Kuningas Katri, Rantanen Terhi, Ukonaho Telle, Lövgren Timo, Soukka Tero
Department of Biotechnology, University of Turku, Finland.
Anal Chem. 2005 Nov 15;77(22):7348-55. doi: 10.1021/ac0510944.
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.
上转换光致发光可以消除基于均相发光分析中与自发荧光和散射激发光相关的问题，而无需时间分辨。我们展示了一种基于发光共振能量转移的分析方法，该方法利用无机上转换（UPC）镧系磷光体作为供体，荧光蛋白作为受体。UPC磷光体在近红外光激发下，在可见波长处具有窄带反斯托克斯发射，能够以最小的背景测量邻近依赖性敏化发射。仅受体在近红外激发下在较短波长处不会产生任何直接发射。构建了一种生物素竞争模型分析方法，使用与链霉亲和素偶联的掺铒、镱的UPC磷光体作为供体，生物素化的藻胆蛋白作为受体。UPC磷光体在近红外（980 nm）激发下，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（600 nm）处测量敏化受体发射。检测下限在亚纳摩尔浓度范围内。与时间分辨荧光法相比，所开发的分析技术使仪器简化。近红外激发和红色波长发射使该技术也适用于对强颜色和荧光样品的分析，这在临床免疫分析和高通量筛选中经常是需要关注的。
