Voltammetric determination of a fluoroquinolone antibiotic based on multilayer reduced graphene oxide sensor prepared directly, promptly by electrochemically expanding graphite electrode surface

ABSTRACT Ciprofloxacin (Cip), a fluoroquinolone antibiotic, was determined by linear sweep adsorptive stripping voltammetric method using multilayer reduced graphene oxide electrode (m-rGO). The m-rGO electrode was fabricated directly, simply and promptly from graphite platform by potentiostatic technique without further modification. The produced electrode exhibited excellent performance for ciprofloxacin (Cip) determination with a much higher signal of Cip than that on the initial graphite electrode (13.4 times). This dominance of the m-rGO electrode resulted from the large electrochemically active surface area, electrochemical catalysis, suitable functional groups on the electrode surface. Experimental conditions such as the electrode fabrication time, the supporting solution, and accumulation time were optimised to obtain the Cip highest signal. A Cip analytical method established under the optimal conditions had a good linear relationship between the Cip signal with its concentration range from 0.5 to 20 µM (R2 = 0.9953). The method showed a low detection limit (LOD = 0.078 µM), satisfactory reproducibility (RSD = 1.50%), and high accuracy. Cip concentration in a spiked lake water sample determined by the proposed method with acceptable precision (RSD = 5.59%) and trueness (recovery = 94.2–106.3%) was comparable with LC-MS/MS method.


Introduction
Ciprofloxacin belongs to the quinolone antibiotic class which has been applied to protect human beings and livestock against bacterial infection.In humans, ciprofloxacin is used in the treatment of infectious diseases such as tissue urinary, tissue, digestive, pulmonary infections and gastrointestinal diseases caused by both gram-positive and gram-negative bacteria [1,2].In livestock, ciprofloxacin is one of the most common antimicrobials used to fight against E. coli, Salmonella, Pasteurella and Mycoplasma infections [3].However, the overuse of this medicine can give rise to allergic, bacterial resistance, which negatively affects human health and the ecosystem [4].In the environment, the ciprofloxacin concentration has been found in the range from ng/L to mg/ L. It is reported that the levels of ciprofloxacin in the surface water range from 0.0018 nM/L to 19,617 nM/L [5].In the Vito Rizzi's report [6], the concentration of ciprofloxacin in the wastewater from hospital and drug production areas reached 150 µg/L and 31 mg/L, respectively.The accumulation of antibiotics has been considered the primary reason for the change in the microbial genes, promoting antibioticresistant human diseases [7].It is announced that in India and Thailand, antibiotic resistance causes 58,000 and 38,000 deaths per year.In the US and EU, more than 20,000 deaths are originated from antimicrobial resistance [8].
Some organisations in the world have issued guidelines on the use of antibiotics as well as the regulation of antibiotic residues in food.Therefore, the detection of antibiotic residues in food and the environment is essential.Various methods have been employed in antibiotic quantification, such as HPLC -fluoresce detection, HPLC-UV detection, HPLC-MS/MS, UPLC-TOF/MS, electrochemiluminescence detection [9]; however, they are known to be expensive, time-consuming, sophisticated in the operation and sample pre-treatment.These days, electrochemical methods have attracted significant attention in the sensing of antibiotics.The methods not only overcome drawbacks of the other methods but also have high sensitivity and selectivity.
Nanocarbon, especially graphene-based materials, is an excellent material for electrode modification to enhance electrochemical analysis performance, especially for organic compounds detection, including antibiotics [1,10].In recent decades, many studies have used this kind of electrode to analyse antibiotics in drug samples, food samples, and environmental samples due to their excellent properties such as high surface area, high mechanical stability, adaptability, and functionality, environmentfriendly material [11].Currently, the application of this material in the electrochemical analysis process is still being exploited, bringing remarkable results in electrochemical sensing.
Typically, the preparation of the graphene material electrode as a sensor experiences different methods with complex steps; then, the materials are attached to electrode substrate by various ways such as mixing with carbon paste, coating the substrate electrode surface, electrodeposition of produced materials on the target surface electrode [12].These methods have some disadvantages: the high cost of commercial nanocarbon, long time of material fabrication, and complicated production procedure.Besides, these types of modified electrodes are mainly produced by indirect methods such as dip-coating and drop-casting methods leading to the loosen linking between modifying layers.In consequence, it can negatively affect the response signals of the analyte.
In our previous study, we proposed a new method to synthesise a reduced graphene oxide electrode directly from the graphite-based electrode by electrochemical expansion; and the modified electrode was then immediately used for electrochemical measurements.Although it is difficult to control the number of formed graphene layers, the method is a direct, fast, convenient and cost-effective way of making graphene electrodes.The method does not require complicated techniques, expensive equipment.Moreover, the sensor can be immediately applied for analysis and still ensure high sensitivity.In this study, we continue to develop the application of the multilayer reduced graphene oxide electrode as a ciprofloxacin sensor with an effective performance.
A 0.5 M H 2 SO 4 solution used as a reagent for graphite surface modification to get reduced graphite oxide nanosheets electrode was diluted from 98% H 2 SO 4 with doubledistilled water.The buffer solutions were studied to choose a suitable supporting electrolyte for Cip detection, including phosphate buffer (PBS), acetate buffer (ABS), Britton-Robinson buffer (BRBS), KCl-HCl solutions.The pH of buffer solution was controlled by using a different ration of buffer components and pH was also adjusted by adding alkaline or acid solutions, respectively, if necessary.0.5 mM and 5 mM Cip stock solutions were prepared by dissolving Cip powder in double-distilled water.Analysis solutions were prepared daily just before use by dilution of the stock solution in the buffer.
All electrochemical measurements were performed by using Potentiogalvanostat manufactured in the Institute of Chemistry, Vietnam Academy of Science and Technology, Hanoi, Vietnam, with three electrode-system.The modified electrode, platinum and Ag/AgCl electrodes were used as a working electrode, counter electrode and reference electrode, respectively.

