Cyclic voltammetry, square wave voltammetry, electrochemical impedance spectroscopy and colorimetric method for hydrogen peroxide detection based on chitosan/silver nanocomposite

2.1 Chemicals and reagents

Chitosan (CS, MW = 400,000 g mol⁻¹, degree of acetylation DA = 70%) was prepared by deacetylation of chitin (Tran et al., 2010). H₂O₂, phosphate buffered saline (PBS), glucose, ascorbic acid and galactose were purchased from Sigma Aldrich. All chemicals were of analytical grade. Glassy carbon electrodes (GCE, 3 mm diameter, S = 0.07 cm⁻²) were purchased from BAS Inc (Japan).

2.2 Synthesis of CS/AgNPs

CS/AgNPs were synthesized following (Tran et al., 2010) with minor modifications. 25 mL of a fresh solution of 0.1 M AgNO₃ was added into 100 mL of 1% w/w CS dissolved in 1% w/w acetic acid/water mixture. This colorless solution was heated to 90 °C for 6 h to obtain an orange-yellow CS/AgNPs colloid, then cooled down to room temperature and centrifuged at 10,000 rpm for 30 min to eliminate the biggest CS/AgNPs particles. The supernatant constitutes the CS/AgNPs stock solution with a AgNPs concentration of 2.16 g L⁻¹. Before use, 10 mL of stock solution was diluted in 90 mL of deionized (DI) water.

2.3 Immobilization of CS/AgNPs on GCE

Glassy carbon electrodes (GCEs) were first polished on wet silicon carbide paper using 1 μm and 0.05 μm Al₂O₃ powders sequentially, and then washed with DI water followed by ethanol for 2 min. GCEs were afterward modified with chitosan/silver nanoparticles (CS/AgNPs) hybrid by drop-casting: a 5 μL drop of diluted CS/AgNPs solution was casted onto the GCE surface and allowed to dry at ambient temperature.

2.4 Electrochemical measurements

All electrochemical experiments were performed using a PGSTAT30 potentiostat (Metrohm Autolab B.V.) coupled with GPES software. A conventional three electrode cell configuration was employed. CS/AgNPs-modified GCEs were used as working electrodes, while a saturated Ag/AgCl electrode (Radiometer Analytical SAS, France) and a platinum wire served as reference and counter electrodes, respectively. Prior to electrochemical measurements, solutions were deoxygenated by purging with pure argon. Cyclic voltammetry (CV) and square wave voltammetry (SWV) were used for electrochemical measurements. For CV, the potential was scanned from −0.1 V to −1 V (vs. SCE) at a scan rate of 50 mV s⁻¹ in 0.1 M degassed PBS. For SWV, the following parameters were used: pulse height 50 mV, pulse width 50 ms, scan increment 2 mV, and frequency 12.5 Hz.

EIS was performed using an Autolab PGSTAT30 equipped with the FRA module. Impedance spectra were recorded in 0.1 M PBS buffer at room temperature at a fixed potential within a frequency range from 10 kHz to 100 mHz with a perturbation amplitude of 10 mV. As a pretreatment before each experiment, a constant potential corresponding to the one used for EIS was imposed, for 120 s.

All electrochemical experiments were performed at 25 °C under stirring. Electrochemical signals (CV, SWV, EIS) of CS/AgNPs-modified GC electrodes were first recorded in pure PBS, and then H₂O₂ was added and let to react for 10 s before another electrochemical measurement was done.

2.5 Colorimetric measurements

First, the UV-Vis spectrum of 3 mL of the diluted CS/AgNPs solution was recorded. Then, 50 μL of the H₂O₂ solution was added into the quartz cuvette. The solution was stirred for 10 s and then a UV-Vis spectrum was recorded. The optical density at 425 nm (OD₄₂₅) of the CS/AgNPs solution before and after addition of various H₂O₂ quantities was used to draw a calibration curve, i.e. ΔA/A₀ vs. [H₂O₂] (here, ΔA = A₀ − A_C where A₀ and A_C are OD₄₂₅ of the CS/AgNPs solution before and after H₂O₂ addition, respectively).

3.1 Characterization of CS/AgNPs and surface morphology of CS/AgNPs-modified GCE

After heating at 90 °C for 6 h, the AgNO₃/chitosan mixture turned dark yellow (Fig. SI.1), indicating the formation of AgNPs into the chitosan matrix. As shown by the UV-vis spectra of CS/AgNPs (Fig. 1a, curve i), the adsorption band at 425 nm is attributed to the characteristic surface plasmon absorption of AgNPs, while no absorption is observed for the control sample (chitosan without AgNO₃ in the mixture), where no AgNPs are formed (Fig. 1a, curve ii). TEM micrographs of CS/AgNPs (Fig. 1b) show that the AgNPs have a spherical shape, a smooth surface morphology and particle sizes from 5 nm to 20 nm. No aggregation of AgNPs was evidenced, which demonstrates the stabilizing role of chitosan. A diffractogram of CS/AgNPs is shown in Fig. 1b (insert), which evidences the typical diffraction planes (1 1 1), (2 0 0), (2 2 0) of the fcc lattice of AgNPs. The diffractogram does not exhibit any diffraction peak for CS only. Using the Scherrer’s formula, crystalline size D was calculated to be in the range of 10–15 nm, consistent with the particle size evidenced on the TEM picture (Fig. 1a).

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Figure 1

(a) UV-Vis spectrum of CS/AgNPs obtained as described in the text; (b) TEM of the corresponding CS/AgNPs (insert: XRD of CS/AgNPs); (c) AFM of CS-modified GCE and (d) CS/AgNPs-modified GCE.

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The morphologies of CS-modified and CS/AgNPs-modified GCE were characterized by AFM (Fig. 1c and d, respectively). Fig. 1c shows a flat and homogeneous layer of CS, whereas Fig. 1d shows small dots corresponding to AgNPs. Fig. SI.2 shows the height profile of CS-modified and CS/AgNPs-modified GCE, consistent with the TEM picture. Fig. 1d also shows that AgNPs are evenly dispersed in the CS matrix.

The results confirmed the successful preparation of the CS/AgNPs layer. Chitosan plays the role of binder between AgNPs and the electrode surface to prevent their release, so that CS/AgNPs films can be stored for a long period and be stable in aqueous solutions.

3.2 Spectrometric assay for hydrogen peroxide detection

It is clearly shown that in situ growth of AgNPs in the chitosan matrix results in a strong absorption band at 425 nm (Fig. 2A, curve a), characteristic of surface plasmon absorption, responsible for the yellowish color. An obvious color fading was observed in the presence of H₂O₂, more pronounced for increased H₂O₂ concentration (Fig. 2B, insert). This behavior provides a potential for quantitative detection of H₂O₂. Fig. 2A shows the UV-vis absorption spectra of a CS/AgNPs solution in the presence of various amounts of H₂O₂. The color fading was attributed to the oxidation of AgNPs in the presence of H₂O₂, the standard potential of Ag⁺/Ag being lower than that of H₂O₂/H₂O (E⁰_Ag+/Ag = 0.8 V < E⁰_H2O2/H2O = 1.77 V) in water at pH = 7. This reaction is described by Eq. (1) below. The detection mechanism is illustrated in Fig. 2C.

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Figure 2

(A) UV-vis absorption spectra of CS/AgNPs hybrid solution in the presence of 0 (curve a), 100, 200, 300, 400, 500, 600, 700, 750, 800, 850, 900, 950, 1000 and 1200 μM H₂O₂ and corresponding calibration curve (B) Plotting ΔA ∗ 100/A₀ versus H₂O₂ concentration (ΔA = A₀ − A_c with the following: A₀ the absorbance at 425 nm without H₂O₂, and A_c the absorbance after adding H₂O₂ (insert: photographs of CS/AgNPs solutions in the presence of (a–h) 0, 200, 400, 600, 800, 900, 1000 and 1200 μM H₂O₂, respectively); (C) Possible detection mechanism of H₂O₂ by AgNPs using absorbance measurements at 425 nm.

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The reaction rate of Eq. (1) is very high and equilibrium constant of 10^32.88; therefore, the concentration of Ag⁰ in the chitosan matrix ((CS)-Ag⁰ or CS-AgNPs) is depleted, which explain the fading of the CS/AgNPs solution when H₂O₂ is added. Therefore, the H₂O₂ concentration can be quantified by monitoring the decrease in the AgNPs surface plasmon resonance at 425 nm. A linear calibration was obtained by plotting 100 ∗ (A₀ − A_c)/A₀ versus H₂O₂ concentration (A₀ is the absorbance at 425 nm without H₂O₂ and A_c the absorbance after adding H₂O₂) within a concentration range from 0 to 1200 μM (ordinate at origin = 3.7 ± 1.1; slope = 0.066 ± 0.002, R² = 0.9927). The detection limit was estimated around 10 μM (Fig. 2B). For immediate and qualitative detection, this reaction can also be monitored by naked eyes. Our colorimetric strategy based on the peroxidase-like property of CS/AgNPs is comparable to the other methods reported in the recently published articles (Zhang et al., 2014; Wang et al., 2015; Chen et al., 2013; Ding et al., 2016; Li et al., 2016; Ge et al., 2015) (Table 1).

The UV-vis absorption spectra of a CS/AgNPs solution in the presence of glucose, galactose and ascorbic acid at various concentrations are shown in Fig. SI.3. Only a small decrease in absorbance at 425 nm at high concentrations from 500 μM to 1 mM of glucose, galactose and ascorbic acid can be observed. These results demonstrate the good selectivity of out colorimetric sensor for hydrogen peroxide.

3.3 Electrochemical detection of hydrogen peroxide

3.3.1

3.3.1 Detection of H₂O₂ by cyclic voltammetry and square wave voltammetry

CVs of CS/AgNPs/GCE in PBS are shown in Fig. 3A. For Ar-saturated PBS without H₂O₂, no reduction peak was observed (Fig. 3A, curve a). On the contrary, when H₂O₂ was added, an increase in the reduction current at −0.7 V vs. SCE was observed, attributed to the electrocatalytic behavior of AgNPs to reduce H₂O₂ into water and dioxygen, as illustrated by Eq. (2); at a sufficiently reducing potential, dioxygen is in turn reduced into H₂O₂ on the electrode, as previously reported (Zhao et al., 2009; Šljukić et al., 2005; He et al., 2012; Flätgen et al., 1999; Wang et al., 2013) (Eq. (3)).

(2)

$${\text{H}}_2{\text{O}}_2\stackrel{\text{CS}/\text{AgNPs}}{\to }{\text{H}}_2\text{O}+\frac{1}{2}{\text{O}}_2$$

(3)

$$\frac{1}{2}{\text{O}}_2+2{\text{e}}^{-}+{\text{H}}_2\text{O}\stackrel{\text{Electrode}}{\to }{\text{H}}_2{\text{O}}_2$$

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Figure 3

(A) Cyclic voltammograms of CS/AgNPs-modified GCE as a function of H₂O₂ concentration from 0 to 1500 μM; (B) Cyclic voltammograms of CS/AgNPs-modified GCE in PBS in the presence of (a–c) 500 μM of Glucose (Glu), Galactose (Gal) and Ascorbic acid (AA), respectively; and (d) after adding 500 μM H₂O₂ in the same solution; (C) Possible detection mechanism of H₂O₂ by AgNPs using electrochemical method. All experiments were performed in Ar-saturated phosphate buffer (pH = 7.0) at a scan rate of 50 mV s⁻¹. (C) Possible detection mechanism of H₂O₂ by AgNPs using cyclic voltammetry.

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CVs performed on the same electrode but without H₂O₂ did not present any peak reduction (Fig. 3B, curve a). Interferences were investigated; CVs stayed unchanged by adding 500 μM glucose (Glu) (Fig. 3B, curve b), galactose (Gal) (Fig. 3B, curve c) or ascorbic acid (AA) (Fig. 3B, curve d). Conversely, when 500 μM H₂O₂ was added, a strong reducing peak at −0.7 V was observed on the CV (Fig. 3B, curve e). These results clearly demonstrate that CS/AgNPs have specific catalytic behavior to reduce H₂O₂.

Square wave voltammetry (SWV) was also used. As shown in Fig. 4A, the peak current showed a linear relationship over a hydrogen peroxide concentration range from 100 μM to 1 mM; I_peak (μA) = −500 [H₂O₂] − 179 (R² = 0.9973). Fig. 4B, curve (i), shows the calibration curve obtained for H₂O₂; the detection limit was estimated around 50 μM. No dependency of the current with galactose (Gal), glucose (Glu) and ascorbic acid (AA) was identified, which demonstrates the specificity of the sensor for H₂O₂ (see Fig. SI.3 for the corresponding SWV spectra).

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Figure 4

(A) SWV of a GCE modified by chitosan/silver nanoparticles (CS/AgNPs) hybrid recorded in Ar-saturated PBS (pH = 7.4, amplitude 25 mV, step height 5 mV, frequency 50 Hz) with various hydrogen peroxide concentrations; (B) Dependence of the reduction current intensity vs concentration of (i) H₂O₂; (ii) Galactose (Gal); (iii) Glucose (Glu); and (iv) Ascorbic Acid (AA).

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3.3.2

3.3.2 Detection of H₂O₂ by electrochemical impedance spectroscopy

Fig. 5A displays Nyquist plots acquired from a CS/AgNPs/GCE electrode before (a) and 30 s after (b−f) hydrogen peroxide (20 μM to 1.5 mM) was added in PBS. The impedance data were fitted with a Randles equivalent circuit (Fig. 5A, insert) in which the interfacial capacitance (C_dl) is in parallel with the diffusion impedance (W) and the charge transfer resistance (R_ct). The fitting EIS data are given in Table SI.1. The double-layer being not a pure capacitor, we modeled it with a constant phase element. Y₀ represents the capacitance C_dl and the parameter n determines the extent of the deviation from the pure capacitor model. When n = 1, the C_dl represents an ideal capacitor; for n = 0, it is a pure resistor. Here, n stays close to one. Without H₂O₂ in the medium, the resistivity of the film is larger than when H₂O₂ is present (Fig. 5A). The data reported in Table SI.1 clearly show that R_ct decreased from 3940 to 694 Ω as the H₂O₂ concentration increased. Fig. 5B shows the linear relationship of ΔR_ct (ΔR = R₀ − R, where R₀ and R are the electron transfer resistances before and after hydrogen peroxide addition, respectively) with H₂O₂ concentration. The linear fit of these data gives R_ct (kΩ) = (55.3 ± 0.7) − (15.3 ± 0.3) × log [H₂O₂] (R² = 0.9976). The limit of detection was estimated around 5 μM.

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Figure 5

(A) Nyquist plot of CS/AgNPs/GCE in PBS after adding different H₂O₂ concentrations. All measurements were performed in 0.1 M PBS solution, a frequency range from 0.1 Hz to 100 kHz, E = −0.5 V. Inset: Randles equivalent circuit used for data fitting; (B) Relationship between the R_ct and H₂O₂ concentration.

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