Nucleated cell enriching and dyeing integrated equipment and method

Abstract

The invention discloses nucleated cell enriching and dyeing integrated equipment and method, and the integrated equipment comprises an enriching and dyeing unit which comprises a microfluidic device and is used for receiving a blood sample to be detected and enriching a target substance from the blood sample; the blood sample adding unit is used for adding a blood sample to be detected into the microfluidic device; the reagent taking and placing unit is used for sucking a reagent and adding the reagent into the microfluidic device; the microfluidic device comprises: a sample introduction tube comprising a tube main body, the lower end of which is provided with a buckling part protruding from the side wall of the tube main body; the liquid accumulation lining table is arranged below the sample injection pipe; the buckling cover is arranged on the pipe main body in a sleeving manner, the buckling cover spirally descends to be in threaded connection with the liquid accumulation lining table, the buckling part is pressed downwards to be close to the liquid accumulation lining table, and the filtering assembly arranged between the buckling part and the liquid accumulation lining table is pressed tightly. The nucleated cell enriching and dyeing integrated equipment can complete the whole process from blood sample conveying to target substance enriching and dyeing, and is beneficial to improving the detection efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of medical detection technology, and in particular, relates to a nucleated 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] Before detecting components such as cells and biomacromolecules in biological fluids, it is necessary to remove the collected biological fluids from the storage container, and filter and stain the target cells or biomacromolecules for further testing. If an integrated device is designed to enable it to autonomously complete the entire process of transporting biological fluid samples, adding reagents with different functions to the samples, and separating the target substances 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, as well as the research and development of organoids. On the other hand, the integrated device described above is also suitable for replacing various eluents to achieve the application scenario of chromatographic separation and preparation methods, or it can also be applied to the application scenario 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 continuous increase in tumor incidence and mortality has become one of the serious problems in today's society. At present, the clinical discovery and diagnosis of most tumors still rely on imaging examinations, traditional tumor markers and tissue biopsies. Traditional invasive tissue biopsy requires taking a piece of tissue or cell sample from the patient's body. This operation often brings certain risks and pain to the patient. More importantly, tissue biopsy is not suitable for repeated extraction, is prone to complications, and has 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 achieved using this device has poor specificity, and it has not been widely popularized.

[0007] In addition, the existing devices for enriching cells, biomacromolecules, etc. generally include two parts connected by threads, with a microporous filter membrane and water-absorbing fiber for enriching the target substance arranged between the two parts. However, during the process of turning and tightening, the microporous filter membrane and water-absorbing fiber will also rotate and even shift, which will result in the microporous filter membrane and water-absorbing fiber not being able to completely overlap. This will affect the enrichment effect of the target substance and prolong the enrichment process, especially in CTC detection, and may even delay the patient's diagnosis and subsequent treatment due to the impact on the test results.

[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 deficiencies of the prior art and provide an integrated device and method for nucleated cell enrichment and staining, which can complete the entire process from blood sample delivery to target substance enrichment and staining, and ensure the enrichment effect of the microfluidic device on the target substance.

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

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

[0012] A dye enrichment unit, including a microfluidic device, is used to receive a blood sample to be tested and enrich the target substance therefrom;

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

[0014] A reagent taking and placing unit, used for taking the reagent and adding it into the microfluidic device;

[0015] The microfluidic device comprises:

[0016] The sample injection tube comprises a tube body, the upper end of the tube body is opened for receiving the blood sample to be tested, and the lower end of the tube body is provided with a buckling portion protruding from the side wall of the tube body;

[0017] A liquid accumulating liner is arranged below the sample injection tube, a filter assembly for filtering the blood sample to be tested 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;

[0018] The buckle cover is sleeved on the tube body of the sample injection tube, and the buckle cover is spirally descended to be threadedly connected with the liquid accumulation liner, and the buckle part is pressed down to approach the liquid accumulation liner to press the filter component.

[0019] 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 provided on the pressing part for the pipe body to pass through;

[0020] 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.

[0021] Furthermore, a limiting groove which cooperates with the locking portion in a limiting manner is provided on the lower surface of the pressing portion.

[0022] Furthermore, the enrichment unit also includes a buffer device, including:

[0023] a peristaltic pump, connected to the microfluidic device, for pumping the filtrate;

[0024] The buffer component is arranged between the microfluidic device and the peristaltic pump, and is communicated with the microfluidic device and the peristaltic pump respectively.

[0025] Further, the buffer assembly includes a buffer bottle connected to the microfluidic device and the peristaltic pump respectively, and a preset amount of gas is sealed in the buffer bottle;

[0026] Alternatively, the buffer component includes a buffer, the interior of the buffer is hollow to form a buffer chamber, the buffer chamber is connected to the microfluidic device and the peristaltic pump respectively, and at least part of the cavity wall of the buffer chamber is made of a material that can undergo elastic deformation.

[0027] Furthermore, the reagent taking and placing unit comprises:

[0028] A reagent containing device, used for storing the absorbed reagent;

[0029] A vacuum pumping device, comprising a vacuum cylinder connected to the reagent containing device, and a piston assembly disposed inside the vacuum cylinder and capable of reciprocating relative to the vacuum cylinder;

[0030] The driving device is used to drive the vacuum cylinder and the piston assembly to generate relative motion.

[0031] Further, the piston assembly includes a piston and a push rod; the piston is arranged inside the vacuum cylinder and is in sealing contact with the inner wall of the vacuum cylinder; one end of the push rod is connected to the piston, and the other end extends to the outside of the vacuum cylinder;

[0032] The push rod is fixedly arranged; the driving device drives the vacuum cylinder to reciprocate along the axis of the push rod, evacuates air from the reagent containing device and reduces pressure to absorb the reagent into the reagent containing device, and ventilates and pressurizes the reagent containing device to discharge the reagent from the reagent containing device.

[0033] Furthermore, the vacuuming device further comprises a vacuuming fixing frame, and the vacuum cylinder is relatively fixedly mounted on the vacuuming fixing frame;

[0034] The driving device includes a driving motor and a transmission screw, and the vacuum fixing frame is connected to a slider installed on the transmission screw; the driving motor drives the transmission screw to rotate, and the vacuum fixing frame moves along the extension direction of the transmission screw with the slider, driving the vacuum cylinder to move along the extension direction of the transmission screw.

[0035] Furthermore, the reagent taking and placing unit also includes a moving mechanism for driving the reagent containing device to move to above the reagent containing device storing the reagent to be added to absorb the reagent, and driving the reagent containing device to move to above the microfluidic device to add the reagent.

[0036] Furthermore, the blood sample adding unit comprises a pressure generating device and a sealed blood collection tube, the pressure generating device is in communication with the sealed blood collection tube, and the sealed blood collection tube is provided with a blood drainage tube in communication with the outside;

[0037] The pressure generating device ventilates and pressurizes the sealed blood collection tube, and the blood sample to be tested contained in the sealed blood collection tube is discharged along the blood discharge tube and added into the microfluidic device.

[0038] A method for cell enrichment and staining using the above-mentioned integrated device for nucleated cell enrichment and staining comprises the following steps:

[0039] (1) The reagent taking and placing unit adds microporous membrane activation liquid to the microfluidic device;

[0040] (2) adding a blood sample unit to add a blood sample to be tested into the microfluidic device, and the enrichment unit suction-filters the microfluidic device to enrich target cells;

[0041] (3) the reagent taking and placing unit sucks the red blood cell lysis solution and adds it to the microfluidic device to lyse the residual red blood cells, and then the dye enrichment unit performs suction filtration on the microfluidic device;

[0042] (4) The reagent taking and placing unit sucks the cell fixative and adds it to the microfluidic device, and then the dyeing unit performs suction filtration on the microfluidic device;

[0043] (5) the reagent taking and placing unit sucks the buffer solution and adds it to the microfluidic device for cleaning, and then the dyeing unit performs suction filtration on the microfluidic device;

[0044] (6) The reagent taking and placing unit absorbs the fixing liquid on the membrane and adds it to the microfluidic device, and then performs suction filtration on the microfluidic device;

[0045] (7) the reagent taking and placing unit absorbs the permeation liquid and adds it to the microfluidic device, and then the dyeing unit performs suction filtration on the microfluidic device;

[0046] (8) The reagent taking and placing unit absorbs the dyeing solution and adds it to the microfluidic device. After dyeing for a certain period of time, the rich dyeing unit performs suction filtration on the microfluidic device; step (8) is performed twice in total;

[0047] (9) The reagent taking and placing unit absorbs the buffer solution and adds it to the microfluidic device for cleaning, and then the enrichment unit performs suction filtration on the microfluidic device; step (9) is performed one to three times, after which the enriched and stained target cells are obtained.

