Cell enrichment and dyeing integrated equipment and method

Abstract

The invention discloses a cell enrichment and dyeing integrated device and method, and the integrated device comprises a microfluidic device which is used for enriching a target substance from a blood sample; the blood sample adding unit is used for adding a blood sample into the microfluidic device; the reagent taking and placing unit comprises a reagent sucking and placing assembly; the intermediate container is communicated with the reagent absorbing and releasing assembly; the two-way pump is arranged between the middle container and the reagent absorbing and releasing assembly; the washing liquid supply assembly is used for supplying washing liquid into the intermediate container; when the flushing fluid is not stored in the intermediate container, the two-way pump can convey negative pressure or positive pressure to the reagent sucking and releasing assembly, so that a reagent is sucked or discharged to the microfluidic device; after the washing liquid supply assembly conveys washing liquid to the intermediate container, the two-way pump can convey the washing liquid from the intermediate container to the reagent suction and release assembly for washing. The all-in-one device can complete the whole process from blood sample conveying to target substance enrichment and dyeing, can achieve flushing and cleaning without disassembly, and is beneficial to improving the detection efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical instruments, and in particular, relates to a cell enrichment and staining integrated device and method. Background Art

[0002] At present, in the field of clinical testing and biomedicine, the detection of cells, biomacromolecules and other components in biological fluids (such as human blood) has gradually received attention and development. Testing biological fluids can avoid trauma to patients, and test samples are easy to obtain. It can be widely used in immunological research, genetic research or microbial testing, and can even be used in biochip and organoid research and development.

[0003] If an integrated device is designed that can autonomously complete the entire process of transporting biological fluid samples, adding reagents with different functions to the samples, and separating the target substance from the samples, it will play an important role in various tests and research in the biomedical field. For example, in immunological research tests, genetic research tests, or microbial tests, the efficiency of sorting target cells, organisms, or biomacromolecules can be improved, which in turn helps in the research and development of biochips and organoids. On the other hand, the integrated device described above is also suitable for the application scenarios of replacing various eluents to realize the chromatographic separation preparation method, or it can also be applied to the application scenarios of adding samples or reagents at a fixed time and quantity, such as the induced expression of genetic recombinants and product purification.

[0004] One of the important application areas of the integrated device described above is disease detection. Specifically, at present, the discovery and diagnosis of most tumors in clinical practice still rely on imaging examinations, traditional tumor markers and tissue biopsies. Traditional invasive tissue biopsies require taking a piece of tissue or cell sample from the patient's body, and this operation often brings certain risks and pain to the patient. More importantly, tissue biopsies are not suitable for repeated extraction, are prone to complications, and have the risk of spreading cancer cells.

[0005] In recent years, some emerging tumor diagnostic detection technologies have emerged in the field of biomedical engineering, such as circulating tumor DNA (ctDNA) and circulating tumor cell (CTCs) detection methods called liquid biopsy. CTC detection is easy to obtain, which overcomes the disadvantages of inconvenient sampling of tissue pathology and certain damage to patients. According to research, CTCs can be found in peripheral blood before solid tumors form, so CTC detection is very suitable for early screening and early diagnosis of malignant tumors. CTC detection has a good effect on the prognosis of malignant tumors, disease progression monitoring, recurrence prediction, postoperative micro-lesion monitoring of malignant tumors, and the design of targeted drug therapy and monitoring of treatment effects. It is currently an advanced method for early screening and diagnosis of malignant tumors. Since the content of CTCs in peripheral blood is very low, CTC detection requires enrichment of CTCs before detection, among which the capture and identification of CTCs is currently the main challenge.

[0006] In 2004, the U.S. FDA approved the world's first CTC detection device CellSearch for the diagnosis of breast cancer, prostate cancer and colorectal cancer. However, the imported equipment is expensive, the detection method implemented using this equipment has poor specificity, and it has not been widely popularized. At the same time, when using microfluidics to enrich CTCs in blood samples in the prior art, it is often only possible to operate on a few microliters of blood samples. Due to the small volume of blood samples, it is easy to have the problem of not being able to enrich enough CTCs for detection, which leads to a high rate of missed detection. In order to overcome this problem, it is necessary to increase the volume of blood samples that can be processed in a one-time microfluidic enrichment process, thereby reducing the missed detection rate.

[0007] In addition, the CTC detection process requires adding different reagents to the blood sample multiple times. When replacing reagents, the existing device needs to be disassembled and cleaned to avoid cross contamination of the reagents. This is not only inconvenient to use, but also consumes a lot of time, seriously affecting the detection efficiency and even delaying the patient's diagnosis and subsequent treatment.

[0008] In view of this, the present invention is proposed. Summary of the invention

[0009] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide an integrated cell enrichment and staining device and method, which can complete the entire process from blood sample delivery to target substance enrichment and staining, and can complete flushing and cleaning without disassembly, which is conducive to improving detection efficiency.

[0010] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0011] A cell enrichment and staining integrated device, comprising:

[0012] a microfluidic device for receiving a blood sample and enriching a target substance therefrom;

[0013] A blood sample adding unit, used for adding a blood sample into the microfluidic device;

[0014] Reagent loading and unloading unit, including:

[0015] Reagent suction and discharge component, used for sucking / discharging reagents;

[0016] An intermediate container, connected to the reagent suction and discharge assembly;

[0017] A bidirectional pump is arranged between the intermediate container and the reagent suction and discharge assembly;

[0018] A washing liquid supply assembly, used for supplying washing liquid into the intermediate container;

[0019] When the intermediate container does not store the flushing liquid, the bidirectional pump can deliver negative pressure or positive pressure to the reagent suction and discharge component to control the reagent suction and discharge component to absorb the reagent, or discharge the reagent into the microfluidic device;

[0020] After the washing liquid supply component transports the washing liquid to the intermediate container, the bidirectional pump can transport the washing liquid from the intermediate container to the reagent suction and discharge component for washing.

[0021] Furthermore, the reagent suction and discharge assembly includes a reagent containing tube for storing the sucked reagent; the upper end opening of the reagent containing tube is connected to the two-way pump through a pipeline, and the reagent / flushing liquid is discharged from the lower end opening of the reagent containing tube.

[0022] Furthermore, the washing liquid supply assembly comprises:

[0023] A washing liquid storage bottle is used to store washing liquid;

[0024] A washing liquid delivery pipeline, the inlet end of which is connected to the washing liquid storage bottle, and the outlet end of which is connected to the intermediate container;

[0025] The delivery pump is arranged on the washing liquid delivery pipeline and is used for delivering the washing liquid to the intermediate container.

[0026] Furthermore, the inlet end of the washing liquid delivery pipeline is arranged close to the bottom of the washing liquid storage bottle.

[0027] Furthermore, the intermediate container is a washing liquid buffer bottle, the washing liquid buffer bottle is connected to the bidirectional pump through a connecting pipe, and one end of the connecting pipe extends into the bottom of the washing liquid buffer bottle.

[0028] Furthermore, the reagent taking and placing unit includes a plurality of reagent suction and placing components, and a plurality of bidirectional pumps connected to the plurality of reagent suction and placing components in a one-to-one correspondence; the plurality of bidirectional pumps are all connected to the same intermediate container.

[0029] Furthermore, the microfluidic device comprises:

[0030] The sample injection tube comprises a tube body, the upper end of the tube body is opened for receiving a blood sample, and the lower end of the tube body is provided with a buckling portion protruding from the side wall of the tube body;

[0031] A liquid accumulating liner is arranged below the sample injection tube, a filter assembly for filtering the blood sample is arranged between the liquid accumulating liner and the buckling part of the sample injection tube, and the target substance is enriched on the upper side of the filter assembly;

[0032] The buckle cover is sleeved on the tube body of the sample inlet tube. The buckle cover is spirally descended and threadedly connected with the liquid accumulation liner. The buckle part of the liquid inlet tube is pressed down to approach the liquid accumulation liner, thereby pressing the filter assembly.

[0033] Furthermore, the buckle cover comprises a pressing part which is horizontally arranged and used to press down the buckle part, and a through hole is formed on the pressing part for the pipe body to pass through;

[0034] The buckle cover also includes a connecting portion, which is formed by extending downward from the outer circumference of the pressing portion; an internal thread is arranged on the inner side of the connecting portion, and an external thread matching the internal thread is arranged on the liquid accumulating lining.

[0035] Furthermore, the blood sample adding unit comprises:

[0036] A blood delivery assembly, used for receiving a blood sample;

[0037] a pressure generating device, connected to the blood delivery assembly, capable of delivering positive pressure to the blood delivery assembly;

[0038] The blood discharge tube is connected to the blood delivery component. When the pressure generating device delivers positive pressure, the blood sample contained in the blood delivery component is discharged along the blood discharge tube and added to the microfluidic device.

[0039] Furthermore, the blood delivery assembly includes a sealed blood collection tube, which is in sealed arrangement, and the sealed blood collection tube is respectively connected to the pressure generating device and the blood drainage tube, and one end of the blood drainage tube extends into the bottom of the sealed blood collection tube;

[0040] Alternatively, the blood delivery component includes a blood collection tube for holding a blood sample, and an intermediate sealed tube connected to the blood collection tube via a blood extraction tube, the intermediate sealed tube is respectively connected to the pressure generating device and the blood drainage tube, and one end of the blood drainage tube extends to the bottom of the intermediate blood collection tube; the pressure generating device can also transmit negative pressure to the intermediate sealed tube to draw the blood sample in the blood collection tube along the blood extraction tube into the intermediate sealed tube.

[0041] A method for cell enrichment and staining using the above-mentioned integrated cell enrichment and staining device comprises the following steps:

[0042] (1) The bidirectional pump of the reagent loading and unloading unit delivers negative pressure to absorb the microporous membrane activation liquid, and then the bidirectional pump delivers positive pressure to add the microporous membrane activation liquid into the microfluidic device;

[0043] (2) adding a blood sample unit to add a blood sample into the microfluidic device; then, aspirating the microfluidic device to enrich the target cells;

[0044] (3) The bidirectional pump delivers negative pressure to absorb the red blood cell lysis solution, and then the bidirectional pump delivers positive pressure to add the red blood cell lysis solution into the microfluidic device to lyse the residual red blood cells; then, the microfluidic device is suction filtered to enrich the target cells;

[0045] (4) the bidirectional pump delivers negative pressure to absorb the cell fixative, and then the bidirectional pump delivers positive pressure to add the cell fixative into the microfluidic device, and then the microfluidic device is suction filtered;

[0046] (5) the bidirectional pump delivers negative pressure to absorb the buffer solution, and then the bidirectional pump delivers positive pressure to add the buffer solution to the microfluidic device for cleaning, and then the microfluidic device is suction filtered;

[0047] (6) the bidirectional pump delivers negative pressure to absorb the fixing liquid on the membrane, and then the bidirectional pump delivers positive pressure to add the fixing liquid on the membrane to the microfluidic device, and then the microfluidic device is suction filtered;

[0048] (7) the bidirectional pump delivers negative pressure to absorb the permeation liquid, and then the bidirectional pump delivers positive pressure to add the permeation liquid to the microfluidic device, and then the microfluidic device is suction filtered;

[0049] (8) The bidirectional pump delivers negative pressure to absorb the dye solution, and then the bidirectional pump delivers positive pressure to add the dye solution to the microfluidic device. After dyeing for a certain period of time, the microfluidic device is filtered by suction. The operation of adding the dye solution and filtering by suction is repeated twice;

[0050] (9) The bidirectional pump delivers negative pressure to absorb the buffer solution, and then the bidirectional pump delivers positive pressure to add the buffer solution to the microfluidic device for cleaning, and then the microfluidic device is filtered by suction. The operation of adding the buffer solution and filtering by suction is performed one to three times, and then the target cells that have been enriched and stained are obtained;

[0051] Wherein, after step (1), and / or after step (3), and / or after step (4), and / or after step (5), and / or after step (6), and / or after step (7), and / or after step (8), and / or after step (9), the method further includes step (0): the washing liquid supply component supplies a quantitative washing liquid into the intermediate container, and the bidirectional pump operates to transport the washing liquid from the intermediate container to the reagent suction and release component for washing.

[0052] After adopting the above technical scheme, the present invention has the following beneficial effects compared with the prior art.

[0053] In the present invention, the integrated cell enrichment and staining device can complete the whole process from blood sample delivery to cell enrichment and staining, so as to achieve more efficient detection. The reagent taking and placing unit in the integrated device can realize the flushing and cleaning of the reagent suction and placing components therein without disassembly, especially in the detection process that requires multiple additions of different reagents, without user participation, and more convenient to use. In the assembly process of the microfluidic device for enriching the target substance, the buckle cover and the effusion liner are rotated and tightened to press the buckle part of the injection tube to approach the effusion liner, and then press the filter component between the two, so that there is no relative rotation between the injection tube and the effusion liner, ensuring that the filter component does not shift, and then ensuring that the effect of the microfluidic device enriching the target substance from the blood sample is not affected. At the same time, the microfluidic device adopts the above-mentioned structure, and a maximum of 3.2 ml of blood sample can be added at a time, and the target cells or other target substances therein are enriched by micro-flow suction filtration. The volume of blood sample that can be processed at one time has achieved an order of magnitude leap compared to the microliter level in the prior art, which greatly reduces the missed detection rate.

