Electrochemical genosensing of Salmonella, Listeria and Escherichia coli on silica magnetic particles.

A magneto-genosensing approach for the detection of the three most common pathogenic bacteria in food safety, such as Salmonella, Listeria and Escherichia coli is presented. The methodology is based on the detection of the tagged amplified DNA obtained by single-tagging PCR with a set of specific primers for each pathogen, followed by electrochemical magneto-genosensing on silica magnetic particles. A set of primers were selected for the amplification of the invA (278 bp), prfA (217 bp) and eaeA (151 bp) being one of the primers for each set tagged with fluorescein, biotin and digoxigenin coding for Salmonella enterica, Listeria monocytogenes and E. coli, respectively. The single-tagged amplicons were then immobilized on silica MPs based on the nucleic acid-binding properties of silica particles in the presence of the chaotropic agent as guanidinium thiocyanate. The assessment of the silica MPs as a platform for electrochemical magneto-genosensing is described, including the main parameters to selectively attach longer dsDNA fragments instead of shorter ssDNA primers based on their negative charge density of the sugar-phosphate backbone. This approach resulted to be a promising detection tool with sensing features of rapidity and sensitivity very suitable to be implemented on DNA biosensors and microfluidic platforms.

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).

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)).

Biomarker detection of global infectious diseases based on magnetic particles.

Infectious diseases affect the daily lives of millions of people all around the world, and are responsible for hundreds of thousands of deaths, mostly in the developing world. Although most of these major infectious diseases are treatable, the early identification of individuals requiring treatment remains a major issue. The incidence of these diseases would be reduced if rapid diagnostic tests were widely available at the community and primary care level in low-resource settings. Strong research efforts are thus being focused on replacing standard clinical diagnostic methods, such as the invasive detection techniques (biopsy or endoscopy) or expensive diagnostic and monitoring methods, by affordable and sensitive tests based on novel biomarkers. The development of new methods that are needed includes solid-phase separation techniques. In this context, the integration of magnetic particles within bioassays and biosensing devices is very promising since they greatly improve the performance of a biological reaction. The diagnosis of clinical samples with magnetic particles can be easily achieved without pre-enrichment, purification or pretreatment steps often required for standard methods, simplifying the analytical procedures. The biomarkers can be specifically isolated and preconcentrated from complex biological matrixes by magnetic actuation, increasing specificity and the sensitivity of the assay. This review addresses these promising features of the magnetic particles for the detection of biomarkers in emerging technologies related with infectious diseases affecting global health, such as malaria, influenza, dengue, tuberculosis or HIV.

Phagomagnetic immunoassay for the rapid detection of Salmonella.

This work explores the use of the phage P22 in a phagomagnetic immunoassay for the rapid detection of Salmonella. The covalent attachment of wild-type phages was performed on two different magnetic carriers: carboxyl-activated magnetic nanoparticles (300 nm) and tosyl-activated magnetic microparticles (2.8 µm). The bacteria were captured and preconcentrated by the phage-modified magnetic particles, followed by the detection using specific anti-Salmonella antibodies conjugated to horseradish peroxidase as an optical reporter. Outstanding selectivity and sensitivity was obtained with this approach, achieving detection limits of 19 CFU mL(-1) in 2.5 h without any pre-enrichment, in milk samples. Moreover, if the samples were pre-enriched for 6 h, the method was able to detect as low as 1.4 CFU in 25 mL of milk. Therefore, the proposed strategy based on the combined use of phagomagnetic separation with immunological labeling is promising as a rapid and simple method for food safety.

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.

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 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.

Lead(II) ion selective electrodes with PVC membranes based on two bis-thioureas as ionophores: 1,3-bis(N'-benzoylthioureido)benzene and 1,3-bis(N'-furoylthioureido)benzene.

