CN110669660B

CN110669660B - Method for manufacturing gene transfer rate control device based on motor protein

Info

Publication number: CN110669660B
Application number: CN201910996484.2A
Authority: CN
Inventors: 陈昌; 胡春瑞; 豆传国
Original assignee: Shanghai Industrial Utechnology Research Institute
Current assignee: Shanghai Industrial Utechnology Research Institute
Priority date: 2019-10-19
Filing date: 2019-10-19
Publication date: 2023-03-24
Anticipated expiration: 2039-10-19
Also published as: CN110669660A

Abstract

The invention provides a method for manufacturing a gene transfer rate control device based on a motor protein, which comprises the following steps: i) Forming a motor protein with a polyanionic tail; ii) providing a nanopore device comprising a housing, a fluid cavity sealing layer, a flexible substrate nanopore structure, a fluid cavity base; the flexible substrate nanopore structure comprises a metal layer to reduce the nanopore size to 1-100 nm; the motor protein is stabilized in the nanopore; the motor protein is anchored in the nanopore by a metal-sulfur bond, so that the transport rate of the gene strand to be detected through the nanopore is at least slower than 0.1ms per base pair. The invention anchors the motor protein in the nanopore by using a click chemistry method so that the transport speed of the gene chain to be detected through the nanopore is at least slower than 0.1ms per base pair, and the transport speed of the gene chain can be effectively controlled by adjusting the temperature and the pH, so that the transport speed of the gene chain to be detected through the nanopore 51 can be further reduced to tens of even hundreds of milliseconds, and the sensitivity of the spectrum detection is improved.

Description

Method for manufacturing gene transfer rate control device based on motor protein

Technical Field

The invention belongs to the field of gene sequencing and the field of biomolecule sensing, and particularly relates to a manufacturing method of a gene transfer speed control device based on a movement protein.

Background

The measurement of genetic information has revolutionary driving effects in the fields of life sciences and medicine. Future precise medicine and personalized medicine require novel sequencing technologies with lower cost, higher speed, higher precision and longer read length.

The new generation of single molecule real-time sequencing technology solves the requirements of longer read length and higher speed; the recently rapidly developed biological nanopore sequencing technology further addresses the need for lower cost. The biological nanopore sequencing technology does not need to prepare a large number of samples, biological and chemical reagents do not need to be consumed in the sample preparation process, the sequencing cost is greatly reduced, the time for DNA cloning and amplification is saved, and the time cost is saved. A first commercial biological Nanopore sequencer MinION is published by Oxford Nanopore Technologies (ONT) in the UK, has a palm-sized volume and very good portability, and greatly expands the application scene of the sequencer. For example, minION is used for rapid detection and identification of Ebola virus in Africa and sequencing in space by the American aerospace agency.

However, the current biological nanopore is embedded in a lipid bilayer membrane, is sensitive to the environment (pH, temperature, salt concentration and the like), has poor stability and durability and has limited service life; in addition, biological nanopores generally only adopt a detection mechanism of ion blocking current, and a specially-made low-noise current amplification circuit is needed to achieve sufficient sensitivity, so that large-scale matrixing integration of a sequencing unit has great challenge.

In order to overcome the disadvantages of biological nanopores, solid-state nanopores which have good stability and durability, are suitable for large-scale mass production and are easy to integrate with photoelectric detection are widely researched. Current solid state nanopore technology also primarily achieves sequencing by measuring ion blocking current, but it faces a number of challenges: firstly, the transport behavior of a DNA chain in a nanopore is not easy to control, the orientation of a base is not controlled, the randomness is high, and the DNA moving speed is too fast (0.1-1 mu s/bp); the DNA is non-specifically combined with the surface of the nanopore to form a secondary or tertiary structure, so that the nanopore is blocked, and the normal transport behavior of a DNA chain is limited; in conventional biological and solid-state nanopore detection technologies, ion blocking current is generally used to distinguish different base sequences, however, current detection technology has an essential limitation: the electric field around the nanopore can extend to both sides, resulting in an extension of the effective length of the nanopore, limiting the detection resolution. These problems have all severely limited the successful implementation of solid-state nanopore sequencing technologies.

In order to solve the above problems, the present invention provides the following technical solutions to achieve convenient, fast and accurate nanopore sequencing: a novel flexible substrate solid-state nanopore preparation technology; a modular nanopore device real-time rapid assembly mode; the motor protein is used for effectively controlling the transport speed of DNA or RNA in the solid-state nanopore; the spectral measurement technique is integrated into the solid state nanopore measurement, improving the accuracy of the sequence measurement.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a motor protein-based DNA transport rate control device for solving several problems of solid-state nanopores.

In order to achieve the above objects and other related objects, the present invention provides a device for controlling gene transfer rate based on a motor protein, comprising a motor protein, a housing, a fluid chamber sealing layer, a flexible substrate nanopore structure, a fluid chamber base, a power supply, a first electrode and a second electrode,

the first electrode and the second electrode are respectively connected with two poles of the power supply;

the flexible substrate nanopore structure comprises a flexible substrate, a pore substrate and a nanopore structure, wherein the nanopore structure comprises a nanopore cavity and a nanopore; the nano-hole cavity penetrates through the hole substrate, the nano-hole is an opening at one end of the nano-hole cavity, and the nano-hole cavity is a nonlinear reduction cavity or a linear reduction cavity and extends towards the nano-hole;

the nanopore structure further comprises:

a grating surrounding the nanopore cavity, the grating formed on a surface of the pore substrate away from the nanopore, the grating comprising a plurality of groups of grating grooves,

a hole protection film covering the grating and the nano-pore cavity,

a metal layer covering the hole protection film to reduce the size of the nano hole to 1-100 nm;

a fluid chamber formed by the flexible substrate not covering the grating and the nanopore cavity, surrounding the grating and over the nanopore cavity,

the flexible substrate nanopore structure comprises a first liquid flow channel, and the first liquid flow channel is connected with the outside and the fluid cavity so as to input or discharge a solution to the fluid cavity;

the fluid cavity base comprises a lower fluid cavity and a second fluid flow channel, and the second fluid flow channel is connected with the outside and the lower fluid cavity so as to input or discharge solution in the lower fluid cavity;

the fluid cavity sealing layer seals the fluid cavity;

the housing includes flow passage openings corresponding to the first liquid flow passage and the second liquid flow passage, respectively;

the motor protein and the metal layer form a metal-sulfur bond, so that the transport speed of the gene chain to be detected through the nanopore is at least slower than 0.1ms per base pair.

Preferably, the motor protein comprises a DNA polymerase, a DNA helicase or an RNA polymerase.

Preferably, the DNA polymerase is one of DNA polymerase I-V, DNA polymerase α, β, γ, δ, ε, ζ; the DNA helicase is one of superfamily I-III, class DnaB family and class rho family.

Preferably, the metal layer is one of copper, silver, gold, zinc, mercury, cadmium, cobalt, nickel or aluminum.

Preferably, the fluid chamber base is one of polydimethylsiloxane, polymethyl methacrylate, polyisoprene, acrylate, methyl methacrylate, o-azidonaphthoquinone, novolac resin, silicone adhesive, glass cement material, or SU-8 high polymer material.

Preferably, the fluid chamber base is a silicon or silicon dioxide material.

Preferably, the fluid chamber sealing layer is a quartz, glass or transparent material sealing sheet.

Preferably, the first electrode and the second electrode are made of one of platinum, gold, silver, titanium nitride and their derived counter electrode materials, the surfaces of the first electrode and the second electrode are chemically modified to improve the electrochemical stability of the electrode interface, and the chemical modification is to form a ferrocene molecular layer on the surfaces of the first electrode and the second electrode.

Preferably, the power source is located inside the housing, the first electrode is located above the flexible substrate, and the second electrode is located at the bottom of the fluid lower chamber.

Preferably, the first electrode is damascene integrated over the flexible substrate.

Also provided is a method for manufacturing a motor protein-based gene transfer rate control device, comprising the steps of:

i) Forming a motor protein with a polyanionic tail;

ii) providing a nanopore device comprising a housing, a fluid chamber sealing layer, a flexible substrate nanopore structure, a fluid chamber base, a power supply, a first electrode and a second electrode, wherein the first electrode and the second electrode are respectively connected with two poles of the power supply;

the nanopore structure further comprises: the grating surrounds the nano-hole cavity, is formed on the surface of the hole substrate far away from the nano-hole and consists of a plurality of groups of grating grooves; a hole protection film covering the grating and the nano-hole cavity; a metal layer covering the hole protection film to reduce the size of the nano hole to 1-100 nm; a fluid chamber formed by the flexible substrate not covering the grating and the nano-cavity, surrounding the grating and over the nano-cavity;

the fluid cavity base comprises a fluid lower cavity and a second fluid flow channel, and the second fluid flow channel is connected with the outside and the fluid lower cavity so as to input or discharge solution in the fluid lower cavity;

the fluid cavity sealing layer seals the fluid cavity;

the shell comprises a flow passage opening which respectively corresponds to the first liquid flow passage and the second liquid flow passage;

driving the polyanionic tail using an electrophoretic method, the polyanionic tail drawing the motor protein, stabilizing the motor protein in the nanopore;

cleaving the polyanionic tail to expose a thiol group of the cysteine; the sulfhydryl and the surface of the metal layer form a metal-sulfur bond, and the movement protein is anchored in the nanopore through the metal-sulfur bond, so that the transport speed of the gene chain to be detected through the nanopore is at least slower than 0.1ms per base pair.

Preferably, the DNA polymerase is one of DNA polymerase I-V, DNA polymerase α, β, γ, δ, ε, ζ.

Preferably, the DNA helicase is one of superfamily I-III, dnaB-like family, ρ -like family.

Preferably, the DNA polymerase is phi29DNA polymerase.

Preferably, the step i) comprises site-directed mutagenesis of the amino acid residues on the surface of the motor protein by using a genetic engineering method, replacing the amino acid residues with a plurality of cysteines, and connecting a sulfhydryl modified single-stranded oligonucleotide (with the length of 5-100 nt) to part of the cysteines; providing a section of double-stranded DNA (with the length of 50-5000 bp) containing a cohesive end to perform base complementary pairing with the single-stranded oligomer on the single-stranded oligonucleotide so as to form the motor protein with the polyanionic tail.

