Fluid micro-fluidic chip as well as preparation method and use method thereof

Abstract

The invention provides a fluid micro-fluidic chip as well as a preparation method and a use method thereof. The fluid micro-fluidic chip comprises a pre-focusing module, a magnetic field separation module and a glass slide, the pre-focusing module and the magnetic field sorting module are arranged on the glass slide, and the pre-focusing module is connected with the magnetic field sorting module through a first amplification section; the pre-focusing module is used for focusing the microspheres at the same equilibrium position, and the magnetic field sorting module is used for sorting the microspheres with different sizes. The method can realize continuous and sheath-free separation of microspheres with different sizes, and has the advantages of high flux, small structure size, no need of magnetic bead modification, simple operation and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of CTC high-throughput sorting, and in particular to a fluid microfluidic chip and a preparation and use method thereof, in particular to a sheathless iron fluid microfluidic chip for continuously sorting non-magnetic microspheres. Background Art

[0002] Cancer is the leading cause of global disease burden. According to the World Health Organization's International Agency for Research on Cancer, there will be 20 million new cancer cases and 9.7 million deaths in 2022, and about 90% of them will die from cancer metastasis. Prompt treatment of patients in the early stages of cancer metastasis can reduce cancer deaths by about 30%. Therefore, studying the mechanism of cancer metastasis and early precision diagnosis and treatment technologies are key means for humans to fight cancer in the future. However, in the early stages of cancer metastasis, the patient's tumor load is extremely low, and it is difficult to locate metastatic lesions through traditional imaging methods, and it is impossible to extract tumor cells through puncture biopsy technology. Therefore, it is difficult to detect the development and metastasis of the tumor, which greatly limits the molecular diagnosis and precision treatment of metastatic tumors in the early stages.

[0003] Circulating tumor cells (CTCs) refer to tumor cells that fall off from primary or metastatic tumor lesions, undergo epithelial-mesenchymal transition, and enter the peripheral blood circulation. A large number of studies have shown that CTCs already exist in the peripheral blood at the low tumor load stage and can express the biological genetic information of tumor cells from genome to protein function. Therefore, CTC liquid biopsy technology can break the limitation that tissue biopsy is only applicable to solid tumor tissues that can be detected by imaging. At the same time, with its characteristics of being non-invasive, dynamically observable, and able to provide personalized medicine for patients, it has broader application prospects in early cancer diagnosis and treatment guidance.

[0004] However, due to the interference of high-abundance blood cells in peripheral blood, high-throughput and accurate separation of CTCs is still a huge challenge. Active manipulation of cell sorting technology, due to its reliance on external forces, usually requires the use of complex instruments and equipment and the introduction of additional manual operations to complete the sorting of CTCs. Some active manipulation technologies require magnetic bead modification of cells, so active manipulation of cell sorting methods usually face problems such as high technical costs, difficulty in small-scale modularization, and complex operations. Existing passive manipulation cell sorting technology can avoid the disadvantages of the above-mentioned active manipulation methods, but it usually requires sheath flow to assist, which reduces the sorting throughput and portability of operation. In addition, passive sorting technology also has problems such as high cell shear stress and severe clogging.

[0005] The patent document with the public number CN109550531A discloses a magnetic size-dependent microfluidic chip, including a cover sheet and a substrate, the cover sheet is located directly above the substrate, the cover sheet and the substrate are sealed and connected, a magnetophoresis separation structure and a size sorting channel for capturing magnetic microspheres of different sizes are opened on the lower surface of the cover sheet, the magnetophoresis separation structure includes a buffer moving channel, a sample moving channel, a confluence channel, a capture channel and a waste liquid discharge channel, the buffer moving channel and the sample moving channel merge at one end of the confluence channel, and one end of the waste liquid discharge channel and the capture channel merge at the other end of the confluence channel. The other end of the capture channel is connected to the entrance of the size sorting channel, a magnet is provided on the cover sheet, the magnet is located above the confluence channel, and the magnet is located on one side of the projection of the confluence channel on the upper surface of the cover sheet, the capture channel extends in a direction close to the magnet, and the waste liquid discharge channel extends in a direction away from the magnet, and a buffer injection port, a sample injection port, a first discharge port, and a second discharge port are provided on the cover sheet, the buffer injection port is connected to the buffer movement channel, the sample injection port is connected to the sample movement channel, the first discharge port is connected to the waste liquid discharge channel, and the second discharge port is connected to the size sorting channel outlet. However, the patent document still has problems such as high cell shear stress and severe blockage.

