Enrichment and dyeing integrated equipment and method for circulating tumor cells

Abstract

The invention discloses enriching and dyeing integrated equipment and method for circulating tumor cells, and the enriching and dyeing integrated equipment comprises a microfluidic device which 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 blood sample adding unit comprises a blood collection tube used for containing a blood sample to be detected; the blood sample temporary storage assembly is communicated with the blood collection tube; and the pressure generating device is communicated with the blood sample temporary storage assembly, sucks the blood sample to be detected into the blood sample temporary storage assembly from the blood collection tube through air exhaust pressure reduction and ventilation pressure increase, discharges the blood sample from the blood sample temporary storage assembly, and adds the blood sample to the micro-fluidic device. By adopting the enriching and dyeing integrated equipment, after a blood sample to be detected is added into the blood collection tube, the enriching and dyeing integrated equipment can autonomously complete the whole process from blood sample conveying to target substance enriching and dyeing, and the detection efficiency can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical instruments, and in particular, relates to an integrated device and method for enriching and staining circulating tumor cells. 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 was expensive, the detection method achieved using the device had poor specificity, and it was not widely popularized.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide an integrated device and method for enriching and staining circulating tumor cells. After adding the blood sample to be tested, the integrated device for enriching and staining can independently complete the whole process from blood sample transportation to target substance enrichment and staining. The operation is simple, which improves the detection efficiency.

[0009] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0010] An integrated device for enrichment and staining of circulating tumor cells, comprising:

[0011] A microfluidic device, used for receiving a blood sample to be tested and enriching a target substance therefrom;

[0012] A blood sample adding unit, used for adding a blood sample to be tested into the microfluidic device;

[0013] A reagent taking and placing unit, used for taking the reagent and adding it into the microfluidic device;

[0014] The blood sample adding unit comprises:

[0015] Blood collection tube, used to hold the blood sample to be tested;

[0016] A blood sample temporary storage component, connected to the blood collection tube;

[0017] A pressure generating device, connected to the blood sample temporary storage component;

[0018] The pressure generating device evacuates air and reduces pressure from the blood sample temporary storage component, and sucks the blood sample to be tested from the blood collection tube into the blood sample temporary storage component; the pressure generator ventilates and pressurizes the blood sample temporary storage component, and discharges the blood sample to be tested from the blood sample temporary storage component and adds it to the microfluidic device.

[0019] Furthermore, the blood sample temporary storage assembly includes a middle sealed tube, and a blood drawing tube and a blood discharge tube sealedly connected to the middle sealed tube;

[0020] The blood drawing tube is connected to the blood sampling tube, and the blood sample to be tested in the blood sampling tube is sucked into the middle sealed tube along the blood drawing tube; the blood sample to be tested in the middle sealed tube can be discharged along the blood discharge tube and added into the microfluidic device.

[0021] Furthermore, the pressure generating device is connected to the middle sealed tube through a pressurized tube, and one end of the pressurized tube extending into the middle sealed tube is arranged inside the middle sealed tube near the tube opening; the liquid inlet end of the drainage blood tube is arranged near the bottom of the middle sealed tube.

[0022] Furthermore, a blocking structure is provided on the blood drainage tube;

[0023] When the blood sample to be tested enters the blood drainage tube from the middle sealing tube, the blocking structure conducts the blood drainage tube to discharge the blood sample to be tested; after the blood sample to be tested stops entering the blood drainage tube, the blocking structure blocks the blood drainage tube.

[0024] Furthermore, the blood drainage tube has a vertical extension portion; the blocking structure is arranged on the vertical extension portion, and includes a pressure stabilizing chamber with a floating block arranged inside, and the density of the floating block is less than the density of the blood sample;

[0025] The blood sample to be tested enters the pressure stabilizing chamber along the blood drainage tube, and the floating block floats up to keep the blood drainage tube conductive; after the blood sample to be tested stops entering the blood drainage tube, the blood sample in the pressure stabilizing chamber completely flows out, and the floating block falls to block the liquid outlet at the bottom of the pressure stabilizing chamber, thereby blocking the blood drainage tube.

[0026] Furthermore, the blood sample adding unit includes a plurality of blood sampling tubes and a plurality of blood sample temporary storage components, and the plurality of blood sample temporary storage components are connected to the plurality of blood sampling tubes in a one-to-one correspondence; and the plurality of blood sample temporary storage components are all connected to the same pressure generating device.

[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 reagent taking and placing driving device is used to drive the vacuum cylinder and the piston assembly to produce the same movement.

[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 reagent taking and placing driving device drives the vacuum cylinder to move back and forth along the axis of the push rod to absorb the reagent into the reagent containing device, or discharge the reagent from the reagent containing device.

[0033] Furthermore, the microfluidic device comprises:

[0034] 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;

[0035] 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;

[0036] The buckle cover is sleeved on the tube body of the sample injection tube. The buckle cover is spirally descended and threadedly connected with the liquid accumulation liner. The buckle part of the sample injection tube is pressed down to approach the liquid accumulation liner, thereby pressing the filter assembly.