Preparation and characterisation of reduced graphite oxide nanosheets electrode
The reduced graphite oxide nanosheets electrode (m-rGO) was fabricated by the potentiostatic method through two successive steps from a flat, smooth disk graphite electrode (99.9% graphite, Japan, d = 3.5 mm) in 0.5 M H 2 SO 4 solution.First, a 5 V potential (versus Ag/AgCl reference electrode) was applied on the graphite electrode to expand electrode surface layers to form graphite oxide with nanosheets structure (step 1).Secondly, a − 1 V potential was applied for 120 s to reduce graphite oxide layers (step 2).The expanding duration in the first step would be optimised to obtain the highest signal of ciprofloxacin.

Ciprofloxacin analytical procedure
The determination of Cip was performed by linear sweep adsorptive stripping voltammetry (LS-AdSV) in the potential range from 0.5 V to 1.4 V at a scan rate of 0.1 V.s −1 .The experimental conditions were optimised including supporting electrolyte (composition, pH), adsorption time of Cip on the electrode surface.
The Cip concentration was spiked in distilled water, West lake water matrix, then determined by the method of standard addition.
All experiments in this study were performed at room temperature (25 ± 1°C).

Electrochemical properties of ciprofloxacin on reduced graphite oxide nanosheets electrode
Cyclic voltammetry and linear sweep adsorptive stripping voltammetry were carried out in 100 µM ciprofloxacin solution using pristine graphite electrode and m-rGO electrode for evaluation of Cip electrochemical properties.The recorded voltammograms are shown in Figure 1.It is clear that the electrochemical reaction of Cip on these electrodes is irreversible with an obvious oxidation peak at 0.87 V in the forward scanning and no reduction peak in the reverse scanning (Figure 1a).Notably, the LS-AdSV signal of Cip with 120 s adsorption on the m-rGO electrode is much higher than that on the pristine graphite electrode (13.4 times) (Figure 1b).The enhancement of the Cip signal on the developed electrode resulted from the electrode surface modification that leads to the formation of nano multilayer structure, the significant increase of active surface area, and the electrocatalysis of m-rGO electrode.
The nanostructure of the electrode can be clearly seen in the SEM image (Figure 2  (a, b, c, d)).In comparison with the flat, smooth surface of the pristine graphite electrode, the modified electrode surface has a spongy structure.At the higher magnification, it is observed that the graphite surface burst into separate nanolayers with a few nanometres thick.This structure brings about significant development of the electrode active surface area (A eas ).The area can be calculated through the reduction signal of ferri-cyanide recorded in 0.1 M PBS (pH 7) solution containing 5 mM potassium ferri-cyanide.The CV curves acquired in this solution clearly show the reversible redox of the ferri-/ferro-cyanide pair on the fabricated electrode.The anode/cathode peak current ratio is approximately 1 at all potential scanning rates (υ), and the heights of peaks are directly proportional to the square root of the voltage sweep rate (Figure 3(a, b)).This result indicates that the process is diffusioncontrolled.According to the Randle-Sevcik equation [13], based on the slope of the equation for the linear correlation between the peak height and the square root of the potential sweep rate, the calculated A eas of the proposed electrode is approximately 2 times greater than that of the original graphite electrode.
The EDX spectrum of the m-rGO electrode was also investigated, and the obtained results indicate the presence of oxygen functional groups on the electrode surface (Figure 2e).After step 1, the expanded electrode experienced an electrochemical reduction (step 2) to reduce a part of oxygen functional groups such as epoxide, hydroxyl groups, as reported in our previous study [14].Thus, the remaining oxygen amount on the electrode surface is supposed to mainly belong to -OH and -COOH groups at the edges of the nanosheets.The reduction step is beneficial for the restoration of sp 2 conjugated network of the m-rGO electrode surface, which ensures good electrical conductivity and electron transfer.Additionally, -OH and -COOH groups benefit the accumulation of ciprofloxacin (containing NH 2 groups) on the electrode surface, which enhances the electrochemical signal of Cip.The appearance of S element in the EDX spectra can be

