Micro-fluidic chip for preparing artificial cells, artificial cell preparation method and application

Abstract

The invention discloses a micro-fluidic chip for preparing an artificial cell, and a preparation method and application of the artificial cell. According to the present invention, the multiple liquid inlet flow channels in the microfluidic chip cooperate to form the first shearing opening and the second shearing opening, each solution sequentially flows through the first shearing opening and the second shearing opening, and is sheared at the first shearing opening and the second shearing opening so as to generate the stable double-emulsion liquid drop, such that the uniformity and the stability of the subsequent artificial cell generation can be improved. The double-emulsion liquid drops are captured through the capture array, so that the external phase and the buffer solution are stably replaced, the organic solvent in the double-emulsion is removed, the artificial cells are formed, the confluence opportunity between the solutions does not need to be strictly controlled, and the operation is easy. The corresponding preparation method and a solution system (capable of improving a formula, osmotic pressure and interfacial tension) with biocompatibility and stability can effectively solve the problems of nonuniformity, low encapsulation efficiency, poor stability, chip blockage, high operation requirements, sample loss and the like in the artificial cell generation process.

Description

Technical Field

[0001] The present invention relates to the technical field of artificial cell preparation, and in particular to a microfluidic chip for preparing artificial cells, an artificial cell preparation method and applications. Background Art

[0002] Artificial cells (ACs), also known as synthetic cells, are lipid or polymer vesicles of cell size that can simulate one or more cell characteristics. Due to the bottom-up construction method of artificial cells, their components are clear and highly customizable, making artificial cells not only provide a new model for personalized drug screening, but also provide an ideal experimental platform for basic scientific research on protein-lipid interactions.

[0003] Existing methods for preparing artificial cells include: (1) reverse emulsification method, which mainly obtains water-in-oil (W / O) droplets through vortex or scraper, and then passes the water-in-oil droplets through the oil-water interface through centrifugal force to form artificial cells; (2) electro-generation method, which induces the self-assembly of phospholipid bilayers to form vesicles through electric fields; (3) repeated freezing and thawing method, which promotes the formation of phospholipid bilayers through multiple freezing and thawing. However, these traditional methods have obvious shortcomings, such as uneven size of prepared artificial cells and low encapsulation rate of contents. In contrast, the microfluidic method manipulates the fluid in a micron-scale chip, which can generate vesicles with more uniform size and higher encapsulation rate. In the current microfluidic technology, confocal flow channel structure is mainly used to generate vesicles. During the operation, the injection timing of different types of fluids needs to be highly synchronized to ensure sufficient constraints at the confocal confluence point to shear and form stable droplets. Any slight deviation in the injection timing may result in uneven size of the generated droplets or even failure to form. In addition, the current microfluidic method for preparing vesicles has the problem of residual organic solvents. The residual organic solvents in the intermediate phase prevent the self-assembly of the phospholipid bilayer, thereby affecting the transmembrane of membrane proteins and also changing the properties of the phospholipid membrane (such as the division and growth of artificial cells). Therefore, there is still a lack of a method that can efficiently achieve artificial cell preparation on a single chip. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a microfluidic chip for preparing artificial vesicles, which is easy to operate and generates vesicles with high uniformity and stability.

[0005] The present invention also provides a method for preparing artificial cells with the above-mentioned microfluidic chip. The method is efficient, low-cost, and easy to operate, and can generate artificial cells with both biocompatibility and stability.

[0006] The present invention also proposes an application of the microfluidic chip in preparing artificial cells, designing microfluidic droplet systems, verifying membrane protein functions, studying cell interaction mechanisms, and drug screening.

[0007] A microfluidic chip for preparing artificial cells according to the first aspect of the present invention comprises:

[0008] A first liquid inlet flow channel, one end of which is provided with a first liquid inlet, wherein the first liquid inlet is used to introduce the oil phase solution;

[0009] A second liquid inlet channel, one end of which is provided with a second liquid inlet, and the other end is connected with an end of the first liquid inlet channel away from the first liquid inlet to form a first shearing opening, the second liquid inlet is used to pass the inner water phase solution, the inner water phase solution and the oil phase solution form water-in-oil droplets at the first shearing opening;

[0010] A first intermediate flow channel, one end of which is connected to the first shear opening;

[0011] A third liquid inlet flow channel, one end of which is provided with a third liquid inlet, and the other end is connected with an end of the first intermediate flow channel away from the first shearing opening to form a second shearing opening, the third liquid inlet is used to introduce the external aqueous phase solution, and the water-in-oil droplets and the external aqueous phase solution form double emulsion droplets at the second shearing opening;

[0012] A capture array, wherein the capture array is provided with an inlet and an outlet, wherein the inlet is communicated with the second shear opening and is used to capture the double emulsion droplets, and the outlet is used to discharge the solution in the capture array;

[0013] A fourth liquid inlet channel has a fourth liquid inlet at one end and is connected to the capture array at the other end. The fourth liquid inlet is used to introduce a buffer solution, and the buffer solution can replace the external phase of the double emulsion droplets to separate the organic solvent in the double emulsion droplets and form artificial cells.

[0014] The microfluidic chip for preparing artificial cells according to the embodiment of the present invention has at least the following beneficial effects:

[0015] Multiple liquid inlet channels in the microfluidic chip cooperate to form a first shearing port and a second shearing port. When preparing artificial cells, each solution flows through the first shearing port and the second shearing port in sequence and is sheared at the first shearing port and the second shearing port to generate stable double emulsion droplets, which can improve the consistency and stability of subsequently generated artificial cells. In addition, the double emulsion droplets are captured by a capture array, so that the external phase of the double emulsion droplets is stably replaced with the buffer solution, and the organic solvent in the double emulsion is released to form artificial cells. In the process of preparing artificial cells, there is no need to strictly control the convergence timing between the solutions, which is easy to operate.

[0016] According to some embodiments of the present invention, each of the first liquid inlet channel and the second liquid inlet channel includes at least one curved portion.

[0017] According to some embodiments of the present invention, the first liquid inlet channel is perpendicular to the second liquid inlet channel.

[0018] According to some embodiments of the present invention, the third liquid inlet channel is perpendicular to the first intermediate channel.

[0019] According to some embodiments of the present invention, the capture array includes a plurality of capture units arranged at intervals along a first direction and a second direction, the first direction intersects with the second direction, each of the capture units is provided with a capture chamber and a capture port connected to the capture chamber, the capture port is arranged on a side of the capture chamber facing the inlet, and the capture chamber is used to accommodate the double emulsion droplets.

[0020] According to some embodiments of the present invention, the capture port is disposed on one side of the capture unit along the first direction, and projections of the capture units adjacent to each other along the first direction formed along the first direction are staggered in the second direction.

[0021] According to some embodiments of the present invention, the capture unit is further provided with a drainage port, which is connected to the capture chamber, and the capture port is arranged on one side of the capture unit along the first direction, wherein the drainage port is arranged on the side of the capture unit facing away from the capture port, and / or the drainage port is arranged on two opposite sides of the capture unit along the second direction.

[0022] According to some embodiments of the present invention, the opening of the capture port is 30-50 μm.

[0023] According to some embodiments of the present invention, a cross-sectional area of ​​at least a portion of the capture cavity gradually increases from the capture port along the first direction.

[0024] The method for preparing an artificial cell according to the second aspect of the present invention comprises:

[0025] Prepare the above-mentioned microfluidic chip, the oil phase solution, the inner aqueous phase solution, the outer aqueous phase solution and the buffer solution;

[0026] Performing surface treatment on the flow channel inside the microfluidic chip so that the surface of the first intermediate flow channel is hydrophobic and the surface of the second intermediate flow channel connected between the inlet and the second shear opening is hydrophilic;

[0027] The oil phase solution, the inner water phase solution, the outer water phase solution and the buffer solution are introduced into the microfluidic chip to prepare and capture artificial cells.

[0028] The method for preparing artificial cells according to the embodiment of the present invention has at least the following beneficial effects:

[0029] The present invention provides an operation process corresponding to the above-mentioned microfluidic chip, and provides a scheme for adapting the introduction of ultra-trace solutions. The double T structure design of the chip is easier to operate, and the channels for adding capture arrays and buffer solutions remove organic solvents more gently, and the selection of oil phase solvents makes biocompatibility better. The method can effectively solve the problems of heterogeneity, low encapsulation rate, poor stability, chip clogging, high requirements for operators, and loss of samples in the process of generating artificial cells.

[0030] According to some embodiments of the present invention, an oil phase solution and an external water phase solution are introduced into the interior of the microfluidic chip to generate oil-in-water droplets in the second intermediate flow channel. Based on the stability of the oil-in-water droplets, it is decided to continue the step of surface treatment of the flow channel inside the microfluidic chip or to continue to introduce the internal water phase solution.

