CN117431058A - Method and application of monodisperse and surface monofunctional ultra-small gold cluster - Google Patents
Method and application of monodisperse and surface monofunctional ultra-small gold cluster Download PDFInfo
- Publication number
- CN117431058A CN117431058A CN202311202723.5A CN202311202723A CN117431058A CN 117431058 A CN117431058 A CN 117431058A CN 202311202723 A CN202311202723 A CN 202311202723A CN 117431058 A CN117431058 A CN 117431058A
- Authority
- CN
- China
- Prior art keywords
- dna
- cage
- monodisperse
- gold
- ncs
- 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
- 239000010931 gold Substances 0.000 title claims abstract description 125
- 229910052737 gold Inorganic materials 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 40
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims description 47
- 238000003384 imaging method Methods 0.000 claims abstract description 19
- 230000000295 complement effect Effects 0.000 claims abstract description 9
- 239000003446 ligand Substances 0.000 claims abstract description 7
- 150000002343 gold Chemical class 0.000 claims description 38
- 238000006862 quantum yield reaction Methods 0.000 claims description 16
- 238000010226 confocal imaging Methods 0.000 claims description 12
- 238000010276 construction Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 150000001408 amides Chemical class 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 239000002086 nanomaterial Substances 0.000 claims description 3
- 238000012650 click reaction Methods 0.000 claims description 2
- 239000002184 metal Substances 0.000 abstract description 2
- 229910052751 metal Inorganic materials 0.000 abstract description 2
- 230000021615 conjugation Effects 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 40
- 239000002245 particle Substances 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 12
- 239000002105 nanoparticle Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 8
- 238000012512 characterization method Methods 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 108091008104 nucleic acid aptamers Proteins 0.000 description 7
- 230000004048 modification Effects 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 230000008685 targeting Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 238000004020 luminiscence type Methods 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 239000000956 alloy Substances 0.000 description 2
- 229910045601 alloy Inorganic materials 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108091023037 Aptamer Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000002744 anti-aggregatory effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 239000011258 core-shell material Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002091 nanocage Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- -1 phosphoramidite triester Chemical class 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000013643 reference control Substances 0.000 description 1
- 238000007430 reference method Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 229940043267 rhodamine b Drugs 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/58—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing copper, silver or gold
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nanotechnology (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Optics & Photonics (AREA)
- Materials Engineering (AREA)
- Life Sciences & Earth Sciences (AREA)
- Crystallography & Structural Chemistry (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- Manufacturing & Machinery (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Inorganic Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method and application of monodisperse and surface monofunctional ultra-small gold clusters, and relates to the technical field of metal clusters, wherein the method comprises the following steps: at Au 22 (SG) 18 Modifying DNA to obtain gold clusters of surface conjugated DNA; -constructing a DNA Cage having one strand inside, on which only one complementary pairing site with DNA is designed; -encapsulating gold clusters of surface conjugated DNA in DNA Cage by base complementary pairing to obtain monodisperse gold clusters Cage-Au NCs. The method provided by the invention can obtain good dispersibility, high stability, brighter, more resistant to photobleaching,gold nanoclusters capable of univalent conjugation of functional ligands have higher application value; the method is used for STED super-resolution imaging, and has higher resolution.
Description
Technical Field
The invention belongs to the technical field of monodisperse luminescent nano materials, and particularly relates to a method for monodisperse and surface monofunctional ultra-small gold clusters and application thereof.
Background
Gold nanoclusters (Au NCs) are molecular-sized aggregates composed of several to several tens of gold atoms, having a size of 2nm or less, and composed of organic molecules, proteins, nucleic acids, etc. as protecting groups. The Au NCs have the characteristics of metal core-organic shell structure, large specific surface area, good light stability, high fluorescence brightness and the like. Au NCs have wide application prospects in the fields of biosensing and imaging, intracellular drug delivery, living cell imaging and cell tracking, cancer treatment, ultrasensitive molecular diagnosis, image guidance treatment and the like.
