Experimental Determination of Quantum Dot Size Distributions, Ligand Packing Densities, and Bioconjugation Using Analytical Ultracentrifugation Emma E. Lees, †,‡ Menachem J. Gunzburg, † Tich-Lam Nguyen, † Geoffrey J. Howlett, § Julie Rothacker, ‡ Edouard C. Nice, ‡ Andrew H. A. Clayton, ‡ and Paul Mulvaney* ,† School of Chemistry and Bio21 Institute, UniVersity of Melbourne, ParkVille, Victoria 3010, Australia, The Department of Biochemistry and Molecular Biology and Bio21 Institute, UniVersity of Melbourne, ParkVille, Victoria 3010, Australia, and Ludwig Institute for Cancer Research, PO Box 2008, Royal Melbourne Hospital, ParkVille, Victoria 3050, Australia Received June 7, 2008 ABSTRACT Analytical ultracentrifugation (AUC) was used to characterize the size distribution and surface chemistry of quantum dots (QDs). AUC was found to be highly sensitive to nanocrystal size, resolving nanocrystal sizes that differ by a single lattice plane. Sedimentation velocity data were used to calculate the ligand packing density at the crystal surface for different sized nanocrystals. Dihydrolipoic acid poly(ethylene glycol) was found to bind between 66 and 60% of the surface cadmium atoms for CdSe nanocrystals in the 1.54-2.59 nm radius size regime. The surface ligand chemistry was found to affect QD sedimentation, with larger ligands decreasing the sedimentation rate through an increase in particle volume and increase in frictional coefficient. Finally, AUC was used to detect and analyze protein association to QDs. Addition of bovine serum albumin (BSA) to the QD sample resulted in a reduced sedimentation rate, which may be attributed to an associated frictional drag. We calculated that one to two BSA molecules bind per QD with an associated frictional ratio of 1.2. 1. Introduction. Semiconductor nanocrystals were originally proposed as substitutes for dyes in fluorescent biolabeling some 10 years ago. 1,2 Since the original experiments, there has been a plethora of papers describing methods for conjugating nanocrystals to biological samples including peptides, 3-6 proteins, 7-9 antibodies, 10-12 and oligonucle- otides. 13,14 The success of QDs as biological probes requires careful control over their physical properties: their size, shape, composition, and surface chemistry. A key parameter is particle size and size distribution since this determines the spectral position and purity of photoluminescence. Transmission electron microscopy (TEM) is routinely used to size QD samples, providing high-resolution detail of QD shape and structure. However TEM provides only limited, qualitative information on any surface-bound organic mate- rial. Surface chemistry dictates the solubility of QDs in various solvents, governs the method of biofunctionalization, and also has a direct impact on quantum yield 15 and blinking properties. 16 The surface chemistry also contributes to the final hydrodynamic diameter, which is a critical parameter for the use of QDs as potential diagnostic and therapeutic agents. Recent in vivo studies show that QD biodistribution and renal clearance are highly sensitive to hydrodynamic diameter. 17 To date a number of different techniques have been employed to characterize QD surface chemistry, notably X-ray photoelectron spectroscopy, 18 nuclear magnetic reso- nance spectroscopy, 19 and Rutherford backscattering. 20 Here, we use analytical ultracentrifugation (AUC) to characterize the nanocrystal size distribution, composition, and surface chemistry. AUC has previously been used to probe the properties of various nanoparticle systems: the hydrodynamic radii of FePt nanoparticles; 21 size-dependent sedimentation of CdSe, Fe 3 O 4 , and gold nanocrystals; 22 pH-dependent ag- gregation of TiO 2 particles; 23 and conjugation of the DNA * Corresponding author. Telephone: (61)3-8344-2420. Fax: (61)3-9348- 1595. E-mail: mulvaney@unimelb.edu.au. † School of Chemistry and Bio21 Institute, University of Melbourne. ‡ Ludwig Institute for Cancer Research, Royal Melbourne Hospital. § The Department of Biochemistry and Molecular Biology and Bio21 Institute, University of Melbourne. NANO LETTERS 2008 Vol. 8, No. 9 2883-2890 10.1021/nl801629f CCC: $40.75  2008 American Chemical Society Published on Web 07/30/2008