A device and method for the preparation and collection of giant liposomes based on flow rate control

Abstract

The application discloses a kind of based on flow rate control's giant liposome preparation, collection device in microfluidic field, comprising: upper electrode;Lower electrode, surface is equipped with microarray structure;Fluid channel layer is arranged between upper electrode and lower electrode, and is communicated with microarray structure, fluid channel layer is used to pass into fluid to complete collection to liposome;And a kind of preparation, collection method;The beneficial effects of the application are: by setting upper electrode, lower electrode and the fluid channel layer between the two, provide environment for the preparation and collection of liposome, the liposome prepared and collected by the device and method is more complete, the quality of collected liposome is higher, more conducive to subsequent research.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic technology, and in particular to a giant liposome preparation and collection device and method based on flow rate control. BACKGROUND

[0002] Giant unilamellar vesicles are important tools for studying the physical and chemical properties of cell membranes and membrane interactions. They isolate the internal environment from the external solution through a layer of phospholipid bilayer and can be used as microreactors and carriers in the field of drug delivery. The size of giant unilamellar vesicles affects the interaction between the phospholipid membrane and the encapsulated molecules, so controlling the uniformity of the liposome size is a top priority in current preparation methods. Currently, microfluidic methods and micro-patterning methods are commonly used to control the particle size of liposomes. However, although microfluidic methods can obtain liposomes with smaller particle size differences, the phospholipid molecule layer contains residual organic reagents that are difficult to completely remove, which will have a significant impact on subsequent applications. We based on the micro-patterning method to prepare giant unilamellar vesicles, which can obtain giant unilamellar vesicles. However, the giant unilamellar vesicles prepared based on the patterning method are not completely closed, and they are connected to the substrate glass through lipid tubules. The strong adhesion of the lipid tubules to the edges of the micro-pore structure and the ITO electrode makes it difficult for the liposomes to complete the detachment and form a complete closed phospholipid bilayer structure. Currently, methods such as pipette adsorption, ultrasonic oscillation, and application of low-frequency square waves are used to make the liposomes detach. However, the pipette method has too much instantaneous force, which can easily cause the rupture of giant unilamellar vesicles and the poor quality of the collected vesicles; while the ultrasonic and low-frequency square wave methods are time-consuming and do not achieve the desired detachment effect.

[0003] Therefore, we propose a giant liposome preparation and collection device and method based on flow rate control. SUMMARY

[0004] In view of the above shortcomings of the prior art, the present application provides a giant liposome preparation and collection device and method based on flow rate control.

[0005] To achieve the above-mentioned application purposes, the technical solutions adopted by the present application are as follows:

[0006] A giant liposome preparation and collection device based on flow rate control, comprising: an upper electrode; a lower electrode having a microarray structure on its surface, the microarray structure being used for size constraint of the preparation of liposomes, the size including size and position; a fluid channel layer disposed between the upper electrode and the lower electrode and in communication with the microarray structure, the fluid channel layer being used for introducing fluid to complete the collection of liposomes.

[0007] By setting the upper electrode and the lower electrode, and setting the fluid channel layer for preparing and collecting liposomes between the upper electrode and the lower electrode, an alternating current field can be formed by electrifying the upper electrode and the lower electrode to prepare liposomes in the fluid channel layer and the microarray structure, the microarray structure can produce certain constraints on the self-assembly process of the liposomes, and then the liposomes are prepared, the liposomes prepared by depositing phospholipids on a plane glass have a huge difference in particle size, and then the prepared liposomes are separated from the microarray structure by passing a fluid through the fluid channel layer, and the collection of the liposomes is completed, the shear environment in the fluid channel layer is controlled by adjusting the flow rate, and then the liposomes are separated by using the shear effect generated on the vesicles, compared with the liposomes prepared and collected by the existing liposome preparation and collection device, the liposomes are more uniform in size, the collected liposomes are more complete, the vesicle quality is higher, and the subsequent research is facilitated.

[0008] Further limitation, the microarray structure includes a plurality of square-shaped limiting holes, and the plurality of limiting holes are arranged in a rectangular array on the surface of the lower electrode.

