Novel surface plasmon resonance (SPR) immunosensor based on monomolecular layer of physically-adsorbed ovalbumin conjugate for detection of 2,4-dichlorophenoxyacetic acid and atomic force microscopy study

A rapid and simple optical immunosensor for detection of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) has been developed based on SPR technology. Functional sensing surface of the immunosensor is created by immobilizing an ovalbumin conjugate of 2,4-D (2,4-D-OVA) by simple physical adsorption on an SPR thin-film gold chip. It has been established that the Au surface of the sensor chip was completely covered by 2,4-D-OVA up to a monomolecular layer and that the 2,4-D-OVA immobilized sensor chip was highly resistive to non-specific binding of proteins. Selective binding of a monoclonal antibody against 2,4-D (2,4-D-Ab) is followed by an increase in SPR angle. The antibody complexed on the sensor surface could be removed simply by the flow of an acidic buffer (glycine.HCl; 0.2 M, pH 2.0) for less than 1 min, facilitating repeated use of a same sensor chip. A competitive immunosensing method has been applied for the detection of 2,4-D, in which binding of the antibody onto the sensor surface in the presence and absence of 2,4-D is investigated. When 2,4-D is present in sample solution, a competition is set off between 2,4-D in solution and 2,4-D-OVA conjugate on sensor chip for binding to 2,4-D-Ab. A lowest detection limit of 0.1 ng/ml 2,4-D is established. Calibration curve of this analytical system covers a wide concentration range of 0.1–300 ng/ml 2,4-D. One assay could be completed in 18 min (binding, 15 min; acidic eluent and followed carrier buffer, 3 min). Enzyme-linked immunosorbent assay (ELISA) measurements for the detection of 2,4-D using analogous antigen-coat format showed a detection limit of 500 ng/ml. The high sensitivity of the configured SPR immunosensor system and the differences between the performances of SPR and ELISA are discussed. Cross-reactivity of the SPR sensor against a few compounds structurally and environmentally relevant to 2,4-D is examined. The fabricated SPR sensor is found to be highly resistant to interference with a maximum cross-reactivity of only 4% for 2,4,5-trichlorophenoxyacetic acid (2,4,5-T).