The Synthesis Of MEHP Fluorescent Probe And Its In Situ Detection In Target Cells

BackgroundPhthalate ester plasticizers represented by di(2-ethylhexyl)phthalate(DEHP)are recognized as endemic disrupting chemicals(EDCs).Through the human blood-testis barrier,blood-brain barrier,Reproductive system and nervous systems are destroyed by EDCs.DEHP is absorbed by the body,then it can be rapidly hydrolyzed to mono(2-ethylhexyl)phthalate(MEHP).Long-term accumulation in the human body produces reproductive toxicity and estrogen effects.5-Aminofluorescein(5-AF)is a small molecule chemical and is aminofluorescein.The active amino group can bind to the carboxyl group in the biomolecule,and the fluorescence intensity is greatly enhanced after binding,thus,it is widely used in protein analysis and immunology research.AimSynthesis of fluorescently traced MEHP,Designed for use at the cellular and molecular level.Methods1.MEHP derivative fluorescent probe(MEHP-AF)was synthesised by the dehydration condensation reaction of MEHP and 5-aminofluorescein(5-AF).2.The purity and structure of the purified MEHP-AF were determined by liquid chromatography-mass spectrometry(HPLC-MS)and Fourier transform infrared spectroscopy,The fluorescence characteristics of the probes were analyzed with fluorescence steady-state and fluorescence transients.3.The experiment was conducted on Hela cells.Comparing the performance difference between 5-AF and MEHP-AF;studying the characteristics of the entrance of MEHP-AF with time as a variable;localizing Hela cells with phycoerythrin-labeled estrogen receptor(ER)antibody to investigate MEHP-AF and ER interactions.Results1.MEHP-AF is a structurally new compound.The molecular formula is C36H33NO8 and the molecular weight is 607.65.2.Physicochemical characteristics indicate that MEHP-AF is a red-brown oil.Similar to MEHP,it is insoluble in water and dissolves organic solvents such as chloroform and ethanol.The purity of the purified MEHP-AF was 92.4%.3.The performance difference between 5-AF and MEHP-AF indicates that water-soluble 5-AF is difficult to enter into cells at concentrations up to 100 pM,while MEHP-AF can be distributed to the cytoplasm and nucleus at concentrations as low as 1 ?M.At 15 min,MEHP-AF penetrated into the cytoplasm through the cell membrane,and a small amount reached the nucleus.The amount of nucleus entering the cell increases with time.ER is a nuclear receptor and can also be distributed in the cell membrane and cytoplasm.MEHP-AF binds with ER to exhibit yellow fluorescence,and the fluorescence density is stronger with longer exposure time.ConclusionAs a novel fluorescent probe,It has the fat-soluble properties of MEHP and the fluorescence characteristics of 5-AF.The subcellular and molecular toxicity mechanisms of MEHP can be studied in situ,in real time,and visualized in target cells.