CN220304956U

CN220304956U - Nuclear cell enrichment dyeing integrated equipment

Info

Publication number: CN220304956U
Application number: CN202321918909.6U
Authority: CN
Inventors: 肖乐义; 刘元柱; 李东; 许元红; 薛冰; 牛玉生; 杨勤英; 米明仁; 李娟�; 张腾业; 高明
Original assignee: Qingdao Yanding Biomedical Technology Co ltd
Current assignee: Qingdao Yanding Biomedical Technology Co ltd
Priority date: 2023-07-20
Filing date: 2023-07-20
Publication date: 2024-01-05
Anticipated expiration: 2033-07-20

Abstract

The utility model discloses a nucleated cell enrichment and dyeing integrated device, which comprises: a enrichment unit comprising a microfluidic device for receiving a blood sample to be tested and enriching a target substance therefrom; a blood sample adding unit for adding a blood sample to be detected to the microfluidic device; the reagent taking and placing unit is used for sucking the reagent and adding the reagent into the microfluidic device; the microfluidic device includes: the feeding tube comprises a tube main body, and the lower end of the tube main body is provided with a buckling part protruding from the side wall of the tube main body; the liquid accumulation lining table is arranged below the sample injection pipe; the buckling cover is sleeved on the pipe main body, the buckling cover is in spiral descending and is in threaded connection with the hydrops lining table, the buckling part is pressed down to approach the hydrops lining table, and the filtering component arranged between the buckling part and the hydrops lining table is pressed. The nucleated cell enrichment and dyeing integrated device can complete the whole process from blood sample delivery to enrichment and dyeing of target substances, and is beneficial to improving the detection efficiency.

Description

Nuclear cell enrichment dyeing integrated equipment

Technical Field

The utility model belongs to the technical field of medical detection, and particularly relates to a nucleated cell enrichment and dyeing integrated device.

Background

Currently, in the fields of clinical detection and biomedical technology, detection of components such as cells and biological macromolecules in biological fluids (e.g., human blood) is increasingly emphasized and developed. The biological fluid is detected, the trauma to a patient can be avoided, the test sample is convenient to obtain, and the method can be widely applied to immunological research detection, gene research detection or microorganism detection, and even applied to biological chips and organoid research and development.

Before detecting the components such as cells and biomacromolecules in the biological fluid, the collected biological fluid needs to be removed from the storage container, and target substances such as target cells or biomacromolecules are filtered and stained for further detection. If an integrated device is designed, the device can autonomously complete the transportation of biological fluid samples, add reagents with different functions into the samples and separate target substances from the samples, and plays an important role in various detection and research in the field of biological medicine. For example, in immunological research, genetic research or microbial detection, the efficiency of sorting target cells, organisms or biomacromolecules can be improved, and further, the research and development of biochips and the research and development of organoids are facilitated. On the other hand, the integrated equipment is also suitable for changing various eluents to realize the application scene of the chromatographic separation preparation method, or can be applied to the application scene of adding samples or reagents in a timed and quantitative manner, such as the induction expression of gene recombinants and the purification of products.

One important field of application of the integrated device described above is disease detection. In particular, the continual rise in tumor morbidity and mortality has become one of the serious problems in today's society. The current clinical discovery and diagnosis of most tumors still relies on imaging examinations, traditional tumor markers, and tissue biopsies. Traditional invasive tissue biopsies require the removal of a sample of tissue or cells from the patient, which often involves a certain risk and pain to the patient. More critical is that tissue biopsies are not suitable for repeated extractions, are prone to complications, and have the risk of spreading cancer cells.

In recent years, in the field of biomedical engineering, several emerging tumor diagnostic detection techniques, such as circulating tumor DNA (ctDNA), circulating Tumor Cells (CTCs) detection methods called liquid biopsies, have emerged. The CTC detection and the material selection are convenient, and the defect that the histopathological detection and the material selection are inconvenient and have a certain damage to patients is overcome. According to researches, the existence of CTC can be found in peripheral blood before solid tumor formation, so that CTC detection is very suitable for early screening and early diagnosis of malignant tumors, and CTC detection has good effects on prognosis of malignant tumors, disease progress monitoring, recurrence prediction, postoperative tiny focus monitoring of malignant tumors and design and treatment effect monitoring of targeted drug treatment, and is an advanced method for early screening and diagnosis of malignant tumors at present. Since the CTC content in peripheral blood is small, CTC detection requires that CTCs be first enriched and then detected, with CTC capture and recognition being currently the most significant challenge.

In 2004, the us FDA approved the CTC detection device CellSearch for the diagnosis of breast cancer, prostate cancer and rectal cancer, but the import device was expensive, and the detection method achieved using this device was not very specific and was not widely used.

In addition, in the existing device for enriching cells, biomacromolecules and the like, the device generally comprises an upper component and a lower component which are connected through threads, and a microporous filter membrane and a water-absorbing fiber which are used for enriching target substances are arranged between the upper component and the lower component, but in the rotating screwing process, the microporous filter membrane and the water-absorbing fiber can rotate along with the rotating process or even shift, so that the microporous filter membrane and the water-absorbing fiber cannot be completely overlapped. This can affect the effectiveness of the enrichment of the target substance, resulting in prolonged enrichment, especially in CTC assays, and can even delay diagnosis and subsequent treatment of the patient by affecting the results of the assay.

In view of this, the present utility model has been made.

Disclosure of Invention

The utility model aims to overcome the defects of the prior art, provides a nucleated cell enrichment and dyeing integrated device, can complete the whole process from blood sample delivery to enrichment and dyeing of target substances, and ensures the enrichment effect of a microfluidic device on the target substances.

In order to solve the technical problems, the utility model adopts the basic conception of the technical scheme that:

an integrated nucleated cell enrichment staining apparatus comprising:

a enrichment unit comprising a microfluidic device for receiving a blood sample to be tested and enriching a target substance therefrom;

a blood sample adding unit for adding a blood sample to be detected to the microfluidic device;

a reagent taking and placing unit for sucking a reagent and adding the reagent into the microfluidic device;

the microfluidic device comprises:

the sample feeding tube comprises a tube main body, wherein an opening at the upper end of the tube main body is used for receiving a blood sample to be detected, and a buckling part protruding from the side wall of the tube main body is arranged at the lower end of the tube main body;

the liquid accumulation lining table is arranged below the sample injection pipe, a filter assembly for filtering a blood sample to be detected is arranged between the liquid accumulation lining table and the buckling part of the sample injection pipe, and the target substance is enriched on the upper side of the filter assembly;

the buckling cover is sleeved on the pipe main body of the sample inlet pipe, the buckling cover is spirally lowered to be in threaded connection with the effusion lining table, and the buckling part is pressed down to approach the effusion lining table to compress the filter assembly.

Further, the buckle cover comprises a pressing part which is horizontally arranged and used for pressing the buckling part, and a through hole for the pipe main body to pass through is formed in the pressing part;

The buckle cover further comprises a connecting part, wherein the connecting part is formed by downwards extending the periphery of the pressing part; the inner side of the connecting part is provided with an internal thread, and the hydrops lining table is provided with an external thread matched with the internal thread.

Further, a limiting groove in limiting fit with the buckling part is formed in the lower surface of the pressing part.

Further, the enrichment dyeing unit further comprises a buffer device, comprising:

a peristaltic pump communicated with the microfluidic device and used for sucking filtered liquid;

the buffer component is arranged between the microfluidic device and the peristaltic pump and is respectively communicated with the microfluidic device and the peristaltic pump.

Further, the buffer assembly comprises a buffer bottle which is respectively communicated with the microfluidic device and the peristaltic pump, and a preset amount of gas is sealed in the buffer bottle;

alternatively, the buffer assembly comprises a buffer, a buffer chamber is formed in the buffer, the buffer chamber is respectively communicated with the microfluidic device and the peristaltic pump, and at least part of the wall of the buffer chamber is made of a material capable of undergoing elastic deformation.

Further, the reagent pick-and-place unit includes:

reagent holding means for holding the sucked reagent;

The vacuumizing device comprises a vacuum cylinder communicated with the reagent accommodating device and a piston assembly which is arranged inside the vacuum cylinder and can reciprocate relative to the vacuum cylinder;

and the driving device is used for driving the vacuum cylinder and the piston assembly to generate relative motion.

Further, the piston assembly includes a piston and a pushrod; 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 with the piston, and the other end of the push rod extends out of the vacuum cylinder;

the push rod is fixedly arranged; the driving device drives the vacuum cylinder to reciprocate along the axis of the push rod, the reagent is sucked into the reagent accommodating device by air suction and decompression in the reagent accommodating device, and the reagent is discharged from the reagent accommodating device by ventilation and pressurization in the reagent accommodating device.

Further, the vacuumizing device further comprises a vacuumizing fixing frame, and the vacuum cylinder is relatively fixedly arranged on the vacuumizing fixing frame;

the driving device comprises a driving motor and a transmission screw rod, and the vacuumizing fixing frame is connected with a sliding block arranged on the transmission screw rod; the driving motor drives the transmission screw rod to rotate, and the vacuumizing fixing frame moves along the extending direction of the transmission screw rod along with the sliding block to drive the vacuum cylinder to move along the extending direction of the transmission screw rod.

Further, the reagent taking and placing unit further comprises a moving mechanism, and the moving mechanism is used for driving the reagent accommodating device to move to the position above the reagent accommodating device for accommodating the reagent to be added to absorb the reagent, and driving the reagent accommodating device to move to the position above the microfluidic device to add the reagent.

Further, the blood adding unit comprises a pressure generating device and a sealed blood collection tube, wherein the pressure generating device is communicated with the sealed blood collection tube, and the sealed blood collection tube is provided with a blood discharge tube communicated with the outside;

the pressure generating device ventilates and pressurizes the sealed blood collection tube, and the blood sample to be detected contained in the sealed blood collection tube is discharged along a blood discharge tube and added into the microfluidic device.

By adopting the technical scheme, compared with the prior art, the utility model has the following beneficial effects.

According to the utility model, through the nucleated cell enrichment and dyeing integrated device, the whole process from blood sample delivery to cell enrichment and dyeing can be completed, so that more efficient detection can be realized. In the nucleated cell enrichment dyeing integrated equipment, the microfluidic device for enriching target substances comprises a sample inlet pipe, a liquid accumulation lining table and a buckle cover, wherein the buckle cover and the liquid accumulation lining table are rotated and screwed up, and the buckling part of the sample inlet pipe can be pressed down to approach the liquid accumulation lining table so as to compress a filter assembly between the sample inlet pipe and the liquid accumulation lining table. Because no relative rotation exists between the sample inlet pipe and the liquid accumulation lining table, the displacement of the filter assembly can be avoided, and the effect of enriching target substances from the blood sample by the microfluidic device is not influenced.

In the utility model, the peristaltic pump is used for sucking filtered liquid from the microfluidic device, so that low-speed extraction is easy to realize, and the problem of hardening of blood samples caused by overlarge suction force is avoided. The buffer is arranged between the peristaltic pump and the microfluidic device, so that the influence of pulse flow generated during operation of the peristaltic pump on blood sample filtration can be reduced, the cell enrichment effect is further ensured, and the accuracy of a detection result is improved.

