Oliveira Et Al-2018-Chemistryopen

DOI:10.1002/open.201700135

Green and Red Fluorescent Dyes for Translational Applications in Imaging and Sensing Analytes:ADual- Color Flag Elisabete Oliveira,[a, b] Emilia BØrtolo,[c] Cristina NﬄÇez,[d] Viviane Pilla,[e] HugoM.Santos,[a, b] Javier Fernµndez-Lodeiro,[a, b] Adrian Fernµndez-Lodeiro,[a, b] Jamila Djafari,[a, b] JosØ Luis Capelo,[a, b] and CarlosLodeiro*[a, b]

ChemistryOpen 2018, 7,9–52 9 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Red and green are two of the most-preferred colors from the the fieldsofbio-, chemo- and nanoscience. The review focuses entire chromatic spectrum,and red and green dyes are widely on fluorescent dyes containing chromophores such as fluores- used in biochemistry,immunohistochemistry,immune-staining, cein, rhodamine, cyanine,boron–dipyrromethene (BODIPY), 7- and nanochemistry applications. Selectivedyes with green and nitobenz-2-oxa-1,3-diazole-4-yl,naphthalimide, acridine red excitable chromophores can be used in biological environ- orange, perylene diimides, coumarins, rosamine, Nile red, ments,such as tissues and cells, and can be irradiated with naphthalene diimide, distyrylpyridinium, benzophosphole P- visible light withoutcell damage. This critical review,covering oxide, benzoresorufins, and tetrapyrrolicmacrocycles. Metal aperiod of five years, provides an overview of the most-rele- complexes and nanomaterials with thesedyes are also vant results on the use of red and green fluorescent dyes in discussed.

1. Introduction histochemistry,immunostaining, and nanochemistry.For example, translational applications have arisen in the field of imag- Due to their sensitivity,technical simplicity,and fast response ing of biological tissues to minimize cellular autofluorescence time, fluorescent probes, also known as fluorescent chemosen- and colocalization in confocal microscopy in multicolored sors, have emerged as very useful tools in analytical sensing experiences.[5,6] and opticalimaging.[1] The main parts of afluorescent probe To achieveabetter understanding of how biological systems are the signalingunit (chromophore), the spacer(chemical work, researchers need to be able to visualize and quantify bridge), and the binding unit (receptor);manipulating these eventshappening at the cellular level with high levels of spa- three key componentsallows the design of probes specifically tial and temporal resolution.[7] Despite great advances in the tailoredtoparticulartargets.Organicdyes that absorb light in field, creatingselectiveand sensitive fluorescent probesre- the visible region of the spectrum (l=400–700nm) can be mains achallenge and generally requires along process of trial used as chromophores. These dyes can contain different auxo- and error.There are many requirements that afluorescent chromes andfunctional groups such as amino, carboxylic acid, probe must meet to be used in biological systems, such as carbonyl, hydroxy,sulfonic acid, and nitro groups, which nontoxicity,specificity,and solubility in aqueous solutions. modify the ability of the chromophoretoabsorb light.[2] These Moreover,probes to be used in intracellular labeling need to auxochromes can increasethe intensity of the color and/or be able to cross plasma membranes.[8] Theoretical modelsspe- shift the emitted color in the spectra as well as increase the cific to particular chromophores have been created to facilitate solubility of the dye.[3,4] Selective probes with red and green the design of better probes, for example, to design boron–di- emission, two of the most-desired colors from the entire elec- pyrromethene (BODIPY)[9] and benzothiazole derivatives.[10] The tromagnetic spectrum,are used in biochemistry,immuno- majority of the current fluorescent probeshave been designed by using alimited number of core chromophores, with cou-

[a] Dr.E.Oliveira, Dr.H.M.Santos, Dr.J.Fernµndez-Lodeiro, marin, BODIPYs, cyanines, fluoresceins, rhodamines, and phe- [11,12] A. Fernµndez-Lodeiro, J. Djafari, Prof. Dr.J.L.Capelo,Prof. Dr.C.Lodeiro noxazines among the most-popular ones. The most-stud- BIOSCOPE Group, UCIBIO-LAQV-REQUIMTE ied green dyes are fluoresceins,[13] Oregon green 488 and Departamento de Química, Faculdade de CiÞncias eTecnologia 514,[14] perylene diimide,[15–19] the rhodamine green family,[20] Universidade NOVAdeLisboa [21] [22] 2829-516 Lisboa (Portugal) chlorophyll, and eosin. Red dyes typically come from rhod- [20,11] [23] [24,25] E-mail:[email protected] amines, porphyrins, and corroles. [b] Dr.E.Oliveira, Dr.H.M.Santos, Dr.J.Fernµndez-Lodeiro, Red and green pigmentshave long attracted the interest of A. Fernµndez-Lodeiro, J. Djafari, Prof. Dr.J.L.Capelo,Prof. Dr.C.Lodeiro researchers. For example, the ability of primates to discriminate Proteomass Scientific Society,Rua dos Inventores between redand green has been linked to foragingadvantag- Madan Park, 2829-516 Caparica (Portugal) es, allowing animals to detect more easily ripe fruit and young [c] Prof. Dr.E.BØrtolo Biomolecular ResearchGroup, School of Human and Life Sciences leaves against maturefoliage. Research also suggestsitmay Canterbury Christ Church University,Canterbury CT1 1QU (UK) help intraspecies sociosexual communication in primates by [26] [d] Dr.C.NﬄÇez aiding them in the selection of their reproductive partner. Research Unit, Hospital Universitario Lucus Augusti (HULA) The primaryfunctionofpigments in plants is the process of Servizo Galego de Saﬄde (SERGAS), 27003, Lugo (Spain) photosynthesis, in which chlorophyllsplay akey role.[27] Chlor- [e] Dr.V.Pilla ophylls are agroup of natural pigmentsbased on achlorin Instituto de Física, Universidade Federal de Uberlândia-UFU Av.Jo¼oNaves de vila 2121, Uberlândia, MG, 38400-902 (Brazil) magnesium macrocycle ring that absorbs yellow and blue [28,29] The ORCID identification number(s) for the author(s) of this article can wavelengths and reflects green color. Chlorophyll is pres- be found under https://doi.org/10.1002/open.201700135. ent in photosynthetic organisms(e.g. plants, algae, and cyano- 2017 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA. bacteria).[30] Other red and yellow pigments can help chloro- This is an openaccessarticleunder the termsofthe Creative Commons phyll capture light and convert it in energy.There are many Attribution-NonCommercial-NoDerivs License, which permits useand colored natural plant pigments, such as porphyrins, anthocya- distribution in any medium, provided the original work is properly cited, [31] the use is non-commercial and no modifications or adaptations are nins, carotenoids, and betalains. In the food industry,em- made. phasis hasbeen placed on replacing synthetic colorants with

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 10 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Elisabete Oliveira graduated in 2006 in Viviane Pilla received her Ph.D. degree in Applied Chemistry from FCT-University Applied Physics Sciences in 2001 from NOVAofLisbon (Portugal), obtained her the University of S¼oPaulo (Brazil). She Master’s degree in Biotechnology in performed postdoctoral research in Ap- 2007, and completed aPh.D. degree in plied Physics in Nonlinear Optics at the Biotechnology in 2010 at the same uni- Federal University of Pernambuco (Brazil) versity.In2013, she obtained asecond and State University of Campinas (Brazil). Ph.D. degree in Food Science and Tech- She later completed apostdoctoral nology from the Science Faculty of Our- period in Physical Chemistry Applications ense Campus in the University of Vigo in the BIOSCOPE research group at the (Spain). Her scientific interests are fo- University NOVAofLisbon (Portugal). Cur- cused on the synthesis of new bioin- rently,she is Adjunct Physics Professor IV spired emissive peptide as fluorescence at the Physics Institute in the Federal Uni- chemosensors;supramolecular chemistry versity of Uberlândia UFU (Brazil) and re- (photophysics and photochemistry);mul- searcher of the group of Optical and tifunctionalapplication of chemosensors in vitro (solution and solid Thermal Properties of Materials of thePhysics Institute UFU (Brazil). Dr. studies) and in vivo (cell-imaging studies);the synthesis of new emis- Pilla has experience in the optical and spectroscopic properties of ma- sive nanomaterials, such as quantum dots and silica for drug delivery; terials, acting mainly on the followingsubjects: photothermal effects and biomarker discovery in biological samples. and thermo-opticalcharacterization of different materials as quantum dots for biological applications;crystal and glasses;and biomaterials (biofluids, naturaldyes, and dental resin composites).

Emilia BØrtolo is aPrincipal Lecturer Hugo M. Santos graduatedinApplied (Chemistry) at Canterbury Christ Church Chemistry from UniversityNOVAof University (CCCU, UK)and previously Lisbon (Portugal)and completed aPh.D. worked in the DepartmentofMaterials, degree in Biochemistryfrom the same ImperialCollege London (UK). She ob- university in 2010. During his time as a tained her Ph.D. and B.Sc. degrees in Ph.D. candidate, he spent six months at Chemistry (Inorganic)from the University the Turku Centre for Biotechnology (Fin- of Santiago de Compostela (Spain). Addi- land) working with state-of-the-art mass tionally,she obtained her M.Sc. degree in spectrometry(MS) instrumentation for Environmental Technology from Imperial biomedical research. He worked as a College London (UK). She also has aPost- postdoctoral researcher at the University graduateCertificate in Learning and of Vigo (Spain) followed by amove to Teaching in Higher Education (CCCU, UK) the Institute of Biomedicine and Biotech- and aPostgraduate Certificate in Online nology (Barcelona, Spain) to advance bio- and Distance Education (Open Universi- medical applications of mass spectrome- ty). Her main focus is on the synthesis and applications of fluorescence try and translational research. In 2011, Dr.Santos joined FCT NOVA(Por- chemosensors. Her other research interests are in the field of pedagogi- tugal) to continue his research in biological MS. Currently, he is Assis- cal research, with special focus on enhancingthe teaching-research tant Researcher—FCT InvestigatorProgrammeatUCIBIO-REQUIMTE nexus in the undergraduate curriculum (undergraduate students as pro- FCT NOVA(Portugal). His scientificinterests focus on the identification ducers of research and scholarship). of molecules involved in complexbiologicalprocesses, characterizing their structure and monitoring how their abundance may change during these processestogain insight into the underlying molecular mechanisms; nanoproteomics and nanomedicine;application of chemosensors to the detection/quantificationofmetals;and MS analysis of organic molecules, metal complexes, and supramolecular systems.

Cristina NﬄÇez obtainedher Ph.D. degree Javier Fernµndez-Lodeiro received his in Chemistry in 2009 from the University Ph.D. degree in 2012 from the University of Santiago de Compostela (Spain). She of Vigo (Spain).In2013, he was apost- spent short periods of time performing doctoral researcheratthe Faculty of Sci- research at the Universidade NOVAde ence and Technology at the University Lisboa, REQUIMTE (Portugal)during 2007 NOVAofLisbon (Portugal)inthe RE- and 2008. In 2010, she started her post- QUIMTE-UCIBIO, working in the BIO- doctoral research periodbetween the SCOPE research group. He then moved University NOVAofLisbon (UNL,Portugal) to the Institute of Chemistry at the Uni- and the Canterbury Christ Church Univer- versity of Sao Paulo (Brazil), working in sity (UK), finishing in 2015. Dr.NﬄÇez is a the LOCSIN research groupand focusing researcher at the Research Unit of the on the synthesis and applicationofchalc- Hospital Universitario Lucus Augusti ogen molecules for construction of fluo- (HULA, Spain). rescence nanoprobes. Since September 2014, he has been workinginthe Faculty of Science and Technology University NOVAofLisbon(Portugal)atthe REQUIMTE-UCIBIO in the BIOSCOPE research group as apostdoctoral researcher.His research interests focus on the synthesis of new nanoparticles of Au, Ag, Pt, Fe, and quantum dots;application of new synthetic methodologies in nanomaterials using chalcogen atoms(Se and Te); and new molecular probes for biochemical and proteomics applications.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 11 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim natural pigments.[32] In other fields such as environmental from the amino acid tyrosine and are also soluble in water. monitoring and biomedical diagnosis, smartmaterials have They are presentinplants belonging to the caryophyllales been synthesized to be used as colorimetric biosensors:color families, such as cactus plants,ice plants, amaranth, and carniv- changes can be seen by the naked eye, whichmakes them orous plants. Betalainsfrom red beets are often used in the easier to visualize and reduces the need for expensive or so- food industry as anatural colorant.[39] phisticated instrumentation.[33] It is well known that if white light goes through asubstance, Some of the most useful red, orange, and yellow pigments the substance will absorb particular wavelengths.The residual are carotenoids, also called tetraterpenoids.[2,34] The most- light being reflected will then result in the color complementa- commoncarotenoids are carotene, the orange pigmentpres- ry to the wavelength that was absorbed. The color wheel ent in carrots;lutein,the yellow pigment present in fruits and showninFigure 1demonstrates this relationship.Here, com- vegetables;and lycopene, the red pigment found in toma- plementary colors are diametrically opposite each other.Con- toes.[35] Anthocyanins,atype of flavonoid pigment, are mostly sequently,absorption at l=420–430nmmakes asubstance responsible for the purple and blue color of flowers and are yellow,and absorption at l= 500–520 nm makesitred. Inter- soluble in water.Covalentbonding of anthocyaninstoorganic estingly, green is aunique color,asitcan be created by ab- acids, other flavonoids, or aromatic acyl groups can result in sorptionatabout l= 400 nm as wellasnear l=800 nm. changes to the color intensity and hue.[36–38] Betalains (which Several reviewshavedescribed the performance of fluores- can have red or yellow color) are indole derivatives derived cent probes with specific applications, for example, used as in-

Adrian Fernµndez-Lodeiro graduatedin JosØ L. Capelo received his Ph.D. degree Chemistry in 2013from the University of from the University of Vigo (2002), com- SantiagodeCompostela (Spain)and is pleted postdoctoral researchatthe IST in now aPh.D. candidate in the Green Lisbon (Portugal), and was later appoint- Chemistry Ph.D. Programatthe RE- ed as Researcher at REQUIMTE (FCT-UNL, QUIMTE-UCIBIO, BIOSCOPE Group, Facul- Portugal). He then moved to the Universi- ty of Science and Technology of the Uni- ty of Vigo (Spain) as IPP (Isidro Parga versity NOVAofLisbon (Portugal). His sci- Pondal) ResearchLecturer.Hewas ap- entific interests are focused on nanosyn- pointed Assistant ProfessoratFCT-UNL thesis;metallicand polymeric nanoparti- (Portugal) in 2012, where he is currently cles, including rods and quantum dots, based. Prof. Capelo has developed re- nano-biomedicine;and fluorescent and search on the followingtopics:quantifi- colorimetricdyes for sensing. cation of metal and metalsspecies in en- vironmentaland food samples,new methods to speed protein identification by using mass spectrometrybased workflows, accurate bottom-up protein quantification, bacterial identification through mass spectrometry, fast determination of steroids in human samples, biomarkerdiscovery, application of sensors and chemosensors to the detection/quantification of metals, and nanoproteomics and nanomedicine.

