Reusable Microfluidic Chambers for Single-Molecule Microscopy

Maintaining a consistent environment in single molecule microfluidic chambers containing surface-bound molecules requires laborious cleaning and surface passivation procedures. Despite such efforts, variations in non-specific binding and background signals commonly occur across different chambers. Being able to reuse the chambers without degrading the surface promises significant practical and fundamental advantages; however, this necessitates removing the molecules attached to the surface, such as DNA, proteins, lipids or nanoparticles. Biotin-streptavidin attachment is widely used for such attachments as biotin can be readily incorporated to these molecules. In this study, we present single-molecule fluorescence experiments that demonstrate effective resetting and recycling of the chambers at least 10 times using photocleavable biotin (PC-biotin) and UV light exposure. This method differs from alternatives as it does not utilize any harsh chemical treatment of the surface. We show that all bound molecules (utilizing various PC-biotin attachment chemistries) can be removed from the surface by a 5-min UV exposure of a specific wavelength. Non-optimal wavelengths and light sources showed varying degrees of effectiveness. Our approach does not result in any detectable degradation of surface quality, as assessed by non-specific binding of fluorescently labeled DNA and protein samples, and recovery of DNA secondary structure and protein activity. The speed and efficiency of the resetting process, the cost-effectiveness of the procedure, and the widespread use of biotin-streptavidin attachment make this approach adaptable for a wide range of single-molecule applications.