Effective delivery of anti-miRNA DNA oligonucleotides by functionalized gold nanoparticles

MicroRNAs (miRNAs) are gaining recognition as essential regulators involved in many biological processes, and they are emerging as therapeutic targets for treating disease. Here, we introduce a method for effective delivery of anti-miRNA oligonucleotides (AMOs) using functionalized gold nanoparticles (AuNPs). To demonstrate the ability of AMOs to silence miRNA, we selected miR-29b, which is known to downregulate myeloid cell leukemia-1 (MCL-1), a factor responsible for promoting cell survival. We first generated AuNPs coated with cargo DNA, which was then coupled to complementary DNA linked to an antisense miR-29b sequence. When the AuNPs were delivered into HeLa cells, MCL-1 protein and mRNA levels were increased significantly. Furthermore, apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was inhibited, proving that AMOs targeting miR-29b were effectively delivered by our innovative AuNP. In addition, we provided evidence that AuNP could deliver other AMOs against miR-21 into two independent cell lines, KGN and 293T, suggesting that the AuNP conjugates can be versatile for any AMO and cell type.     

Femtomolar DNA detection by parallel colorimetric darkfield microscopy of functionalized gold nanoparticles

We introduce a sensing platform for specific detection of DNA based on the formation of gold nanoparticles dimers on a surface. The specific coupling of a second gold nanoparticle to a surface bound nanoparticle by DNA hybridization results in a red shift of the nanoparticle plasmon peak. This shift can be detected as a color change in the darkfield image of the gold nanoparticles. Parallel detection of hundreds of gold nanoparticles with a calibrated true color camera enabled us to detect specific binding of target DNA. This enables a limit of detection below 1.0×10(-14) M without the need for a spectrometer or a scanning stage.     

Streptavidin conjugates with gold nanoparticles for visualization of single DNA interactions on the silicon surface

Applicability of scanning electron microscopy (SEM) for visualization of individual acts of DNA hybridization with oligonucleotide probes has been investigated using gold nanoparticles as a label. DNA or oligonucleotides were labeled with biotin molecules, which were then detected in DNA duplexes using a streptavidin conjugate with gold nanoparticles. Effective imaging of DNA duplexes was possible using the conjugate prepared by covalent binding. The detection limit of the model oligonucleotide of 19 bases was 20 pg.     

Toxicity of gold nanoparticles functionalized with cationic and anionic side chains

The structure and properties of gold nanoparticles make them useful for a wide array of biological application. Toxicity, however, has been observed at high concentrations using these systems. MTT, hemolysis, and bacterial viability assays were used to explore differential toxicity among the cell types used, using 2 nm core particles. These studies show that cationic particles are moderately toxic, whereas anionic particles are quite nontoxic. Concentration-dependent lysis mediated by initial electrostatic binding was observed in dye release studies using lipid vesicles, providing the probable mechanism for observed toxicity with the cationic MMPCs.     

Gold DNA-conjugates: ion specific self-assembly of gold nanoparticles via the dG-quartet

The Oxytricha telomere DNA hairpin 5'-d(G4T4G4) immobilized on 13 nm gold nanoparticles forms a supramolecular assembly via dGC-quartets, as determined by the color change and by SEM. The aggregation is ion-dependent and selective for sodium ions. K+ is less efficient while Li+ and Cs+ do not drive the aggregation. This work is the first effort exploring the use of secondary structures of DNA (quadruplexes) for producing self-assemblies of gold nanoparticles.     

Human anti-gold antibodies: biofunctionalization of gold nanoparticles and surfaces with anti-gold antibodies

The interface molecules designed to exhibit molecular recognitions between different species have become attractive tools for the bottom-up fabrication and hybridization of nanostructured units. Here, we focus on antibodies with high binding ability and specificity to construct a novel biomolecule interface for recognizing an inorganic material. Careful selection from a phage-displayed library of variable region heavy and light Fv chains of human antibodies using enzyme-linked immunosorbent assay and surface plasmon resonance assay resulted in the identification of an antibody fragment, A14P-b2, with high affinity (KD = 1.7 nm) and specificity for gold materials. Our results indicated the potential usefulness of human antibody libraries and the effectiveness of the antibody framework for recognizing bulk material surfaces. Construction of bivalent and bispecific antibodies on the A14P-b2 platform with high affinity by means of fusion technology enabled the functionalization of gold nanoparticles and allowed selective protein accumulation on gold spots patterned on a silicon substrate. This type of antibody engineering is potentially applicable to bio-inspired materials and nanobiosensing.     

