Rationally Manipulating Aptamer Binding Affinities in a Stem-Loop Molecular Beacon

Single-stranded DNA sequences that are highly specific for a target ligand are called aptamers. While the incorporation of aptamer sequences into stem-loop molecular beacons has become an essential tool in optical biosensors, the design principles that determine the magnitude of binding affinity and its relationship to placement of the aptamer sequence in the stem-loop architecture are not well defined. By controlled placement of the aptamer along the loop region of the molecular beacon, it is observed that the binding affinity can be tuned over 4 orders of magnitude (1.3 nM – 203 μM) for the Huizenga and Szostak ATP DNA aptamer sequence. It is observed that the <i>K</i>_d is enhanced for the fully exposed sequence, with reduced binding affinity when the aptamer is part of the stem region of the beacon. Analysis of the Δ<i>G</i> values indicate a clear correlation between the aptamer hybridized length in the stem and its observed <i>K</i>_d. The use of a nanometal surface energy transfer probe method for monitoring ATP binding to the aptamer sequence allows the observation of negative cooperativity between the two ATP binding events. Maintenance of the high binding affinity of this ATP aptamer and the observation of two separate <i>K</i>_d’s for ATP binding indicate NSET as an effective, nonmanipulative, optical method for tracking biomolecular changes

Ligand Passivated Core–Shell FePt@Co Nanomagnets Exhibiting Enhanced Energy Product

Systematic growth of a soft-magnet Co shell (0.6 to 2.7 nm thick) around a hard-magnet Fe_0.65Pt_0.35 core (5 nm in diameter) has been achieved in a one-pot microwave synthesis. This controlled growth led to a 4-fold enhancement in the energy product of the core–shell assembly as compared to the energy product of bare Fe_0.65Pt_0.35 nanoparticles. The simultaneous enhancement of coercivity and saturation moment reflects the onset of theoretically predicted exchange-spring behavior. The demonstration of nanoscale exchange-spring magnets can lead to improved high-performance magnet design for energy applications

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Leaving Förster Resonance Energy Transfer Behind: Nanometal Surface Energy Transfer Predicts the Size-Enhanced Energy Coupling between a Metal Nanoparticle and an Emitting Dipole

The interaction of a fluorescent molecule with a gold nanoparticle is complex and can lead to excited-state enhancement or quenching. Many attempts have been made to explain the observed interaction when in close proximity to the metal surface; yet no single model has been capable of explaining the observations. In this work, we show that by accurately describing the interaction in terms of an induced image dipole modified within the gold nanoparticle by the size-dependent changes in absorptivity and dielectric constant, the oscillator interaction can be fully described in terms of a surface-moderated interaction. Comparison of experimental and theoretical data confirms the validity of the model for a selected range of separation distances, nanoparticle radii, and fluorescent molecule selection. The results of the study illustrate the importance of nonradiative pathways for modifying the decay of a fluorescent molecule by coupling to the image dipole, thus providing a firm understanding of the reported variance in behavior for an emitting species in close proximity to nanometal surfaces. A more significant impact of the results is the ability to apply nanometal surface energy transfer methods as a molecular ruler to probe physical questions at much greater distances (>400 Å) than previously achievable

Synthesis of Highly Uniform Nickel Multipods with Tunable Aspect Ratio by Microwave Power Control

As the importance of anisotropic nanostructures and the role of surfaces continues to rise in applications including catalysis, magneto-optics, and electromagnetic interference shielding, there is a need for efficient and economical synthesis routes for such nanostructures. The article describes the application of cycled microwave power for the rapid synthesis of highly branched pure-phase face-centered cubic crystalline nickel multipod nanostructures with >99% multipod population. By controlling the power delivery to the reaction mixture through cycling, superior control is achieved over the growth kinetics of the metallic nanostructures, allowing formation of multipods consisting of arms with different aspect ratios. The multipod structures are formed under ambient conditions in a simple reaction system composed of nickel acetylacetonate (Ni­(acac)₂), oleylamine (OAm), and oleic acid (OAc) in a matter of minutes by selective heating at the (111) overgrowth corners on Ni nanoseeds. The selective heating at the corners leads to accelerated autocatalytic growth along the ⟨111⟩ direction through a “lightning rod” effect. The length is proprtional to the length and number of microwave (MW)-on cycles, whereas the core size is controlled by continuous MW power delivery. The roles of heating mode (cycling <i>versus</i> variable power <i>versus</i> convective heating) during synthesis of the materials is explored, allowing a mechanism into how cycled microwave energy may allow fast multipod evolution to be proposed

Bimodal Gold Nanoparticle Therapeutics for Manipulating Exogenous and Endogenous Protein Levels in Mammalian Cells

A new advance in cell transfection protocol using a bimodal nanoparticle agent to selectively manipulate protein expression levels within mammalian cells is demonstrated. The nanoparticle based transfection approach functions by controlled release of gene regulatory elements from a 6 nm AuNP (gold nanoparticle) surface. The endosomal release of the regulatory elements from the nanoparticle surface results in endogenous protein knockdown simultaneously with exogenous protein expression for the first 48 h. The use of fluorescent proteins as the endogenous and exogenous signals for protein expression enables the efficiency of codelivery of siRNA (small interfering RNA) for GFP (green fluorescent protein) knockdown and a dsRed-express linearized plasmid for induction to be optically analyzed in CRL-2794, a human kidney cell line expressing an unstable green fluorescent protein. Delivery of the bimodal nanoparticle in cationic liposomes results in 20% GFP knockdown within 24 h of delivery and continues exhibiting knockdown for up to 48 h for the bimodal agent. Simultaneous dsRed expression is observed to initiate within the same time frame with expression levels reaching 34% after 25 days although cells have divided approximately 20 times, implying daughter cell transfection has occurred. Fluorescence cell sorting results in a stable colony, as demonstrated by Western blot analysis. The simultaneous delivery of siRNA and linearized plasmid DNA on the surface of a single nanocrystal provides a unique method for definitive genetic control within a single cell and leads to a very efficient cell transfection protocol

