CN104198464A - Method for building surface enhanced Raman scattering detection system - Google Patents
Method for building surface enhanced Raman scattering detection system Download PDFInfo
- Publication number
- CN104198464A CN104198464A CN201410491747.1A CN201410491747A CN104198464A CN 104198464 A CN104198464 A CN 104198464A CN 201410491747 A CN201410491747 A CN 201410491747A CN 104198464 A CN104198464 A CN 104198464A
- Authority
- CN
- China
- Prior art keywords
- nano silver
- dna
- silver grain
- add
- raman
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 14
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 title claims abstract description 12
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims abstract description 99
- 238000001069 Raman spectroscopy Methods 0.000 claims abstract description 47
- 229910052709 silver Inorganic materials 0.000 claims abstract description 34
- 239000004332 silver Substances 0.000 claims abstract description 33
- 229930027917 kanamycin Natural products 0.000 claims abstract description 25
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims abstract description 25
- 108020004414 DNA Proteins 0.000 claims abstract description 23
- 108091023037 Aptamer Proteins 0.000 claims abstract description 14
- 239000002245 particle Substances 0.000 claims abstract description 6
- 239000002105 nanoparticle Substances 0.000 claims description 32
- 238000006243 chemical reaction Methods 0.000 claims description 29
- 239000000243 solution Substances 0.000 claims description 29
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 26
- 238000010276 construction Methods 0.000 claims description 21
- 238000000429 assembly Methods 0.000 claims description 19
- 230000000712 assembly Effects 0.000 claims description 19
- 150000001875 compounds Chemical class 0.000 claims description 17
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 13
- 239000007864 aqueous solution Substances 0.000 claims description 12
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 10
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 10
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 10
- WCDSVWRUXWCYFN-UHFFFAOYSA-N 4-aminobenzenethiol Chemical compound NC1=CC=C(S)C=C1 WCDSVWRUXWCYFN-UHFFFAOYSA-N 0.000 claims description 8
- 239000002131 composite material Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 8
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 8
- 239000012279 sodium borohydride Substances 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 230000009514 concussion Effects 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000000376 reactant Substances 0.000 claims description 4
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 4
- 229940038773 trisodium citrate Drugs 0.000 claims description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 4
- 239000012498 ultrapure water Substances 0.000 claims description 4
- 239000004615 ingredient Substances 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- 238000004383 yellowing Methods 0.000 claims description 2
- 230000008859 change Effects 0.000 abstract description 6
- 102000053602 DNA Human genes 0.000 abstract 2
- 230000002708 enhancing effect Effects 0.000 abstract 1
- 229960000318 kanamycin Drugs 0.000 abstract 1
- 229930182823 kanamycin A Natural products 0.000 abstract 1
- 238000004904 shortening Methods 0.000 abstract 1
- 238000001237 Raman spectrum Methods 0.000 description 8
- 229910052751 metal Inorganic materials 0.000 description 6
- 239000002184 metal Substances 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 230000002776 aggregation Effects 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 230000007274 generation of a signal involved in cell-cell signaling Effects 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007788 roughening Methods 0.000 description 1
- 150000003378 silver Chemical class 0.000 description 1
- 238000004557 single molecule detection Methods 0.000 description 1
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention belongs to the field of analytical chemistry and relates to a method for building a surface enhanced Raman scattering detection system. The method comprises the following steps: firstly, embedding the aptamer sequences of a target object into six DNA (deoxyribonucleic acid) edges of a nanosilver assembly body; secondly, shortening the distance between nanosilver particles through specific identification of the target object and the aptamers of the target object; thirdly, forming a high-density electrical magnetic field region between the silver particles within the very short distance; fourthly, enhancing Raman signals of beacon molecules in the region; finally, detecting the target object through the change of the Raman signals of the beacon molecules. According to the method, the target object, especially kanamycin can be specifically, quickly and ultra-sensitively detected.
Description
Technical field
The invention belongs to analytical chemistry field, relate to a kind of construction method of Surface enhanced raman spectroscopy detection system.
Background technology
Ramam effect also claims Raman scattering, is the inelastic scattering phenomenon of photon.When light is from an atom or molecular scattering out time, most photons, is all elastic scattering, and this is called Rayleigh scattering.Under Rayleigh scattering, the photon scattering out, follows the photon while injecting, and its energy, frequency are identical with wavelength.Yet, there is the photon one of (be approximately 10,000,000 minutes) of sub-fraction scattering, the frequency after scattering can change, the photon frequency when injecting normally, reason is, between incident photon and medium molecule, energy exchange occurs.This is Raman scattering.Raman spectrum (Raman spectra) is a kind of scattering spectrum.Raman spectrum analysis method is the Raman scattering effect of finding based on India scientist C.V. Raman (Raman), scattering spectrum analyze different from incident light frequency obtained to molecular vibration, rotation aspect information, and be applied to a kind of analytical approach of molecular structure research.
