CN103911432A

CN103911432A - Rapid screening method for monoclonal surface-displayed nucleic acid aptamers

Info

Publication number: CN103911432A
Application number: CN201410076295.0A
Authority: CN
Inventors: 杨朝勇; 朱志; 宋彦龄; 李聪; 邹远; 朱玲; 安源
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2014-03-04
Filing date: 2014-03-04
Publication date: 2014-07-09
Anticipated expiration: 2034-03-04
Also published as: CN103911432B

Abstract

The invention provides a rapid screening method for monoclonal surface-displayed nucleic acid aptamers, which relates to a nucleic acid aptamer screening technology. The rapid, high-efficiency and novel screening method for monoclonal surface-displayed nucleic acid aptamers is established. The method comprises the following concrete steps: (1) coupling an amino-modified forward primer onto the surfaces of NHS-modified microspheres through an NHS-amino reaction; (2) amplifying an oligonucleotide library onto the surfaces of the microspheres with coupled forward primer by using liquid-drop single-molecular microemulsion PCR in a monoclonal manner, wherein each microsphere only displays a nucleic acid sequence; (3) through dependent interaction between a monoclonal microsphere library and a target, visually inspecting the bonding degree of surface display sequences and the target; and (4) directly choosing monoclonal microspheres capable of bonding with target molecules and acquiring DNA sequences on the microspheres so as to obtain high-quality nucleic acid aptamers. The method is rapid, highly efficient and economic, achieves visual monitoring of molecular recognition, realizes rapid screening of nucleic acid aptamers in an enrichment library under the conditions of no cloning, sequencing and synthesizing and obtains nucleic acid aptamers with good selectivity and strong affinity.

Description

A kind of mono-clonal surface display aptamer rapid screening method

Technical field

The present invention relates to a kind of mono-clonal surface display aptamer rapid screening method for multiple target, belong to aptamer triage techniques development field.

Background technology

Aptamer, as the novel analysis of a class, diagnosis molecule, since occurring, has been subject to the extensive concern of researcher.Because of its many characteristic that are better than traditional protein monoclonal antibody, aptamer has become emerging research field, progressively replaces traditional antibody class diagnosis and biological technology products at aspects such as scientific research, medical diagnosis on disease and treatment reagent.(1.G.Mayer, The chemical biology of aptamers, Angew Chem Int Ed Engl[J] .2009,48,2672-2689; 2.M.Mascini, I.Palchetti, S.Tombelli, Nucleic acid and peptide aptamers:fundamentals and bioanalytical aspects, Angew Chem Int Ed Engl[J] .2012,51,1316-1332.) aptamer refer to from single stranded DNA/RNA library of synthetic that screening obtains can be with high specificity with high-affinity the single stranded oligonucleotide of being combined with target molecules.Aptamer forms special three-dimensional structure by weak force between hydrogen bond, Van der Waals force, hydrophobic interaction equimolecular, as hair clip, false knot, bulge loop, the G-tetramer etc., even affects its biological activity thereby identify specifically target material.The distinctive biochemical characteristic of aptamer itself makes it have many advantages in biomedical applications field, and as wide in target molecule scope, avidity and high specificity, synthetic modification fast and easy, molecular weight, good bio-compatibility, nontoxic, vitro stability is good etc.

Most aptamers of people's research at present and report all obtain by SELEX technology.SELEX technology is part index concentration phyletic evolution technology (Systematic Evolution of Ligands by Exponential Enrichment), mainly based on Darwinian evolutionsim thought: make a variation, select, copy, evolutionary process in test tube in simulating natural environment, filters out the aptamer that meets particular requirement in a short period of time.The method is proposed in nineteen ninety by Larry Gold, Jack Szostak and Gerald Joyce the earliest.(3.C.Tuerk, L.Gold, Systematic evolution of ligands by exponential enrichment:RNA ligands to bacteriophage T4DNA polymerase, Science[J] .1990,249,505-510; 4.A.D.Ellington, J.W.Szostak, Invitro Selection of RNA Molecules That Bind Specific Ligands, Nature[J] .1990,346,818-822.) be accompanied by impact and the application prospect that functional nucleic acid progressively expands at aspects such as biology, medicine and pharmacology, environment and disease detection, obtaining more functional nucleic acid how accurately, efficiently, economically also becomes the focus that people pay close attention to.In two ten years so far, the continuous studied person's innovation of SELEX technology, innovation, it is versatile and flexible that screening mode becomes especially, and the target molecule of screening has also been extended to the complicated target such as cell, tissue from metal ion, organic molecule, protein etc., has shown the powerful application space of this technology.

