CN105624165B - The biomolecule detecting method of cascade amplification strategy based on self-locking aptamer probe - Google Patents

The biomolecule detecting method of cascade amplification strategy based on self-locking aptamer probe Download PDF

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CN105624165B
CN105624165B CN201610005486.7A CN201610005486A CN105624165B CN 105624165 B CN105624165 B CN 105624165B CN 201610005486 A CN201610005486 A CN 201610005486A CN 105624165 B CN105624165 B CN 105624165B
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王磊
姜玮
李伟
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Shandong University
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Abstract

The invention discloses the biomolecule detecting method of the cascade amplification strategy based on self-locking aptamer probe, which includes at least two parts：There is the aptamer sequence at 3 ' ends of specific recognition and the signal transduction sequence at 5 ' ends to object, the signal transduction sequence at the 5 ' end hybridizes with part aptamer sequence, 5 ' end stem-loop structures are formed, the signal transduction sequence at the 5 ' end includes the recognition site of nicking restriction endonuclease.The probe marriage chain replaces amplification (SDA) and double indexing type rolling circle amplification (DE-RCA) strategy, realizes protein-PDGF-BB and small molecule-adenosine hypersensitive and high specific detection, detection limit is respectively up to 3.8 × 10‑16Mol/L and 4.8 × 10‑8mol/L。

Description

The biomolecule detecting method of cascade amplification strategy based on self-locking aptamer probe
Technical field
The present invention relates to a kind of nucleic acid detection technique fields, and in particular to a kind of cascade expansion based on self-locking aptamer probe Increase highly sensitive, high specific the detection method that strategy is used for protein and small-molecule substance.
Background technique
Aptamer is a kind of artificial synthesized, short single strand oligonucleotide acid sequence, is commonly used for bio-sensing, diagnosing and treating Equal fields.Aptamer usually has specific and compact second level or tertiary structure, has high affinity to its corresponding object And selectivity.Compared with antibody or antibody fragment, aptamer have the characteristics that stablize, be readily synthesized and modify, object it is extensive, Object includes albumen, small molecule, metal ion even cell etc..Therefore, aptamer has been widely used for the building of molecular probe.
Molecular probe based on aptamer mainly includes two parts：Aptamer sequence and signal sequence.Aptamer sequence can The second level or tertiary structure being fold into, while specific recognition its object.Once aptamer sequence is in conjunction with object, letter Number sequence can be released generation signal.However, aptamer probe is usually excessive in practical aptamer sensing system.It is theoretical Calculate display, aptamer in unbonded object, also can occurred conformation transformation to generate interference signal influence the standard of detection True property.In order to solve this problem, a kind of method is fixed on aptamer probe on one out-phase surface, will be extra by washing Aptamer probe wash away.This method can effectively avoid the generation of interference signal, but the introducing of biphase interface will lead to line Property range is limited and the joint efficiency of object, probe reduces defect.In order to avoid these problems, realize based on the equal of aptamer It mutually detects, another method is exactly the retardance DNA molecular for introducing one section short, hybridizes it with the active part of aptamer sequence, shape At a closed aptamer probe of retardance chain, so that the non-specific of aptamer be inhibited to fold.However, since the retardance chain is shorter (12-15nt), the unstability of duplex structure will lead to retardance chain leakage (falling off from aptamer probe), and then cause interference with letter Number generation.Therefore, in order to be further reduced interference signal, improve detection accuracy, construct one it is new, more stable suitable Body probe is highly important.
Summary of the invention
The present invention is to solve above-mentioned the deficiencies in the prior art, provide a kind of detection protein and biological micromolecule substance from Locking-type aptamer probe and cascade amplification strategy based on self-locking aptamer probe for protein and small-molecule substance it is highly sensitive, The detection method of high specific.
The technical solution adopted by the present invention is as follows：
A kind of self-locking aptamer probe for detecting protein and biological micromolecule, the probe include at least two portions Point：There is the aptamer sequence at 3 ' ends of specific recognition and the signal transduction sequence at 5 ' ends, the signal at the 5 ' end to object Transduction sequence hybridizes with part aptamer sequence, forms 5 ' end stem-loop structures, the signal transduction sequence at the 5 ' end includes in nicking The recognition site of enzyme cutting, the recognition site of the nicking restriction endonuclease are located at 3 ' ends of the signal transduction sequence at 5 ' ends.
