Microfluidic device and method for preparing lipidosome through reversed-phase emulsification

Abstract

The invention provides a microfluidic device and method for preparing lipidosome through reversed-phase emulsification. The microfluidic device comprises: a first microfluidic chip comprising at least one first micro-channel, each first micro-channel being used for receiving a continuous phase and a dispersed phase and emulsifying the continuous phase and the dispersed phase into single emulsion droplets; the second micro-fluidic chip is bonded with the first micro-fluidic chip, the second micro-fluidic chip comprises at least one reverse emulsion column, each reverse emulsion column is connected with the corresponding first micro-channel, and the reverse emulsion columns are used for reversely emulsifying the single emulsion droplets of the corresponding first micro-channels into corresponding lipidosome. The microfluidic device and method provided by the invention can be used for rapidly and controllably preparing lipidosome with uniform size and better integrity at high flux.

Description

Technical Field

[0001] The invention relates to the technical field of microfluidics, and in particular to a microfluidics device and method for preparing liposomes by reverse emulsification. Background Art

[0002] Liposomes are a closed and ordered combination of natural amphiphilic molecules represented by phospholipids that spontaneously form a double-layer structure when dispersed in water. The special structure of liposomes allows them to selectively allow some ions and small molecules to move across the membrane while isolating the internal and external water environments. It plays an important role in synthetic biology, drug release, artificial single cell preparation and other fields.

[0003] Currently, the main method for preparing liposomes is the inverse emulsified oil method. This method is simple to operate and has a high liposome yield, but the size uniformity of the prepared liposomes is poor and there is a possibility of content leakage. Summary of the invention

[0004] In view of this, the present invention provides a microfluidic device and method for preparing liposomes by inversion emulsification, which can quickly, controllably and high-throughput prepare liposomes with uniform size and good integrity.

[0005] On the one hand, an embodiment of the present invention provides a microfluidic device for preparing liposomes by inverse emulsification, comprising: a first microfluidic chip, comprising at least one first microchannel, each first microchannel is used to receive a continuous phase and a dispersed phase, and emulsify the continuous phase and the dispersed phase into single emulsion droplets; a second microfluidic chip, the second microfluidic chip is bonded to the first microfluidic chip, the second microfluidic chip comprises at least one reverse emulsification column, each reverse emulsification column is connected to the corresponding first microchannel, and the reverse emulsification column is used to reverse emulsify the single emulsion droplets of the corresponding first microchannel into corresponding liposomes.

[0006] According to an embodiment of the present invention, the microfluidic device also includes: at least one first pipe, each first pipe is connected to a corresponding first microchannel, and each first pipe is used to inject a continuous phase into the connected first microchannel; at least one second pipe, each second pipe is connected to a corresponding first microchannel, and each second pipe is used to inject a dispersed phase into the connected first microchannel; and multiple flow rate control components, each first pipe and each second pipe is connected to a flow rate control component, and the flow rate control component is used to control the flow rate of the continuous phase and the dispersed phase in the respective pipes.

[0007] According to an embodiment of the present invention, the flow rate control component includes a syringe connected to a corresponding first pipe or a second pipe, and the syringe is used to inject a continuous phase and a dispersed phase into the first pipe and the second pipe respectively.

[0008] According to an embodiment of the present invention, the second microfluidic chip also includes multiple second microchannels, each second microchannel is connected to a corresponding reverse emulsification column, and the second microchannel is used to flow out liposomes; the microfluidic device also includes a flow control module for controlling the on and off of each second microchannel.

[0009] According to an embodiment of the present invention, the flow control module includes: a film, bonded to the second microfluidic chip, and the film is attached to multiple second microchannels; a third microfluidic chip, bonded to the film, and the film is located between the second microfluidic chip and the third microfluidic chip, and an airway is prepared on the third microfluidic chip, and the airway leads to the film; a gas control component, connected to the airway, the gas control component is used to control the gas pressure in the airway, so as to deform the film, and then control the on and off of each second microchannel.

[0010] According to an embodiment of the present invention, the microfluidic device also includes: a fourth microfluidic chip, a capture structure is prepared on the upper surface of the fourth microfluidic chip, the capture structure is connected to each second microchannel, and the fourth microfluidic chip is used to drive the liposomes to move to the corresponding predetermined position in the capture structure.

[0011] According to an embodiment of the present invention, the fourth microfluidic chip includes a glass sheet, the capture structure is prepared on the upper surface of the glass sheet, and the material of the glass sheet includes indium tin oxide.

[0012] According to an embodiment of the present invention, a plurality of storage areas are provided on the glass sheet, each storage area stores a corresponding reagent, each storage area is connected to a capture structure, and the fourth microfluidic chip is also used to drive each reagent to move to a predetermined position in the capture structure.

[0013] According to an embodiment of the present invention, the microfluidic device further includes: a microscope imaging module, including a microscope, and the microscope is aligned with the first microfluidic chip, the second microfluidic chip or the fourth microfluidic chip.

[0014] On the other hand, an embodiment of the present invention also provides a method for preparing liposomes using the microfluidic device of the above embodiment, comprising: injecting a continuous phase and a dispersed phase into at least one first microchannel of a first microfluidic chip, so that the matching continuous phase and dispersed phase are emulsified into at least one single emulsion droplet; introducing each single emulsion droplet into a corresponding reverse emulsification column of a second microfluidic chip, so that the single emulsion droplet is reverse emulsified into a corresponding liposome.

