CN103439296B - Based on the aptamer sensor construction method of the two amplifying technique of Au NPs and DNA circulation and the application in adenosine detects thereof - Google Patents
Based on the aptamer sensor construction method of the two amplifying technique of Au NPs and DNA circulation and the application in adenosine detects thereof Download PDFInfo
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Abstract
The invention discloses a kind of aptamer sensor based on the two amplifying technique of Au NPs and DNA circulation to build and application in adenosine detects, belong to aptamers field of sensing technologies.This detection system comprise adenosine aptamers, with the DNA probe of aptamers partial hybridization, hairpin structure DNA that Au NPs marks and the hairpin structure DNA that is assembled in gold plaque surface.When there is no adenosine, Au NPs marker DNA keep hairpin structure and can not with the hair clip DNA hybridization on gold plaque surface, Au NPs cannot be captured gold plaque surface.When there is adenosine, adenosine is combined with its aptamers, DNA probe then discharges and makes its open loop with the hair clip DNA hybridization that Au NPs marks from aptamers/DNA probe double-strand, Au NPs captures on gold plaque by the Au NPs marker DNA after open loop and the DNA hybridization on gold plaque surface, the DNA probe replaced out causes next round DNA chain and replaces reaction, circulation like this, single adenosine molecule can cause a large amount of Au NPs and be assembled on gold plaque, obtain the spr signal strengthened, for the highly sensitive detection to adenosine.
Description
Technical field
The present invention relates to a kind of aptamer sensor based on the two amplifying technique of Au NPs and DNA circulation to build and application in adenosine detects, belong to aptamers field of sensing technologies.
Background technology
Aptamers refers to that the Fas lignand system evolution technology in-vitro screening through index concentration synthesizes the few core former times acid sequence of a bit of strand obtained, and has high affinity and specific recognition capability to target molecule.Compared with being recognition component with antibody, aptamers has the advantages such as part extensively, easily synthesizes, easy mark, chemical stability are good, is widely used in identification and the detection of target molecule.Up to now, existing various take aptamer as the report of the colorimetric sensor of recognition element, fluorescent optical sensor, electrochemical sensor, luminescence sensor, quartz crystal microbalance sensor, surface plasma body resonant vibration (SPR) sensor.Wherein, based on the aptamer sensor of SPR detection method because having a good application prospect without the need to advantages such as mark, highly sensitive and Real-Time Monitorings.Major part SPR aptamer sensor adopts sandwich method of inspection, target to cause conformational change or target to cause chain and replace and the detection competed the principle such as to combine and realize biomolecule.Although the detection of these sensors to target molecule has higher selectivity and sensitivity, their mostly press signal of 1:1: object ratio output signal, cannot realize the highly sensitive detection of low concentration material particularly Small molecular and nucleic acid etc.Therefore, high-sensitive SPR sensing technology is developed significant for detecting low concentration biomolecule.
In recent years, amplified nucleic acid molecule technology is widely used in improving the detection sensitivity of aptamer sensor, and e.g., target induction is replaced polymerization, rolled ring amplification, polymerase chain reaction, signal iodine etc. based on endonuclease.Although these methods improve detection sensitivity, they often need to use proteinase, not only increase analysis cost, also need the reaction conditions accurately controlling proteinase, greatly limit application.Develop without enzyme amplification method particularly important for the highly sensitive detection of biomolecule.Based on hybridization and chain replace without enzyme amplifying technique (as hybridization chain amplified reaction, entropy drive catalysis, intended catalyzed hair clip assembles), because pattern is intrinsic, be easy to expanding, without the need to using the features such as proteinase, having broad application prospects.But, there is not yet so far based on the SPR aptamer sensor without enzyme amplifying technique for the report of the highly sensitive detection of Small molecular.
Summary of the invention
The object of the present invention is to provide a kind of aptamer sensor based on Au NPs and the two amplifying technique of DNA circulation to build and application in adenosine detects, it has detects sensitive and that selectivity is good advantage.
