Fluorescein Derivatives As Fluorescent Probes for Ph Monitoring Along Recent Biological Applications

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International Journal of Molecular Sciences

Review Fluorescein Derivatives as Fluorescent Probes for pH Monitoring along Recent Biological Applications

Florent Le Guern 1,* , Vanessa Mussard 1, Anne Gaucher 1 , Martin Rottman 2,3 and Damien Prim 1

1 Institut Lavoisier de Versailles, CNRS, UVSQ, Université Paris-Saclay, 78035 Versailles, France; [email protected] (V.M.); [email protected] (A.G.); [email protected] (D.P.) 2 Faculté de Médecine Simone Veil, Université de Versailles St Quentin, INSERM UMR U1173, 2 Avenue de la Source de la Bièvre, 78180 Montigny le Bretonneux, France; [email protected] 3 Hôpital Raymond Poincaré, AP-HP, GHU Paris Saclay, 104 Bd Poincaré, 92380 Garches, France * Correspondence: ﬂ[email protected]

Received: 3 November 2020; Accepted: 1 December 2020; Published: 3 December 2020

Abstract: Potential of hydrogen (pH) is one of the most relevant parameters characterizing aqueous solutions. In biology, pH is intrinsically linked to cellular life since all metabolic pathways are implicated into ionic flows. In that way, determination of local pH offers a unique and major opportunity to increase our understanding of biological systems. Whereas the most common technique to obtain these data in analytical chemistry is to directly measure potential between two electrodes, in biological systems, this information has to be recovered in-situ without any physical interaction. Based on their non-invasive optical properties, fluorescent pH-sensitive probe are pertinent tools to develop. One of the most notorious pH-sensitive probes is fluorescein. In addition to excellent photophysical properties, this fluorophore presents a pH-sensitivity around neutral and physiologic domains. This review intends to shed new light on the recent use of fluorescein as pH-sensitive probes for biological applications, including targeted probes for specific imaging, flexible monitoring of bacterial growth, and biomedical applications.

Keywords: ﬂuorescein; pH-sensitive; probe; dyad; imaging; organelle; cell; bacteria; application

1. Introduction In chemistry, potential of hydrogen, or pH, is a data describing the acidity or the basicity of a medium [1]. Logarithmically obtained from H+ ion concentration, pH is one of the main physical characteristics used to describe an aqueous solution. Since the development of pH-meters, its measurement became inevitable across scientific fields, such as drinking water [2], industrial waste [3,4], global health [5], and agronomy [6]. As acidic or basic compounds are continuously released as outputs of cellular life, pH monitoring offered a unique opportunity to easily acquired data from biologic systems [7]. The relevance of pH in biological systems can be observed at different scales: the pH of biological fluids is well described and its regulation essential to the proper function of organs, since abnormal values are both the sign and cause of disease developments. pH regulation within biological systems relies on a sensitive equilibrium, called pH homeostasis. At the organism level, pH regulation is performed by the lungs through the elimination of CO2 and the kidney through the filtration of the HCO3− ion. In cells, where organic acids, such as lactic, pyruvic, or beta-hydroxybutyrate acids are produced along metabolic pathways, some membrane proteins compensate the decrease of pH by transporting protons outside the cytosol [8]. Thus, cellular pH monitoring leads to the understanding of key milestones within cells, such as proliferation [9], ion transport [10], or carcinogenesis [11]. For example, unchecked

