Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels.

Area of Science:

• Nanotechnology
• Biophysics
• Surface Chemistry

Background:

• Zero mode waveguides (ZMWs) enable nanoscale light confinement for single molecule fluorescence analysis.
• Non-specific adhesion of fluorescent molecules to ZMW surfaces hinders experimental outcomes.
• Optimization of ZMW surface passivation protocols is crucial but underexplored.

Purpose of the Study:

• To investigate the influence of fluorescent dyes on non-specific DNA adhesion to ZMW surfaces.
• To benchmark various surface passivation methods for preventing DNA adsorption in ZMWs.
• To identify optimal strategies for enhancing ZMW applications in single molecule fluorescence.

Main Methods:

• Monitoring non-specific adhesion of double-stranded DNA labeled with different fluorescent dyes (Alexa Fluor 546/647, Atto 550/647N).
• Evaluating surface passivation techniques including bovine serum albumin (BSA), polyethylene glycol (PEG), and polyvinylphosphonic acid (PVPA).
• Quantitatively assessing passivation efficiency using fluorescence correlation spectroscopy.

Main Results:

• Non-specific DNA adhesion is significantly mediated by the choice of fluorescent dye; Atto dyes show higher adhesion than Alexa Fluor dyes.
• Surface charge and hydrophobicity of the fluorescent label play a critical role in DNA affinity for ZMWs.
• PVPA and 1000 Da PEG-silane passivation protocols effectively prevent non-specific adsorption of Atto 647N-labeled DNA.

Conclusions:

• The selection of fluorescent dyes and surface passivation strategies are critical for successful ZMW-based single molecule fluorescence applications.
• Dye properties (charge, hydrophobicity) directly impact DNA adsorption onto ZMW surfaces.
• PVPA and PEG offer robust solutions for ZMW surface passivation, minimizing experimental interference.