CN115228407B - Preparation device and preparation method of lipid vesicles - Google Patents
Preparation device and preparation method of lipid vesicles Download PDFInfo
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- CN115228407B CN115228407B CN202110444007.2A CN202110444007A CN115228407B CN 115228407 B CN115228407 B CN 115228407B CN 202110444007 A CN202110444007 A CN 202110444007A CN 115228407 B CN115228407 B CN 115228407B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J19/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
- B01J19/087—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing electric or magnetic energy
Abstract
The preparation device comprises at least one group of electrode groups, chambers and an insulating isolation structure, wherein each group of electrode groups comprises a first electrode plate and a second electrode plate, an electric field space is formed between the first electrode plate and the second electrode plate, the chambers are all positioned in the corresponding electric field spaces, at least one preparation chamber for containing preparation solution is arranged in each chamber, and the insulating isolation structure is used for enabling the preparation solution in a single preparation chamber to be in contact with only one electrode plate of the first electrode plate and the second electrode plate; when in preparation, only the lipid is coated on the electrode plates, the preparation solution is added into the preparation chamber, and electric signals are applied to each electrode plate. According to the invention, by adding the insulating isolation structure, the lipid vesicle can be prepared in the ionic solution in an electric forming mode, and the preparation yield of the lipid vesicle in the ionic solution is improved.
Description
Technical Field
The invention relates to preparation of vesicles, in particular to a preparation device and a preparation method of lipid vesicles.
Background
Lipid Vesicles, particularly Giant Vesicles (GV), have been widely used as simple model systems for studying certain physicochemical properties of biological cell activities, e.g. in cell membrane models, cell models, bioreactors, etc., while playing an important role in studying lipid domain formation, lipid kinetics, membrane growth, budding, fission and membrane fusion. As a model system for studying the structure and function of biological cells, complex physiological solution conditions must be based to obtain practical results and reliable conclusions. As a bioreactor, it is only interesting to encapsulate complex and diverse substances. Therefore, the preparation of GV in a wide range of solution conditions (e.g., high ion concentration) has great scientific application value.
Common GV preparation methods comprise a hydration method, an SUV (small unilamellar vesicle) fusion method, a microfluidic method, an emulsion method and an electroforming method, and the yields of the first 3 methods are low; the emulsion method has higher yield, but is limited by organic solvent residues, and can not completely simulate a lipid membrane; GV films prepared by the electroforming method have no organic volume residue and high yield, and are widely used at present. However, for physiological solutions containing ions, the electric field distribution is affected, but the method is not suitable for preparing lipid vesicles by an electric forming method, and many researches are focused on improving the yield of the lipid vesicles prepared by electric forming in the ionic solution, but the effect is very little.
Disclosure of Invention
The invention mainly aims to provide a preparation device and a preparation method for lipid vesicles, which are used for preparing the lipid vesicles in an ionic solution in an electro-formation mode and improving the preparation yield of the lipid vesicles in the ionic solution.
To achieve the above object and other related objects, the present invention provides the following technical solutions:
a preparation device of lipid vesicles, comprising:
at least one group of electrode groups, each group of electrode groups comprises a first electrode plate and a second electrode plate, and an electric field space is formed between the first electrode plate and the second electrode plate;
the chambers are all positioned in corresponding electric field spaces, and at least one preparation chamber for containing preparation solution is arranged in each chamber;
an insulating isolation structure for contacting the preparation solution in the single preparation chamber with only one of the first electrode plate and the second electrode plate.
Optionally, the types of the preparation chambers include one or two of a first preparation chamber and a second preparation chamber;
a part of cavity walls of the first preparation cavity are surrounded by the first electrode plate, and the insulating isolation structure separates the first preparation cavity from the second electrode plate;
and part of the cavity wall of the second preparation cavity is surrounded by the second electrode plate, and the insulating isolation structure separates the second preparation cavity from the first electrode plate.
Optionally, the insulating isolation structure includes the insulation board, first electrode plate the insulation board with the second electrode plate stacks gradually and sets up, be provided with the hollow groove on the insulation board, be provided with an at least insulating barrier in the hollow groove, insulating barrier segmentation the hollow groove and form at least one preparation cavity.
