Microfluidic device and method for liposome preparation

Abstract

The present invention provides a microfluidic device and method for liposome preparation. The microfluidic device includes: a microfluidic chip for preparing at least one liposome using a variety of reagents; an automated control module configured with a trained machine learning model, the automated control module being used to analyze the imaging data of the liposome using the machine learning model and generate a first control instruction for regulating the liposome; a pressure control module connected to the microfluidic chip and the automated control module, the pressure control module being used to adjust the pressure value of at least one reagent during the process of the microfluidic chip preparing the liposome according to the first control instruction so as to obtain the desired liposome.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic technology, and particularly relates to a microfluidic device and method for liposome preparation. Background Art

[0002] Liposomes are closed molecular ordered assemblies formed by lipid bilayers, which can separate the internal water environment from the surrounding water environment, and at the same time allow some small molecules and ions to selectively penetrate, playing an important role as carriers for biological reactions in fields such as artificial synthesis of single cells.

[0003] Using the microfluidic method, uniform and stable liposomes can be prepared in high throughput, and at the same time, bioactive reactants can be encapsulated inside the liposomes, providing the possibility for subsequent biological reaction experiments. However, traditional microfluidic devices include a pressure pump-reservoir system, where the liquid in the reservoir is driven into the microfluidic chip by compressed air, resulting in a large amount of liquid required in the reservoir. And for some reagent systems, the cost is high, the total amount of reagents is small, and the dead volume of the microfluidic chip channels and connecting pipelines is large, resulting in a high experimental cost. In addition, it is difficult to regulate the pressure of each phase reagent in traditional microfluidic devices, which poses a high requirement for the experimental ability of experimental personnel. Summary of the Invention

[0004] In view of the above problems, the present invention provides a microfluidic device and method for liposome preparation, which can realize the high-throughput preparation of micron-sized liposomes using trace reagents, and at the same time introduce a multi-modal machine learning model to realize the automated preparation of liposomes.

[0005] One aspect of an embodiment of the present invention provides a microfluidic device for liposome preparation, including: a microfluidic chip for preparing at least one liposome using a variety of reagents; an automated control module configured with a trained machine learning model, and the automated control module is used to analyze the imaging data of liposomes using the machine learning model and generate a first control instruction for adjusting liposomes in real time; a pressure control module connected to the microfluidic chip and the automated control module, and the pressure control module is used to adjust the pressure value of at least one reagent in the process of preparing liposomes by the microfluidic chip in real time according to the first control instruction to obtain the desired liposomes.

[0006] According to an embodiment of the present invention, the microfluidic chip includes a negative mold and a hydrophobic film. The negative mold includes opposite first and second surfaces, and the hydrophobic film is bonded to the first surface. A plurality of through holes and microchannels are formed on the first surface. The plurality of through holes communicate the first surface and the second surface, and each through hole stores one reagent, and the microchannels connect each through hole; the pressure control module is connected to each through hole on the second surface and transmits pressure to the corresponding reagent in each through hole.

[0007] According to an embodiment of the present invention, a liposome purification structure and a liposome capture structure are further formed on the first surface of the negative mold. The liposome purification structure is connected to the microchannel, and the liposome capture structure is connected to the microchannel.

[0008] According to an embodiment of the present invention, the microfluidic chip further includes a cover glass, and the cover glass is bonded to the hydrophobic film; the microfluidic device further includes a microscopic imaging module, the microscopic imaging module is connected to the automatic control module, the microscopic imaging module is close to the cover glass, and the microscopic imaging module is used to obtain a microscopic image of the liposome and transmit it to the automatic control module in real time.

[0009] According to an embodiment of the present invention, the automatic control module includes: a machine learning module in which a machine learning model is configured; a first input unit for inputting size data of the microchannel into the machine learning module; a second input unit for inputting component data and concentration data of each reagent into the machine learning module; a third input unit for inputting a microscopic image into the machine learning module; a fourth input unit for inputting a pressure value in the pressure control module into the machine learning module; a first output unit for outputting a first control instruction to the pressure control module; a second output unit for outputting a second control instruction to the microscopic imaging module, and the second control instruction is used to control imaging parameters for the microscopic imaging module to obtain a microscopic image, wherein the first control instruction and the second control instruction are generated by the machine learning model according to the data input by the first input unit, the second input unit, the third input unit, and the fourth input unit.

[0010] According to an embodiment of the present invention, the pressure control module includes a pressure pump, a plurality of hoses, and a plurality of connectors. The input end of the pressure pump is connected to the automatic control module, the output end of the pressure pump is connected to the plurality of connectors through the hoses, each connector is connected to a corresponding through hole, and the pressure pump is used to generate gases with different pressure values and act on the connectors.

[0011] According to an embodiment of the present invention, the microfluidic chip includes a fixed hole and groove layer, the fixed hole and groove layer is bonded to the second surface, there are a plurality of holes and grooves on the fixed hole and groove layer, and each hole and groove is coaxial with the corresponding through hole; each connector includes a flat plate joint and a flat plate base, the flat plate joint is connected to the flat plate base, the flat plate joint is connected to the corresponding hose, and the flat plate base is fitted and arranged in the hole and groove.

[0012] According to an embodiment of the present invention, the connector includes a first joint, a second joint, and a through pipe. The first joint is connected to the second joint, the first joint is connected to the hose, the second joint is connected to the through hole, the inlet of the through pipe is arranged inside the first joint, and the outlet of the through pipe is arranged inside the second joint.

[0013] According to an embodiment of the present invention, the caliber of the inlet of the through pipe is greater than or equal to the caliber of the outlet.

