Full Paper Development of a Progesterone Immunosensor Based on a Colloidal Gold-Graphite-Teflon Composite Electrode V. Carralero, A. Gonza ´lez-Corte ´s, P. Ya ´n ˜ez-Seden ˜o, J. M. Pingarro ´n* Department of Analytical Chemistry, Faculty of Chemistry, University Complutense of Madrid. 28040-Madrid, Spain *e-mail: pingarro@quim.ucm.es Received: October 3, 2006 Accepted: November 20, 2006 Abstract A novel amperometric immunosensor for the determination of progesterone, based on direct attachment of antiprogesterone on a gold nanoparticles-modified graphite-Teflon composite electrode is reported. Competitive assay involving progesterone labeled with alkaline phosphatase (AP), and 1-naphtyl phosphate as the enzyme substrate, was employed. The product of the enzyme reaction, 1-naphtol was detected amperometrically at þ 0.3 V vs. Ag/AgCl. Variables involved in the immunosensor preparation such as the colloidal gold loading and the amount of anti-Prog immobilized, as well as in the immunosensing event (pH, Prog/Prog-AP ratio and volume) were optimized. A calibration plot was obtained for progesterone with a linear range between 0 and 30 ng mL 1 , and a slope value,  1.2 nA ng 1 mL, which is remarkably higher than that reported for other progesterone biosensors. A good repeatability of the amperometric measurements, RSD ¼ 3.5% (n ¼ 10), and an acceptable fabrication reproducicility, RSD ¼ 8.2% (n ¼ 3), were obtained. The developed anti-Prog-Au coll -graphite-Teflon immunosensor was applied to the progesterone determination in milk. Recovery at the 3.5 ng mL 1 progesterone concentration level was of 101  6% (n ¼ 7). Keywords: Electrochemical immunosensor, Gold nanoparticles, Composite electrodes, Progesterone DOI: 10.1002/elan.200603794 1. Introduction Voltammetric detection methods for immunoassays have beendevelopedsincemorethan20years[1,2].Inparticular, electrochemical immunosensors combine selectivity of the antibody-antigen recognition processes with the excellent capabilities of electrochemical transduction such as sensi- tivity, low cost, speed of analysis and possibility of portable screening. Of course, immobilization of immunoactive compounds on the electrode surface without significant losses of their activity and stability is an important goal to achieve in the preparation of immunosensors. In this context, the use of gold nanoparticles has demonstrated to be an useful tool for the development of electrochemical immunosensors. As it is well known, biological molecules retain their activity when adsorbed on gold nanoparticles [3]. Modification of electrodes with these nanoparticles provides a microenvironment similar to that of the proteins in native systems, with gold nanocrystals acting as conduct- ing tunnels and facilitating electron transfer [4, 5]. In fact, very recently, various configurations of electrochemical immunosensors using gold nanoparticles have been report- ed. Amperometric enzyme immunosensors have been developed for the detection of a-1-fetoprotein based on layer-by-layer assembly of gold nanoparticles and thionine on a Nafion modified electrode by electrostatic adsorption [6]. Gold nanoparticles supported on a glassy carbon electrode modified with toluidine blue-polysulfanilic acid havebeenalsousedfortheconstructionofanamperometric immunosensor for determination of carcinoembryonic antigen [7]. Furthermore, an electrochemical immunoassay for carcinoma antigen-125 based on antigen immobilization oncolloidalgoldstabilizedwithcelluloseacetatemembrane on a glassy carbon electrode has been reported [8]. More- over, various immunosensor designs prepared from hybrid materials such as sol-gel and/or self-assembled monolayers and gold nanoparticles, which exhibit high sensitivity and long-termstability,havebeenalsorecentlyreported[9–12]. On the other hand, colloidal gold-graphite-Teflon compo- sites have demonstrated to be an excellent material for the preparation of biosensors [13]. Gold nanoparticles and biomolecules can be incorporated into the graphite-Teflon matrix by simple physical inclusion, thus making the electrode fabrication procedure easy, fast and cheap, and giving rise to bioelectrodes easily renewable by polishing. Until now, no electrochemical immunosensors based on this type of config- uration are found in the literature, although an immunosensor based on a thionine and gold nanoparticles-modified carbon paste electrode has been reported very recently [14]. Monitoring of progesterone levels in cow)s milk has a great interest because it constitutes a reliable approach to predict ovulation, and to detect pregnacy and fertility problems[15].Electrochemicalbiosensorsforprogesterone using screen-printed carbon electrodes (SPCE) coated with 853 Electroanalysis 19, 2007, No.7-8, 853–858 # 2007 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim