DNA Base Pair Resolution Measurements Using Resonance Energy Transfer Efficiency in Lanthanide Doped Nanoparticles

Lanthanide-doped nanoparticles are of considerable interest for biodetection and bioimaging techniques thanks to their unique chemical and optical properties. As a sensitive luminescence material, they can be used as (bio) probes in Förster Resonance Energy Transfer (FRET) where trivalent lanthanide ions (La³⁺) act as energy donors. In this paper we present an efficient method to transfer ultrasmall (ca. 8 nm) NaYF₄ nanoparticles dispersed in organic solvent to an aqueous solution via oxidation of the oleic acid ligand. Nanoparticles were then functionalized with single strand DNA oligomers (ssDNA) by inducing covalent bonds between surface carboxylic groups and a 5’ amine modified-ssDNA. Hybridization with the 5’ fluorophore (Cy5) modified complementary ssDNA strand demonstrated the specificity of binding and allowed the fine control over the distance between Eu³⁺ ions doped nanoparticle and the fluorophore by varying the number of the dsDNA base pairs. First, our results confirmed nonradiative resonance energy transfer and demonstrate the dependence of its efficiency on the distance between the donor (Eu³⁺) and the acceptor (Cy5) with sensitivity at a nanometre scale.     

Interaction mechanism of insulin with ZnO nanoparticles by replica exchange molecular dynamics simulation

The interaction of ZnO nanoparticles with biological molecules such as proteins is one of the most important and challenging problems in molecular biology. Molecular dynamics (MD) simulations are useful technique for understating the mechanism of various interactions of proteins and nanoparticles. In the present work, the interaction mechanism of insulin with ZnO nanoparticles was studied. Simulation methods including MD and replica exchange molecular dynamics (REMD) and their conditions were surveyed. According to the results obtained by REMD simulation, it was found that insulin interacts with ZnO nanoparticle surface via its polar and charged amino acids. Unfolding insulin at ZnO nanoparticle surface, the terminal parts of its chains play the main role. Due to the linkage between chain of insulin and chain of disulfide bonds, opposite directional movements of N terminal part of chain A (toward nanoparticle surface) and N termini of chain B (toward solution) make insulin unfolding. In unfolding of insulin at this condition, its helix structures convert to random coils at terminal parts chains.     

A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles^1,2. The simple, robust and scalable technique efficiently functionalizes metallic nanoparticles with thiol-ligands in a miscible water/organic solvent mixture allowing for rapid grafting of thiol groups onto the gold nanoparticle surface. The hydrophobic ligands on the nanoparticles then quickly phase separate the nanoparticles from the aqueous based suspension and confine them to the air-fluid interface. This drives the ligand-capped nanoparticles to form monolayer domains at the air-fluid interface.  The use of water-miscible organic solvents is important as it enables the transport of the nanoparticles from the interface onto template-free substrates.  The flow is mediated by a surface tension gradient^3,4 and creates macroscopic, high-density, monolayer nanoparticle-ligand films.  This self-assembly technique may be generalized to include the use of particles of different compositions, size, and shape and may lead to an efficient assembly method to produce low-cost, macroscopic, high-density, monolayer nanoparticle films for wide-spread applications.     

Directional conjugation of antibodies to nanoparticles for synthesis of multiplexed optical contrast agents with both delivery and targeting moieties

Molecular optical imaging has shown promise in visualizing molecular biomarkers with subcellular resolution both noninvasively and in real-time. Here, we use gold nanoparticles as optical probes to provide meaningful signal in the presence of targeted biomarkers. We present a novel conjugation technique to control the binding orientation of antibodies on the surface of gold nanoparticles to maximize antibody functionality. Briefly, a heterobifunctional linker, hydrazide-polyethylene glycol-dithiol, is used to directionally attach the Fc, or nonbinding region of the antibody, to the gold nanoparticle surface. The conjugation strategy allows for multiplexing various glycosylated antibodies on a single nanoparticle. We present a method to prepare multifunctional nanoparticles by incorporating targeting and delivery moieties on the same nanoparticle that addresses the challenge of imaging intracellular biomarkers. The time estimate for the entire protocol is approximately 6 h.     