[0037] Furthermore, the buckle cover comprises a pressing part which is horizontally arranged and used to press down the buckle part, and a through hole is formed on the pressing part for the pipe body to pass through;

[0038] 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.

[0039] A method for cell enrichment and staining using the above-mentioned integrated device for enrichment and staining of circulating tumor cells comprises the following steps:

[0040] (1) The reagent taking and placing unit adds microporous membrane activation liquid to the microfluidic device;

[0041] (2) the pressure generating device of the blood sample unit evacuates air and reduces pressure from the blood sample temporary storage component, and the blood sample to be tested is sucked into the blood sample temporary storage component from the blood collection tube, and then the pressure generating device ventilates and pressurizes the blood sample temporary storage component, and the blood sample to be tested is discharged from the blood sample temporary storage component and added to the microfluidic device; the microfluidic device is suction filtered to enrich the target cells;

[0042] (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;

[0043] (4) The reagent taking and placing unit sucks the cell fixative and adds it to the microfluidic device, and then performs suction filtration on the microfluidic device;

[0044] (5) The reagent taking and placing unit sucks the buffer solution and adds it to the microfluidic device for cleaning, and then performs suction filtration on the microfluidic device;

[0045] (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;

[0046] (7) the reagent taking and placing unit absorbs the permeation liquid and adds it to the microfluidic device, and then performs suction filtration on the microfluidic device;

[0047] (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 microfluidic device is filtered by suction; step (8) is performed twice in total;

[0048] (9) The reagent taking and placing unit absorbs the buffer solution and adds it to the microfluidic device for cleaning, and then the microfluidic device is filtered; step (9) is performed one to three times, after which the enriched and stained target cells are obtained.

[0049] After adopting the above technical scheme, the present invention has the following beneficial effects compared with the prior art.

[0050] In the present invention, the operator only needs to add the blood sample to be tested into the blood collection tube, and then the integrated enrichment and staining device can independently complete the whole process from blood sample transportation to cell enrichment and staining, so as to achieve more efficient detection. The blood sample transportation process is controlled by the vacuum decompression and ventilation pressurization operation of the pressure generating device. The pressure generating device is connected to the blood sample temporary storage component, so that the blood sample to be tested is first sucked into the blood sample temporary storage component by the blood collection tube, and then discharged from the blood sample temporary storage component. The blood collection tube itself does not need to be sealed, so after the operator adds the blood sample to be tested therein, there is no need to perform additional sealing operations, which simplifies the operator's use procedures and reduces the workload.

[0051] In the present invention, a sealing structure that can be opened and closed automatically is provided on the blood drainage tube to control the on-off state of the blood drainage tube, especially when the same pressure generating device is connected to multiple intermediate sealed tubes, if the blood samples in some of the intermediate sealed tubes are completely discharged, the corresponding blood drainage tube can be blocked by the sealing structure, thereby preventing the positive pressure introduced by the pressure generating device from leaking from the blood drainage tube and affecting the process of other intermediate sealed tubes receiving the positive pressure to discharge the blood samples outward.

[0052] In the present invention, the reagent taking and placing unit is provided with a reagent containing device for storing the sucked reagent, and the vacuum cylinder is only used to cooperate with the piston assembly to provide positive pressure or negative pressure, and the sucked 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.

[0053] In the present invention, the microfluidic device for enriching the target substance comprises an injection tube, a liquid effusion liner and a buckle cover. During the process of rotating and tightening the buckle cover and the liquid effusion liner, the buckle part of the injection tube can be pressed down to approach the liquid effusion liner, thereby pressing the filter assembly between the two. Since there is no relative rotation between the injection tube and the liquid effusion liner, it can be ensured that the filter assembly does not move, thereby ensuring that the effect of the microfluidic device in enriching the target substance from the blood sample is not affected.

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

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

[0056] Figure 1 is a schematic structural diagram of a vacuum pumping device in an embodiment of the present invention;

[0057] Figure 2 is a schematic diagram of a first limiting plate in an embodiment of the present invention;

[0058] 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;

[0059] Figure 4 is a schematic diagram of the installation of a reagent containing device in an embodiment of the present invention;

[0060] Figure 5 is a schematic structural diagram of a reagent containing device and a moving mechanism in an embodiment of the present invention;

[0061] Figure 6 is an exploded view of a microfluidic device according to an embodiment of the present invention;

[0062] Figure 7 is a schematic diagram of an assembled microfluidic device in an embodiment of the present invention;

[0063] Figure 8 is a partial cross-sectional view of a sample injection tube in an embodiment of the present invention;

[0064] Fig. 9 is a top view of the sample injection tube in an embodiment of the present invention;

[0065] Fig.10 is a partial cross-sectional view of a buckle cover in an embodiment of the present invention;

[0066] Fig.11 The present invention Fig.10 A-direction schematic diagram of the middle buckle cover;

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

[0068] Fig.13 is a top view of a liquid accumulation liner in an embodiment of the present invention;

[0069] Fig.14 is a top view of the lining mesh in an embodiment of the present invention;

[0070] Fig.15 is a schematic diagram of the connection between the bottom pipe and the rubber hose in an embodiment of the present invention;

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

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

[0073] Fig.18 is a side view of the connecting bottom pipe installed on the fixing platform in the embodiment of the present invention;

[0074] 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;

[0075] Fig. 20 is a schematic structural diagram of a blood sample adding unit in Embodiment 1 of the present invention;

[0076] Fig.21 It is a schematic diagram of the overall structure of the blood sample adding unit in Embodiments 1 and 2 of the present invention;

[0077] Fig. 22 It is a partial structural schematic diagram of a blood sample adding unit in the second embodiment of the present invention;

[0078] Fig.23 The present invention Fig. 22 A magnified schematic diagram of point B in the middle.

