On-chip colorimetric detection of Cu2+ ions via density-controlled plasmonic core-satellites nanoassembly.

We report on an on-chip colorimetric method for the detection and analysis of Cu(2+) ions via the targeted assembly of plasmonic silver nanoparticles (2.6 nm satellites) on density-controlled plasmonic gold nanoparticles (50 nm cores) on a glass substrate. Without any ligand modification of the nanoparticles, by directly using an intrinsic moiety (carboxylate ion, COO(-)) surrounded with nanoparticles, the method showed a high selectivity for Cu(2+), resulting in a nearly 2 times greater optical response compared to those of other metal ions via the targeted core-satellites assembly. By modulating the surface chemistry, it was possible to control the density of core gold nanoparticles on the surface, thus permitting easy tuning of the optical responses induced by plasmon coupling generated between each core-satellites nanostructure. Using chips with a controlled optimal core density, we observed the remarkable scattering color changes of the chips from green to yellow and finally to orange with the increase of Cu(2+) concentration. The detection limits of the fabricated chips with controlled core densities (ca. 1821 and 3636 particles/100 µm(2)) are 10 nM and 10 pM, respectively, which are quite tunable and below the level of 20 µM (or 1.3 ppm) defined by the United States Environmental Protection Agency. The findings suggest that the method is a potentially promising protocol for detecting small molecules with target selectivity and the tunability of the detection limits by replacing with ligands and adjusting core densities.

Lipid molecules induce the cytotoxic aggregation of Cu/Zn superoxide dismutase with structurally disordered regions.

Cu/Zn-superoxide dismutase (SOD1) is present in the cytosol, nucleus, peroxisomes and mitochondrial intermembrane space of human cells. More than 114 variants of human SOD1 have been linked to familial amyotrophic lateral sclerosis (ALS), which is also known as Lou Gehrig's disease. Although the ultimate mechanisms underlying SOD1-mediated cytotoxicity are largely unknown, SOD1 aggregates have been strongly implicated as a common feature in ALS. This study examined the mechanism for the formation of SOD1 aggregates in vitro as well as the nature of its cytotoxicity. The aggregation propensity of SOD1 species was investigated using techniques ranging from circular dichroism spectroscopy to fluorescence dye binding methods, as well as electron microscopic imaging. The aggregation of SOD1 appears to be related to its structural instability. The demetallated (apo)-SOD1 and aggregated SOD1 species, with structurally disordered regions, readily undergo aggregation in the presence of lipid molecules, whereas metallated (holo)-SOD1 does not. The majority of aggregated SOD1s that are induced by lipid molecules have an amorphous morphology and exhibit significant cytotoxicity. The lipid binding propensity of SOD1 was found to be closely related to the changes in surface hydrophobicity of the proteins, even at very low levels, which induced further binding and assembly with lipid molecules. These findings suggest that lipid molecules induce SOD1 aggregation under physiological conditions and exert cytotoxicity, and might provide a possible mechanism for the pathogenesis of ALS.

Core-satellites assembly of silver nanoparticles on a single gold nanoparticle via metal ion-mediated complex.

We report core-satellites (Au-Ag) coupled plasmonic nanoassemblies based on bottom-up, high-density assembly of molecular-scale silver nanoparticles on a single gold nanoparticle surface, and demonstrate direct observation and quantification of enhanced plasmon coupling (i.e., intensity amplification and apparent spectra shift) in a single particle level. We also explore metal ion sensing capability based on our coupled plasmonic core-satellites, which enabled at least 1000 times better detection limit as compared to that of a single plasmonic nanoparticle. Our results demonstrate and suggest substantial promise for the development of coupled plasmonic nanostructures for ultrasensitive detection of various biological and chemical analytes.

Picomolar selective detection of mercuric ion (Hg(2+)) using a functionalized single plasmonic gold nanoparticle.

A highly sensitive method for the selective detection and quantification of mercuric ions (Hg(2+)) using single plasmonic gold nanoparticle (GNP)-based dark-field microspectroscopy (DFMS) is demonstrated. The method is based on the scattering property of a single GNP that is functionalized with thiolated molecules, which is altered when analytes bind to the functionalized GNP. The spectral resolution of the system is 0.26 nm and a linear response to Hg(2+) was found in the dynamic range of 100 pM-10 microM. The method permits Hg(2+) to be detected at the picomolar level, which is a remarkable reduction in the detection limit, considering the currently proscribed Environmental Protection Agency regulation level (10 nM, or 2 ppb) and the detection limits of other optical methods for detecting Hg(2+) (recently approx. 1-10 nM). In addition, Hg(2+) can be sensitively detected in the presence of Cd(2+), Pb(2+), Cu(2+), Zn(2+) and Ni(2+), which do not interfere with the analysis. Based on the findings reported herein, it is likely that single-nanoparticle-based metal ion sensing can be extended to the development of other chemo- and biosensors for the direct detection of specific targets in an intracellular environment as well as in environmental monitoring.