The design is so that self-locking aptamer probe is in self-locking state in no object, to make aptamer sequence in no mesh It is not folded when marking object.Self-locking aptamer probe molecule keeps the signal transduction sequence at 5 ' ends and part suitable by folded back on itself Complementary base-pair meets in body sequence, is formed made of Hydrogenbond, referred to as hairpin structure (or stem-loop structure).
The aptamer sequence at the 3 ' end is for combining target object, it is the Fas lignand system evolution technology by index concentration What (abbreviation SELEX technology) repeated screening from the random oligonucleotide sequences library synthesized outside prosthesis obtained can be with high affine The segment oligonucleotide sequence of power and specificity in conjunction with object.
The self-locking aptamer probe that the present invention designs can be used to detect various bioprotein molecules and biological micromolecule, institute Stating bioprotein molecule is platelet-derived growth factor-BB (PDGF-BB), and biological micromolecule is adenosine.By changing aptamer sequence Column and design probe, this method may be alternatively used for the detection of other biomolecule.
It preferably, should when the base of 5 ' end of aptamer sequence is not suitable for forming hydrogen bond with 3 ' end of signal transduction sequence Probe further includes the catenation sequence for connecting aptamer sequence and signal transduction sequence, and the catenation sequence is used to participate in formation 5 ' Stem-loop structure is held, guarantees that signal transduction sequence can hybridize to form stem-loop structure with part aptamer sequence.
Preferably, which is also connected with cooperation sequence, the alkali of the cooperation sequence in 3 ' ends of the aptamer sequence at 3 ' ends Base number is 1~5, it is therefore an objective to the hairpin structure for holding 3 ' end aptamer sequence smooth openings 5 '.
The present invention also provides a kind of application of above-mentioned probe in detection protein and biological micromolecule content of material.
The method of cascade amplification strategy detection protein or biological micromolecule substance based on self-locking aptamer probe, including Following steps：
(1) chain replaces amplified reaction：First by the object to be detected containing object in conjunction with self-locking aptamer probe, fold At three-dimensional helical structure, opens 5 ' end signal transduction sequences and the stem-loop structure of aptamer sequence formation is held in part 3 '；Above-mentioned system In the presence of archaeal dna polymerase, nicking restriction endonuclease and dNTP, the three-dimensional helical structure at 3 ' ends is generated as primer triggering SDA reaction A large amount of primer sequence 1；
(2) double indexing type expands rolling circle amplification：Primer sequence 1 in step (1) is hybridized with template 1, in archaeal dna polymerase Under the action of trigger first order exponential type RCA amplified reaction, generate a large amount of primer sequence 2；Primer sequence 2 hybridizes with template 2, Exponential type RCA amplified reaction in the second level is triggered under the action of archaeal dna polymerase, generates a large amount of G- tetraploid sequence, is inserted into glimmering After optical molecule, fluorescence signal is generated, quantitative survey is carried out to protein or biological micromolecule substance by the fluorescence signal detected It is fixed.
Specific step is as follows：Primer sequence 1 hybridizes with template 1, and linear RCA reaction is triggered under the action of archaeal dna polymerase, Generate a long single-stranded DNA product；The product can hybridize with template 1 excessive in system, expose nicking restriction endonuclease Recognition site, to generate a large amount of primer sequence 2 by nicking enzyme restriction endonuclease nicking；And free 1/ template of primer, 1 compound Next polymerization, nicking circulation are carried out, first order exponential type RCA amplified reaction is completed；Finally, primer sequence 2 and template 2 are miscellaneous It hands over, exponential type RCA amplified reaction in the second level is triggered under the action of archaeal dna polymerase, generates a large amount of G- tetraploid sequence, be inserted into After fluorescent molecule, fluorescence signal is generated by the fluorescence signal detected, quantitative survey is carried out to protein or biological micromolecule substance It is fixed.
