CN104789674A - Probe based on double-signal amplification triggered by target and application of probe - Google Patents
Probe based on double-signal amplification triggered by target and application of probe Download PDFInfo
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Abstract
The invention provides a probe based on double-signal amplification triggered by a target and application of the probe. The probe comprises a first hairpin, a second hairpin and a third hairpin, wherein the first hairpin, the second hairpin and the third hairpin are formed by pairing hybridization of complementary bases in folding areas after single-chain linear molecules are folded back; the parts, forming double-chain structures in the partial areas, of the hairpins are stem areas, and the parts, not forming the double-chain structures and folded back, are ring-shaped areas. According to the probe provided by the invention, the self-assembly of hairpins is realized through target protein catalysis, signal amplification is achieved under the assistance of an excision enzyme III, obvious amplification of a detection signal is realized, and the detection sensitivity is greatly improved further; since the probe does not depend on template replication in the detection process, the problem that false positive occurs due to cross contamination in the detection process is avoided, generation of a false positive signal is effectively prevented and background noise is reduced greatly.
Description
Technical field
The invention belongs to molecular bioinformatics field, be specifically related to the probe of the dual signal amplification that a kind of based target triggers and detect method of protein and application.
Background technology
The detection by quantitative of protein plays very important effect in biological study and pharmacodiagnosis field.But, because protein is often in low-down concentration, often need high-sensitive analyzing and testing method to be detected, therefore, it is possible to be the direction that people study to the detection of protein molecule high sensitivity, highly selective, simple and fast always.At present, in the quantitative detecting method of protein, enzyme linked immunosorbent assay (ELISA) is the most frequently used method, although the method can reach the object of quantitative analysis, by the restriction of enzyme-substrate reactions, its detection sensitivity still cannot detect the protein molecule of trace, thus its using value in disease early diagnosis is limited, again due to its operation steps more complicated, cause the many factors affecting result, as operating time and operating process require stricter; Linearity range is narrower; Specificity is poor, and in serum, the interference of various material can affect reaction result; Defining of yin and yang attribute is fuzzyyer, needs revision test; Manual operations is loaded down with trivial details, is difficult to automatization and mass; The response difference of different batches is comparatively large, the difficult normalization method of result.Therefore, now frequent amplification of signal strategy is used for protein molecule detect biosensor structure in.In the past few decades, the amplification strategy of quite a few has been incorporated in the detection of protein, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), rolling circle amplification (RCA).These amplification techniques drastically increase the susceptibility of protein detection, but because they depend critically upon template duplicating, add the risk occurring crossed contamination in amplification procedure, cause the appearance (J.Dong of false positive results, X.Cui, Y.Deng, Z.Tang, Biosens.Bioelectron., 2012).So the exploitation for protein sensing amplification strategy remains and needs further exploratory development.
Recently, intended catalyzed hair clip self-assembly is exempted from amplification of signal strategy as a kind of promising enzyme and is in the news for the detection of biomolecules, and exempts from usually to use DNA enzymatic in the optical biosensor of amplification of signal Strategy Design according to enzyme.DNA enzymatic (also claiming DNAzyme or catalytic dna) is single strand oligodeoxynucleotide, there is the ability of catalyzed chemical reaction, compared with traditional protease, DNA enzymatic has high thermostability, the simplification of nucleic acid synthesis, identified region is enrolled the handiness of DNA enzymatic sequence, these character make DNA enzymatic become the ideal chose of bio-sensing.In the past in 10 years, G-tetra-serobila DNA enzymatic by design ap-plication widely to many optical biosensors.G-tetra-serobila in conjunction with cofactor protohemine, can form G-tetra-serobilas/protohemine DNA enzymatic, at H
2o
2amino-2, the 3-bis-chloro-Isosorbide-5-Nitraes-phenyl-diformyl hydrazine (luminol,3-aminophthalic acid cyclic hydrazide) of the lower catalyzed oxidation 5-of mediation, 2,2 '-Lian nitrogen-bis-(3-ethyl-benzothiazole-6-sulfonic acid) (ABTS
2-), or VitB1, and be attended by chemical light emission, color change or fluorescent emission.Therefore, chemoluminescence is established, the biological sensing system of colorimetric or fluorescence based on G-tetra-serobila DNA enzymatic.Wherein, chemiluminescence detection causes more concern, and this depends primarily on, and it is easy and simple to handle, highly sensitive, and does not need expensive analytical instrument.So far, some biosensors based on this strategy are successfully developed, however this wherein most biosensor mainly for the detection in nucleic acid.
