CN107937257A - A kind of circulating tumor cell separating chips and its detection method - Google Patents

Abstract

A circulating tumor cell separation chip and a detection method thereof belong to the technical field of circulating tumor cell separation, and solve the problems of complex existing devices, many transmission mechanisms, and difficulty in ensuring precision. When the chip rotates, the sample liquid moves radially outward rapidly and is pumped through the transition piece to the filter chamber, the cells larger than the pore size of the cell size selective membrane are captured, and the cells smaller than the pore size of the cell size selective membrane are transferred to the filter chamber and flow into the waste chamber . The invention is small in size, light in weight, and easy to carry. It adopts a cell separation membrane with liquid-filled holes and has the advantages of non-blocking, high sensitivity, selectivity, and speed. The capillary is triggered by the liquid stored in the fluid-assisted chamber below the membrane. , remains fully wetted throughout the filtration process so that liquid flow requires minimal pressure differential and filtration occurs more uniformly across the membrane; this method provides uniform, non-clogging ultra-fast cell separation technology with higher pressure values than conventional size 1kPa in filtration is much smaller.

Description

A circulating tumor cell separation chip and its detection method

technical field

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

Background technique

A major obstacle in the study of circulating tumor cells (CTCs) is that the amount of whole blood is extremely rare, with 1-10 CTCs per 10 ⁶ -10 ⁷ blood cells, and the blood volume that can be processed is limited. Due to the rarity of CTCs, developing an efficient collection process has become a critical step in the detection and characterization of CTCs, which occur at a frequency of 1–10 cells per 1 mL of blood. In this regard, microfluidic platforms have emerged as a possible solution, as the fine structure of the device allows precise fluidic control and can also provide a biocompatible environment for cells. Although various CTC detection platforms using microfluidics have been reported so far, all of these platforms have some major limitations. So far, proposed microfluidic chip-based CTC isolation techniques have been rooted in cancer cell biological properties (i.e., surface marker expression) or physical properties (i.e., size, density, dielectric properties, etc.).

CTCs shed into peripheral blood from primary and metastatic tumors are important biomarkers in the early detection of invasive cancers and in personalized cancer therapy. Due to the extremely low concentration of CTCs, it is a great challenge to obtain high recovery and high purity CTCs. In addition, CTCs are very fragile, so it is strongly recommended to analyze them within 6-8 hours after blood collection from the patient. Therefore, there is an urgent need to develop rapid, efficient and stable CTC isolation techniques that can be widely deployed in clinical settings. In previous experiments to selectively isolate CTCs from peripheral blood, the most common immunoaffinity-based detection method has relatively high sensitivity and specificity, but the detection time is long (>1-4 hours) .

After investigating the existing technologies, Chinese patent 201520684448.X discloses a cell separation method similar to the principle of the patent of the present invention. Although the device has a certain ability to separate cells, the design of the device is relatively complicated, the transmission mechanism is too many, and the accuracy is not easy to guarantee; and it is bulky, complicated to operate, and not easy to carry. It cannot be used for hospital laboratory and bedside monitoring, 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 chip and its detection method, using the technology of separating CTC by size, and adopting a photolithographic film with regular aperture and shape to improve the recovery rate and purity of CTC , through the liquid phase diffusion that fills the chamber under the cell separation membrane before and throughout the entire filtration process, the entire membrane area can be filtered and the clogging problem is greatly alleviated.

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

A circulating tumor cell separation chip, comprising a base and an upper cover, 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 is provided with a sampling hole in an area corresponding to the loading chamber 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 passes through The channel communicates with the waste chamber, 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 in the center of the chip; when using a separation chip, the loading chamber is injected through the sample injection hole, and the chip is installed On a centrifugal device, when the chip rotates, the sample liquid moves radially outward rapidly and is pumped through the transition piece. On the filter chamber, cells larger than the pore size of the cell size selective membrane are captured, and cells smaller than the pore size of the cell size selective membrane are transferred to the filter chamber. The filter chamber flows into the waste chamber.