[0015] The microfluidic device and method for preparing liposomes by reverse emulsification provided in the embodiment of the present invention utilize a microfluidic chip to prepare single emulsion droplets and utilize the single emulsion droplets to prepare liposomes, which can achieve high-throughput preparation of liposomes, and because the size of the single emulsion droplets is uniform and controllable, the size of the liposomes finally obtained is also uniform and controllable, which has a great advantage over the liposomes of uneven size obtained by centrifugation or scraping centrifuge tubes. By gently reverse emulsifying the single emulsion droplets in the reverse emulsification column on the microfluidic chip, the leakage of the contents in the traditional reverse emulsification process can be avoided, thereby reducing the interference caused by the background containing the same components as the contents. The microfluidic device and method of the embodiment of the present invention can be further integrated with a digital microfluidic chip to efficiently and conveniently manipulate subsequent liposome-related biochemical experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The above and other objects, features and advantages of the present invention will become more apparent through the following description of the embodiments of the present invention with reference to the accompanying drawings, in which:

[0017] Figure 1 The structure of a microfluidic device for preparing liposomes by inversion emulsification according to an embodiment of the present invention is schematically shown;

[0018] Figure 2 A schematic top view of a first microfluidic channel according to an embodiment of the present invention is shown;

[0019] Figure 3 The structure of a microfluidic device for preparing liposomes by inversion emulsification according to another embodiment of the present invention is schematically shown;

[0020] Figure 4 Schematically shows a structural diagram of a flow rate control assembly according to an embodiment of the present invention;

[0021] Figure 5 A schematic diagram of a flow control module according to an embodiment of the present invention is shown;

[0022] Figure 6 A schematic diagram showing the structure of a fourth microfluidic chip according to an embodiment of the present invention;

[0023] Figure 7 A schematic diagram of a capture structure according to an embodiment of the present invention is shown;

[0024] Figure 8 A schematic diagram of a functional area plan of a fourth microfluidic chip integrated with a liposome capture structure according to an embodiment of the present invention is shown;

[0025] Fig. 9 Schematically shows a droplet driving path on a fourth microfluidic chip according to an embodiment of the present invention;

[0026] Fig.10 Schematically showing an experimental diagram of high-throughput generation of single emulsion droplets according to an embodiment of the present invention;

[0027] Fig.11 A flow chart schematically shows a method for preparing liposomes using a microfluidic device according to an embodiment of the present invention;

[0028] Fig.12 The flowchart of a method for preparing liposomes using a microfluidic device according to another embodiment of the present invention is schematically shown.

[0029] Description of reference numerals:

[0030] 1. Single emulsion droplet inversion emulsification module; 101. First microfluidic chip; 102. First microfluidic channel; 103. Continuous phase inlet; 104. Dispersed phase inlet #1; 105. Dispersed phase inlet #2; 106. Dispersed phase inlet #3; 107. Dispersed phase inlet #4; 108. Second microfluidic chip; 109. Inversion emulsification column; 110. Second microfluidic channel; 2. Flow rate control module; 201. Syringe; 202. Syringe propulsion device; 203. Hose; 3. Flow control module; 301. Third microfluidic chip; 302. Airway; 303. Film; 304. Syringe; 305. Syringe propulsion device; 306. Hose; 4. Fourth microfluidic chip Fluidic chip; 401, glass slide; 402, DMF chip body; 403, DMF chip support; 404, liposome inlet #1; 405, liposome inlet #2; 406, liposome inlet #3; 407, liposome inlet #4; 408, liposome capture area #1; 409, liposome capture area #2; 410, liposome capture area #3; 411, liposome capture area #4; 412, reagent storage area #1; 413, reagent storage area #2; 414, reagent storage area #3; 415, reagent storage area #4; 5, microscope imaging module; 501, microscope; 502, CCD camera; 6, control module. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0033] Unless otherwise specifically stated, the relative arrangement of the parts and steps described in these embodiments, numerical expressions and numerical values ​​do not limit the scope of the present invention. At the same time, it should be understood that, for ease of description, the sizes of the various parts shown in the accompanying drawings are not drawn according to the actual proportional relationship. The technology, methods and equipment known to ordinary technicians in the relevant field may not be discussed in detail, but in appropriate cases, the technology, methods and equipment should be regarded as a part of the authorization specification. In all examples shown and discussed here, any specific value should be interpreted as being merely exemplary, rather than as a limitation. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters represent similar items in the following drawings, so once a certain item is defined in an accompanying drawing, it does not need to be further discussed in subsequent drawings.

[0034] In the description of the present invention, it is necessary to understand that the directions or positional relationships indicated by directional words such as "front, back, up, down, left, right", "lateral, vertical, perpendicular, horizontal" and "top, bottom" are usually based on the directions or positional relationships shown in the drawings. They are only for the convenience of describing the present invention and simplifying the description. Unless otherwise specified, these directional words do not indicate or imply that the devices or elements referred to must have a specific direction or be constructed and operated in a specific direction. Therefore, they cannot be understood as limiting the scope of protection of the present invention. The directional words "inside and outside" refer to the inside and outside relative to the contours of each component itself.

[0035] For ease of description, spatially relative terms such as "above", "above", "on the upper surface of", "above", etc. may be used here to describe the spatial positional relationship between a device or feature and other devices or features as shown in the figure. It should be understood that spatially relative terms are intended to include different orientations of the device in use or operation in addition to the orientation described in the figure. For example, if the device in the accompanying drawings is inverted, the device described as "above other devices or structures" or "above other devices or structures" will be positioned as "below other devices or structures" or "below other devices or structures". Thus, the exemplary term "above" may include both "above" and "below". The device may also be positioned in other different ways (rotated 90° or in other orientations), and the spatially relative descriptions used here are interpreted accordingly.

[0036] In addition, it should be noted that the use of terms such as "first" and "second" to limit components is only for the convenience of distinguishing the corresponding components. If not otherwise stated, the above terms have no special meaning and therefore cannot be understood as limiting the scope of protection of the present invention.

[0037] Figure 1 The structure of a microfluidic device for preparing liposomes by inversion emulsification according to an embodiment of the present invention is schematically shown; Figure 2 A top view of a first microfluidic channel according to an embodiment of the present invention is schematically shown.

