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WO2023092892A1 - Préparation d'une sonde de nanoagrégat d'or à matrice d'adn ayant un nombre précis d'atomes et son utilisation dans une analyse unicellulaire - Google Patents

Préparation d'une sonde de nanoagrégat d'or à matrice d'adn ayant un nombre précis d'atomes et son utilisation dans une analyse unicellulaire Download PDF

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Publication number
WO2023092892A1
WO2023092892A1 PCT/CN2022/080041 CN2022080041W WO2023092892A1 WO 2023092892 A1 WO2023092892 A1 WO 2023092892A1 CN 2022080041 W CN2022080041 W CN 2022080041W WO 2023092892 A1 WO2023092892 A1 WO 2023092892A1
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Prior art keywords
gold
dna
atoms
ions
nanocluster
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PCT/CN2022/080041
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Chinese (zh)
Inventor
任晓君
王施政
高学云
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Beijing University of Technology
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Beijing University of Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12
    • 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
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/626—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas

Definitions

  • the invention relates to the technical field of preparation of nucleic acid detection materials, in particular to a preparation method of gold nanoclusters that can be used for nucleic acid detection and its application at the single cell level.
  • Nanoscale, atomically precise matter composed of noble metals is a new class of materials with many exceptional properties. More than 100 molecular formulas of these molecules are known, such as Au 25 (SR) 18 , Au 38 (SR) 24 and Au 102 (SR) 44 and Ag 25 (SR) 18 , Ag 29 (S 2 R) 12 and Ag 44 (SR) 30 (usually with some counterions to compensate for charge). They can be reproducibly fabricated using robust synthetic protocols, yielding colored solutions, yielding powders or diffractive crystals. They differ markedly from nanoparticles in spectral properties such as optical absorption and emission, exhibiting as well-defined signatures as molecules.
  • Metal nanoclusters contain a few to a few hundred atoms and range in size from subnanometer to nanometer, occupying intermediate-sized domains linking larger plasmonic nanoparticles and smaller metal complexes. Due to strong quantum confinement, metal nanoclusters exhibit molecule-like properties. Fluorescent gold nanocluster (AuNC) has the advantages of ultra-small size, strong luminescence, good photostability, and good biocompatibility. It is a new type of high-performance sensor and fluorescent probe for bioimaging. It has good applications in anions, small biological molecules, proteins, nucleic acids, drug molecules, pH and temperature. Although DNA-templated metal nanoclusters are still in their infancy, they are expected to emerge as a new class of functional nanomaterials with broad applications in biology and energy science.
  • the present invention proposes a synthesis method of gold nanoclusters based on DNA as a template and its application in nucleic acid detection.
  • the specific plan is as follows:
  • DMAB dimethylamine borane
  • the designed DNA is an oligonucleotide molecule with a specific hairpin structure, including a complementary region that can spontaneously form a hairpin structure, a gold atom/ion region and a nucleic acid targeting region, and its sequence (5'-3') is : TATCCGTCCCCCCCCCACGGATATTTTTAATCCTCCTCAATGCTGG.
  • the reaction temperature is 20-30° C., and the pH condition is 4.4-7.1.
  • the concentration of the DNA is 1 ⁇ M ⁇ 10 mM
  • the concentration of the Au compound is 1 ⁇ M ⁇ 1M.
  • the Au compound is a trivalent inorganic compound of Au, such as chloroauric acid; wherein the Au ions in the trivalent compound are reduced to Au atoms or monovalent Au ions.
  • Oxidation-reduction reaction occurs in the mixed solution, after the color of the solution changes, it is stirred at 20-30° C. for 3-24 hours, and purified by an ultrafiltration tube for 5-11 hours to obtain the gold nanoclusters.
  • the hydrated particle size of the gold nano clusters is 1-4nm.
