CN117470912A - Ultrasensitive nanopore structure, chip, analysis device, amino acid and protein detection method and application - Google Patents
Ultrasensitive nanopore structure, chip, analysis device, amino acid and protein detection method and application Download PDFInfo
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- CN117470912A CN117470912A CN202210909213.0A CN202210909213A CN117470912A CN 117470912 A CN117470912 A CN 117470912A CN 202210909213 A CN202210909213 A CN 202210909213A CN 117470912 A CN117470912 A CN 117470912A
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Abstract
The invention discloses an ultrasensitive nanopore structure, a chip, an analysis device, an amino acid and protein detection method and application, wherein the ultrasensitive nanopore structure is a nanopore, the aperture of the nanopore is in the range of 0.35-5nm, the length of a sensing area of the nanopore is in the range of 0.3-2nm, and the size of the sensing area is similar to that of a single amino acid molecule. The invention discloses an ultrasensitive nanopore chip, which comprises a substrate with the aperture of 5-500nm and is used for supporting ultrasensitive nanopores, wherein the ultrasensitive nanopore analysis device is used for realizing single-molecule detection limit smaller than 1 dalton, realizing detection of amino acid molecules on single-molecule level, completing direct detection of 20 natural amino acid molecules and posttranslational chemically modified amino acids, and providing a strategy for realizing protein sequencing by technical fusion of ultrasensitive nanopore structures and biological enzymes.
Description
Technical Field
The invention relates to the field of single-molecule detection, in particular to an ultrasensitive nanopore structure, a chip, an analysis device, an amino acid and protein detection method and application.
Background
Protein is an extremely important living substance, and development of protein sequencing technology can not only provide key information for research of protein structure and function, but also bring about revolutionary progress for proteomics research and medical research. Edman degradation and mass spectrometry are currently the dominant methods of protein sequencing, and are deficient in terms of detection speed, read length, or achieving routine, complete proteomic quantification at low abundance.
The nanopore technology has extremely high resolution, and has important significance in the fields of gene sequencing, pathogen sequencing and the like. Currently, nanopore technology has been making attention to DNA sequencing, but the development in protein sequencing is lagging, mainly due to the very complex structure of proteins. The protein consists of 20 amino acids, the DNA consists of 4 bases, and the detection of 20 distinguishable signals is a difficult problem, and in addition, the characteristics of the protein such as multi-stage structure, nonuniform charge and the like also bring a plurality of challenges to the sequencing of the protein.
Detection of proteins and short peptide chains has been achieved using nanopore technology at present, but de novo sequencing of proteins still faces two crucial challenges: 1. time resolution, controlling the speed at which the peptide chain passes through the nanopore can increase the number of valid data points, and by adopting methods such as introducing enzyme to pull the peptide chain through the nanopore, sufficient time resolution can be provided for nanopore protein sequencing; 2. spatial resolution in recent years, several literature reports methods for increasing protein detection sensitivity in nanopores have achieved differentiation of amino acid substitutions, different sizes of homogeneous charged short isopeptides and post-translational modifications in carrier polymers, however, spatial resolution remains a major bottleneck for directly resolving individual amino acid residues, limiting the development of nanopore protein sequencing. It is extremely difficult to distinguish the signals generated by the small differences between the 20 natural amino acids.
The length of the sensing region of the nanopore, i.e. the number of amino acids read simultaneously by the nanopore as it passes linearly through the polypeptide chain, is crucial for the improvement of spatial resolution of the nanopore. The sensing region length of biological nanopores in widespread use today is several amino acids, for example, mspA biological nanopores are 8 amino acids in length (h.brinkerhoff et al, science,374 (6574), 1509-1513.), which limits their further improvement in spatial resolution. The invention provides an ultrasensitive nanopore structure, namely a nanopore structure with a sensing area length smaller than the shortest sensing area length (8 amino acids) which can be provided by a traditional biological nanopore. The ultrasensitive nanopore chip and the ultrasensitive nanopore analysis device are designed based on the ultrasensitive nanopore structure. The ultrasensitive nanopore structure provides a basis for the identification of single amino acid molecules and de novo sequencing of proteins, since the length of the sensing region is smaller than the shortest sensing region that can be provided by conventional biological nanopores. In addition, based on the methods reported in the prior literature that utilize biological enzymes to control the passage of peptide chains through nanopores to improve time resolution (h. Brinkerhoff et al, science,374 (6574), 1509-1513), the present invention also proposes strategies for the technical fusion of ultrasensitive nanopore structures with biological enzymes to achieve protein sequencing.
Currently, challenges for achieving nanopore protein sequencing are:
1. and (5) improving the spatial resolution. In view of the problem of overlarge sensing area length faced by the current biological nanopores, it is important to develop an ultrasensitive nanopore with sensing area length equivalent to the size of a single amino acid molecule.
2. And controlling the aperture of the nano hole. The electron beam perforation method mentioned in patent US10648965B2 allows to achieve nanopore diameters in the range of 3-20 nm, but this size is still large for single amino acid detection. Controlling the nanopore size in the sub-nanoscale to 2 nanometer range will bring new opportunities for amino acid detection and protein sequencing.
3. Control of the amino acid recognition signal-to-noise ratio. Because of the small structural differences of different amino acids, it is a necessary condition to ensure a high signal to noise ratio during single molecule experiments. Conventional artificial nanopores face the problem of excessive noise, and noise reduction is a great challenge to the development of artificial nanopores.
Disclosure of Invention
The invention provides an ultrasensitive nanopore structure, a chip, an analysis device, an amino acid and protein detection method and application, which are used for overcoming the defects of the prior art, and the invention is realized by adopting the following technical scheme:
An ultrasensitive nanopore structure is characterized in that the ultrasensitive nanopore structure is a nanopore, the aperture of the nanopore is in the range of 0.35-5nm, the length of a sensing area of the nanopore is in the range of 0.3-2nm, and the size of the sensing area is similar to that of a single amino acid molecule.
As a further improvement, the aperture of the nano-pore is in the range of 0.35-2nm, so that the detection and identification of single amino acid molecules can be realized.
As a further improvement, the aperture of the nano-pore is in the range of 1-5nm, and detection and recognition of the post-translational chemically modified amino acid molecules, detection of polypeptides, differentiation of polypeptide amino acid sequences and protein sequencing can be realized.
As a further improvement, the nanopore according to the present invention is an artificial nanopore or a biological nanopore.
As a further refinement, the material sensing region of the nanopore of the present invention is less than 2nm in length and has an atomic-scale thickness, including molybdenum disulfide, molybdenum diselenide, molybdenum ditelluride, tungsten diselenide, tungsten ditelluride, titanium disulfide, titanium diselenide, titanium ditelluride, zirconium disulfide, zirconium diselenide, zirconium ditelluride, hafnium diselenide, hafnium ditelluride, vanadium disulfide, vanadium ditelluride, niobium disulfide, niobium diselenide, niobium ditelluride, tantalum disulfide, tantalum diselenide, tantalum ditelluride, technetium diselenide, technetium distelluride, rhodium disulfide, rhodium diselenide, rhodium ditelluride, rhenium diselenide, rhenium ditelluride, platinum disulfide, platinum diselenide, platinum ditelluride, palladium diselenide, palladium ditelluride, graphene carbon nitride, boron nitride, black phosphorus, layered metal oxide, layered metal carbide, layered metal oxide, metal nitride, two-dimensional organic framework materials, and layered organic framework materials.
