CN117779058A

CN117779058A - Preparation method and device of amphiphilic molecular layer

Info

Publication number: CN117779058A
Application number: CN202311813882.9A
Authority: CN
Inventors: 杨秉达; 李梓琦; 赵长健
Original assignee: Guangzhou Kongque Gene Technology Co ltd
Current assignee: Guangzhou Kongque Gene Technology Co ltd
Priority date: 2022-04-14
Filing date: 2022-04-14
Publication date: 2024-03-29
Also published as: CN114807982B; CN114807982A

Abstract

The invention relates to a preparation method and a device of an amphiphilic molecular layer, wherein the method comprises the steps of providing a preparation device for preparing the amphiphilic molecular layer, wherein at least one micropore and a first channel and a second channel which can enable a solution to flow through the micropore are arranged in the preparation device, and the first end and the second end of the micropore are respectively communicated with the first channel and the second channel, wherein the cross section of the first channel is square or square-like; s2, adding a first polar solution into the second channel, so that the first polar solution enters at least one micropore; s3, sequentially adding a film solution and a second polar solution into the first channel, so that the film solution forms a meniscus in the first channel; s4, adding a second polar solution into the first channel based on a preset speed, so that the second polar solution pushes the membrane solution to move and flow through the micropores, and the membrane solution forms an amphiphilic molecular layer at the micropores; wherein the first channel material has a surface contact angle of about 65 ° to about 120 °. The method is simple to operate and good in repeatability.

Description

Preparation method and device of amphiphilic molecular layer

The application is a divisional application of Chinese patent application based on application number 202210436809.3 and application date 2022, 04 and 14, and the invention name is 'a preparation method and a preparation device of an amphiphilic molecule layer'.

Technical Field

The invention relates to the field of amphiphilic molecular films, in particular to a preparation method and a device of an amphiphilic molecular layer.

Background

The nanopore protein needs to be stably embedded in a phospholipid or polymer film, such as an amphiphilic molecular layer (namely an amphiphilic molecular film), so that a DNA sequence to be detected can pass through, and the aim of detecting the DNA sequence is fulfilled. However, some existing preparation devices and corresponding methods for preparing amphiphilic molecule layers still have some problems in application.

The existing film forming methods mainly include a folding double layer forming method (Montal & Mueller method), a tip dipping method, a coating method, a patch clamp method, a water-in-oil droplet interface method, and the like. The above methods such as folding double layer method, tip dipping method, coating double layer method, etc. tend to form a thick film for the first time, and require thinning treatment such as evaporation of an organic solvent, or spreading by physical application, or pressing by air pressure, etc. The thinning process is important for control of film thickness, but the operation process is complicated, and the thinning step is difficult to control (for example, it is difficult to ensure uniform spreading during physical application). The droplet interface rule often needs to form droplets one by one, and needs to control the volume, the position and the like of the droplets, so that the operation has certain difficulty, and when a plurality of amphiphilic molecule layers need to be formed, a plurality of droplets need to be formed first, and the operation is complex. Meanwhile, the existing film forming methods generally involve pretreatment of preparation devices, such as fluorine plasma treatment, silanization treatment, etc., which require high-risk chemicals for the pretreatment operations, and are carried out in laboratories with high construction costs and high maintenance costs. There is a potential threat to the life safety of the operator and the surrounding environment. In addition, the pretreatment also needs to accurately calculate the used paint dosage, and too little paint can affect the test effect. The preparation device of the amphiphilic molecular film is usually a small chip, and the size of the internal structure (such as micropores and the like) is tiny, so that the amount of the selected paint is very small, and the operation of smearing a very small amount of paint on the tiny structure is not simple, so that the problems of too much and too little smearing are difficult to avoid in the smearing process.

For example, as disclosed in chinese patent application No. 201480056839.5, the invention discloses a film forming method for a biochip, the film forming method comprising the steps of: a liquid comprising lipid molecules (i.e. amphiphilic molecules) is added to the chip surface, which is then separated by air bubbles, such that the lipid molecules are distributed over the chip surface and the lipids are thinned by the air bubbles. However, the bubble generation often requires manual operation control (such as preparation of bubbles by a pipette), and this process is difficult to automatically control, and the size of bubbles is difficult to control by manual operation, and the stability of the bubble morphology is difficult to ensure, so that the repeatability of the method is also poor. And the method also requires pretreatment.

For another example, chinese invention of application No. CN200880126160.3 discloses a method of forming a layer separating two volumes of aqueous solution by flowing an aqueous solution containing amphiphilic molecules through a body to cover grooves (i.e., micropores) so that the aqueous solution can form amphiphilic molecules across the grooves, although the technique of flowing the aqueous solution through the grooves to form amphiphilic molecules is easily achieved, the film produced is thicker, requiring a subsequent thinning treatment, and the method involves a pretreatment step.

In addition, there are some problems in the existing preparation apparatuses on the market, for example, in order to achieve mass production and ensure uniformity of the tank body (i.e. a flow channel for adding the solution sample) (i.e. reduce production errors), a casting process (i.e. through a mold) is often selected to manufacture the tank body, so that in order to meet more experimental requirements, the internal structure of the tank body is generally set larger, and thus, when some experiments only need a small amount of solution sample, unnecessary waste is caused to the sample. If the waste of the sample is to be avoided, sample grooves with different sizes need to be prepared according to different experimental requirements (different sequencing experiments need different volumes and sizes of groove bodies), so that corresponding molds (namely, molds need to be opened for each production) need to be prepared according to the sample grooves with different sizes, and the cost is high in the industrial production process.

Disclosure of Invention

In order to partially solve or partially alleviate the above technical problems, the present invention provides a method for preparing an amphiphilic molecule layer, including:

s1, providing a preparation device for preparing an amphiphilic molecular layer, wherein at least one micropore for providing a growth platform for the amphiphilic molecular layer and a first channel and a second channel capable of enabling a solution to flow through the micropore are arranged in the preparation device, and a first end and a second end of the micropore are respectively communicated with the first channel and the second channel, wherein the cross section of the first channel is square or square-like;

S2, adding a first polar solution into the second channel, so that the first polar solution enters at least one micropore;

s3, sequentially adding a film solution and a second polar solution into the first channel, so that the film solution forms a meniscus in the first channel;

s4, adding a second polar solution into the first channel based on a preset speed, so that the second polar solution pushes the membrane solution to move and flow through the micropores, and the membrane solution forms an amphiphilic molecular layer at the micropores;

wherein the contact angle of the inner surface of the first channel is about 65 ° to about 120 °.

In some embodiments, the film solution comprises: a nonpolar solution, and an amphiphilic molecule.

In some embodiments, the amphiphilic molecule optionally comprises: a phospholipid, or a polymer, or a mixture of a phospholipid and a polymer.

In some embodiments, the first polar solution comprises: an electrolyte, and/or a polyelectrolyte.

In some embodiments, the first polar solution comprises: redox couples, and/or redox couples that may be partially oxidized or reduced to provide a redox couple combination.

In some embodiments, the first polar solution comprises: crosslinked agarose gel, and/or crosslinked sodium alginate gel.

In some embodiments, the first polar solution comprises: buffers for adjusting the pH.

In some embodiments, the second polar solution comprises: an electrolyte, and/or a polyelectrolyte.

In some embodiments, the second polar solution comprises: redox couples, and/or redox couples that may be partially oxidized or reduced to provide a redox couple combination.

In some embodiments, the second polar solution comprises: crosslinked agarose gel, and/or crosslinked sodium alginate gel.

In some embodiments, the second polar solution comprises: buffers for adjusting the pH.

In some embodiments, in S3, the second polar solution is injected at a rate of about 5 μl/min to about 10 μl/min;

in some embodiments, in S4, the preset speed of the second polar solution is about 10 μl/min to about 20 μl/min.

