CN114908358A

CN114908358A - Preparation method and device of amphiphilic molecular layer

Info

Publication number: CN114908358A
Application number: CN202210391473.3A
Authority: CN
Inventors: 杨秉达
Original assignee: Guangzhou Kongque Gene Technology Co ltd
Current assignee: Kong Que (Chengdu) Technology Co.,Ltd.
Priority date: 2022-04-14
Filing date: 2022-04-14
Publication date: 2022-08-16
Anticipated expiration: 2042-04-14

Abstract

The invention relates to a preparation method and a preparation device for rapidly preparing an amphiphilic molecular layer, which comprises the following steps: s1 provides a preparation device for preparing a plurality of amphiphilic molecule layers, a plurality of micropores are arranged in the preparation device, a flow channel enabling a solution to flow to the micropores is arranged in the preparation device, an electrode layer is further arranged in the preparation device, and the solution flowing to the micropores can be in contact with 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 with the electrode layer; s3, sequentially adding the film solution and the second polarity solution into the flow channel, so that the film 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 flow through at least one pore of the first polar solution contacting the electrode layer, and the amphiphilic molecules in the membrane solution form a layer of amphiphilic molecules on the corresponding pore. The method provided by the invention is simple and safe to operate, and the selected preparation device is low in production cost.

Description

Preparation method and device of amphiphilic molecular layer

Technical Field

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

Background

The nanopore protein needs to be stably embedded in a thin film formed by phospholipid or macromolecules, such as an amphiphilic molecule layer (namely, an amphiphilic molecule film), so that a DNA sequence to be detected can pass through the nanopore protein, and the aim of detecting the DNA sequence is fulfilled. However, some of the current methods for preparing layers of amphiphilic molecules (i.e., membrane methods) and devices still have problems in application.

The existing film forming methods mainly include a folding bilayer formation method (Montal & Mueller method), a dipping tip method, a coating method, a patch clamp method, a water-in-oil droplet interface method, and the like. The above methods such as the folding bilayer method, the dipping method, the coating bilayer method, and the like, often form a thick film for the first time, and require a thinning treatment such as volatilization of an organic solvent, spreading by physical application, or pressing by air pressure or the like. The thinning process is important for controlling the 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 painting). Meanwhile, the existing film forming methods generally involve pretreatment of the manufacturing apparatus, such as fluorine plasma treatment, silanization treatment, etc., which require high-risk chemicals and are carried out in laboratories with high construction cost and high maintenance cost. Potentially threatening the life safety of the operator and the surrounding environment. In addition, the pretreatment needs to accurately calculate the amount of the used coating, and the test effect is affected when too much coating is used. The preparation device of the amphiphilic molecular membrane is usually a small chip, the size of the internal structure (such as micropores) is tiny, so the dosage of the selected coating is very small, and the operation of coating a tiny amount of coating on the tiny structure is not simple, so the problems of too much coating and too little coating are difficult to avoid in the coating process.

For example, as in the chinese patent application No. 201480056839.5, the invention discloses a method for forming a film on a biochip, and the method comprises the steps of: a liquid containing lipid molecules (i.e., amphipathic molecules) is added to the chip surface, and then the liquid is partitioned by air bubbles, so that the lipid molecules are distributed on the chip surface, and the lipids are thinned by the air bubbles. However, the bubble generation often requires manual control (for example, preparing bubbles by using a pipette), and the process is difficult to be controlled automatically, because the manual control is difficult to control the size of bubbles and ensure the stable form of bubbles, so the repeatability of the method is poor. And also this method requires pretreatment.

As another example, chinese invention, application No. CN200880126160.3, discloses a method of forming a layer separating two volumes of aqueous solution by flowing an aqueous solution comprising amphipathic molecules through a body to cover grooves (i.e., micropores) such that the aqueous solution can form amphipathic molecules across the grooves, although the technique of flowing an aqueous solution through grooves to form amphipathic molecules is easily achieved, the resulting membrane is thick, requires subsequent thinning, and involves a pretreatment step.

Disclosure of Invention

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

s1 provides a device for preparing a plurality of amphiphilic molecule layers, wherein the device has a plurality of pores and a flow channel for allowing a solution to flow into the pores, the device further includes an electrode layer for allowing the solution flowing into the pores to contact the electrode layer, and 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 film solution and a second polarity solution into the flow channel, so that the film 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 pore where the first polar solution contacts the electrode layer, and the amphiphilic molecules in the membrane solution form a layer of amphiphilic molecules on the corresponding pore;

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

In some embodiments, before the S2, the method further includes the step of: wetting at least one of the micropores;

in some embodiments, said S2 includes the steps of: and after adding the first polar solution into the flow channel, carrying out ultrasonic treatment on the preparation device.

In some embodiments, the wetting treatment comprises: and (5) performing electrowetting treatment.

In some embodiments, the wetting treatment comprises: and (5) liquid wetting treatment.

In some embodiments, the membrane solution comprises: a non-polar solution, and an amphiphilic molecule.

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

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

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

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

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

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

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

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

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

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

In some embodiments, the preparation device is further provided with a common electrode, and the common electrode is contacted with the second polar solution, and correspondingly, the method further comprises the following steps:

and electrifying 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 polarity solution is about 10 μ L/min to about 50 μ L/min;

in some embodiments, in step S4, the injection rate of the second polarity solution is about 200 μ L/min to about 500 μ L/min.

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

The invention also provides a preparation device of the amphiphilic molecule layer, wherein a plurality of micropores and a flow channel capable of enabling a solution to flow into the micropores are arranged in the preparation device, an electrode layer is also arranged in the device, the solution flowing into the micropores can be in contact with the electrode layer, the cross section of the flow channel is square or square-like, and the inner surface material of the flow channel can be polyformaldehyde optionally.

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

The beneficial technical effects are as follows:

the invention provides a preparation method (namely a film forming method) and a preparation device for rapidly preparing a plurality of amphiphilic molecule layers (namely amphiphilic molecule films, also called membranes for short). Unlike the prior art, the preparation method of the present invention proposes a new film forming method, i.e., a film solution is made to form 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 a layer (film) of amphiphilic molecules is formed on the corresponding micropores.

