Improved Method for Counting DNA Molecules on Biofunctionalized Nanoparticles

Filip Delport† , Ania Deres‡, Jun-ichi Hotta‡, Jeroen Pollet† , Bert Verbruggen† , Bert Sels§, Johan Hofkens‡ and Jeroen Lammertyn* †

Langmuir, 2010, 26 (3), pp 1594-1597

DOI: 10.1021/la904702j

Publication Date (Web): January 5, 2010

Copyright © 2010 American Chemical Society

*To whom correspondence should be addressed. E-mail: Jeroen.Lammertyn@biw.kuleuven.be.

In order to accurately determine low numbers (1−100) of immobilized ssDNA molecules at a single, silica 250 nm nanoparticle surface, we hereby propose an integrated approach combining classic single molecule confocal microscopy (SMCM), that is, stepwise photobleaching of labeled ssDNA, with modified total internal reflection fluorescence microscopy (mTIRF). We postulate that SMCM alone is unable to exactly account for all labeled ssDNA because of inherent laser polarization effects; that is, perpendicularly oriented molecules to the sample surface are not (or are only slightly) susceptible to laser excitation and thus are invisible in a classic photobleaching experiment. The SMCM method accounts for at best two-thirds (68%) of the present ssDNA molecules. The principle of the mTIRF technique, which relies on the creation of highly inclined illumination combined with part of the laser remaining in normal Köhler illumination, enables accurate counting of SMCM invisble molecules. The combined approach proposed here circumvents the polarization issue and allows a complete single molecule counting on individual nanoparticles, fully in line with bulk measurements, as will be demonstrated.