The gene transport speed control device based on the motor protein is characterized in that a solution containing a gene chain to be detected and having a temperature and a pH value is provided, loaded into the fluid cavity through the first fluid flow channel, driven into the nanopore cavity by spontaneous diffusion or electrophoresis, and after being captured by the motor protein, the transport speed of the gene chain to be detected in the nanopore 51 is reduced to 0.1-999 ms per base pair.

Preferably, the gene chain is a DNA chain, an RNA chain, a polypeptide chain, and the temperature is 25 ℃ to-200 ℃.

Preferably, the gene strand is a DNA strand, an RNA strand, a polypeptide chain, and the pH is 1-13.

The gene transfer speed control device based on the motor protein and the manufacturing method and the using method thereof provided by the invention anchor the motor protein in the nanopore by using a click chemistry method, so that the transfer speed of a gene chain to be detected passing through the nanopore 51 is at least slower than 0.1 ms/base pair, the temperature and the pH are adjusted to effectively control the transfer speed of the gene chain, the transfer speed of the gene chain to be detected passing through the nanopore 51 can be further reduced to tens of even hundreds of milliseconds, and the sensitivity of spectral detection is improved; the local plasmon enhanced electric field on the surface of the metal layer is exponentially attenuated with the distance, and the sub-nanometer spatial resolution can be provided, so that the detection of the sub-nanometer spatial resolution can be realized only by using the plasmon enhanced electric field on the surface regardless of the size of the nanopore.

Drawings

Fig. 1a is a top view of an array of well substrates with pre-formed nanopore structures on a wafer.

Fig. 1b is a perspective view of an array of well substrates with pre-formed nanopore structures on a wafer.

Fig. 2 a-2 f are cross-sectional views of a sub-step of a process for forming a nanopore in a silicon nitride pore substrate.

Fig. 3 a-3 f are cross-sectional views of a process of forming a nanopore in a silicon pore substrate in steps.

FIGS. 4 a-4 i are top views of combinations of 3 grating structures and 3 nanopore structures.

Fig. 5 a-5 f are top views of combinations of 2 grating structures and 3 nanopore structures.

Fig. 6a to 6c are cross-sectional views of 3 kinds of grating grooves.

Fig. 7 a-7 c are cross-sectional views of process steps for forming a flexible substrate on an aperture substrate.

Fig. 8a to 8b are plan views of fig. 7 a.

FIG. 8c is a schematic top view of a blind via formed in a via substrate. .

Fig. 9 a-9 c are cross-sectional views of process steps for forming a flexible substrate nanopore.

FIGS. 10 a-10 c are top views of process steps for forming a flexible substrate nanopore.

Fig. 11 is a perspective view of a nanopore array of a flexible substrate.

Fig. 12 is a flow chart of a method of fabricating a nanopore of a flexible substrate.

Fig. 13 is a schematic cross-sectional view of the assembly of a modular assembly nanopore device.

FIGS. 14 a-b are schematic views showing the assembly of a sealing member for a flow path opening and sectional structures thereof

FIGS. 15 a-b are schematic cross-sectional views and top views of a modularly assembled nanopore device.

FIGS. 16 a-b are schematic cross-sectional views of an assembled structure.

FIGS. 17 a-c are schematic diagrams of the procedure for modifying the long-stranded tail of DNA on a motor protein.

FIG. 18 is a schematic of the procedure for anchoring a motor protein to a metal layer.

FIG. 19 is a Raman spectroscopy biomolecule sequencing system.

FIG. 20a is a schematic representation of surface enhanced Raman spectroscopy DNA sequencing.

FIG. 20b is a schematic diagram of ultra-fast coherent Raman spectroscopy DNA sequencing.

FIG. 21 shows the sequence information of DNA directly read by Raman spectroscopy.

Description of the element reference numerals

1. Substrate 74 fluid chamber base

2. Sacrificial layer 75 power supply

3. Bore base 76 flow passage system

4. Flexible substrate 91 laser Raman microscope

5. Nano hole cavity 92 spectrum measuring device

6. Blind hole 93 data acquisition and analysis device

7. Nanopore device 711 assembly structure

8. Kinetin 721 fluid lumen

21. Downhole microcavity 741 fluid lower chamber

30. Hole-protecting sacrificial layer 751 first electrode

31. Second electrode of grating groove 752

32. Hole protection film 761 first flow channel opening seal

33. Metal layer 762 second flow channel open seal

40. Through hole 7110 assembling pivot

51. Nanopore 7111 first assembly

71. Shell 7112 second assembly

72. Flexible substrate nanopore structure 7610 first liquid flow channel

73. Fluid chamber sealing layer 7620 second fluid flow channel

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Please refer to fig. 1 to 21. It should be noted that the drawings provided in the present embodiment are only for illustrating the basic idea of the present invention in a schematic manner, and the drawings only show the components related to the present invention rather than the number, shape and size of the components in actual implementation, and the type, quantity and proportion of each component in actual implementation may be changed arbitrarily, and the layout of the components may be more complicated.

The present invention provides a flexible substrate nanopore structure, as shown in fig. 9c, the flexible substrate nanopore structure 72 comprises a flexible substrate 4 and a nanopore structure (not labeled), the nanopore structure being formed on a pore substrate 3, the nanopore structure comprising a nanopore cavity 5, a nanopore 51; the nanopore structure prepared on the flexible substrate 4 can reduce the impact of the external environment on the nanopore structure, is not easy to be broken by external force, and can meet the sequencing work in severe environment.

In the present embodiment, the flexible substrate 4 is a high molecular polymer material, including at least one of polydimethylsiloxane, polymethyl methacrylate, polyisoprene, acrylate, methyl methacrylate, o-azidonaphthoquinone, novolac resin, silicone adhesive, glass cement material, and SU-8 high molecular polymer; in this embodiment, the thickness of the flexible substrate 4 is 0.1-1000 μm, so that the impact of the external environment on the nanopore structure can be reduced, the nanopore structure is not easily broken by an external force, and the sequencing operation under a bad environment can be satisfied.

Preferably, the thickness of the flexible substrate 4 is 1 to 10 μm.

In the present embodiment, the hole substrate 3 is a silicon nitride or silicon material, but the invention is not limited thereto, and in other embodiments, the hole substrate may be other silicon-containing materials, or may be a semiconductor material without silicon, such as compound semiconductor, e.g. gallium nitride, gallium arsenide, etc.

As shown in fig. 9c, the nano-pore cavity 5 penetrates through the pore substrate 3, the nano-pore 51 is an opening at one end of the nano-pore cavity 5, and the nano-pore cavity 5 is a non-linear reduced cavity or a linear reduced cavity extending towards the nano-pore 51.

The nanopore structure further comprises a grating (not labeled) surrounding the nanopore cavity 5, the grating being formed on the upper surface of the pore substrate 3 and remote from the nanopore 51, the grating being formed by grating grooves 31, i.e. the grating grooves 31 define a grating.

The method is mainly used in the field of gene sequencing, particularly in the field of fourth-generation solid nanopore sequencing, and the main functional units are the grating and the nanopore 51, wherein when the solid nanopore 51 is in a working state by the spectroscopy, long-chain biomolecules containing sequence information such as DNA (deoxyribonucleic acid) pass through the nanopore 51, the nanopore 51 is used as antenna optics, a highly local plasmon electric field is generated on the lower surface under laser irradiation, the specific vibration/rotation/absorption/reflection spectrum of base molecules is excited, and the grating reflects back a spectral signal generated by the nanopore 51, so that the propagation loss is reduced, and the technical effect of enhancing the spectral signal intensity is achieved.

As shown in fig. 4a to 5f, the present invention provides various combinations of gratings and nanopores 51: the size of the nano-pores 51 is 1-100nm, the size of the pore substrate is 1-1000 μm, the size is smaller, and the technical effect that mechanical fracture is not easy to occur is achieved. As shown in fig. 4a to 5f, the present invention provides different structures of the nanopore cavity 5 and the nanopore 51, in this embodiment, the nanopore cavity 5 is a cylindrical shape with a wide top and a narrow bottom, i.e. the cross-sectional shape of the nanopore 51 is circular; in other embodiments, the nanopore cavity 5 has an inverted pyramid shape with a wide top and a narrow bottom, i.e., the cross-sectional shape of the nanopore 51 may be square or rectangular.

As shown in fig. 4a to 5f, the grating (not labeled) for cooperating with the nanopore 51 is defined by a plurality of grating grooves 31, the width of the upper opening of the grating grooves 31 is 10-500nm, the distance between the grating grooves 31 is 10-5000nm, and preferably, the distance between the grating grooves 31 is 100-1000nm.

As shown in fig. 4a to 5f, the present invention provides a grating structure defined by different grating grooves 31. In this embodiment, the cross section of the grating groove 31 has a plurality of concentric ring groove groups, and in other embodiments, the cross section of the grating groove 31 has a symmetrical parallel straight line groove group or a symmetrical arc line groove group;

as shown in fig. 6a to 6c, the present invention provides a cross-sectional groove structure of the grating groove 31, in this embodiment, the cross-section of the grating groove 31 is an inverted triangle, and in other embodiments, the cross-section of the grating groove 31 may be a square or a trapezoid.

As shown in fig. 4a to 4i, the present invention provides a combination in which the grating defined by the grating grooves 31 is disposed around the nanopore 51, in this embodiment, the grating defined by the grating grooves 31 is disposed in a manner of surrounding the nanopore 51, and in other embodiments, as shown in fig. 5a to 5f, the grating defined by the grating grooves 31 is disposed in two sets of symmetric patterns around the nanopore 51. In the way of combining the grating defined by the grating groove 31 and the nanopore 51, when long-chain biomolecules such as DNA containing sequence information pass through the nanopore 51, the nanopore 51 is used as antenna optics, a highly localized plasmon electric field is generated on the lower surface under laser irradiation, a specific vibration/rotation/absorption/reflection spectrum of a base molecule is excited, and a spectral signal generated by the nanopore 51 is reflected back by the grating, so that the technical effects of reducing light propagation loss and enhancing spectral signal intensity are achieved.

The nanopore structure further comprises a pore protection film 32 covering the grating and the nanopore cavity 5 for protecting the nanopore cavity 5 from defects in the manufacturing process.

