CN104694649A - Nanopore sequencing method for nucleic acid molecules with low perforation rate and special nanopore device thereof - Google Patents
Nanopore sequencing method for nucleic acid molecules with low perforation rate and special nanopore device thereof Download PDFInfo
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Abstract
The invention discloses a nanopore sequencing method for nucleic acid molecules with low perforation rate and a special nanopore device thereof. The method comprises the following steps: adding to-be-detected nucleic acid molecules into a nanopore sequencing device filled with electrolyte, wherein the nanopore sequencing device comprises an electrolytic tank provided with a positive electrode and a negative electrode and a solid-state nanopore film which separates the positive electrode and the negative electrode of the electrolytic tank and covers an agarose gel layer; adding the nucleic acid molecules into a negative electrode chamber of the electrolytic tank; applying voltage between the positive electrode and the negative electrode during measurement to serve as a driving electric field of nucleic acid molecule perforation. The nanopore device consists of the solid-state nanopore film and the agarose gel layer covered by the film. According to the method disclosed by the invention, the signal to noise ratio of a measurement signal is not influenced, the motion speed of the nucleic acid molecules in the nanopores is effectively reduced, and the time resolution is improved; the high signal to noise ratio of the measurement signal is maintained, the problem that the signal to noise ratio is sacrificed to slow down DNA sequencing is solved, and a foundation is laid for realizing accurate DNA sequencing by utilizing solid-state nanopores; and the method is simple and easy to operate.
Description
Technical field
The present invention relates to the nanoporous sequence measurement of the low punching rate of a kind of nucleic acid molecule and special nanoporous device thereof, belong to testing of materials field.
Background technology
DNA single molecular detection analysis based on solid nano hole device is considered to one of technological line being hopeful most to realize third generation fast and low-cost Human genome order-checking (spending less than the 1000 dollars gene order-checkings realizing single people in 24 hours), become the focus that current investigation and application is explored, except Harvard University, Boston University, Brown University, Caltech, outside the research groups such as TU Delft polytechnical university, in October, 2009, IBM Corporation also announces that adding human genome of future generation surveys sequence ?1000 dollars plan, with the sequencing technologies based on nanoporous device for implementation method, the research of nanoporous is made to move towards to apply (D.Branton by fundamental research, et al.Nature Biotechnology26 (10), 1146 (2008), M.Zwolak and M.Di Ventra, Reviews of Modern Physics 80 (1), 141 (2008) .).Drive molecule can realize single-molecule detection based on nanoporous device and analysis ability through the hole of a nanoscale by electrophoresis in the solution.Can be analyzed fast a large amount of molecule by various means in the finite space of nanoporous, when high-polymer molecular is through nanoporous, the structural information of high-polymer molecular and the signal characteristic of detection have one-to-one relationship.Utilize this characteristic directly to characterize the single strand dna of many kilobases to length, avoid amplification or labelling experiment preparation link, make fast and low-cost DNA sequencing technology become possibility.The greatest difficulty of this technology tremendous development of current obstruction is that, under electric field driven voltage, DNA punching rate is too fast, beyond the resolving power of universal apparatus, thus cannot realize identifying the difference of single base.
Experimental technique comparatively conventional in the world at present adds a voltage at nanoporous two ends, pass through electric field driven, DNA molecular is made to pass through nanoporous from one end, hole electrophoresis, the ion(ic)current collected at external circuit there will be a decline suddenly, the unexpected drop-out value of electric current and residence time, can the biological information of corresponding DNA.But use electric field adjusting merely, the punching rate of DNA is too fast, substantially in the magnitude of base one microsecond, and the minimum resolution of instrument is 4 microseconds at present, that is, if want to distinguish the difference between different base, by existing means, temporal resolution is beyond one's reach.Improve temporal resolution, the aperture time of DNA molecular just must be made to extend, effectively slow down the movement velocity of DNA in hole.Slow down the punching rate of DNA, just likely realizes the resolution to different base.The solution salt concentration that the people such as the Amit Meller of U.S. Boston College fill by changing Cis and Trans chamber, can in silication nitrogen (SiN) nanoporous of 5nm, realize the perforation procedure (M.Wanunu of DNA molecular millisecond magnitude, W.Morrison, Y.Rabin, A.Y.Grosberg and A.Meller, Nature Nanotechnology, 5,160, (2010)).But they are unclear to the mechanism explain producing this phenomenon, and the nanoporous that they use is very little, and the interaction between DNA and nanometer hole wall may be worked, and this has a greatly reduced quality to the controllability of experiment.And the very little nanoporous of diameter implements very difficult in experiment, success ratio is very low.
