RT Journal Article
SR Electronic
T1 Modular Fluorescent Nanoparticle DNA Probes for Detection of Peptides and Proteins
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.07.30.454524
DO 10.1101/2021.07.30.454524
A1 Cassandra M. Stawicki
A1 Torri E. Rinker
A1 Markus Burns
A1 Sonal S. Tonapi
A1 Rachel P. Galimidi
A1 Deepthi Anumala
A1 Julia K. Robinson
A1 Joshua S. Klein
A1 Parag Mallick
YR 2021
UL http://biorxiv.org/content/early/2021/07/31/2021.07.30.454524.abstract
AB Fluorescently labeled antibody and aptamer probes are used in biological studies to characterize binding interactions, measure concentrations of analytes, and sort cells. Fluorescent nanoparticle labels offer an excellent alternative to standard fluorescent labeling strategies due to their enhanced brightness, stability and multivalency; however, challenges in functionalization and characterization have impeded their use. This work introduces a straightforward approach for preparation of fluorescent nanoparticle probes using commercially available reagents and common laboratory equipment. Fluorescent polystyrene nanoparticles, Thermo Fisher FluoSpheres™, were used in proof-of-principle studies. Particle passivation was achieved by covalent attachment of amine-PEG-azide to carboxylated particles, neutralizing the surface charge from -47 to -17 mV. A conjugation-annealing handle and DNA aptamer probe was attached to the azide-PEG nanoparticle surface either through reaction of pre-annealed handle and probe or through a stepwise reaction of the nanoparticles with the handle followed by aptamer annealing. Nanoparticles functionalized with DNA aptamers targeting histidine tags and VEGF protein had high affinity (EC50s ranging from 2-7 nM) and specificity, and were more stable than conventional labels. This protocol for preparation of nanoparticle probes relies solely on commercially available reagents and common equipment, breaking down the barriers to use of nanoparticles in biological experiments.Competing Interest StatementThe authors have declared no competing interest.