In this embodiment, the hole protection film 32 comprises a silicon dioxide layer, preferably at least two silicon dioxide layers, to form a step protection structure to protect the nanopore cavity 5 from defects in the manufacturing process. In other embodiments, the hole protection film 32 may further include at least two other oxide layers, such as a metal oxide layer, to form a step protection structure, and the nano-hole cavity 5 is protected from defects generated in the manufacturing process.

Preferably, the hole protection film 32 includes at least two atomic layers of silicon dioxide or a silicon dioxide film.

The nanopore structure further comprises a metal layer 33, the metal layer 33 covers the pore protection film 32, the thickness of the metal layer 33 is 10-500nm, the size of the nanopore 51 is reduced to 1-100nm, the spectrum nanopore 51 with a plasmon enhancement effect is formed, the local plasmon enhancement electric field and the distance on the surface of the metal layer 33 are exponentially attenuated, sub-nanometer spatial resolution can be provided, when long-chain biomolecules such as DNA (deoxyribonucleic acid) containing sequence information pass through the nanopore 51, the nanopore 51 serves as antenna optics, a highly local plasmon electric field is generated on the surface under laser irradiation, a specific vibration/rotation/absorption/reflection spectrum of basic group molecules is excited, and a grating reflects spectral signals generated by the nanopore 51, so that the technical effects of reducing light propagation loss and enhancing spectral signal intensity are achieved.

In this embodiment, the metal layer 33 is a gold material, and in other embodiments, the metal layer 33 may be an aluminum, lead, silver, copper, platinum, or nickel material.

The nanopore structure further comprises a fluid chamber 721, as shown in fig. 9c, formed by the flexible substrate 4 not covering the grating and the nanopore cavity 5, surrounding the grating and the nanopore cavity 5, for loading the detected molecule solution and performing nanopore detection.

In this embodiment, the hole substrate 3 further includes a blind hole 6 array, and the flexible substrate 4 fills the blind hole 6 array, so that the contact area between the flexible substrate 4 and the nanopore structure is increased, and good sealing performance is ensured.

Preferably, as shown in fig. 8a to 8c, the cross-sectional shape of the blind hole 6 may be square, rectangular, circular, cross-shaped, pentagonal star-shaped or polygonal, so as to increase the contact area between the flexible substrate 4 and the nanopore structure and ensure good sealing performance.

As shown in fig. 11, the present invention further provides a nanopore array of a flexible substrate, wherein a plurality of nanopore structures as described above are disposed in the flexible substrate 4 in an array form, and the nanopore structures in the array are the same as those described above, which are not repeated herein. Wherein the spacing between the arrays is 0.01-10mm. It should be noted that the nanopore structure can be used for the characteristic spectrum detection and sequencing work of DNA molecules, RNA molecules and protein molecules. The nanopore arrays of the flexible substrate can be cut according to requirements, so that nanopore arrays with different shapes and numbers can be obtained, and the method is used for sequencing or detection application of different scenes.

The nanopore 51, the flexible substrate nanopore structure 72 and the flexible substrate nanopore array need to be completely wetted before use, otherwise bubbles are easily generated at the nanopore 51, and long-chain biomolecules such as DNA or detected molecules cannot enter the nanopore 51.

The invention also provides a method for keeping the nanopore 51, the flexible substrate nanopore structure 72 or the flexible substrate nanopore array, which comprises the following steps: the nano-pores 51 are slowly immersed in the mixed liquid of the alcohol substances and the water to completely wet the nano-pores 51, the water flows from the low surface tension to the high tension by utilizing the surface tension gradient generated by the evaporation of the alcohol substances, the nano-pores 51 are completely wetted, and bubbles are prevented from being generated at the nano-pores 51. The wetted nanopore 51, the flexible substrate nanopore structure 72 or the flexible substrate nanopore array is placed in the mixed solution of the alcohol substance and water for waiting use, so that bubbles are prevented from being generated at the nanopore 51.

In this example, the volume ratio 1 of the alcohol substance to water in the mixed solution of the alcohol substance and water was such that water flowed from a low surface tension to a high tension by the surface tension gradient generated by evaporation of the alcohol substance, thereby completely wetting the nanopore 51 and avoiding the formation of air bubbles in the nanopore 51. After being wetted, the nanopore 51, the flexible substrate nanopore structure 72 or the flexible substrate nanopore array are placed in a mixed solution of alcohol substances and water for waiting use, so that bubbles are prevented from being generated at the nanopore 51.

Preferably, the alcohol is isopropanol.

The present invention also provides a method for manufacturing a flexible substrate nano-pore structure, which is not easy to be broken mechanically considering that the size of a single nano-pore is relatively small (usually below ten microns). If a single or a little brittle solid-state nanopore is inlaid and sealed in a flexible high polymer material, the preparation of the flexible substrate solid-state nanopore can be realized, and the impact of the external environment on a brittle nanopore device is reduced. Therefore, as shown in fig. 12 and fig. 1 to 10c, a manufacturing method is proposed in which a solid nanopore structure is first prepared, and then a flexible substrate is damascene-bonded to the nanopore structure.

The manufacturing method of the flexible substrate nano-pore structure comprises the following steps:

step 100, forming a nanopore structure;

step 200, forming a nano-pore structure of the flexible substrate 4;

wherein step 100 comprises:

1001. providing a substrate 1, wherein the substrate 1 is a silicon wafer;

1002. forming a sacrificial layer 2 on the substrate 1, the sacrificial layer 2 being a silicon-containing compound;

1003. forming a hole base layer (not shown) on the sacrificial layer 2;

1004. the hole base layer forms a hole base 3;

1005. forming a nanopore structure (not labeled) on the pore substrate 3, wherein the nanopore structure comprises a nanopore cavity 5, a nanopore 51 and a grating (not labeled), the grating (not shown) is composed of a grating groove 31, the nanopore cavity 5 penetrates through the pore substrate 3, the nanopore 51 is an opening at one end of the nanopore cavity 5, the nanopore cavity 5 extends towards the nanopore 51 in a nonlinear narrowing cavity or a linear narrowing cavity, the grating surrounds the nanopore cavity 5, and the grating is formed on the upper surface of the pore substrate 3 and is far away from the nanopore 51;

1006. forming a hole protective film 32 covering the grating and the nano-hole cavity 5, and forming a metal layer 33 covering the grating and the nano-hole cavity 5 to reduce the size of the nano-hole 51 to 1-100 nm;

as shown in fig. 7a to 7c and fig. 9a to 9c (fig. 10a to 10c are plan views corresponding to fig. 9a to 9 c), the step 200 includes:

2001. forming a hole protection sacrificial layer 30 covering the grating and the nano-cavity 5 to protect the grating and the nano-cavity 5 from contamination by the flexible substrate 4 and to protect the plasmonic properties of the nano-hole structure, as shown in fig. 7 b; in the present embodiment, the hole-protecting sacrificial layer 30 is an Al, cu or Ti material with a thickness of 1-500nm to protect the plasmon properties of the nanopore structure.

Preferably, the method further comprises forming a blind hole 6 on the hole substrate 3 to increase the contact area between the flexible substrate 4 and the hole substrate 3.

2002. As shown in fig. 7c, a flexible substrate 4 is formed covering the sacrificial layer 2 and the hole substrate 3, that is, a flexible substrate 4 is formed covering the sacrificial layer 2, the hole substrate 3, and the hole-protecting sacrificial layer 30;

it should be noted that, in order to ensure that the flexible substrate 4 and the hole substrate 3 are tightly attached, enough contact regions are reserved on the periphery of the grating groove 31 (grating) on the hole substrate 3 to attach to the flexible substrate 4, and the area of the contact regions needs to be larger than the area of the nanopore structure (i.e., the grating groove 31 and the nanopore cavity 5) to ensure tight attachment.

The flexible substrate 4 is a flexible high polymer material, and includes at least one of polydimethylsiloxane, polymethyl methacrylate, polyisoprene, acrylate, methyl methacrylate, o-azidonaphthoquinone, novolac resin, silicone adhesive, glass cement material, and SU-8 high polymer; in this embodiment, the process for preparing the flexible substrate 4 is described by taking polydimethylsiloxane PDMS as an example: uniformly mixing polydimethylsiloxane PDMS solution and a curing agent according to a certain proportion, preferably 6; after the hole protection sacrificial layer 30 is formed, uniformly coating a flexible substrate solution on the surfaces of the hole protection sacrificial layer 30, the blind hole 6 and the hole substrate 3, heating the flexible substrate solution at the temperature of 40-180 ℃ for 10-180min to ensure that molecules in the flexible substrate solution are subjected to cross-linking polymerization, and forming a flexible substrate 4 with uniform thickness on the surfaces of the hole protection sacrificial layer 30, the blind hole 6 and the hole substrate 3, wherein the thickness of the flexible substrate 4 is 0.1-1000 mu m, so that the impact of an external environment on a nano-hole structure can be reduced, the nano-hole structure is not easily broken by an external force, and the sequencing work under a severe environment can be met; preferably, the thickness of the flexible substrate 4 is 1-10 μm.

2003. As shown in fig. 9a and 10a, forming a plurality of through holes 40 on the flexible substrate 4 to expose the sacrificial layer 2, in this embodiment, photoetching the flexible substrate 4 to form an array of a plurality of through holes 40, where the cross section of each through hole 40 is a square with a side length of 0.01-100 μm, and the bottom end of each through hole 40 directly exposes the surface of the sacrificial layer 2;

2004. as shown in fig. 9b and 10b, BHF is added through the through hole 40 to completely etch the sacrificial layer 2, a flexible base nanopore structure 72 is formed at the upper part of the sacrificial layer 2, and the lower part of the sacrificial layer 2, i.e. the substrate 1, falls off and can be recycled;

prior to step 2005 may also include: a layer of flexible polymer material is coated on the surface of the flexible substrate 4 (containing the through hole 40) again, and the through hole 40 is blocked by heating and polymerizing.

2005. As shown in fig. 9c and 10c, the flexible substrate 4 covering the grating and the nanopore cavity 5 and the hole protection sacrificial layer 30 are removed, and a fluid cavity 721 is formed, i.e. the fluid cavity 721 is formed by the flexible substrate 4 not covering the grating and the nanopore cavity 5 and surrounding the grating and the nanopore cavity 5, so as to load the detected molecule solution and perform nanopore sequencing work.

