Counting ssDNA on a single nanoparticle

This paper describes the immobilization and quantification of 15base ssDNA on 250 nm silica nanoparticles. 1 up to 2400 amine functionalized ssDNA are coupled to a carboxyl functionalized nanoparticle using EDC/NHS chemistry. The amount of ssDNA on the nanoparticles is quantified by bulk fluorescence measurements in a microtiterplate reader on both the nanoparticles in dense solution and the supernatant. To determine few molecules of ssDNA on each nanoparticle Single Molecule detection techniques were applied. Single molecule confocal microscopy focuses a laser on one nanoparticle and photobleaches fluorescent dyes stochastically, thus enabling a precise counting from 1 up to 6 molecules on a single nanoparticle. These measurements revealed 2/3th of the ssDNA present in comparison with the bulk measurements. Wide field total internal reflection fluorescence microscopy showed the 1/3th missing ssDNA which is immobilized perpendicular to the sample surface. Thus, this method suggests a precise, complete and oriented counting of molecules from single molecule up to bulk level on nanoparticles.