Surface enhanced raman spectroscopy technology (SERS) technology has overcome the shortcoming of the inherent weak output signal of traditional Raman spectrum, can be so that raman scattering intensity increases several orders of magnitude.Its enhancer can up to 1014-1015 doubly, be enough to detect the Raman signal of individual molecule.SERS can be for trace material analysis, flow cytometry and some other application, and these are all that sensitivity and the measuring speed of traditional Raman has been not enough to.SERS can occur on any surface, only on limited several metal surfaces, can effectively produce.Conventionally that application is Ag and Au, and having found now also has SERS effect on Pt, Cu, Al and alkaline metal surface.But, be not to have suitable metal just can produce SERS.SERS only betides the metal surface through roughening, in smooth metal surface, does not observe SERS.
Utilize the strong-electromagnetic field of the formation between metal nano material, original Raman signal intensity can be improved to 10
5-10
7doubly, thereby SERS has very high sensitivity, can be for Single Molecule Detection.But current Raman detection is mainly the strong-electromagnetic field based on producing after the random gathering of nano particle amplifies signal, due to cause nanoparticle aggregate because have a lot (ionic strength, temperature, PH etc.), thereby the specificity of this method is bad, tend to produce false positive.It is a kind of novel detection technique that Raman detection is carried out in structural change based on nanometer assembly, in whole process, nanometer assembly remains good disperse state, in system, there is not large stretch of aggregation, effectively avoided the signal causing because of extraneous factor to strengthen, the specificity and the sensitivity that have greatly improved Raman detection method.At present, not nanoparticle assemblies almost not having for kanamycins Raman detection.
Summary of the invention
The object of the invention is to solve blank of the prior art, a kind of construction method of Surface enhanced raman spectroscopy detection system is provided, thereby nanoparticle assemblies is used for to kanamycins Raman detection.
Object of the present invention is achieved through the following technical solutions
1. the invention provides a kind of construction method of Surface enhanced raman spectroscopy detection system, it is skeleton that the method be take the three-dimensional silver nanoparticle assemblies with pyramid structure, the DNA of skeleton partly has the aptamers sequence of object, and object can be combined with aptamers sequence and be formed DNA loop-stem structure.
Three-dimensional silver nanoparticle assemblies is space pyramid structure, and four tops are four silver nano-grains, and six limits are the DNA double chain of complementary hybridization.Each length of side of pyramid structure is in 8nm left and right.
2. above-mentioned 1 construction method providing, take 4-aminothiophenol as Raman beacon molecule, and the ingredient of modifying three-dimensional silver nanoparticle assemblies is the surface of Nano silver grain-DNA compound.
3. above-mentioned 2 construction methods that provide, described object is kanamycins, its aptamers sequence is: TGGGGGTTGAGGCTAAGCCGA.
4. above-mentioned 3 construction methods that provide, three-dimensional silver nanoparticle assemblies is assembled by four kinds of different Nano silver grain-DNA compounds, that is: Ag-T1, Ag-T2, Ag-T3, Ag-T4;
Wherein, T1 sequence as shown in SEQ ID NO.1, T2 sequence as shown in SEQ ID NO.2, T3 sequence as shown in SEQ ID NO.3 and T4 sequence as shown in SEQ ID NO.4.
5. above-mentioned 4 construction methods that provide, T1 wherein, T2, tri-single stranded DNAs of T3 have embedded respectively the aptamer sequence of kanamycins.
6. above-mentioned 5 construction methods that provide, wherein, Nano silver grain particle diameter is 17-23nm.
7. above-mentioned 6 construction methods that provide, the method specifically comprises the steps:
1) Nano silver grain finishing DNA:
Initial volume is that the silver nano-particle solution of 200-400uL, 20nM is under 7500-8000r/min at rotating speed, centrifugal 10-15min, add 10mM tris-hydrochloride buffer and return to initial volume, equal-volume divides and installs in four different pipes, and four pipes add the T1 of 1-2uL, 10uM, T2 successively, T3 and T4, in four parts of systems, add respectively 1.25-5uL 2M NaCl solution, room temperature concussion reaction is spent the night, and forms Nano silver grain-DNA compound;
2) Nano silver grain-DNA composite surface is modified Raman beacon molecule:
Take 4-aminothiophenol as beacon molecule is to step 1) composite surface of Nano silver grain-DNA of preparing modifies, and the final concentration that makes beacon molecule is 3-8uM;
3) preparation of Nano silver grain assembly
Step 2) concentration of preparing is the compound of four kinds of Nano silver grain-DNA of 20nM: Ag-T1, Ag-T2, Ag-T3, Ag-T4, equal-volume adds respectively the NaCl solution that 5-15uL, concentration are 1M, room temperature reaction 20-36h, forms and has the three-dimensional silver nanoparticle assemblies of pyramid structure, thereby add kanamycins to set up Surface enhanced raman spectroscopy detection system.
