Fluorogenic Response Research Articles

Direct Real-Time Monitoring of Prodrug Activation by Chemiluminescence.

The majority of theranostic prodrugs reported so far relay information through a fluorogenic response generated upon release of the active chemotherapeutic agent. A chemiluminescence detection mode offers significant advantages over fluorescence, mainly due to the superior signal-to-noise ratio of chemiluminescence. Here we report the design and synthesis of the first theranostic prodrug monitored by a chemiluminescence diagnostic mode. As a representative model, we prepared a prodrug from the chemotherapeutic monomethyl auristatin E, which was modified for activation by β-galactosidase. The activation of the prodrug in the presence of β-galactosidase is accompanied by emission of a green photon. Light emission intensities, which increase with increasing concentration of the prodrug, were linearly correlated with a decrease in the viability of a human cell line that stably expresses β-galactosidase. We obtained sharp intravital chemiluminescent images of endogenous enzymatic activity in β-galactosidase-overexpressing tumor-bearing mice. The exceptional sensitivity achieved with the chemiluminescence diagnostic mode should allow the exploitation of theranostic prodrugs for personalized cancer treatment.

Polyamine-Functionalized Polydiacetylene (PDA) Vesicles for Colorimetric Sensing of Carbon Dioxide

Ocean acidification resulting from anthropogenic CO2 has led to severe threats to marine biodiversity and ecosystems. Therefore, an effective CO2 sensing system is necessary for marine environment monitoring. In this study, polyaminefunctionalized polydiacetylene was synthesized via the conjugation of 10,12-tricosadiynoic acid (TRCDA) monomers with diethylenetriamine (DETA), and its capability for CO2 detection was demonstrated. The structure of TRCDA-DETA was confirmed using nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. Then, the colorimetric and fluorogenic responses of TRCDA-DETA vesicles were examined by applying CO2. A specific color transition with a colorimetric response (CR%) of 34.39±1.46 was observed within 3 min of CO2 exposure as well as detecting fluorescent response upon CO2 detection coincidently. These results indicated that TRCDA-DETA vesicles can be an effective tool for CO2 detection, and their unique properties may have potential applications in multiple fields.

Real time detection of the nerve agent simulant diethylchlorophosphate by nonfluorophoric small molecules generating a cyclization-induced fluorogenic response.

Chemical warfare agents (CWA) are some of the most nefarious weapons, and their possible use in terrorist attacks has led to growing interest in the development of reliable and accurate methods to detect these lethal chemicals. In this paper, we have prepared three nonfluorophores containing 2-(2-hydroxybenzylidene)-malononitrile with various 5-substituents as chemosensor probes for a nerve-agent simulant, diethylchlorophosphate (DCP). The phenolic group of the probes, as the active site, can be rapidly phosphorylated by DCP via nucleophilic substitution and then undergo a simultaneous intramolecular cyclization reaction within 45 s, which can respond to DCP in turn-on fluorescence mode. To the best of our knowledge, this is the first report on the utilization of non-fluorophoric small molecule species to generate a fluorogenic response from DCP along with an investigation of substituent effects on the sensing response. The detection process can be visualized by the naked eye and under a UV lamp the probe HNBM exhibits a strong green fluorescence upon interaction with DCP. Among the three chemosensors, probe HNBM displayed several beneficial attributes such as an extremely fast response time along with high selectivity, sensitivity and the lowest limit of detection (0.10 μM) with DCP in solution. TDDFT calculations were performed in order to demonstrate the electronic properties of the probe and the cyclized product. Furthermore, the probe was used to develop a low cost portable cellulose paper strip for real-time visual detection of DCP vapor. Also, the probe has been extensively used to detect DCP in live cells.