Two PVC membrane ion selective electrodes for Pb(II) ion based on two bis-thioureas: 1,3-bis(N'-benzoylthioureido)benzene and 1,3-bis(N'-furoylthioureido)benzene as ionophores, are reported. A first membrane formulated using 1,3-bis(N'-benzoylthioureido)benzene as carrier exhibited a Nernstian response to Pb(II) over a wide concentration range (4.0x10(-6) to 1.0x10(-2)M) with a slope of 31.5+/-1.6 mV/dec. It showed a fast response time (t(90%)=14 s) and could be used for 10 weeks without any divergence in potentials. The membrane formulated using 1,3-bis(N'-furoylthioureido)benzene as carrier exhibited a Nernstian response in the concentration range (5.0x10(-6) to 1.0x10(-2) M), with a slope of 30.0+/-1.3 mV/dec. Its response time was t(90%)=14 s, and it could be used for 14 weeks without any divergence in potentials. The two proposed potentiometric sensors revealed acceptable selectivities for Pb(II) over a wide variety of other metal ions and could be used in a pH range of 2.2-6.0. Both electrodes were assayed in direct potentiometric determination of lead in soils (10-30 mg/kg range) with very good performance (0.99935 correlation coefficient in the comparison against ICP-MS method).

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

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 (AU)

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Rapid detection of Salmonella in milk by electrochemical magneto-immunosensing.

A very simple and rapid method for the detection of Salmonella in milk is reported. In this approach, the bacteria are captured and preconcentrated from milk samples with magnetic beads through an immunological reaction. A second polyclonal antibody labeled with peroxidase is used as serological confirmation with electrochemical detection based on a magneto-electrode. The 'IMS/m-GEC electrochemical immunosensing' approach shows a limit of detection of 5 x 10(3) and 7.5 x 10(3)CFU mL(-1) in LB and in milk diluted 1/10 in LB broth, respectively, in 50 min without any pretreatment. If the skimmed-milk is preenriched for 6h, the method is able to detect as low as 1.4 CFU mL(-1), while if it is preenriched for 8h, as low as 0.108 x CFU mL(-1) (2.7 x CFU in 25 g of milk, in 5 samples of 5 mL) are detected accordingly with the legislation. Moreover, the method is able to clearly distinguish between food pathogenic bacteria such as Salmonella and Escherichia coli. The features of this approach are discussed and compared with classical culture methods.

Magneto immunoseparation of pathogenic bacteria and electrochemical magneto genosensing of the double-tagged amplicon.

A rapid and sensitive method for the detection of food pathogenic bacteria is reported. In this approach, the bacteria are captured and preconcentrated from food samples with magnetic beads by immunological reaction with the specific antibody against Salmonella. After the lysis of the captured bacteria, further amplification of the genetic material by PCR with a double-tagging set of primers is performed to confirm the identity of the bacteria. Both steps are rapid alternatives to the time-consuming classical selective enrichment and biochemical/serological tests. The double-tagged amplicon is then detected by electrochemical magneto genosensing. The "IMS/double-tagging PCR/m-GEC electrochemical genosensing" approach is used for the first time for the sensitive detection of Salmonella artificially inoculated into skim milk samples. A limit of detection of 1 CFU mL(-1) was obtained in 3.5 h without any pretreatment, in LB broth and in milk diluted 1/10 in LB. If the skim milk is pre-enriched for 6 h, the method is able to feasibly detect as low as 0.04 CFU mL(-1) (1 CFU in 25 g of milk) with a signal-to-background ratio of 20. Moreover, the method is able to clearly distinguish between pathogenic bacteria such as Salmonella and Escherichia coli. The features of this approach are discussed and compared with classical culture methods and PCR-based assay.

Electrochemical detection of DNA hybridization using micro and nanoparticles.

A novel, rapid, and sensitive protocol for the electrochemical detection of DNA hybridization that take the advantage of a magnetic separation/mixing process and the use of monomaleimido-gold nanoparticles of 1.4 nm diameter as label is presented. A sandwich-type assay is formed in this protocol by the capture probe DNA immobilized on the surface of magnetic beads and the double hybridization of the target (cystic fibrosis related DNA), first with the immobilized probe, and then with signaling probe DNA labeled with monomaleimido-gold nanoparticles. When the assay is completed, the final conjugate is transferred onto genomagnetic sensor surface (graphite epoxy composite electrode with a magnet inside) used as working electrode, and then the direct determination of gold nanoparticles by differential pulse voltammetry striping technique is carried out. This protocol is quite promising for numerous applications in different fields as clinical analysis, environmental control as well as other applications.