However, gold clusters are easily agglomerated into large nano particles due to higher specific surface energy, so that uneven luminescence is caused, and the luminous efficiency, imaging result analysis and the like are affected. An effective method to prevent agglomeration is to encapsulate gold clusters in other materials such as porous materials (cof\mof) or core-shell structures (silica microspheres), etc. However, the size of these carrier molecules cannot be controlled, and it is not possible to encapsulate only one gold nanocluster. In recent years, the use of DNA nanocages to encapsulate nanoparticles with a diameter d >5nm has been reported. There are mainly two methods: 1. the nanoparticle is confined in the DNA Cage by hybridization complementary pairing of the extended strand on the DNA Cage and the DNA strand on the nanoparticle. 2. Two half DNA cage were designed and nanoparticles were randomly encapsulated in DNA cage by DNA hybridization. But neither method is suitable for ultra-small gold nanoclusters (diameter d <2 nm). The main reasons are as follows: 1. gold nanoclusters limit the anchoring of multiple DNA strands (typically 1-3) due to the ultra-small surface area, but at least 10-12 DNA strands on the particle are required by DNA cage and nanoparticle hybridization confinement; 2. the DNA cage encapsulates small particles, with or without DNA strand restrictions, the number of encapsulated particles generally depends on the relative sizes of the DNA cage lumen and the size of the gold clusters. Because the diameter of the gold clusters is similar to the width of the DNA double strand, it is difficult to achieve single gold nanocluster encapsulation with DNA cage.
Disclosure of Invention
The invention aims to provide a method for monodisperse and surface monofunctional ultra-small gold clusters and application thereof, wherein the method can accurately distribute the monodisperse gold clusters in the center of a DNA Cage so as to realize high dispersibility and colloid stability and has higher application value; the method is used for STED super-resolution imaging, and higher imaging resolution is obtained.
The technical scheme adopted by the invention for achieving the purpose is as follows:
a method for isolating single monodisperse gold clusters on a DNA Cage surface comprising:
-modifying DNA on Au NCs to obtain gold clusters of surface conjugated DNA (Au NCs-DNA);
-constructing a DNA Cage having one strand inside, on which only one complementary pairing site with DNA is designed;
-encapsulating Au NCs-DNA in DNA Cage by base complementary pairing to obtain monodisperse gold clusters Cage-Au NCs.
Further specifically, the method for isolating single monodisperse gold clusters on the surface of DNA Cage comprises the following steps:
(1) Synthesis of Au NCs;
(2) Modifying DNA on Au NCs to obtain gold cluster Au NCs-DNA of surface conjugated DNA;
(3) Constructing a DNA Cage, synthesizing the DNA Cage with one chain inside, and designing only one complementary pairing site with the DNA on the inner chain;
(4) DNA Cage surface isolates individual gold clusters: and (3) encapsulating the gold cluster Au NCs-DNA of the surface conjugated DNA in the DNA Cage through base complementation pairing to obtain the monodisperse gold cluster Cage-AuNCs.
The invention synthesizes Au NCs (Au 22 (SG) 18 ) The DNA is then modified on the gold cluster surface glutathione ligand by an amide reaction. Au (gold) and method for producing the same 22 (SG) 18 Is the thiol-protected gold cluster with the highest quantum yield at present, and the quantum yield is 7.5%. The quantum yield of the gold clusters of the DNA template prepared at present is generally low and is less than 0.1 percent. The invention is realized by the method that the alloy is prepared by the method of preparing the alloy in Au 22 (SG) 18 The direct modification of DNA can improve the quantum yield by 3 times to 19.1 percent, which is DNA-Au NCs with highest quantum yield at present. And quantum yield can be further improved by further increasing the ligand (DNA or co-modification of DNA and other ligands) modification density or optimizing the sequence.
Next, DNA Cage was constructed by design and individual Au was isolated with the DNA Cage surface 22 (SG) 18 Gold nanoclusters; and the surface of a single gold nanocluster is chemically modified by adopting 15nt-DNA with a nucleotide sequence, and the Au NCs-DNA is complemented into a DNA cage, so that the fluorescence quantum yield is further improved to 21.4%. The principle of the method for isolating single monodisperse gold clusters on the surface of the DNA Cage is that the single gold clusters are limited in one DNA Cage through the interaction force between DNA bases, so that the aggregation of gold nanoclusters can be effectively prevented, and the method has good dispersibility and long-time stability; and the method is applied to STED super-resolution imaging, and has higher imaging resolution. Meanwhile, after surface isolation is carried out on the single gold nanocluster, the single gold nanocluster has important application potential in the fields of industrial catalysis, biosensing, imaging, in-vivo and in-vitro micro-nano device construction and the like, can be used for improving the application efficiency in the fields of sensing, catalysis, imaging, nano device construction and the like, and is suitable for popularization and application. The method provided by the invention provides a new idea for isolating single other ultra-small functional particles on the surface.
