CN104711347A - Label-free fluorescence aptamer sensor detection adenosine based on double-amplification strategy construction - Google Patents
Label-free fluorescence aptamer sensor detection adenosine based on double-amplification strategy construction Download PDFInfo
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- 239000002126 C01EB10 - Adenosine Substances 0.000 title claims abstract description 90
- 229960005305 adenosine Drugs 0.000 title claims abstract description 90
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 38
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 31
- 238000001514 detection method Methods 0.000 title claims abstract description 19
- 238000010276 construction Methods 0.000 title claims abstract description 13
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Abstract
The invention discloses a label-free fluorescence aptamer sensor detection adenosine based on double-amplification strategy construction. A method comprises the following steps: (1), preparation of a streptavidin magnetic sphere probe; (2), DNA circulation and HCR reaction assisted by EXo III; (3), fluorescence measurement: after the HCR reaction is completed, adding NMM and KCl to reaction products, and carrying out fluorescence measurement after incubation to obtain a fluorescence value; (4), calculation: calculating the concentration of adenosine, in an object to be detected, containing the adenosine by utilizing the following linear equation according to the measured fluorescence value. The combination of two signal amplification strategies provides higher sensitivity for the detection of low adenosine level in a body fluid; the method can also prominently distinguish the content of adenosine in the urine of a cancer patient with the content of adenosine in the urine of a normal person, and the method can provide a novel reliable strategy for quantifying the adenosine for medical research and early clinical diagnosis.
Description
Technical field
The mark fluorescent aptamer sensor of exempting from that the present invention relates to the dual amplification construction of strategy of a kind of DNA circulation of assisting based on exonucleaseⅢ and cross chain reaction detects adenosine.
Background technology
Adenosine is a kind of endogenous nucleoside, is also ATP degraded product, and it plays very important effect [1,2] in central nervous system, peripheral nervous system unify immunity system.Recently, increasing evidence shows that adenosine has the effect [3-5] promoting tumor promotion.Therefore, as a kind of potential tumor markers, the Sensitive Detection of adenosine is to understanding it at tumor cell proliferation with further illustrate it functionally have important effect in cancer diagnosis and treatment.Traditional method comprises high performance liquid chromatography (HPLC) [6,7], capillary electrophoresis [8] and radioimmunoassays [9], but these methods have some limitation, the harm of such as loaded down with trivial details sample pre-treatments, larger sample and reagent dosage, muting sensitivity and radioelement.Alternatively, fluorescence aptamer sensor because it is easy, quick, the advantage of highly selective and sensitivity causes increasing concern.In order to obtain fluorescent signal, investigator establishes some common methods [10-12] being labeled as basis.Dong seminar has enough built a kind of fluorescence aptamer sensor [12] increased based on graphene oxide and exonucleaseⅢ subsidiary signal.This aptamer sensor achieves the Sensitive Detection to adenosine, but the step of mark may reduce the performance of signal probe.In addition, complicacy and high cost also can limit the generally application of the method.
Test to simplify and reduce costs, investigator establishes some fluorescence aptamer sensor of exempting to mark for detecting adenosine [13-15].Lu seminar establishes a kind of aptamer sensor based on malachite green fluorescence, realizes exempting from marker detection [13] to adenosine, but because target compound and signal are the ratios of 1:1, the sensitivity of this method is relatively low, and detection is limited to 2.0 × 10
-5mol L
-1.In order to improve sensitivity, the strategy of some amplification of signal is introduced in the structure of exempting from mark fluorescent aptamer sensor, for the detection analysis [16-18] of adenosine.In the middle of these methods, our seminar report the self-assembly of a kind of based target thing catalysis hair fastener exempt from mark aptamer sensor for the augmentation detection [17] of adenosine, by adopting amplification of signal, the sensitivity of the method improves, and detects and is limited to 6.0 × 10
-6mol L
-1but this does not still reach the detection level [19-21] of low adenosine content in body fluid.Therefore, the sensitivity that adenosine detects needs to improve further.
In addition, the aptamer sensor reported in patent " aptamer sensor triggering chain type hybridization based on colocalization detects adenosine application number: 20140344669.2 " based on a kind of chain type hybridization of being induced by colocalization detects adenosine, it mainly utilizes HCR and SYBR Green I to produce the fluorescent signal of dual amplification, simultaneously in conjunction with colocalization and the centrifugation of magnetic ball to realize low background.But, because this invention will identify that the fit chain of adenosine is divided into two portions, this reduces fit binding constant own, affect combination that is fit and target compound.And the present invention is the fluorescence sense design adopting different principle---the DNA circulation that Exo III assists and the novel fluorescence aptamer sensor that HCR builds, realize the Sensitive Detection to adenosine; The present invention simultaneously adopts complete aptamer structure, avoids the problem that above-mentioned binding constant is low.
In in the past 10 years, many amplification of signal strategies are established [22-25] as a kind of instrument of effective raising target compound detection sensitivity.The DNA circulation method that Exo III assists is one of common method.Be different from nicking restriction endonuclease, Exo III, can optionally catalysis double-stranded DNA without the need to special recognition site, from its 3 ' overhang or built-in end, be progressively hydrolyzed into mononucleotide [23,26-28].By the DNA circulation reaction that Exo III is auxiliary, a large amount of single stranded DNAs can be obtained for amplification of signal.But, adopt Exo III to assist most of work of DNA circulation all to relate to markers step, be such as marked with fluorophore for optical detection, or be marked with oxidizing reducing group for electrochemical analysis [12,27,29,30].
Summary of the invention
For the deficiencies in the prior art, the object of this invention is to provide and a kind ofly exempt from mark fluorescent aptamer sensor based on dual amplification construction of strategy, this aptamer sensor is used for the analyzing and testing of adenosine.
In order to obtain exempt from mark assist DNA circulation method for detecting adenosine based on Exo III, combine cross chain reaction (HCR) and produce a large amount of G-tetrad structures, obtain effective signal by the binding of many N-methyl porphyrins dipropionic acid IX (NMM) and export and amplification of signal [31-33].By these two kinds of amplification of signal strategies, construct the fluorescence aptamer sensor of a kind of Novel free mark and dual amplification for Sensitive Detection adenosine.First, three functional probes that comprises catalysis chain, fit chain and streptavidin magnetic ball are constructed.The function of this streptavidin magnetic ball is that target compound is carried out to enrichment and is separated thus obtains low background signal.After target compound is by fit chain identification, catalysis chain can be released immediately.Then, catalysis chain triggers the auxiliary DNA circulation of Exo III, produces a large amount of DNA fragmentations, thus obtains first time amplification.Subsequently, DNA fragmentation causes HCR as triggering chain, forms the duplex with many G-tetrad structures, obtains second time amplification.Finally, NMM is inserted in G-tetrad structure, produces the fluorescence response strengthened, and achieves the detection exempting to mark.In the method, a small amount of adenosine can be converted to a large amount of DNA and trigger chain, causes the amplification of target compound.The method has high sensitivity, detects and is limited to 4.2 × 10
-7mol L
-1, this mainly gives the credit to Exo III and assists the dual amplification effect of DNA circulation and HCR and the low background signal of streptavidin magnetic ball.What is more important, the method can distinguish the content of adenosine in cancer patient and normal people's urine sample significantly, shows that our method can provide a kind of reliable strategy quantitative for adenosine newly for medical research and early clinical diagnosis.
The present invention is achieved by the following technical solutions:
Exempt from a mark fluorescent aptamer sensor based on dual amplification construction of strategy, comprise catalysis chain, fit chain and streptavidin magnetic ball three functional probe;
Wherein, described catalysis chain (cDNA) is 5 '-ACT GCA TCG CAA TAC TCG ATT TTT T-3 ' (nucleotide sequence is as shown in SEQ ID NO.2);
Described fit chain (Bio-Apt) is 5 '-biotin-TTT TTT TTT ACC TGG GGG AGT ATT GCG GAGGAA GTT-3 ' (as shown in SEQ ID NO.1);
Described streptavidin magnetic ball, be conventional products existing in prior art, density is 1.343g mL
-1, diameter is 350nm.
Wherein, the Streptavidin magnetic ball of interpolation is suspension, and concentration is 3.324 × 10
11beads mL
-1, the volume of interpolation accounts for 1/11 of overall solution volume, and in the solution, the concentration of catalysis chain is 4.55 μMs, and the concentration of fit chain is 4.55 μMs.
Describedly exempt from mark fluorescent aptamer sensor based on dual amplification construction of strategy, as follows for the process detecting adenosine:
(1) preparation of streptavidin magnetic ball-probe: because streptavidin magnetic ball contains exhibiting high surface promoting agent, in order to prevent the gathering of magnetic ball, needs these tensio-active agents to wash away, to activate the function of magnetic ball before use.Concrete steps are as follows:
First, with TTL damping fluid to the washing of streptavidin magnetic ball, the streptavidin magnetic ball after activating is obtained.
Then, by point Bio-Apt and cDNA an installed annealing, room temperature is cooled to; Subsequently both mixing are blended in incubated at room temperature with the magnetic ball of streptavidin after activation.
Finally, the streptavidin magnetic ball PBS buffer solution after hatching, to remove unnecessary cDNA, obtains streptavidin magnetic ball-probe.Washing steps all in this work all completes under additional the action of a magnetic field.
Consisting of of described TTL damping fluid: be made up of Tris-HCl, Tween20, LiCl and water, wherein, the concentration of each component is: 0.1mol L
-1tris-HCl, pH 8.0,0.1% (v/v) Tween-20,1.0mol L
-1liCl.
Consisting of of described PBS damping fluid: by NaCl, NaH
2pO
4, Na
2hPO
4, and water composition, wherein, the concentration of each component is: 0.15mol L
-1naCl, 7.6mmol L
-1naH
2pO
4, 2.4mmol L
-1na
2hPO
4, pH 7.4.
Further, it is 5 times with the number of times that TTL damping fluid washs streptavidin magnetic ball.
Further, annealing temperature is 95 DEG C, and the time is 5min.
Further, incubation time is 2h.
Further, the number of times of streptavidin magnetic ball PBS buffer solution is 2 times.
(2) DNA circulation that Exo III is auxiliary and HCR reaction:
First, the thing to be detected containing adenosine and TNaK damping fluid are joined in test tube, at room temperature hatches; Subsequently, under the action of a magnetic field, isolate streptavidin magnetic ball, and retain in reaction solution to new centrifuge tube.
Then, get probe HP respectively, Exo III and 10 × Exo III damping fluid join in centrifuge tube.Carrying out ExoIII after mixing assists DNA circulation to react.After being hydrolyzed, heat the activity stopping enzyme.
Finally, add hair fastener probe H1 and H2, HCR reaction is carried out in mixing.
Described probe HP is 5 '-GCA ATA CTC GAA CAC GTT ACC-3 ' (nucleotide sequence is as shown in SEQ IDNO.3);
Described probe H1 is 5 '-CTC GAA CAC GTT ACC GGG TAG GGC GTT AGG AGG TAACGT GTT CGA GTA TTG C-3 ' (nucleotide sequence is as shown in SEQ ID NO.11).
