(294g) The Impact of Dissociation Constant On the Detection Sensitivity of Polymerization-Based Signal Amplification Reactions

Authors: 
Kaastrup, K., Massachusetts Institute of Technology
Chan, L., Massachusetts Institute of Technology
Sikes, H. D., Massachusetts Institute of Technology



Many studies have demonstrated free-radical polymerization as a simple and inexpensive signal amplification technique for the detection of molecular recognition events indicative of disease states. Of the various chemistries that have been implemented, photopolymerization has shown promise as a point-of-care technology for its ability to generate micron-scale hydrogels in as little as 35 seconds. Photopolymerization-based amplification requires the localization of photoinitiators at a surface as a function of molecular recognition events. Much of the previous work developing this technique has explored the case in which the signal amplification rather than the binding kinetics of the molecular recognition is limiting. We provide the first systematic study of how dissociation constant impacts assay sensitivity using proteins with affinities typical of those encountered in molecular diagnostic applications involving protein-protein binding. We use experimental results to validate a mass-action kinetic model that may be used to predict assay performance by serving as an alternative/supplement to the currently employed empirical approach to polymerization-based amplification assay development. We show that this model, given the surface probe density and protein binding affinity, is able to predict whether or not the minimal surface initiator density required for polymerization will be exceeded at a particular protein concentration.  In addition, the polymerization-based amplification assays were performed on novel activated agarose surfaces, an inexpensive alternative to the SAM modified glass surfaces typically used.