A droplet preparation device and method based on emulsification in porous media

Abstract

The application discloses a kind of liquid drop preparation device and method based on porous medium emulsification, including water-in-oil droplet generation part and droplet classification part, water-in-oil droplet generation part includes channel piece, compressed sponge body loaded in channel piece and fixing piece, and the droplet classification part is gradient sandwich plate assembly with stepped slit channel, the droplet generated by water-in-oil droplet generation part is transferred to droplet classification part by pipettor and is classified and arranged, the flux, speed and availability of droplet formation are greatly improved using common materials, and the uniformity of high-throughput droplets is significantly improved by gradient slit classification, which can provide extremely low-cost, portable and automated droplet formation and detection components for microbial culture, micro-nano synthesis reaction, biochemical analysis and digital absolute quantitative detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemical detection, and in particular to a droplet preparation device and method based on porous medium emulsification. BACKGROUND

[0002] Microdroplets have been widely used in biological, chemical detection analysis, medical diagnosis, theoretical calculation, material synthesis and other fields due to their small volume, high flux, uniform size, internal stability and controllability. In particular, high-throughput ultra-fast droplet generation technology has become a key limiting factor for POCT in digital PCR and single-molecule non-amplification absolute quantitative detection. Traditional droplet generation is based on microfluidic chips, which requires precise syringe pumps to strictly control the flow rate of oil and water phase fluids in the channel to generate droplets, and the operation needs to be precisely controlled. The existing droplet method still needs complex chip structure design, and the processing is complex. Similarly, the device also needs a centrifugal device, which is difficult to miniaturize and is not convenient for POCT detection. It is worth noting that droplet generation needs complex hydrophilic and hydrophobic treatment of chip materials or capillary tubes to make droplets free from surface tension to complete droplet preparation. In summary, due to the structural complexity or difficulty in operation of the existing droplet generation method, the driving force based on micro-vibration or centrifugal force seriously restricts the need for simplicity and portability in droplet POCT detection.

[0003] There are also some methods in the prior art that can simply form droplets, such as oscillation method, ultrasonic atomization method, etc. However, the droplets generated by these methods have the characteristics of too large variation coefficient (CV>60%) and are difficult to use in some fields that use droplet detection technology for digital quantification.

[0004] In summary, the current droplet generation technology is difficult to meet the characteristics of simplicity, efficiency, speed and uniformity. Therefore, it is necessary to design a simple structure and easy-to-operate ultra-fast large-scale droplet preparation and observation device and method based on hydrophobic porous medium emulsification. SUMMARY

[0005] Therefore, the present application provides a droplet preparation device and method based on porous medium emulsification, which effectively solves the problem of miniaturization and portability of ultra-high-throughput droplet rapid preparation.

[0006] To achieve the above technical purposes, the present application adopts the following technical solutions:

[0007] In a first aspect, the application provides a droplet preparation device based on porous medium emulsification, comprising a water-in-oil droplet generating part and a droplet grading part. The water-in-oil droplet generating part comprises a channel member, a compressed sponge body loaded in the channel member, and a fixing member for fixing the compressed sponge body in the channel member. The droplet grading part is a gradient sandwich plate assembly provided with a stepped slit channel, wherein the stepped slit channel is provided with a plurality of throat portions, and the two sides of the gradient sandwich plate assembly are sealed. The droplets generated by the water-in-oil droplet generating part are transferred to the droplet grading part by a pipettor for grading arrangement.

[0008] Preferably, the gradient sandwich plate assembly comprises a flat sheet, an obtuse angle bent sheet, a first limiting member, and a second limiting member. The bottom of the flat sheet is attached to the bottom of the obtuse angle bent sheet. The first limiting member is used to fix the top of the flat sheet and the top of the obtuse angle bent sheet. The second limiting member is used to fix the middle of the flat sheet and the middle of the obtuse angle bent sheet. The width gradient of the slit channel formed from the top to the bottom of the gradient sandwich plate assembly decreases.

[0009] Preferably, the first limiting member and the second limiting member are adhesive members with different thicknesses. The first limiting member corresponds to the adhesive flat sheet top side and the obtuse angle bent sheet top side. The second limiting member corresponds to the adhesive flat sheet middle side and the obtuse angle bent sheet middle side. The thickness of the first limiting member is 50-500 mm, and the thickness of the second limiting member is less than or equal to 0.02 mm.