Alternatively to this, the method may comprise,
the number of the insulating partition boards is one, the groove walls of the hollow grooves, the insulating partition boards and the first electrode plates are enclosed to form a first preparation chamber, and the groove walls of the hollow grooves, the insulating partition boards and the second electrode plates are enclosed to form a second preparation chamber;
or (b)
The number of the insulating partition plates is one, and the insulating partition plates are abutted against the first electrode plate or the second electrode plate to form a second preparation chamber or one of the first preparation chambers;
or (b)
The number of the insulating partition plates is at least two, and the first preparation chamber is defined by the wall of the hollow groove, the first electrode plate and the insulating partition plate nearest to the first electrode plate; the wall of the hollow groove, the second electrode plate and the insulating partition plate closest to the second electrode plate enclose the second preparation chamber.
Optionally, the insulating plate includes a first insulating plate and a second insulating plate, the first electrode plate, the first insulating plate, the second insulating plate and the second electrode plate are sequentially stacked, the hollow grooves are formed in the first insulating plate and the second insulating plate, the insulating partition plates are arranged in the hollow grooves, so that the hollow grooves of the first insulating plate are divided into a first preparation chamber, and the hollow grooves of the second insulating plate are divided into a second preparation chamber; the first preparation chamber back and the second preparation chamber are separated by the insulating partition.
Optionally, the preparation device of the lipid vesicle further comprises an insulating sealing member, the first preparation chamber and the second preparation chamber are all communicated from one side of the insulating plate to form a liquid injection opening, and the insulating sealing member is used for sealing the liquid injection opening.
Optionally, the number of the electrode groups is one group;
or (b)
The number of the electrode groups is at least two, each electrode group is arranged in a lamination way, and two adjacent electrode groups share a first electrode plate or a second electrode plate;
or (b)
The number of the electrode groups is at least two, each electrode group is arranged in a lamination way, and two first electrode plates of two adjacent electrode groups are integrally arranged or two second electrode plates of two adjacent electrode groups are integrally arranged.
Optionally, the electrode plate is an opaque conductive plate;
or (b)
The electrode plate is a transparent conductive plate.
Optionally, the electrode plate is an ITO conductive glass plate.
Correspondingly, the invention also provides a preparation method of the lipid vesicle, when the lipid vesicle is prepared, the preparation device of the lipid vesicle is used, and the preparation method comprises the following steps:
forming a lipid layer so that the lipid layer covers the region of each electrode plate serving as the cavity wall of the preparation chamber
Adding a preparation solution into the preparation chamber;
and connecting a signal generator to the first electrode plate and the second electrode plate to enable the polarities of the first electrode plate and the second electrode plate to be opposite.
According to the invention, by adding the insulating isolation structure, the lipid vesicle can be prepared in the ionic solution in an electric forming mode, and the preparation yield of the lipid vesicle in the ionic solution is improved.
Drawings
FIG. 1 is a schematic view showing an exemplary three-dimensional structure of a manufacturing apparatus of the present invention;
FIG. 2 is an exploded view of FIG. 1;
FIG. 3 is a schematic diagram of an exemplary structure of an insulating isolation structure;
FIG. 4 is a schematic diagram of an exemplary structure of an insulating isolation structure (with insulating spacers removed);
FIG. 5 shows another exemplary structural schematic of an insulating isolation structure;
FIG. 6 is a schematic view of an exemplary manufacturing apparatus of the present invention partially broken away;
FIG. 7 is a schematic view of a further exemplary manufacturing apparatus of the present invention shown partially in section;
FIG. 8 is a schematic view of a further exemplary manufacturing apparatus of the present invention shown partially in section;
fig. 9 is a schematic structural view showing a further exemplary manufacturing apparatus of the present invention;
fig. 10 is a schematic view showing the structure of another exemplary manufacturing apparatus of the present invention;
FIG. 11 shows the effect of a prior art device for GV preparation in aqueous solution (observed by fluorescence microscopy);
FIG. 12 is a graph showing the effect of the prior art device for GV preparation in physiological saline solution (observed by fluorescence microscopy);
FIG. 13 is a graph showing the effect of the production apparatus of the present invention on GV production in an aqueous solution (observed by a fluorescence microscope);
FIG. 14 is a graph showing the effect of GV produced by the production apparatus of the present invention in physiological saline (observed by a fluorescence microscope).
