Biomedical subjects

S Alegret

Publications and source records attributed to S Alegret.

At least 19 recordsLinked to original sources

PubMed2006

Electrochemical magneto immunosensing of antibiotic residues in milk.

A novel electrochemical immunosensing strategy for the detection of sulfonamide antibiotics in milk based on magnetic beads is presented. Among the different strategies for immobilizing the class-specific anti-sulfonamide antibody to the magnetic beads--such as those based on the use of Protein A or carboxylate modified magnetic beads - ,the best strategy was found to be the covalent bonding on tosyl-activated magnetic beads. The immunological reaction for the detection of sulfonamide antibiotics performed on the magnetic bead is based on a direct competitive assay using a tracer with HRP peroxidase for the enzymatic labelling. After the immunochemical reactions, the modified magnetic beads can be easily captured by a magneto sensor made of graphite-epoxy composite (m-GEC), which is also used as the transducer for the electrochemical immunosensing. The electrochemical detection is thus achieved through a suitable substrate for the enzyme HRP and an electrochemical mediator. The electrochemical approach is also compared with a novel magneto-ELISA with optical detection. The performance of the electrochemical immunosensing strategy based on magnetic beads was successfully evaluated using spiked milk samples, and the detection limit was found to be 1.44 microg L(-1) (5.92 nmol L(-1)) for raw full cream milk. This strategy offers great promise for rapid, simple, cost-effective and on-site analysis of biological, food and environmental samples.

Animals↗

PubMed2006

Electrochemical biosensing of pesticide residues based on affinity biocomposite platforms.

A novel and very sensitive electrochemical immunosensing strategy for the detection of atrazine based on affinity biocomposite transducers is presented. Firstly, the graphite-epoxy composite transducer was bulk-modified with different universal affinity biomolecules, such as avidin and Protein A. Two strategies for the immobilization of the anti-atrazine antibodies on both biocomposite transducers were evaluated: 'wet-affinity' and 'dry-assisted affinity' immobilization. Finally, the performance of a novel anti-atrazine immunocomposite bulk-modified with anti-atrazine antibodies was also evaluated. The better immobilization performance of the anti-atrazine antibodies was achieved by 'dry-assisted affinity' immobilization on Protein A (2%) graphite-epoxy biocomposite (ProtA(2%)-GEB) as a transducer. The immunological reaction for the detection of atrazine performed on the ProtA(2%)-GEB biosensors is based on a direct competitive assay using atrazine-HRP tracer as the enzymatic label. The electrochemical detection is thus achieved through a suitable substrate and a mediator for the enzyme HRP. This novel strategy was successfully evaluated using spiked orange juice samples. The detection limit for atrazine in orange juices using the competitive electrochemical immunosensing assay was found to be 6 x 10(-3) microgL-1 (0.03 nmolL-1) thus this biosensing method accomplishes by far the LODs required for the European Community directives for potable water and food samples (0.1 microgL-1). This strategy offers great promise for rapid, simple, cost effective, and on-site biosensing of biological, food, and environmental samples.

Atrazine↗

PubMed2006

In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens.

A sensitive and selective genomagnetic assay for the electrochemical detection of food pathogens based on in situ DNA amplification with magnetic primers has been designed. The performance of the genomagnetic assay was firstly demonstrated for a DNA synthetic target by its double-hybridization with both a digoxigenin probe and a biotinylated capture probe, and further binding to streptavidin-modified magnetic beads. The DNA sandwiched target bound on the magnetic beads is then separated by using a magneto electrode based on graphite-epoxy composite. The electrochemical detection is finally achieved by an enzyme marker, anti-digoxigenin horseradish peroxidase (HRP). The novel strategy was used for the rapid and sensitive detection of polymerase chain reaction (PCR) amplified samples. Promising resultants were also achieved for the DNA amplification directly performed on magnetic beads by using a novel magnetic primer, i.e., the up PCR primer bound to magnetic beads. Moreover, the magneto DNA biosensing assay was able to detect changes at single nucleotide polymorphism (SNP) level, when stringent hybridization conditions were used. The reliability of the assay was tested for Salmonella spp., the most important pathogen affecting food safety.

Biosensing Techniques↗

PubMed2006

Electrochemical genosensors for biomedical applications based on gold nanoparticles.

