Microfluidic device and method for preparing lipidosome

Abstract

The invention provides a microfluidic device and a microfluidic method for preparing lipidosome. The microfluidic device comprises: a microfluidic chip for preparing at least one liposome by using a plurality of reagents; the automatic control module is configured with a trained machine learning model, and the automatic control module is used for analyzing the imaging data of the lipidosome by using the machine learning model and generating a first control instruction for adjusting the lipidosome; the pressure control module is connected with the micro-fluidic chip and the automatic control module, and the pressure control module is used for adjusting the pressure value of at least one reagent in the process of preparing the lipidosome by the micro-fluidic chip according to the first control instruction so as to obtain the expected lipidosome.

Description

Technical Field

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

[0002] Liposomes are closed, ordered molecular assemblies formed by lipid bilayers, which can separate the internal water environment from the surrounding water environment while allowing some small molecules and ions to pass selectively. They play an important role as carriers of biological reactions in fields such as artificial synthesis of single cells.

[0003] Microfluidic methods can be used to prepare uniform and stable liposomes with high throughput, and biologically active reactants can be encapsulated inside the liposomes, providing the possibility for subsequent biological reaction experiments. However, traditional microfluidic devices include a pressure pump-liquid reservoir system, which drives the liquid in the reservoir into the microfluidic chip through compressed air, resulting in a large amount of liquid required in the reservoir, and some reagent systems are expensive, with a small amount of reagents, and a large dead volume in the microfluidic chip channels and connecting pipes, resulting in high experimental costs; in addition, traditional microfluidic devices are difficult to regulate the pressure of each phase of reagents, which places high demands on the experimental capabilities of experimental personnel. Summary of the invention

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

[0005] On the one hand, an embodiment of the present invention provides a microfluidic device for preparing liposomes, comprising: a microfluidic chip, used to prepare at least one liposome using multiple reagents; an automation control module, configured with a trained machine learning model, the automation control module is used to use the machine learning model to analyze imaging data of the liposomes, and generate a first control instruction for adjusting the liposomes in real time; a pressure control module, connecting the microfluidic chip and the automation control module, 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, so as to obtain the desired liposomes.

[0006] According to an embodiment of the present invention, a microfluidic chip includes a negative mold and a hydrophobic film, the negative mold includes a first surface and a second surface relative to each other, 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 connect the first surface and the second surface, each through hole stores a reagent, and the microchannels connect each through hole; the pressure control module connects 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 also 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 also includes a cover glass, which is bonded to the hydrophobic film; the microfluidic device also includes a microscopic imaging module, which is connected to the automation control module and is close to the cover glass. The microscopic imaging module is used to obtain a microscopic image of the liposome and transmit it to the automation control module in real time.

[0009] According to an embodiment of the present invention, the automation control module includes: a machine learning module, in which a machine learning model is configured; a first input unit, used to input the size data of the microchannel into the machine learning module; a second input unit, used to input the component data and concentration data of each reagent into the machine learning module; a third input unit, used to input the microscopic image into the machine learning module; a fourth input unit, used to input the pressure value in the pressure control module into the machine learning module; a first output unit, used to output a first control instruction to the pressure control module; a second output unit, used to output a second control instruction to the microscopic imaging module, the second control instruction being used to control the microscopic imaging module to obtain imaging parameters of the microscopic image, wherein the first control instruction and the second control instruction are generated by the machine learning model based on data processing 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, multiple hoses and multiple connectors. The input end of the pressure pump is connected to the automation control module, and the output end of the pressure pump is connected to multiple connectors through a hose. Each connector is connected to a corresponding through hole. The pressure pump is used to generate gas 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 slot layer, which is bonded to the second surface, and has multiple holes slots on the fixed hole slot layer, each hole slot is coaxial with the corresponding through hole; each connecting part 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 fitly arranged in the hole slot.

[0012] According to an embodiment of the present invention, the connecting member 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 diameter of the inlet of the through pipe is greater than or equal to the diameter of the outlet.

[0014] On the other hand, an embodiment of the present invention also provides a method for preparing liposomes using the microfluidic device of the above embodiment, comprising: using multiple reagents to prepare at least one liposome on a microfluidic chip; using a machine learning model in an automation control module to analyze imaging data of the liposomes and generate a first control instruction for adjusting the liposomes; executing the first control instruction in a pressure control module to adjust the pressure value of at least one reagent in the process of preparing liposomes on the microfluidic chip to obtain the desired liposomes.

[0015] The microfluidic device and method for preparing liposomes provided by the embodiment 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 bubbles during the experiment; at the same time, the dead volume of the sample entering the channel is effectively reduced, 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 reagent to consume this part of the dead volume, thereby improving the preparation efficiency of the 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 height difference between the reservoir and the microfluidic chip and the influence of the hose connecting the reservoir and the microfluidic chip on pressure control and feedback, making pressure control and feedback more accurate; the multimodal machine learning model is used to automatically adjust the pressure parameters of each phase to realize the automated preparation of liposomes, reducing the requirements for the operating technology and operating experience of the experimenter for the microfluidic preparation of liposomes. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0017] Figure 1 The structure diagram of the microfluidic device according to an embodiment of the present invention is schematically shown;

[0018] Figure 2 A schematic diagram of the structure of a microfluidic chip according to an embodiment of the present invention is shown;

[0019] Figure 3 A schematic diagram of a negative mold structure according to an embodiment of the present invention is shown;

[0020] Figure 4 A schematic diagram of a flat plate base and a flat plate joint structure according to an embodiment of the present invention is shown;

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

[0022] Figure 6A schematic diagram showing a structure in which a centrifuge tube with a cut-off bottom and a pressure pump matching joint are used to replace a flat base and a flat joint according to an embodiment of the present invention;

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

[0024] Figure 8 A schematic diagram of a supporting base structure for supporting a pressure pump matching joint according to an embodiment of the present invention is shown;

[0025] Fig. 9 A schematic diagram showing a structure of a centrifuge tube with a special truncated cone-shaped bottom and an open lower end and a pressure pump matching joint replacing a flat base and a flat joint according to an embodiment of the present invention;

[0026] Fig.10 A schematic diagram of a centrifuge tube structure with a special bottom portion in a truncated cone shape and an open lower end according to an embodiment of the present invention is shown;

[0027] Fig.11 The experimental pictures of the liposomes prepared in Example 1 are shown;

[0028] Fig.12 A flow chart of a method for preparing liposomes using a microfluidic device according to an embodiment of the present invention is shown.

[0029] Description of reference numerals:

[0030] 1. Microfluidic chip; 101. Negative mold; 102. Hydrophobic film; 103. Cover glass; 104. Through hole; 105. Through hole; 106. Through hole; 107. Outlet; 108. Microfluidic channel; 109. Liposome purification structure; 110. Liposome capture structure; 111. Fixed pore layer; 2. Flat base; 3. Flat connector; 4. No. 1 hose; 5. Pressure pump; 6. Microscope lens; 7. CCD camera; 8. Automation control module; 801. Chip parameter input; 802. Reagent parameter input; 803. Fluid mechanics 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, focal length and objective lens magnification adjustment; 9, centrifuge tube with the bottom cut off; 10, pressure pump matching connector; 1001, upper part of the flat connector; 1002, lower part of the flat connector; 11, support base; 12, No. 2 hose; 13, centrifuge tube with a truncated cone-shaped bottom and an open lower end; 14, glue; 15, pressure control module; 16, microscopic imaging module; 1111, hole groove. DETAILED DESCRIPTION

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

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

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

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

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

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

[0037] Figure 1 The structure diagram of the microfluidic device according to an embodiment of the present invention is schematically shown; Figure 2 A schematic diagram of the structure of a microfluidic chip according to an embodiment of the present invention is shown; Figure 3 A schematic diagram of a negative mold structure according to an embodiment of the present invention is shown; Figure 4 A schematic diagram of a flat plate base and a flat plate joint structure according to an embodiment of the present invention is shown; Figure 5 The functional diagram of the automation control module according to an embodiment of the present invention is schematically shown.

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

[0039] The microfluidic chip 1 can be used to prepare at least one liposome using multiple reagents. The automated control module 8 is configured with a trained machine learning model, and the automated control module 8 is used to analyze the imaging data of the liposome using the machine learning model to generate a first control instruction for adjusting the liposome. The process of the automated control module 8 generating the first control instruction according to the imaging data of the liposome can be real-time. The pressure control module 15 connects the microfluidic chip 1 and the automated control module 8, and the pressure control module 15 can be used to adjust the pressure value of at least one reagent in the process of preparing the liposome by the microfluidic chip 1 according to the first control instruction 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, and the preparation efficiency of the liposome is further improved; the pressure of the output port of the pressure control module directly acts on the reagent liquid, making the pressure control and feedback more accurate; the multimodal machine learning model is used to automatically adjust the pressure parameters of each phase in real time to realize the automated preparation of liposomes, which reduces the requirements for the operation technology and operation experience of the experimenter for the microfluidic preparation of liposomes.

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

[0041] As an example, a hole puncher can be used to punch out through holes 104, 105, and 106 with a hole diameter of 1 to 4 mm to store different reagents. The through holes 104, 105, and 106 can be used as an external continuous aqueous inlet, an intermediate dispersed oily reagent inlet, and an internal dispersed aqueous reagent inlet, respectively, to flow different reagents into the microchannel 108 to prepare liposomes. For example, a microinjector can be used to directly inject the reagent into the corresponding inlet; a microchannel 108 with a depth of 5 to 20 μm and a width of 5 to 20 μm can be prepared using technologies including but not limited to soft lithography, nanoimprinting, and 3D printing; and oxygen plasma can be used to bond the negative mold 101 and the hydrophobic film 102 together.

[0042] like Figure 2 and Figure 3 As shown, a liposome purification structure 109 and a liposome capture structure 110 may 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 the liposomes are stably produced, the liposome purification structure 109 can use liquid metal materials including but not limited to gallium as electrodes based on the difference in dielectric properties between the liposomes and the byproducts, and use multiple dielectrophoresis to purify the liposomes; the liposome capture structure 110 can use a fluid mechanics capture structure to capture and observe the liposomes in situ. In this embodiment, experiments on multiple liposomes can be completed on a microfluidic chip that integrates liposome preparation, capture, and in situ observation, thereby improving the efficiency and accuracy of the experiment. In some embodiments, the microchannel 108 of the prepared microfluidic chip 1 can be partially modified so that the hydrophilicity of the modified part is improved. The exemplary steps of modification can be referred to in the embodiments below.

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

[0044] like Figure 1 and Figure 5As shown, the automation 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. The second input unit 802, which can be used as a reagent parameter input, is used to input the component data and concentration data of each reagent into the machine learning module 807; the third input unit 804, which can be used as a microscope real-time image input, is used to input the microscopic image into the machine learning module 807; the fourth input unit 805, which can be used as a real-time pressure input, is used to input the pressure value in the pressure control module 15 into the machine learning module 807. The first output unit 808, which can be used as a pressure control output, is used to output a first control instruction to the pressure control module 15; the second output unit 809, which can be used as a microscope control output, is used to output 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 can be, for example, a control instruction for adjusting the focal length objective lens magnification 810. The image data set 806 collected during the experiment can be used to train the machine learning model 807 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 are input to the machine learning model 807, and the first control instruction and the second control instruction are generated in combination with the fluid mechanics formula 803. In the automatic control module 8, the control and adjustment process of each of the above modules can be real-time. This embodiment uses a multimodal machine learning model to automatically adjust the pressure parameters of each phase in real time, which can realize the automated preparation of liposomes and reduce the requirements for the operation technology and operation experience of the experimenter in the microfluidic preparation of liposomes.

[0045] like Figure 1 and Figure 2 As shown, the pressure control module 15 may include a pressure pump 5, a plurality of hoses 4 and a plurality of connectors. The hose 4 may be regarded as a No. 1 hose. The input end of the pressure pump 5 is connected to the automation 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 being connected to a corresponding through hole 104, 105, 106. The pressure pump 5 is used to generate gas with different pressure values ​​and act on the connectors.