[0010] Preferably, the first limiting member is an adhesive member, and the second limiting member is an elastic fastener. The first limiting member corresponds to the adhesive flat sheet top side and the obtuse angle bent sheet top side. The thickness of the first limiting member is 50-500 mm. The second limiting member is wound around the middle of the flat sheet and the middle of the obtuse angle bent sheet and is pressed and fixed to form a slit of less than or equal to 0.01 mm between the middle of the flat sheet and the middle of the obtuse angle bent sheet.

[0011] Preferably, the compressed sponge body is compressed from one or more of polyester sponge, PDMS sponge, and natural sponge. The density of the compressed sponge body is 5-100 kg / m 3 .

[0012] Preferably, the channel member is one of a funnel-shaped pipette tip and a pipette tip with a pear-shaped channel. The channel member is used to connect a single-channel pipettor or a multi-channel pipettor.

[0013] Preferably, the middle to the bottom of the flat sheet and the obtuse angle bent sheet are modified by roughness or hydrophobicity, or the flat sheet and the obtuse angle bent sheet are provided with flow channels for controlling the flow speed and path of the oil phase. The end of the flow channel is connected with a liquid storage tank for controlling the flow and volume of the oil phase.

[0014] Preferably, the area of the capillary sandwich formed by the lower part to the bottom of the second limiting member increases, and the capillary sandwich can be cut into any shape.

[0015] In a second aspect, the application provides a method for preparing emulsion droplets based on porous medium, comprising the following steps:

[0016] S1. sequentially sucking the oil phase and the water phase into a water-in-oil droplet generator to obtain water-in-oil droplets;

[0017] S2. transferring the water-in-oil droplets into a vertically placed droplet grading unit by using a pipette, and automatically grading and arranging under the action of gravity and capillary force;

[0018] The oil phase is one or more of mineral oil, fluorinated oil or fatty hydrocarbon polyester oil phase containing a surfactant.

[0019] Preferably, the oil phase includes a stabilizer.

[0020] Preferably, before step S1, it further includes pouring n-hexadecane diluted PDMS into the compressed sponge body, and then blowing and drying.

[0021] The application has the following advantages:

[0022] 1. The operation is extremely simple, and no special training and experience of traditional microfluidics are required, and the generation is convenient, and no strict control of high-precision sample pumps is required;

[0023] 2. The droplet generation speed is superfast, can meet the ul-ml level droplet production, completely bypasses the solution volume limitation, and can complete 50 microliters of ultra-high throughput droplet preparation in 30 seconds, while the traditional droplet microfluidic needs at least five minutes or more, and its preparation speed is only about 3000 droplets per second;

[0024] 3. The droplet size is adjustable, the distribution range of the droplets can be effectively controlled by using sponges with different hardness, the operation is simple, the material cost is extremely low, the production is convenient, the throughput is high, and the droplets are relatively uniform;

[0025] 4. The sample has no dead volume and saves sample, and almost all water phase solutions can generate droplets with uniform size;

[0026] 5. In the application, not only oil phases with a density greater than that of the water phase can be used, but also oil phases with a density less than that of the water phase can be used, thereby expanding the application range;

[0027] 6. The stability is high, and once the used porous sponge medium and the extrusion degree are determined, the droplet size will not change;

[0028] 7. Batch production is possible, the structure is simple, and the cost is low; compared with microfluidic chips, MEMS process production, bonding, packaging and complex process are required, and the cost is high;

[0029] 8、The method can completely get rid of the limitation of the centrifuge or the injection pump, has small structure and low cost, and is very suitable for POCT detection application;

[0030] 9、More importantly, by the gradient sandwich plate, high-throughput droplets can be quickly and massively assembled, and the problem of relatively large variation of the generated droplet size is greatly overcome, the droplet monolayer arrangement is relatively uniform, and the requirements of single-molecule biochemical detection, digital absolute quantification and the like can be met.

