Site-Specific Multivalent Carbohydrate Labeling of Quantum Dots and Magnetic Beads Xue-Long Sun,* [a] Wanxing Cui, [a] Carolyn Haller, [a] and Elliot L. Chaikof* [b] Cell-surface carbohydrates act as receptors for a variety of pro- tein ligands and thereby play a significant role in a wide range of biological processes, including immune-recognition events [1] and the interaction of viruses and bacteria with host cells [2] as well as tissue growth and repair. [3] As such, binding interactions of carbohydrates and proteins provide a starting point for the development of novel diagnostic agents and a framework for new therapies. [4] It is notable that the low affinity and specifici- ty that are typical of monomeric carbohydrate–protein interac- tions are dramatically enhanced when the carbohydrate com- ponent is presented as a multivalent ligand; a phenomenon referred to as the “cluster-glycoside effect”. [5–7] In response to this observation, considerable effort has focused on the design of unique, multivalent carbohydrate ligands in the form of linear polymers, [8–13] liposomes, [14,15] dendrimers, [16–18] beads, [19,20] or nanoparticles. [21–23] In this regard, we have re- cently described a useful route for the synthesis of glycopoly- mers by a cyanoxyl-mediated free-radical polymerization scheme that can be performed under aqueous condition and is tolerant of a wide range of monomer functionalities, includ- ing ÀOH, ÀCOOH, ÀNH 2 , and ÀOSO 3 H groups. [24] Conveniently, this synthetic approach facilitates selective derivatization of the polymer-chain terminus. [25] Herein, we report site-specific multivalent carbohydrate labeling of nanocrystal (quantum- dot) and magnetic-bead surfaces using a biotin chain-end- functionalized glycopolymer and demonstrate the potential value of these multivalent carbohydrate polymers in both imaging and biocapture applications (Figure 1). Semiconductor nanocrystals are a new class of size-tunable optical probe. [26,27] Recently, nanocrystal surfaces have been functionalized with DNA, [28] peptides, [29] proteins, [30] and other small ligands [31] with intended applications as biological re- agents and probes. Nanocrystal–streptavidin conjugates, for example, have been used to stain tissues, cells, and intracellu- lar organelles. [32,33] Likewise, nanocrystal–avidin–antibody con- jugates have improved the sensitivity of conventional fluoroim- munoassays. [34] To the best of our knowledge, carbohydrate- conjugated nanocrystals have yet to be explored in bioimaging applications although a few nanocrystal–carbohydrate conju- gates have been reported (see also note added in proof). [35,36] In the present study, nanocrystal–multivalent carbohydrate conjugates were produced by incubating nanocrystal–strepta- vidin (50 mL, 120 mgmL À1 streptavidin in phosphate buffered saline (PBS), Qdot TM 565 streptavidin conjugate, Quantum Dot Corp., Hayward, CA) with biotin end-terminated glycopolymer 1 (50 mL, 1mgmL À1 in PBS) bearing ten pendant lactose groups for one hour at room temperature. RCA 120 is a lectin that binds to terminal b-d-galactose. [37] As a model system, RCA 120 -immobilized agarose beads (100 mL, 2 mgmL À1 , Sigma) were incubated with nanocrystal–carbohydrate conjugates in PBS (100 mL) for 1 h at room temperature and subsequently washed three times with PBS. Confocal microscopy confirmed fluorescent staining of the lectin-modified bead surfaces (Fig- ure 2A). Of particular interest was that staining intensity was dramatically enhanced by the initial exposure of RCA 120 beads to biotin end-terminated glycopolymer 1 followed by incuba- tion of the mixture with streptavidin–nanocrystal conjugates (Figure 2B). The weak-intensity staining observed when using the first approach might have been due to the presence of free glycopolymer along with the nanocrystal–carbohydrate conjugates. As a two-step procedure, the sensitivity of staining was increased through the formation in situ of nanocrystal– carbohydrate complexes on the bead surface without the need to purify the conjugate. The absence of staining on treat- ment with streptavidin nanocrystals alone or with the use of nonbiotinylated glycopolymer 2 confirms the necessity and specificity of both carbohydrate–lectin and streptavidin–biotin interactions (Figure 2C). Biotin end-terminated glycopolymers were also used to extend the versatility of magnetic-bead-based biocapture assays that have been employed for the rapid isolation of a va- riety of lectin-bearing cells and biomolecules. [38] Indeed, Rye and Bovin have demonstrated that glycopolymer-derivatized magnetic beads provide a useful tool for the selection of cells expressing a specific carbohydrate-binding phenotype. [39] Bundy and Fenselau have also reported that glycopolymer- based affinity capture surfaces are more sensitive than lectin- based systems for microbial capture. [40] While in both reports glycopolymers were effectively attached to the bead and [a] Dr. X.-L. Sun, Dr. W. Cui, Dr. C. Haller Departments of Surgery and Biomedical Engineering Emory University School of Medicine Atlanta, GA 30322 (USA) E-mail: xsun@emory.edu [b] Dr. E. L. Chaikof Department of Surgery and Biomedical Engineering Emory University School of Medicine Atlanta, GA 30322 (USA) Fax: (+ 1)404-727-3660 E-mail: echaiko@emory.edu Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. ChemBioChem 2004, 5, 1593–1596 DOI: 10.1002/cbic.200400137 # 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1593