Development of an electrochemical immunoassay for detection of gatifloxacin in swine urine

Development of an electrochemical immunoassay for detection of gatifloxacin in swine urine 1 first-author 2 1 3 1 1 1 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:jintliu@sdu.edu.cnxirimo2000@yahoo.com †E-mail:jintliu@sdu.edu.cnxirimo2000@yahoo.com jintliu@sdu.edu.cn 2 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:jintliu@sdu.edu.cnxirimo2000@yahoo.com †E-mail:jintliu@sdu.edu.cnxirimo2000@yahoo.com jintliu@sdu.edu.cn School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100080, China Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100080, China To detect gatifloxacin (GAT) residue in swine urine, an electrochemical immunoassay was established. An indirect competitive immunoassay was developed, in which the coating antigen is immobilized in an enzyme-linked immunosorbent assay (ELISA) plate and GAT residue from the sample competes with the limited binding sites in added anti-GAT antibody. Horseradish peroxidase (HRP) conjugated to goat anti-rabbit IgG was used as the enzymatic label. A carbon fiber working electrode was constructed and current signals were detected by using hydrogen peroxide as a substrate and hydroquinone as an electrochemical mediator. The electrochemical immunoassay was evaluated by analysis of GAT in buffer or swine urine and an average value of half inhibition concentration (IC 50 ) of 8.9 ng/ml was obtained. Excellent specificity of the antibody was achieved with little cross-reaction with lomefloxacin (3.0%), ciprofloxacin (3.0%), and ofloxacin (1.9%) among commonly used (fluoro)quinolones. In conclusion, the immunoassay system developed in this research can be used as a rapid, powerful and on-site analytical tool to detect GAT residue in foods and food products. Gatifloxacin Drug residue Antibody Carbon fiber electrode Electrochemical immunoassay Introduction 1 Gatifloxacin (GAT, Fig. 1 1 ) is a fourth generation synthetic antibacterial (fluoro)quinolone agent. It has been widely used in clinical medicine and as a veterinary drug in animal husbandry (Hooper, 1998 1998 ; Blondeau et al., 2000 2000 ; Weigel et al., 2002 2002 ; Kays et al., 2005 2005 ) for the treatment of acute bacterial exacerbation of chronic bronchitis, acute sinusitis, community acquired pneumonia, uncomplicated urinary tract infections (cystitis), and complicated urinary tract infection (Perry et al., 1999 1999 ). However, the presence of GAT in food samples constitutes a potential hazard for consumers and may cause abnormalities in glucose metabolism (Bhasin et al., 2005 2005 ). As a consequence, the US Food and Drug Administration (FDA) has claimed that the potential risk associated with GAT administration must be emphasized in the drug description. In May 2006, Bristol-Myers Squibb (BMS), the developer of GAT, even asked suppliers to stop selling GAT due to increasing concerns about the side effects. In China, GAT was found to inhibit hens from laying eggs (Amin et al., 2007 2007 ). Due to the important effects of GAT on human and animal health, extensive research has been conducted to generate sensitive and specific methods for rapid and cost-effective GAT detection in food and biological samples. The main analytical techniques used for GAT analysis in foodstuffs include high pressure liquid chromatography (HPLC) (Overholser et al., 2003 2003 ), liquid chromatography-mass spectrometry (LC-MS) (Vishwanathan et al., 2001 2001 ), high-performance thin layer chromatography (HPTLC) (Suhagia et al., 2006 2006 ), liquid chromatography (LC) (Amin et al., 2007 2007 ), capillary electrochromatography (Fierens et al., 2000 2000 ), and fluorescence methods (Espinosa-Mansilla et al., 2006 2006 ). The chromatographic techniques usually require extraction, per-concentration steps, and complicated cleanup procedures. Moreover, expensive and sophisticated equipment is required, which must be run by skilled operators. As a consequence, routine and efficient residue monitoring by the official agencies has become problematic. To protect consumer health, official laboratories should be able to test a large number of samples using rapid, sensitive, cost-effective, high-throughput and on-site analytical methods. Immunoassay, on the other hand, has proven to be an