[0014] Another aspect of the embodiments of the present invention further provides a method for preparing liposomes using the microfluidic device of the above embodiments, including: preparing at least one liposome using various reagents on a microfluidic chip; analyzing the imaging data of the liposomes using a machine learning model in an automated control module to generate a first control instruction for adjusting the liposomes; and executing the first control instruction in a pressure control module to adjust the pressure value of at least one reagent during the process of preparing liposomes on the microfluidic chip, so as to obtain the desired liposomes.

[0015] The microfluidic device and method for liposome preparation provided by the embodiments of the present invention can complete the preparation of liposomes on a microfluidic chip using only microliter-level reagents, avoiding the use of a large amount of reagents to ensure that the liquid in the reservoir completely covers the hose inlet, thereby avoiding the introduction of air bubbles during the experiment; at the same time, it effectively reduces the dead volume of the sample entering the channel, including the dead volume in the hose connecting the reservoir and the microfluidic chip and the dead volume in the through holes on the surface of the microfluidic chip, effectively reducing the time required for the dead volume of reagent consumption, thereby improving the preparation efficiency of liposomes; the pressure at the output port of the pressure control module directly acts on the reagent liquid stored in the through holes on the surface of the microfluidic chip, rather than on the reagent liquid in the reservoir, reducing the influence of the height difference between the reservoir and the microfluidic chip and the hose connecting the reservoir and the microfluidic chip on pressure control and feedback, making pressure control and feedback more accurate; using a multi-modal machine learning model to automatically adjust the pressure parameters of each phase to achieve the automated preparation of liposomes, reducing the requirements for the operation skills and operation experience of experimental personnel in microfluidic preparation of liposomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Through the following description of the embodiments of the present invention with reference to the drawings, the above and other objects, features and advantages of the present invention will become clearer. In the drawings:

[0017] Figure 1 Schematically shows the structural diagram of the microfluidic device according to an embodiment of the present invention;

[0018] Figure 2 Schematically shows the structural diagram of the microfluidic chip according to an embodiment of the present invention;

[0019] Figure 3 Schematically shows the structural diagram of the negative mold according to an embodiment of the present invention;

[0020] Figure 4 Schematically shows the structural diagram of the flat base and the flat joint according to an embodiment of the present invention;

[0021] Figure 5 Schematically shows the functional diagram of the automated control module according to an embodiment of the present invention;

[0022] Figure 6Schematically shows a structural diagram according to an embodiment of the present invention, using a centrifuge tube with the lower bottom cut off and a matching joint of a pressure pump to replace a flat base and a flat joint;

[0023] Figure 7 Schematically shows a structural diagram of a centrifuge tube with the lower bottom cut off and a matching joint of a pressure pump according to an embodiment of the present invention;

[0024] Figure 8 Schematically shows a structural diagram of a support base for supporting a matching joint of a pressure pump according to an embodiment of the present invention;

[0025] Figure 9 Schematically shows a structural diagram according to an embodiment of the present invention, using a special centrifuge tube with a frustum-shaped lower bottom and an unclosed lower end and a matching joint of a pressure pump to replace a flat base and a flat joint;

[0026] Figure 10 Schematically shows a structural diagram of a special centrifuge tube with a frustum-shaped lower bottom and an unclosed lower end according to an embodiment of the present invention;

[0027] Figure 11 Shows an experimental photograph of the liposomes prepared in Example 1;

[0028] Figure 12 Shows a flowchart of a method for preparing liposomes using a microfluidic device according to an embodiment of the present invention.

[0029] Description of reference numerals:

[0030] 1. Microfluidic chip; 101. Negative mold; 102. Hydrophobic thin film; 103. Cover glass; 104. Through hole; 105. Through hole; 106. Through hole; 107. Outlet; 108. Microchannel; 109. Liposome purification structure; 110. Liposome capture structure; 111. Fixed hole groove layer; 2. Flat base; 3. Flat joint; 4. First hose; 5. Pressure pump; 6. Microscope lens; 7. CCD camera; 8. Automatic control module; 801. Chip parameter input; 802. Reagent parameter input; 803. Hydrodynamics formula; 804. Microscope real-time image input; 805. Real-time pressure input; 806. Machine learning module; 807. Image data set; 808. Pressure control output; 809. Microscope control output; 810. Focus and objective magnification adjustment; 9. Centrifuge tube with the lower bottom cut off; 10. Matching joint of pressure pump; 1001. Upper part of flat joint; 1002. Lower part of flat joint; 11. Support base; 12. Second hose; 13. Centrifuge tube with a frustum-shaped lower bottom and an unclosed lower end; 14. Glue; 15. Pressure control module; 16. Microscopic imaging module; 1111. Hole groove. Detailed implementation manners

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a 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 in no way restricts the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to 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 "comprising" and / or "including" 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 arrangements, numerical expressions, and numerical values of the components and steps set forth in these embodiments do not limit the scope of the present invention. At the same time, it should be understood that for the sake of convenience of description, the dimensions of the various parts shown in the drawings are not drawn in actual proportional relationships. Technologies, methods, and devices known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, the technologies, methods, and devices should be regarded as part of the authorized specification. In all the examples shown and discussed here, any specific value should be interpreted as merely exemplary and not as a limitation. Therefore, other examples of the exemplary embodiments may have different values. It should be noted that like reference numerals and letters denote like items in the following drawings, and thus, once an item is defined in one drawing, further discussion thereof is not required in subsequent drawings.