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Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 23 biological processes occurring in malignant cells release a consequent amount of acid derivatives, leadingtissues [12]. to a pHIn microbiology, decrease in tumoral proliferations tissues [12 of]. aero In microbiology,bic bacteria also proliferations lead to a massive of aerobic production bacteria also of leadacidic to metabolites, a massive production which quickly of acidic induce metabolites, pH variations which quickly in medium induce pH[13]. variations Whereas in global medium pH [13 in]. Whereascytosol has global to be pHregulated, in cytosol each has organelle to be regulated, is fully effective each organelle in specific is fullyionic eenvironments,ffective in specific which ionic are environments,often correlated which to local are functions often correlated [14]. For to example, local functions the average [14]. pH For in example, lysosomes, the Golgi average network, pH in lysosomes,and mitochondria Golgi network, are 4.7, 6.7, and and mitochondria 8 respectively. are 4.7,Thus, 6.7, pH and monitoring 8 respectively. of each Thus, organelle pH monitoring has helped of eachto determine organelle their has helpedfunction. to At determine an atomic their scale, function. all metabolism At an atomic pathways scale, allare metabolism directly correlated pathways to arepH directlysince enzymatic correlated activities to pH since and enzymatic kinetics activitiesdepend andon ionic kinetics environments. depend on ionicAnother environments. common Anotherexample commonis the classification example is of the essential classification amino of acids, essential according amino their acids, acidic according or basic their trends. acidic Thus, or basic pH trends.is an important Thus, pH physicochemical is an important datum, physicochemical which can attest datum, of biological which can activities attest of at biological different activitiesscales. In atorder diff erentto monitor scales. these In order fluctuations to monitor without these fluctuationsany physical without contact, anyfluorescent physical pH-sensitive contact, fluorescent probes pH-sensitivewere developed probes during were the developed last century. during Under the last specific century. light, Under these specific molecular light, theseprobes molecular re-emit probesphotons re-emit at another photons wavelength at another for wavelength which related for whichintensity related depends intensity on the depends surrounding on the pH surrounding [7]. Since pHoptical [7]. devices Since opticalare continuously devices are becoming continuously more becomingprecise, especially more precise, with the especially broad dissemination with the broad of disseminationoptic fibers, pH-sensitive of optic fibers, molecular pH-sensitive probes molecular are recurrent probes across are biological recurrent acrossstudies. biological studies. One of the most used pH probes is fluorescein fluorescein 1; its chemical structure is composed of a tricyclic xanthene flankedflanked by two hydroxyl groups and a bicyclic fused lactone fragment linked by a spiro carbon atom (Figure1 1).).

Figure 1. Chemical structure of fluorescein ﬂuorescein 1.

This familyfamily ofof molecules molecules was was discovered discovered in in 1871 1871 by by Adolf Adolf von von Baeyer, Baeyer, and and has becomehas become one of one most of ubiquitousmost ubiquitous probes probes in biological in biological studies, studies, because be ofcause its intense of its fluorescence, intense fluorescence, reversible pHreversible sensitivity, pH chemicalsensitivity, stability, chemical and lackstability, of cytotoxicity and lack at of working cytotoxicity concentrations. at working For years,concentrations. fluorescein For1 has years, been usedfluorescein as a starting 1 has been material used to as create a starting novel material fluorescent to crea probeste novel revealing fluorescent specific probes biological revealing activities specific such asbiological enzymatic activities cleavage such [15 as]. enzymatic Fluorescein cleavage1 is still [15]. the focusFluorescein of interest 1 is fromstill the the focus scientific of interest community from forthe itsscientific intense community fluorescence for and its its intense sensitivity fluorescence to pH variations and its aroundsensitivity neutral to pH domain variations [16]. Asaround most biologicalneutral domain systems [16]. are As fully most effective biological at physiological systems are conditions fully effective (pH~7.3), at physiological fluorescein 1conditionsbecame a benchmark(pH~7.3), fluorescein for monitoring 1 became pH fluctuationsa benchmark in for cell monitoring cultures. Indeed, pH fluctuations every year, in acell substantial cultures. amountIndeed, ofevery new year, articles a substantial describes biologicalamount of studies new articles using describes fluorescein biological1 as pH-sensitive studies using probe fluorescein (Figure2)[ 117 as]. InpH-sensitive this review, probe we propose (Figure an 2) overview[17]. In this of fluoresceinreview, we1 proposeand its mainan overview derivatives of fluorescein complemented 1 and by its a surveymain derivatives of recent studies complemented using fluorescein by a surv1eyas of pH recent sensors studies for biological using fluorescein applications. 1 as pH sensors for biological applications.

Figure 2. Number of articles with keywords “fluorescein, pH, probe, biology” during last fifty years [17].

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 23 tissues [12]. In microbiology, proliferations of aerobic bacteria also lead to a massive production of acidic metabolites, which quickly induce pH variations in medium [13]. Whereas global pH in cytosol has to be regulated, each organelle is fully effective in specific ionic environments, which are often correlated to local functions [14]. For example, the average pH in lysosomes, Golgi network, and mitochondria are 4.7, 6.7, and 8 respectively. Thus, pH monitoring of each organelle has helped to determine their function. At an atomic scale, all metabolism pathways are directly correlated to pH since enzymatic activities and kinetics depend on ionic environments. Another common example is the classification of essential amino acids, according their acidic or basic trends. Thus, pH is an important physicochemical datum, which can attest of biological activities at different scales. In order to monitor these fluctuations without any physical contact, fluorescent pH-sensitive probes were developed during the last century. Under specific light, these molecular probes re-emit photons at another wavelength for which related intensity depends on the surrounding pH [7]. Since optical devices are continuously becoming more precise, especially with the broad dissemination of optic fibers, pH-sensitive molecular probes are recurrent across biological studies. One of the most used pH probes is fluorescein 1; its chemical structure is composed of a tricyclic xanthene flanked by two hydroxyl groups and a bicyclic fused lactone fragment linked by a spiro carbon atom (Figure 1).