Two gold nanoparticles-based genomagnetic sensors designs for detection of DNA hybridization are described. Both assays are based on a magnetically induced direct electrochemical detection of gold tags on magnetic graphite-epoxy composite electrodes. The first design is a two strands assay format that consists of the hybridization between a capture DNA strand which is linked with paramagnetic beads and another DNA strand related to BRCA1 breast cancer gene used as a target which is coupled with streptavidin-gold nanoparticles. The second genomagnetic sensor design is a sandwich assay format with more application possibilities. A cystic fibrosis related DNA strand is used as a target and sandwiched between two complementary DNA probes: the first one linked with paramagnetic beads and a second one modified with gold nanoparticles via biotin-streptavidin complexation reactions. The electrochemical detection of gold nanoparticles by differential pulse voltammetry was performed in both cases. The developed genomagnetic sensors provide a reliable discrimination against noncomplementary DNA as well against one and three-base mismatches. Optimization parameters affecting the hybridization and analytical performance of the developed genosensors are shown for genomagnetic assays of DNA sequences related with the breast cancer and cystic fibrosis genes.

Biosensing Techniques↗

PubMed2006

Impedimetric genosensors for the detection of DNA hybridization.

Impedance spectroscopy is proposed as the transduction principle for detecting the hybridization of DNA complementary strands. In our experiments, different DNA oligonucleotides were used as model gene substances. The gene probe is first immobilized on a graphite-epoxy composite working electrode based genosensor. Detection principle is based on changes of impedance spectra of a redox marker, the ferro/ferricyanide couple, after hybridization with target DNA. Resistance offered to the electrochemical reaction serves as the working signal, allowing for an unlabelled gene assay.

Electric Impedance↗

PubMed2006

A sequential injection electronic tongue employing the transient response from potentiometric sensors for anion multidetermination.

Intelligent and automatic systems based on arrays of non-specific-response chemical sensors were recently developed in our laboratory. For multidetermination applications, the normal choice is an array of potentiometric sensors to generate the signal, and an artificial neural network (ANN) correctly trained to obtain the calibration model. As a great amount of information is required for the proper modelling, we proposed its automated generation by using the sequential injection analysis (SIA) technique. First signals used were steady-state: the equilibrium signal after a step-change in concentration. We have now adapted our procedures to record the transient response corresponding to a sample step. The novelty in this approach is therefore the use of the dynamic components of the signal in order to better discriminate or differentiate a sample. In the developed electronic tongue systems, detection is carried out by using a sensor array formed by five potentiometric sensors based on PVC membranes. For the developed application we employed two different chloride-selective sensors, two nitrate-selective sensors and one generic response sensor. As the amount of raw data (fivefold recordings corresponding to the five sensors) is excessive for an ANN, some feature extraction step prior to the modelling was needed. In order to attain substantial data reduction and noise filtering, the data obtained were fitted with orthonormal Legendre polynomials. In this case, a third-degree Legendre polynomial was shown to be sufficient to fit the data. The coefficients of these polynomials were the input information fed into the ANN used to model the concentrations of the determined species (Cl-, NO3- and HCO3-). Best results were obtained by using a backpropagation neural network trained with the Bayesian regularisation algorithm; the net had a single hidden layer containing three neurons with the tansig transfer function. The results obtained from the time-dependent response were compared with those obtained from steady-state conditions, showing the former superior performance. Finally, the method was applied for determining anions in synthetic samples and real water samples, where a satisfactory comparison was also achieved.

Algorithms↗

PubMed2006

Electrochemical magnetoimmunosensing strategy for the detection of pesticides residues.

A novel electrochemical immunosensing strategy for the detection of atrazine based on magnetic beads is presented. Different coupling strategies for the modification of the magnetic beads with the specific anti-atrazine antibody have been developed. The immunological reaction for the detection of atrazine performed on the magnetic bead is based on a direct competitive assay using a peroxidase (HRP) tracer as the enzymatic label. After the immunochemical reactions, the modified magnetic beads can be easily captured by a magnetosensor made of graphite-epoxy composite, which is also used as the transducer for the electrochemical immunosensing. The electrochemical detection is thus achieved through a suitable substrate and mediator for the enzyme HRP. The electrochemical approach is also compared with a novel magneto-ELISA based on optical detection. The performance of the electrochemical immunosensing strategy based on magnetic beads was successfully evaluated using spiked real orange juice samples. The detection limit for atrazine using the competitive electrochemical magnetoimmunosensing strategy with anti-atrazine-specific antibody covalent coupled with tosyl-activated magnetic beads was found to be 6 x 10(-3) microg L(-1) (0.027 nmol L(-1)). This strategy offers great promise for rapid, simple, cost-effective, and on-site analysis of biological, food, and environmental samples.

