CN102590314A - Method for decelerating nucleic acid molecules in solid nanopore - Google Patents
Method for decelerating nucleic acid molecules in solid nanopore Download PDFInfo
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- CN102590314A CN102590314A CN2012100398556A CN201210039855A CN102590314A CN 102590314 A CN102590314 A CN 102590314A CN 2012100398556 A CN2012100398556 A CN 2012100398556A CN 201210039855 A CN201210039855 A CN 201210039855A CN 102590314 A CN102590314 A CN 102590314A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
Abstract
The invention discloses a method for reducing perforation speed of nucleic acid molecules in a nanopore sequencing method. The method comprises the following steps of: adding the nucleic acid molecules to be measured into a nanopore sequencing device filled with electrolyte, wherein the nanopore sequencing device comprises an electrolytic cell provided with an anode and a cathode, and a solid nanopore film for separating the anode from the cathode of the electrolytic cell; placing the nucleic acid molecules to be measured in a cathode cavity of the electrolytic cell,; and when the nucleic acid molecules are measured, applying the voltage between the anode and the cathode, and introducing a reverse external field taking a pressure intensity external field as an electric field simultaneously. According to the method, the pressure intensity external field is reversely applied under the drive of an additional electric field, so that the speed of DNA (Deoxyribonucleic Acid) molecules is reduced when the DNA molecules pass through the solid nanopore, and the speed is reduced by about 50% to 80%, thus the time resolution of a DNA unimolecule detection technology is greatly improved. The method has very bright application prospect on a solid nanopore DNA molecule sequencing device.
Description
Technical field
The present invention relates to the retarding method of a kind of nucleic acid molecules in the solid nano hole.
Background technology
Dna single molecular detection analysis based on solid nano hole device is considered to be hopeful most one of technology path of realizing third generation fast and low-cost human gene order-checking (cost realizes single people's gene order-checking below 1000 dollars in 24 hours); Become present research and use the focus of exploring; Except that research groups such as Harvard University, Boston University, Brown University, California Institute of Technology, TU Delft polytechnical university; IBM Corporation also announces to add-1000 dollars of plans of human genome order-checking of future generation in October, 2009; With the sequencing technologies based on the nano-pore device is implementation method; Make the research of nano-pore begin to move towards to use (D.Branton, et al.Nature Biotechnology26 (10), 1146 (2008) by fundamental research; M.Zwolak and M.Di Ventra, Reviews of Modern Physics 80 (1), 141 (2008) .).Nano-pore is meant the mesoporous of diameter 5-10 nanometer on the nano thin-film.In electrolytic solution, pass nano-pore to realize single-molecule detection and analysis ability based on the nano-pore device through electrophoresis driving biomolecule such as DNA.In the finite space of nano-pore, can analyze fast a large amount of molecules through various means, when high-polymer molecular passed nano-pore, the structural information of high-polymer molecular and signals detected characteristic had one-to-one relationship.Utilize this characteristic directly the logarithm kilobase single strand dna of length is characterized, avoided amplification or labelling experiment to prepare link, make fast and low-cost dna sequencing technology become possibility.The greatest difficulty that hinders this technological tremendous development at present is that under electric field driven voltage, the DNA punching rate is too fast, has exceeded the resolution of all purpose instrument, thereby can't realize the difference identification to single base.