The Jiali Li of University of Arkansas of the U.S. etc. are by adding glycerine in the solution, the aperture time of DNA can be increased, but requirement for experiment condition is very harsh, very high (D.Fologea, J.Uplinger, B.Thomas are required to experimental technique, D.S.McNabb, and J.L.Li, Nano Letters 5,1734 (2005)).And along with the increase of glycerine, causing solution viscosity factor to increase, DNA retardation of current value amplitude of boring a hole declines, and the signal to noise ratio decline of test like this, causes measuring error to increase.Can, by reducing voltage, DNA punching rate is slowed down, but loss of voltage cause current signal to reduce in addition, signal to noise ratio be deteriorated, and causes measurement very difficult.Can see that the way of the existing DNA punching rate that much slows down is all just likely realize on the basis of sacrificing signal to noise ratio, like this great difficulty be added to accurate measurement signal, be not easy to obtain accurately relevant biological information.
Summary of the invention
The object of this invention is to provide the nanoporous sequence measurement of the low punching rate of a kind of nucleic acid molecule and special nanoporous device thereof, the present invention ensure that under the prerequisite not affecting measurement signal signal to noise ratio, solid nano hole film is modified as deceleration device using sepharose, effectively slowed down the speed that nucleic acid molecule moves in nanoporous, to improve the temporal resolution of nanoporous device.
The nanoporous sequence measurement of the low punching rate of nucleic acid molecule provided by the invention, comprise the steps: nucleic acid molecule to be measured to join to fill in the nanoporous sequencing device of electrolytic solution, described nanoporous sequencing device comprises: the electrolyzer being provided with positive pole and negative pole and the one side separating described electrolyzer positive pole and negative pole are coated with the solid nano hole film of sepharose layer; Described nucleic acid molecule is placed in the cathode chamber of described electrolyzer; During mensuration, between positive pole and negative pole, apply voltage, as the driving electric field of nucleic acid molecule perforation.
Above-mentioned method, described nucleic acid molecule can be DNA, RNA or peptide nucleic acid(PNA), and described DNA can be the DNA of strand or the DNA of double-strand.
Above-mentioned method, the preparation method of sepharose layer, comprises the steps: the sepharose aqueous solution to be coated in the one side of described solid nano hole film, namely obtains described sepharose layer after the described sepharose aqueous solution solidifies; Detailed process, for will cool after sepharose powder and deionized water Hybrid Heating, namely obtains sepharose solution, by transfer flow, can realize the surface-coated to described solid nano hole film.
In the present invention, described solid nano hole thin film coated has the one side of described sepharose layer can be relative with the negative or positive electrode of electrolyzer; Preferably relative with the negative pole of electrolyzer, namely described sepharose layer is in cathode chamber.
Above-mentioned method, the mass percentage concentration of the described sepharose aqueous solution can be 0.5 ~ 2%, specifically can be 1%, and the thickness that the described agarose that described solid nano hole film applies coagulates layer can be 10 ~ 100 μm, specifically can be 100 μm.
Above-mentioned method, described sepharose layer uniform fold is on the film of described solid nano hole.
Above-mentioned method, the diameter of the nanoporous of described solid nano hole film is 10 ~ 20nm, and specifically can be 10nm, hole depth is 20 ~ 100nm, specifically can be 60nm;
Described nucleic acid molecule to be measured in mensuration process is subject to the decelerating effect of the grid of the outer described sepharose layer of nanoporous simultaneously when entering the nanoporous on the film of described solid nano hole.