The basis on which the nanopore structure is formed is the pore base 3, and pore bases 3 of different materials are important contributing factors to the differences in the process of step 100. In the present invention, the inventors will describe the step 100 of forming the nanopore structure with the silicon nitride substrate 3 and the silicon pore substrate 3 separately, regarding that the pore substrate 3 is made of silicon nitride or silicon material to form the nanopore structure, and in other embodiments may be made of other silicon-containing materials, or may be made of non-silicon-containing semiconductor materials, such as compound semiconductors like gallium nitride, gallium arsenide, etc.

(I) silicon nitride nano-pore structure process

1001. Providing a substrate 1, wherein the substrate 1 is a silicon wafer, the invention adopts a silicon wafer with a crystal orientation 110, but is not limited to a silicon wafer with a (100) crystal plane, and the size can be 4, 6, 8 or 12 inches, but is not limited to 4 sizes;

1002. forming a sacrificial layer 2 on the substrate 1, the sacrificial layer 2 being a silicon-containing compound; the sacrificial layer 2 is a silicon dioxide layer, the step uses oxidation or chemical vapor deposition to form the sacrificial layer 2, the method for forming the sacrificial layer 2 is not limited to the above method, the thickness of the sacrificial layer 2 is 100-2000nm, preferably, the thickness of the sacrificial layer 2 is 500nm, but not limited to this range;

1003. forming a hole base layer (not shown), which is a silicon nitride layer, on the sacrificial layer 2 using a Low Pressure Chemical Vapor Deposition (LPCVD), a Plasma Enhanced Chemical Vapor Deposition (PECVD) deposition or an inductively coupled plasma enhanced vapor deposition (ICPCVD) method, the hole base layer forming method being not limited to the above-described method, the hole base layer having a thickness of 100 to 2000nm, but not limited thereto, and preferably, 700nm;

1004. the hole substrate layer is formed with a hole substrate 3, specifically, a photoresist is coated on the surface of a silicon nitride layer in a spinning way, a mask is manufactured by electron beam exposure, a silicon nitride hole substrate 3 is formed by using Reactive Ion Etching (RIE), and a square silicon nitride hole substrate 3 with the side width of 1-1000 μm is formed, and preferably, the silicon nitride hole substrate 3 has the side width of 1 μm, 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm or 1000 μm according to different actual detection fields;

1005. forming a nanopore structure (not labeled) on the pore substrate 3 by using reactive ion etching, wherein the nanopore structure comprises a nanopore cavity 5, a nanopore 51 and a grating (not labeled), the grating (not shown) is composed of grating grooves 31, the nanopore cavity 5 penetrates through the pore substrate 3, the nanopore 51 is an opening at one end of the nanopore cavity 5, the nanopore cavity 5 extends towards the nanopore 51 in a nonlinear narrowing cavity or a linear narrowing cavity, the grating surrounds the nanopore cavity 5, and the grating is formed on the upper surface of the pore substrate 3 and is far away from the nanopore 51.

As shown in fig. 2 a-2 f, the aperture substrate layer is etched using reactive ions to form the grating, the nano-cavities 5 in turn.

First, a grating is fabricated on a silicon nitride porous substrate 3, as shown in fig. 2a to 2 b: the silicon nitride porous substrate 3 is spin-coated with photoresist, a mask is prepared by photolithography, and a grating defined by grating grooves 31, which is a bragg grating, is prepared by Reactive Ion Etching (RIE), the groove surfaces of the grating grooves 31 define bragg reflectors, the width of the opening on the grating grooves 31 is 10-500nm, the distance between the grating grooves 31 is 10-5000nm, and preferably, the distance between the grating grooves 31 is 100-1000nm.

Second, a nano-pore 5 is fabricated in the center of the grating, as shown in fig. 2 c: spin-coating photoresist, preparing a nano-pore cavity 5 at the middle position of the Bragg reflector by using Reactive Ion Etching (RIE), wherein the width of an upper opening of the nano-pore cavity 5 is 0.1-5 mu m, a nano-pore 51 is formed at a lower opening of the nano-pore cavity 5, the size of the nano-pore 51 is controlled to be 10-5000nm, and preferably, the size of the nano-pore 51 is controlled to be 50-500nm.

1006. Forming a hole protection film 32 covering the grating and the nano-hole cavity 5, specifically as shown in fig. 2 d-e, adding BHF through the nano-hole cavity 5 to partially etch the sacrificial layer 2 to form a hole lower micro-cavity 21, wherein the hole lower micro-cavity 21 is an elliptical hole, and the hole protection film 32 is formed by using an atomic deposition method, and the hole protection film 32 comprises at least one silicon dioxide layer; forming a metal layer 33 covering the grating and the nano-cavity 5, specifically as shown in 2f, forming the metal layer 33 by using a sputtering or evaporation process to reduce the size of the nano-hole to 1-100nm, forming a spectrum nano-hole 51 with a plasmon enhancement effect, wherein the local plasmon enhancement electric field and the distance on the surface of the metal layer 33 are exponentially attenuated, and the sub-nanometer spatial resolution can be provided.

The silicon nitride nanopore is combined with the grating to prepare an integrated optical chip which is used for a high-flux on-chip optical detection mode.

(II) silicon nano-pore structure process

1002. forming a sacrificial layer 2 on the substrate 1, the sacrificial layer 2 being a silicon-containing compound; the sacrificial layer 2 is a silicon dioxide layer, the sacrificial layer 2 is formed by evaporation through oxidation or chemical vapor deposition, the method for forming the sacrificial layer 2 is not limited to the above method, the thickness is 100-2000nm, but not limited to this range, and preferably, the thickness of the sacrificial layer 2 is 500nm;

1003. forming a hole base layer (not shown) on the sacrificial layer 2, wherein the hole base layer is a silicon hole base layer, and the silicon hole base layer is formed by using a chemical vapor deposition method and has a thickness of 100-2000nm, but is not limited to this range, and preferably, the silicon hole base layer has a thickness of 700nm;

1004. the hole substrate layer is used for forming a hole substrate 3, specifically, photoresist is coated on the surface of a silicon layer in a spinning mode, a mask is manufactured through electron beam exposure, a silicon hole substrate 3 is formed through Reactive Ion Etching (RIE), and the silicon hole substrate 3 with the square edge width of 1-1000 mu m is formed;

1005. forming a nanopore structure (not labeled) on the pore substrate 3 by using potassium hydroxide or tetramethylammonium hydroxide wet etching, wherein the nanopore structure comprises a nanopore cavity 5, a nanopore 51 and a grating (not labeled), the grating (not shown) is composed of grating grooves 31, the nanopore cavity 5 penetrates through the pore substrate 3, the nanopore 51 is an opening at one end of the nanopore cavity 5, the nanopore cavity 5 extends towards the nanopore 51 in a nonlinear or linear shrinking cavity, the grating surrounds the nanopore cavity 5, and the grating is formed on the upper surface of the pore substrate 3 and is far away from the nanopore 51.

As shown in fig. 3a to 3f, the aperture base layer is wet etched using potassium hydroxide or tetramethylammonium hydroxide to simultaneously form the grating, the nano-cavities 5.

As shown in fig. 3a to 3 c: spin-coating photoresist on the silicon hole substrate 3, preparing a mask by photoetching, and etching the silicon hole substrate 3 by using potassium hydroxide or tetramethylammonium hydroxide wet method to form grating grooves 31 and nano-hole cavities 5, wherein the grating grooves 31 define gratings which are Bragg gratings, the groove surfaces of the grating grooves 31 define Bragg reflectors, the width of the openings on the grating grooves 31 is 10-500nm, the distance between every two grating grooves 31 is 10-5000nm, and preferably, the distance between every two grating grooves 31 is 100-1000nm. The upper opening of the nano-pore cavity 5 has a width of 0.1-5 μm, and the lower opening of the nano-pore cavity 5 forms a nano-pore 51, wherein the size of the nano-pore is controlled to be 10-5000nm, preferably, the size of the nano-pore 51 is controlled to be 50-500nm.

1006. Forming a hole protection film 32 covering the grating and the nano-hole cavity 5, as shown in fig. 3 d-e, adding BHF through the nano-hole cavity 5 to partially etch the sacrificial layer 2 to form a hole lower micro-cavity 21, wherein the hole lower micro-cavity 21 is an elliptical hole, the hole protection film 32 is formed by using an atomic deposition method, and the hole protection film 32 comprises at least one silicon dioxide layer; forming a metal layer 33 covering the grating and the nano-cavity 5, specifically as shown in fig. 3f, forming the metal layer 33 using a sputtering or evaporation process to reduce the nano-hole size to 1-100nm, forming a spectral nano-hole 51 having a plasmon enhancement effect. The surface localized plasmon enhanced electric field of the metal layer 33 decays exponentially with distance, and sub-nanometer spatial resolution can be provided.

In other embodiments, step 100 or step 200 may further include making appropriate markings on each nanopore structure to facilitate searching, alignment, and the like.

The invention also provides a method for manufacturing a nanopore array of a flexible substrate, wherein in step 1004, an array consisting of a plurality of nanopore substrates 3 is formed on the nanopore substrate (not labeled). Specifically, as shown in fig. 1a to 1b, the hole substrate layer is etched into a plurality of square hole substrates 3 with the side widths of 1 to 1000 μm by using reactive ion etching, the square hole substrates 3 form an array, the array interval is 0.01 to 10mm, and the subsequent process of forming a nanopore structure on each hole substrate 3 in the array is shown in fig. 2a to 10c, and the specific implementation steps and technical details are the same as those of the flexible substrate nanopore structure 72, which is not described herein again.

In summary, each nanopore structure comprises a plasmon spectrum nanopore covered with a gold film, bragg grating reflectors are arranged around the nanopore, the preparation of the flexible substrate nanopore structure or the flexible substrate nanopore array is realized, the size of a single nanopore is small, mechanical fracture is not easy to occur, impact of an external environment on a nanopore device is reduced, the nanopore is not easy to be fractured by an external force, and the beneficial effect of sequencing work under a severe environment is met; the local plasmon enhanced electric field on the surface of the metal layer is exponentially attenuated with the distance, and the sub-nanometer spatial resolution can be provided, so that the detection of the sub-nanometer spatial resolution can be realized only by using the plasmon enhanced electric field on the surface regardless of the size of the nanopore.

