Precise liquid treatment system and method based on multi-channel liquid distribution valve and gas replacement

Abstract

The invention discloses a precise liquid treatment system and method based on a multi-channel liquid separation valve and gas replacement, and belongs to the technical field of biomedical detection equipment. The system comprises a multi-channel liquid distribution valve and an intelligent control system, the liquid distribution valve is provided with a plurality of channels which are respectively connected with four groups of coloring agent bottles, sheath liquid bottles, waste liquid bottles and gas bottles; a liquid level detection sensor is arranged between the liquid distribution valve and the peristaltic pump, and an outlet of the peristaltic pump is communicated with the dyeing pool. The method comprises the steps of cancer cell acquisition, fixed permeation treatment, system pre-filling, multiple fluorescent staining and fluorescent imaging. High-precision distribution of micro-liquid is realized through cooperative control of liquid separation valve channel switching and inert gas replacement, and high-precision and high-repeatability distribution of micro-upgrading liquid can be realized; a gas isolation mechanism significantly reduces the risk of cross contamination, multi-channel parallel processing improves the detection efficiency, and the device is suitable for circulating tumor cell multi-staining, flow analysis and other biomedical scenes needing high-precision liquid treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical detection equipment, and particularly relates to a precise liquid processing system and method based on a multi-channel dispensing valve and gas replacement. The system is particularly suitable for detection scenarios that require high-precision micro-liquid dispensing, such as circulating tumor cell immunostaining, flow cytometry analysis, etc. BACKGROUND

[0002] In the field of biomedical detection, the accurate dispensing of staining agents and sheath fluid is a key factor in ensuring the reliability of detection results. Traditional liquid dispensing systems face the following technical bottlenecks: 1) insufficient dispensing accuracy of micro-liquid (microliter level), making it difficult to meet the needs of high-sensitivity detection; 2) cross-contamination problems caused by pipeline residue are difficult to completely solve; 3) bubbles seriously affect the repeatability and stability of dispensing; 4) liquid boundaries are blurred during multi-channel switching, affecting detection accuracy.

[0003] Although the double-pump compensation system, microfluidic chip, and gas-liquid isolation technology in existing solutions can improve the performance of liquid dispensing to some extent, they still have obvious limitations in actual clinical application scenarios: the double-pump compensation system is complex in structure and has high maintenance costs; the microfluidic chip is prone to clogging, has high manufacturing costs, and is difficult to implement multi-reagent parallel processing; and the traditional gas-liquid isolation technology faces problems such as high gas consumption and insufficient interface control precision. Therefore, it is imperative to develop a new type of liquid dispensing solution that combines high-precision dispensing capability and multi-channel processing advantages. SUMMARY

[0004] The present application provides a precise liquid processing system and method based on a multi-channel dispensing valve and gas replacement, aiming to solve the technical problems of micro-liquid dispensing in the field of biomedical detection. The system innovatively integrates multi-channel fluid control and gas replacement technology, achieving high-precision and high-repeatability dispensing of microliter-level liquid.

[0005] A precise liquid processing system based on a multi-channel dispensing valve and gas replacement includes a multi-channel dispensing valve and an intelligent control system. The dispensing valve is a multi-channel rotary valve with a total of eight channels. A first staining agent bottle is connected to a first channel, a second staining agent bottle is connected to a second channel, a third staining agent bottle is connected to a third channel, a fourth staining agent bottle is connected to a fourth channel, a sheath fluid bottle is connected to a fifth channel, a waste liquid bottle is connected to a sixth channel, a gas bottle is connected to a seventh channel, a liquid level detection sensor is arranged on the pipeline between the eighth channel and the peristaltic pump, and the outlet of the peristaltic pump is connected to a staining pool.

[0006] Further, the gas bottle contains inert gas.

[0007] Further, the inert gas is high-purity nitrogen.

[0008] Further, the application also provides a multi-channel liquid distribution valve and a gas-replaced precision liquid processing method, comprising the following steps: S1: obtaining cancer cells; S2: cancer cell fixation treatment and permeation treatment; S3: system pre-filling treatment; S4: multi-fluorescence staining; S5: cancer cell fluorescence imaging.

