Coral reef-inspired hierarchical supersandwich on test strip for ultrasensitive dual-mode detection of cystatin C

Conventional gold nanoparticle (AuNP)-based lateral flow assays enable rapid point-of-care testing but suffer from limited sensitivity and a sole reliance on colorimetric readout. Here we developed a dual-mode lateral flow immunoassay (LFIA) that synergistically amplifies both colorimetric and fluorescent signals for the ultrasensitive detection of cystatin C. The key innovation is a UV-excitable DNA fluorescent "adhesive" composed of biotinylated tetrahedral DNA nanostructures bearing G-quadruplex motifs at each vertex. Upon chelation with N-methylmesoporphyrin IX（NMM）, the structure generates strong fluorescence with a large Stokes shift (Ex/Em = 399/610 and 670 nm), effectively minimizing background interference and avoiding fluorescence quenching by AuNPs. Furthermore, inspired by coral reef growth, we established a bioinspired hierarchical assembly strategy. This DNA "adhesive" enables the sequential tethering of multiple layers of AuNP reporters onto the test line via streptavidin-biotin binding, forming a hierarchical Tet/AuNP superstructure that co-amplifies dual signals. The dual-mode strategy allows result cross-validation and achieves low limits of detection (LoD) for cystatin C at 5 ng/mL (fluorescence) and 25 ng/mL (colorimetric), representing a 50-fold sensitivity improvement over conventional LFIA (LoD: 250 ng/mL). Clinical validation using 40 human serum samples demonstrated excellent correlation with the standard particle-enhanced turbidimetric immunoassay and exhibited no cross-reactivity. By integrating a bioinspired assembly mechanism with a low-cost fluorescent adhesive, this work provides a robust and versatile platform for sensitive point-of-care detection of kidney injury biomarkers.