8. above-mentioned 7 construction methods that provide, wherein, the synthesis step of Nano silver grain is:
Reaction bulb bottle is placed in ice bath, adds successively 20mL ultrapure water, the polyvinylpyrrolidone of 5mL 1%, 0.5mL 0.01mol/L sodium borohydride and 0.5mL 1% trisodium citrate aqueous solution; Speed by the silver nitrate aqueous solution of the polyvinylpyrrolidone aqueous solution of 5mL 1% and 5mL 0.1% with 30mL/h joins in the reaction bulb in ice bath simultaneously, add while stirring until react end, solution is from colourless yellowing, reactant is placed in to 80 ℃ of reaction 2h and removes unreacted sodium borohydride, last solution becomes glassy yellow, makes Nano silver grain.
Yellow and glassy yellow are according to artificial judgement, as long as there is yellow, the depth of color is big or small relevant to Nano silver grain.
9. above-mentioned 8 construction methods that provide, the method specifically comprises the steps:
1) Nano silver grain finishing DNA
Silver nano-particle solution is in rotating speed 8000r/min, centrifugal 10min, and adding 10mM tris-hydrochloride buffer to Nano silver grain final concentration is 10nM, respectively get 50uL in 4 reaction tubes, each pipe adds respectively the T1 of 1uL 10uM/L, T2, T3 and T4, mix and in backward each system, add 1.25uL 2M NaCl solution, after room temperature concussion reaction is spent the night, rotating speed 8000r/min, centrifugal 10min, remove supernatant, add 10mM tris-hydrochloride buffer.
2) Nano silver grain-DNA composite surface is modified Raman beacon molecule
Take 4-aminothiophenol as Raman beacon molecule, to step 1) compound of four kinds of Nano silver grain-DNA preparing carries out finishing, the final concentration that makes beacon molecule is 3uM, mixing rear reaction spends the night, again with the centrifugal 10min of 8000r/min, remove supernatant, then carry out resuspended with 10mM tris-hydrochloride buffer;
3) preparation of Nano silver grain assembly
Step 2) concentration of preparing is the compound of tetra-kinds of Nano silver grain-DNA of 20nM: Ag-T1, Ag-T2, Ag-T3, Ag-T4; Respectively get 50uL and be mixed in reaction tube, add 5uL 1M NaCl solution, room temperature reaction spends the night, and forms and has the three-dimensional silver nanoparticle assemblies of pyramid structure, thereby add kanamycins to form Surface enhanced raman spectroscopy detection system.
Beneficial effect:
1. utilize the specific recognition between aptamers and object, thereby by the distance of controlling between silver nano-grain that adds of object, make to produce strong Raman signal between nano particle, significantly improved specificity and the sensitivity of detection method; In DNA molecular, insert different aptamers and can detect different target products.
2. in whole process, the DNA skeleton of silver nano-grain does not dissociate, and does not occur large stretch of agglomeration in system, thereby has effectively avoided, because of the interference of non-specific aggregation to Raman signal generation, having reduced false-positive appearance.
(3) utilize silver nano-grain apart from changing the strong Raman signal producing, set up the Raman sensing detection new method of kanamycins, the minimum detectable concentration of kanamycins is 0.02ng/ml, and sensitivity is at 0.05ng/ml-10ng/ml.Than general enzyme linked immunosorbent detection method (Development of an enzyme-linked immunoassay for the detectionof gentamicin in swine tissues, Food Chemistry 108 (2008) 304 – 309, detection line is 0.098ug/ml), its sensitivity has improved 10
3doubly.
Accompanying drawing explanation
Fig. 1: Nano silver grain photoscope figure
Fig. 2: the Raman spectrogram of Nano silver grain assembly and finishing Raman beacon molecule;
Fig. 3: add the Raman change curve after variable concentrations kanamycins in Nano silver grain assembly; The concentration of kanamycins is followed successively by 0ng/ml, 0.05ng/ml, 0.1ng/ml, 0.5ng/ml, 1ng/ml, 5ng/ml, 10ng/ml;
Fig. 4: the canonical plotting of kanamycins Raman detection;
Fig. 5: Nano silver grain assembly pyramid structure figure
Embodiment
Below in conjunction with embodiment, the present invention will be further described, and the experimental technique of unreceipted actual conditions in the following example, conventionally according to the known approaches of this area.
Embodiment 1
(1) Nano silver grain is synthetic
The preparation method of Nano silver grain: the conical flask of getting a cleaning is placed in ice bath, adds polyvinylpyrrolidone (PVP), 0.5mL 0.01mol/L sodium borohydride and the 0.5mL1% trisodium citrate aqueous solution of 20mL ultrapure water, 5mL 1% successively.Then, with the needle tubing of two 50mL, fill respectively the polyvinylpyrrolidone aqueous solution of 5mL 1% and the silver nitrate aqueous solution of 5mL 0.1%, two are fixed on the both sides of miniflow syringe pump by all means, speed with 30mL/h joins two kinds of solution in the conical flask in ice bath simultaneously, add while stirring until react end, solution is from the colourless buff that becomes, reactant is placed in to 80 ℃ of reaction 2h and removes unreacted sodium borohydride, last solution becomes glassy yellow, transmission electron microscope shows that particle diameter is 20 ± 3nm, as shown in Figure 1.
