Fluorescent Probe for Investigating the Mitochondrial Viscosity and Hydrogen Peroxide Changes in Cerebral Ischemia/Reperfusion Injury.

Cerebral ischemia-reperfusion injury (CIRI), a cause of cerebral dysfunction during cerebral infarction treatment, is closely associated with mitochondrial viscosity and hydrogen peroxide (H2O2). However, the accurate measurement of mitochondrial viscosity and H2O2 levels in CIRI is challenging because of the lack of sufficient selectivity and blood-brain barrier (BBB) penetration of existing monitoring tools related to CIRI, hampering the exploration of the role of mitochondrial viscosity and H2O2 in CIRI. To address this issue, we designed an activatable fluorescent probe, mitochondria-targeting styryl-quinolin-ium (Mito-IQS), with excellent properties including high selectivity, mitochondrial targeting, and BBB penetration, for the visualization of mitochondrial viscosity and H2O2 in the brain. Based on the real-time monitoring capabilities of the probe, bursts of mitochondrial viscosity and H2O2 levels were visualized during CIRI. This probe can be used to monitor the therapeutic effects of butylphthalein treatment. More importantly, in vivo experiments further confirmed that CIRI was closely associated with the mitochondrial viscosity and H2O2 levels. This discovery provides new insights and tools for the study of CIRI and is expected to accelerate the process of CIRI diagnosis, treatment, and drug design.

A turn-on near-infrared fluorescent probe for visualization of endogenous alkaline phosphatase activity in living cells and zebrafish.

Alkaline phosphatase (ALP) is an essential hydrolase and widely distributed in living organisms. It plays important roles in various physiological and pathological processes. Herein, a turn-on near-infrared (NIR) fluorescent probe (DXMP) was developed for sensitive detection of ALP activity both in vitro and in vivo based on the intramolecular charge transfer (ICT) mechanism. Upon incubation with ALP, DXMP exhibited a strong fluorescence increment at 640 nm, which was attributed to the fact that ALP-catalyzed cleavage of the phosphate group in DXMP induced the transformation of DXMP into DXM-OH. The probe exhibited prominent features including outstanding selectivity, high sensitivity, and excellent biocompatibility. More importantly, it has been successfully used to detect and image endogenous ALP in living cells and zebrafish.

A near-infrared excitation/emission fluorescent probe for imaging of endogenous cysteine in living cells and zebrafish.

The fluorescence imaging technique provides an essential tool for studying biological systems. However, due to the interference of autofluorescence of biological tissues, the application of short-wavelength fluorescent probes in biological imaging was limited. The near-infrared (NIR) excitation/emission fluorescent probe possesses unique advantages in optical imaging in vivo, including less light scattering, minimal photo-damage to biological samples, deep tissue penetration, and weak autofluorescence interference from complicated biological systems. In this work, a convenient fluorophore (E)-2-[2-(6-hydroxy-2,3-dihydro-1H-xanthen-4-yl)vinyl]-3- methylbenzo[d]thiazol-3-ium iodide (DXM-OH) with NIR excitation and emission was rationally designed and developed. What's more, DXM-OH was applied to construct an "OFF-ON" fluorescent probe (E)-2-{2-[6-(acryloyloxy)-2,3-dihydro-1H- xanthen-4-yl]vinyl}-3-methylbenzo[d]thiazol-3-ium iodide (DXM) for sensitive and selective detection of cysteine (Cys). The experimental results showed that DXM had the advantages of good cell permeability, low toxicity, and excellent optical properties (NIR excitation/emission) and it was successfully applied to image Cys of living cells and zebrafish.

Hydrogen peroxide sensing in body fluids and tumor cells via in situ produced redox couples on two-dimensional holey CuCo2O4 nanosheets.

A novel nanomaterial of two-dimensional holey CuCo2O4 (2D HCCO) nanosheets was synthesized via a general template-directed method and employed for the first time to construct an effective electrochemical platform for H2O2 sensing with the combination of cerium oxide (CeO2). During the electrocatalytic reduction of H2O2, the synergetic catalysis of CeO2/HCCO/MWCNTs/GCE owing to the naturally holey frameworks and the mediator of CeO2 results in the ultra-sensitive detection of H2O2. The current was greatly enhanced owing to the unique holey structure that can minimize the charge transfer distance and provide more active sites to boost the signals, and the dual oxidation state of Ce3+/Ce4+ on the surface of 2D HCCO nanosheets can promote the in situ production of Cu2+/Cu+ and Cu+/Cu and further amplify the detection signal. The CeO2/HCCO/MWCNTs/GCE showed a wide linear range from 1 µM to 7.31 mM using chronoamperometry at the potential of - 0.25 V and a relatively low detection limit of 0.16 µM in physiological environment, which was also utilized for tracking the trace H2O2 released from Hela cells. This study shows great promise for the emerging application of holey HCCO-based biosensors in bioanalysis and early cancer diagnosis. Graphical abstract.

