Calibration curve-free electrochemical quantitation by micro-nano multi-scale gap devices.

Quantitation without relying on the calibration curve has long been an issue of overcoming analytical problems accompanied with the inherent limitations of the calibration curve fitting errors. Here, we report on a calibration curve-free method for electrochemical quantitation based on a multi-scale gap device (MGD). The MGD is an integrated device having a series of interdigitated electrodes (IDE) with micro-to-nano gap distances. The device shows a gap-dependent redox current of the analyte when subjected to the electrochemical cycling between the two facing electrodes of its componential IDEs. Based on the fact that the current increases as the gap distance decreases, the analyte concentration could be directly estimated: the rate of increase in the current was directly proportional to the analyte concentration. The calibration curve was not necessary for the quantitation. The accuracy of this MGD approach was better than that of an IDE collection of the same gap distance, which was deteriorated at the larger gap distances particularly. The MGD-based quantitation of dopamine, potassium ferricyanide, and aminophenol was demonstrated in a relatively broad range of concentrations (100 nM-5 mM).

Development and validation of an LC-MS/MS method for profiling 39 urinary steroids (estrogens, androgens, corticoids, and progestins).

Abnormal production or metabolism of steroid hormones is responsible for the development of endocrine diseases. Thus, accurate quantification of steroid hormones is needed for both research into clinical conditions and diagnostic and monitoring purposes. An improved analytical method for profiling 39 steroids in urine using LC-MS/MS was developed. As a pre-treatment procedure prior to LC-tandem mass spectrometry (LC-MS/MS) analysis, hydrolysis using ß-glucuronidase and solid-phase extraction for purifying the samples were performed. Steroids were separated using Waters ACQUITY BEH C18 column (2.1 × 100 mm, 1.7 µm) and a mobile phase consisting of eluent A (0.01% formic acid and 1 mm ammonium formate in water) and eluent B (0.01% formic acid and 1 mm ammonium formate in methanol) with a gradient program at a flow rate of 0.4 mL/min. Under the optimized method, the linearity of calibration curves was higher than 0.992. The limits of detection at signal-to-noise ratio of 3 were 0.03-90 ng/mL. The developed novel LC-MS/MS method can quantitatively profile 39 steroids in a single analytical run. Steroid profiling based on quantitative results could improve the diagnosis and monitoring of hormone-dependent diseases.

Electrical Detection of Pneumococcus through the Nanoparticle Decoration Method.

A simple method of nanoparticle decoration can be used in the detection of pneumococcus. After the pneumococcal bacteria were captured by an antibody (pneumococcal C-polysaccharide (PnC) antibody) between the interdigitated electrodes, the gold nanoparticles conjugated with the PnC antibodies were let to bind onto an outer membrane of the bacteria. Upon successfully dense decoration, the bacteria surface will become conductive owing to the metal nanoparticles, and a distinctive conductance change between the electrodes can be observed. Since this success ratio, or the probability of the conductance change, reflects the concentration of the analyte, a number of repeated measurements can be used in the quantification of the bacteria. In this way, we have successfully detected S. pneumoniae in the range of 10-108 CFU/mL. The limit of detection in this work is lower than that in the commercial detection kit. We hope that the nanoparticle decoration method will play a role in the facile detection of various bacteria.

Synergistic Effect of Detection and Separation for Pathogen Using Magnetic Clusters.

Early diagnosis of infectious diseases is important for treatment; therefore, selective and rapid detection of pathogenic bacteria is essential for human health. We report a strategy for highly selective detection and rapid separation of pathogenic microorganisms using magnetic nanoparticle clusters. Our approach to develop probes for pathogenic bacteria, including Salmonella, is based on a theoretically optimized model for the size of clustered magnetic nanoparticles. The clusters were modified to provide enhanced aqueous solubility and versatile conjugation sites for antibody immobilization. The clusters with the desired magnetic property were then prepared at critical micelle concentration (CMC) by evaporation-induced self-assembly (EISA). Two different types of target-specific antibodies for H- and O-antigens were incorporated on the cluster surface for selective binding to biological compartments of the flagella and cell body, respectively. For the two different specific binding properties, Salmonella were effectively captured with the O-antibody-coated polysorbate 80-coated magnetic nanoclusters (PCMNCs). The synergistic effect of combining selective targeting and the clustered magnetic probe leads to both selective and rapid detection of infectious pathogens.