[0054] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The accompanying drawings are part of the present invention and are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an improper limitation of the present invention. Obviously, the drawings described below are only some embodiments. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the accompanying drawings:

[0056] Figure 1 Schematic diagram of the reagent taking and placing device in an embodiment of the present invention (reagent suction / discharge process);

[0057] Figure 2 Schematic diagram of the reagent taking and placing device in an embodiment of the present invention (flushing process);

[0058] Figure 3 is an overall schematic diagram of a reagent taking and placing device in an embodiment of the present invention;

[0059] Figure 4 It is a schematic diagram of the structure of the reagent taking and placing component and the moving mechanism in an embodiment of the present invention;

[0060] Figure 5 is an exploded view of a microfluidic device according to an embodiment of the present invention;

[0061] Figure 6is a schematic diagram of an assembled microfluidic device in an embodiment of the present invention;

[0062] Figure 7 is a partial cross-sectional view of a sample injection tube in an embodiment of the present invention;

[0063] Figure 8 is a top view of the sample injection tube in an embodiment of the present invention;

[0064] Fig. 9 is a partial cross-sectional view of a buckle cover in an embodiment of the present invention;

[0065] Fig.10 The present invention Fig. 9 A-direction schematic diagram of the middle buckle cover;

[0066] Fig.11 is a partial cross-sectional view of a liquid accumulation liner in an embodiment of the present invention;

[0067] Fig.12 is a top view of a liquid accumulation liner in an embodiment of the present invention;

[0068] Fig.13 is a top view of the lining mesh in an embodiment of the present invention;

[0069] Fig.14 is a schematic diagram of the connection between the bottom pipe and the rubber hose in an embodiment of the present invention;

[0070] Fig.15 is a top view of the connecting bottom pipe in an embodiment of the present invention;

[0071] Fig.16 is a top view of the connecting bottom pipe installed on the fixing platform in the embodiment of the present invention;

[0072] Fig.17 is a side view of the connecting bottom pipe installed on the fixing platform in the embodiment of the present invention;

[0073] Fig.18 is a schematic diagram of the connection between the buffer device and the microfluidic device in the first embodiment of the present invention;

[0074] Fig.19 is a schematic structural diagram of a blood sample adding unit in Embodiment 1 of the present invention;

[0075] Fig. 20 is a top view of the blood sample adding unit in the first embodiment of the present invention;

[0076] Fig.21 is a schematic diagram of the connection between the buffer device and the microfluidic device in the second embodiment of the present invention;

[0077] Fig. 22 is a schematic diagram of a partially cutaway buffer film in a buffer according to a second embodiment of the present invention;

[0078] Fig.23 is a schematic diagram of the structure of the blood sample adding unit in the third embodiment of the present invention;

[0079] Fig.24 The present invention Fig.23 The enlarged schematic diagram of point B in the middle;

[0080] Fig.25 is a schematic structural diagram of a blood sample adding unit in Embodiment 4 of the present invention;

[0081] Fig.26 is a schematic diagram of the overall structure of the blood sample adding unit in the fourth and fifth embodiments of the present invention;

[0082] Fig. 27 It is a schematic diagram of the partial structure of the blood sample adding unit in the fifth embodiment of the present invention.

[0083] It should be noted that these drawings and textual descriptions are not intended to limit the conceptual scope of the present invention in any way, but are intended to illustrate the concept of the present invention for those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0084] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0085] In the description of the present invention, it should be noted that the directions or positional relationships indicated by terms such as “upper”, “lower”, “front”, “back”, “left”, “right”, “vertical”, “inside” and “outside” are based on the directions or positional relationships shown in the accompanying drawings and cannot be understood as limitations on the present invention.

[0086] like Figures 1 to 27 As shown, an embodiment of the present invention provides an integrated cell enrichment and staining device for realizing the complete detection process of transporting a sample to be detected (such as a blood sample to be detected), adding one or more reagents, and enriching a target substance from the sample to be detected.

[0087] Specifically, the cell enrichment and staining integrated device of the embodiment of the present invention includes a reagent taking and placing unit, an enrichment and staining unit and a blood sample adding unit. The blood sample adding unit adds the blood sample to the enrichment and staining unit; the reagent taking and placing unit can absorb the reagent and add it to the enrichment and staining unit, thereby enriching the target substance in the blood sample in the enrichment and staining unit; the reagent taking and placing unit can also add a reagent with a staining function to the enrichment and staining unit, thereby staining the enriched target substance for subsequent detection and identification procedures.

[0088] Embodiment 1

[0089] like Figures 1 to 20 As shown, the integrated cell enrichment and staining device described in this embodiment includes a reagent taking and placing unit, an enrichment and staining unit, and a blood sample adding unit. Specifically, the integrated cell enrichment and staining device of this embodiment is used to achieve enrichment and staining of target cells, wherein the sample to be detected is a blood sample collected from a patient, and the target substance to be enriched is the cells in the blood sample, specifically nucleated pathological cells. More specifically, the integrated cell enrichment and staining device can be used to achieve CTC detection, in which case the target substance enriched from the blood sample is circulating tumor cells (CTC).

[0090] In this embodiment, the reagent taking and placing unit of the integrated device is used to absorb the reagent and then add the absorbed reagent to the dyeing unit. Figures 1 to 4 As shown, the reagent taking and placing unit at least includes a reagent suction and placing assembly 110, an intermediate container 140, a two-way pump 141 and a washing liquid supply assembly 150. The reagent suction and placing assembly 110 is used to absorb or discharge the reagent, the intermediate container 140 is connected to the reagent suction and placing assembly 110, and the two-way pump 141 is arranged between the intermediate container 140 and the reagent suction and placing assembly 110 to realize two-way operation. The washing liquid supply assembly 150 is connected to the intermediate container 140 and is used to supply flushing liquid into the intermediate container 140.

[0091] In the above scheme, the reagent taking and placing unit can not only absorb and discharge the reagent, thereby adding it to the microfluidic device 200, but also autonomously transport the flushing liquid to the reagent suction and placement component 110 after the reagent is added, so as to flush and clean the reagent suction and placement component 110 in preparation for the next absorption of different reagents. During the flushing and cleaning process, the reagent suction and placement component 110 does not need to be disassembled, and the user does not need to participate, so that the integrated device of this embodiment has a high degree of automation and is easy to use.

[0092] Specifically, in this embodiment, when the integrated device absorbs and discharges the reagent, as shown in FIG. Figure 1 As shown, no flushing liquid is stored in the intermediate container 140. At this time, the two-way pump 141 delivers negative pressure to the reagent suction and release component 110, that is, the reagent suction and release component 110 is evacuated to reduce pressure, and the reagent suction and release component 110 can use the internal negative pressure environment to absorb the required reagent. After the reagent is absorbed, the two-way pump 141 delivers positive pressure to the reagent suction and release component 110, that is, the reagent suction and release component 110 is ventilated and pressurized. Due to the increase in pressure, the reagent suction and release component 110 can discharge the internally absorbed reagent, thereby completing the reagent adding process. When the reagent suction and release component 110 needs to be flushed, as shown in FIG. Figure 2As shown, the washing liquid supply component 150 first supplies the washing liquid to the intermediate container 140, and a certain amount of washing liquid is stored in the intermediate container 140. Then the two-way pump 141 is operated (the operation direction of the two-way pump 141 is the same as that when delivering positive pressure), and the washing liquid can be delivered from the intermediate container 140 to the reagent suction and discharge component 110 for washing.

[0093] By the setting of the intermediate container 140, when the flushing liquid is not stored therein, the two-way pump 141 can be directly communicated with the atmosphere through the intermediate container 140, so that negative pressure or positive pressure can be delivered to the reagent suction and release assembly 110. When it is necessary to flush, the flushing liquid is injected into the intermediate container 140, and the flushing operation of the reagent suction and release assembly 110 can be completed by the two-way pump 141. In this way, the absorption and discharge of the reagent, and the process of delivering the flushing liquid to the reagent suction and release assembly 110 can be controlled by the two-way pump 141, and the structure is simpler. At the same time, the reagent picking and placing unit can be controlled to switch between the absorption and discharge of the reagent and the two working states of the flushing reagent suction and release assembly 110 by whether the flushing liquid is stored in the intermediate container 140, without the need to disassemble the device for the demand of flushing, which is more convenient to operate.

[0094] In a further solution of the present embodiment, the reagent suction and discharge assembly 110 includes a reagent holding tube 111 for temporarily storing the absorbed reagent. The reagent holding tube 111 is vertically arranged, and its upper end opening is connected to the two-way pump 141 through a pipeline. The lower end of the reagent holding tube 111 also has an opening, and the absorbed reagent and the flushing liquid entering the reagent holding tube 111 are all discharged from the lower end opening. Under the driving action of the two-way pump 141, the flushing liquid enters the tube from the upper end of the reagent holding tube 111 and is then discharged from the lower end. The flushing liquid flows downward in the reagent holding tube 111 under the action of gravity, and can more fully flush the tube wall of the reagent holding tube 111, achieving a more thorough and effective flushing effect.

[0095] Further, the reagent suction and release assembly 110 further includes a needle 115, which is connected to the lower end opening of the reagent holding tube 111, and the reagent is sucked into or discharged from the reagent holding tube 111 through the needle 115. Specifically, in this embodiment, the bidirectional pump 141 is a peristaltic pump, one end of which is connected to the reagent holding tube 111 of the reagent suction and release assembly 110 through the second connecting tube 145, and the other end is connected to the intermediate container 140 through the first connecting tube 144. More specifically, the lower end opening of the reagent holding tube 111 is connected to a connecting hose 114, and the lower end of the connecting hose 114 is connected to the needle 115.

[0096] With the above structure, when the reagent taking and placing device is working, the lower end of the needle 115 is immersed below the liquid level of the reagent to be absorbed, and then the reagent containing tube 111 can be depressurized by the positive rotation of the motor of the peristaltic pump, and the reagent can be sucked into the reagent containing tube 111 along the needle 115. Afterwards, the reagent containing tube 111 is ventilated and pressurized by the reverse rotation of the motor of the peristaltic pump, and the reagent therein can be discharged along the needle 115 again, so as to realize the addition of the reagent to the microfluidic device 200. When it is necessary to flush the reagent suction and placing assembly 110, the motor of the peristaltic pump is reversed, and the flushing liquid in the intermediate container 140 passes through the first connecting tube 144, the peristaltic pump and the second connecting tube 145 in sequence under the extraction action of the peristaltic pump, enters the tube from the upper end of the reagent containing tube 111, and then flows out from the lower end and is finally discharged through the needle 115, so that the reagent containing tube 111, the connecting rubber tube 114 and the needle 115 can be fully flushed.

[0097] Furthermore, in this embodiment, the intermediate container 140 is a washing liquid buffer bottle 146, and the first connecting pipe 144 extends into the bottle from the bottle mouth of the washing liquid buffer bottle 146. One end of the first connecting pipe 144 extending into the interior of the washing liquid buffer bottle 146 is arranged at a position close to the bottom of the washing liquid buffer bottle 146, preferably against the bottom of the washing liquid buffer bottle 146. In this way, the washing liquid transported to the washing liquid buffer bottle 146 by the washing liquid supply assembly 150 can be fully pumped out along the first connecting pipe 144 under the action of the two-way pump 141, so as to avoid residual washing liquid in the washing liquid buffer bottle 146.

[0098] In a further solution of this embodiment, the washing liquid supply assembly 150 includes a washing liquid storage bottle 153, a washing liquid delivery pipeline 151 and a delivery pump 152. The washing liquid storage bottle 153 is used to store the washing liquid, the inlet end of the washing liquid delivery pipeline 151 is connected to the washing liquid storage bottle 153, and the outlet end is connected to the washing liquid buffer bottle 146. The delivery pump 152 is arranged on the washing liquid delivery pipeline 151, and is used to deliver the washing liquid in the washing liquid storage bottle 153 to the washing liquid buffer bottle 146 along the washing liquid delivery pipeline 151.

[0099] It is understandable that the washing solution storage bottle 153 and the washing solution buffer bottle 146 in this embodiment are not sealed, but can be connected to the external atmosphere to ensure the smooth operation of reagent suction and discharge, as well as the delivery process of the flushing liquid.

[0100] As a specific scheme of this embodiment, a peristaltic pump is used as the delivery pump 152. When in use, the volume of the washing liquid delivered from the washing liquid storage bottle 153 to the washing liquid buffer bottle 146 can be controlled, so that the washing liquid entering the washing liquid buffer bottle 146 can be completely consumed in the subsequent washing process, thereby not affecting the subsequent reagent absorption and discharge process.

[0101] Further, in the present embodiment, the inlet end of the washing liquid delivery pipeline 151 extends into the bottle by the bottle mouth of the washing liquid storage bottle 153, and is arranged at a position close to the bottom of the washing liquid storage bottle 153. Preferably, the inlet end of the washing liquid delivery pipeline 151 contacts and counteracts the bottom of the washing liquid storage bottle 153. In this way, the flushing liquid stored in the washing liquid storage bottle 153 can be completely extracted, avoiding the situation that there is still flushing liquid remaining in the washing liquid storage bottle 153, but the inlet end of the washing liquid delivery pipeline 151 has been separated from the liquid level of the flushing liquid, and the flushing liquid cannot be further extracted. In this way, after the operator adds flushing liquid to the washing liquid storage bottle 153 once, the added flushing liquid can be fully utilized to complete multiple flushing processes, avoiding the trouble of frequently adding flushing liquid.

[0102] In a further embodiment of this embodiment, Figure 3 As shown, the reagent taking and placing unit includes a plurality of reagent suction and placing components 110, and a plurality of bidirectional pumps 141 connected to the plurality of reagent suction and placing components 110 in a one-to-one correspondence. The plurality of bidirectional pumps 141 are all connected to the same intermediate container 140, that is, the same washing liquid buffer bottle 146. Specifically, a plurality of second connecting tubes 145 are also provided, and the plurality of reagent containing tubes 111 are connected to the plurality of bidirectional pumps 141 in a one-to-one correspondence through the plurality of second connecting tubes 145.

[0103] In the present embodiment, by arranging multiple reagent suction and release components 110, reagent can be added to multiple microfluidic devices 200 at the same time, which is suitable for the rapid detection of large batches of samples. And each reagent suction and release component 110 is connected to a two-way pump 141 one by one, and then each reagent suction and release component 110 whether to carry out the absorption / discharge operation of reagent, and whether to need to rinse can be independently controlled, without interfering with each other. In this way, the number of reagent suction and release components 110 used can be selected according to the actual number of samples, and other reagent suction and release components 110 that do not need to be used do not need to be unloaded, easy to use, and can be more flexibly adapted to different application scenarios. The reagent pick-up and release unit with the above structure can also control multiple reagent suction and release components 110 to absorb different reagents at the same time, and then control multiple reagent suction and release components 110 that absorb reagent to discharge reagent successively, perform the operation of absorbing reagent once, and can add multiple different reagents successively in microfluidic device 200, which can further improve detection efficiency.