The invention also provides a nanopore device 7 which is assembled in a modularization mode, so that maintenance and part replacement procedures of nanopore equipment are simplified, self-service maintenance and part replacement of a user are realized, the time cost of the user is saved, each module can be stored for standby application, rapid assembly and measurement can be realized when needed, and the flexibility and the portability of use are greatly improved. As shown in fig. 13 to 16b, the nanopore device 7 includes a housing 71, a fluid cavity sealing layer 73, a flexible substrate nanopore structure 72, a fluid cavity base 74, a power supply 75, a first electrode 751 and a second electrode 752, wherein the first electrode 751 is integrated on the flexible substrate 4 in an inlaid manner, the second electrode 752 is integrated on the bottom of the fluid lower cavity 741 in an inlaid manner, and the first electrode 751 and the second electrode 752 are respectively connected to two poles of the power supply 75 for driving a solution to be detected (such as a solution containing DNA fragments) to pass through a nanopore by using an electrophoresis technique; the housing 71 comprises a first housing (upper housing, not labeled) and a second housing (lower housing, not labeled) that are separate, each comprising an assembly structure 711 for assembly; the flexible substrate nanopore structure 72 comprises a flexible substrate 4, a pore substrate 3, and a nanopore structure (not labeled) comprising a nanopore cavity 5, a nanopore 51; the nano-hole cavity 5 penetrates through the hole substrate 3, the nano-hole 51 is an end opening of the nano-hole cavity 5, and the nano-hole cavity 5 is a nonlinear reduced cavity or a linear reduced cavity and extends towards the nano-hole 51; the nanopore structure further comprises: a grating (not shown) surrounding the nanopore cavity 5, the grating being formed on the upper surface of the pore substrate 3 and far away from the nanopore 51, the grating being composed of a grating groove 31, a pore protection film 32 covering the grating and the nanopore cavity 5, a metal layer 33 covering the pore protection film 32 to reduce the size of the nanopore 51 to 1-100nm to form a spectral nanopore 51 with plasmon enhancement effect, the surface local plasmon enhancement electric field of the metal layer 33 decaying exponentially with distance, and providing sub-nanometer spatial resolution; a fluid chamber 721 formed by the flexible substrate 4 not covering the grating and the nano-cavities 5, surrounding the grating and above the nano-cavities 5. The remaining technical details of the flexible substrate nanopore structure 72 are fully set forth above and will not be described further herein by the inventors.

The flexible substrate nanopore structure 72 includes a first liquid flow channel 7610 and a first flow channel alignment mark (not labeled) and a second module alignment mark (not labeled), the first liquid flow channel 7610 connecting the outside with the fluid chamber 721 to input or discharge a solution to or from the fluid chamber 721; the fluid chamber base 74 includes a lower fluid chamber 741, a second fluid flow path 7620, a second flow path alignment mark (not shown), and a second module alignment mark (not shown), wherein the fluid flow path 7620 connects the outside with the lower fluid chamber 741 to input or discharge a solution in the lower fluid chamber 741; the fluid chamber sealing layer 73 seals the fluid chamber 721 and includes a module alignment mark (not labeled) and a third module alignment mark (not labeled); the housing 71 includes flow passage openings (not labeled) corresponding to the first and second liquid flow passages 7610 and 7620, respectively; the first and second flow channel alignment marks are used for alignment assembly of the liquid flow channel and the flow channel opening sealing member so as to enable the liquid flow channel and the flow channel opening sealing member to achieve rapid alignment and sealing, and the first and second module alignment marks and the third module alignment mark are aligned up and down so as to achieve alignment assembly of the flexible substrate nanopore structure 72, the fluid chamber base 74 and the fluid chamber sealing layer 73 up and down. The first housing and the second housing both include a fourth module alignment mark (not labeled) to align up and down with the first module alignment mark, the second module alignment mark, and the third module alignment mark to achieve up-down alignment and assembly of the housing 71, the flexible substrate nanopore structure 72, the fluid chamber base 74, and the fluid chamber sealing layer 73, so that the housing 71, the fluid chamber sealing layer 73, the flexible substrate nanopore structure 72, and the fluid chamber base 74 are aligned sequentially from top to bottom to facilitate assembly, and at the same time, the first electrode 751, the fluid chamber 721, the nanopore 51, the fluid lower chamber 741, and the second electrode 752 are aligned sequentially from top to bottom, thereby simplifying maintenance and component replacement procedures of the nanopore device, achieving user self-service maintenance and component replacement, saving time cost of a user, enabling each module to be stored separately for use, enabling rapid assembly and measurement to be achieved when needed, and greatly improving flexibility and portability of use.

As shown in fig. 13, the module alignment marks are located on a module alignment line, and when the flexible substrate nanopore structure 72, the fluid chamber base 74, and the fluid chamber sealing layer 73 are standardized and have consistent cross-sectional areas, the first electrode 751, the fluid chamber 721, the nanopore 51, the fluid lower chamber 741, and the second electrode 752 can be precisely aligned from top to bottom in sequence.

In this embodiment, as shown in fig. 16 a-b, the assembly structure 711 is a multi-stage structure, wherein as shown in fig. 16a, the assembly structure 711 includes a second assembly component 7112 on the lower housing and a first assembly component 7111 on the upper housing, and the first assembly component 7111 is rotated by an assembly pivot 7110 to be engaged with the second assembly component 7112 under the condition that the upper and lower housings are pressed to make the internal modules tightly fit. As shown in fig. 16b, the assembly structure 711 includes a second assembly component 7112 on the lower housing and a first assembly component 7111 on the upper housing, and the upper and lower housings are pressed to engage the first assembly component 7111 with the second assembly component 7112 in a staggered manner until the internal modules are tightly attached, so that maintenance and component replacement procedures of the nanopore device are simplified, self-service maintenance and component replacement of a user are realized, time cost of the user is saved, rapid assembly and measurement can be realized when necessary, and flexibility and portability of use are greatly improved.

In this embodiment, as shown in fig. 14a, a first flow path opening seal 761 and a second flow path opening seal 762 are further included, and together with the flow path opening (not labeled), the first liquid flow path 7610 and the second liquid flow path 7620, a flow path system 76 is formed. The first and second flow channel opening seals 761 and 762 are respectively connected to the first and second

liquid flow channels

7610 and 7620 through the flow channel openings, the first and second flow channel opening seals 761 and the flow channel openings are coaxial with the flow channel alignment line of the first liquid flow channel 7610, the second flow channel opening seal 762 is coaxial with the flow channel alignment line of the second liquid flow channel 7620 to sealingly engage with the exterior and the first and second

liquid flow channels

7610 and 7620, so as to prevent liquid from leaking into the gaps between the housing 1 and the flexible substrate nanopore structure 72 and the fluid chamber base 74, causing contamination and detecting liquid loss.

To achieve accurate horizontal alignment, preferably, as shown in fig. 13, the housing 1 further includes a third housing (middle housing) on which the flow channel opening is located to align with the first liquid flow channel 7610 in the flexible substrate nanopore structure 72, and an assembly structure 711 assembled with the first housing (upper housing) and the second housing (lower housing) thereon, so as to avoid the problem that the two housing assembly flow channel openings and the first liquid flow channel 7610 cannot be aligned horizontally accurately.

Preferably, as shown in fig. 14b, the second flow passage opening seal 762 is a wedge-shaped pipe joint, a step-gradual pipe joint, or a step-pipe joint.

In this embodiment, as shown in fig. 13, for more precise alignment, the first housing (upper housing) and the second housing (lower housing) each include a module alignment mark (not shown) for up-down alignment assembly, so as to achieve precise alignment of the housing 71, the first electrode 751, the fluid chamber 721, the nanopore 51, the fluid lower chamber 741, and the second electrode 752 from top to bottom in sequence.

In this embodiment, the fluid chamber sealing layer 73 is a quartz sealing piece. In other embodiments, the fluid chamber sealing layer 73 may be a transparent sealing layer, such as glass, transparent material, etc., with light transmittance suitable for spectrum detection in molecular detection (e.g., long-chain biomolecule detection containing sequence information, such as DNA).

In this embodiment, the first electrode 751 and the second electrode 752 are silver or silver chloride electrodes. In other embodiments, the first electrode 751 and the second electrode 752 may also be other metal or conductive electrode, such as one of conductive materials of platinum, gold, silver, titanium nitride, and their derivative counter electrode materials, and the surfaces of the first electrode and the second electrode may be chemically modified to improve the electrochemical stability of the electrode interface, where the chemical modification is to form a ferrocene molecular layer on the surfaces of the first electrode and the second electrode.

In the present embodiment, as shown in fig. 13 and 15a, the power supply 75 is located inside the housing 71 and integrated inside the housing 71, and the first electrode 751 is located above the flexible substrate 4, and preferably, the first electrode 751 is embedded and integrated above the flexible substrate 4, that is, embedded near the upper surface of the flexible substrate 4; the second electrode 752 is located at the bottom of the lower fluid cavity 741, and preferably, the second electrode 752 is embedded and integrated at the bottom of the lower fluid cavity 741.

In practical use, the component that generally needs to be replaced frequently is the flexible substrate nanopore structure 72, and therefore, preferably, as shown in fig. 13, the fluid chamber base 74 and the power supply 75 are integrated in the second housing (lower housing) at the same time, the second flow channel alignment mark and the module alignment mark on the second housing (lower housing) can be aligned in advance, so as to reduce assembly errors, and the power supply 75 and the second electrode 752 are fixedly connected, i.e., connected by a metal wire under normal conditions, so as to avoid dynamic connection, so as to avoid the problems of poor contact and even failure in use due to abrasion of the second electrical connection structure (not labeled) between the second electrode 752 and the power supply 75 during assembly.

Preferably, the first electrode 751 and a first electrical connection structure (not labeled) for connecting the power supply 75 are partially adhered to the surface of the flexible substrate 4 or are embedded above the flexible substrate 4, and preferably, the first electrode 751 is embedded above the flexible substrate 4, that is, embedded near the upper surface of the flexible substrate 4. In the case where the power source 75 is integrated in the second housing (lower housing), in the actual process of assembling the modular-assembled nanopore device 7, the first electrical connection structure between the power source 75 and the first electrode 751 is dynamic connection, and there is an effective connection range, in order to avoid poor contact between the power source 75 and the first electrical connection structure, in this embodiment, a portion of the first electrical connection structure extending out of the flexible substrate 4 may be set to have a length long enough in the up-down direction, so as to ensure good contact with the power source 75.