If do not contain object in object to be detected, chain does not occur and replaces amplified reaction and double indexing type amplification rolling ring Amplified reaction does not generate fluorescence signal then after being inserted into fluorescent molecule.
Specifically include following steps：
(1) chain of binding induction replaces amplification (SDA)
Object to be detected containing object and above-mentioned self-locking aptamer probe are subjected to first time incubation reaction, after incubation The archaeal dna polymerase with strand-displacement activity, nicking restriction endonuclease, dNTPs, buffer and water is added and carries out second of incubation reaction, Make SDA reaction terminating finally by heating；Specific reaction step is as follows：
Object and self-locking aptamer probe to be detected (50nM, 5 μ L) containing object is incubated for 1h at 37 DEG C；Then add Enter 0.4 μ L KF polymerase, 0.2 μ L Nt.BbvCI, 4 μ L 2mM dNTPs, 2 μ L Cutsmart and 3.4 μ L water, is incubated at 37 DEG C Educate 2.5h；Make SDA reaction terminating finally by 80 DEG C of heating 10min.
(2) connection reaction
Addition template 1 in amplified production in step (1), T4DNA ligase, T4DNA ligase buffer solution and water, into The reaction of 1 loop connecting of row template；Specific reaction step is as follows：
1.4 μ L, 10 μM of templates 1,0.3 μ L T4 DNA ligase, 3.0 μ L are added into above-mentioned 20 μ L SDA amplified production T4 DNA ligase buffer and 5.3 μ L water react 40min at 37 DEG C.
(3) double indexing type rolling circle amplification (DE-RCA)
Take the connection product in step (2), be added template 2, archaeal dna polymerase, nicking restriction endonuclease, buffer and dNTPs and Water carries out amplification reaction, and makes DE-RCA reaction terminating finally by heating；Specific reaction step is as follows：
Above-mentioned 30 μ L connection reaction product is taken, 1.4 μ L, 10 μM of templates 2,0.4 μ L phi 29DNA polymerase, 0.4 μ are added LNt.BbvCI, 5 μ L Cutsmart, 10 μ L dNTPs and 22.8 μ L water react 5h at 37 DEG C；Step reaction passes through 80 DEG C of heating 10min is terminated.
(4) fluorescence detection
The amplified production in step (3) is taken, N- methyl porphyrin dipropionic acid IX (NMM) is added and is reacted, finally using glimmering Light instrument carries out fluoremetry；Specific reaction step is as follows：
It takes above-mentioned DE-RCA product, 5 μ L 2mM KCl and 5 μ L 0.08mM NMM is added, react 40min at 37 DEG C.Finally Fluoremetry is carried out with Hitachi F-7000 luminoscope, excitation wavelength selects 399nm, the capture range selection of launch wavelength Fluorescence intensity at 550~680nm, final choice 612nm investigates the sensitivity of method.
Preferably, the signal transduction sequence such as SEQ ID No at the 5 ' end：Shown in 1.
Preferably, the signal transduction sequence at the 5 ' end is 5 '-GCT GTG GAT ACT GCT GAG GCC A-3 ', such as SEQ ID No：Shown in 1.
It is different according to the object of detection, replace corresponding 3 ' end aptamer sequence and catenation sequence.When object is and cancer When relevant platelet derived growth factor BB (PDGF-BB) of disease, it is preferred that the aptamer sequence of PDGF-BB is 5 '-CA GGC TAC GGC ACG TAG AGC ATC ACC ATG ATC CTG-3 ', such as SEQ ID No：Shown in 2, catenation sequence 5 '-CCA-3 ', Cooperation sequence is 5 '-TG-3 '.When object is adenosine, it is preferred that the aptamer sequence of adenosine is 5 '-AC CTG GGG GAG TAT TGC GGA GGA AGG T-3 ', such as SEQ ID No：Shown in 3, catenation sequence 5 '-CCACAG-3 ', cooperation sequence be 5’-CTGT-3’。
Preferably, the nicking restriction endonuclease is nicking enzyme Et.BbvCI, the recognition site of corresponding nicking enzyme Et.BbvCI Sequence is 5 '-GCT GAG G-3 '.