The most basic screening approach of SELEX is: first, jumbo random oligonucleotide sequence library (1013～1015 random oligonucleotide sequences) and target molecule are hatched jointly, by various separating pathways, target molecules-oligonucleotide complex is separated with unconjugated oligonucleotide sequence.Obtain the oligonucleotide sequence of being combined with target molecule by types of elution such as thermally denatures again, and these sequences are carried out to polymerase chain reaction (PCR), amplification generates secondary library, screens for lower whorl.So after the repeated screening of number wheel, obtain enriched library, this enriched library is carried out to bacterium mono-clonal order-checking, the oligonucleotide sequence that final acquisition can specific recognition target molecule, i.e. aptamer.The two ends that are used for the random oligonucleotide storehouse of screening are generally fixed sequence program, mate with the primer sequence of pcr amplification; Centre is the stochastic sequence of 15～60 bases, has almost contained all possible three-dimensional conformation, can filter out specific aptamer for any target molecule in theory.

SELEX technology is the process of a relative complex, and it is filtered into power and is subject to many factors impact, as the specific aim of the design in library, isolation technique and universality, the textural property of target molecule, type and the contained ionic species etc. of damping fluid.Not yet there is at present the standard SELEX screening method of report for any target.Conventionally protein or small molecules target must carry out the screening of 8-12 wheel, and cell, tissue etc. even needs 20 to take turns, and expends a large amount of manpowers and time, has also limited the development of aptamer simultaneously.

In recent years, some new aptamer triage techniqueses are continually developed.In SELEX screening process, effectively isolating to be the step of most critical with the sequence of target molecule identification.Some new technology for example based on capillary electrophoresis, flow cytometer, micro-fluidic chip, etc. separation method be suggested, for the screening of aptamer provides new developing direction.(5.S.D.Mendonsa, M.T.Bowser, In vitro evolution of functional DNA using capillary electrophoresis, J Am Chem Soc[J] .2004,126,20-21; 6.M.S.Raddatz, A.Dolf, E.Endl; et al.Enrichment of cell-targeting and population-specific aptamers by fluorescence-activated cell sorting; Angew Chem Int Ed Engl[J] .47,28,5190-5193; 7.X.Lou, J.Qian, Y.Xiao, et al.Micromagnetic selection of aptamers in microfluidic channels, Proc Natl Acad Sci U S A[J] .2009,106,2989-2994.) these new technology can effectively improve separation efficiency, reduce screening wheel number.On the other hand, after traditional SELEX end of processing, the enrichment storehouse finally obtaining need be carried out to bacterial clone order-checking, choose from tens to hundreds of candidate sequences by modes such as structural analyses again accounting example at most, there is secondary structure in conjunction with potential quality most as candidate sequence, with very strong artificial subjectivity.Then synthesize these sequences by chemical process, calculate separately the avidity of each sequence and target, finally obtain optimum aptamer.Subsequent processes is tediously long, take time and effort and cost higher.And this improvement on the one hand rarely has report.

We have reason to believe, along with the development and improvement of SELEX technology, aptamer will be taken on prior role at aspects such as clinical detection, medicine and bioseparation.

Summary of the invention

The problems such as existing aptamer screening method complexity, loaded down with trivial details, poor efficiency, costliness, the first object of the present invention be to provide one fast, simply, efficiently, mono-clonal surface display aptamer screening method cheaply.