Directing noble metal ion chemistry within a designed ferritin protein

Human H ferritin (HuHF) assembles from 24 four-helix bundles to form an approximately 500 kDa protein with an 8 nm internal cavity. HuHF provides a useful model for studying the transport of metal ions in solution to buried reaction sites in proteins. In this study, HuHF was redesigned to facilitate noble metal ion (Au(3+), Ag(+)) binding, reduction, and nanoparticle formation within the cavity. Computationally determined amino acid substitutions were targeted at four external and four internal surface sites. A variant with a total of 96 cysteines and histidines removed from the exterior surface and 96 non-native cysteines added to the interior surface retained wild-type stability and structure, as confirmed by X-ray crystallography, and promoted the formation of silver or gold nanoparticles within the protein cavity. Crystallographic studies with HuHF variants provide insight into how ferritins control access of metal ions to interior residues that perform chemistry.     

Electrochemiluminescence observing the surface features of Ru‐doped silica nanoparticles based on nanoparticle–ultramicroelectrode collision

We present an innovative and sensitive electrogenerated chemiluminescence (ECL) strategy for observing the surface feature of a single silica nanoparticle based on its collision with an ultramicroelectrode (UME). As an ECL luminophore, Ru(bpy)₃²⁺ molecules are doped into silica nanoparticles. The stochastic collision events of Ru(bpy)₃²⁺‐doped silica nanoparticles (RuSNPs) can be tracked by observing the ECL ‘blips’ from the ECL reaction of Ru(bpy)₃²⁺ with a coreactant in solution. When RuSNPs collided with UME, Ru(bpy)₃²⁺ molecules that only exist near the collision site of silica nanoparticles (NPs) were electrochemically oxidized to form Ru(bpy)₃³⁺, and then emitted light, because silica NPs are insulated. The inhomogeneous properties of silica nanoparticle surfaces will produce diverse ECL blips in intensity and shape. In addition, distribution gradients from the he Ru(bpy)₃²⁺ in a silica matrix also affect ECL blips. Some information on the surface properties of silica NPs can be obtained by observation of single silica collision events.     

Protein solution structure calculations in solution: Solvated molecular dynamics refinement of calbindin D9k

The three-dimensional solution structures of proteins determinedwith NMR-derived constraints are almost always calculated in vacuo. Thesolution structure of (Ca²⁺)_{_2}-calbindinD_9k has been redetermined by new restrained molecular dynamics(MD) calculations that include Ca²⁺ ions and explicit solventmolecules. Four parallel sets of MD refinements were run to provide accuratecomparisons of structures produced in vacuo, in vacuo withCa²⁺ ions, and with two different protocols in a solvent bathwith Ca²⁺ ions. The structural ensembles were analyzed interms of structural definition, molecular energies, packing density,solvent-accessible surface, hydrogen bonds, and the coordination of calciumions in the two binding loops. Refinement including Ca²⁺ ionsand explicit solvent results in significant improvements in the precisionand accuracy of the structure, particularly in the binding loops. Theseresults are consistent with results previously obtained in free MDsimulations of proteins in solution and show that the rMD refinedNMR-derived solution structures of proteins, especially metalloproteins, canbe significantly improved by these strategies.     

Correlated Optical Spectroscopy and Electron Microscopy Studies of the Slow Ostwald-Ripening Growth of Silver Nanoparticles under Controlled Reducing Conditions

Metallic nanoparticles display distinct localized surface plasmon resonance (LSPR) properties that depend on their size, shape, and composition and that can be monitored to characterize their growth. Utilizing LSPR properties, we report the first investigation of ambient temperature formation of trioctylamine (TOA)-stabilized spherical silver nanoparticles (AgNPs) of ～3.0-nm diameter by mild reduction of AgClO₄ with the weak reducing agent heptamethyltrisiloxane in organic solvent. The appropriate choice of experimental conditions caused slow reduction, which allowed the study of the nanoparticle growth process by time-resolved UV–visible spectroscopy and transmission electron microscopy (TEM). The linear nanoparticle growth kinetics from 50 min to end of the reaction derived from LSPR changes, the absence of a bimodal size distribution during the initial stage of the reduction process from TEM analysis, and the single crystallinity of the resulting AgNPs suggested a diffusion-controlled Ostwald-ripening growth process. It was also found that in addition to its stabilizing ability, TOA acted as a catalyst and facilitated Ag⁺ reduction. Furthermore, a modest increase in reaction temperature caused a substantial enhancement in the AgNP formation rate, and low concentration of stabilizing ligand yielded an increase in size and dispersity.     