In this embodiment, the fluid chamber base 74 is a flexible polymer material, and includes at least one of polydimethylsiloxane, polymethyl methacrylate, polyisoprene, acrylate, methyl methacrylate, o-azidonaphthoquinone, novolac resin, silicone adhesive, glass adhesive material, and SU-8 high molecular polymer. In other embodiments, the fluid chamber base 74 may be a silicon-based material, and preferably may be a silicon or silicon dioxide material.

In this embodiment, the first electrode 751 and the second electrode 752 are made of a conductive material such as platinum, gold, silver, titanium nitride, and a derivative counter electrode material thereof, and the surfaces of the first electrode and the second electrode may be chemically modified (such as a ferrocene molecular layer), so as to improve the electrochemical stability of the electrode interface.

The modular assembled nanopore device provided by the invention can be flexibly assembled, the maintenance and the part replacement of nanopore equipment are simplified, the self-service maintenance and the part replacement of a user are realized, the time cost of the user is saved, the sequencing and the detection application of multiple scenes can be realized, each module can be respectively stored for later use, the rapid assembly and the measurement can be realized when needed, and the flexibility and the portability of the use are greatly improved; the local plasmon enhanced electric field on the surface of the metal layer is exponentially attenuated with the distance, and the sub-nanometer spatial resolution can be provided, so that the detection of the sub-nanometer spatial resolution can be realized only by using the plasmon enhanced electric field on the surface no matter the size of the nanopore.

The transport speed of biological macromolecular chains such as DNA or RNA in the nanopore 51 is very high, the transport speed of gene chains in the nanopore 51 is generally 0.1-1 mus/bp (0.1-1 mus/base pair), and accurate spectroscopic sequencing is difficult to meet.

The invention also provides a gene transfer speed control device based on the movement protein, which comprises a nanopore device 7 and the movement protein 8, wherein the nanopore device 7 comprises a shell 71, a fluid cavity sealing layer 73, a flexible substrate nanopore structure 72, a fluid cavity base 74, a power supply 75, a first electrode 751 and a second electrode 752, and the first electrode 751 and the second electrode 752 are respectively connected with two poles of the power supply 75; the flexible substrate nanopore structure 72 comprises a flexible substrate 4, a pore substrate 3, and a nanopore structure (not labeled) comprising a nanopore cavity 5, a nanopore 51; the nano-hole cavity 5 penetrates through the hole substrate 3, the nano-hole 51 is an opening at one end of the nano-hole cavity 5, and the nano-hole cavity 5 is a nonlinear reduction cavity or a linear reduction cavity and extends towards the nano-hole 51; the nanopore structure further comprises: a grating (not shown) surrounding the nanopore cavity 5, the grating being formed on the upper surface of the pore substrate 3 and far away from the nanopore 51, the grating being composed of a grating groove 31, a pore protection film 32 covering the grating and the nanopore cavity 5, a metal layer 33 covering the pore protection film 32 to reduce the size of the nanopore to 1-100nm to form a spectral nanopore 51 with a plasmon enhancement effect, the local plasmon enhancement electric field on the surface of the metal layer 33 decays exponentially with distance, and sub-nanometer spatial resolution can be provided; a fluid chamber 721 formed by the flexible substrate 4 not covering the grating and the nanopore chamber 5, surrounding the grating and the nanopore chamber 5, the flexible substrate nanopore structure 72 including a first liquid flow channel 7610 connecting an outside with the fluid chamber 721 to input or discharge a solution to the fluid chamber 721; the fluid chamber base 74 comprises a lower fluid chamber 741 and a second fluid flow path 7620, wherein the second fluid flow path 7620 connects the outside with the lower fluid chamber 741 so as to input or discharge a solution in the lower fluid chamber 741; the fluid chamber sealing layer 73 seals the fluid chamber 721; the housing 71 includes flow passage openings (not labeled) corresponding to the first and second liquid flow passages 761 and 762, respectively; as shown in fig. 18c, the motor protein 8 forms a metal-sulfur bond with the metal layer 33, so that the transport speed of the gene strand to be detected (e.g. DNA strand) through the nanopore 51 is at least slower than 0.1ms per base pair. The technical features of the above-mentioned details have been set forth in the preceding description of the modularly assembled nanopore device 7, and the inventors are not repeated here.

In this embodiment, the motor protein 8 includes a DNA polymerase, a DNA helicase, or an RNA polymerase to control the speed of a DNA strand or an RNA strand through the nanopore 51.

Preferably, the DNA polymerase is one of DNA polymerase I-V, DNA polymerase alpha, beta, gamma, delta, epsilon, zeta; the DNA helicase is one of superfamily I-III, class DnaB family and class rho family. Preferably, the motor protein 8 is phi29DNA polymerase.

In the present embodiment, the metal layer 33 is one of copper, silver, gold, zinc, mercury, cadmium, cobalt, nickel or aluminum. Preferably, the metal layer 33 is a gold layer, and the gold layer forms a gold-sulfur bond with the kinetin 8.

In the present embodiment, the fluid chamber base 74 is a flexible polymer material, and preferably, the fluid chamber base 74 is one of polydimethylsiloxane, polymethyl methacrylate, polyisoprene, acrylate, methyl methacrylate, o-azidonaphthoquinone, novolac resin, silicone adhesive, glass adhesive material, or SU-8 polymer material. In other embodiments, the fluid chamber base 74 may be a silicon-based material, and preferably may be a silicon or silicon dioxide material.

In order to manufacture the gene chain transport speed control device based on the motor protein, the invention provides a click chemistry method for guiding and modifying the motor protein on the inner surface of the nanopore 51, and the speed of controlling the via hole of the gene chain is at least slower than 0.1 ms/base pair. The description is as follows:

as shown in fig. 17a to 18c, the method includes the steps of:

i) 17 a-17 c, a motor protein 8 with a polyanionic tail was formed. The sports egg 8 has been described in detail above, and the inventors will not repeat this description, in this embodiment, phi29DNA polymerase is taken as an example, and the metal layer 33 is taken as a gold layer.

As shown in FIG. 17a, the specific amino acid residues on the surface of phi29DNA polymerase protein are site-directed mutated to replace cysteine by genetic engineering methods, and preferably, the amino acid residues G410 and P562 on the surface of phi29DNA polymerase protein are selected to be site-directed mutated to replace cysteine.

As shown in fig. 17b, providing a thiol-modified single-stranded oligonucleotide, wherein the length of the single-stranded oligonucleotide is 5-100nt, and linking the thiol-modified single-stranded oligonucleotide with cysteine; preferably, the single-stranded oligonucleotide is 8-50nt in length.

As shown in fig. 17c, cysteine not bound to the single stranded oligonucleotide was protected with triphenylchloromethane or iodoacetic acid; providing a double-stranded DNA containing a section of cohesive end, wherein the length of the double-stranded DNA is 50-5000bp (base pairs), and the cohesive end and the single-stranded oligonucleotide are subjected to base complementary pairing to form phi29DNA polymerase protein with a polyanion tail.

ii) anchoring the motor protein 8 in the nanopore 5 via a metal-sulfur bond,

as shown in fig. 18a, a nanopore device 7 is provided, the nanopore device 7 being described in detail above and not described herein again by the inventors; the polyanionic tail is driven using an electrophoretic method, the polyanionic tail pulls the phi29DNA polymerase protein into the nanopore 5 and stabilizes the phi29DNA polymerase protein in the nanopore 5 near the nanopore 51 or in the nanopore 51.

As shown in fig. 18b to 18c, the disulfide bond is reduced to a thiol group to cut off the polyanion tail, exposing the thiol group of cysteine, the thiol group of cysteine is combined with the surface of the gold layer 33 to form a gold-sulfur bond, and the phi29DNA polymerase protein is anchored in the nanopore cavity 5 near the nanopore 51 or stabilized in the nanopore 51 by the gold-sulfur bond, so that the transport speed of the DNA strand to be detected through the nanopore 51 is at least slower than 0.1ms per base pair.

As shown in fig. 18d, the DNA strand to be detected is loaded into the fluid chamber 721 through the first fluid flow channel 761, spontaneously diffused or electrophoretically driven into the nanopore chamber 5, and after being captured by the motor protein 8, the DNA strand to be detected is ratcheted and transported in the nanopore in units of single base, and the moving speed thereof can be reduced to be at least slower than 0.1ms per base pair.

Further, the present invention also provides a method for using a device for controlling gene chain transport rate based on a motor protein, as shown in fig. 18d, a solution containing a gene chain to be detected with controllable temperature and pH is provided, loaded into the fluid chamber 721 through the first fluid flow channel 761, driven into the nanopore cavity 5 by spontaneous diffusion or electrophoresis, captured by the motor protein 8, and subjected to ratchet motion, and transported in the nanopore 51 with a single base as a unit, and the transport rate of the gene chain to be detected in the nanopore 51 is controlled to be reduced to tens or hundreds of ms/base pair, i.e., 10 to 999 ms/base pair, by controlling the temperature or pH of the solution, the transport rate of the gene chain to be detected is reduced, so that the intensity of a spectral signal is greatly improved, and more accurate sequencing can be achieved.

Preferably, the temperature range is 0 ℃ to-200 ℃, the pH value range is 1-13, the transport speed of the gene chain to be detected is reduced to tens or hundreds of ms/base pair, the intensity of a spectrum signal is greatly improved, and more accurate sequencing can be realized.

It should be noted that the gene transfer rate control device based on motor protein shown in FIG. 18c can be stored in a low temperature environment, preferably-20 ℃ to-200 ℃, when not in use, to ensure the activity of the motor protein 8.

Preferably, the gene chain is a DNA chain, and may be an RNA chain or a polypeptide chain.

