Antibody immobilisation on fibre optic TIRF sensors Lorena Tedeschi a , Claudio Domenici a, *, Arti Ahluwalia a , Francesco Baldini b , Andrea Mencaglia c a Istituto di Fisiologia Clinica del C.N.R., Pisa e Centro Interdipartimentale di Ricerca ‘E. Piaggio’, Universita ` di Pisa, Pisa, Italy b Istituto per la Ricerca sulle Onde Elettromagnetiche ‘N. Carrara’  / C.N.R., Florence, Italy c Prodotec s.r.l., Florence, Italy Received 13 December 2001; received in revised form 17 October 2002; accepted 31 March 2003 Abstract A study of antibody immobilisation techniques on quartz and fibre optic surfaces for immunosensors has been carried out. Methods of covalent antibody immobilisation which have not previously been applied to optical fibres were investigated, and compared with classical methods found in the literature. Preliminary experiments on covalent immobilisation methods on planar quartz surfaces were conducted to enable us to choose the most suitable protein immobilisation technique for sensor applications. The immobilisation studies were directed in particular towards obtaining a high density of binding sites for the analyte of interest. Two of the most promising methods, antibody immobilisation on surfaces coated with dextran based hydrogel and F(ab?)-SH fragments bound to silanised glass, which resulted in surface densities of active sites of above 0.45 pmol/cm 2 , were selected for further experiments on a fibre optic total internal reflection fluorescence immunosensor and gave satisfactory responses to changes in analyte concentrations of the order of 10 8 M. The efficiency of polar organic solvents, such as dimethylsulfoxide, in dissociating the antigen  /antibody complex and hence to regenerate the immunosensor surface was also evaluated. # 2003 Elsevier B.V. All rights reserved. Keywords: Antibody immobilisation; Fibre optic sensor; Fluorescence; Immunosensor; Surface regeneration 1. Introduction In the wide and heterogeneous field of biosensors, immunosensors are very interesting for their many potential applications such as in medical diagnostics (Vo-Dinh and Cullum, 2000; Stefan et al., 2000), environmental monitoring (Van Emon, 2001) and in the food industry (Rasooly, 2001; Bjurling et al., 2000; Strachan et al., 1997). In this context, total internal reflection fluorescence (TIRF) sensors are very promis- ing, both for their sensitivity and because they can avoid complex separation and extraction steps (Schuderer et al., 2000; Schult et al., 1999). A TIRF immunosensor can be therefore a good measurement device to detect small amounts of a target molecule in a complex mixture. However, despite the technological leaps made in the past decade, this type of sensor, based on the selective interaction between antigen and antibody, has eluded commercialisation because of the problems associated with the biological sensing layer. To date, protein or antibody immobilisation techniques have been neither standardised nor optimised, and the regeneration of immunosensing surfaces is still achieved by primitive methods such as acid rinsing (Wijesuriya et al., 1994). The choice of immobilisation technique must allow a stable bound between sensing surface and the biorecep- tor, without interfering with the biological activity of the system. Furthermore, high sensitivity and an adequate operating range are also essential requisites for an immunosensor for diagnostic applications and in prin- ciple these can be met by ensuring a high density of active antibodies on the optical sensing surface (Dome- nici et al., 1995). Among the methods commonly used, physical ad- sorption has been shown to maintain adequate protein activity, but the forces involved can produce only bland * Corresponding author. Instituto di Fisiologia Clinica CNR, Via G Moruzzi 1, 56124 Pisa (Italy). Tel.:  /39-50-315-2470; fax:  /39-50- 315-2166. E-mail address: domenici@ifc.cnr.it (C. Domenici). Biosensors and Bioelectronics 19 (2003) 85 /93 www.elsevier.com/locate/bios 0956-5663/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0956-5663(03)00173-8