(2) Nano silver grain finishing DNA
Nano silver grain 200uL prepared by step (1), 7500r/min, centrifugal 15min, adds 15mM tris-hydrochloride buffer and returns to 200uL, and now the final concentration of Nano silver grain is 10nM.The Nano silver grain preparing is respectively got 50uL in 4 PCR pipes, add successively the T1 sequence of 2uL 10uM/L as shown in SEQ ID NO.1, T2 sequence is as shown in SEQ ID NO.2, T3 sequence as shown in SEQ ID NO.3 and T4 sequence as shown in SEQ ID NO.4, mix and in backward each system, add 5uL 2M NaCl solution, after room temperature concussion reaction is spent the night, 8000r/min, centrifugal 10min, removes supernatant, adds 10mM tris-hydrochloride buffer to 200uL.
(3) Nano silver grain-DNA composite surface is modified Raman beacon molecule
The compound of four kinds of Nano silver grain-DNA that the 4-aminothiophenol of usining prepares step (2) as Raman beacon molecule carries out finishing, the final concentration that makes beacon molecule is 8uM, mixing rear reaction spends the night, again with the centrifugal 10min of 8000r/min, remove supernatant, then carry out resuspended with 10mM tris-hydrochloride buffer.
(4) preparation of Nano silver grain assembly
The compound of four kinds of Ag-DNA that concentration prepared by step (3) is 20nM (Ag-T1, Ag-T2, Ag-T3, Ag-T4); Respectively getting 50uL is mixed in 1.5ml centrifuge tube, add 15uL 1M NaCl solution, room temperature reaction spends the night, formation has the three-dimensional silver nanoparticle assemblies of pyramid structure, add object to be detected, object can be in being embedded in DNA skeleton the aptamer of self be combined and form DNA loop-stem structure, make to produce between Nano silver grain strong " focus " region, thereby the signal of the Raman beacon molecule in this region is obviously strengthened, according to the Raman signal intensity at beacon molecule 4-ATP place and object concentration Criterion curve.
Embodiment 2
(1) Nano silver grain is synthetic
The preparation method of Nano silver grain: the conical flask of getting a cleaning is placed in ice bath, adds 20mL ultrapure water successively, the polyvinylpyrrolidone of 5mL 1% (PVP) and 0.5mL 0.01mol/L sodium borohydride and 0.5mL1% trisodium citrate aqueous solution.Then, with the needle tubing of two 50mL, fill respectively the polyvinylpyrrolidone aqueous solution of 5mL 1% and the silver nitrate aqueous solution of 5mL 0.1%, two are fixed on the both sides of miniflow syringe pump by all means, speed with 30mL/h joins two kinds of solution in the conical flask in ice bath simultaneously, add while stirring until react end, solution is from the colourless buff that becomes, reactant is placed in to 80 ℃ of reaction 2h and removes unreacted sodium borohydride, last solution becomes glassy yellow, transmission electron microscope shows that particle diameter is 20 ± 3nm, as shown in Figure 1.
(2) Nano silver grain finishing DNA
Nano silver grain 8000r/min prepared by step (1), centrifugal 10min, adds 10mM tris-hydrochloride buffer and returns to original volume, and now the final concentration of Nano silver grain is 10nM.The Nano silver grain preparing is respectively got 50uL in 4 PCR pipes, add successively the T1 sequence of 1uL 10uM/L as shown in SEQ ID NO.1, T2 sequence is as shown in SEQ ID NO.2, T3 sequence as shown in SEQ ID NO.3 and T4 sequence as shown in SEQ ID NO.4, mix and in backward each system, add 1.25uL 2M NaCl solution, after room temperature concussion reaction is spent the night, 8000r/min, centrifugal 10min, removes supernatant, adds 10mM tris-hydrochloride buffer to original volume.
(3) Nano silver grain-DNA composite surface is modified Raman beacon molecule
The compound of four kinds of Nano silver grain-DNA that the 4-aminothiophenol of usining prepares step (2) as Raman beacon molecule carries out finishing, the final concentration that makes beacon molecule is 3uM, mixing rear reaction spends the night, again with the centrifugal 10min of 8000r/min, remove supernatant, then carry out resuspended with 10mM tris-hydrochloride buffer.Silver nanoparticle ion surface is modified to beacon molecule and carry out Raman spectrum detection, as shown in Figure 2
(4) preparation of Nano silver grain assembly
The compound of four kinds of Nano silver grain-DNA prepared by step (3) (Ag-T1, Ag-T2, Ag-T3, Ag-T4); Respectively get 50uL and be mixed in 1.5ml centrifuge tube, add 5uL 1M NaCl solution, room temperature reaction spends the night, and the three-dimensional silver nanoparticle assemblies with pyramid structure of formation Raman beacon molecular modification is (silver nanoparticle assembly+4-ATP) as shown in Figure 2.