Preferably, the sequence of the template 1, which is formed, includes the complementary series of one section of primer sequence 1 after ring-type and draws with two The complementary series of object sequence 2, between the complementary series of each primer sequence 2, the complementary series of primer sequence 2 and primer sequence 1 Complementary series between be connected by nicking endonuclease recognized site sequence.Preferably 5 '-TAC TGC TGA GGG AGT TGA GTG CTG AGG GAG TTG AGT GCT GAG GCT GTG GA-3 ', such as SEQ ID No：Shown in 4, wherein crossing Sequence is the recognition site of nicking restriction endonuclease.Wherein, the primer sequence 1 is the product of above-mentioned SDA reaction, the primer sequence 2 be the product of above-mentioned first order exponential type RCA amplified reaction.
Preferably, the sequence of the template 2 forms complementary series and two G- tetra- including one section of primer sequence 2 after ring-type The complementary series of times body sequence, between each G- tetraploid complement thereof, the complementary series and primer of G- tetraploid sequence It is connected by nicking endonuclease recognized site sequence between the complementary series of sequence 2.Preferably 5 '-AGTGCT GAG GAA ACC CAA CCC GCC CTA CCC GCT GAG GAA ACC CAA CCC GCC CTA CCC GCT GAG GGA GTT G-3 ', such as SEQ ID No：Shown in 5, wherein drawing the complementary series that single line sequence is G- tetraploid, drawing two-wire sequence is to cut Carve the recognition site of restriction endonuclease.
Preferably, the fluorescent molecule is NMM (N- methyl porphyrin dipropionic acid IX).
Preferably, the archaeal dna polymerase used in the SDA reaction is the archaeal dna polymerase with strand-displacement activity, such as Klenow Fragment polymerase.
Preferably, the archaeal dna polymerase used in rolling circle amplification reaction is phi 29DNA polymerase.
The present invention also provides a kind of kits for detecting protein and biological micromolecule substance, including：
(1) self-locking aptamer probe according to any one of claims 1 to 3；
(2) archaeal dna polymerase, nicking restriction endonuclease, T4DNA ligase；
(3) dNTPs, fluorescent molecule and KCl solution；
(4) buffer of Cutsmart buffer, T4DNA ligase；
(5) above-mentioned template 1 and template 2.
Preferably, the archaeal dna polymerase includes Klenow Fragment polymerase and phi 29DNA polymerase.
The beneficial effects of the invention are as follows：
The present invention constructs a self-locking aptamer probe.The probe has triple functions：First is that based on its 3 ' end aptamer The molecular recognition function of sequence；Second is that being based on the signal transduction functionality of its 5 ' terminal signal sequence；Third is that based on signal sequence and fitting The self-locking function of body sequence hybridization.Since signal sequence is intramolecular hybridization with hybridizing for aptamer sequence, so this self-locking Aptamer probe is stablized than the aptamer probe of retardance chain retardance, therefore is more conducive to reducing the folding of aptamer non-specificity and interference Signal.In addition, the expansion of the cascade based on the self-locking aptamer probe has also been devised in the present invention for the sensitivity of ensuring method Increasing strategy, realizes the highly sensitive and high specific detection of platelet derived growth factor BB (PDGF-BB), detection limits up to 3.8 × 10-16Mol/L, the range of linearity are more than 6 orders of magnitude.By changing aptamer sequence, which is also employed successfully in small molecule-gland The Sensitive Detection of glycosides illustrates that the self-locking aptamer probe has good versatility, has at the actually detected aspect of biomolecule There is very big application potential.
The present invention reacts the strategy combined with SDA reaction and DE-RCA using self-locking aptamer probe, realizes biological egg High sensitivity and the high specific detection of white molecule and biological micromolecule substance.
Detailed description of the invention
Fig. 1：The cascade amplification strategy that self-locking aptamer probe mediates is used for sensitive, specific detection the principle of protein Figure.
Fig. 2 (A)：The fluorogram spectrogram of various concentration PDGF-BB；Wherein, a → l is followed successively by blank, 2 × 10-15M、5× 10-15M、1×10-14M、1×10-13M、1×10-12M、1×10-11M、1×10-10M、1×10-9M、1×10-8M、2.0×10- 8M、5×10-8M。
Fig. 2 (B)：The linear relationship chart of fluorescence intensity and PDGF-BB concentration.