[0048] After adopting the above technical scheme, the present invention has the following beneficial effects compared with the prior art.

[0049] In the present invention, the whole process from blood sample delivery to cell enrichment and staining can be completed through the integrated nucleated cell enrichment and staining device, so that more efficient detection can be achieved. In the integrated nucleated cell enrichment and staining device, the microfluidic device for enriching the target substance includes an injection tube, a fluid effusion liner and a buckle cover. During the process of rotating and tightening the buckle cover and the fluid effusion liner, the buckle part of the injection tube can be pressed down to approach the fluid effusion liner, thereby pressing the filter assembly between the two. Since there is no relative rotation between the injection tube and the fluid effusion liner, it can be ensured that the filter assembly does not shift, thereby ensuring that the effect of the microfluidic device in enriching the target substance from the blood sample is not affected.

[0050] In the present invention, the peristaltic pump is used to draw the filtrate from the microfluidic device, which can easily achieve low-speed extraction, thereby avoiding the problem of blood sample hardening caused by excessive suction force. A buffer is arranged between the peristaltic pump and the microfluidic device to reduce the influence of the pulse flow generated by the peristaltic pump during operation on the blood sample filtration, thereby further ensuring the cell enrichment effect and facilitating the improvement of the accuracy of the detection results.

[0051] In the present invention, the reagent taking and placing unit is provided with a reagent containing device for storing the absorbed reagent, and the vacuum cylinder is only used to cooperate with the piston assembly to provide positive pressure or negative pressure, and the absorbed reagent will not enter the vacuum cylinder. In this way, only the reagent containing device needs to be replaced or cleaned, and there is no need to disassemble and clean the vacuum device, which is more convenient to use. The vacuum device is driven by a screw, which can more accurately control the moving distance of the vacuum cylinder, and then accurately control the absorption or addition amount of the reagent. The reagent taking and placing unit is provided with a moving mechanism, so that the reagent containing device can move between the position for absorbing the reagent and the position for adding the reagent, which can improve the efficiency of adding the reagent.

[0052] 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

[0053] 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:

[0054] Figure 1 is a schematic structural diagram of a vacuum pumping device in an embodiment of the present invention;

[0055] Figure 2 is a schematic diagram of a first limiting plate in an embodiment of the present invention;

[0056] Figure 3 is a left side view of the vacuum pumping device in the embodiment of the present invention without the first limiting plate and the second limiting plate;

[0057] Figure 4 is a schematic diagram of the installation of a reagent containing device in an embodiment of the present invention;

[0058] Figure 5 is a schematic structural diagram of a reagent containing device and a moving mechanism in an embodiment of the present invention;

[0059] Figure 6 is an exploded view of a microfluidic device according to an embodiment of the present invention;

[0060] Figure 7is a schematic diagram of an assembled microfluidic device in an embodiment of the present invention;

[0061] Figure 8 is a partial cross-sectional view of a sample injection tube in an embodiment of the present invention;

[0062] Fig. 9 is a top view of the sample injection tube in an embodiment of the present invention;

[0063] Fig.10 is a partial cross-sectional view of a buckle cover in an embodiment of the present invention;

[0064] Fig.11 The present invention Fig.10 A-direction schematic diagram of the middle buckle cover;

[0065] Fig.12 is a partial cross-sectional view of a liquid accumulation liner in an embodiment of the present invention;

[0066] Fig.13 is a top view of a liquid accumulation liner in an embodiment of the present invention;

[0067] Fig.14 is a top view of the lining mesh in an embodiment of the present invention;

[0068] Fig.15 is a schematic diagram of the connection between the bottom pipe and the rubber hose in an embodiment of the present invention;

[0069] Fig.16 is a top view of the connecting bottom pipe in an embodiment of the present invention;

[0070] Fig.17 is a top view of the connecting bottom pipe installed on the fixing platform in the embodiment of the present invention;

[0071] Fig.18 is a side view of the connecting bottom pipe installed on the fixing platform in the embodiment of the present invention;

[0072] Fig.19 is a schematic diagram of the connection between the buffer device and the microfluidic device in the first embodiment of the present invention;

[0073] Fig. 20 is a schematic structural diagram of a blood sample adding unit in Embodiment 1 of the present invention;

[0074] Fig.21 is a top view of the blood sample adding unit in the first embodiment of the present invention;

[0075] Fig. 22 is a schematic diagram of the connection between the buffer device and the microfluidic device in the second embodiment of the present invention;

[0076] Fig.23 is a schematic diagram of a partially cutaway buffer film in a buffer according to a second embodiment of the present invention;

[0077] Fig.24 is a schematic diagram of the structure of the blood sample adding unit in the third embodiment of the present invention;

[0078] Fig.25 The present invention Fig.24 A magnified schematic diagram of point B in the middle.

[0079] In the figure: 101, vacuum device; 1011, vacuum cylinder; 1012, driving motor; 1013, transmission screw; 1014, push rod; 1015, first limit plate; 1016, second limit plate; 1017, fixing part; 1018, fixing plate; 1019, holder; 1020, first connecting pipe; 1021, operation panel; 1022, groove; 110, reagent containing device; 1101, reagent containing tube; 1102, first support frame; 1103, second support frame; 1104, second connecting pipe; 1105, needle; 1106, first screw; 1107, second screw; 1108, fixing seat; 1109, reagent containing device; 1110, first motor; 1111, second motor;

[0080] 200, microfluidic device; 201, injection tube; 2011, tube body; 2012, buckle part; 202, buckle cover; 2021, pressing part; 2022, connecting part; 203, liquid lining platform; 2031, threaded part; 2032, operating part; 204, mounting groove; 205, lining platform net; 2051, long strip opening; 206, guide strip; 207, limit groove; 208, guide cone; 209, connecting bottom pipe; 210, fixing platform; 212, microfiltration membrane; 213, water absorption layer; 214, filter assembly; 215, guide hole;

[0081] 310, buffer bottle; 311, first pipeline; 3111, one-way valve; 312, second pipeline; 313, sealing cover; 330, peristaltic pump; 340, buffer; 343, bracket; 3431, support rod; 344, buffer membrane; 345, end wall;

[0082] 410. Sealed blood collection tube; 411. Blood collection tube body; 412. Sealing cover; 420. Pressure generating device; 421. Pressure generator; 422. Voltage divider; 430. Blood collection tube; 437. Pressure stabilizing chamber; 438. Float; 439. Funnel-shaped structure; 440. Pressurizing tube; 441. Main switch; 442. Sub-switch; 450. Sampling fixing table; 451. Table; 452. Threaded hole; 453. Motor; 454. Third lead screw; 455. Guide rail; 456. Support frame; 457. Positioning hole.

[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 are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore cannot be understood as a limitation on the present invention.

[0086] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0087] like Figures 1 to 25 As shown, an embodiment of the present invention provides a nucleated cell enrichment and staining integrated device for realizing the delivery of a sample to be detected (such as a blood sample to be detected), the addition of one or more reagents, and a complete detection process of enriching a target substance from the sample to be detected.

[0088] Specifically, the integrated nucleated cell enrichment and staining 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 be tested into 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 to be tested 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.

[0089] Embodiment 1

[0090] like Figures 1 to 21 As shown, the integrated nucleated 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.

[0091] Specifically, the integrated nucleated 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 nucleated cells in the blood sample, such as nucleated pathological cells. More specifically, the integrated nucleated 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).

[0092] 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 5 As shown, the reagent taking and placing unit at least includes a reagent containing device 110, a vacuum pumping device 101 and a driving device.

[0093] The reagent containing device 110 is used to store the absorbed reagent. The vacuum device 101 includes a vacuum cylinder 1011 connected to the reagent containing device 110, and a piston assembly disposed inside the vacuum cylinder 1011 and capable of reciprocating relative to the vacuum cylinder 1011. The driving device is used to drive the vacuum cylinder 1011 and the piston assembly to produce relative movement, thereby evacuating the pressure from the reagent containing device 110 or ventilating and pressurizing the reagent containing device 110 to achieve the suction or discharge of the reagent.

