Science in China Series B: Chemistry

© 2007 SCIENCE IN CHINA PRESS

Springer

www.scichina.com www.springerlink.com Sci China Ser B-Chem | Aug. 2007 | vol. 50 | no. 4 | 547-553

A capacitive sensor based on molecularly imprinted polymers and poly(p-aminobenzene sulfonic acid) film for detection of pazufloxacin mesilate

ZHOU Lu, YE GuangRong, YUAN Ruo†, CHAI YaQin & CHEN SuMing

College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China

A novel capacitive sensor for pazufloxacin mesilate (pazufloxacin) determination was developed by electropolymerizing p-aminobenzene sulfonic (p-ABSA) and molecularly imprinted polymers (MIPs), which was synthesized through thermal radical copolymerization of metharylic acid (MAA) and ethyl-ene glycol dimethacrylate (EGDMA) in the presence of pazufloxacin template molecules, on the gold electrode surface. Furthermore, 1-dedecanethiol was used to insulate the modified electrode. Alter-nating current (ac) impedance experiments were carried out with a Model IM6e to obtain the capaci-tance responses. Under the optimum conditions, the sensor showed linear capacitance response to pazufloxacin in the range of 5 ng·mL−1 to 5 μg·mL−1 with a relative standard deviation (RSD) 5.3% (n=7) and a detection limit of 1.8 ng·mL−1. The recoveries for different concentration levels of pazufloxacin samples varied from 94.0% to 102.0%. Electrochemical experiments indicated the capacitive sensor exhibited good sensitivity and selectivity and showed excellent parameters of regeneration and stabil-ity.

capacitive sensor, molecularly imprinted polymers, p-aminobenzene sulfonic acid, pazufloxacin mesilate

Pazufloxacin mesilate (pazufloxacin)[(-)-(S)-10-(1-ami- nocyclopropyl)-9-fluoro-3-Methyl-7-oxo-2,3-dihydro-7H- pyrid[1,2,3-de][1,4]benzoxazine-6-carboxylic acid mo- nomethanesulfonate] was a novel fourth generation qui-nolone antimicrobial agent[1]. It was reported firstly in 2002 in Japan and its antimicrobial activity results from a selective antagonism between host DNA and bacterial DNA without interfering with eukaryotic topoisom-erases, which have the similar effect mechanism with ofloxacin and ciprofloxacin. This compound is currently widely used for the treatment of infections, such as res-piratory organs infection, genitourinary organ infection, skin and parenchyma infection, and surgical infection, which is caused by Gram-negative and positive bacte-ria[2]. The chemical structure of pazufloxacin is shown in Figure 1.

Chemiluminescence and chromatography methods were employed for the determination of pazufloxacin[3－5].

Figure 1 The structure of pazuflouxacin mesilate. However, these methods have suffered several disad-vantages, such as low sensitivity and long detection pe- Received August 7, 2006; accepted October 16, 2006 doi: 10.1007/s11426-007-0035-7 †Corresponding author (email: yuanruo@swu.edu.cn) Supported by the National Natural Science Foundation of China (Grant No. 20675064), the Natural Science Foundation of Chongqing City (Grant No. CSTC-2004BB4149 and 2005BB4100) and High Technology Project Foundation of Southwest University (Grant No. XSGX02).

548 ZHOU Lu et al. Sci China Ser B-Chem | Aug. 2007 | vol. 50 | no. 4 | 547-553

riod. As a result, an efficient and rapid method for the determination of pazufloxacin with acceptable assay capability is highly necessary for academic research and practical application. Molecularly imprinted polymers (MIPs) method was an important analytical tool, which showed good mechanical, thermal and chemical proper-ties, as well as low cost. All the merits mentioned above makes these synthetic materials appear ideal chemo- receptors[6]. Recently, some sensors based on MIPs, such as chemiluminescence sensor[7,8], quartz crystal microbalance (QCM) sensor[9,10], and electrochemical sensor[11,12], were reported. Among these sensors, ca-pacitive sensor based on MIPs evoked much attention because it possessed the merits of high sensitivity and label free. Nevertheless, the reported fabrications of the capacitive sensor based on MIPs were complex and ex-pensive[13,14]. Thus, it is important to develop a highly sensitive and cheap capacitive sensor based on MIPs.

