Giant liposome preparation and collection device and method based on flow velocity control

Abstract

The invention discloses a giant liposome preparation and collection device based on flow velocity control in the field of microfluidics. The giant liposome preparation and collection device comprises an upper electrode; a microarray structure is arranged on the surface of the lower layer electrode; the fluid channel layer is arranged between the upper-layer electrode and the lower-layer electrode and communicated with the microarray structure, and the fluid channel layer is used for introducing fluid to collect the lipidosome; the invention also provides a preparation and collection method. The device and the method have the beneficial effects that an environment is provided for preparation and collection of the liposome by arranging the upper-layer electrode, the lower-layer electrode and the fluid channel layer located between the upper-layer electrode and the lower-layer electrode, the liposome prepared and collected by the device and the method is more complete, the quality of the collected liposome is higher, and subsequent research is more facilitated.

Description

Technical Field

[0001] The invention 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 Art

[0002] Giant unilamellar liposomes 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 bilayers and can be used as microreactors and carriers in the field of drug delivery. The size of giant unilamellar liposomes affects the interaction between the phospholipid membrane and the encapsulated molecules. Therefore, controlling the uniformity of the liposome scale is the top priority in the current preparation method research. At present, microfluidics and micropatterning methods are often used to control the particle size of liposomes. However, although the microfluidics method can obtain liposomes with smaller particle size differences, the phospholipid molecular layer contains residual organic reagents, which are difficult to completely remove, which will have a great impact on subsequent applications. We prepared giant liposomes based on the micropatterning method and obtained giant unilamellar liposomes. However, the giant liposomes 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 lipid tubules to the edge of the microporous structure and the ITO electrode makes it difficult for the liposomes to fall off and form a complete closed phospholipid bilayer structure. At present, liposomes are mainly detached by pipette adsorption, ultrasonic vibration and low-frequency square wave. However, the pipette method has too much instantaneous force, which can easily cause the giant unilamellar liposomes to rupture and the quality of the collected vesicles is poor; while the ultrasonic and low-frequency square wave methods are time-consuming and do not achieve the ideal detachment effect.

[0003] To this end, we propose a giant liposome preparation and collection device and method based on flow rate control. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a giant liposome preparation and collection device and method based on flow rate control.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] A giant liposome preparation and collection device based on flow rate control comprises: an upper electrode; a lower electrode, the surface of which is provided with a microarray structure, the microarray structure is used to impose scale constraints on the preparation of the liposome, the scale including size and position; a fluid channel layer, which is arranged between the upper electrode and the lower electrode and is connected to the microarray structure, the fluid channel layer is used to pass fluid to complete the collection of the liposome.

[0007] By setting an upper electrode and a lower electrode, and setting a fluid channel layer for preparing and collecting liposomes between the upper electrode and the lower electrode, an alternating electric field can be formed by electrifying the upper electrode and the lower electrode, and liposomes are prepared in the fluid channel layer and the microarray structure. The microarray structure can exert certain constraints on the self-assembly process of the liposomes, and then uniform liposomes are prepared. The particle sizes of liposomes prepared by depositing phospholipids on flat glass vary greatly. Then, a fluid pair is passed through the fluid channel layer to separate the prepared liposomes from the microarray structure, and the liposomes are collected. The shear environment in the fluid channel layer is controlled by adjusting the flow rate, and then the shearing effect generated on the vesicles is used to make them fall off. Compared with the existing liposome preparation and collection devices, the liposomes prepared by the existing liposome preparation and collection devices are more uniform in size, the collected liposomes are more complete, and the vesicles are of higher quality, which is conducive to subsequent research.

[0008] It is further defined that the microarray structure includes a plurality of square limiting holes, and the plurality of limiting holes are arranged in a rectangular array on the surface of the lower electrode.

[0009] It is further defined that the fluid channel layer includes an injection chamber, an inflow channel, a preparation chamber, an outflow channel and a collection chamber; the bottom of the preparation chamber is hollowed out and connected to the microarray structure, and the preparation chamber and the microarray structure are overlapped up and down, the injection chamber is connected to the preparation chamber through the inflow channel, and the preparation chamber is connected to the collection chamber through the outflow channel, the heights of the inflow channel and the outflow channel are both lower than the height of the preparation chamber, and the bottom surface is flush with the bottom surface of the preparation chamber; since the vesicles grow on the microarray structure at the bottom of the preparation chamber, this arrangement can effectively increase the shear force at the bottom, which is more conducive to the detachment of liposomes.

[0010] It is further defined that the preparation cavity is a circular cavity, and the inner diameter of the preparation cavity is larger than the outer diameter of the microarray structure.

[0011] It is further defined that the upper electrode is located above the preparation cavity and completely covers the preparation cavity.