[0046] like Figure 1 , Figure 2 , Figure 4As shown, the microfluidic chip 1 may include a fixed hole slot layer 111, the fixed hole slot layer 111 is bonded to the second surface of the negative mold 101, and a plurality of holes 1111 are provided on the fixed hole slot layer 111, and each hole slot 1111 is coaxial with the corresponding through hole 104, 105, 106. Each connector may include a flat plate connector 3 and a flat plate base 2, the flat plate connector 3 and the flat plate base 2 may be connected by internal and external threads, the flat plate connector 3 is connected to the corresponding hose 4, and the flat plate base 2 is fitted in the hole slot 111, which can ensure good sealing performance.

[0047] Figure 6 A schematic diagram showing a structure in which a centrifuge tube with a cut-off bottom and a pressure pump matching joint are used to replace a flat base and a flat joint according to an embodiment of the present invention; Figure 7 The schematic diagram shows a structure diagram of a centrifuge tube with its lower bottom cut off and a matching joint for a pressure pump according to an embodiment of the present invention; Figure 8 A schematic diagram of the structure of a support base for supporting a pressure pump matching joint according to an embodiment of the present invention is shown.

[0048] like Figure 6 As shown, in the microfluidic device of this embodiment, the centrifuge tube with the bottom cut off (i.e., the through tube 9) and the pressure pump matching connector 10 can be used to replace the flat base 2 and the flat connector 3 of the above embodiment. In addition to the different connectors, Figure 6 The microfluidic device of the embodiment shown is Figure 1 The other parts of the microfluidic device of the illustrated embodiment are the same and have the same numbering, so they will not be described in detail.

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

[0050] As an example, the bottom of the through tube 9 can be directly attached to the position corresponding to the through hole on the upper surface of the microfluidic chip 1 body by using AB glue, and the sample can be injected into the corresponding through hole on the microfluidic chip 1 body by using a micro-liquid injector (not shown in the figure), and the through tube 9 is directly connected to the pressure pump matching connector 10, and the through tube 9 is stabilized by using the supporting 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 can be equal to the caliber of the outlet.

[0051] Fig. 9 A schematic diagram showing a structure of a centrifuge tube with a special truncated cone-shaped bottom and an open lower end and a pressure pump matching joint replacing a flat base and a flat joint according to an embodiment of the present invention; Fig.10 The structure of a centrifuge tube with a special bottom portion in the shape of a truncated cone and an open lower end according to an embodiment of the present invention is schematically shown.

[0052] like Fig. 9 and Fig.10 As shown, in this embodiment, the through pipe 9 can use a special centrifuge tube 13 with a truncated cone bottom and an open lower end. Fig. 9 The microfluidic device of the embodiment shown is Figure 6 The other parts of the microfluidic device of the illustrated embodiment are the same and have the same numbering, so they will not be described in detail.

[0053] like Fig. 9 and Fig.10 As shown, the special centrifuge tube 13 with a truncated cone-shaped bottom and an open lower end can be directly inserted into the cylindrical groove of the microfluidic chip 1 body, and AB glue or UV curing glue is spin-coated at the junction of the upper surface (that is, the second surface) of the microfluidic chip 1 body and the centrifuge tube 13 to ensure its sealing performance. At the same time, the special centrifuge tube 13 with a truncated cone-shaped bottom and an open lower end 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 pipe 9 can be larger than the caliber of the outlet, which can form a better sealing effect.

[0054] According to the microfluidic devices of the above-mentioned multiple embodiments, the present invention analyzes and identifies a large amount of experimental data based on a deep reinforcement learning model, and combines the microfluidic chip parameters and reagent parameters to automatically adjust the preparation of liposomes according to the fluid mechanics formula, thereby greatly reducing the operational difficulty of the microfluidic experiment; by directly connecting the connector to the upper surface of the chip and injecting the reagent directly into the liquid reservoir 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 the liposomes, and a liposome capture structure is integrated to achieve in situ observation of the 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. The method has the same technical features and beneficial effects as the microfluidic device, which will not be described in detail here.