Electropolymerization is a good approach to immobi-lize polymers to prepare polymer-modified electrodes (PMEs) as adjusting the electrochemical parameters can control film thickness and excellent stability characteris-tics, which has been widely used for the fabrication of electrode in batches. In this present work, a capacitive sensor was developed for the determination of pazu-floxacin based on MIPs technology and capacitive transducer technology. MIPs could be immobilized into the poly(p-ABSA) film on the gold electrode surface as recognition elements. Also, 1-dedecanethiol was used to insulate the modified electrode. The capacitance of the electrode changed linearly over a certain concentration of pazufloxacin. The capacitive sensor exhibited fast potentiometric response (≤ 3 min). Regeneration of the capacitive sensor was achieved with CH3OH-HAc (9︰1, V/V). The results of the experiment demonstrated that the proposed capacitive sensor had high stability and good regeneration. Furthermore, the results of recovery experiments demonstrated that it was a feasible alterna-tive tool for determining pazufloxacin in human urine. Moreover, this kind of capacitive sensor was easy to prepare with low cost.

1 Experimental

1.1 Reagents and chemicals

L-pazufloxacin mesilate and D.L-pazufloxacin mesilate (bulk pharmaceutical) were purchased from Changzhou

Yabang Pharmaceutical Research Institute Co., Ltd. L-pazufloxacin mesilate, ofloxacin, ciprofloxacin (pri-mary standard) were obtained from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Methacrylic acid (MAA) and 2.2′- azobisisobutyronitrile (AIBN) were obtained from Shanghai Chemical Reagent Company (Shanghai, China). Ethylene glycol dimethacrylate (EGDMA) was obtained from Guangzhou Trade Ltd. (Guangzhou, China). All the reagents were of analytical grade except that AIBN was of chemical purity grade. Doubly dis-tilled water was used throughout the experiments. EGDMA and MAA were distilled and AIBN was recrystallized before use.

1.2 Apparatus

Alternating current (AC) impedance experiments were carried out with a Model IM6e (ZAHNER Elektrik, Germany). Cyclic voltammetric (CV) measurements were carried out with a CHI 660A electrochemistry workstation (Shanghai CH Instruments, China). The pH measurements were made with a pH meter (MP 230, Mettler-Toledo Switzerland). Bransonic 200 ultrasonic cleaner was used in the experiments (BRANSON Ul-traschall Co., Ltd. Germany).

All electrochemical experiments were carried out with a conventional three-electrode system with gold electrodes (φ = 4 mm) unmodified or covered by differ-ent layers as working electrodes, a platinum wire as aux-iliary electrode and a saturated calomel reference elec-trode (SCE) as reference electrode in 0.02 mol·L−1 phosphate buffer (pH = 7.4).

1.3 Synthesization of MIPs

The MIPs were synthesized according to ref. [15] with some modifications as in the following description: 1 mmol template (pazufloxacin) and 12 mmol MAA monomer were dissolved in 10 mL dichloromethane sol- vent in a 25 mL glass ampoule. The mixture was surged ultrasonically for 1 h. Then, 40 mmol EGDMA cross- linker and 0.3 mmol AIBN initiator were added into the mixture. After bubbling with N2 for 20 min and sonicat-ing for 5 min, the ampoule was sealed and controlled at 60℃ for 24 h in a water bath curved by a thermostat.

The dried polymeric monolith was thoroughly ground in a mortar and sieved with 280-mesh griddle. Part of MIPs particles were washed with CH3OH-HAc (9︰1, V/V) solvent until the absence of pazufloxacin, and then

ZHOU Lu et al. Sci China Ser B-Chem | Aug. 2007 | vol. 50 | no. 4 | 547-553 549

Figure 2 The preparation of pazufloxacin imprinted polymer. vacuum dried. The schematic diagram of the MIPs synthesization is shown in Figure 2.

1.4 Preparation of capacitive sensor

The gold electrode (φ = 4 mm) was carefully polished with alumina slurries (1.0, 0.3 μm) on microcloth pads, immersed in the piranha solution for 10 min, and then rinsed with water. Afterward, the resulting electrode was cleaned through sonication in acetone and doubly dis-tilled water for 5 min, respectively. Finally, the electrode was etched in 0.5 mol·L−1 H2SO4 solution by cycling electrode potential from −0.3 to +1.5 V until a repro-ducible voltammetric response was obtained. The elec-trode was then dried in air at room temperature.