[0104] Since a single reagent suction and release component 110 requires a smaller volume of flushing liquid, in this embodiment, when multiple reagent suction and release components 110 and two-way pumps 141 are provided, multiple two-way pumps 141 are connected to the same washing liquid buffer bottle 146, which can both meet the flushing requirements and simplify the overall structure of the equipment.

[0105] Further, the first connecting pipe 144 includes a main pipe 142 and a plurality of shunt pipes 143. The main pipe 142 is connected to the washing liquid buffer bottle 146, and the plurality of shunt pipes 143 are respectively connected to the main pipe 142, and are connected to each bidirectional pump 141 one by one. Specifically, one end of the main pipe 142 extends into the bottom of the washing liquid buffer bottle 146 and abuts against the bottom of the washing liquid buffer bottle 146, thereby ensuring that the flushing liquid in the washing liquid buffer bottle 146 can be completely extracted. The main pipe 142 and the plurality of shunt pipes 143 can be connected through a three-way joint or other similar joints to achieve the main pipe 142 and the plurality of shunt pipes 143 are respectively connected.

[0106] With the above structure, when the two-way pump 141 starts to extract the flushing liquid in the washing liquid buffer bottle 146, the flushing liquid first enters the main pipe 142, and then can be diverted along the main pipe 142, entering the diversion pipe 143 of the connected two-way pump 141 which is in a working state, and finally enters the corresponding reagent holding tube 111, thereby realizing the flushing of the reagent suction and release component 110.

[0107] In a further scheme of the present embodiment, the reagent taking and placing unit also includes a moving mechanism, which is used to drive the reagent suction and placing assembly 110 to move in both horizontal and vertical directions. Specifically, the reagent required to be added is stored in the reagent holding device 130 with a top opening, and the reagent holding device 130 is pre-placed below the reagent suction and placing assembly 110. The moving mechanism drives the reagent suction and placing assembly 110 to move in the vertical direction, so that the lower end of the needle 115 can be immersed below the liquid level of the reagent to absorb, and after the absorption is completed, it rises again to the top opening of the lower end of the needle 115 higher than the reagent holding device 130. The microfluidic device 200 is also arranged below the reagent suction and placing assembly 110, and the moving mechanism drives the reagent suction and placing assembly 110 to move in the horizontal direction, so that the needle 115 can be moved to the top of the microfluidic device 200, and then the reagent is discharged by the two-way pump 141, so that the reagent can be added to the microfluidic device 200. More specifically, the plurality of reagent suction and discharge components 110 in this embodiment are fixed on a support frame 116 , and the support frame 116 is connected to a moving mechanism, and the moving mechanism can drive the support frame 116 to move, thereby causing the plurality of reagent suction and discharge components 110 to move synchronously.

[0108] As a specific solution of this embodiment, Figure 3 and Figure 4 As shown, the moving mechanism includes a first moving mechanism and a second moving mechanism. The first moving mechanism is used to drive the support frame 116 to move in the vertical direction. The first moving mechanism is installed on the second moving mechanism, and the second moving mechanism can drive the first moving mechanism and the support frame 116 to move in the horizontal direction as a whole.

[0109] In detail, the first mobile mechanism comprises a first motor 123 and a first lead screw 121. The first lead screw 121 is vertically arranged and rotatably mounted on a bracket, and the upper end of the first lead screw 121 is connected to the first motor 123 and rotates under the drive of the first motor 123. A slider is installed on the first lead screw 121, and a support frame 116 is connected to the slider. The first motor 123 drives the first lead screw 121 to rotate, so that the slider moves up and down along the first lead screw 121, and then the reagent suction and release assembly 110 can be driven to move in the vertical direction. The second mobile mechanism comprises a second motor 124 and a second lead screw 122. The second lead screw 122 is horizontally arranged, and the right end is connected to the second motor 124 and rotates under the drive of the second motor 124. A fixed seat is installed on the second lead screw 122, and a bracket for installing the first lead screw 121 is fixed on the fixed seat. The second motor 124 drives the second lead screw 122 to rotate, driving the fixed seat to move horizontally along the second lead screw 122, thereby driving the reagent suction and discharge assembly 110 to move in the horizontal direction.

[0110] In this embodiment, the initial height of the reagent suction and release assembly 110 in the vertical direction needs to meet the following requirements: the lower end of the needle 115 is higher than the top opening of the reagent holding device 130. During the operation of the integrated cell enrichment and staining device, when it is necessary to add reagents to the microfluidic device 200, the second motor 124 is started first to control the reagent suction and release assembly 110 to move horizontally, so that the needle 115 moves to the reagent holding device 130 where the required reagent is stored. Then the first motor 123 is started to control the reagent suction and release assembly 110 to descend, so that the needle 115 extends into the reagent holding device 130 until the lower end of the needle 115 is immersed below the liquid surface of the reagent.

[0111] Afterwards, the motor of the two-way pump 141 rotates forward, evacuates air from the reagent holding tube 111 to reduce pressure, and sucks the reagent into the reagent holding tube 111 along the needle 115. Then, the first motor 123 runs in the reverse direction to control the reagent suction and release assembly 110 to rise until the lower end of the needle 115 is a certain distance above the top opening of the reagent holding device 130. Then, the second motor 124 is started again to control the reagent suction and release assembly 110 to move horizontally to the right until the needle 115 moves to the top of the microfluidic device 200, and then the second motor 124 is turned off. At this time, the motor of the two-way pump 141 is reversed, and ventilation and pressurization are increased in the reagent holding tube 111, so that the reagent therein is discharged along the needle 115 under the pressure, and the reagent can be added to the microfluidic device 200 below.

[0112] After reagent addition is completed, delivery pump 152 starts, and transports the required rinsing fluid of a flushing operation in washing liquid buffer bottle 146.Rinsing fluid is in the washing liquid buffer bottle 146 delivery process, or after delivery is completed, mobile mechanism also needs to drive reagent suction and release assembly 110 to move to the waste liquid collection container (not shown) top that is used to collect waste liquid.Afterwards, the motor reversal of two-way pump 141 transports the flushing fluid in washing liquid buffer bottle 146 to reagent suction and release assembly 110, and flushing fluid flows through reagent holding tube 111, communication hose 114 and syringe needle 115 successively from top to bottom, and finally is discharged along syringe needle 115 lower ends and is collected in the waste liquid collection container.

[0113] The above process can be repeated multiple times to achieve the addition of different reagents, and after each reagent addition operation is completed, the reagent suction and release component 110 is automatically flushed to effectively avoid cross contamination between different reagents.

[0114] Furthermore, if Figures 5 to 18 As shown, the enrichment and staining unit of the cell enrichment and staining integrated device in this embodiment includes a microfluidic device 200 and a buffer device. Among them, the microfluidic device 200 is used to receive the blood sample, and can also receive the reagent added by the reagent taking and placing unit. The buffer device is connected to the microfluidic device 200 to provide a negative pressure of appropriate size to extract the blood sample and the reagent, and the target cells in the blood sample are intercepted by the microfluidic device 200, thereby achieving the enrichment and staining of the target cells in the microfluidic device 200.

[0115] In this embodiment, the microfluidic device 200 includes a sample injection tube 201 and a liquid accumulating support 203. The sample injection tube 201 includes a tube body 2011 with an upper opening, and the blood sample and the reagent are added into the microfluidic device 200 through the upper opening of the tube body 2011. The liquid accumulating support 203 is arranged below the sample injection tube 201, and a filter assembly 214 is arranged between the liquid accumulating support 203 and the sample injection tube 201. The lower end of the tube body 2011 also has an opening, and the blood sample can pass through the tube body 2011 of the sample injection tube 201 and fall on the filter assembly 214, and the filter assembly 214 plays a filtering role on the blood sample, thereby enriching the target cells on the upper side of the filter assembly 214.

[0116] Further, the lower end of the tube body 2011 is provided with a buckle part 2012 protruding from the outer wall of the tube body 2011, and the filter assembly 214 is provided between the buckle part 2012 and the liquid accumulating liner 203. The microfluidic device 200 also includes a buckle cover 202, which is sleeved on the tube body 2011 and threadedly connected with the liquid accumulating liner 203. Specifically, during the assembly process of the microfluidic device 200, the filter assembly 214 is first placed between the buckle part 2012 of the injection tube 201 and the liquid accumulating liner 203 below, and then the buckle cover 202 is sleeved from the upper end of the tube body 2011. After the buckle cover 202 is installed to the lower end of the tube body 2011, it contacts the buckle part 2012, and then spirally descends to be threadedly connected with the liquid accumulating liner 203, thereby pressing the buckle part 2012 down to approach the liquid accumulating liner 203, and pressing the filter assembly 214 between the two. In this way, during the assembly of the microfluidic device 200, the sample injection tube 201 and the liquid accumulation base 203 only approach each other in the vertical direction, and no relative rotation occurs between the two, thereby ensuring that the filter component 214 between the two does not shift, thereby avoiding the position deviation of the filter component 214 affecting the enrichment effect on the target cells.

[0117] In a further solution of this embodiment, the buckle cover 202 includes a horizontally arranged pressing portion 2021 for pressing down the buckle part 2012, and a through hole is provided on the pressing portion 2021 for the pipe body 2011 to pass through. The buckle cover 202 also includes a connecting portion 2022, which is formed by extending downward from the outer periphery of the pressing portion 2021. An internal thread is arranged on the inner side of the connecting portion 2022, and an external thread matching the internal thread is arranged on the liquid accumulation liner 203. Specifically, the liquid accumulation liner 203 includes a threaded portion 2031, and an operating portion 2032 arranged at the lower part of the threaded portion 2031. The threaded portion 2031 is provided with an external thread for threaded connection with the connecting portion 2022 of the buckle cover 202. The horizontal cross-sectional area of ​​the operating portion 2032 is larger than the area enclosed by the outer diameter of the threaded portion 2031, so that a horizontal stop surface is formed at the junction of the threaded portion 2031 and the operating portion 2032.

[0118] Furthermore, the lower surface of the pressing part 2021 is provided with a limiting groove 207 which is limitedly matched with the buckle part 2012. The through hole on the pressing part 2021 of the buckle cover 202 for the tube body 2011 to pass through can be interference-fitted with the tube body 2011, ensuring that the buckle cover 202 and the injection tube 201 can rotate relative to each other. The buckle cover 202 is put on the tube body 2011 from top to bottom. When it is installed downward in place, the buckle part 2012 is embedded in the limiting groove 207 to limit the relative position of the buckle cover 202 and the injection tube 201. The limiting groove 207 is an annular groove, so that the injection tube 201 and the buckle cover 202 can rotate relative to each other, but will not produce relative displacement in the horizontal direction. The two always remain coaxial, further ensuring that the relative position between the injection tube 201 and the effusion liner 203 remains stable during the assembly process of the microfluidic device 200.

[0119] In the specific scheme of this embodiment, the filter component 214 includes a microfiltration membrane 212 and a water-absorbing layer 213. The water-absorbing layer 213 is made of a water-absorbing material and can be provided with one or more layers. Among them, the microfiltration membrane 212 is used to achieve a filtering effect and intercept target cells in the blood sample. The water-absorbing layer 213 is arranged on the lower side of the microfiltration membrane 212, and is used to absorb the filtrate passing through the microfiltration membrane 212. By providing a buckle cover 202 threadedly connected to the effusion liner 203, the two do not rotate relative to each other during the process of the sample inlet tube 201 and the effusion liner 203 approaching each other, thereby avoiding relative displacement between the microfiltration membrane 212 and the water-absorbing layer 213, which affects the enrichment effect.

[0120] In detail, in this embodiment, in order to achieve the enrichment of CTCs in the blood sample, the microfiltration membrane 212 is a polycarbonate membrane with a diameter of 25 mm, on which micropores with a diameter of 8 μm are randomly distributed. The lower side of the microfiltration membrane 212 is lined with one or more water-absorbing layers 213, preferably three water-absorbing layers 213. The diameter of the water-absorbing layer 213 is the same as that of the microfiltration membrane 212, and the pore size on the water-absorbing layer 213 is 15 to 20 μm.

[0121] In this embodiment, the target cells enriched in the microfluidic device 200 are CTCs. The nuclei of CTCs are deformed with multiple irregular nucleoli. The rigidity of the oversized nuclei makes it impossible for CTCs to pass through the micropores on the microfiltration membrane 212. However, the white blood cells in the blood sample are smaller in size, and the red blood cells have no nuclei, so they can pass through the micropores on the microfiltration membrane 212 directly or by changing their shape. In this way, after the blood sample is added to the microfluidic device 200, the CTCs therein can be screened out.

[0122] It has been found through experiments that the three-layer water-absorbing layer 213 provided on the lower side of the microfiltration membrane 212 helps to support the microfiltration membrane 212 to maintain its posture. At the same time, the three-layer water-absorbing layer 213 can quickly absorb the filtered sample filtrate or reagents used for cell staining, etc., to avoid the problem of blurred background of the staining result caused by staining of the microfiltration membrane 212. Furthermore, the three-layer water-absorbing layer 213 can effectively disrupt the flow direction of the microfluid passing through the micropores on the microfiltration membrane 212, avoiding the damage of cell morphology caused by the shear force of the microfluid laminar flow. In addition, the microfluid passing through the micropores can also encounter resistance and form countless vortices, ensuring that the cells are evenly dispersed, which is helpful for subsequent detection procedures.

[0123] Through the scheme of this embodiment, a maximum of 3.2 ml of blood sample can be added to the microfluidic device 200 at a time, and CTCs therein can be enriched by micro-flow suction filtration, and a good enrichment effect is achieved. Since the amount of blood sample added is greatly increased compared to the microliter level in the prior art, a leap in the order of magnitude of the processed blood sample volume is achieved, so that more CTCs may be enriched, thereby greatly reducing the missed detection rate.

