CN107058299B - Near-infrared fluorescent nucleic acid silver nanocluster and preparation method and application thereof - Google Patents
Near-infrared fluorescent nucleic acid silver nanocluster and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of cell imaging and preparation of nano nucleic acid quantum dot reagents, and particularly relates to a stable near-infrared silver nucleic acid nano cluster, a method thereof and application thereof in cell imaging. Using nucleic acid sequence as template, in sodium citrate buffer solution and using NaBH4Reducing AgNO3Silver nucleic acid nanoparticles with near-infrared fluorescence are synthesized. The silver nucleic acid nanoparticles can emit fluorescence of 800nm under the excitation wavelength of 750 nm. The size of the silver nucleic acid nanocluster is 70 nm. The nucleic acid silver has high stability and can be used for fluorescence imaging of tumor cells.
Description
Technical Field
The invention belongs to the field of cell imaging and preparation of nano nucleic acid quantum dot reagents, and particularly relates to a stable near-infrared silver nucleic acid nano cluster, a method thereof and application thereof in cell imaging.
Background
The preparation of inorganic nano materials and nanoclusters by using DNA as a template has become a new application of DNA. The silver nanoclusters taking DNA as a template have potential application in biosensing. Compared with quantum dots and organic dyes, the nucleic acid silver nanoclusters (DNA-AgNCs) have the advantages of easiness in synthesis, excellent optical performance, adjustable emission and the like. However, most DNA-AgNCs have short fluorescence lifetime, reduced fluorescence intensity with time, and quenching in a short time, which limits their application in bioprobes (Zhou Z X and Dong S J, Protein-DNA interactions: A novel approach to improve the fluorescence stability of DNA/Agnacusters, Nanoscale,2014,10, 1-4). Imaging in the visible region (400-. In the near infrared region, the background of absorption, scattering and autofluorescence of tissue is low, and the near infrared light source can reach the maximum penetration depth in biological tissue and can image deep tissue, so this band range is called "near infrared tissue transparent window" (Jiang W, Sinkal A, Kim B Y S., et al, assembling near-infrared tissue dots for deep tissue applications, organ, and animal imaging applications, JALA,2008,13, 6-12). At present, near-infrared imaging mainly adopts near-infrared fluorescent dye and near-infrared gold quantum dots, but the targeting property to tumor tissues is lacked. Therefore, the preparation of the near-infrared emission silver nucleic acid with high stability can obtain the quantum dot with the nucleic acid identification performance, can solve the problem of targeting in tumor imaging, and is a novel near-infrared dye with low toxicity, biocompatibility and targeting.
Disclosure of Invention
The present invention designs a novel deoxyribonucleic acid (DNA) sequence: 5 'CCCACCCACCCTCCCAACAACAGAGGAG 3'. Secondly, the DNA is taken as a template, and NaBH is utilized in sodium citrate buffer solution4Reducing AgNO3And a method for interacting tryptophan and BSA, and successfully synthesizes a stable novel fluorescent nucleic acid silver nanocluster (Trp-DNA-AgNCs @ BSA). The excitation wavelength of the silver nucleic acid nanocluster (Trp-DNA-AgNCs @ BSA) is set to be 750nm during fluorescence measurement, near-infrared fluorescence with the emission wavelength of 800nm is obtained, and the size of the silver nucleic acid nanocluster is 70 nm. The near infrared fluorescence can be used for cell imaging and detection of nucleic acid in blood.
A preparation method for improving the stability of silver nucleic acid for cell imaging comprises the following steps:
DNA (sequence 5 'CCCACCCACCCTCCCAACAACAGAGGAG 3'), tryptophan (Trp) and AgNO were added to a sodium citrate buffer using the DNA sequence as a template3Mixing, placing in water bath, heating at 20-71 deg.C for 1-3min, preferably 2min, and slowly cooling to room temperature for 1-2 hr, preferably 1.5 hr; adding NaBH4The solution is mixed with Trp-DNA-Ag+Reducing into Trp-DNA-Ag, transferring to 4 deg.C environment, standing in dark for 1-3 days, preferably 2 days, standing for 1-3 days, adding Bovine Serum Albumin (BSA) solution,and obtaining the stable near-infrared nucleic acid silver nanocluster.
Wherein the pH value of the sodium citrate buffer solution is 5.0-6.0, the concentration is 10-20mM, the optimal condition is pH5.0, the concentration is 10mM, and 5-10 mu mol of DNA is added into each 1mL of the sodium citrate buffer solution.