Fig. 3：The specificity of method investigates schematic diagram.
Fig. 4 (A)：Adenosine detection principle diagram.
Fig. 4 (B)：The fluorescence response figure of various concentration adenosine；Wherein, a → i is followed successively by blank, 1 × 10-7M、5×10- 7M、1×10-6M、5×10-6M、1×10-5M、5×10-5M、8×10-5M、1×10-4M。
Fig. 4 (C)：The linear relationship chart of various concentration adenosine and fluorescence intensity.
Specific embodiment
Embodiment 1
1. experimental section
1.1 reagents and material
DNA (sequence such as table 1) used and dNTPs are by Sheng Gong bioengineering Co., Ltd (China, Shanghai) synthesis in experiment And purifying；Platelet derived growth factor (PDGF-BB), immunoglobulin G (Ig G), huamn tumor necrosis factory alpha (TNF-α), Human interferon gamma (IFN-γ) and fibrin ferment are purchased from ProSpec-Tany company (Nai Siciaona, Israel)；Adenosine is purchased from Sigma-Aldrich company (St. Louis, the U.S.)；Klenow Fragment polymerase, nicking enzyme Et.BbvCI, T4DNA connect Enzyme and 29 archaeal dna polymerase of phi are connect purchased from New England Biolabs company (U.S.)；NMM is purchased from J&K Scientific Company (Beijing, China)；Other reagents (analysis is pure) are purchased from standard suppliers.
HEPES buffer solution used includes 50mM NaCl and 25mM HEPES (pH 7.0) in experimentation；PBS buffer solution Include 0.15M NaCl, 2.4mM NaH2PO4With 7.6mM Na2HPO4(PH 7.4)；TE buffer include 10mM Tris and 1.0mM Na2EDTA(PH 8.0)。
DNA sequence dna used in the experiment of table 1
Note：Double-crossed sequence is PDGF-BB aptamer sequence difference, the aptamer sequence of chain-dotted line sequence adenosine；Single scribing line sequence Nicking enzyme Et.BbvCI recognition site, wave sequence are the complementary series of G- tetraploid.
The chain of 1.2 binding inductions replaces amplification (SDA)
PDGD-BB (10nM, 5 μ L) and self-locking aptamer probe (50nM, 5 μ L) are incubated for 1h at 37 DEG C.Then it is added 0.4 μ L KF polymerase, 0.2 μ L Nt.BbvCI, 4 μ L 2mM dNTPs, 2 μ L Cutsmart and 3.4 μ L water are incubated at 37 DEG C 2.5h.Make SDA reaction terminating finally by 80 DEG C of heating 10min.
1.3 connection reactions
1.4 μ L, 10 μM of templates 1,0.3 μ L T4 DNA ligase, 3.0 μ L T4 are added into above-mentioned 20 μ L SDA product DNA ligase buffer and 5.3 μ L water react 40min at 37 DEG C.
1.4 double indexing type rolling circle amplifications (DE-RCA)
Above-mentioned 30 μ L connection reaction product is taken, 1.4 μ L, 10 μM of templates 2,0.4 μ L phi, 29 polymerase, 0.4 μ are added LNt.BbvCI, 5 μ L Cutsmart, 10 μ L dNTPs and 22.8 μ L water react 5h at 37 DEG C.Step reaction passes through 80 DEG C of heating 10min is terminated.
1.5 fluorescence detection
It takes above-mentioned DE-RCA product, 5 μ L 2mM KCl and 5 μ L 0.08mM NMM is added, react 40min at 37 DEG C.Finally Fluoremetry is carried out with Hitachi F-7000 luminoscope, excitation wavelength selects 399nm, the capture range selection of launch wavelength Fluorescence intensity at 550~680nm, final choice 612nm investigates the sensitivity of method.