The present invention is achieved like this, at the hairpin structure DNA(Au-H-DNA1 comprising adenosine aptamers, mark with the DNA probe (c-DNA1) of aptamers partial hybridization, Au NPs), the sulfhydrylation hairpin structure DNA(H-DNA2 be pre-assembled on SPR gold plaque) experimental system in, when there is no target molecule adenosine, H-DNA1 on Au-H-DNA1 keeps hairpin structure and can not hybridize with the hairpin structure H-DNA2 being pre-assembled in gold plaque surface, Au NPs cannot be captured gold plaque surface.But, when there is target molecule adenosine, adenosine and aptamers specific binding impel aptamers to occur to fold and conformational change, c-DNA1 is discharged from aptamers/c-DNA1 double-strand to hybridize with Au-H-DNA1 to make its open loop, Au-H-DNA1 after open loop is hybridized with the H-DNA2 being pre-assembled in gold plaque surface and is captured on gold plaque by Au NPs, replace out c-DNA1, the c-DNA1 replaced out then causes next round DNA chain and replaces circulation simultaneously.Replace circulation by DNA chain like this, single adenosine molecule then can cause a large amount of Au-H-DNA1 and be assembled on SPR gold plaque.Au NPs, to the enlarge-effect of spr signal, makes SPR measuring-signal greatly strengthen, is low to moderate pM rank to the detectability of adenosine.Meanwhile, the high specific of aptamers, makes sensor have good antijamming capability.The present invention is based on Au NPs and the SPR aptamer sensor that the dual amplification construction of strategy of circulation replaced by DNA chain is caused to spr signal coupling amplification effect and object, for the highly sensitive of low concentration biological micromolecule and selective enumeration method provide universality platform, have a good application prospect.
For successfully building described aptamer sensor, the present invention by the following technical solutions:
Aptamer sensor structure based on Au NPs and the two amplifying technique of DNA circulation comprises the following steps:
(1) preparation of Au NPs: be the HAuCl of 0.01% by the mass percent of 50 mL
4solution joins in 100 mL round-bottomed flasks, after being heated to boiling, the mass percentage concentration adding rapidly 1 mL is under mechanical stirring the citric acid three sodium solution of 5%, continue to stir and keep boiling, solution colour stops heating when becoming burgundy look by yellow, naturally cool to room temperature under stirring, namely obtain mean grain size and be the stable of 13 nm and monodispersed Au NPs, save backup in 4 ° of C;
(2) preparation of Au NPs-H-DNA1: after sulfhydrylation H-DNA1 activated post with the 1 mM dithiothreitol (DTT) solution that 100 μ L newly prepare, the Au NPs solution prepared with 1 mL step (1) mixes, oscillation incubation 12 h on shaking table, be scattered in by product in phosphate (PBS) buffer solution, 25 ° of C place 40 h; Centrifugal 10 min under 14000 rpm, abandoning supernatant, the Red oil precipitation PBS buffer solution after centrifugal cleans and centrifugal 3 times, the H-DNA1 that removing is not reacted with Au NPs; The Au NPs-H-DNA1 of acquisition is resuspended in PBS buffer solution, saves backup under 4 ° of C;
(3) structure of SPR aptamers sensing interface: by the H of gold plaque at volume ratio 7:3
2sO
4: H
2o
2soak 2 min in mixed solution, rinse well with intermediate water, immerse 2 h in the sulfydryl hexanol solution of 10 mM, rinse with intermediate water and after drying up with nitrogen, load SPR detection cell; Inject 50 μ L, sulfhydrylation hairpin structure H-DNA2 solution reaction 2 h of 1.2 μMs, the obtained pre-assembled sensing interface of H-DNA2;
In above-mentioned steps, in step (2), described dithiothreitol (DTT) solution concentration to be 170 mM, pH be 8.0 phosphate buffered solution preparation.Described phosphate buffered solution concentration is 10 mM, pH is 7.4, containing 0.1 M NaCl.In step (3), described sulfydryl hexanol solution absolute ethyl alcohol is prepared.Described sulfhydrylation hairpin structure DNA solution concentration is that 10 mM, pH are 7.4 and phosphate buffered solution containing 0.1 M NaCl is prepared.
