We used analytical-grade chemicals from Fisher, Acros Organics, and Sigma-Aldrich (USA). Supporting electrolytes were prepared using HNO₃ or NaCl. Stock solution of 10 × 10⁻⁴ M Hg²⁺ was prepared using mercury nitrate [Hg(NO₃)₂] and used after further dilution. All aqueous solutions were prepared using deionized water with 18.2 MΩ cm (EMD, Millipore). The PEDOT:PSS sample was received from CIDETEC research group (San Sebastian, Spain) (Istamboulie et al., 2010).

The po-Gr flakes were obtained by electrochemical exfoliation of graphite sheet. Briefly, a two-electrode cell was used with a piece of flexible graphite sheet (Graphitestore, Inc., USA) as a working electrode and a platinum (Pt) wire were placed parallel to and about 10 mm away from the graphite electrode which served as a counter electrode. The electrodes were connected to a DC power supply (Tektronix PS 280). About 10 mm of the working and counter electrodes were immersed into 25 mL containing 0.1 M HClO₄ and 0.1 M NaCl which served as an electrolyte. The exfoliation was performed by applying DC bias on the working electrode. Initially, a potential of −2.5 V was applied for 60 min to facilitate the electrochemical expansion of graphite electrode by intercalation of Na⁺ ions into graphite layers; after which, a potential of +10 V was applied for another 60 min to achieve exfoliation. The electrolyte solution containing the exfoliated po-Gr flakes was vacuum filtrated (using membrane with a pore size 0.4 μm) and washed with deionized water many times to remove the residual electrolyte.

The obtained po-Gr flakes were dried at 60°C for 30 min, and then dispersed in 10 mL PEDOT:PSS solution by a probe sonicator (Sonics, VibraCell VCX130) for 15 min. Later, the po-Gr/PEDOT:PSS dispersion was centrifuged for 30 min at 2,000 rpm (Sorvall Super T21) and the supernatant was used for further characterization. The po-Gr flakes were also dispersed in water (without PEDOT:PSS) for 15 min by probe sonicator and centrifuged at 2,000 rpm for 30 min to collect supernatant for control studies. All experiments were performed at room temperature (25 ± 3°C).

A 10 μL sample of po-Gr/PEDOT:PSS or po-Gr dispersion was placed on a well cleaned (after mirror-like polishing with alumina powder) glassy carbon electrode (GCE) surface and dried in an air-oven for 30 min to evaporate solvents. By this method, po-Gr/PEDOT:PSS or po-Gr film-coated electrode was obtained and gently washed by immersing in water for about 5 min to remove unbounded materials from the electrode surface. For Raman measurements, the po-Gr film prepared on glass substrate was reduced with hydrazine in pH 9.0 water solution at 80°C for 2 h (Park et al., 2011).

Electrochemical measurements were performed by using an electrochemical workstation (660D, CH Instruments). A 10-mL volume, three-electrode system was used with GCE, Ag/AgCl (3 M KCl), and Pt wire as working, reference, and counter electrode, respectively. The GCE was used either bare or after modification with po-Gr film, PEDOT:PSS film, or po-Gr/PEDOT:PSS film. The electrochemical responses of the bare GCE and modified GCE’s toward Hg²⁺ were examined with cyclic voltammetry (CV) in 0.05 M HNO₃ as an electrolyte.

Electrochemical impedance spectroscopy (EIS) measurements were performed in a solution containing 2.5 mM [Fe(CN)₆]^4-/3- and 0.1 M KCl supporting electrolyte in the frequency range of 1–106 Hz. Differential pulse stripping voltammetry (DPSV) was performed by applying deposition and then stripping steps with the following parameters: initial potential, −0.2 V; final potential, 0.8 V; amplitude, 50 mV; pulse width, 0.2 s; pulse period, 0.5 s; sample width, 0.0169 s; and deposition time, 2 min. During the deposition period, the solution was stirred at 800 rpm, and the potential was held at −0.30 V. The DPSV voltammograms were recorded upon injection of Hg²⁺ ions in the range of 0.2–14.0 μM in 0.05 M HNO₃. The interferences of some metal ions (Ca²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cd²⁺, Zn²⁺, Cr⁶⁺, and Pb²⁺) on the determination of 6.0 μM Hg²⁺ in 0.05 M HNO₃ were also investigated by adding their respective nitrate salts.

