A fluorescent probe for ultrarapid H2O2 detection during reagent-stimulated oxidative stress in cells and zebrafish.

Hydrogen peroxide(H2O2), as a reliable signaling biomolecule for oxidative stress, its accurate detection during agent-stimulated oxidative stress plays a vital role in pathological and physiological mechanism exploration for disease theranostics. It's necessary to develop an efficient method for their detection. In view of the advantages of fluorescent probes, we rationally constructed a novel fluorescent probe Compound 2 based on 4-(Bromomethyl)benzeneboronic acid pinacol ester_Herein, a small molecule fluorescent probe was fabricated using isoflore nitrile as fluorescent group, phenylboronic acid pinacol ester as the response group, to detect H2O2. The probe Compound 2 has a strong fluorescence intensity at 575 nm, indicating that the structure of the probe molecule is reasonably designed, and the Stokes shift is up to 172 nm. While the detection time is as low as 30 s and the LOD of the probe for H2O2 is as low as 3.7 µmol/L,the quantum yield is Φ = 40.31 %. It has been successfully used for imaging detection of H2O2 in HepG2 cells and zebrafish for its low toxicity. It can be found that this small molecule fluorescent probe can identify H2O2 in tumor cells significantly and efficiently, which would realize the early diagnosis of tumor.

An activatable fluorescent-photoacoustic dual-modal probe for highly sensitive imaging of nitroxyl in vivo.

Nitroxyl (HNO) plays a vital role in various biological functions and pharmacological activities, so the development of an excellent near-infrared fluorescent (NIRF) and photoacoustic (PA) dual-modality probe is crucial for understanding HNO-related physiological and pathological progression. Herein, we proposed and synthesized a novel NIRF/PA dual probe (QL-HNO) by substituting an indole with quinolinium in hemicyanine for the sensitive detection of exogenous and endogenous HNO in vivo. The designed probe showed the highest sensitivity in NIRF mode and a desirable PA signal-to-noise ratio for HNO detection in vitro and was further applied for NIRF/PA dual-modal imaging of HNO with high contrast in living cells and tumor-bearing animals. Based on the excellent performance of QL-HNO, we believe that this study provides a promising molecular tool for further understanding of HNO-related physiological and pathological progression.

Golgi apparatus-targeting fluorescent probe for the imaging of superoxide anion (O2•-) in living cells during ferroptosis.

Ferroptosis is an emerging iron-dependent oxidative cell death type, and recently has been demonstrated to show close relation with Golgi apparatus (GA). Exploring the fluctuation of superoxide anion (O2•-) level in GA during ferroptosis is of great significance to profoundly study the biological functions of GA in ferroptosis. Here, we present a GA-targeting probe (N-GA) to monitor cellular O2•- during ferroptosis. N-GA employed a triflate group and a tetradecanoic amide unit as the recognition site for O2•- and GA-targeting unit, respectively. After the response of N-GA to O2•-, the triflate unit of N-GA converted into hydroxyl group with strong electron-donating ability, generating bright green fluorescence under UV light. N-GA exhibited excellent sensitivity and selectivity towards O2•-. Fluorescence imaging results showed that N-GA could be applied as a GA-targeting probe to monitor cellular O2•-. The stimulation of cells with PMA and rotenone could result in the massive generation of endogenous O2•- in GA. Erastin-induced ferroptosis can markedly induce the increase of O2•- level in GA. Similar to Fer-1 and DFO, dihydrolipoic acid (DHLA) and rutin were demonstrated to inhibit the enormous production of O2•- in GA of the living cells during ferroptosis.

A novel donor-acceptor fluorescent probe for the fluorogenic/ chromogenic detection and bioimaging of nitric oxide.

