Microfluidic device and method for preparing liposomes by inversion emulsification

Abstract

The present invention provides a microfluidic device and method for preparing liposomes by reverse emulsification. The microfluidic device comprises: a first microfluidic chip including at least one first microchannel, each of which is used to receive a continuous phase and a dispersed phase and emulsify the continuous and dispersed phases into single emulsion droplets; a second microfluidic chip, bonded to the first microfluidic chip, and comprising at least one reverse emulsification column, each of which is connected to a corresponding first microchannel and is used to reverse emulsify the single emulsion droplets in the corresponding first microchannel into corresponding liposomes. The microfluidic device and method provided by the present invention can rapidly, controllably, and high-throughput prepare liposomes of uniform size and good integrity.

Description

Technical Field

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

[0002] Liposomes are closed, ordered structures with a bilayer structure that spontaneously form when naturally occurring amphiphilic molecules, such as phospholipids, are dispersed in water. Their unique structure allows them to isolate the internal and external aqueous environments while selectively allowing the transmembrane movement of some ions and small molecules. This plays a crucial role in synthetic biology, drug delivery, and the preparation of artificial single cells.

[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 being 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 being bonded to the first microfluidic chip, the second microfluidic chip comprising at least one reverse emulsification column, each reverse emulsification column being connected to a corresponding first microchannel, the reverse emulsification column being 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; 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 their respective pipes.

[0007] According to an embodiment of the present invention, the flow rate control component includes a syringe connected to the corresponding first pipe or one of the second pipes, and the syringe is used to inject the continuous phase and the 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 thin film, bonded to the second microfluidic chip, and the thin film is adhered to multiple second microchannels; a third microfluidic chip, bonded to the thin film, and the thin 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 thin film; a gas control component, connected to the airway, the gas control component is used to control the gas pressure in the airway, causing the thin film to deform, and thereby controlling 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] Another embodiment of the present invention 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 performing a gentle reverse emulsification on 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 A schematic diagram showing the structure of a microfluidic device for preparing liposomes by inversion emulsification according to one embodiment of the present invention;

[0018] Figure 2 Schematically shows a top view of a first microfluidic channel according to an embodiment of the present invention;

[0019] Figure 3 A schematic diagram showing the structure of a microfluidic device for preparing liposomes by inversion emulsification according to another embodiment of the present invention;

[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 Schematically shows a structural diagram of a flow control module according to an embodiment of the present invention;

[0022] Figure 6 Schematically shows a structural diagram 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 Schematically showing a plan view of functional areas of a fourth microfluidic chip integrated with a liposome capture structure according to an embodiment of the present invention;

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

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

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

[0028] Figure 12 The flowchart of the 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 reverse 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. Reversed 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 accompanying 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 in no way 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 making creative efforts 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, they indicate 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, numerical expressions and numerical values ​​set forth in these embodiments 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 drawings are not drawn according to actual proportional relationships. The techniques, methods and equipment known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, the techniques, methods and equipment should be considered as part of the authorized specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as being merely exemplary and not as limitations. Therefore, other examples of the exemplary embodiments may have different values. It should be noted that similar numbers and letters represent similar items in the following figures, and therefore, once an item is defined in one figure, it does not need to be further discussed in subsequent figures.

[0034] In the description of the present invention, it should be understood that the directions or positional relationships indicated by directional words such as "front, back, up, down, left, right", "horizontal, vertical, perpendicular, horizontal" and "top, bottom" are usually based on the directions or positional relationships shown in the accompanying 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 device or element 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 outline of each component itself.

[0035] For ease of description, spatially relative terms such as "above," "above," "on the upper surface of," and "upper" may be used herein to describe the spatial positional relationship of a device or feature to other devices or features as shown in the figures. It should be understood that spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in a drawing is inverted, a device described as "above" or "on top of" another device or structure would then be positioned as "below" or "below" the other device or structure. Thus, the exemplary term "above" can include both the "above" and "below" orientations. The device may also be positioned in other different ways (rotated 90° or in other orientations), and the spatially relative descriptions used herein should be 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. Unless 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 A schematic diagram showing the structure of a microfluidic device for preparing liposomes by inversion emulsification according to one embodiment of the present invention; Figure 2 The figure schematically shows a top view of a first microfluidic channel according to an embodiment of the present invention.

