Critical flocculation concentrations, binding isotherms, and ligand exchange properties of peptide-modified gold nanoparticles studied by UV-visible, fluorescence, and time-correlated single photon counting spectroscopies

Protocols for modifying gold nanoparticles with peptide-bovine serum albumin (BSA) conjugates are described within. The resulting constructs were characterized using a number of techniques including static fluorescence spectroscopy and time-correlated single photon counting spectroscopy (TCSPC) in order to quantify peptide-BSA binding isotherms, exchange rates, critical flocculation concentrations, and the composition of mixed peptide-BSA monolayers on gold nanoparticles. TCSPC has proven to be a powerful technique for observing the microenvironment of protein-gold nanoparticle conjugates because it can distinguish between surface-bound and solution-phase species without the need for separation steps. Full characterization of the composition and stability of peptide-modified metal nanoparticles is an important step in their use as intracellular delivery vectors and imaging agents.     

Ligand-installed PEGylated bionanosphere

The synthesis of poly(ethylene glycol)-b-poly(2-N,N-dimethylaminoethylmethacrylate) processing an acetal group at the PEG chain end (acetal-PEGPAMA) is reported. The obtained acetal-PEGPAMA block copolymer was found to reduce tetrachloroauric acid at room temperature to produce gold nanoparticles. The size of these nanoparticles was controllable in the range of 6 to 13 nm by changing the initial Au3+: polymer ratio. In addition to the reduction of tetrachloroauric acid, acetal-PEGPAMA bonds on the surface of the obtained gold nanoparticles to improve their dispersion stability in an aqueous medium even at a salt concentration as high as two. Biotinyl-PEGPAMA-anchored gold nanoparticles undergo specific aggregation in the presence of streptavidin thereby revealing their promising utility as colloidal sensing systems for use in biological systems. Biotin-PEGPAMA can also be utilised for the preparation of a functionally PEGylated quantum dot (QD). When CdCl2 and Na2S were mixed in aqueous media in the presence of the biotin-PEGPAMA, a CdS QD with an approximately 5 nm size was prepared. The polyamine segment was anchored onto the surface of the formed CdS nanoparticle, whereas the PEG segment was tethered onto the surface to form a hydrophilic palisade, thus improving the dispersion stability in aqueous media even under a high salt concentration condition. An effective fluorescent resonance energy transfer (FRET) was observed by the specific interaction of the biotin-PEGPAMA stabilised CdS QD with TexasRed-labelled streptavidin with the physiological ionic strength of 0.15 M. The extent of the energy transfer was in proportion to the concentration of the TexasRed-streptavidin. This FRET system using the PEGylated CdS QD coupled with fluorescent-labelled protein can be utilised as a highly sensitive bioanalytical system.     

How to Control AFM Nanoxerography for the Templated Monolayered Assembly of 2 nm Colloidal Gold Nanoparticles

This paper reports on the directed monolayered assembly of 2 nm colloidal gold nanoparticles onto charge patterns written by atomic force microscopy (AFM) on poly(methylmethacrylate) thin films with a sub-100-nm spatial resolution. The impact of key experimental parameters (surface potential of charge patterns, immersion time in the colloidal solution, nanoparticle concentration, rinsing time) on the nanoparticle assembly was quantified for the first time. This study reveals that the high level of control of this so-called AFM nanoxerography process will allow one to construct promising colloid-based devices integrating localized nanoparticle monolayers of desired density.     

pH-Dependent gold nanoparticle self-organization on functionalized Si/SiO2 surfaces

The self-organization of citrate- and acrylate-stabilized gold nanoparticles onto SiO₂/hydroxyl-, amino- and nitro-terminated surfaces was investigated as a function of pH. Bare clean Si/SiO₂ substrates were used as the SiO₂/hydroxyl-terminated surfaces and self-assembled monolayers (SAM) of (3-aminopropyl)trimethoxysilane (APTMS) and 3-(4-nitrophenoxy)-propyltrimethoxysilane (NPPTMS) on Si/SiO₂ were employed as the amino- and nitro-terminated surfaces, respectively. All the surfaces were fully characterized by contact angle, atomic force microscopy (AFM), ellipsometry and X-ray photoelectron spectroscopy (XPS). Citrate- and acrylate-stabilized gold nanoparticle stability was also investigated as a function of pH by UV–visible absorption spectroscopy and Z-potentiometry. The gold nanoparticle surface coverage of the substrates was independently estimated by AFM and XPS. The results show that colloid deposition on bare SiO₂/OH surfaces and on NPPTMS monolayers is negligible with the exception of acrylate-stabilized gold nanoparticles which were found to be immobilized on nitro-terminated surfaces at pH lower than 3.5. Nevertheless, APTMS monolayers interact strongly with citrate- and acrylate-stabilized gold nanoparticles exhibiting a dependence of the surface coverage from the pH of the colloidal solution.     