Nitric oxide (NO) plays an essential role in regulating various physiological and pathological processes. This has spurred various efforts to develop feasible methods for the detection of NO. Herein we designed and synthesized a novel donor-acceptor fluorescent probe Car-NO for the selective and specific detection of NO. Reaction of Car-NO with NO generated a new donor-acceptor structure with strong intramolecular charge transfer (ICT) effect, and led to remarkable chromogenic change from yellow to blue and dramatic fluorescence quenching. Car-NO exhibited high selectivity, excellent sensitivity, and rapid response for the detection of NO. In addition, the nanoparticles prepared from Car-NO (i.e., Car-NO NPs) showed strong NIR emission and high selectivity/sensitivity. Car-NO NPs was successfully employed to image both endogenous and exogenous NO in HeLa and RAW 264.7 cells. The present findings reveal that Car-NO is a promising probe for the detection and bioimaging of NO.

A Fluorescent Probe with Zwitterionic ESIPT Feature for Ratiometric Monitoring of Peroxynitrite In Vitro and In Vivo.

Peroxynitrite (ONOO-), as a short-term reactive biological oxidant, could lead to a series of effects in various physiological and pathological processes due to its subtle concentration changes. In vivo monitoring of ONOO- and relevant physiological processes is urgently required. Herein, we describe a novel fluorescent probe termed HBT-Fl-BnB for the ratiometric detection of ONOO- in vitro and in vivo. The probe consists of an HBT core with Fl groups at the ortho and para positions responding to the zwitterionic excited-state intramolecular proton-transfer (zwitterionic ESIPT) process and a boronic acid pinacol ester with dual roles that block the zwitterionic ESIPT and recognize ONOO-. Thanks to the specificity as well as low cytotoxicity, success in imaging of endogenous and exogenous ONOO- in living cells by HBT-Fl-BnB was obtained. Additionally, the applicability of HBT-Fl-BnB to tracking the abnormal expression of ONOO- in vivo induced by inactivated Escherichia coli was also explored. This is the first report of a fluorescent probe for ONOO- sensing via a zwitterionic ESIPT mechanism.

Turn-On Fluorescent Probe for BSA Detection Constructed by Supramolecular Assembly.

The fluorescent probe method has attracted significant research attention due to its high sensitivity and reproducibility in detecting bovine serum albumin (BSA). In this study, we constructed a fluorescent probe for BSA detection by assembling an amphiphilic organic fluorescent molecule, termed 2-(2'-hydroxyphenyl) benzothiazole (HBT-11), with BSA. In an aqueous solution, HBT-11 exhibited a weak fluorescence emission at 501 nm. However, the addition of BSA substantially enhanced the fluorescence emission at 501 nm, indicating that the assembly was driven by electrostatic interactions between HBT-11 and BSA. HBT-11, serving as a fluorescent probe for BSA detection, demonstrated a limit of detection (LOD) as low as 3.92 nmol L-1, excellent photostability, high selectivity, and robust anti-interference capability. Notably, we successfully applied HBT-11 for detecting BSA in fetal bovine serum and selectively imaging BSA in HeLa cells.

A human serum albumin-binding-based fluorescent probe for monitoring hydrogen sulfide and bioimaging.

In this work, a fluorescent probe, TPABF-HS, was developed for detecting hydrogen sulfide (H2S) using a human serum albumin (HSA)-binding-based approach for amplifying the fluorescence signal and extending the linear correlation range. Compared to the most recent probes for H2S, the most interesting feature of the detection system developed herein was the especially wide linear range (0-1000 µM (0-100 eq.)), which covered the physiological and pathological levels of H2S. TPABF-HS could be used in applications high sensitivity and selectivity with an LOD value of 0.42 µM. Further, site-competition experiments and molecular docking simulation experiments indicated that signal amplification was realized by the binding of the TPABF fluorophore to the naproxen-binding site of HSA. Moreover, the extension of the measurement span could allow for applications in living cells and Caenorhabditis elegans for imaging both exogenous and endogenous H2S. This work brings new information to the strategy of signal processing by exploiting fluorescent probes.

Rational design of a near-infrared dual-emission fluorescent probe for ratiometric imaging of glutathione in cells.