A fluorogenic probe for in vitro and in vivo detection of biothiols and vitamin-C with an in-depth mechanistic understanding

Thiol-containing biomolecules like Cysteine (Cys), Homocysteine (Hcy), and reduced Glutathione (rGSH) are involved in various biological processes. In addition, vitamin-C (ascorbic acid, AA), being a common antioxidant, plays a significant role in various biological reactions. In vivo detection of these biologically relevant small molecules by using a fluorogenic probe provides a pivotal tool to unravel real-time biomolecular dynamics. The development of a cell-permeable, water-soluble and non-toxic fluorogenic molecules are always advantageous for their spatiotemporal sensitivity and accuracy. Although the application of fluorogenic probes is widely studied, the deeper investigation of underlying mechanisms has remained curtained. In this report, a fluorogenic probe, 4-azido-7-nitrobenzo-2-oxa-1,3-diazole (NBD-N3), has been developed for detection of AA, Cys, Hcy among a range of biologically relevant molecules including rGSH with an extensive mechanistic investigation. The weakly fluorescent NBD-N3 transforms into a highly fluorescent NBD-NH2 with the action of AA by redox mechanism. However, Cys, and Hcy convert NBD-N3 into highly fluorescent NBD-NHR (R=Cys/Hcy) via aromatic nucleophilic substitution (SNAr) followed by S-N rearrangement. The reaction mechanisms for the fluorogenic response have also been authenticated by UV–vis., fluorescence, NMR spectroscopy and mass spectrometry. Furthermore, the probe has also been employed to validate in vitro and in vivo monitoring of biothiols and AA in mammalian cancer cells and in multicellular living Caenorhabditis elegans.

Spirohydrazine rhodamine as a fluorescent chemodosimeter for the selective detection of Cu(II) ions and its application in live cell imaging

A rhodamine-based fluorescent chemdosimeter SHR for Cu2+ has been developed based on the spirocyclic ring-opening mechanism. SHR is colorless and nonfluorescent due to intramolecular spirocyclization. Upon incubation with Cu2+ in aqueous media, a highly efficient ring opening of SHR occurs via Cu2+-promoted phenylhydrazine oxidation, causing a dual chromogenic and fluorogenic response. The fluorescent intensity at 580nm increases linearly with Cu2+ concentration and as low as 10nM of Cu2+ can be readily detected. The proposed chemodosimeter shows high selectivity toward Cu2+ over other metal ions and proves to be able to image Cu2+ levels in live cells.

Generation of Triplet Excited States via Photoinduced Electron Transfer in meso-anthra-BODIPY: Fluorogenic Response toward Singlet Oxygen in Solution and in Vitro.

Heavy atom-free BODIPY-anthracene dyads (BADs) generate locally excited triplet states by way of photoinduced electron transfer (PeT), followed by recombination of the resulting charge-separated states (CSS). Subsequent quenching of the triplet states by molecular oxygen produces singlet oxygen (1O2), which reacts with the anthracene moiety yielding highly fluorescent species. The steric demand of the alkyl substituents in the BODIPY subunit defines the site of 1O2 addition. Novel bis- and tetraepoxides and bicyclic acetal products, arising from rearrangements of anthracene endoperoxides were isolated and characterized. 1O2 generation by BADs in living cells enables visualization of the dyads distribution, promising new imaging applications.

Extending photophysical behavior of Schiff base tripod for the speciation of iron and fabrication of INHIBIT type molecular logic gate for fluorogenic recognition of Zn(II) and Cd(II) ions

A tripodal Schiff base derived by the condensation of tris(2-aminoethyl)amine and 2-hydroxynaphthaldehyde, and characterized by single crystal X-ray diffraction has been explored herein as a chemosensor for metallic species. Although, its fluorogenic response towards Zn2+ (in 95:5 ethanol–water solvent) has been already reported, however, its utilization for the naked eye speciation of iron and fluorogenic response towards Cd2+ ions needs to be explored. It exhibits pronounced fluorescence signaling for Zn2+ and Cd2+ as well as chromogenic response towards Fe3+, Fe2+, Zn2+ and Cd2+ ions. The addition of metallic cations induces a remarkable colorimetric response from yellow to colorless (for Zn2+, Cd2+), purple (for Fe2+) and light green (for Fe3+) visible to naked eye. Interestingly, the sensor can easily differentiate two states of iron (Fe3+ from Fe2+) by revealing distinctive colors and different absorption maxima. The detection limit of the sensor towards Zn2+/Cd2+ is low down to nanomolar concentration (86nM and 50nM, respectively). Moreover, the sensor can be applied for the fabrication of fluorogenic molecular switch, which turns “ON” upon selective binding with Zn2+/Cd2+ ions and turns “OFF” in the simultaneous presence of Zn2+/Cd2+ and IO4- ion. Thus, it is potentially significant sensor and should be explored further for environmental applications.