Described probe H2 is 5 '-TGG GTT CCT AAC GCC CTA CCC GGT AAC GTG TTC GAG GCAATA CTC GAA CAC GTT ACC GGG TAG GGC GGG-3 ' (nucleotide sequence is as shown in SEQ ID NO.12).
Consisting of of described TNaK damping fluid: Tris, NaCl, KCl and water composition, wherein the concentration of each component is: 20mmolL
-1tris, 600mmol L
-1naCl, 5mmol L
-1kCl, pH 7.5.Addition is 15 μ L.
Consisting of of described 10 × Exo III damping fluid: Tris-HCl, MgCl
2, DTT and water composition, wherein, the concentration of each component is: 0.5mol L
-1tris-HCl, 50mmol L
-1mgCl
2, 10mmol L
-1dTT.
The concentration of described probe HP is 5 μMs, and consumption is 5 μ L.
The consumption of described Exo III is 0.20 ~ 0.40U mL
-1.
The concentration of described hair fastener probe H1 is 0.8 × 10
-6~ 1.2 × 10
-6m, preferred concentration 1.0 × 10
-6m, consumption is 5 μ L.
The concentration of described hair fastener probe H2 is 1.2 × 10
-6~ 1.8 × 10
-6m, preferred concentration 1.5 × 10
-6m, consumption is 5 μ L.
Further, the urine that the described thing to be detected containing adenosine is behaved, addition is 5 μ L.
Further, incubation time is 2h.
Further, put after mixing and carry out Exo III to 37 DEG C and assist DNA circulation to react.
Further, probe HP, H1 and H2 anneal before the use, and object guarantees the formation of hairpin structure.The step of annealing is: probe HP, H1, H2 anneal 5min at 95 DEG C, and are cooled to room temperature.
Further, after being hydrolyzed, heat the heat-up time of the activity stopping enzyme for 20min, temperature is 75 DEG C.
Further, the temperature of HCR reaction is 37 DEG C.
(3) fluorescence measurement:
After HCR has reacted, in reaction product, add NMM and KCl, carry out fluorescence measurement after hatching, obtain fluorescent value.
The concentration of described NMM is 1.5 × 10
-6~ 2.5 × 10
-6m, preferred concentration is 2.0 × 10
-6m, consumption is 2 μ L.
The concentration of described KCl is 1M, and consumption is 8 μ L.
Further, the temperature of hatching is 37 DEG C, and the time is 20 ~ 40min.
Further, in described fluorescence measurement, the basic setup of fluorophotometer is: under room temperature condition, and excitation wavelength is 399nm, and sweep limit is 550 ~ 680nm, and slit width is 10nm, PMT voltage 700V.
(4) calculate: according to above-mentioned measured fluorescent value, utilize following linear equation to calculate the concentration of adenosine in the thing to be detected containing adenosine.
Further, following linear equation is utilized to calculate the concentration of adenosine:
ΔF＝15.12+143.58×10
5C，r＝0.998；
Wherein, Δ F represents fluorescence net signal value, and C represents the concentration of adenosine, and unit is mol L
-1; The linearity range of this linear equation is 1.0 × 10
-6mol L-1 ~ 1.0 × 10
-4mol L
-1between.
The present invention is based on the principle of design exempting from mark fluorescent aptamer sensor of dual amplification construction of strategy: this Novel free marks and the fluorescence aptamer sensor of Sensitive Detection adenosine forms primarily of three parts, as shown in schematic diagram 1, first the fit chain of adenosine (Bio-Apt) of biotinylation mark is connected on streptavidin magnetic ball, another strand-catalysis chain (cDNA) is complementary with this fit chain, so just constructs three functional probes by catalysis chain, fit chain and streptavidin magnetic ball.Under adenosine existence condition, fit chain is combined with adenosine, and induction aptamer conformation changes, and makes double-stranded DNA unstable, finally causes the release of cDNA.Secondly, cDNA opens hair fastener HP by sticky end, forms the double-stranded DNA with 3 ' flat end.Exo III from 3 ' flush end progressively catalytic hydrolysis double-stranded DNA generation mononucleotide, can discharge cDNA simultaneously and is formed and trigger chain.The cDNA of release binds the cutting process that another HP molecule triggers a new round further, meanwhile, takes turns in cutting process all discharge triggering chain at each.Finally, trigger chain and open hair fastener probe H1 by the strand replacement reaction that sticky end mediates; Similarly, the H1 opened exposes the sequence made new advances can open H2, and H2 includes triggering chain identical sequence, and therefore H2 can open H1 further, causes the autonomous alternately hybridization of two hair fastener probe H1 and H2, forms the duplex with otch.It should be noted that G-tetrad sequence is present in stem and the end of H2, when H2 is in hair fastener state, part G-tetrad sequence is hidden in the stem of H2, therefore can not form G-tetrad structure.But reacted by HCR, G-tetrad sequence self-assembly can form the G-tetrad structure integrated.Therefore, the band otch duplex comprising many G-tetrad structures is defined.At this, select NMM as signal reporter, it has obvious structure selectivity to G-tetrad, inoperative to triploid, double-strand or single stranded form.Once add this reporter to this system, just can by realizing a fluorescence response strengthened with the binding of many G-tetrad structures.
The invention has the beneficial effects as follows:
The present invention establish a kind of exempt to mark and sensitive dual amplification fluorescent aptamer sensor for the detection of adenosine.Assist DNA circulation reaction and HCR to combine by ExoIII, this novel method mainly has following advantage: the detection being combined into low adenosine level in body fluid of (i) two kinds of amplification of signal strategies provides higher sensitivity; (ii) the G-tetrad structure formed in HCR process provides the detection exempting to mark, and make this system easier, and cost is lower; (iii) the method can also distinguish the content of adenosine in cancer patient and normal people's urine significantly, shows that the method can provide a kind of reliable strategy quantitative for adenosine newly for medical research and early clinical diagnosis.
Accompanying drawing explanation
Fig. 1 is the fluorescence aptamer sensor principle of work schematic diagram of DNA circulation and the cross chain reaction of assisting based on Exo III.
The fluorescence pattern of Fig. 2 model experiment, wherein, (a) H1+H2, (b) HP+Exo III+H1+H2, (c) Bio-Apt+cDNA+HP+Exo III+H1+H2, (d) Bio-Apt+cDNA+HP+Exo III+H1+H2+adenosine (1.0 × 10
-3mol L
-1).
Fig. 3 shows: the design of H1 and H2 in HCR.
Fig. 4 is that different Exo III concentration affects schematic diagram to △ F.
Fig. 5 is that different H1 concentration affects schematic diagram to △ F.
Fig. 6 is that different H2 concentration affects schematic diagram to △ F.
Fig. 7 different N MM concentration affects schematic diagram to △ F.
Fig. 8-1 is the response schematic diagram of system fluorescence intensity to adenosine concentration.Wherein, (a) background, (b) 5.0 × 10
-6molL
-1, (c) 1.0 × 10
-5mol L
-1, (d) 2.0 × 10
-5mol L
-1, (e) 5.0 × 10
-5mol L
-1, (f) 8.0 × 10
-5mol L
-1, (g) 1.0 × 10
-4mol L
-1.
Fig. 8-2 is linear relationship schematic diagram of Δ F and adenosine concentration.
Fig. 9 is the selectivity experimental result schematic diagram of adenosine and its analogue.
Embodiment
Below in conjunction with drawings and Examples, the present invention is further described.
In following embodiment, the reagent of not detailed description, method are conventional reagent, the method in affiliated field.
Embodiment 1 exempts from mark fluorescent aptamer sensor based on dual amplification construction of strategy, is used for detecting adenosine.
1. material and reagent
The present embodiment all nucleic acid probes used are by Sangon Biotech's synthesis and purify, and base sequence is as shown in table 1.DNA stock solution is TE Buffer (10mM Tris-HCl, pH 8.0,1mMEDTA-2Na), and is deposited in-20 DEG C.Adenosine is purchased from Sigma-Aldrich (Shanghai, China).Cytidine, uridine, guanosine, Exo III and DMSO are all purchased from the raw work in Shanghai.(diameter 350nm, 0.05%Tween-20, and 10 μMs of EDTA, concentration is 3.324 × 10 to the magnetic ball of streptavidin
11beads mL
-1, 1.343g mL
-1), Tris (> 99.8%) is purchased from AmrescoInc.NMM (N-methylmesoporphyrinIX) is purchased from J & KScientificLtd. (Beijing, China).NMM stock solution adopt DMSO dissolve and stored protected from light at-20 DEG C.In test, all reagent (analytical pure) provides by standard reagent supplier.All solution is prepared by Millipore Milli-Q water (18.25M Ω).Clinical serum and urine sample are all taken from Hospital of Shandong University.In literary composition, M refers to mol/L, and mM refers to mmol/L, μM finger μm ol/L.
Oligonucleotide sequence used in table 1 the present invention
2 experimental procedures
The preparation of 2.1 streptavidin magnetic ball-probes
Because streptavidin magnetic ball contains exhibiting high surface promoting agent, in order to prevent the gathering of magnetic ball, need these tensio-active agents to wash away before use, to activate the function of magnetic ball.First, with the TTL damping fluid (0.1M Tris-HCl, pH8.0,0.1% (v/v) Tween-20,1.0M LiCl) of 200 μ L, 5 times are washed to 1 μ L streptavidin magnetic ball, obtain the streptavidin magnetic ball after activating; Then, anneal point 5 μ L Bio-Apt (10 μMs) installed and 5 μ L cDNA (10 μMs) at 95 DEG C 5min, is cooled to room temperature; Subsequently both mixed and be blended in incubated at room temperature 2h with the streptavidin magnetic ball after activation.Finally, streptavidin magnetic ball PBS damping fluid (0.15M NaCl, the 7.6mM NaH after hatching
2pO
4, 2.4mM Na
2hPO
4, pH 7.4) wash 2 times, to remove unnecessary cDNA.Washing steps all in this work all completes under additional the action of a magnetic field.
Wherein, TTL damping fluid to streptavidin magnetic ball washing methods is: in centrifuge tube, 1. add the TTL damping fluid of 200 μ L and the magnetic ball of 1 μ L streptavidin, after fully shaking washing (shaking about 5min), centrifuge tube is placed on magnetic ball separator frame and leaves standstill 7min, remove solvent portions (with the careful sucking-off solution of liquid-transfering gun, discarding);
2. repeat above-mentioned operation 1. 5 times, finally obtain the magnetic ball (1 μ L) of the streptavidin after activating, stand-by.