[0038] like Figure 1 As shown, the microfluidic device of this embodiment may include a first microfluidic chip 101 and a second microfluidic chip 108. The second microfluidic chip 108 is bonded to the first microfluidic chip 101.

[0039] The first microfluidic chip 101 includes at least one first microfluidic channel 102, each of which is used to receive a continuous phase and a dispersed phase, and emulsify the continuous phase and the dispersed phase into single emulsion droplets. The first microfluidic chip 101 can be called a single emulsion droplet preparation microfluidic chip. The second microfluidic chip 108 includes at least one reverse emulsification column 109, each reverse emulsification column 109 is connected to the corresponding first microfluidic channel 102, and the reverse emulsification column 109 is used to reverse emulsify the single emulsion droplets of the corresponding first microfluidic channel 102 into corresponding liposomes. The second microfluidic chip 108 can be called a reverse emulsification microfluidic chip.

[0040] In this embodiment, single emulsion droplets are prepared using microfluidic chips 101 and 108, and liposomes are prepared using single emulsion droplets, so that high-throughput preparation of liposomes can be achieved, and since the size of the single emulsion droplets is uniform and controllable, the size of the liposomes finally obtained is also uniform and controllable. By gently inverting emulsification of the single emulsion droplets in the inverted emulsification column 109 on the microfluidic chip 108, leakage of contents in the traditional inverted emulsification process can be avoided.

[0041] like Figure 1 and Figure 2 As shown, as an example, the continuous phase #1 can be injected into the microfluidic device through the first pipe 103, and the dispersed phases #1, #2, #3, and #4 can be injected into the microfluidic device through the second pipes 104, 105, 106, and 107, so that the continuous phase #1 is emulsified with the dispersed phases #1, #2, #3, and #4 into four different single emulsion droplets. Then, through four reverse emulsification columns 109, the four different single emulsion droplets are reversely emulsified into four different liposomes, and are drawn out through the second microchannel 110. In this way, a variety of different liposomes can be obtained simultaneously in the same microfluidic device, thereby improving the efficiency of obtaining liposomes. The drawn liposomes can have a variety of uses, including but not limited to in situ observation of liposomes, biochemical experiments, etc., which will be illustrated later.

[0042] Each microchannel 102 can be injected with a continuous phase and a dispersed phase, and multiple microchannels 102 can be arranged in parallel on the first microfluidic chip 101. Figure 1 and Figure 2 As shown, in order to reduce the number of microchannels 102 in the first microfluidic chip 101, the multiple microchannels 102 can be designed in a grid shape. That is, when the dispersed phases #1, #2, #3, and #4 are emulsified with the same continuous phase #1, multiple branches can be set at one end of the microchannel 102 where the continuous phase #1 is injected, and the continuous phase #1 is introduced from the first pipeline 103 into the four branches connected to the second pipelines 104, 105, 106, and 107.

[0043] It should be noted that Figure 1 and Figure 2 The number of pipes, microchannels, and reverse emulsification columns shown is only an example and does not constitute a limitation of the present invention. The specific number of pipes, microchannels, and reverse emulsification columns can be reasonably set according to the types of continuous phase and dispersed phase. The microfluidic chips 101 and 108 can be prepared by soft lithography, nanoimprinting, 3D printing, etc., but the present invention is not limited thereto.

[0044] Figure 3 The structure of a microfluidic device for preparing liposomes by phase inversion emulsification according to another embodiment of the present invention is schematically shown. In the drawings, the same parts are numbered the same.

[0045] like Figure 1 and Figure 3 As shown, the first microfluidic chip 101 and the second microfluidic chip 108 can be regarded as a single emulsion droplet inversion emulsification module 1 of the microfluidic device. In some embodiments, the microfluidic device further includes a plurality of flow rate control components 201, and the plurality of flow rate control components 201 and the first and second pipelines controlled by them constitute a flow rate control module 2. For example, in Figure 1 and Figure 3 In the embodiment, five pipes 103, 104, 105, 106, and 107 can be provided for injecting the continuous phase or the dispersed phase, and the number of the corresponding flow rate control components 201 can be five, that is, one flow rate control component 201 is provided on each pipe, so as to control the flow rate and frequency of the solution in each pipe.

[0046] Figure 4 The structure of the flow rate control assembly according to an embodiment of the present invention is schematically shown.

[0047] like Figure 4 As shown, in some embodiments, the flow rate control component 201 may include a syringe 201, and each syringe 201 may be connected to the corresponding first pipeline 103 or a second pipeline 104, 105, 106, 107 through a hose 203. The syringe 201 can be used to inject the continuous phase #1 into the first pipeline 103, and can inject the dispersed phases #1, #2, #3, and #4 into the second pipelines 104, 105, 106, and 107, respectively. The syringe 201 can be connected to a syringe propulsion device 202, and the syringe propulsion device 202 can be used to accurately control the injection speed of the syringe 201. This embodiment uses a syringe pump to prepare a single emulsion droplet, which can further save experimental costs and operational difficulties.

[0048] Please continue reading Figure 1 and Figure 3 The microfluidic device further comprises a flow control module 3. The flow control module 3 can be used to control the on-off of each second microchannel 110. In other words, the flow control module 3 can control the outflow flow of each liposome.

[0049] Figure 5 The structure diagram of the flow control module according to the embodiment of the present invention is schematically shown.

[0050] like Figure 3 and Figure 5 As shown, in some embodiments, the flow control module 3 may include a film 303, a third microfluidic chip 301 and a gas control component 304. The third microfluidic chip 301 may be regarded as a gas control chip.

