Phenoxazine‐based Near‐infrared Fluorescent Probes for the Specific Detection of Copper (II) Ions in Living Cells

Abstract : It is well known that copper ions play a critical role in various physiological processes. However, a variety of human diseases are tightly correlated with copper overload. Although there are numerous fluorescent probes capable of detecting copper ions, most of them are “turn‐off” probes owing to copper (II) ions fluorescence quenching effect, resulting in poor sensitivity. Herein, a novel “turn‐on” near‐infrared (NIR) fluorescent probe PZ‐N based on phenoxazine was designed and synthesized for the selective detection of copper (II) ions (Cu²⁺). Upon the addition of Cu²⁺, the probe could quickly react with Cu²⁺ and emit strong fluorescence, along with colour change from colourless to obvious blue. Moreover, the probe PZ‐N showed good water solubility, high selectivity, and excellent sensitivity with low limit of detection (1.93 nM) towards copper (II) ions. More importantly, PZ‐N was capable of effectively detecting Cu²⁺ in living cells.     

A General Strategy for the Construction of NIR‐emitting Si‐rhodamines and Their Application for Mitochondrial Temperature Visualization

Si‐rhodamine (SiR) is an ideal fluorophore because it possesses bright emission in the NIR region and can be implemented flexibly in living cells. Currently, several promising approaches for synthesizing SiR are being developed. However, challenges remain in the construction of SiR containing functional groups for bioimaging application. Herein, we introduce a general and simple approach by a condensation reaction of diarylsilylether and arylaldehyde in o‐dichlorobenzene to synthesize a series of SiRs bearing various functional substituents. These SiRs have moderate to high quantum efficiency, tolerance to photobleaching, and high water solubility as well as NIR emitting, and their NIR fluorescence properties can be controlled through the photoinduced electron transfer (PET) mechanism. Fluorescence OFF‐ON switching effect is observed for SiR 9 in the presence of acid, which is rationalized by DFT/TDDFT calculations. Moreover, reversible stimuli response toward temperature is achieved. Since positive charge enables mitochondrial targeting ability and chloromethyl unit can covalently immobilize the dyes onto the mitochondrial via click reaction between the benzyl choride and protein sulfhydryls, SiR 8 is identified as a valuable fluorescent marker to visualize the morphology and monitor the temperature change of mitochondria with high photostability.     

Stable J‐Aggregation of an aza‐BODIPY‐Lipid in a Liposome for Optical Cancer Imaging

Organic building blocks are the centerpieces of “one‐for‐all” nanoparticle development. Herein, we report the synthesis of a novel aza‐BODIPY‐lipid building block and its self‐assembly into a liposomal nanoparticle (BODIPYsome). We observed optically stable NIR J‐aggregation within the BODIPYsome that is likely attributed to J‐dimerization. BODIPYsomes with cholesterol showed enhanced colloidal stability while maintaining a high extinction coefficient (128 mm ^?1 cm^?1) and high fluorescence quenching (99.70±0.09 %), which enables photoacoustic (PA) properties from its intact structure and recovered NIR fluorescence properties when it is disrupted in cancer cells. Finally, its capabilities for optical imaging (PA/fluorescence) were observed in an orthotopic prostate tumor mouse model 24 h after intravenous administration. Overall, the BODIPYsome opens the door for engineering new building blocks in the design of optically stable biophotonic imaging agents.     

Fluorescence Quenching over Short Range in a Donor‐DNA‐Acceptor System

A new donor‐DNA‐acceptor system has been synthesized containing Nile red‐modified 2′‐deoxyuridine as charge donor and 6‐N,N‐dimethylaminopyrene‐modified 2′‐deoxyuridine as acceptor to investigate the charge transfer in DNA duplexes using fluorescence spectroscopy and time‐resolved femtosecond pump‐probe techniques. Fluorescence quenching experiments revealed that the quenching efficiency of Nile red depends on two components: 1) the presence of a charge acceptor and 2) the number of intervening CG and AT base pairs between donor and acceptor. Surprisingly, the quenching efficiency of two base pairs (73 % for CG and the same for AT) is higher than that for one base pair (68 % for CG and 37 % for AT), while at a separation of three base pairs less than 10 % quenching is observed. A comparison with the results of time‐resolved measurements revealed a correlation between quenching efficiency and the first ultrafast time constant suggesting that quenching proceeds via a charge transfer from the donor to the acceptor. All transients are satisfactorily described with two decays: a rapid charge transfer with 600 fs (～10¹² s^?1) that depends strongly and in a non‐linear fashion on the distance between donor and acceptor, and a slower time constant of a few picoseconds (～10¹¹ s^?1) with weak distance dependence. A third time constant on a nanosecond time scale represents the fluorescence lifetime of the donor molecule. According to these results and time‐dependent density functional theory (TDDFT) calculations a combination of single‐step superexchange and multistep hopping mechanisms can be proposed for this short‐range charge transfer. Furthermore, significantly less quenching efficiency and slower charge transfer rates at very short distances indicate that the direct interaction between donor and acceptor leads to a local structural distortion of DNA duplexes which may provide some uncertainty in identifying the charge transfer rates in short‐range systems.     

