CN207680633U - A kind of centrifugal type microfludic chip for Water-In-Oil drop formation - Google Patents

Abstract

The utility model discloses a centrifugal microfluidic chip for generating water-in-oil droplets. Its chip structure includes a channel layer and a sealing layer. Its microfluidic chip channel structure includes a water phase storage area, a connecting channel, a droplet generation area and a droplet collection area. The operation steps include: (1) adding a certain volume of water phase sample to the water phase area, and adding a certain volume of oil phase to the droplet collection area. (2) Centrifuge to generate droplets, which are automatically collected in the droplet storage area. The utility model can be applied to research and application fields such as biology, chemistry, and medical diagnosis.

Description

A Centrifugal Microfluidic Chip for Water-in-Oil Droplet Generation

technical field

The utility model relates to a centrifugal microfluidic chip for generating water-in-oil droplets, which is applied to the fields of microfluidic theoretical calculation and analysis, biological and chemical detection and analysis, medical diagnosis and the like.

Background technique

Micro-droplets have the advantages of small size, high throughput, uniform size, internal stability, and controllability, and are widely used in biological and chemical detection and analysis, medical diagnosis, theoretical calculation, material synthesis and other fields. Traditional droplet generation is based on a fluid-focusing chip. This droplet generation requires a precision syringe pump to strictly control the flow rate of the channel fluid, which is very difficult to operate. In 1997, M.Nakajima's research group proposed a stepped chip structure for droplet generation. Its working principle is that when the fluid flows through the three-dimensional steps, it will break and generate droplets due to static instability. Although this droplet generation method reduces The influence of the fluid flow rate on the droplet is reduced, but the generation of the droplet is highly dependent on the complex step structure, and the chip processing is difficult. In 2015, Du Wenbin's research group proposed a micro-vibration at the end of the capillary to achieve droplet generation. This droplet generation depends on the flow rate of the aqueous fluid and the frequency of capillary vibration, which is more difficult to control than microfluidic chips. In 2016, Huang Yanyi's research group proposed a method of generating droplets based on capillary arrays combined with inertial force drive. However, the production batches of capillary arrays vary greatly, which limits its application. Based on the unavoidable disadvantages of complex structure or difficult control in the existing droplet generation methods, the utility model designs a centrifugal microfluidic chip with simple structure and easy operation for droplet generation.

Utility model content

The utility model aims at the shortcomings of the existing cross-shaped or T-shaped fluid focusing droplet generation method that the fluid is unstable and requires a precision injection pump to strictly control the fluid flow rate in the channel; the step-shaped droplet generation method chip reported in the literature The structure is complex, and the three-dimensional structure of the microfluidic channel needs to be realized through cumbersome processing steps, and the production cost of the chip is high; and the existing published or published centrifugal droplet generation microfluidic chip is not suitable for applications requiring magnetic particles, microspheres, The combination of single cells and other small particles that are easily affected by centrifugal force is a medium problem, and a centrifugal-based water-in-oil droplet generation chip has been developed.

The technical scheme of the utility model is as follows:

A centrifugal microfluidic chip for generating water-in-oil droplets, characterized in that it includes a channel layer and a sealing layer, and the channel layer includes an aqueous phase storage area, a connecting channel, a droplet generation area, and a droplet collection area, wherein the position of the aqueous phase storage area away from the end of the centrifugal shaft is closer to the centrifugal axis than the position of the droplet storage area near the end of the centrifugal shaft; the position of the droplet storage area near the end of the centrifugal shaft is closer to the centrifugal axis than the position of the droplet generation area;

The connection channel connects the water phase storage area and the droplet generation area, and the droplet generation area connects the droplet collection area and one end of the connection channel; a pressure balance channel is also connected between the water phase storage area and the droplet collection area ; The axis of the opening direction of the droplet generating area and the direction of the centrifugal force form an included angle of 30-150 degrees.

When in use, add a certain volume of oil phase solution to the droplet collection area, add a certain volume of water phase sample to the water phase area, and centrifuge to generate droplets in the droplet generation area, and the droplets are automatically collected in the droplet storage area .

Preferably, the axis of the opening direction of the droplet generating area and the direction of the centrifugal force form an included angle of 45-135 degrees.