[0056] Fig.12 A flow chart of a method for preparing liposomes using a microfluidic device according to an embodiment of the present invention is shown.

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

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

[0059] In some embodiments, step S121 may include: injecting multiple reagents into different through holes 104, 105, 106 of the negative mold 101 of the microfluidic chip 1, allowing 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: treating the liposomes through the liposome purification structure 109 and the liposome capture structure 110 on the negative mold 101 , wherein the liposome purification structure 109 and the liposome capture structure 110 are connected to the microchannel 108 .

[0061] In some embodiments, after step S121 , the following step may be included: using the microscopic imaging module 16 to acquire a microscopic image of the liposomes, and transmitting the image to the automation control module 8 .

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

[0063] In some embodiments, step S122 may include: calling a machine learning model to generate a first control instruction and a second control instruction based on data input by the first input unit, the second input unit, the third input unit, and the fourth input unit, the second control instruction being used to control the microscopic imaging module 16 to obtain imaging parameters of the microscopic image, wherein a machine learning model is configured in the machine learning module, the first input unit being used to input size data of the microchannel 108 into the machine learning module, the second input unit being used to input component data and concentration data of each reagent into the machine learning module, the third input unit being used to input a microscopic image into the machine learning module, and the fourth input unit being used to input a 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 in the process of preparing liposomes by the microfluidic chip 1 to obtain the desired liposomes.

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

[0066] Example 1

[0067] See also Figures 1 to 5 As shown, an embodiment of a liposome microfluidic preparation, purification, and capture device and method is provided. This embodiment uses an n-octanol-deionized water reagent system for automated liposome preparation, and the operation steps can be as follows:

[0068] Step S1, design the required microfluidic chip 1 structure, and prepare it using soft lithography technology, nanoimprinting technology, 3D printing technology, etc. to prepare a microchannel 108 with a depth of 5~20 μm and a width of 5~20 μm, and use a puncher to punch through holes 104, 105, and 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, and then use oxygen plasma to bond the fixed hole groove layer 111, the negative mold 101, the hydrophobic film 102 and the glass cover glass 103 from top to bottom in this order; use liquid metal including but not limited to gallium and other materials to prepare the dielectrophoresis effect separation electrode.

[0069] Step S2, partially modifying the prepared chip to improve the hydrophilicity of the modified part, and the modification method includes but is not limited to:

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

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

[0072] (3) Add 1:1 hydrogen peroxide (30 wt%):hydrochloric acid (37 wt%) solution, deionized water, polydiallyldimethylammonium chloride (5 wt%), deionized water, poly(sodium 4-styrene sulfonate) (5 wt%), and deionized water in sequence for 2 min each.

[0073] Step S3, preparing the required reagents, including an external continuous aqueous reagent, an intermediate dispersed oily reagent, and an intermediate dispersed oily reagent, the components of which may be as follows:

[0074] Internal dispersion aqueous reagent: 0~5% wt / vol poloxamer 188 (P188), 0~15% vol / vol glycerol aqueous solution is used as the internal dispersion aqueous reagent;

[0075] Intermediate dispersing oily reagent: 2-20 mg / mL 1,2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC) n-octanol solution is used as the intermediate dispersing oily reagent;

[0076] External continuous aqueous reagent: 0~5% wt / vol P188, 0~15% vol / vol glycerol aqueous solution as external continuous aqueous;

[0077] Optionally, fluorescent dyes of appropriate concentrations may be added to the internally dispersed aqueous reagent, 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;

[0078] Optionally, an appropriate concentration of fluorescent dye can be added to the intermediate dispersed oily reagent, including but not limited to 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocyanine perchlorate, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt), Nile Red, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt), etc.