Carboxymethyl chitosan as a matrix material for platinum, gold, and silver nanoparticles

Carboxymethyl chitosan (CMC) was evaluated for its use in the synthesis and stabilization of catalytic nanoparticles for the first time. Many studies have reported on the ability of chitosan to bind with metal ions and support metal nanoparticles. CMC has a higher reported chelation capacity than chitosan, which has potential implications for improved catalyst formation and immobilization. Platinum, gold, and silver nanoparticles were synthesized in both chitosan and CMC. Particle size, morphology, and aggregation were examined using transmission electron microscopy (TEM). Complexation of nanoparticles was studied through Fourier transform infrared spectroscopy (FTIR). Similar nanoparticle size distributions were observed in the two polymers; however, CMC was observed to have higher rates of aggregation. This indicates that the carboxymethyl groups did not change nanoparticle formation; however, poor cross-linking and a limited anchoring ability of CMC led to the inability to immobilize the catalyst materials effectively.     

Simulation Modelling Study of Self-Assembled Nanoparticle Coatings for Retinal Implants

The electrode resolution of current retinal prostheses is still far from matching the densities of retinal neurons. Decreasing electrode diameter increases impedance levels thus deterring effective stimulation of neurons. One solution is to increase the surface roughness of electrodes, which can be done via nanoparticle coatings. This paper explores a Lattice Gas Model of the drying-mediated self-assembly of nanoparticle mixtures. The model includes representations for different types of nanoparticles, solvent, vapour, substrate and the energetic relationships between these elements. The dynamical aspect of the model is determined by energy minimization, stochastic fluctuations and physical constraints. The model attempts to unravel the relationships between different experimental conditions (e.g. evaporation rate, substrate characteristics and solvent viscosity) and the surface roughness of resulting assemblies. Some of the main results include the facts that the assemblies formed by nanoparticles of different sizes can boost roughness in specific circumstances and that the optimized assemblies can exhibit walled or stalagmite structures. This study provides a set of simulation modelling experiments that if confirmed in the laboratory may result in new and useful materials.     

Determining the Locations of Ions and Water around DNA from X-Ray Scattering Measurements

Nucleic acids carry a negative charge, attracting salt ions and water. Interactions with these components of the solvent drive DNA to condense, RNA to fold, and proteins to bind. To understand these biological processes, knowledge of solvent structure around the nucleic acids is critical. Yet, because they are often disordered, ions and water evade detection by x-ray crystallography and other high-resolution methods. Small-angle x-ray scattering (SAXS) is uniquely sensitive to the spatial correlations between solutes and the surrounding solvent. Thus, SAXS provides an experimental constraint to guide or test emerging solvation theories. However, the interpretation of SAXS profiles is nontrivial because of the difficulty in separating the scattering signals of each component: the macromolecule, ions, and hydration water. Here, we demonstrate methods for robustly deconvoluting these signals, facilitating a more straightforward comparison with theory. Using SAXS data collected on an absolute intensity scale for short DNA duplexes in solution with Na⁺, K⁺, Rb⁺, or Cs⁺ counterions, we mathematically decompose the scattering profiles into components (DNA, water, and ions) and validate the decomposition using anomalous scattering measurements. In addition, we generate a library of physically motivated ion atmosphere models and rank them by agreement with the scattering data. The best-fit models have relatively compact ion atmospheres when compared to predictions from the mean-field Poisson-Boltzmann theory of electrostatics. Thus, the x-ray scattering methods presented here provide a valuable measurement of the global structure of the ion atmosphere that can be used to test electrostatics theories that go beyond the mean-field approximation.     

Biorecovery of gold by Escherichia coli and Desulfovibrio desulfuricans

Microbial precipitation of gold was achieved using Escherichia coli and Desulfovibrio desulfuricans provided with H2 as the electron donor. No precipitation was observed using H2 alone or with heat-killed cells. Reduction of aqueous AuIII ions by both strains was demonstrated at pH 7 using 2 mM HAuCl4 solution and the concept was successfully applied to recover 100% of the gold from acidic leachate (115 ppm of AuIII) obtained from jewelry waste. Bioreductive recovery of gold from aqueous solution was achieved within 2 h, giving crystalline Au0 particles (20-50 nm), in the periplasmic space and on the cell surface, and small intracellular nanoparticles. The nanoparticle size was smaller (red suspension) at acidic pH (2.0) as compared to that obtained at pH 6.0 and 7.0 (purple) and 9.0 (dark blue). Comparable nanoparticles were obtained from AuIII test solutions and jewelry leachate.     