Figure 1. Chemical structure of fluorescein 1.

This family of molecules was discovered in 1871 by Adolf von Baeyer, and has become one of most ubiquitous probes in biological studies, because of its intense fluorescence, reversible pH sensitivity, chemical stability, and lack of cytotoxicity at working concentrations. For years, fluorescein 1 has been used as a starting material to create novel fluorescent probes revealing specific biological activities such as enzymatic cleavage [15]. Fluorescein 1 is still the focus of interest from the scientific community for its intense fluorescence and its sensitivity to pH variations around neutral domain [16]. As most biological systems are fully effective at physiological conditions (pH~7.3), fluorescein 1 became a benchmark for monitoring pH fluctuations in cell cultures. Indeed, every year, a substantial amount of new articles describes biological studies using fluorescein 1 as pH-sensitive probe (Figure 2) [17]. In this review, we propose an overview of fluorescein 1 and its mainInt. J. Mol.derivatives Sci. 2020, 21 complemented, 9217 by a survey of recent studies using fluorescein 1 as pH sensors3 offor 23 biological applications.

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 23 FigureFigure 2. Number of articlesarticles withwith keywords keywords “ﬂuorescein, “fluorescein, pH, pH, probe, probe, biology” biology” during during last last ﬁfty fifty years years [17]. 2. Fluorescein and Derivatives as Notorious pH Sensors [17]. 2. Fluorescein and Derivatives as Notorious pH Sensors

2.1. Fluorescein: Synthesis and Properties 2.1. Fluorescein: Synthesis and Properties Originally obtained by condensing phtalic anhydride with phenol in acidic conditions by Von Baeyer,Originally this preparation obtained of by fluorescein condensing 1 phtalicis nowadays anhydride based with on phenolFriedel–Crafts in acidic reactions conditions (Figure by Von 3). Baeyer,Thus, the this fluorophore preparation can of be fluorescein easily obtained1 is nowadays by mixing based phtalic on anhydride, Friedel–Crafts resorcinol reactions and (Figure ZnCl23 or). Thus,methanesulfonic the fluorophore acid can[18,19]. be easily Large obtained scale manufactured, by mixing phtalic fluorescein anhydride, 1 has resorcinol been included and ZnCl in 2theor methanesulfonicWorld Health Organization acid [18,19]. Large(WHO)’s scale model manufactured, list of essential fluorescein medicines1 has been [20] included for its in thebiological World Healthapplications, Organization but it is also (WHO)’s used in model other listfields, of essentialsuch as petrochemistry medicines [20] for for leak its biologicaldetection and applications, cosmetic butformulations. it is also used in other fields, such as petrochemistry for leak detection and cosmetic formulations.

Figure 3. General synthetic pathway to fluorescein. fluorescein. Under basic conditions (pH > 8), fluorescein 1 absorbs blue light with a maxima absorption peak Under basic conditions (pH > 8), fluorescein 1 absorbs blue light with a maxima absorption around 490 nm, and emits a green light around 515 nm [21]. Due to phenolic fragments and equilibrium peak around 490 nm, and emits a green light around 515 nm [21]. Due to phenolic fragments and between a carboxylic function and a lactone, ionic charge and chemical structure of fluorescein evolve equilibrium between a carboxylic function and a lactone, ionic charge and chemical structure of depending on the surrounding pH, leading to fluctuations of photophysical properties (Figure4)[ 22]. fluorescein evolve depending on the surrounding pH, leading to fluctuations of photophysical Whereas the fluorescence quantum yield under an excitation at 490 nm is very high under basic properties (Figure 4) [22]. Whereas the fluorescence quantum yield under an excitation at 490 nm is conditions (ΦF: 0.95 in NaOH 0.1 M), an acidification of the solution progressively leads to the very high under basic conditions (ΦF: 0.95 in NaOH 0.1 M), an acidification of the solution fluorescence extinction [23]. This fluctuation is due to the transition of the di-anionic form of fluorescein progressively leads to the fluorescence extinction [23]. This fluctuation is due to the transition of the into an anionic equilibrium, which has a lower absorbance associated to a blue-shifting [23]. di-anionic form of fluorescein into an anionic equilibrium, which has a lower absorbance associated The most problematic drawback of fluorescein is its photobleaching when exposed to light [24]. to a blue-shifting [23]. Indeed, the fluorescent emission from this probe progressively decreases under intense light irradiations, which is an issue since pH monitoring is based on this intensity, and repeated measurements cause the signal to fade. Because of this photobleaching, all fluorescein derivatives must be stored in the dark, and experiments using this probe have to occur quickly. Spectral bands of fluorescein can also induce issues with some optical systems. Depending on the purpose of the application, its relatively broad fluorescent emission can be considered as an advantage (for example, in case of the Förster resonance energy transfer (FRET) system), but also as a drawback for multi fluorescent probing experiences. Fluorescein presents also a weak Stokes shift, the interval between highest absorption and emission wavelength, which is a potential issue for devices using a single optical path, such as entry-level plate readers. Like most intense fluorescent probes, self-quenching can occur in case of aggregation (ACQ)

