Spatially Uniform and Quantitative Surface-Enhanced Raman Scattering under Modal Ultrastrong Coupling Beyond Nanostructure Homogeneity Limits.

We developed a substrate that enables highly sensitive and spatially uniform surface-enhanced Raman scattering (SERS). This substrate comprises densely packed gold nanoparticles (d-AuNPs)/titanium dioxide/Au film (d-ATA). The d-ATA substrate demonstrates modal ultrastrong coupling between localized surface plasmon resonances (LSPRs) of AuNPs and Fabry-Pérot nanocavities. d-ATA exhibits a significant enhancement of the near-field intensity, resulting in a 78-fold increase in the SERS signal for crystal violet (CV) compared to that of d-AuNP/TiO2 substrates. Importantly, high sensitivity and a spatially uniform signal intensity can be obtained without precise control of the shape and arrangement of the nanoscale AuNPs, enabling quantitative SERS measurements. Additionally, SERS measurements of rhodamine 6G (R6G) on this substrate under ultralow adsorption conditions (0.6 R6G molecules/AuNP) show a spatial variation in the signal intensity within 3%. These findings suggest that the SERS signal under modal ultrastrong coupling originates from multiple plasmonic particles with quantum coherence.

Size Segregation of Gold Nanoparticles into Bilayer-like Vesicular Assembly.

Size segregation of nanoparticles with different sizes into highly ordered, unique nanostructures is important for their practical applications. Herein, we demonstrate spontaneous self-assembly of the binary mixtures of small and large gold nanoparticles (GNPs; 5/15, 5/20, or 10/20 in diameter) in the presence of a tetra(ethylene glycol)-terminated octafluoro-4,4'-biphenol ligand, namely, TeOFBL, resulting in a size-segregated assembly. The outer single layer of large GNPs forming a gold nanoparticle vesicle (GNV) encapsulated the inner vesicle-like assembly composed of small GNPs, which is referred to as bilayer-like GNV and similar to the molecular bilayer structure of a liposome. The size segregation was driven by the solvophobic feature of the TeOFBLs on the surface of GNPs. A time-course study indicated that size segregation occurred instantaneously during the mixing stage of the self-organization process. The size-segregated precursors quickly fused with each other through the inner-inner and outer-outer layer fashion to form the bilayer-like GNV. This study provides a new approach to creating biomimetic bilayer capsules with different physical properties for potential applications such as surface-enhanced Raman scattering and drug delivery.

Hysteresis in the Thermo-Responsive Assembly of Hexa(ethylene glycol) Derivative-Modified Gold Nanodiscs as an Effect of Shape.

Anisotropic gold nanodiscs (AuNDs) possess unique properties, such as large flat surfaces and dipolar plasmon modes, which are ideal constituents for the fabrication of plasmonic assemblies for novel and emergent functions. In this report, we present the thermo-responsive assembly and thermo-dynamic behavior of AuNDs functionalized with methyl-hexa(ethylene glycol) undecane-thiol as a thermo-responsive ligand. Upon heating, the temperature stimulus caused a blue shift of the plasmon peak to form a face-to-face assembly of AuNDs due to the strong hydrophobic and van der Waals interactions between their large flat surfaces. Importantly, AuNDs allowed for the incorporation of the carboxylic acid-terminated ligand while maintaining their thermo-responsive assembly ability. With regard to their reversible assembly/disassembly behavior in the thermal cycling process, significant rate-independent hysteresis, which is related to their thermo-dynamics, was observed and was shown to be dependent on the carboxylic acid content of the surface ligands. As AuNDs have not only unique plasmonic properties but also high potential for attachment due to the fact of their flat surfaces, this study paves the way for the exploitation of AuNDs in the development of novel functional materials with a wide range of applications.

Visualization of DNA Replication in Single Chromosome by Stable Isotope Labeling.

Among the inheritance of cellular components during cell division, deoxyribonucleic acid (DNA) and its condensate (chromosome) are conventionally visualized using chemical tag-labeled nucleotide analogs. However, associated mutagenesis with nucleotide analogs in the visualization of chromosomes is cause for concern. This study investigated the efficiency of using stable isotope labels in visualizing the replicating cultured human cell-chromosomes, in the absence of analog labels, at a high spatial resolution of 100 nm. The distinct carbon isotope ratio between sister chromatids reflected the semi-conservative replication of individual DNA strands through cell cycles and suggested the renewal of histone molecules in daughter chromosomes. Thus, this study provides a new, powerful approach to trace and visualize cellular components with stable isotope labeling.Key words: stable isotope, chromosome replication, semi-conservative replication, imaging, mass spectrometry.

