CN101799447A - Nano-pore tester for rapidly analyzing unimolecule - Google Patents
Nano-pore tester for rapidly analyzing unimolecule Download PDFInfo
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- CN101799447A CN101799447A CN 201010100860 CN201010100860A CN101799447A CN 101799447 A CN101799447 A CN 101799447A CN 201010100860 CN201010100860 CN 201010100860 CN 201010100860 A CN201010100860 A CN 201010100860A CN 101799447 A CN101799447 A CN 101799447A
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Abstract
The invention discloses a nano-pore tester capable of rapidly analyzing unimolecule, comprising a sample operation system and a signal processing system, wherein the sample operation system is used for guiding an analyzed sample to stretch out and pass through a nano-pore channel system at a constant speed without entanglement, and comprises a micro nano-pore channel system and a dynamic detection and regulation system; and the signal processing system is composed of a signal acquisition system, a signal analyzing system and a computer display system. The nano-pore tester provided by the invention can be applicable to gene super-fast sequencing, screening, separating and counting biological unimolecule, and screening and analyzing drug molecules at a nanometer order.
Description
Technical field
The present invention relates to a kind of nano-pore tester that can be used in the research of unimolecule multifrequency nature, this tester can be applied to gene supper-fast order-checking, be used for screening, separation, the counting of biologic single molecular (DNA, RNA, protein molecule etc.), can be used in the screening and the analysis of the drug molecule of nanometer scale size.
Background technology
At present, the monomolecular method of detection of biological, especially the common method that detects the dna molecular ordering has " Sanger method " and " shotgun method ", and method---the pyrophosphoric acid sequencing of the JonathanRothberg of " 454 Life Sciences Corp. " and its colleague invention.These sequence measurements, ultimate principle and method all are to sample " segmentation ", " order-checking ", " splicing " at last more earlier.Use these technology that a kind of gene of biology is checked order fully, its cost is very expensive, and required time is long.
The gene sequencing technology further develops with popularization and application need research and develop new sequence measurement, reduces the order-checking cost, improves order-checking speed.The innovation target of a new generation's gene sequencing technology mainly concentrates on the following aspects: (1) order-checking cost is low; (2) order-checking speed (surpassing) is fast; (3) accuracy height as a result; (4) to read base length big at every turn.At present, the research and development of new-generation sequencing technology are guiding theory with the single-molecule sequencing technology, and major technique has: synthetic sequencing technologies, the sequencing by hybridization technology connects sequencing technologies, the nano-pore sequencing technologies.Wherein, the nano-pore sequencing technologies of proposition such as the Daniel of Harvard University Branton, Jene Golovchenko and Dave professor Deamer of University of California is the at a low price efficient ultrafast full gene sequencing technology of the most potential realization.
But, utilize existing nano-pore method of production to make nano-pore, yield rate is not high; The thickness of nano-pore is generally also bigger, has about 10nm; And the nano-pore of being developed is an individualism, and promptly single nano-pore is fabricated on the center (protein alpha hemolysin biological nano hole also is to be positioned on the bigger insulation plane of area) of the bigger solid insulation film of area.Single nano-pore has many shortcomings in order-checking is used.At first, the dna molecular of perforation is the result who competes at random; Can not control an independent dna molecular and pass through nano-pore.Secondly, the dna molecular speed of perforation is very big, and is not uniform motion; Can not control the traverse velocity of dna molecular.These factors cause obtaining the accurate information of perforation dna molecular, thereby cause the erroneous judgement to gene order, seriously restrict the practicability development of nano-pore technology.
Chinese invention patent application " a kind of synthetic solid lateral nano-pore " (application number: 2007100729863) disclose a kind of synthetic solid lateral nano-pore, its objective is in order effectively to guide single DNA molecules not have and tangle, pass through evenly nano-pore, and to its accurate order-checking.Its weak point is the effective selection that lacks the single sample molecule.
