CN118835255A - Preparation method and device for multiple amphiphilic molecule layers - Google Patents

Abstract

The present invention relates to a preparation method and a preparation device for rapidly preparing an amphiphilic molecule layer, comprising the steps of: S1 providing a preparation device for preparing multiple amphiphilic molecule layers, wherein the preparation device is provided with multiple micropores and a flow channel capable of allowing a solution to flow to the micropores, and the preparation device also includes an electrode layer, so that the solution flowing into the micropores can contact the electrode layer; S2 adding a first polar solution into the flow channel, so that the first polar solution enters at least one micropore and contacts the electrode layer; S3 sequentially adding a membrane solution and a second polar solution into the flow channel, so that the membrane solution forms a meniscus in the flow channel; S4 adding a second polar solution into the flow channel, so that the second polar solution pushes the membrane solution to move and flows through at least one micropore where the first polar solution contacts the electrode layer, and the amphiphilic molecules in the membrane solution form an amphiphilic molecule layer on the corresponding micropores. The method provided by the present invention is simple and safe to operate, and the selected preparation device has low production cost.

Description

A method and device for preparing multiple amphiphilic molecular layers

Divisional application

This application is a divisional application based on the Chinese invention patent application with application number CN2022103914733, application date April 14, 2022, and invention name “A method and device for preparing an amphiphilic molecular layer”.

Technical Field

The invention relates to the field of amphiphilic molecular films, and in particular to a preparation method and a preparation device for rapidly forming a plurality of amphiphilic molecular layers.

Background Art

Nanopore proteins need to be stably embedded in a thin film formed of phospholipids or polymers, such as an amphiphilic molecule layer (i.e., an amphiphilic molecule membrane), in order to allow the DNA sequence to pass through, so as to achieve the purpose of detecting the DNA sequence. However, some current preparation methods (i.e., film-forming methods) and devices for preparing amphiphilic molecule layers still have some problems in application.

The existing film forming methods mainly include folding double layer formation method (Montal & Mueller's method), tip dipping method, coating method, patch clamp method and oil-in-water droplet interface method, etc. The above methods such as folding double layer method, tip dipping method, coating double layer method, etc. often form thick films for the first time, and need to be thinned, such as by volatilization of organic solvents, or by physical spreading, or by air pressure extrusion, etc. Thinning treatment is very important for controlling 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 spreading). At the same time, the existing film forming methods usually involve pretreatment of the preparation device, such as fluorine plasma treatment, silanization treatment, etc. These pretreatment operations require the use of high-risk chemicals and need to be carried out in laboratories with high construction costs and high maintenance costs. There is a potential threat to the life safety of operators and the surrounding environment. In addition, the pretreatment also requires accurate calculation of the amount of coating used. Too much or too little coating will affect the test results. The preparation devices of amphiphilic molecular membranes are usually small chips, and their internal structures (such as micropores) are tiny in size, so the amount of paint selected is also very small. It is not easy to apply a very small amount of paint on a tiny structure. Therefore, it is difficult to avoid the problem of over- or under-application during the coating process.

For example, the Chinese invention patent with application number 201480056839.5 discloses a biochip, and the invention also discloses a film-forming method for the biochip, which includes the steps of: adding a liquid containing lipid molecules (i.e., amphiphilic molecules) to the surface of the chip, and then separating the liquid with bubbles so that the lipid molecules are distributed on the surface of the chip, and the bubbles make the lipids thinner. However, bubble generation often requires manual operation control (such as preparing bubbles with a pipette), and this process is difficult to automate because it is difficult to control the size of the bubbles manually and it is difficult to ensure the stability of the bubble shape, so the repeatability of this method is also poor. And this method also requires pretreatment.

For another example, the Chinese invention with application number CN200880126160.3 discloses a method for forming a layer of aqueous solution separating two volumes. The method allows an aqueous solution containing amphiphilic molecules to flow through a main body to cover the grooves (i.e., micropores), so that the aqueous solution can cross the grooves to form amphiphilic molecules. Although the technology of allowing the aqueous solution to flow through the grooves to form amphiphilic molecules is easy to implement, the prepared membrane is relatively thick and requires subsequent thinning treatment, and the method also involves a pretreatment step.

Summary of the invention

In order to partially solve or partially alleviate the above technical problems, the first aspect of the present invention is to propose a method for preparing an amphiphilic molecule layer, the method comprising the following steps:

S1 provides a preparation device for preparing multiple amphiphilic molecule layers, wherein the preparation device is provided with multiple micropores and flow channels that enable solutions to flow into the micropores, and the preparation device also includes an electrode layer so that the solutions flowing into the micropores can contact the electrode layer, wherein the cross section of the flow channel is square or quasi-square;

S2: adding a first polar solution into the flow channel, so that the first polar solution enters at least one of the micropores and contacts the electrode layer;

S3 sequentially adding a membrane solution and a second polar solution into the flow channel, so that the membrane solution forms a meniscus in the flow channel;

S4: adding the second polar solution into the flow channel based on a preset injection speed, so that the second polar solution pushes the membrane solution to move and flow through at least one micropore where the first polar solution contacts the electrode layer, and the amphiphilic molecules in the membrane solution form an amphiphilic molecule layer on the corresponding micropore;

Wherein, the contact angle of the inner surface of the flow channel is about 65°-about 120°.

In some embodiments, before S2, the method further includes: performing a wetting treatment on at least one of the micropores;

In some embodiments, S2 includes the step of: after adding the first polar solution into the flow channel, ultrasonically treating the preparation device.

In some embodiments, the wetting process includes: an electrowetting process.

In some embodiments, the wetting treatment includes: liquid wetting treatment.

In some embodiments, the membrane solution includes: a non-polar solution, and amphiphilic molecules.

In some embodiments, the amphiphilic molecule optionally includes: phospholipids, or polymers, or a mixture of phospholipids and polymers.

In some embodiments, the non-polar solution optionally includes: an alkane organic solvent.

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

In some embodiments, the first polar solution includes: a redox pair, and/or a combination of redox pairs that can be partially oxidized or reduced to provide a redox pair.

In some embodiments, the first polar solution includes: cross-linked agarose gel, and/or cross-linked sodium alginate gel.

In some embodiments, the first polar solution includes: a buffer for adjusting pH.

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

In some embodiments, the second polar solution includes: a redox pair, and/or a combination of redox pairs that can be partially oxidized or reduced to provide a redox pair.

In some embodiments, the second polar solution includes: cross-linked agarose gel, and/or cross-linked sodium alginate gel.

In some embodiments, the second polar solution includes: a buffer for adjusting pH.

In some embodiments, the preparation device is further provided with a common electrode, and the common electrode is in contact with the second polar solution. Accordingly, the method further comprises the steps of:

Electricity is supplied to the first polar solution and the second polar solution through the electrode layer and the common electrode to insert the nanopore protein into the amphiphilic molecule layer.

In some embodiments, in step S3, the injection rate of the second polar solution is about 10 μL/min to about 50 μL/min;

In some embodiments, in step S4, the injection rate of the second polar solution is about 200 μL/min to about 500 μL/min.

In some embodiments, the inner surface material of the flow channel is polyoxymethylene.

The second aspect of the present invention is to provide a device for preparing an amphiphilic molecular layer, wherein the device is provided with a plurality of micropores and a flow channel that enables the solution to flow into the micropores. The device is also provided with an electrode layer so that the solution flowing into the micropores can contact the electrode layer, wherein the cross-section of the flow channel is square or quasi-square, and the inner surface material of the flow channel can optionally be polyformaldehyde.

