CN107058299A - A kind of near-infrared fluorescent nucleic acid silver nanoclusters and its production and use - Google Patents

A kind of near-infrared fluorescent nucleic acid silver nanoclusters and its production and use Download PDF

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CN107058299A
CN107058299A CN201610837758.XA CN201610837758A CN107058299A CN 107058299 A CN107058299 A CN 107058299A CN 201610837758 A CN201610837758 A CN 201610837758A CN 107058299 A CN107058299 A CN 107058299A
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acid silver
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陈秋云
穆威宇
肖新新
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Shanghai Boni Medical Laboratory Co ltd
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    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract

The invention belongs to cell imaging and nano nucleic acid quantum dot reagent preparation field, a kind of stable near-infrared nucleic acid silver nanoclusters and its method and the utilization in cell imaging are refered in particular to.Using nucleotide sequence as template, in sodium citrate buffer solution, NaBH is utilized4Reduce AgNO3The nucleic acid silver nano-grain with near-infrared fluorescent is synthesized.The nucleic acid silver nano-grain can launch 800nm fluorescence in the case where excitation wavelength is 750nm.The size of nucleic acid silver nanoclusters is 70nm.This nargol stability is high, the fluorescence imaging available for tumour cell.

Description

A kind of near-infrared fluorescent nucleic acid silver nanoclusters and its production and use
Technical field
The invention belongs to cell imaging and nano nucleic acid quantum dot reagent preparation field, a kind of stable near-infrared nucleic acid is refered in particular to Silver nanoclusters and its method and the utilization in cell imaging.
Background technology
Inorganic nano material and nano-cluster are prepared by template of DNA turns into the applications of DNA newly.Wherein, using DNA as template Silver nanoclusters have potential application in terms of bio-sensing.Compared with quantum dot, organic dyestuff, nucleic acid silver nanoclusters (DNA- AgNCs) have and be readily synthesized, excellent optical property and the advantages of launch adjustable.But, most of DNA-AgNCs fluorescence longevity Life is short, and fluorescence intensity is reduced with the time, is quenched in the short time, limits its application (Zhou Z X and in bioprobe Dong S J,Protein-DNA interactions:A novel approach to improve the fluorescent stability of DNA/Agnanoclusters,Nanoscale,2014,10,1-4).At visible region (400-700nm) Imaging there are problems that it is many, such as can be by endogenous material in biological tissue (aerobic, anaerobic hemoglobin, melanin, water With bilirubin etc.) influence (Sevick-Muraca E M, the Houston J P and to optical imagery such as absorption, scattering Gurfinkel M,Fluorescence-enhanced,near infrared diagnostic imaging with contrast agents,Curr.Opin.Chem.Biol.,2002,6,642-650).In near infrared region, the absorption of tissue, Scattering and autofluorescence background are all than relatively low, and near-infrared light source can reach maximum penetration in biological tissue, and can carry out Deep tissues are imaged, thus this wavelength band is called " near infrared tissue transparent window " (Jiang W, Sinkal A, Kim B Y S.,et al,Assessing near-infrared quantum dots for deep tissue,organ,and animal imaging applications,JALA,2008,13,6-12).Current near infrared imaging is mainly glimmering using near-infrared Photoinitiator dye and the golden quantum dot of near-infrared, but it is a lack of the targeting to tumor tissues.So, near infrared emission and the high core of stability The preparation of sour silver can obtain the quantum dot of nucleic acid recognizing performance, it is possible to resolve sex chromosome mosaicism is targetted in tumor imaging, is a kind of new tool There is the nir dye of hypotoxicity, biocompatibility and targeting.
The content of the invention
The present invention devises a kind of new picodna (DNA) sequence：5’ CCCACCCACCCTCCCAACAACAGAGGAG3’.Secondly using this DNA as template, in sodium citrate buffer, utilize NaBH4Reduce AgNO3And the method that tryptophan interacts with BSA, successfully synthesize the novel fluorescence nucleic acid silver nanoparticle of stability Cluster (Trp-DNA-AgNCs@BSA).This nucleic acid silver nanoclusters (Trp-DNA-AgNCs BSA) are set in fluoremetry to be excited Wavelength is 750 nm, obtains launch wavelength 800nm near-infrared fluorescent, the size of nucleic acid silver nanoclusters is 70nm.Its near-infrared Fluorescence can be used for the detection of cell imaging and nucleic acids in blood.
