的三个实施例，液路系统还包括废液池211，注射泵209还包括第三动力口，第三动力口与废液池211连通。
参考图12，在一些实施例中，流体动力单元包括注射泵214和第二换向阀，注射泵包括第一动力口，第二换向阀具有第一口、第二口和第三口，第一口和第二口分别和第一分流通道和第二分流通道连接，第三口与注射泵214的第一动力口连接，第二换向阀动作以控制第三口与第一口或第二口连通。
以图12示出的第四实施例的基因测序仪的液路系统为例来具体说明。从图12可见，本实施例相对第一实施例的改变是在三通管202后端增设一个换向阀，具体为两位三通电磁阀213。该电磁阀的常开端与第二分流通道207流体连接，常闭端与第一分流通道206流体连接。另外，本例中的注射泵214只有两个可选动力口，一端与电磁阀213流体连接，另一端与第三管道210以及废液池211流体连接。
在本例中，试剂回收的步骤与第一实施例稍有不同，具体为：
1)试剂选择元件203切换至与第一储存容器流体连接，电磁阀213切换为通电状态，注射泵214抽取40μL合成试剂221以高流速(如2000μL/min，下同)经过测序载片201。在此步中，合成试剂221替代原本在第一管道204和流动池中的缓冲液224；
2)流动池中进行持续一段时间的合成反应；
3)注射泵214将30μL试剂沿第一分流通道206反向推送至上游(见图12中虚线箭头)，合成试剂221中浓度被稀释的部分回流至试剂选择元件203与试剂储存容器208之间的管道，但不会进入到试剂储存容器208中；
4)试剂选择元件203切换至与第四储存容器流体连接，注射泵214抽取50μL缓冲液224经过测序载片201，替代第一管道204和流动池中残余的合成试剂221；
5)试剂选择元件203切换至与第二储存容器流体连接，注射泵214抽取40μL扫描试剂222经过测序载片201，完成后光学系统(图中未标示)对测序载片201进行检测；
6)试剂选择元件203切换至与第四储存容器流体连接，注射泵214抽取50μL缓冲液224经过测序载片201，替代第一管道204和流动池中的扫描试剂222；
7)试剂选择元件203切换至与切除试剂223流体连接，电磁阀213切换至断电状态，注射泵214抽取40μL切除试剂223经过测序载片201并进入到第二分流通道207内；
8)流动池中进行持续一段时间的切除反应；
9)试剂选择元件203切换至与第四储存容器流体连接，注射泵214抽取50μL缓冲液224经过测序载片201，替代第一管道204和流动池中的切除试剂223；
10)注射泵214切换至与第三管道210和废液储存容器211流体连接，将注射器中的液体排空。
在一些实施例中，液路系统还包括废液池211，注射泵214还包括第二动力口，注射泵214的第二动力口与废液池连通。如图12所示，注射泵214包括两个动力口，一个动力口与换向阀(两位三通电磁阀213)连接，另一个动力口通过第三管道210与废液池211。
在一些实施例中，参考图13，流体动力单元包括第一蠕动泵216和第二蠕动泵217。液路系统还包括废液池211，第一分流通道206和第二分流通道207均与废液池211连通，第一蠕动泵216设置在第一分流通道206上，第二蠕动泵217设置在第二分流通道207上。
图13示出第五实施例的基因测序仪的液路系统。如图13所示，本实施例为第三实施例的一个变形设计，主要变化在于将注射泵更换为两个蠕动泵216和217。从图13可见，电磁阀213的常开端通过第一分流通道206与第一蠕动泵216流体连接，常闭端通过第二分流通道207与第二蠕动泵217流体连接。
在本例中，试剂回收的步骤与第三实施例稍有不同，具体为：
1)试剂选择元件203切换至与第一储存容器流体连接，电磁阀213保持断电状态，注射泵217抽取40μL合成试剂221以高流速(如2000μL/min，下同)经过测序载片201。在此步中，合成试剂221替代原本在第一管道204和流动池中的缓冲液224；
2)流动池中进行持续一段时间的合成反应；
3)第一蠕动泵216将30μL试剂沿第一分流通道206反向推送至上游(见图14中虚线箭头)，合成试剂221中浓度被稀释的部分回流至试剂选择元件203与试剂储存容器208之间的管道，但不会进入到试剂储存容器208中；
4)试剂选择元件203切换至与第四储存容器流体连接，第二蠕动泵217抽取50μL缓冲液224经过测序载片201，替代第一管道204和流动池中残余的合成试剂221；
5)试剂选择元件203切换至与第二储存容器流体连接，第二蠕动泵217抽取40μL扫描试剂222经过测序载片201，完成后光学系统(图中未标示)对测序载片201进行检测；