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

Step 1: place the sample liquid in a culture medium supplemented with fetal calf serum and antibiotics/antimycotics, and cultivate it in an incubator under a _CO environment;

Step 2: passivating the surface of the chip with bovine serum albumin through the loading chamber;

Step 3: After incubation, the chip is driven by a centrifugal device to rotate at a certain speed, so that the fetal bovine serum solution in the loading chamber is moved to the waste chamber;

Step 4: The washing buffer is added from the injection hole to the loading chamber, and the speed of the chip in step 3 is maintained, and the washing buffer passes through the cell size selection membrane and passes through the filter chamber to the waste chamber;

Step 5: Keep the centrifugal device working at the speed of step 3, add the sample liquid to the loading chamber through the sample hole; separate the cells in the filter chamber, and then wash the filter chamber with washing buffer;

Step 6: After the separation, remove the cell size selection membrane and install it on a glass slide for staining, observe with a fluorescence microscope and record the data.

The beneficial effects of the present invention are: the patent of the present invention is small in size, light in weight and convenient to carry, and can detect not only a large number of CTCs of patients with cancer metastasis, but also CTCs can be detected from patients at a relatively early stage without cancer symptoms, which shows that It is possible that potential CTCs can be used as early diagnostic markers. Using a cell separation membrane with liquid-filled pores, which has the advantages of non-blocking, high sensitivity (95.9±3.1% recovery), selectivity (>2.5log depletion of leukocytes), and rapidity (>3mL/min), the capillary is stored in The liquid in the fluid-assisted chamber located below the membrane initiates and remains fully wetted throughout the filtration process so that liquid flow requires minimal pressure differential and filtration occurs more uniformly across the membrane; and using a fluid-assisted separation technique A lab-independent chip system that enables label-free isolation of live CTCs from whole blood without prior sample processing. Numerical simulations and experiments demonstrate that this method provides uniform, clog-free ultrafast cell separation at pressure values much smaller than 1kPa in conventional size filtration.

Description of drawings

Fig. 1 is an exploded diagram of a circulating tumor cell separation chip of the present invention.

Fig. 2 is a structure diagram of the back surface of a circulating tumor cell separation chip of the present invention.

Fig. 3 is a schematic diagram of a circulating tumor cell separation chip of the present invention.

In the figure 1. Sealing silicone pad, 2. Cell size selection membrane, 3. Top cover, 4. Top cover shading sticker, 5. Strong water-absorbing membrane, 6. Base shading sticker, 7. Base, 8. Sealing O-ring and 9. Cell size selection membrane gland.

Detailed ways

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

As shown in Figure 1 and Figure 2, a circulating tumor cell separation chip, the chip includes: sealing silica gel pad 1, cell size selection membrane 2, upper cover 3, upper cover light-shielding sticker 4, strong water-absorbing membrane 5, base Shading sticker 6, base 7, sealing O-ring 8 and cell size selection membrane gland 9. The chip is composed of three separate filter units, which can process three different blood samples at the same time, each unit includes a sample loading chamber, a transition piece and a filter chamber, and the three filter units share a waste chamber; the upper cover 3 and The substrate 7 is bonded by a double-sided pressure-sensitive adhesive tape to realize the tight assembly of the chip; the upper cover 3 has a sampling hole for sampling and ventilation, and the sampling liquid is sent into the loading chamber, and the loading The chamber is provided with a baffle and a filter element corresponding to the position of the baffle. Seen from the position close to the baffle, the filter element is a slope structure from low to high; the base 7 is created with a channel, and a main fluid chamber is formed on the base 7; An outlet channel is created on the base 7 and an area for placing the cell size selection membrane 9 is set, and the sealing O-ring 8, the cell size selection membrane gland 9, and the sealing silica gel pad 1 are fastened to form a filter chamber. The height of the transition piece is greater than or equal to the position of the cell size selective membrane 9 and is closely adjacent to the cell size selective membrane 9 . The cell size selection membrane cover 9 is easy to disassemble, and it can be installed on a glass slide for subsequent image analysis and molecular characterization. The cell size selection membrane gland 9 is provided with a sample hole for adding coating solution; the chip waste chamber is bonded by a strong water-absorbing film 5 through a double-sided pressure-sensitive adhesive tape, and the strong water-absorbing film 5 can Absorb part of the waste liquid; the upper cover shading sticker 4 and the base shading sticker 6 are respectively adhered to the upper cover 3 and the base 7 by double-sided pressure-sensitive adhesive tape, so that the sample liquid is not disturbed by the external environment; the middle of the chip There are placement holes for connecting with the centrifugal device. In this example, the above-mentioned upper cover 3, base 7 and cell size selection membrane gland 9 are made of polycarbonate.