Summary of the invention
The purpose of this invention is to provide and a kind ofly can select the single sample molecule, and guide its nothing to tangle, at the uniform velocity enter the nano-pore tester for rapidly analyzing unimolecule of nano-pore, be used for biomolecule such as DNA, RNA, protein, ribose, even the multifrequency nature research of cell and drug molecule etc., solve fast measuring to the single sample molecular property.
Implementation procedure of the present invention is as follows:
A kind of nano-pore tester for rapidly analyzing unimolecule, comprise sample manipulations system and signal processing system, wherein: the sample manipulations system is used to guide analyzed sample not have to tangle, trail, pass through evenly the nano-pore channel system, and it comprises micro-nano hole path system and detection of dynamic regulator control system; Signal processing system comprises signal acquiring system, Signal Analysis System and calculator display organization,
Micro-nano hole path system is made by high insulating material and forms, and it is selected single analytic sample, and guarantees that analytic sample does not have winding, passes in and out and pass through whole passage evenly;
Signal acquiring system is used for the passage blocking effect signal that causes when the real-time collecting sample is flowed through channel system under external force;
Signal Analysis System is used for the signal that the received signal acquisition system collects, and carries out data preparation, simultaneously, receives the detected sample of detection of dynamic regulator control system and transports information, according to analysis result, gives the detection of dynamic regulator control system with information feedback;
Calculator display organization in conjunction with the biophysics mathematical model, is determined the special properties of analytic sample according to the information that Signal Analysis System transmits;
The sample that detection of dynamic regulator control system acknowledge(ment) signal acquisition system collects transports information, according to the analysis result of Signal Analysis System, and the direction of motion and the speed of control sample.
Described micro-nano hole path system has adopted two sections sample access ways of " T " font structure, has deposited in initial sample intake passage and selecting monomolecular a plurality of detections regulation and control electrodes (general 2～5), be provided with the electrode of adjusting sample direction of motion in the passage, use software control multi-electrode cooperation group right.Micro-nano hole path system includes the sample reservoir, the sample recovery pond, and initial sample intake passage, sample intake passage, auxiliary sample output passage, sample output passage selects single DNA molecules and detection and regulation and control sample to transport the electrode and the nano-pore of state.Micro-nano hole path system includes the aperture and can be as small as 1.5 nanometers, the hole depth nano-pore to monatomic level.
In above-mentioned micro-nano hole path system, be integrated with many group (4～8 groups) electrodes, be provided with the movement position that is used for the detection analysis sample and the electrode of guiding sample direction of motion in the fork on the road of channel system.
The sample detection of dynamic of nano-pore detector system and regulator control system adopt integrated circuit (IC) chip to realize.
The signals collecting of nano-pore detector partly adopts accurate little current tester.
Detection of dynamic regulator control system, signal acquiring system, Signal Analysis System and calculator display organization are modularized integrated structure, and each module realizes the function interlock by software control.
Advantage of the present invention and good effect:
Nano-pore detector provided by the present invention system, the passage internal diameter is reduced to nanometer gradually by micron structure combines with multi-electrode, has guaranteed the selection to single analytic sample; The sample intake passage and the sample output passage of the long enough that directly links to each other with nano-pore (can to centimetre-sized) guarantee the disposable turnover nano-pore of long-chain biomacromolecule analytic sample, need not to cut apart; Long enough, the aperture that directly links to each other with nano-pore little to 50nm sample intake passage and sample output passage guaranteed sample molecule at the uniform velocity, do not have the turnover of tangling and pass through nano-pore; The little nano-pore to 1.5nm, the extremely monatomic level of hole depth in aperture detects the high accuracy of electric current when having guaranteed analytic sample; Sample manipulations system and signal processing system realize the function interlock by software, guaranteed the real-time monitoring of analytic sample motion state is regulated, and then realization are to the fast detecting of individual molecule special properties.