[0124] It is understandable that by replacing the microfiltration membrane 212 with micropores of different diameters, the integrated cell enrichment and staining device of this embodiment can be used to enrich other target substances, such as target organisms or biomacromolecules in the enriched sample.

[0125] In a further solution of this embodiment, the lower surface of the buckle part 2012 is provided with a mounting groove 204 which surrounds the circumference of the tube body 2011, and is used to provide a space for accommodating the filter assembly 214. The top of the threaded part 2031 of the effusion liner 203 has a concave cavity, in which a liner net 205 is provided, and the liner net 205 is clamped on the top of the threaded part 2031, so as to cooperate with the mounting groove 204 to realize the placement of the filter assembly 214. The liner net 205 has a dense mesh structure, so that it can realize the support function of the filter assembly 214, and will not block the filtrate passing through the microfiltration membrane 212.

[0126] Furthermore, a diversion cavity is formed between the bottom wall of the cavity and the lining mesh 205, and the filtered sample filtrate, dyeing liquid and other filtrate are collected in the diversion cavity, and then drawn out with the help of the negative pressure provided by the buffer device, and sent to the waste liquid bottle for collection. Specifically, the bottom wall of the cavity gradually decreases from the periphery to the middle to form a funnel-shaped structure, and a diversion hole 215 is provided at the lowest point of the funnel-shaped structure for discharging the filtrate. More specifically, the effusion lining 203 is hollow inside, and the bottom wall of the cavity is formed by the middle part of the inner wall of the threaded portion 2031 extending obliquely downward toward the central axis, and a circular diversion hole 215 is formed at the lowest point. The periphery of the diversion hole 215 extends vertically downward to a certain extent, and then extends obliquely in the direction close to the central axis to form a diversion cone 208, and the filtrate can be discharged outward along the diversion cone 208.

[0127] Furthermore, the guide cone 208 is connected to the connecting bottom pipe 209, and the connecting bottom pipe 209 is connected to the buffer device through the first pipeline 311, so that negative pressure can be provided to the microfluidic device 200 through the buffer device. Specifically, the lower end of the guide cone 208 is open and extends vertically downward, and is sealed and inserted into the connecting bottom pipe 209. The first pipeline 311 is a rubber hose, which facilitates the sealed connection between the connecting bottom pipe 209 and the first pipeline 311.

[0128] In this embodiment, the connecting bottom tube 209 is installed on the fixing platform 210. Specifically, the upper surface of the fixing platform 210 is provided with a through hole, and the connecting bottom tube 209 is inserted into the through hole. Specifically, the fixing platform 210 is provided with a plurality of through holes, so that a plurality of connecting bottom tubes 209 can be installed correspondingly, so that a plurality of microfluidic devices 200 can be installed at the same time.

[0129] In a further solution of this embodiment, a raised guide bar 206 is provided on the bottom wall of the concave cavity in the liquid accumulating liner 203. The guide bar 206 is in the shape of a rod and extends radially along the liquid accumulating liner 203, and the raised height gradually increases from the periphery to the center. Specifically, a plurality of guide bars 206 are distributed at intervals along the periphery of the liquid accumulating liner 203. After the filtrate passes through the filter assembly 214 and the liner net 205, it can flow out of the guide hole 215 more quickly under the guiding effect of the guide bar 206. The raised height of the guide bar 206 gradually increases from the periphery to the center, so that when the filtrate enters the diversion cavity in a dripping state, the dripping filtrate contacts the guide bar 206, and the filtrate flows on the surface of the guide bar 206 and the funnel-shaped structure in the direction of the gradually increasing raised height of the guide bar 206 under the action of gravity and liquid tension.

[0130] Furthermore, a plurality of long strip openings 2051 are provided on the surface of the lining mesh 205, and the setting position and shape of at least some of the long strip openings 2051 match the guide strip 206, so that the top of each guide strip 206 can be embedded in one of the long strip openings 2051, thereby playing an auxiliary positioning role for the lining mesh 205.

[0131] In this embodiment, the outer circumference of the upper end of the threaded portion 2031 of the effusion liner 203 protrudes upward to form an annular protrusion structure, and the liner net 205 is radially limited by the protrusion structure. Furthermore, the filter component 214 is at least partially located on the inner side of the annular protrusion structure, thereby limiting the filter component 214 in the radial direction, further preventing the filter component 214 from being displaced during the assembly process of the microfluidic device 200. It should be noted that the height of the protrusion structure is not less than the thickness of the liner net 205, which is used to avoid the liner net 205 from being installed unstably in the protrusion structure, and also to avoid the filter component 214 from sliding when contacting the upper surface of the liner net 205, which affects the installation.

[0132] In a further solution of this embodiment, at least two sealing gaskets (not shown in the figure) are installed in the microfluidic device 200, which can prevent liquid leakage on the one hand and air leakage on the other hand, thereby helping the negative pressure provided by the buffer device to more fully play a suction role.

[0133] Specifically, one of the sealing gaskets is sleeved on the threaded portion 2031 of the liquid accumulating liner 203. When the buckle cover 202 and the liquid accumulating liner 203 are tightened, the lower end of the connecting portion 2022 of the buckle cover 202 presses the sealing gasket against the horizontal stop surface where the threaded portion 2031 and the operating portion 2032 of the liquid accumulating liner 203 meet, thereby achieving sealing. Another sealing gasket is pre-installed in the mounting groove 204 at the lower side of the buckle part 2012 of the injection tube 201. When the buckle cover 202 is tightened, the buckle part 2012 is pressed downward, and the sealing gasket in the mounting groove 204 cooperates with the liquid accumulating liner 203 below to press the filter assembly 214. Alternatively, another sealing gasket can also be inserted downward from the upper end of the tube body 2011 and placed on the upper surface of the buckle part 2012. During the tightening process of the buckle cover 202, its downward pressing part 2021 and the buckle part 2012 of the sample injection tube 201 press the sealing gasket to achieve sealing.

[0134] In a further embodiment of the present invention, the buffer device of the enrichment unit includes a peristaltic pump 330 and a buffer assembly. The peristaltic pump 330 is connected to the microfluidic device 200 to provide negative pressure to suck the filtrate. The buffer assembly is arranged between the microfluidic device 200 and the peristaltic pump 330, and is connected to the microfluidic device 200 and the peristaltic pump 330 respectively.

[0135] In this embodiment, the blood sample is filtered in the microfluidic device 200 to enrich the nucleated pathological cells therein. Since the blood is difficult to filter and has hardened when too much external force is applied, the peristaltic pump 330 is used to provide negative pressure to achieve low-speed extraction, thereby avoiding the problem of excessive suction force. However, the peristaltic pump 330 is prone to generate pulse flow during operation. By setting the buffer component, the pulse can be offset at the buffer component, thereby avoiding the influence of the pulse flow on the cell enrichment effect in the microfluidic device 200.

[0136] In a specific solution of this embodiment, the buffer assembly includes a buffer bottle 310 connected to the microfluidic device 200 and the peristaltic pump 330 respectively, and a preset amount of gas is sealed in the buffer bottle 310. The buffer bottle 310 is sealedly connected with a first pipeline 311 and a second pipeline 312, wherein the first pipeline 311 is connected to the microfluidic device 200, and the second pipeline 312 is connected to the peristaltic pump 330.

[0137] Furthermore, the top of the buffer bottle 310 has a bottle mouth, and a sealing cap 313 is provided at the bottle mouth to seal the bottle mouth, and one end of the first pipeline 311 is connected to the connecting bottom pipe 209, so as to realize the communication between the microfluidic device 200 and the buffer bottle 310. The other end of the first pipeline 311 passes through the sealing cap 313 and extends into the buffer bottle 310. Similarly, one end of the second pipeline 312 passes through the sealing cap 313 and extends into the buffer bottle 310, and the other end is connected to the peristaltic pump 330.

[0138] During the operation of the peristaltic pump 330, the gas in the buffer bottle 310 can be pumped outward, so that a negative pressure is formed inside the buffer bottle 310. Since the buffer bottle 310 is in a sealed state, the negative pressure environment in the buffer bottle 310 will generate suction on the first pipeline 311, thereby sucking the filtrate passing through the filter assembly 214 in the microfluidic device 200 into the first pipeline 311, and flowing into the buffer bottle 310 along the first pipeline 311. When the peristaltic pump 330 generates a pulse, due to the presence of the buffer bottle 310, the pulse flow can be filled in the buffer bottle 310, thereby achieving a buffering effect, so that the suction force on the microfluidic device 200 will not have an obvious peak. Especially when the filtrate in the microfluidic device 200 is only a few milliliters, the generation of the pulse flow will have a great impact on the experimental results. By setting the buffer bottle 310, the influence of the pulse on the filtration process can be significantly reduced, thereby ensuring the enrichment effect of the cells in the microfluidic device 200.

[0139] Specifically, the peristaltic pump 330 in this embodiment is a BT100F-1 distribution intelligent peristaltic pump with a multi-channel card-type pump head, which can realize simultaneous driving of multiple pump heads. In this way, a peristaltic pump 330 can be connected to multiple microfluidic devices 200 at the same time, and realize simultaneous suction of multiple microfluidic devices 200 to extract the filtered liquid.

[0140] In the preferred solution of this embodiment, the length of the first pipeline 311 extending into the buffer bottle 310 is less than half the height of the buffer bottle 310, so as to prevent the filtrate from entering the buffer bottle 310 in small amounts and then submerging the lower end of the first pipeline 311, thereby affecting the buffer bottle 310's effect of resisting the pulse flow. The liquid filtered out of the microfluidic device 200 is drawn out along the first pipeline 311 and enters the buffer bottle 310, so that the buffer bottle 310 also plays the role of collecting the filtered liquid. Since the first pipeline 311 is connected to the microfluidic device 200, only by ensuring that the lower end of the first pipeline 311 is always connected to the air and not immersed under the liquid surface, can it play the role of resisting the pulse flow inside the buffer bottle 310. If the first pipeline 311 contacts the liquid in the buffer bottle 310, since the pressure of the liquid is greater than the suction force of the negative pressure, one end of the first pipeline 311 in the bottle will be blocked by the liquid, and the blood in the microfluidic device 200 cannot be sucked. The length of the first pipeline 311 extending into the buffer bottle 310 is as short as possible, so as to avoid the problem that the lower end of the first pipeline 311 contacts the liquid in the bottle.

[0141] In a further solution of this embodiment, the length of the second pipeline 312 inside the buffer bottle 310 is greater than half the height of the buffer bottle 310. During the suction process, when more and more liquid is collected in the buffer bottle 310, the volume of the gas in the bottle decreases, and the density of the liquid is much greater than the density of the gas, and thus it will not play a buffering role. When the gas volume decreases, the buffering effect on the pulse flow also decreases. By extending the second pipeline 312 to more than half the height of the buffer bottle 310, the peristaltic pump 330 can be sucked out when the liquid surface contacts the second pipeline 312, thereby ensuring the volume of the gas in the buffer bottle 310, and then ensuring the buffering effect of the buffer bottle 310. More preferably, the length of the second pipeline 312 extending into the buffer bottle 310 is greater than the length of the first pipeline 311 extending into the buffer bottle 310, so that when the liquid level in the buffer bottle 310 rises and contacts the second pipeline 312, the peristaltic pump 330 can suck out the liquid in the bottle, and the liquid level will no longer continue to rise, which can effectively prevent the liquid accumulated in the buffer bottle 310 from submerging the lower end of the first pipeline 311. Optimally, the second pipeline 312 is configured to extend to the bottom of the buffer bottle 310, and the first pipeline 311 is configured to extend to the mouth of the buffer bottle 310. When the liquid is sucked into the bottle, the second pipeline 312 contacts the liquid surface, and the liquid is sucked out under the action of the peristaltic pump 330. Regardless of whether the peristaltic pump 330 sucks liquid or gas, the volume of the liquid or gas sucked out is the same, which will not affect the negative pressure state in the bottle, which not only ensures the negative pressure state in the bottle, but also greatly reduces the frequency of cleaning the liquid in the bottle.

[0142] Furthermore, the mouth of the buffer bottle 310 is arranged lower than the microfluidic device 200, so that the filtrate discharged from the microfluidic device 200 flows downward along the first pipeline 311 into the buffer bottle 310, avoiding the situation where the liquid in the first pipeline 311 may flow back to the microfluidic device 200.

[0143] In another specific solution of this embodiment, two or more buffer bottles can be arranged between the microfluidic device and the peristaltic pump, and each buffer bottle is connected in sequence through a pipeline. Specifically, at least a first buffer bottle and a second buffer bottle are arranged between the microfluidic device and the peristaltic pump, the bottle mouth of the first buffer bottle is sealed and connected to the first pipeline, the first pipeline is connected to the connecting bottom pipe below the microfluidic device, the bottle mouth of the second buffer bottle is sealed and connected to the second pipeline, and the second pipeline is connected to the peristaltic pump.

[0144] When only the first buffer bottle and the second buffer bottle are provided, the first buffer bottle and the second buffer bottle are connected through an intermediate pipeline. Alternatively, at least one intermediate buffer bottle is provided between the first buffer bottle and the second buffer bottle, in which case any two adjacent buffer bottles are connected through an intermediate pipeline.

[0145] Furthermore, in the present embodiment, during the process of enriching CTCs and staining in the microfluidic device 200, it is necessary to aspirate the filtrate for multiple times, especially when one aspiration process is completed and the peristaltic pump 330 stops working, the problem of liquid reflux will inevitably occur. Therefore, in the present embodiment, a one-way valve 3111 is provided on the first pipeline 311 between the buffer bottle 310 and the microfluidic device 200, and the one-way valve 3111 is unidirectionally connected from the microfluidic device 200 to the buffer bottle 310. In this way, when the peristaltic pump 330 stops working, the filtrate in the first pipeline 311 cannot flow back to the microfluidic device 200 through the one-way valve 3111 in the reverse direction, thereby avoiding the influence of the filtrate reflux on the enrichment effect and further improving the accuracy of the detection result.

