Fluorescence activation imaging of localization, distribution, and level of miRNA in various organelles inside cells.

This work reports an approach for imaging the localization, distribution, and level of miRNA in different organelles based on an activated fluorescence signal triggered by an alteration of the specific binding-induced conformation of the designed activatable probe. We selected miR-150 as an miRNA example to image its localization, distribution, and level in human cervical cancer cells (HeLa cells). The results indicate that miR-150 is localized and distributed in different subcellular organelles (mainly in mitochondria and lysosomes) and that its levels (actually its concentrations) in lysosomes are higher than those in mitochondria in both HeLa and MCF-7 cells. Moreover, the level of miRNA in cells is displayed in a height-dependent (in z-direction) manner. This approach can also be used to image the localization and distribution of various miRNAs (such as miR-150 and miR-214) in different organelles in cancer cells simultaneously. The probes exhibit high resistance to cellular endo- and exonucleases, with high specificity; the capability of avoiding false signals, with a high signal-to-background ratio; and a good ability to operate in complicated environments. The developed approach may provide a useful tool for studying the localization and distribution and evaluating the level of multiple tumor-related miRNAs in cells.

Real-time fluorescence assay of alkaline phosphatase in living cells using boron-doped graphene quantum dots as fluorophores.

This work reports a convenient and real-time assay of alkaline phosphatase (ALP) in living cells based on a fluorescence quench-recovery process at a physiological pH using the boron-doped graphene quantum dots (BGQDs) as fluorophore. The fluorescence of BGQDs is found to be effectively quenched by Ce3+ ions because of the coordination of Ce3+ ions with the carboxyl group of BGQDs. Upon addition of adenosine triphosphate (ATP) into the system, the quenched fluorescence can be recovered by the ALP-positive expressed cells (such as MCF-7 cells) due to the removal of Ce3+ ions from BGQDs surface by phosphate ions, which are generated from ATP under catalytic hydrolysis of ALP that expressed in cells. The extent of fluorescence signal recovery depends on the level of ALP in cells, which establishes the basis of ALP assay in living cells. This approach can also be used for specific discrimination of the ALP expression levels in different type of cells and thus sensitive detection of those ALP-positive expressed cells (for example MCF-7 cells) at a very low abundance (10±5 cells mL-1). The advantages of this approach are that it has high sensitivity because of the significant suppression of the background due to the Ce3+ ion quenching the fluorescence of BGQDs, and has the ability of avoiding false signals arising from the nonspecific adsorption of non-target proteins because it operates via a fluorescence quench-recovery process. In addition, it can be extended to other enzyme systems, such as ATP-related kinases.