A dual-modal probe for NIR fluorogenic and ratiometric photoacoustic imaging of Cys/Hcy in vivo

Biothiols, such as cysteine(Cys) and homocysteine(Hcy), play vital roles in biological homeostasis and are closely related to various pathological and physiological processes in the living systems. Therefore, the in vivo detection of biothiols is of great importance for early diagnosis of diseases and assessment of disease progression. In this work, we developed a near-infrared(NIR) fluorescence and photoacoustic dual-modal molecular probe(NIR-S) that can be specifically activated by Cys or Hcy. The aryl-thioether substituted cyanine probe can undergo nucleophilic substitution and Smiles rearrangement reaction, resulting in specific turn-on NIR fluorescence and ratiometric photoacoustic responses for Hcy/Cys. Thus, NIR-S not only realizes the specific NIR fluorescence and photoacoustic dual mode imaging to detect Hcy/Cys in solution, but also can be applied to living cells and mice to detect Hcy/Cys. This work provided a practical tool to detect Hcy/Cys levels in vivo, which would be beneficial for the early diagnosis and progress of diseases.     

A ratiometric fluorescence probe for selective visual sensing of Zn2+

A simple ratiometric fluorescence probe based on vinylpyrrole end-capped bipyridine for the visual sensing of Zn2+ under aqueous physiological pH (6.8-7.4) is described. The fluorophores 3a-c showed strong emission around 537 nm in acetonitrile with a quantum yield of 0.4. In buffered (HEPES, pH 7.2) acetonitrile-water mixture (9:1 v/v), titration of transition metal salts to 3c showed strong quenching of the emission at 547 nm except in the case of Zn2+, which resulted in a red-shifted emission at 637 nm. Alkali and alkaline earth metal salts could not induce any considerable changes to the emission behavior of 3a-c. The binding of Zn2+ was highly selective in the presence of a variety of other metal ions. Though Cu2+ quenches the emission of 3c, in the presence of Zn2+, a red emission prevails, indicating the preference of 3c toward Zn2+. Job plot and Benesi-Hildebrand analysis revealed a 1:1 complexation between the probe and the metal ion. The selective visual sensing of Zn2+ with a red emission is ideally suited for the imaging of biological specimens.     

Design of a ratiometric fluorescent probe for benzenethiols based on a thiol-sulfoxide reaction

A novel thiol-sulfoxide reaction based ratiometric probe for benzenethiols was synthesized and evaluated. The probe features a chemospecific reduction over a pH range of 1-10 by benzenethiols with a marked emission color change, enabling the highly selective detection and is promising for applications.     

A new fluorescent probe with ultralow background fluorescence for imaging of endogenous cellular selenol under oxidative stress

A new fluorescent probe (Rhod-Sec) for selenol detection with ultralow background fluorescence have been developed in this paper, which showed a 380-fold off-on fluorescence response, and can be applied to visualize the fluctuation of selenol in HepG2 cells through LPS-induced cells oxidation resistance.     

Activatable nanoscale metal-organic framework for ratiometric photoacoustic imaging of hydrogen sulfide and orthotopic colorectal cancer in vivo

Nanoscale metal-organic frameworks(nano MOFs) have emerged as a promising biomedical nanoplatform because of their unique properties. However, the exploration of nano MOFs in photoacoustic(PA) imaging is still limited. Here, a novel hydrogen sulfide(H2 S)-activated nano copper-based MOF(Cu-MOF) was developed as a near-infrared(NIR) ratiometric PA probe for in vivo monitoring of endogenous H2 S level and orthotopic colorectal cancer imaging via in situ reaction of nano Cu-MOFs with endogenous H_2 S that is closely associated with tumor growth and proliferation in colon cancer. The synthesized nano Cu-MOFs displayed excellent PA responsiveness towards tumor H_2 S level with high selectivity and rapid kinetics. The result suggests the developed probe may provide a unique opportunity to investigate the malignant behaviors of H_2 S-associated events in vivo.     