[0006] The patent document with publication number US20220379312A1 discloses a magnetic separation microfluidic chip, and specifically discloses a magnetic separation microfluidic chip, including a substrate, a chip model material layer, a microchannel unit and a magnetic separation unit, the chip model material layer is arranged on the substrate, the microchannel unit and the magnetic separation unit are both arranged in the chip model material layer, the microchannel unit includes a separation channel and a magnetic pole channel; the separation channel has multiple separation channel entrances and multiple separation channel exits; the magnetic separation unit includes a permanent magnet, a high magnetic permeability alloy and a magnetic pole array arranged in the magnetic pole channel, and the high magnetic permeability alloy is used to conduct the magnetic field of the permanent magnet to the magnetic pole array, so that the magnetic pole array generates a magnetic field with opposite polarity at the left and right positions of the separation channel. However, the patent document still has problems such as high cell shear stress and severe blockage. Summary of the invention

[0007] In view of the defects in the prior art, the object of the present invention is to provide a fluid microfluidic chip and a preparation and use method thereof.

[0008] A fluid microfluidic chip for sorting non-magnetic microspheres provided by the present invention comprises: a pre-focusing module, a magnetic field sorting module and a glass slide;

[0009] The pre-focusing module and the magnetic field sorting module are arranged on the glass slide, and the pre-focusing module is connected to the magnetic field sorting module via a first amplification section;

[0010] The pre-focusing module is used to focus the microspheres at the same equilibrium position, and the magnetic field sorting module is used to sort the microspheres of different sizes.

[0011] Preferably, the pre-focusing module comprises: a first inlet, a first straight channel section and a serpentine channel section;

[0012] The first inlet is connected to one end of the first straight channel section, and the other end of the first straight channel section is connected to one end of the serpentine channel section;

[0013] The other end of the serpentine channel section is connected to one end of the first amplifying section, and the other end of the first amplifying section is connected to the magnetic field sorting module.

[0014] Preferably, the serpentine channel section is an asymmetric structure, and the serpentine channel section includes a plurality of semicircles connected in sequence, two adjacent semicircles are connected by an arc segment, and the diameters of the two adjacent semicircles are different.

[0015] Preferably, the magnetic field sorting module comprises: a second straight channel section, a magnet, a second amplification section, a first outlet and a second outlet;

[0016] The other end of the first amplifying section is connected to one end of the second straight channel section, the other end of the second straight channel section is connected to one end of the second amplifying section, and the first outlet and the second outlet are connected to the other end of the second amplifying section;

[0017] The magnet is located on one side of the second straight channel section and is disposed adjacent to the second straight channel section.

[0018] The present invention also provides a method for preparing a fluid microfluidic chip, which is used to prepare the above-mentioned fluid microfluidic chip for sorting non-magnetic microspheres, comprising the following steps:

[0019] Step 1: Mix polydimethylsiloxane and polydimethylsiloxane curing agent according to a preset mass ratio, and stir with a glass rod for a preset time to fully mix them;

[0020] Step 2: placing the polydimethylsiloxane mixed solution in step 1 in a vacuum drying dish, and removing bubbles in the polydimethylsiloxane mixed solution by vacuuming;

[0021] Step 3: placing the silicon wafer with the channel pattern of the microfluidic chip in a cell culture dish, and slowly introducing the polydimethylsiloxane mixture after the bubbles are removed in step 2, and after the polydimethylsiloxane mixture completely and evenly covers the surface of the silicon wafer, placing it in a vacuum drying dish again to evacuate and remove the bubbles between the silicon wafer and the bottom of the cell culture dish;

[0022] Step 4: placing the cell culture dish in step 3 in an oven for curing;

[0023] Step 5: Take out the cell culture dish of step 4, separate the solidified polydimethylsiloxane from the silicon wafer, cut it into a preset shape, cut out a space for placing the magnet, and use a puncher to punch holes to form a first inlet, a first outlet, and a second outlet;

[0024] Step 6: Use transparent tape to remove the pattern surface of the polydimethylsiloxane and the surface of the glass slide in step 5, and put the polydimethylsiloxane and the glass slide into a plasma cleaning machine;

[0025] Step 7: Turn on the vacuum pump connected to the plasma cleaning machine in step 6. When the pressure in the chamber of the plasma cleaning machine drops to a preset pressure, stop evacuating and turn on the glow. When a purple-red glow appears in the vacuum chamber, start timing. After the preset time, stop the glow and take out the two.

[0026] Step 8: The patterned surface of the polydimethylsiloxane in step 7 is tightly attached to the glass slide, and the bubbles between the surfaces are gently pressed to be removed, and then the surface is placed on a heating table to further strengthen the bonding effect, and a polytetrafluoroethylene capillary is inserted into the punched hole of the microfluidic chip, and a polydimethylsiloxane mixture is poured at the position, and the surface is placed in an oven to strengthen the sealing performance of the inlet and outlet positions;

[0027] Step 9: Embed the magnet into the microfluidic chip.

[0028] Preferably, in step 1, the amounts of polydimethylsiloxane and polydimethylsiloxane curing agent used are 30 g and 3 g respectively.

[0029] Preferably, in step 2, the vacuuming time is 30 minutes to 50 minutes.

[0030] Preferably, in step 3, the vacuuming time is 10 minutes to 20 minutes.

[0031] Preferably, in step 4, the oven temperature is set to 90° C. and the time is 30 to 50 minutes;

[0032] And / or, in step 8, the temperature of the heating stage is set to 85° C., and the time is set to 40 minutes to 50 minutes.

[0033] The present invention also provides a method for using a fluid microfluidic chip, based on the above-mentioned fluid microfluidic chip for sorting non-magnetic microspheres, comprising the following steps:

[0034] Step a: adding a preset percentage of Tween to the nano-ferroferric oxide suspension, placing the suspension in an ultrasonic cleaner and oscillating for a preset time to obtain an iron fluid sample stock solution;

[0035] Step b: dispersing two polystyrene microspheres with different diameters in a PBS solution at a preset concentration to prepare microsphere samples;

[0036] Step c, mixing the iron fluid sample stock solution and the microsphere sample to prepare a sample with a ferroferric oxide concentration of a preset percentage, and placing the sample in an ultrasonic cleaner for oscillation for a preset time;

[0037] Step d, using a syringe pump to inject a mixed sample of the microsphere sample and the ferrofluid sample into the microfluidic chip from the first inlet at a preset flow rate;

[0038] Step e: collecting two types of microspheres with different diameters from the first outlet and the second outlet respectively.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1. The microfluidic chip of the present invention focuses polystyrene microspheres of different sizes in the middle of the channel by introducing an asymmetric serpentine channel, which not only provides a consistent equilibrium position for subsequent sorting, but also reduces the damage caused by the collision between the microspheres and the wall of the microfluidic chip.

[0041] 2. The magnetic field sorting module of the microfluidic chip of the present invention uses the ferrofluidic method to sort microspheres, without the need for magnetic bead labeling, thereby reducing the time and cost associated with labeling. In addition, the module is simple to operate and low in cost, and only a permanent magnet is required to achieve sorting.

[0042] 3. The microfluidic chip of the present invention has a simple structure and a smaller area compared to the traditional spiral microfluidic chip structure, which is conducive to further improving the sorting throughput through parallel design.

[0043] 4. The microfluidic chip of the present invention uses the force of the fluid on the microspheres to achieve sorting, which effectively reduces the probability of direct collision between the microspheres and the microstructure, can reduce the damage to the microspheres during the sorting process, and improve the integrity of the microspheres after sorting. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0045] Figure 1 Schematic diagram of the structure of a sheathless ferrofluidic microfluidic chip for continuously sorting non-magnetic microspheres in an embodiment of the present invention.

[0046] Figure 2 Schematic diagram of velocity flow field and force of microspheres in an asymmetric serpentine channel in an embodiment of the present invention.

[0047] Figure 3-4Schematic diagram of the movement of microspheres in a microfluidic chip channel in an embodiment of the present invention.

[0048] Figure 5 Schematic diagram of the overall structure of the microfluidic chip in an embodiment of the present invention.

[0049] Figure 6 Schematic diagram of the local structure of the asymmetric serpentine channel of the microfluidic chip in an embodiment of the present invention.

[0050] Figure 7 Schematic diagram of the local structure of the microfluidic chip at the second amplification section in an embodiment of the present invention.

[0051] The figure shows:

[0052] Pre-focusing module 1 Second straight channel section 31

[0053] First inlet 11 Magnet 32

[0054] First straight channel section 12 Second enlarged section 33

[0055] Serpentine channel section 13 first outlet 34

[0056] First amplification section 2 Second outlet 35

[0057] Magnetic field sorting module 3 Slide 4 DETAILED DESCRIPTION

[0058] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0059] Embodiment 1:

[0060] like Figure 1-7 As shown, this embodiment provides a fluid microfluidic chip, including: a pre-focusing module 1, a magnetic field sorting module 3 and a glass slide 4; the pre-focusing module 1 and the magnetic field sorting module 3 are arranged on the glass slide 4, and the pre-focusing module 1 is connected to the magnetic field sorting module 3 through the first amplification section 2; the pre-focusing module 1 is used to focus the microspheres at the same equilibrium position, and the magnetic field sorting module 3 is used to sort microspheres of different sizes.

[0061] The magnetic field sorting module 3 includes: a second straight channel section 31, a magnet 32, a second enlargement section 33, a first outlet 34 and a second outlet 35; the other end of the first enlargement section 2 is connected to one end of the second straight channel section 31, the other end of the second straight channel section 31 is connected to one end of the second enlargement section 33, and the first outlet 34 and the second outlet 35 are connected to the other end of the second enlargement section 33; the magnet 32 ​​is located on one side of the second straight channel section 31, and is arranged adjacent to the second straight channel section 31.

[0062] The pre-focusing module 1 includes: a first inlet 11, a first straight channel section 12 and a serpentine channel section 13; the first inlet 11 is connected to one end of the first straight channel section 12, and the other end of the first straight channel section 12 is connected to one end of the serpentine channel section 13; the other end of the serpentine channel section 13 is connected to one end of the first amplifying section 2, and the other end of the first amplifying section 2 is connected to the magnetic field sorting module 3. The serpentine channel section 13 is an asymmetric structure, and the serpentine channel section 13 includes a plurality of semicircles connected in sequence, and two adjacent semicircles are connected by an arc segment, and the diameters of the two adjacent semicircles are different.

