A widely used CRP aptamer does not bind CRP, revealing a general route to surface-driven pseudoaffinity in biosensors

ABSTRACT Aptamer sequences are often reused in biosensors and diagnostics without independent confirmation of binding activity, even though rigorous validation requires quantitative solution-phase testing by orthogonal methods. Here, we re-examined a DNA sequence reported in 2010 to bind C-reactive protein (CRP) with a K d of 3.5 nM by surface plasmon resonance (SPR) and subsequently adopted in many CRP aptasensor studies. We tested this sequence by two orthogonal solution-phase methods and by SPR. In solution-phase assays, we found no detectable binding of the reported aptamer to native CRP. In SPR, we found that CRP binds strongly to streptavidin-containing sensor surfaces even in the absence of immobilized DNA, whereas the reported sequence produced no measurable ligand-specific SPR response under lower-background capture conditions. Thus, the originally reported affinity is best explained by surface-driven pseudoaffinity rather than true aptamer-CRP recognition. More broadly, this case does not merely add another example of aptamer nonbinding, but reveals a general route by which nonbinding aptamers can acquire apparent affinity through surface-driven pseudoaffinity and then be propagated through the biosensor literature as validated molecular recognition reagents. This study underscores the need for orthogonal solution-phase validation before aptamers are reused in biosensor development and related analytical applications.