Colorimetric and electrochemical genosensors for the detection of Escherichia coli DNA without amplification in seawater.
Monitoring seawater, particularly recreational water, for indicator bacteria presence is required to protect the public from exposure to fecal pollution and to guarantee the safety of the swimming areas. Two methods for the detection and quantification of Escherichia coli DNA were developed: a colorimetric assay in a microplate and an electrochemical biosensor. These assays were based on the double hybridization recognition of a single-strand DNA capture probe immobilized onto the microplate or the screen-printed carbon electrode to its complementary ssDNA, which is hybridized with an ssDNA signal probe labeled with horseradish peroxidase enzyme. The hybridization recognition step used the colorimetric monitoring of the oxidation state of the 3,3',5,5'-tetramethylbenzidine. The electrochemical monitoring of the oxidation state of 5 methyl-phenazinium methyl sulfate was allowed when the horseradish-peroxidase was in the presence of the mediator (5 methyl-phenazinium methyl sulfate and hydrogen peroxide). These approaches allow for the detection and quantification of 10(2) to 10(3) cells of E. coli in 5l of seawater samples in less than 5h. Detection was achieved without a nucleic acid amplification step. The specificity of the two methods against E. coli was demonstrated by testing a panel of bacteria. The two methods can be used for on-site monitoring of seawater quality.
UPMC Univ Paris 06, UMR 7621, LOMIC, Observatoire Océanologique, F-66650, Banyuls/mer, France; CNRS, UMR 7621, LOMIC, Observatoire Océanologique, F-66650, Banyuls/mer, France. Electronic address: nathalie.paniel@obs-banyuls.fr.
Nathalie
N
Paniel
Julia
J
Baudart
D001560:Benzidines / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods* / Q000592:standards; D003124:Colorimetry / Q000379:methods* / Q000592:standards; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000737:chemistry; D004566:Electrodes; D004784:Environmental Monitoring; D004800:Enzymes, Immobilized / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification*; D006735:Horseradish Peroxidase / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D008773:Methylphenazonium Methosulfate / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D010084:Oxidation-Reduction; D012623:Seawater / Q000032:analysis* / Q000382:microbiology
D001560:Benzidines; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004800:Enzymes, Immobilized; D008773:Methylphenazonium Methosulfate; C021758:3,3',5,5'-tetramethylbenzidine; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase
Colorimetric assay; DNA; Electrochemical biosensor; Escherichia coli; Sandwich hybridization assay; Seawater