Jamila Djafari graduated in 2013 in Carlos Lodeiro graduated in Chemistry in Chemical Biologyfrom the University 1995 and received his Ph.D. degree in Paris-Saclay (France). In 2015, she ob- Chemistry in 1999 from the University of tained adouble Master’s degree in Santiago de Compostela(Spain).In1999, Chemical Scienceand Engineering at the he moved to the University NOVAof Ecole Nationale SupØrieuredeChimie de Lisbon (UNL, Portugal) as European Marie Paris (ENSCP,France) and in Molecular Curie PostdoctoralResearcher in aproject Chemistry from the University Pierre et concerningmoleculardevices and ma- Marie-Curie,Paris (France). She is now chines;in2004, he became aFellow Re- working towards aPh.D. degree in searcher and Invited Assistant Lecturer at Chemistry focusing on antibiotic-func- the REQUIMTE-CQFB, Chemistry Depart- tionalizednanomaterials at the RE- ment (UNL, Portugal).In2008, he re- QUIMTE-UCIBIO, BIOSCOPE Group, Faculty ceived habilitationinChemistry in Spain, of Scienceand Technology of the Univer- and ayear later he movedtothe Univer- sity NOVAofLisbon (Portugal). Her scien- sity of Vigo, Faculty of SciencesofOur- tific interests are centered around the synthesis of nanomaterials, or- ense (FCOU,Spain) as IPP (Isidro Parga Pondal) Researcher–Lecturer.He ganic chemistry,and biochemistry. is currently an Assistant Professor in the Chemistry Department UCIBIO- REQUIMTELaboratory in the Faculty of Science and Technology,Univer- sity NOVAofLisbon (Portugal). In 2017, he received habilitationinInor- ganic Analytical Chemistry in Portugal at the FCT-UNL. His research interests include physical organic and physical inorganic chemistry of fluorescence chemosensors;the synthesis of functionalizednanoparti- cles, nanocomposites, and nanomaterials;applications of nanomaterials in environmental research;application of nanomaterials in biomedical research;supramolecular analytical proteomics;and onco- and nanoproteomics.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 12 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim tation wavelength (l 500 nm) and high fluorescencequantum yields and extinction coefficients, and thus, they are widely used in biochemistry,cell imaging, cell biology,clinical diagnosis, and drug delivery.[50–52] Green fluorescent dyes show an emission wavelength in the l= 500–550 nm range. Fluoro- phores with green fluorescenceinclude fluorescein, rhodamine, cyanine, BODIPY-FL, 7-nitrobenz-2-oxa-1, 3-diazole-4-yl, naphthalimide (lucifer yellow), and acridine orange. This section provides ageneral overview of the key advances in the design and applicationsofthese types of probes.

2.1. Fluorescein Probes containing fluoresceinhave been well studied:they Figure 1. Color wheel. produce abright signal, are nontoxic, can be obtained on a gram scale, and have many possible reactive sites in their skel- etons.[53–57] Fluorescein has several sites at which modifications tracellular pH indicators;sensors for reactiveoxygen and nitro- can be introduced, such as the xantheneunit (at positions 4 gen species, for metal ions, and for anions;and as diagnostic and/or 5, 3, and/or6), the hydroxy groups, and the phenylring imaging tools.[40–42] However,most of the reviewspublished so (positions 4’ and/or 5’). Derivatization of the carboxylic acid is far tend to be either exclusively focusedonsensors for aspe- very common, as it leads to spirolactam-basedchemosensors, cific target or centeredaroundaspecific chromophore.[43–46] which in their open ring form result in highly emissive probes. The aim of this reviewistoprovide acomprehensive, critical, Most fluoresceinderivatives are synthesized by substitution on and readable overall overview of the latest research on green the benzene unit, which leaves the 3’-and 6’-positions avail- and red fluorescent probesand their application in the fields able forconjugation and the ability to form the strongly emis- of bio-, chemo-, and nanoscience. This review focusesonre- sive fluorescentdianion.[58] Modification of both hydroxy search published over afive-year period and looks at both the groups to form the methyl ester of fluorescein also yields a structureofthe different probesand their applications. Sec- highly fluorescent compound.[59] tion 2provides ageneraloverview of key advances in the The excitation wavelength of fluorescein is about l= design and applicationsofgreen dyesderived from fluores- 494 nm, which is close to the l=488 nm spectralline of an cein, rhodamine, cyanine, boron–dipyrromethene (BODIPY-FL), argon laser.Thus, fluoresceincan be agood fluorophore for 7-nitobenz-2-oxa-1,3-diazole-4-yl, naphthalimide, acridine, and probesused in confocal laser-scanning microscopy and flow perylenediimide. Section 3presents some recent examples of cytometry applications. However,theseprobes are not without red probesbased on cyanine, BODIPY,coumarin, xanthene, problems. Fluorescein can be susceptible to photobleaching Nile red, naphthalene diimide, distyrylpyridinium dyes, benzo- (photodegradation,which can eventually lead to the destruc- phosphole P-oxide scaffold derivatives,and benzoresorufins. tion of the probe), and it has abroad fluorescenceemission Metal complexes with lanthanides, iridium, and ruthenium are spectrum.Moreover,ithas the tendency to self-quench if con- discussed in Section 4. Section5 discusses briefly different jugated with polymers and high degrees of substitution, and nanomaterials such as quantum dots, fluorescent metallic consequently,fluorescein derivatives have limitations in multi- nanoclusters,and semiconductor nanocrystals. color applications.[60] Fluoresceinisalso highly pH dependent: basic pH resultsinring openingoffluorescein, which becomes 2. Green Fluorescent Dyes strongly emissive and shows avery intense greenish-yellow color.[61] Depending on the pH, fluorescein can exist in four ion- Considerable effort has been focusedonthe design of new flu- izationforms:cationic (pH 2), neutral (pH <5), monoanionic orescentprobes with the aim to synthesize increasingly more (pH 6–7), anddianionic(pH>8).[62] sophisticated structures to enhance further their properties Oliveira and co-workers[58] report on fluorescent alanine–flu- and applications.[47] High sensitivity and specificity and the abil- orescein probes 1 and 2 with green emission (see Figure 2). ity to fine-tune the opticalproperties (e.g. lifetime, emission Probe 1 shows colorimetric and “turn-off” fluorescencebehav- and excitation spectra,intensity,and anisotropy) are some of ior for both the HgII ion and for the neurotransmitter dopa- the advantages of fluorescent probes. Probes bearing visible mine in HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic excitable chromophores are particularly appealing, because acid] buffer at pH 8; achange in color is observed from yellow they can be applied in biological models, such as in cells to pink for HgII and to orange fordopamine. Probes 1 and 2 and tissues,and can be irradiated with light without cell are highlydependent on pH. The absorption spectraof1 show damage.[48] an increase in the absorbance at l= 490 nm, as well as acolor Fluorescein and rhodaminedyesare the ones most com- change from colorless to yellow at high pH values. Different monly used to develop biological sensingprobes.[49] These speciesinequilibrium are presentdepending on the pH:a probespossessexcellent opticalproperties such as along exci- neutralspecies (N)isdetected below pH 5, amonoanionic spe-

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 13 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 2. Chemical structures of fluorescein in compounds 1 and 2.Pictures of compounds 1 or 2 at pH 6–7 andpH8a) under the naked eye and b) under a UV lamp at l= 365 nm. a) Spectrophotometric and b) spectrofluorimetric titrationsofcompound 1 and c) spectrophotometric and d) spectrofluorimetric titrations of compound 2 as afunction of pH in ethanol/Milli-Q water (40:60) solution. The insets in panels b, dshow the emission fluorescenceintensity at 5 6 l= 520 nm for 1,free fluorescein(panelb), and 2 (panel d). ([Fluorescein]= [1]=1.010À m,[2]=4.0 10À m, lex =490 nm, T=298 K). Adapted with permission from Ref. [58].Copyright2013 Elsevier B. V.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 14 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim cies (M)isobserved at pH 6–7, and adianionic species (D)is similarapproach, Buccella, Horowitz, and Lippard[71] have de- seen above pH 8(see Figure 2). Acationic form at pH 2isnot signed the new probe ZPP1 (14)byincorporating carboxylate/ observed. The emissionintensity for 1 reaches amaximum at ester groups in the 6-position of fluorescein;these structural pH 8(dianionic form) and then decreases. For comparative changes allow better control of the intra/extracellular distribu- purposes,the researchers have performed asimilar pH study tion of the probe while maintaining the ZnII bindingproperties with free fluorescein:the free chromophoredoes not present of ZP1 (3). adecline in the emissionsignalabove pH 8. Theauthorscon- Ashortcoming of theseprobes is that they suffer from un- clude that quenching of the emission above pH 8can be at- predictable cellular localization.Inanattempt to design a tributed to the presenceofthe protected amino-acid unit.[63] more accurate delivery system, Radford and co-workers[72] have Extensive work has been performed to design ion-selective published apeptide-based targeting strategy,inwhich aseries fluorescencesensors for alkali, alkali-earth, and transition-metal of targeting peptides are attached to the diacetylated Zinpyr ions. “Turn-on”sensors are the mostappealing, as they can be sensor 15 for intracellular detection of ZnII (see Figure 4). Modi- used as fluorescentsensors in cellsand tissues. Zinc(II) is the fication of probe 15 with amitochondrial-targetingpeptide second most-abundant d-block metal ion in the human allows the use of the sensorinconcentrations four times lower brain,[64] and its closed-shell nature produces achelation en- than previously reported for the non-acetylated probe. hancement of fluorescence(CHEF). Zinc(II) has been widely Most fluorescent probesexhibit aquenching response upon studied by Lippard and co-workers, who have synthesized sev- chelation to paramagnetic transition-metal ions;however, eral CHEF families of fluorescein derivatives that are able to some probes with a“turn-on” effect have been reported for detect ZnII in biological systems (see Figure3): Zinpyr sensors those metal ions. Abebe and Sinn[73] report on “turn-on”and 3–5,[65,66] Zinspysensors 6–9,[67,68] and QZ sensors 10 and 11.[69] colorimetricfluorescein derivativeprobes 16 and 17 for CoII Zinpyr sensors ZP1 (3)and ZP2 (4)are able to detect ZnII in and NiII,two metal ions that are usuallyfluorescencequench- sub-nanomolar concentrations: chelation with the ZnII ion at ers. The probes are capable of detecting both ions in aqueous the di(2-picolyl)amine (DPA) ligand causes an enhancementin solutionsquickly and selectively.Coordination occurs through the fluorescence. X-ray crystallography reveals a1:2 (ligand/ the carbonyl Oatom and the two Natoms and is reversible metal) stoichiometry for the 3/ZnII complex,with abipyramidal (see Figure5). trigonal geometry for both ZnII centers.[65] ZP3 (5)isasymmetri- CuII,ametal ionwith an unfilleddshell, is also well known cal, and coordination by the ZnII ion gives asixfold enhance- for quenching fluorescenceupon binding to fluorescent ment in the fluorescence; the 5/ZnII complex has a1:1 stoichi- probes. Like CoII and NiII,itusually produces colored metal ometry,which results in adistorted octahedral geometry.[66] complexes due to d–delectronic transitions. Probes 18, 19, Zinspy (ZS) sensors ZS1 (6), ZS2 (7), ZS3 (8), and ZS4 (9)have and 20 are fluorescein derivatives with ahighly selective“off– sulfur units. Due to the fact that sulfur has lower affinity for on” behavior towards CuII in aqueous solution (for 18 and 19) ZnII than nitrogen and oxygen, only a1.4–2.0-fold enhance- and acetonitrile(for 20).[75,76] For 18 and 19,ayellow color ap- ment in the fluorescence is observed;interestingly,thesecom- pears upon coordination with CuII due to the opening of the pounds also show a“turn-on” effect in the presence of CdII ring in the fluoresceinunit, which resultsinthe formationof ions. Chemosensors QZ1 (10)and QZ2 (11)contain one and complexes with a1:1 (ligand/metal) stoichiometry.Probe 20 is two 8-aminoquinoline units, respectively.QZ1 is asymmetrical formed by acalix[4]arene derivative with fluoresceinsubstitu- and showsa42-foldenhancement in the fluorescenceupon ents. The addition of CuII to an acetonitrile solution of the the addition of ZnII.QZ2 (11)binds two metal ions with a150- probe results in acolor change from colorless to yellow; if the fold “turn-on” effect for ZnII.Chemosensors QZ are ZnII selec- reaction is performed in aqueous solution, the colorchanges tive and can detect this ion in the presence of other biological- from colorless to purple. In acetonitrile, the probe also produ- ly relevant metal ions such as NaI,KI,MgII,and CaII. ces an enhancement in the fluorescenceintensity and is able StudiesbyNolan et al. on these ZnII sensors, performed in to detect nanomolar concentrationsofCuII ;this “turn-on” living cells and brain tissue, showthat tertiary amine probes 9 effect is not observed in aqueous solution.In2014, rapid are more cell permeable than aniline probes 5 and 11.Imaging “turn-on”probe 21 for CuII detection in water was reported by studies in HeLa cells show that ZP probe 3 accumulates pri- Muthuraj et al.[74] (see Figure 5). Probe 21,with an indole-3-car- marily within the Golgi apparatus, and ZS probe 9 accumulates boxaldehyde-functionalized fluorescein hydrazine, can selec- in the mitochondria(see Figure 3).[68] Although QZ2 (11)can tively bind CuII both in vivo and in vitro. CuII coordination is detect higherconcentrations of free ZnII reversiblyand without the resultofinduced Forster resonance energy transfer (FRET): probe saturation, it has the limitation of not being cell trappa- in the presence of CuII,interaction between the donor (indole- ble;toaddress this limitation, McQuade and Lippard[70] have 3-carboxaldehyde) and the acceptor fluorophore(xanthene) modified the structure to develop new “turn-on”probes QZ2E leads to intramolecular FRET between the two. The in vivo re- (12)and QZ2A (13). Twoesters are added to the quinolone sults performed in RAW264.7 cells show bright fluorescencein rings of QZ2 to produce QZ2E (12). The esters allow cell per- the presence of CuII,with no interferencefrom other metal meability until the probe is inside the cells. After that, intracel- ions;research has revealed that both probe 21 and its CuII lular esterases hydrolyze the esters to carboxylates and QZ2A complex are nontoxic in the cellular system and show consid- (13)isformed. QZ2A (13)isnegatively charged, which pre- erable potential in biomedical applications(see Figure 5). vents the probe from diffusing out of the cells. Following a

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 15 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 3. Chemical structures of ZnII probes:Zinpyr sensors 3–5,Zinspy sensors 6–9,QZsensors 10 and 11,QZ2E (12), QZ2A (13), ZPP1 (14), 14a,and 14b; subcellular compartmentalization of ZS4 (9)(mitochondrial) in HeLa cells. Adaptedwith permission from Ref. [68] Copyright 2006 American Chemical Society.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 16 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 4. Illustrative scheme of the two zinc-sensingmechanismsoperating with peptideconstructs. Deconvoluted fluorescence microscopy images of live HeLa cells pretreated with ZP1 (3)-R9 (5 mm)orDA-ZP1 (15)-R9 (2.5 mm)and the indicated organelle stain. Top, ZP1-R9) a) differential interference contrast (DIC) image, b) signal from ZP1-R9, c) signal from Lyso-Tracker Red, d) overlayofthe images in panels band c. Pearson’s r=0.42 0.16 (n=8). Bottom,DA- Æ ZP1-R9) e) DIC, f) signal from DA-ZP1-R9 after treatment with 25 mm zinc pyrithione(ZnPT), g) signal from Hoechst 33258, h) overlay of the images in panels f and g. Scale bar= 25 mm. Reproduced from Ref. [72] with permission from The RoyalSocietyofChemistry.