In some embodiments, the method further comprises the step of:

inserting a first electrode and a second electrode into the first channel and the second channel, respectively, and adding a protein solution into the first channel and/or the second channel, the protein solution comprising: a nanopore protein;

And applying voltage to the amphiphilic molecule layer through the first electrode and the second electrode to insert the nanopore protein.

The invention also provides a preparation device of the amphiphilic molecule layer, which comprises the following steps: a microwell for providing a growth platform for the amphiphilic molecule layer, and a first channel and a second channel capable of allowing a solution to flow through the microwell, and a first end and a second end of the microwell being in communication with the first channel and the second channel, respectively;

wherein the cross section of the first channel is square or square-like, and the inner surface material of the first channel is optionally: polyoxymethylene, polytetrafluoroethylene, polymethyl methacrylate, epoxy resin, polycarbonate.

In some embodiments, the first channel comprises: a first flow path, the preparation device comprising:

the first flow channel is arranged on the first surface of the first structural layer, and two ends of the first flow channel are respectively provided with a first opening and a second opening, wherein the first opening is used for sample adding, and the second opening is used for sample discharging;

the second channel is arranged on the first surface of the second structural layer, and a third opening is formed in the second channel and used for sample adding;

A third structural layer, at least one of the micropores being disposed on the third structural layer;

the first, third and second structural layers are sequentially and tightly connected, so that the first runner is matched with the first surface of the second structural layer to form a first channel for solution to flow, and the first end and the second end of the micropore are respectively communicated with the first channel and the second channel.

In some embodiments, the first surface of the third structural layer is further provided with a groove, and the micropores are disposed on the groove, and when the first structural layer and the third structural layer are tightly connected, the groove and the first flow channel cooperate together to form a first channel for the solution to flow.

In some embodiments, the microwells have an inner diameter between 100 microns and 200 microns.

In some embodiments, the first opening and/or the second opening are provided in a top-down size.

The beneficial technical effects are as follows:

the invention provides a preparation method (namely a film forming method) and a preparation device for preparing an amphiphilic molecular layer (or an amphiphilic molecular film, also called as a film for short). Specifically, the present invention proposes a novel film forming method that causes a film solution to form a meniscus in a first channel, and pushes the meniscus by a polar solution (e.g., a second polar solution), thereby enabling the meniscus to move in the first channel and pass through micropores, and then forming an amphiphilic molecular layer (film) on the corresponding micropores.

Specifically, the preparation device provided by the invention selects a material with certain hydrophobicity, namely the contact angle of the material on the inner surface of the first channel in the preparation device is about 65-120 degrees, so that the film solution added into the first channel can form a meniscus under the combined action of the inner surface of the first channel, the polar solution (second polar solution) and the air in the first channel. The cross section of the first channel is preferably square or square-like, at this time, the first channel generates a certain resistance to the movement of the film solution (or the meniscus formed by the film solution), so that the movement speed of the film solution in the first channel is not too fast, and the movement speeds of the positions on the film solution are relatively uniform (or the differences of the flow speeds of the positions have little influence on the stability of the meniscus), so that the meniscus can maintain a stable form during the movement.

The film forming mode that the meniscus (namely the film solution) is pushed by the polar solution to form a film at the micropores can control the moving speed of the film solution more accurately (for example, the injection speed of the polar solution is controlled by a pipette or a syringe pump, so that the moving speed of the film solution is controlled), thereby enabling the residence time of the film solution at the micropores to be controllable and avoiding the film solution from staying for too long in the micropores to form a thick film or moving too fast in the micropores to form a film successfully. Therefore, the invention can directly prepare the amphiphilic molecular layer with the film thickness meeting the use requirement by precisely controlling the moving speed of the film solution, and thinning treatment (such as high-voltage breakdown and multiple film forming) of the film after film forming is not needed. In other words, the film forming method of the present invention has better controllability (as compared with the bubble extrusion method of the prior art), and can form a film at one time.

Therefore, in the practical application scenario, after the parameters such as the concentration of the film solution, the moving speed and the like are selected (for example, determined by pre-experiment or by operation experience of a worker), multiple experiments can be performed based on the same experimental conditions and parameters, and the results (for example, film forming conditions) obtained by the multiple experiments have small difference, that is, the repeatability of the method is better. In other words, the method provided by the invention can avoid or reduce the influence of manual operation of staff on experimental operation, so as to avoid or reduce uncontrollable factors in the operation process (namely, the method has better controllability), thereby having better repeatability.

In addition, as the inner surface of the first channel and the inner surface of the micropore of the preparation device are made of materials with certain hydrophobicity (such as polyformaldehyde), the film forming method provided by the invention does not need pretreatment, is simpler, and ensures the safety of workers in the operation process (the operation and the use of dangerous goods are not involved).

Furthermore, the injection speed of the polar solution can be controlled by the injection pump (or other devices capable of controlling the injection speed of the solution), and the injection pump can be automatically controlled by electronic equipment (such as a computer, etc.) (namely, the film forming method provided by the invention can realize automatic control), so that the manual operation steps of staff can be further reduced, and correspondingly, errors possibly caused by the manual operation of the staff are avoided, so that the stability and uniformity of film forming are further ensured.

The film forming method and the device provided by the invention can be applied to various detection requirements in a laboratory (such as application scenes of different scientific research institutions, commercial detection companies and the like), and further, the film forming method and the device provided by the invention are preferably applied to application scenes with small data volume (such as test research and development stage in which only a small amount of amphiphilic molecule layers are formed at one time).

It can be understood that the method and the device provided by the invention can be matched with the existing automatic control to realize the automation of the flow, thereby further precisely controlling the operation flow and improving the accuracy and the reliability of the experimental result. However, when there is less demand for data (e.g., when only one or a few amphiphilic molecule layers are needed), the use cost of automated operations is relatively high. At this time, the time and the cost are comprehensively considered, and the manual operation is more suitable for actual demands. The method provided by the invention has simple flow, can form a film once by controlling the moving speed of the film solution (with better controllability), does not need to pretreat the device or thin the film, and reduces the operation difficulty and the film forming time. Therefore, even though the whole process is manually operated by a worker, the film with proper thickness can be prepared, and the technical effect which is the same as or similar to the automatic control can be achieved.

Further, unlike the prior art, the preparation device of the present invention is provided with channels (i.e., a first channel and a second channel) for introducing a solution at both ends of the microwells. Therefore, the preparation device can flexibly insert electrodes at two sides of the micropore without presetting the electrodes at the micropore (in the prior art, the electrodes are required to be processed and arranged on a small chip, the production process is complex), and the production process of the preparation device is simpler and the production cost is lower. Meanwhile, the first channel and the second channel are respectively arranged at the two ends of the micropore, so that the inside of the device (such as the micropore) and the electrode can be cleaned more conveniently, for example, cleaning liquid is respectively introduced into the first channel and the second channel, so that the cleaning of the first end and the second end of the micropore can be realized, the cleaning maintenance of the inside of the device is realized, and the service life of the device is prolonged (meanwhile, the subsequent experiment is not comprehensively influenced by cleaning is avoided).

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. Like elements or portions are generally identified by like reference numerals throughout the several figures. In the drawings, elements or portions thereof are not necessarily drawn to scale. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the invention and that other drawings may be derived from these drawings without inventive faculty.