[0051] The film 303 is bonded to the second microfluidic chip 108, and the film 303 is attached to the second microfluidic channel 110. The third microfluidic chip 301 is bonded to the film 303, and the film 303 is located between the second microfluidic chip 108 and the third microfluidic chip 301. An airway 302 is prepared on the third microfluidic chip 301, and the airway 303 leads to the film 303. The gas control component 304 is connected to the airway 302, for example, the airway 302 can be connected through a hose 306. The gas control component 304 can be used to control the gas pressure in the airway 303, so that the film 303 is deformed, and then the opening and closing of each second microfluidic channel 110 is controlled. For example, when the gas control component 304 introduces a certain pressure of gas into the airway 302, the gas can lift the film 303 in contact, so that the film 303 controls the size of the channel of the second microfluidic channel 110, thereby realizing the flow control of the liposome.

[0052] like Figure 4 and Figure 5 As shown, the gas control component 304 may also adopt a structure similar to the flow rate control component 201 , that is, the gas control component 304 may be a syringe 304 , and the injection speed of the syringe 304 may be controlled by a syringe propulsion device 305 .

[0053] Please continue reading Figure 3 The microfluidic device may further include a fourth microfluidic chip 4. A capture structure (not shown in the figure) is prepared on the upper surface of the fourth microfluidic chip 4, and the capture structure is connected to each second microchannel 110 through a specific driving path. The fourth microfluidic chip 4 can be used to drive the liposome to move to a corresponding predetermined position in the capture structure. The fourth microfluidic chip 4 can be a digital micro fluidics (DMF) chip. The fourth microfluidic chip 4 can be used as a DMF chip module.

[0054] Figure 6 The structure of a fourth microfluidic chip according to an embodiment of the present invention is schematically shown.

[0055] See also Figure 3 and Figure 6 The fourth microfluidic chip 4 may include a glass sheet 401, the capture structure is prepared on the upper surface of the glass sheet 401, and the material of the glass sheet may be indium tin oxide (ITO). The DMF chip may include a DMF chip body 402 and a DMF chip support 403. The glass sheet 401 is arranged on the DMF chip body 402.

[0056] By integrating the DMF chip on the microfluidic device, the difficulty of operating the droplet movement can be reduced. At the same time, the optional capture structure on the ITO glass surface in the DMF chip module makes it possible to capture and observe liposomes in situ on the DMF chip.

[0057] Figure 7 The structure diagram of the capture structure according to the embodiment of the present invention is schematically shown.

[0058] like Figure 7 As shown, a capture structure formed on an ITO glass sheet 401 is shown. The capture structure formation method includes but is not limited to soft lithography technology, 3D printing technology, etc. It should be emphasized that Figure 7 The capture structure shown is only an example and does not constitute a specific limitation on the capture structure.

[0059] Figure 8 A schematic diagram of a functional area plan of a fourth microfluidic chip integrated with a liposome capture structure according to an embodiment of the present invention is shown; Fig. 9 The figure schematically shows a droplet driving path on a fourth microfluidic chip according to an embodiment of the present invention.

[0060] like Figure 8 As shown, a plurality of storage areas 412, 413, 414, 415 may be provided on the glass sheet 401, each of which may store corresponding reagents #1, #2, #3, #4, each of which may be connected to a capture structure, and the fourth microfluidic chip 4 may also be used to drive each reagent #1, #2, #3, #4 to move to a predetermined position in the capture structure. The predetermined position may be, for example, the liposome capture areas 408, 409, 410, 411 shown in the figure.

[0061] like Figure 8 and Fig. 9 As shown, liposome #1, #2, #3, #4 can be moved to liposome capture areas 408, 409, 410, 411 along a straight path, and liposome #1, #2, #3, #4 should enter the liposome capture area from the front to achieve efficient capture of liposome. Reagent #1, #2, #3, #4 can be moved to liposome capture areas 408, 409, 410, 411 along a broken line path, and reagent #1, #2, #3, #4 should also enter the liposome capture area from the front to avoid lipids leaving the capture structure. Thus, liposomes can be observed in biochemical experiments in liposome capture areas 408, 409, 410, 411.

[0062] Please continue reading Figure 3For example, the entire process of liposome generation, flow and use can be observed through the microscope imaging module 5. The microscope imaging module 5 may include a microscope 501 and a charge coupled device (CCD) camera 502. The microscope 501 may be an upright microscope. The observation lens of the upright microscope is aimed at the first microfluidic chip 101, the second microfluidic chip 108 or the fourth microfluidic chip 4. The microscope imaging module 5 can obtain the generation of oil-in-water single emulsion droplets, the generation of liposomes, and liposome-related biochemical experiments in real time.

[0063] like Figure 3 As shown, the microfluidic device may further include a control module 6, which may be used to receive real-time flow rate values, real-time images of single emulsion droplet generation, real-time images of liposome generation, and real-time status of liposome-related biochemical experiments.

[0064] Fig.10 The experimental diagram of high-throughput generation of single emulsion droplets according to an embodiment of the present invention is schematically shown.

[0065] like Fig.10 As shown, the left side of the figure shows the continuous phase or dispersed phase injected into the microchannel, and the right side of the figure shows the emulsification process. It can be seen that the microfluidic device of the embodiment of the present invention can stably produce single emulsion droplets at high throughput, and can achieve high-throughput preparation of liposomes. Moreover, since the size of the single emulsion droplets is uniform and controllable, the size of the liposomes finally obtained is also uniform and controllable.

[0066] Based on the microfluidic device of the above embodiment, the present invention also provides a method for preparing liposomes using the microfluidic device of the above embodiment. The method has the same technical features and beneficial effects as the microfluidic device, which will not be described in detail here.

[0067] Fig.11 The flowchart of the method for preparing liposomes using a microfluidic device according to an embodiment of the present invention is schematically shown.

[0068] like Fig.11 As shown, the method for preparing liposomes using a microfluidic device may include operations S111 to S112.

[0069] In operation S111, a continuous phase and a dispersed phase are injected into at least one first microchannel 102 of the first microfluidic chip 101 to emulsify the matched continuous phase and dispersed phase into at least one single emulsion droplet. Some details of operation S111 can be found in the above description of the microfluidic device and will not be repeated here.

