CN112126588A

CN112126588A - Separation capture system for specific cells in liquid sample

Info

Publication number: CN112126588A
Application number: CN201910547940.5A
Authority: CN
Inventors: 吴波
Original assignee: Tecrup Pte Ltd
Current assignee: Tecrup Pte Ltd
Priority date: 2019-06-24
Filing date: 2019-06-24
Publication date: 2020-12-25

Abstract

The invention relates to a system for separating and capturing specific cells in a liquid sample, which comprises a liquid sample processing tube and an instrument main body matched with the liquid sample processing tube. The instrument main body is internally provided with a liquid treatment control mechanism for treating the liquid sample in the sample treatment tube, a magnet control mechanism and various related sensors. The sample processing tube contains a microporous filter membrane, and can separate and capture cells with specific sizes and morphologies. Immune antibody magnetic beads aiming at specific cell surface antigens are added into the sample processing tube, and the movement of the magnetic beads and the cells can be controlled by utilizing a magnet control mechanism, so that the aim of further separating and purifying the specific cells is fulfilled. The system provides a cell separation system which is simple and convenient to operate, combines physical characteristic separation and immunomagnetic bead separation of specific cells, and can be used for relevant clinical detection and scientific research.

Description

Separation capture system for specific cells in liquid sample

Technical Field

The invention relates to the technical field of cell separation, in particular to a system for separating and capturing specific cells in a liquid sample.

Background

The liquid sample containing the cells is a common clinical detection sample, and comprises blood, urine, ascites and other liquid samples, and the efficient separation and capture of specific cells in the liquid sample can assist clinical diagnosis and treatment. In addition, cell culture fluid is also a common fluid sample, and the separation and capture of cells or specific cells can assist related scientific research or improve the production efficiency of biological products.

Various cell separation methods can be broadly classified into two types, including an immunoaffinity method by recognizing a cell surface-specific antigen and a screening method based on the physical properties of the recognized cell. Immune affinity method is widely used in clinical diagnosis, and the method comprises two strategies of positive enrichment and negative enrichment. The positive enrichment separates and captures cells aiming at the specific antigen related to the target cells, and the obtained cells have higher purity. However, due to the expression and degree of expression of cell-specific antigens, positive enrichment strategies may lead to lower isolation efficiency and higher probability of missed detection in clinical diagnosis, i.e. false negative results. The negative enrichment strategy separates and captures target cells by removing non-target cells and intercepting residual cells, and the method does not rely on antibody selection for separating the target cells, and has high capture efficiency but relatively low purity. The second method separates cells by using physical properties such as size, shape, density, and charging characteristics of target cells. Such as the current application of filter membrane separation methods in biological products and clinical tests. The method is simple and feasible, but the separation purity of the target cells is relatively low. The gradient centrifugation method is also a common cell separation technology and has higher cell separation purity. But the operation is complex and time-consuming, and the higher clinical detection flux requirement cannot be met.

Here, the separation of circulating tumor cells in the medical field is taken as an example. Circulating tumor cells are tumor cells released into the blood circulation system from solid tumors. Part of the circulating tumor cells are directly or indirectly involved in the process of tumor metastasis and recurrence. In clinical practice, needle biopsies cannot be performed for patients with weak physical conditions or particular lesion locations. The method has small invasiveness in separating, capturing and detecting circulating tumor cells in blood, is suitable for early screening, risk classification and recurrence prognosis of tumors, can carry out multiple detections, and dynamically monitors curative effect and evaluates medication effect in real time. However, the clinical blood samples have very low circulating tumor cell content, and even in the blood samples of metastatic tumor patients with relatively high circulating tumor cell content, the appearance rate is less than one million parts of normal white blood cells. Therefore, the separation and capture of the circulating tumor cells are extremely difficult. Currently, the clinical separation of circulating tumor cells mainly depends on manual operation. Due to the complex and tedious operation process, human misoperation and difference exist in the process. And the manual operation time is long, the efficiency is low, the single-day sample processing quantity is small, and the requirements of various medical institutions cannot be met. In addition, cell separation and capture technologies using microfluidic technology have begun to appear, but due to technical limitations and other factors, such methods all use only one of immunomagnetic bead capture or physical characteristic separation, and the method limitations such as low capture efficiency or low purity, which are inherent to these methods, cannot be avoided. It follows that there is a wide need for efficient, high purity cell separation techniques that are simple to operate.

Disclosure of Invention

The invention mainly solves the problems of complicated manual operation, low efficiency and low purity of separating specific cells in a liquid sample. Provides a convenient and automatic system for separating and capturing specific cells with high efficiency.

