As a binding system, Pertuzumab and the Fab fragment of Herceptin (FabHER) were selected as a matched pair for binding the HER2 antigen. Pertuzumab and Herceptin are cost-effective FDA-approved antibodies that are known to bind HER2 via different epitopes, with low nanomolar binding affinities, providing an adequate matched pair for R&D purposes. The capture protein FabHER was modified with biotin via multiple strategies allowing us to study the influence of linker length, flexibility, and modification approach when evaluating the efficiency of 'capture-and-release'. A range of thiolcleavable commercially available lysine-reactive linkers including sulfo-NHS-SS-biotin, NHS-SS-PEG4-biotin were employed in reaction with FabHER in a non-site-selective modification strategy, with NHS-PEG12-biotin as a non-cleavable control (Figure , parts i-iii). This generated FabHER-linker-biotin conjugates 1-3. A further strategy utilised pyridazinedione (PD) chemistry to site-specifically modify the FabHER's single solvent accessible disulfide using Br2PD-BCN to site-selectively install a single strained alkyne (bicyclo[6.1.0]non-4-yne, BCN), which was then reacted with an N3-PEG3-SS-PEG4-biotin linker (see Figure for synthesis) via a quantitative 'click' chemistry reaction to generate FabHER-PD-PEG3-SS-PEG4-biotin (4) (Figure ). Representing the other half of the immunoassay, Pertuzumab was employed as a detection antibody for the functionalisation of gold nanoparticle (AuNP) labels. Aside from binding the HER2 antigen, our design criteria comprised a particle label which could be orthogonally re-bound at the test line of the detection strip. The method of particle rebinding itself was anticipated to necessitate a high-affinity interaction to ensure maximal recovery of particle complexes -following release by linker cleavage -and detection with superior signal-to-noise, when compared to initial analyte capture. To develop a 'dual affinity' nanoparticle label, Pertuzumab was first pre-modified with fluorescein isothiocyanate (FITC) to install specific and orthogonal affinity tags. The resulting fluorescein-Pertuzumab (FluoroPer) conjugates were then used to functionalise 40 nm AuNPs via passive physisorption (Figure ). The physisorption of FluoroPer to citrate capped 40 nm AuNPs was confirmed by UV-Vis (Figure ) and dynamic light scattering (DLS) (Figure ) where upon binding of the protein, a peak plasmon shift and increase in hydrodynamic diameter of 12.1 nm respectively were observed. The increase in hydrodynamic diameter reflected the thickness of a protein layer consistent with expected antibody dimensions. 40 nm FluoroPerAuNPs blocked with BSA were shown to have a further increased hydrodynamic diameter suggestive of corona formation. Zeta potential measurements (Figure ) indicated the blocked FluoroPerAuNPs exhibited a negative surface charge -22.8 mV, comparable with the bare citrate capped AuNPs, however during the process of FluoroPer physisorption, an intermediate surface charge closer to neutral was measured. To evaluate the affinity of these particles as a re-binding target, LFA studies were performed to benchmark the performance of FluoroPerAuNPs against a biotin-streptavidin model AuNP system. Here, nitrocellulose membranes printed with either anti-FITC or PSA test lines were used to bind and detect 40 nm FluoroPerAuNPs and biotinylated-AuNPs, respectively. As a general procedure, test line signal measurements were achieved by imaging test strips under controlled lighting conditions and by using ImageJ software to extract pixel intensity values (full procedure described in the Materials & Methods, as well as in Supplementary Information Figure ). In these experiments, 40 nm FluoroPerAuNPs were shown to be detected down to concentrations of 274 fM (95% CI of 150 to 533 fM), and 40 nm biotinylated-AuNPs down to concentrations of 94.8 fM (95% CI of 52.6 to 169 fM). Whilst lesser than its model comparison, the detection of our FluoroPerAuNPs down to femtomolar sensitivities likely reflects a significant degree of decoration on the particle surface by FluoroPer antibodies.