In some embodiments, the spacing between adjacent microholes is greater than about 0.4 mm.

Beneficial technical effects:

The present invention provides a preparation method (i.e., film-forming method) and a preparation device for rapidly preparing multiple amphiphilic molecular layers (i.e., amphiphilic molecular membranes, also referred to as "membranes"). Different from the prior art, the preparation method of the present invention proposes a new film-forming method, that is, a membrane solution forms a meniscus in a flow channel, and the meniscus is pushed by a polar solution, so that the meniscus can move in the flow channel and pass through micropores, and then an amphiphilic molecular layer (membrane) is formed on the corresponding micropores.

Specifically, the preparation device proposed in the present invention uses a hydrophobic material that meets the film-forming conditions, that is, the contact angle of the material on the inner surface of the flow channel in the preparation device is about 65°-120°, so that the membrane solution added to the flow channel can form a meniscus under the joint action of the inner surface of the flow channel, the polar solution and the air in the flow channel. Among them, the cross-section of the flow channel is preferably square or quasi-square. At this time, the flow channel will produce a certain resistance to the movement of the membrane solution (or the meniscus formed by the membrane solution), so that the movement speed of the membrane solution in the flow channel will not be too fast, and the movement speed of each position on the membrane solution is relatively uniform (or the difference in flow speed at each position has little effect on the stability of the meniscus), so that the meniscus can maintain a stable shape during the movement.

By using a polar solution to push the meniscus (membrane solution) to form a film at the micropores, the moving speed of the membrane solution can be controlled more accurately (for example, the injection speed of the polar solution is controlled by a pipette gun or a syringe pump, so as to control the moving speed of the membrane solution), so that the membrane solution stays at different micropores for the same or similar time, avoiding the membrane solution staying in some micropore areas for too long to form a thick film, or moving too fast in some micropore areas and failing to successfully form a film. Therefore, the present invention can efficiently and directly form an amphiphilic molecular layer with a suitable thickness (or a film thickness that meets the use requirements) by more accurately controlling the moving speed of the membrane solution, without the need to thin the film after film formation (such as high-voltage breakdown, multiple film formation). In other words, the film forming method of the present invention can form a film in one time.

Therefore, in actual application scenarios, after the concentration of the membrane solution, the moving speed and other parameters are selected (such as through preliminary experiments, or determined by the operating experience of the staff), multiple experiments can be performed based on the same experimental conditions and parameters, and the results obtained from multiple experiments (such as film formation) are very different, that is, the repeatability of this method is good. In other words, the method provided by the present invention can avoid or reduce the impact of the manual operation of the staff on the experimental operation, so as to avoid or reduce the uncontrollable factors in the operation process (that is, the controllability of this method is better), so as to have good repeatability.

Furthermore, the film forming method proposed in the present invention does not require pretreatment, the film forming method is simpler, and the safety of the workers during the operation is better guaranteed (it does not involve the operation and use of dangerous goods).

Furthermore, the injection speed of the polar solution can be controlled by a syringe pump, and the syringe pump can be automatically controlled by an electronic device (such as a computer, etc.) (that is, the film formation method proposed in the present invention can be automatically controlled), thereby further reducing the manual operation steps of the staff, and correspondingly avoiding the errors that may be caused by manual operation of the staff, so as to further ensure the stability and uniformity of the film formation.

The film forming method (and device) provided by the present invention can be applied to various detection needs in the laboratory (such as different application scenarios applicable to scientific research units, commercial detection companies, etc.), and because the present invention can form multiple films at one time, it can better meet the commercial or market detection needs (such as human genome sequencing, virus sequencing, etc.) in actual use. The detection cycle of this type of detection is relatively long and the amount of data required is large. At this time, the probability of staff making misoperations during long-term operation will also increase. The method provided by the present application can be coordinated with the automated control in the prior art to further improve the accuracy and reliability of the test results.

In the prior art, in order to ensure that the solution containing amphiphilic molecules can cover (or flow through) all or multiple micropores to form multiple amphiphilic molecule layers, those skilled in the art usually choose to add more solutions containing amphiphilic molecules to avoid the existence of some micropores that cannot form amphiphilic molecule layers, so the generated film is also relatively thick. In response to this technical problem, those skilled in the art usually consider how to further process the film (i.e., thinning treatment) after the film is formed to make the film thinner, such as requiring high-voltage breakdown to form multiple films. Unlike the prior art, the present application adopts a different technical route and proposes a new film-forming method, which can form a film at one time and is simpler in the operating process. And because it avoids time-consuming operations such as pretreatment and thinning, the method of the present invention is less time-consuming and more efficient.

Moreover, based on the existing preparation devices, it is difficult for those skilled in the art to think of the film forming method proposed in this application. First, the preparation device in the prior art does not provide the conditions for the formation of a meniscus. Similarly, the methods and devices in the prior art are also difficult to ensure that the meniscus can move stably in the flow channel. Moreover, since the devices in the prior art use different materials from those in this application, and both require pretreatment of the devices, the pretreatment may make it more difficult to form a meniscus in the flow channel in the device.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

FIG. 1a is a schematic cross-sectional view of a preparation device in an exemplary embodiment of the present invention;

FIG1b is a schematic diagram of an optional cross-sectional structure of a flow channel of a preparation device in an exemplary embodiment of the present invention;

FIG1c is a schematic cross-sectional view of a preparation device in another exemplary embodiment of the present invention;

FIG2 is a schematic structural diagram of a preparation device in another exemplary embodiment of the present invention;

FIG3 is a schematic structural diagram of a preparation device in an exemplary embodiment of the present invention;

FIG4a is a schematic diagram of a first state of a first polar liquid at a micropore of a preparation device in an exemplary embodiment of the present invention;

FIG4 b is a schematic diagram of a second state of the first polar liquid at the micropores of the preparation device in an exemplary embodiment of the present invention;

FIG4c is a schematic diagram of a third state of the first polar liquid at the micropores of the preparation device in an exemplary embodiment of the present invention;

FIG5 is a schematic flow diagram of a preparation method in an exemplary embodiment of the present invention;

FIG6 is a schematic diagram of the structure of an amphiphilic molecule;

FIG7a is a schematic diagram of a first structure of a meniscus in a flow channel;

FIG7 b is a schematic diagram of a second structure of a meniscus in a flow channel;

FIG8 is a schematic diagram of the structure of an amphiphilic molecular membrane formed at a micropore;

Figure 9a shows the relationship between the contact angle and solid, liquid, and gas;

FIG9 b shows the process of liquid wetting the capillary;

FIG10 is a schematic diagram showing the structure of membranes of different thicknesses formed at micropores;

FIG. 11a shows a schematic structural diagram of a flow channel in a preparation device in an exemplary embodiment of the present invention;

FIG. 11 b is a schematic diagram showing the shape change of the meniscus during the movement in an exemplary embodiment of the present invention.

1 is the first structural layer, 11 is the flow channel, 12 is the first opening, 13 is the second opening, 14 is the electrode insertion port, 15 is the first flow channel, 16 is the third opening, 2 is the second structural layer, 21 is the micropore, 3 is the third structural layer, 31 is the electrode layer, 32 is the electrode protrusion, 4 is the meniscus, 5 is the amphiphilic molecule, 51 is the hydrophobic end, 52 is the hydrophilic end, 6 is the first polar solution, 7 is the second polar solution, 8 is air, L is the solution, G is the gas, and S is the solid.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Herein, suffixes such as "module", "component" or "unit" used to represent elements are only used to facilitate the description of the present invention, and have no specific meanings by themselves. Therefore, "module", "component" or "unit" can be used mixedly.

In this document, the terms "upper", "lower", "inner", "outer", "front end", "rear end", "two ends", "one end", "the other end" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

In this document, unless otherwise clearly specified and limited, the terms "installed", "provided with", "connected", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a wireless connection or a wireless communication connection; it can be a direct connection or an indirect connection through an intermediate medium, and it can be the internal connection of two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

Herein, "amphiphilic" molecules refer to compounds having both hydrophilic and lipophilic properties, which have a hydrophilic head and a hydrophobic tail. The hydrophilic head is generally composed of polar groups, such as choline, ammonium salts; the hydrophobic tail is generally composed of long fatty chains. Herein, "amphiphilic" and "amphiphilic" are used synonymously. Amphiphilic molecules can be lipid molecules. A typical amphiphilic molecular membrane (or amphiphilic molecular layer, also referred to as "membrane") can be a lipid bilayer, which is a bilayer formed by two opposing lipid monolayers, and the two lipid monolayers are arranged through self-assembly so that the hydrophobic tails face each other to form a hydrophobic interior, while the hydrophilic heads face the outside (each side is a polar hydrophilic environment). The lipids forming the lipid bilayer can include any suitable lipids, such as 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine, diphytylphosphatidylcholine (DPhPC). Amphiphilic molecules can be chemically modified or functionalized to promote the coupling of polynucleotides. The amphiphilic molecule may also be a polymer material synthesized by physical and chemical methods, such as ABA triblock copolymer (PMOXA-PDMS-PMOXA dimethyloxazoline-polydimethylsiloxane-dimethyloxazoline). The amphiphilic molecule may be a mixture.

Herein, "non-polar solvent" or "non-polar solution" refers to a compound or mixture of compounds that is immiscible with water. The non-polar solvent can be an oil, more specifically, a pure paraffin, 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 oil. More specifically, the oil can be methylphenyl silicone oil AR20, hydroxyl-terminated polydimethylsiloxane PDMS-OH.

Herein, "polar aqueous solution" or "polar solution" refers to an aqueous solution containing water that is easily miscible with water and other polar solvents. The polar aqueous solution may include one or more solutes. For example, a buffer 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 a polyelectrolyte to effectively enhance the ion exchange life. The polar aqueous solution may also include a redox pair or a combination of redox pairs that may be partially oxidized or reduced to provide a redox pair, such as iron/ferrocyanide. The polar aqueous solution may also be a cross-linked agarose gel and sodium alginate gel.

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

Herein, "square" includes polygons whose adjacent sides have an angle of about 90° or close to 90°, for example, the square is a quadrilateral, such as a square, or a rectangle (as shown in a in Figure 1b), etc. Of course, the "square" in this article does not necessarily need to be set as a standard square, rectangle or other polygon. For example, the opposite sides of the "quadrilateral" in the square can be set in parallel or non-parallel, and correspondingly, the adjacent sides of the "quadrilateral" can be set perpendicular to each other or non-perpendicular. "Quasi-square" includes a square with chamfers at the adjacent side angles, or a square with adjacent sides connected by arcs, for example, a quasi-quadrilateral, such as a quasi-rectangle (as shown in b in Figure 1b, the top corners of the rectangle are replaced with arc structures), a quasi-square (such as replacing the top corners of the square with arc structures), etc. For example, here, "the cross-section of the flow channel is square or quasi-square" means that the cross-section of the flow channel can be set as a rectangle, or a square, or a quasi-rectangle, or a quasi-square, etc. In order to avoid defects such as burrs on the inner surface of the flow channel during processing, preferably, the cross-section of the flow channel is set to be square-like, such as rectangular, square, etc.

Herein, the terms "about" and "approximately" are typically expressed as +/-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 indicate a value that is understood by those skilled in the art to include the customary range of error in the art.

Herein, some embodiments may be disclosed in a format that is in a certain range. It should be understood that the description of this "being in a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation to the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent digital values within this range, such as the contact angle of "about 65 degrees to about 120 degrees" can be understood as having disclosed the following range: the contact angle is about 65-95 degrees, 95-105 degrees, 105-120 degrees, etc., and also discloses independent digital values within this range, such as 65 degrees, 69 degrees, 75 degrees, 80 degrees, 90 degrees, 100 degrees, 110 degrees, 120 degrees.

Embodiment 1

The first aspect of the present invention is to provide a preparation device for quickly forming an amphiphilic molecular membrane (amphiphilic molecular layer), referring to Figures 1a to 4, wherein the preparation device is provided with a plurality of micropores 21, and a flow channel 11 that enables the solution to flow into the micropores 21, and the preparation device also includes: an electrode layer 31, so that the solution (such as a polar solution, i.e., an electrolyte) flowing into the micropores 21 can contact the electrode layer 31, wherein the cross-section of the flow channel is preferably square or quasi-square. Of course, as long as the cross-section of the flow channel enables the membrane solution to form a meniscus and move in the form of a meniscus, it belongs to the protection scope of the present invention. Among them, the micropores provide a growth platform for the membrane solution to form an amphiphilic molecular layer (amphiphilic molecular membrane, also referred to as "membrane").

In some embodiments, the inner surface of the flow channel is made of a polymer material, and the contact angle of the material selected for the inner surface of the flow channel (ie, the contact angle between the material and pure water) is about 65 degrees to about 120 degrees.

Preferably, in some embodiments, the contact angle of the inner surface of the flow channel is about 75 degrees to 95 degrees.

Preferably, in some embodiments, the material selected for the inner surface of the flow channel is polyoxymethylene (delrin).

Specifically, in some embodiments, the material selected for the inner surface of the flow channel and the micropore surface (micropore side wall) is polyformaldehyde, so that the membrane solution added to the flow channel can form a meniscus in the flow channel and can form an amphiphilic molecular layer at the micropores. Therefore, the preparation device in this embodiment does not need to be pretreated when used.

Preferably, in some embodiments, the material selected for preparing the device is polyoxymethylene.

For those skilled in the art, it is possible to select suitable preparation materials through preliminary experiments. Specifically, the contact angles of different materials can be tested by contact angle testing methods (such as appearance image analysis methods, or weighing methods) to select suitable preparation materials. Specifically, when the contact angle of the material is about 65°-120° (of course, as long as the flow channel prepared by the selected material can form a meniscus), it is considered that the flow channel prepared by the material has the ability to form a meniscus.

Furthermore, in some embodiments, the flow channel includes a first opening for adding a sample (ie, adding a liquid, such as a membrane solution and a polar solution) into the flow channel.

Furthermore, in some embodiments, the flow channel further includes: a second opening for discharging the liquid in the flow channel.

Furthermore, in some embodiments, the flow channel is also provided with an electrode insertion port for inserting a common electrode.

Specifically, in some embodiments, referring to FIG. 1 , the device includes: a body, the body includes a first structure layer 1, a second structure layer 2 and a third structure layer 3 arranged in sequence, wherein a flow channel 11 is provided on the first structure layer 1, and a first opening 12 is provided at one end of the flow channel 11 for adding samples, a plurality of micropores 21 are provided at intervals on the second structure layer 2, and an electrode layer (equivalent to the first electrode) is provided on the third structure layer 3;

When a solution (such as a first polar solution) is added to the flow channel and the solution flows through at least one micropore, the solution replaces the air in at least one micropore (for example, the solution can fill at least one micropore, see Figure 4a, the first polar solution 6 fills the micropore) and contacts the electrode layer, so that the solution (such as the first polar solution) can conduct electricity under the action of the electrode.

