Fluidic microfluidic chips and methods of making and using the same

Abstract

The application provides a fluid micro-fluidic chip and a preparation and use method thereof, which comprises a pre-focusing module, a magnetic field sorting 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 microspheres at the same equilibrium position, and the magnetic field sorting module is used for sorting microspheres with different sizes. The application can realize continuous and sheath-free sorting of microspheres with different sizes, and has the advantages of high throughput, small structure size, no need for magnetic bead modification and simple operation.

Description

Technical Field

[0001] This invention relates to the field of CTC high-throughput sorting technology, specifically to a fluid microfluidic chip and its preparation and use methods, particularly a sheathless iron fluid microfluidic chip for continuous sorting of non-magnetic microspheres. Background Technology

[0002] Cancer is the leading cause of the global disease burden. According to the International Agency for Research on Cancer (IARC) of the World Health Organization, in 2022, there were 20 million new cancer cases and 9.7 million cancer deaths worldwide, with approximately 90% of these deaths due to metastasis. Providing timely treatment in the early stages of cancer metastasis can reduce cancer deaths by about 30%. Therefore, researching the mechanisms of cancer metastasis and developing early precision diagnosis and treatment technologies are crucial for humanity's future fight against cancer. However, in the early stages of cancer metastasis, the tumor burden is extremely low, making it difficult to locate metastatic lesions using traditional imaging methods and to extract tumor cells through biopsy. This makes it difficult to detect tumor development and metastasis, significantly limiting molecular diagnosis and precision treatment of early metastatic tumors.

[0003] Circulating tumor cells (CTCs) are tumor cells that detach from primary or metastatic tumor lesions and enter the peripheral blood circulation through epithelial-mesenchymal transition. Numerous studies have shown that CTCs exist in the peripheral blood even during low tumor burden stages and can express biological genetic information from the genome to protein function. Therefore, CTC liquid biopsy technology can overcome the limitation of tissue biopsy being only applicable to solid tumor tissues detectable by imaging. Furthermore, with its non-invasive, dynamic monitoring capabilities and ability to provide personalized medical care, it has a broader application prospect in early cancer diagnosis and treatment guidance.

[0004] However, high-throughput and precise separation of central cytokines (CTCs) remains a significant challenge due to interference from the high abundance of blood cells in peripheral blood. Active manipulator cell sorting technologies, relying on external forces, typically require complex instruments and additional manual intervention to complete CTC sorting. Some active manipulator techniques also require magnetic bead modification of cells. Therefore, active manipulator cell sorting methods generally suffer from high technical costs, difficulty in miniaturization and modularization, and operational complexity. Existing passive manipulator cell sorting technologies avoid the disadvantages of active manipulator methods, but they usually require sheath flow assistance, reducing sorting throughput and operational portability. Furthermore, passive sorting technologies also suffer from high cell shear stress and severe clogging.

[0005] Patent document CN109550531A discloses a magnetic size-dependent microfluidic chip, including a cover plate and a substrate. The cover plate is located directly above the substrate, and the cover plate and the substrate are sealed together. A magnetophoretic separation structure and size sorting channels for capturing magnetic microspheres of different sizes are formed on the lower surface of the cover plate. The magnetophoretic separation structure includes a buffer flow channel, a sample flow channel, a confluence channel, a capture channel, and a waste discharge channel. The buffer flow channel and the sample flow channel converge at one end of the confluence channel, and the waste discharge channel and the capture channel converge at the other end of the confluence channel. The confluence channel is connected at one end to the inlet of the size sorting channel. A magnet is mounted on the cover plate, positioned above the confluence channel and on one side of the projection of the confluence channel onto the upper surface of the cover plate. The confluence channel extends towards the magnet, while the waste discharge channel extends away from the magnet. The cover plate has a buffer injection port, a sample injection port, a first discharge port, and a second discharge port. The buffer injection port is connected to a buffer movement channel, the sample injection port is connected to a sample movement channel, the first discharge port is connected to the waste discharge channel, and the second discharge port is connected to the size sorting channel outlet. However, this patent document still suffers from problems such as high cell shear stress and severe clogging.

