Analytica Chimica Acta 835 (2014) 29–36
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Electrocatalytic oxidation and voltammetric determination of ciprofloxacin employing poly(alizarin red)/graphene composite film in the presence of ascorbic acid, uric acid and dopamine Xin Zhang, Youli Wei, Yaping Ding * Department of Chemistry, Shanghai University, Shanghai 200444, PR China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
PAR/EGR composite film was prepared for the first time. The sensor can be applied to determinate CPFX in the presence of AA, UA and DA. The sensor indicated the feasibility in drug samples and biological media.
An electrochemical sensor based on PAR/EGR/GCE via a cooperation of the potentiostatic technique and cyclic voltammetry was first fabricated for the determination of CPFX with satisfied detecting result of real samples.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 March 2014 Received in revised form 9 May 2014 Accepted 14 May 2014 Available online 17 May 2014
A glassy carbon electrode modified with poly(alizarin red)/electrodeposited graphene (PAR/EGR) composite film was prepared and applied to detect ciprofloxacin (CPFX) in the presence of ascorbic, uric acid and dopamine. The morphology and interface property of PAR/EGR films were examined by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). The electrocatalytic oxidation of CPFX on AR/EGR was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The linearity ranged from 4 108 to 1.2 104 M with a detection limit (S/N = 3) of 0.01 mM. The modified electrode could be applied to the individual determination of CPFX as well as the simultaneous determination of CPFX, ascorbic acid, uric acid and dopamine. This method proved to be a simple, selective and rapid way to determine CPFX in pharmaceutical preparation and biological media. ã 2014 Published by Elsevier B.V.
Keywords: Alizarin red Potentiostatically electrodeposited graphene Ciprofloxacin Simultaneously detection
1. Introduction Quinolones are broad spectrum synthetic antimicrobial agents against both gram-positive and gram-negative aerobic pathogens [1,2]. They could be used to inhibit the action of bacterial DNA gyrase
* Corresponding author. Tel.: +86 21 66134734; fax: +86 21 66132797. E-mail address: [email protected] (Y. Ding). http://dx.doi.org/10.1016/j.aca.2014.05.020 0003-2670/ ã 2014 Published by Elsevier B.V.
enzymes, thus preventing DNA replication [3,4]. Ciprofloxacin (CPFX) [1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7(piperazinyl) quinolone-3-carboxylic acid], a second-generation fluoroquinolone antibiotic, has been approved for exclusive use in humans [5]. However, the recent misuse of antibiotics in food producing animals has caused a wide concern among the public due to the transfer of antibiotic-resistant bacteria from animals to human beings [6]. ]Over the previous years, a wide range of techniques have been used for the determination of CPFX, such as adsorptive stripping
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voltammetry [7], spectrophotometry [8–10], spectrofluorometry [11–14], capillary electrophoresis [15], high-performance liquid chromatography (HPLC) [16,17] and electrochemical analysis [18– 21]. Among all the above, electrochemical technique may be the most widely applied owing to its advantages of low cost, quick response, simple instruction, high sensitivity, facile miniaturization and low power requirement. Recently, conductive polymer film modified electrode has received an increasing attention because of its easier fabrication and better reproducibility compared with monolayer modified electrodes. Besides, different polymer film-modified electrodes were constructed to enhance the sensitivity and selectivity of electrode [22,23]. Alizarin red (AR), an anthraquinone derivative extracted from madder (a natural dye), is electrochemically active [24–26]. As a 3-substituted derivative of 1,2-dihydroxy-9, 10-anthraquinone, the central quinone functionality of the adsorbed AR undergoes quasi reversible reduction is to produce a stable polymeric product [27]. Owing to the benzoquinonyl and hydroxyl group in the molecule, PAR can serve as a receptor of protons, which facilitates the balance of reaction and promotes the transfer of charge between the electrode and the analytes [28]. Graphene (GR), a novel carbon material, is a closely-packed honeycomb two-dimensional lattice, which has exhibited many excellent properties including high conductivity, huge surface area and favorable mechanical strength [29–31]. Up to now, such methods as drop-casting method [32,33], cyclic voltammetry [34], screen printing [35] and potentiostatic deposition [36] have been used to modify graphene onto electrode. Among them, potentiostatic method not only ensures the similar thicknesses of films, but also minimizes the unwanted side reactions which might occur during the polymerization [37]. However, there has been few literature concerning electrochemical sensors based on potentiostatically manufactured GR hybrids up to the present. In this paper, a novel method was employed to prepare PAR/EGR composite film via the cooperation of potentiostatic technique and cyclic voltammetry for the determination of CPFX, where the strong electron transfer ability of PAR was combined with the unique structure of electrodeposited graphene. To the best of our knowledge, it has been the first attempt to integrate combine polymer film and electrodeposited GR modified GCE for the determination of CPFX. Poly(alizarin red)/electrodeposited grapheme composite film modified glassy carbon electrode PAR/EGR/ GCE could be applied to the individual determination of CPFX as well as the simultaneous determination of CPFX, ascorbic acid, uric acid and dopamine. Moreover, this proposed method also indicated its feasibility in pharmaceutical formulations and biological media. 2. Experimental 2.1. Reagents and apparatus Ciprofloxacin was obtained from Wuhan Yuancheng Gongchuang Technology Co., Ltd. (China). Dopamine (DA), ascorbic acid (AA), uric acid (UA), glycine, alanine, phenylalanine and leucine were purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). Graphene (GR) was bought from XFNANO, INC (Nanjing, China). Graphite powder (spectral reagent), HCl, HNO3, C2H5OH, glucose, sucrose and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The drug sample of CPFX was purchased from a local pharmacy, while the fresh serum samples extracted from healthy persons were provided by Shanghai University Hospital.