Figure 4. Ionic forms of fluorescein 1 according the pH domains and their relative fluorescence intensities. At neutral pH and under excitation at 490 nm, the most fluorescent di-anionic form of fluorescein takes prominence over other forms. Below pH = pKa~6.4, mono-anionic fluorescein displays a blue-shifted absorption followed by drastic decrease of fluorescence. At even lower pH, neutral and further cationic forms of fluorescein becomes non-fluorescent under irradiation at 490 nm.

The most problematic drawback of fluorescein is its photobleaching when exposed to light [24]. Indeed, the fluorescent emission from this probe progressively decreases under intense light

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2. Fluorescein and Derivatives as Notorious pH Sensors

2.1. Fluorescein: Synthesis and Properties Originally obtained by condensing phtalic anhydride with phenol in acidic conditions by Von Baeyer, this preparation of fluorescein 1 is nowadays based on Friedel–Crafts reactions (Figure 3). Thus, the fluorophore can be easily obtained by mixing phtalic anhydride, resorcinol and ZnCl2 or methanesulfonic acid [18,19]. Large scale manufactured, fluorescein 1 has been included in the World Health Organization (WHO)’s model list of essential medicines [20] for its biological applications, but it is also used in other fields, such as petrochemistry for leak detection and cosmetic formulations.

Figure 3. General synthetic pathway to fluorescein.

Under basic conditions (pH > 8), fluorescein 1 absorbs blue light with a maxima absorption peak around 490 nm, and emits a green light around 515 nm [21]. Due to phenolic fragments and equilibrium between a carboxylic function and a lactone, ionic charge and chemical structure of fluorescein evolve depending on the surrounding pH, leading to fluctuations of photophysical properties (Figure 4) [22]. Whereas the fluorescence quantum yield under an excitation at 490 nm is Int. J. Mol. Sci. 2020, 21, 9217 4 of 23 very high under basic conditions (ΦF: 0.95 in NaOH 0.1 M), an acidification of the solution progressively leads to the fluorescence extinction [23]. This fluctuation is due to the transition of the ordi-anionic high degrees form ofof surface fluorescein substitution into an [anionic25]. Hopefully, equilibrium, due to which low the has concentration a lower absorbance used in associated biological systems,to a blue-shifting this drawback [23]. has little relevance.

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 23 irradiations, which is an issue since pH monitoring is based on this intensity, and repeated measurements cause the signal to fade. Because of this photobleaching, all fluorescein derivatives must be stored in the dark, and experiments using this probe have to occur quickly. Spectral bands of fluorescein can also induce issues with some optical systems. Depending on the purpose of the application, its relatively broad fluorescent emission can be considered as an advantage (for example, in case of the Förster resonance energy transfer (FRET) system), but also as a drawback for multi fluorescent probing experiences. Fluorescein presents also a weak Stokes shift, the interval between highest absorption and emission wavelength, which is a potential issue for devices using a Figure 4. Ionic forms of fluorescein 1 according the pH domains and their relative fluorescence singleFigure optical 4. Ionicpath, forms such of asfluorescein entry-level 1 according plate readers.the pH domainsLike most and theirintense relative fluorescent fluorescence probes, intensities.intensities. At At neutral pH and under excitation at 490490 nm,nm, thethe mostmost fluorescentfluorescent di-anionicdi-anionic formform ofof self-quenching can occur in case of aggregation (ACQ) or high degrees of surface substitution [25]. fluoresceinfluorescein takes takes prominence prominence over over other other forms. forms. Below Below pH = pKa~6.4,pH = pKa~6.4, mono-anionic mono-anionic fluorescein fluorescein displays Hopefully, due to low the concentration used in biological systems, this drawback has little adisplays blue-shifted a blue-shifted absorption absorption followed byfollowed drastic decreaseby drastic of decrease fluorescence. of fluorescence. At even lower At pH,even neutral lower andpH, relevance. furtherneutral cationicand further forms cationic of fluorescein forms of becomes fluorescein non-fluorescent becomes non-fluorescent under irradiation under at 490 irradiation nm. at 490 nm. 2.2. Most Used Fluorescein DerivativesDerivatives and Their Properties SeveralThe most fluoresceinfluorescein problematic derivatives drawback haveof fluorescein been developed is its photobleaching in order to when fitfit withwith exposed thethe downstreamdownstream to light [24]. applications.Indeed, the fluorescentThe The best-known best-known emission derivatives derivatives from this are are prbased basedobe progressivelyon the modificationmodification decreases of benzo-fusedbenzo-fusedunder intense lactonelactone light moiety in order to add reactive chemical functions such as isothiocyanate, carboxylic acid, or amine. TheThe outcomingoutcoming probesprobes areare namednamed fluoresceinfluorescein isothiocyanateisothiocyanate (FITC)(FITC) (Figure(Figure5 5-- 22),), carboxyfluoresceincarboxyfluorescein (5(6)-FAM(5(6)-FAM oror CF)CF) (Figure(Figure5 -5-33),), andand fluoresceinaminefluoresceinamine (FA) (FA) (Figure (Figure5 -5-44),), respectively. respectively.