[0006] US Patent No. 20220379312A1 discloses a magnetic separation microfluidic chip, specifically including a substrate, a chip model material layer, microchannel units, and a magnetic separation unit. The chip model material layer is disposed on the substrate, and the microchannel units and magnetic separation units are both disposed within the chip model material layer. The microchannel units include separation channels and magnetic pole channels; the separation channels have multiple separation channel inlets and multiple separation channel outlets; the magnetic separation unit includes a permanent magnet, a high-permeability alloy, and a magnetic pole array disposed in the magnetic pole channels. The high-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 magnetic fields of opposite polarities at the left and right positions of the separation channels. However, this patent still has problems such as high cell shear stress and severe clogging. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide a fluid microfluidic chip and its preparation and usage methods.

[0008] A fluid microfluidic chip for sorting non-magnetic microspheres according to the present invention includes: 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 disposed on the glass slide, and the pre-focusing module is connected to the magnetic field sorting module through a first magnification 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 microspheres of different sizes.

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

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

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

[0014] Preferably, the serpentine channel segment has an asymmetrical structure, comprising a plurality of semicircles connected in sequence, with adjacent semicircles connected by an arc segment, and the diameters of adjacent semicircles being different.

[0015] Preferably, the magnetic field sorting module includes: 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 segment is connected to one end of the second straight channel segment, the other end of the second straight channel segment is connected to one end of the second amplifying segment, and the first outlet and the second outlet are connected to the other end of the second amplifying segment;

[0017] The magnet is located on one side of the second straight channel segment, adjacent to the second straight channel segment.

[0018] This invention also provides a method for fabricating a fluid microfluidic chip, 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 the preset mass ratio, and stir with a glass rod for a preset time to ensure that they are fully mixed;

[0020] Step 2: Place the polydimethylsiloxane mixture from Step 1 in a vacuum desiccator and remove air bubbles from the polydimethylsiloxane mixture by vacuuming.

[0021] Step 3: Place the silicon wafer with the microfluidic chip channel pattern into the cell culture dish, and slowly pour in the polydimethylsiloxane mixture after removing the air bubbles 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 remove the air bubbles between the silicon wafer and the bottom of the cell culture dish.

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

[0023] 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 to place the magnet, and use a hole punch to make holes to form a first inlet, a first outlet and a second outlet.

[0024] Step 6: Use transparent tape to remove the patterned surface of the polydimethylsiloxane and the surface of the glass slide from Step 5, and put the polydimethylsiloxane and the glass slide together into a plasma cleaner.

[0025] Step 7: Turn on the vacuum pump connected to the plasma cleaner in Step 6. When the pressure inside the plasma cleaner chamber drops to the preset pressure, stop evacuating and turn on the glow discharge. Start timing when a purple-red glow appears in the vacuum chamber. After the preset time, stop the glow discharge and remove both items.

[0026] Step 8: The patterned surface of polydimethylsiloxane from step 7 is tightly bonded to the glass slide. After gently pressing to remove air bubbles between the surfaces, it is placed on a heating table to further enhance the bonding effect. A polytetrafluoroethylene capillary is inserted into the perforated part of the microfluidic chip, and a polydimethylsiloxane mixture is poured into the position. The chip is then placed in an oven to enhance the sealing performance at 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 are 30g and 3g, respectively.

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

[0030] Preferably, in step 3, the vacuuming time is 10 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 table is set to 85°C and the time is 40 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: Add a preset percentage of Tween to the nano-Fe3O4 suspension, place the suspension in an ultrasonic cleaner and vibrate for a preset time to obtain the iron fluid sample stock solution;

[0035] Step b: Disperse two types of polystyrene microspheres with different diameters in PBS solution at a preset concentration to obtain microsphere samples;

[0036] Step c: Mix the iron fluid sample stock solution and the microsphere sample to prepare a sample with a ferric oxide concentration of a preset percentage, and place it in an ultrasonic cleaner and vibrate for a preset time;

[0037] Step d: Use an injection pump to mix the microsphere sample and the ferrofluid sample. Inject the sample into the microfluidic chip through the first inlet at a preset flow rate.

[0038] Step e: Collect 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 balance position for subsequent sorting, but also reduces the damage caused by 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 utilizes ferrohydrodynamics to sort microspheres, eliminating the need for magnetic bead marking and thus reducing the time and cost associated with marking. Furthermore, this module is simple to operate and inexpensive, requiring only a permanent magnet for sorting.

