A new nanoparticle-based electrical detection of DNA hybridization, based on electrochemical stripping detection of the colloidal gold tag, is described. In this protocol, the hybridization of a target oligonucleotide to magnetic bead-linked oligonucleotide probes is followed by binding of the streptavidin-coated metal nanoparticles to the captured DNA, dissolution of the nanometer-sized gold tag, and potentiometric stripping measurements of the dissolved metal tag at single-use thick-film carbon electrodes. An advanced magnetic processing technique is used to isolate the DNA duplex and to provide low-volume mixing. The influence of relevant experimental variables, including the amounts of the gold nanoparticles and the magnetic beads, the duration of the hybridization and gold dissolution steps, and the parameters of the potentiometric stripping operation upon the hybridization signal, is examined and optimized. Transmission electron microscopy micrographs indicate that the hybridization event leads to the bridging of the gold nanoparticles to the magnetic beads. Further signal amplification, and lowering of the detection limits to the nanomolar and picomolar domains, are achieved by precipitating gold or silver, respectively, onto the colloidal gold label. The new electrochemical stripping metallogenomagnetic protocol couples the inherent signal amplification of stripping metal analysis with discrimination against nonhybridized DNA, the use of microliter sample volumes, and disposable transducers and, hence, offers great promise for decentralized genetic testing.
An electrochemical impedance spectroscopy method of detection for aptamer-based array electrodes is reported in which the binding of aptamers immobilized on gold electrodes leads to impedance changes associated with target protein binding events. Human IgE was used as a model target protein and incubated with the aptamer-based array consisting of single-stranded DNA containing a hairpin loop. To increase the binding efficiency for proteins, a hybrid modified layer containing aptamers and cysteamine was fabricated on the photolithographic gold surface through molecular self-assembly. Atomic force microscopy analysis demonstrated that human IgE could be specifically captured by the aptamer and stand well above the self-assembled monolayer (SAM) surface. Compared to immunosensing methods using anti-human IgE antibody as the recognition element, impedance spectroscopy detection could provide higher sensitivity and better selectivity for aptamer-modified electrodes. The results of this method show good correlation for human IgE in the range of 2.5-100 nM. A detection limit of 0.1 nM (5 fmol in a 50-microL sample) was obtained, and an average of the relative standard deviation was <10%. The method herein describes the first label-free detection for arrayed electrodes utilizing electrochemical impedance spectroscopy.
We report on a novel method for detecting DNA hybridization, based on the precipitation of silver on gold nanoparticle tags and a subsequent electrochemical stripping detection of the dissolved silver. Such coupling of a nanoparticle-promoted silver precipitation with the remarkable sensitivity of stripping metal analysis offers a dramatic enhancement of the hybridization response. An efficient magnetic isolation of the duplex is used for discriminating against nonhybridized DNA, including an excess of mismatched oligonucleotides. The new silver-enhanced colloidal gold stripping detection strategy holds great promise for the detection of DNA hybridization and represents an attractive alternative to indirect optical affinity assays of nucleic acids and other biomolecules.
A magnetic triggering of a solid-state electrical transduction of DNA hybridization is described. Positioning of an external magnet below the thick-film electrode attracts the DNA/particle network and enables the solid-state electrochemical stripping detection of the silver tracer. TEM imaging indicates that the hybridization event results in a three-dimensional aggregate structure in which duplex segments link the metal nanoparticles and magnetic spheres, and that most of this assembly is covered with the silver precipitate. This leads to a direct contact of the metal tag with the surface (in connection to the magnetic collection) and enables the solid-state electrochemical transduction (without prior dissolution and subsequent electrodeposition of the metal), using oxidative dissolution of the silver tracer. No such aggregates (and hence magnetic "collection") are observed in the presence of noncomplementary DNA, that is, without the linking hybrid. The new method couples high sensitivity of silver-amplified assays with effective discrimination against excess of closely related nucleotide sequences (including single-base imperfections). Such direct electrical detection of DNA/metal-particle assemblies can bring new capabilities to the detection of DNA hybridization, and could be applied to other bioaffinity assays.
A sensing platform based on the fluorescence quenching ability of polydopamine nanospheres was developed for the assay of biomolecules.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.