CN107988042A - A kind of circulating tumor cell separator and its detection method - Google Patents

Abstract

A circulating tumor cell separation device and its detection method belong to the technical field of cancer circulating tumor cell detection. The device includes a touch display module, a single-chip microcomputer, a rotation speed control module, a rotating motor, a circulating tumor cell separation disc, a casing and a power supply module; the present invention A circulating cell separation device was designed to rapidly and label-free separate high-purity circulating tumor cells by using the hydrodynamic drive method, the effective combination of centrifugal force and microfluidics, and the mechanical filtration method based on size screening. Utilizing centrifugal force to rapidly transfer unprocessed whole blood samples from one chamber to another, the Circulating Tumor Cell Separation Disc allows for more efficient fluid control because it requires no external interconnectors and only requires a simple rotary motor to drive the fluid . These advantages of the device can reduce manual handling steps in the filtration, staining and detection process.

Description

A circulating tumor cell separation device and detection method thereof

technical field

The invention belongs to the technical field of cancer circulating tumor cell detection, and in particular relates to a circulating tumor cell separation device and a detection method thereof.

Background technique

The dissemination and metastasis of cancer cells is the main cause of cancer death. During metastasis, primary tumor cells, as well as pre-existing metastatic tumor cells, undergo a series of dissemination steps from the cell's original site to distant organs in the body. Initially, tumor cells disseminated from solid tumors undergo a process known as epithelial-mesenchymal transition, achieving their migratory and invasive properties. These tumor cells, known as circulating tumor cells, then enter the peripheral blood by means of metastasis and travel along the bloodstream until they reach suitable secondary sites. Circulating tumor cells in the peripheral blood of cancer patients serve as excellent diagnostic tools and markers for prevention. Therefore, quantification and characterization of these malignant cells can provide important clinical information for patients with metastatic cancer, potentially providing effective and personalized cancer therapy. Therefore, it is imperative that clinicians have reliable diagnostic tools to detect circulating tumor cells to improve clinical outcomes.

A major obstacle in the study of circulating tumor cells is that they are extremely rare in whole blood (every 10 ⁶ -10 ⁷ blood cells contain 1-10 circulating tumor cells), and the blood volume that can be processed is limited. Due to the rarity of circulating tumor cells, developing an efficient collection process has become a critical step in the detection and characterization of circulating tumor cells, which occur at a frequency of 1-10 cells per 1 mL of blood. In this regard, microfluidic technology has become a possible solution. After investigating the existing technologies, Chinese Patent No. 201510177966.7 discloses a circulating tumor cell detection device similar to the principle of the patent of the present invention. The device consists of a centrifuge, A fluorescent staining box, a magnetic bead separation mechanism, and a display mechanism, and a casing for supporting the centrifuge, the fluorescent staining box, the magnetic bead separation mechanism, and the display mechanism. The detection principle of the device is to separate the red blood cells in the blood sample through a centrifuge, and then use the magnetic bead method to separate the circulating tumor cells, label the circulating tumor cells with a fluorescent dyeing machine, and observe the labeled circulating tumor cells with a fluorescent microscope. Although the device has the function of detecting circulating tumor cells, it is unable to improve the probability of capturing circulating tumor cells, and the design of the device is too cumbersome and requires external equipment, which is not conducive to popularization and application.

Contents of the invention

In order to solve the problems existing in the prior art, the present invention provides a circulating tumor cell separation device and detection method. The present invention adopts a hydrodynamic driving method, utilizes the effective combination of centrifugal force and microfluidic mechanics, and selects mechanical filtration based on size screening. approach for rapid and label-free isolation of high-purity circulating tumor cells.

