CN116698541A - Protein pretreatment method to be sequenced for sub-nanopore microfluidic biochip - Google Patents
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Classifications
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- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- G01N1/44—Sample treatment involving radiation, e.g. heat
Abstract
The application belongs to the field of nanopore protein sequencing, and particularly discloses a protein pretreatment method to be sequenced for a sub-nanopore microfluidic biochip; the technical scheme of the application is as follows: mixing 0.001-0.1% w/v surfactant and 0.1-5mM reducer to form micro denaturant, mixing with 50-500pM target protein in 50-500mM sodium chloride solution, heating at 80-100deg.C for 1-3 hr to obtain final concentration; after the protein is pretreated by the method disclosed by the application, SDS anions with equal quantity can be uniformly coated on the surface of the protein, so that an advantage is created for realizing transmembrane translocation of a protein single molecule in a sub-nanopore in an electrophoresis manner, and an ionic current signal for collecting a denatured protein single molecule transmembrane translocation event can be recorded by using a microfluidic biochip based on a solid-state nanopore, so that conditions are created for single-molecule sequencing of the protein.
Description
Technical Field
The application belongs to the field of nanopore protein sequencing, and particularly discloses a protein pretreatment method to be sequenced for a sub-nanopore microfluidic biochip.
Background
Proteins are major components of life, whose function is largely dependent on its primary structure, i.e., the order of amino acids in a polypeptide chain. Protein diversity in cells provides a great deal of key information for exploring physiological and pathological processes, so developing a protein sequencing method has great significance for basic research and clinical application. However, only a few techniques are available for determining protein sequences, such as mass spectrometry (Mass Spectrometry). Unfortunately, these methods have limitations such as the need for protein samples of sufficient purity and concentration, and low sensitivity, high cost, and short read length. The new generation of single molecule sequencing technology is gradually innovating proteomics research, and provides a new thought for detecting low-abundance proteins and researching single cell secretion proteomics. To date, a nanopore sequencing technique based on electrophoretic force driven, electrical signal feedback (Nanopore Sequencing Technology) has been established that uses uniformly charged surface DNA or RNA single molecules to pass through the nanopore and induce ionic current changes under the force of an electric field generated by a transmembrane voltage, and then directly reads the sequence information of the nucleotide by identifying fluctuation level changes in the blocking current (j.kasianowicz et al 1996,Proc.Natl.Acad.Sci.USA 93:13770-13773, a.meller et al 2001,Physical Review Letter 86:3435-3438). The Minion nanopore sequencer (C.ip et al 2015, F1000 Research 4:1075) proposed in 2014, oxford Nanopore Technologies Inc is representative, and the portable sequencing device can perform ultra-long reading and on-site rapid detection, so that the gene sequencing method is thoroughly changed and rapidly becomes the main stream of the market. After completion of the sequencing work of the highly repetitive and complex DNA fragments left behind by the human genome project (The Human Genome Project), the first human whole genome sequence was published, nanopore single molecule sequencing technology began to strive towards protein sequencing (C.Dekker et al 2018,Nature Nanotechnology 13:786-796).
Continuing to use current nanopore gene sequencing technology to read the primary structure of a protein can be too sensitive to resolve the amino acid sequence due to the effects of the amino acid averaging volume and protein denaturing agents. Specifically, the pore size of the shrinkage region of the prior biological nanopore is about 1.5 nanometers, which is suitable for carrying out nucleotide sequencing, however, the volume of amino acids is only about one tenth of that of nucleotides, so that for protein sequencing, the prior nanopore can lose the signal to noise ratio due to the oversized pore size. In addition, protein molecules in a natural state have complex and diverse three-dimensional structures, and the surface charge distribution of polypeptide chains is uneven, which makes it difficult to realize transmembrane translocation of proteins in nanopores by using an electrophoresis method. Furthermore, in order to be able to detect the primary structure of proteins, some chemicals are usually added to promote the denaturation of proteins, and these chemicals have an irreversible adverse effect on the biological nanopores and lipid bilayers. Therefore, a new generation of sequencing technology is needed to realize de novo sequencing of proteins, and the acquisition of electric signals is completed by expanding the proteins and attaching uniform charges on the surfaces of the proteins to force the 'rod' -shaped polypeptide chains to pass through the nanopores in an electrophoretic manner, which has important strategic significance for deep exploration of proteomics.