Functionalization of gold and glass surfaces with magnetic nanoparticles using biomolecular interactions

Advances in nanotechnology have enabled the production and characterization of magnetic particles with nanometer-sized features that can be functionalized with biological recognition elements for numerous applications in biotechnology. In the present study, the synthesis of and interactions between self-assembled monolayers (SAMs) on gold and glass surfaces and functionalized magnetic nanoparticles have been characterized. Immobilization of 10-15 nm streptavidin-functionalized nanoparticles to biotinylated gold and glass surfaces was achieved by the strong interactions between biotin and streptavidin. Fluorescent streptavidin-functionalized nanoparticles, biotinylated surfaces, and combinations of the two were characterized by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and electron and fluorescent microscopy to confirm that little or no functionalization occurred in nonbiotinylated regions of the gold and glass surfaces compared to the biotinylated sites. Together these techniques have potential use in studying the modification and behavior of functionalized nanoparticles on surfaces in biosensing and other applications.     

Radiosensitization of DNA by gold nanoparticles irradiated with high-energy electrons

Zheng, Y., Hunting, D. J., Ayotte, P. and Sanche, L. Radiosensitization of DNA by Gold Nanoparticles Irradiated with High-Energy Electrons. Radiat. Res. 168, 19-27 (2008). Thin films of pGEM-3Zf(-) plasmid DNA were bombarded by 60 keV electrons with and without gold nanoparticles. DNA single- and double-strand breaks (SSBs and DSBs) were measured by agarose gel electrophoresis. From transmission electron micrographs, the gold nanoparticles were found to be closely linked to DNA scaffolds, probably as a result of electrostatic binding. The probabilities for formation of SSBs and DSBs from exposure of 1:1 and 2:1 gold nanoparticle:plasmid mixtures to fast electrons increase by a factor of about 2.5 compared to neat DNA samples. For monolayer DNA adsorbed on a thick gold substrate, the damage increases by an order of magnitude. The results suggest that the enhancement of radiosensitivity is due to the production of additional low-energy secondary electrons caused by the increased absorption of ionizing radiation energy by the metal, in the form of gold nanoparticles or of a thick gold substrate. Since short-range low-energy secondary electrons are produced in large amounts by any type of ionizing radiation, and since on average only one gold nanoparticle per DNA molecule is needed to increase damage considerably, targeting the DNA of cancer cells with gold nanoparticles may offer a novel approach that is generally applicable to radiotherapy treatments.     

Oligonucleotide and polymer functionalized nanoparticles for amplification-free detection of DNA

Sensitive and quantitative nucleic acid testing from complex biological samples is now an important component of clinical diagnostics. Whereas nucleic acid amplification represents the gold standard, its utility in resource-limited and point-of-care settings can be problematic due to assay interferants, assay time, engineering constraints, and costs associated with both wetware and hardware. In contrast, amplification-free nucleic acid testing can circumvent these limitations by enabling direct target hybridization within complex sample matrices. In this work, we grew random copolymer brushes from the surface of silica-coated magnetic nanoparticles using azide-modified and hydroxyl oligo ethylene glycol methacrylate (OEGMA) monomers. The azide-functionalized polymer brush was first conjugated, via copper-catalyzed azide/alkyne cycloaddition (CuAAC), with herpes simplex virus (HSV)-specific oligonucleotides and then with alkyne-substituted polyethylene glycol to eliminate all residual azide groups. Our methodology enabled control over brush thickness and probe density and enabled multiple consecutive coupling reactions on the particle grafted brush. Brush- and probe-modified particles were then combined in a 20 min hybridization with fluorescent polystyrene nanoparticles modified with HSV-specific reporter probes. Following magnetic capture and washing, the particles were analyzed with an aggregate fluorescence measurement, which yielded a limit of detection of 6 pM in buffer and 60 pM in 50% fetal bovine serum. Adoption of brush- and probe-modified particles into a particle counting assay will result in the development of diagnostic assays with significant improvements in sensitivity.     

Time-resolved spectral observations of cadmium-enriched cadmium sulfide nanoparticles and the effects of DNA oligomer binding

We measured the steady-state and time-resolved fluorescence spectral properties of cadmium-enriched nanoparticles (CdS-Cd2+). These particles displayed two emission maxima, at 460 and 580 nm. The emission spectra were independent of excitation wavelength. Surprisingly, the intensity decays were strongly dependent on the observation wavelength, with longer decay times being observed at longer wavelengths. The mean lifetime increased from 150 to 370 ns as the emission wavelength was increased from 460 to 650 nm. The wavelength-dependent lifetimes were used to construct the time-resolved emission spectra, which showed a growth of the long-wavelength emission at longer times, and decay-associated spectra, which showed the longer wavelength emission associated with the longer decay time. These nanoparticles displayed anisotropy values as high as 0.35, depending on the excitation and emission wavelengths. Such high anisotropies are unexpected for presumably spherical nanoparticles. The anisotropy decayed with two correlation times near 5 and 370 ns, with the larger value probably due to overall rotational diffusion of the nanoparticles. Addition of a 32-base pair oligomer selectively quenched the 460-nm emission, with less quenching being observed at longer wavelengths. The time-resolved intensity decays were minimally affected by the DNA, suggesting a static quenching mechanism. The wavelength-selected quenching shown by the nanoparticles may make them useful for DNA analysis.     