  • the gold nanoclusters have good fluorescence and precise atomic number, and can visualize ribonucleic acid splicing variants in vitro/cell/tissue section/in vivo through fluorescence, and can be used in an inductively coupled plasma mass spectrometer through precise atomic number Quantification of ribonucleic acid splice variants on (ICP-MS).
  • Figure 1 is a schematic diagram of the synthesis of (Au) 6 (DNA) 1 gold nanoclusters of the present invention
  • Fig. 2 is the ultraviolet absorption and fluorescence spectrum of (Au) 6 (DNA) 1 gold nanoclusters in Example 1 of the present invention
  • Fig. 3 is the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) figure of (Au) 6 (DNA) 1 gold nanoclusters of embodiment 1 of the present invention
  • Figure 4 is the DLS particle size distribution diagram of (Au) 6 (DNA) 1 gold nanoclusters in Example 1 of the present invention
  • Figure 5 is a confocal imaging diagram of (Au) 6 (DNA) 1 gold nanoclusters in Example 2 of the present invention
  • LA-ICP-MS laser ablation inductively coupled plasma mass spectrometry
  • Embodiment 1 Preparation of DNA synthesis accurate atomic number gold nanocluster (Au) 6 (DNA) 1
  • the composition is (Au) 6 (DNA) 1 .
  • the hydrated particle size of (Au) 6 (DNA) 1 measured by DLS is about 3.0 nm.
  • RAW 264.7 cells were seeded on confocal culture dishes, divided into two groups, and incubated at 37°C for 24 hours in the presence or absence of LPS (1 ⁇ g mL-1). Then, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min, washed with PBS. Treat with 0.5% Triton X-100 for 5 minutes, then wash with PBS.
  • Hybridization of (Au) 6 (DNA) 1 to the target mRNA splice variant MyD88 L was performed in a volume of 20 ⁇ L containing 2 ⁇ L of 20 ⁇ sodium citrate buffer (SSC), 2 ⁇ L of (Au) 6 (DNA) 1 probe (20 ⁇ M), 1 ⁇ L DTT (100 mM), 2 ⁇ L yeast transfer RNA (10 mg mL-1), 2 ⁇ L 10 ng ⁇ L-1 salmon sperm DNA, and 0.5 ⁇ L RiboLock RNase inhibitor (40 U ⁇ L-1) were incubated at 37°C for 60 min. The samples were then imaged after washing with PBS-T (DEPC-PBS containing 0.05% Tween-20) for 3 minutes at room temperature.
  • PBS-T DEPC-PBS containing 0.05% Tween-20
  • Example 3 Quantitative analysis of MyD88 L on single RAW 264.7 cells by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS).
  • LA-ICP-MS measurements were performed using a NWR 213 laser ablation system and a NexION 300D ICP-MS instrument (PerkinElmer, Norwalk, CT, USA). Helium was used as the ablative gas. The flow rate of helium is 0.6L min-1. After cell ablation, argon gas is injected through the Y-piece. During NIST 612 glass ablation, the 115In signal intensity was adjusted to the maximum and the UO/U ratio was kept low. Signal strength was recorded as a function of time (counts per second (CPS)). We seeded RAW 264.7 cells (2 ⁇ 10 4 ) on coverslips. Cells were incubated with the (Au) 6 (DNA) 1 probe for 60 minutes.
  • the single signal intensity peak is the RNA splicing variant MyD88 L in situ in a single cell.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Materials Engineering (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Electrochemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne la préparation d'une sonde de nanoagrégat d'or à matrice d'ADN ayant un nombre précis d'atomes et son utilisation. Le procédé de préparation comprend : (1) La conception d'une séquence d'ADN spécifique ayant une structure en épingle à cheveux ; (2) le mélange d'une solution d'ADN avec une solution de sel d'or pour former une solution mixte ; et (3) l'ajout d'un agent réducteur de diméthylamine borane (DMAB) dans une certaine condition pour permettre au système de solution mélangée d'être soumis à une réaction d'oxydation-réduction, de telle sorte que des ions or à valence élevée dans le sel d'or sont réduits en atomes d'or ou en ions or, et de l'or est lié à la cytosine dans la structure en épingle à cheveux conçue dans l'ADN pour former un nanoagrégat d'or à matrice d'ADN. Le nanoagrégat d'or a une bonne fluorescence et un nombre précis d'atomes, des variants d'épissage d'acide ribonucléique peuvent être visualisés in situ au moyen de la propriété de fluorescence et des variants d'épissage d'acide ribonucléique à cellule unique peuvent être quantifiés sur une spectrométrie de masse à plasma à couplage inductif par ablation laser (LA-ICP-MS) au moyen du nombre précis de propriétés d'atomes.
PCT/CN2022/080041 2021-11-23 2022-03-10 Préparation d'une sonde de nanoagrégat d'or à matrice d'adn ayant un nombre précis d'atomes et son utilisation dans une analyse unicellulaire Ceased WO2023092892A1 (fr)

