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1.
Chem Commun (Camb) ; 53(70): 9721-9724, 2017 Aug 29.
Article in English | MEDLINE | ID: mdl-28782763

ABSTRACT

An on-surface isothermal helicase-dependent amplification is devised for simple, point-of-need quantification of bacterial genomes. The method relies on the enzyme-extension of a thiol-modified reverse primer anchored to indium tin oxide electrodes, which shows strikingly high thermal and storage stability. Amplification and electrochemical detection of only 10 genomes are thus performed on the same platform without thermal cycling.


Subject(s)
DNA Helicases/metabolism , Electrochemical Techniques , Enzymes, Immobilized/metabolism , Nucleic Acid Amplification Techniques , Oligonucleotides/chemistry , Salmonella/isolation & purification , Tin Compounds/chemistry , Electrodes , Salmonella/genetics
2.
Talanta ; 164: 261-267, 2017 Mar 01.
Article in English | MEDLINE | ID: mdl-28107927

ABSTRACT

Monitoring of genetically modified organisms in food and feed demands molecular techniques that deliver accurate quantitative results. Electrochemical DNA detection has been widely described in this field, yet most reports convey qualitative data and application in processed food and feed samples is limited. Herein, the applicability of an electrochemical multiplex assay for DNA quantification in complex samples is assessed. The method consists of the simultaneous magnetic entrapment via sandwich hybridisation of two DNA sequences (event-specific and taxon-specific) onto the surface of magnetic microparticles, followed by bienzymatic labelling. As proof-of-concept, we report its application in a transgenic food/feed survey where relative quantification (two-target approach) of Roundup Ready Soybean® (RRS) was performed in food and feed. Quantitative coupling to end-point PCR was performed and calibration was achieved from 22 and 243 DNA copies spanning two orders of magnitude for the event and taxon-specific sequences, respectively. We collected a total of 33 soybean-containing samples acquired in local supermarkets, four out of which were found to contain undeclared presence of genetically modified soybean. A real-time PCR method was used to verify these findings. High correlation was found between results, indicating the suitability of the proposed multiplex method for food and feed monitoring.


Subject(s)
DNA, Plant/chemistry , DNA, Plant/genetics , Electrochemistry/methods , Enzymes/metabolism , Magnets/chemistry , Plants, Genetically Modified , Surveys and Questionnaires , Animals , Base Sequence , DNA, Plant/metabolism , Microspheres , Polymerase Chain Reaction
3.
Biosens Bioelectron ; 26(5): 2396-401, 2011 Jan 15.
Article in English | MEDLINE | ID: mdl-21067909

ABSTRACT

Reactive oxygen species (ROS) are produced as a consequence of normal aerobic metabolism and are able to induce DNA oxidative damage. At the cellular level, the evaluation of the protective effect of antioxidants can be achieved by examining the integrity of the DNA nucleobases using electrochemical techniques. Herein, the use of an adenine-rich oligonucleotide (dA(21)) adsorbed on carbon paste electrodes for the assessment of the antioxidant capacity is proposed. The method was based on the partial damage of a DNA layer adsorbed on the electrode surface by OH radicals generated by Fenton reaction and the subsequent electrochemical oxidation of the intact adenine bases to generate an oxidation product that was able to catalyze the oxidation of NADH. The presence of antioxidant compounds scavenged hydroxyl radicals leaving more adenines unoxidized, and thus, increasing the electrocatalytic current of NADH measured by differential pulse voltammetry (DPV). Using ascorbic acid (AA) as a model antioxidant species, the detection of as low as 50 nM of AA in aqueous solution was possible. The protection efficiency was evaluated for several antioxidant compounds. The biosensor was applied to the determination of the total antioxidant capacity (TAC) in beverages.


Subject(s)
Antioxidants/analysis , Antioxidants/chemistry , Beverages/analysis , Biosensing Techniques/instrumentation , Conductometry/instrumentation , DNA/chemistry , Food Analysis/instrumentation , Catalysis , DNA Damage , Equipment Design , Equipment Failure Analysis
4.
Anal Bioanal Chem ; 378(8): 1922-8, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15064901

ABSTRACT

This article summarises our work on the development of voltammetric sensors based on molecularly imprinted polymers. Several recognition elements and integration strategies were used:1.membranes electropolymerised at the electrode surface; 2.casting of polymeric membranes by drop-coating a solution of pre-formed polymer (polyphosphazene) and template in a low-boiling-point solvent on to the electrode surface; 3.preparation of composite membranes containing conductive material (graphite or carbon black), acrylic-type molecularly imprinted polymers (small particle size), and PVC as binder; and 4.in-situ polymerisation of a thin layer of acrylic imprinted polymer deposited on the electrode surface by spin coating. All the options evaluated offer the possibility of controlling electrode characteristics such as hydrophobic/hydrophilic character, permeability, or film thickness, which are essential for obtaining good sensor performance.


