Selective turn-on fluorescent probes for imaging hydrogen sulfide in living cells

Hydrogen sulfide (H(2)S) is an important biological messenger but few biologically-compatible methods are available for its detection. Here we report two bright fluorescent probes that are selective for H(2)S over cysteine, glutathione and other reactive sulfur, nitrogen, and oxygen species. Both probes are demonstrated to detect H(2)S in live cells.     

Sulfonate-based fluorescent probes for imaging hydrogen peroxide in living cells

Based on the mechanism of H₂O₂-mediated hydrolysis of sulfonates, two fluorescein disulfonates compounds (FS-1 and FS-2) were designed and synthesized as the highly selective and sensitive fluorescent probes for imaging H₂O₂ in living cells. The probes were detected with elemental analysis, IR, ¹H NMR and ¹³C NMR. Upon reaction with H₂O₂, the probes exhibit strong fluorescence responses and high selectivity for H₂O₂ over other reactive oxygen species and some biological compounds. Furthermore, the sulfonate-based probes, as novel fluorescent reagents, are cell-permeable and can detect micromolar changes in H₂O₂ concentrations in living cells by using confocal microscopy. Supported by the National Basic Research Program of China (Grant No. 2007CB936000), the National Natural Science Funds for Distinguished Young Scholar (Grant No. 20725518), Major Program of the National Natural Science Foundation of China (Grant No. 90713019), the National Natural Science Foundation of China (Grant No. 20875057), the Natural Science Foundation of Shandong Province, China (Grant No. Y2007B02), and the Science and Technology Development Programs of Shandong Province, China (Grant No. 2008GG30003012)     

基于磺酸酯的荧光探针用于活细胞内过氧化氢的成像检测

基于过氧化氢（H2O2）特异性催化水解磺酸酯,设计合成了新型绿色荧光探针：荧光素二磺酸酯（FS—1）和二氯荧光素二磺酸酯（FS-2）两种螺环内酯型化合物,用于活细胞内过氧化氢的检测．探针结构由元素分析、IR、^1H NMR及^13C NMR表征．实验表明：探针FS-1和FS-2在模拟生物体系中检测过氧化氢具有良好的选择性和灵敏度,且线性范围较宽．细胞成像显示：探针FS-1和FS-2用于PMA刺激或外加不同浓度H2O2孵育的小鼠腹膜巨噬细胞均呈现明亮的绿色荧光,且能对细胞内H2O2微摩尔级浓度变化产生响应,证明两探针均具有良好的膜渗透性、高的选择性及良好的灵敏度．该方法的建立对研究生物体内H2O2的产生,H2O2导致的各种疾病机制及H2O2介导的信号转导途径具有重要的理论及实际意义．     

Reaction-based genetically encoded fluorescent hydrogen sulfide sensors

The detection of hydrogen sulfide (H(2)S), a toxic gas and an important biological signaling molecule, has been a long-time challenge. Here we report genetically encoded fluorescent protein (FP)-based probes that can selectively detect H(2)S. By expanding the genetic codes of E. coli and mammalian cells, FP chromophores were modified with the sulfide-reactive azide functional group. These structurally modified chromophores were selectively reduced by H(2)S, resulting in sensitive fluorescence enhancement detectable by spectroscopic and microscopic techniques. Exploration of a circularly permuted FP led to an improved sensor with faster responses, and the feasibility of using such a genetically encoded probe to monitor H(2)S in living mammalian cells has also been demonstrated.     

Reaction based fluorescent probes for hydrogen sulfide

A reaction based fluorescence turn-on strategy for hydrogen sulfide (H(2)S) was developed. This strategy was based on a H(2)S-specific Michael addition-cyclization sequence. Other biological thiols such as cysteine and glutathione did not pursue the reaction and therefore did not turn on the fluorescence/consume the substrates. The probes showed good selectivity and sensitivity for hydrogen sulfide.     

A retrievable and highly selective fluorescent probe for monitoring sulfide and imaging in living cells

A novel selective fluorescent chemosensor based on an 8-hydroxyquinoline-appended fluorescein derivative (L1) was synthesized and characterized. Once combined with Cu(2+), it displayed high specificity for sulfide anion. Among the various anions, only sulfide anion induced the revival of fluoresecence of L1, which was quenched by Cu(2+), resulting in "off-on"-type sensing of sulfide anion. What's more, the sensor was retrievable to indicate sulfide anions with Cu(2+), and S(2-), in turn, increased. With the addition of Cu(2+), compound L1 could give rise to a visible pink-to-yellow color change and green fluorescence quenching. The resulting yellow solution could change to pink and regenerate to green fluorescence immediately upon the addition of sulfide anion; however, no changes were observed in the presence of other anions, including CN(-), P(2)O(7)(4-), and other forms of sulfate, making compound L1 an extremely selective and efficient sulfide chemosensor. The signal transduction occurs via reversible formation-separation of complex L1Cu and CuS. What's more, the biological imaging study has demonstrated that the chemosensor can detect sulfur anions in biological systems at a relatively low concentration.     