All electrochemical experiments were carried out with a CHI 660D electrochemical workstation (Chenhua Corp., Shanghai, China). A conventional three-electrode system was employed throughout the experiments, with a glassy carbon electrode (GCE) as the working electrode, a platinum electrode being the counter electrode and a saturated calomel electrode as the reference electrode. The scanning electron micrograph (SEM) was made on scanning electron microscope (JSM-6700F, 15.0 kV). 2.2. Preparation of modified electrodes Before modification, the bare GCE was polished successively on chamois leather with 0.3 and 0.05 mM Al2O3 slurry until a mirrorlike surface was acquired. Then, the GCE was ultrasonicated in 1:1 HNO3 solution, ethanol and double distilled water for 5 min each. The electrodeposited graphene modified GCE (EGR/GCE) was gained via electrodeposition by applying a potentiostatic potential of +1.7 V in 10 mL 0.1 M KCl containing 200 mL 1 mg mL1 GR. Also, a dropped-graphene modified GCE (DGR/GCE) was prepared for comparison, which was obtained by dropping 6 mL GR (1 mg mL1) on the GCE and then being dried under an infrared lamp. After that, the electrodes were immersed into 10 mM of AR solution by cyclic voltammetry from 1.4 to 1.8 V at 100 mV s1 for 10 cycles in order to obtain the PAR/GCE, PAR/DGR/GCE and PAR/EGR/GCE respectively. Besides, the as-prepared electrodes were rinsed carefully with double distilled water and preserved in a refrigerator at 4 C for later use. 2.3. Tablets and human serum samples The developed sensor was employed for drug tablet determination. Six tablets were weighed and powdered. An equivalent to one tablet was weighed, dissolved into double distilled water and transferred into a 100 mL volumetric flask to be diluted. The sample was stored at 4 C. For 1 mL blood sample, 0.15 mL perchloric acid was added, vortex-mixed for 1 min and centrifuged at 2500 rpm for 15 min. And then, the supernatant was directly injected into 0.1 M phosphate buffer solution (PBS, pH 5.5) to get a total volume of 10 mL for the drug determination in human serum. 3. Results and discussion 3.1. Characterization of PAR/GR modified electrode The surface morphology of the modified electrodes was characterized by SEM. Fig. 1(A–C) illustrates the SEM images of DGR, EGR and the novel PAR/EGR composite. As shown in Fig. 1(A), the DGR exhibited a sheet-like appearance with typical transparency and corrugated morphology, indicating the large surface area inside. Compared with DGR in Fig 1(B), EGR showed a finer crimpled structure. It was predicted that the EGR film exhibited a different status from that of DGR and other reported electrochemical synthesized GR [38,39] would support. From Fig 1(C), PAR film had a brunch-like structure; when composited with EGR, it turned smooth and homogenous, evidencing the successful polymerization process of AR and the electrodeposition process of GR onto the surface of GCE. EIS, as a valid method to monitor the features of a surfacemodified electrode, includes a semicircular portion and a linear portion. The semicircle portion corresponds to the limited process of electron transfer at a higher frequency, while the diameter in the EIS equals to the electron transfer resistance (Ret) at the electrode surface [40]. Fig. 1(D) gives a clear illustration to the impedance change with the modification of DGR, EGR, PAR/EGR and bare GCE. Ret of DGR (82 V) reduced compared with that of bare GCE (130 V),
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Fig. 1. SEM images of DGR (A), EGR (B) and PAR/EGR (C), inset is the SEM image of PAR, Nyquist plots of EIS(D) for bare GCE, DGR/GCE, EGR/GCE and PAR/EGR/GCE in 5 mM K3Fe(CN)6/K4Fe(CN)6 solution containing 0.1 M KCL, inset is the equivalent circuit diagram.
while EGR exhibited an evidently large semicircle (647 V), which was caused by the defects and disorder of the graphitized structures owing to the electrodepositon [41,42]. The Ret of PAR/ EGR/GCE was increased to 1048 V, which might be ascribed to the electrostatic repulsion force between the negatively charged [Fe (CN)6]3/4 and PAR film. 3.2. Electrochemical study of EGR and DGR Cyclic voltammetry (CV) can provide useful information on the barrier changes of the electrode surface during the fabrication process. CV of 5 mM [Fe(CN)6]3/4 containing 0.1 M KCl was chosen as a marker to investigate the changes of electrode behaviors before and after modification. Fig. 2(A) suggests a higher electron transport rate on DGR/GCE than EGR/GCE, echoing the result of EIS, which proved that electrodepostion process generated remarkable structure defects of GR accompanied by the loss in electrical conductivity [43]. To further explore the sensing performance of CPFX, differential pulse voltammetry (DPV) was implemented in 10 mL PBS (pH 5.5) containing 100 mM CPFX for PAR/DGR/GCE, PAR/EGR/GCE and bare GCE (Fig. 2(B)). A slightly broader and larger oxidation peak of CPFX was obtained on PAR/DGR/GCE compared with that on bare GCE, while the current response dramatically peaked at PAR/EGR/GCE. This demonstrated that PAR/EGR/GCE exhibited a more desirable sensitivity to CPFX than PAR/DGR/GCE. 3.3. Electrochemical behavior of PAR/EGR modified electrode The PAR/EGR composite modified electrode was firstly used to determine CPFX alone. Fig 3(A) shows the CV curves of bare GCE,
Fig. 2. (A) CV curves of GCE, DGR/GCE and EGR/GCE in 5 mM K3Fe(CN)6/K4Fe(CN)6 solution containing 0.1 M KCL; (B) DPVs of 100 mM CPFX on GCE, PAR/DGR/GCE and PAR/EGR/GCE in 0.1 M PBS (pH 5.5).
