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Hemoglobin on phosphonic acid terminated self-assembled monolayers at a gold electrode: immobilization, direct electrochemistry, and electrocatalysis.

Phosphonic acid (--PO(3)H(2)) terminated self-assembled monolayers (SAMs) on a gold surface were used as a functional interface to immobilize hemoglobin (Hb). In situ surface-enhanced infrared absorption spectroscopy (SEIRAS) measurements show that Hb immobilization is a sluggish process due to formation of multilayer Hb structures on the PO(3)H(2)-terminated SAMs, as revealed by ellipsometry, atomic force microscopy (AFM), and cyclic voltammetry (CV). In the multilayered Hb film, the innermost Hb molecules can directly exchange electrons with the electrode, whereas Hb beyond this layer communicates electronically with the electrode via protein-protein electron exchange. In addition, electrochemical measurements indicate that immobilization of Hb on the PO(3)H(2)-terminated SAMs is not driven by the electrostatic interaction, but likely by hydrogen-bonding interaction. The immobilized Hb molecules show excellent bioelectrocatalytic activity towards hydrogen peroxide, that is, the PO(3)H(2)-terminated SAMs are promising for construction of third-generation biosensors.