Another metal that often produces aquenching effect in the yellow to pink. These different spectral behaviors are relatedto emission of the probe is PdII.However,fluoresceinderivative the different bindingmodes of the AgI ion in both systems. 22 published by Wei and co-workers[77] can selectively sense TwoAgI ions chelate to probe 24 through the sulfur atoms of PdII in the presence of CuII,with CuII acting as asynergic trig- the thiomorphine moieties andthe phenyl oxygen atoms of ger.The addition of PdII and CuII to asolution of 22 in acetoni- fluorescein; this leads to photoinduced electron transfer (PET) trile turns the solution from colorless to green, acolor change from the released tertiary aminegroups, which before were visible to the naked eye; the reactionconditions are 2h at blocked by hydrogen bondingofthe phenolic hydrogen room temperature with an excitation wavelength of 492 nm. atoms. For probe 23,only one AgI ion coordinatestothe The two metal ions hydrolyze the alkyne groups of 22,which probe through the nitrogen donoratoms in the morpholine leads to asignificant fluorescent enhancement at l= 514 nm. and benzylic amines, which blocks the PET phenomena;thus, Swamy and co-workers[78] have designed fluoresceinprobes “turn on” of fluorescenceoccurs (Figure 6). Recently,fluores- 23, 24,and 25 by Mannich reactionof2’,7’-dichlorofluorescein cein spirolactam derivative 26 was design by Lin and co-work- with morpholine, thiomorpholine, and 1-methylpiperazine, re- ers;[79] these authors have used this probe as a“turn-on” fluo- spectively.pHstudies conducted with the three probesshow rescence probe for the detection of AgI ions in aqueoussolu- that, unlike the free chromophore, the three probes exhibit in- tions (detection limit 0.08 mm), including tap, river,and lake tense fluorescenceatacidic pH values and weak fluorescence waters;the resultsobtained are in excellent agreementwith at basic pH values. Probes 23 and 24 showselectivity towards those obtained by studying the samples by flame atomic ab- AgI ions in aqueous solution at pH 7.4;anenhancement in the sorptionspectrometry (Figure 6). emission intensity is only observed for probe 23,whereas Water-soluble “turn-on”fluorescein derivative 27 has been quenching is seen for 24,followed by acolor changefrom reported by Nolan and Lippard.[80] Probe 27 is highly sensitive

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 17 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 5. Chemical structures of probes 16–22.Fluorescence imaging of 21 in RAW264.7 cells in the presence of CuII RAW264.7 loaded with 21 and treatedwith copper for 1hat 378C. Adapted with permission from Ref. [74]. Figure 6. Chemical structuresand mechanismsofprobes 23–30.Adapted Copyright 2014 American ChemicalSociety. from Refs. [78–83].

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 18 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim and selective to HgII ions over alkali (LiI,NaI,and RbI), alkali- earth (MgII,CaII,SrII,and BaII), and transition-metal ions (CrIII, MnII,FeII,CoII,NiII,CuII,ZnII,CdII,and PbII). Coordination at the aniline nitrogen atoms by HgII blocks the PET mechanism and afivefold increase in the emission intensity is observed. The same authors have published new asymmetrical fluorescein derivative 28,which selectively coordinates to HgII ions (see Figure 6);this probe shows a“turn on” of the emission intensity and is able to detect parts per billion levelsofthis metal ion in aqueous solutions.[81] Probe 29,afluoresceinthiol ester derivative,has been synthesized by Fernµndez-Lodeiro and co-workers[82] and has been graftedonto the surfaceofsilver nanoparticles (AgNPs). This new hybrid system (i.e. AgNPs@29)shows an intense enhancement in fluorescencedue to the presence of silver.The interaction of AgNPs@29 with HgII (16 mm)leads to ablueshift and an increaseinthe surface plasmon resonance (SPR) band from around l= 410 to 398 nm as well as an enhancement in the emission band at about l=534 nm in toluene. Choi and co- workers[83] report on dichlorofluorescein probe 30,which shows an intense emission band at l=528 nm;this band is quenched upon the addition of HgII with acolor change from yellowish green to orange, visible by the naked eye. These changes are due to selectivemercuration of the xanthenering at positions 4and 5. Wang et al.[84] report on fluorescein-based sensor 31 bearing anitroolefin that is able to detect biological thiols such as cysteine and glutathione. An increase in the intensity of the ab- sorptionband at l= 497 nm as well as an enhancement in the fluorescenceintensity at l=520 nm are noticedupon the addition of both thiols. Thespectral changes are due to Michael addition of the thiols to the doublebond of the nitroolefin moiety,which leads to the formation of 32 (see Figure7). This mechanism has also been demonstrated in the imaging of thiols in PC-12 cells. The amino acid l-histidine in its free form acts as atridentate ligand towards transition-metal ions such as CuII.Wang and co-workers[85] report on water-soluble fluorescent probe 33,which selectively binds to CuII ions;this binding causes quenching of the emission intensity through photoinduced electron transfer (limit of detection, LOD= 1.6 mm). The 33–CuII complex can selectively detect histidine (LOD =1.6 mm)inthe presenceofthe other naturallyoccurring amino acids and shows a“turn on” of the fluorescence signal. This behavior has also been verifiedinbiological environments such as living HepG2 cells (see Figure 7). Figure 7. Chemical structuresand reaction mechanism of probes 31–33. Aseries of CuII complexes with fluorescein probes 34 to 38 Adapted from Refs. [84,85] have been synthesized by Lim and co-workers[86] (see Figure 8). The structures suggest that CuII is coordinated with one oxygen and two nitrogen atoms to form nonfluorescentcom- in SK-N-SH neuroblastoma cellsand in Raw 264.7 murinemac- plexes. The addition of nitrogen monoxide (NO) to abuffered rophages.[87] The 38–CuII complex has significant advantages aqueous solution of the probes (pH 7.0) produces the “turn such as brightness,cell-membrane permeability,minimal cyto- on” of the emission. The reduction of CuII to CuI produces the toxicity, and selectivity.Italso shows arapid fluorescent en- NO+ cation, which in turns reacts with the probestoform ni- hancement in the presence of NO, with an immediate 11-fold trosated probes (34–38)–NO. Probes 34–38 are selective to NO “turn on” in the emission intensity (see Figure 8). To be used over other biologically important species, such as H2O2,NO2À , successfully in biological tissues, the signalingagentmust be II NO3À ,HNO, ONOOÀ ,and ClOÀ .The coppercomplex of probe retained insidecells;unfortunately,the 38–Cu complex shows 38 has been used to detect NO as abiological signaling agent atendency to diffuse out of cells. To overcomethis problem,

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 19 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 8. Chemical structure of probes 34–38.a)CuFL1 detection of NO in SK-N-SH neuroblastoma cells. [CuFL1]=1 mm,[17b-estradiol]=100 nm.Fromleft to right) 25 minexposure to CuFL1 without 17b-estradiol and 5, 10, 15, and 25 min exposure to CuFL1 and 17b-estradiol.Top) fluorescence images. Bottom)DIC 1 images. Scale bars = 50 mm. b) CuFL1 detection of NO in Raw 264.7 murine macrophages.[CuFL1] =1 mm,[lipopolysaccharide (LPS)] =500 ng mLÀ ,[interferon- g (IFN-g)] =250 UmÀ.From left to right) 12 hexposuretoCuFL1 withoutLPS/IFN-g and 6, 8, 10, and 12 hexposure to CuFL1 and LPS/IFN-g.Top) fluorescence images. Bottom) DIC images.Scale bars=50 mm. Reprinted with permission from Ref. [87] Copyright 2010 American Chemical Society.

McQuade and Lippardhave prepared three new probes, 39, have synthesized probe 40,abiocompatible hydrophilic poly- 39E, and 39A, by employing the ester/acid strategy to improve (ethyleneglycol) (PEG) polymer attached to afluorescein deriv- the ability of the probestostay inside cells.[70] ative;the role of the PEG polymer is to guarantee water solu- The development of methods that can quickly,sensitively, bility and biocompatibility (see Figure 9). Quenching of fluores- and selectively detect fluoride anions in aqueous samples is of cence is observedupon linkageoftert-butyldiphenylsilyl great importance because of the impact of fluorideanionson (TBDPS) groups.However,the addition of fluoride anions leads human healthand the environment. Zheng and co-workers[88] to selective fluoride-mediatedcleavage of the Si Obond, À

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 20 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 9. Chemical structures and/or mechanismsofcompounds 40–49.Adapted from Refs. [84–101].

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 21 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim 2 which causesa“turn on” of the fluorescenceemission;the de- probe is able to detect S À anions by showinganintense tection limit for fluoride is 19 ppb. This effect is also observed green fluorescence(see Figure 9). in “real-life” samples such as running water,urine, andserum, as well as in HeLa and L929 cells. 2.2. Rhodamine Several fluorescein derivatives for fluoride detection have been synthesized;[89–93] however,these probes do not selective- The spirolactam structure of rhodamine is nonfluorescent and ly detect fluoride, as they suffer from interference from other colorless and exhibits highselectivity for metal ions;the rhoda- anions. Asthanaand co-workers[94] reportthe synthesis of fluo- mine ring in its openform has strongfluorescence. Zhang and ride-selective “turn-on”fluoresceinprobe 41 containing nitro- co-workers[102] have reported rhodamine derivative 50 contain- benzenesulfonyl chloride as amaskingunit (see Figure9). The ing an ethylenediamine-N,N-diacetic acid moiety (see addition of FÀ releases the NBS group from the fluorescein Figure 10). Experiments performed in acetonitrileshow that unit, which leaves the ring open. In the absorption spectrum,a probe 50 is CuII selective.The addition of up to 6equivalents new intense band appearsatl=517 nm;moreover,achange of CuII to 50 produces a49-fold enhancement in the fluores- in colorfrom colorless to yellow is observed, followedbya60- cence, followed by ablueshift of Dl=45 nm, which results in fold enhancement in emission at l=530 nm (green emission). intense green fluorescenceatl=530 nm. However,the addi- II Probe 41 detectsFÀ over sulfide anionsand thiols (detection tion of Hg results in only aminimal enhancement in the emis- limit 27.5 nm); however,the probe works in an organic solvent, sion. Recently,Park and co-workers[103] have described “turn- acetonitrile, not in aqueous solution. on” fluorescence sensor 51 based on rhodamine that selective- Regarding the cyanideanion, “turn-on” fluorescence probe ly detectsCuII ions overothers metal ions such as HgII,CoII,KI, 42 hasbeen reported by Kwon and co-workers.[95] Probe 42 is CsI,AgI,PbII,ZnII,MgII,FeIII,NiII,LiI,and AlIII.Probe 51 shows un- able to detect selectively CNÀ ions over other anionsin precedented colorimetric (from colorless to pink) and fluoro-

CH3CN/HEPES (9:1, v/v), at pH 7.4. The CNÀ anion attacks the metric (from colorless to yellowish green) changes in the pres- aldehydegroup in the salicylaldehyde unit, which resultsinin- ence of CuII on the basis of the ring-opening mechanism of tramolecular hydrogen transfer with the phenol proton(see the rhodamine spirolactam with a1:1 (ligand/metal)binding Figure 9). This reactionmechanism implies that 42 behaves ratio. The detection limit is 0.14 mm,and the solventused is like acolorimetric (colorless to yellow) and off–on green fluo- CH3CN/H2O(9:1, v/v). Taking advantage of the same reaction rescent sensorfor cyanideand can be used forlive-cell imag- mechanism, Lee and co-workers[104] have developed “turn-on” ing in HaCaT cells. Lv et al.[96] also report ratiometric fluores- fluorescencerhodamine 6G phenylthiourea derivative 52 to II II cein-based probe 43 for CNÀ detection in water.The probe detect Hg ions in acetonitrile. The addition of Hg opens the acts by aFRET mechanism, with 4-(N,N-dimethylamino)benza- spirolactam ring of the rhodamine moiety,which leads to a mide as the donor, fluorescein as the receptor, and the partici- 700-fold increase in the fluorescence emission(detection limit pation of the salicylaldehyde hydrazone unit. The CNÀ anion 0.45 mm); the authors also prove the reversibility of this system activatesthe hydrazone functionality,which is followed by fast with the addition of ethylenediaminetetraacetic acid (EDTA) proton transfer of the phenol to the developing nitrogen (see Figure 10). Lozano and co-workers[105] have immobilizeda anion of the probe, generating the open form of fluorescein rhodamine 6G spirocyclic phenylthiosemicarbazidederivative (see Figure 9). Unfortunately,the probe is not exclusively CNÀ on anylon membrane to form probe 53,which shows intense II selectiveand can also weakly senseFÀ ,AcOÀ ,and H2PO4À green emissioninthe presence of Hg (see Figure10). This be- anions. havior can be attributed to mercury-promoted ring opening of Knowingthe amazing ability of cyanidetoreact with copper the spirolactam in the rhodamine moiety (detection limit 1 ions to form very stable Cu(CN2)species, Chung and co-work- 0.4 ngmLÀ ). This mechanism has been tested through arecov- [97] ers describe selectiveoff–on-type CNÀ sensing probe 44, ery study by using severalspiked water samples from different which is based on the CuII complex of afluoresceinderivative. locations. The fluorescenceofthe system is quenched by the CuII ion: Highly sensitivefluorescenceprobes for proteases and gly- [106] CNÀ added in aqueous solution (pH 7.4) coordinates with the cosidases have been developed by Sakabeand co-workers metal, whichenhances the fluorescencesignal (Figure 9). by replacing the acetyl group of rhodamine derivative 54, Regarding off–on anion sensing, fluoresceinprobes 45[84] which is colorless and nonfluorescent, with atarget enzyme and 46[98] show intense green emission upon the addition of substrate moiety R(see Figure 10), leadingtoahydroxymethyl the hypochlorite anion (ClOÀ)inaqueous solutionwith very rhodamine derivative highly fluorescent.The addition of the low detection limits (7.3 and 40 nm,respectively);similar be- targetenzyme to the probe generates an open nonspirocyclic havior is observed in studies performed in living cells. Probes structure that is strongly fluorescent. The authors have synthe- 47[99] and 48[100] show high selectivity and sensitivity to the sul- sized nonfluorescent probes for leucine aminopeptidase (Leu- 2 fide anion (S À)with an enhancement in the emission signal in 54), fibroblast activation protein (Ac-GlyPro-54), and b-galacto- aqueous solutions, even in real samples such as white wine sidase (bGal-54);inall cases, upon the addition of the en- (47)and MGC-803 cells (48). In the presence of CuII,sulfide zymes, agreen fluorescenceemission is observed. They have forms very stable CuS species, and this has allowed Hou and also conducted imaging studies in livingcells (see Figure 10). co-workers[101] to design anonemissive CuII complex with fluo- Oliveira and co-workers[107] report an unusual green-lumines- rescein probe 49 containing a8-hydroxyquinoline unit. This cent IrIII complex based on alissamine chromophore (see

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 22 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 10. Chemical structuresand reaction mechanisms of probes 50–54.Panel I) Photographs of 53-doped nylonmembranes irradiated with a) visible and II 1 II 1 II b) UV lamps, after treatment with 10 mL of 1) Hg -free water,2)5ngmLÀ Hg solution, and 3) 12 ngmLÀ Hg solution. The bars indicate the diameters of the nylon membrane and the area exposed to the solution flow.Reproduced from Ref.[105] with permission from The Royal Society of Chemistry.Panel II) Fluor- escence confocal imagingofa)HEK 293/lacZ cellsand b) HEK 293 cells loaded with bGal-HMRG. Scale bar represents50mm. Reprinted (adapted) with permission from Ref. [106].Copyright 2013 AmericanChemical Society.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 23 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 11). Probe 55,alissamine rhodamine Bsulfonyl derivative bearingalanine, produces orangeemission in water;the addition of IrIII at room temperatureresults in ahighly emissive green Ir@55 complex, with afluorescencequantum yield of 0.47. NMRspectroscopy and theoretical studies suggest coordination of the IrIII ion at the alanine moiety through involve- ment of the amide and carbonyl groups.This complex is selective towards HgII,which results in quenching of the green emission;the addition of cysteine resultsinrecovery of the emission signal. TheIr@55 complex can detect minimal amountsofHgII [(13.63 0.11) mm]and cysteine [(6.56 Æ Æ 0.10) mm].

