CN108485944A - The gene sequencing device and sequencing approach sprayed using pulse static - Google Patents
The gene sequencing device and sequencing approach sprayed using pulse static Download PDFInfo
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- CN108485944A CN108485944A CN201810249718.2A CN201810249718A CN108485944A CN 108485944 A CN108485944 A CN 108485944A CN 201810249718 A CN201810249718 A CN 201810249718A CN 108485944 A CN108485944 A CN 108485944A
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Abstract
The invention discloses a kind of gene sequencing device and sequencing approach sprayed using pulse static, device includes spraying flow path by the DNA molecular solution that liquid storage pool and thin film channel form；It is connected and inserted into the filamentary silver in liquid storage pool by electrostatic power sources, with electrostatic power sources, and the electrostatic circuit that the Pt extraction electrodes being connect with electrostatic power sources cathode form；Base measure loop and control loop.Sequencing approach is：The DNA molecular solution for flowing into liquid storage pool is sprayed by thin film channel；Electrostatic circuit applies driving voltage by the filamentary silver being inserted into liquid storage pool to DNA molecular；The injection orifices of thin film channel bottom end form continually changing conical cavities, and DNA molecular is discontinuously sprayed from cone mouth, forms impulse jet pattern；Different bases distinguish different bases by causing the variation of voltage when thin-film capacitor channel according to different voltages in DNA chain.Compared with prior art, the present invention is high to the discrimination of different bases, improves accuracy of detection, reduces detection time.
Description
Technical field
The present invention relates to gene sequencing, biomolecule detection technologies, more particularly to are surveyed using the gene of pulse static injection
Sequence device and sequencing approach.
Background technology
Nanometer, biology and information are considered as influencing three big science technologies of social development, and wherein nanosecond science and technology are prominent with it
The intercrossing and integration gone out, directly influences the development of biotechnology and information technology.The Human Genome Project, gene core
Piece, the diagnosis of individual character chemoattractant molecule, biological cloud computing all have close association with gene sequencing.For each bion,
Genome contains the hereditary information of entire organism.Gene sequencing technology can truly reflect the whole on a chromosome
Hereditary information, and then the complexity and diversity of genome can be comprehensively disclosed, therefore in life science, gene is surveyed
Sequence plays a highly important role.
Gene sequencing technology develops by now, has undergone three generations.First generation gene sequencing technology is mainly Sanger et al.
The classical dideoxy nucleotide end of proposition terminates the chemical degradation method that PCR sequencing PCR and Gilbert et al. propose, by cutting DNA
Be cut into the DNA chain of random-length, with specified chemical reagent demarcate base, then by gel electrophoresis and autoradiograph after, can
With according to the DNA sequence dna of the location determination testing molecule of electrophoresis band.Second generation gene sequencing technology is mainly shown as on a large scale
Parallel polymerization sequencing instrument, not only maintains the high accuracy of first generation sequencing technologies, but also greatly reduce gene sequencing
Cost simultaneously greatly improve sequencing speed.But still it is defective, it before changing sequencing substantially without needing to sample
The problem of being expanded.Third generation gene sequencing technology is nano-pore sequencing technology, it uses electrophoretic techniques, is driven by electrophoresis
Individual molecule realizes sequencing by nano-pore one by one.The technology be not necessarily to DNA cloning, fluorescent marker, and greatly save the time and
Money is spent.The operation principle of nano-pore gene sequencing is mainly that ion blocking current detecting principle and tunnel current detection method are former
Reason.Ion blocking electric current testing is that a container for filling buffer solution is divided by a nano-pore into two, in nano-pore both sides
Apply bias voltage, when DNA molecular passes through nano-pore, due to the physics occupy-place of DNA molecular, it will cause the resistance of nanochannel
Variation, to cause the variation of ionic current.Tunnel current detection method is to make two parallel poles in nano-pore inner wall both sides,
Since the radial dimension of nano-pore and the diameter of biomolecule are suitable, when biomolecule passes through, if it is possible to measure cross
To the electric current for flowing through biomolecule, in the case where electrode size is sufficiently small, what size of current reflected is the knot of the single bases of DNA
Structure feature.
Invention content
Goal of the invention：The object of the present invention is to provide a kind of gene sequencing device using pulse static injection and sequencing sides
Method, the invention can significantly improve gene sequencing efficiency and measurement accuracy.
