Homogeneous assay technology based on upconverting phosphors
Homogeneous assay technology based on upconverting phosphors
复制标题
DOI：
10.1021/ac0510944
复制
发表时间：
2005-11-15
影响因子：
7.4
通讯作者：
Soukka, T
中科院分区：
文献类型：
--
作者：
Kuningas, K;Rantanen, T;Soukka, T
上转换光致发光可在基于均相发光的检测中消除与自发荧光和散射激发光相关的问题，且无需时间分辨。我们展示了一种基于发光共振能量转移的检测方法，该方法使用无机上转换（UPC）镧系磷光体作为供体，荧光蛋白作为受体。UPC磷光体在近红外光下被激发，它们在可见波长处具有窄带反斯托克斯发射，从而能够在最小背景下测量与距离相关的敏化发射。在近红外激发下，单独的受体在较短波长处不会产生任何直接发射。利用链霉亲和素偶联的掺铒、镱的UPC磷光体作为供体，生物素化藻胆蛋白作为受体，构建了一种生物素的竞争模型检测方法。UPC磷光体在近红外（980nm）处被激发，通过使用配备红外激光二极管以及合适的激发和发射滤光片的微量滴定板荧光计，在红色波长（600nm）处测量敏化受体发射。检测下限处于亚纳摩尔浓度范围。与时间分辨荧光测定法相比，所开发的检测技术使仪器简化。近红外激发和红色波长发射使得该技术也适用于对颜色深和有荧光的样品进行分析，这在临床免疫测定和高通量筛选中常常是需要关注的问题。
Upconversion photoluminescence can eliminate problems associated with autofluorescence and scattered excitation light in homogeneous luminescence-based assays without need for temporal resolution. We have demonstrated a luminescence resonance energy-transfer-based assay utilizing inorganic upconverting (UPC) lanthanide phosphor as a donor and fluorescent protein as an acceptor. UPC phosphors are excited at near-infrared and they have narrow-banded anti-Stokes emission at visible wavelengths enabling measurement of the proximity-dependent sensitized emission with minimal background. The acceptor alone does not generate any direct emission at shorter wavelengths under near-infrared excitation. A competitive model assay for biotin was constructed using streptavidin-conjugated Er3+,Yb3+-doped UPC phosphor as a donor and biotinylated phycobiliprotein as an acceptor. UPC phosphor was excited at near-infrared (980 nm) and sensitized acceptor emission was measured at red wavelength (600 nm) by using a microtitration plate fluorometer equipped with an infrared laser diode and suitable excitation and emission filters. Lower limit of detection was in the subnanomolar concentration range. Compared to time-resolved fluorometry, the developed assay technology enabled simplified instrumentation. Excitation at near-infrared and emission at red wavelengths render the technology also suitable to analysis of strongly colored and fluorescent samples, which are often of concern in clinical immunoassays and in high-throughput screening.