[0063] The first enlargement section 2 is a linear expansion, and the lower wall of the channel is enlarged along the 1100 μm length of the channel, and the channel width is expanded from 40 μm to 500 μm, while the position of the upper wall of the channel remains unchanged. In the second enlargement section 33, the upper and lower walls of the channel are symmetrically enlarged along the 530 μm length of the channel, and the channel width is expanded from 500 μm to 1110 μm.

[0064] The serpentine channel is composed of two semicircular structures of different diameters connected alternately, with inner diameters of 100 μm and 60 μm respectively, and a channel width of 40 μm. Figure 2 The channels shown are called a group of serpentine channels, and the pre-focusing module 1 is composed of 45 groups of the same serpentine channels.

[0065] The first amplification section 2 is a linear expansion.

[0066] This embodiment also provides a method for preparing a fluid microfluidic chip, which is used to prepare the above-mentioned fluid microfluidic chip, comprising the following steps:

[0067] Step 1: Mix polydimethylsiloxane and polydimethylsiloxane curing agent according to a preset mass ratio, and stir with a glass rod for a preset time to fully mix them; the amounts of polydimethylsiloxane and polydimethylsiloxane curing agent are 30g and 3g respectively;

[0068] Step 2: placing the polydimethylsiloxane mixed solution in step 1 in a vacuum drying dish, and removing bubbles in the polydimethylsiloxane mixed solution by vacuuming; the vacuuming time is 30 minutes to 50 minutes;

[0069] Step 3: Place the silicon wafer with the channel pattern of the microfluidic chip in a cell culture dish, and slowly introduce the polydimethylsiloxane mixture after the bubbles are removed in step 2. After the polydimethylsiloxane mixture completely and evenly covers the surface of the silicon wafer, place it in a vacuum drying dish again to evacuate and remove the bubbles between the silicon wafer and the bottom of the cell culture dish; the evacuation time is 10 minutes to 20 minutes;

[0070] The silicon wafer with the microfluidic chip channel pattern has a first straight channel section 12, a serpentine channel section 13, a first enlarged section 2, a second straight channel section 31, and a second enlarged section 33. The pattern is formed on the surface of the silicon wafer by photolithography. The chip with the above structure is formed by pouring polydimethylsiloxane on the silicon wafer and curing it.

[0071] Step 4: Place the cell culture dish in step 3 in an oven for curing; the oven temperature is set to 90°C for 30 to 50 minutes;

[0072] Step 5: Take out the cell culture dish from step 4, separate the solidified polydimethylsiloxane from the silicon wafer, cut it into a preset shape, cut out a space for placing the magnet, and use a puncher to punch holes to form a first inlet 11, a first outlet 34, and a second outlet 35;

[0073] Step 6: Use transparent tape to remove the pattern surface of the polydimethylsiloxane and the surface of the glass slide 4 in step 5, and put the polydimethylsiloxane and the glass slide 4 into a plasma cleaning machine;

[0074] Step 7: Turn on the vacuum pump connected to the plasma cleaning machine in step 6. When the pressure in the chamber of the plasma cleaning machine drops to the preset pressure, stop evacuating and turn on the glow. When a purple-red glow appears in the vacuum chamber, start timing. After the preset time, stop the glow and take out the two.

[0075] Step 8: The patterned surface of polydimethylsiloxane in step 7 is tightly attached to the glass slide 4, and the bubbles between the surfaces are gently pressed to remove them, and then placed on a heating table to further strengthen the bonding effect. The polytetrafluoroethylene capillary is inserted into the punched hole of the microfluidic chip, and the polydimethylsiloxane mixture is poured at this position, and then placed in an oven to strengthen the sealing performance of the inlet and outlet positions; the temperature of the heating table is set to 85°C, and the time is 40 minutes to 50 minutes;

[0076] Step 9: Embed the magnet 32 ​​into the microfluidic chip.

[0077] This embodiment also provides a method for using a fluid microfluidic chip, based on the above-mentioned fluid microfluidic chip, comprising the following steps:

[0078] Step a: adding a preset percentage of Tween to the nano-ferroferric oxide suspension, placing the suspension in an ultrasonic cleaner and oscillating for a preset time to obtain an iron fluid sample stock solution;

[0079] Step b: dispersing two polystyrene microspheres with different diameters in a PBS solution at a preset concentration to prepare microsphere samples;

[0080] Step c, mixing the iron fluid sample stock solution and the microsphere sample to prepare a sample with a ferroferric oxide concentration of a preset percentage, and placing the sample in an ultrasonic cleaner for oscillation for a preset time;

[0081] Step d, using a syringe pump to inject a mixed sample of the microsphere sample and the ferrofluid sample into the microfluidic chip from the first inlet 11 at a preset flow rate;

[0082] Step e: collecting two types of microspheres with different diameters from the first outlet 34 and the second outlet 35 respectively.