Antibodies↗

PubMed2005

Electrochemical biosensing based on universal affinity biocomposite platforms.

Rigid conducting biocomposites are versatile and effective transducing materials for the construction of a wide range of amperometric biosensors such as immunosensors, genosensors and enzymosensors, particularly if the transducer is bulk-modified with universal affinity biomolecules. The strept(avidin)-graphite-epoxy biocomposite could be considered as an universal immobilization platform whereon biotinylated DNAs, oligonucleotides, enzymes or antibodies can be captured by means of the highly affinity (strept)avidin-biotin reaction. Universal affinity biocomposite-based biosensors offer many potential advantages compared to more traditional electrochemical biosensors commonly based on a biologically surface-modified transducer. The integration of many materials into one matrix is their main advantage. As biological bulk-modified materials, the conducting biocomposites act not only as transducers, but also as reservoir for the biomaterial. After its use, the electrode surface can be renewed by a simple polishing procedure, establishing a clear advantage of these approaches relative to classical biosensors and other common biological assays. Moreover, the same material is useful for the analysis of many molecules whose determinations are based on genetic, enzymatic or immunological reactions. The different strategies for electrochemical genosensing, immunosensing and enzymosensing, all of them being dependent on the presence of a redox enzyme marker for the generation of the electrochemical signal, based on this universal affinity biocomposite platform are all presented and discussed.

Biosensing Techniques↗

PubMed2005

Sensitive stripping voltammetry of heavy metals by using a composite sensor based on a built-in bismuth precursor.

A new graphite-epoxy composite electrode (GECE) containing Bi(NO(3))(3) as a built-in bismuth precursor for simultaneous and individual anodic stripping analysis of heavy trace metals like lead and cadmium is reported. The developed Bi(NO(3))(3)-GECE is compatible with bismuth film electrodes reported previously including the composite electrodes (Bi-GECE) recently reported by our group. Bi(NO(3))(3)-GECE displays the ability for the detection of both individual and simultaneous determination of heavy trace metals and exhibits well defined, reproducible and sharp stripping signals. The sensitive response is combined with the minimal toxicity of Bi(NO(3))(3). This novel sensor would be an appropriate alternative tool to sensors using bismuth in solution during their utilization in environmental quality monitoring as well as other applications.

Bismuth↗

PubMed2005

Determination of phenolic compounds by a polyphenol oxidase amperometric biosensor and artificial neural network analysis.

The determination of phenolic compounds is significant given its toxicity, even at very low concentration levels. Amperometric determination of phenols is a simple technique available. Direct oxidation of phenols can be used, but another possibility is the use of polyphenol oxidase (tyrosinase) enzyme biosensors that oxidises the phenolic compounds into their corresponding quinones. Reduction of the resulting quinones accomplishes the amplification of the amperometric signal, as long as the result of the reduction process is the corresponding cathecol, this being able to be oxidised again by the polyphenol oxidase immobilized on the surface of the biosensor. In this communication, simultaneous determination of different phenols was carried out combining biosensor measurements with chemometric tools, in what is known as electronic tongue. The departure information used was the overlapped reduction voltammogram generated with the amperometric biosensor based on polyphenol oxidase. Artificial Neural Networks (ANN) were used for extraction and quantification of each compound. Phenol, cathecol and m-cresol formed the three-analyte study case resolved in this work. Good prediction ability was attained, and so, the separate quantification of these three phenols was accomplished.

Algorithms↗

PubMed2004

Renewable Protein A modified graphite-epoxy composite for electrochemical immunosensing.