Experimental technique comparatively commonly used in the world at present is to add a voltage at the nano-pore two ends; Pass through electric field driven; Make dna molecular pass through nano-pore from the Kong Yiduan electrophoresis; The ion-conductance of collecting at external circuit fails to be convened for lack of a quorum and occurs one and descend suddenly, the unexpected drop-out value and the residence time of electric current, biological information that can corresponding DNA.But use electric field adjusting merely, the punching rate of DNA is too fast, basic magnitude base one microsecond; And the minimum resolution of instrument is 4 microseconds at present, that is to say, if want to distinguish the difference between the different bases; Through existing means, temporal resolution is to be beyond one's reach.Improve temporal resolution, the aperture time of dna molecular is prolonged, the movement velocity of DNA in the hole effectively slows down.The people such as Amit Meller of U.S. Boston College can realize the perforation procedure (M.Wanunu of dna molecular millisecond magnitude through changing the solution salt concentration that fill in Cis and Trans chamber in the SiN of 5nm nano-pore; W.Morrison, Y.Rabin, A.Y.Grosberg and A.Meller; Nature Nanotechnology; 5,160, (2010)).But they are unclear to the mechanism explain that produces this phenomenon, and the nano-pore that they use is very little, and the interaction between DNA and the nanometer hole wall may be worked, and this controllability to experiment is had a greatly reduced quality.And the very little nano-pore of diameter implements very difficulty in experiment, and success ratio is very low.People such as the Mark of Harvard University are through change the method for direction of an electric field fast; Dna molecular through nano-pore is captured once more, returns and pass once more nano-pore again, near the motion (M.Gershow of the control DNA of implementation part in the hole and the hole; J.A.Golovchenko; Nature Nanotechnology, 2,775 (2007)).But this method experiment condition is loaded down with trivial details, and success ratio is very low, and controllability is bad, very difficult realization practical value truly.The Jiali Li of U.S. University of Arkansas etc. can increase the aperture time of DNA through in solution, adding glycerine, but requirement for experiment condition is very harsh; Experimental technique is required very high (D.Fologea, J.Uplinger, B.Thomas; D.S.McNabb; And J.L.Li, Nano Letters 5,1734 (2005)).And, cause the solution coefficient of viscosity to increase along with the increase of glycerine, and DNA perforation retardance current value amplitude descends, and the signal to noise ratio (S/N ratio) of test descends like this, causes the measuring error increase.Can the DNA punching rate slowed down through reducing voltage in addition, but the voltage reduction causes current signal to reduce, the signal to noise ratio (S/N ratio) variation causes measuring very difficulty.
Summary of the invention
The purpose of this invention is to provide the method that reduces the nucleic acid molecules punching rate in a kind of nano-pore PCR sequencing PCR.
The method of reduction nucleic acid molecules punching rate provided by the present invention; Be based on existing solid nano hole PCR sequencing PCR; Comprise the steps: nucleic acid molecules to be measured is joined in the nano-pore sequencing device that fills electrolytic solution; Said nano-pore sequencing device comprises: be provided with anodal and the electrolytic cell of negative pole and the solid nano hole film of separating said electrolytic cell positive pole and negative pole; Said nucleic acid molecules to be measured places the negative pole chamber of said electrolytic cell, between positive pole and negative pole, applies voltage during mensuration; Its improvement is: introduce the reverse outfield of a pressure outfield as electric field during mensuration simultaneously, thereby the perforation that realizes nucleic acid molecules is slowed down.
Nucleic acid molecules described in the present invention comprises two strands or single stranded DNA, RNA and peptide nucleic acid.
The pressure that the pressure outfield of being introduced produces can be 0.5-2.5 standard atmospheric pressure.
During concrete mensuration, punching rate or aperture time and the concentration of electrolyte and the magnitude of voltage that can will reach according to institute, in above-mentioned pressure through the limited number of time test, definite best pressure that is applied.
During mensuration, the concentration of the electrolytic solution that is adopted is 0.1-3.2mol/L, and the pH value is 8-10; The voltage that is applied is 100-250mV.Said electrolytic solution can be the electrolytic solution that is adopted in the nano-pore PCR sequencing PCR.Electrolytic solution when adopting Klorvess Liquid as dna sequencing usually, all the other are such as NaCl, and LiCl is fine.
The diameter of nano-pore can be 10-20nm in the film of said solid nano hole, and hole depth can be 20-100nm.
Method of the present invention is simple; Under the prerequisite that does not reduce the measuring-signal signal to noise ratio (S/N ratio); Use the SiN solid nano hole about 10nm; Effectively reduce the speed that dna molecular passes the nano-pore device, improve the temporal resolution of work on hand, lay a solid foundation for using solid nano hole device to carry out dna sequencing.
A further object of the present invention is based on above-mentioned retarding method a kind of nano-pore sequencing device is provided.
