研究目的
To develop a biomineralization strategy for synthesizing Y-DNA@CuP hybrid nanoflowers as efficient gene carriers for fluorescence imaging of tumor-related mRNA in living cells, enabling the distinction of tumor cells from normal ones and assessment of drug effectiveness.
研究成果
The Y-DNA@CuP hybrid nanoflowers successfully enable intracellular imaging of TK1 mRNA, distinguishing tumor cells from normal cells and assessing drug effects. This strategy provides a novel approach for synthesizing biocompatible nanomaterials for biomedical applications.
研究不足
The encapsulation efficiency of Y-DNA in hybrid nanoflowers is low (around 13.6%), and the size control may depend on specific conditions. Potential cytotoxicity at higher concentrations and limitations in real-time application in vivo are not fully addressed.
1:Experimental Design and Method Selection:
A biomineralization strategy using Y-shaped DNA as a template to synthesize hybrid nanoflowers for gene delivery and mRNA detection. Methods include fluorescence spectroscopy, SEM, TEM, XRD, XPS, and cell imaging.
2:Sample Selection and Data Sources:
Oligonucleotides from Sangon Biotech, cell lines (HeLa, HepG2, MCF-7, L02), and drugs (b-estradiol, tamoxifen) from Sigma-Aldrich.
3:List of Experimental Equipment and Materials:
Instruments include Hitachi S-4800 SEM, JEM-2010F TEM, X'Pert diffractometer, ESCALAB 250 Xi XPS spectrometer, Hitachi F-4600 fluorometer, Eclipse Ti fluorescence microscope, BD Accuri C6 flow cytometer, Leica TCS SP8 confocal microscope, iMark Microplate Reader. Materials include DNase I, MTT, Trizol, DMEM, FBS, etc.
4:Experimental Procedures and Operational Workflow:
Preparation of Y-DNA via annealing, biomineralization with CuSO4 and PBS, characterization, encapsulation and release studies, cytotoxicity assay, cell culture, fluorescence imaging, flow cytometry, and RT-qPCR analysis.
5:Data Analysis Methods:
Fluorescence intensity measurements, statistical analysis using ANOVA, and fold change calculation for mRNA expression.
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Scanning Electron Microscope
S-4800
Hitachi
Acquiring SEM images for morphological characterization of nanoflowers.
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Transmission Electron Microscope
JEM-2010F
JEOL
Obtaining TEM images for detailed structural analysis of particles.
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X-ray Diffractometer
X'Pert
Panalytical
Performing XRD measurements to identify chemical components.
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X-ray Photoelectron Spectrometer
ESCALAB 250 Xi
Thermo Scientific
Conducting XPS measurements for elemental analysis.
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Fluorometer
F-4600
Hitachi
Measuring fluorescence spectra for detection and analysis.
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Confocal Microscope
TCS SP8
Leica
Executing confocal fluorescence imaging for co-localization studies.
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Fluorescence Microscope
Eclipse Ti
Nikon
Performing fluorescence imaging of cells.
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Flow Cytometer
Accuri C6
BD
Analyzing cell fluorescence for quantitative assessment.
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Microplate Reader
iMark
Bio-Rad
Measuring absorbance for cytotoxicity assays.
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Thermal Cycler
T100
Bio-Rad
Performing cDNA conversion and qPCR.
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Thermal Cycler
C1000 Touch
Bio-Rad
Carrying out qPCR reactions.
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