[0031] According to some embodiments of the present invention, the solvent of the oil phase solution is oleic acid.

[0032] According to some embodiments of the present invention, the osmotic pressure of the inner aqueous phase solution is not greater than the osmotic pressure of the outer aqueous phase solution, and the osmotic pressure of the inner aqueous phase solution is not less than 20% of the osmotic pressure of the outer aqueous phase solution.

[0033] According to some embodiments of the present invention, the interfacial tension (γ W1-O ) and the interfacial tension between the external water phase and the oil phase (γ W2-O ) is less than 2mN / m.

[0034] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0036] Figure 1 A schematic diagram of an embodiment of a microfluidic chip for preparing artificial cells according to the present invention;

[0037] Figure 2 Schematic diagram of flow channel distribution in one embodiment of a microfluidic chip;

[0038] Figure 3 is a schematic diagram of flow channel distribution in another embodiment of a microfluidic chip;

[0039] Figure 4is a schematic diagram of flow channel distribution in another embodiment of a microfluidic chip;

[0040] Figure 5 is a schematic diagram of an embodiment of a capture array;

[0041] Figure 6 A flow chart of the method for preparing artificial cells;

[0042] Figure 7 Schematic diagram and corresponding physical pictures for chip fabrication and artificial cell generation;

[0043] Figure 8 Schematic diagram of the uniformity results generated for artificial cells;

[0044] Fig. 9 Schematic diagram of imaging of artificial cells encapsulating 1 mg / mL calcein;

[0045] Fig.10 Schematic diagram of imaging of the artificial cell-encapsulated in vitro transcription and translation system.

[0046] Reference numerals:

[0047] A first shear cut 10, a second shear cut 20, a bending portion 30, a first liquid inlet channel 100, a first liquid inlet port 110; a second liquid inlet channel 200, a second liquid inlet port 210; a first intermediate channel 300; a third liquid inlet channel 400, a third liquid inlet port 410, a fourth liquid inlet channel 500, a fourth liquid inlet port 510; a capture array 600, an inlet 610, an outlet 620, a capture unit 630, a capture chamber 631, a capture port 632, a discharge port 633; ​​a second intermediate channel 700, and an annular channel 800. DETAILED DESCRIPTION

[0048] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0049] In the description of the present invention, it should be understood that descriptions involving orientations, such as up, down, front, back, left, right, etc., and orientations or positional relationships indicated are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0050] In the description of the present invention, "several" means more than one, "many" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, and "above", "below", "within", etc. are understood to include the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing the technical features, and cannot be understood as indicating or implying the relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0051] In the description of the present invention, unless otherwise clearly defined, terms such as setting, installing, connecting, etc. should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in the present invention based on the specific content of the technical solution.

[0052] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0053] Reference Figure 1 In an embodiment of the present invention, a microfluidic chip for preparing artificial cells (hereinafter referred to as the microfluidic chip) is disclosed. The microfluidic chip includes a first liquid inlet channel 100, a second liquid inlet channel 200, a first intermediate channel 300, a third liquid inlet channel 400, a fourth liquid inlet channel 500 and a capture array 600. A first liquid inlet port 110 is provided at one end of the first liquid inlet channel 100. The first liquid inlet port 110 is used to introduce an oil phase solution into the first liquid inlet channel 100. A second liquid inlet port 210 is provided at one end of the second liquid inlet channel 200, and the other end is connected to an end of the first liquid inlet channel 100 away from the first liquid inlet port 110 to form a first shearing port 10. The second liquid inlet port 210 is used to introduce an inner water phase solution into the second liquid inlet channel 200. The inner water phase solution and the oil phase solution converge at the first shearing port 10, and the inner water phase oil is sheared by the oil phase oil to form oil-in-water droplets. One end of the first intermediate flow channel 300 is connected to the first shear opening 10, and one end of the third liquid inlet channel 400 is provided with a third liquid inlet 410, and the other end is connected to the end of the first intermediate flow channel 300 away from the first shear opening 10 to form a second shear opening 20. The third liquid inlet 410 is used to introduce the external aqueous phase solution into the third liquid inlet channel 400. The external aqueous phase solution and the oil-in-water droplets converge at the second shear opening 20, and the oil-in-water droplets are sheared by the external aqueous phase oil to form double emulsion droplets coated with the external aqueous phase-oil phase-inner aqueous phase in sequence.

[0054] The capture array 600 is provided with an inlet 610 and an outlet 620. The inlet 610 is connected to the second shear opening 20. Double emulsion droplets can enter the capture array 600 through the inlet 610 and be captured by the capture array 600. A fourth liquid inlet 510 is provided at one end of the fourth liquid inlet channel 500, and the other end is connected to the capture array 600. The fourth liquid inlet 510 is used to introduce a buffer solution into the capture array 600. After the buffer solution enters the capture array 600, the surfactant concentration in the buffer solution is low and has a higher interfacial tension. In the process of increasing interfacial tension between the external aqueous phase and the oil phase, phospholipid molecules assemble to form a double layer. Due to the dewetting effect, the organic solution in the middle and the excess phospholipids are separated from the middle phase to form artificial cells and oil droplets. The waste liquid generated during the preparation of the artificial cells (such as satellite droplets generated with vesicles, oil droplets removed due to interfacial tension, etc.) or the artificial cells can be discharged from the capture array 600 from the outlet 620 after the preparation, performance observation and preparation are completed.

[0055] The multiple liquid inlet channels in the microfluidic chip cooperate to form a first shearing port 10 and a second shearing port 20. When preparing artificial cells, each solution flows through the first shearing port 10 and the second shearing port 20 in sequence, and is sheared at the first shearing port 10 and the second shearing port 20 to generate stable double emulsion droplets, which can improve the consistency and stability of subsequently generated artificial cells. In addition, the double emulsion droplets are captured by the capture array 600, so that the external phase of the double emulsion droplets is stably replaced with the buffer solution, and the organic solvent in the double emulsion is released to form artificial cells. In the process of preparing artificial cells, there is no need to strictly control the convergence timing between the solutions, which is easy to operate.

[0056] In addition, the microfluidic chip in the present invention integrates the generation and capture structure of artificial cells. By adding a buffer solution channel, the external phase solution can be replaced, so that the surfactant concentration is reduced in sequence, thereby adjusting the interfacial tension to remove the organic solvent. The double emulsion can directly perform external phase replacement in the capture array 600 and generate artificial cells. The artificial cells do not need to be transferred between different devices during the preparation process, which can reduce the sample consumption of the artificial cells during the transfer process.

[0057] It should be noted that the inner aqueous phase solution can also be added to the in vitro transcription-translation system (IVTT system). Phospholipids are dissolved in the oil phase solution. During the formation of artificial cells, the organic solvent that dissolves the phospholipids is removed, and finally a phospholipid bilayer vesicle with cell size is formed, which can simulate the structural characteristics of natural cells. IVTT is encapsulated in the vesicle, so that proteins can be synthesized in the vesicle and perform biological tasks similar to natural cells in function. However, there is glycerol in the IVTT system encapsulated in the vesicle, and there is a high interfacial tension between the inner phase and the oil phase, resulting in an unstable interface during encapsulation. The present invention adopts a method of replacing the buffer solution with the outer phase solution. The surfactant added to the outer aqueous phase solution reduces the interfacial tension between the outer phase and the oil phase. The buffer solution dilutes the concentration of the surfactant, increases the interfacial tension between the outer phase and the oil phase, and restores the interfacial tension between the outer phase and the oil phase. The organic solvent is removed gently and controllably to better match the physicochemical characteristics of the IVTT system and improve the stability of the vesicle.

[0058] In addition, the fourth liquid inlet 510 can also be used to introduce metabolic substrates such as lipid micelles, amino acids, and nucleotides. After the artificial cells are prepared, the metabolic substrates can be introduced into the fourth liquid inlet channel 500 through the fourth liquid inlet 510, and the metabolic substrates enter the capture array 600 to provide nutrition for the growth and research of the artificial cells.

[0059] In this way, in the present invention, by setting up a capture array 600, double emulsion droplets can be captured, so that the position of the double emulsion droplets is fixed, and a buffer solution is introduced into the capture array 600 through the fourth liquid inlet channel 500. The external phase of the double emulsion droplets can be stably replaced with the buffer solution to separate the organic solvent and form artificial cells; the artificial cells are always in a state of being captured by the capture unit 630, which is convenient for later observation and research of the artificial cells; the fourth liquid inlet channel 500 can also introduce nutrients into the capture array 600 for further growth and observation of the artificial cells.