The surface morphology of po-Gr flakes was studied by scanning electron microscopy (SEM) (LEO1530, Gemini FESEM, Carl Zeiss). Further characterizations were performed on dry samples using Raman spectroscopy (LabRAM Aramis Horiba Jobin Yvon Confocal Raman Microscope, wavelength: 532 nm) and attenuated total reflectance-Fourier transform-infrared spectroscopy (ATR-FT-IR) (Spectrum 100, PerkinElmer). UV–Vis spectra of po-Gr and po-Gr/PEDOT:PSS dispersed in water were obtained using a spectrophotometer (Lambda 25, PerkinElmer). X-ray diffraction (XRD) pattern of samples were measured using Bruker D8 Discover diffractometer.

The scheme for electrochemical exfoliation of po-Gr flakes and modification of GCE surface with po-Gr/PEDOT:PSS dispersion for Hg²⁺ detection using DPSV is shown in Figure 1. After successful exfoliation, po-Gr flakes were dispersed separately in water, and PEDOT:PSS solution (Figure 2A). The po-Gr solution (Figure 2A, image a) was light yellowish brown in color and the po-Gr dispersed in PEDOT:PSS was light bluish (Figure 2A, image b). UV–Vis spectra of po-Gr solution showed a strong absorption band at 261 nm, which can be assigned to the partially oxidized graphene sheets. It has been shown that graphene oxide (GO) and rGO have absorption peaks at 230 nm, and 270 nm (π–π* transition of aromatic C–C bonds), respectively (Figure 2B) (Li et al., 2008; Choi et al., 2010).

The optical absorption peak of our synthesized po-Gr solution (after centrifugation) is in the range between that for GO and rGO; thus, we describe it as po-Gr. However, UV–Vis spectra of po-Gr/PEDOT:PSS solution showed two major absorption bands, first band observed at 261 nm and second broad band centered at 870 nm. The first peak corresponds to the optical absorption of the po-Gr and the second peak corresponds to the oxidized PEDOT:PSS (light blue) (Gustafsson-Carlberg et al., 1995; Pettersson et al., 1999; Tarabella et al., 2012). For comparison, we also measured UV–Vis spectra of PEDOT:PSS solution, which shows a broad optical absorption band centered at 870 nm (Figure 2B). This study provides evidence that po-Gr sheets are stabilized in PEDOT:PSS solution.

Raman spectra of the (a) pristine graphite sheet, (b) as-synthesized po-Gr flakes, (c) po-Gr film prepared from water dispersion after centrifugation, and (d) po-Gr film after reduction with hydrazine are presented in Figure 3. The spectra show D, G, and 2D bands for all samples (Table 1). For pristine graphite sheet, almost insignificant D band was observed, because of highly crystalline structure of graphite without defects (Figure 3A). However, Raman spectrum of the other three samples (Figures 3B–D) showed significant changes upon exfoliation, following redispersion in water and after reduction with hydrazine (Table 1). The G band is characteristic of sp2-hybridized C = C bonds in graphene sheets (Childres et al., 2013); whereas, the D band is associated with structural defects and partially disordered structures of the sp2 domain (Childres et al., 2013). The 2D band located at 2718 cm⁻¹ originates from a double-resonance process (Krauss et al., 2009; Yan et al., 2011). The calculated values of I_D/I_G listed in Table 1. Su et al. (2011) show that it changed significantly from 1.073 to 1.234 after reduction with hydrazine, indicating that restoration of C–C bonds in the graphene lattice, and a decrease in the average size of graphene domains (Lee et al., 2014). This study supports our understanding that as synthesized graphene flakes contain significant defects, due to functional groups generated upon electrochemical exfoliation at high voltage (Morales et al., 2011). I_D/I_G ratio (0.310) of as-synthesized graphene flakes is relatively small compared to po-Gr film (after centrifugation), which may be due to the presence of large graphene flakes without complete exfoliation (Figure 3B). Therefore, it was necessary to disperse po-Gr flakes in a suitable dispersant with a probe sonicator to achieve complete exfoliation of graphene sheets. In this work, we used PEDOT:PSS to disperse po-Gr flakes in water.

The as-synthesized po-Gr flakes, po-Gr film (after centrifugation), PEDOT:PSS film, and po-Gr/PEDOT:PSS film were characterized by FT-IR (Figure 4). For as-synthesized po-Gr flakes, no significant bands were observed, which may be due to the incomplete exfoliation process. Indeed, after successful redispersion in water and following centrifugation, po-Gr film on glass showed significant bands at ~3430 (OH stretch), 2337 (CO₂ stretch), 1722 (C = O stretch), and 1637 cm⁻¹(OH bending and C = C stretch) (Pham et al., 2011). The FT-IR spectrum of PEDOT:PSS showed peaks at 1372, 1289, 1124, 1023, 1002 cm⁻¹, which are derived from PEDOT:PSS (Alemu Mengistie et al., 2013; Yoo et al., 2014). The IR bands at 1160 and 1023 cm⁻¹ are assigned to SO³⁻ of the PSS. Further, the FT-IR spectrum of po-Gr/PEDOT:PSS consists bands of PEDOT:PSS, which proves that po-Gr sheets wrapped with the polymer structure (Figure 4).

Surface morphology of po-Gr sheets was also studied by SEM. SEM images of the exfoliated po-Gr film show wrinkled or folded thin sheets with the lateral dimension of 1–3 μm (Figure 5A). The XRD spectrum of graphite sheet exhibits an intense peak at 26.5°, corresponding to d-spacing of 0.34 nm (Figure 5B). However, after electrochemical exfoliation, po-Gr flakes exhibit a broad peak at ~16 to 23° and another intense peak at 26.52°. The broad peak at ~16 to 23° indicates the presence of functional groups containing oxygen, which is formed during electrochemical exfoliation (Figure 5B) (Fang et al., 2009). There is also slight shift in the peak position of po-Gr flakes from graphite after electrochemical exfoliation from 26.50 to 26.52 (inset of Figure 5B), which may due to intercalation of ions into the graphene layers.

Figure 6 is the Nyquist plot of the modified GCE’s in 2.5 mM [Fe(CN)₆]^4-/3- in 0.1 M KCl. The semicircular part in the high-frequency region represents electron-transfer-limiting process with its effective diameter equal to Faradaic charge transfer resistance (R_ct), which is responsible for the electron transfer kinetics of redox reactions at the electrode-electrolyte interface (Kumar et al., 2010; Yang and Gunasekaran, 2013). The R_ct values of po-Gr/GCE (R_ct = 144 Ω), PEDOT:PSS/GCE (R_ct = 65 Ω), and po-Gr/PEDOT:PSS/GCE (R_ct = 54 Ω), modified electrodes were lower than that for bare electrode (R_ct = 228 Ω), indicating higher conductivity as a result of modification processes (Figure 6). Generally poor conductivity of GO-modified electrode is due to the presence of excessive oxygenated species, which accentuates the insulating characteristics (Yang and Gunasekaran, 2013). The R_ct for po-Gr film is also higher than for PEDOT:PSS/GCE and po-Gr/PEDOT:PSS/GCE’s perhaps due to the presence of oxygenated species associated with po-Gr, which may affect conductivity of the electrode. However, when po-Gr sheets present in PEDOT:PSS, it improves the conductivity of the electrode (Figure 6).

The influence of various common interfering metal cations (Ca²⁺, Zn²⁺, Ni²⁺, Cr⁶⁺, As³⁺, Cd²⁺, Co²⁺, Fe²⁺, and Cu²⁺) in the presence of 6.0 μM Hg²⁺ in 0.05 M HNO₃ were tested using the po-Gr/PEDOT:PSS-modified GCE. The interference effect was calculated as: Interference(%)=IHg−IHg+interferentIHg×100 where, I_Hg = peak current for Hg²⁺, I_{Hg+ interferent} = peak current for Hg²⁺ plus added interferent. The data in Table 2 indicate that the performance of the po-Gr-PEDOT:PSS-modified GCE was unaffected by the tested interferents. The stripping peak current of Hg²⁺ exhibited no change in the presence of Zn²⁺, Cd²⁺, Ca²⁺, As³⁺, or Ni²⁺ ions even at concentrations each at more than 300 times that of Hg²⁺. Assuming an acceptable interference of ±5% (Fifield and Kealey, 2000), only Cu²⁺ at 16 times that of Hg²⁺ and Fe²⁺, at 10 times that of Hg²⁺, can be considered as interferents. DPSV of a solution containing Fe²⁺, Cu²⁺, and Hg²⁺, exhibited oxidation peaks for Fe²⁺ at 0.44 V and for Cu²⁺ at −0.024 V vs. Ag/AgCl (Figure S1 in Supplementary Material).

The performance of the po-Gr/PEDOT:PSS-modified GCE was evaluated by comparing test results with those determined according to US Environmental Protection Agency (EPA) Method 245.1 (EPA, 2007a) using CVAAS performed at the Wisconsin State Laboratory of Hygiene. The data obtained by measuring Hg²⁺ content in three unknown laboratory waste samples (Table 3) provide an excellent validation of proposed method.