excellent on-site analytical strategy as a sensitive, timely, inexpensive and high-throughput technique that can be used to detect veterinary drugs in a variety of food matrices (Zhou et al., 2005 2005 ; Liu et al., 2007 2007 ). Immunoanalytical techniques have been reported to detect GAT by an enzyme-linked immunosorbent assay (ELISA) using ultraviolet (UV) or fluorescence absorbance as a quantitative parameter (Santoro et al., 2006 2006 ; Zhao et al., 2007 2007 ). In recent years, electrochemical immunoassays have attracted attention because of their excellent sensitivity, simplicity, inexpensive construction, suitability for mass production, and their variety of labels, including enzymes and electro-active molecules. For the development of hand-held devices which can be used for daily measurements, electrochemical immunoassay is a promising alternative to the existing ELISA tests. Also, electrochemical immunoassays are preferable for opaque matrices (Laschi et al., 2003 2003 ; Conneely et al., 2007 2007 ; Piermarini et al., 2007 2007 ). The aim of this work was to develop an electrochemical immunoassay to detect GAT in biological samples. To our knowledge, there are no published reports of an electrochemical immunoassay for GAT detection. In this research, an indirect competitive immunoassay was established in which the coating antigen is immobilized onto an ELISA plate and GAT from a sample competes with the limited binding sites in the anti-GAT antibody. Horseradish peroxidase (HRP) conjugated to goat anti-rabbit IgG was added as an enzymatic label. A carbon fiber working electrode was fabricated to detect current signals on the ELISA plate caused by the electroreaction with hydrogen peroxide as the substrate and hydroquinone (HQ) as the electrochemical mediator. The current response was used to indicate the competition of GAT with coating antigen for binding with the anti-GAT antibody (Ni et al., 2006 2006 ). The electrochemical immunoassay was evaluated by analysis of GAT in buffer or swine urine. This electrochemical immunoassay shows good performance in terms of sensitivity and specificity and could be used as a screening method to detect GAT residues in foods and food products. Materials and methods 1 Chemicals and materials 2 GAT, lomefloxacin, ofloxacin, ciprofloxacin, fleroxacin, enrofloxacin, enoxacin, norfloxacin, pefloxacin, sarafloxacin, danofloxacin and pipemidic acid were obtained from the Institute of Veterinary Medicine of China (Beijing, China). 1-Ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC), N -hydroxysuccinimide (NHS), bovine serum albumin (BSA), and ovalbumin (OVA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Goat anti-rabbit IgG-HRP conjugate was provided by the Military Medical Institute (Beijing, China). HQ was purchased from Hongye Chemical Technology (Tianjin, China). Dimethylformamide (DMF), hydrogen peroxide [H 2 O 2 , 30% (mass fraction)], and other reagents were purchased from Guangmang Chemical Co. (Jinan, China). Sodium phosphate buffer (PBS, pH 7.4) consisted of NaCl (138 mmol/L), KH 2 PO 4 (1.5 mmol/L), Na 2 HPO 4 ·12H 2 O (7 mmol/L), and KCl (2.7 mmol/L). Washing buffer (PBST) comprised the PBS solution containing 0.05% (v/v) Tween 20. Carbonate buffer (pH 9.6) containing 15 mmol/L Na 2 CO 3 and 35 mmol/L NaHCO 3 was used as a coating buffer. The blocking buffer was 0.1 mol/L PBS mixed with 0.01 g/ml of OVA and 0.05% of Tween 20. Ultrapure deionized water with resistivity > 18 MΩ∙cm (Milli-Q) was used throughout this study. The 2.0 mmol/L H 2 O 2 was used as the substrate solution. The mediator solution was 2.0 mmol/L HQ solution, prepared by dissolving 17.6 mg HQ in 20 ml of PBS. Apparatus 2 Amperometric signal was monitored by an electrochemical potentiometer (CHI 800, Shanghai Chenhua Instrument Ltd., China). The electrochemical system consisted of a three-electrode system in which a carbon fiber electrode was used as a working electrode (Fig. 2 2 ), a platinum wire was used as an auxiliary electrode, and filamentous silver was used as a reference electrode. The electrochemical reaction was carried out in one well of a polystyrene 12-well microtiter plate (Bio Basic Inc., Ontario, Canada). Construction of carbon fiber working electrode 2 Carbon fiber working electrodes are widely used as probes for