A kind of nargol stability that improves is used for the preparation method of cell imaging, carries out as steps described below：
Using DNA sequence dna as template, (sequence is 5 ' to addition DNA in sodium citrate buffer solution CCCACCCACCCTCCCAACAACAGAGGAG3 '), tryptophan (Trp) and AgNO3, it is placed into after being well mixed in water-bath, 20- 71 DEG C heating 1-3min, optimal 2min, after be slowly dropped to room temperature, temperature fall time is 1-2h, optimal 1.5h；Add NaBH4Solution By Trp-DNA-Ag+Trp-DNA-Ag is reduced into, avoid light place 1-3 days in 4 DEG C of environment is transferred to, optimal 2 days, places 1-3 days Bovine serum albumin (BSA) solution is added afterwards, you can the near-infrared nucleic acid silver nanoclusters stablized.
The pH value of wherein described sodium citrate buffer is 5.0-6.0, and concentration is 10-20mM, optimum condition PH5.0, concentration is 10mM, and 5-10 μm of ol DNA is added per 1mL sodium citrate buffer solutions.
Wherein described DNA and Ag+Mol ratio be 1:20-1:40, optimum mole ratio is 1:33.33.
Wherein described tryptophan and Ag+Mol ratio be 1:1-1:2, optimum mole ratio is 1:1.33.
Wherein described NaBH4With Ag+Mol ratio be 1:1-4:1, optimum mole ratio is 2:1.
Wherein described BSA and Ag+Mol ratio be 1:50-1:80, optimum mole ratio is 1:66.67.
Wherein described temperature is 20-71 DEG C, and optimum temperature is 71 DEG C.
Fluorescence nargol in document it has been reported that Chinese patent 201510686946.2 " a kind of fluorescence nargol and its Prepare and apply " in, add BSA its main purposes and be to Fe3+The detection of ion, is carried to nargol fluorescent stability without larger Height, fluorescence is reduced within a short period of time；The present invention is prepared for the nargol containing tryptophan and BSA first, hydrophobic by introducing Acidic amino acid, by hydrophobic effect, obtains new using BSA as shell, Tryptophan fluorescence nargol is glimmering for the near-infrared of core with BSA Photoemissive nano-cluster (Fig. 1), this nargol stability is high, and room temperature places fluorescence and is held essentially constant (Fig. 2), the nargol Available for the fluorescence imaging (Fig. 3) of tumour cell, the method for this stability for improving nucleic acid silver nanoclusters is to report first.
Brief description of the drawings
Fig. 1 is Trp-DNA-AgNCs@BSA (CDNA=1 μM, the aqueous solution) fluorescence excitation and launching light spectrogram.
Fig. 2 be Trp-DNA-AgNCs BSA in sodium citrate buffer solution (pH=7.0,10mM) fluorescence with time covariant The figure of change.
Fig. 3 is tumour cell fluorescence imaging figure.
Embodiment
Trp-DNA-AgNCs@BSA synthesis
Embodiment 1 (Trp-DNA-AgNCs@BSA most preferably prepare scheme)：Using DNA sequence dna as template, in 1mL sodium citrates In cushioning liquid (pH 5.0,10mM), (DNA sequence dna is " 5 ' to 6 μm of ol of addition DNA CCCACCCACCCTCCCAACAACAGAGGAG3 ' ") (sequence is synthesized by our oneself design with marine growth Sheng Gong company DNA Instrument synthesize, purified by HPLC, mass spectrum checking sequence be 5 ' CCCACCCACCCTCCCAACAACAGAGGAG3 '), 200 μm of ol AgNO3(wherein DNA and Ag+Mol ratio be 1:And 150 μm of ol tryptophans (wherein tryptophan and Ag 33.33)+Mol ratio be 1:1.33) it is placed into after, being well mixed in water-bath, after 71 DEG C of thermal-stable 2min, starts cooling, temperature-fall period is 2h, is down to Room temperature.At room temperature, NaBH is added into reaction solution4Solution (wherein NaBH4With Ag+Mol ratio be 2:1) it is, static in room temperature 10min is placed, by Trp-DNA-Ag+Trp-DNA-Ag is reduced into, reaction solution avoid light place 2 days in 4 DEG C of environment are transferred to, so 3 μm of ol BSA (wherein BSA and Ag are added afterwards+Mol ratio be 1:66.67), you can the near-infrared nucleic acid silver nanoparticle stablized Cluster.To excite 750nm to obtain 800nm near-infrared fluorescent.1 μM of Trp-DNA-AgNCs@BSA fluorescence intensity is 225.