[0079] Step S4, use a micro-injector to directly inject the reagent into the corresponding inlet of the corresponding chip surface, use UV curing glue or AB glue to adhere the flat base 2 to the corresponding hole grooves 1111 of each phase reagent inlet on the upper surface of the negative mold 101 and fix and seal it, use the flat joint 3 and No. 1 hose 4 to connect the pressure control module 15, manually adjust the pressure to prepare liposomes, and collect experimental data and experimental images.

[0080] Step S5, using the image data collected during the experiment, train the machine learning model in the automation 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 automation control module 8, so that the machine learning model can adjust the pressure pump output and the focal length and magnification of the microscope in real time according to the real-time pressure value and the real-time image of the microscope.

[0081] Step S6, the microfluidic chip 1 prepared and modified according to the steps above is injected with the required reagents, the pressure control module 15 and the microscope imaging device 16 are connected, and the pressure control module 15 and the microscope imaging device 16 are started. At this time, the automatic control module 8 receives the pressure value and the microscope image in real time, and uses the trained multimodal machine learning model to automatically control the output of the pressure control module 15 and the adjustment of the microscope imaging device 16 until the liposomes are stably produced.

[0082] Step S7, after the liposomes are stably generated, the byproducts are separated and the obtained liposomes are purified by using the multiple dielectrophoresis effect, and the liposomes are captured and observed in situ by using the fluid dynamics capture structure.

[0083] The liposomes prepared by Example 1 can be Fig.11 As shown. Fig.11 It can be seen that the embodiments of the present invention can prepare liposomes of cell size (1-10 μm).

[0084] Example 2

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

[0086] Step S1, design the required microfluidic chip 1 structure, and prepare it using soft lithography technology, nanoimprinting technology, 3D printing technology, etc. to prepare a microchannel 108 with a depth of 5~20 μm and a width of 5~20 μm, and use a puncher to punch through holes 104, 105, and 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, and then use oxygen plasma to bond the fixed hole groove layer 111, the negative mold 101, the hydrophobic film 102 and the glass cover glass 103 from top to bottom in this order; use liquid metal including but not limited to gallium and other materials to prepare the dielectrophoresis effect separation electrode.

[0087] Step S2, partially modifying the prepared chip to improve the hydrophilicity of the modified part, and the modification method includes but is not limited to:

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

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

[0090] (3) Add 1:1 hydrogen peroxide (30 wt%):hydrochloric acid (37 wt%) solution, deionized water, polydiallyldimethylammonium chloride (5 wt%), deionized water, poly(sodium 4-styrene sulfonate) (5 wt%), and deionized water in sequence for 2 min each.

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

[0092] Internal dispersion aqueous reagent: PBS solution containing 0-5% wt / vol P188, 0-15% vol / vol glycerol, and 0-500 mM sucrose was used as the internal dispersion aqueous reagent;

[0093] Intermediate dispersing oily reagent: 2~20 mg / mL DOPC in n-octanol solution is used as the intermediate dispersing oily reagent;

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

[0095] Optionally, fluorescent dyes of appropriate concentrations may be added to the internally dispersed aqueous reagent, 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;

[0096] Optionally, an appropriate concentration of fluorescent dye can be added to the intermediate dispersed oily reagent, including but not limited to 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocyanine perchlorate, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt), Nile Red, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt), etc.

[0097] Step S4, use a micro-injector to directly inject the reagent into the corresponding inlet of the corresponding chip surface, use UV curing glue or AB glue to adhere the flat base 2 to the corresponding hole grooves 1111 of each phase reagent inlet on the upper surface of the negative mold 101 and fix and seal it, use the flat joint 3 and No. 1 hose 4 to connect the pressure control module 15, manually adjust the pressure to prepare liposomes, and collect experimental data and experimental images.

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

[0099] Step S6, the microfluidic chip 1 prepared and modified according to the steps above is injected with the required reagents, the pressure control module 15 and the microscope imaging device 16 are connected, and the pressure control module 15 and the microscope imaging device 16 are started. At this time, the automatic control module 8 receives the pressure value and the microscope image in real time, and uses the trained multimodal machine learning model to automatically control the output of the pressure control module 15 and the adjustment of the microscope imaging device 16 until the liposomes are stably produced.

