Rapid Amperometric Verification of PCR Amplification of DNA

Lumley-Woodyear, Thierry De; Campbell, Charles N.; Freeman, Esti; Freeman, Amihay; Georgiou, George; Heller, Adam

doi:10.1021/ac980770h

Cited by 46 publications

(51 citation statements)

References 12 publications

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“…When the target hybridizes with the probe, the hybrid formation can be electrochemically transduced by a number of indicator-based or indicator-free approaches. The former approach relies on the binding of intercalators [4, 6 ± 9], redox enzymes [10,11], or metal nanoparticles [12 ± 17] onto the hybrid while the latter approach is based on the intrinsic electroactivity of DNA [18,19] or changes in interfacial properties [20 ± 22].…”

Section: Introductionmentioning

confidence: 99%

Electrochemical Detection of PCR Amplicons Using Electroconductive Polymer Modified Electrode and Multiple Nanoparticle Labels

Cai

Lee

et al. 2004

Electroanalysis

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We report the utilization of an oligonucleotide-functionalized electroconducting polymer, coupled with a nanoparticle-based hybridization indicator for a sensitive detection of multi-labeled PCR amplicons. The electroconducting polymer permits the immobilization of an active probe onto the transducer surface. In this work, 2-aminobenzoic acid is electropolymerized onto an indium tin oxide electrode. The coated film has carboxyl groups for the immobilization of amino-terminated probe, mediated by coupling agents of water-soluble carbodiimide and Nhydroxysulfosuccinimide. This coupling chemistry has minimal nonspecific adsorption of the gold nanoparticle indicator, leading to a low background signal allowing a more sensitive hybridization detection. Signal transduction of the hybridization event is achieved through a series of steps: i) the binding of the gold nanoparticle label to the hybridized target based on streptavidin-biotin interaction, ii) the catalytic silver deposition onto the gold label (i.e., the silver enhancement) and iii) potentiometric stripping analysis of the deposited silver. In addition to signal amplification by silver enhancement, multiple-labeling of the amplicons by the incorporation of biotin-16-dUTP is employed to further enhance the signal to background ratio. The sequence-specific identification of both symmetric and asymmetric PCR amplicons is also demonstrated.

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Section: Introductionmentioning

confidence: 99%

Electrochemical Detection of PCR Amplicons Using Electroconductive Polymer Modified Electrode and Multiple Nanoparticle Labels

Cai

Lee

et al. 2004

Electroanalysis

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“…The amplicon can be attached directly onto a GEC surface by simple ™dry-adsorption∫ (the simplest immobilization method and the easiest to automate), avoiding the use of procedures based on previous activation of the surface transducer and subsequent immobilization, which are tedious, expensive and time-consuming [37]. The DNA binds strongly to GEC in a way that prevents the strands from self -associating, while at the same time permitting its hybridization to complementary DNA in connection with a short alkaline denaturing treatment.…”

Section: Discussionmentioning

confidence: 99%

PCR‐Genosensor Rapid Test for Detecting Salmonella

et al. 2003

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A PCR-genosensor based assay for detecting the IS200 DNA sequence specific to Salmonella spp is reported. This rapid test is based on PCR, where amplicon detection is achieved electrochemically using GEC (graphite-epoxy composite) genosensor. The amplicon is immobilized onto GEC electrodes by simple dry-adsorption and the detection is carried out using an enzymatic labeling system. Results demonstrate that this new electrochemical genosensor fulfils the requirements desired for these devices: easy preparation ± as dry-adsorption of DNA is very simple ±, robustness, sensitivity, low cost, simple use and fast response. Additionally, the assay can be produced as a kit format, increasing its commercial potentiality. Also, the electrode material can be implemented for screen-printing procedures for the mass production of genosensors.

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“…[34] A few groups have used this ECD technique for nucleic acid amplification product detection. [34][35][36][37] In 2003, Lee et al reported the first integrated microchip-based PCR with ECD functionality.…”

Section: Post-pcr Detectionmentioning

confidence: 99%

Flow through PCR module of BioBriefcase

et al. 2005

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The BioBriefcase is an integrated briefcase-sized aerosol collection and analysis system for autonomous monitoring of the environment, which is currently being jointly developed by Lawrence Livermore and Sandia National Laboratories. This poster presents results from the polymerase chain reaction (PCR) module of the system. The DNA must be purified after exiting the aerosol collector to prevent inhibition of the enzymatic reaction. Traditional solid-phase extraction results in a large loss of sample. In this flow-through system, we perform sample purification, concentration and amplification in one reactor, which minimizes the loss of material. The sample from the aerosol collector is mixed with a denaturation solution prior to flowing through a capillary packed with silica beads. The DNA adheres to the silica beads allowing the environmental contaminants to be flushed to waste while effectively concentrating the DNA on the silica matrix. The adhered DNA is amplified while on the surface of the silica beads, resulting in a lower limit of detection than an equivalent eluted sample. Thus, this system is beneficial since more DNA is available for amplification, less reagents are utilized, and contamination risks are reduced.

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Rapid Amperometric Verification of PCR Amplification of DNA

Cited by 46 publications

References 12 publications

Electrochemical Detection of PCR Amplicons Using Electroconductive Polymer Modified Electrode and Multiple Nanoparticle Labels

Electrochemical Detection of PCR Amplicons Using Electroconductive Polymer Modified Electrode and Multiple Nanoparticle Labels

PCR‐Genosensor Rapid Test for Detecting Salmonella

Flow through PCR module of BioBriefcase

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