A method and apparatus for preparing a plurality of layers of amphiphilic molecules

Abstract

This invention relates to a method and apparatus for rapidly preparing amphiphilic molecular layers, comprising the following steps: S1, providing an apparatus for preparing multiple amphiphilic molecular layers, the apparatus having multiple micropores and channels allowing solution to flow into the micropores, and including an electrode layer so that the solution flowing into the micropores can contact the electrode layer; S2, adding a first polar solution into the channels, 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 channels, so that the membrane solution forms a meniscus within the channels; S4, adding the second polar solution into the channels, 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, thereby forming amphiphilic molecular layers on the corresponding micropores. The method provided by this invention is simple and safe to operate, and the selected apparatus has low production costs.

Description

[0001] Divisional application

[0002] This application is a divisional application of Chinese invention patent application with application number CN2022103914733, application date April 14, 2022, entitled "A method and apparatus for preparing an amphiphilic molecular layer". Technical Field

[0003] This invention relates to the field of amphiphilic molecular membranes, and more specifically to a preparation method and apparatus for rapidly forming multiple amphiphilic molecular layers. Background Technology

[0004] Nanoporous proteins require stable embedding on a phospholipid or polymeric thin film, such as an amphiphilic molecular layer (i.e., an amphiphilic molecular membrane), to allow the DNA sequence to pass through, thus achieving the purpose of DNA sequence detection. However, some current methods (i.e., film-forming methods) and devices for preparing amphiphilic molecular layers still have some problems in application.

[0005] Existing film formation methods mainly include the folded bilayer formation method (Montal & Mueller's method), dip-tip method, coating method, patch-clamp method, and water-in-oil microdrop interface method, etc. These methods, such as the folded bilayer method, dip-tip method, and coating bilayer method, often produce initially thick films that require thinning treatments, such as organic solvent evaporation, physical spreading, or air pressure extrusion. Thinning treatment is crucial for controlling film thickness, but the process is complex and difficult to control (e.g., ensuring uniform spreading during physical spreading is challenging). Furthermore, existing film formation methods typically involve pretreatment of the preparation equipment, such as fluorine plasma treatment and silanization treatment. These pretreatment operations require the use of highly hazardous chemicals and must be conducted in laboratories with high construction and maintenance costs, posing potential threats to the safety of operators and the surrounding environment. In addition, pretreatment requires precise calculation of the amount of coating used; too much or too little coating will affect the experimental results. The devices for preparing amphiphilic molecular membranes are usually small chips with tiny internal structures (such as micropores). Therefore, the amount of coating used is also very small. Applying a very small amount of coating to such a tiny structure is not simple to operate. As a result, it is difficult to avoid problems of applying too much or too little coating during the coating process.

[0006] For example, Chinese invention patent application number 201480056839.5 discloses a biochip and a film-forming method for the biochip. The film-forming method includes the steps of adding a liquid containing lipid molecules (i.e., amphiphilic molecules) to the chip surface, then separating the liquid with bubbles to distribute the lipid molecules on the chip surface, and thinning the lipid layer by the bubbles. However, bubble generation often requires manual control (e.g., preparing bubbles with a pipette). This process is difficult to automate because manual operation makes it difficult to control the bubble size and ensure bubble morphology stability, resulting in poor repeatability. Furthermore, this method also requires pretreatment.

[0007] For example, Chinese invention application number CN200880126160.3 discloses a method for forming a layer of aqueous solution separating two volumes. This method involves allowing an aqueous solution containing amphiphilic molecules to flow through a substrate to cover grooves (i.e., micropores), so that the aqueous solution can cross the grooves to form amphiphilic molecules. Although the technique of allowing the aqueous solution to flow through the grooves to form amphiphilic molecules is easy to implement, the prepared film is relatively thick and requires subsequent thinning treatment. In addition, this method also involves a pretreatment step. Summary of the Invention

[0008] To partially solve or alleviate the aforementioned technical problems, the first aspect of this invention is to provide a method for preparing an amphiphilic molecular layer, comprising the following steps:

[0009] S1 provides a preparation apparatus for preparing multiple amphiphilic molecular layers. The preparation apparatus has multiple micropores and channels that allow a solution to flow into the micropores. The preparation apparatus also includes an electrode layer, which allows the solution flowing into the micropores to contact the electrode layer. The cross-section of the channels is square or quasi-square.

[0010] S2 adds 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;

[0011] S3 sequentially adds a membrane solution and a second polar solution into the flow channel, causing the membrane solution to form a meniscus within the flow channel;

[0012] S4 adds the second polar solution into the flow channel based on a preset injection rate, so that the second polar solution pushes the membrane solution to move and flow through at least one micropore where the first polar solution is in contact with the electrode layer, and the amphiphilic molecules in the membrane solution form an amphiphilic molecular layer on the corresponding micropore;

[0013] The contact angle of the inner surface of the flow channel is approximately 65° to approximately 120°.

[0014] In some embodiments, prior to step S2, the step further includes: wetting at least one of the micropores;

[0015] In some embodiments, step S2 includes: after adding a first polar solution into the flow channel, ultrasonic treatment of the preparation device.

[0016] In some embodiments, the wetting treatment includes: electrowetting treatment.

[0017] In some embodiments, the wetting treatment includes: liquid wetting treatment.

[0018] In some embodiments, the membrane solution comprises: a nonpolar solution and an amphiphilic molecule.

[0019] In some embodiments, the amphiphilic molecule may optionally include: phospholipids, or polymers, or a mixture of phospholipids and polymers.

[0020] In some embodiments, the nonpolar solution may optionally include: alkane-based organic solvents.

[0021] In some embodiments, the first polar solution comprises an electrolyte and / or a polyelectrolyte.

[0022] In some embodiments, the first polar solution comprises: a redox pair, and / or a combination of redox pairs that can be partially oxidized or reduced to provide a redox pair.

[0023] In some embodiments, the first polar solution comprises: cross-linked agarose gel, and / or cross-linked sodium alginate gel.

[0024] In some embodiments, the first polar solution includes a buffer for adjusting the pH.

[0025] In some embodiments, the second polar solution comprises an electrolyte and / or a polyelectrolyte.

[0026] In some embodiments, the second polar solution comprises: a redox pair, and / or a combination of redox pairs that can be partially oxidized or reduced to provide a redox pair.

[0027] In some embodiments, the second polar solution comprises: cross-linked agarose gel, and / or cross-linked sodium alginate gel.

[0028] In some embodiments, the second polar solution includes a buffer for adjusting the pH.

[0029] In some embodiments, the preparation apparatus is further provided with a common electrode, which is in contact with the second polar solution. Accordingly, the method further includes the step of:

[0030] By passing an electric current through the electrode layer and the common electrode to the first polar solution and the second polar solution, the nanoporous protein is inserted into the amphiphilic molecular layer.

[0031] In some embodiments, in step S3, the injection rate of the second polar solution is from about 10 μL / min to about 50 μL / min;

[0032] In some embodiments, in step S4, the injection rate of the second polar solution is from about 200 μL / min to about 500 μL / min.