[0012] It is further defined that 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 comprises the following steps:

[0014] S1. Prepare a chip, which includes a lower electrode, a fluid channel layer and an upper electrode from bottom to top, and prepare a microarray structure on the lower electrode;

[0015] S2. Chip processing: Plasma treatment is performed on the chip, and a phospholipid solution is dripped onto the plasma-treated microarray structure to deposit the phospholipid solution in the limiting holes of the microarray structure. After evaporation for a period of time, a vacuum treatment is performed to remove excess phospholipid membrane, and then the fluid channel layer is placed on the lower electrode, and the preparation cavity overlaps 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 injection: after the chip is assembled, deionized water is slowly introduced into the injection chamber at a flow rate of 200 μl / min. The introduction is stopped when the preparation chamber is filled with deionized water and the deionized water contacts the upper electrode;

[0017] S4. Prepare liposomes, apply electric signals to the upper and lower electrodes to form a sinusoidal alternating electric field with a frequency of 10 Hz and a peak-to-peak value of 2 Vpp, and continue the power treatment for 20 min;

[0018] S5. Collect the sample, disconnect the alternating current on the upper electrode and the lower electrode, adjust the flow rate of the injection chamber so that the liposomes prepared on the microarray structure in the preparation chamber fall off the microarray structure and flow into the collection chamber. The flow rate is 300-400 μl / min, and the liposomes are collected in the collection chamber.

[0019] According to the present invention, deionized water is introduced from the injection chamber at a flow rate of 200 μl / min to create an environment for the preparation of liposomes. The introduction of deionized water is stopped during the hydration process of the liposomes. After the preparation of the liposomes is completed, deionized water is continued to be introduced from the injection chamber at a flow rate of 300-400 μl / min. The deionized water introduced at a faster flow rate can cause the liposomes to separate from the microarray structure and be brought into the collection chamber 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] It is further defined that the plasma treatment time in step S2 is 15 s.

[0021] It is further defined that the phospholipid solution in step S2 is prepared from lecithin and chloroform, with a concentration of 0.2 mg / ml and a dropwise addition amount of 4 ul.

[0022] It is further defined that the vacuum treatment time in step S2 is 30 minutes.

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

[0024] Figure 1 The present invention does not cover the top view of the upper electrode state;

[0025] Figure 2 It is a schematic diagram of the internal structure of the present invention from a front view perspective;

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

[0027] Figure 4 A comparison diagram of the liposomes prepared by the present invention and the liposomes prepared on a glass plane;

[0028] Figure 5 A comparison diagram of liposomes collected by the method of the present invention and liposomes collected by a pipette;

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

[0030] The symbols of the components are as follows:

[0031] Upper electrode 1, lower electrode 2, microarray structure 21, limiting hole 211, fluid channel layer 3, injection chamber 31, inflow channel 32, preparation chamber 33, outflow channel 34, collection chamber 35. DETAILED DESCRIPTION

[0032] The specific implementation modes of the present invention are described below to facilitate those skilled in the art to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0033] Example:

[0034] like Figure 1-Figure 6As shown, a giant liposome preparation and collection device based on flow rate control includes an upper electrode 1, a lower electrode 2 and a fluid channel layer 3; a microarray structure 21 is provided on the surface of the lower electrode 2, and the microarray structure 21 is used to perform scale constraints on the preparation of liposomes, and the scale includes size and position; the microarray structure 21 includes a plurality of square limiting holes 211, and the plurality of limiting holes 211 are arranged in a rectangular array on the surface of the lower electrode 2, the limiting holes 211 are square micropores with a circumscribed circle diameter of 15 μm, and the spacing between adjacent limiting holes 211 is 1 / 16 of the radius of its circumscribed circle, the outer diameter of the microarray structure 21 is a square shape of 2.7 mm*2.7 mm, and the height of the microarray structure 21 is 4-6 μm; the fluid channel layer 3 is arranged between the upper electrode 1 and the lower electrode 2, and is connected to the microarray structure 21, and the fluid channel layer 3 is used to pass fluid to complete the collection of liposomes; the fluid channel Layer 3 includes an injection chamber 31, an inflow channel 32, a preparation chamber 33, an outflow channel 34 and a collection chamber 35; the bottom of the preparation chamber 33 is hollowed out and connected to the microarray structure 21, and the preparation chamber 33 and the microarray structure 21 are overlapped up and down, the injection chamber 31 is connected to the preparation chamber 33 through the inflow channel 32, and the preparation chamber 33 is connected to the collection chamber 35 through the outflow channel 34, the heights of the inflow channel 32 and the outflow channel 34 are both lower than the height of the preparation chamber 33, and the bottom surfaces are flush with the bottom surface of the preparation chamber 33, and the inflow channel 32 and the outflow channel 34 are both circumferentially closed and open at both ends; the preparation chamber 33 is a circular cavity with a diameter of 6 mm, the thickness of the preparation chamber 33 is 1.2 mm, and the heights of the inflow channel 32 and the outflow channel 34 are 0.6 mm; the upper electrode 1 is located above the preparation chamber 33 and completely covers the preparation chamber 33; the upper electrode 1 and the lower electrode 2 are both indium tin oxide glass electrodes.