Based on the application of the aptamer sensor of the two amplifying technique of Au NPs and DNA circulation, refer to it adenosine detect in application: 20 μ L, the adenosine aptamers of 1.0 μMs and the DNA probe of same ratio hatch 2 h at 37 ° of C, formed aptamers/c-DNA1 double-strand; Add the adenosine solution of 20 μ L variable concentrations, hatch 2 h at 37 ° of C, adenosine and its aptamers specific binding and DNA probe is discharged from aptamers/c-DNA1 double-strand; Get the above-mentioned solution of 25 μ L to join in 25 μ L Au NPs-H-DNA1 solution, this mixed solution is injected in SPR detection cell and makes it react 1 h with the H-DNA2 being pre-assembled in sensing interface; With peristaltic pump reaction solution discharged and inject 50 μ L intermediate waters and clean; Along with the increase of adenosine concentration, the Au NPs capturing sensing interface increases gradually, and SPR response signal strengthens rapidly, and SPR Angular measures adenosine concentration is good linear relationship within the scope of 0.5-50 pM, detection is limited to 0.21 pM, can be used for the hypersensitive to adenosine and super low concentration detection.
Technique effect of the present invention is: the present invention causes DNA chain in conjunction with Au NPs to the coupling amplification effect of spr signal and target and replaces amplifying technique, develops a kind of without enzyme SPR aptamer sensor, for the highly sensitive of adenosine and selective enumeration method.Advantage is as follows: (1) adopts target to cause DNA chain and replaces circulation amplify technology, and by repeatedly circulation amplify, single adenosine molecule can cause a large amount of Au NPs and be assembled on SPR sensing interface; (2) Au NPs has high molecule mass and high-k, and SPR gold film and Au NPs also have field coupled resonance effect, and these all make Au NPs can greatly improve SPR response signal, and then improve detection sensitivity; (3) utilize dual amplification method of the present invention, the detection of pM level adenosine can be realized, than amplifying 3 orders of magnitude as low in the detectability of sensor to adenosine that Au NPs amplifies or DNA chain replacement circulation amplify effect builds based on substance; (4) aptamers is to the high specific of its object, and the sensor that the present invention is built has good selectivity to adenosine.
Accompanying drawing explanation
Fig. 1 is the schematic diagram that SPR aptamer sensor builds and detects adenosine.
Fig. 2 is the uv-visible absorption spectra figure of (a) Au NPs and (b) Au-H-DNA1.
Fig. 3 is (a) bare electrode, (b) MCH, (A) cyclic voltammetry curve of (c) H-DNA2 and (d) Au-H-DNA1/H-DNA2 modified electrode and (B) AC impedance curve.Interior illustration is equivalent circuit diagram; R
s, Z
w, R
etand C
dlbe respectively solution resistance, Warburg resistance, electro transfer impedance and double-layer capacitance.
Fig. 4 is that cycling time replaced by (A) chain, and (B) H-DNA2 concentration, (C) H-DNA1 concentration is on the impact of spr sensor performance.
Fig. 5 is the SPR response curve that different modes detects 50 pM adenosines: circulation amplify replaced by (a) chain, and (b) Au NPs amplifies, and the dual amplification that circulates replaced by (c) Au NPs and chain.
Fig. 6 is (A) naked gold plaque, and the SEM on the gold plaque surface after the gold plaque after the dual amplification detection 50 pM adenosine that circulates replaced by (B) Au NPs and chain and (C) Au NPs amplification detection 50 pM adenosine schemes.
Fig. 7 is the SPR curve that two amplification detection adenosines that circulate replaced by (A) Au NPs and chain: a – i is respectively the adenosine of 0,0.5,1,5,10,30,40,50 and 100 pM.(B) adenosine detects calibration curve: two amplification of circulating replaced by (a) Au NPs and chain, and (b) Au NPs amplifies and circulation amplify replaced by (c) chain.
