Microbial Enrichment Liquid Flow Control System and Method

Abstract

The present invention discloses a microbial enrichment liquid flow control system, belonging to the field of water area microbial analysis. The exhaust branch can discharge the air in the first liquid storage bag, the sampling branch can extract samples into the first liquid storage bag, the staining branch can extract staining liquid to stain the samples in the first liquid storage bag, the liquid supply branch is connected to the inlet of the microfluidic chip and pumps the stained samples in the first liquid storage bag to the microfluidic chip, the waste liquid discharge branch is communicated with the waste liquid port of the microfluidic chip to discharge the waste liquid of the microfluidic chip; the collector is communicated with the sorting outlet of the microfluidic chip and collects the microorganisms sorted by the microfluidic chip. The cleaning liquid assembly, the fifth pump and the collector form a cleaning branch to wash the microorganisms collected on the collector; the cleaning liquid assembly, the fifth pump and the collector can also form a transfer branch to transfer the microorganisms from the collector to the storage end outlet. The present application also relates to a microbial enrichment liquid flow control method implemented by using the above control system.

Description

Technical Field

[0001] The present invention relates to the field of water microbial analysis, and particularly to a microbial enrichment liquid flow control system and method. Background Art

[0002] A large number of documents show that one of the most commonly used research methods for studying the microbial ecology in lakes or oceans is based on sampling, analyzing the microbial population at the sampling time point, or bringing the sample back to the laboratory for enrichment culture and analysis through simulation techniques. For example, patents CN202022765498 or CN202021014863 disclose water microbial collectors, that is, directly collecting and storing water samples in water. After the water samples are collected, they are generally directly stored anaerobically in cold storage, but the in-situ pressure cannot be guaranteed during the processes before high-pressure culture, such as in-situ fidelity sampling, sample preservation and transfer, and laboratory sample segmentation. Especially, the period from sampling on a scientific research ship to culturing is relatively long, and many piezophilic microorganisms die during this period and cannot be isolated, which also seriously affects the analysis of the in-situ ecosystem.

[0003] Microfluidic chip technology can integrate basic operation units such as sample preparation, reaction, separation, and detection in the biological, chemical, and medical analysis processes onto a chip with a micron scale, and can complete the whole process of analysis. The acoustic tweezer technology based on the principle that particles move towards the nodes in a standing wave sound field has the function of fixing the particle position and can realize the focusing and sorting functions of particles in a fluid. For example, the microfluidic chips in the publicly disclosed patents CN201911348149 and CN201910493156, although the chips have the functions of analyzing and enriching microorganisms, the external liquid flow system supporting the microfluidic chips generally adopts manual control or semi-automatic control, and it is difficult to complete the long-term in-situ analysis and enrichment of microorganisms in lakes or oceans. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, one of the purposes of the present invention is to provide an automated liquid flow control system based on a microfluidic chip, which includes full-process control such as bubble removal, quantitative sampling, biological reagent staining, analysis and sorting, sample cleaning, and sample storage for water samples in lakes or oceans.

[0005] In order to overcome the deficiencies of the prior art, the second purpose of the present invention is to provide an automated liquid flow control method based on a microfluidic chip, which includes full-process control such as bubble removal, quantitative sampling, biological reagent staining, analysis and sorting, sample cleaning, and sample storage for water samples in lakes or oceans.

[0006] One of the purposes of the present invention is achieved by adopting the following technical solutions:

[0007] A microbial enrichment liquid flow control system includes a microfluidic chip, a front-end liquid flow structure, and a rear-end liquid flow structure. The front-end liquid flow structure includes a first liquid storage bag, a sampling branch, a staining branch, a liquid supply branch, and an exhaust branch. The sampling branch, the staining branch, the liquid supply branch, and the exhaust branch are respectively connected to the first liquid storage bag. The exhaust branch can discharge the air in the first liquid storage bag. The sampling branch can extract a sample into the first liquid storage bag. The staining branch can extract a staining solution to stain the sample in the first liquid storage bag. The liquid supply branch is connected to the inlet of the microfluidic chip and pumps the stained sample in the first liquid storage bag into the microfluidic chip. The rear-end liquid flow structure further includes a waste liquid discharge branch, a collector, a fifth pump, and a cleaning liquid assembly. The waste liquid discharge branch is connected to the waste liquid port of the microfluidic chip to discharge the waste liquid of the microfluidic chip. The collector is connected to the sorting outlet of the microfluidic chip and collects the microorganisms sorted by the microfluidic chip. The cleaning liquid assembly, the fifth pump, and the collector form a cleaning branch to wash the microorganisms collected on the collector. The cleaning liquid assembly, the fifth pump, and the collector can also form a transfer branch. The water flow in the transfer branch in the collector is opposite to the water flow in the cleaning branch in the collector. The transfer branch transfers the microorganisms from the collector to the storage end outlet.