[0146] Furthermore, if Fig.19 and Fig. 20 As shown, the cell enrichment and staining integrated device of this embodiment adds blood samples to the microfluidic device 200 through a blood sample adding unit, and the blood sample adding unit includes a blood delivery component for accommodating the blood sample, and a pressure generating device 420 and a blood drain 430 respectively connected to the blood delivery component. The pressure generating device 420 can deliver positive pressure to the blood delivery component, so that the blood sample contained in the blood delivery component is discharged along the blood drain 430 and added to the microfluidic device 200.

[0147] Specifically, the blood delivery assembly includes a sealed blood collection tube 410, which is in sealed arrangement. The sealed blood collection tube 410 is connected to a pressure generating device 420 and a blood drainage tube 430, respectively. A blood sample to be tested is placed in advance in the sealed blood collection tube 410, and the pressure generating device 420 ventilates and pressurizes the sealed blood collection tube 410, and the blood sample contained in the sealed blood collection tube 410 is discharged along the blood drainage tube 430 under the action of positive pressure. The liquid outlet of the blood drainage tube 430 is arranged above the microfluidic device 200, so that the blood sample is added to the microfluidic device 200. More specifically, in this embodiment, the sealed blood collection tube 410 includes a tube body 411 and a sealing cover 412. After the blood sample is collected, the collected blood sample is added to the tube body 411, and then the tube mouth of the tube body 411 is blocked with the sealing cover 412, so as to form a sealed blood collection tube 410 with an airtight structure relative to the outside.

[0148] Further, the pressure generating device 420 is connected to the sealed blood collection tube 410 through the pressurizing tube 440. The pressure generating device 420 of this embodiment can be a pressure generating device 420 that can be automatically controlled, or it can be a pressure generating device 420 that needs to be manually operated. When the pressure generating device 420 is a pressure generating device 420 that can be automatically controlled, preferably, the pressure generating device 420 is a peristaltic pump. The peristaltic pump includes a driver and a pump head. The driver drives the pump head to squeeze the pressurizing tube 440, forming a unidirectional airflow in the pressurizing tube 440. The unidirectional airflow flows into the sealed blood collection tube 410 through the pressurizing tube 440 to achieve ventilation and pressurization of the sealed blood collection tube 410. During the pressurization process, the gas flowing into the sealed blood collection tube 410 only passes through the pressurizing tube 440 and does not contact the internal structure of the peristaltic pump itself, so there is no need to worry about the peristaltic pump contaminating the blood sample. When the pressure generating device 420 is a pressure generating device 420 that needs to be manually operated, preferably, a syringe-type air pump that is manually pushed and pulled is used.

[0149] In the preferred solution of this embodiment, the blood sample adding unit includes a plurality of sealed blood collection tubes 410, each of which is provided with a blood discharge tube 430, and each of which is connected to the same pressure generating device 420 through a pressurizing tube 440. The liquid outlets of the plurality of blood discharge tubes 430 are respectively provided above the plurality of microfluidic devices 200 in a one-to-one correspondence, so that blood samples contained in different sealed blood collection tubes 410 can be added to different microfluidic devices 200.

[0150] By adopting the above solution, the pressure generating device 420 can ventilate and pressurize the multiple sealed blood collection tubes 410 to discharge the blood samples. This solution does not require a separate pressure generating device 420 for each sealed blood collection tube 410, which not only reduces the number of pressure generating devices 420 and reduces costs, but also simplifies the structure of the blood sample adding unit and reduces the difficulty of repair and maintenance.

[0151] In a further solution of this embodiment, the pressure generating device 420 includes a pressure generator 421 and a pressure divider 422, wherein the pressure divider 422 includes an air inlet end and an air outlet end, and the air outlet end includes a plurality of air outlets, each of which is connected to the air inlet end. Each of the plurality of sealed blood collection tubes 410 is connected to the plurality of air outlets in a one-to-one correspondence through a pressurizing tube 440.

[0152] As a specific implementation of this embodiment, the pressure generator 421 is a peristaltic pump, which distributes the positive pressure generated by itself to each sealed blood collection tube 410 through the pressure divider 422. There is no need to change the structure of the pressure generator 421 itself due to the number of sealed blood collection tubes 410, thereby reducing the difficulty and cost of using the equipment.

[0153] Furthermore, a main switch 441 is provided between the pressure generator 421 and the voltage divider 422 to block the connection between the pressure generator 421 and the voltage divider 422, so as to control the start and stop of each sealed blood collection tube 410 to discharge blood outward.

[0154] Furthermore, a sub-switch 442 is provided between each gas outlet of the voltage divider 422 and the corresponding sealed blood collection tube 410. Specifically, the sub-switch 442 can be provided on each pressurized tube 440. The sub-switch 442 can block the on-off of the airflow between each gas outlet and its corresponding sealed blood collection tube 410, and each sub-switch 442 is opened and closed independently of each other without affecting each other.

[0155] The sub-switch 442 can be a manual switch that needs to be manually operated, or an automatic switch, which automatically controls the opening and closing of the switch according to whether each pressurized tube 440 is connected to the sealed blood sampling tube 410. Specifically, the sub-switch 442 on the pressurized tube 440 connected to the sealed blood sampling tube 410 or the sealed blood sampling tube 410 connected with the blood sample is controlled to be opened, so that the corresponding pressurized tube 440 is turned on; and the sub-switch 442 on the pressurized tube 440 not connected to the sealed blood sampling tube 410 or the sealed blood sampling tube 410 connected with the blood sample is controlled to be closed, so that the corresponding pressurized tube 440 is cut off.

[0156] In the above scheme, each pressurizing tube 440 is respectively provided with a sub-switch 442 that can be opened and closed independently. When some pressurizing tubes 440 are not connected to the sealed blood collection tube 410, or there is no blood sample in the connected sealed blood collection tube 410, this part of the pressurizing tube 440 can be blocked to prevent it from leaking the positive pressure generated by the pressure generator 421, thereby ensuring that other sealed blood collection tubes 410 containing blood samples can effectively receive the positive pressure, thereby discharging the blood sample.

[0157] In this way, the blood sample adding unit can add the blood sample to the corresponding microfluidic device 200 without waiting for all the pressurizing tubes 440 to be connected to the sealed blood collection tubes 410 containing the blood sample, so that the blood sample adding unit can be more flexibly adapted to different needs, without the need to wait for a long time to gather the number of sealed blood collection tubes 410, thereby improving work efficiency and saving patients' waiting time.

[0158] In a further solution of this embodiment, the air outlet of the pressurizing tube 440 communicating with the sealed blood collection tube 410 is arranged near the tube outlet in the sealed blood collection tube 410. When adding a blood sample into the sealed blood collection tube 410, the liquid level of the blood sample should be lower than the height of the air outlet of the pressurizing tube 440. In this way, the pressurizing tube 440 is never in direct contact with the blood sample, and even if the pressure generating device 420 is back-sucked when it stops working, the blood sample will not be sucked into the pressurizing tube 440, thus avoiding the problem of the blood sample entering the pressure generating device 420 and causing a malfunction.

[0159] One end of the drain tube 430 connected to the sealed blood collection tube 410 extends to the bottom of the sealed blood collection tube 410. Specifically, the liquid inlet port of the drain tube 430 abuts against the bottom of the sealed blood collection tube 410. During the operation of the pressure generating device 420, the sealed blood collection tube 410 is ventilated and pressurized through the pressure tube 440, so that the air pressure in the sealed blood collection tube 410 increases, and the blood sample therein can be pressed into the drain tube 430 from the liquid inlet port of the drain tube 430. The liquid inlet port abuts against the bottom of the sealed blood collection tube 410, so that the blood sample inside the sealed blood collection tube 410 can all enter the drain tube 430 to avoid blood sample residue.

[0160] In a further solution of this embodiment, the blood sample adding unit further includes a sampling fixing table 450, and the liquid outlet of the blood drainage tube 430 is fixed by the sampling fixing table 450. Specifically, the sampling fixing table 450 includes a horizontally arranged table 451, and a plurality of positioning holes 457 extending vertically are provided on the table 451, and the liquid outlet of the blood drainage tube 430 passes through the positioning holes 457 of the table 451 from top to bottom. The aperture of the positioning hole 457 is the same as the outer diameter of the blood drainage tube 430, or slightly larger than the outer diameter of the blood drainage tube 430.

[0161] The blood drain tube 430 in this embodiment is a hose, and by providing a platform 451 with a positioning hole 457, the position of the liquid outlet of the blood drain tube 430 can be fixed to ensure that it is stably maintained directly above the microfluidic device 200, thereby ensuring that the blood sample discharged along the blood drain tube 430 can be added to the microfluidic device 200. In this way, leakage of blood samples or mixing of blood samples is avoided.

[0162] Furthermore, when the number of microfluidic devices 200 that can be set in the integrated cell enrichment and staining device is greater than the number of sealed blood collection tubes 410, the liquid outlets of the same row of blood vessels 430 may need to be moved above different microfluidic devices 200. When the positioning holes 457 on the table 451 are set one-to-one with each row of blood vessels 430, the table 451 needs to be moved as a whole to drive the liquid outlets of the blood collection tubes 430 to move.

[0163] To this end, in this embodiment, the blood sample adding unit further includes a fixing frame, and the two sides of the sampling fixing platform 450 are provided with guide rails 455 arranged in parallel in the horizontal direction, and the fixing frame includes a support frame 456 matched with the two guide rails 455 of the sampling fixing platform 450, and the guide rails 455 and the support frame 456 are slidably connected. The support frame 456 specifically includes two symmetrically arranged parts, which are respectively located on the two sides of the sampling fixing platform 450 provided with the guide rails 455, and the support frame 456 is provided with a slide rail or a pulley matched with the guide rails 455.

[0164] In the above scheme, the guide rail 455 of the sampling fixing table 450 cooperates with the support frame 456, so that the sampling fixing table 450 can move along the extension direction of the guide rail 455, and then adjust the position of the positioning hole 457, so that the same positioning hole 457 can be aligned with different microfluidic devices 200, and then adjust the liquid outlet of the blood drainage tube 430 to above different microfluidic devices 200.

[0165] In a further solution of this embodiment, the blood sample adding unit further includes a driving mechanism for driving the sampling fixing table 450 to reciprocate along the extension direction of the guide rail 455. In this way, the movement of the sampling fixing table 450 can be controlled by the driving mechanism, without the user having to adjust the position of the sampling fixing table 450 by manually pushing and pulling, which is more convenient.

[0166] As a specific implementation, the driving mechanism includes a third lead screw 454 and a motor 453. A slider is installed on the third lead screw 454. The third lead screw 454 rotates under the driving action of the motor 453, so that the slider moves along the third lead screw 454. The third lead screw 454 is arranged in parallel with the guide rail 455 of the sampling fixed platform 450, and the sampling fixed platform 450 is fixedly connected to the slider on the third lead screw 454. Through the third lead screw 454 and the threaded hole 452 on the slider, when the third lead screw 454 rotates under the drive of the motor 453, the slider can move along the extension direction of the third lead screw 454, thereby driving the sampling fixed platform 450 to move along the third lead screw 454. The motor 453 is connected to the support frame 456. When the motor 453 drives the third lead screw 454 to rotate in different directions, the sampling fixed platform 450 will reciprocate with the slider on the third lead screw 454.

[0167] This embodiment also provides a method for cell enrichment and staining using the above-mentioned cell enrichment and staining integrated device, which specifically includes the following steps:

[0168] (1) The bidirectional pump 141 of the reagent loading and unloading unit delivers negative pressure to absorb the microporous membrane activation liquid, and then the bidirectional pump 141 delivers positive pressure to add the microporous membrane activation liquid into the microfluidic device 200;

[0169] (2) adding a blood sample to the microfluidic device 200 through a blood sample adding unit; then, aspirating the microfluidic device 200 to enrich target cells;

[0170] (3) The bidirectional pump 141 delivers negative pressure to absorb the red blood cell lysis solution, and then the bidirectional pump 141 delivers positive pressure to add the red blood cell lysis solution into the microfluidic device 200 to lyse the residual red blood cells; then, the microfluidic device 200 is filtered to enrich the target cells;

[0171] (4) The bidirectional pump 141 delivers negative pressure to absorb the cell fixative, and then the bidirectional pump 141 delivers positive pressure to add the cell fixative into the microfluidic device 200, and then the microfluidic device 200 is filtered;

[0172] (5) The bidirectional pump 141 delivers negative pressure to absorb the buffer solution, and then the bidirectional pump 141 delivers positive pressure to add the buffer solution to the microfluidic device 200 for cleaning, and then the microfluidic device 200 is filtered by suction;

[0173] (6) The bidirectional pump 141 delivers negative pressure to absorb the fixing liquid on the membrane, and then the bidirectional pump 141 delivers positive pressure to add the fixing liquid on the membrane into the microfluidic device 200, and then the microfluidic device 200 is suction filtered;

[0174] (7) The bidirectional pump 141 delivers negative pressure to absorb the permeation liquid, and then the bidirectional pump 141 delivers positive pressure to add the permeation liquid to the microfluidic device 200, and then the microfluidic device 200 is filtered;

[0175] (8) The bidirectional pump 141 delivers negative pressure to absorb the dye solution, and then the bidirectional pump 141 delivers positive pressure to add the dye solution to the microfluidic device 200. After dyeing for a certain period of time, the microfluidic device 200 is filtered by suction. The operation of adding the dye solution and filtering by suction is repeated twice.

[0176] (9) The bidirectional pump 141 delivers negative pressure to absorb the buffer solution, and then the bidirectional pump 141 delivers positive pressure to add the buffer solution to the microfluidic device 200 for cleaning. The microfluidic device 200 is then filtered. The operation of adding the buffer solution and filtering is repeated one to three times, and then the target cells that have been enriched and stained are obtained.