biochemical detection in the wells of microplates (Jin et al., 2003 2003 ; He et al., 2003 2003 ). Our electrode was constructed as described in Fig. 2 2 . First, 20–30 carbon fibers (6 μm diameter) soaking in acetone were carefully inserted into a fused silica capillary (250 μm i.d., 375 μm o.d., 0.6 cm length) by gluing end A using ethyl α-cyanoacrylate. The fused silica capillary was then inserted into a glass capillary (2 mm i.d., 3 mm o.d., 3 cm length) with end B sealed using epoxy AB adhesive. Mercury (2.0 cm height) was then injected into one end of the glass capillary while at the other end, copper wire was inserted and sealed with epoxy AB adhesive. Before each use, the end A was sonicated for 10 min in ethanol and deionized water (Milli-Q water, > 18 MΩ∙cm). Preparation of polyclonal antibody for GAT 2 The immunogen cationized BSA (cBSA)-GAT and coating antigen cOVA-GAT for GAT were prepared via the carbodiimide-modified active ester method, with EDC and NHS used as crosslinking agents. Two male New Zealand white rabbits were subcutaneously immunized to obtain polyclonal antibody for GAT. The synthesis procedure and immunization design were the same as those reported in our previous study (Zhao et al., 2007 2007 ). The purified antiserum was aliquotted and stored at −70 °C. Immunoassay procedure 2 The immunoassay was developed on the basis of an indirect competition scheme (Fig. 3 3 ), in which GAT and goat anti-rabbit IgG-HRP compete for the binding sites of the anti-GAT antibody. First, the coating antigen cOVA-GAT (10 μg/ml, 100 μl/well) was immobilized onto the surface of ELISA plates for the capture of the antibody. The microplates were incubated overnight at 4 °C and were then blocked with 250 μl/well of blocking buffer, followed by incubation for 2 h at 37 °C. Between each step, the microplates were washed three times with washing buffer. Then, 100 μl of anti-GAT antibody was added, followed by addition of buffer or competitor solution (GAT samples or similar compounds analyzed), and the plates were incubated for 2 h. Goat anti-rabbit IgG-HRP (1:1000, 100 μl/well) was added. The competitive immunoreaction was then allowed to proceed for 70 min at 37 °C. Then HQ mediator solution and H 2 O 2 substrate solution were added (50 μl/well). The HRP enzymatic reaction progressed for another 10 min at room temperature. Then, the carbon fiber working electrode was inserted and amperometric signals were determined at −0.15 V. The result was expressed in percent inhibition as follows: I / I 0 ×100%, where I is the peak current in the linear sweep voltammetry (LSV) profile for the well containing competitor and I 0 is that for the well without competitor. Results and discussion 1 Optimization of the assay 2 As ideal chemical sensors, microelectrodes have attracted widespread research interest and have been studied intensively in many fields (Wightman et al., 1988 1988 ; Huang et al., 2001 2001 ) to facilitate rapid measurement of chemical compounds. Carbon fiber microelectrodes are important components of the wider microelectrode family. Several electrochemical assays for rapid analysis with carbon fiber microelectrodes have been described, including amperometry, differential pulse voltammetry, and cyclic voltammetry (Pihel et al., 1996 1996 ; Blanco et al., 1999 1999 ; Netchiporouk et al., 2001 2001 ). The carbon fiber electrodes used in this study were fabricated using a traditional preparation method. The competitive immunoassay was performed on ELISA microplate wells, in which GAT from the sample and the immobilized coating antigen compete for the binding sites of the antibody. When the analyte molecular recognition event happens, the extent of the reaction is evaluated by the electrochemical detection of the mediator (HQ) based directly on the activity of the enzyme tracer (goat-anti-rabbit IgG-HRP). Hydrogen peroxide was used to facilitate the enzymatic reaction (Lei et al., 2004 2004 ; Hervás et al., 2009 2009 ). After the reaction was allowed to proceed for 10 min, the electrode was inserted and LSV evaluation was employed to examine current signals for each group with the potential fixed. The response was determined at −0.15 V (previously optimized) on the carbon fiber working electrode where the reduction of the enzymatically oxidized mediator HQ took place. The concentrations of substrate (H 2 O 2 ), mediator (HQ), and enzyme tracer (HRP) are important factors that affect the sensitivity of the immunoassay, and were optimized by testing current values for each group. The tested concentration of H 2 O 2 was in the range of 0.4−3.2 mmol/L, using 1 μl/ml of IgG-HRP and 2.0 mmol/L of HQ (Fig. 4a 4a ). The current response increased gradually when the concentration of H 2 O 2 increased until a peak was reached at 2.0 mmol/L, then the current measured started to decrease as the concentration of H 2 O 2 continued to increase (Fig. 4a 4a ). As the concentration of H 2 O 2 increased, electron transfer was enhanced. However, when the concentration of H 2 O 2 increased further from 2.0 to 3.2 mmol/L, the reaction was inhibited by the excess of H 2 O 2 . Thus, a concentration of 2.0 mmol/L of H 2 O 2 was selected as the optimal concentration throughout the research. The optimization result for the peroxide concentration is in good agreement with Ordóñez and Fàbregas ( 2007 2007 ). The influences of (a) H 2 O 2 (2.0 mmol/L HQ), (b) HQ (2.0 mmol/L H 2 O 2 ), (c) incubation time (2.0 mmol/L HQ and 2.0 mmol/L H 2 O 2 ), and (d) ultrasonic for the working electrode (2.0 mmol/L HQ, 2.0 mmol/L H 2 O 2 , and 10 min for incubation) on current response in the competitive assay are shown. The concentration of IgG-HRP conjugate was 1 μl/ml for protocols (a) to (c). The experiments were carried out in PBST (pH 7.4) containing NaCl (138 mmol/L), KH 2 PO 4 (1.5 mmol/L), Na 2 HPO 4 ·12H 2 O (7 mmol/L), KCl (2.7 mmol/L), and Tween 20 (0.05%), with the applied potential of −0.15 V It was reported that reducing substrates, such as HQ, have a low specificity for catalyzing the oxidation of H 2 O 2 (Zhang et al., 2008 2008 ). To find out the most suitable quantity of HQ, a series of concentrations in the range of 0.4–3.2 mmol/L of HQ were evaluated, with the concentrations of H 2 O 2 and IgG-HRP fixed at 2.0 mmol/L and 1 μl/ml, respectively. The maximum current was observed when the concentration of HQ was 2.0 mmol/L (Fig. 4b 4b ). The decrease in current when the concentration of HQ was more than 2.0 mmol/L was probably caused by the limited enzyme-substrate kinetics (Lei et al., 2004 2004 ). The optimized concentration of HQ, therefore, was determined to be 2.0 mmol/L and was used throughout this research. The effect of the immunochemical incubation time was also investigated due to its importance. The concentrations of both H 2 O 2 and HQ were fixed at 2.0 mmol/L, and the concentration of goat-anti-rabbit IgG-HRP conjugate was fixed at 1 μl/ml. The current was measured at 10 min intervals (Fig. 4c 4c ). Although, the current measured can be increased to a high value, we eventually chose a 10-min incubation time because the current measured at this time was high enough to make a judgment. More importantly, the experimental time can thereby be shortened significantly. Non-specific adsorption due to the direct adsorption of enzyme tracer onto the working electrode surface could produce a residual background signal when the electrode is moved to the next well. This effect was evaluated by comparing the relationship of current response with IgG-HRP concentration while ultrasonic and control electrodes were used. The results (Fig. 4d 4d ) showed that non-specific adsorption in non-ultrasonic groups markedly affected the linear relationship for GAT detection. Therefore, in our research, the working electrode was sonicated in PBST for 10 min to stabilize the background before each measurement. Sensitivity and specificity of the test 2 The electrochemical immunoassay was developed under the optimized condition (2.0 mmol/L H 2 O 2 , 2.0 mmol/L HQ, and 1 μl/ml IgG-HRP). Antiserum with a dilution factor of 1:8 000 was utilized as the primary antibody and co-incubated with GAT from 0.01 to 1 000 ng/ml to compete with the coating antigen for binding the antiserum. The competitive inhibition curve and standard calibration curve ( Y =0.86−0.31 X , R 2 =0.99) are shown in Figs. 5a and 5b 5a and 5b , respectively. The GAT concentration with 50% inhibition (IC 50 value) was estimated to be 10.5 ng/ml and the linear concentration range was from 1 to 100 ng/ml. c GAT : concentration of GAT (ng/ml) The specificity of the antibody was evaluated by testing the cross-reactivity with various (fluoro) quinolones. The cross-reactivity studies were carried out with the electrochemical immunoassay by adding various competitors in concentrations from 0.01 to 1000 ng/ml (Lu et al., 2006 2006 ). The results confirmed that the anti-GAT antibody was highly specific and showed little cross-reactivity with lomefloxacin, ciprofloxacin, or ofloxacin (Table 1 1 ). Compound IC 50 (ng/ml) Cross-reactivity (%) Gatifloxacin 10.5 100.0 Lomefloxacin 280 3.0 Ciprofloxacin 345 3.0 Ofloxacin 548 1.9 Enrofloxacin >1000 <1 Sarafloxacin >1000 <1 Flerfloxacin >1000 <1 Pipemidic acid >1000 <1 Pefloxacin >1000 <1 Norfloxacin >1000 <1 Enoxacin >1000 <1 Detection of GAT in swine urine 2 Urine, composed of various electrochemical interferents, such as uric acid, ascorbic xanthine, ascorbic acid, and p -aminophenol (Jamal et al., 2005 2005 ), was selected to study the matrix effect (Yang et al., 2006 2006 ). Swine urine was centrifuged at 15 000 r/min for 15 min and the supernatant obtained was diluted with PBST. The competitive curves with final GAT concentrations of 0.01, 0.1, 1, 5, 10, 50, 100, 1 000 and 10 000 ng/ml were run in PBST and in various dilutions (1:2, 1:5, 1:10, and 1:20) of the extraction to determine the matrix effect (Fig. 6 6 ). The IC 50 values with GAT as the competitor were 66.07, 29.36, 12.37, and 12.59 ng/ml when the dilution factors were 1:2, 1:5, 1:10, and 1:20, respectively. It was possible that electrochemical interferents would restrain the electron transfer, resulting in an increase in background current signal. The results were in good agreement with those observed in a milk system (Zhao et al., 2007 2007 ). The average IC 50 value of a 1:10 dilution was close to that in PBST (8.9 ng/ml), and was chosen for performance of the immunoassay. Based on the standard curve obtained in 1:10 dilution, swine urine was used as a real system to test the applicability of the electrochemical immunoassay (Table 2 2 ). A series of GAT solutions with concentrations of 1, 5, 10, 50, and 100 ng/ml were added to the urine sample, and the recovery rate and coefficient of variation (CV) were calculated. The CVs were below 13.38% and the recovery rates were between 93% and 118%, indicating the reliable accuracy of the test when applied to swine urine (Table 2 2 ). c GAT : concentration of GAT (ng/ml) GAT level (ng/ml) n Measured GAT level (ng/ml) Recovery (%) CV (%) 1 3 1.13±0.09 112.54 8.16 5 3 4.69±0.62 93.72 13.38 10 3 11.74±0.38 117.36 3.27 50 3 48.30±1.62 96.60 3.35 100 3 105.06±2.27 105.06 2.16 Conclusions 1 In this work, a carbon fiber working electrode was prepared and a sensitive electrochemical immunoassay was developed to detect GAT residue in swine urine. The average IC 50 value for this test was found to be 8.9 ng/ml, and there was little cross-reactivity with other common (fluoro)quinolones. The utilization of the assay was also tested by using it to detect GAT in spiked swine urine samples. Satisfactory results were obtained (CV < 13.38%; recovery rates < 20%). In summary, the potential application of the electrochemical immunoassay for the detection of GAT residue in swine urine was demonstrated. The method developed could be commercialized in the form of a hand-held device to detect GAT residue in foods. References AS Amin , AAEF Gouda , R El-Sheikh , F Zahran . Spectrophotometric determination of gatifloxacin in pure form and in pharmaceutical formulation . Spectrochim Acta A , 2007 . 67 ( 5 ): 1306 - 1312 . DOI: 10.1016/j.saa.2006.09.041 http://doi.org/10.1016/j.saa.2006.09.041 . Spectrophotometric determination of gatifloxacin in pure form and in pharmaceutical formulation R Bhasin , FC Arce , R Pasmantier . Hypoglycemia associated with the use of gatifloxacin . Am J Med Sci , 2005 . 330 ( 5 ): 250 - 253 . 16284487 DOI: 10.1097/00000441-200511000-00010 http://doi.org/10.1097/00000441-200511000-00010 . Hypoglycemia associated with the use of gatifloxacin MH Blanco , LH Quintana , L Hernandez . Determination of dihydrozeatin riboside in apples by anodic stripping voltammetry with a carbon fiber microelectrode . Fresenius J Anal Chem , 1999 . 