[0042] 3. The microfluidic chip structure of the present invention is simple and has a smaller area compared with 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 fluid acting on microspheres to achieve sorting, which effectively reduces the probability of microspheres directly colliding with microstructures, reduces damage to microspheres during the sorting process, and improves the integrity of microspheres after sorting. Attached Figure Description

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

[0045] Figure 1 This is a schematic diagram of the sheathless iron fluid microfluidic chip structure for continuously sorting non-magnetic microspheres in an embodiment of the present invention.

[0046] Figure 2 This is a schematic diagram of the velocity flow field and force of the microsphere in the asymmetric serpentine channel in an embodiment of the present invention.

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

[0048] Figure 5 This is a schematic diagram of the overall structure of the microfluidic chip in an embodiment of the present invention.

[0049] Figure 6 This is a schematic diagram of a partial structure of the microfluidic chip in an asymmetric serpentine channel in an embodiment of the present invention.

[0050] Figure 7 This is a partial structural diagram of the microfluidic chip in the second amplification section in an embodiment of the present invention.

[0051] The diagram shows:

[0052] Prefocusing module 1 Second straight channel segment 31

[0053] First entrance 11, magnet 32

[0054] First straight channel segment 12; Second enlarged segment 33

[0055] Serpentine tunnel section 13, first exit 34

[0056] First enlarged section 2, second exit 35

[0057] Magnetic field sorting module 3 Glass slide 4 Detailed Implementation

[0058] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0059] Example 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 disposed on the glass slide 4, and the pre-focusing module 1 is connected to the magnetic field sorting module 3 through a first amplification section 2; the pre-focusing module 1 is used to focus 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 amplification section 33, a first outlet 34, and a second outlet 35; the other end of the first amplification section 32 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 amplification section 33, and the first outlet 34 and the second outlet 35 are connected to the other end of the second amplification section 33; the magnet 32 ​​is located on one side of the second straight channel section 31 and is disposed adjacent to the second straight channel section 31.

[0062] The pre-focusing module 1 includes: a first inlet 11, a first straight channel segment 12, and a serpentine channel segment 13; the first inlet 11 is connected to one end of the first straight channel segment 12, and the other end of the first straight channel segment 12 is connected to one end of the serpentine channel segment 13; the other end of the serpentine channel segment 13 is connected to one end of the first amplification segment 2, and the other end of the first amplification segment 2 is connected to the magnetic field sorting module 3. The serpentine channel segment 13 has an asymmetrical structure, comprising multiple semicircles connected in sequence, with adjacent semicircles connected by arc segments, and the diameters of adjacent semicircles being different.

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

[0064] The serpentine channel consists of alternating semicircular structures of two different diameters, with inner diameters of 100 μm and 60 μm respectively, and a channel width of 40 μm. For example... Figure 2 The channel shown is called a set of serpentine channels. The prefocus module 1 is composed of 45 repeated sets of the same serpentine channels connected together.

[0065] The first enlarged segment 2 is a linear enlargement.

[0066] This embodiment also provides a method for fabricating a fluid microfluidic chip, which includes the following steps:

[0067] Step 1: Mix polydimethylsiloxane and polydimethylsiloxane curing agent according to the preset mass ratio, and stir with a glass rod for a preset time to ensure thorough mixing; the amount of polydimethylsiloxane and polydimethylsiloxane curing agent used are 30g and 3g respectively;

[0068] Step 2: Place the polydimethylsiloxane mixture from Step 1 in a vacuum desiccator and remove air bubbles from the polydimethylsiloxane mixture by vacuuming; the vacuuming time is 30 to 50 minutes.

[0069] Step 3: Place the silicon wafer with the microfluidic chip channel pattern into the cell culture dish, and slowly pour in the polydimethylsiloxane mixture after removing the air bubbles 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 and evacuate to remove the air bubbles between the silicon wafer and the bottom of the cell culture dish; the evacuation time is 10 to 20 minutes.

[0070] The silicon wafer with the microfluidic chip channel pattern has a first straight channel segment 12, a serpentine channel segment 13, a first amplified segment 2, a second straight channel segment 31, and a second amplified segment 33. The pattern is formed on the surface of the silicon wafer by photolithography. After polydimethylsiloxane is poured into the silicon wafer and cured, a chip with the above structure is formed.

[0071] Step 4: Place the cell culture dish from Step 3 in an oven for curing; set the oven temperature to 90℃ and the time to 30-50 minutes.

