研究目的
To construct a sensitive and simple fluorescence aptasensor for the detection of Aflatoxin M1 (AFM1) in milk samples using an Exonuclease III-assisted signal amplification strategy.
研究成果
The developed aptasensor provides a highly sensitive, selective, and simple method for AFM1 detection in milk, with a low detection limit of 9.73 ng kg?1, meeting regulatory standards. It offers advantages over traditional methods like lower cost and faster operation, and shows potential for broader analyte detection by adapting the aptamer system.
研究不足
The sensor may have limitations in complex matrices beyond milk, potential interference from other substances not tested, and reliance on specific enzymes and DNA sequences which could affect reproducibility. Optimization was done for specific conditions, and real-world application might require further validation.
1：Experimental Design and Method Selection:
The aptasensor is designed with a two-part system: AFM1-induced strand release on magnetic nanobeads and an Exo III-assisted signal amplification recycle. It uses DNA hybridization, enzymatic digestion, and fluorescence enhancement via G-quadruplex formation with NMM dye.
2：Sample Selection and Data Sources:
Milk samples (whole milk, skimmed milk, juice milk) were commercially purchased and spiked with AFM1 standards. Negative control milk samples were pre-tested to ensure no AFM1 contamination.
3：List of Experimental Equipment and Materials:
Includes magnetic nanobeads, DNA strands, enzymes (Exo III), fluorescent dye (NMM), buffers (Tris-HCl, MgCl2, KCl), and mycotoxin standards. Specific equipment: NanoDrop for DNA concentration measurement, metal bath for temperature control, Cary Eclipse fluorescence spectrophotometer for signal detection.
4：Experimental Procedures and Operational Workflow:
Assembly involves immobilizing biotinylated aptamers on streptavidin-coated magnetic nanobeads, hybridization with complementary DNA, incubation with AFM1 to release C-strand, magnetic separation, addition to G/T duplex and Exo III for amplification, incubation with NMM, and fluorescence measurement. Optimization of parameters like NMM concentration, reaction times.
5：Data Analysis Methods:
Fluorescence intensities were recorded and analyzed using calibration curves. Statistical analysis included recovery percentages and comparison with ELISA kit results.
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magnetic nanobeads
500 nm
Ocean Nanotech
Serve as a nanocarrier for aptamers, provide large surface area for DNA loading, enable easy magnetic separation to remove uncaptured strands and improve assay stability.
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NanoDrop
Thermo-Fisher Scientific
Used to redefine the concentration of DNA strands and aptamers.
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metal bath
Thermo-Fisher Scientific
Control the temperature of the recycle amplification process.
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Cary Eclipse fluorescence spectrophotometer
Agilent Technologies
Record fluorescence intensities at 612 nm for signal detection.
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ELISA kit
Sangon Biotech Co., Ltd.
Used for verification of the aptasensor performance by comparing results.
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Millipore water system
Millipore
Prepare Milli-Q water for experiments.
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Exonuclease III
Genesearch Pty Ltd.
Act as an amplifying biocatalyst to digest duplex DNAs and facilitate signal amplification.
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N-methyl mesoporphyrin IX
NMM
Frontier Scientific
Fluorescent dye that binds to G-quadruplex structures to generate amplified fluorescence signals.
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