[0100] Step S7, after the liposomes are stably generated, the byproducts are separated and the obtained liposomes are purified by using the multiple dielectrophoresis effect, and the liposomes are captured and observed in situ by using the fluid dynamics capture structure.

[0101] Example 3

[0102] Based on Example 1, combined with reference to Figures 1 to 5 As shown, an embodiment of a device and method for preparing, purifying, capturing, and in-situ observing protein synthesis in liposomes using an automated micro-reagent system is provided, and the operation steps may be as follows:

[0103] Step S1, design the required microfluidic chip 1 structure, and prepare it using soft lithography technology, nanoimprinting technology, 3D printing technology, etc. The prepared microchannel 108 is 5~20 μm deep and 5~20 μm wide, and through holes 104, 105, and 106 with a diameter of 1~4 mm are punched out 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 using a puncher, and then the fixed hole groove layer 111, the negative mold 101, the hydrophobic film 102 and the glass cover glass 103 are bonded from top to bottom using oxygen plasma; and the dielectrophoresis effect separation electrode is prepared using liquid metal of materials including but not limited to gallium.

[0104] Step S2, partially modifying the prepared chip to improve the hydrophilicity of the modified part, and the modification method includes but is not limited to:

[0105] (1) Modification with 2.5~5 wt% PVA solution for 5 min;

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

[0107] (3) Add 1:1 hydrogen peroxide (30 wt%):hydrochloric acid (37 wt%) solution, deionized water, polydiallyldimethylammonium chloride (5 wt%), deionized water, poly(sodium 4-styrene sulfonate) (5 wt%), and deionized water in sequence for 2 min each.

[0108] Step S3, prepare the required reagents, including external continuous aqueous reagent, intermediate dispersed oily reagent and intermediate dispersed oily reagent, the specific components are as follows:

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

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

[0111] Intermediate dispersing oily reagent: 2~20 mg / mL DOPC in n-octanol solution is used as the intermediate dispersing oily reagent;

[0112] External continuous water: 0~5% wt / vol P188, 0~15% vol / vol glycerol aqueous solution was used as the external continuous water;

[0113] Optionally, an appropriate concentration of fluorescent dye can be added to the intermediate dispersed oily reagent, including but not limited to 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocyanine perchlorate, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt), Nile Red, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt), etc.

[0114] Step S4, use a micro-injector to directly inject the reagent into the corresponding inlet of the corresponding chip surface, use UV curing glue or AB glue to adhere the flat base 2 to the corresponding hole grooves 1111 of each phase reagent inlet on the upper surface of the negative mold 101 and fix and seal it, use the flat joint 3 and No. 1 hose 4 to connect the pressure control module 15, manually adjust the pressure to prepare liposomes, and collect experimental data and experimental images.

[0115] Step S5, using the image data collected during the experiment, train the machine learning model in the automation 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 automation control module 8, so that the machine learning model can adjust the pressure pump output and the focal length and magnification of the microscope in real time according to the real-time pressure value and the real-time image of the microscope.

[0116] Step S6, the microfluidic chip 1 prepared and modified according to the steps above is injected with the required reagents, the pressure control module 15 and the microscope imaging device 16 are connected, and the pressure control module 15 and the microscope imaging device 16 are started. At this time, the automatic control module 8 receives the pressure value and the microscope image in real time, and uses the trained multimodal machine learning model to automatically control the output of the pressure control module 15 and the adjustment of the microscope imaging device 16 until the liposomes are stably produced.

[0117] Step S7, after the liposomes are stably generated, the by-products are separated and the liposomes obtained are purified by using the multiple dielectrophoresis effect, and the liposomes are captured and observed in situ using the fluid dynamics capture structure. When the liposomes enter the liposome capture structure, the inverted microscope lens is adjusted to the corresponding area to observe the situation after the liposomes are captured. At the same time, the room temperature is adjusted to 23°C, and the fluorescence image of the liposomes is taken every 15 minutes within 3 hours using the inverted microscope lens and the CCD camera green fluorescence module to observe the change trend of the liposome fluorescence image. In theory, the gradually enhanced green fluorescence inside the liposome can be seen.