Vesicle Formation by the Self-Assembly of Gold Nanoparticles Covered with Fluorinated Oligo(ethylene glycol)-Terminated Ligands and Its Stability in Aqueous Solution.

Water-stable gold nanoparticle vesicles (GNVs) with hollow interiors have attracted attention due to their great potential for biological applications; however, their preparation through the self-assembly approaches has been restricted due to the limited understanding of their critical mechanistic issues. In this paper, we demonstrate that a fluorinated tetra (ethylene glycol) (FTEG)-terminated tetra (ethylene glycol) (EG4), namely, FTEG-EG4, ligand can self-assemble with gold nanoparticles (5 and 10 nm) into GNVs with a hollow structure in THF due to the solvophobic feature of the ligand. Time-dependent studies showed that the GNVs with a closely packed surface derived from the incomplete and irregular GNVs, but not through the fusion of the GNV precursors. After dialysis in water, the assemblies retained vesicular structures in water, even though GNVs aggregated together, which was initiated by the hydrophobic interactions between the FTEG heads of the surface ligands on GNVs. This study provides a new insight into the design of novel small surface ligands to produce water-stable GNVs for biological applications.

Molecular configuration-mediated thermo-responsiveness in oligo(ethylene glycol) derivatives attached on gold nanoparticles.

Biomolecular systems actively control their local environment on a sub-nm scale via changes in molecular configuration from their flexible structures and derive emergent functions. Although this functional emergence based on local environmental control is attracting a great deal of attention in chemistry, it remains challenging to realize this artificially. Herein, we report the tuning of the thermo-responsive properties of oligo(ethylene glycol) (OEG) derivatives attached on gold nanoparticles via local environmental control not only by the hydrophobic moiety at their terminus but also by their molecular configuration. OEG-attached alkane thiol-modified AuNPs showed thermo-responsive assembly/disassembly in water through the hydration/dehydration of the OEG portions in a manner dependent both on the hydrophobicity at their terminus and the surface curvature of the core nanoparticles. Further, the assembly temperature (T A) was also tuned by ligand mixing with a non-thermo-responsive ligand with a shorter OEG length. Molecular dynamics simulations show that the distribution of the hydrophobic terminus in the normal direction along the gold surface varied in accordance with the surface curvature, indicating variations in molecular configuration. It is expected that a bent configuration could accelerate the thermo-responsiveness of OEG by allowing them greater accessibility to the hydrophobic terminus. Experimental and simulation results support the notion that local OEG density tuning by surface curvature or ligand mixing with a different OEG length leads to different degrees of accessibility to the hydrophobic terminus via changes in molecular configuration, promoting local environmental control-directed assembly temperature tuning.

Micro-liquid enclosure array and its semi-automated assembling system for x-ray free-electron laser diffractive imaging of samples in solution.

We developed micro-liquid enclosure arrays (MLEAs) for holding solution samples in coherent diffractive imaging (CDI) using x-ray free-electron lasers (XFELs). Hundreds of fully isolated micro-liquid enclosures are arranged in a single MLEA chip for efficient measurement, where each enclosure is destroyed after exposure to a single XFEL pulse. A semi-automated MLEA assembling system was also developed to enclose solution samples into MLEAs efficiently at high precision. We performed XFEL-based CDI experiments using MLEAs and imaged in-solution structures of self-assembled gold nanoparticles. The sample hit rate can be optimized by adjusting solution concentration, and we achieved a single-particle hit rate of 31%, which is not far from the theoretical upper limit of 37% derived from the Poisson statistics. MELAs allow us to perform CDI measurement under controlled solution conditions and will help reveal the nanostructures and dynamics of particles in solution.

Intrahelical Interactions in an α-Helical Coiled Coil Determine the Structural Stability of Tropomyosin.

Tropomyosin (Tpm) is a two-stranded parallel α-helical coiled-coil protein, and studying its structure is crucial for understanding the nature of coiled coils. Previously, we found that the N-terminal half of the human skeletal muscle α-Tpm (α-Tpm 140) was less structurally stable in the presence of phosphate ions than the coiled-coil protein carrier (CCPC) 140 variant with 18 mutated residues, in which all amino acid residues located at the interface between the two α-helices were completely conserved. A classical hypothesis explains that interhelical interactions stabilize the coiled-coil structure. In this study, we tested the hypothesis that the structural stability of Tpm and its variant is governed by the binding of multivalent ions that form a bridge between charged side chains located at positions b, c, and f of the heptad repeat on a single α-helical chain. We found that the structural stability of α-Tpm 140 and CCPC 140 markedly increased upon addition of divalent cations and divalent anions, respectively. We also clarified that the structural stability of the α-Tpm 140/CCPC 140 heteromeric coiled-coil molecule was governed by the stability of a less stable α-helical chain. These results demonstrated that the entire structural stability of Tpm is determined by the stability of a single α-helix. Our findings provide new insights into the study of the structure of coiled-coil proteins.