[0060] In one embodiment, the second shear cut 20 is connected to the inlet 610 through the second intermediate flow channel 700. The inner wall of the first intermediate flow channel 300 is hydrophobic. The hydrophobic surface of the first intermediate flow channel 300 is conducive to the formation of droplets of the inner aqueous phase solution in the oil phase solution, thereby generating oil-in-water droplets. The inner wall of the second intermediate flow channel 700 is hydrophilic. The hydrophilic surface helps the oil phase solution to form droplets in the outer aqueous phase solution, thereby generating double emulsion droplets of water-in-oil and then water-in-water.

[0061] In one embodiment, the first liquid inlet channel 100 and the second liquid inlet channel 200 each include at least one curved portion 30. After the oil phase solution and the inner water phase solution flow through the curved portion 30, they converge at the first shear cut 10. The curved portion 30 can intercept the residual glue generated when the microfluidic chip is punched, preventing the residual glue at the first liquid inlet 110 and the second liquid inlet 210 from clogging the internal flow channel of the microfluidic chip, while providing flow resistance to maintain stable droplet generation.

[0062] It should be noted that the curved portion 30 is not limited to being arranged in an arc shape, a zigzag shape, etc. The first liquid inlet flow channel 100 and the second liquid inlet flow channel 200 can both be provided with a plurality of curved portions 30, and the curved portions 30 are sequentially connected along the extension direction of the first liquid inlet flow channel 100 and the second liquid inlet flow channel 200 to form a serpentine flow channel, so as to further enhance the interception effect of residual glue.

[0063] In addition, the flow channel diameters of the first liquid inlet flow channel 100 and the second liquid inlet flow channel 200 can be set to be smaller than the flow channel diameters of the third liquid inlet flow channel 400 and the fourth liquid inlet flow channel 500, so that the microfluidic chip can meet the requirements of micro-amount and ultra-micro-amount liquid inlet and is conducive to obtaining droplets of different sizes. It is understandable that the third liquid inlet flow channel 400 and the fourth liquid inlet flow channel 500 can also be provided with a curved portion 30 to intercept the residual glue hitting the third liquid inlet port 410 and the fourth liquid inlet port 510.

[0064] It should be noted that if Figure 2 As shown, there is a preset angle a between the first liquid inlet channel 100 and the second liquid inlet channel 200, and between the third liquid inlet channel 400 and the first intermediate channel 300, 60°≤a≤120°, so that the oil phase solution and the inner water phase solution converged at the first shearing opening 10 have different flow directions and are sheared, and the oil-in-water liquid and the outer water phase solution converged at the second shearing opening 20 have different flow directions and are sheared. a is not limited to being set to 60°, 70°, 80°, 85°, 90°, 95°, 100°, 110°, 120°, etc. In one embodiment, as Figure 1 As shown, a=90°, that is, the first liquid inlet channel 100 is perpendicular to the second liquid inlet channel 200, and the third liquid inlet channel 400 is perpendicular to the first intermediate channel 300, so that a channel structure with high shear force is formed at the first shear opening 10 and the second shear opening 20 to form stable double emulsion droplets in the microfluidic chip.

[0065] In one embodiment, if Figure 3As shown, the microfluidic chip includes two second liquid inlet channels 200, which are arranged on opposite sides of the first liquid inlet channel 100 and are both perpendicular to the first liquid inlet channel 100. Two oil phase solutions and one inner water phase solution converge at the first shear opening 10, and the two symmetrical oil phase solutions flow toward each other and squeeze the inner water phase solution at the same time, so that the inner water phase solution breaks to form droplets, and generates oil-in-water droplets.

[0066] In another embodiment, if Figure 4 As shown, the microfluidic chip also includes an annular flow channel 800, and one end of the first liquid inlet channel 100 away from the first liquid inlet port 110 is connected to the annular flow channel 800, and one end of the second liquid inlet channel 200 away from the second liquid inlet port 210 is connected to the annular flow channel 800 at the first shear cut 10. The annular flow channel 800 is symmetrical with respect to the second liquid inlet channel 200. After the oil phase solution enters the annular flow channel 800 through the first liquid inlet channel 100, it flows in two opposite directions. The two oil phase solutions converge with the inner water phase solution at the first shear cut 10, and the two symmetrical oil phase solutions flow in opposite directions and squeeze the inner water phase solution at the same time, so that the inner water phase solution breaks to form droplets, and oil-in-water droplets are generated.

[0067] Reference Figure 5 The capture array 600 includes a plurality of capture units 630 arranged at intervals along a first direction and a second direction. The first direction intersects with the second direction, that is, a plurality of capture units 630 are arranged along different directions. The capture units 630 are used to capture double emulsion droplets. Each capture unit 630 is provided with a capture chamber 631 and a capture port 632 connected to the capture chamber 631. The capture port 632 is arranged on a side of the capture chamber 631 facing the inlet 610. The double emulsion droplets entering the capture array 600 through the inlet 610 flow into the capture chamber 631 from the capture port 632. The double emulsion contained in the capture chamber 631 is in a captured state, and the external phase of the double emulsion droplets is then replaced with the buffer solution. Since a plurality of capture units 630 are arranged along the first direction and the second direction, the double emulsion droplets can pass through the gaps between adjacent capture units 630 and flow to different capture units 630 to be captured by different capture units 630, thereby increasing the number of captured double emulsion droplets. In some embodiments, hundreds or thousands of capture units 630 are arranged in the capture array 600, which can complete the generation and capture of hundreds or thousands of artificial cells.

[0068] In one embodiment, the first direction is perpendicular to the second direction, the spacing between every two adjacent capture units 630 in the first direction is the same, and the spacing between every two adjacent capture units 630 in the second direction is the same. The distribution of the capture units 630 is more uniform, which can increase the probability of double emulsion droplets being captured.

[0069] Furthermore, the capture port 632 is arranged on one side of the capture unit 630 along the first direction, and the double emulsion droplets entering the capture array 600 through the inlet 610 flow along the first direction and enter the capture chamber 631 from the capture port 632. The projections formed by the capture units 630 adjacent to each other in the first direction along the first direction are staggered in the second direction, that is, the capture unit 630 in the first direction is directly opposite to the gap between two capture units 630 arranged along the second direction. Therefore, when the double emulsion droplets gradually flow to different rows of capture units 630 along the first direction, the double emulsion droplets can flow directly from the gap between the two capture units 630 in the previous row to the capture units 630 in the next row, and enter the capture chamber 631 through the capture port 632. The capture units 630 in the next row are just located on the flow path of the double emulsion droplets flowing out from the gap between the capture units 630 in the previous row, making the capture of the double emulsion more accurate and convenient.

[0070] It is also possible to set the cross-sectional area of ​​at least part of the capture chamber 631 to gradually increase from the capture port 632 along the first direction, that is, the opening of the capture port 632 is smaller than the opening of the cross section of the capture chamber 631 parallel to the second direction. On the one hand, the capture chamber 631 can provide sufficient accommodation space for double emulsion droplets, and on the other hand, the double emulsion droplets entering the capture chamber 631 are not easy to overflow from the capture port 632, so that the vesicles can be stably captured. Further, in one embodiment, the drainage port 633 and the capture port 632 are respectively located on two opposite sides of the capture chamber 631 along the first direction, and the cross-sectional area of ​​at least part of the capture chamber 631 gradually increases from the drainage port 633 to the capture port 632. Therefore, the capture chamber 631 close to the drainage port 633 has a diversion function, and the excess liquid in the capture chamber 631 is diverted to the drainage port 633 for discharge, and the liquid in the capture chamber 631 is discharged more thoroughly.

[0071] In one embodiment, the opening of the capture port 632 is set to 30-50 μm, and the opening of the capture port 632 is not limited to 30 μm, 32 μm, 35 μm, 38 μm, 40 μm, 45 μm, 48 μm, 50 μm, etc. On the one hand, the double emulsion can enter the capture chamber 631 through the capture port 632 and be captured by the capture unit 630, and on the other hand, bubbles in the fluid are prevented from entering the capture chamber 631 to affect the capture of the double emulsion and the external phase replacement.

[0072] The capture unit 630 is also provided with a drainage port 633, which is in communication with the capture chamber 631. The drainage port 633 is used to discharge the excess liquid that enters the capture chamber 631 along with the double emulsion, so as to form a fluid potential well inside the capture unit 630, so that the vesicle is always contained in the capture chamber 631 and is stably captured. A plurality of drainage ports 633 may be provided. In one embodiment, the capture port 632 is located on one side of the capture unit 630 along the first direction and faces the inlet 610, and the drainage port 633 is located on the side of the capture unit 630 that is away from the capture port 632, that is, the excess liquid follows the flow of the double emulsion and enters the capture chamber 631, and the liquid that enters the capture chamber 631 still has a tendency to flow along the first direction and is directly discharged from the drainage port 633 on the other side of the capture unit 630. Alternatively, in another embodiment, the drainage port 633 is disposed on two opposite sides of the capture unit 630 along the second direction, so that the excess liquid entering the capture chamber 631 can flow out from the drainage port 633 on the side of the capture unit 630. It is understandable that the drainage port 633 can be disposed on the side of the capture unit 630 facing away from the capture port 632, as well as on two opposite sides of the capture unit 630 along the second direction, and the excess liquid can flow out of the capture chamber 631 from the drainage ports 633 at different positions, so that the liquid can be quickly discharged, which is beneficial to the full exchange of the buffer solution.