As, a kind of method that Chinese patent literature CN102827836A discloses oligonucleotide probe and detects target molecule with it, above-mentioned probe is DNA a pair hairpin with sticky end based on target molecule design, and essence is the product that the hairpin of hybridization chain reaction combines with G-tetra-serobila sequence.When there is not target molecule in reaction system, two hairpins keep metastable hairpin structure, when there is target molecule in reaction system, target molecule just can with first hairpin specific binding, destroy its secondary structure and discharge remaining G-tetra-serobila sequence, the G-freely tetra-serobila sequence of first hair clip release can be hybridized with second hairpin, destroy second hair clip and discharge the nucleotide sequence with target molecule with phase same-action, also can with the target molecule specific recognition part specific binding of first hair clip, thus cause two hair clip cascade hybridization, so go round and begin again, the high polymer of the similar double-stranded DNA of final formation, d/d a large amount of G-tetra-serobila sequence freely can combine with protohemine (hemin), catalyzed oxidation H
2o
2the ABTS of mediation, thus detect target molecule.But during above-mentioned probe in detecting target molecule, there is following problem:
(1) ground unrest is comparatively large, sensitivity is poor.Specifically, in reaction system, hairpin structure is keep running balance with the straight chain not forming hairpin structure, straight chain does not form closed ring and protected, G-tetra-serobila sequence is caused not to be that target molecule is opened first hair clip and discharges and then formed with second clamps hybridise entirely, also there is part to be sequence and second hair clip direct cross of straight chain, thus form G-tetra-serobila sequence freely, therefore cannot realize comparatively sensitive detection;
(2) utilize the hybridization coordination of base to carry out the detection of target molecule, in fact only can realize the detection of DNA, and the detection of protein cannot be realized.
Given this, a kind of simple to operate, cost is low, low ground unrest has highly sensitive detection protein molecule simultaneously probe is urgently proposed at present.
Summary of the invention
For this reason, technical problem to be solved by this invention is in prior art low for the probe sensitivity detecting protein molecule, and ground unrest is large, and then the probe of the dual signal amplification providing the based target of the high and low ground unrest of a kind of sensitivity to trigger.
Second technical problem to be solved by this invention is in prior art high to the detection method of protein length consuming time, complicated operation and cost, and then provides a kind of method of detection protein molecule of simple to operate, low cost.
For solving the problems of the technologies described above, the invention provides the probe of the dual signal amplification that a kind of based target triggers, comprising the first hair clip, the second hair clip and the 3rd hair clip; After described first hair clip, the second hair clip and the 3rd hair clip are strand linear molecule folded back on itself, complementary base pairing hybridization in fold domain forms, it is stem district that its regional area forms the part of duplex structure, does not form duplex structure and the part of inflection is ring-shaped area.
The probe of the dual signal amplification that described based target triggers, described first hair clip comprises: can specific recognition target protein adaptive tagma (I) and form the first stem district (II) of hairpin structure with described adaptive tagma (I) partial hybridization;
Described second hair clip comprises: can with the first base complementrity district (I ') of adaptive tagma (I) partial hybridization of described first hair clip, the second base complementrity district (II ') that can hybridize with described first stem district (II), and form the second stem district (III) of hairpin structure with described first base complementrity district (I ') and the second base complementrity district (II ') partial hybridization;
Described 3rd hair clip comprises: with the 3rd base complementrity district (III ') of described second stem district (III) partial hybridization and can form the G-tetra-serobila sequence area (IV) of hairpin structure with described 3rd base complementrity district (III ') partial hybridization.
The probe of the dual signal amplification that described based target triggers, the 3 ' end in described first stem district (II) and described second stem district (III) is not easily by critical sequences that excision enzyme is sheared.
The probe of the dual signal amplification that described based target triggers, described first stem district (II) is the 1-4 base from 3 ' end is the sequence of T-T-T-T; Described second stem district (II) is the 1-4 base from 3 ' end is the sequence of T-T-A-A.
The probe of the dual signal amplification that described based target triggers, the ring-shaped area being connected to form described first hair clip is held in 5 ' end portion sequence and the described adaptive tagma (I) 3 ' in described first stem district (II).
The probe of the dual signal amplification that described based target triggers, the part that the 5 ' end in described first stem district (II) is positioned at described ring-shaped area be hold from 3 ' 1-4 base be the sequence of T-T-T-T.
The probe of the dual signal amplification that described based target triggers, described G-tetra-serobila sequence area (IV) has the sequential structure as shown in SEQ ID No.1.
Present invention also offers a kind of method detecting protein molecule, the probe of the dual signal amplification that the based target described in utilization triggers carries out fluoroscopic examination.
Described method, comprises the steps:
Probe and the excision enzyme III of getting the dual signal amplification that described based target triggers respectively join in solution to be measured, 80-100 minute is hatched at 20-40 DEG C, subsequently to wherein adding protohemine and HEPES damping fluid, at 20-40 DEG C, hatching 50-70 minute, then adding luminol,3-aminophthalic acid cyclic hydrazide and H wherein
2o
2, carry out chemoluminescence measurement, observe or detect luminescence or the discoloration of mixed solution.