[0094] In the above scheme, the vacuum cylinder 1011 cooperates with the piston assembly to provide an air pressure difference, thereby controlling the suction and discharge of the reagent. The sucked reagent is stored in the reagent containing device 110, and does not enter the vacuum cylinder 1011. When the type of reagent changes, or the reagent taking and placing unit needs to be cleaned after long-term use, it is only necessary to remove the reagent containing device 110 for replacement or cleaning, and there is no need to disassemble and clean the vacuum device 101 connected to the driving device, which is more convenient to operate.

[0095] In the specific scheme of this embodiment, the piston assembly is fixedly arranged, and the driving device is connected to the vacuum cylinder 1011, which can drive the vacuum cylinder 1011 to reciprocate relative to the piston assembly to achieve the above-mentioned air extraction and decompression or ventilation and pressurization.

[0096] It can be understood that, in this embodiment, the vacuum cylinder can also be fixedly arranged, and the driving device is connected to the piston assembly to drive the piston assembly to reciprocate relative to the vacuum cylinder.

[0097] The scheme of this embodiment is described in detail below according to the fixed arrangement of the piston assembly and the movement of the vacuum cylinder 1011 under the driving action of the driving device.

[0098] In this embodiment, the piston assembly includes a piston and a push rod 1014. The piston is disposed inside the vacuum cylinder 1011 and is in sealing contact with the inner wall of the vacuum cylinder 1011. One end of the push rod 1014 is connected to the piston, and the other end extends to the outside of the vacuum cylinder 1011.

[0099] The push rod 1014 is fixedly arranged, and the driving device drives the vacuum cylinder 1011 to reciprocate along the axis of the push rod 1014. Figure 1 When the vacuum cylinder 1011 is driven to move to the right, air can be evacuated and pressure can be reduced from the reagent containing device 110, thereby absorbing the reagent into the reagent containing device 110. When the vacuum cylinder 1011 is driven to move to the left, air can be ventilated and pressurized into the reagent containing device 110, thereby discharging the reagent from the reagent containing device 110.

[0100] Further, the push rod 1014 is located at one end outside the vacuum cylinder 1011 (i.e. Figure 1 The left end of the push rod 1014 is provided with a fixing portion 1017, and the cross-sectional area of ​​the fixing portion 1017 in the direction perpendicular to the axis of the push rod 1014 is larger than the cross-sectional area of ​​the main part of the push rod 1014. The vacuum pumping device 101 also includes a limiting component, which is limited and matched with the fixing portion 1017 to keep the push rod 1014 fixed.

[0101] In a specific solution of this embodiment, the limiting component includes a first limiting plate 1015 that is fixedly arranged, and the fixing portion 1017 is fixed to the first limiting plate 1015 . For example, the fixing portion 1017 can be fixed by sticking it to the first limiting plate 1015 with glue.

[0102] Further, the first limiting plate 1015 is arranged in a direction perpendicular to the movement direction of the vacuum cylinder 1011. The first limiting plate 1015 is provided with a groove 1022 with an opening, and the main body of the push rod 1014 is inserted in the groove 1022. The groove 1022 is preferably opened upward to facilitate the installation of the push rod 1014.

[0103] Specifically, the vacuum device 101 is disposed above the reagent containing device 110, and a horizontally disposed operation panel 1021 is provided above the reagent containing device 110, and the vacuum device 101 is integrally mounted on the operation panel 1021. The first limiting plate 1015 is disposed vertically relative to the operation panel 1021, and the lower side of the first limiting plate 1015 is bent to form a horizontal flange, and the first limiting plate 1015 is fixed by the flange.

[0104] In another specific scheme of this embodiment, the limiting component includes a first limiting plate 1015 and a second limiting plate 1016 that are fixedly arranged. The first limiting plate 1015 and the second limiting plate 1016 are arranged opposite to each other, and the fixing portion 1017 is clamped between the first limiting plate 1015 and the second limiting plate 1016 to maintain fixation.

[0105] In the above scheme, the first limiting plate 1015 and the second limiting plate 1016 are arranged in a direction perpendicular to the movement direction of the vacuum cylinder 1011, and the first limiting plate 1015 also has an upwardly open groove 1022, so that the main part of the push rod 1014 can be inserted into the groove 1022. The spacing distance between the first limiting plate 1015 and the second limiting plate 1016 is substantially consistent with the thickness of the fixing portion 1017 along the length direction of the push rod 1014, so that during the movement of the vacuum cylinder 1011, the fixing portion 1017 is fixed between the first limiting plate 1015 and the second limiting plate 1016 and cannot move, so that the positions of the push rod 1014 and the piston are kept fixed. At this time, there is no need to additionally fix the fixing portion 1017 to the first limiting plate 1015 or the second limiting plate 1016.

[0106] More specifically, the vacuum device 101 is arranged above the reagent containing device 110, and a horizontally arranged operation panel 1021 is provided above the reagent containing device 110, and the vacuum device 101 is integrally mounted on the operation panel 1021. The first limiting plate 1015 and the second limiting plate 1016 are arranged vertically relative to the operation panel 1021, and the lower side of the first limiting plate 1015 is bent toward the second limiting plate 1016 to form a horizontal flange, and the first limiting plate 1015 is fixed by the flange. The second limiting plate 1016 is connected and fixed to the first limiting plate 1015 by screws or other connecting members, so as to cooperate with the first limiting plate 1015 to clamp the fixing portion 1017 at the end of the push rod 1014.

[0107] In a further solution of this embodiment, the vacuum device 101 further includes a vacuum fixing frame, and the vacuum cylinder 1011 is relatively fixedly mounted on the vacuum fixing frame. The driving device includes a driving motor 1012 and a transmission screw 1013, and the vacuum fixing frame is connected to a slider mounted on the transmission screw 1013. The driving motor drives the transmission screw 1013 to rotate, and the vacuum fixing frame moves along with the slider along the extension direction of the transmission screw 1013, thereby driving the vacuum cylinder 1011 to move along the extension direction of the transmission screw 1013.

[0108] Specifically, the vacuum fixing frame includes a fixing plate 1018, on which a holder 1019 for mounting a vacuum cylinder 1011 is disposed, and the vacuum cylinder 1011 is fixed in the holder 1019, so that the vacuum fixing frame moves synchronously with the vacuum fixing frame under the driving action of the driving screw 1013. The bottom of the fixing plate 1018 is fixedly connected to a slider having a threaded hole, and the driving screw 1013 is disposed below the fixing plate 1018 and penetrates the threaded hole on the slider.

[0109] Through the above structure, the driving motor 1012 drives the transmission screw 1013 to rotate, and the slider moves along the transmission screw 1013, which can drive the vacuum cylinder 1011 to move left and right. At the same time, by adopting the screw transmission method, the moving distance of the vacuum cylinder 1011 can be accurately controlled by the rotation angle of the driving motor 1012, and then the amount of reagent sucked or discharged by the reagent containing device 110 can be accurately controlled. Especially when adding reagents to the dyeing unit, the amount of reagent added can be accurately controlled to improve the accuracy of detection.

[0110] In this embodiment, a plurality of holders 1019 may be arranged side by side on the fixing plate 1018, and a vacuum cylinder 1011 is installed in each holder 1019. The push rods 1014 connected to the pistons in each vacuum cylinder 1011 are fixed by the first limit plate 1015, or are clamped and fixed by the first limit plate 1015 and the second limit plate 1016. Therefore, the first limit plate 1015 is provided with the same number of grooves 1022 as the number of vacuum cylinders 1011.

[0111] Further, the reagent containing device 110 includes a reagent containing tube 1101 for temporarily storing reagents, and a needle 1105 connected to the reagent containing tube 1101, and the reagent is sucked into or discharged from the reagent containing tube 1101 through the needle 1105. The combination of the reagent containing tube 1101 and the needle 1105 is also arranged in multiple groups, wherein the multiple reagent containing tubes 1101 are connected to the multiple vacuum cylinders 1011 in a one-to-one correspondence. The dyeing unit includes a microfluidic device 200 for receiving a blood sample to be tested, and the nucleated cells in the blood sample are enriched and dyed in the microfluidic device 200. The microfluidic device 200 is also arranged in multiple groups, and the reagents in the multiple reagent containing tubes 1101 can be added to the multiple microfluidic devices 200 in a one-to-one correspondence.

[0112] In the above scheme, the driving motor 1012 and the transmission screw 1013 can drive multiple vacuum cylinders 1011 to move simultaneously, thereby synchronously sucking reagents into multiple reagent containing tubes 1101, and then synchronously adding the sucked reagents to multiple microfluidic devices 200 for cell enrichment and staining one by one. In this way, the detection process of multiple blood samples can be completed synchronously, further improving the detection efficiency.