[0033] In some embodiments, the inner surface material of the flow channel is polyoxymethylene.

[0034] A second aspect of the present invention is that an apparatus for preparing an amphiphilic molecular layer is also provided. The apparatus has a plurality of micropores and a flow channel that allows a solution to flow into the micropores. The apparatus also has an electrode layer that allows the solution flowing into the micropores to contact the electrode layer. The flow channel has a square or near-square cross-section, and the inner surface material of the flow channel may optionally be polyoxymethylene.

[0035] In some embodiments, the spacing between adjacent micropores is greater than approximately 0.4 mm.

[0036] Beneficial technical effects:

[0037] This invention provides a method (or film-forming method) and apparatus for rapidly preparing multiple amphiphilic molecular layers (i.e., amphiphilic molecular membranes, also simply "membranes"). Unlike existing technologies, this invention proposes a novel film-forming method where the membrane solution forms a meniscus within a flow channel, and a polar solution pushes the meniscus, allowing it to move within the flow channel and pass through micropores, thereby forming amphiphilic molecular layers (membranes) on the corresponding micropores.

[0038] Specifically, the preparation apparatus proposed in this invention uses a hydrophobic material that meets the film-forming conditions. Specifically, the contact angle of the material on the inner surface of the flow channel within the apparatus is approximately 65°-120°, allowing the membrane solution added into the flow channel to form a meniscus under the combined action of the inner surface of the flow channel, the polar solution, and the air within the flow channel. Preferably, the cross-section of the flow channel is square or near-square. In this case, the flow channel generates a certain resistance to the movement of the membrane solution (or the meniscus formed by the membrane solution), preventing the membrane solution from moving too quickly within the flow channel and ensuring that the movement speed at various positions on the membrane solution is relatively uniform (or, in other words, the difference in flow speed at different positions has little impact on the stability of the meniscus), thus enabling the meniscus to maintain a stable shape during movement.

[0039] This film-forming method, which uses a polar solution to propel a meniscus (membrane solution) to form a film at micropores, allows for precise control of the membrane solution's movement speed (e.g., controlling the injection rate of the polar solution using a pipette or syringe pump, thereby controlling the membrane solution's movement speed). This ensures that the membrane solution remains at different micropores for similar or equal times, preventing the membrane solution from remaining in some micropore areas for too long and forming a thick film, or moving too quickly in other micropore areas and failing to form a film. Therefore, this invention can efficiently and directly produce amphiphilic molecular layers of suitable thickness (or film thickness meeting usage requirements) by precisely controlling the membrane solution's movement speed, without requiring post-film thinning treatments (such as high-voltage breakdown or multiple film formation processes). In other words, the film-forming method of this invention can form a film in a single step.

[0040] Therefore, in practical applications, once parameters such as the concentration and migration speed of the membrane solution are selected (e.g., through preliminary experiments or based on the operator's experience), multiple experiments can be conducted under the same conditions and parameters, and the results (e.g., film formation) obtained from these multiple experiments show very little difference, indicating good repeatability of this method. In other words, the method provided by this invention can avoid or reduce the influence of manual operation on the experimental process, thereby avoiding or reducing uncontrollable factors during operation (i.e., this method has better controllability) and thus exhibits good repeatability.

[0041] Furthermore, the film-forming method proposed in this invention does not require pretreatment, making the film-forming method simpler and ensuring greater safety for workers during operation (as it does not involve the handling or use of hazardous materials).

[0042] Furthermore, the injection rate of the polar solution can be controlled by an injection pump, which can be automatically controlled by electronic devices (such as computers) (i.e., the film-forming method proposed in this 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, so as to further ensure the stability and uniformity of the film.

[0043] The film-forming method (and apparatus) provided by this invention can be applied to various testing needs in laboratories (e.g., suitable for different application scenarios such as research institutions and commercial testing companies). Because this invention can form multiple membranes at once, it can better meet commercial or market testing needs (e.g., human genome sequencing, virus sequencing). These types of tests have relatively long testing cycles and require large amounts of data, which increases the probability of operator errors during long-term operation. Furthermore, the method provided in this application can be combined with existing automated control systems to further improve the accuracy and reliability of the test results.

[0044] In existing technologies, to ensure that the solution containing amphiphilic molecules can cover (or flow through) all or multiple micropores to form multiple amphiphilic molecular layers, those skilled in the art typically add a larger amount of solution containing amphiphilic molecules to avoid some micropores failing to form amphiphilic molecular layers, thus resulting in a relatively thick film. To address this technical problem, those skilled in the art usually consider how to further process the film after formation (i.e., thinning) to make the film thinner, for example, by using high-pressure breakdown multiple film formation processes. Unlike existing technologies, this application adopts a different technical approach, proposing a novel film formation method. This method can form a film in one step, is simpler in operation, and avoids time-consuming operations such as pretreatment and thinning, resulting in a faster and more efficient method.

[0045] Furthermore, given the existing fabrication apparatus, those skilled in the art would find it difficult to conceive of the film-forming method proposed in this application. Firstly, the existing fabrication apparatus does not provide the conditions for meniscus formation; similarly, the existing methods and apparatus cannot guarantee the stable movement of the meniscus within the flow channel. Moreover, since the existing apparatus uses different materials than those used in this application and requires pretreatment, this pretreatment may make it even more difficult to form a meniscus within the flow channel. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0047] Figure 1a This is a schematic cross-sectional view of the preparation apparatus in an exemplary embodiment of the present invention;

[0048] Figure 1b This is a schematic diagram of an optional cross-sectional structure of the flow channel of the preparation apparatus in an exemplary embodiment of the present invention;

[0049] Figure 1c This is a schematic cross-sectional view of the preparation apparatus in another exemplary embodiment of the present invention;

[0050] Figure 2 This is a schematic diagram of the preparation apparatus in another exemplary embodiment of the present invention;

[0051] Figure 3 This is a schematic diagram of the preparation apparatus in an exemplary embodiment of the present invention;

[0052] Figure 4a This is a schematic diagram of the first state of the first polar liquid at the micropore of the preparation device in an exemplary embodiment of the present invention;

[0053] Figure 4b This is a schematic diagram of the second state of the first polar liquid at the micropore of the preparation device in an exemplary embodiment of the present invention;

[0054] Figure 4c This is a schematic diagram of the third state of the first polar liquid at the micropore of the preparation device in an exemplary embodiment of the present invention;

[0055] Figure 5 This is a schematic flowchart of a preparation method in an exemplary embodiment of the present invention;

[0056] Figure 6 This is a schematic diagram of the structure of an amphiphilic molecule;

[0057] Figure 7a This is a schematic diagram of the first structure of the meniscus within the flow channel;

[0058] Figure 7b This is a schematic diagram of the second structure of the meniscus within the flow channel;

[0059] Figure 8This is a schematic diagram of the structure of the amphiphilic molecular membrane formed at the micropores;

[0060] Figure 9a The relationship between contact angle and solid, liquid, and gas is shown;

[0061] Figure 9b This illustrates the process of liquid wetting a capillary tube;

[0062] Figure 10 A schematic diagram of the structure of membranes of different thicknesses formed at the micropores is shown;

[0063] Figure 11a A schematic diagram of the flow channel structure within the preparation apparatus according to an exemplary embodiment of the present invention is shown;

[0064] Figure 11b A schematic diagram showing the shape change of the meniscus during movement in an exemplary embodiment of the present invention is shown.

