研究目的
To construct a graphene-based steganographicly aptasensing system for multifunctional applications in information computing, encryption and hiding, fluorescent sensing and in vivo imaging of fish pathogens.
研究成果
The graphene-based steganographicly aptasensing system was successfully constructed for multipurpose applications, including logic computing, fluorescent sensing, in vivo imaging of fish pathogens, and information encryption and hiding. It provides a novel nano-biosensing assay and demonstrates a prototype for molecular-level information security, promoting the development of multifunctional molecular-level devices.
研究不足
The sensitivity and reproducibility of the direct detection method may be attributed to poor bacterial dispersion and unstable bacterial activity (especially for A. hydrophila at a relatively higher concentration) which deserves special attention in future work.
1:Experimental Design and Method Selection:
The system was constructed using specific molecular recognition and information encoding abilities of DNA aptamers (Aeromonas hydrophila and Edwardsiella tarda-binding aptamers) and the selective adsorption and fluorescence quenching capacities of graphene oxide (GO). The experimental design involved self-assembly of aptamers with GO, followed by pathogen-induced release and fluorescence recovery.
2:Sample Selection and Data Sources:
Two FAM-labeled aptamers (Apt1 and Apt2) were used as models, synthesized by Sangon Biotech. Co., Ltd. Bacteria (A. hydrophila, E. tarda, and interfering bacteria) were cultured in Luria-Bertani broth medium and counted using the agar plate-counting method. Freshwater fish Carassius auratus were used for in vivo imaging.
3:List of Experimental Equipment and Materials:
Materials included aptamers, GO (purchased from XFNANO Co., Ltd.), Tris, NaCl, MgCl2, KCl, tryptone, yeast extract, agar, and double-distilled water. Instruments included atomic force microscope (Bruker Multimode 8 AFM/SPM), UV-Vis spectrophotometer (Hach DR6000), fluorescence microplate spectrophotometer (SpectraMax M5), zeta-potential analyzer (Zetasizer Nano ZS), fluorescence microscope (Zeiss Axio Observer A1), and in vivo imaging system (IVIS SPECTRUM).
4:Experimental Procedures and Operational Workflow:
Aptamer solutions were diluted in binding buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM KCl, 1 mM MgCl2, pH
5:4). GO (02 mg/mL) was incubated with aptamer (200 nM) for 8 min to form aptamer-GO complexes. Pathogens at varying concentrations were added and incubated for 30 min, followed by fluorescence measurement. For in vivo imaging, fish were injected with pathogens and aptamer-GO complexes, then imaged after 6 h. Data Analysis Methods:
Fluorescence intensity changes (F-F0)/F0 were analyzed for sensitivity and selectivity. AFM images were processed with Gwyddion software. UV-Vis spectra, zeta potentials, and hydrodynamic sizes were measured. Logic gates were abstracted based on matter and fluorescence outputs.
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Atomic Force Microscope
Multimode 8 AFM/SPM
Bruker
Characterization of GO and aptamer-GO complexes via AFM imaging and height analysis
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Zeta Potential Analyzer
Zetasizer Nano ZS
Malvern Instruments
Measuring zeta potentials of GO and aptamer-GO complexes
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Fluorescence Microscope
Axio Observer A1
Zeiss
Fluorescence imaging of bacteria after incubation with aptamer-GO complexes
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In Vivo Imaging System
IVIS SPECTRUM
Caliper-PE
In vivo fluorescence imaging of fish infected by pathogens
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UV-Vis Spectrophotometer
DR6000
Hach
Recording UV-Vis absorption spectra of GO, aptamers, and complexes
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Fluorescence Microplate Spectrophotometer
SpectraMax M5
Molecular Devices
Measuring fluorescence emission spectra and intensity for sensing and logic computing
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Graphene Oxide
XFNANO
Used as a cover for steganography and for fluorescence quenching in aptasensing
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Aptamers
Apt1, Apt2
Sangon Biotech
Used as information carriers, for molecular recognition, and in logic computing
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