[0113] In the specific scheme of this embodiment, the vacuum cylinder 1011 is connected to the reagent holding tube 1101 through the first connecting tube 1020. The reagent holding tube 1101 is vertically arranged, and has an opening at the upper end, and the opening is sealed and connected to one end of the first connecting tube 1020. The lower end of the reagent holding tube 1101 is sealed and connected to the second connecting tube 1104, and is connected to the needle 1105 through the second connecting tube 1104. The first connecting tube 1020 and the second connecting tube 1104 are both rubber hoses, which can more flexibly match the relative position relationship between the vacuum cylinder 1011, the reagent holding tube 1101 and the needle 1105.

[0114] Since the vacuum device 101 is installed on the operation board 1021 located above the reagent containing device 110, in order to realize that the vacuum cylinder 1011 and the reagent containing tube 1101 can be connected through the first connecting tube 1020, the operation board 1021 has a hollow structure, thereby forming a through hole that passes through from top to bottom. One end of the first connecting tube 1020 is connected to the vacuum cylinder 1011, and then passes through the through hole on the operation board 1021, so that the other end is connected to the reagent containing tube 1101 below the operation board 1021.

[0115] Preferably, the operating plate 1021 has a plurality of through holes extending vertically therethrough, and the plurality of first connecting tubes 1020 are respectively disposed in one of the through holes.

[0116] In a further solution of this embodiment, the reagent taking and placing unit further includes a moving mechanism for driving the reagent containing device 110 to move. Specifically, a reagent holding device 1109 is provided below the reagent containing device 110, in which the reagent to be added is stored. The microfluidic device 200 of the dyeing unit is also provided below the reagent containing device 110.

[0117] The moving mechanism can drive the reagent containing device 110 to move above the reagent containing device 1109 to absorb the reagent therefrom. The moving mechanism can also drive the reagent containing device 110 to move above the microfluidic device 200 to add the absorbed reagent to the microfluidic device 200.

[0118] In the specific solution of this embodiment, the reagent containing device 110 further includes a first support frame 1102 and a second support frame 1103, the reagent containing tube 1101 is fixed on the first support frame 1102, and the needle 1105 is fixed on the second support frame 1103. The moving mechanism can drive the first support frame 1102 and the second support frame 1103 to move synchronously, so that the reagent containing device 110 moves as a whole.

[0119] Further, the first support frame 1102 includes a horizontally arranged plate-like structure, the plate-like structure is provided with a fixing hole, and the reagent containing tube 1101 is inserted into the fixing hole. There can be multiple fixing holes, so that multiple reagent containing tubes 1101 can be fixed in each fixing hole one by one.

[0120] Preferably, at least one side edge of the plate-like structure is provided with an upward or downward flange, which can play a blocking role when the reagent containing tube 1101 topples over, thereby keeping the reagent containing tube 1101 stable during the movement.

[0121] Further, the second support frame 1103 includes a first support plate and a second support plate, wherein the first support plate is vertically arranged. The second support plate is formed by bending a plate-like structure, and includes a horizontal portion that is horizontally arranged and perpendicular to the first support plate, and a vertical portion that is parallel or nearly parallel to the first support plate. The first support plate and the second support plate are relatively fixedly connected, so that the needle 1105 is clamped and fixed by the first support plate and the second support plate.

[0122] In detail, the second support plate is formed by bending a metal plate by 90 degrees, and the vertical portion of the second support plate is connected and fixed to the first support plate by screws or other connecting members. In this way, the needle 1105 can be pressed and fixed on the first support plate at the position where the horizontal portion and the vertical portion of the second support plate meet.

[0123] Preferably, a protective structure is provided in the area where the needle 1105 contacts the first support plate and the second support plate. Since the needle 1105, the first support plate and the second support plate in this embodiment are all metal parts, in order to more stably fix the needle 1105 between the first support plate and the second support plate, a protective structure is provided on the periphery of the needle 1105, and the protective structure may be a flexible material with a certain deformation amount, or a material that can more firmly fix the needle 1105, such as an adhesive tape.

[0124] In a further solution of this embodiment, the moving mechanism includes a first moving mechanism and a second moving mechanism. The first moving mechanism is used to drive the first support frame 1102 and the second support frame 1103 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, the first support frame 1102 and the second support frame 1103 to move in the horizontal direction as a whole.

[0125] Specifically, the first moving mechanism includes a first motor 1110 and a first lead screw 1106. The first lead screw 1106 is vertically arranged and rotatably mounted on a bracket. The upper end of the first lead screw 1106 is connected to the first motor 1110 and rotates under the drive of the first motor 1110. A slider is installed on the first lead screw 1106, and the first support frame 1102 and the second support frame 1103 are respectively connected to the slider. The first motor 1110 drives the first lead screw 1106 to rotate, so that the slider moves up and down along the first lead screw 1106, and then drives the reagent containing device 110 to move in the vertical direction.

[0126] The second moving mechanism includes a second motor 1111 and a second lead screw 1107. The second lead screw 1107 is horizontally arranged, and the right end is connected to the second motor 1111, and rotates under the drive of the second motor 1111. A fixing seat 1108 is installed on the second lead screw 1107, and a bracket for installing the first lead screw 1106 is fixed on the fixing seat 1108. The second motor 1111 drives the second lead screw 1107 to rotate, driving the fixing seat 1108 to move horizontally along the second lead screw 1107, and then driving the reagent containing device 110 to move in the horizontal direction.

[0127] In this embodiment, the initial height of the reagent containing device 110 in the vertical direction needs to satisfy: the lower end of the needle 1105 is higher than the upper opening of the reagent containing device 1109. During the operation of the integrated nucleated cell enrichment and staining device, when it is necessary to add reagents to the microfluidic device 200, the second motor 1111 is first started to control the reagent containing device 110 to move horizontally, so that the needle 1105 moves to the top of the reagent containing device 1109 where the required reagent is stored. Then the first motor 1110 is started to control the reagent containing device 110 to descend, so that the needle 1105 extends into the reagent containing device 1109 until the lower end of the needle 1105 is submerged below the liquid surface of the reagent.

[0128] Afterwards, the driving motor 1012 is started, driving the vacuum cylinder 1011 to move, extracting air from the reagent containing tube 1101 to reduce pressure, and sucking the reagent into the reagent containing tube 1101 through the needle 1105. Then the first motor 1110 runs in the reverse direction, controlling the reagent containing device 110 to rise, until the lower end of the needle 1105 is a certain distance above the upper opening of the reagent containing device 1109. Then, the second motor 1111 is started again, controlling the reagent containing device 110 to move horizontally to the right, until the needle 1105 moves to the top of the microfluidic device 200, and then the second motor 1111 is turned off.

[0129] At this time, the driving motor 1012 moves in the reverse direction, driving the vacuum cylinder 1011 to move in the reverse direction, ventilating and pressurizing the reagent holding tube 1101, so that the reagent therein is discharged along the needle 1105 under the action of pressure, and the reagent can be added to the microfluidic device 200 below.

[0130] The above process can be repeated multiple times to achieve the addition of different reagents, or multiple additions of the same reagent.

[0131] The enrichment and staining unit of the integrated device for enriching and staining nucleated cells in this embodiment is described in detail below.

[0132] Specifically, Figures 6 to 19 As shown, the dye enrichment unit described in this embodiment includes a microfluidic device 200 and a buffer device. The microfluidic device 200 is used to receive the blood sample to be tested, and can also receive the reagent added by the reagent placement 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 nucleated cells in the blood sample are intercepted by the microfluidic device 200, thereby achieving the enrichment and dyeing of the nucleated cells in the microfluidic device 200.

[0133] In this embodiment, the microfluidic device 200 includes a sample injection tube 201 and a liquid effusion support 203. The sample injection tube 201 includes a tube body 2011 with an upper opening, and the blood sample to be tested and the reagent are added into the microfluidic device 200 through the upper opening of the tube body 2011. The liquid effusion support 203 is arranged below the sample injection tube 201, and a filter assembly 214 is arranged between the liquid effusion support 203 and the sample injection tube 201. The lower end of the tube body 2011 also has an opening, and the blood sample to be tested can pass through the tube body 2011 of the sample injection tube 201 and fall onto the filter assembly 214, and the filter assembly 214 plays a filtering role on the blood sample, thereby enriching the nucleated cells on the upper side of the filter assembly 214.

[0134] Furthermore, 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 further includes a buckle cover 202, which is sleeved on the tube body 2011 and is threadedly connected to the liquid accumulating liner 203.