The compound of four kinds of Nano silver grain-DNA prepared by step (2) (Ag-T1, Ag-T2, Ag-T3, Ag-T4); Respectively getting 50uL is mixed in 1.5ml centrifuge tube, add 5uL 1M NaCl solution, room temperature reaction spends the night, and forms the three-dimensional silver nanoparticle assemblies with pyramid structure without Raman beacon molecular modification, carry out Raman spectrum detection, as shown in Figure 2 (silver nanoparticle assembly).
By transmission electron microscope, Raman spectrum, three-D space structure and the optical property of carrying out the Nano silver grain assembly of Raman beacon molecular modification characterize: three-dimensional silver nanoparticle assemblies is space pyramid structure, four tops are four silver nano-grains, and six limits are the DNA double chain of complementary hybridization.Each length of side of pyramid structure is in 8nm left and right, as shown in Figure 5.
(5) foundation of kanamycins Raman detection system
Structure based on Nano silver grain assembly kanamycins SERS detection system, concrete steps are: in the three-dimensional silver nanoparticle assemblies with pyramid structure of the Raman beacon molecular modification of preparing, add variable concentrations (0ng/ml, 0.05ng/ml, 0.1ng/ml, 0.5ng/ml, 1ng/ml, 5ng/ml, kanamycins solution 10ng/ml), as shown in Figure 3, now, kanamycins can be in being embedded in DNA skeleton the aptamer of self be combined and form DNA loop-stem structure, make to produce between Nano silver grain strong " focus " region, thereby the signal of the Raman beacon molecule in this region is obviously strengthened, according to beacon molecule 4-ATP 1096cm
-1raman signal intensity and the kanamycins concentration Criterion curve at place.As shown in Figure 4, in this experiment, the detection of kanamycins is limited to 0.02ng/ml, range of linearity 0.05ng/ml-10ng/ml.As shown in Figure 2, utilize Raman spectrum to adding the Nano silver grain assembly of the finishing beacon molecule after kanamycins to detect (silver nanoparticle assembly+4-ATP+ kanamycins), compare with the three-dimensional silver nanoparticle assemblies with pyramid structure (silver nanoparticle assembly) without Raman beacon molecular modification, the three-dimensional silver nanoparticle assemblies with pyramid structure (silver nanoparticle assembly+4-ATP) of Raman beacon molecular modification, illustrate and add kanamycins can produce very strong Raman signal simultaneously.
The sequence of DNA used in table 1 experiment
Can know; the illustrative embodiments that above-described embodiment only adopts for inventive principle is described; yet the present invention is not limited only to this; those skilled in the art are not departing under real situation of the present invention; can make various improvement and change, these improvement and change also belong to protection scope of the present invention.
Claims (9)
1. the construction method of a Surface enhanced raman spectroscopy detection system, it is characterized in that: the method be take three-dimensional silver nanoparticle assemblies as skeleton, the DNA of skeleton partly has the aptamers sequence of object, and object can be combined with aptamers sequence and be formed DNA loop-stem structure.
2. construction method claimed in claim 1, is characterized in that: take 4-aminothiophenol as Raman beacon molecule, modify the ingredient of Nano silver grain assembly, i.e. the surface of Nano silver grain-DNA compound.
3. construction method claimed in claim 2, is characterized in that: described object is kanamycins, and its aptamer sequence is: TGGGGGTTGAGGCTAAGCCGA.
4. construction method claimed in claim 3, is characterized in that: three-dimensional silver nanoparticle assemblies is assembled by four kinds of different Nano silver grain-DNA compounds, that is: Ag-T1, Ag-T2, Ag-T3, Ag-T4;
Wherein, T1 sequence as shown in SEQ ID NO.1, T2 sequence as shown in SEQ ID NO.2, T3 sequence as shown in SEQ ID NO.3 and T4 sequence as shown in SEQ ID NO.4.
5. construction method claimed in claim 4, is characterized in that: T1 wherein, and T2, tri-single stranded DNAs of T3 have embedded respectively the aptamer sequence of kanamycins.
6. construction method claimed in claim 5, is characterized in that: Nano silver grain particle diameter is 17-23nm.