Sensors for which the output signal is an intensity change for a single-emission peak are easily disturbed by many factors, such as the stability of the instrument, intensity of the excitation light, and biological background. However, for ratiometric fluorescence sensors, the output signal is a change in the intensity ratio of two or more emission peaks. The fluorescence intensity of these emission peaks is similarly affected by external factors; thus, these sensors have the ability to self-correct, which can greatly improve the accuracy and reliability of the detection results. To accurately image glutathione (GSH) in cells, gold nanoclusters (AuNCs) with intrinsic double emission at wavelengths of 606 nm and 794 nm were synthesized from chloroauric acid. With the emission peak at 606 nm as the recognition signal and the emission peak at 794 nm as the reference signal, a near-infrared dual-emission ratio fluorescence sensing platform was constructed to accurately detect changes in the GSH concentration in cells. In vitro and in vivo analyses showed that the ratiometric fluorescent probe specifically detects GSH and enables ultrasensitive imaging, providing a new platform for the accurate detection of active small molecules.

A Zn-modified PCN-224 fluorescent nanoprobe for selective and sensitive turn-on detection of glutathione.

Monitoring endogenous glutathione (GSH) levels in living cells is essential for cancer diagnose and treatment. In this work, GSH responsive fluorescent nanoprobe with turn-on property was constructed using Zn-modified porphyrinic metal-organic frameworks (PCN-224-Zn). The introduced Zn2+ could quench the fluorescence of PCN-224 by the metallization of organic ligand (TCPP) and serves as sensing site for GSH. When exposed to GSH, the strong binding affinity of GSH generates the formation of Zn-GSH complex, eliminating the fluorescence quenching effect of Zn2+. Based on the constructed PCN-224-Zn nanoprobe, selective determination of GSH was achieved in the range of 0.01-6 µM with a detection limit of 1.5 nM. Furthermore, the constructed nanoprobe can realize the fluorescence imaging of endogenous GSH in MCF-7 and HeLa cells. Meanwhile, PCN-224-Zn could also monitor GSH in cell lysate with recovery rates from 93.8 % to 102.3 %. The performance of PCN-224-Zn demonstrates its capacities in the application of fluorescence sensing and bio-imaging fields.

A highly sensitive and selective rhodamine-semicarbazide based fluorescent sensor for Cu2+ detection in real water samples and fluorescence bioimaging in HepG2 cells.

A colorimetric and fluorescent sensor, selective for Cu2+ ions, was synthesized in two steps using a rhodamine-based compound attached to the semicarbazide-picolylamine moiety (RBP). Spectroscopic measurements, including UV-Vis absorption and fluorescence emission, were conducted in the semi-aqueous medium containing acetonitrile/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, denoted as MeCN/HEPES buffer (2:8, v/v, pH 7.0). The sensor exhibited high selectivity towards Cu2+ ions compared to other cations and demonstrated remarkable sensitivity towards Cu2+ ions, with a limit of detection at the nanomolar level. The calculated transitions indicated a 1:1 stoichiometric binding of RBP to Cu2+ ions based on a 4-coordination mode involving additional chelation in the semi-aqueous medium. The sensing mechanism for the detection of Cu2+ ions was investigated using high-resolution mass spectroscopy. The sensor could be employed as a real-time chemosensor for monitoring Cu2+ ions. Furthermore, the sensor has the potential for utilization in the detection of Cu2+ ions in actual water samples with the high precision and accuracy, as indicated by the small relative standard derivation values. The 50th percentile cytotoxicity concentration of RBP was found to be 22.92 µM. Additionally, the fluorescence bioimaging capability of RBP was demonstrated for the detection of Cu2+ ions in human hepatocellular carcinoma (HepG2) cells.

Construction of a super large Stokes shift near-infrared fluorescent probe for detection and imaging of superoxide anion in living cells, zebrafish and mice.