Selective aggregation of polyanion-coated gold nanorods induced by divalent metal ions in an aqueous solution.

A simple, accurate method for detecting metal ions in an aqueous solution using functionalized gold nanorods (AuNRs) is described. The method involves the complexing of divalent metal ions with poly(acrylic acid) (PAA) and, the localized surface plasmon resonance (LSPR) phenomena of AuNRs. Changes in the longitudinal surface plasmon bands (LSPBs) were monitored using aggregates of PAA-coated AuNRs with various divalent metal ions via UV-vis spectroscope. Functionalized AuNRs underwent robust aggregate formation by chelation with divalent metal ions (e.g., Cu2+, Zn2+, Cd2+, and Fe2+). Copper ions formed largest aggregates within 2 h, because complexation between Cu2+ and dicarboxylate has the highest deltaH and -deltaG values. This process represents an easy and useful method for detecting certain divalent metal ions, and the aggregates are also, in some cases, clearly visible to the naked eye.

Sensitive and colorimetric detection of the structural evolution of superoxide dismutase with gold nanoparticles.

The detection and characterization of protein aggregates are critical in terms of advanced diagnostic applications and investigations of protein stability. A variety of analytical methods (e.g., circular dichroism, size exclusion chromatography, and fluorescence microscopy) have been used in this regard, but they are limited in the trace detection of the structural evolution of protein aggregation. Here we report the gold nanoparticle (AuNP)-based highly sensitive and colorimetric detection of the temporal evolution of superoxide dismutase (SOD1) aggregates implicated in the pathology of amyotrophic lateral sclerosis (ALS). For the temporal discrimination of SOD1 aggregation, AuNPs were conjugated with SOD1 monomers (SOD1-AuNPs). Upon exposure of the probes (SOD1-AuNPs) with SOD1 aggregates, significant changes in both surface plasmon resonance spectra and concomitant colors were observed which are attributed to the formation of probe aggregates of variable sizes onto the SOD1 aggregates.

Surface-specific overgrowth of platinum on shaped gold nanocrystals.

Controlling the shape and composition of metal nanocrystals can be beneficial for tuning the optical or catalytic properties in a variety of applications. In this study, surface-specific overgrowth of platinum was found on shaped gold nanocrystals of cubes, octahedra and spheres. Platinum overgrowth was observed on the planar faces of gold cubes, while the overgrowth occurred at the vertices of gold octahedra, indicating that platinum was selectively reduced on the Au (100) surface for each gold nanocrystal shape. The platinum nuclei covered the entire surface for gold spheres, which don't have well-defined surfaces. As the Pt/Au ratio increased, a full platinum shell was formed. Solution-based UV-Vis absorption spectra of the composite nanocrystals showed that the absorption peak was red-shifted with increasing platinum coverage. The optical response of a single composite nanocrystal was measured by dark field microscope, which also demonstrated a red shift in the scattering spectrum with increasing platinum coverage. The extent of a red shift depended on the shape and composition of the composite nanocrystals.

Construction of pcAFM module to measure photoconductance with a nanoscale spatial resolution.

A photoconductive atomic force microscopy (pcAFM) module was designed and the performance was tested. This module consisted of three units: the conductive mirror-plate, the steering mirror and the laser source. The module with a laser irradiation unit was equipped to a conventional conducting probe atomic force microscopy (CP-AFM) instrument to measure photoconductance in a nanoscale resolution. As a proof-of-concept experiment, the photoconductance of aggregated fullerene on indium tin oxide (ITO) substrate was measured with this module. The electrical signals (currents) of aggregated fullerene under the conditions of laser on/off at about -10 V sample bias voltage were -100 to -160 nA and 0 to -20 nA, respectively. Results indicated that the pcAFM with this module allowed one to observe photoinduced changes of electrical properties in nanodevices with nanoscale spatial resolution.

Solution based, on chip direct growth of three-dimensionally wrinkled gold nanoparticles for a SERS active substrate.

This work reports a solution-based method for on chip growth of wrinkled gold nanoparticles. During the growth process, time-resolved scattering profiles were measured, which permitted one to collect information regarding the growth kinetics. Finally, using the fabricated substrate, a 30 times stronger SERS enhancement was achieved than a spherical nanoparticle immobilized substrate.

Real-time analysis and direct observations of different superoxide dismutase (SOD1) molecules bindings to aggregates in temporal evolution step.

The misfolding and intracellular aggregation of Cu-Zn superoxide dismutase (SOD1) is pathologically key feature of amyotrophic lateral sclerosis (ALS). Although details of the mechanisms continue to be unclear, there are key steps in the possible pathway to the development of ALS. This study focuses on interactions between different SOD1 molecules (A4V apo/holo, and WT apo/holo) and homogeneous aggregates in the temporal evolution step, and a determination of whether any of the SOD1 molecules are reactive to the aggregates with the extent of binding, as determined by surface plasmon resonance (SPR) measurements. Using a kinetic binding model, the association constant of A4V apo was found to be three times larger than that for the WT apo species. Differences in the extent of the interactions were also simultaneously measured and visualized by means of SPR imaging techniques. The SPR-based approach suggests direct correlation between SPR signal and the extent of molecular binding, which can identify the significant contributors to the formation of macroaggregates of SOD1 in the temporal evolution step.