In the step (1), au NCs are preferably Au 22 (SG) 18 。
In the step (1), au NCs (Au 22 (SG) 18 ) The synthesis of (2) is carried out with reference to the prior art, the operation of which is conventional as will be appreciated by those skilled in the art.
The molecular sequence of the DNA in step (2) includes: HOOC-CACTGTTAAGAGATG is not limited to this sequence nor to the length of the DNA sequence nor to the modification by mixing with other ligands.
Further, the DNA is covalently modified on the gold cluster ligand by a click reaction.
The dimensions of the DNA Cage constructed in the step (3) include (3.4 to 10.5 nm) × (3.4 to 10.5 nm), preferably 7.1nm ×7.1nm ×7.1 nm.
The invention also discloses the monodisperse gold cluster Cage-Au NCs obtained by the method.
The invention discloses application of the monodisperse gold cluster Cage-Au NCs with uniform luminescence in preparation of a biological probe for confocal imaging. The gold cluster Cage-Au NCs prepared by the method can be used as a biological probe for monovalent biological marking, and can be applied to confocal imaging to show excellent targeting.
It should be noted that the gold cluster Cage-Au NCs can modify a specific single number of targeted nucleic acid aptamers, and then confocal imaging is performed.
It is a further object of the present invention to provide the use of gold cluster Cage-Au NCs in STED super resolution.
The invention also discloses the monodisperse gold cluster Cage-Au NCs used for sensing, catalysis, imaging and construction of nano devices.
The invention further discloses the application of the electron-rich structure such as DNA/PNA and the like to the gold cluster directly by utilizing a click or amide chemical reaction method to improve the quantum yield of the gold cluster through modifying the electron-rich structure.
The invention further discloses application of HOOC-CACTGTTAAGAGATG in preparation of monodisperse gold clusters, and the quantum yield and the photobleaching resistance of the gold clusters are improved by modifying DNA on the gold clusters through an amide reaction.
The invention further discloses application of the DNA Cage in improving the quantum yield of gold clusters and the photobleaching resistance.
The invention also discloses application of the method in preparing other monodisperse or surface monofunctional monodisperse nano materials.
Compared with the prior art, the invention has the following beneficial effects:
the invention develops a surface physical isolation method of a single monodisperse gold cluster, has a good anti-aggregation effect on the gold cluster, ensures that the gold cluster is not aggregated after encapsulation, increases the stability of the gold cluster, has a stability time longer than 6 months, and is suitable for popularization and application. Meanwhile, the single monodisperse gold cluster can be used as a biomarker probe and applied to confocal imaging, and shows excellent targeting. Cage-Au NCs were placed on a slide glass and were more dispersed and luminescence was more uniform than the control particle Au NCs-DNA. For STED imaging, higher resolution is provided.
Therefore, the invention provides a method and application of monodisperse and surface monofunctional ultra-small gold clusters, and has higher application value; the method is used for STED super-resolution imaging, and has higher imaging resolution and more uniform imaging effect.
Drawings
FIG. 1 is a flow chart of a mechanism for preparing monodisperse gold cluster Cage-Au NCs according to the present invention;
FIG. 2 is an ultraviolet absorption spectrum and a fluorescence spectrum (a is an ultraviolet absorption spectrum, b is a fluorescence spectrum) of Au NCs synthesized in the present invention;
FIG. 3 is a representation of the PAGE of gold clusters of surface conjugated DNA obtained in the present invention;
FIG. 4 is a representation of Zate potential of Au NCs, au NCs-DNA and DNA synthesized in the present invention;
FIG. 5 is a PAGE characterization of monodisperse gold cluster Cage-Au NCs synthesized in the present invention;
FIG. 6 is a TEM and DLS characterization of Au NCs, au NCs-DNA, cage-Au NCs synthesized in the present invention;
FIG. 7 is a comparison of fluorescence intensity, quantum yield and photobleaching resistance of Au NCs, au NCs-DNA and Cage-Au NCs synthesized in the present invention;
FIG. 8 is a confocal imaging of monodisperse gold cluster Cage-Au NCs modified nuclear-targeted nucleic acid aptamers of the present invention;
FIG. 9 is a STED image and cross-sectional profile of monodisperse gold cluster Cage-Au NCs particles according to the invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the following describes in detail various embodiments of the present invention with reference to the embodiments. However, those of ordinary skill in the art will understand that in various embodiments of the present invention, numerous technical details have been set forth in order to provide a better understanding of the present application. However, the technical solutions claimed in the present application can be implemented without these technical details and with various changes and modifications based on the following embodiments.
