CN106970032B - A kind of preparation method and application visualizing gold nanoparticle probe - Google Patents
A kind of preparation method and application visualizing gold nanoparticle probe Download PDFInfo
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Abstract
The present invention provides a kind of preparation method and application for visualizing gold nanoparticle probe, and the present invention is commonly assembled on gold nanoparticle surface with adenosine aptamers and MUA, and the gold nanoparticle after assembling is in reticular structure, and solution is blue.The addition of adenosine will lead to the variation of aptamer structure, and adenosine aptamers is made to get off from gold nanoparticle surface dissociation, while gold nanoparticle becomes free dispersed structure from reticular structure, and solution becomes red from blue.Cr is added into solution3+, utilize Cr3+With the complexing of-COOH in MUA, the particle after dispersion can be reassembled, solution colour becomes blue from red again.The present invention has double sequence detection adenosine and Cr3+Function, and high sensitivity, selectivity is good, is not required to large-scale instrument, it can be achieved that quickly detection in situ, testing result is intuitive, can naked eye.It can be applied to adenosine and Cr in practical biological sample3+Detection.
Description
Technical field
The present invention relates to nano-probe fields, and in particular to a kind of gold for visualizing sequence detection adenosine and trivalent chromic ion
The application of the preparation method of nanoparticle probes and prepared probe in detection adenosine and trivalent chromic ion.
Background technique
Gold nanoparticle has unique electrical and optical properties, is widely used in analysis detection and biomedicine etc.
Field.The local surface plasma resonance absorption peak of gold nanoparticle can be controllably adjusted by changing size and shape,
Its solution is set to show different colours, only naked eye can distinguish.DNA has been constructed for the first time from Mirkin in 1996 etc.
(DNA), since gold nanoparticle (AuNPs) biological nano compound system, in recent years using AuNPs as optical biosensor
As research hotspot (C.A.Mirkin, R.L.Letsinger, R.C.Mucic and J.J.Storhoff, Nature, 1996,
382,607-609.)。
If the method for the typical detection lead ion of one kind of Liu et al. exploitation in 2006 is by two kinds of DNA modifications to AuNPs table
On face, nanoparticle is connected with aptamers using base pair complementarity principle, thus assemble AuNPs, solution colour
For blue.After ligand to be detected is added, due to the specific binding of ligand and aptamers, aptamers is made to fall off from the surface AuNPs,
And then make the AuNPs depolymerization of aggregation, solution become it is red (J.W.Liu and Y.Lu, Angew.Chem.Int.Ed., 2006,
45,90-94.).This property by the unique optical property of AuNPs and aptamers specific binding ligand combines development
Visible detection method be extended to other detection fields, such as the inspection to metal ion, DNA, protein, cell
It surveys.However using Liu et al. as a series of gold nanoparticle visible detection methods that representative develops can only be single detect a kind of object
Matter, therefore in order to promote the utilization rate of AuNPs visualization probe solution to the maximum extent, develop one kind can be realized it is dual visual
The new method for changing detection is particularly important.
Summary of the invention
It is an object of that present invention to provide a kind of gold nanoparticle probes for visualizing sequence detection adenosine and trivalent chromic ion
Preparation method, this method is easy to operate, and reaction condition is mild, gained gold nanoparticle probe uniform particle sizes, good dispersion, energy
Detection for adenosine and trivalent chromic ion in practical biological sample.