The technical problem of the invention is mainly solved by the following technical scheme: a specific cell separation and capture system comprises a liquid sample processing tube and an instrument main body matched with the liquid sample processing tube, wherein a functional unit for processing a liquid sample in the sample processing tube is arranged in the instrument main body, and the functional unit comprises a control circuit board, a liquid control mechanism, a magnet control mechanism and various related sensors. The sample processing tube is mounted in a sample processing tube slot linked to the functional unit. The sample processing tube is a disposable plastic device and is used for containing a liquid sample containing cells, and the sample is filtered, cleaned and uniformly mixed by the instrument through a functional unit and a liquid control mechanism. The sample processing tube comprises a tube body, a tube buckle, a tube cover, a filter membrane supporting head and a tube plug. The upper part of the tube body is a sample inlet. The pipe cover is provided with a hydrophobic and air-permeable air inlet. Microporous filter membranes are arranged at the lower part of the pipe body and in the middle of the pipe buckle. The bottom of the pipe buckle is provided with a sample outlet which is sealed by a soft rubber pipe plug. The sample outlet is connected with a liquid control mechanism of the instrument. The liquid control mechanism is used for extracting and injecting liquid into the sample processing tube, cleaning and filtering cells in the tube and uniformly mixing the cells with the reagent; the magnet control mechanism is used for operating the magnetic bead reagent added into the sample processing tube. The system provides a cell separation device which is simple and convenient to operate, and solves the problems of low specific cell separation and capture rate, low purity and complex manual operation in the prior art.

As a preferable mode of the above, the sample processing tube includes a tube body, a tube cap, a tube buckle, a filter membrane support head, and a tube plug. The tube cap is placed at a sample inlet on the upper portion of the tube body, a hollow part is arranged on the tube cap, and a hydrophobic breathable film is arranged in the hollow part. The hydrophobic breathable film can be isolated from liquid through gas, the hydrophobic breathable film can be a Teflon hydrophobic breathable film, and the Teflon hydrophobic breathable film can be fixed at the hollow part by ultrasonic bonding to form an air vent. The sample outlet is arranged at the opening of the filter membrane supporting head. The opening is sealed by a soft rubber plug to prevent liquid leakage. The pipe buckle is nested in the filter membrane supporting head to be fixed at the bottom of the pipe body. The microporous filter membrane is arranged between the pipe body and the filter membrane supporting head, and the barb and the buckle structure on the pipe body and the pipe buckle apply pressure on the rubber sealing rings on two sides of the microporous filter membrane, so that liquid in the pipe is ensured to be free of leakage. The outlet sample is connected to the fluid control mechanism when the sample processing tube is placed on the sample processing tube holder of the instrument.

As a preferable mode of the above, the microfiltration membrane is, but not limited to, a micromachined microfiltration membrane or a plastic microfiltration membrane. The micropores may be circular or elongated through holes. The plurality of through holes are arranged in an array to form a filtering area with the same size as the flow passage. The size and shape of the through-holes are such that cells of similar size and shape, including the particular cell, are trapped on the filter membrane during the filtration operation and can be easily backflushed.

As a preferable mode of the above, the instrument main body includes an instrument case, a structural frame, a power supply module, and a functional unit. The power module and the functional unit are arranged in the structural framework, the structural framework is placed in the instrument shell, and the touch display screen and the sample processing tube slot are arranged on the instrument shell. The touch display screen is used for operating the instrument and displaying the working state of the instrument. The sample processing tube slot is used for placing a sample processing tube. In addition, a liquid path inlet, a liquid path outlet, a liquid level sensor connector, an instrument switch and a power supply access port are also arranged on the shell. The liquid path inlet and the liquid path outlet are respectively connected with the treatment liquid bottle and the waste liquid bottle, two liquid level sensor joints are arranged, the liquid level sensors for detecting the liquid levels of the treatment liquid bottle and the waste liquid bottle are connected, and the liquid levels in the treatment liquid bottle and the waste liquid bottle are respectively detected. When the treating liquid in the treating liquid bottle is less than a certain volume, the corresponding liquid level sensor prompts early warning. When the volume of the waste liquid in the waste liquid bottle is more than a certain volume, the corresponding liquid level sensor prompts early warning. The instrument switch is used to turn the instrument on and off. The functional unit comprises a control circuit board, a liquid control mechanism and a magnet control mechanism. The operation flow and the steps of the sample contained in the sample processing tube are defined by the microcontroller code in the control circuit board according to the sample processing method and the flow, and after one step is finished, the microcontroller on the control circuit board executes the next step until the operation flow is finished.