The reference numerals in the embodiments include:
a first electrode plate 110, a second electrode plate 120;
insulation plate 200, hollow groove 201, first preparation chamber 201a, second preparation chamber 201b, lower insulation portion 210, left insulation portion 220, sharp corner 221, right insulation portion 230, insulation seal 240, groove 241;
an insulating spacer 300;
lipid layer 400.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the invention may be practiced without one or more of these details. In other instances, well-known features have not been described in detail in order to avoid obscuring the invention.
It should be understood that the present invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In the drawings, like numbers refer to like elements throughout.
Referring to fig. 1 to 10 in combination, the preparation device of the lipid vesicle of the present invention comprises at least one group of electrode groups, each group of electrode groups comprising a first electrode plate 110 and a second electrode plate 120, an electric field space is formed between the first electrode plate 110 and the second electrode plate 120, the chambers are all in the corresponding electric field spaces, at least one preparation chamber (201 a, 201b in the drawings) for containing a preparation solution is arranged in each chamber, the insulating isolation structure is used for enabling the preparation solution in a single preparation chamber to be in contact with only one electrode plate of the first electrode plate 110 and the second electrode plate 120, the region corresponding to the preparation chamber on the electrode plates is covered with a lipid layer 400, and the region serving as the inner wall of the preparation chamber on the electrode plates is referred to herein as the "corresponding region". The preparation solution may be water, an ionic solution, or the like.
In fig. 1, fig. 2, fig. 3, and fig. 6, the number of the chambers is two, and both the two chambers (i.e. 201a and 201 b) are preparation chambers, in the practical implementation process, referring to fig. 8, the number of the preparation chambers may be one, referring to fig. 5 and fig. 7, and the number of the preparation chambers may also be multiple; in addition, in the practical implementation process, all the chambers may be preparation chambers, or only part of the chambers may be preparation chambers, for example, in fig. 7, three chambers are disposed between the first electrode plate 110 and the second electrode plate 120, but only the chamber adjacent to the first electrode plate 110 (i.e. 201 a) and the chamber adjacent to the second electrode plate 120 (i.e. 201 b) are preparation chambers.
When the lipid vesicles are prepared by using the preparation apparatus described above and below, the lipid layer 400 is formed so as to cover the area of each electrode plate used as the cavity wall of the preparation chamber, and then the preparation solution is added into the preparation chamber; and the signal generator is connected to the first electrode plate 110 and the second electrode plate 120, so that the polarities of the first electrode plate 110 and the second electrode plate 120 are opposite. The first electrode plate 110 and the second electrode plate 120 form the electric field space after being electrified, and preparation is started, and the signal generator can adopt a current source, a voltage source and the like.
In a conventional manufacturing apparatus, there is only one chamber (also a manufacturing chamber), and both the first electrode plate and the second electrode plate are used to form the walls of the chamber. Fig. 11 and fig. 12 show graphs of the effects of the conventional apparatus for producing lipid vesicles GV in an aqueous solution and a physiological saline solution, and fig. 13 and fig. 14 show graphs of the effects of the apparatus for producing lipid vesicles GV in an aqueous solution and a physiological saline solution, respectively. Comparing fig. 11 and 13, it can be seen that when GV is prepared by the preparation device of the present invention and GV is prepared by the existing device, if the solution in the preparation chamber is an aqueous solution, the GV yields are equivalent; comparing fig. 12 and 14, it can be seen that when the GV is prepared by the preparation device of the present invention and the GV is prepared by the existing device, if the solution in the preparation chamber is a physiological saline solution, the GV cannot be formed by the conventional preparation device, and more lipid vesicles GV can be formed by the preparation device of the present invention.
In some embodiments, referring to fig. 6 and 7, the types of preparation chambers include a first preparation chamber 201a and a second preparation chamber 201b, wherein a part of a cavity wall of the first preparation chamber 201a is surrounded by the first electrode plate 110, and an insulating isolation structure separates the first preparation chamber 201a and the second electrode plate 120; a portion of the cavity wall of the second preparation chamber 201b is surrounded by the second electrode plate 120, and an insulating isolation structure separates the second preparation chamber 201b from the first electrode plate 110. In practical implementation, the type of preparation chamber may also be one of the first preparation chamber 201a or the second preparation chamber 201b, for example, in fig. 8, only the second preparation chamber 201b is provided.
