Improved method for counting DNA molecules on biofunctionalized nanoparticles.

Area of Science:

• Nanotechnology
• Biophysics
• Analytical Chemistry

Background:

• Accurate quantification of immobilized single-stranded DNA (ssDNA) at the single-molecule level is crucial for various applications.
• Classic single molecule confocal microscopy (SMCM) faces limitations in counting all molecules due to laser polarization effects, potentially missing up to 32% of ssDNA.

Purpose of the Study:

• To develop and validate an integrated approach for precise counting of low numbers (1-100) of immobilized ssDNA molecules on individual silica nanoparticles.
• To overcome the limitations of SMCM by incorporating modified total internal reflection fluorescence microscopy (mTIRF).

Main Methods:

• Combining single molecule confocal microscopy (SMCM) with stepwise photobleaching of labeled ssDNA.
• Utilizing modified total internal reflection fluorescence microscopy (mTIRF) to detect ssDNA molecules missed by SMCM due to polarization.
• Implementing a dual illumination strategy within mTIRF (highly inclined and normal Kohler illumination).

Main Results:

• The proposed integrated SMCM and mTIRF approach accurately accounts for all immobilized ssDNA molecules on nanoparticles.
• The combined method successfully circumvents laser polarization issues inherent in SMCM.
• Demonstrated complete single-molecule counting on individual nanoparticles, aligning with bulk measurement results.

Conclusions:

• The integrated SMCM and mTIRF technique provides a robust solution for accurate quantification of immobilized ssDNA at the single-molecule level.
• This method enhances the reliability of ssDNA counting on nanoparticles, crucial for nanotechnology and biophysical studies.
• The approach offers a significant improvement over traditional SMCM for precise molecular enumeration.