Suseung Lee

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.

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.

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.

Picomolar selective detection of mercuric ion (Hg2 + ) 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.

Automated quantification of retinal nerve fiber layer atrophy in fundus photograph

Quantitative analysis of the retinal nerve fiber layer (RNFL) defect is prerequisite in the early detection and management of glaucoma. A new automatic quantification method to evaluate the degree of RNLF defect has been proposed in this paper. Simple image processing technique is applied to locate optic disc and intensity of the pixels around optic disc is plotted. The area with RNFL defect can be easily determined by comparing the intensity plot of the RNFL and the first derivative of the intensity plot. Through analysis of the plot, thickness of RNFL also can be postulated.

Supplementation with spermine during in vitro maturation of porcine oocytes improves early embryonic development after parthenogenetic activation and somatic cell nuclear transfer

Spermine plays an important role in protection from reactive oxygen species (ROS) in bacteria, yeast, and mammalian cells, but there are few studies on the effects of spermine on porcine oocyte maturation and subsequent embryo development. The aim of this study was to determine the effects of spermine on in vitro maturation (IVM) of porcine oocytes and their developmental competence after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). We evaluated nuclear maturation, intracellular glutathione (GSH), and ROS levels in oocytes, and their subsequent embryonic development, as well as gene expression in mature oocytes, cumulus cells, and PA blastocysts. After treatment with various concentrations of spermine in IVM culture medium, there was no significant difference in nuclear maturation rate. However, spermine treatment groups (10- 500 µM) showed significantly increased intracellular GSH levels and decreased ROS levels compared to the control ( < 0.05). Furthermore, 10 µM spermine supported significantly higher blastocyst formation rates after PA than the control group ( < 0.05). According to the optimal condition from the PA results, we investigated the effects of 10 µM spermine on SCNT, and it also significantly improved blastocyst formation rates compared with the control group ( < 0.05). In evaluating the effects of 10 µM spermine on gene expression, there was significantly lower expression of a proapoptotic gene () and higher expression of an antiapoptotic gene () in cumulus cells ( < 0.05). was increased in spermine-treated oocytes. Levels of transcription for and were significantly increased in PA blastocysts. In conclusion, 10 µM spermine supplementation during IVM improved the development of porcine PA and SCNT embryos by increasing intracellular GSH, scavenging ROS levels, and regulating gene expression.

Simultaneous Optical Monitoring of the Overgrowth Modes of Individual Asymmetric Hybrid Nanoparticles

Nanoparticles that combine domains with different composi-tions (e.g., noble metals, iron oxide, organic compounds, orpolymer) have attracted considerable interest for biomedicalapplications, because two or more important functions, forexample, targeting, imaging, and therapy, can be combinedinto a single nanoparticle, and such a particle would increasethe precision and efficacy of diagnosis and treatment.

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.

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.

Sensitive and molecular size-selective detection of proteins using a chip-based and heteroliganded gold nanoisland by localized surface plasmon resonance spectroscopy.

A highly sensitive and molecular size-selective method for the detection of proteins using heteroliganded gold nanoislands and localized surface plasmon resonance (LSPR) is described. Two different heteroligands with different chain lengths (3-mercaptopionicacid and decanethiol) were used in fabricating nanoholes for the size-dependent separation of a protein in comparison with its aggregate. Their ratios on gold nanoisland were optimized for the sensitive detection of superoxide dismutase (SOD1). This protein has been implicated in the pathology of amyotrophic lateral sclerosis (ALS). Upon exposure of the optimized gold nanoisland to a solution of SOD1 and aggregates thereof, changes in the LSPR spectra were observed which are attributed to the size-selective and covalent chemical binding of SOD1 to the nanoholes. With a lower detection limit of 1.0 ng/ml, the method can be used to selectively detect SOD1 in the presence of aggregates at the molecular level.