FIG. 1 is a top view of a manufacturing apparatus in an exemplary embodiment of the invention;

FIG. 2 illustrates a schematic cross-sectional structure of first, second, and third structural layers in an exemplary embodiment of the present invention;

fig. 3 is a schematic cross-sectional structure of a manufacturing apparatus in still another exemplary embodiment of the present invention;

FIG. 4 is a schematic cross-sectional view of a first channel and a third channel in accordance with yet another exemplary embodiment of the present invention;

FIG. 5a shows the relationship between contact angle and solid, liquid, gas;

FIG. 5b shows a process of liquid wetting a capillary;

FIG. 5c is a schematic view of a first configuration of the meniscus within the flow channel;

FIG. 6 is a schematic structural diagram of an amphiphilic molecule;

FIG. 7a is a schematic diagram of a process for forming an amphiphilic membrane;

FIG. 7b is a schematic structural diagram of an amphiphilic membrane;

fig. 8 is a first perspective view of a manufacturing apparatus in another exemplary embodiment of the present invention;

fig. 9 is a schematic structural view of a manufacturing apparatus in another exemplary embodiment of the present invention;

fig. 10 is a second perspective view of a manufacturing apparatus in another exemplary embodiment of the present invention;

fig. 11 is a third perspective view of a manufacturing apparatus in another exemplary embodiment of the present invention;

fig. 12 shows a schematic structural view of films of different thicknesses formed at micropores.

1 is a first structural layer, 11 is a first channel, 11a is a first section, 11b is a second section, 12 is a first opening, 13 is a second opening, 14 is a first flow channel, 2 is a second structural layer, 21 is a second channel, 22 is a third opening, 23 is a fourth opening, 3 is a third structural layer, 31 is a micropore, 32 is a groove, 4 is an amphiphilic molecule, 41 is a hydrophobic end, 42 is a hydrophilic end, 5 is a membrane solution (meniscus), 6 is a polar solution, and 7 is air.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In this document, suffixes such as "module", "component", or "unit" used to represent elements are used only for facilitating the description of the present invention, and have no particular meaning in themselves. Thus, "module," "component," or "unit" may be used in combination.

The terms "upper," "lower," "inner," "outer," "front," "rear," "both ends," "one end," "the other end," and the like herein refer to an orientation or positional relationship based on that shown in the drawings, merely for convenience of description and to simplify the description, rather than to indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be construed as limiting the invention. Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

The terms "mounted," "configured," "connected," "coupled," and the like, herein, are to be construed broadly as, for example, "connected," either permanently connected, detachably connected, or integrally connected, unless otherwise specifically indicated and defined; can be mechanically or electrically connected; the wireless communication connection can be adopted; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.

As used herein, an "amphiphilic" molecule refers to a compound that has both hydrophilic and lipophilic properties, with a hydrophilic head portion and a hydrophobic tail portion. Hydrophilic heads (i.e., hydrophilic ends) generally consist of polar groups, such as choline, ammonium salts; the hydrophobic tail (i.e., hydrophobic end) generally consists of a long fatty chain. Herein, "amphiphilic" and "amphipathic" are used synonymously. The amphiphilic molecule may be a lipid molecule. A typical amphiphilic layer (or amphiphilic membrane, also referred to simply as a "membrane") may be a lipid bilayer that is a bilayer formed of two opposing lipid monolayers that are self-assembled so that the hydrophobic tails face each other to form a hydrophobic interior, while the hydrophilic heads face the exterior (polar hydrophilic environment on each side). The lipid forming the lipid bilayer may comprise any suitable lipid, for example 1, 2-biphytoyl-sn-glycero-3-phosphatidylcholine, di-phytoyl phosphatidylcholine (DPhPC). Amphiphilic molecules may be chemically modified or functionalized to facilitate coupling of polynucleotides. The amphiphilic molecules may also be polymeric materials synthesized by physicochemical means, such as ABA triblock copolymers (PMOXA-PDMS-PMOXA dimethyl oxazoline-polydimethylsiloxane-dimethyl oxazoline). The amphiphilic molecules may be a mixture.

As used herein, "non-polar solvent" or "non-polar solution" refers to a compound or mixture of compounds that is not miscible with water. The non-polar solvent may be an oil, more specifically, may be a pure alkane such as n-hexadecane, n-decane, n-pentane, n-hexane, n-heptane, n-octane, carbon tetrachloride. Other types of oils are also possible, for example, silicone oils. More specifically, the oil may be methylphenyl silicone oil AR20, hydroxy-terminated polydimethylsiloxane PDMS-OH.

As used herein, "polar aqueous solution" or "polar solution" refers to an aqueous solution containing water that is readily miscible with water and other polar solvents. The polar aqueous solution may include one or more solutes. For example, buffers capable of adjusting the pH of the polar aqueous solution may be included. The buffer may include any suitable buffer, such as Phosphate Buffer (PBS), 4-bis-2-ethanesulfonic acid buffer PIPES, N-2-hydroxyethylpiperazine-N' -ethanesulfonic acid buffer (HEPES). The polar aqueous solution may also be an electrolyte or polyelectrolyte to effectively enhance ion exchange lifetime. The polar aqueous solution may also contain a redox pair or a combination of redox pairs that may be partially oxidized or reduced to provide a redox pair, such as iron/ferricyanide. The polar aqueous solution may also be a cross-linked agarose gel or sodium alginate gel.

As used herein, "self-assembly" refers to the ability of a molecule to spontaneously assemble or organize in a suitable environment to form a highly ordered structure, such as a membrane of amphiphilic molecules.

Herein, "square" includes polygons having adjacent side angles of about 90 ° or nearly 90 °, for example, squares are quadrilaterals, such as squares, rectangles, or the like. Of course, the "square" herein does not necessarily need to be set as a standard square or rectangle or other polygon, for example, opposite sides of the "quadrangle" in the square may be set in parallel or may be set in non-parallel, and accordingly, adjacent sides of the "quadrangle" may be set in mutually perpendicular or may be set in non-perpendicular. The "square-like" includes a square with a chamfering process at the angle between adjacent sides, or a square with adjacent sides connected by an arc, for example, a square-like shape such as a rectangle (the top corner of the rectangle is replaced with an arc structure), a square-like shape (the top corner of the square is replaced with an arc structure), or the like. For example, herein, "the cross section of the flow channel is square or square-like" means that the cross section of the flow channel may be set to be rectangular, square, rectangular-like, square-like, or the like. In order to avoid the occurrence of burrs and the like on the inner surface of the runner during the processing, it is preferable to set the cross section of the runner to be square-like, such as rectangular-like, square-like, or the like.

The terms "about", "approximately" are used herein to denote +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, even more typically +/-0.5% of the value, or values that are understood by those of skill in the art to include ranges of error that are conventional in the art.

Certain embodiments may be disclosed herein in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered as having specifically disclosed all possible subranges and individual numerical values within that range, e.g., a contact angle of "about 65 degrees to about 120 degrees" is to be understood as having disclosed the following ranges: contact angles between about 65-95 degrees, 95-105 degrees, 105-120 degrees, etc., are also disclosed, as are individual numerical values within this range, such as 65 degrees, 69 degrees, 75 degrees, 80 degrees, 90 degrees, 100 degrees, 110 degrees, 120 degrees.

Example 1

In a first aspect of the present invention, there is provided an amphiphilic preparation device comprising: a microwell for providing a growth platform for the amphiphilic molecule layer, and a first channel and a second channel capable of allowing a solution to flow through the microwell, and a first end and a second end of the microwell being in communication with the first channel and the second channel, respectively;

wherein the cross section of the first channel is square or square-like, and the inner surface material of the first channel is optionally: polyoxymethylene, teflon (polytetrafluoroethylene), polymethyl methacrylate, epoxy, polycarbonate, and the like.

In this embodiment, the first and second electrodes can be flexibly inserted into the two ends of the micropore through the first and second channels, so that an electrode layer does not need to be preset in the preparation device (such as near the micropore), and the structure of the preparation device is simpler, and the production cost is greatly reduced. Meanwhile, as the two sides of the micropore are provided with the channels for the solution to flow, the cleaning of the internal structure of the device is more convenient, for example, the first end and the second end of the micropore can be comprehensively cleaned by introducing cleaning liquid (such as distilled water and the like) into the first channel and the second channel.

It is understood that the polarities of the first electrode and the second electrode are opposite.