[0079] Fig.24 is a schematic diagram of the connection between the buffer device and the microfluidic device in the third embodiment of the present invention;

[0080] Fig.25 is a schematic diagram of a partially cutaway buffer film in a buffer according to a third embodiment of the present invention;

[0081] 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;

[0082] 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;

[0083] 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;

[0084] 411. Blood collection tube; 413. First one-way valve; 415. Blood collection tube; 420. Pressure generating device; 421. Pressure generator; 422. Voltage divider; 430. Blood drainage tube; 431. Middle sealing tube; 432. Middle tube; 433. Middle sealing cover; 434. Second one-way valve; 435. Control valve; 437. Pressure stabilizing chamber; 438. Float; 439. Funnel-shaped structure; 440. Pressurizing tube; 443. Pressurizing switch; 450. Sampling fixing table; 457. Positioning hole.

[0085] 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

[0086] 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.

[0087] 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.

[0088] 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.

[0089] like Figures 1 to 25 As shown, an embodiment of the present invention provides an integrated enrichment and staining device for realizing the complete detection process of transporting a sample to be detected (such as a blood sample to be detected), adding one or more reagents, and enriching a target substance from the sample to be detected.

[0090] Specifically, the enrichment and staining integrated device of the embodiment of the present invention includes a reagent taking and placing unit, an enrichment and staining unit and a blood sample adding unit. The blood sample adding unit adds the blood sample to be detected 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 detected 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.

[0091] Embodiment 1

[0092] like Figures 1 to 21 As shown, the integrated 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.

[0093] Specifically, the enrichment and staining integrated 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 exfoliated cells in the blood sample, such as nucleated pathological cells. More specifically, the enrichment and staining integrated device described in this embodiment is used to achieve CTC detection, in which case the target substance enriched from the blood sample is circulating tumor cells (CTC).

[0094] In this embodiment, the reagent taking and placing unit of the enrichment and dyeing integrated device is used to absorb the reagent and then add the absorbed reagent to the enrichment and 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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 enrichment unit includes a microfluidic device 200 for receiving a blood sample to be tested, and the CTC in the blood sample is enriched and stained 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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 enrichment and staining integrated 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 reagent containing device 1109 directly above the required reagent. 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.

[0130] 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.

[0131] 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.

[0132] The above process can be repeated multiple times to achieve the addition of different reagents, or multiple additions of the same reagent.

[0133] The enrichment and dyeing unit of the integrated enrichment and dyeing device described in this embodiment is described in detail below.

[0134] Specifically, Figures 6 to 19 As shown, the enrichment unit described in this embodiment includes a microfluidic device 200 and a buffer device. Among them, the microfluidic device 200 is used to receive the blood sample 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 CTC in the blood sample is intercepted by the microfluidic device 200, thereby achieving the enrichment and staining of CTC in the microfluidic device 200.

[0135] In this embodiment, the microfluidic device 200 includes an injection tube 201 and a liquid accumulating support 203. The 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 accumulating support 203 is arranged below the injection tube 201, and a filter assembly 214 is arranged between the liquid accumulating support 203 and the 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 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 CTCs on the upper side of the filter assembly 214.

[0136] 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.

[0137] 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 accumulating support 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 at 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 accumulating support 203, thereby pressing the snap-fit ​​portion 2012 to approach the liquid accumulating support 203, and pressing the filter assembly 214 between the two. In this way, during the assembly of the microfluidic device 200, the sample injection tube 201 and the liquid accumulating support 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 CTCs.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] In the specific solution of this embodiment, the filter assembly 214 includes a microfiltration membrane 212 and a water-absorbing layer 213. The water-absorbing layer 213 is made of a water-absorbing material and can be provided with one or more layers. Among them, the microfiltration membrane 212 is used to achieve a filtering effect and intercept CTCs in the blood sample. The water-absorbing layer 213 is provided on the lower side of the microfiltration membrane 212 to absorb the filtrate passing through the microfiltration membrane 212.

[0142] 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.

[0143] In this embodiment, the target cells enriched in the microfluidic device 200 are CTCs, and 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.

[0144] 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.

[0145] It is understandable that by replacing the microfiltration membrane 212 with micropores of different diameters, the enrichment and staining integrated device of this embodiment can be used to enrich other target substances, such as target organisms or biomacromolecules in the enriched sample.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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 .

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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 .

