We have investigated various aspects of a gap mode plasmon to establish it as an analytical tool. First, markedly large (10⁷ – 10⁹) enhancement factors for the Raman scattering intensity from a thiophenol (TP) monolayer sandwiched by Ag films on a prism and silver nanoparticles (AgNPs) were obtained under attenuated total reflection (ATR) geometry. Second, AgNPs with a radius of ∼20 nm were optically trapped and immobilized on TP-covered Ag films under a gap mode resonance with extremely weak laser power density of ∼1 μW/μm² at 532 nm. The observed optical trapping and immobilization were theoretically rationalized using a dipole-dipole coupling and van der Waals interaction between AgNPs and Ag films. Third, p-alkyl TP molecules such as p-methyl TP, p-ethyl TP, p-isopropyl TP, and p-tertiary butyl TP were photocatalytically oxidized into p-carboxyl TP, whereas o- and m-methyl TP did not show such reactions.

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Potential-modulated attenuated total reflectance (PM-ATR) spectroscopy is a spectroelectrochemical method that utilizes the potential modulation approach and a waveguide ATR geometry. This unique combination enables measurements of electron-transfer (ET) kinetics of monolayer to submonolayer thin films on waveguide electrode surfaces. Selective probing of molecular subpopulations in a film can be achieved by choosing appropriate combinations of applied potential, wavelength, and polarization of light, which allows subpopulation structure to be correlated with ET kinetics. In this review, the basic theory of PM-ATR is introduced, and examples illustrating characterization of the structure and ET kinetics of organic semiconductor monolayers on electrode surfaces are presented, demonstrating the capabilities and applications of the PM-ATR technique.

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A novel methodology has been developed to determine electron-transfer rate in electrically driven redox reactions. Based on a widely adopted electrical circuit describing faradaic processes in an electrochemical cell, the approach uses a combination of impedance data from optical and electrical measurements that are simultaneously acquired in a spectroelectrochemical experiment. Once the consistency of our methodology was experimentally corroborated, it was put to practice for investigating electron-transfer rate of cytochrome c adsorbates at very low concentrations on an indium tin oxide electrode by using a highly sensitive, single-mode, electro-active, integrated optical waveguide platform. Different surface densities of redox species on the electrode interface and different ionic strengths in the electrolyte solution were studied. Higher surface densities and higher ionic strengths are shown to slow down the electron-transfer process between the redox molecules and the working electrode.

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Analysis of NH₃ gas under various humidity conditions was conducted using a waveguide surface plasmon resonance (SPR) sensor with dual sensing parts. Two pairs of Ag films/sensing polymer films were prepared separately on a waveguide core of BK-7 slide glass. Poly(acrylic acid) (PAA) and poly(vinyl alcohol) (PVA) were used as sensing materials. A white light was guided through the core by illuminating the substrate edge, and the SPR property was investigated by observing the output light spectrum. The thicknesses of PAA and PVA films were adjusted to induce SPR at different wavelengths. PAA exhibited remarkable response against NH₃ gas, but it also exhibited a strong dependence on humidity. In contrast, PVA responded to humidity but hardly responded to NH₃ gas below 20 ppm. The dual sensing would allow us to conduct precise NH₃ measurements under various humidity conditions.

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A stable two-phase sheath flow using tetrahydrofuran (THF) for an inner flow and water for an outer flow was formed in a glass capillary, and worked as a stable liquid-core/liquid-cladding optical waveguide (THF/water LLW). Although THF and water were miscible with any ratio, the length of the stable THF/water LLW at 0.9 – 2.1 cm s⁻¹ reached at least 150 mm. The THF/water LLW was applied to the observation of extraction behavior of solvatochromic fluorescence dye, 1-anilino-8-naphtalene sulfonate (ANS), through the THF/water interface. ANS was added to the water phase (clad solution) and its fluorescence, which was excited with the guided light (355 nm) through the LLW, was observed by changing the position of the detector. While the ANS stayed in the region of 70% THF to the end of the LLW without the addition of cationic surfactant, hexadecyltrimethylammonium ion (CTA⁺) at pH 3 and 11, the ion-pair of ANS and CTA⁺ was extracted into the higher concentration region of THF with the addition of CTA⁺ at pH 11.

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Early and rapid detection of bovine tuberculosis (bTB) is critical to controlling the spread of this disease in cattle and other animals. In this study, we demonstrate the development of an immunoassay for the direct detection of the bovine bTB biomarker, lipomannan (LM) in serum using a waveguide-based optical biosensor. We apply an ultra-sensitive detection strategy developed by our team, termed lipoprotein capture, that exploits the pull-down of high-density lipoprotein (HDL) nanodiscs from cattle blood that allows for the recovery and detection of associated LM. We also profile the change in the expression of these TB biomarkers as a function of time from a small set of samples collected from studies of bovine TB-infected cattle. We demonstrate for the first time the direct detection of bovine LM in serum, and clearly show that the biomarker is expressed in detectable concentrations during the entire course of the infection.