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Fig. 3. (A) CV curves of bare GCE, PAR/GCE, EGR/GCE, PAR/EGR/GCE in 0.1 M PBS (pH 5.5) containing 100 mM CPFX; (B) CV curves of PAR/EGR/GCE in 0.1 M PBS containing 50 mM CPFX at scan rate from 20 to 180 mV s1. Insets are the linear relationship of v vs. I and log v vs. Ep.
PAR/GCE, EGR/GCE and PAR/EGR/GCE in 0.1 M PBS containing 100 mM CPFX at a scan rate of 100 mV s1. It can be seen that PAR/ EGR/GCE displayed an obvious increase in the peak current. This phenomenon indicated that PAR/EGR/GCE could be utilized to effectively determine CPFX. In order to investigate the reaction mechanism of CPFX, the effect of scan rate was investigated and shown in Fig. 3(B). The peak current (Ip) varied linearly as the scan rates (v) ranged from 20 to 180 mV s1 with the linear equation of: Ip ðmAÞ ¼ 1:3182 þ 152:0583v ðVs1 ÞðR ¼ 0:997Þ
(1)
validating a typical adsorption-controlled process. The Ep shifted positively along with the increase of scan rate, showing a linear relationship with log v, which was further constructed with the equation: (2) Ep ðVÞ ¼ 1:1566 þ 0:0501logv Vs1 ðR ¼ 0:998Þ The equation can be described as follows [44,45]: Ep ¼ A þ
2:303RT logv ð1 aÞnF
(3)
A is a constant related to the formal electrode potential (E0) and standard rate constant at E0; a is the transfer coefficient characterizes the effect of electrochemical potential on activation energy of an electrochemical reaction; n is the number of electrons involved in the rate-controlling step; R, T and F are the gas constant,
Fig. 4. Effect of electro-polymerization cycles (A) and pH (B) on the peak currents of 25 mM CPFX. Inset is the linear relationship between pH value and Ep.
temperature and Faraday constant respectively. On the basis of the slope being equal to 2.303RT/(1a)nF, the transferred electron is calculated to be 2.3 (a = 0.5), suggesting that two electrons were involved in the oxidation reaction. 3.4. Optimization of experimental parameters The number of electro-polymerization cycles was investigated (Fig. 4(A)). The oxidation current increased dramatically as the polymerization cycles were raised from 6 to 10 and then decreased after 10 cycles. The phenomenon could be associated with the increasing thickness of the composite film, which hindered the transfer of electron on the electrode surface. Therefore, 10 electropolymerization cycles were selected in this project. The effect of the solution pH on the electrochemical behaviors of CPFX at the PAR/EGR modified GCE was also investigated (Fig. 4(B)). The experiment revealed that the peak currents increased from 4.5 to 5.5 and then decreased. Therefore, PBS with pH 5.5 was used as the supporting electrolyte in all determinations. Meanwhile, the oxidation peak potentials of CPFX shifted negatively with the increase of solution pH. The relationship between the anodic peak potential (Ep) and the pH could be fit to the regression equation of Ep ðV Þ ¼ 1:4221 0:0679pH
(4)
with a correlation coefficient of R = 0.995. The slope of 67.9 mV per pH unit was close to the anticipated theoretical value of 59 mV,
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Scheme 1. The mechanism of CPFX oxidation.
which means that the electrocatalytic oxidation of CPFX at the modified electrode was an equal electron and proton process [46]. Corresponding to the result of Section 3.3, two electrons and two protons were involved in the oxidation reaction. The mechanism of CPFX oxidation was inferred as follows (Scheme 1), which proved consistent with the reported literature [18,19].
ranges were obtained. The regression equations could be described as: Ip ðmAÞ ¼ 2:5741 þ 0:0366cðmMÞðR ¼ 0:998Þ
(5)
for 0.04–10 mM and Ip ðmAÞ ¼ 0:2396 þ 0:2462cðmMÞðR ¼ 0:998Þ
(6)
3.5. Determination of CPFX on PAR/EGR/GCE
for 10–120 mM with a detection limit of 0.01 mM (S/N = 3).
Under the above optimized condition, the electrochemical response of PAR/EGR/GCE towards CPFX in 0.1 M PBS (pH 5.5) was performed by DPV. As shown in Fig 5, the oxidation peak current increased linearly with the concentration of CPFX. Two linear
3.6. Simultaneous determination of CPFX, ascorbic acid, uric acid and dopamine Some important biological substances often coexist with drugs in the biological samples. The linear detection of CPFX in the presence of AA, UA and DA was implemented by DPV in 0.1 M PBS (pH 5.5). Fig. 6(A) shows the DPV curves of PAR/EGR/GCE at different concentrations of AA (100–100 mM) and the constant concentration of CPFX (10 mM). The result indicated that the presence of AA had no influence on CPFX detection. However, with the simultaneous change in the concentration of AA and CPFX, two distinctive peaks were observed in Fig. 6(B). The calibration curves of AA and CPFX were shown in Fig. 6(C). The linear regression equations were described as: Ip ðmAÞ ¼ 0:2154 þ 0:0072cðmMÞðR ¼ 0:998Þ
(7)
for AA (100–1000 mM) and Ip ðmAÞ ¼ 1:2653 þ 0:0406cðmMÞðR ¼ 0:998Þ
(8)
for CPFX (10–100 mM) respectively. The simultaneous detection of uric acid, dopamine and CPFX was also investigated. Fig. 7(A) describes the DPV curves of PAR/ EGR/GCE at different concentrations of UA (30–270 mM), DA (10– 50 mM) and CPFX (10–100 mM) in 0.1 M PBS (pH 5.5). It can be seen that three well-separated peaks presented at the detached potentials, indicating that AA and DA had no interference with the detection of CPFX. In addition, the peak currents of three compounds increased synchronously with concentrations of CPFX, UA and DA, implying that AR/EGR/GCE could also be applied to the simultaneous determination of CPFX, UA and DA. The calibration curves of UA (inset), DA and CPFX were shown in Fig. 7(B). The linear regression equations were described as: IP ðmAÞ ¼ 0:6170 þ 0:0374cðmMÞðR ¼ 0:998Þ
(9)
for DA (10–50 mM), Ip ðmAÞ ¼ 0:0048 þ 0:0049cðmMÞðR ¼ 0:998Þ
(10)
for UA (30–270 mM) and Fig. 5. (A) DPV curves of PAR/EGR/GCE at different concentrations of CPFX in 0.1 M PBS (pH 5.5); (B) the corresponding calibration curve of CPFX with different concentration.