Technical solution：The gene sequencing device sprayed using pulse static, including：
DNA molecular solution sprays flow path, including liquid storage pool and the thin film channel positioned at liquid storage pool bottom end；
Electrostatic circuit, including electrostatic power sources and electrostatic power sources anode are connected and inserted into the filamentary silver in liquid storage pool, and and electrostatic power sources
The extraction electrode of cathode connection；
Base measure loop, include detection DNA molecular different bases capacitor thin film channel, and with capacitor thin film channel
Voltmeter in parallel；
Serial communication controlling circuit, including computer, the voltmeter being connect by agreement with computer, signal generator and
Pico-ampere table.
Electrostatic circuit controls electrostatic power sources by signal generator and generates loop voltage.
Electrostatic circuit further includes pico-ampere table, the DNA molecular being ejected into detection on extraction electrode.
Liquid storage pool is made by wet-etching technology, the silicon nitride film silicon layer of two outgrowths of etching.
Thin film channel is made by focused ion beam sputtering liquid storage pool bottom end silicon nitride film.
There are insulating layer in capacitor thin film channel and extraction electrode both sides.
The channel dimension in capacitor thin film channel ranging from 0~5nm.
Use pulse static spray gene sequencing device sequencing approach for：Flow into liquid storage pool DNA molecular solution by
Thin film channel sprays；Electrostatic circuit applies driving voltage by the filamentary silver being inserted into liquid storage pool to DNA molecular；Thin film channel bottom
The injection orifices at end form continually changing conical cavities, and DNA molecular is discontinuously sprayed from cone mouth, forms DNA molecular impulse jet
Pattern；Different bases distinguish different alkali by causing the variation of voltage when thin-film capacitor channel according to different voltages in DNA chain
Base.
When the tip subtended angle maximum curvature of conical cavities, no DNA molecular sprays.
Operation principle：The present invention carries out gene sequencing using pulse static jet mode, and electrostatic spraying is a kind of using quiet
Principle of hydrodynamics makes the method that charged liquid is sprayed out of pipeline.By giving fluid in certain size hole to apply electric field and stream
Under ram compression power, fluid can be ejected from aperture, and the injection of " taylor cone " shape can be formed in aperture.In certain application voltage and stream
Under ram compression power, causes charge migration amount insufficient since flow velocity supply is very few, the imbalance of electric field force and surface tension can be caused, into
And it is reflected as the variation of " taylor cone " shape.It is " meniscus " structure when maximum curvature, is sprayed at this time without fluid, curvature gradually becomes
Hour forms taper nozzle, and fluid sprays at this time.A critical value is reached, surface tension is better than electric field force, and curvature becomes larger, cone
Shape is gradually condensed to " meniscus ".This pulse mode provides method to the abiotic formula feeding pattern of DNA single chain molecules, passes through
Change and applies voltage, thus it is possible to vary pulse frequency, to realize that pulse feed distance is consistent with DNA base length.In addition, in height
Under electric field strength, charged dna molecule is interfered since electric field force effect will be straightened, between base to be weakened, and is easy to capture detection
Signal.For gene order surveying method, using capacitance detecting method.When single base passes through nano-pore, its own electrically charged meeting of institute
The quantity of electric charge on nano-capacitors is caused to change, so as to detect the variation of voltage, by the electrically charged difference of base institute,
Caused voltage change also can difference, to realize DNA sequencing.
Advantageous effect：Compared with prior art, the present invention has the following advantages：(1) the non-life of pulse static injection is used
Object formula feeding mode, reduces detection time；(2) ion blocking electric current detecting method is compared, the present invention is surveyed using capacitor plate
DNA Via signals are measured, via electric current is easy to analyze signal up to nA ranks；(3) present invention is to DNA chain different bases
Discrimination it is high, greatly improve accuracy of detection；(4) the configuration of the present invention is simple, it is at low cost, it is easy to commercialization.
Description of the drawings
Fig. 1 is gene sequencing device overall schematic；
Fig. 2 is DNA detection method schematic diagrames；
Fig. 3 is liquid storage pool and thin film channel machining sketch chart；
Fig. 4 is capacitor thin film passageway machining schematic diagram；
Fig. 5 is DNA molecular injection apparatus structural schematic diagram.
Specific implementation mode
As shown in Figure 1, gene sequencing device includes the DNA molecular solution injection flow path being connected with DNA solution transfer pipeline e
A, DNA single chain molecules electrostatic circuit b, base measure loop c and serial communication controlling circuit d；Wherein DNA molecular solution injection stream
Road a, including liquid storage pool 2 and the thin film channel 3 positioned at 2 bottom end of liquid storage pool；Electrostatic circuit b, including electrostatic power sources 7 and electrostatic power sources 7 are just
Pole is connected and inserted into the filamentary silver 4 in liquid storage pool 2, and the Pt extraction electrodes 15 being connect with 7 cathode of electrostatic power sources；Electrostatic circuit b is logical
It crosses signal generator 8 and controls the generation loop voltage of electrostatic power sources 7；Base measure loop c, including capacitor thin film channel 11 and and capacitance
The voltmeter 10 in parallel of thin film channel 11, there are SiO in wherein 11 both sides of capacitor thin film channel2Insulating layer 12；Serial communication controlling is returned
Road d, including computer 9, the voltmeter 10, signal generator 8 and the pico-ampere table 6 that are connect by agreement with computer 9.
