Coupling Molecular Beacons to Barcoded Metal Nanowires for Multiplexed, Sealed Chamber DNA Bioassays

Abstract

We have combined molecular beacon (MB) probes with barcoded metal nanowires to enable nowash, sealed chamber, multiplexed detection of nucleic acids. Probe design and experimental parameters important in nanowire-based MB assays are discussed. Loop regions of 24 bases and 5 base pair stem regions in the beacon probes gave optimal performance. Our results suggest that thermodynamic predictions for secondary structure stability of solution-phase MB can guide probe design for nanowire-based assays. Dengue virus-specific probes with predicted solution-phase ΔG of folding in 500 mM buffered NaCl of approximately −4 kcal/mol performed better than those with ΔG > −2 or < −6 kcal/mol. Buffered 300-500 mM NaCl was selected after comparison of several buffers previously reported for similar types of assays, and 200-500 mM NaCl was found to be the optimal ionic strength for the hybridization temperatures (25 and 50 °C) and probe designs used here. Target binding to the surface as a function of solution concentration fit a Sips isotherm with K d = 1.7 ± 0.3 nM. The detection limit was ∼100 pM, limited by incomplete quenching. Single base mismatches could be discriminated from fully complementary targets. Oligonucleotide target sequences specific for human immunodeficiency, hepatitis C, and severe acute respiratory viruses were assayed simultaneously in a no-wash, sealed chamber, multiplexed experiment in which each of three probe sequences was attached to a different pattern of encoded nanowires. Finally, we demonstrated that probe-coated nanowires retain their selectivity and sensitivity in a triplexed assay after storage for over 3 months.

Self‐Assembled Monolayer Mediated Silica Coating of Silver Triangular Nanoprisms

“…[3][4][5] Gold and silver nanostructures are of particular interest due to their unusual optical properties that are dependent on size and shape. [6][7][8] Triangular silver nanoprisms, in particular, exhibit highly tunable architecture-dependent optical properties.…”

Cationic Polyelectrolyte Amplified Bead Array for DNA Detection with Zeptomole Sensitivity and Single Nucleotide Polymorphism Selectivity

“…The shape of curve is similar to previous reports on microarray based detection. [41][42][43][44] This yields a detection limit of 1 × 10 − 17 M , calculated based on 3 × the standard deviation of background. Since 300 μ L of target has been used for hybridization, the sensitivity for Cy5-ssDNA c detection is thus at the zeptomole level.…”

Visual Scanometric Detection of DNA through Silver Enhancement Regulated by Gold‐Nanoparticle Aggregation with a Molecular Beacon as the Trigger

“…Recently, MBs have received considerable interest because of the inherent signaling mechanism by energy transfer and the high selectivity in the detection of DNA sequences and proteins. [22][23][24][25][26][27] Different from regular MBs that contain stems based on the Watson-Crick system, MBs based on a non-Watson-Crick system show greater selectivity, control, and resistance toward nuclease digestion than Abstract: A convenient and label-free scanometric approach for DNA assay was designed by integrating a metalion-mediated conformational molecular beacon (MB) and silver-signal amplification regulated by gold-nanoparticle (AuNP) aggregation. The strategy was based on displacing the interaction between the target DNA sequence and a competitor Hg 2 + ion with a link DNA sequence.…”