High throughput detection apparatus based on nanopore proteins

Abstract

This invention relates to a high-throughput detection device based on nanoporous proteins, comprising a biochip, a bilayer membrane, microwell arrays, microwells, and a pool structure. One or more microwell arrays are disposed on the biochip; each microwell array includes multiple microwells; the pool structure is a cylindrical structure with openings at the top and bottom; the pool structure is laid flat on the upper surface of the biochip and tightly connected to it; the inner cavity of the pool structure communicates with multiple microwells; a polar medium containing porous proteins is injected into the inner cavity of the pool structure; a bilayer membrane is disposed at the pore opening of each microwell; and the porous proteins are inserted into the bilayer membrane. This invention provides a high-throughput detection device based on nanoporous proteins that can improve film formation throughput, increase the types and efficiency of simultaneous film formation, and accommodate multiple porous proteins.

Description

Technical Field

[0001] This invention belongs to the technical field of electrochemical analysis and measurement equipment, and relates to a high-throughput detection device, particularly a high-throughput detection device based on nanoporous proteins. This application is a divisional application of application number 2023102118859, entitled "High-throughput Detection Device Based on Nanoporous Proteins". Background Technology

[0002] A bilayer membrane is a bilayer membrane constructed in vitro, composed of amphiphilic molecules. This membrane is impermeable to hydrophilic molecules and ions but allows transmembrane proteins to insert. After insertion, the nanoscale channels of these transmembrane proteins become the sole pathway connecting the solutions on both sides of the membrane. By applying a bias voltage across the nanopores, the specific current signal generated when biomolecules pass through the channels can be obtained. This nanopore sensing technology has advantages such as label-free operation, high sensitivity, and high specificity, enabling DNA or protein sequencing, drug screening, and analytical detection, and has great potential for wide-ranging applications. Currently, some commercial products based on nanopore sensing technology already exist, such as MinION manufactured by Oxford Nanopore Technologies. TM The device is capable of sequencing, characterized by containing only one type of pore for high-throughput sequencing of a single DNA molecule sample. However, such products can only detect one sample substance at a time and cannot accommodate multiple pore proteins simultaneously or detect more than one sample substance at a time. These common pore protein measurement products also cannot perform site-specific membrane formation operations and cannot fully utilize each detection channel on the array chip. Multiple channels can only form a single bilayer membrane. These defects limit the further development and application of this technology to some extent.

[0003] To achieve widespread application of nanopore sensing technology, a device that facilitates film formation is needed, possessing advantages such as high throughput, multifunctionality, and site-specific film formation. This would allow for the embedding of two or more porous proteins on a single chip, enabling the simultaneous analysis of two or more sample types (such as proteins, DNA, and drugs) to improve experimental efficiency. Bilayer membranes are typically formed by amphiphilic molecules dissolved in a nonpolar medium, sandwiched between two polar media. Existing film formation devices employ a two-chamber design, with each chamber containing a polar medium and connected by a micron-sized pore made of hydrophobic material. This pore can be used to support the bilayer membrane, typically formed using folding or painting methods (P Kongsuphol, et al. Sensor Actuat. B-Chem. 2013, 185, 530). However, these methods struggle to improve experimental throughput and result in low experimental efficiency. Leptihn et al. proposed a droplet film-forming device (S Leptihn, et al. Nat. Protoc. 2013, 8(6), 1048–1057) that constructs a bilayer film in the middle of the oil-water two-phase interface, reducing reagent consumption. For example, the invention patent with authorization announcement number CN104918696B discloses an arrayed droplet film-forming device that combines microfluidic technology to achieve high-throughput film formation. However, such droplet film-forming devices have complex chip designs, require a long incubation time to form the film, and are difficult to change the liquid to achieve multifunctional detection. The Orbit 16 instrument manufactured by Nanion Technologies, when paired with a rotor film-forming device, can achieve high-throughput multi-channel simultaneous film formation with high efficiency and easy liquid change. However, the device has limited throughput and can only form films uniformly for all channels, making it difficult to achieve point-to-point operation and different channels cannot form films separately. For example, the invention patent with authorization announcement number CN104254619B discloses a method for generating a bilayer, which forms a bilayer membrane by guiding bubbles through a flow channel to lubricate the lipid layer. The device is simple to operate, has high throughput and low cost, but it cannot achieve point-to-point film formation and can only form one type of phospholipid membrane, with multiple porous proteins embedded on a chip, etc.

[0004] Clearly, achieving high-throughput, multifunctional, and point-to-point film formation is a technical problem that urgently needs to be solved. Summary of the Invention

[0005] Purpose of the invention: In order to solve the above-mentioned technical problems in the background art, the present invention provides a high-throughput detection device based on nanoporous proteins that can improve film formation flux, increase the types and efficiency of simultaneous film formation, and accommodate a variety of porous proteins.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A high-throughput detection device based on nanoporous proteins is disclosed. The device comprises a biochip, a bilayer membrane, microwell arrays, microwells, and a pool structure. One or more microwell arrays are disposed on the biochip, each microwell array containing multiple microwells. The pool structure is a cylindrical structure with openings at both ends. The pool structure is laid flat on the upper surface of the biochip and is tightly connected to it. The inner cavity of the pool structure communicates with multiple microwells. A polar medium containing nanoporous proteins is injected into the inner cavity of the pool structure. A bilayer membrane is disposed at the opening of each microwell. The nanoporous proteins are inserted into the bilayer membrane.