As one of the major reactive oxygen species (ROS), superoxide anion (O2•-) is engaged in maintaining redox homeostasis in the cell microenvironment. To identify the pathological roles in related disorders caused by abnormal expression of O2•-, it is of great significance to monitor and track the fluctuation of O2•- concentration in vivo. However, the low concentration of O2•- and the interference caused by tissue autofluorescence make the development of an ideal detection methodology full of challenges. Herein, a "Turn-On" chemical response near-infrared (NIR) fluorescence probe Dcm-Cu-OTf for O2•- detection in inflamed models, was constructed by conjugating the NIR fluorophore (dicyanisophorone derivative) with an O2•- sensing moiety (trifluoromethanesulfonate). Dcm-Cu-OTf exerted about 140-fold fluorescence enhancement after reacting 200 µM O2•- with an excellent limited of detection (LOD) as low as 149 nM. Additionally, Dcm-Cu-OTf exhibited a super large Stokes shift (260 nm) and high selectivity over other bio-analytes in stimulated conditions. Importantly, Dcm-Cu-OTf showed low toxicity and enabled imaging of the generation of O2•- in the Lipopolysaccharide (LPS)-stimulated HeLa cells, zebrafish, and LPS-induced inflamed mice. The present study provided a potential and reliable detection tool to inspect the physiological and pathological progress of O2•- in living biosystems.

Rational design and comparison of three curcumin-based fluorescent probes for viscosity detection in living cells and zebrafish.

Viscosity is a crucial indicator of the cellular microenvironment, which can affect the normal level of cellular metabolism. Aberrant levels of viscosity can result in the emergence of a variety of physiological problems including diabetes, Parkinson's disease, inflammation, etc. Therefore, it is crucial to exploit effective assays that can detect viscosity levels in living cells and organisms. Three new nitrogen-containing heterocyclic fluorescent probes, CNO, CNN and CNNB, were designed and prepared by coupling curcumin with isoxazole, pyrazole, and phenylpyrazole rings, respectively. The fluorescence response properties of these probes to the viscosity level were analyzed in parallel. All the probes, CNO, CNN and CNNB, exhibited a significantly enhanced fluorescence response to viscosity in a broad pH range with excellent photostability, sensitivity and anti-interference ability. The sensing mechanisms of these probes for viscosity were verified by DFT calculations. In addition, these probes were successfully employed for detecting viscosity levels in living HeLa cells and zebrafish. This research compares the viscosity-responsive capabilities of curcumin-based fluorescent probes containing different nitrogen-containing heterocyclic structures, and provides a new design strategy and guidance for developing curcumin-based fluorescent probes for viscosity analysis.

Long-Term Imaging of Cys in Cells and Tumor Mice by a Solid-State Fluorescence Probe.

Cysteine is an important biological thiol and is closely related to cancer. It remains a challenge to develop a probe that can provide long-term fluorescence detection and imaging of Cys in cells as well as in living organisms. Here, a solid-state fluorophore HTPQ is combined with an acrylate group to construct a solid-state fluorescent probe HTPQC for Cys recognition. The fluorescence of the probe is quenched when the photoinduced electron transfer (PET) process is turned on and the excited-state intramolecular proton transfer (ESIPT) process is turned off. In the presence of Cys, an obvious solid-state fluorescence signal can be observed. The double quenching mechanism makes the probe HTPQC have the advantages of high sensitivity, good selectivity, and high contrast of biological imaging. Due to low cytotoxicity, the probe HTPQC can be used to detect exogenous and endogenous Cys in living cells and is capable of imaging over long periods of time. By making full use of long wavelengths, the probe can be applied for the detection of Cys levels in tumor mice and equipped with the ability to conduct long-term imaging in vivo.

Fluorescence probe for real-time malonaldehyde detection in epilepsy model.

Oxidative stress, a condition involving an imbalance between reactive oxygen species (ROS) and antioxidants, is closely linked to epilepsy, contributing to abnormal neuronal excitability. This study introduces a novel fluorescent probe, the MDP probe, designed for the efficient detection of malondialdehyde (MDA), a critical biomarker associated with oxidative stress. The MDP probe offers several key advantages, including high sensitivity with a low detection limit of 0.08 µM for MDA, excellent selectivity for MDA even in the presence of interfering substances, and biocompatibility, making it suitable for cell-based experiments. The probe allows for real-time monitoring of MDA levels, enabling dynamic studies of oxidative stress. In vivo experiments in mice demonstrate its potential for monitoring MDA levels, particularly in epilepsy models, which could have implications for disease research and diagnosis. Overall, the MDP probe represents a promising tool for studying oxidative stress, offering sensitivity and specificity in cellular and in vivo settings. Its development opens new avenues for exploring the role of oxidative stress in various biological processes and diseases, contributing to advancements in healthcare and biomedical research.

