CN102703454B - 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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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 detection technique field of albumen, relate in particular to a kind of screening method and application of aptamer, this aptamer for Protein Detection.
Background technology
Myohaemoglobin (Myo) is a kind of oxygen set albumen being present in the red tissue of Mammals flesh, 153 amino-acid residues, consists of, and 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 Healthy Human Serum.When muscle injury, a large amount of myohaemoglobin just can enter into blood, causes its content in blood sharply to rise.Myocardial myoglobin is at present as the mark of early diagnosis myocardial infarction.In recent years, the relation of myohaemoglobin and myocardial infarction is subject to people's attention day by day.At present, detect the antigen antibody reaction that the most conventional method of myohaemoglobin is still utilized myohaemoglobin.Yet the detection technique based on antibody still exists certain limitation now.Therefore, design a kind of high-affinity and high specific identification myohaemoglobin nucleic acid molecular probe for after optimize its detection technique, avoid limiting to and providing the foundation due to antibody response.
Aptamer (aptamer), also referred to as aptamer or nucleic acid aptamer, 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 energy specific combination target material obtaining from random single strain oligonucleotide library, can be that DNA can be also RNA, length be generally 25～60 Nucleotide.The appearance of aptamer probe, having broken in traditional sense is the understanding of genetic information storage and transport vehicle about nucleic acid, utilizes the diversity of nucleic acid construct, can make aptamer probe efficiently, specifically in conjunction with various target molecules.Due to aptamer probe, there is the advantages such as easily synthetic, easy modification, easy storage, at aspects such as the exploitation of the analyzing and testing of protein, biosensor, molecular switch, medical diagnosis treatments, have very large application prospect, this is also for the concrete detection application of myohaemoglobin provides prerequisite and basis.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the 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 based on the integrated micro-fluidic chip of positive and negative sieve and with the fit screening method of this cDNA microarray aforementioned nucleic acid, the concrete application that utilizes this aptamer to carry out myohaemoglobin detection is also provided.
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 described 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:
5’- GACATCTATCGCCTCGATTTCCTTTGGTGAGATCGGCTCC-3’；
5’- CCCTCCTTTCCTTCGACGTAGATCTGCTGCGTTGTTCCGA-3’；
5’- ACGCACAATTCCTTGTCCAATTAGGAAATTCTACGCGGAT-3’；
5’- CGAGTACTTCTTTGCTAGTTCGCGAGATACGTTGGCTAGG-3’；
5’ - TTCCTTGTCCAATTAGGAA-3’。
The above-mentioned aptamer that can be used for detecting myohaemoglobin, a certain position that the nucleotides sequence of described aptamer lists can be phosphorylated, methylates, amination, sulfhydrylation or isotropic substance.
The above-mentioned aptamer that can be used for detecting myohaemoglobin, the nucleotides sequence of described aptamer lists 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 described derivative comprises any one in following four kinds:
(1) base A, T, G or C on above-mentioned aptamer optional position are replaced as to rare base and methylate after purine, dihydrouracil or xanthoglobulin, the nucleic acid aptamer derivative obtaining;
(2) phosphorothioate backbone that the skeleton of the nucleotide sequence of above-mentioned aptamer derives;
(3) peptide nucleic acid(PNA) that above-mentioned aptamer is transformed into;
(4) the lock nucleic acid that above-mentioned aptamer is transformed into.
As a total technical conceive, the present invention also provides a kind of micro-fluidic chip that can be used for screening aptamer, described micro-fluidic chip comprises injection port, outlet and the sample introduction runner that is connected the two, described sample introduction runner is provided with two contraction sections to form two narrowed flow path, two contraction sections are separated into front runner by sample introduction runner, middle runner, three stream sections of rear runner, in described front runner, fill at least thousands of finishinges and have the protein modified microballon of anti-sieve of anti-sieve albumen to form anti-sieve unit, in described middle runner, filling at least thousands of finishinges has the protein modified microballon of just sieve that just sieves albumen just sieving unit to form, the particle diameter of the protein modified microballon of described anti-sieve is greater than the width of the narrowed flow path that is communicated with described front runner and middle runner, the described particle diameter that is just sieving protein modified microballon is greater than the width of the narrowed flow path that is communicated with described middle runner and rear runner.
