Department of Molecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia.
School of Physics, The University of Sydney, Sydney, NSW, 2006, Australia.
Mikrochim Acta. 2019 May 11;186(6):346. doi: 10.1007/s00604-019-3466-x.
The authors report on a simplified approach to encapsulate upconversion nanoparticles (UCNPs) in polystyrene spheres by mini-emulsion polymerisation. The resulting particles (PS-UCNP) are hydrophilic, stable and suitable for biomolecular recognition and biosensing applications. Also, a strategy was developed for bioconjugation of antibodies onto the surface of the PS-UCNPs by using the bifunctional fusion protein linker-protein G (LPG). LPG mediates the functionalisation of PS-UCNPs with antibodies against digoxigenin allowing for specific labelling of convective PCR (cPCR) amplicons. Lambda DNA was amplified using cPCR on a heat block for 30 min using the digoxigenin labelled forward and biotin labelled reverse primers. The antibody functionalised PS-UCNPs bind to the digoxigenin end of the cPCR amplicons. Finally, the streptavidin labelled magnetic beads were used to selectively capture the PS-UCNP-labelled cPCR amplicons and the upconversion signal was detected at 537 nm under 980 nm excitation. This sandwich approach enables direct recognition of the target lambda DNA with a detection limit of 10 copies μL. The upconversion signal decreased proportionally to the concentration of the lambda DNA with a linear response between 10 and 10 copies of DNA. Graphical abstract Schematic representation of polystyrene-encapsulated upconversion nanoparticles (PS-UCNPs) prepared by mini-emulsion polymerisation. The PS-UCNPs were functionalised with anti-digoxigenin antibody using the fusion protein linker-protein G (LPG). Detection of digoxigenin-labelled amplicons is achieved (a) by using the antibody-functionalised LPG@PS-UCNP labels; (b) magnetic separation, and (c) 980 nm laser light for detection of the green upconversion luminescence peaking at 537 nm.
作者报告了一种通过微乳液聚合将上转换纳米粒子（UCNPs）封装在聚苯乙烯球中的简化方法。所得的粒子（PS-UCNP）具有亲水性、稳定性，适用于生物分子识别和生物传感应用。此外，还开发了一种通过双功能融合蛋白接头蛋白 G（LPG）将抗体偶联到 PS-UCNP 表面的策略。LPG介导了 PS-UCNP 与针对地高辛的抗体的功能化，允许对对流 PCR（cPCR）扩增子进行特异性标记。使用标记有地高辛的正向引物和标记有生物素的反向引物，在热块上通过 cPCR 对 lambda DNA 进行 30 分钟的扩增。抗体功能化的 PS-UCNP 与 cPCR 扩增子的地高辛端结合。最后，使用链霉亲和素标记的磁性珠选择性捕获 PS-UCNP 标记的 cPCR 扩增子，并在 980nm 激发下在 537nm 处检测上转换信号。这种三明治方法能够直接识别目标 lambda DNA，检测限为 10 拷贝μL。上转换信号与 lambda DNA 的浓度成比例下降，DNA 浓度在 10 到 10 拷贝之间呈线性响应。示意图说明通过微乳液聚合制备的聚苯乙烯封装的上转换纳米粒子（PS-UCNPs）。使用融合蛋白接头蛋白 G（LPG）将 PS-UCNP 功能化抗地高辛抗体。通过使用抗体功能化的 LPG@PS-UCNP 标记物（a）、磁分离（b）和 980nm 激光光（c）检测地高辛标记的扩增子，检测到绿色上转换荧光，峰值为 537nm。