In the utility model, the reagent taking and placing unit is provided with the reagent accommodating device for storing the sucked reagent, the vacuum cylinder is only used for being matched with the piston assembly to provide positive pressure or negative pressure, and the sucked reagent cannot enter the vacuum cylinder, so that the reagent accommodating device is only required to be replaced or cleaned, the vacuumizing device is not required to be disassembled and cleaned, and the use is more convenient. The vacuum pumping device can more accurately control the moving distance of the vacuum cylinder through screw transmission, and further accurately control the sucking or adding amount of the reagent. The reagent is got and is put the unit and set up moving mechanism for reagent holding device can remove between the position of absorbing reagent and the position of adding reagent, can improve the efficiency of adding reagent.

The following describes the embodiments of the present utility model in further detail with reference to the accompanying drawings.

Drawings

The accompanying drawings, which are included to provide a further understanding of the utility model and are incorporated in and constitute a part of this specification, illustrate embodiments of the utility model and together with the description serve to explain the utility model. It is evident that the drawings in the following description are only examples, from which other drawings can be obtained by a person skilled in the art without the inventive effort. In the drawings:

FIG. 1 is a schematic view of a vacuum pumping apparatus according to an embodiment of the present utility model;

FIG. 2 is a schematic view of a first limiting plate according to an embodiment of the present utility model;

FIG. 3 is a left side view of an embodiment of the utility model in which the vacuum apparatus does not include a first limiting plate and a second limiting plate;

FIG. 4 is a schematic view of the installation of a reagent containing device according to an embodiment of the present utility model;

FIG. 5 is a schematic view of the structure of the reagent holding apparatus and the moving mechanism in the embodiment of the present utility model;

FIG. 6 is an exploded view of a microfluidic device in an embodiment of the present utility model;

fig. 7 is a schematic diagram of an assembled microfluidic device according to an embodiment of the present utility model;

FIG. 8 is a partial cross-sectional view of a sample tube in an embodiment of the present utility model;

FIG. 9 is a top view of a sample tube in an embodiment of the present utility model;

FIG. 10 is a partial cross-sectional view of a clasp cover in an embodiment of the utility model;

FIG. 11 is a schematic view of the clasp of FIG. 10 in the A direction in accordance with the utility model;

FIG. 12 is a partial cross-sectional view of a liquid product liner station in accordance with an embodiment of the utility model;

FIG. 13 is a top view of a liquid product liner table in an embodiment of the utility model;

FIG. 14 is a top view of a backing web in an embodiment of the utility model;

FIG. 15 is a schematic view of a connecting bottom tube with a rubber hose according to an embodiment of the present utility model;

FIG. 16 is a top view of a communication bottom tube in an embodiment of the present utility model;

FIG. 17 is a top view of a communication bottom tube mounted to a stationary table in an embodiment of the utility model;

FIG. 18 is a side view of a communication bottom tube mounted to a stationary table in an embodiment of the utility model;

FIG. 19 is a schematic diagram of a buffer device connected to a microfluidic device according to a first embodiment of the present utility model;

FIG. 20 is a schematic diagram showing the structure of a blood sample applying unit according to the first embodiment of the present utility model;

FIG. 21 is a top view of a blood sample addition unit according to a first embodiment of the present utility model;

FIG. 22 is a schematic diagram of a buffer device connected to a microfluidic device according to a second embodiment of the present utility model;

FIG. 23 is a schematic view of a buffer film in a buffer according to a second embodiment of the present utility model, partially cut away;

FIG. 24 is a schematic view showing the structure of a blood sample applying unit in accordance with the third embodiment of the present utility model;

fig. 25 is an enlarged schematic view of the present utility model at B in fig. 24.

In the figure: 101. a vacuum pumping device; 1011. a vacuum cylinder; 1012. a driving motor; 1013. a transmission screw; 1014. a push rod; 1015. a first limiting plate; 1016. a second limiting plate; 1017. a fixing part; 1018. a fixing plate; 1019. a clamping seat; 1020. a first communication pipe; 1021. an operation panel; 1022. a groove; 110. a reagent accommodating device; 1101. a reagent accommodating tube; 1102. a first support frame; 1103. a second support frame; 1104. a second communicating pipe; 1105. a needle; 1106. a first lead screw; 1107. a second lead screw; 1108. a fixing seat; 1109. a reagent holding device; 1110. a first motor; 1111. a second motor;

200. a microfluidic device; 201. a sample inlet tube; 2011. a tube body; 2012. a buckling part; 202. a buckle cover; 2021. a pressing part; 2022. a connection part; 203. a liquid accumulation lining table; 2031. a threaded portion; 2032. an operation unit; 204. a mounting groove; 205. a backing net; 2051. a strip-shaped opening; 206. a flow guiding strip; 207. a limit groove; 208. a diversion frustum; 209. a communicating bottom pipe; 210. a fixed table; 212. microfiltration membrane; 213. a water-absorbing layer; 214. a filter assembly; 215. a deflector aperture;

310. A buffer bottle; 311. a first pipeline; 3111. a one-way valve; 312. a second pipeline; 313. sealing cover; 330. a peristaltic pump; 340. a buffer; 343. a bracket; 3431. a support rod; 344. a buffer film; 345. an end wall;

410. sealing the blood collection tube; 411. a blood collection tube body; 412. sealing cover; 420. a pressure generating device; 421. a pressure generator; 422. a voltage divider; 430. discharging blood vessels; 437. a pressure stabilizing bin; 438. a floating block; 439. a funnel-shaped structure; 440. a pressurizing tube; 441. a main switch; 442. a sub-switch; 450. a sampling fixing table; 451. a platen; 452. a threaded hole; 453. a motor; 454. a third lead screw; 455. a guide rail; 456. a support frame; 457. and positioning holes.

It should be noted that these drawings and the written description are not intended to limit the scope of the inventive concept in any way, but to illustrate the inventive concept to those skilled in the art by referring to the specific embodiments.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present utility model more apparent, the technical solutions in the embodiments will be clearly and completely described with reference to the accompanying drawings in the embodiments of the present utility model, and the following embodiments are used to illustrate the present utility model, but are not intended to limit the scope of the present utility model.

In the description of the present utility model, it should be noted that the directions or positional relationships indicated by the terms "upper", "lower", "front", "rear", "left", "right", "vertical", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, are merely for convenience of describing the present utility model and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present utility model.

In the description of the present utility model, it should be noted that, unless explicitly specified and limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be either fixedly connected, detachably connected, or integrally connected, for example; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium. The specific meaning of the above terms in the present utility model will be understood in specific cases by those of ordinary skill in the art.

As shown in fig. 1 to 25, an embodiment of the present utility model provides a nucleated cell enrichment and staining integrated device for implementing the transportation of a sample to be detected (for example, a blood sample to be detected), the addition of one or more reagents, and the complete detection process of enriching a target substance from the sample to be detected.

Specifically, the nucleated cell enrichment and staining integrated device of the embodiment of the utility model comprises a reagent taking and placing unit, an enrichment and staining unit and a blood sample adding unit. Wherein the blood adding unit adds a blood sample to be detected into the enrichment unit; the reagent taking and placing unit can absorb the reagent and add the reagent into the enrichment unit, so that the target substances in the blood sample to be detected are enriched in the enrichment unit; the reagent taking and placing unit can also add a reagent with a dyeing function to the enrichment dyeing unit so as to dye the enriched target substances for subsequent detection and identification procedures.

Example 1

As shown in fig. 1 to 21, the integrated nucleated cell enrichment and staining apparatus according to the present embodiment includes a reagent taking and placing unit, an enrichment and staining unit, and a blood sample adding unit.

Specifically, the integrated nucleated cell enrichment and staining device of the present embodiment is used for enriching and staining target cells, wherein the sample to be detected is a blood sample collected from a patient, and the target substance to be enriched is nucleated cells, such as nucleated pathological cells, in the blood sample. More specifically, the integrated nucleated cell enrichment and staining device can be used for CTC detection, and at this time, the target substance enriched from the blood sample is Circulating Tumor Cells (CTCs).

In this embodiment, the reagent picking and placing unit of the integrated apparatus is configured to aspirate a reagent, and then add the aspirated reagent to the enrichment unit. Specifically, as shown in fig. 1 to 5, the reagent pick-and-place unit includes at least a reagent accommodating device 110, a vacuum-pumping device 101, and a driving device.

Wherein the reagent holding device 110 is used for storing the sucked reagent. The evacuating device 101 includes a vacuum cylinder 1011 communicating with the reagent accommodating device 110, and a piston assembly provided inside the vacuum cylinder 1011 and reciprocally movable with respect to the vacuum cylinder 1011. The driving device is used for driving the vacuum cylinder 1011 and the piston assembly to generate relative motion, so that the suction and the decompression in the reagent containing device 110 or the ventilation and the pressurization in the reagent containing device 110 are realized, and the suction or the discharge of the reagent is realized.

In the above scheme, the vacuum cylinder 1011 is matched with the piston assembly to provide air pressure difference, thereby controlling the suction and discharge of the reagent. The sucked reagent is stored in the reagent holding device 110 without entering the vacuum cylinder 1011. When the reagent type is changed or the reagent taking and placing unit needs to be cleaned after long-time use, only the reagent accommodating device 110 needs to be taken down for replacement or cleaning, and the vacuumizing device 101 connected with the driving device does not need to be disassembled for cleaning, so that the operation is more convenient.

In the specific scheme of this embodiment, the piston assembly is fixedly arranged, and the driving device is connected with the vacuum cylinder 1011 and can drive the vacuum cylinder 1011 to reciprocate relative to the piston assembly, so as to realize the air extraction, decompression or ventilation and pressurization.

It will be appreciated that in this embodiment, the vacuum cylinder may be fixedly disposed, and the driving device is connected to the piston assembly to drive the piston assembly to reciprocate relative to the vacuum cylinder.

The scheme of this embodiment will be specifically described below in terms of the fixed arrangement of the piston assembly, in which the vacuum cylinder 1011 moves under the driving action of the driving device.

In this embodiment, the piston assembly includes a piston and a pushrod 1014. Wherein, the piston is arranged 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 outside the vacuum cylinder 1011.

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. Referring to fig. 1, the vacuum cylinder 1011 is driven to move rightward, and is capable of exhausting air from the reagent accommodating apparatus 110 and decompressing, thereby sucking the reagent into the reagent accommodating apparatus 110, and when the vacuum cylinder 1011 is driven to move leftward, it is capable of ventilating and pressurizing the reagent accommodating apparatus 110, thereby discharging the reagent from the reagent accommodating apparatus 110.

Further, one end of the push rod 1014 located outside the vacuum cylinder 1011 (i.e., the left end in fig. 1) 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 body portion of the push rod 1014. The vacuum pumping apparatus 101 further includes a limiting member, which is in a limiting fit with the fixing portion 1017, so that the push rod 1014 remains stationary.

In a specific scheme of this embodiment, the limiting component includes a first limiting plate 1015 fixedly disposed, the fixing portion 1017 is fixed on the first limiting plate 1015, and for example, the fixing portion 1017 may be adhered to the first limiting plate 1015 by using glue for fixing.