Long dsRNA-mediated RNA interference and immunostimulation: a targeted delivery approach using polyethyleneimine based nano-carriers.

RNA oligonucleotides capable of inducing controlled immunostimulation combined with specific oncogene silencing via an RNA interference (RNAi) mechanism provide synergistic inhibition of cancer cell growth. With this concept, we previously designed a potent immunostimulatory long double stranded RNA, referred to as liRNA, capable of executing RNAi mediated specific target gene silencing. In this study, we developed a highly effective liRNA based targeted delivery system to apply in the treatment of glioblastoma multiforme. A stable nanocomplex was fabricated by complexing multimerized liRNA structures with cross-linked branched poly(ethylene imine) (bPEI) via electrostatic interactions. We show clear evidence that the cross-linked bPEI was quite effective in enhancing the cellular uptake of liRNA on U87MG cells. Moreover, the liRNA-PEI nanocomplex provided strong RNAi mediated target gene silencing compared to that of the conventional siRNA-PEI complex. Further, the bPEI modification strategy with specific ligand attachment assisted the uptake of the liRNA-PEI complex on the mouse brain endothelial cell line (b.End3). Such delivery systems combining the beneficial elements of targeted delivery, controlled immunostimulation, and RNAi mediated target silencing have immense potential in anticancer therapy.

Nanoscale biomemory composed of recombinant azurin on a nanogap electrode.

We fabricate a nanoscale biomemory device composed of recombinant azurin on nanogap electrodes. For this, size-controllable nanogap electrodes are fabricated by photolithography, electron beam lithography, and surface catalyzed chemical deposition. Moreover, we investigate the effect of gap distance to optimize the size of electrodes for a biomemory device and explore the mechanism of electron transfer from immobilized protein to a nanogap counter-electrode. As the distance of the nanogap electrode is decreased in the nanoscale, the absolute current intensity decreases according to the distance decrement between the electrodes due to direct electron transfer, in contrast with the diffusion phenomenon of a micro-electrode. The biomemory function is achieved on the optimized nanogap electrode. These results demonstrate that the fabricated nanodevice composed of a nanogap electrode and biomaterials provides various advantages such as quantitative control of signals and exclusion of environmental effects such as noise. The proposed bioelectronics device, which could be mass-produced easily, could be applied to construct a nanoscale bioelectronics system composed of a single biomolecule.

Calcination-temperature-Dependent shape and crystal structure of TiO2 nanomaterials synthesized by hard-template method.

The shape and crystal structure of TiO2 nanomaterials synthesized by the hard-template method can be controlled by simply changing the calcination temperature. In this work, SiO2 nanoparticles were used as a hard template and TiO2 was coated onto the surface of the silica core, resulting in core-shell nanoparticles, which were then calcined at various temperatures to induce shape transformation and crystallization of the TiO2 shell. After etching of the silica cores, spherical hollow nanocapsules with anatase crystal phase were obtained by calcination at 400-1000 degrees C, while urchin-like hollow capsules and small-sized particulates were obtained at temperatures below 400 degrees C and above 1000 degrees C, respectively. The core-shell nanoparticles exhibited greatly enhanced anatase phase stability (up to approximately 1200 degrees C), which was attributable to the effect of the core material. The phase stability was found to be dependent on the shell thickness of the nanocapsules, also supporting the effect of the core material.

Preparation of an aqueous suspension of stabilized TiO2 nanoparticles in primary particle form.