The above-mentioned chip is installed in a centrifugal device, as shown in Figure 3, when the chip rotates, the sample liquid located in the loading chamber is quickly pumped radially outward, and the sample liquid passes through the baffle and filter element arranged in the loading chamber. Gently pushed into the filter chamber at the base 7, the larger sized cells are captured by the cell size selective membrane 2 while the smaller sized cells pass through the cell size selective membrane 2 for transfer to the waste chamber. During the whole filtration process, in each unit composed of the upper cover 3 and the base 7, the filter chamber under the cell size selective membrane 2 is filled with liquid. The filter element is provided with an inclined surface, which is used to prevent the cells from being damaged or ruptured due to excessive pressure due to centrifugal force, and the pressure drop ΔP across the cell size selective membrane 2 is kept below the threshold to prevent cell damage. important. Calculate the pressure drop across the cell size selective membrane 2 by using the following equation:

Where μ is the coefficient of dynamic viscosity, L is the thickness of the cell size selective membrane 2, d is the pore diameter of the cell size selective membrane 2, Q is the flow rate on the cell size selective membrane 2, and N is the number of pores on the cell size selective membrane 2.

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

Step 1: All the cells of the injected liquid were cultured in RPMI medium supplemented with 5% FBS and 1% antibiotic/antimycotic in an incubator set at 37°C and under 5% _CO2 environment, For spiking experiments, cells are pre-labeled with fluorescent dyes.

Step 2: before cell size selection, passivate the surface of the chip with BSA, add 1% BSA solution from the sample hole in the upper cover 3 to each unit composed of the upper cover 3 and the base 7, to Passivates the surface to prevent cell adsorption.

Step 3: After incubation for 30 minutes, the chip is rotated at a certain speed by the centrifugal device, which moves the BSA solution in the sample loading chamber to the waste chamber, but the filter chamber below the membrane remains full of liquid.

Step 4: Add 1mL of washing buffer to the loading chamber from the sample hole in the upper cover 3, and the chip maintains the rotation speed of the previous step. The washing buffer passes through the cell size selection membrane 2 to the waste chamber, and the cell size selection membrane 2 is below The BSA solution in the chamber is replaced by freshly added wash buffer. After the surface is passivated, add 3mL of sample solution to the loading chamber from the sample injection hole in the upper cover 3, without any sample pretreatment steps.

Step 5: Separate the cells in the loading chamber by rotating the chip, then wash the loading chamber twice with 1 mL of washing buffer, and keep the original working speed of the centrifugal device for work after each liquid addition.

Step 6: After the separation, the cell size selection membrane 2 was removed and installed on a glass slide for staining treatment, observed by a fluorescence microscope and recorded data.

In this example, the pores distributed on the cell size selective membrane 2 in the above steps are non-uniform and randomly distributed. Both the base 7 and the cell size selection membrane gland 9 have screw holes, which are fastened by screws. The chip described in the above steps is filled with the liquid phase under the cell size selective membrane 2, and the pressure gradient generated in the submembrane chamber and the pressure gradient in the upper chamber in the detection medium have good transmissibility, resulting in a relatively uniform pressure drop, so the liquid Uniform flow through cell size selective membrane 2.

