Demonstration of labeless detection of food pathogens using electrochemical redox probe and screen printed gold electrodes S. Susmel c , G.G. Guilbault a , C.K. O’Sullivan b, * a Laboratory of Sensor Development, Department of Chemistry, National University of Ireland, Cork, Ireland b Bioelectrochemistry and Bioengineering Group, Department of Chemical Engineering, Universidad Rovira i Virgili, Tarragona 43006, Spain c Analytical Chemistry Group, Department of Chemical Science and Technology, University of Udine, 33100 Udine, Italy Received 16 November 2001; accepted 6 August 2002 Abstract The demonstration of a labeless immunosensor for the detection of pathogenic bacteria using screen printed gold electrodes (SPGEs) and a potassium hexacyanoferrate(II) redox probe is reported. Gold electrodes were produced using screen printing and the gold surfaces were modified by a thiol based self assembled monolayer (SAM) to facilitate antibody immobilisation. SAMs based on the use of thioctic acid (TA), mercaptopropionic acid (MPA) and mercaptoundecanoic acid (MUA) were evaluated. Following antibody immobilisation via the optimum SAM, the redox behaviour and diffusion co-efficient (D ) of the potassium hexacyanoferrate(II) probe was monitored in the absence and presence of analyte. In the presence of analyte, a change in the apparent diffusion co-efficient of the redox probe was observed, attributable to impedance of the diffusion of redox electrons to the electrode surface due to the formation of the antibody-bacteria immunocomplex. No change in the diffusion co-efficient was observed when a non-specific antibody (mouse IgG) was immobilised and antigen added. The system has been demonstrated with Listeria monocytogenes and Bacillus cereus . # 2002 Elsevier Science B.V. All rights reserved. Keywords: Self-assembled monolayer; Labeless detection; Electrochemical immunosensor; Screen printed electrodes; Food pathogens 1. Introduction The exploitation of thiol based self assembled mono- layers (SAMs) for the immobilisation of antibodies in immunosensing has been widely reported (Rickter et al., 1996; Yu et al., 1999). The strong affinity of thiols towards gold surfaces produces a fast initial adsorption followed by a slower process of reorganisation of the S/ SH molecules on the gold surface (Yang et al., 1996). This strong affinity and self assembly facilitates the preparation of structured oriented layers containing functional groups well ordered at the monolayer-air (or liquid) interface (Bain and Whitesides, 1988). The length of the thiol chain and the chemical nature of the functional group engenders the control of some of the macroscopic properties of the film (e.g. wettability; Bain et al., 1989). Moreover, binding of biological compo- nents through these functional group allows control of their orientation (Caruso et al., 1996; Choen et al., 1996). As has been previously reported, immobilisation of biocomponents can have a deleterious affect on activity, presumed to be attributable to hindering of access to the active site (in the case of enzymes) or the antigenic binding site (in the case of antibodies; Spitz- nagel and Clark, 1993; Lu et al., 1996). Several strategies can be tested to obtain optimum coupling of biocom- ponent for the particular system under investigation (Longone, 1978; Shriver-Lake et al., 1997). Abbreviations: ALP, alkaline phosphatase; EDC, 1-ethyl-3-[3- dimethylamino)propyl]carbodiimide hydrochloride; MPA, 3- mercaptopropionic acid; MUA, 11-mercaptoundecanoic acid; NHS, N -hydroxysulphosuccinimide; SAM, self assembled monolayer; SPGE, screen printed gold electrode; TA, thioctic acid. * Corresponding author. Tel.: /34-977-55-8174; fax: /34-977-55- 9667 E-mail address: ckosulli@etse.urv.es (C.K. O’Sullivan). Biosensors and Bioelectronics 18 (2003) 881 /889 www.elsevier.com/locate/bios 0956-5663/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII:S0956-5663(02)00214-2