An asymmetrical flavylium based probe with large Stokes shift and near infrared emission for highly sensitive detecting and visualizing cellular drug induced H2S fluctuations.

Hydrogen sulfide (H2S) has been recognized as an important gaseous signaling molecule in living systems, and is of great significance in many pathological and physiological processes. Misregulation of endogenous H2S is implicated in various diseases in the neuronal, gastrointestinal, circulatory, and endocrine systems. Fluorescent probe with large Stokes shift and near infrared emission, is ideal candidate for imaging applications to prevent excitation scattering, autofluorescence interference, matrix absorption caused signal loss, and sample destruction. In this study, a dual-side expansion approach was performed to develop spectra tunable hydroxyl functional flavylium derivative named HN8 with enlarged Stokes shift of 81 nm, lengthened emission of 671 nm, satisfied quantum yield of 0.23, and good fluorescence enhancement factor of 14.3-fold. Moreover, based on HN8, the screened probe HN8DNP displayed 225-fold fluorescence enhancement containing linear correlations to H2S from 0 to 50 µM with good limit of detection (LOD) of 0.31 µM. Therefore, HN8DNP was then applied for imaging exogenous H2S and drug induced enzymatic H2S generation in living cells with satisfied results, revealing the relationship between intracellular H2S levels and related enzyme activities. In a word, the present work provided a potential fluorescence probe for highly selective and sensitive detecting H2S in vitro and in living cells. And the promising dual-side expansion strategy for regulation optical feature of traditional fluorophore may meet the increasing requirements of sensing and imaging applications.

Optimized self-immolative near-infrared probe based on hemicyanine for highly specific monitoring thiophenols in living systems.

In this paper, utilizing the same recognition group dinitrophenyl and hydroxyl functional NIR fluorophore hemicyanine, directly-linked probe CyNO2 and self-immolative probe CyBNO2 were developed for evaluation of sensing PhSH. Though CyNO2 was easily synthesized and sensitive to mercapto, the probe CyBNO2 showed higher selectivity, broader linear range from 1.0 × 10-7 to 7.0 × 10-6 M with lower detection limit of 22 nM for PhSH. Moreover, CyBNO2 was successfully applied for monitoring PhSH in living cells and in vivo, indicating the great potential of self-immolative probes.

A novel weak acid activated probe for highly selective monitoring selenocysteine in living cells.

Selenocysteine (Sec, pKa 5.8) is genetically encoded 21st amino acid into the active site of selenoproteins, which have broad functions relevant to various diseases, tissues or organs and subcellular organelles. However, many selenoproteins involved cellular functions still remains unclear. In addition, since biothiols such as glutathione (GSH, pKa 8.3), possessing similar chemical properties with Sec, commonly exist in living systems at high levels. Thus, it is of great importance and high challenge to identify novel probes for selectively monitoring Sec over biothiols. In this paper, we proposed a smart strategy which allowed us to develop a lysosome targetable probe for specifically sensing Sec. By restricting weak acidic microenvironment, the probe shows a specific detection for Sec with 85-fold fluorescence enhancement owing to the remaining high activity of Sec at pH 5.0. Moreover, being low cytotoxicity to the cells verified by MTS assay, the probe was then successfully applied for imaging exogenous and endogenous Sec in lysosomes, indicating its potential for the biological investigation of Sec in subcellular organelles.

A structure optimized fluorescent probe for highly sensitive monitoring drug induced lysosomal pH value changes.

Lysosomes generally maintain the weak acidic microenvironment, to ensure highly efficient activity and functions of hydrolytic enzymes and proteins. Aberrations of the lysosomal pH may result in cellular functional changes and influence human physiology, possibly causing serious diseases. Small-molecular fluorescent probes based imaging techniques capable of providing information on target locations are considerably appreciated. Herein, by reducing the size of the typical lysosome targetable group 4-(2-aminoethyl) morpholine, we rationally designed a rhodamine analogue probe Ly-HN2AM with N-Aminomorpholine as the ring-closed switch and the lysosome targetable moiety for visualizing lysosomal pH changes. With the benefit of constructing multi-pentacyclic intramolecular hydrogen bond when binding with the H+, Ly-HN2AM gives a highly sensitive response towards pH values ranging from 4.79 to 6.07, with a remarkable higher pKa 5.35 over the typical 4-(2-aminoethyl) morpholine modified probes. The new probe was successfully applied to visualize pH value changes in lysosome-associated physiological and pathological processes with excellent photostability and low cytotoxicity, indicating the potential applications of lysosome specific bioimaging.

A near-infrared fluorescent probe based on photostable Si-rhodamine for imaging hypochlorous acid during lysosome-involved inflammatory response.

Hypochloric acid (HClO) is mainly distributed in acidic lysosomes of phagocytes and closely associated with numerous physiological and pathological processes, especially inflammatory response. Fluorescent probe has become an important tool for imaging HClO in lysosomes, but suffered from interference from autofluorescence in vivo, phototoxicity to biosamples and photobleaching phenomenon due to their short-wavelength excitation and emission. Unfortunately, up to now, no near-infrared (NIR) lysosome-targetable fluorescent probe has been reported for imaging HClO. In this paper, a near-infrared fluorescent probe Lyso-NIR-HClO for imaging lysosomal HClO was reported for the first time. Lyso-NIR-HClO based on Si-rhodamine is consisted of a morpholine unit as a lysosome-targetable group and a HClO-mediated cyclization reaction site as a response group, which was applied for highly selective and sensitive detection and imaging for endogenous and exogenous HClO in lysosomes, with a linear range from 5.0 × 10-8 to 1.0 × 10-5 M and a detection limit of 2.0 × 10-8 M in vitro. Attributed to NIR emission and excellent photostability of Si-rhodamine, Lyso-NIR-HClO exhibits excellent performances in vivo, such as low interference from intracellular autofluorescence, stable and persistent fluorescence signal and good tissue penetration, which are in favor of accurate, time-lapse and long-term imaging for HClO. Finally, we applied the probe Lyso-NIR-HClO to visualize endogenous HClO during lysosome-involved inflammatory response including bacteria-infected cells and inflamed mouse model with satisfactory results. The above results proved that Lyso-NIR-HClO would be a potentially useful tool for the study of biological functions and pathological roles of HClO in lysosomes, especially role of lysosome in the inflammatory response.

A novel two-photon fluorescent probe with long-wavelength emission for monitoring HClO in living cells and tissues.

Hypochlorous acid (HClO), one of the most important reactive oxygen species (ROS), is a potent antimicrobial agent for the immune system against invasive bacteria and a wide range of pathogens. Therefore, it is critical to develop sensitive and selective methods for visualization of HClO in biological samples. In this work, a two-photon fluorescent probe HN2-TP) with long-wavelength emission (far-red: 630 nm) based on rhodamine analogue for bioimaging HClO was developed. Owing to a specific HClO induced cyclization reaction, the new probe shows large fluorescence enhancement (about 106-fold), good linear range with high sensitivity (detection limit: 40 nM), high selectivity and fast response when monitoring HClO in vitro. More importantly, by successfully imaging HClO in living cells and tissues, this kind of two-photon fluorescent probe with long-wavelength emission is expected for accurate sensing in complex biosystems, which could eliminate undesired autofluorescence and self-absorption.

A unique approach toward near-infrared fluorescent probes for bioimaging with remarkably enhanced contrast.

Near-infrared (NIR) fluorescent probes are attractive molecular tools for bioimaging because of their low autofluorescence interference, deep tissue penetration, and minimal damage to sample. However, most previously reported NIR probes exhibit small Stokes shift, typically less than 30 nm, and low fluorescence quantum yield, strictly limited contrast and spatial resolution for bioimaging. Herein, by expanding the π-conjugated system of rhodamine B, while, at the same time, keeping its rigid and planar structure, we reported an efficient NIR dye, HN7, with large stokes shift of 73 nm and fluorescence quantum yield as high as 0.72 in ethanol, values superior to those of such traditional cyanine NIR dyes as Cy5. Using HN7, living cells, tissues and mice were imaged, and the results showed significantly enhanced contrast, improved spatial resolution, and satisfactory tissue imaging depth when compared to Cy5. Moreover, the nonfluorescent spirocyclic structure of rhodamine B is an inherent component of HN7; therefore, our strategy provided a universal platform for the design of efficient NIR turn-on bioimaging probes for various targets. As a proof-of-concept, two different NIR probes, HN7-N2 and HN7-S for NO and Hg2+, respectively, were designed, synthesized, and successfully applied for the imaging of NO and Hg2+ in living cells, tissues and mice, respectively, demonstrating the potential bioimaging applications of the new probes. In sum, this new type of dye may present new avenues for the development of efficient NIR fluorescent probes for contrast-enhanced imaging in biological applications.

Rhodamine-based fluorescent probe for direct bio-imaging of lysosomal pH changes.

Intracellular pH plays a pivotal role in various biological processes. In eukaryotic cells, lysosomes contain numerous enzymes and proteins exhibiting a variety of activities and functions at acidic pH (4.5-5.5), and abnormal variation in the lysosomal pH causes defects in lysosomal function. Thus, it is important to investigate lysosomal pH in living cells to understand its physiological and pathological processes. In this work, we designed a one-step synthesized rhodamine derivative (RM) with morpholine as a lysosomes tracker, to detect lysosomal pH changes with high sensitivity, high selectivity, high photostability and low cytotoxicity. The probe RM shows a 140-fold fluorescence enhancement over a pH range from 7.4 to 4.5 with a pKa value of 5.23. Importantly, RM can detect the chloroquine-induced lysosomal pH increase and monitor the dexamethasone-induced lysosomal pH changes during apoptosis in live cells. All these features demonstrate its value of practical application in biological systems.

