Abstract
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.
Highlights
In the field of nanobiotechnology, the electrochemical study of biomolecules plays an important role in understanding the electron transfer mechanism of living organisms [1,2]
Within a living organism H2 O2 is a reactive oxygen by-product which related with key elements as a regulating diverse biological stress
The oxidative stress related with H2 O2 has been linked to cytotoxic effects and immune cell activation, as well as intracellular thermogenesis [12]
Summary
In the field of nanobiotechnology, the electrochemical study of biomolecules plays an important role in understanding the electron transfer mechanism of living organisms [1,2]. An electrochemical-based biosensor composed of protein can be a very effective tool to characterize a target analyte [5]. Sensors 2016, 16, 660 types of electrochemical-based biosensor composed of protein such as antibody and enzyme have been reported for environmental, biomedical, and food industry applications [6,7,8,9]. The oxidative stress related with H2 O2 has been linked to cytotoxic effects and immune cell activation, as well as intracellular thermogenesis [12]. For these reasons the detection of H2 O2 at low concentration is considered to be an important analyte. Various enzymes such as peroxidases, lipoxygenase, and catalases are used to detect H2 O2 for biosensor applications [13,14,15]
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