Rapid, sequence-specific detection of unpurified PCR amplicons via a reusable, electrochemical sensor

Abstract

We report an electrochemical method for the sequence-specific detection of unpurified amplification products of the gyrB gene of Salmonella typhimurium. Using an asymmetric PCR and the electrochemical E-DNA detection scheme, single-stranded amplicons were produced from as few as 90 gene copies and, without subsequent purification, rapidly identified. The detection is specific; the sensor does not respond when challenged with control oligonucleotides based on the gyrB genes of either Escherichia coli or various Shigella species. In contrast to existing sequence-specific optical- and capillary electrophoresis-based detection methods, the E-DNA sensor is fully electronic and requires neither cumbersome, expensive optics nor high voltage power supplies. Given these advantages, E-DNA sensors appear well suited for implementation in portable PCR microdevices directed at, for example, the rapid detection of pathogens.

Fig. 1.

An E-DNA-based PCR sensor fabricated by self-assembly of a MB-labeled DNA probe on a gold electrode surface. In the absence of a target, the stem-loop structure holds the MB tag in proximity to the electrode surface, thus enabling efficient electron transfer. Upon hybridization with the target PCR amplicon, a large change in the reduction peak current of MB is observed. A room temperature distilled water wash is sufficient to disrupt hybridization and reset this reagentless, electrochemical sensor.

Fig. 2.

The E-DNA sensor is sensitive, reusable, and highly sequence-specific. (Left) Shown are baseline-subtracted AC voltammograms for the E-DNA sensor before hybridization, after incubation with 2 μM of a low-identity target DNA, and after challenge with 400 nM of two synthetic DNAs (S1 and S2) equivalent to the Salmonella-specific gyrB PCR amplicons we are investigating here. (Right) The sequence specificity of E-DNA is sufficient for species-specific detection. Shown are baseline-subtracted AC voltammograms for the E-DNA sensor before and after incubation with 200 nM (each) of target DNA comprised of sequences from the gyrB genes of S. flexneri, S. sonnei, and E. coli. Hybridization time was fixed at 30 min for all experiments.

Fig. 3.

E-DNA: A reagentless, reusable means for the electrochemical detection of unpurified PCR amplicons. Shown are baseline-subtracted AC voltammograms from the E-DNA sensor before use, after incubation with PCR products containing the relevant 17-base recognition element at their 3′-end (illustrated in Inset), and after regeneration via a simple, room temperature rinse with distilled water. Of note, these data were collected directly in the PCR buffer without any purification of the PCR products.

Fig. 4.

The E-DNA sensor readily detects unpurified PCR products by targeting an internal 17-base recognition element (illustrated in Inset). Shown are baseline-subtracted AC voltammograms for the E-DNA sensor before use, after incubation with PCR products in which the 17-base recognition element is 48 bases from the 3′ end, and after regeneration.

Fig. 5.

The E-DNA sensor response time is rapid when compared to the tens of minutes typically required for PCR amplification. The slower hybridization observed for the int-PCR target may be due to steric hindrance arising from the 48 bases overhang at this target’s 3′ end.