Tetracyclines (TTCs) are one of the most extensively used veterinary medicines for their broad antibacterial spectrum and low production cost. Currently,TTCs are increasingly applied in personal care products. The large consumption of this antibiotic inevitably leads to the existence of the unmodified parent compounds or metabolized products in surface water,groundwater and sewage treatment plants,which can be directly toxic or spread drug-resistant genes ^{[1, 2]}. So far,several techniques and methods have been developed for the determination of TTC,including immunoassay,microbiological methods,and chemical-physical techniques (e.g.,high-performance liquid chromatography/glass carbon electrode and capillary zone electrophoresis) ^{[3, 4]}. However,these are expensive,time- consuming,or demand complicated sample pre-treatments. Most importantly,they are not suitable for in-situ or routine analysis.

Electrochemical methods are proposed to be promising for pollutant detection owing to their simple and reliable procedure,low cost,fast investigation,and high sensitivity ^{[5, 6]}. It has been reported that TTC can be electrochemically oxidized by several materials,especially carbon nanotubes ^{[7, 8, 9, 10, 11]}.

As a competitor to carbon nanotubes,graphene has exhibited superior performance in electrochemical sensor application ^[12]. However,graphene oxide (GO),as a precursor of graphene,has many oxygen-containing functional groups on the basal plane and the sheet edge,which lead to its insulation and disorder. These functional groups provide the unique chemical functionality of GO owing to its heterogeneous electronic structure,which contains a mixture of sp²- and sp³-hybridized carbon atoms ^[13]. Owing to some unique characteristics,interest among researchers has increasingly shifted from investigating GO as a precursor for graphene to determining the properties of GO. It has been found that the oxygen-containing functional groups on the basal plane and sheet edge allow GO to interact with a wide variety of organic and inorganic materials. Furthermore,a more efficient reduction treatment for GO could result in further interesting properties by the creation of new sp² clusters and the adjustment of surface functional groups through the removal of oxygen ^[14]. It is known that the electrochemistry of a variety of compounds is sensitive to surface defects and surface functional groups. Therefore,reduced GO may be potentially used as an electrocatalyst in various electrochemical reactions ^{[12, 15, 16, 17, 18]}. However,investigations on the development of graphene-based materials/devices for electroanalysis and electrocatalysis are still limited ^[15].

Among the different techniques in reduction chemistry,electrochemical reduction provides a green and fast method for GO reduction,and shows promise for controlling the extent and process of reduction ^{[6, 12, 19, 20]}. Moreover,electrochemically reduced GO (ERGO) has shown a better performance than chemically reduced GO (CRGO) in several previous reports ^{[5, 6, 17, 18, 21]}. The tunability of the sp² and sp³ fractions by the electrochemical reduction is a powerful way to tune its bandgap and transform GO from an insulator to a semiconductor ^[15]. The obtained functionalized graphene material has an abundance of structural defects and functional groups,which are beneficial for electrochemical applications ^{[22, 23]}. Researchers have reported that the ERGO exhibits enhanced activity for the electrocatalytic reduction of O₂ and H₂O₂,and higher electrochemical capacitance for the potential application in ultracapacitors ^[18] or other applications ^{[24, 25, 26]}. Most electrochemical reduction is performed at the potential of -1.0 to -1.5 V,where a variety of the oxygen-containing functional groups can be removed ^{[6, 16, 27]}. However,the electrocatalytic activity of GO may also disappear along with the functional groups.

In this work,the electrochemical reduction of GO was applied through cyclic voltammetry to adjust the surface defects and surface functional groups. The obtained ERGO shows unique electrocatalytic activity toward TTC detection compared with GO and CRGO. The structural features of ERGO,GO,and CRGO were investigated by Raman,Fourier transform infrared (FT-IR),and X-ray photoelectron spectroscopy (XPS) techniques,and were correlated with their electrochemical behavior toward TTC detection. The possible electrocatalytic mechanism of ERGO toward TTC oxidation was proposed.

Graphite powder (99.95% purity) was purchased from Aladdin (Shanghai,China). TTC was obtained from Sigma-Aldrich. Other chemicals,such as hydrazine hydrate and N,N-dimethylformamide (DMF),were all of analytical grade and obtained commercially. All the solutions were prepared using double-distilled water.

A whirlpool mixer (QL-901,Haimen Kylin-Bell Lab Instruments Co.,Ltd) was employed to disperse TTC in 0.10 mol/L sodium tartrate solutions (pH = 3.0). The cyclic voltammetric (CV) experiments were performed on a CHI 760D electrochemical workstation (Chenhua Co.,Shanghai,China) in a conventional three-electrode system,including a modified glassy carbon electrode (GC,3 mm in diameter) as a working electrode,an SCE as a reference electrode,and a platinum plate (1 cm×1.5 cm) as an auxiliary electrode. All the potential values were reported against SCE.

