Toshihiko Imato

A surface plasmon resonance immunosensor for detecting a dioxin precursor using a gold binding polypeptide

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 degrees 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.

Preparation and characterization of a polyclonal antibody from rabbit for detection of trinitrotoluene by a surface plasmon resonance biosensor

A polyclonal antibody against trinitrophenyl (TNP) derivatives was raised in rabbit, and the antibody was applied to detection of trinitrotoluene (TNT) using a surface plasmon resonance (SPR) biosensor. TNP-keyhole limpet hemocyanine (TNP-KLH) conjugate was injected into a rabbit, and a polyclonal anti-TNP antibody was realized after purification of the sera using protein G. Aspects of the anti-TNP antibody against various nitroaromatic compounds, such as cross-reactivities and affinities, were characterized. The temperature dependence of the affinity between the anti-TNP antibody and TNT was also evaluated. The quantification of TNT was based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-beta-alanine-ovalbumin (TNP-beta-ala-OVA) and anti-TNP antibody was inhibited in the presence of free TNT in solution. TNP-beta-ala-OVA was immobilized to the dextran matrix on the Au surface by amine coupling. The addition of a mixture of free TNT to the anti-TNP antibody was found to decrease the incidence angle shift due to the inhibitory effect of TNT. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range of 3x10(-11) to 3x10(-7)g/ml. To increase the sensitivity of the sensor, anti-rabbit IgG antibody was used. After flowing the mixture of free TNT and anti-TNP antibody, anti-rabbit IgG antibody was injected, and the incidence angle shift was measured. Amplification of the signal was observed and the detection limit was improved to 1x10(-11)g/ml.

A ratiometric fluorescent probe for imaging hydroxyl radicals in living cells.

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.

Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads

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 20min. A concave calibration curve was obtained between Vg concentration and chemiluminescence intensity when various concentrations of standard Vg samples (2-100ngmL(-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 2ngmL(-1).

Sequential injection chemiluminescence immunoassay for anionic surfactants using magnetic microbeads immobilized with an antibody.

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 (0-500ppb) under optimum conditions. The time required for analysis is less than 15min.

A novel surface plasmon resonance immunosensor for 2,4,6-trinitrotoluene (TNT) based on indirect competitive immunoreaction: a promising approach for on-site landmine detection

A surface plasmon resonance (SPR) immunosensor for the determination of 2,4,6-trinitrotoluene (TNT) has been developed based on the principle of indirect competitive immunoreaction. 2,4,6-trinitrophenol-bovine serum albumin (TNP-BSA) conjugate was immobilized onto a SPR gold chip by means of simple physical adsorption. Binding of anti-TNP antibody with TNP-BSA conjugate was detected based on an increase in resonance angle due to antigen-antibody interaction. Preincubation of anti-TNP antibody with TNT suppresses its interaction with immobilized TNP-BSA conjugate, which leads to a decrease in resonance angle shift. Following the dependence of the resonance angle shift, concentration of TNT was detected. Pepsin solution was used for the regeneration of the sensing surface. The response time for TNT measurement is about 22 min. The immunosensor showed excellent sensitivity to TNT in a wide concentration range from 60 ppt to 1000 ppb with good selectivity, stability, and reproducibility. The proposed system is promising for future application for the on-site detection of landmines.

Methodology of reversible protein labeling for ratiometric fluorescent measurement

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 palm-sized surface plasmon resonance sensor with microchip flow cell

A small-sized surface plasmon resonance (SPR) sensor with a microchip flow cell has been developed for the purpose of enhancing the sensitivity of the SPR detector for low molecular weight compounds. This portable differential SPR detector consisted of an LED, two cylindrical lenses, a round prism, a divided mirror, a CCD, electronics, and a polydimethylsiloxane/gold microchip with two flow paths (10mm long, 1mm wide, 20-100mum deep). 3-Mercaptopropyltrimethoxysilane was used for sealing the microchip. The performance of the on-site orientated SPR detector was estimated using sucrose and IgA. A drastic change in the SPR intensity appeared. The depth of the flow cell was in inverse proportion to the SPR intensity. Compared to a conventional flow cell having the size of 10mm (L)x1mm (W)x1mm (D), its sensitivity to 10% sucrose and 0.9nM IgA increased about 11 and 39 times, respectively. This phenomenon seemed to be due to the increase in the substance on the SPR sensor based on its size effect. These results showed that the application of the microchip sensor for SPR measurement has the possibility for improvement of the SPR intensity for low molecular substances.

Flow immunoassay of trinitrophenol based on a surface plasmon resonance sensor using a one-pot immunoreaction with a high molecular weight conjugate

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 25ppt to 25ppb, and the coefficient of variation of the SPR signals for the 25ppb 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.

Potentiometric flow injection determination of manganese(II) by using a hexacyanoferrate(III)–hexacyanoferrate(II) potential buffer

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 1x10(-7) M. The sampling rate and relative standard deviation were 20 h(-1) and 1.9% (n=8) for 6x10(-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.