Described method, probe and the excision enzyme III of getting the dual signal amplification that described based target triggers respectively join in solution to be measured, hatch 90 minutes, subsequently to wherein adding protohemine and HEPES damping fluid at 37 DEG C, hatch 60 minutes at 30 DEG C, then add luminol,3-aminophthalic acid cyclic hydrazide and H wherein
2o
2, carry out chemoluminescence measurement, observe or detect luminescence or the discoloration of mixed solution.
Present invention also offers the probe of the dual signal amplification that a kind of described based target triggers in the purposes detecting protein molecule or photodetector system field.
Described purposes, described protein molecule is the protein that can be combined with DNA.
Technique scheme of the present invention has the following advantages compared to existing technology,
(1) probe that the dual signal that based target of the present invention triggers increases, comprise the first hair clip, the second hair clip and the 3rd hair clip, after described first hair clip, the second hair clip and the 3rd hair clip are strand linear molecule folded back on itself, complementary base pairing hybridization in fold domain forms, it is stem district that its regional area forms the part of duplex structure, does not form duplex structure and the part of inflection is ring-shaped area, first hair clip comprises: can specific recognition target protein adaptive tagma I and form the first stem district II of hairpin structure with adaptive tagma I partial hybridization, second hair clip comprises: can with the first base complementrity district I ' of adaptive tagma I partial hybridization of the first hair clip, the second base complementrity district II ' that can hybridize with the first stem district II, and form the second stem district III of hairpin structure with the first base complementrity district I ' and the second base complementrity district II ' partial hybridization, described 3rd hair clip comprises: can form the G-tetra-serobila sequence area IV of hairpin structure with the 3rd base complementrity district III ' of the second stem district III partial hybridization and with the 3rd base complementrity district III ' partial hybridization, above-mentioned is a kind of simple and exempt from the probe based on G-tetra-serobila DNA enzymatic that marks, it can pass through the above-mentioned hair clip self-assembly of target protein catalysis, and be issued to amplification of signal excision enzyme III is auxiliary, realize the obvious amplification of detection signal, and then significantly improve the susceptibility of detection, use zymoplasm as Proof of Concept analysis, this sensor-based system can detect zymoplasm with high specificity, detectability is low to moderate 0.92pM, and because it does not rely on template duplicating in testing process, avoid the risk occurring crossed contamination in testing process, and then cause false positive produced problem, effectively prevent the generation of false positive signal, substantially reduce ground unrest, improve the sensitivity of detection greatly,
(2) probe that the dual signal that based target of the present invention triggers increases, 3 ' the end in the first stem district (II) and the second stem district (III) is for not easily by critical sequences that excision enzyme is sheared, when ensureing there is not target compound in reaction system, first hair clip, the second hair clip can keep hairpin structure, keep steady state, do not produce signal, effectively prevent the generation of false positive signal, substantially reduce ground unrest;
(3) probe that the dual signal that based target of the present invention triggers increases, the part that the 5 ' end in the first stem district (II) is positioned at described ring-shaped area be hold from 3 ' 1-4 base be the sequence of T-T-T-T, when ensureing there is not target compound in reaction system, first hair clip can keep hairpin structure, keeps steady state;
(4) method of detection protein molecule of the present invention, the probe in detecting protein molecule of the dual signal amplification that the based target described in utilization triggers, compared with detecting the method for protein molecule with the fluorescently-labeled hairpin probe of needs, probe of the present invention without any need for chemically modified, whole reaction simultaneously can be carried out in the homogeneous phase solution of isothermal, make this detection method very simple and economical and efficient, what is more important, because multiple recognition unit can incorporate probe, can be used for detecting the multiple protein that can be combined with DNA specifically, and can explore at biochemical field and apply more widely.
(5) method of detection protein molecule of the present invention, probe and the excision enzyme III of the dual signal amplification triggered by based target join in solution to be measured, 80-100 minute is hatched at 20-40 DEG C, subsequently to wherein adding protohemine and HEPES damping fluid, hatch 50-70 minute at 20-40 DEG C, then add luminol,3-aminophthalic acid cyclic hydrazide and H wherein
2o
2, aforesaid method detects simple to operate, condition and is easy to control and consuming time shorter.
Accompanying drawing explanation
In order to make content of the present invention be more likely to be clearly understood, below according to a particular embodiment of the invention and by reference to the accompanying drawings, the present invention is further detailed explanation, wherein
Fig. 1 is the Cleaning Principle schematic diagram of the probe of the dual signal amplification that the based target of embodiment 1 triggers; Wherein, in figure, Reference numeral is expressed as: I-adaptive tagma; II-the first stem district; I '-the first base complementrity district; II '-the second base complementrity district; III-the second stem district; III '-three base complementrity district; IV-G-four serobila sequence area.