GO was synthesized from graphite powder (99%,40 nm,Aladdin) by Hummers’ method ^[28]. CRGO was obtained by the chemical reduction of GO,where GO was reduced by ammonia water and hydrazine hydrate. GO (50 mg) was first dispersed in 250 mL of water by ultrasonication. Consequently,250 µL of 25% ammonia water and 250 µL of hydrazine hydrate were added to the solution. The solution was maintained at 95 °C in a water bath for 1 h,followed by filtration and washing with water.

The GO-modified GC (GC/GO) electrode was prepared with a 5-µL GO aqueous dispersion solution (0.05 wt%) dropped on the electrode,and then dried in air. A similar method was used to prepare the GC/CRGO electrode,with a 5-µL CRGO DMF dispersion solution (0.05 wt %) dropped on the electrode. Prior to use,each of the modified electrodes was carefully rinsed with water to remove the loosely attached materials.

The GC/ERGO_-0.8V electrode was obtained by electrochemical reduction of the GC/GO electrode. CV was applied to the GC/GO electrode in 0.1 mol/L sodium tartrate buffer solutions in the potential range of -0.8 to +1.4 V at a scan rate of 10 mV/s for five cycles,until the cyclic voltammetric curve was stable. The GC/ERGO_-1.2V electrode was obtained by applying a potential in the range of -1.2 to +1.4 V to the GC/GO electrode under the same conditions.

CV was applied in a series of 0.1 mol/L sodium tartrate buffer solutions containing different TTC concentrations on the above prepared electrodes. The electrode was placed into a 0.1 mol/L sodium tartrate buffer solution and scanned at a rate of 50 mV/s between -0.8 and +1.4 V for four cycles after each experiment. The third cycle of the voltammogram was recorded for each sample.

A 5-µL GO aqueous dispersion solution was dropped on the conductive glass as the sample,and then CV was used to reduce GO,which was characterized by FT-IR,Raman,and XPS. XPS measurements were carried out on a Thermo ESCALAB 250 X-ray photoelectron spectrometer. FT-IR spectra were recorded on a Thermo NEXUS 670 transform infrared spectrophotometer (frequency region between 2000 and 600 cm⁻¹). Raman spectra were obtained using a T64000 Raman spectrometer with excitation from an argon-ion laser beam (λ = 514.5 nm) employing a backscattering geometry.

The electrochemical behavior of TTC was investigated on ERGO_-0.8V,ERGO_-1.2V (GO applied to a negative potential of -0.8 V and -1.2 V,respectively),GO,CRGO-modified GC,and bare GC electrodes by CV in sodium tartrate buffer (pH = 3.0),as shown in Fig. 1. Two pairs of redox peaks were observed for the GC/ERGO_-0.8V electrode at 0.2 and 0.3 V in Fig. 1(f),but the redox peaks at approximately 0.2 V decreased when the TTC concentration was in the range of 0-1.0 mg/L (Fig. 1(e)),or more than 10 mg/L. As the TTC concentration was increased,the current of these redox peaks increased,and the separation between the oxidation and reduction peaks became larger. This suggests that these two pairs of redox peaks are related to the electrochemical behavior of TTC. For example,the second oxidation peak at about 0.3 V shows a good relationship between peak current and TTC concentration in the regions of 0.1-1.0,1-10,and 10-160 mg/L,although the correlation varied in the different concentration ranges. When the TTC concentration is higher than 160 mg/L,the electrochemical signal is unstable. A possible reason for this may be that the electrocatalytic activity was destroyed by the high reaction rate. The cyclic voltammograms of TTC on the GC,GC/ERGO_-1.2V,GC/CRGO,and GC/GO electrodes are shown in Fig. 1 (a),(b),(d),and (g),respectively. The bare GC electrode shows an oxidation peak at about 1.2 V,and the GC/ERGO_-1.2V electrode shows two oxidation peaks at about 0.7 and 1.2 V compared with the blank control experiment,which are related to the oxidation of TTC. The GC/GO electrode shows two oxidation peaks at 1.0 and 1.2 V,and the latter disappeared as the concentration was increased. The peak current at 1.0 V shows a correlation to the TTC concentration only in a limited range of approximately 20-80 mg/L. However,a correlation of the oxidation peak current at 1.0 V versus TTC concentration on the GC/CRGO electrode was not evident (data not shown). Meanwhile,all of these electrodes did not show a corresponding reduction peak. CV analysis revealed that the electrocatalytic activity of the GC/ERGO_-0.8V electrode toward TTC detection was greatly improved compared with the GC,GC/ERGO_-1.2V,GC/GO,and GC/CRGO electrodes. At the same time,it should be noted that the background current caused by oxygen evolution exists at the potential region higher than 0.8 V. Hence the potential range of 0-0.5 V is more stable and more suitable for the electrochemical detection of TTC. As reported previously,carbon nanotubes,as important nano-carbon nanomaterials,have shown similar electrochemical activities. Comparatively,the potential of the TTC oxidation peak from the carbon nanotube-modified GC electrode ^{[8, 10]} is about 1.0 V,which is also more positive than that observed from the GC/ERGO_-0.8V electrode.

