Liquid drop capturing micro-column array micro-fluidic chip and application thereof

Abstract

The invention discloses a droplet capture microcolumn array microfluidic chip, which comprises a chip main body and a substrate, the chip main body comprises a support layer, a channel structure layer and an anti-evaporation layer arranged between the support layer and the channel structure layer; the channel structure layer internally comprises a liquid drop capturing chamber area, the liquid drop capturing chamber area is provided with a micro-column array formed by regularly arranging a plurality of micro-columns with the same height and the same diameter, and a capturing chamber is formed by a plurality of adjacent micro-columns; if the height of the droplet capturing chamber area is set as H, the height of the micro-column is set as h, and the diameter of the micro-column is set as d, h is more than H / 6 and less than or equal to H / 2; h / d is less than 1.5. According to the micro-fluidic chip disclosed by the invention, the depth-to-width ratio of the micro-column array is relatively low, the micro-column array is easy to manufacture, and meanwhile, the micro-fluidic chip still has the functions of separating and capturing liquid drops; the height of the micro-column is lower than that of the liquid drop capturing chamber area, so that the liquid drops can be captured and fixed, and good moving fluency is achieved; in addition, the chip is provided with an anti-evaporation layer, so that the evaporation loss of a sample in liquid drops can be reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic chips, and in particular to a droplet capture microcolumn array microfluidic chip and its application. Background Art

[0002] Droplet microfluidics, as an important branch of microfluidic technology, mainly uses two immiscible fluids as the continuous phase and the dispersed phase respectively. Through the adjustment of the flow rates of the two phases and the design of the microchannel structure, a series of operations can be carried out, such as controlling the generation, sorting, fusion, splitting, capture, etc. of droplets. Compared with the continuous flow system, each droplet can serve as an individual reaction chamber, separating the samples therein, providing an independent and stable microenvironment for biochemical reactions, and the parallel processing of a large number of droplets helps to achieve ultra-high throughput and is conducive to achieving high-precision analysis. The characteristics of droplet microfluidics make it widely used in many fields such as nucleic acid detection, single-cell sequencing, bacterial research, protein / enzyme detection, etc. However, the requirements and challenges for droplet microfluidic technology in different application fields are also different. Therefore, chip design and fluid manipulation need to be carried out according to different application requirements.

[0003] Taking digital polymerase chain reaction (dPCR) as an example, as the third-generation PCR detection technology, dPCR technology has advantages such as high sensitivity, no need for a standard curve, and absolute quantification, and has received increasing attention and application. Currently, the mainstream dPCR is divided into droplet-based dPCR (ddPCR) and chamber-based dPCR (cdPCR). Among them, ddPCR has advantages such as flexible and controllable droplet generation quantity, simple chip processing, and low cost, and has received more and more attention, and multiple commercial platforms have been developed. However, the current ddPCR technology still faces some problems. One of the main problems is that during the thermal cycling process, droplets will randomly move in the continuous phase, resulting in collisions between droplets, which are likely to cause droplet breakage or fusion. At the same time, droplet movement is also not conducive to real-time monitoring of individual droplets, ultimately affecting the accuracy of nucleic acid quantification.

[0004] Regarding the problem of droplet movement and collision, a micro-column array microfluidic chip for lossless droplet capture, its preparation method and application are disclosed in the Chinese patent document with the application publication number CN113318798A. This technical solution was the research success of the inventor of the present invention. Based on further applications, it was found that in this micro-column array microfluidic chip, the height of the micro-columns is the same as that of the chamber, and the bottom of the micro-columns is sealed and bonded to the base layer. On the one hand, this design makes the micro-columns have a relatively high aspect ratio, that is, the ratio of the height of the micro-columns to the diameter of the micro-columns is relatively large, and it is relatively difficult to process the micro-column structure, and the micro-column structure is prone to fracture during demolding. On the other hand, the micro-columns with the same height as the chamber make the droplets can only squeeze and deform through the gaps between adjacent micro-columns and enter the capture chamber. After generating droplets using a low-viscosity oil phase such as fluorinated oil, the deformation ability of the droplets is poor, it is difficult to enter the micro-column array and the smoothness of movement in the micro-column array is limited. Another problem is that due to the small volume and large specific surface area of micro-droplets, the samples in the droplets are easily evaporated, thus affecting the detection reaction.

[0005] Therefore, there is still an urgent need to develop a microfluidic chip with functions of droplet separation and capture and fixation, low aspect ratio of micro-structures, easy processing and complete demolding, smooth droplet capture, and good anti-evaporation performance. Summary of the Invention

[0006] In view of the above problems, the present invention discloses a droplet capture micro-column array microfluidic chip, which has both droplet generation and droplet collection functions. The designed micro-column array has a relatively low aspect ratio and is easy to fabricate. At the same time, it still has the function of separating and capturing droplets. The height of the micro-columns is lower than the height of the droplet capture chamber area, so that the droplets can be captured and fixed, and can also have better smoothness of movement under the push of the subsequently generated droplets. In addition, the chip is provided with an anti-evaporation layer, which can reduce the evaporation loss of the samples in the droplets.