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

Through the film forming mode that the polar solution pushes the meniscus (film solution) to form a film at the micropores, the moving speed of the film solution can be controlled more accurately (for example, the injection speed of the polar solution is controlled through a pipette gun or a syringe pump, etc., so as to control the moving speed of the film solution), so that the residence time of the film solution at different micropores is the same or similar, and the film solution is prevented from being left in part of the micropore area for a long time to form a thick film, or from moving too fast to form a film unsuccessfully in part of the micropore area. Therefore, the invention can efficiently and directly prepare the amphiphilic molecular layer with proper thickness (or the film thickness meets the use requirement) by relatively accurately controlling the moving speed of the film solution, and does not need to thin the film after film formation (such as high-voltage breakdown and multiple film formation). In other words, the film forming method of the present invention can form a film at one time.

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

Moreover, the film forming method provided by the invention does not need pretreatment, is simpler, and ensures the safety of workers in the operation process (does not relate to the operation and use of dangerous goods).

Further, the injection speed of the polar solution can be controlled by the syringe pump, and the syringe pump can be automatically controlled by electronic equipment (such as a computer, etc.) (that is, the film forming method provided by the invention can realize automatic control), so that the manual operation steps of workers can be further reduced, errors possibly caused by manual operation of the workers can be correspondingly avoided, and the stability and uniformity of film formation can be further ensured.

The film forming method (and the device) provided by the invention can be applied to various detection requirements in a laboratory (such as being suitable for different application scenes of scientific research units, commercial detection companies and the like), and because the film forming method can form a plurality of films at one time, the detection requirements (such as human genome sequencing, virus sequencing and the like) on the commercial or market can be better met in the actual use process, the detection period of the detection is relatively long, the required data volume is large, and at the moment, the probability of misoperation of a worker in the long-term operation process can be increased. The method provided by the application can be matched with automatic control in the prior art, so that the accuracy and the reliability of the detection result are further improved.

In the prior art, in order to ensure that the solution containing the amphiphilic molecules can cover (or flow through) all or a plurality of micropores to form a plurality of amphiphilic molecule layers, a person skilled in the art usually chooses to add more solution containing the amphiphilic molecules to avoid the existence of part of micropores which can not form amphiphilic molecule layers, so that the resulting membrane is relatively thick. In response to this technical problem, a person skilled in the art generally considers how to further process (i.e. thin) the film after the film formation to thin the film, for example, a high voltage breakdown is required for forming the film multiple times. Different from the prior art, the method adopts different technical routes, provides a new film forming method, can form a film at one time, is simpler in operation process, and has less time consumption and higher efficiency because the operations of pretreatment, thinning and the like which take longer time are avoided.

Further, it is difficult for those skilled in the art to think of the film forming method proposed in the present application based on the conventional production apparatus. First, the preparation apparatus of the prior art does not provide the conditions for meniscus formation, and similarly, the method and apparatus of the prior art have difficulty in ensuring that the meniscus can be stably moved in the flow channel. Also, because prior art devices have chosen different materials than those used in the present application, and all require pre-treatment of the device, pre-treatment may make it more difficult for the flow channels within the device to form a meniscus.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale. It is obvious that the drawings in the following description are some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive exercise.

FIG. 1a is a schematic cross-sectional view of a manufacturing apparatus in an exemplary embodiment of the invention;

FIG. 1b is a schematic cross-sectional view of an alternative configuration of a flow channel of a production apparatus in an exemplary embodiment of the invention;

FIG. 1c is a schematic cross-sectional view of a manufacturing apparatus in another exemplary embodiment of the invention;

FIG. 2 is a schematic view of the structure of a manufacturing apparatus in still another exemplary embodiment of the present invention;

FIG. 3 is a schematic view of the construction of a manufacturing apparatus in an exemplary embodiment of the invention;

FIG. 4a is a schematic view of a first state of a first polar liquid at the micro-pores of a preparation apparatus in an exemplary embodiment of the invention;

FIG. 4b is a schematic view of a second state of the first polar liquid at the micro-pores of the manufacturing apparatus in an exemplary embodiment of the invention;

FIG. 4c is a schematic view of a third state of the first polar liquid at the micro-pores of the production apparatus in an exemplary embodiment of the invention;

FIG. 5 is a schematic flow diagram of a method of preparation in an exemplary embodiment of the invention;

FIG. 6 is a schematic diagram of the structure of an amphipathic molecule;

figure 7a is a schematic view of a first configuration of a meniscus within a flow channel;

figure 7b is a schematic view of a second configuration of the meniscus within the channel;

FIG. 8 is a schematic diagram of the structure of an amphiphilic molecular membrane formed at micropores;

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

FIG. 9b shows the process of wetting the capillary with liquid;

FIG. 10 shows a schematic of the structure of films of different thickness formed at the micropores;

FIG. 11a shows a schematic structural view of a flow channel in a preparation apparatus in an exemplary embodiment of the invention;

figure 11b shows a schematic view of the change of shape of the meniscus during movement in an exemplary embodiment of the invention.

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

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Herein, suffixes such as "module", "part", or "unit" used to denote elements are used only for facilitating the description of the present invention, and have no specific meaning in itself. Thus, "module", "component" or "unit" may be used mixedly.

Herein, the terms "upper", "lower", "inner", "outer", "front", "rear", "both ends", "one end", "the other end", and the like indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of describing the present invention and simplifying the description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

As used herein, unless otherwise expressly specified or limited, the terms "mounted," "disposed," "connected," "coupled" and the like are to be construed broadly and include, for example, "coupled," which can be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; wireless connection or wireless communication connection; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

As used herein, an "amphiphilic" molecule refers to a compound having both hydrophilic and lipophilic properties, with 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 "amphiphilicity" are used synonymously. The amphiphilic molecule may be a lipid molecule. A typical amphiphilic molecular membrane (or layer of amphiphilic molecules, also referred to simply as a "membrane") may be a lipid bilayer, which is a bilayer formed by two opposing lipid monolayers that are self-assembled such that the hydrophobic tails face each other to form a hydrophobic interior, and the hydrophilic heads face the exterior (each side being a polar hydrophilic environment). The lipid forming the lipid bilayer may comprise any suitable lipid, for example 1, 2-diphytanoyl-sn-glycero-3-phosphatidylcholine, diphytanoylphosphatidylcholine (DPhPC). The amphipathic molecule may be chemically modified or functionalized to facilitate coupling of the polynucleotides. The amphiphilic molecules may also be polymeric materials synthesized by physicochemical methods, such as ABA triblock copolymers (PMOXA-PDMS-PMOXA dimethyl oxazoline-polydimethylsiloxane-dimethyl oxazoline). The amphiphilic molecules may be a mixture.