To achieve the above object, a kind of gold nano visualizing sequence detection adenosine and trivalent chromic ion provided by the invention
The preparation method of particle probe, step include:
(1) 50-150mL 1-3mM chlorauric acid solution is added in the flask with reflux unit, stirring is heated to boil
It rises, is then quickly added into 5-15mL 35-40mM sodium citrate solution, reflux becomes claret until solution.Flask is removed into heat
Source is continued to stir, is cooled to room temperature, and saves backup at 0-5 DEG C；
It (2) is 1:20-1:80 according to the ratio of the amount of substance by 100-200 μM of 5-10 μ L sulfydryl DNA1 and DNA2 and TCEP
It is uniformly mixed, is placed at room temperature for 0.5-2 hours.The sequence of the DNA1 and DNA2 are respectively:
5’-CCCAGGTAAAAAAAAAA-HS-3’
5’-HS-AAAAAAAAAAACCTTCC-3’
(3) the solution of gold nanoparticles 500-1000 μ L for taking step (1) to obtain respectively is separately added into two glass tubes
DNA1, the DNA2 and 100-200 μM of 5-10 μ L MUA that step (2) processing obtains, secondary water, which is added, makes final solution volume 1-
2mL is mixed, the solution in two glass tubes is placed in darkroom 15-20 hours；
(4) solution into two glass tubes that step (3) obtains respectively adds 1-2M Tris-HCl (pH 7.0-7.5) to buffer
Liquid 5-10 μ L and 1-2M NaCl solution 50-100 μ L, gently concussion shake up be placed in darkroom stay overnight；
(5) solution into two glass tubes that step (4) obtains respectively adds 1-2M Tris-HCl (pH 7.0-7.5) to buffer
Liquid 1-5 μ L and 1-2M NaCl solution 10-50 μ L, gently concussion shake up be placed in darkroom stay overnight；
(6) each 200-1000 μ L of solution in two glass tubes for taking step (5) to obtain respectively is in two 1.5mL centrifuge tubes
In, be placed in desk centrifuge and be centrifuged 10-20 minutes with the revolving speed of 10000-15000rpm, it is as much as possible remove supernatant with
Remove the DNA drifted in solution.200-1000 μ L NaCl containing 100-200mM, 15-25mM Tris-HCl pH 7.0- is added
7.5 buffer disperses gold nanoparticle again；
(7) two kinds of solution for obtaining step (6) are uniformly mixed, and the adenosine aptamers of 1-2 μ L 100-200 μ Μ are added,
Make aptamers and the ratio 0.2-1.0 of DNA；
(8) solution for obtaining step (7) reacts 5-20 minutes at 50-70 DEG C, is slowly cooled to room temperature, and in 2-7
It is saved at DEG C.
The concentration of gold chloride and sodium citrate is respectively preferably 1mM and 38.8mM in step (1), and storage temperature is preferably 4
℃；
The concentration and volume of sulfydryl DNA1 and sulfydryl DNA2 are preferably 100 μM of 6.6 μ L in step (2), are placed at room temperature for the time
Preferably 1 hour；
Solution of gold nanoparticles volume is preferably the concentration of 600 μ L, MUA in step (3) and volume is preferably 100 μM 6.6
μ L, final volume are preferably 1mL, and being placed in the darkroom time is preferably 16 hours；
8.4 μ L and 1M NaCl solution of Tris-HCl buffer, the 76 μ L of preferred 1M pH 7.5 in step (4)；
4.2 μ L and 1M NaCl solution of Tris-HCl buffer, the 42 μ L of preferred 1M pH 7.5 in step (5)；
Step (6) preferably liquor capacity is 500 μ L, is placed in desk centrifuge and is centrifuged 15 points with the revolving speed of 13000rpm
Clock.The buffer that 500 μ L NaCl containing 1500mM, 20mM Tris-HCl pH 7.0 are added disperses gold nanoparticle again；
The volume of preferred adenosine aptamers and concentration are 1.75 μ L, 100 μ Μ respectively in step (7), aptamers and DNA's
Ratio is 0.6；
Preferable reaction temperature is 65 DEG C in step (8), and the reaction time is 15 minutes.
The visualization nano-probe of the method for the present invention preparation can be used for detecting adenosine and Cr in practical biological sample3+。
Compared with the prior art, the advantages of the present invention are as follows:
(1) visualization probe provided by the invention can detect adenosine and Cr with double sequence3+, realize " one is dual-purpose "；
(2) Visual retrieval probe high sensitivity provided by the invention, selectivity are good, to adenosine and Cr3+Detection limit point
It Wei 1.8 × 10-8M、1.7×10-11M, at least an order of magnitude lower than the detection limit of existing method；
(3) large-scale instrument is not needed, it, can recognition detection result by naked eye or comparison spectrum；
(4) agents useful for same of the present invention and operating process have no toxic side effect；
(5) the method for the present invention is simple, quick, easy to operate, can carry out live quickly detection in situ.
