Electrochemical antioxidant screening and evaluation based on guanine and chitosan immobilized MoS₂ nanosheet modified glassy carbon electrode (guanine/CS/MoS₂/GCE)

2.1 Chemicals and reagents

Guanine, chitosan, MoS₂ powder, ferrous sulfate and ethylenediaminetetraacetic acid (EDTA) were purchased from Merck KGaA (Darmstadt, Germany). Quercetin, fisetin and catechin were acquired from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). Voltammetric measurements were carried out in phosphate buffered solutions (PBS, 50 mM) as supporting electrolytes. Stock solutions of guanine (1 g L^-1) were prepared by dissolving a certain amount of guanine in NaOH (0.1 M). Stock solutions of flavonoids (quercetin, fisetin and catechin) were prepared by dissolving the solid in ethanol. The working standard solutions were prepared daily by diluting stock solutions with supporting electrolytes to certain concentration. Fe²⁺-EDTA solution was prepared daily by adding definite amount of ferrous sulfate to EDTA solution (1 mM). Fenton solution for ·OH generation was carried out in a mixture of hydrogen peroxide (20 mM) and Fe²⁺-EDTA. Ultra-pure water (18.25 MΩ·cm, 25 °C) was obtained from a Millipore system (Millipore Corporation, Milford, MA, USA). All other chemicals were of analytical grade without further purification.

2.2 Apparatus

All electrochemical measurements were performed on a CHI 660E Electrochemical Workstation (Chenhua Instrument Company of Shanghai, Shanghai, China) with a standard three electrodes system including a glassy carbon electrode (GCE, a diameter of 3 mm) as the working electrode, a platinum wire as the auxiliary electrode and an Ag/AgCl (KCl 3.0 M) electrode as the reference electrode. All the potentials obtained in this study are reported against Ag/AgCl/KCl electrode. The morphology of MoS₂ was obtained by using a scanning electron microscopy (SEM, JSM 6610Lv, JEOL, Japan) operated at 30 kV. An ultrasonic processor FS-250 (20% amplitude; Shanghai Shengxi, Shanghai, China) was used for synthesizing the MoS₂ nanosheet. All the experiments were carried out at room temperature.

Scheme 1

The preparation process of the modified electrode and the detection method for guanine damage: SWV.

2.3 Preparation of MoS2 nanosheet

The MoS₂ nanosheet was prepared by MoS₂ powder according to literature [16] with some modifications. Briefly, 300 mg of MoS₂ powders were dispersed in 100 mL of mixtures consisted of acetone and water (v:v, 89:11) to obtain MoS₂ suspension (3 mg mL^-1). Afterwards, the suspension was ultrasonically treated in iced water for 1 h using an ultrasonic processor FS-250 with pulsed ultrasonic irradiation mode (Power, 35 W; Pulse frequency: 20 s on and 10 s off) to attain MoS₂ nanosheet. The pulsed ultrasonic irradiation mode could reduce solvent heating and avoid degradation of produced MoS₂ nanosheet. Finally, the dispersion was centrifuged at 3000 rpm for 30 min (Beckman Coulter Allegra 64R, Brea, CA, USA). The supernatant was collected and dried in a vacuum oven for further use.

2.4 Preparation of the guanine/CS/MoS2/GCE

The MoS₂ suspension (0.5 mg mL^-1) was obtained by dispersing MoS₂ nanosheet in water. The MoS₂/GCE was obtained by dropping 6 μL of MoS₂ suspension on the surface of a GCE and dried at room temperature to form a MoS₂ film on the GCE. Then, chitosan solution (1.0 mg mL^-1, in 1% acetic acid) was added to a guanine solution (0.6 mg mL^-1) at a volume ratio of 1:5 (v/v), and then 6 μL of the mixture added on to the surface of MoS₂/GCE and dried at room temperature. Ultimately, we obtained a guanine/CS/MoS₂/GCE.

2.5 Antioxidant capacity assays

Hydroxyl (·OH) radicals could be produced by adding hydrogen peroxide into freshly prepared Fe²⁺-EDTA solution in PBS (50 mM, pH 3.5). Damage of guanine was performed by immersing the guanine/CS/MoS₂/GCE in Fenton solution. After a certain incubation time, the guanine/CS/MoS₂/GCE was washed with water and immersed in PBS (50 mM, pH 1.5) immediately to perform the SWV detection. The oxidation peak of guanine in SWV peaks was measured at about 1.20 V.

For antioxidant capacity assay, antioxidant (quercetin, fisetin and catechin) with a certain concentration was firstly added into freshly prepared Fenton solution. Then, the guanine/CS/MoS₂/GCE was immersed in the Fenton solution containing antioxidant. After the same procedures as mentioned before, the SWV detection of guanine/CS/MoS₂/GCE was performed as well. The oxidation peak of guanine in SWV peaks could be observed and used to calculate the antioxidant activity of sample. The antioxidant capacity of sample was calculated using the following equation:

Where I₀ is the oxidation peak current of guanine/CS/MoS₂/GCE before damage in Fenton solution, I_c is the oxidation peak current after damage in Fenton solution without sample, and I_s is the oxidation peak current after damage in Fenton solution with sample.

Ethical approval: The conducted research is not related to either human or animal use.

3.1 Characterization of MoS2 nanosheet

The morphologies of MoS₂ powder (Figure 1A) and MoS₂ nanosheet (Figure 1B) were characterized by scanning electron microscopy (SEM). It could be observed in Figure 1A that the MoS₂ powders were agglomerated by a large number of particles with 2 μm of diameter, and the particles were uniform in morphology. Figure 1B showed that the exfoliated MoS₂ nanosheet with a typical layered and wrinkled structure. The SEM characterization confirmed that MoS₂ nanosheet was successfully prepared from MoS₂ powders.

Figure 1

SEM images of MoS₂ powder (A) and MoS₂ nanosheet (B).

3.2 Electrochemical study of the guanine/CS/MoS2/GCE

Figure 2 showed the SWV results of guanine/GCE (a), guanine/CS/GCE (b) and guanine/CS/MoS₂/GCE (c) in PBS (50 mM, pH 1.5). It can be noted from the picture that an oxidation peak potential of guanine appeared at about 1.20 V. When the guanine molecules were previously mixed with chitosan and then immobilized together on the electrode, the corresponding oxidation peak current got a clear improvement (Figure 2b). It indicated that chitosan could effectively adsorb guanine to the surface of electrode. Moreover, the oxidation peak current of guanine on the guanine/CS/MoS₂/GCE is 42.6 μA, which is nearly 3 times than that on the guanine/GCE (15.2 μA). This might be due to the good adsorption performance and biocompatibility of MoS₂ nanosheet.

Figure 2

SWVs obtained at the guanine/GCE (a), guanine/CS/GCE (b) and guanine/CS/MoS₂/GCE (c) in PBS (50 mM, pH 1.5).

In order to get a better understanding about the interaction between chitosan and guanine, the geometry of chitosan and guanine was optimized with the density functional theory method (B3LYP/6-31G*). The density functional theory [17] B3LYP/6-31G* basis set was employed to optimize the guanine and chitosan without any constraints via the Gaussian software [18]. The B3LYP functional includes Becke’s three-parameter-exchange functional and Lee-Young-Parr correlation functional [19, 20, 21]. Figure 3 showed the optimized structures and the electrostatic potential surface of aromatic systems of guanine (a) and chitosan (b). Electrostatic potentials were caused by the interactions of electrons or nuclei with electrons [22, 23]. The blue area corresponded to the negative charges. The red region expressed the positive charges. As shown in Figure 3, the N₆ and N₁₁ atom in guanine had negative charges. However, the positive charges have been found in the amino unit of chitosan which was consisted well with previous experiment [24]. It indicated that chitosan and guanine could bind together well with electrostatic interaction.

Figure 3

The optimized structure of guanine (a) and chitosan (b) and electrostatic potential surface of aromatic systems calculated at B3LYP/ 6-31G*.

3.3 Effect of pH and immobilized concentration

The effect of pH value on the electrochemical response of guanine on guanine/CS/MoS₂/GCE was studied by SWV and the results were shown in Figure 4A. An insert displayed a plot of guanine electro-oxidation signals with the change of pH. The oxidation peak currents decreased gradually with the increasing of pH values. The response reached the maximum value at pH 1.5. The oxidation peak potentials obeyed the equation in the range of pH from 1.5 to 4.0 (Figure 4B), Ep = 1.288 – 0.0696 pH (R² = 0.9861). The calculated slope (69.6 mV pH^-1) was closed to the theoretical value of 59 mV indicating that direct electrooxidation of guanine is an isoelectron and isoproton process [25, 26]. Therefore, 50 mM phosphate buffered solution with pH 1.5 was used as the supporting electrolyte in all electrochemical determinations during this research.

Figure 4

(A) Effect of pH on the electrochemical response of guanine on the guanine/CS/MoS₂/GCE. Insert: relationship between oxidant peak current and pH. (B) The calibration plot of potential against pH. The experimental conditions were incubated in Fenton solution (1 mM EDTA, 2.0 mM Fe²⁺ ions and 20 mM H₂O₂) for 300 s.

The amount of modifiers on the surface of electrode is one of the important factors that influence the voltammetric response of guanine. As shown in Figure 5, the peak currents increased gradually with the increase of guanine/CS concentrations (from 0.1 to 0.6 mg mL^-1) and decreased rapidly when the concentration was higher than 0.6 mg mL^-1. This result might indicate that the thicker film of guanine/CS blocked the electron transfer between guanine and electrode. Therefore, 0.6 mg mL^-1 of guanine/CS suspension was chosen as the optimum concentration to modify the GCE.

Figure 5

Effect of immobilized concentration on the electrochemical response of guanine on the guanine/CS/MoS₂/GCE. The experimental conditions were incubated in Fenton solution (1 mM EDTA, 2.0 mM Fe²⁺ ions and 20 mM H₂O₂) for 300 s.

3.4 Optimization of electrochemical assays

Hydroxyl radicals (·OH) are part of reactive oxygen species in living systems, which are generated from the metal ion-dependent breakdown of hydrogen peroxide. The ·OH produced by Fenton’s reaction plays a major role during oxidative damage in vivo. EDTA is usually used as a chelating agent in Fenton solution to increase the solubility of Fe²⁺ ions. Meanwhile, MoS₂ nanosheet could enhance the efficiency of ·OH generation as a co-catalyst. Therefore, Fenton solution containing EDTA was used to generate ·OH for the damage of guanine on electrode in this study.

Reported studies indicated that the concentration of Fe²⁺ ions had a close relationship with the production of ·OH and affected the damage of guanine [27]. The relationship between concentration of Fe²⁺ ions and electrochemical oxidation peak current of guanine/CS/MoS₂/GCE was studied. As shown in Figure 6, it could be observed that the peak currents decreased with increasement of Fe²⁺ ions. When the concentration of Fe²⁺ ions is higher than about 2.0 mM, the change of peak current tended to gentle.

Figure 6

(A) The oxidation peak current of the guanine/CS/MoS₂/GCE after incubation for 300 s in Fenton solution (1 mM EDTA and 20 mM H₂O₂) with different concentration of Fe²⁺ ions (a-i): 0, 0.3, 0.5, 0.8, 1.0, 1.5, 2.0, 2.5 and 3.0 mM. (B) The relationship between oxidant peak current and the concentration of Fe²⁺ ions.

Incubation time could affect the damage degree of guanine. Therefore, the effect of incubation time was also investigated in this study (Figure 7). With the incubation time increased, the peak currents decreased gradually until the incubation time reached 300 s. It indicated that 300 s incubation was enough for guanine damage. Therefore, 2.0 mM Fe²⁺ ions and 300 s incubation time were chosen as experimental conditions for future studies.

Figure 7

Effect of incubation time of the guanine/CS/MoS₂/GCE with the Fenton solution (1 mM EDTA, 2.0 mM Fe²⁺ ions and 20 mM H₂O₂).

3.5 Antioxidant capacity assays

The ·OH generated by Fenton solution could attack electrode surface and damage guanine molecules. When antioxidants were added into Fenton solution, the produced ·OH was scavenged by antioxidants and correspondingly the damage of guanine was reduced. Through the electrochemical detection, a higher oxidation peak current of guanine could be observed and the antioxidant capacity could be then calculated.

As a well-known and powerful antioxidant, ascorbic acid (AA) is regarded as an important water-soluble ROS scavenger in human body [28]. In this work, AA was selected as a model antioxidant to evaluate the antioxidant capacity performance in developed electrochemical biosensor. Figure 8A showed the SWV curves of guanine/CS/MoS₂/GCE incubated for 300 s in Fenton solution (1 mM EDTA, 2.0 mM Fe²⁺ ions and 20 mM H₂O₂) with different concentrations of AA. Figure 8B showed the plots of peak currents against AA concentrations. It was noted that the presence of AA significantly affected the oxidation peak current, and the peak current increased with increasing concentration of AA. A linear relationship of peak current and AA concentration was also observed (Figure 8B) when the concentration of AA was between 0.5 and 4.0 mg L^-1. The corresponding linear function was I_p (μA) = -4.4395 C_AA – 18.2693 with a correlation coefficient of R² = 0.9959. The detection limit was calculated to be 0.33 mg L^-1 (S/N = 3). Based on these results, it could be considered the developed sensor showed good linear range and detection limit.

Figure 8

(A) The oxidation peak current of the guanine/CS/MoS₂/GCE after incubation for 300 s in Fenton solution (1 mM EDTA, 2.0 mM Fe²⁺ ions and 20 mM H₂O₂) with different concentration of AA (a-i): 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mg L^-1. (B) The calibration plot of oxidant peak current against the concentration of AA.

In order to confirm the abilities in antioxidant capacity detection and antioxidant screening of proposed sensor, the antioxidant capacities of several flavonoids were analyzed by guanine/CS/MoS₂/GCE. The concentration of antioxidant in this assay was set at 2.0 mg L^-1. Figure 9 presented the results of antioxidant capacities for three flavonoids and AA. AA displayed the highest antioxidant capacity (51.84%), then followed by quercetin (45.82%), fisetin (34.39%) and catechin (16.99%). Three investigated flavonoids showed different antioxidant capacities, which showed this sensor could be used for evaluation of samples.

Figure 9

Antioxidant screening of several antioxidant compounds using the guanine/CS/MoS₂/GCE electrode.

3.6 Reproducibility and stability tests

The reproducibility and stability of modified electrode were also evaluated by detecting AA (2.0 mg L^-1) in Fenton solution. As shown in Figure 10, Different electrodes were used for 10 times of parallel measurements (Black plots), and it showed a good reproducibility with the relative standard deviation is 2.9%. The same electrode was also tested for 10 times of parallel measurements (Red plots), and it showed a better reproducibility with the relative standard deviation of 1.2%. The modified electrode was stored at the room temperature (25°C) for a week. The current of electrode was reduced about 1.8%. The modified electrode displayed acceptable reproducibility and stability, indicating that the guanine/CS/MoS₂/GCE is suitable for antioxidant screening.

Figure 10

The reproducibility of guanine/CS/MoS₂/GCE (Black: different electrodes; Red: the same electrode) in detection of AA (2.0 mg L^-1). The experimental conditions were incubated in Fenton solution (1 mM EDTA, 2.0 mM Fe²⁺ ions and 20 mM H₂O₂) for 300 s.