[0007] The specific technical solution is as follows:

[0008] A droplet capture micro-column array microfluidic chip, comprising a chip body and a base sealed to the chip body:

[0009] The chip body includes a support layer, a channel structure layer, and an anti-evaporation layer with a water vapor barrier function disposed between the support layer and the channel structure layer;

[0010] The channel structure layer includes a droplet capture chamber area, and the droplet capture chamber area is provided with a micro-column array composed of a plurality of micro-columns with the same height and the same diameter regularly arranged. Adjacent micro-columns form a capture chamber for realizing the separation and capture of droplets;

[0011] If the height of the droplet capture chamber area is set as H, the height of the micro-columns is set as h, and the diameter of the micro-columns is set as d, then:

[0012] H / 6 < h ≤ H / 2;

[0013] h / d < 1.5, where h / d is the depth-to-width ratio.

[0014] The present invention discloses a microcolumn array microfluidic chip, which has both droplet generation and droplet collection functions. The continuous phase and the dispersed phase are respectively introduced into the injection area. Stable droplets with uniform sizes can be generated through the droplet generation area, and after being dispersed through the droplet dispersion area, they enter the droplet capture chamber area and are captured in the microcolumn array, enabling on-chip detection reactions without additional droplet transfer and collection. The barrier of the microcolumn array in the chip confines the droplets in the capture chamber, and they can only enter the next capture chamber by being pushed by subsequent droplets. The height of the microcolumn array being lower than the height of the droplet capture chamber area allows the droplets to move with good fluidity. By reasonably controlling the number of generated droplets, efficient and lossless droplet capture can be achieved. At the same time, the chip is designed with an anti-evaporation layer, which can effectively reduce the evaporation loss of the samples in the droplets.

[0015] If the gap value between adjacent microcolumns is set as L and the diameter of the droplet to be captured is set as D, then:

[0016] D > H - h and L < D ≤ d + 2L.

[0017] This parameter limitation ensures that the droplet to be captured will definitely be blocked by the microcolumns when moving in the droplet capture chamber area and cannot pass directly through the microcolumn gaps or below without deformation. It can only squeeze and deform to enter the next capture chamber under the push of subsequent droplets.

[0018] Preferably:

[0019] The depth-to-width ratio h / d ≤ 1;

[0020] More preferably, 0.5 ≤ h / d ≤ 1;

[0021] By controlling the depth-to-width ratio within the above range and slowly demolding the chip along one side, the integrity of the microcolumns can be maintained.

[0022] Preferably:

[0023] In the present invention, a capture chamber is composed of 6 adjacent microcolumns and is in a regular hexagon shape.

[0024] Preferably:

[0025] The diameter D of the droplet to be captured is selected from 40 - 80 μm;

[0026] More preferably:

[0027] h = 20 μm, d = 30 μm, H = 60 μm, L = 20 μm.

[0028] The microcolumn array designed with the above parameters can capture droplets with D selected from 40 to 80 μm.

[0029] More preferably, the diameter D of the droplet to be captured is selected from 60 to 70 μm.

[0030] It has been experimentally found that when the droplet diameter is selected from this range, it is more matched with the microcolumn array designed with this size, and the capture and separation of a single droplet can be achieved.

[0031] Preferably:

[0032] A number of support columns are also provided in the droplet capture chamber area to strengthen the sealing strength with the substrate, and the height of the support columns is the same as the height of the droplet capture chamber area.

[0033] Preferably:

[0034] The channel structure layer also includes a sample injection area, a droplet generation area, a droplet dispersion area, and a sample outlet area;

[0035] A continuous phase injection port and a dispersed phase injection port are provided in the sample injection area;

[0036] More preferably, rectangular microcolumn filtering structures are provided behind both the continuous phase injection port and the dispersed phase injection port to prevent solid impurities from blocking the channels.

[0037] The droplet generation area includes a droplet generation structure that cuts the continuous phase to disperse the dispersed phase to form droplets;

[0038] More preferably, the droplet generation structure is specifically a flow focusing type channel structure.

[0039] The droplet dispersion area includes a multi-stage binary structure that enables the formed droplets to enter the droplet capture chamber area simultaneously after being dispersed through several channels.

[0040] A sample outlet is provided in the sample outlet area.

[0041] Preferably:

[0042] The material of the anti-evaporation layer is selected from one or more of polypropylene (PP), polyethylene terephthalate (PET), polycarbonate (PC), and polyvinylidene chloride (PVDC); the presence of the anti-evaporation layer can block the diffusion of water vapor and effectively reduce the evaporation loss of the sample in the droplet.

[0043] Adhesives are provided on both the upper and lower surfaces of the anti-evaporation layer for fixed connection with the support layer and the channel structure layer.

[0044] More preferably:

[0045] The material of the anti-evaporation layer is selected from PP and its composite materials;

[0046] The adhesive is selected from pressure-sensitive adhesives.

[0047] Preferably:

[0048] The support layer is composed of a plastic support and a photocurable elastic support;

[0049] The plastic support completely covers the droplet capture chamber area within the channel structure layer;

[0050] The photocurable elastic support completely covers other areas within the channel structure layer except the droplet capture chamber area;

[0051] With the support layer adopting the above combination method, using a plastic support above the droplet capture chamber area can provide stronger support force to reduce deformation caused by pressure inside and outside the chamber; using a photocurable elastic support above the sample inlet and outlet areas can facilitate the punching operation better.

[0052] Preferably:

[0053] The material of the plastic support is selected from one or more of polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), polyethylene terephthalate (PET), acrylonitrile-styrene copolymer (SAN), poly-4-methylpentene-1 (TPX), cycloolefin copolymer (COC), epoxy-propylene copolymer (COP);

[0054] The photocurable elastic support is made by photocuring a photocurable material with ultraviolet light;

[0055] The photocurable material includes raw materials containing photocurable functional groups, and the photocurable functional groups are selected from one or more of acrylate functional groups, methacrylate functional groups, vinyl ether functional groups, mercapto functional groups, alkenyl functional groups, epoxy functional groups.