Detailed description of the invention
Fig. 1 is the mechanism of action schematic diagram of visualization nano-probe prepared by the present invention
Fig. 2 is that visualization nano-probe solution prepared by the present invention is absorbed with incubation temperature, its UV-Vis of the variation of time
Spectral ratio A522/A650Variation
Fig. 3 is visualization nano-probe solution prepared by the present invention with its UV-Vis absorption spectrum of the variation of adenosine concentration
Variation
Fig. 4 is the linear relationship between visualization nano-probe solution and various concentration adenosine solution prepared by the present invention
Fig. 5 is visualization nano-probe solution prepared by the present invention with Cr3+Its UV-Vis absorption spectrum of the variation of concentration
Variation
Fig. 6 is visualization nano-probe solution prepared by the present invention and various concentration Cr3+Linear relationship between solution
Specific embodiment
The present invention is that solution of gold nanoparticles is prepared in the method for classical reduction of sodium citrate gold chloride, is passed through
Au-S chemical bond is by the common functionalization of sulfydryl modification DNA and 11- Mercaptoundecanoic acid (MUA) on gold nanoparticle surface.It utilizes
DNA complementary pairing of the nucleic acid base complementary pairing principle by adenosine aptamers and modification on gold nanoparticle surface, makes Jenner
Rice corpuscles has the ability of the specific recognition adenosine of aptamers and the complexing Cr of MUA simultaneously3+Ability, and for practical biology
Adenosine and Cr in sample3+Detection.Below by specific embodiment combination attached drawing, the invention will be further described.
Embodiment 1
Visualize the preparation of gold nanoparticle probe:
(1) 100mL 1mM chlorauric acid solution is added in the flask with reflux unit, stirring is heated to boil, then
10mL 38.8mM sodium citrate solution is rapidly joined, reflux becomes claret until solution.Flask is removed into heat source, continues to stir
It mixes, is cooled to room temperature, saved backup at 4 DEG C；
(2) 100 μM of 6.6 μ L sulfydryl DNA1 and DNA2 and TCEP is mixed according to the ratio that the mass ratio of the material is 1:50 equal
It is even, it is placed at room temperature for 1 hour；
(3) the 600 μ L of solution of gold nanoparticles for taking step (1) to obtain respectively is separately added into step in two glass tubes
(2) obtained DNA1, DNA2 and 100 μM of 6.6 μ L MUA is handled, secondary water, which is added, makes final solution volume 1mL, mixes, will
Solution in two glass tubes is placed in darkroom 16 hours；
(4) solution into two glass tubes that step (3) obtains respectively adds 8.4 μ of 1M Tris-HCl (pH 7.5) buffer
76 μ L of L and 1M NaCl solution, gently concussion shake up be placed in darkroom stay overnight；
(5) solution into two glass tubes that step (4) obtains respectively adds 4.2 μ of 1M Tris-HCl (pH 7.5) buffer
42 μ L of L and 1M NaCl solution, gently concussion shake up be placed in darkroom stay overnight；
(6) each 500 μ L of solution in two glass tubes for taking step (5) to obtain respectively is set in two 1.5mL centrifuge tubes
It is centrifuged 15 minutes in desk centrifuge with the revolving speed of 13000rpm, it is as much as possible to remove supernatant and be free in solution to remove
In DNA.The buffer that 500 μ L NaCl containing 150mM, 20mM Tris-HCl pH 7.0 are added divides gold nanoparticle again
It dissipates；
(7) two kinds of solution for obtaining step (6) are uniformly mixed, and the adenosine aptamers of 1.75 μ L, 100 μ Μ are added, make to fit
The ratio of ligand and DNA are 0.6；
(8) solution for obtaining step (7) reacts 15 minutes at 65 DEG C, is slowly cooled to room temperature, and protects at 4 DEG C
It deposits.
The mechanism of action schematic diagram of the visualization nano-probe of preparation is shown in Fig. 1.
Embodiment 2
The influence for the visualization nano-probe solution reaction that incubation time and temperature prepare adenosine and embodiment 1:
The visualization nano-probe solution for taking 500 μ L embodiments 1 to prepare, is added 150 μ L 0.01M adenosines thereto, 2 ×
10-3M MgCl2, add secondary water that final solution volume is made to reach 750 μ L.Solution is reacted at 25,30,35,40,45 DEG C respectively
It 2,4,6,8,10,12,14 minutes, detects under different temperatures different time to UV-Vis absorption spectrum ratio A522/A650Influence.
Different temperatures different time is to visualization nano-probe solution UV-Vis absorption spectrum ratio A522/A650Influence see
Fig. 2: under the differential responses time, absorption spectrum ratio A522/A650It is constant；As temperature constantly increases, constant value is not
It is disconnected to reduce, illustrate that visualization nano-probe solution prepared by the present invention reacts very fast (less than 2 minutes) with adenosine and in room
Reaction can be completed in (25 DEG C) under temperature.
