Sinks Louise E, Roussakis Emmanuel, Esipova Tatiana V, Vinogradov Sergei A
Department of Biochemistry and Biophysics, University of Pennsylvania, USA.
J Vis Exp. 2010 Mar 3(37):1731. doi: 10.3791/1731.
Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO(2;)). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (Förster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.
通过磷光猝灭法进行氧测量[1,2]包括以下步骤:1)将探针置于感兴趣的介质中(如血液或组织液);2)用适当波长的光照射目标物,以使探针激发到三重态;3)收集发射的磷光,并分析其时间进程以得出磷光寿命,然后将其转换为氧浓度(或分压,pO₂)。探针不得与生物环境相互作用,在某些情况下,测量完成后必须从介质中4)排出。这些步骤中的每一步都对磷光探针的分子设计提出了要求,而磷光探针是测量方案中唯一的侵入性组件。在此,我们综述用于生物系统中氧测量的树枝状磷光纳米传感器的设计。这些探针由基于铂或钯卟啉的聚芳基甘氨酸(AG)树枝状大分子组成,其外围用聚乙二醇(PEG)残基修饰。为了实现有效的双光子激发,树枝状大分子的末端可用双光子天线发色团修饰,这些发色团捕获激发能量并通过分子内荧光共振能量转移(FRET)将其传递到探针的三重态核心。我们描述了探针的关键光物理性质,并给出了详细的校准方案。