Further preferably, the axis of the opening direction of the droplet generating area and the direction of the centrifugal force form an included angle of 80-100 degrees.

In the present invention, the droplet generating area can be in various shapes, such as square. But as a preference, the structure of the droplet generating area is a nozzle-type structure with a narrow inlet and a wide outlet. The angle of inclination of the bell mouth can range from 15 to 165 degrees.

The sealing layer can be provided with an upper sealing layer or a lower sealing layer or both exist simultaneously according to actual needs;

The sealing layer and channel layer are made of silicon, glass, quartz or organic polymer. Organic polymers such as polydimethylsiloxane (polydimethylsiloxane, PDMS), polyurethane, epoxy resin, polymethyl methacrylate (polymethyl methacrylate, PMMA), polycarbonate (polycarbonate, PC), cycloolefin copolymer ( One or more of cycloolefincoplymer, COC), polystyrene (polystyrene, PS), polyethylene (polyethylene, PE), acrylic, rubber, fluoroplastic, etc.

The channel layer includes a main body microfluidic channel for droplet generation, and forms a sealed channel by bonding with the sealing layer;

According to the material requirements of the channel layer, the microfluidic channel can be fabricated by the following methods: pouring method, hot pressing method, photolithographic polymerization method and etching method;

One end of the pressure balance channel is located in the droplet collection area, which is at the same distance from the droplet generating area as the center shaft; the other end is located in the liquid phase storage area, and the distance between the channel and the centrifugal shaft is smaller than the distance between the connecting channel and the centrifugal shaft.

The volume of the aqueous phase storage area is: 0.1μL-1mL, which is used for storage of aqueous phase samples;

The water phase storage area is provided with an opening near the end of the centrifugal shaft, which can be used for injecting the water phase sample solution;

The length of the connecting channel is 1 mm-100 mm, the width is 10 μm-10 mm, and the depth is 1 μm-1 mm, which is used to connect the water phase storage area and the droplet generation area, so that the aqueous phase solution can smoothly flow into the droplet generation area during centrifugation;

The shape of the connecting channel can be the same as the centrifugal direction, and the shape of the channel can also be adjusted according to actual needs;

The droplet generating area is a nozzle-shaped opening with a narrow inlet and a wide outlet. Preferably, the width of the inlet end is 1 μm-200 μm, the width of the outlet end of the nozzle is 10 μm-1 mm, and the depth of the nozzle is 1 μm-200 μm. The droplet storage area is directly connected to the end of the droplet generation area, and its depth is 1 μm-1 mm.

The droplet storage area is characterized in that the droplet storage area should be filled with the oil phase solution before centrifugation to generate droplets; the depth of the droplet collection area is 1 μm-200 μm.

The droplet storage area has an opening near the end of the centrifugal shaft, which can inject or discharge the oil phase solution;

The water phase can be: pure water, aqueous buffer, blood, body fluid, saliva, urine, and cell lysate.

The microfluidic channels in the channel layer are treated with superhydrophobic treatment. The method of superhydrophobicity can be chemical covalent bonding method, physical adsorption method or a combination of the two methods. The reagents used in the chemical covalent method are fluorosilanes such as dimethyloctadecylchlorosilane, octadecyltrichlorosilane, trimethoxypropylsilane, trimethoxypropyltrichlorosilane, 1H, 1H, 2H,2H-Perfluorooctylsilane, 1H,1H,2H,2H-Perfluorooctyldimethylchlorosilane, 1H,1H,2H,2H-Perfluorooctyltrichlorosilane, 1H,1H,2H, 2H-perfluorododecyltrichlorosilane, triperfluoromethylchlorosilane, trimethylchlorosilane. The reagents used in the physical adsorption method are fluoro oils such as EGC-1700, EGC-1720, EGC-1702, EGC-1704, FC-722, FC-724, FC-725, and FC-732.

