Huatang Zhang

A FRET-based Ratiometric Fluorescent Probe for Nitroxyl Detection in Living Cells

HNO has recently been found to possess distinct biological functions from NO. Studying the biological functions of HNO calls for the development of sensitive and selective fluorescent probes. Herein, we designed and synthesized a FRET-based ratiometric probe to detect HNO in living cells. Our studies revealed that the probe is capable of detecting HNO in a rapid and ratiometric manner under physiological conditions. In bioimaging studies, the probe displayed a clear color change from blue to green when treated with HNO.

A reaction-based near-infrared fluorescent sensor for Cu2+ detection in aqueous buffer and its application in living cells and tissues imaging

Copper (II) is one of the most of important cofactors for numerous enzymes and has captured broad attention due to its role as a neurotransmitters for physiological and pathological functions. In this article, we present a reaction-based fluorescent sensor for Cu2+ detection (NIR-Cu) with near-infrared excitation and emission, including probe design, structure characterization, optical property test and biological imaging application. NIR-Cu is equipped with a functional group, 2-picolinic ester, which hydrolyzes in the presence of Cu2+ with high selectivity over completed cations. With the experimental conditions optimized, NIR-Cu (5μM) exhibits linear response for Cu2+ range from 0.1 to 5μM, with a detection limit of 29nM. NIR-Cu also shows excellent water solubility and are highly responsive, both desirable properties for Cu2+ detection in water samples. In addition, due to its near-infrared excitation and emission properties, NIR-Cu demonstrates outstanding fluorescent imaging in living cells and tissues.

An iminocoumarin benzothiazole-based fluorescent probe for imaging hydrogen sulfide in living cells.

Hydrogen sulfide (H2S) has recently been identified as the third gaseous signaling molecule that is involved in regulating many important cellular processes. We report herein a novel fluorescent probe for detecting H2S based on iminocoumarin benzothiazole scaffold. The probe displayed high sensitivity and around 80-fold increment in fluorescence signal after reacting with H2S under physiological condition. The fluorescent intensity of the probe was linearly related to H2S concentration in the range of 0-100 μM with a detection limit of 0.15 μM (3σ/slope). The probe also showed excellent selectivity towards H2S over other biologically relevant species, including ROS, RSS and RNS. Its selectivity for H2S is 32 folds higher than other reactive sulfur species. Furthermore, the probe has been applied for imaging H2S in living cells. Cell imaging experiments demonstrated that the probe is cell-permeable and can be used to monitor the alteration of H2S concentrations in living cells. We envisage that this probe can provide useful tools to further elucidate the biological roles of H2S.

Fluorescent probes for detecting monoamine oxidase activity and cell imaging

A series of new fluorogenic probes for monoamine oxidases (MAOs) were reported based on an oxidation and β-elimination mechanism. The limits of detection of the probes for MAO-A and -B were determined to be 3.5 and 6.0 μg mL(-1) respectively. These probes displayed strong activity towards MAOs, especially MAO-B. Cellular imaging studies were also successfully conducted with MCF-7 cells.

Reaction-Based Off-On Near-infrared Fluorescent Probe for Imaging Alkaline Phosphatase Activity in Living Cells and Mice

Alkaline phosphatases are a group of enzymes that play important roles in regulating diverse cellular functions and disease pathogenesis. Hence, developing fluorescent probes for in vivo detection of alkaline phosphatase activity is highly desirable for studying the dynamic phosphorylation in living organisms. Here, we developed the very first reaction-based near-infrared (NIR) probe (DHXP) for sensitive detection of alkaline phosphatase activity both in vitro and in vivo. Our studies demonstrated that the probe displayed an up to 66-fold fluorescence increment upon incubation with alkaline phosphatases, and the detection limit of our probe was determined to be 0.07 U/L, which is lower than that of most of alkaline phosphatase probes reported in literature. Furthermore, we demonstrated that the probe can be applied to detecting alkaline phosphatase activity in cells and mice. In addition, our probe possesses excellent biocompatibility and rapid cell-internalization ability. In light of these prominent properties, we envision that DHXP will add useful tools for investigating alkaline phosphatase activity in biomedical research.