Electrochemical immunosensing using micro and nanoparticles.

A model immunosensor based on a labeling method using gold nanoparticles (AuNPs) and electrochemical detection is developed. Microparamagnetic beads (MB) as primary antibody immobilization platforms and AuNPs modified with a secondary antibody as high sensible electrochemical labels have been used. The carbon electrode used as transducer incorporates a magnet that allows the collection/ immobilization on its surface of the immunological sandwich attached to the MB. Briefly, the sandwich type assay consists in the incubation of streptavidin-coupled-MB with an antihuman IgG biotin conjugate, and then, the immunological reaction with the human IgG antigen takes place. After that, a gold labeled anti-human IgG reacts with the antigen, and finally the AuNPs are electrochemically detected. This approach allows the obtaining of an immunosensor with a low antigen detection limit with special interest for several applications in protein analysis.

Double-tagging polymerase chain reaction with a thiolated primer and electrochemical genosensing based on gold nanocomposite sensor for food safety.

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.

Lab-on-a-chip for ultrasensitive detection of carbofuran by enzymatic inhibition with replacement of enzyme using magnetic beads.

In this paper an ultrasensitive method to determine toxicity due to pesticides in a glass lab-on-a-chip by means of enzymatic inhibition of acetylcholinesterase immobilised on magnetic beads is described. The reproducible insertion of a controlled amount of enzyme-coupled magnetic beads inside the chip channel and their immobilisation in a capture region with the aid of a magnetic field has been optimised. This procedure enables the easy renewal of the biosensing material after each determination in a highly reproducible manner. Several operational parameters such as the working potential for the selective detection of thiocholine (TCh) on a platinum disc electrode, the TCh detection reproducibility and sensitivity, the electroosmotic flow driving voltage and the inhibition time were also evaluated or optimised. The detection of carbofuran (one of the most toxic carbamate pesticides) has been achieved down to the nanomolar level.

Nutrient solution monitoring in greenhouse cultivation employing a potentiometric electronic tongue.

This work investigates the use of electronic tongues for monitoring nutrient solution compositions in closed soilless systems. This is a horticultural technique in which the nutrient solution is continuously recirculated and an automatic recomposition system maintains the concentration of the different ions in the optimum range for the plants. Electronic tongues used in this study comprised an array of potentiometric sensors and complex data processing by artificial neural networks. A first experiment was able to carry out the simultaneous inline monitoring of ammonium, potassium, sodium, chloride, and nitrate ions during the winter. In the second and third applications, done during summer, some changes were introduced in the sensor array to improve its response toward chloride ions and to incorporate phosphate in the model. This electronic tongue was validated with real greenhouse samples and was also able to detect the variations in the ion concentrations caused by an incorrect configuration of the recomposition system.

Bioelectronic tongue for the simultaneous determination of urea, creatinine and alkaline ions in clinical samples.

Urea and creatinine biosensors based on urease and creatinine deiminase, respectively, covalently immobilized onto ammonium selective electrodes, were included in an array together with sensors sensitive to ammonium, potassium and sodium. Generic sensors to alkaline ions were also included. All the sensors used were of all-solid-state type, employing polymeric membranes and having rather nonspecific response characteristics. A response model based on artificial neural networks was built and tested for the simultaneous determination of urea, creatinine, ammonium, potassium and sodium. The results show that it is possible to obtain a good multivariate calibration model. In this way, the developed bioelectronic tongue was successfully applied to multidetermination of the five species in raw and spiked urine samples. Predicted concentrations showed a good agreement with reference methods of analysis, allowing a simple direct method for determining urea and creatinine in real samples. At the same time, this method permitted to obtain the concentrations of the alkaline interferences (endogenous ammonium, potassium and sodium) without the need of eliminating them.

Double-codified gold nanolabels for enhanced immunoanalysis.

