PT  - JOURNAL ARTICLE
AU  - Cassandra M. Stawicki
AU  - Torri E. Rinker
AU  - Markus Burns
AU  - Sonal S. Tonapi
AU  - Rachel P. Galimidi
AU  - Deepthi Anumala
AU  - Julia K. Robinson
AU  - Joshua S. Klein
AU  - Parag Mallick
TI  - Modular Fluorescent Nanoparticle DNA Probes for Detection of Peptides and Proteins
AID  - 10.1101/2021.07.30.454524
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.07.30.454524
4099  - http://biorxiv.org/content/early/2021/07/31/2021.07.30.454524.short
4100  - http://biorxiv.org/content/early/2021/07/31/2021.07.30.454524.full
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.