The gene transfer speed control device based on the motor protein and the manufacturing method thereof provided by the invention anchor the motor protein in the nanopore by using a click chemistry method, so that the transfer speed of the gene chain to be detected passing through the nanopore 51 is at least slower than 0.1ms per base pair, the temperature and the pH are adjusted to effectively control the transfer speed of the gene chain, the transfer speed of the gene chain to be detected passing through the nanopore 51 can be further reduced to tens or even hundreds of milliseconds, and the sensitivity of spectral detection is improved; the local plasmon enhanced electric field on the surface of the metal layer is exponentially attenuated with the distance, and the sub-nanometer spatial resolution can be provided, so that the detection of the sub-nanometer spatial resolution can be realized only by using the plasmon enhanced electric field on the surface no matter the size of the nanopore.

On the basis of the above technologies, as shown in fig. 19 to 21, the present invention further provides a raman spectroscopy biomolecule sequencing system, which can provide a vibration spectrum of molecule-specific fingerprint information by using raman spectroscopy, has excellent discrimination and chemical sensitivity compared with the conventional ion current biomolecule sequencing technology, and can rapidly sequence biomolecules, and the following is introduced into the sequencing system:

as shown in fig. 19, the raman spectroscopy biomolecule sequencing system includes a nanopore device 7, a laser raman microscope 91, a spectroscopic measurement device 92, and a data acquisition and analysis device 93, the nanopore device 7 being disposed below the laser raman microscope 91.

The nanopore device 7 comprises a shell 71, a fluid cavity sealing layer 73, a flexible substrate nanopore structure 72, a fluid cavity base 74, a power supply 75, a first electrode 751 and a second electrode 752, wherein the first electrode 751 and the second electrode 752 are respectively connected with two poles of the power supply 75; the flexible substrate nanopore structure 72 comprises a flexible substrate 4, a pore substrate 3, and a nanopore structure (not labeled) comprising a nanopore cavity 5, a nanopore 51; the nano-hole cavity 5 penetrates through the hole substrate 3, the nano-hole 51 is an opening at one end of the nano-hole cavity 5, and the nano-hole cavity 5 is a nonlinear reduction cavity or a linear reduction cavity and extends towards the nano-hole 51; the nanopore structure further comprises: a grating (not shown) surrounding the nanopore cavity 5, the grating being formed on the upper surface of the pore substrate 3 and far away from the nanopore 51, the grating being composed of a grating groove 31, a pore protection film 32 covering the grating and the nanopore cavity 5, a metal layer 33 covering the pore protection film 32 to reduce the size of the nanopore to 1-100nm, forming a spectral nanopore 51 with plasmon enhancement effect, the surface localized plasmon enhancement electric field of the metal layer 33 decaying exponentially with distance, and providing sub-nanometer spatial resolution; a fluid chamber 721 formed by the flexible substrate 4 not covering the grating and the nanopore chamber 5, surrounding the grating and the nanopore chamber 5, the flexible substrate nanopore structure 72 including a first liquid flow channel 7610, the first liquid flow channel 7610 connecting the outside with the fluid chamber 721 to input or discharge a solution to the fluid chamber 721; the fluid chamber base 74 comprises a lower fluid chamber 741 and a second fluid flow passage 7620, wherein the second fluid flow passage 7620 connects the outside with the lower fluid chamber 741 to input or discharge a solution in the lower fluid chamber 741; the fluid chamber sealing layer 73 seals the fluid chamber 721; the housing 71 includes flow passage openings (not labeled) corresponding to the first and second liquid flow passages 761 and 762, respectively. The above technical features included in the nanopore device 7 have been described in detail above, and are not described in detail herein by the inventors. It should be noted that the nanopore 51 should be kept completely wet, and the method for keeping completely wet is described in detail above, and will not be described herein again.

The solution containing the biomolecule to be measured enters the fluid cavity 721 through the first fluid flow channel 7610, and is driven into the nano-cavity 5 by spontaneous diffusion or electrophoresis; when the biomolecules pass through the nanopore 51, the laser raman microscope 91 emits laser to the solution to generate raman spectrum signals of characteristic vibration peak positions of the biomolecules, the raman spectrum signals have excellent discrimination and chemical sensitivity, the spectrum measuring device 92 measures the raman spectrum signals to obtain measurement data, and the data acquisition and analysis device 93 analyzes the measurement data and outputs results.

As shown in fig. 19 to 20a, the thickness of the metal layer 33 is 10 to 500nm to reduce the size of the nanopore 51 to 1 to 100nm, thereby forming a spectral nanopore 51 with plasmon enhancement effect, the localized plasmon enhancement electric field on the surface of the metal layer 33 is exponentially attenuated with distance, and a sub-nanometer spatial resolution can be provided, when a biomolecule passes through the nanopore 51, the nanopore 51 is used as antenna optics, a highly localized plasmon electric field is generated on the lower surface under laser irradiation, so as to excite a specific vibration/rotation/absorption/reflection spectrum of the biomolecule, and a grating reflects a spectral signal generated by the nanopore 51, thereby having technical effects of reducing light propagation loss and enhancing spectral signal intensity.

Preferably, the size of the nanopore 51 is 1-100nm, so as to form a spectral nanopore with better plasmon enhancement effect, and further enhance the spectral signal intensity.

In the present embodiment, the biomolecule is DNA, RNA, but is not limited to such biomolecules. As shown in fig. 19, it is necessary to anchor the motor protein 8 in the nanopore 51 to reduce the transport speed of DNA strands and RNA strands in the nanopore 51, and the motor protein 8 forms a metal-sulfur bond with the metal layer 33. The remaining technical details of the motor protein 8 are described in detail above and will not be described further by the inventors.

When electrophoresis driving is used, a bias voltage of 0.01-10V is applied between the first electrode 751 and the second electrode 752, DNA and RNA in a solution are driven to move towards the nanopore 51 through electrophoresis, the probability of capturing the DNA strand and the RNA strand by the motor protein 8 is increased, the laser Raman microscope 91 emits laser to the DNA strand and the RNA strand, and a base generates a Raman spectrum signal of a characteristic vibration peak position, so that the high-sensitivity electrophoresis driving device has excellent discrimination and chemical sensitivity.

In other embodiments, the biomolecule is a polypeptide chain, which can be a polypeptide chain formed after protein processing.

Preferably, the laser wavelength emitted by the laser raman microscope 91 is 200 to 1000nm, and preferably, the laser wavelength emitted by the laser raman microscope 91 is 400 to 800nm; the grating of the spectral measuring device 92 is 150-2400 lines/row; the time for collecting Raman spectrum signals is 1 mu s-1s.

Preferably, as shown in fig. 20b, in order to shorten the spectrum collection time and improve the detection efficiency, the spectrum collection time is greatly reduced by using the ultrafast coherent raman spectroscopy, and the laser raman microscope 91 emits a pump laser (not shown), a stokes laser (not shown) and a probe laser to the biomolecules to generate a coherent raman spectrum signal, which has excellent discrimination and chemical sensitivity. The ultra-fast coherent Raman spectroscopy includes stimulated Raman scattering spectroscopy, coherent anti-Stokes Raman spectroscopy and two-photon comb coherent Raman spectroscopy. Preferably, the ultrafast coherent raman spectroscopy method uses a laser source including an ultrafast laser light source and a continuous wave laser light source.

As shown in FIG. 21, this is a spectrum obtained by the present Raman spectroscopy biomolecule sequencing system, and the nucleotide sequence can be read very clearly.

The invention also provides a biomolecule sequencing method by Raman spectroscopy, as shown in FIGS. 19 to 21, comprising the steps of:

step I, providing a nanopore device 7, wherein the nanopore device 7 comprises a shell 71, a fluid cavity sealing layer 73, a flexible substrate nanopore structure 72, a fluid cavity base 74, a power supply 75, a first electrode 751 and a second electrode 752, and the first electrode 751 and the second electrode 752 are respectively connected with two poles of the power supply 75; the flexible substrate nanopore structure 72 comprises a flexible substrate 4, a pore substrate 3, and a nanopore structure (not labeled) comprising a nanopore cavity 5, a nanopore 51; the nano-hole cavity 5 penetrates through the hole substrate 3, the nano-hole 51 is an opening at one end of the nano-hole cavity 5, and the nano-hole cavity 5 is a nonlinear reduction cavity or a linear reduction cavity and extends towards the nano-hole 51; the nanopore structure further comprises: a grating (not shown) surrounding the nanopore cavity 5, the grating being formed on the upper surface of the pore substrate 3 and far away from the nanopore 51, the grating being composed of a grating groove 31, a pore protection film 32 covering the grating and the nanopore cavity 5, a metal layer 33 covering the pore protection film 32 to reduce the size of the nanopore to 1-100nm, and the local plasmon enhancement electric field on the surface of the metal layer 33 being exponentially attenuated with distance, which can provide sub-nanometer spatial resolution; a fluid chamber 721 formed by the flexible substrate 4 not covering the grating and the nano-wells 5, surrounding the grating and the nano-wells 5, the flexible substrate nano-well structure 72 comprising a first liquid flow channel 7610, the first liquid flow channel 7610 connecting the outside with the fluid chamber 721 to input or discharge a solution to the fluid chamber 721; the fluid chamber base 74 includes a lower fluid chamber 741 and a second fluid flow path 7620, and the second fluid flow path 7620 connects the outside with the lower fluid chamber 741 to input or discharge a solution in the lower fluid chamber 741; the fluid chamber sealing layer 73 seals the fluid chamber 721; the housing 71 includes flow path openings (not labeled) corresponding to the first and second liquid flow paths 761 and 762, respectively. The above technical features included in the nanopore device 7 have been described in detail above, and are not described in detail herein by the inventors. It should be noted that the nanopore 51 should be kept completely wet, and the method for keeping completely wet is described in detail above, and will not be described in detail herein by the inventors.

Inputting a solution containing a biomolecule to be detected into the fluid chamber 721;

step II, the biological molecules move towards the nano holes 51, the laser Raman microscope 91 emits laser towards the nano holes 51, and when the biological molecules pass through the nano holes 51, raman spectrum signals are generated, so that excellent discrimination and chemical sensitivity are achieved;

step III, measuring the Raman spectrum signal by the spectrum measuring device 92 to obtain measurement data;

step IV, the data acquisition and analysis device 93 analyzes the measured data and outputs a result;

in this embodiment, as shown in fig. 19 to 20a, the thickness of the metal layer 33 is 10 to 500nm to reduce the size of the nanopore 51 to 1 to 100nm, so as to form a spectroscopic nanopore 51 with plasmon enhancement effect, the localized plasmon enhancement electric field on the surface of the metal layer 33 is exponentially attenuated with distance, so as to provide sub-nanometer spatial resolution, when the biomolecule passes through the nanopore 51, the nanopore 51 acts as antenna optics, a highly localized plasmon electric field is generated on the laser irradiation lower surface, so as to excite the specific vibration/rotation/absorption/reflection spectrum of the biomolecule, and the grating reflects the spectroscopic signal generated by the nanopore 51, so as to reduce the light propagation loss and enhance the spectral signal intensity.