Further, referring to fig. 1 to 3, the present invention provides an amphiphilic molecular layer preparation apparatus (i.e., preparation apparatus) capable of rapidly forming an amphiphilic molecular layer (i.e., amphiphilic molecular film), comprising:

the first structure layer 1, a first channel 11 is arranged on the first structure layer 1, and a first opening 12 and a second opening 13 are respectively arranged at two ends of the first channel 11, wherein the first opening is used for sample adding (namely adding solution), and the second opening is used for sample discharging (namely discharging redundant liquid) and observing the height of the solution;

a second channel 21 is arranged on the second structural layer 2, and a third opening for sample addition is arranged on the second channel 21;

wherein the first and second structural layers are tightly connected in sequence, and the first channel 11 and the second channel 21 are communicated through at least one micropore.

Specifically, in some embodiments, the micropores are formed by communicating micropores disposed on the first channel 11 with micropores disposed on the second channel (in this case, the first and second channels are disposed inside the first and second structural layers, respectively).

Further, in some embodiments to reduce processing difficulty, the preparation apparatus further includes: a third structural layer 3, and the micropores 31 are disposed on the third structural layer, and penetrate through the first surface (i.e., the side connected to the first structural layer) and the second surface (i.e., the side connected to the second structural layer) of the third structural layer, and accordingly, a first channel is disposed on the first surface of the first structural layer and a second channel is disposed on the first surface (i.e., the side connected to the third structural layer) of the second structural layer; the third structural layer is arranged between the first structural layer and the second structural layer (namely, the first structural layer, the third structural layer and the second structural layer are sequentially and tightly connected), and the first channel and the second channel are communicated through the micropores.

Specifically, in some embodiments, referring to fig. 2, the first channel includes: a first flow passage 14, the preparation device comprising:

the first flow channel 14 is arranged on the first surface of the first structural layer, and two ends of the first flow channel are respectively provided with a first opening and a second opening, wherein the first opening is used for sample adding, and the second opening is used for sample discharging;

a second structure layer 2, wherein the second channel 21 is arranged on the first surface of the second structure layer, and a third opening is arranged on the second channel and is used for loading samples;

Further, in some embodiments, the first surface of the third structural layer is further provided with a groove 32, and the micropores 31 are disposed on the groove 32, and when the first structural layer and the third structural layer are tightly connected, the groove and the first flow channel cooperate together to form a first channel through which the solution can flow.

In some embodiments, the microwells have an inner diameter between 100 microns and 200 microns, e.g., the microwells have a first end (the end in communication with the first channel) with an inner diameter size between 100 microns and 200 microns.

Further, in some embodiments, the microwells have an inner diameter between 150 microns and 200 microns, e.g., the first ends of the microwells have an inner diameter size between 150 microns and 200 microns.

It is possible for the person skilled in the art to choose suitable starting materials by preliminary experiments. Specifically, contact angles of different materials can be obtained through a contact angle test method (such as a contour image analysis method or a weighing method) so as to select a proper preparation material. Specifically, a flow channel made of a selected material is considered to have the ability to form a meniscus when the contact angle of the material is about 65 ° -120 ° (of course, as long as the flow channel made of the material is capable of forming a meniscus).

In this embodiment, the processing difficulty of the manufacturing apparatus is reduced by providing the third structural layer. The thickness of the first and second structural layers is also small because the overall volume of the manufacturing device is relatively small. At this time, if the first channel and the second channel are to be arranged inside the first structural layer and the second structural layer, and corresponding micropores are to be formed on the first channel and the second channel, the processing difficulty is high. The third structural layer is additionally arranged, and the micropores are formed in the third structural layer, so that on one hand, the first and second channels can be directly arranged on the surfaces of the first and second structural layers, and the processing difficulty is greatly reduced; on the other hand, the third structural layer is also simpler to directly form micropores than the first channel or the second channel. Specifically, after the first, third and second structural layers are tightly connected in sequence, the first runner and the second channel are respectively matched with the first surface and the second surface of the third structural layer to form two channels inside the preparation device, namely the first and second channels achieve the effect of being arranged inside the first and structural layers, and the third structural layer plays a role in sealing the first and second channels.

In practical applications, the amount of sample (i.e., the volume of the membrane solution) required for use in different experiments is not the same. In order to avoid wasting the sample, the size of the first channel needs to be correspondingly set according to the experimental scenes of different sample usage amounts, so that the method is suitable for experiments of different sample amounts. In this embodiment, since the preparation device may be formed by combining the first, third and second structural layers, the cross section of the first channel may be flexibly adjusted by directly changing the thicknesses of the third structural layer and the groove, so as to prepare preparation devices with different sizes. For example, in the actual production process, the sizes of the first structural layer and the second structural layer do not need to be adjusted, and the first structural layer and the second structural layer can be uniformly processed to improve the production efficiency. At this time, only the third structural layer is subjected to size adjustment, namely, the first structural layer and the second structural layer are respectively matched with the third structural layer with different thickness by processing the third structural layer with different thickness (corresponding to the grooves with different thickness), so that the preparation devices with different sizes (the sizes of the first channels are different) are prepared. In the production process, the preparation device in the embodiment can simply prepare different devices by adjusting the thickness of the third structural layer, and the first, second and third structural layers are simple to process, low in production cost and easier to realize in process.

Of course, in other embodiments, the size of the first channel of the device may be changed during the device manufacturing process by adjusting the size of the first channel on the first structural layer.

Further, in some embodiments, a fourth opening for sample ejection is further provided on the second channel.

Further, in some embodiments, referring to fig. 3, the first channel comprises: the first section 11a and the second section 11b are communicated, wherein the first section 11a is a part of the first channel close to the first opening, and the second section 11b is a part formed by the first flow channel and the groove.

Further, in some embodiments, the first segment has a square or square-like cross section with sides ranging from about 5mm to about 20mm, such as a rectangle; the second section has a square or square-like, e.g., rectangular, cross-section with sides of about 5mm to about 20 mm.

Further, in some embodiments, the depth of the first section (i.e., the length of the first section) is about 5mm to about 10mm.

Likewise, in some embodiments, the depth of the second section (i.e., the length of the first section) is about 5mm to about 10mm.

In some embodiments, the inner diameter of the second section is greater than the inner diameter of the first section, i.e., the cross-sectional area of the second section is greater than the cross-sectional area of the first section.

In this embodiment, since the inner diameter of the first section is smaller than that of the second section, when the film solution is added into the first channel through the first opening, the film solution first enters the first section with smaller inner diameter, and at this time, the inner space of the first channel (i.e. the first section of the first channel) is narrower, the meniscus formed by the film solution is thicker, and the morphology is also relatively stable. Thus, the movement speed of the film solution in the early stage can be set faster. When the film solution gradually moves to the second section, the meniscus formed by the film solution gradually becomes thinner, and at this time, when the difference between the inner diameters of the second section and the first section is larger, the moving speed of the film solution can be properly reduced, so that the stability of the meniscus in the moving process can be ensured.

In some embodiments, if the inner diameter of the second segment is only slightly larger than the inner diameter of the first segment (i.e. the difference between the inner diameters of the first and second segments is relatively small), the meniscus will also change to a lesser extent (or the thinning of the meniscus will be correspondingly slower), and therefore the movement speed of the meniscus may not be adjusted.

Further, the trend of the change from the first segment to the second segment may be set to be slower, such as the inner diameter at the junction of the first segment and the second segment gradually becoming larger from smaller to larger (specifically, along the direction from the first segment to the second segment), so that the film solution can form a thicker and more stable meniscus first, and gradually becomes thinner during the movement in the first channel, and the meniscus can continue to maintain a stable state during the thinning process due to the slower change of the inner diameter from the first segment to the second segment, thereby enabling the meniscus to maintain a relatively stable state during the formation and movement thereof.

Of course, in other embodiments, the first channel may be disposed with equal diameters, i.e., the inner diameters of the first and second sections are the same or similar.