[0008] Further, the sampling branch includes a pre-filter, a first pump, and a first two-way valve connected in sequence through pipelines. The front end of the pre-filter is a sample inlet, and the end of the first two-way valve is connected to the first liquid storage bag.

[0009] Further, the sampling branch further includes a multi-stage filter, and the multi-stage filter is located between the pre-filter and the first pump.

[0010] Further, the staining branch includes a second two-way valve, a second pump, and a second liquid storage bag connected in sequence through pipelines. The second liquid storage bag stores a staining agent, and the second two-way valve is connected to the first liquid storage bag.

[0011] Further, the liquid supply branch includes a first steering valve and a third pump. The first steering valve is respectively connected to the first liquid storage bag, the third pump, and the microfluidic chip. The third pump is an injection pump, and the third pump can extract the sample in the first liquid storage bag and send the sample to the microfluidic chip.

[0012] Further, the waste liquid discharge branch includes a first one-way valve and a three-way joint connected to the first one-way valve. The first one-way valve is connected to the waste liquid port of the microfluidic chip.

[0013] Further, the backend liquid flow structure further includes a second steering valve, a third steering valve, a fourth steering valve, a fifth steering valve, and a third one-way valve. The second steering valve is respectively connected to the sorting outlet of the microfluidic chip, the collector inlet, and the fourth steering valve. The third steering valve is connected to the collector outlet, the three-way joint, the fifth steering valve, and the cleaning liquid assembly. The fourth steering valve is connected to the second steering valve, the third one-way valve, and the fifth pump. The fifth steering valve is connected to the collector inlet, the fifth pump, and the cleaning liquid assembly.

[0014] Further, the microbial enrichment liquid flow control system further includes a housing, which is a cage structure. The housing includes a top cover and a plurality of connectors provided on the top cover. The plurality of connectors are respectively connected to the sample inlet of the sampling branch, the exhaust end of the exhaust branch, the waste liquid end outlet of the waste liquid discharge branch, and the storage end outlet of the transfer branch.

[0015] The second object of the present invention is achieved by the following technical solutions:

[0016] A microbial enrichment liquid flow control method implemented by using the above microbial enrichment liquid flow control system includes the following steps:

[0017] Bubble removal: Remove the air from the microbial enrichment liquid flow control system;

[0018] Quantitative sampling: Open the sampling branch, and extract a quantitative sample from the research water area to the first liquid storage bag;

[0019] Biological reagent staining: Open the staining branch, extract the staining reagent from the second liquid storage bag to the first liquid storage bag, and stain and incubate the microorganisms in the sample;

[0020] Analysis and sorting: Open the liquid supply branch, transport the sample after staining and incubation in the first liquid storage bag to the microfluidic chip. The microfluidic chip sorts the sample and transports the sorted sample to the collector, and the waste liquid is discharged through the waste liquid discharge branch;

[0021] Sample cleaning: Open the cleaning branch, and the fifth pump extracts the cleaning liquid and enters the collector from the collector inlet to clean the sample on the collector;

[0022] Sample storage: Open the transfer branch, the fifth pump extracts the cleaning liquid and enters the collector from the collector outlet, and leaves the collector from the collector outlet, and transfers the microbial sample on the collector to the storage end outlet.

