Shi-Li Shen

A ratiometric lysosomal pH probe based on the naphthalimide–rhodamine system

In this study, a novel ratiometric pH probe RNL based on fluorescence resonance energy transfer (FRET) was well developed. It was fabricated by integrating the naphthalimide moiety as an FRET donor with the rhodamine moiety as an FRET acceptor. Meanwhile, 4-(2-aminoethyl)morpholine, which was a lysosome-locating group, was introduced. The sensing mechanism was the integration of PET and FRET processes and the comprehensive effect led to the simultaneous intensity enhancement of naphthalimide and rhodamine along with the pH value decrease. With a pKa of 4.82, the fluorescence intensity ratio (I529/I580) of the probe changed significantly within the pH range from 4.50 to 5.50. The probe showed excellent selectivity among various metal cations, amino acids and ATP. Moreover, RNL has been successfully applied in HeLa cells, and the results demonstrated that it could be used to detect lysosomal pH changes. The probe could also selectively stain lysosome in HeLa cells. Besides, the probe exhibited low cytotoxicity and satisfactory photostability in living HeLa cells.

A rhodamine B-based lysosomal pH probe

A novel rhodamine B-based fluorescent probe (RML) for lysosomal pH was developed by integrating a 4-(2-aminoethyl)morpholine moiety, which is a lysosome-targetable group, into a rhodamine B fluorophore, which is associated with rhodamine B dyes possessing spirocyclic (non-fluorescent) and ring-opening (fluorescent) forms with response to pH. The probe responded to acidic pH at low concentration in a short amount of time. In addition, RML showed good membrane permeability and brilliant selectivity among various amino acids and metal cations. RML exhibited an 80-fold increase in fluorescence intensity at 583 nm throughout the pH range of 7.40-4.00 with a pKa of 5.16, which indicates that RML is valuable for studying intracellular acidic organelles. Moreover, RML has been successfully applied in HeLa cells, and the results demonstrated that RML could selectively stain lysosomes in living HeLa cells. Note that RML could be used to detect the pH increase in lysosomes induced by bafilomycin A1 within HeLa cells.

Novel pyrazoline-based fluorescent probe for detecting glutathione and its application in cells

A novel compound, 2-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl acrylate (probe L), was designed and synthesized as a highly sensitive and selective fluorescent probe for recognizing and detecting glutathione among cysteine, homocysteine and other amino acids. The structures of related compounds were characterized using IR, NMR and HRMS spectroscopy analysis. The probe is a non-fluorescent compound. On being mixed with glutathione in buffered EtOH:PBS=3:7 solution at pH 7.4, the probe exhibited the blue emission of the pyrazoline at 474 nm and a 83-fold enhancement in fluorescence intensity. This probe is very sensitive and displayed a linear fluorescence off-on response to glutathione with fluorometric detection limit of 8.2 × 10(-8)M. The emission of the probe is pH independent in the physiological pH range. Live-cell imaging of HeLa cells confirmed the cell permeability of the probe and its ability to selectively discriminate GSH from Cys and Hcy in cells. The toxicity of the probe was low in cultured HeLa cells.

A ratiometric lysosomal pH probe based on the coumarin–rhodamine FRET system

In this study, we developed a coumarin–rhodamine based fluorescence resonance energy transfer (FRET) system RC1 as a ratiometric pH probe. The probe with a pKa of 4.98 was constructed by integrating a coumarin moiety as an FRET donor with a rhodamine moiety as an FRET accepter. Upon addition of H+, the coumarin emission at 477 nm decreased and the rhodamine emission at 582 nm increased simultaneously. The fluorescence intensity ratio (I477/I582) displayed excellent pH-dependent performance and responded linearly to minor pH changes in the range of 4.20–6.00. The probe exhibited brilliant selectivity among different amino acids, metal cations and the ATP. Moreover, it has been successfully applied in fluorescence imaging in HeLa cells and the results indicated that the probe could selectively stain lysosomes with low cytotoxicity and excellent photostability. We also applied RC1 to monitor intracellular pH variations induced by dexamethasone. Therefore, RC1 could act as a practical tool for the detection of pH in weakly acidic conditions and provide essential information in medicinal analysis and real biological systems.

A novel pyrazoline-based selective fluorescent probe for detecting reduced glutathione and its application in living cells and serum

A new fluorescent probe, N-(4-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-2,4-dinitrobenzenesulfonamide (probe 3), was designed and synthesized as a highly sensitive and selective fluorescent probe for recognizing and detecting glutathione among biological thiols in aqueous media. Probe 3 is a nonfluorescent compound. On being mixed with biothiols under neutral aqueous conditions, the 2,4-dinitrobenzenesulfoyl moiety can be cleaved off by glutathione, and the blue emission of the pyrazoline at 464 nm is switched on, with a fluorescence enhancement of 488-fold for glutathione. Furthermore, probe 3 was highly selective for glutathione without interference from some biologically relevant analytes. The detection limit of glutathione was 4.11 × 10(-7) M. The emission of the probe is pH independent in the physiological pH range. Moreover, the probe can be used for fluorescent imaging of cellular glutathione and can be used for detecting glutathione in calf serum.