[0073] It should be noted that the microfluidic chip in the present invention can also be used in conjunction with an emulsion generation device, and the capture array 600 is placed on the surface of a conductive glass. After voltage is applied, the solution generates artificial cells under the action of alternating current to limit the size distribution range of the generated artificial cells and improve the consistency of the artificial cells. Alternatively, the microfluidic chip can be used in conjunction with a microneedle or micropore template, and the capture array 600 is placed on the template to control the morphology and size of the artificial cells and improve the uniformity of the artificial cells.

[0074] The present invention also proposes a method for preparing artificial cells, which is performed using the above-mentioned microfluidic chip, including the following steps: making a mask plate and a mother plate of a microfluidic chip in sequence; making a microfluidic chip and performing partial surface treatment on the flow channel in the microfluidic chip; preparing an oil phase solution, an inner water phase solution, an outer water phase solution and a buffer solution; adjusting the osmotic pressure and interfacial tension of the solution; introducing each phase solution into the microfluidic chip to prepare and capture artificial cells; and verifying performance. The flowchart of the above-mentioned method for preparing artificial cells is shown in the figure. Figure 6 The technical route mainly includes: (1) chip preparation before artificial cell generation and capture, as well as solution preparation and debugging, (2) artificial cell generation and capture operation, and (3) quality detection after artificial cell generation and capture (including evaluation of uniformity, encapsulation efficiency and stability).

[0075] In one embodiment, manufacturing a mask plate and a mother plate of a microfluidic chip includes the following steps:

[0076] The steps for making the mask plate include: using a maskless photolithography machine to read the chip structure and directly write the pattern on a chrome plate coated with photoresist; then using a chrome etching solution to etch it to form the corresponding mask plate.

[0077] The steps of making the motherboard include: repeatedly cleaning the wafer with distilled water and anhydrous ethanol in turn to remove organic impurities on the surface of the wafer, then rinsing with distilled water and drying with high-pressure nitrogen; finally, placing the wafer in a constant temperature drying oven at 80℃ for drying. Apply photoresist on the cleaned wafer, and then use a glue spreader to spread it at the required speed to form a uniform photoresist thin layer (20μm channel structure can use SU-8 3025 series, 40μm structure can use SU-8 3050 series photoresist to control the stability of chip height). Place the wafer that has been spun in an oven and heat it at 65℃ for 2min, then raise the temperature to 95℃ and heat it for 5min to dry the photoresist and increase the adhesion between the photoresist and the wafer. The mask plate and the wafer coated with photoresist are aligned and overlapped from top to bottom, and exposed under the photolithography machine. The exposure time is adjusted according to the power requirements. Place the exposed substrate in an oven and heat it at 65°C for 1 min, then raise the temperature to 95°C and heat it for 5 min. Place the exposed positive mold in a developer until the structure is clear, then rinse it with deionized water. Use a step profiler to measure the height of the photoresist structure to confirm that the height of the microfluidic chip meets expectations.

[0078] The microfluidic chip is made of PDMS (polydimethylsiloxane) material. PDMS itself has hydrophobic properties. The area where the second intermediate flow channel 700 is located can be hydrophilicized to make the inner wall of the second intermediate flow channel 700 hydrophilic. The hydrophobic surface of the first intermediate flow channel 300 is conducive to the formation of droplets of the inner aqueous phase solution in the oil phase solution, thereby generating oil-in-water droplets. The hydrophilic surface of the second intermediate flow channel 700 helps the oil phase solution to form droplets in the outer aqueous phase solution, thereby generating water-in-oil and then water-in-water double emulsion droplets. The formation of droplets is closely related to the interfacial tension of the oil-water interface. For oil-in-water droplets (W 1 / O), the hydrophobic surface of the channel helps the water phase to form droplets in the oil phase; while for oil-in-water droplets (O / W 2 ), the hydrophilic surface helps the oil phase to form droplets in the water phase. Appropriate surface treatment can reduce the energy barrier during droplet formation, thereby achieving stable droplet generation.

[0079] In one embodiment, manufacturing a microfluidic chip and performing partial surface treatment on a flow channel in the microfluidic chip comprises the following steps:

[0080] (1) Casting and curing: PDMS prepolymer and curing agent were mixed in a ratio of 10:1, and bubbles were removed by vacuum pump. The mixture was cast upside down on the master, placed in an oven at 80°C, and cured for 45 min. The cured PDMS structure was then peeled off from the master with a scalpel to obtain a fluid microchannel substrate.

[0081] (2) Drilling: Use a punch to vertically drill holes at the channel inlet (0.3 mm) and outlet (0.7 mm) on the fluid microchannel substrate;

[0082] (3) Bonding: Place the punched fluid microchannel substrate in deionized water for 30 minutes for ultrasonic treatment to remove the debris caused by the punching. After drying with nitrogen, place it in a plasma cleaner for 45 seconds, quickly take it out, and bond it with another flat PDMS. Then place it in an 80°C oven and heat dry it for 2 hours to increase the strength of the bond. After drying, a microfluidic chip is obtained;

[0083] (4) Examine the microfluidic chip under an inverted fluorescence microscope and place the second cut structure in the center of the field of view;

[0084] (5) Prepare three 1.5 mL liquid reservoirs connected to an air pump, wherein one of the liquid reservoirs is added with 3 wt.% polyvinyl alcohol (PVA), and the other two are connected to air; preferably, the alcoholysis degree of PVA is 89% to 90%, and the molecular weight is 30,000 to 70,000.

[0085] (6) Connect the air-connected liquid reservoir to the second liquid inlet (for introducing the inner water phase (W 1 )) and the first liquid inlet (for introducing the oil phase (O)), and the pressure was increased to about 50 mbar; the third liquid inlet was connected to the PVA solution, and a pressure of 50 mbar was also applied, and the introduction was stopped after 30 seconds;

[0086] (7) Connect a negative pressure of -160 mbar at the outlet to ensure that the air discharges all the liquid in the pipeline; wipe off the liquid at the outlet to ensure that no liquid remains;

[0087] (8) The microfluidic chip was placed in an oven at 80°C and baked for 30 min to fix the PVA on the channel surface.

[0088] The flow channel inside the microfluidic chip is partially surface treated to make the surface of the first intermediate flow channel hydrophobic and the surface of the second intermediate flow channel connected between the inlet and the second shear opening hydrophilic.

[0089] In one embodiment, the solvent of the oil phase solution is oleic acid. At present, most microfluidic technologies use n-octanol as a solvent for phospholipids. N-octanol is slightly soluble in water and difficult to be completely removed from the solution system, and has certain cytotoxicity and instability; in addition, there are a few that use squalene to dissolve phospholipids, but its solubility in phospholipids is low, and it is easily oxidized, saponified in the chip and blocks the channel. Based on this, the embodiment of the present invention uses oleic acid solution as a solvent for phospholipids. Oleic acid not only has good biocompatibility, but can also serve as a substrate for cell metabolic reactions.

[0090] In one embodiment, preparing the oil phase solution, the inner aqueous phase solution, the outer aqueous phase solution and the buffer solution comprises the following steps:

[0091] (1) Preparation of oil phase solution: Add phospholipids to oleic acid to prepare an oil phase solution.

[0092] Specifically, take the phospholipid dissolved in chloroform, put it into a dry vacuum pump, extract the chloroform, and obtain a dry phospholipid film; then add the required volume of oleic acid to the phospholipid film and adjust it to a concentration of 4 mg / mL to 15 mg / mL, preferably 5 mg / mL; after sealing, put the container into an ultrasonic water bath at 45°C for 45 minutes to ensure that the phospholipid is completely dissolved and evenly dissolved.

[0093] (2) Preparation of internal water phase (W 1 ) solution: add an appropriate volume of in vitro transcription and translation system to the sucrose solution to obtain an inner aqueous phase (W 1 ) solution.

[0094] Specifically, the final concentration of the sucrose solution is 0 to 500 mM, preferably 200 to 400 mM, and more preferably about 300 mM.

[0095] Specifically, the internal water phase (W 1 ) The osmotic pressure of the solution is 0 to 3000 mOsm / kg, preferably 100 to 500 mOsm / kg, and more preferably about 300 mOsm / kg (theoretically, the osmotic pressure should be restored to the osmotic pressure of the cells themselves as much as possible).

[0096] During the entire operation, sterile and enzyme-free conditions must be strictly maintained to ensure the purity of the experimental environment and the reliability of the results. The in vitro transcription and translation system can be homemade or commercial in vitro translation system, such as In vitro protein synthesis kit ( In Vitro Protein Synthesis Kit), add 30 μL (containing 400 ng of DNA template corresponding to the target protein, including T7 promoter and terminator).

[0097] (3) Prepare the external water phase (W 2) solution: Glucose, nucleoside triphosphates (NTPs), amino acid composition, surfactant, PVA and other small molecules are added to the buffer to obtain an external aqueous phase (W 2 ) solution.

[0098] Specifically, the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES) with a final concentration of 100 mM.

[0099] Specifically, the final concentration of glucose is 0 to 500 mM, preferably 100 to 300 mM, and more preferably about 200 mM.

[0100] Specifically, the surfactant is multi-block copolymer F-68 (Pluronic F-68), and the amount used is 0.1 to 10 wt.%, preferably 1 to 5 wt.%, and more preferably about 2 wt.%.

[0101] Specifically, the amount of PVA is 0-10 wt.%, preferably 0.1-1 wt.%, and more preferably about 0.5 wt.%. Preferably, the alcoholysis degree of PVA is 89%-90%, and the molecular weight is 30,000-70,000.

[0102] Specifically, other small molecules include: spermidine (final concentration is 0-10mM, preferably 1-3mM, more preferably about 1.5mM), dithiothreitol (DTT) (final concentration is 0-10mM, preferably 1-3mM, more preferably about 1.5mM), folic acid (Folinic Acid) (final concentration is 0-1mM, preferably 0.01-0.05mM, more preferably about 0.02mM), potassium glutamate (Potassium Glutamate) (final concentration is 0-500mM, preferably 200-400mM, more preferably about 280mM), magnesium glutamate (Magnesium Glutamate) (final concentration is 0-200mM, preferably 1-50mM, more preferably about 20mM).

[0103] Specifically, nucleoside triphosphates (NTPs) include: adenosine triphosphate (ATP) (final concentration is 0-100 mM, preferably 1-10 mM, more preferably about 6 mM), cytidine triphosphate (CTP) (final concentration is 0-100 mM, preferably 1-10 mM, more preferably about 4 mM), uridine triphosphate (UTP) (final concentration is 0-100 mM, preferably 1-10 mM, more preferably about 4 mM), guanosine triphosphate (GTP) (final concentration is 0-100 mM, preferably 1-10 mM, more preferably about 6 mM).

[0104] Specifically, the amino acids include a combination of one or more essential amino acids, and the final concentration of each amino acid is 0 to 50 mM, preferably 0.1 to 2 mM, and more preferably about 0.5 mM.

[0105] In a specific embodiment, the external aqueous phase solution comprises:

[0106] Nucleoside triphosphates (NTPs) solution (including 6mM adenosine triphosphate (ATP), 4mM cytidine triphosphate (CTP), 4mM uridine triphosphate (UTP), 6mM guanosine triphosphate (GTP)); amino acid solution (including 0.5mM alanine (Ala), 0.5mM arginine (Arg), 0.5mM aspartic acid (Asp), 0.5mM asparagine (Asn), 0.5mM cysteine ​​(Cysteine, Cys), 0.5mM glutamic acid (Glutamic acid, GTP)). Acid, Glu), 0.5mM Glutamine (Glutamine, Gln), 0.5mM Glycine (Gly), 0.5mM Histidine (Histidine, His), 0.5mM Isoleucine (Ile), 0.5mM Leucine (Leucine, Leu), 0.5mM Lysine (Lys), 0.5mM Methionine (Met), 0.5mM Phenylalanine (Phe), 0.5mM Proline (Proline, Pro), 0.5mM Serine (Serine, Ser), 0.5mM Threonine (Threonine, Thr), 0.5mM Tryptophan (Tryptophan, Trp), 0.5mM Tyrosine (Tyrosine, Tyr), 0.5mM Valine (Val), 1.5mM Spermidine (Spermidine), 1.5mM Dithiothreitol (Dithiothreitol, DTT), 0.02mM Folic acid (Folinic Acid), 280mM potassium glutamate, 20mM magnesium glutamate, 100mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES), 200mM glucose, 2wt.% multi-block copolymer F-68 (Pluronic F-68), 0.5wt.% PVA, pH adjusted to 7.6. The osmotic pressure of the external aqueous phase was measured to be approximately 1600mOsm / kg.

[0107] (4) Preparation of buffer solution: Glucose, nucleoside triphosphates (NTPs), amino acid composition, surfactant, PVA and other small molecules are added to the buffer solution to obtain a buffer solution.

[0108] Specifically, the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES) with a final concentration of 100 mM.

[0109] Specifically, the final concentration of glucose is 0 to 500 mM, preferably 100 to 300 mM, and more preferably about 200 mM.

[0110] Specifically, the surfactant is multi-block copolymer F-68 (Pluronic F-68), and the amount used is 0.1 to 10 wt.%, preferably 0.1 to 2 wt.%, and more preferably about 0.5 wt.%.

[0111] Specifically, the amount of PVA is 0-10 wt.%, preferably 0.1-1 wt.%, and more preferably about 0.5 wt.%. Preferably, the alcoholysis degree of PVA is 89%-90%, and the molecular weight is 30,000-70,000.

[0112] In a specific embodiment, the buffer solution comprises:

[0113] Nucleoside triphosphates (NTPs) solution (including 6mM adenosine triphosphate (ATP), 4mM cytidine triphosphate (CTP), 4mM uridine triphosphate (UTP), 6mM guanosine triphosphate (GTP)); amino acid solution (including 0.5mM alanine (Ala), 0.5mM arginine (Arg), 0.5mM aspartic acid (Asp), 0.5mM asparagine (Asn), 0.5mM cysteine ​​(Cysteine, Cys), 0.5mM glutamic acid (Glutamic acid, GTP)). Acid, Glu), 0.5mM Glutamine (Glutamine, Gln), 0.5mM Glycine (Gly), 0.5mM Histidine (Histidine, His), 0.5mM Isoleucine (Ile), 0.5mM Leucine (Leucine, Leu), 0.5mM Lysine (Lys), 0.5mM Methionine (Met), 0.5mM Phenylalanine (Phe), 0.5mM Proline (Proline, Pro), 0.5mM Serine (Serine, Ser), 0.5mM Threonine (Threonine, Thr), 0.5mM Tryptophan (Tryptophan, Trp), 0.5mM Tyrosine (Tyrosine, Tyr), 0.5mM Valine (Val), 1.5mM Spermidine (Spermidine), 1.5mM Dithiothreitol (Dithiothreitol, DTT), 0.02mM Folic acid (Folinic Acid), 280mM Potassium Glutamate, 20mM Magnesium Glutamate, 100mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES), 200mM glucose, 0.5wt.% multi-block copolymer F-68 (Pluronic F-68), 0.5wt.% PVA, and the pH value was adjusted to 7.6.

[0114] In one embodiment, the osmotic pressure of the inner aqueous phase solution is not greater than that of the outer aqueous phase solution, and the osmotic pressure of the inner aqueous phase solution is not less than 20% of the osmotic pressure of the outer aqueous phase solution. Preferably, the osmotic pressure difference between the inner aqueous phase solution and the outer aqueous phase solution does not exceed 10%.

[0115] In one embodiment, the interfacial tension (γ W1-O ) and the interfacial tension between the external water phase and the oil phase (γ W2-O ) is less than 2mN / m.

[0116] In one embodiment, adjusting the osmotic pressure and interfacial tension of the solution comprises the following steps:

[0117] Adjustment of osmotic pressure: measure the osmotic pressure of the inner aqueous phase and the outer aqueous phase, take the osmotic pressure of the inner aqueous phase as the standard, and match the osmotic pressure of the inner aqueous phase by increasing or decreasing the concentration of glucose in the outer aqueous phase. The pressure difference shall not exceed 10%.

[0118] Adjustment of interfacial tension: The interfacial tension is adjusted by adjusting the concentration of Pluronic F-68 in the external water phase. The interfacial tension between the internal water phase and the oil phase (γ W1-O ) and the interfacial tension between the external water phase and the oil phase (γ W2-O ) should be less than 2mN / m. The interfacial tension determines the stability of the vesicles and can be measured by the hanging drop method. W1-O ) value is too high, the concentration of Pluronic F-68 in the external water phase is increased to reduce the external phase-oil phase interfacial tension (γ W2-O ) (mass fraction adjustment range is 1% to 10%, try not to exceed 5%); if the sum of interfacial tension (γ W1-O +γ W2-O ) value is too high, which leads to instability of the vesicles. The concentration of PVA can be increased to increase stability. Pluronic F-68 and PVA are both macromolecules, and their concentration changes have little effect on the osmotic pressure of the solution.