In the present invention, the specific gravity of the oil phase solution used can be greater than that of the water phase, and can also be smaller than that of the water phase. For example, the oil phase is: fluorine oil (such as HFE-7100, HFE-7200, HFE-7500, HFE-71DA, HFE-71DE, HFE-72DA, HFE-72DE, HFE-71IPA, FC-3251, FC -3252, FC-3255, FC-3275, FC-3280, FC-3283, FC-3284, FC-40, FC-41, FC-43, FC-5311, FC-5312, FC-5320, FC-6003 , FC-6064, FC-6047, FC-70, FC-72, FC-726, FC-75, FC-77, FC-770, FC-8270, FC-84, FC-87, GH135), mineral oil Or silicone oil (such as fluorosilicone, cyclopentasiloxane, aliphatic silicone, oligomeric dimethylsiloxane). To promote the stabilization of the water-in-oil interface, the surfactants that can be used are: perfluorinated surfactants, E2K0660, FS-D, FS-P, Tween and Span.

The main operation steps of the droplet generation method include: (1) adding the water phase to the water phase storage area, filling the oil phase in the droplet storage area; (2) centrifuging, and automatically capturing and storing in the droplet storage area.

The utility model uses medium and low-speed centrifugation as the driving force for the generation of droplets, and the size of the droplets has nothing to do with the centrifugal speed within a certain range, thereby ensuring the uniformity and stability of the droplets generated by the chip. At the same time, through the ingenious design of the structure of the microfluidic channel, the method realizes the stable and high-speed generation of droplets with a single-nozzle two-dimensional microfluidic channel, and overcomes the problems of complex structure and high production cost of the stepped droplet generation method.

The advantages of the utility model for generating liquid droplets are: 1. The generation is fast and convenient, without strict control of a high-precision sampling pump. 2. The droplet generation speed is fast. 3. The droplet size is uniform. 4 The sample volume is small, only 2ul sample can be used to generate droplets. 5. The sample has no dead volume, and almost all aqueous solutions can generate uniform-sized droplets. 6. Due to the design of a certain angle between the droplet generation area and the centrifugal force direction, granular substances such as magnetic beads, microspheres, and cells are allowed to exist in the solution. Compared with other centrifugal microfluidic chips, the utility model can adapt to the analysis and application of more single molecule and single cell levels. 7. The design on the two-dimensional scale of the droplet generation area significantly reduces the cost of chip production. 8. In the prior art, the oil phase used must be denser than the water phase, but in the utility model, not only the oil phase with a density greater than the water phase can be used, but also the oil phase with a density smaller than the water phase can be used, which expands the application range.

Description of drawings

Below in conjunction with accompanying drawing and embodiment the utility model is further described.

FIG. 1 shows a schematic diagram of the channel structure of the chip in Embodiment 1.

FIG. 2 is a schematic diagram of the structure of the droplet generation region in Embodiment 1. FIG.

FIG. 3 is a schematic diagram showing the structure of the PDMS chip in Embodiment 1.

Fig. 4 depicts a schematic structural view of the PMMA chip of Embodiment 2.

Fig. 5 is a schematic diagram of the structure of the glass chip in Example 3.

FIG. 6 is a schematic diagram of the chip channel structure of Embodiment 4.

Fig. 7 is the result figure of embodiment 1;

Fig. 8 is the result figure of embodiment 2

Fig. 9 is the result figure of embodiment 3

Fig. 10 is the result figure of embodiment 4

1. Aqueous phase storage area

2. Connection channel

3. Droplet generation zone

4. Droplet storage area

5. Air pressure balance channel

6. Nozzle-shaped channel

7. Aqueous phase inlet

8. Oil phase injection port

9. Upper sealing layer (glass)

10. Flatten PDMS film (channel layer, PDMS)

11. PDMS film containing channels (channel layer, PDMS)

12. Lower sealing layer (glass)

13. Upper sealing layer (PMMA)

14. Lower sealing layer (PMMA)

15. Upper sealing layer (glass)

16. Channel layer (glass)

Detailed ways

Example 1 Fabrication and Application of PDMS Material Microfluidic Chip

1 to 3, the chip structure includes an upper sealing layer 9, a lower sealing layer 12, and channel layers 10 and 11 between the upper and lower sealing layers. The channel structure includes an aqueous phase storage area 1, a connecting channel 2, and a liquid droplet. Generation zone 3 , droplet collection zone 4 (droplet storage zone) and air pressure equalization channel 5 .

In this embodiment, the upper sealing layer 9 and the lower sealing layer 12 are both rectangular glass.