Influence of the expansion duration on the signal of Cip
The duration of positive potential application (t exp ) to expand the graphite layers on the initial electrode surface significantly influences the structure of the m-rGO electrode surface; thereby, affecting the electrochemical signals of Cip. Figure 4a describes the change of Cip peak current recorded on m-rGO electrodes fabricated in different expansion time from 1 s ÷ 5 s.Since high voltage (5 V) was applied for a very short time (several seconds), the oxidation reactions occurred suddenly and intensely on the graphite surface.During the expansion, the powerful generation of gases such as CO 2 , O 2 , . . .burst the graphite layers to form a porous surface [15].However, the longer the voltage application duration is applied, the more surficial graphite layers are exfoliated from the surface; consequently, the surface is extremely destroyed.Thus, the bond between the layers on the surface would gradually loosen, resulting in the restriction of electron transfer among reduced graphene oxide layers.Furthermore, as longer t exp is applied, more graphene layers are oxidised intensely, leading to strongly breaking down the π conjugate system so that it is more difficult to restore.Consequently, the high electron transfer resistance on the electrode reduces the recorded signal of Cip.Indeed, when the modification potential was applied for 1 s, the highest Cip signal was recorded.Meanwhile, increasing t exp caused a remarkable decrease in the response signal.The active surface area of the electrodes fabricated in different t exp calculated from the cyclic voltammograms in the ferri-/ferro-cyanide solution is consistent with the changing trend of the Cip signals on these electrodes (Figure 4b).Therefore, a t exp of 1 s was selected to expand the graphite surface to produce modified electrodes used for further studies.

Influence of scan rate on the peak current of Cip
The relationship between electrochemical behaviours of Cip and potential scan rate ranging from 10 to 100 mV is represented in Figure S2.It is evident that the peak current of Cip linearly relates to the potential scan rate, which indicates that the electrochemical process was controlled by adsorption of Cip on the m-rGO surface in the scan rate range (Figure S2a).In addition, Figure S2b also demonstrates a linear correlation between the logarithm of peak current and logarithm of scan rate with the regression equation: log I p = 0.080 + 0.822 × log ʋ.The slope of the linear line is 0.822, close to the theoretical value of 1.00 for an adsorption controlled process [16].

