Immobilization-driven enhancement of CNT-binding peptide affinity: substantial shift from the free-state

Abstract

Three carbon-binding peptide aptamers, Y1 (FKQDAWEAVDIR), Y2 (SYTHLLHRSLPG), and Y3 (SLHNAEMANFRA) were identified via phage display. Their adsorption efficiency was analyzed in the free state and immobilized on the cage-shaped protein Dps. Cyclic voltammetry (CV) showed that Y1 and Y2 had high affinities, while Y3 had lower affinity in its free state. Dps mutant proteins presenting Y1, Y2, and Y3 on the outer surface (Y1Ct, Y2Ct, Y3Ct) were then analyzed. Unexpectedly, Y3Ct exhibited a strong affinity for carbon surfaces. Quartz crystal microbalance (QCM) measurements, conducted in both batch and open-flow modes, quantified the adsorbed mass and dissociation constants (K_d). Y3Ct had a much lower K_d (48 nM) than Y1Ct (1.04 µM) and Y2Ct (2.18 µM). These findings confirmed that aptamer adsorption efficiency depended on whether the aptamer was free or immobilized. Although the mechanism is unclear, a plausible explanation is that hydrophobic and electrostatic interactions near the C-terminus might have contributed to Y3Ct's enhanced affinity for the carbon surface.