[0034] In the description of the present invention, it should be understood that the orientation terms such as "front, rear, upper, lower, left, right", "lateral, vertical, perpendicular, horizontal", and "top, bottom" generally refer to the orientation or position relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description. Without contrary instructions, these orientation terms do not indicate and imply that the device or element referred to must have a specific orientation or be constructed and operated in a specific orientation, and thus should not be construed as limiting the scope of protection of the present invention; the orientation terms "inside, outside" refer to the inside and outside relative to the contour of each component itself.

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

[0036] In addition, it should be noted that the use of terms such as "first", "second" etc. to define components is only for the convenience of differentiating the corresponding components. Without additional statements, the above terms have no special meanings, and thus should not be construed as limiting the protection scope of the present invention.

[0037] Figure 1 Schematically shows a structural diagram of a microfluidic device according to an embodiment of the present invention; Figure 2 Schematically shows a structural diagram of a microfluidic chip according to an embodiment of the present invention; Figure 3 Schematically shows a structural diagram of a negative mold according to an embodiment of the present invention; Figure 4 Schematically shows a structural diagram of a flat base and a flat joint according to an embodiment of the present invention; Figure 5 Schematically shows a functional diagram of an automatic control module according to an embodiment of the present invention.

[0038] As Figure 1 shown, the microfluidic device can include a microfluidic chip 1, an automatic control module 8, and a pressure control module 15.

[0039] The microfluidic chip 1 can be used to prepare at least one kind of liposome using a variety of reagents. A trained machine learning model is configured in the automatic control module 8. The automatic control module 8 is used to analyze the imaging data of the liposome using the machine learning model and generate a first control instruction for regulating the liposome. The process of the automatic control module 8 generating the first control instruction based on the imaging data of the liposome can be real-time. The pressure control module 15 is connected to the microfluidic chip 1 and the automatic control module 8. The pressure control module 15 can be used to adjust the pressure value of at least one reagent during the process of the microfluidic chip 1 preparing the liposome according to the first control instruction, so as to obtain the desired liposome. The process of the pressure control module 15 adjusting the pressure value according to the first control instruction can be real-time. In this embodiment, only microliter-level reagents can be used to complete the preparation of liposomes on the microfluidic chip; at the same time, the dead volume of the sample entering the channel is effectively reduced, further improving the preparation efficiency of liposomes; the pressure at the output port of the pressure control module directly acts on the reagent liquid, making the pressure control and feedback more precise; the multi-modal machine learning model is used to automatically adjust the pressure parameters of each phase in real-time to realize the automatic preparation of liposomes, reducing the requirements for the operation technology and operation experience of experimental personnel for microfluidic preparation of liposomes.

[0040] As Figure 2 shown, the microfluidic chip 1 may include a negative mold 101 and a hydrophobic thin film 102. The negative mold 101 includes an opposite first surface and a second surface. The hydrophobic thin film 102 is bonded to the first surface. A plurality of through holes 104, 105, 106 and microchannels 108 are formed on the first surface. The plurality of through holes 104, 105, 106 communicate the first surface and the second surface. Each of the through holes 104, 105, 106 stores a reagent, and the microchannel 108 connects each of the through holes 104, 105, 106. As Figure 1 and Figure 2 shown, the pressure control module 15 is connected to each of the through holes 104, 105, 106 on the second surface and transmits the pressure to the corresponding reagent in each of the through holes 104, 105, 106.

[0041] As an example, through holes 104, 105, 106 with a pore diameter of 1-4 mm can be punched out using a puncher to store different reagents. The through holes 104, 105, 106 can be used as an external continuous aqueous inlet, an intermediate dispersed oily reagent inlet, and an internal dispersed aqueous reagent inlet respectively. Different reagents are flowed into the microchannel 108 to prepare liposomes. For example, a reagent can be directly injected into the corresponding inlet using a microsyringe; a microchannel 108 with a depth of 5-20 μm and a width of 5-20 μm can be prepared using, including but not limited to, soft lithography technology, nanoimprint technology, 3D printing technology, etc.; the negative mold 101 and the hydrophobic thin film 102 can be bonded together using oxygen plasma.

[0042] As shown Figure 2 and Figure 3 shown, a liposome purification structure 109 and a liposome capture structure 110 can also be formed on the first surface of the negative mold 101. The liposome purification structure 109 is connected to the microchannel 108, and the liposome capture structure 110 is connected to the microchannel 108. After liposomes are stably produced, the liposome purification structure 109 can purify liposomes by dielectrophoresis based on the difference in the dielectric properties between liposomes and by-products, using a liquid metal material such as, but not limited to, gallium as an electrode; the liposome capture structure 110 can capture and in-situ observe liposomes by using a hydrodynamic capture structure. In this embodiment, multiple liposome experiments can be completed on a microfluidic chip integrating liposome preparation, capture, and in-situ observation, improving the efficiency and accuracy of the experiments. In some embodiments, part of the microchannel 108 of the prepared microfluidic chip 1 can be modified to improve the hydrophilicity of the modified part. Exemplary steps of the modification can be found in the embodiments described later in this document.

[0043] As shown Figure 1 and Figure 2 shown, the microfluidic chip 1 may further include a cover glass 103, and the cover glass 103 is bonded to the hydrophobic thin film 102. For example, oxygen plasma bonding can be used. The microfluidic device may further include a microscopic imaging module 16. The microscopic imaging module 16 is connected to the automatic control module 8. The microscopic imaging module 16 is close to the cover glass 103. The microscopic imaging module 16 is used to obtain microscopic images of liposomes and transmit them to the automatic control module 8. The microscopic imaging module 16 may be composed of a microscope lens 6 and a CCD camera 7, for example. The microscope lens 6 may be an inverted microscope lens. The microscope lens 6 is aligned with the cover glass 103 and can be specifically aligned with the liposome generation position or the liposome capture position to track and analyze the biochemical behavior of liposomes. The CCD camera 7 can be used to capture real-time images observed by the inverted microscope lens 6 and simultaneously transmit the above real-time images to the automatic control module 8.

