Fluorescein Derivatives Research Articles

A turn-on TADF chemosensor for sulfite with a microsecond-scale luminescence lifetime.

A reaction-based luminescence chemosensor was synthesized for sulfite detection based on a fluorescein derivative with thermally activated delayed fluorescence (TADF). The chemosensor exhibited a fluorescence turn-on effect on sulfite with good sensitivity and selectivity. Importantly, utilizing the long luminescence lifetime of the TADF compound, the chemosensor realized photoluminescence lifetime imaging for sulfite in living cells with the luminescence lifetime distribution mainly around 14 μs.

Фотофизические процессы в молекулах галогенпроизводных флуоресцеина в анионных обратных мицеллах

Studies of photophysical processes in aqueous micellar solutions of halogen derivatives of fluorescein: Eosin( E), Erythrosin (ER) and Bengal Rose (BR) by methods of dynamic light scattering, stationary and time-resolved fluorescence spectroscopy were carried out. It was found that the introduction of dye molecules into reverse AOT micelles causes an increase in their hydrodynamic radii Rh. The time-resolved fluorescence of the studied dye molecules in reverse micelles was measured. A decrease in the average time of the excited state with an increase in Rh for E, ER, and BR was found, which is associated with an increase in the mobility of water molecules and a decrease in the effect of geometric restriction of dye molecules. The degrees of anisotropy of the fluorescence r of dye molecules in reverse micelles were measured. It was shown that in micellar systems r is greater than in aqueous solutions and decreases with increasing Rh. The rotational correlation time for the studied dye molecules in micellar systems is determined, which decreases for all the studied dyes with an increase in Rh, indicating a decrease in the microviscosity of the confined aqueous medium inside the micelle. In this case , i.e., the value of the time of the rotational correlation is affected by the "heavy atom effect".

Aggregation-induced emission and solid fluorescence of fluorescein derivatives.

We report herein for the first time that the aggregation-induced emission and strong solid fluorescence of fluorescein derivatives can be realized by slightly modifying their structure, which provides a new option for AIEgens and solid fluorescent materials.

A spiropyran functionalized fluorescent probe for mitochondria targeting and imaging of endogenous hydrogen sulfide in living cells.

A turn-on spiropyran functionalized fluorescein derivative (FMC) is developed for targeting HS- in mitochondria. FMC exhibits very weak fluorescence at 525 nm under the excitation of 470 nm in aqueous solution due to its colorless spiropyran form; upon addition of HS-, a strong fluorescence enhancement by 6.4-fold is observed with spirocycle-opened merocyanine form and rapid trapping kinetics for HS-. FMC has good biocompatibility and high selectivity towards HS- with a detection limit of 88.2 nM and is very sensitive among the reported H2S fluorescent probes. Moreover, the significant colocalization of FMC with Mito Tracker® Deep Red FM in human laryngeal epidermoid carcinoma (HEp-2) cells and the Pearson correlation coefficient of 0.87 together demonstrate that FMC can target and image the endogenous H2S in the mitochondria of living cells.

Cancer cell recognition by a Cys-reactive turn-on significant enhanced fluorescent emission targeting biotin receptors

Cancer is a leading cause of death worldwide. The discrimination of cancer cells from normal cells is of importance for early diagnosis. Designing and synthesizing fluorescent probes that precisely target cancer cells can greatly aid treatment efforts. Biotin can target over-expressed receptors on the surface of cancer cells. In addition, the abundant free biothiols in cells play important roles in many physiological and pathological processes. We therefore developed a fluorescein derivative probe possessing an acryloyl group as a reaction site for detecting biothiol and biotin as a cancer cell-targeting agent. Thus, cancer cells over-expressing the biotin receptor are labeled via a fluorescence signal significant enhancement from the reaction product of the probe and Cys, enabling the probe (Ac) to distinguish cells via imaging. The probe is successfully applied to distinguish among four cell types, Hela, B16, RAW264.7 and NIH-3T3.