[0135] Specifically, during the assembly of the microfluidic device 200, the filter assembly 214 is first placed between the snap-fit ​​portion 2012 of the sample injection tube 201 and the liquid effusion liner 203 below, and then the snap-fit ​​cover 202 is mounted on the upper end of the tube body 2011. After the snap-fit ​​cover 202 is installed to the lower end of the tube body 2011, it contacts the snap-fit ​​portion 2012, and then spirally descends to be threadedly connected with the liquid effusion liner 203, thereby pressing the snap-fit ​​portion 2012 to approach the liquid effusion liner 203, and compressing 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 effusion liner 203 only approach each other in the vertical direction, and there is no relative rotation between the two, thereby ensuring that the filter assembly 214 between the two is not displaced, and avoiding the position offset of the filter assembly 214 affecting the enrichment effect of nucleated cells.

[0136] In a further solution of this embodiment, the buckle cover 202 includes a horizontally arranged pressing portion 2021 for pressing down the buckle portion 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 provided on the inner side of the connecting portion 2022, and an external thread matching the internal thread is provided on the liquid accumulating liner 203.

[0137] Specifically, the liquid accumulating liner 203 includes a threaded portion 2031 and an operating portion 2032 disposed at the lower portion 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.

[0138] 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.

[0139] In the specific scheme of this embodiment, the filter assembly 214 includes a microfiltration membrane 212 and a water absorption layer 213. The water absorption 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 nucleated cells in the blood sample. The water absorption layer 213 is provided on the lower side of the microfiltration membrane 212 to absorb the filtrate passing through the microfiltration membrane 212.

[0140] In detail, in this embodiment, in order to achieve the enrichment of CTCs in the blood sample to be detected, 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, and preferably three water-absorbing layers 213 are provided. The water-absorbing layer 213 is a medium-speed neutral filter paper, and its diameter 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.

[0141] In this embodiment, the nucleated 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 to be tested is added to the microfluidic device 200, the CTCs therein can be screened out.

[0142] It was found through experimental tests that the three-layer water-absorbing layer 213 is provided on the lower side of the microfiltration membrane 212, which helps to support the microfiltration membrane 212 to maintain its posture, thereby ensuring the enrichment effect on CTC. At the same time, the three-layer water-absorbing layer 213 can quickly absorb the filtered sample filtrate or staining liquid (i.e., the reagent used for cell staining), avoiding the accumulation of liquid on the microfiltration membrane 212 due to liquid tension or viscosity, resulting in staining of the microfiltration membrane 212, and then causing the problem of blurred background of the staining result. Furthermore, the three-layer water-absorbing layer 213 can effectively disrupt the flow direction of the microfluid passing through each micropore on the microfiltration membrane 212, avoiding cell morphological damage caused by the laminar shear force of the microfluid. In addition, the microfluid passing through each micropore can also encounter resistance and form countless vortices, ensuring that the cells are evenly dispersed, which is helpful for subsequent detection procedures.

[0143] It is understandable that by replacing the microfiltration membrane 212 with micropores of different diameters, the integrated nucleated 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.

[0144] In this embodiment, by providing a buckle cover 202 threadedly connected to the liquid accumulation liner 203, the two will not rotate relative to each other when the sample injection tube 201 and the liquid accumulation liner 203 are approaching each other, thereby avoiding relative displacement between the microfiltration membrane 212 and the water absorption layer 213, which affects the enrichment effect.

[0145] In a further solution of this embodiment, the lower surface of the buckle portion 2012 is provided with a mounting groove 204 surrounding the circumference of the tube body 2011, which is used to provide a space for accommodating the filter assembly 214. The top of the threaded portion 2031 of the effusion lining 203 has a concave cavity, in which a lining net 205 is arranged, and the lining net 205 is clamped on the top of the threaded portion 2031, so as to cooperate with the mounting groove 204 to realize the placement of the filter assembly 214.

[0146] The lining mesh 205 has a dense mesh structure, so that it can support the filter assembly 214 without blocking the filtrate passing through the microfiltration membrane 212, further ensuring the enrichment effect of the cells on the upper side of the microfiltration membrane 212.

[0147] Furthermore, a diversion cavity is formed between the bottom wall of the cavity and the lining mesh 205, and 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.

[0148] Specifically, the bottom wall of the cavity gradually decreases from the outer circumference to the middle to form a funnel-shaped structure, and a guide hole 215 is provided at the lowest point of the funnel-shaped structure for discharging the filtrate.

[0149] More specifically, the interior of the liquid accumulating liner 203 is hollow, and the bottom wall of the concave cavity is formed by the middle of the inner wall of the threaded portion 2031 extending obliquely downward toward the central axis, and a circular guide hole 215 is formed at the lowest point. The outer periphery of the guide hole 215 extends vertically downward to a certain extent, and then extends obliquely toward the direction close to the central axis to form a guide cone 208, and the filtrate can be discharged outward along the guide cone 208.

[0150] 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 the buffer device provides negative pressure to the microfluidic device 200. 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.

[0151] 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.

[0152] 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 accumulation liner 203. The guide bar 206 is rod-shaped and extends radially along the liquid accumulation liner 203. The raised height gradually increases from the periphery to the center.

[0153] Specifically, a plurality of guide strips 206 are distributed at intervals along the circumference of the liquid lining 203 . After the filtrate passes through the filter assembly 214 and the lining mesh 205 , it can flow out from the guide holes 215 more quickly under the guiding effect of the guide strips 206 .

[0154] In the above scheme, the protrusion height of the guide bar 206 gradually increases from the periphery to the center. The purpose is that when the filtrate enters the guide cavity in a dripping state, the dripping filtrate contacts the guide bar 206. Under the action of gravity and liquid tension, the filtrate flows on the surface of the guide bar 206 and the funnel-shaped structure in the direction of gradually increasing protrusion height of the guide bar 206.

[0155] 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.

[0156] In this embodiment, the outer circumference of the upper end of the threaded portion 2031 of the liquid accumulating liner 203 protrudes upward to form an annular protruding structure, and the liner mesh 205 is radially limited by the protruding structure.

[0157] Furthermore, the filter assembly 214 is at least partially located inside the annular protruding structure, thereby limiting the filter assembly 214 in the radial direction, and further preventing the filter assembly 214 from being displaced during the assembly process of the microfluidic device 200 .

[0158] It should be noted that the height of the raised structure is not less than the thickness of the lining mesh 205, which is used to prevent the lining mesh 205 from being unstable when installed in the raised structure, and to prevent the filter component 214 from sliding when contacting the upper surface of the lining mesh 205, affecting the installation.

[0159] 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.

[0160] Specifically, one of the sealing gaskets is mounted on the threaded portion 2031 of the liquid accumulation liner 203. During the process of tightening the buckle cover 202 and the liquid accumulation liner 203, 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 on the liquid accumulation liner 203 meet, thereby achieving sealing.

[0161] Another sealing gasket is pre-installed in the installation groove 204 at the lower side of the buckle part 2012 of the sample injection tube 201. When the buckle cover 202 is tightened, the buckle part 2012 is pressed down, and the sealing gasket in the installation 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. When the buckle cover 202 is tightened, its pressing part 2021 and the buckle part 2012 of the sample injection tube 201 press the sealing gasket to achieve sealing.

[0162] 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.

[0163] In this embodiment, the blood sample is filtered in the microfluidic device 200 to enrich the nucleated 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.

[0164] 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.

[0165] Further, 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 passes through the peristaltic pump 330 to communicate with the atmosphere.

[0166] During the operation of the peristaltic pump 330, the gas in the buffer bottle 310 can be pumped out to form a negative pressure 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 in the microfluidic device 200 that passes through the filter assembly 214 into the first pipeline 311, and flowing along the first pipeline 311 into the buffer bottle 310.

[0167] When the peristaltic pump 330 generates pulses, 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 received by 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 impact of the pulse on the filtration process can be significantly reduced, thereby ensuring the enrichment effect of the cells in the microfluidic device 200.

[0168] 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 avoid that a small amount of filtrate enters the buffer bottle 310 and immediately submerges the lower end of the first pipeline 311, thereby affecting the buffer bottle 310's charging effect on the pulse flow.

[0169] In this embodiment, the liquid filtered out of the microfluidic device 200 is drawn out along the first pipeline 311 into 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 below 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, it will also cause one end of the first pipeline 311 in the bottle to 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 to avoid the problem of the lower end of the first pipeline 311 contacting the liquid in the bottle.