The invention discloses an ultrasensitive nanopore chip comprising an ultrasensitive nanopore structure, which comprises a lower substrate and an upper nano film containing the ultrasensitive nanopore structure.
As a further improvement, the substrate provided by the invention is provided with the supporting holes for supporting the ultrasensitive nanopores, and the aperture of the supporting holes is in the range of 5-500 nm.
As a further improvement, the substrate according to the invention comprises a silicon layer with a resistance higher than 2700 ohm cm.
As a further improvement, the substrate comprises five layers of silicon nitride, silicon oxide, silicon oxide and silicon nitride from top to bottom, wherein the thickness of the silicon layer is 300-500 mu m, the surface layer is an insulating layer, and the supporting holes are formed in the surface layer silicon nitride layer.
As a further improvement, the chip of the invention can realize background noise lower than 20 pA. Dielectric noise is reduced and a signal to noise ratio sufficient to resolve individual amino acid molecules can be provided.
The invention discloses an ultrasensitive nanopore analysis device of an ultrasensitive nanopore chip, which comprises a fluid tank filled with ion solution, the ultrasensitive nanopore chip dividing the fluid tank into a cis-side cavity and a trans-side cavity, an electrode connected with the fluid tank solution or the chip, a current amplifier and a power supply connected with the electrode in series, and digital-to-analog conversion equipment and a computer connected with the current amplifier.
The invention discloses a method for preparing an ultrasensitive nanopore structure by an ultrasensitive nanopore analysis device, which comprises the steps of injecting saline solution into chambers at two sides of a fluid tank, directly applying voltage within the range of 0-10V to a nano film through a power supply and a current amplifier, etching to obtain a nanopore, and controlling electrochemical reaction by controlling the size and duration of the applied voltage, so that the size of the aperture can be controlled; and obtaining real-time data of the transmembrane conductance of the nanopore, and stopping applying voltage when the conductance value reaches the corresponding value of the target aperture to finish the preparation process of the nanopore.
The invention discloses a method for rapidly detecting amino acid in a nanopore by using an ultrasensitive nanopore analysis device, wherein the aperture of the nanopore is in the range of 0.35-2nm, and the method comprises the following steps:
1) Dissolving an amino acid sample in a buffer;
2) Adding an amino acid sample into the fluid tank solution, and enabling the sample to pass through the nano holes under the action of an electric field;
3) Recording a current perforation map through a current amplifier;
4) The detected amino acids are analyzed by analyzing the amperometric pattern and/or the relative amperometric block conductance decrease and/or the perforation time and/or the capture rate obtained by statistical analysis of the amperometric pattern.
The invention discloses a method for rapid protein sequencing of nanopores by using an ultrasensitive nanopore analysis device, wherein the aperture of the nanopores is in the range of 1-5nm, and the method comprises the following steps:
1) Designing and synthesizing a compound of a peptide chain and a nucleotide chain, a primer chain and a blocking chain, mixing and annealing the three substances according to a required proportion before each experiment to obtain a sample to be detected;
2) Adding biological enzyme, deoxyribonucleoside triphosphates (dNTPs) and a sample to be tested into the cis-side chamber;
3) Applying voltage, wherein the sample passes through the nano hole under the action of an electric field, and recording a current perforation map through a current amplifier;
4) The protein sequence was resolved by analysis of the amperometric punch pattern.
As a further improvement, the biological enzymes described herein include phi29 DNA polymerase, tth DNA polymerase, pfu HS DNA polymerase, T4 DNA polymerase, bst DNA polymerase, taq II DNA polymerase, taq plus DNA polymerase, LA Taq DNA polymerase, ventR DNA polymerase, phusion DNA polymerase, KOD DNA polymerase, iaroof DNA polymerase, hel308 helicase, T4 helicase, dnaB helicase, rep protein helicase, pcrA helicase, tte UvrD helicase, tth UvrD helicase, T4 Dda helicase, T4 gp41 helicase, recQ helicase, NS3 HCV helicase, elF4A helicase, WRN helicase, TRCF helicase, LTag helicase, E1 helicase, T7 gp4 helicase, rho protease, clx protease.
The invention discloses an ultrasensitive nanopore chip applied to the detection of amino acids in nanopores, wherein the aperture of the nanopores is in the range of 0.35-2nm, so as to realize the detection and differentiation of 20 natural amino acids.
The invention discloses an ultrasensitive nanopore chip applied to detection of chemically modified amino acid of a nanopore after translation, wherein the aperture of the nanopore is in the range of 1-5nm, so that detection and differentiation of the modified amino acid after translation are realized.
The invention discloses an ultrasensitive nanopore chip applied to polypeptide detection, wherein the aperture of a nanopore is in the range of 1-5nm, so that the detection of polypeptide and the distinction of polypeptide amino acid sequences are realized.
The invention discloses an ultrasensitive nanopore chip applied to protein sequencing, wherein the pore diameter of a nanopore is in the range of 1-5nm, so that protein sequencing is realized.
The invention designs an ultrasensitive nanopore structure, and an ultrasensitive nanopore chip and an ultrasensitive nanopore analysis device based on the ultrasensitive nanopore structure. The sensing sensitivity length of the ultra-sensitive nanopore structure is smaller than the shortest sensing area length which can be provided by the traditional biological nanopore, and the aperture size can be controlled between 0.35 and 5nm. The ultrasensitive nanopore chip can provide background noise of less than 20 pA. By utilizing the ultrasensitive nanopore analysis device, the single-molecule detection limit of less than 1 dalton is realized, the detection of amino acid molecules on the single-molecule level is realized, the direct detection of 20 natural amino acid molecules and post-translational chemical modification amino acids is completed, and a strategy for realizing protein sequencing by technical fusion of ultrasensitive nanopore structures and biological enzymes is provided. The invention provides a direct solution for improving the spatial resolution of nanopore detection.
Further, the invention has the following beneficial effects:
(1) The ultra-sensitive nano-pore is prepared by adopting an electrochemical etching method, the pore diameter is precisely controllable within the range of 0.35-5nm, and the operation is simple. Compared with a scanning electron microscope (TEM) electron beam punching method, the method has the advantages that compared with a scanning electron microscope (TEM) electron beam punching method capable of achieving a minimum aperture of 2nm (Storm, A.et al Nature Mater 2,537-540 (2003)), the electrochemical etching punching method can be used for preparing nano holes smaller than 2nm, and the method is simple in operation and low in cost. The nanopore can be accurately designed and prepared according to the size of a sample to be detected, the amplitude of a generated current signal is improved, and the nanopore has the capability of identifying different types of amino acids.