Fig. 2 is the variation relation figure between luminous intensity and target concentration of thrombin detected in embodiment 2;
Fig. 3 is the index curve diagram between luminous intensity and target concentration of thrombin detected in embodiment 2;
Fig. 4 is the schematic diagram of the assay method of embodiment 3;
Fig. 5 is to the result schematic diagram that the colorimetric reaction of sensor-based system detects in embodiment 4;
Fig. 6 is the luminous intensity detected result figure in embodiment 5;
Fig. 7 is the luminous intensity detected result figure in embodiment 6;
Fig. 8 is the luminous intensity detected result figure in embodiment 7;
Fig. 9 is the luminous intensity detected result figure in embodiment 8
Embodiment
The material used in the following embodiment of the present invention and reagent are commercially available prod, adopt the following material of different model and manufacturer and reagent for effect of the present invention and indifference:
The oligonucleotide that PAGE is pure is bought in Genscript company (oligonucleotide sequence is listed in table 1 below);
People's α-zymoplasm, people's immunity ball albumin G (IgG), human serum albumin and bovine serum albumin are bought in Sigma-Aldrich company (U.S., Constantine, St. Louis);
Excision enzyme III and protohemine are bought in Sangon Biotech (Shanghai) Co., Ltd. (China, Shanghai);
HEPES (HEPES), three (methylol) aminomethane (Tris), luminol,3-aminophthalic acid cyclic hydrazide and hydrogen peroxide are bought in Aladdin reagent (Shanghai) Co., Ltd. (China, Shanghai);
Other chemical reagent are bought in Chemical Reagent Co., Ltd., Sinopharm Group (China, Shanghai);
Redistilled water is obtained by Milli-Q purification system (U.S., the state of Massachusetts, the Bill's profit card) preparation of resistivity 18M Ω cm.
The probe design of the dual signal amplification that embodiment 1 based target triggers
The probe of the dual signal amplification that the based target described in the present embodiment triggers comprises: the first hair clip H1, the second hair clip H2 and the 3rd hair clip H3, and each composition sequence is as shown in table 1 below; The zymoplasm that target protein to be detected is behaved.
The probe sequence of the dual signal amplification that table 1 based target triggers
After described first hair clip, the second hair clip and the 3rd hair clip are strand linear molecule folded back on itself, complementary base pairing hybridization in fold domain forms, it is stem district that its regional area forms the part of duplex structure, does not form duplex structure and the part of inflection is ring-shaped area;
Described first hair clip comprises: can specific recognition target protein adaptive tagma I and form the first stem district II of hairpin structure with described adaptive tagma I partial hybridization;
Described second hair clip comprises: can with the first base complementrity district I ' of adaptive tagma I partial hybridization of described first hair clip, the second base complementrity district II ' that can hybridize with described first stem district II, and form the second stem district III of hairpin structure with described first base complementrity district I ' and the second base complementrity district II ' partial hybridization;
Described 3rd hair clip comprises: can form the G-tetra-serobila sequence area IV of hairpin structure with the 3rd base complementrity district III ' of described second stem district III partial hybridization and with described 3rd base complementrity district III ' partial hybridization.
Its Cleaning Principle is illustrated in figure 1:
First cyclic amplification reaction:
(1) adding along with target protein, the specific binding of the I pair of target protein in adaptive tagma that described first hair clip H1 is contained by it and being opened, discharges described first stem district II with single stranded form;
(2) the first stem district II be released hybridizes with described second hair clip H2 immediately, forms mixture, and;
(3) because above-mentioned mixture is unstable, target protein is got off from displacement described first hair clip H1 by described second hair clip H2 by the process that is similar to DNA molecular migration, leaves the DNA duplex H1-H2 of the number of base complementation with 3 '-convex end; Wherein, the new H1-H2 duplex with 3 '-convex end formed can not be sheared by excision enzyme III, and described excision enzyme III can only shear DNA double chain that is flat or recessed 3 '-end specifically;
(4) the first hair clip H1 that replaced target protein out can be new with another is combined, the cyclic amplification reaction of triggering coagulation enzyme (circulation I), every part of target protein can trigger the multiple self-assembling reactions between described first hair clip H1 and described second hair clip H2;
Second cyclic amplification reaction
(5) the described second stem district III described H1-H2 double-strand is released can hybridize with described 3rd hair clip H3, forms the DNA duplex H2-H1-H3 of a number of base complementation further;
(6) by after identifying described H2-H1-H3 duplex, excision enzyme III only catalysis shears the described 3rd hair clip H3 with 3 '-recessed end, by the 3rd hair clip H3 in described H1-H2-H3 double-strand with 3 '-to-5 ' direction from 3 '-recessed end, be progressively hydrolyzed mononucleotide, after shearing completes, discharge the G-tetra-serobila sequence area IV that described H1-H2 duplex and described 3rd hair clip H3 are not sheared;
(7) the described H1-H2 duplex discharged then freely is combined with another the 3rd hair clip H3 and triggers new excision enzyme III cleavage reaction, thus constitutes the second circulation (circulation II); According to this mechanism, every a target protein can open multiple hairpin probe to trigger multiple circulating reaction, generates a large amount of G-tetra-serobila sequences;
(8) the G-tetra-serobila sequence discharged is folded into G-tetra-stranded structure in the basic conditions, and is combined with cofactor protohemine, forms a large amount of imitative peroxidase dna enzymes with catalytic activity, at H
2o
2mediation lower catalyzed oxidation luminol,3-aminophthalic acid cyclic hydrazide, thus the chemoluminescence amplification detection of realize target thing.