[0056] Preferably:

[0057] The channel structure layer is made by photocuring a photocurable material with ultraviolet light; the photocurable material includes raw materials containing photocurable functional groups, and the photocurable functional groups are selected from one or more of acrylate functional groups, methacrylate functional groups, vinyl ether functional groups, mercapto functional groups, alkenyl functional groups, epoxy functional groups.

[0058] Preferably:

[0059] The substrate is a composite layer, including a photocurable sealing layer and a base layer;

[0060] The photocuring sealing layer is used for sealing with the channel structure layer; the photocuring sealing layer is also made of a photocuring material cured by ultraviolet light; the photocuring material includes raw materials containing photocuring functional groups, and the photocuring functional groups are selected from one or more of acrylate functional groups, methacrylate functional groups, vinyl ether functional groups, mercapto functional groups, alkenyl functional groups, and epoxy functional groups.

[0061] The base layer plays a supporting role, and preferably a base layer made of glass is used.

[0062] The present invention also discloses an application method of the droplet capture microcolumn array microfluidic chip, including:

[0063] (1) The continuous phase and the dispersed phase are respectively introduced into the microcolumn array microfluidic chip through the injection areas in the channel structure layer, and the injection rates of the continuous phase and the dispersed phase are respectively adjusted to generate stable droplets in the droplet generation area. After the droplets flow through the droplet dispersion area, they enter the droplet capture chamber area and are captured in the capture chamber. After the injection of the dispersed phase is complete, the injection is stopped. After the fluid is stable, the generation and capture of droplets are completed;

[0064] (2) Seal the injection port and the outlet port of the microcolumn array microfluidic chip;

[0065] (3) Heat the microcolumn array microfluidic chip, set the heating temperature according to the application requirements, and finally obtain the detection result.

[0066] Preferably:

[0067] The continuous phase includes an oil phase raw material, and the dispersed phase includes an aqueous phase raw material;

[0068] The oil phase raw material is selected from one or more of mineral oil, silicone oil, fluorinated oil, and photocuring oil.

[0069] The specific injection process is as follows:

[0070] First, the continuous phase is introduced. After the continuous phase fills the entire flow channel structure of the microfluidic chip, the dispersed phase is introduced. First, adjust and fix the flow rate of the continuous phase, and then adjust the flow rate of the dispersed phase to control the droplet generation size. After the droplets are generated, stop the injection of the dispersed phase first. At this time, the continuous phase continues to be injected until all the droplets in the droplet generation area and the droplet dispersion area are pushed into the droplet capture chamber area, and then stop the injection of the continuous phase;

[0071] When the oil phase raw material is selected from photocuring oil, after the droplet capture is completed and stable, the photocuring oil phase can also be cured by ultraviolet light, so that the droplets are completely fixed and will not move, fuse, or break even during the PCR thermal cycle.

[0072] Further preferably, the photocuring oil is an acrylate-based oil phase.

[0073] Compared with the prior art, the present invention has the following beneficial effects:

[0074] The present invention discloses a droplet capture microcolumn array microfluidic chip, which has both droplet generation and droplet collection functions. In the droplet generation area, the dispersed phase is sheared into uniform and stable droplets by the continuous phase, enters the droplet capture chamber area after passing through the droplet dispersion area, and then undergoes on-chip reactions without additional droplet collection operations. The designed microcolumn array can separate and capture droplets without loss. The arrangement of droplets in an array is more conducive to image recognition and data statistics. The height of the microcolumn array is lower than the height of the droplet capture chamber area, enabling the droplets to be captured and having good movement fluency. Even droplets with poor deformation ability in a low-viscosity oil phase can quickly enter the capture chamber. By reasonably controlling the number of generated droplets, high-efficiency and lossless droplet capture can be achieved.

[0075] A droplet capture microcolumn array microfluidic chip disclosed by the present invention is mainly prepared using a photocurable material. Compared with PDMS commonly used in laboratories, the preparation is simpler and faster. At the same time, there is a rich variety of photocurable materials with various different physical and chemical properties, and different photocurable materials can be selected according to application requirements. A combined structure of a photocurable elastic support and a plastic support is used in the support part, enabling the chip to perform better punching operations in the sample inlet and outlet areas and providing better support for the chamber to prevent obvious deformation of the chamber under pressure.

[0076] The microcolumn array chip disclosed by the present invention is also provided with an anti-evaporation layer, which can block water vapor and reduce the evaporation loss of the sample in the droplet, thereby ensuring the accuracy of detection within the chip. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 is a schematic structural diagram of the droplet capture microcolumn array microfluidic chip disclosed by the present invention;

[0078] Figure 2 is a mask diagram of the channel structure layer designed in Example 1, where (A) is the first layer and (B) is the second layer;

[0079] Figure 3 is a schematic process flow diagram of the preparation of the microfluidic chip disclosed by the present invention;

[0080] In the figure, 1 - chip body, 2 - substrate, 3 - sample inlet area, 4 - droplet generation area, 5 - droplet dispersion area, 6 - droplet capture chamber area, 7 - sample outlet area;

[0081] 11 - Support layer, 12 - Anti - evaporation layer, 13 - Channel structure layer, 21 - Photo - curable sealing layer, 22 - Glass base layer; 31 - Continuous - phase injection port, 32 - Dispersed - phase injection port, 33 - Filter structure, 41 - Flow - focusing structure, 51 - Multi - stage binary dispersion structure, 61 - Micro - column array, 62 - Support column, 71 - Sampling port;

