Cell centrifugal sorting micro-fluidic chip integrated with micro-valve and micro-filter

Abstract

The invention relates to an integrated method for integrating a micro-fluidic chip and cell centrifugal sorting, in particular to a cell centrifugal sorting micro-fluidic chip integrating a micro valve and a micro filter. The opening and closing function of the micro valve in the chip and the size-based cell sorting function of the micro filter are realized by adjusting the direction and the rotating speed. The system comprises a driving control subsystem and a centrifugal separation subsystem, the centrifugal separation subsystem comprises a micro-fluidic chip, a driving disc, a short column and other components, a micro-column structure is designed in the micro-fluidic chip, and the micro-column structure and the short column on the driving disc cooperatively play the functions of a micro valve and a micro filter through the centrifugal effect. By setting the rotating speed of the driving control subsystem and designing the structure of the micro-fluidic chip, the aim of quickly and accurately sorting the circulating tumor cells at low cost is fulfilled.

Description

Technical Field

[0001] The present invention relates to an integrated method of integrating a microfluidic chip with cell centrifugal sorting, specifically a cell centrifugal sorting microfluidic chip integrating a microvalve and a microfilter, which generates a centrifugal effect by driving a control subsystem, and adjusts the direction and rotation speed to realize the opening and closing function of the microvalve in the microfluidic chip and the size-based cell sorting function of the microfilter. Background Art

[0002] Malignant tumors are one of the major diseases with the highest mortality rate in my country. More than 90% of cancer patients die due to the metastasis of circulating tumor cells. Invasion and metastasis are one of the most significant characteristics of malignant tumors. In recent years, a large number of studies have shown that the detection of circulating tumor cells can help to detect tumor micrometastasis at an early stage, monitor postoperative recurrence, evaluate efficacy and prognosis, and select appropriate individualized treatment, thus providing a non-invasive method.

[0003] The current diagnostic methods mainly include ultrasound examination, MRI examination, liver puncture, etc., which have the problems of strong radiation, high equipment cost, great pain for patients during detection, and difficulty in early detection. The US Food and Drug Administration has approved the separation and counting of circulating tumor cells, Cell Search, into clinical practice, but the system still has shortcomings in clinical application, such as low automation, low detection efficiency, and the inability of captured circulating tumor cells to survive.

[0004] Microfluidic technology can precisely control the parameters of microfluidic devices at the cellular scale, efficiently process complex cell fluids with minimal damage, and sort circulating tumor cells with the advantages of high automation, high detection efficiency, and the survival of captured circulating tumor cells. Cell sorting technology includes active sorting technology, passive sorting technology, and centrifugal sorting technology. Active sorting technology allows more precise control of target cells, but the flow rate is always limited because the external force field must overcome the hydrodynamic resistance to achieve effective function. Passive sorting technology is simple and easy to operate and can work at relatively high flow rates, but it has high requirements for microchannel structure and dimensional accuracy. Therefore, it is of great significance for tumor treatment to study a detection system that can quickly, accurately, and low-costly sort circulating tumor cells from human blood. Summary of the invention

[0005] In view of the problem that active and passive cell sorting has high requirements on external instruments and microfluidic chip structure, the present invention provides a cell centrifugal sorting microfluidic chip integrating microvalve and microfilter, which can achieve the following purposes: (1) adjusting the rotation speed of the drive control subsystem to realize the opening and closing function of the microvalve; (2) the microfilter in the microfluidic chip realizes the cell sorting function.

[0006] The present invention is implemented by adopting the following technical scheme: a cell centrifugal sorting microfluidic chip integrating a microvalve and a microfilter comprises a drive control subsystem and a centrifugal sorting subsystem, wherein the centrifugal sorting subsystem comprises a microfluidic chip, a microfluidic chip fixing disk, a drive disk, a short column, an upper connecting block, a lower connecting block, a connecting rod, a spring and a flying ball, wherein the microfluidic chip fixing disk is fixed on the shaft body of the connecting shaft of the drive control subsystem, the microfluidic chip is fixed on the bottom surface of the microfluidic chip fixing disk, the connecting shaft passes through the drive disk and the upper connecting block, and the drive The disk is located below the microfluidic chip fixing disk, the short column is fixed on the top surface of the driving disk, the upper connecting block is located below the driving disk, the lower connecting block is fixed on the connecting shaft, the spring is arranged on the shaft body between the upper connecting block and the lower connecting block of the connecting shaft, the connecting rod is hinged to the upper connecting block and the lower connecting block respectively, and the end of the connecting rod is fixed with the flying ball; the microfluidic chip comprises a microfluidic channel, the two ends of the microfluidic channel are respectively provided with an inlet and an outlet, a microcolumn array structure is arranged in the microfluidic channel, the top surface of the microcolumn in the microcolumn array structure is connected to the microfluidic channel, and the bottom surface is suspended.