2. results and discussion
2.1 principle
Such as Fig. 1, designs first and construct a self-locking aptamer probe.The probe includes two parts：The aptamer at 3 ' ends The signal transduction sequence of sequence and 5 ' ends.In addition, the signal transduction sequence can hybridize with aptamer Sequence, make probe in nothing Self-locking state is in when object, from without folding, the low background of ensuring method.In this method, select cancer relevant PDGF-BB is as model analysis object.In the presence of having PDGF-BB in system, aptamer probe is folded into three in conjunction with PDGF-BB To helical structure, the hairpin structure at 5 ' ends is opened.Next, in the presence of KF polymerase, nicking enzyme Et.BbvCI and dNTP, 3 ' The three-dimensional helical structure at end generates a large amount of primer 1 as primer triggering SDA reaction.Then, primer 1 and 1 Eclectics of template, Linear RCA reaction is triggered under the action of 29 polymerase of phi, generates a long single-stranded DNA product.The product can be with system In excessive template 1 hybridize, expose the recognition site of nicking enzyme, to be carved by nicking digestion, generate a large amount of primer 2.And Free 1/ template of primer, 1 compound have can carry out it is next polymerization, nicking circulation, complete the first order exponential type RCA amplification.Most Afterwards, primer 2 hybridizes with template 2, and triggering second level exponential amplification reaction generates a large amount of G- tetraploid sequence, after being inserted into NMM, Generate the fluorescence signal of enhancing.
2.2 condition optimizing
It is dense to SDA reaction time, DE-RCA reaction time, the polymerization of phi 29 enzyme dosage, Et.BbvCI dosage and NMM respectively Degree is optimized.The final choice SDA time be 2.5h, DE-RCA reaction time be 5h, phi 29 polymerize enzyme dosage be 4U, Et.BbvCI dosage is final concentration of 5 μM of 4U, NMM.
2.3 sensitivity are investigated
In optimal conditions, the sensitivity of method is investigated, such as Fig. 2,2.0 × 10-15Mol/L~1.0 × 10-8Within the scope of mol/L, the linear relationship of fluorescence intensity and target concentration is good, and linear equation is Δ F=2548.4+ 713.0lgC, detection are limited to 3.8 × 10-16mol/L。
2.4 specificity are investigated
Using Ig G, TNF-α, IFN-γ and fibrin ferment as jamming target object, the specificity of method is investigated.Such as figure 3, this method only has strong fluorescence response to PDGF-BB, to the fluorescence response very little of other albumen, illustrates that this method has very Good specificity.
2.5 versatilities are investigated
In order to investigate the versatility of method, the aptamer part of probe is replaced with to the aptamer sequence of adenosine, is realized to gland The Sensitive Detection of glycosides.Such as Fig. 4, method is 1.0 × 10-7Mol/L~1.0 × 10-4Within the scope of mol/L, fluorescence intensity is dense with adenosine The linear relationship of degree is good, and detection is limited to 4.8 × 10-8mol/L。
In addition, the aptamer sequence of probe to be changed to the aptamer sequence of other biomolecule, it can also be carried out highly sensitive It spends and detects with high specificity.
3. summarizing
In this work, the present invention constructs a self-locking aptamer probe, expands in conjunction with the cascade of SDA and DE-RCA, Protein and the highly sensitive of small molecule, high specific detection are realized, detection limit is respectively up to 3.8 × 10-16Mol/L and 4.8 × 10-8mol/L。
Bibliography：
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Claims (4)

1. the method for the cascade amplification strategy detection protein based on self-locking aptamer probe, characterized in that the method is used for Non-treatment and non-diagnostic purpose, include the following steps：
(1) chain replaces amplified reaction：By the object to be detected containing object in conjunction with self-locking aptamer probe, it is folded into three-dimensional spiral shell Structure is revolved, 5 ' end signal transduction sequences is opened and the stem-loop structure of aptamer sequence formation is held in part 3 '；Above-mentioned system is poly- in DNA In the presence of synthase, nicking restriction endonuclease and dNTP, chain occurs and replaces amplified reaction, generates a large amount of primer sequence 1；