In some embodiments, the second structure layer is bonded together with corresponding electrodes on the electrode layer by bonding, patterned cross-linking equipment, or AZ4620, su-8, and the flow channel above is only provided with (inserted with) one common electrode.

Since the materials used for the inner surface of the flow channel and the side walls of the micropores (i.e., the inner surface of the micropores) have a certain degree of hydrophobicity, if the inner surface of the flow channel and the side walls of the micropores are not pre-wetted, in some embodiments, the polar solution may not be able to enter the micropores smoothly, or in other embodiments, only a portion of the polar solution can enter the interior of the micropores and cannot fill the micropores.

Therefore, in some embodiments, the first polar solution added first may not be able to completely fill the micropores. For example, referring to FIG. 4b , the first polar solution 6 forms droplets suspended at the first end of the micropores, and the droplets formed by the first polar solution 6 are in contact with the electrode layer 31 .

Furthermore, in some embodiments, in order to ensure that the droplets formed by the first polar solution 6 can contact the electrode layer, at least one electrode bump 32 (such as a probe, etc.) is provided on the electrode layer 31, so that when the volume of the droplets formed by the first polar solution is small, they can also contact the electrode layer.

Furthermore, in some embodiments, in order to enable the droplets formed by the first polar solution to contact the electrode layer, an electrode layer 31 for conducting electricity may be provided on the sidewall 22 of the micropore. Furthermore, an electrode bump 32 may be provided on the electrode layer 31.

Alternatively, in some other embodiments, referring to FIG. 4c, the first polar solution firstly introduced into the flow channel only forms a thin solution film (layer) at the first end of the micropore. At this time, in order to enable the first polar solution to contact the electrode layer, an electrode bump 32 (such as a probe, etc.) can also be provided on the electrode layer, thereby ensuring that the first polar solution can contact the electrode layer.

Specifically, in some embodiments, the inner diameter of the first end of the micropore (i.e., the upper end, i.e., the end connected to the flow channel) is larger than the inner diameter of the second end of the micropore (i.e., the other end of the micropore). In this embodiment, the micropore is designed to be small at the top and large at the bottom, so that there is enough space inside the micropore to accommodate the polar solution (such as the first polar solution first added to the micropore) without affecting the size of the first end of the micropore (the inner diameter of the first end of the micropore is closely related to the formation of the amphiphilic molecule layer, so the inner diameter of the first end of the micropore is usually fixed between about 100 microns and 200 microns). Specifically, when the meniscus flows through the micropore, the amphiphilic molecule layer in the meniscus is formed at the narrowest part of the micropore, that is, the amphiphilic molecules self-assemble at the first end of the micropore to form an amphiphilic molecule layer.

For example, in some embodiments, referring to FIG. 1 a and FIG. 4 , the microholes are arranged in a trumpet shape that is small at the top and large at the bottom.

In other embodiments, the micropores may be arranged in a cylindrical shape (i.e., the upper and lower ends are equal in size), or arranged in a shape of being larger at the top and smaller at the bottom. Of course, the micropores may also be arranged in other shapes, as long as the shape of the micropores can successfully form an amphiphilic molecule layer, they are all within the scope of protection of the present invention.

Furthermore, in some embodiments, the flow channel is provided with a second opening for discharging the liquid in the flow channel.

In some embodiments, the second opening may also be used to insert a second electrode (ie, a common electrode). Therefore, in some embodiments, an electrode insertion port may not be provided on the flow channel, thereby simplifying the structure of the device.

Furthermore, in some embodiments, referring to FIG. 1 a - FIG. 3 , an electrode insertion port 14 is further provided on the flow channel. Specifically, the second electrode is inserted into the flow channel through the electrode insertion port 14 .

Furthermore, in some embodiments, a mating portion mating with the second electrode is provided on the second structural layer, and when the second electrode is inserted into the flow channel, the first end (ie, the insertion end) of the second electrode mating with the mating portion (eg, contacting each other).

Furthermore, in order to fix the second electrode and improve the stability of the preparation device during operation, in some embodiments, the matching portion is a groove (i.e., a groove is provided on the first surface of the second structural layer), and when the second electrode matches the matching portion, the first end of the second electrode is inserted into the groove. In this embodiment, since the second electrode is inserted into the groove for fixing, on the one hand, it is convenient to position and install the second electrode (avoiding the second electrode from being offset during the insertion process), and at the same time, the second electrode will not be displaced or shaken in the horizontal direction during use, thereby improving the working stability of the second electrode.

Furthermore, the second electrode (ie, the common electrode) in this embodiment can be set to be detachable, that is, it can be installed (inserted) and removed by the staff themselves, so that the second electrode can be easily replaced and cleaned.

It is understandable that the polarities of the first and second electrodes are opposite, for example, in some embodiments, the first electrode is a positive electrode and the second electrode is a negative electrode. For another example, in other embodiments, the first electrode is a negative electrode and the second electrode is a positive electrode.

Preferably, in some embodiments, the third structural layer is an ASIC circuit layer, the electrode layer part is a TSV through-hole electrode, and the selected electrode material is pure gold. A chip (i.e., the second structural layer) containing a plurality of micropores (e.g., an array of 8*32=256 micropores) is bonded to the third structural layer by hot pressing bonding, a permanent adhesive bonding machine, and photoresist AZ4620 or su-8. The common electrode is preferably a gold-plated copper column inserted into the groove of the chip. The first opening (i.e., the sample inlet) and the second opening (i.e., the sample outlet) of the flow channel are used for adding samples (i.e., adding corresponding liquids, such as polar solutions or membrane solutions) and discharging samples using a microfluidic dedicated pipeline, and a syringe pump (e.g., a reciprocating syringe pump) is used to control the flow rate of the polar solution to control the movement speed of the membrane solution.

Preferably, in some embodiments, the selected reciprocating syringe pump model is Harvard Apparatus 4400.

Further, in some embodiments, the pore size of the micropore is between about 100 microns and about 200 microns, for example, the inner diameter of the first end of the micropore is between about 100 microns and about 200 microns.

Preferably, in some embodiments, the pore size of the micropore is between about 150 microns and 200 microns, for example, the inner diameter of the first end of the micropore is between about 150 microns and 200 microns.

Preferably, in some embodiments, in order to facilitate processing, referring to FIG. 2 , the microholes on the second structural layer are arranged in multiple arrays, for example, in a 2*8 arrangement, or in an 8*32 arrangement.

Preferably, in some embodiments, for ease of processing, referring to FIG. 3 , the length L1 of the preparation device is approximately between 40 mm and 51 mm, the width L2 of the preparation device is approximately between 16 mm and 27 mm, the maximum width L3 inside the flow channel is approximately between 4.0-12.0 mm, the width L4 at the first and second openings of the flow channel (the minimum width inside the flow channel) is approximately between 0.3 mm and 0.8 mm, and the length L5 of the micropore array is approximately between 10.5-20.5 mm.

Preferably, in some embodiments, the distance L6 between adjacent microwells in the microwell array is greater than about 0.4 mm.

Further, in some embodiments, the distance L6 between adjacent microwells in the microwell array is greater than about 0.5 mm.

