Zuokai Wang

Highly Specific and Ultrasensitive Two-Photon Fluorescence Imaging of Native HOCl in Lysosomes and Tissues Based on Thiocarbamate Derivatives

Hypochlorous acid (HOCl) acts as a weak acid distributed mainly in acidic organelle lysosomes of phagocytes and plays a crucial role in the immune defense. The elaborate interrelation between the variations of HOCl levels in lysosomes and different physiological and pathological processes remains unclear. Thus, the accurate determination of lysosomal HOCl in living cells and in vivo is very important. Because of extremely low concentration and difficulty in distinguishing HOCl from OCl- under the physiological environment, it is still a great challenge to specifically monitor the intracellular intrinsic HOCl levels without exogenous stimulation, which impedes an exact understanding of its biological roles. In this paper, based on the electrophilic addition of Cl+ to sulfide moiety, we have developed a two-photon fluorescent probe O-(N-butyl-1,8-naphthalimide)-4-yl-N,N-dimethylthiocarbamate (NDMTC) for the specific determination of HOCl over OCl- and other bioactive molecules. Our results show that NDMTC possesses a detection limit of 7.6 pM, and it is the first fluorescent probe for detecting HOCl at the picomolar level. Furthermore, by introducing an alkylmorpholine group to the NDMTC framework, the lysosome-targetable derivative Lyso-NDMTC was obtained, and its ability to image HOCl in the lysosome organelles was clearly confirmed. Combined with two-photon fluorescence imaging of background suppression and deeper tissue penetration, NDMTC and Lyso-NDMTC were used to successfully visualize intracellular native HOCl and discern tumor tissue in mice. This study offers two perfect fluorescence imaging probes for further investigation of pathological roles of HOCl in various diseases.

A highly specific and ultrasensitive fluorescent probe for basal lysosomal HOCl detection based on chlorination induced by chlorinium ions (Cl

The development of techniques for detecting HOCl at the subcellular level is very important to elucidate its cellular functions. Due to its relatively low concentration, it is still a great challenge to specifically track the basal HOCl in normal cells. In this paper, based on the unique chlorination of HOCl by the initiation of chlorinium ions (Cl+) in an acidic medium, we have developed a simple pH-mediated lysosome-targetable fluorescent probe Lyso-HOCl for the specific detection of HOCl over other bioactive molecules at higher concentration (500 μM). Our results show that Lyso-HOCl possesses a detection limit of 8.0 pM, and can quantitatively detect HOCl at the picomolar level. The ultrasensitive and ultrafast response property of probe Lyso-HOCl offers a good opportunity to monitor the basal HOCl and the fluctuation of endogenous HOCl levels in the lysosomes of macrophages (Raw 264.7 cells), and we thus anticipate that this probe would provide a promising tool for further unraveling the biological functions of HOCl in subcellular lysosomes.

A highly specific and ultrasensitive near-infrared fluorescent probe for imaging basal hypochlorite in the mitochondria of living cells

The development of highly specific and ultrasensitive fluorescent probes for tracking basal mitochondrial hypochlorite is very important to unravel its diverse cellular functions in the mitochondria of living cells. In this paper, we have developed a water-soluble, mitochondria-targeted near-infrared fluorescent probe NB-OCl for selectively measuring OCl- in the presence of higher concentration (500 μM) other biologically important substances. Surprisingly, the obtained results demonstrated that probe NB-OCl could sensitively determine OCl- in the range of 0-200 pM with the detection limit of 10.8 pM. To the best of our knowledge, NB-OCl is the first fluorescent probe for the specific determination of OCl- at the picomolar level. Moreover, probe NB-OCl exhibits a fast response for OCl- (< 5 s), which would be in favor of tracking the highly reactive and short-lived OCl- in the living systems. The preeminent recognition properties of probe NB-OCl enable its applications in the monitoring of basal OCl- and the fluctuations of endogenous/exogenous OCl- levels in the mitochondria of living cells.

A highly selective ratiometric fluorescent probe for the sensitive detection of hypochlorous acid and its bioimaging applications

The development of simple techniques for monitoring hypochlorous acid (HOCl) in living cells is urgently needed to disclose the important roles of HOCl in biological systems. Although a number of fluorescent probes have been exploited to detect HOCl, most of them are easily influenced by other competing reactive oxygen species (ROS) and reactive nitrogen species (RNS), and disturbed by many factors including variabilities in excitation, probe distribution and environmental conditions due to the recognition of HOCl based on the fluorescence changes in single wavelength. Herein, in this work, we present a simple fluorescent probe AETU-HOCl containing a new recognition group (2-amino-ethyl)-thiourea (AETU) for the ratiometric detection of HOCl in living cells. Probe AETU-HOCl exhibits outstanding selectivity for HOCl over other ROS/RNS at higher concentrations (0.5 mM, 100 equiv.). More importantly, the rapid response (1 min) and high sensitivity (a detection limit of 3.9 nM) allow it to successfully visualize the intracellular HOCl levels by the ratiometric imaging. The present study would offer a simple, ratiometric, rapid, and ultrasensitive assay for the accurate determination of HOCl in the biological systems.

A highly selective and ultrasensitive ratiometric fluorescent probe for peroxynitrite and its two-photon bioimaging applications

In this work, taking full advantage of the intramolecular charge transfer (ICT) mechanism, a hydroxynaphthalimide-based ratiometric two-photon fluorescent probe RTP-PN was synthesized to detect ONOO-. Probe RTP-PN could accurately detect ONOO- in the range of 1.4 nM-1.4 μM with the detection limit of 1.4 nM by a ratiometric fluorescence spectroscopy method. Additionally, probe RTP-PN exhibited an ultrafast response for ONOO- than other various species including H2O2 and ClO-. Finally, probe RTP-PN was successfully adopted to detect intracellular ONOO- by the two-photon excitation microscopy.

A highly specific and sensitive ratiometric fluorescent probe for carbon monoxide and its bioimaging applications

The sensitive and accurate detection of intracellular gasotransmitter carbon monoxide (CO) is of great significance for unraveling its diverse cellular functions. Herein, we report a simple highly specific and sensitive 4-hydroxynaphthalimide-based ratiometric fluorescent probe Ratio-CO for monitoring CO levels in living cells. Probe Ratio-CO could quantitatively detect CO in the range of 0–50 μM by the ratiometric fluorescence spectroscopy method and the detection limit was measured to be about 17.9 nM. Additionally, probe Ratio-CO has been proven to possess preeminent selectivity towards CO over other bioactive species. Most importantly, the excellent response properties of probe Ratio-CO enable its applications in the monitoring of the fluctuations of CO levels in living cells by the ratiometric fluorescence imaging technique, and we thus anticipate that this probe would be a novel tool for further elucidating the biological functions of CO in living systems.