Ip ðmAÞ ¼ 1:2482 þ 0:0511cðmMÞðR ¼ 0:998Þ for CPFX (10–50 mM) separately.
(11)
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Fig. 6. (A) DPV curves of PAR/EGR/GCE at different concentration of AA (100–1000 mM) and constant concentration of CPFX (10 mM) in 0.1 M PBS (pH 5.5); (B) DPV curves of PAR/EGR/GCE at different concentration of AA (100–1000 mM) and CPFX (10–100 mM) respectively in 0.1 M PBS (pH 5.5); (C) calibration curves for the simultaneous determination of CPFX. Inset is the calibration curve of AA.
Table 1 gives a comparison of different electrochemical methods that have been used in the determination of CPFX over recent years. Compared with most methods stated in other reported papers, this proposed sensor showed a lower detection limit and wider linear range. It has also been utilized for the simultaneous determination of CPFX, ascorbic acid, uric acid and dopamine. Given the advantages of electrochemical methods, the PAR/EGR composite modified electrode would be a feasible sensor for CPFX detection. 3.7. Interference, reproducibility and stability studies The effects of some foreign species for investigating the antiinterference ability of PAR/EGR/GCE on the detection of CPFX (10 mM) were evaluated in detail. The result indicated that Table 1 The comparison of different methods for the determination of ciprofloxacin. Methods
Dynamic ranges (mM)
Detection limit (mM)
Refs.
MWCNT/GCEa P-b-CD-L-arg/CPEb MgFe2O4–MWCNT electrodec HRP-rotating GCEd AR/EGR/GCE
40–1000 0.05–100 0.1–1000
6 0.01 0.03
[18] [19] [20]
0.01–65 0.04–10 10–120
N 0.01
[21] This work
a
MWCNT/GCE: multiwalled carbon nanotubes modified glassy carbon electrode. P-b-CD-L-arg/CPE: polymerization of b-cyclodextrin and L-arginine modified carbon paste electrode. c MgFe2O4–MWCNT electrode: MgFe2O4-multiwall carbon nanotubes electrode. d HRP-rotating GCE: horseradish peroxidase-rotating GCE. b
common metal ions such as K+, Na+, Ca2+, and Zn2+ almost have no interference. 20-fold bovine serum albumin, L-cysteine, Lphenylalanine, glucose and sucrose may exist in biological sample also had no effect on the response of CPFX (signal change blow 5%). All these indicated that the proposed method had good selectivity to the determination of CPFX. The reproducibility of the same PAR/EGR/GCE was examined by measuring the current response of six samples containing 10 mM CPFX using the same modified electrode. The relative standard deviation was calculated to be 1.89%, which indicated good reproducibility. The stability of PAR/EGR/GCE was also tested after preserving the sensor in the refrigerator for one week. It was found that the peak current intensities only decreased 6.6% for CPFX, reflecting the excellent stability. 3.8. Analytical applications In order to verify the applicability of the sensor in clinical applications, PAR/EGR/GCE was utilized for detect CPFX in tablet samples and human serum. The results were listed in Table 2, indicating a good precision of this method which can meet the requirement of microanalysis. 4. Conclusions In summary, a facile and novel PAR/EGR composite film has been synthesized via potentiostatic deposition and cyclic voltammetry techniques. The EGR was partially oxidized and formed a unique structure compared with DGR. As a result, the electrochemical sensor for CPFX based on PAR/EGR was
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References
Fig. 7. (A) DPV curves of PAR/EGR/GCE at different concentration of UA (30– 270 mM), DA (10–50 mM) and CPFX (10–50 mM) in 0.1 M PBS (pH 5.5); (B)
Table 2 Detection of CPFX in pharmaceutical products and human serum samples (n = 3). Analytes
Added (mM)
Found (mM)
Recovery (%)
RSD (%)
Tablets align="center" align="center" Human serum align="center"
5 10 20 5 10 20
4.89 10.23 21.08 4.93 9.91 19.43
97.8% 102.3% 105.4% 98.6% 99.1% 97.2%
2.1 1.0 3.6 0.4 0.3 0.8
fabricated with desirable detection results in pharmaceutical formulation as well as in human serum samples. The further investigation of simultaneous determination of AA, UA, DA and CPFX was also satisfactory. The strategy of PAR/EGR for the determination of CPFX exhibited wide liner concentration range, low detection limit and high selectivity. In addition, this method can be applied in pharmaceutical preparations and biological media. Acknowledgements This work is supported by the National Natural Science Foundation of China (No. 21271127, 61171033), the Nano-Foundation of Science and Techniques Commission of Shanghai Municipality (No. 12nm0504200, 12dz1909403).