[0177] Among them, after step (1), and / or after step (3), and / or after step (4), and / or after step (5), and / or after step (6), and / or after step (7), and / or after step (8), and / or after step (9), preferably after each step of adding reagent, that is, after step (1) and steps (3) to (9), it also includes step (0): the washing liquid supply component 150 supplies a quantitative flushing liquid into the intermediate container 140, and the two-way pump 141 is operated to transport the flushing liquid from the intermediate container 140 to the reagent suction and release component 110 for flushing.

[0178] In detail, the specific operation process of cell enrichment staining using the above-mentioned cell enrichment and staining integrated device is as follows.

[0179] In the preparation stage, the operator adds the blood sample to be tested into the sealed blood collection tube 410 and seals the sealed blood collection tube 410. Wherein, the blood sample to be tested is pretreated whole blood, and the pretreatment can adopt the pretreatment method of the blood sample collected from the patient before the test when the nucleated pathological cells such as CTC are enriched in the prior art. The reagents that need to be added later, such as microporous membrane activation liquid, red blood cell lysis liquid, cell fixative, buffer, staining liquid, etc., are added to the reagent holding device 130 below the reagent suction and release component 110. It is also necessary to assemble the microfluidic device 200 in advance and install it on the connecting bottom tube 209 fixed on the fixed platform 210. When assembling the microfluidic device 200, the water absorption layer 213 therein needs to be pre-wetted. In addition, it is also necessary to add a sufficient amount of flushing liquid to the washing liquid storage bottle 153.

[0180] After the preparation stage is completed, the integrated device can be started to work. First, the reagent suction and release assembly 110 in the reagent loading and unloading unit is controlled to move, and the motor of the two-way pump 141 is rotated forward to deliver negative pressure, thereby absorbing the microporous membrane activation liquid, and then the reagent suction and release assembly 110 is moved above the microfluidic device 200, and the motor of the two-way pump 141 is reversed to deliver positive pressure, and the absorbed microporous membrane activation liquid is added to the microfluidic device 200 to pre-treat the microfiltration membrane 212.

[0181] After the addition is completed, the pressure generating device 420 starts to operate to pass positive pressure into the sealed blood collection tube 410, thereby adding a blood sample to the microfluidic device 200. After the blood sample is added, the peristaltic pump 330 is started to perform micro-flow suction filtration on the microfluidic device 200, thereby enriching the target cells in the blood sample, that is, CTC. In the process of adding the blood sample and enriching the target cells, the delivery pump 152 is started to deliver a quantitative flushing solution to the washing solution buffer bottle 146, and control the reagent suction and release assembly 110 to move to the top of the waste liquid collection container. After moving into place, the motor of the two-way pump 141 is reversed to deliver flushing solution to the reagent suction and release assembly 110 for flushing until the flushing solution in the washing solution buffer bottle 146 is completely consumed.

[0182] Afterwards, a similar process is used to control the reagent placement unit again, so that the motor of the two-way pump 141 rotates forward, and an appropriate amount of red blood cell lysing solution is absorbed through the reagent suction and release component 110. The motor of the two-way pump 141 is reversed again, and the red blood cell lysing solution is added to the microfluidic device 200 to lyse the remaining small amount of red blood cells. After the addition is completed, a quantitative flushing solution is delivered to the washing solution buffer bottle 146 again, and the motor of the two-way pump 141 is reversed to deliver the flushing solution to flush the reagent suction and release component 110. During the flushing process, the peristaltic pump 330 is started to perform micro-flow suction filtration on the microfluidic device 200 to extract the filtered liquid.

[0183] Then the motor of the two-way pump 141 is controlled to rotate forward, and the cell fixative is sucked into the reagent suction and release assembly 110, and then the motor of the two-way pump 141 is reversed to add the cell fixative to the microfluidic device 200 to fix the target cells. After the addition is completed, the reagent suction and release assembly 110 is flushed by the control of the delivery pump 152 and the two-way pump 141. After the cell fixative is added to the microfluidic device 200, the peristaltic pump 330 is also started to perform micro-flow suction filtration on the microfluidic device 200 to extract the filtered liquid.

[0184] Then, the motor of the bidirectional pump 141 is controlled to rotate forward to absorb an appropriate amount of buffer in the reagent suction and release component 110, and then the motor of the bidirectional pump 141 is reversed to add buffer to the microfluidic device 200. After the addition is completed, the reagent suction and release component 110 is rinsed. After the buffer is added to the microfluidic device 200, the peristaltic pump 330 is started to perform micro-flow suction filtration on the microfluidic device 200 to extract the filtered liquid, thereby cleaning and filtering once.

[0185] Afterwards, the motor of the bidirectional pump 141 is controlled to rotate forward to absorb the membrane fixing liquid in the reagent suction and release assembly 110, and then the motor of the bidirectional pump 141 is reversed to add the membrane fixing liquid to the microfluidic device 200. After the addition is completed, the reagent suction and release assembly 110 is rinsed. After the membrane fixing liquid is added to the microfluidic device 200, the peristaltic pump 330 is started to perform micro-flow suction filtration on the microfluidic device 200 to extract the filtered liquid.

[0186] Then, the motor of the bidirectional pump 141 is controlled to rotate forward to absorb the permeation liquid in the reagent suction and release assembly 110, and then the motor of the bidirectional pump 141 is reversed to add the permeation liquid to the microfluidic device 200. After the addition is completed, the reagent suction and release assembly 110 is rinsed. After the permeation liquid is added to the microfluidic device 200, the peristaltic pump 330 is started to perform micro-flow suction filtration on the microfluidic device 200 to extract the filtered liquid.

[0187] Then, the cells are dyed, including: controlling the motor of the two-way pump 141 to rotate forward, adding dyeing liquid to the reagent suction and release component 110, and then reversing the motor of the two-way pump 141 to add dyeing liquid to the microfluidic device 200. After dyeing for a certain period of time, the peristaltic pump 330 is started to perform micro-flow suction filtration on the microfluidic device 200 to extract the liquid. After the suction filtration is completed, the dyeing process is performed again. After the second addition of the dyeing liquid is completed, the reagent placement unit is rinsed again. It can be understood that when performing the dyeing process of the cells, the required amount of dyeing liquid can be sucked twice at one time, and then added in two times; or after the first addition, the dyeing liquid can be sucked again for the second addition.

[0188] Finally, the reagent taking and placing unit is controlled to add an appropriate amount of buffer to the microfluidic device 200, and then the peristaltic pump 330 is started to perform micro-flow suction filtration on the microfluidic device 200 to extract the filtered liquid. The above-mentioned steps of adding buffer and then performing micro-flow suction filtration can be performed only once, or can be repeated twice or three times, and after the last addition of buffer is completed, the reagent taking and placing unit is flushed again. It can be understood that when it is necessary to add two or three times of buffer, the amount of buffer required for multiple additions can be drawn in the reagent holding tube 111 at one time, and then added in batches according to the amount added each time; or after each addition, the buffer can be drawn again for the next addition.

[0189] After the above operation process is completed, the stained target cells are enriched on the microfiltration membrane 212. The operator can disassemble the microfluidic device 200 and remove the microfiltration membrane 212 therefrom for microscopic examination or other subsequent detection procedures.

[0190] The above operation process can be automatically controlled by a pre-set program, so that the integrated cell enrichment and staining device of this embodiment can automatically complete the whole process from blood sample delivery to cell enrichment and staining without manual intervention. In addition, it can process several milliliters of blood samples at a time, achieving a leap in the order of magnitude of the volume of processed blood samples, solving the problem that the blood sample volume is too small to be enriched to a sufficient number of cells, and greatly reducing the missed detection rate. The operator only needs to add the blood sample, the required reagents and the flushing solution in advance, and then directly take out the microfiltration membrane 212 for microscopic examination after the integrated cell enrichment and staining device is completed. At the same time, there is no need to disassemble and clean the reagent placement unit after the integrated device finishes working, which is convenient for use.

[0191] Embodiment 2

[0192] like Fig.21 and Fig. 22As shown, the difference between this embodiment and the above-mentioned embodiment 1 is that: the buffer assembly includes a buffer 340, the interior of the buffer 340 is hollow to form a buffer chamber, the buffer chamber is connected to the microfluidic device 200 and the peristaltic pump 330 respectively, and at least part of the cavity wall of the buffer chamber is made of a material that can undergo elastic deformation. Since part of the cavity wall of the buffer chamber can undergo elastic deformation, during the operation of the peristaltic pump 330, the air in the buffer chamber can be evacuated to reduce the pressure, so that the cavity wall of the buffer chamber undergoes inward concave deformation. When the peristaltic pump 330 generates a pulse flow, the cavity wall of the buffer chamber will be further concave, so that the volume inside the buffer chamber is reduced, thereby achieving the effect of buffering pulses.

[0193] In the specific scheme of this embodiment, the buffer 340 includes a bracket 343 with a hollow interior, and a buffer membrane 344 that can undergo elastic deformation. Among them, a plurality of through holes are provided on the bracket 343, and the buffer membrane 344 at least covers all of the through holes. In this way, the buffer membrane 344 and the bracket 343 together enclose the buffer chamber, and the portion of the buffer membrane 344 covering the through hole forms a cavity wall that can undergo elastic deformation. In this way, during the operation of the peristaltic pump 330, the portion of the buffer membrane 344 covering the through hole can be recessed into the interior of the bracket 343 through the through hole. When the peristaltic pump 330 generates a pulse flow, the degree to which the buffer membrane 344 is recessed into the interior of the bracket 343 increases, thereby reducing the internal volume of the buffer chamber and playing a role in buffering pulses.

[0194] In a specific solution of this embodiment, the bracket 343 is a hollow column structure, preferably a cylindrical structure. The through hole is arranged on the side wall of the column structure, and the buffer film 344 is sleeved on the side wall of the column structure to cover the through hole.

[0195] More specifically, when the bracket 343 is a hollow cylindrical structure, the buffer film 344 is tubular, and its inner diameter in the initial state is slightly smaller than the outer diameter of the bracket 343. In this way, the buffer film 344 is in a stretched state when it is mounted on the bracket 343, which can enhance its sealing performance on the through hole and prevent the buffer 340 from leaking. Preferably, the buffer film 344 can be bonded and fixed at both ends of the bracket 343.

[0196] Further, the bracket 343 includes two end walls 345 arranged at intervals relative to each other, and a plurality of support rods 3431 for connecting the two end walls 345. One end of the support rod 3431 is connected to the outer periphery of one of the end walls 345, and the other end is connected to the outer periphery of the other end wall 345. The plurality of support rods 3431 are arranged at intervals along the outer periphery of the end wall 345, and the through hole is formed between two adjacent support rods 3431. Preferably, the support rods 3431 are evenly distributed along the outer periphery of the end wall 345, and the support rods 3431 are parallel to each other. More preferably, the support rods 3431 are extended in a direction parallel to the axis of the column structure.

[0197] In this embodiment, the length of the buffer film 344 along the axial direction of the bracket 343 is preferably greater than the axial length of the bracket 343. In this way, after the buffer film 344 is mounted on the bracket 343, the two ends of the buffer film 344 can wrap around the outer periphery of the end wall 345 under the action of its own elasticity, ensuring that no air leakage occurs at the connection between the end wall 345 and the support rod 3431.

[0198] In another specific solution of this embodiment, a plurality of support rods are provided, and any two support rods may cross or not cross each other and be arranged irregularly, as long as through holes are ensured to be left on the side wall of the bracket.

[0199] In a further solution of this embodiment, an air port is respectively provided on the two end walls 345. One of the air ports is connected to the first pipeline 311, thereby communicating with the microfluidic device 200 through the first pipeline 311. The other air port is connected to the second pipeline 312, and communicates with the peristaltic pump 330 through the second pipeline 312. Further, the air port is configured as a trumpet-shaped structure extending outward from the end wall 345 with a gradually decreasing diameter. The first pipeline 311 and the second pipeline 312 are both hoses, which are mounted at the end of the trumpet-shaped structure, thereby reducing the amplitude of the sudden change of the cross-sectional area on the path passing through the first pipeline 311, the buffer 340 and the second pipeline 312 in sequence.

[0200] In this embodiment, the peristaltic pump 330 operates to provide negative pressure inside the buffer 340, thereby extracting the filtrate in the microfluidic device 200. When the peristaltic pump 330 generates a pulse flow, the buffer membrane 344 wrapped on the bracket 343 can be deformed and contracted toward the inside of the bracket 343, so that the volume inside the buffer chamber is reduced to offset or partially offset the sudden change in the negative pressure generated by the pulse flow, so that the suction force at the microfluidic device 200 will not have an obvious peak, thereby avoiding the influence of the pulse flow on the experimental results.

[0201] In a further solution of this embodiment, in order to avoid the problem of liquid reflux when the peristaltic pump 330 stops working, similar to the above-mentioned embodiment 1, a one-way valve 3111 is also provided between the microfluidic device 200 and the peristaltic pump 330. Specifically, the one-way valve 3111 is provided on the second pipeline 312, and is unidirectionally connected from the buffer 340 to the peristaltic pump 330. When the peristaltic pump 330 stops working, the liquid in the second pipeline 312 is intercepted by the one-way valve 3111 and will not flow back to the buffer 340. During the operation of the peristaltic pump 330, the buffer 340 is subjected to the negative pressure provided by the peristaltic pump 330, and the buffer membrane 344 always keeps the state of inward contraction, and then after the peristaltic pump 330 stops working, the buffer membrane 344 returns to its original state under its own elasticity, and can continue to provide a certain negative pressure to the microfluidic device 200, ensuring that the liquid in the first pipeline 311 will not flow back into the microfluidic device 200.

[0202] In this embodiment, a buffer 340 composed of a bracket 343 and an elastic buffer membrane 344 is arranged between the microfluidic device 200 and the peristaltic pump 330. The buffer membrane 344 can deform itself when the pressure changes to achieve a buffering effect on the pulse flow, thereby ensuring the accuracy of the detection.