Wherein in the solution of DNA single chain molecules 13 liquid storage pool 2 made of the inflow MEMS micro Process of flow control valve 1；
7 anode of electrostatic power sources is applied the voltage in DNA solution, electrostatic power sources 7 is negative by being connect with the special filamentary silver 4 being inserted into liquid storage pool 2
Pole connects Pt extraction electrodes 15 to form high field, and DNA single chain molecules 13 are thin by the submicron-scale of 2 bottom end of liquid storage pool
When membrane channels 3 flow out, is sprayed under electric field driven, pass through 6 measure loop electric current of pico-ampere table；Different bases pass through work in DNA chain
For detect DNA Via signals tool gate oxide film layer capacitance channel 11 when, cause two lateral capacitances opposed polarity change draw
Different voltages variation is played, is obtained by the detection of voltmeter 10, the base sequence of DNA chain known to analysis.
Within the scope of voltage and velocity pressure, 3 bottom end of submicron-scale silicon nitride film channel can form " taylor cone " shape
Cavity 5 causes charge migration amount insufficient, leads to the imbalance of electric field force and surface tension, be reflected as since flow velocity supply is very few
The variation of 5 shape of cavity is " meniscus " structure when maximum curvature, is sprayed at this time without DNA molecular；Shape when curvature tapers into
At taper nozzle, at this time DNA molecular ejection.A critical value is reached, surface tension is better than electric field force, and curvature becomes larger, taper
It is gradually condensed to " meniscus ".Under the impulse jet pattern, DNA single chain molecules 13 are fed since electric field force effect is straightened
Mode feeds for abiotic formula.Wherein, electrostatic power sources 7 is adjusted for charge injection, the generation of electrostatic field and voltage.
As shown in Fig. 2, DNA single chain molecules include a lot of ATCG bases arranged in certain sequence, wherein A is that gland is fast
Purine, T are thymidine, and C is cytimidine, and G is guanine；Chains of the L between DNA single chain molecule adjacent bases is over long distances.DNA into
It is abiotic formula pulse feed to pattern, when electric field force is more than surface tension, a large amount of charge migrations to " taylor cone " face, tip
Curvature tapers into, and single DNA single chain molecule bases adenine A is sprayed from tip, and leading portion adenine A is pushed to enter gate oxide
Thin film channel 11 detects.Later, surface tension increased dramatically, and since charge is run out of with DNA molecular, surface tension is more than electric field
Power, tip curvature become larger, and " taylor cone " is by " point " change " blunt ", at this point, being filled again in cavity has enough charges, under promotion
One base thymine T is sprayed from tip.
As shown in figure 3, for the machining sketch chart of liquid storage pool 2 and submicron-scale silicon nitride film channel 3, in base silicon 18
Both sides deposition silicon nitride film 17, upper silicon nitride film reserve window by photo etched mask, are obtained by wet etching silicon 18
MEMS micro Process liquid storage pool 2.Submicron-scale thin film channel 3 is obtained by focused ion beam sputtering lower layer silicon nitride film, and
And be bonded with the Pt extraction electrodes 15 processed, when processing, should be noted that insulation, and reserve electrode lead portion.
Fig. 4 is the machining sketch chart of gate oxide capacitance thin film channel 11, and two layers of grid oxygen is gradually deposited in base silicon 18
Change layer 11 and SiO2Insulating layer 14, upper layer gate oxide thickness are 5nm, and lower layer's gate oxide thickness is 20nm.Lower substrate silicon
18 dig out trapezoidal channel from back side using wet etching, and it is thin that upper layer using focused ion beam sputtering obtains 5nm gate oxide capacitances
Membrane channels 11, when processing, reserve lead portion.
As shown in figure 5, wherein 2 be MEMS micro Process liquid storage pools, 3 be submicron-scale thin film channel, and 5 be " taylor cone " shape
Cavity, 11 be gate oxide film layer capacitance channel, and 14 be SiO2Insulating layer, 15 be Pt extraction electrodes, and 17 be silicon nitride film, 18
For base silicon.Submicron-scale thin film channel 3 sputters 2 bottom end silicon nitride of MEMS micro Process liquid storage pool by focused ion beam FIB
Film obtains；Gate oxide capacitance thin film channel 11 using deposit gate oxide film layer obtain, channel dimension in 5nm hereinafter,
Both sides should have insulating layer, reserve lead portion using photo etched mask, constitute measure loop；Pt extraction electrodes 15 are by insulating layer
Deposit obtains on 14, and lead portion is reserved using photo etched mask.