The DNA circulation that 2.2Exo III is auxiliary and HCR
This process is carried out in TNaK damping fluid.Anneal probe HP, H1, H2 at 95 DEG C 5min, and be cooled to room temperature to guarantee the formation of hairpin structure.First 5 μ L adenosines and 15 μ L TNaK damping fluids are joined in test tube, at room temperature hatch 2h; Subsequently, under the action of a magnetic field, isolate streptavidin magnetic ball, and retain in reaction solution to new centrifuge tube.Then, 5 μ L HP (5 μMs) are got respectively, Exo III (0.4U mL
-1) and 5 μ L 10 × Exo III damping fluids (0.5M Tris-HCl, pH 8.0,50mM MgCl
2, 10mM DTT) join in centrifuge tube.Put after mixing and carry out Exo III to 37 DEG C and assist DNA circulation to react, the time is 1h.After being hydrolyzed, at 75 DEG C, heat 20min to stop the activity of enzyme.Finally, add 5 μ L H1 (1.0 μMs) and 5 μ L H2 (1.5 μMs), mix and carry out HCR reaction at 37 DEG C, the time is 3.5h.
2.3 fluorescence measurements and instrument
After HCR has reacted, in reaction product, add 2 μ L NMM (5.0 × 10
-5m) and 8 μ L KCl (1M), measure after hatching 30min under 37 DEG C of conditions.All fluorescence measurements all carry out under room temperature at Hitachi F-2500 spectrophotofluorometer (Hitachi, Japan).The basic setup of fluorophotometer is excitation wavelength is 399nm, and sweep limit is 550 ~ 680nm, and slit width is 10nm, PMT voltage 700V.
3. result and discussion
The principle of design of 3.1 fluorescence aptamer sensor
This Novel free marks and the fluorescence aptamer sensor of Sensitive Detection adenosine forms primarily of three parts, as shown in schematic diagram 1, first the fit chain of adenosine (Bio-Apt) of biotinylation mark is connected on streptavidin magnetic ball, another strand-catalysis chain (cDNA) is complementary with this fit chain, so just constructs three functional probes by catalysis chain, fit chain and streptavidin magnetic ball.Under adenosine existence condition, fit chain is combined with adenosine, and induction aptamer conformation changes, and makes double-stranded DNA unstable, finally causes the release of cDNA.Secondly, cDNA opens hair fastener HP by sticky end, forms the double-stranded DNA with 3 ' flat end.Exo III from 3 ' flush end progressively catalytic hydrolysis double-stranded DNA generation mononucleotide, can discharge cDNA simultaneously and is formed and trigger chain (nucleotide sequence is as shown in SEQ ID NO.4).The cDNA of release binds the cutting process that another HP molecule triggers a new round further, meanwhile, takes turns in cutting process all discharge triggering chain at each.Finally, trigger chain and open hair fastener probe H1 by the strand replacement reaction that sticky end mediates; Similarly, the H1 opened exposes the sequence made new advances can open H2, and H2 includes triggering chain identical sequence, and therefore H2 can open H1 further, causes the autonomous alternately hybridization of two hair fastener probe H1 and H2, forms the duplex with otch.It should be noted that G-tetrad sequence is present in stem and the end of H2, when H2 is in hair fastener state, part G-tetrad sequence is hidden in the stem of H2, therefore can not form G-tetrad structure.But reacted by HCR, G-tetrad sequence self-assembly can form the G-tetrad structure integrated.Therefore, the band otch duplex comprising many G-tetrad structures is defined.At this, we select NMM as signal reporter, and it has obvious structure selectivity to G-tetrad, inoperative to triploid, double-strand or single stranded form.Once add this reporter to this system, just can by realizing a fluorescence response strengthened with the binding of many G-tetrad structures.
The feasibility of 3.2 designs
In order to verify now methodical feasibility, We conducted the fluorescence emission spectrum under different condition, as shown in Figure 2.In Fig. 2, curve a represents fluorescence spectrum when only having H1 and H2, shows that the design of two kinds of hairpin structures is revealed very little, enough stable; Curve b represents the fluorescence pattern containing HP, Exo III, H1 and H2 in system, can be observed faint fluorescence and increases; Curve c is a little growth fluorescent value produced add Bio-Apt and cDNA again in system after, and at this, we define it for background signal.The leakage of fuel hairpin structure when this background signal mainly to come between Bio-Apt and cDNA-breathes ‖ effect and do not trigger agent.When adding adenosine in system, showing a fluorescence response obviously strengthened, as shown in curve d in Fig. 2, showing that this adenosine detection system has significant amplification capability.
The optimization of sequences Design in 3.3HCR
In HCR process, the acquisition of sequences Design to signal of hair fastener probe (H1 and H2) has vital effect.Rational hairpin structure design effectively can reduce the leakage of signal.At this, in order to obtain stronger fluorescent signal, We conducted four kinds of different sequences Design, G-tetrad sequence is incorporated in hair fastener probe, their sequences Design is as shown in table 1: the G-tetrad sequence of (1) 1/2 is hidden in the stem of H2, and residue 1/2 is at the end of H2; The G-tetrad sequence of (2) 3/4 is hidden in the stem of H2, and residue 1/4 is at the end of H2; The G-tetrad sequence of (3) 1/2 is hidden in the stem of H2, and residue 1/2 is at the end of H2, but the stem of H2 increases to some extent; The G-tetrad sequence of (4) 3/4, at the 3 ' end of H2, remains 1/4 and holds at 5 ' of H2.As can be seen from Fig. 3 we, design 1 (H1-1 and H2-1; Nucleotide sequence is as shown in SEQ ID NO.5 and SEQ ID NO.6) and design 2 (H1-2 and H2-2; Nucleotide sequence is as shown in SEQ ID NO.7 and SEQ ID NO.8) create higher background signal, may be form G-tetrad structure because hair fastener probe a little when not triggering chain is opened, thus be combined with NMM and create fluorescent signal.In order to reduce background signal further, the stem of H2 in design 1 increases by we, designs 3 (H1-3 and H2-3 by Fig. 3; Nucleotide sequence is as shown in SEQ ID NO.9 and SEQ ID NO.10) known, negative signal obtains and effectively reduces, but unfortunately, positive signal is also suppressed.Therefore We conducted further design (design 4 (H1-4 and H2-4; Nucleotide sequence is as shown in SEQ ID NO.11 and SEQ ID NO.12)), in design 4, G-tetrad sequence is present in the two ends of H2, and background signal obtains obvious suppression.Consider above-mentioned four kinds of designs, final selection design 4 is as optimal design.