A highly sensitive naked-eye fluorescent probe for trace hydrazine based on 'C-CN' bond cleavage.

In this work, a colorimetric and ratiometric fluorescent probe, 2,3-bis(((E)-4-(diethylamino)-2-hydroxybenzylidene)amino)maleonitrile (DHM), was developed based on N2H4-induced bond cleavage. Two cyanide groups of DHM were applied to sense hydrazine (N2H4) for the first time, which was demonstrated using 1H NMR and HRMS spectra. The specific 'C-CN' bond cleavage induced by N2H4 showed an excellent fluorescence ratiometric behavior and a visible colorimetric change from purple to yellow. Meanwhile, it showed a low detection limit of 58.1 nM (1.65 ppb), which is far below the safety level (10 ppb) set by the U.S. EPA. Moreover, the probe was successfully employed for the quantitative detection toward N2H4 in real water samples.

Dual-site lysosome-targeted fluorescent sensor for fast distinguishing visualization of HClO and ONOO- in living cells and zebrafish.

As two of important highly reactive species / nitrogen species, hypochloric acid (HClO) and peroxynitrite (ONOO-) are involved in various pathological and physiological processes, which are important factors that affect and reflect the functional state of lysosome. Nevertheless, many of their roles are still indefinite because of lack of suitable analytical methods for HClO and ONOO- detection in lysosome. Herein, we designed a lysosome-targeted probe to monitor HClO and ONOO-, which was a hydrid of the benzothiazole derivative, methyl thioether (HClO recognition site) and morpholino hydrazone (ONOO- recognition and lysosome target site). The probe exhibited high sensitivity, good selectivity and fast response toward HClO and ONOO- without spectral crosstalk, and can be employed for quantitative monitoring HClO and ONOO- with LOD of 63 and 83 nM, respectively. In addition, the dual-site probe was lysosome targetable and could be used for detection of HClO and ONOO- in living cells. Furthermore, the excellent behavior made it was suitable for imaging of HClO and ONOO- in zebrafish. Thus, the present probe provides a potent tool for distinguishing monitoring HClO and ONOO- and exploring the role of HClO and ONOO- in biological systems.

Visualization of cysteine in AD mouse with a high-quantum yield NIR fluorescent probe.

Alzheimer's disease (AD) has gradually received enthusiastic attention with the aging process, and studying its biological relevance is expected. Excitingly, fluorescence probes were considered to be powerful tools for exploring biological correlations. Therefore, a highly selective near-infrared (NIR) fluorescent probe (DCM-Cl-Acr) for imaging cysteine (Cys) in AD was designed and synthesized. Through structural optimization, the probe exhibited high fluorescence quantum yield and low detection limit (20 nM) towards Cys. Meanwhile, based on the high selectivity and high sensitivity response exhibited by the probe to Cys, it was successfully applied to visualize endogenous and exogenous Cys in living cells and zebrafish, and showed good discrimination from homocysteine (Hcy) and glutathione (GSH). Further, the correlation between AD and Cys concentration was clarified by imaging studies in hippocampus tissue of AD mouse, and the abnormal accumulation of Cys in the hippocampus of AD brain was demonstrated.

An activatable fluorescence probe for rapid detection and in situ imaging of ß-galactosidase activity in cabbage roots under heavy metal stress.

ß-Galactosidase (ß-gal), an enzyme related to cell wall degradation, plays an important role in regulating cell wall metabolism and reconstruction. However, activatable fluorescence probes for the detection and imaging of ß-gal fluctuations in plants have been less exploited. Herein, we report an activatable fluorescent probe based on intramolecular charge transfer (ICT), benzothiazole coumarin-bearing ß-galactoside (BC-ßgal), to achieve distinct in situ imaging of ß-gal in plant cells. It exhibits high sensitivity and selectivity to ß-gal with a fast response (8 min). BC-ßgal can be used to efficiently detect the alternations of intracellular ß-gal levels in cabbage root cells with considerable imaging integrity and imaging contrast. Significantly, BC-ßgal can assess ß-gal activity in cabbage roots under heavy metal stress (Cd2+, Cu2+, and Pb2+), revealing that ß-gal activity is negatively correlated with the severity of heavy metal stress. Our work thus facilitates the study of ß-gal biological mechanisms.

