NDLI: Streptavidin conjugation and quantification—a method evaluation for nanoparticles

Content Provider	Springer Nature Link
Author	Quevedo, Pablo Darío Behnke, Thomas Resch Genger, Ute
Copyright Year	2016
Abstract	Aiming at the development of validated protocols for protein conjugation of nanomaterials and the determination of protein labeling densities, we systematically assessed the conjugation of the model protein streptavidin (SAv) to 100-, 500-, and 1000-nm-sized polystyrene and silica nanoparticles and dye-encoded polymer particles with two established conjugation chemistries, based upon achievable coupling efficiencies and labeling densities. Bioconjugation reactions compared included EDC/sulfo NHS ester chemistry for direct binding of the SAv to carboxyl groups at the particle surface and maleimide-thiol chemistry in conjunction with heterobifunctional PEG linkers and aminated nanoparticles (NPs). Quantification of the total and functional amounts of SAv on these nanomaterials and unreacted SAv in solution was performed with the BCA assay and the biotin–FITC (BF) titration, relying on different signal generation principles, which are thus prone to different interferences. Our results revealed a clear influence of the conjugation chemistry on the amount of NP crosslinking, yet under optimized reaction conditions, EDC/sulfo NHS ester chemistry and the attachment via heterobifunctional PEG linkers led to comparably efficient SAv coupling and good labeling densities. Particle size can obviously affect protein labeling densities and particularly protein functionality, especially for larger particles. For unstained nanoparticles, direct bioconjugation seems to be the most efficient strategy, whereas for dye-encoded nanoparticles, PEG linkers are to be favored for the prevention of dye–protein interactions which can affect protein functionality specifically in the case of direct SAv binding. Moreover, an influence of particle size on achievable protein labeling densities and protein functionality could be demonstrated.
Starting Page	4133
Ending Page	4149
Page Count	17
File Format	PDF
ISSN	16182642
Journal	Analytical and Bioanalytical Chemistry
Volume Number	408
Issue Number	15
e-ISSN	16182650
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2016-04-01
Publisher Place	Berlin, Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Protein quantification BCA assay Biotin–FITC titration Polystyrene nanoparticles Streptavidin Staining procedure Analytical Chemistry Biochemistry Laboratory Medicine Characterization and Evaluation of Materials Food Science Environmental Monitoring/Analysis
Content Type	Text
Resource Type	Article
Subject	Analytical Chemistry Biochemistry

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