[0008] Preferably, when the microwell group provided by the present invention is multiple, the pool structure is provided with left and right partitions; the partitions divide the inner cavity of the pool structure into independent small chambers, and the number of small chambers is the same as the number of microwell groups; the pore protein in different small chambers is different.

[0009] Preferably, the microwell groups and microwells provided by the present invention are arranged in an array.

[0010] Preferably, the biochip provided by the present invention includes a substrate layer, an electrode layer, and a support layer; the support layer is laid flat on the substrate layer and is tightly connected to the substrate layer; through holes are formed along the thickness direction of the support layer; the through holes and the substrate layer form a microwell; the electrode layer is placed at the bottom of the microwell; the diameter of the microwell is 1~1000 μm, preferably 50~150 μm.

[0011] Preferably, the high-throughput detection device based on nanoporous proteins provided by the present invention further includes a detection circuit and a central control unit connected to the detection circuit; the number of detection circuits is the same as the number of microwell groups; one end of the detection circuit is connected to the polar medium in the pool structure, and the other end is connected to the polar medium in the microwell.

[0012] Preferably, the detection circuit provided by the present invention includes a counter electrode, a preamplifier, an analog-to-digital converter (ADC), and a digital-to-analog converter (DAC); one end of the counter electrode extends into the pool structure and is in communication with the polar medium within the pool structure, and the other end of the counter electrode is connected to the ADC via the DAC; the polar medium within the microwell is connected via the preamplifier and the ADC; the ADC is connected to a central control unit; and the central control unit is an FPGA.

[0013] A site-specific film formation system for realizing the high-throughput detection device based on nanoporous proteins as described above, the site-specific film formation system comprising a hollow pipe, a fluid mechanical control module, and a microfluidic control system; the hollow pipe is connected to the fluid mechanical control module and moves synchronously with the fluid mechanical control module; the microfluidic control system is connected to the hollow pipe and injects fluid into the hollow pipe; the fluid is a liquid or a gas.

[0014] Preferably, the fluid machinery control module provided by the present invention is a pipette or a fluid pump; the hollow pipe is a pipette tip or a conduit.

[0015] A method for fabricating a bilayer membrane for a high-throughput detection device based on nanoporous proteins, as described above, using the site-specific film formation system, the method comprising the following steps:

[0016] 1) Fabrication of biochips;

[0017] 2) Prepare a site-specific film formation system;

[0018] 3) The ends of the hollow pipes in the fixed-point film formation system are coated with a nonpolar medium containing amphiphilic molecules;

[0019] 4) Insert the hollow pipe into the polar medium inside the pool structure;

[0020] 5) Move the hollow pipe through the fluid mechanical control module of the fixed-point film formation system, so that the hollow pipe gradually approaches the micro well opening of the micro well.

[0021] 6) Activate the microfluidic control system and inject fluid into the hollow pipe through the microfluidic control system;

[0022] 7) As the fluid flows through the hollow pipe and reaches the end of the hollow pipe, it gradually expands the end of the hollow pipe, which is coated with a non-polar medium containing amphiphilic molecules, and forms a fluid bubble.

[0023] 8) The fluid bubble is squeezed at the microwell opening, and the nonpolar medium containing amphiphilic molecules remains at the microwell opening to form a bilayer film;

[0024] Preferably, step 8) is followed by:

[0025] 9) Test the bilayer film formed in step 8). Based on the test results, retain the formed bilayer film or repeat steps 3) to 8) until a bilayer film with usable function is formed.

[0026] Application of high-throughput detection devices based on nanoporous proteins in the detection of high-throughput small biological molecules.

[0027] The advantages of this invention are:

[0028] This invention provides a high-throughput detection device based on nanoporous proteins, comprising a biochip, a bilayer membrane, microwell arrays, microwells, and a pool structure. One or more microwell arrays are disposed on the biochip; each microwell array includes multiple microwells. The pool structure is a cylindrical structure with openings at the top and bottom; the pool structure is laid flat on the upper surface of the biochip and tightly connected to it; the inner cavity of the pool structure communicates with multiple microwells; a polar medium containing nanoporous proteins is injected into the inner cavity of the pool structure; a bilayer membrane is disposed at the pore opening of each microwell; the nanoporous proteins are inserted into the bilayer membrane. The microwells used in this invention contain an electrode layer at their bottom, and both the electrode layer and the counter electrode located within the pool are connected to an external circuit for detecting current and applying voltage. The microwells are grouped and arrayed, and each group of microwells can form a membrane independently. Simultaneously, a specialized robotic arm or fluid pump controls the fluid to pass precisely through the microwell pores filled with a polar medium. The fluid is a gas or liquid containing amphiphilic molecules and a non-polar medium. A membrane forms at the microwell pore opening, and the nanoporous insertion membrane enables sensing. In short, because different bilayer membranes can be formed in different microwell groups, high-throughput detection of different small biological molecules can be performed simultaneously, making it very convenient and highly efficient. Attached Figure Description

[0029] Figure 1 This is a side view of the biochip with a three-layer structure provided by the present invention;

[0030] Figure 2 This is a top view of the 16-channel biochip provided by the present invention;

[0031] Figure 3 This is a schematic diagram of a biochip with an open-cell structure and its external circuitry.