S C N O O COOH O NH2

O O O

HO O OH HO O OH HO O OH

2 - FITC 4 - FA 3 - FAM Figure 5. Figure 5. Most used reactive derivatives of fluorescein.fluorescein. FITC 2 is one of most used derivatives due to its properties and good reactivity for conjugation. FITC 2 is one of most used derivatives due to its properties and good reactivity for conjugation. In comparison to fluorescein, FITC 2 presents a slight decrease of fluorescence quantum yield In comparison to fluorescein, FITC 2 presents a slight decrease of fluorescence quantum yield (ΦF (Φ (excitation: 500 nm): 0.75 in 10 mM Phosphate-Buffered Saline) [26]. However, in comparison (excitation:F 500 nm): 0.75 in 10 mM Phosphate-Buffered Saline) [26]. However, in comparison with with other fluorophores, FITC 2 still has an intense fluorescence and can be used as a pH-sensitive other fluorophores, FITC 2 still has an intense fluorescence and can be used as a pH-sensitive probe probe around the neutral domain. FITC 2 can be easily associated to any kind of chemical structures around the neutral domain. FITC 2 can be easily associated to any kind of chemical structures bearing an amine fragment, such as fluorophores [27], targeting agents [28], proteins [29], polymers [30], bearing an amine fragment, such as fluorophores [27], targeting agents [28], proteins [29], polymers or nanoparticles [31]. Resulting bioconjugation linkage is based on a thiourea function for which [30], or nanoparticles [31]. Resulting bioconjugation linkage is based on a thiourea function for stability depends on physiochemical surroundings due to its strong H-bond interactions [32–34]. which stability depends on physiochemical surroundings due to its strong H-bond interactions [32– Using such derivatives offers a unique opportunity to create specific pH sensors, and particularly, 34]. Using such derivatives offers a unique opportunity to create specific pH sensors, and for biological systems where all proteins are bearing a terminal amine. Even if FITC 2 is still the most particularly, for biological systems where all proteins are bearing a terminal amine. Even if FITC 2 is still the most prolific derivative in literature (Figure 6), new fluorescein reactive derivatives are still described with optimized properties [35].

84% 2% 14% FAM FITC BCECF and others

Figure 6. Average repartition of used fluorescein derivatives in literature the last decade [17].

2.2. Most Used Fluorescein Derivatives and Their Properties Several fluorescein derivatives have been developed in order to fit with the downstream applications. The best-known derivatives are based on the modification of benzo-fused lactone moiety in order to add reactive chemical functions such as isothiocyanate, carboxylic acid, or amine. The outcoming probes are named fluorescein isothiocyanate (FITC) (Figure 5-2), carboxyfluorescein (5(6)-FAM or CF) (Figure 5-3), and fluoresceinamine (FA) (Figure 5-4), respectively.

S C N O O COOH O NH2

O O O

HO O OH HO O OH HO O OH

2 - FITC 4 - FA 3 - FAM Figure 5. Most used reactive derivatives of fluorescein.

FITC 2 is one of most used derivatives due to its properties and good reactivity for conjugation. In comparison to fluorescein, FITC 2 presents a slight decrease of fluorescence quantum yield (ΦF (excitation: 500 nm): 0.75 in 10 mM Phosphate-Buffered Saline) [26]. However, in comparison with other fluorophores, FITC 2 still has an intense fluorescence and can be used as a pH-sensitive probe around the neutral domain. FITC 2 can be easily associated to any kind of chemical structures bearing an amine fragment, such as fluorophores [27], targeting agents [28], proteins [29], polymers [30], or nanoparticles [31]. Resulting bioconjugation linkage is based on a thiourea function for Int.which J. Mol. stability Sci. 2020 ,depends21, 9217 on physiochemical surroundings due to its strong H-bond interactions5 [32– of 23 34]. Using such derivatives offers a unique opportunity to create specific pH sensors, and particularly, for biological systems where all proteins are bearing a terminal amine. Even if FITC 2 is proliﬁcstill the derivativemost prolific in literature derivative (Figure in literature6), new (Figure ﬂuorescein 6), new reactive fluorescein derivatives reactive are stillderivatives described are with still optimizeddescribed with properties optimi [35zed]. properties [35].