A mitochondrion targetable dimethylphosphorothionate-based far-red and colorimetric fluorescent probe with large Stokes shift for monitoring peroxynitrite in living cells.

Peroxynitrite (ONOO-) is a biological oxidant that is related to numerous physiological and pathological processes. An overdose of ONOO- is the cause of various serious diseases. Some evidence demonstrates that mitochondria are the major sites of ONOO- production. Therefore, monitoring mitochondrial ONOO- is important to understand the related pathological processes in living systems. Herein, a colorimetric and far-red fluorescent sensing probe (PCPA) for the determination of ONOO- was constructed based on a dicyanoisophorone skeleton using dimethylphosphorothionate as the recognition group and pyridine salt as the mitochondrion-targeting unit. PCPA showed a far-red fluorescence response to ONOO- accompanied by a distinct color change from colorless to yellow via the ONOO- induced deprotection of dimethylphosphorothionate. In addition, PCPA exhibited a large Stokes shift (200 nm), high selectivity detection and high sensibility (LOD = 39 nM). Furthermore, PCPA was successfully employed for imaging ONOO- and tracing ONOO- in mitochondria. PCPA presents a new recognition group and has potential applications in the biology field.

A turn-on NIR fluorescent probe for risk-assessing oxidative stress in cabbage roots under abiotic stress.

Oxidative stress is closely related to the crop health status under stress conditions. H2O2 is an important signaling molecule in plants under stress. Therefore, monitoring H2O2 fluctuations is of great significance when risk-assessing oxidative stress. However, few fluorescent probes have been reported for the in situ tracking of H2O2 fluctuations in crops. Herein, we designed a "turn-on" NIR fluorescent probe (DRP-B) to detect and in situ-image H2O2 in living cells and crops. DRP-B exhibited good detection performance for H2O2 and could image endogenous H2O2 in living cells. More importantly, it could semi-quantitatively visualize H2O2 in cabbage roots under abiotic stress. Visualization of H2O2 in cabbage roots revealed H2O2 upregulation in response to adverse environments (metals, flood, and drought). This study provides a new method for risk-assessing oxidative stress in plants under abiotic stress and is expected to provide guidance for the development of new antioxidant defense strategies to enhance plant resistance and crop productivity.

Mitochondria-targeting fluorescent sensor with high photostability and permeability for visualizing viscosity in mitochondrial malfunction, inflammation, and AD models.

Viscosity changes in mitochondria are closely associated with numerous cellular processes and diseases. Currently available fluorescence probes used in mitochondrial viscosity imaging are not very photostable or sufficiently permeable. Herein, a highly photostable and permeable mitochondria-targeting red fluorescent probe (Mito-DDP) was designed and synthesized for viscosity sensing. Viscosity was imaged in living cells using a confocal laser scanning microscope, and the results suggested that Mito-DDP penetrated the membrane and stained the living cells. Importantly, practical applications of Mito-DDP were demonstrated: viscosity visualization was realized for mitochondrial malfunction, cellular and zebrafish inflammation, and Drosophila Alzheimer's disease models, i.e., for subcellular organelles, cells, and organisms. The excellent analytical and bioimaging performance of Mito-DDP in vivo makes it an effective tool for exploring the physiological and pathological effects of viscosity.

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.

An N-nitrosation reaction-based fluorescent probe for detecting nitric oxide in living cells and inflammatory zebrafish.

Nitric oxide (NO), an essential biological messenger molecule, participates in various physiological and pathological processes. The sensitive and specific detection of NO is of great significance for understanding the biological function of NO. Here, we synthesized a fluorescent probe (Rho-NO) for highly selective detection of NO both in vitro and in vivo. The high selectivity of Rho-NO is attributed to the fact that NO is easily replaced by electron donor amino group to form N-nitrosation products, causing rhodamine spiro ring open and fluorescence emit. Rho-NO showed a good linear response to NO (0-100 µM) with a low detection limit (0.06 µM). Importantly, it exhibited excellent specificity for NO detection in human serum and was also applied for imaging NO in living cells and inflammatory model of zebrafish. This work proves the potential of Rho-NO in pathological research and disease diagnosis.