[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 referred to as a single emulsion droplet preparation microfluidic chip. The second microfluidic chip 108 includes at least one reverse emulsification column 109, each of which is connected to a corresponding first microfluidic channel 102, and the reverse emulsification column 109 is used to reverse emulsify the single emulsion droplets in the corresponding first microfluidic channel 102 into corresponding liposomes. The second microfluidic chip 108 can be referred to as a reverse emulsification microfluidic chip.

[0040] In this embodiment, single emulsion droplets are prepared using microfluidic chips 101 and 108, and liposomes are prepared from these single emulsion droplets, enabling high-throughput liposome preparation. Furthermore, because the size of the single emulsion droplets is uniform and controllable, the size of the resulting liposomes is also uniform and controllable. Gentle inverse emulsification of the single emulsion droplets in reverse emulsification column 109 on microfluidic chip 108 prevents leakage of contents during conventional reverse emulsification processes.

[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, multiple 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, conducting biochemical experiments, etc., which will be explained with examples 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, to reduce the number of microchannels 102 in the first microfluidic chip 101, multiple microchannels 102 can be designed in a grid pattern. That is, when dispersed phases #1, #2, #3, and #4 are emulsified with the same continuous phase #1, multiple branches can be provided at the end of the microchannel 102 where the continuous phase #1 is injected. The continuous phase #1 is introduced from the first channel 103 into the four branches connected to the second channels 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 for illustrative purposes only and does not constitute a limitation of the present invention. The specific number of pipes, microchannels, and reverse emulsification columns can be appropriately determined based on the types of continuous and dispersed phases. Microfluidic chips 101 and 108 can be fabricated using methods such as soft lithography, nanoimprinting, and 3D printing, 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 accompanying 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 pipes 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 set up 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 set on each pipe 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 assembly 201 may include a syringe 201, each of which may be connected to a corresponding first conduit 103 or one of the second conduits 104, 105, 106, 107 via a flexible tube 203. The syringe 201 may be used to inject the continuous phase #1 into the first conduit 103 and to inject the dispersed phases #1, #2, #3, and #4 into the second conduits 104, 105, 106, and 107, respectively. A syringe propulsion device 202 may be connected to the syringe 201 to precisely control the injection speed of the syringe 201. This embodiment utilizes a syringe pump to prepare single emulsion droplets, further reducing experimental costs and operational difficulty.

[0048] Please continue reading Figure 1 and Figure 3 The microfluidic device further includes 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 flow rate of each liposome outflow.

[0049] Figure 5 The structure 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 microchannel 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, it can be connected to the airway 302 through a hose 306. The gas control component 304 can be used to control the gas pressure in the airway 303, causing the film 303 to deform, thereby controlling the opening and closing of each second microchannel 110. 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 microchannel 110, thereby achieving 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 syringe propulsion device 305 may be used to control the injection speed of the syringe 304 .

[0053] Please continue reading Figure 3 The microfluidic device may further include a fourth microfluidic chip 4. A capture structure (not shown) is fabricated on the upper surface of the fourth microfluidic chip 4. The capture structure is connected to each second microchannel 110 via a specific drive path. The fourth microfluidic chip 4 can be used to drive liposomes to corresponding predetermined positions within the capture structure. The fourth microfluidic chip 4 may be a digital microfluidics (DMF) chip. The fourth microfluidic chip 4 can serve 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, with a capture structure formed on the upper surface of the glass sheet 401. The glass sheet may be made of 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 disposed on the DMF chip body 402.