Disclosure of Invention
In order to solve the problems, the application discloses a protein pretreatment method to be sequenced for a sub-nanopore microfluidic biochip.
In order to achieve the above purpose, the application adopts the following technical scheme:
the protein pretreatment method to be sequenced for the sub-nanopore microfluidic biochip at least comprises the following steps: mixing 0.001-0.1% w/v surfactant and 0.1-5mM reducer to form micro denaturant, mixing with 50-500pM target protein in 50-500mM sodium chloride solution, and heating at 80-100deg.C for 1-3 hr to obtain final concentration.
Furthermore, in the method for preprocessing the protein to be sequenced for the sub-nanopore microfluidic biochip, the surfactant is sodium dodecyl sulfate.
Furthermore, in the method for preprocessing the protein to be sequenced for the sub-nanopore microfluidic biochip, the reducing agent is 2-mercaptoethanol.
Preferably, the surfactant sodium dodecyl sulfate is mainly 0.1% w/vSodium Dodecyl SThe protein denaturation treatment can be completed by mixing a micro-denaturant consisting of a sodium sulfate (SDS) and 10mM of a reducing agent 2-Mercaptoethanol (BME) with a target protein (50-500 pM) in a 250mM sodium chloride solution and heating at 85 ℃ for 2 hours.
Furthermore, the protein to be sequenced pretreatment method for the sub-nanopore-oriented microfluidic biochip is characterized in that the target protein is selected from human histone H3.3 and beta-amyloid Native Abeta 1-42 Or an interleave-disrupted version of variant Scrambled aβ 1-42 。
Further, the method for preprocessing the protein to be sequenced for the sub-nanopore microfluidic biochip further comprises a verification step after preprocessing, wherein the verification step comprises the following steps: round dichroism was used to examine whether the protein molecule had lost its higher order structure. Round dichroism (Circular Dichroism Spectroscopy) was used to examine whether the protein molecule had lost its higher order structure, such as the alpha-helix, etc. In the round secondary chromatography of the denatured protein, the characteristic peaks of the original secondary structure at 208nm and 222nm ultraviolet wavelengths disappear.
Furthermore, according to the protein pretreatment method to be sequenced for the micro-fluidic biochip with the sub-nano holes, the sub-nano holes are processed by a transmission electron microscope, and the diameter of the sub-nano holes is smaller than 1 nm. In the previous work of the present inventors, sub-nanopores having a diameter of less than 1nm have been simply and reliably processed on a pure silicon inorganic thin film having a thickness of 5nm.
Further, the protein pretreatment method to be sequenced for the sub-nanopore microfluidic biochip comprises the following steps:
1) Developing design work of a microfluidic device, and then manufacturing a resin mold by adopting a stereolithography 3D printing technology;
2) The silicon-based material bearing the inorganic film window, the PDMS mold and the glass slide are integrated by using a plasma bonding method, so that the single-molecule detection function of the microfluidic biochip is realized.
In some embodiments, the geometric design of the microfluidic device is first completed by AutoCAD drawing software, then the fabrication of the mold on the resin material is completed by using stereolithography 3D printing technology, and finally the individual units are integrated together using a plasma cleaner and a micro-stage.
Further, the above method for pretreating protein to be sequenced for a sub-nanopore microfluidic biochip, wherein the step 2) comprises the following specific steps:
a. cleaning the glass slide by acetone, isopropanol and deionized water respectively;
b. preparing a PDMS mould, and punching a small hole in the middle by using a flat needle under a microscope;
c. the glass slide and PDMS mold were placed in a plasma cleaner with the adhesive side facing up, run O 2 Plasma cleaning for 15-45s; the bonded glass slide and PDMS mould are put back into the plasma cleaner, and the silicon-based material is put into the plasma cleaner, and the second round of O is operated 2 Plasma cleaning;
d. connecting a vacuum pump probe to a micro-stage on a microscope stage, opening the vacuum pump to enable the silicon-based material to be adsorbed to the vacuum pump probe, and adjusting the micro-stage to bond the silicon-based material to an opening of a PDMS mold;
e. after bonding, using a magnet to provide additional adhesion between the silicon-based material and the glass slide, and placing in a vacuum oven, and baking at 70-80 ℃ for 10-20min;
f. and (3) taking down the magnet, coating uncured PDMS on the edge of the silicon-based material, putting the device back into an oven, and baking at 70-80 ℃ for 20-40min to realize the assembly of the silicon-based material bearing the inorganic film window and the microfluidic device, thereby forming the sub-nano-pore microfluidic biochip.