Green Synthesis of Red‐Emitting Carbon Nanodots as a Novel “Turn‐on” Nanothermometer in Living Cells

Temperature measurements in biology and medical diagnostics, along with sensitive temperature probing of living cells, is of great importance; however, it still faces significant challenges. Herein, a novel “turn‐on” carbon‐dot‐based fluorescent nanothermometry device for spatially resolved temperature measurements in living cells is presented. The carbon nanodots (CNDs) are prepared by a green microwave‐assisted method and exhibit red fluorescence (λ_em=615 nm) with high quantum yields (15 %). Then, an on–off fluorescent probe is prepared for detecting glutathione (GSH) based on aggregation‐induced fluorescence quenching. Interestingly, the quenched fluorescence could be recovered by increasing temperature and the CNDs–GSH mixture could behave as an off–on fluorescent probe for temperature. Thus, red‐emitting CNDs can be utilized for “turn‐on” fluorescent nanothermometry through the fluorescence quenching and recovery processes, respectively. We employ MC3T3‐E1 cells as an example model to demonstrate the red‐emitting CNDs can function as “non‐contact” tools for the accurate measurement of temperature and its gradient inside a living cell.     

Double Metal Ions Competitively Control the Guest‐Sensing Process: A Facile Approach to Stimuli‐Responsive Supramolecular Gels

A facile approach to the design of stimuli‐responsive supramolecular gels (SRSGs) termed double‐metal‐ion competitive coordination control is reported. By this means, the fluorescence signals and guest‐selective responsiveness of the SRSGs are controlled by the competitive coordination of two different metal ions with the gelators and the target guest. To demonstrate this approach, a gelator G2 based on multiple self‐assembly driving forces was synthesized. G2 could form Ca²⁺‐coordinated metallogel CaG with strong aggregation‐induced emission (AIE). Doping of CaG with Cu²⁺ results in AIE quenching of CaG and formation of Ca²⁺‐ and Cu²⁺‐based metallogel CaCuG. CaCuG could fluorescently detect CN^? with specific selectivity through the competitive coordination of CN^? with the Cu²⁺ and the coordination of Ca²⁺ with G2 again. This approach may open up routes to novel stimuli‐responsive supramolecular materials.     

A New Fluorescent Chemosensor for Metal Ions Based upon 1,8‐Naphthalimide and 8‐Hydroxyquinoline

A new fluorescent chemosensor based upon 1,8‐naphthalimide and 8‐hydroxyquinoline was synthesized, and its fluorescent properties in the presence of different metal cations (Hg²⁺, Ag⁺, Zn²⁺, Fe²⁺, Cd²⁺, Pb²⁺, Ca²⁺, Cu²⁺, Mg²⁺, and Ba²⁺) were investigated. It displayed fluorescence quenching with some heavy and transition metal (HTM) ions, and the quenching strongly depended on the nature of HTM ions.     

Excimer‐Based On‐Off Bis(pyreneamide) Macrocyclic Chemosensors

A series of bis(pyreneamide) macrocycles, synthesized in two steps from THF, THP, oxepane and 1,4‐dioxane, are tested as chemosensors for a large range of mono‐, di‐ and trivalent cations. In their native states, these macrocycles exhibit a strong excimer fluorescence that is quenched upon the addition of the metal ions (alkaline, alkaline earth, p‐, d‐, and f‐block metals). UV‐Vis spectrophotometric titrations, cyclic voltammetry, excimer fluorescence quenching, and transient absorption spectroscopy experiments helped characterize the On‐Off changes occurring upon binding and demonstrate that the highest stability constants are obtained with divalent cations Ca²⁺ and Ba²⁺ specifically.     