Description of drawings
Fig. 1 nano-pore tester for rapidly analyzing unimolecule The general frame of the present invention;
The micro-nano hole path system architecture of Fig. 2 synoptic diagram;
Fig. 3 Digital Signal Analysis and Processing software flow pattern.
1-sample reservoir 2-sample recovery pond 3-sample recovery pond
4-electrode 5-electrode 6-electrode
7-electrode 8-electrode 9-electrode
The initial sample intake passage 12-of 10-electrode 11-sample intake passage
13-assists sample output passage 14-sample output passage 15-nano-pore
Embodiment
Fig. 1 is a nano-pore tester system principle diagram provided by the invention.As shown in Figure 1, it comprises five chief components: micro-nano hole path system, signal acquiring system, Signal Analysis System, sample detection of dynamic regulator control system and calculator display organization.
Fig. 2 is micro-nano hole path system architecture synoptic diagram, it can be selected single DNA molecules and handle its directed movement, include sample reservoir (1), sample recovery pond (2), sample recovery pond (3), initial sample intake passage (11), sample intake passage (12), auxiliary sample output passage (13), sample output passage (14) is selected single DNA molecules and is detected and regulate electrode (4), electrode (5), electrode (6), electrode (7), electrode (8), electrode (9), electrode (10) and the nano-pore (15) that sample transports state.
Micro-nano hole path system includes the passage entity that insulating material forms and is deposited on wherein electrode, under the signal instruction control of information analysis system and dynamic regulation system, voltage on each electrode is adjusted in real time, the guiding sample moves in passage, and combine with passage, select single analytic sample, uniform motion in passage.The present invention adopted two sections sample access ways of " T " font structure, deposit a plurality of detections regulation and control electrodes of selecting single sample in initial sample intake passage, be provided with the electrode special of adjusting sample direction of motion etc. in the passage.
In preferred embodiment of the present invention, the microelectrode system of selecting to be provided with in the initial sample intake passage of single sample has 3,2 millimeters of the length of passage, and the aperture of passage is to dwindle, be taper structure gradually.Certainly, also can choose the passage that compositions such as passage that 1～2 or more electrode system and aperture equate are selected single sample.Electrode among Fig. 2 (10) is arranged on the fork on the road of channel system, and effect has two, and the one, be used for the movement position of detection analysis sample, the one, the direction of motion of guiding sample.
Signal processing system of the present invention comprises signal acquiring system, Signal Analysis System and calculator display organization.Signal processing system is used for the fluid in the passage is applied specific External Electrical Field, and the motion of sample in passage, certain characteristic that the nano-pore sample is passed through in analysis are handled in guiding.In preferred embodiment, the information handling system various piece adopts integrated circuit to realize separately.Detecting electrode (7), electrode (8), electrode (9) and electrode (10) and sample reservoir (1), electrode (4) in sample recovery pond (2) and the sample recovery pond (3), electrode (5), electrode (6) constitutes the loop, whether the monitoring analysis sample molecule is by this place, by the real-time acquired signal of information acquisition system electrode, and feed back to information analysis system, the result who obtains by the analytic system analysis, judge the motion conditions of specimen in passage, and detect and regulator control system sends instruction to passage, it is positive and negative to adjust each electrode, adjust direction of an electric field, the control sample moves in passage.
According to the information Control flow process of describing in the Digital Signal Analysis and Processing software flow pattern shown in Figure 3, realize the function interlock between signal acquiring system, Signal Analysis System, detection of dynamic regulator control system, the calculator display organization, nano-pore tester of the present invention is as follows to the order-checking experiment basic process of single DNA molecules:
At first an amount of damping fluid is injected in the sample reservoir (1), because the mobile and capillarity of liquid, damping fluid will pass through initial sample intake passage (11), initial sample output passage (13) continuously, enter sample recovery pond (2); Simultaneously, damping fluid by initial sample intake passage (11), sample intake passage (12), nano-pore (15), sample output passage (14), enters sample recovery pond (3) continuously at last.
Then, sample DNA solution is injected in the sample reservoir (1), connects electrode (4), electrode (5), the former is a positive electrode for negative, the latter.DNA enters initial sample intake passage (11) under extraneous electric field force effect, and moves to sample recovery pond (2) direction.Behind sample admission passage (11), the signal analysis control system is sent instruction to dynamic monitoring system, and detecting electrode (7) is open-minded, and forms the electric field that stops sample to continue directed movement with electrode (4), forbids that subsequent sample continues admission passage (11); Simultaneously, electrode (7) continues to guide sample to move in passage (11) with the electric field of electrode (5) composition.According to same principle, the information analysis control system is sent instruction to dynamic monitoring system, and spaced apart successively electrify electrode (8), electrode (9) are formed the electric field force of different size and Orientations, in passage (11), stop more movement of sample stage by stage, select single DNA molecules to cross electrode (9).
Subsequently, information acquisition system passes to the analysis and Control system with information, and the latter sends instruction to dynamic monitoring system, and the control system circuit is opened electrode (10), electrode (6), simultaneously closed electrode (5).The movement velocity that electric field between electrode (9) and the electrode (10) will slow down dna molecular, form the electric field leading sample between electrode (6) and electrode (9), the electrode (10) and enter sample intake passage (12), pass through nano-pore (15), sample output passage (14), enter recovery pond (3) at last.
In preferred embodiment of the present invention, the aperture＜50nm of sample intake passage (12), the best is～5nm, length long enough, greater than the length of sample.The single DNA molecules that enters in the sample intake passage (12) trails at the uniform velocity mobile.Sample enters sample output passage (14) after nothing enters and pass through nano-pore (15) kinkily under electric field action.In preferred embodiment of the present invention, nano-pore (15) aperture can be changed with different sample volumes, and the aperture of test single strand dna (gene) is 1.5nm, the thick 0.3nm in hole.Sample output passage (14) is identical with structure, the size of sample intake passage (12).
At last, as shown in Figure 3, information analysis system accurately passes to information display system with the information that the acquisition system transmission comes, and after the express-analysis of latter's process system, detection signal is changed into putting in order of analytic sample dna molecular constituent nucleotide.
More than narration only is the exemplary implementation example of the present invention, and according to essence spirit of the present invention and method, the structure of tester system is formed, and multiple variation and combination can be arranged, and they are limited by claims of the present invention.
Claims (10)
1. a nano-pore tester for rapidly analyzing unimolecule comprises sample manipulations system and signal processing system, it is characterized in that:
The sample manipulations system is used to guide analyzed sample not have to tangle, trail, pass through evenly the nano-pore channel system, and it comprises micro-nano hole path system and detection of dynamic regulator control system; Signal processing system comprises signal acquiring system, Signal Analysis System and calculator display organization,
Micro-nano hole path system is made by high insulating material and forms, and it is selected single analytic sample, and guarantees that analytic sample does not have winding, passes in and out and pass through whole passage evenly;
Signal acquiring system is used for the passage blocking effect signal that causes when the real-time collecting sample is flowed through channel system under external force;
Signal Analysis System is used for the signal that the received signal acquisition system collects, and carries out data preparation, simultaneously, receives the detected sample of detection of dynamic regulator control system and transports information, according to analysis result, gives the detection of dynamic regulator control system with information feedback;
Calculator display organization in conjunction with the biophysics mathematical model, is determined the special properties of analytic sample according to the information that Signal Analysis System transmits;
The sample that detection of dynamic regulator control system acknowledge(ment) signal acquisition system collects transports information, according to the analysis result of Signal Analysis System, and the direction of motion and the speed of control sample.
2. express-analysis unimolecule nano-pore tester according to claim 1, it is characterized in that: micro-nano hole path system has adopted two sections sample access ways of " T " font structure, has deposited in initial sample intake passage and select monomolecular a plurality of detections regulation and control electrodes, is provided with the electrode of adjusting sample direction of motion in the passage.