Solvatochromic and Fluorogenic Dyes as Environment-Sensitive Probes: Design and Biological Applications.

Fluorescent environment-sensitive probes are specially designed dyes that change their fluorescence intensity (fluorogenic dyes) or color (e.g., solvatochromic dyes) in response to change in their microenvironment polarity, viscosity, and molecular order. The studies of the past decade, including those of our group, have shown that these molecules become universal tools in fluorescence sensing and imaging. In fact, any biomolecular interaction or change in biomolecular organization results in modification of the local microenvironment, which can be directly monitored by these types of probes. In this Account, the main examples of environment-sensitive probes are summarized according to their design concepts. Solvatochromic dyes constitute a large class of environment-sensitive probes which change their color in response to polarity. Generally, they are push-pull dyes undergoing intramolecular charge transfer. Emission of their highly polarized excited state shifts to the red in more polar solvents. Excited-state intramolecular proton transfer is the second key concept to design efficient solvatochromic dyes, which respond to the microenvironment by changing relative intensity of the two emissive tautomeric forms. Due to their sensitivity to polarity and hydration, solvatochromic dyes have been successfully applied to biological membranes for studying lipid domains (rafts), apoptosis and endocytosis. As fluorescent labels, solvatochromic dyes can detect practically any type of biomolecular interactions, involving proteins, nucleic acids and biomembranes, because the binding event excludes local water molecules from the interaction site. On the other hand, fluorogenic probes usually exploit intramolecular rotation (conformation change) as a design concept, with molecular rotors being main representatives. These probes were particularly efficient for imaging viscosity and lipid order in biomembranes as well as to light up biomolecular targets, such as antibodies, aptamers and receptors. The emerging concepts to achieve fluorogenic response to the microenvironment include ground-state isomerization, aggregation-caused quenching, and aggregation-induced emission. The ground-state isomerization exploits, for instance, polarity-dependent spiro-lactone formation in silica-rhodamines. The aggregation-caused quenching uses disruption of the self-quenched dimers and nanoassemblies of dyes in less polar environments of lipid membranes and biomolecules. The aggregation-induced emission couples target recognition with formation of highly fluorescent dye aggregates. Overall, solvatochromic and fluorogenic probes enable background-free bioimaging in wash-free conditions as well as quantitative analysis when combined with advanced microscopy, such as fluorescence lifetime (FLIM) and ratiometric imaging. Further development of fluorescent environment-sensitive probes should address some remaining problems: (i) improving their optical properties, especially brightness, photostability, and far-red to near-infrared operating range; (ii) minimizing nonspecific interactions of the probes in biological systems; (iii) their adaptation for advanced microscopies, notably for superresolution and in vivo imaging.

Sensitive in vivo Visualization of Breast Cancer Using Ratiometric Protease-activatable Fluorescent Imaging Agent, AVB-620