Wherein said DNA is bound to Ag+The molar ratio of (A) is 1:20-1:40, and the optimal molar ratio is 1: 33.33.
Wherein said tryptophan is reacted with Ag+The molar ratio of (A) is 1:1-1:2, and the optimal molar ratio is 1: 1.33.
Wherein said NaBH4With Ag+The molar ratio of (A) is 1:1-4:1, and the optimal molar ratio is 2: 1.
Wherein the BSA and Ag+The molar ratio of (A) is 1:50-1:80, and the optimal molar ratio is 1: 66.67.
Wherein the temperature is 20-71 ℃, and the optimal temperature is 71 ℃.
In the literature, it has been reported that in the Chinese patent 201510686946.2 "a fluorescent silver nucleic acid and its preparation and application", BSA is added for the main purpose of Fe3+The detection of ions does not greatly improve the fluorescence stability of the nucleic acid silver, and the fluorescence is reduced in a short time; the invention prepares the silver nucleic acid containing tryptophan and BSA for the first time, obtains a novel near-infrared fluorescence emission nano-cluster (figure 1) taking the BSA as a shell and the tryptophan fluorescent silver nucleic acid as a core by introducing hydrophobic amino acid and the BSA through hydrophobic interaction, has high stability, basically keeps the fluorescence unchanged when placed at room temperature (figure 2), can be used for the fluorescence imaging of tumor cells (figure 3), and is reported for the first time.
Drawings
FIG. 1 shows Trp-DNA-AgNCs @ BSA (C)DNA1 μ M, aqueous solution) fluorescence excitation and emission spectra.
FIG. 2 is a graph of the change in fluorescence with time of Trp-DNA-AgNCs @ BSA in sodium citrate buffer (pH 7.0, 10 mM).
FIG. 3 is a photograph of tumor cell fluorescence images.
Detailed Description
Synthesis of Trp-DNA-AgNCs @ BSA
Example 1(Trp-DNA-AgNCs @ BSA optimal preparation): using the DNA sequence as a template, 6. mu. mol of DNA (the DNA sequence was "5 'CCCACCCACCCTCCCAACAACAGAGGAG 3'") (the sequence was designed by us, synthesized with DNA synthesizer of Shanghai Biotech, and purified by HPLC, and the sequence was 5 'CCCACCCACCCTCCCAACAACAGAGGAG 3'), 200. mu. mol of AgNO were added to 1mL of sodium citrate buffer solution (pH 5.0, 10mM) in the presence of a DNA sequence (the DNA sequence was "5 'CCCACCCACCCTCCCAACAACAGAGGAG 3'")3(wherein the DNA is bound to Ag+In a molar ratio of 1:33.33) and 150. mu. mol tryptophan (wherein tryptophan is bound to Ag)+The molar ratio of (1: 1.33), uniformly mixing, placing in a water bath, heating at 71 ℃ for 2min for stabilization, cooling for 2h, and cooling to room temperature. Adding NaBH into the reaction solution at room temperature4Solution (in which NaBH is added to the solution)4With Ag+At a molar ratio of 2:1), standing at room temperature for 10min, and adding Trp-DNA-Ag+Reducing to Trp-DNA-Ag, transferring the reaction solution to 4 ℃ environment, keeping away from light for 2 days, and adding 3 mu mol BSA (wherein BSA and Ag+The molar ratio of 1:66.67), and the stable near-infrared nucleic acid silver nanocluster can be obtained. To excite 750nm to obtain 800nm near infrared fluorescence. The fluorescence intensity of 1. mu.M Trp-DNA-AgNCs @ BSA was 225.
Example 2: using the DNA sequence as a template, 10. mu. mol of DNA (the DNA sequence was "5 'CCCACCCACCCTCCCAACAACAGAGGAG 3'") (the sequence was designed by us, synthesized with DNA synthesizer of Shanghai Biotech, and purified by HPLC, and the sequence was 5 'CCCACCCACCCTCCCAACAACAGAGGAG 3'), 200. mu. mol of AgNO were added to 1mL of sodium citrate buffer solution (pH 5.0, 10mM) in the presence of a DNA sequence (the DNA sequence was "5 'CCCACCCACCCTCCCAACAACAGAGGAG 3'")3(wherein the DNA is bound to Ag+In a molar ratio of 1:20) and 200. mu. mol tryptophan (wherein tryptophan is bound to Ag)+The molar ratio of (1: 1), uniformly mixing, placing in a water bath, heating at 71 ℃ for 2min, cooling for 2h, and cooling to room temperature. Adding NaBH into the reaction solution at room temperature4Solution (in which NaBH is added to the solution)4With Ag+At a molar ratio of 1:1), standing at room temperature for 10min, and adding Trp-DNA-Ag+Reducing to Trp-DNA-Ag, transferring the reaction solution to 4 deg.C environment, standing in dark for 2 days, and adding 4 μmol BSA (where BSA is associated with Ag)+The molar ratio of the silver nitrate to the silver nitrate is 1:50), and the stable near-infrared nucleic acid silver nano cluster can be obtained. To excite 750nm to obtain 800nm near infrared fluorescence. The fluorescence intensity of 1. mu.M Trp-DNA-AgNCs @ BSA was 150.