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] In this embodiment, the blood sample is filtered in the microfluidic device 200 to enrich the CTC 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] Furthermore, if Fig. 20 and Fig.21 As shown, the integrated 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.

[0182] like Fig. 20 As shown, the blood sample adding unit includes a blood sampling tube 411, a blood sample temporary storage component and a pressure generating device 420. The blood sample to be tested is placed in the blood sampling tube 411, the blood sample temporary storage component is connected to the blood sampling tube 411, and the pressure generating device 420 is connected to the blood sample temporary storage component. When a blood sample needs to be added, the pressure generating device 420 first evacuates and decompresses the blood sample temporary storage component, so that the blood sample to be tested is sucked into the blood sample temporary storage component from the blood sampling tube 411. Then, the pressure generating device 420 ventilates and pressurizes the blood sample temporary storage component, so that the blood sample to be tested is discharged from the blood sample temporary storage component and added to the microfluidic device 200.

[0183] Specifically, in this embodiment, the blood sample temporary storage assembly includes an intermediate sealed tube 431, and a blood sampling tube 415 and a blood discharge tube 430 that are sealed and connected to the intermediate sealed tube 431. The blood sampling tube 415 is connected to the blood sampling tube 411, and the blood sample to be tested in the blood sampling tube 411 is sucked into the intermediate sealed tube 431 along the blood sampling tube 415. The liquid outlet end of the blood discharge tube 430 is located above the microfluidic device 200. When the pressure generating device 420 ventilates and pressurizes the intermediate sealed tube 431, the blood sample to be tested in the intermediate sealed tube 431 is discharged along the blood discharge tube 430, and is thus added to the microfluidic device 200.

[0184] More specifically, the middle sealing tube 431 includes an open middle tube 432 and a middle sealing cover 433. The middle sealing cover 433 can adopt a cover structure that can wrap the tube mouth of the middle tube 432 and the outer wall adjacent to the tube mouth, or a plug structure that can be inserted into the tube mouth and tightly abut against the inner wall of the tube mouth.

[0185] In this embodiment, the pressure generating device 420 is connected to the middle sealing tube 431 through the pressurizing tube 440, and can provide both positive pressure and negative pressure. Preferably, the pressure generating device 420 in this embodiment is a peristaltic pump that can provide both positive pressure and negative pressure.

[0186] With the blood sample adding unit of the above structure, since the blood sampling tube 411 does not need to receive positive pressure to discharge the blood sample, the blood sampling tube 411 can be set open without being sealed. The operator puts the blood sample to be tested collected from the patient's body into the blood sampling tube 411, and then inserts the blood extraction tube 415 from the upper open side of the blood sampling tube 411, and then starts the pressure generating device 420 to transport the blood sample, without the need for additional sealing treatment of the blood sampling tube 411, thereby simplifying the operation process. In addition, during the blood sample transportation process, the pressure generating device 420 is only used to provide positive pressure and negative pressure, and the blood sample itself does not flow through the pressure generating device 420, thereby avoiding contamination of the blood sample.

[0187] Furthermore, the middle sealing cover 433 includes a puncture portion for connecting the blood drainage tube 430, the blood extraction tube 415 and the pressurizing tube 440, and the puncture portion is opposite to the tube mouth of the middle tube 432. The puncture portion is made of a material with a certain elasticity, such as rubber, so as to maintain airtightness when the blood drainage tube 430, the blood extraction tube 415 and the pressurizing tube 440 are connected with the middle sealing tube 431.

[0188] The puncture portion may be integrally provided with the intermediate sealing cover 433 , or may be a gasket provided between the intermediate sealing cover 433 and the intermediate tube 432 .

[0189] Furthermore, the liquid outlet end of the blood drawing tube 415, the liquid inlet end of the blood draining tube 430, and one end of the pressurizing tube 440 extending into the middle sealed tube 431 are all provided with a blood collection needle, and the blood collection needle penetrates the puncture part of the middle sealed tube 431, so that the blood drawing tube 415, the blood draining tube 430 and the pressurizing tube 440 are connected with the inside of the middle sealed tube 431.

[0190] In a further solution of this embodiment, a first one-way valve 413 is provided on the blood collection tube 415 to conduct one-way communication from the blood collection tube 411 to the middle sealing tube 431, and a second one-way valve 434 is provided on the blood discharge tube 430 to conduct one-way communication from the inside of the middle sealing tube 431 to the outside.

[0191] By providing the first one-way valve 413 and the second one-way valve 434, when the pressure generating device 420 performs air extraction and pressure reduction, the internal pressure of the intermediate sealed tube 431 decreases, and the first one-way valve 413 can conduct the blood sampling tube 415 under the pressure difference, so that the blood sample is sucked into the intermediate sealed tube 431 along the blood sampling tube 415, while the second one-way valve 434 cannot conduct from the outside to the inside, so the negative pressure environment in the intermediate sealed tube 431 will not leak through the blood discharge tube 430. Similarly, when the pressure generating device 420 performs ventilation and pressure increase, the internal pressure of the intermediate sealed tube 431 increases, and the second one-way valve 434 conducts the blood discharge tube 430 under the pressure difference, so that the blood sample can be discharged outward along the blood discharge tube 430, while the first one-way valve 413 cannot conduct from the intermediate sealed tube 431 to the blood sampling tube 411, and thus the positive pressure environment in the intermediate sealed tube 431 will not leak through the blood sampling tube 415, thereby ensuring the discharge efficiency of the blood sample.

