Display of Potassium Channel–Blocking Tertiapin‐Q Peptides on Gold Nanoparticles Enhances Depolarization of Cellular Membrane Potential

The use of nanoparticle (NP) systems to control cellular physiology, including membrane potential, can facilitate furthered understanding of many disparate cellular processes ranging from cellular proliferation to tissue regeneration. A gold NP (AuNP) bioconjugate system based on the honeybee venom peptide, tertiapin‐Q (AuNP‐TPN‐Q), that depolarizes membrane potential by targeting inward rectifier potassium channels (Kir), is developed. The conjugate elicits, in a peptide concentration–dependent manner, a greater and more rapid depolarization response compared to the free peptide alone. The specificity of the interaction of the AuNP‐TPN‐Q conjugate with the Kir channel using immunocytochemistry and competition binding assays is confirmed. It is further shown that membrane depolarization is photothermally reversible via the laser‐induced plasmonic heating of the AuNP, providing a level of control over Kir channels not afforded by currently available drugs. The functional nanobioconjugate described herein provides a new research tool for understanding the intricacies of ion channel activity and the modulation of cellular membrane potential.     

Direct Synthesis of Gold Nanoparticle‐Over‐Nanosheet for Sensitive SERS Detection

A simple ethanol sol‐based method for the synthesis of gold nanosheets (AuNSs) and gold nanoparticle‐over‐nanosheet (AuNP/NS) is developed. Gold nanoparticles (AuNPs) with average sizes of ≈8 nm are grown in situ on the surface of the AuNS, which forms a NP/NS structure that obtains strong, significantly improved, surface‐enhanced Raman spectroscopy activity with the magnitude ≈2 and ≈6 orders higher than the simplex AuNP and AuNS, respectively. This performance is mainly attributed to uniform AuNPs that are closely packed over AuNS and coupled with NP–NS and NP–NP interactions. The NP–NS–GP (the gap between NP–NS) is narrower than NP–NP–GP in which much stronger and steadier plasmon resonance is obtained that can significantly enhance the Raman signal. The results show that single‐crystalline AuNS is an ideal substrate, which can be further coated with other metallic NPs to form a new flexible, high‐activity and AuNS‐based nanocomposite for a wide variety of applications.     

Blood Compatibility of Multilayered Polyelectrolyte Films Containing Immobilized Gold Nanoparticles

Surface material functionalization including layer‐by‐layer (LbL) polyelectrolyte films with incorporated nanoparticles is a growing field with a wide range of biomedical applications: drug reservoirs, medical devices, or tissue engineering. In parallel, gold nanoparticles (AuNPs) can be grafted by drugs and sensitive molecules using simple protocols. This study shows that AuNP behavior is modified when they are entrapped into three partner LbL films in comparison to the colloidal solution. A polycationic (polyallylamine hydrochloride (PAH)) and a polyanionic (polyacrylic acid (PAA)) polymer is used to build films based on three cycles ((PAH/AuNP/PAA)₃). To investigate the interaction with biomolecules and cells, three different films are developed changing the outer layer (either PAH or AuNP or PAA) with the same number of AuNP deposit. The best biocompatibility is observed with a polyacrylic acid outer layer. Due to the high capacity of drug grafting on gold nanoparticles, the results seem promising for the development of nanostructured biomedical devices.     

Interfacial ketene via the photo‐Wolff rearrangement for the modification of monolayer protected gold nanoparticles

An interfacial diazoketone‐modified gold nanoparticle (AuNP) was prepared and characterized by ¹H NMR and IR spectroscopy, TGA and TEM. Irradiation of the diazoketone leads to loss of nitrogen and the formation an interfacial ketene–AuNP via the photo‐Wolff rearrangement, evidenced by the loss of the characteristic C = N = N signal at 2068 cm^?1 in the IR spectrum and the growth of a new signal at 2100 cm^?1 indicative of the ketene. This ketene is relatively stable in the absence of added nucleophiles, but reacts quickly with oxygen nucleophiles illustrating the potential use of this ketene–AuNP as a template for a wide range of surface modifications. Copyright © 2013 John Wiley & Sons, Ltd.     