[0035] By setting an upper electrode 1 and a lower electrode 2, and setting a fluid channel layer 3 for preparing and collecting liposomes between the upper electrode 1 and the lower electrode 2, an alternating electric field can be formed by energizing the upper electrode 1 and the lower electrode 2, and liposomes can be prepared in the fluid channel layer 3 and the microarray structure 21. The microarray structure 21 can exert certain constraints on the self-assembly process of the liposomes, thereby preparing uniform liposomes. The particle sizes of liposomes prepared by depositing phospholipids on a flat glass vary greatly. Then, a fluid pair is introduced through the fluid channel layer 3 to separate the prepared liposomes from the microarray structure 21, and the liposomes are collected. The shear environment in the fluid channel layer 3 is controlled by adjusting the flow rate, and the shearing effect generated on the vesicles is used to make them fall off. Compared with the existing liposome preparation and collection device, the liposomes prepared by the existing liposome preparation and collection device are more uniform in size, the collected liposomes are more complete, and the vesicles are of higher quality, which is conducive to subsequent research. Since the vesicles grow on the microarray structure 21 at the bottom of the preparation cavity 33, such a setting can effectively increase the shear force at the bottom, which is more conducive to the separation of the liposomes.

[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. Prepare a chip, the chip is composed of a lower electrode 2, a fluid channel layer 3 and an upper electrode 1 from bottom to top, and a microarray structure 21 is formed on the surface of the lower electrode 2 by a photolithography process;

[0038] S2. Chip treatment: plasma treatment is performed on the chip for 15 seconds, and a phospholipid solution is dripped onto the plasma-treated microarray structure 21, so that the phospholipid solution is deposited in the limiting hole 211 of the microarray structure 21. The phospholipid solution is prepared from lecithin and chloroform, with a concentration of 0.2 mg / ml and a dripping amount of 4 ul. After the phospholipid solution evaporates for 2 minutes, a vacuum treatment is performed to remove excess chloroform, and the vacuum treatment time is 30 minutes. Then, the height difference between the bottom and the top of the limiting hole 211 in the microarray structure 21 is used to stick the excess dry phospholipid film on the surface with a PET film, and a uniform dry phospholipid film is formed in the micropores. Then, the fluid channel layer 3 is placed on the lower electrode 2, and the preparation cavity 33 overlaps with the microarray structure 21, and then the upper electrode 1 is covered on the preparation cavity 33 to seal the top of the preparation cavity 33, and the chip assembly is completed.

[0039] S3. Chip injection: after the chip is assembled, deionized water is slowly introduced into the injection chamber 31 at a flow rate of 200 μl / min. The introduction is stopped after the preparation chamber 33 is filled with deionized water and the deionized water contacts the upper electrode 1;

[0040] S4. Prepare liposomes, apply an electric signal to the upper electrode 1 and the lower electrode 2 to form a sinusoidal alternating electric field with a frequency of 10 Hz and a peak-to-peak value of 2 Vpp, and continue the power treatment for 20 min;

[0041] S5. Collect the sample, disconnect the alternating current on the upper electrode 1 and the lower electrode 2, adjust the flow rate of the injection chamber 31 so that the liposomes prepared on the microarray structure 21 in the preparation chamber 33 fall off the microarray structure 21 and flow into the collection chamber 35. The flow rate is 300 μl / min, and the liposomes are collected in the collection chamber 35.

[0042] Through the present invention, deionized water is introduced from the injection chamber 31 at a flow rate of 200 μl / min to create an environment for the preparation of liposomes. The introduction of deionized water is stopped during the liposome hydration process. After the liposome preparation is completed, deionized water is continued to be introduced from the injection chamber 31 at a flow rate of 300-400 μl / min. The deionized water introduced at a faster flow rate can cause the liposomes to detach from the microarray structure 21 and be brought into the collection chamber 35 by the deionized water to complete the collection. Compared with the existing collection method using a pipette, the liposomes collected by this method are more complete, the quality of the collected vesicles is better, and the collection efficiency is also higher.

[0043] Liposome homogeneity test:

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

[0045] Collect liposomes for scale testing:

[0046] The liposomes prepared on the device were collected by the collection method of the present invention and the pipette method, respectively. The average particle size of the liposomes collected by the collection scheme of the present invention was 18.49±1.04μm, while the average particle size of the vesicles obtained by the pipette method was 12.21±3.582μm, and the vesicles were smaller in size and had a larger particle size distribution. In addition, it was found that the liposomes collected by the pipette had a larger proportion of multi-chamber vesicles, which was not conducive to the collection of giant unilamellar liposomes. The collection results of the two are as follows: Figure 5 As shown, the upper part shows liposomes collected by the method of the present invention, and the lower part shows liposomes collected by a pipette.