[0192] In a further solution of this embodiment, one end of the pressurizing tube 440 extending into the middle sealed tube 431 is arranged at a position near the tube opening inside the middle sealed tube 431. The maximum volume of the blood sample to be tested in the blood collection tube 411 should satisfy the following requirement: when all the blood sample to be tested in the blood collection tube 411 is sucked into the middle sealed tube 431, the liquid level of the blood sample in the middle sealed tube 431 is lower than the height of the tube opening of the pressurizing tube 440. In this way, it can be ensured that the pressurizing tube 440 is never in direct contact with the blood sample, and it can be avoided that the pressurizing tube 440 sucks in the blood sample when the pressure generating device 420 is evacuating and reducing pressure, thereby avoiding malfunction caused by the blood sample being sucked in by the pressurizing tube 440.

[0193] On the other hand, the mouth of the pressurizing tube 440 does not directly contact the blood sample, and when the pressure generating device 420 performs ventilation and pressurization, it can also prevent the pressurized airflow from flowing into the blood sample in the middle sealing tube 431 to generate bubbles and affect the effect of blood sample discharge. When a large number of bubbles are generated in the blood sample, it is not easy to discharge them completely, and the blood sample discharged in the form of bubbles has a reduced filtration efficiency, which affects the subsequent process of cell enrichment in the microfluidic device 200.

[0194] The liquid inlet end of the blood drainage tube 430 is arranged close to the bottom of the middle sealed tube 431. Preferably, the liquid inlet port of the blood drainage tube 430 abuts against the bottom of the middle sealed tube 431. When the pressure generating device 420 ventilates and pressurizes the middle sealed tube 431, the air pressure in the middle sealed tube 431 rises, and the blood sample therein can be pressed into the blood drainage tube 430 from the liquid inlet port of the blood drainage tube 430. The liquid inlet port of the blood drainage tube 430 abuts against the bottom of the middle sealed tube 431, so that the blood sample sucked into the middle sealed tube 431 can all enter the blood drainage tube 430, avoiding blood sample residue.

[0195] Furthermore, the liquid inlet of the blood sampling tube 415 is arranged close to the bottom of the blood sampling tube 411. Preferably, the liquid inlet of the blood sampling tube 415 abuts the bottom of the blood sampling tube 411. When the pressure generating device 420 evacuates air and reduces pressure through the middle sealed tube 431, the air pressure in the middle sealed tube 431 decreases, and the blood sampling tube 411 is connected to the atmospheric pressure, and then the blood sample therein can be pressed into the blood sampling tube 415 from the liquid inlet of the blood sampling tube 415 by the atmospheric pressure, and then transported to the middle sealed tube 431. The liquid inlet of the blood sampling tube 415 abuts the bottom of the blood sampling tube 411, so that all the blood samples contained in the blood sampling tube 411 can be sent into the middle sealed tube 431 to avoid blood sample residue.

[0196] In the preferred solution of this embodiment, the liquid outlet of the blood sampling tube 415 is arranged close to the tube opening of the middle sealed tube 431, and will never touch the liquid surface of the blood sample in the middle sealed tube 431 during the blood sample transportation process. In this way, when the pressure generating device 420 evacuates the pressure from the middle sealed tube 431, the liquid outlet of the blood sampling tube 415 is always directly exposed to the negative pressure environment inside the middle sealed tube 431, avoiding the problem of increasing the resistance of the blood sample being discharged from the liquid outlet of the blood sampling tube 415 when the liquid outlet is immersed in the blood sample.

[0197] In a further embodiment of this embodiment, Fig.21 As shown, the blood sample adding unit includes a plurality of blood sampling tubes 411 and a plurality of blood sample temporary storage components. The plurality of blood sample temporary storage components are connected to the plurality of blood sampling tubes 411 in a one-to-one correspondence, and the plurality of blood sample temporary storage components are all connected to the same pressure generating device 420 .

[0198] Specifically, the blood sample adding unit includes a combination of multiple sets of blood sampling tubes 411 and intermediate sealed tubes 431, and each intermediate sealed tube 431 is connected to the same pressure generating device 420 through a respective pressurizing tube 440. More specifically, the pressure generating device 420 includes a pressure generator 421 and a pressure divider 422, and the pressure generator 421 is used to provide positive pressure and negative pressure, and can be a peristaltic pump, for example. The air inlet end of the pressure divider 422 is connected to the pressure generator 421, and the air outlet end includes a plurality of air outlets, each of which is respectively connected to the air inlet end, and a plurality of pressurizing tubes 440 are connected to each air outlet in a one-to-one correspondence.

[0199] In the above scheme, the same pressure generator 421 provides positive pressure and / or negative pressure to several intermediate sealed tubes 431 through the pressure divider 422 at the same time. There is no need to set up a separate pressure generator 421 for each intermediate sealed tube 431, which simplifies the structure of the blood sample adding unit and reduces production costs and maintenance difficulties.

