Characterization of Recombinant His-Tag Protein Immobilized onto Functionalized Gold Nanoparticles.

The recombinant polyhistidine-tagged hemoglobin I ((His)₆-rHbI) from the bivalve is an ideal biocomponent for a hydrogen sulfide (H₂S) biosensor due to its high affinity for H₂S. In this work, we immobilized (His)₆-rHbI over a surface modified with gold nanoparticles functionalized with 3-mercaptopropionic acid complexed with nickel ion. The attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) analysis of the modified-gold electrode displays amide I and amide II bands characteristic of a primarily α-helix structure verifying the presence of (His)₆-rHbI on the electrode surface. Also, X-ray photoelectron spectroscopy (XPS) results show a new peak after protein interaction corresponding to nitrogen and a calculated overlayer thickness of 5.3 nm. The functionality of the immobilized hemoprotein was established by direct current potential amperometry, using H₂S as the analyte, validating its activity after immobilization. The current response to H₂S concentrations was monitored over time giving a linear relationship from 30 to 700 nM with a corresponding sensitivity of 3.22 × 10 nA/nM. These results confirm that the analyzed gold nanostructured platform provides an efficient and strong link for polyhistidine-tag protein immobilization over gold and glassy carbon surfaces for a future biosensors development.