[0007] The microfluidic chip structure contains a microcolumn array structure, which works together with the short columns on the drive disk to play the role of microvalve and microfilter through centrifugal action. By setting the speed of the drive control subsystem and designing the microfluidic chip structure, cell sorting of different sizes can be achieved.

[0008] The above-mentioned cell centrifugal sorting microfluidic chip with integrated microvalve and microfilter, the drive control subsystem includes a servo motor, a servo driver, a pulse generator, an AC-DC converter power supply, a diaphragm coupling and a connecting shaft. The connecting shaft is connected to the servo motor through the diaphragm coupling, the pulse generator is connected to the servo driver, the servo driver is connected to the servo motor, and the AC-DC converter power supply supplies power to the pulse generator and the servo motor.

[0009] The above-mentioned cell centrifugal sorting microfluidic chip with integrated microvalve and microfilter has 6 microchannel circles, a microchannel width of 500μm, a spacing of 600μm between every two microchannel circles, a microchannel height of 120μm, a microcolumn height of 40μm, and an inlet and outlet diameter of 3000μm; the microfluidic chip is made of polydimethylsiloxane material using soft lithography technology.

[0010] In the above-mentioned cell centrifugal sorting microfluidic chip integrating microvalve and microfilter, the short columns on the driving disk and the microcolumn array structure in the microfluidic chip cooperate to play the functions of microvalve and microfilter. When the system is not rotating, the microvalve is closed, the gap between the microcolumns is between the diameters of circulating tumor cells and other cells, and other cells are collected at the outlet; when the system is rotating, the microvalve is opened, and circulating tumor cells are collected at the outlet.

[0011] The present invention utilizes centrifugal force to drive fluid and cell movement. Compared with other existing active and passive microfluidic driving methods, it has the advantages of easy processing, wide range of driven fluids, pulsation-free fluid flow, and ease of high-throughput analysis. It replaces traditional injection pumps and is suitable for cell sorting and in vitro diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the composition diagram of the drive control subsystem.

[0013] Figure 2 This is a functional schematic diagram of the centrifugal sorting subsystem.

[0014] Figure 3 Schematic diagram of microfluidic chip cell sorting.

[0015] Figure 4 Schematic diagram of a microfilter.

[0016] Figure 5 Schematic diagram of microvalve function.

[0017] Figure 6 This is a diagram of the microfluidic chip structure dimensions.

[0018] In the figure: 1-connecting shaft, 2-microfluidic chip fixing plate, 3-driving plate, 4-upper connecting block, 5-connecting rod, 6-lower connecting block, 7-flying ball, 8-spring, 9-microfluidic chip, 10-short column, 11-screw.

[0019] 91 - inlet, 92 - outlet, 93 - micro-pillar array structure, 94 - micro-channel. DETAILED DESCRIPTION

[0020] The cell centrifugal sorting microfluidic chip integrating microvalves and microfilters is divided into a drive control subsystem and a centrifugal sorting subsystem.

[0021] The drive control subsystem consists of a servo motor, a servo driver, a pulse generator, an AC-DC converter, a motor fixing bracket, a diaphragm coupling, a connecting shaft 1 and other components. The opening and slot size of the lower end of the diaphragm coupling are determined according to the size of the servo motor shaft. The size of the connecting shaft 1 is determined according to the opening size of the upper end of the diaphragm coupling and is made of steel to ensure its rotation stability. Figure 1 As shown, after each device is correctly wired, the encoded pulse generator sends a command to the servo driver to adjust the speed and direction. The servo driver drives the servo motor and drives the centrifugal sorting subsystem to rotate at a specified speed and direction through the connecting shaft 1.