Biological synthesis of platinum nanoparticles using Diopyros kaki leaf extract

The leaf extract of Diopyros kaki was used as a reducing agent in the ecofriendly extracellular synthesis of platinum nanoparticles from an aqueous H₂PtCl₆·6H₂O solution. A greater than 90% conversion of platinum ions to nanoparticles was achieved with a reaction temperature of 95°C and a leaf broth concentration of >10%. A variety of methods was used to characterize the platinum nanoparticles synthesized: inductively coupled plasma spectrometry, transmission electron microscopy, energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and Fourier-transform infrared spectroscopy (FTIR). The average particle size ranged from 2 to 12 nm depending on the reaction temperature and concentrations of the leaf broth and PtCl₆ ²⁻. FTIR analysis suggests that platinum nanoparticle synthesis using Diopyros kaki is not an enzyme-mediated process. This is the first report of platinum nanoparticle synthesis using a plant extract.     

Silver Nanoparticles for the Adsorption of Manganese from Biological Samples

In this study, silver nanoparticles were prepared and used for separation and preconcentration of manganese from biological samples. The technical feasibility of silver nanoparticles for manganese removal was investigated under batch studies. The effects of different parameters such as pH of solution, time (t), amounts of PAN (E), and silver nanoparticles (N) on the adsorption of manganese by silver nanoparticle were investigated using factorial design and response surface methodology based on Box–Behnken design. Thermodynamic parameters indicate the adsorption process to be exothermic. The limit of detection of the proposed method followed by inductively coupled plasma was found to be 0.08?µg L^?1. The method was applied to determine of manganese in biological samples.     

Various physicochemical and surface properties controlling the bioactivity of cerium oxide nanoparticles

Amidst numerous emerging nanoparticles, cerium oxide nanoparticles (CNPs) possess fascinating pharmacological potential as they can be used as a therapeutic for various oxidative stress-associated chronic diseases such as cancer, inflammation and neurodegeneration due to unique redox cycling between Ce³⁺ and Ce⁴⁺ oxidation states on their surface. Lattice defects generated by the formation of Ce³⁺ ions and compensation by oxygen vacancies on CNPs surface has led to switching between CeO₂ and CeO_2–x during redox reactions making CNPs a lucrative catalytic nanoparticle capable of mimicking key natural antioxidant enzymes such as superoxide dismutase and catalase. Eventually, most of the reactive oxygen species and nitrogen species in biological system are scavenged by CNPs via an auto-regenerative mechanism in which a minimum dose can exhibit catalytic activity for a longer duration. Due to the controversial outcomes on CNPs toxicity, considerable attention has recently been drawn towards establishing relationships between the physicochemical properties of CNPs obtained by different synthesis methods and biological effects ranging from toxicity to therapeutics. Unlike non-redox active nanoparticles, variations in physicochemical properties and the surface properties of CNPs obtained from different synthesis methods can significantly affect their biological activity (inactive, antioxidant, or pro-oxidant). Moreover, these properties can influence the biological identity, cellular interactions, cellular uptake, biodistribution, and therapeutic efficiency. This review aims to highlight the critical role of various physicochemical and the surface properties of CNPs controlling their biological activity based on 165 cited references.     

Ligand density effect on biorecognition by PEGylated gold nanoparticles: regulated interaction of RCA120 lectin with lactose installed to the distal end of tethered PEG strands on gold surface

PEGylated gold nanoparticles (diameter: 20 nm) possessing various functionalities of lactose ligand on the distal end of tethered PEG ranging from 0 to 65% were prepared to explore the effect of ligand density of the nanoparticles on their lectin binding property. UV-visible spectra of the aqueous solution of the nanoparticles revealed that the strong steric stabilization property of the PEG layer lends the nanoparticles high dispersion stability even under the physiological salt concentration (ionic strength, I = 0.15 M). The number of PEG strands on a single particle was determined to be 520 from thermogravimetric analysis (TGA). Scanning electron microscopy (SEM) observation under controlled acceleration voltage revealed the thickness of the PEG layer on the nanoparticle to be approximately 7 nm. The area occupied by a single lactose molecule on the surface of PEGylated gold nanoparticles was then calculated based on TGA and SEM results and was varied in the range of 10-34 nm2 depending on the lactose functionality (65 approximately 20%). PEGylated gold nanoparticles with 40% and 65% lactose functionality showed a selective and time-dependent aggregation in phosphate buffer with the addition of Ricinus communis agglutinin (RCA120) lectin, a bivalent galactose-specific protein. The aggregates can be completely redispersed by adding an excess amount of galactose. Time-lapse monitoring of UV-visible spectra at 600-750 nm revealed that the aggregation of PEGylated gold nanoparticles was accelerated with an increase in both RCA120 concentration in the solution and the lactose density of the nanoparticles. Furthermore, the sensitivity of lectin detection could be controlled by the regulation of lactose density on the particle surface. Interestingly, there was a critical lactose density (>20%) observed to induce detectable particle aggregation, indicating that the interaction between the particles is triggered by the multimolecular bridging via lectin molecules.     