Unravelling Malaria Antigen Binding to Antibody‐Gold Nanoparticle Conjugates

Conjugates formed by antibody adsorption to gold nanoparticles (AuNP) have found extensive utilization in immunoassays due to the high surface area and interesting optical and electronic properties of the nanomaterials. Nevertheless, the mechanism of formation of antibody‐AuNP conjugates and their antigen binding characteristics have not been sufficiently explored in terms of specificity and consequent clinical applicability. Dynamic light scattering and related techniques have been successfully employed to detect antigen binding to antibody‐AuNP complexes. Here, a range of different techniques from the bionanotechnology realm have been applied to obtain a detailed picture of a competitive immunoassay for malaria antigen detection, based on fluorescence‐quenching by AuNPs. Both agarose gel electrophoresis and differential centrifugal sedimentation (DCS) analyses provide binding constants in the same order of magnitude, for antibody binding to AuNP and for antigen binding to antibody‐AuNP conjugates. Both techniques are also able to reveal inhibition of antigen binding in the presence of a major blood plasma protein, transferrin (via competitive binding). DCS is further used to show inhibition of the binding of the antigen in the presence of human plasma, a realistic testing condition, of high relevance to the implementation of immunoassays at the clinical level.     

Flow Synthesis of Plasmonic Gold Nanoshells via a Microreactor

Gold nanoshells with tunable surface plasmon resonances are a promising material for optical and biomedical applications. They are produced through seed‐mediated growth, in which gold nanoparticles (AuNPs) are seeded on the core particle surface followed by growth of the gold seeds into a shell. However, synthetic gold nanoshell production is typically a multistep, time‐consuming batch‐type process, and a simple and scalable process remains a challenge. In the present study, a continuous flow process for the seed‐mediated growth of silica–gold nanoshells is established by exploiting the excellent mixing performance of a microreactor. In the AuNP‐seeding step, the reduction of gold ions in the presence of core particles in the microreactor enables the one‐step flow synthesis of gold‐decorated silica particles through heterogeneous nucleation. Flow shell growth is also realized using the microreactor by selecting an appropriate reducing agent. Because self‐nucleation in the bulk solution phase is suppressed in the microreactor system, no washing is needed after each step, thus enabling the connection of the microreactors for the seeding and shell growth steps into a sequential flow process to synthesize gold nanoshells. The established system is simple and robust, thus making it a promising technology for producing gold nanoshells in an industrial setting.     

The Competitive Dynamic Binding of Some Blood Proteins Adsorbed on Gold Nanoparticles

Nanoparticle (NP) surfaces are modified immediately by the adsorption of proteins when injected into human blood, leading to the formation of a protein corona. The protein‐coated NPs may be recognized by living cells. Furthermore, the adsorption of serum proteins is a continuous competitive dynamic process that is the key to exploring the bioapplication and biosafety of NPs. In this study, the competitive dynamic adsorption of some serum proteins on gold nanoparticles (AuNPs) is investigated by fluorescence emission, dynamic light scattering, and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Serum proteins with different AuNPs binding affinities are used to address the competitive dynamic process of protein‐AuNP interactions in vitro. The results show that more abundant serum proteins, such as human serum albumin, adsorb on AuNPs first, and then the higher binding affinity and lower concentration serum proteins, such as fibrinogen (FIB), replace the abundant and lower binding affinity serum proteins. However, the lower binding affinity serum proteins, such as hemoglobin, do not replace the higher binding affinity proteins from the protein‐AuNP conjugates. During the dynamic exchange process, the larger the binding affinities difference between two proteins, the faster the exchange rate. This dynamic exchange process usually takes longer in inner protein‐AuNP conjugates (hard corona) than the external surface of protein‐AuNP conjugates (soft corona).     

Raman spectroscopy of combinatory anticancer drug release from gold nanoparticles inside a single leukemia cell

Combinatory anticancer drug release from gold nanoparticles (AuNPs) in K562 human myeloid leukemia cells was performed using Raman spectroscopy. We fabricated the anticancer drug of imatinib as a BCR‐ABL tyrosine kinase inhibitor on AuNP surfaces along with a transferrin (Tf)‐targeting moiety to treat the leukemia cells. DNA topoisomerase I inhibitor topotecan was also assembled to monitor its fluorescence onto AuNPs. The linker group of 4‐carboxylic benzoic acid was used to conjugate to targeting the Tf protein. Our Raman data indicated that the drug molecules were not detached in the cell culture media but released after treatment with glutathione (2 mM). Intracellular distribution and release of the anticancer drug–AuNP conjugates in K562 cells were examined by both fluorescence microscopy and dark‐field microscopy with surface‐enhanced Raman scattering. Copyright © 2013 John Wiley & Sons, Ltd.     