Further, the first limiting plate 1015 is disposed in a direction perpendicular to the moving direction of the vacuum cylinder 1011, a groove 1022 having an opening is disposed on the first limiting plate 1015, and a main body portion of the push rod 1014 is inserted into the groove 1022. The grooves 1022 are preferably upwardly open to facilitate installation of the push rod 1014.

Specifically, the evacuation device 101 is disposed above the reagent containing device 110, and there is a horizontally disposed operation panel 1021 above the reagent containing device 110, and the evacuation device 101 is integrally mounted on the operation panel 1021. The first limiting plate 1015 is vertically arranged relative to the operation plate 1021, the lower side of the first limiting plate 1015 is bent to form a horizontal flanging, and the first limiting plate 1015 is fixed through the flanging.

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 disposed, the first limiting plate 1015 and the second limiting plate 1016 are disposed opposite to each other, and the fixing portion 1017 is clamped between the first limiting plate 1015 and the second limiting plate 1016 and is kept fixed.

In the above-mentioned scheme, the setting direction of the first limiting plate 1015 and the second limiting plate 1016 is perpendicular to the moving direction of the vacuum cylinder 1011, and the first limiting plate 1015 is also provided with a groove 1022 that is opened upwards, so that the main body portion of the push rod 1014 can be inserted into the groove 1022. The distance between the first limiting plate 1015 and the second limiting plate 1016 is substantially identical to the thickness of the fixing portion 1017 along the length direction of the push rod 1014, so that the fixing portion 1017 is fixed between the first limiting plate 1015 and the second limiting plate 1016 in the moving process of the vacuum cylinder 1011, and the positions of the push rod 1014 and the piston are kept unchanged. At this time, the fixing portion 1017 and the first limiting plate 1015 or the second limiting plate 1016 do not need to be additionally fixed.

More specifically, the evacuation device 101 is disposed above the reagent containing device 110, and there is a horizontally disposed operation panel 1021 above the reagent containing device 110, and the evacuation device 101 is integrally mounted on the operation panel 1021. The first limiting plate 1015 and the second limiting plate 1016 are vertically arranged relative to the operation plate 1021, the lower side of the first limiting plate 1015 is bent towards the second limiting plate 1016 to form a horizontal flanging, and the first limiting plate 1015 is fixed through the flanging. The second limiting plate 1016 is fixedly connected with the first limiting plate 1015 through a screw or other connecting member, so as to cooperate with the first limiting plate 1015 to clamp the fixing portion 1017 of the end portion of the push rod 1014.

In a further aspect of this embodiment, the vacuumizing device 101 further includes a vacuumizing fixing frame, and the vacuum cylinder 1011 is relatively fixedly mounted on the vacuumizing fixing frame. The driving device comprises a driving motor 1012 and a transmission screw 1013, and the vacuumizing fixing frame is connected with a sliding block arranged on the transmission screw 1013. The driving motor drives the transmission screw 1013 to rotate, and the vacuumizing fixing frame moves along the extending direction of the transmission screw 1013 along with the sliding block, so as to drive the vacuum cylinder 1011 to move along the extending direction of the transmission screw 1013.

Specifically, the vacuum fixing frame includes a fixing plate 1018, a clamping seat 1019 for mounting the vacuum cylinder 1011 is provided on the fixing plate 1018, and the vacuum cylinder 1011 is fixed in the clamping seat 1019, so as to move synchronously with the vacuum fixing frame under the transmission action of a transmission screw 1013. A slider with a threaded hole is fixedly connected to the bottom of the fixed plate 1018, and a driving screw 1013 is disposed below the fixed plate 1018 and penetrates through the threaded hole on the slider.

Through the structure, the driving motor 1012 drives the transmission screw 1013 to rotate, and the sliding block moves along the transmission screw 1013, so that the vacuum cylinder 1011 can be driven to move left and right. Meanwhile, by adopting a lead screw transmission mode, the moving distance of the vacuum cylinder 1011 can be precisely controlled by the rotating angle of the driving motor 1012, and the amount of the reagent sucked or discharged by the reagent accommodating device 110 can be precisely controlled. Especially when adding reagent to the enrichment unit, can accurate control reagent's addition, improve the accuracy of detection.

In this embodiment, a plurality of clamping bases 1019 may be disposed on the fixing plate 1018 side by side, and a vacuum cylinder 1011 is installed in each clamping base 1019. The push rods 1014 connected to the pistons in the vacuum cylinders 1011 are all fixed by the first limiting plate 1015, or are all clamped and fixed by the first limiting plate 1015 and the second limiting plate 1016, so that grooves 1022 with the same number as the vacuum cylinders 1011 are arranged on the first limiting plate 1015.

Further, the reagent holding device 110 includes a reagent holding tube 1101 for temporarily storing a reagent, and a needle 1105 communicating with the reagent holding tube 1101, and the reagent is sucked into or discharged from the reagent holding tube 1101 through the needle 1105. The combination of the reagent holding tube 1101 and the needle 1105 is also provided with a plurality of groups, wherein the plurality of reagent holding tubes 1101 are connected with the plurality of vacuum cylinders 1011 in a one-to-one correspondence. The enrichment unit comprises a microfluidic device 200 for receiving a blood sample to be detected, and the enrichment and staining of nucleated cells in the blood sample are realized in the microfluidic device 200. The microfluidic device 200 is also provided in plural, and the reagents in the plural reagent holding tubes 1101 can be added to the plural microfluidic devices 200 in one-to-one correspondence.

In the above scheme, the driving motor 1012 and the driving screw 1013 can drive the plurality of vacuum cylinders 1011 to move simultaneously, so as to synchronously suck the reagents into the plurality of reagent holding pipes 1101, and then synchronously add the sucked reagents into the plurality of microfluidic devices 200 for cell enrichment and staining in a one-to-one correspondence. Therefore, the detection flow of a plurality of blood samples can be synchronously completed, and the detection efficiency is further improved.

In the embodiment, the vacuum cylinder 1011 and the reagent holding tube 1101 are connected by a first communication tube 1020. The reagent holding tube 1101 is arranged vertically, and has an opening at an upper end thereof, which is hermetically connected to one end of the first communication tube 1020. A second communication tube 1104 is connected to the lower end of the reagent holding tube 1101 in a sealed manner, and communicates with the needle 1105 via the second communication tube 1104. The first communication pipe 1020 and the second communication pipe 1104 are rubber hoses, and can be more flexibly matched with the relative position relationship among the vacuum cylinder 1011, the reagent holding pipe 1101 and the needle 1105.

Since the vacuum suction device 101 is mounted on the operation board 1021 above the reagent holding device 110, in order to achieve communication between the vacuum cylinder 1011 and the reagent holding tube 1101 through the first communication pipe 1020, the operation board 1021 has a hollow structure, thereby forming a through hole penetrating up and down. One end of the first communication pipe 1020 is connected to the vacuum cylinder 1011 and then passed through a through hole in the operation plate 1021, so that the other end is connected to the reagent holding tube 1101 under the operation plate 1021.

Preferably, the operation board 1021 has a plurality of through holes penetrating up and down, and a plurality of first communication pipes 1020 are respectively disposed in one of the through holes.

In a further aspect of this embodiment, the reagent picking and placing unit further includes a moving mechanism, configured to drive the reagent accommodating device 110 to move. Specifically, a reagent holding device 1109 is provided below the reagent holding device 110, in which a reagent to be added is stored. The microfluidic device 200 of the enrichment unit is likewise arranged below the reagent holding device 110.

The moving mechanism can drive the reagent holding device 110 to move above the reagent holding device 1109 so as to suck the reagent therefrom. The moving mechanism may also move the reagent holding device 110 over the microfluidic device 200, thereby adding the aspirated reagent into the microfluidic device 200.

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

Further, the first support frame 1102 includes a horizontally disposed plate-like structure provided with fixing holes in which the reagent holding tubes 1101 are inserted. The plurality of fixing holes may be provided so that a plurality of reagent holding tubes 1101 may be fixed in each fixing hole in one-to-one correspondence.

Preferably, at least one side edge of the plate-like structure is provided with an upward or downward flange that can act as a stop when reagent holding tube 1101 is tipped over, thereby stabilizing reagent holding tube 1101 during movement.

Further, the second support frame 1103 includes a first support plate and a second support plate, wherein the first support plate is disposed vertically. The second support plate is formed by bending a plate-shaped structure and comprises a horizontal part which is horizontally arranged and vertical relative to the first support plate and a vertical part which 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.

In detail, the second support plate is formed by bending a metal plate by 90 °, and the vertical portion of the second support plate is fixedly connected with the first support plate by a screw or other connecting member. In this way, the needle 1105 can be pressed firmly against the first support plate by the position where the horizontal portion and the vertical portion of the second support plate meet.

Preferably, the area of the needle 1105 in contact with the first support plate and the second support plate is provided with a protective structure. Since the needle 1105, the first support plate and the second support plate are all metal members in this embodiment, in order to fix the needle 1105 more stably between the first support plate and the second support plate, a protective structure is provided on the outer periphery of the needle 1105, and the protective structure may be a flexible material having a shape-variable or a material capable of fixing the needle 1105 more firmly, such as an adhesive tape.

In a further aspect of this embodiment, the moving mechanism includes a first moving mechanism and a second moving mechanism. The first moving mechanism is used for driving the first support frame 1102 and the second support frame 1103 to move in the vertical direction. The first moving mechanism is mounted on the second moving mechanism, and the second moving mechanism can drive the first moving mechanism, and the first supporting frame 1102 and the second supporting frame 1103 integrally move in the horizontal direction.

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 installed on a bracket, the upper end of the first lead screw 1106 is connected with the first motor 1110, and the first lead screw 1106 is driven by the first motor 1110 to rotate. A slider is mounted on the first screw 1106, and the first support 1102 and the second support 1103 are respectively connected with the slider. The first motor 1110 drives the first screw 1106 to rotate, so that the slider moves up and down along the first screw 1106, and further drives the reagent accommodating device 110 to move in the vertical direction.

The second moving mechanism includes a second motor 1111 and a second lead screw 1107. The second screw 1107 is horizontally arranged, the right end of the second screw is connected with the second motor 1111, and the second screw is driven by the second motor 1111 to rotate. A fixing base 1108 is mounted on the second screw 1107, and a bracket for mounting the first screw 1106 is fixed on the fixing base 1108. The second motor 1111 drives the second screw 1107 to rotate, and drives the fixing base 1108 to move horizontally along the second screw 1107, so as to drive the reagent accommodating device 110 to move horizontally.

In this embodiment, the initial height of the reagent holding device 110 in the vertical direction needs to satisfy: the lower end of the needle 1105 is open above the upper side of the reagent holding device 1109. In the operation process of the integrated nucleated cell enrichment and staining apparatus, when a reagent needs to be added to the microfluidic device 200, the second motor 1111 is started first, and the reagent accommodating device 110 is controlled to move horizontally, so that the needle 1105 moves to a position right above the reagent accommodating device 1109 where the required reagent is stored. The first motor 1110 is then activated to control the reagent holding device 110 to descend so that the needle 1105 extends into the reagent holding device 1109 until the lower end of the needle 1105 is submerged below the liquid surface of the reagent.