Low-concentration trypsin detection from a metal-enhanced fluorescence (MEF) platform: Towards the development of ultra-sensitive and rapid detection of proteolytic enzymes.

Proteolytic enzymes, which serve to degrade proteins to their amino acid building blocks, provide a distinct challenge for both diagnostics and biological research fields. Due to their ubiquitous presence in a wide variety of organisms and their involvement in disease, proteases have been identified as biomarkers for various conditions. Additionally, low-levels of proteases may interfere with biological investigation, as contamination with these enzymes can physically alter the protein of interest to researchers, resulting in protein concentration loss or subtler polypeptide clipping that leads to a loss of functionality. Low levels of proteolytic degradation also reduce the shelf-life of commercially important proteins. Many detection platforms have been developed to achieve low-concentration or low-activity detection of proteases, yet many suffer from limitations in analysis time, label stability, and ultimately sensitivity. Herein we demonstrate the potential utility of fluorescein derivatives as fluorescent labels in a new, turn-off enzymatic assay based on the principles of metal-enhanced fluorescence (MEF). For fluorescein sodium salt alone on nano-slivered 96-well plates, or Quanta Plates™, we report up to 11,000x enhancement for fluorophores within the effective coupling or enhancement volume region, defined as ~100 nm from the silver surface. We also report a 9% coefficient of variation, and detection on the picomolar concentration scale. Further, we demonstrate the use of fluorescein isothiocyanate-labeled YebF protein as a coating layer for a MEF-based, Quanta Plate™ enzymatic activity assay using trypsin as the model enzyme. From this MEF assay we achieve a detection limit of ~1.89 ng of enzyme (2.8 mBAEE activity units) which corresponds to a minimum fluorescence signal decrease of 10%. The relative success of this MEF assay sets the foundation for further development and the tuning of MEF platforms for proteolytic enzyme sensing not just for trypsin, but other proteases as well. In addition, we discuss the future development of ultra-fast detection of proteases via microwave-accelerated MEF (MAMEF) detection technologies.

A dual-targeted theranostic photosensitizer based on a TADF fluorescein derivative

Specific diagnosis and therapy of cancer is still a challenge in biomedical research. Photodynamic therapy (PDT) has emerged as a novel therapeutic modality for cancer treatment. However, the traditional PDT photosensitizers often exhibit low specific selectivity. In this study, we have reported a dual-targeted theranostic photosensitizer FL-RGD by covalently conjugating tumor marker cyclic arginine-glycine-aspartic acid tripeptide (RGD) and a fluorescein derivative FL which has a property of thermally activated delayed fluorescence (TADF) and a long triplet lifetime for efficient PDT. The FL-RGD can target tumor tissues and further locate lysosomes of tumor cells to concurrently achieve the cancers' specific diagnosis and efficient treatment. The mechanism of its highly efficient PDT was attributed to the damage of lysosome via 1O2. Besides, FL-RGD has the potential to be utilized in depth imaging and treatment by two-photon excitation. The actual diagnosis performance of FL-RGD was proved by fluorescence imaging of living cells and tumor bearing mice. The therapy performance was proved by MTT assays, fluorescence-activated cell sorting (FACS) analysis and PDT experiments on tumor bearing mice. The research obviously exhibited the potential of FL-RGD for tumor theranostics in vivo and in vitro.

Fluorescent Phosphorus Dendrimers: Towards Material and Biological Applications.

Fluorescent derivatives of phosphorhydrazone dendrimers are reviewed. Diverse types of fluorophores have been used, such as pyrene, naphthol, anthracene, dansyl, diketone, phthalocyanine, maleimide, julolidine, rhodamine, fluorescein, or fluorene derivatives. The fluorescent groups can be located either as terminal groups on the surface, at the core, linked to the core (off-center), or to the branches of the dendritic structure. After fundamental research on their synthesis, these compounds have been used in the fields of catalysis, nanomaterials, OLEDs, sensors and biology/nanomedicine, in particular for monitoring transfection, or for their anti-inflammatory or anti-cancer properties.