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

As used herein, "polar aqueous solution" or "polar solution" refers to an aqueous solution containing water that is readily miscible with water and other polar solvents. The polar aqueous solution may include one or more solutes. For example, 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 polyelectrolyte to effectively enhance ion exchange lifetime. The polar aqueous solution may also comprise a redox couple or a combination of redox couples which may be partially oxidised or reduced to provide a redox couple, for example iron/ferricyanide. The polar aqueous solution may also be a cross-linked agarose gel and a sodium alginate gel.

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 membranes.

Herein, "square" includes polygons whose adjacent sides are at an angle of about 90 ° or close to 90 °, for example, squares are quadrangles, such as squares, or rectangles (as shown in a in fig. 1 b), etc. Of course, the "square" in this document does not necessarily need to be arranged as a standard square or rectangle or other polygon, for example, opposite sides of the "quadrangle" in the square may be arranged in parallel or not in parallel, and correspondingly, adjacent sides of the "quadrangle" may be arranged in perpendicular or not in perpendicular. The "square-like" includes a square with chamfers at the included corners of the adjacent sides, or a square with the adjacent sides connected by arcs, for example, a square-like shape, such as a rectangle-like shape (as shown in b in fig. 1b, the vertex angle of the rectangle is replaced by an arc structure), a square-like shape (such as a square with the vertex angle replaced by an arc structure), and the like. For example, herein, the phrase "the cross section of the flow channel is square or square-like" means that the cross section of the flow channel may be configured to be rectangular, square, rectangular-like, square-like, or the like. In order to avoid the inner surface of the flow channel from generating burrs and other defects during the machining process, the cross section of the flow channel is preferably shaped like a square, such as a rectangle, a square or the like.

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

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

Example one

In a first aspect of the present invention, there is provided a manufacturing apparatus for rapidly forming an amphiphilic molecular membrane (amphiphilic molecular layer), referring to fig. 1a to 4, the manufacturing apparatus having a plurality of micropores 21 therein and a flow channel 11 capable of allowing a solution to flow into the micropores 21, the manufacturing apparatus further comprising: an electrode layer 31, so that a solution (e.g., a polar solution, i.e., an electrolyte) flowing into the pores 21 can contact the electrode layer 31, wherein the cross section of the flow channel is preferably square or quasi-square. Of course, the cross section of the flow channel is within the scope of the present invention as long as it enables the film solution to form a meniscus and move in the form of a meniscus. Wherein, the micropores provide a growth platform for forming an amphiphilic molecular layer (amphiphilic molecular membrane, also called as 'membrane') by the membrane solution.

In some embodiments, the inner surface of the flow channel is formed by a polymer material, and the contact angle of the material selected for the inner surface of the flow channel (i.e., 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 between about 75 degrees and 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 surface of the micropores (the side walls of the micropores) is polyoxymethylene, so that the membrane solution added into the flow channel can form a meniscus in the flow channel and can form a layer of amphiphilic molecules at the micropores.

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

It is within the ability of the person skilled in the art to select suitable preparation materials by preliminary experiments. Specifically, the contact angles of different materials can be measured by a contact angle measuring method (such as a shape image analysis method, or weighing method) to select a suitable preparation material. In particular, a channel prepared from a material is considered to have the ability to form a meniscus when the contact angle of the material is between about 65 ° and 120 ° (of course, as long as the channel prepared from the selected material is capable of forming a meniscus).

Further, in some embodiments, the flow channel includes a first opening for loading (i.e., adding liquid, such as a membrane solution and a polar solution) into the flow channel.

Further, in some embodiments, the flow channel further comprises: and a second opening for discharging the liquid in the flow passage.

Further, in some embodiments, the flow channel is further provided with an electrode insertion opening for inserting a common electrode.

Specifically, in some embodiments, referring to fig. 1, the apparatus comprises: the sample injection device comprises a body, wherein the body comprises a first structural layer 1, a second structural layer 2 and a third structural layer 3 which are sequentially arranged, a flow channel 11 is arranged on the first structural layer 1, a first opening 12 is formed in one end of the flow channel 11 and used for sample injection, a plurality of micropores 21 are formed in the second structural layer 2 at intervals, and an electrode layer (equivalent to a first electrode) is arranged on the third structural layer 3;

when a solution (e.g., a first polar solution) is added to the flow channel and the solution flows through the at least one pore, the solution displaces air in the at least one pore (e.g., the solution may fill the at least one pore, see fig. 4a, with the first polar solution 6), and contacts the electrode layer, such that the solution (e.g., the first polar solution) can conduct electricity under the action of the electrode.

In some embodiments, the second structural layer is bonded, patterned and cross-linked, or the AZ4620, su-8 is bonded to a corresponding electrode on the electrode layer, and only one common electrode is provided (inserted) in the flow channel.

Because the inner surface of the flow channel and the sidewalls of the wells (i.e., the inner surface of the wells) are made of hydrophobic materials, the polar solution may not be able to smoothly enter the wells in some embodiments, or only a portion of the polar solution may enter the interior of the wells and not fill the wells in other embodiments, if the inner surface of the flow channel and the sidewalls of the wells are not wetted in advance.

Therefore, in some embodiments, the first polar solution added first may not completely fill the micro-pores, for example, referring to fig. 4b, the first polar solution 6 forms a droplet suspended at the first end of the micro-pores at the micro-pores, and the droplet formed by the first polar solution 6 and the electrode layer 31 are brought into contact with each other.

Further, in some embodiments, in order to ensure that the droplets formed by the first polar solution 6 can contact with the electrode layer, at least one electrode bump 32 (such as a probe or the like) is further disposed on the electrode layer 31, so that the droplets formed by the first polar solution can contact with the electrode layer when the volume of the droplets is small.

