Disposable Magnetic DNA Sensors for the Determination at the Attomolar Level of a Specific Enterobacteriaceae Family Gene O ´ scar A. Loaiza, † Susana Campuzano, † Marı ´a Pedrero, † M. Isabel Pividori, ‡ Pedro Garcı ´a, § and Jose ´ M. Pingarro ´ n* ,† Departamento de Quı ´mica Analı ´tica, Facultad de CC. Quı ´micas, Universidad Complutense de Madrid, E-28040 Madrid, Spain, Grup de Sensors i Biosensors, Departament de Quı ´mica, Universitat Auto `noma de Barcelona, E-08193 Bellaterra, Barcelona, Spain, and Departamento de Microbiologı ´a Molecular, Centro de Investigaciones Biolo ´ gicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain Disposable magnetic DNA sensors using an enzyme- amplified strategy for the specific detection of a gene related to the Enterobacteriaceae bacterial family, based on the coupling of streptavidin-peroxidase to biotinylated lacZ gene target sequences, has been developed. A bioti- nylated 25-mer capture probe was attached to streptavi- din-modified magnetic beads and hybridization with the biotinylated target was allowed to proceed. Then, a streptavidin-peroxidase polymer was attached to the bi- otinylated target, and the resulting modified magnetic beads were captured by a magnetic field on the surface of tetrathiafulvalene (TTF) modified gold screen-printed electrodes (Au/SPEs). The amperometric response ob- tained at -0.15 V after the addition of hydrogen peroxide was used to detect the hybridization process. In order to improve the sensitivity of the determination and reduce the assay time, different variables of the assay protocol were optimized. A low detection limit (5.7 fmol) with good stability (RSD ) 7.1%, n ) 10) was obtained. The DNA nonspecific adsorption at the magnetic beads was negli- gible, the obtained results thus demonstrating the pos- sibility to detect the hybridization event with great speci- ficity and sensitivity. The developed method was used for the analysis of Escherichia coli DNA fragments (326 bases) in polymerase chain reaction (PCR) amplicons extracted from a cell culture. As low as 2.5 aM asymmetric PCR product could be detected with the developed methodology. Detection of pathogenic bacteria is of great importance from the health and safety point of view. The most popular analytical methods used in pathogen detection still are those based on culture and colony counting methodologies because, although time-consuming, they offer good sensitivity, selectivity and reli- ability. 1 However, the need for 7-8 days to yield analytical results by these methods has resulted in the search for new rapid methods based on the use of novel technologies. Although biosensors are of great interest in this field, revealing equally reliable results in much shorter times, they still need to reach at least the same detection levels as traditional methodologies (10-100 cfu mL -1 ). In particular, DNA biosensors, also called genosensors, based on the integration of a sequence-specific probe (usually a short synthetic oligonucleotide) and an electrochemical signal transducer are nowadays considered as the most attractive due to their simplicity, low instrumentation costs, and possibility for real-time accurate detection with very low detection limits. 2 Moreover, PCR amplification of the sample is still necessary due to the low abundance and extreme complexity of the nonamplified targets, and improved probe designs and sample pretreatments must be developed to allow efficient biorecognition events to occur at the transducer-solution interface. Magnetic beads (MBs) offer a versatile tool for electrochemical DNA and protein biosensing. 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Chem. 2008, 80, 8239–8245 10.1021/ac801319b CCC: $40.75  2008 American Chemical Society 8239 Analytical Chemistry, Vol. 80, No. 21, November 1, 2008 Published on Web 10/07/2008