Preferably, the size of the nanopore 51 is 1 nm to 100nm, so as to form a spectral nanopore with better plasmon enhancement effect, and further enhance the spectral signal intensity.

In this embodiment, step I further includes inputting a buffer solution into the lower fluid cavity 741, where the buffer solution may be one of deionized water, KCl solution, KNO3 solution, TE buffer solution, PBS buffer solution, HEPES buffer solution, or the like.

The biomolecule is a polypeptide chain, which can be a polypeptide chain formed after protein processing:

step I, also including the process of processing protein to form the polypeptide chain, processing the protein solution to be detected by using 8mol/L urea or 6mol/L guanidine hydrochloride, and splitting into polypeptide chains; using excessive dithiothreitol, beta-mercaptoethanol or tris (2-chloroethyl) phosphate to break disulfide bonds, and using iodoacetic acid to protect the sulfhydryl groups from re-forming disulfide bonds; the processed protein is decomposed into polypeptide chains and loaded into the nanopore device 7;

in step II, a microfluidic control polypeptide chain is used to pass through nanopore 51; the laser Raman microscope 91 emits laser to excite amino acid residues in polypeptide chains to obtain Raman spectrum signals with characteristic vibration peak positions, and the Raman spectrum signals have excellent discrimination and chemical sensitivity;

in step III, the spectral measurement device 92 can directly measure and obtain the position information of the N-terminal, C-terminal and disulfide bond;

in step IV, the data acquisition and analysis device 93 performs data analysis to obtain amino acid sequence information of the polypeptide chain.

The biomolecule is DNA, RNA or methylated DNA, but is not limited to such biomolecules:

as shown in fig. 19, it is necessary to anchor the motor protein 8 in the nanopore 51 to reduce the transport speed of DNA strands and RNA strands in the nanopore 51, and the motor protein 8 forms a metal-sulfur bond with the metal layer 33. The remaining technical details of motor protein 8 are described in detail above and will not be described further by the inventors.

For the biomolecule to be DNA:

in the step I, loading a solution containing DNA to be detected into a nanopore device 7, electrophoretically driving a DNA chain to be captured by a movement protein 8, and carrying out base-by-base stepping ratchet motion on the DNA with the aid of the movement protein 8;

preferably, the motor protein 8 is a DNA polymerase.

In the step II, the DNA chain enters the nanopore 51 and is excited by laser emitted by the laser Raman microscope 91, different base components in the chain emit Raman spectrum signals with characteristic vibration peak positions, the base spectrum signals are not overlapped, and the DNA chain has excellent division and chemical sensitivity;

in step III, the spectrum measuring device 92 measures a raman spectrum signal that changes with time;

in step IV, the data acquisition and analysis device 93 analyzes the Raman spectrum signals, calculates the transport speed of the DNA in the nanopore 51, and converts the species attribution into the sequence information of the DNA according to the characteristic Raman spectrum of the four bases.

For the biomolecule being RNA:

in step I, a solution containing RNA to be detected is filled into a nanopore device 7, and an RNase inhibitor is additionally added to prevent RNA degradation; the electrophoresis drives the RNA chain to be captured by the movement protein 8, and the RNA generates ratchet motion of stepping one by one base under the assistance of the movement protein 8;

preferably, the motor protein 8 is an RNA polymerase.

In the step II, the RNA chain enters the nanopore 51 and is excited by laser emitted by the laser Raman microscope 91, different base components in the chain emit Raman spectrum signals with characteristic vibration peak positions, the base spectrum signals are not overlapped, and the excellent division and chemical sensitivity are realized;

in step IV, the data acquisition and analysis device 93 analyzes the Raman spectrum signals, calculates the transport speed of RNA in the nanopore 51, and converts species attribution into RNA sequence information according to the characteristic Raman spectrum of the four bases.

For DNA in which the biomolecule is methylated:

in the step I, loading a DNA solution containing methylation to be detected into a nanopore device 7, electrophoretically driving a DNA chain to be captured by a motor protein 8, and carrying out base-by-base stepping ratchet motion on the DNA with the aid of the motor protein;

preferably, the motor protein 8 is a DNA polymerase. In the step II, the DNA chain enters the nanopore 51 and is excited by laser emitted by a laser Raman microscope, methylated basic group components in the chain emit Raman spectrum signals with methylation characteristic vibration peak positions, the basic group spectrum signals are not overlapped, and the DNA chain has excellent discrimination and chemical sensitivity;

in step IV, the data acquisition and analysis device 93 analyzes the Raman spectrum signals, calculates the transport speed of the DNA in the nanopore 51, and converts the type attribution into the methylation sequence information of the DNA according to the characteristic Raman spectrum of the four bases.

The electrophoresis driving is to add a bias voltage of 0.01-10V between the first electrode 751 and the second electrode 752 to electrophoretically drive DNA and RNA in a solution to move towards the nanopore 51, so that the probability of capturing the DNA chain and the RNA chain by the motor protein 8 is increased, the laser Raman microscope 91 emits laser to the DNA chain and the RNA chain, and the base generates a Raman spectrum signal with a characteristic vibration peak position.

Preferably, the laser raman microscope 91 emits a laser wavelength of 200-1000nm; the grating of the spectral measuring device 92 is 150-2400 lines/row; the time for collecting the Raman spectrum signal is 1 mu s-1s.

Preferably, as shown in fig. 20b, in order to shorten the spectrum collection time and improve the detection efficiency, the spectrum collection time is greatly reduced by using the ultra-fast coherent raman spectroscopy, and the laser raman microscope 91 emits pump laser (not shown), stokes laser (not shown) and probe laser to the biomolecules to generate coherent raman spectrum signals, including stimulated raman scattering spectrum signals, coherent anti-stokes raman spectrum signals and double-optical comb coherent raman spectrum signals. Preferably, the ultrafast coherent raman spectroscopy method uses a laser source including an ultrafast laser source and a continuous wave laser source.

As shown in FIG. 21, it is a spectrogram obtained by the present Raman spectroscopy biomolecule sequencing method, and the base sequence can be read very clearly.

The Raman spectrum measurement mode can be replaced by a fluorescence spectrum, infrared spectrum, absorption spectrum and reflection spectrum measurement mode;

the Raman spectrum biological molecule sequencing system and the method thereof provided by the invention use the detection strategy of surface enhanced Raman spectrum, and have the technical effects that: the local plasmon enhanced electric field on the surface of the metal layer is exponentially attenuated with the distance, and sub-nanometer spatial resolution can be provided, so that the detection of the sub-nanometer spatial resolution can be realized only by using the plasmon enhanced electric field on the surface no matter the size of the nanopore; compared with the traditional ion current biomolecule sequencing technology, the characteristic current signals of 20 amino acid R groups and different basic groups have larger overlapping regions, so that the distinguishing sensitivity is limited, while the Raman spectrum can provide the vibration spectrum of the molecule specificity fingerprint information, the spectrum signals of all the basic groups are not overlapped, and the distinguishing degree and the chemical sensitivity are excellent.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be accomplished by those skilled in the art without departing from the spirit and scope of the present invention as set forth in the appended claims.

Claims

1. A method for manufacturing a motor protein-based gene transfer rate control device, comprising the steps of:

i) Forming a motor protein with a polyanionic tail;

the nanopore structure further comprises: the grating surrounds the nano-hole cavity, the grating is formed on the surface of the hole substrate far away from the nano-hole, and the grating is composed of a plurality of groups of grating grooves; the hole protection film covers the grating and the nano hole cavity, and comprises a silicon dioxide layer, at least two silicon dioxide layers or at least two metal oxide layers; the metal layer covers the hole protection film to reduce the size of the nano hole to 1 to 100nm; a fluid chamber formed by the flexible substrate not covering the grating and the nano-pore cavity, surrounding the grating and over the nano-pore cavity;

the fluid cavity sealing layer seals the fluid cavity;

2. The method according to claim 1, wherein the motor protein comprises a DNA polymerase, a DNA helicase, or an RNA polymerase.

3. The method according to claim 2, wherein the DNA polymerase is one of DNA polymerase I-V and DNA polymerase α, β, γ, δ, ε, ζ.

4. The method of claim 2, wherein the DNA helicase is one of superfamily I-III, class DnaB family, and class rho family.

5. The method according to claim 2, wherein the DNA polymerase is phi29DNA polymerase.

6. The method of manufacturing of claim 1, wherein the metal layer is one of copper, silver, gold, zinc, mercury, cadmium, cobalt, nickel, or aluminum.

7. The method according to claim 1, wherein the step i) comprises performing site-directed mutagenesis on the amino acid residues on the surface of the motor protein by using a genetic engineering method, replacing the amino acid residues with a plurality of cysteines, and connecting thiol-modified single-stranded oligonucleotides (5 to 100nt in length) to a part of the cysteines; providing a section of double-stranded DNA (with the length of 50-5000 bp) containing a cohesive end to perform base complementary pairing with the single-stranded oligomer on the single-stranded oligonucleotide to form the motor protein with the polyanionic tail.

8. A use method of a gene transfer rate control device based on a motor protein, which uses the gene transfer rate control device based on the motor protein manufactured by the manufacturing method of claims 1 to 7, is characterized in that a solution containing a gene chain to be detected with temperature and pH value is provided, loaded into the fluid cavity through the first liquid flow channel, spontaneously diffused or electrophoretically driven into the nanopore cavity, and after being captured by the motor protein, the transfer rate of the gene chain to be detected in the nanopore 51 is reduced to 0.1 to 999ms per base pair.

9. The use method according to claim 8, wherein the gene strand is a DNA strand or an RNA strand, and the temperature is 25 ℃ to 200 ℃.

10. The use method of claim 8, wherein the gene strand is a DNA strand or an RNA strand, and the pH value is 1 to 13.