2.3. Cyanine Cyaninedyes are fluorescent molecules commonly used as labels or probesinproteomics, imaging, andbiomolecular labeling.[108–111] They have high biocompatibility,are bright, and have high molar absorptivity;they also have the advantage of coveringawide spectralrange, from blue to near infrared.[60] Cyaninedye families are mostly formed by apolymethine chain linked to two nitrogen-containing heterocycles, such as indoles,benzothiazoles, benzoxazoles, or quinolones.[112] These probeshave excellent ability to bind DNA through noncova- lent interactions by intercalation between base pairs.[113] Probe 56,ahexacyclic acridine–monomethine cyanine dye, was designed by Mahmoodand co-workers[114] (see Figure 11). The probe exhibitsgreen emission with aquantum yield close to 1 for dimethyl sulfoxide, methanol, and glyceroland 0.5 for water.The ability of 56 to bind to DNA has led the researchers to studyits potential as an intracellular DNA stain by using human breast carcinoma cells, MD231 and ECACC;the results show that the probe accumulates primarily in the nucleus. Taking advantage of the ability of cyaninetobind DNA, Boh- händerand Wagenknecht[115] have synthesized green fluorescent cyanine styryl probe 57,which can be further incorporat- ed into oligonucleotides through “click”-typechemistry to be used as an energy donor in DNA. Energy transfer from 57 to a red emitter leads to asignificant wavelength shift from green to red, with both emittersworkingas“DNA traffic lights”(see Figure 11). Colorimetric “turn-on”fluorescent probe 58,with a coumarin–hemicyanine structure, is selectivefor the cyanide [116] anion in HEPES buffer (pH 9.3). The addition of the CNÀ anion to 58 leads to agradual decrease in the absorption band at l=603 nm with the emergence of anew band at l= 440 nm;atthe same time, aremarkablefluorescenceenhance- ment at l=502 nm occurs. The cyanideanion is highly reactive to indolium groups;thus, nucleophilic attack of CNÀ to the indolium group blocks p conjugation between this group and the coumarin, which leads to the observed spectral and color changes (see Figure 11). Figure 11. Chemical structures and/or mechanisms of probes 55–58.a)Spec-

trofluorometric spectra and image under aUVlamp (lex =365 nm) of 55, 55–IrIII, 55–IrIII +HgII, 55–IrIII +HgII +cysteine in aqueous solution, 2.4. BODIPY-FL lex =510 nm. Side and top view of the lowest energy coordination mode of IrIII to the lissamine receptor,with the inset highlighting the interaction sites Boron–dipyrromethene (BODIPY)dyesare very stable against and coordination distances (color code:Cl, green; S, yellow;O,pink;C,gray; light degradation and chemical attack;they have very high ex- Ir,dark blue;H,white).Reproducedfrom Ref. [107]with permission from tinctioncoefficients, high fluorescence quantum yields, and are The Royal Society of Chemistry.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 24 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim soluble in most organic solvents. By simply varying the sub- H2O2 without interference from other reactive oxygen species stituents in the different positions, awide range of emissive (ROS) and reactivenitrogen species(RNS) under physiologic fluorophores can be synthesized, covering the blue to the conditions. The “turn on” in the fluorescencesignal can be at- near-IRpartthe electromagnetic spectrum. Probe 59 (see tributed to the formation of probe 69a,which is aproduct of

Figure 12) shows bright-greenfluorescence with an emission oxidation by H2O2 (see Figure 12). band at l=512 nm in acetonitrile/watersolutions. This probe To design probes that are able to detect gas molecules, shows highselectivity towards CuII cations, which causes Gotor and co-workers[124] have developed probes 70 and 70 a, quenching of the fluorescenceemission. The 59–Cu complex is which are able to detect the nerve gases diethylcyanophos- selectivetocysteine (Cys) and homocysteine (Hcy) over other phonate (DCNP) and diisopropylfluorophosphate (DFP) in buf- amino acids such as Thr,Asp, Leu, Iso, Pro, Met, Glu, Try, Gly, fered water/acetonitrile solutions(see Figure 13). Initially,both Ser,Asn, Phe, Gln, Tyr, Arg, Lys, His, Ala, and Val; recovery of probeshave no emission due to intramolecular charge transfer the emissionwith asixfold enhancementinthe intensity is ob- (ICT) processes promoted by the nonbonding electrons on the served.[117] Probes 60 and 61,selective towards Cys, are report- nitrogen atom. The addition of the nerve agentsleads to a ed by Guo and co-workers.[118] These probes are not emissive, large enhancementinthe corresponding emission bands due but the presence of Cys leads to a300-fold increaseinthe to the formation of ammoniumsalts through adisplacement emission intensity at l=516 nm for probe 60 and a54-fold in- reaction. To assess the potential use of theseprobesinhand- crease at l=512 nm for 61,generating green-emissive species held sensing kits, the authors have also immobilizedthe 60a and 61a (Figure 12). Cysteine causes cleavage of the 2,4- probesonasolid support;anenhancementinthe fluores- dinitrobenzenesulfonyl (DNBS) groupsinthe probes, which cence is also observed in this case. Pan and co-workers[125] generates the new species. These probe have been used for reportoff–on fluorescence BODIPY derivative 71,which can fluorescenceimaging of intracellular thiols in NCI-H446 cells. detectcarbon dioxidegas. Probe 71 is highly sensitive to pH Aseries of green-fluorescent BODIPY–piperazine conjugates with a500-fold enhancement in the fluorescence signal at separated by alkyl spacers (probes 62–65)have been synthe- acidic pH values (pH 1.42 to 4.12) on the basis of aPET mecha- sized by Singh and co-workers[119] (see Figure 12). The sensing nism. The authors postulate that the green fluorescenceob- ability of these probestowards avariety of metal ions has served in the presence of CO2 gas can be attributedeither to been studied. Probes 62–64 are inactive towards NaI,KI,CaII, the reaction of the tethered phenylamino group of 71 with II I II II II II II II III III III Mg ,Ag,Ni,Zn,Cd,Cu,Pb,Hg,Al,Fe,and Cr in aceto- CO2 with the formation of carbamic acid or to the formation of nitrile/water solutions. However,probe 65 shows selectivity to- the protonated phenylammonium species of probe 71,which II wards Hg ,with a“turn off” in the fluorescencesignal at l= induces the conversion of CO2 into carbonic acid (see 521 nm. This quenching of the fluorescencesignal is due to Figure 13). HgII coordination with the donor atoms in the piperazine unit and the triazlyl nitrogen atom, which leads to the formation of 2.5. 7-Nitrobenz-2-oxa-1,3-diazole-4-yl a 65–HgII complex. Wang et al.[120] have prepared BODIPY- based chemosensors 66 and 67 (see Figure 12). In experiments 7-Nitrobenz-2-oxa-1,3-diazole-4-yl (NBD), achromophore with performed in ethanol, probe 66 is selectivetoFeIII with little in- high cell permeability and long wavelength absorbance, is terference from AlIII and CrIII ;inaqueous solution, probe 67 is often used in biological applicationssuch as the fluorescence selectivetoAlIII over CrIII and FeIII.Probe 66 shows the typical labeling of proteins and to study structuraland conformational bands of BODIPYdyes, with absorption maximum at l= modifications in proteins. Xu and co-workers[126] have synthe- 337 nm and an emission band at l= 571 nm. Upon the addi- sized NBD-based probe 72 for the selective detection of ZnII. tion of FeIII,the band at l= 571 nm decreases and anew in- In aqueous solution and upon the addition of ZnII,probe 72 tense band at l=502 nm appears;achange in color from pink developsared-to-yellow color change and an enhancement in to yellow occurs, which can be observed by the nakedeye.[120] the fluorescence signal due to the combination of ICT and PET Probe 67 showeda“turn-on”effect on the fluorescence signal mechanisms caused by the formation of the 72–ZnII chelate at l=515 nm upon metal coordination. Coordination takes (see Figure 13). Using the NDB chromophoreasthe signaling place with the N-methyl-N-(2-hydroxyethyl) moiety at the lone unit, probes 73[127] and 74[128] have been developed (see pair of electronsonthe nitrogen atom, which blocks the PET Figure 13). Probe 73,formedbyaddition of (1,3-alternate- process and gives rise to an enhancement in the emission in- 25,27-bis(2-aminoethoxy)-26,28-bis(2-methoxyethyl)thiacalix[4]- tensity.[121] Also taking advantage of aPET mechanism, Li and arene) to NBD-Cl, is acolorimetricand fluorescent sensor for [122] I co-workers have designed probe 68,a“turn-on” green fluo- Ag and AcOÀ in water/THF (1/4, v/v;pH7.5);the addition of rescenceBODIPY derivative bearing an indole moiety;the these ions causes quenching of the emission signal. Sensor 74 probe detects hydrogen sulfate (HSO4À)anions in acetonitrile containsaN-(2-aminoethyl)picolinamide moiety.Studies per- and aqueous solutions. In acetonitrile, the PET effect is blocked formed in aqueous solutionsshow that upon binding to CuII, due to coordination between the HSO4À anion and the amine the green emission of the probe at l=544 nm is completely groups;inaqueous solutions, blocking is due to protonation. quenched;CaII,ZnII,MgII,CdII,CoII,FeII,HgII,MnII,and NiII are Li and co-workershave also published interesting N-alkylated not detected by this probe.The 74–CuII complex has 1:1 [123] BODIPYderivative 69. This probe shows a“turnon” of the (ligand/metal) stoichiometry,with an association constant(Ka) 3 1 II fluorescencesignal in the presence of H2O2 and is selective to of 1.2210 mÀ .Regardingthe detection of Hg by using NBD

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 25 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 12. Chemical structuresand mechanisms of compounds 59–69.Panel I) a) In vivo fluorescenceimages of 20-day-old angelfish treated with 10 mm probe 69 for 30 minat258Cand b) the bright-field image. c) Probe-stained angelfish treatedwith 100 mm H2O2 for 120 minat258Cand d) the bright-field image.Panel II) a) Fluorescence imagesofHepG2 cells incubated with 10 mm probe 69 for 30 min at 378Cand b) the bright-field overlay to confirm viability. c) Probe-stained HepG2cells treated with 100 mm H2O2 for 90 min at 378Cand d) the bright-field overlay.e)Probe-stainedHepG2 cells stimulated with 1 1 mgmLÀ (PMA=phorbol myristate acetate) for 90 min at 378Cand f) the bright-field overlay.g)Fluorescence imagesofLO2 cells incubated with 10 mm probe 69 for 30 minat378Cand h) the bright-field overlay. i) Probe-stained LO2 cells treatedwith 100 mm H2O2 for 90 min at 378Cand j) the bright-field 1 overlay.k)Probe-stained LO2 cells stimulated with 1 mgmLÀ PMA for 90 min at 378Cand l) the bright-field overlay. Reproduced with permission from Ref. [123].Copyright 2014 Elsevier B. V.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 26 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim probesbehave as on–off fluorescencesensors in the presence of HgII.For probe 77,besides achange in the fluorescence emission, acolorimetric effect is also observed, and the color changes from yellow to colorless. The stoichiometry of the 77– II 9 1 Hg complex is 1:2(ligand/metal) with Ka =8.610 mÀ . Tsui and co-workers[131] describe chemosensors 78–80 containing NBD and 4-[(4-methoxyphenyl)diazenyl]benzoic acid moieties for cyanide detection in aqueoussolution.The recognition of CNÀ is due to the strong affinity of this anion to the carbonyl Catom of the amide group;nucleophilic attack occurs, which is followed by protontransfer of the amide hydrogen atom, and this leads to the formation of the alkoxide anions of 78–80.Acolorimetric effect, with acolor change from yellow to red, is produced by the enhancement in the charge-transfer interactions between the electron-rich and electron-deficient units, and a“turn on” in the emission intensity is also observed. Niu and co-workers[132] report the ability of NBD-Cl probe 81 (see Figure 14) to detect thiols on the basis of an intramolecular displacement mechanism. Probe 81 can detectselectively intracellular cysteine and homocysteine in HeLa cells.[133] Chen and co-workers[134] have designed NBD-SCN fluorogenic agent 82 for accurate detection of cysteineand homocysteine. The thiocyanate group presentinthe structure of 82 increases its push–pull characteristics, which results in an enhancement in the emission intensity.Atl= 550 nm, probe 82 shows a470- fold enhancement in the fluorescence signal for cysteineand a 745-fold enhancement in the fluorescencesignal for homocysteine;the detection limits are 2.99 and 1.43 nm,respectively. The efficiency and cell permeability of 82 have been studied in Raw 264.7 cells (see Figure14).

2.6. Naphthalimide Derivatives and Acridine Orange 1,8-Naphthalimide (NP) derivatives have apure-blue fluorescence emission at l=460 nm;the introductionofdifferent electron-donating substituents at the 4-position allows fine- tuning of the emission with colors ranging from blue to yellowish green. Lucifer yellow is an example of asubstituted NP derivative, and it is widely used in cell biology to visualize fixed and living cells.[135] Xu and co-workers report fluorescent probes 83[136] and 84[137] based on naphthalimide that exhibit highly selective “turn-on”fluorescencefor AgII and HgII ions, respectively,inaqueousmedia. Probe 83 has ahydroxyquinoline receptor,and it is responsible for coordination to the AgI metal ion. PET from the electron-rich receptor to the excited naphthalimide fluorophore makes the probe weakly fluorescent;coordination with AgI inhibits transfer,which leads to asignificant enhancement in fluorescenceatl= 533 nm (see Figure14). The 83–AgI complex has a1:1 (ligand/metal) ratio 5 1 and an association constant of 9.010 mÀ .Cell permeability I Figure 13. Chemical structuresofcompounds 70–80. and imaging of Ag in living cells by probe 83 have also been tested.Upon replacing the hydroxyquinoline moiety by amor- pholine one, the same authors have designed “turn-on”fluo- derivatives, NBD-based probes 75 and 76 containing triazole rescence probe 84,whichisselective for HgII.The mechanism units[129] and probe 77 containing a2-pyridylmethyl-(2-quino- is similartothat of probe 83 and involves inhibition of the PET lylmethyl)amine unit[130] are shown in Figure 13. These three mechanism, which leads to an 11-fold enhancement in the

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 27 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 14. Chemical structuresand/ormechanisms of probes 81–84.Panel I) a) Fluorescenceimages of HeLa cells incubated with only probe 4 and b) followed by incubation with Cys for 30 min. Reproduced with permission from Ref. [134]. Copyright 2014 Elsevier B. V. Panel II) Bright-field (top) and fluorescence (bottom) images of Raw 264.7 macrophages a,b) in the absenceand c,d) in the presence of NBD-SCN (5 mm). e,f) Bright-field and fluorescenceimages of Raw 264.7 macrophages, whichwere pretreated with NEM (12.5 mm)for 30 min and then incubated with NBD-SCN for 30 min, respectively.The scale bar represents 50 mm. GSH =glutathione;NEM=N-ethylmaleimide. Reproduced with permission from Ref. [134].Copyright 2014 ElsevierB.V.

