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

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.

Keywords: Au micro-gap; cyclic voltammetry; electrochemical biosensor; gold nanoparticle; hemoglobin.

Figure 1

Schematic diagram of fabricated hemoglobin/gold nanoparticle heterolayer immobilized on the micro-gap for H₂O₂ detection.

Figure 2

(a) Schematic diagram of micro-gap electrode; (b) Optical image of fabricated micro-gap electrode with working chamber for H₂O₂ biosensor application; (c) Optical image of zoomed micro-gap.

Figure 3

Surface morphology investigation of (a) hemoglobin; (b) hemoglobin/gold nanoparticle on 6-mercaptohexanoic acid (6-MHA) layer; (c) Surface roughness analysis of the hemoglobin, hemoglobin/gold nanoparticle; (d) Raman spectra of hemoglobin (Brown line); (b) hemoglobin/gold nanoparticle on 6-MHA layer (Blue line).

Figure 4

Cyclic voltammogram of (a) hemoglobin (Brown line) and hemoglobin/gold nanoparticle (Purple line) immobilized on bulk Au electrode, respectively; (b) hemoglobin (Red line) and hemoglobin/gold nanoparticle (Blue line) immobilized on micro-gap Au electrode, respectively.

Figure 5

(a) Cyclic voltammogram of hemoglobin/gold nanoparticle (Blue line) immobilized on Au micro-gap electrode in 10 mM PBS (pH = 7.4) at different scan rate (mV/s) (Red line: 10 mV/s, Orange line: 20 mV/s, Yellow line: 30 mV/s, Green line: 40 mV/s, Blue line: 50 mV/s); (b) Plots of anodic and cathodic peaks currents vs. scan rates; (c) Cyclic voltammogram of hemoglobin/gold nanoparticle (Blue line) immobilized on Au micro-gap electrode containing (Red line: 0, Orange line: 10 nmol, Yellow line: 30 nmol, Green line: 50 nmol, Blue line: 100 nmol H₂O₂ at 50 mV/s); (d) Plots of anodic and cathodic peaks currents vs. addition of H₂O₂; (e) Table of current values corresponding to H₂O₂ concetrations.