Minimizing Nonspecific Cellular Binding of Quantum Dots with Hydroxyl-Derivatized Surface Coatings Brad A. Kairdolf, Michael C. Mancini, Andrew M. Smith, and Shuming Nie* Departments of Biomedical Engineering and Chemistry, Emory University and Georgia Institute of Technology, 101 Woodruff Circle, Suite 2001, Atlanta, Georgia 30322 Quantum-dot (QD) nanocrystals are promising fluores- cent probes for multiplexed staining assays in biological applications. However, nonspecific QD binding to cellular membranes and proteins remains a limiting factor in detection sensitivity and specificity. Here we report a new class of hydroxyl (-OH)-coated QDs for minimizing nonspecific cellular binding and for overcoming the bulky size problems encountered with previous surface coatings. The hydroxylated QDs are prepared from carboxylated (-COOH) dots via a hydroxylation and cross-linking process. With a compact hydrodynamic size of 13-14 nm (diameter), they are highly fluorescent (>60% quantum yields) and stable under both basic and acidic conditions. By using human cancer cells, we have evaluated their superior nonspecific binding properties against that of carboxylated, protein-coated, and poly(ethylene glycol) (PEG)-coated QDs. Quantitative cellular staining data indicate that the hydroxylated QDs result in a dramatic 140-fold reduction in nonspecific binding relative to that of carboxylated dots and a still significant 10-20-fold reduction relative to that of PEG- and protein-coated dots. Semiconductor quantum dots (QDs) are a new class of fluorescent labeling agents and have recently been used for a broad range of biological applications. 1-11 This broad interest is driven by their unique optical and electronic properties such as size-tunable light emission, superior signal brightness, resistance to photobleaching, and simultaneous excitation of multiple fluo- rescence colors. 7-10 Recent advances have led to highly bright and stable QD probes that are well-suited for multiplexed molec- ular profiling of intact cells and clinical tissue specimens. 12-18 In contrast to in vivo clinical imaging where the potential toxicity of cadmium-containing QDs is a major concern, histological and cellular staining is performed on in vitro or ex vivo clinical patient samples. As a result, the use of multicolor QD probes for cellular staining is likely one of the most important and clinically relevant applications in the near term. 13-15 However, a major problem for this application is that QD probes tend to be “sticky” and often bind nonspecifically to cellular membranes, proteins, and extra- cellular matrix materials. In particular, nanoparticles with highly charged surface groups, such as carboxylic acids and amines, have been shown to exhibit strong nonspecific binding to various cells and tissues. 19-22 This nonspecific binding problem causes a high level of background fluorescence that degrades the signal-to-noise ratio and limits tagging specificity and detection sensitivity. A number of surface encapsulation methods have been used for QD solubilization and bioconjugation, including direct ligand- exchange reactions and indirect surface encapsulation using silica, * To whom correspondence should be addressed. E-mail: snie@ emory.edu. (1) Bruchez, M. 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