Highly Sensitive and Selective Fluorescence Probe for 2,4‐Dinitrophenylhydrazine Detection in Wastewater Using Water‐Soluble CdTe QDs

A simple, cheap, sensitive and selective probe for determination of DNPH in wastewater using thioglycolic acid (TGA)‐coated CdTe QDs (TGA‐QDs) as fluorescence probe has been established, and the properties of CdTe QDs were characterized by TEM, FT‐IR, DLS, XRD and zeta potentials. CdTe QDs fluorescence is highly efficiently quenched after adding DNPH on account of electron transfer effect, and the fluorescence quenching behavior of CdTe QDs interaction with DNPH is static quenching process. A good linear relationship is observed between the relative fluorescence intensity (F₀/F) and 0.06–10 ng mL^?1 of DNPH. As compared with some of reported methods, LOD of this method for analysis of DNPH (0.23 ng mL^?1) is the lowest. Masking agents of DDTC and NH₄OH can eliminate the interference of Cu²⁺, Ag⁺ and Hg²⁺. Hence, DNPH can be selectively and accurately detected and the established method was successfully used for detecting DNPH in wastewater with acceptable recovery of 90.6–102%.     

A DNA-Stabilized Ag1812+ Cluster with Excitation-Intensity-Dependent Dual Emission

DNA-stabilized silver nanoclusters (DNA-AgNCs) are easily tunable emitters with intriguing photophysical properties. Here, a DNA-AgNC with dual emission in the red and near-infrared (NIR) regions is presented. Mass spectrometry data showed that two DNA strands stabilize 18 silver atoms with a nanocluster charge of 12+. Besides determining the composition and charge of DNA₂[Ag₁₈]¹²⁺, steady-state and time-resolved methods were applied to characterize the picosecond red fluorescence and the relatively intense microsecond-lived NIR luminescence. During this process, the luminescence-to-fluorescence ratio was found to be excitation-intensity-dependent. This peculiar feature is very rare for molecular emitters and allows the use of DNA₂[Ag₁₈]¹²⁺ as a nanoscale excitation intensity probe. For this purpose, calibration curves were constructed using three different approaches based either on steady-state or time-resolved emission measurements. The results showed that processes like thermally activated delayed fluorescence (TADF) or photon upconversion through triplet-triplet annihilation (TTA) could be excluded for DNA₂[Ag₁₈]¹²⁺. We, therefore, speculate that the ratiometric excitation intensity response could be the result of optically activated delayed fluorescence.     

Tm3+‐Sensitized NIR‐II Fluorescent Nanocrystals for In Vivo Information Storage and Decoding

In vivo fluorescence imaging in the second near‐infrared window (NIR‐II) affords deep‐tissue penetration and high spatial resolution. Herein, we present a new type of Tm³⁺‐sensitized lanthanide nanocrystals with both excitation (1208 nm) and emission (1525 nm) located in the NIR‐II window for in vivo optical information storage and decoding. Taking advantage of the tunable fluorescence lifetimes, the optical multiplexed encoding capacity is enhanced accordingly. Micro‐devices with QR codes featuring the NIR‐II fluorescence‐lifetime multiplexed encoding were implanted into mice and were successfully decoded through time‐gated fluorescence imaging technology.     

Salicylaldimine‐functionalized poly(m‐phenyleneethynylene) as turn‐on chemosensor for ferric ion

A new turn on fluorescent probe for ferric ion based on poly(m‐phenyleneethynylene salicylaldimine) ( PPE‐IM ) has been developed. The preparation of PPE‐IM involves post‐polymerization functionalization of the corresponding polymeric amine, PPE‐AM , via the condensation with salicylaldehyde. The degree of polymerization of both PPE‐IM and PPE‐IM is 17 with polydispersity index of 1.5. In aqueous solution, the polymeric PPE‐IM is highly stable unlike its small molecule analog which is gradually hydrolyzed. The weak fluorescence of initial PPE ‐ IM (λ_em = 470) is greatly enhanced by 300 folds upon the addition of Fe³⁺. The ¹H NMR reveals that the fluorescence enhancement is caused by Fe³⁺‐induced hydrolysis of the imine group. The sensing system shows a detection limit of 0.14 μM of Fe³⁺. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 1155–1161     

A General Strategy for Development of Near‐Infrared Fluorescent Probes for Bioimaging