(2) Previously, nanopores having atomic scale thicknesses have proven useful for resolving individual nucleotide molecules, and the present invention further reduces the nanopore aperture compared to this work, enabling detection and resolution of smaller size amino acids in the 0.35-2nm range of aperture. Compared with the patent US10648965B2, the invention improves the performance of the nano-pore from the direct detection of 4 nucleotide molecules to the direct detection of 20 amino acid molecules. In addition, the identification of molecules which are isomers can be realized, and the identification of single group differences in the molecules can improve the resolution capability of the ultrasensitive nanopore structure to less than 1 dalton.
(3) The ultrasensitive nanopore structure with the sensing area length of 0.3-2nm can provide sufficient spatial resolution for amino acid detection and protein sequencing.
(4) The substrate structure of the ultrasensitive nanopore chip adopted in the invention is shown in fig. 2, and a sandwich structure is formed by silicon nitride, silicon oxide and silicon. The thickness of the silicon layer is 380+/-25 mu m, and the silicon layer has a higher resistance value (more than 2700ohm cm); the thickness of the silicon oxide layer is 60+/-6 nm, and the silicon oxide layer is prepared by dry thermal oxidation; the thickness of the silicon nitride layer is 20+/-4 nm, and the silicon nitride with low stress is used for facilitating the subsequent preparation of the nano-scale support holes. The design of the sandwich structure greatly reduces dielectric noise, and the ultra-sensitive nanopore analysis device adopting the sandwich structure can realize background noise lower than 20pA, and has lower noise compared with the prior literature report (Feng, J.et., 2015, nature nanotechnology,10 (12), 1070-1076).
(5) The size of the supporting hole on the ultrasensitive nanopore chip substrate is controllable within the range of 5-500nm, and the mechanical noise caused by the vibration of the upper layer of the nanometer film in the detection process can be improved by using the size of the supporting hole smaller than 60 nm.
(6) The ultrasensitive nanopore analysis device has high spatial resolution and high signal to noise ratio, can realize direct detection of 20 natural amino acids, does not need modification and pretreatment of molecules to be detected, and is simple in operation. And the analysis device has a single molecule detection limit of less than 1 dalton, and provides a basis for nanopore protein sequencing.
(7) The ultrasensitive nanopore analysis device can identify posttranslational chemical modification amino acid within the range of 1-5nm of the pore diameter of the nanopore, and is beneficial to the development of medical research and disease diagnosis because posttranslational modification of protein is closely related to diseases.
(8) The ultrasensitive nanopore analysis device can realize the detection of polypeptide and the differentiation of the amino acid sequences of hybrid polypeptides within the range of 1-5nm of the pore diameter of the nanopore.
(9) The ultrasensitive nanopore analysis device can realize protein sequencing within the range of 1-5nm of the pore diameter of the nanopore. Designing and synthesizing a connection complex of a target peptide chain and a nucleotide chain, introducing biological enzymes (such as phi29 DNA polymerase and Hel308 helicase), enabling the target chain to be in contact with the nanopore and the biological enzymes, controlling the target chain to move through the nanopore by the enzymes, and obtaining the sequence of the target peptide chain by measuring and analyzing a current map generated in the perforation process.
(10) The ultrasensitive nanopore structure has high stability, can be stored for a plurality of weeks and can finish detection of thousands of perforation events.
Drawings
FIG. 1 is a schematic diagram of an analysis device according to the present invention;
in the figure, 1 is a power supply, 2 is a current amplifier, 3 is a digital-to-analog conversion device and a computer, 4 is an electrode, 5 is a substrate, 6 is a supporting hole, 7 is a nano film, 8 is a nano hole, 9 is a fluid tank, 10 is a sample to be measured, 11 is a cis-side chamber, and 12 is a trans-side chamber.
FIG. 2 is a schematic illustration of a substrate;
FIG. 3 is a schematic illustration of a fluid tank;
the fluid channel structure used in this embodiment is shown in fig. 3 and is composed of two parts, the only difference between which is that the part shown at 16 contains internal threads, while the part shown at 15 is a smooth inner wall, which facilitates assembly of the fluid channel with screws. The illustration 13 accommodates insertion of a silver/silver chloride electrode, the illustrations 11 and 12 being chambers, the annular groove in the illustration 14 fitting an O-ring.
FIG. 4 is a representation of a single layer of molybdenum disulfide;
FIG. 5 is a schematic diagram of detection of a single amino acid in a molybdenum disulfide nanopore;
a. schematic of the experimental setup (not to scale); b. current tracks collected during glutamic acid perforation; c. the right side of the current track collected in the alanine perforation process shows a fitting curve of the normalized current histogram; d.G, GG, GGG produce a thermal map of dwell time and blocking current of the signal in the same device; e. a flow chart for identifying amino acids using deep learning; f. ΔI/I caused by glycine (G) and alanine (A) in the same device 0 Is a histogram of (1); g. for the data in F, utilizing the accuracy, recall and score map of F1 obtained by deep learning;
FIG. 6 is a graph showing the effect of voltage on nanopore detection amino acids.
a. A graph of the perforation frequency of serine at different voltages; b-c. blocking current values ΔI and ΔI/I due to perforation of glycine at different voltages 0 A drawing.
FIG. 7 is a graph showing the results of detection of all 20 natural amino acids;
the figure shows the delta I/I obtained by carrying out a nanopore experiment on 20 amino acids 0 The histogram is displayed sequentially by charged amino acids (a-b), hydrophobic non-aromatic amino acids (c-d), polar uncharged amino acids (e-f), hydrophobic aromatic amino acids (g) and special amino acids (h). A corresponding confusion matrix map is attached under each histogram.
FIG. 8 is a graph of the results of molybdenum disulfide nano Kong Shibie chemical groups;
a-c are aspartic acid (D) and asparagine (N), glutamic acid (E) and glutamine (Q), respectively, asparagine (N) and leucine (L) induce a penetration in nanopore experimentsΔI/I of pore event 0 A histogram of values; d-f. heat maps of serine (S) and threonine (T), phenylalanine (F) and tyrosine (Y), leucine (L) and isoleucine (I), respectively. The width of the boxes in figures d-f represents the standard deviation.
FIG. 9 is a graph showing the recognition result of phosphorylated amino acids;
a. chemical structure of tyrosine (Y) and phosphorylated tyrosine (p-Y). b. Delta I/I of perforation event of tyrosine and phosphorylated tyrosine in same device 0 And (5) a histogram. c. And b, obtaining the precision, recall rate and F1 score map after deep learning the data in b.
FIG. 10 is a graph showing the results of single molecule detection of peptide chains;
typical current signal patterns of peptide chains EEEGEEE and EEEWEEE during the course of the experiment are shown.
FIG. 11 is a schematic diagram of the introduction of enzymes to effect protein sequencing.