[0119] In one embodiment, introducing various phase solutions into a microfluidic chip to prepare and capture artificial cells comprises the following steps:

[0120] When using a microfluidic chip to prepare and capture artificial cells, first fix the microfluidic chip on the stage of a microscope, and use a microscope to observe whether the flow channel in the microfluidic chip is blocked or has sharp protrusions, etc., to ensure that the microfluidic chip is in a normal usable state. Then, the inner water phase solution, the oil phase solution, and the outer water phase solution are respectively loaded into their respective reservoirs, and the reservoirs are connected to different liquid inlets through polytetrafluoroethylene microtubes. The outer water phase pressure is adjusted to 40mbar. After the outer water phase solution fills the pipeline, the oil phase channel is treated in the same way. After the oil phase solution and the outer water phase solution flow stably, water-in-oil droplets are generated in the second intermediate flow channel 700; observe whether the generation of water-in-oil droplets is stable through a microscope. If the generation of oil-in-water droplets is unstable, check the surface treatment steps of the microfluidic chip.

[0121] If the generation of water-in-oil droplets is stable, the inner aqueous phase solution is connected. The initial adjustment pressure of each solution can be set as 40 mbar for the outer aqueous phase oil, 30 mbar for the oil phase solution, and 30 mbar for the inner aqueous phase solution. On this basis, the pressure is adjusted in steps of 2 mbar to fine-tune the size of the generated droplets and accurately adjust the distribution of the vesicle size. In this process, the inner aqueous phase solution and the oil phase solution are sheared at the first shearing port 10, and water-in-oil droplets are generated in the first intermediate flow channel 300. The water-in-oil droplets are then sheared with the outer aqueous phase oil at the second shearing port 20, and double emulsion droplets of water-in-oil and then water-in-water are generated in the second intermediate flow channel 700. After the double emulsion droplets are stably generated, the waste liquid discharged from the outlet 620 is wiped with a dust-free cloth, and 2 μL of isotonic glucose solution is added to wet the outlet 620 to help the oily waste liquid float and separate from the vesicles. After the double emulsion droplets are generated, they enter the capture array 600 under the action of fluid propulsion and are captured by the capture unit 630. A buffer solution is introduced into the capture array 600 at 20 mbar, and the temperature of the microfluidic chip is set at about 30° using a temperature control device. The buffer solution replaces the external phase of the double emulsion droplets to generate artificial cells.

[0122] In addition, in order to reduce the volume consumption of the IVTT system in the pipeline and avoid the entry of bubbles, the pipeline is set to a 0.25 mm polytetrafluoroethylene hose to reduce the volume consumption in the pipeline and reduce the amount used in a single experiment. The volume consumed in the pipeline is about 10 μL, which can ensure that the amount used in each experiment is less than 30 μL.

[0123] In one embodiment, the performance verification includes the following steps:

[0124] The vesicle morphology is observed under a microscope, and vesicle images are taken. The area, perimeter and other data of each vesicle are counted according to the vesicle boundaries shown in the image to evaluate the uniformity of the vesicle. Alternatively, the microscope field of view is fixed for 24 hours, and the same field of view is scanned every 1 hour to count the data and size distribution of the vesicles to evaluate the stability of the vesicles. Alternatively, 1 mg / mL of calcein is encapsulated in the vesicles, and the fluorescence distribution after vesicle generation is observed to evaluate the encapsulation efficiency of the vesicles.

[0125] More specifically, assessing the homogeneity of the vesicles involves the following steps: Observe the vesicles through a microscope and take a picture of the vesicles to ensure that the image is of high quality and clearly shows the vesicle boundaries. Convert it to a grayscale image using Image>Type>8-bit to help with subsequent thresholding. To better identify cell boundaries, perform some preprocessing operations first, use Process>Noise>Despeckle to remove noise, and use Process>Filters>Gaussian Blur to apply Gaussian blur to smooth the image. Adjust the threshold using Image>Adjust>Threshold to separate the cell area from the background. Adjust the slider until the cells are highlighted. Then click Apply to apply the threshold. If the vesicles are connected to each other, you can use Process>Binary>Watershed to segment them to better separate individual vesicles. Perform cell size statistics using Analyze>Analyze Particles. In the pop-up dialog box, you can set the minimum and maximum sizes to exclude unwanted particles. Check Display Results to display the size data for each cell, and check Show Outlines to display the outline of the analysis area. After Analyze Particles is completed, use ImageJ to generate a result table to count the area, perimeter and other data of each vesicle to evaluate the uniformity of the vesicles.

[0126] The present invention also proposes an application of the microfluidic chip as described above in preparing artificial cells, designing microfluidic droplet systems, verifying membrane protein functions, studying cell interaction mechanisms, expressing different types of proteins, and using them for drug screening.

[0127] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0128] Example 1 Preparation of microfluidic chip

[0129] (1) According to the present invention Figure 1The mask plate is processed according to the drawing: a maskless photolithography machine is used to read the chip structure and the pattern is directly written on a chrome plate coated with photoresist; a chrome etching solution is used for etching to form a corresponding mask plate.

[0130] (2) Master processing

[0131] ① Clean the wafer: Clean the wafer repeatedly with distilled water and anhydrous ethanol in turn to remove organic impurities on the surface; then rinse it with distilled water and blow it dry with high-pressure nitrogen; finally, place the wafer in a constant temperature drying oven at 80°C for drying.

[0132] ② Spinning: Apply the photoresist on the cleaned wafer, and then use a coating machine to spread it at the required speed to form a uniform thin layer of photoresist.

[0133] ③ Pre-baking: Place the wafer with the photoresist on it in an oven and heat it at 65℃ for 2 minutes, then raise the temperature to 95℃ and heat it for 5 minutes to dry the photoresist and increase the adhesion between the photoresist and the wafer.

[0134] ④ Exposure: The mask and the wafer coated with photoresist are aligned and overlapped from top to bottom, and exposed under the photolithography machine. The exposure time is adjusted according to the power requirements.

[0135] ⑤ Post-baking: Place the exposed substrate in an oven and heat it at 65°C for 1 min, then raise the temperature to 95°C and heat it for 5 min.

[0136] ⑥ Development: Place the exposed positive mold in the developer until the structure is clear, and then rinse it with deionized water.

[0137] ⑦Profile measurement: Use a step profiler to measure the height of the photoresist structure and confirm that the height on the chip meets expectations.

[0138] (3) Fabricating a microfluidic chip and performing surface treatment on the flow channel in the microfluidic chip:

[0139] ① Casting and curing: Mix the PDMS prepolymer and curing agent in a ratio of 10:1, remove bubbles with a vacuum pump, cast it upside down on the master, place it in an oven at 80°C, cure it for 45 minutes, and then peel the cured PDMS structure from the master with a scalpel to obtain a fluid microchannel substrate;

[0140] ② Drilling: Use a puncher to vertically drill holes at the channel inlet (0.3 mm) and outlet (0.7 mm) on the fluid microchannel substrate;

[0141] ③ Bonding: Place the punched fluid microchannel substrate in deionized water for 30 minutes to remove the debris caused by the punching. After drying with nitrogen, place it in a plasma cleaner for 45 seconds, quickly take it out, and bond it with another flat PDMS. Then place it in an 80℃ oven and heat dry it for 2 hours to increase the strength of the bond.

[0142] ④ Check the microfluidic chip under an inverted fluorescence microscope and place the second cut structure in the center of the field of view;

[0143] ⑤ Prepare three 1.5 mL reservoirs and connect them to the air pump. Add 3 wt.% polyvinyl alcohol (PVA, alcoholysis degree of 89% to 90%, molecular weight of 30,000 to 70,000) to one of the reservoirs, and connect the other two to air.

[0144] ⑥ Connect the liquid reservoir connected to the air to the second liquid inlet (for introducing the inner water phase (W 1 )) and the first liquid inlet (for introducing the oil phase (O)), and the pressure was increased to about 50 mbar; the third liquid inlet was connected to the PVA solution, and a pressure of 50 mbar was also applied, and the introduction was stopped after 30 seconds;

[0145] ⑦ Connect a negative pressure of -160mbar at the outlet to ensure that the air discharges all the liquid in the pipeline; wipe off the liquid at the outlet to ensure that no liquid remains;

[0146] ⑧Put the microfluidic chip in an oven at 80°C and bake for 30 minutes to fix the PVA on the channel surface.