[0170] 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.

[0171] In a better solution of this embodiment, 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. In this way, 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.

[0172] 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 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, and will not affect the negative pressure state in the bottle. This not only ensures the negative pressure state in the bottle, but also greatly reduces the frequency of cleaning the liquid in the bottle.

[0173] 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.

[0174] In another specific solution of this embodiment, two or more buffer bottles may be arranged between the microfluidic device and the peristaltic pump, and the buffer bottles are connected in sequence through pipelines.

[0175] Specifically, at least a first buffer bottle and a second buffer bottle are arranged between the microfluidic device and the peristaltic pump. The mouth of the first buffer bottle is sealed and connected to a first pipeline, and the first pipeline is connected to a connecting bottom pipe below the microfluidic device. The mouth of the second buffer bottle is sealed and connected to a second pipeline, and the second pipeline is connected to the peristaltic pump.

[0176] 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.

[0177] Furthermore, in this embodiment, during the process of enriching CTCs and staining in the microfluidic device 200, it is necessary to aspirate the filtrate multiple times, especially when one aspiration process ends and the peristaltic pump 330 stops working, the problem of liquid reflux is inevitable. Therefore, in this 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 conducted from the microfluidic device 200 to the buffer bottle 310.

[0178] Thus, when the peristaltic pump 330 stops working, the filtrate in the first pipeline 311 cannot flow back through the one-way valve 3111 to the microfluidic device 200, thereby avoiding the influence of the filtrate backflow on the enrichment effect and further improving the accuracy of the detection result.

[0179] Furthermore, if Fig. 20 and Fig.21 As shown, the integrated nucleated cell enrichment and staining device of this embodiment adds the blood sample to be detected into the microfluidic device 200 through the blood sample adding unit. The blood sample adding unit is described in detail below.

[0180] The blood sample adding unit includes a pressure generating device 420 and a sealed blood sampling tube 410. The pressure generating device 420 is connected to the sealed blood sampling tube 410. The sealed blood sampling tube 410 is provided with a blood drain tube 430 connected to the outside. The pressure generating device 420 ventilates and pressurizes the sealed blood sampling tube 410, and the blood sample to be tested contained in the sealed blood sampling tube 410 is discharged along the blood drain tube 430. The liquid outlet of the blood drain tube 430 is arranged above the microfluidic device 200, so that the blood sample to be tested is added to the microfluidic device 200.

[0181] Specifically, in this embodiment, the sealed blood collection tube 410 includes a blood collection tube body 411 and a sealing cover 412. The sealing cover 412 can be a cap-type structure that can wrap the tube opening and the outer tube wall adjacent to the tube opening, or a plug-type structure that plugs into the tube opening and tightly abuts against the inner tube wall adjacent to the tube opening. After collecting the blood sample to be tested, the collected blood sample is added to the blood collection tube body 411, and then the tube opening of the blood collection tube body 411 is blocked with the sealing cover 412, thereby forming a sealed blood collection tube 410 with an airtight structure relative to the outside world.

[0182] Preferably, the sealing cover 412 includes a puncture portion for connecting the blood collection tube 430, and the puncture portion is directly opposite to the tube opening of the blood collection tube body 411. The puncture portion is made of a material with a certain elasticity, such as rubber, to ensure that the blood collection tube 430 and the sealed blood collection tube 410 are kept airtight when connected.

[0183] The puncture portion may be integrally formed with the sealing cover 412 , or may be a gasket disposed between the sealing cover 412 and the blood collection tube body 411 .

[0184] The pressure generating device 420 is connected to the sealed blood collection tube 410 via a pressurizing tube 440. The pressure generating device 420 of this embodiment may be a pressure generating device 420 that can be automatically controlled, or may be a pressure generating device 420 that needs to be manually operated.

[0185] When the pressure generating device 420 is an automatically controllable pressure generating device 420, preferably, the pressure generating device 420 is a peristaltic pump. The peristaltic pump includes a driver and a pump head, and the driver drives the pump head to squeeze the pressurizing tube 440, forming a unidirectional airflow in the pressurizing tube 440, and the unidirectional airflow flows into the sealed blood collection tube 410 through the pressurizing tube 440, thereby achieving 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.

[0186] When the pressure generating device 420 is a pressure generating device 420 that needs to be manually operated, preferably, a manually pushed and pulled syringe-type air pump is used.

[0187] The air outlet of the pressurizing tube 440 connected to the sealed blood collection tube 410 and the liquid inlet of the blood discharge tube 430 connected to the sealed blood collection tube 410 are both provided with a blood collection needle, and the blood collection needle includes an internal hollow needle head for puncturing the puncture part of the sealing cover 412, so that the pressurizing tube 440, the blood discharge tube 430 and the sealed blood collection tube 410 are connected.

[0188] 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 the blood samples to be tested contained in different sealed blood collection tubes 410 can be added to different microfluidic devices 200.

[0189] By adopting the above solution, the pressure generating device 420 can ventilate and pressurize the multiple sealed blood sampling tubes 410 respectively, so that the blood samples to be tested in the sealed blood sampling tubes 410 are discharged along the respective connected blood discharge tubes 430. This solution does not need to separately set a pressure generating device 420 for each sealed blood sampling tube 410, which not only reduces the number of pressure generating devices 420 and reduces the cost, but also simplifies the structure of the blood sampling unit and reduces the difficulty of repair and maintenance.

[0190] 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.

[0191] 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.

[0192] Furthermore, a main switch 441 is provided between the pressure generator 421 and the voltage divider 422 , which can block the connection between the pressure generator 421 and the voltage divider 422 .

[0193] Specifically, the pressure generator 421 and the voltage divider 422 are connected by a hose, and the main switch 441 is a device that can apply pressure to the hose. When the main switch 441 applies pressure to the hose, the hose is compressed and flattened, so that its inner wall is fitted, and the airflow cannot flow, which hinders the positive pressure of the pressure generator 421 from being transmitted to the voltage divider 422. When the main switch 441 releases the hose, the hose returns to its original state, and the airflow can flow normally, so that the positive pressure of the pressure generator 421 can be transmitted to the voltage divider 422.

[0194] Alternatively, the pressure generator 421 and the voltage divider 422 may be connected by a hard pipe, or a joint directly connected to each other may be provided between the pressure generator 421 and the voltage divider 422. The main switch 441 may be a switch device that can be inserted into the hard pipe and block the flow of gas in the hard pipe.

[0195] In the above scheme, a main switch 441 is provided to control the on-off between the voltage divider 422 and the pressure generator 421, and to control the start and stop of each sealed blood collection tube 410 to discharge blood outward.

[0196] 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 pressurizing tube 440 .

[0197] The sub-switches 442 can block the flow of air between each gas outlet and its corresponding sealed blood collection tube 410, and each sub-switches 442 can be opened and closed independently of each other. That is, the multiple sub-switches 442 can be all opened or all closed, or some can be opened and others closed.

[0198] The sub-switch 442 can be a manual switch, that is, the on / off state of each switch is set by manually opening and closing one by one. It can also be an automatic switch, that is, the opening and closing of the switch is automatically controlled according to whether each pressurized tube 440 is connected to the sealed blood collection tube 410. Specifically: the sub-switch 442 on the pressurized tube 440 connected to the sealed blood collection tube 410 or the connected sealed blood collection tube 410 with a 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 collection tube 410 or the connected sealed blood collection tube 410 without a blood sample is controlled to be closed, thereby cutting off the corresponding pressurized tube 440.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] The liquid inlet port of the drain tube 430 communicating with the sealed blood collection tube 410 is arranged near the bottom of the sealed blood collection tube 410. Preferably, the liquid inlet port of the drain tube 430 abuts 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 pressurizing tube 440, so that the air pressure in the sealed blood collection tube 410 rises, 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 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.

[0203] As a specific embodiment, the air outlet is provided with a blood collection needle, which penetrates the sealing cover 412 of the sealed blood collection tube 410. The needle tip of the blood collection needle is located inside the sealed blood collection tube 410 near the tube mouth, and this position is higher than the highest page of the blood sample in the sealed blood collection tube 410, that is, the needle tip of the blood collection needle will not touch the blood sample.

[0204] As another specific embodiment, the sealing cover 412 of the sealed blood collection tube 410 is provided with a connecting port for inserting the blood drainage tube 430 and the pressurizing tube 440. The blood drainage tube 430 and the pressurizing tube 440 are sealed with the connecting port and are plugged into the sealed blood collection tube 410 through the connecting port.

[0205] 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 .

