Detection of ∼10³ Copies of DNA by an Electrochemical Enzyme-Amplified Sandwich Assay with Ambient O₂ as the Substrate

Abstract

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.

Determination of Surface-Immobilized Double-Stranded DNA Using a Metallointercalator and Catalytic Voltammetry

“…Most recently, a ferrocene-tagged ''hairpin'' type DNA probe was developed for the reagentless detection of a specific DNA sequence with a detection limit of 5 fmol [24]. Another group of frequently used covalent labels is that of enzymes, which have produced the most sensitive DNA assays to date (1 fmol L À1 ) thanks to the power of enzymatic amplification [25][26][27]. Apart from molecular labels, various nanoparticles have also been shown to be promising materials in DNA detection [28][29][30].…”

Design requirements for integrated biosensor arrays

“…In recent years, there have been great advances in the electronic detection of the biological entities [18][19][20][21][22] . Detection is based on a change in an electrical quantity such as voltage, current impedance or charge by a biochemical reaction.…”

Polystyrene Microspheres Coupled with Hybridization Chain Reaction for Dual-Amplified Chemiluminescence Detection of Specific DNA Sequences

“…Up to now, a broad range of strategies such as optical (fluorescence [8][9][10][11], chemiluminescence (CL) [12][13][14][15][16], colorimetry [17,18], dynamic light scattering [19] surface plasmon resonance [20,21], etc. ), piezoelectric [22] and electronic transduction techniques [23,24] have been developed. Among the many methods devised for the detection of sequence-specific DNA, amplification is one of the most important concepts since it permits the highest analytical sensitivity [25][26][27][28][29][30].…”