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

[0073] Step 6: Use transparent tape to remove the patterned surface of polydimethylsiloxane and the surface of glass slide 4 from step 5, and put polydimethylsiloxane and glass slide 4 together into a plasma cleaner.

[0074] Step 7: Turn on the vacuum pump connected to the plasma cleaner in Step 6. When the pressure inside the plasma cleaner chamber drops to the preset pressure, stop evacuating and turn on the glow discharge. Start timing when a purple-red glow appears in the vacuum chamber. After the preset time, stop the glow discharge and remove both items.

[0075] Step 8: Firmly adhere the patterned surface of the polydimethylsiloxane from Step 7 to the glass slide 4, gently press to remove air bubbles between surfaces, and place it on a heating stage to further enhance the bonding effect. Insert the polytetrafluoroethylene capillary into the perforated area of ​​the microfluidic chip, and pour the polydimethylsiloxane mixture into this position. Place it in an oven to enhance the sealing performance at the inlet and outlet positions. The temperature of the heating stage is set to 85℃, and the time is 40 to 50 minutes.

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

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

[0078] Step a: Add a preset percentage of Tween to the nano-Fe3O4 suspension, place the suspension in an ultrasonic cleaner and vibrate for a preset time to obtain the iron fluid sample stock solution;

[0079] Step b: Disperse two types of polystyrene microspheres with different diameters in PBS solution at a preset concentration to obtain microsphere samples;

[0080] Step c: Mix the iron fluid sample stock solution and the microsphere sample to prepare a sample with a ferric oxide concentration of a preset percentage, and place it in an ultrasonic cleaner and vibrate for a preset time;

[0081] Step d: Use an injection pump to mix the microsphere sample and the iron fluid sample and inject the sample into the microfluidic chip through the first inlet 11 at a preset flow rate;

[0082] Step e: Collect 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 uses an asymmetric serpentine channel to focus polystyrene microspheres of different sizes in the middle of the channel. This not only provides a consistent balance position for subsequent sorting but also reduces damage caused by collisions between the microspheres and the wall of the microfluidic chip.

[0084] In this embodiment, the magnetic field sorting module of the microfluidic chip uses ferrohydrodynamics to sort microspheres, eliminating the need for magnetic bead marking and thus reducing the time and cost associated with marking. Furthermore, this module is simple to operate and inexpensive, requiring only permanent magnets for 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 beneficial for further improving the sorting throughput through parallel design.

[0086] In this embodiment, the microfluidic chip uses the force of fluid acting on the microspheres to achieve sorting, which effectively reduces the probability of microspheres directly colliding with microstructures, reduces damage to microspheres during the sorting process, and improves the integrity of microspheres after sorting.

[0087] Example 2:

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

[0089] This embodiment provides a cascaded ferrofluid microfluidic chip, which includes three parts: an asymmetric serpentine channel segment 13 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 segment 2, and an asymmetric serpentine channel segment 13. The magnetic field sorting module 3 is composed of a second straight channel segment 31, a neodymium iron boron permanent magnet 32, a second amplification segment 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 amplification segment 2.

[0090] The microfluidic chip has a channel height of 80 μm. The length and width of the first straight channel segment 2 are 450 μm and 40 μm, respectively. The inner diameters of the asymmetric serpentine channel segments 13 are 100 μm and 60 μm, and the width is 40 μm, totaling 45 groups. The length and width of the second straight channel segment 31 are 20 mm and 500 μm, respectively. The first amplified segment 2 is a linear expansion used for connection. The length and width of the second amplified segment 33 are 1060 μm and 1110 μm, respectively. The radius of the first inlet 1 is 1.2 mm, and the radii of the two outlets are 1 mm.

[0091] The ferrous fluid at the first inlet 1 is a suspension of magnetite nanoparticles with a diameter of 10 nm-20 nm and a suspension concentration of 1.2% w / v. The flow rate at 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, providing technical support for the sorting of micron-sized biological particles such as CTC, and is a microfluidic chip for continuous sorting of non-magnetic microspheres of different sizes without sheath fluid.

[0093] A sample composed of ferrofluid and polystyrene microspheres flows into the pre-focusing module 1 from the first inlet 1 and is focused to 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, small-sized microspheres and large-sized microspheres flow out from the first outlet 34 and the second outlet 35, respectively.

[0094] The microfluidic chip is prepared using polydimethylsiloxane.