With the goal of improving intraoperative cancer visualization, we have developed AVB-620, a novel intravenously administered, in vivo fluorescent peptide dye conjugate that highlights malignant tissue and is optimized for human use. Matrix metalloproteinases (MMPs) hydrolyze AVB-620 triggering tissue retention and a ratiometric fluorescence color change which is visualized using camera systems capable of imaging fluorescence and white light simultaneously. AVB-620 imaging visualizes primary tumors and demonstrated high in vivo diagnostic sensitivity and specificity (both >95%) for identifying breast cancer metastases to lymph nodes in two immunocompetent syngeneic mouse models. It is well tolerated and single-dose toxicology studies in rats determined a no-observed-adverse-effect-level (NOAEL) at >110-fold above the imaging and estimated human dose. Protease specificity and hydrolysis kinetics were characterized and compared using recombinant MMPs. To understand the human translation potential, an in vitro diagnostic study was conducted to evaluate the ability of AVB-620 to differentiate human breast cancer tumor from healthy adjacent tissue. Patient tumor tissue and healthy adjacent breast tissue were homogenized, incubated with AVB-620, and fluorogenic responses were compared. Tumor tissue had 2-3 fold faster hydrolysis than matched healthy breast tissue; generating an assay sensitivity of 96% and specificity of 88%. AVB-620 has excellent sensitivity and specificity for identifying breast cancer in mouse and human tissue. Significant changes were made in the design of AVB-620 relative to previous ratiometric protease-activated agents. AVB-620 has pharmaceutical properties, fluorescence ratio dynamic range, usable diagnostic time window, a scalable synthesis, and a safety profile that have enabled it to advance into clinical evaluation in breast cancer patients.

An ultrasensitive fluorogenic probe for revealing the role of glutathione in chemotherapy resistance.

Unveiling the detailed roles of glutathione (GSH) in chemoresistance necessitates a reliable assay for its detection in intact live specimens. Herein, by taking advantage of the susceptibility of electron-poor Csp2 -Ssufinyl bond to GSH nucleophilic attack, we developed a naphthalimide-sulfoxide based fluorogenic probe (Na-8) applicable for tracking endogenous GSH fluctuation in live cells. Na-8 features a high degree of sensitivity towards GSH as demonstrated by its utmost 2200-fold fluorogenic response. As a proof of concept, Na-8 has been applied to image GSH in liver cancer HepG2 cells with the normal L02 cell counterparts serving as a control, and elevated GSH level was observed in HepG2 in contrast to L02. Further experiments showed that this elevated GSH level was involved in doxorubicin-resistance but not in cisplatin-resistance. Noteworthy, monitoring GSH change in HepG2 and L02 cells in response to doxorubicin treatment revealed that while normal cells showed a burst of GSH in adaption to doxorubicin treatment, no significant change was detected in HepG2 cells, suggesting that HepG2 cells have been preconditioned by their intrinsic oxidative stress which confers drug-resistance. Given the observed sensitivity and spatiotemporal resolution, Na-8 should hold promise for future study on the detailed roles of GSH in drug-resistance.

Red-Green-Blue Trichromophoric Nanoparticles with Dual Fluorescence Resonance Energy Transfer: Highly Sensitive Fluorogenic Response Toward Polyanions.

A red-green-blue (RGB) trichromophoric fluorescent organic nanoparticle exhibiting multi-colour emission was constructed; the blue-emitting cationic oligofluorene nanoparticle acted as an energy-donor scaffold to undergo fluorescence resonance energy transfer (FRET) to a red-emitting dye embedded in the nanoparticle (interior FRET) and to a green-emitting dye adsorbed on the surface through electrostatic interactions (exterior FRET). Each FRET event occurs independently and is free from sequential FRET, thus the resultant dual-FRET system exhibits multi-colour emission, including white, in aqueous solution and film state. A characteristic white-emissive nanoparticle showed visible responses upon perturbation of the exterior FRET efficiency by acceptor displacement, leading to highly sensitive responses toward polyanions in a ratiometric manner. Specifically, our system exhibits high sensitivity toward heparin with an extremely low detection limit.

Red-Fluorescent Activatable Probes for the Detection of Hydrogen Peroxide in Living Cells.