Fig. 8 replaces sensor that the dual amplification detection mode that circulates builds to the SPR response curve of adenosine, uridine, guanosine and cytidine based on Au NPs and chain.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is further elaborated, and the present invention is not limited to this;
embodiment 1
(1) preparation of Au NPs: be the HAuCl of 0.01% by the mass percent of 50 mL
4solution joins in 100 mL round-bottomed flasks, after being heated to boiling, the mass percentage concentration adding rapidly 1 mL is under mechanical stirring the citric acid three sodium solution of 5%, continue to stir and keep boiling, solution colour stops heating when becoming burgundy look by yellow, naturally cool to room temperature under stirring, namely obtain mean grain size and be the stable of 13 nm and monodispersed Au NPs, save backup in 4 ° of C;
(2) preparation of Au NPs-H-DNA1: after sulfhydrylation H-DNA1 activated post with the 1 mM dithiothreitol (DTT) solution that 100 μ L newly prepare, the Au NPs solution prepared with 1 mL step (1) mixes, oscillation incubation 12 h on shaking table, be scattered in by product in PBS buffer solution, 25 ° of C place 40 h; Centrifugal 10 min under 14000 rpm, abandoning supernatant, the Red oil precipitation PBS buffer solution after centrifugal cleans and centrifugal 3 times, the H-DNA1 that removing is not reacted with Au NPs; The Au NPs-H-DNA1 of acquisition is resuspended in PBS buffer solution, saves backup under 4 ° of C;
Adopt ultraviolet-visible spectrum to characterize the preparation that Au NPs marks H-DNA1 probe, result as shown in Figure 2.In curve a, the absorption peak at 520 nm places shows Au NPs particle diameter about 13 nm synthesized.When sulfhydrylation H-DNA1 being modified behind Au NPs surface, absorption peak red shift to 526 nm at 520 nm places, show that H-DNA1 and the Au NPs of sulfhydrylation there occurs interaction, define Au-H-DNA1 probe, and the Au-H-DNA1 of formation is highly stable in the NaCl solution of 0.1 M in concentration.
embodiment 2
The preparation process of SPR aptamer sensor
(1) by the H of gold plaque at volume ratio 7:3
2sO
4: H
2o
2soak 2 min in mixed solution, rinse well with intermediate water, immerse 2 h in the sulfydryl hexanol solution of 10 mM, rinse with intermediate water and after drying up with nitrogen, load SPR detection cell.Inject 50 μ L, H-DNA2 solution reaction 2 h of 1.2 μMs, the obtained pre-assembled sensing interface of H-DNA2;
(2) 20 μ L, the adenosine aptamers of 1.0 μMs and the c-DNA1 of same ratio are hatched 2 h at 37 ° of C, add the adenosine solution of 20 μ L variable concentrations, hatch 2 h at 37 ° of C; Get the above-mentioned solution of 25 μ L to join in 25 μ L Au NPs-H-DNA1 solution, this mixed solution is injected in SPR detection cell and makes it react 1 h with the H-DNA2 being pre-assembled in sensing interface; With peristaltic pump reaction solution discharged and inject 50 μ L intermediate waters and clean;
Cyclic voltammetry and the preparation process of electrochemical AC impedance method to spr sensor is adopted to characterize.From Fig. 3 A, naked gold electrode presents a pair reversible [Fe (CN)
6]
3-/4-redox peak (curve a); After assembling one deck MCH at electrode surface, [Fe (CN)
6]
3-/4-redox peak current reduce (curve b); Modify by gold-mercapto key after gold electrode surfaces by H-DNA2, peak current reduces further (curve c); After 50 pM adenosines and adenosine aptamers/c-DNA double-strand react 2 h, add Au NPs-H-DNA1 and inject SPR detection cell and hatch 1 h, [Fe (CN)
6]
3-/4-reduce further (curve d) at the redox current of electrode surface, this is that the H-DNA2 being fixed in SPR gold plaque surface due to a large amount of Au-H-DNA1 catches, and adds the negative charge density of electrode surface, hinders [Fe (CN)
6]
3/4to the electro transfer of electrode surface.Fig. 3 B is that the EIS of different modifying electrode characterizes, the electron transmission impedance of naked gold electrode (
r et) be only 0.2 K Ω (curve a); When after MCH in electrode surface assembling,
r etbe increased to 12 K Ω (curve b); And then by after gold-upper H-DNA2 of mercapto key assembling,
r etbe increased to 16.8 K Ω (curve c); When 50 pM adenosines exist, DNA chain is replaced circulation and is activated, and makes a large amount of Au NPs-H-DNA1 open loop and is captured to SPR gold plaque surface,
r etenlarge markedly to 24.4 K Ω (curve d).EIS and CV result is consistent, shows that sensing interface successfully builds according to predetermined scheme.