Fig. 1. Cyclic voltammograms of TTC with different concentrations recorded on bare GC (a),GC/ERGO-1.2V (b),GC/ERGO-0.8V (c,e,and f),GC/CRGO (d),and GC/GO (g) electrodes in a 0.1 mol/L sodium tartrate buffer (pH 3.0) at scan rate of 10 mV/s. Insets of (e),(f),and (g) are curves of TTC concentration (c) versus peak current of (e),(f),and (g),respectively.

The FT-IR spectra (Fig. 2) were used to characterize the functional groups on ERGO_-0.8V,ERGO_-1.2V,GO,and CRGO. A wide band at 3200-3500 cm^-1,related to the -OH stretching vibration,is observed for both GO and ERGOs. Compared with GO,ERGOs exhibited a decrease in the band at 1728 cm^-1,corresponding to the C=O stretching vibration in the carbonyl groups,and the peak at 2930 cm^-1 arises from the strong association between carboxyl C=O and hydroxyl O-H ^[6]. These signals reflect the lower number of C(=O)-OH bonds in GO after electrochemical reduction. The decrease of the peaks at 1200 and 1054 cm^-1,corresponding to hydroxyl C-OH vibration,reflects that the number of hydroxyl C-OH bonds on ERGO_-0.8V decreases after electrochemical reduction. It also exhibits that the C=C (1627 cm^-1) and epoxy C-O (1416 cm^-1) peaks of ERGOs become more evident relative to the other peaks originating from oxygen-containing functional groups. Compared with ERGO_-0.8V,the quantity of C-O (O-C-O) bonds lowers significantly in ERGO_-1.2V. In summary,the oxygen-containing functional groups on the ERGO decrease,and the functional groups become less varied but still numerous,which are mainly hydroxyl,epoxy,and carboxyl,while CRGO only shows a few low-intensity bands,such as at 1728,1200,and 1050 cm^-1,corresponding to carboxyl C=O,hydroxyl C-OH,and epoxy C-O-C groups,respectively. This reflects that the oxygen-containing functional groups of GO are mostly reduced by chemical reduction.

Fig. 2. FT-IR spectra of GO (1),ERGO-0.8V (2),ERGO-1.2V (3),and CRGO (4).

Figure 3 shows the Raman spectra of ERGO_-0.8V,GO,and CRGO. An increased D/G intensity ratio of ERGO_-0.8V is observed compared with GO. This change indicates that the electrochemical reduction increases the defect density and decreases the average number of sp² domains. This is because the reduction process created numerous new graphitic domains that were smaller in size than those in GO. However,a much lower D/G intensity ratio of CRGO is obtained,which shows that the residual defects cannot be removed,although the oxygen-containing functional groups of GO are mostly reduced by chemical reduction.

Fig. 3. Raman spectra of GO (1),ERGO-0.8V (2),and CRGO (3).

XPS was performed on GO,ERGO_-0.8V,and ERGO_-1.2V to further analyze the change of the oxygen-containing functional groups during electrochemical reduction. As the C 1s XPS spectra of GO and ERGOs (Fig. 4) show,four deconvoluted peaks at 288.16 (O=C-O),287.5 (C=O/O-C-O),286.6 (C-O),and 284.7 (C-C) eV are resolved by fitting the experimental line profile. Comparably,the strong intensity of the three peaks,consistent with oxygen-containing functional groups in both GO and ERGOs,indicates an abundance of oxygen-containing functional groups,such as carboxyl,hydroxyl C-OH,and epoxy C-O-C,still existed after the electrochemical reduction. Comparatively,the amounts of O-C=O and C-O decrease and that of C=O increases with a negative potential applied to GO,and further shifted from -0.8 to -1.2 V. The tunability of oxygen-containing functional groups is proposed as follows,in reference to previous research ^[13]. The amount of C-O decreases with the desorption of epoxy and hydroxyl,and carboxyl groups form at the holes in the reduced GO. Meanwhile,part of the O=C-O was reduced to C=O during the electrochemical reduction of GO. Meanwhile,the C/O ratios in GO,ERGO_-0.8V,and ERGO_-1.2V were found to be 1.35,1.69,and 2.00,respectively,which reflects that new sp² carbons may form in ERGO,and the reductive degree of GO was increased as the potential became more negative. Combined with the FT-IR results,the quantity of oxygen-containing functional groups decreases as the applied potential is more negative. Therefore,the decrease of oxygen-containing functional groups,especially O=C-O on the edge,is likely to be the main reason for the lowered electrochemical activity of ERGO_-1.2V toward TTC compared with ERGO_-0.8V.