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

[0096] Step 1: Mix polydimethylsiloxane and polydimethylsiloxane curing agent at a mass ratio of 10:1, and stir with a glass rod for 8 to 10 minutes to ensure thorough mixing;

[0097] Step 2: Place the polydimethylsiloxane mixture from Step 1 in a vacuum desiccator and remove air bubbles from the polydimethylsiloxane mixture by vacuuming.

[0098] Step 3: Place the silicon wafer with the microfluidic chip channel pattern into a cell culture dish, and slowly introduce the polydimethylsiloxane mixture after removing the air bubbles 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 remove the air bubbles between the silicon wafer and the bottom of the cell culture dish.

[0099] Step 4: Place the cell culture dish from Step 3 in an oven for curing;

[0100] Step 5: Take out the cell culture dish from Step 4, separate the solidified polydimethylsiloxane from the silicon wafer, cut it into a regular cuboid shape, cut out a space to place the magnet 32, and use a punch 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 patterned surface of polydimethylsiloxane and the surface of glass slide 4 from step 5, and put polydimethylsiloxane and glass slide 4 together into a plasma cleaner.

[0102] Step 7: Turn on the vacuum pump connected to the plasma cleaner as described in Step 6. When the pressure inside the plasma cleaner chamber drops to 200 Pa, stop evacuating and turn on the glow discharge. Start timing when a purplish-red glow appears in the vacuum chamber. Stop the glow discharge after 60 seconds and remove both items.

[0103] Step 8: Firmly adhere the patterned surface of the polydimethylsiloxane from Step 7 to the glass slide 4, gently press to remove air bubbles between surfaces, and then place it on a heating stage to further enhance the bonding effect. Insert the polytetrafluoroethylene capillary into the perforated area of ​​the microfluidic chip, and pour a small amount of polydimethylsiloxane mixture into this position. Place it in an oven to enhance the sealing performance at the inlet and outlet positions.

[0104] Step 9: Embed magnet 32 ​​into the microfluidic chip;

[0105] In step 1, the amounts of polydimethylsiloxane and polydimethylsiloxane curing agent are 30g and 3g respectively. In step 2, the vacuuming time is 30-50 minutes. In step 3, the vacuuming time is 10-20 minutes. In step 4, the oven temperature is set to 90℃ for 30-50 minutes. In step 8, the heating platform temperature is set to 85℃ for 40-50 minutes.

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

[0107] Step a: Add 0.5% (v / v) Tween 20 to the commercial nano-Fe3O4 suspension, place the suspension in an ultrasonic cleaner and vibrate for 5 minutes to obtain the iron fluid sample stock solution;

[0108] Step b: Polystyrene microspheres with diameters of 5 μm and 15 μm are respectively loaded at 2 × 10⁻⁶. 5 -3×10 5 Microspheres were prepared by dispersing them at a concentration of 1 per milliliter in PBS solution.

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

[0110] Step d: The microsphere sample and the iron fluid sample are mixed and injected into the microfluidic chip using an injection pump. The flow rate of the first inlet 1 is 210 μL / min.

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

[0112] This embodiment provides a cascaded ferrofluid microfluidic chip capable of continuously and sheath-less sorting of non-magnetic microspheres of different sizes. It comprises three parts: a pre-focusing module 1 with an asymmetric serpentine channel section 13, a magnetic field sorting module 3, and a glass slide 4. The pre-focusing module 1 consists of a first inlet 1, a first straight channel section 2, and an asymmetric serpentine channel section 13. The magnetic field sorting module 3 consists of a second straight channel section 31, a neodymium iron boron permanent magnet 32, a second amplification 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 using the first amplification section 2. A sample composed of ferrofluid and polystyrene microspheres of two sizes flows into the pre-focusing module 1 from the first inlet 1 and is focused to 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 achieve continuous, sheathless sorting of microspheres of different sizes, and has advantages such as high throughput, small structural size, no need for magnetic bead modification, and simple operation.

[0114] Example 3:

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

[0116] like Figure 1 As shown, this embodiment proposes a sheathless iron fluid microfluidic chip for continuous sorting of non-magnetic microspheres. It mainly includes three parts: an asymmetric serpentine channel pre-focusing module 1, a magnetic field sorting module 3, and a glass slide 4. The pre-focusing module 1 consists of a first inlet 11, a first straight channel segment 12, and an asymmetric serpentine channel segment 13. The magnetic field sorting module 3 consists of a second straight channel segment 31, a neodymium iron boron permanent magnet 32, a second amplification segment 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 amplification segment 2.