[0009] Further limitation, the fluid channel layer includes a sample inlet cavity, an inflow channel, a preparation cavity, an outflow channel and a collection cavity; the bottom of the preparation cavity is hollow and is arranged in communication with the microarray structure, and the preparation cavity and the microarray structure are arranged in an upper-lower overlapping manner; the sample inlet cavity is in communication with the preparation cavity through the inflow channel, and the preparation cavity is in communication with the collection cavity through the outflow channel; the height of the inflow channel and the outflow channel is lower than the height of the preparation cavity, and the bottom surface is flush with the bottom surface of the preparation cavity; since the vesicles grow on the microarray structure at the bottom of the preparation cavity, the setting can effectively improve the shear force at the bottom, and is more beneficial to the separation of the liposomes.

[0010] Further limitation, the preparation cavity is a circular cavity, and the inner diameter of the preparation cavity is greater than the outer diameter of the microarray structure.

[0011] Further limitation, the upper electrode is located above the preparation cavity and completely covers the preparation cavity.

[0012] Further limitation, the height of the microarray structure is 4-6 μm, the thickness of the preparation cavity is 1.2 mm, the height of the inflow channel and the outflow channel is 0.6 mm, and the upper electrode and the lower electrode are both indium tin oxide glass electrodes.

[0013] A preparation and collection method using the above-mentioned giant liposome preparation and collection device based on flow rate control includes the following steps:

[0014] S1. Preparing a chip, the chip is sequentially arranged from bottom to top as the lower electrode, the fluid channel layer and the upper electrode, and the microarray structure is prepared on the lower electrode;

[0015] S2. Chip processing, the chip is subjected to plasma treatment, and a phospholipid solution is added to the microarray structure subjected to the plasma treatment, so that the phospholipid solution is deposited in the limiting hole of the microarray structure, and after a period of evaporation, vacuum treatment is performed and the excess phospholipid film is removed, then the fluid channel layer is placed on the lower electrode, and the preparation cavity is overlapped with the microarray structure, and then the upper electrode is covered on the preparation cavity to seal the top of the preparation cavity, and the chip assembly is completed;

[0016] S3. Chip sampling, after the chip assembly is completed, deionized water is slowly introduced into the sampling cavity at a flow rate of 200ul / min, and the introduction is stopped after the preparation cavity is filled with deionized water and the deionized water contacts the upper electrode;

[0017] S4. Preparation of liposomes, a sinusoidal alternating current field with a frequency of 10Hz and a peak-to-peak value of 2Vpp is applied to the upper electrode and the lower electrode for 20min.

[0018] S5. Collecting sample, the alternating current on the upper electrode and the lower electrode is disconnected, the flow rate of the sampling cavity is adjusted so that the liposomes prepared on the microarray structure in the preparation cavity fall off the microarray structure and flow into the collection cavity, and the flow rate is 300-400ul / min, and the collection of the liposomes in the collection cavity is completed.

[0019] Through the present application, deionized water is introduced into the sampling cavity at a flow rate of 200ul / min to create an environment for the preparation of liposomes, and the hydration process of the liposomes is stopped by introducing deionized water, and after the preparation of the liposomes is completed, deionized water is continuously introduced from the sampling cavity at a flow rate of 300-400ul / min, and the deionized water introduced at a faster flow rate can make the liposomes separate from the microarray structure and be brought into the collection cavity by the deionized water to complete the collection. Compared with the existing collection method using a pipette, the liposomes collected by the present method are more complete, the quality of the collected vesicles is better, and the collection efficiency is also higher.

[0020] Further limited, the plasma treatment time in step S2 is 15s.

[0021] Further limited, the phospholipid solution in step S2 is configured by lecithin and chloroform, with a concentration of 0.2mg / ml and a drop amount of 4ul.

[0022] Further limited, the vacuum treatment time in step S2 is 30min.

