Cascade Signal Amplification of Time-Resolved Fluorescence Immunoassay through Enzyme Catalysis Coupled with Dissolution Enhancement

Time-resolved fluorescence immunoassay (TRFIA) has found wide application in various fields due to its low background noise characteristics. However, the limited attachment of lanthanide ion complexes onto antibodies restricts the signal intensities, making it challenging to detect analytes with low abundance. To overcome this challenge, we developed a cascade signal amplification strategy that combines enzyme-catalyzed tyramine signal amplification (TSA) with the dissolution enhancement of lanthanide fluoride nanoprobes (NPs). The TSA process (primary signal amplification) enriches biotin moieties around the immunocomplex in a conventional enzyme-linked immunosorbent assay (ELISA), followed by the attachment of streptavidin-modified NaEuF₄ NPs. When exposed to acidic conditions, NaEuF₄ NPs dissociate into a large quantity of Eu³⁺ ions. Each Eu³⁺ ion can then form an emissive lanthanide ion complex by chelation with enhancer agents, leading to secondary signal amplification. As a proof-of-concept, this approach is used to detect anti-Müllerian hormone (AMH), achieving a detection limit of 8.6 × 10^–13 g/mL, which is superior to commercially available kits. The proposed TRFIA with cascade signal amplification benefits from the low background noise of TRFIA, simultaneously harnessing enhanced signals through dual amplification. This approach shows great potentials in improving the sensitivity of immunoassays and also achieving dual-mode detection capabilities.