[0044] As shown Figure 1 and Figure 5As shown in the figure, the automatic control module 8 may include: a machine learning module 807 in which a machine learning model is configured; a first input unit 801, which can be used as a chip parameter input for inputting the size data of the microchannel 108 into the machine learning module 807. A second input unit 802, which can be used as a reagent parameter input for inputting the component data and concentration data of each reagent into the machine learning module 807; a third input unit 804, which can be used as a real-time microscope image input for inputting a microscopic image into the machine learning module 807; a fourth input unit 805, which can be used as a real-time pressure input for inputting the pressure value in the pressure control module 15 into the machine learning module 807. A first output unit 808, which can be used as a pressure control output for outputting a first control instruction to the pressure control module 15; a second output unit 809, which can be used as a microscope control output for outputting a second control instruction to the microscopic imaging module 16, and the second control instruction is used to control the microscopic imaging module 16 to obtain the imaging parameters of the microscopic image. The second control instruction may be, for example, a control instruction for adjusting the focal length and objective magnification 810. The machine learning model 807 can be trained using the image dataset 806 collected during the experiment so that it can accurately identify liposomes, and then the data input by the first input unit 801, the second input unit 802, the third input unit 804, and the fourth input unit 805, etc. are input into the machine learning model 807. At the same time, in combination with the hydrodynamic formula 803, the first control instruction and the second control instruction are generated. In the automatic control module 8, the control and adjustment processes of the above-mentioned modules can be real-time. In this embodiment, the multi-modal machine learning model is used to automatically adjust the pressure parameters of each phase in real time, which can realize the automatic preparation of liposomes and reduce the requirements for the operation technology and operation experience of experimental personnel for the preparation of liposomes by microfluidics.

[0045] As Figure 1 and Figure 2 shown in the figure, the pressure control module 15 may include a pressure pump 5, a plurality of hoses 4, and a plurality of connectors. The hose 4 can be regarded as the first hose. The input end of the pressure pump 5 is connected to the automatic control module 8, and the output end of the pressure pump 5 is connected to a plurality of connectors through the hose 4. Each connector is connected to the corresponding through holes 104, 105, 106. The pressure pump 5 is used to generate gases with different pressure values and act on the connectors.

[0046] As Figure 1 , Figure 2 , Figure 4As shown, the microfluidic chip 1 may include a fixed hole and groove layer 111, which is bonded to the second surface of the negative mold 101. There are a plurality of holes and grooves 1111 on the fixed hole and groove layer 111, and each hole and groove 1111 is coaxial with the corresponding through holes 104, 105, and 106. Each connecting member may include a flat joint 3 and a flat base 2. The flat joint 3 and the flat base 2 may be connected by internal and external threads. The flat joint 3 is connected to the corresponding hose 4, and the flat base 2 is disposed in the hole and groove 111 in a fitting manner, which can ensure good sealing performance.

[0047] Figure 6 Schematically shows a structural diagram of using a centrifuge tube with the bottom cut off and a pressure pump matching joint to replace the flat base and the flat joint according to an embodiment of the present invention; Figure 7 Schematically shows a structural diagram of a centrifuge tube with the bottom cut off and a pressure pump matching joint according to an embodiment of the present invention; Figure 8 Schematically shows a structural diagram of a support base for supporting a pressure pump matching joint according to an embodiment of the present invention.

[0048] As Figure 6 shown, in the microfluidic device of this embodiment, a centrifuge tube with the bottom cut off (i.e., the through tube 9) and a pressure pump matching joint 10 can be used to replace the flat base 2 and the flat joint 3 in the above embodiment. Except for the different connecting members, Figure 6 shown in the embodiment Figure 1 The microfluidic device of the embodiment is the same as other parts of the microfluidic device shown in

[0049] As Figure 6 、 Figure 7 and Figure 8 shown, the connecting member may include a first joint 1001, a second joint 1002, and a through tube 9. The through tube 9 may be, for example, a centrifuge tube with the bottom cut off. The first joint 1001 is connected to the second joint 1002 to form a pressure pump matching joint 10. The first joint 1001 is connected to the second hose 12, the second joint 1002 is connected to the through holes 104, 105, and 106, the inlet of the through tube 9 is disposed inside the first joint 1001, and the outlet of the through tube 9 is disposed inside the second joint 1002.

[0050] As an example, the bottom of the through tube 9 can be directly pasted on the upper surface of the microfluidic chip 1 body at a position corresponding to the through hole by AB glue. A micro-syringe (not shown in the figure) is used to inject the sample into the corresponding through hole on the microfluidic chip 1 body. The through tube 9 is directly connected to the pressure pump matching joint 10. At the same time, the through tube 9 is stabilized by the support base 11, and the microfluidic chip 1 is placed on the inverted microscope stage. In this embodiment, the caliber of the inlet of the through tube 9 may be equal to the caliber of the outlet.