To prepare stabilized TiO2 nanoparticles (TiO2 NPs) in aqueous media as a suspension of the primary particles, we attempted to optimize the conditions for dispersing stable, aggregated TiO2 NPs (A-TiO2 NPs) in aqueous HCl/NaOH solutions or 5 mM pH buffered aqueous solutions. The A-TiO2 NPs with a hydrodynamic diameter (or DLS size) of 150 +/- 20 nm could be dispersed at high concentration (63.5 +/- 0.5 mg/ml) in a 5 mM phosphate buffer (PB) solution of pH 8, and a primary TiO2 (P-TiO2) NP suspension (1.2 +/- 0.3 mg/ml) with DLS size of 30 +/- 5 nm could be separated from the highly concentrated A-TiO2 NP suspension by sonication and subsequent centrifugation. It was observed by comparing the UV-Vis absorption spectra of the A-TiO2 and P-TiO2 NP suspensions that the extinction coefficient of the TiO2 NPs in the aqueous suspension depended on the degree of aggregation. The stabilized P-TiO2 NP suspension in aqueous solution can be used to study nanotoxicity as well as to characterize the physicochemical properties of TiO2 NPs.

Fabrication of nanoporous TiO2 hollow capsules using core-shell silica nanoparticle templates.

We report on the fabrication of the nanoporous TiO2 hollow capsules using core-shell silica nanoparticle templates. The thickness of the capsules can be simply controlled by varying the amount of the TiO2 precursor. The resulting nanoporous capsules exhibited the high specific surface area and the large pore volume of 103-180 m2/g and 0.40-0.86 cm3/g, respectively. Photocatalytic activity of the TiO2 hollow capsules was also investigated and compared to that of the commercial TiO2 nanoparticles.

Nanogap-based electrical PNA chips for the detection of genetic polymorphism of cytochrome P450 2C19.

PNA chips for the detection of the genetic polymorphism of Cytochrome P450 2C19 (CYP2C19), a well-known enzyme related to the metabolism of therapeutic drugs, were electrically-interfaced with interdigitated nanogap electrodes (INEs). The average gap distance and effective length of the INEs were about approximately 70 nm and approximately 140/m, respectively. Those INEs having the aspect ratio of about 2000, were prepared by the combination of the photolithography (for the formation of initial electrodes) and the surface-catalyzed chemical deposition (for the gap narrowing), without the e-beam lithography. The PNA probes for the detection of CYP2C19 were immobilized in the gap region of INEs via Schiff base formation. The I-V characteristics clearly showed a sharp increase in the conductance between the nanogap electrodes upon the PNA-DNA hybridization, followed by the adsoprtion of functionalized Au nanoparticles. Four different target DNAs for the diagnosis of CYP2C19 polymorphism were successfully detected and discriminated with the INE-based PNA chips.

Effect of Secondary Structures on the Adsorption of Peptides onto Hydrophobic Solid Surfaces Revealed by SALDI-TOF and MD Simulations.

The adsorption of peptides and proteins on hydrophobic solid surfaces has received considerable research attention owing to their wide applications to biocompatible nanomaterials and nanodevices, such as biosensors and cell adhesion materials with reduced nanomaterial toxicity. However, fundamental understandings about physicochemical hydrophobic interactions between peptides and hydrophobic solid surfaces are still unknown. In this study, we investigate the effect of secondary structures on adsorption energies between peptides and hydrophobic solid surfaces via experimental and theoretical analyses using surface-assisted laser desorption/ionization-time-of-flight (SALDI-TOF) and molecular dynamics (MD) simulations. The hydrophobic interactions between peptides and hydrophobic solid surfaces measured via SALDI-TOF and MD simulations indicate that the hydrophobic interaction of peptides with random coil structures increased more than that of peptides with an α-helix structure when polar amino acids are replaced with hydrophobic amino acids. Additionally, our study sheds new light on the fundamental understanding of the hydrophobic interaction between hydrophobic solid surfaces and peptides that have diverse secondary structures.

Superhydrophobic thin films with nanostructured surface prepared with Au nanoparticle masks.

The hydrophobicity of a perfluoropolyether bisurethane methacrylate polymer film was investigated along with the formation of nano-hairs on its surface through reactive ion etching using gold nanoparticles (Au NPs) as masks. It was found that the hydrophobicity of the polymer film was strongly dependent on the number density of the nano-hairs which was determined by that of the Au NPs. The superhydrophobic surface was obtained when the number density was higher than 250 microm(-2). The effects of surface functionalization, Au NP immobilization, and etching time on the hydrophobicity of the polymer film were also examined extensively and discussed based on the results of the contact angle measurements and the scanning electron microscopy.