[0023] Further, the bubble removal includes the following steps:

[0024] Remove bubbles from the sampling branch;

[0025] Bubble exhaust for the dyeing branch

[0026] Bubble exhaust for the waste liquid discharge branch

[0027] Bubble exhaust for the cleaning branch

[0028] Bubble exhaust for the transfer branch

[0029] Bubble exhaust for the first liquid storage bag

[0030] Compared with the prior art, the microbial enrichment liquid flow control system of the present invention can achieve full-process and automated control such as bubble exhaust, quantitative sampling, biological reagent dyeing, analysis and sorting, sample cleaning, and sample storage during the process of sorting water samples from lakes or oceans by a microfluidic chip, and can complete long-term in-situ analysis and enrichment of microorganisms in lakes or oceans. Description of the Drawings

[0031] Figure 1 It is a schematic structural diagram of the first embodiment of the microbial enrichment liquid flow control system of the present invention;

[0032] Figure 2 It is Figure 1 a schematic diagram of the front-end liquid flow structure of the microbial enrichment liquid flow control system of

[0033] Figure 3 It is Figure 1 a schematic diagram of the rear-end liquid flow structure of the microbial enrichment liquid flow control system of

[0034] Figure 4 It is a three-dimensional diagram of the microbial enrichment liquid flow control system of the present invention;

[0035] Figure 5 It is a schematic structural diagram of the second embodiment of the microbial enrichment liquid flow control system of the present invention.

[0036] In the figure: 10, front-end liquid flow structure; 11, first liquid storage bag; 12, sampling branch; 120, pre-filter; 121, multi-stage filter; 122, first pump; 123, first two-way valve; 13, dyeing branch; 130, second two-way valve; 131, second pump; 132, second liquid storage bag; 14, liquid supply branch; 140, first steering valve; 141, third pump; 15, exhaust branch; 150, third two-way valve; 151, fourth pump; 20, microfluidic chip; 30, rear-end liquid flow structure; 31, waste liquid discharge branch; 310, first one-way valve; 311, three-way joint; 32, collector; 33, cleaning liquid assembly; 330, cleaning liquid storage bag; 331, second one-way valve; 34, fifth pump; 35, third one-way valve; 36, second steering valve; 37, third steering valve; 38, fourth steering valve; 39, fifth steering valve; 40, housing; 41, top cover; 42, joint. Detailed implementation manners

[0037] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0038] It should be noted that when a component is referred to as being "fixed to" another component, it can be directly on the other component or there can also be another intermediate component through which it is fixed. When a component is considered to be "connected" to another component, it can be directly connected to the other component or there may be another intermediate component at the same time. When a component is considered to be "disposed on" another component, it can be directly disposed on the other component or there may be another intermediate component at the same time. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0040] As Figures 1 to 4 shown, it is the first embodiment of the microbial enrichment liquid flow control system of the present invention, including a front-end liquid flow structure 10, a microfluidic chip 20, and a rear-end liquid flow structure 30.

[0041] The front-end liquid flow structure 10 includes a first liquid storage bag 11, a sampling branch 12, a staining branch 13, a liquid supply branch 14, and an exhaust branch 15.

[0042] The sampling branch 12 is used to extract a cleaning liquid (clean water in this embodiment) or a sample of the research water area (water in a lake or ocean in this embodiment). The sampling branch 12 includes a pre-filter 120, a multi-stage filter 121, a first pump 122, and a first two-way valve 123 connected in sequence through pipelines. The inlet of the pre-filter 120 is the sample inlet. The outlet of the first two-way valve 123 is connected to the first liquid storage bag 11. The pre-filter 120 uses a stainless steel or nylon or polytetrafluoroethylene (PTFE) material filter screen with a pore size of 0.5 - 2 mm.

[0043] The dyeing branch 13 is used to add a dyeing agent to the first liquid storage bag 11 to dye the sample. The dyeing branch 13 includes a second two-way valve 130, a second pump 131, and a second liquid storage bag 132 that are connected in sequence through pipelines. The second liquid storage bag 132 stores the dyeing agent, and SYBR Green I can be used as the dyeing agent for measuring heterotrophic bacteria. The specific measurement method can refer to Patent CN201410171582.X. The outlet of the second two-way valve 130 is connected to the first liquid storage bag 11.

[0044] The liquid supply branch 14 is used to extract the sample in the first liquid storage bag 11 and send the sample to the microfluidic chip 20. The liquid supply branch 14 includes a first steering valve 140 and a third pump 141. The first steering valve 140 is respectively connected to the first liquid storage bag 11, the third pump 141, and the microfluidic chip 20. The third pump 141 is an injection pump, and the third pump 141 can extract the sample in the first liquid storage bag 11 and store it briefly, and then send the sample to the microfluidic chip 20.

[0045] The exhaust branch 15 is used to discharge the air in the first liquid storage bag 11. The exhaust branch 15 includes a third two-way valve 150 and a fourth pump 151. The third two-way valve 150 and the fourth pump 151 are connected through a pipeline. The other end of the third two-way valve 150 is connected to the first liquid storage bag 11, and an exhaust end is provided at the outlet of the fourth pump 151.

