Susana Liébana

Phagomagnetic separation and electrochemical magneto-genosensing of pathogenic bacteria.

This paper addresses the use of bacteriophages immobilized on magnetic particles for the biorecognition of the pathogenic bacteria, followed by electrochemical magneto-genosensing of the bacteria. The P22 bacteriophage specific to Salmonella (serotypes A, B, and D1) is used as a model. The bacteria are captured and preconcentrated by the bacteriophage-modified magnetic particles through the host interaction with high specificity and efficiency. DNA amplification of the captured bacteria is then performed by double-tagging polymerase chain reaction (PCR). Further detection of the double-tagged amplicon is achieved by electrochemical magneto-genosensing. The strategy is able to detect in 4 h as low as 3 CFU mL(-1) of Salmonella in Luria-Bertani (LB) media. This approach is compared with conventional culture methods and PCR-based assay, as well as with immunological screening assays for bacteria detection, highlighting the outstanding stability and cost-efficient and animal-free production of bacteriophages as biorecognition element in biosensing devices.

Immunomagnetic separation of Salmonella with tailored magnetic micro and nanocarriers. A comparative study.

This paper addresses a comparative study of immunomagnetic separation of Salmonella using micro and nano-sized magnetic carriers. In this approach, nano (300 nm) and micro (2.8 μm) sized magnetic particles were modified with anti-Salmonella antibody to pre-concentrate the bacteria from the samples throughout an immunological reaction. The performance of the immunomagnetic separation on the different magnetic carriers was evaluated using classical culturing, confocal and scanning electron microscopy to study the binding pattern, as well as a magneto-actuated immunosensor with electrochemical read-out for the rapid detection of the bacteria in spiked milk samples. In this approach, a second polyclonal antibody labeled with peroxidase as electrochemical reporter was used. The magneto-actuated electrochemical immunosensor was able to clearly distinguish between food pathogenic bacteria such as Salmonella enterica and Escherichia coli, showing a limit of detection (LOD) as low as 538 CFU mL(-1) and 291 CFU mL(-1) for magnetic micro and nanocarriers, respectively, in whole milk, although magnetic nanoparticles showed a noticeable higher matrix effect and higher agglomeration effect. These LODs were achieved in a total assay time of 1h without any previous culturing pre-enrichment step. If the samples were pre-enriched for 8 h, the magneto immunosensor based on the magnetic nanoparticles was able to detect as low as 1 CFU in 25 mL of milk (0.04 CFU mL(-1)).

Multiplexed detection of foodborne pathogens based on magnetic particles

This paper addresses the novel approaches for the multiplex detection of food poisoning bacteria, paying closer attention to three of the most common pathogens involved in food outbreaks: Salmonella enterica, Escherichia coli O157:H7 and Listeria monocytogenes. End-point and real-time PCR, classical immunological techniques, biosensors, microarrays and microfluidic platforms, as well as commercial kits for multiplex detection of food pathogens will be reviewed, with special focus on the role of magnetic particles in these approaches. Although the immunomagnetic separation for capturing single bacteria from contaminating microflora and interfering food components has demonstrated to improve the performance on these approaches, the integration of magnetic particles for multiplex detection of bacteria is still in a preliminary stage and requires further studies.

Electrochemical immunosensors, genosensors and phagosensors for Salmonella detection

This review discusses the current state of the art Salmonella detection methods. In this perspective, emphasis is given to the recent developments in biosensors, in particular electrochemical immunosensors, genosensors and phagosensors. Different aspects of biosensors development have been summarised and discussed in detail. The integration of new materials into biosensors such as magnetic particles is also fully revised. More importantly, the advantages of using magnetic particles in magnetic separation of bacteria coupled with different detection techniques are also reviewed. This article also deals with the latest developments in simultaneous detection of several foodborne pathogenic bacteria. Accordingly, research opportunities and future development trends in these areas are finally discussed.

Simultaneous electrochemical magneto genosensing of foodborne bacteria based on triple-tagging multiplex amplification

Simultaneous detection of Salmonella enterica, Listeria monocytogenes and Escherichia coli based on triple-tagging multiplex PCR and electrochemical magneto genosensing on silica magnetic particles is reported. A set of tagging primers were selected for the specific amplification of yfiR (375 bp), hlyA (234 bp) and eaeA (151bp), being one of the primers for each set labelled with fluorescein, biotin and digoxigenin coding for S. enterica, L. monocytogenes and E. coli, respectively. Afterwards, electrochemical magneto genosensing of the bacteria was achieved by using silica magnetic particles as a carrier and three different electrochemical reporters, specific for each pathogen. This method was able to clearly distinguish among the pathogenic bacteria tested within 50 min, with detection limits ranging from 12 to 46 pg μL(-1).

A novel strategy for screening-out raw milk contaminated with Mycobacterium bovis on dairy farms by double-tagging PCR and electrochemical genosensing

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 bacteriums DNA in milk samples. The obtained results are discussed and compared with respect to those of inter-laboratory PCR assays and tuberculin skin testing.