Determining the conformation of thiolated poly(ethylene glycol) on Au nanoshells by surface-enhanced Raman scattering spectroscopic assay

The packing density of thiolated poly(ethylene glycol) (PEG) adsorbates on Au nanoshells is determined by exploiting the surface-enhanced Raman scattering response of individual nanoshell substrates. By incorporating the linker molecule p-mercaptoaniline (pMA), the number of 2000 MW and 5000 MW PEG molecules on each nanoparticle is determined by interpolation of the Langmuir isotherm for pMA. We conclude that both PEG adsorbates maintain a compact "brush" rather than an extended "mushroom" configuration on nanoshell surfaces.     

Influence of the shape of nanostructured metal surfaces on adsorption of single peptide molecules in aqueous solution

Self-assembly and function of biologically modified metal nanostructures depend on surface-selective adsorption; however, the influence of the shape of metal surfaces on peptide adsorption mechanisms has been poorly understood. The adsorption of single peptide molecules in aqueous solution (Tyr(12) , Ser(12) , A3, Flg-Na(3) ) is investigated on even {111} surfaces, stepped surfaces, and a 2 nm cuboctahedral nanoparticle of gold using molecular dynamics simulation with the CHARMM-METAL force field. Strong and selective adsorption is found on even surfaces and the inner edges of stepped surfaces (-20 to -60 kcal/mol peptide) in contrast to weaker and less selective adsorption on small nanoparticles (-15 to -25 kcal/mol peptide). Binding and selectivity appear to be controlled by the size of surface features and the extent of co-ordination of epitaxial sites by polarizable atoms (N, O, C) along the peptide chain. The adsorption energy of a single peptide equals a fraction of the sum of the adsorption energies of individual amino acids that is characteristic of surface shape, epitaxial pattern, and conformation constraints (often β-strand and random coil). The proposed adsorption mechanism is supported and critically evaluated by earlier sequence data from phage display, dissociation constants of small proteins as a function of nanoparticle size, and observed shapes of peptide-stabilized nanoparticles. Understanding the interaction of single peptides with shaped metal surfaces is a key step towards control over self-organization of multiple peptides on shaped metal surfaces and the assembly of superstructures from nanostructures.     

Determination of the intracellular stability of gold nanoparticle monolayers using mass spectrometry

Monolayer stability of core-shell nanoparticles is a key determinant of their utility in biological studies such as imaging and drug delivery. Intracellular thiols (e.g., cysteine, cysteamine, and glutathione) can trigger the release of thiolate-bound monolayers from nanoparticles, a favorable outcome for controllable drug release applications but an unfavorable outcome for imaging agents. Here, we describe a method to quantify the monolayer release of gold nanoparticles (AuNPs) in living cells using parallel measurements by laser desorption/ionization (LDI) and inductively coupled plasma (ICP) mass spectrometry. This combination of methods is tested using AuNPs with structural features known to influence monolayer stability and on cells types with varying concentrations of glutathione. On the basis of our results, we predict that this approach should help efforts to engineer nanoparticle surface monolayers with tunable stability, providing stable platforms for imaging agents and controlled release of therapeutic monolayer payloads.     

Enhanced x-ray irradiation-induced cancer cell damage by gold nanoparticles treated by a new synthesis method of polyethylene glycol modification

We explored a very interesting gold nanoparticle system-pegylated gold in colloidal solution-and analyzed its uptake by mice colorectal adenocarcinoma CT26 tumor cells and the impact on the cell's response to x-ray irradiation. We found that exposure to polyethylene glycol (PEG) modified ('pegylated') 4.7 ± 2.6?nm gold nanoparticles synthesized by a novel synchrotron-based method enhances the response of CT26 cells to x-ray irradiation. Transmission electron microscopy (TEM) and confocal microscopy revealed that substantial amounts of such nanoparticles are taken up and absorbed by the cells and this conclusion is supported by quantitative induced coupled plasma (ICP) results. Standard tests indicated that the internalized particles are highly biocompatible but strongly enhance the cell damage induced by x-ray irradiation. Synchrotron radiation Fourier transform infrared (SR-FTIR) spectromicroscopy analyzed the chemical aspects of this phenomenon: the appearance of C = O stretching bond spectral features could be used as a marker for cell damage and confirmed the enhancement of the radiation-induced toxicity for cells.     

