Mechanism of surface plasmon resonance sensing by indirect competitive inhibition immunoassay using Au nanoparticle labeled antibody.

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.