Then, the driving motor 1012 is started to drive the vacuum cylinder 1011 to move, and the reagent is sucked into the reagent accommodating tube 1101 through the needle 1105 by sucking air from the reagent accommodating tube 1101 and decompressing. The first motor 1110 is then operated in reverse to control the reagent holding device 110 to rise until the lower end of the needle 1105 is located above the upper side of the reagent holding device 1109 by a certain distance. Then, the second motor 1111 is activated again, and the reagent holding device 110 is controlled to move horizontally rightward until the second motor 1111 is turned off after the needle 1105 moves right above the microfluidic device 200.

At this time, the driving motor 1012 moves reversely to drive the vacuum cylinder 1011 to move reversely, and ventilate and pressurize the reagent containing tube 1101, so that the reagent in the reagent containing tube is discharged along the needle 1105 under the pressurizing action, and the reagent can be added into the microfluidic device 200 below.

The above process can be repeated multiple times, thereby realizing the addition of different reagents, or the multiple addition of the same reagent.

The enrichment unit of the integrated nucleated cell enrichment and staining apparatus of this embodiment will be described in detail.

Specifically, as shown in fig. 6 to 19, the enrichment unit according to the present embodiment includes a microfluidic device 200 and a buffer device. The microfluidic device 200 is configured to receive a blood sample to be detected, and may also receive a reagent added by the reagent taking and placing unit. The buffer device is communicated with the microfluidic device 200 and is used for providing negative pressure with proper size to extract blood samples and reagents, and nucleated cells in the blood samples are intercepted by the microfluidic device 200, so that enrichment and staining of the nucleated cells in the microfluidic device 200 are realized.

In this embodiment, the microfluidic device 200 includes a sample tube 201 and a liquid-product liner 203. The sample tube 201 includes a tube body 2011 with an upper end opening, and a blood sample and a reagent to be detected are added to the microfluidic device 200 through the upper end opening of the tube body 2011. The effusion lining table 203 is arranged below the sample feeding pipe 201, and a filter assembly 214 is arranged between the effusion lining table 203 and the sample feeding pipe 201. The lower end of the tube main body 2011 is also provided with an opening, and the blood sample to be detected can fall on the filter assembly 214 through the tube main body 2011 of the sample inlet tube 201, and the filter assembly 214 plays a role in filtering the blood sample, so that nucleated cells are enriched on the upper side of the filter assembly 214.

Further, a buckling part 2012 protruding from the outer side wall of the pipe body 2011 is provided at the lower end of the pipe body 2011, and the filter assembly 214 is provided between the buckling part 2012 and the liquid accumulation lining table 203. The microfluidic device 200 further comprises a cap 202, wherein the cap 202 is sleeved on the tube main body 2011 and is in threaded connection with the liquid accumulation lining table 203.

Specifically, in the process of assembling the microfluidic device 200, the filter assembly 214 is placed between the fastening portion 2012 of the sample tube 201 and the liquid accumulation lining 203 below, and then the fastening cover 202 is sleeved on the upper end of the tube main body 2011. The buckling cover 202 is mounted to the lower end of the pipe main body 2011, contacts with the buckling part 2012, and then is spirally lowered to be in threaded connection with the liquid accumulation lining table 203, so that the buckling part 2012 is pressed close to the liquid accumulation lining table 203, and the filter assembly 214 between the buckling part and the liquid accumulation lining table is pressed. In this way, in the process of assembling the microfluidic device 200, the sample injection tube 201 and the hydrops lining table 203 are only close to each other in the vertical direction, and no relative rotation occurs between the sample injection tube 201 and the hydrops lining table, so that the filter assembly 214 between the sample injection tube and the hydrops lining table can be ensured not to displace, and the influence of the position deviation of the filter assembly 214 on the enrichment effect of nucleated cells is avoided.

In a further aspect of this embodiment, the fastening cover 202 includes a pressing portion 2021 horizontally disposed for pressing the fastening portion 2012, and a through hole through which the pipe main body 2011 passes is formed in the pressing portion 2021. The buckle cover 202 further includes a connecting portion 2022, and the connecting portion 2022 is formed by extending the outer periphery of the pressing portion 2021 downward. An internal thread is provided on the inner side of the connection portion 2022, and an external thread matching the internal thread is provided on the liquid accumulation table 203.

Specifically, the liquid product pad 203 includes a screw portion 2031, and an operation portion 2032 provided below the screw portion 2031. The screw portion 2031 is provided with external screw threads for screw-coupling with the coupling portion 2022 of the buckle cover 202. The horizontal cross-sectional area of the operation portion 2032 is larger than the area surrounded by the outer diameter of the screw portion 2031, so that a horizontal stop surface is formed at the junction of the screw portion 2031 and the operation portion 2032.

Further, a limiting groove 207 in limiting engagement with the buckling portion 2012 is provided on the lower surface of the pressing portion 2021. The through hole of the pressing part 2021 of the buckle cover 202 for the pipe main body 2011 to pass through can be in interference fit with the pipe main body 2011, so that relative rotation between the buckle cover 202 and the sample injection pipe 201 can be ensured. The buckle cover 202 is sleeved on the tube main body 2011 from top to bottom, and when the buckle cover is installed in place downwards, the buckling part 2012 is embedded into the limiting groove 207 to limit the relative positions of the buckle cover 202 and the sample injection tube 201. The limiting groove 207 is an annular groove, so that the sample inlet pipe 201 and the buckle cover 202 can relatively rotate, but cannot generate relative displacement in the horizontal direction, and the sample inlet pipe 201 and the buckle cover 202 are always coaxial, so that the relative position between the sample inlet pipe 201 and the liquid accumulation lining table 203 is further ensured to be stable in the assembling process of the microfluidic device 200.

In the embodiment, the filter assembly 214 includes the micro-filtration membrane 212 and the water absorbing layer 213, and the water absorbing layer 213 is made of a water absorbing material, and may be provided in one to more layers. The micro-filtration membrane 212 is used for achieving the filtering effect and intercepting nucleated cells in the blood sample. A water-absorbing layer 213 is provided on the underside of the micro-filtration membrane 212 for absorbing the filtrate passing through the micro-filtration membrane 212.

In detail, in this example, to achieve enrichment of CTC in a blood sample to be tested, the microfiltration membrane 212 is a 25mm diameter polycarbonate membrane on which microwells with a diameter of 8 μm are randomly distributed. The underside of the microfiltration membrane 212 is lined with one layer to a plurality of water-absorbing layers 213, preferably three water-absorbing layers 213. The water-absorbing layer 213 is medium-speed neutral filter paper, the diameter of which is the same as that of the micro-filtration membrane 212, and the pore diameter on the water-absorbing layer 213 is 15-20 μm.

In this embodiment, the nucleated cells enriched in the microfluidic device 200 are CTCs, the nuclei of the CTCs have a plurality of irregular nuclei to be deformed, and the oversized nuclei have rigidity such that the CTCs cannot pass through the micropores on the microfiltration membrane 212. While the white blood cells in the blood sample are smaller in volume and the red blood cells have no nuclei, so that they can pass through the micropores in the microfiltration membrane 212 either directly or by changing shape. Thus, after a sample of blood to be tested is added to the microfluidic device 200, CTCs therein can be screened.

Experiments show that the three layers of water-absorbing layers 213 are arranged on the lower side of the micro-filtration membrane 212, so that the micro-filtration membrane 212 is supported to maintain the posture, and the enrichment effect on CTC is further ensured. Meanwhile, the three-layer water-absorbing layer 213 can absorb the filtered sample filtrate or staining solution (i.e. the reagent for cell staining) rapidly, so as to avoid the problem of fuzzy staining result background caused by staining of the microfiltration membrane 212 due to accumulation of liquid on the microfiltration membrane 212 caused by liquid tension or viscosity. Furthermore, the three water-absorbing layers 213 can effectively disturb the flow direction of the microfluid passing through each micropore on the microfiltration membrane 212, and avoid the damage of cell morphology caused by the laminar shear force of the microfluid. In addition, the micro-fluid passing through each micropore can encounter resistance to form countless eddies, so that uniform dispersion of cells is ensured, and the subsequent detection procedure is facilitated.

It will be appreciated that by replacing the microfiltration membrane 212 with a different diameter of the micropores, the nucleated cell enrichment staining apparatus of the present embodiment can be used to enrich other target substances, such as target organisms or biomacromolecules in the sample.

In this embodiment, by arranging the fastening cover 202 in threaded connection with the liquid accumulation lining table 203, the sample injection tube 201 and the liquid accumulation lining table 203 do not rotate relatively in the process of approaching each other, so that the relative displacement between the micro-filtration membrane 212 and the water absorption layer 213 can be avoided, and the enrichment effect is prevented from being affected.

In a further aspect of this embodiment, a lower surface of the fastening portion 2012 is provided with a mounting groove 204 circumferentially surrounding the tube main body 2011 for providing a space for accommodating the filter assembly 214. The top of the threaded portion 2031 of the liquid accumulation liner 203 has a concave cavity in which the liner web 205 is disposed, and the liner web 205 is clamped on the top of the threaded portion 2031, so as to cooperate with the mounting groove 204 to realize placement of the filter assembly 214.

The lining table mesh 205 has a compact net structure, so that the supporting effect on the filter assembly 214 can be realized, the filtered liquid passing through the micro-filtration membrane 212 can not be blocked, and the enrichment effect of cells on the upper side of the micro-filtration membrane 212 is further ensured.

Further, a flow guiding cavity is formed between the bottom wall of the concave cavity and the lining table mesh 205, filtered liquid such as filtered sample filtrate and dyeing liquid is collected in the flow guiding cavity, and is pumped out by negative pressure provided by the buffer device and sent into the waste liquid bottle to be collected.

Specifically, the bottom wall of the concave cavity gradually decreases from the periphery to the middle to form a funnel-shaped structure, and a deflector hole 215 is arranged at the lowest part of the funnel-shaped structure for discharging the filtered liquid.

More specifically, the liquid accumulation liner 203 is hollow, and the bottom wall of the cavity is formed by extending the middle of the inner wall of the threaded portion 2031 obliquely downward toward the central axis, and a circular diversion hole 215 is formed at the lowest position. The outer portion Zhou Shuzhi of the deflector hole 215 extends downward to a certain extent and then extends obliquely in a direction approaching the central axis to form a deflector cone 208, and the filtered liquid can be discharged outwards along the deflector cone 208.

Further, the flow guiding frustum 208 is connected to the communicating bottom pipe 209, and the communicating bottom pipe 209 is communicated with 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 flow guiding frustum 208 is open and extends vertically downward, and is inserted into the communication bottom pipe 209 in a sealing manner. The first pipeline 311 is a rubber hose, so that the sealing connection between the bottom pipe 209 and the first pipeline 311 is convenient to realize.

In this embodiment, the communication bottom tube 209 is mounted on the stationary stage 210. Specifically, the upper surface of the fixing table 210 is provided with a through hole, and the communication bottom pipe 209 is inserted into the through hole. Specifically, the fixing stage 210 is provided with a plurality of through holes so as to correspondingly mount a plurality of communication bottom pipes 209, so that a plurality of microfluidic devices 200 can be mounted at the same time.

In a further aspect of this embodiment, a raised guide strip 206 is disposed on the bottom wall of the cavity in the liquid accumulation lining table 203, and the guide strip 206 extends radially along the liquid accumulation lining table 203 in a rod shape, and the height of the raised portion gradually increases from the outer circumferential center.

