CN102703454A - Nucleic acid aptamer capable of detecting myohemoglobin, microfluidic chip for screening and screening method and application - Google Patents
Nucleic acid aptamer capable of detecting myohemoglobin, microfluidic chip for screening and screening method and application Download PDFInfo
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- CN102703454A CN102703454A CN2012102149967A CN201210214996A CN102703454A CN 102703454 A CN102703454 A CN 102703454A CN 2012102149967 A CN2012102149967 A CN 2012102149967A CN 201210214996 A CN201210214996 A CN 201210214996A CN 102703454 A CN102703454 A CN 102703454A
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Abstract
The invention discloses a nucleic acid aptamer capable of detecting myohemoglobin and a derivative of the nucleic acid aptamer. The nucleotide sequence of the nucleic acid aptamer comprises a DNA segment shown by any one sequence in the sequences from sequence 1-sequence 5 shown in the description. The invention discloses a microfluidic chip which comprises a sample inlet, a sample outlet and a sample feeding channel. The sample feeding channel is divided by two narrow channels into front, middle and rear channels. The front channel is filled with reverse screening protein modified microspheres, and the middle channel is filled with positive screening protein modified microspheres, wherein the grain size of microspheres is greater than width of the narrow channels. The invention further discloses a screening method for screening the nucleic acid aptamer by using the microfluidic chip. The method comprises the main steps of optimizing a nucleic acid library, screening initially, purifying, and circulating. The detection method provided by the invention has the advantages of high affinity and specificity.
Description
Technical field
The present invention relates to a kind of proteic detection technique field, relate in particular to of screening method and the application of a kind of Protein Detection with aptamer, this aptamer.
Background technology
Myohaemoglobin (Myo) is a kind of oxygen set albumen that is present in the red tissue of Mammals flesh, is made up of 153 amino-acid residues, contains protoheme and oxyphorase homology.Under normal circumstances with the micro-form (male sex: 20～80 μ g/L; Women: 10～70 μ g/L) be present in the healthy subjects serum.When muscle injury, a large amount of myohaemoglobin just can enter into blood, causes its content in blood sharply to rise.The cardiac muscle myohaemoglobin is at present as the mark of early diagnosis myocardial infarction.In recent years, the relation of myohaemoglobin and myocardial infarction receives people's attention day by day.At present, detect myohaemoglobin the most conventional method still utilize the antigen antibody reaction of myohaemoglobin.Yet, now still exist certain limitation based on the detection of antibodies technology.Therefore, design a kind of high-affinity and high specific the identification myohaemoglobin nucleic acid molecular probe for after optimize its detection technique, avoid limiting to due to the antibody response providing the foundation.
Aptamer (aptamer); Be also referred to as nucleic acid and know body or nucleic acid aptamer; Be through a kind of novel in-vitro screening technology---phyletic evolution technology (SELEX) screening of index concentration aglucon obtains; The single strain oligonucleotide of the ability specific combination target material that from single strain oligonucleotide library at random, obtains can be that DNA also can be RNA, and length is generally 25～60 Nucleotide.The appearance of aptamer probe, having broken on the traditional sense about nucleic acid is the understanding of genetic information storage and transport vehicle, utilizes the variety of nucleic acid construct, can make the aptamer probe combine various target molecules efficiently, specifically.Because aptamer probe has advantages such as being prone to synthesize, being prone to modify, being prone to storage; At aspects such as the exploitation of proteinic analyzing and testing, biosensor, molecular switch, medical diagnosis treatments very big application prospect is arranged, this also uses for the concrete detection of myohaemoglobin prerequisite and basis is provided.
Summary of the invention
The technical problem that the present invention will solve is the deficiency that overcomes prior art; A kind of aptamer that can be used for detecting myohaemoglobin with high-affinity and high specific is provided; Also provide a kind of, also provide and utilize this aptamer to carry out the concrete application that myohaemoglobin detects based on the integrated micro-fluidic chip of positive and negative sieve and with the fit screening method of this chip screening aforementioned nucleic acid.
