Detection of approximately 10(3) copies of DNA by an electrochemical enzyme-amplified sandwich assay with ambient O(2) as the substrate.
The electrochemical sandwich-type, enzyme-amplified assay of Zhang, Kim, and Heller (Anal. Chem. 2003, 75, 3267-3269) was simplified by replacing the amplifying horseradish peroxidase with bilirubin oxidase (BOD). BOD catalyzes the reduction of ambient O(2) to water and obviates the need for adding H(2)O(2). Femtomolar (10(-)(15) M) concentrations of DNA were detected at a 10-microm-diameter tip of a carbon fiber electrode. Correspondingly, a few thousand copies of DNA were detected in approximately 5-microL samples. The sandwich is formed in an electron-conducting redox hydrogel, to the polymer of which a DNA capture sequence is bound. Capture of the analyte DNA and its hybridization with a BOD-labeled complementary DNA sequence, electrically connects the BOD label to the electron-conducting redox polymer, which is in electrical contact with the electrode. Placing the BOD in contact with the redox polymer thus converts the noncatalytic base layer into a catalyst for the electroreduction of O(2) to water at +0.12 V (vs Ag/AgCl) (Figure 1). In an exemplary assay, approximately 3000 copies of the iron transporting sequence of the sit gene of Shigella flexneri were detected without PCR amplification.
Zhang
Yongchao
Y
Department of Chemical Engineering, University of Texas, Austin, Texas 78712, USA.
Pothukuchy
Arti
A
Shin
Woonsup
W
Kim
Yousung
Y
Heller
Adam
A
D000818:Animals; D004247:DNA / Q000032:analysis*; D004563:Electrochemistry / Q000379:methods; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D012763:Shigella flexneri / Q000235:genetics
D004247:DNA; D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase; D010100:Oxygen