Specifically, the plurality of flow guide strips 206 are circumferentially and alternately distributed along the effusion lining table 203, and after the filtered liquid passes through the filtering component 214 and the lining table mesh 205, the filtered liquid can flow out from the flow guide holes 215 more quickly under the flow guide action of the flow guide strips 206.

In the above solution, the protrusion height of the guide strip 206 is gradually increased from the outer circumferential center, so that when the filtrate is less to drop into the guide cavity, the dropped filtrate contacts the guide strip 206, and the filtrate flows along the direction in which the protrusion height of the guide strip 206 is gradually increased on the surfaces of the guide strip 206 and the funnel-shaped structure under the action of gravity and liquid tension.

Further, the surface of the backing net 205 is provided with a plurality of elongated openings 2051, and at least part of the elongated openings 2051 are arranged in a position and shape matching with the guide strips 206, so that the top of each guide strip 206 can be embedded into one of the elongated openings 2051, thereby playing a role in assisting positioning of the backing net 205.

In this embodiment, the upper end of the screw portion 2031 of the liquid accumulation backing 203 is protruded upward to form an annular protrusion structure, and the backing net 205 is radially restrained by the protrusion structure.

Further, the filter assembly 214 is at least partially located inside the annular protrusion structure, so as to also limit the filter assembly 214 in the radial direction, and further prevent the filter assembly 214 from being displaced during the assembly process of the microfluidic device 200.

It should be noted that, the height of the protruding structure is not lower than the thickness of the backing net 205, so as to avoid unstable installation of the backing net 205 in the protruding structure, and also avoid the easy sliding of the filter assembly 214 when contacting with the upper surface of the backing net 205, which affects the installation.

In a further aspect of this embodiment, at least two sealing gaskets (not shown in the drawings) are installed in the microfluidic device 200, so that on one hand, leakage of the liquid is prevented, and on the other hand, leakage of the air is avoided, so that the negative pressure provided by the buffer device is facilitated to perform the pumping function more fully.

Specifically, one of the sealing gaskets is sleeved on the threaded portion 2031 of the liquid accumulation lining table 203, and the lower end of the connecting portion 2022 of the buckle cover 202 presses the sealing gasket on a horizontal stop surface at the joint of the threaded portion 2031 and the operating portion 2032 on the liquid accumulation lining table 203 in the screwing process of the buckle cover 202 and the liquid accumulation lining table 203, so that sealing is achieved.

The other sealing gasket is pre-installed in the installation groove 204 below the buckling part 2012 of the sample injection pipe 201, and in the screwing process of the buckling cover 202, the buckling part 2012 is pressed down, and the filtering component 214 is pressed by matching the sealing gasket in the installation groove 204 with the liquid accumulation lining table 203 below. Alternatively, another sealing gasket may be sleeved downwards from the upper end of the tube main body 2011 and placed on the upper side surface of the buckling part 2012, and the pressing part 2021 and the buckling part 2012 of the sample feeding tube 201 compress the sealing gasket to realize sealing in the screwing process of the buckling cover 202.

In a further aspect of this embodiment, the buffer device of the enrichment unit includes a peristaltic pump 330 and a buffer assembly. Wherein peristaltic pump 330 is in communication with microfluidic device 200 for providing negative pressure to aspirate the filtrate. The buffer assembly is disposed between the microfluidic device 200 and the peristaltic pump 330 and is in communication with the microfluidic device 200 and the peristaltic pump 330, respectively.

In this embodiment, the blood sample is filtered in the microfluidic device 200 to enrich nucleated cells therein, and the peristaltic pump 330 is used to provide negative pressure to achieve low-speed extraction, so as to avoid the problem of excessive suction force due to the difficulty in filtering and hardening of the blood when excessive external force is applied. However, the peristaltic pump 330 is easy to generate pulse flow in the operation process, and by the arrangement of the buffer assembly, the pulse can be counteracted at the buffer assembly, so that the influence of the pulse flow on the cell enrichment effect in the microfluidic device 200 is avoided.

In this embodiment, the buffer assembly includes a buffer bottle 310 respectively connected to the microfluidic device 200 and the peristaltic pump 330, and a predetermined amount of gas is sealed in the buffer bottle 310. The buffer bottle 310 is connected with a first pipeline 311 and a second pipeline 312 in a sealing way, wherein the first pipeline 311 is communicated with the micro-fluidic device 200, and the second pipeline 312 is communicated with the peristaltic pump 330.

Further, the top of the buffer bottle 310 is provided with a bottle mouth, a sealing cover 313 for sealing the bottle mouth is arranged at the bottle mouth, and one end of the first pipeline 311 is connected with the communicating bottom pipe 209, so that the communication between the microfluidic device 200 and the buffer bottle 310 is realized. The other end of the first pipe 311 extends into the buffer bottle 310 through the sealing cap 313. Similarly, the second tube 312 has one end extending into the interior of the buffer bottle 310 through the sealing cap 313 and the other end communicating with the atmosphere through the peristaltic pump 330.

During operation of peristaltic pump 330, the gas within buffer bottle 310 may be drawn out, creating a negative pressure within buffer bottle 310. Since the buffer bottle 310 is in a sealed state, the negative pressure environment in the buffer bottle 310 generates suction force on the first pipeline 311, so that the filtered liquid passing through the filter assembly 214 in the microfluidic device 200 is sucked into the first pipeline 311 and flows into the buffer bottle 310 along the first pipeline 311.

When the peristaltic pump 330 generates pulses, the pulse flow can be stopped in the buffer bottle 310 due to the buffer bottle 310, so as to realize a buffer effect, and the pumping force applied to the microfluidic device 200 does not have a significant peak value. Especially, in the case that the filtered liquid in the microfluidic device 200 has only a few milliliters, the generation of the pulse flow can greatly affect the experimental result, and the impact of the pulse on the filtering process can be remarkably reduced by arranging the buffer bottle 310, so that the enrichment effect of cells in the microfluidic device 200 is ensured.

In the preferred scheme of this embodiment, the length of the first pipeline 311 extending into the buffer bottle 310 is less than half of the height of the buffer bottle 310, so as to avoid the influence of the buffer bottle 310 on the filling effect of the pulse flow.

In this embodiment, the filtered liquid in the microfluidic device 200 is pumped into the buffer bottle 310 along the first pipeline 311, so that the buffer bottle 310 also plays a role in collecting the filtered liquid. Since the first pipeline 311 is communicated with the microfluidic device 200, the buffer bottle 310 can be filled with pulse flow only by ensuring that the lower end of the first pipeline 311 is always communicated with air and is not immersed below the liquid level. If the first tube 311 contacts the liquid in the buffer bottle 310, the pressure of the liquid is greater than the suction force of the negative pressure, which may cause the end of the first tube 311 in the bottle to be blocked by the liquid, so that the blood in the microfluidic device 200 cannot be sucked. The length of the first pipe 311 extending into the buffer bottle 310 is as short as possible, so that the problem that the lower end of the first pipe 311 contacts the liquid in the bottle can be avoided.

In a further aspect of this embodiment, the second conduit 312 is located within the buffer bottle 310 at a length greater than half the height of the buffer bottle 310. During the pumping process, as more and more liquid is collected in the buffer bottle 310, the volume of the gas in the bottle is reduced, the density of the liquid is far greater than that of the gas, and thus the buffer effect on the pulse flow is reduced when the volume of the gas is reduced. By extending the second conduit 312 to more than half the height of the buffer bottle 310, the liquid surface can be sucked out by the peristaltic pump 330 when contacting the second conduit 312, thereby ensuring the volume of the gas in the buffer bottle 310 and further ensuring the buffer effect of the buffer bottle 310.

In a more preferred 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, so that when the liquid level in the buffer bottle 310 rises to contact with the second pipeline 312, the peristaltic pump 330 can suck out the liquid in the bottle, the liquid level does not rise any more, and the liquid accumulated in the buffer bottle 310 can be effectively prevented from immersing the lower end of the first pipeline 311.

Optimally, the second pipeline 312 is arranged to extend into the bottle bottom of the buffer bottle 310, the first pipeline 311 is arranged to extend into the bottle mouth of the buffer bottle 310, when liquid is sucked into the bottle, the second pipeline 312 contacts with the liquid surface, the liquid is sucked out under the action of the peristaltic pump 330, no matter whether the peristaltic pump 330 sucks liquid or gas, the volume of the sucked liquid or gas is the same, the negative pressure state in the bottle is not influenced, the negative pressure state in the bottle is ensured, and the frequency of cleaning the liquid in the bottle is greatly reduced.

Further, the bottle mouth of the buffer bottle 310 is lower than the microfluidic device 200, so that the filtered liquid discharged from the microfluidic device 200 flows down along the first pipeline 311 into the buffer bottle 310, and the situation that the liquid in the first pipeline 311 may flow back to the microfluidic device 200 is avoided.

In another specific scheme of the embodiment, two or more buffer bottles can be further arranged between the microfluidic device and the peristaltic pump, and each buffer bottle is sequentially communicated through a pipeline.

Specifically, at least a first buffer bottle and a second buffer bottle are arranged between the microfluidic device and the peristaltic pump. The bottle mouth of the first buffer bottle is connected with a first pipeline in a sealing mode, and the first pipeline is connected with a communicating bottom pipe below the microfluidic device. The bottleneck of the second buffer bottle is connected with a second pipeline in a sealing way, and the second pipeline is communicated with the peristaltic pump.

When only the first buffer bottle and the second buffer bottle are arranged, the first buffer bottle and the second buffer bottle are communicated through the intermediate pipeline. Or at least one middle buffer bottle is further arranged between the first buffer bottle and the second buffer bottle, and at the moment, any two adjacent buffer bottles are communicated through a middle pipeline.

Further, in this embodiment, in the process of enriching CTCs and staining in the microfluidic device 200, the filtration liquid needs to be pumped multiple times, especially when the peristaltic pump 330 stops working at the end of one pumping process, which inevitably causes the problem of liquid backflow. Therefore, in this embodiment, the check valve 3111 is disposed on the first line 311 between the buffer bottle 310 and the microfluidic device 200, and the check valve 3111 is unidirectional connected to the buffer bottle 310 by the microfluidic device 200.

Thus, when the peristaltic pump 330 stops working, the filtered liquid in the first pipeline 311 cannot reversely flow back to the microfluidic device 200 through the one-way valve 3111, so that the influence of the filtered liquid backflow on the enrichment effect is avoided, and the accuracy of the detection result is further improved.

Further, as shown in fig. 20 and 21, the nucleated cell enrichment staining integrated apparatus of the present embodiment adds a blood sample to be detected to the microfluidic device 200 through a blood sample adding unit, which will be described in detail below.

The blood adding unit includes a pressure generating device 420 and a sealing blood collection tube 410, the pressure generating device 420 is communicated with the sealing blood collection tube 410, and the sealing blood collection tube 410 is provided with a blood discharge tube 430 communicated with the outside. The pressure generating device 420 ventilates and pressurizes the sealed blood collection tube 410, and the blood sample to be detected contained in the sealed blood collection tube 410 is discharged along the blood discharge tube 430. The outlet orifice of the blood drain tube 430 is disposed above the microfluidic device 200 so as to add a blood sample to be detected to the microfluidic device 200.

