Anal. Chem. 2009, 81, 1332–1339
Double-Tagging Polymerase Chain Reaction with a Thiolated Primer and Electrochemical Genosensing based on Gold Nanocomposite Sensor for Food Safety Paulo R. Brasil de Oliveira Marques,† Anabel Lermo,‡ Susana Campoy,§ Hideko Yamanaka,† Jordi Barbe ´ ,§ Salvador Alegret,‡ and M. Isabel Pividori*,‡ Grup de Sensors i Biosensors, Departament de Quı´mica, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Catalonia, Spain, Unitat de Microbiologia, Departament de Gene`tica i de Microbiologia, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Catalonia, Spain, and UNESP, Universidade Estadual Paulista, Instituto de Quı´mica, Laborato´rio de Eletroanalı´tica, Campus de Araraquara, Sa˜o Paulo, Brazil A novel material for electrochemical biosensing based on rigid conducting gold nanocomposite (nano-AuGEC) is presented. Islands of chemisorbing material (gold nanoparticles) surrounded by nonreactive, rigid, and conducting graphite epoxy composite are thus achieved to avoid the stringent control of surface coverage parameters required during immobilization of thiolated oligos in continuous gold surfaces. The spatial resolution of the immobilized thiolated DNA was easily controlled by merely varying the percentage of gold nanoparticles in the composition of the composite. As low as 9 fmol (60 pM) of synthetic DNA were detected in hybridization experiments when using a thiolated probe. Moreover, for the first time a double tagging PCR strategy was performed with a thiolated primer for the detection of Salmonella sp., one of the most important foodborne pathogens affecting food safety. This assay was performed by double-labeling the amplicon during the PCR with a -DIG and -SH set of labeled primers. The thiolated end allows the immobilization of the amplicon on the nano-AuGEC electrode, while digoxigenin allows the electrochemical detection with the antiDIG-HRP reporter in the femtomole range. Rigid conducting gold nanocomposite represents a good material for the improved and oriented immobilization of biomolecules with excellent transducing properties for the construction of a wide range of electrochemical biosensors such as immunosensors, genosensors, and enzymosensors. The oriented and improved immobilization of single-stranded DNA to solid substrates, followed by hybridization, has gained importance over the past decade, due to its use as a genomic * To whom correspondence should be addressed. E-mail: isabel.pividori@ uab.es. † UNESP, Universidade Estadual Paulista. ‡ Departament de Quı´mica, Universitat Auto`noma de Barcelona. § Departament de Gene`tica i de Microbiologia, Universitat Auto`noma de Barcelona.
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detection tool in DNA arrays,1 DNA biosensors,2 and selfassembled molecular electronic circuits.3 In some cases, immobilization on a conducting metal surface is necessary. Because of the strong affinity of thiols to metal surfaces, sulfur chemistry is widely employed when attaching DNA, especially to continuous gold films4 or, instead, to gold nanoparticles-modified electrodes, to increase the electroactive surface.5 Furthermore, the use of gold nanoparticles as a label for biosensing devices is also gaining importance.6 The standard way for thiolating a strand of DNA is to attach a HS(CH2)6- linker molecule to either the 3′ or the 5′ end phosphate group. Although the linker keeps the DNA away from the gold surface and makes the binding event sterically more favorable, it also acts as an electrical insulator between the conducting surface and the DNA molecule.3 Although van der Waals attraction drives the assembly and ordering in typical SAMs (self-assembled monolayers), DNA immobilization is subject to strong electrostatic repulsion.7 As a result, in hybridization experiments, although a spacer arm being used, (1) (a) Schena, M. DNA Microarrays: A Practical Approach; Oxford University Press: Oxford, U.K., 1999. (b) Polsky, R.; Harper, J. C.; Wheeler, D. R.; Brozik, S. M. Electroanalysis 2008, 20, 671–679. (2) Lucarelli, F.; Marrazza, G.; Turner, A. P. F.; Mascini, M. Biosens. Bioelectron. 2004, 19, 515–530. (3) Wirtz, R.; Wa¨lti, C.; Tosch, P.; Pepper, M. Langmuir 2004, 20, 1527–1530. (4) (a) Pang, D. W.; Abrun ˜a, H. D. Anal. Chem. 1998, 70, 3162–3169. (b) Kerman, K.; Ozkan, D.; Kara, P.; Meric, B.; Gooding, J. J.; Ozsoz, M. Anal. Chim. Acta 2002, 462, 39–47. (c) Mannelli, I.; Minunni, M.; Tombelli, S.; Wang, R. H.; Spiriti, M. M.; Mascini, M. Bioelectrochemistry 2005, 66, 129– 138. (d) Steichen, M.; Buess-Herman, C. Electrochem. Commun. 2005, 7, 416–420. (5) (a) Cai, H.; Xu, C.; He, P.; Fang, Y. J. Electroanal. Chem. 2001, 510, 78– 85. (b) Wang, M.; Sun, C.; Wang, L.; Ji, X.; Bai, Y.; Li, T.; Li, J. J. Pharm. Biomed. Anal. 2003, 33, 1117–1125. (c) Jin, B.; Ji, X.; Nakamura, T. Electrochim. Acta 2004, 50, 1049–1055. (d) Fu, Y.; Yuan, R.; Xu, L.; Chai, Y.; Zhong, X.; Tang, D. Biochem. Eng. J. 2005, 23, 37–44. (e) Liu, S.-F.; Li, Y.-F.; Li, J.-R.; Jiang, L. Biosens. Bioelectron. 