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Journal article
NeutrAvidin Functionalization of CdSe/CdS Quantum Nanorods and Quantification of Biotin Binding Sites using Biotin-4-Fluorescein Fluorescence Quenching
Bioconjugate chemistry, v 27(3), pp 562-568
01 Mar 2016
PMID: 26722835
Abstract
We developed methods to solubilize, coat, and functionalize with NeutrAvidin elongated semiconductor nano crystals (quantum nanorods, QRs) for use in single molecule polarized fluorescence microscopy. Three different ligands were compared with regard to efficacy for attaching NeutrAvidin using the "zero-length cross-linker" 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Biotin-4-fluorescene (B4F), a fluorophore that is quenched when bound to avidin proteins, was used to quantify biotin binding activity of the NeutrAvidin coated QRs and biotin binding activity of commercially available streptavidin coated quantum dots (QDs). All three coating methods produced QRs with NeutrAvidin coating density comparable to the streptavidin coating density of the commercially available quantum dots (QDs) in the B4F assay. One type of QD available from the supplier (ITK QDs) exhibited similar to 5-fold higher streptavidin surface density compared to our QRs, whereas the other type of QD (PEG QDs) had 5-fold lower density. The number of streptavidins per QD increased from similar to 7 streptavidin tetramers for the smallest QDs emitting fluorescence at 525 nm (QD525) to similar to 20 tetramers for larger, longer wavelength QDs (QD655, QD705, and QD800). QRs coated with NeutrAvidin using mercaptoundecanoicacid (MUA) and QDs coated with streptavidin bound to biotinylated cytoplasmic dynein in single molecule TIRF microscopy assays, whereas Poly(maleic anhydride-alt-1-ocatdecene) (PMAOD) or glutathione (GSH) QRs did not bind cytoplasmic dynein. The coating methods require optimization of conditions and concentrations to balance between substantial NeutrAvidin binding vs tendency of QRs to aggregate and degrade over time.
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Details
- Title
- NeutrAvidin Functionalization of CdSe/CdS Quantum Nanorods and Quantification of Biotin Binding Sites using Biotin-4-Fluorescein Fluorescence Quenching
- Creators
- Lisa G. Lippert - University of PennsylvaniaJeffrey T. Hallock - Raymond and Ruth Perelman School of Medicine at the University of PennsylvaniaTali Dadosh - Weizmann Institute of ScienceBenjamin T. Diroll - University of PennsylvaniaChristopher B. Murray - University of PennsylvaniaYale E. Goldman - University of PennsylvaniaChristopher B Rodell - School of Biomedical Engineering, Science, and Health Systems (1997-)
- Publication Details
- Bioconjugate chemistry, v 27(3), pp 562-568
- Publisher
- American Chemical Society; Washington, DC
- Number of pages
- 7
- Grant note
- T32GM008275 / NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of General Medical Sciences (NIGMS) P01-GM086352 / NIH; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA P01AR051174 / NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Arthritis & Musculoskeletal & Skin Diseases (NIAMS) DMR04-25780 / NSF Nanotechnology Science and Engineering Center
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- School of Biomedical Engineering, Science, and Health Systems
- Web of Science ID
- WOS:000372478600009
- Scopus ID
- 2-s2.0-84961744760
- Other Identifier
- 991019320609904721
InCites Highlights
Data related to this publication, from InCites Benchmarking & Analytics tool:
- Collaboration types
- Domestic collaboration
- International collaboration
- Web of Science research areas
- Biochemical Research Methods
- Biochemistry & Molecular Biology
- Chemistry, Multidisciplinary
- Chemistry, Organic