[0070] In some embodiments, operation S111 may include: injecting a continuous phase into the connected first microfluidic channel 102 through a first pipe; injecting a dispersed phase into the connected first microfluidic channel 102 through a second pipe; and controlling the flow rates of the continuous phase and the dispersed phase in the respective pipes through respective flow rate control components on the first pipe and the second pipe.

[0071] In some embodiments, controlling the flow rates of the continuous phase and the dispersed phase in the respective pipes through the respective flow rate control components on the first pipe and the second pipe may include: injecting the continuous phase and the dispersed phase into the first pipe and the second pipe respectively through the injector 201.

[0072] In operation S112, each single emulsion droplet is introduced into the corresponding reverse emulsification column 109 of the second microfluidic chip 108, so that the single emulsion droplet is reversely emulsified into corresponding liposomes. Some details of operation S112 can be found in the above description of the microfluidic device, which will not be repeated here.

[0073] Fig.12 The flowchart of a method for preparing liposomes using a microfluidic device according to another embodiment of the present invention is schematically shown.

[0074] like Fig.12 As shown, the method for preparing liposomes using a microfluidic device may include operations S121 to S124.

[0075] In operation S121 , a continuous phase and a dispersed phase are injected into at least one first microchannel 102 of the first microfluidic chip 101 , so that the matched continuous phase and dispersed phase are emulsified into at least one single emulsion droplet.

[0076] In operation S122, each single emulsion droplet is introduced into the corresponding reverse emulsification column 109 of the second microfluidic chip 108, so that the single emulsion droplet is reversely emulsified into the corresponding liposome. Some details of operation S122 can be found in the above description of the microfluidic device, which will not be repeated here. The processes of operations S121-S122 and operations S111-S112 can be the same, which will not be repeated here.

[0077] In operation S123 , the reverse emulsification column 109 is connected to the second microchannel 110 of the second microfluidic chip 109 , and the on-off of the second microchannel 110 is controlled by the flow control module 3 .

[0078] In some embodiments, controlling the on-off of the second microchannel 110 through the flow control module 3 may include: bonding the second microfluidic chip 109 to the film 303 and the third microfluidic chip 301 in sequence, so that the airway on the third microfluidic chip 301 leads to the film 303; controlling the gas pressure in the airway 302 through the gas control component to deform the film 303, thereby controlling the on-off of the second microchannel 110.

[0079] In operation S124, the second microfluidic channel 110 is connected to the capture structure on the upper surface of the fourth microfluidic chip 4 to drive the liposomes to move to the corresponding predetermined positions.

[0080] In some embodiments, the capture structure is prepared on the upper surface of the glass sheet 401, and the material of the glass sheet includes indium tin oxide.

[0081] In some embodiments, a plurality of storage areas are provided on the glass sheet 401, each storage area stores a corresponding reagent, and each storage area is connected to the capture structure. Operation S124 may also include: driving each reagent to move to a predetermined position in the capture structure.

[0082] Example 1

[0083] In combination with the above embodiments and drawings, a microfluidic device and method system for preparing liposomes by inversion emulsification is provided below to explore the denaturation temperature of α-hemolysin. The specific steps may include:

[0084] Step S1, designing the structure of the single emulsion droplet preparation microfluidic chip 101 and the reverse emulsion microfluidic chip 108 in the single emulsion droplet reverse emulsification module 1, and preparing the structure by methods including but not limited to soft lithography, nanoimprinting, 3D printing, etc., to prepare the microchannel 102; a hole puncher can be used to punch through holes at the corresponding positions of the continuous phase inlet 103, the dispersed phase inlet #1 104, the dispersed phase inlet #2 105, the dispersed phase inlet #3 106, the dispersed phase inlet #4 107 and the reverse emulsification column 109; designing the structure of the gas control chip 301 in the flow control module 3, and preparing the structure by methods including but not limited to soft lithography, nanoimprinting, 3D printing, etc.; designing the structure of the DMF body chip 402 in the DMF chip module 4 and preparing it;

[0085] Step S2, prepare the required reagents and load the reagents into the syringe 201 of the flow rate control module 2, the reverse phase emulsification column 109 and the corresponding areas of the DMF chip 4:

[0086] Continuous phase: For example, a 5-15 mg / mL 1-palmitoyl-2-(9Z-oleoyl)-sn-glycero-3-phosphocholine (POPC) mineral oil solution is used as the continuous phase;

[0087] Optionally, fluorescent dyes may be added to the continuous phase at appropriate concentrations, including but not limited to 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocyanine perchlorate, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt), Nile Red, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt), etc.;

[0088] Dispersed phase #1: 0~5% wt / vol poloxamer 188 (P188), 100~500 mM sucrose in water as dispersed phase #1;

[0089] Dispersed phase #2: 0-5% wt / vol P188, 100-500 mM sucrose in water as dispersed phase #2;

[0090] Dispersed phase #3: 0-5% wt / vol P188, 100-500 mM sucrose in water as dispersed phase #3;

[0091] Dispersed phase #4: 0-5% wt / vol P188, 100-500 mM sucrose in water as dispersed phase #4;

[0092] Adding appropriate concentrations of fluorescent dyes into the dispersed phase, including but not limited to calcein, 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt, green fluorescent protein, fluorescein isothiocyanate-carboxymethyl-dextran, etc.;

[0093] Reverse phase emulsion column: 0-5% wt / vol P188, 100-500 mM glucose in water solution as the reverse phase emulsion column lower layer reagent, 5-15 mg / mL POPC in mineral oil solution as the reverse phase emulsion column upper layer reagent;

[0094] Reagent storage area #1: 2 μM α-hemolysin in water;

[0095] Reagent storage area #2: 2 μM α-hemolysin aqueous solution (heated at 40 °C for 1 h);

[0096] Reagent storage area #3: 2 μM α-hemolysin aqueous solution (heated at 60 °C for 1 h);

