Novel Electrochemical Biosensor for Simultaneous Detection of Adenine and Guanine Based on Cu2O Nanoparticles

Procedia Engineering 47 ( 2012 ) 702 – 705 1877-7058 © 2012 The Authors. Published by Elsevier Ltd. Selection and/or peer-review under responsibility of the Symposium Cracoviense Sp. z.o.o. doi: 10.1016/j.proeng.2012.09.244 Proc. Eurosensors XXVI, September 9-12, 2012, Kraków, Poland Novel Electrochemical Biosensor for Simultaneous Detection of Adenine and Guanine Based on Cu2O Nanoparticles Jana Chomouckaa,b,*, Jan Praseka,b, Petra Businovaa,b, Libuse Trnkovac, Jana Drbohlavovaa,b, Jan Pekareka,b, Radim Hrdya,b and Jaromir Hubaleka,ba a Department of Microelectronics, Brno University of Technology, Technicka 3058/10, Brno, CZ-616 00,Czech Republic bCentral European Institute of Technology, Brno University of Technology, Technicka 3058/10, Brno, CZ-616 00,Czech Republic cDepartment of Chemistry, Masaryk University, Kamenice 5, Brno ,CZ-625 00,Czech Republic Abstract In this paper Cu2O nanoparticles were prepared and used for the construction of novel electrochemical voltammetric biosensor for the simultaneous detection of adenine and guanine. Cu2O nanoparticles were synthesized via simple wet chemical route, where glucose was used as a reductant. The nanoparticles were characterized by SEM and XRD analysis, which showed the presence of spherical aggregations with diameter of 1000 nm. These nanoparticles were successfully used for fabrication of spray-coated and screen-printed working electrodes on alumina substrate for the electrochemical detection of purine bases. We observed that Cu(I) reacts with adenine to form insoluble complex that accumulates on the electrode surface and causes the decrease of current response. In the case of guanine, we did not observed any significant decrease of current response which is probably caused by adsorption of guanine on the electrode surface. © 2012 Published by Elsevier Ltd. Keywords: Thick film technology; electrochemistry; cyclic voltametry; copper(I) oxide; purines 1. Introduction Cuprous oxide (Cu2O) is a p–type metal oxide semiconductor with a direct band gap of 2.0–2.2 eV. It has attracted increasing interest due to its promising application in magnetic devices, solar energy conversion and catalysts [1]. New types of solid electrodes are necessary for small device technologies which may replace the standard electrochemical analysis, where toxic mercury drop electrodes are commonly used. The * Corresponding author. Tel.: +420-541-146-163; fax: +420-541-146-298. E-mail address: [email protected]. Available online at www.sciencedirect.com © 2012 The Authors. Published by Elsevier Ltd. Selection and/or peer-review under responsibility of the Symposium Cracoviense Sp. z.o.o. Open access under CC BY-NC-ND license. Open access under CC BY-NC-ND license. 703 Jana Chomoucka et al. / Procedia Engineering 47 ( 2012 ) 702 – 705 performance of solid electrode is determined by its surface modification to make it sensitive and selective towards a certain analyte [2]. Solid electrodes can be fabricated using thick–film technology (TFT). The advantage of TFT is its flexibility, low production costs, good reproducibility and good electrical and mechanical properties of electrodes. Adenine and guanine are components of DNA. Most of the current electroanalytical protocols for DNA detection are based on these electroactive purine bases. The abnormal changes in the concentration of these bases in organisms indicate the efficiency and/or mutation of the immune system, and may be the sign of various diseases. Hence, the determination of individual concentrations of these components, or their ratios in DNA is of great interest in bioscience and clinical diagnosis [3]. 2. Experimental 2.1. Cu2O nanoparticles preparation The preparation method of Cu2O nanoparticles is based on the procedure reported in [4]. Nanoparticles were prepared by two-step synthesis. At first, 0.0035 mol Cu(CH3COO)2+2O was dissolved in 100 mL absolute ethanol under ultrasonic to form the deep green solution. The solution was heated to 60 °C. Than 50 mL glucose aqueous solution (0.1 mol/L) was slowly added into the solution under vigorous stirring. After that, 50 mL NaOH aqueous solution (0.5 mol/L) was also added to the solution at the same speed. Then some light yellow precipitates was occurred. The particles in the suspension were then separated by centrifugation at 4000 rpm for 8 min. The particles were resuspended in absolute ethanol followed by distilled water. The centrifugation was repeated thrice to remove CH3COOí, glucose and NaOH. The light yellow precipitate was then dried under nitrogene atmosphere overnight. 