研究目的
To develop a BRET-based dual-colour (visible/near-infrared) molecular imaging probe using a quantum dot (QD) and luciferase protein conjugate for highly sensitive detection of membrane receptors in cancer cells.
研究成果
The BRET-based molecular imaging probe consisting of a NIR-emitting QD and EGFP–RLuc fusion protein was successfully developed for highly sensitive detection of membrane receptors in cancer cells. The probe demonstrated higher detection sensitivity for HER2 and EGFR compared to fluorescence-based molecular imaging and can be used for dual-colour molecular imaging at the visible and NIR region.
研究不足
The BRET-based QD probe has a dose-dependent cytotoxicity, and significant cytotoxicities were not found under the concentration of QDs (<50 nM) used for the BRET-based cellular imaging. The application of bioluminescence imaging has been restricted to protein expression and protein–protein interaction using their reporter genes.
1:Experimental Design and Method Selection:
The study designed a BRET-based molecular imaging probe consisting of a NIR-emitting QD and EGFP–RLuc fusion protein. The probe was tested for its ability to emit dual-colour luminescence and bind to antibodies for targeted imaging.
2:Sample Selection and Data Sources:
Human breast tumour cells (KPL-4) and human skin cancer cells (A431) were used to test the probe's imaging capabilities.
3:List of Experimental Equipment and Materials:
CdSeTe/CdS QDs, EGFP–RLuc fused proteins, antibodies (Herceptin, Erbitux), and coelenterazine (CTZ) were used.
4:Experimental Procedures and Operational Workflow:
The binding of proteins to QDs was confirmed by agarose gel electrophoresis. BRET efficiency was measured, and the probe's ability to bind to antibodies was examined using fluorescence correlation spectroscopy (FCS) and fluorescence activated cell sorter (FACS). BRET and fluorescence imaging were performed on cell pellets.
5:Data Analysis Methods:
BRET efficiency was calculated from emission intensities. FCS was used to analyze diffusion times of QDs in solution.
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