ORIGINAL PAPER Competitive amperometric immunosensor based on covalent linking of a protein conjugate to dendrimer-functionalised nanogold substrate for the determination of 2,4,6-trinitrotoluene Marco Giannetto & Elisabetta Maiolini & Elida Nora Ferri & Stefano Girotti & Giovanni Mori & Maria Careri Received: 13 March 2012 / Revised: 11 May 2012 / Accepted: 22 May 2012 # Springer-Verlag 2012 Abstract A new amperometric immunosensor for 2,4,6- trinitrotoluene based on the working principle of competi- tive enzyme-linked immunosorbent assay was developed and characterised. An electrodeposited nanogold substrate was functionalised by deposition of self-assembled mono- layers of 2-aminoethanethiol as linkers for the subsequent immobilisation of polyamidoaminic dendrimers. Our ap- proach makes use of those dendrimers to anchor a trinitrobenzene-ovalbumin conjugate on the electrode surface. The immunosensor was tested and validated for the determi- nation of 2,4,6-trinitrotoluene showing high selectivity with respect to other nitroaromatic compounds, a limit of detection of 4.8 ng/mL and a limit of quantitation of 6 ng/mL. The immunosensor was tested for the quantification of the analyte in spiked soils and in a real sample of post-blast soil, evidenc- ing a good recovery rate (113 %). Keywords Biosensors . Immunoassay . Nanoparticles . TNT . Explosives . Dendrimer . SAM Introduction 2,4,6-trinitrotoluene (TNT) is one of the most common bulk explosives used in military munitions and in civilian mining and quarrying activities [1, 2]. TNT was first used on a wide scale during World War I and is still used today. Nitroaromatic compounds also form an important group of refractory xenobiotics [3], considering that only a few aro- matic compounds, bearing one nitro group as a substituent of the aromatic ring, are produced as secondary metabolites by microorganisms. The majority of nitroaromatics in the biosphere are industrial chemicals such as explosives, dyes, polyurethane foams, herbicides, insecticides and solvents. For these reasons, the development of selective and sensi- tive devices capable of detecting traces of their presence is a challenge. The increasingly urgent need for rapid and reli- able analytical methods for the determination of explosives covers both forensic investigations, as the analysis of post- blast scenarios, and prevention of terrorist acts through security checks at strategic locations such as airports. Another important characteristic of nitroaromatic com- pounds is their ability to rapidly penetrate the skin [4]. They can cause the formation of methemoglobin on acute exposures and anaemia on chronic exposures. Additionally, local irritation, liver damage and bladder tumours have also been identified [5]. These compounds are generally recalci- trant to biological treatment and remain in the biosphere, where they constitute a source of pollution due to both toxic Published in the special issue Analytical Science in Italy with guest editor Aldo Roda. Electronic supplementary material The online version of this article (doi:10.1007/s00216-012-6137-x) contains supplementary material, which is available to authorized users. M. Giannetto (*) : G. Mori : M. Careri Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Università di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy e-mail: marco.giannetto@unipr.it E. Maiolini : E. N. Ferri : S. Girotti Dipartimento di Scienza dei Metalli, Elettrochimica e Tecniche Chimiche, Università di Bologna, Via S. Donato 15, 40127 Bologna, Italy Anal Bioanal Chem DOI 10.1007/s00216-012-6137-x