Nanoparticle Probes for Quantifying Supramolecular Determinants of Biosurface Affinity

Interactions between macromolecular systems and biosurfaces are complicated by the complexity of these multivalent interactions and challenges in quantifying affinities. In this study, a library of gold nanoparticles (AuNPs) with different functional head groups as multivalent probes to quantify biosurface affinity, using hair as a model targeted substrate, is used. The adhesion of the AuNPs is quantified by inductively coupled plasma mass spectrometry. Using this method it is demonstrated that multiple supramolecular forces affect affinity. As expected, electrostatic interaction is a strong driving force for adhesion of the nanoparticle tags onto hair in aqueous solution, evidenced by a much higher level of gold adsorption for cationic AuNPs compared to anionic or neutral AuNPs. Functionalized cationic AuNPs are synthesized with systematically varied terminal groups and are screened for deposition onto hair. AuNP adhesion onto hair in water generally decreases as a function of increasing hydrophobicity; however, electron‐rich aromatic rings provide significantly enhanced attachment. Although the intact, healthy hair cuticle is considered negatively charged and hydrophobic, the findings indicate that hydrophobic interactions are not as critical to deposition of AuNPs onto hair as the electrostatic component from the presence and accessibility of the cationic moieties, which are the greatest drivers for deposition onto hair.     

A Correlative Analysis of Gold Nanoparticles Internalized by A549 Cells

Fluorescently labeled nanoparticles are widely used to investigate nanoparticle cell interactions by fluorescence microscopy. Owing to limited lateral and axial resolution, nanostructures (<100 nm) cannot be resolved by conventional light micro­scopy techniques. Especially after uptake into cells, a common fate of the fluorescence label and the particle core cannot be taken for granted. In this study, a correlative approach is presented to image fluorescently labeled gold nanoparticles inside whole cells by correlative light and electron microscopy (CLEM). This approach allows for detection of the fluorescently labeled particle shell as well as for the gold core in one sample. In this setup, A549 cells are exposed to 8 nm Atto 647N‐labeled gold nanoparticles (3.3 × 10⁹ particles mL^?1, 0.02 μg Au mL^?1) for 5 h and are subsequently imaged by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Eight fluorescence signals located at different intracellular positions are further analyzed by TEM. Five of the eight fluorescence spots are correlated with isolated or agglomerated gold nanoparticles. Three fluorescence signals could not be related to the presence of gold, indicating a loss of the particle shell.     

Nuclear Uptake of Gold Nanoparticles Deduced Using Dual‐Angle X‐Ray Fluorescence Mapping

Studies into the cell nucleus' incorporation of gold nanoparticles (AuNPs) are often limited by ambiguities arising from conventional imaging techniques. Indeed, it is suggested that to date there is no unambiguous imaging evidence for such uptake in whole cells, particularly at the single nanoparticle level. This shortcoming in understanding exists despite the nucleus being the most important subcellular compartment in eukaryotes and gold being the most commonly used metal nanoparticle in medical applications. Here, dual‐angle X‐ray flouresence is used to show individually resolved nanoparticles within the cell nucleus, finding them to be well separated and 79% of the intranuclear population to be monodispersed. These findings have important implications for nanomedicine, illustrated here through a specific exemplar of the predicted enhancement of radiation effects arising from the observed AuNPs, finding intranuclear dose enhancements spanning nearly five orders of magnitude.     

界面自组装的金/氧化石墨烯复合材料的表面增强拉曼散射行为研究

采用Frens法制备金纳米粒子溶胶,通过界面自组装技术在掺磷的非晶碳衬底表面构筑三维的金/氧化石墨烯/金复合结构.以罗丹明B为探针分子,考察金/氧化石墨烯/金复合材料的表面增强拉曼散射活性.结果表明,由于氧化石墨烯的化学增强和金纳米粒子的电磁场增强的协同作用,在该三维复合材料上获得了很强的罗丹明B拉曼信号.所设计的三维金/氧化石墨烯/金复合材料在生物分析、环境监测、疾病防控、食品安全等领域具有潜在的应用价值.     