[0083] In this embodiment, the microfluidic chip focuses polystyrene microspheres of different sizes in the middle of the channel by introducing an asymmetric serpentine channel, which not only provides a consistent equilibrium position for subsequent sorting, but also reduces the damage caused by the collision between the microspheres and the wall of the microfluidic chip.

[0084] In this embodiment, the magnetic field sorting module of the microfluidic chip uses ferrofluidic dynamics to sort microspheres, without the need for magnetic bead labeling, thereby reducing the time and cost associated with labeling. In addition, the module is simple to operate and low in cost, and only requires a permanent magnet to achieve sorting.

[0085] In this embodiment, the microfluidic chip has a simple structure and a smaller area compared to the traditional spiral microfluidic chip structure, which is conducive to further improving the sorting throughput through parallel design.

[0086] In this embodiment, the microfluidic chip uses the force of the fluid on the microspheres to achieve sorting, which effectively reduces the probability of direct collision between the microspheres and the microstructure, can reduce the damage to the microspheres during the sorting process, and improve the integrity of the microspheres after sorting.

[0087] Embodiment 2:

[0088] Those skilled in the art may understand this embodiment as a more specific description of Embodiment 1.

[0089] The present embodiment provides a cascaded ferrofluidic microfluidic chip, which includes three parts: an asymmetric serpentine channel section 13, a pre-focusing module 1, a magnetic field sorting module 3 and a glass slide 4. The pre-focusing module 1 is composed of a first inlet 1, a first straight channel section 2 and an asymmetric serpentine channel section 13. The magnetic field sorting module 3 is composed of a second straight channel section 31, a neodymium iron boron permanent magnet 32, a second amplifying section 33, a first outlet 34 and a second outlet 35. The pre-focusing module 1 and the magnetic field sorting module 3 are cascaded in series using the first amplifying section 2.

[0090] The channel height in the microfluidic chip is 80 μm, the length and width of the first straight channel section 2 are 450 μm and 40 μm respectively, the inner diameter of the asymmetric serpentine channel section 13 is 100 μm and 60 μm respectively, the width is 40 μm, and there are 45 groups in total, the length and width of the second straight channel section 31 are 20 mm and 500 μm respectively, the first enlarged section 2 is linearly enlarged for connection, and the length and width of the second enlarged section 33 are 1060 μm and 1110 μm respectively. The radius of the first inlet 1 is 1.2 mm, and the radius of the two outlets is 1 mm.

[0091] The ferrofluid at the first inlet 1 is a suspension of ferroferric oxide nano-magnetic beads, the diameter of the magnetic beads is 10nm-20nm, the concentration of the suspension is 1.2% w / v, and the flow rate of the first inlet 1 is 210 μL / min.

[0092] This embodiment provides a microfluidic chip that can sort micron-sized particles without sheath fluid and with simple operation, provides technical support for the sorting of micron-sized biological particles such as CTCs, and is a microfluidic chip that can continuously sort non-magnetic microspheres of different sizes without sheath.

[0093] The sample composed of ferrofluid and polystyrene microspheres flows into the pre-focusing module 1 from the first inlet 1 and is focused at the same equilibrium position by the asymmetric serpentine channel section 13. After passing through the first amplification section 2, the microspheres enter the second straight channel section 31 of the sorting module. Under the action of the ferrofluid and the magnetic field, the microspheres move in the opposite direction of the magnetic field gradient. After passing through the second amplification section 33, the small-sized microspheres and the large-sized microspheres flow out from the first outlet 34 and the second outlet 35, respectively.

[0094] The microfluidic chip is prepared by using polydimethylsiloxane.

[0095] This embodiment provides a method for preparing the above-mentioned microfluidic chip, comprising the following steps:

[0096] Step 1, mix polydimethylsiloxane and polydimethylsiloxane curing agent in a mass ratio of 10:1, and stir with a glass rod for 8 minutes to 10 minutes to fully mix;

[0097] Step 2, placing the polydimethylsiloxane mixed solution in step 1 in a vacuum drying dish, and removing bubbles in the polydimethylsiloxane mixed solution by vacuuming;

[0098] Step 3, placing the silicon wafer with the channel pattern of the microfluidic chip in a cell culture dish, and slowly introducing the polydimethylsiloxane mixture after the bubbles are removed in step 2, and after the polydimethylsiloxane mixture completely and evenly covers the surface of the silicon wafer, placing it in a vacuum drying dish again to evacuate and remove bubbles between the silicon wafer and the bottom of the cell culture dish;

[0099] Step 4, placing the cell culture dish in step 3 in an oven for curing;

[0100] Step 5, taking out the cell culture dish of step 4, separating the solidified polydimethylsiloxane from the silicon wafer, cutting it into a regular rectangular shape, cutting out a space for placing the magnet 32, and using a puncher to punch holes at the positions of the first inlet 1, the first outlet 34, and the second outlet 35;