Preferably, the sample processing tube slot is disposed on the instrument housing. The lower end of the slot is provided with a limit switch for detecting whether the sample processing tube is inserted into the slot. The top of the slot is provided with a positioning device, so that the sample processing tube can be vertically inserted into the slot. A socket connected to the liquid control mechanism is arranged at the bottom of the slot, a liquid guide needle is arranged on the socket, the liquid guide needle is hollow, and an opening is formed in the side face of the liquid guide needle. When the sample processing tube is inserted to the bottom of the slot, the liquid guide needle penetrates into the soft rubber plug of the sample processing tube, so that the sample processing tube is linked with the liquid control mechanism. In the scheme, the socket and the liquid guide needle are made of metal processing without limitation.

As a preferable mode of the above, the magnet control mechanism is disposed in the functional unit. The sample treatment device is characterized in that a linear stepping motor which moves in the direction perpendicular to the side face of the slot is arranged at the position close to the slot of the sample treatment tube, a permanent magnet support is arranged on a lead screw of the linear stepping motor, a permanent magnet is fixed on the permanent magnet support, a linear guide rail or an optical axis which is parallel to the execution stepping motor is further arranged on the structural framework, a sliding block is arranged on the linear guide rail or the optical axis, and the permanent magnet support is fixed with the sliding block. Two limit switches are respectively arranged at the positions of the linear stepping motor, which are close to one section of the motor and the slots, and respectively define the positions of the magnet, which are far away from and close to the sample processing tube. The linear stepping motor can drive the permanent magnet to be close to and far away from the sample processing tube in the slot so as to control the reaction and the movement of the magnetic beads in the sample processing tube. When the immunomagnetic bead reagent is added into the sample processing tube and then the magnetic beads need to be uniformly mixed in the sample processing tube, the permanent magnet is placed at a position far away from the slot of the sample processing tube; when the magnetic beads need to be collected on the inner wall of the sample processing tube, the permanent magnet is placed at a position close to the side wall of the slot. The linear slide rail and the sliding block can ensure the smooth movement of the permanent magnet support and also play a role in guiding the movement of the permanent magnet support.

In a preferred embodiment of the present invention, the liquid control mechanism includes a peristaltic pump, a liquid two-way valve, a flow path substrate, a flow path tube detection sensor, and a liquid path joint. The peristaltic pump and the flow path substrate are fixed to a holder of the functional unit. The four liquid two-way valves are arranged on the flow path substrate, the four liquid two-way valves are sequentially connected through a channel, a first liquid path interface, a second liquid path interface, a third liquid path interface, a fourth liquid path interface and a fifth liquid path interface are arranged on the flow path substrate, the first liquid path interface is connected to the treatment liquid bottle, the second liquid path interface is connected to the sample treatment pipe, the third liquid path interface is connected to the waste liquid bottle, and the fourth liquid path interface and the fifth liquid path interface are connected to two liquid path joints of the peristaltic pump through liquid path pipes. The first liquid path interface, the second liquid path interface, the third liquid path interface, the fourth liquid path interface, the fifth liquid path interface and the four liquid two-way valves are communicated through a plurality of channels to form three flow paths from the processing liquid bottle to the waste liquid bottle, from the processing liquid bottle to the sample processing pipe and from the sample processing pipe to the waste liquid bottle, and the flow path pipe detection sensor is integrated on the control circuit board and used for detecting the liquid filling states of the three flow paths. The liquid control module is used for carrying out operations of adding treatment liquid, discharging waste liquid, cleaning cells, uniformly mixing reagents and the like on the sample treatment tube. The liquid two-way valve comprises a first liquid two-way valve, a second liquid two-way valve, a third liquid two-way valve and a fourth liquid two-way valve, the first liquid two-way valve first port is connected with a first liquid path interface through a channel, the first liquid two-way valve second port is connected with a second liquid two-way valve second port through a pipeline, the fourth liquid path interface is connected on the pipeline between the first liquid two-way valve second port and the second liquid two-way valve second port, the second liquid two-way valve first port is connected with a third liquid two-way valve first port through a pipeline, the second liquid path interface is connected on the pipeline between the second liquid two-way valve first port and the third liquid two-way valve first port, the third liquid two-way valve second port is connected with the fourth liquid two-way valve second port through a pipeline, the fifth liquid path interface is connected to a pipeline between the second port of the third liquid two-way valve and the second port of the fourth liquid two-way valve, and the first port of the fourth liquid two-way valve is connected with the third liquid path interface through the pipeline. When the first liquid two-way valve and the fourth liquid two-way valve are opened, a flow path from the treatment liquid bottle to the waste liquid bottle is opened, and the flow path can be filled with liquid or cleaned. When the first liquid two-way valve and the third liquid two-way valve are opened, the flow path from the processing liquid bottle to the sample processing tube is opened, and the processing liquid can be injected into the sample processing tube. When the second liquid two-way valve and the fourth liquid two-way valve are opened, a flow path of the waste liquid bottle from the sample processing pipeline is opened, and the waste liquid bottle can be used for discharging waste liquid from the sample processing pipe. The flow rate of the liquid is controlled by the rotating speed of a stepping motor of the peristaltic pump. The three flow path pipe detection sensors respectively detect the liquid filling state from the liquid path substrate to the sample processing pipe, the processing liquid bottle and the waste liquid bottle hose, and perform corresponding processing operation on the sample in the sample processing pipe by matching with the liquid two-way valve and the peristaltic pump.