Further, in some embodiments, in order to enable the droplets formed by the first polar solution and the electrode layer to come into contact with each other, an electrode layer 31 for conducting electricity may be further provided on the side wall 22 of the micro-hole. Further, an electrode bump 32 may be further provided on the electrode layer 31.

Alternatively, in other embodiments, referring to fig. 4c, the first polar solution first introduced into the flow channel forms a thin solution film (layer) only at the first end of the micro-hole, and at this time, in order to enable the first polar solution to contact the electrode layer, an electrode bump 32 (such as a probe or the like) may also be disposed on the electrode layer, so as to ensure that the first polar solution can contact the electrode layer.

Specifically, in some embodiments, the first end of the micro-wells (i.e., the upper end, i.e., the end in communication with the flow channels) has an inner diameter that is greater than the inner diameter of the second end of the micro-wells (i.e., the other end of the micro-wells). In this embodiment, the pores are designed to be small at the top and large at the bottom, so that there is enough room inside the pores to accommodate a polar solution (e.g., a first polar solution that is first added into the pores) without affecting the size of the first end of the pores (the size of the inner diameter of the first end of the pores is closely related to the formation of the layer of amphiphilic molecules, and thus, the size of the inner diameter of the first end of the pores is usually fixedly set to be between about 100 micrometers and 200 micrometers). In particular, when the meniscus passes through the micropores, the layer of amphiphilic molecules in the meniscus forms at the narrowest point of the micropores, i.e. the amphiphilic molecules self-assemble at the first end of the micropores to form the layer of amphiphilic molecules.

For example, in some embodiments, as shown in fig. 1a and 4, the micro-holes are arranged in a trumpet shape with a small top and a large bottom.

In other embodiments, the micro-holes may be arranged in a cylindrical shape (i.e., the upper and lower ends are equal), or in a large-size manner. Of course, the micropores may be configured in other shapes, and it is within the scope of the present invention that the shape of the micropores is configured to successfully form the amphiphilic molecule layer.

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

In some embodiments, the second opening may also be used for inserting a second electrode (i.e., a common electrode), and thus, in some embodiments, an electrode insertion opening may not be provided on the flow channel, thereby simplifying the structure of the device.

Further, in some embodiments, referring to fig. 1 a-3, an electrode insertion opening 14 is further provided on the flow channel, and specifically, the second electrode is inserted into the flow channel through the electrode insertion opening 14.

Further, in some embodiments, the second structural layer is provided with a matching portion for matching with the second electrode, and when the second electrode is inserted into the flow channel, the first end (i.e., the insertion end) of the second electrode matches (e.g., contacts) with the matching portion.

Further, in order to fix the second electrode and improve the stability of the manufacturing apparatus 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 the first end of the second electrode is inserted into the groove when the second electrode is matched with the matching portion. In this embodiment, because the second electrode is inserted into the groove for fixing, on one hand, the second electrode is convenient to position and mount (the second electrode is prevented from shifting in the inserting process), and meanwhile, the second electrode is prevented from shifting and shaking in the horizontal direction in the using process, so that the working stability of the second electrode is improved.

In addition, the second electrode (i.e., the common electrode) in this embodiment may be set in a detachable state, i.e., may be installed (inserted) and removed by a worker, so as to facilitate replacement and cleaning of the second electrode.

It will be appreciated that the first and second electrodes are of opposite polarity, 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 portion is a TSV electrode, and the selected electrode material is pure gold. A chip (i.e., the second structural layer) having a plurality of micro-holes (e.g., an array of 256 micro-holes 8 × 32) is bonded to the third structural layer by thermocompression bonding, a permanent glue bonder, and a photoresist AZ4620 or su-8. The common electrode is preferably inserted into the recess of the chip using a gold plated copper post. The first opening (i.e. the sample inlet) and the second opening (i.e. the sample outlet) of the flow channel adopt a microfluidic dedicated pipeline for sample adding (i.e. adding corresponding liquid, such as polar solution or membrane solution) and sample outlet, and an injection pump (such as a reciprocating injection pump) is used for controlling the flow rate of the polar solution so as to control the moving speed of the membrane solution.

Preferably, in some embodiments, the reciprocating syringe pump model selected is harvard @ appaatus 4400.

Further, in some embodiments, the pore size of the microwells is between about 100 microns and about 200 microns, for example, the first ends of the microwells have an inner diameter size of about 100 microns to 200 microns.

Preferably, in some embodiments, the pore size of the microwells is between about 150 microns and 200 microns, for example, the first ends of the microwells have an inner diameter size of about 150 microns to 200 microns.

Preferably, in some embodiments, for ease of processing, referring to fig. 2, the micropores of the second structural layer are arranged in a multi-array, such as 2 x 8, or 8 x 32.

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

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

Further, in some embodiments, adjacent microwell spacing L6 within the array of microwells is greater than about 0.5 mm.

Surprisingly, the preparation device provided by the invention selects polyformaldehyde as a raw material to prepare structures such as a flow passage, micropores and the like of the device, and adopts a new size design for the space between adjacent micropores. The processing technology of the device is simplified, the device can be prepared by various types of processing modes (such as mechanical processing, laser processing and the like), the production cost of the device is greatly reduced, and the requirement for preparing a plurality of amphiphilic molecule layers at one time can be met. Moreover, according to the preparation device provided by the invention, the micropores in the preparation device do not need to be pretreated in the film forming process, and the film forming method is simpler.

It is understood that the apparatus provided by the present invention can be applied to both the new film forming method proposed by the present invention and the existing film forming method such as coating method.

In some embodiments, the raw material of the preparation device may be teflon, pmma, delrin (polyoxymethylene, i.e., polyoxymethylene) or parylene.

Of course, in other embodiments, the raw material of the preparation device may also be pmma (polymethyl methacrylate), epoxy resin, PC (polycarbonate), PVC (polyvinyl chloride), COC (cyclic olefin copolymer), polyimide, etc.

Further, in some embodiments, the preparation apparatus may adopt processing modes including: machining, laser machining, micro-injection molding, 3d printing, casting, electroporation, and the like.

