Bio-Electroanalysis Performance of Heme Redox-Center for π- π Interaction Bonding of a Methylene Blue-Graphene Modified Electrode

Abstract

Hemeprotein detection has motivated extensive research on the direct reaction of a heme molecule and a redox dye. The present study used methylene blue as both donor and acceptor for a redox reaction. First, the solid phases of methylene blue (MB) and graphene (GP) formed a π-π interaction bond at the aromatic rings. The conductivity of GP was better than that of carbon in a carbon electrode (CE). Then, the working CE was modified using strong adsorption of MB/GP on the electrode surface. The surface of the electrode was investigated using a modified and an unmodified electrode. The electrode's properties were studied using voltammograms of redox couple K₃[Fe(CN)₆]^3-/4-. Its reaction was used to find the active area of the modified electrode, which was 1.76 times bigger than that of the unmodified electrode. The surface coverage values of the modified and unmodified electrodes were 8.17 × 10^-6 and 1.53 × 10^-5 mol/cm², respectively. This research also studied the application of hemeprotein detection. Hemoglobin (Hb), myoglobin (Mb), and cytochrome c (Cyt. C) were studied by the reaction of Fe (III/II) at the heme-redox center. The electrocatalytic reaction between MB/GP and hemeproteins produced an anodic peak at 0.35 V for Hb, Mb, and Cyt. C. This nanohybrid film enhanced electron transfer between protein molecules and the modified carbon electrode. The amperometric measurements show that the limit of detection was 0.2 µM, 0.3 µM, and 0.1 µM for Hb, Mb, and Cyt. C, respectively. The measurement spanned a linear range of 0.2 µM to 5 µM, 0.3 µM to 5 µM, and 0.1 µM to 0.7 µM for Hb, Mb, and Cyt. C, respectively. Hb showed the lowest sensitivity compared with Mb and Cyt. C due to the role of steric hindrance in the hemeprotein specificity structure. This study offers a simple and efficient fabrication platform for electrochemical sensors for hemeproteins. When compared to other complex immobilization processes, the fabrication method for this sensor has many benefits, including no need for special chemicals and easy preparation and electrode modification-both of which are crucial for the development of electrochemical sensing devices.

Keywords: electrochemical sensor; graphene; heme; methylene blue; modified electrode.

Figure 1

A normal heme complex (Fe protoporphyrin IX) showing the attachment and release of an oxygen molecule.

Figure 2

SEM images of unmodified SPCE (a,b), modified MP/GP nanohybrids on SPCE (c,d), and modified MP/GP nanohybrids on SPCE that was scanned by CV for 100 cycles (e,f).

Figure 3

The XPS spectra of MB/GP (a) C 1s, (b) N 1s, (c) S 2p, MB/GP at 10th cycle of CV scan (d) C 1s, (e) N 1s, (f) S 2p, and GP (g) C 1s.

Figure 4

(a) UV spectra of MB (red), GP (green), and MB/GP nanohybrids (blue) in DI water. (b) Particle sizes of GP (green) and MB/GP nanohybrids (blue) measured by DLS. (c) Zeta potential of MB (red), GP (green), and MB/GP (blue) nanohybrids measured by ELS.

Figure 5

FT-IR spectra of GP (bottom), MB (middle), and MB/GP (top) in DI water (a) at 500–5000 cm⁻¹ and (b) at 600–2000 cm⁻¹. FT-IR spectra of SPCE (bottom), 1 μL of Mb/GP (middle), and 4 μL of Mb/GP (top) modified on SPCE (c) at 500–4000 cm⁻¹ and (d) at 1000–2000 cm⁻¹.

Figure 6

Cyclic voltammogram of the modified and unmodified electrode in 5 mM K₃Fe(CN)₆ and adding 0.1 M KCl scanned at 20 mV/s.

Figure 7

Measurement of oxidation peak current, for unmodified and modified electrodes, as a function of the applied scan rate. The measurements were obtained using a solution of 5 mM K₃[Fe(CN)₆] and 0.1 M KCl. This graph was used for the calculation of the total active area.

Figure 8

Measurements of cathodic peak current for unmodified and modified electrodes as a function of the applied scan rate. The measurements were obtained using a solution of 5 mM K₃[Fe(CN)₆] and 0.1 M KCl. This graph is used for the calculation of surface coverage.

Figure 9

(a) Cyclic voltammograms of the MB/GB-modified electrode SPCE on scanning 100 cycles in 0.1 M PBS pH 7.0 at a scan rate of 20 mV/s. (b) Plots of the percentage decrease in redox current response from the initial response over n cycles (n = 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100).

Figure 10

Cyclic voltammograms (10th scan cycles) at 20 mV/s in pH 7 buffers of (black) an MB/GP-modified electrode without hemeproteins and when adding 0.5 μM of (dark green) Hb, (light green) Mb, and (red) Cyt. C.

Figure 11

Catalytic current ratio (I_n/I_o) of hemeproteins versus t^1/2 where (red) Hb, (black) Mb, and (blue) Cyt. C reacted at the MB/GP film electrode in a pH 7 buffer solution.

Figure 12

Electrocatalytic reduction of hemeproteins at MB/GP modified electrode.

Figure 13

The current response of Hb (red), Mb (black), and Cyt. C (blue) vs. concentration of hemeproteins. The scan rate was used at 20 mV/s. Inset illustrates the LSV of the MB/GP-modified electrode in 0.1 M buffer solution containing 0.1 M KCl in the presence of 0.5 μM of each of Hb (red), Mb (black), and Cyt. C (blue).