Colorimetric tracking of protein structural evolution based on the distance-dependent light scattering of embedded gold nanoparticles.

In this communication, we describe a new, simplified colorimetric method for in situ tracking of structural evolution of Cu/Zn-superoxide dismutase (SOD1) aggregates, based on changes in plasmonic coupling between gold nanoparticles (GNPs) embedded along the structural backbone of the SOD1 aggregates.

Top-down shaping of metal nanoparticles in solution: partially etched Au@Pt nanoparticles with unique morphology.

Metallic nanoparticles with a unique morphology were produced by combining bottom-up nucleation/growth and top-down etching for Au core-Pt shell nanoparticles. Interesting optical and electrocatalytic properties were observed for the newly formed shapes.

The sensitive, anion-selective detection of arsenate with poly(allylamine hydrochloride) by single particle plasmon-based spectroscopy.

The use of single gold nanoparticle plasmon-based spectroscopy for the sensitive, anion-selective detection of arsenate is described. The method is based on the selective formation of electrostatic complexes between arsenate and poly(allylamine hydrochloride) (PAH) and changes in the single particle plasmon in Rayleigh scattering profiles. PAH, when modified with gold nanoparticles, binds arsenate via its amine-functionalities. The scattering properties of the resulting selectively formed complexes are altered, leading to significant changes in the surface plasmon resonance wavelength. The limit of detection of the method was determined to be 10 nM, which is ca. 13 times more sensitive than U.S. EPA regulation levels. The response is essentially linear in the concentration range of 50-300 nM. The method also shows good selectivity for arsenate in the presence of other environmentally relevant anions, including H(2)PO(4)(-), SO(4)(2-), NO(3)(-), and Cl(-).

Direct observation of defects and increased ion permeability of a membrane induced by structurally disordered Cu/Zn-superoxide dismutase aggregates.

Interactions between protein aggregates and a cellular membrane have been strongly implicated in many protein conformational diseases. However, such interactions for the case of Cu/Zn superoxide dismutase (SOD1) protein, which is related to fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS), have not been explored yet. For the first time, we report the direct observation of defect formation and increased ion permeability of a membrane induced by SOD1 aggregates using a supported lipid bilayer and membrane patches of human embryonic kidney cells as model membranes. We observed that aggregated SOD1 significantly induced the formation of defects within lipid membranes and caused the perturbation of membrane permeability, based on surface plasmon resonance spectroscopy, atomic force microscopy and electrophysiology. In the case of apo SOD1 with an unfolded structure, we found that it bound to the lipid membrane surface and slightly perturbed membrane permeability, compared to other folded proteins (holo SOD1 and bovine serum albumin). The changes in membrane integrity and permeability were found to be strongly dependent on the type of proteins and the amount of aggregates present. We expect that the findings presented herein will advance our understanding of the pathway by which structurally disordered SOD1 aggregates exert toxicity in vivo.

Fast image scanning method in liquid-AFM without image distortion.

High speed imaging by atomic force microscopy (AFM) allows one to directly observe the dynamic behavior of a sample surface immersed in liquid media; thus, it has been considered to be an indispensable tool for nanobiotechnology and is used in many research fields, including molecular biology and surface science. For real-time observation of a certain behavior, the high speed imaging technique should be accompanied with a high resolution imaging technique to identify target materials. To improve the image quality at a high scanning rate, we developed a variable-controlled fast scanning method, which originated from the modified squeeze-drag superposition model in liquid media. A collection of non-distorted images was accomplished after proper modification of the operating conditions in a viscous fluid, via the simple handling of loading force and cantilever length. Consequently, a speeded-up AFM imaging process was achieved in the liquid environment at up to 200 µm s(-1), without attachment of additional devices. The reliability of the proposed method was verified by the characterization of a grating sample immersed in three types of liquid media. In addition, the results were visualized for elastic biomolecules submerged in a liquid with high kinematic viscosity.

Directed positioning of single cells in microwells fabricated by scanning probe lithography and wet etching methods.

Scanning probe microscopy has emerged as a powerful technique for mapping the surface morphology of biological specimens, including proteins and cells. In addition to providing measurements of topographic images, it enables the fabrication of micro-/nanostructures with a high spatial resolution. Herein, we demonstrate a simple and reliable method for the preparation of single Escherichia coli bacterial cell arrays using pre-fabricated microwell structures. Using a <100>-oriented silicon substrate, microwell arrays with inclined sidewalls were fabricated by scanning probe lithography and sequential chemical wet etching. The trapping efficiency of single cells was optimized by controlling the geometries of the microwells. These data suggest that single-cell arrays may be applicable in a variety of areas, including drug testing and toxicology, as well as basic cell biology.