3.1. Morphological and electrochemical characterization of DCell-CB//Ag/δ-FeOOH
Figure 1A presents the SEM image of the DCell-CB//Ag/δ-FeOOH. The image reveals a well-coated mixture of CB and Ag/δ-FeOOH on the working electrode, displaying a heterogeneous surface with porous topography. Notably, the modified electrode exhibits increased surface irregularities compared to the unmodified DCell previously reported[
19], which can enhance conductivity and the analytical response. EDS analysis in
Figure 1B shows C, Ag, and Fe, validating the successful modification of DCell’s working electrode.
EIS and CV were used to evaluate the performance of the modified and unmodified DCell in a ferri-ferro cyanide solution.
Figure 2A shows EIS spectra for DCell (a), DCell modified with Ag/δ-FeOOH (b), and DCell modified with CB//Ag/δ-FeOOH (c). The bare DCell exhibited an Rct of 1.4 × 10
4 Ω, which significantly decreased after modification with Ag/δ-FeOOH (389 Ω) and CB//Ag/δ-FeOOH (130 Ω). These findings indicate that Ag/δ-FeOOH and CB//Ag/δ-FeOOH play a crucial role as promoters of electron transfer in the ferri/ferro-cyanide redox system at the working electrode surface. The CV results in
Figure 2B align well with the EIS findings. DCell-CB//Ag/δ-FeOOH demonstrated better current response (I
oxi = 38.10 µA and I
red = -36.31 µA, ΔEp = 110 mV) compared to DCell-Ag/δ-FeOOH (I
oxi = 18.83 µA and I
red = -23.22 µA, ΔEp = 110 mV) and bare DCell (I
oxi = 7.01 µA and I
red = -7.01 µA, ΔEp = 270 mV). The ΔEp value of more than 58 mV (the expected value for one-electron Nernstian-precess) suggests a quasi-reversible electrochemical response. The significant improvement observed can be attributed to the specific properties of CB, such as high surface area and excellent conductivity[
13]. Previous studies have reported that screen-printed electrodes modified with CB by drop-casting exhibit lower peak-to-peak separation and higher intensity peak current in a ferri-ferro cyanide solution, aligning with our findings[
13,
21].
Furthermore, the magnitudes of the voltammetric peak currents plotted against the square root of the applied scan rate (υ
1/2) ranging from 10 – 200 mV s
-1, exhibited a linear relationship for the bare DCell, DCell-Ag/δ-FeOOH, and DCell-CB//Ag/δ-FeOOH, indicating a diffusion-controlled process at the electrode surface (Figure SI 1). The apparent heterogeneous electron-transfer rate constant, Κ
0app, for the quasi-reversible system was determined using the Nicholson method[22-24]. The rate constants calculated in the ferri-ferro cyanide solution were 3.21 x 10
-4±9.36 x 10
-6 cm s
-1, 1.66 x 10
-3±6.10 x 10
-5 cm s
-1, and 3.06 x 10
-3±1.10 x 10
-4 cm s
-1, for the bare Dcell, DCell-Ag/δ-FeOOH, and DCell-CB//Ag/δ-FeOOH, respectively. These results demonstrate that DCell-CB//Ag/δ-FeOOH exhibits superior performance, with a rate constant almost twice that of DCell-Ag/δ-FeOOH. The slower electron transfer rate observed for the bare DCell in the ferri-ferro cyanide solution is consistent with the higher Rct value obtained from the EIS data. Our experimental findings indicate significant improvements in the electrochemical performance of DCell-CB//Ag/δ-FeOOH using CB, highlighting the advantages of incorporating CB to enhance the magnitude of Κ
0app [
13].
Additionally, the working electrodes areas for bare DCell, DCell-Ag/δ-FeOOH, and DCell-CB//Ag/δ-FeOOH were determined experimentally using the Randles-Sevčík equation[
19,
25]. The electroactive areas, evaluated using the ferri-ferro cyanide solution, were found to be 0.021±0.002 cm
2, 0.190±0.010 cm
2, and 0.239±0.009 cm
2, respectively. The DCell modified with CB and Ag/δ-FeOOH exhibited a larger electroactive area, providing more sites for electrochemical reactions, consistent with the superior electrochemical behavior observed for DCell-CB//Ag/δ-FeOOH.
3.2. Electrochemical behavior of H2O2 in DCell-CB//Ag/δ-FeOOH
Cyclic voltammetry (CV) was utilized to inquire the electrochemical characteristics of DCell-CB//Ag/δ-FeOOH for H
2O
2 reduction. The voltammograms of DCell (a), DCell – CB (b), DCell - CB//δ-FeOOH (c), and DCell – CB//Ag/δ-FeOOH (d) in N
2-saturated 0.2 M PBS at pH 7.2 with 500 μM of H
2O
2, recorded at a scan rate of 100 mV s
-1, are presented in
Figure 3A. Curves a and b show no distinct response to H
2O
2 in the -1.0 to 1.0 V range for DCell and DCell-CB, respectively. Curve c, corresponding to DCell - CB//δ-FeOOH, demonstrates an anodic current of 16µA, and a cathodic current of 19 µA at -0.15V and -0.75 V, respectively, in the presence of H
2O
2, indicating the redox process of H
2O
2 on δ-FeOOH[
7]. Curve d, representing the electrochemical profile of DCell-CB//Ag/δ-FeOOH in H
2O
2 solution, displays an anodic current of 134.0 µA and 39.4 µA at 0.08V and –0.15 V, respectively, as well as a cathodic current of 100.0 µA and 57.0 µA at -0.3V and -0.75V, respectively.
The sharp oxidation peak at 0.08 V can be assigned to the oxidation of silver nanoparticles coverage on δ-FeOOH, and the reduction peak at -0.3 V may arise from the reduction of silver halides or silver oxide formed during the forward scan on δ-FeOOH. Similar observations were reported by Plowman et al. for gold-silver alloy nanoparticles in KCl solution, where an oxidation peak of silver nanoparticles at 0.14 V and a reduction peak at 0.01 V in the reverse scan were attributed to the reduction of silver chloride formed in the forward scan[
26]. The peak currents at -0.15 V and -0.75V can be attributed to the redox process of H
2O
2 on CB//Ag/δ-FeOOH.
Figure 3B illustrates the voltammograms of DCell–Ag/δ-FeOOH (a) and DCell–CB//Ag/δ-FeOOH (b) along with their respective background voltammograms in N
2-saturated 0.2 M PBS at pH 7.2, with and without 500 μM H
2O
2, recorded at a scan rate of 100 mV s
-1. Notably, CB on the working electrode catalyzes the redox process of silver on δ-FeOOH in PBS, leading to anodic and cathodic peaks at 0.08 V and -0.3 V, respectively, which are absent in the voltammogram of DCell–Ag/δ-FeOOH. Furthermore, a 33% increase in the cathodic peak current at -0.75 V in 500 μM H
2O
2/ PBS solution is observed for DCell–CB//Ag/δ-FeOOH compared to DCell–Ag/δ-FeOOH. These results can be assigned to the superior electrochemical behavior and higher electroactive area of the working electrode in DCell-CB//Ag/ δ-FeOOH. The enhancement of electroanalytical performance for H
2O
2 observed in working electrodes modified with CB has been reported in previous studies, indicating improved electrochemical activity due to the high conductivity and large specific surface area provided by CB[
27,
28].
After confirming that DCell-CB//Ag/δ-FeOOH exhibited the best electrochemical characteristic for H
2O
2 detection, we investigated the peak potential of cyclic voltammetry for detecting H
2O
2 at different concentrations. Amperometry measurements were conducted in N
2-saturated 0.2 M PBS at pH 7.2, under magnetic agitation, using H
2O
2 concentrations of 100, 500, and 1000 µM. As shown in
Figure 4, an increase in current is observed at -0.75 V with increasing H
2O
2 concentration. However, no significant changes in current were observed at -0.3 V, -0.15 V, and 0.08V, indicating the absence of an electrochemical process for H
2O
2 at those potentials. Consequently, these potentials were not effective for electroanalysis. Considering the maximum current achieved at -0.75 V, we selected -0.75 V as the potential for H
2O
2 detection using DCell-CB//Ag/ δ-FeOOH.
Figure 5A depicts the amperometry response of DCell-CB//Ag/δ-FeOOH at -0.75V for H
2O
2 detection in N
2-saturated 0.2 M PBS (pH 7.2) under continuous stirring. The response of DCell-CB//Ag/δ-FeOOH for H
2O
2 reduction is rapid, reaching a steady-state signal quickly upon H
2O
2 addition. As shown in
Figure 5B, the current changes linearly with increasing H
2O
2 concentration from 70 µM to 6000 µM. Typically, the concentration of H
2O
2 in a human cell is less than 10 nM, and in human plasma, it ranges from 1 to 5 µM. However, during inflammation, the H
2O
2 concentration in plasma can exceed 50 μM[
29,
30]. In our experiments, the limit of detection (LOD) was calculated to be 22 μM (S/N = 3), which reliably covers the concentration range in plasma during inflammation. The sensitivity of the method was 214 μA mM
-1 cm
-2. Our results demonstrate that the proposed electroanalytical method exhibits comparable features to those previously reported (see Table SI1).
3.4. Repeatability, Interference Studies, and Biological Sample Analysis
We assessed the performance of DCell-CB//Ag/δ-FeOOH in generating consistent electrochemical results in PBS containing H
2O
2. The estimated relative standard deviation (RSD) for five independent DCell-CB//Ag/δ-FeOOH measurements was approximately 4.78%, demonstrating the reliability of the process fabrication. Furthermore, we evaluated the interference effects of common biomolecules in physiologic samples, such as ascorbic acid, uric acid, and dopamine[
31,
32], on DCell-CB//Ag/δ-FeOOH. Electrochemical measurements (
Figure 6) were carried out in N
2-saturated 0.2 M PBS (pH 7.2) at -0.75V under continuous stirring. DCell-CB//Ag/δ-FeOOH displayed no amperometric signal in 100 μM of dopamine, uric acid, or ascorbic acid. However, significant amperometric responses were observed upon adding 100 μM of H
2O
2 in the initial and final steps. Importantly, there was no change in the current signal of H
2O
2 after introducing interfering agents, indicating excellent selectivity of the proposed sensor. These characteristics, coupled with the reliable response of the DCell-CB//Ag/δ-FeOOH, make it suitable for detecting H
2O
2 levels in biological samples.
Fetal bovine serum (FBS) is commonly added as a supplement to the basal medium in cell culture. Cells can release H
2O
2 when stimulated in a cell culture medium containing 10% fetal bovine serum[
33,
34]. Therefore, for application in biological samples, it assesses its performance in a solution containing FBS. To this end, we applied DCell-CB//Ag/δ-FeOOH to determine H
2O
2 levels in a 10% fetal bovine serum disinfected solution diluted in 0.2 M PBS (pH 7.2). The samples were spiked with different concentrations of H
2O
2 standard solution (
Table 1). The calculated H
2O
2 recoveries fell within the range of 92 to 103%. These results highlight the potential of DCell-CB//Ag/δ-FeOOH for monitoring H
2O
2 in biological samples.