[0082] 611 - Capture chamber;

[0083] Figure 4 Is a physical picture of the droplet - capture micro - column - array microfluidic chip prepared in Example 1;

[0084] Figure 5 Are the side - view microscope pictures of the micro - column arrays in the microfluidic chips prepared in Examples 1 - 5 and Comparative Example 1 respectively, where (a) d = 20μm, h = 20μm, (b) d = 30μm, h = 20μm, (c) d = 40μm, h = 20μm, (d) d = 20μm, h = 30μm, (e) d = 30μm, h = 30μm, (f) d = 40μm, h = 30μm;

[0085] Figure 6 Are the capture pictures of droplets with different diameters generated in Application Example 2 and Application Example 3 respectively;

[0086] Figure 7 Are the microscope pictures of droplet generation (a), droplet dispersion (b), and droplet capture (c) in Application Example 3;

[0087] Figure 8 Are the fluorescence microscope pictures of the droplet array prepared in Application Example 3 before (a) and after (b) PCR thermal cycling after photo - curing;

[0088] Figure 9 Are the microscope pictures of the photo - cured droplet array prepared by the same method as in Application Example 3 after PCR thermal cycling when the microfluidic chip has no anti - evaporation film;

[0089] Figure 10 Are the capture pictures of the droplets generated by photo - curing oil 2 in Application Example 4 (a), Application Example 5 (b), and Comparative Application Example 2 (c). Detailed implementation manners

[0090] To further understand the present invention, the present invention will be specifically described below in conjunction with the drawings and embodiments. However, the present invention is not limited to these embodiments. Non - essential improvements and adjustments made by those skilled in the art under the core guiding ideology of the present invention still fall within the protection scope of the present invention.

[0091] Figure 1Schematic diagram of the structure of a droplet capture microcolumn array microfluidic chip disclosed in the present invention, including a chip body 1 and a substrate 2.

[0092] The chip body 1 includes a support layer 11, an anti-evaporation layer 12, and a channel structure layer 13. The support layer 11 is composed of a photocurable elastic support 111 that is easy to punch for sample introduction / removal and a plastic support 112 with strong supporting force to prevent the deformation of the chamber. It is fabricated using soft lithography technology. First, a mold with a corresponding microchannel structure is fabricated by photolithography, and then a thin layer with the microchannel structure is obtained by replicating the mold using a photocurable material, thus obtaining the channel structure layer 13, which is convenient for subsequent detection.

[0093] The anti-evaporation layer 12 is located between the support layer 11 and the channel structure layer 13, and its main function is to block water vapor to prevent sample evaporation.

[0094] The substrate 2 is a composite layer, including a photocurable sealing layer 21 for sealing with the channel structure layer 13 and a glass substrate layer 22 that provides support.

[0095] Figure 2 The mask diagram of the channel structure layer adopted in Example 1 is given. Since the height of the microcolumns in the present invention is less than the total height of the droplet capture chamber area, two-layer masks are used. Figure (A) is the first-layer mask diagram, and Figure (B) is the second-layer mask diagram; both figures include a sample introduction area 3, a droplet generation area 4, a droplet dispersion area 5, a droplet capture chamber area 6, and a sample removal area 7. The difference is that a microcolumn array 61 structure is provided in the droplet capture chamber area 6 of the second-layer mask diagram in Figure (B).

[0096] A continuous phase sample introduction port 31 and a dispersed phase sample introduction port 32 are provided in the sample introduction area 3, and a filtering structure 33 is provided on the flow path behind the sample introduction ports. The continuous phase and the dispersed phase are respectively introduced through their respective sample introduction ports. If there are large solid impurities in the fluid, they will be blocked by the filtering structure and cannot flow into the subsequent flow path. The two-phase fluids converge in the droplet generation area 4, and the dispersed phase is sheared by the continuous phase to generate droplets.

[0097] The droplet generation structure in the droplet generation area 4 specifically adopts a flow focusing structure 41.

[0098] The droplets generated in the droplet generation area 4 then flow into the droplet dispersion area 5. A multi-stage binary dispersion structure 51 is adopted in the droplet dispersion area. The droplets enter from one path and become two paths after passing through the dispersion structure. After multiple dispersions, they enter the droplet capture chamber area 6.

[0099] The droplet capture chamber region 6 is provided with a micro-column array 61 structure. The micro-column array 61 is composed of a number of micro-columns with the same height and diameter regularly arranged. Six adjacent micro-columns can form a regular hexagonal capture chamber 611. The droplets flowing into the droplet capture chamber region 6 can be captured by the capture chamber 611 and separated. There are also 9 support columns 62 with diameters much larger than those of the micro-columns in the droplet capture chamber region 6, which are used to strengthen the sealing between the channel structure layer 13 and the substrate 2 and prevent the droplet capture chamber from collapsing or bulging.

[0100] The sample output region 7 is provided with a sample output port 71, and the excess continuous phase fluid flows out from the sample output port 71.

[0101] Figure 3 It is a schematic diagram of the preparation process flow of the microfluidic chip disclosed in the present invention.