Above-mentioned method, described voltage can be 100 ~ 300mV;
The volumetric molar concentration of described electrolytic solution can be 0.1 ~ 3.2mol/L, specifically can be 1.6mol/L, and described electrolytic solution can be Klorvess Liquid, sodium chloride solution or lithium chloride solution;
The pH value of described electrolytic solution can be 8 ~ 10, specifically can be 9.
Present invention also offers the special nanoporous device of above-mentioned method, it is made up of solid nano hole film and the sepharose layer covered on the film of described solid nano hole thereof.
Above-mentioned nanoporous device, the diameter of the nanoporous of described solid nano hole film can be 10 ~ 20nm, and specifically can be 10nm, hole depth can be 20 ~ 100nm, specifically can be 60nm;
The thickness of described sepharose layer can be 10 ~ 100 μm, specifically can be 100 μm;
The mass percentage concentration preparing the sepharose aqueous solution that described sepharose layer uses can be 0.5 ~ 2%, and specifically can be 1%, described sepharose covers on the film of described solid nano hole uniformly with same thickness.
The present invention still further provides a kind of method reducing nucleic acid molecule punching rate in nanoporous sequencing, comprises and separates with solid nano hole film the electrolyzer being provided with positive pole and negative pole; Described solid nano hole film is coated with sepharose layer.
Due to three dimensional network structure that sepharose layer is the porous be made up of sepharose fiber in the present invention, channel diameter average out to 200nm (during 1% mass concentration), therefore this grid is under the prerequisite not affecting ion current signal, can produce the DNA entering nanoporous via this grid and interact, thus realize the deceleration to DNA.And the nanoporous sequencing reported at present detects nucleic acid molecule and only has an electric field driven, to slow down DNA by reducing electric field action, greatly reducing measurement signal to noise ratio like this, being unfavorable for signal measurement.The introducing of gel decorative layer introduces nanoporous especially innovatively and interacts outward as the principle of molecule deceleration, and this has no precedent in report before.Utilize aperture deceleration principle to be introduce in nanoporous to interact, the method needs the aperture used to prepare extremely difficult, and controllability is bad, and aperture is also very easily blocked by DNA simultaneously before.Sepharose is slowing down DNA punching rate while as the benefit of deceleration device, maintain very high measurement signal to noise ratio, and can maintain higher DNA catch rate, simultaneously we can select diameter to be the nanoporous of about 10nm, effectively ensure that experiment success rate.
The present invention uses simple method, solid nano hole film is introduced sepharose layer as deceleration device, under the prerequisite not reducing measurement signal signal to noise ratio, use the silicon nitride solid nano hole of about 10nm, effective reduction DNA molecular passes the speed of nanoporous device, more than the order of magnitude that punching rate can be slowed down; Significantly improve the temporal resolution of work on hand, carry out DNA sequencing for using solid nano hole device and lay a solid foundation.Both high current signal signal to noise ratio can have been kept, can avoid again introducing the DNA molecular owing to using very little nanoporous (diameter is less than 5nm) to bring and the uncontrollable INTERACTION PROBLEMS of nanometer hole wall, and test simple, repeatability and controllability are all significantly increased than prior art.In addition, when the length of detected DNA molecular is longer, method slowing effect used in the present invention is more remarkable, can adapt to solid nano hole sequencing technologies well and read long requirement to DNA molecular, and the reading length improving DNA molecular just gene sequencing of future generation need one of direction captured; And the experimental installation of this kind of method is simple, controllability, repeatable high, signal to noise ratio is very high.
The present invention has the following advantages:
1, the problem that temporal resolution is low perplexs one of maximum obstacle of solid nano hole DNA sequencing just, under the prerequisite not affecting signal to noise ratio, using sepharose decorative layer as deceleration device, effectively slowed down the movement velocity of DNA molecular in nanoporous, substantially increases temporal resolution.