The second object of the present invention is to provide that a kind of mono-clonal surface display aptamer screening method is for the fit screening of nucleus fast, simply, efficiently, at low cost.

The 3rd object of the present invention is to provide one, and mono-clonal surface display aptamer screening method is for the screening of albumen and micromolecular aptamer fast, simply, efficiently, at low cost.

Described mono-clonal surface display aptamer screening method comprises the steps: that (1) is coupled to by the amino reaction of NHS-the microsphere surface that NHS modifies by amido modified forward primer; (2) oligonucleotide library is had to the microsphere surface of forward primer by drop unit molecule micro emulsion PCR with the coupling of increasing of monoclonal form, on each microballoon, only show a kind of nucleotide sequence; (3), by mono-clonal microballoon storehouse and target dependent interaction, investigate visually the combination degree of surface display sequence and target; (4) directly select acquisition can and the mono-clonal microballoon of target molecules combination, obtain the DNA sequence dna on microballoon, can obtain high-quality nucleic acid fit.

In step (1), described microballoon is the agarose microbeads that NHS modifies.Also can utilize other coupling chemistry.

In step (2), described drop by vibrating, stirring or micro-fluidic chip mode generate.The formation of mono-clonal microballoon depends on oligonucleotide library is diluted to single molecules level, and guaranteeing only has 0.3-1 bar DNA in each drop.

Wherein, while adopting mode of oscillation to generate the water-in-oil drop that contains microballoon, preferably to screen required oligonucleotide library as pcr amplification template, add coupling to have the microballoon of forward primer and the required reagent of PCR as water, oil phase consist of 40% (w/w) DOW CORNING 5225C, 30% (w/w) DOW CORNING 749,30% (w/w) silicone oil Ar20.

Target of the present invention can be cell, protein or small molecules, while being small molecules or protein, preferably target is coupled to the surface of solid phase carriers such as microballoon so that observe and separate as target.

In step (3), preferably adopt the interaction of examining under a microscope mono-clonal microballoon storehouse and target.

In step (4), preferably directly choose the microballoon of being combined with target by wicking action with kapillary.

The invention has the advantages that: first, by micro emulsion drop PCR, each sequence in oligonucleotide library is showed in to mono-clonal microsphere surface with hundreds of copy, changes the oligonucleotide library of former SELEX timing into microballoon library; Secondly, the affinity interaction of each nucleotide sequence and target independently displays by clone's microballon separately, and the interaction of nucleotide sequence and target can directly be observed; Again, can directly obtain aptamer sequence from mono-clonal microballoon by selecting microballoon; In addition,, by changing washing dynamics, can regulate as required the affinity of obtained aptamer.Compared with traditional SELEX technology, the method is more quick, economical, efficient, realize molecular recognition visualizing monitor, and can under without clone, order-checking, comparison, synthetic condition, realize the fit rapid screening of enrichment storehouse amplifying nucleic acid, obtain that selectivity is good, aptamer that avidity is strong, open up a shortcut by screening for prosthesis, in the road of evolution function nucleic acid outward.

Accompanying drawing explanation

Fig. 1 is the chemical coupling (A) of NHS-agarose microbeads and amino forward primer; Be associated with the agarose microbeads of forward primer by being combined and carrying out coupling sign (B) with fluorescently-labeled forward primer complementary DNA; The fluorescence imaging of (D) and flow cytometry result after (C) and coupling before agarose microbeads coupling.

Fig. 2 is the aptamer screening of EpCAM albumen.(A) be combined with target cell KatoIII in the microballoon library of coupling oligonucleotide library; (B) microballoon of coupling forward primer is not combined with target cell; (C) sequence on select 7 mono-clonal microballoons and the bonding force of target protein are all better than the 5th enriched library of taking turns; (D) sequence on two microballoons that take out from the supernatant solution washing away is not combined with target protein.

Fig. 3 is the aptamer EpCAM-5 of order-checking acquisition and the combination situation of EpcAM albumen.Flow cytometry characterization result, aptamer EpCAM-5 can specific binding EpCAM-microballon (A), and be not combined with reference to Ni-microballon (B).Aptamer EpCAM-5 is 33 ± 3nM(C with the combination dissociation constant of EpCAM albumen).

