Lippert Lisa G, Hallock Jeffrey T, Dadosh Tali, Diroll Benjamin T, Murray Christopher B, Goldman Yale E
Electron Microscopy Unit, Department of Chemical Research Support, Weizmann Institute of Science , Rehovot 7610001, Israel.
Bioconjug Chem. 2016 Mar 16;27(3):562-8. doi: 10.1021/acs.bioconjchem.5b00577. Epub 2016 Jan 14.
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.
我们开发了用于溶解、包覆和用中性抗生物素蛋白对细长半导体纳米晶体（量子纳米棒，QRs）进行功能化的方法，以用于单分子偏振荧光显微镜。使用“零长度交联剂”1-乙基-3-[3-(二甲基氨基)丙基]碳二亚胺（EDC），比较了三种不同配体连接中性抗生物素蛋白的效果。生物素-4-荧光素（B4F）是一种与抗生物素蛋白结合时会被淬灭的荧光团，用于量化中性抗生物素蛋白包覆的QRs的生物素结合活性以及市售链霉抗生物素蛋白包覆的量子点（QDs）的生物素结合活性。在B4F分析中，所有三种包覆方法产生的带有中性抗生物素蛋白包覆密度的QRs与市售量子点（QDs）的链霉抗生物素蛋白包覆密度相当。供应商提供的一种类型的QD（ITK QDs）与我们的QRs相比，链霉抗生物素蛋白表面密度高约5倍，而另一种类型的QD（PEG QDs）密度低5倍。每个QD的链霉抗生物素蛋白数量从发射525nm荧光的最小QD（QD525）的约7个链霉抗生物素蛋白四聚体增加到较大的、较长波长QD（QD655、QD705和QD800）的约20个四聚体。在单分子全内反射荧光显微镜分析中，使用巯基十一烷酸（MUA）包覆中性抗生物素蛋白的QRs和包覆链霉抗生物素蛋白的QDs与生物素化的细胞质动力蛋白结合，而聚（马来酸酐-alt-1-十八碳烯）（PMAOD）或谷胱甘肽（GSH）包覆的QRs不与细胞质动力蛋白结合。包覆方法需要优化条件和浓度，以在大量中性抗生物素蛋白结合与QRs随时间聚集和降解的趋势之间取得平衡。
