Institute of Macromolecular Chemistry of the Czech Academy of Sciences, Heyrovského nám. 2, 162 06 Prague 6, Czech Republic.
Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
Biomacromolecules. 2020 Nov 9;21(11):4502-4513. doi: 10.1021/acs.biomac.0c00459. Epub 2020 May 29.
Lanthanide-doped upconversion nanoparticles (UCNPs) display highly beneficial photophysical features for background-free bioimaging and bioanalysis; however, they are instable in high ionic strength buffers, have no functional groups, and are nonspecifically interacting. Here, we have prepared NIR-excitable UCNPs that are long-term colloidally stable in buffered media and possess functional groups. Heterobifunctional poly(ethylene glycol) (PEG) linkers bearing neridronate and alkyne or maleimide were attached to UCNPs via a ligand exchange. Streptavidin (SA)-conjugates were prepared by click reaction of UCNP@PEG-alkyne and SA-azide. Antihuman serum albumin pAbF antibody was modified with azide groups and conjugated to UCNP@PEG-alkyne via click reaction; alternatively, the antibody, after mild reduction of its disulfide bonds, was conjugated to UCNP@PEG-maleimide. We employed these nanoconjugates as labels for an upconversion-linked immunosorbent assay. SA-based labels achieved the lowest LOD of 0.17 ng/mL for the target albumin, which was superior compared to a fluorescence immunoassay (LOD 0.59 ng/mL) or an enzyme-linked immunoassay (LOD 0.56 ng/mL).
镧系掺杂上转换纳米粒子（UCNPs）在无背景生物成像和生物分析中表现出高度有益的光物理特性；然而，它们在高离子强度缓冲液中不稳定，没有官能团，并且是非特异性相互作用的。在这里，我们制备了在缓冲介质中具有长期胶体稳定性且具有官能团的近红外激发 UCNPs。通过配体交换将带有奈立膦酸和炔基或马来酰亚胺的杂双官能聚乙二醇（PEG）接头连接到 UCNPs 上。通过 UCNP@PEG-alkyne 和 SA-叠氮化物的点击反应制备了链霉亲和素（SA）-缀合物。抗人血清白蛋白 pAbF 抗体用叠氮基团修饰，并通过点击反应与 UCNP@PEG-alkyne 缀合；或者，在温和还原其二硫键后，将抗体与 UCNP@PEG-马来酰亚胺缀合。我们将这些纳米复合物用作上转换免疫吸附测定的标记物。基于 SA 的标记物实现了针对靶蛋白的最低检测限为 0.17ng/mL，优于荧光免疫测定（LOD 0.59ng/mL）或酶联免疫吸附测定（LOD 0.56ng/mL）。
