title: Highly sensitive electrochemical detection of DNA hybridisation by coupling the chemical reduction of a redox label to the electrode reaction of a solution phase mediator.
abstract: We have described a highly sensitive method for detecting DNA hybridisation using a redox-labeled stem loop probe. The redox labels were poly(styrene-co-acrylic) (PSA) spheres of 454 nm diameter, modified by methylene blue (MB) deposited alternatively with poly(sodium 4-styrene sulphonate) (PSS) in a layer-by-layer process. Each PSA sphere carried approx. 3.7 × 10(5) molecules of MB, as determined optically. DIG-tagged stem loop probes were immobilised on screen printed electrodes bearing anti-DIG antibodies. Binding with the target enabled straightening of the stem loop, which made attachment to the MB-coated PSA spheres possible. For measuring the current from the direct reduction of MB by differential pulse voltammetry, a 30 mer DNA target common to 70 strains of Escherichia coli was calibrated across the range 1.0 fM to 100 pM (gradient = 3.2 × 10(-8) A (log fM)(-1), r(2) = 0.95, n = 60), with an LOD of ∼58 fM. By using Fe(CN)6(3-/4-) as a solution phase mediator for the MB reduction, we were able to lower the LOD to ∼39 aM (gradient = 5.95 × 10(-8) A (log aM)(-1), r(2) = 0.96, n = 30), which corresponds to the detection of 0.76 ag (∼50 molecules) in the 2 μL analyte sample. We hypothesise that the lowering of the LOD was due to the fact that not all the MB labels were able to contact the electrode surface.
authors.affiliation: School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bang Khun Thian, Bangkok 10150, Thailand. werasak.sur@kmutt.ac.th.
authors.forename: Umphan
authors.initials: U
authors.lastname: Ngoensawat
authors.forename: Patsamon
authors.initials: P
authors.lastname: Rijiravanich
authors.forename: Mithran
authors.initials: M
authors.lastname: Somasundrum
authors.forename: Werasak
authors.initials: W
authors.lastname: Surareungchai
mesh_terms: D001483:Base Sequence; D002138:Calibration; D004269:DNA, Bacterial / Q000737:chemistry*; D055664:Electrochemical Techniques / Q000592:standards*; D004566:Electrodes; D004926:Escherichia coli / Q000235:genetics; D057230:Limit of Detection; D009693:Nucleic Acid Hybridization; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet
chemical_list: D004269:DNA, Bacterial