For solving the problems of the technologies described above; The technical scheme that the present invention proposes is a kind of aptamer that can be used for detecting myohaemoglobin; The nucleotide sequence of said aptamer comprises the dna fragmentation shown in any sequence in following sequence 1～sequence 5, and sequence 1～sequence 5 quilts are called after Myo40-7-23, Myo40-7-27, Myo40-7-34, Myo40-7-69 and Myo19-7-34 respectively:
Sequence 1 Myo40-7-23:
5’-?GACATCTATCGCCTCGATTTCCTTTGGTGAGATCGGCTCC-3’；
5’-?CCCTCCTTTCCTTCGACGTAGATCTGCTGCGTTGTTCCGA-3’；
5’-?ACGCACAATTCCTTGTCCAATTAGGAAATTCTACGCGGAT-3’；
5’-?CGAGTACTTCTTTGCTAGTTCGCGAGATACGTTGGCTAGG-3’；
5’?-?TTCCTTGTCCAATTAGGAA-3’。
The a certain position that the above-mentioned aptamer that can be used for detecting myohaemoglobin, the nucleotides sequence of said aptamer list can be by phosphorylation, methylate, amination, sulfhydrylation or isotropic substanceization.
The above-mentioned aptamer that can be used for detecting myohaemoglobin, the nucleotides sequence of said aptamer list and can be combined with vitamin H, digoxin, fluorescent substance, nano luminescent material or enzyme labelling.
As a total technical conceive, the present invention also provides a kind of nucleic acid aptamer derivative that can be used for detecting myohaemoglobin, and said verivate comprises any one in following four kinds:
(1) base A, T, G or C on the above-mentioned aptamer optional position are replaced as rare base and methylate behind purine, dihydrouracil or the xanthoglobulin nucleic acid aptamer derivative that obtains;
(2) phosphorothioate backbone that derives of the skeleton of the nucleotide sequence of above-mentioned aptamer;
(3) PNAG3 that is transformed into of above-mentioned aptamer;
(4) the lock nucleic acid that is transformed into of above-mentioned aptamer.
As a total technical conceive; The present invention also provides a kind of micro-fluidic chip that can be used for screening aptamer; Said micro-fluidic chip comprises injection port, outlet and is connected the sample introduction runner of the two; Said sample introduction runner is provided with two contraction sections to form two narrowed flow path; Two contraction sections are separated into preceding runner, middle runner, three stream sections of back runner with the sample introduction runner; Fill thousands of finishinges at least before said in the runner and the protein modified microballon of the proteic anti-sieve of anti-sieve is arranged to form anti-sieve unit; Filling at least thousands of finishinges in said in the runner has just to sieve and is proteicly just sieving protein modified microballon and just sieving the unit to form, the particle diameter of the protein modified microballon of said anti-sieve greater than be communicated with said before the width of narrowed flow path of runner and middle runner, the said particle diameter that is just sieving protein modified microballon greater than be communicated with said in the width of runner and the narrowed flow path of back runner.
In the above-mentioned micro-fluidic chip; As further improvement; Also be provided with elutriant fluid inlet and effluent flow channel on the said micro-fluidic chip in addition; Said effluent flow channel one end connects said elutriant fluid inlet, and the other end then is intersected on the said middle runner said effluent flow channel and sample introduction runner are communicated with.
In the above-mentioned micro-fluidic chip, preferably more than 150 μ m, the height of sample introduction runner is preferably more than 50 μ m for the width of said sample introduction runner.The height of the narrowed flow path that said contraction section forms is preferably 10 μ m～18 μ m.The preferred polystyrene microbeads of said microballon, the particle diameter of said microballon are preferably 20 μ m～40 μ m.
In the above-mentioned micro-fluidic chip; Said micro-fluidic chip is preferably the micro-fluidic chip of the above-mentioned aptamer of screening; Said anti-sieve albumen preferably includes at least a in bovine serum albumin (BSA), human serum albumin (HSA), the c reactive protein (CRP), and the said albumen that just sieving is myohaemoglobin.