Further, the method for preprocessing the protein to be sequenced for the sub-nanopore microfluidic biochip further comprises the following steps of: the inorganic thin film in the nanopore microfluidic biochip was immersed in a 250mM sodium chloride solution ph=6.7±0.1 as a standard electrolyte for sub-nanopore conductivity measurement, the conductivity of the sub-nanopore processed by transmission electron microscopy was measured on a picoampere level using a patch clamp amplifier, and an I-V curve was plotted.
The quality of the nanopore microfluidic device is assessed based on the information of conductivity, surface charge density, ion current noise, etc. of the nanopore. The conductivity of nanopores is generally in positive correlation with the size of the pores, so intermittent observations of the I-V curve can be used to determine the integrity of the inorganic thin film and the sealability of the device. The nanopore surface charge density is estimated by measuring the dependence of the nanopore conductivity on the concentration of ions in the electrolyte. Since protein molecules translocate through the membrane in the pores too rapidly, we will increase the sampling rate and bandwidth as much as possible, which inevitably enhances noise in the data. For this purpose, an insulating material (PDMS, κ=2.3-2.8) is applied to the film surface to reduce parasitic capacitance and thereby suppress electrical noise inherent in the dielectric element, which is advantageous for signal compensation.
The application has the following beneficial effects:
currently, a major obstacle faced by nanopore single molecule sequencing technology when applied in proteomics is that polypeptide chains do not possess a uniform distribution of surface charges on similar nucleic acid chains.
The present application discloses a method for producing target proteins, such as human Histone (Histone H3.3) and beta-amyloid (Native Abeta) 1-42 ) And its interweaving scrambling version variants (arranged Aβ 1-42 ) The pretreatment method mainly comprises the step of promoting the denaturation of protein molecules by increasing the temperature so as to lose the higher structure. In addition, the denaturation treatment also wraps the protein surface with an equal amount of SDS anions, which creates an advantage for the protein single molecule to realize transmembrane translocation in the sub-nanometer hole in an electrophoresis manner.
By denaturing the protein and attaching surfactant anions to its surface, not only is the protein unfolded, but an equal amount of negative charge is wrapped, forcing the protein molecules to electrophoretically pass through the nanopore, which opens the door for sequencing the protein using the nanopore. Meanwhile, the preparation method of the matched microfluidic biochip is provided, and powerful support is provided for realizing the transformation of the high-throughput protein sequencing platform product based on the nanopore single-molecule sequencing technology.
Drawings
Fig. 1: round two chromatography of human histone H3.3 before and after denaturation;
fig. 2:
(a) The figure is a TEM photograph of a sub-nanopore with a diameter of 0.3 nm;
(b) The figure shows a schematic of a mold of a microfluidic device designed by AutoCAD software;
(c) FIG. is a schematic diagram of a resin mold fabricated using stereolithography 3D printing techniques and a PDMS mold fabricated from the mold of the microfluidic device;
(d) The figure shows the assembled nano-pore microfluidic biochip by using the plasma bonding technology;
(e) The figure is a cross-sectional view of a nanopore microfluidic biochip;
(f) The graph shows the measurement of the conductivity of sub-nanopores in 250mM sodium chloride solution after plasma bonding using a patch clamp amplifier;
(g) The figure is an electric noise figure before and after a layer of insulating material is overlapped on the surface of the film, and 1/f noise is obviously suppressed;
fig. 3: the (a) Native Abeta collected by the protein pretreatment method of the application 1-42 And (b) a scissored Abeta 1-42 A profile of transmembrane translocation events of the protein in a solid state sub-nanopore;
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
The reagents or instruments used in the examples of the present application were not manufacturer-identified and were conventional reagent products commercially available.
Example 1
Denaturation treatment of human histone H3.3.
