ORIGINAL PAPER Towards the development of a single-step immunosensor based on an electrochemical screen-printed electrode strip coupled with immunomagnetic beads G. Volpe & U. Sozzo & S. Piermarini & E. Delibato & G. Palleschi & D. Moscone Received: 5 January 2012 / Revised: 17 May 2012 / Accepted: 23 May 2012 / Published online: 27 June 2012 # Springer-Verlag 2012 Abstract This work investigates the behaviour of two alternative systems that model the crucial event involved in any ELISA test, i.e. the molecular recognition between an antigen and its specific antibody on a solid phase, and its measurement. Each approach is devised with the goal of making possible a single-step, separation and wash-free amperometric magneto-immunosensor. Magnetic particles (MBs) are used as support for the immobilization of rabbit IgGs that are recognized by the specific anti-rabbit IgG- HRP. The assay protocol is based on the use of a series of small “reservoirs” containing phosphate buffer, hydroqui- none, anti-rabbit IgG-HRP and an appropriate amount of MB-rabbit IgG. After a brief incubation, the content of each “reservoir” is transferred to one of the wells of a 8-well magnetized-screen-printed electrode strip. The resulting MB-IgG-anti-IgG-HRP chain, is then concentrated on the working electrode surface for electrochemical measurement. Two different approaches to monitor this immunological reaction are investigated. The first one is based on the enzyme-channeling principle (ECP) and involves the use of a second enzyme, glucose oxidase (GOD), immobilized on the working electrode previously modified with Prussian Blue. Since the H 2 O 2 produced by GOD is the co-substrate of the HRP enzyme, glucose is added into the well and the current, generated by the residual H 2 O 2 , is measured. The second, more direct, approach is performed without exploit- ing ECP (no GOD enzyme), by adding H 2 O 2 into the well and measuring the current generated by the HRP product on a pristine screen-printed electrode. Both approaches yielded a typical sigmoidal binding curve, illustrating the discrimi- nation between the signal produced by the immuno-bound HRP concentrated on the electrode surface, and the back- ground signal due to HRP in the bulk solution. Keywords Single-step amperometric immunosensor . 8-well/sensor strip . Magnetic beads . Enzyme channelling principle . SPE-based immunoassay Introduction The enzyme-linked immunosorbent assay (ELISA) is a bio- chemical technique widely used in clinical, pharmaceutical, environmental and food analysis to detect the presence of antibodies or antigens in complex sample matrices. ELISA is commonly implemented on a 96-well spectrophotometric plate suitable for automation and thus resulting in high- throughput bioanalysis. However, this approach still involves multiple incubation, washing and separation steps that result, in the end, in long analysis times. A possible alternative use of electrochemical techniques to develop sensitive immunosensors, with similar or even better performances than the classic spectrophotometric ELISA assay, has been demonstrated by our and other research groups [1–6]. Despite the advantages of electro- chemical measurements over spectrophotometric ones, such Published in the special issue Analytical Science in Italy with guest editor Aldo Roda. G. Volpe (*) : U. Sozzo : S. Piermarini : G. Palleschi : D. Moscone Dipartimento di Scienze e Tecnologie Chimiche, Università degli Studi di Roma “Tor Vergata”, Via della Ricerca Scientifica 1, 00133 Rome, Italy e-mail: giulia.volpe@uniroma2.it E. Delibato Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare, Istituto Superiore di Sanità, V.le Regina Elena 299, 00161 Rome, Italy Anal Bioanal Chem (2013) 405:655–663 DOI 10.1007/s00216-012-6141-1 11974 Nilde 514890 06_03_2013