Specifically, in this embodiment, the sealed blood collection tube 410 includes a blood collection tube body 411 and a sealing cover 412, and the sealing cover 412 may be a cover structure that can wrap the tube opening and the outer tube wall adjacent to the tube opening, or may be a plug structure that plugs into the tube opening and tightly abuts the inner tube wall adjacent to the tube opening. After collecting a blood sample to be detected, the collected blood sample is added into the blood collection tube body 411, and then the tube orifice of the blood collection tube body 411 is plugged by the sealing cap 412, so that the sealing blood collection tube 410 with an airtight structure relative to the outside is formed.

Preferably, the sealing cap 412 includes a piercing portion for communicating with the blood discharge vessel 430, the piercing portion facing the nozzle of the blood collection tube body 411. The piercing portion is made of a material having a certain elasticity, such as rubber, etc., to ensure that the sealing of the drainage tube 430 and the sealing blood collection tube 410 is maintained when they are communicated.

The puncture part may be integrally provided with the sealing cap 412, or may be a spacer provided between the sealing cap 412 and the blood collection tube body 411.

The pressure generating device 420 is communicated with the sealed blood collection tube 410 through a pressurizing tube 440, and the pressure generating device 420 of the present embodiment may be a pressure generating device 420 capable of being automatically controlled, or may be a pressure generating device 420 requiring manual operation.

When the pressure generating means 420 is an automatically controllable pressure generating means 420, preferably the pressure generating means 420 is a peristaltic pump. The peristaltic pump comprises a driver and a pump head, wherein the driver drives the pump head to squeeze the pressurizing tube 440, unidirectional air flow is formed in the pressurizing tube 440, and the unidirectional air flow flows into the sealed blood collection tube 410 through the pressurizing tube 440, so that ventilation and pressurization of the sealed blood collection tube 410 are realized. During pressurization, the gas flowing into sealed blood collection tube 410 passes through pressurization tube 440 only and does not contact the internal structure of peristaltic pump itself, without fear of contaminating the blood sample with the peristaltic pump.

When the pressure generating means 420 is a pressure generating means 420 requiring manual operation, it is preferable to use a syringe type air pump which is pushed and pulled manually.

The outlet orifice of the pressurizing tube 440 communicated with the sealed blood collection tube 410 and the inlet orifice of the discharging tube 430 communicated with the sealed blood collection tube 410 are respectively provided with a blood collection needle, and the blood collection needle comprises a hollow needle head inside and is used for puncturing the puncturing part of the sealing cover 412 so that the pressurizing tube 440 and the discharging tube 430 are communicated with the sealed blood collection tube 410.

In a preferred embodiment of the present embodiment, the blood adding unit includes a plurality of sealed blood collection tubes 410, each sealed blood collection tube 410 is provided with a blood discharge tube 430, and each sealed blood collection tube 410 is respectively communicated with the same pressure generating device 420 through a pressurizing tube 440. The liquid outlet orifices of the plurality of blood discharge tubes 430 are arranged above the plurality of microfluidic devices 200 in a one-to-one correspondence manner, so that the blood samples to be detected contained in the different sealed blood collection tubes 410 can be added into the different microfluidic devices 200.

With the above-described scheme, the pressure generating device 420 may ventilate and pressurize the plurality of sealed blood collection tubes 410, respectively, so that the blood sample to be detected in the sealed blood collection tubes 410 is discharged along the respective connected blood discharge tubes 430. The scheme does not need to separately arrange the pressure generating devices 420 for each sealed blood collection tube 410, so that the number of the pressure generating devices 420 is reduced, the cost is reduced, the structure of the blood sample adding unit is simplified, and the difficulty of maintenance is reduced.

In a further aspect of this embodiment, the pressure generating device 420 includes a pressure generator 421 and a voltage divider 422, where the voltage divider 422 includes an air inlet end and an air outlet end, the air outlet end includes a plurality of air outlets, and each air outlet is respectively communicated with the air inlet end. The plurality of sealed blood collection tubes 410 are respectively communicated with the plurality of air outlets in a one-to-one correspondence manner through the pressurizing tubes 440.

As a specific implementation manner of this embodiment, the pressure generator 421 is a peristaltic pump, and the pressure generator distributes the positive pressure generated by itself to each sealed blood collection tube 410 through the voltage divider 422, so that the structure of the pressure generator 421 itself does not need to be changed due to the number of the sealed blood collection tubes 410, and the use difficulty and the use cost of the device are reduced.

Further, a main switch 441 is provided between the pressure generator 421 and the pressure divider 422 to block communication between the pressure generator 421 and the pressure divider 422.

Specifically, the pressure generator 421 and the pressure divider 422 are connected by a hose, and the main switch 441 is a device capable of applying pressure to the hose, when the main switch 441 applies pressure to the hose, the hose is pressed and flattened, so that the inner wall of the hose is attached, air cannot circulate, and the positive pressure of the pressure generator 421 is prevented from being transmitted to the pressure divider 422. When the main switch 441 releases the hose, the hose returns to its original state, and the air flow can normally flow, so that the positive pressure of the pressure generator 421 can be supplied to the pressure divider 422.

Alternatively, a rigid pipe connection may be used between pressure generator 421 and pressure divider 422, or a joint may be provided between pressure generator 421 and pressure divider 422 in direct communication with each other. The main switch 441 may be a switch device that may be inserted into the interior of the rigid tube and block the flow of gas within the rigid tube.

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

Further, a separate switch 442 is disposed between each air outlet of the voltage divider 422 and the corresponding sealed blood collection tube 410. Specifically, a separate switch 442 may be provided on each of the pressurizing pipes 440.

The sub-switches 442 can block the on-off of the air flow between each air outlet and its corresponding sealed blood collection tube 410, and each sub-switch 442 is opened and closed independently without affecting each other. That is, the plurality of sub-switches 442 may be all open, all closed, or a portion may be open while another portion is closed.

The sub-switch 442 may be a manual switch, i.e., the open/close state of each switch is set by manually operating to open/close one by one. The switch may be automatically turned on or off, i.e., the switch may be automatically controlled according to whether or not each pressurizing tube 440 is connected to the sealing blood collection tube 410. The method comprises the following steps: the sub-switch 442 on the pressurizing tube 440 connected with the sealed blood collection tube 410 or the blood sample in the connected sealed blood collection tube 410 is controlled to be opened, so that the corresponding pressurizing tube 440 is conducted; and the sub-switch 442 on the pressurizing tube 440, which is not connected to the sealing blood collection tube 410 or to which no blood sample is connected in the sealing blood collection tube 410, is controlled to be closed, thereby cutting off the corresponding pressurizing tube 440.

In the above-mentioned scheme, the separate switches 442 that can be opened and closed independently are respectively disposed on each pressurizing tube 440, and when a part of the pressurizing tubes 440 is not connected to the sealed blood collection tube 410 or there is no blood sample in the connected sealed blood collection tube 410, the part of the pressurizing tubes 440 can be blocked to prevent the leakage of the positive pressure generated by the pressure generator 421, so that other sealed blood collection tubes 410 containing blood samples can be ensured to effectively receive the positive pressure, thereby discharging the blood samples.

In this way, the blood sample adding unit can add the blood sample into the corresponding microfluidic device 200 without waiting for all the pressurizing pipes 440 to be connected with the sealed blood collection pipe 410 containing the blood sample, so that the blood sample adding unit can more flexibly adapt to different requirements, and the number of the sealed blood collection pipes 410 is not required to be increased by waiting for a long time, thereby improving the working efficiency and saving the waiting time of patients.

In a further aspect of this embodiment, the outlet orifice of the pressurizing tube 440 in communication with the sealed blood collection tube 410 is disposed within the sealed blood collection tube 410 near the orifice. When blood is added to sealed blood collection tube 410, the level of the blood sample should be below the level of the outlet orifice of pressurized tube 440. In this way, the pressurizing tube 440 is not in direct contact with the blood sample all the time, and even if the pressure generating device 420 is in reverse suction when the operation is stopped, the blood sample is not sucked into the pressurizing tube 440, so that the problem that the blood sample enters the pressure generating device 420 to cause faults is avoided.

The inlet port of the drainage tube 430 communicating with the sealing blood collection tube 410 is disposed near the bottom of the sealing blood collection tube 410, preferably, the inlet port of the drainage tube 430 abuts against the bottom of the sealing blood collection tube 410. During operation of the pressure generating device 420, the pressurizing tube 440 is used to ventilate and pressurize the sealed blood collection tube 410, so that the air pressure in the sealed blood collection tube 410 is increased, and the blood sample therein can be pressed into the blood discharge tube 430 from the liquid inlet of the blood discharge tube 430. The liquid inlet pipe is abutted to the bottom of the sealed blood collection tube 410, so that the blood sample in the sealed blood collection tube 410 can completely enter the blood discharge tube 430, and the blood sample residue is avoided.

As a specific embodiment, the outlet nozzle is provided with a lancet, the lancet penetrates through the sealing cover 412 of the sealed blood collection tube 410, and the needle of the lancet is located inside the sealed blood collection tube 410 near the nozzle, and this position is higher than the highest page of the blood sample in the sealed blood collection tube 410, i.e. the needle of the lancet does not contact the blood sample.

As another specific embodiment, the sealing cover 412 of the sealing blood collection tube 410 is provided with a communication port for inserting the blood discharge tube 430 and the pressurizing tube 440, the blood discharge tube 430 and the pressurizing tube 440 are in sealing fit with the communication port, and the communication port is inserted into the sealing blood collection tube 410.

In a further aspect of this embodiment, the blood sample adding unit further includes a sampling fixing table 450, and the liquid outlet of the blood discharge tube 430 is fixed by the sampling fixing table 450.

Specifically, the sampling fixing table 450 includes a platen 451 horizontally arranged, and a plurality of positioning holes 457 penetrating up and down are formed in the platen 451, and the liquid outlet of the drainage tube 430 passes through the positioning holes 457 of the platen 451 from top to bottom. The positioning hole 457 has the same diameter as the outer diameter of the drainage tube 430 or slightly larger than the outer diameter of the drainage tube 430.

The drainage tube 430 in this embodiment is a hose, and by providing the platen 451 with the positioning hole 457, the position of the outlet of the drainage tube 430 can be fixed, so as to ensure that the outlet is stably maintained directly above the microfluidic device 200, and further ensure that the blood sample discharged along the drainage tube 430 can be added into the microfluidic device 200. In this way, leakage or mixing of the blood sample is avoided.

Further, when the number of the microfluidic devices 200 that can be disposed in the integrated apparatus for enrichment and staining of nucleated cells is greater than the number of the sealed blood collection tubes 410, the liquid outlet orifices of the same row of blood collection tubes 430 may need to be moved above different microfluidic devices 200. When the positioning holes 457 on the platen 451 are arranged in a one-to-one correspondence with the blood drain tubes 430, the platen 451 needs to be moved integrally to drive the liquid outlet of the blood drain tube 430 to move.

For this purpose, in this embodiment, the blood sample adding unit further comprises a fixing frame (not shown in the drawing), the two sides of the sampling fixing table 450 are provided with guide rails 455 arranged in parallel along the horizontal direction, the fixing frame comprises a supporting frame 456 matched with the two guide rails 455 of the sampling fixing table 450, and the guide rails 455 and the supporting frame 456 are slidingly connected.

Specifically, support 456 includes two symmetrically disposed portions, one on each side of sampling fixture 450 where rail 455 is disposed, and support 456 is provided with a rail or pulley that mates with rail 455.

In the above-mentioned scheme, the guide rail 455 of the sampling fixing table 450 is matched with the supporting frame 456, so that the sampling fixing table 450 can move along the extending direction of the guide rail 455, and then the position of the positioning hole 457 is adjusted, so that the same positioning hole 457 can be aligned to different microfluidic devices 200, and then the liquid outlet of the blood drainage tube 430 is adjusted to be above different microfluidic devices 200.

In a further aspect of this embodiment, the blood sample adding unit further includes a driving mechanism for driving the sampling fixture 450 to reciprocate along the extending direction of the guide rail 455. Thus, the movement of the sampling fixing table 450 can be controlled by the driving mechanism, and the user does not need to adjust the position of the sampling fixing table 450 in a manual pushing and pulling manner, so that the sampling fixing table is more convenient.

As a specific embodiment, the driving mechanism comprises a third screw rod 454 and a motor 453, a sliding block is mounted on the third screw rod 454, and the third screw rod 454 is driven by the motor 453 to rotate so as to enable the sliding block to move along the third screw rod 454. The third screw 454 is arranged in parallel with the guide rail 455 of the sampling fixing stage 450, and the sampling fixing stage 450 is fixedly connected with the slider on the third screw 454.

In the above scheme, the third screw 454 is matched with the threaded hole 452 on the slider, and when the third screw 454 is driven by the motor 453 to rotate, the slider can move along the extending direction of the third screw 454, so as to drive the sampling fixing table 450 to move along the third screw 454. The motor 453 is connected to the support frame 456, and when the motor 453 drives the third screw 454 to rotate in different directions, the sampling fixing table 450 reciprocates along with the slider on the third screw 454.

The embodiment also provides a method for carrying out cell enrichment and staining by adopting the nucleated cell enrichment and staining integrated equipment, which specifically comprises the following steps:

(1) The reagent taking and placing unit adds microporous membrane activating solution into the microfluidic device;

(2) The blood sample adding unit adds a blood sample to be detected into the microfluidic device, and the enrichment unit performs suction filtration on the microfluidic device to enrich target cells;

(3) The reagent taking and placing unit absorbs the erythrocyte lysate and adds the erythrocyte lysate into the microfluidic device, lyses residual erythrocyte, and then the enrichment unit carries out suction filtration on the microfluidic device;

(4) The reagent taking and placing unit absorbs the cell fixing liquid and adds the cell fixing liquid into the microfluidic device, and then the enrichment unit carries out suction filtration on the microfluidic device;

(5) The reagent taking and placing unit absorbs the buffer solution and adds the buffer solution into the microfluidic device for cleaning, and then the enrichment unit carries out suction filtration on the microfluidic device;

(6) The reagent taking and placing unit absorbs the fixed liquid on the membrane and adds the fixed liquid into the microfluidic device, and then suction filtration is carried out on the microfluidic device;

(7) The reagent taking and placing unit absorbs the penetrating fluid and adds the penetrating fluid into the microfluidic device, and then the enrichment unit carries out suction filtration on the microfluidic device;

(8) The reagent taking and placing unit absorbs the dyeing liquid and adds the dyeing liquid into the microfluidic device, and the enrichment dyeing unit carries out suction filtration on the microfluidic device after dyeing for a certain time; step (8) is carried out twice in total;

(9) The reagent taking and placing unit absorbs the buffer solution and adds the buffer solution into the microfluidic device for cleaning, and then the enrichment unit carries out suction filtration on the microfluidic device; and (3) performing the step (9) for one to three times, and then obtaining the target cells after enrichment dyeing.

In detail, the specific operation procedure of performing cell enrichment staining by using the above-mentioned integrated equipment for cell enrichment staining is as follows.

In the preparation phase, the operator adds a blood sample to be tested to the sealed blood collection tube 410 and seals the sealed blood collection tube 410. Wherein, the blood sample to be detected is whole blood after pretreatment, and the pretreatment can be a pretreatment mode of blood sample collected from a patient body before detection when enrichment of nucleated pathological cells such as CTC and the like is carried out in the prior art. The reagent containing device 1109 below the reagent containing device 110 is filled with a reagent to be added later, such as a microporous membrane activating solution, a red blood cell lysate, a cell fixing solution, a buffer solution, a staining solution, and the like. It is also necessary to assemble the microfluidic device 200 in advance and mount it on the communication bottom tube 209 that has been fixed on the fixing stage 210. The water-absorbing layer 213 therein needs to be pre-wetted when assembling the microfluidic device 200.

After the preparation phase is completed, the integrated equipment can start to work. First, the reagent accommodating device 110 in the reagent taking and placing unit is controlled to move, and the driving motor 1012 is used for controlling the sucking of the microporous membrane activating solution, then the reagent accommodating device 110 moves to the position above the microfluidic device 200, the driving motor 1012 is reversely operated to add the sucked microporous membrane activating solution into the microfluidic device 200, and the microfiltration membrane 212 is pretreated.

After the addition is completed, the pressure generating device 420 is activated to apply positive pressure to the sealed blood collection tube 410, thereby adding a blood sample to the microfluidic device 200. The blood sample addition is completed, peristaltic pump 330 is activated and microfluidic device 200 is subjected to microfluidic suction filtration to enrich the blood sample for target cells, i.e., CTCs.

Then, the reagent taking and placing unit is controlled again by adopting a similar control flow, so that the reagent accommodating device 110 absorbs a proper amount of erythrocyte lysate, the erythrocyte lysate is added into the microfluidic device 200, a small amount of residual erythrocyte is lysed, and then the peristaltic pump 330 is started to perform micro-flow suction filtration on the microfluidic device 200, and the filtered liquid is pumped out.

And then controlling the reagent taking and placing unit to add a proper amount of cell fixing liquid into the microfluidic device 200 to fix target cells, starting the peristaltic pump 330, performing micro-flow suction filtration on the microfluidic device 200, and extracting the filtered liquid.

Then, the reagent taking and placing unit is controlled to add a proper amount of buffer solution into the microfluidic device 200, then the peristaltic pump 330 is started, the microfluidic device 200 is subjected to micro-flow suction filtration, and the filtered liquid is pumped out, so that the microfluidic device is cleaned and filtered once.

Then, the reagent taking and placing unit is controlled to add the fixed liquid on the membrane into the microfluidic device 200, and then the peristaltic pump 330 is started to perform micro-flow suction filtration on the microfluidic device 200, and the filtered liquid is pumped out.

Then, the reagent taking and placing unit is controlled to add the penetrating fluid into the microfluidic device 200, and then the peristaltic pump 330 is started to perform micro-flow suction filtration on the microfluidic device 200, and the filtered fluid is pumped out.

Then, the cells were stained, including: by controlling the reagent taking and placing unit to add the dye liquid into the microfluidic device 200, after a certain period of dyeing, the peristaltic pump 330 is started to perform micro-flow suction filtration on the microfluidic device 200, and the liquid is pumped out. After the suction filtration is completed, the dyeing treatment is performed once again.

Finally, by controlling the reagent taking and placing unit to add a proper amount of buffer solution into the microfluidic device 200, then starting the peristaltic pump 330, performing micro-flow suction filtration on the microfluidic device 200, and pumping out the filtered liquid. The step of adding the buffer solution and then performing micro-flow suction filtration can be performed only once, or can be performed repeatedly for two or three times.

After the above procedure is completed, the micro-filtration membrane 212 is enriched with stained target cells, and the operator can disassemble the microfluidic device 200 and remove the micro-filtration membrane 212 therefrom for microscopic examination or other subsequent detection procedures.

The operation process can be automatically controlled through a preset program, so that the whole process from blood sample delivery to cell enrichment and staining can be fully automatically completed by the nucleated cell enrichment and staining integrated device of the embodiment under the condition of no manual intervention. The operator can directly take out the micro-filtration membrane 212 for microscopic examination after the integrated equipment for enrichment and staining of nucleated cells is finished by adding the blood sample to be detected and the required reagent in advance.

Example two

As shown in fig. 22 and 23, this embodiment differs from the first embodiment in that: the buffer assembly comprises a buffer 340, wherein a buffer chamber is formed in the buffer 340, the buffer chamber is respectively communicated with the microfluidic device 200 and the peristaltic pump 330, and at least part of the cavity wall of the buffer chamber is made of a material capable of undergoing elastic deformation.

In this embodiment, since part of the wall of the buffer chamber may be elastically deformed, during the operation of the peristaltic pump 330, the buffer chamber may be pumped and depressurized, so that the wall of the buffer chamber may be deformed to be concave inwards. When peristaltic pump 330 generates pulse flow, the walls of the buffer chamber are further recessed, so that the volume inside the buffer chamber is reduced, and the effect of buffering pulses is achieved.

In the embodiment of the present invention, the buffer 340 includes a hollow bracket 343 and a buffer film 344 that can be elastically deformed. Wherein, the bracket 343 is provided with a plurality of through holes, and the buffer film 344 at least covers the through holes entirely. Thus, the buffer membrane 344 and the support 343 together enclose the buffer chamber, and the portion of the buffer membrane 344 covering the through hole forms a chamber wall capable of being elastically deformed.

With the above structure, during operation of the peristaltic pump 330, the portion of the buffer film 344 covered on the through hole may be recessed into the bracket 343 through the through hole. And when the peristaltic pump 330 generates a pulse flow, the buffer membrane 344 is recessed toward the inside of the holder 343 to reduce the internal volume of the buffer chamber, thereby playing a role of buffering the pulse.

In one embodiment of the present invention, the bracket 343 is a hollow cylinder structure, preferably a cylinder structure. The through holes are formed on the side walls of the column structure, and the buffer film 344 is sleeved on the side walls of the column structure to cover the through holes.

More specifically, when the holder 343 has a hollow cylindrical structure, the buffer film 344 has a tubular shape, and an inner diameter thereof in an initial state is slightly smaller than an outer diameter of the holder 343. Thus, the buffer film 344 is stretched when being sleeved on the bracket 343, so that the sealing performance of the buffer film 344 covering the through holes can be enhanced, and the buffer 340 is prevented from generating air leakage faults. More preferably, the buffer film 344 may be adhesively fixed to both ends of the bracket 343.

Further, the bracket 343 includes two end walls 345 disposed at opposite intervals, 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 circumference of one of the end walls 345, the other end is connected to the outer circumference of the other end wall 345, and a plurality of support rods 3431 are arranged at intervals along the outer circumference of the end wall 345, and the through holes are formed between the adjacent two support rods 3431.

Preferably, the struts 3431 are uniformly distributed along the outer circumference of the end wall 345 with the individual struts 3431 being parallel to one another. More preferably, the support rods 3431 are arranged extending in a direction parallel to the axis of the cylinder structure.