[0200] Furthermore, each drainage tube 430 connected to each intermediate sealing tube 431 is provided with a control valve 435 that can cut off the drainage tube 430. The multiple control valves 435 are independent of each other and can be opened or closed at the same time, or partially opened or partially closed.

[0201] Specifically, the plurality of control valves 435 may be manually opened and closed one by one by a user, or may be automatically opened and closed by a program, or may be automatically opened and closed depending on the structure of the second switch 435 itself.

[0202] In this embodiment, since the volume of the blood samples in each blood collection tube 411 may be different, the volume of the blood samples drawn into the middle sealed tube 431 may also be different. When the blood samples are discharged outward through the blood discharge tube 430, the blood samples in some middle sealed tubes 431 may be completely discharged, while blood samples may still be in other middle sealed tubes 431. At this time, the blood discharge tube 430 connected to the middle sealed tube 431 from which the blood samples have been completely discharged is emptied, and the pressurized air flow from the pressure generator 421 will flow out of the blood discharge tube 430, causing pressure leakage, which affects the discharge of blood samples in other middle sealed tubes 431.

[0203] By providing an openable and closable control valve 435 on each blood drainage tube 430, when the blood sample in a part of the intermediate sealed tube 431 is drained, the control valve 435 on the blood drainage tube 430 connected to this part of the intermediate sealed tube 431 is controlled to be closed, thereby blocking these blood drainage tubes 430 and preventing the positive pressure in this part of the intermediate sealed tube 431 from leaking, thereby ensuring that the other intermediate sealed tubes 431 can continue to obtain positive pressure and discharge the blood sample inside normally.

[0204] In this embodiment, the blood sample adding unit has a fixed number of intermediate sealed tubes 431, but not every time it is used, the same number of blood samples to be tested are placed in different blood collection tubes 411 waiting for transportation. Therefore, in a further solution, a pressurizing switch 443 that can block conduction is respectively provided on each pressurizing tube 440. The multiple pressurizing switches 443 are independent of each other and can be turned on or off at the same time, or partially turned on or off.

[0205] During use, when the blood sampling tube 415 connected to the middle sealed tube 431 is inserted into the blood sampling tube 411 containing the blood sample to be tested, the pressurization switch 443 on the corresponding pressurization tube 440 is controlled to be turned on. If the blood sampling tube 415 is not inserted into the blood sample, or the blood sample in part of the blood sampling tube 411 is drained in advance during the blood sample delivery process, the pressurization switch 443 on the corresponding pressurization tube 440 is turned off, thereby cutting off the pressurization tube 440. In this way, when the pressure generator 421 only delivers negative pressure to the middle sealed tube 431 that is inserted into the blood sample of the blood sampling tube 415, it is avoided that the middle sealed tube 431 that is directly connected to the atmosphere causes negative pressure leakage, resulting in insufficient negative pressure in other middle sealed tubes 431, affecting the extraction of blood samples.

[0206] In a further solution of this embodiment, the blood sample adding unit further includes a sampling fixing platform 450, on which a plurality of positioning holes 457 are provided, which extend vertically and vertically, and the liquid outlet of the drainage tube 430 passes through the positioning holes 457 from top to bottom, thereby fixing the drainage tube 430. 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 sampling fixing platform 450 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] This embodiment also provides a method for cell enrichment staining using the above-mentioned enrichment and staining integrated device, which specifically includes the following steps:

[0209] (1) The reagent taking and placing unit adds microporous membrane activation liquid into the microfluidic device 200;

[0210] (2) the pressure generating device of the blood sample unit evacuates air and reduces pressure from the blood sample temporary storage component, and the blood sample to be tested is sucked into the blood sample temporary storage component from the blood collection tube, and then the pressure generating device ventilates and pressurizes the blood sample temporary storage component, and the blood sample to be tested is discharged from the blood sample temporary storage component and added to the microfluidic device; the microfluidic device is suction filtered to enrich the target cells;

[0211] (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;

[0212] (4) The reagent taking and placing unit sucks the cell fixative and adds it to the microfluidic device, and then performs suction filtration on the microfluidic device;

[0213] (5) The reagent taking and placing unit sucks the buffer solution and adds it to the microfluidic device for cleaning, and then performs suction filtration on the microfluidic device;

[0214] (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;

[0215] (7) the reagent taking and placing unit absorbs the permeation liquid and adds it to the microfluidic device, and then performs suction filtration on the microfluidic device;

[0216] (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 microfluidic device is filtered by suction; step (8) is performed twice in total;

[0217] (9) The reagent taking and placing unit absorbs the buffer solution and adds it to the microfluidic device for cleaning, and then the microfluidic device is filtered; step (9) is performed one to three times, after which the enriched and stained target cells are obtained.

[0218] In detail, the specific operation process of enrichment and staining of circulating tumor cells using the above-mentioned integrated enrichment and staining device is as follows.

