Unique Fluorescent Probe Research Articles

Naturally Occurring Flavonol, Quercetagetin 5,6,7,3',4'-Pentamethyl Ether (Marionol), as a Nontoxic Plant-Based Fluorescent Probe for Rapid, Sensitive, and Selective Detection of Cu2+ in Water.

Herein, we report a naturally occurring flavonol, quercetagetin 5,6,7,3',4'-pentamethyl ether (known as marionol), as a nontoxic fluorescent probe for rapid, sensitive, and selective detection of Cu2+ in water. The interaction between marionol and Cu2+ ions is studied by various state-of-the-art spectroscopic techniques such as 1H NMR, UV-vis, fluorescence, and high-resolution mass spectrometry (HRMS). Marionol shows strong fluorescence quenching in the presence of Cu2+, whereas other interfering cations such as Fe3+, Co2+, Al3+, Pb2+, Hg2+, Zn2+, Mg2+, Mn2+, Ca2+, Cu+, Ag+, Na+, and K+ produce negligible changes in fluorescence. The binding stoichiometry between marionol and Cu2+ is found to be 2:1, according to Job's plot and HRMS analyses. The binding constant (K a) of Cu2+ with marionol is calculated to be 2.89 × 109 M-2, and the limit of detection is found to be 0.15 μM. In addition, marionol is further used for the quantification of Cu2+ in natural spring water samples. The good recoveries (92-110% with RSD <5%) and its low toxicity to MCF-7 human breast cancer cells make marionol a unique plant-based fluorescent probe for the detection of Cu2+ ions.

Ultrafast detection of bisulfite by a unique quinolinium-based fluorescent probe and its applications in smartphone-assisted food detection and bioimaging

Sulfur dioxide (SO2) is one of the major pollutants in the atmosphere, which is highly susceptible to inhalation by the human body and is converted into its derivatives (HSO3−/SO32−), which is hazardous to both human health and the ecological environment. Therefore the detection of SO2 derivatives (HSO3−/SO32−) is very important. In this work, we have prepared ID-QL, a water-soluble fluorescent probe based on the intramolecular charge transfer (ICT) mechanism, it exhibits colorimetric and fluorescent dual-channel response to HSO3− with ultrafast, highly selective and sensitive detection. In particular, ID-QL can be used for quantitative detection of HSO3− in real food samples. We developed a portable test strip for ID-QL and successfully combined it with smartphone to achieve convenient, low-cost and portable detection of HSO3− in real samples. The probe displays good mitochondrial targeting ability and can be used for visual monitoring and imaging of sulfites in live cells and zebrafish.

A dual-response fluorescent probe to reveal the role of ferroptosis in drug-induced liver injury

Drug-induced liver injury (DILI) has already been recognized as a formidable obstacle in global health issues. Despite the proposed common cellular death types in DILI, exploration of alternative mechanisms such as ferroptosis for prevention and therapy remains limited. Visualization of the role of ferroptosis in DILI holds profound promise in advancing diagnostic and therapeutic strategies for DILI. To address this, herein, a unique multi-functional fluorescent probe, VIS-HNO, was elaborately built up and endowed with the extraordinary performance of excellent selectivity and high sensitivity toward HNO and viscosity. Employing the probe VIS-HNO with the confocal fluorescence imaging, the up-regulations of HNO and viscosity in the process of DILI and ferroptosis were disclosed. Then the remediation of DILI was achieved by VIS-HNO under the treatment of ferrostatin1 (Fer-1), indicating its therapeutic value for DILI. In addition, taking into account the significance of HNO in determining the role of ferroptosis in DILI, the pathway of HNO production in DILI was further explored, which presented essential insights into the role of ferroptosis in DILI. Overall, these findings demonstrated that VIS-HNO may be a potent tool to aid in diagnosing ferroptosis and DILI, more importantly, to understand the role of ferroptosis in DILI by evaluating HNO and viscosity level, which will facilitate the exploration of unique strategies for DILI treatment.