[0032] Figure 4 This is a schematic diagram of a biochip with a covered pool structure and its external circuitry.

[0033] Figure 5 This is a schematic diagram of the process of a robotic arm manipulating fluid to form a film at a specific point;

[0034] Figure 6 This is a schematic diagram of the process of a microfluidic control system manipulating fluid to form a film at a specific point;

[0035] Figure 7 This is an instrument architecture diagram of a high-throughput detection device for nanoporous proteins;

[0036] Figure 8 This is a graph showing the test results of the open-circuit potential of the Ag / AgCl electrode;

[0037] Figure 9 This is a physical image of a coverless pool structure with bubble film formation at a fixed point;

[0038] Figure 10 This is a current-time plot of a microwell array formed into a film and with inserted pore proteins;

[0039] Figure 11 It is a current-time graph for the simultaneous detection of two small biomolecules by two pore proteins;

[0040] Figure 1-11 middle:

[0041] 1-Biochip; 2-Site-based film formation system; 3-Bilayer membrane; 4-Polar medium; 5-Amphiphilic molecule; 6-Nonpolar medium; 7-Microwell; 8-Microwell orifice; 9-Base layer; 10-Electrode layer; 11-Support layer; 12-Microwell group; 13-Pool structure; 14-Uncovered pool; 15-Covered pool; 16-Microchannel; 17-Buffer solution; 18-Pore protein; 19-Small biomolecule; 20-Counter electrode; 21-Common counter electrode; 22-External circuit; 23-Detection circuit; 24-Preamplifier; 25-Analog-to-digital converter; 26-Digital-to-analog converter; 27-Central control section; 28-Fluid control structure; 29-Microfluidic control system; 30-Fluid; 31-Mechanical device; 32-Microchannel device; 33-Hollow tube; 34-Fluid mechanical control module; 35-Pipette; 36-Fluid pump; 37-Pipette tip; 38-Conduit; 39-Bubble; 40-Robotic arm; 41-Optical observation platform; 42-Feedback control. Detailed Implementation

[0042] The following describes a specific embodiment of a high-throughput detection device based on nanoporous proteins according to the present invention, with reference to the accompanying drawings. However, it should be noted that the implementation of the present invention is not limited to the following embodiments.

[0043] As attached Figure 1 To be continued Figure 11 As shown: A high-throughput detection device based on nanoporous proteins, comprising:

[0044] A biochip 1 for supporting the membrane includes an array of microwells 7, with an electrode layer 10 at the bottom of each microwell 7. The microwells 7 are filled with a polar medium 4, which makes good contact with the electrode layer 10. A controllable, site-specific film-forming system 2 independently controls the directional flow of fluid in a hollow conduit 33 through a specific array of microwells 12, forming a bilayer membrane 3 at the microwell openings 8 of the specific microwell array 12. The bilayer membrane 3 is typically formed between two cavities capable of containing the polar medium 4.

[0045] To construct a bilayer membrane 3 in vitro, a biomimetic cell membrane with inserted bio-nanoporous proteins 18 for sensing and detection can be achieved by adding an amphiphilic molecule 5 between two polar media 4. The amphiphilic molecule 5 can dissolve in a nonpolar medium 6. Therefore, two chambers need to be designed to accommodate the polar media 4 respectively, and a film-forming region is used to form the bilayer membrane 3. A microwell 7 can serve as one chamber to accommodate the polar media 4, and the area above the microwell 7 can serve as the other chamber. The microwell opening 8 can be used to support the bilayer membrane 3. Furthermore, during detection, an electric field needs to be applied and current changes recorded; electrodes need to be inserted into the polar media 4 of each of the two chambers to form a current loop.

[0046] Biochip 1 has a three-layer structure, such as Figure 1 As shown, the substrate includes a substrate 9 with a smooth surface and a low dielectric constant to minimize noise introduced by the substrate. In some embodiments, the substrate 9 can be SiO2, which provides a smooth surface to form a flat support, and the low dielectric constant of the substrate can reduce system noise. In addition, due to the transparency of SiO2, an inverted microscope can be mounted on the bottom of the biochip 1 to observe fluid movement and film formation. Alternatively, the substrate 9 can also be a PCB, glass, silicon wafer with a thick oxide layer, etc.