Embodiment 2：Using DNA sequence dna as template, in 1mL sodium citrate buffers (pH 5.0,10mM), 10 μ are added Mol DNA (DNA sequence dna is " 5 ' CCCACCCACCCTCCCAACAACAGAGGAG3 ' ") (by our oneself design used by sequence The DNA synthesizer synthesis of marine growth Sheng Gong companies, is purified by HPLC, and mass spectrum checking sequence is 5 ' CCCACCCACCCTCCCAACAACAGAGGAG3’)、200μmol AgNO3(wherein DNA and Ag+Mol ratio be 1:20) with 200 μm ol tryptophans (wherein tryptophan and Ag+Mol ratio be 1:1) it is placed into after, being well mixed in water-bath, 71 DEG C of thermal-stables After 2min, start cooling, temperature-fall period is 2h, is down to room temperature.At room temperature, NaBH is added into reaction solution4Solution is (wherein NaBH4With Ag+Mol ratio be 1:1), in the static placement 10min of room temperature, by Trp-DNA-Ag+Trp-DNA-Ag is reduced into, will Reaction solution is transferred to avoid light place 2 days in 4 DEG C of environment, then adds 4 μm of ol BSA (wherein BSA and Ag+Mol ratio be 1: 50), you can the near-infrared nucleic acid silver nanoclusters stablized.To excite 750nm to obtain 800nm near-infrared fluorescent.1μM Trp-DNA-AgNCs@BSA fluorescence intensities are 150.
Embodiment 3：Using DNA sequence dna as template, in 1mL sodium citrate buffers (pH 5.0,10mM), 5 μ are added Mol DNA (DNA sequence dna is " 5 ' CCCACCCACCCTCCCAACAACAGAGGAG3 ' ") (by our oneself design used by sequence Upper marine growth Sheng Gong companies DNA synthesizer synthesis, is purified by HPLC, and mass spectrum checking sequence is 5 ' CCCACCCACCCTCCCAACAACAGAGGAG3’)、200μmol AgNO3(wherein DNA and Ag+Mol ratio be 1:40) with 100 μm ol tryptophans (wherein tryptophan and Ag+Mol ratio be 1:2) it is placed into after, being well mixed in water-bath, 71 DEG C of thermal-stables After 2min, start cooling, temperature-fall period is 2h, is down to room temperature.At room temperature, NaBH is added into reaction solution4Solution is (wherein NaBH4With Ag+Mol ratio be 4:1), in the static placement 10min of room temperature, by Trp-DNA-Ag+Trp-DNA-Ag is reduced into, will Reaction solution is transferred to avoid light place 2 days in 4 DEG C of environment, then adds 2.5 μm of ol BSA (wherein BSA and Ag+Mol ratio be 1: 80), you can the near-infrared nucleic acid silver nanoclusters stablized.To excite 750nm to obtain 800nm near-infrared fluorescent.1μM Trp-DNA-AgNCs@BSA fluorescence intensities are 100.
Embodiment 4：Using DNA sequence dna as template, in 1mL sodium citrate buffers (pH 5.0,10mM), 6 μ are added Mol DNA (DNA sequence dna is " 5 ' CCCACCCACCCTCCCAACAACAGAGGAG3 ' ") (by our oneself design used by sequence The DNA synthesizer synthesis of marine growth Sheng Gong companies, is purified by HPLC, and mass spectrum checking sequence is 5 ' CCCACCCACCCTCCCAACAACAGAGGAG3’)、200μmol AgNO3(wherein DNA and Ag+Mol ratio be 1:33.33) and 150 μm of ol tryptophans (wherein tryptophan and Ag+Mol ratio be 1:1.33) it is placed into after, being well mixed in water-bath, 20 DEG C add After thermally-stabilised 2min, start cooling, temperature-fall period is 2h, is down to room temperature.At room temperature, NaBH is added into reaction solution4Solution (wherein NaBH4With Ag+Mol ratio be 2:1), in the static placement 10min of room temperature, by Trp-DNA-Ag+It is reduced into Trp-DNA- Ag, avoid light place 2 days in 4 DEG C of environment are transferred to by reaction solution, then add 3 μm of ol BSA (wherein BSA and Ag+Mol ratio For 1:66.67), you can the near-infrared nucleic acid silver nanoclusters stablized.To excite 750nm to obtain 800nm near-infrared fluorescent. 1 μM of Trp-DNA-AgNCs@BSA fluorescence intensity is 120.
Stable near-infrared nargol is used for cell imaging
Step one, takes synthetic Trp-DNA-AgNCs@BSA, is made into 18 μM of DNA-AgNCs-Trp@BSA DMSO Solution, is put in 4 DEG C of light protected environments.
Trp-DNA-AgNCs@BSA are diluted to 6 μM using nutrient solution and add incubation in HepG-2 liver cancer cells by step 2 1h。
Step 3 rinses the compound that culture medium removing is introduced into cell using PBS cushioning liquid.
Step 4 uses the microscope observed and recordeds of Nikon Ti-E 2000.Excitation wavelength is 750nm, and launch wavelength is 800nm。
Step 5 measurement results are shown in the cell imaging figure that the Fig. 3 of accompanying drawing 3. is Trp-DNA-AgNCs@BSA.
Sequence table
<110>Jiangsu University
<120>A kind of near-infrared fluorescent nucleic acid silver nanoclusters and its production and use
<160> 1
<210> 1
<211> 28
<212> DNA
<213>Artificial sequence
<400> 1
cccacccacc ctcccaacaa cagaggag 28

