Organic Coatings and Interfacial Characterization
Enhanced Immunofluorescent Staining via Polymerization.
Modern immunofluorescent techniques are often limited by the ability to distinguish the presence of specific signalfrom background non-specific staining. The focus of my work is the demonstration of free radical polymerization as a new technology that is appropriate for improving the sensitivity of immunofluorescent techniques. Specifically, probes are labeled with polymerization photoinitiators, and exposure to light and monomers containing fluorescent moieties results in a highly fluorescent film (Figure 1A). Our studies demonstrate a fluorescent polymerization-based amplification method (PBA) that is shown to enable similar sensitivities and signal intensities as the highly sensitive, enzyme-based tyramide signal amplification approach, yet does not suffer from diffusion-related loss of signal localization or non-specific staining of endogenous enzymes, as occurs with enzyme-based approaches (Figure 1B).
A) Schematic illustration of polymerization-based amplification. B) Multicolor PBA staining. Human endothelial cells are stained against nuclear pore complex using Nile red NPs (red), followed by a second round of staining against vimentin using yellow/green NPs (green).
Enzymatically Initiated Polymerization for Signal Amplification.
In addition to my work on polymerization enhanced immunofluorescent staining, I am contributing to an ongoing effort to use immobilized enzymes to initiate polymerization. This approach uses glucose oxidase as a label conjugated to a biological probe, where the enzyme is used to subsequently initiate polymerization which can be monitored by various means (profilometry, plate reader, fluorescent scanner). Glucose oxidase can be used in conjunction with glucose, Fe2+, and O2 to initiate radical chain polymerizations by the following reaction scheme:
• glucose → δ-gluconolactone (by glucose oxidase)
• O2 → H2O2 (by glucose oxidase)
• H2O2 + Fe2+ + H+ → H2O + Fe3+ + .OH
We determined glucose oxidase concentration specific polymerization rate dependencies for iron and glucose, to discover a critical balance of initiation by Fe2+and inhibition by Fe3+. This fundamental knowledge allowed us to reduce the minimum glucose oxidase concentration for polymerization by three orders of magnitude, sufficiently low that we are now using surface-immobilized glucose oxidase to develop several assay formats.
Polymeric Coatings on Biological Substrates.
The manipulation of biological function on the cellular and subcellular level holds great potential for innovation in medical diagnostics and treatment, power generation and applied materials. Central to this manipulation is the interface of the biological species and its surroundings. Cells often exist in a naturally immobilized state, and emerging biotechnology will require a similar immobilization for the protection of the cell from shear and immunological attack as well as other hostile conditions. Polymers and organic coatings are obvious choices for biocompatible interfaces as they can mimic the biological environment in structure and chemical functionality and are readily tuned owing to their wide variation in architectures, mechanical properties, surface energy and transport properties.
Mechanisms of Instability in Surface Initiated ROMP Coatings.
Surface-initiated ring opening metathesis polymerization (ROMP) is a coating technology which enables growth in ambient conditions and the formation of >1 µm thick films on complex metal shapes in 15 minutes. These are among the thickest coatings grown by surface-initiated polymerization and are grown >10-fold faster than other surface-initiated chemistries. While these coatings are highly promising for protecting many materials against corrosion by salt and water, they are uniquely unstable towards many common solvents, despite strong chemical linkages. Our research will investigate multiple modes of stabilization of these structures, and elucidate the underlying mechanism for coating degradation.