364 ( 3 ): 254 - 260 . DOI: 10.1007/s002160051333 http://doi.org/10.1007/s002160051333 . Determination of dihydrozeatin riboside in apples by anodic stripping voltammetry with a carbon fiber microelectrode JM Blondeau , R Laskowski , J Bjarnason , C Stewart . Comparative in vitro activity of gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin and trovafloxacin against Gram-negative and Gram-positive organisms . Int J Antimicrob Agents , 2000 . 14 ( 1 ): 45 - 50 . 10717500 DOI: 10.1016/S0924-8579(99)00143-0 http://doi.org/10.1016/S0924-8579(99)00143-0 . Comparative in vitro activity of gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin and trovafloxacin against Gram-negative and Gram-positive organisms G Conneely , D O′Mahony , H Lu , GG Guilbault , M Pravda , M Aherne . An immunosensor for the detection of stanozolol in bovine urine . Anal Lett , 2007 . 40 ( 7 ): 1280 - 1293 . DOI: 10.1080/00032710701326650 http://doi.org/10.1080/00032710701326650 . An immunosensor for the detection of stanozolol in bovine urine A Espinosa-Mansilla , AM de la Pena , DG Gomez , FS Lopez . Determination of fluoroquinolones in urine and serum by using high performance liquid chromatography and multiemission scan fluorimetric detection . Talanta , 2006 . 68 ( 4 ): 1215 - 1221 . 18970452 DOI: 10.1016/j.talanta.2005.07.035 http://doi.org/10.1016/j.talanta.2005.07.035 . Determination of fluoroquinolones in urine and serum by using high performance liquid chromatography and multiemission scan fluorimetric detection C Fierens , S Hillaert , W van den Bossche . The qualitative and quantitative determination of quinolones of first and second generation by capillary electrophoresis . J Pharmaceut Biomed Anal , 2000 . 22 ( 5 ): 763 - 772 . DOI: 10.1016/S0731-7085(99)00282-4 http://doi.org/10.1016/S0731-7085(99)00282-4 . The qualitative and quantitative determination of quinolones of first and second generation by capillary electrophoresis ZH He , N Gao , WR Jin . Capillary electrophoretic enzyme immunoassay with electrochemical detection using a noncompetitive format . J Chromatogr B , 2003 . 784 ( 2 ): 343 - 350 . DOI: 10.1016/S1570-0232(02)00823-1 http://doi.org/10.1016/S1570-0232(02)00823-1 . Capillary electrophoretic enzyme immunoassay with electrochemical detection using a noncompetitive format M Hervás , MA López , A Escarpa . Electrochemical immunoassay using magnetic beads for the determination of zearalenone in baby food: an anticipated analytical tool for food safety . Anal Chim Acta , 2009 . 653 ( 2 ): 167 - 172 . DOI: 10.1016/j.aca.2009.09.024 http://doi.org/10.1016/j.aca.2009.09.024 . Electrochemical immunoassay using magnetic beads for the determination of zearalenone in baby food: an anticipated analytical tool for food safety DC Hooper . Clinical applications of quinolones . BBA-Gene Struct Expr , 1998 . 1400 ( 1-3 ): 45 - 61 . DOI: 10.1016/S0167-4781(98)00127-4 http://doi.org/10.1016/S0167-4781(98)00127-4 . Clinical applications of quinolones WH Huang , DW Pang , H Tong , ZL Wang , JK Cheng . A method for the fabrication of low-noise carbon fiber nanoelectrdes . Anal Chem , 2001 . 73 ( 5 ): 1048 - 1052 . 11289416 DOI: 10.1021/ac0008183 http://doi.org/10.1021/ac0008183 . A method for the fabrication of low-noise carbon fiber nanoelectrdes M Jamal , MA Crowe , E Magner . Characterization of the composition of bovine urine and its effect on the electrochemical analysis of the model mediator, p -aminophenol . Anal Chim Acta , 2005 . 554 ( 1-2 ): 79 - 85 . DOI: 10.1016/j.aca.2005.08.065 http://doi.org/10.1016/j.aca.2005.08.065 . Characterization of the composition of bovine urine and its effect on the electrochemical analysis of the model mediator, p-aminophenol WR Jin , XJ Li , N Gao . Simultaneous determination of tryptophan and glutathione in individual rat hepatocytes by capillary zone electrophoresis with electrochemical detection at a carbon fiber bundle-Au/Hg dual electrode . Anal Chem , 2003 . 75 ( 15 ): 3859 - 3864 . 14572054 DOI: 10.1021/ac0207022 http://doi.org/10.1021/ac0207022 . Simultaneous determination of tryptophan and glutathione in individual rat hepatocytes by capillary zone electrophoresis with electrochemical detection at a carbon fiber bundle-Au/Hg dual electrode MB Kays , BR Overholser , S Lagvankar , M Goldman , KM Sowinski . Effect of ensure on the oral bioavailability of gatifloxacin in healthy volunteers . Pharmacotherapy , 2005 . 25 ( 11 ): 1530 - 1535 . 