Fluorescently encoded microbeads are in demand for multiplexed applications in different fields. Compared to organic dye-based commercially available Luminex’s xMAP technology, upconversion nanoparticles (UCNPs) are better alternatives due to their large anti-Stokes shift, photostability, nil background, and single wavelength excitation. Here, we developed a new multiplexed detection system using UCNPs for encoding poly(ethylene glycol) diacrylate (PEGDA) microbeads as well as for labeling reporter antibody. However, to prepare UCNPs-encoded microbeads, currently used swelling-based encapsulation leads to non-uniformity, which is undesirable for fluorescence-based multiplexing. Hence, we utilized droplet microfluidics to obtain encoded microbeads of uniform size, shape, and UCNPs distribution inside. Additionally, PEGDA microbeads lack functionality for probe antibodies conjugation on their surface. Methods to functionalize the surface of PEGDA microbeads (acrylic acid incorporation, polydopamine coating) reported thus far quench the fluorescence of UCNPs. Here, PEGDA microbeads surface was coated with silica followed by carboxyl modification without compromising the fluorescence intensity of UCNPs. In this study, droplet microfluidics-assisted UCNPs-encoded microbeads of uniform shape, size, and fluorescence were prepared. Multiple color codes were generated by mixing UCNPs emitting red and green colors at different ratios prior to encapsulation. UCNPs emitting blue color were used to label the reporter antibody. Probe antibodies were covalently immobilized on red UCNPs-encoded microbeads for specific capture of human serum albumin (HSA) as a model protein. The system was also demonstrated for multiplexed detection of both human C-reactive protein (hCRP) and HSA protein by immobilizing anti-hCRP antibodies on green UCNPs.

Open image in new window

荧光编码的微珠在不同领域中需要多重应用。与基于有机染料的市售Luminex xMAP技术相比，上转换纳米粒子（UCNP）具有更好的抗斯托克斯频移，光稳定性，零背景和单波长激发，因此是更好的选择。在这里，我们开发了一种使用UCNPs的新型多重检测系统，用于编码聚（乙二醇）二丙烯酸酯（PEGDA）微珠以及标记报道抗体。但是，为了制备UCNPs编码的微珠，目前使用的基于膨胀的封装会导致不均匀，这对于基于荧光的多路复用是不希望的。因此，我们利用液滴微流控技术获得内部大小，形状和UCNPs分布均一的编码微珠。此外，PEGDA微珠缺乏探针抗体在其表面缀合的功能。迄今为止，已报道了使PEGDA微珠表面功能化的方法（丙烯酸掺入，聚多巴胺涂层）淬灭UCNPs的荧光。在这里，PEGDA微珠表面涂有二氧化硅，然后进行羧基修饰而不影响UCNPs的荧光强度。在这项研究中，制备了均匀形状，大小和荧光的液滴微流体辅助的UCNPs编码微珠。在封装之前，通过混合以不同比例发射红色和绿色的UCNP来生成多个颜色代码。发出蓝色的UCNP用于标记报告抗体。将探针抗体共价固定在红色UCNPs编码的微珠上，以特异性捕获人类血清白蛋白（HSA）作为模型蛋白。该系统还通过将抗hCRP抗体固定在绿色UCNP上，用于人C反应蛋白（hCRP）和HSA蛋白的多重检测。

在新窗口中打开图像