This collaboration will develop a next generation imaging technology for broad application across all fields of biology and medicine. This project aims to apply many well understood principles of polymerization to the development of a new strategy for immunofluorescent labeling. Our approach, termed polymerization- based amplification (PBA), is based on obtaining a specific and highly amplified response unique to polymerization in response to an initiating molecule that is coupled through a biorecognition event to the cell or sub-cellular feature of interest. Specifically, we will develop the approach to utilize the coupling of a polymerization initiating molecule to an antibody to generate polymerization in localized regions, based on protein expression. We tailor the ~100 nm polymer film to incorporate numerous fluorescent labels through entrapment of fluorescent nanoparticles in the polymer network. Preliminary findings demonstrate over a three order of magnitude reduction in primary antibody utilization is achievable with our PBA technology. The entrapment based immobilization of the label enables the use of numerous commercially available fluorophores, without the need for chemical modification of the fluorescent nanoparticle. We seek to develop this technology to address problems spanning biology and clinical research. To that end, we will develop PBA systems to address cultured cell, multi-target / multi-color, tissue, and flow-based applications. The development of this technology will enable the detection of low abundance proteins with inexpensive imaging instrumentation. In addition, a three order of magnitude reduction in antibody consumption would provide a drastic decrease in financial requirements for basic biological and clinical research. This cost savings would translate not only to the individual research lab, but also to research funding agencies. This application establishes a project-appropriate pairing of established investigators, each with an ongoing record of accomplishments in their respective field. Dr. Bowman is an expert in photopolymerization reaction engineering, and has extensive physical resources for the development and characterization of the advanced polymer systems required by this project. Dr. Balasubramaniam is an active researcher in pediatric pulmonology and brings expertise in immunofluorescent techniques and histology. Dr. Balasubramaniam is the Director of the Pediatric Heart Lung Lab at the University of Colorado - Denver Health Sciences Center, and has access to all of the biological characterization resources required by this collaboration.
This project synergistically combines uses polymer chemistry and biology to facilitate the detection of biological species that are present in amounts much lower than previously possible. The ability to detect small amounts of biological species will allow researchers in biology to inexpensively explore new scientific aspects of cells and tissues.
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