[0065] 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 bump, 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 solution, G is gas, and S is solid. Detailed Implementation

[0066] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0067] In this document, suffixes such as "module," "part," or "unit" used to denote elements are used only for the purpose of illustrative purposes and have no specific meaning in themselves. Therefore, "module," "part," or "unit" may be used interchangeably.

[0068] In this document, the terms "upper," "lower," "inner," "outer," "front end," "rear end," "both ends," "one end," and "the other end," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the present invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0069] In this document, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "connected," and "linked" should be interpreted broadly. For example, "linked" can refer to 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 connection; it can be a direct connection or an indirect connection through an intermediate medium; or it can refer to the internal connection between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0070] In this article, an "amphiphilic" molecule refers to a compound that possesses both hydrophilic and lipophilic properties, having a hydrophilic head and a hydrophobic tail. The hydrophilic head is generally composed of polar groups, such as choline or ammonium salts; the hydrophobic tail is generally composed of long fatty acid chains. In this article, "amphiphilic" and "amphiphilic" are used synonymously. Amphiphilic molecules can be lipid molecules. A typical amphiphilic molecular membrane (or amphiphilic molecular layer, also simply "membrane") can be a lipid bilayer, which is a bilayer formed by two opposing lipid monolayers. The two lipid monolayers are self-assembled so that the hydrophobic tails face each other to form a hydrophobic interior, while the hydrophilic heads face outwards (each side is a polar hydrophilic environment). The lipids forming the lipid bilayer can include any suitable lipid, such as 1,2-diphydanyl-sn-glycerol-3-phosphatidylcholine or diaphytylphosphatidylcholine (DPhPC). Amphiphilic molecules can be chemically modified or functionalized to promote polynucleotide coupling. Amphiphilic molecules can also be polymers synthesized through physicochemical methods, such as ABA triblock copolymers (PMOXA-PDMS-PMOXA dimethyloxazoline-polydimethylsiloxane-dimethyloxazoline). Amphiphilic molecules can be mixtures.

[0071] In this article, "nonpolar solvent" or "nonpolar solution" refers to a compound or mixture of compounds that is immiscible with water. A nonpolar solvent can be an oil, and more specifically, a pure alkane, such as n-hexadecane, n-decane, n-pentane, n-hexane, n-heptane, n-octane, or carbon tetrachloride. Other types of oil are also possible, such as silicone oil. More specifically, the oil can be methylphenyl silicone oil AR20 or hydroxyl-terminated polydimethylsiloxane PDMS-OH.

[0072] In this document, "polar aqueous solution" or "polar solution" refers to an aqueous solution containing water that is readily miscible with water and other polar solvents. A polar aqueous solution may include one or more solutes. For example, it may include a buffer capable of adjusting the pH of the polar aqueous solution. The buffer may include any suitable buffer, such as phosphate buffer (PBS), 4-bis-2-ethanesulfonic acid buffer (PIPES), or N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid buffer (HEPES). Polar aqueous solutions may also be electrolytes or polyelectrolytes to effectively enhance ion exchange lifetime. Polar aqueous solutions may also contain redox pairs or combinations of redox pairs that can be partially oxidized or reduced to provide redox pairs, such as iron / ferricyanide. Polar aqueous solutions may also be cross-linked agarose gels and sodium alginate gels.

[0073] In this article, "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 membranes.

[0074] In this article, "square" includes polygons whose adjacent sides form an angle of approximately 90° or close to 90°. For example, a square is a quadrilateral, such as a square, or a rectangle (e.g., ...). Figure 1b (as shown in a) etc. Of course, the "square" in this article does not necessarily need to be a standard square, rectangle, or other polygon. For example, the opposite sides of the "quadrilateral" within a square can be parallel or non-parallel; correspondingly, the adjacent sides of the "quadrilateral" can be perpendicular or non-perpendicular. "Square-like" includes squares with chamfered corners at the included angles of adjacent sides, or squares with adjacent sides connected by arcs, such as quadrilateral-like shapes, like rectangle-like shapes (e.g.,...). Figure 1b As shown in b, the top corners of the rectangle can be replaced with rounded arcs, or the top corners of a square can be replaced with rounded arcs, etc. For example, in this article, "the cross-section of the flow channel is square or square" means that the cross-section of the flow channel can be set as a rectangle, a square, a rectangle-like structure, or a square-like structure, etc. In order to avoid defects such as burrs on the inner surface of the flow channel during processing, it is preferable to set the cross-section of the flow channel as square, such as a rectangle-like structure or a square-like structure, etc.

[0075] In this document, the terms “about” or “approximately” typically mean + / -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 mean a value that includes the error range conventionally understood in the art.

[0076] In this document, some embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered as having specifically disclosed all possible sub-ranges and independent numerical values ​​within these ranges. For example, "contact angle from approximately 65 degrees to approximately 120 degrees" can be understood as having disclosed ranges such as contact angles of approximately 65-95 degrees, 95-105 degrees, 105-120 degrees, etc., and also disclosing independent numerical values ​​within these ranges, such as 65 degrees, 69 degrees, 75 degrees, 80 degrees, 90 degrees, 100 degrees, 110 degrees, and 120 degrees.

[0077] Example 1

[0078] The first aspect of this invention is that it provides an apparatus for rapidly forming an amphiphilic molecular film (amphiphilic molecular layer), see [link to previous section]. Figure 1a Figure 4 shows that the preparation device has multiple micropores 21 and flow channels 11 that allow the solution to flow into the micropores 21. The preparation device also includes an electrode layer 31, allowing the solution flowing into the micropores 21 (such as a polar solution, i.e., an electrolyte) to contact the electrode layer 31. Preferably, the cross-section of the flow channel is square or near-square. Of course, any cross-section of the flow channel that allows the membrane solution to form a meniscus and move in a meniscus shape is within the scope of protection of this invention. The micropores provide a growth platform for the membrane solution to form an amphiphilic molecular layer (amphiphilic molecular membrane, also referred to as "membrane").

[0079] In some embodiments, the inner surface of the flow channel is made of a polymer material, and the contact angle (i.e., the contact angle between the material and pure water) of the material selected for the inner surface of the flow channel is between approximately 65 degrees and approximately 120 degrees.

[0080] Preferably, in some embodiments, the contact angle of the inner surface of the flow channel is between approximately 75 degrees and 95 degrees.

[0081] Preferably, in some embodiments, the material used for the inner surface of the flow channel is polyoxymethylene (delrin).

[0082] Specifically, in some embodiments, the material used for the inner surface of the flow channel and the surface of the micropores (micropore sidewalls) is polyoxymethylene, which allows the membrane solution added into the flow channel to form a meniscus within the flow channel and to form an amphiphilic molecular layer at the micropores. Therefore, the preparation device in this embodiment does not require pretreatment during use.

