光激化学发光免疫检测探针材料的设计与制备

Design and Preparation of AlphaLISA Probe Materials

摘要:

光激化学发光免疫检测具有无背景荧光干扰、均相免洗的优势，在体外诊断快速检测领域中具有重要的应用前景。然而，该检测技术所用探针材料的稳定可控制备仍是该技术产业化应用的主要难点之一。采用PVP调控的共聚法，制备的负载铕螯合物Eu(DBM)₃Phen和硫氧杂环己烯衍生物PCU的聚苯乙烯纳米球作为受体球。采用介孔二氧化硅包覆，制备的负载光敏剂Ce6的Fe₃O₄@mSiO₂纳米球作为磁控驱动的供体球。利用扫描电子显微镜、透射电子显微镜、能谱仪等手段对受体球和磁控供体球进行表征。结果表明：受体球呈规则球形且尺寸均一，平均粒径约为200 nm，易分散于水，能与单线态氧作用而发光，最大发射波长约为615 nm；磁控供体球呈球形核壳结构，平均粒径约为700 nm，易分散于水中，在波长680 nm光照下能产生单线态氧，可用磁铁分离。分别用兔IgG和羊抗兔IgG修饰磁控供体球和受体球，结果显示两者之间能发生有效的特异性结合，用磁铁分离去除非特异性结合的探针球后，成功测得光激化学发光信号，证明了两种探针材料的成功制备。

Abstract:

AlphaLISA has the advantages of no background fluorescence interference and a wash-free homogeneous process， which has important application prospects in the field of in vitro diagnostic rapid tests. However， the stable and controllable preparation of the probe materials used in this detection technology is still one of the main difficulties in the industrialization and application of this technology. In this study， polystyrene nanospheres loaded with the europium chelate Eu（DBM）₃Phen and the thioxene derivative PCU were prepared as the acceptor beads， and Fe₃O₄@mSiO₂ nanospheres loaded with the photosensitizer Ce6 were prepared as the magnetically controlled donor beads， respectively. Comprehensive characterizations including SEM， TEM， and EDS were performed on both beads. The results show that the acceptor beads are regular spheres of uniform size， with an average particle size of about 200 nm， which are readily dispersed in water and can emit light with singlet molecular oxygen， with a maximum emission wavelength of about 615 nm. The magnetically controlled donor beads are a core-shell structure with an average particle size of about 700 nm， which are readily dispersed in water and produce singlet molecular oxygen under 680 nm light， and can be separated by the magnet. After the modification of rabbit IgG and goat anti-rabbit IgG on the magnetically controlled donor beads and acceptor beads respectively， the results show that effective specific binding can occur between the two， and the AlphaLISA signals were successfully measured after removing the non-specifically bound probe beads with the magnet， proving the successful preparation of the two probe materials.