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

[0118] like Figure 2 As shown, within the curved channel of the asymmetric serpentine channel segment 13, the microsphere is mainly subjected to inertial lift F generated by the inertial migration effect. L ~a p 4 / D h 2 And the Dean drag F caused by the Dean flow across the cross section D ~a p D h 2 , where a p It is the diameter of the microsphere, D h = 2hw / (h+w), where h and w are the height and width of the channel, respectively. Inertial lift F L This typically causes the microspheres to migrate towards their equilibrium position near the center of the long channel wall, while the Dean resistance F D This typically accelerates the migration of microspheres and alters their original equilibrium position within the straight channel. For example... Figure 3 As shown, the microspheres are randomly distributed when they enter the first straight channel segment 12 from the first inlet 11. Subsequently, they are subjected to the repeatedly rotating Dean flow and inertial migration in the asymmetric serpentine channel segment 13, migrating towards the direction perpendicular to the central plane. Upon reaching the vertical central plane, the Dean resistance F generated by the channel... D Sufficient to overcome inertial lift F L This propels the microspheres to their equilibrium position near the outer curved wall.

[0119] like Figure 4As shown, after passing through the pre-focusing module 1 and the first amplification section 2, the sample enters the second straight channel section 31 of the magnetic field sorting module 3. Under the action of the magnetic field, the nano-iron oxide particles in the sample move in the direction of the magnetic field gradient, and while colliding with the microspheres, a negative magnetic force is applied to them, causing them to move in the opposite direction of the magnetic field gradient. Therefore, the main force experienced by the microspheres during their movement is the negative magnetic force F. m ~a p 3 And Stokes Traction F d ~a p Since the negative magnetic force is cubically related to the microsphere diameter, larger microspheres experience a much greater negative magnetic force than smaller ones. The effect of the first amplification section 2 reduces the fluid velocity in the second straight channel section 31. Therefore, the negative magnetic force on larger microspheres can overcome the Stokes drag force, causing them to be repelled more quickly towards the lower wall of the channel, creating a lateral positional difference with the smaller microspheres. This difference further increases upon entering the second amplification section 33, resulting in 5μm diameter microspheres entering the first outlet 34 and 15μm diameter microspheres entering the second outlet 35, thus separating the two sizes of microspheres.

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

[0121] Working principle:

[0122] After a mixture of 5μm and 15μm diameter microspheres and iron fluid enters the microfluidic chip through the first inlet 11, the microspheres are focused to the same equilibrium position within the asymmetric serpentine channel section 13. Upon entering the first amplification section 2, the microspheres flow at a slower rate into the second straight channel section 31. In the second straight channel section 31, under the influence of a magnetic field and the negative magnetic force of the iron oxide nanoparticles, the two types of microspheres exhibit a difference in lateral position, with the 15μm diameter microspheres moving closer to the lower wall of the channel. This difference is further amplified in the second amplification section 33, causing the 5μm diameter microspheres to exit from the first outlet 34 and the 15μm diameter microspheres to exit from the second outlet 35.

[0123] This invention enables continuous, sheath-free sorting of microspheres of different sizes, and has advantages such as high throughput, small structural size, no need for magnetic bead modification, and simple operation.

[0124] In the description of this application, it should be understood that the terms "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0125] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. A fluidic microfluidic chip, characterized by, include: The pre-focusing module (1), the magnetic field sorting module (3), and the glass slide (4) are included. The pre-focusing module (1) and the magnetic field sorting module (3) are mounted on the glass slide (4), and the pre-focusing module (1) is connected to the magnetic field sorting module (3) through the first magnification 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; The pre-focusing module (1) includes: a first inlet (11), a first straight channel segment (12), and a serpentine channel segment (13). The first entrance (11) is connected to one end of the first straight channel segment (12), and the other end of the first straight channel segment (12) is connected to one end of the serpentine channel segment (13); The other end of the serpentine channel segment (13) is connected to one end of the first amplified segment (2), and the other end of the first amplified segment (2) is connected to the magnetic field sorting module (3); The serpentine channel segment (13) is an asymmetrical structure. The serpentine channel segment (13) includes multiple semicircles connected in sequence. Adjacent semicircles are connected by arc segments, and the diameters of adjacent semicircles are different. The magnetic field sorting module (3) includes: a second straight channel section (31), a magnet (32), a second amplification section (33), a first outlet (34), and a second outlet (35); The other end of the first amplified 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 amplified section (33), and the first outlet (34) and the second outlet (35) are connected to the other end of the second amplified section (33). The first enlarged segment (2) is a linear enlargement, which enlarges the lower wall of the channel along the length of the channel while keeping the position of the upper wall of the channel unchanged; the second enlarged segment (33) is a symmetrical enlargement of the upper and lower walls of the channel along the length of the channel. The magnet (32) is located on one side of the second straight channel segment (31) and is disposed adjacent to the second straight channel segment (31).