[0083] Preferably, in some embodiments, the material used in the preparation apparatus is polyoxymethylene.

[0084] For those skilled in the art, selecting suitable preparation materials through preliminary experiments is feasible. Specifically, the contact angles of different materials can be measured using contact angle testing methods (such as shape image analysis or weighing methods) to select a suitable preparation material. Specifically, when the contact angle of a material is approximately 65°-120° (of course, as long as the selected material can form a meniscus in the flow channel it prepares), the flow channel prepared by that material is considered to have the ability to form a meniscus.

[0085] Furthermore, in some embodiments, the flow channel includes a first opening for adding a sample (i.e., adding a liquid, such as a membrane solution or a polar solution) into the flow channel.

[0086] Furthermore, in some embodiments, the flow channel further includes a second opening for discharging liquid from the flow channel.

[0087] Furthermore, in some embodiments, the flow channel is also provided with an electrode insertion port for inserting a common electrode.

[0088] Specifically, in some embodiments, referring to FIG1, the device includes: a body, the body including a first structural layer 1, a second structural layer 2 and a third structural layer 3 arranged sequentially, wherein the first structural layer 1 is provided with a flow channel 11 and one end of the flow channel 11 is provided with a first opening 12 for sample addition, the second structural layer 2 is provided with a plurality of micropores 21 spaced apart, and the third structural layer 3 is provided with an electrode layer (equivalent to the first electrode).

[0089] When a solution (such as a first polar solution) is added into the flow channel and the solution flows through at least one micropore, the solution displaces the air in at least one micropore (e.g., the solution can fill at least one micropore, see [reference]). Figure 4a The first polar solution 6 is filled in the micropores and is in contact with the electrode layer, so that the solution (such as the first polar solution) can conduct electricity under the action of the electrode.

[0090] In some embodiments, the second structural layer is bonded together with corresponding electrodes on the electrode layer by bonding, patterned cross-linking devices, or AZ4620, SU-8, and the flow channel on the upper layer is provided with only one common electrode.

[0091] Because the materials used for the inner surface of the flow channel and the sidewalls 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 sidewalls 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.

[0092] Therefore, in some embodiments, the first polar solution added may not completely fill the micropores, for example, see Figure 4b The first polar solution 6 forms droplets suspended at the first end of the micropore, and the droplets formed by the first polar solution 6 come into contact with the electrode layer 31.

[0093] Furthermore, in some embodiments, in order to ensure that the droplets formed by the first polar solution 6 can make contact with the electrode layer, at least one electrode bump 32 (such as a probe) is provided on the electrode layer 31, so that even when the droplets formed by the first polar solution are small in volume, they can still make contact with the electrode layer.

[0094] Furthermore, in some embodiments, in order to enable the droplets formed by the first polar solution to come into contact with the electrode layer, an electrode layer 31 for conducting electricity can be provided on the sidewall 22 of the micropore. Furthermore, electrode bumps 32 can be provided on the electrode layer 31.

[0095] Alternatively, in other embodiments, see Figure 4c First, the first polar solution introduced into the flow channel forms a thin solution film (layer) only at the first end of the micropore. At this time, in order to enable the first polar solution to contact the electrode layer, electrode bumps 32 (such as probes) can also be set on the electrode layer to ensure that the first polar solution can contact the electrode layer.

[0096] Specifically, in some embodiments, the inner diameter of the first end (i.e., the upper end, which is connected to the flow channel) of the micropore is larger than the inner diameter of the second end (i.e., the other end of the micropore). In this embodiment, the micropore is designed to be smaller at the top and larger at the bottom, so that there is enough space inside the micropore to accommodate the polar solution (such as the first polar solution added into the micropore first) 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 molecular layer, therefore, the inner diameter of the first end of the micropore is usually fixed between about 100 micrometers and 200 micrometers). Specifically, when the meniscus flows through the micropore, the amphiphilic molecular layer in the meniscus forms at the narrowest point of the micropore, that is, the amphiphilic molecules self-assemble to form the amphiphilic molecular layer at the first end of the micropore.

[0097] For example, in some embodiments, see Figure 1aAs shown in Figure 4, the micropores are arranged in a funnel shape, with the top being smaller and the bottom being larger.

[0098] In other embodiments, the micropores may also be cylindrical (i.e., equal in size at the top and bottom) or wider at the top and narrower at the bottom. Of course, the micropores may also be configured in other shapes, as long as the shape of the micropores can successfully form an amphiphilic molecular layer, they are all within the scope of protection of this invention.

[0099] Furthermore, in some embodiments, the flow channel is also provided with a second opening for discharging liquid from the flow channel.

[0100] In some embodiments, the second opening can also be used to insert a second electrode (i.e., 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.

[0101] Furthermore, in some embodiments, see Figures 1a-3 An electrode insertion port 14 is also provided on the flow channel. Specifically, the second electrode is inserted into the flow channel through the electrode insertion port 14.

[0102] Furthermore, in some embodiments, the second structural layer is provided with a mating portion that cooperates with the second electrode. When the second electrode is inserted into the flow channel, the first end (i.e. the insertion end) of the second electrode cooperates with the mating portion (e.g., they come into contact with each other).

[0103] Furthermore, to fix the second electrode and improve the stability of the preparation device during operation, in some embodiments, the mating part is a groove (i.e., a groove is provided on the first surface of the second structural layer). When the second electrode mates with the mating part, the first end of the second electrode is inserted into the groove. In this embodiment, because the second electrode is fixed by inserting it into the groove, it facilitates the positioning and installation of the second electrode (avoiding displacement of the second electrode during insertion) and prevents horizontal displacement and shaking of the second electrode during use, thus improving the working stability of the second electrode.

[0104] Furthermore, the second electrode (i.e., the common electrode) in this embodiment can be set to a detachable state, that is, it can be installed (inserted) and removed by the staff themselves, thereby facilitating the replacement and cleaning of the second electrode.

[0105] It is understood that the first and second electrodes have opposite polarities. For example, in some embodiments, the first electrode is the positive electrode and the second electrode is the negative electrode. In other embodiments, the first electrode is the negative electrode and the second electrode is the positive electrode.

[0106] Preferably, in some embodiments, the third structural layer is an ASIC circuit layer, and the electrode layer is a TSV via electrode, with the selected electrode material being pure gold. A chip containing multiple micropores (e.g., an array of 8*32=256 micropores) (i.e., the second structural layer) is bonded to the third structural layer via thermoforming, permanent adhesive bonding, and photoresist AZ4620 or SU-8. The common electrode is preferably a gold-plated copper pillar inserted into the chip's recess. The first opening (i.e., the inlet) and the second opening (i.e., the outlet) of the flow channel are fed (i.e., the corresponding liquid, such as a polar solution or membrane solution) and discharged using a microfluidic-specific tubing. 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.

[0107] Preferably, in some embodiments, the selected reciprocating syringe pump is a Harvard apparatus 4400.

[0108] Furthermore, in some embodiments, the pore size of the micropore is between approximately 100 micrometers and approximately 200 micrometers; for example, the inner diameter of the first end of the micropore is between approximately 100 micrometers and 200 micrometers.

