Laboratory of Biophysics and Medicity, University of Turku, Tykistökatu 6A, FI-20520 Turku, Finland.
Anal Chem. 2010 Nov 15;82(22):9282-8. doi: 10.1021/ac1017869. Epub 2010 Oct 18.
We have developed easy-to-use homogeneous methods utilizing time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence quenching for quantification of eukaryotic cells. The methods rely on a competitive adsorption of cells and fluorescently labeled protein onto citrate-stabilized colloidal gold nanoparticles or carboxylate-modified polystyrene nanoparticles doped with an Eu(III) chelate. In the gold nanoparticle sensor, the adsorption of the labeled protein to the gold nanoparticles leads to quenching of the fluorochrome. Eukaryotic cells reduce the adsorption of labeled protein to the gold particles increasing the fluorescence signal. In the Eu(III) nanoparticle sensor, the time-resolved fluorescence resonance energy transfer between the nanoparticles and an acceptor-labeled protein is detected; a decrease in the magnitude of the time-resolved energy transfer signal (sensitized time-resolved fluorescence) is proportional to the cell-nanoparticle interaction and subsequent reduced adsorption of the labeled protein. Less than five cells were detected and quantified with the nanoparticle sensors in the homogeneous microtiter assay format with a coefficient of variation of 6% for the gold and 12% for the Eu(III) nanoparticle sensor. The Eu(III) nanoparticle sensor was also combined with a cell impermeable nucleic acid dye assay to measure cell viability in a single tube test with cell counts below 1000 cells/tube. This sensitive and easy-to-use nanoparticle sensor combined with a viability test for a low concentration of cells could potentially replace existing microscopic methods in biochemical laboratories.
我们开发了易于使用的均相方法，利用时间分辨荧光共振能量转移（TR-FRET）和荧光猝灭来定量真核细胞。这些方法依赖于细胞和荧光标记蛋白通过竞争吸附到柠檬酸稳定的胶体金纳米颗粒或掺杂 Eu(III)螯合物的羧基化聚苯乙烯纳米颗粒上。在金纳米颗粒传感器中，标记蛋白吸附到金纳米颗粒上会导致荧光团猝灭。真核细胞减少了标记蛋白对金颗粒的吸附，从而增加了荧光信号。在 Eu(III)纳米颗粒传感器中，检测到纳米颗粒与受体标记蛋白之间的时间分辨荧光共振能量转移；时间分辨能量转移信号（敏化时间分辨荧光）幅度的减小与细胞-纳米颗粒相互作用以及随后标记蛋白吸附减少成正比。在均相微量滴定分析格式中，使用纳米颗粒传感器可以检测到少于五个细胞并进行定量，金纳米颗粒传感器的变异系数为 6%，Eu(III)纳米颗粒传感器的变异系数为 12%。Eu(III)纳米颗粒传感器还与一种细胞不可渗透的核酸染料测定法结合使用，可在单个试管测试中测量细胞活力，每个试管中的细胞计数低于 1000 个细胞。这种灵敏且易于使用的纳米颗粒传感器与低浓度细胞的活力测试相结合，可能会替代生化实验室中现有的显微镜方法。
