ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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).
ABSTRACT
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.
ABSTRACT
In order to simplify the procedure for assembling a surface-plasmon resonance (SPR) sensor, a refractive index matching polymer film was prepared as an alternative to the conventionally used matching oil. The refractive index matching polymer film, the refractive index of which was nearly equal to the prism and sensor chip material (a cover glass) of the SPR sensor, was prepared by casting a tetrahydrofuran solution of poly (vinyl chloride) (PVC) containing equal weights of dioctyl phthalate and tricresyl phosphate. The refractive index matching polymer film was found to have a refractive index of 1.516, which is identical to that of the prism and the cover glass used for the present SPR sensor. The utility of the matching polymer film for the SPR sensor was confirmed by the detection of anti-human albumin, based on an antigen-antibody reaction.
Subject(s)
Antibodies/analysis , Surface Plasmon Resonance/methods , Antigen-Antibody Reactions , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , Calibration , Equipment Design , Humans , Polyvinyl Chloride/chemistry , Refractometry , Serum Albumin/immunology , Surface Plasmon Resonance/instrumentationABSTRACT
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.