Further, in some embodiments, the micro-holes in the preparation device are preferably machined using a laser. The micropores formed by laser processing are round and smooth in inner wall, and an amphiphilic molecular layer is easily formed.

Of course, in other embodiments, the micro-holes may be machined, electroporated, etc. It is understood that the micropores are processed such that the micropores are circular and have smooth sidewalls, and an amphiphilic molecule layer is easily formed.

Further, in some embodiments, the first, second, and third structural layers of the manufacturing apparatus body may be processed in layers and then assembled, or may be integrally formed (e.g., integrally injection molded), so that the manufacturing apparatus obtained by processing in layers and then assembling or integrally forming is within the scope of the present invention.

For example, in some embodiments, the teflon material can be micro-injection molded to make the device; the apparatus may be manufactured by pma, delrin, parylene, by physical vapour deposition, e.g. depositing parylene onto a surface of another processed material, preferably to a thickness of 5 microns

In some embodiments, the raw material is selected from materials with certain hydrophobicity and lipophilicity, so that pretreatment steps can be reduced in the process of preparing the amphiphilic molecular membrane, and the membrane forming method is simplified.

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

Further, in some embodiments, when the number of the micro holes is set to be larger, at least one third opening is further provided on the flow channel.

Because the lowest part of the third opening (i.e. the second end of the third opening connected with the flow channel) is higher than the highest part of the liquid level in the flow channel, or the second end of the third opening is flush with the liquid level in the flow channel, the liquid level of the meniscus in the flow channel can be kept unchanged in the moving process, and the stability of the form can be ensured without being influenced by the third opening.

Further, in some embodiments, to facilitate sample application, the first opening and the third opening can be arranged in a vertical size, for example, a first end (arranged on the surface of the preparation device and used for sample application) of the first opening is larger than a second end (connected to the flow channel) of the first opening.

In particular, in some embodiments, referring to fig. 1c, the third opening 13 is disposed on a side adjacent to the first opening 12. This embodiment is equivalent to providing two sample inlets (i.e. two sample inlets, namely, a first opening and a third opening) on the flow channel. At this time, the first opening (corresponding to the first inlet) is used for adding the membrane solution and for injecting/withdrawing the second polarity solution by the reciprocating syringe pump, and the second opening (corresponding to the second inlet) is used for adding the membrane solution.

For example, in some embodiments, when the micropores through which the meniscus passes are not film-forming, this indicates that the concentration of amphiphilic molecules in the meniscus is too low. At this point, the second polarity solution is pumped back at the first opening by the reciprocating syringe pump, causing the meniscus to move between the first opening and the third opening. During this process the meniscus will again flow through the pores that have already formed a film, and since the layer of amphiphilic molecules (i.e. the film) is itself a very stable arrangement, the thickness of the layer of amphiphilic molecules on the corresponding pores will not increase after the layer of amphiphilic molecules has been formed on the pores, even if the meniscus is repeatedly passed through the pores a number of times thereafter. That is, the process of moving the meniscus back towards the first opening does not affect the previously formed film.

Further, when the meniscus moves between the first opening and the third opening, a certain amount of membrane solution is added into the third opening (corresponding to the second sample inlet), then the meniscus between the first opening and the third opening is controlled by the injection pump to continue moving along the direction from the first opening to the third opening and to be combined with the newly added membrane solution to form a new meniscus, and the new meniscus continues to pass through the micropores which are not formed with membranes under the push of the second polarity solution and forms membranes on the micropores.

Therefore, one or more sample inlets may be correspondingly disposed on the flow channel based on the number of the micropores, for example, when the number of the micropores is relatively small, and the influence of the concentration change of the amphipathic molecules on the film formation is relatively small (or the influence caused by the concentration change can be ignored in the practical application process), only one sample inlet may be disposed on the flow channel, for example, only the first opening for sample injection is disposed.

For another example, when the number of micropores is set relatively large, the concentration of amphipathic molecules in the meniscus gradually decreases as the meniscus passes through a certain number of micropores, and thus film formation at the following micropores is not possible. In this case, a plurality of sample inlets may be provided on the flow channel, for example, referring to fig. 1c, a third opening may be added on the flow channel for adding the liquid sample. That is to say, can be through set up the introduction port on the runner, and then increase micropore quantity for preparation facilities can once only form more amphipathic molecular layer.

Of course, in other embodiments, the membrane solution may be added directly through the third opening, the first opening being used only for injecting/withdrawing the second polarity solution through the reciprocating syringe pump.

In other embodiments, a new meniscus may be formed directly through the third opening (such that the previously formed meniscus is between the first and third openings and is no longer involved in subsequent film formation) and such that the newly formed meniscus moves through and forms a film on the non-film-forming microvia.

Preferably, in order to control the above process more accurately, it can be realized by an automatic control (e.g. a program controlled by computer software).

Example two

Based on the first embodiment, referring to fig. 5, the present invention also provides a method for preparing a novel amphiphilic molecular membrane (membrane forming method), comprising the following steps:

s1 provides a preparation apparatus for preparing a plurality of amphiphilic molecular membranes, the preparation apparatus having a plurality of micropores therein, and a flow channel capable of allowing a solution to flow into the micropores, the preparation apparatus further having an electrode layer therein, 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 with the electrode layer;

s3 sequentially adding the film solution and the second polarity solution into the flow channel such that the film solution forms a meniscus within the flow channel (see fig. 7a and 7 b);

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 pore where the first polar solution contacts the electrode layer, and the amphiphilic molecules in the membrane solution form an amphiphilic molecule layer (specifically, the amphiphilic molecules form an amphiphilic molecule layer based on self-assembly capability) on the corresponding pore (i.e., the pore where the first polar solution is located and the first polar solution contacts the electrode layer). Wherein the contact angle of the inner surface of the flow channel is about 65 ° to 120 °.

It is understood that, in some embodiments, the steps S3 and S4 may be performed continuously (or, in an actual operation, the steps S3 and S4 may be one step), and of course, in other embodiments, the steps S3 and S4 may be performed step by step.