2.2. Electrodes fabrication Working mikroelectrodes were fabricated using standard thick-film technology process on the alumina substrate. Thick-film pastes used for contact and covering layers were ESL 9562-G and ESL 4917 both from ESL Electroscience, UK. Carbon paste BQ 221 (Dupont) was screen-printed over Ag/Pd/Pt based contact layer to avoid the contact layer to be present in electrochemical reaction. The working electrode was fabricated by spray-coating and screen printing deposition using Cu2O nanoparticles powder as the filling material. For spray coating deposition Cu2O particles were dispersed in N-Methyl-Pyrrolidone. To ensure good shape of the electrode a precise template was used for the deposition and the substrate was heated to 250 °C. The spray-coating process was repeated until the carbon layer was totally covered with the nanotubes to prevent later impact to electrochemical analysis. In the case of screen-printed deposition method, Cu2O nanoparticles were well homogenised with vehicle. Prepared paste were screen-printed on an alumina substrate and dried. 2.3. Electrochemical measurement Electrochemical measurements were performed with PalmSens handheld potentiostat/galvanostat (Palm Instruments BV, Netherlands). The device was connected to a personal computer for measurement setup and response evaluation. A three–electrode system was used, Cu2O electrode was employed as the working electrode, an Ag/AgCl/3M KCl electrode served as the reference electrode and Pt electrode was used as the auxiliary electrode. Cyclic voltammetry (CV) were carried out in the presence of 20 ml 0,2 M acetate buffer pH 5.0 and in the presence of various concentration of adenine or guanine. CV parameters: scan rate 100 mV/s, potential range -0.4 to 0.4 V. 704 Jana Chomoucka et al. / Procedia Engineering 47 ( 2012 ) 702 – 705 3. Results and discussion In our experiment, glucose was used as reductant. Reduction of Cu(II) tartrate complex by glucose can yield stable sols of cuprous oxide, whose particle size and morphology are strongly dependent on the concentration of reactants As shown in Fig. 1, sample has a spherical aggregation with a diameter of 1000 nm. But they are conglomerated by smaller particles with the mean size of 150 nm. According to the XRD measurement, we prepared Cu2O/CuO nanoparticles consisted of 70 % Cu2O and 30 % CuO. Fig. 1. SEM image of Cu2O spherical aggregations (left) and detail of Cu2O nanoparticles (right). The electrochemical oxidation of adenine, guanine or other purine derivatives at carbon electrode is well known. Formation of complexes of these compounds with metals including copper has been studied. Cu(II) ions can be reduced to Cu(I) which reacts with adenine to form insoluble compounds that accumulate on the electrode surface and cause the decrease of current response (Fig. 2). This approach can be applied for the detection of oligodeoxynucleotide (ODN) after acid hydrolysis during which the purine bases are released from the ODN chain [5]. Fig. 2. Cyclic voltammograms of Cu2O spray-coating working electrode (left) and screen-printed working electrode (right) in the presence of adenine. 2 ȝP 2 nP 705 Jana Chomoucka et al. / Procedia Engineering 47 ( 2012 ) 702 – 705 Fig. 3. Cyclic voltammograms of Cu2O spray-coating working electrode (left) and screen-printed working electrode (right) in the presence of guanine. In the case of guanine, we did not observed any significant decrease of current response which is probably caused by adsorption of guanine on the electrode surface (Fig. 3). 4. Conclusion Cu2O nanoparticles for preparation of thick film pastes were prepared and characterized. Prepared NPs were sprayed or screen-printed on previously prepared electrode substrate. These electrodes were successfully used as the working electrodes for electrochemical detection of adenine and guanine. Acknowledgements This work has been supported by Grant Agency of the Academy of Sciencies of the Czech Republic under the contract GAAV KAN208130801 and by project CEITEC CZ.1.07/2.3.00/20.0027. References [1] Zahmakiran M, Ozkar S, Kodaira T, Shiomi T. A novel, simple, organic free preparation and characterization of water dispersible photoluminescent Cu2O nanocubes. 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