Materials Science and Engineering C 37 (2014) 113–119
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Modified Au nanoparticles-imprinted sol–gel, multiwall carbon nanotubes pencil graphite electrode used as a sensor for ranitidine determination B. Rezaei ⁎, H. Lotfi-Forushani, A.A. Ensafi Department of Chemistry, Isfahan University of Technology, Isfahan 84156–83111 I.R., Iran
a r t i c l e
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Article history: Received 13 July 2013 Received in revised form 19 December 2013 Accepted 27 December 2013 Available online 7 January 2014 Keywords: Ranitidine Imprinted sol–gel polymers Pencil graphite electrode Multiwall carbon nanotubes Au-nanoparticles
a b s t r a c t A new, simple, and disposable molecularly imprinted electrochemical sensor for the determination of ranitidine was developed on pencil graphite electrode (PGE) via cyclic voltammetry (CV). The PGEs were coated with MWCNTs containing the carboxylic functional group (f-MWCNTs), imprinted with sol–gel and Au nanoparticle (AuNPs) layers (AuNP/MIP-sol–gel/f-MWCNT/PGE), respectively, to enhance the electrode's electrical transmission and sensitivity. The thin film of molecularly imprinted sol–gel polymers with specific binding sites for ranitidine was cast on modified PGE by electrochemical deposition. The AuNP/MIP-sol–gel/f-MWCNT/PGE thus developed was characterized by electrochemical impedance spectroscopy (EIS) and CV. The interaction between the imprinted sensor and the target molecule was also observed on the electrode by measuring the current response of 5.0 mM K3[Fe(CN)6] solution as an electrochemical probe. The pick currents of ranitidine increased linearly with concentration in the ranges of 0.05 to 2.0 μM, with a detection limit of (S/N = 3) 0.02 μM. Finally, the modified electrode was successfully employed to determine ranitidine in human urine samples. © 2014 Elsevier B.V. All rights reserved.
1. Introduction N-[2-[[[5-[(dimethylamino)methyl]-2-furanyl]-methyl]thio]ethyl]N′-methyl-2-nitro-1,1′-ethylenediamine, named ranitidine, was introduced to the pharmaceutical market in 1981. It is a histamine H2 receptor antagonist and has been used for the short-term treatment of active duodenal ulcer [1–6]. The chemical structure of ranitidine is shown in Scheme 1. The analytical methods reportedly used for the determination of ranitidine include high performance liquid chromatography (HPLC) [7–14], spectrophotometry [15–23], spectrometry [24], chemiluminescence [25,26], capillary electrophoresis [27–29], fluorimetry [30,31], and electrochemistry [32–40] among others. Most of these methods, however, are associated with such disadvantages as long response time, complicated procedure, interferences of matrix or similar structure molecules, and requirement of expensive instruments and toxic solvents. Molecularly imprinted polymers (MIPs) have nowadays attracted more attention because of their remarkable selectivity due to specific recognition site in shape and size toward analytes [41]. MIPs are typically prepared based on the polymerization of a functional monomer and a cross linking agent in the presence of a target molecule as a template. Once the template is removed, a molecular
⁎ Corresponding author. Tel.: +98 3113912351; fax: +98 3113912350. E-mail addresses: [email protected], [email protected] (B. Rezaei). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.12.036
recognition site of appropriate size and chemical functionality is produced to rebind the template. MIP has been widely used in such fields as solid phase extraction [42], chromatography [43], and electrochemical sensor technology [44]. The incorporation of electrochemical devices and MIPs has attracted increasing attention owing to their advantages of low cost, excellent stability, and especially high specificity [45–48]. They, however, suffer from such limitations as long response time, low density of imprinted sites, slow diffusion of the analytes across the MIP film, heterogeneous distribution of imprinted sites, and low sensitivity [49–51]. An excellent design for their improvement is the formation of a thin MIP layer on the surface of nanomaterials with a high surface to volume ratio [52–57]. The present study investigates the construction of an electrochemical sensor for ranitidine detection using an imprinted sol–gel network. The electrochemical sensor was fabricated by the electrochemical deposition of a sol–gel layer on the surface of the modified PGE for the easy control of the thickness of the deposited sol–gel film [58]. To improve the sensitivity of the electrochemical sensor, multiwall carbon nanotubes containing carboxylic functional group layer (f-MWCNTs) and AuNPs were used, respectively, as modified layers before and after the formation of the imprinted layer. The combined imprinted sol–gel and nanomaterials led to enhanced selectivity and sensitivity of the sensor. EIS and CV were then used to characterize the electrochemical behavior of the sensor thus prepared and it exhibited high recognition capacity toward ranitidine and was successfully employed for ranitidine detection in human urine samples.
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Scheme 1. Ranitidine structure.