[0009] Further, step S1 is specifically as follows: S11: aspirating old culture medium in a cancer cell culture bottle.

[0010] S12: adding 2-3 mL PBS buffer to rinse the cells and aspirating the PBS.

[0011] S13: adding 1-2 mL 0.25% trypsin to cover the bottom of the bottle, standing at room temperature or 37°C for 1-2 minutes, and gently tapping the bottle wall to promote cell shedding.

[0012] S14: adding complete culture medium containing serum to terminate the digestion reaction and gently blowing to completely suspend the cells.

[0013] S15: centrifuging the cell suspension and discarding the supernatant.

[0014] S16: resuspending the cell pellet with fresh culture medium, adjusting the cell concentration to 1×10 6 -5×10 6 cells / mL, and the viable cell rate is >90%.

[0015] Further, step S2 is specifically as follows: S21: preparing a sterile 6-well plate and selecting one well to add 1-2 mL of culture medium.

[0016] S22: aspirating 10-20 μL of cancer cell suspension obtained in step S1 and adding it to the culture medium in the well.

[0017] S23: incubating in a cell culture incubator overnight to allow the cells to adhere.

[0018] S24: discarding the culture medium in the well.

[0019] S25: adding 1-2 mL of PBS to rinse the cells and aspirating the PBS. Repeat this rinsing step once.

[0020] S26: adding 1-2 mL of 4% paraformaldehyde solution, fixing at room temperature for 15-20 minutes, and discarding the fixing solution.

[0021] S27: rinsing the cells with PBS buffer 2-3 times.

[0022] S28: Add 1-2 mL of 0.2% Triton X-100 solution, incubate at room temperature for 10-15 minutes for permeation, and discard the permeation liquid.

[0023] S29: Rinse the cells with PBS buffer for 3 times.

[0024] S210: Add 1-2 mL of 5% BSA solution, block at room temperature for 1 hour, after blocking, absorb the BSA solution, and rinse once with PBS for standby.

[0025] Further, step S3 is specifically: S31: Add the first staining agent, the second staining agent, the third staining agent, the fourth staining agent, and the sheath liquid into the first staining agent bottle, the second staining agent bottle, the third staining agent bottle, the fourth staining agent bottle, and the sheath liquid bottle respectively, and the sheath liquid can be PBS.

[0026] S32: Start the intelligent control system, switch the dispensing valve to the seventh channel to open, and blow the pipeline with 0.3-0.5 bar high-purity nitrogen for 30-60 seconds, and the waste liquid is discharged to the waste liquid bottle through the sixth channel.

[0027] S33: Switch the dispensing valve to the first channel, the second channel, the third channel, the fourth channel, and the fifth channel in turn to open. The peristaltic pump extracts the corresponding liquid at a flow rate of 5-10 mL / min. When the high-sensitivity liquid level detection sensor detects that the liquid surface reaches the predetermined position, the pump stops automatically, ensuring that the pipeline is full and free of bubbles.

[0028] S34: Switch the dispensing valve to the sixth channel to open, and drive the peristaltic pump in reverse to discharge excess liquid in the pipeline.

[0029] Further, step S4 is specifically: S41: Stain and wash with the first staining agent: Switch the dispensing valve to the first channel to open, and the peristaltic pump extracts 300-600 μL of the first staining agent from the first staining agent bottle, then immediately switch the dispensing valve to the seventh channel to open, and push the gas at a flow rate of 5-10 μL / s to inject the first staining agent into the cancer cell culture well in S2 which has been fixed and treated, and then incubate at room temperature for 30-60 minutes in the dark; switch the dispensing valve to the fifth channel to open, and the peristaltic pump extracts 500-1000 μL of PBS sheath liquid to flush the incubated cancer cells, and after standing for 1-2 minutes, switch the dispensing valve to the sixth channel to open, and discharge the waste liquid to the waste liquid bottle. Repeat this washing process 1-2 times.