Subject(s)
Chemistry Techniques, Analytical/instrumentation , Chemistry Techniques, Analytical/methods , Polymers/chemistry , Polymers/chemical synthesis , Electrochemistry , Electrodes , Molecular Structure , Organophosphorus Compounds/chemistry , Plastics/chemical synthesis , Plastics/chemistry
5.
Biosens Bioelectron ; 18(4): 353-62, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12604252

ABSTRACT

Despite the increasing number of applications of molecularly imprinted polymers (MIPs) in analytical chemistry, the construction of a biomimetic voltammetric sensor remains still challenging. This work investigates the development of a voltammetric sensor for vanillylmandelic acid (VMA) based on acrylic MIP-modified electrodes. Thin layers of MIPs for VMA have been prepared by spin coating the surface of a glassy carbon electrode with the monomers mixture (template, methacrylic acid, a cross-linking agent and solvent), followed by in situ photopolymerisation. After extraction of the template molecule, the peak current recorded with the imprinted sensor after rebinding was linear with VMA concentration in the range 19-350 microg ml(-1), whereas the response of the control electrode is independent of incubation concentration, and was about one-tenth of the value recorded with the imprinted sensor at the maximum concentration tested. Under the conditions used, the sensor is able to differentiate between VMA and other closely structural-related compounds, such as 3-methoxy-4-hydroxyphenylethylene glycol (not detected), or 3,4- and 2,5-dihydroxyphenilacetic acids, which are adsorbed on the bare electrode surface but not at the polymer layer. Homovanillic acid was detected with the imprinted sensors after incubation, indicating that the presence of both methoxy and carboxylic groups in the same position as in VMA is necessary for effective binding in the imprinted sites. Nevertheless, both species can be differentiated by the oxidation potential. It can be concluded that MIP-based voltammetric electrodes are very promising analytical tool for the development of highly selective analytical sensors.


Subject(s)
Biomimetic Materials/chemical synthesis , Coated Materials, Biocompatible/chemical synthesis , Electrochemistry/instrumentation , Electrodes , Methacrylates , Transducers , Vanilmandelic Acid/analysis , Electrochemistry/methods , Equipment Design , Equipment Failure Analysis , Polymers , Reproducibility of Results , Sensitivity and Specificity
6.
Anal Chem ; 72(3): 520-7, 2000 Feb 01.
Article in English | MEDLINE | ID: mdl-10695137

ABSTRACT

The electrochemical oxidation of the adenine moiety in NAD+ and other adenine nucleotides at carbon paste electrodes gives rise to redox-active products which strongly adsorb on the electrode surface. Carbon paste electrodes modified with the oxidation products of NAD+ show excellent electrocatalytic activity toward NADH oxidation, reducing its overpotential by about 400 mV. The rate constant for the catalytic oxidation of NADH, determined by rotating disk electrode measurements and extrapolation to zero concentration of NADH, was found to be 2.5 x 10(5) M-1 s-1. The catalytic oxidation current allows the amperometric detection of NADH at an applied potential of +50 mV (Ag/AgCl) with a detection limit of 4.0 x 10(-7) M and linear response up to 1.0 x 10(-5) M NADH. These modified electrodes can be used as amperometric transducers in the design of biosensors based on coupled dehydrogenase enzymes and, in fact, we have designed an amperometric biosensor for glycerol based on the glycerol dehydrogenase (GlDH) system. The enzyme GlDH and its cofactor NAD+ were co-immobilized in a carbon paste electrode using an electropolymerized layer of nonconducting poly(o-phenylenediamine) (PPD). After partial oxidation of the immobilized NAD+, the modified electrode allows the amperometric detection of the NADH enzymatically obtained at applied potential above 0 V (Ag/AgCl). The resulting biosensor shows a fast and linear response to glycerol within the concentration range of 1.0 x 10(-6)-1.0 x 10(-4) M with a detection limit of 4.3 x 10(-7) M. The amperometric response remains stable for at least 3 days. The biosensor was applied to the determination of glycerol in a plant-extract syrup, with results in good agreement with those for the standard spectrophotometric method.


Subject(s)
Biosensing Techniques/methods , Glycerol/analysis , NAD/analysis , Carbon/chemistry , Catalysis , Electrodes , Feasibility Studies , NAD/chemistry , Oxidation-Reduction , Oxidoreductases/chemistry , Plant Extracts/chemistry
7.
Biosens Bioelectron ; 12(8): 739-47, 1997.
Article in English | MEDLINE | ID: mdl-9421885

ABSTRACT

A glutamate biosensor was developed by electropolymerizing o-phenylenediamine on a dehydrogenase-NAD+ modified carbon paste electrode. The amperometric response to glutamate was obtained by means of the electrocatalytic oxidation of the enzymatically produced NADH, at an applied potential close to 0 V (Ag/AgCl). After optimizing carbon paste composition, polymer thickness and operating variables, a linear amperometric response to glutamate was obtained within the concentration range 5.0 x 10(-6)-7.8 x 10(-5) M with a detection limit of 3.8 x 10(-6) M. The biosensor was applied to the determination of glutamate in chicken bouillon cubes. Good accuracy was found with respect to a reference enzymatic spectrophotometric method.


Subject(s)
Biosensing Techniques , Glutamic Acid/analysis , Phenylenediamines , Carbon , Electrodes
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