Conjugation of Transferrin to Azide-Modified CdSe/ZnS Core-Shell Quantum Dots using Cyclooctyne Click Chemistry
Conjugation of Transferrin to Azide-Modified CdSe/ZnS Core-Shell Quantum Dots using Cyclooctyne Click Chemistry
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DOI：
10.1002/anie.201202876
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发表时间：
2012-01-01
影响因子：
16.6
通讯作者：
Mulvaney, Paul
中科院分区：
文献类型：
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作者：
Schieber, Christine;Bestetti, Alessandra;Mulvaney, Paul
量子点(QD)是一种半导体纳米晶体，具有独特的光学性质，有别于常见的有机荧光团。例如，它们具有尺寸相关的发射光谱、高的光致发光量子产率和大的摩尔消光系数，这为单分子检测提供了可能性。它们还具有宽的吸收截面，这使得能够同时激发混合的量子点群体。此外，与传统染料相比，量子点更耐化学降解，受光漂白的影响更小。[1]量子点在诊断、生物传感和生物标记方面的许多应用都可以受益于量子点的光学性质。[2]任何方法的第一步都是制备水溶性量子点，已经开发了各种策略来生成水溶性纳米颗粒。[3]将生物分子，如蛋白质、单抗或酶附着到水溶性量子点上并不是一件微不足道的事情，到目前为止，还没有广泛适用、容易实现、在不损害量子点功能的情况下可重复性。[4-6]在出现的最强大的生物偶联反应中，有一组生物正交化学反应，称为点击反应。铜(I)催化的叠氮化物-末端烯烃环加成(CuAAC)反应[7-9]提供了精致的官能团选择性，并且不存在标准偶联反应(例如，肟/肼连接、酯化和硫醇-马来酰亚胺加成反应)的缺点，这些反应往往容易水解和/或交联。[10]CuAAC反应已被广泛用于金、二氧化硅、氧化铁纳米颗粒以及碳纳米管的功能化。将铜(I)添加到CdSe半导体量子点中，完全且不可逆地猝灭量子点的光致发光(参见支持信息)。[15-17]一个有吸引力的替代方案是叠氮化合物与应变的环辛炔的应变促进的叠氮化物-炔环加成(SPAAC)反应，该反应发生得很快，并且不需要铜催化剂。我们报道了一种使用先前报道的量子点聚合物包裹技术来制备叠氮修饰量子点的策略。[20]我们开发了一种基于双官能团连接物(L)的模块化且广泛适用的连接策略，该策略能够将环辛炔基团结合到金属蛋白转铁蛋白上，并使环辛炔修饰的转铁蛋白与叠氮修饰的量子点发生SPAAC共轭(图1)。最后，我们通过监测荧光QD-转铁蛋白结合物在表达转铁蛋白受体(TFR)的肿瘤细胞中的摄取，证明了QD-蛋白质结合物具有生物活性。利用连续离子层吸附反应(SILAR)在十八烯中合成了CdSe/ZnS核壳量子点。[21]SILAR提供了三辛基膦/三辛基膦氧化物(TOP/TOPO)键合在纳米晶表面的有机可溶量子点。以有机可溶量子点为原料，分两步制备叠氮修饰的水溶性量子点(图1a)。用过量的低相对分子质量聚苯乙烯-马来酸酐共聚物(PSMA，Mw 1700)处理量子点会导致聚合物包覆量子点。用亲水性氨基聚乙二醇胺M1000处理PSMA包裹的量子点，会导致酸酐的氨解并自发转移到水相。通过将不同数量的叠氮化修饰的H_2N±PEG±N3混合到Jeffamine M1000中，可以实现叠氮化官能团在QD表面上的可控呈现。由此产生的…
Quantum dots (QDs) are semiconductor nanocrystals with unique optical properties that distinguish them from common organic fluorophores. For example, they give size-dependent emission spectra, high photoluminescence quantum yields, and large molar extinction coefficients, which gives the potential for single-molecule detection. They also have broad absorption cross-sections, which enables simultaneous excitation of mixed populations of QDs. Additionally, QDs are more resistant to chemical degradation and are less affected by photo-bleaching than conventional dyes.[1] Many applications in diagnostics, biosensing, and biolabeling could benefit from the optical properties of QDs.[2] The first step of any method is the preparation of water soluble QDs, and various strategies have been developed to generate water-soluble nanoparticles.[3] The attachment of biomolecules such as proteins, monoclonal antibodies, or enzymes to water-soluble QDs is not trivial, and to date there is no generic conjugation method that is broadly applicable, easily implemented, and reproducible without compromising the function of the QDs.[4–6] Among the most powerful bioconjugation reactions to emerge are a set of bioorthogonal chemical reactions referred to as click reactions. The copper (I)-catalyzed azide-terminal alkyne cycloaddition (CuAAC) reaction [7–9] provides exquisite functional-group selectivity and does not suffer the drawbacks of standard coupling reactions (for example, oxime/hydrazone ligation, esterification, and thiol-maleimide addition reactions) that are often susceptible to hydrolysis and/or cross-linking.[10] The CuAAC reaction has been extensively used for the functionalization of gold, silica, and iron oxide nanoparticles, as well as carbon nanotubes.[11–14] Unfortunately, addition of copper (I) to CdSe semiconductor QDs completely and irreversibly quenches QD photoluminescence (see Supporting Information).[15–17] An attractive alternative is the strainpromoted azide–alkyne cycloaddition (SPAAC) reaction of azides with strained cyclooctynes, which occurs rapidly and does not require a copper catalyst.[18, 19] Herein, we report a strategy to prepare azide-modified QDs using a previously reported QD polymer-encapsulation technique.[20] We have developed a modular and broadly applicable conjugation strategy based on a bifunctional linker (L) that enables incorporation of cyclooctyne groups onto the metalloprotein transferrin and the SPAAC conjugation of the cyclooctyne-modified transferrin to azide-modified QDs (Figure1). Finally, we demonstrate that the QD–protein conjugates are biologically active by monitoring the uptake of fluorescent QD–transferrin conjugates in transferrin-receptor (TfR) expressing tumor cells.CdSe/ZnS core–shell QDs were synthesized in octadecene using the SILAR (successive ion-layer adsorption reaction) process.[21] SILAR affords organic-soluble QDs with trioctylphosphine/trioctylphosphine oxide (TOP/TOPO) bound to the nanocrystal surface. Azide-modified water-soluble QDs were prepared from organic-soluble QDs in two steps (Figure1a). Treatment of the QDs with an excess of low molecular weight polystyrene-co-maleic anhydride polymer (PSMA, MW 1700) results in polymer encapsulation of the QD. Treatment of the PSMA-encapsulated QDs with a hydrophilic amino poly (ethylene glycol)(amino-PEG), Jeffamine M1000, results in aminolysis of the anhydride and spontaneous transfer to the aqueous phase. Controllable presentation of azide functional groups on the QD surface can be achieved by blending various amounts of an azide-modified H2NÀPEGÀN3 into Jeffamine M1000. The resulting …