title: Electrochemical detection of synthetic DNA and native 16S rRNA fragments on a microarray using a biotinylated intercalator as coupling site for an enzyme label.
abstract: The direct electrochemical detection of synthetic DNA and native 16S rRNA fragments isolated from Escherichia coli is described. Oligonucleotides are detected via selective post-labeling of double stranded DNA and DNA-RNA duplexes with a biotinylated intercalator that enables high-specific binding of a streptavidin/alkaline phosphatase conjugate. The alkaline phosphatase catalyzes formation of p-aminophenol that is subsequently oxidized at the underlying gold electrode and hence enables the detection of complementary hybridization of the DNA capture strands due to the enzymatic signal amplification. The hybridization assay was performed on microarrays consisting of 32 individually addressable gold microelectrodes. Synthetic DNA strands with sequences representing six different pathogens which are important for the diagnosis of urinary tract infections could be detected at concentrations of 60 nM. Native 16S rRNA isolated from the different pathogens could be detected at a concentration of 30 fM. Optimization of the sensing surface is described and influences on the assay performance are discussed.
authors.affiliation: FRIZ Biochem, Floriansbogen 2-4, D-82061 Neuried, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Zimdars
authors.affiliation: Analytical Chemistry - Center for Electrochemical Sciences (CES), Ruhr-Universität Bochum, Universitätsstr. 150, D-44780 Bochum, Germany.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Gebala
authors.affiliation: FRIZ Biochem, Floriansbogen 2-4, D-82061 Neuried, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Hartwich
authors.affiliation: Analytical Chemistry - Center for Electrochemical Sciences (CES), Ruhr-Universität Bochum, Universitätsstr. 150, D-44780 Bochum, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Neugebauer
authors.affiliation: Analytical Chemistry - Center for Electrochemical Sciences (CES), Ruhr-Universität Bochum, Universitätsstr. 150, D-44780 Bochum, Germany. Electronic address: wolfgang.schuhmann@rub.de.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schuhmann
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D019921:Biotinylation; D004247:DNA / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D004563:Electrochemistry; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D007364:Intercalating Agents / Q000737:chemistry*; D057230:Limit of Detection; D020411:Oligonucleotide Array Sequence Analysis; D011370:Proflavine / Q000737:chemistry; D012336:RNA, Ribosomal, 16S / Q000032:analysis* / Q000737:chemistry; D019809:Streptavidin / Q000378:metabolism
chemical_list: D015342:DNA Probes; D007364:Intercalating Agents; D012336:RNA, Ribosomal, 16S; D004247:DNA; D019809:Streptavidin; D011370:Proflavine; D000469:Alkaline Phosphatase
keywords: DNA hybridization; DNA–RNA assay; Electrochemical DNA analysis; Intercalation; Label-free; Urinary tract infection