The molecular sequence of the DNA used in the embodiment of the invention is HOOC-CACTGTTAAGAGATG, which is custom-made and is specifically synthesized by Shanghai Biotechnology, inc., and the synthesis method is a solid-phase phosphoramidite triester method conventionally understood by those skilled in the art.
The DNA Cage sequences used in the examples of the present invention were synthesized by Shanghai Bioengineering Co., ltd:
reference [ Xiaoyi Fu, guoling Ke, fangqi Peng, et al Size-selective molecular recognition based on a confined DNAmolecular sieve using cavity-tunable framework nucleic acids NATURE COMMUNICATIONS (2020) 11:1518|https:// doi.org/10.1038/s41467-020-15297-7 ].
Example 1:
the method for isolating single monodisperse gold clusters on the surface of DNA Cage comprises the following steps:
（1）Au NCs（Au 22 (SG) 18 ) 230mL of ultrapure water was taken in the flask, and 20mM HAuCl was added thereto 4 12.5mL and 50mM glutathione 7.5mL, rapidly stirred for 2min until the pale yellow solution became cloudy; adjusting the pH of the solution to about 12.0 by using 1M NaOH, wherein the solution is yellow in color; 0.1mL of 3.5mM NaBH was added dropwise 4 The solution slowly turned orange over 15 min; after 30min, naBH was quenched by adjusting the pH to 2.5 with 1M HCl 4 Activity; stirring at room temperature and 150rpm for 6 hours; evaporating the solution to 10mL by using a rotary evaporator, and stopping evaporation; adding 12mL of isopropanol to induce precipitation of nanoclusters, namely Au NCs; the synthesized product was characterized by ultraviolet spectrum (shown in fig. 2 a) and fluorescence spectrum (shown in fig. 2 b), and the results are shown in fig. 2;
(2) Modification of DNA on Au NCs to obtain gold clusters of modified DNA, specifically:
100. Mu.M DNA was taken in 100. Mu.L, 10mM EDC 100. Mu.L and 10mM NHS 100. Mu.L were added thereto, and stirred for 40min to activate-COOH on the DNA; then, 10. Mu.M Au NCs 100. Mu.L was added thereto and stirred for 4 hours; unmodified DNA, EDC, NHS and gold clusters were removed with a 10KD ultrafiltration tube to obtain Au NCs-DNA; the results of the PAGE characterization and the Zate potential characterization are shown in fig. 3 and 4, and further confirm that the Au NCs-DNA is successfully prepared;
（3）construction of DNA Cage: mixing equimolar amounts of DNA Cage sequences in TAE-Mg 2+ （20mM Tris，2mM EDTA，12.5mMMgCl 2 pH 7.4) in a buffer solution, annealing to assemble DNA Cage, synthesizing DNA Cage with the size of 7.1nm multiplied by 7.1nm and one strand inside, and designing only one complementary pairing site with DNA on the inner strand;
(4) DNA Cage encapsulates individual gold clusters: encapsulating the Au NCs in the DNA Cage through base complementation pairing to obtain monodisperse gold cluster Cage-Au NCs; the results of the PAGE characterization are shown in FIG. 5, which shows the DNA Cage step-by-step assembly process and the isolation of individual gold clusters from the structure.
Example 2:
TEM and DLS characterization
TEM and DLS characterization were performed on Au NCs, au NCs-DNA, cage-Au NCs prepared in example 1, and the results are shown in FIG. 6. As can be seen from the analysis of the graph, the particle distribution interval of Cage-Au NCs is obviously larger than that of Au NCs and Au NCs-DNA groups, and the better dispersibility of the Cage-AuNCs is proved by the interval between gold clusters.
