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New Nile Blue Derivatives As NIR Fluorescent Probes and Antifungal Agents †
Proceedings New Nile Blue Derivatives as NIR Fluorescent Probes † and Antifungal Agents Rui P. C. L. Sousa 1,2, João C. C. Ferreira 1,2, Maria João M. F. Sousa 2 and M. Sameiro T. Gonçalves 1,* 1 Centre of Chemistry, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal 2 Centre of Molecular and Environmental Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal * Correspondence: [email protected] † Presented at the 22nd International Electronic Conference on Synthetic Organic Chemistry, 15 November– 15 December 2018; Available Online: https://sciforum.net/conference/ecsoc-22. Published: 14 November 2018 Abstract: The synthesis of four new Nile Blue derivatives with hydrogen, propyl and/or aminopropyl groups as substituents of the amines of 5- and 9-positions is described. Photophysical properties were evaluated in acidified ethanol and aqueous solution at physiological pH. Antifungal activity is also studied through the obtention of MIC values. Keywords: benzo[a]phenoxazines; Nile Blue derivatives; NIR fluorescent probes; antifungal agents 1. Introduction The development of new near-Infrared (NIR) fluorescent probes is a very important issue due to the wide range of applications [1–4]. These probes are an excellent choice to label biological material since its emission will not interfere with the natural fluorescence of biological compounds. Benzo[a]phenoxazinium salts, with Nile Blue being the best known, display fluorescence at around 600 nm and have been used as covalent and non-covalent fluorescent probes for amino acids, proteins and DNA, among other biological material [5–10]. In addition, applications as sensors or agents for photodynamic therapy (PDT) have been described [11,12]. -
The Green Fluorescent Protein
P1: rpk/plb P2: rpk April 30, 1998 11:6 Annual Reviews AR057-17 Annu. Rev. Biochem. 1998. 67:509–44 Copyright c 1998 by Annual Reviews. All rights reserved THE GREEN FLUORESCENT PROTEIN Roger Y. Tsien Howard Hughes Medical Institute; University of California, San Diego; La Jolla, CA 92093-0647 KEY WORDS: Aequorea, mutants, chromophore, bioluminescence, GFP ABSTRACT In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrin- sically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the rela- tion between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories. CONTENTS NATURAL AND SCIENTIFIC HISTORY OF GFP .................................510 Discovery and Major Milestones .............................................510 Occurrence, Relation to Bioluminescence, and Comparison with Other Fluorescent Proteins .....................................511 PRIMARY, SECONDARY, TERTIARY, AND QUATERNARY STRUCTURE ...........512 Primary Sequence from -
Microenvironment-Triggered Dual-Activation of a Photosensitizer
www.nature.com/scientificreports OPEN Microenvironment‑triggered dual‑activation of a photosensitizer‑ fuorophore conjugate for tumor specifc imaging and photodynamic therapy Chang Wang1, Shengdan Wang1, Yuan Wang1, Honghai Wu1, Kun Bao2, Rong Sheng1* & Xin Li1* Photodynamic therapy is attracting increasing attention, but how to increase its tumor‑specifcity remains a daunting challenge. Herein we report a theranostic probe (azo‑pDT) that integrates pyropheophorbide α as a photosensitizer and a NIR fuorophore for tumor imaging. The two functionalities are linked with a hypoxic‑sensitive azo group. Under normal conditions, both the phototoxicity of the photosensitizer and the fuorescence of the fuorophore are inhibited. While under hypoxic condition, the reductive cleavage of the azo group will restore both functions, leading to tumor specifc fuorescence imaging and phototoxicity. The results showed that azo‑PDT selectively images BEL‑7402 cells under hypoxia, and simultaneously inhibits BEL‑7402 cell proliferation after near‑infrared irradiation under hypoxia, while