[0101] Step 6: Use transparent tape to remove the pattern surface of the polydimethylsiloxane and the surface of the glass slide 4 in step 5, and put the polydimethylsiloxane and the glass slide 4 into a plasma cleaning machine;

[0102] Step 7, turn on the vacuum pump connected to the plasma cleaning machine in step 6, and when the pressure in the plasma cleaning machine chamber drops to 200Pa, stop evacuating and turn on the glow. Start timing when a purple-red glow appears in the vacuum chamber, stop the glow after 60 seconds and take out the two;

[0103] Step 8: The patterned surface of polydimethylsiloxane in step 7 is tightly attached to the glass slide 4, and the bubbles between the surfaces are gently pressed to remove them, and then placed on a heating table to further strengthen the bonding effect. A polytetrafluoroethylene capillary is inserted into the punched hole of the microfluidic chip, and a small amount of polydimethylsiloxane mixed liquid is poured at this position, and then placed in an oven to strengthen the sealing performance of the entrance and exit positions;

[0104] Step 9, embedding the magnet 32 ​​into the microfluidic chip;

[0105] In step 1, the amount of polydimethylsiloxane and polydimethylsiloxane curing agent used is 30g and 3g respectively. In step 2, the vacuuming time is 30 minutes to 50 minutes. In step 3, the vacuuming time is 10 minutes to 20 minutes. In step 4, the oven temperature is set to 90°C for 30 minutes to 50 minutes. In step 8, the temperature of the heating table is set to 85°C for 40 minutes to 50 minutes;

[0106] This embodiment provides a method for separating non-magnetic microspheres of different sizes from a sample using the above-mentioned microfluidic chip, which mainly includes the following steps:

[0107] Step a, adding 0.5% (v / v) Tween 20 to a commercial nano-ferroferric oxide suspension, placing the suspension in an ultrasonic cleaner and vibrating for 5 minutes to obtain a ferrofluid sample stock solution;

[0108] Step b: Polystyrene microspheres with diameters of 5 μm and 15 μm were placed at 2×10 5 -3×10 5 The concentration of each milliliter was dispersed in PBS solution to prepare microsphere samples;

[0109] Step c: Mix the iron fluid sample stock solution and the microsphere sample to prepare a sample with a ferroferric oxide concentration of 1.2% and place it in an ultrasonic cleaner for oscillation for 5 minutes.

[0110] Step d, injecting a mixed sample of the microsphere sample and the ferrofluid sample into the microfluidic chip using a syringe pump, wherein the flow rate of the first inlet 1 is 210 μL / min;

[0111] Step e: collecting 5 μm microspheres and 15 μm microspheres from the first outlet 34 and the second outlet 35 respectively.

[0112] The present embodiment provides a cascaded ferrofluid microfluidic chip capable of continuously and sheathlessly sorting non-magnetic microspheres of different sizes, comprising three parts: an asymmetric serpentine channel section 13, a pre-focusing module 1, a magnetic field sorting module 3, and a glass slide 4. The pre-focusing module 1 is composed of a first inlet 1, a first straight channel section 2, and an asymmetric serpentine channel section 13; the magnetic field sorting module 3 is composed of a second straight channel section 31, a NdFeB permanent magnet 32, a second amplifying section 33, a first outlet 34, and a second outlet 35; the pre-focusing module 1 and the magnetic field sorting module are cascaded in series by using the first amplifying section 2. The sample consisting of ferrofluid and polystyrene microspheres of two sizes flows from the first inlet 1 into the pre-focusing module 1 and is focused at the same equilibrium position by the asymmetric serpentine channel section 13. After passing through the first amplification section 2, the microspheres enter the second straight channel section 31 of the sorting module. Under the action of the ferrofluid and the magnetic field, the microspheres move in the opposite direction of the magnetic field gradient. After passing through the second amplification section 33, the small-sized microspheres and the large-sized microspheres flow out from the first outlet 34 and the second outlet 35 respectively.

[0113] This embodiment can realize continuous and sheath-free sorting of microspheres of different sizes, and has the advantages of high throughput, small structure size, no need for magnetic bead modification, and simple operation.

[0114] Embodiment 3:

[0115] Those skilled in the art may understand this embodiment as a more specific description of Embodiment 1.

[0116] like Figure 1 As shown, this embodiment proposes a sheathless ferrofluidic microfluidic chip for continuously sorting non-magnetic microspheres, which mainly includes an asymmetric serpentine channel pre-focusing module 1, a magnetic field sorting module 3 and a glass slide 4. The pre-focusing module 1 is composed of a first inlet 11, a first straight channel section 12 and an asymmetric serpentine channel section 13. The magnetic field sorting module 3 is composed of a second straight channel section 31, a neodymium iron boron permanent magnet 32, a second amplifying section 33, a first outlet 34 and a second outlet 35. The pre-focusing module and the magnetic field sorting module are cascaded in series using the first amplifying section 2.