Detailed Description
The invention discloses an ultrasensitive nanopore 8 structure, wherein the ultrasensitive nanopore 8 structure is a nanopore 8, the aperture of the nanopore 8 is in the range of 0-50nm, the length of a sensing area of the nanopore 8 is in the range of 0.3-2nm, and the size of the sensing area is similar to that of a single amino acid molecule. The aperture of the nanopore 8 is further limited in the range of 0.35-2nm, so that detection and identification of single amino acid molecules can be realized, and the nanopore 8 is an artificial nanopore 8 or a biological nanopore 8. When the nanopore 8 is made by a person, the nanopore 8 material has a sensing region length of less than 2nm and an atomic-scale thickness, including molybdenum disulfide, molybdenum diselenide, molybdenum ditelluride, tungsten diselenide, tungsten ditelluride, titanium disulfide, titanium diselenide, titanium ditelluride, zirconium disulfide, zirconium diselenide, zirconium ditelluride, hafnium diselenide, hafnium ditelluride, vanadium diselenide, vanadium ditelluride, niobium disulfide, niobium diselenide, niobium ditelluride, tantalum disulfide, tantalum diselenide, tantalum ditelluride, technetium diselenide, technetium distelluride, rhodium disulfide, rhodium diselenide, rhodium ditelluride, rhenium diselenide, rhenium ditelluride, platinum diselenide, platinum ditelluride, palladium diselenide, palladium ditelluride, graphene carbon nitride, boron nitride, black phosphorus, layered metal oxide, layered metal carbide, layered metal oxide, metal nitride, two-dimensional organic framework material, or layered organic framework material.
The invention discloses an ultrasensitive nanopore 8 chip, which comprises a lower substrate 5 and an upper nano film 7 containing an ultrasensitive nanopore 8 structure, wherein the substrate 5 is provided with a supporting hole 6 for supporting the ultrasensitive nanopore 8, the aperture of the supporting hole 6 is in the range of 5-500nm, the substrate 5 comprises a silicon layer, the resistance value of the silicon layer is higher than 2700 ohm cm, the substrate 5 comprises five layers of silicon nitride, silicon oxide and silicon nitride from top to bottom, the thickness of the silicon layer is 300-500 mu m, the surface layer is an insulating layer, the supporting hole 6 is arranged on the surface layer silicon nitride layer, and the chip can realize background noise lower than 20 pA. Dielectric noise is reduced and a signal to noise ratio sufficient to resolve individual amino acid molecules can be provided.
The invention discloses an ultrasensitive nanopore 8 analysis device, which comprises a fluid tank 9 filled with ion solution, an ultrasensitive nanopore 8 chip dividing the fluid tank 9 into a cis-side chamber 11 and a trans-side chamber 12, an electrode 4 connected into the fluid tank 9 solution or the chip, a current amplifier 2 and a power supply 1 which are connected in series with the electrode 4, a digital-to-analog conversion device connected with the current amplifier 2 and a computer 3.
The invention discloses a method for preparing an ultrasensitive nanopore 8 structure by an ultrasensitive nanopore 8 analysis device, which comprises the steps of injecting saline solution into chambers at two sides of a fluid tank 9, directly applying voltage within the range of 0-10V to a nano film 7 through a power supply 1 and a current amplifier 2, etching to obtain a nanopore 8, and controlling electrochemical reaction by controlling the size and duration of the applied voltage, so that the size of an aperture can be controlled; and obtaining real-time data of the transmembrane conductance of the nanopore 8, and stopping applying voltage when the conductance value reaches the corresponding value of the target aperture to finish the preparation process of the nanopore 8.
The invention discloses a method for rapidly detecting amino acid in a nanopore 8 by using an ultrasensitive nanopore 8 analysis device, which comprises the following steps:
1) Dissolving an amino acid sample in a buffer;
2) Adding an amino acid sample into the solution in the fluid tank 9, and enabling the sample to pass through the nano holes 8 under the action of an electric field;
3) Recording a current perforation map through a current amplifier 2;
4) The detected amino acids are analyzed by analyzing the amperometric pattern and/or the relative amperometric block conductance decrease and/or the perforation time and/or the capture rate obtained by statistical analysis of the amperometric pattern.
The invention discloses a method for rapid protein sequencing of a nanopore 8 by using an ultrasensitive nanopore 8 analysis device, which comprises the following steps:
1) Designing and synthesizing a compound of a peptide chain and a nucleotide chain, a primer chain and a blocking chain, and mixing and annealing the three substances according to a required proportion before each experiment to obtain a sample 10 to be detected;
2) Adding biological enzyme, deoxyribonucleoside triphosphates (dNTPs) and a sample to be detected 10 into the cis-side chamber 11;
3) Applying voltage, wherein a sample passes through the nano hole 8 under the action of an electric field, and a current perforation map is recorded through the current amplifier 2;
4) The protein sequence was resolved by analysis of the amperometric punch pattern.
Wherein the biological enzyme comprises phi29 DNA polymerase, tth DNA polymerase, pfu HS DNA polymerase, T4 DNA polymerase, bst DNA polymerase, taq II DNA polymerase, taq plus DNA polymerase, LA Taq DNA polymerase, ventR DNA polymerase, phusion DNA polymerase, KOD DNA polymerase, iproof DNA polymerase, hel308 helicase, T4 helicase, dnaB helicase, rep protein helicase, pcra helicase, tte UvrD helicase, tth UvrD helicase, T4 Dda helicase, T4 gp41 helicase, recQ helicase, NS3 HCV helicase, elF4A helicase, WRN helicase, TRCF helicase, LTag helicase, E1 helicase, T7 gp4 helicase, rho helicase, clpX protease.
The invention discloses an ultrasensitive nanopore 8 chip which is applied to the detection of amino acids in a nanopore 8 to realize the detection and the distinction of 20 natural amino acids.
The invention discloses an ultrasensitive nanopore 8 chip applied to detection of chemically modified amino acid of a nanopore 8 after translation, and detection and differentiation of the post-translation modified amino acid are realized.
The invention discloses an ultrasensitive nanopore 8 chip applied to polypeptide detection, which realizes the detection of polypeptide and the distinction of polypeptide amino acid sequences.
The invention discloses an ultrasensitive nanopore 8 chip applied to protein sequencing, which realizes protein sequencing.
The technical scheme of the invention is further described by the specific embodiments with reference to the attached drawings.
Example 1: 20 natural amino acids are detected by adopting molybdenum disulfide nano holes 8.
FIG. 1 is a schematic diagram of an analysis device according to the present invention; comprises a fluid tank 9 filled with ion solution, an ultrasensitive nanopore 8 chip dividing the fluid tank 9 into a cis-side chamber 11 and a trans-side chamber 12 an electrode 4 connected to the fluid tank 9 solution or chip, a current amplifier 2 and a power supply 1 connected in series with the electrode 4, a digital-to-analog conversion device connected with the current amplifier 2 and a computer 3. The substrate 5 is provided with a supporting hole 6, the nano film 7 is provided with an ultrasensitive nano hole 8, the nano film 7 adopted in the embodiment is single-layer molybdenum disulfide, the thickness is about 0.65 nanometer, the aperture of the ultrasensitive nano hole 8 is controllable within the range of 0-50nm, and the substrate 5 is a sandwich structure composed of silicon nitride, silicon oxide and silicon.