The invention realizes the operation flow of separating and capturing specific cells by utilizing a sample processing tube and a matched instrument, and the system can separate the cells with specific sizes by utilizing the filter membrane in the sample processing tube only by inserting the processing tube added with the sample into the slot of the instrument. The system can further carry out the immunomagnetic bead separation on cells with specific antigens by combining the immunomagnetic bead reagent and a magnet control mechanism in the instrument. The system of the invention automatically standardizes the original complicated manual operation without professional training personnel, thereby avoiding the human misoperation and the difference of manual operation. The system can carry out filter membrane filtration and immunomagnetic bead purification on the liquid sample containing the cells, and combines the respective advantages of the two methods. The separated specific cells can be easily taken out from the sample processing tube, so that various subsequent analyses, detection and culture can be conveniently carried out, and the application openness is high.

Drawings

FIG. 1 is a schematic view of an instrument and sample processing tube of the present invention;

FIG. 2 is a schematic diagram of one configuration of a sample processing tube of the present invention;

FIG. 3 is a schematic representation of two configurations of the filter membrane of the present invention;

FIG. 4 is a schematic diagram of a structure of the structural frame and internal apparatus of the present invention;

FIG. 5 is a schematic diagram of a functional unit according to the present invention;

FIG. 6 is a schematic diagram of one embodiment of a liquid circuit substrate and liquid two-way valve combination of the present invention;

FIG. 7 is a schematic view of the structure of the side of the apparatus according to the invention;

FIG. 8 is a schematic diagram of the operation of the present invention for isolating and capturing circulating tumor cells.

Detailed Description

The technical solution of the present invention is further specifically described below by way of examples with reference to the accompanying drawings.

The present embodiment is a system for separating and capturing specific cells in a liquid sample, and includes an instrument body 100 and a sample processing tube 107. As shown in fig. 1, the main body of the apparatus includes an apparatus housing 101 and a structural frame, and a functional unit and a power module disposed in the structural frame for processing a sample in the sample processing tube. The instrument housing 101 is provided with a touch display screen 102 for displaying the working state of the instrument, the operating parameters and the touch keys required for operating the instrument. When the system is used to process a liquid sample loaded into a sample processing tube 107 and to separate specific cells therein, the cover 103 of the instrument is opened and the positioning device 106 is inserted into a slot 105 provided in the housing of the instrument up to a receptacle 104 at the bottom of the slot. The positioning device 106 and the receptacle 104 are made of, but not limited to, metal. The positioning device 106 contains symmetrically placed spring steel balls for enabling the sample processing tube to be inserted vertically into the slot 105 when the sample processing tube 107 is inserted, and for enabling the liquid guide needle on the socket 104 to penetrate into the rubber stopper at the lower end of the sample processing tube, communicating the sample processing tube with the liquid control mechanism in the instrument. The hollow side opening of the catheter on the receptacle 104 is not blocked when the stopper of the sample processing tube is pierced.

FIG. 2 shows a block diagram and partial cross-sectional view of a sample processing tube 200 according to a preferred embodiment of the present invention. The main structure of the sample processing tube is a tube cover 201, a tube body 202, a tube buckle 204, a filter membrane support head 205, a plug 406, a sealing ring 207 and a microporous filter membrane 208. Wherein the tube cap 201, the tube body 202, the tube buckle 204 and the filter membrane supporting head 205 are made of rigid plastics. The pipe cover is provided with a hollow-out 203, and the hollow-out is covered by a breathable and waterproof Teflon film, so that the operation of liquid in the device is facilitated. The tube 202 may be, but is not limited to, a transparent plastic, and the tube is marked with a liquid volume scale 209 for easy use and observation. The inside of the pipe buckle 204 is provided with a clamping groove structure, the bottom of the pipe body 202 is provided with a corresponding barb structure, when the pipe buckle is arranged at the bottom of the pipe body, the clamping groove and the barb structure are locked, the pipe buckle 204 presses the filter membrane supporting head 205 at the bottom of the pipe body 202, and the two sealing rings 207 clamp the microporous filter membrane 208 in the middle and seal the microporous filter membrane. The plug 206 is made of silica gel or rubber material and is used for sealing the liquid path channel of the filter membrane support head. In specific use, the tube cap 201 is first opened, and a liquid sample containing cells, such as blood, tissue fluid or cell culture fluid, is added into the tube body. The sample processing tube 200 is then inserted into the socket of the instrument 100 designed for the apparatus and the introducer needle of the receptacle 104 of the device pierces the stopper 206 to allow the connection of the interior of the sample processing tube to the fluid handling mechanism of the device. Because the plug is made of soft rubber material, the plug can play a sealing effect to prevent liquid leakage after the liquid guide needle pierces the plug. After filtration of the liquid sample containing cells, the tube clamp 204 is opened and the filter 208 with the specific cells is removed for detection, analysis or culture.