[0022] The centrifugal sorting subsystem includes components such as a microfluidic chip 9, a microfluidic chip fixing disk 2, a driving disk 3, a short column 10, an upper connecting block 4, a lower connecting block 6, a connecting rod 5, a spring 8 and a flying ball 7. The microfluidic chip structure contains a microcolumn array structure 93, which cooperates with the short column 10 on the driving disk to play the function of a microvalve and a microfilter through centrifugal action. By setting the speed of the drive control subsystem and designing the microfluidic chip structure, cells of different sizes can be sorted. The microfluidic chip fixing plate 2 is fixed on the shaft body of the connecting shaft 1 by screws 11, the microfluidic chip 9 is fixed on the bottom surface of the microfluidic chip fixing plate 2, the connecting shaft 1 passes through the driving plate 3 and the upper connecting block 4, the driving plate 3 is located below the microfluidic chip fixing plate 2, the short column 10 is fixed on the top surface of the driving plate 3, the upper connecting block 4 is located below the driving plate 3 and connected to the driving plate 3, the lower connecting block 6 is fixed on the connecting shaft 1 by screws, the spring 8 is arranged on the shaft body between the upper connecting block 4 and the lower connecting block 6 of the connecting shaft, the two ends of the connecting rod 5 are respectively hinged on the upper connecting block 4 and the lower connecting block 6, and the other end of the connecting rod 5 is fixed to the flying ball 7. The microfluidic chip 9 includes a microfluidic channel 94, the two ends of the microfluidic channel 94 are respectively provided with an inlet 91 and an outlet 92, a microcolumn array structure 93 is arranged in the microfluidic channel 94, the top surface of the microcolumn in the microcolumn array structure 93 is connected to the microfluidic channel 94, and the bottom surface is suspended. The short column 10 is located directly below the microcolumn array structure in the microfluidic chip.

[0023] The flying ball 7 is made of an iron ball with a mass between 0.5-1.0kg, and the connecting rod 5, the connecting block, the driving disk 3, the microfluidic chip fixing disk 2, and the short column 10 are made of acrylic material with a low density. When the connecting shaft 1 is not rotating, the compression spring 8 pushes the connecting block 4 and the driving disk 3 upward under the action of the restoring force, and the short column 10 on the driving disk 3 presses the microfluidic chip 9 upward, and the microchannel 94 contacts the bottom surface of the microcolumn array structure 93, and the microvalve is closed. Figure 2 As shown in (a) and (b), when the connecting shaft 1 rotates, the centrifugal force drives the flying ball 7 to move outward, and the driving disk 3 moves downward along the connecting shaft 1. Figure 2 As shown in (c) and (d), the spring 8 is further compressed, the short column 10 is separated from the microfluidic chip 9, and the microvalve is opened.

[0024] Centrifugal force drive is a microfluidic chip ( Figure 3 ) When the servo motor drives the microfluidic chip to make circular motion, the centrifugal force generated is used as the driving force of the fluid. By changing the rotation speed of the chip and designing different flow channel structures, flow rate regulation and cell sorting can be achieved. The microfluidic chip of the present invention contains a microcolumn array structure 93, which plays the role of a microfilter and a microvalve. The height of a single microcolumn structure is designed to be 40μm, the top surface of the microcolumn is connected to the microchannel 94, and the bottom surface is suspended. Figure 5As shown, the micro-column gap is between the diameters of circulating tumor cells and other cells. When the system is not rotating, the micro-valve is closed, and the micro-column array structure 93 plays the role of a micro-filter, injecting the cell mixture, and other cells except circulating tumor cells reach the outlet through the micro-column gap; when the system rotates, the micro-valve is opened, and circulating tumor cells reach the outlet, realizing the sorting function of cells of different sizes.

[0025] The present invention uses a microfluidic chip 9 with integrated microvalve and microfilter. In order to reduce the velocity loss caused by the wall surface to the particle movement, the microchannel 94 is designed as a 6-turn spiral microchannel. In order to achieve the goal of high throughput and avoid cell clogging, the microchannel structure size is relatively large, such as Figure 6 As shown, the microchannel 94 has a width of 500 μm, a spacing of 600 μm between each two circles, a height of 120 μm, a microcolumn height of 40 μm, and an inlet and outlet diameter of 3000 μm.

[0026] The components in the centrifugal separation subsystem are Figure 2 As shown, it is fixed. When the connecting shaft 1 does not rotate, the force of the spring 8 on the upper connecting block 4 causes the short column 10 to squeeze the microcolumn array structure 93 in the microfluidic chip upward, and the microvalve is closed. The gap between the microcolumns is between the diameters of circulating tumor cells and other cells. After the cell mixture is injected using a microinjection pump, only other cells can reach the outlet 92 through the gap between the microcolumns, and other cells are collected at the outlet. The microchannel 94 is rinsed with phosphate buffered saline to remove other cells in the channel wall to ensure that other cells reach the outlet. The drive control subsystem is started, and the centrifugal sorting subsystem moves at a specified speed and direction. The flying ball 7 moves downward under the action of centrifugal force, pulling the drive disk 3 and the short column 10 to move downward along the connecting shaft 1. The spring 8 is further compressed, and the short column 10 is separated from the microfluidic chip 9. The microvalve is opened, and the circulating tumor cells reach the outlet 92 (when rotating, the tumor cells gather at the outlet, and are sucked out through the catheter after stopping rotation). Finally, phosphate buffered saline is injected, and the drive control subsystem is restarted to remove the circulating tumor cells attached to the microchannel wall to ensure that all circulating tumor cells reach the outlet.