3.0 mg MIP was added into 2 mL 2×10−3 mol·L−1 (p-ABSA) solution, and then ultrasonically vibrated for 20 min to prepare the polymerization solution. The elec-tropolymerization was carried out with a conventional three-electrode system according to ref. [16]. The pre-pared electrode was dried in air for 12 h. Then, the elec-trode was treated with 5% 1-dedecanethiol for occluding the defects of the polymer film for 5 h. Finally, the re-sulted electrode was stored at room temperature for the further use.

2 Results and discussion

2.1 Selection of fabrication condition for sensor

The poly(p-ABSA) film doped with MIPs was obtained

by electropolymerizing p-ABSA in the presence of MIPs. The cyclic voltammograms of electropolymerization are shown in Figure 3. From the diagram, it was found that the values of anodic peak and cathodic peak almost kept constant. The poly(p-ABSA) film containing MIPs with scattered structure probably improved the conductive ability of the polymer film and resulted in the constant CV peak current. In this experiment, we also investi-gated the effect of different MIPs concentrations and the potential scan number of electropolymerization on the characteristics of the electrode (Table 1). The prepared capacitive electrode showed the best analytical per-

Figure 3 Repetitive cyclic voltammograms of 2.0 × 10−3 mol·L−1 p-ABSA and MIPs in 0.1 mol·L−1 NaCl solution. Terminal potential: +2.5 V; initial potential: −1.5 V; sensitivity: 1.0×10−4 A·V−1; scan rate: 100 mV·s−1.

550 ZHOU Lu et al. Sci China Ser B-Chem | Aug. 2007 | vol. 50 | no. 4 | 547-553

Table 1 Parameters of electropolymerization and the analytical performance of the capacitive sensor

Electrode MIP (mg) Electropolymerization number

Capacitance shift (nf·cm−2)

Linear range (μg·mL−1)

Detect limit (μg·mL−1)

A 1 6 −87.3 8.7×10−3~0.93 2.90×10−3 B 1 8 −90.7 7.7×10−3~1.13 2.56×10−3 C 1 10 −95.1 7.3×10−3~1.22 2.43×10−3 D 1 12 −92.2 7.4×10−3~1.07 2.46×10−3 E 2 6 −100.5 7.1×10−3~0.52 2.36×10−3 F 2 8 −105.2 6.0×10−3~0.70 2.00×10−3 G 2 10 −107.1 5.9×10−3~0.87 1.96×10−3 H 2 12 −109.5 6.3×10−3~1.15 2.10×10−3 I 3 6 −111.3 5.7×10−3~3.61 1.90×10−3 J 3 8 −114.7 6.4×10−3~3.97 2.13×10−3 K 3 10 −117.7 5.0×10−3~5.00 1.80×10−3 L 3 12 −104.3 6.1×10−3~4.73 2.03×10−3 M 4 6 −121.7 8.2×10−3~3.12 2.73×10−3 N 4 8 −118.5 8.3×10−3~4.20 2.76×10−3 O 4 10 −119.1 8.0×10−3~4.00 2.70×10−3 P 4 12 −115.5 9.5×10−3~2.75 3.16×10−3 Q 5 6 −116.7 6.7×10−3~3.55 2.23×10−3 R 5 8 −110.3 7.5×10−3~4.07 2.50×10−3 S 5 10 −112.6 7.7×10−3~4.40 2.56×10−3 T 5 12 −113.6 7.2×10−3~4.50 2.40×10−3

formances when 10 cycles and MIPs with 3 mg were used.

2.2 CV characterization of sensor

In order to characterize the insulating property of the electrode at each modification stage, CV was performed in 5 mol·L−1 K3Fe(CN)6/K4Fe(CN)6 solution (pH 7.4). Compared with the CV redox couple of bare gold elec-trode (Figure 4, curve a), the reversible peaks current of the redox couple of the electrode modified with poly (p-ABSA) film containing MIPs dropped significantly (Figure 4, curve b). However, the Faraday current can still be observed because the polymer film could not overlay the surface of the gold electrode absolutely. Furthermore, the permeable redox couple can still pene-trate the modified polymer film. In order to reduce the background current, 1-dodecanethiol was employed to block the defects in the polymer film. The peak current declined more after the treatment (Figure 4, curve c). The phenomena demonstrated that 1-dodecanethiol can penetrate the polymer film and occlude the defects of the polymer film.