[0097] Reagent storage area #4: 2 μM α-hemolysin aqueous solution (heated at 80 °C for 1 h);

[0098] Step S3, bonding single emulsion droplets from top to bottom in sequence to prepare microfluidic chip 101, reverse emulsification microfluidic chip 108, film 303, and gas control chip 301, using 1 wt% bovine serum albumin (BSA) solution to seal the glass cover slip at the corresponding position of reverse emulsification column 109 overnight, and then washing away the residual BSA with a large amount of deionized water; connecting flow rate control module 2, DMF chip module 4, and microscope imaging module 5, and connecting control module 6 with flow rate control module 2, flow control module 3, DMF chip module 4, and microscope imaging module 5;

[0099] Step S4, start the flow rate control module 2, the flow control module 3, the DMF chip module 4, and the microscope imaging module 5, and stably generate oil-in-water single emulsion droplets in the single emulsion droplet inversion emulsification module 1 with high throughput, and then the oil-in-water single emulsion droplets are inverted emulsified to form liposomes;

[0100] Step S5, when enough liposomes are generated, the flow control module 3 is controlled to allow the liposomes to enter the upper surface of the indium tin oxide (ITO) glass 401 in the DMF chip module 4;

[0101] Step S6, using the DMF chip body 402 to drive the movement of liposomes and reagents in each region to perform liposome biochemical reactions, may include:

[0102] Step S601, driving the liquid in the liposome inlet #1 404 into the liposome capture area #1 408;

[0103] Step S602, driving the liquid in the liposome inlet #2 405 into the liposome capture area #2 409;

[0104] Step S603, driving the liquid in the liposome inlet #3 406 into the liposome capture area #3 410;

[0105] Step S604, driving the liquid in the liposome inlet #4 407 into the liposome capture area #4 411;

[0106] Step S605, driving the liquid in the reagent storage area #1 412 forward into the liposome capture area #1 408;

[0107] Step S606, driving the liquid in the reagent storage area #2 413 forward into the liposome capture area #2 409;

[0108] Step S607, driving the liquid in the reagent storage area #3 414 forward into the liposome capture area #3 410;

[0109] Step S608, driving the liquid in the reagent storage area #4 415 forward into the liposome capture area #4 411;

[0110] Step S7, recording the curve of the change of the internal fluorescence intensity of the liposomes captured in each liposome capture area over time, and judging the denaturation temperature of α-hemolysin based on the curve.

[0111] Example 2

[0112] In combination with the above embodiments and drawings, a microfluidic device and method system for preparing liposomes by inversion emulsification is provided below to explore the dynamic reconstruction morphology between liposomes and oil droplets. The specific steps may include:

[0113] Step S1, designing the structure of the single emulsion droplet preparation microfluidic chip 101 and the reverse emulsion microfluidic chip 108 in the single emulsion droplet reverse emulsification module 1, preparing the structure by methods including but not limited to soft lithography, nanoimprinting, 3D printing, etc., and preparing the microchannel 102; punching through holes at the corresponding positions of the continuous phase inlet 103, the dispersed phase inlet #1 104, the dispersed phase inlet #2 105, the dispersed phase inlet #3 106, the dispersed phase inlet #4 107 and the reverse emulsification column 108 with a puncher; designing the structure of the gas control chip 301 in the flow control module 3, and preparing the structure by methods including but not limited to soft lithography, nanoimprinting, 3D printing, etc.; designing the structure of the DMF body chip 402 in the DMF chip module 4 and preparing it;

[0114] Step S2, prepare the required reagents and load the reagents into the syringe 201 of the flow rate control module 2, the reverse phase emulsification column 109 and the corresponding area of ​​the DMF chip module 4:

[0115] Continuous phase: 5-15 mg / mL POPC in mineral oil solution was used as the continuous phase;

[0116] Optionally, fluorescent dyes may be added to the continuous phase at appropriate concentrations, including but not limited to 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocyanine perchlorate, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt), Nile Red, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt), etc.;

[0117] Dispersed phase #1: 0-5% wt / vol P188, 100-500 mM sucrose in water as dispersed phase #1;

[0118] Dispersed phase #2: 0-5% wt / vol P188, 100-500 mM sucrose in water as dispersed phase #2;

[0119] Dispersed phase #3: 0-5% wt / vol P188, 100-500 mM sucrose in water as dispersed phase #3;

[0120] Dispersed phase #4: 0-5% wt / vol P188, 100-500 mM sucrose in water as dispersed phase #4;

[0121] Optionally, fluorescent dyes of appropriate concentrations may be added to the dispersed phase, including but not limited to calcein, 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt, green fluorescent protein, fluorescein isothiocyanate-carboxymethyl-dextran, etc.;

[0122] Reverse phase emulsion column: 0-5% wt / vol P188, 100-500 mM glucose in water solution as the reverse phase emulsion column lower layer reagent, 5-15 mg / mL POPC in mineral oil solution as the reverse phase emulsion column upper layer reagent;

[0123] Reagent storage area #1: Disperse 1:4 ~1:10 vol / vol n-octanol: 5% wt / vol P188 aqueous solution using ultrasound;

[0124] Reagent storage area #2: Disperse 1:4 ~1:10 vol / vol oleic acid: 5% wt / vol P188 aqueous solution using ultrasound;

[0125] Reagent storage area #3: Disperse 1:4 ~1:10 vol / vol chloroform, hexane (36:64 vol / vol): 5% wt / vol P188 aqueous solution using ultrasound;

[0126] Reagent Storage Area #4: 5% wt / vol P188 in water;

