Formation of Silver Nanostructures by Rolling Circle Amplification Using Boranephosphonate-Modified Nucleotides.

We investigate the efficiency of incorporation of boranephosphonate-modified nucleotides by phi29 DNA polymerase and present a simple method for forming large defined silver nanostructures by rolling circle amplification (RCA) using boranephosphonate internucleotide linkages. RCA is a linear DNA amplification technique that can use specifically circularized DNA probes for detection of target nucleic acids and proteins. The resulting product is a collapsed single-stranded DNA molecule with tandem repeats of the DNA probe. By substituting each of the natural nucleotides with the corresponding 5'-(α-P-borano)deoxynucleosidetriphosphate, only a small reduction in amplification rate is observed. Also, by substituting all four natural nucleotides, it is possible to enzymatically synthesize a micrometer-sized, single-stranded DNA molecule with only boranephosphonate internucleotide linkages. Well-defined silver particles are then readily formed along the rolling circle product.

Gold nanowire based electrical DNA detection using rolling circle amplification.

We present an electrical sensor that uses rolling circle amplification (RCA) of DNA to stretch across the gap between two electrodes, interact with metal nanoparticle seeds to generate an electrically conductive nanowire, and produce electrical signals upon detection of specific target DNA sequences. RCA is a highly specific molecular detection mechanism based on DNA probe circularization. With this technique, long single-stranded DNA with simple repetitive sequences are produced. Here we show that stretched RCA products can be metalized using silver or gold solutions to form metal wires. Upon metallization, the resistance drops from TΩ to kΩ for silver and to Ω for gold. Metallization is seeded by gold nanoparticles aligned along the single-stranded DNA product through hybridization of functionalized oligonucleotides. We show that combining RCA with electrical DNA detection produces results in readout with very high signal-to-noise ratio, an essential feature for sensitive and specific detection assays. Finally, we demonstrate detection of 10 ng of Escherichia coli genomic DNA using the sensor concept.

Sensitive detection of bacterial DNA by magnetic nanoparticles.

This work presents sensitive detection of bacterial genomic DNA using a magnetic nanoparticle-based substrate-free method. For the first time, such a method is employed for detection of a clinically relevant analyte by implementing a solid-phase-based molecular probing and amplification protocol that can be executed in 80 min. The molecular detection and amplification protocol is presented and verified on samples containing purified genomic DNA from Escherichia coli cells, showing that as few as 50 bacteria can be detected. This study moves the use of volume-amplified magnetic nanoparticles one step further toward rapid, sensitive, and selective infectious diagnostics.

Real-space transmission electron microscopy investigations of attachment of functionalized magnetic nanoparticles to DNA-coils acting as a biosensor.

The present work provides the first real-space analysis of nanobead-DNA coil interactions. Immobilization of oligonucleotide-functionalized magnetic nanobeads in rolling circle amplified DNA-coils was studied by complex magnetization measurements and transmission electron microscopy (TEM), and a statistical analysis of the number of beads hybridized to the DNA-coils was performed. The average number of beads per DNA-coil using the results from both methods was found to be around 6 and slightly above 2 for samples with 40 and 130 nm beads, respectively. The TEM analysis supported an earlier hypothesis that 40 nm beads are preferably immobilized in the interior of DNA-coils whereas 130 nm beads, to a larger extent, are immobilized closer to the exterior of the coils. The methodology demonstrated in the present work should open up new possibilities for characterization of interactions of a large variety of functionalized nanoparticles with macromolecules, useful for gaining more fundamental understanding of such interactions as well as for optimizing a number of biosensor applications.

Investigation of immobilization of functionalized magnetic nanobeads in rolling circle amplified DNA coils.

Immobilization characteristics for single-stranded oligonucleotide-functionalized magnetic beads with nominal sizes of 40, 80, 130, and 250 nm in rolling circle amplified (RCA) DNA coils is investigated by employing complex magnetization measurements, dynamic light scattering and fluorescence microscopy. It was found that larger beads in a polydisperse bead size distribution more easily immobilize in the RCA DNA coils than do smaller beads. This may be related to a higher oligonucleotide surface coverage for the larger beads. Furthermore, it was concluded that both bead size and oligonucleotide surface coverage determine whether beads immobilize to give isolated coils with beads or larger clusters of beads and coils. A small bead size and a low oligonucleotide surface coverage favor the first kind of immobilization behavior, whereas a large bead size and a high oligonucleotide surface coverage favor the other. The present findings could be used to optimize both size and surface functionalization of beads employed in substrate-free magnetic biosensors.