Claims (7)

1. a kind of near-infrared fluorescent nucleic acid silver nanoclusters, labeled as Trp-DNA-AgNCs BSA, it is characterised in that：With DNA sequence dna 5 ' CCCACCCACCCTCCCAACAACAGAGGAG3 ' are template, in sodium citrate buffer, utilize NaBH4Reduce AgNO3 And the method that tryptophan and BSA interact, synthesis obtains；This nucleic acid silver nanoclusters set excitation wavelength in fluoremetry For 750nm, launch wavelength 800nm near-infrared fluorescent is obtained, the size of nucleic acid silver nanoclusters is 70nm, and its near-infrared fluorescent can Detection for cell imaging and nucleic acids in blood.
2. a kind of preparation method of near-infrared fluorescent nucleic acid silver nanoclusters as claimed in claim 1, it is characterised in that specific step It is rapid as follows：
With the CCCACCCACCCTCCCAACAACAGAGGAG3 ' of DNA sequence dna 5 ' for template, DNA is added in sodium citrate buffer solution Sequence, tryptophan (Trp) and AgNO3, it is well mixed after be placed into water-bath, 20-71 DEG C of heating 1-3min, after be slowly dropped to room Temperature, temperature fall time is 1-2h, adds NaBH4Solution is by Trp-DNA-Ag+Trp-DNA-Ag is reduced into, is transferred in 4 DEG C of environment and keeps away Light is placed 1-3 days, adds bovine serum albumin (BSA) solution after placing 1-3 days, you can the near-infrared nucleic acid silver nanoparticle stablized Cluster.
3. a kind of preparation method of near-infrared fluorescent nucleic acid silver nanoclusters as claimed in claim 2, it is characterised in that described The pH value of sodium citrate buffer is 5.0-6.0, and concentration is 10-20mM, and 5-10 μm of ol is added per 1mL sodium citrate buffer solutions DNA。
4. a kind of preparation method of near-infrared fluorescent nucleic acid silver nanoclusters as claimed in claim 3, it is characterised in that described The pH value of sodium citrate buffer is 5.0, and concentration is 10mM, and 6 μm of ol DNA are added per 1mL sodium citrate buffer solutions.
5. a kind of preparation method of near-infrared fluorescent nucleic acid silver nanoclusters as claimed in claim 2, it is characterised in that described DNA and Ag+Mol ratio be 1:20-1:40；Described tryptophan and Ag+Mol ratio be 1:1-1:2；Described NaBH4With Ag+Mol ratio be 1:1-4:1；Described BSA and Ag+Mol ratio be 1:50-1:80.
6. a kind of preparation method of near-infrared fluorescent nucleic acid silver nanoclusters as claimed in claim 5, it is characterised in that described DNA and Ag+Mol ratio be 1:33.33；Described tryptophan and Ag+Mol ratio be 1:1.33；Described NaBH4With Ag+'s Mol ratio is 2:1；Described BSA and Ag+Mol ratio be 1:66.67.
7. a kind of preparation method of near-infrared fluorescent nucleic acid silver nanoclusters as claimed in claim 2, it is characterised in that 71 DEG C add Hot 2min；Temperature fall time is 1.5h；It is transferred to avoid light place 2 days in 4 DEG C of environment.
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Cited By (4)

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CN109266333A (en) * 2018-10-23 2019-01-25 山西大学 A kind of preparation method and application of Fluorescent silver nanocluster probe
CN110095519A (en) * 2019-04-29 2019-08-06 中国科学院苏州生物医学工程技术研究所 The preparation method of silver nanoclusters based composites and nucleic acid detection method based on the composite material
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
CN113456670A (en) * 2021-07-02 2021-10-01 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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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109266333A (en) * 2018-10-23 2019-01-25 山西大学 A kind of preparation method and application of Fluorescent silver nanocluster probe
CN110095519A (en) * 2019-04-29 2019-08-06 中国科学院苏州生物医学工程技术研究所 The preparation method of silver nanoclusters based composites and nucleic acid detection method based on the composite material
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
CN113456670A (en) * 2021-07-02 2021-10-01 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
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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