Search

We have developed a fluorescence enhancement system for biomolecular detection using a monolithic waveguide sensing plate. The plate consists of a thermally grown amorphous SiO2 layer, a Si single-crystal layer, and bulk amorphous SiO2. Waveguide-mode excitation in this plate produces an enhanced electric field on the plate surface, and therefore, signals from analyte-labeling fluorescent dyes are enhanced. In the present paper, we elucidate the impact of the system from viewpoints of the electric field enhancement factor calculated numerically and the analysis of the limit of detection estimated from experimental results. Optimal layer thicknesses of the plates to achieve the highest sensitivity are also discussed. Further, an interesting phenomenon that accompanied the waveguide-mode excitation, namely, photoluminescence from the amorphous SiO2 layer itself, is also reported. [ABSTRACT FROM AUTHOR]

Discrimination of closely related strains is a key issue, particularly for infectious diseases whose incidence fluctuates according to variations in the season and evolutionary changes. Among infectious diseases, influenza viral infections are a worldwide cause of pandemic disease and mortality. With the emergence of different influenza strains, it is vital to develop a method using antibodies that can differentiate between viral types and subtypes. Ideally, such a system would also be user friendly. In this study, a polyclonal antibody generated against A/Udorn/307/1972 (H3N2) was used as a probe to distinguish between influenza H3N2 viruses based on the interaction between the antibody and hemagglutinin, demonstrating its applicability for viral discrimination. Clear discrimination was demonstrated using an evanescent-field-coupled waveguide-mode sensor, which has appealing characteristics over other methods in the viewpoint of improving the sensitivity, measurement time, portability and usability. Further supporting evidence was obtained using enzyme-linked immunosorbent assays, hemagglutination-inhibition assays, and infectivity neutralization assays. The results obtained indicate that the polyclonal antibody used here is a potential probe for distinguishing influenza viruses and, with the aid of a handheld sensor it could be used for influenza surveillance. [ABSTRACT FROM AUTHOR]

Gold nanoparticles were conjugated to an antibody (immuno-AuNP) against A/Udorn/307/1972 (H3N2) influenza virus to detect viruses on a sensing plate designed for an evanescent field-coupled waveguide-mode sensor. Experiments were conducted using human influenza A/H3N2 strains, and immuno-AuNP could detect 8×105 PFU/ml (40 pg/µl) intact A/Udorn/307/1972 and 120 pg/µl A/Brisbane/10/2007. Furthermore, increased signal magnitude was achieved in the presence of non-ionic detergent, as the virtual detection level was increased to 8×104 PFU/ml A/Udorn/307/1972. Immuno-AuNPs were then complexed with viruses to permit direct observation, and they formed a ring of confined nanodots on the membrane of both intact and detergent-treated viruses as directly visualized by scanning electron microscopy. With this complex the detection limit was improved further to 8×103 PFU/ml on anti-rabbit IgG immobilized sensing plate. These strategies introduce methods for observing trapped intact viruses on the sensing plates generated for optical systems. [ABSTRACT FROM AUTHOR]

An evanescent-field-coupled waveguide-mode (EFC-WM) sensor utilizes monolithic SiO2/Si/SiO2 sensing plates having a multilayered structure and is used to evaluate a blocking agent comprising poly(ethylene glycol)-based block copolymers. Factor IX (FIX) protein was detected using its aptamer, viz. FIX was immobilized on a glutaraldehyde-modified silica surface, and then treated with a biotinylated aptamer. The quantitative analysis of FIX was carried out using streptavidin-conjugated gold nanoparticles (SA-GNPs). The blocking polymer, poly(ethylene glycol)-b-poly(acrylic acid) (PEG-b-PAAc), was found to mask unreacted amine and glutaraldehyde (Glu) moieties on the SiO2 surface, and it completely prevented the non-specific binding of SA-GNPs. By exploiting the strong blocking effect of PEG-b-PAAc, we achieved high ligand–analyte interaction sensitivity (sensitive down to 100 pM). To improve the sensitivity further, we also used pentaethylenehexamine-terminated PEG (N6-PEG) on GNPs. The improvement in sensitivity was found to be 1000-fold (to 100 fM), which was substantiated by the observation of higher numbers of GNPs on the sensing surface in the results of the scanning electron microscopic examination. Based on the competition assay of free biotin premixed with SA-GNPs, it was concluded that some active biotin-binding sites on the streptavidin were blocked by N6-PEG, which improved the binding ability to the biotinylated sensing surface. An optimum number of binding sites on the SA-GNPs might improve their binding affinity. The strategy shown with dual polymers, viz. blocking of the sensor chip surface and coating of SA-GNPs, is recommended for developing sensors with higher sensitivity and reliability. Selective binding of the aptamer to a very small amount of FIX in the mixed sample containing FXIa and FVIIa, or albumin, makes this the optimal strategy for detecting a FIX deficiency in human blood samples. [ABSTRACT FROM AUTHOR]