Photoswitchable oligonucleotide-modified gold nanoparticles: controlling hybridization stringency with photon dose

We describe a new class of stimulus-responsive DNA-functionalized gold nanoparticles that incorporate azobenzene-modified oligonucleotides. Beyond the classic directed assembly and sensing behaviors associated with oligonucleotide-modified nanoparticles, these particles also exhibit reversible photoswitching of their assembly behavior. Exposure to UV light induces a trans-cis isomerization of the azobenzene which destabilizes the DNA duplex, resulting in dissociation of the nanoparticle assemblies. The isomerization is reversible upon exposure to blue light, resulting in rehybridization and reassembly of the DNA-linked nanoparticle clusters. We show that perfectly complementary and partially mismatched strands exhibit clearly distinguishable photoinduced melting properties, and we demonstrate that photon dose can thus be used in place of temperature or ionic strength to control hybridization stringency with the ability to discriminate single-base mismatches.     

In vitro safety toxicology data for evaluation of gold nanoparticles-chronic cytotoxicity, genotoxicity and uptake

Safety and toxic effects of nanoparticles are still largely unexplored due to the multiple aspects that influence their behaviour toward biological systems. Here, we focus the attention on 12 nm spherical gold nanoparticle coated or not with hyaluronic acid compared to its precursor counterpart salt. Results ranging from the effects of a 10-days exposure in an in vitro model with BALB/c 3T3 fibroblast cells show how 12 nm spherical gold nanoparticles are internalized from 3T3 cells by endo-lysosomal pathway by an indirect measurement technique; and how gold nanoparticles, though not being a severe cytotoxicant, induce DNA damage probably through an indirect mechanism due to oxidative stress. While coating them with hyaluronic acid reduces gold nanoparticles cytotoxicity and slows their cell internalization. These results will be of great interest to medicine, since they indicate that gold nanoparticles (with or without coating) are suitable for therapeutic applications due to their tunable cell uptake and low toxicity.     

Anionic functionalized gold nanoparticle continuous full filling separations: importance of sample concentration

Electrically driven separations which contain nanoparticles offer detection and separation advantages but are often difficult to reproduce. To address possible sources of separation inconsistencies, anionic functionalized gold nanoparticles are thoroughly characterized and subsequently included in continuous full filling capillary electrophoresis separations of varying concentrations of three small molecules. Citrate stabilized gold nanospheres are functionalized with 11-mercaptoundecanoic acid, 6-mercaptohexanoic acid, or thioctic acid self-assembled monolayers (SAMs) and characterized using dynamic light scattering, extinction spectroscopy, zeta potential, and X-ray photoelectron spectroscopy prior to use in capillary electrophoresis. Several important trends are noted. First, the stability of these anionic nanoparticles in the capillary improves with increased ligand packing density as indicated by a ratio of absorbance collected at 520 to 600 nm. Second, increasing nanoparticle concentration from 0 to 2 nM (0-0.002(5)%, w/w) minimally impacts analyte migration times; however, when higher nanoparticle concentrations are included within the capillary, nanoparticle aggregation occurs which induces separation inconsistencies. Third, analyte peak areas are most significantly impacted as their concentration decreases. These trends are attributed to both sample enrichment and electrostatic interactions between the anionic carboxylic acid functionalized gold nanoparticles and sample. These important findings suggest that sample concentration-induced conductivity differences between the sample matrix and separation buffer as well as SAM packing density are important parameters to both characterize and consider when nanoparticles are used during continuous full filling separations and their subsequent use to enhance spectroscopic signals to improve in-capillary analyte detection limits.     

Patterned self-assembly of gold nanoparticles on chemical templates fabricated by soft UV nanoimprint lithography

Chemical templates for the patterned immobilization of gold nanoparticles were fabricated by soft UV nanoimprint lithography. The template structures were fabricated by means of the consecutively performed process steps of nanoimprint lithography, reactive ion etching, chemical functionalization with amino groups, and lift-off of imprint resist. These chemical templates were used for the defined assembly of 20 nm diameter citrate stabilized gold nanoparticles from aqueous solution. By reducing the ionic strength of the solution, one- and zero-dimensional particle assemblies were generated on sub-100-nm template structures. By this means, the pattern resolution predefined by the lithography process could be easily enhanced by dilution of the nanoparticle solution.     

