RESUMO
A facile silica coating significantly enhances the thermal stability and polymerase chain reaction (PCR) compatibility of oligonucleotide-gold nanoparticle conjugates, thus enabling colorimetric detection of PCR results in a closed-tube format. This method is specific, sensitive, and generally applicable. Its simplicity, visual readout, and carryover contamination-free features hold promise for point-of-care or on-site DNA testing.
Assuntos
Colorimetria/métodos , DNA/análise , DNA/genética , Nanopartículas Metálicas , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Primers do DNA/química , Primers do DNA/genética , Ouro , Dióxido de SilícioAssuntos
Colorimetria/métodos , Nanopartículas Metálicas , Técnicas de Amplificação de Ácido Nucleico/métodos , Bacteriófago lambda/genética , Sequência de Bases , Primers do DNA/genética , DNA Viral/genética , Ácidos Graxos/química , Ouro , Nanopartículas Metálicas/química , Nanotecnologia , Compostos de Sulfidrila/química , Ressonância de Plasmônio de SuperfícieRESUMO
Gold nanoparticles have proven to be promising for decentralized nucleic acid testing by virtue of their simple visual readout and absorbance-based quantification. A major challenge toward their practical application is to achieve ultrasensitive detection without compromising simplicity. The conventional strategy of thermocycling amplification is unfavorable (because of both instrumentation and preparation of thermostable oligonucleotide-modified gold nanoparticle probes). Herein, on the basis of a previously unreported co-precipitation phenomenon between thiolated poly(ethylene glycol)/11-mercaptoundecanoic acid co-modified gold nanoparticles and magnesium pyrophosphate crystals (an isothermal DNA amplification reaction byproduct), a new ultrasensitive and simple DNA assay platform is developed. The binding mechanism underlying the co-precipitation phenomenon is found to be caused by the complexation of carboxyl and pyrophosphate with free magnesium ions. Remarkably, poly(ethylene glycol) does not hinder the binding and effectively stabilizes gold nanoparticles against magnesium ion-induced aggregation (without pyrophosphate). In fact, a similar phenomenon is observed in other poly(ethylene glycol)- and carboxyl-containing nanomaterials. When the gold nanoparticle probe is incorporated into a loop-mediated isothermal amplification reaction, it remains as a red dispersion for a negative sample (in the absence of a target DNA sequence) but appears as a red precipitate for a positive sample (in the presence of a target). This results in a first-of-its-kind gold nanoparticle-based DNA assay platform with isothermal amplification and real-time monitoring capabilities.