A near-infrared fluorescent probe for detecting hydrogen sulfide with high selectivity in cells and ulcerative colitis in mice.

Although hydrogen sulfide (H2S) is a well-known toxic gas, its vital role as a gas transmitter in various physiological and pathological processes of living systems cannot be ignored. Relevant investigations indicate that endogenous H2S is involved in the development of ulcerative colitis pathology and is overexpressed in ulcerative colitis, and hence can be considered as an ulcerative colitis biomarker. Herein, an isophorone-xanthene-based NIR fluorescent probe (IX-H2S) was constructed to image H2S. Owing to its large conjugated structure, the probe exhibits a near-infrared emission wavelength of 770 nm with a large Stokes shift (186 nm). Moreover, IX-H2S has excellent selectivity for the detection of H2S without interference from other analytes including thiols. In addition, the probe has been successfully applied not only in fluorescence imaging of endogenous and exogenous H2S in living cells, but also in imaging of H2S in normal and ulcerative colitis mice. Encouraged by the eminent performance, IX-H2S is expected to be a potent "assistant" for the diagnosis of ulcerative colitis.

Bifunctional near-infrared fluorescent probe for the selective detection of bisulfite and hypochlorous acid in food, water samples and in vivo.

We report the development of a bifunctional near-infrared fluorescent probe (QZB) for selective sensing of bisulfite (HSO3-) and hypochlorous acid (HOCl). The synergistic detection of HSO3- and HOCl was achieved via a C=C bond recognition site. In comparison with the red-fluorescence QZB, two different products with non-fluorescence and paleturquoise fluorescence were produced by the recognition of QZB towards HSO3- and HOCl respectively, which can realize effectively the dual-functional detection of HSO3- and HOCl. QZB features prominent preponderances of dual-function response, near-infrared emission, reliability at physiological pH, low cytotoxicity and high sensitivity to HSO3- and HOCl. The detection of HSO3- in actual food samples has been successfully achieved using QZB. Utilization of QZB-based test strip to semi-quantitatively detect HSO3- and HOCl in real-world water samples by the "naked-eye" colorimetry are then demonstrated. Simultaneously, the determination of HSO3- and HOCl in real-world water sample has been achieved by smartphone-based standard curves. Additionally, the applications of QZB for imaging HSO3- and HOCl in vivo are successfully demonstrated. Consequently, the successful development of QZB could be promising as an efficient tool for researching the role of HSO3-/HOCl in the regulation of redox homeostasis regulation in vivo and complex signal transduction and for future food safety evaluation.

A lysosome-targeted triazole near-infrared cyanine fluorescent probe for in vivo long-term cell tracking.

In vivo visualization of cell migration and engraftment in small animals provides crucial information for the development and clinical translation of cell-based therapies. Therefore, a good quality near-infrared (NIR) fluorescent probe with high optical properties and excellent cellular retention ability is desired for in vivo cell tracking. Herein, we designed and synthesized a lysosome-targeted triazole NIR cyanine fluorescent probe, named IR780-NT-NH2, for in vivo long-term cell tracking. For the design, the heptamethine cyanine dye IR780 was used as the NIR fluorescent skeleton to ensure that the absorption and emission wavelengths fall within the NIR window. The substituent N-triazole group endowed the probe with high photostability and brightness. It has a quantum yield of 17.3% and the brightness remained above 85% after continuous illumination for 30 min. Due to the primary amine docking group, IR780-NT-NH2 has excellent lysosomal targeting and retention abilities as it becomes protonated in an acidic environment. The strong signal strength of IR780-NT-NH2 was maintained in well-shaped cells after an additional 12 h incubation. Moreover, this NIR probe exhibited ideal cellular permeability and biosafety. Finally, we realized long-term cell tracking with IR780-NT-NH2 labeled PC-3 cells using a NIR imaging system. The present study provides evidence that IR780-NT-NH2 exhibits ideal optical properties, excellent cellular permeation and retention, and good biosafety, which are useful for in vivo long-term observation of cells, and thus it shows promising potential for visualization in cell-based therapy.