[0023] The present application has the beneficial effects that: by setting the upper electrode, the lower electrode and the fluid channel layer located between the two, an environment is provided for the preparation and collection of liposomes, and the liposomes prepared and collected by the present device and method are more complete, the quality of the collected liposomes is higher, and it is more conducive to subsequent research. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The top view of the upper electrode state is not covered by the present application;

[0025] Figure 2 The internal structure diagram of the front view of the present application;

[0026] Figure 3 The top view of the microarray structure;

[0027] Figure 4 The contrast diagram of the liposome prepared by the present application and the liposome prepared by the glass plane;

[0028] Figure 5 The contrast diagram of the liposome collected by the method of the present application and the liposome collected by the pipette gun;

[0029] Figure 6 The Figure 5 The statistical histogram of the liposome size obtained by two methods.

[0030] The symbols of each component are as follows:

[0031] The upper electrode 1, the lower electrode 2, the microarray structure 21, the limiting hole 211, the fluid channel layer 3, the sample cavity 31, the inflow channel 32, the preparation cavity 33, the outflow channel 34, and the collection cavity 35. DETAILED DESCRIPTION

[0032] The specific embodiments of the present application are described below to facilitate the understanding of the present application for those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0033] Embodiment:

[0034] As Figures 1-6As shown, a kind of giant liposome preparation based on flow rate control, collection device, including upper electrode 1, lower electrode 2 and fluid channel layer 3;Lower electrode 2 surface is equipped with microarray structure 21, and microarray structure 21 is used to scale constraint for the preparation of liposome, scale includes size and position;Microarray structure 21 includes several square-shaped limiting holes 211, several limiting holes 211 are arranged in the surface of lower electrode 2 in rectangular array, limiting hole 211 is square micropore with 15 μm circumscribed circle diameter, and the spacing between adjacent limiting holes 211 is 1 / 16 of the radius of its circumscribed circle, the outer diameter of microarray structure 21 is square shape with 2.7mm*2.7mm, and the height of microarray structure 21 is 4-6 μm;Fluid channel layer 3 is arranged between upper electrode 1 and lower electrode 2 and is communicated with microarray structure 21, and fluid channel layer 3 is used to pass into fluid to complete the collection of liposome;Fluid channel layer 3 includes sample injection cavity 31, inflow channel 32, preparation cavity 33, outflow channel 34 and collection cavity 35;The bottom of preparation cavity 33 is hollow, and is communicated with microarray structure 21, and preparation cavity 33 is arranged in upper and lower overlapping with microarray structure 21, sample injection cavity 31 is communicated with preparation cavity 33 through inflow channel 32, preparation cavity 33 is communicated with collection cavity 35 through outflow channel 34, the height of inflow channel 32 and outflow channel 34 is lower than the height of preparation cavity 33, and the bottom surface is flush with the bottom surface of preparation cavity 33, and inflow channel 32 and outflow channel 34 are both arranged as circumferentially closed two-end opening;Preparation cavity 33 is circular cavity with 6mm diameter, the thickness of preparation cavity 33 is 1.2mm, and the height of inflow channel 32 and outflow channel 34 is 0.6mm;Upper electrode 1 is located above preparation cavity 33 and completely covers preparation cavity 33;Upper electrode 1 and lower electrode 2 are both indium tin oxide glass electrodes.

[0035] By setting upper electrode 1 and lower electrode 2, and setting fluid channel layer 3 for preparing and collecting liposome between upper electrode 1 and lower electrode 2, alternating current field can be formed by electrifying upper electrode 1 and lower electrode 2, liposome is prepared in fluid channel layer 3 and microarray structure 21, microarray structure 21 can produce certain constraint to liposome self-assembly process, and then uniform liposome is prepared, the particle size of liposome prepared by depositing phospholipid on plane glass is greatly different, and then fluid is passed into fluid channel layer 3 to make prepared liposome separate from microarray structure 21, and liposome is collected, the shear environment in fluid channel layer 3 is controlled by adjusting flow rate, and then liposome is made to fall off by using shear effect on vesicle, compared with the liposome prepared by existing liposome preparation and collection device, the scale of liposome is more uniform, the collected liposome is more complete, the quality of vesicle is higher, and subsequent research is facilitated;Since vesicle grows on microarray structure 21 at the bottom of preparation cavity 33, the bottom shear force can be effectively improved by this setting, and liposome is more easily separated.

