A new controlled concept of immune-sensing platform for specific detection of Alzheimer’s biomarkers M. Ammar a,b,n , C. Smadja c , L. Giang Thi Phuong c , M. Azzouz a,b , J. Vigneron d , A. Etcheberry d , M. Taverna c , E. Dufour-Gergam a,b a Univ. Paris Sud, Institut d’Electronique Fondamentale, UMR CNRS 8622, Orsay F-91405, France b CNRS, Orsay F-91405, France c Univ. Paris Sud, Institut Galien Paris-Sud, UMR CNRS 8612, Chatenay-Malabry F-92296, France d Univ. Saint-Quentin en Yvelines, Institut Lavoisier de Versailles, UMR 8180, Versailles F-78035, France article info Available online 13 August 2012 Keywords: Immunoassay Silanisation XPS AFM Amyloid peptide Alzheimer abstract We propose a concept of very specific immune-sensing platform dedicated to the quantification of biomarkers of Alzheimer disease (AD) in biological fluids. High sensitivity is required for the earliness of AD diagnostic, mainly based on clinical evaluation at present time. Accordingly, a controlled and adaptative surface functionalization of a silicon wafer with carboxylated alkyltrichlorosilane has been developed. The surface has extensively been characterized by AFM and X-ray Photoemissive Spectroscopy. The surface modification has been chemically assessed by XPS at each functionalization step. The survey spectra of silicon surface, after, 1, 3, 6 and 24 h of silanisation, highlight a significant enhancement of the functionalization efficiency upon time. The oxidation reaction has also been investigated by XPS and showed components related to the carboxylic group. AFM measurements pointed out a morphological modification consistent with a homogenous development of the carboxylic group and an almost protein monolayer on the surface. Moreover, we evaluated the biological activity of the grafted antibodies involved in (AD) biomarker detection onto this silanized surface by fluorescent microscopy. A sandwich immunoassay dedicated to the sensitive detection of one biomarker of Alzheimer disease (AD), the amyloid peptide 1–42 (Ab 1–42), was carried out. The results demonstrated that the controlled silanized surface provides a novel and viable way to detect biomarkers with high specificity and open the route to an original development of immune-sensing applications on such surfaces. & 2012 Elsevier B.V. All rights reserved. 1. Introduction Self-assembled monolayers of organosilanes have focused much attention in the fields of immune-sensor technology. Organosilanes have been reported in the literature to be used for protein immo- bilization (Kanan et al., 2002; Gabriel et al., 2007; Rusmini et al., 2007; Hervas et al., 2012). They generally offer favorable properties such as biocompatibility of the surface and protein protection from denaturation during the immobilization process (Rusmini et al., 2007; Buchapudi et al., 2011). On the other hand, the main difficulty for protein immobilization on a solid support, particularly for anti- bodies, is to maintain their three dimensional conformation and orientation. Indeed, antibody orientation is crucial for an immunoas- say in which the antibody is linked to a solid surface. A proper orientation allows the exposition of the binding sites to the sample solution and ensures a specific recognition. Most of the immobilization strategies, lead to random immobilization of the antibodies that can limit the antigen binding capacity (Caruso et al., 1996; Pei et al., 2010; Seitz et al., 2011). Among these, one of the most popular strategies employed consists of an amine coupling on carboxylated or active ester substrates via their amino groups (Rusmini et al., 2007). This method has been widely used on flat carboxylated surfaces (Pei et al., 2010; Samanta and Sarkar, 2011). However, the grafting density, the orientation and the biological function of the grafted antibodies, following the immobilization strategy described previously, on silane modified silicon surfaces have to be investigated. In this way, a fine understanding and control of the chemical surface preparation are needed (Faucheux et al., 2004; Popat et al., 2002). In this paper, we present an extensive characterization of chemically silanized silicon surface by contact angle, XPS and AFM. All these techniques constitute essential tools to control the chemical surface modification in view of a successful bio-functionalization (Boussert et al., 2009; Inoue and Ishihara, 2010). We also aimed at clarifying the immobilization mechanism relying on self-assembled silanes for antibodies and silicon surface. In this perspective, the grafting density and orientation of antibodies on the prepared Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics 0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.07.072 n Corresponding author at: Univ. Paris Sud, Institut d’Electronique Fondamentale, UMR CNRS 8622, F-91405 Orsay, France. Tel.: þ33 169 153 970. E-mail address: mehdi.ammar@u-psud.fr (M. Ammar). Biosensors and Bioelectronics 40 (2013) 329–335