6 1 emission spectra with an association constantof6.06 10 mÀ . selectivedye that emits green light if linked to double-strand- The authors conclude that, for both probes, coordination of ed DNA (dsDNA) with an emission wavelength of 520 nm and the pyrazinenitrogen atom that links the naphthalimidering is emits red if it is coordinated to single-stranded DNA (ssDNA) involved in chelation of the AgI and HgII ions and that this in- or RNA (l=650 nm). Due to its metachromatic properties, acri- volvement is crucial for the “turn-on” effect;reactionofprobe dine orange is often used in flow cytometry analysis, fluores- 84 with CuII produces a“turn-off”effect, which indicates that cence microscopy,and cellular physiology.[138,139] this nitrogen atom is not involved in coordination to this metal ion. 2.7. Perylene Diimides Acridine orange is another green-emissive dye used for cellular stainingdue to its good cell permeability and its capabili- Perylene diimide (PDI) is avery attractive chromophoreand ty to bind nucleic acids through electrostatic interaction. It is a has been widely studied as an active component in solar cells,

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 28 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim field-effect transistors (OFETs), and organic light-emitting the red region are readily available. Red emittersare of interest diodes (OLEDs). The dye has great opticalproperties such as a in numerous fields such as opticalcommunications[142] and high fluorescencequantum yield andahigh extinction coeffi- energy conversion;[143] they are also extremelyuseful tools in cient, and it has good chemical stability,photostability,and biomedical analysis, for example, geneticanalysis, DNA se- thermostability.[18,19] The absorption and emission maximaof quencing, in vivo imaging, and proteomics.[144, 145] Dyes with PDI are above l=500 nm, which avoids problems with auto- red emission are employed in the areas of OLEDs,[146] protein fluorescencebackground in experiments with living cells. How- tracking,[147] multicolor imaging,[148] and far-field optical nano- ever,because of its tendency to form aggregates, its water sol- scopy.[149] In OLEDs, red-emitting dyes can be used to comple- ubility is poor.Inanattempt to address solubility problems in ment blue[150] and green[151] dyes in the construction of dis- aqueous solutions, Sun and co-workers[18] have published a plays. Red-emitting dyes are particularly valued in the imaging review on the synthesis and applications of PDI derivatives of biological tissues,due to their ability to produce emission with improved aqueous solubility.These probes are obtained signals that are distinguishable from the autofluorescence by the insertion of multiple polar groups into the bay region, background of the tissues[152] and to produce images with high in the imide or ortho positions of the PDI dyes; these substitu- spatialand temporal resolution.[153] ents hinder aggregationand improve the solubility of the Organic red-emitting fluorophores belongtovarious dye probesinwater.Studies performed on the potentialofthese families. As red emission is linked to extensive p-electron con- water-soluble PDIs to be used as biosensors and bioimaging jugation, fluorophores are usually large polycyclic aromatic hy- agents in living cells, tissues, and the body are promising, and drocarbons, porphyrin-typecompounds,orvery polar push– the authors proposethat PDIs can be used as carriers for pull heteroaromatic compounds. Developing red fluorophores gene/drug delivery in gene therapy or chemotherapy.[18] that can be used as pure organic materials is particularly chal- Heek et al.[15] have designed aPDI derivativethat can be lenging. Because of their structure, they all show atendency used as amembrane marker,and it is able to track polyglycer- towardsaggregationdue to intermolecular p stacking or at- ol-boundbioactive compounds in both artificial and cellular tractive dipole–dipoleinteractions.Aggregation is extremely membranes. Upon dissolving the probe in water,itforms mi- detrimental to fluorescence,[154] and most red-emitting fluoro- cellar self-aggregatesand the fluorescencesignal is quenched; phores becomevery weakly emissive, or not emissive at all,at if the probe is in alipophilic environment, such as abiological high concentrationand/orinthe solid state;thus, they are membrane, the probe remainsinmonomeric form and produ- generally used in solution at lowconcentrations or after dis- ces strong green fluorescence. Montalti and co-workers[16] have persion in organic or inorganic matrices. However,the search taken advantage of the ability of PDIderivatives to change for red dyes that can be used in the solid phase is essential to their fluorescent color and have synthesized nonfluorescent be able to synthesize self-assemblingfluorescent organic nanoparticles (NPs) of anew strongly fluorescent perylenede- micro- and nanoparticles.These materials present distinct ad- rivativethat can be dispersed in water.The NPs are used as vantages in optics[155] and biomedical imaging[156] because of multicolor fluorescent imaging agentsinyeast cells;bycon- their increased brightness, chemical and photochemical stabili- trolling the dosage, the researchers are able to produce green ty,low cost, high structural flexibility,low toxicity,and bioavail- or red fluorescence, and by photoirradiating the samples, the ability. authorsare able to achieve amulticolor experience.

3. Red Fluorescent Dyes 3.1. Cyanines Fluorescent probes are very useful tools for the analysis of Cyaninederivatives are akey type of NIR fluorescent probes physiological events such as ion-channel activity,localization of for biological applications, because of their high molar extinc- metal ions in biological samples, and enzyme activity;this can tion coefficients, moderate-to-highfluorescencequantum be achieved by following changes in their optical properties yields, and abroad wavelength tunability.[157] Guo and co-work- (fluorescenceintensity and excitation/emissionwavelength) as ers[158] describe the five-stage synthesisofcyanine-based fluo- aresult of specific interactions with the target molecules.[140] rescence probe 85 composed of atricarbocyaninefluorophore This section presents some recent examples of novel probes and tris(2-pyridylmethyl)amine (TMPA);this probe is selective based on red dyes. Fluorescent dyes that emit in the red and towardsZnII ions (see Figure 15). Upon binding of the TMPA near-infrared (NIR) regionsofthe electromagnetic spectrum moiety to the ZnII ion, the amine attached to the center of the can be particularly useful for imaging living cells and tissues, polymethine chain of the tricarbocyanine is deprotonated to as the fluorescenceemission in the longerwavelength region form an imine, which is cross-conjugatedwith the less-delocal- can reduce autofluorescence effects from the biological matrix, ized diaminotetraene group. Reduced delocalization resultsin encourage deeper tissue penetration in vivo, and avoid visible- alarge hypsochromic shift in the emission maxima of 85 light absorption;moreover,typically they causeless photo- (Figure 15) and leads to lowering of the background signal and damage.[141] The opticalsetup used for working in the red an increase in the signal fidelity for ZnII.Inaddition, 85 has a region of the electromagnetic spectrum is simpler than that very strong binding affinity for ZnII,and it exhibits high sensi- used for workinginthe green region, as the scattering effect tivity and selectivity towards ZnII over other metal ions, with a for the red region is minimal and sources such as dye lasers in detection limit in the nanomolar range. Probe 85 has also

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 29 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 15. Chemical structuresofcompounds 85–88.Panel I) Normalized fluorescence emission spectra of a) PMI (87)(lex =530 nm) and b) PDMI (86)

(lex =425 nm) beforeand after reaction with cyanide. Inset) Corresponding photographs of PMI (87)and PDMI(86)bothinHEPES buffer/DMSO (7:3, v/v, pH 7.4) solution under aUVlamp at l= 365 nm. Panel II) Confocal laserscanningimaging of HeLa cells with PMI (87)incubated in RPMI-1640,pH7.4, 378C. First row)a)bright-field,b)dark-field, and c) merged images of HeLa cells incubated with PMI (10 mm)for 30 min. Second row)d)bright-field, e) dark-field, and f) mergedimages with following incubated with CNÀ (2.0 equiv.) for another 30 min. lex =543 nm. Emissionwas collected by the red channelfrom l= 650 to 750 nm. (For interpretation of the references to color in this figure legend, the reader is referredtothe web version of the article.). Reproduced with permission from Ref. [159]. Copyright 2014 Elsevier B. V.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 30 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim been successfully used in live mammalian cells as an imaging cules, such as glutathione and amino acids (see Figure 16). tool for ZnII ions released during apoptosis. Probe 89,astyryl BODIPY/DNBS dyad, shows intenseabsorp- To improvethe solubility of phenazine–cyanines in water, tion at l= 556 nm and is nonfluorescent;however,the DNBS Yang and co-workers[159] have introducedavinylindolium moiety is cleaved by thiols, which resultsina46-fold enhance- moiety into the phenazineskeleton to synthesize phenazine– ment in the red emission band centered at around l= 590 nm. cyaninesderivatives PMDI(with two indolium moieties on The probe has been used for the fluorescent imaging of cellu- both sides of phenazine) 86 and PMI (with one indolium lar thiols, with the advantage that it is pH independent in the group) 87 (see Figure 15). The quenching effect on the phenazine–cyanine fluorophorebystrongintramolecular charge transfer (ICT) from the phenazine (donor) to the indolium (receptor) moieties makes both 86 and 87 nonemissive. Both compounds behaveaschemodosimeters for CNÀ detection, with detection limits of 1.4 mm and 200 nm,respectively.Upon coordination to the CNÀ anion through the N-methylindolium group, the ICT effect disappears, which leads to adramatic “off–on” enhancement in the fluorescencesignal with emission maximaatl= 580 nm for 86 and l=630 nm for 87.Probe 87 seems to be apromising candidate for monitoring intracellular CNÀ in HeLa cells (see Figure 15, panel II). The measurement of intracellular CaII has becomeanimpor- tant topic in biological and medicalresearch.[160] Zhu and co- workers[161] have developed new visible-light-excited andred- emitting probe 88,which is used as aratiometric fluorescence probe for detecting intracellular CaII (see Figure15). Probe 88 is made of 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid, which acts as the CaII-chelating moiety,and two benzo- thiazolium hemicyanine dyes acting as fluorophores. An approximately 48-foldenhancement in the intensity of the fluo- rescencesignaland ablueshift of Dl=20 nm (from l= 600 to 580 nm) in the emission spectrum are observed upon adding the CaII ion;probe 88 has successfully been used to distinguish simultaneously between CaII changes in the cytosoland nuclei of living cells.

3.2. BODIPY Boron dipyrromethene (BODIPY)derivatives can be used as molecular probes,[162] in photodynamictherapy,[163] as laser dyes,[164] in nonlinearoptics,[165] in dye-sensitized solar cells,[166] and as part of electrogenerated chemiluminescence (ECL) emit- ters for the study of organic and inorganic materials.[167] The use of BODIPY derivatives in those applicationsisbased on their narrow absorption andemission bands,high fluorescence quantum yields (even in aqueous media), large molar absorption coefficients, pH-independent emissions, and excellent photostability;the characteristic emission of the original BODIPY fluorophore is centered at around l=520 nm with a small Stokes shift (l 20 nm).[168] Interest in BODIPYs hasbeen mostly on red- or near-infrared-emitting probes constructed by the following:1)intramolecular rigidification of the molecular structure by B Oring for- À mation;[169] 2) fusion of aryl or heteroaryl moieties into the BODIPY structure;[170] 3) using tetraaryl-substituted azadipyrro- methenes;[171] and 4) with styryl linkers in the 3,5- or 1,3,5,7-positions and, more recently,inthe 8-position.[172] Following this last strategy,Shao and co-workers[173] have synthesized 89 for the specific detection of cysteine among other biological mole- Figure 16. Chemical structures of compounds 89–92.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 31 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim physiological pH range. Hiruta and co-workers[174] report on two distyryl-BODIPY-based NIR red-shifting ratiometric fluorescent probes: KBHR-1 (90)for pH and KBAHgR-1 (91)for AgI and HgII (see Figure 16). Probe 90 shows aredshifting ratiometric response to pH in the NIR region. The use of adifferent dialkylamino moiety as the recognitionunit for probe 91 results in aratiometric response to AgI and HgII,also in the NIR region. Hirata and co-workers[175] have developed probe B3TAC (92) (see Figure 16), which is synthesized by conjugating 2-triaza- cryptand[2,2,3]-1-(2-methoxyethoxy)benzene (TAC) to the 3- positionofthe BODIPY fluorophore through astyryl linkage. Probe 92 is highly selectivetoKI in the physiological concentration range over NaI and other metal ions by using water/ acetonitrile as the solvent. Although 92 shows good potential as aselectiveprobe forthe detection of intracellular or extracellular KI,furthermodificationsofthe structure are neededto improveits solubility in water to avoid the need for an organic solvent. BODIPY derivatives 93–98 (see Figure 17), prepared by Fu and co-workers,showsimilar solubility problemsinwater.[176] The presence of trihexylsilyl (THS) and trimethylsilyl (TMS) groups makesthem sensitive to fluoride ions, which induces a strong blueshift (Dl=70–117 nm) and asignificant increase (6- to 40-fold) in the fluorescenceintensityinthe emission spectra. It is likely that the extended p system in all of these probesallows for the large shifts in the absorption and fluorescence bands in the presence of FÀ .Inparticular,probe 98a turns from red to yellow upon adding FÀ ions (see Figure 17), but no color change is observed in the presenceofClÀ ,BrÀ , 2 2 IÀ ,SCNÀ ,NO3À ,HCO3À ,CH3COOÀ ,H2PO4À ,CO3 À,and SO4 À. The chemosensing abilities of 98 a have also been tested by monitoring the emission spectra of the probe in acetone. The probe exhibits the largestblueshift in the fluorescenceband if in the presence of FÀ (Dl=117nm), along with a40-fold enhancement in the fluorescenceintensity.Again,the promising sensing capability of these probes is somewhat hampered by the fact that they cannot be used in aqueous solution. Major effort has been devoted to improving the solubility of BODIPY dyes in water,sothat they can be used in biological applicationssuch as intracellular and tissue imaging. As an alternative to red-emitting 3,5-distyryl-BODIPYs, 3,5-dithienyl- Figure 17. Chemical structures of compounds 93–98.Relative fluorescence BODIPYscan provideagood compromise between solubility, spectralchanges of probe 8a (98 a)(10 mm)after treatment with 50 equiva- [177] stability, and hydrophobicity.Poirel and co-workers report lents of various anionsinacetone at lem =570 nm. Insets) correspondingsol- the synthesis of red-emittingwater-soluble thienyl-BODIPYs utionsuponirradiationwith aUVlampatl=365 nm (top) and underambi- 99–102 (see Figure 18). The trimethyl(propargyl)ammonium ent light (bottom). Reproduced with permission from Ref. [176].Copyright 2015 Elsevier B. V. group is chosen to improve watersolubility.One or two cationic arms are introduced either in the 2-position of the thienyl unit or in the 4-positiononthe boronatom. Probes 99–102 bioimaging probes, and for the synthesis of organic light-emit- have strong absorption (at l 600 nm) and intense emission ting materials. Probe 103c shows aremarkable capacity to (at l 650 nm) in water. change color due to protonation effects and has been used as Zhu and co-workers[178] have designednew aza–boron–di- apHsensorboth in solution and in the solid state;the probe, quinomethene complexes 103a–e containing different N-aryla- supported on filter paper,isable to act as apHsensor upon mines (see Figure 18). Photoluminescence studies reveal that exposure to acid and base vapors. all of the probes show an intense and tunable luminescence Chen and co-workers[179] have synthesized two novel bis(me- signal from blue to red and good emission quantum yields. thoxyphenyl)–BODIPY fluorescentprobes, 104 and 105,for the These probes show considerable potentialaspHsensors, as detection of nitric oxide (NO) (see Figure19). The probesshow

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 32 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim red fluorescence(l=590 and 620 nm, respectively) and large Stokes shifts (Dl=38 nm) in the far-visible/NIRspectralregion. Both probes are highly selective towardsNOinthe presence of other reactive oxygen species and reactive nitrogen species. Besides studying their fluorescentproperties, the researchers have used both probes for NO imaginginliving cells (RAW264.7);the cytotoxicity studies are performed in amix- ture of acetonitrile/water,asthe probes are not soluble in water.Both dyes are promisingcandidates for fluorescence imaging of NO due to low background interference and high detection sensitivity.

3.3. Coumarins Probes containing coumarin, afluorophore known to be highly fluorescent and with moderate to good quantum yields, have been used in awide range of biological applications such as the fluorescent labeling of proteins,cellular imaging, and lasers.[180–183] Different strategies can be employed to ensure that coumarin derivatives have enough charge transfer in the coumarin molecule to push the absorptionand emission in the red region:1)introducing an electron donor in the 5- and 7- positions;2)introducing an electron-withdrawing group at the 3- and 4-positions;3)increasing the rigidity of the donor and acceptorgroups;and 4) extending the conjugation. Khema- khem and co-workers[184] show the value of using substituents with low molecular weights to prepare new orange- and red- emitting fluorescent materials based on coumarin. In their work, the researchers explore the fluorescenceproperties of four derivatives of 3-thienyl-2-(N-dicyanovinyl)iminocoumarin, that is, 106–109,bearing adiethylamino group in the 7-position (as in 106)oramethoxy group in the 6-, 7-, and 8-positions (as in 107–109); studies have been performed both in solution, using arange of organic solvents as well as water,and in the solid state (see Figure 19). The fluorescence emission spectra of probe 106 ranges from yellow/orange to red;the compound emits strongly in dilute solutionsand shows remarkable solvatochromic behavior.Stability studies have been conducted on probe 106,which shows the best solubility in aqueous solutions, and this probe resists pH changes without hydrolysis; 106 has successfully been used to stain the cytoplasm of HCT-116 colon cancercells. The fluorescenceemission spectra of probes 107–109 range from yellow to green. The presence of the methoxygroup in the structure makes the probessuitable for solid-state emission;additionally,probes 107 and 109 exhibit crystallization-enhanced emission. More- over,the researchers also prepare nano- and microsized parti- cles for all of the probes, including millimeter-long microfibers, which exhibit clear wave-guiding properties;this makes them promising candidates as optical guides forpotential biomedical applications. Hou andco-workers[185] have synthesized red-fluorescence

probe 110,with lmax(emission)=616 nm. The probe is able to detectFÀ in aqueous solution on the basis of FÀ-triggered Si À Figure 18. Chemical structuresofcompounds 99–103.Reproduced from Obond cleavage, whichleads to acyclization reaction in the Ref. [178] with permission from The RoyalSocietyofChemistry. probe and the formation of iminocoumarin dye 111.The reaction has arapid response time (within10min), is highly selec-

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 33 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 19. Chemical structuresofprobes 104–115.Synthesisroute to probe 110 and its proposed reaction with NaF to give fluorophore 111.Structures of the isaindigotone framework,colorimetric probe 112,and upgraded colorimetric and fluorescent dual probe 113.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 34 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim tive towards FÀ in the presenceofother anions, and can 3.5. Rosamine detect FÀ in concentrations ranging from 1.0 mm to a10mm. End-product 111 emits red photons, which help to minimize Differences in pH can be used to differentiate cancercells interference from endogenous chromophores (Figure 19), and (pH 4.5–5.5) from normal cells (pH 5.0–6.0);[192] thus, with the exhibitsarelatively large Stokes shift (Dl= 143 nm), which is use of fluorescence probessensitive to pH it is possible to vis- beneficial for signaldetection in fluorescencemicroscopy.Fluo- ualize the acidic lysosomal lumen of cancer cells. Sun and co- rescence microscopy experiments have established the useful- workers[193] have developed rosamine-based pH probe 116 (see ness of probe 110 to detect FÀ in living cells, andthe probe Figure20), which is capable of intracellular pH imaging in live has been used to monitorand image FÀ in living HaCaT cells in cells. The presence of an electron-donating side chain contain- aqueous media. ing an amino group makes the probe nonfluorescent in neutral In biochemistry and clinicaldiagnosis, it is of key importance environments as aresult of PET;however,inacidic pH environ- to develop highly sensitive and selective probes to detect nu- ments, the probe behaves as an off–on probe with a400-fold cleic acids,[186] in particular G-quadruplex structures.[187] To enhancement in the fluorescencesignal upon binding with H+ detect G-quadruplexstructures, Yanand co-workers[188] have ;other physiologically important cations do not interfere with developedcolorimetric probe 112 by merging thiazole orange the recognition process. Research performed in live HeLa cells into an isaindigotone framework(see Figure 19);the sensitivity indicates that the probe has good cell permeability,and co- of the probe and its further application are restricted due to staining with the commercial lysosome-staining probe Lyso- weak fluorescenceemission. Buildingonthis work, Yanand co- Tracker Green DND-26shows good agreement betweenboth workers[189] have synthesized asimilarprobe, 113,this time by the commercial and the experimental dyes. The research thus merging acoumarin–hemicyanine fluorophore into the isaindi- showsthat 116 can be successfully used as red-emitting lyso- gotoneframework. Probe 113 is successful in detecting G- some-specific probe. quadruplesstructures, and amarked “turn-on” effect in the fluorescencesignal is observed. Remarkably,the probe also 3.6. Nile Red shows acolor change from pink to blue, which is visible by the naked eye, so it could also act as acolorimetricprobe.The Nile red is well established as an environment-sensitive fluores- researchers show the selectivity of 113 towards G-quadruplex- cent dye for labeling and sensing biomolecules, because of its es in the presence of interference analytes such as ssDNA, high fluorescence quantum yield, long-wavelength emission, dsDNA,and the bovine serum albumin (BSA) protein. Intracel- and good photostability.[194] Tang and co-workers[195] have de- lular applications of 113 in live andfixed cells have been per- signed long-wavelength-emitting fluorescent “turn-on” probe formed by using HeLa and A549 cells. The results reveal that 117 for H2Sdetection(see Figure 20). The detection mecha- 113 accumulatesinthe nucleus, mainly bound to the rDNA nism is based on the thiolysis of the dinitrophenyl ether,which regions. is added to the 2-position of Nile red to form 117.The fluorescence of the free probe is quenched by the PET process between the fluorophore and the dinitrophenylether group. In experiments performed in aqueous solution, the probe shows 3.4. Rhodamine a17-foldincrease in the fluorescencesignal in the presence of 7 Because of their high photostability,high fluorescent quantum H2S(detection limit of 2.7 10À m), with no interference from yields, high extinction coefficients, andlow degree of triplet variousbiologically relevant species. The probe shows good formation, probes derivedfrom rhodamine are widely used as membrane permeability and has successfully been used for laser dyes and fluorescent markers for the labeling of biomole- the fluorescenceimaging of H2SinMCF-7 cells (human breast cules.[11,190] Kolmakovand co-workers[191] have developed novel carcinoma). red fluorescent dyes for imaging and labeling applicationsin the red optical region (see Figure 19). The probes have been 3.7. Naphthalene Diimide Dyad used to image arange of biological substrates such as various animal antibodies and sphingomyelin lipids. Probe 114 and de- Naphthalene diimide (NDI) derivatives have been successfully rivatives are lipophilic, whereas probes 115 and 115D are solu- used in awide range of applications, including the construc- ble in water and aqueous buffers. The probes are very photo- tion of supramolecular materials, such as rotaxanes and cate- stable, have alow tendency to aggregate, and have relatively nanes, and as molecular sensors.[196] Doria and co-workers[197] long excited-state lifetimes (t=3.4 ns). High-resolution GSDIM reportthe synthesis of 118,awater-soluble dimeric NDI result- (ground-state depletion with individual molecular return) ing from the conjugation of two monomeric NDIs, red trisub- images and live-cell STED-FCS(stimulated emission depletion- stituted dye 119 and blue tetrasubstituted 120,through a fluorescencecorrelation spectroscopy) experiments performed (CH2)7 flexible spacer.Probe 118 is nonfluorescent, but in the on labeled microtubules and lipids show the capability of the presence of G-quadruplex DNA, aturn-on effect in fluores- probestobeused in fluorescencemicroscopy and nanoscopy. cence is observed with the subsequent complex emitting in the red/NIRregion. Although the probe exhibits good selectivity to G-quadruplexDNA in the presence of dsDNA,itisnot able to select between different G-quadruplex structures.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 35 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim 3.8. Distyrylpyridinium

Styryl (or arylvinyl) dyes represent an important class of func- tional dyes that can be used in optical recording media, laser dyes, and optical sensitizers and as fluorescent probes in bio- medicalapplications.[198] Xie and co-workers[199] have synthesized three red-emittingcationic distyryl probes (see Figure20): two of them, 121a and 121 b,are derived from the asymmetric2,4-bis(4-dimethylaminostyryl)-1-methylpyridinium ion, and the third one,122a,isderived from the symmetric 2,6- bis(4-dimethylaminostyryl)-1-methylpyridinium ion. The re- searchershave studied the ability of the probes to detectsev- eral quadruplex and duplex nucleic acids. Dyes 121a and 121b are able to distinguish between quadruplex DNA and dsDNAstructures, both by astrong“light-up” effect in the presence of quadruplex DNA (80–100-fold “turn on” of the fluorescencesignal),aswell as by the positionofthe emission maximainthe fluorescence spectra.Symmetric analogue 122a as not abletodistinguish between quadruplex and duplex DNA, and the enhancementinthe fluorescencesignalistypi- cally 20–40-fold.

3.9. Benzophosphole P-Oxide Scaffold Ratiometric fluorescent probesare powerful diagnostic tools for the quantitative detection of metal ions in living systems. Using benzophosphole P-oxide (123)asafluorophore, Taki and co-workers[200] have developed ratiometric fluorescent probes 124 and 124-AM for NaI detection (see Figure21). These probes are examples of d-p-A systems(D=electron-donating moiety;A=electron-accepting moiety). Typically, d-p-A systemsare fluorescent because of the existence of an ICT process. Once the probesinteract with the target metal ion, the ICT character is reduced and hypsochromic shifts can be observed for both the absorption and emission maxima. This behavior is observed for probe 124;after excitation under visible I light (lex = 405 nm), and in the presence of Na ,the probe exhibits ahypsochromic shift in its emission spectrumupon + + complexation. Experimentsconsisting in blocking the Na –KÀ pump in living mammalian cells show that probe 124-AM (the membrane-permeable form) can successfully be used for the ratiometric visualization of intracellular Na+ dynamics. The results suggest that this probe can potentially be used as an imaging tool to study NaI dynamics, for example, imaging of potential-evoked NaI influx between axon and soma in neuro- nal cells.

3.10. Benzoresorufins It is well knownthat ZnII and reactive nitrogen species(RNS) such as nitric oxide (NO) and peroxynitrite (ONOOÀ)are impli- cated in some neurological dysfunctions and play important [201] Figure 20. Chemical structuresofprobes 116–122.Mechanism of H2Ssens- physiological roles in the nervoussystem. Fluorescein-based ing of compound 117.Structureofdimeric 118 (resulting from the merging sensors can be used to study thesespecies, but they are limit- of monomeric 119 and 120), and its quaternaryammonium salt 118a,as ed by their high-energyabsorptions and small Stokesshifts. the iodide. The use of sensors emitting in the red or NIR region can avoid those shortcomings.With this aim, Linand co-workers[202]

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 36 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim reportanew family of red-emitting fluorescent sensors 127– 129 that can be used to detect labile ZnII (see Figure21). The probesare based on abenzoresorufin dyefunctionalized with dipicolylamine or picolylamine moieties as the metal-binding groups.The researchers have also studied probes 125 and 126,which contain identicalbinding moieties (andthus the

same [N3O] metal-binding motif) but have fluoresceinasthe fluorophore. Benzoresorufin-based dyes 127–129 perform well in terms of their binding capabilities towards ZnII and show binding affinities similartothose of their fluorescein-based counterparts. Asignificant advantage of probes 127–129 is their ability to monitororganelle-specific ZnII levels in live cells. This research shows that the benzoresorufin platform is a promising dye that can serve as the basis for the design of new red-emittingsensors with improvedperformance.

