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This work addresses the design of an Ebola diagnostic test involving a simple, rapid, specific and highly sensitive procedure based on isothermal amplification on magnetic particles with electrochemical readout. Ebola padlock probes were designed to detect a specific L-gene sequence present in the five most common Ebola species. Ebola cDNA was amplified by rolling circle amplification (RCA) on magnetic particles. Further re-amplification was performed by circle-to-circle amplification (C2CA) and the products were detected in a double-tagging approach using a biotinylated capture probe for immobilization on magnetic particles and a readout probe for electrochemical detection by square-wave voltammetry on commercial screen-printed electrodes. The electrochemical genosensor was able to detect as low as 200 ymol, corresponding to 120 cDNA molecules of L-gene Ebola virus with a limit of detection of 33 cDNA molecules. The isothermal double-amplification procedure by C2CA combined with the electrochemical readout and the magnetic actuation enables the high sensitivity, resulting in a rapid, inexpensive, robust and user-friendly sensing strategy that offers a promising approach for the primary care in low resource settings, especially in less developed countries.

La convergència de tecnologies com la nanotecnologia, la biotecnologia i les tecnologies de la informació estan obrint nous camins en el camp dels biosensors electroquímics. La integració de materials microestructurats i nanoestructuratsen dispositius biosensors (com micropartícules de grafit, bioreceptors, nanopartícules d’or o micropartícules o nanopartícules magnètiques) proporciona característiques excel·lents per a l’anàlisi de residus alimentaris (plaguicides o antibiòtics), additius alimentaris (àcid fòlic), al·lergògens alimentaris (gliadina) i patògens transmesos pels aliments. Una de les contribucions clau en el camp de la biodetecció electroquímica es basa en el disseny de transductors nous, no solament amb propietats transductores realçades, sinó també amb la immobilització millorada de biomolècules, que retenen l’ activitat biològica. La integració de partícules magnètiques és un avenç molt clar en la detecció de residus alimentaris en matrius complexes, que millora les respostes electroquímiques perquè augmenta l’àrea activa transductora. En aquesta contribució es descriuen sistemes immunosensors i genosensors electroquímics desenvolupats recentment en el nostre laboratori com a mètodes de detecció, d’anàlisi i de diagnòstic ràpids, sensibles i automatitzables per als contaminants alimentaris, principalment residus alimentaris i patògens., The convergence of new technologies, including nanotechnology, biotechnology, and information technology, has opened new horizons in electrochemical biosensors. The integration of micro- and nanostructured materials within biosensing devices (graphite microparticles, bioreceptors, gold nanoparticles, and magnetic micro- or nanoparticles) has provided excellent analytical performances in the detection of food residues (pesticides, antibiotics), food additives (folic acid) and allergens (gliadin), and food-borne pathogens. One of the key contributions in the electrochemical biosensing field relies on the design of novel transducers, not only with enhanced transducing features but also with improved immobilization of biomolecules while preserving their biological activity. The integration of magnetic particles provides further improvements in the detection of food residues in a complex matrix, achieving better electrochemical response due to the increased size of the transducing active area. Electrochemical immunosensors and genosensors systems recently developed in our laboratories are described in this contribution. These sensitive, hand-held, user-friendly devices provide new analytical approaches for the in-field detection of food contaminants, mainly food residues and pathogens.

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. [Int Microbiol 2010; 13(2):91-97]