Please cite this article in press as: G. Merola, et al., Simple and suitable immunosensor for -lactam antibiotics analysis in real matrixes: Milk, serum, urine, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.08.005 ARTICLE IN PRESS G Model PBA-9680; No. of Pages 11 Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxx–xxx Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis j o ur na l ho mepage: www.elsevier.com/locate/jpba Simple and suitable immunosensor for -lactam antibiotics analysis in real matrixes: Milk, serum, urine Giovanni Merola, Elisabetta Martini, Mauro Tomassetti, Luigi Campanella ∗ Department of Chemistry, “Sapienza” University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy a r t i c l e i n f o Article history: Received 5 May 2014 Received in revised form 31 July 2014 Accepted 1 August 2014 Available online xxx Keywords: Penicillin G -Lactam antibiotics Competitive assay Immunosensor Drugs and real biological matrixes analysis a b s t r a c t The anti-penicillin G was conjugated to avidin–peroxidase and biotin to obtain immunogen and competi- tor which were then used to develop a competitive immunosensor assay for the detection of penicillin G and other -lactam antibiotics, with K aff values of the order of 10 8 M -1 . The new immunosensor appears to afford a number of advantages in terms of sensitivity, possibility of “in situ” analysis, but especially of simplicity and lower costs, compared with other existing devices, or different chemical instrumen- tal methods reported in the literature and used for the analysis of -lactam compounds. Satisfactory results were found in the analysis of real matrixes and good recoveries were obtained by applying the standard addition method to spiked milk, urine, serum and drug samples. The new device uses an amper- ometric electrode for hydrogen peroxide as transducer, the BSA–penicillin G immobilized on polymeric membrane overlapping the amperometric transducer and the peroxidase enzyme as marker. It proved to be highly sensitive, inexpensive and easily reproducible; LOD was of the order of 10 -11 M. Lastly, the new immunosensor displayed low selectivity versus the entire class of -lactam antibiotics and higher selectivity toward other classes of non--lactam antibiotics. © 2014 Elsevier B.V. All rights reserved. 1. Introduction -Lactam antibiotics are frequently used to prevent bacterial infections in humans and cattle. But improper use of -lactam antibiotics can leave residues in cow milk and cause serious prob- lems to human health. The intake of -lactam antibiotics, which may be partly involuntary, since, as seen above, the food chain itself leads to its ingestion, or voluntary, in order to combat bacterial infections, more and more frequently causes bacterial resistance toward -lactam antibiotics. It is also well known that the intake of -lactam antibiotics can cause allergic potential in presensi- tized people. This is causing growing alarm among healthcare specialists as it can offset or prevent the use of one of the most important drugs ever discovered and produced by pharmaceuti- cal chemistry. This explains the need felt for frequent, fast, simple and cheap tests that can be applied not only to human biolog- ical fluids but also performed directly in dairies on the cow’s milk produced and used directly in human food. Of course there is no lack of analytical methods to test for -lactam antibiotics, such as penicillins and cephalosporins. These tests comprise in ∗ Corresponding author. Tel.: +39 06 49913744; fax: +39 06490631. E-mail address: luigi.campanella@uniroma1.it (L. Campanella). particular chromatography [1–5], mass spectrometry [6–13] and capillary electrophoresis techniques [14], circular dichroism [15], but also tests based on microbial screening [16–18], including a good number of imprinted-polymer methods [19–21]. Nevertheless the need for rapid tests with low LOD values has also encouraged the use of sensor, biosensor and immunosensor methods. In this field also relatively sophisticated sensors have been developed, such as bioFET [22,23], optical sensor [24,25], evanescent wave [26], based fluorescence [27–30], luminescence [31–33], or plasmon resonance sensor (SPR) [34,35] and sophisti- cated immunological microarray [36–41]. However, over the past year our research group developed [42] no complex sensors or devices suitable for array systems, but rather a single use, simple and inexpensive immunosensor that is easy to build, as it is made of materials readily obtainable on the market, such as the classi- cal amperometric hydrogen peroxide sensor used as transducer. This new Immunodevice was recently optimized by our research group and then successfully applied to the testing for antibiotics in wastewater in rivers of central Italy [42]. The present article reports on the latest developments in this research, consisting in both the development of a competitive format based on BSA–penicillin G conjugated immobilized on a polymeric active membrane, and the application of a new immun- odevice to the analysis of real samples, such as cow urine and http://dx.doi.org/10.1016/j.jpba.2014.08.005 0731-7085/© 2014 Elsevier B.V. All rights reserved.