[0109] Preferably, in some embodiments, the pore size of the micropore is between approximately 150 micrometers and 200 micrometers, for example, the inner diameter of the first end of the micropore is between approximately 150 micrometers and 200 micrometers.

[0110] Preferably, in some embodiments, for ease of processing, see [reference needed]. Figure 2 The micropores on the second structural layer are arranged in multiple arrays, such as 2*8 or 8*32.

[0111] Preferably, in some embodiments, for ease of processing, see [reference needed]. Figure 3 The length L1 of the preparation device is approximately 40 mm to 51 mm, the width L2 of the preparation device is approximately 16 mm to 27 mm, the maximum width inside the flow channel L3 is approximately 4.0-12.0 mm, the width at the first and second openings of the flow channel (the minimum width inside the flow channel) L4 is approximately 0.3 mm to 0.8 mm, and the length L5 of the micropore array is approximately 10.5-20.5 mm.

[0112] Preferably, in some embodiments, the spacing L6 between adjacent micropores in the micropore array is greater than approximately 0.4 mm.

[0113] Furthermore, in some embodiments, the spacing L6 between adjacent micropores within the micropore array is greater than approximately 0.5 mm.

[0114] Surprisingly, the preparation apparatus of this invention, by using polyoxymethylene as a raw material to fabricate the flow channels and micropores, and by adopting a new dimensional design for the spacing between adjacent micropores, simplifies the fabrication process. This allows the apparatus to be fabricated using various processing methods (such as machining and laser processing), significantly reducing production costs, while still meeting the requirement of preparing multiple amphiphilic molecular layers at once. Furthermore, based on the preparation apparatus provided by this invention, pretreatment of the micropores within the apparatus is unnecessary during film formation, making the film formation method simpler.

[0115] It is understood that the device provided by the present invention can be applied to the new film-forming method proposed in the present invention, as well as existing film-forming methods, such as coating methods.

[0116] In some embodiments, the raw materials for the preparation device can be selected from materials such as Teflon, PMMA, Delrin (polyoxymethylene), and Parylene.

[0117] Of course, in other embodiments, the raw materials for the preparation device can also be selected from PMMA (polymethyl methacrylate), epoxy resin, PC (polycarbonate), PVC (polyvinyl chloride), COC (cyclic olefin copolymer), polyimide, and other materials.

[0118] Furthermore, in some embodiments, the processing methods that the fabrication apparatus can select include: machining, laser processing, micro-injection molding, 3D printing, casting, electroporation, etc.

[0119] Furthermore, in some embodiments, the micropores in the fabrication apparatus are preferably formed using laser processing. Laser-processed micropores are circular with smooth inner walls, facilitating the formation of amphiphilic molecular layers.

[0120] Of course, in other embodiments, the micropores can also be fabricated using machining, electroporation, etc. It is understood that the fabrication of the micropores should satisfy the requirement that they be circular with smooth sidewalls, facilitating the formation of amphiphilic molecular layers.

[0121] 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 they can be integrally molded (such as integral injection molding). Therefore, the preparation device obtained by either layered processing and assembly or integral molding is within the protection scope of this invention.

[0122] For example, in some embodiments, the device can be manufactured using Teflon material via micro-injection molding; PMMA can be manufactured using machining or micro-injection molding; Delrin can be manufactured using machining or micro-injection molding; and Parylene can be manufactured using physical vapor deposition, such as depositing Parylene onto the surface of another pre-processed material, with a preferred deposition thickness of 5 micrometers.

[0123] In some embodiments, the raw materials are selected from materials with certain hydrophobicity and lipophilicity, thereby reducing the pretreatment steps and simplifying the film formation method in the process of preparing amphiphilic molecular films. For example, polyoxymethylene is preferably used to make the preparation device. The hydrophobicity and lipophilicity of polyoxymethylene meet the film formation requirements, so no pretreatment is required.

[0124] In some embodiments, the electrode material in the electrode layer can be silver, gold, platinum, and titanium electrodes, and the electrode layer can be fabricated by magnetron sputtering or PCB surface treatment processes.

[0125] Furthermore, in some embodiments, when the number of micropores is set to be large, at least one third opening is also provided on the flow channel.

[0126] 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 surface in the flow channel, or in other words, the second end of the third opening is level with the liquid surface in the flow channel, the meniscus in the flow channel can maintain a constant liquid surface height during movement, thus ensuring the stability of its shape and not being affected by the third opening.

[0127] Furthermore, in some embodiments, to facilitate sample addition, the first opening and the third opening may be arranged with the top being larger than the bottom. For example, the first end of the first opening (the end disposed on the surface of the preparation device and used for sample addition) is larger than the second end of the first opening (the end connected to the flow channel).

[0128] Specifically, in some embodiments, see [link to relevant documentation]. Figure 1c The third opening 16 is located on the side close to the first opening 12. In this embodiment, it is equivalent to setting two injection ports (i.e., setting two injection ports, the first opening and the third opening) on ​​the flow channel. At this time, the first opening (equivalent to the first injection port) is used to add the membrane solution and to inject / retract the second polar solution through a reciprocating syringe pump, and the second opening (equivalent to the second injection port) is used to add the membrane solution.

[0129] For example, in some embodiments, if no film forms in the micropores through which the meniscus passes, it indicates that the concentration of amphiphilic molecules in the meniscus is too low. In this case, a second polar solution is drawn back at the first opening using a reciprocating syringe pump, causing the meniscus to move between the first and third openings. During this process, the meniscus flows again through the already formed film-forming micropores. Since the amphiphilic molecular layer (i.e., the film) itself is a very stable arrangement, once the amphiphilic molecular layer has formed on the micropore, even if the meniscus repeatedly passes through the micropore multiple times, the thickness of the amphiphilic molecular layer on the corresponding micropore will not increase. That is to say, the process of the meniscus moving back towards the first opening does not affect the previously formed film.

[0130] Furthermore, when the meniscus moves between the first and third openings, a certain amount of membrane solution is added to the third opening (equivalent to the second injection port). Then, the injection pump controls the meniscus located between the first and third openings to continue moving in the direction from the first opening to the third opening, and combines 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.

[0131] Therefore, one or more injection ports can be set on the flow channel according to the number of micropores. For example, when the number of micropores is relatively small, the change in the concentration of amphiphilic molecules has a relatively small impact on film formation (or the impact of concentration change can be ignored in actual application), only one injection port needs to be set on the flow channel, such as only setting the first opening for injection.

[0132] For example, when the number of micropores is set relatively high, as the meniscus passes through a certain number of micropores, the concentration of amphiphilic molecules in the meniscus gradually decreases, making film formation impossible at subsequent micropores. In this case, multiple inlets can be set in the flow channel, for example, see [reference needed]. Figure 1c A third opening is added to the flow channel for adding liquid samples. In other words, by adding an inlet to the flow channel, the number of micropores can be increased, allowing the preparation device to form more amphiphilic molecular layers at once.