[0046] The structure of the microfluidic chip 20 is as shown in the publicly disclosed patents CN201911348149 and CN201910493156. The acoustic tweezer technology based on the principle that particles move towards the nodes in a standing wave sound field has the function of fixing the particle position and can realize the functions of focusing and sorting particles in a fluid.

[0047] The rear-end liquid flow structure 30 includes a waste liquid discharge branch 31, a collector 32, a cleaning liquid assembly 33, a fifth pump 34, a third one-way valve 35, a second steering valve 36, a third steering valve 37, a fourth steering valve 38, and a fifth steering valve 39.

[0048] The waste liquid discharge branch 31 includes a first one-way valve 310 and a three-way joint 311 connected to the first one-way valve 310. The first one-way valve 310 is connected to the waste liquid port of the microfluidic chip 20. The end of the three-way joint 311 is the waste liquid end outlet.

[0049] The collector 32 uses a stainless steel or nylon or polytetrafluoroethylene (PTFE) material filter membrane with a pore size of 0.2 - 5 μm.

[0050] The cleaning liquid assembly 33 is used to provide a water source for cleaning microorganisms and a medium for transferring and storing microorganisms. The cleaning liquid assembly 33 includes a cleaning liquid storage bag 330 and a second one-way valve 331 communicating with the cleaning liquid storage bag 330. The cleaning liquid storage bag 330 can place a buffer solution as the cleaning liquid according to actual application requirements, such as phosphate buffered saline (PBS). In the sample cleaning process of Embodiment 1, it is used to clean each pipeline, pipe joint or steering valve before and after the collector 32, so that the target microorganisms sorted in the collector 32 are suspended in the buffer solution. The second one-way valve 331 also communicates with the third steering valve 37 and the fourth steering valve 38.

[0051] The fifth pump 34 is used to provide power for cleaning microorganisms and transferring and storing microorganisms. Both ends of the fifth pump 34 are respectively connected to the fourth steering valve 38 and the fifth steering valve 39.

[0052] The third one-way valve 35 is connected to the fourth steering valve 38, and the end of the third one-way valve 35 is the storage end outlet.

[0053] The second steering valve 36 is respectively communicated with the sorting outlet of the microfluidic chip 20, the inlet of the collector 32 and the fourth steering valve 38. The third steering valve 37 is communicated with the outlet of the collector 32, the three-way joint 42311, the fifth steering valve 39 and the cleaning liquid assembly 33. The fourth steering valve 38 is communicated with the second steering valve 36, the third one-way valve 35 and the fifth pump 34. The fifth steering valve 39 is communicated with the inlet of the collector 32, the fifth pump 34 and the cleaning liquid assembly 33.

[0054] The pipelines used for connection in the microorganism enrichment liquid flow control system are made of polyether ether ketone (PEEK) or polytetrafluoroethylene (PTFE). The outer diameter of the pipeline is 1.6 - 3.2 mm, and the inner diameter is 0.2 - 1.0 mm.

[0055] The first liquid storage bag 11, the second liquid storage bag 132 and the cleaning liquid storage bag 330 have good tensile properties and biocompatibility, and the material is preferably polyvinyl chloride (PVC) or polypropylene (PP).

[0056] The first pump 122, the second pump 131, the third pump 141, the fourth pump 151 and the fifth pump 34 adopt constant flow pumps such as syringe pumps, peristaltic pumps or diaphragm pumps.

[0057] The front-end liquid flow structure 10, the microfluidic chip 20 and the rear-end liquid flow structure 30 are installed inside the housing 40. The housing 40 is a cage structure. The housing 40 includes a top cover 41 and several connectors 42 arranged on the top cover 41. The several connectors 42 are respectively communicated with the sample inlet of the sampling branch 12, the exhaust end of the exhaust branch 15, the waste liquid end outlet of the waste liquid discharge branch 31 and the storage end outlet of the transfer branch.

[0058] When using the microbial enrichment liquid flow control system of the present invention, exhaust gas is carried out first.

