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

Abstract

This paper describes the formation and characterization of a regenerable surface plasmon resonance (SPR) immunosensor for the detection of benzo( a)pyrene (B aP) by employing a self-assembled mixed monolayer presenting B aP 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-B aP antibody (B aP-Ab) with the monolayer is studied by SPR angle interrogation method. With B aP head groups exposing out of the self-assembled monolayer, B aP-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 B aP. In this method, B aP-Ab is passed together with the free analyte over the sensor chip using a flow system. With the presence of B aP together in the analyte solution, competition for binding to B aP-Ab occurs between B aP molecules in solution and the B aP functional groups present on the sensor chip and thus the binding of B aP-Ab with the sensor surface is found inhibited. Extent of the inhibition of immunoreaction by the presence of B aP is followed in determining the concentration of B aP. Calibration curves corresponding to analyte concentrations ranging from 50 ppt (pg/ml) to 100 ppb (ng/ml) B aP 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 B aP, 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 B aP concentration repeatedly for not less than 20 cycles without significant decrease in the sensor activity.