Liquid drop micro-fluidic chip

Abstract

The invention relates to the technical field of microfluidics, in particular to a droplet micro-fluidic chip. Comprising a main pipeline, a first pipeline and a second pipeline, the first pipeline and the second pipeline are respectively communicated with the main pipeline, and the first pipeline and the second pipeline are respectively positioned on two opposite sides of the main pipeline; the same liquid flows in the first pipeline and the second pipeline. The hydrogel microspheres loaded with the hiPSCs not only are beneficial to transfer and control of the hydrogel microspheres loaded with the hiPSCs, but also can load different types of cells in the shell and core space of the microspheres, thereby being beneficial to research on the interaction relationship between cells or organoid derived from the cells.

Description

Technical Field

[0001] The present application relates to the field of microfluidics technology, and in particular to a droplet microfluidics chip. Background Art

[0002] At present, two hydrogel materials, matrigel and alginate, are widely used in the cultivation of organoids due to their high biocompatibility. Matrigel is the most commonly used extracellular matrix material in organoid culture. It is in liquid state at 4°C and gradually solidifies as the temperature rises. Due to the low mechanical strength of matrigel, it makes it difficult to operate and transfer microspheres. Alginate is easily available and inexpensive and is widely used to support the growth of cell clumps / organoids, but it is difficult to use in stem cell-derived organoid culture because it cannot provide attachment points for the growth of single cells. However, in another strategy, the material can be used for the shell of the hydrogel, and the mechanical properties of the shell can be adjusted by adjusting its concentration to facilitate automated operations such as the transfer of microspheres.

[0003] Since the cross-linking modes of the two hydrogels are different, the preparation of core-shell structured hydrogel microspheres based on the two hydrogels has not yet been discovered. Summary of the invention

[0004] In this application, we propose a simple, rapid, and high-throughput method for preparing alginate-matrigel-oil dual hydrogel droplets, thereby achieving high-throughput preparation of shell-core structure composite hydrogel microspheres. The preparation of the microspheres is not only conducive to the subsequent high-throughput culture of stem cell-derived organoids and their automated operation (such as transfer of microspheres and removal of the oil phase), but also conducive to the study of cell-cell or cell-derived organoid interactions, providing reliable technical support for research in the fields of drug development, regenerative medicine, and precision medicine.

[0005] The high-throughput generation of hydrogel microspheres has brought opportunities for high-throughput uniform culture of organoids. Currently, most hydrogel microspheres used to load cells are solid hydrogel microspheres prepared from a single hydrogel material or hollow hydrogel microcapsules formed by droplet microfluidics. These two forms of hydrogel microspheres / microcapsules are not universal for most organoid cultures that require 3D scaffold support or co-culture of different types of cells / organoids. At present, the cultivation of a variety of organoids has been achieved through matrix gel. However, the mechanical strength of matrix gel is relatively low and it is easy to depolymerize and collapse during long-term culture. In order to provide a stable 3D growth environment for organoids, we designed a droplet microfluidic chip and proposed the preparation of alginate-matrix gel-oil dual hydrogel droplets for the first time. Through solidification under certain conditions, the high-throughput preparation of core-shell composite hydrogel microspheres was achieved.

[0006] In order to solve the problems existing in the prior art, the present application provides a droplet microfluidic chip.

[0007] The specific technical solutions of this application are as follows:

[0008] 1. A droplet microfluidic chip, comprising a main pipeline with liquid flowing therein, and a first pipeline and a second pipeline; the first pipeline and the second pipeline are respectively connected to the main pipeline, and the first pipeline and the second pipeline are respectively located at two opposite sides of the main pipeline; the same liquid flows in the first pipeline and the second pipeline;

[0009] A third pipeline and / or a fourth pipeline are intersected and connected with the main pipeline;

[0010] The third pipe and / or the fourth pipe can divide the continuously flowing liquid in the main pipe into droplets;

[0011] The third pipeline and / or the fourth pipeline are arranged at a downstream position of the first pipeline and the second pipeline.

[0012] 2. According to the chip described in item 1, the intersection between the first pipeline and the main pipeline forms a first connection, and the second pipeline and the main pipeline form a second connection at the intersection; preferably, the first connection and the second connection overlap.

[0013] 3. According to the chip described in item 1 or 2, the diameter of the main pipeline gradually increases from upstream to downstream.

[0014] 4. According to the chip described in item 1, the diameters of the first pipe and the second pipe are equal or similar.

[0015] 5. According to the chip of item 2, a core liquid flows in the main channel, a first liquid flows in the first channel, and a second liquid flows in the second channel; preferably, the first liquid and the second liquid both flow toward the inside of the main channel; further preferably, the core liquid and the first liquid and the second liquid merge at the first connection and / or the second connection to form a fifth liquid;

[0016] More preferably, the flow rate of the inner core liquid in the main pipeline is 1 to 20 μL / min; more preferably, the flow rate of the inner core liquid in the main pipeline is 4 to 10 μL / min.

[0017] 6. In the chip according to item 5, the fifth liquid flows in the main channel.

[0018] 7. According to the chip of item 5, the flow rate of the first liquid is equal to or similar to the flow rate of the second liquid; preferably, the cross-sectional diameter and flow rate of the first liquid and the second liquid are equal to or similar;

[0019] More preferably, the flow rate of the first liquid in the first pipe is 1 to 10 μL / min; More preferably, the flow rate of the first liquid in the first pipe is 2 to 5 μL / min;

[0020] More preferably, the flow rate of the second liquid in the second pipe is 1 to 10 μL / min; more preferably, the flow rate of the second liquid in the second pipe is 2 to 5 μL / min.