Claims

1. A giant liposome preparation and collection device based on flow rate control, characterized in that: include: Upper electrode (1); The lower electrode (2) has a microarray structure (21) on its surface, and the microarray structure (21) is used to constrain the scale of the liposome preparation, and the scale includes size and position; The fluid channel layer (3) is arranged between the upper electrode (1) and the lower electrode (2) and is connected to the microarray structure (21). The fluid channel layer (3) is used to pass fluid to complete the collection of liposomes.

2. The giant vesicle preparation and collection device based on flow rate control according to claim 1, characterized in that: The microarray structure (21) comprises a plurality of square limiting holes (211), and the plurality of limiting holes (211) are arranged in a rectangular array on the surface of the lower electrode (2).

3. The giant vesicle preparation and collection device based on flow rate control according to claim 2, characterized in that: The fluid channel layer (3) comprises an injection chamber (31), an inflow channel (32), a preparation chamber (33), an outflow channel (34) and a collection chamber (35); the bottom of the preparation chamber (33) is hollowed out and is connected to the microarray structure (21), and the preparation chamber (33) and the microarray structure (21) are arranged to overlap each other, the injection chamber (31) is connected to the preparation chamber (33) through the inflow channel (32), and the preparation chamber (33) is connected to the collection chamber (35) through the outflow channel (34); the heights of the inflow channel (32) and the outflow channel (34) are both lower than the height of the preparation chamber (33), and the bottom surfaces are flush with the bottom surface of the preparation chamber (33).

4. The giant vesicle preparation and collection device based on flow rate control according to claim 3, characterized in that: The preparation cavity (33) is a circular cavity, and the inner diameter of the preparation cavity (33) is greater than the outer diameter of the microarray structure (21).

5. The giant vesicle preparation and collection device based on flow rate control according to claim 4, characterized in that: The upper electrode (1) is located above the preparation cavity (33) and completely covers the preparation cavity (33).

6. The giant vesicle preparation and collection device based on flow rate control according to claim 4, characterized in that: The microarray structure (21) has a height of 4-6 μm, the preparation cavity (33) has a thickness of 1.2 mm, the inflow channel (32) and the outflow channel (34) have a height of 0.6 mm, and the upper electrode (1) and the lower electrode (2) are both indium tin oxide glass electrodes.

7. A preparation and collection method, using the giant vesicle preparation and collection device based on flow rate control according to any one of claims 1 to 6, characterized in that: The steps include: S1. Prepare a chip, the chip comprises a lower electrode (2), a fluid channel layer (3) and an upper electrode (1) from bottom to top, and prepare a microarray structure (21) on the lower electrode (2); S2. Chip processing: Plasma treatment is performed on the chip, and a phospholipid solution is dripped onto the plasma-treated microarray structure (21), so that the phospholipid solution is deposited in the limiting hole (211) of the microarray structure (21). After evaporation for a period of time, a vacuum treatment is performed to remove excess phospholipid membrane, and then the fluid channel layer (3) is placed on the lower electrode (2), and the preparation cavity (33) overlaps with the microarray structure (21). Then, the upper electrode (1) is covered on the preparation cavity (33) to seal the top of the preparation cavity (33), and the chip assembly is completed; S3. Chip injection: after the chip is assembled, deionized water is slowly introduced into the injection chamber (31) at a flow rate of 200 μl / min. The introduction is stopped after the preparation chamber (33) is filled with deionized water and the deionized water contacts the upper electrode (1); S4. Prepare liposomes, apply an electric signal to the upper electrode (1) and the lower electrode (2) to form a sinusoidal alternating electric field with a frequency of 10 Hz and a peak-to-peak value of 2 Vpp, and continue to apply the power for 20 min; S5. Collect the sample, disconnect the alternating current on the upper electrode (1) and the lower electrode (2), adjust the flow rate of the injection chamber (31) so that the liposomes prepared on the microarray structure (21) in the preparation chamber (33) fall off the microarray structure (21) and flow into the collection chamber (35), the flow rate is 300-400 μl / min, and the liposomes are collected in the collection chamber (35).

8. The preparation and collection method according to claim 7, characterized in that: The plasma treatment time in step S2 is 15 seconds.

9. The preparation and collection method according to claim 7, characterized in that: The phospholipid solution in step S2 is prepared from lecithin and chloroform, with a concentration of 0.2 mg / ml and a dropwise addition amount of 4 ul.

10. The preparation and collection method according to claim 7, characterized in that: The vacuum treatment time in step S2 is 30 minutes.

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