Polymerization-based amplification (PBA), which combines bio-recognition events with polymerization reactions on surfaces, provides visual, sensitive, and cost-effective detection of biological interactions, particularly at extremely low levels of the targeted biological moiety. This study characterizes assay parameters that enhance the utility of PBA to detect nucleic acid and protein biomarkers. Here, we successfully employ PBA on surfaces that contain uniform, high density, immobilized capture molecules, including three-dimensional nitrocellulose-coated substrates. Optimized assay and polymerization conditions are used to characterize the dynamic polymer film heights on glass substrates that result from solutions of KRAS proto-oncogene biomarker targets at concentrations between 5 nM and 500 pM. Differing aqueous monomer formulations are utilized to produce 20 nm films at the 500 pM DNA detection limit.