[0021] 8. The chip according to any one of items 1 to 7, wherein a third pipeline and a fourth pipeline are intersecting and connected on the main pipeline;

[0022] Preferably, the third pipeline and the fourth pipeline are relatively arranged on both sides of the main pipeline.

[0023] 9. According to the chip described in item 8, the diameter of the third pipe is equal to or similar to the diameter of the fourth pipe; preferably, the diameters of the third pipe and the fourth pipe are both less than or equal to the diameter of the main pipe.

[0024] 10. According to the chip described in item 8, the intersection of the third pipeline and the main pipeline forms a third connection, and the intersection of the fourth pipeline and the main pipeline forms a fourth connection; preferably, the third connection and the fourth connection overlap.

[0025] 11. The chip according to item 10, wherein a third liquid flows inside the third channel, a fourth liquid flows inside the fourth channel, and the third liquid and the fourth liquid can shear the liquid in the main channel into droplets;

[0026] Further preferably, the flow rate of the third liquid in the third pipe is 1 to 5 μL / min;

[0027] Further preferably, the flow rate of the fourth liquid in the fourth pipe is 1-5 μL / min.

[0028] 12. The chip according to item 8, wherein the main pipeline is connected to a fifth pipeline and / or a sixth pipeline;

[0029] Preferably, a fifth pipeline and a sixth pipeline are connected to the main pipeline in an intersecting state;

[0030] Preferably, the fifth pipeline and the sixth pipeline are relatively arranged on both sides of the main pipeline;

[0031] Further preferably, the fifth pipeline and the sixth pipeline are both located downstream of the third pipeline and the fourth pipeline.

[0032] 13. According to the chip of item 12, a sixth liquid flows inside both the fifth channel and the sixth channel.

[0033] 14. The chip according to any one of items 1 to 13, further comprising a first introduction pipe disposed upstream of the main pipe, through which the liquid flows into the main pipe;

[0034] Preferably, the chip further comprises a second introduction pipe, wherein the second introduction pipe is in communication with the first pipe and the second pipe;

[0035] Preferably, the chip further comprises a third introduction pipe, wherein the third introduction pipe is connected to the third pipe and the fourth pipe;

[0036] Preferably, the chip further comprises a fourth introduction pipe, and the fourth introduction pipe is connected with the fifth pipe and the sixth pipe.

[0037] Beneficial Effects

[0038] The droplet microfluidic chip of the present application is composed of three cross-channels. The core phase of the chip is a mixture of matrix gel and hiPSCs, and the shell phase is a mixture of sodium alginate and Ca-EDTA. Since the Reynolds number of the two hydrogel mixtures flowing in the chip channels is low, the core phase and the shell phase form incompatible laminar flows. Then, when passing through the second cross channel, it is cut by the oil phase to form monodisperse droplets consisting of an aqueous core (matrigel is liquid at 4°C) and a hydrogel shell. When the droplets pass through the third cross channel, the acidic oil phase triggers the release of calcium ions in the sodium alginate shell from the chelate; subsequently, the divalent Ca 2+ Combined with the two carboxyl groups of alginate, a cross-linked 3D network structure shell is formed. Subsequently, the temperature is raised from 4°C to 37°C, and the core phase matrix gel is solidified into a 3D network structure.

[0039] This method is not only conducive to the transfer and manipulation of hydrogel microspheres loaded with hiPSCs, but also can load different types of cells in the shell and core space of the microspheres, which is conducive to the study of the interaction between cells and cells or cell-derived organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a schematic diagram of the principle of the chip of this application;

[0041] Figure 2 This is a structural diagram of the chip of this application.

[0042] In the figure, 1, the first pipeline; 2, the second pipeline; 3, the third pipeline; 4, the fourth pipeline; 5, the fifth pipeline; 6, the sixth pipeline; 7, the main pipeline. DETAILED DESCRIPTION

[0043] The present application is described in detail below. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0044] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the functional differences of components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0045] refer to Figure 1 and Figure 2 The present application provides a droplet microfluidic chip, comprising a main pipeline 7 with liquid flowing therein, and a first pipeline 1 and a second pipeline 2; the first pipeline 1 and the second pipeline 2 are respectively connected to the main pipeline 7, and the first pipeline 1 and the second pipeline 2 are respectively located at two opposite sides of the main pipeline 7; the same liquid flows in the first pipeline 1 and the second pipeline 2;

[0046] The main pipeline 7 is intersected and connected with a third pipeline 3 and / or a fourth pipeline 4;

[0047] The third pipe 3 and / or the fourth pipe 4 can divide the continuously flowing liquid in the main pipe 7 into droplets;

[0048] The third pipeline 3 and / or the fourth pipeline 4 are arranged at a downstream position of the first pipeline 1 and the second pipeline 2 .

[0049] refer to Figure 1 and Figure 2 There is a continuously flowing liquid in the main pipeline 7, and then the first pipeline 1, the second pipeline 2, the third pipeline 3 and the fourth pipeline 4 which are respectively connected to the main pipeline 7 work together to input the liquid into the main pipeline 7 to achieve the effects of wrapping, cutting and solidifying the liquid in the main pipeline 7.

[0050] In the present application, the first pipe 1 and the second pipe 2 are connected to the main pipe 7, and the liquid in the first pipe 1 and the second pipe 2 can be transported to the main pipe 7, and the first pipe 1 and the second pipe 2 are respectively located on both sides of the main pipe 7, so that the liquid in the first pipe 1 and the second pipe 2 can wrap the liquid in the main pipe 7. When the liquid in the first pipe 1 and the second pipe 2 wraps the liquid in the main pipe 7, the liquid in the first pipe 1 can first wrap a part of the liquid in the main pipe 7, and then the liquid in the second pipe 2 wraps the remaining part. It is also possible that the liquid in the first pipe 1 and the second pipe 2 wrap the liquid in the main pipe 7 at the same time.