Its conditional is C
trigger=5.0 × 10
-7mol L
-1, C
h1=1.0 × 10
-6mol L
-1, C
h2=1.5 × 10
-6mol L
-1, C
nMM=2.0 × 10
-6mol L
-1, temperature of reaction 37 DEG C.
The optimization of 3.4 reaction conditionss
In order to obtain the optimal performance of this sensor, we adopted net signal △ F (△ F=F2-F1) as standard survey reaction terms and conditions, be that target compound does not exist the fluorescence intensity with G-tetrad-NMM mixture under existence condition respectively at this F1 and F2.
It is reported that the concentration of Exo III has very important impact to the performance that Exo III assists DNA circulation to react.Shown in Fig. 4, along with the increase of Exo III concentration, △ F value increases fast, to 0.40U mL
-1time reach maximum value then held stationary.Therefore, we select 0.40U mL
-1as the consumption that Exo III is final.
Meanwhile, we have also investigated the concentration of probe to obtain optimum letter multiple proportions.In our study, H1 and H2 is amplification probe, and NMM is signal probe.The concentration of these probes is all at adenosine concentration 1.0 × 10
-3mol L
-1carry out under condition measuring, shown in Fig. 5-7, when the concentration of H1, H2 and NMM is respectively 1.0 × 10
-6mol L
-1, 1.5 × 10
-6molL
-1, 2.0 × 10
-6mol L
-1time, △ F reaches maximum value, therefore selects these concentration as optimal concentration.
Wherein, during the optimization of Exo III concentration, condition is: C
adenosine=1.0 × 10
-3mol L
-1, C
bio-Apt=C
cDNA=1.0 × 10
-6mol L
-1, C
hP=5.0 × 10
-7mol L
-1, C
h1=1.0 × 10
-6mol L
-1, C
h2=1.5 × 10
-6mol L
-1, C
nMM=2.0 × 10
-6mol L
-1, temperature of reaction 37 DEG C.
During the optimization of H1 concentration, condition is: C
adenosine=1.0 × 10
-3mol L
-1, C
bio-Apt=C
cDNA=1.0 × 10
-6molL
-1, C
hP=5.0 × 10
-7mol L
-1, C
h2=1.5 × 10
-6mol L
-1, C
nMM=2.0 × 10
-6mol L
-1, Exo III=0.40U mL
-1, temperature of reaction 37 DEG C.
During the optimization of H2 concentration, condition is: C
adenosine=1.0 × 10
-3mol L
-1, C
bio-Apt=C
cDNA=1.0 × 10
-6molL
-1, C
hP=5.0 × 10
-7mol L
-1, C
h1=1.0 × 10
-6mol L
-1, C
nMM=2.0 × 10
-6mol L
-1, Exo III=0.40U mL
-1, temperature of reaction 37 DEG C.
The optimal conditions of NMM concentration is: C
adenosine=1.0 × 10
-3mol L
-1, C
bio-Apt=C
cDNA=1.0 × 10
-6molL
-1, C
hP=5.0 × 10
-7mol L
-1, C
h1=1.0 × 10
-6mol L
-1, C
h2=1.5 × 10
-6mol L
-1, Exo III=0.40U mL
-1, temperature of reaction 37 DEG C.
Other conditions do not mentioned, all carry out according to the condition on-2 experimental procedure ‖ herein.
The analytical performance of 3.5 aptamer sensor
We analyze the fluorescence aptamer sensor performance of this detection by quantitative adenosine.The different fluorescence response values that Fig. 8-1 is corresponding under showing different adenosine concentration condition.When the concentration of adenosine is increased to 1.0 × 10 gradually from 0
-4mol L
-1time, fluorescence intensity is theatrical enhancing thereupon also.Under optimum experiment condition, the method is linearly good, and as shown in Fig. 8-2, linearity range is 1.0 × 10
-6mol L
-1-1.0 × 10
-4mol L
-1between, relation conefficient is 0.998.Detection is limited to 4.2 × 10
-7mol L
-1.What in contrast to that other have reported exempts from mark and amplification strategy [16-18], and our method provides higher sensitivity.
In order to investigate the specific recognition effect that this aptamer sensor detects adenosine, we test adenosine and analogue (uridine, guanosine and cytidine) thereof.As shown in Figure 9, system shows faint fluorescence response to neplanocin, and its fluorescence intensity is almost identical with background signal.But due to fit to the specific recognition effect of target compound, after adding adenosine, fluorescence has obvious enhancing.Meanwhile, the fluorescence response that we have also demonstrated adenosine and four kinds of mixture of nucleosides does not almost have difference.The aptamer sensor that above-mentioned phenomenon demonstrates us has good selectivity, effectively can distinguish adenosine and its analogue.