6)试剂选择元件203切换至与第四储存容器流体连接，第二蠕动泵217抽取50μL缓冲液224经过测序载片201，替代第一管道204和流动池中的扫描试剂222；
7)试剂选择元件203切换至与第三储存容器流体连接，电磁阀213切换至通电状态，此时第二蠕动泵217抽取40μL切除试剂223经过测序载片201并进入到第二分流通道207内；
8)流动池中进行持续一段时间的切除反应；
9)试剂选择元件203切换至与缓冲液224流体连接，第一蠕动泵216抽取50μL缓冲液224经过测序载片201，替代第一管道204和流动池中的切除试剂223。
在一些实施例中，流体动力单元包括注射泵214。液路系统还包括废液池211、试剂选择元件203，第一分流通道206和第二分流通道207均与废液池211连通，注射泵214包括动力口，试剂选择元件203包括公共孔和多个分支孔，公共孔可选择地与多个分支孔中的一个连通，多个分支孔包括与至少两个试剂储存容器对应连通的至少两个试剂分支孔以及与流动池连通的流动池分支孔，注射泵214的动力口与公共孔连接。
图14示出第六实施例的基因测序仪的液路系统。该液路系统是图11示出的第三实施例的变形实施例。与第三实施例相比，主要变化在于将注射泵前置到试剂选择元件后，使正向的试剂流动变为正压驱动方式。从图14可见，注射泵214通过管道215与试剂选择元件203的公共孔位流体连接，而公共管道204改为与试剂选择元件203的一个分支孔位流体连接。另外，电磁阀213的分流通道206、207均与废液池211流体连接。在应用时，试剂回收的步骤与第三实施例基本一致，只是每次正向泵液时，注射泵214先从试剂选择元件203的公共孔位抽取试剂到管道215中，试剂选择元件203再切换至与第一管道204流体连接，最后注射泵214向第一管道204推送试剂。当需要回收试剂时，注射泵214先从第一管道204抽取使用过的试剂至管道215中，试剂选择元件203再切换至与该试剂对应的孔位，最后注射泵214向该试剂孔位推送回收的试剂。
本申请实施例还提供一种基因测序仪，包括测序载片和上述液路系统，流动池设置在测序载片上。
最后应当说明的是：以上实施例仅用以说明本申请的技术方案而非对其限制；尽管参照较佳实施例对本申请进行了详细的说明，所属领域的普通技术人员应当理解：依然可以对本申请的具体实施方式进行修改或者对部分技术特征进行等同替换；而不脱离本申请技术方案的精神，其均应涵盖在本申请请求保护的技术方案范围当中。

Claims (21)

  1. 一种液路系统，包括：
    至少两个试剂储存容器，所述至少两个试剂储存容器用于分别储存至少两种不同的试剂；
    流动池，用于容纳样本，且所述流动池与至少两个试剂储存容器连接；
    分流模块，包括分流结构和至少两个分流通道，所述分流结构具有与所述流动池流体连通的合流口以及与所述至少两个分流通道对应设置的至少两个分流口；和
    流体动力单元，与所述分流模块连接，所述流体动力单元可选择地与所述至少两个分流通道中的一个分流通道连通，且所述流体动力单元被配置为驱动试剂从所述试剂储存容器朝所述分流模块的方向正向流动，且所述流体动力单元还被配置为驱动试剂从所述分流模块朝所述试剂储存容器的方向反向流动。
  2. 根据权利要求1所述的液路系统，其中，所述至少两个分流通道和所述至少两个试剂储存容器一一对应设置。
  3. 根据权利要求1所述的液路系统，其中，所述至少两个分流通道包括第一分流通道和第二分流通道，所述分流结构包括三通管，所述三通管包括与所述流动池流体连通的合流口、与所述第一分流通道连通的第一分流口以及与所述第二分流通道连通的第二分流口。
  4. 根据权利要求3所述的液路系统，其中，所述分流结构还包括通断控制阀，所述通断控制阀设置在第一分流通道和/或所述第二分流通道上。
  5. 根据权利要求1所述的液路系统，其中，所述至少两个分流通道包括第一分流通道和第二分流通道，所述分流结构包括第一换向阀，所述第一换向阀具有第一口、第二口和第三口，所述第一口形成合流口，所述第二口形成与所述第一分流通道连通的第一分流口，所述第三口形成与所述第二分流通道连通的第二分流口，所述第一换向阀动作以控制所述第一口与所述第二口或所述第三口连通。
  6. 根据权利要求3至5中任一项所述的液路系统，所述流体动力单元包括注射泵，所述注射泵包括第一动力口和第二动力口，所述第一动力口与所述第一分流通道流体连接，所述第二动力口与所述第二分流通道流体连接。
  7. 根据权利要求6所述的液路系统，所述液路系统还包括废液池，所述注射泵还包括第三动力口，所述第三动力口与所述废液池连通。