Nile-red and Nile-blue-based near-infrared fluorescent probes for in-cellulo imaging of hydrogen sulfide

Hydrogen sulfide has recently been identified as a biologically responsive species. The design and synthesis of fluorescence probes, which are constructed with Nile-red or Nile-blue fluorophores and a fluorescence-controllable dinitrophenyl group, for hydrogen sulfide are reported in this paper. The Nile-red–dinitrophenyl-ether-group-based probe (1a) is essentially non-fluorescent because of the inhibition of the photo-induced electron-transfer process; when the dinitrobenzene moiety is removed by nucleophilic substitution with the hydrosulfide anion, probe 1a is converted into hydroxy Nile red, eliciting a H₂S-induced fluorescence turn-on signal. Furthermore, probe 1a has high selectivity and sensitivity for the hydrosulfide anion, and its potential for biological applications was confirmed by using it for real-time fluorescence imaging of hydrogen sulfide in live HeLa cells. The Nile-blue–dinitrobenzene-based probe (1b) has gradually diminishing brightness in the red-emission channel with increased hydrogen-sulfide concentration. Thus, this paper reports a comparative study of Nile-red and Nile-blue-based hydrogen-sulfide probes. Graphical Abstract

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A selective fluorescent probe for carbon monoxide imaging in living cells

A genetically encoded fluorescent probe is capable of selectively detecting carbon monoxide inside living cell. The probe, named COSer (CO sensor), consists of a circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the bacterial CO-sensing protein, CooA, which gives the probe its selective CO-binding property.     

A selective turn-on fluorescent sensor for imaging copper in living cells

We present the synthesis, properties, and biological applications of Coppersensor-1 (CS1), a new water-soluble, turn-on fluorescent sensor for intracellular imaging of copper in living biological samples. CS1 utilizes a BODIPY reporter and thioether-rich receptor to provide high selectivity and sensitivity for Cu+ over other biologically relevant metal ions, including Cu2+, in aqueous solution. This BODIPY-based probe is the first Cu+-responsive sensor with visible excitation and emission profiles and gives a 10-fold turn-on response for detecting this ion. Confocal microscopy experiments further establish that CS1 is membrane-permeable and can successfully monitor intracellular Cu+ levels within living cells.     

香豆素类荧光探针对硫化氢检测的研究进展

硫化氢(H2S)是目前人们发现的第三类生物内源性“气体信使分子”。其及时检测对人类的健康有着非常大的意义。随着荧光探针技术的发展,有机小分子荧光探针受到广大学者的关注。其中,香豆素因其结构简单,荧光量子产率高以及易于功能化而备受青睐。本文根据探针的识别机理综述近三年来报道的香豆素类H2S荧光探针代表性研究成果,并对其进行了展望,为后续设计开发更具实用价值的H2S荧光探针提供一点有益的参考。     

Boronate-based fluorescent probes for imaging cellular hydrogen peroxide

The syntheses, properties, and biological applications of the Peroxysensor family, a new class of fluorescent probes for hydrogen peroxide, are presented. These reagents utilize a boronate deprotection mechanism to provide high selectivity and optical dynamic range for detecting H2O2 in aqueous solution over similar reactive oxygen species (ROS) including superoxide, nitric oxide, tert-butyl hydroperoxide, hypochlorite, singlet oxygen, ozone, and hydroxyl radical. Peroxyresorufin-1 (PR1), Peroxyfluor-1 (PF1), and Peroxyxanthone-1 (PX1) are first-generation probes that respond to H2O2 by an increase in red, green, and blue fluorescence, respectively. The boronate dyes are cell-permeable and can detect micromolar changes in H2O2 concentrations in living cells, including hippocampal neurons, using confocal microscopy and two-photon microscopy. The unique combination of ROS selectivity, membrane permeability, and a range of available excitation/emission colors establishes the potential value of PR1, PF1, PX1, and related probes for interrogating the physiology and pathology of cellular H2O2.     

Fluorescein-derived fluorescent probe for cellular hydrogen sulfide imaging

In this work, a fluorescein-derived fluorescent probe for H2 S based on the thiolysis of dinitrophenyl ether is reported. This probe exhibits turn-on fluorescence imaging of H2 S in living cells and bulk solutions with excellent selectivity. The reaction mechanism was explained by means of absorption, fluorescence and HPLC–MS.     