2. Experimental 2.1. Chemicals Ranitidine, warfarin, trisodium citrate, hydrogen chloride, potassium chloride, nitric acid, sodium hydroxide, potassium hexacyanoferrate (III), methyl trimethoxysilane (MTMOS), and gold chloride (HAuCl4) were purchased from Sigma–Aldrich. Furazolidone, amoxicillin, and chlorpromazine hydrochloride were supplied from Fluka. Dimethylformamide (DMF), tetraethylorthosilicate (TEOS), phosphoric acid, acetic acid, and HPLC-grade methanol were purchased from Merck. All chemicals and reagents used were of analytical grade and doubly distilled water was used throughout. MWCNTs (synthesized by chemical vapor deposition (CVD) with a purity of 95%, an average wall thickness of about 40 nm, an average length of 5 micrometers) were obtained from Iran's Research Institute of Petroleum Industry. pH effect was studied using phosphate buffer solution (PBS, 0.03 M) with different pH values. 2.2. Apparatus Electrochemical measurements were carried out using a potentiostat– galvanostat μAutoLab (Echo Chemie, B.V., Netherlands, NOVA software) and a conventional three-electrode arrangement consisting of an AuNP/ MIP-sol–gel/f-MWCNT/PGE or a non-imprinted polymer electrode (NIPE) as the working electrode, a platinum wire as the auxiliary electrode, and a saturated calomel electrode (SCE) as the reference electrode. EIS measurement was performed with the Autolab system (PGSTAT 12, Eco Chemie B.V, Utrecht, Netherlands). pH was measured with Metrohm pH/mV-meter (model 827). 2.3. Pencil graphite electrode pretreatment In this study, the CV method was used for electrochemical treatment of PGE surface. A rotring pencil (model T 0.7, Germany) was used to hold graphite leads. All the leads were about 60 mm in total length and 0.7 mm in diameter. The pencil leads were obtained from “Know”, Japan (black lead of degree 2B). Electrical contact was established with the leads by wrapping a metallic wire around the metallic part of the pencil. The leads were vertically dipped by about 5 mm in the probe solution per measurement to give an active geometrical electrode surface area of about 11.37 mm2. The surface of the PGE was immersed in NaoH (1.0 M) solution and pretreated by using 7 cycles in the potential range between −0.40 and +1.00 V at a scan rate of 50.0 mV s−1. Then, the electrode was washed with doubly distilled water and dried at room temperature. 2.4. Preparation of Au-nanoparticles AuNPs were prepared by following a previously reported procedure [59]. Sodium citrate (4.0 mL 1.0%, w/v) was added rapidly to the solution obtained by dissolving 1.0 mL 1.0% (w/v) HAuCl4 and 99 mL water. The mixture was then kept in an oven at 60 °C for 90 min. The color of the solution first changed from pale yellow to blue and then
to red–violet, indicating the formation of AuNPs. The prepared solution containing HAuCl4 (1.5 × 10−4 M) was stored in a dark bottle at 4 °C for later use. 2.5. Preparation of the modified-imprinted sensor The procedure for the fabrication of the modified imprinted sensor is presented in Fig. 1.To achieve better dispersion and compatibility with MWCNTs, carboxylic group (COOH) was first imbedded on the surface of MWCNTs as follow: 500 mg of crude MWCNTs was added into a glass reactor containing 60 mL of HNO3. The mixture was then kept under ultrasonic for 15 min. It was then refluxed at 80 °C for 22 h before being cooled, filtered, washed by passing through about 5 L of distilled water, and finally dried. The prepared f-MWCNTs were used as a supporting material for the formation of the polymer on the surface of PGE to enhance both accessible sites and electron transfer sensitivity. After that, 5.0 mg of f-MWCNTs was added and dispersed into 2.5 mL DMF by ultrasonic for 6 min. The treated PGE was immersed into the prepared solution for 45 min to fabricate the f-MWCNT- layer (f-MWCNT-PGE). A quantity of 3.0 mL MTMOS as the monomer was mixed with 3.0 mL TEOS as the cross-linker, 1.0 mL 0.1 M HCl as the catalyst, 3.0 mL methanol as the homogenizer, and 1.0 mL of H2O. The mixture was stirred for 1 h to obtain a homogeneous sol at room temperature. Then, 10.0 mL of the sol was mixed with 1.0 mL (1.0 mM) ranitidine in an aqueous solution for an additional 1 h. This solution was used for ranitidine imprinted sol–gel film (MIP-sol–gel/f-MWCNT/PGE), while the original solution (without ranitidine) was used for the preparation of the non-imprinted reference sol–gel film. CV was used for electro-formation of the imprinted sol–gel on the surface of f-MWCNT/ PGE (Fig. 2) in the potential range of − 0.40 V to 1.0 V in 5 cycles and at a scan rate of 50 mV s−1. Then, the prepared MIP-sol–gel/fMWCNT/PGE was dried overnight at room temperature. Finally, potentiostatic deposition was employed to prepare the AuNP modified electrode (AuNP/MIP-sol–gel/f-MWCNT/PGE) by immersing the MIP-sol–gel/f-MWCNT/PGE into a solution of 1.5 × 10−4 M HAuCl4 containing 0.1 M KNO3 at the deposition potential of − 0.20 V for 6 min [60]. Removal of the template molecule (ranitidine) was achieved by immersing the obtained modified electrode in a glacial acetic acid solution for 90 min under gentle stir. 2.6. Electrochemical measurements CV and EIS measurements were carried out using a threeelectrode system. In each measurement, the sensor was immersed into the ranitidine solution at pH = 5.5 for 420 s. Then, electrochemical measurements were accomplished in a solution containing 5.0 mM K3[Fe(CN)6] and 0.1 M KCl as a probe at pH = 6.8 with PBS [61]. CV curves were obtained under a potential range of − 0.10 to 0.50 V and at a scan rate of 50 mV s− 1. For impedance measurements, a frequency range of 100 kHz to 5.0 mHz was employed. The potential and AC voltage amplitude were 0.20 V and 5.0 mV, respectively. All the electrochemical measurements were performed at room temperature.
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Fig. 1. The simplified sketch for the fabrication process of AuNP/MIP-sol–gel/f-MWCNT/PGE.
