Abstract
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.
Highlights
Zero mode waveguide (ZMW) nanoapertures efficiently confine the light down to the nanometer scale and overcome the diffraction limit in single molecule fluorescence analysis
While extensive literature exists for passivating glass surfaces for single molecule fluorescence microscopy[45,46,47,48,49,50,51,52], little is known about the surface passivation of ZMWs and the comparative analysis of different passivation strategies
We have demonstrated that despite the small size of the fluorescent label as compared to the 51 base pairs DNA molecule, the choice of the dye and its surface charge after labelling plays a key role in promoting the adhesion of DNA molecules in the ZMW surface
Summary
Zero mode waveguide (ZMW) nanoapertures efficiently confine the light down to the nanometer scale and overcome the diffraction limit in single molecule fluorescence analysis. Another major advantage of ZMWs concerns their ability to enhance the fluorescence brightness per molecule thanks to the enhanced local excitation intensity inside the ZMW and the modification of the fluorescence photokinetics decay rates[7,8,9,10,11,12,13,14,15,16] Since their inception in 2002, ZMWs have been widely used for a large range of biophysical and biochemical applications, including DNA sequencing[17,18,19], enzymatic reaction monitoring[20,21,22], protein-protein interaction[23,24,25,26,27], nanopore sensing[28,29,30,31,32], Förster resonance energy transfer[33,34,35], biomembrane investigations[36,37,38,39,40], and nano-optical trapping[41,42,43,44]. It was shown that the surface passivation of the aluminum layer can greatly improve the chemical stability in water buffers[58,59,60], which points out another advantage for the surface passivation of aluminum ZMWs
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