[0127] Step S3, bonding single emulsion droplets from top to bottom in sequence to prepare microfluidic chip 101, reverse emulsification microfluidic chip 108, film 303, and gas control chip 301, using 1 wt% BSA solution to seal the glass cover slip at the corresponding position of reverse emulsification column 109 overnight, and then washing away the residual BSA with a large amount of deionized water; connecting flow rate control module 2, DMF chip module 4, and microscope imaging module 5, and connecting control module 6 with flow rate control module 2, flow control module 3, DMF chip module 4, and microscope imaging module 5;

[0128] Step S4, start the flow rate control module 2, the flow control module 3, the DMF chip module 4, and the microscope imaging module 5, and stably generate oil-in-water single emulsion droplets in the single emulsion droplet inversion emulsification module 1 with high throughput, and then the oil-in-water single emulsion droplets are inverted emulsified to form liposomes;

[0129] Step S5, when enough liposomes are generated, the flow control module 3 is controlled to allow the liposomes to enter the upper surface of the ITO glass 401 in the DMF chip module 4;

[0130] Step S6, using the DMF chip body 402 to drive the movement of liposomes and reagents in each region to perform liposome biochemical reactions, the specific steps include:

[0131] Step S601, driving the liquid in the liposome inlet #1 404 into the liposome capture area #1 408;

[0132] Step S602, driving the liquid in the liposome inlet #2 405 into the liposome capture area #2 409;

[0133] Step S603, driving the liquid in the liposome inlet #3 406 into the liposome capture area #3 410;

[0134] Step S604, driving the liquid in the liposome inlet #4 407 into the liposome capture area #4 411;

[0135] Step S605, driving the liquid in the reagent storage area #1 412 forward into the liposome capture area #1 408;

[0136] Step S606, driving the liquid in the reagent storage area #2 413 forward into the liposome capture area #2 409;

[0137] Step S607, driving the liquid in the reagent storage area #3 414 forward into the liposome capture area #3 410;

[0138] Step S608, driving the liquid in the reagent storage area #4 415 forward into the liposome capture area #4 411;

[0139] Step S7, control the environmental temperature change (room temperature - 40°C - room temperature cycle) and observe the dynamic reconstruction between the captured liposomes and oil droplets.

[0140] Example 3

[0141] With reference to Example 2, in combination with the accompanying drawings, according to an embodiment of the present invention, a microfluidic device and method system for preparing liposomes by inversion emulsification is provided to study the dynamic reconstruction morphology between liposomes and oil droplets, and the specific steps include:

[0142] Step S1, designing the structure of the single emulsion droplet preparation microfluidic chip 101 and the reverse emulsion microfluidic chip 108 in the single emulsion droplet reverse emulsification module 1, preparing the structure by methods including but not limited to soft lithography, nanoimprinting, 3D printing, etc., and preparing the microchannel 102; punching through holes at the corresponding positions of the continuous phase inlet 103, the dispersed phase inlet #1 104, the dispersed phase inlet #2 105, the dispersed phase inlet #3 106, the dispersed phase inlet #4 107 and the reverse emulsification column 108 with a puncher; designing the structure of the gas control chip 301 in the flow control module 3, and preparing the structure by methods including but not limited to soft lithography, nanoimprinting, 3D printing, etc.; designing the structure of the DMF body chip 402 in the DMF chip module 4 and preparing it;

[0143] Step S2, prepare the required reagents and load the reagents into the syringe 201 of the flow rate control module 2, the reverse phase emulsification column 109 and the corresponding area of ​​the DMF chip module 4:

[0144] Continuous phase: 5-15 mg / mL POPC in mineral oil solution was used as the continuous phase;

[0145] Optionally, fluorescent dyes may be added to the continuous phase at appropriate concentrations, including but not limited to 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocyanine perchlorate, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt), Nile Red, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt), etc.;

[0146] Dispersed phase #1: 0-5% wt / vol P188, 100-500 mM sucrose in water as dispersed phase #1;

[0147] Dispersed phase #2: 0-5% wt / vol P188, 100-500 mM sucrose in water as dispersed phase #2;

[0148] Dispersed phase #3: 0-5% wt / vol P188, 100-500 mM sucrose in water as dispersed phase #3;

[0149] Dispersed phase #4: 0-5% wt / vol P188, 100-500 mM sucrose in water as dispersed phase #4;

[0150] Optionally, fluorescent dyes of appropriate concentrations may be added to the dispersed phase, including but not limited to calcein, 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt, green fluorescent protein, fluorescein isothiocyanate-carboxymethyl-dextran, etc.;

[0151] Reverse phase emulsion column: 0-5% wt / vol P188, 100-500 mM glucose in water solution as the reverse phase emulsion column lower layer reagent, 5-15 mg / mL POPC in mineral oil solution as the reverse phase emulsion column upper layer reagent;

[0152] Reagent storage area #1: Disperse 1:4 ~1:10 vol / vol n-octanol:deionized water solution using ultrasound;

[0153] Reagent storage area #2: Disperse 1:4 ~1:10 vol / vol n-octanol: 2.5% wt / vol P188 aqueous solution using ultrasound;

[0154] Reagent storage area #3: Disperse 1:4 ~1:10 vol / vol n-octanol: 5% wt / vol P188 aqueous solution using ultrasound;

[0155] Reagent storage area #4: Disperse 1:4 ~1:10 vol / vol n-octanol: 10% wt / vol P188 aqueous solution using ultrasound;

[0156] Step S3, bonding single emulsion droplets from top to bottom in sequence to prepare microfluidic chip 101, reverse emulsification microfluidic chip 108, film 303, and gas control chip 301, using 1 wt% BSA solution to seal the glass cover slips at the corresponding positions of the reverse emulsification column overnight, and then washing away the residual BSA with a large amount of deionized water; connecting the flow rate control module 2, DMF chip module 4, and microscope imaging module 5, and connecting the control module 6 with the flow rate control module 2, the flow control module 3, the DMF chip module 4, and the microscope imaging module 5;