embodiment 3
SPR aptamer sensor is to the detection of adenosine
(1) cycling time replaced by chain, H-DNA2 concentration, the optimization of H-DNA1 concentration
When Fig. 4 A is the different chain replacement circular response time, the SPR of sensor to 50 pM and 0 pM adenosine responds.As seen from the figure, to 50 pM adenosines, along with the prolongation of circular response time replaced by chain, SPR response intensity strengthens thereupon, tends towards stability after 1 h; When not having adenosine, along with the prolongation in reaction time, SPR response intensity slightly strengthens thereupon.Therefore, selecting chain to replace circular response is 1 h.Fig. 4 B is the impact of H-DNA2 concentration on SPR sensing response.When H-DNA2 concentration is 1.2 μMs, the signal to noise ratio (S/N ratio) of sensor is best, further increase H-DNA2 concentration, SPR response slightly declines, although this increases owing to being assembled in capture probe density on gold plaque, highdensity hairpin structure probe adds sterically hindered and is unfavorable for the hybridization of H-DNA2 and Au-H-DNA1.Therefore, experimental selection 1.2 μMs is the optium concentration of H-DNA2.Fig. 4 C is the impact of H-DNA1 concentration on SPR sensing response.Along with the increase (0.5-0.9 μM) of H-DNA1 concentration, the background signal of sensor and its spr signal of 50 pM adenosines is all increased, further increase H-DNA1 concentration, spr signal declines, this is that H-DNA1 owing to being modified on Au NPs is overstocked, and its space steric effect inhibits hybridization efficiency.Therefore, the optium concentration selecting H-DNA1 is 0.9 μM.
(2) in order to verify the amplification effect that Au NPs and chain are replaced two enlarge-effect that circulates and detected adenosine, to respond the SPR of 50 pM adenosines the sensor only replacing circulation amplify technique construction based on Au NPs or chain and investigate, result as shown in Figure 5.As seen from the figure, the SPR response of chain replacement circulation amplify mode to 50 pM adenosines only has 14.1 m
o(curve a), and Au NPs to amplify the SPR response of mode to 50 pM adenosines be 80.3 m
o(curve b) is replace circulation amplify response based on chain 5.7 times; It is 996 m(curve c that Au NPs and chain replace that the two SPR of amplification mode to 50 pM adenosines that circulate respond), be amplify response based on Au NPs 12.5 times, show that Au NPs and chain are replaced the dual amplification mode that circulates and detected adenosine and have obvious amplification and strengthen effect.This replaces circulation pattern due to chain to impel a large amount of Au-H-DNA1 to be assembled into SPR gold plaque surface, and Au NPs and the intermembranous beam coupling effect of gold, make spr signal efficiently be amplified.And in Au NPs amplification mode, a short chain DNA(c-DNA2) instead of hair clip H-DNA2 be fixed on SPR gold plaque surface.C-DNA2 contains 12 bases, can hybridize with 3 ' 12 bases of holding of closing on of Au-H-DNA1, but, c-DNA1 chain can not discharge by this crossover process from Au-H-DNA1/c-DNA1 double-strand, thus can not cause DNA chain and replace circulation, therefore Au-H-DNA1 and c-DNA2 is fixed on SPR gold plaque by the stoichiometric proportion hybridization of 1:1, cannot realize the further amplification to spr signal;
Adopt scanning electron microscope to characterize the gold plaque surface topography after three kinds of different amplification modes detect 50 pM adenosines, result as shown in Figure 6.SPR gold plaque surface comparatively smooth (Fig. 6 A), when employing Au NPs and chain are replaced after the dual amplification mode that circulates detects 50 pM adenosines, a large amount of Au NPs is carried on gold plaque surface (Fig. 6 B) equably, shows impel the H-DNA2 on Au-H-DNA1 a large amount of in solution and gold plaque surface to occur to hybridize by chain replacement circulation pattern and capture on SPR gold plaque.After employing Au NPs mode of amplifying detects 50 pM adenosines, a small amount of Au NPs(Fig. 6 C is only had) on gold plaque surface, coverage rate is far below Fig. 6 B, this is because Au-H-DNA1 and the short chain c-DNA2 being fixed on gold plaque surface can only be hybridized with the form of 1:1, limits Au-H-DNA1 and is fixed to amount on gold plaque.