[0051] Figure 9 Schematically shows a structural diagram according to an embodiment of the present invention, which uses a centrifuge tube with a special frustum-shaped lower bottom and an unclosed lower end and a pressure pump matching joint to replace a flat base and a flat joint; Figure 10 Schematically shows a structural diagram of a special centrifuge tube with a frustum-shaped lower bottom and an unclosed lower end according to an embodiment of the present invention.

[0052] As Figure 9 and Figure 10 shown, in this embodiment, the through-tube 9 can use a special centrifuge tube 13 with a frustum-shaped lower bottom and an unclosed lower end. Except for the different through-tubes, Figure 9 the microfluidic device of the shown embodiment is the same as other parts of the Figure 6 microfluidic device of the shown embodiment and uses the same numbers, so it will not be described in detail.

[0053] As Figure 9 and Figure 10 shown, the special centrifuge tube 13 with a frustum-shaped lower bottom and an unclosed lower end can be directly inserted into the cylindrical groove of the microfluidic chip 1 body. At the junction of the upper surface (i.e., the second surface) of the microfluidic chip 1 body and the centrifuge tube 13, AB glue or ultraviolet curable glue is spin-coated to ensure its sealing performance. At the same time, the support base 11 is used to stabilize the special centrifuge tube 13 with a frustum-shaped lower bottom and an unclosed lower end, and the microfluidic chip 1 is placed on an inverted microscope stage. In this embodiment, the caliber of the inlet of the through-tube 9 can be larger than that of the outlet, which can form a better sealing effect.

[0054] For the microfluidic device according to the above-mentioned multiple embodiments, the present invention analyzes and identifies a large amount of experimental data based on a deep reinforcement learning model, combines the parameters of the microfluidic chip and the reagent parameters, and automatically adjusts the preparation of liposomes according to the hydrodynamics formula, greatly reducing the operation difficulty of microfluidic experiments; by directly connecting the joint to the upper surface of the chip and directly injecting the reagent into the liquid storage tank on the upper surface of the chip, the total amount of reagent required is effectively reduced; a liquid metal electrode is integrated at the rear end of the liposome preparation structure to separate by-products and purify liposomes, and a liposome capture structure is integrated to realize in-situ observation of liposomes.

[0055] 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.

[0056] Figure 12 Shows a flowchart of a method for preparing liposomes using the microfluidic device according to an embodiment of the present invention.

[0057] As Figure 12 shown, the method for preparing liposomes may include steps S121 to S123.

[0058] In step S121, at least one kind of liposome is prepared on the microfluidic chip 1 by using a variety of reagents.

[0059] In some embodiments, step S121 may include: injecting a variety of reagents into different through holes 104, 105, 106 of the negative mold 101 of the microfluidic chip 1, enabling the reagents to flow from the through holes 104, 105, 106 into the microchannel 108 on the negative mold 101, and preparing liposomes in the microchannel 108.

[0060] In some embodiments, step S121 may further include: processing the liposomes through the liposome purification structure 109 and the liposome capture structure 110 on the negative mold 101, and the liposome purification structure 109 and the liposome capture structure 110 are connected to the microchannel 108.

[0061] In some embodiments, after step S121, it may include: acquiring a microscopic image of the liposome by using the microscopic imaging module 16 and transmitting it to the automated control module 8.

[0062] In step S122, the imaging data of the liposome is analyzed by using the machine learning model in the automated control module 8 to generate a first control instruction for regulating the liposome.

[0063] In some embodiments, step S122 may include: according to the data input by the first input unit, the second input unit, the third input unit, and the fourth input unit, calling the machine learning model to generate a first control instruction and a second control instruction, and the second control instruction is used to control the imaging parameters for the microscopic imaging module 16 to acquire a microscopic image. Among them, a machine learning model is configured in the machine learning module, the first input unit is used to input the size data of the microchannel 108 into the machine learning module, the second input unit is used to input the component data and concentration data of each reagent into the machine learning module, the third input unit is used to input the microscopic image into the machine learning module, and the fourth input unit is used to input the pressure value in the pressure control module 15 into the machine learning module.

[0064] In step S123, the first control instruction is executed in the pressure control module 15 to adjust the pressure value of at least one reagent during the process of preparing liposomes on the microfluidic chip 1 to obtain the desired liposomes.

[0065] In some embodiments, step S123 may include: using the automated control module 8 to control the pressure pump 5 to generate gases with different pressure values, and applying the gases with different pressure values to the connectors connected to the through holes 104, 105, 106 to control the pressure value of the reagent.

[0066] Embodiment 1

[0067] See in combinationFigures 1 to 5 As shown, an embodiment of a liposome microfluidic control preparation, purification, and capture device and method is provided. In this embodiment, a reagent system based on n-octanol-deionized water is used for automated liposome preparation, and the operation steps can be as follows:

[0068] Step S1, design the structure of the required microfluidic chip 1, and use techniques including but not limited to soft lithography, nanoimprinting, 3D printing, etc. for preparation. Prepare a microchannel 108 with a depth of 5 - 20 μm and a width of 5 - 20 μm, and punch through holes 104, 105, 106 with a diameter of 1 - 4 mm at the corresponding positions of the external continuous aqueous reagent inlet, the middle dispersed oily reagent inlet, the internal dispersed aqueous reagent inlet, and the outlet 107. Then, use oxygen plasma to bond the fixed hole groove layer 111, the negative mold 101, the hydrophobic thin film 102, and the glass cover slip 103 from top to bottom in sequence; prepare dielectrophoresis effect separation electrodes using liquid metal including but not limited to materials such as gallium.