The specific experimental operation of this example is as follows:
(1) Processing of the device support substrate 5
To obtain a device with a higher signal to noise ratio, the substrate 5 is prepared using a wafer with a sandwich structure, and the structure of the substrate 5 is shown in fig. 2. The sandwich structure is composed of silicon nitride, silicon oxide and silicon, wherein the thickness of the silicon layer is 380+/-25 mu m, and the sandwich structure has a higher resistance value (more than 2700ohm cm); the thickness of the silicon oxide layer is 60+/-6 nm, and the silicon oxide layer is prepared by dry thermal oxidation; the thickness of the silicon nitride layer is 20 + -4 nm, and the use of silicon nitride with low stress facilitates the subsequent preparation of the nano-sized support holes 6.
Firstly, writing a square pattern of 0.5cm multiplied by 0.5cm on a 4-inch wafer by utilizing laser direct writing, then performing reactive ion etching and wet etching to etch away part of silicon nitride, silicon oxide and silicon to obtain the silicon nitride suspension structure shown in fig. 2, and finally preparing a support hole 6 on a silicon nitride support window by utilizing a Focused Ion Beam (FIB). Wherein the size of the support window is 15-20 μm, and the size of the support hole 6 is controlled to be about 60 nm. The background noise of the ultrasensitive nanopore 8 assay device employing this sandwich structure was about 18pA.
(2) Preparation of molybdenum disulfide
A Chemical Vapor Deposition (CVD) process is used to prepare a monolayer of molybdenum disulfide. Molybdenum trioxide powder was used as a molybdenum source, sulfur powder was used as a sulfur source, high purity argon gas was used as a shielding gas and a carrier gas, and a single-sided polished silica wafer was used as the substrate 5 for deposition of the sample. 2mg of molybdenum trioxide powder and 0.15g of sulfur powder were respectively loaded into two quartz boats, and the substrate 5 was placed on the quartz boat loaded with molybdenum trioxide powder in a polishing surface-down direction. The quartz boat with molybdenum trioxide and substrate 5 is placed in the central heating zone of the quartz tube of the tube furnace, and the quartz boat with sulfur powder is placed at the edge of the quartz tube of the tube furnace. And (3) checking the tightness of the tube furnace, pumping out air in the tube by using a mechanical pump, and then introducing argon as shielding gas. And (3) heating to 700 ℃ at a constant speed under the condition of argon flow of 100sccm, wherein the heating rate is about 15 ℃/min, heating at a constant temperature after heating to 700 ℃ for 10 minutes, stopping heating, and naturally cooling to room temperature, wherein a single-layer molybdenum disulfide film is deposited on the oxide layer of the substrate 5. FIG. 4 shows characterization results of a single layer of molybdenum disulfide, wherein graph a is at SiO 2 And b is an image of the single-layer molybdenum disulfide obtained by growth on the surface of Si under a high-resolution spherical aberration correction transmission electron microscope (AC-TEM), c is an electron diffraction diagram of the single-layer single-crystal molybdenum disulfide obtained under a transmission electron microscope (200 kV), d is an image of a molybdenum disulfide sample transferred onto a sapphire sheet under an atomic force microscope, and the thickness of the sample is about 0.78nm.
(3) Transfer of molybdenum disulfide
Taking a small piece of single-layer molybdenum disulfide sample with good crystallinity, uniformly spin-coating PMMA (polymethyl methacrylate) glue at a rotating speed of 2500r/s by using a glue homogenizing machine, and baking for 15min at 180 ℃. The samples were then divided into dimensions of 2mm by 2mm and wet etched in 30% KOH solution at 80℃until the PMMA film floated. And cleaning the PMMA film obtained by etching in pure water for 2-3 times, transferring the PMMA film to a silicon nitride wafer containing 60nm support holes 6 by means of a micro-operation system, and baking the PMMA film for 20min at 180 ℃ after the water is evaporated. And removing PMMA gel on the silicon nitride surface by adopting an acetone solution at the temperature of 60 ℃, and then, quenching for 4 hours in an argon atmosphere at the temperature of 200 ℃.
(4) Ultrasensitive nanopore 8 preparation
And (3) injecting KCl solution with the concentration of 1mol/L into the chambers at the two sides of the fluid tank 9 by adopting an electrochemical etching perforation mode, directly applying voltage within the range of 0-10V to the nano film 7 through the power supply 1 and the current amplifier 2, etching to obtain the nano hole 8, controlling electrochemical reaction by controlling the magnitude and duration of the applied voltage, stopping applying the voltage when the conductivity value reaches the corresponding value of the target aperture, and controlling the aperture within the range of 0.35-2 nm.
(5) Assembly of fluid tank 9
The fluid channel 9 is made of PMMA material, the structure of the fluid channel 9 is shown in fig. 3, and the fluid channel 9 mainly consists of two parts, wherein the only difference is that the part shown in the figure 16 contains internal threads, and the figure 15 is a smooth inner wall, and the design is convenient for assembling the fluid channel 9 by using screws. The illustration 13 accommodates insertion of the silver/silver chloride electrode 4, the illustrations 11 and 12 being chambers, the annular groove in the illustration 14 fitting an O-ring. The chip was sealed between the two-part fluid grooves 9 with O-rings, and the device was secured by 4 screws.
(6) Solution preparation
A Tris-EDTA-KCl buffer solution was prepared at pH=7.8, wherein the Tris concentration was 10mmol/L, the EDTA concentration was 1mmol/L, and the KCl concentration was 1mol/L. The amino acid solid powder is dissolved in a buffer solution to prepare a corresponding amino acid solution, and the concentration of a sample is 2 mu mol/L.
(7) Construction of experimental device
A patch clamp (Axiopatch 200B amplifier) low-noise system is adopted for testing, and an NI PXI-1042Q acquisition card is adopted for data acquisition. Two sections of silver wire, approximately 3cm long, were taken and polished with sandpaper to remove the oxide layer from the surface. Three-quarters of the silver wire length and platinum electrode 4 (as anode and cathode, respectively) were immersed in 1M KCl solution. A voltage of 2V was applied for a period of time to prepare an Ag/AgCl electrode 4. Then, two Ag/AgCl electrodes 4 are respectively connected to the probes of the patch clamp to serve as a positive electrode and a ground wire. The patch clamp needs to be calibrated by a model cell before use, so that errors are eliminated.
(8) Measurement of
Prior to each experiment, a current-voltage sweep was performed in KCl solution at a concentration of 1mol/L to determine the pore size of the device. In the formal experiment, corresponding buffer solutions are injected into the cis-side chamber 11 and the trans-side chamber 12 of the fluid tank 9, a bias voltage of 200mV is applied to the trans-side chamber 12, the positive and negative of the voltage depend on the relative magnitudes of isoelectric points of different amino acids and pH values of the buffer solutions, for example, the isoelectric point of the amino acid to be detected is smaller than 7.8, and when the amino acid is negatively charged in the buffer solution environment, the applied bias voltage is positive. In a blank experiment without the addition of amino acid molecules, an average value of I can be detected 0 Is passed through the nanopore 8. After injection of the amino acid-containing buffer solution into the cis-sided chamber 11, a short interruption of the ion current can be detected. Each current interruption corresponds to the perforation behaviour of a single amino acid molecule in the nanopore 8, characterized by a relative current drop ΔI/I 0 And a residence time deltat. The results of each set of experiments were normalized.
(9) Data analysis
And processing and analyzing the data by adopting a Matlab program, and integrating the data to present experimental results. The experimental data were further analyzed in conjunction with deep learning.
As shown in fig. 5a, the amino acid molecule passes through the molybdenum disulfide nanopore 8 under the driving of the electrophoretic force, when the molecule to be detected passes through the molybdenum disulfide nanopore 8, the pore channel is temporarily blocked, so that the current in the pore channel can be changed, and the amplitude and duration of the current change and the current waveform are analyzed, so that the corresponding molecular information can be obtained. In this example, a current signal generated by 20 natural amino acids in the via hole can be detected and distinguished for different amino acid molecules in a molybdenum disulfide nanopore 8 with a diameter in the range of 0.35-2nm, as shown in FIG. 5bA typical current signal and can be obtained by relatively blocking the current value DeltaI/I of the signal 0 Histogram distribution statistics were performed and fitted to distinguish amino acid species (fig. 5 f). The choice of the size of the molybdenum disulphide nanopores 8 is very important, as the size of the nanopores 8 is close to the size of the amino acid molecule to be measured, the ion current can be reduced to 0nA (fig. 5 c). When the size of the nanopore 8 is greater than 2nm, the detection sensitivity for amino acid molecules decreases.
In this example, to determine that the resulting signal was due to amino acid perforation, a set of perforation experiments at different voltages were performed. The result shows that as the voltage increases, the value of the blocking current delta I increases, and the relative blocking current delta I/I 0 The value of (c) is substantially unchanged (fig. 6b, c), and the frequency of perforation events occurs with increasing voltage, rapidly increasing and then saturating (fig. 6 a).
In this example, the detection sensitive area of the molybdenum disulfide nanopore 8 is equivalent to the size of a single amino acid, and in order to prove this, experiments were performed using short chains G, GG, GGG of different lengths composed of the same amino acid, and the blocking current values generated by the three in the same nanopore 8 are very close (fig. 5 d), which is quite different from the results generated in the biological nanopore 8 reported previously.
In a specific experimental process, since the baseline current of the molybdenum disulfide nanopore 8 has a certain instability, in order to ensure that experimental data is not affected by the change of the baseline current, 20 amino acids are discussed in groups according to the category to which the 20 amino acids belong, namely charged amino acids, polar uncharged amino acids, hydrophobic aromatic amino acids, hydrophobic non-aromatic amino acids and special amino acids.
A deep learning model is introduced in the analysis process of the data, and a neural network diagram is shown in fig. 5 e. The deep learning model learns the characteristic value and waveform of the current signal, and each group of experimental results are displayed in the form of a confusion matrix.
For charged amino acids, considering that the Tris-EDTA buffer employed in the experiment had pH=7.8, under this condition, the pI values for glutamic acid (E, 147.13 Da), aspartic acid (D, 133.11 Da) and histidine (H, 155.15 Da) were smallAt the pH of the solution, lysine (K, 146.19 Da) and arginine (R, 174.20 Da) are negatively charged in solution, and vice versa, and are positively charged in solution, so experiments were performed in two groups according to actual charge conditions. Delta I/I for lysine and arginine 0 The value and the relative mass of the molecules are positively correlated (as shown in fig. 7 a), and the discrimination accuracy after deep learning analysis reaches 82.18%. The relative current blocking values of glutamic acid and aspartic acid are similar (FIG. 7 b), ΔI/I of histidine molecules 0 The value distribution is obviously distinguished from glutamic acid and aspartic acid (as shown in figure 7 b), and the distinguishing accuracy of the glutamic acid, the aspartic acid and the histidine after deep learning of the obtained data reaches 82.08%.
Of the five amino acids contained in the hydrophobic non-aromatic, methionine (M, 149.21 Da) has a high probability of blocking the pore channels during the experiment, resulting in interruption of the experiment, probably due to the fact that it contains sulfur atoms, which are prone to interaction with molybdenum disulfide. It was also found in this set of experiments that the isomeric amino acids leucine (L) and isoleucine (I) with the same molecular mass produced different relative blocking currents (fig. 7 d). In 7 molybdenum disulfide nanopore 8 devices (pore size 0.6-1.4 nm), ΔI/I shown in 5 devices was observed 0 The value is higher than L but the result is opposite in the other 2 devices. The latter occurs in nanopores 8 of about 0.6nm diameter. Attributing this variation to the size of the nanopores 8 may affect the pore entry direction of the amino acids, and the resolution accuracy of these two isomeric amino acids reaches 87.25% by deep learning.
For polar uncharged amino acids, serine (S, 105.09 Da), threonine (T, 119.10 Da), asparagine (N, 132.12 Da) and glutamine (Q, 146.15 Da) produce ΔI/I in the pore canal 0 The value becomes larger as the molecular mass increases (see fig. 7e, f). The molecular structures of serine and threonine, asparagine and glutamine all differ by only one group, and the detection results of the group of amino acids show that the molybdenum disulfide nanopore 8 can recognize the difference of single chemical groups with the molecular weight of only 14.01 Da.
For hydrophobic aromatic amino acids, the relative current blocking values of phenylalanine (F, 165.20 Da), tyrosine (Y, 181.19 Da) and tryptophan (W, 204.20 Da) also show a positive correlation with the relative molecular masses (as in fig. 7 g), where it is notable that there is only one hydroxyl difference between phenylalanine and tyrosine, but the current distribution of both can be distinguished, which once demonstrates that the molybdenum disulfide nanopore 8 has a very high sensitivity to the detection of chemical groups.
For a particular class of amino acids, glycine (G, 75.07 Da), cysteine (C, 121.16 Da) and proline (P, 115.13 Da), where proline produces the greatest relative current blocking value in the experiment (as in fig. 7 h), because proline contains a five-membered ring in its molecular structure, maximizing its molecular volume in a particular group of amino acids. Cysteine also has a sulfur atom, which may block the nanopore 8 during the experiment. Through the above experiments, as a whole, the relative blocking current value generated by the amino acid molecule when passing through the molybdenum disulfide nanopore 8 increases with the increase in its volume.
In order to further demonstrate the high sensitivity of the molybdenum disulfide nanopores 8 to amino acids, separate experiments were performed on aspartic acid (D) and asparagine (N) with a relative molecular mass difference of only 1Da, and the results indicate that the two can be distinguished by amperometric analysis (as in fig. 8 a), which also demonstrates that the prepared molybdenum disulfide nanopores 8 achieve a resolution of 1Da, which is the highest resolution in the current nanopore 8 field.
In recent years, the final resolution of the nanopore 8 for detecting polypeptides has been continuously updated. FraC biological nanopore 8 can distinguish polypeptides differing by only one amino acid residue, with a resolution of 44Da (Huang, g.et., (2019). Nature communications,10 (1), 835). The aerolysin nanopore 8 shows a higher sensitivity for detecting peptide chains formed by linking a polycationic polymer to an amino acid (Ouldali, h.et., (2020). Nature biotechnology,38 (2), 176-181.). However, these measurements all distinguish individual amino acids in an indirect manner, and the difference in current is measured by substitution of amino acid residues. The invention is based on the experimental foundation that can distinguish single amino acid, further study the limit resolution of the present molybdenum disulfide nanopore 8 system. For aspartic acid and asparagine, or glutamic acid and glutamine, two by two The relative molecular mass difference of (2) is only 0.99Da, and the molecular skeleton is the same except the terminal chemical groups, wherein the molecular structure of the charged amino acid (aspartic acid, glutamic acid) is terminated by-OH (17.01 Da), and the molecular structure of the polar uncharged amino acid (asparagine, glutamine) is terminated by-NH 2 (16.02 Da). Although the molecular weight and volume of each pair of amino acids are very close, the difference in functional groups results in a difference in their molecular configuration and charge, resulting in a different current blocking across the molybdenum disulfide nanopore 8 (fig. 8a, b). Leucine and asparagine, which have a relative molecular mass difference of 0.94Da, also produce a distinguishable relative current blocking during perforation due to their large structural differences (fig. 8 c). This is known to be the highest resolution (distinguishing the poor relative molecular mass of less than 1 Da) of the experimentally reported nanopore 8.
Example 2: and detecting posttranslational modification of amino acid by adopting a molybdenum disulfide nanopore 8.
The invention can be applied to detect post-translational modifications (PTMs) of amino acids. Recognition of amino acid post-translational modifications is of great importance in disease research, for example, phosphorylation of tyrosine (Y) plays a key regulatory role in cellular activity, and abnormal phosphorylation of tyrosine is closely related to cancer.
In this example, tyrosine (Y) and phosphotyrosine (p-Y) are detected and identified by using molybdenum disulfide nanopore 8, and fig. 9a is a molecular structure diagram of tyrosine (Y) and phosphotyrosine (p-Y). The experimental procedures (1), (2), (3), (4), (5), (6), (7), (8), (9) described in example 1 were repeated, wherein the sample solutions formulated in procedure (5) were a tyrosine solution and a phosphorylated tyrosine solution.
As shown in FIG. 9b, tyrosine and phosphorylated tyrosine cause ΔI/I in the experiment 0 The distribution of (c) can be distinguished, and the recognition accuracy is 80.67%. This result suggests that molybdenum disulfide nanopore 8 has the potential to recognize post-translationally modified amino acids.
Example 3: and detecting a short peptide chain by adopting the molybdenum disulfide nano hole 8.
The invention can be applied to detection of short peptide chains.
In this example, two peptide chains were designed, each of which was EEEGEEE and EEEWEEE, and the experimental procedures (1), (2), (3), (4), (5), (6), (7), (8) and (9) described in example 1 were repeated, wherein the sample solution prepared in the procedure (6) was two peptide chain molecular solutions of EEEGEEE and EEEWEEE, and the voltage applied in the procedure (8) was 300mV.
Current waveforms as shown in the box in fig. 10 were observed in the experiment. The waveform shown at the bottom of fig. 10 is an ideal waveform of the actual measured current signal without any noise. The larger volume of W produces a larger current blocking, while the smaller volume of G produces a smaller current blocking, which results are consistent with expectations.
Based on the current research progress of the present invention, nanopore 8 resolution below 1Da and detection sensitive regions comparable to single amino acid molecule size have theoretically been able to provide sufficient spatial resolution for protein sequencing. It is proposed that the introduction of enzymes into the molybdenum disulfide nanopore 8 system to precisely control the rate of peptide chain passage through the pore channel is expected to provide sufficient time resolution to achieve de novo sequencing of proteins. Experiments for controlling the movement of polypeptides by introducing enzymes into biological nanopores 8 are now widely used, and many documents report the compatibility between DNA polymerase and solid nanopore 8 systems, so this strategy is viable.
Example 4: protein sequencing was performed using enzyme and molybdenum disulfide nanopores 8.
The invention can be applied to protein sequencing. The detection principle is shown in fig. 11.
The complex in this example in which the peptide chain was designed and synthesized to be linked to the nucleotide chain was HOOC-GEPGEPDDGDD-5 '-X-AGAACTTTAGAACTTTTCAGATCTCACTATCGCATTCTCATGCAGGTCGTAGCC-3', the primer chain was 5'-GCGTACGCCTACGGTTTTCCGTAGGCGTACGCGGCTACGACCTGCATGAGAATGC-3' and the blocking chain was 5 '-GATAGTGAGAGTGAATCTGAXXXXXXXXXZ-3'. X in the sequence represents an abasic spacer and Z represents a 3' spacer. These three materials were mixed at 1:1:2 and annealing for 20 minutes at room temperature to obtain the sample 10 to be measured. HEPES buffer solution was prepared at pH=7.5, where KCl was at a concentration of 1mol/L and HEPES was at a concentration of 10mmol/L ，MgCl 2 At a concentration of 10mmol/L, (NH) 4 ) 2 SO 4 The concentration of (C) was 10mmol/L and the concentration of DTT was 4mmol/L. The experimental operations (1), (2), (3), (4), (5), (7) described in example 1 were repeated. A buffer solution containing phi29 DNA polymerase at a concentration of 5nmol/L, deoxyribonucleoside triphosphates (dNTPs) at a concentration of 1mmol/L and a sample 10 to be tested at a concentration of 10nmol/L was added to the cis-side chamber 11, and a blank buffer solution was added to the trans-side. And applying a voltage in the range of +200-300mV, and passing through the nanopore 8 under the action of an electric field after the sample is contacted with phi29 DNA polymerase and the nanopore 8. The current perforation map is recorded by a current amplifier 2, and the sequence of the target protein is obtained by analyzing the current perforation map.
It should be clear that the above description is not intended to limit the embodiments, but that various modifications can be made by a person skilled in the art on the basis of the above description, which modifications remain within the scope of the invention.
Claims (19)
1. The ultrasensitive nanopore (8) structure is characterized in that the ultrasensitive nanopore (8) structure is a nanopore (8), the aperture of the nanopore (8) is in the range of 0.35-5nm, and the length of a sensing area of the nanopore (8) is in the range of 0.3-2nm and is similar to the size of a single amino acid molecule.
2. Ultrasensitive nanopore (8) structure according to claim 1, characterized in that the pore size of the nanopore (8) is in the range of 0.35-2 nm.
3. Ultrasensitive nanopore (8) structure according to claim 1, characterized in that the pore size of the nanopore (8) is in the range of 1-5 nm.
4. The ultrasensitive nanopore (8) structure of claim 1, wherein the nanopore (8) is an artificial nanopore (8) or a biological nanopore (8).
