Biosensors and Bioelectronics 20 (2005) 1780–1787 A separation-free amperometric immunosensor for vitellogenin based on screen-printed carbon arrays modified with a conductive polymer Farzana Darain 1 , Doeg Su Park, Jang-Su Park, Seung-Cheol Chang, Yoon-Bo Shim ∗ Department of Chemistry and Center for Innovative Bio-physio Sensor Technology, Pusan National University, Pusan 609-735, South Korea Received 1 May 2004; received in revised form 5 July 2004; accepted 7 July 2004 Available online 12 August 2004 Abstract A disposable amperometric immunosensor was studied for the rapid detection of carp (Carassius auratus) Vitellogenin (Vtg). The sensor was fabricated based on screen-printed carbon arrays (SPCAs) containing eight carbon working and an integrated carbon counter electrodes. To construct the sensor, a conducting polymer (poly-terthiophene carboxylic acid) was electropolymerized on the surface of working electrodes and the polymer-coated SPCAs was characterized by SEM. Horseradish peroxidase (HRP) and a monoclonal antibody (anti-Vtg) specific to carp Vtg were covalently attached onto the polymer modified SPCAs. The immobilization of HRP and anti-Vtg onto the polymer-coated SPCAs was examined using cyclic voltammetry and quartz crystal microbalance studies. In order to detect the amount of Vtg, glucose oxidase (GOx)-labelled Vtg bound to the sensor surface under competition with the Vtg analyte was quantified amperometrically using glucose as a substrate. The performance of the eight sensors in arrays was evaluated by obtaining the calibration plots for Vtg. The sensor arrays exhibit a linear range of the Vtg concentration from 0.25 to 7.8 ng/ml and the detection limit was determined to be 0.09 ng/ml. Furthermore, the performance of the immunosensor for the determination of Vtg was evaluated by a standard addition method performed in fish serum samples. © 2004 Elsevier B.V. All rights reserved. Keywords: Screen-printed carbon arrays; Vitellogenin; Conducting polymer; Amperometric immunosensor; Mediatorless system; Enzyme label 1. Introduction Vitellogenin (Vtg) is a precursor protein of egg yolk syn- thesised by the mature female fish liver under estrogen stimu- lation and secreted in response to circulating estrogen. More recently, Vtg has been proposed as a biomarker for xenobi- otics estrogen (Hansen et al., 1998; Sol´ e et al., 2001; Sumpter and Jobting, 1995). Hence, the detection of Vtg could serve as an important marker causing endocrine disruption. However, conventional immunoassay techniques such as enzyme linked immunosorbant assays (ELISAs), radio immunoassay (RIA) and western blot techniques are mostly tedious and require ra- dioactive chemicals or sophisticated instrumentations. In this connection, searching for new, simple and sensitive methods ∗ Corresponding author. Tel.: +82 51 510 2244; fax: +82 51 514 2430. E-mail address: ybshim@pusan.ac.kr (Y.-B. Shim). 1 On leave from Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka-1205, Bangladesh. for the detection of Vtg in real fish serum samples with a low cost is of prime importance. Electrochemical immunosensors combine the analytical power of electrochemical techniques with the specificity of biological recognition processes, which constitute an evident alternative to the already existing traditional methods (Heineman and Halsall, 1985). Upto now, to our knowledge, there has been no published description of an electrochemical immunosensor for the detection of Vtg except a preliminary investigation currently reported by our group using direct assay with impedance measurements (Darain et al., 2004). Furthermore, there is still no single electrochemical enzyme immunoassay approach in the literature that enables the detection of Vtg in real samples. Amperometric methods based on ELISAs generally in- volve the combination of the merits of the highly sensitive electrochemical detection with the specificity of immunoin- teraction. These techniques are particularly well suited due to their ease and sensitivity in detecting minute amounts of 0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2004.07.006