In some embodiments, referring to fig. 1 to 4 and 6 to 8 in combination, the insulating isolation structure includes an insulating plate 200, and the first electrode plate 110, the insulating plate 200, and the second electrode plate 120 are sequentially stacked, wherein a hollow groove 204 is provided on the insulating plate 200, at least one insulating partition 300 is provided in the hollow groove 201, and the insulating partition 300 partitions the hollow groove 204 and forms at least one preparation chamber. Of course, in actual implementation, the insulating plate may be replaced by an insulating block, and in actual implementation, the insulating spacer 300 may be integrally formed in the hollow groove 201, or may be fixed in the hollow groove 201 by bonding or the like.
In some embodiments, referring to fig. 6, the number of insulating spacers 300 is one, the walls of the hollow groove 201, the insulating spacers 300 and the first electrode plate 110 enclose a first preparation chamber 201a, and the walls of the hollow groove 201, the insulating spacers 300 and the second electrode plate 120 enclose a second preparation chamber 201b.
In other embodiments, referring to fig. 7, the number of insulating spacers 300 is at least two, and the walls of the hollow groove 201, the first electrode plate 110, and the insulating spacer 300 nearest to the first electrode plate 110 enclose a first preparation chamber 201a; the walls of the hollow groove 201, the second electrode plate 120, and the insulating separator 300 nearest to the second electrode plate 120 enclose a second preparation chamber 201b.
In still other embodiments, referring to fig. 8, the number of insulating spacers 300 is one, and the insulating spacers 300 are abutted against the first electrode plate or the second electrode plate to form one of the second preparation chamber or the first preparation chamber.
In still other embodiments, the insulating plate includes a first insulating plate and a second insulating plate, the first electrode plate, the first insulating plate, the second insulating plate and the second electrode plate are sequentially stacked (not shown), hollow grooves are formed in the first insulating plate and the second insulating plate, insulating partition plates are arranged in the hollow grooves, the hollow grooves of the first insulating plate are divided to form a first preparation chamber, the hollow grooves of the second insulating plate are divided to form a second preparation chamber, and the first preparation chamber and the second preparation chamber are separated by the insulating partition plates.
In some embodiments, referring to fig. 3, the preparation device of the lipid vesicle further comprises an insulating seal 240, the first preparation chamber 201a and the second preparation chamber 201b each form a liquid injection opening therethrough from one side of the insulating plate 200, and the insulating seal 240 is used to seal the liquid injection opening, so that the preparation chambers are sealed. The provision of the opening facilitates the injection of the preparation solution into the preparation chamber, and in actual practice, the insulating sealing member 240 may be fixed to the insulating plate 200 using an adhesive or a specific structure.
In some embodiments, referring to fig. 3, an anti-slip structure for preventing the sealing member from sliding relative to the insulating plate 200 is provided between the insulating plate 200 and the sealing member, which is beneficial to improving the structural reliability of the whole device in the process of preparing the vesicle.
Specifically, for example, in fig. 3, the bottom of the hollow groove 201 of the insulating plate 200 is the lower insulating portion 210, the left insulating portion 220 and the right insulating portion 230 are respectively disposed on two sides of the hollow groove 201, the insulating seal 240 is disposed above the hollow groove 201, the butt joint position of the insulating seal 240 and the left insulating portion 220 and the butt joint position of the insulating seal 240 and the right insulating portion 230 are respectively provided with a concave-convex butt joint structure, in fig. 3, the upper ends of the left insulating portion 220 and the right insulating portion 230 are respectively provided with a sharp angle 221, and the bottom of the insulating seal 240 is correspondingly provided with a groove 241 in which the sharp angle 221 is embedded.
In some embodiments, referring to fig. 1-3, the number of electrode sets is one set; in practical implementation, the number of electrode groups may be at least two, and the electrode groups may be stacked, for example, in fig. 9, in two adjacent electrode groups, the first electrode plate 110 of one electrode group and the first electrode plate 110 of the other electrode group are integrally disposed, or in the manner of fig. 10, two adjacent electrode groups may share the first electrode plate 110, and of course, the electrode plate that is shared or integrally disposed may also be the second electrode plate 120.
In the practical implementation process, the electrode plate can be an opaque conductive plate or a transparent conductive plate, preferably a transparent conductive plate, and the whole preparation device has only one group of electrode groups, and in the preparation process, the reaction process can be observed through the transparent conductive plate. For example, each electrode can be made of ITO conductive glass plates, so that an electric field space can be formed, and the observation requirement can be met.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (10)
1. A device for preparing lipid vesicles, comprising:
at least one group of electrode groups, each group of electrode groups comprises a first electrode plate and a second electrode plate, and an electric field space is formed between the first electrode plate and the second electrode plate;
the chambers are all positioned in corresponding electric field spaces, and at least one preparation chamber for containing preparation solution is arranged in each chamber;
an insulating isolation structure for contacting the preparation solution in the single preparation chamber with only one of the first electrode plate and the second electrode plate.
2. The apparatus for producing lipid vesicles according to claim 1 wherein: the types of the preparation chambers comprise one or two of a first preparation chamber and a second preparation chamber;
a part of cavity walls of the first preparation cavity are surrounded by the first electrode plate, and the insulating isolation structure separates the first preparation cavity from the second electrode plate;
and part of the cavity wall of the second preparation cavity is surrounded by the second electrode plate, and the insulating isolation structure separates the second preparation cavity from the first electrode plate.
3. The apparatus for producing lipid vesicles according to claim 2 wherein: the insulation isolation structure comprises an insulation plate, and the first electrode plate, the insulation plate and the second electrode plate are sequentially stacked; the insulating plate is provided with a hollow groove, at least one insulating partition plate is arranged in the hollow groove, and the insulating partition plate divides the hollow groove and forms at least one preparation chamber.
4. The apparatus for producing lipid vesicles as claimed in claim 3 wherein:
the number of the insulating partition boards is one, the groove walls of the hollow grooves, the insulating partition boards and the first electrode plates are enclosed to form a first preparation chamber, and the groove walls of the hollow grooves, the insulating partition boards and the second electrode plates are enclosed to form a second preparation chamber;
or (b)
The number of the insulating partition plates is one, and the insulating partition plates are abutted against the first electrode plate or the second electrode plate to form a second preparation chamber or one of the first preparation chambers;
or (b)
The number of the insulating partition plates is at least two and more, and the first preparation chamber is defined by the wall of the hollow groove, the first electrode plate and the insulating partition plate nearest to the first electrode plate; the wall of the hollow groove, the second electrode plate and the insulating partition plate closest to the second electrode plate enclose the second preparation chamber.
5. The apparatus for producing lipid vesicles as claimed in claim 3 wherein: the insulation board includes first insulation board and second insulation board, first electrode plate first insulation board the second insulation board with the second electrode plate stacks gradually, all be provided with on the first insulation board with on the second insulation board hollow groove, each hollow inslot all is provided with insulating barrier, makes the hollow groove of first insulation board is cut apart and is formed first preparation cavity, the hollow groove on the second insulation board is cut apart and is formed the second preparation cavity, makes first preparation cavity with the second preparation cavity is separated by insulating barrier.
6. The apparatus for producing lipid vesicles according to any one of claims 3 to 5 wherein: the insulating sealing piece is used for sealing the liquid injection opening, and the preparation cavity is sealed.
7. The apparatus for producing lipid vesicles according to claim 1 wherein:
the number of the electrode groups is one group;
or (b)
The number of the electrode groups is at least two, each electrode group is arranged in a lamination way, and two adjacent electrode groups share a first electrode plate or a second electrode plate;
or (b)
The number of the electrode groups is at least two, each electrode group is arranged in a lamination way, and two first electrode plates of two adjacent electrode groups are integrally arranged or two second electrode plates of two adjacent electrode groups are integrally arranged.
8. The apparatus for producing lipid vesicles according to claim 1 wherein:
the electrode plate is an opaque conductive plate;
or (b)
The electrode plate is a transparent conductive plate.
9. The apparatus for producing lipid vesicles according to claim 1 wherein: the electrode plate is an ITO conductive glass plate.
10. A method for preparing lipid vesicles, which is characterized in that: the lipid vesicle production apparatus according to any one of claims 1 to 6, wherein the lipid vesicle production apparatus is used for producing the lipid vesicle, and the production method comprises:
forming a lipid layer such that the lipid layer covers a region of each electrode plate serving as a cavity wall of the preparation chamber;
adding a preparation solution into the preparation chamber;
and connecting a signal generator to the first electrode plate and the second electrode plate to enable the polarities of the first electrode plate and the second electrode plate to be opposite.
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