A novel,cell-permeable,fluorescent probe for ratiometric imaging of zinc ion

Zn(2+) plays important roles in various biological systems; as a result, the development of tools that can visualize chelatable Zn(2+) has attracted much attention recently. We report here newly synthesized fluorescent sensors for Zn(2+), ZnAF-Rs, whose excitation maximum is shifted by Zn(2+) under physiological conditions. Thus, these sensors enable ratiometric imaging, which is a technique to reduce artifacts by minimizing the influence of extraneous factors on the fluorescence of a probe. Ratiometric measurement can provide precise data, and some probes allow quantitative detection. ZnAF-Rs are the first ratiometric fluorescent sensors for Zn(2+) that enable quantitative analysis under physiological conditions. ZnAF-Rs also possess suitable K(d) for applications, and high selectivity against other biologically relevant cations, especially Ca(2+). Using these probes, changes of intracellular Zn(2+) concentration in cultured cells were monitored successfully. We believe that these probes will be extremely useful in studies on the biological functions of Zn(2+).     

A two-photon fluorescent probe for ratiometric imaging of hydrogen peroxide in live tissue

We report a two-photon fluorescent probe (PN1) that can be excited by 750 nm femto-second pulses, shows high photostability and negligible toxicity, and can visualize H(2)O(2) distribution in live cells and tissue by two-photon microscopy.     

Development of an ICT-based ratiometric fluorescent hypochlorite probe suitable for living cell imaging

We have judiciously constructed a novel ICT-based ratiometric OCl(-) probe capable of ratiometric imaging in the live cells based on the new OCl(-)-promoted de-diaminomaleonitrile reaction.     

Cu nanoclusters-based ratiometric fluorescence probe for ratiometric and visualization detection of copper ions

Copper is a highly toxic environmental pollutant with bioaccumulative properties. Therefore, sensitive detection of Cu²⁺ is very important to prevent over-ingestion, and visual detection is preferred for practical applications. In this work, we developed a simple and environmental friendly approach to synthesize hyperbranched polyethyleneimine-protected copper nanoclusters (hPEI-Cu NCs) with great stability against extreme pH, high ionic strength, thiols etching and light illumination, which were then conjugated to the surface of silica coated CdSe quantum dots (QDs) to design a ratiometric fluorescence probe. In the presence of different amounts of Cu²⁺ ions, the fluorescence of Cu NCs can be drastically quenched, while the emission from QDs stayed constant to serve as a reference signal and the color of the probe changed from yellow-green to red, resulting in ratiometric and visualization detection of Cu²⁺ ion with high accuracy. The detection limit for Cu²⁺ was estimated to be 8.9 nM, much lower than the allowable level of Cu²⁺ in drinking water (∼20 μM) set by U.S. Environmental Protection Agency. Additionally, this probe can be also applied for the determination of Cu²⁺ ion in complex real water samples.     

A ratiometric optical imaging probe for intracellular pH based on modulation of europium emission

A set of three pH-responsive ratiometric Eu(III)complexes has been synthesised incorporating a coordinated azathioxanthone sensitiser and a pH dependent alkylsulfonamide moiety. Emission properties, anion binding affinities, pH response curves and protein binding constants were studied in detail in aqueous media, and solutions containing various concentrations of interfering anions and protein were also examined. The complex, [EuL3] exhibited some interference from protein and endogenous anions, e.g. lactate and hydrogen carbonate, but possessed a protonation constant of 7.2 in human serum solution. A suitable calibration curve was obtained and was used to determine the local pH using a 680/589 nm intensity ratio vs. pH plot. Confocal fluorescence microscopy images revealed fast uptake of the complex and a well distributed localisation within the cell; fast egress also occurred. Ribosomal localisation, with a high concentration within the protein-dense nucleoli was observed, in a similar manner to structurally related complexes bearing the same coordinated sensitising moiety. An IC(50) value of 67 (+/-20) microM was estimated using an MTT assay. Selected emission band ratio versus pH plots allow pH measurement in the range 6 to 8, enabling intracellular pH to be measured by microscopy. A value of 7.4 was estimated for NIH 3T3 cells in the protein rich regions of the nucleolus and ribosomes.     