[0069] Step S2, perform partial modification on the prepared chip to improve the hydrophilicity of the modified part. The modification methods include but not limited to:

[0070] (1) Modify with a 2.5 - 5 wt% polyvinyl alcohol (PVA) solution for 5 min;

[0071] (2) Modify with a 1 wt% PVA solution for 15 min;

[0072] (3) Sequentially introduce a 1:1 hydrogen peroxide (30 wt%):hydrochloric acid (37 wt%) solution, deionized water, poly(diallyldimethylammonium chloride) (5 wt%), deionized water, poly(sodium 4-styrenesulfonate) (5 wt%), and deionized water for 2 min each.

[0073] Step S3, prepare the required reagents, including the external continuous aqueous reagent, the middle dispersed oily reagent, and the internal dispersed aqueous reagent. The components can be as follows:

[0074] Internal dispersed aqueous reagent: An aqueous solution containing 0 - 5% wt / vol poloxamer 188 (P188) and 0 - 15% vol / vol glycerol is used as the internal dispersed aqueous reagent;

[0075] Middle dispersed oily reagent: A n-octanol solution containing 2 - 20 mg / mL 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) is used as the middle dispersed oily reagent;

[0076] External continuous aqueous reagent: An aqueous solution containing 0 - 5% wt / vol P188 and 0 - 15% vol / vol glycerol is used as the external continuous aqueous reagent;

[0077] Optionally, a fluorescent dye at an appropriate concentration can be added to the internal aqueous dispersion reagent, including but not limited to calcein, trisodium 8-hydroxy-1,3,6-pyrenetrisulfonate, green fluorescent protein, fluorescein isothiocyanate-carboxymethyl-dextran, etc.;

[0078] Optionally, a fluorescent dye at an appropriate concentration can be added to the intermediate oil dispersion reagent, including but not limited to 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine 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.

[0079] Step S4: Use a micropipette to directly inject the reagent into the corresponding inlet on the surface of the corresponding chip, fix and seal the flat base 2 in the corresponding hole grooves 1111 corresponding to the reagent inlets on the upper surface of the negative mold 101 by using ultraviolet curable glue or AB glue, connect the pressure control module 15 with the flat joint 3 and the first hose 4, manually adjust the pressure to prepare liposomes, and collect experimental data and experimental images.

[0080] Step S5: Use the image data collected during the experiment to train the machine learning model in the automatic control module 8 so that it can accurately identify liposomes, and connect the pressure control module 15 and the microscope imaging device 16 to the automatic control module 8, so that the machine learning model can adjust the output of the pressure pump, the focal length and magnification of the microscope in real time according to the real-time pressure value and the real-time microscope image.

[0081] Step S6: According to the microfluidic chip 1 prepared and modified in the previous steps, inject the required reagent, connect the pressure control module 15 and the microscope imaging device 16, start the pressure control module 15 and the microscope imaging device 16. At this time, the automatic control module 8 receives the pressure value and the microscope image in real time, and automatically controls the output of the pressure control module 15 and the adjustment of the microscope imaging device 16 by using the trained multi-modal machine learning model until liposomes are stably produced.

[0082] Step S7: After liposomes are stably produced, use the multiple dielectrophoresis effect to separate by-products and purify the obtained liposomes, and use the hydrodynamic capture structure to capture and in-situ observe liposomes.

[0083] The liposomes prepared in Example 1 can be as Figure 11 shown. It can be known from Figure 11 that the embodiments of the present invention can prepare liposomes with a cell size of 1-10 μm.

[0084] Example 2

[0085] Based on Example 1, with reference to Figures 1 to 5 as shown, another embodiment of a liposome microfluidic control preparation, purification, and capture device and method is provided. In this embodiment, a reagent system based on n-octanol - phosphate buffered saline (PBS) solution (1×, pH = 7.4) is used for automated liposome preparation, and the operation steps can be as follows:

[0086] Step S1, design the structure of the required microfluidic chip 1, and prepare it using techniques including but not limited to soft lithography, nanoimprinting, 3D printing, etc. Prepare a microchannel 108 with a depth of 5 - 20 μm and a width of 5 - 20 μm, and punch through holes 104, 105, 106 with a diameter of 1 - 4 mm at the corresponding positions of the external continuous aqueous reagent inlet, the intermediate dispersed oily reagent inlet, the internal dispersed aqueous reagent inlet, and the outlet 107. Then, bond the fixed hole groove layer 111, the negative mold 101, the hydrophobic thin film 102, and the glass cover slip 103 from top to bottom in sequence; prepare dielectrophoretic effect separation electrodes using liquid metals including but not limited to gallium and other materials.

[0087] Step S2, perform partial modification on the prepared chip to improve the hydrophilicity of the modified part. The modification methods include but are not limited to:

[0088] (1) Modify with 2.5 - 5 wt% PVA solution for 5 min;

[0089] (2) Modify with 1 wt% PVA solution for 15 min;

[0090] (3) Sequentially introduce 1:1 hydrogen peroxide (30 wt%): hydrochloric acid (37 wt%) solution, deionized water, poly(diallyldimethylammonium chloride) (5 wt%), deionized water, poly(sodium 4-styrenesulfonate) (5 wt%), and deionized water for 2 min each.