2. A method for fabricating a fluidic microfluidic chip, comprising: The method for fabricating the fluid microfluidic chip according to claim 1 includes the following steps: Step 1: Mix polydimethylsiloxane and polydimethylsiloxane curing agent according to the preset mass ratio, and stir with a glass rod for a preset time to ensure that they are fully mixed; Step 2: Place the polydimethylsiloxane mixture from Step 1 in a vacuum desiccator and remove air bubbles from the polydimethylsiloxane mixture by vacuuming. Step 3: Place the silicon wafer with the microfluidic chip channel pattern into the cell culture dish, and slowly pour in the polydimethylsiloxane mixture after removing the air bubbles 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 remove the air bubbles between the silicon wafer and the bottom of the cell culture dish. Step 4: Place the cell culture dish from Step 3 in an oven for curing; 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 to place the magnet, and use a punch to make holes to form a first inlet (11), a first outlet (34) and a second outlet (35). Step 6: Use transparent tape to remove the patterned surface of polydimethylsiloxane and the surface of the glass slide (4) in step 5, and put the polydimethylsiloxane and the glass slide (4) together into a plasma cleaner; Step 7: Turn on the vacuum pump connected to the plasma cleaner in Step 6. When the pressure inside the plasma cleaner chamber drops to the preset pressure, stop evacuating and turn on the glow discharge. Start timing when a purple-red glow appears in the vacuum chamber. After the preset time, stop the glow discharge and remove both items. Step 8: The patterned surface of polydimethylsiloxane in step 7 is tightly attached to the glass slide (4). After gently pressing to remove the air bubbles between the surfaces, it is placed on a heating table to further enhance the bonding effect. The polytetrafluoroethylene capillary is inserted into the hole of the microfluidic chip, and polydimethylsiloxane mixture is poured into the position. The chip is then placed in an oven to enhance the sealing performance of the inlet and outlet positions. Step 9: Embed the magnet (32) into the microfluidic chip.

3. The method of claim 2, wherein the fluidic microfluidic chip is prepared by a method comprising: In step 1, the amounts of polydimethylsiloxane and polydimethylsiloxane curing agent are 30g and 3g, respectively.

4. The method of claim 2, wherein the fluidic microfluidic chip is prepared by a method comprising: In step 2, the vacuuming time is 30 to 50 minutes.

5. The method of claim 2, wherein the fluidic microfluidic chip is prepared by a method comprising: In step 3, the vacuuming time is 10 to 20 minutes.

6. The method of claim 2, wherein the fluidic microfluidic chip is prepared by a method comprising: In step 4, the oven temperature is set to 90℃ and the time is 30 to 50 minutes. And / or, in step 8, the temperature of the heating table is set to 85°C and the time is 40 to 50 minutes.

7. A method of using a fluidic microfluidic chip, comprising: The fluid microfluidic chip according to claim 1 includes the following steps: Step a: Add a preset percentage of Tween to the nano-Fe3O4 suspension, place the suspension in an ultrasonic cleaner and vibrate for a preset time to obtain the iron fluid sample stock solution; Step b: Disperse two types of polystyrene microspheres with different diameters in PBS solution at a preset concentration to obtain microsphere samples; Step c: Mix the iron fluid sample stock solution and the microsphere sample to prepare a sample with a ferric oxide concentration of a preset percentage, and place it in an ultrasonic cleaner and vibrate for a preset time; Step d: Use an injection pump to mix the microsphere sample with the iron fluid sample and inject the sample into the microfluidic chip through the first inlet (11) at a preset flow rate; Step e: Collect two types of microspheres with different diameters from the first outlet (34) and the second outlet (35), respectively.

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