Preferably, in some embodiments, the first channel 11 may be provided in a curved arrangement, such as in a U-shaped channel configuration.

Preferably, in some embodiments, the second channel 12 may be provided in a curved arrangement, such as in a U-shaped channel configuration.

In this embodiment, referring to fig. 3, the first and second channels are all U-shaped grooves, where the first channel is set in a U-shaped groove, so that a worker can control the height and the movement rate of the membrane solution (specifically, observe the height of the membrane solution through the second opening) by controlling the addition amount and the addition rate of the polar solution to the first opening during the experimental operation, thereby controlling the addition amount of the polar solution and helping the worker to determine whether the amphiphilic molecular layer is generated.

In some embodiments, the microwells may be provided in one or two or more. In some embodiments, when the preparation device is in the use state (i.e., the working state), the first, second, third and fourth openings are all located upward. The micropores are arranged in a vertical state or in a nearly vertical state (namely, the included angle between a film formed on the micropores and the horizontal plane is 90 degrees or nearly 90 degrees), and particularly, the axial directions of the micropores and the horizontal plane are parallel or nearly parallel to each other.

In order to more clearly explain the technical scheme provided by the embodiment, the structure of the preparation device is further described by combining an operation method;

adding an amount of a polar solution (e.g., 30 microliters of the polar solution) to the second channel through the third opening 22 such that the level of the polar solution is above the at least one microwell;

1 microliter of a nonpolar solution of an appropriate amount of a membrane solution (e.g., 25mg/mL concentration) or a mixture of an appropriate amount of a nonpolar solution and a nonpolar solution of an appropriate amount of 25mg/mL concentration) is added to the first channel through the first opening 12;

continuing to add an amount of polar solution (e.g., 5 microliters of polar solution) into the first channel through the first opening 12 such that the membrane solution forms a meniscus (specifically, the membrane solution forms a meniscus under the surface tension provided by the polar solution, air (surface tension of the polar solution, interfacial action of liquid and air)) and moves along the internal path of the first channel under the urging of the polar solution and passes through the at least one microwell;

when the meniscus formed by the membrane solution is pushed by the polar solution to pass through the micropores, amphiphilic molecules in the membrane solution form amphiphilic molecule membranes at the micropores.

Wherein, the film forming principle of the amphipathic molecule is as follows:

as shown in fig. 6, amphiphilic molecule 4 comprises a hydrophilic end 42 and a hydrophobic end 41.

As shown in fig. 7a, when the membrane solution 5 (nonpolar solution in which amphiphilic molecules are dissolved) is added to the first channel, the two sides of the membrane solution 5 form a meniscus, and when the membrane solution flows (or passes) through the micropores (specifically, through one end of the micropores), and at this time the other end of the micropores is already provided with polar solution, the hydrophilic end 42 of the amphiphilic molecules 4 in the membrane solution 5 will be directed to and contact with the polar solution, and the hydrophobic end 41 will combine with the hydrophobic end 41 of another layer of amphiphilic molecules 4, thereby forming an amphiphilic molecular membrane at the micropores (as shown in fig. 7 a). By controlling the flow rate of the membrane solution, the amphiphilic molecules 4 cannot be stacked thicker and thicker to cause incapability of embedding holes.

After the amphiphilic molecular film is formed at the microwells in the preparation apparatus, the first electrode and the second electrode may be inserted into one of the openings (first opening or second opening) of the first channel and one of the openings (third opening or fourth opening) of the second channel, respectively, so that the nanopore protein is inserted onto the amphiphilic molecular film (specifically, a protein solution including the nanopore protein may be added into the first channel or the second channel, and then a voltage is applied to the solution using the first and second electrodes) for subsequent DNA sequencing. That is, the electrode can be flexibly inserted into the two ends of the preparation device, so that the electrode layer for conducting is not required to be preset in the preparation device, the structure and the processing technology of the amphiphilic molecular film preparation device are simplified, and the production cost of the preparation device is reduced.

It will be appreciated that the concentration of the membrane solution (i.e. the concentration of the amphiphilic molecules in the membrane solution) added to the preparation device is related to the size of the first channel (i.e. the cross-sectional size of the first channel), when the concentration of the membrane solution is too high, the formed amphiphilic molecule membrane is too thick to be inserted further into the protein later (i.e. the channel is not opened), whereas too low a concentration may not form the amphiphilic molecule membrane.

In some embodiments, the membrane solution is a nonpolar solution containing amphiphilic molecules.

Further, in some embodiments, the amphiphilic molecule may be a phospholipid, may be a polymer, for example, the amphiphilic molecule may be a polymer material synthesized by a physicochemical method, an ABA triblock copolymer (PMOXA-PDMS-PMOXA dimethyl oxazoline-polydimethylsiloxane-dimethyl oxazoline), or a mixture of a phospholipid and a polymer.

Further, in some embodiments, the non-polar solution is an alkane-based organic solvent comprising decane, hexadecane, pentane, or a mixture of two or more of the above organic solvents, and is used to dissolve phospholipids or polymeric triblock copolymers to form a membrane solution.

In some embodiments, the polar solution is an electrolyte during gene sequencing, comprising an electrolyte or polyelectrolyte effective to enhance ion exchange lifetime, and may also comprise a redox pair or a combination of redox pairs that may be partially oxidized or reduced to provide a redox pair, such as iron/ferricyanide; crosslinked agarose gels and sodium alginate gels are also possible; buffers may also be included to adjust the pH of the aqueous medium, suitable buffers include, but are not limited to, phosphate buffered saline, PBS; 4-bis-2-ethanesulfonic acid buffer PIPES, N-2-hydroxyethylpiperazine-N' -ethanesulfonic acid buffer (HEPES), and the like.

Specifically, in some embodiments, the first end (i.e., the upper end, i.e., the end in communication with the first channel) of the microwell has an inner diameter that is greater than the inner diameter of the second end (i.e., the other end of the microwell, in communication with the second channel) of the microwell. In this embodiment, the microwells are designed to be small at the top and large at the bottom, so that there is enough space within the microwells to accommodate the polar solution (e.g., to accommodate the first polar solution that is first introduced into the microwells) without affecting the size of the first end of the microwells (the size of the inner diameter of the first end of the microwells is closely related to the formation of the amphiphilic molecule layer, and thus, the inner diameter of the first end of the microwells is typically fixedly positioned between about 100 microns and 200 microns). Specifically, when the meniscus flows through the microwells, the amphiphilic molecule layer in the meniscus is formed at the narrowest of the microwells, i.e., the amphiphilic molecules self-assemble at the first end of the microwells to form the amphiphilic molecule layer.

In other embodiments, the micropores may be configured in a cylindrical shape (i.e., with equal upper and lower ends), or in a shape with equal upper and lower ends. Of course, the micropores may be formed in other shapes, and all shapes are within the scope of the present invention as long as the micropores are formed in a shape that can successfully form the amphiphilic molecule layer.

It will be appreciated that where the cross-sectional shape of the first channel is constant (the shape of the first cross-section, the dimensions are constant), for a correspondingly shaped first channel, the amount of membrane solution that needs to be added to meet certain requirements for the formation of a meniscus in the first channel, preferably in some embodiments, when the cross-section of the first channel is in a rectangular configuration with sides of about 5mm to about 20mm, the addition of 1 microliter to 5 microliter of membrane solution can form a more desirable meniscus; when the first channel has different numbers of micropores and the number of the amphiphilic molecule membranes to be prepared is different, the concentration requirements on the amphiphilic molecules contained in the membrane solution are also different. For the simple test operation, the mixed solution of amphiphilic molecules and nonpolar solutions with different amounts is usually selected, namely, the membrane solutions with different concentrations are prepared.