Electrochemical impedance immunosensor based on gold nanoparticles and aryl diazonium salt functionalized gold electrodes for the detection of antibody

Electrodes modified with passivating organic layers have been shown to, here and previously, to exhibit good Faradaic electrochemistry upon attachment of gold nanoparticles (AuNP). Due to their low background capacitances these constructs have good potential in electrochemical sensing. Herein is reported the application of these electrode constructs for impedance based immunosensing. The immunosensor was constructed by modifying a gold electrode with 4-thiophenol (4-TP) passivating layers by diazonium salt chemistry. Subsequently, the attachment of AuNP and then a biotin derivative as a model epitope to detect anti-biotin IgG were carried out. The interfacial properties of the modified electrodes were evaluated in the presence of Fe(CN)(6)(4-/3-) redox couple as a probe by cyclic voltammetry and electrochemical impedance spectroscopy. The impedance change, due to the specific immuno-interaction at the immunosensor surface was utilized to detect anti-biotin IgG. The increase in charge-transfer resistance (R(ct)) was linearly proportional to the concentration of anti-biotin IgG in the range of 5-500 ng mL(-1), with a detection limit of 5 ng mL(-1).     

Kinetics and thermodynamics of DNA hybridization on gold nanoparticles

Hybridization of single-stranded DNA immobilized on the surface of gold nanoparticles (GNPs) into double stranded DNA and its subsequent dissociation into ssDNA were investigated. Melting curves and rates of dissociation and hybridization were measured using fluorescence detection based on hybridization-induced fluorescence change. Two distribution functions, namely the state distribution and the rate distribution, were proposed in order to take interfacial heterogeneity into account and to quantitatively analyze the data. Reaction and activation enthalpies and entropies of DNA hybridization and dissociation on GNPs were derived and compared with the same quantities in solution. Our results show that the interaction between GNPs and DNA reduces the energetic barrier and accelerates the dissociation of adhered DNA. At low surface densities of ssDNA adhered to GNP surface, the primary reaction pathway is that ssDNA in solution first adsorbs onto the GNP, and then diffuses along the surface until hybridizing with an immobilized DNA. We also found that the secondary structure of a DNA hairpin inhibits the interaction between GNPs and DNA and enhances the stability of the DNA hairpin adhered to GNPs.     

Tetrahedron DNA dendrimers and their encapsulation of gold nanoparticles

DNA dendrimers have achieved increasing attention recently. Previously reported DNA dendrimers used Y-DNA as monomers. Tetrahedron DNA is a rigid tetrahedral cage made of DNA. Herein, we use tetrahedron DNA as monomers to prepare tetrahedron DNA dendrimers. The prepared tetrahedron DNA dendrimers have larger size compared with those made of Y-DNA. In addition, thanks to the central cavity of tetrahedron DNA monomers, some nanoscale structures (e.g., gold nanoparticles) can be encapsulated within tetrahedron DNA monomers. Tetrahedron DNA encapsulated with gold nanoparticles can be further assembled into dendrimers, guiding gold nanoparticles into clusters.     

The enhancement effect of gold nanoparticles as a surface modifier on DNA sensor sensitivity

The enhancement of a single strain DNA probe linked to the sensor surface is of crucial importance in DNA molecule recognition. By means of nanogold modification of the sensor surface in addition to the nanogold amplifier, DNA detection sensitivity higher than 10(-16)mol/L was obtained in a Quartz Crystal microbalance (QCM) system, much higher than the ordinary QCM sensor without surface modification by nanogold.     

DNA and RNA oligomer thermodynamics: The effect of mismatched bases on double-helix stability

The thermodynamic parameters for the double-strand formation of the molecules rCAmG + rCU_nG, m,n = 5–7, and dCA_mG + dCT_nG, m,n = 5,6, were measured from optical melting curves. Normal helices are formed when m = n. The deoxyoligomers are more stable than the ribo-oligomers, due to a more favorable enthalpy. Double helices with mismatched bases can be formed by mixing oligomers with m ≠ n. Such helices may form several possible structures. A structure with a dangling base is favored over a structure with a bulged base. The destabilization of the double strands by the formation of a bulged base was determined to be greater than 1.6 kcal/mol at 10°C. The extent of aggregation in the oligomer double strand rCA₇G·rCU₇G was determined using ultracentrifugation equilibrium. The possible effects of aggregation on the determination of the thermodynamic parameters for double-strand formation are discussed.     

Fluorescence life‐time imaging and steady state polarization for examining binding of fluorophores to gold nanoparticles

Nanocomposites as multifunctional agents are capable of combing imaging and cell biology technologies. The conventional methods used for validation of the conjugation process of nanoparticles (NPs) to fluorescent molecules such as spectroscopy analysis and surface potential measurements, are not sufficient. In this paper we present a new and highly sensitive procedure that uses the combination of (1) fluorescence spectrum, (2) fluorescence lifetime, and (3) steady state fluorescence polarization measurements. We characterize and analyze gold NPs with Lucifer yellow (LY) surface coating as a model. We demonstrate the ability to differentiate between LY‐GNP (the conjugated complex) and a mixture of coated NP and free dyes. We suggest the approach for neuroscience applications where LY is used for detecting and labeling cells, studying morphology and intracellular communications.

Histograms of Fluorescence lifetime imaging (FLIM) of free LY dye (Left) in comparison to the conjugated dye to gold nanoparticles, LY‐GNP (Middle) enable the differentiation between LY‐GNP (the conjugated complex) and a mixture of coated NP and free dyes (Right).     

Functional gold nanoparticles for studying the interaction of lectin with glycosyl complex on living cellular surfaces

In this study, lectin-conjugated gold nanoparticles (GNPs) were prepared by standard biotin-streptavidin chemistry. The lectin-conjugated GNPs can be used as an indicator for studying the interaction of lectin with glycosyl complex on living cellular surfaces due to the high affinity of the lectin with saccharides. The interactions of two well-known lectins (Ricinus communis agglutinin and concanavalin A) and three different cell lines (HeLa, 293, and 293T) were selected here to establish this assay. Highly binding affinity of R. communis agglutinin with cells was demonstrated by conventional microscopic and UV-visible spectroscopic studies. In addition, the binding process can be inhibited by galactose, giving further proof of the binding mechanism.     

Radioprotective effects produced by the condensation of plasmid DNA with avidin and biotinylated gold nanoparticles

The treatment of aqueous solutions of plasmid DNA with the protein avidin results in significant changes in physical, chemical, and biochemical properties. These effects include increased light scattering, formation of micron-sized particles containing both DNA and protein, and plasmid protection against thermal denaturation, radical attack, and nuclease digestion. All of these changes are consistent with condensation of the plasmid by avidin. Avidin can be displaced from the plasmid at higher ionic strengths. Avidin is not displaced from the plasmid by an excess of a tetra-arginine ligand, nor by the presence of biotin. Therefore, this system offers the opportunity to reversibly bind biotin-labeled species to a condensed DNA–protein complex. An example application is the use of biotinylated gold nanoparticles. This system offers the ability to examine in better detail the chemical mechanisms involved in important radiobiological effects. Examples include protein modulation of radiation damage to DNA, and radiosensitization by gold nanoparticles     

Preparation of gold nanoparticles/functionalized multiwalled carbon nanotube nanocomposites and its glucose biosensing application

Gold nanoparticles stabilized by amino-terminated ionic liquid (Au-IL) have been in situ noncovalently deposited on poly(sodium 4-styrene-sulfonate) (PSS)-functionalized multiwalled carbon nanotubes (MWCNTs) to form a MWCNTs/PSS/Au-IL nanocomposite. PSS can interact with MWCNTs through hydrophobic interaction. Amino-terminated ionic liquid was applied to reduce aqueous HAuCl(4), and the resulting gold nanoparticles were attached to the PSS-functionalized MWCNTs simultaneously. Most gold nanoparticles dispersed well on the functionalized MWCNTs. Transmission electron microscopy, Raman and X-ray photoelectron spectroscopy were used to confirm the composition and structure of the nanocomposites. The resulting MWCNTs/PSS/Au-IL composite exhibits good electrocatalysis toward oxygen and hydrogen peroxide reduction. And good biocompatibility with glucose oxidase was also demonstrated due to its good biocatalysis toward glucose substrate, which offered a friendly environment for the immobilization of biomolecules. Such bionanocomposite provides us potential applications in fabrication of biosensors. The resulting biosensor exhibits good response to glucose with a low detection limit 25 microM. It also has excellent reproducibility, satisfied operational stability and good storage stability.