A thiol fluorescent probe reveals the intricate modulation of cysteine's reactivity by Cu(II).

In this study, Cu(II)-mediated differential alteration of cysteine (Cys) reactivity is reported by using a Cys-specific fluorescent probe. The probe could react with Cys to give out strong fluorescence. When Cys was preincubated with Cu(II), the fluorescence of the probe was decreased due to the inhibition of Cyss reactivity by Cu(II). Remarkably, experimental results reveal that the probe could detect Cu(II) at subnanomolar concentrations. In contrast, Cu(II) could only partially inhibit the reaction between Cys and Ellmans reagent (DTNB). Furthermore, selectivity experiments show that Cu(II) is a much more potent inhibitor for Cys compared to other metal ions. Cell imaging experiments also confirm the inhibitory effects of Cu(II) on Cyss reactivity in living cells. We envision that the probe could add a useful tool for sensitive and selective detection of Cu(II) for biomedical research.

Design and Synthesis of Near-infrared Fluorescent Probes for Imaging of Biological Nitroxyl.

Nitroxyl (HNO), the reduced and protonated form of nitric oxide (NO), has recently been identified as an interesting and important signaling molecule in biological systems. However, research on its biosynthesis and bioactivities are hampered by the lack of versatile HNO detection methods applicable to living cells. In this report, two new near-infrared (NIR) probes were designed and synthesized for HNO imaging in living cells. One of the probes was found to display high sensitivity towards HNO, with up to 67-fold of fluorescence increment after reaction with HNO. The detection limit was determined to be as low as 0.043 μM. The probe displayed high selectivity towards HNO over other biologically related species including metal ions, reactive oxygen species, reactive nitrogen species and reactive sulfur species. Furthermore, the probe was shown to be suitable for imaging of exogenous and endogenous HNO in living cells. Interestingly, the probe was found to be mainly localized in lysosomes. We envision that the new NIR probe described here will serve as a useful tool for further elucidation of the intricate roles of HNO in living cells.

A general colorimetric method for detecting protease activity based on peptide-induced gold nanoparticle aggregation

Herein we describe a modularly designed colorimetric approach for facile and sensitive detection of proteases. The method employs non-crosslinking AuNP aggregation induced by peptide treated with specific protease. A visible colour change of the AuNPs solution from wine-red to violet-blue is readily observed when the protease-digested peptide is added. This method allows facile and visualized assay of the enzyme activity without any sophisticated instrument, affording both convenience and simplicity. The platform can be readily extended to detect virtually any protease.

Construction of an alkaline phosphatase-specific two-photon probe and its imaging application in living cells and tissues

Alkaline phosphatase (ALP) is a family of enzymes involved in the regulation of important biological processes such as cell differentiation and bone mineralization. Monitoring the activity of ALP in serum can help diagnose a variety of diseases including bone and liver diseases. There has been growing interest in developing new chemical tools for monitoring ALP activity in living systems. Such tools will help further delineate the roles of ALP in biological and pathological processes. Previously reported fluorescent probes has a number of disadvantages that limit their application, such as poor selectivity and short-wavelength excitation. In this work, we report a new two-photon fluorescent probe (TP-Phos) to selectively detect ALP activity. The probe is composed of a two-photon fluorophore, a phosphate recognition moiety, and a self-cleavable adaptor. It offers a number of advantages over previously reported probes, such as fast reaction kinetics, high sensitivity and low cytotoxicity. Experimental results also showed that TP-Phos displayed improved selectivity over DIFMUP, a commonly utilized ALP probe. The selectivity is attributed to the utilization of an ortho-functionalised phenyl phosphate group, which increases the steric hindrance of the probe and the active site of phosphatases. Moreover, the two-photon nature of the probe confers enhanced imaging properties such as increased penetration depth and lower tissue autofluorescence. TP-Phos was successfully used to image the endogenous ALP activity of hippocampus, kidney and liver tissues from rat.