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.E-DNA ͉ methylene blue ͉ Salmonella gyrB ͉ polymerase chain reaction T he species-specific identification of pathogenic bacteria poses a pressing problem with impacts ranging from food safety to the detection of biowarfare agents. For example, it has been shown that the early identification of bacterial pathogens can significantly reduce the breadth and severity of outbreaks of food-borne diseases (1), outbreaks that are responsible for Ϸ76 million illnesses, 325,000 hospitalizations, and 5,000 deaths per year in the United States alone (2). Current methods for the detection and identification of bacteria, however, are complex and slow; because the minimum infectious doses of food-borne pathogens such as Escherichia coli, Listeria monocytogenes, and Salmonella are very low (3, 4), the detection of clinically relevant levels of contamination generally requires amplification of the infectious organism via laboratory culturing over the course of 1 to several days (5, 6).The PCR-based amplification of pathogen-specific DNA, rather than the pathogens themselves, offers a potentially promising means of avoiding cumbersome, time-consuming culturing steps and achieving the rapid and reliable identification of microbes. Unfortunately, however, the methods traditionally used for the detection of PCR-amplified DNA, which include Southern blots and capillary electrophoresis (CE), are rather unwieldy, and, thus, PCR-based assays are typically limited to laboratory settings. In response to this problem, a number of more convenient, field-portable PCR detection schemes have been described in recent years (7,8). In particular, because microfluidic techniques allow the miniaturization of PCR reactions to single-chip dimensions, there has been much interest in the development of a PCR-amplification͞detection platform integrated onto a single integrated microdevice (9). The first such approach used the optical detection of PCR products via intercalating dyes that report on the presence of double-stranded DNA (10, 11). PCR, however, is often promiscuous, producing spurious amplification products (12, 13) that produce false pos...

Effect of Molecular Crowding on the Response of an Electrochemical DNA Sensor

“…13,24 In the absence of the target DNA, the sensor gives a sharp, well-defined ACV peak at ~260 mV (vs Ag/AgCl), consistent with the formal potential of the MB redox moiety used in this work ( Figure 1). Upon hybridization, binding-induced changes in the conformation of the stem-loop probe result in a decrease of the signal.…”

“…For example, while it is likely that the signaling properties of these sensors depend sensitively on the density of immobilized probe DNA molecules on the sensor surface (measured in molecules of probe per square centimeter) [see, e.g., refs 5 and [29][30][31][32][33][34][35][36] ], no systematic study of this effect has been reported. Similarly, while it appears that the size of the target and the location of the recognition element within the target sequence affect signal suppression, 24 this effect, too, has seen relatively little study. Here we detail the effects of probe surface density, target length, and other aspects of molecular crowding on the signaling properties, specificity, and response time of the E-DNA sensor.…”

“…Both E-DNA sensors [13][14][15][16] and related sensors based on the binding-induced folding of DNA aptamers [23][24][25][26][27][28] have been extensively studied in recent years. Nevertheless, key issues in their fabrication and use have not yet been explored in detail.…”

Scaffolded biosensors with designed DNA nanostructures

“…By using this strategy, Lubin et al 43 successfully demonstrated an E-DNA sensor-based approach for detecting DNA authentication tags that are associated with paper or drugs. Lai et al 44 achieved the sequence-specific detection of unpurified amplification products of the gyrB gene of Salmonella typhimurium, which may open the path toward effective, field-portable sample-to-answer pathogen identification.…”

Bio-surface engineering with DNA scaffolds for theranostic applications

“…This E-DNA thus provides a costeffective and efficient technique for reagent-less and reusable detection of picomolar DNA, which holds great potential in medical and military applications. By using the E-DNA scheme, Lai et al [80] achieved sequencespecific detection of unpurified amplification products of the gyrB gene of Salmonella typhimurium, which may open the path toward effective and field-portable sampleto-answer pathogen identification.…”