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MICROFLUIDIC IMMUNOSENSOR FOR DETERMINATION OF CLENBUTEROL USING GOLD NANOPARTICLES AND MAGNETIC MICRO PARTICLES AS BIOAFFINITY PLATFORM Regiart DMGa, Pereira SVa, Fernández-Baldo MA, Aranda Pa, Spotorno VGb, Bertolino FAa, Raba Ja a Departamento de Química, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis. Chacabuco 917. D5700BWS. San Luis, Argentina. b Instituto de Recursos Biológicos, IRB, CIRN. Instituto Nacional de Tecnología Agropecuaria, INTA. C.C. 77 Morón B1708WAB. Buenos Aires, Argentina. Introduction Clenbuterol (CLB) is a β2-adrenergic agonist used in animal production which is banned in Argentina and in the European Union (1). CLB has been used illegally in animal production although the accumulation of their residues in animal tissue can cause symptoms of acute poisoning in humans. Several cases of human food poisoning have been reported (2). This β-agonist represents a potential risk and has to be carefully monitored to avoid the illegal use of high amounts as a growth-promoting agent in meat production. Since then, it is being extensively controlled as a veterinary drug residue in food safety (3). In this work, we report the development of a microfluidic immunosensor performed on a screen-printed carbon electrode (SPCE) (4), which was modified by electrodeposition of gold nanoparticles (AuNPs), and characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and cyclic voltammetry (CV). This electrochemical detection system was applied to the quantification of low concentrations of CLB present in bovine hair samples. The great advantage of hair analysis is the high affinity of CLB for melanin. This fact prevents metabolic clearance, and provides an ??in vitro?? piece of evidence prior to sacrifice, facilitating the action of veterinary inspectors. Materials and methods The main body of the sensor was made of polymethylmethacrylate (PMMA). The sensor has a flow-through central channel (CC) with a diameter of 100 µm. The SPCE placed at the end of the CC is made up of a graphite working electrode, a graphite counter electrode and an Ag pseudo-reference electrode. The CLB capture procedure was performed employing as bioaffinity support the magnetic micro particles amino functionalized (MMPAF), which was modified by the immobilization of anti-CLB Ab with Glutaraldehyde. For the electrodeposition procedure of AuNPs, the SPCE was immersed into HAuCl4 solution, after that a constant potential value of ?0.2 V vs. Ag was applied. The microﬂuidic device was prepared by injection of anti?CLB Ab?MMPAF in the flow system. A permanent magnet was used to attract the beads at specific area near AuNPs/SPCE. The samples competed with CLB?AP conjugate. The immunosensor was then washed with PBS. The substrate solution (p-APP) was injected into the carrier stream and the enzymatic product (p-AP) was measured on the surface of AuNPs/SPCE. Results Linearity and range of the developed method were studied by analyzing different concentrations of the standard solution of CLB on the matrix. The calibration curve was obtained with a linear relation between 0.027?800 ng mL-1. The data were analyzed by linear regression least-squares ﬁt method. The calibration graph was described by the calibration equation ΔI (nA) = 143.01 ? 35.29 log CCLB with a correlation coefficient of 0.998. The standard deviation (SD) for the calibration curve was 3.33. The coefficient of variation (CV) for the determination of 6.40 ng mL-1 CLB was below 5.32% (n=6). For the electrochemical detection procedure, the LOD and LOQ were 0.008 and 0.027 ng mL−1, respectively. The assay showed good precision; the CV% within-assay values were below 4% and the between-assay values were below 6%. The total assay time for the determination of the CLB concentration was 18 min. The F-test value for the biosensor was 0.39 (the F-test value is 2.26 at a 95% conﬁdence level), suggesting that the method has a linear behavior. Conclusions In this work we have developed a microfluidic immunosensor coupled to electrochemical detection for the quantification of CLB in bovine hair samples. The great advantage of hair analysis is that the high affinity of CLB for melanin prevents metabolic clearance. Our proposed system is coupled to SPCE with electrodeposited gold nanoparticles; this modiﬁcation procedure allowed us to obtain an important increment in the sensitivity of our system. The use of magnetic microparticles as bioafﬁnity platform for the immobilization of antibodies enhances the amount of immobilized antibodies on the particle surface. This device provides a simple design with a high sensitivity and selectivity for the detection of CLB residues in low times and represents a significant tool for an automated and portable determination for its application in food safety. Acknowledgments The authors wish to thank the financial support from the San Luis National University (UNSL), the San Luis Chemistry Institute (INQUISAL), the National Council of Scientific and Technical Research (CONICET) and the National Institute of Agricultural Technology (INTA). References 1) Mitchell GA, Dunnavan G. Illegal Use of β-Adrenergic Agonists in the United States. J. Anim. Sci. 1998; 76:208-211. 2) Chan T. Health hazards due to clenbuterol residues in food. Clin. Toxicol. 1999; 37:517-519. 3) Gallo P, Brambilla G, Neri B, Fiori M, Testa C, Serpe L. Purification of clenbuterol-like β2-agonist drugs of new generation from bovine urine and hair by α1-acid glycoprotein affinity chromatography and determination by gas chromatography-mass spectrometry. Anal. Chim. Acta. 2007; 587:67-74. 4) Pereira SV, Bertolino FA, Fernández-Baldo MA, Messina GA, Salinas E, Sanz MI, Raba J. A microfluidic device based on a screen-printed carbon electrode with electrodeposited gold nanoparticles for the detection of IgG anti-Trypanosoma cruzi antibodies. Analyst. 2011; 136:4745-4751.

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