Surprisingly, the preparation device of the present invention uses polyoxymethylene as a raw material to prepare the flow channel and micropores of the device, and adopts a new size design for the spacing between adjacent micropores. It simplifies the processing technology of the device, so that the device can be prepared by various types of processing methods (such as mechanical processing, laser processing, etc.), greatly reducing the production cost of the device, and can also meet the needs of preparing multiple amphiphilic molecular layers at one time. In addition, based on the preparation device provided by the present invention, there is no need to pre-treat the micropores in the preparation device during the film forming process, and the film forming method is simpler.

It can be understood that the device provided by the present invention can be applied to the new film-forming method proposed by the present invention, and can also be applied to existing film-forming methods, such as coating methods.

In some embodiments, the raw materials for preparing the device can be selected from materials such as teflon, pmma (plexiglass), delrin (polyoxymethylene, also known as polyoxymethylene) and parylene (polyparaxylene).

Of course, in other embodiments, the raw materials for preparing the device can also be PMMA (polymethyl methacrylate), epoxy resin, PC (polycarbonate), PVC (polyvinyl chloride), COC (cycloolefin copolymer), polyimide and other materials.

Furthermore, in some embodiments, the processing methods that can be used to prepare the device include: mechanical processing, laser processing, micro injection molding, 3D printing, casting, electroporation, etc.

Furthermore, in some embodiments, the micropores in the preparation device are preferably processed by laser. The micropores formed by laser processing are circular and have smooth inner walls, which are easy to form an amphiphilic molecule layer.

Of course, in other embodiments, the micropores may also be processed by mechanical processing, electroporation processing, etc. It is understood that the processing of the micropores only requires that the micropores are round and have smooth side walls, and that the amphiphilic molecule layer is easily formed.

Furthermore, in some embodiments, the first, second, and third structural layers of the preparation device body can be processed in layers and then assembled, or can be integrally formed (such as integral injection molding). Therefore, whether the preparation device is obtained by layered processing and then assembly, or integrally formed, it falls within the protection scope of the present invention.

For example, in some embodiments, teflon material can be manufactured by micro-injection molding; pmma can be manufactured by machining or micro-injection molding, delrin can be manufactured by machining or micro-injection molding, and parylene can be manufactured by physical vapor deposition, such as depositing parylene on the surface of other processed materials, with a deposition thickness of preferably 5 microns.

In some embodiments, the raw materials are selected from materials with certain hydrophobicity and lipophilicity, thereby reducing the pretreatment steps and simplifying the film-forming method in the process of preparing the amphiphilic molecular membrane. For example, polyformaldehyde is preferably used to make the preparation device. The hydrophobicity and lipophilicity of polyformaldehyde meet the film-forming requirements, so no pretreatment is required.

In some embodiments, the material of the electrodes in the electrode layer can be silver, gold, platinum and titanium electrodes, and the electrode layer can be manufactured by magnetron sputtering or PCB surface treatment process.

Furthermore, in some embodiments, when the number of microholes is large, at least one third opening is also provided on the flow channel.

Since the lowest point of the third opening (i.e., the second end of the third opening connected to the flow channel) is higher than the highest point of the liquid level in the flow channel, or in other words, the second end of the third opening is flush with the liquid level in the flow channel, the meniscus in the flow channel can maintain a constant liquid level during movement, thereby ensuring the stability of the shape without being affected by the third opening.

Furthermore, in some embodiments, in order to facilitate sample loading, the first opening and the third opening may be arranged to be larger at the top and smaller at the bottom. For example, the first end of the first opening (an end arranged on the surface of the preparation device and used for sample loading) is larger than the second end of the first opening (an end connected to the flow channel).

Specifically, in some embodiments, referring to FIG. 1c , the third opening 16 is disposed on a side close to the first opening 12. This embodiment is equivalent to providing a double injection port on the flow channel (i.e., providing two injection ports, the first opening and the third opening). At this time, the first opening (equivalent to the first injection port) is used to add the membrane solution, and to inject/withdraw the second polar solution through the reciprocating injection pump, and the second opening (equivalent to the second injection port) is used to add the membrane solution.

For example, in some embodiments, when the micropores through which the meniscus passes do not form a film, it indicates that the concentration of amphiphilic molecules in the meniscus is too low. At this time, the second polar solution is withdrawn at the first opening by a reciprocating syringe pump, so that the meniscus moves between the first opening and the third opening. In this process, the meniscus will flow through the micropores that have already formed a film again. Since the amphiphilic molecule layer (i.e., the film) itself is a very stable arrangement structure, after the amphiphilic molecule layer has been formed on the micropores, even if the meniscus repeatedly passes through the micropores many times, the thickness of the amphiphilic molecule layer on the corresponding micropores will not increase. In other words, the process of the meniscus moving back to the first opening does not affect the previously formed film.

Furthermore, when the meniscus moves to between the first opening and the third opening, a certain amount of membrane solution is added to the third opening (equivalent to the second injection port), and then the meniscus located between the first and third openings is controlled by the injection pump to continue to move in the direction from the first opening to the third opening, and is combined with the newly added membrane solution to form a new meniscus. The new meniscus continues to pass through the unfilmed micropores under the push of the second polar solution and forms a film on the micropores.

Therefore, one or more injection ports can be correspondingly arranged on the flow channel based on the number of micropores. For example, when the number of micropores is relatively small and the change in the concentration of amphiphilic molecules has a relatively small effect on film formation (or the effect of the concentration change can be ignored in actual application), only one injection port is required on the flow channel, such as only the first opening for injection.

For another example, when the number of micropores is set relatively large, as the meniscus passes through a certain number of micropores, the concentration of amphiphilic molecules in the meniscus gradually decreases, so that it is impossible to form a film at the subsequent micropores. In this case, multiple injection ports can be set on the flow channel. For example, referring to FIG. 1c, a third opening is added to the flow channel for adding liquid samples. In other words, by adding injection ports to the flow channel, the number of micropores can be increased, so that the preparation device can form more amphiphilic molecule layers at one time.

Of course, in other embodiments, the membrane solution may be directly added through the third opening, and the first opening is only used to inject/withdraw the second polar solution through a reciprocating syringe pump.

In other embodiments, a new meniscus can be directly formed through the third opening (so that the previously formed meniscus is located between the first opening and the third opening and no longer participates in subsequent film formation), and the newly generated meniscus moves and passes through the unfilmed micropores and forms a film on the micropores.

Preferably, in order to more accurately control the above process, it can be achieved through automated control (such as a program controlled by computer software).

Embodiment 2

Based on Example 1, referring to FIG. 5 , the present invention further provides a novel method for preparing an amphiphilic molecular film (film forming method), comprising the following steps:

S1 provides a preparation device for preparing a plurality of amphiphilic molecular membranes, wherein the preparation device is provided with a plurality of micropores and a flow channel capable of allowing a solution to flow into the micropores, and the preparation device also includes an electrode layer, so that the solution flowing into the micropores can contact the electrode layer, wherein the cross section of the flow channel is preferably square or quasi-square;

S2: adding a first polar solution into the flow channel, so that the first polar solution enters at least one micropore and contacts the electrode layer;

S3: adding the membrane solution and the second polar solution into the flow channel in sequence, so that the membrane solution forms a meniscus in the flow channel (see FIG. 7a and FIG. 7b );

S4: adding a second polar solution into the flow channel, so that the second polar solution pushes the membrane solution to move and flow through at least one micropore where the first polar solution contacts the electrode layer, and the amphiphilic molecules in the membrane solution form an amphiphilic molecule layer on the corresponding micropore (i.e., the micropore where the first polar solution is contained and the first polar solution contacts the electrode layer) (specifically, the amphiphilic molecules form an amphiphilic molecule layer based on self-assembly ability). The contact angle of the inner surface of the flow channel is about 65° to 120°.