Fig. 4 is the combination situation of aptamer EpCAM-5 and cell.Flow cytometry characterization result, aptamer EpCAM-5 not with the cell of not expressing EpCAM albumen as HEK-293(A) and Ramos(B) combination, and specifically in conjunction with the cell of EpCAM high expression level as T47D(C) and KatoIII(D).(E) fluorescence co-focusing characterization result, aptamer EpCAM energy specific recognition KatoIII cell, in conjunction with Ramos cell.

Fig. 5 is the experiment flow that flavacin is coupled to amido modified microsphere surface.

Fig. 6 is the aptamer screening of flavacin.(A) structural formula of flavacin; (B) the microballoon library of coupling oligonucleotide library and the combination of flavacin microballoon; (C) sequence on select 3 clone's microballoons and the bonding force of target flavacin are all better than the 5th enriched library of taking turns; (D) aptamer AFB ₁-1 with the binding constant (K of flavacin _d) characterize.

Embodiment

The coupling of embodiment 1NHS-agarose microbeads and amino forward primer

NHS-agarose microbeads (Hi Trap NHS-activated HP) is generally held in precaution of hydrolysis inactivation in 100% Virahol, needs to remove Virahol also with cold HCl activation before use.Weigh 0.2g NHS-agarose microbeads in 1.5mL centrifuge tube, add the cold HCl of 500mL0.1M, vibration mixes 10s, and the centrifugal supernatant of abandoning, repeats once.Add the cold ultrapure water of 500mL, vibration mixes 10s, and the centrifugal supernatant of abandoning, repeats fully to wash away residual HCl twice.Add 1M PBS damping fluid (10 ×) 100 μ L, amino forward primer (the 5 '-NH of 50 μ M ₂-AGCGTCGAATACCACTACAG-3 ') 90 μ L, be placed in room temperature rotation and mix the 8h that spends the night.The centrifugal supernatant of abandoning, adds ultrapure water repeatedly to clean microballoon, removes responseless amino forward primer forward primer.Forward primer-agarose microbeads good coupling is kept in 750 μ L ultrapure waters in 4 ℃, and every μ L storage liquid is approximately containing 1.7 × 10 ⁴individual forward primer-agarose microbeads.(Figure 1A)

Embodiment 2 utilizes flow cytometer and laser confocal fluorescence microscope checking coupling effect

Get the good forward primer-agarose microbeads of 5 μ L coupling, join the fluorescently-labeled forward primer complementary DNA of 15 μ L50 μ M (FAM-FP-cDNA, 5 '-FAM-CTGTAGTGGTATTCGACGCT-3 ') in the aqueous solution, then add 20 μ L binding buffer liquid (PBS damping fluid, MgCl ₂5mM, pH7.4) mix.Be placed in 95 ℃ of heating 5min on dry bath and make DNA sex change, wrap tinfoil lucifuge, room temperature rotation renaturation 2h, makes the complementary combination of primer of coupling in FAM-FP-cDNA and agarose microbeads.The solution having reacted is pushed to homemade filter core rifle head, filter out reaction solution, and clean microballoon 3 times with 100 μ L PBS, fully to wash the FAM-FP-cDNA that there is no combination off.Microballoon is released in 300 μ L PBS, and with flow cytometry analysis, numeration is greater than 3000 microballoons.Remaining sample is got to 10 μ L points and on slide, carry out laser confocal fluorescence microscope observation.(Figure 1B-D) calculates the concentration of forward primer on microballoon and is approximately 300amole/ microballoon.