In above-mentioned micro-fluidic chip, as further improvement, on described micro-fluidic chip, be also separately provided with elutriant fluid inlet and effluent flow channel, described effluent flow channel one end connects described elutriant fluid inlet, and the other end is intersected on described middle runner described effluent flow channel and sample introduction runner are communicated with.
In above-mentioned micro-fluidic chip, the width of described sample introduction runner is preferably more than 150 μ m, and the height of sample introduction runner is preferably more than 50 μ m.The height of the narrowed flow path that described contraction section forms is preferably 10 μ m～18 μ m.The preferred polystyrene microbeads of described microballon, the particle diameter of described microballon is preferably 20 μ m～40 μ m.
In above-mentioned micro-fluidic chip, described micro-fluidic chip is preferably the micro-fluidic chip of the above-mentioned aptamer of screening, described anti-sieve albumen preferably includes at least one in bovine serum albumin (BSA), human serum albumin (HSA), c reactive protein (CRP), and the described 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 use to screen the screening method of above-mentioned aptamer, comprises the following steps:
(1) optimize nucleic acid library: the short sequence of ,Yu two ends, predefined initial nucleic acid library PCR primer sequence complementation is hatched respectively, primer sequence is sealed to avoid participating in the secondary structure forming when myohaemoglobin is combined; Described initial nucleic acid library is 5 '-FAM-GACAGGCAGGACACCGTAAC-N
40-CTGCTACCTCCCTCCTCTTC-3 ', (capacity was preferably 10
14above), described and short sequence two ends PCR primer sequence complementation is:
Primer RP-Biotin:5 '-Biotin-GAAGAGGAGGGAGGTAGCAG-3 ';
Primers F P-C:5 '-GTTACGGTGTCCTGCCTGTC-3 ';
(2) primary dcreening operation: the solution in described initial nucleic acid library is injected from the injection port of described micro-fluidic chip, make the solution in the described initial nucleic acid library described anti-sieve unit of first flowing through, in initial nucleic acid library, there is the nucleic acid molecule of avidity to be caught by the protein modified microballon of described anti-sieve with described anti-sieve albumen, residue nucleic acid library flows to described just sieve unit through narrowed flow path, in residue nucleic acid library, there is the part nucleic acid molecule of avidity to be combined with the myohaemoglobin just sieving on protein modified microballon with described myohaemoglobin, then by described sample introduction runner is rinsed, wash-out, collection obtains the primary dcreening operation nucleic acid library of being combined with myohaemoglobin,
(3) purifying: the primary dcreening operation nucleic acid library that step (2) is obtained is carried out pcr amplification, and the affinity column that amplified production is filled by Streptavidin microballon carries out separation, then unwind, form after desalination time one-level nucleic acid library by alkaline denaturation;
(4) circulation: substitute described initial nucleic acid library and repeat above-mentioned step (2)～step (3) (each circulation time should be modified microballon again in micro-fluidic chip) with obtained above one-level nucleic acid library, until obtain including the nucleic acid library of the aptamer of being combined with myohaemoglobin high-affinity and high specific.
In above-mentioned screening method, the primer sequence of using during described pcr amplification is:
Primers F P-FAM:5 '-FAM-GACAGGCAGGACACCGTAAC-3 ';
Primer RP-Biotin:5 '-Biotin-GAAGAGGAGGGAGGTAGCAG-3 ';
The processing condition of described pcr amplification are preferably: 94 ℃ of 5min, and 94 ℃ of 30sec, 60.5 ℃ of annealing 30sec, 72 ℃ are extended 30sec, increase 10 and circulate, and 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 identifying, detecting myohaemoglobin or are preparing the application in the test kit that detects myohaemoglobin.
Compared with prior art, the invention has the advantages that: utilize aptamer of the present invention, can identify quickly and efficiently myohaemoglobin, other homologous proteins are not had to 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 being conducive to.