2, method of the present invention maintains higher measurement signal signal to noise ratio, introduce sepharose layer as after deceleration device, do not need by reducing voltage, DNA to be slowed down, such signal to noise ratio is not affected, and solves to sacrifice signal to noise ratio to the problem making DNA slow down.
3, method is simple, easy handling.Method of the present invention is very simple, cools, namely obtains sepharose solution, by simple transfer flow, can realize the finishing to solid nano hole film after sepharose powder and deionized water Hybrid Heating.This method favorable repeatability, is very beneficial to popularization, does not need complicated circuit layout and programdesign, and do not need to introduce complicated system and manufacture craft, not high to technical requirements, experiment success rate is very high, substantially increases conventional efficient.
4, the present invention effectively can reduce DNA movement velocity in nanoporous, while significantly improving nanoporous device time resolving power, effectively reduce the velocity fluctuation that DNA molecular moves in nanoporous, improve the time tolerance range of solid nano hole device detection DNA molecular, realize accurate DNA sequencing for utilizing solid nano hole and have laid a good foundation.
Accompanying drawing explanation
Fig. 1 is nanoporous sequencing device in the present invention, and wherein, Fig. 1 (a) is ion(ic)current measurement and nanoporous surface sepharose schematic diagram; Fig. 1 (b) is the schema modifying sepharose at solid nano hole film surface.
Fig. 2 is the embodiment of the present invention 1 middle punch conductance-aperture time distribution plan, wherein, Fig. 2 (a) is 1.6M KCl, 48.5kb DNA, gel is not being had to modify (open-hole) with under the 1% concentration agarose gel condition of modification, the perforation conductance-aperture time distribution plan under 300mV voltage respectively; Fig. 2 (b) is 1.6M KCl, 48.5kb DNA, and gel-free modifies (open-hole) with under the 1% concentration agarose gel condition of modification, and 300mV voltage profiles versus of lower aperture time schemes; Fig. 2 (c) is 1.6M KCl, 48.5kb DNA, and gel-free modifies (open-hole) with under the 1% concentration agarose gel condition of modification, the typical pattern comparison diagram of event of boring a hole under 300mV voltage.
Fig. 3 is the embodiment of the present invention 1 middle punch conductive test data plot, wherein, Fig. 3 (a) is 1.6M KCl, under the nano-porous thin film finishing condition of 1% concentration agarose gel, the DNA of 5kb, 10kb, 48.5kb to bore a hole the figure of statistical average value of conductance respectively under voltage is 100mV, 200mV, 300mV condition; Fig. 3 (b) 1.6M KCl, under nano-porous thin film surface nothing is modified (open-hole) and has been modified the condition of 1% concentration agarose gel, the figure of 5kb, 10kb, 48.5kb DNA Statistical Comparison of aperture time under voltage is 200mV condition.
Fig. 4 is at 1.6M KCl in the embodiment of the present invention 1, and under the nano-porous thin film finishing condition of 1% concentration agarose gel, 5kb, 10kb, 48.5kb DNA is the figure of 100mV, 200mV, 300mV condition mean value of lower aperture time respectively at voltage.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment, sepharose powder is purchased from Sigma Aldrich, CAS:9012-36-6.
The method of nucleic acid molecule punching rate is reduced in embodiment 1, nanoporous sequencing
1, chip device is prepared
Utilize high energy in transmission electron microscope to assemble electron beam and punch operation is carried out to device free standing structure film, prepare chip device.This which includes the micro-nano technology technique related in a series of conventional semiconductors complete processing, and relate to the nanoscale processing technology in a series of modern forward position.