The technical solution adopted by the present invention to solve technical problems is as follows:

A circulating tumor cell separation device, which includes a touch display module, a single-chip microcomputer, a rotational speed control module, a rotating motor, a circulating tumor cell separation disc, a casing, and a power supply module; input a preset rotational speed on the touch display module, The touch screen module transmits the information to the single-chip microcomputer, and the single-chip microcomputer controls the speed of the rotation speed control module according to the instruction, and the rotation speed control module controls the rotation motor to drive the circulating tumor cell separation disc installed on the rotation motor to rotate according to the preset rotation speed , while feeding the speed information to the touch display module, which is displayed by the touch display module; the touch display module, single-chip microcomputer, speed control module, rotating motor, circulating tumor cell separation disc and power module are all installed in the shell .

A method for separating and detecting circulating tumor cells, comprising the steps of:

Step 1: Prepare reagents, prepare blood samples, turn the circulating tumor cell separation disc to the back, ensure that one of the holes is closed, add cell membrane penetrant from the other hole and seal the injection hole, and click Wash on the touch screen module model;

Step 2: Add the cell membrane penetrating agent from the first sample port on the front of the circulating tumor cell separation disc, put the circulating tumor cell separation disc into the device, and click the washing process on the touch screen module;

Step 3: Put the circulating tumor cell separation disk into the device from the blood sample of the lung cancer patient pretreated by the first sampling port, and click the filter mode on the touch screen module;

Step 4: After the filtration is completed, press the stop button of the touch display module; observe whether there are still samples in the sample chamber: if yes, repeat step 3; if not, add cell membrane penetrant from the second sample injection port to separate circulating tumor cells Put the disk into the device, and click the start button on the touch display module;

Step 5: Click the stop button on the touch screen module to execute the dyeing mode;

Step 6: Click the stop button on the touch screen module, add a small amount of cell membrane penetrant from the first sample port, put the circulating tumor cell separation disc into the device, put it flat and wait for 5 minutes, click start on the touch screen module button; click the stop button on the touch screen module, add phosphate buffered saline solution from the first sample port, put the circulating tumor cell separation disc into the device, and click the start button on the touch screen module; click the touch screen module Stop button on the top, add a small amount of blocking agent from the first sample port, put the circulating tumor cell separation disc into the device, wait for 20 minutes, add phosphate buffered saline solution from the first sample port, click on the touch screen module the start button;

Step 7: Click the stop button on the touch screen module, add a small amount of antibody from the first sample port, put the circulating tumor cell separation disc into the device, put it flat and wait for 20 minutes, and click the start button on the touch screen module; Click the stop button on the touch display module, add cell buffer from the first sample port, put the circulating tumor cell separation disc into the device, click the start button on the touch display module; click stop on the touch display module button, add a small amount of circulating tumor cell positive antibody mixture from the first sample port, put the circulating tumor cell separation disk into the device, wait for 20 minutes, click the stop button on the touch screen module, and add cells from the first sample port Buffer, click the start button on the touch display module;

Step 8: Click the stop button on the touch screen module, take out the circulating tumor cell separation disk, open the back sample hole, put the circulating tumor cell separation disk into the device, click the start button on the touch screen module; The cell size selection membrane of the cell separation disc is placed on a glass slide to observe the number and shape of circulating tumor cells in lung cancer;

The beneficial effects of the present invention are: the present invention uses centrifugal force to quickly transfer untreated whole blood samples from one chamber to another chamber, and the circulating tumor cell separation disc can control the fluid more effectively because it does not require external interconnectors, only A simple rotary motor is required to drive the fluid. These advantages of the device can reduce manual handling steps in the filtration, staining and detection process.

Description of drawings

Fig. 1 is a cross-sectional view of a device for separating circulating tumor cells of the present invention.

Fig. 2 is a perspective view of a device for separating circulating tumor cells of the present invention.

Fig. 3 is a principle diagram of a circulating tumor cell separation disc of the present invention.

Fig. 4 is a flowchart of a method for separating and detecting circulating tumor cells of the present invention.