FIG. 3 is a schematic diagram of the structure of a circular microfiltration membrane 300 and an elongated microfiltration membrane 303 according to a preferred embodiment of the invention. The microporous filter membrane is made of metal and plastic, but not limited to, and has a thickness of 5um to 10 um. The micro-holes may be either circular 302 or elongated 304. The micropores 301 are arranged equidistantly and regularly on the filter membrane structure surface 301. As shown in fig. 3a, the micro-cells may be arranged, but are not limited to, according to three vertices of an equilateral triangle. With a center spacing of, but not limited to, 20 um. The number of the micropores on the filter membrane with the fixed area can be adjusted by adjusting the hole spacing under the condition that the size of the circular micropores is not changed. As shown in FIG. 3b, the elongated micropores 304 are arranged in a regular pattern on the filter membrane structure surface 305. The width of the elongated micro-holes is but not limited to 5um or 10um, and the length is but not limited to 20um or 40 um. In order to enhance the pressure resistance and the mechanical structure stability of the structural surface of the filter membrane, the micropores can be arranged in a hexagonal area, and a plurality of hexagonal areas are spliced at equal intervals to form the filter membrane. The interval of the hexagonal areas is but not limited to 50-100 um. The filtration area covered by the micropores on the filter membrane should be not less than the cross-sectional area of the liquid path channel of the filter membrane support head 205.

As shown in fig. 4, the structural frame 400 of the instrument has mounted thereon a power module 402 and a functional unit 403. The structural frame 400 is made by, but not limited to, laser cutting a sheet of steel and then bending the sheet metal. The power module 402, power switch and power inlet are mounted on the power bracket 401. The bracket of the functional unit 403 is provided with a control circuit board, a fluid control mechanism, a magnet control mechanism and related sensors for performing corresponding operations on the liquid sample, cells and reagents in the sample processing tube. The structural frame 400 is provided with a hollow 405 to reduce the weight of the instrument without affecting the structural stability. Mounting holes 404 in the mechanism frame are used to mount the mechanism frame tightly within the instrument housing.