Optimization of Gold Nanoparticle-Based DNA Detection for Microarrays

DNA microarrays are promising tools for fast and highly parallel DNA detection by means of fluorescence or gold nanoparticle labeling. However, substrate modification with silanes (as a prerequisite for capture DNA binding) often leads to inhomogeneous surfaces and/or nonspecific binding of the labeled DNA. We examined both different substrate cleaning and activating protocols and also different blocking strategies for optimizing the procedures, especially those for nanoparticle labeling. Contact angle measurements as well as fluorescence microscopy, atomic force microscopy (AFM), and a flatbed scanner were used to analyze the multiple-step process. Although the examined different cleaning and activating protocols resulted in considerably different contact angles, meaning different substrate wettability, silanization led to similar hydrophobic surfaces which could be revealed as smooth surfaces of about 2–4 nm roughness. The two examined silanes (3-glycidoxypropyltrimethoxysilane (GOPS) and 3-aminopropyltriethoxysilane (APTES)) differed in their DNA binding homogeneity, maximum signal intensities, and sensitivity. Nonspecific gold binding on APTES/PDC surfaces could be blocked by treatment in 3% bovine serum albumin (BSA).     

Spectroscopic investigations on the interaction between cadmium telluride semiconductor nanoparticle and bovine alkaline phosphatase

The interaction of enzymes with nanoparticles is important in the field of biotechnology and medicine. Due to the various uses of cadmium telluride nanoparticle in protein science, biotechnology and biophysical chemistry, drug delivery, and cellular imaging, study of this nanoparticle interaction with protein seems to be necessary. Therefore, the interaction between cadmium telluride semiconductor nanoparticle and bovine alkaline phosphatase, a clinical marker enzyme, were investigated by assaying kinetic parameters and fluorescence absorption, UV–vis absorption spectra, and circular dichroism spectroscopic techniques. Obtained results showed that cadmium telluride nanoparticle could quench the fluorescence signal of bovine alkaline phosphatase effectively with a static quenching mechanism. Moreover, the binding of cadmium telluride nanoparticle to the enzyme was spontaneous and van der Waals and hydrogen bonding forces played a key role in the complex stabilization. Circular dichroism spectra measurements indicated that cadmium telluride nanoparticle decreased α-helical content and increased the β-sheet structure of bovine alkaline phosphatase. These findings suggest that cadmium telluride nanoparticle changes the structure and activity of bovine alkaline phosphatase.     

The Effect of ζ‐Potential and Hydrodynamic Size on Nanoparticle Interactions in Hydrogels

The ζ‐potential and hydrodynamic size (d_h) of nanoparticles (NPs) are systematically controlled by capping gold NPs (AuNPs) with polymers having different charges and treating them in NaCl solutions of diverse concentrations. Interactions between AuNPs in hydrogel are caused by chemical reactions induced by 1,4‐dithiothreitol. The effect of ζ‐potential is clear, as negatively charged AuNPs can be aggregated in neutral agarose gel, but the amount of aggregation is significantly affected by the magnitude of the negative surface charge on the AuNPs. However, all positively charged AuNPs show negligible aggregation in agarose gel with slightly negative polarity. The effect of d_h on AuNP aggregation is different from that of ζ‐potential. Although AuNPs with small d_h generally show more aggregation than those with large d_h, the amount of AuNP capping layer is critical. Thus, the amount of polymer present on NP surface needs to be considered to investigate the effect of d_h on AuNP aggregation. Through extended Derjaguin, Landau, Verwey, Overbeek (XDLVO) theory, it is shown that the charges of the AuNPs and the hydrogel, as well as the d_h of the NPs, are related to electrostatic repulsion and steric hindrance, which affect AuNP aggregation in hydrogel.     

ICP-MS: a powerful technique for quantitative determination of gold nanoparticles without previous dissolving

A direct and simple inductively coupled plasma mass spectroscopy (ICP-MS) method for the determination of gold nanoparticles (AuNP) with different particle sizes ranging from 5 to 20 nm and suspended in aqueous solutions is described. The results show no significant difference compared to the determination of the same AuNPs after digestion, as claimed by the literature. The obtained limit of quantification of the method is 0.15 μg/L Au(III) that corresponds to 4.40 × 10⁹ AuNP/L, considering spherical AuNPs 15 nm sized. Spike recovery experiments have shown that the sample matrix is a significant factor influencing the accuracy of the measurement. Spike recoveries from 93% to 95% are found for AuNP samples prepared in trisodium citrate, while for deionized H₂O a spike recovery of around 80% was obtained. The sample preparation mode along with the ICP-MS parameters have been optimized and found to be crucial so as to achieve the required accuracy for the direct quantification of AuNP suspensions. The effect of the nanoparticle size upon the ICP-MS signal also was studied, and only significant differences due to the chemical environment and not to the AuNPs size were found.     