Furthermore, compared with the particle size of the sample stored for 6 months, the Au NCs-DNA and the Cage-Au NCs are compared, and the Cage-Au NCs are not aggregated, so that the gold cluster isolated by the DNA Cage has better long-time stability.
Example 3:
the Au NCs, au NCs-DNA, cage-Au NCs prepared in example 1 were tested for fluorescence intensity, quantum yield and photo-bleaching resistance: the excitation wavelength is set to 520nm and the emission wavelength is set to 535-750 nm. Wherein the quantum yield test is obtained according to a reference method (reference control: rhodamine B quantum yield in ethanol is 89%). The results are shown in FIG. 7. As can be seen from the graph, the quantum yields of the Au NCs, the AuNCs-DNA and the Cage-Au NCs are respectively 7.5%, 19.1% and 21.4%, and the brightness of the Cage-Au NCs is obviously higher than that of the free Au NCs; the method comprises the steps of carrying out a first treatment on the surface of the The photobleaching resistance is obviously enhanced compared with Au NCs-DNA.
Example 4:
application of monodisperse gold cluster Cage-Au NCs in confocal imaging
Cage-Au NCs surface modification targeting nucleic acid aptamer: constructing one strand of the DNA Cage aiming at the outer layer, extending the strand by 15 bases, and complementarily pairing the strand with an extended strand of a nucleic acid aptamer sequence of a targeted cell nucleus to enable the nucleic acid aptamer to hybridize to the DNA Cage, wherein the hybridization condition is specifically 30 ℃, 300rpm and 4 hours;
confocal imaging:
1) HeLa cells (commercially available) were selected, and the cells were seeded in confocal dishes at 3X 10 cells per well 4 Individual cells, 37 ℃, 5% CO 2 Allowing the cells to culture on the wall overnight under the condition;
2) Fixing the cells overnight at-20deg.C with 90% ethanol solution, and washing the cells with PBS buffer solution after fixing;
3) Incubating the cells with the aptamer-modified Cage-Au NCs at 4 ℃ for 40min, washing the cells with PBS solution for 3 times to remove the nonspecifically adsorbed probes, and adding 100 mu LPBS solution for confocal imaging.
Meanwhile, a comparison experiment is set: confocal imaging was performed using gold cluster Au NCs.
Analysis of results:
the confocal imaging results are shown in fig. 8. From the analysis in the figure, it can be seen by confocal imaging: after adding the targeting nucleic acid aptamer, the probes are targeted to the cell nucleus part, and gold clusters without the nucleic acid aptamer are distributed in the whole cell without targeting; the results show that the Cage-Au NCs prepared by the method can easily modify the aptamer and show excellent targeting.
Example 5:
application exploration on STED super resolution
STED imaging was performed on the gold clusters Cage-Au NCs and the control particles Au NCs-DNA, and resolution analysis was performed (the specific methods and procedures are conventional as understood by those skilled in the art).
Analysis of results:
the results are shown in FIG. 9. From the analysis of the figure, figure 9 a is the image of Cage-Au NCs particles under a multiplied by 100 oil lens, and figures b and c are the images of CLSM and STED after the enlargement of nanoparticles in red circles in a, and the figure shows that the nanoparticles imaged under STED have obvious narrowing effect. In fig. 9 d is a cross-sectional view of the nanoparticle under b and c imaging, with a resolution of 43nm. e is to image Au NCs-DNA particles under a X100 oil lens, and the particles are uneven in size and particle diameter. The smaller nanoparticles in e are amplified to obtain CLSM and STED imaging, namely figures f and g, and the nanoparticles imaged under STED can be seen to have obvious narrowing effect. The cross-sectional analysis, panel g, gave 56nm of resolution that could be achieved for the control particles, indicating that there was a potential for aggregation of the control particles.
The conventional technology in the above embodiments is known to those skilled in the art, and thus is not described in detail herein.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. A method of monodisperse and surface unifunctionalized ultra-small gold clusters comprising:
-modifying DNA on Au NCs to obtain gold clusters of surface conjugated DNA;
-constructing a DNA Cage having one strand inside, on which only one complementary pairing site with DNA is designed;
and encapsulating the gold clusters of the surface conjugated DNA in DNA Cage by base complementary pairing to obtain monodisperse stable gold clusters Cage-Au NCs.
2. The method of monodisperse and surface monofunctional ultra-small gold cluster according to claim 1, wherein the DNA is covalently modified on the gold cluster ligand by a click reaction.
