Figure 2.

(A) Protease-sensitive synthetic biomarkers release reporter 1 (R1; structure fluorescein-PEG_5kDa-Biotin) from carrier NWs upon proteolytic cleavage of thrombin substrate. (B) Renal clearance control R2 is protease insensitive and uses Alexa Fluor 488 as capture ligand. (C) SiMoA assay with αR1-coated beads and increasing concentrations of R1 resulted in higher proportions of active fluorescent wells. (D) SiMoA assay for R1 demonstrated a linear relationship between active beads and R1 concentration from 5 to 200 fM and was unaffected by incubation with 1% control mouse urine. (E) The R2 SiMoA assay resulted in a similar linear relationship from 50 to 1000 fM. (F) Combination of R1 or R2 with αR1- or αR2-coated beads results in signal only from appropriately paired reporters and capture antibodies. Scale bar is 50 μm.