[0047] Electrode layer 10 is a conductive metal thin film electrode with good electrochemical stability, providing a stable potential. Electrode layer 10 needs to provide a stable potential for nanopore sensing and also possess good conductivity to connect to the external circuit 22. In this embodiment, an Ag / AgCl electrode is used to provide a stable potential, and an Au electrode is used as a wire to connect to the external circuit 22. Specifically, the wire pattern is first constructed by photolithography, and Cr (10 nm) / Au (100~200 nm) wires are deposited by electron beam evaporation. Excess metal is removed by a stripping process. The photoresist can be a dedicated stripping negative adhesive ROL-7133. The substrate is first cleaned sequentially with isopropanol and deionized water, dried with N2, and pretreated by heating at 120 ℃ for 10 min. A spin coater is used to obtain a sacrificial layer with a thickness of ~2 μm by spin coating at 600 rpm for 10 s and 1000 rpm for 30 s. Pre-baking is performed at 110 ℃ for 2 min, followed by UV exposure for 6.5 s (9 mW / cm²). 2After drying at 110 ℃ for 2 min, the mixture was developed with ZX-238 developer for 70 s, rinsed with deionized water, and dried with N2. Cr was used as the adhesion layer for Au during vapor deposition. To obtain a uniform and dense Au layer, a suitable vapor deposition rate, such as 0.1 nm / s, was required. Next, Ag electrodes were prepared. Since Ag has lower conductivity than Au and is easily oxidized, Au was used as the conductive wire, and Ag was only used as an electrode at the bottom of microwell 7 to provide potential. Silver could be prepared by electroplating, but the resulting silver layer would have different morphologies due to the different impedances of each channel. Therefore, photolithography lift-off and electron beam vapor deposition were used to obtain the Ag layer. A thick Ag layer is difficult to prepare, while a thin layer has a short service life. Therefore, vapor deposition of Ag of 200 nm to 1 μm is more suitable. Since Ag is easily oxidized, it should be chlorinated as soon as possible after preparation. NaClO or FeCl3 can be used for chlorination to obtain AgCl. The prepared Ag / AgCl electrode exhibits good electrochemical stability. When the Ag layer thickness is 500 nm and NaClO oxidation takes 3 min, the electrochemical stability is as follows: Figure 8 As shown, the open-circuit potential of the self-made electrode was tested using CHI. A commercial Ag / AgCl reference electrode was used for electrode 20 and the reference electrode. The results showed that, except for the initial charge and discharge, the self-made electrode fluctuated by less than 2 mV within 8 hours, and the electrode potential was stable.

[0048] The support layer 11 has good hydrophobic properties, a low dielectric constant, and a smooth surface, making it easy to support the bilayer film 3. The support layer 11 is 1~100 μm thick. The support layer 11 is patterned to obtain microwells 7, such as by electron beam etching, ultraviolet lithography, or laser etching. In one embodiment, the support layer 11 is SU-8, which has a low dielectric constant, good hydrophobicity, and is easy to pattern, making it a good film support layer 11. The microwells 7 are prepared by photolithography. SU-8 is further spin-coated onto the chip containing the electrode layer 10 at 600 rpm for 10 s / 1600 rpm for 40 s to obtain a support layer 11 of ~25 μm. Pre-baking at 95 ℃ for 4 min and exposure for 9 s (9 mW / cm²) are then performed. 2 After drying at 95 ℃ for 3 min and developing for 4 min, the microwells were rinsed with isopropanol and dried to obtain microwells with a diameter of 1~1000 μm, preferably 50~150 μm.

[0049] To increase experimental throughput, microwells 7 were grouped together, and each group of microwells 7 was further arranged to form a microwell group 12 array. For example... Figure 2 In the illustrated embodiment, each microwell group 12 contains 2×2 microwells 7, and each biochip 1 contains an array of 2×2 microwell groups 12, forming a 16-channel array of microwells 7. Each group of microwells 7 can be individually controlled to form a film. Each microwell 7 within a group of microwells 7 can be individually controlled to form a film, or they can be controlled uniformly to achieve point-operable film formation.

[0050] Above the biochip 1 is a pool structure 13. Each group of microwells 7 shares one pool structure 13. The pool can serve as another chamber for containing polar media 4, used to contain the same biomolecule. The pools in different microwell groups 12 are not interconnected, and each pool can contain different biomolecules. In one embodiment, pool structure A contains pore protein 18A and nucleic acid a, pool structure B contains pore protein 18A and nucleic acid b, pool structure C contains pore protein 18B and polypeptide a, and pool structure D contains pore protein 18B and polypeptide b, achieving the purpose of simultaneous detection of pore protein 18 and simultaneous detection of small biomolecules 19 (such as nucleic acid a, nucleic acid b, polypeptide a, and polypeptide b as previously described). Figure 2 In the embodiment shown, there are 4 pool structures, and each pool structure 13 corresponds to 4 sets of micro wells 7.

[0051] In one embodiment, the pool structure 13 can be an uncovered pool 14 (e.g., Figure 3 As shown, the PMMA is laser-cut, forming a through-structure in the corresponding microwell group 12. One side of the PMMA is fixed to the biochip 1 using double-sided tape, forming an open-top pool 14. Alternatively, the pool can be implemented using a clamp to hold the biochip 1, silicone pad, and PMMA in a three-layer structure. The pool structure can be rectangular or circular, with dimensions sufficient to accommodate all the microwells 7 of the microwell group 12 while allowing fluid displacement. Figure 2 In the embodiment shown, the diameter of the pool structure 13 is 10 mm and the depth of the pool structure 13 is 5 mm.

