Yuzuru Takamura

A localized surface plasmon resonance based immunosensor for the detection of casein in milk

Abstract In this research, a localized surface plasmon resonance (LSPR) immunosensor based on gold-capped nanoparticle substrate for detecting casein, one of the most potent allergens in milk, was developed. The fabrication of the gold-capped nanoparticle substrate involved a surface-modified silica nanoparticle layer (core) on the slide glass substrate between bottom and top gold layers (shell). The absorbance peak of the gold-capped nanoparticle substrate was observed at ∼520 nm. In addition, the atomic force microscopy (AFM) images demonstrated that the nanoparticles formed a monolayer on the slide glass. After immobilizing anti-casein antibody on the surface, our device, casein immunosensor, could be applied easily for the detection of casein in the raw milk sample without a difficult pretreatment. Under the optimum conditions, the detection limit of the casein immunosensor was determined as 10 ng/mL. Our device brings several advantages to the existing LSPR-based biosensors with its easy fabrication, simple handling, low-cost, and high sensitivity.

Localized surface plasmon resonance based optical biosensor using surface modified nanoparticle layer for label-free monitoring of antigen–antibody reaction

Abstract In recent years, label-free biosensors not requiring external modifications have been receiving intense attention. A label-free optical biosensor, which retains many of the desirable features of conventional surface plasmon resonance (SPR) reflectometry, namely, the ability to monitor the kinetics of biomolecular interactions in real-time without a label has been developed with several important advantages: the biosensor device is easy to fabricate, and simple to implement, requiring only an UV–Vis spectrophotometer or flatbed scanner. Importantly, the label-free optical biosensor can be easily multiplexed to enable high-throughput monitoring of biomolecular interactions in an arraybased format. In this research,the development of a localized surface plasmon resonance (LSPR)-based label-free optical biosensor using a surface modified nanoparticle layer is aimed. This optical detection method promises to offer a massively parallel detection capability in a highly miniaturized package. The two-dimensional nanoparticle layer was formed by the surface modified silica nanoparticles. The optical properties and surface analysis of nanoparticle layer substrate were characterized through transmissionmeasurements and atomic force microscopy (AFM). Simultaneously, the nanoparticle layer substrate was applied to the optical LSPR-based biosensor for label-free monitoring of the antigen–antibody reaction. The anti-fibrinogen antibody wasimmobilized onto the nanoparticle layer substrate surface. Different concentrations of fibrinogen were introduced to the anti-fibrinogen antibody immobilized nanoparticle layer substrate surface, and the change in the absorption spectrum, caused by the antigen–antibody reaction, was observed. By using this anti-fibrinogen antibody immobilized nanoparticle layer substrate; the detection limit of this optical LSPR-based biosensor was 10 ng/ml.

Quantum dot-based immunosensor for the detection of prostate-specific antigen using fluorescence microscopy

A sensitive optical method based on quantum dot (QD) technology is demonstrated for the detection of an important cancer marker, total prostate-specific antigen (TPSA) on a disposable carbon substrate surface. Immuno-recognition was carried out on a carbon substrate using a sandwich assay approach, where the primary antibody (Ab)-protein A complex covalently bound to the substrate surface, was allowed to capture TPSA. After the recognition event, the substrate was exposed to the biotinylated secondary Abs. After incubation with the QD streptavidin conjugates, QDs were captured on the substrate surface by the strong biotin-streptavidin affinity. Fluorescence imaging of the substrate surface illuminated the QDs, and provided a very sensitive tool for the detection of TPSA in undiluted human serum samples with a detection limit of 0.25ng/mL. The potential of this method for application as a simple and efficient diagnostic strategy for immunoassays is discussed.