This device comprises: be provided with anodal and the electrolytic cell of negative pole and the solid nano hole film of separating said electrolytic cell positive pole and negative pole, it also comprises an air supply system, and said air supply system links to each other through conduit with the anode chamber of electrolytic cell.
The diameter of nano-pore is 10-20nm in the film of said solid nano hole, and hole depth is 20-100nm.
Method of the present invention is under extra electric field drives; Through oppositely applying the pressure outer field action; The speed of dna molecular through the solid nano hole time is effectively reduced, realize about 50～80% deceleration, thereby improve the temporal resolution of dna single molecular detection technology greatly.This method both can keep high current signal signal to noise ratio (S/N ratio); Can avoid again introducing because dna molecular and the uncontrollable INTERACTION PROBLEMS of nanometer hole wall of using very little nano-pore (diameter is less than 5nm) to bring; And test simple, repeatability and controllability all be significantly increased than prior art.In addition; Through regulating suitable concentration of electrolyte; Voltage swing and pressure size; Can detect the perforation procedure that length is lower than the DNA short chain molecule of 1kb, this technology has significantly been liberated the length restriction that the solid nano hole is surveyed dna molecular, the DNA length that the same apertures nano-pore can't detect basically before can detecting.And one of direction that short chain dna molecular gene sequencing of future generation just need be captured.
The present invention compares and existing report to based on the solid nano hole dna molecular being surveyed; The advantage of concentrating is embodied in: 1, under the prerequisite that does not influence signal to noise ratio (S/N ratio); The introducing of the reverse pressure movement velocity of dna molecular in nano-pore that effectively slowed down improved the temporal resolution of existing report greatly.And the low problem of temporal resolution perplexs one of maximum obstacle of solid nano hole dna sequencing just, and conventional a lot of ways can't realize this purpose.
2, method is simple, easy operating.Compare the bibliographical information that other slow down DNA, the method that we use is very simple, only needs to buy a pressure and shows meter and a gas cylinder, also has through simple to connect, and can realize the introducing to pressure.This method favorable repeatability is beneficial to popularization very much, does not need complex circuit design and program design.Need not introduce complicated system and manufacture craft, not high to technical requirement, experiment success rate is very high, has improved conventional efficient greatly.
3, use this technology can detect the shorter DNA of length, and when not introducing pressure, the nano-pore in same aperture is difficult to detect the DNA below the 3kb.Utilization should technology, and we can successfully detect 3kb, 1kb, or even the perforation procedure of the DNA short chain molecule of 600bp.This has improved the investigative range of solid nano hole to DNA length greatly, has expanded its range of application, for realizing that early utilizing the solid nano hole that DNA is carried out single-molecule sequencing has laid good basis.
Description of drawings
Fig. 1 is the figure of the photo mask board in the used solid nano of the present invention hole; Wherein, (a) be the photo mask board figure of 4 inches silicon chip designs, (b) to the partial enlarged drawing of Fig. 1 a, what the white circular porose area was corresponding is the circular transmission region in each 3mm*3mm chip.(c), can know the circular transmission region of seeing corresponding to corresponding in each 3mm*3mm chip to the further enlarged drawing of 1b.(d) for convenient from 4 inches big silicon chips the single little chip of cleavage, and the scribe line of particular design is represented with white dashed line.
Fig. 2 is that nano-pore device (3mm*3mm chip) is made schematic flow sheet; (a) whirl coating; Exposure is developed, and removes photoresist; Going out figure (b) reactive ion beam etching (RIBE) carve to pass through the monox and the silicon nitride (c) of one side and removes photoresist (d) KOH solution anisotropy rot eating away silicon; Exposing the unsettled film of monox and silicon nitride (e), to use FIB to carve a degree of depth at monox be that little pane (f) the RCA reaction of 1.5 μ m will remain 0.5 μ m silicon oxide layer and remove, and it is big or small only to expose 2 μ m * 2 μ m fenestella lattice, and thickness is the unsettled film of the silicon nitride of 70nm.
Fig. 3 (a) gas current is measured with reverse pressure and is loaded synoptic diagram; (b) swimming pool and electrode charger figure; (c) pressure charger figure; (d) pressure shows meter.
