Surface plasmon resonance detection of endocrine disruptors using immunoprobes based on self-assembled monolayers

This paper describes the formation and characterization of a regenerable surface plasmon resonance (SPR) immunosensor for the detection of benzo(a)pyrene (BaP) by employing a self-assembled mixed monolayer presenting BaP head groups as the sensor immunoprobe. The mixed monolayer was used as the base layer in the fabrication of immunoprobe to assure stability to the immunoprobe and inertness to non-selective haptens and proteins. Interfacial interaction of a monoclonal anti-BaP antibody (BaP-Ab) with the monolayer is studied by SPR angle interrogation method. With BaP head groups exposing out of the self-assembled monolayer, BaP-Ab undergoes selective immunoreaction with the immunoprobe and binds firmly. Flow of high concentrations of bovine serum albumin (BSA) showed negligible response indicating high resistivity of the sensor surface to non-selective adsorption of proteins. An indirect inhibition immunoassay method is employed for analyzing sensor responses to BaP. In this method, BaP-Ab is passed together with the free analyte over the sensor chip using a flow system. With the presence of BaP together in the analyte solution, competition for binding to BaP-Ab occurs between BaP molecules in solution and the BaP functional groups present on the sensor chip and thus the binding of BaP-Ab with the sensor surface is found inhibited. Extent of the inhibition of immunoreaction by the presence of BaP is followed in determining the concentration of BaP. Calibration curves corresponding to analyte concentrations ranging from 50 ppt (pg/ml) to 100 ppb (ng/ml) BaP are obtained. The lowest detection limit of the present SPR-based immunosensor is 50 ppt (pg/ml) with a response time of ca. 20 min. After the use in determination of BaP, active sensor surface is generated again for repeated use by dissociating the bound antibody from the sensor chip with the treatment of a pepsin solution. A same sensor chip is reusable in the determination of BaP concentration repeatedly for not less than 20 cycles without significant decrease in the sensor activity.