[0056] By integrating the DMF chip on the microfluidic device, the difficulty of manipulating 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 an 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. Methods for forming the capture structure include but are 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 Schematically showing a plan view of functional areas of a fourth microfluidic chip integrated with a liposome capture structure according to an embodiment of the present invention; Figure 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, and 415 can be provided on the glass sheet 401. Each storage area 412, 413, 414, and 415 can store a corresponding reagent #1, #2, #3, and #4. Each storage area #1, #2, #3, and #4 is connected to a capture structure. The fourth microfluidic chip 4 can also be used to drive each reagent #1, #2, #3, and #4 to a predetermined position in the capture structure. The predetermined position is, for example, the liposome capture areas 408, 409, 410, and 411 shown in the figure.

[0061] like Figure 8 and Figure 9 As shown, liposomes #1, #2, #3, and #4 can, for example, be moved to liposome capture regions 408, 409, 410, and 411 along a straight path. Liposomes #1, #2, #3, and #4 should enter the liposome capture region from the front to achieve efficient capture of the liposomes. Reagents #1, #2, #3, and #4 can, for example, be moved to liposome capture regions 408, 409, 410, and 411 along a broken line path. Reagents #1, #2, #3, and #4 should also enter the liposome capture region from the front to avoid causing lipids to leave the capture structure. Thus, liposomes can be subjected to biochemical experimental observations in liposome capture regions 408, 409, 410, and 411.

[0062] Please continue reading Figure 3For example, the entire process of liposome production, flow, and use can be observed using 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 capture the production of oil-in-water single emulsion droplets, the production of liposomes, and the results of liposome-related biochemical experiments in real time.

[0063] like Figure 3 As shown, the microfluidic device may further include a control module 6, which can 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] Figure 10 Schematic diagram of an experiment for high-throughput generation of single emulsion droplets according to an embodiment of the present invention.

[0065] like Figure 10 As shown, the left image shows the injection of a continuous or dispersed phase into the microchannel, while the right image shows the emulsification process. As can be seen, the microfluidic device of the present invention can stably produce single emulsion droplets at high throughput, enabling high-throughput liposome preparation. Furthermore, because the size of the single emulsion droplets is uniform and controllable, the size of the resulting liposomes 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. This method has the same technical features and beneficial effects as the microfluidic device, and will not be described in detail here.

[0067] Figure 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 Figure 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, and the matched continuous phase and dispersed phase are emulsified into at least one single emulsion droplet. Details of operation S111 can be found in the above description of the microfluidic device and are not 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 syringe 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 reverse emulsified into the corresponding liposome. Some details of operation S112 can be found in the above description of the microfluidic device and will not be repeated here.

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

[0074] like Figure 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, where it undergoes reverse emulsification into the corresponding liposome. Details of operation S122 can be found in the above description of the microfluidic device and are not repeated here. The processes of operations S121-S122 and S111-S112 are similar and are not 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 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 further 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 figures, 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 structures 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 structures using methods including but not limited to soft lithography, nanoimprinting, 3D printing, etc. to prepare the microchannel 102; a puncher can be used to punch through holes at the corresponding positions of the continuous phase inlet 103, dispersed phase inlet #1 104, dispersed phase inlet #2 105, dispersed phase inlet #3 106, dispersed phase inlet #4 107, and reverse emulsion column 109; designing the structure of the gas control chip 301 in the flow control module 3, and preparing the structure using 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 them into the syringe 201 of the flow rate control module 2, the reverse phase emulsion 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) solution in mineral oil is used as the continuous phase;

[0087] Alternatively, fluorescent dyes may be added to the continuous phase at appropriate concentrations, including but not limited to 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine 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) and 100-500 mM sucrose in water.

[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.