[0206] Specifically, the sampling fixing table 450 includes a horizontally arranged table 451, on which a plurality of vertically penetrating positioning holes 457 are provided, and the liquid outlet of the drainage tube 430 passes through the positioning holes 457 of the table 451 from top to bottom. The aperture of the positioning holes 457 is the same as the outer diameter of the drainage tube 430, or slightly larger than the outer diameter of the drainage tube 430.

[0207] 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.

[0208] Furthermore, when the number of microfluidic devices 200 that can be set in the integrated nucleated 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 row of blood vessels 430 to move.

[0209] To this end, in this embodiment, the blood sample adding unit also includes a fixed frame (not shown in the figure), and the two sides of the sampling fixed table 450 have guide rails 455 arranged in parallel in the horizontal direction. The fixed frame includes a support frame 456 that cooperates with the two guide rails 455 of the sampling fixed table 450, and the guide rails 455 and the support frame 456 are slidably connected.

[0210] Specifically, the support frame 456 includes two symmetrically arranged parts, which are respectively located on both sides of the sampling fixing table 450 where the guide rail 455 is arranged. The support frame 456 is provided with a slide rail or a pulley that cooperates with the guide rail 455 .

[0211] 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.

[0212] 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.

[0213] As a specific implementation, the driving mechanism includes a third lead screw 454 and a motor 453. A slider is mounted 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 parallel to the guide rail 455 of the sampling fixing platform 450, and the sampling fixing platform 450 is fixedly connected to the slider on the third lead screw 454.

[0214] In the above solution, the third lead screw 454 cooperates with the threaded hole 452 on the slider, and 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 fixing platform 450 to move along the third lead screw 454. The motor 453 is connected to the support frame 456, and when the motor 453 drives the third lead screw 454 to rotate in different directions, the sampling fixing platform 450 will reciprocate with the slider on the third lead screw 454.

[0215] This embodiment also provides a method for cell enrichment and staining using the above-mentioned integrated device for nucleated cell enrichment and staining, which specifically includes the following steps:

[0216] (1) The reagent taking and placing unit adds microporous membrane activation liquid to the microfluidic device;

[0217] (2) adding a blood sample unit to add a blood sample to be tested into the microfluidic device, and the enrichment unit suction-filters the microfluidic device to enrich target cells;

[0218] (3) the reagent taking and placing unit sucks the red blood cell lysis solution and adds it to the microfluidic device to lyse the residual red blood cells, and then the dye enrichment unit performs suction filtration on the microfluidic device;

[0219] (4) The reagent taking and placing unit sucks the cell fixative and adds it to the microfluidic device, and then the dyeing unit performs suction filtration on the microfluidic device;

[0220] (5) the reagent taking and placing unit sucks the buffer solution and adds it to the microfluidic device for cleaning, and then the dyeing unit performs suction filtration on the microfluidic device;

[0221] (6) The reagent taking and placing unit absorbs the fixing liquid on the membrane and adds it to the microfluidic device, and then performs suction filtration on the microfluidic device;

[0222] (7) the reagent taking and placing unit absorbs the permeation liquid and adds it to the microfluidic device, and then the dyeing unit performs suction filtration on the microfluidic device;

[0223] (8) The reagent taking and placing unit absorbs the dyeing solution and adds it to the microfluidic device. After dyeing for a certain period of time, the rich dyeing unit performs suction filtration on the microfluidic device; step (8) is performed twice in total;

[0224] (9) The reagent taking and placing unit absorbs the buffer solution and adds it to the microfluidic device for cleaning, and then the enrichment unit performs suction filtration on the microfluidic device; step (9) is performed one to three times, after which the enriched and stained target cells are obtained.

[0225] In detail, the specific operation process of cell enrichment staining using the above-mentioned integrated device for nucleated cell enrichment and staining is as follows.

[0226] 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. The blood sample to be tested is pretreated whole blood, and the pretreatment can be carried out by 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 solution, red blood cell lysis solution, cell fixative, buffer solution, staining solution, etc., are added to the reagent holding device 1109 below the reagent containing device 110. It is also necessary to assemble the microfluidic device 200 in advance and install it on the connecting bottom tube 209 that has been fixed on the fixing table 210. When assembling the microfluidic device 200, the water absorption layer 213 therein needs to be pre-wetted.

[0227] After the preparation stage is completed, the integrated device can start working. First, the reagent containing device 110 in the reagent taking and placing unit is controlled to move, and the microporous membrane activation liquid is sucked by the driving motor 1012, and then the reagent containing device 110 moves to the top of the microfluidic device 200, and the driving motor 1012 runs in reverse to add the sucked microporous membrane activation liquid to the microfluidic device 200, so as to pretreat the microfiltration membrane 212.

[0228] 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 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, namely CTCs.

[0229] Afterwards, a similar control process is used to control the reagent placement unit again, so that the reagent containing device 110 absorbs an appropriate amount of red blood cell lysis solution and adds it to the microfluidic device 200 to lyse the small amount of remaining red blood cells. Then the peristaltic pump 330 is started to perform micro-flow suction filtration on the microfluidic device 200 to extract the filtered liquid.

[0230] Then, the reagent taking and placing unit is controlled to add an appropriate amount of cell fixative to the microfluidic device 200 to fix the target cells, and then the peristaltic pump 330 is started to perform micro-flow suction filtration on the microfluidic device 200 to extract the filtered liquid.

[0231] Then, the reagent taking and placing unit is controlled to add a proper amount of buffer solution 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, thereby cleaning and filtering once.

[0232] Afterwards, the fixing liquid on the membrane is added to the microfluidic device 200 by controlling the reagent taking and placing unit, and then the peristaltic pump 330 is started to perform micro-flow suction filtration on the microfluidic device 200 to extract the filtered liquid.

[0233] Then, the reagent taking and placing unit is controlled to add permeation liquid into 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.

[0234] Then, the cells are stained, including: adding staining liquid to the microfluidic device 200 by controlling the reagent taking and placing unit, and after a certain period of staining, starting the peristaltic pump 330 to perform micro-flow suction filtration on the microfluidic device 200 to extract the liquid. After the suction filtration is completed, the staining process is performed again.

[0235] 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 steps of adding buffer and then performing micro-flow suction filtration can be performed only once, or can be repeated twice or three times.

[0236] 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.

[0237] The above operation process can be automatically controlled by a pre-set program, so that the integrated nucleated 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. The operator only needs to add the blood sample to be tested and the required reagents in advance, and then the microfiltration membrane 212 can be directly taken out for microscopic examination after the integrated nucleated cell enrichment and staining device is completed.

[0238] Embodiment 2

[0239] like Fig. 22 and Fig.23 As shown, the difference between this embodiment and the above-mentioned embodiment 1 is that the buffer component 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.

[0240] In this embodiment, since part of the wall of the buffer chamber can be elastically deformed, during the operation of the peristaltic pump 330, the air in the buffer chamber can be evacuated to reduce the pressure, causing the wall of the buffer chamber to deform inward. When the peristaltic pump 330 generates a pulse flow, the wall of the buffer chamber will be further concave, reducing the volume inside the buffer chamber, thereby achieving the effect of buffering pulses.

[0241] In the specific solution of this embodiment, the buffer 340 includes a bracket 343 with a hollow interior and a buffer film 344 that can undergo elastic deformation. The bracket 343 is provided with a plurality of through holes, and the buffer film 344 at least covers all of the through holes. Thus, the buffer film 344 and the bracket 343 together enclose the buffer chamber, and the portion of the buffer film 344 covering the through holes forms a cavity wall that can undergo elastic deformation.

[0242] With the above structure, during the operation of the peristaltic pump 330, the portion of the buffer film 344 covering the through hole can be recessed into the inside of the bracket 343 through the through hole. When the peristaltic pump 330 generates a pulse flow, the degree to which the buffer film 344 is recessed into the inside of the bracket 343 increases, thereby reducing the internal volume of the buffer chamber and playing a role in buffering pulses.

[0243] 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.

[0244] 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.

[0245] Furthermore, 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.

[0246] Preferably, the support rods 3431 are evenly distributed along the outer circumference 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.

[0247] 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.

[0248] 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.

[0249] In a further solution of this embodiment, a gas port is respectively provided on the two end walls 345. One of the gas ports is connected to the first pipeline 311, thereby communicating with the microfluidic device 200 through the first pipeline 311. The other gas port is connected to the second pipeline 312, and communicates with the peristaltic pump 330 through the second pipeline 312.

