We demonstrate that novel oligonucleotide-modified gold nanoparticle probes hybridized to fluorophore-labeled complements can be used as both transfection agents and cellular "nanoflares" for detecting mRNA in living cells. Nano-flares take advantage of the highly efficient fluorescence quenching properties of gold, cellular uptake of oligonucleotide nanoparticle conjugates without the use of transfection agents, and the enzymatic stability of such conjugates, thus overcoming many of the challenges to creating sensitive and effective intracellular probes. Nano-flares exhibit high signaling, have low background fluorescence, and are sensitive to changes in the number of RNA transcripts present in cells.Probes to visualize and detect intracellular RNA including those used for in situ staining, 1 molecular beacons, 2 and fluorescent resonance energy transfer (FRET) pairs 3 are important tools to measure and quantify activity in living systems in response to external stimuli. 4 However, these probes are often difficult to transfect, require additional agents for cellular internalization, and can be unstable in cellular environments. These factors can lead to a high background signal and the inability to detect targets. Here we show how novel oligonucleotide-modified gold nanoparticle probes hybridized to fluorophore complements can be used as both transfection agents and cellular "nano-flares" for visualizing and quantifying RNA in living cells. Nano-flares take advantage of the highly efficient fluorescence quenching properties of gold, 5 cellular uptake of oligonucleotide nanoparticle conjugates without the use of transfection agents, and the enzymatic stability of such conjugates, 6 thus overcoming many of the challenges to creating sensitive and effective intracellular probes. Specifically, nano-flares exhibit high signaling, have low background fluorescence, and are sensitive to changes in the number of RNA transcripts present in cells. The discovery and subsequent development of the oligonucleotide-nanoparticle conjugate have led to a variety of new opportunities in molecular diagnostics 11 and materials design. 12 Recently, it has been demonstrated that oligonucleotide-functionalized nanoparticles enter cells and can act as antisense agents to control gene expression. 6 These "antisense particles" are not only delivery vehicles, 13 but also single entity regulation and transfection agents that undergo facile cellular internalization, resist enzymatic degradation, and bind intracellular targets with affinity constants that are as much as two orders of magnitude greater than free oligonucleotides. 14 Moreover, they can be easily modified with potent designer materials such as locked nucleic acids 15 and are nontoxic under conditions required for gene regulation.The nano-flares described herein are oligonucleotide functionalized nanoparticle conjugates designed to provide an intracellular fluorescence signal that correlates with the relative amount of a specific intracellular RNA. By utilizing nanopart...

We show that by correlating the radius of curvature of spherical gold nanoparticles of varying sizes with their respective thiol terminated oligonucleotide loading densities that a mathematical relationship can be derived for predicting the loading of oligonucleotides on anisotropic gold nanomaterials. This mathematical relationship was tested with gold nanorods (radius 17.5 nm, length 475 nm) where the measured number of oligonucleotides per rod (3330 ±110) was within experimental error of the predicted loading of 3244 oligonucleotides from the derivation. Additionally, we show that once gold nanoparticles reach a diameter of approximately 60 nm (and certainly 100 nm) the local surface experienced by the oligonucleotide is highly similar to that of a planar surface.

The recently developed bio-barcode assay for the detection of nucleic acid and protein targets without PCR has been shown to be extraordinarily sensitive, showing high sensitivity for both nucleic acid and protein targets. Two types of particles are used in the assay: (i) a magnetic microparticle with recognition elements for the target of interest; and (ii) a gold nanoparticle (Au-NP) with a second recognition agent (which can form a sandwich around the target in conjunction with the magnetic particle) and hundreds of thiolated single-strand oligonucleotide barcodes. After reaction with the analyte, a magnetic field is used to localize and collect the sandwich structures, and a DTT solution at elevated temperature is used to release the barcode strands. The barcode strands can be identified on a microarray via scanometric detection or in situ if the barcodes carry with them a detectable marker. The recent modification to the original bio-barcode assay method, utilizing DTT, has streamlined and simplified probe preparation and greatly enhanced the quantitative capabilities of the assay. Here we report the detailed methods for performing the ligand exchange bio-barcode assay for both nucleic acid and protein detection. In total, reagent synthesis, probe preparation and detection require 4 d.

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DNA-functionalized gold nanoparticles can be used to induce the formation and control the unit cell parameters of highly ordered face-centered cubic crystal lattices. Nanoparticle spacing increases linearly with longer DNA interconnect length, yielding maximum unit cell parameters of 77 nm and 0.52% inorganic-filled space for the DNA constructs studied. In general, we show that longer DNA connections result in a decrease in the overall crystallinity and order of the lattice due to greater conformational flexibility.