2.7. Sample preparation Different concentrations of human urine samples including ranitidine were prepared by adding accurate amounts of ranitidine hydrochloride and human urine samples in volumetric flasks. All the samples were determined by standard addition method. The sample analysis experiments were performed by square wave voltammetry (SWV) measurement. SWV parameters were as follows: amplitude, 50 mV; and frequency, 25 Hz. 3. Results and discussion 3.1. Preparation and characterization of the imprinted sol–gel sensor CV method was employed to electrodeposit the imprinted sol–gel on the surface of f-MWCNTs/PGE electrode. The optimum parameters for this step were: a potential range from − 0.40 to 1.00 V, a scan rate of 50 mV s−1, and 5 CV cycles (Fig. 2). The results show that peak current (Ip) obviously decreased with increasing cycle number (from the 1st to
the 5th), indicating that the surface of the f-MWCNTs/PGE electrode was continuously covered by a polymeric insulation layer. Fig. 3 shows the electrochemical behavior of the probe in each modification step. Firstly, the electrochemical treatment of PGE in NaOH (1.0 M) was performed and the results (in the insert of Fig. 3) showed that Ip increased, compared to the bare PGE, and that the difference between cathodic (Epc) and anodic (Epa) peak potentials (ΔEp) also decreased due to the greater reversibility of the electrochemical probe. Then, the f-MWCNT solution was introduced at the surface of the treated PGE to increase the surface area accessible for the formation of the sol–gel layer. Fig. 3b shows the electrochemical behavior of the modified electrode in this step in the presence of redox probe. It is obvious that the current response at the treated f-MWCNT/PGE became larger and more reversible than the treated PGE. The polymeric sol–gel film was then used to coat the surface of the f-MWCNT/PGE and its electrochemical performance in the redox probe was studied by CV (Fig. 3c). As can be seen, Ip decreased due to the formation of the polymeric layer while introducing AuNPs on the polymeric film (AuNP/MIP-sol–gel/f-MWCNT/PGE) caused an increase in the magnitude of the peak current (Fig. 3d). This was attributed to the enhancement of the electrode conductivity by the deposited AuNPs. The template (ranitidine) was extracted from the imprinted sensor that caused the redox peak currents of probe to occur again (Fig. 3e). This is due to the formation of vacant recognition sites after the removal of template molecules from the imprinted sol–gel layer. The CV of the modified electrode incubated in ranitidine (1.0 μM) and in PBS (pH 5.5) was obtained in the probe solution, exhibiting the capability of the sensor to recognize the analyte (Fig. 3f). As can be seen, the oxidation peak of the probe reduced again due to the occupation of the recognition sites in the polymeric layer. 3.2. EIS measurements
Fig. 2. CV curves taken during the electro-polymerization of the imprinted sol–gel on the PGE surface. Conditions: Scan rate, 50 mV s−1; number of scans, 5; potential range, −0.4 to 1.0 V; ranitidine concentration, 1.0 mM.
EIS is a useful method for monitoring the features of modified electrodes. In impedance spectra, the semicircle diameter equals the electron transfer resistance (Rct) at the electrode surface. Fig. 4 shows the Nyquist diagrams of bare PGE (a), f-MWCNT/PGE (b), MIP-sol–gel/fMWCNT/PGE (c), and AuNP/MIP-sol–gel/f-MWCNT/PGE (d) in the probe solution. As can be seen, the Rct is lower for f-MWCNT/PGE than it is for the bare PGE. Also, Rct significantly increased after modifying
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Fig. 3. Cyclic voltammograms of 5.0 mM K3[Fe(CN)6] and 0.1 M KCl at: (a) bare PGE, (b) f-MWCNT/PGE, (c) MIP-sol–gel/f-MWCNT/PGE, (d) AuNP/MIP-sol–gel/f-MWCNT/PGE, (e) AuNP/ MIP-sol–gel/f-MWCNT/PGE after removing ranitidine from polymeric network; and (f) the same electrode as (e) after incubation for 420 s in 1.0 μM ranitidine solution. Inset: treated PGE compared to bare PGE. Conditions: Potential scan range, −0.10 V to +0.50 V; Scan rate, 50 mV s−1 in PBS (pH 6.8).
the electrode surface with imprinted sol–gel, which indicates a surface passivation caused by the MIP film (Fig. 4c). After potentiostatic deposition of AuNPs, the diameter of the high frequency semicircle obviously reduced due to AuNP deposition on the surface of the electrode. This plays a key role in enhancing conductivity and tunneling effects (Fig. 4d). AuNPs provide electric conducting pathways to facilitate electron transfer of the redox probe between the solution and the electrode, which leads to a decrease in Rct and, thereby, enhances detection sensitivity. The mechanism by which the AuNPs enhance electrochemical reactivity of the passivated electrode is not completely understood [62]. It is believed that AuNPs act as electron conducting tunnels or conducting wires to provide the necessary conducting pathways between the analyte and the electrode surface. The Rct of f-MWCNT/PGE further reduced compared to that of AuNP/MIP-sol–gel/f-MWCNT/PGE as a result of removing the template (ranitidine) from the polymeric network (Fig. 4e). Once the template had been extracted, the modified electrode was incubated in the 1.0 μM ranitidine solution and PBS (at pH 5.5). The results showed
that the resistance substantially increased due to the rebinding of ranitidine in imprinted cavities that blocked some arrival channels of the probe onto the electrode surface (Fig. 4f). 3.3. Effect of sol–gel to template ratio During the preparation of the solution used for producing the ranitidine imprinted sol–gel film, the ratio of homogeneous sol to template was observed to affect the affinity and imprint efficiency of the imprinted layer. The performance of the AuNP/MIP-sol–gel/f-MWCNT/ PGE electrode was examined by changing this ratio over the range of 1:1, 5:1, 10:1, and 15:1 to prepare four types of the imprinted sensors. The results show that the electrode prepared with a ratio of 10:1 exhibited the greatest current response. It can, therefore, be used as the electrochemical sensor capable of detecting ranitidine. 3.4. Influence of pH Solution pH was found to have effects on ranitidine adsorption and oxidation at the surface of the modified electrode. However, solution pH needs to be optimized in the incubation step in order to make a
Fig. 4. EIS of 5.0 mM K3[Fe(CN)6] and 0.1 M KCl at: (a) bare PGE; (b) f-MWCNT/PGE; (c) MIP-sol–gel/f-MWCNT/PGE; (d) AuNP/MIP-sol–gel/f-MWCNT/PGE; (e) AuNP/MIPsol–gel/f-MWCNT/PGE after extraction of ranitidine from polymeric network; and (f) the same electrode as (e) after incubation for 420 s in 1.0 μM ranitidine solution. Conditions: Potential, 0.20 V; frequency range, 5.0 mHz to 100 kHz; and AC voltage amplitude, 5.0 mV.
Fig. 5. Effect of pH of the working solution on ranitidine adsorption in MIP cavities. The response was measured through CV of the probe solution (5.0 mM K3[Fe(CN)6] and 0.1 M KCl) after immersion of the electrode in a 1.0 μM ranitidine solution for 7.0 min. Scan range = −0.1 to +0.5 V, scan rate = 50 mV s−1.