As a total technical conceive, the present invention also provides the above-mentioned micro-fluidic chip of a kind of usefulness to screen the screening method of above-mentioned aptamer, may further comprise the steps:
(1) optimizes nucleic acid library:, primer sequence is sealed with in the secondary structure of avoiding participating in formation when combining with myohaemoglobin with predefined initial nucleic acid library, hatch respectively with the short sequence of two ends PCR primer sequence complementary; Said initial nucleic acid library is 5 '-FAM-GACAGGCAGGACACCGTAAC-N
40(capacity is preferably 10 for-CTGCTACCTCCCTCCTCTTC-3 '
14More than), the short sequence of said and two ends PCR primer sequence complementary is:
Primer RP-Biotin:5 '-Biotin-GAAGAGGAGGGAGGTAGCAG-3 ';
Primers F P-C:5 '-GTTACGGTGTCCTGCCTGTC-3 ';
(2) primary dcreening operation: the solution in the said initial nucleic acid library injection port from said micro-fluidic chip is injected; Make the solution in the said initial nucleic acid library said anti-sieve unit of at first flowing through; There is the nucleic acid molecule of avidity to be caught with said anti-sieve albumen in the initial nucleic acid library by the protein modified microballon of said anti-sieve; The residue nucleic acid library flows to the described unit that just sieving through narrowed flow path; Have the part nucleic acid molecule of avidity to combine with said myohaemoglobin in the residue nucleic acid library with myohaemoglobin on just sieving protein modified microballon, then through said sample introduction runner is washed, wash-out, collection obtains and myohaemoglobin bonded primary dcreening operation nucleic acid library;
(3) purifying: the primary dcreening operation nucleic acid library that step (2) obtains is carried out pcr amplification, and the affinity column that amplified production is filled through the Streptavidin microballon separates, and unwinds, forms time one-level nucleic acid library after the desalination through alkaline denaturation again;
(4) circulation: substitute said initial nucleic acid library and repeat above-mentioned step (2)～step (3) (circulation time should be modified microballon again in micro-fluidic chip at every turn) with the above-mentioned inferior one-level nucleic acid library that obtains, until the nucleic acid library that obtains including with myohaemoglobin high-affinity and high specific bonded aptamer.
In the above-mentioned screening method, the primer sequence of using during said pcr amplification is:
Primers F P-FAM:5 '-FAM-GACAGGCAGGACACCGTAAC-3 ';
Primer RP-Biotin:5 '-Biotin-GAAGAGGAGGGAGGTAGCAG-3 ';
The processing condition of said pcr amplification are preferably: 94 ℃ of 5min, and 94 ℃ of 30sec, 60.5 ℃ of annealing 30sec, 72 ℃ are extended 30sec, 10 circulations of increasing, last 72 ℃ are extended 7min.
As a total technical conceive, the present invention also provides a kind of above-mentioned aptamer or above-mentioned nucleic acid aptamer derivative perhaps to prepare the application in the test kit that detects myohaemoglobin at identification, detection myohaemoglobin.
Compared with prior art, the invention has the advantages that: utilize aptamer of the present invention, can discern myohaemoglobin quickly and efficiently, other homologous proteins are not had recognition function.Aptamer good stability, the nontoxicity that the present invention filters out, be easy to modify, and sequence length is shorter, the design of molecular probe after helping.
In addition; The present invention creatively will just sieve the unit and be integrated on the micro-fluidic chip with anti-sieve unit with respect to existing micro-fluidic chip SELEX method, and micro-fluidic chip of the present invention is not only simple in structure; Easy to make; And positive and negative sieve process can be carried out simultaneously, and further reduced the flow process of screening, improved screening efficiency.
Above technical scheme of the present invention will impel aptamer in the detection of myohaemoglobin, to bring into play enormous function.
Description of drawings
Fig. 1 utilizes the integrated micro-fluidic chip of positive and negative sieve to be used to screen the fit process flow sheet of myohaemoglobin specific nucleic acid in the embodiment of the invention.
Fig. 2 is the structural representation of micro-fluidic chip in the embodiment of the invention, and wherein, a representes protein modified microballon to the first contraction section of fixing anti-sieve; B representes that fixedly myohaemoglobin is modified microballon to the second contraction section; C representes to feed the library that is dissolved in binding buffer liquid and flows to the path; D representes to feed the path that flows to behind dcq buffer liquid and the elution buffer.
Fig. 3 is the variation diagram of detected every wheel surface plasma resonance (SPR) signal in the myohaemoglobin aptamer Cycle Screening process of the embodiment of the invention.