[0052] Alternatively, the pool body is a covered pool body 15 containing a microchannel structure 16 (e.g., Figure 4 As shown, a mold is prepared by photolithography, PDMS is poured in, and after the PDMS is cured, it is cut into a specified shape. A hole is punched directly above the channel to connect the hollow tube 33. After being treated with Plasma and soaked in APTES silanizing agent, the PDMS is bonded to the biochip 1 containing SU-8 to form a microchannel 16 with a channel depth of 20~80 μm.

[0053] Both the microwell 7 and the pool structure 13 are filled with a polar medium 4. The polar medium 4 may contain a conductive buffer solution 17, pore proteins 18, and the target biomolecules 19. The pore proteins 18 and the target biomolecules 19 can be added separately after the bilayer membrane 3 is formed, or they can be premixed in the buffer solution. In this embodiment, a 1 M Tris-KCl, 1 mM EDTA, pH 8.0 buffer solution 17 is used as the polar medium 4 to fill the microwell 7 and the pool structure. After the bilayer membrane 3 is formed, pore proteins 18, such as Aerolysin, α-Hemolysin, MspA, OmpF, OmpG, CsgG, etc., and the target biomolecules 19, such as DNA, peptides, and glycans, are added to the pool.

[0054] One possible scenario is that when the polar medium 4 is added to the chip, the air inside the microwell 7 is difficult to remove due to its small size, making it difficult for the polar medium 4 solution to enter the microwell 7 and form a good contact with the electrode, thus preventing the formation of a current loop. In this case, the air can be expelled by applying voltage, bubbling agitation, or turning on the fluid control pump to increase the pressure inside the microchannel, so that the microwell 7 is filled with the polar medium 4.

[0055] The tank structure 13 facilitates liquid replacement. Without damaging the membrane structure, new biomolecules can be added to the polar medium 4 as needed, or the original polar medium 4 can be replaced with another polar medium 4.

[0056] like Figure 3 as well as Figure 4 As shown, in order to form a current loop, an electrode is inserted into each cell structure as the counter electrode 20 of the microwell group 12. The counter electrode 20 is an Ag / AgCl reference electrode. The counter electrodes 20 of each microwell group 12 are interconnected to form the common counter electrode 21 of the device.

[0057] Both electrode layer 10 and common counter electrode 21 are connected to external circuit 22, forming a dual-electrode system. Electrode layer 10 within each microwell group 12 is connected to a detection circuit 23 for current signal monitoring and bias voltage application. External circuit 22 includes a preamplifier section, an analog-to-digital converter section, and a central control section 27. In this embodiment, a sixteen-channel microcurrent amplifier is used for measurement. Electrode layers 10 within the same microwell group 12 are connected to the same detection circuit 23. Detection circuit 23 includes a preamplifier 24 for amplifying and transmitting the current signal from each microwell 7 (e.g., using a transimpedance amplifier); an analog-to-digital converter 25 for converting analog voltage signals into digital voltage signals for convenient signal transmission and processing; and a digital-to-analog converter 26 for converting digital voltage into analog voltage for applying bias voltage. Figures 3-4In the example shown, each detection circuit 23 is four-channel, with at least the preamplifier and analog-to-digital converter being four-channel. The digital-to-analog converter can be single-channel, in which case each microwell 7 in the group is uniformly charged with voltage. Alternatively, the digital-to-analog converter can also be four-channel, with each microwell 7 individually controlled in terms of voltage. The external circuit 22 also includes a central control section 27 for controlling the data transmission and processing of the sixteen microwells 7, such as using an FPGA programmable chip to implement logic control.

[0058] Furthermore, the present invention also provides a controllable site-specific film formation system 2, which includes a fluid control structure 28, a microfluidic control system 29, and a fluid 30. The fluid 30 can be a liquid or a gas; this embodiment uses a gas as an example. The fluid contains a small amount of amphiphilic molecules 5 and a nonpolar medium 6, which are used to form a bilayer film 3 at the microwell opening 8.

[0059] The fluid control structure consists of a fluid control structure 28 for each microwell group 12, enabling individual control of the fluid in each microwell group 12. In this embodiment, the four microwell groups 7 have four independent fluid control structures 28, capable of controlling the fluid within the four pool structures 13. In one embodiment, the fluid control structure 28 is a mechanical device 31; in another embodiment, the fluid control structure 28 is a microchannel device 32.

[0060] See Figure 7 The microfluidic control system 29 includes a hollow tube 33 and a fluid mechanical control module 34. The fluid mechanical control module 34 can be a pipette 35 (such as...). Figure 3 (as shown) or fluid pump 36 (e.g.) Figure 4 (As shown). The pipette 35 can be a pipette or a multi-pipette, etc., and the fluid pump 36 can be a pipette, a multi-pipette, a pressure pump, a peristaltic pump, or a syringe pump, etc.

[0061] Fluid 30 is located within hollow tubing 33, which is formed by pipette tip 37 connected to pipette 35 (e.g., Figure 3 (As shown). Alternatively, the hollow pipe 33 can also be constructed from a conduit 38 capable of connecting to the fluid pump 36 (as shown). Figure 4 (As shown).