(3) with optimal conditions, detect adenosine according to shown in Fig. 1, SPR curve as shown in Figure 7 A.SPR angle change (Δ Angle) increases along with the concentration increase of adenosine, and Δ Angle and adenosine concentration (C) have good linear relationship within the scope of 0.5-50 pM, and equation of linear regression is Angle(m
o)=164.28+16.31 C(R=0.9949), detect and be limited to 0.21 pM.Under same experimental conditions, investigated and only replaced circulation amplify mode to the response of adenosine based on Au NPs or chain, result as shown in Figure 7 B.As seen from the figure, replace circulation amplify mode based on Au NPs or chain and to respond the SPR that adenosine detects and be starkly lower than the SPR that Au NPs and chain replace the dual amplification mode that circulates and respond, detectability is respectively 0.52 nM and 1.13 nM.Visible, the highly sensitive detection that the dual amplification mode that circulates can be used for adenosine replaced by Au NPs and chain.
(4) with other three kinds of nucleosides (uridine, guanosine and cytidine) of nucleosides family for chaff interference, investigated the selectivity that sensor detects adenosine, result is as shown in Figure 8.As seen from the figure, under identical experiment condition, the SPR response of sensor to 50 pM adenosines is maximum, and does not almost respond 50 pM uridines, guanosine and cytidine, shows that the sensor that the present invention builds has good selectivity to adenosine detection.
Claims (6)
1., based on the aptamer sensor construction method of the two amplifying technique of Au NPs and DNA circulation, it is characterized in that described structure comprises the following steps:
(1) preparation of Au NPs: be the HAuCl of 0.01% by the mass percent of 50 mL
4solution joins in 100 mL round-bottomed flasks, after being heated to boiling, the mass percentage concentration adding rapidly 1 mL is under mechanical stirring the citric acid three sodium solution of 5%, continue to stir and keep boiling, solution colour stops heating when becoming burgundy look by yellow, naturally cool to room temperature under stirring, namely obtain mean grain size and be the stable of 13 nm and monodispersed Au NPs, save backup in 4 ° of C;
(2) Au NPs marks the preparation of hairpin structure DNA: after sulfhydrylation DNA activated post with the 1 mM dithiothreitol (DTT) solution that 100 μ L newly prepare, the Au NPs solution prepared with 1 mL step (1) mixes, oscillation incubation 12 h on shaking table, product is scattered in phosphate buffered solution, places 40 h for 25 DEG C; Centrifugal 10 min under 14000 rpm, abandoning supernatant, the Red oil precipitation phosphate buffered solution after centrifugal is cleaned and centrifugal 3 times, the DNA that removing is not reacted with Au NPs; The hairpin structure DNA that the Au NPs of acquisition marks is resuspended in phosphate buffered solution, saves backup at 4 DEG C;
(3) structure of SPR aptamer sensor: by the H of gold plaque at volume ratio 7:3
2sO
4: H
2o
2soak 2 min in mixed solution, rinse well with intermediate water, immerse 2 h in the sulfydryl hexanol solution of 10 mM, rinse with intermediate water and after drying up with nitrogen, load SPR detection cell; Inject 50 μ L, the sulfhydrylation hairpin structure DNA solution of 1.2 μMs reacts 2 h, the obtained pre-assembled sensing interface of sulfhydrylation hairpin structure DNA; The DNA probe of adenosine aptamers and same ratio is hatched 2 h at 37 DEG C, forms aptamers/DNA probe double-strand; Add adenosine solution, hatch 2 h at 37 DEG C, adenosine and its aptamers specific binding and DNA probe is discharged from aptamers/DNA probe double-strand; Add Au NPs mark hairpin structure DNA solution prepared by step (2), mixed solution is injected in SPR detection cell and makes the sensing interface of itself and pre-assembled sulfhydrylation hairpin structure DNA react 1 h, with peristaltic pump reaction solution discharged and inject intermediate water and clean.