[0203] Embodiment 3

[0204] like Fig.23 and Fig.24 As shown, this embodiment is a further limitation of the above-mentioned embodiment 1 or 2, and an openable and closable blocking structure is provided on the blood drainage tube 430. When the blood sample enters the blood drainage tube 430, the blocking structure can conduct the blood drainage tube 430, so that the blood sample can be discharged along the blood drainage tube 430 and added to the microfluidic device 200 below. When there is no blood sample in the blood drainage tube 430, the blocking structure automatically blocks the blood drainage tube 430. When the blood sample in any sealed blood collection tube 410 is completely discharged, the blood drainage tube 430 connected to the sealed blood collection tube 410 is automatically blocked by the blocking structure, and then the positive pressure provided by the pressure generating device 420 will not leak to the outside along the blood drainage tube 430, ensuring that other sealed blood collection tubes 410 can discharge blood samples normally.

[0205] In a specific solution of this embodiment, the blood drainage tube 430 has a vertical extension portion, and the blocking structure includes a pressure stabilizing chamber 437 disposed on the vertical extension portion, and a floating block 438 disposed inside the pressure stabilizing chamber 437. The density of the floating block 438 is less than or equal to the density of the blood sample, so that the floating block 438 can be suspended in the blood sample. The liquid inlet end of the pressure stabilizing chamber 437 is connected to the blood drainage tube 430, and a liquid outlet is disposed at the bottom of the pressure stabilizing chamber 437, and the floating block 438 can block the liquid outlet.

[0206] More specifically, the pressure stabilizing chamber 437 has a barrel-shaped structure, and its inner diameter is larger than the inner diameter of the drainage tube 430. The floating block 438 is spherical, and the liquid outlet of the pressure stabilizing chamber 437 is circular with a diameter smaller than the diameter of the floating block 438. The height of the pressure stabilizing chamber 437 is larger than the diameter of the floating block 438, ensuring that the floating block 438 has space to move up and down in the pressure stabilizing chamber 437. The inner diameter of the pressure stabilizing chamber 437 is larger than the diameter of the floating block 438, ensuring that the floating block 438 will not be restricted by the inner wall of the pressure stabilizing chamber 437 when being transported in the pressure stabilizing chamber 437.

[0207] In the initial state, the float 438 is held at the bottom of the pressure stabilizing chamber 437 by gravity, blocking the liquid outlet. When the sealed blood collection tube 410 receives positive pressure, the blood sample is pressed into the blood discharge tube 430, and after entering the pressure stabilizing chamber 437 along the blood discharge tube 430, the float 438 floats up and separates from the liquid outlet, thereby opening the liquid outlet, and the blood sample can be discharged along the blood discharge tube 430 and added to the microfluidic device 200. When all the blood samples in the sealed blood collection tube 410 are discharged, there is no more blood sample in the pressure stabilizing chamber 437, and the float 438 is blocked again by gravity by gravity, thereby cutting off the blood discharge tube 430. At this time, the positive pressure provided by the pressure generating device 420 will not leak out through the blood discharge tube 430.

[0208] In the above scheme, the opening and closing of the liquid outlet of the pressure stabilizing chamber 437 is controlled by the floating block 438, and the lifting and lowering movement of the floating block 438 is controlled by the buoyancy provided by the blood sample, without manual intervention, thereby realizing automatic control of the opening and closing of the blood drainage tube 430. The spherical floating block 438 can avoid the problem of poor sealing with the liquid outlet of the pressure stabilizing chamber 437 due to its movement and rolling in the pressure stabilizing chamber 437, thereby improving the reliability of the automatic opening and closing of the pressure stabilizing chamber 437.

[0209] In a further solution of this embodiment, the lower part of the pressure stabilizing chamber 437 has a funnel-shaped structure 439 whose diameter gradually decreases from top to bottom, and the liquid outlet of the pressure stabilizing chamber 437 is located at the bottom of the funnel-shaped structure 439. The upper part of the pressure stabilizing chamber 437 is a cylindrical structure, and the upper edge of the funnel-shaped structure 439 is connected to the lower edge of the cylindrical structure of the pressure stabilizing chamber 437, that is, the maximum diameter of the funnel-shaped structure 439 is the same as the diameter of the cylindrical structure of the pressure stabilizing chamber 437, and the minimum diameter of the funnel-shaped structure 439 is the same as the diameter of the liquid outlet of the pressure stabilizing chamber 437. When the blood sample in the pressure stabilizing chamber 437 is completely discharged and the floating block 438 descends under the action of gravity, the floating block 438 will sink along the inner wall of the peripheral side of the funnel-shaped structure 439 to the liquid outlet position of the pressure stabilizing chamber 437, thereby avoiding the problem of positive pressure leakage caused by the inability to completely block the liquid outlet of the pressure stabilizing chamber 437 due to the deviation of the landing point of the floating block 438 when it descends.

[0210] In this embodiment, when the pressure generating device 420 is connected to multiple sealed blood collection tubes 410 at the same time, it can simultaneously ventilate and pressurize multiple sealed blood collection tubes 410, press blood samples out of each sealed blood collection tube 410, and add them to the corresponding microfluidic device 200. Since the amount of blood samples in each sealed blood collection tube 410 may be different, and the viscosity of different blood samples often also varies, the time when the blood samples in each sealed blood collection tube 410 are drained out may be different. By providing a pressure stabilizing chamber 437 with a floating block 438 inside on the blood drainage tube 430, the corresponding blood drainage tube 430 can be automatically cut off after the blood sample in the sealed blood collection tube 410 is drained out, preventing the blood drainage tube 430 from leaking the positive pressure provided by the pressure generating device 420, and avoiding affecting the process of adding blood samples to other sealed blood collection tubes 410.

[0211] Embodiment 4

[0212] like Fig.25 and Fig.26 As shown, the difference between this embodiment and the above embodiment is that: the blood delivery component includes a blood sampling tube 414 and an intermediate sealed tube 431, the blood sampling tube 414 and the intermediate sealed tube 431 are connected through a blood drawing tube 415, and the intermediate sealed tube 431 is connected to the pressure generating device 420 and the blood drain tube 430 respectively. The pressure generating device 420 can deliver both positive pressure and negative pressure. The blood sampling tube 414 contains a blood sample, and the pressure generating device 420 delivers negative pressure to the intermediate sealed tube 431, which can draw the blood sample in the blood sampling tube 414 along the blood drawing tube 415 into the intermediate sealed tube 431. Then, the pressure generating device 420 delivers positive pressure to the intermediate sealed tube 431, which can press the blood sample out along the blood drain tube 430 and add it to the microfluidic device 200.

[0213] Specifically, the middle sealing tube 431 of this embodiment includes an open middle tube 432 and a middle sealing cover 433. The pressure generating device 420 is also connected to the pressurizing tube 440, and communicates with the middle sealing tube 431 through the pressurizing tube 440. Preferably, the pressure generating device 420 of this embodiment is a peristaltic pump capable of providing positive pressure and negative pressure.

[0214] With the blood sample adding unit of the above structure, since the blood sampling tube 414 does not need to receive positive pressure to discharge the blood sample, the blood sampling tube 414 can be set open without being sealed. The operator puts the blood sample to be tested in the blood sampling tube 414, and then inserts the blood extraction tube 415 from the upper open side of the blood sampling tube 414, and then starts the pressure generating device 420 to transport the blood sample, without the need for additional sealing of the blood sampling tube 414, thereby simplifying the operation process.

[0215] In a further solution of this embodiment, a first one-way valve 413 is provided on the blood sampling tube 415 for one-way communication from the blood sampling tube 414 to the middle sealed tube 431, and a second one-way valve 434 is provided on the blood discharge tube 430 for one-way communication from the inside of the middle sealed tube 431 to the outside. By providing the first one-way valve 413 and the second one-way valve 434, when the pressure generating device 420 delivers negative pressure, the pressure inside the middle sealed tube 431 decreases, and the first one-way valve 413 can conduct the blood sampling tube 415 under the pressure difference, so that the blood sample is sucked into the middle sealed tube 431 along the blood sampling tube 415, while the second one-way valve 434 cannot conduct from the outside to the inside, so the negative pressure environment in the middle sealed tube 431 will not leak through the blood discharge tube 430. Similarly, when the pressure generating device 420 delivers positive pressure, the internal pressure of the middle sealed tube 431 increases, and the second one-way valve 434 conducts the blood discharge tube 430 under the pressure difference, so that the blood sample can be discharged outward along the blood discharge tube 430, while the first one-way valve 413 cannot conduct from the middle sealed tube 431 to the blood collection tube 414, and thus the positive pressure environment in the middle sealed tube 431 will not leak through the blood collection tube 415, thereby ensuring the discharge efficiency of the blood sample.

[0216] In a further solution of this embodiment, one end of the pressurizing tube 440 extending into the middle sealed tube 431 is arranged at a position near the tube mouth of the middle sealed tube 431. The maximum volume of the blood sample in the blood collection tube 414 should satisfy the following requirement: when all the blood samples in the blood collection tube 414 are sucked into the middle sealed tube 431, the liquid level of the blood sample in the middle sealed tube 431 is lower than the height of the tube mouth of the pressurizing tube 440. In this way, it can be ensured that the pressurizing tube 440 is never in direct contact with the blood sample, and the blood sample is not sucked into the pressurizing tube 440 when the pressure generating device 420 delivers negative pressure, thereby preventing the pressurizing tube 440 from sucking the blood sample and causing a malfunction. On the other hand, the tube mouth of the pressurizing tube 440 is not in direct contact with the blood sample, and when the pressure generating device 420 delivers positive pressure, it can also prevent the pressurized air flow from flowing into the blood sample in the middle sealed tube 431 to generate bubbles and affect the effect of blood sample discharge. When a large number of bubbles are generated in the blood sample, it is not easy to discharge them completely, and the blood sample discharged in the form of bubbles has a reduced filtration efficiency, which affects the subsequent process of enriching cells in the microfluidic device 200.

[0217] Furthermore, one end of the blood drainage tube 430 connected to the middle sealed tube 431 extends into the bottom of the middle sealed tube 431, and specifically, the liquid inlet port of the blood drainage tube 430 abuts against the bottom of the middle sealed tube 431. When the pressure generating device 420 delivers positive pressure, the air pressure in the middle sealed tube 431 rises, and the blood sample therein can be pressed into the blood drainage tube 430 from the liquid inlet port of the blood drainage tube 430. The liquid inlet port of the blood drainage tube 430 abuts against the bottom of the middle sealed tube 431, so that the blood sample sucked into the middle sealed tube 431 can all enter the blood drainage tube 430, avoiding blood sample residue.

[0218] Furthermore, the liquid inlet of the blood sampling tube 415 is arranged close to the bottom of the blood sampling tube 414. Preferably, the liquid inlet of the blood sampling tube 415 abuts the bottom of the blood sampling tube 414. When the pressure generating device 420 delivers negative pressure, the air pressure in the middle sealed tube 431 decreases, and the blood sampling tube 414 is connected to the atmospheric pressure, and then the blood sample therein can be pressed into the blood sampling tube 415 from the liquid inlet of the blood sampling tube 415 by the atmospheric pressure, and then delivered to the middle sealed tube 431. The liquid inlet of the blood sampling tube 415 abuts the bottom of the blood sampling tube 414, so that all the blood samples contained in the blood sampling tube 414 can be delivered into the middle sealed tube 431 to avoid blood sample residue.

[0219] In the preferred solution of this embodiment, the liquid outlet of the blood sampling tube 415 is arranged close to the tube opening of the middle sealed tube 431, and will never touch the liquid surface of the blood sample in the middle sealed tube 431 during the blood sample transportation process. When the pressure generating device 420 evacuates the pressure from the middle sealed tube 431, the liquid outlet of the blood sampling tube 415 is always directly exposed to the negative pressure environment inside the middle sealed tube 431, thus avoiding the problem of increasing the resistance of the blood sample from being discharged from the liquid outlet of the blood sampling tube 415 when the liquid outlet is immersed in the blood sample.

[0220] In a further embodiment of this embodiment, Fig.26 As shown, the blood sample adding unit includes a plurality of blood sampling tubes 414 and a plurality of intermediate sealed tubes 431, and the plurality of intermediate sealed tubes 431 are connected to the plurality of blood sampling tubes 414 in a one-to-one correspondence, and the plurality of intermediate sealed tubes 431 are connected to the same pressure generating device 420 through the respective connected pressurizing tubes 440. Specifically, the pressure generating device 420 includes a pressure generator 421 and a pressure divider 422, and the pressure generator 421 is used to provide positive pressure and negative pressure, and can be a peristaltic pump, for example. The air inlet end of the pressure divider 422 is connected to the pressure generator 421, and the air outlet end includes a plurality of air outlets, each of which is connected to the air inlet end, and the plurality of pressurizing tubes 440 are connected to each air outlet in a one-to-one correspondence. Since the same pressure generator 421 simultaneously provides positive pressure and / or negative pressure to the plurality of intermediate sealed tubes 431 through the pressure divider 422, it is not necessary to separately set a pressure generator 421 for each intermediate sealed tube 431, which simplifies the structure of the blood sample adding unit and reduces the production cost and maintenance difficulty.

[0221] Furthermore, each of the drainage tubes 430 connected to the intermediate sealing tubes 431 is provided with a control valve 435 that can cut off the drainage tube 430. The multiple control valves 435 are independent of each other and can be opened or closed at the same time, or partially opened or partially closed. The multiple control valves 435 can be manually opened or closed by the user one by one, or can be automatically opened or closed by a program, or can be automatically opened or closed by the structure of the second switch 435 itself.