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Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Electrocatalytic oxidation and voltammetric determination of ciprofloxacin employing poly(alizarin red)/graphene composite film in the presence of ascorbic acid, uric acid and dopamine Xin Zhang, Youli Wei, Yaping Ding * Department of Chemistry, Shanghai University, Shanghai 200444, PR China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
PAR/EGR composite film was prepared for the first time. The sensor can be applied to determinate CPFX in the presence of AA, UA and DA. The sensor indicated the feasibility in drug samples and biological media.
An electrochemical sensor based on PAR/EGR/GCE via a cooperation of the potentiostatic technique and cyclic voltammetry was first fabricated for the determination of CPFX with satisfied detecting result of real samples.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 March 2014 Received in revised form 9 May 2014 Accepted 14 May 2014 Available online 17 May 2014
A glassy carbon electrode modified with poly(alizarin red)/electrodeposited graphene (PAR/EGR) composite film was prepared and applied to detect ciprofloxacin (CPFX) in the presence of ascorbic, uric acid and dopamine. The morphology and interface property of PAR/EGR films were examined by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). The electrocatalytic oxidation of CPFX on AR/EGR was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The linearity ranged from 4 108 to 1.2 104 M with a detection limit (S/N = 3) of 0.01 mM. The modified electrode could be applied to the individual determination of CPFX as well as the simultaneous determination of CPFX, ascorbic acid, uric acid and dopamine. This method proved to be a simple, selective and rapid way to determine CPFX in pharmaceutical preparation and biological media. ã 2014 Published by Elsevier B.V.
Keywords: Alizarin red Potentiostatically electrodeposited graphene Ciprofloxacin Simultaneously detection
1. Introduction Quinolones are broad spectrum synthetic antimicrobial agents against both gram-positive and gram-negative aerobic pathogens [1,2]. They could be used to inhibit the action of bacterial DNA gyrase
* Corresponding author. Tel.: +86 21 66134734; fax: +86 21 66132797. E-mail address: [email protected] (Y. Ding). http://dx.doi.org/10.1016/j.aca.2014.05.020 0003-2670/ ã 2014 Published by Elsevier B.V.
enzymes, thus preventing DNA replication [3,4]. Ciprofloxacin (CPFX) [1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7(piperazinyl) quinolone-3-carboxylic acid], a second-generation fluoroquinolone antibiotic, has been approved for exclusive use in humans [5]. However, the recent misuse of antibiotics in food producing animals has caused a wide concern among the public due to the transfer of antibiotic-resistant bacteria from animals to human beings [6]. ]Over the previous years, a wide range of techniques have been used for the determination of CPFX, such as adsorptive stripping
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voltammetry [7], spectrophotometry [8–10], spectrofluorometry [11–14], capillary electrophoresis [15], high-performance liquid chromatography (HPLC) [16,17] and electrochemical analysis [18– 21]. Among all the above, electrochemical technique may be the most widely applied owing to its advantages of low cost, quick response, simple instruction, high sensitivity, facile miniaturization and low power requirement. Recently, conductive polymer film modified electrode has received an increasing attention because of its easier fabrication and better reproducibility compared with monolayer modified electrodes. Besides, different polymer film-modified electrodes were constructed to enhance the sensitivity and selectivity of electrode [22,23]. Alizarin red (AR), an anthraquinone derivative extracted from madder (a natural dye), is electrochemically active [24–26]. As a 3-substituted derivative of 1,2-dihydroxy-9, 10-anthraquinone, the central quinone functionality of the adsorbed AR undergoes quasi reversible reduction is to produce a stable polymeric product [27]. Owing to the benzoquinonyl and hydroxyl group in the molecule, PAR can serve as a receptor of protons, which facilitates the balance of reaction and promotes the transfer of charge between the electrode and the analytes [28]. Graphene (GR), a novel carbon material, is a closely-packed honeycomb two-dimensional lattice, which has exhibited many excellent properties including high conductivity, huge surface area and favorable mechanical strength [29–31]. Up to now, such methods as drop-casting method [32,33], cyclic voltammetry [34], screen printing [35] and potentiostatic deposition [36] have been used to modify graphene onto electrode. Among them, potentiostatic method not only ensures the similar thicknesses of films, but also minimizes the unwanted side reactions which might occur during the polymerization [37]. However, there has been few literature concerning electrochemical sensors based on potentiostatically manufactured GR hybrids up to the present. In this paper, a novel method was employed to prepare PAR/EGR composite film via the cooperation of potentiostatic technique and cyclic voltammetry for the determination of CPFX, where the strong electron transfer ability of PAR was combined with the unique structure of electrodeposited graphene. To the best of our knowledge, it has been the first attempt to integrate combine polymer film and electrodeposited GR modified GCE for the determination of CPFX. Poly(alizarin red)/electrodeposited grapheme composite film modified glassy carbon electrode PAR/EGR/ GCE could be applied to the individual determination of CPFX as well as the simultaneous determination of CPFX, ascorbic acid, uric acid and dopamine. Moreover, this proposed method also indicated its feasibility in pharmaceutical formulations and biological media. 2. Experimental 2.1. Reagents and apparatus Ciprofloxacin was obtained from Wuhan Yuancheng Gongchuang Technology Co., Ltd. (China). Dopamine (DA), ascorbic acid (AA), uric acid (UA), glycine, alanine, phenylalanine and leucine were purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). Graphene (GR) was bought from XFNANO, INC (Nanjing, China). Graphite powder (spectral reagent), HCl, HNO3, C2H5OH, glucose, sucrose and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The drug sample of CPFX was purchased from a local pharmacy, while the fresh serum samples extracted from healthy persons were provided by Shanghai University Hospital.