100. Mu.L, 0.2. Mu.g/. Mu.L of human histone H3.3 solution, 100. Mu.L of deionized water (18M. OMEGA.), 0.5mM 2-mercaptoethanol, and 0.05% (w/v) sodium dodecyl sulfate were mixed together. After thoroughly mixing, the mixture was heated to 85℃and held for 2 hours, followed by cooling to 20 ℃. To confirm that the secondary structure of the protein has been developed, its circular dichroism spectrum was acquired using an AVIV 202SF spectrometer and the absorbance difference in the ultraviolet range of 195-260nm was measured. To enhance the signal, the protein concentration can be increased from 1 μg/mL to 400 μg/mL. Each sample was scanned three times and data was recorded in 1nm wavelength increments. The circular dichroism spectrum of the protein before denaturation has two negative peaks around 208nm and 222nm, which are typical signs of alpha helix structures, and after denaturation, the characteristic peaks disappear as the secondary structure is destroyed. Circular dichroism spectrum of human histone H3.3 before and after denaturation is shown in FIG. 1.
Example 2
And assembling the microfluidic biochip based on the solid sub-nano holes.
The design work of the microfluidic device is carried out by AutoCAD drawing software, and then a resin mold is manufactured by adopting a stereolithography 3D printing technology. The silicon-based material bearing the inorganic film window, the PDMS mould and the glass slide glass (75 mm multiplied by 25mm multiplied by 1 mm) are integrated by a plasma bonding method, so that the single-molecule detection function of the microfluidic biochip is realized. The specific steps are as follows, the glass slide is cleaned by acetone, isopropanol and deionized water respectively. A PDMS mold was prepared and a small hole was punched in the middle with a flat needle under a microscope. The glass slide and PDMS mold were placed in a plasma cleaner with the adhesive side facing up, run O 2 Plasma cleaning for 30 seconds. The bonded slide-PDMS device is replaced in the cleaning machine, and the film window is placed in the plasma cleaning machine, and the second round O is operated 2 And (5) plasma cleaning. The vacuum pump probe is connected to a micro stage on the microscope stage. And (3) opening a vacuum pump to enable the silicon chip to be adsorbed to a probe of the vacuum pump, and adjusting the micro-stage to bond the chip to an opening of the PDMS mould. Adhesive tapeAfter closing, a magnet was used to provide additional adhesion between the chip and the glass slide and placed in a vacuum oven and baked at 75 ℃ for 15 minutes. And (3) taking down the magnet, coating uncured PDMS on the edge of the silicon chip, putting the device back into an oven, and baking at 75 ℃ for 30 minutes to finally realize the assembly of the film chip and the microfluidic device. The present application uses 250mM sodium chloride solution (pH. Apprxeq. 6.7.+ -. 0.1) as the standard electrolyte for sub-nanopore conductivity measurement. Immersing an inorganic film in a sodium chloride solution, measuring the conductivity of a sub-nano hole processed by a transmission electron microscope on a picoampere level by using a patch clamp amplifier (Axiopatch 200B), and drawing an I-V curve, wherein the experimental results are shown in a graph (a) shown in a scanning transmission electron microscope (FEI Titan), an accelerating voltage of 300kV, a beam diaphragm aperture of 70 mu m, a spot size of 6, a current of 0.429nA and a drilling time of 25s, so that a TEM picture of the sub-nano hole with the diameter of 0.3nm is obtained; (b) The figure shows a schematic of a mold of a microfluidic device designed by AutoCAD software; (c) FIG. is a schematic diagram of a resin mold fabricated using stereolithography 3D printing techniques and a PDMS mold fabricated from the mold of the microfluidic device; (d) The method comprises the steps of performing plasma bonding on a chip and a PDMS microfluidic device by using a plasma bonding process in combination with an optical microscope and a micropositioner, and sealing the PDMS microfluidic device on a glass sheet with the thickness of 1mm by using the same bonding strategy to finally form a microfluidic channel containing sub-nano holes; (e) drawing is a cross-sectional view of the nanopore microfluidic biochip; (f) The graph shows the measurement of the conductivity of sub-nanopores in 250mM sodium chloride solution after plasma bonding using a patch clamp amplifier; (g) The figure is an electric noise figure before and after a layer of insulating material is overlapped on the surface of the film, and 1/f noise is obviously suppressed.
Example 3
Native Aβ 1-42 Pretreatment of protein and nanopore test.