[0031] DRAWINGS

[0032] Figure 1 Structure diagram of the droplet preparation device based on porous medium emulsification;

[0033] Figure 2 Front view of the water-in-oil droplet generating part (the channel piece is a funnel-shaped pipette tip);

[0034] Figure 3 Front view of the water-in-oil droplet generating part (the channel piece is a funnel-shaped pipette tip);

[0035] Figure 4 Front view of the water-in-oil droplet generating part (the channel piece is a pipette tip with a pear-shaped channel at the head);

[0036] Figure 5 Side view of the droplet grading part;

[0037] Figure 6 Structure diagram of the sponge body (a) before compression and (b) after compression.

[0038] In the figure: 1, water-in-oil droplet generating part; 2, droplet grading part; 11, channel piece; 12, compressed sponge body; 21, flat plate; 22, obtuse angle bending plate; 23, first limiting piece; 24, second limiting piece. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present application clearer and more apparent, the present application will be further described in detail below in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0040] The present application is directed to the problems of the prior art microfluidic chip-based droplet method, such as complex structure, the need for complex processing steps to achieve microfluidic channel structure, high chip production cost, the need for some special surface material hydrophobic treatment, difficulty in mass production, complex fluid control, the need for precise syringe pumps to strictly control the flow rate of the fluid in the channel, and the like; and the problems of the prior art centrifugal droplet generation microfluidic chip, which cannot get rid of the complex chip design and manufacturing problems, and the like, and these devices can only be used for a small amount of droplet generation (about 20 microliters) and cannot meet the process of a large amount of droplet generation, and the like, resulting in that the existing droplet generation system is still difficult to realize simple and portable application, and is difficult to play a role in POCT detection, and is also difficult to be used for mass production.

[0041] The present application fills the extruded porous sponge-like medium in the channel piece 11 to form a compressed sponge body 12, then sequentially sucks the oil phase and the water phase, the water phase is efficiently cut into droplets in the porous medium, and more importantly, a gradient sandwich plate assembly is further constructed, which can arrange the above-mentioned droplets in the sandwich gap in stages, and realizes a droplet production system with simple structure without additional complex devices; the device does not need complex chip design, completely gets rid of the droplet preparation restrictions of the injection pump or centrifugal force and complex processing and manufacturing equipment in the traditional microfluidic droplet system, effectively solves the miniaturization and portability problems of ultra-high-throughput droplet rapid preparation; further, the multi-channel pipette displacement gun or the automatic high-throughput pipette can realize ultra-fast multi-sample droplet processing, and plays a key role in high-throughput digital detection.

[0042] Based on the above, the present application is created, and the specific content is as follows:

[0043] As shown in Figure 1 The present application provides a droplet preparation device based on porous medium emulsification, which comprises a water-in-oil droplet generation part 1 and a droplet grading part 2, the water-in-oil droplet generation part 1 comprises a channel piece 11, a compressed sponge body 12 loaded in the channel piece 11, a fixing piece for fixing the compressed sponge body in the channel piece, the compressed sponge body 12 is a porous sponge loaded in the front end of the channel piece 11, and is fixed after being compressed into a porous medium of several microns by extrusion, Figure 6 (a) a structure schematic diagram of the uncompressed compressed sponge body, Figure 6 (b) a structure schematic diagram of the compressed sponge, the fixing piece can be a silica gel gasket, in order to prevent the compressed sponge from moving, the silica gel ring is further filled behind the sponge body to fix the compressed sponge body by friction, the oil phase is first sucked to occupy the sponge medium, then the water phase is sucked, and when the water phase passes through the sponge pores, it is efficiently dispersed into high-throughput water-in-oil droplets, and the process is repeated twice to complete the efficient preparation of droplets; asFigure 5 As shown, the droplet grading part 2 is a gradient sandwich plate assembly provided with a stepped slit channel provided with a plurality of throats, and the two sides of the gradient sandwich plate assembly are sealed, which can be adhesive or integrally formed sealing, convenient for detection observation, and the water-in-oil droplet generating part 1 generates high-throughput droplets to transfer to the top opening of the gradient sandwich plate assembly. The oil phase and droplets in the channel piece 11 are quickly transferred to the gradient sandwich plate assembly under the action of capillary force and gravity, and continue to migrate under the action of capillary force of the gradient sandwich plate assembly, and different size droplets are independently arranged in the gradient sandwich plate assembly. It can be used for observation and detection.

