Gold Nanoparticles with Asymmetric Polymerase Chain Reaction for Colorimetric Detection of DNA Sequence

Abstract

We developed a novel strategy for rapid colorimetric analysis of a specific DNA sequence by combining gold nanoparticles (AuNPs) with an asymmetric polymerase chain reaction (As-PCR). In the presence of the correct DNA template, the bound oligonucleotides on the surface of AuNPs selectively hybridized to form complementary sequences of single-stranded DNA (ssDNA) target generated from As-PCR. DNA hybridization resulted in self-assembly and aggregation of AuNPs, and a concomitant color change from ruby red to blue-purple occurred. This approach is simpler than previous methods, as it requires a simple mixture of the asymmetric PCR product with gold colloid conjugates. Thus, it is a convenient colorimetric method for specific nucleic acid sequence analysis with high specificity and sensitivity. Most importantly, the marked color change occurs at a picogram detection level after standing for several minutes at room temperature. Linear amplification minimizes the potential risk of PCR product cross-contamination. The efficiency to detect Bacillus anthracis in clinical samples clearly indicates the practical applicability of this approach.

Magnetic barcode assay for genetic detection of pathogens

“…For the ssDNA samples, highest signal could be obtained when the target ssDNA was first captured by beads and then labeled with MNPs; this approach presumably minimized competitive binding to ssDNA between capture beads and MNPs. 21 Based on these results, we adopted the asymmetric PCR and sequential labeling strategy for target DNA production and its magnetic targeting, respectively.…”

Surface plasmon resonance biosensor for enzyme-free amplified microRNA detection based on gold nanoparticles and DNA supersandwich

“…Since gold nanoparticles (AuNPs) (~13 nm) with good monodispersity spherical shape, and narrow size distribution are easy to prepare and modify [30,31], AuNPs (~13 nm) were used here. AuNPs were prepared by the citrate reduction of HAuCl 4 following a literature procedure [32].…”

Simplified detection of the asymmetric polymerase chain reaction-amplified DNA and its application in the target identification

“…The asymmetric PCR (aPCR) that relies on the differing primer concentration [10] or the dissimilar Tm of the primers [11] has been used to produce the targetspecific single-stranded DNA (ssDNA) along with the double-stranded target DNA (dsDNA). The products of the aPCR can be detected by the methods such as the probe hybridization and fluorescence polarization [12], solid phase assay [13], or gold nanoparticle-based colorimetric assay [14,15].…”