Unique fluorescent probe for the recognition of late apoptosis via translocation from plasma membrane to nucleus

Cell apoptosis is a crucial physiological process playing central roles in key biological and pathological activities. However, the current fluorescent probes for the detection of late apoptosis were “off–on” probes, which were facilely interfered by false positive signals caused by inhomogeneous staining and other factors. Herein, a unique fluorescent probe (NPn) discriminating late apoptosis from early apoptosis and heathy status with two different sets of fluorescent signals have been prepared, to overcome the possible false positive signals. NPn was designed impermeable to biomembranes and simultaneously with high affinity to DNA/RNA, which localized on the plasma membranes of living and early apoptotic cells, while relocated to the nucleus in late apoptotic cells. The hydrophilic amine unit and small ion radius were responsive for its membrane impermeability, which was confirmed with two control molecules without amine group. Using the probe, we have successfully evaluated the cell apoptosis induced by ultraviolet irradiation, rotenone, colchicine, and paclitaxel, demonstrating its potential application in biological researches.

A unique near-infrared fluorescent probe based on dual-DNP binding sites for rapid monitoring of hydrogen sulfide in food samples and living cells.

A new NIR fluorescent probe (DCIQ-2DNP), which combined the thiolysis of dinitrophenyl (DNP) ether and DNP-marked electron-deficient quaternary carbon, was reported for the first time for detection of H2S. The DCIQ-2DNP probe showed an NIR emission signal (740 nm), a large Stokes shift (128 nm), and rapid monitoring of hydrogen sulfide (within 60 s) in food samples and living cells.

A unique phenothiazine-based fluorescent probe using benzothiazolium as a reactivity regulator for the specific detection of hypochlorite in drinking water and living organisms

As a common disinfectant and an essential reactive oxygen species (ROS), hypochlorite (ClO−) plays vital roles in both water treatment and cell metabolism, but its abnormal level can cause serious harm to human health. Therefore, quantifying ClO− level in drinking water and living organisms is extremely significant. Herein, we decorated different cationic heterocycles on phenothiazine core to construct three fluorescent probes for ClO−. According to the results, only benzothiazolium moiety reasonably adjusted the electron cloud density at sulfur atom of phenothiazine core for the specific oxidation with ClO−, thus endowing the prepared probe PT-BT with a perfect selectivity for ClO−. Meanwhile, PT-BT exhibited a low detection limit (38 nM) and a fast response (within 20 s) toward ClO−. Furthermore, this probe was utilized to fabricate a ready-to-use test strip, which could quantitatively measure ClO− level in real water samples by a portable smartphone sensing platform. Notably, PT-BT targeted mitochondria efficiently, and successfully visualized endogenous ClO− in living cells and zebrafish larvae. Especially, PT-BT was able to monitor the dynamic change of ClO− level in inflammatory mice. These results strongly manifested that probe PT-BT was a promising tool for detecting ClO− in drinking water and living organisms.

A novel activatable fluorescent probe for revealing the dynamic of esterase and viscosity during ferroptosis and DILI

Esterase, a hydrolase, is present in a vast variety of animals, plants, and microbes. It has the potential to promote the hydrolysis of diverse esters while also could function as a valid indicator for assessing cell health. Besides, viscosity is a crucial metric for determining the smooth flow state of various biological macromolecules including proteins, polysaccharides, and lipids in the biological system, which is essential for intracellular signaling and interplay among multiple biological elements. Nevertheless, investigating the relationship between esterase activity, and viscosity in several relevant physiological and pathological processes remains challenging, owing to a lack of an appropriate tool. Herein, EaV is a unique multifunctional fluorescent probe that could measure local microviscosity and esterase in live cells via green and red channels at the same time. The utility of EaV for cell imaging was proven by measuring the esterase and viscosity changes in different cell states. More notably, we studied the changes in esterase and viscosity during erastin-induced ferroptosis, detecting for the first time a considerable reduction in esterase activity and an evident elevation in viscosity. In addition, EaV was used to image the changes of viscosity and esterase in the APAP-induced liver damage model. This fluorescence imaging tool revealed a flexible platform capable of highlighting variations of esterase and viscosity in many cellular processes and was also meaningful in further actually applied in more related early diagnosis and medical intervention.

A unique ratiometric fluorescent probe for detection of SO2 derivatives in living cells and real food samples

Abnormal levels of SO2 in organisms can cause cardiovascular disease and respiratory allergies. In addition, the amount of SO2 derivatives used as food preservatives is strictly controlled, and excessive addition can also be harmful to health. Therefore, it is essential to develop a highly sensitive method for the detection of SO2 and its derivatives in biological systems and real food samples. In this work, a new fluorescent probe (TCMs) with high selectivity and sensitivity for the detection of SO2 derivatives was reported. The TCMs could quickly identify SO2 derivatives. It has been successfully used to detect exogenous and endogenous SO2 derivatives. Furthermore, the TCMs has high sensitivity to SO2 derivatives in food samples. Moreover, the prepared test strips could be evaluated for the content of SO2 derivatives in aqueous solutions. This work provides a potential chemical tool to detect SO2 derivatives in living cells and real food samples.

Esterase Specific Fluorescent Probe: Mechanistic Understanding Using QM/MM Calculation and Cell States Discrimination.

Esterases enzymes regulate the body's homeostasis by catalyzing the hydrolysis of various esters. These are also involved in protein metabolism, detoxification, and signal transmission. Most importantly, esterase plays a significant role in cell viability and cytotoxicity assays. Hence, developing an efficient chemical probe is essential for monitoring the esterase activity. Several fluorescent probes for esterase have also been reported targeting cytosol and lysosomes. However, the ability to create efficient probes is constrained due to a lack of understanding of the esterase's active site for hydrolyzing the substrate. In addition, the fluorescent turn-on may limit efficient monitoring. Herein, we have developed a unique fluorescent probe, PM-OAc, to monitor mitochondrial esterase enzyme activity ratiometrically. This probe exhibited a bathochromic wavelength shift with esterase enzyme in alkaline pH (pH∼8.0) due to an intramolecular charge transfer (ICT) process. The phenomenon is well supported by TD-DFT calculation. Moreover, the substrate (PM-OAc) binding at the active site of esterase and its catalytic mechanism to hydrolyze the ester bond are elucidated by molecular dynamics (MD) simulation and QM/MM (Quantum mechanics/molecular mechanics) calculations, respectively. Fluorescent image-based analysis of the cellular environment reveals that our probe can distinguish between live and dead cells based on esterase enzyme activity.

Endoplasmic Reticulum Peroxynitrite Fluctuations in Hypoxia-Induced Endothelial Injury and Sepsis with a Two-Photon Fluorescence Probe.

Sepsis is a serious systemic inflammatory disease that frequently results in death. Early diagnosis and timely targeted interventions could improve the therapeutic effect. Recent work has revealed that the reactive oxygen species (ROS) in the endoplasmic reticulum (ER) and hypoxia-induced endothelial injury play significant roles in sepsis. However, the relationship between the levels of peroxynitrite (ONOO-) and hypoxia-induced endothelial injury as well as different states of sepsis remain unexplored. Herein, we developed a unique two-photon fluorescent probe (ER-ONOO-) for detecting ONOO- in aqueous solution that has high sensitivity, high selectivity, and ultrafast response time. In addition, ER-ONOO- was successfully used to evaluate the levels of ONOO- at the ER with three kinds of methods in a hypoxia-induced endothelial injury model. Furthermore, ER-ONOO- is capable of monitoring the changes in organ fluorescence through ONOO- variation in different stages of a cecum ligation and puncture (CLP) mouse model. Moreover, we also confirmed that the endoplasmic reticulum stress and oxidative stress participated in the CLP model. Consequently, this research can provide a reliable tool for studying ONOO- fluctuation in sepsis and provide new insights into the pathogenic and therapeutic mechanisms involved.