In the figure: 1. Touch display module, 2. Speed control module, 3. Rotating motor, 4. Circulating tumor cell separation disc, 5. Shell, 6. Power module and 7. Flexible feet.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

As shown in Figures 1 and 2, a circulating tumor cell separation device includes: a touch display module 1, a rotational speed control module 2, a rotating motor 3, a circulating tumor cell separation disc 4, a casing 5, a power supply module 6, Single-chip microcomputer and flexible ground 7. The single-chip microcomputer is respectively connected with the touch display screen module 1 and the speed control module 2; the speed control module 2 is connected with the rotating motor 3; connected; the touch display screen module 1, the speed control module 2, the rotating motor 3, the power supply module 6, and the single-chip microcomputer are all installed in the shell 5, and the shell 5 is provided with corresponding mounting holes, which are fastened and installed by screws; The touch display module 1 includes a touch display and a control circuit board, and the control circuit board is fastened to the back of the touch display by screws; 1 and the instructions issued by the single-chip microcomputer, the speed control module 2 controls the speed and direction of rotation of the rotating motor 3; the upper end of the housing 5 is provided with a shaft hole for the rotating motor 3, and the rotating motor 3 is coaxially installed with the hole; the circulating tumor The cell separation disc 4 is installed on the 3 shafts of the rotating motor, the 3 shafts of the rotating motor are provided with an installation sleeve, and the installation sleeve is provided with an installation key, and a mounting hole is opened in the middle of the circulating tumor cell separation disc 4, and a mounting hole is arranged in the hole. There is an installation key, which is installed in cooperation with the installation sleeve; the upper end of the shell 5 is provided with a protective cover for the circulating tumor cell separation disc 4, which is used to protect the normal operation of the rotating motor 3 and the circulating tumor cell separation disc 4, wherein the protective cover It is connected with the housing 5 itself by a flexible hinge. ; The rear end of the device shell 5 is provided with a power socket hole and a device switch hole for installing a power socket and a device switch. Input the preset speed on the touch screen module 1, the touch screen module 1 transmits the information to the single-chip microcomputer, and the single-chip microcomputer controls the speed of the speed control module 2 according to the instruction, and the speed control module controls the rotating motor 3 Drive the circulating tumor cell separation disc 4 installed on the rotating motor 3 to rotate according to the preset rotation speed, while feeding the rotation speed information to the touch display module 1 for display by the touch display module 1;

The device also includes several enameled wires. One end of the enameled wire is welded to the touch display module 1 and the power supply module 6 , and the other end is welded to the speed control module 2 . The device also includes flexible feet 7 for flexibly supporting the device. A layer of protective film is glued on the touch display screen module 1, and the glue used is epoxy resin glue. The rotating electrical machine 3 is a pulse signal low-speed motor, and its rotational speed and direction are adjustable.

The circulating tumor cell separation disc 4 includes a base and an upper cover, and the base and the upper cover are sealed; the base includes: a loading chamber, a transition piece, a filter chamber and a waste chamber; the upper cover corresponds to the area of the loading chamber for sample injection hole; a baffle is set in the loading chamber, and a transition piece is arranged at the corresponding place of the baffle; the structure of the transition piece near the baffle is a slope from low to high; the upper end of the filter chamber is a cell size selection membrane, and the space at the lower end The passage is communicated with the waste chamber, and the height of the transition piece is greater than or equal to the position of the cell size selection membrane; a centrifugal hole is set at the center of the chip; when using the separation disc 4, the loading chamber is fed through the sample injection hole, such as As shown in Figure 3, the separation disc 4 is installed on the rotating motor 3. When the separation disc 4 rotates, the sample liquid moves radially outward quickly and is pumped to the filter chamber through the transition piece. Cells with larger cell size select the membrane pore size. Captured cells smaller than the pore size of the cell size selective membrane are transferred to the filter chamber and flow into the waste chamber.

In this example, the cell size selective membrane of the circulating tumor cell separation disc 4 is a disassembled membrane, and the bottom and top of the disc body are equipped with supporting backboards, so that it can be easily installed on a glass slide for subsequent image analysis and molecular characterization. There are two sample holes on the front, namely hole 1 and hole 2, and two sample holes on the back, namely hole A and hole B. There is a circulating tumor cell filter membrane inside, and a membrane pressure plate and pressure ring on the back, which are fastened around by four screws.

