Attomole DNA electrochemical sensor for the detection of Escherichia coli O157.
Enterohemorrhagic Escherichia coli O157, a verocytotoxin (VT1/2)-producing pathogen, can be deadly because it can induce acute or chronic renal failure. To speed up the clinical diagnosis of related syndromes caused by E. coli O157, there is an urgent need for rapid, simple, and reliable analytical tools for its quantitation. In this study, we developed a novel electrochemical competitive genosensor, featuring gold-electrodeposited screen-printed electrodes (nanoAu/SPE) modified with a self-assembled monolayer of thiol-capped single-stranded DNA (capture probe), for the detection of the rfbE gene, which is specific to E. coli O157. This assay functions based on competition between the target gene (complementary to the capture probe DNA) and reporter DNA-tagged, hexaammineruthenium(III) chloride-encapsulated liposomes. The current signal of the released liposomal Ru(NH(3))(6)(3+) was measured using square wave voltammetry, yielding a sigmoidally shaped dose-response curve whose linear portion was over the range from 1 to 10(6) fmol. This liposomal competitive assay provides an amplification route for the detection of the rfbE gene at ultratrace levels; indeed, we could detect as little as 0.75 amol of the target rfbE DNA (equivalent to the amount present in 5 microL of a 0.15 pM solution).
Liao
Wei-Ching
WC
BioAnalytical Laboratory, Department of Chemistry, National Tsing Hua University, Hsinchu 30013 Taiwan.
Ho
Ja-An Annie
JA
D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D002238:Carbohydrate Epimerases / Q000032:analysis / Q000235:genetics; D015342:DNA Probes / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004563:Electrochemistry; D004566:Electrodes; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D017930:Genes, Reporter; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D009942:Organometallic Compounds / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D012428:Ruthenium / Q000737:chemistry; D013194:Staining and Labeling; D020349:Surface Plasmon Resonance; D000637:Transaminases / Q000032:analysis / Q000235:genetics
D015342:DNA Probes; D004269:DNA, Bacterial; D008081:Liposomes; D009942:Organometallic Compounds; D012428:Ruthenium; D000637:Transaminases; D002238:Carbohydrate Epimerases; C061272:perosamine synthetase