Tatsuro Endo

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.

Stimuli-responsive hydrogel-silver nanoparticles composite for development of localized surface plasmon resonance-based optical biosensor.

In this paper, the development of a localized surface plasmon resonance (LSPR)-based optical enzyme biosensor using stimuli-responsive hydrogel-silver nanoparticles composite is described. This optical enzyme biosensor was constructed by immobilizing glucose oxidase (GOx) into the stimuli-responsive hydrogel. When a sample solution such as glucose was applied to the surface of this optical enzyme biosensor, the interparticle distances of the silver nanoparticles present in the stimuli-responsive hydrogel were increased, and thus the absorbance strength of the LSPR was decreased. Furthermore, hydrogen peroxide, which was produced by the enzymatic reaction, induced the degradation of highly clustered silver nanoparticles by the decomposition of hydrogen peroxide. Hence, a drastic LSPR absorbance change, which depends on the glucose concentrations, could be observed. On the basis of the abovementioned mechanism, the characterization of the LSPR-based optical enzyme biosensor was carried out. It was found that the LSPR-based optical enzyme biosensor could be used to specifically determine glucose concentrations. Furthermore, the detection limit of this biosensor was 10 pM. Therefore, this LSPR-based optical enzyme biosensor has the potential to be applied in cost-effective, highly simplified, and highly sensitive test kits for medical applications.

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.

Super-sensitivity in label-free protein sensing using a nanoslot nanolaser

Microphotonic sensors have been actively studied with increasing demands for label-free biosensing in medical diagnoses and life sciences. For high-throughput and low-cost sensing, a high sensitivity is crucial for eliminating the pre-concentration process, while a simple setup of sensors is also desirable. This paper demonstrates a super-sensitivity for protein, which satisfies these requirements. The key device is a photonic crystal nanolaser, in particular with a nanoslot. Even using a simple setup, the nanolaser achieves an extraordinary-low detection limit for BSA protein, i.e. 255 fM on an average, which cannot be explained by its bulk index sensitivity. The specific adsorption of the protein is observed only around the nanoslot with strong laser intensity. This suggests that the super-sensitivity arises from the effective trapping of protein in the nanoslot.

Label-free cell-based assay using localized surface plasmon resonance biosensor

For an understanding of the life activities, the analysis of cells, which are the smallest units of life form, has a significant impact on biology and biotechnology. In this study, we propose a novel label-free cell-based assay that is based on localized surface plasmon resonance (LSPR) biosensor, which is excited using core-shell structured nanoparticle layer substrate. To demonstrate the promising properties of our LSPR-based label-free cell-based assay, we performed the detection of the cell metabolites using the isolated cells from mouse thymus. For detection of the cellular metabolites, the refractive index change by the specific interaction between the antigen and antibody was detected on the antibody immobilized LSPR-based biosensor. Using our LSPR-based biosensor, the optical characteristics were monitored for the detection of specific reactions between antibody and cell metabolites. As a result, the detection limit of this antibody immobilized LSPR-based biosensor was 10 pg mL(-1). Furthermore, the time-course analysis of cell metabolisms using the isolated cells from mouse thymus was also achieved. From these results, the LSPR-based biosensor 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.

Au nanoparticle-modified DNA sensor based on simultaneous electrochemical impedance spectroscopy and localized surface plasmon resonance.

Electrochemical impedance spectroscopy (EIS) and localized surface plasmon resonance (LSPR) were performed on the same Au nanoparticle (AuNP)-modified indium tin oxide (ITO) coated glass surfaces. Cyclic voltammetry was applied to electrodeposit AuNPs on ITO surface directly. The surface plasmon band characterization of AuNPs was initially studied by controlling the electrodeposition conditions. It was found that the size of AuNP clusters was significantly affected by the applied potential and KCl concentration in solution. The dual-detection platform was applied to detect DNA hybridization related to a specific point mutation in apolipoprotein E gene (ApoE), which was related to the progression of Alzheimers disease. The preliminary results facilitate the development of a versatile biosensor that can be easily miniaturized and integrated into a high-throughput diagnostic device.

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.

Printed two-dimensional photonic crystals for single-step label-free biosensing of insulin under wet conditions

Two-dimensional photonic crystals (2D-PCs) fabricated on a cyclo-olefin polymer (COP) film using a printable photonics technology based on nano-imprint lithography (NIL) were used for label-free biosensing of insulin under wet conditions. In general, 2D-PC-based biosensing involves a complicated dry-up procedure after biosensing reactions on the 2D-PCs to obtain a high sensitivity through the large difference in refractive index. Therefore, it can be difficult to achieve simple operation involving single-step analysis. Performance of the biosensing under wet conditions would simplify the operational procedure. For label-free biosensing of insulin under wet conditions, the Fresnel reflection intensity change was used instead of the wavelength shift, which is the commonly used sensing signal. By detecting changes in refractive index caused by specific interactions between the antigen and antibody as the Fresnel reflection intensity changes, physiologically important concentrations of insulin could be detected, even under wet conditions. These results suggest that low-cost printed 2D-PCs offer great potential for single-step label-free biosensing through the introduction of a sample solution.

Fabrication of core-shell structured nanoparticle layer substrate for excitation of localized surface plasmon resonance and its optical response for DNA in aqueous conditions

LSPR from nanostructured noble metals such as gold and silver offers great potential for biosensing applications. In this study, a core-shell structured nanoparticle layer substrate was fabricated and the localized surface plasmon resonance (LSPR) optical characteristics were investigated for DNA in aqueous conditions. Factors such as DNA length dependence, concentration dependence, and the monitoring of DNA aspects (ssDNA or dsDNA) were measured. Different lengths and concentrations of DNA solutions were introduced onto the surface of the substrate and the changes in the LSPR optical characteristics were measured. In addition, to monitor the changes in LSPR optical characteristics for different DNA aspects, a DNA solutions denatured by means of heat or alkali were introduced onto the surface, after which optical characterization of the core-shell structured nanoparticle substrate was carried out. With this core-shell structured nanoparticle layer for the excitation of LSPR, the dependence upon specific DNA conditions (length, concentration, and aspect) could be monitored. In particular, the core-shell structured nanoparticle layer substrate could detect DNA of length 100-5000 bp and 400-bp DNA at a concentration of 4.08 ng mL(-1) (1 x 10(7) DNA molecules mL(-1)). Furthermore, the changes in LSPR optical characteristics with DNA aspect could be monitored. Thus, LSPR-based optical detection using a core-shell structured nanoparticle layer substrate can be used to determine the kinetics of biomolecular interactions in a wide range of practical applications such as medicine, drug delivery, and food control.

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.