[0250] Furthermore, the gas port is configured as a trumpet-shaped structure extending outward from the end wall 345 and gradually decreasing in diameter, and the first pipeline 311 and the second pipeline 312 are both hoses, which are mounted on the ends of the trumpet-shaped structure. Since the cross-sectional area of ​​the buffer 340 is larger than the cross-sectional area of ​​the first pipeline 311 and the second pipeline 312, by configuring the trumpet-shaped structure, the amplitude of the sudden change of the cross-sectional area is reduced on the path passing through the first pipeline 311, the buffer 340 and the second pipeline 312 in sequence.

[0251] 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 film 344 wrapped on the bracket 343 can be deformed and contracted toward the inside of the bracket 343, so that the internal volume of the buffer chamber is reduced, thereby offsetting or partially offsetting the sudden change in the negative pressure generated by the pulse flow, thereby preventing the suction force on the microfluidic device 200 from having an obvious peak, thereby avoiding the influence of the pulse flow on the experimental results.

[0252] 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 .

[0253] Specifically, in this embodiment, the one-way valve 3111 is arranged on the second pipeline 312, and the buffer 340 is unidirectionally connected 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. 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.

[0254] 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.

[0255] Embodiment 3

[0256] like Fig.24 and Fig.25 As shown, this embodiment is a further limitation of the above-mentioned embodiment 1 or 2, and the blood drainage tube 430 is provided with an openable and closable sealing structure.

[0257] Specifically, when the blood sample to be tested 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. In this way, 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 normally discharge blood samples to the outside.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] Furthermore, the upper part of the pressure stabilizing chamber 437 is a barrel-shaped structure, and the upper edge of the funnel-shaped structure 439 is connected to the lower edge of the barrel-shaped 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 barrel-shaped 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.

[0264] With the above structure, when the blood sample in the pressure stabilizing chamber 437 is completely discharged and the float 438 descends due to gravity, the float 438 will sink along the inner wall of the surrounding side of the funnel-shaped structure 439 to the liquid outlet of the pressure stabilizing chamber 437, thereby avoiding the problem of deviation in the landing point of the float 438 when it descends, resulting in the failure to completely block the liquid outlet of the pressure stabilizing chamber 437 and causing positive pressure leakage.

[0265] In this embodiment, when the pressure generating device 420 is connected to multiple sealed blood collection tubes 410 at the same time, the multiple sealed blood collection tubes 410 can be ventilated and pressurized at the same time, and the blood samples can be pressed out of each sealed blood collection tube 410 and added to the corresponding microfluidic device 200. Since the amount of blood samples to be tested 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, so as to prevent the blood drainage tube 430 from leaking the positive pressure provided by the pressure generating device 420, and avoid affecting the process of adding blood samples to other sealed blood collection tubes 410.

[0266] 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 nucleated cell enrichment and staining integrated device, characterized in that: include: A dye enrichment unit, including a microfluidic device, is used to receive a blood sample to be tested and enrich the target substance therefrom; A blood sample adding unit, used for adding a blood sample to be tested into the microfluidic device; A reagent taking and placing unit, used for taking the reagent and adding it into the microfluidic device; The microfluidic device comprises: The sample injection tube comprises a tube body, the upper end of the tube body is opened for receiving the blood sample to be tested, 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 to be tested 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, and the buckle cover is spirally descended to be threadedly connected with the liquid accumulation liner, and the buckle part is pressed down to approach the liquid accumulation liner to press the filter component.

2. The integrated nucleated cell enrichment and staining device according to claim 1, 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.

3. The integrated nucleated cell enrichment and staining device according to claim 1 or 2, characterized in that: The enrichment unit also includes a buffer device, including: a peristaltic pump, connected to the microfluidic device, for pumping the filtrate; The buffer component is arranged between the microfluidic device and the peristaltic pump, and is communicated with the microfluidic device and the peristaltic pump respectively.

4. The integrated nucleated cell enrichment and staining device according to claim 3, characterized in that: The buffer assembly comprises a buffer bottle respectively connected to the microfluidic device and the peristaltic pump, wherein a preset amount of gas is sealed in the buffer bottle; Alternatively, the buffer component includes a buffer, the interior of the buffer is hollow to form a buffer chamber, the buffer chamber is connected to the microfluidic device and the peristaltic pump respectively, and at least part of the cavity wall of the buffer chamber is made of a material that can undergo elastic deformation.

5. The integrated nucleated cell enrichment and staining device according to claim 1 or 2, characterized in that: The reagent taking and placing unit comprises: A reagent containing device, used for storing the absorbed reagent; A vacuum pumping device, comprising a vacuum cylinder connected to the reagent containing device, and a piston assembly disposed inside the vacuum cylinder and capable of reciprocating relative to the vacuum cylinder; The driving device is used to drive the vacuum cylinder and the piston assembly to produce relative movement, to evacuate air from the reagent containing device to reduce pressure or to ventilate and increase pressure in the reagent containing device.

6. The integrated nucleated cell enrichment and staining device according to claim 5, characterized in that: The piston assembly comprises a piston and a push rod; the piston is arranged inside the vacuum cylinder and is in sealing contact with the inner wall of the vacuum cylinder; one end of the push rod is connected to the piston, and the other end extends to the outside of the vacuum cylinder; The push rod is fixedly arranged; the driving device drives the vacuum cylinder to reciprocate along the axis of the push rod, evacuates air from the reagent containing device and reduces pressure to absorb the reagent into the reagent containing device, and ventilates and pressurizes the reagent containing device to discharge the reagent from the reagent containing device.

7. The integrated nucleated cell enrichment and staining device according to claim 6, characterized in that: The vacuum device also includes a vacuum fixing frame, and the vacuum cylinder is relatively fixedly mounted on the vacuum fixing frame; The driving device includes a driving motor and a transmission screw, and the vacuum fixing frame is connected to a slider installed on the transmission screw; the driving motor drives the transmission screw to rotate, and the vacuum fixing frame moves along the extension direction of the transmission screw with the slider, driving the vacuum cylinder to move along the extension direction of the transmission screw.

8. The integrated nucleated cell enrichment and staining device according to claim 5, characterized in that: The reagent taking and placing unit also includes a moving mechanism for driving the reagent containing device to move to above the reagent containing device storing the reagent to be added to absorb the reagent, and driving the reagent containing device to move to above the microfluidic device to add the reagent.

9. The integrated nucleated cell enrichment and staining device according to claim 1 or 2, characterized in that: The blood sample adding unit comprises a pressure generating device and a sealed blood sampling tube, wherein the pressure generating device is in communication with the sealed blood sampling tube, and the sealed blood sampling tube is provided with a blood drain tube in communication with the outside; The pressure generating device ventilates and pressurizes the sealed blood collection tube, and the blood sample to be tested contained in the sealed blood collection tube is discharged along the blood discharge tube and added into the microfluidic device.

10. A method for cell enrichment and staining using the integrated nucleated cell enrichment and staining device according to any one of claims 1 to 9, characterized in that: The following steps are involved: (1) The reagent taking and placing unit adds microporous membrane activation liquid to the microfluidic device; (2) adding a blood sample unit to add a blood sample to be tested into the microfluidic device, and the enrichment unit suction-filters the microfluidic device to enrich target cells; (3) the reagent taking and placing unit sucks the red blood cell lysis solution and adds it to the microfluidic device to lyse the residual red blood cells, and then the dye enrichment unit performs suction filtration on the microfluidic device; (4) The reagent taking and placing unit sucks the cell fixative and adds it to the microfluidic device, and then the dyeing unit performs suction filtration on the microfluidic device; (5) the reagent taking and placing unit sucks the buffer solution and adds it to the microfluidic device for cleaning, and then the dyeing unit performs suction filtration on the microfluidic device; (6) The reagent taking and placing unit absorbs the fixing liquid on the membrane and adds it to the microfluidic device, and then performs suction filtration on the microfluidic device; (7) the reagent taking and placing unit absorbs the permeation liquid and adds it to the microfluidic device, and then the dyeing unit performs suction filtration on the microfluidic device; (8) The reagent taking and placing unit absorbs the dyeing solution and adds it to the microfluidic device. After dyeing for a certain period of time, the rich dyeing unit performs suction filtration on the microfluidic device; step (8) is performed twice in total; (9) The reagent taking and placing unit absorbs the buffer solution and adds it to the microfluidic device for cleaning, and then the enrichment unit performs suction filtration on the microfluidic device; step (9) is performed one to three times, after which the enriched and stained target cells are obtained.

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