[0044] It can be known from the existing droplet forming method that the droplet forming needs a certain driving force to overcome the surface tension of water to be cut by the flowing oil phase, and the size of the droplet needs to be controlled. In the present application, the speed and particle size of droplet formation are well controlled by extruding the porous sponge medium and controlling the pore diameter; the driving force for droplet generation is provided by the suction process of the channel piece 11 to complete the efficient and rapid division of droplets in the oil-containing porous medium and complete the preparation of droplets; the suction process of the channel piece 11 can be easily prepared by using a common professional pipette or a simple device such as a spring rebounding syringe on a push rod; in addition, the gradient sandwich plate assembly can automatically grade different size droplets under the action of capillary force, realize the ultrafast high-throughput and uniformity of the droplet generation process, and achieve efficient balance.

[0045] The gradient sandwich plate assembly in the present application includes a flat sheet 21, an obtuse angle bent sheet 22, a first limiting piece 23, and a second limiting piece 24. The bottom of the flat sheet 21 is attached to the bottom of the obtuse angle bent sheet 22. The first limiting piece 23 is used to fix the top of the flat sheet 21 and the top of the obtuse angle bent sheet 22. The second limiting piece 24 is used to fix the middle of the flat sheet 21 and the middle of the obtuse angle bent sheet 22. The slit channel formed from the top to the bottom of the gradient sandwich plate assembly has a decreasing width gradient. The length-width ratio of the flat sheet 21 and the obtuse angle bent sheet 22 is about 1:4. The flat sheet 21 and the obtuse angle bent sheet 22 are made of plastic or glass sheets with a certain hardness or closed molds made by 3D printing technology. The first limiting piece and the second limiting piece can be integrally injection molded with the flat sheet and the obtuse angle bent sheet to obtain the gradient sandwich plate assembly. The first limiting piece and the second limiting piece can also be separate structures and can be combined into the gradient sandwich plate assembly by adhesion, compression, pressing, etc. Preferably, the gradient sandwich plate assembly according to different limiting pieces provides the following structures:

[0046] 1. The first limiting member 23 and the second limiting member 24 are adhesive members with different thicknesses, which have the functions of limiting, adhering and supporting. The first limiting member 23 corresponds to the top side of the adhesive flat sheet 21 and the top side of the obtuse angle bent sheet 22. The second limiting member 24 corresponds to the middle side of the adhesive flat sheet 21 and the middle side of the obtuse angle bent sheet 22. The adhesive member can be double-sided tape with different thicknesses. The thickness and position of the double-sided tape directly determine the separation and distribution position of the liquid droplets in the slit. The bottom is tightly attached to form a stepped slit channel for the hierarchical arrangement of liquid droplets. The high-throughput liquid droplets are transferred to the interlayer plate with a height gradient. Different size liquid droplets are arranged in the interlayer plate for digital absolute quantitative observation and detection with high uniformity requirements. In this scheme, the flat sheet 21 and the obtuse angle bent sheet 22 are both glass sheets. The thickness of the first limiting member 23 is 50-500mm, and the thickness of the second limiting member 24 is 0.01mm. The area of the double-sided tape is about 1cm, which is distributed on both sides of the glass sheet. The middle is a capillary force channel. The oil phase liquid is dragged by gravity and capillary force. The oil phase and liquid droplets migrate downward. When passing through the slit with different heights, different size liquid droplets are fixed at a specific position, thereby realizing the rapid arrangement of liquid droplets.

[0047] 2. The middle and lower sections of the clamp plate can provide additional capillary pumping force for liquid flow, enabling liquid droplets to complete gradient assembly quickly. In this structure, the first limiting member 23 is an adhesive member, and the second limiting member 24 is an elastic fastener. The first limiting member 23 corresponds to the top side of the adhesive flat sheet 21 and the top side of the obtuse angle bent sheet 22. The thickness of the first limiting member 23 is 50-500mm. The second limiting member 24 is wrapped around the middle of the flat sheet 21 and the middle of the obtuse angle bent sheet 22 and is pressed and fixed to form a 0.01mm slit between the middle of the flat sheet 21 and the middle of the obtuse angle bent sheet 22. For example, an elastic rubber band can be used to fix the clamp plate in the middle to form a similar segmented stepped gap.