Generation and the breaking method of embodiment 3 water-in-oil drops

Can adopt vibration, stirring or micro-fluidic chip mode to generate the water-in-oil drop that contains microballoon.For example, adopt the mode that manually vibration mixes to generate the water-in-oil drop that contains agarose microbeads.First prepare oil phase, prepare the round-bottomed flask of clean dried, add in proportion 40% (w/w) DOW CORNING 5225C, 30% (w/w) DOW CORNING 749,30% (w/w) silicone oil Ar20, magneton stirs 30min.In centrifuge tube, add the 150 μ L aqueous solution, add 6 μ L agarose microbeads (to be total to approximately 10 ⁵individual), vibration mixes, and covers thereon immediately 400 μ L mixing silicone oil, on Labnet vortex vibrator with intermediate speed vibration 5s.The water-in-oil drop having vibrated is creamy white.

Mix silicone oil water insoluble, be soluble in the organic reagent such as acetone, Virahol.Condition by cleaning solvent and centrifugal rotational speed is groped, and finally adopts successively polarity acetone, Virahol, water from low to high to clean water-in-oil emulsion, regains agarose microbeads thereby remove oil phase.Step is as follows: to the acetone that adds 10 times of volumes in water-in-oil emulsion, on vortex vibrator, mix, in whizzer, with the centrifugal 4min of rotating speed 500rpm, inhale and abandon upper organic phase, repeated washing once after; Add the Virahol of 10 times of volumes also by same condition washed twice; Finally, add the ultrapure water of 10 times of volumes to wash away Virahol, improve centrifugal rotational speed to 7000rpm.The agarose microbeads finally obtaining is kept in ultrapure water.

The formation of embodiment 4 mono-clonal microballoons and unit molecule micro emulsion drop PCR

The initial capacity of nucleic acid library is 10 ¹⁴, two ends are 20 nucleotide primers, centre comprises the stochastic sequence of 40 Nucleotide.Library is as follows: 5 '-AGCGTCGAATACCACTACAG-(N) ₄₀-CTAATGGAGCTCGTGGTCAG-3 '.There is the Ni microballon of EpCAM-His albumen to screen as target has carried out 5 tradition of taking turns for this nucleic acid library take coupling, take turns enrichment storehouse as template, template amount approximately 4 × 10 using the 5th of acquisition the ⁵bar, forward primer-agarose microbeads approximately 10 ⁵individual, free forward primer be on microballoon coupling primer total amount ten thousand/(calculate approximately 5 × 10 ⁷), free reverse primer is for excessive greatly, and water condition is as shown in table 1.

Table 1: the water condition of micro emulsion drop

By the 150 μ L water vibration 30s that prepare, rotation mixes 2min, and each component is fully contacted, and balance is distributed.On water, cover 400 μ L mixing silicone oil, on vortex vibrator with intermediate speed vibration 5s.The water-in-oil emulsion obtaining is divided in 200 μ L PCR pipes, and every pipe 100 μ L carry out PCR.PCR reaction conditions: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 30s, and circulation is taken turns in amplification 35; Last 72 ℃ are extended 5min, 4 ℃ of 3min eventually.Product is carried out breakdown of emulsion recovery and uses NaOH single stranded.Centrifugally remove most of supernatant liquor, add the NaOH of 100 μ L0.1M to hatch 1min the sex change of DNA double chain is dissociated, microballoon is pushed to self-control filter core rifle head, with 100 μ L binding buffer liquid filtration washing three times, obtain being connected with the microballoon of single stranded DNA.Carry out quantitatively with fluorescently-labeled reverse primer, obtain the concentration of DNA on microballoon and be approximately 70amole/ microballoon.

Embodiment 5 nucleic acid mono-clonals show that microballoon is for cell screening

Take Kato III as target cell, when the cell in culture dish grows to approximately 100,000, inhale and abandon cell culture fluid and change binding buffer liquid into, the single-chain nucleic acid of handling well is above shown to microballoon all adds, culture dish is placed in to shaking table, at room temperature hatch 30min.Carefully siphon away the liquid in substratum, add binding buffer liquid washing 5min, repeat 3 times.Tissue Culture Dish is put and examined under a microscope (Fig. 2 A-B).Most of nucleic acid shows that microballoon is sucked away, and only has the nucleic acid that sub-fraction avidity is higher to show that microballoon stays, and is combined on cell.Adjacent nucleic acid on cell is shown to microballoon sucks kapillary by wicking action, guarantee that every capillary sucks a nucleic acid and shows microballoon.In PCR pipe, add 10 μ L ultrapure waters, the liquid in kapillary is blown in PCR pipe with rubber suction bulb, in each PCR pipe, contain a nucleic acid and show microballoon.In each test tube, supply PCR reagent, carry out PCR reaction, and single stranded, obtain with fluorescently-labeled single stranded DNA, quantitatively concentrated.