A mitochondria-localized two-photon fluorescent probe for ratiometric imaging of hydrogen peroxide in live tissue

We report a two-photon fluorescent probe (SHP-Mito) which can ratiometrically detect mitochondrial H(2)O(2) in live cells and intact tissues at >100 μm depth through the use of two-photon microscopy.     

In vivo monitoring of tissue regeneration using a ratiometric lysosomal AIE probe

Tissue regeneration is a crucial self-renewal capability involving many complex biological processes. Although transgenic techniques and fluorescence immunohistochemical staining have promoted our understanding of tissue regeneration, simultaneous quantification and visualization of tissue regeneration processes is not easy to achieve. Herein, we developed a simple and quantitative method for the real-time and non-invasive observation of the process of tissue regeneration. The synthesized ratiometric aggregation-induced-emission (AIE) probe exhibits high selectivity and reversibility for pH responses, good ability to map lysosomal pH both in vitro and in vivo, good biocompatibility and excellent photostability. The caudal fin regeneration of a fish model (medaka larvae) was monitored by tracking the lysosomal pH change. It was found that the mean lysosomal pH is reduced during 24–48 hpa to promote the autophagic activity for cell debris degradation. Our research can quantify the changes in mean lysosomal pH and also exhibit its distribution during the caudal fin regeneration. We believe that the AIE-active lysosomal pH probe can also be potentially used for long-term tracking of various lysosome-involved biological processes, such as tracking the stress responses of tissue, tracking the inflammatory responses, and so on.

An AIE-active ratiometric probe for the first time achieved the long-term quantification of lysosomal pH during the medaka larva''s caudal fin regeneration.     

Highly selective sensing of cyanide by a benzochromene-based ratiometric fluorescence probe

Enone-functionalized benzochromene chemodosimeter (1) was prepared through the Baylis-Hillman condensation reaction and was utilized as a ratiometric fluorescence probe for cyanide anions in aqueous buffer. The probe has shown a selective and sensitive response to cyanides over other various anions through the Michael addition and a subsequent [1,3]-sigmatropic rearrangement reaction. When cyanide anions were added, a prominent ratiometric fluorescence change of 1 was observed thus allowing to detect the micromolar concentration of cyanides by the naked eye.     

A ratiometric fluorescent probe for real-time monitoring of intracellular glutathione fluctuations in response to cisplatin

Real-time imaging of fluctuations in intracellular glutathione (GSH) concentrations is critical to understanding the mechanism of GSH-related cisplatin-resistance. Here, we describe a ratiometric fluorescence probe based on a reversible Michael addition reaction of GSH with the vinyl-functionalized boron-dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or BODIPY) 1. The probe was applied for real-time monitoring of the fluctuations in GSH levels in cells under cisplatin treatment. Notably, in cellular cisplatin-sensitive A549 cells, GSH concentrations rose until cell death, while in cisplatin-resistant cell lines, GSH levels first rose to the maximum then fell back to the initial concentration without significant apoptosis. These results indicate that different trends in GSH fluctuation can help distinguish cisplatin-resistant from cisplatin-sensitive cells. As such, this study has shown that probe 1 may potentially be used for real-time monitoring of intracellular GSH levels in response to therapeutics.

Real-time imaging of intracellular glutathione in response to cisplatin by a ratiometric fluorescent probe reveals that the different trends in intracellular GSH levels is crucial in distinguishing cisplatin-resistant from cisplatin-sensitive cells.     

Rational design of a highly reactive ratiometric fluorescent probe for cyanide

A novel highly reactive ratiometric fluorescent cyanide probe was judiciously designed based on 2-formylacrylonitrile moiety as a new cyanide reaction site. A DFT study was conducted to rationalize the extremely high reactivity nature of the ratiometric fluorescent cyanide probe.     

Design of quinoline-based fluorescent probe for the ratiometric detection of cadmium in aqueous media

A new fluorescent probe,DQCd2,based on 4-piperidinoquinoline has been synthesized as a fluorescent probe for Cd²⁺.It can ratiometrically respond to Cd²⁺ in PBS buffer by a remarkable emission intensity enhancement（88-fold） and wavelength shift（70 nm）.     