The storing solution (100 μMs) of the oligonucleotide of the present embodiment is by Tris-HCl damping fluid (the 200mM NaCl of 20mM, 2mM MgCl2, with 20mM KCl, pH=7.4) dilution form, the above-mentioned first hair clip H1 of synthetic, the second hair clip H2 and the 3rd hair clip H3.
The probe that the dual signal that embodiment 2 based target triggers increases is for the detection of different concns target protein molecules
The probe of the dual signal amplification of the based target triggering of the present embodiment, for the detection method of target protein molecules, comprises the steps:
First, get the solution of described first hair clip, the second hair clip, the 3rd hair clip, be heated to 95 DEG C, keep 5min, be more slowly down to room temperature, for subsequent use.Get 10ml methyl-sulphoxide (DMSO) and 6.5mg protohemine, the protohemine solution of preparation 1mM, and be stored in dark place at-20 DEG C, for subsequent use.Preparation 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) damping fluid is stand-by, and this damping fluid comprises: the KCl of the NaCl of the HEPES of 25mM, 200mM, 20mM, the DMSO of 1%, and pH value is 7.4.
Preparation Tris-HCl damping fluid is stand-by, and this damping fluid comprises: the MgCl of the KCl of the NaCl of the Tris-HCl of 20mM, 200mM, 20mM, 2mM
2, pH value is 7.4.Getting the concentration that the above-mentioned Tris-HCl damping fluid of people's α-zymoplasm is diluted to target protein molecules is 1pM (i.e. pmol/L, down together), the solution of 2pM, 5pM, 10pM, 20pM, 50pM, 100pM, 200pM, 500pM, 1000pM, and do not add the damping fluid of target protein molecules, as solution to be measured.Wherein target protein molecules is zymoplasm.
Respectively in Example 1 concentration of design be the first hair clip H1 solution of 200nM, the concentration second hair clip H2 solution that is 200nM, the concentration each 10 μ L of the 3rd hair clip H3 solution that are 200nM, and the excision enzyme III 10 μ L of 2U/ μ L, be placed in the liquid to be measured of 10 μ L, form the solution of 50 μ L, hatch 90 minutes for 37 DEG C, be the protohemine of 100nM and the HEPES damping fluid of 40 μ L subsequently to the concentration wherein adding 10 μ L, hatch 60 minutes for 30 DEG C, thus make the G-tetra-serobila sequence discharged form G-tetra-serobilas/protohemine DNA enzymatic fully;
Get the above-mentioned mixed solution of 100 μ L, add the H of the luminol,3-aminophthalic acid cyclic hydrazide of the 0.125mM of 50 μ L and the 0.125mM of 50 μ L
2o
2composition cumulative volume is that the liquid to be detected of 200 μ L is placed in the chemiluminescent measurement use multi-functional microplate reader (U.S. of SpectraMax M5e, California, Molecular Devices) in detect, and record data, determined wavelength scope is 360nm to 530nm, and step footpath is 1nm.The result that the target zymoplasm of probe to different concns that the dual signal using based target to trigger increases measures as shown in Figure 2,3.Wherein, in fig. 2, the mixed solution of curve a to be target concentration of thrombin be 0pM, the mixed solution of curve b to be target concentration of thrombin be 1pM, the mixed solution of curve c to be target concentration of thrombin be 2pM, the mixed solution of curve d to be target concentration of thrombin be 5pM, the mixed solution of curve e to be target concentration of thrombin be 10pM, the mixed solution of curve f to be target concentration of thrombin be 20pM, the mixed solution of curve g to be target concentration of thrombin be 50pM, the mixed solution of curve h to be target concentration of thrombin be 100pM, the mixed solution of curve i to be target concentration of thrombin be 200pM, the mixed solution of curve j to be target concentration of thrombin be 500pM, the mixed solution of curve k to be target concentration of thrombin be 1000pM.
