Naoki Nagatani

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.

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.

Gold nanoparticle based immunochromatography using a resin modified micropipette tip for rapid and simple detection of human chorionic gonadotropin hormone and prostate-specific antigen

Abstract A novel bioanalysis system based on immunochromatography was developed in connection with a nitrocellulose resin-modified micropipette tip, namely as ZipTip®. The sandwich-type immunoassay was applied to our bioananalysis system. The first antibodies that were aspirated by the micropipette were immobilized on the immunochromatographic resin at the edge of micropipette tip. The blocking solution was also aspirated in the same fashion. The measurement operation was performed by aspirating the sample solution, and then the gold colloidal nanoparticles (Au naps) conjugated secondary antibody solution. Since this bioanalysis system utilizes a micropipette, it is possible to increase the sample volume, which would enable the detection of antigen at low concentrations. In addition, the washing procedure can also be performed easily to reduce the background level. After the antigen-antibody reaction, the color intensity of Au naps could be observed by the naked eye. For analytical evaluation, the color intensity was captured by a scanner, and processed by analysis software. We have achieved the detection of human chorionic gonadotropin hormone (hCG) and total prostate-specific antigen (TPSA), which are well-known as fundamental indicators of pregnancy and cancer. The limit of detection (LOD) for hCG was 8 ng/ml (0.8 ng/tip), which is comparable to that of other conventional systems based on immunochromatography. Moreover, the LOD for TPSA was improved over the existing systems with the application of different sample volumes, such as 5 ng/μl (1 ng/tip) in 200 μl sample volume, and 2 ng/ml (0.6 ng/tip) in 300 ml sample volume. Since our bioanalysis system requires a small amount of immobilized antigen, it would be greatly useful in basic research for screening the antigen–antibody reactions. Besides, our bioanalysis system can be applied to on-field screening, since its operation is simple, and the visual results can be obtained rapidly.

Electrochemical biosensors for biocontaminant detection consisting of carbon nanotubes, platinum nanoparticles, dendrimers, and enzymes.

The presented approach provides the advanced development of effective, rapid, and versatile electrochemical sensors for a small amount of analytes on potential, cheap, and disposable printed chips. The electrocatalytic activity of this biosensor revealed the feasible detection of hydrogen peroxide at low potential (~0.09 V) and the detection of a biocontaminant inhibitor (organophosphorus pesticide) in a wide range of concentrations. This efficiency comes from the chemical immobilization of catalysts (Pt nanoparticles) and electron transfer-enlarging materials (carbon nanotubes) on an electrode. Especially, dendrimers raise the stable conjugation of enzymes (acetylcholinesterase/choline oxidase/peroxidase) as well as nanoparticles and carbon nanotubes on an electrode.

Semi-real time imaging of the expression of a maize polyubiquitin promoter-GFP gene in transgenic rice

We have introduced the green fluorescent protein gene under the control of the maize ubiquitin promoter into rice embryos by microprojectile bombardment. Transient expression of GFP on the surface cells in the rice embryos depended on the localization of DNA coated gold particles. Bialaphos-resistant rice calli were produced by bombardment with a two plasmid mixture of pUbi-GFP and pDM302 carrying the coding region of the bar gene. Microscopic fluorescent image analyses indicated that the area of GFP expression increased and spreaded on the transgenic rice calli after heat stress.

Excitation of localized surface plasmon resonance using a core–shell structured nanoparticle layer substrate and its application for label-free detection of biomolecular interactions

The novel characteristics of nanomaterials enable highly sensitive and specific applications in electronics, optics and biotechnology. In particular, nanomaterials have become the preferable tools for monitoring biomolecular interactions on a biochip without labelling procedures using enzymes and fluorescent dyes. In this report, label-free detection of hybridization between nucleic acids using localized surface plasmon resonance (LSPR) based on a core–shell structured nanoparticle layer substrate is described. The core–shell structured nanoparticle layer substrate could be excited using LSPR phenomena, and its LSPR characteristics were controlled by applying different fabrication conditions. Using our LSPR label-free biochip, the optical characteristics were monitored for the detection of specific DNA–DNA and PNA–DNA hybridization reactions. Furthermore, the detection limit of the LSPR label-free biochip was 1 pM. The highly sensitive label-free detection of DNA hybridization was possible in a short analysis time. As a result, the LSPR label-free biochip provides a promising platform with attractive advantages for the detection of biomolecular interactions at low cost in a simplified experimental set-up with a low sample volume.

Scanning Near-Field Optical/Atomic Force Microscopy for Fluorescence Imaging and Spectroscopy of Biomaterials in Air and Liquid: Observation of Recombinant Escherichia coli with Gene Coding to Green Fluorescent Protein*

We have developed a system of scanning near-field optical/atomic force microscopy (SNOM/AFM) for fluorescence imaging and spectroscopy of biomaterials in air and liquid. SNOM/AFM uses a bent optical fiber simultaneously as a dynamic force AFM cantilever and a SNOM probe. Optical resolution of SNOM images shows about 50 nm in an illumination mode for a standard sample of a patterned chromium layer of 20 nm thickness on a quartz glass plate. The SNOM/AFM system contains a photon counting system and polychrometer/ICCD (intensified charge coupled device) system for observation of the fluorescence image and spectrograph of micro areas, respectively. The gene coding to green fluorescence protein (GFP) was cloned in recombinantEscherichia coli (E. coli). Topography, fluorescence image and spectrograph of recombinantE. coli by SNOM/AFM showed a difference in fluorescence in individualE. coli. Fluorescence activity of GFP can thus be used as a convenient indicator of transformation. SNOM/AFM is also applicable to observe immobilizedE. coli on a glass plate in water with a liquid chamber and may allow the viewing of observation of floating organisms.

Synthesis, oxygen radical absorbance capacity, and tyrosinase inhibitory activity of glycosides of resveratrol, pterostilbene, and pinostilbene

The stilbene compound resveratrol was glycosylated to give its 4′-O-β-D-glucoside as the major product in addition to its 3-O-β-D-glucoside by a plant glucosyltransferase from Phytolacca americana expressed in recombinant Escherichia coli. This enzyme transformed pterostilbene to its 4′-O-β-D-glucoside, and converted pinostilbene to its 4′-O-β-D-glucoside as a major product and its 3-O-β-D-glucoside as a minor product. An analysis of antioxidant capacity showed that the above stilbene glycosides had lower oxygen radical absorbance capacity (ORAC) values than those of the corresponding stilbene aglycones. The 3-O-β-D-glucoside of resveratrol showed the highest ORAC value among the stilbene glycosides tested, and pinostilbene had the highest value among the stilbene compounds. The tyrosinase inhibitory activities of the stilbene aglycones were improved by glycosylation; the stilbene glycosides had higher activities than the stilbene aglycones. Resveratrol 3-O-β-D-glucoside had the highest tyrosinase inhibitory activity among the stilbene compounds tested. Graphical abstract Synthesis and evaluation of stilbene glucosides.

Fabrication and Characterization of Planar Screen-Printed Ag/AgCl Reference Electrode for Disposable Sensor Strip

An accurate disposable planar Ag/AgCl reference electrode with an internal electrolyte was successfully fabricated by the screen-printing process. The internal electrolyte layer was also printed by using an electrolyte paste of sodium alginate containing KCl. The potential stability of the electrode was investigated at different operation times and Cl- concentrations in test solutions. Results show that the electrode has long-term potential stability (approximately 60 min), and that its performance does not depend on the Cl- concentration. This electrode can be used to provide various promising applications in sensing techniques based on disposable strips for sensing purposes.