Full Paper Development of Streptavidin Carrying Magnetic Nanoparticles and TheirApplicationsinElectrochemicalNucleicAcidSensorSystems Arzum Erdem, a * Filiz Sayar, b Hakan Karadeniz, a Guldem Guven, b Mehmet Ozsoz, a Erhan Piskin b * a Ege University, Faculty of Pharmacy, Analytical Chem. Dept., 35100 Bornova, Izmir, Turkey *e-mail: arzum.erdem@ege.edu.tr b Hacettepe University, Faculty of Engineering, Chem. Eng. Dept and Bioengin Div., and TUBITAK-Biyomedtek: Center for Biomedical Technologies, Beytepe, 06000 Ankara, Turkey *e-mail: piskin@hacettepe.edu.tr, erhanpiskin@biyomedtek.com Received: August 25, 2006 Accepted: November 22, 2006 Abstract The aim of this study is to investigate the applicability of streptavidin carrying magnetic nanoparticles in electrochemical nucleic acid sensor systems. The magnetite (Fe 3 O 4 ) nanoparticles were prepared by co-precipitation technique, and then they were coated with a carboxylic acid containing polymer layer. Streptavidin molecules were immobilized onto the surfaces of these magnetic nanoparticles (with two different average diameter of 125 and 225 nm) via these functional groups. An electrochemical nucleic acid sensor system composed of a disposable graphite sensor, pencil graphite electrode (PGE) and differential pulse voltammetry (DPV) was used for measurement of guanine oxidation signal observed at þ 1.0 V. In the hybridization tests biotinylated inosine substituted probe was immobilized onto the magnetic nanoparticles and hybridization with its complementary target at the surface of particles within the medium was exhibited successfully by using this electrochemical sensor system. For electro- chemical detection of sequence specific DNA hybridization and its selectivity studies, the biotinylated hepatitis B Virus (HBV) wild type probe was immobilized onto the nanoparticles. The guanine oxidation signals were measured in the solutions containing no target, the complementary target, a single-base mismatch target, and noncomple- mentary target oligonucleotides. A significant guanine oxidation signal (71 nA) was observed with the complementary target due to complete hybridization, while with single-base mismatch target, the signal at the same conditions was quite low about 14 nA. However, in the case of the noncomplementary target, there was a low but observable mean signal about 9 nA (showing the nonspecific interaction). The detection limit (S/N ¼ 3) was found as 43.11 pmol/mL target concentration obtained in 20 min hybridization time. Keywords: Magnetic nanoparticles, Streptavidin, Electrochemical nucleic acid sensors, Guanine oxidation signal DOI: 10.1002/elan.200603774 1. Introduction Electrochemical nucleic acid sensors have been attracted a greatattentionasveryspecificsensorsindiversebiomedical and related applications [1 – 4]. An increasing interest has appeared in the development of simple, rapid and user- friendly electrochemical detection systems based on DNA sequence and mutant gene analysis, for instance early and precise diagnosis of infectious agents, for routine clinical tests [5–10]. These systems have been exploited for monitoring sequence-specific hybridization events directly [7 – 9, 11 – 13] based on the oxidation signal of guanine and/ or adenine or detecting by using DNA intercalators or bis- intercalators such as, some antibiotics which form com- plexes with the nitrogenous bases of DNA [14 – 16]. The development of advanced biosensors based on novel nanomaterials have been considered as important tools in the new approaches in the field of genomics, medical diagnosis, and drug-DNA interactions [5, 17–19]. Electro- chemical nucleic acid sensors based on magnetic particles, nanoparticles labeled with metal tags, nanotubes and other nanomaterials have been recently investigated [7, 9, 11, 13, 20–28]. The electrochemical detection of DNA based on mag- netic particles [7, 9, 11, 13, 20 – 25] as labeling with an enzyme [23, 24], or using label free system [7, 9, 11], or combining with metal nanoparticles [20, 25], brings the sequence specific detection of DNA hybridization observed in exceedingly low detection limits as resulting in efficient magnetic separation. Wang et al. was reported a novel genomagnetic electrochemical assay related to BRCA1 breast-cancer gene based on label-free detection [7]. In another study performed by Wang)s group [24], an enzyme- linked sandwich hybridization was studied combined with electrochemical detection of DNA sequences related to BRCA1 gene by using magnetic particles labeled probe hybridizing to a biotinylated DNA target capturing a streptavidin-alkaline phosphatase (AP) enzyme, and con- sequently, 1-naphthol was measured as a product of enzymatic reaction in the presence of DNA hybridization. The application of genomagnetic assay connected with a metal nanoparticle was presented for electrochemical DNA 798 Electroanalysis 19, 2007, No.7-8, 798–804 # 2007 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim