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In the present study, we fabricated a hemoglobin/gold nanoparticle (Hb/GNP) heterolayer immobilized on the Au micro-gap to confirm H₂O₂ detection with a signal-enhancement effect. The hemoglobin which contained the heme group catalyzed the reduction of H₂O₂. To facilitate the electron transfer between hemoglobin and Au micro-gap electrode, a gold nanoparticle was introduced. The Au micro-gap electrode that has gap size of 5 µm was fabricated by conventional photolithographic technique to locate working and counter electrodes oppositely in a single chip for the signal sensitivity and reliability. The hemoglobin was self-assembled onto the Au surface via chemical linker 6-mercaptohexanoic acid (6-MHA). Then, the gold nanoparticles were adsorbed onto hemoglobin/6-MHA heterolayers by the layer-by-layer (LbL) method. The fabrication of the Hb/GNP heterolayer was confirmed by atomic force microscopy (AFM) and surface-enhanced Raman spectroscopy (SERS). The redox property and H₂O₂ detection of Hb/GNP on the micro-gap electrode was investigated by a cyclic voltammetry (CV) experiment. Taken together, the present results show that the electrochemical signal-enhancement effect of a hemoglobin/nanoparticle heterolayer was well confirmed on the micro-scale electrode for biosensor applications.

Investigation of Hemoglobin/Gold Nanoparticle Heterolayer on Micro-Gap for Electrochemical Biosensor Application