Enzyme-based impedimetric detection of PCR products using oligonucleotide-modified screen-printed gold electrodes. 2005

Fausto Lucarelli, and Giovanna Marrazza, and Marco Mascini
Department of Chemistry, University of Florence, 50019 Sesto Fiorentino, Florence, Italy.

This paper describes the optimisation and the analytical performances of an enzyme-based electrochemical genosensor, developed using disposable oligonucleotide-modified screen-printed gold electrodes. The immobilisation of a thiol-tethered probe was qualitatively investigated by means of faradic impedance spectroscopy. Impedance spectra confirmed that the thiol moiety unambiguously drives the immobilisation of the oligonucleotide probe. Furthermore, both probe surface densities and hybridisation efficiencies were quantified through chronocoulometric measurements. Electrochemical transduction of the hybridisation process was also performed by means of faradic impedance spectroscopy, after coupling of a streptavidin-alkaline phosphatase conjugate and bio-catalysed precipitation of an insoluble and insulating product onto the sensing interface. Chronocoulometric results allowed discussion of the magnitude of hybridisation signals in terms of probe surface densities and their corresponding hybridisation efficiency. The genosensor response varied linearly (r2 = 0.9998) with the oligonucleotide target concentration over three orders of magnitude, between 12 pmol/L and 12 nmol/L. The estimated detection limit was 1.2 pmol/L (i.e., 7.2 x 10(6) target molecules in 10 microL of sample solution). The analytical usefulness of the impedimetric genosensor was finally demonstrated analysing amplified samples obtained from the pBI121 plasmid and soy and maize powders containing 1 and 5% of genetically modified product. Sensing of such unmodified amplicons was achieved via sandwich hybridisation with a biotinylated signaling probe. The electrochemical enzyme-amplified assay allowed unambiguous identification of all genetically modified samples, while no significant non-specific signal was detected in the case of all negative controls.

UI MeSH Term Description Entries
D004247 DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D004563 Electrochemistry The study of chemical changes resulting from electrical action and electrical activity resulting from chemical changes. Electrochemistries
D004867 Equipment Design Methods and patterns of fabricating machines and related hardware. Design, Equipment,Device Design,Medical Device Design,Design, Medical Device,Designs, Medical Device,Device Design, Medical,Device Designs, Medical,Medical Device Designs,Design, Device,Designs, Device,Designs, Equipment,Device Designs,Equipment Designs
D015374 Biosensing Techniques Any of a variety of procedures which use biomolecular probes to measure the presence or concentration of biological molecules, biological structures, microorganisms, etc., by translating a biochemical interaction at the probe surface into a quantifiable physical signal. Bioprobes,Biosensors,Electrodes, Enzyme,Biosensing Technics,Bioprobe,Biosensing Technic,Biosensing Technique,Biosensor,Electrode, Enzyme,Enzyme Electrode,Enzyme Electrodes,Technic, Biosensing,Technics, Biosensing,Technique, Biosensing,Techniques, Biosensing
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D017097 Electric Impedance The resistance to the flow of either alternating or direct electrical current. Bioelectrical Impedance,Electric Resistance,Impedance,Ohmic Resistance,Biolectric Impedance,Electrical Impedance,Electrical Resistance,Impedance, Bioelectrical,Impedance, Biolectric,Impedance, Electric,Impedance, Electrical,Ohmic Resistances,Resistance, Electric,Resistance, Electrical,Resistance, Ohmic,Resistances, Ohmic
D017403 In Situ Hybridization A technique that localizes specific nucleic acid sequences within intact chromosomes, eukaryotic cells, or bacterial cells through the use of specific nucleic acid-labeled probes. Hybridization in Situ,Hybridization, In Situ,Hybridizations, In Situ,In Situ Hybridizations
D019544 Equipment Failure Analysis The evaluation of incidents involving the loss of function of a device. These evaluations are used for a variety of purposes such as to determine the failure rates, the causes of failures, costs of failures, and the reliability and maintainability of devices. Materials Failure Analysis,Prosthesis Failure Analysis,Analysis, Equipment Failure,Analysis, Materials Failure,Analysis, Prosthesis Failure,Analyses, Equipment Failure,Analyses, Materials Failure,Analyses, Prosthesis Failure,Equipment Failure Analyses,Failure Analyses, Equipment,Failure Analyses, Materials,Failure Analyses, Prosthesis,Failure Analysis, Equipment,Failure Analysis, Materials,Failure Analysis, Prosthesis,Materials Failure Analyses,Prosthesis Failure Analyses
D020411 Oligonucleotide Array Sequence Analysis Hybridization of a nucleic acid sample to a very large set of OLIGONUCLEOTIDE PROBES, which have been attached individually in columns and rows to a solid support, to determine a BASE SEQUENCE, or to detect variations in a gene sequence, GENE EXPRESSION, or for GENE MAPPING. DNA Microarrays,Gene Expression Microarray Analysis,Oligonucleotide Arrays,cDNA Microarrays,DNA Arrays,DNA Chips,DNA Microchips,Gene Chips,Oligodeoxyribonucleotide Array Sequence Analysis,Oligonucleotide Microarrays,Sequence Analysis, Oligonucleotide Array,cDNA Arrays,Array, DNA,Array, Oligonucleotide,Array, cDNA,Arrays, DNA,Arrays, Oligonucleotide,Arrays, cDNA,Chip, DNA,Chip, Gene,Chips, DNA,Chips, Gene,DNA Array,DNA Chip,DNA Microarray,DNA Microchip,Gene Chip,Microarray, DNA,Microarray, Oligonucleotide,Microarray, cDNA,Microarrays, DNA,Microarrays, Oligonucleotide,Microarrays, cDNA,Microchip, DNA,Microchips, DNA,Oligonucleotide Array,Oligonucleotide Microarray,cDNA Array,cDNA Microarray

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