Two chemodosimeters for fluorescence recognition of biothiols in aqueous solution and their bioimaging application

Two fluorescein derivatives containing 2,4-dinitrobenzenesulfonyl group have been developed as fluorescent probes to detect the biothiols (Cys, Hcy and GSH) in aqueous solution. Probes 1 and 2 can distinguish these biothiols in the presence of other amino acids. While probe 1 can recognize the biothiols in PBS/DMSO (v:v = 95:5, pH = 7.40) solution, notably probe 2 could be used in PBS buffer solution (pH = 7.40). The detection limit of Cys for probe 2 reached at 0.021 μM in aqueous solution, which was lower than the intracellular concentration of Cys. In the recognition process, a reaction between the probes and the biothiols occurred, in which the S–O bond was cleaved to remove 2, 4-dinitrobenzenesulfonyl group. The data of 1H NMR, MS and DFT/TD-DFT calculation further confirmed the detection mechanism. Moreover, two probes were successfully applied to the HeLa cell imaging.

Abstract 217: Development of a rapid and efficient methodology to quantitate thiol detection in antibody-drug conjugates (ADCs)

Abstract Antibody-Drug Conjugates (ADCs) are comprised of three distinct components, an antibody, a payload (usually a cytotoxic agent) and a linker (a synthetic chemical chain joining the antibody and payload). Payloads can be conjugated to antibodies in either a stochastic or site-specific manner, with stochastic conjugations usually accomplished through either amine groups of lysine residues or through thiol groups in disulfide bonds located in the hinge region of antibodies. In the latter case, an electrophilic Michael acceptor grouping (e.g., maleimide) of a linker-payload construct reacts with a nucleophilic thiol group of the antibody, forming the ADC. In order for this to occur, disulfide bonds need to be reduced to free thiols. However, knowing the concentration of reactive thiols at different stages during the reduction process can be challenging, and so a more accurate assessment of thiol concentration may allow greater control of the desired average Drug-Antibody Ratio (DAR). Currently, different spectrophotometric (i.e. Ellman’s reagent) and fluorometric assays are used to monitor thiol concentration, but these methods can be expensive, time consuming and inaccurate. To address these issues, a rapid and efficient fluorescence-based method has been developed to allow the reduction of thiols to be monitored. The method is based on a fluorescein derivative produced via a four-step synthesis starting from commercially available fluorescein sodium salt. In its unreacted form, this novel probe exists in a lactone form. However, once it reacts with a free thiol group, it converts stoichiometrically via opening of the lactone ring to a fluorescent form, thus allowing quantitative determination of the concentration of thiol groups present in solution. The three antibodies used to trial this procedure (i.e., Trastuzumab, Cetuximab and Rituximab) were reduced using TCEP over a 120 minute period. Aliquots of solution were collected at various time intervals, and incubated with the fluorescein probe for 5 minutes, monitoring fluorescence with a simple fluorimeter. Using this approach, it was possible to accurately assess thiol concentration at each time interval, and relate the overall reduction profile of each antibody to the concentration of reducing agent used. Payloads were then conjugated to the three antibodies at specific timepoints (selected through monitoring thiol reduction) in an effort to generate species with a defined DAR. Overall, the resulting ADCs were found to have average DAR profiles consistent with the measured number of thiols in the reduced antibodies. When compared with other assays, the developed method gave results with improved reproducibility. Furthermore, the fluorescein probe was less complex and faster to use than other fluorometric kits, and was easy to synthesize, making it a useful new tool in the generation of ADCs. Citation Format: Ilona Pysz, Paolo Andriollo, Paul J. Jackson, K M. Rahman, David E. Thurston. Development of a rapid and efficient methodology to quantitate thiol detection in antibody-drug conjugates (ADCs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 217.