In order to investigate the application of this aptamer sensor in actual biological specimen, having carried out the experiment of the adenosine rate of recovery in complex biological sample, having comprised human serum sample and urine sample, result is as shown in table 2 and 3.The rate of recovery of adenosine in serum is between 96.2%-107.0%, and relative standard deviation is at 1.6%-3.3%.Meanwhile, the rate of recovery of adenosine in urine sample is between 97.8%-109.2%, and relative standard deviation is 1.7%-3.6%.Result show the precision of the method in complex biological sample and the rate of recovery satisfactory.
Sample recovery rate in table 2 human serum
Sample recovery rate in table 3 people urine sample
Subsequently, carried out the detection of adenosine content to the urine sample of three groups of normal peoples and cancer patient respectively, as shown in table 4, the adenosine concentration of three groups of normal people's urine samples concentrates on 3.4 × 10
-6mol L
-1to 4.8 × 10
-6mol L
-1, adenosine concentration scope (2.0 × 10 normal with people's urine sample of bibliographical information
-6mol L
-1to 7.0 × 10
-6mol L
-1) conform to [19,21].In addition, the adenosine concentration of three groups of normal people's urine samples concentrates on 2.2 × 10
-5mol L
-1to 4.3 × 10
-5mol L
-1.Bibliographical information adenosine content in cancer patient urine sample 5 ~ 10 times [34] higher than normal people.Therefore result shows, the inventive method has potential using value in the clinical diagnosis of actual sample.
Table 4 the inventive method is to the actual sample analysis of normal people and cancer patient
By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.
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Claims (10)
1. exempt from a mark fluorescent aptamer sensor based on dual amplification construction of strategy, it is characterized in that: comprise catalysis chain, fit chain and streptavidin magnetic ball three functional probe;
Wherein, described catalysis chain is: 5 '-ACT GCA TCG CAA TAC TCG ATT TTT T-3 ', and nucleotide sequence is as shown in SEQ ID NO.2;
Described fit chain is: 5 '-biotin-TTT TTT TTT ACC TGG GGG AGT ATT GCG GAG GAA GTT-3 ', nucleotide sequence is as shown in SEQ ID NO.1.
2. as claimed in claim 1 exempt from mark fluorescent aptamer sensor based on dual amplification construction of strategy, it is characterized in that: the Streptavidin magnetic ball of interpolation is suspension, and concentration is 3.324 × 10
11beads mL
-1, the volume of interpolation accounts for 1/11 of overall solution volume, and in the solution, the concentration of catalysis chain is 4.55 μm of ol L
-1, the concentration of fit chain is 4.55 μm of ol L
-1.
3. the mark fluorescent aptamer sensor of exempting from based on dual amplification construction of strategy according to claim 1 is detecting the application in adenosine.
4. the method exempting from mark fluorescent aptamer sensor detection adenosine based on dual amplification construction of strategy according to claim 1, it is characterized in that, concrete steps are as follows:
(1) preparation of streptavidin magnetic ball-probe:
First, with TTL damping fluid, the magnetic ball of streptavidin is washed, obtain the magnetic ball of the streptavidin after activation;
Then, point fit chain installed and catalysis chain are annealed, after annealing, is cooled to room temperature; Subsequently both mixed and be blended in incubated at room temperature with the streptavidin magnetic ball after activation;
Finally, the streptavidin magnetic ball PBS buffer solution after hatching, obtained streptavidin magnetic ball-probe;
(2) DNA circulation that Exo III is auxiliary and HCR reaction:
First, the thing to be detected containing adenosine and TNaK damping fluid are joined in test tube, at room temperature hatches; Subsequently, under the action of a magnetic field, isolate streptavidin magnetic ball, and retain in reaction solution to new centrifuge tube;
Then, get probe HP respectively, Exo III and 10 × Exo III damping fluid join in centrifuge tube; Carrying out Exo III after mixing assists DNA circulation to react; After being hydrolyzed, heat the activity stopping enzyme;
Finally, add hair fastener probe H1 and probe H2, HCR reaction is carried out in mixing;
(3) fluorescence measurement
After HCR has reacted, in reaction product, add NMM and KCl, carry out fluorescence measurement after hatching, obtain fluorescent value;
(4) calculate: utilize following linear equation to calculate the concentration of adenosine in the thing to be detected containing adenosine according to above-mentioned measured fluorescent value.
5. the method detecting adenosine as claimed in claim 4, is characterized in that: in step (1), all washing steps all complete under additional the action of a magnetic field;
The magnetic ball washing times of TTL damping fluid to streptavidin is 5 times; Described annealing temperature is 95 DEG C, and the time is 5min; Described incubation time is 2h; The streptavidin magnetic ball number of times 2 times of PBS buffer solution;
Consisting of of described TTL damping fluid: be made up of Tris-HCl, Tween20, LiCl and water, wherein, the concentration of each component is: 0.1mol L
-1tris-HCl, pH 8.0,0.1% (v/v) Tween-20,1.0mol L
-1liCl;
Consisting of of described PBS damping fluid: by NaCl, NaH
2pO
4, Na
2hPO
4, and water composition, wherein, the concentration of each component is: 0.15mol L
-1naCl, 7.6mmol L
-1naH
2pO
4, 2.4mmol L
-1na
2hPO
4, pH 7.4.