[0027] The invention uses SU-8 3050 negative photoresist and polydimethylsiloxane material and adopts soft lithography technology to prepare a microfluidic chip. The main preparation processes include exposure, mold turning and bonding. The specific process is as follows: a clean 4-inch silicon wafer is treated with hexamethyldisilazane, and SU-8 3050 negative photoresist is spin-coated on the polished surface of the silicon wafer; a propylene glycol methyl ether acetate solution and an isopropanol solution are used to develop the exposed silicon wafer; a polydimethylsiloxane prepolymer and a curing agent are mixed and stirred at a mass ratio of 10:1, and then poured on a silicon mold, and heated to a solid state after vacuum treatment; and the polydimethylsiloxane chip after cutting and punching and a glass sheet are subjected to oxygen plasma treatment to achieve irreversible bonding.

Claims

1. A cell centrifugal sorting microfluidic chip integrating a microvalve and a microfilter, characterized in that: The centrifugal sorting subsystem comprises a drive control subsystem and a centrifugal sorting subsystem. The centrifugal sorting subsystem comprises a microfluidic chip (9), a microfluidic chip fixing disk (2), a drive disk (3), a short column (10), an upper connecting block (4), a lower connecting block (6), a connecting rod (5), a spring (8) and a flying ball (7). The microfluidic chip fixing disk (2) is fixed on the shaft body of the connecting shaft (1) of the drive control subsystem. The microfluidic chip (9) is fixed on the bottom surface of the microfluidic chip fixing disk (2). The connecting shaft (1) passes through the drive disk (3) and the upper connecting block (4). The drive disk (3) is located below the microfluidic chip fixing disk (2). The short column (10) is fixed on the drive disk (3). On the top surface of the driving disk (3), an upper connecting block (4) is located below the driving disk (3), a lower connecting block (6) is fixed on the connecting shaft (1), a spring (8) is arranged on the shaft body between the upper connecting block (4) and the lower connecting block (6) of the connecting shaft, a connecting rod (5) is hinged to the upper connecting block and the lower connecting block respectively, and a flying ball (7) is fixed to the end of the connecting rod (5); the microfluidic chip (9) comprises a microfluidic channel (94), an inlet (91) and an outlet (92) are respectively arranged at both ends of the microfluidic channel (94), a microcolumn array structure (93) is arranged in the microfluidic channel, and the top surface of the microcolumns in the microcolumn array structure (93) is connected to the microfluidic channel (94), and the bottom surface is suspended.

2. The cell centrifugal sorting microfluidic chip integrating a microvalve and a microfilter according to claim 1, characterized in that: The drive control subsystem comprises a servo motor, a servo driver, a pulse generator, an AC-DC power supply, a diaphragm coupling and a connecting shaft. The connecting shaft (1) is connected to the servo motor through the diaphragm coupling, the pulse generator is connected to the servo driver, the servo driver is connected to the servo motor, and the AC-DC power supply supplies power to the pulse generator and the servo motor.

3. The cell centrifugal sorting microfluidic chip integrating a microvalve and a microfilter according to claim 1 or 2, characterized in that: The number of microchannels (94) of the microfluidic chip (9) is 6, the width of the microchannels (94) is 500 μm, the interval between every two microchannels (94) is 600 μm, the height of the microchannels (94) is 120 μm, the height of the microcolumns is 40 μm, and the diameters of the inlet (91) and the outlet (92) are both 3000 μm; the microfluidic chip (9) is made of polydimethylsiloxane material using a soft lithography process.

4. The cell centrifugal sorting microfluidic chip integrating a microvalve and a microfilter according to claim 1 or 2, characterized in that: The short columns (10) on the driving disk (3) and the microcolumn array structure in the microfluidic chip (9) cooperate to play the functions of a microvalve and a microfilter. When the system is not rotating, the microvalve is closed, the gap between the microcolumns is between the diameters of circulating tumor cells and other cells, and the outlet collects other cells; when the system is rotating, the microvalve is opened, and the outlet collects circulating tumor cells.

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