Triggered “Capture-and-Release” Enables a High-Affinity Rebinding Strategy for Sensitivity Enhancement in Lateral Flow Assays

Lateral flow assays (LFAs) are point-of-care devices that are known for their affordability, speed, and simplicity. However, LFA sensitivity is often limited by the need for fast associative rates between the assay components. This work presents a strategy toward reducing the demand for fast test line associative kinetics via a "capture-and-release" approach. Using HER2 protein as a model biomarker system, this methodology─termed the "AmpliFold" approach─involves the initial sequestration of analyte-bound complexes, which undergo triggered release and are rebound, using high-affinity hapten interactions, resulting in 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 bioconjugation 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 the 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 LFA sensitivity. The AmpliFold approach was further shown to address the poor diffusivity and surface binding kinetics of large nanoparticles in LFA systems. Using high capture receptor densities and a 150 nm AuNP example, a 12-fold sensitivity enhancement was achieved when using AmpliFold to detect the target analyte spiked into both buffer and human serum samples. Incorporated into a folding "two-strip" LFA design and performed via a multistep (capture, wash, and linker cleavage) workflow, the AmpliFold approach represents a proof-of-concept strategy that utilizes established protein modification chemistries to provide a rapid (<30 min), equipment-free, and tractable route toward enhancing LFA kinetics and sensitivity.