16232015 DOI: 10.1592/phco.2005.25.11.1530 http://doi.org/10.1592/phco.2005.25.11.1530 . Effect of ensure on the oral bioavailability of gatifloxacin in healthy volunteers S Laschi , M Mascini , G Scortichini , M Franek , M Mascini . Polychlorinated biphenyls (PCBs) detection in food samples using an electrochemical immunosensor . J Agric Food Chem , 2003 . 51 ( 7 ): 1816 - 1822 . 12643636 DOI: 10.1021/jf0208637 http://doi.org/10.1021/jf0208637 . Polychlorinated biphenyls (PCBs) detection in food samples using an electrochemical immunosensor CX Lei , SQ Hu , N Gao , GL Shen , RQ Yu . An amperometric hydrogen peroxide biosensor based on immobilizing horseradish peroxidase to a nano-Au monolayer supported by sol-gel derived carbon ceramic electrode . Bioelectrochemistry , 2004 . 65 ( 1 ): 33 - 39 . 15522690 DOI: 10.1016/j.bioelechem.2004.06.002 http://doi.org/10.1016/j.bioelechem.2004.06.002 . An amperometric hydrogen peroxide biosensor based on immobilizing horseradish peroxidase to a nano-Au monolayer supported by sol-gel derived carbon ceramic electrode Y Liu , CZ Zhang , XY Yu , ZY Zhang , X Zhang , RR Liu , XJ Liu , ZM Gong . Development and evaluation of immunoassay for zeranol in bovine urine . J Zhejiang Univ-Sci B , 2007 . 8 ( 12 ): 900 - 905 . 18257125 DOI: 10.1631/jzus.2007.B0900 http://doi.org/10.1631/jzus.2007.B0900 . Development and evaluation of immunoassay for zeranol in bovine urine SX Lu , YL Zhang , JT Liu , CB Zhao , W Liu , RM Xi . Preparation of anti-pefloxacin antibody and development of an indirect competitive enzyme-linked immunosorbent assay for detection of pefloxacin residue in chicken liver . J Agric Food Chem , 2006 . 54 ( 19 ): 6995 - 7000 . 16968054 DOI: 10.1021/jf061309q http://doi.org/10.1021/jf061309q . Preparation of anti-pefloxacin antibody and development of an indirect competitive enzyme-linked immunosorbent assay for detection of pefloxacin residue in chicken liver L Netchiporouk , N Shram , D Salvert , R Cespuglio . Brain extracellular glucose assessed by voltammetry throughout the rat sleep-wake cycle . Eur J Neursci , 2001 . 13 ( 7 ): 1429 - 1434 . DOI: 10.1046/j.0953-816x.2001.01503.x http://doi.org/10.1046/j.0953-816x.2001.01503.x . Brain extracellular glucose assessed by voltammetry throughout the rat sleep-wake cycle YN Ni , YR Wang , S Kokot . Simultaneous determination of three fluoroquinolones by linear sweep stripping voltammetry with the aid of chemometrics . Talanta , 2006 . 69 ( 1 ): 216 - 225 . 18970557 DOI: 10.1016/j.talanta.2005.09.032 http://doi.org/10.1016/j.talanta.2005.09.032 . Simultaneous determination of three fluoroquinolones by linear sweep stripping voltammetry with the aid of chemometrics SS Ordóñez , E Fàbregas . New antibodies immobilization system into a graphite-polysulfone membrane for amperometric immunosensors . Biosens Bioelectron , 2007 . 22 ( 6 ): 965 - 972 . DOI: 10.1016/j.bios.2006.03.022 http://doi.org/10.1016/j.bios.2006.03.022 . New antibodies immobilization system into a graphite-polysulfone membrane for amperometric immunosensors BR Overholser , MB Kays , KM Sowinski . Determination of gatifloxacin in human serum and urine by high-performance liquid chromatography with ultraviolet detection . J Chromatogr B , 2003 . 798 ( 1 ): 167 - 173 . DOI: 10.1016/j.jchromb.2003.09.020 http://doi.org/10.1016/j.jchromb.2003.09.020 . Determination of gatifloxacin in human serum and urine by high-performance liquid chromatography with ultraviolet detection CM Perry , JA Barman Balfour , HM Lamb . Gatifloxacin . Drugs , 1999 . 58 ( 4 ): 683 - 696 . 10551438 DOI: 10.2165/00003495-199958040-00010 http://doi.org/10.2165/00003495-199958040-00010 . Gatifloxacin S Piermarini , L Micheli , NHS Ammida , G Palleschi , D Moscone . Electrochemical immunosensor array using a 96-well screen-printed microplate for aflatoxin B-1 detection . Biosens Bioelectron , 2007 . 22 ( 7 ): 1434 - 1440 . 16893640 DOI: 10.1016/j.bios.2006.06.029 http://doi.org/10.1016/j.bios.2006.06.029 . Electrochemical immunosensor array using a 96-well screen-printed microplate for aflatoxin B-1 detection K Pihel , QD Walker , RM Wightman . Overoxidized polypyrrole-coated carbon fiber microelectrodes for dopamine measurements with fast-scan cyclic voltammetry . Anal Chem , 1996 . 68 ( 13 ): 2084 - 2089 . 