Detecting and Imaging of γ‑Glutamytranspeptidase Activity in Serum, Live Cells, and Pathological Tissues with a High Signal-Stability Probe by Releasing a Precipitating Fluorochrome

γ-Glutamytranspeptidase (GGT) is a significant tumor-related biomarker that overexpresses in several tumor cells. Accurate detection and imaging of GGT activity in serum, live cells, and pathological tissues hold great significance for cancer diagnosis, treatment, and management. Recently developed small molecule fluorescent probes for GGT tend to diffuse to the whole cytoplasm and then translocate out of live cells after enzymatic reaction, which make them fail to provide high spatial resolution and long-term imaging in biological systems. To address these problems, a novel fluorescent probe (HPQ-PDG) which releases a precipitating fluorochrome upon the catalysis of GGT is designed and synthesized. HPQ-PDG is able to detect GGT activity with high spatial resolution and good signal-stability. The large Stokes shift of the probe enables it to detect the activity of GGT in serum samples with high sensitivity. To our delight, the probe is used for imaging GGT activity in live cells with the ability of discriminating cancer cells from normal cells. What’s more, we successfully apply it for pathological tissues imaging, with the results indicating that the potential application of HPQ-PDG in histopathological examination. All these results demonstrate the potential application of HPQ-PDG in the clinic

A Dual-Response Fluorescent Probe for the Detection of Viscosity and H₂S and Its Application in Studying Their Cross-Talk Influence in Mitochondria

Intracellular viscosity is an essential microenvironmental parameter and H₂S is a critical gaseous signaling molecule, which are both related to various physiological processes. It is reported that the change of viscosity and an imbalance of H₂S production in the mitochondria are both associated with overexpression of amyloid betapeptide (Aβ), which is thought to play a central role in the pathogenesis of Alzheimer’s disease (AD). However, to our best knowledge, no fluorescent probe is found for dual detection of mitochondrial viscosity and H₂S. Herein, a dual-response fluorescent probe (Mito-VS) is designed and synthesized to monitor the level of viscosity and H₂S, respectively. Mito-VS itself is nonfluorescent due to a free intramolecular rotation between dimethylaniline and pyridine. After the increase of viscosity, the rotation is prohibited and an intense red fluorescence is released. Upon the addition of H₂S, the probe can react with H₂S to form compound <b>3</b> and a strong green fluorescence can be observed. Moreover, the probe possesses a good mitochondrion-targeting ability and is applied for imaging the change of viscosity on the red channel and visualizing the variation of exogenous and endogenous H₂S concentration on the green channel in mitochondria. Most importantly, the probe is capable of studying the cross-talk influence of viscosity and H₂S in mitochondria, which is very beneficial for knowing the pathogenesis of AD

Efficient Two-Photon Fluorescent Probe for Nitroreductase Detection and Hypoxia Imaging in Tumor Cells and Tissues

Hypoxia plays an important role in tumor progression, and the development of efficient methods for monitoring hypoxic degree in living systems is of great biomedical importance. In the solid tumors, the nitroreductase level is directly corresponded with the hypoxic status. Many one-photon excited fluorescent probes have been developed for hypoxia imaging in tumor cells via the detection of nitroreductase level. However, two-photon excited probes are more suitable for bioimaging. In this work, a two-photon probe 1 for nitroreductase detection and hypoxic status monitoring in living tumor cells and tissues was reported for the first time. The detection is based on the fact that the nitro-group of probe 1 could be selectively reduced to an amino-group by nitroreductase in the presence of reduced NADH, following by a 1,6-rearrangement-elimination to release the fluorophore, resulting in the enhancement of fluorescence. The probe exhibited both one-photon and two-photon excited remarkable fluorescence enhancement (∼70-fold) for nitroreductase, which afforded a high sensitivity for nitroreductase, with a detection limit of 20 ng/mL observed. Moreover, the applications of the probe for fluorescent bioimaging of hypoxia in living cells and two-photon bioimaging in tissues were carried out, with tissue-imaging depths of 70–160 μm observed, which demonstrates its practical application in complex biosystems

A Bioluminescent Probe for Imaging Endogenous Peroxynitrite in Living Cells and Mice

Peroxynitrite (ONOO^–), an extremely reactive nitrogen species (RNS), is implicated in diverse pathophysiological conditions, including cancer, neurodegenerative diseases, and inflammation. Sensing and imaging of ONOO^– in living systems remains challenging due to the high autofluorescence and the limited light penetration depth. In this work, we developed a bioluminescent probe <b>BP-PN</b>, based on luciferase–luciferin pairs and the ONOO^–-responded group α-ketoamide, for highly sensitive detection and imaging of endogenous ONOO^– in living cells and mice for the first time. Attributed to the BL without external excitation, the probe <b>BP-PN</b> exhibits a high signal-to-noise ratio with relatively low autofluorescence. Furthermore, we examine the application of the probe <b>BP-PN</b> using the mice model of inflammation, and <b>BP-PN</b> shows high sensitivity for imaging endogenous ONOO^– in inflamed mice. This newly developed bioluminescent probe would be a potentially useful tool for in vivo imaging of ONOO^– in wider physiological and pathological processes