Reasonable construction of a unique near-infrared fluorescent probe with improved performance for imaging β-galactosidase in living cells and in vivo

β-Galactosidase is closely related to primary ovarian cancer and highly expressed in metastatic ovarian cancer. Therefore, the development of effective tool for in situ detecting β-gal is urgently needed for further studies in ovarian cancer. Inspired by the advantages of near-infrared (NIR) fluorescence imaging, a NIR fluorescent probe (DCM-CHO-βgal) with improved near-infrared fluorescence emission and detection performance was reasonably designed for the quantification and visualization of β-gal, employing structurally modified dicyanobenzopyran derivative (DCM-CHO-OH) as fluorophore. DCM-CHO-βgal showed a rapid response (<20 min) towards β-gal accompanied by nearly 40-fold fluorescence enhancement at 665 nm. DCM-CHO-βgal can quantitatively detect β-gal ranging from 0.2 to 3 U/mL with a low detection limit (3.15 × 10−5 U/mL), and can selectively detect β-gal in the presence of other possible interferences. In view of excellent optical, analytical performance and good biocompatibility, DCM-CHO-βgal was applied to the visualization of endogenous β-gal in ovarian cancer cells and zebrafish, which provided a powerful tool for the in-depth study of the role of β-gal in β-gal-related diseases.

In vivo imaging of heart failure with preserved ejection fraction by simultaneous monitoring of cardiac nitric oxide and glutathione using a three-channel fluorescent probe

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) remains unclear, making the diagnosis and treatment challenging. Cardiac oxidative and nitrative stress are strongly implicated in the pathogenesis of HFpEF. Herein, we present a unique three-channel fluorescent probe for evaluating cardiac oxidative and nitrative stress in HFpEF by simultaneous detection of NO and GSH. The probe exhibits a native green fluorescence (probe channel), while the presence of GSH and NO can sensitively turn the native green fluorescence into red fluorescence (GSH channel) and near-infrared fluorescence (NO channel), respectively. The probe clearly reveals that both GSH and NO levels are upregulated in cardiomyocytes and heart tissue with HFpEF. Moreover, it uncovers that the enhancement in NO and GSH levels are closely associated with increased level of iNOS (inducible nitric oxide synthase) and activation of the Keap1 (Kelch-like ECH-associated protein 1)/Nrf2 (nuclear factor erythroid 2–related factor 2)/ARE (antioxidant response element) signaling pathway in cardiomyocytes, respectively. This work proposes a promising approach for distinguishing normal heart and HFpEF heart by in vivo noninvasive imaging of both GSH and NO, and greatly contributing to the improvement of the diagnosis and treatment of HFpEF.

Revealing the Effects of Endoplasmic Reticulum Stress on Ferroptosis by Two-Channel Real-Time Imaging of pH and Viscosity.

Endoplasmic reticulum (ER) is sensitive to changes in the intracellular environment such as pH and viscosity, and slight changes may trigger stress response. Besides, different from apoptosis and necrosis, ferroptosis is the result of lipid peroxidation accumulation. There is evidence that ferroptosis is closely related to endoplasmic reticulum stress (ERS). However, the possible changes in the pH and viscosity of the ER during the ferroptosis process have not yet been studied. Therefore, we used a new type of ER-targeted dual-excitation fluorescent probe (DSPI-3) to investigate the possible changes of pH and viscosity of ER during the ferroptosis. The novel probe DSPI-3 exhibited a highly sensitive and selective response to pH and viscosity. During the bioimaging process, it was found that the ER acidified and viscosity increased during the ferroptosis process induced by erastin, while the cells treated with ferrostatin-1 did not alter significantly. In addition, when dithiothreitol (DTT) and erastin stimulated the cells at the same time, we discovered that ER was acidified considerably at short notice, but the pH was slightly increased in the later stage. Besides, the change of the viscosity enhanced slowly with the passage of time, and there was a noteworthy decline in the later stage, demonstrating that the DTT-induced ERS accelerated the process of ferroptosis. We hope that this unique fluorescent probe can provide an effective method for studying the relationship between ERS and ferroptosis.

Construction of a unique two-photon fluorescent probe and the application for endogenous CO detection in live organisms.