3.11. Tetrapyrrolic Macrocycles Metal-ion probes based on porphyrins can presentlarge Stokes shifts, high fluorescent quantum yields, and long lifetimes, andthus designing this type of probe constitutes an important area of research.[203,204] Our research group, in collabo- ration with the University of Aveiro, has explored the sensing ability of porphyrin-based probes 130–145.Probes 130–134 are benzoporphyrins, and probes 135–139 are porphyrin-2-yl- pyridines (see Figure 22).[205] Probe 141 is a3,5-disubstituted pyridine with two porphyrin moieties (Figure22).[206] Porphyrin- based probes 143a–e contain an a,b-unsaturated ketone unit in a b-pyrrolic position,[207] and probes 145a–d are pyrazole– porphyrin conjugates (see Figure 23).[208] Probes 135–139,which form complexes with a2:1 (ligand/ metal) stoichiometry,show higher stabilityconstants than probes 130–134,with a1:1 stoichiometry;itissuggested that asubstituent in the 2-positionimproves the host–guest interaction and thus allows the formation of amore stable complex. Importantly,benzoporphyrin-based probes 130–134 show significant changes upon titrationwith HgII.First, there is adecrease in the two bands at l=658 and 718 nm due to free-base porphyrin emission Q(0–0) and Q(0–0)(0–1).[209] Moreover,anew band appears at l=687 nm:this new emission band, which increases with the addition of HgII,isattribut- ed to metal-to-ligand charge transfer and indicates the genera- tion of anew fluorophore arising from metal–porphyrin complexation. Upon titration with HgII,the colorofthe solution changes from yellowish-brown to green, and this is accompa- nied by the appearance of ablueshifted emission and acolor change in the emission from red to intense orange. The high-

est association constantisobtained for probe 130 [log Ka = 3 (8.71 4.81)10À ]; this probe is able to quantify 32 ppb of Æ HgII. The resultsofthe spectrophotometric and spectrofluorimetric studies for probe 141 are shown in Figure22. Probes 141 Figure 21. Structuresoffluorescentprobes based on abenzophosphole P- and 143b are able to quantify 79 and 80 ppb of ZnII,respec- oxide scaffold. Environment-sensitive probe 123.Ratiometric NaI probe 124 and membrane-permeable form 124-AM. Chemical structuresofprobes tively.The ligand/metal stoichiometries of the complexes 125–129. formed are 1:3for the 141–ZnII complex and 1:1for the (143a–e)–ZnII complexes;probe 141 shows the highest associ-

ation constant (log Ka =15.50). The possibility of using the

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 37 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 22. Chemical structuresand mechanism of compounds 130–143.Panel I) a) Spectrophotometric and b) spectrofluorimetric titrations of 141 in chloro- form as afunction of added ZnII in acetonitrileat298 K. The inset of panel ashowsthe absorptionatl=418 and 425 nm, and the inset of panelbshows the 6 normalized fluorescenceintensity at l=608, 652, and 716 nm ([141]= 1.0010À m, lex =536 nm). The inset photograph in panel ashows asolution of 141 II in CHCl3 before(left) and after (right) the addition of Zn under visible light;the inset photograph in panelbshowsasolutionof141 in CHCl3 before (left) and after (right) the addition of ZnII afterexcitationatl =365 nm by aUVlamp. PanelII) Visual changes observed upon spraying aPMMAfilm with chemo- sensor 141 with an aqueous solution containingHgII or ZnII undervisible light and after excitationatl=365 nm by using aUVlamp (top). Emissionspectra of the PMMAfilm doped with 141 and after spraying with ZnII or HgII at room temperature (bottom). Adapted with permission from Ref. [149]. Copyright 2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 38 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 23. Chemical structuresand mechanism for the synthesis of compounds 143–145. probesasmetal-ion chemosensors in the solid state has also very strong red emission and apurple (for 146)orblue color been investigated. The probes, supported in polymethylmeth- (for 150)tothe naked eye. Upon supporting 146 on polyacryl- acrylate (PMMA) and made into films, show promising amide, the resulting gel is not emissive;however,the probe is results in the solidphase;inparticular, compounds 130, 141, able to switch on the emission after being submerged in aso- II and 143d in PMMA are abletodistinguish between Zn and lution containing FÀ ,probablydue to the large pores in the HgII. gel, which allow entry of the anion. An enhancement in the From the porphyrinoid family,corroles have merited special emissionintensity with time is also observed (see Figure 24). attentioninrecent years because of their high fluorescence Similar behavior is observed in the presence of CNÀ ,and the quantum yields and high molar extinction coefficients;more- acrylamide polymer doped with 146 is able to detect about over,the corrolecore can accommodate differentmetal ions 70.0 ppb of CNÀ in water.Santos and co-workersreport the that can act as active centers. In experimentsperformed in tol- sensing ability of probes 152 and 153 (Figure24) towards dif- uene, the sensing abilities of probes 146–151 towards arange ferent anions by using absorption and emission spectrosco- of biologically andenvironmentally relevant anions have been py.[211] Probe 152 is acolorimetric probe capable of detecting [210] studied (Figure 24). Spherical (FÀ ,BrÀ ,and ClÀ)and linear CNÀ with achange in color from green to colorless (Figure 24); (CNÀ)anions have been investigated;bulky anionssuch as the detection limit is around1.00 mm. [212] CH3COOÀ and H2PO4À have also been studied. Probe 146 with Santos and co-workers have synthesized probes 154–161 FÀ shows the highest associationconstant, and this probe is (see Figure 25). Probes 154–159 are porphyrin–coumarin deriv- able to quantify 0.69 ppm of this anion.Probes 146 and 147 atives;probes 160 and 161 are corrole–coumarin derivatives, show high sensitivity towards CNÀ ,and complexation with for which the coumarin moiety is inserted to improvesolubility CNÀ produces aredshift in the absorption and emission spec- in aqueous solution.This approachissomewhat successful, tra and an increase in the emission intensity;the probesare and both probes 160 and 161 are soluble in ethanol/water able to quantify 1.43 ppm of this anion. (50:50) mixtures. Studies on the ability of the probestodetect Probes 146 and 150 have been used to preparelow-costing arange of anionsand metal ions have been performed.Probe solid polymers supported on PMMA and polyacrylamide. The 158 shows high selectivity for HgII both in solution andinthe PMMA films prepared with compounds 146 and 150 show solid state if supported on cellulose paper (i.e. filter paper).

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 39 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 24. Reagents and conditionsfor Scheme I: a) i) GaCl3,pyridine,reflux, ii)1.36% HCl, 2. aq NaHCO3 ;b)Vilsmeier–Haack reagent;c)CH3PPh3Br,NaH in

THF,RT, N 2.Reagents and conditionsfor Scheme II:a)reflux, toluene, N2;b)reflux,toluene, N2.Chemical structures of compounds 146–153.Panel III) a) Emis- sion spectra of acrylamide gel dopedwith compound 146 in the presence of fluoride as afunction of time(T=298 K, lex =570 nm). b) Polymethylmethac- rylate film with 146 and c) polyacrylamidegel of 146 in the presenceoffluoride(FÀ). Reproduced from Ref. [210] with permissionfrom The Royal Society of Chemistry.Panel IV) a) Spectrophotometric and b) spectrofluorimetric titration of compound 152 with the additionofCNÀ in toluene.The inset represents 5 a) the absorption at l=605 and 630 nm and b) the emission intensity at l =616 and 637 nm ([152]=110À m, lex =590 nm, T=298 K). Reproduced with permission from Ref. [211].Copyright 2015Elsevier B. V.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 40 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim tinine (165)(see Figure 26).[213] Spectrophotometric and fluoro- metric experiments,performed in ethanol, show a1:1 162/al- kaloid stoichiometry in all cases. The ability of probe 162 to detectthesealkaloids in real environmental samples has been studied, and the probe is able to detect (2.5 0.3) mm of coti- Æ nine in samples of dam water.

3.12. Photostability The optical properties, low toxicity,and high selectivity to ac- cumulate in cancer cellsmakeporphyrins very useful in photodynamic therapy (PDT).[214,215] In photodynamic therapy agood photosensitizer must have ahigh quantum yield for triplet formation, has to produce singlet oxygen very efficiently (pre- dominant cytotoxic agent in PDT),[216,217] and must have an excitation wavelength to the first singlet state in the l= 700– 800 nm region.Indeed,wavelengths longer than 800 nm are preferable to penetrate deeper into the tissue; however,they do not provide enough triplet energy to excite oxygen to its singlet state. Unfortunately,during this process,molecules tend to loss their fluorescenceand are photobleached. Photo- bleaching occurs in green and red fluorophores, especially if they are irradiatedwith light. As an example, Benson and co- workers[218] report the different rates of photobleaching of acridine orange bound to DNA and RNA with green and red fluorescence. Photobleaching can be caused by photodynamic interactions between the excited fluorophores and molecular oxygen in the media. This generatessinglet oxygen and other types of damaging oxygen free radicals and leads to photo- damage. To avoid such an issue, it is very important to select a fluorophore with high photostability.Chemicals capable of quenching singlet oxygen can also be employedtoreduce the effects of photobleaching. Agood strategy is to design oxygen-reactive protective molecules includingvitamin Eana- logues, vitamin C, glutathione imidazole, cysteamine, and histidine.[219]

4. Metal Complexes The usefulness of organic molecules as probescan sometimes be hampered by the need for complicatedsynthetic proce- dures to preparethem.Also, organic probescan suffer from short fluorescence lifetimes (sometimes in the range of nanoseconds), narrow energy gaps, andinterference problems causedbyautofluorescence from surrounding biological envi- ronmentsorlight scattering.[220] To address these shortcomings, considerable effort has been focused on synthesizingflu- orescentsensors based on metal complexes.

4.1. Lanthanide Complexes Fluorescent sensors based on lanthanide complexes can be used in awiderange of applications, including as temperature Figure 25. Chemical structuresofcompounds 154–161. sensors,molecular sensors, and bioimaging agents. The sharp emission lines arising from the characteristic 4f electronic tran- Probe 162,aZnII complex of acoumarin–porphyrinunit, is sitions from the lanthanide ions and the analyte-inducedhy- sensitivetothe alkaloids caffeine (163), nicotine (164), and co- perfine energy transfer or change in coordination environment

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 41 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 27. Proposed binding modes of HSO4À and H2PO4À with sensor 166. In the absence of the anion, excitationofPQC followed by energy transfer to the lanthanide enablesbright-red luminescence centeredatl =617 nm. Interactionofanions favorsquenching of the antenna and, consequentially, also that of the EuIII luminescence. Reproduced from Ref. [223] with permission from The Royal SocietyofChemistry.

anions, with very low detection limits (15.3 and 8.3 nm,respectively).The luminescence signal of 166 is quenched in the presence of these anions throughhydrogenbondingfor

HSO4À and through coordination to the metal ion for H2PO4À . Probe 166 has been successfully used as aluminescent sensor for three nucleoside phosphates, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine mono- phosphate (AMP), in mixed aqueous solution. Due to extremelyweak absorption from f–f transitions, lan- thanideions possesslow molar absorptivity.However,their luminescence can be increased by using organic antenna chromophores, whichcoordinate to the metal ion and act as sensitizers.[224] If the organic antennachromophorechosen as asen- sitizinggroup is also sensitivetopH, it is possible to design pH probesthat detect pH in two independentpHwindows. Figure 26. Structural formula of {2-(5-oxo-2,3,4,5-tetrahydro-2H-pyrano-[3,2- [225] c]chromen-2-yl)-5,10,15,20-tetraphenylporphyrinato}zinc(II) (162), caffeine Zhang and co-workers have selected pH-sensitivefluoro- (163), nicotine (164), and cotinine (165). a, b) Naked-eye picture and under a phores such as hydroxyquinoline derivatives and rhodamine III UV lamp (lex =365 nm) of 162 (left) and 162 +cotinine(right). c) Hypotheti- moieties as the binding sites to form Eu complexes cal coordination structure of cotinine as an exampleofthe alkaloids studied (Figure 28). The researchers have developed Eu(TTA) -DSQ and probe 162. 2 (167)and Eu(TTA)3-DR1 (168){DSQ=5-(dimethylamino)-N-(4- {2-[(8-hydroxyquinolin-2-yl)methylene]hydrazinecarbonyl}phe- mechanism imply that lanthanide-based fluorescencesensors nyl)naphthalene-1-sulfonamide;DR1=N1-[4-(dimethylamino)- can offer considerable advantages over typical luminescent benzylidene]-N2-(rhodamine6G) lactamethylene-diamine; complexes.Lanthanide complexes have high luminous efficien- TTA=thiophentrifluoroacetone} probes. Probe 167 shows high cy,large Stokes shifts, and long excited-state lifetimes (up to sensitivity to pH changes in neutralaqueous solution,and milliseconds). Moreover,their high sensitivity to changes in the background fluorescenceisnegligible. For probe 168,the EuIII surrounding local environmentallows their use in time-re- ion acts as ared emitter,and the rhodamine 6G fluorophore solved fluorescence(TRF) measurements.[221,222] acts as agreen emitter.Bothcomponents of the probe are pH [223] III Xu and co-workers report the synthesis of lanthanide sensitive, with pKa values of 7.2 (Eu moiety) and 5.0 (rhoda- III III III III III complexes,Ln2PQC6 (Ln =La ,Pr,Nd,Sm,and Eu ), derived mine moiety). Luminescencetitrationsshow the ability of the from the quinolinecarboxylate ligand 2-phenyl-4-quinolinecar- probe to detectpHchanges in two different ranges, and this boxylic acid (PQC). Probe Eu2PQC6 (166)(see Figure 27) shows allows 168 to measure pH in both near-neutral pH and acidic intense red emission both in solution and in the solid state. pH ranges (see Figure 28);the probe is also able to detect pH

The probe displays high affinity towards HSO4À and H2PO4À in both cultured cells and in vivo.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 42 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim 4.2. Iridium Complexes

Cyclometalated IrIII complexes have received considerable at- tention due to their high phosphorescent quantum yields, excellent color-tuning capability,and large Stokes shifts. They also have long lifetimes (of the orderofmicroseconds), especially if compared to the lifetimes of organic fluorescent probes(typically in the nanoseconds range).[226,227] These properties make them promising candidates to be used as phosphorescent emittersinOLEDs,[228] in arange of biological applications as chemosensors,[229] as cellularimaging probes,[230] in vivo tumor imaging,[231] andasphotosensitizers forthe pro- 1 [232] ductionofsinglet oxygen ( O2). Kando and co-workers[233] have published aseries of pH-ac- tivable IrIII complexes 169–178 (Figure29) that can be used for tumor imaging. The emission intensity of these IrIII complexes is considerably enhanced upon protonation of their basic groups in aqueous solution. Astrong orange-red emission of 171 and 176 has also been reported. These probeshave been successfully used for live-cell imaging of HeLa-S3cells. More- over,byphotoirradiatingprobes 176–178 at l= 465 nm the 1 researchers are able to generate singlet oxygen ( O2)from trip- 3 II let oxygen ( O2). Photoirradiation of the Ir probesisalso able to induce necrosis-like cell death in HeLa-S3cells (Figure 30 a). Fischer and co-workers[234] report on the preparation and cal- ibration of adual sensorfor barometric pressure and temperature. The sensorismade by combining two organometallic IrIII probes, green-emitting complex 179 to measure temperature and red-emitting complex 180 to functionasabarometric (due to its oxygen-sensing ability) probe (Figure 30). Probe 179 is then appliedtopoly(acrylonitrile) (PAN) microparticles; these 179/PAN microparticles are dispersed intoaTHF solution of cellulose acetate butyrate (CAB) also containing oxygen probe 180.The mixture is then spread onto solid poly(ethyle- neterephthalate) (PET);once the solvent evaporates, asensor film approximately 6 mmthick results. Due to adifference of about 75 nm in the emission maxima of both probes, both signals can be separated by using optical filters. The dual sensor can be successfully calibrated and has potentialfor luminescence lifetime imaging of temperature and barometric pressure.