[0091] Step S3, prepare the required reagents, including external continuous aqueous reagents, intermediate dispersed oily reagents, and internal dispersed aqueous reagents. The specific components are as follows:

[0092] Internal dispersed aqueous reagent: Use a PBS solution with 0 - 5% wt / vol P188, 0 - 15% vol / vol glycerol, and 0 - 500 mM sucrose as the internal dispersed aqueous reagent;

[0093] Intermediate dispersed oily reagent: Use a n-octanol solution with 2 - 20 mg / mL DOPC as the intermediate dispersed oily reagent;

[0094] External continuous aqueous phase: Use a PBS solution containing 0 - 5% wt / vol P188, 0 - 15% vol / vol glycerol, and 0 - 500 mM sucrose as the external continuous aqueous phase;

[0095] Optionally, an appropriate concentration of fluorescent dyes can be added to the internal dispersion aqueous reagent, including but not limited to calcein, trisodium 8-hydroxy-1,3,6-pyrenetrisulfonate, green fluorescent protein, fluorescein isothiocyanate-carboxymethyl-dextran, etc.;

[0096] Optionally, an appropriate concentration of fluorescent dyes can be added to the intermediate dispersion oily reagent, including but not limited to 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine 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.

[0097] Step S4: Use a micropipette to directly inject the reagent into the corresponding inlet on the surface of the corresponding chip, fix and seal the flat base 2 in the corresponding hole grooves 1111 on the upper surface of the negative mold 101 with ultraviolet curing glue or AB glue, connect the pressure control module 15 with the flat joint 3 and the first hose 4, and manually adjust the pressure to prepare liposomes, and collect experimental data and experimental images.

[0098] Step S5: Use the image data collected during the experiment to train the machine learning model in the automatic control module 8 so that it can accurately identify liposomes, and connect the pressure control module 15 and the microscope imaging device 16 to the automatic control module 8, so that the machine learning model can adjust the output of the pressure pump, the focal length and magnification of the microscope in real time according to the real-time pressure value and the real-time microscope image;

[0099] Step S6: According to the microfluidic chip 1 prepared and modified in the previous steps, inject the required reagents, connect the pressure control module 15 and the microscope imaging device 16, start the pressure control module 15 and the microscope imaging device 16. At this time, the automatic control module 8 receives the pressure value and the microscope image in real time, and uses the trained multi-modal machine learning model to automatically control the output of the pressure control module 15 and the adjustment of the microscope imaging device 16 until liposomes are stably produced.

[0100] Step S7: After liposomes are stably produced, use the multi-frequency dielectrophoresis effect to separate by-products and purify the obtained liposomes, and use the hydrodynamic capture structure to capture and in-situ observe liposomes.

[0101] Example 3

[0102] Based on Example 1, with reference to Figures 1 to 5 As shown, an embodiment of a liposome microfluidic control preparation, purification, capture, and in-situ observation of protein synthesis device and method in an automated microscale reagent system is provided, and the operation steps can be as follows:

[0103] Step S1, design the structure of the required microfluidic chip 1, and prepare it using techniques including but not limited to soft lithography, nanoimprinting, 3D printing, etc. The prepared microchannels are 1085 - 20 μm deep and 5 - 20 μm wide, and through holes 104, 105, 106 with a diameter of 1 - 4 mm are punched at the corresponding positions of the external continuous aqueous reagent inlet, the middle dispersed oil reagent inlet, the internal dispersed aqueous reagent inlet, and the outlet 107. Then, the fixed hole groove layer 111, the negative mold 101, the hydrophobic film 102, and the glass cover slip 103 are bonded successively from top to bottom using oxygen plasma; prepare dielectrophoretic effect separation electrodes using liquid metal including but not limited to materials such as gallium.

[0104] Step S2, perform partial modification on the prepared chip to improve the hydrophilicity of the modified part. The modification methods include but not limited to:

[0105] (1) Modify with a 2.5 - 5 wt% PVA solution for 5 min;

[0106] (2) Modify with a 1 wt% PVA solution for 15 min;

[0107] (3) Sequentially introduce a 1:1 hydrogen peroxide (30 wt%): hydrochloric acid (37 wt%) solution, deionized water, poly(diallyldimethylammonium chloride) (5 wt%), deionized water, poly(sodium 4-styrenesulfonate) (5 wt%), and deionized water for 2 min each.

[0108] Step S3, prepare the required reagents, including external continuous aqueous reagents, middle dispersed oil reagents, and internal dispersed aqueous reagents. The specific components are as follows:

[0109] Internal dispersed aqueous reagent: An aqueous solution containing 0 - 5% wt / vol P188, 0 - 15% vol / vol glycerol, and RTS 500 E. coli Disulfide Kit is used as the internal dispersed aqueous reagent;

[0110] Among them, the RTS 500 E. coli Disulfide Kit includes lysis solution, reagents, chaperone proteins, and buffers;

[0111] Middle dispersed oil reagent: A 2 - 20 mg / mL DOPC n-octanol solution is used as the middle dispersed oil reagent;

[0112] External continuous aqueous phase: An aqueous solution containing 0 - 5% wt / vol P188 and 0 - 15% vol / vol glycerol is used as the external continuous aqueous phase;

[0113] Optionally, a fluorescent dye with an appropriate concentration can be added to the intermediate dispersed oily reagent, 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.

[0114] Step S4: Use a micropipette to directly inject the reagent into the corresponding inlet on the surface of the corresponding chip. Fix and seal the flat base 2 in the corresponding hole grooves 1111 of the upper surface of the negative mold 101 with ultraviolet curing glue or AB glue. Connect the pressure control module 15 with the flat joint 3 and the first hose 4. Manually adjust the pressure to prepare liposomes, and collect experimental data and experimental images.

[0115] Step S5: Use the image data collected during the experiment to train the machine learning model in the automatic control module 8 so that it can accurately identify liposomes. Connect the pressure control module 15 and the microscope imaging device 16 to the automatic control module 8, so that the machine learning model can adjust the output of the pressure pump, the microscope focal length, and the magnification in real time according to the real-time pressure value and the real-time microscope image.