Influence of Microwave Frequency and Power on Nanometal Growth

The rapid heating rates (Δ<i>T</i>/δ<i>t</i>) achieved in a microwave (MW) reactor has been shown to accelerate reaction rates due to the direct power absorbed (<i>P</i>_abs) into the reactants leading to faster kinetics. The <i>P</i>_abs is proportional to the dielectric cross section of the materials as defined by the real (ε′) and imaginary (ε″) components. In a nanocrystal, the dielectric cross-section will be frequency dependent as well as size dependent. In this work, the frequency dependent growth of nickel nanocrystals at frequencies of 2.45, 15.50, and 18.00 GHz at constant Δ<i>T</i>/δ<i>t</i> was studied to evaluate the frequency dependence on MW growth of Ni. A scaling law behavior for growth rates is observed that is shown to depend on the MW electric field strength. A relationship is derived between the “configurational energy” of the precursor molecules and the final nanoparticle size. The study provides a clear description of a microwave effect that is dependent on the frequency and power of the microwave and offers further insight into the physical chemistry of microwave applications to nanomaterial synthesis

Eu³⁺-Doped ZnB₂O₄ (B = Al³⁺, Ga³⁺) Nanospinels: An Efficient Red Phosphor

This paper describes the synthesis of Eu­(III)-doped ZnB₂O₄ (B = Al­(III) or Ga­(III)) nanospinels with Eu­(III) concentrations varying between 1% and 15.6%. The synthesis was achieved through a microwave (MW) synthetic methodology producing 3 nm particles by the thermal decomposition of zinc undecylenate (UND) and a metal 2,4-pentanedionate (B­(acac)₃, B = Al³⁺ or Ga³⁺) in oleylamine (OAm). The nanospinels were then ligand exchanged with the β-diketonate, 2-thenoyltrifluoroacetone (tta). Using tta as a ligand on the surface of the particles resulted in soluble materials that could be embedded in lens mimics, such as poly­(methyl methacrylate) (PMMA). Through a Dexter energy transfer mechanism, tta efficiently sensitized the Eu­(III) doped within the nanospinels, resulting in red phosphors with intrinsic quantum efficiencies (QEs) and QEs in PMMA as high as 50% when excited in the UV. Optical measurements on the out of batch and tta-passivated nanospinels were done to obtain absorption, emission, and lifetime data. The structural properties of the nanospinels were evaluated by ICP-MS, pXRD, TEM, FT-IR, EXAFS, and XANES

Plasmid Transfection in Mammalian Cells Spatiotemporally Tracked by a Gold Nanoparticle

Recent advances in cell transfection have suggested that delivery of a gene on a gold nanoparticle (AuNP) can enhance transfection efficiency. The mechanism of transfection is poorly understood, particularly when the gene is appended to a AuNP, as expression of the desired exogenous protein is dependent not only on the efficiency of the gene being taken into the cell but also on efficient endosomal escape and cellular processing of the nucleic acid. Design of a multicolor surface energy transfer (McSET) molecular beacon by independently dye labeling a linearized plasmid and short duplex DNA (sdDNA) appended to a AuNP allows spatiotemporal profiling of the transfection events, providing insight into package uptake, disassembly, and final plasmid expression. Delivery of the AuNP construct encapsulated in Lipofectamine2000 is monitored in Chinese hamster ovary cells using live-cell confocal microscopy. The McSET beacon signals the location and timing of the AuNP release and endosomal escape events for the plasmid and the sdDNA discretely, which are correlated with plasmid transcription by fluorescent protein expression within the cell. It is observed that delivery of the construct leads to endosomal release of the plasmid and sdDNA from the AuNP surface at different rates, prior to endosomal escape. Slow cytosolic diffusion of the nucleic acids is believed to be the limiting step for transfection, impacting the time-dependent expression of protein. The overall protein expression yield is enhanced when delivered on a AuNP, possibly due to better endosomal escape or lower degradation prior to endosomal escape

Triangulating Nucleic Acid Conformations Using Multicolor Surface Energy Transfer

Optical ruler methods employing multiple fluorescent labels offer great potential for correlating distances among several sites, but are generally limited to interlabel distances under 10 nm and suffer from complications due to spectral overlap. Here we demonstrate a multicolor surface energy transfer (McSET) technique able to triangulate multiple points on a biopolymer, allowing for analysis of global structure in complex biomolecules. McSET couples the competitive energy transfer pathways of Förster Resonance Energy Transfer (FRET) with gold-nanoparticle mediated Surface Energy Transfer (SET) in order to correlate systematically labeled points on the structure at distances greater than 10 nm and with reduced spectral overlap. To demonstrate the McSET method, the structures of a linear B-DNA and a more complex folded RNA ribozyme were analyzed within the McSET mathematical framework. The improved multicolor optical ruler method takes advantage of the broad spectral range and distances achievable when using a gold nanoparticle as the lowest energy acceptor. The ability to report distance information simultaneously across multiple length scales, short-range (10–50 Å), mid-range (50–150 Å), and long-range (150–350 Å), distinguishes this approach from other multicolor energy transfer methods