1：Experimental Design and Method Selection:

The study designed a single-stranded DNA with an appended block for synthesizing fluorescent Ag nanoclusters (AgNCs) and an anchoring block (Poly A) for binding to gold nanoparticles (GNPs) to quench fluorescence. A sandwich ELISA complex was used for detection.

2：Sample Selection and Data Sources:

E. coli O157:H7 samples were used, with concentrations ranging from 0 to 2.6 × 10^6 CFU/mL. Milk samples (low-fat and whole milk) and PBS solutions were spiked with known concentrations for recovery experiments.

3：6 × 10^6 CFU/mL. Milk samples (low-fat and whole milk) and PBS solutions were spiked with known concentrations for recovery experiments. List of Experimental Equipment and Materials:

3. List of Experimental Equipment and Materials: Instruments included an F-380 fluorescence spectrophotometer (Gangdong Co., Ltd.), K5600 Micro-Spectrophotometer (Kaiao Co.), Varioskan LUX multimode microplate reader (Thermo Fisher Scientific Co.), and JEM-2100 Electron Microscope (JEOL Co. Ltd). Materials included HAuCl4·3H2O, trisodium citrate, Bovine serum albumin (BSA), AgNO3, Sodium borohydride (NaBH4), Polyethylene glycol (PEG) 20,000, skim milk, Ovalbumin, E. coli O157:H7 monoclonal and polyclonal antibodies, oligonucleotides, black 96-well microplates, and other chemicals.

4：Experimental Procedures and Operational Workflow:

AgNCs were synthesized using Dickson's method. GNPs were prepared by reducing HAuCl4 with sodium citrate. GNPs were conjugated with antibodies via electrostatic adsorption. ELISA was performed by coating plates with pAb, blocking, adding E. coli samples, adding mAb-GNPs mixture, and then adding AgNCs to measure fluorescence quenching after 35 minutes.

5：Data Analysis Methods:

Fluorescence intensity was measured, and quenching efficiency (1-F/F0) was calculated. Statistical analysis used GraphPad Prism v7.01 with t-Student test, considering P < 0.05 significant. Linear regression equations were derived for quantitative detection.