[0048] The compressed sponge body 12 is compressed from one or more of hydrophobic polyester sponge, PDMS sponge, and natural sponge. The density of the compressed sponge body 12 is 18-45kg / m 3 The size of the generated liquid droplets is adjusted by the selection of the hardness, softness and porosity of the compressed sponge body 12. Preferably, the sponge raw material is cut into 3mm cubes. The compressed sponge body 12 with a size of about 1mm is extruded in the channel, and the rubber ring is pressed against the compressed sponge body to fix it at the front end of the channel.

[0049] The channel member 11 is one of a funnel-shaped pipette tip and a pipette tip with a micro-pear-shaped channel at the head, Figure 2 When the channel member is a funnel-shaped pipette tip, the front view of the water-in-oil droplet generating part; Figure 3Fig. 2 is a top view of the oil-in-water droplet generating part when the channel member is a funnel-shaped pipette tip; Figure 4 Fig. 2 is a front view of the oil-in-water droplet generating part when the channel member is a pipette tip with a pear-shaped channel; more specifically, the pipette tip with a micro-pear-shaped channel has a tapered tip extending about 2 mm, contains 3 micro-0.5 mm protrusions or pear-shaped bellies at 1 mm from the tip, and the compressed sponge body 12 can be well fixed at the tip of the channel, preventing the sponge from loosening during the suction process. The channel member 11 is used to connect a single-channel pipettor or a multi-channel pipettor, and the filtered suction is connected through a conventional multi-channel pipettor or a syringe containing a rebound spring. The connector can connect multiple filtered tips to achieve rapid sample droplet generation of multiple or high-throughput different samples, and can quickly achieve high-throughput absolute quantitative detection of samples, further realizing automatic droplet preparation and detection.

[0050] The middle to bottom of the flat sheet 21 and the obtuse angle bent sheet 22 are modified in roughness or hydrophobicity, or the flat sheet and the obtuse angle bent sheet are provided with flow channels for controlling the flow speed and path of the oil phase. This scheme can accelerate the capillary speed of the oil phase and strengthen the hierarchical arrangement speed of the droplets in the oil phase droplets. The bottom of the gradient sandwich plate assembly is provided with a liquid storage tank for adjusting the containing volume of the oil phase.

[0051] In a second aspect, the present application provides a method for preparing droplets based on porous medium emulsification, comprising the following steps:

[0052] S1. sequentially sucking the oil phase and the water phase into the oil-in-water droplet generating part 1 to obtain oil-in-water droplets;

[0053] S2. transferring the oil-in-water droplets to the vertically placed droplet hierarchical part 2 using a pipettor for automatic hierarchical arrangement;

[0054] The oil phase is one or more of a mineral oil containing a surfactant, a fluorinated oil, or a fatty hydrocarbon polyester oil phase.

[0055] In some embodiments, the oil phase includes a stabilizer.

[0056] In some embodiments, before step S1, the compressed sponge body 12 is also poured with n-hexadecane diluted PDMS, and then blown and dried, and solidification can further reduce the number of micro-pores and increase the uniformity of the droplets.

[0057] The present application is further described below through specific embodiments.

[0058] Embodiment 1

[0059] A kind of based on porous medium emulsion's droplet preparation device, including water-in-oil droplet generation part 1 and droplet classification part 2, water-in-oil droplet generation part 1 includes channel piece 11, compressed sponge body 12 loaded in channel piece 11, droplet classification part 2 is gradient sandwich plate assembly with stepped slit channel, the droplet generated by water-in-oil droplet generation part 1 is transferred to droplet classification part 2 by pipette and is classified and arranged.