In addition, the present invention is with respect to existing micro-fluidic chip SELEX method, creatively will just sieve He Fanshai unit, unit is integrated on a micro-fluidic chip, 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 huge effect.
Accompanying drawing explanation
Fig. 1 utilizes the integrated micro-fluidic chip of positive and negative sieve for screening the process flow sheet that myohaemoglobin specific nucleic acid is fit in the embodiment of the present invention.
Fig. 2 is the structural representation of micro-fluidic chip in the embodiment of the present invention, and wherein, a represents protein modified microballon to the first contraction section of fixing anti-sieve; B represents fixation muscles Lactoferrin modification microballon to the second contraction section; C represents to pass into the library that is dissolved in binding buffer liquid and flows to path; D represents to pass into the path that flows to after dcq buffer liquid and elution buffer.
Fig. 3 is the variation diagram of every wheel surface plasma resonance (SPR) signal of detecting in the myohaemoglobin aptamer Cycle Screening process of the embodiment of the present invention.
Fig. 4 is the aptamer of the myohaemoglobin that filters out in the embodiment of the present invention RU value variation diagram when change in concentration.
Fig. 5 is that in the embodiment of the present invention, fixed sequence program method detects the RU value variation diagram of myohaemoglobin when protein concn changes.
Fig. 6 is the fit specific investigation result to myohaemoglobin of embodiment of the present invention amplifying nucleic acid.
Marginal data:
1, injection port; 2, outlet; 3, sample introduction runner; 31, front runner; 32, middle runner; 33, rear 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 invention will be 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:
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 the present embodiment, the a certain position that its nucleotides sequence lists can be phosphorylated, methylates, amination, sulfhydrylation or isotropic substance, 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 the 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 the present embodiment are replaced as to rare base and methylate after purine, dihydrouracil or xanthoglobulin, the nucleic acid aptamer derivative obtaining;
(2) phosphorothioate backbone that the skeleton of the nucleotide sequence of the aptamer of the present embodiment derives;
(3) peptide nucleic acid(PNA) that the aptamer of the present embodiment is transformed into;
(4) the lock nucleic acid that the aptamer of the present embodiment is transformed into.
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 the sample introduction runner 3 that is connected injection port 1 and outlet 2, sample introduction runner 3 is provided with two contraction sections to form two narrowed flow path 4, two contraction sections are separated into front runner 31 by sample introduction runner, middle runner 32, 33 3 stream sections of rear runner, in front runner 31, filling approximately 8000 finishinges has anti-sieve albumen (to comprise bovine serum albumin, human serum albumin and c reactive protein) the protein modified microballon 5 of anti-sieve to form anti-sieve unit, the protein modified microballon 6 of just sieve that filling approximately 8000 finishinges in middle runner 32 has myohaemoglobin is just sieving unit to form.On the micro-fluidic chip of the present embodiment, be also separately provided with elutriant fluid inlet 7 and effluent flow channel 8, effluent flow channel 8 one end connect elutriant fluid inlet 7, and the other end is intersected on middle runner 32 effluent flow channel 8 and sample introduction runner 3 are communicated with.In the micro-fluidic chip of the 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 the present embodiment is glass material, adopts photoetching technique, wet etching and the high temperature bonding mode of standard to prepare.The microballon of the present embodiment (the protein modified microballon of positive and negative sieve) is polystyrene microbeads, and the particle diameter of microballon is 25 μ m.When making the micro-fluidic chip of the present embodiment, the modification of microballon realizes by following operation: first myohaemoglobin and anti-sieve albumen are hatched with polystyrene microbeads respectively, obtain just sieving protein modified microballon 6 and the protein modified microballon 5 of anti-sieve, then will instead sieve protein modified microballon from injection port 1 dispose into, make it fixedly be filled in the first contraction section place and form and just sieve unit; To just sieve again albumen (being myohaemoglobin) modify microballon from elutriant fluid inlet 7 dispose into, make it fixedly be filled in the second contraction section place and form and just sieve unit.