3. The method of monodisperse and surface monofunctional ultrafine gold clusters according to claim 1, wherein the molecular sequence of the DNA includes, but is not limited to: HOOC-CACTGTTAAGAGATG.
4. The method of monodisperse and surface monofunctional ultrafine gold clusters according to claim 1, wherein the step DNA Cage has a size of (3.4-10.5 nm) x (3.4-10.5 nm).
5. Monodisperse gold cluster Cage-Au NCs obtained by the method of claim 1.
6. The use of the monodisperse gold cluster Cage-Au NCs of claim 5 for the preparation of a bioprobe for confocal imaging.
7. The use of the monodisperse monofunctional gold cluster Cage-Au NCs of claim 5 in STED super resolution.
8. The use of the monodisperse gold cluster Cage-Au NCs of claim 5 in biosensing, imaging and construction of internal and external micro-nano devices.
9. Use of HOOC-CACTGTTAAGAGATG for the preparation of ultra-small gold clusters, characterized in that carboxylated DNA is used to increase the quantum yield and photobleaching resistance of gold clusters by modifying the DNA on the gold clusters using an amide reaction.
10. Use of the method of claim 1 for the preparation of other monodisperse or surface mono-functionalized ultra-small nanomaterials.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311202723.5A CN117431058B (en) | 2023-12-18 | 2023-12-18 | Method and application of monodisperse and surface monofunctional ultra-small gold cluster |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311202723.5A CN117431058B (en) | 2023-12-18 | 2023-12-18 | Method and application of monodisperse and surface monofunctional ultra-small gold cluster |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117431058A true CN117431058A (en) | 2024-01-23 |
| CN117431058B CN117431058B (en) | 2024-06-18 |
Family
ID=89557304
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311202723.5A Active CN117431058B (en) | 2023-12-18 | 2023-12-18 | Method and application of monodisperse and surface monofunctional ultra-small gold cluster |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117431058B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110033706A1 (en) * | 2009-08-04 | 2011-02-10 | Yamuna Krishnan | Nanocapsules and methods for modular assembly |
| CN106754894A (en) * | 2017-02-28 | 2017-05-31 | 临沂大学 | A kind of multifunction magnetic DNA nanospheres and preparation method and application |
| CN107470648A (en) * | 2017-07-11 | 2017-12-15 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | A kind of DNA functionalization gold nano cluster and preparation method thereof |
| CN107556999A (en) * | 2017-07-28 | 2018-01-09 | 安徽师范大学 | Gold nano cluster and its preparation method and application |
| CN111849467A (en) * | 2020-08-11 | 2020-10-30 | 苏州大学 | Infrared II-region fluorescence gold nanocluster and preparation and application thereof |
| US20210222063A1 (en) * | 2019-03-27 | 2021-07-22 | Qingdao University | Method for preparing nanohybrid used for ratiometric fluorescence and ratiometric electrochemical sensing simultaneously |
| CN115558706A (en) * | 2021-07-02 | 2023-01-03 | 中国科学院上海微系统与信息技术研究所 | DNA nano cage fluorescent probe for in-situ detection of miRNA in exosome and application thereof |
| WO2023092892A1 (en) * | 2021-11-23 | 2023-06-01 | 北京工业大学 | Preparation of dna-templated gold nanocluster probe having precise number of atoms, and application thereof in single-cell analysis |
| CN116765387A (en) * | 2023-06-22 | 2023-09-19 | 河南工业大学 | Preparation method of DNA nanoflower in-situ synthesis gold nanocluster and manganese metal organic framework fluorescent aptamer sensor |
-
2023
- 2023-12-18 CN CN202311202723.5A patent/CN117431058B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110033706A1 (en) * | 2009-08-04 | 2011-02-10 | Yamuna Krishnan | Nanocapsules and methods for modular assembly |
| CN106754894A (en) * | 2017-02-28 | 2017-05-31 | 临沂大学 | A kind of multifunction magnetic DNA nanospheres and preparation method and application |
| CN107470648A (en) * | 2017-07-11 | 2017-12-15 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | A kind of DNA functionalization gold nano cluster and preparation method thereof |