Efficient Two-Photon Fluorescent Probe for Glutathione S‑Transferase Detection and Imaging in Drug-Induced Liver Injury Sample

Drug-induced liver injury (DILI) is a potential complication of any prescribed medication. So far, the diagnosis of DILI is still a clinical challenge due to the lack of efficient diagnosis method. Glutathione S-transferase (GST), with a high concentration in liver cytosol, can reduce toxicity and facilitate urinary excretion by catalyzing the conjugation of glutathione (GSH) with reactive metabolites in liver. When liver is seriously damaged, GST and GSH will be released into plasma from liver cytosol, which caused a lower GST activity in liver cytosol. Therefore, monitoring the level of GST activity in liver tissue may be a potential strategy for diagnosis of DILI. Here, we reported a two-photon probe <b>P-GST</b> for GST activity detection for the first time. In the proposed design, a donor-π-acceptor (D-π-A) structured naphthalimide derivative with efficient two-photon properties was chosen as the fluorescent group, and a 2,4-dinitrobenzenesulfonate group was employed as the GST recognition unit, which also acted as the fluorescence quencher. In the present of GST and GSH, the recognition unit was removed and the fluorophore was released, causing a 40-fold enhancement of fluorescence signal with a detection limit of 35 ng/mL. At last, <b>P-GST</b> was successfully applied in two-photon imaging of GST in cells and DILI samples, which demonstrated its practical application in complex biosystems as a potential method for diagnosis of DILI

Visualization of Endoplasmic Reticulum Aminopeptidase 1 under Different Redox Conditions with a Two-Photon Fluorescent Probe

Endoplasmic reticulum aminopeptidase 1 (ERAP1), a metallopeptidase belonging to the M1 peptidase family, plays an important role in antigen processing in vivo. Additionally, many diseases are caused by ERAP1 perturbation. Thus, an efficient method for monitoring its content is extremely important for disease diagnosis and treatment. However, few fluorescent probes have been reported for efficiently monitoring ERAP1 in living cells and tissues. In this work, a two-photon fluorescent probe (<b>SNCL</b>) containing 1,8-naphthalimide (two-photon fluorophore), l-leucine (trigger moiety), and a methyl sulfonamide moiety (endoplasmic reticulum-targeting group) for imaging ERAP1 activity in living cells is reported for the first time. The optimized probe exhibited high sensitivity toward ERAP1, with about a 95-fold fluorescence enhancement at 550 nm. Herein, we monitored ERAP1 with <b>SNCL</b> by introducing interferon-γ to induce ERAP1 activity in living cells. The content of ERAP1 was dependent on the redox state of the endoplasmic reticulum, which was demonstrated by using <b>SNCL</b> to monitor the enzymatic activity of ERAP1 under different redox conditions. Excitingly, <b>SNCL</b> was also successfully applied for monitoring ERAP1 in tumor tissue with an imaging depth of 50–120 μm. In conclusion, <b>SNCL</b> not only can be used for the sensitive detection of endogenous ERAP1 in living cells and tumor tissues but also can serve as a potentially useful tool to reveal ERAP1-related diseases

Ratiometric Two-Photon Fluorescent Probe for in Vivo Hydrogen Polysulfides Detection and Imaging during Lipopolysaccharide-Induced Acute Organs Injury

Acute organ injury observed during sepsis, caused by an uncontrolled release of inflammatory mediators, such as lipopolysaccharide (LPS), is quite fatal. The development of efficient methods for early diagnosis of sepsis and LPS-induced acute organ injury in living systems is of great biomedical importance. In living systems, cystathionine γ-lyase (CSE) can be overexpressed due to LPS, and H₂S_<i>n</i> can be formed by CSE-mediated cysteine metabolism. Thus, acute organ injury during sepsis may be correlated with H₂S_<i>n</i> levels, making accurate detection of H₂S_<i>n</i> in living systems of great physiological and pathological significance. In this work, our previously reported fluorescent platform was employed to design and synthesize a FRET-based ratiometric two-photon (TP) fluorescent probe TPR-S, producing a large emission shift in the presence of H₂S_<i>n</i>. In this work, a naphthalene derivative two-photon fluorophore was chosen as the energy donor; a rhodol derivative fluorophore served as the acceptor. The 2-fluoro-5-nitrobenzoate group of probe TPR-S reacted with H₂S_<i>n</i> and was selectively removed to release the fluorophore, resulting in a fluorescent signal decrease at 448 nm and enhancement at 541 nm. The ratio value of the fluorescence intensity between 541 and 448 nm (<i>I</i>_541 nm/<i>I</i>_448 nm) varied from 0.13 to 8.12 (∼62-fold), with the H₂S_<i>n</i> concentration changing from 0 to 1 mM. The detection limit of the probe was 0.7 μM. Moreover, the probe was applied for imaging H₂S_<i>n</i> in living cells, tissues, and organs of LPS-induced acute organ injury, which demonstrated its practical application in complex biosystems as a potential method to achieve early diagnosis of LPS-induced acute organ injury