Fig. 4 is the RU value variation diagram of aptamer when concentration change of the myohaemoglobin that filters out in the embodiment of the invention.
Fig. 5 is that the fixed sequence program method detects the RU value variation diagram of myohaemoglobin when protein concn changes in the embodiment of the invention.
Fig. 6 is the fit specific investigation result to myohaemoglobin of embodiment of the invention amplifying nucleic acid.
Marginal data:
1, injection port; 2, outlet; 3, sample introduction runner; 31, preceding runner; 32, middle runner; 33, back runner; 4, narrowed flow path; 5, instead sieve protein modified microballon; 6, just sieving protein modified microballon; 7, elutriant fluid inlet; 8, effluent flow channel.
Embodiment
Below in conjunction with Figure of description and specific embodiment the present invention is further described.
Embodiment:
A kind of aptamer that can be used for detecting myohaemoglobin of the present invention; The nucleotide sequence of this aptamer comprises the dna fragmentation shown in any sequence in following sequence 1～sequence 5, and sequence 1～sequence 5 quilts are called after Myo40-7-23, Myo40-7-27, Myo40-7-34, Myo40-7-69 and Myo19-7-34 respectively:
Sequence 1 Myo40-7-23:
5’-?GACATCTATCGCCTCGATTTCCTTTGGTGAGATCGGCTCC-3’；
5’-?CCCTCCTTTCCTTCGACGTAGATCTGCTGCGTTGTTCCGA-3’；
5’-?ACGCACAATTCCTTGTCCAATTAGGAAATTCTACGCGGAT-3’；
5’-?CGAGTACTTCTTTGCTAGTTCGCGAGATACGTTGGCTAGG-3’；
5’?-?TTCCTTGTCCAATTAGGAA-3’。
The aptamer that can be used for detecting myohaemoglobin of present embodiment; The a certain position that its nucleotides sequence lists can be by phosphorylation, methylate, amination, sulfhydrylation or isotropic substanceization, and its nucleotides sequence lists also can be combined with vitamin H, digoxin, fluorescent substance, nano luminescent material or enzyme labelling.
The above-mentioned aptamer of present embodiment also can derive the following nucleic acid aptamer derivative that can be used for detecting myohaemoglobin:
(1) base A, T, G or C on the aptamer optional position of present embodiment are replaced as rare base and methylate behind purine, dihydrouracil or the xanthoglobulin nucleic acid aptamer derivative that obtains;
(2) phosphorothioate backbone that derives of the skeleton of the nucleotide sequence of the aptamer of present embodiment;
(3) PNAG3 that is transformed into of the aptamer of present embodiment;
(4) the lock nucleic acid that is transformed into of the aptamer of present embodiment.
A kind of micro-fluidic chip that can be used for screening aptamer of the present invention as shown in Figure 2; This micro-fluidic chip comprises injection port 1, outlet 2 and is connected injection port 1 and the sample introduction runner 3 of outlet 2; Sample introduction runner 3 is provided with two contraction sections to form two narrowed flow path 4; Runner 31, middle runner 32,33 3 stream sections of back runner before two contraction sections are separated into the sample introduction runner; The protein modified microballon 5 of anti-sieve that about 8000 finishinges of filling have anti-sieve albumen (comprising bovine serum albumin, human serum albumin and c reactive protein) in the preceding runner 31 is to form anti-sieve unit, and the protein modified microballon 6 of just sieve that filling about 8000 finishinges in the middle runner 32 has myohaemoglobin is just sieving the unit to form.Also be provided with elutriant fluid inlet 7 and effluent flow channel 8 on the micro-fluidic chip of present embodiment in addition, effluent flow channel 8 one ends connect elutriant fluid inlet 7, and effluent flow channel 8 and sample introduction runner 3 are communicated with.In the micro-fluidic chip of present embodiment, the width of sample introduction runner 3 is 200 μ m, and the height of sample introduction runner 3 is 70 μ m; The width of effluent flow channel 8 is 200 μ m, and the height of effluent flow channel 8 is 70 μ m; The height of the narrowed flow path 4 that contraction section forms is 15 μ m.The micro-fluidic chip of present embodiment is a glass material, adopts photoetching technique, wet etching and the high temperature bonding mode of standard to prepare.The microballon of present embodiment (the protein modified microballon of positive and negative sieve) is a polystyrene microbeads, and the particle diameter of microballon is 25 μ m.When making the micro-fluidic chip of present embodiment; The modification of microballon realizes through following operation: at first myohaemoglobin and anti-sieve albumen are hatched with polystyrene microbeads respectively; Just sieved protein modified microballon 6 and the protein modified microballon 5 of anti-sieve; To instead sieve protein modified microballon then and dispose, and make it fixedly be filled in the first contraction section place and form and just sieve the unit from injection port 1; To just sieve albumen (being myohaemoglobin) again and modify microballon and dispose, and make it fixedly be filled in the second contraction section place and form and just sieve the unit from elutriant fluid inlet 7.