It can be understood that, in some embodiments, the above steps S3 and S4 can be performed continuously (or, in actual operation, steps S3 and S4 can be one step). Of course, in other embodiments, steps S3 and S4 can also be performed in steps.

In this embodiment, the preparation device preferably uses a material with a certain hydrophobicity, and the contact angle of the material on the inner surface of the flow channel in the preparation device is about 65°-120°, so that the membrane solution added to the flow channel can form a meniscus under the joint action of the inner surface of the flow channel, the polar solution and the air in the flow channel. Among them, the cross-section of the flow channel is preferably square or quasi-square. At this time, the flow channel will produce a certain resistance to the movement of the membrane solution (or the meniscus formed by the membrane solution), so that the movement speed of the membrane solution in the flow channel will not be too fast, and the movement speed of each position on the membrane solution is relatively uniform (or the difference in flow speed at each position has little effect on the stability of the meniscus), so that the meniscus can maintain a stable shape during the movement.

Specifically, the inner surface of the flow channel and the surface of the micropore (i.e., the side wall of the micropore) are both made of a material with a contact angle between approximately 65° and 120° (preferably, made of polyformaldehyde), so that when the membrane solution moves to the corresponding micropores, an amphiphilic molecular membrane can be smoothly formed at the micropores (specifically, at the first end of the micropores) without the need to pre-treat the device (such as the micropores arranged at the flow channel) in advance.

Among them, in the process of formation of meniscus and movement of meniscus by membrane solution, the amount (volume) of membrane solution added and the movement speed of the meniscus formed by the membrane solution are also very important to the formation and movement process of the meniscus, and for technicians in this field, it is achievable to select appropriate amount of membrane solution added and movement speed through preliminary experiments.

For example, in the step of selecting or predicting the amount of membrane solution added, the main parameters to be considered are: Reynolds number (Re), which is a dimensionless number that can be used to characterize the flow of fluid. The calculation method of Reynolds number is: Re = ρvd/μ, where v, ρ, and μ are the flow velocity (equivalent to the movement velocity of the membrane solution), density, and viscosity coefficient of the fluid, respectively, and d is the characteristic length (equivalent to the length of the flow channel). Among them, the density and viscosity coefficient of the membrane solution can be obtained through experimental measurement. To maintain the stability of the meniscus, the Reynolds coefficient of the fluid needs to be small (the smaller the Reynolds number, the more stable the fluid flow), that is, the laminar flow state.

Therefore, the present invention preferably makes the Re of the fluid <2300, thereby, the Reynolds number can be used to make a preliminary restriction on the relationship between the length of the flow channel and the movement speed of the membrane solution (therefore, when the membrane solution is selected and the flow channel length of the device (the flow channel length is related to the setting of the micropore array) is determined, the movement speed of the membrane solution can be preliminarily restricted based on the Reynolds number, that is, the movement speed of the membrane solution is constrained by the membrane solution and the flow channel length). In other words, for those skilled in the art, after the membrane solution is selected, a preparation device is selected on this basis for preliminary experiments, and a more suitable movement speed of the membrane solution can be screened out. Based on the movement speed screened out in the preliminary experiment, the injection speed of the polar solution (such as the second polar solution) is designed accordingly, and the preset injection speed of the second polar solution (such as the injection speed of the second polar solution in steps S3 and S4) can be obtained. Of course, the injection speed of the second polar solution can also provide the operating experience of the staff to make a preliminary estimate, and then conduct a preliminary experiment to obtain a suitable injection speed.

Further, in some embodiments, the contact angle of the material selected for the inner surface of the flow channel is about 75° to about 95°. Specifically, in some embodiments, a plurality of micropores are disposed on the inner surface of the flow channel, that is, the contact angle of the material selected for the inner surface of the flow channel and the micropore surface is about 75° to about 95°.

Preferably, the inner surface of the flow channel and the microporous surface are both made of polyoxymethylene.

Further, in some embodiments, during the process of forming the meniscus (i.e., step S3), the injection rate of the second polar solution is about 10 μL/min to about 50 μL/min, and during the process of passing through the micropore array after the meniscus is formed (i.e., step S4), the injection rate of the second polar solution is about 200 μL/min to about 500 μL/min.

In order to more clearly illustrate the technical solution adopted by the present invention, the formation of the contact angle and the meniscus is briefly described below:

Referring to Figure 9a, the contact angle refers to the angle between the solid-liquid interface, the liquid L, and the gas G at the junction of the three phases, from the solid-liquid interface through the liquid to the gas-liquid interface. If θ<90°, the liquid is easier to wet the solid, and the smaller the angle, the better the wettability; if θ>90°, the liquid is not easy to wet the solid and is easy to move on the surface. Specifically, when θ=0, it is completely wetted; when θ<90°, it is partially wetted or wetted; when θ=90°, it is the dividing line between wettability and non-wetting; when θ>90°, it is non-wetting; when θ=180°, it is completely non-wetting.

Referring to FIG9b, the liquid pressure rising along the capillary wall in the figure is Among them, 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 acceleration of gravity, and h is the height of the liquid surface. Based on the definition of contact angle, we can know that: For example, when the contact angle θ is less than 90°, the solution can form a meniscus in the capillary, and the meniscus formed at this time is concave. At the same time, it can be known that the radius of curvature of the formed meniscus is related to the radius r of the capillary. When the contact angle θ is greater than 90°, the solution can form a convex meniscus in the capillary.

Specifically, the surface tension of the polar solution (such as the second polar solution) added to the inside of the flow channel causes the membrane solution to spread on the liquid surface of the polar solution (i.e., the end in contact with the membrane solution). Since the inner surface of the flow channel is made of a material with a certain hydrophobicity, the inner surface of the flow channel has a certain adsorption effect on the membrane solution. At this time, referring to FIG7b, one side of the membrane solution is the polar solution (the second polar solution 2), and the other side is air 8. Among them, the liquid surface has a contraction force, and the molecules in the interface layer where the membrane solution contacts the air are subjected to a pulling force directed toward the inside of the liquid, so that the membrane solution gradually forms a meniscus 4 inside the flow channel, as shown in FIG7b).

Referring to FIG. 7a, amphiphilic molecules 5 are dissolved in the membrane solution, wherein the amphiphilic molecules 5 have a hydrophilic end 52 and a hydrophobic end 51 (see FIG. 6). Since the amphiphilic molecules have the ability to self-assemble, when the membrane solution flows through the micropores, the hydrophilic end 52 of the amphiphilic molecules inside will point to and contact the first polar solution, while the hydrophobic end 51 will combine with the hydrophobic end 51 of another layer of amphiphilic molecules to form an amphiphilic molecule layer, as shown in FIG. 7a and FIG. 8. In this embodiment, by controlling the movement speed of the membrane solution, it is possible to avoid the amphiphilic molecules from accumulating thicker and thicker at the micropores (as shown in FIG. 10 (a)), which makes it impossible to embed the pores.