[0051] The third pipe 3 and the fourth pipe 4 are also connected to the main pipe 7. When the liquid in the main pipe 7 flows to the position of the third pipe 3 and the fourth pipe 4, the liquid in the third pipe 3 and the fourth pipe 4 also flows into the main pipe 7. Then, when the liquid in the third pipe 3 and the fourth pipe 4 flows into the main pipe 7, shear force is generated on the liquid in the main pipe 7, thereby cutting the liquid in the main pipe 7 into droplets.

[0052] refer to Figure 1 and Figure 2 In the present application, by connecting the first pipe 1 and the second pipe 2 on the main pipe 7, the liquid in the main pipe 7 can be wrapped by the liquid in the first pipe 1 and the second pipe 2, and then under the addition and action of the liquid in the third pipe 3 and the fourth pipe 4, the liquid in the main pipe 7 is sheared, thereby achieving the inclusion and cutting of the liquid flowing inside the main pipe 7 into droplets. Thus, the liquid in the main pipe 7 can meet the requirements of subsequent processing.

[0053] The intersection between the first pipeline 1 and the main pipeline 7 forms a first connection, and the intersection between the second pipeline 2 and the main pipeline 7 forms a second connection; preferably, the first connection and the second connection coincide with each other.

[0054] In a specific embodiment, the first connection point and the second connection point are staggered, and the first connection point is located upstream of the second connection point.

[0055] In another specific embodiment, the first connection and the second connection are staggered, and the first connection is located downstream of the second connection.

[0056] In a specific embodiment, the first connection point and the second connection point are arranged to overlap.

[0057] The liquid in the first pipe 1 and the second pipe 2 is used to wrap the liquid in the main pipe 7, so the first connection point and the second connection point can be staggered so that the liquid in the first pipe 1 and the second pipe 2 can wrap the liquid inside the main pipe 7 on both sides.

[0058] refer to Figure 1 and Figure 2 In a specific embodiment, the diameter of the main pipeline 7 gradually increases from the upstream to the downstream.

[0059] Since the liquid inside the first pipe 1 and the second pipe 2 is connected to the main pipe 7 and flows into the main pipe 7, the amount of liquid in the main pipe 7 increases, so that the diameter of the main pipe 7 gradually increases. When the liquid flows in the main pipe 7, as the diameter of the main pipe 7 increases, the liquid flow rate in the main pipe 7 will also decrease accordingly. Thus, the liquid flow rate in the main pipe 7 can be controlled within a reasonable range, making it easier to wrap or cut the liquid.

[0060] refer to Figure 1 and Figure 2 , the diameters of the first pipe 1 and the second pipe 2 are equal or similar.

[0061] Since the first pipe 1 and the second pipe 2 are respectively located at the two sides of the main pipe 7 and respectively input liquid into the main pipe 7, in order to make the first pipe 1 and the second pipe 2 evenly wrap the liquid in the main pipe 7 when inputting liquid into the main pipe 7, the diameters of the first pipe 1 and the second pipe 2 are set to be equal or similar. This makes it possible for the liquid in the first pipe 1 and the second pipe 2 to wrap the liquid in the subject more evenly, and also realizes the encirclement and holding of the liquid in the main pipe 7.

[0062] The diameter of the first pipe or the second pipe is 50 μm to 150 μm.

[0063] Specifically, the diameter of the first pipe or the second pipe is: 50μm, 51μm, 52μm, 53μm, 54μm, 55μm, 56μm, 57μm, 58μm, 59μm, 60μm, 61μm, 62μm, 63μm, 64μm, 65μm, 66μm, 67μm, 68μm, 69μm, 70μm, 71μm , 72μm, 73μm, 74μm, 75μm, 76μm, 77μm, 78μm, 79μm, 80μm, 81μm, 82μm, 83μm, 84μm m, 85μm, 86μm, 87μm, 88μm, 89μm, 90μm, 91μm, 92μm, 93μm, 94μm, 95μm, 96μm, 97μm m, 98μm, 99μm, 100μm, 101μm, 102μm, 103μm, 104μm, 105μm, 106μm, 107μm, 108μm m, 109μm, 110μm, 111μm, 112μm, 113μm, 114μm, 115μm, 116μm, 117μm, 118μm, 119 μm, 130μm, 131μm, 132μm, 133μm, 134μm, 135μm, 136μm, 137μm, 138μm, 139μm, 1 40μm, 141μm, 142μm, 143μm, 144μm, 145μm, 146μm, 147μm, 148μm, 149μm, 150μm.

[0064] refer to Figure 1 and Figure 2 , a core liquid flows in the main pipeline 7, a first liquid flows in the first pipeline 1, and a second liquid flows in the second pipeline 2; preferably, the first liquid and the second liquid both flow toward the inside of the main pipeline 7; further preferably, the core liquid and the first liquid and the second liquid merge at the first connection and / or the second connection to form a fifth liquid.

[0065] The inner core liquid flows in the main pipeline 7 at an upstream position of the first pipeline 1 and the second pipeline 2, and then after passing through the first pipeline 1 and the second pipeline 2, the inner core liquid is wrapped by the first liquid and the second liquid to form a fifth liquid.

[0066] In one embodiment, the first liquid and the second liquid are the same liquid.

[0067] In another embodiment, the first liquid and the second liquid may be different liquids.

[0068] The first liquid and the second liquid are both liquids that are not easily soluble in the inner core liquid, so the first liquid and the second liquid can wrap the inner core liquid at the same time.

[0069] The flow rate of the inner core liquid in the main pipeline is 1 to 20 μL / min; preferably, the flow rate of the inner core liquid in the main pipeline is 2 to 10 μL / min.

[0070] Specifically, the flow rate of the inner core liquid in the main pipeline is: 1μL / min, 2μL / min, 3μL / min, 4μL / min, 5μL / min, 6μL / min, 7μL / min, 8μL / min, 9μL / min, 10μL / min, 11μL / min, 12μL / min, 13μL / min, 14μL / min, 15μL / min, 16μL / min, 17μL / min, 18μL / min, 19μL / min, and 20μL / min.

[0071] The flow rate of the first liquid in the first pipe is 1 to 10 μL / min; more preferably, the flow rate of the first liquid in the first pipe is 2 to 5 μL / min.

[0072] Specifically, the flow rate of the first liquid in the first pipeline is 1 μL / min, 2 μL / min, 3 μL / min, 4 μL / min, 5 μL / min, 6 μL / min, 7 μL / min, 8 μL / min, 9 μL / min, and 10 μL / min.

[0073] The flow rate of the second liquid in the second pipe is 1 to 10 μL / min; more preferably, the flow rate of the second liquid in the second pipe is 2 to 5 μL / min.

[0074] Specifically, the flow rate of the second liquid in the second pipe is 1 μL / min, 2 μL / min, 3 μL / min, 4 μL / min, 5 μL / min, 6 μL / min, 7 μL / min, 8 μL / min, 9 μL / min, and 10 μL / min.

[0075] refer to Figure 1 and Figure 2 When the core liquid and the first liquid and the second liquid are used to prepare the droplet microspheres at different flow rates, when the flow rate of the core liquid is relatively large, the size of the core phase of the prepared droplet microspheres is larger, and the thickness of the shell phase is smaller. Therefore, in the present application, the flow rate of the core liquid and the flow rates of the first liquid and the second liquid are controlled within a reasonable range, so that the core phase and the shell phase of the prepared droplet microspheres have appropriate sizes.

[0076] refer to Figure 1 and Figure 2 , the fifth liquid flows in the main pipeline 7.

[0077] After the inner core liquid is wrapped to form the fifth liquid, it continues to flow downstream in the main pipeline 7, and then the third pipeline 3 and / or the fourth pipeline 4 connected to the main pipeline 7 can cut the fifth liquid at the downstream position of the fifth liquid, so that the fifth liquid can form droplets (i.e., microspheres).

[0078] refer to Figure 1 and Figure 2 , the flow rate of the first liquid is equal to or similar to the flow rate of the second liquid; preferably, the cross-sectional diameter and flow rate of the first liquid and the second liquid are equal to or similar.

[0079] In order to make the first liquid and the second liquid wrap the inner core liquid more uniformly, the flow diameters of the first liquid and the second liquid are controlled to be equal or close to each other. In this way, after the first liquid and the second liquid flow into the main pipeline 7, they will be evenly diffused to the periphery of the inner core liquid and evenly wrap the inner core liquid.

[0080] The third pipeline 3 and the fourth pipeline 4 are intersecting and communicating with each other on the main pipeline 7;

[0081] Preferably, the third pipeline 3 and the fourth pipeline 4 are relatively arranged on both sides of the main pipeline 7 .

[0082] In a specific embodiment, the main pipeline 7 is only connected to the third pipeline 3 .

[0083] In a specific embodiment, the main pipeline 7 is only connected to the fourth pipeline 4 .

[0084] In a specific embodiment, the main pipeline 7 is connected to the third pipeline 3 and the fourth pipeline 4 .

[0085] Since the third pipe 3 and the fourth pipe 4 are used to cut the fifth liquid in the main pipe 7, when only one of the third pipe 3 or the fourth pipe 4 is provided, the cutting effect can also be achieved on the liquid in the main pipe 7. Preferably, the third pipe 3 and the fourth pipe 4 are provided on the main pipe 7 at the same time, and the third pipe 3 and the fourth pipe 4 are relatively provided on both sides of the main pipe 7, and the third pipe 3 and the fourth pipe 4 are used to cut the liquid in the subject at the same time, so as to improve the cutting efficiency, and further, the flow of the liquid in the third pipe 3 and the fourth pipe 4 can be controlled to realize the control of the state such as the interval or size of the droplets.

[0086] refer to Figure 1 and Figure 2 , the diameter of the third pipeline 3 is equal to or close to the diameter of the fourth pipeline 4 ; preferably, the diameters of the third pipeline 3 and the fourth pipeline 4 are both less than or equal to the diameter of the main pipeline 7 .

[0087] The intersection of the third pipeline 3 and the main pipeline 7 forms a third connection point, and the intersection of the fourth pipeline 4 and the main pipeline 7 forms a fourth connection point; preferably, the third connection point and the fourth connection point overlap.

[0088] A third liquid flows inside the third pipe 3 , and a fourth liquid flows inside the fourth pipe 4 . The third liquid and the fourth liquid can shear the liquid in the main pipe 7 into droplets.

[0089] The diameter of the third pipe or the fourth pipe is 50 μm to 200 μm.

[0090] Specifically, the diameter of the third pipe or the fourth pipe is: 50μm, 51μm, 52μm, 53μm, 54μm, 55μm, 56μm, 57μm, 58μm, 59μm, 60μm, 61μm, 62μm, 63μm, 64μm, 65μm, 66μm, 67μm, 68μm, 69μm, 70μm, 71μm, 72μm, 73μm, 74μm, 75μm, 76μm, 77μm, 78μm, 79μm, 80μm, 81μm, 82μm, 83μm, 84μm, 85μm, 86μm , 87μm, 88μm, 89μm, 90μm, 91μm, 92μm, 93μm, 94μm, 95μm, 96μm, 97μm, 98μm, 99μm, 100μm, 101μm, 102μm, 103μm, 104μm, 105μm, 106μm, 107μm, 108μm, 109μm, 110μm, 111μm, 112μm, 113μm, 114μm, 115μm, 116μm, 117μm, 118μm, 119μm, 130μm, 131μm, 132μm, 133μm, 134μm, 135μm, 136μm, 137μm, 138μm, 139μm, 140μm, 141μm, 142μm, 143μm, 144μm, 145μm, 146μm, 147μm, 148μm, 149μm, 150μm, 151μm, 152μm, 153μm, 154μm, 155μm, 156μm, 157μm, 158μm, 159μm, 160μm, 161μm, 162μm, 163μm, 164μm, 165μm, 166μm, 167μm, 168μm, 169μm, 170μm, 171μm, 172μm, 173μm, 174μm, 175μm, 176μm, 177μm, 178μm, 179μm, 180μm, 181μm, 182μm, 183μm, 184μm, 185μm, 186μm, 187μm, 188μm, 189μm, 190μm, 191μm, 192μm, 193μm, 194μm, 195μm, 196μm, 197μm, 198μm, 199μm, 200μm.

[0091] refer to Figure 1 and Figure 2 In one embodiment, the diameters of the third pipe and the fourth pipe are larger than the diameters of the first pipe and the second pipe.

[0092] The third pipe 3 and the fourth pipe 4 are formed by passing liquid into the main pipe 7, so that the fifth liquid in the main pipe 7 is cut by the impact of the third liquid and the fourth liquid, thereby forming a droplet state.

[0093] In order to improve the cutting effect of the fifth liquid, the diameters of the third pipe 3 and the fourth pipe 4 are set to be equal or similar, so that the liquids in the third pipe 3 and the fourth pipe 4 can flow into the main pipe 7 at equal flow rates and exert equal cutting effects on the fifth liquid, thereby reducing the possibility that the fifth liquid in the main pipe 7 is scattered by the impact of the third liquid or the fourth liquid due to the difference in liquid flow in the third pipe 3 or the fourth pipe 4.

[0094] The distance from the first pipeline to the third pipeline or the fourth pipeline is 500 μm to 1500 μm; more preferably, the distance from the first pipeline to the third pipeline or the fourth pipeline is 800 μm to 1200 μm.

[0095] Specifically, the distance from the first pipeline to the third pipeline or the fourth pipeline is: 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm, 1000μm, 1050μm, 1100μm, 1150μm, 1200μm, 1250μm, 1300μm, 1350μm, 1400μm, 1450μm, 1500μm.

[0096] The flow rate of the third liquid in the third pipe is 1 to 5 μL / min;

[0097] Specifically, the flow rate of the third liquid in the third pipe is: 1 μL / min, 1.5 μL / min, 2 μL / min, 2.5 μL / min, 3 μL / min, 3.5 μL / min, 4 μL / min, 4.5 μL / min, and 5 μL / min.

[0098] The flow rate of the fourth liquid in the fourth pipe is 1-5 μL / min.

[0099] Specifically, the flow rate of the fourth liquid in the fourth pipe is 1 μL / min, 1.5 μL / min, 2 μL / min, 2.5 μL / min, 3 μL / min, 3.5 μL / min, 4 μL / min, 4.5 μL / min, and 5 μL / min.

[0100] The main pipeline 7 is connected with a fifth pipeline 5 and / or a sixth pipeline 6;

[0101] refer to Figure 1 and Figure 2Preferably, a fifth pipeline 5 and a sixth pipeline 6 are connected to the main pipeline 7 in an intersecting state;

[0102] Preferably, the fifth pipeline 5 and the sixth pipeline 6 are relatively arranged on both sides of the main pipeline 7;

[0103] Further preferably, the fifth pipeline 5 and the sixth pipeline 6 are both located downstream of the third pipeline 3 and the fourth pipeline 4 .

[0104] The sixth liquid flows inside both the fifth pipe 5 and the sixth pipe 6 .

[0105] In a specific embodiment, the main pipeline 7 is only connected to the fifth pipeline 5 .

[0106] In a specific embodiment, the main pipeline 7 is only connected to the sixth pipeline 6 .

[0107] In a specific embodiment, the main pipeline 7 is connected to the fifth pipeline 5 and the sixth pipeline 6 at the same time.

[0108] refer to Figure 1 and Figure 2 After the fifth liquid is cut into droplets by the third liquid and the fourth liquid, a sixth liquid can be arranged at the downstream position of the fifth liquid, and the droplets of the fourth liquid are treated with the sixth liquid, so that the first liquid and the second liquid wrap the liquid of the core liquid for secondary treatment. The droplets of the fifth liquid can achieve the target effect. At the same time, in order to reduce the damage effect of the sixth liquid on the droplets in the fifth pipe 5 and the sixth pipe 6, the fifth pipe 5 and the sixth pipe 6 are respectively arranged on opposite sides of the main pipe 7. Reduce the impact effect of the sixth liquid on the liquid.

[0109] The diameters of the fifth pipe and the sixth pipe are 200-400 μm.

[0110] Specifically, the diameters of the fifth pipe and the sixth pipe are: 200μm, 201μm, 202μm, 203μm, 204μm, 205μm, 206μm, 207μm, 208μm, 209μm, 210μm, 211μm, 212μm, 213μm, 214μm, 215μm, 216μm, 217μm, 218μm, 219μm, 230μm, 231μm, 232μm, 233μm, 234μm, 235μm, 236μm, 237μm, 238μm, 239μm, 240μm, 241μm, 242μm, 243μm, 244μm, 245μm, 246μm, 247μm, 248 μm, 249μm, 250μm, 251μm, 252μm, 253μm, 254μm, 255μm, 256μm, 257μm, 258μm , 259μm, 260μm, 261μm, 262μm, 263μm, 264μm, 265μm, 266μm, 267μm, 268μm, 2 69μm, 270μm, 271μm, 272μm, 273μm, 274μm, 275μm, 276μm, 277μm, 278μm, 279 μm, 280μm, 281μm, 282μm, 283μm, 284μm, 285μm, 286μm, 287μm, 288μm, 289μm, 290μm, 291μm, 292μm, 293μm, 294μm, 295μm, 296μm, 297μm, 298μm, 299μm, 30 0μm, 301μm, 302μm, 303μm, 304μm, 305μm, 306μm, 307μm, 308μm, 309μm, 310μm m, 311μm, 312μm, 313μm, 314μm, 315μm, 316μm, 317μm, 318μm, 319μm, 320μm, 321μm, 322μm, 323μm, 324μm, 325μm, 326μm, 327μm, 328μm, 329μm, 330μm, 331 μm, 332μm, 333μm, 334μm, 335μm, 336μm, 337μm, 338μm, 339μm, 340μm, 341μm , 342μm, 343μm, 344μm, 345μm, 346μm, 347μm, 348μm, 349μm, 350μm, 351μm, 3 52μm, 353μm, 354μm, 355μm, 356μm, 357μm, 358μm, 359μm, 360μm, 361μm, 362 μm, 363μm, 364μm, 365μm, 366μm, 367μm, 368μm, 369μm, 370μm, 371μm, 372μm,373μm, 374μm, 375μm, 376μm, 377μm, 378μm, 379μm, 380μm, 381μm, 382μm, 383μm, 384μm, 385μm, 386μm, 387μm, 388μm, 389μm, 390μm, 391μm, 392μm, 393μm, 394μm, 395μm, 396μm, 397μm, 398μm, 399μm, 400μm. ,

[0111] In a specific embodiment, the diameters of the fifth and sixth pipes are greater than the diameters of the third and fourth pipes.

[0112] The distance from the third pipeline to the fifth pipeline or the sixth pipeline is 500 μm to 1500 μm; further preferably, the distance from the first pipeline to the third pipeline or the fourth pipeline is 800 μm to 1200 μm.

[0113] Specifically, the distance from the first pipeline to the third pipeline or the fourth pipeline is: 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm, 1000μm, 1050μm, 1100μm, 1150μm, 1200μm, 1250μm, 1300μm, 1350μm, 1400μm, 1450μm, 1500μm.

[0114] The flow rate (or flow rate) of the fifth liquid in the fifth conduit is 1 to 5 μL / min;

[0115] Specifically, the flow rate of the fifth liquid in the fifth pipe is: 1 μL / min, 1.5 μL / min, 2 μL / min, 2.5 μL / min, 3 μL / min, 3.5 μL / min, 4 μL / min, 4.5 μL / min, and 5 μL / min.

[0116] The flow rate (or flow rate) of the sixth liquid in the sixth pipe is 1 to 5 μL / min;

[0117] Specifically, the flow rates of the sixth liquid in the sixth pipe are: 1 μL / min, 1.5 μL / min, 2 μL / min, 2.5 μL / min, 3 μL / min, 3.5 μL / min, 4 μL / min, 4.5 μL / min, and 5 μL / min.

[0118] The chip further comprises a first introduction pipe arranged upstream of the main pipe 7, through which the liquid flows into the main pipe 7;

[0119] Preferably, the chip further comprises a second introduction pipe, wherein the second introduction pipe is connected to the first pipe 1 and the second pipe 2;

[0120] Preferably, the chip further comprises a third introduction pipe, and the third introduction pipe is connected with the third pipe 3 and the fourth pipe 4;

[0121] Preferably, the chip further comprises a fourth introduction pipe, and the fourth introduction pipe is connected with the fifth pipe 5 and the sixth pipe 6 .

[0122] refer to Figure 1 and Figure 2 The first introduction pipe, the second introduction pipe, the third introduction pipe and the fourth introduction pipe are respectively used to introduce liquid into each pipe on the chip, so that the core liquid can smoothly enter the main pipe 7. The first liquid can enter the first pipe 1, the second liquid can enter the second pipe 2, the third liquid can enter the third pipe 3, the fourth liquid can enter the fourth pipe 4, and the sixth liquid can enter the fifth pipe 5 and the sixth pipe 6.

[0123] refer to Figure 1 and Figure 2 In one embodiment, the first liquid and the second liquid are the same liquid, and thus can be introduced simultaneously using the second introduction pipe. The third liquid and the fourth liquid are the same liquid, and can be introduced simultaneously using the third introduction pipe. The difference between the first liquid and the second liquid and the difference between the third liquid and the fourth liquid are further reduced. The purity of the manufactured liquid is improved, and impurities are reduced.

[0124] The diameter of the main pipeline gradually increases along the first pipeline, the third pipeline, and the fifth pipeline.

[0125] Specifically, the diameter of the main pipeline between the first pipeline and the third pipeline is larger than the diameter of the main pipeline at the position where the first pipeline is away from the third pipeline (ie, the main pipeline is at the upstream position of the first pipeline).

[0126] Similarly, the diameter of the main conduit between the third and fifth conduits is greater than the diameter of the main conduit between the first and third conduits. The diameter of the main conduit downstream of the fifth conduit is greater than the diameter of the main conduit between the third and fourth conduits.

[0127] The diameter of the main pipeline at the upstream position of the first pipeline is 200 μm to 250 μm;

[0128] Specifically, the diameter of the main pipeline at the upstream position of the first pipeline is: 200μm, 201μm, 202μm, 203μm, 204μm, 205μm, 206μm, 207μm, 208μm, 209μm, 210μm, 211μm, 212μm, 213μm, 214μm, 215μm, 216μm, 217μm, 218μm m, 219μm, 230μm, 231μm, 232μm, 233μm, 234μm, 235μm, 236μm, 237μm, 238μm, 239μm , 240μm, 241μm, 242μm, 243μm, 244μm, 245μm, 246μm, 247μm, 248μm, 249μm, 250μm.

[0129] The diameter of the main pipeline between the first pipeline and the third pipeline is: 200 μm to 300 μm;

[0130] Specifically, the diameter of the main pipeline between the first pipeline and the third pipeline is: 200μm, 201μm, 202μm, 203μm, 204μm, 205μm, 206μm, 207μm, 208μm, 209μm, 210μm, 211μm, 212μm, 213μm, 214μm, 215μm, 216μm, 217μm, 218μm, 219μm m, 230μm, 231μm, 232μm, 233μm, 234μm, 235μm, 236μm, 237μm, 238μm, 239μm, 240μm, 241 μm, 242μm, 243μm, 244μm, 245μm, 246μm, 247μm, 248μm, 249μm, 250μm, 251μm, 252μm, 253 μm, 254μm, 255μm, 256μm, 257μm, 258μm, 259μm, 260μm, 261μm, 262μm, 263μm, 264μm, 26 5μm, 266μm, 267μm, 268μm, 269μm, 270μm, 271μm, 272μm, 273μm, 274μm, 275μm, 276μm, 2 77μm, 278μm, 279μm, 280μm, 281μm, 282μm, 283μm, 284μm, 285μm, 286μm, 287μm, 288μm, 289μm, 290μm, 291μm, 292μm, 293μm, 294μm, 295μm, 296μm, 297μm, 298μm, 299μm, 300μm.

[0131] The diameter of the main pipeline between the third pipeline and the fifth pipeline is 250 μm to 350 μm.

[0132] Specifically, the diameter of the main pipeline between the third pipeline and the fifth pipeline is 251μm, 252μm, 253μm, 254μm, 255μm, 256μm, 257μm, 258μm, 259μm, 260μm, 261μm, 262μm, 263μm, 264μm, 265μm, 266μm, 267μm, 268μm, 269μm, 270μm, 271μm, 272μm, 273μm, 274μm, 275μm, 276μm, 277μm, 278μm, 279μm, 280μm, 281μm, 282μm, 283μm, 284μm, 285μm, 286μm, 287μm, 288μm, 289μm, 290μm, 291μm, 292μm, 293μm, 294μm, 295μm, 296μm, 297μm, 298μm, 299μm, 300μm, 301μm, 302μm, 303μm, 304μm, 305μm, 306μm, 307μm, 308μm, 309μm, 310μm, 311μm, 312μm, 313μm, 314μm, 315μm, 316μm, 317μm, 318μm, 319μm, 320μm, 321μm, 322μm, 323μm, 324μm, 325μm, 326μm, 327μm, 328μm, 329μm, 330μm, 331μm, 332μm, 333μm, 334μm, 335μm, 336μm, 337μm, 338μm, 339μm, 340μm, 341μm, 342μm, 343μm, 344μm, 345μm, 346μm, 347μm, 348μm, 349μm, 350μm.

[0133] The diameter of the main pipeline at the downstream position of the fifth pipeline is 400 μm to 600 μm.

[0134] Specifically, the diameter of the main pipeline at the downstream position of the fifth pipeline is: 400μm, 405μm, 410μm, 415μm, 420μm, 425μm, 430μm, 435μm, 440μm, 445μm, 450μm, 455μm, 460μm, 465μm, 470μm, 475μm, 480μm, 485μm, 490 μm, 495μm, 500μm, 505μm, 510μm, 515μm, 520μm, 525μm, 530μm, 535μm, 540μm, 545μm m, 550μm, 555μm, 560μm, 565μm, 570μm, 575μm, 580μm, 585μm, 590μm, 595μm, 600μm.

[0135] In summary, the present application provides a droplet microfluidic chip. When in use, the core liquid to be wrapped is input into the main pipeline 7, and then the first liquid and the second liquid to form the shell are transported to the second pipeline 2 and the third pipeline 3, and then at the position of the first connection, the first liquid and the second liquid wrap the core liquid, thereby forming a fifth liquid that wraps the core liquid. The fifth liquid continues to flow in the main pipeline 7 until the position of the third pipeline 3 and the fourth pipeline 4, and the third liquid and the fourth liquid flowing into the third pipeline 3 and the fourth pipeline 4 flow into the main pipeline 7, and produce a shearing effect on the main pipeline 7, and the fifth liquid in the main pipeline 7 is sheared into a droplet shape. And as the third liquid and the fourth liquid continue to flow. Subsequently, the sixth liquid flowing in the fifth pipeline 5 and the sixth pipeline 6 enters the main pipeline 7, and the sixth liquid wraps the liquid of the fifth liquid, so that the droplets of the fifth liquid achieve the expected effect.

[0136] Example

[0137] The main pipeline, the first pipeline, the second pipeline, the third pipeline, the fourth pipeline, the fifth pipeline and the sixth pipeline are pipelines formed in the chip substrate. The first pipeline and the second pipeline are coaxial, the third pipeline and the fourth pipeline are coaxial, and the fifth pipeline and the sixth pipeline are coaxial. The size of the chip substrate is 3cm*4cm; the thickness of the substrate is 1-10mm, and the material used for the chip substrate is PDMS.

[0138] The main pipeline formed in the chip substrate is a pipeline with a circular or horseshoe-shaped cross section, and its cross section is 225-500 μm and gradually increases. The first pipeline and the second pipeline formed in the chip substrate are pipelines with a circular or horseshoe-shaped cross section, and their cross-sectional diameter is 100 μm. The third pipeline and the fourth pipeline formed in the chip substrate are pipelines with a circular or horseshoe-shaped cross section, and their cross-sectional diameter is 125 μm. The fifth pipeline and the sixth pipeline formed in the chip substrate are pipelines with a circular or horseshoe-shaped cross section, and their cross-sectional area is 280 μm.

[0139] The flow rate of the inner core liquid in the main pipeline is 4-10 μL / min; the flow rate of the first liquid and the second liquid is 4 μL / min; the flow rate of the third liquid and the fourth liquid is 2 μL / min; the flow rate of the fifth liquid and the sixth liquid is 2 μL / min.

[0140] The main pipeline is connected to the first pipeline, the second pipeline, the third pipeline, the fourth pipeline, the fifth pipeline and the sixth pipeline respectively. The first pipeline and the second pipeline are located upstream of the third pipeline and the fourth pipeline, and the third pipeline and the fourth pipeline are located upstream of the fifth pipeline and the sixth pipeline. The first pipeline and the second pipeline are 950 μm away from the third pipeline and the fourth pipeline. The third pipeline and the fourth pipeline are 1050 μm away from the fifth pipeline and the sixth pipeline.

[0141] The diameter of the main pipe upstream of the first pipe is 225 μm; the diameter of the main pipe between the first and third pipes is 250 μm; the diameter of the main pipe between the third and fifth pipes is 300 μm; and the diameter of the main pipe downstream of the fifth pipe is 500 μm.

[0142] The first introduction pipe is connected to a peristaltic pump A, the second introduction pipe is connected to a peristaltic pump B, the third introduction pipe is connected to a peristaltic pump C, and the fourth introduction pipe is connected to a peristaltic pump D. The peristaltic pumps A to D are connected to corresponding liquid solutions, respectively.

[0143] Peristaltic pump A is connected to a tank containing matrix gel and a tank containing hiPSCs.

[0144] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A droplet microfluidic chip, characterized in that: It comprises a main pipeline with liquid flowing therein, and a first pipeline and a second pipeline; the first pipeline and the second pipeline are respectively connected to the main pipeline, and the first pipeline and the second pipeline are respectively located at two opposite sides of the main pipeline; the same liquid flows in the first pipeline and the second pipeline; A third pipeline and / or a fourth pipeline are intersected and connected with the main pipeline; The third pipe and / or the fourth pipe can divide the continuously flowing liquid in the main pipe into droplets; The third pipeline and / or the fourth pipeline are arranged at a downstream position of the first pipeline and the second pipeline.

2. The chip according to claim 1, characterized in that: The intersection between the first pipeline and the main pipeline forms a first connection, and the intersection between the second pipeline and the main pipeline forms a second connection; preferably, the first connection and the second connection coincide with each other.

3. The chip according to claim 1 or 2, characterized in that: The diameter of the main pipeline gradually increases from upstream to downstream.

4. The chip according to claim 1, characterized in that: The diameters of the first pipe and the second pipe are equal or similar.

5. The chip according to claim 2, characterized in that: A core liquid flows in the main pipe, a first liquid flows in the first pipe, and a second liquid flows in the second pipe; preferably, the first liquid and the second liquid both flow toward the inside of the main pipe; further preferably, the core liquid and the first liquid and the second liquid merge at the first connection and / or the second connection to form a fifth liquid; More preferably, the flow rate of the inner core liquid in the main pipeline is 1 to 20 μL / min; more preferably, the flow rate of the inner core liquid in the main pipeline is 4 to 10 μL / min.

6. The chip according to claim 5, characterized in that: The fifth liquid flows in the main pipe.

7. The chip according to claim 5, characterized in that: The flow rate of the first liquid is equal to or similar to the flow rate of the second liquid; preferably, the cross-sectional diameter and flow rate of the first liquid and the second liquid are equal to or similar; More preferably, the flow rate of the first liquid in the first pipe is 1 to 10 μL / min; More preferably, the flow rate of the first liquid in the first pipe is 2 to 5 μL / min; More preferably, the flow rate of the second liquid in the second pipe is 1 to 10 μL / min; more preferably, the flow rate of the second liquid in the second pipe is 2 to 5 μL / min.

8. The chip according to any one of claims 1 to 7, characterized in that: A third pipeline and a fourth pipeline are intersected and connected on the main pipeline; Preferably, the third pipeline and the fourth pipeline are relatively arranged on both sides of the main pipeline.

9. The chip according to claim 8, characterized in that: The diameter of the third pipeline is equal to or similar to the diameter of the fourth pipeline; preferably, the diameters of the third pipeline and the fourth pipeline are both smaller than or equal to the diameter of the main pipeline.

10. The chip according to claim 8, characterized in that: The intersection of the third pipeline and the main pipeline forms a third connection, and the intersection of the fourth pipeline and the main pipeline forms a fourth connection; preferably, the third connection and the fourth connection overlap.

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