[0059] The exhaust gas step is specifically as follows:

[0060] (1) The first pump 122 and the first two-way valve 123 are opened, and the rest of the valves and pumps are closed. According to specific needs, it runs for a period of time; a certain volume of water sample is extracted from the sample inlet to remove the air in each pump, valve or pipeline on the left side of the first liquid storage bag 11 (sampling branch 12), and prepare for filling the pumps, valves or pipelines behind to remove the air therein.

[0061] (2) The first pump 122, the first two-way valve 123, the second two-way valve 130 and the second pump 131 are opened, and the rest of the valves and pumps are closed. Water samples are respectively extracted from the sample inlet and the second liquid storage bag 132 into the first liquid storage bag 11 to remove the air in each pump, valve or pipeline on the lower side of the first liquid storage bag 11 (dyeing branch 13).

[0062] (3) The third pump 141 starts sampling, and the other two-way valves, steering valves or pumps are closed; the third pump 141 extracts a certain amount of water sample from the first liquid storage bag 11 to prepare for exhausting air later.

[0063] (4) The first steering valve 140 and the third pump 141 start sampling, and the other two-way valves, steering valves or pumps are closed; the third pump 141 is used to make the water sample flow through the microfluidic chip 20, the first one-way valve 310, the three-way joint 311, the second steering valve 36, the collector 32, the third steering valve 37 and the air in the pipelines between these devices, and discharge it at the waste liquid end outlet.

[0064] (5) The second steering valve 36 and the fifth pump 34 are opened, and the other two-way valves, steering valves or pumps are closed; a water sample is extracted from the cleaning liquid storage bag 330 to remove the air in the cleaning pipeline and discharge it at the waste liquid end outlet.

[0065] (6) The fifth pump 34, the second steering valve 36, the third steering valve 37, the fourth steering valve 38 and the fifth steering valve 39 are opened, and the other two-way valves, steering valves or pumps are closed; a water sample is extracted from the cleaning liquid storage bag 330 to remove the air in other devices and pipelines in the rear-end liquid flow structure 30 (transfer branch) and discharge it at the storage end outlet.

[0066] (7) The third two-way valve 150 and the fourth pump 151 are opened, and the other two-way valves, steering valves or pumps are closed; it is mainly used to remove the air in the first liquid storage bag 11.

[0067] (8) All two-way valves, steering valves or pumps are closed.

[0068] In the exhaust step, it is determined whether the exhaust of bubbles is completed mainly by observing whether there is a sample flowing out of the exhaust port and no more bubbles flowing out as the judgment criterion. For automated judgment, a bubble detector can be connected to the rear end of the exhaust port. If there are no bubbles within a certain period of time, it can be determined that the process of exhausting bubbles is completed.

[0069] Perform the quantitative sampling step:

[0070] The first pump 122 and the first two-way valve 123 are opened, and the rest of the valves and pumps are closed. According to actual application requirements, a sample is quantitatively extracted, and the water sample to be tested is extracted from a lake or ocean into the first liquid storage bag 11.

[0071] Biological reagent staining step:

[0072] (1) The second two-way valve 130 and the second pump 131 are opened, and the rest of the valves and pumps are closed. According to actual application requirements, a dye is quantitatively extracted; the pre-stored dye is extracted from the second liquid storage bag 132 into the first liquid storage bag 11.

[0073] (2) All two-way valves or steering valves or pumps are closed. According to actual application requirements, the microorganisms in the sample are stained and incubated.

[0074] Analysis and sorting step:

[0075] (1) The third pump 141 starts sampling, and the other two-way valves or steering valves or pumps are closed; the third pump 141 extracts a certain amount of sample from the first liquid storage bag 11;

[0076] (2) The first steering valve 140 and the third pump 141 start sample injection, and the other two-way valves or steering valves or pumps are closed; the sample extracted by the third pump 141 is injected into the microfluidic chip 20 for microorganism analysis or sorting, and the waste liquid after detection is discharged from the waste liquid end outlet.

[0077] (3) According to actual application requirements, the processes of (1) and (2) are cycled.

[0078] Sample cleaning step:

[0079] The fifth pump 34 and the second steering valve 36 are opened, and the other two-way valves or steering valves or pumps are closed. The pre-stored specific buffer solution is extracted from the cleaning liquid storage bag 330 to clean the sample sorted in the collector 32 (mainly for ocean samples). The specific microorganisms sorted are intercepted in front of the filter screen in the collector 32, and the waste liquid is discharged from the waste liquid end outlet.