2005, 21, 789–795. (f) Yang, J.; Yang, T.; Feng, Y.; Jiao, K. Anal. Biochem. 2007, 365, 24–30. (g) Kang, J.; Li, X.; Wu, G.; Wang, Z.; Lu, X. Anal. Biochem. 2007, 364, 165–170. (6) (a) Gonza´lez-Garcı´a, M. B.; Ferna´ndez-Sa´nchez, C.; Costa-Garcı´a, A. Biosens. Bioelectron. 2000, 15, 315–321. (b) Dequaire, M.; Degrand, C.; Limoges, B. Anal. Chem. 2000, 72, 5521–5528. (c) Wang, J.; Polsky, R.; Xu, D. Langmuir 2001, 17, 5739–5741. (d) Erdem, A. Talanta 2007, 74, 318– 325. (e) Valentini, F.; Palleschi, G. Anal. Lett. 2008, 41, 479–520. (7) Petrovykh, D. Y.; Pe´rez-Dieste, V.; Opdahl, A.; Kimura-Suda, H.; Sullivan, J. M.; Tarlov, M. J.; Himpsel, F. J.; Whitman, L. J. J. Am. Chem. Soc. 2006, 128, 2–3. 10.1021/ac801736b CCC: $40.75 2009 American Chemical Society Published on Web 01/26/2009
tightly packed and negatively charged SAM are obtained, which impedes hybridization with the cDNA probe due to both steric as well as electrostatic effects.8 As such, a stringent control of the surface coverage of DNA is an important factor in maximizing hybridization efficiency,9 which can be performed by using auxiliary reagents such as lateral spacer thiols and mixed monolayers to obtain bioactive gaps.10 In order to avoid the stringent control of surface coverage parameters during immobilization of thiolated oligos on continuous gold films, the use of gold nanoparticles in a graphite-epoxy composite (nano-AuGEC) is presented in this paper. The aim of this work is to obtain islands of chemisorbing material (gold nanoparticles) surrounded by rigid, nonchemisorbing, conducting, graphite-epoxy composite. With this arrangement in the electrochemical transducer, the resulting less-packed surface provides improved hybridization features with a complementary probe minimizing steric and electrostatic repulsion. In order to prove the utility of this material, electrochemical genosensing of synthetic DNA sequences was performed with a thiolated probe and a digoxigenin complementary oligonucleotide and using antiDIG-HRP as an electrochemical label. Moreover, for the first time, a double tagging PCR strategy was designed with a thiolated primer for the detection of Salmonella sp. The rapid electrochemical verification of the amplicon coming from the pathogenic genome of Salmonella was performed by double-labeling the amplicon during the PCR with a set of two labeled primers, one of them with digoxigenin and the other one with thiol moieties. The thiolated end allows the immobilization of the amplicon on the nano-AuGEC electrode and digoxigenin, for the electrochemical detection, by attaching an AntiDIG antibody modified with HRP enzyme. Rigid conducting gold nanocomposite represents a simple method for the oriented immobilization of biomolecules. The microscopic and electrochemical characterization of this material is presented in this paper, as well as its utility in electrochemical genosensing for food safety. EXPERIMENTAL SECTION Instrumentation. Temperature-controlled incubations were done in a temperature-controlled mixer Eppendorf Thermomixer 5436. The PCR reaction was carried out in an Eppendorf Mastercycler Personal thermocycler. The Leica MZ FLIII fluorescence stereomicroscope (Leica, Heidelberg, Germany) and the Jeol JSM6300 scanning electron microscope (Jeol LTD, Tokio, Japan) coupled with an EDX detector which allows a characteristic X-ray spectrum (Oxford instruments, Bucks, England) were used to study the distribution of the gold nanoparticles on the electrode surface. (8) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481–4483. (9) (a) Hianik, T.; Gajdos, V.; Krivanek, R.; Oretskaya, T.; Metelev, V.; Volkov, E.; Vadgama, P. Bioelectrochemistry 2001, 53, 199–204. (b) Keighley, S. D.; Li, P.; Estrela, P.; Migliorato, P. Biosens. Bioelectron. 2008, 23, 1291–1297. (10) (a) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916–8920. (b) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787–9792. (c) Zhao, Y.-D.; Pang, D.-W.; Hu, S.; Wang, Z.-L.; Cheng, J.-K.; Dai, H.-P. Talanta 1999, 49, 751–756. (d) Petrovykh, D. Y.; Kimura-Suda, H.; Whitman, L. J.; Tarlov, J. M. J. Am. Chem. Soc. 2003, 125, 5219–5226. (e) Carpini, G.; Lucarelli, F.; Marrazza, G.; Mascini, M. Biosens. Bioelectron. 2004, 20, 167–175. (f) Loaiza, O. A.; Campuzano, S.; Pedrero, M.; Pingarro´n, J. M. Talanta 2007, 73, 838–844.
Amperometric measurements were performed with an LC-4C amperometric controller (BAS Bioanalytical Systems Inc.). Voltammetric characterization was carried out using an Autolab PGSTAT Eco-chemie (The Netherlands). A three-electrode setup was used comprising a platinum auxiliary electrode (Crison 5267 1, Spain), a double junction Ag/AgCl reference electrode (Orion 900200) with 0.1 mol L-1 KCl as the external reference solution, and a working electrode (the GEC or nanoAu-GEC electrodes). Chemicals and Biochemicals. Composite electrodes were prepared using 50 µm particle size graphite powder (Merck, U.K.) and Epotek H77 epoxy resin and hardener (both from Epoxy Technology). The gold nanoparticles (nanopowder,