With the above-mentioned fluorescently-labeled single stranded DNA of 100nM and approximately 5 × 10 ⁴30min is hatched in individual EpCAM-microballon room temperature rotation.Utilize self-control filter core rifle head binding buffer liquid washing 3 times, remove unconjugated DNA.Protein microsphere-DNA mixture is resuspended in 200 μ L binding buffer liquid and carries out flow cytometry.(Fig. 2 C-D), and record respectively the combination dissociation constant (table 2) of Clone1-7 and EpCAM-microballon

The combination dissociation constant of table 2:EpCAM Clone1-7 and EpCAM protein microsphere

Choose the wherein EpCAM Clone5 of dissociation constant minimum, carry out cloning and sequencing, obtain aptamer sequence EpCAM-5:5 '-AGC GTC GAA TAC CAC TAC AGC TCC GGG GTT TTT TGG GGT TTTTTC GGG GTT TTT TGG GGG CTA ATG GAG CTC GTG GTC AG-3 '.Fig. 3 is the combination situation of EpCAM-5 and EpCAM-microballon and Ni-microballon, and the combination dissociation constant that records EpCAM-5 and EpCAM albumen is 33 ± 3nM.Fig. 4 is the aptamer EpCAM-5 that obtains of order-checking and the combination situation of cell.Flow cytometry characterization result, aptamer EpCAM-5 not with the cell of not expressing EpCAM albumen as HEK-293(A) and Ramos(B) combination, and specifically in conjunction with the cell of EpCAM high expression level as T47D(C) and KatoIII(D).(E) fluorescence co-focusing characterization result, aptamer EpCAM energy specific recognition KatoIII cell, in conjunction with Ramos cell.

The aptamer screening of embodiment 6 flavacin

Using flavacin as micromolecular example, carry out the screening of flavacin aptamer.First flavacin is fixed on to bead surface.Fig. 5 is the experiment flow that flavacin is coupled to amido modified microsphere surface.First by carboxylated a group on flavacin, then react the oxime Acibenzolar form that generates flavacin with NHS, DCC.Simultaneously NHS-microballon generates amino-microballon with reacting ethylenediamine, then with the coupling of flavacin-oxime Acibenzolar, generation flavacin-microballon.

The initial capacity of nucleic acid library is 10 ¹⁴, two ends are 19 nucleotide primers, centre comprises the stochastic sequence of 40 Nucleotide.Library is as follows: 5 '-CAGCTTATTCAATTGTGCA-(N) ₄₀-AGATAGTAAGTGCAATCTA-3 '.Carry out the 5 tradition screenings of taking turns for this nucleic acid library take flavacin-microballon as target, taken turns enrichment storehouse as template, template amount approximately 4 × 10 using the 5th of acquisition the ⁵bar, as described in example 4, builds mono-clonal aptamer and shows microballoon.

Aptamer is shown to microballoon and flavacin-microballon at room temperature with culture dish hatch 30min.Carefully siphon away the liquid in culture dish, add binding buffer liquid washing 5min, repeat 3 times.Culture dish is put and examined under a microscope (Fig. 6 B).Most of nucleic acid shows that microballoon is sucked away, and only has the nucleic acid that sub-fraction avidity is higher to show that microballoon stays, and is combined with flavacin-microballon.These nucleic acid are shown to microballoon sucks kapillary by wicking action, guarantee that every capillary sucks a nucleic acid and shows microballoon.In PCR pipe, add 10 μ L ultrapure waters, the liquid in kapillary is blown in PCR pipe with rubber suction bulb, in each PCR pipe, contain a nucleic acid and show microballoon.In each test tube, supply PCR reagent, carry out PCR reaction, and single stranded, obtain with fluorescently-labeled single stranded DNA, quantitatively concentrated.