[0080] Sample storage step:

[0081] (1) The fifth pump 34, the second steering valve 36, the third steering valve 37, the fourth steering valve 38, and the fifth steering valve 39 are opened, and the other two-way valves, steering valves, or pumps are all closed; the pre-stored specific buffer solution is extracted from the cleaning liquid storage bag 330, and the microorganisms in front of the filter screen in the collector 32 are transferred to the storage end outlet.

[0082] (2) All two-way valves, steering valves, or pumps are closed.

[0083] Please continue to refer to Figure 5 , which is the second embodiment of the present invention. In this embodiment, the structure of the microorganism enrichment liquid flow control system of the present invention is the same as that of the first embodiment. The difference is that in this embodiment, the cleaning liquid assembly 33 does not include the cleaning liquid storage bag 330, and the second one-way valve 331 is communicated with the outlet of the multi-stage filter 121, so that the sampling branch 12 can provide buffer solution to the cleaning branch and the transfer branch.

[0084] Please continue to refer to Figure 5 , the present invention also relates to a microorganism enrichment liquid flow control method implemented by using the above microorganism enrichment liquid flow control system, including the following steps:

[0085] Air bubble removal: Remove the air in the microorganism enrichment liquid flow control system;

[0086] Quantitative sampling: The sampling branch 12 is opened, and a quantitative sample is extracted from the research water area to the first liquid storage bag 11;

[0087] Biological reagent staining: The staining branch 13 is opened, and the staining reagent is extracted from the second liquid storage bag 132 to the first liquid storage bag 11 to stain and incubate the microorganisms in the sample;

[0088] Analysis and sorting: The liquid supply branch 14 is opened, and the sample after staining and incubation in the first liquid storage bag 11 is transported to the microfluidic chip 20. The microfluidic chip 20 sorts the sample and transports the sorted sample to the collector 32, and the waste liquid is discharged through the waste liquid discharge branch 31;

[0089] Sample cleaning: The cleaning branch is opened, and the fifth pump 34 extracts the cleaning liquid and enters the collector 32 from the inlet of the collector 32 to clean the sample on the collector 32;

[0090] Sample storage: The transfer branch is opened, and the fifth pump 34 extracts the cleaning liquid and enters the collector 32 from the outlet of the collector 32 and leaves the collector 32 from the outlet of the collector 32, and transfers the microbial sample on the collector 32 to the storage end outlet.

[0091] Furthermore, the air bubble removal includes the following steps:

[0092] Air bubble removal from the sampling branch 12;

[0093] The dyeing branch 13 discharges air bubbles;

[0094] The waste liquid discharge branch 31 discharges air bubbles;

[0095] The cleaning branch discharges air bubbles;

[0096] The transfer branch discharges air bubbles;

[0097] The first liquid storage bag 11 discharges air bubbles.

[0098] The present invention can overcome the problem in the prior art that microbial analysis and enrichment cannot be carried out in situ, and provides an automated liquid flow control system based on a microfluidic chip, which includes full-process control such as air bubble discharging, quantitative sampling, biological reagent dyeing, analysis and sorting, sample cleaning, and sample storage for water samples in lakes or oceans. The liquid flow control system of the present invention can realize in-situ microbial analysis and enrichment of existing microfluidic chips in lake or ocean environments. In addition, the liquid flow system of the present invention does not need to carry additional sheath fluid or buffer solution, which is beneficial to the long-term underwater operation of the system.

[0099] The above embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made. These are all equivalent modifications and evolutions based on the essence of the present invention to the above embodiments, and all of these belong to the protection scope of the present invention.