[0060] Compressed sponge body 12 is made: compressed sponge body 12 is compressed by one or several of polyester sponge, PDMS sponge, natural sponge, and the density of compressed sponge body 12 is 5-100 kg / m 3 The porous structure of sponge can become dense microchannels with specific pore distribution under appropriate extrusion, and when the oil phase liquid is filled, the water phase can be efficiently cut into specific water-in-oil droplets when the water phase is introduced, the degree of compression can be controlled by different soft and hard conditions of sponge, and media with different pore sizes can be constructed to further regulate the size of generated droplets, and the size of the sponge material for compression is preferably 3mm spherical or cubic;

[0061] Water-in-oil droplet generation part 1 assembly: channel piece 11 is used to fix compressed sponge, pipe for sucking and storing oil phase and / or water phase liquid, channel piece 11 is one of funnel-shaped pipette gun head, pipette gun head with pear-shaped channel in head, pipette gun head with pear-shaped channel in head has a 2mm extension at the tip, and contains 3 micro 0.5mm protrusions at 1mm from the tip, 3mm cubic sponge is extruded to 1mm sponge in the channel, which can be well fixed in the channel tip by three protrusion points to prevent the loosening of the sponge during the syringe suction process;

[0062] After the assembly of water-in-oil droplet generation part 1 is completed, a single-channel or multi-channel syringe or pipette gun is used to fix the gun head loaded with sponge, and the oil phase is preferentially sucked to fill the porous medium, then the gun head is moved to the oil-water interface or aqueous solution, and slow suction is continued, the aqueous solution is dispersed into high-throughput droplets under the protection of the oil phase, and in order to further homogenize the droplets, two times of pumping can be repeated.

[0063] Manufacture of gradient sandwich plate assembly: in order to eliminate the non-uniformity of droplets at the time of observation, the generated droplets are sorted by using the gradient sandwich plate assembly, which comprises a flat sheet 21, an obtuse angle bending sheet 22, a first limiting piece 23, a second limiting piece 24, the bottom of the flat sheet 21 is attached to the bottom of the obtuse angle bending sheet 22, the first limiting piece 23 is used to fix the top of the flat sheet 21 and the top of the obtuse angle bending sheet 22, and the second limiting piece 24 is used to fix the middle of the flat sheet 21 and the middle of the obtuse angle bending sheet 22. The width of the slit channel formed from the top to the bottom of the gradient sandwich plate assembly decreases in gradient, the length-width ratio of the flat sheet 21 and the obtuse angle bending sheet 22 is about 1:4, which is a certain hardness plastic or glass sheet or a closed mold made of 3D printing technology; the first limiting piece 23 and the second limiting piece 24 are adhesive pieces with different thicknesses, at this time the adhesive piece has the functions of limiting, bonding and supporting, the first limiting piece 23 corresponds to the bonding of the top side edge of the flat sheet 21 and the top side edge of the obtuse angle bending sheet 22, and the second limiting piece 24 corresponds to the bonding of the middle side edge of the flat sheet 21 and the middle side edge of the obtuse angle bending sheet 22. The adhesive can be double-sided tape with different thicknesses, and the thickness and position of the double-sided tape directly determine the separation and distribution position of the droplets in the slit, and the bottom is tightly attached to support the sandwich plate to form a certain capillary force gradient for the graded arrangement of droplets. The high-throughput droplets are transferred to the sandwich plate with a height gradient, and different size droplets are arranged in the sandwich plate in a graded manner, which is used for digital absolute quantitative observation and detection with high uniformity requirement. In the present scheme, the thickness of the first limiting piece 23 is 50-500mm, the thickness of the second limiting piece 24 is 0.01mm, the area of the double-sided tape is about 1cm distributed on both sides of the glass sheet, the interval between the double-sided tapes is about 2mm, one or more throat channels can be set, and the oil phase liquid is pulled down by gravity and capillary force. After passing through the capillary channel of the middle and lower segment of the sandwich plate, the oil phase continues to migrate downward, and different size droplets are fixed at a certain position when passing through a slit of different height, so as to realize the sorting of droplets.

[0064] A method for preparing droplets based on porous medium emulsion, comprising the following steps:

[0065] S1. Use high / medium / low density (45kg / m 3 , 30kg / m 3 , 18kg / m 3 ) sponge unfilled material to make droplets; the channel piece 11 is selected as a 0.25ml dispensing gun head, the sponge is cut into 3mm cubes, and is filled into the top of the gun head. It is not suitable to be squeezed too much, and it can be stopped at the bottom. The bottom is a funnel-shaped cross section with a diameter of about 1mm. At this time, the sponge is squeezed to about 1mm in size, and a water-in-oil droplet generating part 1 is obtained.

