Talanta 79 (2009) 62–67 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta Development of an enrofloxacin immunosensor based on label-free electrochemical impedance spectroscopy Ching-Chou Wu a,b,∗ , Chia-Hung Lin a , Way-Shyan Wang c a Department of Bio-industrial Mechatronics Engineering, National Chung Hsing University, No. 250, Kuo Kuang Road, Taichung 402, Taiwan b Institute of Biomedical Engineering, National Chung Hsing University, No. 250, Kuo Kuang Road, Taichung 402, Taiwan c Department of Veterinary Medicine, National Chung Hsing University, No. 250, Kuo Kuang Road, Taichung 402, Taiwan article info Article history: Received 30 December 2008 Received in revised form 26 February 2009 Accepted 3 March 2009 Available online 14 March 2009 Keywords: Enrofloxacin Electrochemical impedance spectroscopy Label-free Equivalent circuit Immunosensor abstract Enrofloxacin is the most widespread antibiotic in the fluoroquinolone family. As such, the development of a rapid and sensitive method for the determination of trace amounts of enrofloxacin is an important issue in the health field. The interaction of the enrofloxacin antigen to a specific antibody (Ab) immobilized on an 11-mercapto-undecanoic acid-coated gold electrode was quantified by electrochemical impedance spectroscopy. Two equivalent circuits were separately used to interpret the obtained impedance spec- tra. These circuits included one resistor in series with one parallel circuit comprised of a resistor and a capacitor (1R//C), and one resistor in series with two parallel RC circuits (2R//C). The results indicate that the antigen-antibody reaction analyzed using the 1R//C circuit provided a more sensitive resistance increment against the enrofloxacin concentration than that of the 2R//C circuit. However, the 2R//C cir- cuit provided a better fitting for impedance spectra, and therefore supplies more detailed results of the enrofloxacin-antibody interaction, causing the increase of electron transfer resistance selectively to the modified layer, and not the electrical double layer. The antibody-modified electrode allowed for analysis of the dynamic linear range of 1–1000 ng/ml enrofloxacin with a detection limit of 1 ng/ml. The reagent- less and label-free impedimetric immunosensors provide a simple and sensitive detection method for the specific determination of enrofloxacin. © 2009 Elsevier B.V. All rights reserved. 1. Introduction A wide variety of antibiotics have been used to treat or prevent bacterial infection in food-producing animals. The use of antibiotics can effectively control the occurrence of disease and maintain the growth of animals. Enrofloxacin, a specific type of fluoroquinolone antibacterial, possesses a broad spectrum of activity against most Gram-negative pathogens, including Pseudomonas aeruginosa and Enterobacteriaceae, and some Gram-positive bacteria [1]. It is fre- quently used to treat urinary, pulmonary and digestive infections in veterinary medicine due to a long half-life and its wide distribu- tion to different tissues [2–5]. According to a report of the World Health Organization (WHO) meeting in 1998, enrofloxacin was the most extensively approved antibiotic of the fluoroquinolones fam- ily for livestock, poultry, fish, or pet animals globally [6]. However, with increasing use of enrofloxacin, the sanitary issues, including drug residues in edible tissues of food-producing animals and the bacterial resistance being transferred from animals to human, have occurred significantly [7,8]. In order to protect consumers’ health, ∗ Corresponding author. Tel.: +886 4 2285 1268; fax: +886 4 2287 9351. E-mail address: ccwu@dragon.nchu.edu.tw (C.-C. Wu). the European Union has established maximum residue limit (MRL) for enrofloxacin, which is fixed at 100 g/kg in several edible animal tissues [9]. Moreover, the USA Food and Drug Administration (FDA) withdrew the approval of enrofloxacin for poultry in 2005, because scientific data have indicated that the use of enrofloxacin causes the emergence of resistance in Campylobacter, that could result in the ineffective treatment of human diseases [10]. As such, the develop- ment of precise, specific, and sensitive methods for the detection of enrofloxacin in veterinary and medical fields has become an impor- tant issue. Different detection strategies have been employed for the determination of enrofloxacin levels. These include adsorptive stripping voltammetry [11], spectrophotometry [12–14], capillary electrophoresis with diode-array detector [15–17] or mass spec- trometry [18], high performance liquid chromatography (HPLC) using ultraviolet adsorption [19–21] or fluorescence detection [21–25], and enzyme-linked immunosorbent assay (ELISA) with UV–vis spectrometry [26–28] or fluorescence detection [29]. Among these, the lowest detection limit of enrofloxacin was 0.1 ng/ml, obtained with the ELISA method with fluorescence detec- tion [29]. ELISA is a sensitive, fast, and specific method for the detection of enrofloxacin in the veterinary field by means of the interaction of an antigen and an antibody (Ab). However, in direct or 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.03.006