Example 3: using the DNA sequence as a template, 5. mu. mol of DNA (the DNA sequence was "5 'CCCACCCACCCTCCCAACAACAGAGGAG 3'") (the sequence was designed by us, synthesized with DNA synthesizer of Shanghai Biotech, and purified by HPLC, and the sequence was 5 'CCCACCCACCCTCCCAACAACAGAGGAG 3'), 200. mu. mol of AgNO were added to 1mL of sodium citrate buffer solution (pH 5.0, 10mM) in the presence of a DNA sequence (the DNA sequence was "5 'CCCACCCACCCTCCCAACAACAGAGGAG 3'")3(wherein the DNA is bound to Ag+In a molar ratio of 1:40) and 100. mu. mol tryptophan (wherein tryptophan is bound to Ag)+The molar ratio of (1: 2), uniformly mixing, placing in a water bath, heating at 71 ℃ for 2min, cooling for 2h, and cooling to room temperature. Adding NaBH into the reaction solution at room temperature4Solution (in which NaBH is added to the solution)4With Ag+At a molar ratio of 4:1), standing at room temperature for 10min, and adding Trp-DNA-Ag+Reducing to Trp-DNA-Ag, transferring the reaction solution to 4 ℃ environment, keeping away from light for 2 days, and adding 2.5 mu mol BSA (wherein BSA and Ag+The molar ratio of the silver salt to the silver salt is 1:80), and the stable near-infrared nucleic acid silver nano cluster can be obtained. To excite 750nm to obtain 800nm near infrared fluorescence. The fluorescence intensity of 1. mu.M Trp-DNA-AgNCs @ BSA was 100.
Example 4: using the DNA sequence as a template, 6. mu. mol of DNA (the DNA sequence was "5 'CCCACCCACCCTCCCAACAACAGAGGAG 3'") (the sequence was designed by us, synthesized with DNA synthesizer of Shanghai Biotech, and purified by HPLC, and the sequence was 5 'CCCACCCACCCTCCCAACAACAGAGGAG 3'), 200. mu. mol of AgNO were added to 1mL of sodium citrate buffer solution (pH 5.0, 10mM) in the presence of a DNA sequence (the DNA sequence was "5 'CCCACCCACCCTCCCAACAACAGAGGAG 3'")3(wherein the DNA is bound to Ag+In a molar ratio of 1:33.33) and 150. mu. mol tryptophan (wherein tryptophan is bound to Ag)+The molar ratio of (1: 1.33), uniformly mixing, placing in a water bath, heating and stabilizing at 20 ℃ for 2min, then beginning to cool, wherein the cooling process is 2h, and cooling to room temperature. Adding NaBH into the reaction solution at room temperature4Solution (in which NaBH is added to the solution)4With Ag+At a molar ratio of 2:1), standing at room temperature for 10min,mixing Trp-DNA-Ag+Reducing to Trp-DNA-Ag, transferring the reaction solution to 4 ℃ environment, keeping away from light for 2 days, and adding 3 mu mol BSA (wherein BSA and Ag+The molar ratio of 1:66.67), and the stable near-infrared nucleic acid silver nanocluster can be obtained. To excite 750nm to obtain 800nm near infrared fluorescence. The fluorescence intensity of 1. mu.M Trp-DNA-AgNCs @ BSA was 120.
Stable near infrared silver nucleic acid for cell imaging
Taking the synthesized Trp-DNA-AgNCs @ BSA, preparing a DMSO solution of 18 mu M DNA-AgNCs-Trp @ BSA, and placing the DMSO solution in a dark environment at 4 ℃.
