Oligonucleotide-Functionalized Gold Nanoparticles as Probes in a Dry-Reagent Strip Biosensor for DNA Analysis by Hybridization

Abstract

The highly specific molecular recognition properties of oligonucleotides are combined with the unique optical properties of gold nanoparticles for the development of a dry-reagent strip-type biosensor that enables visual detection of double stranded DNA within minutes. The assay does not require instrumentation and avoids the multiple incubation and washing steps performed in most current assays. Gold nanoparticle reporters with oligo(dT) attached to their surface form an integral part of the strip. Biotinylated PCR products (233 bp or 495 bp) are hybridized (5 min) with a poly(dA)-tailed oligo and applied on the strip, which is then immersed in the appropriate buffer. As the buffer migrates upward, it rehydrates the nanoparticles that are linked to the target DNA through poly(dA)/(dT) hybridization. Capture of the hybrids by immobilized streptavidin in the test zone of the strip generates a characteristic red band. A second red band is formed, by hybridization, in the control zone of the strip to indicate proper test performance. The sensor offers at least 8 times higher detectability than ethidium bromide staining of agarose gels and provides confirmation of the amplified fragments. Quantitative data are obtained by densitometric analysis of the bands. As low as 2 fmol of amplified DNA were detectable by the strip sensor. Also, 500 copies of prostate-specific antigen cDNA were detected by combining PCR and the strip sensor. The sensor was used successfully for detection of hepatitis C virus in plasma samples from 20 patients. The strip detected 16 out of 16 positive samples and gave no signal for 4 samples that were negative for the virus. To our knowledge, this is the first dry-reagent system that makes use of oligonucleotide-conjugated gold nanoparticles as probes.

Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications

“…[254] Sensitive naked-eye detection of immunoglubulin G by the enlargement of Au NPs has been reported. [255] Au NPs were also used as spectroscopic enhancers for in vitro studies on single viruses.…”

Quantitative Immunochromatographic Strip Biosensor for the Detection of Carcinoembryonic Antigen Tumor Biomarker in Human Plasma

“…The principle of IST relies on the migration of test samples and antibody-gold nanoparticle (Ab-Au-NP) conjugates along membrane strips on which the binding interactions take place. The results of IST could be examined visually, thus providing fast and simple qualititative detection without the need of skilled personnel [24][25][26][27][28]. However the ISTs are used for qualititative or semi-quantitative detection, and fail to offer the quantitative information of the interested target in the samples.…”

Dual-allele dipstick assay for genotyping single nucleotide polymorphisms by primer extension reaction

“…20 Streptavidin, anti-Dig antibody and poly(dA) oligonucleotide were immobilized at the test zone 2, the test zone 1 and the control zone of the nitrocellulose membrane, respectively. Streptavidin diluted in water containing 2% sucrose was loaded at a density of 1.6 mg per 4 mm (width) strip.…”

“…The strips were stored dry at room temperature. Poly(dT)-functionalized gold nanoparticles were prepared as described previously, 20 and loaded on the conjugate pad at a density of 6.25 fmol per 4 mm width.…”