Taking Native Abeta with molecular weight of 4514.4Da 1-42 Protein 0.5mg, 40. Mu.L of 1.0% NH 4 The lyophilized polypeptide was dissolved in OH, and then the solution was diluted with 44mL of deionized water to give a 2.52. Mu.M stock polypeptide solution, which was gently vortexed for mixing. 200. Mu.L of 2.52. Mu.M polypeptide stock was added to 9.8mL DI, dilutedRelease was 50-fold, yielding a 50nM protein solution. Re-diluted 10-fold with deionized water, 100. Mu.L of 50nM solution was added to 900. Mu.L of deionized water in a 1.5mL centrifuge tube to a final sample concentration of 5nM (2.26X 10) -5 μg/μl). Then carrying out protein denaturation treatment according to the following steps:
a. 20. Mu.L of protein solution was withdrawn from 5nM aliquots;
b. 50. Mu.L (0.04 g SDS in 40mL deionized water) was removed from the 0.1% w/v SDS inventory;
c. remove 50 μl from 10mM BME stock (add about 30 μl of 14.3M pure BME liquid in 40mL deionized water);
d. 120. Mu.L of the solution was removed from 500mM NaCl;
e. 760. Mu.L from 250mM NaCl;
f. after mixing the above, 100pM target protein, 0.005% SDS and 500. Mu.M BME were obtained in 1mL 250mM NaCl buffer in a 1.5mL centrifuge tube;
g. the tube was heated at 85℃for 2 hours to denature the protein and to give it a uniform negative charge on its surface.
Data collection was performed using this denatured protein solution in a nanopore microfluidic biochip.
Example 4
Scrambled Aβ 1-42 Pretreatment of protein and nanopore test.
Taking the Scramble Abeta 1-42 Protein 0.5mg, 40. Mu.L of 1.0% NH 4 OH was dissolved in the lyophilized polypeptide and the solution was then diluted with 44mL deionized water to give a 2.52. Mu.M stock polypeptide solution, which was gently vortexed for mixing. 200. Mu.L of the 2.52. Mu.M stock polypeptide was added to 9.8mL DI and diluted 50-fold to give a 50nM protein solution. A further 10-fold dilution with deionized water was performed and 100. Mu.L of 50nM solution was added to 900. Mu.L of deionized water in a 1.5mL centrifuge tube to give a final sample concentration of 5nM. Then carrying out protein denaturation treatment according to the following steps:
a. 20. Mu.L of protein solution was withdrawn from 5nM aliquots;
b. 50. Mu.L (0.04 g SDS in 40mL deionized water) was removed from the 0.1% w/v SDS inventory;
c. remove 50 μl from 10mM BME stock (add about 30 μl of 14.3M pure BME liquid in 40mL deionized water);
d. 120. Mu.L of the solution was removed from 500mM NaCl;
e. 760. Mu.L from 250mM NaCl;
f. after mixing the above, 100pM target protein, 0.005% SDS and 500. Mu.M BME were obtained in 1mL 250mM NaCl buffer in a 1.5mL centrifuge tube;
g. the tube was heated at 85℃for 2 hours to denature the protein and to give it a uniform negative charge on its surface.
Data collection was performed using this denatured protein solution in a nanopore microfluidic biochip. FIG. 3 shows (a) Native Abeta collected by the protein pretreatment method of the present application 1-42 And (b) a scissored Abeta 1-42 Transmembrane translocation event profile of proteins in solid state sub-nanopores. (a) And (b) are respectively Native Abeta 1-42 And Scramble Abeta 1-42 Normalized two-dimensional speckle heat map of Probability Density Function (PDF) of dataset, wherein I 0 For the aperture current, Δi is the blocking current amplitude and Duration is the blocking Duration. Each translocation event is based on its blockage rate (ΔI/I) relative to the pore current 0 ) And the occlusion duration is mapped to a point in the graph, the red contour is represented by a graph having 50% x PDF max The connection of these points of value represents a distribution characteristic of a typical event.
The above examples show that the present application, by denaturing the protein and attaching surfactant anions to its surface, not only spreads the protein but also encapsulates the same amount of negative charge forcing the protein molecules to electrophoretically pass through the nanopore, which opens the door for sequencing the protein using the nanopore. Meanwhile, the application also discloses a preparation method of the matched microfluidic biochip, which provides powerful support for realizing the transformation of the high-throughput protein sequencing platform product based on the nanopore single-molecule sequencing technology.
