Osteoblastic exosomes. A non-destructive quantitative approach of alkaline phosphatase to assess osteoconductive nanomaterials.

Alkaline phosphatase (ALP) is an essential biomarker of osteoblastic activity. Currently, ALP activity has been used to study bone mineralization mechanisms and osteoactive biomaterials among others. The ALP quantification is usually performed by destructive methods either on growing cells or cells lysate in which the osteoconductive biomaterial is being assessed. This work addresses the evaluation of a non-destructive colorimetric approach for the determination of ALP activity on osteoblast-derived exosomes from culture supernatants. The efficiency of the method was evaluated on osteoconductive electrospun scaffolds of PCL compounded with ZnO as a reference biomaterial. The results demonstrated that the osteoblast cell line mineralization induced by osteoconductive scaffolds can be monitorized over time by the non-destructive measurement of ALP activity on osteoblast derived exosomes. Consequently, this non-destructive approach suggested to be a reliable alternative technique for the quantification of biomaterials osteoconductivity or even evaluation of osteoblastic response at stem cells.

An electrochemical magneto immunosensor (EMIS) for the determination of paraquat residues in potato samples.

An electrochemical magneto immunosensor for the detection of low concentrations of paraquat (PQ) in food samples has been developed and its performance evaluated in a complex sample such as potato extracts. The immunosensor presented uses immunoreagents specifically developed for the recognition of paraquat, a magnetic graphite-epoxy composite (m-GEC) electrode and biofunctionalized magnetic micro-particles (PQ1-BSAMP) that allow reduction of the potential interferences caused by the matrix components. The amperometric signal is provided by an enzymatic probe prepared by covalently linking an enzyme to the specific antibodies (Ab198-cc-HRP). The use of hydroquinone, as mediator, allows recording of the signal at a low potential, which also contributes to reducing the background noise potentially caused by the sample matrix. The immunocomplexes formed on top of the modified MP are easily captured by the m-GEC, which acts simultaneously as transducer. PQ can be detected at concentrations as low as 0.18 ± 0.09 µg L(-1). Combined with an efficient extraction procedure, PQ residues can be directly detected and accurately quantified in potato extracts without additional clean-up or purification steps, with a limit of detection (90% of the maximum signal) of 2.18 ± 2.08 µg kg(-1), far below the maximum residue level (20 µg kg(-1)) established by the EC. The immunosensor presented here is suitable for on-site analysis. Combined with the use of magnetic racks, multiple samples can be run simultaneously in a reasonable time.

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.

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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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.

Disposable magnetic DNA sensors for the determination at the attomolar level of a specific enterobacteriaceae family gene.

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 biotinylated 25-mer capture probe was attached to streptavidin-modified magnetic beads and hybridization with the biotinylated target was allowed to proceed. Then, a streptavidin-peroxidase polymer was attached to the biotinylated 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 obtained 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 negligible, the obtained results thus demonstrating the possibility to detect the hybridization event with great specificity 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.

Graphite-epoxy composites as a new transducing material for electrochemical genosensing.

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.