Preferably, in some embodiments, the preparation material of the preparation device is selected from materials having certain hydrophobicity and lipophilicity, for example, materials having a contact angle between about 75 ° and about 120 °, such as teflon, delrin (polyoxymethylene), pmma (organic glass, i.e., polymethyl methacrylate), epoxy, PC (polycarbonate), PDMS (polydimethylsiloxane), and the like. In this embodiment, a preparation device may be processed from a raw material having a certain hydrophobicity and lipophilicity (such as polyoxymethylene), so as to meet the requirements of hydrophobicity and lipophilicity at the first channel and the micropores in the test operation process, so that pretreatment operation on the preparation device is not required, and the operation flow is simplified.

In some embodiments, the preparation device may employ a processing manner including: machining, casting, laser machining, electroporation, micro-injection molding, 3d printing, and the like.

Further, in some embodiments, the processing manner of the micro-hole includes: laser machining, electroporation, and the like.

Preferably, in order to ensure that the cross-section of the microwells is circular and that the inner walls are smooth, in some embodiments, the microwells are laser machined.

In some embodiments, referring to fig. 4, the preparation device is internally provided with a first channel 11 and a second channel 21, wherein the first channel is configured as a U-shaped groove flow channel, the inner volume of the first channel is set between about 40 microliters and about 80 microliters, and the first channel with different volumes can be manufactured by using plates with different thicknesses according to actual user requirements.

Preferably, in some embodiments, the second opening 13 (i.e. the sample outlet of the first flow channel) is provided as a horn with a large top and a small bottom.

If the inner diameter of the second opening is smaller, the solution does not directly flow out when flowing out from the second opening, but forms a small liquid drop first, and finally breaks along with the gradual enlargement of the small liquid, and in the breaking process of the small liquid drop, stronger shearing stress is generated on the liquid in the first channel, so that the film forming effect can be influenced. In this embodiment, the solution can be rapidly and smoothly discharged from the second opening by enlarging the inner diameter of the second opening at one end of the surface of the preparation device, so that the formation and breaking processes of the liquid drops are avoided, and adverse effects on the film forming effect are correspondingly avoided.

In other embodiments, the first opening may be configured to be larger and smaller.

Further, in some embodiments, the third and fourth openings may be in a top-down configuration.

Preferably, in some embodiments, the first channel inner diameter L1 in the preparation device is set between about 0.5mm-2.0mm, and the inner diameter of the flow channel portion L2 formed by the first channel and the third channel together is between about 1.0mm-5.0mm, the first end inner diameter of the first opening L3 is sized between about 1.0mm-4.0mm (the inner diameters of the second, third and fourth openings can be seen in the first opening), the length L4 of the third channel (i.e., the portion of the first channel and the third channel that cooperate) is between about 5.0 mm-10.0 mm, the overall height L5 of the preparation device is between about 15 mm-25 mm, and the width L6 of the preparation device is between about 23mm-32 mm.

Preferably, in some embodiments, the location of the micro-holes may be adaptively designed according to the structure of the different first channels, such as the micro-holes being located at a distance of about 1/3 to 2/3L from the bottom end of the first channel, where L is the length of the opening of the first channel from the bottom end. Preferably, the thickness of the first structural layer and the second structural layer together is about 2mm.

Example two

Referring to fig. 8 to 11, the present invention also provides an apparatus for rapidly forming an amphiphilic molecular film, which comprises:

the first structure layer 1, a first channel 11 is arranged on the first structure layer 1, and two ends of the first channel 11 are respectively provided with a first opening 12 and a second opening 13, wherein the first opening is used for sample adding (i.e. adding solution), and the second opening is used for sample discharging (i.e. discharging redundant liquid) and for observing the height of the solution;

wherein the first and second structural layers are sequentially connected, and the first channel 11 and the second channel 21 are communicated through at least one micropore.

Specifically, in some embodiments, when the preparation device is in a use state (i.e., a working state), the micropores are horizontally disposed or nearly horizontally disposed, that is, the amphiphilic molecular membrane formed on the micropores is parallel or nearly parallel to the horizontal plane (for example, the included angle between the plane of the membrane and the horizontal plane is 0 degrees or nearly 0 degrees), and the first channel is located above the second channel.

Specifically, in some embodiments, the second channel is provided with a third opening for loading the sample and a fourth opening for unloading the sample, respectively.

Further, in some embodiments, the first structural layer and the second structural layer are sequentially arranged from top to bottom, so that the first channel is located above the second channel, and the first opening and the second opening of the first channel and the third opening and the fourth opening on the second channel are all arranged upwards, thereby being convenient for the operation and sample application of staff and convenient for inserting electrodes.

Further, in some embodiments, the second opening is in a top-down sized configuration.

If the inner diameter of the opening is smaller, the solution can not directly flow out when flowing out from the corresponding opening, but a small liquid drop is formed first, the small liquid drop gradually becomes large and small along with the small liquid drop to be finally broken, and stronger shearing stress is generated on the liquid in the first channel in the breaking process of the small liquid drop, so that the film forming effect can be influenced. In this embodiment, the solution can be rapidly and smoothly discharged from the opening by enlarging the inner diameter of the opening at one end of the surface of the preparation device, so that the formation and breaking processes of liquid drops are avoided, and adverse effects on the film forming effect are correspondingly avoided.

Further, in some embodiments, the third opening and the fourth opening are arranged in a manner of being big in top and small in bottom, wherein the inner diameters of the first opening and the second opening of the second channel are larger, so that sample adding can be facilitated.

In this embodiment, the preparation device may select a layered processing mode to process the first structural layer and the second structural layer respectively, and then assemble the first structural layer and the second structural layer into the preparation device. Of course, the preparation device can also be integrated into one piece, and the first channel, the second channel and related structures can be directly processed on one plate. Therefore, the preparation device prepared by the integral molding (such as integral injection molding) or the layering processing mode is covered in the protection scope of the application.

In some embodiments, the second channel (also referred to as the lower flow channel) is used to add the polar solution and the first channel (also referred to as the upper flow channel) is used to add the membrane solution, since the second channel is located below the first channel when the device is in operation. In order to further explain the technical scheme adopted in this embodiment, the following describes the method of using the preparation device and the film forming process of the amphiphilic molecular film in this embodiment:

firstly, adding an appropriate amount of membrane solution into a first opening of a first channel (an upper flow channel) and an appropriate amount of polar solution into a third opening of a second channel (a lower flow channel) respectively;

Continuing to add an amount of the membrane solution (e.g., 1 microliter of the nonpolar solution having a concentration of 25mg/mL or a mixture of an amount of the nonpolar solution and 1 microliter of the nonpolar solution having a concentration of 25 mg/mL) to the first channel through the first opening, and continuing to add an amount of the polar solution (e.g., 5 microliters of the polar solution) to the first channel through the first opening such that a meniscus is formed in the first channel by the membrane solution;

the meniscus formed by the membrane solution moves along the internal path of the first channel under the pushing of the polar solution and passes through at least one micropore (i.e. gradually approaches and reaches the micropore under the pushing of the polar solution, and then continues to move forward and gradually moves away from the micropore under the pushing of the polar solution), and when the membrane solution moves to the micropore, amphiphilic molecules in the membrane solution form an amphiphilic molecular membrane at the micropore. Wherein the formed amphiphilic molecular film is parallel or nearly parallel to the horizontal plane direction.

In this example, the membrane solution, polar solution and nonpolar solution are selected as described in the above examples.

In some embodiments, based on different test requirements, one micropore, two micropores or a plurality of micropores can be arranged between the first channel and the second channel, so that one amphiphilic molecular film, two amphiphilic molecular films or a plurality of amphiphilic molecular films can be prepared at one time.

Similarly, the overall size of the preparation device is smaller, and the size of the internal channel and the size of the micropores are also smaller, so that the requirement on processing precision is higher. In order to further simplify the preparation process, a third structural layer is further arranged between the first structural layer and the second structural layer, at least one micropore is arranged on the third structural layer, and the first structural layer, the third structural layer and the second structural layer are sequentially and tightly connected, so that the first channel and the second channel are communicated through the micropores arranged on the third structural layer.