84% 2% 14% FAM FITC BCECF and others Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 23 Figure 6. Average repartition of used fluoresceinfluorescein derivativesderivatives inin literatureliterature thethe lastlast decadedecade [[17].17]. When fluorescein was used for intracellular pH (pHin) monitoring, a high leakage rate appeared fromWhen the cells, fluorescein which made was usedaccurate for intracellular pH determination pH (pH difficult.in) monitoring, Fluorescein a high 1 leakagewas then rate replaced appeared by carboxyfluoresceinfrom the cells, which 3 (FAM made or accurate CF) as pH the determination main pHin probe, diffi cult.since Fluoresceinits main advantage1 was then was replaced its low leakingby carboxyfluorescein rate through cell3 (FAM membranes. or CF) asIndeed, the main due pHto itsin probe,additional since carboxylic its main advantageacid function, was the its supplementarylow leaking rate anionic through charge cell membranes. significantly Indeed, reduces due solubility to its additional of FAM carboxylic 3 in lipid acid structures function, composingthe supplementary cell membranes anionic charge [36] significantlyMoreover, FAM reduces 3 has solubility similar of photophysica FAM 3 in lipidl structuresproperties composing to FITC 2 [26].cell membranesThus, FAM [336 has] Moreover, become one FAM of3 thehas most similar used photophysical probes for characterization properties to FITC of tumoral2 [26]. tissue Thus, [37].FAM FAM3 has 3 becomecan alsoone be used of the for most preparation used probes of biocon for characterizationjugates using carbodiimide/NHS of tumoral tissue activation. [37]. FAM In3 thiscan alsocase, be resulting used for amide preparation function of is bioconjugates stable due to usingits omnipresence carbodiimide in/ NHSbiological activation. systems. In Diacetate this case, derivativesresulting amide of fluorescein function is stableand dueFAM to 3 its, omnipresencerespectively named in biological fluorescein systems. di-acetate Diacetate (FDA) derivatives and carboxyfluoresceinof fluorescein and FAMdi-ace3,tate respectively (CFDA) named(Figure fluorescein7-5), were di-acetatealso been (FDA) used andacross carboxyfluorescein several studies [38,39].di-acetate Di-ester (CFDA) analogs (Figure present7- 5), were a quenching also been of used the acrossfluorescence, several but, studies under [38 ,esterase39]. Di-ester intracellular analogs presentactivities, a quenchingemissions are of the restored fluorescence, [40]. Thus, but, underthese derivatives esterase intracellular are used to activities, estimate emissions intracellular are enzymaticrestored [40 activities]. Thus, [41]. these Moreover, derivatives in comparison are used to of estimate FAM 3, intracellular which is used enzymatic for its weak activities membrane [41]. permeability,Moreover, in comparisondiacetate derivatives of FAM 3 ,ar whiche cell ispermeable used for itsprobes. weak Thus, membrane CFDA permeability, 5 is one of best diacetate pHin sensorsderivatives because are cellFAM permeable 3 is directly probes. released Thus, underCFDA intracellular5 is one of enzymatic best pHin activitiessensors becauseand is not FAM able3 tois leakdirectly outside released cells. under intracellular enzymatic activities and is not able to leak outside cells.

Figure 7. DifferentDiﬀerent fluorescein ﬂuorescein derivatives used as pH-sensitive probes.

