An ideal linker cleavage was envisaged to have high efficiency (i.e. nearly quantitative release of captured material) and to occur in a rapid timeframe (10 minutes). To appraise both cleavage efficiency and kinetics, a simple, multi-step procedure was performed where, in short, a pre-mixture of HER2 antigen, FabHER-linker-biotin and 40 nm FluoroPerAuNPs were carried up PSA-printed membranes to generate a HER2 antigen dependent test line signal. Following a wash step, biotin anchored immunocomplexes were then cleaved using various concentrations of DTT in a pH 10 cleavage buffer (see Supplementary Information Figure for pH optimisation). After 10 minutes, a thiolreactive, N-methylmaleimide solution was then wicked through the membrane to quench the reaction. A control in the absence of DTT was used to assess the release of bound complexes, via triggered linker cleavage (Figure ). Interestingly, linker cleavage was not as facile as anticipated. In examples featuring complexes captured via FabHER-SSbiotin (2) (a short linker conjugate), a maximal signal loss of only 40.6% (± 0.9%) was observed despite a DTT concentration of 100 mM being used. When compared to supplementary studies using mass spectrometry (Supplementary Information Figures ), quantitative linker cleavage of unbound FabHER-SS-biotin (2) proceeded at a 30fold lower concentration of DTT within the same timeframe was observed. Based on the differences between bound and unbound linker cleavage efficiencies, steric hinderance at the linker cleavage site may have been induced between the interface of this large proteins -in this case, the Fab fragment and PSA. Further cleavage experiments involving an alternative modified fragment with a longer linker to the biotin entity, i.e., FabHER-SS-PEG4-biotin (3), showed an improved maximal cleavage of 63.1% (± 2.5%). Here, the elongation of the PEG spacer in the linker was thought to provide some flexibility, thus alleviating some steric hinderance and improving cleavage efficiency. In an effort to achieve near-quantitative linker cleavage, FabHER-PD- PEG3-SS-PEG4-biotin conjugate (4) was also employed. Cleavage experiments featuring this conjugate demonstrated the highest cleavage efficiency within 10 minutes with a reduction of signal by 82.1% (± 7.4%) upon elution with 100 mM DTT. As with previous iterations, reduction of steric hinderance by virtue of linker elongation may be offered as an explanation for improved linker cleavage, yet the drastic enhancement in cleavage efficiency is also theorised to be the result of site-specific linker installation. Previous FabHER-linker-biotin iterations conjugated through lysine modifications were observed by mass spectrometry to have a large distribution of FabHER-to-linker loading ratios which may enable a single complex to bind numerous PSA receptors multivalently, such that multiple cleavages are required per complex for release. Using PD modification, linker installation occurs at the site of a disulfide bridge which, for a Fab fragment, results in a 1:1 loading ratio and a single cleavage target per complex. In this way, the release of captured antigen-bound complexes is expected to be relatively facile -compared to constructs with the potential for multivalent anchoring to the immobilised PSA test-lineand may further explain the marked difference in performance between the studied examples.