Metal–Polymer Hybrid Nanoparticles for Correlative High‐Resolution Light and Electron Microscopy

The combination of fluorescence microscopy and electron microscopy promises a deeper insight into the ultrastructural features of cell organelles, e.g., after drug administration. Both methods complement each other and provide, as a correlative approach, a keen insight into the fate of nanoparticles within the cell. Moreover, it represents a promising tool to determine alterations of the cellular environment as a response to particle uptake. However, the availability of suitable correlative markers is mandatory for such correlative approaches. In this contribution, the utilization of poly(ethylene imine) based metal–polymer hybrid particles labeled with small gold nanoparticles and Rhodamine B facilitating the observation of the particles by means of fluorescence as well as by transmission electron microscopy is suggested. Correlative light and electron microscopy is used to study uptake and intracellular fusion processes of endosomal/lysosomal structures.     

Electrochemical and surface characterisation of gold nanoparticle decorated multi-walled carbon nanotubes

A novel type of gold nanoparticle/multi-walled carbon nanotube (AuNP/MWCNT) composite electrodes is presented. The electrochemical reduction of oxygen on these hybrid electrodes was studied using the rotating disk electrode (RDE) method. The AuNP/MWCNT nanocomposites were prepared by sputter deposition of gold in argon atmosphere on MWCNTs followed by heat-treatment of the catalyst at different temperatures. High-resolution scanning electron microscopy (HR-SEM), glancing incidence angle X-ray powder diffraction (GIXRD) and small-angle X-ray scattering (SAXS) techniques were employed to characterise the surface structure and morphology of catalyst materials. Au nanoparticles with diameter around 20 nm were dispersed at the tips and on the sidewalls of nanotubes. Electrochemical measurements were performed to demonstrate the electrocatalytic properties of the composite catalysts towards O₂ reduction in acid media. The successful preparation of AuNP/MWCNT nanocomposites by magnetron sputtering opens up the possibility of making an efficient dispersion of nanoparticles for electrocatalyst design.     

Gold Nanoparticles Stabilized by Single Tripodal Ligands

In order to coat the entire surface of gold nanoparticles (AuNPs) by a single ligand, tripodal macromolecules comprising benzylic thioethers coordinating to the AuNP surface are synthesized and their abilities to stabilize AuNPs are investigated. Out of the five studied ligands 1 – 5 , the tetraphenylmethane‐based oligomers 4 and 5 display excellent AuNP coating features. Both ligand structures are able to control the dimensions of the AuNPs by stabilizing particles of narrow size distributions during their syntheses (1.05 ± 0.28 nm for Au‐4 , and 1.15 ± 0.34 nm for Au‐5 ). Closer inspection of these AuNPs by transmission electron microscopy and thermogravimetric analyses suggests that single ligands 4 and 5 are able to stabilize entire AuNPs. These particles Au‐4 and Au‐5 are obtained in good yields and display promising thermal stabilities (110 °C for Au‐4 , and 95 °C for Au‐5 ), making them interesting nanoscale inorganic–organic building blocks for further functionalization/processing by wet chemistry.     

Development of a Liver‐Targeting Gold–PEG–Galactose Nanoparticle Platform and a Structure–Function Study

For the specific liver parenchymal cell delivery, a series of short heterobifunctional poly(ethylene glycol) (PEG) derivatives containing dimercapto and galactose (Gal) terminals is synthesized for the preparation of gold conjugates. The Gal density on the surface of all gold conjugates can be well controlled and the prepared gold conjugates are stable in various media, even in the presence of serum. For the liver targeting and reflectance imaging applications, the structure–function relationships of this platform, including the influence of the PEG molecular weight and the Gal ligand coverage of hybrid particles on the cytotoxicity and cellular recognition of tumor cells in vitro and on their liver‐targeting ability in small animals, are studied. Biocompatibility results show that HepG2 cells are more sensitive than HeLa cells to gold conjugates. Cellular uptake studies demonstrate that a lower PEG molecular weight, a higher Gal density, or a higher gold concentration can increase the cellular uptake efficiency of these hybrid particles in HepG2 cells when the other parameters are constant. The results reveal the importance of parameter modulation for the design and control of nanoprobes and the gold conjugates with short PEG chains and a high Gal density are a potential vector for active‐targeting therapy.