Strategy for Finely Aligned Gold Nanorod Arrays Using Polymer Brushes as a Template.

The development of a strategy for the assembly of nanoscale building blocks, in particular, anisotropic nanoparticles, into desired structures is important for the construction of functional materials and devices. However, control over the orientation of rod-shaped nanoparticles on a substrate for integration into solid-state devices remains challenging. Here, we report a strategy for the fabrication of finely aligned gold nanorod (GNR) arrays using polymer (DNA) brushes as a nanoscale template. The gold nanorods modified with cationic surface ligands were electrostatically adsorbed onto the DNA brush substrates under various conditions. The orientational behavior of the GNRs was examined by spectral analyses and transmission electron microtomography (TEMT). As a result, we found several important factors, such as moderate interaction between GNRs and polymers and polymer densities on the substrate, related to the vertical alignment of GNRs on the substrates. We also developed a purification method to remove the undesired adsorption of GNRs onto the arrays. Finally, we have succeeded in the fabrication of extensive vertical GNR arrays of high quality via the easy bottom-up process.

Reversible changes in the orientation of gold nanorod arrays on polymer brushes.

Nanoparticles exhibit a number of unique properties such as localized surface plasmon resonance (LSPR). As this LSPR is sensitive to geometrical or spatial conditions, the arrangement of nanoparticles, in particular the active arrangement of plasmonic structures, is an important issue. In this study, gold nanorod (GNR) arrays were prepared by GNR attachment on anionic polymer (DNA) brushes via electrostatic interactions and their stimuli-responsive changes in orientation were investigated. As a result, the orientation of GNR arrays on DNA brushes reversibly changed by the modulation of electrostatic interactions between GNRs and polymers via changes in the solution pH. As these extensive GNR arrays are prepared via easy bottom-up processes, GNR surface properties are easily tuned by simple modification, and DNAs could be replaced with various synthetic polymers, we believe that this study will lead to the development of next-generation materials and devices with actively tunable structures.

Correction to "DNA Brush-Directed Vertical Alignment of Extensive Gold Nanorod Arrays with Controlled Density".

[This corrects the article DOI: 10.1021/acsomega.7b00303.].

Nanoscale uniformity in the active tuning of a plasmonic array by polymer gel volume change.

Active plasmonic tuning is an attractive but challenging research subject, leading to various promising applications. As one of the approaches, nanostructures are placed in or on soft matter, such as elastomers and gels, and their gap distances are tuned by the mechanical extension or volume change of the supporting matrices. As hydrogels possess various types of stimuli-responsiveness with large volume change and biocompatibility, they are good candidates as supporting materials for active nanostructure tuning. However, it remains unclear how accurately we can control their nanogap distance changes using polymer gels with a low deviation due to major difficulties in the precise observation of nanostructures on the gels. Here, we prepared gold arrays with sub-100 nm dots on silicon substrates by electron beam lithography and transferred them onto the hydrogel surface. Then, their nanopattern was actively tuned by the changes in gel size in water and their structural changes were confirmed by optical microscopy, microspectroscopy, and atomic force microscopy (AFM). Further, we successfully prepared ionic liquid (IL) gels with various degrees of swelling via solvent exchange. Scanning electron microscopy (SEM) observation of the IL gels provided clear pictures at nanoscale resolution. Finally, we calculated the plasmonic spectra using a finite difference time domain (FDTD) simulation based on the SEM images and compared them with the measured spectra. The results in this study totally support the notion that active changes in plasmonic nanodot patterns via volume changes in the hydrogel are quite homogenous on a several nanometer scale, making them ideal for precise active surface plasmon tuning.

Size-Defined Cracked Vesicle Formation via Self-Assembly of Gold Nanoparticles Covered with Carboxylic Acid-Terminated Surface Ligands.