Simultaneous visualization and quantification of copper (II) ions in Alzheimer's disease by a near-infrared fluorescence probe.

The abnormal accumulation of copper ions (Cu2+) is considered to be one of the pathological factors of Alzheimer's disease (AD), but the internal relationship between Cu2+ and AD progression is still not fully clear. In this work, a sensitive and selective near-infrared fluorescent copper ion probe (DDP-Cu) was designed for quantification and visualization of Cu2+ level in lysates, living cells, living zebrafish and brain tissues of drosophila and mice with AD. By using this probe, we demonstrated that the content of Cu2+ in the brains of AD mice and drosophila enhanced nearly 3.5-fold and 4-fold than that of normal mice and drosophila, respectively. More importantly, pathogenesis analysis revealed that elevated Cu2+ led to changes in factors closely associated with AD, such as the increasing of reactive oxygen species(ROS), the aggregation of amyloid-ß protein (Aß) and nerve cell cytotoxicity. These findings could promote the understanding of the roles between Cu2+ and AD.

A turn-on red-emitting fluorescent probe for determination of copper(II) ions in food samples and living zebrafish.

Herein, we developed a turn-on red-emitting fluorescent probe for the sensitive and selective detection of copper ions (Cu2+) in food samples and living zebrafish. The probe employs a hemicyanine scaffold as the fluorophore and a 2-pyridinecarbonyl group as the recognition receptor and quenching moiety. The 2-pyridinecarbonyl moiety can be specifically cleaved by Cu2+ and results in an approximately 18-fold fluorescence enhancement of the probe, thereby providing a fluorescence turn-on assay for Cu2+. Additionally, the probe exhibited excellent selectivity, high sensitivity, a broad linear relationship (0.020 to 8.0 µM), and a low limit of detection (4.0 nM, S/N = 3) for Cu2+. Concomitantly, the probe exhibited satisfactory analytical performance when used with actual food samples. Moreover, the probe could be used for in situ determination of Cu2+ in both living plant tissues and in living zebrafish.

A colorimetric and near-infrared ratiometric fluorescent probe for hydrazine detection and bioimaging.

Hydrazine (N2H4) is extensively used in industry but highly toxic; hence, highly sensitive detection of N2H4 is extremely meaningful. Herein, a colorimetric and near-infrared (NIR) ratiometric fluorescent probe named DXM-OH was rationally designed and synthesized based on oxanthrene malononitrile derivative for the specific detection of N2H4. The dicyanovinyl group in DXM-OH was served as the recognition unit for N2H4. DXM-OH showed high sensitivity to N2H4 in the range of 1-900 µM, with the limit of detection (LOD) of 0.09 µM (2.87 ppb), which is much lower than the U.S. Environmental Protection Agency standard (10 ppb). Furthermore, the practical applications of DXM-OH in detecting N2H4 in real water samples and imaging of N2H4 in living cells were demonstrated, indicating its potential utility for N2H4 sensing in environmental and biological samples.

Visualization of endogenous ß-galactosidase activity in living cells and zebrafish with a turn-on near-infrared fluorescent probe.

ß-Galactosidase (ß-gal) is an important biomarker for primary ovarian cancers. Developing noninvasive bioimaging probes for studying the activity of ß-gal is highly desirable for cancer diagnosis. Herein, a turn-on near-infrared (NIR) fluorescent probe， 2-((6-(((2S, 3R, 4S, 5R, 6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran -2-yl)oxy)-2,3-dihydro-1H-xanthen-4-yl)methylene)malononitrile named DXM-ßgal, was rationally designed based on enzymatic reaction for the detection of ß-gal activity both in vitro and in vivo. Upon incubating with ß-gal, DXM-ßgal displayed a significant fluorescence enhancement at 640 nm, accompanying by a color change of solution color from red to purple. DXM-ßgal exhibited high selectivity and sensitively to ß-gal with low limit of detection (2.92 × 10-4 U mL-1). Besides, based on its advantages of long-wavelength emission and excellent biocompatibility, DXM-ßgal was successfully applied to imaging ß-gal in living cells and zebrafish. Given these prominent properties, we believe that DXM-ßgal will be a potential tool for investigating ß-gal activity in biomedical research.

A near-infrared fluorescent probe for monitoring and imaging of ß-galactosidase in living cells.

ß-Galactosidase (ß-gal) which is overexpressed in primary ovarian cancer can be employed as a valuable biomarker for ovarian cancer. Thus, monitoring and imaging endogenous ß-gal in living cells is of great importance. Herein, a dicyanoisophorone-based near-infrared (NIR) fluorescent probe 2-(5,5-dimethyl-3-((E)-4-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)styryl)cyclohex-2-en-1-ylidene)malononitrile named DP-ßgal, was rationally designed and synthesized for the monitoring of ß-gal activity in living cells. In the presence of ß-gal, with the breaking of the glycosidic bond, the NIR fluorescence of the dicyanoisophorone derivative gradually recovered, enabling the fluorescence "off-on" quantitative determination of ß-gal activity. DP-ßgal has the advantages of good selectivity and high sensitivity for the detection of ß-gal, with the limit of detection (LOD) of 3.2 × 10-3 U. Furthermore, based on its advantages of long-wavelength emission and excellent biocompatibility, the practical applications of DP-ßgal in NIR imaging of ß-gal in living ovarian cancer cells (SKOV-3) were demonstrated.

Molecular structure regulation and enzyme cascade signal amplification strategy for upconversion ratiometric luminescent and colorimetric alkaline phosphatase detection.

Herein, molecular structure regulation and enzyme cascade signals amplification (MRECAmp) strategy was proposed to construct a novel upconversion ratiometric fluorescence and colorimetric dual-readout assay platform for highly sensitive and selective detection of alkaline phosphatase (ALP) activity. The detection strategy is divided into three aspects. Firstly, Ag+ oxidates o-phenylenediamine (OPD) to 2,3-diaminophennazine (OPDox), which can significantly quench upconversion fluorescence at 487 nm based on inner filter effects (IFE) while the upconversion emission at 668 nm was essentially unchanged for Zn2+-doped NaYF4:Yb3+,Er3+,Tm3+ upconversion nanoparticles (UCNPs) probes under the near-infrared (NIR) irradiation. Secondly, ALP catalyzes the hydrolysis of the substrate l-ascorbic acid 2-phosphate (AAP) to ascorbic acid (AA) which can consume part of Ag+ to inhibit the generation of OPDox and the AA was oxidized to dehydroascorbic acid (DHAA). The specific performance of DHAA molecular structure regulation, due to the condensation reaction between dicarbonyl group of DHAA and diamine group of OPD, that is confirmed by ESI-MS and 1H NMR spectra analysis, can remarkably enhance the selectivity of ALP detection, which is conceptually different from the previously reported ALP fluorescent assays. Thirdly, the generated DHAA reacts with OPD to form 3-(dihydroxyethyl)furo [3,4-b]quinoxaline-1-one (DFQ) that further inhibits the generation of OPDox, which makes the enzyme-controlled cascade signal amplification (ECAmp) can be realized with a great fluorescence recovery (60.04%) at 487 nm. Therefore, the fluorescence response signal ((I487/I668)0/(I487/I668)) is amplified by ECAmp strategy, allowing the quantitative analysis of ALP with a detection limit of 0.03 mU/mL. The detection of ALP activity in human serum samples over the conventional methods demonstrates that the MRECAmp method provides a sensing platform for probing ALP activity and shows promising outlook in biomedical studies.

Aggregation-induced emission fluorescent probe for monitoring endogenous alkaline phosphatase in living cells.

Alkaline phosphatase (ALP) is a non-specific phosphate monoesterase and often regarded as an important biomarker of hypothyroidism and hepatobiliary diseases in medical diagnosis. In-situ detection of endogenous ALP and exploration of the distribution of ALP in cells are of great importance for the diagnosis of diseases associated with ALP. In this work, we designed and synthesized an aggregation-induced emission (AIE) fluorescent probe, (E)-2-(((9H-fluoren-9-ylidene) hydrazono)methyl)phenyl dihydrogen phosphate (FAS-P), that can respond to ALP with a remarkable large Stokes shift (>200 nm) based on excited state intramolecular proton transfer (ESIPT) mechanism. The probe FAS-P has high selectivity and sensitivity to the detection of ALP. And there is a linear relationship between the fluorescence intensity of FAS-P and ALP activity in the range of 1-100 U L-1, the limit of detection (LOD) is as low as 0.6 U L-1. More importantly, we successfully applied FAS-P to detect ALP in living cells and the monitoring of ALP in real time.