[0219] In the preparation stage, the operator adds the blood sample to be tested into the blood collection tube 411, inserts the blood collection tube 415 into the blood collection tube 411 through the tube mouth of the blood collection tube 411, and makes the liquid inlet tube mouth of the blood collection tube 415 abut the bottom of the blood collection tube 411. Wherein, the blood sample to be tested is whole blood that has been pretreated, and the pretreatment can be the pretreatment method of the blood sample collected from the patient before the test when CTC enrichment is performed 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.

[0220] After the preparation stage is completed, the enrichment and staining integrated device can start working. First, the reagent holding device 110 in the reagent taking and placing unit is controlled to move, and the microporous membrane activation liquid is controlled to be sucked by the driving motor 1012, and then the reagent holding 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, and the microfiltration membrane 212 is pretreated.

[0221] After the addition is completed, the pressure generating device 420 starts to operate, firstly evacuates the air from the middle sealed tube 431 to reduce the pressure, and the blood sample is sucked from the blood collection tube 411 into the middle sealed tube 431, and then the middle sealed tube 431 is ventilated and pressurized, and the sucked blood sample is pressed out along the blood discharge tube 430 and added to the microfluidic device 200. After the blood sample is added, the peristaltic pump 330 is started to pump the microfluidic device 200, thereby enriching the target cells in the blood sample, namely CTCs.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] The above operation process can be automatically controlled by a pre-set program. After the operator collects and processes the blood sample from the patient, he inserts the blood sampling tube 415 into the blood sampling tube 411 to start the enrichment and staining integrated device of this embodiment. The enrichment and staining integrated device can automatically complete the whole process from blood sample delivery to cell enrichment and staining without manual intervention. After the enrichment and staining integrated device is completed, the operator can directly take out the microfiltration membrane 212 for microscopic examination, which greatly reduces manual operation and improves detection efficiency.

[0231] Embodiment 2

[0232] like Figure 21 to Figure 23 As shown, the difference between this embodiment and the above-mentioned embodiment 1 is that: the drainage blood vessel 430 is provided with a blocking structure that can be automatically controlled to open and close, thereby eliminating the setting of the control valve on the drainage blood vessel 430.

[0233] Specifically, in this embodiment, when the blood sample to be tested enters the blood drainage tube 430 from the middle sealed tube 431, the blocking structure is opened, thereby opening the blood drainage tube 430 to discharge the blood sample to be tested. After the blood sample to be tested stops entering the blood drainage tube 430, the blocking structure can be automatically closed, thereby blocking the blood drainage tube 430. In this way, when the blood sample in any middle sealed tube 431 is completely discharged, the blood drainage tube 430 connected to the middle sealed tube 431 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 the other middle sealed tubes 431 can normally discharge blood samples to the outside.

[0234] In a specific solution of this embodiment, the blood drainage tube 430 has a vertical extension portion, and the blocking structure is arranged on the vertical extension portion, including a pressure stabilizing chamber 437 with a floating block 438 arranged therein, wherein the sealing 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 in a conductive state with the blood drainage tube 430, and a liquid outlet is arranged at the bottom of the pressure stabilizing chamber 437, and the floating block 438 can block the liquid outlet.

[0235] When the pressure generating device 420 provides positive pressure to the middle sealing tube 431, the blood sample to be tested enters the pressure stabilizing chamber 437 along the blood drainage tube 430, so that the floating block 438 floats up to keep the blood drainage tube 430 open. When the blood sample to be tested stops entering the blood drainage tube 430, the blood sample in the pressure stabilizing chamber 437 completely flows out, and the floating block 438 falls to block the liquid outlet at the bottom of the pressure stabilizing chamber 437, thereby blocking the blood drainage tube 430.

[0236] 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.

[0237] In this embodiment, 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 middle sealed tube 431 receives positive pressure, the blood sample in the tube is pressed into the blood drain 430 and enters the pressure stabilizing chamber 437 along the blood drain 430. At this time, the float 438 floats up and separates from the liquid outlet due to the buoyancy of the blood sample, thereby opening the liquid outlet, and the blood sample can be discharged along the blood drain 430 and added to the microfluidic device 200. When all the blood samples in the middle sealed tube 431 are discharged, there is no more blood sample in the pressure stabilizing chamber 437, and the float 438 re-blocks the liquid outlet of the pressure stabilizing chamber 437 under the action of gravity, thereby blocking the blood drain 430. At this time, the positive pressure provided by the pressure generating device 420 will not leak out through the blood drain 430.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] In this embodiment, the pressure generating device 420 is connected to multiple intermediate sealed tubes 431 at the same time, and can simultaneously ventilate and pressurize multiple intermediate sealed tubes 431, so as to press blood samples out of each intermediate sealed tube 431 and add them to the corresponding microfluidic device 200. Since the amount of blood sample drawn from the blood collection tube 411 by each intermediate sealed tube 431 may be different, and the viscosity of different blood samples often also varies, the time when the blood samples in each intermediate sealed tube 431 are completely discharged may be different. By providing a pressure stabilizing chamber 437 with a floating block 438 inside on each blood discharge tube 430, the corresponding blood discharge tube 430 can be automatically blocked after the blood sample in the intermediate sealed tube 431 is completely discharged, so as to prevent the blood discharge tube 430 from leaking the positive pressure provided by the pressure generating device 420, and avoid affecting the process of adding blood samples to the microfluidic device 200 by other intermediate sealed tubes 431.