From above-mentioned detected result, along with the increase of target concentration of thrombin, chemiluminescent intensity also progressively strengthens.This is consistent with theoretical fact, and namely the concentration of zymoplasm is higher, will produce more G-tetra-serobilas/protohemine DNA enzymatic, at H
2o
2the lower catalyzed oxidation luminol,3-aminophthalic acid cyclic hydrazide of effect, causes the progressively enhancing of chemiluminescence intensity.The relation (as shown in Figure 3) that exponent pair is answered is there is between chemiluminescence intensity and concentration of thrombin.Further, on logarithmic graph, chemiluminescence intensity and concentration of thrombin (from 1pM to 1000pM) present good linear relationship (as illustration Fig. 3).Equation of linear regression is I=13479.42+28186.74log
10c, wherein I and C represents chemiluminescence intensity and concentration of thrombin respectively, and linearly dependent coefficient is 0.9893, detects and is limited to 0.92pM.This detection sensitivity has exceeded 21 times of (A.X.Zheng, J.R.Wang, J.Li compared with the fluorometry (20pM) of based target catalysis hair clip self-assembly, X.R.Song, G.N.Chen, H.H.Yang, Biosens.Bioelectron., 2012); Exceed compared with the electrochemical methods (5pM) increased with the autocatalysis target circulation of assisting based on exonucleaseⅢ 5 times (S.Liu, Y.Lin, L.Wang, T.Liu, C.Cheng, W.Wei, B.Tang, Anal.Chem., 2014); Suitable with colorimetry (1.5pM) sensitivity of increasing based on DNA enzymatic and restriction endonuclease, but analyzing and testing step more simple (Y.Huang, J.Chen, S.Zhao, M.Shi, Z.F.Chen, H.Liang, Anal.Chem., 2013).The detection sensitivity of the inventive method improves the ingenious combination mainly owing to intended catalyzed hair clip self-assembly and the amplification of exonucleaseⅢ subsidiary signal.Therefore, use the method for probe in detecting target protein molecules of the present invention not only simple to operate, and there is superior sensitivity.
The amplification of signal proof test of the probe of the dual signal amplification that embodiment 3 based target triggers
In the present embodiment, prepare following solution according to the method in embodiment 2, and number consecutively a, b, c, d:
Sample a: the concentration of described first hair clip H1, the second hair clip H2, the 3rd hair clip H3 is the solution of 200nM;
Sample b: the concentration of described first hair clip H1, the second hair clip H2, the 3rd hair clip H3 is 200nM, the concentration of described exonucleaseⅲ is the solution of 20U;
Sample c: the concentration of described first hair clip H1, the second hair clip H2, the 3rd hair clip H3 is 200nM, the concentration of target zymoplasm is the solution of 1nM;
Sample d: the concentration of described first hair clip H1, the second hair clip H2, the 3rd hair clip H3 is 200nM, the concentration of described exonucleaseⅲ is 20U, and the concentration of target zymoplasm is the solution of 1nM;
Cultivate 75min at above-mentioned each solution is placed in 37 DEG C, the sample solution then getting equivalent respectively adds the protohemine of equivalent, HEPES damping fluid, luminol,3-aminophthalic acid cyclic hydrazide and H
2o
2, react.Method in the same manner as in Example 2 is adopted to carry out chemiluminescent detection to sample a, b, c, d.
Above-mentioned detected result as shown in Figure 4, when not having target zymoplasm, can only observe very little chemoluminescence peak (curve b), substantially with the chemiluminescence intensity of blank solution close to (curve a).After adding target zymoplasm, an obvious signal can be observed and increase (curve c), this increase mainly due to the target Thrombin specificity that adds combine adaptive tagma I in described first hair clip H1, thus open described first hair clip H1 and the first stem district II of described first hair clip H1 is discharged, then hybridize with described second hair clip H2 and described 3rd hair clip H3, opened by described 3rd hair clip H3 and discharged G-tetra-serobila sequence, G-tetra-serobila sequence is combined with cofactor protohemine and forms G-tetra-serobilas/protohemine DNA enzymatic, at H
2o
2effect under, DNA enzymatic catalyzed oxidation luminol,3-aminophthalic acid cyclic hydrazide, thus cause the increase of chemiluminescence intensity.Further, when excision enzyme III is incorporated into after in this sensing system, it can shear H2-H1-H3 duplex release H1-H2 double-strand, the continuous circulation shear of H1-H2 double-strand just constitutes the circulation II shown in Fig. 1, more G-tetra-serobila sequence is released, and further enhancing chemiluminescent intensity (curve d).Therefore, the probe of the dual signal amplification that based target of the present invention triggers can the amplification detection target protein molecules of high sensitivity, has significant sensitivity.
