Ho, Chapman; (2025) Harnessing cleavable linkers for the development of ‘capture-and-release’ sensitivity enhancement strategies in lateral flow assays. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Lateral flow assays (LFAs) represent Point-of-Care (PoC) diagnostics that are cheap, rapid, and require minimal training and equipment. LFAs have been used to diagnose a variety of diseases with simple colorimetric readouts, yet typically fall short of the sensitivities of molecular tests. To address the shortfalls of LFA sensitivity, this thesis explores cleavable linkers and the development of the “AmpliFold” approach – a methodology whereby HER2, a model biomarker protein, is captured over a large area of capture receptors, controllably eluted, and re-bound for detection with high signal- to-noise and sensitivity across a ‘two-strip’ design. This work involves the modification of an anti-HER2 Fab fragment using biotin-linkers featuring cleavable internal disulfide bonds. Linker length, flexibility, and bioconjugation strategy are investigated to achieve a cleavable anti-HER2 capture conjugate that enables efficient target immunocomplexes release (~85%) from polystreptavidin (PSA) functionalised capture lines in the ‘first’ test strip. This work also explores the development of FluoroPerAuNP conjugates, anti-HER2 antibody-gold nanoparticle conjugates modified with fluorescein tags. When utilised in AmpliFold assays and combined with cleavable capture conjugates, FluoroPerAuNPs were shown to enable high-affinity re-binding of eluted immunocomplexes, following linker cleavage, by anti-fluorescein detection lines in the ‘second’ test strip. Optimisation of AmpliFold buffer, washing, and blocking parameters, as well as details of the design and assembly of the ‘two-strip’ format, are reported in this thesis. AmpliFold capabilities are validated using an idealised system – where low PSA capture receptor density is used to model poor binding kinetics – resulting in a 16-fold sensitivity enhancement compared to traditional LFA tests. The application of the AmpliFold format is further shown by overcoming the limited diffusivity and capture kinetics of larger nanoparticles, ultimately improving upon the sensitivity of traditional LFA tests by 12-fold when detecting HER2 spiked into both buffer and human serum samples. This thesis establishes the AmpliFold methodology as a rapid sensitivity enhancement technique with the potential to empower PoC diagnostics towards more timely responses to emerging diseases.

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