A simple and pH-independent and ultrasensitive fluorescent probe for the rapid detection of Hg2+.

Development of fluorescent probes for Hg(2+) has become a hot topic in modern chemical research due to its high toxicity. In this paper, we for the first time report the synthesis and application of a thioether spirocyclic rhodamine B derivative (TR) as an efficient fluorescent probe for Hg(2+). TR was synthesized using a simple procedure under mild condition. By employing a thioether spirocycle instead of classic spirolactam as recognition unit, our proposed probe TR is acidity-insensitive, and exhibits a pH-independent and ultrasensitive response to Hg(2+). The probe works well within a wide pH range from 3.5 to 11.5, and exhibits a 350-fold fluorescence enhancement upon 0.5 equiv of Hg(2+) triggered, with a detection limit of 2.5 nM estimated for Hg(2+). In virtue of the strong thiophilic characteristic of Hg(2+), the response of the probe to Hg(2+) is instantaneous and highly selective, which make it favorable for cellular Hg(2+) imaging applications. It has been preliminarily used for highly sensitive monitoring of Hg(2+) level in living cells with satisfying resolution, demonstrating its value of the practical applications in biological systems.

Through bond energy transfer: a convenient and universal strategy toward efficient ratiometric fluorescent probe for bioimaging applications.

Fluorescence resonance energy transfer (FRET) strategy has been widely applied in designing ratiometric probes for bioimaging applications. Unfortunately, for FRET systems, sufficiently large spectral overlap is necessary between the donor emission and the acceptor absorption, which would limit the resolution of double-channel images. The through-bond energy transfer (TBET) system does not need spectral overlap between donor and acceptor and could afford large wavelength difference between the two emissions with improved imaging resolution and higher energy transfer efficiency than that of the classical FRET system. It seems to be more favorable for designing ratiometric probes for bioimaging applications. In this paper, we have designed and synthesized a coumarin-rhodamine (CR) TBET system and demonstrated that TBET is a convenient strategy to design an efficient ratiometric fluorescent bioimaging probe for metal ions. Such TBET strategy is also universal, since no spectral overlap between the donor and the acceptor is necessary, and many more dye pairs than that of FRET could be chosen for probe design. As a proof-of-concept, Hg(2+) was chosen as a model metal ion. By combining TBET strategy with dual-switch design, the proposed sensing platform shows two well-separated emission peaks with a wavelength difference of 110 nm, high energy transfer efficiency, and a large signal-to-background ratio, which affords a high sensitivity for the probe with a detection limit of 7 nM for Hg(2+). Moreover, by employing an Hg(2+)-promoted desulfurization reaction as recognition unit, the probe also shows a high selectivity to Hg(2+). All these unique features make it particularly favorable for ratiometric Hg(2+) sensing and bioimaging applications. It has been preliminarily used for a ratiometric image of Hg(2+) in living cells and practical detection of Hg(2+) in river water samples with satisfying results.

Bifunctional fluoroionphore-ionic liquid hybrid for toxic heavy metal ions: improving its performance via the synergistic extraction strategy.

Several heavy metal ions (HMIs), such as Cd(2+), Pb(2+), and Hg(2+), are highly toxic even at very low concentrations. Although a large number of fluoroionphores have been synthesized for HMIs, only a few of them show detection limits that are below the maximum contamination levels in drinking water (usually in the nM range), and few of them can simultaneously detect and remove HMIs. In this work, we report a new fluoroionphore-ionic liquid hybrid-based strategy to improve the performance of classic fluoroionphores via a synergistic extraction effect and realize simultaneous instrument-free detection and removal of HMIs. As a proof-of-concept, Hg(2+) was chosen as a model HMI, and a rhodamine thiospirolactam was chosen as a model fluoroionphore to construct bifunctional fluoroionphore-ionic liquid hybrid 1. The new sensing system could provide obviously improved sensitivity by simply increasing the aqueous-to-ionic liquid phase volume ratio to 10:1, resulting in a detection limit of 800 pM for Hg(2+), and afford extraction efficiencies larger than 99% for Hg(2+). The novel strategy provides a general platform for highly sensitive detection and removal of various HMIs in aqueous samples and holds promise for environmental and biomedical applications.

An efficient rhodamine thiospirolactam-based fluorescent probe for detection of Hg2+ in aqueous samples.