In this embodiment, the preparation device is preferably made of 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 about 65-120 degrees, so that the film solution added into the flow channel can form a meniscus under the combined action of the inner surface of the flow channel, the polar solution and the air in the flow channel. The cross section of the flow channel is preferably square or quasi-square, and at this time, the flow channel generates a certain resistance to the movement of the film solution (or the meniscus formed by the film solution), so that the moving speed of the film solution in the flow channel is not too fast, and the moving speed of each position on the film solution is relatively uniform (or the difference of the flowing speeds of each position has little influence on the stability of the meniscus), so that the meniscus can maintain a stable form during the moving process.

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

In the meniscus formation and meniscus movement of the film solution, the amount (volume) of the film solution and the speed of movement of the meniscus formed by the film solution are also important to the meniscus formation and movement, and it is possible for those skilled in the art to select the appropriate amount and speed of film solution through preliminary experiments.

For example, in the selection or prediction step of the amount of addition of the membrane solution, the main parameters considered are: reynolds number (Re), which is a dimensionless number that can be used to characterize a fluid flow condition. The Reynolds number calculation method comprises the following steps: where v, ρ, and μ are the flow velocity of the fluid (corresponding to the moving velocity of the membrane solution), the density, and the viscosity coefficient, respectively, and d is the characteristic length (corresponding to the length of the flow channel). The density and viscosity coefficient of the film solution can be obtained through experimental measurement, and the stability of the meniscus needs to be kept so that the Reynolds coefficient of the fluid is smaller (the smaller the Reynolds number is, the more stable the fluid flow is), namely, the laminar state is obtained.

Therefore, the present invention preferably makes Re of the fluid <2300, whereby the relationship between the length of the flow channel and the moving speed of the membrane solution can be preliminarily limited by the reynolds number (therefore, when the membrane solution is selected and the length of the flow channel of the device (the length of the flow channel is related to the arrangement of the micro well array) is determined, the moving speed of the membrane solution can be preliminarily limited based on the reynolds number, that is, the moving speed of the membrane solution is restricted by the membrane solution and the length of the flow channel). That is, for those skilled in the art, after the membrane solution is selected, the preparation device is selected for a preliminary experiment, so that a moving speed of the selected membrane solution can be selected appropriately. The injection speed of the polar solution (e.g., the second polar solution) is correspondingly designed based on the movement speed screened out in the preliminary experiment, so as to obtain the preset injection speed of the second polar solution (e.g., the injection speed of the second polar solution in steps S3 and S4). Of course, the injection speed of the second polarity solution can also provide the operation experience of the operator to make a preliminary estimate, and then perform a preliminary experiment to obtain the appropriate injection speed.

Further, in some embodiments, the contact angle of the selected material for the inner surface of the flow channel is between about 75 ° and about 95 °. Specifically, in some embodiments, the plurality of micropores are disposed at the inner surface of the flow channel, i.e., the inner surface of the flow channel and the surface of the micropores, at a contact angle of the material selected to be between about 75 ° and about 95 °.

Preferably, the inner surface of the flow passage and the surface of the micropore are both made of polyformaldehyde.

Further, in some embodiments, the second polarity solution is injected at a rate of about 10 μ L/min to about 50 μ L/min during formation of the meniscus (i.e., in step S3), and about 200 μ L/min to about 500 μ L/min during passage through the microwell array after formation of the meniscus (i.e., in step S4).

In order to more clearly illustrate the technical solution adopted by the present invention, the following is a brief description of the formation of the contact angle and the meniscus:

referring to fig. 9a, the contact angle (contact angle) refers to the angle from the solid-liquid interface to the gas-liquid interface through the liquid interior at the intersection of the solid S, the liquid L and the gas G. If theta is less than 90 degrees, namely the liquid is easier to wet the solid, the smaller the angle is, the better the wettability is represented; if θ >90 °, the liquid does not readily wet the solid and moves easily over the surface. Specifically, when θ is 0, complete wetting; partial wetting or wetting when θ <90 °; when theta is 90 degrees, the boundary line between wetting and non-wetting is formed; when theta is greater than 90 degrees, the material is not wetted; when θ is 180 °, it is completely non-wetting.

Referring to FIG. 9b, the liquid pressure rising along the capillary wall in the figure is

Wherein Δ p is the pressure of the liquid rising along the capillary wall, σ is the surface tension, R is the radius of curvature of the liquid surface, ρ is the liquid density, g is the gravitational acceleration, and h is the height at which the liquid level rises. Based on the definition of contact angle:

for example, when the contact angle θ is less than 90 °, the solution may form a meniscus within the capillary, and the meniscus formed at this time is concave. At the same time, the radius of curvature of the meniscus formed is related to the radius r of the capillary, and 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 (e.g., the second polar solution) introduced into the flow channel causes the membrane solution to diffuse out at the liquid surface of the polar solution (i.e., the end in contact with the membrane solution). Because the inner surface of the flow channel is made of a material with certain hydrophobicity, the inner surface of the flow channel has certain adsorption effect on the membrane solution. At this time, referring to fig. 7b, the membrane solution is a polar solution (second polar solution 2) on one side and air 8 on the other side. Wherein the surface of the liquid has a contractile force and the molecules of the interface layer of the membrane solution in contact with air are subjected to a pulling force directed towards the inside of the liquid, so that the membrane solution gradually forms a meniscus 4 inside the flow channel, see fig. 7 b).

Referring to fig. 7a, amphipathic molecules 5 are dissolved in the membrane solution, wherein amphipathic molecules 5 have hydrophilic ends 52 and hydrophobic ends 51 (see fig. 6). Due to the self-assembly ability of the amphiphilic molecules, when the membrane solution flows through the micropores, the hydrophilic ends 52 of the amphiphilic molecules inside will point to and contact the first polar solution, and the hydrophobic ends 51 will combine with the hydrophobic ends 51 of another layer of amphiphilic molecules, thereby forming a layer of amphiphilic molecules, as shown in fig. 7a and fig. 8. In this embodiment, by controlling the moving speed of the membrane solution, it is possible to avoid that the amphiphilic molecules are accumulated thicker and thicker at the micropores (as shown in fig. 10 (a)) and cannot be embedded.