While FITC 2 and FAM 3 are the most prominent fluorescein derivatives in the literature, While FITC 2 and FAM 3 are the most prominent fluorescein derivatives in the literature, other other probes were developed for specific and optimized applications at physiological pH. Introduced by probes were developed for specific and optimized applications at physiological pH. Introduced by Roger Tsien in 1982, 2 ,7 -bis-(2-carboxyethyl)-carboxyfluorescein (BCECF) (Figure7- 6) was developed Roger Tsien in 1982,0 0 2′,7′-bis-(2-carboxyethyl)-carboxyfluorescein (BCECF) (Figure 7-6) was in order to fit more precisely with pH variation around neutral domain [42]. Indeed, due to its pKa developed in order to fit more precisely with pH variation around neutral domain [42]. Indeed, due near 7, weak acidification of medium directly induces a significant reduction of fluorescence intensity. to its pKa near 7, weak acidification of medium directly induces a significant reduction of Moreover, the absorption spectrum of BCECF 6 possesses an isosbestic point, where its absorbance fluorescence intensity. Moreover, the absorption spectrum of BCECF 6 possesses an isosbestic point, is pH-independent. This property allows the establishment of ratiometric analyses for which a ratio where its absorbance is pH-independent. This property allows the establishment of ratiometric of fluorescence is calculated from excitations at two different wavelengths (generally at maxima and analyses for which a ratio of fluorescence is calculated from excitations at two different wavelengths isosbestic point). These protocols greatly overcome common hurdles that are dye loading, leakage, (generally at maxima and isosbestic point). These protocols greatly overcome common hurdles that optical imprecisions, or photobleaching [43]. are dye loading, leakage, optical imprecisions, or photobleaching [43]. In order to fit with acidic organelles, present in yeast [44] for example, fluorinated derivatives of In order to fit with acidic organelles, present in yeast [44] for example, fluorinated derivatives of fluorescein, such as Oregon Green 7, have been prepared for acidic pH monitoring (Figure7- 7)[45]. fluorescein, such as Oregon Green 7, have been prepared for acidic pH monitoring (Figure 7-7) [45]. Introduction of electron-withdrawing atoms within the xanthene structure leads to a decrease of pKa Introduction of electron-withdrawing atoms within the xanthene structure leads to a decrease of around 4.7. Then, pH-sensitivity of such compounds is suitable with cellular components operating at pKa around 4.7. Then, pH-sensitivity of such compounds is suitable with cellular components pH around 5. operating at pH around 5.

3. Recent Studies Using Fluorescein as pH Sensors for Biological Applications

3.1. Cellular pH Imaging

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3. Recent Studies Using Fluorescein as pH Sensors for Biological Applications

3.1. Cellular pH Imaging

3.1.1. Molecular pH Sensor Most of recent developed pH-sensitive probes are based on ratiometric determination. This strategy spread across biological studies because previous developments using simple fluorescence monitoring wereInt. J. Mol. subject Sci. 2020 to several, 21, x FOR interferences PEER REVIEW such as the quality of the optical devices used, the biological6 of and 23 physical environments, and compounds stability (such as photobleaching or chemical degradation). Tohave overcome been successfully these issues andadopted. increase The probe most precision, convenie ratiometricnt way to measurements use this method have beenis to successfullyassociate a adopted.pH-dependent The most probe convenient to one (dyad) way toor usemore this fluorophores method is to emitting associate at a different pH-dependent wavelengths probe tounder one (dyad)the same or excitation. more fluorophores The ratio emitting of fluorescence at different is th wavelengthsen calculated under in which the samepH-independent excitation. The probes ratio are of fluorescenceused as internal is then control. calculated in which pH-independent probes are used as internal control. Novel chemicalchemical associations associations based based on fluoresceinon fluorescein1 were 1 recentlywere recently elaborated elaborated as dyads. as For dyads. example, For Zhangexample, et Zhang al. proposed et al. proposed a ratiometric a ratiometri probec basedprobe onbased the on chemical the chemical association association8 of an8 of iridium an iridium (III) complex(III) complex with with FITC FITC2 (Figure 2 (Figure8) for 8) pHfor pHin monitoringin monitoring [27 [27].]. Under Under a a single single wavelengthwavelength excitationexcitation at 488 nm, ratiometric determination of pHin isis possible possible from from 5.2 to 7.8, using nearnear infraredinfrared emissionemission from iridium (III)(III) complex.complex.

Figure 8. Iridium (III) complex-ﬂuoresceincomplex-fluorescein dyaddyad 8 as intracellular pHpH (pH(pHinin)) probe probe by Zhang [27]. [27].