This paper described the optimized design, synthesis and application of a novel rhodamine thiospirolactam derivative as an 'off-on' fluorescent probe for the detection of Hg(2+) in aqueous samples. The 'off-on' fluorescence and color signal change of the probe is based on an Hg(2+)-triggered domino reaction which brings on the opened-ring form of the rhodamine spirolactam to regain the conjugated system of the rhodamine skeleton. In the well designed probe, the thiospirolactam serves as both Hg(2+) binding unit and electron-defect carbon centre, a phenolic hydroxyl with very strong nucleophilicity after deprotonation is chosen as the attacking unit, and a benzene ring is introduced on the linker to afford steric effects, which benefits an efficient nucleophilic reaction, with a high sensitivity towards Hg(2+). It exhibits a stable response for Hg(2+) from 1.0 × 10(-8) to 1.0 × 10(-6) M, with a detection limit of 3.0 × 10(-9) M. The response of the probe to Hg(2+) is highly selective and pH-insensitive, with a fast response time. All these unique features make it particularly favorable for cellular Hg(2+) imaging applications. It has been preliminarily used for highly sensitive monitoring of Hg(2+) levels in living cells with satisfying resolution.

Clicking fluoroionophores onto mesoporous silicas: a universal strategy toward efficient fluorescent surface sensors for metal ions.

Mesoporous SBA-15 silica is an excellent support for constructing fluorescent surface sensors. In this letter, we reported a two-step surface reaction involved strategy to construct efficient fluorescent surface sensors for metal ions by clicking fluoroionophores onto azide-functionalized SBA-15. Our experimental results indicate that such a strategy exhibits an obviously higher loading efficiency within commercial SBA-15 than a previously reported strategy. As a proof-of-concept, a newly designed alkyne-functionalized Hg(2+) fluoroionophore was grafted onto SBA-15 to form a fluorescent Hg(2+) surface sensor. It shows improved sensitivity and selectivity than the fluoroionophore itself working in the solution phase with a detection limit of 2.0 x 10(-8) M for Hg(2+).

Efficient fluorescence resonance energy transfer-based ratiometric fluorescent cellular imaging probe for Zn(2+) using a rhodamine spirolactam as a trigger.

This letter described the design and synthesis of a novel fluorescein-appended rhodamine spirolactam derivative and its preliminary application as a ratiometric fluorescent cellular imaging probe for Zn(2+). The ratiometric fluorescent signal change of the probe is based on an intramolecular fluorescence resonance energy transfer (FRET) mechanism modulated by a specific metal ion induced ring-opening process of the rhodamine spirolactam (acting as a trigger). In the new developed sensing system, the emission peaks of the two fluorophores are well-resolved, which can avoid the emission spectra overlap problem generally met by spectra-shift type probes and benefits for observation of fluorescence signal change at two different emission wavelengths with high resolution. It also benefits for a large range of emission ratios, thereby a high sensitivity for Zn(2+)detection. Under optimized experimental conditions, the probe exhibits a stable response for Zn(2+) over a concentration range from 2.0 x 10(-7) to 2.0 x 10(-5) M, with a detection limit of 4.0 x 10(-8) M. Most importantly, the novel probe has well solved the problem of serious interferences from other transition metal ions generally met by previously reported typical fluorescent probes for Zn(2+) with the di(2-picolyl)amine moiety as the receptor (in this case, the fluorescence response induced by Cd(2+)is even comparable to that of Zn(2+)) and shows a reversible and fast response toward Zn(2+). All these unique features make it particularly favorable for ratiometric cellular imaging investigations. It has been preliminarily used for ratiometric imaging of Zn(2+) in living cells with satisfying resolution.

A ratiometric fluorescent sensor for zinc ions based on covalently immobilized derivative of benzoxazole.

In the present paper, we describe the fabrication and analytical characteristics of fluorescence-based zinc ion-sensing glass slides. To construct the sensor, a benzoxazole derivative 4-benzoxazol-2'-yl-3-hydroxyphenyl allyl ether (1) with a terminal double bond was synthesized and copolymerized with 2-hydroxyethyl methacrylate (HEMA) on the activated surface of glass slides by UV irradiation. In the absence of Zn(2+) at pH 7.24, the resulting optical sensor emitted fluorescence at 450 nm via excited-state intramolecular proton transfer (ESIPT). Upon binding with Zn(2+), the ESIPT process was inhibited resulting in a 46 nm blue-shift of fluorescence emission. Thus, the proposed sensor can behave as a ratiometric fluorescent sensor for the selective detection of Zn(2+). In addition, the sensor shows nice selectivity, good reproducibility and fast response time. Cd(2+) did not interfere with Zn(2+) sensing. The sensing membrane demonstrates a good stability with a lifetime of at least 3 months. The linear response range covers a concentration range of Zn(2+) from 8.0x10(-5) to 4.0x10(-3) mol/L and the detection limit is 4.0x10(-5) mol/L. The determination of Zn(2+) in both tap and river water samples shows satisfactory results.