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An automated solution exchange (SE) mechanism has been introduced to slab optical waveguide spectroscopy to estimate the protein immobilizing ability of a slab optical waveguide (SOWG) surface. In each SE process, SOWG spectral change in absorbance at peak position of cytochrome c (cyt.c) Soret band at 409 nm was observed to analyze the desorption ratio of cyt.c adsorbed on SOWG surface. Continuous SE processes for 100 times have successfully brought us a kind of master desorption curve of cyt.c, which was well fitted by a double exponential equation, indicating the existence of three kinds of adsorbed states, including weakly adsorbed, strongly adsorbed, and immobilized cyt.c. The present results showed that around 30 times SE processes were enough to anticipate the ratio of desorbed and immobilized amounts of cyt.c adsorbed on SOWG surface.

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The adsorption-desorption equilibrium of sublimed molecules has been analyzed dynamically by optical waveguide (OWG) spectroscopy. Dynamic change of the transition moment during adsorption behavior was successfully detected using polarized incident light. With this technique, sublimed planer aromatic molecules, such as azobenzene, were revealed to stack parallel to the solid surface at the initial stage followed by adsorption with relatively random orientation.

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To immobilize cytochrome c (cyt.c) on an ITO electrode while keeping its direct electron transfer (DET) functionality, the ITO electrode surface was modified with 11-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}undecylphosphonic acid (CH₃O (CH₂CH₂O)₃C₁₁H₂₂PO(OH)₂, M-EG₃-UPA) self-assembled monolayer (SAM) film. After a 100-times washing process to exchange a phosphate buffer saline solution surrounding cyt.c and ITO electrode to a fresh one, an in situ observation of visible absorption spectral change with slab optical waveguide (SOWG) spectroscopy showed that 87.7% of the cyt.c adsorbed on the M-EG₃-UPA modified ITO electrode remained on the ITO electrode. The SOWG absorption spectra corresponding to oxidized and reduced cyt.c were observed with setting the ITO electrode potential at 0.3 and –0.3 V vs. Ag/AgCl, respectively, while probing the DET reaction between cyt.c and ITO electrode occurred. The amount of cyt.c was evaluated to be about 19.4% of a monolayer coverage based on the coulomb amount in oxidation and reduction peaks on cyclic voltammetry (CV) data. The CV peak current maintained to be 83.4% compared with the initial value for a M-EG₃-UPA modified ITO electrode after 60 min continuous scan with 0.1 V/s between 0.3 and –0.3 V vs. Ag/AgCl.

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Mesoporous silica is considered as promising host material for enzymes due to its uniform pore size of enzyme dimensions and tunable surface chemical properties. In this study, we applied nanoporous waveguide (NPWG) spectroscopy to observe adsorption dynamics of heme proteins with different molecular size within mesoporous silica film modified with different surface functional groups. Since NPWG spectroscopy provides kinetic information and rough quantification of adsorption amount, it is useful to study the adsorption process of enzymes within inorganic nanoporous materials.

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Infrared waveguide spectroscopy using a sapphire rod coated with an amorphous fluoropolymer (Cytop, Asahi Glass Co., ltd, Japan) has been developed in order to directly observe CO₂ in aqueous solutions. Since the amorphous fluoropolymer has a relatively high gas-permeability and hydrophobic feature, the aqueous CO₂ transmits into the amorphous fluoropolymer coating film, but water cannot penetrate into the film. Good linearity of calibration curves for CO₂ in the gas and the aqueous solution were obtained.

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In order to confirm the correlation between interfacial properties of modified surface plasmon resonance (SPR) chips and their SPR responses to immobilized anti-IgG, SPR chips were modified by mercaptoundecanoic acid, poly(ethylene glycol) diacrylate (PEG), PEG-based copolymer and cyclodextrin coupled PEG using self-assembled or radical polymerization methods. The resulting interfacial properties such as film thickness and hydrophilicity were characterized by AFM, elliptic polarization scanners and contact angle meter. Immobilization of human IgG on the modified chips was achieved by EDC/NHS activation through an amide bond. The association between fixed IgG and free anti-IgG was reflected by the variation of SPR responses and the binding ability was evaluated by Langmuir isotherms. As observed, the adsorption between IgG and anti-IgG was affected by the interfacial properties of different modifiers, such that a chip with a thinner and more hydrophilic layer may result in a higher SPR response, producing a larger adsorption equilibrium constant for protein interaction.

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A simple and rapid in situ method for the determination of hydrazine based on the concentration of aldazine compound formed by the reaction of hydrazine with p-dimethylaminobenzaldehyde was developed. This method was based on solid-phase extraction using a Sep-Pak C18 cartridge, followed by the quantification of hydrazine using a spectrophotometric method. To a sample solution of environmental water, p-dimethylaminobenzaldehyde solution was added to form aldazine by the reaction with hydrazine. The solution was passed through a Sep-Pak C18 cartridge for the adsorption of aldazine. In the laboratory, the aldazine adsorbed on the Sep-Pak C18 cartridge was eluted by passing a hydrochloric acid–ethanol (1:10) solution through the cartridge, and the color intensity of the solution was measured at 457 nm. The limit of detection for the new method was 0.2 mgN L⁻¹ of hydrazine. The determination of hydrazine in solution was not influenced even by hydrogen sulfide and organic matter. This method was then applied to the brackish water of Lake Nakaumi in the eastern area of Shimane Prefecture, Japan. This method was used to determine hydrazine in freshwater, seawater and wastwater.

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Chalcones are the proverbial precursors of many naturally occurring compounds and possess a variety of biological activities and a broad spectrum of pharmacological properties. The interaction mechanism between three chalcones, 2′,4′,4-trihydroxyflavone (also called isoliquiritigenin, 2′,4′,4-triHC), 2′,4′-dihydroxyflavone (2′,4′-diHC), and 4-hydroxyflavone (4-HC) and human serum albumin (HSA) was investigated using fluorescence quenching, fluorescence enhancement and UV absorption spectra. The binding parameters of chalcone-HSA complexes were evaluated by fluorescence quenching measurements, and the results were consistent with those obtained from fluorescence enhancement methods. The binding affinities of three chalcones with HSA at pH 7.4 were ranked in the order (the binding constants in the range 0.28 – 2.39 × 10⁵ L mol⁻¹), 2′,4′,4-triHC > 2′,4′-diHC > 4-HC, indicating that the three chalcones displayed tight affinities for HSA and the hydroxyl group in chalcones played a key role in their binding affinities with HSA. The results of the UV absorption and the fluorescence enhancement elucidated that the chalcones may lead to micro-environmental and conformational changes of HSA. The binding site of the chalcone 2′,4′,4-triHC on the HSA was explored by the spectroscopic properties of the 2′,4′,4-triHC-HSA complex at pH 7.4 and 3.5, and the results demonstrated that 2′,4′,4-triHC bound within the hydrophobic pockets of subdomain IIA of HSA, namely site I, and the electrostatic force and ionic interactions played a crucial role in the binding interaction between chalcones and protein. The obtained results provided scientific evidence for the binding property of the chalcones and protein, which would be helpful for development of novel drugs with the skeleton of chalcones.

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We present a colorimetric method for the detection of aminoglycoside antibiotics such as neomycin (NEO) using a reddish anionic dye, amaranth (AR³⁻). Under acidic conditions, at which NEO exists in fully protonated form (NEOH⁶⁺), the AR³⁻ anion associates with the NEOH⁶⁺ cation to form a precipitate, NEOH(AR)₂. The precipitate was soluble in a buffer solution of pH 8.5, yielding a reddish solution with an absorption maximum at around 520 nm. Tobramycin and gentamycin, which exist as pentavalent cations under acidic conditions, gave almost the same results. On the other hand, kanamycin, amikacin and streptomycin, which would exist as tri- and tetravalent cations, were not precipitated. Thus, the AR³⁻ anion could be considered to be an analytical reagent for specific aminoglycosides with polycationic functionality. However, since the precipitation reaction was considerably affected by other anions, a separation method using the tetraphenylborate anion was employed as a pretreatment. The separation method involves precipitating the polycationic aminoglycosides with the tetraphenylborate anion, washing the precipitate with acetonitrile, and re-precipitating the aminoglycosides as hydrochloride salts. Thus, the present method was applied to a microtiter plate assay of the products in an NEO-producing culture broth.

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To achieve a highly sensitive and onsite analysis of a small amount samples, a microplasma-based micro total analysis systems (μ-TAS) device was developed. A dielectric barrier discharge (DBD) that can generate a stable plasma at atmospheric pressure was generated in a microchip and used as the plasma source. The use of DBD suppresses the temperature rise of the electrodes and enables operation for long times because of a reduction of the electrode damage due to suppression of the current via dielectric interposing between the electrodes. It is expected that the analytical system can be miniaturized because helium plasma is generated in the microchannel contained in the microchip. Emissions from gaseous Cl, Br, and I were analyzed using the plasma source, and it was found that the detection limits for these analytes were 0.22, 0.18, and 0.14 ppm, respectively. The calibration curves for gaseous Cl, Br, and I were also plotted obtaining correlation coefficients of 0.975, 0.955 and 0.986, respectively, and showing good linearity for the developed plasma source.

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An optical assay for the rapid determination and chemical speciation of Fe²⁺/Fe³⁺ species has been proposed for the first time on a polyether sulfone (PES) membrane platform. The small pore size and low wettability (θ ∼82°) of the membrane disallowed the dissipation of analyte droplets on the surface, thus localizing it onto nanoliter arrayed 1,10-phenanthroline spots. Under optimized conditions and within ∼5 min, an acceptable limit of detection (0.1 μg mL⁻¹) and linear dynamic range (1 – 100 μg mL⁻¹) were achieved. The proposed method was also successfully applied for indirect determination of Fe³⁺ ions in synthetic samples after reduction to Fe²⁺ using SO₂. The performance of the proposed sensor was validated for its robustness and stability. Due to high selectivity and accuracy, the method was satisfactorily applied for the analysis of Fe²⁺/Fe³⁺ species in marine water samples. The proposed method is an easy and low-cost system coupled with good reproducibility and ruggedness, applicable for point-of-use testing.

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Incoherent broadband cavity-enhanced absorption spectroscopy (IBBCEAS) is a useful technique for measuring trace gaseous species in the atmosphere. Recently, IBBCEAS was used to measure concentrations of nitrous acid (HONO) in the troposphere to resolve controversies related to its formation and loss. Here, measurements of HONO and a mixture of HONO and NO₂ using IBBCEAS were validated by comparing them with those obtained with a NOx analyzer. Good agreement was found between these methods, given their respective experimental uncertainties. The detection limit of our IBBCEAS instrument was 0.2 ppbv, with a signal-to-noise ratio of 1, and a 5-min integration time.

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Here, we report that a single-cell microwell array based on photocrosslinked hydrogel can be used to screen cells exhibiting a defective regulatory volume decrease (RVD) in high-throughput. The RVD is a regulatory function of cells that maintains cell volume homeostasis in a hypotonic medium. Single Madin–Darby canine kidney (MDCK) cells grown in the microwells were loaded with a volume-sensitive fluorescence dye. Changes in the volume of discrete single cells were traced for 20 min in a hypotonic solution using a wide-field fluorescence microscopy. The volume changes of more than 100 single cells were analyzed simultaneously using time-lapse fluorescence micrographs. Cells showing erratic RVD could be easily screened from the image analysis. Nearly 40% of the MDCK single cells exhibited weak, or no, RVD. Since other previously reported methods could not detect as many changes in the volume of discrete singles cells as the method used in this report, we anticipate that our reported method will provide an efficient way of elucidating the RVD mechanisms of cells that have not yet been completely understood.

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A single-step electrochemical immunochromatography has been developed: the device was based on two pieces of nitrocellulose membrane, a sample pad with anti-mouse IgG antibody labeled with glucose oxidase (GOx-labeled antibody), a conjugate pad with glucose, and a Pt working electrode. Either antibody or antigen was immobilized on the membrane. The addition of a solution containing mouse IgG, a model target, allows for the dissolution of GOx-labeled antibody in the sample pad to form an immunocomplex. The produced immunocomplex was automatically separated by capturing to the antibody immobilized on the membrane with the sandwich structure or by passing through the membrane modified with an antigen for the competitive reaction. The separated GOx label arrived at the conjugate pad with glucose to undergo the enzyme reaction. Hydrogen peroxide generated by this reaction was detected at the Pt electrode prepared on the second nitrocellulose membrane downstream from the conjugate pad. The results demonstrated that the designed immunochromatography can be applied to quantitative detection with a single-step procedure, because both the GOx-labeled antibody for revealing the immunoreactions and the substrate for the enzyme reaction were prepared in the device. Moreover, the initial concentration of the GOx-labeled antibody permitted control of the detectable concentration for mouse IgG.

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Laser ablation in liquid (LAL) sampling method transformed hard-to-digest materials to soluble particles, and thus allowed for smooth decomposition by acid digestion. LAL sampling is useful to generate nanoparticles from samples with less contamination. After acid digestion, trace elements in the LAL-sampled particles were analyzed by solution nebulization inductively coupled plasma mass spectrometry (ICPMS). For the first time we demonstrated that LAL-ICPMS can be used to determine trace elements in hard-to-digest samples; sintered SiC and single-crystal SiC. Results obtained by laser ablation ICPMS and LAL-ICPMS were compared in terms of accuracy and detection limits. The detection limits of LAL-ICPMS were 0.04 – 0.4 μg g⁻¹ for Al, Ti, Cr, Mn, Fe, Co, Ni, Cu, Sr, Y, Zr, and W. LAL-ICPMS is expected to be used to control contamination in the manufacturing of semiconductor devices.

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