SYNTHESIS OF LANTHANIDE ENCODED MICROSPHERES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 新墨西哥大学正在开发一些基于乳液聚合和气雾剂合成的微球生产方法。这些方法将适用于形成均匀的微珠群体，这些微珠的直径是单分散的，并且含有已知浓度的发光稀土元素，如Tb和Eu络合物。直接合成单分散微粒(而不是多分散微粒的粒度分级)将是非常有利的，因为许多稀土络合物的成本相对较高，并将为生产流式细胞仪试剂提供足够的生产能力。由于稀土元素在水合过程中发光效率降低的敏感性，我们将开发一种将它们包裹在极性无机和非极性有机微球主体中的方法，如下所述。在大多数情况下，包裹与共轭分子“天线”络合的稀土离子以增加光致发光截面可能是有利的[54]。许多Tb和Eu的络合物(Aldrich，Strem)以及镧系元素的标记都可以在商业上获得。(Invitgen)，具有一系列的激发特性。 稀土元素在无机基质中的包覆。我们的第一个目标将是开发简单和可重复生产单分散二氧化硅颗粒的方法，这种颗粒以明确定义的浓度封装发光稀土离子。Lopez等人。在UNM，最近开发了从气溶胶液滴中生产单分散二氧化硅微粒的溶胶-凝胶法(见图14)。[55]在该方法中，使用振动孔板气溶胶发生器产生具有所需前体的均匀气溶胶液滴，该方法已被证明有利于将无机和有机主体结合到颗粒中。使用这种方法，我们将介绍已知浓度的光致发光Tb和Eu络合物。我们将优化分子水平的分散和总粒子发光，以便能够使用NFCR开发的新型声聚焦流动细胞仪直接检测稀土光致发光。通过已建立的硅烷偶联化学，这些硅基微珠将很容易进行表面生物功能化[56]。 稀土元素在有机聚合物微球中的包覆。由于上述问题与水合稀土元素潜在的低发光有关，我们的第二个目标将是开发基于有机单体乳液聚合的方法，以方便地制备包含稀土离子的单分散粒子。基于乳液的聚合是众所周知的，但本体乳液方法通常会导致颗粒具有多分散尺寸(通常为对数正态尺寸分布)[57]。对于流式细胞术的应用，使用大小均匀的粒子群是非常理想的，因此，我们将开发直接形成包裹稀土元素的单分散聚合物粒子的方法。为此，我们将使用哈佛大学开发的微流控注入方法来形成稳定的单分散乳液滴[58]。通过国家科学基金会资助的一项合作，形成这种单分散液滴的方法最近已转移到UNM。图14所示的液滴是在UNM制造和使用的微流控装置中产生的。我们将开发稳定这类乳液滴的方法，将有机单体如苯乙烯、甲基丙烯酸甲酯和甲基丙烯酸丁酯与发光稀土络合物一起负载到这些液滴中，并引发聚合反应以形成均匀的微球。这种用于形成微球的乳液聚合方法特别强大，因为它能够一步形成核壳结构，其中表面覆盖有生物分子反应性官能团。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A number of methods for the production of microspheres based on emulsion polymerization and aerosol synthesis are being developed at the University of New Mexico. These methods will be adapted for the formation of uniform populations of microbeads that are monodisperse in diameter and that incorporate known concentrations of luminescent lanthanides, such as terbium and europium complexes. Direct synthesis of mondisperse microparticles (as opposed to size fractionation of polydisperse particles) will be highly advantageous because of the relatively high cost of many lanthanide complexes and will provide sufficient throughput for the production of flow cytometry reagents. Because of the susceptibility of lanthanides reduction in luminescence yield by hydration, we will develop a method for their encapsulation in both polar inorganic and apolar organic microspherical hosts as described below. In most cases it may be advantageous to encapsulate lanthanide ions that are complexed to conjugated molecular "antennae" to increase photoluminescence cross-section [54]. A number of terbium and europium complexes are commercially available (Aldrich, Strem) as are lanthanide labels. (Invitrogen) that have a range of excitation characteristics. Encapsulation of Lanthanides in Inorganic Hosts. Our first aim will be to develop methods for the facile and reproducible production of monodisperse silica particles that encapsulate luminescent lanthanide ions in well-defined concentrations. Lopez, et al. at UNM have recently developed sol-gel methods for production of monodisperse silica microparticles from aerosol droplets (see Fig. 14). [55] In this method, which has been shown to be conducive to the incorporation of inorganic and organic hosts in the particles, uniform aerosol droplets with the desired precursors are generated using a vibrating orifice aerosol generator. Using this method we will introduce known concentrations of photoluminescent terbium and europium complexes. We will optimize the molecular level dispersal and total particle lumimescence to enable direct detection of lanthanide photoluminescence using new acoustically focused flow cytometers developed by the NFCR. These silica-based beads will be readily amenable to surface biofunctionalization by established silane based coupling chemistry [56]. Encapsulation of Lanthanides in Organic Polymeric Microbeads. Because of the issue identified above related to the potential low luminescence of hydrated lanthanides, our second aim will be to develop methods based on emulsion polymerization of organic monomers for the facile preparation of monodisperse particles that incorporate lanthanide ions. Emulsion based polymerizations are well known, but bulk emulsion methods generally result in particles with polydisperse sizes (generally in a log-normal size distribution) [57]. For flow cytometry applications it is highly desirable to use particle populations of uniform size, and thus, we will develop methods for direct formation of monodispersed polymeric particles encapsulating lanthanides. To do so we will use microfluidic injection methods for forming stable monodisperse emulsion droplets developed at Harvard University [58]. Through an NSF funded collaboration, the methods for forming such monodisperse droplets have recently been transferred to UNM. The droplets shown in Fig. 14 were generated in microfluidic devices fabricated and utilized at UNM. We will develop methods of stabilization of such emulsion droplets, of loading them with organic monomers such as styrene, methyl methacrylate and n-butyl methacrylate, together with luminescent lanthanide complexes, and initiating polymerization reactions to form uniform microspheres. Such emulsion polymerization methods for forming microspheres are especially powerful because of the ability to form core-shell architectures in which are surface coated with biomolecule-reactive functional groups in one step.