[0116] Step S6: Prepare and modify the microfluidic chip 1 according to the previous steps, inject the required reagent, connect the pressure control module 15 and the microscope imaging device 16, start the pressure control module 15 and the microscope imaging device 16. At this time, the automatic control module 8 receives the pressure value and the microscope image in real time, and automatically controls the output of the pressure control module 15 and the adjustment of the microscope imaging device 16 using the trained multi-modal machine learning model until liposomes are stably produced.

[0117] Step S7: After liposomes are stably produced, use the multiple dielectrophoresis effect to separate by-products and purify the obtained liposomes. Use the hydrodynamic capture structure to capture and in-situ observe the liposomes. When the liposomes enter the liposome capture structure, adjust the inverted microscope lens to the corresponding area to observe the situation after the liposomes are captured. At the same time, adjust the indoor temperature to 23°C. Use the inverted microscope lens and the green fluorescence module of the CCD camera to take fluorescence images of the liposomes every 15 minutes within 3 hours, and observe the change trend of the liposome fluorescence images. Theoretically, the gradually enhanced green fluorescence inside the liposomes can be seen.

[0118] Note that the above is only a preferred embodiment of the present application and the technical principles applied. Those skilled in the art will understand that the present application is not limited to the specific embodiments here, and various obvious changes, re-adjustments 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 has been described in more detail through the above embodiments, the present application is not limited to the above embodiments. Without departing from the concept of the present application, more other equivalent embodiments can be included, all of which fall within the protection scope of the present application.

Claims

1. A microfluidic device for liposome preparation, characterized in that, Comprising: A microfluidic chip for preparing at least one liposome using multiple reagents; An automated control module configured with a trained machine learning model, the automated control module being used to analyze the imaging data of the liposome using the machine learning model and generate a first control instruction for adjusting the liposome; A pressure control module connected to the microfluidic chip and the automated control module, the pressure control module being used to adjust the pressure value of at least one of the reagents during the process of the microfluidic chip preparing the liposome according to the first control instruction to obtain the desired liposome; the microfluidic chip includes a negative mold, a hydrophobic thin film and a cover glass, the negative mold includes opposite first and second surfaces, the hydrophobic thin film is bonded to the first surface, a plurality of through holes and microchannels are formed on the first surface, the plurality of through holes communicate the first surface and the second surface, each through hole stores one of the reagents, the microchannels connect each through hole, the pressure control module is connected to each through hole on the second surface and transmits pressure to the corresponding reagent in each through hole, a liposome purification structure and a liposome capture structure are also formed on the first surface of the negative mold, the liposome purification structure is connected to the microchannels, the liposome capture structure is connected to the microchannels, and the cover glass is bonded to the hydrophobic thin film; And A microscopic imaging module connected to the automated control module and close to the cover glass, the microscopic imaging module being used to acquire a microscopic image of the liposome and transmit it to the automated control module.

2. The microfluidic device according to claim 1, wherein The automated control module includes: A machine learning module in which the machine learning model is configured; A first input unit for inputting the size data of the microchannels into the machine learning module; A second input unit for inputting the component data and concentration data of each of the reagents into the machine learning module; A third input unit for inputting the microscopic image into the machine learning module; A fourth input unit for inputting the pressure value in the pressure control module into the machine learning module; A first output unit for outputting the first control instruction to the pressure control module; A second output unit for outputting a second control instruction to the microscopic imaging module, the second control instruction being used to control the imaging parameters of the microscopic imaging module for acquiring the microscopic image; wherein the first control instruction and the second control instruction are generated by the machine learning model according to the data input by the first input unit, the second input unit, the third input unit and the fourth input unit.

3. The microfluidic device according to claim 1, wherein The pressure control module includes a pressure pump, a plurality of hoses and a plurality of connectors, the input end of the pressure pump is connected to the automated control module, the output end of the pressure pump is connected to the plurality of connectors through the hoses, each connector is connected to the corresponding through hole, and the pressure pump is used to generate gases with different pressure values and act on the connectors.

4. The microfluidic device according to claim 3, characterized in that, The microfluidic chip includes a fixed hole and groove layer, the fixed hole and groove layer is bonded to the second surface, and there are a plurality of holes and grooves on the fixed hole and groove layer, and each hole and groove is coaxial with the corresponding through hole; Each of the connectors includes a flat plate joint and a flat plate base, the flat plate joint is connected to the flat plate base, the flat plate joint is connected to the corresponding hose, and the flat plate base is disposed in the hole and groove in a fitting manner.

5. The microfluidic device according to claim 3, characterized in that, The connector includes a first joint, a second joint and a through pipe, the first joint is connected to the second joint, the first joint is connected to the hose, the second joint is connected to the through hole, the inlet of the through pipe is disposed inside the first joint, and the outlet of the through pipe is disposed inside the second joint.

6. The microfluidic device according to claim 5, wherein, The caliber of the inlet of the through pipe is greater than or equal to the caliber of the outlet.

7. A method for preparing liposomes using the microfluidic device according to any one of claims 1 to 6, characterized in that, Including: Preparing at least one liposome with a variety of reagents on the microfluidic chip; Using a machine learning model in the automatic control module to analyze the imaging data of the liposome and generating a first control instruction for adjusting the liposome; Executing the first control instruction in the pressure control module to adjust the pressure value of at least one of the reagents during the process of preparing the liposome by the microfluidic chip so as to obtain the desired liposome.

Images