Fig. 4. C 1s XPS spectra of GO (a),ERGO-0.8V (b),and ERGO-1.2V (c).

Physical characterization analyses above demonstrated the stability of carboxyl,hydroxyl C-OH,and epoxy C-O-C groups during the electrochemical reduction of GO at the potential of -0.8 V. Therefore,we can correlate the unique electrocatalysis of ERGO toward TTC detection with the adjustment of oxygen-containing groups as the main factor. However,in contrast,GO contains similar functional groups to the ERGO. Thus,the changes in defect density and new sp² domains may also contribute to the electrocatalytic behavior of ERGO. It is known that the tunability of the ratio of the sp² and sp³ fraction can be a powerful method to tune its bandgap and tailor the electrical,optical and/or chemical properties of GO ^[14]. Herein,it can be presumed that the tunability of defect density and the sp² domains size could substantially affect the electrocatalytic activity of ERGO.

The redox peaks at 0-0.5 V on the GC/ERGO_-0.8V electrode in blank buffer without TTC (Fig. 1(c)) may have contributed to the transformation between carboxyl C=O and hydroxyl C-OH groups ^[29]. Similar redox peaks were also observed on the ERGO_-1.2V,GO,and CRGO-modified electrode. When the TTC was added into the buffer solution,the redox reaction of TTC occurred at the potential of 0-0.5 V on the GC/ERGO_-0.8V electrode. Hence,the redox of TTC on the GC/ERGO_-0.8V electrode may correspond to the oxygen-containing functional groups transforming between C=O and C-OH. Figure 4 shows that ERGO_-1.2V has a greater number of C=O groups,but its electrocatalytic activity toward TTC decreases compared with ERGO_-0.8V. A possible reason for this is that the newly formed C=O bonds occur at the holes in the basal plane ^[13],where the electron-transfer rate is slow compared with that at the edges ^[30]. Moreover,the TTC oxidation peak at 1.0 V is observed for the bare GC (Fig. 1(a)),GC/ERGO_-1.2V (Fig. 1(b)),GC/CRGO electrodes (Fig. 1(d)),and GC/GO (Fig. 1(g)) and completely disappeared for the GC/ERGO_-0.8V electrode (Fig. 1(c)). This may suggest that the TTC oxidation peak at about 0-0.5 V on the ERGO_-0.8V electrode corresponds to the negative shift of the TTC oxidation potential on GC,ERGO_-1.2V,GO,and CRGO,instead of a new electrocatalytic reaction.

As previous studies reported,TTC on a carbon nanotube-modified GC electrode showed an oxidation peak at 1.0-1.8 V,similar to the GC/ERGO_-1.2V,GC/GO,and GC/CRGO electrodes ^[8]. The electrochemical reaction mechanisms,as well as those of phenol ^[31] and paracetamol ^[32],were proposed to arise from the structural changes of hydroquinone to quinone. Thus,the redox of TTC at the GC/ERGO electrode may correspond to the transformation between hydroquinone and quinone. A possible mechanism of TTC redox at the GC/ERGO electrode is proposed in Scheme 1.

Scheme 1. The possible mechanism of TTC redox at the GC/ERGO electrode.

This study shows the unique electrocatalytic activity of the GC/ERGO electrode toward TTC detection by electrochemical reduction of GO at -0.8 V. Physical characterization analyses demonstrated that the oxygen-containing functional groups were adjusted but were still numerous,which are mainly as carboxyl,hydroxyl C-OH,and epoxy C-O-C groups during the electrochemical reduction of GO at this potential. Meanwhile,the defect density and the size of sp² domains were changed by the formation of new sp² carbons. Therefore,we can correlate the unique electrocatalysis of ERGO with the adjustment of oxygen-containing groups as the main factor,and the defect density and sp² domains also exert a profound influence. The possible mechanism of TTC redox reactions at the GC/ERGO_-0.8V electrode is also assumed. This work not only provides a new potential material for TTC electrochemical detection,but also demonstrates that electrochemical reduction is an effective method to establish new catalytic activities for GO with the setting of appropriate parameters.