Claims (10)

1. A circulating tumor cell separation chip, comprising a base and an upper cover, the base and the upper cover are sealed; it is characterized in that the base comprises: a loading chamber, a transition piece, a filter chamber and a waste chamber; the upper cover corresponds to the loading chamber Injection holes are set in the region; 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 is a slope from low to high near the baffle; the upper end of the filter chamber is for cell size selection Membrane, the space at the lower end communicates with the waste chamber through a channel, 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 in the center of the chip; when using a separation chip, the loading chamber is provided through the injection hole For sample injection, the chip is installed on the centrifugal device. When the chip rotates, the sample liquid moves radially outward quickly and is pumped to the filter chamber through the transition piece. The cells larger than the pore size of the cell size selection membrane are captured, and the cells smaller than the cell size selection The cells of the membrane pore size are transferred to the filter chamber and flow into the waste chamber. 2 . The circulating tumor cell separation chip according to claim 1 , wherein the substrate and the upper cover are bonded by a double-sided pressure-sensitive adhesive tape. 3 . 3. A circulating tumor cell separation chip according to claim 1, characterized in that the filter chamber is flexibly connected to the base, and the back of the filter chamber is sequentially arranged to be sealed and sealed by a sealing O-ring and a cell size selection membrane gland. Loading. 4 . The circulating tumor cell separation chip according to claim 1 , wherein non-uniform holes are randomly distributed on the cell size selective membrane, and the cell size selective membrane is a detachable structure. 5. A circulating tumor cell separation chip according to claim 4, characterized in that, when processing the sample liquid containing fragile cells, the pressure drop ΔP across the cell size selective membrane is kept below the threshold to prevent cell damage, by Use the following equation to calculate the pressure drop across the cell size selective membrane: where μ is the coefficient of dynamic viscosity, L is the thickness of the cell size selective membrane, d is the pore diameter of the cell size selective membrane, Q is the flow rate of the cell size selective membrane, and N is the number of pores of the cell size selective membrane. 6 . The chip for separating circulating tumor cells according to claim 1 , wherein a light-shielding sticker is provided on the outside of the base and the upper cover, and a strong water-absorbing film is arranged inside the upper cover. 6 . 7. A detection method based on a circulating tumor cell separation chip according to claims 1-6, characterized in that the method comprises the following steps: Step 1: place the sample liquid in a culture medium supplemented with fetal calf serum and antibiotics/antimycotics, and cultivate it in an incubator under a _CO environment; Step 2: passivating the surface of the chip with bovine serum albumin through the loading chamber; Step 3: After incubation, the chip is driven by a centrifugal device to rotate at a certain speed, so that the fetal bovine serum solution in the loading chamber is moved to the waste chamber; Step 4: The washing buffer is added from the injection hole to the loading chamber, and the speed of the chip in step 3 is maintained, and the washing buffer passes through the cell size selection membrane and passes through the filter chamber to the waste chamber; Step 5: Keep the centrifugal device working at the speed of step 3, add the sample liquid to the loading chamber through the sample hole; separate the cells in the filter chamber, and then wash the loading chamber with washing buffer; Step 6: After the separation, remove the cell size selection membrane and install it on a glass slide for staining, observe with a fluorescence microscope and record the data. 8. detection method according to claim 7, is characterized in that, in described step 1, all cells are supplemented with 5% FBS fetal bovine serum 1% antibiotic/antimycotic medium in the culture medium, and set For the 37 °C incubator and 5% _CO2 environment, for spiking experiments, cells were pre-labeled with fluorescent dyes. 9. The detection method according to claim 7, characterized in that, in said step 3, before performing cell size selection, the surface of the chip is passivated with bovine serum albumin, and 1% bovine serum albumin solution is added To the sample from the sample hole in the top cover of the chip. 10. The detection method according to claim 7, characterized in that, in said step 3, keep the filter chamber full of liquid.