A mitochondria-targeted ratiometric fluorescent probe for hypochlorite and its applications in bioimaging

A novel ratiometric probe (RCP) for -OCl was developed based on the fluorescence resonance energy transfer (FRET) platform. The probe was constructed by integrating the coumarin moiety (FRET donor) with the rhodamine moiety (FRET acceptor). Upon treatment with -OCl, the coumarin emission at 483 nm decreased and the rhodamine emission at 570 nm increased, enabling the probe to provide accurate detection of -OCl (in the concentration range of 0-50 μM). The probe exhibited brilliant selectivity and sensitivity, rapid response and low cytotoxicity. More importantly, the introduction of the quaternized pyridine moiety can not only manage to increase the solubility, but also achieve mitochondria-targeting. The probe was applied successfully to imaging endogenous -OCl in mitochondria, highlighting its potential applications in bioanalysis.

A novel ratiometric pH probe for extreme acidity based on FRET and PET

In this study, a novel ratiometric pH probe RC1 was successfully developed. RC1 was constructed by integrating a coumarin fluorophore as a fluorescence resonance energy transfer (FRET) donor into a rhodamine B fluorophore as a FRET acceptor, which is associated with rhodamine B dyes possessing spirocyclic (non-fluorescent) and ring-opening (fluorescent) forms with response to pH. At weak basic pH, the photo-induced electron transfer (PET) process of the N atom of aromatic imino in the rhodamine moiety partly quenches the coumarin emission. At acidic pH, the PET process is gradually inhibited upon acidification, enhancing the fluorescence intensity of coumarin remarkably; at the same time, the spirolactam form of rhodamine changes to a ring-opening form followed by the FRET process between coumarin and rhodamine. Hence, the emission intensities of coumarin and the rhodamine moiety simultaneously increase along with the pH decrease. The sensing mechanism is an integration of the PET and FRET processes. Based on the ratios of fluorescence intensity at 583 nm and 470 nm (I583/I470), RC1 with a pKa of 3.21 could be used in the ratiometric detection of pH in the range 2.20–4.20 with high selectivity. Furthermore, it can be applied to visualize extreme acidity in bacteria. The results demonstrate that RC1 can serve as an ideal probe for extremely acidic pH levels with excellent biological significance.

Synthesis of ferrocenyl pyrazole-containing chiral aminoethanol derivatives and their inhibition against A549 and H322 lung cancer cells

A series of novel ethyl 3-ferrocenyl-1-(2-hydroxy-3-(phenylamino)propyl)-1H-pyrazole-5-carboxylate derivatives with optical activity (4) was synthesized by microwave-assisted reaction of substituted aniline and ethyl 3-ferrocenyl-1-(oxiran-2-ylmethyl)-1H-pyrazole-5-carboxylate that was prepared from ethyl 3-ferrocenyl-1H-pyrazole-5-carboxylate and (R)- or (S)-oxiran-2-ylmethyl 4-methylbenzenesulfonate. Structures of the compounds were characterized by means of IR, (1)H NMR and mass spectroscopy. Preliminary biological evaluation showed that all of the compounds could suppress the growth of A549 and H322 lung cancer cells. Among all of the tested compounds 4a, 4b and 4d were more effective and might perform their action through cell cycle arrest. Moreover, although the inhibition differences between R and S enantiomers are mostly not so significant, (R)-4b displayed more effective inhibition than (S)-4b.

Fluorescence turn-on chemodosimeter for rapid detection of mercury (II) ions in aqueous solution and blood from mice with toxicosis.

The heavy metal mercury (Hg) is a threat to the health of people and wildlife in many environments. Among various chemical forms, Hg(2+) salts are usually more toxic than their counterparts because of their greater solubility in water; thus, they are more readily absorbed from the gastrointestinal tract into circulation. Therefore, new chemical receptors for detecting Hg(2+) ions in circulation are needed. In this study, we developed a rhodamine-based turn-on fluorescence probe to monitor Hg(2+) in aqueous solution and in blood of mice with toxicosis. The chemodosimeter responds to Hg(2+) ions stoichiometrically, rapidly, and irreversibly at room temperature as a result of a chemical reaction that produces strongly fluorescent oxadiazole. The new fluorescent probe shows good fluorescence response, with high sensitivity and selectivity, toward Hg(2+) ions in aqueous solution and in blood from mice with toxicosis and facilitates the naked-eye detection of Hg(2+) ions.

A novel pH probe based on a rhodamine–rhodamine platform

We developed a rhodamine–rhodanine-based pH probe with pKa = 4.85 in buffer solution. The fluorescence intensity exhibited strong pH-dependent performance and responded linearly to minor pH fluctuations within the range of 4.2–5.2. In addition, the fluorescence microscopic images suggested this probe had excellent cell membrane permeability and could image weak acid pH changes of lysosomes in live cells without auto-fluorescence and interference from the complex intracellular environment. The results demonstrated that the probe had great potential in monitoring H+ in vitro and in living cells.