An efficient chemodosimeter for Cu(II) ions based on hydrolysis of fluorescein and its utility in live cell imaging

A new NBD-based chemodosimeter system bearing fluorescein derivative has been synthesized for the detection of Cu(II) ions. The spirocyclic form of molecular system bearing a cleavable active bond performs as a fluorescent probe for Cu2+ ion selectively towards other metal ions. The sensing mechanism of Cu2+ with fluorescein hydrazide supports hydrolytic cleavage of the amide bond, hence causing the release of fluorophore (fluorescein) and enhancement of fluorescence. The proposed chemodosimeter displays the selective detection of Cu2+ ion and has been effectively utilised for live cell imaging of Cu2+ in HeLa cells.

Hybrid fluorophores-based fluorogenic paper device for visually high-throughput detection of Cu2+ in real samples

Paper-based diagnostic assay is quite popular in point-of-care testing (POCT). In this work, we reported a series of hybrid fluorophores based on fluorescein derivatives and investigated their fluorescent properties in detail. To extend the practical application of these hybrid fluorophores, we further constructed the corresponding probes (FN1-FN4) for Cu2+ detection. In comparison with the other three probes, FN4 showed high selectivity and sensitivity, as well as response to Cu2+ within 20 min. Particularly, by introducing this probe into the paper-based device, we could achieve simple, high-throughput, visual and highly sensitive platform for POC detection of Cu2+. Finally, we assessed the paper-based device in real samples such as serum, cytochylema and lake water to demonstrate its promising application. As a result, satisfied performance for Cu2+ detection was achieved, which indicates the great potential of our strategy in the construction of POCT system for detecting analytes.

A Wide Range of pH-Sensitive Fluorescent Probe based on Responsive Double Hydrophilic Block Copolymer for Real-Time Intracellular pH Sensing in Water Samples

In this work, a wide range of pH-sensitive fluorescent probe has been designed by the responsive double hydrophilic block copolymer (DHBCs), which containing the fluorescein (Methacrylic salicylaldoxime, SHMa) and rhodamine (Rhodamine B methacryl hydrazide, RhSAMa) derivatives in the DHBCs. The pH-sensitive fluorescent probe gave blue and red fluorescence emissions at basic pH and acidic pH, respectively. Moreover, the fluorescent probe exhibited changes in fluorescence intensity with an alteration of turbidity of the block copolymer induced by temperature. Furthermore, we have developed a model that combines with the mobile phone for quantitative and qualitative measurement of pH value. The color extract APP for real-time pH measurement in mobile phone was constructed and used to detect the pH of the water samples.

Dark Sweet Cherry phenolics as Dietary Chemopreventive/therapeutic Compounds for Aggressive Breast Cancer Cell Growth with No Toxicity to Normal Breast Cells (FS13-03-19)

ObjectivesDark sweet cherries (DSC) (Prunus avium) contain phenolic compounds with chemopreventive and chemotherapeutic potential. This study aimed to evaluate the effect of DSC extracts in inhibiting the most aggressive breast cancer subtypes without toxicity to normal cells. MethodsDSC phenolic extracts (WE) or its fractions enriched in phenolic acids (F1), anthocyanins (F2), flavonols (F3) or procyanidins (F4) were assessed for their antiproliferative activity against the most aggressive breast cancer BT-474, MDA-MB-453, and MDA-MB-231 cell lines, and MCF-10A noncancer breast epithelial cells using the resazurin assay. Cell levels of oxidative stress were assessed with fluorescein derivative H2DFFDA. Expression of genes linked to cell growth, apoptosis, and metastasis was assessed. ResultsDSC phenolics in whole extract (WE) and fractions enriched in phenolic acids (F1), flavonols (F3) and procyanidins (F4) inhibited MDA-MB-453 with higher potency (lower IC50values) compared to the other cell lines tested (P < 0.05) followed by MDA-MB-231 ∼ BT-474, without toxicity to MCF-10A cells. F3 inhibited BT-474 and MCF-10A cells with similar potency (P > 0.05). F2 inhibited all assessed breast cancer cell lines with similar potencies without toxicity to MCF-10A cells. Mechanisms for WE, F2 and F4 inhibitory activity on MDA-MB-453 cells were mediated by reduction of ROS (down to 28%, 56%, and 58% of control, respectively); which mediate proliferation and other events required for tumor progression. This was accompanied by downregulation of Akt/mTOR, p38, and survivin mRNA, suggesting antiproliferative and apoptotic mechanisms. An enhanced chemopreventive/chemotherapeutic potential of DSC anthocyanins is suggested because mRNA levels of invasive/metastatic biomarkers Sp1, Sp4, and VCAM-1 were downregulated only by F2. ConclusionsThese results demonstrate the chemopreventive/chemotherapeutic potential of DSC phenolics and fractions enriched in anthocyanins and procyanidins as natural compounds to target cell pathways linked to proliferation, apoptosis and invasion in breast cancer. The underlying molecular mechanisms are currently under investigation. Funding SourcesThis work was supported by the Northwest Cherry Growers.