6. the method detecting adenosine as claimed in claim 4, is characterized in that: in step (2),
The urine that the described thing to be detected containing adenosine is behaved, consumption is 5 μ L;
Consisting of of described TNaK damping fluid: Tris, NaCl, KCl and water composition, wherein the concentration of each component is: 20mmolL
-1tris, 600mmol L
-1naCl, 5mmol L
-1kCl, pH 7.5; Addition is 15 μ L;
Described incubation time is 2h;
Described probe HP is: 5 '-GCA ATA CTC GAA CAC GTT ACC-3 ', and nucleotide sequence is as shown in SEQ ID NO.3; Concentration is 5 μm of ol L
-1, consumption is 5 μ L;
Consisting of of described 10 × Exo III damping fluid: Tris-HCl, MgCl
2, DTT and water composition, wherein, the concentration of each component is: 0.5mol L
-1tris-HCl, 50mmol L
-1mgCl
2, 10mmol L
-1dTT; Addition is 5 μ L.
7. the method detecting adenosine as claimed in claim 4, is characterized in that: in step (2),
The consumption of described Exo III is 0.20 ~ 0.40U mL
-1;
Put after mixing and carry out Exo III to 37 DEG C and assist DNA circulation to react;
Described probe HP, H1 and H2 anneal before the use, and the step of annealing is: probe HP, H1, H2 anneal 5min at 95 DEG C, and are cooled to room temperature;
After being hydrolyzed, heat the heat-up time of the activity stopping enzyme for 20min, temperature is 75 DEG C;
The temperature of described HCR reaction is 37 DEG C.
8. the method detecting adenosine as claimed in claim 4, is characterized in that: in step (2),
Described hair fastener probe H1 is: 5 '-CTC GAA CAC GTT ACC GGG TAG GGC GTT AGG AGG TAA CGT GTT CGA GTA TTG C-3 ', and nucleotide sequence is as shown in SEQ ID NO.11; Concentration is 0.8 × 10
-6~ 1.2 × 10
-6mol L
-1, consumption is 5 μ L;
Described probe H2 is: 5 '-TGG GTT CCT AAC GCC CTA CCC GGT AAC GTG TTC GAG GCA ATA CTC GAA CAC GTT ACC GGG TAG GGC GGG-3 ', and nucleotide sequence is as shown in SEQ ID NO.12; Concentration is 1.2 × 10
-6~ 1.8 × 10
-6mol L
-1, consumption is 5 μ L.
9. the method detecting adenosine as claimed in claim 4, it is characterized in that: in step (3), the concentration of described NMM is 1.5 × 10
-6~ 2.5 × 10
-6mol L
-1, consumption is 2 μ L; The concentration of described KCl is 1mol L
-1, consumption is 8 μ L;
In described fluorescence measurement, the basic setup of fluorophotometer is: under room temperature condition, and excitation wavelength is 399nm, and sweep limit is 550 ~ 680nm, and slit width is 10nm, PMT voltage 700V.
10. the method detecting adenosine as claimed in claim 4, is characterized in that: in step (4), utilizes following linear equation to calculate the concentration of adenosine: Δ F=15.12+143.58 × 10
5c, r=0.998;
Wherein, Δ F represents fluorescence net signal value, and C represents the concentration of adenosine, and unit is mol L
-1; The linearity range of this linear equation is 1.0 × 10
-6mol L-1 ~ 1.0 × 10
-4mol L
-1between.
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| CN105624165A (en) * | 2016-01-05 | 2016-06-01 | 山东大学 | Cascade amplifying-strategy biomolecule detecting method based on self-locking aptamer probe |
| CN106048024A (en) * | 2016-06-17 | 2016-10-26 | 山东大学 | Method for detecting adenosine by using fluorescent signal amplification biological sensing platform established based on target object-induced adjacent combination |
| CN108624620A (en) * | 2018-04-02 | 2018-10-09 | 湖南大学 | DNA nanometers of tetrad realizations are efficiently conveyed to nucleic acid and the super sensitivity detection method of miRNA |
| CN110095608A (en) * | 2019-04-12 | 2019-08-06 | 南方医科大学南方医院 | Tumour excretion body nano fluorescent sensor based on Magnetic Isolation and DNA self assembly |
| CN110305770A (en) * | 2019-07-17 | 2019-10-08 | 中国科学院上海高等研究院 | A kind of micro-fluidic chip of DNA nanostructure modification is sensed and its prepared for optical bio and application |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105624165A (en) * | 2016-01-05 | 2016-06-01 | 山东大学 | Cascade amplifying-strategy biomolecule detecting method based on self-locking aptamer probe |
| CN105624165B (en) * | 2016-01-05 | 2018-11-30 | 山东大学 | The biomolecule detecting method of cascade amplification strategy based on self-locking aptamer probe |
| CN106048024A (en) * | 2016-06-17 | 2016-10-26 | 山东大学 | Method for detecting adenosine by using fluorescent signal amplification biological sensing platform established based on target object-induced adjacent combination |
| CN108624620A (en) * | 2018-04-02 | 2018-10-09 | 湖南大学 | DNA nanometers of tetrad realizations are efficiently conveyed to nucleic acid and the super sensitivity detection method of miRNA |
| CN110095608A (en) * | 2019-04-12 | 2019-08-06 | 南方医科大学南方医院 | Tumour excretion body nano fluorescent sensor based on Magnetic Isolation and DNA self assembly |
| CN110305770A (en) * | 2019-07-17 | 2019-10-08 | 中国科学院上海高等研究院 | A kind of micro-fluidic chip of DNA nanostructure modification is sensed and its prepared for optical bio and application |
| CN110305770B (en) * | 2019-07-17 | 2022-07-08 | 中国科学院上海高等研究院 | DNA nanostructure modified micro-fluidic chip for optical biosensing, and preparation and application thereof |
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