Use of Cyclic Olefin Polymer in Single Molecule Total Internal Reflection Fluorescence Microscopy

Abstract

Single molecule imaging is an established technique for many researchers in the biophysical sciences. Often, a fluorescent molecule bound near a glass or quartz surface is excited using total internal reflection fluorescence (TIRF) microscopy. TIRF excitation selectively excites fluorophores at the surface of a well or channel, thereby eliminating background fluorescence from bulk solution. However, nonspecific binding of fluorescent probes and fluorescently labeled molecules can become a limiting factor for protein association experiments. Extensive cleaning is required when using a glass or quartz substrate and the substrate is often coated with polymers such as poly-ethylene-glycol (PEG) to reduce non-specific binding. To eliminate the extensive wash and coating procedure steps we use a plastic cyclic olefin copolymer (COC) substrate that is extremely hydrophobic and can be passively activated to perform specific protein-protein interaction experiments in a very simple manner. Furthermore, COC has optical properties similar to glass making it amenable to TIRF microscopy. We first show simple processes to activate the surface in under a minute while rejecting fluorescent labeled antibody at concentrations of 1 nM. This is confirmed using objective-based TIRF microscopy and off-the-shelf COC microfluidic channels. We also show sufficient signal to noise to observe green fluorescent protein at the single molecule level using TIRF excitation. Finally, activated surfaces are used to perform a protein association experiment by detecting a sandwich of two antibodies bound to a single antigen molecule. We believe fluidic devices made of COC polymer will eliminate many of the preparation steps employed to treat other substrates allowing for easier and significantly faster protein association experiments.