National Research Council Canada , 100 Sussex Drive , Ottawa , Ontario K1N 0R6 , Canada.
Italian Institute of Technology , 30 Via Morego , Genoa 16163 , Italy.
Langmuir. 2019 Apr 9;35(14):4909-4917. doi: 10.1021/acs.langmuir.8b03833. Epub 2019 Mar 26.
Nanoparticles (NPs) functionalized with antibodies on their surface are used in a wide range of research applications. However, the bioconjugation chemistry between the antibodies and the surface of nanoparticles can be very challenging, often accompanied by several undesired effects such as nanoparticle aggregation, antibody denaturation, or poor target recognition of the surface-bound antibodies. Here, we report on a synthesis of fluorescent silica nanoparticle-antibody (NP-Ab) conjugates, in which polycarboxylated dextran is used as the multivalent linker. First, we present a synthetic methodology to prepare polycarboxylated dextrans with molecular weights of 6, 40, and 70 kDa. Second, we used water-soluble, polycarboxylated dextrans as a multivalent spacers/linkers to immobilize antibodies onto fluorescent silica nanoparticles. The prepared NP-Ab conjugates were tested in a direct binding assay format in both phosphate-buffered saline buffer and whole serum to investigate the role of the spacer/linker in the capacity of the NP-Ab to specifically recognize their target in "clean" and also in complex media. We have compared the dextran conjugates with two standards: (a) NP-Ab with antibodies attached on the surface of nanoparticles through the classical physical adsorption method and (b) NP-Ab where an established poly(amidoamine) (PAMAM) dendrimer was used as the linker. Our results showed that the polycarboxylated 6 kDa dextran facilitates antibody immobilization efficiency of nearly 92%. This was directly translated into the improved molecular recognition of the NP-Ab, which was measured by a direct binding assay. The signal-to-noise ratio in buffered solution for the 6 kDa dextran NP-Ab conjugates was 81, nearly 3 times higher than that of PAMAM G4.5 conjugates and 9 times higher than the physically adsorbed NP-Ab sample. In whole serum, the effect of 6 kDa dextran was more hindered due to the formation of protein corona but the signal-to-noise ratio was at least double that of the physically adsorbed NP-Ab conjugates.
纳米粒子 (NPs) 表面功能化的抗体被广泛应用于各种研究应用中。然而，抗体与纳米粒子表面的生物偶联化学可能非常具有挑战性，通常伴随着几种不理想的效应，如纳米粒子聚集、抗体变性或表面结合抗体对靶标的识别不良。在这里，我们报告了荧光二氧化硅纳米粒子-抗体 (NP-Ab) 缀合物的合成，其中多羧基化葡聚糖用作多价连接物。首先，我们提出了一种合成分子量为 6、40 和 70 kDa 的多羧基化葡聚糖的方法。其次，我们使用水溶性多羧基化葡聚糖作为多价间隔物/连接物将抗体固定在荧光二氧化硅纳米粒子上。制备的 NP-Ab 缀合物在磷酸盐缓冲盐水缓冲液和全血清中以直接结合测定的形式进行测试，以研究间隔物/连接物在 NP-Ab 特异性识别其靶标的能力中的作用在“干净”和复杂介质中。我们比较了葡聚糖缀合物与两种标准：(a) 通过经典物理吸附法将抗体附着在纳米粒子表面的 NP-Ab 和 (b) 作为连接物的已建立的聚(酰胺-胺) (PAMAM) 树枝状大分子。我们的结果表明，多羧基化 6 kDa 葡聚糖有利于抗体的固定化效率接近 92%。这直接转化为 NP-Ab 的分子识别能力的提高，这通过直接结合测定来测量。缓冲溶液中 6 kDa 葡聚糖 NP-Ab 缀合物的信噪比为 81，几乎是 PAMAM G4.5 缀合物的 3 倍，是物理吸附 NP-Ab 样品的 9 倍。在全血清中，由于形成蛋白质冠，6 kDa 葡聚糖的作用受到更多阻碍，但信噪比至少是物理吸附 NP-Ab 缀合物的两倍。