As shown in Figure 4, a method for separating and detecting circulating tumor cells, the method includes the following steps:

Step 1: Prepare various reagents, prepare blood samples, turn the circulating tumor cell separation plate 4 to the back, ensure that hole B is closed, add 1ml of cell membrane penetrant from inlet A, and keep the circulating tumor cell separation plate in order not to generate residual air bubbles 4 Stand upright, seal hole A with a specific sticker, put the circulating tumor cell separation disc 4 into the device and cover it with a protective cover, click the washing mode on the touch screen module 1, and make the rotating motor work at a speed of 1200rpm.

Step 2: Use a 1ml pipette gun to add 1ml of cell membrane permeation agent from the sample injection port 1 on the front of the circulating tumor cell separation disc 4, put the circulating tumor cell separation disc 4 into the device and cover the protective cover, and click on the touch screen module 1 In the wash mode on the top, the spin motor works at 1200rpm.

Step 3: Use a 1ml pipette gun to add 5ml of pretreated blood samples from lung cancer patients from the sample injection port 1, put the circulating tumor cell separation disc 4 into the device and cover with the protective cover, and click the filter on the touch screen module 1 mode to make the rotary motor work at 240rpm.

Step 4: After the filtering is completed, press the stop button of the touch display module 1. Observe whether there is still a sample in the sample chamber: if so, repeat step 3, that is, use a 1ml pipette gun to add 1ml of cell membrane penetrant from the sample injection port 1, put the circulating tumor cell separation disc 4 into the device and cover with the protective cover, Click the start button on the touch screen module 1 to filter again; if not, use a 1ml pipette gun to add 1ml cell membrane penetrant from the sample injection port 2, put the circulating tumor cell separation disc into the device, cover the protective cover, and click Touch the start key on the display module 1, wherein the sampling port 1 is a normal sampling port, and the sampling port 2 is a capillary hole.

Step 5: Click the stop button on the touch screen module 1 to execute the staining mode, add 1000 μl 3.5% formaldehyde from the sample injection port 1 with a 1ml pipette gun, put the circulating tumor cell separation disc 4 into the device and cover with the protective cover , wait for 20 minutes, click the start button on the touch screen module 1; click the stop button on the touch screen module 1, add 1ml phosphate buffered saline solution from the sample injection port 1 with a 1ml pipette gun, and separate the circulating tumor cells The disk 4 is put into the device and the protective cover is covered, and the start button on the touch display module 1 is clicked.

Step 6: Click the stop button on the touch screen module 1, add 300 μl of cell membrane penetrant from the sample injection port 1 with a 1ml pipette gun, put the circulating tumor cell separation disc 4 into the device, cover the protective cover, and put it flat to wait After 5 minutes, click the start button on the touch display module 1; click the stop button on the touch display module 1, add 1ml of phosphate buffered saline solution from the sample injection port 1 with a 1ml pipette gun, and separate the circulating tumor cells from the plate 4. Put it into the device and cover it with a protective cover, click the start button on the touch screen module 1; click the stop button on the touch screen module 1, add 300 μl blocking agent from the sample injection port 1 with a 1ml pipette gun to dissolve the circulating tumor Put the cell separation disc 4 into the device and cover it with a protective cover, wait for 20 minutes, add 1ml of phosphate buffered saline solution from the sample injection port 1 with a 1ml pipette gun, and click the start button on the touch screen module 1.