[0102] The preparation of the chip body 1 includes:

[0103] (a) The support layer 11 and the anti-evaporation layer 12 are tightly connected to obtain structure A;

[0104] (b) The channel structure layer to be cured is poured onto the mold 8 with a photo-curing reagent 14;

[0105] (c) The structure A in step (a) is covered on the photo-curing reagent 14 in step (b), and ultraviolet light is applied to cure it to obtain structure B;

[0106] (d) The structure B obtained in step (c) is separated from the mold 8 and punched to obtain the chip body 1;

[0107] The preparation of the substrate 2:

[0108] (e) The photo-curing sealing layer to be cured is dropped onto the hydrophobic glass 9 with a photo-curing reagent 23;

[0109] (f) The glass base layer 22 is covered on the photo-curing reagent 23 in step (e), and ultraviolet light is applied to cure it to obtain structure C;

[0110] (g) The structure C obtained in step (f) is separated from the hydrophobic glass 9 to obtain the substrate 2;

[0111] Sealing and bonding:

[0112] (h) The chip body 1 obtained in step (d) and the substrate 2 obtained in step (g) are closely attached, and then ultraviolet light is applied to seal and bond them to obtain the final microfluidic chip.

[0113] Example 1 Preparation of a photo-cured micro-column array chip based on thiol-ene materials

[0114] a) Mold processing: Take a clean and dry silicon wafer, immediately pour and spin-coat photoresist (Microchem, SU-8 3025) after 30 s of plasma treatment, with a thickness of 40 μm, and pre-bake at 95 °C for 25 min; Place the first layer of mask ( Figure 2 in (A)) closely on the pre-baked photoresist, post-bake at 95 °C for 30 min after exposure with an ultraviolet lithography machine; Spin-coat the second layer of photoresist (Microchem, SU-8 3025), with a thickness of 20 μm, and pre-bake at 95 °C for 25 min; Use the alignment system of the lithography machine to align the second layer of mask ( Figure 2 (B) in, micro-column diameter d = 30 μm) with the corresponding pattern on the exposed first layer of photoresist, also make the mask closely adhere to the photoresist, post-bake at 95 °C for 30 min after exposure with an ultraviolet lithography machine; Remove the excess photoresist with SU-8 developer, transfer the developed mold to a hot plate and hard-bake at 200 °C for 30 min to solidify the mold.

[0115] b) Chip body support layer processing: Mix 95% polyurethane acrylate, 4.9% ethoxyethoxyethyl acrylate, and 0.1% photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone evenly by mass percentage; Place a customized 2-mm-thick frame on the release film for elevation, pour the prepared photocuring reagent into the frame, cover the release film above the frame and press it flat with a glass plate, and cure the photocuring reagent by ultraviolet light irradiation (365 nm, 45 mW / cm 2 , 60 s) to obtain a photocured elastic support; Cut the photocured elastic support and the PMMA plastic support into specified sizes for subsequent chip preparation.

[0116] c) Chip body processing: Mix 59.3% pentaerythritol tetrakis(3-mercaptopropionate), 40.2% triallyl isocyanurate, and 0.5% photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone evenly by mass percentage; Drop the prepared photocuring reagent onto the mold made in step a); Bond the PP anti-evaporation layer with adhesives on both the upper and lower surfaces and the cut support in step b) as Figure 1 shown, then slowly cover it on the dropped photocuring reagent to completely cover the pattern area of the mold with the photocuring reagent, and cure it by ultraviolet light irradiation (365 nm, 45 mW / cm 2 , 2 s) to form a channel structure layer, and the overall demolding forms the chip body.

[0117] d) Chip substrate processing: The preparation of the photocuring reagent is exactly the same as that in step c) above; Drop the prepared photocuring reagent onto a hydrophobically treated glass slide (with a 100-μm elevation on both sides), slowly cover a common glass slide above, and cure it by ultraviolet light irradiation (365 nm, 45 mW / cm 2, (1 s) It is cured to form a photocured sealing layer, and is separated together with a common glass slide to form a substrate.

[0118] e) Chip sealing: The chip body in step c) is tightly attached to the substrate in step d), and after ultraviolet light irradiation (365 nm, 45 mW / cm 2 , (2 s)), they are firmly sealed together to obtain a complete microcolumn array microfluidic chip.

[0119] The physical picture of the chip is as shown in Figure 4 .

[0120] In the microfluidic chip prepared in this embodiment, the height H of the droplet capture chamber area is 60 μm, the height h of the microcolumns is 20 μm, the height of the microcolumns is 1 / 3 of the total height of the chamber, the diameter d of the microcolumns is 30 μm, the aspect ratio h / d = 2 / 3, and the gap value L between adjacent microcolumns is 20 μm. The side view microscope picture of the microcolumn array in this embodiment is shown in Figure 5 (b) in.

[0121] Example 2 Preparation of a Photocured Microcolumn Array Chip Based on Thiol-ene Materials

[0122] The preparation process is basically the same as that in Example 1, except that in the mold processing of step a), the microcolumn diameter in the second layer of mask pattern is replaced with 20 μm. At this time, the gap value L between adjacent microcolumns is 30 μm.

[0123] In the microfluidic chip prepared in this embodiment, the aspect ratio h / d = 1. The side view microscope picture of the microcolumn array in this embodiment is shown in Figure 5 (a) in.

[0124] Example 3 Preparation of a Photocured Microcolumn Array Chip Based on Thiol-ene Materials

[0125] The preparation process is basically the same as that in Example 1, except that in the mold processing of step a), the microcolumn diameter in the second layer of mask pattern is replaced with 40 μm. At this time, the gap value L between adjacent microcolumns is 10 μm.

[0126] In the microfluidic chip prepared in this embodiment, the aspect ratio h / d = 0.5. The side view microscope picture of the microcolumn array in this embodiment is shown in Figure 5 (c) in.