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Fig. 6. Calibration curve corresponding to the response of the MIP sensor to the concentration of ranitidine. The response was measured through SWV of 5 probe solutions after incubation for 7.0 min with different concentrations of ranitidine in the buffer solution. Conditions: Scan range = −0.1 to +0.5 V; scan rate = 50 mV s−1; frequency, 25 Hz; amplitude, 50 mV.
suitable molecular conformation for ranitidine for better penetration in the recognition cavities. For this purpose, a series of PBS solutions in the pH range of 4.0 to 7.5 containing 1.0 μM ranitidine was examined with an incubation time of 420 s. As shown in Fig. 5, the maximum current response was obtained at pH = 5.5, which was chosen as the suitable pH for further studies.
remove ranitidine from the cavities. The relative standard deviation (RSD) was estimated at about 5.7. This electrode was tested several times every day for two weeks. The results (with recovery values higher than 96%) showed that the modified electrode could be used several times without any major change. So, the proposed imprinting sensor was expected to be regenerated with acceptable reproducibility. Table 2 compares the detection limit of the proposed method and
3.5. Effect of incubation time In electrochemical determination, incubation time is a simple and impressive tool for improving the sensitivity of the imprinted sensor. In this study, the effect of incubation time on the response current was investigated by CV measurement. After template molecules had been removed from the imprinted film, the modified electrode was incubated in 1.0 μM ranitidine solution at pH 5.5 for different incubation times and the reduction of the peak current was recorded. The results show that the oxidation peak current of the probe reduced sharply up to 420 s by increasing the incubation time, before it gradually leveled off. Decreasing oxidation peak current indicates that the amount of ranitidine adsorbed in the imprinted film cavities rose with prolonged incubation time. Thus, 420 s was chosen as the optimum incubation time for the determination of ranitidine. 3.6. Figures of merit
Table 2 Comparison of the detection limit of the proposed method and other reported methods. Method
Detection limit
Ref.
Polarography Potentiometry Colourimetry Visible spectroscopy HPTLC LSV Flow injection fluorometry High-performance liquid chromatography SWV
107 ng/mL N3.0 × 105 ng/mL N5.0 × 104 ng/mL N144 ng/mL N30 ng/mL 1.1 × 104 ng/mL N20 ng/mL 5 ng/mL 6.28 ng/mL
[32] [64] [33] [63] [65] [36] [66] [67] this work
Table 3 Chemical structures of the molecules used. Molecule
SVW measurements were performed to study the relationship between peak current and template-free modified electrode (Δi) vs. ranitidine concentration (Fig. 6). The currents linearly increased with analyte concentration in the range of 0.05 to 2.0 μM following the regression equation of Δi (μA) = 9.121C (μM) + 1.529 (R2 = 0.997) and the limit of detection based on S/N = 3 was 0.02 μM ranitidine. In order to evaluate the reproducibility of the imprinted sensor, the change in the net response of the sensor before and after incubation in the presence of 0.1 μM ranitidine solution was measured in five replicates. After each experiment, the elution was performed with acetic acid to
Warfarin
Furazolidone
Amoxicillin
Table 1 Results of ranitidine determination in real samples (n = 3).
Urine sample
Ranitidine added (μM)
Average of ranitidine found (μM)
Recovery (%)
RSD (%)
– 1.0 2.0 5.0
Not detected 0.93 ± 0.08 1.9 ± 0.14 5.3 ± 0.37
– 93.0 95 106
– 4.5 4.1 3.8
Chlorpromazine hydrochloride
Structure
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Fig. 7. Selective recognition for the imprinted or non-imprinted polymer film electrodes by CV of the probe solution (5.0 mM K3[Fe(CN)6] and 0.1 M KCl) after incubation for 7 min in a 1.0 μM solution of ranitidine in the buffer solution.
those of other methods reported in the literature. It is crystal clear that the sensitivity of the method is remarkably more satisfactory compared to previously reported methods.
sensor was evaluated by using it to determine ranitidine in human urine samples. The results revealed that the imprinted sensor fulfilled the simplicity requirements for ranitidine detection and provided possibilities for clinical application in physiological fluids.