[0117] A sample of a mixture of polystyrene microspheres with diameters of 5 μm and 15 μm and ferrofluid was injected into the microfluidic chip from the first inlet 11 by using a syringe pump. The flow rate at the first inlet 11 was 210 μL / min.

[0118] like Figure 2 As shown, in the curved channel of the asymmetric serpentine channel section 13, the microspheres are mainly affected by the inertial lift force F generated by the inertial migration effect. L ~a p 4 / D h 2 , and the Dean drag F caused by the cross-sectional Dean flow D ~a p D h 2 , where a p is the microsphere diameter, D h =2hw / (h+w), h and w are the height and width of the channel respectively. Inertial lift F L Usually, the microspheres migrate to the equilibrium position near the center of the long channel wall, and the Dean resistance F D Usually it accelerates the migration of microspheres and changes the original equilibrium position in the linear channel. Figure 3 As shown, the microspheres are randomly distributed when entering the first straight channel section 12 from the first inlet 11, and then migrate toward the direction perpendicular to the center plane due to the repeated rotation of the Dean flow and inertial migration in the asymmetric serpentine channel section 13. After reaching the vertical center plane, the Dean resistance F generated by the channel D Sufficient to overcome the inertial lift F L , pushing the microsphere to the equilibrium position near the outer curved wall.

[0119] like Figure 4As shown, after the sample passes through the pre-focusing module 1 and the first amplification section 2, it enters the second straight channel section 31 of the magnetic field sorting module 3. Under the action of the magnetic field, the nano-iron tetroxide particles in the sample move in the direction of the magnetic field gradient, and exert negative magnetic force on the microspheres while colliding with them, causing them to move in the opposite direction of the magnetic field gradient. Therefore, the main force on the microspheres during movement is the negative magnetic force F m ~a p 3 and Stokes drag force F d ~a p . Since the negative magnetic force is related to the diameter of the microsphere in cubic terms, the negative magnetic force on the large microsphere is much greater than that on the small microsphere, and the first amplification section 2 reduces the fluid velocity in the second straight channel section 31, so the negative magnetic force on the large microsphere can overcome the Stokes drag force, and the large microsphere will be repelled to a position close to the lower wall of the channel more quickly, resulting in a lateral position difference with the small microsphere. This difference further increases after entering the second amplification section 33, so the microsphere with a diameter of 5μm enters the first outlet 34, and the microsphere with a diameter of 15μm enters the second outlet 35, so that the two sizes of microspheres are separated.

[0120] like Figure 5 – Figure 7 As shown, the length and width of the first straight channel section 12 are 450μm and 40μm respectively, the inner diameter of the asymmetric serpentine channel section 13 is 100μm and 60μm respectively, and the width is 40μm, the length and width of the second straight channel section 31 are 20mm and 500μm respectively, the first enlargement section 2 is a linear expansion for connection, the length and width of the second enlargement section 33 are 1060μm and 1110μm respectively, and the width of the outlet connecting channel is 300μm.

[0121] Working principle:

[0122] After the microspheres with diameters of 5 μm and 15 μm and the iron fluid mixed sample enter the microfluidic chip from the first inlet 11, the microspheres are focused at the same equilibrium position in the asymmetric serpentine channel section 13. After entering the first amplification section 2, the microspheres enter the second straight channel section 31 at a slower flow rate. In the second straight channel section 31, under the action of the magnetic field and the negative magnetic force of the ferroferric oxide nanoparticles, the two types of microspheres have a difference in lateral position, and the microspheres with a diameter of 15 μm move to a position close to the lower wall of the channel. This difference is further amplified in the second amplification section 33, so that the microspheres with a diameter of 5 μm flow out from the first outlet 34 and the microspheres with a diameter of 15 μm flow out from the second outlet 35.

[0123] The present invention can realize continuous and sheath-free sorting of microspheres of different sizes, and has the advantages of high throughput, small structure size, no need for magnetic bead modification, and simple operation.

[0124] In the description of the present application, it should be understood that the terms "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0125] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A fluid microfluidic chip, characterized in that: include: A pre-focusing module (1), a magnetic field sorting module (3) and a glass slide (4); The pre-focusing module (1) and the magnetic field sorting module (3) are arranged on the glass slide (4), and the pre-focusing module (1) is connected to the magnetic field sorting module (3) via a first amplification section (2); The pre-focusing module (1) is used to focus the microspheres at the same equilibrium position, and the magnetic field sorting module (3) is used to sort microspheres of different sizes.

2. The fluid microfluidic chip according to claim 1, characterized in that: The pre-focusing module (1) comprises: a first inlet (11), a first straight channel section (12) and a serpentine channel section (13); The first inlet (11) is connected to one end of the first straight channel section (12), and the other end of the first straight channel section (12) is connected to one end of the serpentine channel section (13); The other end of the serpentine channel section (13) is connected to one end of the first amplifying section (2), and the other end of the first amplifying section (2) is connected to the magnetic field sorting module (3).