[0127] Example 4 Preparation of a Photocured Microcolumn Array Chip Based on Thiol-ene Materials

[0128] The preparation process is basically the same as that in Example 1, except that in the mold processing of step a), the spin-coated thickness of the first layer of photoresist is replaced with 30 μm, and the spin-coated thickness of the second layer of photoresist is replaced with 30 μm.

[0129] In the microfluidic chip prepared in this embodiment, the height H of the droplet capture chamber area is 60 μm, the height h of the micro-columns is 30 μm, the height of the micro-columns is 1 / 2 of the total height of the chamber, the diameter d of the micro-columns is 30 μm, the aspect ratio h / d = 1, and the adjacent micro-column gap value L is 20 μm. The side-view microscope picture of the micro-column array in this embodiment is shown in Figure 5 (e).

[0130] Preparation of a Photocurable Micro-Column Array Chip Based on Thiol-Ene Material in Example 5

[0131] The preparation process is basically the same as that in Example 4, except that in the mold processing of step a), the diameter of the micro-columns in the second layer of mask pattern is replaced with 40 μm.

[0132] In the microfluidic chip prepared in this embodiment, the aspect ratio h / d = 0.75. The side-view microscope picture of the micro-column array in this embodiment is shown in Figure 5 (f).

[0133] In the above embodiments, slowly demolding the chip along one side can maintain the integrity of the micro-columns.

[0134] Preparation of a Photocurable Micro-Column Array Chip Based on Thiol-Ene Material in Comparative Example 1

[0135] The preparation process is basically the same as that in Example 4, except that in the mold processing of step a), the diameter of the micro-columns in the second layer of mask pattern is replaced with 20 μm.

[0136] At this time, in the prepared microfluidic chip, the aspect ratio h / d = 1.5. The side-view microscope picture of the micro-column array is shown in Figure 5 (d).

[0137] In this comparative example, even if the chip is slowly demolded along one side, the micro-columns are prone to breakage and a complete micro-column array cannot be turned out.

[0138] Preparation of a Photocurable Micro-Column Array Chip Based on Thiol-Ene Material in Example 6

[0139] The preparation process is basically the same as that in Example 1, except that in the mold processing of step a), the thickness of the first layer of photoresist spin-coated is replaced with 50 μm, and the thickness of the second layer of photoresist is replaced with 10 μm.

[0140] At this time, in the prepared microfluidic chip, the height of the micro-columns is 1 / 6 of the total height of the chamber, and the aspect ratio h / d = 1 / 3.

[0141] Preparation of a Photocurable Micro-Column Array Chip Based on Polyurethane Acrylate in Example 7

[0142] a) Mold processing: Exactly the same as step a) in Example 1.

[0143] b) Chip body support layer processing: Exactly the same as step a) in Example 1.

[0144] c) Chip body processing: Calculated by mass percentage, mix 99% of polyurethane acrylate and 1% of photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone evenly; drop the prepared photocuring reagent onto the mold made in step a); bond the anti-evaporation layer and the support cut in step b) as Figure 1 shown, then slowly cover it on the dropped photocuring reagent to make the photocuring reagent completely cover the pattern area of the mold, and cure it by ultraviolet light irradiation (365 nm, 45 mW / cm 2 , 10 s) to form a channel structure layer, and the overall demolding forms the chip body.

[0145] d) Chip substrate processing: Basically the same as step d) in Example 1, the only difference is that the ultraviolet light application time is 5 s.

[0146] e) Chip sealing: Basically the same as step e) in Example 1, the only difference is that the ultraviolet light application time is 8 s.

[0147] Preparation of photocured microcolumn array chip based on perfluoropolyether acrylate in Example 8

[0148] a) Mold processing: Exactly the same as step a) in Example 1.

[0149] b) Chip body support layer processing: Exactly the same as step a) in Example 1.

[0150] c) Chip body processing: Calculated by mass percentage, mix 99% of perfluoropolyether acrylate and 1% of photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone evenly; drop the prepared photocuring reagent onto the mold made in step a); bond the anti-evaporation layer and the support cut in step b) as Figure 1 shown, then slowly cover it on the dropped photocuring reagent to make the photocuring reagent completely cover the pattern area of the mold, and cure it by ultraviolet light irradiation (365 nm, 45 mW / cm 2 , 15 s) to form a channel structure layer, and the overall demolding forms the chip body.

[0151] d) Chip substrate processing: Basically the same as step d) in Example 1, the only difference is that the ultraviolet light application time is 8 s.

[0152] e) Chip sealing: Basically the same as step e) in Example 1, the only difference is that the chip is sealed by ultraviolet light under vacuum conditions in a vacuum dryer, and the ultraviolet light application time is 10 s.

[0153] Application Example 1: Droplet Generation and Capture Based on a Fluorinated Oil Continuous Phase

[0154] Use HFE-7500 fluorinated oil (Sphere Fluidics, Pico-Surf (2% (w / w) in Novec 7500)) containing 2 wt% surfactant as the continuous phase and 0.1 mM aqueous calcein solution as the dispersed phase. Set the oil phase flow rate to 400 μL / h and the water phase flow rate to 8 μL / h. Droplets with a diameter of approximately 60 μm can be generated in the microfluidic chip prepared in Example 1. After the generated droplets enter the droplet capture chamber area, they can be separated and captured by the microcolumn array. TM

[0155] Application Example 2: Droplet Generation and Capture Based on a Photocurable Oil 1 Continuous Phase

[0156] 1. Prepare the oil phase: Calculate by mass percentage. First, mix polyurethane acrylate and isobornyl acrylate evenly in a ratio of 3:7 to obtain a mixed reagent. Then, mix 96.5% of the mixed reagent, 3% of the ISOLAN 17 surfactant, and 0.5% of the photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone evenly to obtain photocurable oil 1.

[0157] 2. Droplet generation and capture: Use the photocurable oil 1 prepared in step 1 as the continuous phase and 0.1 mM aqueous calcein solution as the dispersed phase. Set the oil phase flow rate to 90 μL / h and the water phase flow rate to 10 μL / h. Uniform and stable droplets can be generated in the microfluidic chip prepared in Example 1. The droplet diameter is approximately 50 μm. After the generated droplets enter the droplet capture chamber area, they can be separated and captured by the microcolumn array; however, the capture density is relatively low in this application example. The droplet capture diagram is as shown in Figure 6 (a) in. Application Example 3: Droplet Generation and Capture Based on a Photocurable Oil 1 Continuous Phase

[0158] 1. Prepare the oil phase: Exactly the same as step 1 in Application Example 2.

[0159] 2. Droplet generation and capture: Use the photocurable oil 1 prepared in step 1 as the continuous phase and 0.1 mM aqueous calcein solution as the dispersed phase. Set the oil phase flow rate to 90 μL / h and the water phase flow rate to 20 μL / h. Uniform and stable droplets can be generated in the microfluidic chip prepared in Example 1. The droplet diameter is approximately 60 μm. After the generated droplets enter the droplet capture chamber area, they can be separated and captured by the microcolumn array. The capture density is high in this application example. The droplet capture diagram is as shown in Figure 6 (b) in.

[0160] Figure 7 These are the microscope pictures of the droplet generation (a), droplet dispersion (b), and droplet capture process (c) in this application example. ​

[0161] After the droplet capture is completed, the inlet and outlet ports are sealed with acrylate photocurable glue, and ultraviolet light is applied to completely cure the photocurable oil phase, forming a photocured droplet array. The entire chip is transferred to the hot plate of a PCR instrument, and the thermal cycling program is set as follows: thermal denaturation at 95 °C for 10 min; thermal cycling at 95 °C for 30 s and 60 °C for 1 min for a total of 40 cycles; finally cooled to 4 °C. After the reaction is completed, the results are observed under a fluorescence microscope. The results show that the evaporation loss of droplets in the photocured droplet array is less before and after thermal cycling, and the sizes and fluorescence brightnesses of the individual droplets are relatively uniform. For details, see Figure 8 。

[0162] Figure 9 Figure shows the microscope images of the photocured droplet array after thermal cycling under the same conditions when the microfluidic chip has no anti-evaporation layer. It can be seen that under the condition without the anti-evaporation layer, the droplets are almost completely evaporated and lost.

[0163] The preparation of the microfluidic chip without the anti-evaporation layer is basically the same as that in Example 1, except that when processing the chip body in step c), the anti-evaporation layer is not adhered, and only the support cut in step b) is slowly covered on the dropped photocurable reagent, and after the photocurable reagent completely covers the pattern area of the mold, photocuring is carried out.

[0164] Comparison of droplet generation and capture based on photocurable oil 1 continuous phase in Application Example 1

[0165] 1. Preparation of the oil phase: Exactly the same as step 1 in Application Example 2.

[0166] 2. Droplet generation and capture: The photocurable oil 1 prepared in step 1 is used as the continuous phase, and the 0.1 mM aqueous solution of calcein is used as the dispersed phase. The flow rate of the oil phase is set to 90 μL / h, and the flow rate of the water phase is set to 20 μL / h. Uniform and stable droplets can be generated in the microfluidic chip (h = H / 6) prepared in Example 6, and the droplet diameter is about 60 μm. However, at this time, due to the low height of the micro-columns, the droplets cannot be well captured and separated.

[0167] Droplet generation and capture based on photocurable oil 2 continuous phase in Application Example 4

[0168] 1. Preparation of the oil phase: By mass percentage, 15% perfluoropolyether acrylate, 82.5% HFE-7500 fluorinated oil, 2% fluorinated oil surfactant (Sphere Fluidics, (un-dissolved) 008-FluoroSurfactant), and 0.5% photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone are mixed evenly to obtain photocurable oil 2. The viscosity of this oil phase is much lower than that of photocurable oil 1, and the droplet deformation ability therein is poor.

[0169] 2. Droplet Generation and Capture: Use the photocurable oil 2 prepared in Step 1 as the continuous phase and 0.1 mM aqueous calcein solution as the dispersed phase. Set the oil phase flow rate to 400 μL / h and the water phase flow rate to 15 μL / h. Uniform and stable droplets can be generated in the microfluidic chip prepared in Example 2 (micro-columns with d = 20 μm and h = 20 μm). The droplet diameter is about 70 μm. The generated droplets can easily enter the droplet capture chamber area for separation and capture, and the droplets have good mobility in the micro-column array. The droplet capture diagram is as shown in Figure 10 Figure (a) in

[0170] Application Example 5 Droplet Generation and Capture Based on the Continuous Phase of Photocurable Oil 2

[0171] 1. Preparation of the Oil Phase: Exactly the same as Step 1 in Application Example 4.

[0172] 2. Droplet Generation and Capture: Basically the same as Step 2 in Application Example 4, except that the microfluidic chip prepared in Example 1 (micro-columns with d = 30 μm and h = 20 μm) is used. The generated droplets can easily enter the droplet capture chamber area for separation and capture, and the droplets have good mobility in the micro-column array. The droplet capture diagram is as shown in Figure 10 Figure (b) in

[0173] Comparative Application Example 2 Droplet Generation and Capture Based on the Continuous Phase of Photocurable Oil 2

[0174] 1. Preparation of the Oil Phase: Exactly the same as Step 1 in Application Example 4.

[0175] 2. Droplet Generation and Capture: Use the photocurable oil 2 prepared in Step 1 as the continuous phase and 0.1 mM aqueous calcein solution as the dispersed phase. Set the oil phase flow rate to 400 μL / h and the water phase flow rate to 10 μL / h. Uniform and stable droplets can be generated in the microfluidic chip prepared in Example 3 (micro-columns with d = 40 μm and h = 20 μm). The droplet diameter is about 60 μm. The generated droplets can easily enter the droplet capture chamber area, but due to the relatively large diameter of the micro-columns and the small horizontal gap between the micro-columns, the droplets are easily stuck under the micro-columns after being squeezed and cannot enter the capture chamber well for separation. The droplet capture diagram is as shown in Figure 10 Figure (c) in

Claims

1. A droplet capture microcolumn array microfluidic chip, comprising a chip body and a substrate sealed to the chip body, characterized in that: The chip body comprises a support layer, a channel structure layer and an anti-evaporation layer with a water vapor barrier function disposed between the support layer and the channel structure layer; The channel structure layer includes a droplet capture chamber area, and the droplet capture chamber area is provided with a micro-column array composed of a number of micro-columns with the same height and the same diameter arranged regularly, and a number of adjacent micro-columns form a capture chamber for realizing the separation and capture of droplets; If the height of the droplet capture chamber is set to H, the height of the microcolumn is set to h, and the diameter of the microcolumn is set to d, then: H / 6<h≤H / 2; h / d<1.

5.

2. The droplet capture microcolumn array microfluidic chip according to claim 1, characterized in that: If the gap between adjacent microcolumns is set to L, and the diameter of the droplet to be captured is set to D, then: D>Hh and L<D≤d+2L.

3. The droplet capture microcolumn array microfluidic chip according to claim 1, characterized in that: A plurality of support columns are also provided in the droplet capture chamber area to enhance the sealing strength with the substrate, and the height of the support columns is the same as the height of the droplet capture chamber area.

4. The droplet capture microcolumn array microfluidic chip according to claim 1, characterized in that: The channel structure layer also includes a sample inlet area, a droplet generation area, a droplet dispersion area and a sample outlet area.

5. The droplet capture microcolumn array microfluidic chip according to claim 1, characterized in that: The material of the anti-evaporation layer is selected from one or more of polypropylene, polyethylene terephthalate, polycarbonate, and polyvinylidene chloride.

6. The droplet capture microcolumn array microfluidic chip according to claim 1, characterized in that: The support layer is composed of a plastic support body and a light-cured elastic support body; The plastic support completely covers the droplet capture chamber area in the channel structure layer; The photocurable elastic support completely covers the other areas in the channel structure layer except the droplet capture chamber area; The material of the plastic support is selected from one or more of polymethyl methacrylate, polycarbonate, polystyrene, polyethylene terephthalate, acrylonitrile-styrene copolymer, poly-4-methylpentene-1, cycloolefin copolymer, and epoxy-polypropylene copolymer; The photocurable elastic support body is made of a photocurable material after being cured by ultraviolet light; The photocurable material includes raw materials containing photocurable functional groups, and the photocurable functional groups are selected from one or more of acrylate functional groups, methacrylate functional groups, vinyl ether functional groups, thiol functional groups, alkenyl functional groups, and epoxy functional groups.

7. The droplet capture microcolumn array microfluidic chip according to claim 1, characterized in that: The channel structure layer is made of a photocurable material after being cured by ultraviolet light; The photocurable material includes a raw material containing a photocurable functional group, and the photocurable functional group is selected from one or more of an acrylate functional group, a methacrylate functional group, a vinyl ether functional group, a thiol functional group, an alkenyl functional group, and an epoxy functional group.

8. The droplet capture microcolumn array microfluidic chip according to claim 1, characterized in that: The substrate is a composite layer, including a light-curing sealing layer and a substrate layer; The photocurable sealing layer is used to seal with the channel structure layer; The base layer plays a supporting role.

9. An application method of the droplet capture microcolumn array microfluidic chip according to any one of claims 1 to 8, characterized in that: include: (1) introducing a continuous phase and a dispersed phase into a microcolumn array microfluidic chip through the injection area in the channel structure layer, respectively, adjusting the injection rates of the continuous phase and the dispersed phase, respectively, so that stable droplets are generated in the droplet generation area, and the droplets flow through the droplet dispersion area and enter the droplet capture chamber area, and are captured in the capture chamber, and the injection of the dispersed phase is stopped after the injection is complete, and the droplet generation and capture are completed after the fluid is stable; (2) sealing the sample inlet and sample outlet of the microcolumn array microfluidic chip; (3) Heat the microcolumn array microfluidic chip, set the heating temperature according to application requirements, and finally obtain the test results.

10. The application method of the droplet capture microcolumn array microfluidic chip according to claim 9, characterized in that: The continuous phase includes an oil phase raw material, and the dispersed phase includes an aqueous phase raw material; The oil phase raw material is selected from one or more of mineral oil, silicone oil, fluorine oil, and light-curable oil; When the oil phase raw material is selected from photocurable oil, after the droplet capture is completed and stabilized, the oil phase can be UV-cured according to detection requirements.

Images