1：Experimental Design and Method Selection:

A competitive fluorescence ELISA was designed based on alkaline phosphatase (ALP)-triggered fluorescence "turn-on" signals. The method utilizes the electrostatic self-assembly of carbon dots (CDs) and MnO2 nanosheets (NSs) to form a nanoassembly (p-CDs@MnO2) that quenches fluorescence, which is recovered upon ALP-catalyzed reduction by ascorbic acid (AA).

2：Sample Selection and Data Sources:

Chicken meat samples were used, spiked with amantadine at various concentrations for recovery and precision tests. Standard solutions of amantadine and other non-target analytes were prepared for specificity evaluation.

3：List of Experimental Equipment and Materials:

Chemicals included polyethyleneimine (PEI), alkaline phosphatase (ALP), 2-phospho-L-ascorbic acid (AAP), L-ascorbic acid (AA), KMnO4, sodium dodecyl sulfate (SDS), citric acid monohydrate, anti-amantadine monoclonal antibody (mAb), coating antigen (BSA-AMD), and others from Sigma-Aldrich and Sinopharm Chem. Ltd. Apparatus included FS-5 spectrofluorometer, Horiba DeltaFlex for fluorescence lifetime, PerkinElmer Envision plate reader, Tecnai G2 F20 TEM microscope, Dimension Icon AFM, Nicolet IS10 FT-IR spectrometer, Zetasizer Nano ZS, inVia Raman microscope, Escalab 250Xi XPS, and SgaeCapture fluorescence imaging system.

4：Experimental Procedures and Operational Workflow:

The nanoassembly was prepared by hydrothermal synthesis of p-CDs and one-step synthesis of MnO2 NSs, followed by electrostatic assembly. For the immunoassay, a 96-well microplate was coated with BSA-AMD, blocked, and incubated with AMD standards and mAb. ALP-labeled secondary antibody was added, followed by AAP hydrolysis and addition of nanoassembly. Fluorescence intensity was measured after 5 minutes.

5：Data Analysis Methods:

Standard curves were fitted using a four-parameter logistic equation: Y = (A–B)/[1+(X/C)D] + B. Data were analyzed for IC50, LOD, recovery, precision, and specificity using statistical methods and software tools (not specified in detail).