In this embodiment, the membrane solution is pushed to move by the second polar solution, so that the membrane solution passes through the at least one micropore and forms an amphiphilic molecule membrane at the at least one micropore. And the flow rate of the second polar solution (or the sample adding speed/injection speed of the second polar solution) is relatively controllable, so that the moving speed (namely the flowing speed) of the membrane solution in the flow channel can be controlled by controlling the sample adding speed of the second polar solution, and the formation process of the amphiphilic molecular membrane is controlled, thereby preventing the membrane solution from flowing too fast and not successfully forming the amphiphilic molecular membrane, or preventing the membrane solution from flowing too slow and forming an excessively thick amphiphilic molecular membrane.

Through the film forming mode that the polar solution pushes the meniscus (film solution) to form a film at the micropores, the moving speed of the film solution can be controlled more accurately (for example, the injection speed of the polar solution is controlled through a pipette gun or a syringe pump, etc., so as to control the moving speed of the film solution), so that the residence time of the film solution at different micropores is the same or similar, and the film solution is prevented from being left in part of the micropore area for a long time to form a thick film, or from moving too fast to form a film unsuccessfully in part of the micropore area. Therefore, the present invention can directly produce the amphiphilic molecule layer with a film thickness meeting the use requirement by controlling the moving speed of the film solution more precisely, without performing a thinning process on the film after the film formation (for example, thinning the film from a thick to a thin by the thinning process, as shown in (a) to (d) of fig. 10). In other words, the film forming method of the present invention can form a film at one time, and has a good film forming effect, for example, by controlling the injection speed of the polar solution and thus the moving speed of the film solution, a film with a suitable thickness can be directly formed, for example, a film shown in (d) of fig. 10 can be directly prepared.

For example, in some embodiments, the injection rate of the second polarity solution is controlled by a syringe pump to control the rate of movement of the membrane solution. In this embodiment, the method provided is more controllable (e.g., compared to bubble extrusion film of the prior art). Of course, other injection methods can 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 scope of the present invention.

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

Preferably, in some embodiments, before S1, the method includes the steps of: and carrying out wetting treatment on at least one micropore.

In this embodiment, the wetting treatment enables the liquid (e.g., the first polar solution) to spread on the solid surface (e.g., the surface of the micropores), so as to enlarge the contact area between the polar solution and the inner wall of the flow channel and reduce the contact angle, thereby enabling the first polar solution to smoothly enter the at least one micropore and fill the micropore.

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

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

Specifically, in some embodiments, a low surface energy liquid (e.g., ethanol, etc.) is first introduced into the flow channel, the low surface energy liquid can flow into very small pores, such as micropores, and the polar solution can be added to fill the micropores, and if the low surface energy liquid is not added in advance, the polar solution may not flow into the micropores directly. And then introducing a first polar solution into the flow channel, so that the first polar solution fills at least one micropore, and dilutes and discharges the low-surface-energy liquid. The previously introduced low surface energy liquid makes it easier for the first polar solution to fill the pores, wherein the low surface energy liquid comprises: ethanol, and the like.

In other embodiments, step S2 further includes the steps of: and after the first polar solution is added into the flow channel, carrying out ultrasonic treatment on the preparation device, so that the first polar solution enters the micropores under the action of sound waves.

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

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

Further, in some embodiments, the non-polar solution may be an alkane organic solvent comprising decane, hexadecane, pentane, or a mixture thereof, and the non-polar solution is used to dissolve the phospholipid or the polymer triblock copolymer to form a membrane solution.

Further, in some embodiments, the second polar solution and the first polar solution may be the same or different polar solutions. The selected polar solution meets the conductive effect, and the nanopore protein can be inserted into the amphiphilic molecule layer.

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

Of course, in other embodiments, the nanopore protein (e.g., a solution comprising the nanopore protein) may be added to the flow channel after the amphipathic layer is formed.

Further, in some embodiments, the polar solution is an electrolyte in a gene sequencing process, and comprises an electrolyte and/or polyelectrolyte to effectively improve the ion exchange lifetime; a redox couple may also be included, and/or a combination of redox couples that may be partially oxidized or reduced to provide a redox couple, such as iron/ferricyanide; it may also be a cross-linked agarose gel, and/or a cross-linked sodium alginate gel; buffers may also be included 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), and the like.

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

the polar solution is energized through the electrode layer and the common electrode, so that the nanoporous proteins are embedded (intercalated) into the amphiphilic molecule layer under the action of the voltage.

For example, in some embodiments, a common electrode is inserted into a second opening (or first opening or electrode insertion opening) of the device, and then the polar solution is energized through the common electrode and a predetermined electrode layer. For example, the common electrode is a pin, a PET compliant electrode, or an electrode integrally designed with the microfluidic chip holder and connected to the circuit portion.

Specifically, in some embodiments, when the inner diameter of the flow channel is set to be large (or, when the flow channel is wide), in order to enable the meniscus to be successfully formed, the flow channel of the manufacturing apparatus related to the present method includes: the first opening and the second opening are communicated through a first flow passage 15. Referring to fig. 11a, the inner diameter of the first opening 12 is relatively small, and the inner diameter of the first flow passage 15 is relatively wide, so that there is a region of varying size at the first end of the flow passage (i.e., the end connected to the first opening), i.e., the inner diameter of the flow passage gradually increases in the direction from the first opening 12 to the first flow passage 15 (or, the inner diameter of the first end of the flow passage gradually increases in the direction away from the first opening).

When the film solution is added into the flow channel through the first opening, the film solution firstly enters the first end of the flow channel with smaller inner diameter, at the moment, the inner space of the flow channel is narrower, the meniscus formed by the film solution is thicker, and the shape is relatively stable. Therefore, the moving speed of the membrane solution can be set to be fast in the previous stage (specifically, because there is a region with an inner diameter increasing from the entrance of the flow channel to the inside of the flow channel, at this time, the injecting speed of the polar solution is relatively slow, but because the cross section of the flow channel is relatively small, even if the injecting speed of the polar solution is relatively slow, the meniscus can have a relatively fast moving speed), when the membrane solution gradually moves to a region with the largest inner diameter of the flow channel (for example, the middle region between the two ends of the flow channel, i.e., the region with the same inner diameter), the meniscus formed by the membrane solution is gradually stretched and thinned, the stability of the meniscus is relatively reduced, at this time, the moving speed of the membrane solution needs to be appropriately reduced (at this time, because the cross section of the region of the flow channel corresponding to the micro-hole is large, when the polar solution maintains the original injecting speed, the moving speed of the meniscus is also reduced; of course, the speed of the movement of the membrane solution can be further reduced by adjusting the injection speed of the polar solution small). In this embodiment, the gradual change in the dimensions of the channel makes the meniscus relatively stable and less prone to damage during formation and change, wherein the different states of the meniscus during movement in the channel are shown as the first, second and

third states

4a, 4b, 4c of the meniscus in fig. 11 b.