  8. 根据权利要求3所述的液路系统，其中，所述流体动力单元包括注射泵和第二换向阀，所述注射泵包括第一动力口，所述第二换向阀具有第一口、第二口和第三口，所述第一口和所述第二口分别和所述第一分流通道和所述第二分流通道连接，所述第三口与所述注射泵的第一动力口连接，所述第二换向阀动作以控制所述第三口与所述第一口或所述第二口连通。
  9. 根据权利要求8所述的液路系统，所述液路系统还包括废液池，所述注射泵还包括第二动力口，所述注射泵的第二动力口与所述废液池连通。
  10. 根据权利要求5所述的液路系统，所述流体动力单元包括第一蠕动泵和第二蠕动泵，所述液路系统还包括废液池，所述第一分流通道和所述第二分流通道均与所述废液池连通，所述第一蠕动泵设置在所述第一分流通道上，所述第二蠕动泵设置在所述第二分流通道上。
  11. 根据权利要求1至10中任一项所述的液路系统，所述液路系统还包括试剂选择元件，所述试剂选择元件包括公共孔和至少两个分支孔，所述至少两个分支孔与所述至少两个试剂储存容器对应连接，所述公共孔与所述流动池连接，所述公共孔可选择地与所述至少两个分支孔中的一个连通。
  12. 根据权利要求5所述的液路系统，所述流体动力单元包括注射泵，所述液路系统还包括废液池、试剂选择元件，所述第一分流通道和所述第二分流通道均与所述废液池连通，所述注射泵包括动力口，所述试剂选择元件包括公共孔和多个分支孔，所述公共孔可选择地与所述多个分支孔中的一个连通，所述多个分支孔包括与所述至少 两个试剂储存容器对应连通的至少两个试剂分支孔以及与所述流动池连通的流动池分支孔，所述注射泵的动力口与所述公共孔连接。
  13. 根据权利要求1至11中任一项所述的液路系统，所述液路系统还包括用于储存缓冲液的缓冲液储存容器，所述缓冲液储存容器与所述流动池连接，所述流体动力单元被配置为驱动所述缓冲液从所述缓冲液储存容器向所述分流模块的方向正向流动。
  14. 根据权利要求1所述的液路系统，所述流体动力单元被配置为驱动试剂从所述分流模块朝所述试剂储存容器的方向反向流动并回流到与试剂储存容器的出口端连接的管路内。
  15. 一种基因测序仪，包括测序载片和如权利要求1至14中任一项所述的液路系统，所述流动池设置在所述测序载片上。
  16. 一种基于权利要求1至14中任一项所述的液路系统的试剂回收方法，所述至少两种不同的试剂包括第一试剂和第二试剂，所述至少两个分流通道包括第一分流通道和第二分流通道，包括如下步骤：
    控制流体动力单元与第一分流通道连通并驱动第一试剂通过流动池和分流结构进入到所述至少两个分流通道中的第一分流通道中，第一试剂在流动池内与样本发生第一反应；和
    控制流体动力单元动作以驱动第二试剂通过流动池和分流结构，第二试剂在流动池内与样本发生第二反应，并在第二反应后控制流体动力单元驱动第二试剂向所述试剂储存容器的方向回流。
  17. 根据权利要求16所述的试剂回收方法，所述试剂回收方法还包括在第一反应后，控制流体动力单元动作以驱动缓冲液通过流动池和分流结构并进入到第一分流通道中以清洗。
  18. 根据权利要求17所述的试剂回收方法，其中，控制流体动力单元动作以驱动 第二试剂通过流动池和分流结构，第二试剂在流动池内与样本发生第二反应，并在第二反应后控制流体动力单元驱动第二试剂向所述试剂储存容器的方向回流包括：控制流体动力单元与第二分流通道连通并驱动第二试剂通过流动池和分流结构进入到所述至少两个分流通道中的第二分流通道中，并在第二反应后，控制流体动力单元与所述第二分流通道连通并驱动所述第二试剂向所述试剂储存容器的方向回流。
  19. 根据权利要求16所述的试剂回收方法，其中，控制流体动力单元动作以驱动第二试剂通过流动池和分流结构，第二试剂在流动池内与样本发生第二反应，并在第二反应后控制流体动力单元驱动第二试剂向所述试剂储存容器的方向回流包括：控制流体动力单元与第一分流通道连通并驱动第二试剂通过流动池和分流结构进入到所述第一分流通道中，并在第二反应后，控制流体动力单元与所述第二分流通道连通并驱动所述第二试剂向所述试剂储存容器的方向回流。
  20. 根据权利要求16所述的试剂回收方法，还包括在第一反应后，控制流体动力单元与所述第一分流通道连通并驱动所述第一试剂向所述试剂储存容器的方向回流以使得回收的第一试剂回流到与试剂储存容器的出口端连接的管路内。
  21. 根据权利要求16所述的试剂回收方法，控制流体动力单元驱动第二试剂向所述试剂储存容器的方向回流包括：
    控制流体动力单元驱动第二试剂向所述试剂储存容器的方向回流以使得回收的第二试剂回流到与试剂储存容器的出口端连接的管路内。
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