Preferably, in some embodiments, the membrane solution is selected in the same or approximately the same amount based on a fixed flow path (e.g., a flow path of a fixed cross-sectional size) to ensure that the meniscus is formed smoothly and that the state remains stable.

Preferably, in some embodiments, the length M1 of the preparation device is between about 26mm and 36mm, the width M2 of the preparation device is between about 18mm and 28mm, the length M3 of the first structural layer is between about 14.5mm and 24.5mm, the width M4 of the first structural layer is between about 8mm and 18mm, the inner diameter M5 of the first opening is between about 1.0 and 4.0mm, the inner diameters M6 of the first ends of the third and fourth openings are between about 2.0 and 4.0mm, and the inner diameters M7 of the second ends of the third and fourth openings are between about 0.5 and 2.0 mm.

Example III

Based on the device provided by the embodiment, the invention also provides a preparation method of the amphiphilic molecular film, which comprises the following steps:

s1, providing a preparation device for preparing an amphiphilic molecular layer, wherein at least one micropore for providing a growth platform for the amphiphilic molecular layer and a first channel and a second channel which can enable a solution to flow through the micropore are arranged in the preparation device, and a first end and a second end of the micropore are respectively communicated with the first channel and the second channel, wherein the cross section of the first channel is square or square-like;

wherein the first channel inner surface has a selected contact angle of about 65 ° to about 120 °.

It will be appreciated that in some embodiments, steps S3 and S4 may be performed continuously (or, in other embodiments, steps S3 and S4 may be performed as one step during actual operation), and that in other embodiments, steps S3 and S4 may be performed in steps.

In some embodiments, the earlier injection rate of the second polar solution is less than the later injection rate, and since the earlier stage is during meniscus formation, the movement rate of the film solution is less and relatively more stable, favoring meniscus formation.

In this embodiment, the preparation device preferably uses a material having a certain hydrophobicity, and the contact angle of the material on the inner surface of the first channel in the preparation device (i.e., the contact angle between the material on the inner surface and pure water) is about 65 ° -120 °, so that the film solution added into the first channel can form a meniscus under the combined action of the inner surface of the flow channel, the polar solution, and the air in the flow channel. The cross section of the first channel is preferably square or square-like, at this time, the first channel generates a certain resistance to the movement of the film solution (or the meniscus formed by the film solution), so that the movement speed of the film solution in the first channel is not too fast, and the movement speeds of the positions on the film solution are relatively uniform (or the differences of the flow speeds of the positions have little influence on the stability of the meniscus), so that the meniscus can maintain a stable form during the movement.

Specifically, the inner surface of the first channel and the surface of the microwell (i.e., the side wall of the microwell) are made of a material (preferably, polyoxymethylene) with a contact angle of about 65 ° -120 °, so that when the membrane solution moves to the corresponding microwell, an amphiphilic molecular membrane can be smoothly formed on the microwell (specifically, the first end of the microwell) without pre-treating the device (such as the microwell disposed at the flow channel) in advance.

In the process of forming a meniscus by the film solution and moving the meniscus, the addition amount (volume) of the film solution and the moving speed of the meniscus formed by the film solution are also very important to the process of forming and moving the meniscus, and it is possible to realize the selection of the appropriate addition amount and moving speed of the film solution through pre-experiments for those skilled in the art.

For example, in the selection or prediction step of the addition amount of the film solution, parameters mainly considered are: reynolds number (Re), a dimensionless number that can be used to characterize fluid flow. The method for calculating the Reynolds number comprises the following steps: re=ρvd/μ, where v, ρ, μ are the flow rate (corresponding to the moving speed of the membrane solution), density, and coefficient of viscosity of the fluid, respectively, and d is the characteristic length (corresponding to the length of the first channel). The density and viscosity coefficient of the film solution can be obtained through experimental measurement, and the reynolds number of the fluid needs to be smaller (the smaller the reynolds number is, the smaller the fluid flows) to keep the stability of the meniscus, namely, the laminar flow state.

The invention is thus preferably such that Re <2300 of the fluid is such that a preliminary restriction can be made on the relationship of the length of the first channel to the movement speed of the membrane solution by the reynolds number (thus, when the membrane solution is selected and the first channel length of the device is determined, the movement speed of the membrane solution can be initially limited based on the reynolds number, i.e. the movement speed of the membrane solution is constrained by the membrane solution and the first channel length). That is, for those skilled in the art, after the membrane solution is selected, a preparation device is selected to perform a preliminary experiment on the basis of the selected membrane solution, so that a movement speed of the selected membrane solution can be selected appropriately. The injection speed of the polar solution (such as the second polar solution) is correspondingly designed based on the movement speed screened by the pre-experiment, so that the preset injection speed of the second polar solution can be obtained. Of course, the injection rate of the second polar solution may also be estimated initially by providing the operator's experience, and then pre-experiment is performed to obtain a suitable injection rate.

Further, in some embodiments, the contact angle of the material selected for the inner surface of the first channel is about 75 ° to about 95 °. Specifically, in some embodiments, the plurality of micro-holes are disposed on the first channel inner surface, i.e., the contact angle of the selected material on the first channel inner surface and the micro-hole surface is about 75 ° to about 95 °.

In order to more clearly illustrate the technical solution adopted by the present invention, the following will briefly explain the contact angle and the formation of the meniscus:

referring to fig. 5a, the contact angle refers to the angle between the solid S, liquid L, and gas G three-phase interface from the solid-liquid interface through the liquid interior to the gas-liquid interface. If theta is smaller than 90 degrees, namely the liquid is easier to wet the solid, the smaller the angle is, the better the wettability is; if θ >90 °, the liquid does not readily wet the solid and is easily moved over the surface. Specifically, when θ=0, completely wet; when θ <90 °, partially wetted or wetted; when θ=90°, it is the boundary line of wetting or not; when theta >90 deg., no wetting; when θ=180°, no wetting at all.

Referring to FIG. 5b, the liquid pressure rising along the capillary wall in the figure isWherein Δp is the pressure of the liquid rising along the capillary wall, σ is the surface tension, R is the radius of curvature of the liquid surface, ρ is the liquid density, g is the gravitational acceleration, and h is the height of the rising liquid level. The definition based on contact angle can be found that: />For example, when the contact angle θ is less than 90 °, the solution may form a meniscus within the capillary, and the meniscus formed at this time is concave. It is also known that the radius of curvature of the meniscus formed is related to the radius r of the capillary, and that when the contact angle θ is greater than 90 °, the solution can form a convex meniscus within the capillary.

Specifically, the surface tension of the polar solution (e.g., the second polar solution) added to the interior of the first channel causes the membrane solution to spread out over the liquid surface of the polar solution (i.e., the end in contact with the membrane solution). Since the inner surface of the first channel is made of a material with certain hydrophobicity, the inner surface of the first channel has certain adsorption effect on the membrane solution. At this time, referring to fig. 5c, the membrane solution is a polar solution (second polar solution) 6 on one side and air 7 on the other side. Wherein the liquid surface has a shrinking force, and the molecules of the interface layer of the film solution in contact with the air are subjected to a pulling force directed towards the inside of the liquid, so that the film solution gradually forms a meniscus 5 inside the first channel.

Referring to fig. 7a, amphiphilic molecules 4 are dissolved in the membrane solution, since amphiphilic molecules 5 have hydrophilic ends 42 and hydrophobic ends 41 (see fig. 6). Because of the self-assembling ability of the amphiphilic molecules, when the membrane solution flows through the micropores, the hydrophilic ends 42 of the amphiphilic molecules inside will be directed to and contact the first polar solution, while the hydrophobic ends 41 will combine with the hydrophobic ends 41 of another layer of amphiphilic molecules, thereby forming an amphiphilic molecule layer, as shown in fig. 7a and 7 b. In this embodiment, by controlling the movement speed of the membrane solution, the amphiphilic molecules can be prevented from being stacked thicker and thicker at the micropores (as shown in fig. 12 (a)), so that the amphiphilic molecules cannot be embedded into the micropores.