For example, in some embodiments, referring to fig. 11a, the region of the flow channel having a varying inner diameter (i.e., the region of unequal inner diameter, i.e., the first end of the flow channel) comprises: the first change area and the second change area are connected, wherein the curve arrangement of the side wall of the first change area (close to the first opening) is similar to the partial arc of the circle with the radius R1 (or the vertical projection of the side wall of the first change area is in the curve arrangement), and the curve arrangement of the side wall of the second change area is similar to the partial arc of the circle with the radius R2 (or the vertical projection of the side wall of the second change area is in the curve arrangement). Where R1 and R2 are equal (of course, in other embodiments R1 and R2 may not be equal, as long as the inner diameter of the flow channel is such that meniscus formation and movement is achieved).

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

For example, in one embodiment (using a manufacturing device having 8 × 32 microwells), the manufacturing method specifically includes the steps of:

firstly, adding pure ethanol into a flow channel to enable the ethanol to enter at least one micropore, then adding a polar solution into the flow channel to dilute and drain the ethanol, so that the polar solution is filled in at least one micropore and contacts an electrode layer (equivalent to a first electrode);

adding from about 20 microliters to about 50 microliters of the membrane solution into the first opening;

adding about 20 microliters to about 50 microliters of the polar solution into the first opening such that the film solution forms a meniscus within the flow channel;

adding about 300 microliters of polar solution to the first opening by a syringe pump at a preset injection rate (flow rate); specifically, the first 100 microliters of polar solution adopts a flow rate of 10 microliters/minute (in the process, the meniscus is formed and gradually thinned), and the second 200 microliters of polar solution adopts a flow rate of 5 microliters/minute, so that the meniscus can keep the shape stable and flow through the micropore array at a constant speed, and 50% to 80% of micropores on the micropore array can directly form the amphiphilic molecule layer.

Alternatively, in another embodiment (using a manufacturing apparatus provided with 16 microwells), the manufacturing method includes the steps of:

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

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

then adding about 8 to about 12 microliters of the polar solution to the first opening such that the film solution forms a meniscus;

approximately 60 microliters of the polar solution is then added to the first opening based on a preset injection rate (flow rate), specifically, the first 15 microliters of the polar solution adopts a flow rate of 4 microliters/minute, and the last 45 microliters of the polar solution adopts a flow rate of 2 microliters/minute, thereby maintaining the stability of the meniscus shape and enabling the membrane solution to flow through the microwell array at a uniform speed. Wherein 50% to 80% of the micropores in the micropore array can be directly provided with the amphiphilic molecule layer.

Of course, the film forming method and the manufacturing apparatus provided by the present invention do not completely exclude the pretreatment operation, such as based on different detection requirements, and those skilled in the art may also need to add a pretreatment step accordingly.

Further, in some embodiments, the method further comprises the steps of: and detecting the amphiphilic molecule layer.

For example, a triangular wave signal is input to the amphiphilic molecule layer, and then a corresponding square wave signal is obtained, and whether the amphiphilic molecule layer is formed and whether the amphiphilic molecule layer meets the requirement can be determined through the upper peak value and the lower peak value of the square wave signal. Specifically, when the layer of amphiphilic molecules is detected to be excessively thick, the concentration of the membrane solution can be appropriately reduced.

Specifically, a commercial AXON 1550B instrument is selected to detect an electrical signal or output a triangular wave to measure capacitances at two ends of the membrane to determine whether an amphiphilic molecular layer is formed.

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

The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

Claims

1. A method for preparing an amphiphilic molecule layer, comprising the following steps:

2. The method of claim 1, further comprising, before the step of S2: wetting at least one of the micropores;

or, the step of S2 includes: and after adding the first polar solution into the flow channel, carrying out ultrasonic treatment on the preparation device.

3. The production method according to claim 2, wherein the wetting treatment includes: an electrowetting treatment, and/or a liquid wetting treatment.

4. The production method according to claim 1, wherein the film solution includes: a non-polar solution, and an amphiphilic molecule, wherein the amphiphilic molecule optionally comprises: a phospholipid, or a macromolecule, or a mixture of a phospholipid and a macromolecule, the non-polar solution optionally comprising: an alkane organic solvent.

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

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

and/or, the first polar solution comprises: a cross-linked agarose gel, and/or a cross-linked sodium alginate gel;

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

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

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

and/or, the second polar solution comprises: a cross-linked agarose gel, and/or a cross-linked sodium alginate gel;

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

6. The method of claim 1, wherein a common electrode is further provided on the preparation apparatus, the common electrode being in contact with the second polar solution, and accordingly, the method further comprises the steps of:

7. The method of claim 1, wherein in step S3, the injection rate of the second polar solution is about 10 μ L/min to about 50 μ L/min;

and/or, in step S4, the injection speed of the second polarity solution is about 200 μ L/min to about 500 μ L/min.

8. The production method according to claim 1, wherein an inner surface material of the flow channel is polyoxymethylene.

9. The preparation device for the amphiphilic molecule layer is characterized in that a plurality of micropores are formed in the preparation device, a flow channel enabling solution to flow into the micropores is formed in the preparation device, an electrode layer is further arranged in the device, the solution flowing into the micropores can be in contact with the electrode layer, the cross section of the flow channel is square or square-like, and the inner surface of the flow channel is made of polyformaldehyde optionally.

10. The manufacturing apparatus of claim 9, wherein a spacing between adjacent ones of said micro-holes is greater than about 0.4 mm.