1：Experimental Design and Method Selection:

The study employs a three stir-bars assisted target recycling (TSBTR) system for triple signal amplification. The design includes a magnetic stir bar (bar-C) modified with two kinds of long DNA chains from hybridization chain reaction (HCR) products, and two gold stir bars (bar-A and bar-B) modified with Y-shape and duplex DNA probes, respectively. The mechanism involves target-induced strand displacement and recycling for signal amplification, with SYBR green dye used for fluorescence detection.

2：Sample Selection and Data Sources:

Standard CAP solutions were used for calibration, and real food samples (milk and fish) were obtained from a local supermarket in Ningbo, China. Samples were pretreated to remove interferents before analysis.

3：List of Experimental Equipment and Materials:

Key materials include chloramphenicol (CAP), various antibiotics for specificity tests, oligonucleotides (sequences listed in Table 1), SYBR Green I dye, gold nanoparticles (AuNPs), amino-functionalized iron oxide (Fe3O4-NH2), and buffers (PBS, TE). Equipment includes a fluorescence spectrophotometer (RF-6000), UV-vis spectrophotometer (UV-1800), scanning electron microscope (S3400N), transmission electron microscope (H600), and microfluidic electrophoresis system (MultiNA 202).

4：2). Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: Fabrication of TSBTR involved modifying stir bars with DNA probes and HCR products. For detection, bars were incubated with sample solutions for 40 min at 40°C, followed by removal of bars, addition of SYBR green dye, and fluorescence measurement at 497 nm excitation and 523 nm emission. Optimization of parameters like pH, temperature, and reaction time was performed.

5：Data Analysis Methods:

Fluorescence intensity was measured and correlated with CAP concentration for quantitative analysis. Specificity, reproducibility, and recovery tests were conducted using statistical methods, with comparisons to ELISA as a reference.