little efect on BEL‑7402 cell viability was observed under normoxia. These results confrm the feasibility of our design strategy to improve the tumor‑ targeting ability of photodynamic therapy, and presents azo‑pDT probe as a promising dual functional agent. Cancer is one of the most common causes of death, and more and more therapeutic strategies against this fatal disease have emerged in the past few decades. Among these strategies, photodynamic therapy has attracted much attention1. Tis therapy is based on singlet oxygen produced by photosensitizers under the irradiation with light of a specifc wavelength to damage tumor tissues (Fig. 1a). Since the photo-damaging efect is induced by the interaction between a photosensitizer and light, tumor-specifc therapy may be realized by focusing the light to the tumor site. -
Exploring the Efficacy of Nile Red in Microplastic Quantification
Letter Cite This: Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX pubs.acs.org/journal/estlcu Exploring the Efficacy of Nile Red in Microplastic Quantification: A Costaining Approach Thomas Stanton,*,†,§ Matthew Johnson,† Paul Nathanail,‡ Rachel L. Gomes,§ Teresa Needham,† and Amanda Burson† †School of Geography, University of Nottingham, NG7 2RD Nottingham, United Kingdom ‡Land Quality Management Ltd, University of Nottingham Innovation Park, NG7 2TU Nottingham, United Kingdom §Food, Water, Waste Research Group, Faculty of Engineering, University of Nottingham, NG7 2RD Nottingham, United Kingdom *S Supporting Information ABSTRACT: The presence of microplastic particles (<5 mm) in the environment has generated considerable concern across public, political, and scientific platforms. However, the diversity of microplastics that persist in the environment poses complex analytical challenges for our understanding of their prevalence. The use of the dye Nile red to quantify microplastics is increasingly common. However, its use in microplastic analysis rarely accounts for its affinity with the breadth of particles that occur in environmental samples. Here, we examine Nile red’s ability to stain a variety of microplastic particles and common natural and anthropogenic particles found in environmental samples. To better constrain microplastic estimates using Nile red, we test the coapplication of a second stain that binds to biological material, 4′,6- diamidino-2-phenylindole (DAPI). We test the potential inflation of microplastic estimates using Nile red alone by applying this costaining approach to samples of drinking water and freshwater. The use of Nile red dye alone resulted in a maximum 100% overestimation of microplastic particles. These findings are of particular significance for the public dissemination of findings from an emotive field of study. -
Determining Lipid Content in Embryos Using Nile Red Fluorescence
Laboratory Procedures, PJ Hansen Laboratory - University of Florida Determining Lipid Content in Embryos using Nile Red Fluorescence Luciano Bonilla and Peter J. Hansen Dept. of Animal Sciences, University of Florida The following protocol is derived from the protocol described by Genicot et al. (Theriogenology 63: 1181- 1194, 2005) and is based on the fluorescence emitted by Nile Red when in association with lipid (see Fowler and Greenspan, J Histochem Cytochem 33: 833-836, 1985). Equipments and Reagents o 96 Well plate: BD Falcon®,cat. # 353912, or equivalent o Nile Red: Invitrogen Molecular Probles, cat. # N-1142 o Hoescht 33342: Sigma-Aldrich, cat. # B2261 o ProLong® Gold antifage reagent: Invitrogen Molecular Probes, cat. # P36934 o Fluorescence microscope: Fluorescent microscope configured with excitation 400-500 nm and emission 515 nm Solutions o 100 mM PO4, pH7.4 (Stock solution) To prepare 100 mM PO4 pH 7.4, add sodium phosphate monobasic (1a) to 300 ml sodium phosphate dibasic until the pH reaches 7.4. Approximately, 100 ml of monobasic solution will be added. 1a. 100 mM sodium phosphate monobasic (NaH2PO4.2H2O) 13.8 g NaH2PO4 1 L double–distilled (dd) ddH2O 1b. 100 mM sodium phosphate dibasic (Na2HPO4) 14.2g Na2HPO4.2H2O 1 L ddH2O o 10 mM PBS/PVP (Work solution) Dilute 100 mM PO4, pH 7.4 with 800 ml ddH2O, add 9 g of NaCl and 1.0 g polyvinylpyrollidone (PVP), and bring volume to 1000 ml with water. Check pH. o 4% Paraformaldahyde Fixative - add 500 mL 8% (w/v) paraformaldehyde to 500 mL PBS- PVP (1:1 dilution) o Nile Red Stock Solution (1 mg/mL) - dissolve 25 mg of Nile Red (Invitrogen N-1142) in 25 mL of DMSO; Store at room temperature in the dark indefinitely. -