Carbon monoxide (CO) is one of the most significant signal molecules and plays an important role in regulating human physiological and pathological processes. In this study, a novel Pd-based complex (Pd-BNP-OH) was developed for endogenous CO detection. The structure and morphology of Pd-BNP-OH was characterized by SEM, XPS, and NMR analyses. When Pd-BNP-OH was reacted with CO, a strong fluorescence enhancement at 510nm was observed. In addition, Pd-BNP-OH exhibited high stability and selectivity toward CO in PBS buffer. In biological experiments, Pd-BNP-OH exhibited little cytotoxicity in cellular environment, and a bright fluorescence turn on was observed in the presence of exogenous CO and endogenous generated CO. The probe was then applied for CO detection in live zebrafish by both one-photon and two-photon excitation. Significantly, Pd-BNP-OH has excellent two-photon property, controllable structure and high biocompatibility. These features enable the probe to detect endogenously generated carbon monoxide in live organisms successfully.

Construction of a unique fluorescent probe for rapid and highly sensitive detection of glutathione in living cells and zebrafish

A rhodamine-based fluorescent probe (Rho-GSH) was rationally designed and synthesized, using nitrobenzene as recognition group for monitoring and imaging GSH in vitro and in vivo. In the presence of GSH, Rho-GSH undergoes sequential nucleophilic substitution and spiro-ring-opening reaction, resulting the fluorescence increase of probe, which enables quantitative detection of GSH with “off-on” fluorescence. The cascade reaction also enables Rho-GSH to rapidly detect GSH (3 s). In addition, Rho-GSH exhibited outstanding selectivity in detecting GSH vs cysteine (Cys) and homocysteine (Hcy). Based on the outstanding detection performance and superior biocompatibility of Rho-GSH, it was used to visualize GSH in vitro and in vivo, demonstrating its highly selectivity for GSH detection (with respect to Cys and Hcy). Rho-GSH can help visually distinguishing cancer cells from normal cells by fluorescence imaging, demonstrating the overexpression of GSH in cancer cells. Because of the outstanding analytical capabilities of Rho-GSH for GSH detection, Rho-GSH may be useful for cancer diagnosis and clinical evaluation.

A robust dual-channel fluorescence-enhanced probe for duplex imaging of endogenous HClO/ClO- in living cells and in zebrafish

A unique fluorescent probe, MCB, for HClO/ClO- was rationally constructed by integrating methylene blue, 7-hydroxycoumarin, and two HClO-specific reacting moieties into a single molecular structure. Distinct from other published cases, MCB displayed remarkable dual-channel fluorescence enhancement, peaked at 464 and 700 nm respectively, which simultaneously provided two crosstalk-free fluorescence channels, blue and NIR channels, for mutual corroboration, and hence improving the reliability of the detection results. More importantly, MCB exhibited excellent sensing properties for HClO/ClO-, such as fast response (within 90 s), high sensitivity (LOD as low as 3.9 nM based on blue fluorescence channel), and sufficient selectivity. Significantly, MCB was successfully utilized for dual-channel imaging of HClO/ClO-in living cells and zebrafish, thereby holding the potential for research that focused on elucidating the biological roles of HClO/ClO-.

Hot-Band Absorption of a Cationic RNA Probe Enables Visualization of ΔΨm via the Controllable Anti-Stokes Shift Emission

Mitochondrial membrane potential (ΔΨm) is an important biophysical parameter playing central roles in cell apoptosis, mitochondrial dysfunction, and other biological and pathological processes. Herein, we have rationally designed and fabricated a unique fluorescent probe for convenient ΔΨm visualization based on hot-band absorption and controllable anti-Stokes shift emission. The robust probe was excitable via hot-band absorption and emitted anti-Stokes upconversion emission and Stokes downconversion fluorescence simultaneously. The anti-Stokes emission could be efficiently inhibited upon the binding to RNA. The cationic probe targeted mitochondria in living cells with high ΔΨm and displayed both anti-Stokes green emission and ordinary red fluorescence. After the decrease of ΔΨm, the probe immigrated out of mitochondria to RNA and nucleolus, which showed only red emission owing to the inhibition of anti-Stokes fluorescence. In this manner, the ΔΨm could be visualized in dual-color mode. The probe enabled clearly monitoring the reversible changes in ΔΨm and was successfully employed to visualize oxidative damage of living cells. The decrease of ΔΨm in living tissues was also successfully observed with the newly designed probe.