Exploring Biological Interactions: A New Pyrazoline as a Versatile Fluorescent Probe for Energy Transfer and Cell Staining Applications.

Fluorescent dyes are used in biological systems, because they are highly sensitive and selective. In this work, we investigated the fluorescent probe properties of 2-(5-(pyridin-2-yl)-1H-pyrazol-3-yl) phenol (PYDP) in two media [sodium dodecyl sulfate (SDS) and human serum albumin (HSA)]. Energy transfer parameters, photophysical and thermodynamic parameters of probe were determined. We investigated cytotoxicity of PYDP against colorectal adenocarcinoma cell lines (HT-29), breast cancer cell lines (MCF-7) and 3T3-L1 adipocytes (3T3 L1) cells. The cell staining property of PYDP was monitored using a confocal microscope. The results showed that PYDP binds to HSA, bindings are due to electrostatic/ionic interactions, and the binding process is spontaneous. PYDP was found to exhibit negligible cytotoxicity at high concentrations, and confocal microscope images showed that PYDP stained the cytoplasm of MCF-7 cells.

Construction of a Dual-Emissive Probe for Discriminative Visualization of Lysosomal and Mitochondrial Dysfunction.

Discriminatively visualizing mitochondrial and lysosomal dysfunction is crucial for an in-depth understanding of cell apoptosis regulation and relative biology. However, fluorescent probes for the separate visualization of lysosomal and mitochondria damages have not been reported yet. Herein, we have constructed a fluorescent probe [2-(4-hydroxystyryl)-1,3,3-trimethyl-3H-indol-1-ium iodide (HBSI)] for labeling mitochondria and lysosomes in dual emission colors and discriminatively imaging mitochondrial and lysosomal damage in two different sets of fluorescent signals. In living cells, HBSI targeted both lysosomes and mitochondria to give green and red emission, respectively. During mitochondrial damages, HBSI immigrated into lysosomes, and the red emission decreased. During lysosomal damage, HBSI immigrated into mitochondria, and the green emission decreased. With the robust probe, the different damaging sequences of mitochondria and lysosomes under different amounts of H2O2 and chloral hydrate have been revealed. The sequential damage of lysosomes and mitochondria during cell apoptosis induced by rotenone, paclitaxel, and colchicine has been discovered. Furthermore, the regulation of mitochondria, lysosome, and their interplay during autophagy was also observed with the probe.

Using Cu2+ to regulate the emission feature of near-infrared fluorescent sensor with AIE: To detect ascorbic acid in food samples and its application in bioimaging.

Conventional ascorbic acid (AA) detection methods such as chromatography, capillary electrophoresis, colorimetry, electrochemical detection, and enzymatic analysis require expensive equipment and complicated operation. Simple, rapid, and accurate AA detection is essential to inspect food quality, diagnose diseases, and assess immunity in humans. In this study, the first near-infrared fluorescence sensor DBHM with aggregation-induced emission was developed to detect AA under the involvement of Cu2+. The DBHM + Cu2+ sensor showed high sensitivity to AA with a limit of detection of 2.37 µM. The AA detection mechanism was investigated by optical studies, 1H NMR titration, high-resolution mass spectrometry, and infrared spectroscopy. AA was detected qualitatively and quantitatively by the DBHM + Cu2+ sensor in beverages, fruits, and Vitamin C tablets using a dual-mode (fluorescence and smartphone app) sensing platform. The new sensing system also showed low toxicity and excellent bioimaging in HeLa cells, C. elegans, and mice. This sensor could advance AA detection technology in the food industry and has potential bioimaging applications.