[0062] like Figure 5As shown, the head of the hollow pipe 33 is dipped into a small amount of non-polar medium 6 containing amphiphilic molecules 5, and bubbles 39 are pumped out through the hollow pipe 33 located above the micro well 7. The volume of the bubbles 39 is at least enough to cover one or a group of micro well orifices 8, such as 1~3 μL. A thin layer of non-polar medium 6 containing amphiphilic molecules 5 is formed at the interface between the bubbles 39 and the polar medium 4 in the pool. This layer slowly forms a bilayer film 3. The amphiphilic molecule 5 can comprise lipids or block copolymers, or any combination of several molecules, such as phospholipid molecules like diphytylphosphatidylcholine (1,2-dimercapto-sn-glycerol-3-phosphate choline, DPhPC), dipalmitoylphosphatidylcholine (1,2-hexadecanoyl-propane-glycerol-3-phosphate, DPPC), and diblock copolymers (1,2-butadiene)-b-poly(ethylene oxide) (PBD-PEO), etc. The nonpolar medium 6 can be an organic solvent such as n-decane, n-octane, n-hexadecane, squalene, or silicone oil. In this example, a phospholipid solution of 20-30 mg / mL DPhPC / n-decane is prepared by first dissolving DPhPC in chloroform solution, removing the organic solvent by vacuum, and finally adding n-decane to obtain the phospholipid solution. The phospholipid solution can be stored at 4 °C for up to 1 month.

[0063] In such Figure 5 In one embodiment shown, a hollow pipe 33 is manipulated by a mechanical device 31 to achieve point-to-point film formation. The mechanical device 31 is operated by a robotic arm 40. Alternatively, a micromanipulator can be used. This method is suitable for the directional flow and manipulation of fluid in an open-top tank 14 structure. The fluid 30 within the hollow pipe 33 manipulated by the mechanical device 31 can have displacement properties on the plane of the tank. The mechanical device 31 can manipulate the fluid to cover or leave the microwell orifice 8 by dragging the hollow pipe 33. The fluid can be pumped in or out. Specifically, the robotic arm 40 manipulates a pipette 35. In this embodiment, the pipette 35 is a pipette tip. A small amount of nonpolar medium 6 containing amphiphilic molecules 5, such as 200 nL, is taken with the tip 37 of the pipette 35. Figure 5 The robotic arm 40 controls the tip of the gun head 37 to move above the micro well 7. Figure 5 b. A fixed-point pump produces a certain volume of bubbles (39). Figure 5 The fluid volume should be sufficient to cover at least one microwell opening, such as 2 μL. Figure 5 d moves the bubble 39 to cover all microwells 7 within the microwell group 12, and waits for 1-5 seconds. Figure 5 e. Remove bubble 39. Figure 5 After bubbling is completed, a bias voltage is applied to measure the film thickness.

[0064] Figure 9In the structure of the uncovered pool body 14, the pipette tip 37, which is dipped in amphiphilic molecules and nonpolar solvent, is moved above a microwell 7 by the pipette 35 to form a 3 μL bubble 39 at a fixed point, thereby achieving fixed-point bubble formation.

[0065] Figure 10 a~ Figure 10 Film formation and pore formation experiments were conducted using a capless cell structure 14. Experimental data were presented on a microwell group 12 containing four microwells 7, each with a diameter of 50 μm. In a buffer solution 17 containing 1 M Tris-KCl, 1 mM EDTA, and pH 8.0, the pore openings 8 of the four microwells 8 were covered and then removed by bubbles 39 containing 30 mg / mL DPhPC / n-decane. Films were formed in each of the four channels, and wild-type Aerolysin pore protein 18 was inserted. With a bias voltage of +100 mV applied, the current was 0 pA when only the membrane was present, as the membrane allowed almost no ion passage. However, after the insertion of pore protein 18, the nanopores within the pore protein allowed specific ions to pass through, and a current step signal was detected, demonstrating that one or more pore proteins 18 self-assembled and embedded on the membrane.

[0066] Figure 11 For some experimental results of this embodiment, a film-forming device with an open-top cell 14 structure was used for nanopore sensing, enabling multifunctional detection. Experiments were conducted in a polar medium of 1 M Tris-KCl, 1 mM EDTA, and pH 8.0. DPhPC bilayer films 3 were formed on both microwells 7. Figure 11 Experiment 1 was conducted using a 12SiO2-based biochip containing 7 microwells with a diameter of 50 μm. Wild-type Aerolysin was used to detect the current signal of poly(dA)5-SH nucleic acid molecules at +150 mV. Figure 11 b represents the use of a 12SiO2-based biochip with a 100 μm diameter microwell group 1, employing wild-type Aerolysin at +100 mV to detect the current signal of poly(dA)4 nucleic acid molecules. Figure 11 c represents a 12SiO2-based biochip using a microwell group with a diameter of 50 μm (microwell 7). The current signal of poly(dA)4 nucleic acid molecules was detected at +100 mV using mutant K238C Aerolysin pore protein, demonstrating that the membrane formation device has good membrane stability and a high signal-to-noise ratio.