Nanoparticle‐Induced Folding and Fibril Formation of Coiled‐Coil‐Based Model Peptides

Nanomedicine is a rapidly growing field that has the potential to deliver treatments for many illnesses. However, relatively little is known about the biological risks of nanoparticles. Some studies have shown that nanoparticles can have an impact on the aggregation properties of proteins, including fibril formation. Moreover, these studies also show that the capacity of nanoscale objects to induce or prevent misfolding of the proteins strongly depends on the primary structure of the protein. Herein, light is shed on the role of the peptide primary structure in directing nanoparticle‐induced misfolding by means of two model peptides. The design of these peptides is based on the α‐helical coiled‐coil folding motif, but also includes features that enable them to respond to pH changes, thus allowing pH‐dependent β‐sheet formation. Previous studies showed that the two peptides differ in the pH range required for β‐sheet folding. Time‐dependent circular dichroism spectroscopy and transmission electron microscopy are used to characterize peptide folding and aggregate morphology in the presence of negatively charged gold nanoparticles (AuNPs). Both peptides are found to undergo nanoparticle‐induced fibril formation. The determination of binding parameters by isothermal titration calorimetry further reveals that the different propensities of both peptides to form amyloid‐like structures in the presence of AuNPs is primarily due to the binding stoichiometry to the AuNPs. Modification of one of the peptide sequences shows that AuNP‐induced β‐sheet formation is related to the structural propensity of the primary structure and is not a generic feature of peptide sequences with a sufficiently high binding stoichiometry to the nanoparticles.     

Electrochemically programmed release of biomolecules and nanoparticles

The controlled release of molecules or nanoparticle conjugates is an important tool for a wide range of applications in science and engineering. Here we demonstrate electrochemically programmed release of biomolecules and nanoparticles immobilized on patterned gold electrodes using the thiol-gold linkage. This technique exploits the reductive desorption of self-assembled monolayers and allows both spatially controlled release and regeneration of small molecules (e.g., drugs), biopolymers (e.g., peptides, proteins, DNA), protein assemblies (e.g., viruses), and nanoparticles (e.g., particle-DNA conjugates). Fluorescence microscopy is used to image the release of avidin and nanoparticles in phosphate-buffered saline and to determine the kinetics of desorption. We also demonstrate that the electrodes can be regenerated using the same conjugation scheme.     

“Dual‐Template” Synthesis of One‐Dimensional Conductive Nanoparticle Superstructures from Coordination Metal–Peptide Polymer Crystals

Bottom‐up fabrication of self‐assembled structures made of nanoparticles may lead to new materials, arrays and devices with great promise for myriad applications. Here a new class of metal–peptide scaffolds is reported: coordination polymer Ag(I)‐DLL belt‐like crystals, which enable the dual‐template synthesis of more sophisticated nanoparticle superstructures. In these biorelated scaffolds, the self‐assembly and recognition capacities of peptides and the selective reduction of Ag(I) ions to Ag are simultaneously exploited to control the growth and assembly of inorganic nanoparticles: first on their surfaces, and then inside the structures themselves. The templated internal Ag nanoparticles are well confined and closely packed, conditions that favour electrical conductivity in the superstructures. It is anticipated that these Ag(I)‐DLL belts could be applied to create long (>100 μm) conductive Ag@Ag nanoparticle superstructures and polymetallic, multifunctional Fe₃O₄@Ag nanoparticle composites that marry the magnetic and conductive properties of the two nanoparticle types.     

聚乙二醇法在温和条件下制备不同结构的磁性Fe3O4纳米晶

以Fe（acac）3为原料,乙二醇、聚乙二醇1000和聚乙二醇5000为还原剂和溶剂,在温和的溶剂热的条件下制备了不同尺寸的顺磁性Fe3O4纳米颗粒.利用X射线衍射（XRD）、光电子能谱（XPS）、透射电子显微镜（TEM）和磁性测量技术对制备的Fe3O4纳米颗粒的结构、形貌、磁性能进行了表征测试.结果发现,聚乙二醇分子链的长度对Fe3O4纳米颗粒的平均粒径大小、结晶度和饱和磁化强度均有重要影响.以乙二醇、聚乙二醇1000和聚乙二醇5000为还原剂制备的Fe3O4纳米颗粒的尺寸分别为2～3nm、5nm和7～8nm;相应的纳米Fe3O4颗粒饱和磁化强度分别为55.2、61.5和81.3emu/g;同时结晶度也随分子链的增加而增加.随分子链长度的增加,还原剂还原性的逐渐增加是导致Fe3O4纳米颗粒平均粒径大小、结晶度和饱和磁化强度逐渐增大的重要因素.     