In this embodiment, the length of the buffer film 344 along the axial direction of the support 343 is preferably greater than the axial length of the support 343. Thus, after the buffer film 344 is sleeved on the bracket 343, the two ends of the buffer film 344 can wrap the periphery of the end wall 345 under the self elastic action, so that no air leakage occurs at the joint of the end wall 345 and the supporting rod 3431.

In another specific scheme of the embodiment, a plurality of support rods are arranged, and any two support rods can be mutually crossed or not crossed to be irregularly arranged, so long as through holes can be reserved on the side wall of the support.

In a further aspect of this embodiment, two end walls 345 are each provided with an air port. One of the ports is connected to the first conduit 311 so as to communicate with the microfluidic device 200 through the first conduit 311. The other port is connected to a second conduit 312, which communicates through the second conduit 312 to a peristaltic pump 330.

Further, the air port is configured as a trumpet-shaped structure with a gradually reduced diameter extending outwards from the end wall 345, and the first pipeline 311 and the second pipeline 312 are both hoses and sleeved at the tail end of the trumpet-shaped structure. Since the cross-sectional area of the buffer 340 is larger than that of the first and second pipes 311 and 312, by providing the horn-like structure, the magnitude of abrupt change in cross-sectional area is reduced on a path passing through the first and second pipes 311 and 340 and 312 in sequence.

In this embodiment, peristaltic pump 330 operates to provide negative pressure to the interior of buffer 340, thereby drawing out filtrate from microfluidic device 200. When the peristaltic pump 330 generates pulse flow, the buffer membrane 344 wrapped on the support 343 can deform and contract towards the inside of the support 343, so that the internal volume of the buffer chamber is reduced, and then the negative pressure abrupt change generated by the pulse flow is counteracted or partially counteracted, so that the suction force applied to the microfluidic device 200 cannot have obvious peaks, and the influence of the pulse flow on experimental results is avoided.

In a further aspect of this embodiment, to avoid the problem of liquid backflow when the peristaltic pump 330 is stopped, a one-way valve 3111 is also provided between the microfluidic device 200 and the peristaltic pump 330, similar to the embodiments described above.

Specifically, in the present embodiment, the check valve 3111 is disposed on the second conduit 312, and is in unidirectional communication with the peristaltic pump 330 via the buffer 340. When peristaltic pump 330 is deactivated, liquid in second tube 312 is blocked by one-way valve 3111 from flowing back to buffer 340. In the operation process of the peristaltic pump 330, the buffer membrane 344 is always kept in an inwardly contracted state due to the negative pressure provided by the peristaltic pump 330, so that after the peristaltic pump 330 stops operating, the buffer membrane 344 is restored under its own elasticity, and can continuously provide a certain negative pressure to the microfluidic device 200, so as to ensure that the liquid in the first pipeline 311 does not flow back into the microfluidic device 200.

In this embodiment, a buffer 340 composed of a support 343 and an elastic buffer membrane 344 is disposed between the microfluidic device 200 and the peristaltic pump 330, and the buffer function of the buffer membrane 344 on pulse flow is achieved by utilizing the characteristic that the buffer membrane 344 can deform when pressure changes, so that the detection accuracy is ensured.

Example III

As shown in fig. 24 and 25, this embodiment is further defined in the first or second embodiment, and the vessel array 430 is provided with an openable and closable occlusion structure.

Specifically, when a blood sample to be tested enters the drainage tube 430, the blocking structure may conduct the drainage tube 430, so that the blood sample may be discharged along the drainage tube 430 and added to the microfluidic device 200 below. The occlusion structure automatically occludes the drainage tube 430 when there is no blood sample in the drainage tube 430. Thus, after all the blood samples in any one sealed blood collection tube 410 are discharged, the blood discharge tube 430 connected with the sealed blood collection tube 410 is automatically plugged by the plugging structure, so that the positive pressure provided by the pressure generating device 420 can not leak to the outside along the blood discharge tube 430, and the normal outward discharge of the blood samples from other sealed blood collection tubes 410 is ensured.

In this embodiment, the blood drainage tube 430 has a vertical extension portion, and the plugging structure includes a pressure stabilizing bin 437 disposed on the vertical extension portion, and a floating block 438 disposed inside the pressure stabilizing bin 437, where the density of the floating block 438 is less than or equal to the density of the blood sample, so that the floating block 438 can be suspended in the blood sample. The liquid inlet end of the pressure stabilizing bin 437 is kept in a conducting state with the blood drain tube 430, a liquid outlet is arranged at the bottom of the pressure stabilizing bin 437, and the floating block 438 can seal the liquid outlet.

More specifically, the pressure stabilizing cartridge 437 has a barrel-like structure with an inner diameter larger than that of the discharge vessel 430. The floating block 438 is spherical, and the liquid outlet of the pressure stabilizing bin 437 is circular with a diameter smaller than that of the floating block 438. The height of the pressure stabilizing bin 437 is greater than the diameter of the float blocks 438, ensuring that the float blocks 438 have space within the pressure stabilizing bin 437 that can move up and down. The inner diameter of the pressure stabilizing bin 437 is larger than the diameter of the floating block 438, so that the floating block 438 is not limited by the inner wall of the pressure stabilizing bin 437 when conveyed in the pressure stabilizing bin 437.

In the initial state, the floating block 438 is kept at the bottom of the pressure stabilizing bin 437 under the action of gravity, and the liquid outlet is blocked. When the sealed blood collection tube 410 receives positive pressure, the blood sample is pressed into the blood discharge tube 430, and after entering the pressure stabilizing bin 437 along the blood discharge tube 430, the floating block 438 floats upwards to be separated from the liquid outlet, so that the liquid outlet is opened, and the blood sample can be discharged along the blood discharge tube 430 and added into the microfluidic device 200. When the blood sample in the sealed blood collection tube 410 is completely discharged, no blood sample exists in the pressure stabilizing bin 437, and the floating block 438 is used for plugging the liquid outlet of the pressure stabilizing bin 437 again under the action of gravity, so that the blood discharge tube 430 is cut off. The positive pressure provided by the pressure generating device 420 does not leak out through the drainage vessel 430 at this time.

In the above scheme, the floating block 438 controls the opening and closing of the liquid outlet of the pressure stabilizing bin 437, the lifting movement of the floating block 438 is controlled by the buoyancy provided by the blood sample, and manual intervention is not needed, so that the automatic control of the on-off of the blood discharge vessel 430 is realized. The spherical floating blocks 438 can avoid the problem that the sealing between the spherical floating blocks and the liquid outlet of the pressure stabilizing bin 437 is not tight due to movement and rolling in the pressure stabilizing bin 437, and the reliability of automatic opening and closing of the pressure stabilizing bin 437 is improved.

In a further aspect of this embodiment, the lower portion of the pressure stabilizing bin 437 has a funnel-shaped structure 439 with a gradually decreasing diameter from top to bottom, and the liquid outlet of the pressure stabilizing bin 437 is located at the bottom of the funnel-shaped structure 439.

Further, the upper part of the pressure stabilizing bin 437 is in a barrel-shaped structure, and the upper edge of the funnel-shaped structure 439 is connected with the lower edge of the barrel-shaped structure of the pressure stabilizing bin 437, namely, the maximum diameter of the funnel-shaped structure 439 is the same as the diameter of the barrel-shaped structure of the pressure stabilizing bin 437, and the minimum diameter of the funnel-shaped structure 439 is the same as the diameter of a liquid outlet of the pressure stabilizing bin 437.

With the adoption of the structure, when the blood sample in the pressure stabilizing bin 437 is completely discharged, the floating block 438 is sunk to the liquid outlet position of the pressure stabilizing bin 437 along the inner wall of the periphery side of the funnel-shaped structure 439 when the floating block 438 is lowered under the action of gravity, so that the problem that the liquid outlet of the pressure stabilizing bin 437 cannot be completely blocked to cause positive pressure leakage due to deviation of the falling point when the floating block 438 is lowered is avoided.

In this embodiment, when the pressure generating device 420 is simultaneously connected to the plurality of sealed blood collection tubes 410, the plurality of sealed blood collection tubes 410 may be simultaneously ventilated and pressurized, so that the blood sample is pressed out from each sealed blood collection tube 410 and added to the corresponding microfluidic device 200. Since the amounts of blood samples to be tested may vary from sealed blood collection tube 410, and the viscosity of the different blood samples often varies, there is a sequential difference in the time at which the blood samples are drained from sealed blood collection tube 410. Through set up the steady voltage storehouse 437 that has the floating block 438 in the interior on arranging the blood vessel 430, can be in sealed blood sampling tube 410 after the blood specimen is discharged completely automatic cutout corresponding row blood vessel 430, prevent this row blood vessel 430 from revealing the malleation that pressure generating device 420 provided, avoid influencing other sealed blood sampling tube 410 and carry out the blood specimen interpolation process.

The foregoing description is only illustrative of the preferred embodiment of the present utility model, and is not to be construed as limiting the utility model, but is to be construed as limiting the utility model to any and all simple modifications, equivalent variations and adaptations of the embodiments described above, which are within the scope of the utility model, may be made by those skilled in the art without departing from the scope of the utility model.

Claims

1. A nucleated cell enrichment staining integrated device, comprising:

the microfluidic device comprises:

2. The integrated nucleated cell enrichment dyeing device according to claim 1, wherein the fastening cover comprises a pressing part horizontally arranged for pressing the fastening part, and a through hole for the pipe body to pass through is formed in the pressing part;

3. The integrated nucleated cell enrichment and staining device according to claim 2, wherein the lower surface of the pressing part is provided with a limit groove in limit fit with the buckling part.

4. A nucleated cell enrichment staining integrated device according to any of the claims 1-3, wherein the enrichment staining unit further comprises a buffer device comprising:

5. The integrated nucleated cell enrichment staining apparatus of claim 4, wherein the buffer assembly comprises a buffer bottle in communication with the microfluidic device and peristaltic pump, respectively, wherein a predetermined amount of gas is sealed in the buffer bottle;

6. A nucleated cell enrichment staining integrated device according to any of claims 1 to 3, wherein the reagent pick-and-place unit comprises:

reagent holding means for holding the sucked reagent;

and the driving device is used for driving the vacuum cylinder and the piston assembly to generate relative motion, and pumping air from the reagent containing device and reducing pressure or ventilating and pressurizing the reagent containing device.

7. The integrated nucleated cell enrichment staining apparatus of claim 6, wherein 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 with the piston, and the other end of the push rod extends out of the vacuum cylinder;

8. The integrated nucleated cell enrichment dyeing apparatus according to claim 7, wherein said vacuum apparatus further comprises a vacuum fixing frame, said vacuum cylinder being relatively fixedly mounted on said vacuum fixing frame;

9. The integrated nucleated cell enrichment staining apparatus according to claim 6, wherein the reagent storage unit further comprises a moving mechanism for driving the reagent storage device to move to a position above the reagent storage device where the reagent to be added is stored, and driving the reagent storage device to move to a position above the microfluidic device to add the reagent.

10. A nucleated cell enrichment dyeing integrated device according to any one of claims 1 to 3, wherein the blood adding unit comprises a pressure generating means and a sealed blood collection tube, the pressure generating means being in communication with the sealed blood collection tube, the sealed blood collection tube being provided with a blood drainage tube in communication with the outside;