Fluorescent Protein-Based Tools for Neuroscience
!1 !2 Fluorescent protein-based tools Outline for neuroscience An animatd primer on biosensor development Fluorescent proteins (FPs) Robert E. Campbell Department of Chemistry Other fluorophore technologies Single FP-based biosensors Imaging Structure & Function in the Nervous System Cold Spring Harbor, July 31, 2019. Lots of structural Lots of structural information information & Transmitted light Fluorescence microscopy of fluorescent color microscopy of live cells live cells No molecular provides molecular information information (more colors = more information) !5 !6 Fluorescence microscopy requires fluorophores Non-natural fluorophores for protein labelling Trends Bioch. Sci., 1984, 9, 88-91. O O O N O N 495 nm 519 nm 557 nm 576 nm - - CO2 CO2 S O O O N O N C N N C S ϕ = quantum yield - - ε = extinction coefficient CO2 CO2 ϕ Brightness ~ * ε S C i.e., for fluorescein N Proteins of interest ϕ = 0.92 N Fluorescein Tetramethylrhodamine ε = 73,000 M-1cm-1 C S (FITC) (TRITC) A non-natural fluorophore must be chemically linked to Non-natural fluorophores made by chemical synthesis a protein of interest… !7 !8 Getting non-natural fluorophores into a cell Some sea creatures make natural fluorophores Trends Bioch. Sci., 1984, 9, 88-91. O O O N O N Bioluminescent - Fluorescent CO2 CO -- S 2 S C N NHN C H S S Chemically labeled proteins of interest Microinjection with micropipet O O O N O N Fluorescent - CO2 CO - S 2 N NH H S …and then manually injected into a cell Some natural fluorophores are genetically encoded proteins http://www.luminescentlabs.org/and can be transplanted into cells as DNA! 228 OSAMU SHIMOMURA, FRANK H. -
Spectroscopic Studies of Nile Red in Organic Solvents and Polymers
,loerllof AND Pl-loitll:lltg.IX~ A: CHI'~[ISTiiY ELSEVIER Journal of Photochemistry and Photobiology A: Chemistry 93 ( 199b.~ 57-64 Spectroscopic studies of nile red in organic solvents and polymers Ashim Kumar Dutta, Kenji Kamada, Koji Ohta * Photonic Chemistry Section, Department of Optical Materiolv, Osaka National Research h~stitute, AIST. Ikeda, Osaka 563, Japan Received 20 March 1995; accepted 14 June 1995 Abstract We have studied tile spectroscopic properties of nile red (NR), a highly fluorescent laser dye, ill organic solvents, binary solvent mixtures and polymers. Spectroscopic studies reveal remarkable changes in the absorption and emission band positions and intensities as a function of the polarity of the medium, Such large dmnges have been attributed to the twisted intramolecular charge transfer (TICT) state of the molecule in polar ntedium. Experimental results show that the molecule is sensitive to the polarity of its microenvironment and is an excellent probe for systems presenting restricted geometries. We have incorporated NR into thin films of poly ( melhyl methacrylate) (PMMA) and poly ( vinyl alcohol) (PVA); it is Ibund that the micropolarity in PVA is greater than that in PMMA; in pv ~., the micropolarity corresponds to that of a binary mixture of acetonitrile and water, whereas in PMMA, the micropolarity corresponds ctosely to that of pure acetonitrile. Keywords: Nile red; Twisted intratnolecular charge transfer (TICT) states; Steady state fluorescence; Time-resolved fluorescence; Aggregation-induced dual fluorescence 1. Introduction films [ 13,14], microparticles [ 15,16] and zeolites [ 17,18], have provided information on the role of the topology of these systems in controlling the photophysical properties of Organic polymers have largely replaced conventional the probe molecules. -