7. construction method claimed in claim 6, is characterized in that: the method specifically comprises the steps:
1) Nano silver grain finishing DNA:
Initial volume is that the silver nano-particle solution of 200-400uL, 20nM is under 7500-8000r/min at rotating speed, centrifugal 10-15min, add 10mM tris-hydrochloride buffer and return to initial volume, equal-volume divides and installs in four different pipes, and four pipes add the T1 of 1-2uL, 10uM, T2 successively, T3 and T4, in four parts of systems, add respectively 1.25-5uL 2M NaCl solution, room temperature concussion reaction is spent the night, and forms Nano silver grain-DNA compound;
2) Nano silver grain-DNA composite surface is modified Raman beacon molecule:
Take 4-aminothiophenol as beacon molecule is to step 1) composite surface of Nano silver grain-DNA of preparing modifies, and the final concentration that makes beacon molecule is 3-8uM;
3) preparation of Nano silver grain assembly
Step 2) concentration of preparing is the compound of four kinds of Nano silver grain-DNA of 20nM: Ag-T1, Ag-T2, Ag-T3, Ag-T4, equal-volume adds respectively the NaCl solution that 5-15uL, concentration are 1M, room temperature reaction 20-36h, forms and has the three-dimensional silver nanoparticle assemblies of pyramid structure, thereby add kanamycins to set up Surface enhanced raman spectroscopy detection system.
8. construction method claimed in claim 7, is characterized in that: the synthesis step of Nano silver grain is:
Reaction bulb is placed in ice bath, adds successively polyvinylpyrrolidone, 0.5mL 0.01mol/L sodium borohydride and 0.5mL 1% trisodium citrate aqueous solution of 20mL ultrapure water, 5mL 1%; Speed by the silver nitrate aqueous solution of the polyvinylpyrrolidone aqueous solution of 5mL 1% and 5mL 0.1% with 30mL/h joins in the reaction bulb in ice bath simultaneously, add while stirring until react end, solution is from colourless yellowing, reactant is placed in to 80 ℃ of reaction 2h and removes unreacted sodium borohydride, last solution becomes glassy yellow, makes Nano silver grain.
9. construction method claimed in claim 8, is characterized in that: the method specifically comprises the steps:
1) Nano silver grain finishing DNA
Silver nano-particle solution is in rotating speed 8000r/min, centrifugal 10min, and adding 10mM tris-hydrochloride buffer to Nano silver grain final concentration is 10nM, respectively get 50uL in 4 reaction tubes, each pipe adds respectively the T1 of 1uL 10uM/L, T2, T3 and T4, mix and in backward each system, add 1.25uL 2M NaCl solution, after room temperature concussion reaction is spent the night, rotating speed 8000r/min, centrifugal 10min, remove supernatant, add 10mM tris-hydrochloride buffer.
2) Nano silver grain-DNA composite surface is modified Raman beacon molecule
Take 4-aminothiophenol as Raman beacon molecule, to step 1) compound of four kinds of Nano silver grain-DNA preparing carries out finishing, the final concentration that makes beacon molecule is 3uM, mixing rear reaction spends the night, again with the centrifugal 10min of 8000r/min, remove supernatant, then carry out resuspended with 10mM tris-hydrochloride buffer;
3) preparation of Nano silver grain assembly
Step 2) concentration of preparing is the compound of tetra-kinds of Nano silver grain-DNA of 20nM: Ag-T1, Ag-T2, Ag-T3, Ag-T4; Respectively get 50uL and be mixed in reaction tube, add 5uL 1M NaCl solution, room temperature reaction spends the night, and forms and has the three-dimensional silver nanoparticle assemblies of pyramid structure, thereby add kanamycins to form Surface enhanced raman spectroscopy detection system.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410491747.1A CN104198464B (en) | 2014-09-23 | 2014-09-23 | A kind of construction method of surface enhanced raman spectroscopy detection system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410491747.1A CN104198464B (en) | 2014-09-23 | 2014-09-23 | A kind of construction method of surface enhanced raman spectroscopy detection system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104198464A true CN104198464A (en) | 2014-12-10 |
| CN104198464B CN104198464B (en) | 2016-10-05 |
Family
ID=52083789
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410491747.1A Expired - Fee Related CN104198464B (en) | 2014-09-23 | 2014-09-23 | A kind of construction method of surface enhanced raman spectroscopy detection system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104198464B (en) |
Cited By (22)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104568898A (en) * | 2014-12-17 | 2015-04-29 | 浙江理工大学 | Hollow gold nanoparticle-DNA composite base and preparation method thereof |
| CN104914088A (en) * | 2015-06-08 | 2015-09-16 | 江南大学 | Method for detecting supersensitivity of Mucin-1 surface enhanced raman signal based on golden rod nuclear-silver satellite assembling body |
| CN105203524A (en) * | 2015-09-29 | 2015-12-30 | 江南大学 | Method based on aptamer recognition surface enhanced Raman spectroscopy for detecting salmonella in food |
| CN105738342A (en) * | 2016-02-26 | 2016-07-06 | 中国人民解放军军事医学科学院军事兽医研究所 | SERS method for in-situ-synthesizing nano-silver by serving aptamer as support |
| WO2016109911A1 (en) * | 2015-01-09 | 2016-07-14 | 江南大学 | Multiple raman detection method based on silver nanoparticle tetrahedron |