[0157] Step S4, start the flow rate control module 2, the flow control module 3, the DMF chip module 4, and the microscope imaging module 5, and stably generate oil-in-water single emulsion droplets in the single emulsion droplet inversion emulsification module 1 with high throughput, and then the oil-in-water single emulsion droplets are inverted emulsified to form liposomes;

[0158] Step S5, when enough liposomes are generated, the flow control module 3 is controlled to allow the liposomes to enter the upper surface of the ITO glass 401 in the DMF chip module 4;

[0159] Step S6, using the DMF chip body 402 to drive the movement of liposomes and reagents in each region to perform liposome biochemical reactions, the specific steps include:

[0160] Step S601, driving the liquid in the liposome inlet #1 404 into the liposome capture area #1 408;

[0161] Step S602, driving the liquid in the liposome inlet #2 405 into the liposome capture area #2 409;

[0162] Step S603, driving the liquid in the liposome inlet #3 406 into the liposome capture area #3 410;

[0163] Step S604, driving the liquid in the liposome inlet #4 407 into the liposome capture area #4 411;

[0164] Step S605, driving the liquid in the reagent storage area #1 412 forward into the liposome capture area #1 408;

[0165] Step S606, driving the liquid in the reagent storage area #2 413 forward into the liposome capture area #2 409;

[0166] Step S607, driving the liquid in the reagent storage area #3 414 forward into the liposome capture area #3 410;

[0167] Step S608, driving the liquid in the reagent storage area #4 415 forward into the liposome capture area #4 411;

[0168] Step S7, control the environmental temperature change (room temperature-40°C-room temperature cycle) and observe the dynamic reconstruction between the captured liposomes and oil droplets.

[0169] Note that the above are only preferred embodiments of the present application and the technical principles used. Those skilled in the art will understand that the present application is not limited to the specific embodiments herein, and that various obvious changes, readjustments and substitutions can be made by those skilled in the art without departing from the protection scope of the present application. Therefore, although the present application is described in more detail through the above embodiments, the present application is not limited to the above embodiments, and may also include more other equivalent embodiments without departing from the concept of the present application, all of which belong to the protection scope of the present application.

Claims

1. A microfluidic device for preparing liposomes by inversion emulsification, characterized in that: include: A first microfluidic chip comprises at least one first microfluidic channel, each of the first microfluidic channels is used to receive a continuous phase and a dispersed phase, and emulsify the continuous phase and the dispersed phase into a single emulsion droplet; A second microfluidic chip, the second microfluidic chip is bonded to the first microfluidic chip, the second microfluidic chip comprises at least one reverse emulsification column, each of the reverse emulsification columns is connected to the corresponding first microfluidic channel, and the reverse emulsification column is used to reverse emulsify the single emulsion droplets of the corresponding first microfluidic channel into corresponding liposomes.

2. The microfluidic device according to claim 1, characterized in that: The microfluidic device further comprises: At least one first pipeline, each of which is connected to the corresponding first microfluidic channel, and each of which is used to inject one of the continuous phases into the connected first microfluidic channel; at least one second pipeline, each of which is connected to the corresponding first microfluidic channel, and each of which is used to inject one of the dispersed phases into the connected first microfluidic channel; A plurality of flow rate control components are provided, each of the first pipeline and each of the second pipeline is connected with the flow rate control component, and the flow rate control component is used to control the flow rate of the continuous phase and the dispersed phase in the respective pipelines.

3. The microfluidic device according to claim 2, characterized in that: The flow rate control component comprises a syringe, which is connected to the corresponding first pipe or one of the second pipes, and is used to inject the continuous phase and the dispersed phase into the first pipe and the second pipe respectively.

4. The microfluidic device according to claim 1, characterized in that: The second microfluidic chip further comprises a plurality of second microchannels, each of which is connected to a corresponding reverse emulsification column, and the second microchannel is used to flow out the liposomes; The microfluidic device further includes a flow control module for controlling the on-off of each of the second microfluidic channels.

5. The microfluidic device according to claim 4, characterized in that: The flow control module comprises: A film, bonded to the second microfluidic chip, the film being attached to the plurality of second microchannels; a third microfluidic chip, bonded to the film, and the film is located between the second microfluidic chip and the third microfluidic chip, and an airway is prepared on the third microfluidic chip, and the airway leads to the film; A gas control component is connected to the air channel, and is used to control the gas pressure in the air channel to deform the film, thereby controlling the on-off of each of the second microchannels.

6. The microfluidic device according to claim 4, characterized in that: The microfluidic device further comprises: A fourth microfluidic chip, wherein a capture structure is prepared on the upper surface of the fourth microfluidic chip, wherein the capture structure is connected to each of the second microchannels, and the fourth microfluidic chip is used to drive the liposomes to move to corresponding predetermined positions in the capture structure.

7. The microfluidic device according to claim 6, characterized in that: The fourth microfluidic chip comprises a glass sheet, the capture structure is prepared on the upper surface of the glass sheet, and the material of the glass sheet comprises indium tin oxide.

8. The microfluidic device according to claim 7, characterized in that: The glass sheet is provided with a plurality of storage areas, each of which stores a corresponding reagent, and each of which is connected to the capture structure. The fourth microfluidic chip is also used to drive each of the reagents to move to the predetermined position in the capture structure.

9. The microfluidic device according to claim 6, characterized in that: The microfluidic device further comprises: The microscope imaging module comprises a microscope, and the microscope is aimed at the first microfluidic chip, the second microfluidic chip or the fourth microfluidic chip.

10. A method for preparing liposomes using the microfluidic device according to any one of claims 1 to 9, characterized in that: include: Injecting a continuous phase and a dispersed phase into at least one first microchannel of a first microfluidic chip, so that the matched continuous phase and the dispersed phase are emulsified into at least one single emulsion droplet; Each of the single emulsion droplets is introduced into a corresponding reverse emulsification column of a second microfluidic chip to reversely emulsify the single emulsion droplets into corresponding liposomes.

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