3. express-analysis unimolecule nano-pore tester according to claim 2, it is characterized in that: micro-nano hole path system includes the sample reservoir, the sample recovery pond, initial sample intake passage, sample intake passage, auxiliary sample output passage, sample output passage selects single DNA molecules and detection and regulation and control sample to transport the electrode and the nano-pore of state.
4. express-analysis unimolecule nano-pore tester according to claim 3 is characterized in that: micro-nano hole path system includes the aperture and can be as small as 1.5 nanometers, the hole depth nano-pore to monatomic level.
5. express-analysis unimolecule nano-pore tester according to claim 3 is characterized in that: be integrated with multi-group electrode in the micro-nano hole path system.
6. express-analysis unimolecule nano-pore tester according to claim 5 is characterized in that: be provided with movement position that is used for the detection analysis sample and the electrode that guides sample direction of motion in the fork on the road of channel system.
7. express-analysis unimolecule nano-pore tester according to claim 3 is characterized in that: use software control multi-electrode cooperation group right.
8. express-analysis unimolecule nano-pore tester according to claim 1 is characterized in that: the sample detection of dynamic of nano-pore detector system and regulator control system adopt integrated circuit (IC) chip to realize.
9. express-analysis unimolecule nano-pore tester according to claim 1 is characterized in that: the signals collecting of nano-pore detector partly adopts accurate little current tester.
10. express-analysis unimolecule nano-pore tester according to claim 1, it is characterized in that: detection of dynamic regulator control system, signal acquiring system, Signal Analysis System and calculator display organization are modularized integrated structure, and each module realizes the function interlock by software control.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102169088A (en) * | 2010-12-31 | 2011-08-31 | 清华大学 | Monomolecular detection method |
| CN102890474A (en) * | 2011-07-21 | 2013-01-23 | 中国科学院物理研究所 | System and method for controlling movement of single biomolecule in nanopore |
| CN105259229A (en) * | 2015-10-22 | 2016-01-20 | 清华大学 | Single-molecule analysis method for detecting medicine |
| WO2016119584A1 (en) * | 2015-01-30 | 2016-08-04 | 华东理工大学 | Preparation method for aeromonas lysine nano-pore channel and application thereof |
| CN106596645A (en) * | 2016-12-13 | 2017-04-26 | 中国科学院重庆绿色智能技术研究院 | Single-molecule-controlled graphene nanometer-pore DNA sequenator |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101225436A (en) * | 2007-01-19 | 2008-07-23 | 深圳大学 | Synthetic solid lateral nanometer pore |
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2010
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102169088A (en) * | 2010-12-31 | 2011-08-31 | 清华大学 | Monomolecular detection method |
| CN102890474A (en) * | 2011-07-21 | 2013-01-23 | 中国科学院物理研究所 | System and method for controlling movement of single biomolecule in nanopore |
| CN102890474B (en) * | 2011-07-21 | 2014-11-26 | 中国科学院物理研究所 | System and method for controlling movement of single biomolecule in nanopore |
| WO2016119584A1 (en) * | 2015-01-30 | 2016-08-04 | 华东理工大学 | Preparation method for aeromonas lysine nano-pore channel and application thereof |
| US10788450B2 (en) | 2015-01-30 | 2020-09-29 | East China University Of Science And Technology | Preparation method for aerolysin nanopore and application thereof |
| CN105259229A (en) * | 2015-10-22 | 2016-01-20 | 清华大学 | Single-molecule analysis method for detecting medicine |
| CN105259229B (en) * | 2015-10-22 | 2018-04-20 | 清华大学 | A kind of single molecule analysis method for detecting medicine |
| CN106596645A (en) * | 2016-12-13 | 2017-04-26 | 中国科学院重庆绿色智能技术研究院 | Single-molecule-controlled graphene nanometer-pore DNA sequenator |
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