In Situ Localization of Enzyme Activity in Live Cells by a Molecular Probe Releasing a Precipitating Fluorochrome

Abstract

Current enzyme-responsive,f luorogenic probes fail to provide in situ information because the released fluorophores tend to diffuse away from the reaction sites.T he problem of diffusive signal dilution can be addressed by designing ap robe that upon enzyme conversion releases afluorophore that precipitates.Anexcited-state intramolecular proton transfer (ESIPT)-based solid-state fluorophore HTPQ was developed that is strictly insoluble in water and emits intense fluorescence in the solid state,with l ex/em = 410/550 nm, thus making it far better suited to use with ac ommercial confocal microscope.HTPQ was further utilized in the design of an enzyme-responsive,f luorogenic probe (HTPQA), targeting alkaline phosphatase (ALP) as am odel enzyme. HTPQA makes possible diffusion-resistant in situ detection of endogenous ALP in live cells.I tw as also employed in the visualizing of different levels of ALP in osteosarcoma cells and tissue,t hus demonstrating its interest for the diagnosis of this type of cancer.

A New Strategy toward “Simple” Water‐Soluble AIE Probes for Hypoxia Detection

“…To the best of our knowledge, TPE‐2E N‐oxide is the first hypoxia imaging probe capable of staining lipid droplets yet with a simple chemical structure. Meanwhile, such a strategy of releasing the precipitating fluorochrome is also conductive to achieve long‐term imaging benefiting from its diffusion‐resistant feature . Compared with TPE‐2M N‐oxide and TPE‐2M2F N‐oxide, TPE‐2E N‐oxide exhibited better biocompatibility to HeLa cells and COS‐7 cells through MTT assay due to its modest metabolic rate (Figures S25 and S26, Supporting Information).…”

Enzymatic Noncovalent Synthesis of Supramolecular Soft Matter for Biomedical Applications

“…We also provide a perspective on the potential of ENS and hope this review is useful for scientists who develop supramolecular soft matter to address societal needs at various frontiers. We would like to apologize to researchers whose work [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] is not discussed in more detail due to space limitations.…”

“…In addition, several other labs have reported the use of ENS for imaging the activities of ALP on cells. 15,93,94 Imaging Intracellular Molecular Self-Assembly by ENS. Although intracellular molecular self-assembly was reported in 2007, 96,97 imaging ENS inside the cell is challenging.…”

A unique off-on near-infrared cyanine-based probe for imaging of endogenous alkaline phosphatase activity in cells and in vivo

“…The distribution of ALP in the cell is very extensive from the cell membrane to the cytoplasm (endoplasmic reticulum, mitochondria), and even to the nucleus [12,47]. In order to investigate the intracellular distribution of the probe, organelle co-localization assay was carried out.…”

“…However, these detection technologies fail to provide an adequate view of ALP dynamic properties by high sensitivity, real-time detection, and high spatial resolution imaging in live samples [10,13]. Fluorescence imaging-based approaches have emerged to address some of these issues via focusing on the detection of ALP activities in cells at the proteome level [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28]. Among them, small-molecule-based fluorescent probes are highly desirable due to their desirable chemical modification, excellent biocompatibility, and low cost [29].…”