ORIGINAL PAPER Surface plasmon resonance immunosensor for cortisol and cortisone determination Marco Frasconi & Monica Mazzarino & Francesco Botrè & Franco Mazzei Received: 23 April 2009 / Revised: 9 June 2009 / Accepted: 12 June 2009 / Published online: 10 July 2009 # Springer-Verlag 2009 Abstract In this paper, we present a surface-plasmon- resonance-based immunosensor for the real-time detection of cortisol and cortisone levels in urine and saliva samples. The method proposed here is simple, rapid, economic, sensitive, robust, and reproducible thanks also to the special features of the polycarboxylate-hydrogel-based coatings used for the antibody immobilization. The sensor surface displays a high level of stability during repeated regener- ation and affinity reaction cycles. The immunosensor shows high specificity for cortisol and cortisone; furthermore, no significant interferences from other steroids with a similar chemical structure have been observed. The suitability of the hydrogel coating for the prevention of nonspecific binding is also investigated. A good correlation is noticed between the results obtained by the proposed method and the reference liquid chromatography/tandem mass spec- trometry method for the analysis of cortisol and cortisone in urine and saliva samples. Standard curves for the detection of cortisol and cortisone in saliva and urine are character- ized by a detection limit less than 10 μgl -1 , sufficiently sensitive for both clinical and forensic use. Keywords Cortisol . Cortisone . SPR . Immunosensor . Antidoping Introduction Endogenous glucocorticoids are produced in the fascicular zone of the adrenal cortex under the control of the hypothalamo-pituitary-adrenal axis and are transported to their target receptors in the form of protein complexes with albumin and with the specific serum protein transcortin. Their mechanism of action depends on their binding to specific receptors, which are widely distributed in a great variety of biological districts, thus involving many different target tissues [1]. Cortisol is the principal circulating glucocorticoid in man and is secreted in relatively high amounts, 15 mg day -1 . The free fraction is biologically active and represents only 1% of the total cortisol secretion rate [2]. The measurement of the blood, urinary, and salivary free cortisol level is useful in clinical for the adrenal or pituitary gland disorder diagnosis and in forensic field as first-level screening to detect the illicit use of glucocorticoids for the nonphysiological enhancement of sport performance. Unfor- tunately, the free cortisol and cortisone concentration profiles are dependent on many factors, including patient age, gender, sample population, intensive endurance training, severe emotional or physical stress, and medications, such as for example glucocorticoids, lithium, diuretics, ketocona- zole, estrogens, and tricyclic antidepressants, so no reference range are still disposable. The control of the cortisol/ cortisone concentration is operated by two isoforms of the 11 β-hydroxysteroid dehydrogenase (11 β-HSD) that cata- lyze the interconversion of the active cortisol to its inactive metabolite cortisone [3]. One of its two isomers, the 11 β- HSD type 1 acts predominantly as an 11-oxo-reductase (i.e., it converts cortisone to cortisol), while 11 β-HSD type 2 catalyzes 11 β-dehydrogenation (converting cortisol to cortisone). Cushing’ s syndrome of apparent mineralocorti- coid excess is caused by 11 β-HSD deficiency [4, 5]. M. Frasconi : F. Mazzei (*) Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy e-mail: franco.mazzei@uniroma1.it M. Mazzarino : F. Botrè Laboratorio Antidoping Federazione Medico Sportiva Italiana, Largo Giulio Onesti 1, 00197 Rome, Italy Anal Bioanal Chem (2009) 394:2151–2159 DOI 10.1007/s00216-009-2914-6