Embodiment 3
The adenosine solution UV-Vis absorption spectra of various concentration is added into visualization nano-probe solution prepared by embodiment 1
Figure is tested with the variation of adenosine concentration:
80 μ L of visualization nano-probe solution prepared by each Example 1 is added thereto respectively in 0.5mL centrifuge tube
The adenosine of various concentration and 2 × 10-3M MgCl2Solution, secondary water, which is added, makes 120 μ L of final solution volume.Detection is in difference
Under adenosine concentration, the variation of the UV-Vis absorption spectrum of nano-probe solution is visualized.
Fig. 3 is shown in influence of the adenosine solution of various concentration to the UV-Vis absorption spectrum of visualization nano-probe solution: by
Cumulative plus adenosine concentration visualizes absorption peak of the nano-probe solution at 522nm and gradually rises, the absorption peak at 650nm
It gradually decreases.Illustrate that visualization nano-probe solution prepared by the present invention can be realized the detection to different adenosine concentrations.
In addition, the UV-Vis absorption spectrum ratio A of visualization nano-probe solution prepared by the present invention522/A650Variation
It is in a linear relationship with various concentration adenosine solution, as shown in figure 4, with the growth of adenosine concentration, absorption spectrum ratio A522/A650
It gradually increases, the R of linear equation2=0.994.
Embodiment 4
The Cr of various concentration is added into visualization nano-probe solution prepared by embodiment 13+Solution UV-Vis absorption spectra
Figure is tested with the variation of adenosine concentration:
Adenosine solution is added 200 μ L of visualization nano-probe solution prepared by Example 1 thereto, makes its concentration 10- 4M is incorporated as MgCl2Solution makes its concentration 2 × 10-3M, secondary water, which is added, makes 450 μ L of final solution volume.Gradually to molten
Cr is added in liquid3+, detect in different Cr3+Under concentration, the variation of the UV-Vis absorption spectrum of nano-probe solution is visualized.
The Cr of various concentration3+Fig. 5 is shown in influence of the solution to the UV-Vis absorption spectrum of visualization nano-probe solution: gradually
Increase Cr3+Concentration, visualize absorption peak of the nano-probe solution at 522nm and gradually decrease, the absorption peak at 650nm is gradually
It increases.Illustrate that visualization nano-probe solution prepared by the present invention can be realized to different Cr3+The detection of concentration.
In addition, the UV-Vis absorption spectrum ratio A of visualization nano-probe solution prepared by the present invention650/A522Variation
With various concentration Cr3+Solution is in a linear relationship, as shown in fig. 6, with Cr3+The growth of concentration, absorption spectrum ratio A650/A522
It gradually increases, the R of linear equation2=0.991.
Embodiment 5
Visualization nano-probe solution prepared by embodiment 1 is for adenosine and Cr in practical biological sample3+Test experience:
As shown in Table 1 and Table 2, using standard addition method, adenosine and Cr is sequentially added into processed human urine3+。
The visualization nano-probe solution detection adenosine and Cr prepared with embodiment 13+Content, the relative standard deviation measured is respectively less than
5%, illustrate that visualization nano-probe prepared by the present invention can be used for detecting adenosine and Cr in practical biological sample3+。
Table 1 is detection of the visualization nano-probe solution prepared by the present invention for adenosine in practical biological sample
Table 2 is visualization nano-probe solution prepared by the present invention for Cr in practical biological sample3+Detection
Claims (10)
1. a kind of preparation method for visualizing gold nanoparticle probe, which is characterized in that step includes:
1) 50-150mL 1-3mM chlorauric acid solution is added in the flask with reflux unit, stirring is heated to boil, then
5-15mL 35-40mM sodium citrate solution is rapidly joined, reflux becomes claret until solution；Flask is removed into heat source, is continued
It stirs, the gold nanoparticle being cooled to room temperature, is saved backup at 0-5 DEG C；
2) 100-200 μM of 5-10 μ L sulfydryl DNA1 and DNA2 is mixed with TCEP according to the ratio of the amount of substance for 1:20-80 respectively