Fig. 4 is perforation electric current-aperture time distribution plan of the DNA of 10kb for length, wherein, (a) does not add pressure, (b) loads the reverse pressure of atmospheric pressure.
Fig. 5 is the DNA of 10kb corresponding DNA aperture time T0 when not loading pressure for length; (the indigo plant circle is experimental data point with the graph of a relation that loads reverse pressure size with aperture time T ratio after the loading pressure; Blue line, red line correspond to simulation curve under the 1.6M salinity).
Embodiment
Through specific embodiment method of the present invention is described below, but the present invention is not limited thereto.
Experimental technique described in the following embodiment like no specified otherwise, is conventional method; Said reagent and material like no specified otherwise, all can obtain from commercial sources.
Embodiment,
1) preparation chip device
The purpose of this step is that preparation can be put into the solid nano hole device that transmission electron microscope is operated, and high energy convergence electron beam carries out punch operation to the device free standing structure film in the transmission electron microscope thereby realize utilizing, thereby realizes the nano-pore device.This is comprising the micro-nano processing technology that relates in a series of conventional semiconductors processing technologys, and has related to the nanoscale process technology in a series of modern forward positions.
Concrete grammar is following: at first, with 4 inches silicon chips of (100) face, 2 microns monox of growth is taken up in order of priority on the two sides, with the low stress nitride silicon about low-pressure chemical vapor deposition method deposition 150 to 200 nanometers.Make lay photoetching mask plate then, purpose is on 4 inches silicon chip, to make the little substrate period profile of a lot of 3mm * 3mm
(Fig. 1 a), the little substrate centre of figure of each 3mm * 3mm have a diameter be 584 μ m circular transmission region (Fig. 1 b, c).The little substrate that on 4 inches silicon chips, separates each 3mm * 3mm for the assurance later stage; Feed is accurate when guaranteeing to separate scribing; Make silicon chip can breakage not become small pieces at random again; The dash line that has added some printing opacities in each little substrate pattern boundaries: the width of light transmission strip is 5 μ m, and length is 20 μ m (Fig. 1 d).The concrete making parameter of lay photoetching mask plate is: plate-making size: 5 inches; Graphical distribution scope: Φ 100mm circular distribution; Crystal orientation bar: distance center 49mm, long 50mm, wide 0.8mm.Utilize then photoetching (Fig. 2 a) and reactive ion beam etching (RIBE) (Fig. 2 b) silicon dioxide of Si sheet one side is carved thoroughly according to the figure of lay photoetching mask plate with silicon nitride, expose the Si surface.Remove photoresist (Fig. 2 c) subsequently, use the KOH anisotropic etch, (40%KOH, 80 ℃, 6 hours) corrosion silicon, corrosion is carried out along (111) face.The corrosion standard of wearing is: behind the fenestella lattice printing opacity after corrosion a period of time.The oxidation silicon face is very smooth under the optical microscope, does not have island structure.So just realize simultaneously having only the unsettled film of little pane (20～80 μ m) of silicon dioxide and silicon nitride, had silicon substrate to make to support (Fig. 2 d) below.Next, for the big silicon chip from 4 inches separates the little substrate of 3mm * 3mm, need carry out the scribing operation.Need stick the powerful blue glue of one deck during scribing at the silicon chip back side and do protection,, tear unsettled film easily when tearing blue glue, so must properly protect because after scribing.Way is: melt wax treating that stroke silicon chip melts wax with another protection silicon chip with elevated temperature heat and sticks together with elevated temperature heat.Be adhesive in the protection silicon chip back to indigo plant then, put scribing machine into and carry out scribing, the scribing degree of depth is about 200 to 250 microns.After the scribing, tear blue glue.Melt the coherent silicon slice placed