[0067] like Figure 6As shown, in another embodiment, individually controlled fluid can also be achieved through the microchannel device 32. This embodiment is suitable for fluid manipulation of the covered pool structure 15, where the microchannel device 32 controls the directional flow of fluid in the hollow pipe 33 to achieve film formation. The pool structure 13 is a covered system containing the microchannel structure 16. Figure 6 A microfluidic channel has an opening above the microwell 7, and a conduit 38 connected to a fluid pump 36 is inserted into the opening. In this embodiment, the fluid pump 36 is a constant flow injection pump. The conduit 38 is pre-diluted with a small amount of amphiphilic molecules 5 and a nonpolar medium 6. Figure 6 First, pump the polar medium 4 into the microchannel 16. Figure 6 c. A small amount of gas is pumped into the conduit 38 by the fluid pump 36 until... Figure 6 When bubble 39 comes into contact with the microwell opening 8, wait 1-5 seconds. Figure 6 e is then pumped back into the bubble 39 to achieve Figure 6 f. Film formation. The system includes at least two fluid pumps 36, one of which is used to pump in or out a polar medium 4 (such as...). Figure 4 The fluid pump 2 shown is used for pumping in and out bubbles 39 for film formation, such as buffer solutions and biomolecules. Figure 4 The fluid pump shown is 1).

[0068] During the formation of the membrane array, when bubble 39 passes through micro-well opening 8, as... Figure 5 g and Figure 6 As shown in g, the nonpolar medium 6 containing amphiphilic molecules 5 in the fluid will partially remain at the microwell orifice 8, forming an amphiphilic molecular layer. This amphiphilic molecular layer, through diffusion or disturbance by applied stimulation, gradually forms a bilayer film 3. For example... Figure 5 h and Figure 6 As shown in h, the porous protein 18 in the polar medium 4 can be autonomously inserted into the bilayer membrane 3 when the conditions are suitable. When the target biomolecule 19 passes through the nanopores, it generates a characteristic blocking current signal to achieve the detection function.

[0069] During nanopore sensing, a bias voltage is applied via external circuit 22, typically within ±1 V. After the formation of five layers of amphiphilic molecules, a bias voltage within ±500 mV is applied to measure the membrane thickness. If the membrane is thick, a bias voltage beyond ±300 mV can be applied to break it down. When driving the target biomolecule 19 through the pore protein 18, a bias voltage within ±300 mV is applied for measurement.

[0070] To form a suitable bilayer membrane 3, certain stimuli can be applied. These stimuli include applying a ±1 V voltage via external circuit 22 to break the thick membrane, measuring the membrane capacitance using a triangular wave voltage (e.g., ±100 mV, 100 Hz), and verifying that the membrane thickness is suitable if the capacitance is 8~12 pF. Alternatively, applying a stepped voltage (e.g., 0~+500 mV) and measuring the membrane voltage, and verifying that the membrane thickness is suitable if it breaks at +300~+500 mV, may also cause membrane rupture. During electrical stimulation, the membrane may rupture. In this case, the nozzle 37 or conduit 38 used to generate fluid initially can be used to re-explode bubbles 39 to reform the membrane. Stimulation also includes adjusting the membrane thickness through airflow disturbance within the fluid, such as continuously pumping out multiple bubbles 39 to break the thick membrane, or slowly pumping out a single bubble 39 and carefully removing it to finely adjust the membrane thickness or reform the membrane.

[0071] The device is connected to an external system, which can be an external circuit 22 or an optical observation platform 41. The external circuit 22 is connected to the common counter electrode 21 and the electrode layer 10 of the chip. The optical observation platform 41 can be an upright microscope or an inverted microscope. The microscope can be located below the biochip 1, with the focal plane being the microwell opening 8, used to observe the control of the bubbles 39, such as whether the bubbles 39 completely cover the microwell 7 or whether the bubbles 39 have moved to the next microwell 7. This can also be observed through the current signal of the external circuit 22. Based on the observed bubble 39, feedback control 42 is applied to the point-forming film-forming device, such as the robotic arm 40 or the fluid control pump, to adjust the position and size of the bubbles 39 in a timely manner to achieve rapid, point-forming film formation. The feedback control 42 can be included in the external circuit 22, or a separate control device can be designed to digitally control the robotic arm 40, the fluid control pump, etc.

[0072] The present invention can achieve high-throughput, point-to-point formation of bilayer membranes and form different types of bilayer membranes in different microwell groups. It can also embed different types of pore proteins 18 into the bilayer membranes of different microwell groups to form nanopores.

[0073] The membrane array formation device of the present invention is reusable, and the biochip 1 is disposable or at least replaceable.