With the above-mentioned fluorescently-labeled single stranded DNA of 100nM and approximately 5 × 10 ⁴30min is hatched in individual flavacin-microballon room temperature rotation.Utilize self-control filter core rifle head binding buffer liquid washing 3 times, remove unconjugated DNA.Microballoon-DNA mixture is resuspended in 200 μ L binding buffer liquid and carries out flow cytometry.(Fig. 6 C), and record respectively the combination dissociation constant (table 3) of Clone1-3 and flavacin-microballon.

Table 3:AFB ₁the combination dissociation constant of Clone1-3 and flavacin microballoon

Choose the wherein AFB of dissociation constant minimum ₁clone1, carries out cloning and sequencing, obtains aptamer sequence A FB ₁-1:5 '-CAGCTTATTCAATTGTGCAGGGGGAGGGGGAGTGGTGGCTCGCGGTGCGTGGTGGC TGTAGATAGTAAGTGCAATCTA-3 '.And measure aptamer AFB ₁-1 is determined as 0.65 ± 0.11 μ M(Fig. 6 D with the combination dissociation constant of flavacin).

Claims

1. a mono-clonal surface display aptamer rapid screening method, is showed in microsphere surface by DNA library mono-clonal, realizes molecular recognition visualizing monitor; Described method comprises the steps: that (1) is coupled to by the amino reaction of NHS-the microsphere surface that NHS modifies by amido modified forward primer; (2) oligonucleotide library is had to the microsphere surface of forward primer by drop unit molecule micro emulsion PCR with the coupling of increasing of monoclonal form, on each microballoon, only show a kind of nucleotide sequence; (3), by mono-clonal microballoon storehouse and target dependent interaction, investigate visually the combination degree of surface display sequence and target; (4) directly select acquisition can and the mono-clonal microballoon of target molecules combination, obtain the DNA sequence dna on microballoon, acquisition high-quality nucleic acid is fit.

2. mono-clonal surface display aptamer rapid screening method as described in claim 1 and claim 2, it is characterized in that: in step (2), utilize vibration, stirring or micro-fluidic chip mode to generate the water-in-oil drop that contains microballoon, in each drop, comprise at the most an oligonucleotide templates.

3. mono-clonal surface display aptamer rapid screening method as claimed in claim 2, it is characterized in that: in step (2), adopt mode of oscillation to generate the water-in-oil drop that contains microballoon, it is to screen required oligonucleotide library as pcr amplification template, add coupling to have the microballoon of forward primer and the required reagent of PCR as water, oil phase consist of 40% (w/w) DOW CORNING 5225C, 30% (w/w) DOW CORNING 749,30% (w/w) silicone oil Ar20.

4. mono-clonal surface display aptamer rapid screening method as claimed in claim 1, it is characterized in that: in step (2), these are connected with to microballoon breakdown of emulsion, the recovery of nucleic acid, and double-stranded DNA is carried out to sex change single stranded, the final nucleic acid obtaining for screening is shown mono-clonal microballoon.

5. mono-clonal surface display aptamer rapid screening method as claimed in claim 1, is characterized in that: target is cell, protein or small molecules, while being small molecules or protein, target being coupled to surface of solid phase carriers and being convenient to observe and separate as target.

6. mono-clonal surface display aptamer rapid screening method as claimed in claim 1, is characterized in that: step (4) amplifying nucleic acid shows that mono-clonal microballoon and target hatch, and removes uncombined microballoon, chooses under the microscope the microballoon of combination.

7. mono-clonal surface display aptamer rapid screening method as claimed in claim 1, it is characterized in that: in step (4), select combination microballoon carries out pcr amplification in vitro, the aptamer that obtains high-affinity, directly checks order, and obtains sequence information.