As shown in Figure 1, a kind of method of the above-mentioned aptamer with the integrated micro-fluidic chip screening the present embodiment in the positive and negative sieve unit of the present embodiment, comprises the following steps:
1. optimize nucleic acid library
1.1 design initial nucleic acid libraries: initial nucleic acid library capacity is 10
14above, the synthetic two ends of design comprise 20 Nucleotide, tundish is as follows containing the random nucleic acid sequence library of 40 Nucleotide:
5’ - FAM-GACAGGCAGGACACCGTAAC-N
40-CTGCTACCTCCCTCCTCTTC-3’。
1.2 optimize initial nucleic acid library: the primer RP-Biotin of the above-mentioned strand initial nucleic acid library of synthetic 1.34nmol and 1.5 times of amounts and primers F P-C are dissolved in to 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, after 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
2.1 to get 0.5mL diameter be that the polystyrene microbeads of 25 μ m is placed in 1.5mL centrifuge tube, removes supernatant, with borate buffer (0.1mol/L boric acid, pH is 8.5), rinses 3 times.
2.2 myohaemoglobin of getting 20 μ g～30 μ g add in the microballon of step 2.1, and 25 ℃ of night incubation add store buffer liquid (0.1mol/L PBS, 10mg/mL BSA, 0.1% NaN after rinsing 2 times with borate buffer
3, 5% glycerine, pH is 7.4) and 4 ℃ of preservations.Get respectively in addition 25 ℃ of night incubation in the microballon that 10 μ g HSA, BSA, CRP add step 2.1 simultaneously, add store buffer liquid after rinsing 2 times with borate buffer.
2.3 preparations one are the micro-fluidic chip of the present embodiment as shown in Figure 2, and sample introduction runner 3 two ends of micro-fluidic chip are respectively equipped with injection port 1 and outlet 2, is provided with the narrowed flow path 4 of two contraction sections to be formed for stopping that microballon passes through in the middle of sample introduction runner 3; By the protein modified microballon 5 of anti-sieve after step 2.2 is modified with just sieving protein modified microballon 6 and be individually fixed in one end of first, second contraction section by the order of a～b shown in Fig. 2.
3. primary dcreening operation
As Fig. 1, shown in Fig. 2, the optimization initial nucleic acid library obtaining in step 1 is passed in the sample introduction runner 3 of above-mentioned micro-fluidic chip, coutroi velocity 0.5 μ L/min(is by micro-injection pump coutroi velocity), the solution in initial nucleic acid library is first through anti-sieve unit, the nucleic acid molecule that wherein has avidity with anti-sieve albumen is due in conjunction with being sieved protein modified microballon 5 and catch by counter, residue nucleic acid library flows to is just sieving unit (as shown in the c in Fig. 2), wherein there is the part nucleic acid molecule of avidity and just sieve protein modified microballon 6 combinations with myohaemoglobin, align afterwards dcq buffer liquid (binding buffer liquid for sieve unit, 0.05% Tween 20) with flow velocity 2 μ L/min, rinse 30min(as shown in the d in Fig. 2, dcq buffer liquid enters from elutriant fluid inlet 7, the effluent flow channel of flowing through successively 8 and middle runner 32 rinse), finally use elution buffer (20mmol/L Tris-HCl, 4mol/L guanidinium isothiocyanate, 1mmol/L DTT, pH is 8.3) with flow velocity 2 μ L/min, rinse 30min(elution process with dcq buffer liquid), through rinsing, after wash-out, collection obtains the primary dcreening operation nucleic acid library of being combined with myohaemoglobin.
4. purifying
Single stranded DNA in the primary dcreening operation nucleic acid library that step 3 is collected carries out PCR reaction;
Primers F P-FAM:5 '-FAM-GACAGGCAGGACACCGTAAC-3 ';
Primer RP-Biotin:5 '-Biotin-GAAGAGGAGGGAGGTAGCAG-3 '.
PCR reaction conditions is as follows: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 60.5 ℃ of annealing 30s, 72 ℃ are extended 30s, increase 10 and circulate, and last 72 ℃ are extended 7min.
By the product after pcr amplification by the biotin labeled product of affinity column decoupled band of the Agarose microbead that is preinstalled with Streptavidin and modifies, then with the NaOH of 200mmol/L, unwind, the strand screening as next round after desalination chromatogram purification time one-level nucleic acid library.