The foregoing has shown and described the basic principles, principal features and advantages of the application. It will be appreciated by persons skilled in the art that the above embodiments are not intended to limit the application in any way, and that all technical solutions obtained by means of equivalent substitutions or equivalent transformations fall within the scope of the application.
Claims (9)
1. The protein pretreatment method to be sequenced for the sub-nanopore microfluidic biochip is characterized by at least comprising the following steps: mixing 0.001-0.1% w/v surfactant and 0.1-5mM reducer to form micro denaturant, mixing with 50-500pM target protein in 50-500mM sodium chloride solution, and heating at 80-100deg.C for 1-3 hr to obtain final concentration.
2. The method for pretreating protein to be sequenced in a sub-nanopore microfluidic biochip according to claim 1, wherein the surfactant is sodium dodecyl sulfate.
3. The method for pretreating protein to be sequenced in a sub-nanopore microfluidic biochip according to claim 1, wherein the reducing agent is 2-mercaptoethanol.
4. The method for pretreating protein to be sequenced for a sub-nanopore microfluidic biochip according to claim 1, wherein said target protein is selected from human histone H3.3, beta-amyloid Native aβ 1-42 Or an interleave-disrupted version of variant Scrambled aβ 1-42 。
5. The method for pretreating a protein to be sequenced for a sub-nanopore microfluidic biochip of claim 1, further comprising a verification step after pretreatment is completed, the verification step comprising: round dichroism was used to examine whether the protein molecule had lost its higher order structure.
6. The method for pretreating protein to be sequenced in a micro-fluidic biochip with sub-nanopores according to claim 1, wherein the sub-nanopores are sub-nanopores with a diameter of less than 1nm processed by transmission electron microscopy.
7. The method for pretreating protein to be sequenced which is oriented to sub-nanopore microfluidic biochip according to claim 1, wherein the nanopore microfluidic biochip is assembled by the steps of:
1) Developing design work of a microfluidic device, and then manufacturing a resin mold by adopting a stereolithography 3D printing technology;
2) The silicon-based material bearing the inorganic film window, the PDMS mold and the glass slide are integrated by using a plasma bonding method, so that the single-molecule detection function of the microfluidic biochip is realized.
8. The method for pretreating protein to be sequenced in a sub-nanopore-oriented microfluidic biochip of claim 7, wherein said step 2) comprises the specific steps of:
a. cleaning the glass slide by acetone, isopropanol and deionized water respectively;
b. preparing a PDMS mould, and punching a small hole in the middle by using a flat needle under a microscope;
c. the glass slide and PDMS mold were placed in a plasma cleaner with the adhesive side facing up, run O 2 Plasma cleaning for 15-45s; the slide glass and the PDMS mould are replaced in the plasma cleaner, and simultaneously the silicon-based material is placed in the plasma cleaner, and the second round of O is operated 2 Plasma cleaning;
d. connecting a vacuum pump probe to a micro-stage on a microscope stage, opening the vacuum pump to enable the silicon-based material to be adsorbed to the vacuum pump probe, and adjusting the micro-stage to bond the silicon-based material to an opening of a PDMS mold;
e. after bonding, using a magnet to provide additional adhesion between the silicon-based material and the glass slide, and placing in a vacuum oven, and baking at 70-80 ℃ for 10-20min;
f. and (3) taking down the magnet, coating uncured PDMS on the edge of the silicon-based material, putting the device back into an oven, and baking at 70-80 ℃ for 20-40min to realize the assembly of the silicon-based material bearing the inorganic film window and the microfluidic device, thereby forming the sub-nano-pore microfluidic biochip.
9. The method for pretreating protein to be sequenced for a nanopore microfluidic biochip of claim 8, further comprising the step of measuring conductivity after step f, comprising the steps of: the inorganic thin film in the nanopore microfluidic biochip was immersed in a 250mM sodium chloride solution ph=6.7±0.1 as a standard electrolyte for sub-nanopore conductivity measurement, the conductivity of the sub-nanopore processed by transmission electron microscopy was measured on a picoampere level using a patch clamp amplifier, and an I-V curve was plotted.
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