研究目的
To design a MnO2 nanosheet-mediated ratiometric fluorescence biosensor for accurate and sensitive detection and imaging of microRNA (specifically miRNA-21) in living cells, aiming to differentiate expression levels in cancer versus normal cells for potential early cancer diagnosis.
研究成果
The MnO2 nanosheet-mediated ratiometric fluorescence biosensor successfully enables sensitive and accurate detection and imaging of miRNA-21 in living cells, with high selectivity and low cytotoxicity. It differentiates miRNA expression levels between cancer and normal cells, showing promise for early diagnosis of miRNA-related diseases like cancer. Future work could focus on in vivo applications and broader miRNA targets.
研究不足
Potential limitations include dependence on intracellular GSH levels for MnO2 degradation, possible interference from other cellular components, and the need for optimization of probe concentrations and incubation times. The method may be affected by variations in cell types and environmental factors, though ratiometric design mitigates some issues.
1：Experimental Design and Method Selection:
The biosensor uses MnO2 nanosheets as DNA carriers and quenchers, with FAM-labeled hairpin H1 and TAMRA-labeled hairpin H2. Upon cell entry, MnO2 degrades via GSH, releasing probes. Target miRNA-21 triggers catalyzed hairpin assembly (CHA), bringing FAM and TAMRA close for FRET, enabling ratiometric detection.
2：Upon cell entry, MnO2 degrades via GSH, releasing probes. Target miRNA-21 triggers catalyzed hairpin assembly (CHA), bringing FAM and TAMRA close for FRET, enabling ratiometric detection. Sample Selection and Data Sources:
2. Sample Selection and Data Sources: Uses synthetic DNA oligonucleotides, miRNAs (e.g., miRNA-21), and cell lines (HeLa, HepG-2, L02) obtained from specified suppliers.
3：List of Experimental Equipment and Materials:
Includes fluorescence spectrophotometer (Hitachi F-7000), UV-vis spectrophotometer (Hitachi U-2910), TEM (JEOL JEM-1011), Raman spectrometer (Horiba Jobin Yvon LabRAM HR800), fluorescence microscope (Carl Zeiss Axio Observer 3), microplate reader (Tecan), DLS/zeta potential analyzer (Malvern ZetaSizer Nano ZS90), and chemicals from Sangon Biotech and Macklin Biochemical.
4：Experimental Procedures and Operational Workflow:
Synthesis of MnO2 nanosheets via oxidation; in vitro detection by mixing probes with MnO2, GSH, and miRNA-21; PAGE analysis; cell culture; MTT assay for cytotoxicity; fluorescence imaging after probe incubation.
5：Data Analysis Methods:
Fluorescence intensity ratios (FA/FD) calculated, linear regression for sensitivity, RSD for stability, PAGE for reaction verification, and microscopy for imaging.
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fluorescence spectrophotometer
F-7000
Hitachi
Obtain fluorescence emission spectra for detecting FRET signals in the biosensor.
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spectrophotometer
U-2910
Hitachi
Perform ultraviolet-visible (UV-vis) absorption spectroscopy for characterizing MnO2 nanosheets and probes.
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transmission electron microscope
JEM-1011
JEOL
Record TEM images to characterize the lamellar structure of MnO2 nanosheets.
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fluorescence microscope
Axio Observer 3
Carl Zeiss
Obtain fluorescence images of cells for miRNA imaging, using specific filters for FAM and TAMRA.
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ZetaSizer
Nano ZS90
Malvern Instrument
Perform dynamic light scattering (DLS) and zeta potential analysis for characterizing nanosheet size and charge.
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Raman spectrometer
LabRAM HR800
Horiba Jobin Yvon
Perform Raman spectroscopy to identify vibrational features of MnO2 nanosheets.
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microplate reader
Tecan Austria GmbH
Tecan
Measure absorbance in MTT assays for cytotoxicity evaluation.
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UV imaging system
Bio-RAD Laboratories Inc.
Bio-RAD
Visualize polyacrylamide gel electrophoresis (PAGE) results after staining with SYBR gold.
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