The self-assembly of gold nanoparticles (GNPs) into a defined structure, particularly hollow capsule structures, provides great potential for applications in materials science and medicine. However, the complexity of the parameters for the preparation of those structures through self-assembly has limited access to critical mechanistic questions. With this in mind, we have studied GNP vesicle (GNV) formation through self-assembly by the surface modification of GNPs with low-molecular-weight ligands. Here, we successfully prepared GNVs composed of GNPs with a diameter of 30 nm by surface modification with carboxylic acid-terminated fluorinated oligo(ethylene glycol) ligands (CFLs). As the carboxylic acid has two states (protonated and deprotonated), the balance of the attraction and repulsion between GNPs covered with CFLs is tunable. Sodium carboxylate-terminated fluorinated oligo(ethylene glycol) ligands (SCFLs) provided smaller GNVs than did CFLs at 0.8 × 1011 NPs/mL. Time-course study revealed that CFL-covered GNPs quickly form small aggregates and gradually grow to larger GNVs (ca. 200 nm), but no gradual growth was observed for SCFL-covered GNPs. This result indicated that the electrostatic repulsion inhibits fusion of the small GNVs. The size of the GNVs formed with the aid of CFLs was independent of the initial GNP concentration, but the extinction spectra were concentration-dependent. Electron microscopy imaging and simulations supported the defect formation in the assemblies. These results provided new insights into the vesicle formation mechanism.

Self-Priming Enzymatic Fabrication of Multiply Modified DNA.

The self-priming synthesis of multiply modified DNA by the extension of repeating unit duplex "oligoseeds" provides a source of versatile DNA. Sterically-demanding nucleotides 5-Br-dUTP, 7-deaza-7-I-dATP, 6-S-dGTP, 5-I-dCTP as well as 5-(octadiynyl)-dCTP were incorporated into two extending oligoseeds; [GATC]5 /[GATC]5 and [A4 G]4 /[CT4 ]4 . The products contained modifications on one or both strands of DNA, demonstrating their recognition by the polymerase as both template (reading) and substrate (writing). Nucleobase modifications that lie in the major groove were reliably read and written by the polymerase during the extension reaction, even when bulky or in contiguous sequences. Repeat sequence DNA over 500 bp long, bearing four different modified units was produced by this method. The number, position and type of modification, as well as the overall length of the DNA can be controlled to yield designer DNA that offers sequence-determined sites for further chemical adaptations, targeted small molecule binding studies, or sensing and sequencing applications.

Two-Step Assembly of Thermoresponsive Gold Nanorods Coated with a Single Kind of Ligand.

Gold nanorods (GNRs) coated with a single kind of ligand show thermoreponsive two-step assembly to provide a hierarchical structure. The GNRs (33 nm in length × 14 nm in diameter) coated with a hexa(ethylene glycol) (HEG) derivative form side-by-side assemblies at 30 °C (TA1 ) as a steady state through dehydration. By further heating to over 40 °C (TA2 ), larger assemblies, which are composed of the side-by-side assembled units, are formed as hierarchical structures. The dehydration temperature of the HEG derivative varies depending on the free volume of the HEG unit, which corresponds to the curvature of the GNRs. Upon heating, dehydration first occurs from the ligands on the side portions with a lower curvature, and then from the ligands on the edge portions with a higher curvature. The different sized GNRs (33 × 8 and 54 × 15 nm) also show two-step assembly. Both the TA1 and TA2 are dependent on the diameter of the GNRs, but independent of their length. This result supports that the dehydration is dependent on the free volume, which corresponds to the curvature. Anisotropic assembly focusing on differences in curvature provides new guidelines for the fabrication of hierarchical structures.

Shape-dependent adjuvanticity of nanoparticle-conjugated RNA adjuvants for intranasal inactivated influenza vaccines.

Intranasal inactivated influenza vaccines can elicit mucosal immune responses that protect against virus infection. For the development of intranasal inactivated influenza vaccines, effective adjuvants inducing minimal adverse reactions are required. Generally, however, lower toxicity adjuvants have lower adjuvanticity. In this research, we fabricated nanoparticle-based adjuvants to enhance its adjuvanticity. Herein, we focused on low-molecular-weight polyinosinic-polycytidylic acid, referred to as uPIC(40:400), as a weak and less toxic RNA adjuvant. We conjugated uPIC(40:400) with different shaped gold nanoparticles (AuNPs) electrostatically. Conjugation with gold nanorods, but not spherical AuNPs, markedly enhanced the adjuvanticity of uPIC(40:400), leading to the suppression of viral infection in mice. Notably, conjugation with gold nanorods did not increase the inflammatory cytokine production in dendritic cells. These data indicated that gold nanorods can provide a good platform for enhancing the weak adjuvanticity of uPIC(40:400) while maintaining low inflammatory cytokine production toward the development of intranasal inactivated influenza vaccines.

pH-Responsive Coassembly of Oligo(ethylene glycol)-Coated Gold Nanoparticles with External Anionic Polymers via Hydrogen Bonding.

