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[ Instrument Network Instrument Development ] Cell microenvironment changes are closely related to many physiological and pathological processes. The development of non-invasive fluorescent probes to monitor small changes in intracellular biomolecule content or physiological parameters has important biological and medical value. . However, most of the current intracellular fluorescence analysis methods only provide non-quantitative fluorescence imaging, and its sensitivity and accuracy are difficult to meet the actual monitoring needs.
Figure 1. Schematic diagram of intracellular detection based on double-excitation ratio type up-conversion fluorescence (UCL): (a) Composition of 980/808 nm double-excitation ratio type UCL probe and ClO-annihilation sensitization upconversion luminescence mechanism of analyte (b) the energy transfer process from dye to upconversion nanoparticles (UCNPs); (c) the ratiometric detection of intracellular ClO- by probes.
The Chen Xueyuan team of the Key Laboratory of Functional Nanostructure Design and Assembly of the Chinese Academy of Sciences, under the support of the Chinese Academy of Sciences' Strategic Pilot Science and Technology Specialist, the Chinese Academy of Sciences Innovation International Team and the National Natural Science Foundation of China and the Chinese Academy of Sciences Youth Promotion Committee, first proposed the near-infrared double The excitation ratio type up-conversion strategy enables accurate detection of intracellular biomolecules. The team developed a polymer-F127-coated water-soluble dye-sensitized rare earth upconversion fluorescent probe that utilizes the near-infrared dye IR808 to Yb/Er co-doped NaGdF4 to upconvert the nanoparticles for efficient energy transfer and low background fluorescence signals. Ultrasensitive detection of hypochlorite (ClO-) at 808 nm excitation was performed with detection limits as low as 16.1 nM (Figure 1). At the same time, the up-conversion luminescence excited at 980 nm is used as a reference signal, which reduces the detection bias caused by the complex environment in the cell and the uneven distribution of the probe. By designing a novel near-infrared double-excitation confocal microscope system, the team successfully achieved accurate quantitative analysis of endogenous and exogenous ClO- in living cell MCF-7 (Fig. 2). The double-excitation ratio type detection strategy can be extended to the detection of various intracellular biomolecules by changing the response group of the near-infrared dye, and provides an important tool for physiological cell physiological process monitoring and disease diagnosis.
Figure 2. (a) Schematic diagram of the optical path of a near-infrared double-excitation confocal microscopy system; (b) probe-labeled MCF-7 cells are incubated with different concentrations of ClO- (0, 0.5, 1 and 2 μM). UCL spectra at 808 nm excitation; (c) double-excitation UCL ratio (UCLex808/UCLex980), calculated by normalization of the 540 nm luminescence intensity at 980 nm for the corresponding graph (b); at different UCNP concentrations , UCLex808 (d) and UCL ratio (e) concentration-dependent curve of ClO-; (f) establishment and accurate quantification of ClO- standard curve in living cells; black point is the average value of UCL ratio in Figure (e); Red dots (1)-(4) are the UCL ratios of cells after incubation with 0, 0.5, 1 and 2 μM ClO-, respectively.
The results were published online in the full text on September 24, 2019 in Adv. Sci. 2019, 1901874. DOI: 10.1002/advs.201901874, Institute of Physical Education / Shanghai University of Science and Technology, Ph.D. The first author of the paper.