Fabrication of carbon capsules with hierarchical pore structures.

Carbon capsules with hierarchical pore structures were fabricated by using core-shell silica nanoparticles as templates and phenolic resin as a carbon precursor. Carbon capsules with hierarchical pore structures were obtained via in-situ polymerization of the phenolic resin on the surface of the silica nanoparticles followed by the carbonization and removal of the silica templates. The hierarchically pored carbon capsules exhibited multimodal porosity with a high specific surface area (approximately 1834 m2/g) and a large pore volume (approximately 1.83 cm3/g).

Control of nanogap separation by surface-catalyzed chemical deposition.

The nanogap devices, which comprise multiple electrodes separated by a few to a few tens of nanometers, have opened up new possibilities in biomolecular sensing as well as various frontier electronics. One of the key aspects of the nanogap device research is how to control the gap distance following each specific needs of the gap structure. Here, we report the extensive study on the fine control of the gap distance between electrodes within the range of 1-80 nm via surface-catalyzed chemical deposition. The initial gap electrodes were prepared via conventional e-beam lithography, and the gap distance was narrowed to a designed value through the surface-catalyzed reduction of gold ion on the predefined electrode surfaces, by simple dipping of the electrodes into the aqueous solution of gold chloride and hydroxylamine. The final gap distance was controlled by adjusting the repetition number, reductant concentration, reaction time, and reaction temperature. The dependence of the gap-narrowing reaction on these parameters was systematically examined based on the results of field emission scanning electron microscopy and atomic-force microscopy.

Effective Diagnosis of Foot-And-Mouth Disease Virus (FMDV) Serotypes O and A Based on Optical and Electrochemical Dual-Modal Detection.

Foot-and-mouth disease virus (FMDV) is a highly contagious disease that affects cloven-hoofed animals. The traditional diagnostic methods for FMDV have several drawbacks such as cross-reactivity, low sensitivity, and low selectivity. To overcome these drawbacks, we present an optical and electrochemical dual-modal approach for the specific detection of FMDV serotypes O and A by utilizing a magnetic nanoparticle labeling technique with resorufin ß-d-glucopyranoside (res-ß-glc) and ß-glucosidase (ß-glc), without the use of typical lateral flow assay or polymerase chain reaction. FMDV serotypes O and A were reacted with pan-FMDV antibodies that recognize all seven FMDV serotypes (O, A, C, Asia 1, SAT 1, SAT 2, and SAT 3). The antigen-antibody complex was then immobilized on magnetic nanoparticles and reacted with ß-glc-conjugated FMDV type O or type A antibodies. Subsequently, the addition of res-ß-glc resulted in the release of fluorescent resorufin and glucose owing to catalytic hydrolysis by ß-glc. The detection limit of fluorescent signals using a fluorescence spectrophotometer was estimated to be log(6.7) and log(5.9) copies/mL for FMDV type O and A, respectively, while that of electrochemical signals using a glucometer was estimated to be log(6.9) and log(6.1) copies/mL for FMDV type O and A, respectively. Compared with a commercially available lateral flow assay diagnostic kit for immunochromatographic detection of FMDV type O and A, this dual-modal detection platform offers approximately four-fold greater sensitivity. This highly sensitive and accurate dual-modal detection method can be used for effective disease diagnosis and treatment, and will find application in the early-stage diagnosis of viral diseases and next-generation diagnostic platforms.

Electrochemical microgap immunosensors for selective detection of pathogenic Aspergillus niger.