5. circulation
The inferior one-level nucleic acid library obtaining with step (4) substitutes to be optimized nucleic acid library and repeats above-mentioned step 2～step 4, after screening process in, the flow velocity progressively increasing while rinsing strengthens proof strength, and by SPR instrument, detects every signal intensity of taking turns and determine screening terminal; From the detected result shown in Fig. 3, can find out, increase along with 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; Altogether carried out 8 and taken turns circulation, finally obtained including the nucleic acid library of the present embodiment aptamer of being combined with myohaemoglobin high-affinity and high specific.
Finally, the aptamer of above-mentioned sequence and the dissociation constant K of myohaemoglobin to the present embodiment screening gained
dmeasure.Adopt Biacore X bio-molecular interaction instrument, take binding buffer liquid as working fluid, carry out according to the following steps:
First the myohaemoglobin that passes into 90 μ L 20 μ M with the flow velocity of 5 μ L/min is modified in golden film surface, passes into the random ssDNA chain of 70 μ L afterwards seal with 10 μ L/min flow velocitys; After handling well, with 10 μ L/min flow velocitys, pass into successively the screening gained aptamer solution of a series of concentration (3.125nM, 6.25nM, 12.5nM, 25nM, 50nM, 100nM, 200nM) of each 70 μ L, the variation of recording the RU value of each step.The result of gained is mapped with Sigma Plot software, by formula Y=BmaxX/ (Kd+X) the Fitting Calculation, go out the Kd value of five sequences of the present embodiment, in contrast with initial library simultaneously, detected result is shown in Fig. 4, according to above-mentioned equation model, obtains Myo40-7-23, Myo40-7-27, Myo40-7-34, Myo40-7-69, these 5 sequences of Myo19-7-34 K of correspondence successively
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 fixed sequence program method to detect myohaemoglobin and specificity investigation.
First with the flow velocity of 5 μ L/min, pass into 90 μ L 0.05% BSA-Biotin solution and modify in golden film surface, with same flow velocity volume, pass into 0.05% Avidin solution, pass into afterwards biotinylated aptamer sequence.After finishing is good, with 10 μ L/min flow velocitys, pass into successively the 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 each 70 μ L, the variation of recording the RU value of each step.The result of gained is mapped as shown in Figure 5 with Sigma Plot software.Adopt the method for fixed sequence program to investigate the specificity of each aptamer sequence, aptamer modifiedly with 10 μ L/min flow velocitys, pass into successively HSA, BSA, CRP, the IgG(immunoglobulin G while of each 70 μ L 500 nM after good) and Myo solution, record each step RU value variation as shown in Figure 6.In conjunction with shown in Fig. 5 and Fig. 6, can find out, by the aptamer sequence filtering out is fixed to behind golden film surface, can effectively catch the myohaemoglobin in solution, and other reference proteins are not had to signal response substantially.
<110> Hunan University
<120> can be used for detecting aptamer, micro-fluidic chip and screening method and the application for screening of myohaemoglobin
<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>
<210> 7
<211> 20bp
<212> DNA
<213> artificial sequence
<400>
<210> 8
<211> 20bp
<212> DNA
<213> artificial sequence
<400>
Claims (4)
1. for detection of an aptamer for myohaemoglobin, the nucleotides sequence of described aptamer is classified the DNA fragmentation shown in following sequence 1 as:
Sequence 1:
5’- CCCTCCTTTCCTTCGACGTAGATCTGCTGCGTTGTTCCGA-3’。
2. one kind for screening the micro-fluidic chip of aptamer described in claim 1, described micro-fluidic chip comprises injection port, outlet and the sample introduction runner that is connected the two, it is characterized in that: described sample introduction runner is provided with two contraction sections to form two narrowed flow path, two contraction sections are separated into front runner by sample introduction runner, middle runner, three stream sections of rear runner, in described front runner, fill at least thousands of finishinges and have the protein modified microballon of anti-sieve of anti-sieve albumen to form anti-sieve unit, in described middle runner, filling at least thousands of finishinges has the protein modified microballon of just sieve that just sieves albumen just sieving unit to form, the particle diameter of the protein modified microballon of described anti-sieve is greater than the width of the narrowed flow path that is communicated with described front runner and middle runner, the described particle diameter that is just sieving protein modified microballon is greater than the width of the narrowed flow path that is communicated with described middle runner and rear runner,
On described micro-fluidic chip, be also separately provided with elutriant fluid inlet and effluent flow channel, described effluent flow channel one end connects described elutriant fluid inlet, and the other end is intersected on described middle runner described effluent flow channel and sample introduction runner are communicated with.