[0118] Note that the above are only preferred embodiments of the present application and the technical principles used. Those skilled in the art will understand that the present application is not limited to the specific embodiments herein, and that various obvious changes, readjustments and substitutions can be made by those skilled in the art without departing from the protection scope of the present application. Therefore, although the present application is described in more detail through the above embodiments, the present application is not limited to the above embodiments, and may also include more other equivalent embodiments without departing from the concept of the present application, all of which belong to the protection scope of the present application.

Claims

1. A microfluidic device for preparing liposomes, characterized in that: include: A microfluidic chip for preparing at least one liposome using a plurality 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 to generate a first control instruction for regulating the liposome; A pressure control module is connected to the microfluidic chip and the automation control module. The pressure control module is used to adjust the pressure value of at least one of the reagents in the process of preparing the liposomes by the microfluidic chip according to the first control instruction to obtain the desired liposomes.

2. The microfluidic device according to claim 1, characterized in that: The microfluidic chip comprises a negative mold and a hydrophobic film, wherein the negative mold comprises a first surface and a second surface opposite to each other, 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 with the first surface and the second surface, each of the through holes stores a reagent, and the microchannels connect each of the through holes; The pressure control module is connected to each of the through holes on the second surface and transmits pressure to the corresponding reagent in each of the through holes.

3. The microfluidic device according to claim 2, characterized in that: 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 microchannel, and the liposome capture structure is connected to the microchannel.

4. The microfluidic device according to claim 2 or 3, characterized in that: The microfluidic chip further comprises a cover glass, and the cover glass is bonded to the hydrophobic film; The microfluidic device also includes a microscopic imaging module, which is connected to the automation control module and close to the coverslip. The microscopic imaging module is used to obtain a microscopic image of the liposome and transmit it to the automation control module.

5. The microfluidic device according to claim 4, characterized in that: The automation control module comprises: A machine learning module, wherein the machine learning model is configured in the machine learning module; A first input unit, used to input the dimension data of the microchannel into the machine learning module; A second input unit, for inputting 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, configured to input the pressure value in the pressure control module into the machine learning module; A first output unit, configured to output the first control instruction to the pressure control module; a second output unit, configured to output a second control instruction to the microscopic imaging module, wherein the second control instruction is configured to control the microscopic imaging module to obtain imaging parameters of the microscopic image; Among them, the first control instruction and the second control instruction are generated by the machine learning model based on data processing input by the first input unit, the second input unit, the third input unit and the fourth input unit.

6. The microfluidic device according to claim 2, characterized in that: The pressure control module includes a pressure pump, multiple hoses and multiple connectors. The input end of the pressure pump is connected to the automation control module, and the output end of the pressure pump is connected to the multiple connectors through the hoses. Each of the connectors is connected to the corresponding through hole. The pressure pump is used to generate gas with different pressure values ​​and act on the connectors.

7. The microfluidic device according to claim 6, characterized in that: The microfluidic chip comprises a fixed hole slot layer, the fixed hole slot layer is bonded to the second surface, the fixed hole slot layer has a plurality of holes slots, and each hole slot is coaxial with the corresponding through hole; Each of the connecting parts includes a flat plate joint and a flat plate base, wherein 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 in the hole groove.

8. The microfluidic device according to claim 6, characterized in that: The connecting member 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.

9. The microfluidic device according to claim 8, characterized in that: The diameter of the inlet of the through pipe is greater than or equal to the diameter of the outlet.

10. A method for preparing liposomes using the microfluidic device according to any one of claims 1 to 9, characterized in that: include: preparing at least one liposome using a plurality of reagents on a microfluidic chip; Analyze the imaging data of the liposome using a machine learning model in an automated control module to generate a first control instruction for regulating the liposome; The first control instruction is executed in the pressure control module to adjust the pressure value of at least one of the reagents in the process of preparing the liposomes by the microfluidic chip to obtain the desired liposomes.

Images