  Zero mode waveguide (ZMW) nanoapertures efficiently confine the light down to
the nanometer scale and overcome the diffraction limit in single molecule
fluorescence analysis. However, unwanted adhesion of the fluorescent molecules
on the ZMW surface can severely hamper the experiments. Therefore a proper
surface passivation is required for ZMWs, but information is currently lacking
on both the nature of the adhesion phenomenon and the optimization of the
different passivation protocols. Here we monitor the influence of the
fluorescent dye (Alexa Fluor 546 and 647, Atto 550 and 647N) on the
non-specific adhesion of double stranded DNA molecule. We show that the
nonspecific adhesion of DNA double strands onto the ZMW surface is directly
mediated by the organic fluorescent dye being used, as Atto 550 and Atto 647N
show a pronounced tendency to adhere to the ZMW while the Alexa Fluor 546 and
647 are remarkably free of this effect. Despite the small size of the
fluorescent label, the surface charge and hydrophobicity of the dye appear to
play a key role in promoting the DNA affinity for the ZMW surface. Next,
different surface passivation methods (bovine serum albumin BSA, polyethylene
glycol PEG, polyvinylphosphonic acid PVPA) are quantitatively benchmarked by
fluorescence correlation spectroscopy to determine the most efficient
approaches to prevent the adsorption of Atto 647N labeled DNA. Protocols using
PVPA and PEG-silane of 1000 Da molar mass are found to drastically avoid the
non-specific adsorption into ZMWs. Optimizing both the choice of the
fluorescent dye and the surface passivation protocol are highly significant to
expand the use of ZMWs for single molecule fluorescence applications.
