Development of an oxidative dehydrogenation-based fluorescent probe for Cu and its biological imaging in living cells

Based on a boron dipyrromethene (BODIPY) derivative containing an N, O and S tridentate ligand, a Cu²⁺ fluorescent probe BTCu was developed. The detection mechanism was verified as Cu²⁺-promoted oxidative dehydrogenation of an amine moiety, leading to a formation of a fluorescent Cu⁺-Schiff base complex. Free BTCu exhibited a maximum absorption wavelength at 496 nm, and a very weak maximum emission at 511 nm. Upon addition of various metals ions, it showed large fluorescence enhancement toward Cu²⁺ (417-fold in MeCN and 103-fold in MeCN/HEPES solution, respectively) with high selectivity. The detection limits are as low as 1.74 × 10⁻⁸ M and 4.96 × 10⁻⁸ M in the two different solutions, respectively. And BTCu could work in a wide pH range with an extraordinary low pK_a of 1.21 ± 0.06. Using fluorescence microscopy, the probe was shown to be capable of penetrating into living cells and imaging intracellular Cu²⁺ changes.     

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．     

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)     

Balance between fluorescence enhancement and association affinity in fluorescent heteroditopic indicators for imaging zinc ion in living cells

A fluorescent heteroditopic indicator for the zinc(II) ion possesses two different zinc(II) binding sites. The sequential coordination of zinc(II) at the two sites can be transmitted into distinct fluorescence changes. In the heteroditopic ligand system that our group developed, the formations of mono- and dizinc(II) complexes along an increasing gradient of zinc(II) concentration lead to fluorescence enhancement and an emission bathochromic shift, respectively. The extents of these two changes determine the sensitivity and, ultimately, the effectiveness of the heteroditopic indicator in quantifying zinc(II) ion over a large concentration range. In this work, a strategy to increase the degree of fluorescence enhancement upon the formation of the monozinc(II) complex of a heteroditopic ligand under simulated physiological conditions is demonstrated. Fluorination of the pyridyl groups in the pentadentate N,N,N'-tris(pyridylmethyl)ethyleneamino group reduces the apparent pK(a) value of the high-affinity site, which increases the degree of fluorescence enhancement as the monozinc(II) complex is forming. However, fluorination impairs the coordination strength of the high-affinity zinc(II) binding site, which in the triply fluorinated ligand reduces the binding strength to the level of the low-affinity 2,2'-bipyridyl. The potential of the reported ligands in imaging zinc(II) ion in living cells was evaluated. The subcellular localization properties of two ligands in five organelles were characterized. Both benefits and deficiencies of these ligands were revealed, which provides directions for the near future in this line of research.     

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 ratiometric fluorescent probe for imaging hydroxyl radicals in living cells

A novel fluorescent probe, the detection mechanism of which is based on the 'on-off' switching of a FRET triggered by the *OH-induced cleavage of a DNA strand, has been developed for the ratiometric imaging of *OH.     

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.     

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.     

Reaction-based fluorescent probes for selective imaging of hydrogen sulfide in living cells

Hydrogen sulfide (H(2)S) is emerging as an important mediator of human physiology and pathology but remains difficult to study, in large part because of the lack of methods for selective monitoring of this small signaling molecule in live biological specimens. We now report a pair of new reaction-based fluorescent probes for selective imaging of H(2)S in living cells that exploit the H(2)S-mediated reduction of azides to fluorescent amines. Sulfidefluor-1 (SF1) and Sulfidefluor-2 (SF2) respond to H(2)S by a turn-on fluorescence signal enhancement and display high selectivity for H(2)S over other biologically relevant reactive sulfur, oxygen, and nitrogen species. In addition, SF1 and SF2 can be used to detect H(2)S in both water and live cells, providing a potentially powerful approach for probing H(2)S chemistry in biological systems.     

A 4-hydroxynaphthalimide-derived ratiometric fluorescent chemodosimeter for imaging palladium in living cells

A highly selective ratiometric fluorescent chemodosimeter derived from 4-hydroxynaphthalimide was designed and synthesized to image palladium species in living cells by virtue of a palladium-catalyzed depropargylation reaction, and it could monitor three typical palladium species (0, + 2 and + 4) without additional reagents.     

A near-infrared fluorescent probe for monitoring ozone and imaging in living cells

A near-infrared fluorescent probe (Trp-Cy) for endogenous ozone is presented, which exhibited a large stokes shift about 140 nm and a rapid fluorescence response to ozone with high selectivity and sensitivity.     

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）,     

Super-resolution imaging and real-time tracking lysosome in living cells by a fluorescent probe

Lysosomes function as important organelles within cells and their movement associates with diverse biological events, hence the real-time tracking of lysosomal movement is of great significance. However, since most lysosome fluorescent probes suffer from relatively unsatisfactory photostability, tracking lysosomal movement in real-time remains challenging. Here, we report that a naphthalimide-based fluorescent compound, namely NIMS, is a quite promising probe for lysosome imaging. The visualizing mechanism lies in the selective accumulation of NIMS in lysosomes via a protonation reaction, followed by the fluorescence enhancement due to the interactions of NIMS with proteins. Owing to its high selectivity and good photostability, NIMS was successfully applied to capture super-resolution fluorescence images of lysosomes. More importantly, real-time tracking of lysosome movement in a single living cell by NIMS was realized with a confocal laser scanning microscope. Surprisingly, even in normal culture conditions, around 2/3 of the captured lysosomes were observed to move within 5 min, indicative of the highly dynamic features of lysosomes. Thus, this probe may facilitate the understanding of the lysosome dynamics in physiological or pathological conditions.     

Selective BODIPY® based fluorescent chemosensor for imaging Pb ion in living cells

A new fluorescent probe has been designed and synthesized by linking dicarboxylate pseudocrown ether to the BODIPY® (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore. The probe allows determination of free lead ions in living cells.     

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 1,8-naphthalimide-derived turn-on fluorescent probe for imaging lysosomal nitric oxide in living cells

Nitric oxide has played an important role in many physiological and pathological processes as a kind of important gas signal molecules. In this work, a new fluorescent probe LysoNO-Naph for detecting NO in lysosomes based on 1,8-naphthalimide was reported. LysoNO-Naph has sub-groups of o-phenylenediamine as a NO reaction site and 4-(2-aminoethyl)-morpholine as a lysosome-targetable group. This probe exhibited good selectivity and high sensitivity (4.57 μmol/L) toward NO in a wide pH range from 4 to 12. Furthermore, LysoNO-Naph can be used for imaging NO in lysosomes in living cells.     

Squaraine-based colorimetric and fluorescent sensors for Cu-specific detection and fluorescence imaging in living cells

New squaraine-based chemosensors SQ1 and SQ2 functionalized with 2-picolyl units were first synthesized and used as highly selective and sensitive colorimetric and fluorometric dual-channel sensors for Cu²⁺-specific recognition in aqueous systems. Among a series of individual metal ions, only Cu²⁺ could result in dramatic color changes. We also evaluated their capability of biological applications and found that SQ2 could be successfully employed as a Cu²⁺-selective probe in the fluorescence imaging of living cells.