Cell separation by an aqueous two-phase system in a microfluidic device

We generated an aqueous two-phase laminar flow in a microfluidic chip and used the system to isolate leukocyte and erythrocyte cells from whole blood cells. The microfluidic system reduced the effect of gravity in the aqueous two-phase system (ATPS). Poly(ethylene glycol) (PEG) and dextran (Dex) solutions were used as the two phases, and the independent flow rates of the solutions were both 2 microL/min. When hydrophobic and hydrophilic polystyrene beads were introduced into the microfluidic device, the hydrophilic beads moved to the Dex layer and the hydrophobic beads to the interface between the two phases. In the case of living cells, Jurkat cells and erythrocytes moved more efficiently to the PEG and Dex layers, respectively, than they move in a conventional ATPS. When whole blood cells were inserted into the microfluidic chip, leukocytes could be separated from erythrocytes because erythrocytes moved to the Dex layer while leukocytes remained outside of this layer in the microfluidic system. The reported microfluidic chip for the whole blood cell separation can effectively be integrated into a Micro Total Analysis System designed for cell-based clinical, forensic, and environmental analyses.

Gold nanoparticle-based novel enhancement method for the development of highly sensitive immunochromatographic test strips

Abstract The immunochromatographic assay that is in widespread use for pregnancy diagnosis is a method for easy visual judging of the antigen–antibody reaction using gold nanoparticle. The rapid observation of results directly by the naked eye ensures the convenience of performing bioassays on-field. Therefore, gold nanoparticle-based immunochromatographic assays have provided attractive means for developing biosensors without the handling of toxic reagents, while allowing an easy and rapid procedure. However, the detection limit of this method is higher than the conventional method, enzyme-linked immunosorbent assay (ELISA). In this report, we developed a highly sensitive immunochromatographic assay for the detection of human chorionic gonadotropin hormone (hCG) as the model case. In this research, we are reporting the application of a new ‘sensitizer’ that contains gold nanoparticle conjugated primary antibody and the antigen. The sensitizer was added to the membrane after finishing the application of the normal method. The antigen of the sensitizer was captured by the secondary antibodies at the test line on the strip. As a result, the accumulation of the gold nanoparticle increased at the test line, and the sensitivity was higher. The sensitivity of our method could be enhanced by the sensitizer to almost the same level of ELISA assay. The test line intensity of hCG at 25 pg/ml treated with sensitizer was almost equal to the density that we observed at 1.0 ng/ml with the normal method. We also tested the performance of the sensitizer by using the surface plasmon resonance (SPR) technology of BIACORETM. The sensitizer using immunochromatographic assay is a promising candidate for decentralized diagnosis in clinically important fields such as the sensitive detection of cancer markers.

Label-free optical detection of aptamer–protein interactions using gold-capped oxide nanostructures

Optical biosensors based on noble nanostructures currently receive attention due to their highly efficient, simultaneous analysis of a number of important biomolecules from proteomics to genomics. In this study, the combination of localized surface plasmon resonance (LSPR) with interferometry in the relative reflected intensity (RRI) spectrum of the gold-capped oxide nanostructure was thoroughly exploited for label-free detection of aptamer-protein interactions. The fabrication of gold-capped oxide nanostructure involved the deposition of gold on the surface of porous anodic alumina (PAA) layer chip. This novel nanomaterial enabled us to simultaneously monitor the changes in both LSPR and interferometric characteristics since the biomolecular interactions occur. After immobilizing the aptamer I on the chip surface, our sensor could be easily applied for specific detection of thrombin and aptamer II with a limit of detection of 1 nM thrombin in the sample. Our optical biosensing device connecting with the gold-capped oxide nanostructure has a high potential for highly sensitive monitoring of the other biomolecular interactions such as protein-protein interactions, DNA-protein interactions, DNA-DNA hybridizations, and ligand-receptor interactions with a massively parallel detection capability in a high-throughput system.

Label-free detection of melittin binding to a membrane using electrochemical-localized surface plasmon resonance.