Organic nanoparticles formed by aggregation-induced fluorescent molecules for detection of hydrogen sulfide in living cells

Hydrogen sulfide (H₂S) has been found to be the third most important endogenous gaseous signaling molecule after nitric oxide (NO) and carbonic oxide (CO) and plays crucial roles in living organisms and biological systems. Here we use aggregation- induced emission (AIE) of a small organic molecule (TPE-indo) to detect H₂S in both solution and living cells. TPE-indo can target mitochondria and aggregate to fluoresce, which can serve as a sensor for monitoring H₂S in the mitochondria. We regulate the fluorescence of AIE molecules by tuning the viscosity of the solution to form TPE-indo nanoparticles, constructing a probe for H₂S with good selectivity and high sensitivity. The nucleophilic addition of HS^- to the TPE-indo is crucial for the rapid H₂S detection. The imaging and analysis of H₂S in mitochondria of living cells with the probe demonstrate potential biological applications.     

A two-photon fluorescent probe with a large turn-on signal for imaging hydrogen sulfide in living tissues

A two-photon fluorescence turn-on H₂S probe GCTPOC–H₂S based on a two-photon platform with a large cross-section, GCTPOC, and a sensitive H₂S recognition site, dinitrophenyl ether was constructed. The probe GCTPOC–H₂S exhibits desirable properties such as high sensitivity, high selectivity, functioning well at physiological pH and low cytotoxicity. In particular, the probe shows a 120-fold enhancement in the presence of Na₂S (500 μM), which is larger than the reported two-photon fluorescent H₂S probes. The large fluorescence enhancement of the two-photon probe GCTPOC–H₂S renders it attractive for imaging H₂S in living tissues with deep tissue penetration. Significantly, we have demonstrated that the probe GCTPOC–H₂S is suitable for fluorescence imaging of H₂S in living tissues with deep penetration by using two-photon microscopy. The further application of the two-photon probe for the investigation of biological functions and pathological roles of H₂S in living systems is under progress.     

Highly selective two-photon fluorescent probe for imaging of nitric oxide in living cells简

A new two-photon fluorescent probe, ADNO, for nitric oxide （NO） based on intramolecular photoinduced electron transfer （PET） mechanism d/splays a rapid response to NO with a remarkable fluorescent enhancement in PBS buffer. The excellent chemoselectivity of ADNO for NO over other ROS/RNS （reactive oxygen species or nitrogen species） and common metal ions was observed. Moreover, ADNO has been successfully applied in fluorescence imaging of NO of living cells using both one-photon microscopy （OPM） and two-~hoton microscopy （TPM）,     

A tetraphenylimidazole-based fluorescent probe for the detection of hydrogen sulfide and its application in living cells

A novel probe based on the fluorescence off–on strategy was prepared to optically detect hydrogen sulfide (H₂S) via an excited state intramolecular proton transfer (ESIPT) mechanism. The probe shows high sensitivity and excellent selectivity to H₂S. It also displays a large Stokes shift (∼140 nm) and a remarkable quantum yield enhancement (Ф = 0.412) after interaction with H₂S. Moreover, the cellular imaging experiment demonstrated that it has potential utility for H₂S sensing in biological sciences.     

A targetable fluorescent probe for imaging hydrogen peroxide in the mitochondria of living cells

We present the design, synthesis, and biological applications of mitochondria peroxy yellow 1 (MitoPY1), a new type of bifunctional fluorescent probe for imaging hydrogen peroxide levels within the mitochondria of living cells. MitoPY1 combines a chemoselective boronate-based switch and a mitochondrial-targeting phosphonium moiety for detection of hydrogen peroxide localized to cellular mitochondria. Confocal microscopy and flow cytometry experiments in a variety of mammalian cell types show that MitoPY1 can visualize localized changes in mitochondrial hydrogen peroxide concentrations generated by situations of oxidative stress.     

A highly selective fluorescent chemosensor for zinc ion and imaging application in living cells

A new 2,6-bis(5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-4-methylphenol (1) serves as a highly selective and sensitive fluorescent probe for Zn(2+) in a HEPES buffer (50 mM, DMSO:water = 1:9 (v/v), pH = 7.2) at 25 °C. The increase in fluorescence in the presence of Zn(2+) is accounted for by the formation of dinuclear Zn(2+) complex [Zn(2)(C(35)H(25)N(6)O)(OH)(NO(3))(2)(H(2)O)] (2), characterized by X-ray crystallography. The fluorescence quantum yield of the chemosensor 1 is only 0.019, and it increases more than 12-fold (0.237) in the presence of 2 equiv of the zinc ion. Interestingly, the introduction of other metal ions causes the fluorescence intensity to be either unchanged or weakened. By incubation of cultured living cells (A375 and HT-29) with the chemosensor 1, intracellular Zn(2+) concentrations could be monitored through selective fluorescence chemosensing.