Surface modification of carbon quantum dots by fluorescein derivative for dual-emission ratiometric fluorescent hypochlorite biosensing and in vivo bioimaging

Surface modification of carbon quantum dots by fluorescein (FH-GA-CQDs) were synthesized for dual-emission ratiometric fluorescent ClO− biosensing and in vivo bioimaging. In the presence of ClO−, FH-GA-CQDs solution shows gradually decrease in the emission intensity at 413 nm and rapidly increase in the emission at 508 nm, corresponding to the blue-to-green emission color change. The detection mechanism is that the ClO− induces the ring-opening of FH units to form an oxadiazole structure, resulting in energy transferring from CQDs moiety to FH units. The energy transfer efficiency and energy transfer rate were estimated as 0.34 and 4.00 × 10−7 s−1, respectively. The results reveal that the FH-GA-CQDs have high sensitivity and selectivity for ClO− in the linear range extending from 0 to 70 μM with a detection limit as low as 93 nM. Meanwhile, the potency of the FH-GA-CQDs detecting ClO− in real water examples were validated by recoveries from 94% to 106% and semi-quantification determination of ClO− on fluorescent test strips. Most importantly, FH-GA-CQDs display low cytotoxicity and were successfully applied for sensing of ClO− in living cells and imaging of ClO− in nude mice.

Selective two way Cd(II) and Co(II) ions detection by 1,2,3–triazole linked fluorescein derivative

This report presents unique “on-off” system for the detection of Cd(II) and Co(II) metal ion using 1,2,3-triazole linked fluorescein chemosensor (FLT). The studies were performed in 4:1 ethanol/water (v/v) solution and present an excellent “turn-off” and “turn-on” response with the Cd(II) and Co(II) metal ions, respectively. The need to develop FLT sensor was propelled by the high toxicity of Cd(II) and Co(II) ions in water and their competitive detection, with the naked eye, in aqueous solution of metal ion mixture. The high selectivity and specificity of 1,2,3-triazole containing FLT probe, consisting of fluorescein backbone, owes to Photo Electron transfer (PET) mechanism.

Nitroreductase-Activatable Theranostic Molecules with High PDT Efficiency under Mild Hypoxia Based on a TADF Fluorescein Derivative.

High specificity detection and site-specific therapy are still the main challenges for theranostic anticancer prodrugs. In this work, we reported two smart activatable theranostic molecules based on a thermally activated delayed fluorescence fluorescein derivative. Nitroreductase induced by a mild hypoxia microenvironment of a solid tumor was used to activate the fluorescence and photodynamic therapy (PDT) efficiency by employing the intramolecular photoinduced electron transfer mechanism. A high PDT efficiency under 10% oxygen concentration was achieved, which is better than that of porphyrin (PpIX), a traditional photosensitizer. Such an excellent PDT efficiency can be attributed to lysosome disruption because the theranostic molecule can specifically enter the lysosomes of cells. Importantly, the strategy of targeting the mild hypoxic cells in the edge of tumor tissue could heal the "Achilles' heel" of traditional PDT. We believe that this theranostic molecule has a high potential to be applied in clinical investigation as a theranostic anticancer prodrug.