As shown in fig. 5, the function unit 500 is composed of a function unit holder 501, a control circuit board 502, a fluid control mechanism, and a magnet control mechanism. The fluid control mechanism is composed of four liquid two-way valves 503, a flow path substrate 504, a peristaltic pump 505, and three flow path pipe detection sensors 513. The flow tube detection sensor 513 may be integrated on the control circuit board 502. The peristaltic pump 505 is fixed to a peristaltic pump support 506. The four liquid two-way valves 503, in cooperation with the flow channels in the flow channel substrate 504, define three flow channels, including a flow channel one from the processing liquid bottle to the waste liquid bottle, a flow channel two from the processing liquid bottle to the sample processing tube, and a flow channel three from the sample processing tube to the waste liquid bottle. Five flow path link ports on the flow path substrate are respectively linked to two flow path ports of the processing liquid bottle, the sample processing tube, the waste liquid bottle and the peristaltic pump by using flow path hoses. The flow rate in the flow tube is controlled by the rotational speed of the peristaltic pump 506 and the selection of the flow path is controlled by the open and closed states of the four liquid two-way valve combination. The three flow path detection sensors 513 are used to detect the liquid filled state in the flow path tube hoses linking the flow path substrate 504 with the processing liquid bottles, the sample processing tubes, and the waste liquid bottles, so as to monitor the operation state of the fluid control mechanism and the volume of the control liquid injected and discharged. The magnet control mechanism is composed of a linear stepping motor 507, a permanent magnet bracket 508, a permanent magnet carrier 509, a permanent magnet 510 and a linear guide rail 512. The permanent magnet bracket 508 is disposed on the lead screw 511 of the linear stepping motor and linked with the slider on the linear guide 512. A limit switch is installed on one side of a motor of the linear stepping motor 507, a definition magnet is far away from the position of the sample processing tube, the limit switch is installed on the inner side of the instrument shell, close to the position of the sample processing tube, and the definition magnet is close to the position of the sample processing tube. In a specific step of separating specific cells in a liquid sample, cell antibody modified magnetic beads and related reagents are injected into a sample processing tube, and the binding bodies of the magnetic beads and the cells can be collected on the tube wall through a magnet control mechanism. For example, antibody combinations include, but are not limited to, CD45, CD16, CD19, and CD163, and the size of the magnetic beads can be, but is not limited to, 0.2um or 1 um. The magnet control mechanism positions the permanent magnet away from the sample processing tube prior to adding the reagent. The liquid control mechanism opens a flow path from the processing liquid bottle to the sample processing tube, and a certain amount of processing liquid is quickly injected into the sample processing tube by the peristaltic pump to uniformly mix the reagent and the magnetic beads. After a period of incubation, the antibody-modified magnetic beads are allowed to bind to normal hematopoietic cells. And then the magnet control mechanism places the permanent magnet at the position of the sample processing tube, so that the combination body of the magnetic beads and the cells is collected on the tube wall of the sample processing tube. After draining all the liquid from the sample processing tube, circulating tumor cells were collected on a microporous filter membrane. Several washing steps may be added to the procedure to wash the cells. The cleaning step comprises opening the processing liquid bottle to the flow path of the sample processing tube, and injecting the processing liquid into the sample processing tube by the peristaltic pump. And then opening the flow path from the sample processing tube to the waste liquid bottle, and discharging the waste liquid in the sample processing tube by the peristaltic pump. This washing step may be repeated a number of times to wash the cells.

Fig. 6a shows a flow path control mechanism 600 comprising a flow path substrate and a liquid two-way valve in the liquid control mechanism. The four liquid two

valves

602, 603, 604, 605 and the

liquid path ports

606, 607, 608, 609, 610 are mounted on the flow path substrate 601. The fluid interface 606 is connected to a processing fluid bottle, the fluid interface 607 is connected to a sample processing tube, the fluid interface 608 is connected to a waste fluid bottle, and the fluid interface 609 and the fluid interface 610 are connected to two fluid connectors of a peristaltic pump through fluid tubes. The five liquid path interfaces are communicated with the four liquid two-way valves through a plurality of channels to form three flow paths from a treatment liquid bottle to a waste liquid bottle, from the treatment liquid bottle to a sample treatment pipe and from the sample treatment pipe to the waste liquid bottle. The flow path pipe detection sensor is integrated on the control circuit board and used for detecting the liquid filling state of the three flow paths. The liquid control mechanism carries out operations of adding treatment liquid, discharging waste liquid, cleaning cells, uniformly mixing reagents and the like on the sample treatment tube. As shown in fig. 6b, the first port of the fluidic bi-directional valve 602 is connected to the fluidic interface 606 via channel 612, the second port of the fluidic bi-directional valve 602 is connected to the second port of the fluidic bi-directional valve 603 via line 614, the fluidic interface 610 is connected to the line between the second port of the fluidic bi-directional valve 602 and the second port of the fluidic bi-directional valve 603, the first port of the fluidic bi-directional valve 603 is connected to the first port of the fluidic bi-directional valve 604 via line 615, the fluidic interface 607 is connected to the line 616 between the first port of the fluidic bi-directional valve 603 and the first port of the fluidic bi-directional valve 604, the second port of the fluidic bi-directional valve 604 is connected to the second port of the fluidic bi-directional valve 605 via line 618, the fluidic interface 609 is connected to the line 618 between the second port of the fluidic bi-directional valve 604 and the second port of the fluidic bi-directional valve 605, and the first port of the fluidic bi. When the two-way liquid valve 602 and the two-way liquid valve 605 are opened, the flow path from the treatment liquid bottle to the waste liquid bottle is opened, and the flow path can be filled with liquid or cleaned. When the liquid two-way valve 602 and the liquid two-way valve 604 are opened, the flow path from the processing liquid bottle to the sample processing tube is opened, and the processing liquid can be injected into the sample processing tube. When the liquid two-way valve 603 and the liquid two-way valve 605 are opened, the flow path of the waste liquid bottle from the sample processing tube is opened, and the waste liquid can be discharged from the sample processing tube.