In this embodiment, the membrane solution is pushed to move by the second polar solution, so that the membrane solution passes through at least one micropore and forms an amphiphilic molecular membrane at at least one micropore. The flow rate of the second polar solution (or the sampling speed/injection speed of the second polar solution) is relatively controllable, so the movement speed (i.e., flow speed) of the membrane solution in the flow channel can be controlled by controlling the sampling speed of the subsequent addition of the second polar solution, thereby controlling the formation process of the amphiphilic molecular membrane, avoiding the membrane solution flowing too fast and failing to successfully form an amphiphilic molecular membrane, or the membrane solution flowing too slowly and forming an overly thick amphiphilic molecular membrane.

By using a polar solution to push the meniscus (membrane solution) to form a film at the micropore, the moving speed of the membrane solution can be controlled more accurately (for example, the injection speed of the polar solution is controlled by a pipette gun or a syringe pump, so as to control the moving speed of the membrane solution), so that the membrane solution stays at different micropores for the same or similar time, avoiding the membrane solution staying in some micropore areas for too long to form a thick film, or moving too fast in some micropore areas without successfully forming a film. Therefore, the present invention can directly form an amphiphilic molecular layer with a film thickness that meets the use requirements by more accurately controlling the moving speed of the membrane solution, without the need to thin the film after film formation (such as thinning the film from thick to thin, as shown in (a)-(d) in Figure 10). In other words, the film forming method of the present invention can form a film once, and the film forming effect is better. For example, by controlling the injection speed of the polar solution and then controlling the moving speed of the membrane solution, a film of suitable thickness can be directly formed, such as directly preparing the film shown in (d) in Figure 10.

For example, in some embodiments, the injection speed of the second polar solution is controlled by a syringe pump to control the movement speed of the membrane solution. In this embodiment, the method provided is more controllable (for example, compared to the bubble extrusion film forming of the prior art). Of course, other injection methods can also be used to control the injection speed of the solution, and any method that can control the injection speed of the solution belongs to the protection scope of the present invention.

Furthermore, in some embodiments, a reciprocating syringe pump is provided to add (inject) the second polar solution. For example, the syringe pump model selected is Harvard Apparatus 4400.

Preferably, in some embodiments, before S1, a step is included: performing a wetting treatment on at least one micropore.

In this embodiment, the wetting treatment is used to allow the liquid (such as the first polar solution) to spread on the solid surface (such as the micropore surface), thereby expanding the contact area between the polar solution and the inner wall of the flow channel and reducing the contact angle, so that the first polar solution can smoothly enter at least one micropore and fill the micropore.

For example, in some embodiments, the wetting process is an electrowetting process.

For example, in some embodiments, the wetting process is a liquid wetting process.

Specifically, in some embodiments, a low surface energy liquid (such as ethanol, etc.) is first introduced into the flow channel. The low surface energy liquid can flow into very small pores, such as micropores. Then, a polar solution is added to fill the micropores. If a low surface energy liquid is not added in advance, the polar solution may not be able to flow directly into the micropores. Then, a first polar solution is introduced into the flow channel so that the first polar solution fills at least one micropore, and the low surface energy liquid is diluted and discharged. The low surface energy liquid previously introduced makes it easier for the first polar solution to fill the micropores, wherein the low surface energy liquid includes: ethanol, etc.

In some other embodiments, step S2 further includes the step of: after adding the first polar solution into the flow channel, ultrasonically treating the preparation device so that the first polar solution enters the micropores under the action of the sound waves.

Furthermore, in some embodiments, the membrane solution is a non-polar solution containing amphiphilic molecules.

Furthermore, in some embodiments, the amphiphilic molecule can be a phospholipid or a polymer. For example, the amphiphilic molecule can also be a polymer material synthesized by physical and chemical methods, ABA triblock copolymer (PMOXA-PDMS-PMOXA dimethyloxazoline-polydimethylsiloxane-dimethyloxazoline), or a mixture of phospholipids and polymers.

Furthermore, in some embodiments, the non-polar solution can be an alkane organic solvent, including decane, hexadecane, pentane, or a mixture thereof, and the non-polar solution is used to dissolve phospholipids or high molecular triblock copolymers to prepare a membrane solution.

Furthermore, in some embodiments, the second polar solution and the first polar solution may be the same or different polar solutions. The selected polar solution only needs to satisfy the conductive function and enable the nanopore protein to be inserted into the amphiphilic molecule layer.

Furthermore, in some embodiments, the nanopore protein may be pre-added in a polar solution (such as a first polar solution or a second polar solution).

Of course, in other embodiments, nanoporous protein (such as a solution including nanoporous protein) may be added into the flow channel after the amphiphilic molecule layer is formed.

Furthermore, in some embodiments, the polar solution is an electrolyte in the gene sequencing process, comprising electrolytes and/or polyelectrolytes to effectively improve the ion exchange life; it may also comprise redox pairs, and/or combinations of redox pairs that can be partially oxidized or reduced to provide redox pairs, such as iron/ferrocyanide; it may also be a cross-linked agarose gel, and/or a cross-linked sodium alginate gel; it may also comprise a buffer to adjust the pH of the aqueous solution medium, and suitable buffers (buffers) include but are not limited to phosphate buffer PBS, 4-bis-2-ethanesulfonic acid buffer PIPES, N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid buffer (HEPES), etc.

Furthermore, in some embodiments, the method further comprises the steps of:

Electricity is supplied to the polar solution through the electrode layer and the common electrode, so that the nanopore protein is embedded (inserted) into the amphiphilic molecule layer under the action of voltage.

For example, in some embodiments, the common electrode is inserted into the second opening (or the first opening or the electrode insertion port) of the device, and then the polar solution is electrified through the common electrode and the preset electrode layer. For example, the common electrode is a pin, a PET flexible electrode, or an electrode designed integrally with the microfluidic chip fixture connected to the circuit part.

Specifically, in some embodiments, when the inner diameter of the flow channel is set larger (or, the flow channel is wider), in order to successfully form the meniscus, the flow channel of the preparation device involved in the method includes: a first opening, a second opening, and the first opening and the second opening are connected through the first flow channel 15. Referring to FIG. 11a, the inner diameter of the first opening 12 is relatively small, and the inner diameter of the first flow channel 15 is relatively wide. Therefore, there is a region of size change at the first end of the flow channel (i.e., the end connected to the first opening), that is, the inner diameter of the flow channel gradually increases in the direction from the first opening 12 to the first flow channel 15 (or, the inner diameter of the first end of the flow channel gradually increases in the direction away from the first opening).

When the membrane solution is added into the flow channel through the first opening, the membrane solution first enters the first end of the flow channel with a smaller inner diameter. At this time, the internal space of the flow channel is narrow, the meniscus formed by the membrane solution is thicker, and the shape is relatively stable. Therefore, the movement speed of the membrane solution in the early stage can be set faster (specifically, since there is a region where the inner diameter changes from small to large between the entrance of the flow channel and the inside of the flow channel. At this time, the injection speed of the polar solution is relatively slow, but since the cross-section of the flow channel is relatively small, even if the injection speed of the polar solution is relatively slow, the meniscus can have a relatively fast movement speed). When the membrane solution gradually moves to the area with the largest inner diameter of the flow channel (such as the middle area between the two ends of the flow channel, that is, the area with equal inner diameters), the meniscus formed by the membrane solution is gradually stretched and thinned, and the stability of the meniscus is relatively reduced. At this time, it is necessary to appropriately reduce the movement speed of the membrane solution (at this time, since the cross-section of the flow channel corresponding to the micropore area is larger, when the polar solution maintains the original injection speed, the movement speed of the meniscus will also decrease; of course, the injection speed of the polar solution can be reduced to further reduce the movement speed of the membrane solution). In this embodiment, the gradual change in size of the flow channel makes the shape of the meniscus relatively stable during its formation and change, and not easily damaged. The different states of the meniscus during its movement in the flow channel are shown in the first, second, and third states 4a, 4b, and 4c of the meniscus in Figure 11b.