| CN108444538A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of mercury ion content is detected based on aldehyde radical chip and gold nano grain |
| CN108444547A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of mercury ion content is detected based on genetic chip and silver nano-grain |
| CN108444539A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of silver ion content is detected based on silver nano-grain |
| CN108444545A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of silver ion content is detected based on aldehyde radical chip and silver nano-grain |
| CN108444544A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of mercury ion content is detected based on silver nano-grain |
| CN108444548A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of mercury ion content is detected based on gold nano grain |
| CN108444541A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of silver ion content is detected based on aldehyde radical chip and gold nano grain |
| CN108444542A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of silver ion content is detected based on gold silver nanometer particle |
| CN108444546A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of silver ion content is detected based on genetic chip and silver nano-grain |
| CN108444543A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of mercury ion content is detected based on gold silver nanometer particle |
| CN108444540A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of mercury ion content is detected based on aldehyde radical chip and silver nano-grain |
| CN108507623A (en) * | 2018-06-05 | 2018-09-07 | 天津大学 | The environment network of silver ion content is detected based on gold nano grain |
| CN108507624A (en) * | 2018-06-05 | 2018-09-07 | 天津大学 | The environment network of silver ion content is detected based on genetic chip and gold nano grain |
| CN108519128A (en) * | 2018-06-05 | 2018-09-11 | 天津大学 | The environment network of mercury ion content is detected based on genetic chip and gold nano grain |
| CN110567936A (en) * | 2019-09-05 | 2019-12-13 | 上海应用技术大学 | method for detecting cyromazine in milk based on nucleic acid aptamer |
| CN111537492A (en) * | 2020-04-30 | 2020-08-14 | 东南大学 | Preparation method of uniform high-sensitivity surface-enhanced Raman spectrum probe, probe prepared by same and application of probe |
| CN113008867A (en) * | 2021-03-11 | 2021-06-22 | 福建师范大学 | Glass SERS platform substrate with internal standard and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101734617A (en) * | 2009-12-22 | 2010-06-16 | 浙江大学 | A method for preparing tube-network silver nano self-assembled bodies |
| CN103698320A (en) * | 2013-12-17 | 2014-04-02 | 江南大学 | Construction method of chiral sensor for detecting DNA enzymatic assembly of lead ion |
-
2014
- 2014-09-23 CN CN201410491747.1A patent/CN104198464B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101734617A (en) * | 2009-12-22 | 2010-06-16 | 浙江大学 | A method for preparing tube-network silver nano self-assembled bodies |
| CN103698320A (en) * | 2013-12-17 | 2014-04-02 | 江南大学 | Construction method of chiral sensor for detecting DNA enzymatic assembly of lead ion |
Non-Patent Citations (5)
| Title |
|---|
| WENJING YAN ET AL.: "Pyramidal Sensor Platform with Reversible Chiroptical Signals for DNA Detection", 《SMALL》 * |
| 曹丽洁: "基于发卡DNA自组装金胶比色检测DNA及分子识别文库的构建", 《中国优秀硕士学位论文全文数据库 基础科学辑 》 * |
| 赵媛: "基于纳米材料光学性质的DNA超灵敏检测方法的建立", 《中国博士学位论文全文数据库 基础科学辑》 * |
| 赵寅: "基于纳米多孔银的表面增强拉曼散射", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 马伟: "基于纳米组装的DNA增敏传感检测原理与方法", 《中国博士学位论文全文数据库 工程科技Ⅰ辑》 * |
Cited By (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104568898A (en) * | 2014-12-17 | 2015-04-29 | 浙江理工大学 | Hollow gold nanoparticle-DNA composite base and preparation method thereof |
| WO2016109911A1 (en) * | 2015-01-09 | 2016-07-14 | 江南大学 | Multiple raman detection method based on silver nanoparticle tetrahedron |
| CN104914088A (en) * | 2015-06-08 | 2015-09-16 | 江南大学 | Method for detecting supersensitivity of Mucin-1 surface enhanced raman signal based on golden rod nuclear-silver satellite assembling body |
| CN104914088B (en) * | 2015-06-08 | 2017-05-24 | 江南大学 | Method for detecting supersensitivity of Mucin-1 surface enhanced raman signal based on golden rod nuclear-silver satellite assembling body |
| CN105203524A (en) * | 2015-09-29 | 2015-12-30 | 江南大学 | Method based on aptamer recognition surface enhanced Raman spectroscopy for detecting salmonella in food |
| CN105738342A (en) * | 2016-02-26 | 2016-07-06 | 中国人民解放军军事医学科学院军事兽医研究所 | SERS method for in-situ-synthesizing nano-silver by serving aptamer as support |
| CN105738342B (en) * | 2016-02-26 | 2018-09-25 | 中国人民解放军军事医学科学院军事兽医研究所 | It is a kind of using aptamers as the SERS methods of holder fabricated in situ nano silver |
| CN108444541A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of silver ion content is detected based on aldehyde radical chip and gold nano grain |
| CN108444540A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of mercury ion content is detected based on aldehyde radical chip and silver nano-grain |
| CN108444545A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of silver ion content is detected based on aldehyde radical chip and silver nano-grain |
| CN108444544A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of mercury ion content is detected based on silver nano-grain |