Shown in fig. 7 are the front 700, side 701 and back 702 of the instrument. The front side 700 of the instrument shows the insertion of a sample processing tube 703 into a slot 704. On the side 701 of the apparatus,

level sensor ports

705 and 706 are provided, which are respectively disposed at the bottom of the treatment liquid bottle and the top of the waste liquid bottle. When the liquid level sensor on the treatment liquid bottle detects that the treatment liquid is about to be used up, a signal is sent to the microcontroller on the control circuit board; when the liquid level sensor on the waste liquid bottle detects that the waste liquid is nearly full, the microcontroller on the control circuit board is signaled. And the microcontroller gives an alarm prompt and corresponding measures on the touch display screen according to the program definition, such as the standby of a customized process. The liquid path interfaces 707 and 708 are respectively provided with a liquid path hose connected with a treatment liquid bottle and a waste liquid bottle. In order to prevent pollution and backflow, a filter or a one-way valve can be connected between the liquid path interface and the treatment liquid bottle and the waste liquid bottle. On the back side 702 of the instrument is a back cover plate 709 with a number of mounting holes 710 that allow the back cover plate to be mounted to the structural frame and power bracket of the instrument. The power switch 711 and the power inlet 712 are mounted on the power bracket.

FIG. 8 shows an example of the operation of the separation and capture system for circulating tumor cells, which combines the filter membrane filtration method and the antibody magnetic bead negative enrichment method. The method comprises the following steps:

step one, initializing the instrument body after the instrument body is opened. The microcontroller of the control circuit board first queries and records the status information of the various sensors and actuators. Initializing the touch display screen menu. And further has initial state definition, and drives each actuator to the initial state of the workflow.

And step two, performing a self-checking program of the starting state after the sample processing tube is inserted into the instrument slot. First, a limit sensor at the bottom of the slot detects whether the sample processing tube is inserted into the bottom of the slot. Secondly, the liquid level of the treatment liquid in the treatment liquid bottle is detected to check whether the treatment liquid is enough for sample treatment. And thirdly, detecting the liquid level of the waste liquid in the waste liquid bottle, and checking whether the residual volume of the waste liquid bottle is enough to place the waste liquid generated by the sample treatment for one time. If the three-day detection program does not meet the requirement in one day, the sample processing program is stopped, and a warning prompt is displayed on the touch display screen.

And step three, evacuating the liquid in the sample processing tube. And opening the channel from the sample processing tube to the waste liquid bottle, and pumping the liquid in the sample processing tube by the peristaltic pump until the liquid leakage tube sensor detects that no liquid exists in the flow path hose communicated to the waste liquid bottle.

And step four, cleaning the cells. And opening a flow path channel from the sample processing tube to the processing liquid bottle, and injecting a certain volume of processing liquid into the sample processing tube by using the peristaltic pump. And then closing the flow path channel from the sample processing tube to the processing liquid bottle, opening the flow path channel from the sample processing tube to the waste liquid bottle, and pumping the liquid in the sample processing tube by the peristaltic pump until the liquid leakage tube sensor detects that no liquid exists in the flow path hose communicated to the waste liquid bottle. The cell washing step may be repeated a plurality of times.

And step five, purifying the circulating tumor cells by using an antibody magnetic bead method. Normal hematopoietic cells are first modified using antibody-modified magnetic beads. And (3) puncturing the Teflon gas-permeable membrane by the hollow part of the tube cover, and injecting an antibody modified magnetic bead reagent into the sample processing tube. And opening a flow path channel from the sample processing tube to the processing liquid bottle, quickly injecting a certain volume of processing liquid into the sample processing tube by the peristaltic pump, uniformly mixing the reagent, the processing liquid and the cells, and incubating for a period of time. And then placing the permanent magnet and the vicinity of the slot of the sample processing tube for a period of time, and collecting the hematopoietic cells modified by the magnetic beads on the wall of the sample processing tube. And discharging the liquid in the pipe.

And step six, capturing and separating the circulating tumor cells on a filter membrane. And repeating the step four to clean the cells without being modified by other magnetic beads in the tube. Finally, the liquid in the sample processing tube is emptied, and the cells are captured on the filter membrane. And taking out the sample processing tube from the instrument slot, opening the tube buckle, taking out the filter membrane, placing the filter membrane in a six-hole plate or a culture dish, and waiting for the next detection, analysis and culture. And (5) ending the separation and capture process of the circulating tumor cells.

The invention realizes the separation and capture process of the circulating tumor cells with high efficiency and high purity, and the system automatically completes all the operation steps of filtering, cleaning, separating magnetic beads and the like of the cells, thereby avoiding manual operation errors and differences.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

Although the terms sample processing tube, tube body, tube clasp, functional unit, structural frame, instrument housing, flow path substrate, etc. are used more herein, the possibility of using other terms is not excluded. These terms are used merely to more conveniently describe and explain the nature of the present invention; they are to be construed as being without limitation to any additional limitations that may be imposed by the spirit of the present invention.