As shown in Figure 1, a kind of method of screening the above-mentioned aptamer of present embodiment with the integrated micro-fluidic chip in the positive and negative sieve unit of present embodiment may further comprise the steps:
1. optimization nucleic acid library
1.1 design initial nucleic acid library: initial nucleic acid library capacity is 10
14More than, the synthetic two ends of design comprise 20 Nucleotide, centres, and to comprise the random nucleic acid sequence library of 40 Nucleotide following:
5’?-?FAM-GACAGGCAGGACACCGTAAC-N
40-CTGCTACCTCCCTCCTCTTC-3’。
1.2 optimize the initial nucleic acid library: will synthesize the above-mentioned strand initial nucleic acid library of good 1.34nmol and the primer RP-Biotin and the primers F P-C of 1.5 times of amounts and be dissolved in binding buffer liquid (20mmol/L Hepes; 120mmol/L NaCl; 5mmol/L KCl, 1mmol/L CaCl
2, 1 mmol/L MgCl
2, pH 7.35) in, behind 95 ℃ of sex change 5 min, slowly cool to room temperature.
Primer RP-Biotin:5 '-Biotin-GAAGAGGAGGGAGGTAGCAG-3 ';
Primers F P-C:5 '-GTTACGGTGTCCTGCCTGTC-3 '.
2. modify microballon and prepare micro-fluidic chip
Place the 1.5mL centrifuge tube 2.1 get the 0.5mL diameter and be the polystyrene microbeads of 25 μ m, remove supernatant, with borate buffer (0.1mol/L boric acid, pH are 8.5) flushing 3 times.
2.2 get in the microballon that the myohaemoglobin of 20 μ g～30 μ g adds step 2.1,25 ℃ of night incubation add store buffer liquid (0.1mol/L PBS, 10mg/mL BSA, 0.1% NaN with after the borate buffer flushing 2 times
3, 5% glycerine, pH are 7.4) and 4 ℃ of preservations.Get 25 ℃ of night incubation in the microballon that 10 μ g HSA, BSA, CRP add step 2.1 simultaneously in addition respectively, add store buffer liquid with after the borate buffer flushing 2 times.
2.3 prepare the micro-fluidic chip of a present embodiment as shown in Figure 2, sample introduction runner 3 two ends of micro-fluidic chip are respectively equipped with injection port 1 and outlet 2, are provided with the narrowed flow path 4 of two contraction sections to be formed for stopping that microballon passes through in the middle of the sample introduction runner 3; To pass through the protein modified microballon 5 of anti-sieve after step 2.2 is modified and just sieve protein modified microballon 6 is individually fixed in first, second contraction section by the order of an a～b shown in Figure 2 end.
3. primary dcreening operation
Like Fig. 1, shown in Figure 2, the optimization initial nucleic acid library that obtains in the step 1 is fed in the sample introduction runner 3 of above-mentioned micro-fluidic chip control flow velocity 0.5 μ L/min (through micro-injection pump control flow velocity); The solution in initial nucleic acid library is at first through anti-sieve unit; Wherein with anti-sieve albumen the nucleic acid molecule of avidity is arranged, remains nucleic acid library and then flow to and is just sieving unit (shown in the c among Fig. 2) owing to combines to be sieved protein modified microballon 5 and catch by counter, the part nucleic acid molecule that wherein avidity is arranged with myohaemoglobin with just sieve protein modified microballon 6 and combine; Align the sieve unit afterwards with dcq buffer liquid (binding buffer liquid; 0.05% Tween 20) with flow velocity 2 μ L/min flushing 30min (shown in the d among Fig. 2, dcq buffer liquid gets into from elutriant fluid inlet 7, and the effluent flow channel of flowing through successively 8 is washed with middle runner 32); Use elution buffer (20mmol/L Tris-HCl at last; The 4mol/L guanidinium isothiocyanate, 1mmol/L DTT, pH are 8.3) with flow velocity 2 μ L/min flushing 30min (elution process is with dcq buffer liquid); Behind flushing, wash-out, collection obtains and myohaemoglobin bonded primary dcreening operation nucleic acid library.
