Site-specific multivalent carbohydrate labeling of quantum dots and magnetic beads
Site-specific multivalent carbohydrate labeling of quantum dots and magnetic beads
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DOI：
10.1002/cbic.200400137
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发表时间：
2004-11-05
期刊：
影响因子：
3.2
通讯作者：
Chaikof, EL
中科院分区：
文献类型：
--
作者：
Sun, XL;Cui, WX;Chaikof, EL
细胞表面碳水化合物作为各种蛋白质配体的受体，从而在广泛的生物过程中发挥重要作用，包括免疫识别事件[1]和病毒和细菌与宿主细胞的相互作用[2]以及组织生长和修复。[3]因此，碳水化合物和蛋白质的结合相互作用为开发新型诊断剂和新疗法的框架提供了起点。[4]值得注意的是，当碳水化合物组分作为多价配体存在时，单体碳水化合物-蛋白质相互作用的典型低亲和力和特异性显著增强;这种现象称为"簇-糖苷效应"。[5-7]针对这一观察结果，相当多的努力集中在设计独特的多价碳水化合物配体，其形式为线性聚合物、[8 - 13]脂质体、[14，15]树枝状聚合物、[16 - 18]珠粒、[19，20]或纳米颗粒。[21 - 23]在这方面，我们最近已经描述了一种通过氰脲酸介导的自由基聚合方案合成糖共聚物的有用途径，该方案可以在水性条件下进行，并且耐受广泛的单体官能团，包括C2H2OH、C2COOH、C2NH2和C2OSO3H基团。[24]方便地，该合成方法促进聚合物链末端的选择性衍生化。[25]在本文中，我们报告了使用生物素链末端官能化的糖聚合物对生物素（量子点）和磁珠表面进行位点特异性多价碳水化合物标记，并证明了这些多价碳水化合物聚合物在成像和生物捕获应用中的潜在价值（图1）。半导体纳米晶是一类新型的尺寸可调的光学探针。[26[27]最近，生物表面已经用DNA，[28]肽，[29]蛋白质[30]和其他小配体[31]功能化，预期应用为生物试剂和探针。例如，纳米纤维素-链霉亲和素缀合物已用于染色组织、细胞和细胞内细胞器。[32同样地，纳米纤维素-抗生物素蛋白-抗体缀合物提高了常规荧光免疫测定的灵敏度。[34]据我们所知，碳水化合物共轭的纳米晶体尚未在生物成像应用中进行探索，尽管已经报道了一些纳米晶体-碳水化合物共轭物（参见证据中添加的注释）。[35在本研究中，通过将纳米链霉亲和素-链霉亲和素（50 μ L，120 μ g mL/L链霉亲和素在磷酸盐缓冲盐水（PBS）中）、Qdot TM 565链霉亲和素缀合物（Quantum Dot Corp.，海沃德，CA）与带有10个乳糖侧基的生物素封端的糖共聚物1（50 μ L，1 mg mL/L，在PBS中）在室温下反应1小时。RCA120是一种与末端β-D-半乳糖结合的凝集素。[37]作为模型系统，将RCA 120固定的琼脂糖珠粒（100 μ L，2mg/mL，Sigma）与纳米琼脂糖-碳水化合物缀合物在PBS（100 μ L）中在室温下孵育1小时，随后用PBS洗涤三次。共聚焦显微镜证实了凝集素修饰的珠表面的荧光染色（图2A）。特别令人感兴趣的是，通过将RCA 120珠初始暴露于生物素末端封端的糖共聚物1，然后将混合物与链霉亲和素-β-葡聚糖缀合物孵育，染色强度显著增强（图2B）。当使用第一种方法时观察到的弱强度染色可能是由于游离的糖共聚物沿着纳米碳-碳水化合物缀合物的存在。作为一个二...
Cell-surface carbohydrates act as receptors for a variety of protein 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 specificity that are typical of monomeric carbohydrate–protein interactions are dramatically enhanced when the carbohydrate component 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 recently described a useful route for the synthesis of glycopolymers 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, including ÀOH, ÀCOOH, ÀNH2, and ÀOSO3H 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 (quantumdot) and magnetic-bead surfaces using a biotin chain-endfunctionalized 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 reagents and probes. Nanocrystal–streptavidin conjugates, for example, have been used to stain tissues, cells, and intracellular organelles.[32, 33] Likewise, nanocrystal–avidin–antibody conjugates have improved the sensitivity of conventional fluoroimmunoassays.[34] To the best of our knowledge, carbohydrateconjugated nanocrystals have yet to be explored in bioimaging applications although a few nanocrystal–carbohydrate conjugates have been reported (see also note added in proof).[35, 36] In the present study, nanocrystal–multivalent carbohydrate conjugates were produced by incubating nanocrystal–streptavidin (50 μL, 120 μg mLÀ1 streptavidin in phosphate buffered saline (PBS), QdotTM 565 streptavidin conjugate, Quantum Dot Corp., Hayward, CA) with biotin end-terminated glycopolymer 1 (50 μL, 1 mg mLÀ1 in PBS) bearing ten pendant lactose groups for one hour at room temperature. RCA120 is a lectin that binds to terminal β-D-galactose.[37] As a model system, RCA120-immobilized agarose beads (100 μL, 2 mgmLÀ1, Sigma) were incubated with nanocrystal–carbohydrate conjugates in PBS (100 μL) for 1 h at room temperature and subsequently washed three times with PBS. Confocal microscopy confirmed fluorescent staining of the lectin-modified bead surfaces (Figure 2A). Of particular interest was that staining intensity was dramatically enhanced by the initial exposure of RCA120 beads to biotin end-terminated glycopolymer 1 followed by incubation 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 …