Claims

1. A high-throughput detection device based on nanoporous proteins, characterized in that: The high-throughput detection device based on nanoporous proteins includes: a biochip (1) for supporting the membrane, the biochip (1) comprising an array of microwells (7), the bottom of the array of microwells being an electrode layer (10), the microwells being filled with a polar medium (4), the polar medium (4) forming good contact with the electrode layer (10); and a controllable site-specific film formation system (2), the system independently controlling the fluid in the hollow pipe (33) to directionally pass through a specific microwell group (12), forming a dual-phase flow at the microwell opening (8) of the specific microwell group (12). The bilayer membrane (3) is typically formed between two cavities capable of containing polar media (4). The biomimetic cell membrane uses inserted bio-nanoporous proteins (18) for sensing and detection. Amphiphilic molecules (5) are added between the polar media on both sides. The amphiphilic molecules (5) are dissolved in non-polar media (6). The two cavities contain polar media (4) respectively. A film-forming region is used to form the bilayer membrane (3). The microwell (7) serves as one side cavity to contain the polar media (4), and the area above the microwell (7) serves as the other side. The chambers are used to contain the polar medium (4), and the microwell openings (8) are used to support the bilayer membrane (3). Electrodes are inserted into the polar medium (4) in the two chambers to form a current loop. The biochip (1) includes a substrate layer (9), an electrode layer (10), and a support layer (11). The support layer (11) is laid flat on the substrate layer (9) and is tightly connected to the substrate layer (9). Through holes are formed along the thickness direction of the support layer (11). The through holes and the substrate layer (9) together form a microwell (7). The electrode layer (10) The bottom of the microwell (7) is placed; above the biochip (1) is the pool structure (13), and each group of microwells (7) shares a pool structure (13). The pool structure (13) is a cylindrical structure with openings at the top and bottom. The pool structure (13) is laid flat on the upper surface of the biochip (1) and is tightly connected to the biochip (1). The inner cavity of the pool structure (13) is connected to multiple microwells (7). A polar medium (4) including pore protein (18) is injected into the inner cavity of the pool structure (13).

2. The high-throughput detection device based on nanoporous proteins according to claim 1, characterized in that: When there are multiple microwell groups (12), the pool structure (13) is provided with left and right partitions; the partitions divide the inner cavity of the pool structure (13) into independent small chambers, the number of small chambers is the same as the number of microwell groups (12); the pore protein (18) is different in different small chambers.

3. The high-throughput detection device based on nanoporous proteins according to claim 1, 2, or 3, characterized in that, The diameter of the microwell (7) is 1~1000 μm, preferably 50~150 μm.

4. The high-throughput detection device based on nanoporous proteins according to claim 1, characterized in that: The high-throughput detection device based on nanoporous proteins also includes a detection circuit and a central control unit (27) connected to the detection circuit; the number of detection circuits is the same as the number of microwell groups (12); one end of the detection circuit is connected to the polar medium (4) in the pool structure (13), and the other end is connected to the polar medium (4) in the microwell (7).

5. The high-throughput detection device based on nanoporous proteins according to claim 4, characterized in that: The detection circuit includes a counter electrode (20), a preamplifier (24), an analog-to-digital converter (25), and a digital-to-analog converter (26); one end of the counter electrode (20) extends into the pool structure (13) and is connected to the polar medium (4) inside the pool structure (13); the other end of the counter electrode (20) is connected to the analog-to-digital converter (25) through the digital-to-analog converter (26); the polar medium (4) inside the microwell (7) is connected to the preamplifier (24) and the analog-to-digital converter (25); the analog-to-digital converter (25) is connected to the central control unit (27); the central control unit (27) is an FPGA.

6. The high-throughput detection device based on nanoporous proteins according to claim 1, characterized in that: The fixed-point film formation system includes a hollow pipe (33), a fluid mechanical control module (34), and a microfluidic control system (29); the hollow pipe (33) is connected to the fluid mechanical control module (34) and moves synchronously with the fluid mechanical control module (34); the microfluidic control system (29) is connected to the hollow pipe (33) and injects fluid (30) into the hollow pipe (33); the fluid (30) is a liquid or a gas.

7. The high-throughput detection device based on nanoporous proteins according to claim 1, characterized in that: The fluid machinery control module (34) is a pipette (35) or a fluid pump (36); the hollow tube (33) is a pipette tip (37) or a conduit (38).

8. A method for forming a bilayer membrane in a high-throughput detection device based on nanoporous proteins according to any one of claims 1 to 7, characterized in that: The forming method includes the following steps: 1) Prepare the biochip (1); 2) Prepare the site-specific film formation system (2); 3) The end of the hollow pipe (33) in the fixed-point film formation system (2) is dipped with a non-polar medium (6) containing amphiphilic molecules (5); 4) Insert the hollow pipe (33) into the polar medium (4) inside the pool structure (13); 5) Move the hollow pipe (33) by the fluid mechanical control module (34) of the fixed-point film formation system (2) so that the hollow pipe (33) gradually approaches the micro well opening (8) of the micro well (7); 6) Turn on the microfluidic control system (29) and inject fluid (30) into the hollow pipe (33) through the microfluidic control system (29); 7) When the fluid (30) flows in the hollow pipe (33) and reaches the end of the hollow pipe (33), it gradually spreads the end of the hollow pipe (33) with a non-polar medium (6) containing amphiphilic molecules (5) and forms a fluid bubble. 8) The fluid bubble is squeezed into the micro well opening (8) of the micro well (7), and the non-polar medium (6) containing amphiphilic molecules (5) remains at the micro well opening (8) to form a bilayer film (3). Preferably, step 8) is followed by: 9) Test the bilayer film (3) formed in step 8), and retain the formed bilayer film (3) or repeat step 3) to step 8) until a bilayer film (3) with usable function is formed.

9. The application of the high-throughput detection device based on nanoporous proteins as described in any one of claims 1-7 as a high-throughput detection device for small biological molecules (19).

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