[0147] Example 2 Generation of artificial cells

[0148] (1) Preparation of solution:

[0149] Prepare the oil phase solution: take the phospholipid dissolved in chloroform, put it into a dry vacuum pump, extract the chloroform to obtain a dry phospholipid film; then add the required volume of oleic acid and adjust it to a concentration of 5 mg / mL; after sealing, put the container into an ultrasonic water bath at 45°C for 45 minutes to ensure that the phospholipid is completely dissolved and evenly dissolved.

[0150] Prepare the internal water phase (W 1 ) solution: add 30 μL of in vitro transcription and translation system ( In vitro protein synthesis kit, containing 400ng of DNA template corresponding to the target protein, including T7 promoter and terminator), and then adding sucrose solution (final concentration 300mM), the final osmotic pressure value was measured to be about 1500mOsm / kg. Sterile and enzyme-free conditions must be strictly maintained throughout the operation to ensure the purity of the experimental environment and the reliability of the results.

[0151] Prepare the external water phase (W2 ) solution: Nucleoside triphosphates (NTPs) solution (including 6mM adenosine triphosphate (ATP), 4mM cytidine triphosphate (CTP), 4mM uridine triphosphate (UTP), 6mM guanosine triphosphate (GTP)); amino acid solution (including 0.5mM alanine (Ala), 0.5mM arginine (Arg), 0.5mM aspartic acid (Asp), 0.5mM asparagine (Asn), 0.5mM cysteine ​​(Cysteine, Cys), 0.5mM glutamic acid (Glutamic acid, GTP)). Acid, Glu), 0.5mM Glutamine (Glutamine, Gln), 0.5mM Glycine (Gly), 0.5mM Histidine (Histidine, His), 0.5mM Isoleucine (Ile), 0.5mM Leucine (Leucine, Leu), 0.5mM Lysine (Lys), 0.5mM Methionine (Met), 0.5mM Phenylalanine (Phe), 0.5mM Proline (Proline, Pro), 0.5mM Serine (Serine, Ser), 0.5mM Threonine (Threonine, Thr), 0.5mM Tryptophan (Tryptophan, Trp), 0.5mM Tyrosine (Tyrosine, Tyr), 0.5mM Valine (Val), 1.5mM Spermidine (Spermidine), 1.5mM Dithiothreitol (Dithiothreitol, DTT), 0.02mM Folic acid (Folinic Acid), 280mM potassium glutamate, 20mM magnesium glutamate, 100mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES), 200mM glucose, 2wt.% Pluronic F-68, 0.5wt.% PVA, pH adjusted to 7.6. The osmotic pressure of the external aqueous phase was measured to be approximately 1600mOsm / kg.

[0152] Prepare buffer solution: Nucleoside triphosphates (NTPs) solution (including 6mM adenosine triphosphate (ATP), 4mM cytidine triphosphate (CTP), 4mM uridine triphosphate (UTP), 6mM guanosine triphosphate (GTP)); amino acid solution (including 0.5mM alanine (Ala), 0.5mM arginine (Arg), 0.5mM aspartic acid (Asp), 0.5mM asparagine (Asn), 0.5mM cysteine ​​(Cysteine, Cys), 0.5mM glutamic acid (Glutamic acid, GTP)). Acid, Glu), 0.5mM Glutamine (Glutamine, Gln), 0.5mM Glycine (Gly), 0.5mM Histidine (Histidine, His), 0.5mM Isoleucine (Ile), 0.5mM Leucine (Leucine, Leu), 0.5mM Lysine (Lys), 0.5mM Methionine (Met), 0.5mM Phenylalanine (Phe), 0.5mM Proline (Proline, Pro), 0.5mM Serine (Serine, Ser), 0.5mM Threonine (Threonine, Thr), 0.5mM Tryptophan (Tryptophan, Trp), 0.5mM Tyrosine (Tyrosine, Tyr), 0.5mM Valine (Val), 1.5mM Spermidine (Spermidine), 1.5mM Dithiothreitol (Dithiothreitol, DTT), 0.02mM Folic acid (Folinic Acid), 280mM potassium glutamate, 20mM magnesium glutamate, 100mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES), 200mM glucose, 0.5wt.% multi-block copolymer F-68 (Pluronic F-68), 0.5wt.% PVA, and the pH value was adjusted to 7.6.

[0153] (2) Regulation of osmotic pressure and interfacial tension: The osmotic pressure of the inner phase is taken as the standard, and the osmotic pressure of the inner phase is matched by increasing or decreasing the concentration of glucose in the outer phase, and the pressure difference does not exceed 10%; by adjusting the concentrations of Pluronic F-68 and PVA, the interfacial tension (γ W1-O ) and the interfacial tension between the water phase and the oil phase (γ W2-O ) is less than 2mN / m.

[0154] (3) Generation and capture of artificial cells

[0155] ① Fix the chip prepared in Example 1 on the microscope stage and check whether the channel is blocked or has sharp protrusions.

[0156] ②Put the inner water phase, oil phase, and outer water phase solutions into 1.5mL reservoirs respectively. In order to reduce the volume consumption of the IVTT system in the pipeline and avoid the entry of bubbles, a polytetrafluoroethylene hose with an inner diameter of 0.25mm is used. The volume consumed in the pipeline is about 10μL, which can ensure that the amount used in each experiment is less than 30μL.

[0157] ③ Adjust the external water phase pressure to about 40 mbar. After filling the pipeline, treat the oil phase channel in the same way. After the solution stabilizes, water-in-oil droplets are generated.

[0158] ④ Connect the inner water phase channel in the same way as above, adjust the pressure in 2 mbar steps (external water phase: 40 mbar, oil phase: 30 mbar, inner water phase: 30 mbar), and fine-tune the droplet size. The distribution of artificial cell size can be precisely adjusted under the constraint of channel size by adjusting the pressure.

[0159] ⑤ Before reaching stable generation, the volume of the external water phase and the oil phase consumed is less than 1 μL. After reaching equilibrium, wipe the waste liquid with a dust-free paper towel, add 15 μL of isotonic glucose solution to wet the outlet, and help the oily waste liquid float and separate from the artificial cells.

[0160] ⑥ The generated vesicles are captured by the array under the action of fluid propulsion, and the buffer solution is passed into it at a pressure of 20mbar. The temperature is kept constant at 30℃ through a temperature control device to achieve the generation and capture of artificial cells.

[0161] In the above scheme, the schematic diagram of chip production and artificial cell generation and the corresponding physical diagram are as follows Figure 7 As shown in the figure: (1) The tools and physical pictures needed for the production of microfluidic chips; (2) The effect pictures and physical pictures of some surface treatments, for water-in-oil droplets (W 1 / O), the hydrophobic surface of the channel helps the water phase to form droplets in the oil phase; while for oil-in-water droplets (O / W 2), the hydrophilic surface helps the oil phase to form droplets in the water phase. Appropriate surface treatment can reduce the energy barrier when droplets are formed, thereby achieving stable generation of droplets. PDMS itself is a hydrophobic material, so it is necessary to make the channel structure from the external phase solution to the outlet hydrophilic, while keeping the properties of other areas unchanged; (3) Schematic diagram of artificial cell generation in microfluidic chip and corresponding physical picture. At the first shear, the internal phase (W containing the IVTT system) 1 ) is sheared by the oil phase (O) containing phospholipids to form water-in-oil droplets (W 1 / O), and then, at the second shear mouth, the water-in-oil droplets are 2 ) is sheared to form a double emulsion (W 1 / O / W 2 ), the double emulsion is captured by the array, and under the regulation of interfacial tension, the organic solvent (oleic acid) that dissolves phospholipids is removed to form artificial cells, in which satellite droplets accompanying vesicle generation and oil droplets removed due to interfacial tension are not captured by the array due to their small size.

[0162] Example 3

[0163] Detect the uniformity of the artificial cells generated in Example 2: Randomly sample 48 artificial cells, observe them under a microscope, take high-quality pictures, and clearly show the cell boundaries. Convert it to a grayscale image through Image>Type>8-bit; use Process>Noise>Despeckle to remove noise; use Process>Filters>Gaussian Blur to apply Gaussian blur to smooth the image; adjust the threshold through Image>Adjust>Threshold to separate the cell area from the background. Adjust the slider until the cells are highlighted. Then click Apply to apply the threshold. Perform cell size statistics through Analyze>AnalyzeParticles. Check Display Results to display the size data of each cell, and check Show Outlines to display the outline of the analysis area. After Analyze Particles is completed, use ImageJ to generate a result table to count the area, perimeter and other data of each cell.