4.3. Ruthenium Complexes RuII polypyridine complexes[235] have assumed aprominent status thanks to their multichannel sensing abilities.[236] RuII polypyridine complexes can be used in colorimetric(UV/Vis),[237] [238] [239] Figure 28. Chemical structuresofdyes 167 and 168.Top) Fluorescence spec- photoluminescence, electrochemiluminescence, and [240] [241] tra of Eu(TTA)3-168 (10 mm)in0.02m NaCl buffer solutionwith different pH redox measurements. Ji and co-workers have designed II values. The inset shows the fluorescence changes of Eu(TTA)3–168 with dif- phosphorescent thiol probe 181 based on aRu–poly(1,10- ferent pH values and the fluorescence photos of Eu(TTA) –168 at pH 6.5 and 3 phenanthroline) complex (Figure 30). This complex is consid- 4.5;fluorescence intensity was recordedatl=550 nm with excitation at l= 500 nm. Bottom)Fluorescencespectra of Eu(TTA)3–168 (10 mm)in ered agood luminophore candidate, as it shows strong metal- MeCN/water (20:80, v/v) buffer(0.02 m NaClbuffersolution) with different to-ligandcharge-transfer(MLCT) red emission (l 600 nm), a pH values.The inset showsthe fluorescencechanges of Eu(TTA)3–168 with large Stokesshift (Dl 150 nm), and long luminescent life- different pH values and the fluorescence photos of Eu(TTA)3–168 at pH 6.5 times (of the order of microseconds).[242] The luminescent prop- and 8.5;the intensities were recordedatl=612 nm with excitation at II l= 340 nm. Reproducedfrom Ref. [225] with permission from The Royal So- erties of the Ru complex can be modified by introducing a ciety of Chemistry. 2,4-dinitrobenzenesulfonyl (DNBS) moiety into the structure.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 43 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim 4.4. Iridium Complexes as Electrochemiluminescent Sensors

Despite their exciting photophysical and photochemical properties,[243] only afew IrIII-based chemosensors for cation sensing have been reported.[244] Electrochemiluminescence(ECL) has emerged as apowerful alternative to photoluminescence (PL) in awiderange of fields such as clinicaldiagnostics, pharma- ceuticalanalysis, environmental assays in food, and water test- ing.[245, 246] Afew examples of ECL-based sensors for metal-ion detection have been reported,such as those reported by High and co-workers for the detection of Cu ions in water samples.[247] The ECL properties of IrIII complexes appear to be su- periortothose of RuII complexes.[248] Lin and co-workers[249] reportECL probes 182 and 183,two IrIII complexes with azacrown ethers, as probes for metal cations(Figure 31). The probesoperate by an oxidation–reduction ECL process, with tri-n-propylamine as aco-reactant and acetonitrile as the solvent. Complexes 182 and 183 behave as remarkable ECL sensors for BaII and AgI,respectively,with aninefold enhancement in the intensity of the emission signaland aredshift in the II emission lmax.Incontrast, structurally analogousRu complexes 184 and 185 do not display any significant ECL changes, and no wavelength shiftisobserved upon the addition of the same metal ions. This differentbehavior can be explained by the azacrown ether phenanthroline moiety, which is responsible for coordination to the target metal ions;for IrIII complexes 182 and 183,itrepresents the lowestunoccupied molecular orbital(LUMO) but is part of the highest occupied molecular orbital(HOMO)for RuII complexes 184* and 185*.Ifthe azacrown ether moieties are part of the LUMO, the electronic sit- uation producesabathochromically shiftedemission of the IrIII probe in the presence of the target metal ion. This work thus provides generally useful guidelines for improving the design of future ECL sensors based on metal complexes for metal-ion recognition.

Figure 29. Chemical structuresofprobes 169—178,where mpiq = 1-(4’- methylphenyl)isoquinoline), tfpiq = 1-(4’-trifluoromethylphenyl)-isoquino- 5. Nanomaterials line), ampiq = 1-(5’-amino-4’-methylphenyl)isoquinoline), atfpiq = 1-(5’- amino-4’-trifluoromethylphenyl)isoquinoline), deampiq = 1-(5’-diethylamino- 5.1. Quantum Dots 4’-methylphenyl)isoquinoline), gmpiq = 1-(5’-guanidyl-4’-methylphenyl)isoquinoline), imzmpiq = 1-(5’-iminoimidazolidinyl-4’-methylphenyl)isoquino- Quantum dots (QDs) have unique optical properties, including line), and imztfpiq =1-(5’-iminoimidazolidinyl-4’-trifluoromethylphenyl)iso- high quantum yields, symmetric fluorescenceemission spectra, quinoline). wide excitation spectra, light resistance, and tunable spectra.[250] QDs probescan be synthesized by linking QDs to peptides,[251] antibodies,[252] and organic molecules with specific Electron transfer (ET) from the RuII center(astrong electron ability to bind aparticularmetal.[253] QDscan be used as mo- donor) to the N^N coordination ligand is divertedtothe DNBS lecular beacons to monitorenzymatic reactions,[254] to track moiety,astrong intramolecular electron acceptor;this results single vesicles following their endocytic uptake,[255] andfor in quenching of the emissionofthe resulting probe,which be- membrane-diffusion studies of individual QD-tagged recep- comes “switched off”.Cleavage of the DNBS moiety by thiols tors;[256] all these applications are based on the ability of QDs re-establishes the MLCT of the RuII complex. The phosphores- to report molecular position. Addingasensing moiety to the cence probe is “switched on” with a90-fold increaseinthe in- QD adds asensing functionality (e.g. by Fçrster resonance tensity of the signal at l=598 nm, aStokes shift of Dl= energytransfer,FRET) to the localization information. These 143 nm, and aluminescent lifetime of 1.1 ms(Figure 30). This functionalized QD probesbecome very powerful tools that can probe has been successfully used to image intracellularthiols be used in arange of fields such as toxin detection, cell physi- in NCI-H446cells. ology,and pathology.[257]

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 44 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 30. Photographshowing solutions of 175 (1 mm)and 178 (5 mm)indegassed DMSO/100mm buffer(from pH 4to10) at 25 8C. Excitationatl= 365 nm (left). Luminescence microscopy images (BiorevoBZ-9000, Keyence) of HeLa-S3 cells irradiatedatl=465 nm (Twinlight 465, Relyon) for 30 min with 178 (right). deampiq = Ir(III) complexes that contain diethylamino groups on the 1-(4’-methylphenyl)isoquinoline) ligand;imztfpiq = Ir(III) complexesthat contain iminoimidazolidinylgroups on the 1-(4’-methylphenyl)isoquinoline) ligand. Reprinted (adapted) with permissionfrom Ref. [233]. Copyright (2015)American Chemical Society. The chemical structures of compounds 179–181 are also shown.

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 45 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 31. Chemical structure of compounds 182–185.

Zamaleeva and co-workers[258] have designed cell-penetrat- of QD probes to plant cells. Yu et al.[259] have synthesized ing FRET-basedCaII nanobiosensor 186 (Figure 32). The re- probe 187,the result of the conjugation of CdTe/ZnS QDs with searchersuse CANdot565QD as the donor and CaRuby, ared- 2-amino-3-indolepropionic acid, to recognize indole–propionic emitting CaII indicator derived from rhodamine, as the accept- acid (IPA)binding proteins in plant tissues (Figure 33). CdTe/ or.Toimprove cell permeability and cytoplasmic delivery,the ZnS-IPAhas the biological activity of the plant hormone IPA QDs are also functionalized with asmallcell-penetratingpep- and is able to recognize IPAbinding sites in plant tissues.The tide (CPP) derived from hadrucalcin.The ability of 186 to act fluorescenceemission wavelength of red-emitting CdTe/ZnS- as aCaII sensorhas been studied in the concentration range of IPAisl=595 nm, which thus avoidsinterferencefrom the in- 0to2mm by using the relative increase in CaRuby fluores- trinsic yellow-green fluorescence background of plant tissues. cence as ameasure of sensitivity.Incell-imaging experiments, CdTe/ZnS-IPAhas been used for the in situ imaging of IPA the nanobiosensors penetrate inside the cells and distribute binding sites, and it has been revealed that the IPAbinding throughout the cytoplasm. Interestingly,the sensorshows a sites in mung-bean root tissues are concentrated in the mem- pointillistic distribution, that is, it is possible to determine lo- brane of endodermal cells. calized CaII concentrations at discrete points. Imaging studies Promising results have so far been obtained in research con- of intracellular CaII in HEK293 cells expressing N-methyl-d-as- ducted on QD probes. Further research is necessary to develop partatereceptors have also been performed. By furtherfunc- QD probeswith even lower toxicity and better fluorescence tionalization with specific antibodies targeting high-conductivi- stability. The aim is to design probes that do not inhibit the ty CaII channels, new CaII sensors can be developed to allow growth and development of cells and that show greater bio- for optical single-channel recording. compatibility. Research is currently focusedonlabeling mammaliancells with QD probes. However,research on imaging plant cells and tissues is limited, because of concerns of the potential toxicity

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 46 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 32. FRET-based Ca2+ biosensor 186.Step 1) The QD TOP/TOPO passivating layer was replaced by apeptidecoating made by mixing cysteine (SH function) andlysine (NH2 function)terminatedpeptides[pC, Ac-CGSESGGSESG(FCC)3F-amide;and pK, NH2-KGSESGGSESG(FCC)3F-amide,respectively].Both com- ponents(hydrophobic QDs and peptides)were first dissolved in theirrespectivesolvents, pyridine and DMSO.After mixing,surfactant exchange and peptide binding were initiatedbyraising the pH. Step 2) Nanoparticles werefurther functionalized by adding CaRuby (red dots)and cell-penetratingpeptides (CPP, purple wiggles) onto peptide-coated QDs by usingaSH/maleimide linking reaction. Reprinted (adapted) with permission from Ref. [258]. Copyright (2014) American Chemical Society.

size-dependent emission wavelength, satisfactory water solubility,and satisfactory photostability.[262] One of these examples has recently been reported by Ke and co-workers.[263] The re- searchersdescribe the synthesis and characterization of dual- emission probe 188 forthe fluorescent ratiometric sensing of

H2O2 concentration and pH change(Figure 34). Probe 188 contains apH-sensitive dye, fluorescein-5-isothio- cyanate (FITC),the emission intensity of which diminisheswith increasing concentration. FITC is conjugated to the amino groups of BSA protein. This FITC/BSA conjugate is used as a template to synthesize red-emitting gold nanoclusters under alkalineconditions, and thus, probe 188 (FITC/BSA-stabilized gold nanoclusters) is formed. Using l=488 nm as the excitation wavelength, the fluorescence spectrumof188 shows two bandsatl= 525 and 670 nm. The band at l=525 nm is sensi- Figure 33. Structure diagram of CdTe/ZnS-IPA(187). tive to pH changes (0.1 pH-unit change, pH 5.0–8.5), andthe

band at l=670 nm is sensitivetochanges in H2O2 concentra- 5.2. FluorescentMetallic Nanoclusters tion. Thus, this dual-emission probe is able to detect changes

in pH and H2O2 concentration separately. Amongthe different fluorescentmetallic nanoclusters (FNCs), fluorescent gold clusters (FGCs)are gradually emerging as 5.3. Semiconductor Nanocrystals promisingimaging probes, because of their tunable emission in the visible-to-NIRrange.[260,261] The FGC probescurrently Semiconductor nanocrystals (NCs) possessunique photolumi- availablehave severallimitations, including low fluorescence nescent properties that make them excellent candidates for quantum yields, unstable fluorescence, lowsynthesis yields, the design of fluorescenceprobesfor chemo/biosensing appli- and poor functionalization options. Research on improving cations.NCs offer distinct advantages over organic dyes such FGC probescontinues, and these probescould potentially be as high photoluminescence efficiency,broad absorption, used for sensing enzyme-related reactions because of their narrowand symmetric emission,and good photostability.[264]

ChemistryOpen 2018, 7,9–52 www.chemistryopen.org 47 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim with alimit of detection (LOD) of 1.8 mm.Significantly,the re- searchershave also successfully prepared paper-basedtest strips with 189 that allow the rapid and visual detection of DEP residues.Probes based on ratiometricmeasurements present more advantages than NC-based sensors, whichuse either turn-off or turn-on fluorescenceintensityasthe sole respon- sive signal. Ratiometric measurements can eliminate perturba- tions by experimentalfactors, such as fluctuation in probe concentration and instrumental efficiency,and can provide more precise measurements because of their self-referencingcapa- bility,which derives from the use of the intensity ratio of the two emissions as the measurement tool.

6. Final Remarks and Perspectives The aim of this review was to provide an in-depthoverview of key developments in the synthesis of green andred fluores- Figure 34. a) Step-by-step illustration of the procedureused to prepare cent probesover the last few years. The importance of taking FITC/BSA gold nanoclusters (188). b) MALDI-TOF massspectraofa)FITC-BSA arational approachtothe designofprobesthat can selectively and b) BSA. c) Native-PAGE image of BSA (lane 1), FITC-BSA(lane2), BSA detectand/or visualize arange of analytes hasbeen empha- gold nanoclusters (lane3), and FITC/BSA gold nanoclusters (lane4)after sized. stainingwith Coomassie BrilliantBlue. Native-PAGE image of BSA gold nanoclusters (lane 5) and FITC/BSA gold nanoclusters (lane 6) under l =488 nm As amatter of fact, green and red fluorescent dyes are the excitation(laser-based gel scanners) without Coomassie brilliant blue. Repro- most-common fluorophores to sense severalanalytes in bio- duced with permission from Ref. [263].Copyright2015Elsevier B. V. logical media, because of their excellent optical properties, such as long excitation wavelengths,high excitation coeffi- Zhang et al.[265] have developed probe 189 for determination cients, and high fluorescent quantum yields. Such properties of the organophosphate compound diethylphosphorothioate make these probesvery appealing for use in cells and tissues (DEP). Probe 189 is an intrinsic dual-emitting Mn-doped ZnS being irradiated with light without cell damage. However,the nanocrystal-based probe. In the presence of DEP,the electron- use of these chromophores, besides their low cost in some transfer pathway is switched off and red emission of the probe cases and low toxicity, has somelimitations, such as high rate is enhanced, whereas the blue emission is almost unchanged. of photobleaching, pH-sensitive fluorescence, tendency to self- By varying the concentration of DEP,the intensity ratio of the quench, and broad fluorescenceemission spectra,all of which two emissions gradually varies and displays color changes limit the efficiency in multicolor applications.Onthe other from dark-blue to purple to red (Figure 35). Thus, this probe hand, the probability of self-quenching increases in red-fluores- can be used for the quantitative and visual detection of DEP cent probes due to the low solubility and high degree of sub- stitutionofthese compounds, and this leads to adecrease in the fluorescencequantum yield. To overcome such issues, several authors have developedand designed fluorophores con- tainingseveral water-soluble groups, such as amino acids, phosphorus-containing groups,sulfur-containing groups, and vitamin units. The aim is to design new selective, sensitive, and biocompatible fluorescent probeswith increasingly more complex and sophisticated structures that would allow additional fine- tuningoftheir properties and expand the range of translational applications.Indeed,the excellentoptical properties of green and red chromophores make them very valuable for working with biological tissues and cells. The amazingwork conducted by Lippard et al. shows how it is possible to design biocompatible fluorescent “turn-on”selective probes for the detection of metal ions and anions for cellular applications, Figure 35. Illustration for the synthesis of the dual-emitting probe 189 and and much work has been done in this direction. Modifications the mechanism for fluorescence turn-on and ratiometric detection of dieth- to the chromophoreskeleton by the introduction of hetero ylphosphorothioate (DEP). (The bottom panel shows the fluorescent spectral groups such as carboxylic acids, sulfonicacids, and carbonyl, changes of the dual-emitting probe upon exposure to DEP and the corre- amino,nitro, and hydroxy groups can strongly contribute to sponding fluorescence photographs of the probe solution taken under UV il- lumination). Reprinted with permissionfrom Ref. [265]. Copyright (2014) enhancing the intensity of color and to improving solubility. American Chemical Society. Modification of the receptor unit, by changing the nature and

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