2. the aptamer sensor construction method based on the two amplifying technique of Au NPs and DNA circulation according to claim 1, it is characterized in that in step (2), described dithiothreitol (DTT) solution concentration to be 170 mM, pH be 8.0 phosphate buffered solution preparation.
3. the aptamer sensor construction method based on the two amplifying technique of Au NPs and DNA circulation according to claim 1, it is characterized in that in step (2), described phosphate buffered solution is concentration 10 mM, pH 7.4, containing 0.1 M NaCl.
4. the aptamer sensor construction method based on the two amplifying technique of Au NPs and DNA circulation according to claim 1, it is characterized in that in step (3), described sulfydryl hexanol solution absolute ethyl alcohol is prepared.
5. the aptamer sensor construction method based on the two amplifying technique of Au NPs and DNA circulation according to claim 1, it is characterized in that in step (3), described sulfhydrylation hairpin structure DNA solution concentration is that 10 mM, pH are 7.4 and phosphate buffered solution containing 0.1 M NaCl is prepared.
6. based on the application of aptamer sensor in adenosine detects of the two amplifying technique of Au NPs and DNA circulation: it is characterized in that, 20 μ L, the adenosine aptamers of 1.0 μMs and the DNA probe of same ratio hatch 2 h at 37 DEG C, form aptamers/DNA probe double-strand; Add the adenosine solution of 20 μ L variable concentrations, hatch 2 h at 37 DEG C, adenosine and its aptamers specific binding and DNA probe is discharged from aptamers/DNA probe double-strand; Get 25 μ L adenosines and aptamers/reacted solution of DNA probe double-strand to join 25 μ L Au NPs and mark in hairpin structure DNA solution, mixed solution is injected in SPR detection cell and makes it react 1 h with the hairpin structure DNA being pre-assembled in sensing interface; With peristaltic pump reaction solution discharged and inject 50 μ L intermediate waters and clean; Along with the increase of adenosine concentration, the Au NPs capturing SPR sensing interface increases gradually, SPR response signal strengthens rapidly, SPR Angular measures adenosine concentration is good linear relationship within the scope of 0.5-50 pM, detection is limited to 0.21 pM, can be used for the hypersensitive to adenosine and super low concentration detection.
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| KR20130011338A (en) * | 2011-07-21 | 2013-01-30 | 이화여자대학교 산학협력단 | Dna hybridization-detecting sensor, and preparing method of the same |
| CN102914570A (en) * | 2012-06-14 | 2013-02-06 | 青岛科技大学 | Method for detecting glutathione based on nanogold and thionine signal amplification |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007103802A2 (en) * | 2006-03-03 | 2007-09-13 | William Marsh Rice University | Nanorice particles: hybrid plasmonic nanostructures |
| CN101078026A (en) * | 2006-05-24 | 2007-11-28 | 江苏吴中高新技术实业有限公司 | DNA electrochemical sensor and preparation method thereof |
| KR20130011338A (en) * | 2011-07-21 | 2013-01-30 | 이화여자대학교 산학협력단 | Dna hybridization-detecting sensor, and preparing method of the same |
| CN102914570A (en) * | 2012-06-14 | 2013-02-06 | 青岛科技大学 | Method for detecting glutathione based on nanogold and thionine signal amplification |
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| Sensitive Detection of Transcription Factors by Isothermal Exponential Amplification-Based Colorimetric Assay;Yan Zhang et al;《Analytical Chemistry》;20121231;第84卷(第21期);第9544-9549页 * |
| 适配体传感器在微生物检测中的应用;文晓棠等;《生命科学研究》;20111031;第15卷(第5期);第455-459页 * |
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