Aspergillus niger (A. niger) is a well-known allergenic, harmful fungus in the indoor environment that can cause asthmatic symptoms and atopy. Previous immunosensing approach suffers from an insufficient detection limit, mainly because there are no techniques for target amplification. We report an electrochemical immunosensor that selectively quantifies the A. niger based on the detection of extracellular proteins by using a specific interaction with antibody. The sensor was designed to show a decrease in redox current upon binding of the antigens secreted from A. niger onto an antibody-immobilized surface between the interdigitated electrodes. The extracellular proteins were profiled by LC-MS/MS to identify the antigens existing in the A. niger solution. Since the targets of the sensor are the proteins, its sensitivity and selectivity remain almost intact even after filtration of the spores. It was also found that the use of secretion promoter in the sampling stage greatly improved the sensor's limit of detection (LOD) for the spores. By this, the LOD was lowered by a few orders of magnitude so as to reach the value as low as ~101 spores/mL.

Real-space mapping of the strongly coupled plasmons of nanoparticle dimers.

We carried out the near-field optical imaging of isolated and dimerized gold nanocubes to directly investigate the strong coupling between two adjacent nanoparticles. The high-resolution (approximately 10 nm) local field maps (intensities and phases) of self-assembled nanocube dimers reveal antisymmetric plasmon modes that are starkly different from a simple superposition of two monomeric dipole plasmons, which is fully reproduced by the electrodynamics simulations. The result decisively proves that, for the closely spaced pair of nanoparticles (interparticle distance/particle size approximately 0.04), the strong Coulombic attraction between the charges at the interparticle gap dominates over the intraparticle charge oscillations, resulting in a hybridized dimer plasmon mode that is qualitatively different from those expected from a simple dipole-dipole coupling model.

Supramolecular Peptide Hydrogel-Based Soft Neural Interface Augments Brain Signals through a Three-Dimensional Electrical Network.

Recording neural activity from the living brain is of great interest in neuroscience for interpreting cognitive processing or neurological disorders. Despite recent advances in neural technologies, development of a soft neural interface that integrates with neural tissues, increases recording sensitivity, and prevents signal dissipation still remains a major challenge. Here, we introduce a biocompatible, conductive, and biostable neural interface, a supramolecular ß-peptide-based hydrogel that allows signal amplification via tight neural/hydrogel contact without neuroinflammation. The non-biodegradable ß-peptide forms a multihierarchical structure with conductive nanomaterial, creating a three-dimensional electrical network, which can augment brain signal efficiently. By achieving seamless integration in brain tissue with increased contact area and tight neural tissue coupling, the epidural and intracortical neural signals recorded with the hydrogel were augmented, especially in the high frequency range. Overall, our tissuelike chronic neural interface will facilitate a deeper understanding of brain oscillation in broad brain states and further lead to more efficient brain-computer interfaces.

Potential Role of Soluble Metal Impurities in the Acute Lung Inflammogenicity of Multi-Walled Carbon Nanotubes.

Multi-walled carbon nanotubes (MWCNTs) have variable metal impurities, but little is known about the impact of soluble metal impurities on the toxicity of MWCNTs. Here, we evaluated the role of soluble metal impurities to the acute inflammogenic potential of MWCNTs, using five types of high purity MWCNTs (>95%). MWCNTs and their soluble fractions collected at 24 h after incubation in phosphate-buffered saline showed diverse metal impurities with variable concentrations. The fiber-free soluble fractions produced variable levels of reactive oxygen species (ROS), and the iron level was the key determinant for ROS production. The acute inflammation at 24 h after intratracheal instillation of MWCNTs to rats at 0.19, 0.63, and 1.91 mg MWCNT/kg body weight (bw) or fiber-free supernatants from MWCNT suspensions at 1.91 and 7.64 mg MWCNT/kg bw showed that the number of granulocytes, a marker for acute inflammation, was significantly increased with a good dose-dependency. The correlation study showed that neither the levels of iron nor the ROS generation potential of the soluble fractions showed any correlations with the inflammogenic potential. However, the total concentration of transition metals in the soluble fractions showed a good correlation with the acute lung inflammogenic potential. These results implied that metal impurities, especially transitional metals, can contribute to the acute inflammogenic potential of MWCNTs, although the major parameter for the toxicity of MWCNTs is size and shape.

Nanoparticle assembly on nanoplates.

By using dithiol linkage or biological interaction, Au nanoparticles are readily assembled on the single-crystalline Au nanoplates with well-defined geometries.