Triggered ‘capture-and-release’ enables a high-affinity re-binding strategy for sensitivity enhancement in lateral flow assays

Lateral flow assays (LFAs) are point-of-care devices known for their affordability, speed, and simplicity. However, their sensitivity is often limited due to the need for fast associative rates between assay components. This work presents a strategy towards reducing the demand for fast test line associative kinetics via a ‘capture-and-release’ approach. Using a HER2 model antigen system, this methodology – termed the “AmpliFold” approach – involves the initial sequestration of analyte-bound complexes which undergo triggered release and are re-bound, using high-affinity hapten interactions, for enhanced signal-to-noise detection. Using anti-HER2 Fab fragments modified with cleavable biotin linkers to achieve triggered release, the importance of linker length and protein modification strategy on the efficiency of analyte-bound complex release is described. Cleavable Fab fragment conjugates were combined with ‘dual-affinity’ gold nanoparticles (AuNPs) highly decorated with fluorescein-tagged anti-HER2 antibodies to facilitate signal amplification. The utility of the AmpliFold approach is demonstrated by titrating capture receptor density to modulate signal distribution across test lines. Larger capture areas in the AmpliFold approach were shown to overcome poor capture kinetics associated with low receptor densities, achieving up to a 16-fold improvement in limit of detection. The AmpliFold approach was further shown to address the poor diffusivity and surface binding kinetics of large nanoparticles in sensitive LFA systems. Using high capture receptor densities and a 150 nm AuNP example, a 12-fold sensitivity enhancement was achieved when comparing AmpliFold to traditional LFAs for the detecting of antigen spiked into both buffer and human serum samples. Incorporated into a manually-assembled ‘folding’ LFA design, the AmpliFold approach represents a proof-of-concept strategy which utilises established protein modification chemistries to provide a rapid (within 30 minutes), equipment-free and tractable route towards enhancing LFA kinetics and sensitivity.