For example, in some embodiments, referring to FIG. 11a, the region where the inner diameter of the flow channel changes (i.e., the region with unequal inner diameters, i.e., the first end of the flow channel) includes: a first change region and a second change region connected, wherein the curve setting of the side wall of the first change region (near the first opening) is similar to a partial arc of a circle with a radius of R1 (or, the vertical projection of the side wall of the first change region is set in a curve), and the curve setting of the side wall of the second change region is similar to a partial arc of a circle with a radius of R2 (or, the vertical projection of the side wall of the second change region is set in a curve). Among them, R1 and R2 are equal (of course, in other embodiments, R1 and R2 may also be unequal, as long as the inner diameter of the flow channel can achieve the formation and movement of the meniscus).

In some embodiments, the second end of the flow channel connected to the second opening is configured in the same or similar manner as the first end of the flow channel.

For example, in a specific embodiment (using a preparation device with 8*32 micropores), the preparation method specifically includes the steps of:

First, pure ethanol is added into the flow channel so that the ethanol enters at least one micropore, and then a polar solution is added into the flow channel to dilute and drain the ethanol, so that the polar solution fills at least one micropore and contacts the electrode layer (equivalent to the first electrode);

Adding about 20 μl to about 50 μl of membrane solution into the first opening;

Adding about 20 microliters to about 50 microliters of a polar solution into the first opening so that the membrane solution forms a meniscus in the flow channel;

About 300 microliters of polar solution are added to the first opening through a syringe pump at a preset injection speed (flow rate); specifically, the first 100 microliters of polar solution adopts a flow rate of 10 microliters per minute (during this process, a meniscus is formed and gradually becomes thinner), and the last 200 microliters of polar solution adopts a flow rate of 5 microliters per minute, so that the meniscus can maintain a stable shape and flow through the micropore array at a uniform speed, and an amphiphilic molecule layer can be directly formed on 50% to 80% of the micropores on the micropore array.

Alternatively, in another specific embodiment (using a preparation device having 16 microwells), the preparation method comprises the steps of:

First, pure ethanol is added into the flow channel, and then a polar solution is added to dilute and drain the ethanol, so that the micropores are filled with the polar solution and the polar solution is in contact with the electrode layer;

adding about 8 to about 12 microliters of membrane solution to the first opening;

Then, adding about 8 to about 12 microliters of a polar solution to the first opening so that the membrane solution forms a meniscus;

Then, based on the preset injection speed (flow rate), about 60 microliters of polar solution are added to the first opening, specifically, the first 15 microliters of polar solution adopts a flow rate of 4 microliters/minute, and the last 45 microliters of polar solution adopts a flow rate of 2 microliters/minute, so as to maintain the stability of the meniscus shape and enable the membrane solution to flow through the micropore array at a uniform speed. Among them, 50% to 80% of the micropores on the micropore array can directly form an amphiphilic molecule layer.

Of course, the film forming method and preparation device provided by the present invention do not completely exclude pretreatment operations. For example, based on different detection requirements, those skilled in the art may also need to add pretreatment steps accordingly.

Furthermore, in some embodiments, the method further comprises the step of: detecting the amphiphilic molecule layer.

For example, by inputting a triangle wave signal into the amphiphilic molecule layer, a corresponding square wave signal is obtained, and the upper and lower peak values of the square wave signal can be used to determine whether the amphiphilic molecule layer is formed and whether the amphiphilic molecule layer meets the requirements. Specifically, when it is detected that the amphiphilic molecule layer is too thick, the concentration of the membrane solution can be appropriately reduced.

Specifically, a commercial AXON 1550B instrument is used to detect electrical signals or output triangular waves to measure the capacitance at both ends of the membrane to determine whether an amphiphilic molecular layer is formed.

It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and should all be included in the scope of the claims and specification of the present invention.

Claims (10)

1. A method for preparing multiple amphiphilic molecular layers, characterized in that it comprises the following steps: S1 provides a preparation device for preparing multiple amphiphilic molecule layers, wherein the preparation device is provided with multiple micropores and flow channels capable of allowing a solution to flow into the micropores, and the preparation device also includes an electrode layer, so that the solution flowing into the micropores can contact the electrode layer; S2: adding a first polar solution into the flow channel, so that the first polar solution enters at least one of the micropores and contacts the electrode layer; S3 adds the membrane solution and the second polar solution into the flow channel in sequence, so that the membrane solution can be moved under the combined action of the inner surface of the flow channel, the second polar solution and the air in the flow channel. Forming a meniscus; wherein the membrane solution comprises: a non-polar solution, and amphiphilic molecules, wherein the amphiphilic molecules comprise: phospholipids, or polymers, or a mixture of phospholipids and polymers; S4 adds the second polar solution into the flow channel based on a preset injection speed to control the moving speed of the meniscus, so that the second polar solution pushes the meniscus to move and flows through at least one of the micropores where the first polar solution contacts the electrode layer, and the amphiphilic molecules in the meniscus form the amphiphilic molecule layer on the corresponding micropores. 2 . The preparation method according to claim 1 , characterized in that at least one electrode bump is also provided on the electrode layer. 3. The preparation method according to claim 1 is characterized in that the cross-section of the flow channel is square or quasi-square. 4 . The preparation method according to claim 1 , wherein the flow channel comprises a first opening, and an inner diameter of a first end of the flow channel gradually increases in a direction away from the first opening. 5. The preparation method according to claim 1 is characterized in that, before S2, it also includes the step of: wetting at least one of the micropores; or, in S2, it includes the step of: after adding the first polar solution into the flow channel, ultrasonically treating the preparation device. 6. The preparation method according to claim 1, characterized in that a third opening is further provided on the flow channel near the first opening, and correspondingly, the method further comprises: When the meniscus moves to between the first opening and the third opening, the membrane solution is added to the third opening, and then the meniscus is controlled to continue to move in the direction from the first opening to the third opening, and is combined with the newly added membrane solution to form a new meniscus, and the new meniscus continues to pass through the micropore under the push of the second polar solution. 7. A preparation device for multiple amphiphilic molecular layers, characterized in that the preparation device is provided with multiple micropores and flow channels that allow solutions to flow into the micropores, and the device is also provided with an electrode layer so that the solution flowing into the micropores can contact the electrode layer, the flow channel includes a first opening, and the inner diameter of the first end of the flow channel gradually increases in the direction away from the first opening, so that when a membrane solution and a second polar solution are added to the flow channel in sequence, the membrane solution can form a meniscus under the joint action of the inner surface of the flow channel, the second polar solution and the air in the flow channel, and when the second polar solution is added to the flow channel based on a preset injection speed, the second polar solution can control the moving speed of the meniscus; wherein the membrane solution includes: a non-polar solution, and amphiphilic molecules; the amphiphilic molecules include: phospholipids, or polymers, or a mixture of phospholipids and polymers. 8. The preparation device according to claim 7 is characterized in that at least one electrode bump is further provided on the electrode layer; and/or the cross-section of the flow channel is square or quasi-square. 9 . The preparation device according to claim 7 , characterized in that at least one third opening is further provided on a side of the flow channel close to the first opening. 10 . The preparation device according to claim 9 , wherein the first opening and/or the third opening is larger at the top and smaller at the bottom.