A new prodrug-derived ratiometric fluorescent probe for hypoxia: high selectivity of nitroreductase and imaging in tumor cell

Based on the hypoxia prodrug moiety of p-nitrobenzyl, a selective ratiometric fluorescent sensor (RHP) for the detection of microenvironment hypoxia was designed and synthesized. RHP can be selectively activated by bioreductive enzymes (NTR) and results in an evident blue to green fluorescent emission wavelength change in both solution phases and in cell lines, which might be the first fluorescent ratiometric probe for hypoxia in solid tumors.     

Engineering a highly selective probe for ratiometric imaging of H2Sn and revealing its signaling pathway in fatty liver disease

Hydrogen polysulfides (H₂S_n, n > 1) have continuously been proved to act as important signal mediators in many physiological processes. However, the physiological role of H₂S_n and their signaling pathways in complex diseases, such as the most common liver disease, nonalcoholic fatty liver disease (NAFLD), have not been elucidated due to lack of suitable tools for selective detection of intracellular H₂S_n. Herein, we adopted a general and practical strategy including recognition site screening, construction of a ratiometric probe and self-assembly of nanoparticles, to significantly improve the probes'' selectivity, photostability and biocompatibility. The ratiometric probe PPG-Np-RhPhCO selectively responds to H₂S_n, avoiding interaction with biothiol and persulfide. Moreover, this probe was applied to image H₂S_n in NAFLD for the first time and reveal the H₂S_n generation pathways in the cell model of drug-treated NAFLD. The pathway of H₂S_n revealed by PPG-Np-RhPhCO provides significant insights into the roles of H₂S_n in NAFLD and future drug development.

A highly selective probe (PPG-Np-RhPhCO) used for revealing the intracellular H₂S_n signaling pathways in a nonalcoholic fatty liver disease (NAFLD) model.     

A ratiometric fluorescent probe for oxalate based on alkyne-conjugated carboxamidoquinolines in aqueous solution and imaging in living cells

A novel ratiometric fluorescent probe for oxalic acid was designed and synthesized, based on the zinc-containing [DAQZ@2Zn(2+)] complex. It shows highly selective "on-off" fluorescence changes with a more than 20 nm blue shift in wavelength for oxalic acids in aqueous solution. Moreover, it can fluorescently respond to oxalic acid in living cells.     

A two-photon ratiometric fluorescent probe for real-time imaging and quantification of NO in neural stem cells during activation regulation

Developing a novel tool capable of real-time monitoring and accurate quantification of NO is critical to understanding its role in physiological and pathological processes. Herein, a two-photon ratiometric fluorescent probe (NOP) was developed for real-time imaging and quantification of NO based on fluorescence resonance energy transfer-photoinduced electron transfer (FRET-PET). In this developed probe, coumarin (CM) and naphthalimide with o-phenylenediamine (NPM) were rationally designed as a fluorescent donor and acceptor, respectively, to enable a ratiometric fluorescence response to NO. The developed NO probe demonstrated good detection linearity with the concentration of NO in the range of 0.100–200 μM, with a detection limit of 19.5 ± 1.00 nM. Considering the advantages of high selectivity, good accuracy and rapid dynamic response (<15 s), the developed NO probe was successfully applied for real-time imaging and accurate quantification of NO in neural stem cells (NSCs) and different regions of mouse brain tissue with a penetration depth of 350 μm. Using this powerful tool, it was found that NO regulated the activation and differentiation of quiescent NSCs (qNSCs). In addition, NO-induced differentiation of qNSCs into neurons was found to be dose-dependent: 50.0 μM NO caused about 50.0% of qNSCs to differentiate into neurons. Moreover, different regions of the mouse brain were observed to be closely related to the concentration of NO, and the concentration of NO in the DG region was found to be lower than that in the S1BF, CA1, LD and CPu of the Alzheimer''s disease (AD) mouse brain. The symptoms of AD mice were significantly improved through the treatment with NO-activated NSCs in the DG region.

Developing a novel tool capable of real-time monitoring and accurate quantification of NO is critical to understanding its role in physiological and pathological processes.