[0133] Of course, in other embodiments, the membrane solution can be added directly through the third opening, while the first opening is only used for injecting / retracting the second polar solution via a reciprocating syringe pump.

[0134] In other embodiments, a new meniscus can be formed directly through a third opening (so that the previously formed meniscus is located between the first and third openings and no longer participates in subsequent film formation), and the newly generated meniscus can move and pass through the unformed micropores, and form a film on the micropores.

[0135] Preferably, in order to control the above process more accurately, it can be achieved through automated control (such as a program controlled by computer software).

[0136] Example 2

[0137] Based on Example 1, see Figure 5 The present invention also provides a novel method for preparing an amphiphilic molecular membrane (film formation method), comprising the following steps:

[0138] S1 provides a preparation apparatus for preparing multiple amphiphilic molecular films. The preparation apparatus has multiple micropores and channels that allow solution to flow into the micropores. The preparation apparatus also includes an electrode layer so that the solution flowing into the micropores can contact the electrode layer. Preferably, the cross-section of the channel is square or quasi-square.

[0139] S2 adds a first polar solution into the flow channel, so that the first polar solution enters at least one micropore and comes into contact with the electrode layer;

[0140] S3 sequentially adds the membrane solution and the second polar solution into the flow channel, causing the membrane solution to form a meniscus within the flow channel (see...). Figure 7a and Figure 7b );

[0141] S4 introduces a second polar solution into the flow channel, causing the second polar solution to propel the membrane solution and flow through at least one micropore where the first polar solution contacts the electrode layer. Amphiphilic molecules in the membrane solution form an amphiphilic molecular layer on the corresponding micropore (i.e., a micropore containing the first polar solution and in contact with the electrode layer). (Specifically, the amphiphilic molecules form the amphiphilic molecular layer based on their self-assembly capability.) The contact angle of the inner surface of the flow channel is approximately 65° to 120°.

[0142] It is understood that in some embodiments, 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 separate steps.

[0143] In this embodiment, the preparation device preferably uses a material with certain hydrophobicity, and the contact angle of the material on the inner surface of the flow channel in the preparation device is approximately 65°-120°. This allows the membrane solution added into the flow channel to form a meniscus under the combined action of the inner surface of the flow channel, the polar solution, and the air within the flow channel. Preferably, the cross-section of the flow channel is square or near-square. In this case, the flow channel generates a certain resistance to the movement of the membrane solution (or the meniscus formed by the membrane solution), preventing the membrane solution from moving too quickly within the flow channel. Furthermore, the movement speed of the membrane solution at various locations is relatively uniform (or, the difference in flow speed at different locations has little impact on the stability of the meniscus), allowing the meniscus to maintain a stable shape during movement.

[0144] Specifically, the inner surface of the flow channel and the surface of the micropores (i.e., the micropore sidewalls) are both made of a material with a contact angle between approximately 65° and 120° (preferably made of polyoxymethylene), so that when the membrane solution moves to the corresponding micropore, an amphiphilic molecular membrane can be smoothly formed at the micropore (specifically, at the first end of the micropore) without the need for pretreatment of the device (such as the micropores set in the flow channel).

[0145] In the process of the formation and movement of the meniscus by the membrane solution, the amount (volume) of the membrane solution added and the movement speed of the meniscus formed by the membrane solution are very important to the formation and movement of the meniscus. For those skilled in the art, it is possible to select a suitable amount of membrane solution added and movement speed through preliminary experiments.

[0146] For example, in the selection or prediction of the amount of membrane solution added, the main parameters considered are: the Reynolds number (Re), a dimensionless number used to characterize fluid flow. The Reynolds number is calculated as: Re = ρvd / μ, where v, ρ, and μ are the fluid velocity (equivalent to the moving speed of the membrane solution), density, and viscosity coefficient, respectively, and d is the characteristic length (equivalent to the length of the flow channel). The density and viscosity coefficient of the membrane solution can be obtained experimentally. Maintaining the stability of the meniscus requires a relatively small Reynolds number (the smaller the Reynolds number, the more stable the fluid flow), i.e., laminar flow.

[0147] Therefore, this invention preferably achieves a Reynolds number (Re) < 2300 for the fluid. This allows for a preliminary limitation on the relationship between the channel length and the membrane solution's velocity using the Reynolds number. (Thus, when the membrane solution is selected and the channel length (related to the micropore array configuration) is determined, the membrane solution's velocity can be initially limited based on the Reynolds number; that is, the velocity is constrained by both the membrane solution and the channel length). In other words, for those skilled in the art, once the membrane solution is selected, preliminary experiments using the preparation apparatus can screen for a suitable velocity. Based on the velocity selected in the preliminary experiments, the injection velocity of the polar solution (e.g., the second polar solution) can be designed accordingly to obtain a preset injection velocity for the second polar solution (e.g., the injection velocity of the second polar solution in steps S3 and S4). Of course, the injection velocity of the second polar solution can also be estimated based on the operator's experience, followed by preliminary experiments to obtain a suitable injection velocity.

[0148] Furthermore, in some embodiments, the contact angle of the material selected for the inner surface of the flow channel is approximately 75° to approximately 95°. Specifically, in some embodiments, multiple micropores are disposed on the inner surface of the flow channel, i.e., the contact angle of the material selected for the inner surface of the flow channel and the surface of the micropores is approximately 75° to approximately 95°.

[0149] Preferably, both the inner surface of the flow channel and the surface of the micropores are made of polyoxymethylene.

[0150] Furthermore, in some embodiments, during the formation of the meniscus (i.e., in step S3), the injection rate of the second polar solution is from 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., in step S4), the injection rate of the second polar solution is from about 200 μL / min to about 500 μL / min.

[0151] To more clearly illustrate the technical solution adopted in this invention, the formation of the contact angle and meniscus is briefly explained below:

[0152] See Figure 9a The contact angle is the angle between the solid-liquid interface, the liquid interior, and the gas-liquid interface at the interface of the three phases S (solid), L (liquid), and G (gas). If θ < 90°, the liquid easily wets the solid; the smaller the angle, the better the wettability. If θ > 90°, the liquid does not easily wet the solid and tends to move on the surface. Specifically, when θ = 0, complete wetting occurs; when θ < 90°, partial wetting or wetting occurs; when θ = 90°, it is the dividing line between wetting and non-wetting; when θ > 90°, no wetting occurs; and when θ = 180°, no wetting occurs.

[0153] See Figure 9b The liquid pressure that increases along the capillary wall in the diagram is: Where 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 due to gravity, and h is the height the liquid level rises. Based on the definition of the contact angle, we know that: For example, when the contact angle θ is less than 90°, the solution can 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 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 within the capillary.

[0154] Specifically, the surface tension of the polar solution (such as a second polar solution) added inside the flow channel causes the membrane solution to diffuse at 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 degree of hydrophobicity, it has a certain adsorption effect on the membrane solution. See here. Figure 7b The membrane solution is composed of a polar solution (second polar solution 2) on one side and air 8 on the other. The liquid surface exhibits contractile force, and the molecules at the interface between the membrane solution and the air are subjected to a pulling force pointing inwards from the liquid. This causes the membrane solution to gradually form a meniscus 4 within the flow channel, as shown in the diagram. Figure 7b ).

[0155] See Figure 7a The membrane solution contains an amphiphilic molecule 5, which has a hydrophilic end 52 and a hydrophobic end 51 (see [reference]). Figure 6 Because amphiphilic molecules have self-assembly capabilities, when the membrane solution flows through the micropores, the hydrophilic ends 52 of the amphiphilic molecules inside will point towards and contact the first polar solution, while the hydrophobic ends 51 will combine with the hydrophobic ends 51 of another layer of amphiphilic molecules, thereby forming an amphiphilic molecular layer, such as... Figure 7a and Figure 8 As shown. In this embodiment, by controlling the migration speed of the membrane solution, it is possible to prevent the amphiphilic molecules from accumulating thickly at the micropores (e.g., Figure 10 As shown in (a), this results in the inability to insert the hole.

[0156] In this embodiment, the membrane solution is moved by the second polar solution, causing it to pass through at least one micropore and form an amphiphilic molecular membrane at that micropore. Furthermore, the flow rate of the second polar solution (or the sample addition / injection rate) is relatively controllable. Therefore, the movement speed (i.e., flow rate) of the membrane solution in the flow channel can be controlled by adjusting the subsequent sample addition rate of the second polar solution, thereby controlling the formation process of the amphiphilic molecular membrane. This avoids the membrane solution flowing too fast and failing to form an amphiphilic molecular membrane, or the membrane solution flowing too slowly and forming an excessively thick amphiphilic molecular membrane.

[0157] This film-forming method, which uses a polar solution to propel a meniscus (membrane solution) to form a film at micropores, allows for precise control of the membrane solution's movement speed (e.g., controlling the injection rate of the polar solution using a pipette or syringe pump, thereby controlling the membrane solution's movement speed). This ensures that the membrane solution remains at different micropores for similar or equal times, preventing the membrane solution from remaining too long in some micropore areas and forming a thick film, or moving too quickly in other micropore areas and failing to form a film. Therefore, this invention can directly produce amphiphilic molecular layers with the required thickness by precisely controlling the membrane solution's movement speed, without requiring post-formation thinning treatments (e.g., reducing the film thickness). Figure 10 (as shown in (a)-(d)). In other words, the film-forming method of the present invention can form a film in one step with good film-forming effect. For example, by controlling the injection rate of the polar solution and thus the movement rate of the film solution, a film of suitable thickness can be directly formed, such as directly preparing a film. Figure 10 The membrane shown in (d) is shown in the image.

[0158] For example, in some embodiments, the injection rate of the second polar solution is controlled by an injection pump to control the movement speed of the membrane solution. In this embodiment, the provided method is more controllable (e.g., compared to prior art bubble extrusion film formation). Of course, other injection methods can also be used to control the injection rate of the solution, and any method capable of controlling the injection rate of the solution falls within the protection scope of this invention.

[0159] Furthermore, in some embodiments, a reciprocating syringe pump is provided to add (inject) the second polarity solution. For example, a Harvard Apparatus 4400 syringe pump is selected.

[0160] Preferably, in some embodiments, prior to S1, the step of wetting at least one micropore is included.

[0161] In this embodiment, the wetting treatment allows the liquid (such as the first polar solution) to spread on the solid surface (such as the microporous surface), increasing the contact area between the polar solution and the inner wall of the flow channel and reducing the contact angle, thereby enabling the first polar solution to smoothly enter at least one micropore and fill the micropore.

[0162] For example, in some embodiments, the wetting treatment is an electrowetting treatment.

[0163] For example, in some embodiments, the wetting treatment is a liquid wetting treatment.

[0164] Specifically, in some embodiments, a low surface energy liquid (such as ethanol) is first introduced into the flow channel. This low surface energy liquid can flow into very small pores, such as micropores. Adding a polar solution then allows the polar solution to fill the micropores. If the low surface energy liquid is not added beforehand, the polar solution may not be able to flow directly into the micropores. Then, a first polar solution is introduced into the flow channel, filling at least one micropore and diluting and discharging the low surface energy liquid. The previously introduced low surface energy liquid makes it easier for the first polar solution to fill the micropores. The low surface energy liquid includes, for example, ethanol.

[0165] In some other embodiments, step S2 further includes the step of: after adding the first polar solution into the flow channel, subjecting the preparation device to ultrasonic treatment, so that the first polar solution enters the micropores under the action of sound waves.

[0166] Furthermore, in some embodiments, the membrane solution described above is a nonpolar solution containing amphiphilic molecules.

[0167] 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 physicochemical methods, such as an ABA triblock copolymer (PMOXA-PDMS-PMOXA dimethyl oxazoline-polydimethylsiloxane-dimethyl oxazoline), or a mixture of phospholipids and polymers.

[0168] Furthermore, in some embodiments, the nonpolar solution may be an alkane organic solvent, including decane, hexadecane, pentane, or a mixture thereof, which is used to dissolve phospholipids or polymeric triblock copolymers to form a membrane solution.

[0169] Furthermore, in some embodiments, the second polar solution and the first polar solution can be the same or different polar solutions. The selected polar solution should satisfy the requirement of conductivity and enable the nanoporous protein to insert into the amphiphilic molecular layer.

[0170] Furthermore, in some embodiments, the nanoporous protein may be pre-added to a polar solution (such as a first polar solution or a second polar solution).

[0171] Of course, in other embodiments, nanoporous proteins (such as solutions containing nanoporous proteins) may be added into the flow channel after the amphiphilic molecular layer has been formed.

[0172] Furthermore, in some embodiments, the polar solution is an electrolyte used in gene sequencing, containing electrolytes and / or polyelectrolytes to effectively enhance ion exchange lifetime; it may also contain redox pairs, and / or combinations of redox pairs that can be partially oxidized or reduced to provide redox pairs, such as iron / iron ferricyanide; it may also be a cross-linked agarose gel, and / or a cross-linked sodium alginate gel; it may also contain a buffer to adjust the pH of the aqueous medium, and suitable buffers (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), etc.

[0173] Furthermore, in some embodiments, the method further includes the step of:

[0174] By applying current to the polar solution through the electrode layer and the common electrode, the nanoporous protein is embedded (inserted) into the amphiphilic molecular layer under the action of voltage.

[0175] For example, in some embodiments, a common electrode is inserted into a second opening (or a first opening or electrode insertion port) of the device, and then current is applied to the polar solution through the common electrode and a pre-defined electrode layer. For example, the common electrode may be a pin, a PET flexible electrode, or an electrode integrated with a microfluidic chip fixture and connected to the circuitry.

[0176] Specifically, in some embodiments, when the inner diameter of the flow channel is set to be large (or the flow channel is wide), in order to successfully form the meniscus, the flow channel of the preparation apparatus involved in this method includes: a first opening and a second opening, and the first opening and the second opening are connected through the first flow channel 15. See also Figure 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).