of wax to heat and on hot plate, heat, wax melts, and separates silicon chip.Top wax totally gets final product with acetone rinsing.This step can guarantee that smooth cleavage obtains the little substrate of a large amount of 3mm * 3mm.In general, one 4 inches silicon chip can cleavage obtain 800 little substrates of 3mm * 3mm.Afterwards the little substrate of 3mm * 3mm is put into 60 ℃ of heating of hot phosphatase 11 attenuate silicon nitride film thickness, attenuate speed is 1～5nm per minute, is thinned to 70 nanometers or other thickness that needs, and thickness can be through the ellipsometer monitoring.Afterwards in order to reduce the area that is exposed to silicon nitride film in the solution reducing capacitor noise, and reduce that silicon nitride film breaks in solution maybe, we use FIB (FIB; DB 235; FEI) 2 microns monox of etching, ion beam current 300pA, approximately etching is 1 minute; Can below silicon nitride, form the little pane of one 1～2 μ m * 1～2 μ m * 1.5 μ m, 1.5 μ m are degree of depth.We have obtained also remaining the 500nm thick silicon oxide at present, the little pane (Fig. 2 e) of 1～2 μ m * 1～2 μ m.Use the RCA reaction then, remaining 500nm thick silicon oxide is eroded, only remaining 1～2 μ m * 1～2 μ m fenestella lattice size, thickness is the unsettled film of the silicon nitride of 70nm (Fig. 2 f).Wherein the RCA reaction is: NH
3H
2O: H
2O
2=1: 1: 5 (v/v), 70 ℃ were heated 10 minutes, removed the organic contamination on the silicon chip, better soaked into during also for next step BOE corrosion; BOE (HF: NH
4F: H
2O=1: 2: 3) 6 minutes (corrosion rate is 100nm/min) of corrosion can be removed remaining 500nm monox and totally obtained unsettled silicon nitride film; HCl: H
2O
2: H
2O=1: 1: 5 (v/v/v),, 70 ℃, cleaned 10 minutes, remove inorganic impurity.
2) transmission electron microscope carries out the nano-pore making
After the 3mm*3mm chip device processes; Put into transmission electron microscope (FEI Tecnai F30) and carry out punch operation; Enlargement factor is adjusted to 520k-890k, and bundle spot size is: 1, and electron beam gathers minimum or big slightly; About 5 minutes, can obtain about diameter 10nm, the degree of depth is the nano-pore of 60nm.Before and after the punching, specimen holder will be placed on (O in the plasma clean appearance
2: Ar=1: 3, cleaned one minute in v/v), remove organic contamination.After the nano-pore element manufacturing is good, be stored in the vacuum drying chamber subsequent use.
3) ion current signal is measured
We go into Chip Packaging in the tiselius apparatus of being processed by PEEK (polyether-ether-ketone resin) with the PDMS pad through a series of impregnation processes then, and swimming pool is made up of Cis chamber and Trans chamber, only connect through nano-pore between two chambeies, do not have other interface channels.Implantation concentration is 1.6 moles every liter a Klorvess Liquid in two chambeies then.Contain the EDTA of 1mM and the Tris of 10mM (pH=8) in the solution.We use two Ag/AgCl electrodes to insert respectively in Cis chamber and the Trans chamber, apply 100mV voltage at the nano-pore two ends, and the Cis chamber connects negative pole, and the Trans chamber connects positive pole, and (Fig. 3 a).At first before not injecting dna molecular, what the patch clamp amplifier system that we use provided is gas current reference current signal, about several nA.The pore size of gas current reference value and nano-pore, the duct degree of depth is relevant with solion concentration.After obtaining stable benchmark gas current, we inject length to the Cis chamber is the dna molecular solution of 10kb.Because DNA is electronegative in solution, so DNA under electric field driven, is moved to the Trans chamber by the Cis chamber.Can observe the perforation incident of single DNA molecules this moment, and corresponding is the electric current decline suddenly on the reference current basis and replys.This wherein has two important parameters to be used for characterizing the single perforation incident of dna molecular, bores a hole electric current and aperture time.The perforation electric current of corresponding 10kbDNA molecule is 50～100pA, and aperture time mean value is about 300us.