3.7. Selectivity Acknowledgments Furazolidone, amoxicillin, warfarin, and chlorpromazine hydrochloride which have different functional groups similar to the template were selected for assessing the selectivity of the imprinted sensor. The structures of these substances are displayed in Table 3. The MIP sensor (AuNP/MIP/f-MWCNT/PGE) and the NIP electrode (AuNP/NIP/fMWCNT/PGE) were tested (Fig. 7). For the NIP electrode, the electrochemical response for ranitidine and the interfering species were found to be negligible. It could be concluded that there were no active sites on the surface of the NIP electrode to adsorb drugs. The current response of the MIPs electrode for ranitidine was significantly higher than that of the other interfering species. This can be explained by the size, conformation, and functional groups of the cavities that matched ranitidine in the imprinting film. 3.8. Real sample analysis In order to investigate the capability of the prepared sensor for the determination of ranitidine in the complex matrix of real clinical samples, a standard addition method was chosen. Three measurements were performed for each concentration. As can be seen in Table 1, significant recoveries and RSDs were obtained. Therefore, this method may be claimed to have a remarkable capability for the determination of ranitidine in human urine samples. 4. Conclusions In this paper, a novel, simple, disposable, and sensitive molecularly imprinted sol–gel was prepared for the determination of ranitidine using electrochemical deposition of sol–gel imprinted layer and AuNPs for the electro-modification of the electrode. The modified electrode was used to achieve a selective ranitidine sensor with high recoveries. Furthermore, the prepared sensor showed a considerable capability for the detection of ranitidine content as low as 0.02 μM. The imprinted sensor demonstrated fast binding kinetics to the template due to its high ratio of surface imprinted sites as well as high surface to volume ratios (because of introducing MWCNTs and AuNPs). It also showed a great affinity to the template. Eventually, the capability of the proposed
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Modified Au nanoparticles-imprinted sol–gel, multiwall carbon nanotubes pencil graphite electrode used as a sensor for ranitidine determination B. Rezaei ⁎, H. Lotfi-Forushani, A.A. Ensafi Department of Chemistry, Isfahan University of Technology, Isfahan 84156–83111 I.R., Iran
a r t i c l e
i n f o
Article history: Received 13 July 2013 Received in revised form 19 December 2013 Accepted 27 December 2013 Available online 7 January 2014 Keywords: Ranitidine Imprinted sol–gel polymers Pencil graphite electrode Multiwall carbon nanotubes Au-nanoparticles
a b s t r a c t A new, simple, and disposable molecularly imprinted electrochemical sensor for the determination of ranitidine was developed on pencil graphite electrode (PGE) via cyclic voltammetry (CV). The PGEs were coated with MWCNTs containing the carboxylic functional group (f-MWCNTs), imprinted with sol–gel and Au nanoparticle (AuNPs) layers (AuNP/MIP-sol–gel/f-MWCNT/PGE), respectively, to enhance the electrode's electrical transmission and sensitivity. The thin film of molecularly imprinted sol–gel polymers with specific binding sites for ranitidine was cast on modified PGE by electrochemical deposition. The AuNP/MIP-sol–gel/f-MWCNT/PGE thus developed was characterized by electrochemical impedance spectroscopy (EIS) and CV. The interaction between the imprinted sensor and the target molecule was also observed on the electrode by measuring the current response of 5.0 mM K3[Fe(CN)6] solution as an electrochemical probe. The pick currents of ranitidine increased linearly with concentration in the ranges of 0.05 to 2.0 μM, with a detection limit of (S/N = 3) 0.02 μM. Finally, the modified electrode was successfully employed to determine ranitidine in human urine samples. © 2014 Elsevier B.V. All rights reserved.
1. Introduction N-[2-[[[5-[(dimethylamino)methyl]-2-furanyl]-methyl]thio]ethyl]N′-methyl-2-nitro-1,1′-ethylenediamine, named ranitidine, was introduced to the pharmaceutical market in 1981. It is a histamine H2 receptor antagonist and has been used for the short-term treatment of active duodenal ulcer [1–6]. The chemical structure of ranitidine is shown in Scheme 1. The analytical methods reportedly used for the determination of ranitidine include high performance liquid chromatography (HPLC) [7–14], spectrophotometry [15–23], spectrometry [24], chemiluminescence [25,26], capillary electrophoresis [27–29], fluorimetry [30,31], and electrochemistry [32–40] among others. Most of these methods, however, are associated with such disadvantages as long response time, complicated procedure, interferences of matrix or similar structure molecules, and requirement of expensive instruments and toxic solvents. Molecularly imprinted polymers (MIPs) have nowadays attracted more attention because of their remarkable selectivity due to specific recognition site in shape and size toward analytes [41]. MIPs are typically prepared based on the polymerization of a functional monomer and a cross linking agent in the presence of a target molecule as a template. Once the template is removed, a molecular
⁎ Corresponding author. Tel.: +98 3113912351; fax: +98 3113912350. E-mail addresses: [email protected], [email protected] (B. Rezaei). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.12.036
recognition site of appropriate size and chemical functionality is produced to rebind the template. MIP has been widely used in such fields as solid phase extraction [42], chromatography [43], and electrochemical sensor technology [44]. The incorporation of electrochemical devices and MIPs has attracted increasing attention owing to their advantages of low cost, excellent stability, and especially high specificity [45–48]. They, however, suffer from such limitations as long response time, low density of imprinted sites, slow diffusion of the analytes across the MIP film, heterogeneous distribution of imprinted sites, and low sensitivity [49–51]. An excellent design for their improvement is the formation of a thin MIP layer on the surface of nanomaterials with a high surface to volume ratio [52–57]. The present study investigates the construction of an electrochemical sensor for ranitidine detection using an imprinted sol–gel network. The electrochemical sensor was fabricated by the electrochemical deposition of a sol–gel layer on the surface of the modified PGE for the easy control of the thickness of the deposited sol–gel film [58]. To improve the sensitivity of the electrochemical sensor, multiwall carbon nanotubes containing carboxylic functional group layer (f-MWCNTs) and AuNPs were used, respectively, as modified layers before and after the formation of the imprinted layer. The combined imprinted sol–gel and nanomaterials led to enhanced selectivity and sensitivity of the sensor. EIS and CV were then used to characterize the electrochemical behavior of the sensor thus prepared and it exhibited high recognition capacity toward ranitidine and was successfully employed for ranitidine detection in human urine samples.
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Scheme 1. Ranitidine structure.