[0177] 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 relatively narrow, the meniscus formed by the membrane solution is thicker, and the shape is relatively stable. Therefore, the initial moving speed of the membrane solution can be set relatively fast (specifically, since there is a region where the inner diameter gradually increases from the inlet of the channel to the inside of the channel, the injection speed of the polar solution is relatively slow at this time. However, because the cross-section of the channel is relatively small, even if the injection speed of the polar solution is relatively slow, the meniscus can still have a relatively fast moving speed). When the membrane solution gradually moves to the region with the largest inner diameter of the channel (such as the middle region between the two ends of the channel, i.e., the region with equal inner diameter), 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 moving speed of the membrane solution (at this time, since the cross-section of the channel corresponding to the micropore is large, when the polar solution maintains its original injection speed, the moving speed of the meniscus will also decrease; of course, the moving speed of the membrane solution can be further reduced by adjusting the injection speed of the polar solution). In this embodiment, the gradual change in the size of the channel makes the shape of the meniscus relatively stable during the formation and change process, and it is not easy to be damaged. The different states of the meniscus during the movement of the channel are as follows: Figure 11b The first, second, and third states of the meniscus are shown in 4a, 4b, and 4c.

[0178] For example, in some embodiments, see Figure 11a The region where the inner diameter of the flow channel changes (i.e., the region with unequal inner diameters, which is also the first end of the flow channel) includes: a first change region and a second change region connected together. The curve of the sidewall of the first change region (near the first opening) is similar to a partial arc of a circle with radius R1 (or, in other words, the vertical projection of the sidewall of the first change region is curved). The curve of the sidewall of the second change region is similar to a partial arc of a circle with radius R2 (or, in other words, the vertical projection of the sidewall of the second change region is curved). R1 ​​and R2 are equal (of course, in other embodiments, R1 and R2 may be unequal, as long as the inner diameter of the flow channel can achieve the formation and movement of a meniscus).

[0179] 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.

[0180] For example, in one specific embodiment (using a fabrication device with 8*32 micropores), the fabrication method specifically includes the following steps:

[0181] First, pure ethanol is added into the flow channel so that the ethanol enters at least one micropore. 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).

[0182] Add approximately 20 to 50 microliters of membrane solution into the first opening;

[0183] Add approximately 20 μL to approximately 50 μL of polar solution into the first opening, so that the membrane solution forms a meniscus within the flow channel;

[0184] Approximately 300 μL of polar solution is added to the first opening using a syringe pump at a preset injection rate (flow rate). Specifically, the first 100 μL of polar solution is injected at a flow rate of 10 μL / min (during which the meniscus forms and gradually thins), and the subsequent 200 μL of polar solution is injected at a flow rate of 5 μL / min. This allows the meniscus to maintain its shape and flow uniformly through the micropore array, enabling the formation of amphiphilic molecular layers on 50% to 80% of the micropores in the micropore array.

[0185] Alternatively, in another specific embodiment (using a preparation device with 16 micropores), the preparation method includes the following steps:

[0186] 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.

[0187] Add approximately 8 to approximately 12 microliters of membrane solution to the first opening;

[0188] Then, approximately 8 to 12 microliters of polar solution are added to the first opening to allow the membrane solution to form a meniscus.

[0189] Next, approximately 60 μL of polar solution is added to the first opening based on a preset injection rate (flow rate). Specifically, the first 15 μL of polar solution is injected at a flow rate of 4 μL / min, and the subsequent 45 μL of polar solution is injected at a flow rate of 2 μL / min, thereby maintaining the stability of the meniscus shape and allowing the membrane solution to flow uniformly through the micropore array. Amphiphilic molecular layers can be directly formed on 50% to 80% of the micropores in the micropore array.

[0190] Of course, the film-forming method and preparation apparatus 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.

[0191] Furthermore, in some embodiments, the step of detecting the amphiphilic molecular layer is also included.

[0192] For example, by inputting a triangular wave signal into the amphiphilic molecular layer, a corresponding square wave signal is obtained. The upper and lower peak values ​​of the square wave signal can be used to determine whether the amphiphilic molecular layer has formed and whether it meets the requirements. Specifically, when an excessively thick amphiphilic molecular layer is detected, the concentration of the membrane solution can be appropriately reduced.

[0193] 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 has been formed.

[0194] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0195] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for preparing multiple amphiphilic molecular layers, characterized in that, Includes the following steps: S1 provides a preparation apparatus for preparing multiple amphiphilic molecular layers. The preparation apparatus has multiple micropores and channels that allow solution to flow into the micropores. The channels include a first opening for adding a sample into the channels and a second opening for discharging liquid from the channels. The preparation apparatus also includes an electrode layer that allows the solution flowing into the micropores to contact the electrode layer. S2 adds 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 adds a membrane solution and a second polar solution into the flow channel, so that the membrane solution can form a meniscus under the combined action of the inner surface of the flow channel, the second polar solution, and the air in the flow channel; wherein, the membrane solution includes: a nonpolar solution and amphiphilic molecules, wherein the amphiphilic molecules include: 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 rate to control the movement speed of the meniscus, so that the second polar solution pushes the meniscus to move and flow 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 molecular layer on the corresponding micropore. The contact angle of the inner surface of the flow channel is 65° to 120°. A third opening is also provided on the side of the flow channel near the first opening. Correspondingly, the method further includes: When the meniscus moves 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 moving in the direction from the first opening to the third opening, and combines 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.

2. The preparation method according to claim 1, characterized in that, The electrode layer is also provided with at least one electrode bump.

3. The preparation method according to claim 1, characterized in that, The cross-section of the flow channel is square or near-square.

4. The preparation method according to claim 1, characterized in that, The inner diameter of the first end of the flow channel gradually increases in the direction away from the first opening.

5. The preparation method according to claim 1, characterized in that, Before step S2, the method further includes the step of wetting at least one of the micropores; or, step S2 includes the step of ultrasonicating the preparation device after adding the first polar solution into the flow channel.

6. An apparatus for preparing multiple amphiphilic molecular layers, characterized in that, The preparation device is provided with multiple micropores and channels that allow the solution to flow into the micropores. The device also includes an electrode layer, allowing the solution flowing into the micropores to contact the electrode layer. The channel includes a first opening, and the inner diameter of the first end of the channel gradually increases away from the first opening. This allows the membrane solution to form a meniscus under the combined action of the inner surface of the channel, the second polar solution, and the air within the channel when a membrane solution and a second polar solution are sequentially added to the channel. Furthermore, the movement speed of the meniscus can be controlled when the second polar solution is added to the channel at a preset injection rate. The membrane solution includes a nonpolar solution and amphiphilic molecules. The amphiphilic molecules include phospholipids, polymers, or a mixture of phospholipids and polymers. The channel also includes a second opening for discharging liquid from the channel. The contact angle of the inner surface of the channel is 65° to 120°. At least one third opening is also provided on the side of the channel near the first opening.

7. The preparation apparatus according to claim 6, characterized in that, The electrode layer is further provided with at least one electrode bump; and / or, the cross-section of the flow channel is square or quasi-square.

8. The preparation apparatus according to claim 7, characterized in that, The first opening and / or the third opening are arranged with the top being larger than the bottom.

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