In this embodiment, the second polar solution pushes the membrane solution to move, so that the membrane solution passes through at least one micropore, and an amphiphilic molecular membrane is formed at the at least one micropore. And the flow rate of the second polar solution (or the sample adding speed of the second polar solution) is relatively controllable, so that the moving speed (namely the flowing speed) of the membrane solution in the first channel can be controlled by controlling the sample adding speed of the second polar solution, thereby controlling the forming process of the amphiphilic molecular membrane, and avoiding that the membrane solution flows too fast to successfully form the amphiphilic molecular membrane or flows too slow to form the too thick amphiphilic molecular membrane.

The film forming mode that the meniscus (film solution) is pushed by the polar solution to form a film at the micropores can control the moving speed of the film solution precisely (for example, the injection speed of the polar solution is controlled by a pipette or a syringe pump, so that the moving speed of the film solution is controlled), so that the residence time of the film solution at different micropores is the same or similar, and the film solution is prevented from staying for too long in part of the micropores to form a thick film or moving too fast in part of the micropores to form a film successfully. Therefore, the invention can directly prepare the amphiphilic molecular layer with the film thickness meeting the use requirement by controlling the moving speed of the film solution more accurately, and thinning treatment (such as thinning treatment to make the film thinner from thick as shown in (a) - (d) in fig. 12) is not needed after film formation. In other words, the film forming method of the present invention can form a film at one time, and the film forming effect is good, for example, by controlling the injection rate of the polar solution and further controlling the moving rate of the film solution, a film having a suitable thickness can be directly formed (for example, a film as shown in fig. 12 (d)) can be directly formed.

For example, in some embodiments, the injection rate of the second polar solution is controlled by a syringe pump to control the rate of movement of the membrane solution. In this embodiment, the provided method is more controllable (e.g., compared to bubble squeeze film formation of the prior art). Of course, other injection methods may be used to control the injection rate of the solution, and any method capable of controlling the injection rate of the solution is within the scope of the present invention.

Further, in some embodiments, a reciprocating syringe pump is provided to add (inject) the second polarity solution. For example, the syringe pump model harvard apparatus 4400 is selected.

In some embodiments, the method uses a membrane solution, a non-polar solution, as described above, and a first, dipolar solution, as also described above.

For example, in some embodiments, the film solution comprises: a non-polar solution, and an amphiphilic molecule, wherein the amphiphilic molecule optionally comprises: the amphiphilic molecule comprises: a phospholipid, or a polymer, or a mixture of a phospholipid and a polymer.

Further, in order to achieve subsequent DNA sequencing, in some embodiments, the steps are further included:

And a first electrode and a second electrode are respectively inserted into the first channel and the second channel, and the nanopore protein is inserted into the amphiphilic molecule layer through the first electrode and the second electrode.

Specifically, a first electrode (positive electrode or negative electrode) and a second electrode (positive electrode or negative electrode) are respectively inserted into the first polar solution and the second polar solution, so that the nanopore protein in the first polar solution and/or the second polar solution is inserted into the amphiphilic molecular membrane.

Further, in some embodiments, the nanopore protein may be pre-added to a polar solution (e.g., a first polar solution or a second polar solution species).

Of course, in other embodiments, the nanopore protein (e.g., a solution comprising the nanopore protein) may also be added to the first channel after formation of the amphiphilic molecule layer.

In some embodiments, pre-experiments may be performed prior to preparing the amphiphilic molecular film based on the preparation apparatus, i.e., different experiments are performed with different amounts of film solution, respectively, to determine the amount of film solution that stably forms the meniscus.

One or more microwells may be provided on the manufacturing apparatus based on different test requirements. For example, in one embodiment, where the first channel and the second channel are in communication via only one microwell, a suitable meniscus may be formed by adding 30 microliters of polar solution to the second channel (lower channel) to fill the second channel, and adding 1 to 5 microliters of membrane solution to the first channel (upper channel), wherein the concentration of membrane solution is 10mg/ml. Then 5 microliters of polar solution was added to the first channel (upper flow channel) such that the membrane solution formed a meniscus in the first channel, and then another 30 microliters of polar solution was added to push the meniscus through the microwells to form an amphiphilic molecular layer at the microwells. If two micropores are further disposed in the first channel and the second channel, the cross section of the first channel is formed and the size of the first channel is unchanged, so that the volume of the membrane solution required for forming the meniscus stably is constant, but the amount of amphiphilic molecules (such as phospholipids or high-molecular triblock copolymers) required for forming the amphiphilic molecule membrane is increased, that is, the concentration of the amphiphilic molecules in the membrane solution needs to be increased, for example, 12.5mg/ml, i.e., the membrane solution content is kept unchanged, and more amphiphilic molecules are contained in the membrane solution by a concentration increasing method.

Further, in some embodiments, the detection electrode (i.e., the first and second electrodes) is Ag or Ag/Cl, i.e., silver or silver chloride wire. Specifically, the detection electrodes are respectively inserted into the first opening and the third opening, and whether the amphiphilic molecular film is formed or not is judged by detecting electric signals or outputting capacitance at two ends of the triangular wave measurement amphiphilic molecular film by using a commercial AXON 1550B instrument.

In some embodiments, the preparation device in the method may be selected from the preparation devices shown in embodiment one or embodiment two.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

The above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention, and are intended to be included within the scope of the appended claims and description.

Claims

1. A method for preparing an amphiphilic molecular layer, comprising:

2. The method of manufacturing according to claim 1, wherein the film solution comprises: a non-polar solution, and an amphiphilic molecule, wherein the amphiphilic molecule optionally comprises: a phospholipid, or a polymer, or a mixture of a phospholipid and a polymer.

3. The method of preparing according to claim 1, wherein the first polar solution comprises: an electrolyte, and/or a polyelectrolyte;

and/or, the first polar solution comprises: a redox couple, and/or a combination of redox couples that may be partially oxidized or reduced to provide a redox couple;

and/or, the first polar solution comprises: crosslinked agarose gel, and/or crosslinked sodium alginate gel;

And/or, the first polar solution comprises: a buffer for adjusting the pH;

and/or, the second polar solution comprises: an electrolyte, and/or a polyelectrolyte;

and/or, the second polar solution comprises: a redox couple, and/or a combination of redox couples that may be partially oxidized or reduced to provide a redox couple;

and/or, the second polar solution comprises: crosslinked agarose gel, and/or crosslinked sodium alginate gel;

and/or, the second polar solution comprises: buffers for adjusting the pH.

4. The method of claim 1, wherein in S3, the second polar solution is injected at a rate of about 5 μl/min to about 10 μl/min;

and/or, in S4, the preset speed of the second polar solution is about 10 μl/min to about 20 μl/min.

5. The method of manufacturing according to claim 1, further comprising the step of:

6. The preparation device of the amphiphilic molecule layer is characterized by comprising: a microwell for providing a growth platform for the amphiphilic molecule layer, and a first channel and a second channel capable of allowing a solution to flow through the microwell, and a first end and a second end of the microwell being in communication with the first channel and the second channel, respectively;

7. The preparation device of claim 6, wherein the first channel comprises: a first flow path, the preparation device comprising:

8. The apparatus of claim 7, wherein the third structural layer further comprises a groove on the first surface, wherein the micro-hole is disposed on the groove, and wherein the groove cooperates with the first flow channel to form a first channel through which the solution can flow when the first and third structural layers are tightly connected.

9. The apparatus of claim 7, wherein the microwells have an inner diameter between 100 microns and 200 microns.

10. The apparatus of claim 7, wherein the first opening and/or the second opening are provided in a top-down size.