研究目的
To develop an enzyme-free fluorometric assay for sensitive and rapid detection of chloramphenicol in food samples, utilizing dual signal amplification via target recycling and catalyzed hairpin assembly to reduce cost and detection time.
研究成果
The developed enzyme-free fluorometric assay provides a highly sensitive and rapid method for chloramphenicol detection, with a low detection limit of 16 pM and reduced reaction time due to simultaneous dual signal amplification. It shows good specificity, precision, and applicability in real food samples, offering advantages over existing methods. However, it cannot detect multiple antibiotics at once, indicating a need for further development in multiplexing capabilities.
研究不足
The strategy is unable to detect multiple antibiotics simultaneously, which limits its application for multiplexed analysis. Future work aims to develop simultaneous detection methods and more portable, low-cost strategies.
1：Experimental Design and Method Selection:
The assay is based on a dual simultaneous signal amplification strategy using two stirring bars modified with DNA probes. Target recycling and catalyzed hairpin assembly (CHA) occur simultaneously upon addition of chloramphenicol, leading to fluorescence emission from thioflavin T (ThT) binding to G-quadruplex DNA sequences.
2：Sample Selection and Data Sources:
Spiked milk and fish samples were used to evaluate the assay's applicability. Oligonucleotides were synthesized and provided by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.
3：List of Experimental Equipment and Materials:
Equipment includes a H600 transmission electron microscope (Hitachi), S3400 N scanning electron microscope (Hitachi), MultiNA 202 System (Shimadzu), and RF-6000 fluorescence spectrophotometer (Shimadzu). Materials include ThT, MPTMS, CAP, various antibiotics, KCl, HAuCl4, TCEP, MCH, PBS, and oligonucleotides.
4：Experimental Procedures and Operational Workflow:
Gold stirring bars were modified with Au nanoparticles and DNA probes. The assay involved incubating modified bars with H2, H3, ThT, and CAP samples at 37°C for 60 min, followed by fluorescence measurement.
5：Data Analysis Methods:
Fluorescence intensity was measured at 485 nm, and calibration curves were plotted. Limit of detection was calculated based on signal-to-noise ratio. Specificity and interference tests were conducted using other antibiotics and common ions/proteins.
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