Influence of electrolyte on the signal of Cip
The component and pH of the electrolyte solution have a remarkable influence on the performance of Cip detection.Among five investigated solutions (ABS, BRBS, KCl-HCl, PBS), Cip shows the most well-defined peak in PBS solution, while no peak current was obtained in KCl-HCl solution in the examined potential region (Figure 5a).Peak currents of Cip in ABS and BRBS are much lower than in PBS solutions.Thus, the phosphate buffer solution is the most appropriate for recording the LS-AdSV signal of Cip.For analytes containing amino-or/and carboxyl functional groups, their existing form of the analyte depends on the pH values of the media based on their pKa values.The distribution of the Cip existence forms in different pH environments has been shown in the report of Del Peiro et al. [17].Besides, the pH value possibly changes the form of functional groups present on the electrode surface, which relates to the analyte adsorption capacity of the electrode surface.Thereby, it has a significant impact on the recorded Cip signal.
Figure 5b indicates that the peak current of Cip on the LSV curve recorded in the PBS solution is the highest at pH 2. When the pH value increases, the peak current sharply decreases.This could be explained by the fact that the existing form of Cip is CipH + cation at low pH, which favourably forms hydrogen bonding with the carboxyl (-COOH), hydroxyl (-OH) functional groups on the m-rGO surface.As a result, this increased the amount of Cip accumulating on the electrode; thereby, a high response of Cip oxidation was obtained.When the pH value gradually raised to 8, Cip mainly changed to the zwitterion form (Cip±) [17].Besides, in an alkaline environment, the functional groups on the electrode surface also changed.These types of the target and the functional groups are difficult to form the bonding with each other to accumulate Cip on the electrode.As a consequence, the recorded electrochemical signal is much lower than in an acidic environment.Hence, PBS at pH 2 was selected as an optimal media for Cip analysis on m-rGO electrode.
The pH of the electrolyte solution also has a great influence on the oxidation/reduction potential of the analyte since protons are involved in the electrochemical reaction.The peak potential (E p ) depends on the amount of H + according to the Nernst's equation [13]: where m and n are the numbers of protons and electrons exchanged in the electrochemical reaction of the analyte, respectively.
As shown in Figure 5c, the LSV curves in Cip solution exhibit a gradual shift of peak potential to a negative direction when the pH was changed from 1 to 8. The E p values are inversely proportional to the pH values according to the equation: E p = 1.3198 − 0.0591× pH.Thus, the m/n ratio in the Nernst's equation is estimated to be about 1, which means that the number of electrons and protons exchanged in the oxidation reaction of Cip are equal.Therefore, the reaction mechanism is proposed as shown in Scheme 1 which is appropriate with the previous studies [18,19].

The influence of accumulation time on the signal of Cip
The accumulation time (t acc ) of Cip onto the electrode surface plays a crucial role in the Cip signal because Cip is determined by the adsorptive stripping voltammetry method.Figure 6 shows that at a low Cip concentration of 2 µM, the peak currents linearly increased as the t acc was changed from 30 s to 600 s.This is due to the higher Cip concentration enriched at longer t acc .However, when the measurement was performed in higher Cip concentration (10 µM), the Cip signal at t acc of 600 s started falling and deviating from the linearity.At the Cip concentration of 30 µM, the oxidation peak signal increased linearly with t acc in the range from 30 s to 300 s.When the duration was longer than 300 s, the Cip peak currents remained unchanged, showing that the absorption achieved an equilibrium condition on the electrode surface.Thus, t acc of 300 s was chosen to obtain a sufficiently high current at low concentrations and ensure a wide linear range.

Repeatability and Reproducibility of the m-rGO electrode
When the m-rGO electrode experienced ten consecutive measurements in 2 µM and 20 µM Cip solutions, the peak current of Cip gradually reduced (Figure S3a,b).However, the decrease in the peak current in the 2 µM Cip solution was much smaller than that in the 20 µM Cip solution.The peak height in the 2 µM Cip solution at the tenth curve decreased by 12.82% compared to the first one, and the RSD value for ten measurements was 4.93%.Meanwhile, in the 20 µM Cip solution, the signal at the second curve decreased by 15.07%compared to the first one.Moreover, at the tenth curve, the Cip oxidation signal remained only 64.72% (decreased by 35.28%) (Figure S3c).The RSD value of 14.80% for ten measurements was obtained in this case.The decline of peak current can be explained by the fact that that after the oxidation of adsorbed Cip, the product was not removed from the electrode surface.Consequently, it covered a part of the electrode's active area, limiting the Cip adsorption in subsequent measurements especially when measuring in the high concentration of Cip solution.Therefore, the proposed electrode did not provide good repeatability at high Cip concentration solution.At the low concentration (≤2 µM Cip), the repeatability is acceptable with the RSD of 5%.It could be concluded that the electrode surface needs to be renewed after each measurement to get a good reproducibility in the analysis.Regarding the reproducibility, LSV responses measured using eight different newly fabricated electrodes in 2 µM and 20 µM Cip solutions showed relative standard deviations of 5.81% and 1.50%, respectively, which demonstrated satisfactory reproducibility of the electrodes (Figure S4).

