Abstract
We describe a method to generate monovalent quantum dots (QDs) using agarose gel electrophoresis. We passivated QDs with a carboxy-terminated polyethylene-glycol ligand, yielding particles with half the diameter of commercial QDs, which we conjugated to a single copy of a high-affinity targeting moiety (monovalent streptavidin or antibody to carcinoembryonic antigen) to label cell-surface proteins. The small size improved access of QD-labeled glutamate receptors to neuronal synapses, and monovalency prevented EphA3 tyrosine kinase activation.
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Acknowledgements
Funding was provided by the US National Institutes of Health (NIH) (P20GM072029-01), and the McKnight, the Dreyfus and the Sloan Foundations. M.H. was supported by an MIT-Merck fellowship, W.L. by a US National Science Foundation fellowship, M.G.B. by the Army Research Office DAAD 19-03-D0004, and K.D.W. and M.M.S. by the NIH (CA101830) and the MIT Biotechnology Training Program. We thank M. Lackmann, P. Janes, S. Manalis and A. Sparks for advice, J. Chan for technical assistance, and Tanabe for providing biotin.
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M.H., W.L., M.G.B. and A.Y.T. designed the experiments; M.H. and A.Y.T. wrote the paper; M.H., W.L., S.P., L.F.M. and Y.Z. performed the experiments; M.M.S. and K.D.W. generated the PEG-scFv.
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M.H., W.L., M.G.B. and A.Y.T. are authors on a submitted US patent application describing the quantum dots generated in this work.
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Supplementary Text and Figures
Supplementary Figures 1–6, Supplementary Methods (PDF 890 kb)
Supplementary Video 1
Single-molecule imaging of wild-type LDL receptor with small monovalent QDs. COS7 cells transfected with wild-type AP-LDL receptor and BirA-ER were incubated with sQD-mSA1 and single QDs were imaged. The video is speeded up 3-fold. (MOV 963 kb)
Supplementary Video 2
Single-molecule imaging of mutant LDL receptor with small monovalent QDs. COS7 cells transfected with mutant AP-LDL receptor and BirA-ER were incubated with sQD-mSA1 and single QDs were imaged. The video is speeded up 3-fold. (MOV 847 kb)
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Howarth, M., Liu, W., Puthenveetil, S. et al. Monovalent, reduced-size quantum dots for imaging receptors on living cells. Nat Methods 5, 397–399 (2008). https://doi.org/10.1038/nmeth.1206
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DOI: https://doi.org/10.1038/nmeth.1206
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