[0222] In this embodiment, since the volume of the blood samples in each blood collection tube 414 may be different, the volume of the blood samples drawn into the middle sealed tube 431 may also be different. When the blood samples are discharged outward through the blood discharge tube 430, the blood samples in some middle sealed tubes 431 may be completely discharged, while blood samples may still be in other middle sealed tubes 431. At this time, the blood discharge tube 430 connected to the middle sealed tube 431 where the blood samples have been completely discharged is emptied, and the pressurized air flow from the pressure generator 421 will flow out of the blood discharge tube 430, causing pressure leakage, which affects the discharge of blood samples in other middle sealed tubes 431. By respectively providing an openable and closable control valve 435 on each blood drainage tube 430, when the blood sample in a part of the intermediate sealed tube 431 is drained, the control valve 435 on the blood drainage tube 430 connected to this part of the intermediate sealed tube 431 is controlled to be closed, thereby blocking these blood drainage tubes 430 and preventing the positive pressure in this part of the intermediate sealed tube 431 from leaking, thereby ensuring that the other intermediate sealed tubes 431 can continue to obtain positive pressure and discharge the blood sample inside normally.

[0223] In a further solution of this embodiment, a pressurizing switch 443 capable of blocking conduction is respectively provided on each pressurizing tube 440. The multiple pressurizing switches 443 are independent of each other and can be turned on or off at the same time, or partially turned on or off. When in use, when the blood sampling tube 415 connected to the middle sealed tube 431 is inserted into the blood sampling tube 414 containing the blood sample, the pressurizing switch 443 on the corresponding pressurizing tube 440 is controlled to be turned on. If the blood sampling tube 415 is not inserted into the blood sample, or the blood sample in part of the blood sampling tube 414 is drained in advance during the blood sample delivery process, the pressurizing switch 443 on the corresponding pressurizing tube 440 is turned off to cut off the pressurizing tube 440. In this way, when the pressure generator 421 only delivers negative pressure to the middle sealed tube 431 that is inserted into the blood sample by the blood sampling tube 415, the middle sealed tube 431 that is directly connected to the atmosphere is prevented from causing negative pressure leakage, resulting in insufficient negative pressure in other middle sealed tubes 431, which affects the extraction of blood samples.

[0224] Similar to the first embodiment, the blood sample adding unit of this embodiment also includes a sampling fixing platform 450, on which a plurality of positioning holes 457 are provided, and the liquid outlet of the blood drainage tube 430 passes through the positioning holes 457 from top to bottom. In this way, the liquid outlet of the blood drainage tube 430 is fixed to ensure that it is stably maintained above the microfluidic device 200.

[0225] The specific operation process of performing cell enrichment and staining using the integrated cell enrichment and staining device provided in this embodiment is similar to that of the first embodiment, and the only difference is the operation process of adding a blood sample.

[0226] Specifically, in the preparation stage, the operator adds the blood sample into the blood collection tube 414, inserts the blood collection tube 415 into the blood collection tube 414 through the tube opening of the blood collection tube 414, and makes the liquid inlet tube opening of the blood collection tube 415 abut against the bottom of the blood collection tube 414. After the integrated device starts working, when it is necessary to add a blood sample, the pressure generating device 420 starts to operate, firstly evacuates the air from the middle sealed tube 431 to reduce the pressure, and the blood sample is sucked from the blood collection tube 414 into the middle sealed tube 431, and then the middle sealed tube 431 is ventilated and pressurized, and the sucked blood sample is pressed out along the blood discharge tube 430 and added to the microfluidic device 200.

[0227] In this embodiment, the blood sample adding unit includes a blood sampling tube 414 and an intermediate sealing tube 431. The blood sample delivery process is achieved by first delivering negative pressure and then delivering positive pressure through the pressure generating device 420. After the operator adds the blood sample collected from the patient's body to the blood sampling tube 414, the blood extraction tube 415 can be directly inserted into the blood sampling tube 414 without performing additional sealing operations on the blood sampling tube 414, which simplifies the operator's use procedures and reduces the workload.

[0228] Embodiment 5

[0229] like Fig.26 and Fig. 27 As shown, this embodiment is a further limitation of the above-mentioned fourth embodiment, and the blood drainage tube 430 is provided with a blocking structure that can automatically control the opening and closing, thereby eliminating the setting of the control valve on the blood drainage tube 430.

[0230] Specifically, the blocking structure described in this embodiment is the same as the blocking structure described in the third embodiment, including a pressure stabilizing chamber 437 disposed on the vertically extending portion of the blood drainage tube 430, and a floating block 438 disposed inside the pressure stabilizing chamber 437 and having a density less than or equal to the density of the blood sample. The lower part of the pressure stabilizing chamber 437 has a funnel-shaped structure 439 with a diameter that gradually decreases from top to bottom, and the liquid outlet of the pressure stabilizing chamber 437 is located at the bottom of the funnel-shaped structure 439.

[0231] In the initial state, the float 438 is held at the bottom of the pressure stabilizing chamber 437 by gravity, blocking the liquid outlet at the bottom of the pressure stabilizing chamber 437. When the pressure generating device 420 delivers positive pressure and presses the blood sample in the middle sealed tube 431 into the blood drain 430, the blood sample enters the pressure stabilizing chamber 437 along the blood drain 430, and the float 438 floats up and separates from the liquid outlet, thereby opening the liquid outlet, and the blood sample can be discharged along the blood drain 430 and added to the microfluidic device 200. When the middle sealed tube 431, the float 438 floats up and separates from the liquid outlet, thereby opening the liquid outlet, and the blood sample can be discharged along the blood drain 430 and added to the microfluidic device 200.

[0232] When all the blood samples in the middle sealing tube 431 are discharged, there is no more blood sample in the pressure stabilizing chamber 437, and the floating block 438 sinks to the liquid outlet of the pressure stabilizing chamber 437 along the inner wall of the circumference of the funnel-shaped structure 439 under the action of gravity, and re-blocks the liquid outlet of the pressure stabilizing chamber 437, thereby blocking the blood drainage tube 430. At this time, the positive pressure provided by the pressure generating device 420 will not leak out through the blood drainage tube 430.

[0233] In this embodiment, the pressure generating device 420 is connected to multiple intermediate sealed tubes 431 at the same time, and can simultaneously deliver positive pressure to multiple intermediate sealed tubes 431, so as to press blood samples out of each intermediate sealed tube 431 and add them to the corresponding microfluidic device 200. Since the amount of blood sample drawn from the blood collection tube 414 by each intermediate sealed tube 431 may be different, and the viscosity of different blood samples often also varies, the time when the blood samples in each intermediate sealed tube 431 are completely drained may be different. By providing a pressure stabilizing chamber 437 with a floating block 438 inside on each blood draining tube 430, the corresponding blood draining tube 430 can be automatically blocked after the blood sample in the intermediate sealed tube 431 is completely drained, so as to prevent the blood draining tube 430 from leaking the positive pressure provided by the pressure generating device 420, and avoid affecting the process of adding blood samples to the microfluidic device 200 by other intermediate sealed tubes 431.

[0234] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A cell enrichment and staining integrated device, characterized in that: include: a microfluidic device for receiving a blood sample and enriching a target substance therefrom; A blood sample adding unit, used for adding a blood sample into the microfluidic device; Reagent loading and unloading unit, including: Reagent suction and discharge component, used for sucking / discharging reagents; An intermediate container, connected to the reagent suction and discharge assembly; A bidirectional pump is arranged between the intermediate container and the reagent suction and discharge assembly; A washing liquid supply assembly, used for supplying washing liquid into the intermediate container; When the intermediate container does not store the flushing liquid, the bidirectional pump can deliver negative pressure or positive pressure to the reagent suction and discharge component to control the reagent suction and discharge component to absorb the reagent, or discharge the reagent into the microfluidic device; After the washing liquid supply component transports the washing liquid to the intermediate container, the bidirectional pump can transport the washing liquid from the intermediate container to the reagent suction and discharge component for washing.

2. The cell enrichment and staining integrated device according to claim 1, characterized in that: The reagent suction and discharge assembly includes a reagent containing tube for storing the sucked reagent; the upper end opening of the reagent containing tube is connected to the two-way pump through a pipeline, and the reagent / flushing liquid is discharged from the lower end opening of the reagent containing tube.

3. The cell enrichment and staining integrated device according to claim 1, characterized in that: The washing liquid supply assembly comprises: A washing liquid storage bottle is used to store washing liquid; A washing liquid delivery pipeline, the inlet end of which is connected to the washing liquid storage bottle, and the outlet end of which is connected to the intermediate container; The delivery pump is arranged on the washing liquid delivery pipeline and is used for delivering the washing liquid to the intermediate container.

4. The cell enrichment and staining integrated device according to claim 1, characterized in that: The intermediate container is a washing liquid buffer bottle, and the washing liquid buffer bottle is connected with the bidirectional pump through a connecting pipe, and one end of the connecting pipe extends into the bottom of the washing liquid buffer bottle.

5. The cell enrichment and staining integrated device according to claim 1, characterized in that: The reagent taking and placing unit comprises a plurality of reagent suction and placing components, and a plurality of bidirectional pumps connected to the plurality of reagent suction and placing components in a one-to-one correspondence; the plurality of bidirectional pumps are all connected to the same intermediate container.

6. The integrated cell enrichment and staining device according to any one of claims 1 to 5, characterized in that: The microfluidic device comprises: The sample injection tube comprises a tube body, the upper end of the tube body is opened for receiving a blood sample, and the lower end of the tube body is provided with a buckling portion protruding from the side wall of the tube body; A liquid accumulating liner is arranged below the sample injection tube, a filter assembly for filtering the blood sample is arranged between the liquid accumulating liner and the buckling part of the sample injection tube, and the target substance is enriched on the upper side of the filter assembly; The buckle cover is sleeved on the tube body of the sample injection tube. The buckle cover is spirally descended and threadedly connected with the liquid accumulation liner. The buckle part of the sample injection tube is pressed down to approach the liquid accumulation liner, thereby pressing the filter assembly.

7. The integrated cell enrichment and staining device according to claim 6, characterized in that: The buckle cover comprises a pressing part which is horizontally arranged and used to press down the buckle part, and a through hole is provided on the pressing part for the pipe body to pass through; The buckle cover also includes a connecting portion, which is formed by extending downward from the outer circumference of the pressing portion; an internal thread is arranged on the inner side of the connecting portion, and an external thread matching the internal thread is arranged on the liquid accumulating lining.

8. The integrated cell enrichment and staining device according to any one of claims 1 to 5, characterized in that: The blood sample adding unit comprises: A blood delivery assembly, used for receiving a blood sample; a pressure generating device, connected to the blood delivery assembly, capable of delivering positive pressure to the blood delivery assembly; The blood discharge tube is connected to the blood delivery component. When the pressure generating device delivers positive pressure, the blood sample contained in the blood delivery component is discharged along the blood discharge tube and added to the microfluidic device.

9. The cell enrichment and staining integrated device according to claim 8, characterized in that: The blood delivery assembly includes a sealed blood collection tube, which is in sealed arrangement. The sealed blood collection tube is respectively connected to the pressure generating device and the blood drainage tube, and one end of the blood drainage tube extends into the bottom of the sealed blood collection tube; Alternatively, the blood delivery component includes a blood collection tube for holding a blood sample, and an intermediate sealed tube connected to the blood collection tube via a blood extraction tube, the intermediate sealed tube is respectively connected to the pressure generating device and the blood drainage tube, and one end of the blood drainage tube extends to the bottom of the intermediate blood collection tube; the pressure generating device can also transmit negative pressure to the intermediate sealed tube to draw the blood sample in the blood collection tube along the blood extraction tube into the intermediate sealed tube.

10. A method for cell enrichment and staining using the cell enrichment and staining integrated device according to any one of claims 1 to 9, characterized in that: The following steps are involved: (1) The bidirectional pump of the reagent loading and unloading unit delivers negative pressure to absorb the microporous membrane activation liquid, and then the bidirectional pump delivers positive pressure to add the microporous membrane activation liquid into the microfluidic device; (2) adding a blood sample unit to add a blood sample into the microfluidic device; then, aspirating the microfluidic device to enrich the target cells; (3) The bidirectional pump delivers negative pressure to absorb the red blood cell lysis solution, and then the bidirectional pump delivers positive pressure to add the red blood cell lysis solution into the microfluidic device to lyse the residual red blood cells; then, the microfluidic device is suction filtered to enrich the target cells; (4) the bidirectional pump delivers negative pressure to absorb the cell fixative, and then the bidirectional pump delivers positive pressure to add the cell fixative into the microfluidic device, and then the microfluidic device is suction filtered; (5) the bidirectional pump delivers negative pressure to absorb the buffer solution, and then the bidirectional pump delivers positive pressure to add the buffer solution to the microfluidic device for cleaning, and then the microfluidic device is suction filtered; (6) the bidirectional pump delivers negative pressure to absorb the fixing liquid on the membrane, and then the bidirectional pump delivers positive pressure to add the fixing liquid on the membrane to the microfluidic device, and then the microfluidic device is suction filtered; (7) the bidirectional pump delivers negative pressure to absorb the permeation liquid, and then the bidirectional pump delivers positive pressure to add the permeation liquid to the microfluidic device, and then the microfluidic device is suction filtered; (8) The bidirectional pump delivers negative pressure to absorb the dye solution, and then the bidirectional pump delivers positive pressure to add the dye solution to the microfluidic device. After dyeing for a certain period of time, the microfluidic device is filtered by suction. The operation of adding the dye solution and filtering by suction is repeated twice; (9) The bidirectional pump delivers negative pressure to absorb the buffer solution, and then the bidirectional pump delivers positive pressure to add the buffer solution to the microfluidic device for cleaning, and then the microfluidic device is filtered by suction. The operation of adding the buffer solution and filtering by suction is performed one to three times, and then the target cells that have been enriched and stained are obtained; Wherein, after step (1), and / or after step (3), and / or after step (4), and / or after step (5), and / or after step (6), and / or after step (7), and / or after step (8), and / or after step (9), the method further includes step (0): the washing liquid supply component supplies a quantitative washing liquid into the intermediate container, and the bidirectional pump operates to transport the washing liquid from the intermediate container to the reagent suction and release component for washing.

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