Simple Electrochemical Synthesis of Polyethylenimine-Encapsulated Ag Nanoparticles from Solid AgCl Applied in Catalytic Reduction of H 2 O 2

: We report a simple and environmentally friendly synthesis of polyethylenimine (PEI)-encapsulated Ag nanoparticles (AgNPs) by a direct electroreduction of solid AgCl. The AgNPs, characterized by ﬁeld-emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), and energy dispersive X-ray spectroscopy (EDS), revealed that AgNPs diameters (100–500 nm) depended on the loading of the AgCl precursor. Using cyclic voltammetry (CV), it was conﬁrmed that the AgNPs had a catalytic e ﬀ ect on the electrochemical reduction of H 2 O 2 . The obtained AgNPs were subsequently used to construct an electrochemical H 2 O 2 sensor exhibiting a low detection limit (1.66 µ M) and a wide linear response range, with real-life tests indicating an insensitivity to common interferents and conﬁrming the potential use of the developed technique in diverse applications.


Introduction
Silver nanoparticles (AgNPs) have been intensively studied due to their physical and chemical characteristics and are now widely applied in the field of catalysis, biomedicine, and toxicity [1][2][3]. The AgNPs are manufactured by physical methods [4,5], chemical reduction [6,7], electrochemical reduction [8,9], microwave processing [10], thermal decomposition [11], and biosynthesis [12]. Among these methods, the electrochemical method has advantages in that the process is simple, the purity of silver nanoparticles is high, and size control is easy. Moreover, metal particles can be produced by controlling the voltage or current, and an additional reducing agent is not required. In general, except for a few physical methods [4,5], the silver ion precursors are dissolved in a solution state and reduced to metallic silver. The electrochemical reduction of silver ions to produce AgNPs generally employs potentiostatic [13] or galvanostatic methods [14]. For the optimization of AgNPs' fabrication using electrochemical synthesis, it is necessary to consider parameters such as silver ion precursors and supports of AgNPs. The Ag + ions from water soluble silver salt are required, and AgNO 3 is generally used, but pure silver metal is used as a sacrificed anode [15,16]. To apply AgNPs for electrochemical applications, supports are needed to fix it on the electrode surface, and several types of supports have been used for this purpose. Depending on the application field and purpose of AgNPs, organic polymers [17], conductive polymers [18][19][20], and functional nanomaterials [21,22] are frequently used as supports. Electrochemical changes occur at the interface between the solution and the electrode, and silver nanoparticles are formed on the electrode surface through electrochemical reduction processes. However, during the electrochemical reduction of silver ions, only some silver ions that are dissolved in the solution are deposited as silver metal on the electrode surface, while most of the silver ions remain in the solution and are discarded. Discarding most of the silver ion precursors dissolved in the solution is economically and environmentally undesirable. To overcome this problem of the electrochemical method, silver ion precursors are not dissolved in solution but fixed to the electrode surface in a solid state and reduced in situ to metallic silver. For this, it would be pertinent to introduce insoluble precursors rather than soluble silver salts. Unlike typical soluble silver salts (e.g., AgNO 3 ) commonly used in the preparation of silver nanoparticles [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22], silver chloride is insoluble (K sp = 1.8 × 10 −10 ) and is therefore suitable as a solid precursor for silver nanoparticles fabrication. In order to reduce solid silver chloride directly on the electrode surface, a membrane is required to fix it on the electrode surface. Polyethylenimine (PEI) is a hydrophilic polymer, containing primary, secondary, and tertiary amino groups, that is negatively charged in neutral aqueous solutions and widely used as a stabilizer in the fabrication of matrix-embedded metal (e.g., Au, Ag, and Pt) NPs [23][24][25]. One of the important applications of silver nanoparticles is the catalytic effect, which is used to determine H 2 O 2 by electrochemical methods. The excellent catalytic activity exhibited by the AgNPs allows for an inexpensive and effective H 2 O 2 detection, and they have therefore been extensively investigated [26][27][28][29][30]. The analysis of H 2 O 2 based on enzymatic or electrochemical reactions plays an important role in chemistry, biology, clinical control, and environmental protection [30][31][32][33][34].
In the present study, an electrochemical method for fabricating PEI-encapsulated AgNPs by direct electroreduction of solid AgCl was proposed. The characterizations of AgNPs were investigated by employing scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). After fabricating AgNPs on the electrode surface, their electrochemical catalytic effect on the reduction of H 2 O 2 was evaluated by cyclic voltammetry. The application of AgNPs was examined by measuring the catalytic reduction current according to the concentration of H 2 O 2 and interfering species. Finally, the reliability of the AgNPs/PEI-decorated electrode was evaluated by determining the H 2 O 2 concentration in a real sample of human urine. On the first scan to the negative potential, the reduction of Ag + began at around −0.1 V (Figure 1a). During the reverse scan to the positive potential direction, a small current, due to the oxidation of the silver metal initially formed on the electrode surface, occurs around 0.1 V [35,36]. After five repeated scans on the same voltage range, the reduction current decreases, and the oxidation current around 0.5 V increases significantly (Figure 1b). During the repeated scan, the amount of silver metal deposited on the electrode surface increases, and the oxidation current of the silver metal increases. This is a typical phenomenon that occurs when metal ions are deposited to the electrode surface as metal by a reduction process [37]. Therefore, it was confirmed that the solid-state silver ion precursors fixed on the electrode surface were reduced to silver metal. The potential of −0.5 V is sufficient for silver ions of AgCl to be reduced to silver metal, so in further experiments, a constant voltage of −0.5 V was applied for 300 s to produce silver nanoparticles. The AgNPs are formed by AgCl immobilized on the electrode surface, not by silver ions in solution, so the process of generating AgNPs is not significantly affected by stirring or sonication of the solution. In addition, when stirring the solution or applying sonication, the polymer membrane of PEI or AgCl fixed to the electrode surface may fall from the electrode. Therefore, the electrochemical reduction of AgCl/PEI/GCE was performed in a quiescent solution.

Characterization of AgNPs
The morphology and nanostructure of AgNPs were evaluated using SEM, XPS, and EDS techniques. Figure 2 shows the FE-SEM images of AgCl/PEI and AgNPs-decorated PEI after electrochemical reduction at different loadings of solid AgCl. In the AgCl/PEI electrode before AgCl is reduced (Figure 2a  To confirm the formation of AgNPs, the change in the oxidation state of silver before and after the electrochemical reduction was investigated using XPS ( Figure 3A). The XPS spectrum of AgCl-decorated PEI revealed the presence of two different species with Ag 3d 5/2 binding energies of 367.2 and 367.6 eV. The former peak was assigned to silver ions (Ag + ) of AgO, whereas the latter was attributed to those in Ag 2 O (Ag 2+ ). After the electrochemical reduction, the two peaks of 367.2 and 367.6 eV disappeared, and new peaks appeared at 368.4 eV and 374.4 eV. The peaks at 368.4 eV and 374.4 were assigned to Ag 0 , indicating the presence of metallic AgNPs [38][39][40] and indicating that they were shifted to positive binding energies when compared to the values observed for bulk Ag (368.1-368.2 eV). This finding reflects the previously reported dependence of the Ag 3d peak position on the nanoparticle size, with a shift to higher binding energies indicating a decreasing AgNP diameter [41]. From the XPS results, it was confirmed that solid silver chloride was successfully reduced to silver nanoparticles. To verify the composition ratio of Cl to Ag atoms during the electrodeposition, an EDS analysis was performed ( Figure 3B). The % atomic content ratios (Cl: Ag) of AgCl/PEI/GCE and AgNPs/PEI/GCE were 29.42%: 31.58% and 4.16%: 55.84%, respectively. The increasing silver ratio in the AgNPs/PEI/GCE is due to AgNPs being fixed on the electrode surface while Cl − is leached out of the electrode surface into the solution.

Electrochemical Catalytic Effect of AgNPs for H 2 O 2 Reduction
The quantification of H 2 O 2 is important in various biological and industrial processes [42,43], with electrochemical sensing exhibiting the advantages of a fast detection, low cost, low detection limit, and high specificity. The decomposition of hydrogen peroxide (H 2 O 2 + 2H + + 2e − → 2H 2 O) through the reduction reaction is a well-known process and is catalyzed by AgNPs. The electrocatalytic activity of AgNPs for the H 2 O 2 reduction was probed by subjecting bare, PEI-coated, and AgNPs/PEI-decorated GCE to a cyclic voltammetry (CV) experiment in the presence of 0.1 mM H 2 O 2 in 0.1 M PBS at pH 7.0 (Figure 4). At the bare and PEI-coated GCE (Figure 4a  confidence level, signal-to-noise ratio, k = 3) and was determined to be 1.66 µM. To test the operational reproducibility of AgNPs/PEI electrodes, four AgNPs/PEI/GCEs were prepared and examined to detect 0.1 mM H 2 O 2 by CV. These four electrodes showed a resemblance of the current response in the detection of H 2 O 2 with a % relative standard deviation of 4.18%. Based on the analytical results, the analytical characteristics of the proposed electrode were compared with the previously reported AgNPs/organic polymer-coated electrode in Table 1. Unlike other electrodes, the AgNPs of this work was formed by the solid precursor of AgCl, and the result was appreciable, as can be seen from the comparison with other works.
To evaluate the practical determination of hydrogen peroxide, the interfering effects of substances that could influence the analysis of hydrogen peroxide were investigated. Three potential interferents commonly present in human urine were selected, namely ascorbic acid (AA), uric acid (UA), and acetaminophen (AP). The AgNPs/PEI-decorated electrode was operated at −0.4 V, which was a sufficient reduction potential of hydrogen peroxide, and the current response was continuously measured by chronoamperometry. The obtained results ( Figure 6) indicated that the proposed sensor did not respond to the three interferents and could thus be used for H 2 O 2 analysis in the presence of interferents.    H 2 O 2 in the human body has been known as a biomarker of oxidative stress, and in urine it may control catabolism and renal function, as well as acting as an antibacterial agent [52][53][54]. A common level in healthy human urine was found in the micromolar range (1-100 µM), but concentrations lower than 1 µM were observed according to individuals' health conditions [53]. The concentration of inherent hydrogen peroxide in the present urine sample was analyzed using the AgNPs/PEI-decorated electrode, but the current of hydrogen peroxide was not observed. These results are assumed to be due to the presence of hydrogen peroxide below the detection limit in the present urine sample. The reliability of the AgNPs/PEI-decorated electrode was evaluated by determining the H 2 O 2 concentration in a real sample of human urine, where the urine samples were diluted five times with 0.1 M PBS. Chronoamperometry measurements were performed to determine the concentration of H 2 O 2 , and calibration plots were constructed using the standard addition method. A H 2 O 2 standard solution was added to the diluted urine sample, and the concentration of hydrogen peroxide to be analyzed was measured using the AgNPs/PEI-decorated electrode. Table 2 shows the analytical values obtained for three urine samples tested. The average recovery of spiked 2.5-µM H 2 O 2 urine samples was 98.4% for three repeat measurements with 4.67% for the % relative standard deviation (% RSD), which indicated the appreciable practicality of catalytic H 2 O 2 determination in real samples.

Characterization
Cyclic voltammetry (CV) and controlled-potential electrolysis were performed for a conventional three-electrode cell (Ag/AgCl (saturated KCl), Pt wire, and a glassy carbon electrode (GCE, diameter = 3 mm) as the reference, counter, and working electrodes, respectively) using an IVIUM electrochemical workstation (Eindhoven, Netherland). Prior to each experiment, the GCE was polished using 0.3-and 0.05-µm alumina powders, followed by washing with ethanol and distilled water, and nitrogen gas was purged through the solution for 5 min to remove dissolved oxygen. Field emission scanning electron microscopy (FE-SEM) images and EDS data were acquired using FE-SEM SUPRA25 (Carl Zeiss, Oberkochen, Germany). An X-ray photoelectron spectroscopy (XPS) analysis was performed at KBSI (Busan, Korea) on a Theta Probe AR-XPS spectrometer (Thermo Fisher Scientific, East Grinstead, UK) using monochromated Al K α radiation (1486.6 eV) at an X-ray power of 100 W and an energy step of 0.1 eV. The C 1s line, with a binding energy of 284.6 eV, was utilized as an internal reference for calibration. For the SEM, XPS, and EDS experiments, a cylindrical glassy carbon rod (diameter = 3 mm) was cut to a thickness of 2 mm, after which a thin GCE was prepared by directly connecting a peace of rod and Cu wire using conductive ink. Figure 7 shows the schematic of the AgNPs/PEI fabrication process from solid AgCl-supported PEI. Before mixing the solid AgCl and PEI solution, AgCl was ground into fine powder with a mortar. Solid AgCl-supported PEI coatings (AgCl/PEI = 0.05, 0.5, 5.0 wt/vol %) were prepared by directly mixing powdered AgCl with 1.0 wt % aqueous PEI. To prepare a 0.5 wt/vol % AgCl/PEI mixture, 0.5 mg of finely ground solid AgCl was mixed with 100 µL of 1.0 wt % PEI solution over 30 s of sonication, and the GCE surface was drop-coated with a 5-µL aliquot of the above mixture and dried at room temperature for 10 min. After drying, AgCl was electrochemically reduced in PBS (pH 7.0) at a constant potential of −0.5 V applied for 300 s or potential cycling.

Conclusions
We developed a simple method for electrochemically fabricating silver nanoparticles (AgNPs) using solid silver salt of silver chloride. The finely ground solid silver chloride (AgCl) was mixed with a poltethyleneimine (PEI) and coated to the electrode surface of a glassy carbon electrode. A reduction potential or potential cycling techniques were applied, and AgNPs were successfully formed on the electrode surface. The characterization of the synthesized AgNPs was performed via scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and energy dispersive X-ray spectroscopy (EDS). The AgNPs/PEI showed an excellent electrocatalytic activity to H 2 O 2 reduction through a potential shift and by significantly increasing the reduction peak current. Based on the result of the catalytic effect on the H 2 O 2 reduction, the AgNPs/PEI electrode was applied to the electrochemical analysis of the H 2 O 2 reduction. As the concentration of H 2 O 2 increased, the catalytic reduction current of H 2 O 2 increased linearly, and the interfering effect of the interfering substances was also eliminated. The sensitive and specific response to the H 2 O 2 reduction demonstrates that the AgNPs/PEI electrode is suitable for the determination of H 2 O 2 concentrations in a biological system. To confirm the actual performance of the AgNPs/PEI, it was applied to a human urine sample and showed reliable results using a spike recovery test.