Conformationally Gated Electrochemical Gene Detection

Immoos, Chad E.; Lee, Stephen J.; Grinstaff, Mark W.

doi:10.1002/cbic.200400045

Cited by 94 publications

(126 citation statements)

References 28 publications

(21 reference statements)

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“…[13][14][15]27,41,43,44 The latter class of sensors, directly analogous to the E-DNA sensor, is based on the binding-induced folding of DNA aptamers and has been demonstrated for targets ranging from proteins 26,41,42 to small molecules 27 and inorganic ions. 43,44 The results presented here suggest that careful optimization of probe density and measurement techniques will be necessary in order to achieve maximum performance across this broad and increasingly important class of sensors.…”

Section: Discussionmentioning

confidence: 99%

“…Knowledge of the extent to which probe density affects the E-DNA signaling and equilibration time would provide a general tool for the optimization of other similar sensing platforms (e.g., refs [13][14][15] ) and may provide insight into the details of the sensing mechanism.…”

Section: Controlling Probe Surface Densitymentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2007

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E-DNA sensors, the electrochemical equivalent of molecular beacons, appear to be a promising means of detecting oligonucleotides. E-DNA sensors are comprised of a redox-modified (here, methylene blue or ferrocene) DNA stem-loop covalently attached to an interrogating electrode. Because E-DNA signaling arises due to binding-induced changes in the conformation of the stemloop probe, it is likely sensitive to the nature of the molecular packing on the electrode surface. Here we detail the effects of probe density, target length, and other aspects of molecular crowding on the signaling properties, specificity, and response time of a model E-DNA sensor. We find that the highest signal suppression is obtained at the highest probe densities investigated, and that greater suppression is observed with longer and bulkier targets. In contrast, sensor equilibration time slows monotonically with increasing probe density, and the specificity of hybridization is not significantly affected. In addition to providing insight into the optimization of electrochemical DNA sensors, these results suggest that E-DNA signaling arises due to hybridization-linked changes in the rate, and thus efficiency, with which the redox moiety collides with the electrode and transfers electrons.

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Section: Discussionmentioning

confidence: 99%

Section: Controlling Probe Surface Densitymentioning

confidence: 99%

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

et al. 2007

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“…For example, although Hwang et al (13) report an exceptional 0.1 fM detection limit, achieving it required a five-step assay, including an enzyme-linked secondary probe, enzymatic reduction of p-aminophenyl phosphate, the concomitant reductive deposition of silver, and, finally, anodic stripping voltammetry to quantify the deposited silver. In contrast to these relatively cumbersome assays (6-13), we and others have recently described several reagentless, single-step electrochemical DNA detection methods based on immobilized, redox-tagged single-stranded DNA (14,15) and DNA stem-loops (16)(17)(18). The latter strategy, termed E-DNA (16), is based on a redoxtagged DNA stem-loop structure that self-assembles on a gold electrode by a gold-thiol bond.…”

mentioning

confidence: 99%

Single-step electronic detection of femtomolar DNA by target-induced strand displacement in an electrode-bound duplex

Xiao

Lubin

Baker

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

219

198

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We report a signal-on, electronic DNA (E-DNA) sensor that is label-free and achieves a subpicomolar detection limit. The sensor, which is based on a target-induced strand displacement mechanism, is composed of a ''capture probe'' attached by its 5 terminus to a gold electrode and a 5 methylene blue-modified ''signaling probe'' that is complementary at both its 3 and 5 termini to the capture probe. In the absence of target, hybridization between the capture and signaling probes minimizes contact between the methylene blue and electrode surface, limiting the observed redox current. Target hybridization displaces the 5 end of the signaling probe, generating a short, flexible single-stranded DNA element and producing up to a 7-fold increase in redox current. The observed signal gain is sufficient to achieve a demonstrated (not extrapolated) detection limit of 400 fM, which is among the best reported for single-step electronic DNA detection. Moreover, because sensor fabrication is straightforward, the approach appears to provide a ready alternative to the more cumbersome femtomolar electrochemical assays described to date.biosensors ͉ electron transfer ͉ gold electrode ͉ methylene blue ͉ signal-on

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“…In previous work we (20) and others (21)(22)(23) have developed a reagentless, electrochemical biosensor termed E-DNA wherein a redox-labeled DNA stem-loop covalently attached to an interrogating electrode produces an electrochemical signal when hybridized to its target sequence (Fig. 1).…”

mentioning

confidence: 99%

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

Lai

Lagally

Lee

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

178

148

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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...

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Conformationally Gated Electrochemical Gene Detection

Cited by 94 publications

References 28 publications

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

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

Single-step electronic detection of femtomolar DNA by target-induced strand displacement in an electrode-bound duplex

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

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