Nile Blue Is a Basic Dye of the Oxazine Group, Which Has Been Used in Several Microscopic and Histochemical Techniques (4, 6)
ACTA HISTOCHEM. CYTOCHEM. Vol. 16, No. 3, 1983 LETTER TO THE EDITOR NILE BLUE SULFATE STAINING FOR DEMONSTRATION OF LIPIDS IN FLUORESCENCE MICROSCOPY Nile blue is a basic dye of the oxazine group, which has been used in several microscopic and histochemical techniques (4, 6) . A staining method based on application of Nile blue sulfate was introduced early for the cytochemical demon- stration of lipids (3, 8, 9), showing neutral fats and fatty acids in red and blue, respectively. There is evidence (1, 10) that aqueous solutions of Nile blue sulfate contain the blue cation of the dye, a red oxidation product (Nile red), and the orange-red imino base. According to this view, phospholipids and fatty acids stain blue because they react with the Nile blue cation, meanwhile neutral fats appear in red color. Other observations seem to be in disagreement with this staining mecha- nism (5, 6) . A fluorescence reaction in tissues after staining with Nile blue sulfate and brilliant cresyl blue has been found by Bozzo and Campos Vidal (2). During the course of investigations by using oxazine dyes we have observed that Nile blue sulfate produces a strong fluorescence reaction in lipid droplets. The fat body of Drosophila larvae is a continuous tissue mass which has been chosen as test material because of its abundance in lipidic inclusions. Drosophila hydei testes, surrounded by the fat body, were fixed in 5 % formaldehyde for 1-24 hr, washed, and then immersed in 0.1 mg/ml aqueous Nile blue sulfate (Fluka) for 30 min. After staining, the material was briefly washed and mounted with a drop of water. -
Study of Wood Pitch Emulsions – Interactions with Nile Red and Influence of Ph Master of Science Thesis in Chemistry and Bioscience
Study of wood pitch emulsions – interactions with Nile red and influence of pH Master of Science Thesis in Chemistry and Bioscience ANNA PALME Department of Chemical and Biological Engineering Division of Applied Chemistry CHALMERS UNIVERSITY OF TECHNOLOGY Göteborg, Sweden, 2011 Study of wood pitch emulsions – interactions with Nile red and influence of pH ANNA PALME Supervisors: Ron Lai and Daniel Persson Examiner: Krister Holmberg Department of Chemical and Biological Engineering Division of Applied Chemistry CHALMERS UNIVERSITY OF TECHNOLOGY Göteborg, Sweden, 2011 Study of wood pitch emulsions – interactions with Nile red and influence of pH. Anna Palme. © Anna Palme, 2011 Department of Chemical and Biological Engineering Division of Applied Chemistry Chalmers University of Technology SE-412 96 Göteborg Sweden Telephone +46 (0)31 772 1000 This work was carried out at Eka Chemicals in Bohus (Sweden). Cover: Left: A density plot from a measurement of Nile red in model pitch emulsion with flow cytometry, showing the red fluorescence intensity versus the forward scattering intensity. Right: The fluorescent probe Nile red. Göteborg, Sweden 2011 Acknowledgements I would like to express my gratitude toward the following people: Ron Lai and Daniel Persson, my supervisors at Eka Chemicals, for their support and engagement in this project. Krister Holmberg for his engagement in the project and for applying a surface chemistry perspective on pitch deposition. The staff at Eka Chemicals for practical assistance in the lab and good company. Anna Palme, Gothenburg, June 2011 Abstract Deposition of wood pitch causes detrimental effects both on the paper and on the paper machine. In this project, it was investigated how a model pitch emulsion is affected by changing the pH from 4 to 8 and back to 4. -
Fluorophore Referenceguide
Fluorophore Reference Guide Fluorophore Excitation and Emission Data Laser Lines Broad UV Excitation Excitation Maxima Emission Maxima Emission Filters 290-365 nm LP = Long pass filter DF = Band pass filter Excel. ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ DAPI: 359 nm ____ SP = Short pass filter Good ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ GFP (Green Fluorescent Protein): 395 nm ____ 400 nm Good ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Coumarin: 402 nm ____ 425 nm Good ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ AttoPhos: 440 nm ____ ____ 443 nm: Coumarin 450 nm Good ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Acridine Orange: 460/500 nm ____ ____ 461 nm: DAPI Good __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ R-phycoerythrin: 480/565 nm ____ Excel. -
Dna Revealed
Staining DNA on electrophoresis gels dna revealed Ethidium bromide, a potent mutagen Staining DNA on the move Concentrated In research laboratories, ethidium bromide and similar Recently, several commercial products have emerged that DNA Stain Dil e ute wi volum o th an equal f di use stilled water before fluorescent compounds are normally used to visualise DNA enable the DNA to be seen as it moves across the gel. Sto . re eze N at 4 °C. Do not fre AT N IO IO NA AT L C DUC ENTR GY E E FOR BIOTECHNOLO T HE U ING on a gel. Unfortunately, ethidium bromide and its breakdown Suppliers seldom reveal their composition, but several of NIVERSITY OF READ products are potent mutagens and carcinogens and therefore these stains contain Nile blue sulphate (also known as they should not be used in schools. Such dyes are often flat Nile blue A), a dye which had not previously been noted molecules with similar dimensions to DNA base pairs. When for its ability to stain DNA. Adkins and Burmeister (1996) ethidium bromide binds to DNA, it slips between adjacent give useful guidance as to its use as well as hints for identifying base pairs and stretches the double helix. This explains the other dyes which may be useful for visualising DNA. resources Methylene blue dye’s mutagenic effect — the ‘extra bases’ cause errors when the DNA replicates. In addition, short-wavelength UV light All of the dyes used for staining ‘mobile’ DNA are cationic Yung-Sharp, D. and (which itself is harmful) is required for ethidium bromide — that is, they are positively charged in the gel buffer, at Kumar, R. -
Water-Soluble Pyrrolopyrrole Cyanine (Ppcy) NIR Fluorophores† Cite This: Chem
Erschienen in: Chemical Communications ; 2014, 50. - S. 4755-4758 ChemComm View Article Online COMMUNICATION View Journal | View Issue Water-soluble pyrrolopyrrole cyanine (PPCy) NIR fluorophores† Cite this: Chem. Commun., 2014, 50, 4755 Simon Wiktorowski, Christelle Rosazza, Martin J. Winterhalder, Ewald Daltrozzo Received 7th February 2014, and Andreas Zumbusch* Accepted 21st March 2014 DOI: 10.1039/c4cc01014k www.rsc.org/chemcomm Water-soluble derivatives of pyrrolopyrrole cyanines (PPCys) have been dyes, BODIPYs or others.7 Notable are also advances in other fields, synthesized by a post-synthetic modification route. In highly polar like the engineering of GFP-related fluorescing proteins or quantum media, these dyes are excellent NIR fluorophores. Labeling experiments dots, which have resulted in the synthesis of novel systems with NIR show how these novel dyes are internalized into mammalian cells. emission.8 To date, however, only a few water-soluble dyes with strong NIR absorptions and emissions have been known. Apart from the Near-infrared (NIR) light absorbing and emitting compounds have general scarcity of NIR absorbing molecules, the main reason for this attracted a lot of interest since the 1990’s.1 Initially, this was motivated is that NIR absorption is commonly observed in extended p-systems by their use in optical data storage or as laser dyes. Recently, however, which most often are hydrophobic. The incorporation of hydrophilic new applications of NIR dyes have emerged, which has led to a surge of functionalities into