Near‐infrared (NIR) fluorescent dyes with favorable photophysical properties are highly useful for bioimaging, but such dyes are still rare. The development of a unique class of NIR dyes via modifying the rhodol scaffold with fused tetrahydroquinoxaline rings is described. These new dyes showed large Stokes shifts (>110 nm). Among them, WR3, WR4, WR5, and WR6 displayed high fluorescence quantum yields and excellent photostability in aqueous solutions. Moreover, their fluorescence properties were tunable by easy modifications on the phenolic hydroxy group. Based on WR6, two NIR fluorescent turn‐on probes, WSP‐NIR and SeSP‐NIR, were devised for the detection of H₂S. The probe SeSP‐NIR was applied in visualizing intracellular H₂S. These dyes are expected to be useful fluorophore scaffolds in the development of new NIR probes for bioimaging.     

Synthesis and Application of a Full Water‐Soluble and Red‐Emitting Chemosensor Based on Phenoxazinium for Copper(II) Ions

A phenoxazinium‐based chemosensor ( 1 ) bearing di(2‐picolyl)amine unit was successfully synthesized. The result shows that it is a red‐emitting and full water‐soluble chemosensor for the selective detection of Cu²⁺ in pure water. The fluorescence on‐off mechanism was studied by ab initio calculations. To confirm the suitability of 1 for biological applications, it was employed in the fluorescence detection of the intracellular Cu²⁺ with cultured KB cells. The results indicated that 1 had good membrane permeability and could be useful for the fluorescence microscopic imaging.     

A Lysosome‐Compatible Near‐Infrared Fluorescent Probe for Targeted Monitoring of Nitric Oxide

The requirement for nitric oxide (NO) of lysosomes has motivated the development of a sophisticated fluorescent probe to monitor the distribution of this important biomolecule at the subcellular level in living cells. A near‐infrared (NIR) fluorescent Si‐rhodamine (SiRB)‐NO probe was designed based on the NO‐induced ring‐opening process of Si‐rhodamine. The probe exhibits fast chromogenic and fluorogenic responses, and high sensitivity and selectivity toward trace amounts of NO. Significantly, the spirolactam in Si‐rhodamine exhibits very good tolerance to H⁺, which in turn brings extremely low background fluorescence not only in the physiological environment but also under acidic conditions. The stability of the highly fluorescent product in acidic solution provides persistent fluorescence emission for long‐term imaging experiments. To achieve targeted imaging with improved spatial resolution and sensitivity, an efficient lysosome‐targeting moiety was conjugated to a SiRB‐NO probe, affording a tailored lysosome‐targeting NIR fluorescent Lyso‐SiRB‐NO probe. Inheriting the key advantages of its parent SiRB‐NO probe, Lyso‐SiRB‐NO is a functional probe that is suited for monitoring lysosomal NO with excellent lysosome compatibility. Imaging experiments demonstrated the monitoring of both exogenous and endogenous NO in real time by using the Lyso‐SiRB‐NO probe.     

一种新型胞内钙离子指示剂的合成、表征及生物性能研究

本文通过在钙离子的螯合母体的结构上引入一个甲基，合成了一种新型钙离子荧光指示剂 Fluo-3M AM，并通过核磁共振氢谱、红外光谱、气相色谱-质谱及飞行时间质谱对其结构进行了充分的表征。同时，还对其荧光光谱及生物活性进行了研究。结果表明：这种新型荧光指示剂对钙离子有较高的亲和性，并且可以获得较强的荧光信号，这对进一步研究生物系统中钙离子的影响将起到非常重要的作用。     

Chlorin–Bacteriochlorin Energy‐transfer Dyads as Prototypes for Near‐infrared Molecular Imaging Probes: Controlling Charge‐transfer and Fluorescence Properties in Polar Media