2. Experimental 2.1. Chemicals Ranitidine, warfarin, trisodium citrate, hydrogen chloride, potassium chloride, nitric acid, sodium hydroxide, potassium hexacyanoferrate (III), methyl trimethoxysilane (MTMOS), and gold chloride (HAuCl4) were purchased from Sigma–Aldrich. Furazolidone, amoxicillin, and chlorpromazine hydrochloride were supplied from Fluka. Dimethylformamide (DMF), tetraethylorthosilicate (TEOS), phosphoric acid, acetic acid, and HPLC-grade methanol were purchased from Merck. All chemicals and reagents used were of analytical grade and doubly distilled water was used throughout. MWCNTs (synthesized by chemical vapor deposition (CVD) with a purity of 95%, an average wall thickness of about 40 nm, an average length of 5 micrometers) were obtained from Iran's Research Institute of Petroleum Industry. pH effect was studied using phosphate buffer solution (PBS, 0.03 M) with different pH values. 2.2. Apparatus Electrochemical measurements were carried out using a potentiostat– galvanostat μAutoLab (Echo Chemie, B.V., Netherlands, NOVA software) and a conventional three-electrode arrangement consisting of an AuNP/ MIP-sol–gel/f-MWCNT/PGE or a non-imprinted polymer electrode (NIPE) as the working electrode, a platinum wire as the auxiliary electrode, and a saturated calomel electrode (SCE) as the reference electrode. EIS measurement was performed with the Autolab system (PGSTAT 12, Eco Chemie B.V, Utrecht, Netherlands). pH was measured with Metrohm pH/mV-meter (model 827). 2.3. Pencil graphite electrode pretreatment In this study, the CV method was used for electrochemical treatment of PGE surface. A rotring pencil (model T 0.7, Germany) was used to hold graphite leads. All the leads were about 60 mm in total length and 0.7 mm in diameter. The pencil leads were obtained from “Know”, Japan (black lead of degree 2B). Electrical contact was established with the leads by wrapping a metallic wire around the metallic part of the pencil. The leads were vertically dipped by about 5 mm in the probe solution per measurement to give an active geometrical electrode surface area of about 11.37 mm2. The surface of the PGE was immersed in NaoH (1.0 M) solution and pretreated by using 7 cycles in the potential range between −0.40 and +1.00 V at a scan rate of 50.0 mV s−1. Then, the electrode was washed with doubly distilled water and dried at room temperature. 2.4. Preparation of Au-nanoparticles AuNPs were prepared by following a previously reported procedure [59]. Sodium citrate (4.0 mL 1.0%, w/v) was added rapidly to the solution obtained by dissolving 1.0 mL 1.0% (w/v) HAuCl4 and 99 mL water. The mixture was then kept in an oven at 60 °C for 90 min. The color of the solution first changed from pale yellow to blue and then
to red–violet, indicating the formation of AuNPs. The prepared solution containing HAuCl4 (1.5 × 10−4 M) was stored in a dark bottle at 4 °C for later use. 2.5. Preparation of the modified-imprinted sensor The procedure for the fabrication of the modified imprinted sensor is presented in Fig. 1.To achieve better dispersion and compatibility with MWCNTs, carboxylic group (COOH) was first imbedded on the surface of MWCNTs as follow: 500 mg of crude MWCNTs was added into a glass reactor containing 60 mL of HNO3. The mixture was then kept under ultrasonic for 15 min. It was then refluxed at 80 °C for 22 h before being cooled, filtered, washed by passing through about 5 L of distilled water, and finally dried. The prepared f-MWCNTs were used as a supporting material for the formation of the polymer on the surface of PGE to enhance both accessible sites and electron transfer sensitivity. After that, 5.0 mg of f-MWCNTs was added and dispersed into 2.5 mL DMF by ultrasonic for 6 min. The treated PGE was immersed into the prepared solution for 45 min to fabricate the f-MWCNT- layer (f-MWCNT-PGE). A quantity of 3.0 mL MTMOS as the monomer was mixed with 3.0 mL TEOS as the cross-linker, 1.0 mL 0.1 M HCl as the catalyst, 3.0 mL methanol as the homogenizer, and 1.0 mL of H2O. The mixture was stirred for 1 h to obtain a homogeneous sol at room temperature. Then, 10.0 mL of the sol was mixed with 1.0 mL (1.0 mM) ranitidine in an aqueous solution for an additional 1 h. This solution was used for ranitidine imprinted sol–gel film (MIP-sol–gel/f-MWCNT/PGE), while the original solution (without ranitidine) was used for the preparation of the non-imprinted reference sol–gel film. CV was used for electro-formation of the imprinted sol–gel on the surface of f-MWCNT/ PGE (Fig. 2) in the potential range of − 0.40 V to 1.0 V in 5 cycles and at a scan rate of 50 mV s−1. Then, the prepared MIP-sol–gel/fMWCNT/PGE was dried overnight at room temperature. Finally, potentiostatic deposition was employed to prepare the AuNP modified electrode (AuNP/MIP-sol–gel/f-MWCNT/PGE) by immersing the MIP-sol–gel/f-MWCNT/PGE into a solution of 1.5 × 10−4 M HAuCl4 containing 0.1 M KNO3 at the deposition potential of − 0.20 V for 6 min [60]. Removal of the template molecule (ranitidine) was achieved by immersing the obtained modified electrode in a glacial acetic acid solution for 90 min under gentle stir. 2.6. Electrochemical measurements CV and EIS measurements were carried out using a threeelectrode system. In each measurement, the sensor was immersed into the ranitidine solution at pH = 5.5 for 420 s. Then, electrochemical measurements were accomplished in a solution containing 5.0 mM K3[Fe(CN)6] and 0.1 M KCl as a probe at pH = 6.8 with PBS [61]. CV curves were obtained under a potential range of − 0.10 to 0.50 V and at a scan rate of 50 mV s− 1. For impedance measurements, a frequency range of 100 kHz to 5.0 mHz was employed. The potential and AC voltage amplitude were 0.20 V and 5.0 mV, respectively. All the electrochemical measurements were performed at room temperature.
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Fig. 1. The simplified sketch for the fabrication process of AuNP/MIP-sol–gel/f-MWCNT/PGE.