4. purifying
Single stranded DNA in the primary dcreening operation nucleic acid library of step 3 collection is carried out the PCR reaction;
Primers F P-FAM:5 '-FAM-GACAGGCAGGACACCGTAAC-3 ';
Primer RP-Biotin:5 '-Biotin-GAAGAGGAGGGAGGTAGCAG-3 '.
The PCR reaction conditions is following: 94 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 30s, 60.5 ℃ of annealing 30s, 72 ℃ are extended 30s, 10 circulations of increasing, last 72 ℃ are extended 7min.
The biotin labeled product of affinity column decoupled band that product behind the pcr amplification is had the agarose microballon of Streptavidin modification through prepackage; NaOH with 200mmol/L unwinds then, the strand that behind the desalination chromatogram purification, screens as next round time one-level nucleic acid library.
5. circulation
The inferior one-level nucleic acid library that obtains with step (4) substitutes the optimization nucleic acid library and repeats above-mentioned step 2～step 4; After screening process in; Flow velocity when progressively increasing flushing strengthens proof strength, and detects every signal variation of taking turns through the SPR appearance and confirm the screening terminal point; Can find out from detected result shown in Figure 3; Increase along with the screening wheel number; After adding the screening product of same concentrations, spr signal also constantly increases, and the product that the aptamer that every screening of taking turns is described is compared previous round has all increased the avidity with myohaemoglobin; Carried out 8 altogether and taken turns circulation, obtained including nucleic acid library at last with myohaemoglobin high-affinity and high specific bonded present embodiment aptamer.
At last, to the aptamer of the above-mentioned sequence of present embodiment screening gained and the dissociation constant K of myohaemoglobin
dMeasure.Adopting Biacore X bio-molecular interaction appearance, is working fluid with binding buffer liquid, carries out according to the following steps:
The myohaemoglobin that at first feeds 90 μ L, 20 μ M with the flow velocity of 5 μ L/min is modified in golden film surface, afterwards with 10 μ L/min flow velocitys feed 70 μ L at random the ssDNA chain seal; Feed the respectively screening gained aptamer solution of a series of concentration (3.125nM, 6.25nM, 12.5nM, 25nM, 50nM, 100nM, 200nM) of 70 μ L, the variation of writing down the RU value of each step successively with 10 μ L/min flow velocitys after handling well.The result of gained is mapped with Sigma Plot software; Go out the Kd value of five sequences of present embodiment with formula Y=BmaxX/ (Kd+X) The Fitting Calculation; Simultaneously with initial library as contrast; Detected result is seen Fig. 4, obtains the corresponding successively K of Myo40-7-23, Myo40-7-27, Myo40-7-34, Myo40-7-69, these 5 sequences of Myo19-7-34 according to above-mentioned equation model
dValue is respectively 6.19 ± 1.51 nM, 3.20 ± 0.47 nM, 5.47 ± 0.65 nM, 1.92 ± 0.25 nM and 3.70 ± 0.27 nM, and above-mentioned 5 sequences have all shown high-affinity to myohaemoglobin.
utilize the fixed sequence program method to detect myohaemoglobin and specificity is investigated.