All electrochemical experiments were carried out with a CHI 660D electrochemical workstation (Chenhua Corp., Shanghai, China). A conventional three-electrode system was employed throughout the experiments, with a glassy carbon electrode (GCE) as the working electrode, a platinum electrode being the counter electrode and a saturated calomel electrode as the reference electrode. The scanning electron micrograph (SEM) was made on scanning electron microscope (JSM-6700F, 15.0 kV). 2.2. Preparation of modified electrodes Before modification, the bare GCE was polished successively on chamois leather with 0.3 and 0.05 mM Al2O3 slurry until a mirrorlike surface was acquired. Then, the GCE was ultrasonicated in 1:1 HNO3 solution, ethanol and double distilled water for 5 min each. The electrodeposited graphene modified GCE (EGR/GCE) was gained via electrodeposition by applying a potentiostatic potential of +1.7 V in 10 mL 0.1 M KCl containing 200 mL 1 mg mL1 GR. Also, a dropped-graphene modified GCE (DGR/GCE) was prepared for comparison, which was obtained by dropping 6 mL GR (1 mg mL1) on the GCE and then being dried under an infrared lamp. After that, the electrodes were immersed into 10 mM of AR solution by cyclic voltammetry from 1.4 to 1.8 V at 100 mV s1 for 10 cycles in order to obtain the PAR/GCE, PAR/DGR/GCE and PAR/EGR/GCE respectively. Besides, the as-prepared electrodes were rinsed carefully with double distilled water and preserved in a refrigerator at 4 C for later use. 2.3. Tablets and human serum samples The developed sensor was employed for drug tablet determination. Six tablets were weighed and powdered. An equivalent to one tablet was weighed, dissolved into double distilled water and transferred into a 100 mL volumetric flask to be diluted. The sample was stored at 4 C. For 1 mL blood sample, 0.15 mL perchloric acid was added, vortex-mixed for 1 min and centrifuged at 2500 rpm for 15 min. And then, the supernatant was directly injected into 0.1 M phosphate buffer solution (PBS, pH 5.5) to get a total volume of 10 mL for the drug determination in human serum. 3. Results and discussion 3.1. Characterization of PAR/GR modified electrode The surface morphology of the modified electrodes was characterized by SEM. Fig. 1(A–C) illustrates the SEM images of DGR, EGR and the novel PAR/EGR composite. As shown in Fig. 1(A), the DGR exhibited a sheet-like appearance with typical transparency and corrugated morphology, indicating the large surface area inside. Compared with DGR in Fig 1(B), EGR showed a finer crimpled structure. It was predicted that the EGR film exhibited a different status from that of DGR and other reported electrochemical synthesized GR [38,39] would support. From Fig 1(C), PAR film had a brunch-like structure; when composited with EGR, it turned smooth and homogenous, evidencing the successful polymerization process of AR and the electrodeposition process of GR onto the surface of GCE. EIS, as a valid method to monitor the features of a surfacemodified electrode, includes a semicircular portion and a linear portion. The semicircle portion corresponds to the limited process of electron transfer at a higher frequency, while the diameter in the EIS equals to the electron transfer resistance (Ret) at the electrode surface [40]. Fig. 1(D) gives a clear illustration to the impedance change with the modification of DGR, EGR, PAR/EGR and bare GCE. Ret of DGR (82 V) reduced compared with that of bare GCE (130 V),
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Fig. 1. SEM images of DGR (A), EGR (B) and PAR/EGR (C), inset is the SEM image of PAR, Nyquist plots of EIS(D) for bare GCE, DGR/GCE, EGR/GCE and PAR/EGR/GCE in 5 mM K3Fe(CN)6/K4Fe(CN)6 solution containing 0.1 M KCL, inset is the equivalent circuit diagram.
while EGR exhibited an evidently large semicircle (647 V), which was caused by the defects and disorder of the graphitized structures owing to the electrodepositon [41,42]. The Ret of PAR/ EGR/GCE was increased to 1048 V, which might be ascribed to the electrostatic repulsion force between the negatively charged [Fe (CN)6]3/4 and PAR film. 3.2. Electrochemical study of EGR and DGR Cyclic voltammetry (CV) can provide useful information on the barrier changes of the electrode surface during the fabrication process. CV of 5 mM [Fe(CN)6]3/4 containing 0.1 M KCl was chosen as a marker to investigate the changes of electrode behaviors before and after modification. Fig. 2(A) suggests a higher electron transport rate on DGR/GCE than EGR/GCE, echoing the result of EIS, which proved that electrodepostion process generated remarkable structure defects of GR accompanied by the loss in electrical conductivity [43]. To further explore the sensing performance of CPFX, differential pulse voltammetry (DPV) was implemented in 10 mL PBS (pH 5.5) containing 100 mM CPFX for PAR/DGR/GCE, PAR/EGR/GCE and bare GCE (Fig. 2(B)). A slightly broader and larger oxidation peak of CPFX was obtained on PAR/DGR/GCE compared with that on bare GCE, while the current response dramatically peaked at PAR/EGR/GCE. This demonstrated that PAR/EGR/GCE exhibited a more desirable sensitivity to CPFX than PAR/DGR/GCE. 3.3. Electrochemical behavior of PAR/EGR modified electrode The PAR/EGR composite modified electrode was firstly used to determine CPFX alone. Fig 3(A) shows the CV curves of bare GCE,
Fig. 2. (A) CV curves of GCE, DGR/GCE and EGR/GCE in 5 mM K3Fe(CN)6/K4Fe(CN)6 solution containing 0.1 M KCL; (B) DPVs of 100 mM CPFX on GCE, PAR/DGR/GCE and PAR/EGR/GCE in 0.1 M PBS (pH 5.5).