2.7. Sample preparation Different concentrations of human urine samples including ranitidine were prepared by adding accurate amounts of ranitidine hydrochloride and human urine samples in volumetric flasks. All the samples were determined by standard addition method. The sample analysis experiments were performed by square wave voltammetry (SWV) measurement. SWV parameters were as follows: amplitude, 50 mV; and frequency, 25 Hz. 3. Results and discussion 3.1. Preparation and characterization of the imprinted sol–gel sensor CV method was employed to electrodeposit the imprinted sol–gel on the surface of f-MWCNTs/PGE electrode. The optimum parameters for this step were: a potential range from − 0.40 to 1.00 V, a scan rate of 50 mV s−1, and 5 CV cycles (Fig. 2). The results show that peak current (Ip) obviously decreased with increasing cycle number (from the 1st to
the 5th), indicating that the surface of the f-MWCNTs/PGE electrode was continuously covered by a polymeric insulation layer. Fig. 3 shows the electrochemical behavior of the probe in each modification step. Firstly, the electrochemical treatment of PGE in NaOH (1.0 M) was performed and the results (in the insert of Fig. 3) showed that Ip increased, compared to the bare PGE, and that the difference between cathodic (Epc) and anodic (Epa) peak potentials (ΔEp) also decreased due to the greater reversibility of the electrochemical probe. Then, the f-MWCNT solution was introduced at the surface of the treated PGE to increase the surface area accessible for the formation of the sol–gel layer. Fig. 3b shows the electrochemical behavior of the modified electrode in this step in the presence of redox probe. It is obvious that the current response at the treated f-MWCNT/PGE became larger and more reversible than the treated PGE. The polymeric sol–gel film was then used to coat the surface of the f-MWCNT/PGE and its electrochemical performance in the redox probe was studied by CV (Fig. 3c). As can be seen, Ip decreased due to the formation of the polymeric layer while introducing AuNPs on the polymeric film (AuNP/MIP-sol–gel/f-MWCNT/PGE) caused an increase in the magnitude of the peak current (Fig. 3d). This was attributed to the enhancement of the electrode conductivity by the deposited AuNPs. The template (ranitidine) was extracted from the imprinted sensor that caused the redox peak currents of probe to occur again (Fig. 3e). This is due to the formation of vacant recognition sites after the removal of template molecules from the imprinted sol–gel layer. The CV of the modified electrode incubated in ranitidine (1.0 μM) and in PBS (pH 5.5) was obtained in the probe solution, exhibiting the capability of the sensor to recognize the analyte (Fig. 3f). As can be seen, the oxidation peak of the probe reduced again due to the occupation of the recognition sites in the polymeric layer. 3.2. EIS measurements
Fig. 2. CV curves taken during the electro-polymerization of the imprinted sol–gel on the PGE surface. Conditions: Scan rate, 50 mV s−1; number of scans, 5; potential range, −0.4 to 1.0 V; ranitidine concentration, 1.0 mM.
EIS is a useful method for monitoring the features of modified electrodes. In impedance spectra, the semicircle diameter equals the electron transfer resistance (Rct) at the electrode surface. Fig. 4 shows the Nyquist diagrams of bare PGE (a), f-MWCNT/PGE (b), MIP-sol–gel/fMWCNT/PGE (c), and AuNP/MIP-sol–gel/f-MWCNT/PGE (d) in the probe solution. As can be seen, the Rct is lower for f-MWCNT/PGE than it is for the bare PGE. Also, Rct significantly increased after modifying
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Fig. 3. Cyclic voltammograms of 5.0 mM K3[Fe(CN)6] and 0.1 M KCl at: (a) bare PGE, (b) f-MWCNT/PGE, (c) MIP-sol–gel/f-MWCNT/PGE, (d) AuNP/MIP-sol–gel/f-MWCNT/PGE, (e) AuNP/ MIP-sol–gel/f-MWCNT/PGE after removing ranitidine from polymeric network; and (f) the same electrode as (e) after incubation for 420 s in 1.0 μM ranitidine solution. Inset: treated PGE compared to bare PGE. Conditions: Potential scan range, −0.10 V to +0.50 V; Scan rate, 50 mV s−1 in PBS (pH 6.8).
the electrode surface with imprinted sol–gel, which indicates a surface passivation caused by the MIP film (Fig. 4c). After potentiostatic deposition of AuNPs, the diameter of the high frequency semicircle obviously reduced due to AuNP deposition on the surface of the electrode. This plays a key role in enhancing conductivity and tunneling effects (Fig. 4d). AuNPs provide electric conducting pathways to facilitate electron transfer of the redox probe between the solution and the electrode, which leads to a decrease in Rct and, thereby, enhances detection sensitivity. The mechanism by which the AuNPs enhance electrochemical reactivity of the passivated electrode is not completely understood [62]. It is believed that AuNPs act as electron conducting tunnels or conducting wires to provide the necessary conducting pathways between the analyte and the electrode surface. The Rct of f-MWCNT/PGE further reduced compared to that of AuNP/MIP-sol–gel/f-MWCNT/PGE as a result of removing the template (ranitidine) from the polymeric network (Fig. 4e). Once the template had been extracted, the modified electrode was incubated in the 1.0 μM ranitidine solution and PBS (at pH 5.5). The results showed
that the resistance substantially increased due to the rebinding of ranitidine in imprinted cavities that blocked some arrival channels of the probe onto the electrode surface (Fig. 4f). 3.3. Effect of sol–gel to template ratio During the preparation of the solution used for producing the ranitidine imprinted sol–gel film, the ratio of homogeneous sol to template was observed to affect the affinity and imprint efficiency of the imprinted layer. The performance of the AuNP/MIP-sol–gel/f-MWCNT/ PGE electrode was examined by changing this ratio over the range of 1:1, 5:1, 10:1, and 15:1 to prepare four types of the imprinted sensors. The results show that the electrode prepared with a ratio of 10:1 exhibited the greatest current response. It can, therefore, be used as the electrochemical sensor capable of detecting ranitidine. 3.4. Influence of pH Solution pH was found to have effects on ranitidine adsorption and oxidation at the surface of the modified electrode. However, solution pH needs to be optimized in the incubation step in order to make a
Fig. 4. EIS of 5.0 mM K3[Fe(CN)6] and 0.1 M KCl at: (a) bare PGE; (b) f-MWCNT/PGE; (c) MIP-sol–gel/f-MWCNT/PGE; (d) AuNP/MIP-sol–gel/f-MWCNT/PGE; (e) AuNP/MIPsol–gel/f-MWCNT/PGE after extraction of ranitidine from polymeric network; and (f) the same electrode as (e) after incubation for 420 s in 1.0 μM ranitidine solution. Conditions: Potential, 0.20 V; frequency range, 5.0 mHz to 100 kHz; and AC voltage amplitude, 5.0 mV.
Fig. 5. Effect of pH of the working solution on ranitidine adsorption in MIP cavities. The response was measured through CV of the probe solution (5.0 mM K3[Fe(CN)6] and 0.1 M KCl) after immersion of the electrode in a 1.0 μM ranitidine solution for 7.0 min. Scan range = −0.1 to +0.5 V, scan rate = 50 mV s−1.
