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ABSTRACT: SUMMARY: A highly sensitive assay for rapidly screening-out Mycobacterium bovis in contaminated samples was developed based on electrochemical genosensing. The assay consists of specific amplification and double-tagging of the IS6110 fragment, highly related to M. bovis, followed by electrochemical detection of the amplified product. PCR amplification was carried out using a labeled set of primers and resulted in a amplicon tagged at each terminus with both biotin and digoxigenin. Two different electrochemical platforms for the detection of the double-tagged amplicon were evaluated: (i) an avidin biocomposite (Av-GEB) and (ii) a magneto sensor (m-GEC) combined with streptavidin magnetic beads. In both cases, the double- tagged amplicon was immobilized through its biotinylated end and electrochemically detected, using an antiDig-HRP conjugate, through its digoxigenin end. The assay was determined to be highly sensitive, based on the detection of 620 and 10 fmol of PCR amplicon using the Av-GEB and m-GEC strategies, respectively. Moreover, the m-GEC assay showed promising features for the detection of M. bovis on dairy farms by screening for the presence of the bacterium's DNA in milk samples. The obtained results are discussed and compared with respect to those of inter-laboratory PCR assays and tuberculin skin testing.
International Microbiology 06/2010; 13(2):91-7. · 1.80 Impact Factor
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ABSTRACT: 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.
Analytical Chemistry 02/2009; 81(4):1332-9. · 5.86 Impact Factor
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ABSTRACT: The development of novel and sensitive assays for DNA hybridization and detection has become an increasingly important research field. The design and fabrication of DNA‐modified surfaces and materials that are reproducible, stable, and selective to complementary DNA sequences are the main goal in the development of emerging analytical tools such as DNA chips or user‐friendly diagnostic devices for detecting a few DNA sequences such as electrochemical genosensors. Carbon‐based materials are widely used for this task due to their electrochemical, physical, and mechanical properties; their commercial availability; and their compatibility with modern microchip fabrication technology. Various approaches for electrochemical DNA determination are reviewed, in which the common element is the use of rigid carbon composite material as the electrochemical transducer. A stable DNA layer can be easily obtained by physical adsorption of DNA on graphite‐epoxy composite (GEC). Moreover, a universal affinity platform for electrochemical genosensing can be easily achieved by modifying the graphite‐epoxy composite with avidin to obtain an avidin biocomposite (Av‐GEB) whereon biotinylated DNA can be rapidly single‐point attached. Additionally, DNA‐modified magnetic beads are easily attached to magneto graphite‐epoxy composite (m‐GEC). The main strategies for electrochemical genosensing when using rigid carbon composites such as electrochemical transducer are discussed. Parameters such as ease of preparation, robustness, sensitivity, surface regeneration, costs, and transfer to mass production of these different DNA detection strategies are also considered.
Analytical Letters 12/2004; 38(15):2541-2565. · 1.02 Impact Factor
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ABSTRACT: The use of a rigid carbon-polymer composite material as an electrochemical transducer in hybridisation genosensors is reported. Graphite-epoxy composites (GEC) have an uneven surface where DNA can be adsorbed using a simple dry-adsorption procedure. Single-stranded-DNA binds strongly to GEC in a way that prevents the strands from self-associating, while permitting hybridisation with complementary DNA. Hybridisation has been detected through biotin-streptavidin interaction using a streptavidin conjugated to horseradish peroxidase. Non-specific adsorption onto GEC is almost non-existent even when the surface has not been treated by blocking reagents. The analytical signal obtained was higher when compared with other electrochemical genosensors. Results can be achieved in 150 min, and the detection limit is in the order of fmol. Additionally, surface regeneration is possible using a simple polishing procedure, allowing for multiple use. The new genosensor based on GEC fulfils the requirements desired for these devices: ease of preparation as dry-adsorption of DNA is very simple and easily automated, robustness, sensitivity, low cost of production, ease of miniaturisation and simple use and fast response. Additionally, it can be used for field measurements and can be produced as a genosensor kit. Also, this material can be implemented for screen-printing procedures for the mass production of genosensors. The utility of the genosensor based on GEC is also illustrated with the detection of a sequence related to novel determinant of beta-lactamase resistance in Staphylococcus aureus.
Biosensors and Bioelectronics 01/2004; 19(5):473-84. · 5.60 Impact Factor
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ABSTRACT: A PCR-genosensor based assay for detecting the IS200 DNA sequence specific to Salmonella spp is reported. This rapid test is based on PCR, where amplicon detection is achieved electrochemically using GEC (graphite-epoxy composite) genosensor. The amplicon is immobilized onto GEC electrodes by simple dry-adsorption and the detection is carried out using an enzymatic labeling system. Results demonstrate that this new electrochemical genosensor fulfils the requirements desired for these devices: easy preparation – as dry-adsorption of DNA is very simple –, robustness, sensitivity, low cost, simple use and fast response. Additionally, the assay can be produced as a kit format, increasing its commercial potentiality. Also, the electrode material can be implemented for screen-printing procedures for the mass production of genosensors.
Electroanalysis 12/2003; 15(23‐24):1815 - 1823. · 2.87 Impact Factor
