CN104198464A - Method for building surface enhanced Raman scattering detection system - Google Patents
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- 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
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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.
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