The concentration parallel test of the probe of the dual signal amplification that embodiment 4 based target triggers
The present embodiment adopts the method identical with embodiment 2 to carry out the detection of target zymoplasm, difference is only, comprise in the mixed reaction solution of described first hair clip H1, the second hair clip H2, the 3rd hair clip H3, Exo III, solution to be measured (target concentration of thrombin is 1nM), the concentration of described first hair clip H1, the second hair clip H2 and the 3rd hair clip H3 is all followed successively by 50nM, 100nM, 150nM, 200nM, 250nM, 300nM.
Reaction terminate after, its detected result as shown in Figure 5, wherein ratio I/I
0be used to the susceptibility of analysis and assessment test, I and I
0represent that system exists and do not exist the chemiluminescence intensity in target zymoplasm situation respectively.As can be seen from Fig. 5, when hair clip concentration is increased to 200nM from 50nM, I/I
0ratio constantly increase, after the concentration of hairpin probe is more than 200nM, I/I
0ratio start decline.Therefore, determine that the optimal concentration of hairpin probe is 200nM.
The Exo III consumption parallel test of the probe of the dual signal amplification that embodiment 5 based target triggers
The present embodiment adopts the method identical with embodiment 2 to carry out the detection of target zymoplasm, difference is only, comprise in the mixed reaction solution of described first hair clip H1, the second hair clip H2, the 3rd hair clip H3, Exo III, solution to be measured (target concentration of thrombin is 1nM), the consumption of described Exo III is followed successively by 10U, 15U, 20U, 25U, 30U.
After reaction terminates, as shown in Figure 6, chemiluminescence intensity constantly strengthens along with the increase (from 10U to 30U) of the amount of Exo III its detected result, and consider the consumption of excision enzyme III, the optimum consumption of excision enzyme III of 20U is 20U.
The amplified reaction time parallel test of the probe of the dual signal amplification that embodiment 6 based target triggers
The present embodiment adopts the method identical with embodiment 2 to carry out the detection of target zymoplasm, difference is only, comprises the time of hatching at 37 DEG C in the mixed reaction solution of described first hair clip H1, the second hair clip H2, the 3rd hair clip H3, Exo III, solution to be measured (target concentration of thrombin is 1nM) and is followed successively by 30min, 45min, 60min, 75min, 90min, 105min, 120min.
After reaction terminates, as shown in Figure 7, along with the prolongation of amplified reaction time, chemiluminescence intensity is mutually deserved enhancing also for its detected result.Consider the time span of susceptibility and the analysis detected, the peak optimization reaction time is 90 minutes.
The concentration parallel test of the protohemine of the probe of the dual signal amplification that embodiment 7 based target triggers
The present embodiment adopts the method identical with embodiment 2 to carry out the detection of target zymoplasm, difference is only, the concentration comprising the protohemine added in the mixed reaction solution of described first hair clip H1, the second hair clip H2, the 3rd hair clip H3, Exo III, solution to be measured (target concentration of thrombin is 1nM) is followed successively by 50nM, 75nM, 100nM, 125nM, 150nM, 200nM.
Reaction terminate after, its detected result as shown in Figure 8, wherein ratio I/I
0be used to the susceptibility of analysis and assessment test, I and I
0represent that system exists and do not exist the chemiluminescence intensity in target zymoplasm situation respectively.As can be seen from Fig. 8, present best signal background ratio (I/I when the protohemine of 100nM
0), therefore optimum protohemine concentration is 100nM.
The specific detection test of the probe of the dual signal amplification that embodiment 8 based target triggers
The present embodiment is in order to verify the specificity of probe of the present invention for different proteins molecule further, specific as follows:
The probe of the dual signal amplification that based target in Example 1 triggers is according to the method for embodiment 2 respectively to target zymoplasm and non-targeted protein bovine serum albumin (BSA), and human serum albumin (HAS) and immunoglobulin G (IgG) and the damping fluid not adding target zymoplasm detect.
Above-mentioned detected result as shown in Figure 9, as can be seen from Figure 9, because target zymoplasm is to the aptamers high specific identification of described probe, can be easy to distinguish target zymoplasm and other non-targeted protein, under the existence of target zymoplasm, the enhancing of an obvious chemiluminescence intensity can be observed.But other non-targeted protein then do not observe the change of significant chemiluminescence intensity.Therefore, the above results shows that this probe has high detection specificity to zymoplasm.