Interactions of Skin with Gold Nanoparticles of Different Surface Charge,Shape, and Functionality

The interactions between skin and colloidal gold nanoparticles of different physicochemical characteristics are investigated. By systematically varying the charge, shape, and functionality of gold nanoparticles, the nanoparticle penetration through the different skin layers is assessed. The penetration is evaluated both qualitatively and quantitatively using a variety of complementary techniques. Inductively coupled plasma optical emission spectrometry (ICP‐OES) is used to quantify the total number of particles which penetrate the skin structure. Transmission electron microscopy (TEM) and two photon photoluminescence microscopy (TPPL) on skin cross sections provide a direct visualization of nanoparticle migration within the different skin substructures. These studies reveal that gold nanoparticles functionalized with cell penetrating peptides (CPPs) TAT and R₇ are found in the skin in larger quantities than polyethylene glycol‐functionalized nanoparticles, and are able to enter deep into the skin structure. The systematic studies presented in this work may be of strong interest for developments in transdermal administration of drugs and therapy.     

Impedance Spectroscopy of Ionic Ligand‐Modulated Charge Transport of Gold Nanoparticle Films

Assembled monolayer‐protected nanoparticles (NPs) possess unique electrical properties that are determined by the coupled effects of their nano‐sized electroactive inorganic cores that are capable of donating and accepting electrons and the organic shells. Core and ligand engineering for NP conductance modulation has been extensively explored; however, most studies focus on electron transport and not the interplay between the ion and electron transport processes. It is demonstrated here that electronic‐ and ionic‐conducting properties of nanoparticle assemblies can be controlled by engineering the charge and flexibility of the ligand shell. By using impedance spectroscopy, the electronic, mixed ionic and electronic, and responsive conductance of the nanoparticle film and structure‐function correlation are systematically investigated, and this correlation is used to provide a prototype volatile gas sensor based on the combined ionic and electronic conductance behavior of ionic ligand‐functionalized gold NPs.     

Gold nanoparticle-enhanced secondary ion mass spectrometry imaging of peptides on self-assembled monolayers

We demonstrate the use of gold nanoparticles (AuNPs) to enhance the secondary ion emission of peptides in time-of-flight secondary ion mass spectrometry (TOF-SIMS). The signal intensity of peptides adsorbed onto AuNPs was significantly increased when compared to that of self-assembled monolayers (SAMs). This gold nanoparticle-enhanced SIMS, termed NE-SIMS, enabled the sensitive detection of subtle modifications of peptides, such as phosphorylation. From a quantitative analysis of the amounts of adsorbed peptides and AuNPs on SAMs using quartz crystal microbalance and surface plasmon resonance spectroscopy, the ratio of peptide molecule to AuNP on amine-SAMs was revealed to be 18-19:1. When considering the ratio of peptide to matrix (1:10(3)-10(6)) employed in a matrix-enhanced SIMS, the use of AuNPs gave rise to a significantly increased secondary ion emission of peptides. Peptides were adsorbed onto patterned AuNPs on SAMs using a microfluidic system, and well-contrasted molecular ion images were obtained. NE-SIMS is expected to be applied to a chip-based analysis of modification of biomolecules in a label-free manner.     

Combined electrochromic and plasmonic optical responses in conducting polymer/metal nanoparticle films

Poly(3,4-ethylenedioxithiophene)/poly(styrene sulphonate) (PEDOT/PSS) aqueous dispersions were mixed with aqueous gold nanoparticle and aqueous silver nanoparticle colloids. PEDOT/gold nanoparticles (Au NP) and PEDOT/silver nanoparticles (Ag NP) films were obtained by solvent casting the corresponding aqueous solutions. The nanocomposite films showed the optical characteristics associated with both the surface plasmon absorption resonance of the metal nanoparticles and the excitation of the bipolaron band of the conducting polymer. As an interesting application we demonstrate the use of metal nanoparticles to tune the color of PEDOT based electrochromic films from blue to violet in the case of Au NP or green in the case of Ag NP.