Applications Claiming Priority (2)

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CN202111390428.8 2021-11-23
CN202111390428.8A CN114213493B (zh) 2021-11-23 2021-11-23 一种精确原子数的dna为模板金纳米团簇探针的制备及其应用

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117431058A (zh) * 2023-12-18 2024-01-23 天津大学 单分散及表面单功能化超小金团簇的方法及应用

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US20050048546A1 (en) * 2003-07-11 2005-03-03 Sharron Penn Multiplexed molecular beacon assay for detection of human pathogens
US6979729B1 (en) * 1999-09-14 2005-12-27 Yeda Research And Development Co. Ltd. Metal cluster containing nucleotides and nucleic acids, and intermediates therefor
WO2015114127A1 (fr) * 2014-01-31 2015-08-06 Fundación Imdea Nanociencia Nanoparticules métalliques fonctionnalisées et leurs utilisations pour détecter des acides nucléiques
WO2017082517A1 (fr) * 2015-11-13 2017-05-18 고려대학교산학협력단 Procédé de détection en temps réel d'épissage d'arn au moyen de la diffusion de la lumière dans des nanostructures plasmoniques
CN111991561A (zh) * 2020-08-26 2020-11-27 中国科学院上海高等研究院 一种高效穿过血脑屏障的寡聚核苷酸/原子精细纳米团簇复合物及其制备方法以及应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6979729B1 (en) * 1999-09-14 2005-12-27 Yeda Research And Development Co. Ltd. Metal cluster containing nucleotides and nucleic acids, and intermediates therefor
US20050048546A1 (en) * 2003-07-11 2005-03-03 Sharron Penn Multiplexed molecular beacon assay for detection of human pathogens
WO2015114127A1 (fr) * 2014-01-31 2015-08-06 Fundación Imdea Nanociencia Nanoparticules métalliques fonctionnalisées et leurs utilisations pour détecter des acides nucléiques
WO2017082517A1 (fr) * 2015-11-13 2017-05-18 고려대학교산학협력단 Procédé de détection en temps réel d'épissage d'arn au moyen de la diffusion de la lumière dans des nanostructures plasmoniques
CN111991561A (zh) * 2020-08-26 2020-11-27 中国科学院上海高等研究院 一种高效穿过血脑屏障的寡聚核苷酸/原子精细纳米团簇复合物及其制备方法以及应用

Non-Patent Citations (2)

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Title
LIU ZHONGDE, HUANG CHENG ZHI, WANG YI, ZHANG PU: "Oligonucleotide-stabilized silver nanoclusters as fluorescent probes for highly selective detection of thiol-containing drugs", ZHONGGUO KEXUE. HUAXUE - SCIENTIA SINICA CHIMICA, ZHONGGUO KEXUE ZAZHISHE, CN, vol. 41, no. 6, 1 June 2011 (2011-06-01), CN , pages 1037 - 1043, XP093069206, ISSN: 1674-7224, DOI: 10.1360/032010-777 *
SHEN JIA-JIA, CHEN WAN-TING, YA DING: "Preparation and applications of gold nanoclusters with precise atom number", ACTA PHARMACEUTICA SINICA, YAOXUE XUEBAO, CN, vol. 53, no. 9, 13 June 2018 (2018-06-13), CN , pages 1484 - 1492, XP093069205, ISSN: 0513-4870, DOI: 10.16438/j.0513-4870.2018-0350 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117431058A (zh) * 2023-12-18 2024-01-23 天津大学 单分散及表面单功能化超小金团簇的方法及应用

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