[0030] S42: Stain and wash with the second staining agent: The dispensing valve is switched to the second channel to open, the peristaltic pump extracts 300-600 μL of the second staining agent from the second staining agent bottle, and then immediately switches the dispensing valve to the seventh channel to open, pushes in the gas at a flow rate of 5-10 μL / s, injects the second staining agent into the culture hole, and then incubates at room temperature for 15-30 minutes in the dark; the dispensing valve is switched to the fifth channel to open, the peristaltic pump extracts 500-1000 μL of PBS sheath liquid to flush the incubated cancer cells, and after standing for 1-2 minutes, the dispensing valve is switched to the sixth channel to open, and the waste liquid is discharged into the waste liquid bottle. Repeat this washing process 2-3 times.

[0031] S43: Staining with a third staining agent and washing: The dispensing valve is switched to the third channel to open, the peristaltic pump extracts 100-200 μL of the third staining agent from the third staining agent bottle, and then immediately switches the dispensing valve to the seventh channel to open, pushes in the gas at a flow rate of 5-10 μL / s, injects the third staining agent into the culture hole, and then incubates at room temperature for 10-15 minutes in the dark; the dispensing valve is switched to the fifth channel to open, the peristaltic pump extracts 500-1000 μL of PBS sheath liquid to flush the incubated cancer cells, and after standing for 1-2 minutes, the dispensing valve is switched to the sixth channel to open, and the waste liquid is discharged into the waste liquid bottle. Repeat this washing process 2-3 times.

[0032] S44: Staining with a fourth staining agent and washing: The dispensing valve is switched to the fourth channel to open, the peristaltic pump extracts 100-200 μL of the fourth staining agent from the fourth staining agent bottle, and then immediately switches the dispensing valve to the seventh channel to open, pushes in the gas at a flow rate of 5-10 μL / s, injects the fourth staining agent into the culture hole, and then incubates at room temperature for 5-10 minutes in the dark; the dispensing valve is switched to the fifth channel to open, the peristaltic pump extracts 500-1000 μL of PBS sheath liquid to flush the incubated cancer cells, and after standing for 1-2 minutes, the dispensing valve is switched to the sixth channel to open, and the waste liquid is discharged into the waste liquid bottle. Repeat this washing process 2-3 times.

[0033] Further, step S5 is specifically: S51: aspirate most of the liquid in the well.

[0034] S52: add an appropriate amount of mounting medium (containing an anti-quenching agent) to cover the cells.

[0035] S53: use a fluorescence microscope for multi-channel fluorescence imaging acquisition.

[0036] The present application has the following beneficial effects: 1. High dispensing accuracy: through the innovative multi-channel dispensing valve and gas displacement collaborative control technology, high precision and high repeatability of microliter level liquid dispensing is achieved.

[0037] 2. Anti-cross contamination: Using inert gas as a physical barrier between liquids, completely eliminating the risk of cross contamination between different reagents.

[0038] 3. High-throughput processing capability: Multi-channel parallel design and optimized workflow significantly improve work efficiency and greatly shorten the detection period.

[0039] 4. Strong versatility: The system is suitable for blood cell staining, flow cytometry analysis, multiplex immunofluorescence staining and other biomedical detection scenarios.

[0040] Through the cooperation of liquid distribution valve switching and gas displacement technology, pipeline initialization emptying and pre-filling are realized, and high-precision quantitative distribution of microliter-level liquid is completed. Gas medium not only promotes precise injection of liquid into the staining pool, but also isolates different liquids to avoid cross contamination. The system realizes high-precision, high-repeatability quantitative distribution of microliter-level liquid, solves the problems of insufficient precision, bubble interference and residual contamination in traditional liquid distribution, and the dynamic gas displacement mechanism effectively removes pipeline residues to ensure no residual interference between different staining stages. Gas isolation technology ensures strict isolation of different staining reagents (such as triple staining or quadruple staining) during transportation, significantly reducing the cross contamination rate. It has the advantages of simple structure and reliable operation. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The system structure diagram of the present application is shown in the figure; Figure 2 The initialization stage flow chart is shown in the figure; Figure 3 The sample addition stage flow chart is shown in the figure. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the present application clearer and more explicit, the technical scheme of the present application will be described in detail below in combination with the drawings and specific embodiments. It should be noted that the embodiments described herein are only used to illustrate the present application and are not a limitation of the present application. Based on the disclosed content of the present application, all equivalent embodiments or obvious modifications derived by those skilled in the art without creative labor should be covered within the protection scope of the present application.

[0043] The implementation manner and preferred embodiments of each technical scheme of the present application will be described in detail below with reference to the embodiments shown in the drawings.

[0044] Example 1: As Figure 1As shown, a multi-channel dispensing valve and gas displacement precision liquid handling system includes a multi-channel dispensing valve 1 and an intelligent control system. The dispensing valve 1 is a multi-channel rotary valve with eight channels in total. A first dye bottle 81 is connected to a first channel 11, a second dye bottle 82 is connected to a second channel 12, a third dye bottle 83 is connected to a third channel 13, a fourth dye bottle 84 is connected to a fourth channel 14, a sheath liquid bottle 7 is connected to a fifth channel 15, a waste liquid bottle 6 is connected to a sixth channel 16, a gas bottle 2 is connected to a seventh channel 17, and a liquid level detection sensor 3 is arranged on a pipeline between an eighth channel 18 and a peristaltic pump 4. An outlet of the peristaltic pump 4 is connected to a dyeing pool 5.

[0045] The intelligent control system (not shown in the figure) is used to control the switching of the seven channels of the dispensing valve 1, the adjustment of the pump speed of the peristaltic pump 4, the on-off of the gas, and the signal processing of the liquid level detection sensor 3. The gas bottle 2 contains inert gas.

[0046] The system workflow includes an initialization stage and a sample addition stage.

[0047] Initialization stage: 1) Start the intelligent control system, switch the dispensing valve to the seventh channel 17 to open, and blow the pipeline with constant pressure (such as 0.3-0.5 bar) high-purity nitrogen or ammonia for 30-60 seconds to ensure that all residual liquid is discharged into the waste liquid bottle 6 through the sixth channel 16.

[0048] 2) Switch the dispensing valve to the first channel 11, the second channel 12, the third channel 13, the fourth channel 14, and the fifth channel 15 in turn to open, and the peristaltic pump 4 extracts liquid from the corresponding bottle at a set flow rate (such as 5-10 mL / min). When the high-sensitivity liquid level detection sensor 3 detects that the liquid surface reaches a predetermined position, the pump stops automatically to ensure that the pipeline is full and free of bubbles.

[0049] 3) Switch the dispensing valve 1 to the sixth channel 16 to open, and drive the peristaltic pump 4 in reverse to discharge excess liquid in the pipeline to ensure that a continuous liquid column without bubbles is formed in each pipeline.

[0050] Sample addition stage: 1) Switch the dispensing valve 1 to the first channel 11 to open, and quantitatively extract a set volume (such as 300-600 μL) of the first dye through the peristaltic pump 4.

[0051] 2) Immediately switch the dispensing valve 1 to the seventh channel 17 to open, and push the first dye into the dyeing pool at a constant flow rate (such as 5-10 μL / s). Inert gas forms a separation zone before and after the target liquid to accurately inject it into the dyeing pool and contact the sample (such as cancer cells).

[0052] 3) The sample is treated in the dyeing pool under specific conditions (such as room temperature and light-free incubation for 30-60 minutes).

[0053] 4) Repeat steps 1-3 to sequentially push the second, third and fourth stains into the staining chamber for subsequent staining operations, as follows: Switch to the second channel 12 open, draw 300-600 μL of the second stain, immediately switch the dispensing valve 1 to the seventh channel 17 open, push the second stain into the staining chamber at a constant flow rate (such as 5-10 μL / s), incubate at room temperature for 15-30 minutes in the dark; Switch to the third channel 13 open, draw 100-200 μL of the third stain, immediately switch the dispensing valve 1 to the seventh channel 17 open, push the third stain into the staining chamber at a constant flow rate (such as 5-10 μL / s), incubate at room temperature for 10-15 minutes in the dark; Switch to the fourth channel 14 open, draw 100-200 μL of the fourth stain, immediately switch the dispensing valve 1 to the seventh channel 17 open, push the fourth stain into the staining chamber at a constant flow rate (such as 5-10 μL / s), incubate at room temperature for 5-10 minutes in the dark; 5) Switch the dispensing valve to the fifth channel 15 open, inject the sheath liquid (such as PBS) into the staining chamber 5 in three times (such as 500-1000 μL each time), and each time after flushing, the waste liquid can be discharged through the sixth channel 16 to completely remove the unbound dye.

[0054] This example fully verifies the technical advantages of the application in multiple immunofluorescence staining, and is particularly suitable for co-localization research of circulating tumor cell markers that require high precision and high repeatability.

[0055] Example 2 As shown in Figures 2-3 A multi-channel dispensing valve and a precise liquid processing method of gas displacement, the method is based on a multi-channel dispensing valve and a precise liquid processing system of gas displacement in example 1, comprising the following steps: S1: Obtain cancer cells S11: Discard the old culture medium in the cancer cell culture bottle.

[0056] S12: Add 2−3 mL of PBS buffer to rinse the cells, and discard the PBS.

[0057] S13: Add 1−2 mL of 0.25% trypsin to cover the bottom of the bottle, stand at room temperature or 37℃ for 1−2 minutes, and gently tap the bottle wall to promote cell detachment.

[0058] S14: Add complete medium containing serum to terminate the digestion reaction, and gently blow to completely suspend the cells.

[0059] S15: Centrifuge the cell suspension and discard the supernatant.

[0060] S16: Resuspend the cell pellet with fresh medium, adjust the cell concentration to 1 x 10 6 -5 x 10 6 cells / mL, and the viable cell rate > 90%.

[0061] S2: Fixing and permeabilizing the cancer cells S21: Prepare a sterile 6-well plate, and select one well to add 1-2 mL of medium.

[0062] S22: Take 10-20 μL of the cancer cell suspension obtained in step S1 and add it to the medium in the well.

[0063] S23: Incubate in a cell culture incubator overnight to allow the cells to adhere.

[0064] S24: Discard the medium in the well.

[0065] S25: Add 1-2 mL of PBS to rinse the cells, and discard the PBS. Repeat this rinsing step once.

[0066] S26: Add 1-2 mL of 4% paraformaldehyde solution, and fix at room temperature for 15-20 minutes. Discard the fixative.

[0067] S27: Rinse the cells with PBS buffer 2-3 times.

[0068] S28: Add 1-2 mL of 0.2% Triton X-100 solution, and permeabilize at room temperature for 10-15 minutes. Discard the permeabilization solution.

[0069] S29: Rinse the cells with PBS buffer 3 times.

[0070] S210: Add 1-2 mL of 5% BSA solution, and block at room temperature for 1 hour. After blocking, discard the BSA solution, and rinse once with PBS for standby.

[0071] S3: System pre-filling S31: Add the first stain, the second stain, the third stain, the fourth stain, and the sheath solution (which can be PBS) to the first stain bottle 81, the second stain bottle 82, the third stain bottle 83, the fourth stain bottle 84, and the sheath solution bottle 7, respectively.

[0072] S32: Start the intelligent control system, switch the dispensing valve 1 to the seventh channel 17 to open, and pass 0.3-0.5 bar of high-purity nitrogen gas through the pipeline for 30-60 seconds to blow off the waste liquid, which is discharged to the waste liquid bottle 6 through the sixth channel 16.

[0073] S33: Switching the distribution valve to the first channel 11, the second channel 12, the third channel 13, the fourth channel 14 and the fifth channel 15 in turn to open. The peristaltic pump 4 extracts the corresponding liquid at a flow rate of 5-10 mL / min. When the high-sensitivity liquid level detection sensor 3 detects that the liquid surface reaches the predetermined position, the pump operation is automatically stopped to ensure that the pipeline is full and free of bubbles.

[0074] S34: Switching the distribution valve 1 to the sixth channel 16 to open, and driving the peristaltic pump 4 in reverse to discharge excess liquid in the pipeline.

[0075] S4: Multiple fluorescence staining S41: Staining with the first staining agent and washing: Switching the distribution valve 1 to the first channel 11 to open, and extracting 300-600 μL of the first staining agent from the first staining agent bottle 81 by the peristaltic pump 4, then immediately switching the distribution valve 1 to the seventh channel 17 to open, and pushing the gas at a flow rate of 5-10 μL / s to inject the first staining agent into the culture well of the cancer cells in S2 which have been fixed and treated, and then incubating at room temperature for 30-60 minutes in the dark; switching the distribution valve 1 to the fifth channel 15 to open, and extracting 500-1000 μL of PBS sheath liquid by the peristaltic pump 4 to flush the incubated cancer cells, and then standing for 1-2 minutes, and switching the distribution valve 1 to the sixth channel 16 to open to discharge the waste liquid into the waste liquid bottle 6. Repeat this washing process 1-2 times.

[0076] S42: Staining with the second staining agent and washing: Switching the distribution valve 1 to the second channel 12 to open, and extracting 300-600 μL of the second staining agent from the second staining agent bottle 82 by the peristaltic pump 4, then immediately switching the distribution valve 1 to the seventh channel 17 to open, and pushing the gas at a flow rate of 5-10 μL / s to inject the second staining agent into the culture well, and then incubating at room temperature for 15-30 minutes in the dark; switching the distribution valve 1 to the fifth channel 15 to open, and extracting 500-1000 μL of PBS sheath liquid by the peristaltic pump 4 to flush the incubated cancer cells, and then standing for 1-2 minutes, and switching the distribution valve 1 to the sixth channel 16 to open to discharge the waste liquid into the waste liquid bottle 6. Repeat this washing process 2-3 times.

[0077] S43: Staining with the third staining agent and washing: S4: Staining with the third dye and washing:

[0078] S44: Staining with the fourth dye and washing: S4: Staining with the third dye and washing:

[0079] S5: Fluorescent imaging of the cancer cells S51: Remove most of the liquid in the well.

[0080] S52: Add a proper amount of mounting medium (containing an anti-quenching agent) to cover the cells.

[0081] S53: Capture multi-channel fluorescent images using a fluorescent microscope.

[0082] The above description is merely preferred embodiments of the present application but not for limiting the protection range of the present application. Otherwise, any modification, equivalent replacement, optimization or improvement made based on the technical concepts and principles of the present application should be included in the protection range of the present application.

Claims

1. A precision liquid handling system with a multi-channel dispensing valve and gas displacement, characterized in that, It includes a multi-channel dispensing valve (1) and an intelligent control system. The dispensing valve (1) is a multi-port rotary valve with eight channels. The first dye bottle (81) is connected to the first channel (11), the second dye bottle (82) is connected to the second channel (12), the third dye bottle (83) is connected to the third channel (13), the fourth dye bottle (84) is connected to the fourth channel (14), the sheath liquid bottle (7) is connected to the fifth channel (15), the waste liquid bottle (6) is connected to the sixth channel (16), the gas cylinder (2) is connected to the seventh channel (17), and a liquid level detection sensor (3) is installed on the pipeline between the eighth channel (18) and the peristaltic pump (4). The outlet of the peristaltic pump (4) is connected to the dyeing pool (5).

2. The multi-channel dispensing valve and gas-displacement precision liquid handling system as described in claim 1, characterized in that, The gas cylinder (2) contains an inert gas.

3. A method of using a precision liquid handling system based on the multi-channel dispensing valve and gas displacement as described in claim 1 or 2, characterized in that, Includes the following steps: S1: Obtain cancer cells; S2: Cancer cell fixation and permeation treatment; S3: System pre-filling process; S4: Multiple fluorescent staining; S5: Fluorescence imaging of cancer cells.

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