Real time optical immunosensing with flow-through porous alumina membranes

Through the presentation of analytical data from bioassay experiments, measured by polarimetry, we demonstrate for the first time a real time immunoassay within a free standing macroporous alumina membrane. The 200 nm nominal pore diameter of the membrane enables flow-through, thereby providing an ideal fluidic platform for the targeted delivery of analytes to bioreceptors immobilized on the pore walls, enabling fast sensing response times and the use of small sample volumes (<100 μL). For the immunoassay, the pore walls were first coated with the functional copolymer, copoly(DMA-NAS) using a novel coupling process, before immobilization of the allergen protein, β-lactoglobulin, by spotting. The immuno-assay then proceeded with the binding of the primary and secondary antibody cognates, rabbit anti-β-lactoglobulin and anti-rabbit IgG respectively. Through the use of streptavidin coated quantum dots as refractive index signal enhancers, a noise floor for individual measurements of 3.7 ng/mL (25 pM) was obtained, with an overall statistical, or formal assay LOD of 33.7 ng/mL (225 pM), for total assay time below 1 h.