Both channel layers 10 and 11 are made of PDMS, and the channel layer 10 is provided with a water phase storage area 1 on the back near one end (near the upper end in FIG. 1 ), and a droplet storage area 4 is provided on the back near the lower end. The zone 1 is extended downwards with a connecting channel 2, and the connecting channel 2 is vertically downward. The end of the connecting channel 2 is connected to the droplet generating area 3 , the right end thereof is connected to the connecting channel 2 laterally, and the left end is connected to the droplet storage area 4 .

In this embodiment, the channel layer 11 is in a planar shape. In other embodiments, the channel layer 11 may also have a storage area on the upper surface.

The air pressure balance channel 5 is connected to the water phase storage area 1 at one end and to the droplet storage area 4 at the other end. In this embodiment, its connection position with the water phase storage area 1 is the uppermost end on the left, and its connection position with the droplet storage area 4 is the other side at the same height as the droplet generation area 3 .

Preferably, the volume of the aqueous phase storage area 1 is 0.1 μL-1 mL, which is used for storage of aqueous phase samples.

In this embodiment, the end of the centrifugal shaft is the upper end in FIG. 1 . As a preference, the position of the aqueous phase storage area 1 away from the end of the centrifugal shaft should be closer to the centrifugal axis than the position of the droplet storage area 4 near the end of the centrifugal shaft;

The water phase storage area 1 is provided with an opening near the end of the centrifugal shaft, which can be used for injecting the water phase sample solution;

The size of the connecting channel preferably has a length of 1 mm-100 mm, a width of 10 μm-10 mm, and a depth of 1 μm-1 mm, which are used to connect the aqueous phase storage area and the droplet generation area, so that the aqueous phase solution can smoothly flow into the droplet generation during centrifugation Area;

The shape of the centrifugal channel can be the same as the centrifugal direction, and the shape of the channel can also be adjusted according to actual needs;

The droplet generating area 3 is preferably a nozzle-shaped opening with a narrow inlet and a wide outlet. The width of the inlet end is preferably 1 μm-200 μm, the width of the outlet end of the nozzle is 10 μm-1 mm, and the depth of the nozzle is 1 μm-200 μm. The droplet storage area is directly connected to the end of the droplet generation area. The depth of the droplet storage area is preferably 1 μm-1 mm.

The position of the droplet storage area near the end of the centrifugal shaft should be closer to the centrifugal axis than the position of the droplet generation area;

The droplet storage area should be filled with oil phase solution before centrifugation to generate droplets;

The droplet storage area has an opening near the end of the centrifugal shaft, which can inject or discharge the oil phase solution;

The water phase is: pure water or water phase buffer.

In this embodiment, the specific gravity of the oil phase is greater than that of water. Preferably, the oil phase is: fluorine oil (such as HFE-7100, HFE-7200, HFE-7500, HFE-71DA, HFE-71DE, HFE-72DA, HFE-72DE, HFE-71IPA, FC-3251, FC-3252, FC-3255, FC-3275, FC-3280, FC-3283, FC-3284, FC-40, FC-41, FC-43, FC-5311, FC-5312, FC-5320, FC- 6003, FC-6064, FC-6047, FC-70, FC-72, FC-726, FC-75, FC-77, FC-770, FC-8270, FC-84, FC-87, GH135), mineral Oils or silicone oils (such as fluorosilicone, cyclopentasiloxane, aliphatic silicone, oligodimethylsiloxane). To promote the stabilization of the water-in-oil interface, the surfactants that can be used are: E2K0660, FS-D, FS-P, Tween and Span.

After each layer of chip is prepared, 1) use a punching machine to drill the sampling hole of the water phase storage area and the oil phase sampling hole of the droplet storage area at the designated position of the upper sealing layer 9 . 2) Ultrasonic cleaning of the upper sealing layer and the lower sealing layer (glass material) to remove surface impurities. 3) After drying at 135 degrees for 1 hour, align the perforated position of the channel layer with the perforated position of the upper sealing layer and then bond them together to complete the covalent bonding, and then bond the lower sealing layer to complete the fabrication of the chip structure. 4) Inject 1720 perfluorinated solvent into the channel layer, and dry at 75°C to complete the perfluorinated treatment of the channel layer.