Uniformly, 0.5-2 hours are placed at room temperature for；The sequence of the DNA1 and DNA2 are respectively:
5’-CCCAGGTAAAAAAAAAA-HS-3’
5’-HS-AAAAAAAAAAACCTTCC-3’
3) the solution of gold nanoparticles 500-1000 μ L for taking step 1) to obtain respectively is separately added into step 2) in two glass tubes
DNA1, DNA2 and 100-200 μM of obtained 5-10 μ L MUA are handled, secondary water, which is added, makes final solution volume 1-2mL, mixes
It is even, the solution in two glass tubes is placed in darkroom 15-20 hours；
4) solution into two glass tubes that step 3) obtains respectively adds 1-2M Tris-HCl pH 7.0-7.5 buffer 5-10 μ
L and 1-2M NaCl solution 50-100 μ L, gently concussion shake up be placed in darkroom stay overnight；
5) solution into two glass tubes that step 4) obtains respectively add 1-2M Tris-HC pH7.0-7.5 buffer 1-5 μ L and
1-2M NaCl solution 10-50 μ L, gently concussion shake up be placed in darkroom stay overnight；
6) each 200-1000 μ L of solution in two glass tubes for taking step 5) to obtain respectively is set in two 1.5mL centrifuge tubes
It is centrifuged 10-20 minutes in desk centrifuge with the revolving speed of 10000-15000rpm, 200-1000 μ L is added and contains 100-200mM
NaCl, 15-25mM Tris-HCl pH 7.0-7.5 buffer disperse gold nanoparticle again；
7) two kinds of solution for obtaining step 6) are uniformly mixed, and the adenosine aptamers of 1-2 μ L 100-200 μ Μ are added, make to be adapted to
The ratio of body and DNA are 0.2-1.0；
8) solution for obtaining step 7) reacts 5-20 minutes at 50-70 DEG C, is slowly cooled to room temperature, and protects at 2-7 DEG C
It deposits.
2. a kind of preparation method for visualizing gold nanoparticle probe as described in claim 1, which is characterized in that the step
1) concentration of chlorauric acid solution is 1mM in, and the concentration of sodium citrate solution is 38.8mM, and storage temperature is 4 DEG C.
3. a kind of preparation method for visualizing gold nanoparticle probe as described in claim 1, which is characterized in that the step
2) concentration of sulfydryl DNA1 and sulfydryl DNA2 are 100 μM in, volume is 6.6 μ L, and being placed at room temperature for the time is 1 hour.
4. a kind of preparation method for visualizing gold nanoparticle probe as described in claim 1, which is characterized in that the step
3) in solution of gold nanoparticles volume be 600 μ L, MUA concentration be 100 μM, volume is 6.6 μ L, final volume 1mL is placed in
The darkroom time is 16 hours.
5. a kind of preparation method for visualizing gold nanoparticle probe as described in claim 1, which is characterized in that the step
4) solution in into two glass tubes respectively adds 8.4 μ L, 1M NaCl solution of Tris-HCl buffer, the 76 μ L of 1M pH 7.5.
6. a kind of preparation method for visualizing gold nanoparticle probe as described in claim 1, which is characterized in that the step
5) solution in into two glass tubes respectively adds 4.2 μ L and 1M NaCl solution of Tris-HCl buffer, the 42 μ L of 1M pH 7.5.
7. a kind of preparation method for visualizing gold nanoparticle probe as described in claim 1, which is characterized in that the step
6) take each 500 μ L of the solution in two glass tubes in two 1.5mL centrifuge tubes respectively, be placed in desk centrifuge with
The revolving speed of 13000rpm is centrifuged 15 minutes；The buffer of 500 μ L NaCl containing 1500mM, 20mM Tris-HCl pH 7.0 is added
Disperse gold nanoparticle again.
8. a kind of preparation method for visualizing gold nanoparticle probe as described in claim 1, which is characterized in that the step
7) it is 100 μ Μ that the volume of adenosine aptamers, which is 1.75 μ L, concentration, in, and the ratio of aptamers and DNA are 0.6.
9. a kind of preparation method for visualizing gold nanoparticle probe as described in claim 1, which is characterized in that the step
8) reaction temperature is 65 DEG C in, and the reaction time is 15 minutes, and is saved at 4 DEG C.
10. if the visualization gold nanoparticle probe of any the method preparation of claim 1-9 is in sequence detection adenosine and Cr3+
In application.
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| Facile Preparation of High-Quantum-Yield Gold Nanoclusters:Application to Probing Mercuric Ions and Biothiols;Heng-Chia Chang et al.;《Applied materials & interfaces》;20141017;第6卷;第18824-18831页 |
| 一种新型发光探针与ＤＮＡ相互作用的研究;王令纺等;《分析科学学报》;20120831;第28卷(第4期);第450-454页 |
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