The photophysical properties of two energy‐transfer dyads that are potential candidates for near‐infrared (NIR) imaging probes are investigated as a function of solvent polarity. The dyads ( FbC‐FbB and ZnC‐FbB ) contain either a free base (Fb) or zinc (Zn) chlorin (C) as the energy donor and a free base bacteriochlorin (B) as the energy acceptor. The dyads were studied in toluene, chlorobenzene, 1,2‐dichlorobenzene, acetone, acetonitrile and dimethylsulfoxide (DMSO). In both dyads, energy transfer from the chlorin to bacteriochlorin occurs with a rate constant of ～(5–10 ps)^?1 and a yield of >99% in nonpolar and polar media. In toluene, the fluorescence yields (Φ _f = 0.19) and singlet excited‐state lifetimes (τ～5.5 ns) are comparable to those of the benchmark bacteriochlorin. The fluorescence yield and excited‐state lifetime decrease as the solvent polarity increases, with quenching by intramolecular electron (or hole) transfer being greater for FbC‐FbB than for ZnC‐FbB in a given solvent. For example, the Φ _f and τ values for FbC‐FbB in acetone are 0.055 and 1.5 ns and in DMSO are 0.019 and 0.28 ns, whereas those for ZnC‐FbB in acetone are 0.12 and 4.5 ns and in DMSO are 0.072 and 2.4 ns. The difference in fluorescence properties of the two dyads in a given polar solvent is due to the relative energies of the lowest energy charge‐transfer states, as assessed by ground‐state redox potentials and supported by molecular‐orbital energies derived from density functional theory calculations. Controlling the extent of excited‐state quenching in polar media will allow the favorable photophysical properties of the chlorin–bacteriochlorin dyads to be exploited in vivo. These properties include very large Stokes shifts (85 nm for FbC‐FbB , 110 nm for ZnC‐FbB ) between the red‐region absorption of the chlorin and the NIR fluorescence of the bacteriochlorin (λ _f = 760 nm), long bacteriochlorin excited‐state lifetime (～5.5 ns), and narrow (≤20 nm) absorption and fluorescence bands. The latter will facilitate selective excitation/detection and multiprobe applications using both intensity‐ and lifetime‐imaging techniques.     

Cobalt Ion‐Mediated Cysteine Detection With a Hyperbranched Conjugated Polyelectrolyte as a New Sensing Platform

A highly efficient colorimetric and fluorescence turn‐off probe for the sensitive and selective detection of the biologically important amino acid, cysteine (Cys), is demonstrated using a newly synthesized water‐soluble hyperbranched polymer (HP) containing sulfonic acid groups. The detection mechanism involves two steps: (i) the slight quenching of HP in the presence of Co²⁺ in advance; and (ii) the gradual quenching of the HP–Co²⁺ complex according to the concentration of Cys due to the absorption screening effect of the formation of the Cys‐Co²⁺ complex, which prevents HP from absorbing excitation energy.     

Optimized Fluorescent Probe for Specific Imaging of Glutathione S‐Transferases in Living Cells and Mice

GSTP1 has been considered to be a marker for malignancy in many tissues. However, the existing GST fluorescent probes are unfavorable for in vivo imaging because of the limited emission wavelength or insufficient fluorescence enhancement (six‐fold). The limited fluorescence enhancement of GST fluorescent probes is mainly ascribed to the high background signals resulting from the spontaneous reaction between GSH and the probes. In this work, a highly specific GST probe with NIR emission has been successfully developed through optimization of the essential unit of the probe to repress the spontaneous reaction. The novel GST probe exhibits over 100‐fold fluorescence enhancement upon incubation with GSTP1/GSH and high selectivity over other potential interference. In addition, the probe has been proved to be capable of tracking endogenous GST in A549 cells. Finally, the in vivo imaging results demonstrate that the probe can be used for effective imaging of endogenous GST activity in subcutaneous tumor mouse with high contrast.     

A fluorescent chemosensor for Cu2+ ions and its application in cell imaging

A new on-off fluorescent probe 1 for Cu²⁺ based on Schiff base compound was designed and synthesized by one-step reaction. The single probe 1 exhibited strong green fluorescence emission. A fluorescence quenching effect and faint color change were observed as soon as the Cu²⁺ was added to the probe system in H₂O/EtOH (v/v = 8:2, HEPES buffer, 0.05 M, pH = 7.4) solution. Other common metal cations did not cause the changes in the fluorescence and color of the probe 1. The optical properties were studied by the fluorescence emission and UV–Vis spectra. Meanwhile, the geometry optimizations of probe 1 and the [1-Cu²⁺] coordination complexes were also carried out by DFT using the Gaussian 09 program, in which the B3LYP function was used. Based on experimental measurement and theoretical analysis, we can know that the combination ratio of the probe and Cu²⁺ is 2:1 and the limit of detection (LOD) is as low as 5.3 × 10^?9 M Besides, the probe 1 was also used to analyze the Cu²⁺ in living cells.