Localized surface plasmon resonance (LSPR) and electrochemistry measurements connecting to core-shell structure nanoparticle are successfully exploited in a simultaneous detectable scheme. In this work, the surface plasmon band characterizations of this nanostructure type are initially examined by controlling the core size of the silica nanoparticle and shell thickness of the deposited gold. These results clearly show that when the shell thickness is increased, keeping the core size constant, the peak wavelength of the LSPR spectra is shifted to a shorter wavelength and the maximum of peak intensity is achieved at a particular shell thickness. On the basis of this structure, we present a membrane-based nanosensor for optically detecting the binding of peptide toxin melittin to hybrid bilayer membrane (HBM) and electrochemically assessing its membrane-disturbing properties as a function of concentrations. It will open up the way to detect functionally similar protein toxins and other membrane-targeting peptides with the intension of integrating this chip into a microfluid and expanding it into multiarray format.

Structural change at the carbon-nanotube tip by field emission

Carbon-nanotube tips are plastically deformed during field emission. High-resolution transmission electron microscopy and structural simulations suggest that the deformed structure of the closed nanotube is explained by heterogeneous nucleation of the pentagonal and heptagonal carbon ring pairs, and that of the opened one is represented by sp3-like line defects in the hexagonal carbon network. It is considered that the changing of the inclination of the Fowler–Nordheim plots corresponds to the structural change in which a tip becomes sharp. The field ion microscope image and the corresponding field-emission pattern suggest that the electron emission from a closed nanotube is not necessarily from pentagonal carbon rings, but from the protrudent carbon network sites on the tip.

Labelless impedance immunosensor based on polypyrrole–pyrolecarboxylic acid copolymer for hCG detection

In this work, a sensitive label-free impedimetric hCG-immunosensor was constructed by using a commercial screen-printing carbon ink electrode (namely disposable electrochemical printed chip) as the basis. The carbon ink electrode of DEP chip is modified first by deposition of polypyrrole-pyrole-2-carboxylic acid copolymer and thence hCG antibody immobilization via the COOH groups of pyrrole-2-carboxylic acid, which can serve as a linker for covalent biomolecular immobilization. The experimental results exposed that the designed immunosensor is more sensitive than other previously reported immunosensors, in the case of detection limit and linear range for antigen detection. With optimal fabrication parameters, the detection limit for α-hCG was 2.3 pg/mL in 10mM phosphate buffer saline (PBS) solution containing 1% bovine serum albumine (BSA). Moreover, the use of inexpensive DEP chip as a basis for these immunosensors will allow for simple instrumentation, disposable and portable at low cost. This work also demonstrates a new approach to develop a sensitive and label-free impedimetric immunosensor based on screen-printed electrode for applications in clinical diagnosis.

Highly Sensitive Elemental Analysis for Cd and Pb by Liquid Electrode Plasma Atomic Emission Spectrometry with Quartz Glass Chip and Sample Flow

This paper describes the development of a highly sensitive liquid-electrode plasma atomic emission spectrometry (LEP-AES) by combination of quartz glass chip and sample flow system. LEP-AES is an ultracompact elemental analysis method, in which the electroconductive sample solution is put into a microfluidic channel whose center is made narrower (∼100 μm in width). When high voltage pulses (1500 V) are applied at both ends of the channel, the sample evaporates locally at the narrow part and generates plasma. By the emission from the plasma, elemental concentration is analyzed. In this paper, the limits of detection (LODs) were investigated in various conditions of accumulation time, material of the chip, and the sample flow. It was found that the long accumulation using the quartz chip with sample flow was effective to improve LOD. Authors suggested that this was because bubbles remaining after each plasma pulse were removed from the narrow channel by sample flow, resulting in highly reproducible plasma generation, to enable a high accumulation effect. Finally, LODs were calculated from a calibration curve, to be 0.52 μg/L for Cd and 19.0 μg/L for Pb at optimized condition. Sub-ppb level LOD was achieved for Cd.