| CN107556999A (en) * | 2017-07-28 | 2018-01-09 | 安徽师范大学 | Gold nano cluster and its preparation method and application |
| US20210222063A1 (en) * | 2019-03-27 | 2021-07-22 | Qingdao University | Method for preparing nanohybrid used for ratiometric fluorescence and ratiometric electrochemical sensing simultaneously |
| CN111849467A (en) * | 2020-08-11 | 2020-10-30 | 苏州大学 | Infrared II-region fluorescence gold nanocluster and preparation and application thereof |
| CN115558706A (en) * | 2021-07-02 | 2023-01-03 | 中国科学院上海微系统与信息技术研究所 | DNA nano cage fluorescent probe for in-situ detection of miRNA in exosome and application thereof |
| WO2023092892A1 (en) * | 2021-11-23 | 2023-06-01 | 北京工业大学 | Preparation of dna-templated gold nanocluster probe having precise number of atoms, and application thereof in single-cell analysis |
| CN116765387A (en) * | 2023-06-22 | 2023-09-19 | 河南工业大学 | Preparation method of DNA nanoflower in-situ synthesis gold nanocluster and manganese metal organic framework fluorescent aptamer sensor |
Non-Patent Citations (1)
| Title |
|---|
| YANG YU等: "Functionalization of Tile-based DNA Nanocages with Gold Nanoparticles (AuNPs) to Form AuNP Cluster-DNA Cage Hybrids", 《CHEMNANOMAT》, 8 June 2020 (2020-06-08), pages 1175 - 1178 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117431058B (en) | 2024-06-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Metal nanoclusters: new fluorescent probes for sensors and bioimaging | |
| Muhammed et al. | Growth of in situ functionalized luminescent silver nanoclusters by direct reduction and size focusing | |
| Li et al. | Fluorescent metal nanoclusters: from synthesis to applications | |
| Ali et al. | Red fluorescent carbon nanoparticle-based cell imaging probe | |
| Kong et al. | Aptamer‐assembled nanomaterials for biosensing and biomedical applications | |
| Chhabra et al. | Distance-dependent interactions between gold nanoparticles and fluorescent molecules with DNA as tunable spacers | |
| Yang et al. | Quantum dot nanobarcodes: epitaxial assembly of nanoparticle− polymer complexes in homogeneous solution | |
| Liu et al. | Studies on CdSe/L-cysteine quantum dots synthesized in aqueous solution for biological labeling | |
| Zhang et al. | Carbon nanodot-decorated Ag@ SiO2 nanoparticles for fluorescence and surface-enhanced Raman scattering immunoassays | |
| Loukanov et al. | Photosensitizer-conjugated ultrasmall carbon nanodots as multifunctional fluorescent probes for bioimaging | |
| Martins et al. | Quantum dots for cancer-related miRNA monitoring | |
| KR101486697B1 (en) | Spectroscopy-active metal nanoparticles | |
| JP4985541B2 (en) | Nanoparticle-encapsulating silica, biological substance labeling substance and biological substance labeling method using the same | |
| CN106620725B (en) | Optical and photoacoustic integrated bimodal molecular imaging probe and preparation method and application thereof | |
| KR101348074B1 (en) | Method for Preparing Fluorene Copolymer Based Silica Nanoparticles | |
| JPWO2007086501A1 (en) | Fluorescent labeling substance for biological material and fluorescent labeling method for biological substance | |
| He et al. | Silicon nano-biotechnology | |
| Nie et al. | Preparation and biomedical applications of gold nanocluster | |
| Han et al. | Direct attachment of oligonucleotides to quantum dot interfaces | |
| CN109181678B (en) | Method for synthesizing green fluorescent gold nanocluster stabilized by mercapto-beta-cyclodextrin and application of green fluorescent gold nanocluster | |
| Wang et al. | Synthesis and characterization of self-assembled CdHgTe/gelatin nanospheres as stable near infrared fluorescent probes in vivo | |
| Adelt et al. | Morphological changes of silica shells deposited on gold nanorods: implications for nanoscale photocatalysts | |
| Zhao et al. | Metal nanoclusters–based ratiometric fluorescent probes from design to sensing applications | |
| CN114034747B (en) | Cathode photoinduced electrochemical biosensor for detecting beta-amyloid oligomer and construction method thereof | |
| CN113368238B (en) | h-BN/MoS capable of realizing targeted photothermal and chemical synergistic treatment 2 Nano probe and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |