•A compact photometric detector was assembled with an OLED and an OPD.•The OLED based on Eu(DBM)3bath as the emitting layer provides a narrow emission spectrum.•The flat shape of the OLED and the OPD was suitable for a flat shaped microchip for assembling.A compact photometric detector was constructed from an organic light emitting diode (OLED) based on a europium complex, europium(diben-zoylmethanato)3(bathophenanthroline) (Eu(DBM)3bath), as the light source and an organic photodiode (OPD) fabricated from a hetero-junction of two layers of copper phthalocyanine (CuPc)/fullerene (C60) as the photo-detector on a microchip prepared from poly(dimethylsiloxan) (PDMS) and was applied to the determination of phosphate. The OLED and the OPD were fabricated by a vapor deposition method on an indium tin oxide (ITO) coated glass substrate with the following layered structure; Glass (0.7 mm)/ITO (110 nm)/4,4′-bis[N-(1-naphthyl)-N-phenyl amino]-biphenyl (α-NPD) (30 nm)/4,4′-di(N-carbazolyl)biphenyl (CBP): Eu3+ (8 wt%, 30 nm)/bathocuproine (BCP) (30 nm)/aluminum tris(8-hydroxyquinoline) (Alq3) (25 nm)/magnesium and silver (MgAg) (100 nm)/Ag (10 nm) and Glass (0.7 mm)/ITO (110 nm)/CuPc (35 nm)/C60 (50 nm)/BCP (10 nm)/Ag (50 nm), respectively. The OLED based on the europium complex emitted a sharp light at the wavelength of 612 nm with a full width at half maximum (FWHM) of 8 nm. The performance of the photometric detector assembled was evaluated based on measurements of the absorbance of different concentrations of malachite green (MG) solutions for a batch system with 1 cm long path length. The molar absorptive coefficient of the MG solution, calculated from the photocurrent of the OPD, was in good agreement with the value reported in the literature. A microchip with two inlets and one outlet U-shaped channel was prepared by a conventional photolithograph method. The OLED and the OPD were configured so as to face each other through the PDMS microchip in parallel in order to align the light axis of the OLED and the OPD with the flow cell (optical path length of 5 mm), which was located at the end of outlet. For the determination of phosphate, an ion-association reaction between MG and a molybdenum-phosphate complex was utilized and a good linear relationship between the concentration and absorbance was observed in the concentration range 0–0.2 ppm, with a detection limit (S/N=3) of 0.02 ppm. The assembled photometric detector was also applied to the determination of phosphate by the flow injection of river water samples using the reagent solution containing MG and molybdenum ammonium in sulfuric acid. A good recovery (97–99%) for the river water samples, which had been spiked with the standard 0.08 ppm, with an RSD of ca 5% (n=5) was obtained using the constructed system.

•A compact fluorometric detector was assembled with an OLED and an OPD.•The OLED based on Tb(acac)3bath as the emitting layer provides a narrow green emission spectrum.•The OPD with higher photosensitivity was obtained by using the bulk heterojunction of C70 and TPTPA.•The developed fluorometric detector was applied to competitive immunoassays for APE.A compact fluorescence detector was constructed on a microchip from an organic light emitting diode (OLED) as the light source and an organic photodiode (OPD) as the photo-detector and was used in an immunoassay for alkylphenol polyethoxylates (APE). The OLED based on a terbium complex emitted a sharp light at the main wavelength of 546 nm with a full width at half maximum of 9 nm. The incident photo-to-current conversion efficiency (IPCE) of the OPD fabricated with Fullerene 70 (C70) and tris[4-(5-phenylthiopen-2-yl)phenyl]-amine (TPTPA) was approximately 44% for light at a wavelength of 586 nm. The performance of the fluorescence detector was evaluated for the determination of resorufin (λem=586 nm) and the photocurrent of the OPD due to the fluorescence of resorufin was proportional to the concentration of resorufin in the range from 0 to 18 µM with a detection limit (S/N=3) of 0.6 µM. The fluorescence detector was successfully utilized in a competitive enzyme-linked immunosorbent assay for APE, where an anti-APE antibody was immobilized on the surface of the channel of the Polydimethylsiloxane (PDMS) microchip or on the surface of magnetic microbeads. After an immunoreaction with a sample solution of APE containing a horse radish peroxidase (HRP)-labeled APE, the fluorescence of resorufin generated just after introduction of a mixed solution of Amplex Red and H2O2 was measured using the fluorescence detector. The calibration curve for the photocurrent signals of the OPD due to the fluorescence of resorufin against the logarithmic concentration of APE was sigmoidal in shape. The detection limits defined as IC80 were ca. 1 ppb and ca. 2 ppb, respectively, for the methods using the anti-APE antibody immobilized on the surface of the microchannel and in the case where the antibody was immobilized on the surface of magnetic microbeads.

•An organic photodiode was successfully employed for the fluorometric ELISA.•Competitive ELISA was performed at the surface of magnetic microbeads.•Alkylphenol polyethoxylates were determined at the concentration as low as 2 and 4 ppb in batch and flow system, respectively.•Stability constant of the immunoreaction with alkylphenol polyethoxylates and the antibody was estimated from the result.An organic thin film photodiode (OPD) was successfully employed as a portable photodetector in a competitive enzyme-linked immunosorbent assay (ELISA) of a class of nonionic surfactants, namely alkylphenol polyethoxylates (APnEOs) which are an environmental pollutant. Microbeads that were chemically immobilized with an anti-APnEOs antibody were used in the assay. The OPD consisted of a layer of copper phthalocyanine (CuPc), C60 and a second layer of bathocuproine (BCP) with a bulk heterojunction composed of CuPc and C60 prepared by a vapor deposition method on an indium-tin oxide coated glass substrate. The OPD showed an incident photon-current efficiency (IPCE) of approximately 19% for light at a wavelength of 585 nm. This relatively high IPCE at 585 nm makes it suitable for detecting the fluorescence of resorufin (λem=585 nm), the product of the competitive ELISA, produced through the enzymatic reaction of Amplex Red with horseradish peroxidase (HRP) and H2O2. A fluorometric detector was assembled on a microchip by combining the fabricated OPD and a commercial LED as a photodetector and a light source, respectively. The photocurrent of the OPD due to the fluorescence of resorufin was proportional to the concentration of resorufin in the concentration range from 0 to 8 μM. When the fabricated OPD was used as a portable photodetector, the competitive ELISA of APnEOs using HRP labeled APnEOs (HRP–APnEOs) was performed on magnetic microbeads on which surface an anti-APnEOs antibody had been immobilized. A typical sigmoidal calibration curve was obtained and the data were in good agreement with a numerical simulation, where the photocurrent of the OPD was plotted against the concentration of APnEOs, determined via the competitive ELISA. The detection limit of the immunoassay for APnEOs was approximately 2 and 4 ppb in batch and flow system, respectively.

The performance of an organic thin film photodiode (OPD), fabricated from a hetero-junction comprised of two layers of C60 and a phthalocyanine-Cu(II) complex was evaluated by detecting the chemiluminescence generated from the reaction of luminol with horseradish peroxidase in the presence of H2O2, and the fluorescence from resorufin, as an optical detector. The photocurrent of the OPD was linear with respect to the power of light from a commercial LED. The sensitivity of the OPD was sufficient for detecting chemiluminescence with a power 0.1 μW/cm2. The OPD was successfully used in a flow-immunoassay for IgA, a marker of human stress, in which a sandwich immunoassay was carried out on the microchip and the fluorescence from resorufin, produced by the enzymatic reaction, was detected. The detection limits for resorufin and IgA were 5.0 μM and 16 ng/mL, respectively. The photosensitivity of the OPD remained relatively constant for a minimum of one year.

A novel fluorimetric assay for dopamine using calcein blue (CB) complexed with Fe2+ ion as a chemical sensor is described. The fluorescence arising from CB of the CB–Fe2+ complex is quenched by the Fe2+ ion. When dopamine is added to a solution of the CB–Fe2+ complex, a dopamine–Fe2+ complex is formed as the result of a ligand exchange reaction between CB and dopamine which permits the fluorescence from CB to be recovered. The fluorescence intensity at the wavelength of 440 nm (at the excitation wavelength of 340 nm) was found to be proportional to the concentration of the dopamine added to the CB–Fe2+ complex solution, which permits dopamine to be quantitatively determined. The selectivity for dopamine in the presence of other catecholamines and related compounds was good. The calibration curve for dopamine, determined using experimental data was successfully simulated based on the equilibrium of the ligand exchange reaction between CB and dopamine. The working range is from 50 μM to 1 mM and the limit of detection and limit of quantization are ca 10 μM and 50 μM, respectively. The assay is simple and economical, compared with conventional methods such as an enzyme-linked immunosorbent assay (ELISA).Highlights► A fluorimetric determination of dopamine using calcein blue (CB) complexed with Fe2+ ion based on ligand exchange mechanism is the first report. ► The method is simple just mixed the CB–Fe2+ complex solution with a dopamine solution. ► The method is with good selectivity to dopamine.

A simple and sensitive flow injection fluorometric method for the determination of ascorbic acid is described. Perylenebisimide-linked nitroxide (PBILN) is used as a fluorescent reagent, which permits the selective determination of ascorbic acid. The fluorescence of the perylenebisimide moiety in PBILN is quenched by the nitroxide moiety, which is linked to the perylenebisimide. When a stream of a solution of ascorbic acid is merged with a stream of PBILN, the ascorbic acid reacts with the nitroxide moiety of PBILN to form hydroxylamine, and the fluorescence properties of the perylenebisimide moiety are recovered. As a result, a peak-shaped fluorescence signal is produced, which can be observed by a fluorescence detector located downstream. Under optimized conditions, a good linear relationship between the concentration of ascorbic acid and peak height in the concentration range from 0.5 to 10 μmol L−1 was found and the detection limit (S/N = 3) was 0.28 μmol L−1. The relative standard deviation for the determination of 4.0 μmol L−1 ascorbic acid samples was 1.0% (n = 5). The proposed method was applied to the determination of ascorbic acid in several soft drink beverages and the analytical results were in good agreement with those obtained using a conventional method.

The possibility of applying antimony-film modified glassy carbon electrode in sequential-injection analysis (SIA) was investigated with the objective of determining Pb(II) and Cd(II) by anodic stripping voltammetry (ASV). The conditions of antimony-film deposition concerning composition of the plating/carrier solutions, concentrations of Sb(III) and hydrochloric acid, effects of different supporting electrolyte salts, and plating potential were optimized. It was found that the antimony-film deposition on glassy carbon substrate in a sample solution consisting of 750 μg L−1 Sb(III), 0.5 mol L−1 HCl at −1.5 V (vs. Ag/AgCl/3 mol L−1 KCl) yielded a modified electrode suitable for the determination of Pb(II) and Cd(II) at the μg L−1 level. The reproducibility of the analytical signals was characterized by a relative standard deviation lower than 2.8%, and the calculated values of detection limits were 1.2 μg L−1 for Pb(II) and 1.4 μg L−1 for Cd(II). The presence of KSCN in the sample solution offers the possibility of detecting ions with more negative oxidation potentials like Zn(II), Mn(II) or Cr(III). The developed SIA-ASV procedure was compared with the commonly used batch method, and its applicability was tested on a spiked tap water sample.

A novel fluorescent probe composed of two moieties, Nile Red and an iminodiacetic acid–Ni2+ complex, for the detection of histamine in living cells is described. The probe was successfully applied to visualizing histamine in RAW264 cells, representing the first demonstration of the imaging of histamine itself in living cells.

We report on a novel histamine monitoring method by using a fluorescent probe, a complex between Ni2+ and calcein, based on a ligand exchange mechanism. The fluorescence intensity of this probe, which has been reduced due to effective quenching by Ni2+ ion, increases drastically by an addition of histamine. Furthermore, the probe shows high selectivity toward histamine among the various neurotransmitters in 0.1 M phosphate buffer solution (pH 7.4). Biomonitoring studies to detect histamine released from RAW264 cells are successfully represented.

A new type of fluorophore-linked nitroxide for the detection of hydroxyl radical (OH) was synthesized using perylene-3,4,9,10-tetracarboxyl bisimide and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). This new fluorescent probe, indicated here as Spy-OH (swallow-tailed perylene derivative for detecting OH) has its natural fluorescence effectively reduced due to the quenching caused by the presence of the nitroxide radical in its structure. In the presence of OH, in media containing DMSO, a conspicuous increase in the fluorescence intensity was observed. No significant change in the fluorescence intensity was observed upon the addition of other several reactive oxygen species (ROS) except for OH. Spy-OH maxima excitation (λexc) and emission (λem) wavelengths lie in the visible region (520/530 nm), therefore, this fluorescent probe is expected to be useful for a simple and selective detection of OH, especially for biosamples.

A simple and sensitive method for the determination of a specific IgG antibody against ovalbumin (anti-OVA IgG antibody) for diagnosis of egg allergy by use of a surface plasmon resonance (SPR) immunosensor in a flow system is described. An OVA conjugate was immobilized on a sensor chip via a self-assembled monolayer of 11-mercaptoundecanoic acid through an amino coupling method. The determination of the anti-OVA IgG antibody was based on a kind of sandwich immunoassay using an anti-IgG secondary antibody in order to enhance the sensitivity of the SPR immunosensor. The sensitivity and detection limit of the present method for the anti-OVA IgG antibody were 7 mdeg/ppm and 300 ppb, respectively. The present method showed an enhanced sensitivity and detection limit for the determination of the anti-OVA antibody, compared with a direct immunoassay, by which the sensitivity and detection limit were 3 mdeg/ppm and 1 ppm, respectively. By assuming a Langmuir type of adsorption isotherm, the affinity constants of an anti-OVA IgG antibody immunocomplex with the anti-IgG secondary antibody and of the anti-OVA antibody to the OVA conjugate immobilized on the sensor chip were calculated to be 2.1 × 106 M−1 and 2.0 × 106 M−1, respectively. The present flow immunoassay for the anti-OVA IgG antibody by an SPR sensor has potential applications in both research and diagnosis of egg allergy.

A fluorescent photochromic compound, composed of diarylethene, fluorescein and succinimidyl ester units, was developed for the controllable fluorescent labeling of biomolecules based on a small molecule.

A swallow-tailed perylene derivative including a triphenylphosphine moiety was synthesized and applied to the detection and the live-cell imaging of lipid hydroperoxides. The novel probe, named Spy-LHP, reacted rapidly and quantitatively with lipid hydroperoxides to form the corresponding oxide, Spy-LHPOx, which emits extremely strong fluorescence (Φ ∼ 1) in the visible range (λem = 535 nm, 574 nm). Spy-LHP was highly selective for lipid hydroperoxides, and the addition of other reactive oxygen species (ROS) including hydrogen peroxides, hydroxyl radical, superoxide anion, nitric oxide, peroxynitrite, and alkylperoxyl radical, caused no significant increase in the fluorescence intensity. The probe exhibited good localization to cellular membranes and was successfully applied to the confocal laser scanning microscopy (CLSM) imaging of lipid hydroperoxides in live J774A.1 cells, in which lipid peroxidation was proceeded by the stimulation of 2,2-azobis(2-amidinopropane)dihydrochloride (AAPH). These findings establish Spy-LHP as a promising new tool for investigating the physiology of lipid hydroperoxides.

The first fluorescent labeling technology, which can induce not only an increase in the fluorescence intensity but also a shift in the fluorescence spectrum, has been developed for “ratiometric” measurements for a protein by utilizing a newly designed “field-sensitive” fluorescent probe and its corresponding unique amino acid tag.

A novel fluorescent probe, 7-hydroxy-2-oxo-N-(2-(diphenylphosphino)ethyl)-2H-chromene-3-carboxamide (DPPEA-HC) was developed for use in monitoring hydrogen peroxide (H2O2) production. DPPEA-HC, which consists of a diphenylphosphine moiety and a 7-hydroxycoumarin moiety, reacts with H2O2 to form DPPEA-HC oxide, which is analogous to the reaction of triphenylphosphine with hydroperoxides such as H2O2 to form triphenylphosphine oxide. Photoinduced electron transfer (PET) was applied in the design of DPPEA-HC. Since the diphenylphosphine moiety and the 7-hydroxycoumarin moiety would act as the PET donor and the acceptor, respectively, it would be expected that DPPEA-HC would rationally cancel the PET process via the formation of DPPEA-HC oxide, based on the calculated energy levels of the donor and the acceptor moieties using the B3LYP/6-31G*//AM1 method. The fluorescence intensity of DPPEA-HC increased on the addition of a H2O2 solution in 100 mM sodium phosphate buffer (pH 7.4), as predicted from the energy level calculation and a good correlation between increase in the fluorescence of DPPEA-HC and the concentration of H2O2 was observed. DPPEA-HC was also fluoresced by H2O2, which was enzymatically produced in xanthine/xanthine oxidase/superoxide dismutase (XA/XOD/SOD) system. The increase in the fluorescence of DPPEA-HC in the presence of H2O2 immediately ceased on the addition of catalase (CAT), which catalyzes the disproportionation of H2O2. In addition, DPPEA-HC was found to have a much higher selectivity for H2O2 and a greater resistance to autoxidation than 2′,7′-dichlorodihydrofluoresein (DCFH). Time-resolved fluorescence measurements of DPPEA-HC and DPPEA-HC oxide confirmed that the fluorescence off/on switching mechanism of DPPEA-HC is based on the PET on/off control.A novel fluorescent probe for monitoring hydrogen peroxide (H2O2), DPPEA-HC, was developed by utilizing the fluorescence off/on switching mechanism, based on the on/off control of photoinduced electron transfer (PET). DPPEA-HC reacted with H2O2 to form the strongly-fluorescent DPPEA-HC oxide. Because of the high selectivity for H2O2 and the greater resistance to autoxidation, the probe is expected to be useful for the detection of H2O2 in cellular systems.

A surface plasmon resonance (SPR) immunosensor based on a competitive immunoreaction for the determination of trinitrophenol (TNP) is described. A goat anti-mouse IgG (1st antibody), which recognizes an Fc moiety of an antibody, was immobilized on a gold film of an SPR sensor chip by physical adsorption. A TNP solution containing a fixed concentration of a mouse anti-TNP monoclonal antibody (2nd antibody) and a TNP-keyhole limpet hemocyanin (KLH) conjugate was incubated in one-pot and introduced into the sensor chip. The TNP-KLH conjugate competes with TNP for binding with the 2nd antibody. The resulting complex of the 2nd antibody with the TNP-KLH conjugate was bound to the 1st antibody, which is immobilized on the sensor chip. The SPR sensor signal based on resonance angle shift is dependent on the concentration of TNP in the incubation solution in the range from 25 ppt to 25 ppb, and the coefficient of variation of the SPR signals for the 25 ppb TNP solution was determined to be 13% (n = 4). The experimental results for the adsorption constant of the 1st antibody on the sensor chip and the binding constant of the 1st antibody complex with the 2nd antibody are discussed, together with theoretical considerations.

A rapid and sensitive immunoassay for the determination of linear alkylbenzene sulfonates (LAS) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a neodymium magnet. Magnetic beads, to which an anti-LAS monoclonal antibody was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by means of a neodymium magnet and adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an anti-LAS monoclonal antibody on the magnetic beads and the LAS sample and horseradish peroxidase (HRP)-labeled LAS, and was based on the subsequent chemiluminscence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The anti-LAS antibody was immobilized on the beads by coupling the antibody with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactic acid film. The antibody immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the neodymium magnet, an LAS solution containing HRP-labeled LAS at constant concentration and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photon counting unit located at the upper side of the flow cell by collecting the emitted light with a lens. A typical sigmoid calibration curve was obtained, when the logarithm of the concentration of LAS was plotted against the chemiluminescence intensity using various concentrations of standard LAS samples (0–500 ppb) under optimum conditions. The time required for analysis is less than 15 min.

A novel fluorescent probe, the detection mechanism of which is based on the ‘on-off’ switching of a FRET triggered by the ˙OH-induced cleavage of a DNA strand, has been developed for the ratiometric imaging of ˙OH.

A rapid and sensitive immunoassay for the determination of carp vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a samarium-cobalt magnet. An anti-Vg monoclonal antibody, immobilized on magnetic beads, was used as a solid support for the immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by a samarium-cobalt magnet and the flow of the carrier solution. The immunoassay was based on a sandwich immunoreaction of anti-Vg monoclonal antibody (primary antibody) on the magnetic beads, Vg, and the anti-Vg antibody labeled with horseradish peroxidase (HRP) (secondary antibody), and was based on a subsequent chemiluminescence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The magnetic beads to which the primary antibody was immobilized were prepared by coupling the primary antibody with the magnetic beads after an agarose-layer on the surface of the magnetic beads was epoxidized. The primary antibody-immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the samarium-cobalt magnet, a Vg sample solution, an HRP-labeled secondary antibody solution and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photomultiplier located at the upper side of the flow cell. The optimal incubation times both for the first and second immunoreactions were determined to be 20 min. A concave calibration curve was obtained between Vg concentration and chemiluminescence intensity when various concentrations of standard Vg samples (2–100 ng mL−1) were applied to the SIA system under optimal conditions. In spite of a narrow working range, the lower detection limit of the immunoassay was about 2 ng mL−1.

A surface plasmon resonance (SPR) based biosensor was developed for monitoring 2,4-dichlorophenol, a known dioxin precursor, using an indirect competitive immunoassay. The SPR sensor was fabricated by immobilizing a gold-thin layer on the surface of an SPR sensor chip with an anti-(2,4-dichlorophenol) antibody using a gold binding polypeptide (GBP) and protein G. The SPR response based on the antigen–antibody reaction in a flow system was measured by injecting a 2,4-dichlorophenol sample solution into the flow system in which the SPR sensor was located. In a direct immunoassay system using the modified sensor chip, no significant SPR angle shift less than 0.001° was observed when a 25 ppm of 2,4-dichlorophenol solution was injected. In order to improve the sensitivity of the SPR sensor, an indirect competitive immunoassay method was used in conjunction with the SPR sensor system using 2,4-dichlorophenol conjugated with bovine serum albumin (BSA). In the competitive assay, a 350 ppm 2,4-dichlorophenol–BSA conjugate solution containing 2,4-dichlorophenol at various concentrations (10–250 ppb) were injected into the SPR sensor system. The sensitivity of this indirect immunoassay was found to be extremely sensitive, compared to the direct one, and a detection limit of 20 ppb was estimated. Verification that the use of GBP for immobilizing the antibody on the sensor chip enhanced the sensitivity to 2,4-dichlorophenol was obtained by comparing the procedure with another modification, in which BSA was used instead of GBP for immobilizing the antibody on the sensor chip. The affinity constant of 2,4-dichlorophenol and its conjugate to the antibody were estimated form the SPR response.

A highly sensitive potentiometric flow injection analysis method for the determination of manganese(II), utilizing a redox reaction with hexacyanoferrate(III) in near neutral media containing ammonium citrate is described. The analytical method is based on the detection of the change in potential of a flow-through type redox electrode detector, resulting from the composition change of an [Fe(CN)6]3−–[Fe(CN)6]4− potential buffer solution. A linear relationship between the potential change (peak height) and the concentration of manganese(II) was found. Manganese(II) in a wide concentration range from 10−4 to 10−7 M could be determined by appropriately altering the concentration of the potential buffer from 10−3 to 10−5 M. The lower detection limit of manganese(II) was determined to be 1×10−7 M. The sampling rate and relative standard deviation were 20 h−1 and 1.9% (n=8) for 6×10−6 M manganese(II), respectively. The proposed method was successfully applied to the determination of manganese(II) in actual soil samples obtained from tea fields. Analytical results obtained by the proposed method were in good agreement with those obtained by an atomic absorption spectrophotometric method.

A sequential injection analysis (SIA) technique, in which antibody-immobilized microbeads were transferred to a jet ring (JR) cell, was used in determination of carp vitellogenin (Vg). The determination is based on a sandwich immunoassay in which two types of reactions between anti carp Vg antibodies and carp Vg are used. Namely, the antibody for the first reaction step was immobilized on microbeads (Sephadex beads), and an antibody labeled with a horseradish peroxidase (HRP) was used in the second step of the reaction. A mixed solution of hydrogen peroxide and o-phenylenediamine (OPD) was used as the source of the chromophore in the reaction. The microbeads-immobilized antibody, Vg analyte, HRP-labeled anitbody and the color developing solution were introduced automatically into the JR cell of the SIA system in a programmed sequence, and the absorbance of the oxidized OPD product was used to determine the amount of Vg present. The optimal incubation times for the immuno-raction for the first and the second steps were determined at 120 and 60 min, respectively, taking into account the sensitivity to the Vg determination. Under these conditions, a good linear correlation was obtained between Vg concentration and the absorbance of the oxidized OPD. The lower detection limit for the determination of Vg was about 5 ng ml−1 in this system. The method developed here represents a simple, accurate method for the determination method of Vg.

A spectrophotometric FIA method for the determination of the total base number (TBN) in a lubricant was proposed, which involved using an acid–base buffer solution prepared with a nonaqueous solvent. This method is based on measurements of the absorbance change of an indicator contained in the acid–base buffer solution, which is generated due to a neutralization reaction of base in the lubricant with the buffer acid. The sample injected into a stream of a nonaqueous solvent, toluene/H2O/2-propanol (52/1/47 v/v/v), was merged with a stream of perchloric acid (HClO4) solution, and then neutralized with HClO4. An excess of HClO4 was subsequently merged with a stream of tetrabutylammonium salt of trifluoroacetic acid (TFA·TBA) solution containing an indicator (m-cresol purple), which pKa value was found to be similar to pKa value of TFA in the present mixed nonaqueous solvent. The reaction of the excess HClO4 with TFA·TBA gave rise to the composition change of the acid–base buffer solution, TFA/TFA·TBA. Since the indicator, m-cresol purple, behaves similarly to the buffer component, the change in the concentration ratio of TFA/TFA·TBA can be determined by the measurement of the absorbance change of the indicator. The absorbance changes were monitored with a spectrophotometric detector. The sensitivity of the proposed method for several kinds of basic compounds, which were often added to lubricant, was nearly identical irrespective of the basicity of the compounds. The proposed method was successfully applied to the determination of the total base number in lubricant.

The flow injection analysis of hydrogen peroxide is proposed, using a redox electrode and an Fe(II)–Fe(III) potential buffer solution. Influencing factors, such as the concentrations of Fe(II)–Fe(III) and sulfuric acid in the potential buffer on sensitivity of the proposed method are examined. The analysis of high concentrations of hydrogen peroxide up to ∼10 M was conducted successfully with relative standard deviation of 0.7%.

A rapid potentiometric flow injection technique for the simultaneous determination of oxychlorine species such as ClO3−–ClO2− and ClO3−–HClO has been developed, using both a redox electrode detector and a Fe(III)–Fe(II) potential buffer solution containing chloride. The analytical method is based on the detection of a large transient potential change of the redox electrode due to chlorine generated via the reaction of the oxychlorine species with chloride in the potential buffer solution. The sensitivities to HClO and ClO2− obtained by the transient potential change were enhanced 700–800-fold over that using an equilibrium potential. The detection limit of the present method for HClO and ClO2− is as low as 5×10−8 M with use of a 5×10−4 M Fe(III)–1×10−3 M Fe(II) buffer containing 0.3 M KCl and 0.5 M H2SO4. On the other hand, sensitivity to ClO3− was low when a potential buffer solution containing 0.5 M H2SO4 was used, but could be increased largely by increasing the acidity of the potential buffer. The detection limit for ClO3− was 2×10−6 M with the use of a 5×10−4 M Fe(III)–1×10−3 M Fe(II) buffer containing 0.3 M KCl and 9 M H2SO4. By utilizing the difference in reactivity of oxychlorine species with chloride in the potential buffer, a simultaneous determination method for a mixed solution of ClO3−–ClO2− or ClO3−–HClO was designed to detect, in a timely manner, a transient potential change with the use of two streams of potential buffers which contain different concentrations of sulfuric acid. Analytical concentration ranges of oxychlorine species were 2×10−5–2×10−4 M for ClO3−, and 1×10−6–1×10−5 M for HClO and ClO2−. The reproducibility of the present method was in the range 1.5–2.3%. The reaction mechanism for the transient potential change used in the present method is also discussed, based on the results of batchwise experiments. The simultaneous determination method was applied to the determination of oxychlorine species in a tap water sample, and was found to provide an analytical result for HClO, which was in good agreement with that obtained by the o-tolidine method and to provide a good recovery for ClO3− added to the sample.

A swallow-tailed perylene derivative including a triphenylphosphine moiety was synthesized and applied to the detection and the live-cell imaging of lipid hydroperoxides. The novel probe, named Spy-LHP, reacted rapidly and quantitatively with lipid hydroperoxides to form the corresponding oxide, Spy-LHPOx, which emits extremely strong fluorescence (Φ ∼ 1) in the visible range (λem = 535 nm, 574 nm). Spy-LHP was highly selective for lipid hydroperoxides, and the addition of other reactive oxygen species (ROS) including hydrogen peroxides, hydroxyl radical, superoxide anion, nitric oxide, peroxynitrite, and alkylperoxyl radical, caused no significant increase in the fluorescence intensity. The probe exhibited good localization to cellular membranes and was successfully applied to the confocal laser scanning microscopy (CLSM) imaging of lipid hydroperoxides in live J774A.1 cells, in which lipid peroxidation was proceeded by the stimulation of 2,2-azobis(2-amidinopropane)dihydrochloride (AAPH). These findings establish Spy-LHP as a promising new tool for investigating the physiology of lipid hydroperoxides.

A fluorescent photochromic compound, composed of diarylethene, fluorescein and succinimidyl ester units, was developed for the controllable fluorescent labeling of biomolecules based on a small molecule.