Highly sensitive detection of polycyclic aromatic hydrocarbons (PAHs) and association constants of the interaction between PAHs and antibodies using surface plasmon resonance immunosensor

Surface plasmon resonance (SPR) immunoprobes for 2-hydroxybiphenyl (HBP) and benzo(a)pyrene (BaP) are fabricated with a remarkably stable immobilization of HBP–bovine serum albumin (HBP–BSA) conjugate or BaP–BSA on a SPR active material by simple physisorption. Using competitive immunoreactions of antibody between antigen and antigen–protein conjugate, we demonstrate that the SPR sensors for HBP and BaP are capable of determining the concentration of HBP and BaP sensitively in the range 0.1–1000 ppb (parts-per-billion) and 0.1–300 ppb, respectively. The antibodies, anti-BaP–BSA antibody and anti-HBP–BSA antibody, anchored to the chip by antigen–antibody binding are found to be removed on treatment with a pepsin solution; the SPR chips are found to be reusable for more than 50 times with <10% decrease in the sensor signal. Association constants of the biomolecular interaction between the antigens (BaP and HBP) and the respective monoclonal antibodies are determined by considering Langmuir adsorption isotherm model. The association constants are determined to be 3.62×106 and 3.68×107 M−1 for the immunoreactions of anti-HBP–BSA antibody with HBP in analyte and the SPR chip-immobilized HBP–BSA, respectively; similarly, the association constants are determined to be 2.66×106 and 7.18×106 M−1 for the immunoreactions of anti-BaP–BSA antibody with BaP in analyte and the SPR chip-immobilized BaP–BSA, respectively.