Concrete grammar is as follows: first, and by (100) face 4 inch silicon wafer, the silicon oxide of growth 2 microns is taken up in order of priority on two sides, with the low stress SiNx of low-pressure chemical vapor deposition method deposition 150 to 200 ran.Then make lay photoetching mask plate, object is on the silicon chip of 4 inches, to make the little substrate period profile of a lot of 3mm × 3mm, and each 3mm × 3mm little substrate centre of figure has a diameter to be the circular transmission region of 584 μm.For ensureing that the later stage is separated the little substrate of each 3mm × 3mm on 4 inch silicon wafer, when ensureing to be separated scribing, feed is accurate, make again silicon chip breakage can not become random small pieces, add the deshed line of some printing opacities on each little substrate image border: the width of light transmission strip is 5 μm, and length is 20 μm.The concrete making parameter of lay photoetching mask plate is: plate-making size: 5 inches; Graphical distribution scope: Ф 100mm circular distribution; Crystal orientation bar: distance center 49mm, long 50mm, wide 0.8mm.Then utilize photoetching and reactive ion beam etching (RIBE) the silicon-dioxide of Si sheet one side and silicon nitride to be carved thoroughly according to the figure of lay photoetching mask plate, expose Si surface.Remove photoresist material subsequently, use KOH anisotropic etch, (40%KOH, 80 DEG C, 6 hours) corrosion silicon, corrodes and carries out along (111) face.The standard be corroded through is: after corrosion for some time after little pane printing opacity.Be oxidized silicon face under opticmicroscope very smooth, there is no island structure.So just achieve little pane (20 ~ 80 μm) the unsettled film that one side only has silicon-dioxide and silicon nitride, have silicon substrate to support below.Next, in order to be separated the little substrate of 3mm × 3mm from the large silicon chip of 4 inches, need to carry out dicing operation.Need to stick the powerful blue glue of one deck at silicon chip back side during scribing to protect, because after scribing, easily unsettled film is torn when tearing blue glue, so must properly protect.Way is: stick together with another protection silicon chip high-temperature hot wax melting waiting to draw silicon chip by high-temperature hot wax melting.Then indigo plant is adhesive in the back side of protection silicon chip, put scribing machine into and carry out scribing, the scribing degree of depth is 200 to 250 microns.After scribing, blue glue is torn.Coherent for hot wax melting silicon chip is placed on hot plate and heats, wax melts, and silicon chip is separated.Wax above with acetone rinsing totally.This step can ensure that smooth cleavage obtains the little substrate of a large amount of 3mm × 3mm.In general, the silicon chip of 4 inches, cleavage can obtain 800 little substrates of 3mm × 3mm.Afterwards little for 3mm × 3mm substrate is put into the thinning silicon nitride film thickness of hot phosphatase 11 60 DEG C heating, thinning speed is 1 ~ 5nm per minute, and be thinned to 70nm or other thickness needed, thickness can be monitored by ellipsometer.Afterwards in order to reduce expose silicon nitride film in the solution area to reduce capacitor noise, and reduce the possibility that silicon nitride film breaks in the solution, we use focused ion beam (FIB, DB 235, FEI) 2 microns of silicon oxide are etched, ion beam current 300pA, approximately etching 1 minute, can form the little pane of 1 ~ 2 μm × 1 ~ 2 μm × 1.5 μm below silicon nitride, 1.5 μm is the degree of depth.We obtain and also remain 500nm thick silicon oxide, the little pane of 1 ~ 2 μm × 1 ~ 2 μm at present.Then use RCA reaction, eroded by remaining 500nm thick silicon oxide, only remaining 1 ~ 2 μm × 1 ~ 2 μm little pane size, thickness is the unsettled film of silicon nitride of 70nm.Wherein RCA reaction is: NH
3h
2o:H
2o
2=1:1:5 (v/v), 70 DEG C are heated 10 minutes, remove the organic contamination on silicon chip, also in order to better infiltrate when next step BOE corrodes; BOE (HF:NH
4f:H
2o=1:2:3) corrode 6 minutes (erosion rate is 100nm/min), remaining 500nm silicon oxide can be removed and totally obtain unsettled silicon nitride film; HCl:H
2o
2: H
2o=1:1:5 (v/v/v), cleans 10 minutes by 70 DEG C, removes inorganic impurity.
