Magnetically-assisted transport evanescent field fluoroimmunoassay

The immunoassay, based on specific recognition of an antigen by its antibody, has garnered widespread use in clinical analysis as well as application in such areas as food industry and environmental monitoring. Fluoroimmunoassays (FIAs) are especially attractive due to the inherent sensitivity of fluorescence spectroscopy and the availability of a wide range of commercial antibodies and fluorescent labels. In current form, however, FIAs can be cumbersome, multistep procedures and often lack versatility when there is interest in measuring many different target antigens. This report is proof of a concept paper introducing a new FIA approach, Magnetically-Assisted Transport Evanescent Field Fluoroimmunoassays (MATEFFs), which seeks to preserve the advantages of current approaches to FIAs while attempting to address some of the drawbacks. MATEFFs utilize magnetic microspheres as solid supports for the fluoroimmunoassay with direct detection of bound analyte within the sample mixture effected by selectively driving the functionalized beads to a prism surface using an external magnet. An evanescent wave is generated by total internal reflection of a laser beam at the optical interface between the prism and sample and serves to excite the fluorescent species magnetically delivered into the localized field. This technique eliminates wash steps without compromising sensitivity, all the while minimizing interference from fluorescing species present in the sample matrix. Preliminary optimization studies assessing the impact of background interfering agents, incident angle, magnetic field direction, laser power density via focusing, and bead concentration on MATEFFs performance characteristics are discussed herein along with a detailed description of the experimental platform. Utilizing a model sandwich assay system with biotinylated anti-IgG as the capture antibody, rabbit IgG as the antigen, and anti-IgG-R-phycoerythrin as the reporter antibody, we demonstrate a linear dynamic range of 3 orders of magnitude, physiologically relevant detection limits of low nanograms per milliliter, and RSD values of less than 5%.