Step 7: Click the stop button on the touch screen module 1, add 300 μl of antibody from the sample injection port 1 with a 1ml pipette gun, put the circulating tumor cell separation disc 4 into the device, cover the protective cover, and wait for 20 minutes , click the start button on the touch display module 1; click the stop button on the touch display module 1, add 1ml of cell buffer from the sample injection port 1 with a 1ml pipette gun, and put the circulating tumor cell separation disc 4 into the device Put the protective cover on the middle cover, click the start button on the touch screen module 1; click the stop button on the touch screen module 1, add 300 μl circulating tumor cell positive antibody mixture from the sample injection port 1 with a 1ml pipette gun, and put the Put the tumor cell separation disc 4 into the device and cover it with a protective cover, wait for 20 minutes, click the stop button on the touch screen module 1, add 1ml of cell buffer from the sample injection port 1 with a 1ml pipette gun, and touch the touch screen Start key on module 1.

Step 8: Click the stop button on the touch screen module 1, take out the circulating tumor cell separation plate 4, remove the sticker on the hole A on the reverse side, put the circulating tumor cell separation plate 4 into the device and cover the protective cover, and touch the touch screen Start button on module 1; unscrew the 4 screws on the reverse side of the circulating tumor cell separation disc 4, take out the separator and ring, carefully grasp the edge of the membrane with tweezers, take out the membrane, and carefully place the membrane on a glass slide , using a microscope to observe the number and shape of circulating tumor cells in lung cancer.

In this example, the 1 ml pipette was covered with cell membrane permeabilizing agent for 5 minutes in advance. In the eighth step, unscrew the four screws on the reverse side of the circulating tumor cell separation disc 4 in two steps, first unscrew and then unscrew, to prevent the internal membrane from falling off. The circulating tumor cell separation disc 4 must be taken out each time the sample is added, and it must be leveled immediately after adding the sample, and protected from light.

Claims (10)

1. a kind of circulating tumor cell separator, it is characterised in that the device includes touch display screen module, microcontroller, turns Fast control module, electric rotating machine, circulating tumor cell separator disk, shell and power module；In the touch display screen module Default rotating speed is inputted, touch display screen module is passed the information on to microcontroller, and the microcontroller controls rotating speed according to instruction The speed of control module, the rotational speed control module are installed on electric rotating while control electric rotating machine drives according to default rotating speed Circulating tumor cell separation disc spins on machine, while rotary speed information is fed back to touch display screen module, are shown by described touch Display screen module is shown；The touch display screen module, microcontroller, rotational speed control module, electric rotating machine, circulating tumor cell separation Disk and power module are all installed inside the shell.

A kind of 2. circulating tumor cell separator according to claim 1, it is characterised in that the microcontroller respectively with Touch display screen module is connected with rotational speed control module；The rotational speed control module is connected with electric rotating machine；The power module It is connected respectively with touch display screen module, microcontroller, rotational speed control module and electric rotating machine；Circulating tumor cell separator disk is installed On electric rotating machine.

3. a kind of circulating tumor cell separator according to claim 1, it is characterised in that further include described in being arranged on Shell upper end, the protection cap with shell flexibility hinge connection.

A kind of 4. circulating tumor cell separator according to claim 1, it is characterised in that the circulating tumor cell Separator disk, including substrate and upper cover, substrate and the upper cover sealing；Substrate includes：Loading chamber, transition piece, filter chamber and waste Room；The region that the upper cover corresponds to loading chamber sets sample holes；One catch is set in loading chamber, and the catch corresponding position was set Cross part；The transition piece structure is inclined-plane from low to high at catch；The filter chamber upper end is cell size selective membrane, By passage and waste compartment unicom, the height of the transition piece is more than or equal to the position of cell size selective membrane in the space of lower end； In the centrally disposed centrifugal hole of the chip；When using separator disk, loading chamber sample introduction is given by sample holes, separator disk is installed On electric rotating machine, when separating disc spins, sample introduction liquid rapidly radially moves out, and is pumped by transition piece in filter chamber, Cell more than cell size selection membrane aperture is captured, and the cell less than cell size selection membrane aperture is transferred to filter chamber's stream Enter waste compartment.

A kind of 5. circulating tumor cell separator according to claim 1 or 4, it is characterised in that the circulating tumor Cell separator disk front and back has sample holes.