A novel double-codified nanolabel (DC-AuNP) based on gold nanoparticle (AuNP) modified with anti-human IgG peroxidase (HRP)-conjugated antibody is reported. It represents a simple assay that allows enhanced spectrophotometric and electrochemical detection of antigen human IgG as a model protein. The method takes advantage of two properties of the DC-AuNP label: first, the HRP label activity toward the OPD chromogen that can be related to the analyte concentration and measured spectrophotometrically; second, the intrinsic electrochemical properties of the gold nanoparticle labels that being proportional to the protein concentration can be directly quantified by stripping voltammetry. Beside these two main direct determinations of human IgG, a secondary indirect detection was also applicable to this system, exploiting the high molar absorptivity of gold colloids, by which, the color intensity of their solution was proportional to the concentration of the antigen used in the assay. Paramagnetic beads were used as supporting material to immobilize the sandwich-type immunocomplexes resulting in incubation and washing times shorter than those typically needed in classical ELISA tests by means of a rapid magnetic separation of the unbound components. A built-in magnet graphite-epoxy-composite electrode allowed a sensibly enhanced adsorption and electrochemical quantification of the specifically captured AuNPs. The used DC-AuNP label showed an excellent specificity/selectivity, as a matter of fact using a different antigen (goat IgG) a minimal nonspecific electrochemical or spectrophotometric signal was measured. The detection limits for this novel double-codified nanoparticle-based assay were 52 and 260 pg of human IgG/mL for the spectrophotometric (HRP-based) and electrochemical (AuNP-based) detections, respectively, much lower than those typically achieved by ELISA tests. The developed label and method is versatile, offers enhanced performances, and can be easily extended to other protein detection schemes as well as in DNA analysis.

Pesticide determination by enzymatic inhibition and amperometric detection in a low-temperature cofired ceramics microsystem.

Among the several fabrication techniques used to construct microflow systems, the low-temperature cofired ceramics (LTCC) technology, taking advantage of its multilayer approach, is one of the most versatile ones. It permits the integration of several unitary operations of an analytical process in a modular or monolithic way. Moreover, due to its perfect compatibility with screen-printing techniques, it also permits the integration of electronic components used to control the whole system setup. In this work the design, construction, and evaluation of a miniaturized analyzer for pesticide determination that integrates a pretreatment stage, based on two mixers or reactors, and an amperometric detection system to measure the product of an enzymatic inhibition reaction are presented. The detection system was monolithically integrated in the microfluidic platform, and it consisted of a screen-printed reference electrode and two platinum sheets, acting as auxiliary and working electrodes, which were embedded within the ceramic structure. The miniaturized system was characterized and successfully evaluated by determining carbofuran at the nanomolar level.

Carbon nanotube detectors for microchip CE: comparative study of single-wall and multiwall carbon nanotube, and graphite powder films on glassy carbon, gold, and platinum electrode surfaces.

The performance of microchip electrophoresis/electrochemistry system with carbon nanotube (CNT) film electrodes was studied. Electrocatalytic activities of different carbon materials (single-wall CNT (SWCNT), multiwall CNT (MWCNT), carbon powder) cast on different electrode substrates (glassy carbon (GC), gold, and platinum) were compared in a microfluidic setup and their performance as microchip electrochemical detectors was assessed. An MWCNT film on a GC electrode shows electrocatalytic effect toward oxidation of dopamine (E(1/2) shift of 0.09 V) and catechol (E(1/2) shift of 0.19 V) when compared to a bare GC electrode, while other CNT/carbon powder films on the GC electrode display negligible effects. Modification of a gold electrode by graphite powder results in a strong electrocatalytic effect toward oxidation of dopamine and catechol (E(1/2) shift of 0.14 and 0.11 V, respectively). A significant shift of the half-wave potentials to lower values also provide the MWCNT film (E(1/2) shift of 0.08 and 0.08 V for dopamine and catechol, respectively) and the SWCNT film (E(1/2) shift of 0.10 V for catechol) when compared to a bare gold electrode. A microfluidic device with a CNT film-modified detection electrode displays greatly improved separation resolution (R(s)) by a factor of two compared to a bare electrode, reflecting the electrocatalytic activity of CNT.