Many inflammatory processes are associated with an increase in the production of reactive oxygen species (ROS). Chemical probes that specifically detect ROS are potentially useful tools for the early diagnosis of inflammatory diseases as well as cancer. Herein we have developed a library of coumarin hybrids by condensation of various heterocyclic quaternary salts to a 7-hydroxycoumarin scaffold. From our library we identified one benzothiazole-coumarin hybrid as a red-fluorescent compound with emission maxima around 620 nm and a strong fluorogenic response. Furthermore, we proved that this scaffold is suitable for the preparation of activatable probes, such as by modification with a boronate group for selective sensing of hydrogen peroxide (H2O2). In vitro assays confirmed the reactivity and subsequent emission of our probe upon incubation with H2O2 with good selectivity over different ROS and reactive nitrogen species (RNS) as well as minimal toxicity in cells. Finally cell imaging experiments were performed in murine macrophages and validated the utility of the activatable probe for the detection of H2O2 in living cells.

A Simple and Selective Fluorescent Sensor for Zn(2+) and H(+) Ions in Aqueous Solution with OR Logic Gate Function.

The synthesis and properties of a new compound, viz., (N,N'-[1,10-phenanthroline-4,7-diyldi((E)methylylidene)]bis(1,10-phenanthrolin-5-amine) (1), is described. Compound 1 can be used as a selective fluorescent Zn(2+) sensor in buffered solution. Furthermore, 1 induces turn on fluorogenic response to H(+) ions. Finally, it is shown that an OR logic gate can be constructed with 1 by using Zn(2+) and H(+) as two-inputs. Graphical Abstract In this paper, the design, synthesis and physicochemical properties of a new compound 1 based on 1,10-phenanthroline scaffold, is reported. It is noted that 1 can be used as a selective fluorescent Zn(2+) sensor in 0.01M HEPES buffer containing DMF (2%v/v, pH=7.4) at room temperature. Furthermore, the spectrophotometric results suggest that compound 1 can be used as a pH reporter in highly acidic conditions (pH<5). Finally, it was also shown that an OR logic gate can be constructed with 1 by using Zn(2+) and H(+) as two-inputs.

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.

A Fluorescent Hypochlorite Probe Built on 1,10-Phenanthroline Scaffold and its Ion Recognition Features.

In this study, the synthesis of 7-((Hydroxyimino)methyl)-1,10-phenanthroline-4-carbaldehyde oxime (1) in two steps starting from 4,7-dimethyl-1,10-phenanthroline (2) is reported. It is found that compound 1 can be used as a fluorogenic probe for the detection of hypochlorite ion in aqueous solution. NMR and mass spectral analysis indicate that probe 1 undergoes a chemical transformation through its oxime units upon treatment with hypochlorite, which results in a remarkable enhancement of the emission intensity. Also, metal ion recognition properties of probe 1 is investigated. It is noted that compound 1 is responsive to Zn(2+), Cd(2+), Ni(2+) and Cu(2+) metal ions, which reduced the emission intensity under identical conditions. Graphical Abstract The design, synthesis and properties of a new fluorescent hypochlorite probe is described. It is found that probe 1 immediately undergoes an oxidation reaction with NaClO through its oxime units in 0.1M Na2CO3-NaHCO3 buffer containing DMF (pH=9.0, 30:1v/v) at room temperature, which resulted in a remarkable enhancement of the emission intensity. It is noteworthy that this novel probe 1 is highly selective to hypochlorite ion when compared to some other ROS and anions. On the other hand, probe 1 also induces turn-off fluorogenic responses to metal ions such as Zn(2+), Cd(2+), Ni(2+) and Cu(2+) ions under identical conditions.

Photochromic spirooxazine as highly sensitive and selective probe for optical detection of Fe3+ in aqueous solution

A photochromic spirooxazine derivative was synthesized and evaluated for optical response toward heavy metal ions. The spirooxazine derivative was observed to selectively respond to the presence of Fe3+ ions and produced both chromogenic and fluorogenic response. The spirooxazine derivative produced a ∼100nm shift in the UV–visible spectrum and ∼90 fold enhancement in fluorescence intensity in the presence of one equivalent Fe3+ ions. A large stokes shift (110nm) was also observed on addition of one equivalent Fe3+ ions in methanol or methanol:water (1:1) solvent system.

Azobenzene-caged sulforhodamine dyes: a novel class of ‘turn-on’ reactive probes for hypoxic tumor cell imaging

New sulforhodamine-based fluorescent ‘turn-on’ probes have been developed for the direct imaging of cellular hypoxia. Rapid access to this novel class of water-soluble ‘azobenzene-caged’ fluorophores was made possible through an easily-implementable azo-coupling reaction between a fluorescent primary arylamine derived from a sulforhodamine 101 scaffold (named SR101-NaphtNH2) and a tertiary aniline whose N-substituents are neutral, cationic, or zwitterionic. The detection mechanism is based on the bioreductive cleavage of the azo bond that restores strong far-red fluorescence (emission maximum at 625 nm) by regenerating the original sulforhodamine SR101-NaphtNH2. This valuable fluorogenic response was obtained for the three ‘smart’ probes studied in this work, as shown by an in vitro assay using rat liver microsomes placed under aerobic and then under hypoxic conditions. Most importantly, the probe namely SR101-NaphtNH2-Hyp-diMe was successfully applied for imaging the hypoxic status of tumor cells (A549 cells).

Novel C3V-symmetric trindane based tripodal anion receptor with tris(coumarin-urea) extension for optical sensing of bioactive anions

A novel fluorescent C3V-symmetric tris(coumarin-urea) anion receptor from trindane based tripodal scaffold was synthesized, and its anion recognition behavior was explored by optical methods. Receptor showed H2PO4− and F− selective fluorogenic response among the tested anions. 1H NMR titration study in DMSO-d6 solution reveals that the receptor formed 1:1 caviplex with H2PO4− whereas the most basic F− abstract the urea-NH protons.

Ruthenium(II) and osmium(II) vinyl complexes as highly sensitive and selective chromogenic and fluorogenic probes for the sensing of carbon monoxide in air.

The detection of carbon monoxide in solution and air has been achieved using simple, inexpensive systems based on the vinyl complexes [M(CHCHR)Cl(CO)(BTD)(PPh3 )2 ] (R=aryl, BTD=2,1,3-benzothiadiazole). Depending on the nature of the vinyl group, chromogenic and fluorogenic responses signalled the presence of this odourless, tasteless, invisible, and toxic gas. Solutions of the complexes in CHCl3 underwent rapid change between easily differentiated colours when exposed to air samples containing CO. More significantly, the adsorption of the complexes on silica produced colorimetric probes for the naked-eye detection of CO in the gas phase. Structural data for key species before and after the addition of CO were obtained by means of single X-ray diffraction studies. In all cases, the ruthenium and osmium vinyl complexes studied showed a highly selective response to CO with exceptionally low detection limits. Naked-eye detection of CO at concentrations as low as 5 ppb in air was achieved with the onset of toxic levels (i.e., 100 ppm), thus resulting in a remarkably clear colour change. Moreover, complexes bearing pyrenyl, naphthyl, and phenanthrenyl moieties were fluorescent, and greater sensitivities were achieved (through turn-on emission fluorescence) in the presence of CO both in solution and air. This behaviour was explored computationally using time-dependent density functional theory (TDDFT) experiments. In addition, the systems were shown to be selective for CO over all other gases tested, including water vapour and common organic solvents. Supporting the metal complexes on cellulose strips for use in an existing optoelectronic device allows numerical readings for the CO concentration to be obtained and provision of an alarm system.

Colorimetric and fluorometric discrimination of geometrical isomers (maleic acid vs fumaric acid) with real-time detection of maleic acid in solution and food additives.

Heterobis imine Schiff base probe L is able to discriminate geometrical isomers (maleic acid vs fumaric acid) through sharp colorimetric as well as fluorogenic responses even conspicuous with the naked eye. Colorimetric as well as fluorogenic sensing of maleic acid among various carboxylic acids was also demonstrated in ethanol-buffer medium. Sensing behavior of L was corroborated by (1)H NMR spectra, mass spectrometry, and theoretical calculations. Subsequently sensing behavior of L was used to probe maleic acid in starch rich food samples.