3. micro-fluidic chip according to claim 2, is characterized in that: described anti-sieve albumen comprises at least one in bovine serum albumin, human serum albumin, c reactive protein, and the described albumen that just sieving is myohaemoglobin.
4. an aptamer as claimed in claim 1 detects the application in the test kit of myohaemoglobin in preparation.
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| CN103801412A (en) * | 2012-11-08 | 2014-05-21 | 中山大学 | Integrated micro-fluidic chip used for fluorescence detection of enzyme catalysis product and application thereof |
| CN102965378B (en) * | 2012-11-14 | 2014-10-15 | 广西安仁欣生物科技有限公司 | Aptamer of glycosylated hemoglobin and preparation method thereof |
| CN104109675B (en) * | 2012-11-14 | 2016-07-06 | 广西安仁欣生物科技有限公司 | A kind of aptamer of glycolated hemoglobin and preparation method thereof |
| CN104109674B (en) * | 2012-11-14 | 2016-07-06 | 广西安仁欣生物科技有限公司 | A kind of aptamer of glycolated hemoglobin and preparation method thereof |
| CN104164436B (en) * | 2012-11-14 | 2016-10-05 | 广西安仁欣生物科技有限公司 | A kind of aptamer of glycosylated hemoglobin and preparation method thereof |
| CN103436608B (en) * | 2013-08-08 | 2015-02-25 | 中国科学院广州生物医药与健康研究院 | Rapid detection method based on nucleic acid aptamers and kit |
| CN106480039B (en) * | 2016-10-13 | 2019-11-12 | 南京大学 | A method of aptamers are screened using micro-fluidic chip |
| CN109900910A (en) * | 2019-03-11 | 2019-06-18 | 嘉兴学院 | A kind of aptamer fluorescent optical sensor and its construction method detecting myoglobins |
| CN111077301A (en) * | 2020-01-16 | 2020-04-28 | 浙江农林大学 | Micro-fluidic chip and method for detecting interaction between biomolecules |
| BR102020004436A8 (en) | 2020-03-05 | 2022-11-22 | Bioptamers Inc | MICROFLUID DEVICE |
| CN113493932B (en) * | 2021-09-09 | 2021-12-03 | 北京贝瑞和康生物技术有限公司 | Method and kit for constructing capture library with high detection performance |
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| CN102186992A (en) * | 2008-08-15 | 2011-09-14 | 康奈尔大学 | Device for rapid identification of nucleic acids for binding to specific chemical targets |
| CN102229932A (en) * | 2011-06-03 | 2011-11-02 | 湖南大学 | Nucleic acid aptamer capable of identifying HCV E1E2 (hepatitis C virus E1E2), nucleic acid aptamer derivatives and screening method and application thereof |
| CN102321616A (en) * | 2011-09-15 | 2012-01-18 | 湖南大学 | Method for screening protein aptamers by using micro-fluidic chip |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102186992A (en) * | 2008-08-15 | 2011-09-14 | 康奈尔大学 | Device for rapid identification of nucleic acids for binding to specific chemical targets |
| CN102229932A (en) * | 2011-06-03 | 2011-11-02 | 湖南大学 | Nucleic acid aptamer capable of identifying HCV E1E2 (hepatitis C virus E1E2), nucleic acid aptamer derivatives and screening method and application thereof |
| CN102321616A (en) * | 2011-09-15 | 2012-01-18 | 湖南大学 | Method for screening protein aptamers by using micro-fluidic chip |
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