Hong-Wen Liu

Efficient Two-Photon Fluorescent Probe with Red Emission for Imaging of Thiophenols in Living Cells and Tissues

Thiophenols, a class of highly toxic and pollutant compounds, are widely used in industrial production. Some aliphatic thiols play important roles in living organisms. Therefore, the development of efficient methods to discriminate thiophenols from aliphatic thiols is of great importance. Although several one-photon fluorescent probes have been reported for thiophenols, two-photon fluorescent probes are more favorable for biological imaging due to its low background fluorescence, deep penetration depth, and so on. In this work, a two-photon fluorescent probe for thiophenols, termed NpRb1, has been developed for the first time by employing 2,4-dinitrobenzene-sulfonate (DNBS) as a recognition unit (also a fluorescence quencher) and a naphthalene-BODIPY-based through-bond energy transfer (TBET) cassette as a fluorescent reporter. The TBET system consists of a D-π-A structured two-photon naphthalene fluorophore and a red-emitting BODIPY. It displayed highly energy transfer efficiency (93.5%), large pseudo-Stokes shifts upon one-photon excitation, and red fluorescence emission (λem = 586 nm), which is highly desirable for bioimaging applications. The probe exhibited a 163-fold thiophenol-triggered two-photon excited fluorescence enhancement at 586 nm. It showed a high selectivity and excellent sensitivity to thiophenols, with a detection limit of 4.9 nM. Moreover, it was successfully applied for practical detection of thiophenol in water samples with a good recovery, two-photon imaging of thiophenol in living cells, and tissues with tissue-imaging depths of 90-220 μm, demonstrating its practical application in environmental samples and biological systems.

Rhodamine-based fluorescent probe for direct bio-imaging of lysosomal pH changes

Intracellular pH plays a pivotal role in various biological processes. In eukaryotic cells, lysosomes contain numerous enzymes and proteins exhibiting a variety of activities and functions at acidic pH (4.5-5.5), and abnormal variation in the lysosomal pH causes defects in lysosomal function. Thus, it is important to investigate lysosomal pH in living cells to understand its physiological and pathological processes. In this work, we designed a one-step synthesized rhodamine derivative (RM) with morpholine as a lysosomes tracker, to detect lysosomal pH changes with high sensitivity, high selectivity, high photostability and low cytotoxicity. The probe RM shows a 140-fold fluorescence enhancement over a pH range from 7.4 to 4.5 with a pKa value of 5.23. Importantly, RM can detect the chloroquine-induced lysosomal pH increase and monitor the dexamethasone-induced lysosomal pH changes during apoptosis in live cells. All these features demonstrate its value of practical application in biological systems.

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 highly sensitive and reductant-resistant fluorescent probe for nitroxyl in aqueous solution and serum

A novel coumarin-based fluorescent probe, P-CM, for quantitative detection of nitroxyl (HNO) was developed. P-CM exhibits a selective response to HNO over other biological reductants and was also applied for quantitative detection of HNO in bovine serum with satisfactory results.

An efficient two-photon fluorescent probe for monitoring mitochondrial singlet oxygen in tissues during photodynamic therapy

A promising two-photon fluorescent probe MNAH for detecting 1O2 during the PDT process in mitochondria was proposed for the first time. MNAH was successfully applied for two-photon imaging of 1O2 in living cells and tissues during the PDT process with deep-tissue imaging depth. MNAH can be a powerful molecular tool for studying 1O2 generation in mitochondria during the PDT process.

Molecular engineering of two-photon fluorescent probes for bioimaging applications

During the past two decades, two-photon microscopy (TPM), which utilizes two near-infrared photons as the excitation source, has emerged as a novel, attractive imaging tool for biological research. Compared with one-photon microscopy, TPM offers several advantages, such as lowering background fluorescence in living cells and tissues, reducing photodamage to biosamples, and a photobleaching phenomenon, offering better 3D spatial localization, and increasing penetration depth. Small-molecule-based two-photon fluorescent probes have been well developed for the detection and imaging of various analytes in biological systems. In this review, we will give a general introduction of molecular engineering of two-photon fluorescent probes based on different fluorescence response mechanisms for bioimaging applications during the past decade. Inspired by the desired advantages of small-molecule two-photon fluorescent probes in biological imaging applications, we expect that more attention will be devoted to the development of new two-photon fluorophores and applications of TPM in areas of bioanalysis and disease diagnosis.

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 (SNCL) 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 SNCL 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 SNCL to monitor the enzymatic activity of ERAP1 under different redox conditions. Excitingly, SNCL was also successfully applied for monitoring ERAP1 in tumor tissue with an imaging depth of 50-120 μm. In conclusion, SNCL 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.

A red emitting two-photon fluorescent probe for dynamic imaging of redox balance meditated by a superoxide anion and GSH in living cells and tissues

Cellular self-regulation of reactive oxygen species (ROS) stress via antioxidant repair plays an important role in maintaining the redox balance. The redox balance between reducing and oxidizing species within cells is significant in the regulation of a signal pathway and is achieved by a series of elaborate mechanisms. In this work, we employed our previously reported D-π-A-structured naphthalene-BODIPY TBET platform to design an efficient two-photon fluorescent probe for dynamic monitoring of superoxide anion oxidative stress and the GSH reducing repair process. The probe displayed high energy transfer efficiency (91.4%), large pseudo-Stokes shifts upon one-photon excitation, and red fluorescence emission (λem = 596 nm), which is highly desirable for bioimaging applications. The probe exhibits reversibility, rapid response, good photostability, high selectivity and sensitivity for the superoxide anion and GSH. More importantly, the probe was successfully applied for visualizing the redox changes in living cells and tissues.

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 P-GST 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, P-GST 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.

In vivo imaging of alkaline phosphatase in tumor-bearing mouse model by a promising near-infrared fluorescent probe

Alkaline phosphatase (ALP), one of the important hydrolases, is associated with the progress of many diseases as a well-defined biomarker. Fluorescence imaging of ALP in living organisms is of great importance for biological studies. However, in vivo detection of ALP remains a great challenge because current fluorescent probes show short excitation and emission wavelength, which are not desired for in vivo fluorescence imaging. Herein we reported a near-infrared (NIR) fluorescent probe (NALP) for turn-on trapping of ALP activity in living cancer cells and tumors. NALP was composed of a NIR-emitting fluorophore as a reporter and phosphate as a triggered moiety. Phosphate group was directly tethered to the hydroxyl group of fluorophore, which prohibited the fluorescence. The probe exhibited a high selectivity and remarkable fluorescence turn-on response to ALP in aqueous solutions with a detection limit of 0.28U/L. Benefiting from NIR excitation and emission, high contrast on the imaging signal could be achieved in response to endogenous ALP activity. Impressively, not only we successfully used NALP for imaging of endogenous ALP activity in cancer cells, but also, applied it for fluorescence imaging of ALP in tumor tissues and living tumor xenograft in nude mice for the first time. The probe was expected to be promising tool for practical application in disease diagnosis on the roles of ALP in disease.