We investigated the use of a surface plasmon resonance (SPR) biosensor using an antibody (Ab) labeled with Au-nanoparticle (Ab-AuNP conjugate). As clenbuterol is a small molecule, an indirect competitive inhibition immunoassay was used. The SPR immunoassay using Ab-AuNP conjugate had an extremely low limit of the detection (LOD) with a magnitude of 0.05 ppt (0.05pgmL), which was 40 times lower than that of unlabeled Ab. To identify the key factor in determining the LOD of the indirect competitive inhibition immunoassay, affinity constants of the surface immunoreaction (K) and of the premixed solution (αK) were evaluated. We found that the dielectric constant change due to AuNP labeling of Ab did not affect on the affinity constants, because all the amplification magnitude terms canceled out in the equations. Thus, the K and αK values were determined to 3.0×10M and 2.9×10M, respectively, which were three and four orders of magnitude higher, respectively, than those of unlabeled Ab. The simulation plot of LOD with respect to K and αK showed that a K one order of magnitude lower than αK produced a ppt level LOD. Because the affinity constants are determined by the molar concentrations of reactant and product, the molar mass of the Ab or Ab-AuNP conjugate in the sample solution containing 1ppm (1μgmL) highly affects the constants. Consequently, molar mass adjustment can be used to adjust the LOD in an indirect competitive inhibition immunoassay as needed for a practical application.
A rapid, sensitive immunoassay based on a surface plasmon resonance sensor in a flow system for the determination of alkylphenol polyethoxylate (APEO) is described. The method is based on an indirect competitive reaction between an anti-APEO antibody in the sample solution and APEO immobilized on a sensor chip and APEO in the same sample solution. A sensor chip was prepared by immobilizing an APEO-horseradish peroxidase (APEO-HRP) conjugate on the thin gold film of the sensor chip. The adsorption constants for the APEO-HRP conjugate on the sensor chip and the surface density of the APEO-HRP adsorbed on the sensor chip at the saturated state were estimated to be 4.7 × 10 5 M -1 and 5.0 × 10 -14 mol/mm 2 , respectively, using a Langmuir adsorption isotherm equation and results from the adsorption experiments. The affinity constants for the immunocomplexes of the anti-APEO antibody with the APEO conjugate on the sensor chip and for APEO in the sample solution were estimated to 2.0 × 10 6 and 5.1 × 10 6 M -1 , respectively. A typical sigmoid calibration curve for APEO was obtained in the concentration range from 1 ppb to 1000 ppb. The detection limit, defined as the concentration of APEO, at which 85% of the sensor signal was observed, was ca. 10 ppb. The assay was applied to the determination of APEO in tap water in conjunction with a solid phase extraction pretreatment; APEO levels of approximately 50 ppt were successfully determined.
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