A novel rigid and renewable transducing material for electrochemical immunosensing, based on Protein A bulk-modified graphite-epoxy biocomposite (ProtA-GEB) is reported. Protein A is able to bind to the Fc region of antibodies and provide an affinity matrix for antibody immobilisation onto the transducer. The rigid conducting biocomposite acts not only as a transducer, but also as a reservoir for protein A. After use, the electrode surface can be renewed by a simple polishing procedure, highlighting a clear advantage of this new approach with respect to classical immunoassays. The performance of ProtA-GEB transducers was compared with surface-modified transducers based on a simple dry adsorption procedure, where both Protein A and directly the antibody were adsorbed onto the surface of graphite-epoxy composite (ProtA/GEC and IgG/GEC, respectively). The application of the new biocomposite material in electrochemical immunosensing was studied using a model competitive immunoassay. The immunological reaction was detected using an enzymatic-labeling procedure together with the amperometric detection through a suitable substrate (H(2)O(2)) for the enzyme (HRP). The enzymatic labelling was performed using a two-step procedure based on the biotin/streptavidin interaction as well as a one-step procedure using an antibody labelled with the enzyme. Electrochemical and microscopic characterisation of ProtA-GEB transducer, optimisation of the immunosensor design as well as the stability of this material are also reported.

Biosensing Techniques↗

PubMed2003

Rapid electrochemical genosensor assay using a streptavidin carbon-polymer biocomposite electrode.

A sensor capable of detecting a specific DNA sequence was designed by bulk modification of a graphite epoxy composite electrode with streptavidin (2% w/w). Streptavidin is used to immobilise a biotinylated capture DNA probe to the surface of the electrode. Simultaneous hybridisation occurs between the biotin DNA capture probe and the target-DNA and between the target-DNA and a digoxigenin modified probe. The rapid binding kinetic of streptavidin-biotin allows a one step immobilisation/hybridisation procedure. Secondly, enzyme labelling of the DNA duplex occurs via an antigen-antibody reaction between the Dig-dsDNA and an anti-Dig-HRP. Finally, electrochemical detection is achieved through a suitable substrate (H2O2) for the enzyme-labelled duplex. Optimisation of the sensor design, the modifier content and the immobilisation and hybridisation times was attained using a simple nucleotide sequence. Regeneration of the surface is achieved with a simple polishing procedure that shows good reproducibility. The generic use of a modified streptavidin carbon-polymer biocomposite electrode capable of surface regeneration and a one step hybridisation/immobilisation procedure are the main advantages of this approach. In DNA analysis, this procedure, if combined with the polymerase chain reaction, would represent certain advantages with respect to classical techniques, which prove to be time consuming in situations where a simple and rapid detection is required. This innovative developed material may be used for the detection of any analyte that can be coupled to the biotin-streptavidin reaction, as is the case of immunoassays.

Biosensing Techniques↗

PubMed2003

An electronic tongue using potentiometric all-solid-state PVC-membrane sensors for the simultaneous quantification of ammonium and potassium ions in water.

The simultaneous determination of NH(4)(+) and K(+) in solution has been attempted using a potentiometric sensor array and multivariate calibration. The sensors used are rather non-specific and of all-solid-state type, employing polymeric (PVC) membranes. The subsequent data processing is based on the use of a multilayer artificial neural network (ANN). This approach is given the name "electronic tongue" because it mimics the sense of taste in animals. The sensors incorporate, as recognition elements, neutral carriers belonging to the family of the ionophoric antibiotics. In this work the ANN type is optimized by studying its topology, the training algorithm, and the transfer functions. Also, different pretreatments of the starting data are evaluated. The chosen ANN is formed by 8 input neurons, 20 neurons in the hidden layer and 2 neurons in the output layer. The transfer function selected for the hidden layer was sigmoidal and linear for the output layer. It is also recommended to scale the starting data before training. A correct fit for the test data set is obtained when it is trained with the Bayesian regularization algorithm. The viability for the determination of ammonium and potassium ions in synthetic samples was evaluated; cumulative prediction errors of approximately 1% (relative values) were obtained. These results were comparable with those obtained with a generalized regression ANN as a reference algorithm. In a final application, results close to the expected values were obtained for the two considered ions, with concentrations between 0 and 40 mmol L(-1).

Journal Article↗

PubMed2001

Environmental toxicity monitoring using electrochemical biosensing systems.

Environmental monitoring faces the challenge of measuring an increasing number of analytes at ever decreasing concentrations. Since not all species of a given analyte have the same detrimental impact on the environment, new analytical devices and techniques are required to distinguish between the different species of a pollutant or different groups of pollutants. This paper describes analytical techniques based on biomaterials that are toxically sensitive to pollutants. This approach permits the biomonitoring of certain compounds by looking at their toxic properties. Although these techniques are based on a sound analytical strategy, their applications are limited because most of the interactions between the biological material and the analyte are irreversible. Additionally, the immobilised biological material has a limited stability. Several biomonitoring strategies based on electrochemical biosensing are discussed here and how to recover the bioactivity of biosensing system, both in discrete and automated procedures, is also reviewed.