2, solid nano hole film preparation
After 3mm*3mm chip device processes, put into transmission electron microscope (FEI Tecnai F30) and carry out punch operation, magnification adjusts to 520k-890k, bundle spot size is 1, electron beam gathers minimum or slightly large, about 5 minutes, diameter about 10nm can be obtained, the degree of depth is the nanoporous of 60nm.Before and after punching, specimen holder will be placed on (O in plasma clean instrument
2: Ar=1:3, v/v) inner cleaning one minute, remove organic contamination, i.e. obtained solid nano hole film.Be stored in vacuum drying oven for subsequent use.
3, the preparation of the solid nano hole film of sepharose layer is coated with
Flow process preparation shown in Fig. 1 (b), sepharose powder mixes with deionized water by 1:100 in mass ratio, is made into the sepharose solution that mass percentage concentration is 1%, is placed in beaker, heating in water bath to 80 degree Celsius, continues 5 minutes, then 10 μ l sepharose solution are drawn with pipettor, drip in the above-mentioned solid nano hole film prepared, (diameter of nanoporous is specially about 10nm, hole depth is specially 60nm) surface (Cis face), with glass stick rapid roller coating on surface before sepharose solution cooled and solidified, the drop of sepharose solution is made to be uniformly distributed in solid nano hole film surface, left at room temperature 10 seconds, now sepharose solution drips quick solidification, namely sepharose layer (thickness about 100 μm) is obtained, prepare the solid nano hole film being coated with sepharose layer.
4, ion current signal is measured
As shown in Fig. 1 (a), by a series of impregnation process polydimethylsiloxane (PDMS) pad, the solid nano hole thin-film package being coated with sepharose layer is entered in the electrolyzer be made up of polyether-ether-ketone (PEEK), electrolyzer is made up of Cis chamber (corresponding negative pole) and Trans chamber (corresponding positive pole), only connected by the nanoporous be coated with on the solid nano hole film of sepharose layer, without other connecting passages between two chambeies.Then to implantation concentration in two chambeies be the Klorvess Liquid of 1.6mol/L.The Tutofusin tris (English Tris by name) of the ethylenediamine tetraacetic acid (EDTA) (EDTA) containing 1mM in Klorvess Liquid and 10mM regulates pH to be 9.Two Ag/AgCl electrodes are used to insert respectively in Cis chamber and Trans chamber.Inject DNA molecular solution to Cis chamber, then from positive pole on load voltage (100mV-300mV), (Fig. 3 a), can utilize patch clamp amplifier system to provide ion(ic)current measurement signal like this.
5, the DNA molecular under different lengths DNA and different voltage slows down
As shown in Figure 2, average bore time 611 μ s during open-hole, average bore time 2229 μ s after modification sepharose; Conductance of boring a hole when open-hole and modification sepharose condition is substantially identical, and after modifying sepharose, the aperture time is significantly elongated, and event pattern more easily reads and analyzes.
As shown in Figure 3,5kb, 10kb, 48.5kb DNA conductance level of boring a hole under voltage is 100mV, 200mV, 300mV condition is respectively close, consistent with conductance level when not modifying sepharose (open-hole), prove that the present invention can not reduce the signal to noise ratio of DNA detection; Show that DNA molecular is 0.012 μ s/bp through the average bore speed without decorated nanometer hole (open-hole) under above-mentioned condition as calculated, average bore speed through the nanoporous modified on the solid nano hole film of sepharose is 0.1 μ s/bp, and the solid nano hole film modified through sepharose will improve nearly order of magnitude the aperture time.
As shown in Figure 4, to different lengths DNA molecular (5kb, 10kb, 48.5kb) (100mV under different voltage, 200mV, 300mV) carry out slowing down experiment, and with do not have the gel DNA molecular aperture time mean value modified under (open-hole) condition to do to contrast (relative aperture time), sepharose of the present invention is modified will improve nearly order of magnitude the aperture time, DNA molecular length is longer simultaneously, the relative aperture time is larger, illustrates that slowing effect is more remarkable.In addition, under same length and same voltage conditions, DNA molecular fluctuation of perforation rate when there being gel to modify is less, average bore Annual distribution is more concentrated, this effectively improves the time tolerance range of solid nano hole device detection DNA molecular, realizes accurate DNA sequencing have laid a good foundation for utilizing solid nano hole.