At first feed 90 μ L, 0.05% BSA-Biotin solution and modify, feed 0.05% Avidin solution, feed biotinylated aptamer sequence afterwards with same flow velocity volume in golden film surface with the flow velocity of 5 μ L/min.After finishing is good, feed the respectively myohaemoglobin solution (3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM) of a series of concentration of 70 μ L, the variation of writing down the RU value of each step successively with 10 μ L/min flow velocitys.Map as shown in Figure 5 with Sigma Plot software the result of gained.Adopt the method for fixed sequence program that the specificity of each bar aptamer sequence is investigated; Aptamer modified good back feeds HSA, BSA, CRP, IgG (immunoglobulin G while) and the Myo solution of each 70 μ L, 500 nM successively with 10 μ L/min flow velocitys, and the variation of RU value of writing down each step is as shown in Figure 6.Can find out in conjunction with Fig. 5 and shown in Figure 6, after the aptamer sequence that filters out is fixed to golden film surface, can catches the myohaemoglobin in the solution effectively, and other reference proteins are not had signal response basically.
< 110>Hunan University
< 120>can be used for detecting myohaemoglobin aptamer, the screening with micro-fluidic chip and screening method and application
<160> 8
<210> 1
<211> 40bp
<212> DNA
< 213>artificial sequence
<400>
GACATCTATC?GCCTCGATTT?CCTTTGGTGA?GATCGGCTCC 40
<210> 2
<211> 40bp
<212> DNA
< 213>artificial sequence
<400>
CCCTCCTTTC?CTTCGACGTA?GATCTGCTGC?GTTGTTCCGA 40
<210> 3
<211> 40bp
<212> DNA
< 213>artificial sequence
<400>
ACGCACAATT?CCTTGTCCAA?TTAGGAAATT?CTACGCGGAT 40
<210> 4
<211> 40bp
<212> DNA
< 213>artificial sequence
<400>
CGAGTACTTC?TTTGCTAGTT?CGCGAGATAC?GTTGGCTAGG 40
<210> 5
<211> 19bp
<212> DNA
< 213>artificial sequence
<400>
TTCCTTGTCC?AATTAGGAA 19
<210> 6
<211> 20bp
<212> DNA
< 213>artificial sequence
<400>
GAAGAGGAGG?GAGGTAGCAG 20
<210> 7
<211> 20bp
<212> DNA
< 213>artificial sequence
<400>
GTTACGGTGT?CCTGCCTGTC 20
<210> 8
<211> 20bp
<212> DNA
< 213>artificial sequence
<400>
GACAGGCAGG?ACACCGTAAC 20
Claims (10)
1. aptamer that can be used for detecting myohaemoglobin, the nucleotide sequence of said aptamer comprises the dna fragmentation shown in any sequence in following sequence 1～sequence 5:
Sequence 1:
5’-?GACATCTATCGCCTCGATTTCCTTTGGTGAGATCGGCTCC-3’；
Sequence 2:
5’-?CCCTCCTTTCCTTCGACGTAGATCTGCTGCGTTGTTCCGA-3’；
Sequence 3:
5’-?ACGCACAATTCCTTGTCCAATTAGGAAATTCTACGCGGAT-3’；
Sequence 4:
5’-?CGAGTACTTCTTTGCTAGTTCGCGAGATACGTTGGCTAGG-3’；
Sequence 5:
5’?-?TTCCTTGTCCAATTAGGAA-3’。
2. the aptamer that can be used for detecting myohaemoglobin according to claim 1 is characterized in that: a certain position that the nucleotides sequence of said aptamer lists by phosphorylation, methylate, amination, sulfhydrylation or isotropic substanceization.
3. the aptamer that can be used for detecting myohaemoglobin according to claim 1 and 2 is characterized in that: the nucleotides sequence of said aptamer lists and is combined with vitamin H, digoxin, fluorescent substance, nano luminescent material or enzyme labelling.
4. nucleic acid aptamer derivative that can be used for detecting myohaemoglobin, said verivate comprises any one in following four kinds:
(1) base A, T, G or C on the aptamer optional position described in claim 1 or 2 or 3 are replaced as rare base and methylate behind purine, dihydrouracil or the xanthoglobulin nucleic acid aptamer derivative that obtains;
(2) phosphorothioate backbone that the skeleton of the nucleotide sequence of aptamer derives described in the claim 1 or 2 or 3;
(3) PNAG3 that aptamer is transformed into described in the claim 1 or 2 or 3;
(4) the lock nucleic acid that aptamer is transformed into described in the claim 1 or 2 or 3.