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Fig. 3. (A) CV curves of bare GCE, PAR/GCE, EGR/GCE, PAR/EGR/GCE in 0.1 M PBS (pH 5.5) containing 100 mM CPFX; (B) CV curves of PAR/EGR/GCE in 0.1 M PBS containing 50 mM CPFX at scan rate from 20 to 180 mV s1. Insets are the linear relationship of v vs. I and log v vs. Ep.
PAR/GCE, EGR/GCE and PAR/EGR/GCE in 0.1 M PBS containing 100 mM CPFX at a scan rate of 100 mV s1. It can be seen that PAR/ EGR/GCE displayed an obvious increase in the peak current. This phenomenon indicated that PAR/EGR/GCE could be utilized to effectively determine CPFX. In order to investigate the reaction mechanism of CPFX, the effect of scan rate was investigated and shown in Fig. 3(B). The peak current (Ip) varied linearly as the scan rates (v) ranged from 20 to 180 mV s1 with the linear equation of: Ip ðmAÞ ¼ 1:3182 þ 152:0583v ðVs1 ÞðR ¼ 0:997Þ
(1)
validating a typical adsorption-controlled process. The Ep shifted positively along with the increase of scan rate, showing a linear relationship with log v, which was further constructed with the equation: (2) Ep ðVÞ ¼ 1:1566 þ 0:0501logv Vs1 ðR ¼ 0:998Þ The equation can be described as follows [44,45]: Ep ¼ A þ
2:303RT logv ð1 aÞnF
(3)
A is a constant related to the formal electrode potential (E0) and standard rate constant at E0; a is the transfer coefficient characterizes the effect of electrochemical potential on activation energy of an electrochemical reaction; n is the number of electrons involved in the rate-controlling step; R, T and F are the gas constant,
Fig. 4. Effect of electro-polymerization cycles (A) and pH (B) on the peak currents of 25 mM CPFX. Inset is the linear relationship between pH value and Ep.
temperature and Faraday constant respectively. On the basis of the slope being equal to 2.303RT/(1a)nF, the transferred electron is calculated to be 2.3 (a = 0.5), suggesting that two electrons were involved in the oxidation reaction. 3.4. Optimization of experimental parameters The number of electro-polymerization cycles was investigated (Fig. 4(A)). The oxidation current increased dramatically as the polymerization cycles were raised from 6 to 10 and then decreased after 10 cycles. The phenomenon could be associated with the increasing thickness of the composite film, which hindered the transfer of electron on the electrode surface. Therefore, 10 electropolymerization cycles were selected in this project. The effect of the solution pH on the electrochemical behaviors of CPFX at the PAR/EGR modified GCE was also investigated (Fig. 4(B)). The experiment revealed that the peak currents increased from 4.5 to 5.5 and then decreased. Therefore, PBS with pH 5.5 was used as the supporting electrolyte in all determinations. Meanwhile, the oxidation peak potentials of CPFX shifted negatively with the increase of solution pH. The relationship between the anodic peak potential (Ep) and the pH could be fit to the regression equation of Ep ðV Þ ¼ 1:4221 0:0679pH
(4)
with a correlation coefficient of R = 0.995. The slope of 67.9 mV per pH unit was close to the anticipated theoretical value of 59 mV,
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Scheme 1. The mechanism of CPFX oxidation.
which means that the electrocatalytic oxidation of CPFX at the modified electrode was an equal electron and proton process [46]. Corresponding to the result of Section 3.3, two electrons and two protons were involved in the oxidation reaction. The mechanism of CPFX oxidation was inferred as follows (Scheme 1), which proved consistent with the reported literature [18,19].
ranges were obtained. The regression equations could be described as: Ip ðmAÞ ¼ 2:5741 þ 0:0366cðmMÞðR ¼ 0:998Þ
(5)
for 0.04–10 mM and Ip ðmAÞ ¼ 0:2396 þ 0:2462cðmMÞðR ¼ 0:998Þ
(6)
3.5. Determination of CPFX on PAR/EGR/GCE
for 10–120 mM with a detection limit of 0.01 mM (S/N = 3).
Under the above optimized condition, the electrochemical response of PAR/EGR/GCE towards CPFX in 0.1 M PBS (pH 5.5) was performed by DPV. As shown in Fig 5, the oxidation peak current increased linearly with the concentration of CPFX. Two linear
3.6. Simultaneous determination of CPFX, ascorbic acid, uric acid and dopamine Some important biological substances often coexist with drugs in the biological samples. The linear detection of CPFX in the presence of AA, UA and DA was implemented by DPV in 0.1 M PBS (pH 5.5). Fig. 6(A) shows the DPV curves of PAR/EGR/GCE at different concentrations of AA (100–100 mM) and the constant concentration of CPFX (10 mM). The result indicated that the presence of AA had no influence on CPFX detection. However, with the simultaneous change in the concentration of AA and CPFX, two distinctive peaks were observed in Fig. 6(B). The calibration curves of AA and CPFX were shown in Fig. 6(C). The linear regression equations were described as: Ip ðmAÞ ¼ 0:2154 þ 0:0072cðmMÞðR ¼ 0:998Þ
(7)
for AA (100–1000 mM) and Ip ðmAÞ ¼ 1:2653 þ 0:0406cðmMÞðR ¼ 0:998Þ
(8)
for CPFX (10–100 mM) respectively. The simultaneous detection of uric acid, dopamine and CPFX was also investigated. Fig. 7(A) describes the DPV curves of PAR/ EGR/GCE at different concentrations of UA (30–270 mM), DA (10– 50 mM) and CPFX (10–100 mM) in 0.1 M PBS (pH 5.5). It can be seen that three well-separated peaks presented at the detached potentials, indicating that AA and DA had no interference with the detection of CPFX. In addition, the peak currents of three compounds increased synchronously with concentrations of CPFX, UA and DA, implying that AR/EGR/GCE could also be applied to the simultaneous determination of CPFX, UA and DA. The calibration curves of UA (inset), DA and CPFX were shown in Fig. 7(B). The linear regression equations were described as: IP ðmAÞ ¼ 0:6170 þ 0:0374cðmMÞðR ¼ 0:998Þ
(9)
for DA (10–50 mM), Ip ðmAÞ ¼ 0:0048 þ 0:0049cðmMÞðR ¼ 0:998Þ
(10)
for UA (30–270 mM) and Fig. 5. (A) DPV curves of PAR/EGR/GCE at different concentrations of CPFX in 0.1 M PBS (pH 5.5); (B) the corresponding calibration curve of CPFX with different concentration.
Ip ðmAÞ ¼ 1:2482 þ 0:0511cðmMÞðR ¼ 0:998Þ for CPFX (10–50 mM) separately.
(11)
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Fig. 6. (A) DPV curves of PAR/EGR/GCE at different concentration of AA (100–1000 mM) and constant concentration of CPFX (10 mM) in 0.1 M PBS (pH 5.5); (B) DPV curves of PAR/EGR/GCE at different concentration of AA (100–1000 mM) and CPFX (10–100 mM) respectively in 0.1 M PBS (pH 5.5); (C) calibration curves for the simultaneous determination of CPFX. Inset is the calibration curve of AA.
Table 1 gives a comparison of different electrochemical methods that have been used in the determination of CPFX over recent years. Compared with most methods stated in other reported papers, this proposed sensor showed a lower detection limit and wider linear range. It has also been utilized for the simultaneous determination of CPFX, ascorbic acid, uric acid and dopamine. Given the advantages of electrochemical methods, the PAR/EGR composite modified electrode would be a feasible sensor for CPFX detection. 3.7. Interference, reproducibility and stability studies The effects of some foreign species for investigating the antiinterference ability of PAR/EGR/GCE on the detection of CPFX (10 mM) were evaluated in detail. The result indicated that Table 1 The comparison of different methods for the determination of ciprofloxacin. Methods
Dynamic ranges (mM)
Detection limit (mM)
Refs.
MWCNT/GCEa P-b-CD-L-arg/CPEb MgFe2O4–MWCNT electrodec HRP-rotating GCEd AR/EGR/GCE
40–1000 0.05–100 0.1–1000
6 0.01 0.03
[18] [19] [20]
0.01–65 0.04–10 10–120
N 0.01
[21] This work
a
MWCNT/GCE: multiwalled carbon nanotubes modified glassy carbon electrode. P-b-CD-L-arg/CPE: polymerization of b-cyclodextrin and L-arginine modified carbon paste electrode. c MgFe2O4–MWCNT electrode: MgFe2O4-multiwall carbon nanotubes electrode. d HRP-rotating GCE: horseradish peroxidase-rotating GCE. b
common metal ions such as K+, Na+, Ca2+, and Zn2+ almost have no interference. 20-fold bovine serum albumin, L-cysteine, Lphenylalanine, glucose and sucrose may exist in biological sample also had no effect on the response of CPFX (signal change blow 5%). All these indicated that the proposed method had good selectivity to the determination of CPFX. The reproducibility of the same PAR/EGR/GCE was examined by measuring the current response of six samples containing 10 mM CPFX using the same modified electrode. The relative standard deviation was calculated to be 1.89%, which indicated good reproducibility. The stability of PAR/EGR/GCE was also tested after preserving the sensor in the refrigerator for one week. It was found that the peak current intensities only decreased 6.6% for CPFX, reflecting the excellent stability. 3.8. Analytical applications In order to verify the applicability of the sensor in clinical applications, PAR/EGR/GCE was utilized for detect CPFX in tablet samples and human serum. The results were listed in Table 2, indicating a good precision of this method which can meet the requirement of microanalysis. 4. Conclusions In summary, a facile and novel PAR/EGR composite film has been synthesized via potentiostatic deposition and cyclic voltammetry techniques. The EGR was partially oxidized and formed a unique structure compared with DGR. As a result, the electrochemical sensor for CPFX based on PAR/EGR was
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References
Fig. 7. (A) DPV curves of PAR/EGR/GCE at different concentration of UA (30– 270 mM), DA (10–50 mM) and CPFX (10–50 mM) in 0.1 M PBS (pH 5.5); (B)
Table 2 Detection of CPFX in pharmaceutical products and human serum samples (n = 3). Analytes
Added (mM)
Found (mM)
Recovery (%)
RSD (%)
Tablets align="center" align="center" Human serum align="center"
5 10 20 5 10 20
4.89 10.23 21.08 4.93 9.91 19.43
97.8% 102.3% 105.4% 98.6% 99.1% 97.2%
2.1 1.0 3.6 0.4 0.3 0.8
fabricated with desirable detection results in pharmaceutical formulation as well as in human serum samples. The further investigation of simultaneous determination of AA, UA, DA and CPFX was also satisfactory. The strategy of PAR/EGR for the determination of CPFX exhibited wide liner concentration range, low detection limit and high selectivity. In addition, this method can be applied in pharmaceutical preparations and biological media. Acknowledgements This work is supported by the National Natural Science Foundation of China (No. 21271127, 61171033), the Nano-Foundation of Science and Techniques Commission of Shanghai Municipality (No. 12nm0504200, 12dz1909403).
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