Sensors and Actuators B 140 (2009) 616–622 Contents lists available at ScienceDirect Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb Love wave immunosensor for antibody recognition using an innovative semicarbazide surface functionalization Laurent Fertier a , Marc Cretin a,∗ , Marc Rolland a , Jean-Olivier Durand b , Laurence Raehm b , Rémi Desmet c , Oleg Melnyk c , Céline Zimmermann d , Corinne Déjous d , Dominique Rebière d a Institut Européen des Membranes, ENSCM-UM2-CNRS, UMR 5635, 1919 Route de Mende, 34293 Montpellier cedex 5, France b Institut Charles Gerhardt Montpellier, UMR 5253 CNRS-UM2-ENSCM-UM1, Equipe Chimie Moléculaire et Organisation du Solide cc1701 Place Eugène Bataillon, 34095 Montpellier cedex 05, France c Institut de Biologie de Lille, CNRS UMR 8161, Université de Lille Nord de France, 1 rue du Pr Calmette, 59021 Lille cedex, France d Laboratoire IMS, Université de Bordeaux, ENSEIRB, CNRS UMR 5218, 351 cours de la Libération, 33405 Talence cedex, France article info Article history: Received 10 March 2009 Received in revised form 27 April 2009 Accepted 28 April 2009 Available online 20 May 2009 Keywords: SH-SAW SAMs Surface modification Semicarbazide functionalization Real-time antibodies recognition monitoring abstract A new novel and easy functionalization route of a Love wave acoustic sensor based on the -oxo- semicarbazone bond is described. The interest is firstly to observe in real-time the immobilization of the peptide on the semicarbazide surface of the transducer and secondly to monitor the specific binding of antibodies. Site-specific immobilization of antigenic-peptides as well as binding of murine mono- clonal antibodies has been shown by gravimetric measurements. A tetramethylrhodamine-labeled goat antibody directed against murine antibodies was used to further characterize the biomolecular inter- actions by fluorescence microscopy and surface analysis (by AFM). Our data show that the gravimetric monitoring developed from the prepared Love wave immunosensor is a promising alternative route to characterize chemical and biomolecular events. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The conventional techniques for the specific detection of biolog- ical analytes are mainly radioimmuno assay (RIA), enzyme-linked immunosorbent assay (ELISA) and fluoroimmuno assay (FIA). In particular, the detection of antibodies is of prime importance for the detection of pathogen infections and is usually realized using ELISA assays. These techniques are highly sensitive for analysis in clin- ical diagnostic but nevertheless they have some limitations, such as the use of radioactive isotopes or fluorophores, and/or relatively long analysis time. One significant limitation is usually the need for expensive equipment which can be hardly exported out of the laboratory for bedside testing for example. Alternative techniques for the detection of antibodies are emerg- ing. Devices are currently built from the SPR (surface plasmon resonance) technique [1–3] or from piezoelectric sensors using bulk acoustic waves as QCM (quartz crystal microbalance) [4–6] or sur- face acoustic waves as guided SH-SAW (shear horizontal-surface acoustic waves) sensors also called Love wave sensors [7–9]. These immunosensors are developed for immunoassay in clinical sciences ∗ Corresponding author. Tel.: +33 4 67 61 34 10; fax: +33 4 67 04 28 20. E-mail address: Marc.Cretin@iemm.univ-montp2.fr (M. Cretin). as well as environmental analysis of trace pollutants or quality con- trol in the food industry. For piezoelectric detection in liquid media, Love waves are particularly interesting because the use of a horizontal polariza- tion and of a guiding layer at the top of the transducer allows to minimize losses and then to lower the detection limit [10,11]. Another aspect of immunosensors is the possibility of real-time antigens/antibodies binding monitoring as recently shown in the case of the detection of Escherichia coli binding to the anti-O157:H7 layer [12] or of an anti-folic acid immunoassay [11]. We present here an original and efficient route to prepare microsystems for antibodies detection by guided SH-SAW sensors. This approach is derivated from a route recently developed for the site-specific immobilization of peptides on silica surfaces printed on polycarbonate substrate for microfluidic devices using fluo- rescence detection [13]. In our approach, the sensitive layer was obtained in a multi-step process on the silica guiding top-layer of the device. The first stage is a batch preparation of a reactive semicarbazide self-assembled monolayer on the transducer. The second step consists of a gravimetric real-time monitoring of the covalent coupling (-oxo-semicarbazone bond) between the semi- carbazide and the -oxo-aldehyde peptide (HCOCO-peptide). Then the last step is the monitoring of the specific antibody binding to immobilized peptides using the Love wave functionalized sensor. 0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2009.04.069