| CN108444548A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of mercury ion content is detected based on gold nano grain |
| CN108444547A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of mercury ion content is detected based on genetic chip and silver nano-grain |
| CN108444542A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of silver ion content is detected based on gold silver nanometer particle |
| CN108444546A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of silver ion content is detected based on genetic chip and silver nano-grain |
| CN108444543A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of mercury ion content is detected based on gold silver nanometer particle |
| CN108444539A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of silver ion content is detected based on silver nano-grain |
| CN108507623A (en) * | 2018-06-05 | 2018-09-07 | 天津大学 | The environment network of silver ion content is detected based on gold nano grain |
| CN108507624A (en) * | 2018-06-05 | 2018-09-07 | 天津大学 | The environment network of silver ion content is detected based on genetic chip and gold nano grain |
| CN108519128A (en) * | 2018-06-05 | 2018-09-11 | 天津大学 | The environment network of mercury ion content is detected based on genetic chip and gold nano grain |
| CN108444538A (en) * | 2018-06-05 | 2018-08-24 | 天津大学 | The environment network of mercury ion content is detected based on aldehyde radical chip and gold nano grain |
| CN110567936A (en) * | 2019-09-05 | 2019-12-13 | 上海应用技术大学 | method for detecting cyromazine in milk based on nucleic acid aptamer |
| CN111537492A (en) * | 2020-04-30 | 2020-08-14 | 东南大学 | Preparation method of uniform high-sensitivity surface-enhanced Raman spectrum probe, probe prepared by same and application of probe |
| CN113008867A (en) * | 2021-03-11 | 2021-06-22 | 福建师范大学 | Glass SERS platform substrate with internal standard and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104198464B (en) | 2016-10-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104198464A (en) | Method for building surface enhanced Raman scattering detection system | |
| Wang et al. | Single-particle LRET aptasensor for the sensitive detection of aflatoxin B1 with upconversion nanoparticles | |
| Zhang et al. | Integral multielement signals by DNA-programmed UCNP–AuNP nanosatellite assemblies for ultrasensitive ICP–MS detection of exosomal proteins and cancer identification | |
| Zhao et al. | Recent advances in bioapplications of C-dots | |
| Hu et al. | Characterization and analytical separation of fluorescent carbon nanodots | |
| Qian et al. | Aconitic acid derived carbon dots: Conjugated interaction for the detection of folic acid and fluorescence targeted imaging of folate receptor overexpressed cancer cells | |
| Zhou et al. | Electrochemiluminescence resonance energy transfer between CdS: Eu nancrystals and Au nanorods for sensitive DNA detection | |
| CN102145279B (en) | Method for preparing lysozyme molecular imprinting nano particles with magnetic responsiveness and extremely high adsorption capacity | |
| CN103743722B (en) | A kind of based on nano-particle and chemiluminescent aptamer sensor and preparation method and application | |
| Patel et al. | Mn-doped ZnS quantum dots–an effective nanoscale sensor | |
| CN105277528A (en) | Construction of algal toxin Raman sensor adopting gold nanoflower-silver nanoparticle bimetallic nano assembly | |
| Xu et al. | A colorimetric aptasensor for the antibiotics oxytetracycline and kanamycin based on the use of magnetic beads and gold nanoparticles | |
| Liu et al. | Construction of FRET biosensor for off-on detection of lead ions based on carbon dots and gold nanorods | |
| CN104549174A (en) | Phenylboronic acid-modified polyethyleneimine hybrid magnetic nanoparticle and its preparation method and use | |
| CN102866139B (en) | Establishment method based on surface plasma reinforcing energy transferring biosensor | |
| Lee et al. | Detection of glyphosate by quantitative analysis of fluorescence and single DNA using DNA-labeled fluorescent magnetic core–shell nanoparticles | |
| CN106323935B (en) | Magnetic composite SERS substrate with core-shell-satellite three-dimensional structure and preparation method thereof | |
| CN101482554B (en) | Magnetic Raman nano composite material used for biological separation and detection, and production method thereof | |
| Wu et al. | Ratiometric fluorescence sensor for the sensitive detection of Bacillus thuringiensis transgenic sequence based on silica coated supermagnetic nanoparticles and quantum dots | |
| CN102879336A (en) | Method for manufacturing plasma chiral ligand sensor for mercury ions | |
| Tian et al. | Recent advances of plasmonic nanoparticle-based optical analysis in homogeneous solution and at the single-nanoparticle level | |
| CN101108423A (en) | Novel method of manufacturing Fe3O4/Au magnetic complex nano particle | |
| Ma et al. | DNA-functionalized gold nanoparticles: Modification, characterization, and biomedical applications | |
| Zhu et al. | Self-assembled Ag nanoparticles for surface enhanced Raman scattering | |
| CN114888299B (en) | Preparation method and application of broccoli-shaped micro-nano silver |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20161005 |