An ultrasensitive lipid droplet-targeted NIR emission fluorescent probe for polarity detection and its application in liver disease diagnosis.

Compared to normal cells, cancer cells require more energy supply during proliferation and metabolism. In living cells, in addition to mitochondria, lipid droplets are also an important organelle for providing energy. Studies have shown that the number and distribution of lipid droplets change significantly during the production of lesions in cells. At this stage, the predisposing factors for the development of cellular lesions are not clear, thus leading to limitations in the early diagnosis and treatment of diseases such as liver injury, fatty liver, and hepatitis. To meet the urgent challenge, we used a near-infrared emission fluorescent probe SSR-LDs based on the intramolecular charge transfer effect (ICT) to detect polarity changes within intracellular lipid droplets. The probe SSR-LDs has ultra-sensitive polarity sensitivity, excellent chemical stability and photo-stability. In addition, by comparing normal and cancer cells through cell imaging experiments, we found that the robust probe has the ability to sensitively monitor the changes in lipid droplet polarity in the living cells. More importantly, using the constructed fluorescent probe, we have achieved an in vitro fluorescence detection of liver injury and fatty liver, and the detection of hepatitis at the in vivo level. The unique fluorescent probe SSR-LDs is expected to serve as a powerful tool for the medical diagnosis of diseases related to lipid droplet polarity.

A unique fluorescent probe for visualization of cell death via its subcellular immigration from lysosomes to nucleus

Fluorescent probes capable of simultaneous visualization of cell death, lysosomes, and nucleus are significant molecular tools essential for the in-depth understanding on cell death. However, such probes have not been devoted yet. In this work, by modifying two alkalescence groups onto naphthimide and tuning the DNA affinity, we obtained a fluorescent probe enabling visualization of cell death via its subcellular immigration between lysosomes and nucleus. In living cells, the probe exclusive targeted lysosomes to give intense fluorescence. During cell death, the probe was released from lysosomes and immigrated into the nucleus. Since lysosomes localized in cytoplasm which exhibited a clear boundary from the nucleus, the cell death could be conveniently monitored via the subcellular localization of the probe molecules. In this manner, the lysosomes, nucleus, and cell death could be visualized concurrently with the probe. The cell death induced by H2O2 has been imaged, and the cell apoptosis caused by rotenone was also successfully visualized by means of the newly designed fluorescent probe. The probe is potential to deep our understanding on cell death in a new aspect, and is also a desirable tool to study the interaction between cell death, lysosomes, and nucleus.

Charge-Dependent Strategy Enables a Single Fluorescent Probe to Study the Interaction Relationship between Mitochondria and Lipid Droplets.

Cooperation between organelles is essential to maintain the normal operation of the cell. A lipid droplet (LD), a dynamic organelle, is specialized in lipid storage and can interact physically with mitochondria in several cell types. However, an appropriate method for in situ studying the interaction relationships of mitochondria-LDs is still lacking. Herein, a charge-dependent strategy is proposed for the first time by considering adequately the charge difference between mitochondria and LDs. According to the novel strategy, we have developed a unique fluorescent probe Mito-LD based on the cyclization and ring-opening conversion. Mito-LD could simultaneously stain mitochondria and LDs and emit a red and green fluorescence, respectively. More importantly, with the probe Mito-LD, the in situ interaction relationships of mitochondria-LDs were investigated in detail from LD accumulation, mitochondrial dysfunction, lower environmental temperatures, and four aspects of apoptosis. The experimental results showed that mitochondria played an important role in LD accumulation, and the numbers and size of LDs would increase after mitochondrial dysfunction that may be due to excess liposomes. In addition, as an energy storage organelle, LDs played an important role in helping to coordinate mitochondrial energy supply in response to cold. In addition, the Mito-LD revealed that the polarity of mitochondria was higher than that of LDs. In a word, the probe Mito-LD could serve as a potential tool for further exploring mitochondria-LD interaction mechanisms, and importantly, the charge-dependent strategy is valuable for designing robust new probes in imaging multiple organelles.