Binding of nanoparticle receptors to peptide alpha-helices using amino acid-functionalized nanoparticles.

Nanoparticles provide large surface areas and controlled surface functionality and structure, making them excellent scaffolds for peptide recognition. A family of nanoparticles has been fabricated by amino acid functionalization to afford tailored surfaces. These particles are complementary to a tetraaspartate peptide (TAP) featuring cofacial anionic functionality when in the alpha-helical conformation. The functional groups present on these nanoparticle surfaces provide a tool to investigate the contribution of various noncovalent interactions at the nanoparticle-peptide interface. The ability of these particles to enforce the folding of the peptide into an alpha-helix was explored, demonstrating high helicity induction with particles featuring dicationic amino acids such as lysine or histidine, and little or no helix stabilization with hydrophobic amino acid termini.     

Effect of formulation factors on incorporation of the hydrophilic peptide dalargin into PLGA and mPEG-PLGA nanoparticles

The objective of this study was to examine formulation factors that influence the incorporation of the hydrophilic peptide dalargin into poly(D, L-lactic-co-glycolic acid) (PLGA) and methoxy-polyethylene glycol (mPEG)-PLGA nanoparticles. In particular, the effect of ionic additives and nanoparticle method of preparation on the incorporation of dalargin and resultant nanoparticle properties was investigated. Biodegradable nanoparticles were prepared from mPEG-PLGA and PLGA by both solvent evaporation and solvent diffusion methods with inclusion of ionic additives of dextran sulphate (DS), sulfobutyl ether-beta-cyclodextrin (SB-CD), or sodium dodecyl sulfate (SDS). The resultant nanoparticles were analyzed for their mean particle size and size distribution, zeta-potential, peptide loading, yield, and morphology. The inclusion of ionic additives in the nanoparticle formulation significantly influenced dalargin entrapment efficiency (EE). For example, with the PLGA/SDS formulation EE increased from 13.3% to 91.2% and from 4.1% to 68.6% with the solvent diffusion and evaporation methods, respectively. The inclusion of ionic surfactant SDS has also lead to the formation of smaller size of nanoparticles. Isothermal titration microcalorimetry revealed a strong interaction between dalargin and DS, medium level interaction with SDS, and weak interaction with SB-CD. The results of this study suggest that a strong ionic interaction between peptides and additives may lead to enhanced peptide incorporation but also increased particle size. Intermediate ionic interaction, especially when it is associated with the formation of reversed micelles in a hydrophobic polymer solution, could be used to enhance the incorporation of hydrophilic peptides in PLGA and mPEG-PLGA nanoparticles.     

Delivery of expression constructs of secreted frizzled-related protein 4 and its domains by chitosan–dextran sulfate nanoparticles enhances their expression and anti-cancer effects

In malignant mesothelioma (MM) cells, secreted frizzled-related protein 4 (SFRP4) expression is downregulated by promoter methylation. In this study, we evaluated the effect of encapsulated chitosan–dextran (CS–DS) nanoparticle formulations of SFRP4 and its cysteine-rich domain (CRD) and netrin-like domain (NLD) as means of SFRP4-GFP protein delivery and their effects in JU77 and ONE58 MM cell lines. CS–DS formulations of SFRP4, CRD, and NLD nanoparticles were prepared by a complex coacervation technique, and particle size ranged from 300 nm for empty particles to 337 nm for particles containing the proteins. Measurement of the zeta potential showed that all preparations were around 25 mV or above, suggesting stable formulation and good affinity for the DNA molecules. The CS–DS nanoparticle formulation maintained high integrity and entrapment efficiency. Gene delivery of SFRP4 and its domains showed enhanced biological effects in both JU77 and ONE58 cell lines when compared to the non-liposomal FUGENE^® HD transfection reagent. In comparison to the CRD nanoparticles, both the SFRP4 and NLD nanoparticles significantly reduced the viability of MM cells, with the NLD showing the greatest effect. The CS–DS nanoparticle effects were observed at an earlier time point and with lower DNA concentrations. Morphological changes in MM cells were characterized by the formation of membrane-associated vesicles and green fluorescent protein expression specific to SFRP4 and the NLD. The findings from our proof-of-concept study provide a stepping stone for further investigations using in vivo models.