Immunosensor for detection of Legionella pneumophila using surface plasmon resonance Byung-Keun Oh a , Young-Kee Kim b , Woochang Lee a , Young Min Bae a , Won Hong Lee a , Jeong-Woo Choi a, * a Department of Chemical Engineering, Sogang University, C.P.O. Box 1142, Seoul 100-611, South Korea b Department of Chemical Engineering, Hankyong National University, 67 Sukjong-dong, Ansung, Kyonggi-do 456-749, South Korea Received 15 May 2002; received in revised form 2 October 2002; accepted 1 November 2002 Abstract Immunosensor using surface plasmon resonance (SPR) onto self-assembled protein G layer was developed for the detection of Legionella pneumophila . A self-assembled protein G layer on gold (Au) surface was fabricated by adsorbing a mixture of 11- mercaptoundecanoic acid (MUA) and hexanethiol (molar ratio of 1:2) and the activation process for chemical binding between free amine ( Ã /NH 2 ) of protein G and 11-(MUA) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) in series. The formation of self-assembled protein G layer on Au substrate and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analyses of self-assembled protein G layer on Au substrate and monoclonal antibody against L. pneumophila immobilized on protein G were performed by atomic force microscope (AFM). The immunosensor for detection of L. pneumophila using SPR was developed and its detection limit could find up to 10 5 cells/ml. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Immunosensor; Surface plasmon resonance; Protein G; Self-assembled layer; Legionella pneumophila 1. Introduction Legionella pneumophila , the etiological agent of Legionnaire’s disease and Pontiac fever (Glick et al., 1978), is found in natural aquatic habitats, especially in potable water systems, cooling tower systems, heat exchanger systems (Colbourne et al., 1988). Human infection occurs through the inhalation of aerosols contaminated by L. pneumophila . The conventional method for detection of L. pneumophila is complicated procedure involving isolation in a selective medium. However, several problems are encountered with this method, including the presence of viable but non- culturable pathogens, loss of viability after collection and the long time required for culture and confirmation, which took several days. In addition, the detection of L. pneumophila in biocontaminated samples such as waste- water is more difficult, since L. pneumophila can be inhibited and masked by the rapid or abundant growth of other microorganisms (Hussong et al., 1987). For these reasons, in order to avoid the problems encoun- tered with conventional analysis method, alternative method to detect L. pneumophila with high sensitivity and short detection time is needed. The immunosensor can be introduced as an alternative method (Choi et al., 2001b), however, the study of immunosensors for the detection of L. pneumophila has not been reported. Recently, the immunosensor using surface plasmon resonance (SPR) have been developed for measurement of antigens binding to antibody molecules immobilized on the SPR sensor surface, which are capable of detecting analytes in complex biological media with high specificity and sensitivity (Sakai et al., 1998; Darren et al., 1998; Toyama et al., 1998). However, enhancement of sensitivity is required to detect biologi- cal materials because analytes in biological system have very low concentration. * Corresponding author. Tel.:  /82-2-705-8480; fax:  /82-2-711- 0439. E-mail address: jwchoi@ccs.sogang.ac.kr (J.-W. Choi). Biosensors and Bioelectronics 18 (2003) 605 /611 www.elsevier.com/locate/bios 0956-5663/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0956-5663(03)00032-0