Target crosby test in the probe in detecting human serum of the dual signal amplification that embodiment 9 based target triggers
The present embodiment is in order to verify the detection of probe of the present invention for the target zymoplasm in human serum further, specific as follows:
The human serum used is provided by Jiang Yuan hospital, and 4 DEG C of storages are for subsequent use.Get above-mentioned serum Tris-HCl and dilute 10 times, then get people's α-zymoplasm and adopt above-mentioned human serum diluted to be 10pM, 100pM, 1000pM to the concentration of zymoplasm, be numbered 1,2,3 respectively, as solution to be measured.Method in the same manner as in Example 2 is adopted to detect.Above-mentioned detected result is as shown in table 2,
Zymoplasm rate of recovery detected result in table 2 10% human serum
Result display rate of recovery scope is 95.58-104.16%, relative standard deviation is 6.62-8.21% (n=3), demonstrate the feasibility of this detection method in actual biological specimen, indicate it when for protein detection in actual biological sample (such as dilution after human serum), detection by quantitative accurately can be realized.
Therefore, method of the present invention has good practicality, is applicable to the detection of the protein in actual biological sample.
Obviously, above-described embodiment is only for clearly example being described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (12)
1. the probe that the dual signal that based target triggers increases, is characterized in that, comprise the first hair clip, the second hair clip and the 3rd hair clip; After described first hair clip, the second hair clip and the 3rd hair clip are strand linear molecule folded back on itself, complementary base pairing hybridization in fold domain forms, it is stem district that its regional area forms the part of duplex structure, does not form duplex structure and the part of inflection is ring-shaped area.
2. the probe that the dual signal that based target according to claim 1 triggers increases, is characterized in that,
Described first hair clip comprises: can specific recognition target protein adaptive tagma (I) and form the first stem district (II) of hairpin structure with described adaptive tagma (I) partial hybridization;
Described second hair clip comprises: can with the first base complementrity district (I ') of adaptive tagma (I) partial hybridization of described first hair clip, the second base complementrity district (II ') that can hybridize with described first stem district (II), and form the second stem district (III) of hairpin structure with described first base complementrity district (I ') and the second base complementrity district (II ') partial hybridization;
Described 3rd hair clip comprises: with the 3rd base complementrity district (III ') of described second stem district (III) partial hybridization and can form the G-tetra-serobila sequence area (IV) of hairpin structure with described 3rd base complementrity district (III ') partial hybridization.
3. the probe that the dual signal that based target according to claim 1 and 2 triggers increases, it is characterized in that, the 3 ' end in described first stem district (II) and described second stem district (III) is not easily by critical sequences that excision enzyme is sheared.
4. the probe of the dual signal amplification triggered according to the arbitrary described based target of claim 1-3, is characterized in that, the 3 ' end in described first stem district (II) be hold from 3 ' 1-4 base be T-T-T-T; 3 ' the end in described second stem district (II) is the 1-4 base from 3 ' end is the sequence of T-T-A-A.
5. the probe that the dual signal triggered according to the arbitrary described based target of claim 1-4 increases, it is characterized in that, the ring-shaped area being connected to form described first hair clip is held in 5 ' end portion sequence and the described adaptive tagma (I) 3 ' in described first stem district (II).
6. the probe that the dual signal triggered according to the arbitrary described based target of claim 1-5 increases, it is characterized in that, the part that the 5 ' end in described first stem district (II) is positioned at described ring-shaped area be hold from 3 ' 1-4 base be the sequence of T-T-T-T.
7. the probe that the dual signal triggered according to the arbitrary described based target of claim 1-6 increases, it is characterized in that, described G-tetra-serobila sequence area (IV) has the sequential structure as shown in SEQ ID No.1.
8. detect a method for protein molecule, it is characterized in that, the probe of the dual signal amplification utilizing arbitrary described based target in claim 1-7 to trigger detects.
9. method according to claim 8, is characterized in that, comprises the steps:
Probe and the excision enzyme III of getting the dual signal amplification that described based target triggers respectively join in solution to be measured, 80-100 minute is hatched at 20-40 DEG C, subsequently to wherein adding protohemine and HEPES damping fluid, hatch 50-70 minute at 20-40 DEG C, then add luminol,3-aminophthalic acid cyclic hydrazide and H wherein
2o
2, carry out chemoluminescence measurement, observe or detect luminescence or the discoloration of mixed solution.
10. method according to claim 8 or claim 9, it is characterized in that, probe and the excision enzyme III of getting the dual signal amplification that described based target triggers respectively join in solution to be measured, 90 minutes are hatched at 37 DEG C, subsequently to wherein adding protohemine and HEPES damping fluid, hatch 60 minutes at 30 DEG C, then add luminol,3-aminophthalic acid cyclic hydrazide and H wherein
2o
2, carry out chemoluminescence measurement, observe or detect luminescence or the discoloration of mixed solution.
In 11. claim 1-7, the probe of the dual signal amplification that arbitrary described based target triggers is in the purposes detecting protein molecule or photodetector system field.
12. purposes according to claim 11, is characterized in that, described protein molecule is the protein that can be combined with DNA.
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