4. Droplet Generation

1) Fill the droplet storage area with HFE 7500 perfluorinated oil containing 2% E2K0660 through a pipette gun, and add 10 μL of PCR Mix to the aqueous phase storage area. 2) Centrifuge at 2800r/min for 60s to generate droplets and store them in the oil phase storage area.

The results are shown in Figure 7.

Implement 2 PMMA chips

The chip of this embodiment is shown in FIG. 4 , and its basic structure is the same as that of Embodiment 1. The difference is that in this embodiment, the chip only has two upper and lower layers. Comprising an upper sealing layer 13 (PMMA material) positioned at the upper layer and a lower sealing layer 14 (PMMA material) positioned at the lower layer, wherein the lower sealing layer 14 is provided with a microfluidic channel structure on the upper surface (also includes an aqueous phase storage area, a connecting channel, a liquid Droplet generation area, droplet collection area, etc.).

The specific chip manufacturing steps are as follows:

1. Channel layer heat embossing

The 1mm thick PMMA plate is hot-stamped with an electroformed copper template with a channel group. The stamping conditions are: 140 degrees, 600ppsi pressure, 3min hot stamping, 95 degrees demoulding, and the anti-adhesive layer of the template is 1720 perfluorinated solvent.

2. Thermal bonding of sealing layer and channel layer

Hot press at 105°C and 2Mpa for 3 minutes, and cool naturally to room temperature to complete the thermal bonding of the sealing layer and the channel layer.

3. Channel surface perfluorinated treatment

Inject 1720 perfluorinated solvent into the channel layer, dry at 75°C, and repeat 3 times to complete the perfluorinated treatment of the channel layer.

4. Droplet Generation

1) Fill the droplet storage area with HFE 7500 perfluorinated oil containing 2% E2K0660 through a pipette gun, and add 10 μL of PCR Mix to the aqueous phase storage area. 2) Centrifuge at 2800r/min for 60s to generate droplets and store them in the oil phase storage area.

The results are shown in Figure 8.

Example 3 Glass microfluidic chip

This chip is shown in Fig. 5 with reference to, and its basic structure is identical with embodiment 1, and difference is, in this embodiment, chip is only two layers up and down: upper sealing layer 15 (glass material) and channel layer 16 (glass material) ), the upper surface of the channel layer 16 is provided with a microfluidic channel (the channel structure includes an aqueous phase storage area, a connecting channel, a droplet generation area, a droplet collection area, etc.). The specific chip manufacturing steps are as follows:

1. Microfluidic channel etching and fabrication

1) Place the chrome plate on the lifting platform of the single-sided lithography machine, and place the film on the chrome plate.

2) Set the exposure time to 8-12s, suction film, lifting, contact, adhesion, exposure position. (When sliding the chip, the film should be aligned with the glass)

3) Remove the film sheet, and soak the homogeneous chromium plate in NaOH (1.6g to 400ml) for 2min. ---development

4) Take out the chromium plate, wash it with ultrapure water, blow dry with N2, and repeat twice.

5) After drying, put cerium ammonium nitrate-perchloric acid solution (strongly corrosive) to corrode the chromium layer for about 2 minutes until the pattern of the chromium layer is completely corroded. Rinse with water, blow dry, repeat twice.

6) Dry at 85°C for about 20 minutes to remove the solvent in the photoresist.

7) Take the chromium plate and put it into the mixed acid (HF 8.7ml, HNO3 16.875ml, NH3F 9.25g, total 500ml/6.9:7.4:13.5-400ml, corrosion rate at 40°C is about 1.5-2um/min), heated in water bath (according to Heating temperature and etching depth set time), cool slightly, wash with water, and observe chip surface erosion under a microscope. (The erosion width is proportional to the depth, isotropic erosion, at a temperature of 40-45°C, 10min etching 15um)

8) Put the glass chip processed in the previous step into acetone to wash off the surface glue layer, and then wash off the chrome layer with cerium ammonium nitrate solution (until the glass is completely transparent, if there is chrome layer remaining, the photoresist may not be cleaned, you can Put in acetone and wash again), and finally wash away the residual ammonium cerium nitrate solution with water.

9) After washing the chip with water, blow it dry with _N2 , find another glass chip of the same size, sprinkle a layer of rosin on the surface, press the non-channel side of the chip used in the experiment on the rosin, put the two chips on a heating plate greater than 120 ° C to heat and melt rosin.