Claims (10)
1. the nanoporous sequence measurement of the low punching rate of nucleic acid molecule, comprise the steps: nucleic acid molecule to be measured to join to fill in the nanoporous sequencing device of electrolytic solution, described nanoporous sequencing device comprises: the electrolyzer being provided with positive pole and negative pole and the one side separating described electrolyzer positive pole and negative pole are coated with the solid nano hole film of sepharose layer; Described nucleic acid molecule is placed in the cathode chamber of described electrolyzer; During mensuration, between positive pole and negative pole, apply voltage, as the driving electric field of nucleic acid molecule perforation.
2. method according to claim 1, is characterized in that: described nucleic acid molecule is DNA, RNA or peptide nucleic acid(PNA).
3. method according to claim 1 and 2, it is characterized in that: the preparation method of sepharose layer, comprise the steps: the sepharose aqueous solution to be coated in the one side of described solid nano hole film, after the described sepharose aqueous solution solidifies, namely obtain described sepharose layer.
4. according to the method described in claim 3, it is characterized in that: the mass percentage concentration of the described sepharose aqueous solution is 0.5 ~ 2%, the thickness that the described agarose that described solid nano hole film applies coagulates layer is 10 ~ 100 μm.
5. the method according to any one of claim 1-4, is characterized in that: described sepharose layer uniform fold is on the film of described solid nano hole.
6. the method according to any one of claim 1-5, is characterized in that: the diameter of the nanoporous of described solid nano hole film is 10 ~ 20nm, and hole depth is 20 ~ 100nm.
7. the method according to any one of claim 1-6, is characterized in that: described voltage is 100 ~ 300mV;
The volumetric molar concentration of described electrolytic solution is 0.1 ~ 3.2mol/L;
The pH value of described electrolytic solution is 8 ~ 10.
8. the special nanoporous device of the method according to any one of claim 1-7, it is made up of solid nano hole film and the sepharose layer covered on the film of described solid nano hole thereof.
9. nanoporous device according to claim 8, is characterized in that: the diameter of the nanoporous of described solid nano hole film is 10 ~ 20nm, and hole depth is 20 ~ 100nm;
The thickness of described sepharose layer is 10 ~ 100 μm;
The mass percentage concentration preparing the sepharose aqueous solution that described sepharose layer uses is 0.5 ~ 2%.
10. reduce a method for nucleic acid molecule punching rate in nanoporous sequencing, comprise and separate with solid nano hole film the electrolyzer being provided with positive pole and negative pole; It is characterized in that: described solid nano hole film is coated with sepharose layer.
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| CN109000600A (en) * | 2018-07-25 | 2018-12-14 | 上海纳米技术及应用国家工程研究中心有限公司 | The devices and methods therefor of measurement and verifying membrane pore size |
| CN109680052A (en) * | 2019-01-14 | 2019-04-26 | 京东方科技集团股份有限公司 | The preparation method of nano-porous thin film, gene sequencing device and nano-porous thin film |
| WO2021000786A1 (en) | 2019-06-29 | 2021-01-07 | 清华大学 | Method for controlling speed of polypeptide passing through nanopore, and application thereof |
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| CN109000600A (en) * | 2018-07-25 | 2018-12-14 | 上海纳米技术及应用国家工程研究中心有限公司 | The devices and methods therefor of measurement and verifying membrane pore size |
| CN109680052A (en) * | 2019-01-14 | 2019-04-26 | 京东方科技集团股份有限公司 | The preparation method of nano-porous thin film, gene sequencing device and nano-porous thin film |
| CN109680052B (en) * | 2019-01-14 | 2022-07-22 | 京东方科技集团股份有限公司 | Nanopore film, gene sequencing device and preparation method of nanopore film |
| WO2021000786A1 (en) | 2019-06-29 | 2021-01-07 | 清华大学 | Method for controlling speed of polypeptide passing through nanopore, and application thereof |
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