[0092] Adding an appropriate concentration of fluorescent dye 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.;

[0093] Reverse-phase emulsion column: Use 0-5% wt / vol P188 and 100-500 mM glucose in water as the lower layer of the reverse-phase emulsion column reagent, and use 5-15 mg / mL POPC in mineral oil as the upper layer of the reverse-phase emulsion column 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: Single emulsion droplets are bonded sequentially from top to bottom to prepare the microfluidic chip 101, the reverse emulsion microfluidic chip 108, the film 303, and the gas control chip 301. The glass cover slips at the corresponding positions of the reverse emulsion column 109 are sealed overnight with a 1 wt% bovine serum albumin (BSA) solution, and then the residual BSA is washed away with a large amount of deionized water. The flow rate control module 2, the DMF chip module 4, and the microscope imaging module 5 are connected, and the control module 6 is connected to the flow rate control module 2, the flow control module 3, the DMF chip module 4, and the microscope imaging module 5.

[0099] Step S4, turning on the flow rate control module 2, the flow rate control module 3, the DMF chip module 4, and the microscope imaging module 5, and stably generating water-in-oil single emulsion droplets in the single emulsion droplet inversion emulsification module 1 with high throughput, and then forming liposomes by inversion emulsification of the water-in-oil single emulsion droplets;

[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 a liposome biochemical reaction, 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: Record the curve of the change of the fluorescence intensity inside the liposomes captured in each liposome capture area over time, and determine the denaturation temperature of α-hemolysin based on the curve.

[0111] Example 2

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

[0113] Step S1, designing the structures 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 structures using methods including but not limited to soft lithography, nanoimprinting, 3D printing, etc. to prepare the microchannel 102; using a puncher to punch through holes at the corresponding positions of the continuous phase inlet 103, dispersed phase inlet #1 104, dispersed phase inlet #2 105, dispersed phase inlet #3 106, dispersed phase inlet #4 107 and reverse emulsion column 108; designing the structure of the gas control chip 301 in the flow control module 3, and preparing the structure using 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 them into the syringe 201 of the flow rate control module 2, the reverse phase emulsion column 109 and the corresponding areas 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] Alternatively, fluorescent dyes may be added to the continuous phase at appropriate concentrations, including but not limited to 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine 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.

[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, and the like;

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

[0123] Reagent Storage Area #1: Ultrasonic dispersion of a 1:4 to 1:10 vol / vol n-octanol:5% wt / vol P188 aqueous solution.

[0124] Reagent Storage Area #2: Ultrasonic dispersion of 1:4 to 1:10 vol / vol oleic acid: 5% wt / vol P188 aqueous solution;

[0125] Reagent Storage Area #3: Ultrasonic dispersion of a 1:4 to 1:10 vol / vol solution of chloroform and hexane (36:64 vol / vol): 5% wt / vol P188 in water.

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

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

[0128] Step S4, turning on the flow rate control module 2, the flow rate control module 3, the DMF chip module 4, and the microscope imaging module 5, and stably generating water-in-oil single emulsion droplets in the single emulsion droplet inversion emulsification module 1 with high throughput, and then forming liposomes by inversion emulsification of the water-in-oil single emulsion droplets;

[0129] Step S5, when enough liposomes are produced, 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, specifically comprising:

[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] In step S7, the environmental temperature is controlled to change (room temperature - 40°C - room temperature cycle) to observe the dynamic reconstruction between the captured liposomes and the oil droplets.

[0140] Example 3

[0141] With reference to Example 2 and in conjunction 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 reconstitution morphology between liposomes and oil droplets, the specific steps comprising:

[0142] Step S1, designing the structures 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 structures using methods including but not limited to soft lithography, nanoimprinting, 3D printing, etc. to prepare the microchannel 102; using a puncher to punch through holes at the corresponding positions of the continuous phase inlet 103, dispersed phase inlet #1 104, dispersed phase inlet #2 105, dispersed phase inlet #3 106, dispersed phase inlet #4 107 and reverse emulsion column 108; designing the structure of the gas control chip 301 in the flow control module 3, and preparing the structure using 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 them into the syringe 201 of the flow rate control module 2, the reverse phase emulsion column 109 and the corresponding areas 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] Alternatively, fluorescent dyes may be added to the continuous phase at appropriate concentrations, including but not limited to 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine 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.