A kind of 6. circulating tumor cell separator according to claim 1 or 4, it is characterised in that the cell size Selective membrane is detachable structure.

7. a kind of circulating tumor cell separator according to claim 1, it is characterised in that the electric rotating machine is arteries and veins Rush signal rotating speed and direction adjustable motor.

8. based on a kind of detection method of circulating tumor cell separator of claim 1-7 any one, it is characterised in that should Method includes the following steps：

Step 1：Prepare reagent, prepare blood sample, circulating tumor cell separator disk is turned to the back side, ensures one of them Hole is closing, is added from another hole after membrane permeability agent and seals sample holes, the washing on point touching display panel module Pattern；

Step 2：In circulating tumor cell separator disk front from the first adding mouth refinement after birth bleeding agent, by circulating tumor cell Separate in disc loading apparatus, the washing process on point touching display panel module；

Step 3：From the pretreated blood of patients with lung cancer sample of the first adding mouth, circulating tumor cell separator disk is put into dress In putting, the filtered model on point touching display panel module；

Step 4：Touch display screen module is pressed after the completion of filtering to stop key；See whether that also sample is in sample room：Such as Fruit is repeat step three；If not, adding membrane permeability agent from the second adding mouth, circulating tumor cell separator disk is put into In device, the start key on point touching display panel module；

Step 5：Stop key on point touching display panel module, performs staining pattern；

Step 6：Stop key on point touching display panel module, adds a small amount of membrane permeability agent from the first adding mouth, will follow In ring tumour cell separation disc loading apparatus, it is laid flat and waits 5 minutes, the start key on point touching display panel module；Click on and touch The stop key on display panel module is touched, phosphate buffered saline solution is added from the first adding mouth, circulating tumor cell separator disk is put Enter in device, the start key on point touching display panel module；Stop key on point touching display panel module, from the first sample-adding Mouth adds a small amount of blocking agent, and circulating tumor cell is separated in disc loading apparatus, waits 20 minutes, and phosphorus is added from the first adding mouth Acid buffering salting liquid, the start key on point touching display panel module；

Step 7：Stop key on point touching display panel module, a small amount of antibody is added from the first adding mouth, and circulating tumor is thin Born of the same parents are separated in disc loading apparatus, are laid flat and are waited 20 minutes, the start key on point touching display panel module；Point touching display screen Stop key in module, adds Cell Buffer from the first adding mouth, circulating tumor cell is separated in disc loading apparatus, click on Start key in touch display screen module；Stop key on point touching display panel module, adds from the first adding mouth and follows on a small quantity Ring positive tumor cell mixtures of antibodies, circulating tumor cell is separated in disc loading apparatus, is waited 20 minutes, point touching is shown Stop key in display screen module, the start key from the first adding mouth addition Cell Buffer, point touching display panel module；

Step 8：Stop key on point touching display panel module, takes out circulating tumor cell separator disk, opens back side sample-adding Hole, circulating tumor cell is separated in disc loading apparatus, the start key on point touching display panel module；By circulating tumor cell The cell size selective membrane of separator disk is placed into the quantity and form that lung cancer circulating tumor cell is observed on glass slide.

9. a kind of circulating tumor cell method for separating and detecting according to claim 8, it is characterised in that the step 5 Staining pattern is to add 1000 μ l, 3.5% formaldehyde from the first adding mouth, circulating tumor cell is separated in disc loading apparatus, etc. Treat 20 minutes, the start key on point touching display panel module；Stop key on point touching display panel module, from the first sample-adding Mouth adds phosphate buffered saline solution, circulating tumor cell is separated in disc loading apparatus, opening on point touching display panel module Dynamic key.

10. a kind of circulating tumor cell method for separating and detecting according to claim 8, it is characterised in that utilized during sample-adding 1ml liquid-transfering guns, the 1ml liquid-transfering guns are covered 5 minutes with membrane permeability agent in advance.