[0243] Embodiment 3

[0244] like Fig.24 and Fig.25 As shown, the difference between this embodiment and the above-mentioned embodiment one or two 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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.

[0252] 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.

[0253] In another specific solution of this embodiment, multiple support rods 3431 are provided, and any two support rods 3431 may cross each other or not, and be arranged irregularly, as long as through holes are left on the side walls of the bracket 343.

[0254] 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.

[0255] 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.

[0256] 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.

[0257] 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 .

[0258] 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.

[0259] 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.

[0260] 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. An integrated device for enrichment and staining of circulating tumor cells, characterized in that: include: A microfluidic device, used for receiving a blood sample to be tested and enriching a 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 blood sample adding unit comprises: Blood collection tube, used to hold the blood sample to be tested; A blood sample temporary storage component, connected to the blood collection tube; A pressure generating device, connected to the blood sample temporary storage component; The pressure generating device evacuates air and reduces pressure from the blood sample temporary storage component, and sucks the blood sample to be tested from the blood collection tube into the blood sample temporary storage component; the pressure generating device ventilates and pressurizes the blood sample temporary storage component, and discharges the blood sample to be tested from the blood sample temporary storage component and adds it to the microfluidic device.

2. The integrated device for enrichment and staining of circulating tumor cells according to claim 1, characterized in that: The blood sample temporary storage assembly comprises a middle sealed tube, and a blood drawing blood tube and a blood discharge blood tube which are sealedly connected to the middle sealed tube; The blood drawing tube is connected to the blood sampling tube, and the blood sample to be tested in the blood sampling tube is sucked into the middle sealed tube along the blood drawing tube; the blood sample to be tested in the middle sealed tube can be discharged along the blood discharge tube and added into the microfluidic device.

3. The integrated device for enrichment and staining of circulating tumor cells according to claim 2, characterized in that: The pressure generating device is connected to the middle sealing tube through a pressurizing tube, and one end of the pressurizing tube extending into the middle sealing tube is arranged inside the middle sealing tube near the tube opening; the liquid inlet end of the blood drainage tube is arranged near the bottom of the middle sealing tube.

4. The integrated device for enrichment and staining of circulating tumor cells according to claim 2, characterized in that: The blood drainage tube has a vertical extension portion; a pressure stabilizing chamber is arranged on the vertical extension portion, and a floating block having a density less than that of the blood sample is arranged in the pressure stabilizing chamber; The blood sample to be tested enters the pressure stabilizing chamber along the blood drainage tube, and the floating block floats up to keep the blood drainage tube conductive; after the blood sample to be tested stops entering the blood drainage tube, the blood sample in the pressure stabilizing chamber completely flows out, and the floating block falls to block the liquid outlet at the bottom of the pressure stabilizing chamber, thereby blocking the blood drainage tube.

5. The integrated device for enrichment and staining of circulating tumor cells according to any one of claims 1 to 4, characterized in that: The blood sample adding unit comprises a plurality of blood sampling tubes and a plurality of blood sample temporary storage components, wherein the plurality of blood sample temporary storage components are connected to the plurality of blood sampling tubes in a one-to-one correspondence; and the plurality of blood sample temporary storage components are all connected to the same pressure generating device.

6. The integrated device for enrichment and staining of circulating tumor cells according to any one of claims 1 to 4, 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 reagent taking and placing driving device is used to drive the vacuum cylinder and the piston assembly to produce the same movement.

7. The integrated device for enrichment and staining of circulating tumor cells according to claim 6, 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 reagent taking and placing driving device drives the vacuum cylinder to move back and forth along the axis of the push rod to absorb the reagent into the reagent containing device, or discharge the reagent from the reagent containing device.

8. The integrated device for enrichment and staining of circulating tumor cells according to any one of claims 1 to 4, characterized in that: The microfluidic device comprises: The sample injection tube comprises a tube body, the upper end of the tube body is opened for receiving 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. The buckle cover is spirally descended and threadedly connected with the liquid accumulation liner. The buckle part of the sample injection tube is pressed down to approach the liquid accumulation liner, thereby pressing the filter assembly.

9. The integrated device for enrichment and staining of circulating tumor cells according to claim 8, 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.

10. A method for cell enrichment and staining using the integrated device for enrichment and staining of circulating tumor cells 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) The pressure generating device of the blood sample unit evacuates air and reduces pressure from the blood sample temporary storage component, and the blood sample to be tested is sucked from the blood collection tube into the blood sample temporary storage component. Then, the pressure generating device ventilates and pressurizes the blood sample temporary storage component, and the blood sample to be tested is discharged from the blood sample temporary storage component and added to the microfluidic device; performing suction filtration on 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 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 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 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 microfluidic device is filtered by suction; 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 microfluidic device is filtered; step (9) is performed one to three times, after which the enriched and stained target cells are obtained.

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