We propose to explore a novel material that promises to enhance the sensitivity of ELISA assays in multi-well plates by at least an order of magnitude, and uniquely enable lectins to be used to probe glycosylation of biomarkers. This technology would enable earlier detection of cancer. The technology is based on colloidal crystalline film on the bottoms of the wells of multiwell plates. The hypothesis is that these films will impart a high surface area for capture of biomarkers, and also control diffusion to prevent lectins from desorbing during rinsing. The crystallinity is the key to combining high transparency with high surface area. The use of higher surface area means that the sensitivity enhancement is independent of other approaches that enhance detection, such as quantum dot labels, and the two independent approaches can be combined for even higher sensitivity. The compatibility with multiwell plates will allow multiplexing so that panels of biomarkers can be used. The compatibility will also facilitate the widespread adoption of this technology because multiwell plates are widely used today.
Three specific aims are designed to test the proposed multiwell plates: choice of coating for the silica to maximize capture and binding activity while reducing nonspecific binding, choice of nanoparticle diameter to balance high surface area with sufficient pore diameter and controlled diffusion of lectins, and evaluation of the sensitivity of the plates for two types of biomarker assays. The first assay will be the standard sandwich immunoassay, with prostate specific antigen as the model system. The second will be a novel type of assay that uses lectins in place of the secondary antibody to probe the glycosylation of the protein. The model system will again be prostate specific antigen used, for which there is sample evidence that differential glycosylation is indicative of cancer, and a commercial fucose-binding lectin from Aleuria aurentia. The proposed work will determine in two years whether these multiwell plates will be broadly useful as a new tool for early stage cancer diagnostics.
A thin film based on nanotechnology will be studied as a new tool for early and more specific in vitro diagnostics of cancer.
| Koshel, Brooke M; Wirth, Mary J (2012) Trajectory of isoelectric focusing from gels to capillaries to immobilized gradients in capillaries. Proteomics 12:2918-26 |