Claims

1. A microbial enrichment liquid flow control system, including a microfluidic chip, characterized in that: It also includes a front-end liquid flow structure and a rear-end liquid flow structure. The front-end liquid flow structure includes a first liquid storage bag, a sampling branch, a staining branch, a liquid supply branch, and an exhaust branch. The sampling branch, the staining branch, the liquid supply branch, and the exhaust branch are respectively connected to the first liquid storage bag. The exhaust branch can discharge the air in the first liquid storage bag. The sampling branch can extract a sample into the first liquid storage bag. The staining branch can extract a staining solution to stain the sample in the first liquid storage bag. The liquid supply branch is connected to the inlet of the microfluidic chip and pumps the stained sample in the first liquid storage bag to the microfluidic chip. The rear-end liquid flow structure further includes a waste liquid discharge branch, a collector, a fifth pump, a second switching valve, a third switching valve, a fourth switching valve, a fifth switching valve, a third one-way valve, and a cleaning liquid assembly. The waste liquid discharge branch includes a first one-way valve and a tee joint communicated with the first one-way valve. The first one-way valve is communicated with the waste liquid port of the microfluidic chip to discharge the waste liquid of the microfluidic chip. The second switching valve is respectively communicated with the sorting outlet of the microfluidic chip, the inlet of the collector, and the fourth switching valve. The third switching valve is communicated with the outlet of the collector, the tee joint, the fifth switching valve, and the cleaning liquid assembly. The fourth switching valve is communicated with the second switching valve, the third one-way valve, and the fifth pump. The fifth switching valve is communicated with the inlet of the collector, the fifth pump, and the cleaning liquid assembly. The collector is communicated with the sorting outlet of the microfluidic chip and collects the microorganisms sorted by the microfluidic chip. The cleaning liquid assembly, the fifth pump, and the collector form a cleaning branch to wash the microorganisms collected on the collector; The cleaning liquid assembly, the fifth pump, and the collector can also form a transfer branch. The water flow in the collector of the transfer branch is opposite to the water flow in the collector of the cleaning branch. The transfer branch transfers the microorganisms from the collector to the storage end outlet.

2. The microbial enrichment liquid flow control system according to claim 1, wherein: The sampling branch includes a pre-filter, a first pump, and a first two-way valve connected in sequence through pipelines. The front end of the pre-filter is the sample inlet, and the end of the first two-way valve is connected to the first liquid storage bag.

3. The microbial enrichment liquid flow control system according to claim 2, wherein: The sampling branch further includes a multi-stage filter, and the multi-stage filter is located between the pre-filter and the first pump.

4. The microbial enrichment liquid flow control system according to claim 1, wherein: The staining branch includes a second two-way valve, a second pump, and a second liquid storage bag connected in sequence through pipelines. The second liquid storage bag stores the staining agent, and the second two-way valve is connected to the first liquid storage bag.

5. The microbial enrichment liquid flow control system according to claim 1, wherein: The liquid supply branch includes a first switching valve and a third pump. The first switching valve is respectively connected to the first liquid storage bag, the third pump, and the microfluidic chip. The third pump is an injection pump, and the third pump can extract the sample in the first liquid storage bag and send the sample to the microfluidic chip.

6. The microbial enrichment liquid flow control system according to claim 1, wherein: The microbial enrichment liquid flow control system further includes a housing, which is a cage structure. The housing includes a top cover and a plurality of connectors provided on the top cover. The plurality of connectors are respectively communicated with the sample inlet of the sampling branch, the exhaust end of the exhaust branch, the waste liquid outlet of the waste liquid discharge branch, and the storage end outlet of the transfer branch.

7. A method for controlling the liquid flow of microbial enrichment solution implemented by using the microbial enrichment liquid flow control system according to any one of claims 1-6, characterized in that, It includes the following steps: Removing air bubbles: removing the air in the microbial enrichment liquid flow control system; Quantitative sampling: opening the sampling branch, and extracting a quantitative sample from the research water area to the first liquid storage bag; Biological reagent staining: opening the staining branch, extracting the staining reagent from the second liquid storage bag to the first liquid storage bag, and staining and incubating the microorganisms in the sample; Analysis and sorting: opening the liquid supply branch, transporting the sample after staining and incubation in the first liquid storage bag to the microfluidic chip. The microfluidic chip sorts the sample and transports the sorted sample to the collector, and the waste liquid is discharged through the waste liquid discharge branch; Sample cleaning: opening the cleaning branch, the fifth pump extracts the cleaning liquid and enters the collector from the collector inlet to clean the sample on the collector; Sample storage: opening the transfer branch, the fifth pump extracts the cleaning liquid and enters the collector from the collector outlet and leaves the collector from the collector outlet, and transfers the microbial sample on the collector to the storage end outlet.

8. The method for controlling the flow of the microbial enrichment solution according to claim 7, wherein: The step of removing air bubbles includes the following steps: Removing air bubbles from the sampling branch; Removing air bubbles from the staining branch; Removing air bubbles from the waste liquid discharge branch; Removing air bubbles from the cleaning branch; Removing air bubbles from the transfer branch; Removing air bubbles from the first liquid storage bag.

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