Nanostructured ZnO in a Metglas/ZnO/Hemoglobin Modified Electrode to Detect the Oxidation of the Hemoglobin Simultaneously by Cyclic Voltammetry and Magnetoelastic Resonance

Abstract

In the present work, a nanostructured ZnO layer was synthesized onto a Metglas magnetoelastic ribbon to immobilize hemoglobin (Hb) on it and study the Hb's electrochemical behavior towards hydrogen peroxide. Hb oxidation by H₂O₂ was monitored simultaneously by two different techniques: Cyclic Voltammetry (CV) and Magnetoelastic Resonance (MR). The Metglas/ZnO/Hb system was simultaneously used as a working electrode for the CV scans and as a magnetoelastic sensor excited by external coils, which drive it to resonance and interrogate it. The ZnO nanoparticles for the ZnO layer were grown hydrothermally and fully characterized by X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM) and photoluminescence (PL). Additionally, the ZnO layer's elastic modulus was measured using a new method, which makes use of the Metglas substrate. For the detection experiments, the electrochemical cell was performed with a glass vial, where the three electrodes (working, counter and reference) were immersed into PBS (Phosphate Buffer Solution) solution and small H₂O₂ drops were added, one at a time. CV scans were taken every 30 s and 5 min after the addition of each drop and meanwhile a magnetoelastic measurement was taken by the external coils. The CV plots reveal direct electrochemical behavior of Hb and display good electrocatalytic response to the reduction of H₂O₂. The measured catalysis currents increase linearly with the H₂O₂ concentration in a wide range of 25-350 μM with a correlation coefficient 0.99. The detection limit is 25-50 μM. Moreover, the Metglas/ZnO/Hb electrode displays rapid response (30 s) to H₂O₂, and exhibits good stability and reproducibility of the measurements. On the other hand, the magnetoelastic measurements show a small linear mass increase versus the H₂O₂ concentration with a slope of 152 ng/μM, which is probably due to H₂O₂ adsorption in ZnO during the electrochemical reaction. No such effects were detected during the control experiment when only PBS solution was present for a long time.

Keywords: Hemoglobin; Metglas; ZnO nanostructures; characterizations; magnetoelastic resonance; sensors; synthesis.

Figure 1

Ribbon diagram of Bovine Hemoglobin showing the position of the four hemes (blue) taken from the RCSB Protein Data Bank and plotted on BIOvia Discovery Studio Visualizer.

Figure 2

The detection experiment consisted of three electrodes, the sensor working electrode (WE), the Pt counter electrode (CE) and the Ag/AgCl reference electrode (RE), all immersed in a PBS buffer solution inside a glass vial on which a coil was wrapped externally.

Figure 3

XRD patterns: (a) Synthetic ZnO nanoparticles and the standard ZnO wurtzite structure; and (b) synthetic ZnO nanoparticles (NPs), a clean strip of Metglas and the ZnO coated Metglas strip.

Figure 4

SEM images: (a) high magnification showing individual ZnO nanocrystals; and (b) a surface view of the obtained ZnO layer, after six depositions. Insert in image (a) shows particle size distribution.

Figure 5

PL spectrum recorded by using an excitation wavelength at 325 nm over the ZnO film deposited onto the Metglas 2826MB strip.

Figure 6

Schematic representation of the Metglas 2826MB and ZnO layers layout in our resonant devices.

Figure 7

Total Young’s modulus (E″) measured as a function of the ratio h′/h (width of the deposited ZnO layer/width of Metglas 2826MB strip).

Figure 8

Reaction scheme for the direct reduction and oxidation of the immobilized hemes of Hb and the electrocatalytic reduction of H₂O₂ on the sensor (created on Chemdraw).

Figure 9

Cyclic voltammetry curves of different electrodes at a scan rate of 0.1 V/s: (a) Metglas 2826MB; (b) Metglas/ZnO; (c) Metglas/ZnO/Hb; and (d) all three curves plotted together, for comparison purposes.

Figure 10

CVs of a Metglas/ZnO/Hb electrode at a scan rate of 0.1 V/s at specific time intervals (5–50 min) (no H₂O₂ present).

Figure 11

Magnetoelastic resonance measurements: (a) a typical resonance signal received when the Metglas/ZnO/Hb electrode is immersed in the PBS solution. The continuous line is a Gaussian fit to the data; and (b) resonance frequency versus time when the electrode is immersed in PBS solution.

Figure 12

(a) CVs obtained for a Metglas/ZnO/Hb electrode in PBS buffer before and after the addition of increasing amounts (50–350 μM) of H₂O₂ at a scan rate of 0.1 V/s (sensing signals measured 30 s after each addition of H₂O₂); and (b) a plot of peak current values vs. H₂O₂ concentration. Error bars were determined from repeating the measurements on the same electrode at least three times.

Figure 13

Comparison of the control peak current (circles) obtained from the CVs of a Metglas/ZnO/Hb electrode in PBS solution and the corresponding sensing current (triangles) when H₂O₂ is added in the solution.

Figure 14

Magnetoelastic resonance data of a Metglas/ZnO/Hb electrode measured 5 min after the addition of increasing aliquots of H₂O₂.

Figure 15

CVs of a Metglas/ZnO/Hb electrode at a scan rate of 0.1 V/s after the addition of three different concentrations of H₂O₂ measured after 30 s and 5 min after each addition.