Biological self-assembly is a natural process that involves various biomolecules, and finding the missing partner in these interactions is crucial for a specific biological function. Previously, we showed that evanescent-field-coupled waveguide-mode sensor in conjunction with a SiO2 waveguide, the surfaces which contain cylindrical nanometric holes produced by atomic bombardment, allowed us to detect efficiently the biomolecular interactions. In the present studies, we showed that the assembly of biomolecules can be monitored using the evanescent-field-coupled waveguide-mode biosensor and thus provide a methodology in monitoring assembly process in macromolecular machines while they are assembling. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: The control of the coupling properties of a directional coupler formed in a planar lightwave circuit by proton irradiation was examined. Irradiation of the cladding between the two cores that constitute the coupling portion causes an increase in the coupling coefficient, whereas subsequent irradiation of the entire coupling portion decreases the coupling coefficient. The change in the coupling coefficient results in changes to the coupling ratios, which depend on the irradiated area and fluence. By adjusting these, the desired coupling property can be obtained. This method can be applied in rectifying fabrication errors in any kind of optical device formed in a planar lightwave circuit. [Copyright &y& Elsevier]

Abstract: Swift heavy ion irradiation of dielectric materials forms latent tracks and selective etching of them results in the formation of well defined nanopores in the material. Irradiation of Au ions accelerated with 137MeV followed by etching with vapor of hydrofluoric acid successfully formed nanopores with diameter of 30nm and depth of 400nm in a waveguide made of amorphous SiO2. In the present research, the nanopores were applied to improve sensitivity of an evanescent-field-coupled waveguide mode sensor that detects adsorption and/or adhesion of molecules at the surface of the waveguide and in the nanopores. Increase in the sensitivity was confirmed by examining the shift in the angular position necessary to excite the waveguide mode before and after adsorption of streptavidin to biotinyl groups. [Copyright &y& Elsevier]

Abstract: We have previously made several modifications to the sensing chips of waveguide-mode sensors to permit detection of the interactions of several biomolecules at the 50-nM level. We further improved the sensitivity of this sensor by preparing a complex of a colored material with the target biomolecule. By using this strategy, we achieved a detection limit equivalent to 1nM for the interactions of aptamers with proteins in the presence of Coomassie Brilliant Blue; this corresponds to an approximately fiftyfold increase in sensitivity. For comparison, we also evaluated the sensitivity of our system to another fluorescent dye, Hoechst 33258. These studies delineated the suitability of our color-sensitive sensor chips for the detection of interactions of biomolecules at low concentrations. [Copyright &y& Elsevier]

Evanescent-field-coupled (EFC) waveguide-mode sensors can be used to detect nucleic acids or proteins from the changes in the local index of refraction upon adsorption of the target molecule on a waveguide surface. We recently described an EFC waveguide-mode sensor in which nanometric holes on a waveguide film resulted in an improved sensitivity in the analysis of the interactions of biomolecules. In the present study, we have shown that sensitivity depends upon the diameter of the holes, where increase in diameter of holes increases spectral shift resulting in an improved sensitivity. Using this improved EFC waveguide-mode sensor, we could detect interactions between RNA and a small ligand, cyanocobalamin (vita- min B12), and between RNA and a protein (human coagulation factor IXa). These two interactions were monitored on surfaces modified with biotin-streptavidin-biotin and N.(2-trifluoroethanesulfonatoethyl)-N-(methyl)triethoxysilylpropyl.3-amine, respectively. [ABSTRACT FROM AUTHOR]