Single-Molecule Characterization of Nanoparticle Bioconjugates using Microwell Array Analysis*

Nanoparticles functionalized with biomolecules have been studied extensively for their applications in nanomedicine. A need for characterizing these nanoparticle bioconjugates at the single-particle level using optical methods is needed. Microwell array analysis is a useful tool for single-molecule studies in which enzyme molecules are isolated in femtoliter wells; the enzymes convert non-fluorescent substrates into fluorescent products which can be measured using optical microscopy. Gold bipyramids were synthesized and functionalized with a self-assembled-monolayer (SAM). These particles were then bioconjugated using carbodiimide crosslinking with mouse anti-human epidermal growth factor receptor (EGFR)[KM1] antibodies, followed by human EGFR labeled with horseradish peroxidase (HRP). Microwell arrays were loaded with the nanoparticle bioconjugates such only that a small number of wells were occupied according to Poisson statistics. The fluorescence intensity of the microwells was then used to determine the number of antibodies that were conjugated to the gold bipyramids.