Stimuli-responsive assembly of gold nanoparticles (AuNPs) with precise control of the plasmonic properties, assembly size, and stimuli responsivity has shown potential benefits with regard to biosensing devices and drug-delivery systems. Here we present a new pH-responsive coassembly system of oligo(ethylene glycol) (OEG)-coated AuNPs with anionic polymers as an external mediator via hydrogen bonding in water. Hydrogen-bond-driven coassemblies of OEG-AuNPs with poly(acrylic acid) (PAA) were confirmed by the monitoring of plasmonic peaks and hydrodynamic diameters. In this system, the protonation of anionic polymers on change in pH triggered the formation of hydrogen bond between the OEG-AuNPs and polymers, providing sensitive pH responsivity. The plasmonic properties and assembly size are affected by both the ratio of PAA to AuNPs and the molecular weight of PAAs. In addition, the attachment of hydrophobic groups to the surface ligand or anionic polymer changed the responsive pH range. These results demonstrated that the coassembly with an external mediator via hydrogen bonding provides a stimuli-responsive assembly system with tunable plasmonic properties, assembly size, and stimuli responsivity.

DNA Brush-Directed Vertical Alignment of Extensive Gold Nanorod Arrays with Controlled Density.

Control over the orientation of metal nanorods is important for both fundamental and applied research. We show that gold nanorods (GNRs) can be aligned in a single direction by adsorbing positively charged GNRs onto a double-strand DNA-grafted substrate through electrostatic interaction. The ordered structure can be optimized by controlling the density of the positive charges on the surface of the GNRs. We found, in agreement with the results of theoretical simulation, that the resultant structure exhibits plasmonic properties that are dependent on the GNR orientation relative to the direction of an oscillating electric field. Our approach provides new insights into the polymer-assisted self-assembly of rod-shaped nanoparticles utilizing electrostatic interactions.

Reverse Size Dependences of the Cellular Uptake of Triangular and Spherical Gold Nanoparticles.

Gold nanoparticles (GNPs) show promise as both drug and imaging carriers with applications in both diagnosis and therapy. For the safe and effective use of such gold nanomaterials in the biomedical field, it is crucial to understand how the size and shape of the nanomaterials affect their biological features, such as in vitro cellular uptake speed and accumulation as well as cytotoxicity. Herein, we focus on triangular gold nanoparticles (TNPs) of four different sizes (side length 46, 55, 72, and 94 nm; thickness 30 nm) and compare the cellular internalization efficiency with those of spherical nanoparticles (SNPs) of various diameters (22, 39, and 66 nm). Both surfaces were coated with anionic thiol ligands. Inductively coupled plasma-emission spectrometry (ICP-ES) data demonstrated that TNPs with longer sides showed higher levels of uptake into RAW264.7 and HeLa cells. On the other hand, in the case of SNPs, those with smaller diameters showed higher levels of uptake in both cells. Our results support the notion of a reverse size dependence of TNPs and SNPs in terms of cellular uptake. For HeLa cells, in particular, 20-fold more efficient internalization was observed for TNPs with longer sides (72 nm side length) compared to SNPs (66 nm) with a similar surface area. These results highlight the importance of the shape of nanomaterials on their interactions with cells and provide a useful guideline for the use of TNPs.

Fabrication of a Novel Cell Culture System Using DNA-Grafted Substrates and DNase.

In conventional cell culture systems, trypsin is generally used for cell harvesting. However, trypsin damages the cells due to the nonselective degradation of proteins on the cell surface. This is a critical issue for cell culture systems. Therefore, an alternative cell culture system with the lowest possible impact on cells is desired. In this paper, we have focused on DNA as a sacrificial layer and DNase as an alternate enzyme instead of trypsin. DNase ought not to result in damage to or stress on cells as it only hydrolyzes DNAs while the plasma membrane and extracellular matrices are basically composed of lipids, proteins, and glycosides. Therefore, we fabricated DNA-grafted substrates as cell culture dishes and evaluated this novel cell culture system. As a result, we were able to culture several types of mammalian cells on the DNA-grafted substrates, with the cells harvested using DNase with only little damage to the cells. This cell culture system could provide a breakthrough in cell culturing technology.