Detection of single-nucleotide polymorphism on uidA gene of Escherichia coli by a multiplexed electrochemical DNA biosensor with oligonucleotide-incorporated nonfouling surface

Abstract

We report here a practical application of a multiplexed electrochemical DNA sensor for highly specific single-nucleotide polymorphism (SNP) detection. In this work, a 16-electrode array was applied with an oligonucleotide-incorporated nonfouling surfaces (ONS) on each electrode for the resistance of unspecific absorption. The fully matched target DNA templated the ligation between the capture probe assembled on gold electrodes and the tandem signal probe with a biotin moiety, which could be transduced to peroxidase-based catalyzed amperometric signals. A mutant site (T93G) in uidA gene of E. coli was analyzed in PCR amplicons. 10% percentage of single mismatched mutant gene was detected, which clearly proved the selectivity of the multiplexed electrochemical DNA biosensor when practically applied.

Keywords: Escherichia coli; electrochemical biosensor; nonfouling electrode surface; single-nucleotide polymorphism (SNP).

Figure 1.

Amperometric curves for the detection of single base mismatched (dashed line) and complemental probe (solid line) in the presence of the complementary target of 5 nM. (A) Analysis performed on electrodes with CP-W, where the T-W was fully matched and generated an enhanced current signal; (B) Analysis on electrode with CP-M, where T-M produced a higher signal than T-W. Potential of amperometry: 0.1 V.

Figure 2.

Optimization of the total target concentration. The DNA target sample consisted of 10% mutant DNA (T-M) and 90% wild DNA (T-W).

Figure 3.

Results of a multiplexed SNP analysis on a 16-electrode array. The mixed sample consisted of T-M and T-W with a serious concentration ratio. (A) Comparison of current signals from two kinds of electrode when the concentration ratio was changed; (B) The signal difference ratio (R) got higher while the percentage of T-M was increased. The total concentration of target DNA was 5 nM.

Figure 4.

Electrophoretic analysis of the PCR amplicons for the uidA gene of E. coli. The box indicates the 250-bp product (uidA). 1, Negative control (NC); M, DNA size marker; 2, wild type uidA gene; 3, mutant type uidA gene; 4 mixed sample with 10% wild type uidA gene; 5, mixed sample with 50% wild type uidA gene.

Figure 5.

the result of the multiplexed electrochemical analysis of PCR amplicons from samples with different percentage of mutant uidA gene.

Scheme 1.

Schematic illustration of the ligation based electrochemical DNA biosensor for the SNP analysis on an ONS electrode. (1) Target DNA (or PCR amplicons) and ligase; (2) Washing step; (3) Avidin-HRP. Capture probes assembled on the ONS electrode had different end bases at 3′ terminals, only when the target DNA could perfectly hybridize to the capture probes and form a “sandwich” structure with the signal probes, the ligation between two probes could be performed and let the signal probes survive in the following washing step. The avidin-HRP was then combined by the biotin label and catalyzed the redox reaction of the TMB substrate.