9027223 DOI: 10.1021/ac960153y http://doi.org/10.1021/ac960153y . Overoxidized polypyrrole-coated carbon fiber microelectrodes for dopamine measurements with fast-scan cyclic voltammetry MIRM Santoro , NM Kassab , AK Singh , ERM Kedor-Hackmam . Quantitative determination of gatifloxacin, levofloxacin, lomefloxacin and pefloxacin fluoroquinolonic antibiotics in pharmaceutical preparations by high-performance liquid chromatography . J Pharmaceut Biomed Anal , 2006 . 40 ( 1 ): 179 - 184 . DOI: 10.1016/j.jpba.2005.06.018 http://doi.org/10.1016/j.jpba.2005.06.018 . Quantitative determination of gatifloxacin, levofloxacin, lomefloxacin and pefloxacin fluoroquinolonic antibiotics in pharmaceutical preparations by high-performance liquid chromatography BN Suhagia , SA Shah , IS Rathod , HM Patel , DR Shah , BP Marolia . Determination of gatifloxacin and ornidazole in tablet dosage forms by high-performance thin-layer chromatography . Anal Sci , 2006 . 22 ( 5 ): 743 - 745 . 16770055 DOI: 10.2116/analsci.22.743 http://doi.org/10.2116/analsci.22.743 . Determination of gatifloxacin and ornidazole in tablet dosage forms by high-performance thin-layer chromatography K Vishwanathan , MG Bartlett , JT Stewart . Determination of gatifloxacin in human plasma by liquid chromatography/electrospray tandem mass spectrometry . Rapid Commun Mass Spectrom , 2001 . 15 ( 12 ): 915 - 919 . 11400196 DOI: 10.1002/rcm.322 http://doi.org/10.1002/rcm.322 . Determination of gatifloxacin in human plasma by liquid chromatography/electrospray tandem mass spectrometry LM Weigel , GJ Anderson , FC Tenover . DNA gyrase and topoisomerase IV mutations associated with fluoroquinolone resistance in Proteus mirabilis . Antimicrob Agents Chemother , 2002 . 46 ( 8 ): 2582 - 2587 . 12121936 DOI: 10.1128/AAC.46.8.2582-2587.2002 http://doi.org/10.1128/AAC.46.8.2582-2587.2002 . DNA gyrase and topoisomerase IV mutations associated with fluoroquinolone resistance in Proteus mirabilis RM Wightman , LJ May , AC Michael . Detection of dopamine dynamics in the brain . Anal Chem , 1988 . 60 ( 13 ): 769 - 779 . DOI: 10.1021/ac00164a001 http://doi.org/10.1021/ac00164a001 . Detection of dopamine dynamics in the brain ZY Yang , AY Kolosova , WB Shim , DH Chung . Development of monoclonal antibodies against pirimiphos-methyl and their application to IC-ELISA . J Agric Food Chem , 2006 . 54 ( 13 ): 4551 - 4556 . 16786997 DOI: 10.1021/jf0606196 http://doi.org/10.1021/jf0606196 . Development of monoclonal antibodies against pirimiphos-methyl and their application to IC-ELISA HL Zhang , GS Lai , DY Han , AM Yu . An amperometric hydrogen peroxide biosensor based on immobilization of horseradish peroxidase on an electrode modified with magnetic dextran microspheres . Anal Bioanal Chem , 2008 . 390 ( 3 ): 971 - 977 . 18060389 DOI: 10.1007/s00216-007-1748-3 http://doi.org/10.1007/s00216-007-1748-3 . An amperometric hydrogen peroxide biosensor based on immobilization of horseradish peroxidase on an electrode modified with magnetic dextran microspheres C Zhao , W Liu , H Ling , S Lu , Y Zhang , J Liu , R Xi . Preparation of anti-gatifloxacin antibody and development of an indirect competitive enzyme-linked immunosorbent assay for the detection of gatifloxacin residue in milk . J Agric Food Chem , 2007 . 55 ( 17 ): 6879 - 6884 . 17645343 DOI: 10.1021/jf070978g http://doi.org/10.1021/jf070978g . Preparation of anti-gatifloxacin antibody and development of an indirect competitive enzyme-linked immunosorbent assay for the detection of gatifloxacin residue in milk M Zhou , C Guan , G Chen , X Xie , S Wu . Determination of theophylline concentration in serum by chemiluminescent immunoassay . J Zhejiang Univ-Sci B , 2005 . 6 ( 12 ): 1148 - 1152 . 16358370 DOI: 10.1631/jzus.2005.B1148 http://doi.org/10.1631/jzus.2005.B1148 . Determination of theophylline concentration in serum by chemiluminescent immunoassay 10.1631/jzus.B1100073 Project supported by the National High-Tech R&D Program (863) of China (Nos. 07AA10Z435 and 2007AA06A407), the National Natural Science Foundation of China (No. 20675048), the Fundamental Research Funds for the Central Universities (No. 65011121), and the Shandong Provincial Natural Science Foundation (No. Y2008B31), China 2011-03-10 2011-12-10 2011-07-26 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2012 2012 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 13