NTA-Co3+-His6 versus NTA-Ni2+-His6 mediated E-Cadherin surface immobilization enhances cellular traction

Abstract Understanding the biological impact of strategies for protein immobilization onto bioactive surfaces is crucial for the design of biomimetic materials. A common strategy used to immobilize or label recombinant proteins is to exploit the Ni 2+ -mediated interaction of nitrilotriacetic acid (NTA) with the hexahistidine tag (His 6 -tag) present on recombinant proteins. While this method ensures a controlled orientation and functionality of the protein, the kinetically labile nature of the bond ensures only its weak immobilization onto the surface. Recently, it has been shown that the oxidation of Co 2+ to Co 3+ greatly stabilizes the bond between NTA and the His 6 -tagged proteins, making it inert to ligand exchange and resistant to chelators. This approach not only has the potential to improve the quality of biomimetic material functionalization and molecule labeling but could also affect cellular mechanical responses for which the mechanical strength of the protein-surface bond is crucial. Here, we compared gold (Au) nanopatterned polyacrylamide (PAA) hydrogels functionalized with E-cadherin via Co 3+ with those functionalized via Ni 2+ for studying adhesion-mediated responses in keratinocytes. We show that keratinocytes develop higher and a broader range of adhesion forces, leading to extended cell spreading and colony organization on Co 3+ vs. Ni 2+ . This work uniquely shows that stabilizing the NTA/His 6 -tag bond via Co 3+ for protein immobilization significantly impacts cellular phenotype on biomimetic materials by impacting cell signaling.