Claims (9)
1. a kind of gene sequencing device sprayed using pulse static, it is characterised in that：Including：
DNA molecular solution sprays flow path (a), including liquid storage pool (2) and the thin film channel (3) positioned at liquid storage pool (2) bottom end；
Electrostatic circuit (b), including electrostatic power sources (7) and electrostatic power sources (7) anode are connected and inserted into the filamentary silver (4) in liquid storage pool (2),
And the extraction electrode (15) being connect with electrostatic power sources (7) cathode；
Base measure loop (c) includes the capacitor thin film channel (11) of detection DNA molecular different bases, and and capacitor thin film
The voltmeter (10) of channel (11) parallel connection；
Serial communication controlling circuit (d), including computer (9), the voltmeter (10) being connect by agreement with computer (9), letter
Number generator (8) and pico-ampere table (6).
2. the gene sequencing device according to claim 1 sprayed using pulse static, it is characterised in that：The electrostatic returns
Road (b) controls electrostatic power sources (7) by signal generator (8) and generates loop voltage.
3. the gene sequencing device according to claim 2 sprayed using pulse static, it is characterised in that：The electrostatic returns
Road (b) further includes pico-ampere table (6), the DNA molecular being ejected into detection on extraction electrode (15).
4. the gene sequencing device according to claim 1 sprayed using pulse static, it is characterised in that：The liquid storage pool
(2) by wet-etching technology, the silicon nitride film silicon layer of two outgrowths of etching is made.
5. the gene sequencing device according to claim 1 sprayed using pulse static, it is characterised in that：The film is logical
Road (3) is made by focused ion beam sputtering liquid storage pool (2) bottom end silicon nitride film.
6. the gene sequencing device according to claim 1 sprayed using pulse static, it is characterised in that：The capacitance is thin
There are insulating layer in membrane channels (11) and extraction electrode (15) both sides.
7. the gene sequencing device according to claim 1 sprayed using pulse static, it is characterised in that：The capacitance is thin
Channel dimension ranging from 0~5nm of membrane channels (11).
8. a kind of using as described in claim 1 using the sequencing approach of the gene sequencing device of pulse static injection, spy
Sign is：The DNA molecular solution for flowing into liquid storage pool (2) is sprayed by thin film channel (3)；Electrostatic circuit (b) is by being inserted into liquid storage pool
(2) filamentary silver (4) in applies driving voltage to DNA molecular；The injection orifices of thin film channel (3) bottom end form continually changing
Conical cavities (5), DNA molecular are sprayed from cone mouth to form impulse jet pattern；Different bases pass through thin-film capacitor channel (11)
Shi Yinqi voltage changes distinguish different bases according to different voltages.
9. the sequencing approach of the gene sequencing device according to claim 8 using pulse static injection, it is characterised in that：
When tip subtended angle (16) maximum curvature of the conical cavities (5), no DNA molecular sprays.
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| CN201810249718.2A CN108485944A (en) | 2018-03-21 | 2018-03-21 | The gene sequencing device and sequencing approach sprayed using pulse static |
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| CN103376275A (en) * | 2012-04-17 | 2013-10-30 | 国际商业机器公司 | Graphene transistor gated by charges through a nanopore for bio-molecular sensing and dna sequencing |
| US20160187290A1 (en) * | 2013-04-18 | 2016-06-30 | The Board Of Trustees Of The University Of Illinois | Method and apparatus for analyzing a target material |
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2018
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Patent Citations (4)
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|---|---|---|---|---|
| CN103376275A (en) * | 2012-04-17 | 2013-10-30 | 国际商业机器公司 | Graphene transistor gated by charges through a nanopore for bio-molecular sensing and dna sequencing |
| CN102899243A (en) * | 2012-09-21 | 2013-01-30 | 清华大学 | Graphene nanopore-microcavity-solid-state nanopore structure based DNA sequencing device and method |
| CN103193189A (en) * | 2013-02-21 | 2013-07-10 | 东南大学 | Multielectrode nanopore device for DNA detection and production method thereof |
| US20160187290A1 (en) * | 2013-04-18 | 2016-06-30 | The Board Of Trustees Of The University Of Illinois | Method and apparatus for analyzing a target material |
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