Step two, diluting Trp-DNA-AgNCs @ BSA to 6 mu M by using a culture solution, adding the diluted Trp-DNA-AgNCs @ BSA into HepG-2 liver cancer cells, and incubating for 1 h.
And step three, washing the culture medium by using a PBS buffer solution to remove compounds which do not enter the cells.
And step four, observing and recording by using a Nikon Ti-E2000 microscope. The excitation wavelength was 750nm and the emission wavelength was 800 nm.
And the measurement result is shown in the attached figure 3, and the figure 3 is a cell imaging graph of Trp-DNA-AgNCs @ BSA.
Sequence listing
<110> university of Jiangsu
<120> near-infrared fluorescent silver nucleic acid nanocluster and preparation method and application thereof
<160> 1
<210> 1
<211> 28
<212> DNA
<213> Artificial sequence
<400> 1
cccacccacc ctcccaacaa cagaggag 28
Claims (6)
1. A preparation method of a near-infrared fluorescent silver nucleic acid nano cluster is provided, wherein the near-infrared fluorescent silver nucleic acid nano cluster is marked as Trp-DNA-AgNCs @ BSA, a DNA sequence 5 'CCCACCCACCCTCCCAACAACAGAGGAG 3' is used as a template, and NaBH is utilized in a sodium citrate buffer solution4Reducing AgNO3And cyanamideThe acid is synthesized by a method of the interaction of acid and BSA; the silver nucleic acid nanocluster is characterized by comprising the following specific steps of setting the excitation wavelength to be 750nm during fluorescence measurement, obtaining near-infrared fluorescence with the emission wavelength of 800nm, wherein the size of the silver nucleic acid nanocluster is 70nm, and the near-infrared fluorescence can be used for cell imaging and detection of nucleic acid in blood:
adding DNA sequence, tryptophan (Trp) and AgNO into sodium citrate buffer solution by using DNA sequence 5 'CCCACCCACCCTCCCAACAACAGAGGAG 3' as template3Mixing, placing in water bath, heating at 71 deg.C for 1-3min, slowly cooling to room temperature for 1-2 hr, adding NaBH4The solution is mixed with Trp-DNA-Ag+Reducing to Trp-DNA-Ag, transferring to 4 ℃ environment, placing for 1-3 days in a dark place, placing for 1-3 days, and adding Bovine Serum Albumin (BSA) solution to obtain the stable near-infrared nucleic acid silver nanocluster.
2. The method of claim 1, wherein the pH value of the sodium citrate buffer solution is 5.0-6.0, the concentration of the sodium citrate buffer solution is 10-20mM, and 5-10 μmol of DNA is added per 1mL of the sodium citrate buffer solution.
3. The method of claim 2, wherein the pH value of the sodium citrate buffer solution is 5.0, the concentration of the sodium citrate buffer solution is 10mM, and 6 μmol of DNA is added to 1mL of the sodium citrate buffer solution.
4. The method of claim 1, wherein the DNA and Ag are present in the silver nucleic acid nanoclusters+In a molar ratio of 1:20 to 1: 40; the tryptophan and the Ag+The molar ratio of (1: 1) - (1: 2); the NaBH4With Ag+The molar ratio of (1: 1) - (4: 1); the BSA and Ag+In a molar ratio of 1:50 to 1: 80.
5. The method of claim 4, wherein the near-infrared fluorescent silver nucleic acid nanoclusters are prepared byThe DNA and Ag+In a molar ratio of 1: 33.33; the tryptophan and the Ag+In a molar ratio of 1: 1.33; the NaBH4With Ag+In a molar ratio of 2: 1; the BSA and Ag+Is 1: 66.67.
6. The method for preparing near-infrared fluorescent silver nucleic acid nanoclusters according to claim 1, wherein the heating is carried out at 71 ℃ for 2 min; the cooling time is 1.5 h; the cells were transferred to a 4 ℃ environment and left for 2 days in the dark.
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| CN110095519B (en) * | 2019-04-29 | 2021-11-16 | 中国科学院苏州生物医学工程技术研究所 | Method for detecting nucleic acid by using silver nano cluster-based composite material for non-disease diagnosis |
| CN110202128A (en) * | 2019-06-25 | 2019-09-06 | 重庆科技学院 | A kind of gold and silver composite Nano cluster, preparation process and the application in biological thiol detection |
| CN113456670B (en) * | 2021-07-02 | 2022-08-16 | 太原理工大学 | Indole derivative-silver composite nano-particles, preparation method thereof and application of indole derivative-silver composite nano-particles as antibacterial material |
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