10) Take out the glass chip, install the puncher and the punching needle, mark the position to be punched with a marker pen, spray a layer of ultrapure water evenly on it, and adjust the position to punch the hole. When punching holes, ensure that there is rosin in the middle layer, otherwise it should be heated to melt the rosin evenly.

11) Wash off the rosin with acetone after drilling.

2. Glass chip bonding

The channel layer and sealing layer were washed with ultrapure water, fixed with a mold, bonded in a muffle furnace at 650°C for 6 hours, cooled naturally to room temperature, and taken out.

3. Channel surface perfluorinated treatment

Inject 1720 perfluorinated solvent into the channel layer, dry at 75°C, and repeat 3 times to complete the perfluorinated treatment of the channel layer.

4. Droplet Generation

1) Fill the droplet storage area with HFE 7500 perfluorinated oil containing 2% E2K0660 through a pipette gun, and add 10 μL of PCR Mix to the aqueous phase storage area. 2) Centrifuge at 2800r/min for 60s to generate droplets and store them in the oil phase storage area.

The results are shown in Figure 9.

Example 4

Referring to Fig. 6, the basic structure of this embodiment is the same as that of Embodiment 1, the difference is that in this embodiment, the lower half of the droplet storage area 4 (the part below the droplet generation area 3, in Fig. 6 Part A) is longer, the oil phase used is light mineral oil with a lighter density than the water phase, and the surfactant used is 5% Span 80.

The results are shown in Figure 10.

Claims (9)

1. A centrifugal microfluidic chip for the generation of water-in-oil droplets, characterized in that it includes a channel layer and a sealing layer, and the channel layer includes an aqueous phase storage area, a connecting channel, a droplet generation area and a liquid A droplet collection area, wherein the aqueous phase storage area is closer to the centrifugal axis than the droplet storage area is near the centrifugal axis end; the droplet storage area is closer to the centrifugal axis than the droplet generation area; The connection channel connects the water phase storage area and the droplet generation area, and the droplet generation area connects the droplet collection area and one end of the connection channel; a pressure balance channel is also connected between the water phase storage area and the droplet collection area ; The axis of the opening direction of the droplet generating area and the direction of the centrifugal force form an included angle of 30-150 degrees. 2. A kind of centrifugal microfluidic chip for the generation of water-in-oil droplets as claimed in claim 1, characterized in that, the opening direction axis of the droplet generation area and the direction of centrifugal force form a clamp of 45-135 degrees. horn. 3. A kind of centrifugal microfluidic chip for the generation of water-in-oil droplets as claimed in claim 1, characterized in that, the axis of the opening direction of the droplet generation area and the centrifugal force direction form an angle between 80-100 degrees. horn. 4. A kind of centrifugal microfluidic chip for the generation of water-in-oil droplets as claimed in any one of claims 1 to 3, wherein the structure of the droplet generation zone is a nozzle-type structure with a narrow inlet and a wide outlet . 5. A centrifugal microfluidic chip for generating water-in-oil droplets as claimed in claim 4, wherein the nozzle inlet width is 1 μm-200 μm, the nozzle outlet width is 10 μm-1 mm, and the nozzle depth 1μm-200μm. 6. A kind of centrifugal microfluidic chip for water-in-oil drop generation as claimed in claim 1, is characterized in that, described pressure balance channel, its one end is positioned at droplet collection area, and droplet generation area The same distance from the centrifugal shaft; the other end is located in the liquid phase storage area, and the distance between it and the centrifugal shaft is smaller than the distance between the connecting channel and the centrifugal shaft. 7 . A centrifugal microfluidic chip for generating water-in-oil droplets according to claim 1 , wherein the volume of the water-phase storage area is 0.1 μL-1 mL. 8 . A centrifugal microfluidic chip for generating water-in-oil droplets according to claim 1 , wherein the length of the connecting channel is 1 mm-100 mm, the width is 10 μm-10 mm, and the depth is 1 μm-1 mm. 9. A centrifugal microfluidic chip for generating water-in-oil droplets according to claim 1, wherein the depth of the droplet collection area is 1 μm-500 μm.