5. Ultrasensitive nanopore (8) structure according to claim 1, characterized in that the material sensing region of nanopore (8) is less than 2nm long, has atomic-scale thickness, including molybdenum disulfide, molybdenum diselenide, molybdenum ditelluride, tungsten disulfide, tungsten diselenide, tungsten ditelluride, titanium disulfide, titanium diselenide, titanium ditelluride, zirconium disulfide, zirconium diselenide, zirconium ditelluride, hafnium disulfide, hafnium diselenide, hafnium ditelluride, vanadium disulfide, vanadium diselenide, vanadium ditelluride, niobium disulfide, niobium diselenide, niobium ditelluride, tantalum disulfide, tantalum diselenide, tantalum ditelluride, technetium disulfide, technetium diselenide, rhodium disulfide, rhodium diselenide, rhodium ditelluride, rhenium diselenide, rhenium ditelluride, platinum disulfide, platinum ditelluride, palladium disulfide, palladium ditelluride, graphene carbon nitride, boron nitride, black phosphorus, layered metal oxides, layered metal carbides, layered metal nitrides or metal oxynitride composite two-dimensional materials, metal organic framework materials, perovskite framework materials, and covalent organic framework materials.
6. An ultrasensitive nanopore (8) chip comprising the ultrasensitive nanopore (8) structure of any one of claims 1 to 5, characterized in that the chip comprises a lower substrate (5) and an upper nanofilm (7) containing the ultrasensitive nanopore (8) structure.
7. The ultra-sensitive nanopore (8) chip of claim 6, wherein the substrate (5) is provided with a supporting hole (6) for supporting the ultra-sensitive nanopore (8), and the aperture of the supporting hole (6) is in the range of 5-500 nm.
8. The ultrasensitive nanopore (8) chip of claim 6, wherein the substrate (5) comprises a silicon layer having a resistance value higher than 2700ohm cm.
9. The ultra-sensitive nanopore (8) chip of claim 6, wherein the substrate (5) comprises five layers of silicon nitride, silicon oxide and silicon nitride from top to bottom, the thickness of the silicon layer is 300-500 μm, the surface layer is an insulating layer, and the supporting hole (6) is opened on the surface layer silicon nitride layer.
10. Ultrasensitive nanopore (8) chip according to claim 7 or 8 or 9, characterized in that said chip can achieve a background noise below 20 pA.
11. An ultrasensitive nanopore (8) analysis device comprising an ultrasensitive nanopore (8) chip according to any one of claims 6 to 9, characterized in that the device comprises a fluidic tank (9) filled with an ionic solution, an ultrasensitive nanopore (8) chip dividing the fluidic tank (9) into a cis-side chamber (11) and a trans-side chamber (12), an electrode (4) connected to the fluidic tank (9) solution or the chip, a current amplifier (2) and a power supply (1) connected in series with the electrode (4), a digital-to-analog conversion device connected to the current amplifier (2), and a computer (3).
12. A method for preparing a structure of an ultrasensitive nanopore (8) by using the ultrasensitive nanopore (8) analysis device of claim 11, which is characterized in that the ultrasensitive nanopore (8) is prepared by adopting an electrochemical etching method, salt solution is injected into chambers at two sides of a fluid tank (9), voltage in the range of 0-10V is directly applied to a nano film (7) through a power supply (1) and a current amplifier (2), the nanopore (8) is obtained by etching, and the size of the aperture can be controlled by controlling electrochemical reaction by controlling the size and duration of the applied voltage; and (3) obtaining real-time data of transmembrane conductance of the nanopore (8), and stopping applying voltage when the conductance value reaches a target aperture corresponding value to finish the preparation process of the nanopore (8).
13. A method for rapid amino acid detection of nanopores (8) using the ultrasensitive nanopore (8) analysis device of claim 11, characterized in that the pore size of the nanopores (8) is in the range of 0.35-2nm, comprising the steps of:
1) Dissolving an amino acid sample in a buffer;
2) Adding an amino acid sample into the solution in the fluid tank (9), and enabling the sample to pass through the nano-pores (8) under the action of an electric field;
3) Recording a current perforation map by a current amplifier (2);
4) The detected amino acids are analyzed by analyzing the amperometric pattern and/or the relative amperometric block conductance decrease and/or the perforation time and/or the capture rate obtained by statistical analysis of the amperometric pattern.
14. A method for rapid protein sequencing of nanopores (8) using the ultrasensitive nanopore (8) analysis device of claim 11, wherein the pore size of the nanopores (8) is in the range of 1-5nm, comprising the steps of:
1) Designing and synthesizing a compound of a peptide chain and a nucleotide chain, a primer chain and a blocking chain, mixing and annealing the three substances according to a required proportion before each experiment to obtain a sample (10) to be detected;
2) Adding biological enzyme, deoxyribonucleoside triphosphates (dNTPs) and a sample (10) to be tested into the cis-side chamber (11);
3) Applying voltage, wherein a sample passes through the nano hole (8) under the action of an electric field, and a current perforation map is recorded through the current amplifier (2);
4) The protein sequence was resolved by analysis of the amperometric punch pattern.
15. The method of rapid protein sequencing of a nanopore (8) according to claim 14, wherein the biological enzyme comprises phi29 DNA polymerase, tth DNA polymerase, pfu HSDNA polymerase, T4 DNA polymerase, bst DNA polymerase, taq ii DNA polymerase, taq plus DNA polymerase, LA Taq DNA polymerase, ventR DNA polymerase, phusion DNA polymerase, KOD DNA polymerase, iproof DNA polymerase, hel308 helicase, T4 helicase, dnaB helicase, rep protein helicase, pcrA helicase, tte UvrD helicase, tth UvrD helicase, T4 Dda helicase, T4 gp41 helicase, recQ helicase, NS3HCV helicase, elF4A helicase, WRN helicase, cf helicase, LTag 1 helicase, T7 gp4 helicase, clo helicase.
16. An ultrasensitive nanopore (8) chip according to any one of claims 6 to 9 applied to nanopore (8) detection in amino acid, wherein the pore diameter of nanopore (8) is in the range of 0.35-2nm, realizing detection and differentiation of 20 natural amino acids.
17. An ultrasensitive nanopore (8) chip according to any one of claims 6 to 9, applied to detection of chemically modified amino acids after translation of nanopores (8), wherein the pore size of nanopores (8) is in the range of 1 to 5nm, allowing detection and differentiation of post-translational modified amino acids.
18. An ultrasensitive nanopore (8) chip according to any one of claims 6 to 9 for polypeptide detection, wherein the pore size of the nanopore (8) is in the range of 1-5nm, enabling detection of polypeptides and differentiation of polypeptide amino acid sequences.
19. An ultrasensitive nanopore (8) chip according to any of claims 6 to 9 for protein sequencing, wherein the pore size of the nanopore (8) is in the range of 1-5nm, and protein sequencing is achieved.
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| PCT/CN2023/088193 WO2024021686A1 (en) | 2022-07-29 | 2023-04-13 | Ultra-sensitive nanopore structure, chip, analysis device, amino acid detection method, protein detection method, and use |
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