Quantitative monitoring of the cytoplasmic release of NCp7 proteins from individual HIV-1 viral cores during the early steps of infection

Fluorescence microscopy imaging of individual HIV-1 viruses necessitates a specific labeling of viral structures that minimally perturbs the infection process. Herein, we used HIV-1 pseudoviruses containing NCp7 fused to a tetracystein (TC) tag, labeled by a biarsenical fluorescein derivative (FlAsH) to quantitatively monitor the NCp7 protein concentration in the viral cores during the early stages of infection. Single particle imaging of individual pseudoviruses with defined ratios of TC-tagged to non tagged NCp7 proteins, together with theoretical modeling of energy transfer between FlAsH dyes, showed that the high packaging of TC-tagged proteins in the viral cores causes a strong fluorescence quenching of FlAsH and that the fluorescence intensity of individual viral complexes is an appropriate parameter to monitor changes in the amount of NCp7 molecules within the viral particles during infection. Interestingly, we observed a dramatic fluorescence increase of individual FlAsH-labeled pseudoviruses containing 100% TC-tagged NCp7 proteins in infected cells at 8 and 16 h post-infection. This effect was significantly lower for pseudoviruses expressing TC-tagged integrase. Therefore, this fluorescence increase is likely related to the cytoplasmic viral transformation and the release of NCp7 molecules from the viral complexes. This loss of quenching effect is largely reduced when reverse transcriptase is inhibited, showing that NCp7 release is connected to viral DNA synthesis. A spatial analysis further revealed that NCp7-TC release is more pronounced in the perinuclear space, where capsid disassembly is thought to be completed. Quantification of NCp7-TC content based on fluorescence quenching presented in this study evidences for the first time the cytoplasmic release of NCp7 during the remodeling of HIV-1 viral particles on their journey toward the nucleus. The developed approach can be applied to quantify dye concentrations in a wide range of nano-objects by fluorescence microscopy techniques.

Red-to-blue photon up-conversion with high efficiency based on a TADF fluorescein derivative

A new photon up-conversion system with a TADF fluorescein derivative as a photosensitizer was developed to achieve a quite large anti-Stokes shift from red to blue with a fairly high up-conversion emission quantum yield. This TADF photosensitizer has a very small ΔEST to provide a 207 nm anti-Stokes shift photon up-conversion. Meanwhile, it has a quite long triplet state lifetime (22.11 μs) and a large molar extinction coefficient to assure a high up-conversion efficiency of 11.2%. These results fully demonstrated the potential of the TADF fluorescein derivative as the photosensitizer used in photon up-conversion for the first time.

Förster Resonance Energy-Transfer-Based Ratiometric Fluorescent Indicator for Quantifying Fluoride Ion in Water and Toothpaste.

A Förster resonance energy-transfer (FRET)-based ratiometric fluorescent indicator Cou-FITC-Si toward fluoride ion has been designed and synthesized by combining coumarin unit and fluorescein derivative as energy donor and acceptor, respectively. The fluorescein unit is capped with tert-butyldiphenylchlorosilane. The indicator gives out emission responses based on switch-on of the FRET process that triggered by the desilylation mediated by the fluoride ion. The fluorescence emission spectrum of Cou-FITC-Si presents a significant bathochromic shift of 59 nm after the addition of fluoride ion with up to 180-fold increase of the fluorescence intensity ratio. The limit of detection of the Cou-FITC-Si indicator system toward fluoride ion was estimated to be 3.3 ppb. Furthermore, this indicator has been successfully applied for quantifying the fluoride ion of different concentrations from commercially available toothpaste.