NeutrAvidin Functionalization of CdSe/CdS Quantum Nanorods and Quantification of Biotin Binding Sites using Biotin-4-Fluorescein Fluorescence Quenching

Abstract

We developed methods to solubilize, coat, and functionalize with NeutrAvidin elongated semiconductor nanocrystals (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 ~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 ~7 streptavidin tetramers for the smallest QDs emitting fluorescence at 525 nm (QD525) 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.

Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk

“…S1A), indicating that insertion of the biotinylation sites does not disrupt motor activity. NeutrAvidin-coated QRs, which have well-polarized fluorescence emission, were attached bifunctionally to the two biotinylation sites using a biotin-avidin linkage (39,40). Initial experiments with QRs containing much lower NeutrAvidin density yielded highly variable and fluctuating orientations, indicating that bifunctional attachment produces a fixed orientation of the QR relative to the dynein ring.…”

“…Further details of polarization analysis are given in SI Appendix, Supplemental Methods. Quantum nanorods sized 56.3 × 5.6 nm were synthesized in organic solvent and coated with NeutrAvidin as previously described (40).…”

The principles and applications of avidin-based nanoparticles in drug delivery and diagnosis

“…[33, 34] As a result, neutravidin can be coated on the surface of quantum nanorods to stabilize them and prevent aggregation. [35] In addition, neutravidin has been utilized as a bridge between biotinylated moieties and biotin-coated surfaces for the detection of protein-specific antibodies. [36] The physical-chemical properties of avidin, neutravidin, and streptavidin are summarized in Table 1.…”

Single molecule optical measurements of orientation and rotations of biological macromolecules

“…These surfaces can then be modified with a targeting molecule such as streptavidin [79], NeutrAvidin [76], or halo ligand [71, 75]. In either case, the large surface area of nanoparticles accommodates the attachment of many ligands to each particle.…”

“…In either case, the large surface area of nanoparticles accommodates the attachment of many ligands to each particle. This allows rigid multi-site attachment to the biological macromolecule if multiple target sequences are inserted with appropriate spacing [71, 76]. …”