Claims

1. A system for separating and capturing specific cells in a fluid sample, comprising: comprises a sample processing tube and an instrument main body matched with the sample processing tube; the sample processing tube comprises a tube body, a tube cover, a tube buckle, a filter membrane supporting head and a tube plug; the instrument main body comprises an instrument shell, a structural frame, a functional unit and a power supply module; the functional unit comprises a control circuit board, a liquid control mechanism and a magnet control mechanism; in using the system to isolate and capture specific cells in a liquid sample, a sample processing tube is mounted in a slot in the instrument housing and connected to a liquid control mechanism in the functional unit.

2. The system of claim 1, wherein the body of the sample processing tube is narrowed at one end and has a barb structure; the inner side of the pipe buckle is provided with a buckle slot structure corresponding to the pipe buckle; the tube buckle can be nested with the filter membrane supporting head and is arranged at the narrow end of the sample processing tube; the filter membrane is clamped between the filter membrane supporting head and the narrow end of the tube body by two sealing rings; a flow passage in the filter membrane support head is sealed by a soft rubber pipe plug at one end; the tube cap is arranged at one end of the tube body and is provided with a hollow part which is covered by a hydrophobic breathable film.

3. The system of claim 1, wherein the instrument body comprises an instrument housing, a structural frame, a functional unit and a power module; the structural framework is arranged in the instrument shell, and the functional unit and the power supply module are arranged on the structural framework; the functional unit comprises a control circuit board, a liquid control mechanism and a magnet control mechanism; the instrument shell is provided with a touch display screen and a sample processing tube slot, the side surface of the instrument shell is provided with a flow path inlet, a flow path outlet and a liquid level sensor connector, the liquid path inlet and the liquid path outlet are respectively connected with a processing liquid bottle and a waste liquid bottle, and the liquid level sensor connector is connected with a liquid level sensor for detecting the liquid levels of the processing liquid bottle and the waste liquid bottle; an instrument switch and a power supply access are arranged on the rear side of the shell.

4. The system of claim 3, wherein the sample processing tube slot is disposed on the instrument housing, and the sample processing tube can be inserted into the slot vertically from top to bottom; the top of the slot is provided with a correction positioning device, and a plurality of spring steel balls are arranged in the correction positioning device, so that the sample processing tube can be ensured to be vertically inserted into the slot and finally fall on a socket at the bottom of the slot; the socket is provided with a hollow liquid guide needle with an opening on the side surface; the liquid guide needle punctures a tube plug of the sample processing tube, so that the sample processing tube is communicated with a liquid control mechanism of the instrument; the liquid guide needle of the socket is connected with the liquid control mechanism by a hose.

5. The system of claim 3, wherein the fluid control mechanism comprises a peristaltic pump, a fluid bi-directional valve, a flow substrate, a flow channel tube detection sensor, and a fluid channel connector; the liquid two-way valve is arranged on the flow path substrate, three flow paths from the treatment liquid bottle to the waste liquid bottle, from the treatment liquid bottle to the sample treatment tube and from the sample treatment tube to the waste liquid bottle are formed by the channel on the flow path substrate and the switch and the closing of the liquid two-way valve, and the flow path tube detection sensor detects the liquid filling state of the three flow paths; the flow rate of the liquid is controlled by the rotational speed of the peristaltic pump.

6. The system of claim 3, wherein the magnet control mechanism controls the distance of the permanent magnet from the sample processing tube, thereby controlling the movement of the magnetic beads and the cells in the sample processing tube; the magnet control mechanism comprises a linear stepping motor, a permanent magnet bracket, a permanent magnet, a guide rail and a limit sensor; the linear stepping motor is arranged close to the sample processing tube slot, and a lead screw of the linear stepping motor is vertical to the side direction of the sample processing tube slot; the permanent magnet bracket is arranged on a lead screw of the linear stepping motor and is connected with the guide rail to ensure linear motion; the permanent magnet is fixed on the permanent magnet bracket; two spacing sensors define two positions that magnet is far away from sample processing pipe and is close to sample processing pipe respectively, and linear stepping motor can drive the permanent magnet and be close to and keep away from the sample processing pipe in the slot and control the motion of the interior magnetic bead of sample processing pipe and cell.

7. The system according to any one of claims 3, 4, 5 and 6, wherein the cells in the liquid sample are filtered and washed by using a filter membrane in the sample processing tube, the cells are separated according to the size and morphology of the cells, the specific cells are modified by using magnetic beads and specific antibodies, the cells in the sample processing tube are further separated and purified, and finally the separated and captured specific cells are collected on the filter membrane.