3. The fluid microfluidic chip according to claim 2, characterized in that: The serpentine channel section (13) is an asymmetric structure. The serpentine channel section (13) comprises a plurality of semicircles connected in sequence, two adjacent semicircles are connected by an arc segment, and the diameters of the two adjacent semicircles are different.

4. The fluid microfluidic chip according to claim 2, characterized in that: The magnetic field sorting module (3) comprises: a second straight channel section (31), a magnet (32), a second amplifying section (33), a first outlet (34) and a second outlet (35); The other end of the first enlarged section (2) is connected to one end of the second straight channel section (31), the other end of the second straight channel section (31) is connected to one end of the second enlarged section (33), and the first outlet (34) and the second outlet (35) are connected to the other end of the second enlarged section (33); The magnet (32) is located on one side of the second straight channel section (31), and is arranged adjacent to the second straight channel section (31).

5. A method for preparing a fluid microfluidic chip, characterized in that: The method for preparing the fluid microfluidic chip according to claim 4 comprises the following steps: Step 1: Mix polydimethylsiloxane and polydimethylsiloxane curing agent according to a preset mass ratio, and stir with a glass rod for a preset time to fully mix them; Step 2: placing the polydimethylsiloxane mixed solution in step 1 in a vacuum drying dish, and removing bubbles in the polydimethylsiloxane mixed solution by vacuuming; Step 3: placing the silicon wafer with the channel pattern of the microfluidic chip in a cell culture dish, and slowly introducing the polydimethylsiloxane mixture after the bubbles are removed in step 2, and after the polydimethylsiloxane mixture completely and evenly covers the surface of the silicon wafer, placing it in a vacuum drying dish again to evacuate and remove the bubbles between the silicon wafer and the bottom of the cell culture dish; Step 4: placing the cell culture dish in step 3 in an oven for curing; Step 5: Take out the cell culture dish of step 4, separate the solidified polydimethylsiloxane from the silicon wafer, cut it into a preset shape, cut out a space for placing the magnet, and use a puncher to punch holes to form a first inlet (11), a first outlet (34) and a second outlet (35); Step 6: Use transparent tape to remove the pattern surface of the polydimethylsiloxane and the surface of the glass slide (4) in step 5, and put the polydimethylsiloxane and the glass slide (4) into a plasma cleaning machine; Step 7: Turn on the vacuum pump connected to the plasma cleaning machine in step 6. When the pressure in the chamber of the plasma cleaning machine drops to a preset pressure, stop evacuating and turn on the glow. When a purple-red glow appears in the vacuum chamber, start timing. After the preset time, stop the glow and take out the two. Step 8: The patterned surface of the polydimethylsiloxane in step 7 is tightly attached to the glass slide (4), and after lightly pressing to remove bubbles between the surfaces, the surface is placed on a heating table to further strengthen the bonding effect, a polytetrafluoroethylene capillary is inserted into the punched hole of the microfluidic chip, and a polydimethylsiloxane mixture is poured at the position, and the surface is placed in an oven to strengthen the sealing performance of the inlet and outlet positions; Step 9: Embed the magnet (32) into the microfluidic chip.

6. The method for preparing a fluid microfluidic chip according to claim 5, characterized in that: In the step 1, the amounts of polydimethylsiloxane and polydimethylsiloxane curing agent used are 30 g and 3 g respectively.

7. The method for preparing a fluid microfluidic chip according to claim 5, characterized in that: In the step 2, the vacuuming time is 30 minutes to 50 minutes.

8. The method for preparing a fluid microfluidic chip according to claim 5, characterized in that: In step 3, the vacuuming time is 10 minutes to 20 minutes.

9. The method for preparing a fluid microfluidic chip according to claim 5, characterized in that: In step 4, the oven temperature is set to 90° C. and the time is 30 to 50 minutes; And / or, in step 8, the temperature of the heating stage is set to 85° C., and the time is set to 40 minutes to 50 minutes.

10. A method for using a fluid microfluidic chip, characterized in that: The fluid microfluidic chip according to claim 4 comprises the following steps: Step a: adding a preset percentage of Tween to the nano-ferroferric oxide suspension, placing the suspension in an ultrasonic cleaner and oscillating for a preset time to obtain an iron fluid sample stock solution; Step b: dispersing two polystyrene microspheres with different diameters in a PBS solution at a preset concentration to prepare microsphere samples; Step c, mixing the iron fluid sample stock solution and the microsphere sample to prepare a sample with a ferroferric oxide concentration of a preset percentage, and placing the sample in an ultrasonic cleaner for oscillation for a preset time; Step d, using a syringe pump to inject a mixed sample of the microsphere sample and the ferrofluid sample into the microfluidic chip from the first inlet (11) at a preset flow rate; Step e: collecting two types of microspheres with different diameters from the first outlet (34) and the second outlet (35) respectively.

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