[0036] A preparation and collection method using the above-mentioned giant liposome preparation and collection device based on flow rate control comprises the following steps:

[0037] S1. A chip is prepared, which comprises a lower electrode 2, a fluid channel layer 3 and an upper electrode 1 from bottom to top. A microarray structure 21 is formed on the surface of the lower electrode 2 by a photolithography process.

[0038] S2. Chip processing. The chip is subjected to plasma treatment for 15 s, and a phospholipid solution is added to the microarray structure 21 after plasma treatment, so that the phospholipid solution is deposited in the limiting hole 211 of the microarray structure 21. The phospholipid solution is prepared by mixing lecithin and chloroform, with a concentration of 0.2 mg / ml and a drop amount of 4 ul. After the phospholipid solution is evaporated for 2 min, vacuum treatment is performed to remove excess chloroform, with a vacuum treatment time of 30 min. Then, the height difference between the bottom and the top of the limiting hole 211 in the microarray structure 21 is used to remove the excess dry phospholipid film on the surface with a PET film. At this time, a uniform dry phospholipid film is formed in the micropore. Then, the fluid channel layer 3 is placed on the lower electrode 2, and the preparation cavity 33 overlaps the microarray structure 21. The upper electrode 1 is then covered on the preparation cavity 33 to close the top of the preparation cavity 33. The chip assembly is completed.

[0039] S3. Chip sampling. After the chip assembly is completed, deionized water is slowly introduced into the sampling cavity 31 at a flow rate of 200 ul / min. The introduction is stopped after the preparation cavity 33 is filled with deionized water and the deionized water contacts the upper electrode 1.

[0040] S4. Liposome preparation. A sinusoidal alternating current field with a frequency of 10 Hz and a peak-to-peak value of 2 Vpp is applied to the upper electrode 1 and the lower electrode 2, and the power-on treatment is continued for 20 min.

[0041] S5. Sample collection. The alternating current on the upper electrode 1 and the lower electrode 2 is turned off, and the flow rate of the sampling cavity 31 is adjusted so that the liposomes prepared on the microarray structure 21 in the preparation cavity 33 fall off the microarray structure 21 and flow into the collection cavity 35 at a flow rate of 300 ul / min. The collection of liposomes is completed in the collection cavity 35.

[0042] Through the present application, deionized water is introduced into the sample cavity 31 at a flow rate of 200 μl / min to create an environment for the preparation of liposomes. The hydration process of the liposomes stops when the deionized water is introduced. After the preparation of the liposomes is completed, deionized water is continuously introduced into the sample cavity 31 at a flow rate of 300-400 μl / min. The deionized water introduced at a faster flow rate can make the liposomes separate from the microarray structure 21 and be carried into the collection cavity 35 to complete the collection. Compared with the existing collection method using a pipette, the liposomes collected by the present method are more complete, the quality of the collected vesicles is better, and the collection efficiency is also higher.

[0043] Liposome uniformity test:

[0044] Liposomes are prepared by the present method and device, and also prepared on a glass plane by a conventional method. The liposomes prepared by the two devices and methods are compared, and the comparison results are shown in Figure 4 , where the left is the liposomes prepared by the method and device of the present application, and the right is the liposomes prepared on the glass plane.

[0045] Liposome size test:

[0046] The liposomes prepared by the present collection method and the pipette method are collected on the present device. The average particle size of the liposomes collected by the collection scheme in the present application is 18.49±1.04 μm, while the average particle size of the vesicles obtained by using the pipette method is 12.21±3.582 μm. The size of the obtained vesicles is smaller, and the particle size distribution is larger. Moreover, it is found that the liposomes collected by using the pipette have a large proportion of multi-chamber vesicles, which is not conducive to the collection of giant unilamellar liposomes. The collection results of the two are shown in Figure 5 , where the upper is the liposomes collected by the method of the present application, and the lower is the liposomes collected by the pipette.