Biosensing Techniques↗

PubMed2001

Classical dot-blot format implemented as an amperometric hybridisation genosensor.

A new electrochemical hybridisation genosensor has been designed. This genosensor is based on a concept adapted from classical dot-blot DNA analysis, but implemented in an electrochemical biosensor configuration. The use of amperometric transduction and the enzyme label method--that increases the genosensor sensitivity--are the main features of this new approach. The analytical procedure consists of five steps: DNA target immobilisation by adsorption onto a nylon membrane, hybridisation between DNA target and biotin-DNA probe, complexation reaction between biotin-DNA probe and an enzyme (horseradish peroxidase) streptavidin conjugate; integration of the modified membrane onto an electrochemical transducer; and finally, amperometric detection using a suitable substrate for the enzyme labelled duplex. Besides the adapted dot-blot format, a competitive assay in which the target is in solution is reported as well. This procedure, based on amperometric transduction, represents certain advantages with respect to dot-blot analysis: labelled hybrid detection is far simpler, quicker and requires more ordinary or simple reactives; the response obtained is a direct analytical signal via low-cost instrumentation, a nonisotopic labelling is used, and the membranes can be reused. These characteristics are ideal in implementing the procedure developed in kit form.

Adsorption↗

PubMed2001

Dot-blot amperometric genosensor for detecting a novel determinant of beta-lactamase resistance in Staphylococcus aureus.

A new electrochemical hybridisation genosensor for the detection of resistant bacteria has been developed. This device relies on the immobilisation of a 50-mer oligonucleotide target, unique to a novel determinant of beta-lactamase resistance in Staphylococcus aureus, onto an electrochemical transducer. This genosensor is based on a concept adapted from classical dot-blot DNA analysis, but implemented in an electrochemical biosensor configuration. Amperometric transduction and an enzyme label method, that increases the genosensor sensitivity, are the main features of this new approach. In addition to the adapted dot-blot format, a double hybridisation assay, in which two different labelled probes were used, is reported. This procedure, if combined with polymerase chain reaction (PCR), allows determination of the genotype of an antibiotic-resistant organism in a shorter time than that required to perform traditional phenotypic susceptibility testing. Its characteristics are ideal for implementation in a kit form.

Biosensing Techniques↗

PubMed2000

Electrochemical genosensor design: immobilisation of oligonucleotides onto transducer surfaces and detection methods.

The present report reviews immobilisation techniques of purified oligonucleotides on electrochemical transducers and their corresponding detection techniques. Most of the literature reviewed was published in the 1990s. The immobilisation techniques of a DNA probe to the surface of an electrochemical transducer made from carbon, gold, platinum or polypyrrole, ranged from simple adsorption to covalent bonding. Recent efforts to couple the recognition layer containing the immobilised nucleic acid recognition layer with the electrochemical signal transducer are discussed. Special attention is given to hybridisation biosensing based on electroactive indicators.

Adsorption↗

PubMed1999

Determination of lysine in pharmaceutical samples containing endogenous ammonium ions by using a lysine oxidase biosensor based on an all-solid-state potentiometric ammonium electrode.

A new potentiometric method is proposed to determine lysine in pharmaceutical samples. This method is based on a lysine biosensor consisting of a chemically immobilized lysine oxidase membrane attached to an all-solid-state ammonium electrode. Lysine is degraded in the sensor to release ammonium, which is detected by means of the ammonium electrode. The presence of endogenous ammonium in the samples interferes with these determinations, since the response measured corresponds to the sum of the ammonium generated enzymatically and that present in the sample. This is a general drawback for all biosensors based on the detection of ammonium. Study of samples containing both lysine and ammonium showed that concentration ranges exist in which a near-logarithmic relationship between potentials measured and lysine concentrations is found. Therefore, within these ranges, lysine can be determined by using the standard addition method, with the subsequent data treatment involving an iterative linearization procedure. Results obtained with the proposed potentiometric method are consistent with those given by the standard method for amino acid analysis.

Amino Acid Oxidoreductases↗