Analytical performance of the m-rGO electrode
In order to investigate the ciprofloxacin analytical performance on the proposed electrode, LS-AdSV was carried out in Cip solutions with concentrations ranging from 0.5 ÷ 40 µM under the optimal conditions.The target's oxidative response signal increased linearly as its concentration raised from 0.5 to µM with the  regression equation: Ip (µA) = −3.86+ 12.87 × C (µM) (R 2 = 0.9953).When the Cip concentration exceeded 30 µM, the response signal deviated from the linear plot (Figure 7a).The limit of detection (LOD) was calculated using the formula LOD = 3σ/b, where σ is the standard deviation of measured signals, and b is the slope of the regression line for the range 0.3-5 µM (Figure 7b).In order to reduce the detection limit, the adsorption time was extended to 600 s.The peak height values are estimated after background subtraction.The LOD was found as 0.078 µM and LOQ = 3× LOD = 0.234 µM.Table 1 presents the performance of various modified sensors in Cip analysis for comparison.

The accuracy
The accuracy is evaluated through the trueness and precision of the experimental results measured in the standard sample with three individual measurements.Table 2 exhibits that the obtained analytical results at five different times have similar values with a relative standard deviation from 99.5% to 105.5%, proving that the measurement has good precision.
In addition, analytical results using the proposed electrode are in correspondence with the results measured by LC-MS/MS.The sensor exhibits a good recovery of around 100%.So, it can be concluded that the analysis has high accuracy.

Analysis of real samples
A real sample tested by the proposed method is a surface water sample collected at West Lake, Hanoi, Vietnam.Measurement results in Table 2 indicate that the concentration of CIP in West Lake water was out of the quantitative limit of the method.In order to make a spiked sample, a known concentration of Cip was added.The measured Cip concentrations in the spiked sample have good repeatability after three different tests.These determined concentrations are close to the added concentration, with recovery values in the range of 94.2-106.3%.The concentrations are also consistent with the results measured by the LC-MS/MS method.

Conclusions
In this work, nano multilayer reduced graphene oxide sensor produced by potentiostatic method was successfully applied in the determination of ciprofloxacin in aqueous media.Under a rapid and simple production, the electrode surface had a nanostructure and suitable functional groups, which significantly contributed to the improvement of the LS-AdSV signal of ciprofloxacin.The signal of Cip on the fabricated electrode is 13.4 times higher than on the initial graphite electrode.The oxidation reaction of Cip occurred with the participation of H + and the electrochemical process on the electrode was controlled by the absorption step.The sensor provided satisfied LOD (0.078 µM) and LOQ (0.234 µM).The Cip concentration in the spiked lake water sample was determined with good recovery (94.2-106.3%)and was comparable with LC-MS results.

Figure 1 .
Figure 1.Cyclic voltammograms in 100 µM Cip solution at a scan rate of 100 mV/s (a) and LS-AdSVs in 0.1 M PBS at pH 7 containing 20 µM Cip (b).

Figure 2 .
Figure 2. SEM images of the graphite electrode (a) and multilayer reduced graphene oxide electrode (b, c, d) at different magnifications.EDX spectra of m-rGO electrode (e).

Figure 3 .
Figure 3. Cyclic voltammograms of the m-rGO electrode in 5 mM potassium ferri-cyanide/0.1 M PBS (pH 7) solution at various potential scan rate (a), plot of oxidation peak (I pa ) and reduction peak (I pc ) on graphite and m-rGO electrode vs. square root of potential scan rate (b).

Figure 4 .
Figure 4. Influence of expansion time for m-rGO fabrication on Cip peak current (a) and on the active surface area of fabricated (b).

Figure 5 .
Figure 5. Plots of Cip signals in different buffer solutions (a); the influence of pH values on the peak current (b) and the peak potential of Cip (c).

Scheme 1 :
Scheme 1:The proposed mechanism of Cip oxidation.

Figure
Figure Plot of response signals vs. accumulation time in different Cip concentration solutions.

Figure 7 .
Figure 7. Plot of peak current responses vs. Cip concentrations at m-rGO electrode with accumulation times: 300 s (a) and 600 s (b).The LS-AdSVs recorded in solutions of different Cip concentrations are insets.

Table 1 .
Comparison of the data obtained using various electrodes for the determination of ciprofloxacin.

Table 2 .
Analytical results for the Cip determination using m-rGO electrode in different water samples.