[0066] The process of making water-in-oil droplets by using water-in-oil droplet generator 1 is as follows: take 30 microliters of liquid in a PCR tube, then take 80 microliters of fluorinated oil phase to cover, use a gun head to first suck the oil phase, then suck the water phase, the water phase passes through the oil phase filled pore medium, and the preparation of the droplet is completed. In order to overcome the difference in suction method, the suction process can be repeated 2-3 times, and the preparation of the droplet is completed within one minute;

[0067] S2. Install multiple filtering channel pieces 11 on a pipettor capable of multiple suction channels, including 8, 16, 32, 96, pass the water-in-oil droplets through the pipettor to the vertically placed droplet grading part 2, and automatically grade and arrange to process 8, 16, 32, 96 sample droplets in a 96-well plate, realizing ultra-fast large-scale processing of samples.

[0068] Example 2

[0069] Other contents are the same as in Example 1, except that the first limiting piece 23 is an adhesive piece, and the second limiting piece 24 is an elastic fastener. The first limiting piece 23 corresponds to the top side of the adhesive flat sheet 21 and the top side of the obtuse angle bent sheet 22. The thickness of the first limiting piece 23 is 50-500mm. The second limiting piece 24 is wrapped around the middle part of the flat sheet 21 and the obtuse angle bent sheet 22 and is pressed and fixed to form a 0.01mm gap between the middle part of the flat sheet 21 and the middle part of the obtuse angle bent sheet 22. For example, an elastic rubber band can be used to fix the middle part of the clamp. The elastic force of the rubber band can press the clamp to form a similar segmented stepped gap in the middle.

[0070] Example 3

[0071] Other contents are the same as in Example 1, except that the middle to bottom of the flat sheet 21 and the obtuse angle bent sheet 22 are designed through the flow channel and the liquid storage tank. Further modification of roughness or hydrophobicity can increase the speed of droplet assembly and the assembly of larger droplet volume.

[0072] Example 4

[0073] Other contents are the same as in Example 1, except that ethyl laurate is used instead of oil phase, and 7% EM180 is added as a droplet stabilizer. The formed water-in-oil droplets are spread in the gradient interlayer plate assembly, which can significantly improve the coefficient of variation of droplet size and form a uniform particle size distribution gradient change droplet array.

[0074] Example 5

[0075] Other contents are the same as example 1, the difference is that in step S1, the sponge is loaded in the channel piece 11, and then the sponge medium is irrigated by the PDMS and curing agent mixed reagent diluted by n-hexadecane, so that the fine pores in the sponge are filled with PDMS, and the PDMS in the large pores is removed, and then dried at 65°C, to obtain a pore medium with more uniform particle size, and the cured PDMS can fix the sponge in the channel piece 11, simplifying the control of the sponge fixation.

[0076] Example 6

[0077] Other contents are the same as example 1, the difference is that the droplet forming part and the grading part are integrated on a flat plate by one-piece molding manufacturing method, and the bottom end of the grading part contains a suction nozzle. Similarly, droplets are generated by the suction process, and then the suction nozzle is sealed, the droplets formed in the channel piece are inverted, and under the action of gravity, the droplets fill the grading part and assemble, completing the droplet generation and integrated assembly observation.

[0078] The droplet preparation of the prior art can only meet the preparation of a small amount of droplets, and the sample injection amount is limited by the sample injection volume on the chip, and even through the injection pump, the demand for flexible and large-scale preparation of droplets cannot be met. The present application does not need to be precisely introduced by an injection pump, but through a suction process, the water phase flows in the extruded capillary tube to shear and form droplets, and the suction speed has little effect on the formation of droplets, greatly overcoming the professionalism of the droplet generation process, and the operation is simple; through simple manual or automatic motor control injection suction process, the speed, quantity and volume of solution and solution type of droplet preparation can be flexibly controlled, which is suitable for larger scale droplet preparation. During the preparation process, the droplets are obviously not uniform in size, which affects the observation and quantification of the droplets, and we realize the grading assembly of the droplets through the gradient clamp, which can effectively grade and collect and observe different droplet sizes through the flow of the oil phase.

[0079] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A droplet preparation device based on porous media emulsification, characterized in that, The system includes an oil-in-water droplet generating section (1) and a droplet grading section (2). The oil-in-water droplet generating section (1) includes a channel component (11), a compressed sponge (12) loaded in the channel component (11), and a fixing component for fixing the compressed sponge (12) in the channel component (11). The droplet grading section (2) is a gradient sandwich plate assembly with a stepped slit channel. The stepped slit channel has several throats. The gradient sandwich plate assembly is sealed on both sides. The droplets generated by the oil-in-water droplet generating section (1) are transferred to the droplet grading section (2) for grading and arrangement. The gradient sandwich panel assembly includes a flat plate (21), an obtuse-angled bent piece (22), a first limiting member (23), and a second limiting member (24). The bottom of the flat plate (21) is attached to the bottom of the obtuse-angled bent piece (22). The first limiting member (23) is used to fix the top of the flat plate (21) and the top of the obtuse-angled bent piece (22). The second limiting member (24) is used to fix the middle part of the flat plate (21) and the middle part of the obtuse-angled bent piece (22). The width gradient of the slit channel formed by the gradient sandwich panel assembly from top to bottom decreases.

2. The droplet preparation device based on porous media emulsification according to claim 1, characterized in that, The first limiting member (23) and the second limiting member (24) are adhesive members with different thicknesses. The first limiting member (23) is bonded to the top side of the flat plate (21) and the top side of the obtuse angle bent piece (22). The second limiting member (24) is bonded to the middle side of the flat plate (21) and the middle side of the obtuse angle bent piece (22). The thickness of the first limiting member (23) is 50-500mm, and the thickness of the second limiting member (24) is less than or equal to 0.02mm.

3. The droplet preparation device based on porous media emulsification according to claim 1, characterized in that, The first limiting member (23) is an adhesive, and the second limiting member (24) is an elastic fastener. The first limiting member (23) is bonded to the top side of the flat plate (21) and the top side of the obtuse angle bent piece (22). The thickness of the first limiting member (23) is 50-500mm. The second limiting member (24) is wrapped around the middle of the flat plate (21) and the middle of the obtuse angle bent piece (22) and pressed to fix it so that a slit less than or equal to 0.01mm is formed between the middle of the flat plate (21) and the middle of the obtuse angle bent piece (22).

4. The droplet preparation device based on porous media emulsification according to claim 1, characterized in that, The compressed sponge (12) is made of one or more of polyester sponge, PDMS sponge, and natural sponge, and the density of the compressed sponge (12) is 5-100 kg / m³. 3 .

5. The droplet preparation device based on porous media emulsification according to claim 1, characterized in that, The channel component (11) is one of a funnel-shaped pipette tip or a pipette tip with a pear-shaped channel at the head. The channel component (11) is used to connect a single-channel pipette or a multi-channel pipette.

6. The droplet preparation device based on porous media emulsification according to claim 1, characterized in that, The middle to bottom of the flat plate (21) and the obtuse angle bent plate (22) are modified by roughness modification or hydrophobic modification, or the flat plate (21) and the obtuse angle bent plate (22) are provided with flow channels for controlling the flow rate and path of the oil phase.

7. A method for preparing droplets based on porous media emulsification using the apparatus according to any one of claims 1-6, characterized in that, Includes the following steps: S1. The oil phase and the water phase are sequentially drawn into the water-in-oil droplet generating unit (1) to obtain water-in-oil droplets; S2. The water-in-oil droplets are transferred to the vertically placed droplet classification section (2) using a pipette, and automatically classified and arranged under the action of gravity and capillary force; The oil phase is one or more of the following: mineral oil containing surfactants, fluorinated oil, or aliphatic hydrocarbon polyester oil phase.

8. The method as described in claim 7, characterized in that, The oil phase includes a stabilizer.

9. The method as described in claim 7, characterized in that, Prior to step S1, diluted PDMS and curing agent are poured into the compressed sponge (12).

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