Claims

1. A method for preparing and collecting a giant liposome based on flow rate control using a giant liposome preparation and collection device based on flow rate control, characterized by, The preparation and collection device comprises: an upper electrode (1); a lower electrode (2) provided with a microarray structure (21) on a surface, the microarray structure (21) being used for scale constraint of preparation of liposomes, the scale including size and position; a fluid channel layer (3) arranged between the upper electrode (1) and the lower electrode (2) and in communication with the microarray structure (21), the fluid channel layer (3) being used for introduction of fluid to complete collection of liposomes; the microarray structure (21) comprises a plurality of square-shaped limiting holes (211) arranged in a rectangular array on the surface of the lower electrode (2); the fluid channel layer (3) comprises a sample injection cavity (31), an inflow channel (32), a preparation cavity (33), an outflow channel (34) and a collection cavity (35); the bottom of the preparation cavity (33) is hollowed out and arranged in communication with the microarray structure (21), and the preparation cavity (33) is arranged in an upper-lower overlapping manner with the microarray structure (21); the sample injection cavity (31) is in communication with the preparation cavity (33) through the inflow channel (32), the preparation cavity (33) is in communication with the collection cavity (35) through the outflow channel (34), and the heights of the inflow channel (32) and the outflow channel (34) are lower than the height of the preparation cavity (33) and the bottom surfaces thereof are flush with the bottom surface of the preparation cavity (33); the preparation and collection method comprises the following steps: S1. preparing a chip, the chip comprising, from bottom to top, a lower electrode (2), a fluid channel layer (3) and an upper electrode (1), and the microarray structure (21) is prepared on the lower electrode (2); S2. chip processing, plasma processing is performed on the chip, and a phospholipid solution is added dropwise to the microarray structure (21) after the plasma processing, so that the phospholipid solution is deposited in the limiting holes (211) of the microarray structure (21); after evaporation for a period of time, vacuum extraction is performed and excess phospholipid film is removed; then the fluid channel layer (3) is placed on the lower electrode (2) with the preparation cavity (33) overlapping the microarray structure (21), and the upper electrode (1) is covered on the preparation cavity (33) to close the top of the preparation cavity (33), and the chip assembly is completed; S3. chip sample injection, after the chip assembly is completed, deionized water is slowly introduced into the sample injection cavity (31) at a flow rate of 200 μl / min; after the preparation cavity (33) is filled with deionized water and the deionized water contacts the upper electrode (1), the introduction is stopped; S4. preparation of liposomes, a sinusoidal alternating current electric field with a frequency of 10 Hz and a peak-to-peak value of 2 Vpp is introduced on the upper electrode (1) and the lower electrode (2) to form an electric signal, and the power supply is continuously maintained for 20 min. S5. Collecting the sample, disconnecting the alternating current on the upper electrode (1) and the lower electrode (2), adjusting the flow rate of the sample chamber (31) to make the liposomes prepared on the microarray structure (21) in the preparation chamber (33) fall off the microarray structure (21) and flow into the collecting chamber (35), the flow rate being 300-400 μl / min, and completing the collection of the liposomes in the collecting chamber (35).

2. The method of claim 1, wherein the flow rate is controlled by a flow rate controller. The preparation chamber (33) is a circular cavity, and the inner diameter of the preparation chamber (33) is greater than the outer diameter of the microarray structure (21).

3. The method of claim 2, wherein the flow rate is controlled by a flow rate controller. The upper electrode (1) is located above the preparation chamber (33) and covers the preparation chamber (33) completely.

4. The method of claim 3, wherein the flow rate is controlled by a flow rate controller. The height of the microarray structure (21) is 4-6 μm, the thickness of the preparation chamber (33) is 1.2 mm, the height of the inflow channel (32) and the outflow channel (34) is 0.6 mm, and the upper electrode (1) and the lower electrode (2) are both indium tin oxide glass electrodes.

5. The method of claim 1, wherein the flow rate control-based giant liposome preparation and collection method is characterized by, The plasma treatment time in the step S2 is 15 s.

6. The method of claim 1, wherein the flow rate is controlled by a flow rate controller. The phospholipid solution in the step S2 is prepared by dissolving lecithin in chloroform, and the concentration is 0.2 mg / ml, and the dropwise adding amount is 4 ul.

7. The method of claim 1, wherein the flow rate is controlled by a flow rate controller. The vacuumizing treatment time in the step S2 is 30 min.

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