(2) double indexing type expands rolling circle amplification：Primer sequence 1 in step (1) is hybridized with template 1, triggers first order index Type RCA amplified reaction generates a large amount of primer sequence 2；Primer sequence 2 hybridizes with template 2, and triggering second level exponential type RCA expands Increase reaction, generate a large amount of G- tetraploid sequence, after being inserted into fluorescent molecule, generates fluorescence signal and pass through the fluorescence signal detected Protein or biological micromolecule are quantitative determined；
If do not contain object in object to be detected, chain does not occur and replaces amplified reaction and double indexing type amplification rolling circle amplification Reaction, then without fluorescence signal；
The object is protein；The protein is platelet derived growth factor BB；
The self-locking aptamer probe includes at least two parts：There is the aptamer sequence at 3 ' ends of specific recognition to object The signal transduction sequence of the signal transduction sequence of column and 5 ' ends, the 5 ' end hybridizes with part aptamer sequence, forms 5 ' end stem-loops The signal transduction sequence of structure, the 5 ' end includes the recognition site of nicking restriction endonuclease；The self-locking aptamer probe is also wrapped The catenation sequence for connecting aptamer sequence and signal transduction sequence is included, the catenation sequence is used to participate in formation 5 ' and holds stem-loop Structure；Aptamer sequence of the self-locking aptamer probe at 3 ' ends is also connected with cooperation sequence；
The self-locking aptamer probe is the self-locking aptamer probe of platelet derived growth factor BB, sequence：GCT GTG GAT ACT GCT GAG GCC ACA GGC TAC GGCACG TAG AGC ATC ACC ATG ATC CTG TG, such as SEQ ID No：Shown in 6；
1 nucleotides sequence of template is classified as：TAC TGC TGA GGG AGT TGA GTG CTG AGG GAG TTGAGT GCT GAG GCT GTG GA, such as SEQ ID No：Shown in 4；2 nucleotides sequence of template is classified as：AGT GCT GAG GAA ACC CAA CCC GCC CTA CCC GCTGAG GAA ACC CAA CCC GCC CTA CCC GCT GAG GGAGTT G, such as SEQ ID No：Shown in 5.
2. the method as described in claim 1, characterized in that in step (1), SDA react the step of be：Object will be contained Object and self-locking aptamer probe to be detected carries out first time incubation reaction, and being added after being incubated for has the DNA of strand-displacement activity poly- Synthase, nicking restriction endonuclease, dNTPs, buffer and water carry out second of incubation reaction, make SDA reaction terminating finally by heating.
3. the method as described in claim 1, characterized in that specific reaction step is in step (2)：Primer sequence 1 and template 1 Hybridization triggers linear RCA reaction under the action of archaeal dna polymerase, generates a long single-stranded DNA product；The product and template 1 Hybridization, exposes the recognition site of nicking restriction endonuclease, to generate a large amount of primer sequence 2 by nicking restriction endonuclease nicking；And it swims From 1/ template of primer, 1 compound carry out it is next polymerization, nicking circulation, complete the first order exponential type RCA amplification；Finally, drawing Object sequence 2 hybridizes with template 2, and triggering second level exponential amplification reaction generates a large amount of G- tetraploid sequence, is inserted into fluorescent molecule Afterwards, fluorescence signal is generated to quantitative determine protein or biological micromolecule by the fluorescence signal detected.
4. a kind of kit for detecting protein, characterized in that the kit includes：(1) self-locking aptamer probe；It is described from Locking-type aptamer probe sequence：GCT GTG GAT ACT GCT GAG GCC ACA GGC TAC GGCACG TAG AGC ATC ACC ATG ATC CTG TG, such as SEQ ID No：Shown in 6；
(2) archaeal dna polymerase, nicking restriction endonuclease, T4DNA ligase；
(3) dNTPs, fluorescent molecule and KCl solution；
(4) buffer of Cutsmart buffer, T4DNA ligase；
(5) template 1 and template 2；
1 nucleotides sequence of template is classified as：TAC TGC TGA GGG AGT TGA GTG CTG AGG GAG TTGAGT GCT GAG GCT GTG GA, such as SEQ ID No：Shown in 4；2 nucleotides sequence of template is classified as：AGT GCT GAG GAA ACC CAA CCC GCC CTA CCC GCTGAG GAA ACC CAA CCC GCC CTA CCC GCT GAG GGAGTT G, such as SEQ ID No：Shown in 5.
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