5. micro-fluidic chip that can be used for screening aptamer; Said micro-fluidic chip comprises injection port, outlet and is connected the sample introduction runner of the two; It is characterized in that: said sample introduction runner is provided with two contraction sections to form two narrowed flow path; Two contraction sections are separated into preceding runner, middle runner, three stream sections of back runner with the sample introduction runner; Fill thousands of finishinges at least before said in the runner and the protein modified microballon of the proteic anti-sieve of anti-sieve is arranged to form anti-sieve unit; Filling at least thousands of finishinges in said in the runner has just to sieve and is proteicly just sieving protein modified microballon and just sieving the unit to form, the particle diameter of the protein modified microballon of said anti-sieve greater than be communicated with said before the width of narrowed flow path of runner and middle runner, the said particle diameter that is just sieving protein modified microballon greater than be communicated with said in the width of runner and the narrowed flow path of back runner.
6. micro-fluidic chip according to claim 5; It is characterized in that: also be provided with elutriant fluid inlet and effluent flow channel on the said micro-fluidic chip in addition; Said effluent flow channel one end connects said elutriant fluid inlet, and the other end then is intersected on the said middle runner said effluent flow channel and sample introduction runner are communicated with.
7. according to claim 5 or 6 described micro-fluidic chips; It is characterized in that: said micro-fluidic chip is the micro-fluidic chip of the described aptamer of screening claim 1; Said anti-sieve albumen comprises at least a in bovine serum albumin, human serum albumin, the c reactive protein, and the said albumen that just sieving is myohaemoglobin.
8. screening method with the said aptamer of the described micro-fluidic chip of claim 7 screening claim 1 may further comprise the steps:
(1) optimizes nucleic acid library:, primer sequence is sealed with predefined initial nucleic acid library, hatch respectively with the short sequence of two ends PCR primer sequence complementary; Said initial nucleic acid library is:
5’?-?FAM-GACAGGCAGGACACCGTAAC-N
40-CTGCTACCTCCCTCCTCTTC?-?3’；
The short sequence of said and two ends PCR primer sequence complementary is:
Primer RP-Biotin:5 '-Biotin-GAAGAGGAGGGAGGTAGCAG-3 ';
Primers F P-C:5 '-GTTACGGTGTCCTGCCTGTC-3 ';
(2) primary dcreening operation: the solution in the said initial nucleic acid library injection port from said micro-fluidic chip is injected; Make the solution in the said initial nucleic acid library said anti-sieve unit of at first flowing through; There is the nucleic acid molecule of avidity to be caught with said anti-sieve albumen in the initial nucleic acid library by the protein modified microballon of said anti-sieve; The residue nucleic acid library flows to the described unit that just sieving through narrowed flow path; Have the part nucleic acid molecule of avidity to combine with said myohaemoglobin in the residue nucleic acid library with myohaemoglobin on just sieving protein modified microballon, then through said sample introduction runner is washed, wash-out, collection obtains and myohaemoglobin bonded primary dcreening operation nucleic acid library;
(3) purifying: the primary dcreening operation nucleic acid library that step (2) obtains is carried out pcr amplification, and the affinity column that amplified production is filled through the Streptavidin microballon separates, and unwinds, forms time one-level nucleic acid library after the desalination through alkaline denaturation again;
(4) circulation: with the alternative said initial nucleic acid library of the above-mentioned inferior one-level nucleic acid library that obtains and repeat above-mentioned step (2)～step (3), until the nucleic acid library that obtains including with myohaemoglobin high-affinity and high specific bonded aptamer.
9. screening method according to claim 8 is characterized in that, the primer sequence of using during said pcr amplification is:
Primers F P-FAM:5 '-FAM-GACAGGCAGGACACCGTAAC-3 ';
Primer RP-Biotin:5 '-Biotin-GAAGAGGAGGGAGGTAGCAG-3 ';
The processing condition of said pcr amplification are: 94 ℃ of 5min, and 94 ℃ of 30sec, 60.5 ℃ of annealing 30sec, 72 ℃ are extended 30sec, 10 circulations of increasing, last 72 ℃ are extended 7min.
One kind as each described aptamer in the claim 1～3 or nucleic acid aptamer derivative as claimed in claim 4 in identification, detect the application in the test kit that myohaemoglobin or preparation detect myohaemoglobin.
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