[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, and the like;

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

[0152] Reagent Storage Area #1: Ultrasonic dispersion of a 1:4 to 1:10 vol / vol n-octanol:deionized water solution;

[0153] Reagent Storage Area #2: Ultrasonic dispersion of a 1:4 to 1:10 vol / vol n-octanol:2.5% wt / vol P188 aqueous solution.

[0154] Reagent Storage Area #3: Ultrasonic dispersion of a 1:4 to 1:10 vol / vol n-octanol:5% wt / vol P188 aqueous solution.

[0155] Reagent Storage Area #4: Ultrasonic dispersion of a 1:4 to 1:10 vol / vol n-octanol:10% wt / vol P188 aqueous solution.

[0156] Step S3: Prepare the microfluidic chip 101, the reverse emulsion microfluidic chip 108, the film 303, and the gas control chip 301 by bonding single emulsion droplets in sequence from top to bottom. Use a 1 wt% BSA solution to seal the glass cover slips at the corresponding positions of the reverse emulsion column overnight, and then wash away the residual BSA with a large amount of deionized water; connect the flow rate control module 2, the DMF chip module 4, and the microscope imaging module 5; and connect the control module 6 to 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, turning on the flow rate control module 2, the flow rate control module 3, the DMF chip module 4, and the microscope imaging module 5, and stably generating water-in-oil single emulsion droplets in the single emulsion droplet inversion emulsification module 1 with high throughput, and then forming liposomes by inversion emulsification of the water-in-oil single emulsion droplets;

[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, specifically comprising:

[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] In step S7, the environmental temperature was controlled to change (room temperature-40°C-room temperature cycle) to observe the dynamic reconstruction between the captured liposomes and the oil droplets.

[0169] Note that the above are only preferred embodiments of the present application and the technical principles employed. 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 scope of protection of the present application. Therefore, although the present application has been described in more detail through the above embodiments, the present application is not limited to the above embodiments, and may include many other equivalent embodiments without departing from the scope of protection of the present application, all of which fall within the scope of protection 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 being configured 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 being bonded to the first microfluidic chip, the second microfluidic chip comprising at least one reverse emulsification column and a plurality of second microfluidic channels, each of the reverse emulsification columns being connected to a corresponding first microfluidic channel, the reverse emulsification column being used to reverse emulsify the single emulsion droplets in the corresponding first microfluidic channel into corresponding liposomes, each of the second microfluidic channels being connected to a corresponding reverse emulsification column, the second microfluidic channel being used to discharge the liposomes; A fourth microfluidic chip, wherein a capture structure is prepared on the upper surface of the fourth microfluidic chip, and the capture structure is connected to each of the second microchannels. The fourth microfluidic chip is used to drive the liposomes to move to the corresponding predetermined positions in the capture structure. The fourth microfluidic chip includes a glass sheet, and the capture structure is prepared on the upper surface of the glass sheet. The material of the glass sheet includes indium tin oxide. A plurality of storage areas are provided on the glass sheet, each of which stores a corresponding reagent, and each of the storage areas 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.

2. The microfluidic device according to claim 1, wherein The microfluidic device further comprises: at least one first pipe, each of the first pipes being connected to a corresponding first microfluidic channel, and each of the first pipes being used to inject one of the continuous phases into the connected first microfluidic channel; at least one second pipe, each second pipe being connected to the corresponding first microfluidic channel, and each second pipe being 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 pipes and each of the second pipes 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 pipes.

3. The microfluidic device according to claim 2, wherein The flow rate control component includes a syringe connected to the corresponding first pipe or one of the second pipes, and the syringe 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, wherein 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 includes: a film bonded to the second microfluidic chip, wherein the film is 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 1, wherein The microfluidic device further comprises: The microscope imaging module includes a microscope, and the microscope is aligned with the first microfluidic chip, the second microfluidic chip or the fourth microfluidic chip.

7. A method for preparing liposomes using the microfluidic device according to any one of claims 1 to 6, 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, so that the single emulsion droplets are reverse emulsified into corresponding liposomes.

Images