2.3 Determination of capacitance and frequency

When the electrode was immersed in an electrolyte solu-tion, it can generally be described as resembling a ca-pacitor in its ability to store charge at the interface be-tween the electrode and the solution[17]. Based on the theory of the electrical double-layer, the total capaci-tance (Ctot) can be described by the following equation:

1/Ctot = 1/Cins + 1/Crec + 1/CGC. (Cins: the capacitance of the insulating layer; Crec: the capacitance of the recogni-tion layer; CGC: the capacitance of the diffuse layer). When the insulating layer is insulated and the recogni-tion layer covers the surface of the electrode completely, the Crec can dominate the total capacitance.

Figure 4 Cyclic voltammograms of the electrodes in PBS (pH 7.4) containing 5 mmol·L−1 potassium ferricyanide and 0.1 mol·L−1 KCl at different stages. a, Bare gold electrode; b, the electrode modified with poly(p-ABSA) and MIPs; c, the modified electrode treated with 1-dodecanethiol.

The value of capacitance (Ctot/F) is calculated directly from the imaginary component of the impedance (−Z″/Ω) according to the following equation[18]: C = −1/2πfZ″, where f is the ac frequency, Z″ is the imaginary imped-

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ance. According to Randles equivalent circuit model, the precise capacitance detection requires that the measure-ments must be performed in a regime that the transduc-tion layer exhibits a near-ideal capacitor behavior, i.e., the phase angle value close to −90°. In this experiment, the working frequency was chosen in the Bode diagram. In Figure 5, the phase angle value is close to −90° at the frequency of 1 kHz. Thus, 1 kHz was chosen as the working frequency.

Figure 5 Bode plot of the capacitive sensor.

2.4 Optimization of experimental parameters

2.4.1 Influence of different buffer solution. The in-fluence of the buffer solution on the capacitive responses of the prepared electrode was investigated with different pH (4.0－10.0). The prepared electrode was immersed in 0.02 mol·L−1 acetate buffer, 0.02 mol·L−1 phosphate buffer, 0.02 mol·L−1 carbonate buffer, and double dis-tilled water with the same pazufloxacin concentration, respectively. Then, the capacitive responses were meas-ured. The experiment results showed that the capacitive changes were maximal in the 0.02 mol·L−1 phosphate buffer solution. Considering the pH of human body fluid, 0.02 mol·L−1 phosphate buffer (pH = 7.4) was chosen as the measurement buffer solution.

2.4.2 Influence of incubation time. The prepared sensor was incubated in 0.02 mol·L−1 phosphate buffer containing 0.50 μg·mL−1 pazufloxacin. The capacitive changes were monitored from 2 to 30 min. It was found that the capacitive response decreased with the increas-ing time up to 3 min. Therefore, 3 min was selected to evaluate the analytical performance of the sensor in all experiments reported. Compared with the same kind of

capacitive sensors in refs. [19,20], the proposed capaci-tive electrode has merit of fast response time.

2.4.3 Effect of temperature. The modified electrode was immersed into 0.02 mol·L−1 phosphate buffer con-taining 0.50 μg·mL−1 pazufloxacin and was incubated for 3 min. Then, the effect of temperature on the capaci-tive response was examined at the range from 20 to 50℃. According to the study of the temperature influ-ence, we found that the temperature changes had little effect on the capacitive shift of the sensor. Considering the convenience of the daily clinical analysis, room temperature (25±1℃) was chosen as the measurement temperature in our study.

2.5 Selectivity against interferences

In order to evaluate the selectivity of the capacitive sen-sor, the effect of possible components in urine was stud-ied. The tolerable limit of existed species was taken as a relative error less than 5% for 0.50 μg·mL−1. A 0.50 μg·mL−1 pazufloxacin was analyzed by being added with interfering species. It showed that more than 1000-fold excess of K+, Na+, Cl−, 800-fold excess of uric acid, urea, 500-fold excess of starch, glucose, citric acid, and 100-fold excess of ascorbic acid did not interfere.

0.50 μg·mL−1 L-pazufloxacin and 0.50 μg·mL−1 D, L-pazufloxacin were tested by the same electrode, re-spectively. The experiment results showed the sensor had the capacitive changes of −46.21 nf·cm−2 to L-pazufloxacin and −92.37 nf·cm−2 to D, L-pazufloxacin. The capacitive responses value to 0.50 μg·mL−1 D, L-pazufloxacin was 50.03% of its capacitive value to the same concentration of L-pazufloxacin. Moreover, the structural analogs, such as ofloxacin and ciprofloxacin, were also investigated. The results showed that 100-fold excess of ofloxacin and 50-fold excess of ciprofloxacin did not interfere. According to all the experiments men-tioned above, the proposed capacitive sensor could be used directly to determinate L-pazufloxacin selectively.

