Takuo Akimoto

An enzyme-chromogenic surface plasmon resonance biosensor probe for hydrogen peroxide determination using a modified Trinder's reagent.

An absorption-based surface plasmon resonance (SPR(Abs)) biosensor probe has been developed for simple and reproducible measurements of hydrogen peroxide using a modified Trinders reagent (a chromogenic reagent). The reagent enabled the determination of the hydrogen peroxide concentration by the development of deep color dyes (lambda(max)=630 nm) through the oxidative coupling reaction with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate (MAOS; C(13)H(20)NNaO(4)S.H(2)O) and 4-aminoantipyrine (4-AA) in the presence of hydrogen peroxide and horseradish peroxidase (HRP). In the present study, urea as an adduct of hydrogen peroxide for color development could be omitted from the measurement solution. The measurement solution containing 5mM hydrogen peroxide was deeply colored at a high absorbance value calculated as 46.7cm(-1) and was directly applied to the SPR(Abs) biosensing without dilution. The measurement was simply performed by dropping the measurement solution onto the surface of the SPR sensor probe, and the SPR(Abs) biosensor response to hydrogen peroxide was obtained as a reflectivity change in the SPR spectrum. After investigation of the pH profiles in the SPR(Abs) biosensor probe, a linear calibration curve was obtained between 1.0 and 50mM hydrogen peroxide (r=0.991, six points, average of relative standard deviation; 0.152%, n=3) with a detection limit of 0.5mM. To examine the applicability of this SPR(Abs) biosensor probe, 20mM glucose detection using glucose oxidase was also confirmed without influence of the refractive index in the measurement solution. Thus, the SPR(Abs) biosensor probe employing the modified Trinders reagent demonstrated applicability to other analyte biosensing tools.

Fluorescence enhancement and reflection of the excitation light observed with a multilayered substrate

Fluorescence enhancement from a multilayered substrate fabricated with Ag and Al(2)O(3) was investigated using fluorescein, rhodamine B, Cy3, and Cy5 as fluorophores. The change in the fluorescence enhancement with Al(2)O(3) had two peaks and one valley in the range from 0 to 300 nm of Al(2)O(3) thickness, and such peaks and valley were found to appear periodically. Moreover, the reflection of the excitation light from the multilayered substrate was investigated. The reflection of the excitation light periodically changed depending on the Al(2)O(3) thickness as well, and the maximum reflection was observed near the Al(2)O(3) thickness of the peak fluorescence enhancement. It was found that the periodic changes of the fluorescence enhancement and the reflection of the excitation light could be explained, for the most part, with the integral multiples of the lambda/4 derived by a simple interference theory.

A surface plasmon resonance probe without optical fibers as a portable sensing device

A surface plasmon resonance (SPR) sensor integrating a small sensor probe, a laser emission diode, a photo detector, and a polarizer was developed as a portable sensing device. The sensor probe was made with a glass cylinder, 50mm long and 1.5mm in diameter, that was connected directly to a beam splitter without optical fibers. The SPR spectrum obtained with this probe system showed a 10% reflectivity minimum at 690 nm. Shifts of the SPR spectrum induced by refractive index (RI) changes in the sample were measured by detecting the reflection light intensity at 670 nm. When the sensitivity was compared using a BIAcore SPR instrument, the lowest sensor response of 1mV observed with the SPR probe system coincided with 1.4x10(-6) of the RI changes. The RI resolution of the SPR probe was estimated with experimentally evaluated noise on the signal, and, consequently, it was concluded that the RI resolution was 1.2x10(-5). Moreover, immunoreaction was demonstrated with adsorbed bovine serum albumin (BSA) and anti-BSA antibody as an analyte. As a result, 50 ng mL(-1) of the lower detection limit was estimated.

Effect of the polarization and incident angle of excitation light on the fluorescence enhancement observed with a multilayered substrate fabricated by Ag and Al2O3.

The fluorescence from a fluorophore on a multilayered substrate fabricated by a metal and a dielectric is known to be enhanced by more than 100-fold. In the course of this study, we prepared a multilayered substrate with Ag as the metal and Al(2)O(3) as the dielectric and then investigated the effects of the polarization of the excitation light on the enhancement of the multilayered substrate. It was found that the enhancement was attributed to an electric field oscillating parallel to the substrate. Maximum 200-fold enhancement could be achieved with 80 nm thick Al(2)O(3) when an unpolarized excitation light was used with an incident angle of 20 degrees.

High-Contrast Fluorescence Imaging Based on the Polarization Dependence of the Fluorescence Enhancement Using an Optical Interference Mirror Slide

High-contrast fluorescence imaging using an optical interference mirror (OIM) slide that enhances the fluorescence from a fluorophore located on top of the OIM surface is reported. To enhance the fluorescence and reduce the background light of the OIM, transverse-electric-polarized excitation light was used as incident light, and the transverse-magnetic-polarized fluorescence signal was detected. As a result, an approximate 100-fold improvement in the signal-to-noise ratio was achieved through a 13-fold enhancement of the fluorescence signal and an 8-fold reduction of the background light.

High-Contrast Fluorescence Microscopy for a Biomolecular Analysis Based on Polarization Techniques Using an Optical Interference Mirror Slide

Fluorescence microscopy with an improved contrast for fluorescence images is developed using an optical interference mirror (OIM) slide, which can enhance the fluorescence from a fluorophore as a result of the double interference of the excitation light and emission light. To improve the contrast of a fluorescence image using an OIM slide, a linearly-polarized excitation light was employed, and the fluorescence emission polarized perpendicular to the polarization of the excitation light was detected. The image contrast with this optical system was improved 110-fold for rhodamine B spotted on the OIM, in comparison with a glass slide using a general fluorescence microscopy optical system. Moreover, a 24-fold improvement of the image contrast was achieved for the detection of Cy3-labeled streptavidin bound to immobilize biotin.

Enhanced Fluorescence Using an Optical Interference Mirror Overlaid with Silver Island Film

Fluorophores overlaid on an optical interference mirror composed of a metal and thin dielectric layer demonstrate enhanced fluorescence. Fluorescence is also enhanced by silver nanostructures such as silver island films, which excite localized surface plasmon resonance. An optical interference mirror surface was overlaid with a silver island film to amplify the fluorescence enhancement. The optimal thickness of the silver island film (100 nm) was evaluated from transmittance and surface roughness measurements. At this thickness, the fluorescence was amplified sixteen-fold. The thickness of the interference layer was optimized at 40 nm providing a one hundred-sixty fold fluorescence enhancement of rhodamine B. However, only a four-fold improvement in sensitivity was achieved for the determination of a labeled streptavidin using biotin immobilized on the silver island film interference mirror.