Fluorescence Color Gradient Immunochromatographic Assay for Highly-Sensitive, Quantitative, and Simultaneous Detection of Small-Molecule Pollutants

Rapid on-site screening of small-molecule pollutants in complex samples is essential but remains unachieved. In this study, we introduce a universal fluorescence color gradient immunochromatographic assay (FCGICA) utilizing dual-signal superposition to enable ultra-sensitive, wide-range, and simultaneous quantitative detection of multiple small molecules. A red fluorescent nanomembrane (GTQD@Si) is synthesized by the continuous self-assembly of multilayer quantum dots and a SiO₂ shell on a graphene oxide surface. This nanomembrane exhibits high stability in complex environments and provides superior fluorescence along with a larger reactive interface for sensing. The integration of GTQD@Si with green fluorescent microspheres embedded in the test line generates a broad fluorescence color gradient based on variations in target molecule concentrations, thereby significantly enhancing the sensitivity, stability, and quantitative range of the immunochromatographic assay (ICA). By directly reading the ratio of red and green image signals, the proposed FCGICA enables simultaneous, high-sensitivity, and quantitative detection of three different types of small-molecule pollutants including fumonisin B1, imidacloprid, and clenbuterol within 15 min, with a detection range improved by 2–3 orders of magnitude compared with traditional methods. Moreover, the powerful practicality of FCGICA has been verified through comprehensive testing on various real samples, demonstrating its great potential in on-site detection of small molecules.