[0164] Finally, the average particle size of the artificial cells was measured to be 18.34 μm, and the variance of the radius was only 0.32 μm. This result proves the high uniformity of the generated artificial cells (e.g. Figure 8 shown). Figure 8Middle: (1) Image of artificial cells under a 40x bright field objective; (2) Fluorescence imaging of artificial cells; (3) Result of automatic extraction of the edge of artificial cells using Image J; (4) Statistical results of a sample of 48 artificial cells, with an average radius of 18.34 μm and a variance of 0.32 μm.

[0165] Example 4

[0166] Small molecule encapsulation experiment: After encapsulating calcein (at a concentration of 1 mg / mL), the artificial cells of Example 2 were randomly sampled and observed, and it was found that the artificial cells successfully encapsulated small molecules with green fluorescence, and the fluorescence was uniform and the intensity values ​​were close (such as Fig. 9 This indicates that the encapsulation effect of the artificial cells is consistent and uniform. Fig. 9 In the figure, (1) is the fusion imaging; (2) is the phospholipid imaging with red fluorescence; and (3) is the calcein uniformly encapsulated in the artificial cell.

[0167] Example 5

[0168] In vitro transcription and translation system encapsulation experiment: After the artificial cells of Example 2 were encapsulated with the in vitro transcription and translation system, microscopic observation showed that the artificial cells successfully expressed green fluorescent protein (e.g. Fig.10 ), further verifying the functionality and biological activity of the system within artificial cells. Fig.10 In the figure, the upper figure shows the situation when green fluorescent protein is not expressed, and the lower figure shows the situation after expression.

[0169] In summary, the present invention can successfully generate and capture uniform artificial cells, and can be used to encapsulate bioactive substances and complex reaction systems to achieve the expected functions.

[0170] Finally, the present invention provides a microfluidic chip that uses a double T structure to provide a high shear force flow channel structure, and combined with a capture array, thousands of artificial cells can be generated and captured on one chip. The present invention also provides an efficient, low-cost, and easy-to-operate method for partial surface treatment adapted to this chip, and provides a low-consumption liquid inlet device for small samples. In addition, the present invention also proposes a solution system that has both biocompatibility and stability for the generation of artificial cells, and provides a strategy for adjusting the solution under different encapsulation systems on this basis, and proposes a design scheme for improving reagent formulations, osmotic pressure, and interfacial tension parameters for efficient protein synthesis in artificial cells.

[0171] The beneficial effects of the above technical solution include:

[0172] (1) The surface treatment of the chip is simple and easy to operate. Each chip takes less than 5 minutes to process and only requires one solution. The generation and capture of artificial cells takes a short time and has higher stability compared to other microfluidic methods.

[0173] (2) Generation and capture as an integral chip: The generation and capture of artificial cells are treated as an integral structure, and channels for fluid replacement are added to reduce the consumption of samples during the transfer process of artificial cells.

[0174] (3) The artificial cells have good uniformity: The generated artificial cells were randomly sampled and the average particle size was found to be 18.34 μm, with a radius variance of only 0.32 μm, indicating that the generated artificial cells are highly uniform and evenly distributed.

[0175] (4) High encapsulation efficiency: In the experiment of encapsulating small molecule fluorescent substances, the proportion of artificial cells with fluorescence was nearly 100% in random sampling, and the fluorescence values ​​were close, indicating that the present invention has good encapsulation efficiency. During the generation of artificial cells, there is almost no material exchange between the inner phase and the outer phase, which is conducive to controlling the composition and concentration of the inner phase and will not cause the loss of the inner phase.

[0176] (5) Low consumption: Suitable for ultra-trace samples, the dead volume of the chip connection is only 10 μL, reducing sample consumption.

[0177] (6) High-precision and efficient encapsulation of complex systems: The high interfacial tension between the IVTT system and the oil phase may lead to interface instability during encapsulation. To address this problem, the present invention replaces the external phase solution to increase the external phase interfacial tension, thereby gently and controllably removing the organic solvent, better matching the physicochemical properties of the IVTT system, and improving the stability of the artificial cells.

[0178] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A microfluidic chip for preparing artificial cells, characterized in that: include: A first liquid inlet flow channel, one end of which is provided with a first liquid inlet, wherein the first liquid inlet is used to introduce the oil phase solution; A second liquid inlet channel, one end of which is provided with a second liquid inlet, and the other end is connected with an end of the first liquid inlet channel away from the first liquid inlet to form a first shearing opening, the second liquid inlet is used to pass the inner water phase solution, the inner water phase solution and the oil phase solution form water-in-oil droplets at the first shearing opening; A first intermediate flow channel, one end of which is connected to the first shear opening; A third liquid inlet flow channel, one end of which is provided with a third liquid inlet, and the other end is connected with an end of the first intermediate flow channel away from the first shearing opening to form a second shearing opening, the third liquid inlet is used to introduce the external aqueous phase solution, and the water-in-oil droplets and the external aqueous phase solution form double emulsion droplets at the second shearing opening; A capture array, wherein the capture array is provided with an inlet and an outlet, wherein the inlet is communicated with the second shear opening and is used to capture the double emulsion droplets, and the outlet is used to discharge the solution in the capture array; A fourth liquid inlet channel has a fourth liquid inlet at one end and is connected to the capture array at the other end. The fourth liquid inlet is used to introduce a buffer solution, and the buffer solution can replace the external phase of the double emulsion droplets to separate the organic solvent in the double emulsion droplets and form artificial cells.

2. The microfluidic chip for preparing artificial cells according to claim 1, characterized in that: The first liquid inlet channel and the second liquid inlet channel each include at least one curved portion.

3. The microfluidic chip for preparing artificial cells according to claim 1, characterized in that: The first liquid inlet flow channel is perpendicular to the second liquid inlet flow channel; And / or, the third liquid inlet channel is perpendicular to the first intermediate channel.

4. The microfluidic chip for preparing artificial cells according to claim 1, characterized in that: The capture array includes a plurality of capture units arranged at intervals along a first direction and a second direction, wherein the first direction intersects with the second direction, and each of the capture units is provided with a capture chamber and a capture port connected to the capture chamber, wherein the capture port is arranged on a side of the capture chamber facing the inlet, and the capture chamber is used to accommodate the double emulsion droplets.

5. The microfluidic chip for preparing artificial cells according to claim 4, characterized in that: The capture port is arranged at one side of the capture unit along the first direction, and projections of the capture units adjacent to each other along the first direction formed along the first direction are staggered in the second direction.

6. The microfluidic chip for preparing artificial cells according to claim 4, characterized in that: The capture unit is further provided with a drainage port, the drainage port is communicated with the capture chamber, and the capture port is arranged on one side of the capture unit along the first direction, wherein: The drainage port is arranged on a side of the capture unit facing away from the capture port, and / or the drainage port is arranged on two opposite sides of the capture unit along the second direction.

7. The microfluidic chip for preparing artificial cells according to claim 6, characterized in that: The opening of the capture port is 30 to 50 μm; And / or, the cross-sectional area of ​​at least a portion of the capture cavity gradually increases from the capture port along the first direction.

8. A method for preparing an artificial cell, characterized in that: include: Prepare the microfluidic chip, the oil phase solution, the inner aqueous phase solution, the outer aqueous phase solution and the buffer solution according to any one of claims 1 to 7; Performing surface treatment on the flow channel inside the microfluidic chip so that the surface of the first intermediate flow channel is hydrophobic and the surface of the second intermediate flow channel connected between the inlet and the second shear opening is hydrophilic; The oil phase solution, the inner water phase solution, the outer water phase solution and the buffer solution are introduced into the microfluidic chip to prepare and capture artificial cells.

9. The method for preparing an artificial cell according to claim 8, characterized in that: include: The oil phase solution and the external water phase solution are introduced into the microfluidic chip to generate oil-in-water droplets in the second intermediate flow channel. Based on the stability of the oil-in-water droplets, it is decided to continue the step of surface treatment of the flow channel inside the microfluidic chip or to continue to introduce the internal water phase solution.

10. The method for preparing an artificial cell according to claim 8, characterized in that: The solvent of the oil phase solution is oleic acid; and / or, the osmotic pressure of the inner aqueous phase solution is not greater than the osmotic pressure of the outer aqueous phase solution, and the osmotic pressure of the inner aqueous phase solution is not less than 20% of the osmotic pressure of the outer aqueous phase solution; And / or, the sum of the interfacial tension between the inner water phase and the oil phase and the interfacial tension between the outer water phase and the oil phase is less than 2 mN / m.

11. Use of the microfluidic chip according to any one of claims 1 to 7 in preparing artificial cells, designing microfluidic droplet systems, verifying membrane protein functions, studying cell interaction mechanisms, and drug screening.

Images