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Fig. 6. Calibration curve corresponding to the response of the MIP sensor to the concentration of ranitidine. The response was measured through SWV of 5 probe solutions after incubation for 7.0 min with different concentrations of ranitidine in the buffer solution. Conditions: Scan range = −0.1 to +0.5 V; scan rate = 50 mV s−1; frequency, 25 Hz; amplitude, 50 mV.
suitable molecular conformation for ranitidine for better penetration in the recognition cavities. For this purpose, a series of PBS solutions in the pH range of 4.0 to 7.5 containing 1.0 μM ranitidine was examined with an incubation time of 420 s. As shown in Fig. 5, the maximum current response was obtained at pH = 5.5, which was chosen as the suitable pH for further studies.
remove ranitidine from the cavities. The relative standard deviation (RSD) was estimated at about 5.7. This electrode was tested several times every day for two weeks. The results (with recovery values higher than 96%) showed that the modified electrode could be used several times without any major change. So, the proposed imprinting sensor was expected to be regenerated with acceptable reproducibility. Table 2 compares the detection limit of the proposed method and
3.5. Effect of incubation time In electrochemical determination, incubation time is a simple and impressive tool for improving the sensitivity of the imprinted sensor. In this study, the effect of incubation time on the response current was investigated by CV measurement. After template molecules had been removed from the imprinted film, the modified electrode was incubated in 1.0 μM ranitidine solution at pH 5.5 for different incubation times and the reduction of the peak current was recorded. The results show that the oxidation peak current of the probe reduced sharply up to 420 s by increasing the incubation time, before it gradually leveled off. Decreasing oxidation peak current indicates that the amount of ranitidine adsorbed in the imprinted film cavities rose with prolonged incubation time. Thus, 420 s was chosen as the optimum incubation time for the determination of ranitidine. 3.6. Figures of merit
Table 2 Comparison of the detection limit of the proposed method and other reported methods. Method
Detection limit
Ref.
Polarography Potentiometry Colourimetry Visible spectroscopy HPTLC LSV Flow injection fluorometry High-performance liquid chromatography SWV
107 ng/mL N3.0 × 105 ng/mL N5.0 × 104 ng/mL N144 ng/mL N30 ng/mL 1.1 × 104 ng/mL N20 ng/mL 5 ng/mL 6.28 ng/mL
[32] [64] [33] [63] [65] [36] [66] [67] this work
Table 3 Chemical structures of the molecules used. Molecule
SVW measurements were performed to study the relationship between peak current and template-free modified electrode (Δi) vs. ranitidine concentration (Fig. 6). The currents linearly increased with analyte concentration in the range of 0.05 to 2.0 μM following the regression equation of Δi (μA) = 9.121C (μM) + 1.529 (R2 = 0.997) and the limit of detection based on S/N = 3 was 0.02 μM ranitidine. In order to evaluate the reproducibility of the imprinted sensor, the change in the net response of the sensor before and after incubation in the presence of 0.1 μM ranitidine solution was measured in five replicates. After each experiment, the elution was performed with acetic acid to
Warfarin
Furazolidone
Amoxicillin
Table 1 Results of ranitidine determination in real samples (n = 3).
Urine sample
Ranitidine added (μM)
Average of ranitidine found (μM)
Recovery (%)
RSD (%)
– 1.0 2.0 5.0
Not detected 0.93 ± 0.08 1.9 ± 0.14 5.3 ± 0.37
– 93.0 95 106
– 4.5 4.1 3.8
Chlorpromazine hydrochloride
Structure
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Fig. 7. Selective recognition for the imprinted or non-imprinted polymer film electrodes by CV of the probe solution (5.0 mM K3[Fe(CN)6] and 0.1 M KCl) after incubation for 7 min in a 1.0 μM solution of ranitidine in the buffer solution.
those of other methods reported in the literature. It is crystal clear that the sensitivity of the method is remarkably more satisfactory compared to previously reported methods.
sensor was evaluated by using it to determine ranitidine in human urine samples. The results revealed that the imprinted sensor fulfilled the simplicity requirements for ranitidine detection and provided possibilities for clinical application in physiological fluids.
3.7. Selectivity Acknowledgments Furazolidone, amoxicillin, warfarin, and chlorpromazine hydrochloride which have different functional groups similar to the template were selected for assessing the selectivity of the imprinted sensor. The structures of these substances are displayed in Table 3. The MIP sensor (AuNP/MIP/f-MWCNT/PGE) and the NIP electrode (AuNP/NIP/fMWCNT/PGE) were tested (Fig. 7). For the NIP electrode, the electrochemical response for ranitidine and the interfering species were found to be negligible. It could be concluded that there were no active sites on the surface of the NIP electrode to adsorb drugs. The current response of the MIPs electrode for ranitidine was significantly higher than that of the other interfering species. This can be explained by the size, conformation, and functional groups of the cavities that matched ranitidine in the imprinting film. 3.8. Real sample analysis In order to investigate the capability of the prepared sensor for the determination of ranitidine in the complex matrix of real clinical samples, a standard addition method was chosen. Three measurements were performed for each concentration. As can be seen in Table 1, significant recoveries and RSDs were obtained. Therefore, this method may be claimed to have a remarkable capability for the determination of ranitidine in human urine samples. 4. Conclusions In this paper, a novel, simple, disposable, and sensitive molecularly imprinted sol–gel was prepared for the determination of ranitidine using electrochemical deposition of sol–gel imprinted layer and AuNPs for the electro-modification of the electrode. The modified electrode was used to achieve a selective ranitidine sensor with high recoveries. Furthermore, the prepared sensor showed a considerable capability for the detection of ranitidine content as low as 0.02 μM. The imprinted sensor demonstrated fast binding kinetics to the template due to its high ratio of surface imprinted sites as well as high surface to volume ratios (because of introducing MWCNTs and AuNPs). It also showed a great affinity to the template. Eventually, the capability of the proposed
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