2.6 Detection of pazufloxacin

With the optimization of experimental parameters, the resulted capacitive sensor was used to incubate in pazu-floxacin solution of different concentration and was taken to a conventional electrochemical cell of 0.02 mol·L−1 phosphate buffer (pH = 7.4) at room temperature. The linear range covered from 5.0 ng·mL−1 to 5.0

552 ZHOU Lu et al. Sci China Ser B-Chem | Aug. 2007 | vol. 50 | no. 4 | 547-553

μg·mL−1 with a regression equation of the form ΔC (nf·cm−2) = −25.14 lg [L-PFM] − 99.58 (μg·mL−1) and correlation coefficient of 0.9901. The detection limit corresponding to three times the standard deviation of the blank solution was estimated as 1.8 ng·mL−1. Com-pared with those of conventional chemiluminescence methods[3－5], the proposed capacitive sensor showed the lower detection limit.

2.7 Reproducibility of the sensor

The reproducibility of the same capacitive electrode was examined at a pazufloxacin concentration of 0.0050, 0.050, 0.60, 1.20, 3.75 μg·mL−1. Under the optimization of experimental conditions, the electrode showed an ac-ceptable repeatability with a relative standard deviation (RSD) of 5.3% for seven successive assays. The fabrica-tion reproducibility was estimated from the response for 0.50 ng·mL−1 pazufloxacin at five different electrodes, and the RSD was calculated to be 3.7%.

2.8 Regeneration and stability

Regeneration was employed to evaluate the analytical performance of the capacitive sensor. The complex of MIPs and template is highly stable because the interac-tion between MIPs and template rely on the multiple non-covalent bond coupling and multiple active site co-operation. There are two methods to regenerate the sen-sor: one is the template was destroyed because the de-tection was realized through the chemical reaction of the template, and then the template was removed from the MIPs; the other is to break the MIPs-template bond through regeneration solution. In this experiment, CH3CN-0.01 mol·L−1 HNO3, CH3OH-0.01 mol·L−1 HNO3 and CH3OH-HAc (9︰1, V/V) were used to re-generate the sensor, respectively. The resulting sensors were immersed in a stirred regeneration solution and removed to wash. After 20 min, the sensor was rinsed with double distilled water. The experiment results showed that the sensor treated with CH3OH-HAc (9︰1, V/V) provided the best analytical parameters. After 30 times of regenerations, the sensor retained 91% of its initial capacitive value.

The storage stability of the electrode was tested over a 70-d period. The capacitive electrode was stored at

room temperature and measured intermittently (every 3 d). After completing each assay, the sensor was im-mersed in regeneration solution CH3OH-HAc (9︰1, V/V) for 20 min to regenerate the sensor. The experi-ment result showed that it retained 93% of its initial ca-pacitive value over 69 d.

2.9 Recovery experiment

Following these detailed procedures, the proposed method was applied to determine pazufloxacin in the urine sample. A known amount of standard solution added to the 2.5 mL urine sample was spiked with a dif-ferent concentration urine sample, respectively, and the mixture was diluted to 25mL with double distilled water. Under the optimum experimental conditions, the recov-eries for different concentration levels of pazufloxacin varied from 94.0% to 102.0%. The results of the assay of pazufloxacin in the urine sample are listed in Table 2 and indicate that the proposed capacitive sensor could be applied in the clinical experiment. Table 2 Results of sample recovery experiment

Sample Added (μg·mL−1) Founded (μg·mL−1) Recovery (%)1 0.0050 0.0047 94.0 2 0.050 0.051 102.0 3 0.60 0.58 96.7 4 1.20 1.17 97.5 5 3.75 3.76 100.3

3 Conclusion

In this paper, molecularly imprinted polymers technol-ogy was combined with capacitive transducer technol-ogy to construct a new capacitive sensor for the detec-tion of chiral pazufloxacin. The results of the experiment demonstrated that the sensor had high selectivity and sensitivity, relatively wide linear range and long lifetime. Practically no remarkable decrease was observed in ca-pacitance response after 30 measurements. Moreover, the proposed capacitive sensor was easy to prepare with low cost. This method not only determines the lower L-pazufloxacin concentration but also immobilize some other MIPs to detect different drugs. Therefore, this method has a good application perspective for the drug analysis.

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