4) reverse pressure loads and the deceleration of DNA in nano-pore
After having obtained normal dna molecular punch signal, introduce reverse pressure in the direction opposite with direction of an electric field.Concrete introducing method is: with conduit that the Trans chamber links to each other on be connected in the conduit that gas cylinder links to each other, make the pressure of gas cylinder generation be directed in the Trans chamber (Fig. 3 b, c).The registration of introducing pressure shows (Fig. 3 d) through barometer.Through regulating the pressure vacuum meter on the gas cylinder, just can regulate the pressure size that is introduced in the Trans chamber.When the pressure size is 1 atmospheric pressure, the aperture time that dna molecular is corresponding effectively elongated be 400us, speed slowed down 33.3% (Fig. 4 a, b).Further strengthen pressure to 1.5 atmospheric pressure, the aperture time is 500us, and the aperture time prolongs 66.7% (Fig. 5).One big advantage of this technology is: the introducing of pressure does not change magnitude of voltage, and ion current signal does not weaken so, has kept normal measurement signal to noise ratio (S/N ratio), and this is that report before is beyond one's reach.And a lot of reports before need be through the method for the regulation voltage or the coefficient of viscosity, and ion current signal will be weakened, thereby causes the reduction of signal to noise ratio (S/N ratio).Through regulating the size of reverse pressure, we accomplish that effective punching rate with dna molecular reduces, and under two atmospheric reverse pressure actions, reduction of speed can reach more than 1 times.This method operation is very simple, and clear principle does not influence normal measurement signal to noise ratio (S/N ratio), effectively prolongs the DNA aperture time, has promoted the temporal resolution of utilizing the solid nano hole that DNA is surveyed greatly.We have carried out analog computation to the perforation procedure of DNA under pressure action, fine (Fig. 5) that curve values that obtains and our experimental data point coincide.Because in nano-pore, the effect of reverse pressure can balance out a part of electric field force driving action, makes that the dna molecular movement velocity under single electric field force drives slowed down originally.
5) dna molecular of different length slows down
We have not only attempted under the different pressure, the deceleration of dna molecular.We also test the dna molecular of different length.To 3kbDNA, under two atmospheric pressure situation (solution concentration 1.6M, voltage 100mV), successful is increased to 100us with the DNA aperture time by 50us, has prolonged one times.For 1kbDNA because its length is shorter, utilize existing instrument temporal resolution, if do not add pressure, with the punch signal that is difficult to detect DNA because its aperture time be shorter than the minimum resolution of instrument.Applying under two atmospheric situation, the perforation procedure of 1kbDNA is successfully surveyed, and the average aperture time is 50us.This is another big advantage place of our technology, can successfully detect the shorter dna molecular of length, and this is to have the solid nano hole of reporting similar sized apertures now to be difficult to accomplish.
Claims (7)
1. reduce the method for nucleic acid molecules punching rate in the nano-pore PCR sequencing PCR; Comprise the steps: nucleic acid molecules to be measured is joined in the nano-pore sequencing device that fills electrolytic solution; Said nano-pore sequencing device comprises: be provided with anodal and the electrolytic cell of negative pole and the solid nano hole film of separating said electrolytic cell positive pole and negative pole; Said nucleic acid molecules to be measured places the negative pole chamber of said electrolytic cell, between positive pole and negative pole, applies voltage during mensuration; It is characterized in that: during mensuration, introduce the reverse outfield of a pressure outfield as electric field.
2. method according to claim 1 is characterized in that: said nucleic acid molecules is DNA, RNA or peptide nucleic acid.
3. method according to claim 1 and 2 is characterized in that: the pressure that the pressure outfield of being introduced produces is 0.5-2.5 standard atmospheric pressure.
4. according to each described method among the claim 1-3, it is characterized in that: the concentration of said electrolytic solution is 0.1-3.2mol/L, and the pH value is 8-10; Said voltage is 100-250mv.
5. according to each described method among the claim 1-4, it is characterized in that: the diameter of nano-pore is 10-20nm in the film of said solid nano hole, and hole depth is 20-100nm.
6. nano-pore sequencing device; Comprise: be provided with anodal and the electrolytic cell of negative pole and the solid nano hole film of separating said electrolytic cell positive pole and negative pole; It is characterized in that: said nano-pore sequencing device also comprises an air supply system, and said air supply system links to each other through conduit with the anode chamber of electrolytic cell.
7. nano-pore sequencing device according to claim 6 is characterized in that: the diameter of nano-pore is 10-20nm in the film of said solid nano hole, and hole depth is 20-100nm.
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