In additionaddition toto thethe creationcreation of of ratiometric ratiometric probes, probes, the the dyads dyads strategy strategy can can also also be be an an opportunity opportunity to enlargeto enlarge operating operating windows windows of pH-sensitivity of pH-sensitivity of fluorescein. of fluorescein. Since Since large large pH fluctuations pH fluctuations mayoccur may inoccur cells, in duringcells, during mitophagy mitophagy for example, for example, more accurate more accurate and wider and pH-sensitive wider pH-sensitive monitoring monitoring offers a betteroffers understandinga better understanding of cellular of life. cellular However, life. Howe the rangever, the of the range pH eofffi thecient pH interval efficient for interval fluorescein for 1fluoresceinis limited 1 to is 6 limited to 8. Therefore, to 6 to 8. Lee Therefore, et al. put Lee forward et al. put a novel forward dyad a novel based dyad on chemical based on association chemical withassociation rhodamine with Brhodamine (9) (Figure B9 )[(9) 46(Figure]. Inspired 9) [46]. by Inspired previous by works previous using works both using compounds both compounds grafted on nanodotsgrafted on [47 nanodots], rhodamine [47], was rhodamine covalently wa linkeds covalently to fluorescein linked1 into orderfluorescein to create 1 in a pH-sensitive order to create probe a epH-sensitivefficient from probe 4 to 8. efficient The widening from 4 of to the 8. pHThe e ffiwidecientning interval of the is basedpH efficient on rhodamine interval fluorescence,is based on whichrhodamine decreases fluorescence, with alkalization. which decreases Moreover, with both al fluorophoreskalization. Moreover, have similar both absorbance fluorophores properties, have osimilarffering absorbance an opportunity properties, to use offering ratiometric an opportunit calculationy byto use exciting ratiometric both molecules calculation at by 488 exciting nm. Finally, both themolecules dyad was at 488 shown nm. toFinally, be effective the dyad to determine was shown pH into acrossbe effective in vitro to experiments. determine pHin across in vitro experiments.

Figure 9. Chemical structure of rhodamine-fluorescein dyad 9 from Lee [46].

Another approach was proposed by Wu et al. to widen the interval of pH sensitivity [48]. Two pyrene groups were covalently linked to FITC 2 (10) in order to create a time-resolved FRET system (Figure 10). Under an excitation at 358 nm, bispyrene moieties have an emission peak at 459 nm fitting with FITC 2 absorbance. Due to bispyrene lifetime properties, excitation of FITC 2 by FRET occurs following alkalization of the solution. Thus, ratiometric determination based on both

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 23 have been successfully adopted. The most convenient way to use this method is to associate a pH-dependent probe to one (dyad) or more fluorophores emitting at different wavelengths under the same excitation. The ratio of fluorescence is then calculated in which pH-independent probes are used as internal control. Novel chemical associations based on fluorescein 1 were recently elaborated as dyads. For example, Zhang et al. proposed a ratiometric probe based on the chemical association 8 of an iridium (III) complex with FITC 2 (Figure 8) for pHin monitoring [27]. Under a single wavelength excitation at 488 nm, ratiometric determination of pHin is possible from 5.2 to 7.8, using near infrared emission from iridium (III) complex.

Figure 8. Iridium (III) complex-fluorescein dyad 8 as intracellular pH (pHin) probe by Zhang [27].

In addition to the creation of ratiometric probes, the dyads strategy can also be an opportunity to enlarge operating windows of pH-sensitivity of fluorescein. Since large pH fluctuations may occur in cells, during mitophagy for example, more accurate and wider pH-sensitive monitoring offers a better understanding of cellular life. However, the range of the pH efficient interval for fluorescein 1 is limited to 6 to 8. Therefore, Lee et al. put forward a novel dyad based on chemical association with rhodamine B (9) (Figure 9) [46]. Inspired by previous works using both compounds grafted on nanodots [47], rhodamine was covalently linked to fluorescein 1 in order to create a pH-sensitive probe efficient from 4 to 8. The widening of the pH efficient interval is based on rhodamine fluorescence, which decreases with alkalization. Moreover, both fluorophores have similar absorbance properties, offering an opportunity to use ratiometric calculation by exciting both Int.molecules J. Mol. Sci. at2020 488, 21 nm., 9217 Finally, the dyad was shown to be effective to determine pHin across in 7vitro of 23 experiments.

Figure 9. Chemical structure of rhodamine-fluorescein rhodamine-ﬂuorescein dyad 9 from LeeLee [[46].46].

Another approach approach was was proposed proposed by by Wu Wu et etal. al. to widen to widen the interval the interval of pH of sensitivity pH sensitivity [48]. Two [48]. Twopyrene pyrene groups groups were werecovalently covalently linked linked to FITC to 2 FITC (10) in2 (order10) in to order create to a create time-resolved a time-resolved FRET system FRET system(Figure (Figure10). Under 10). Underan excitation an excitation at 358 at nm, 358 nm,bispyr bispyreneene moieties moieties have have an an emission emission peak peak at at 459 459 nm Int.ﬁttingfitting J. Mol. with Sci. 2020 FITC, 21 2, x absorbance. absorbance.FOR PEER REVIEW Due to bispyrene lifetime properties, excitation of FITC 2 by FRET7 of 23 occurs followingfollowing alkalization alkalization of theof solution.the solution. Thus, Thus, ratiometric ratiometric determination determination based on based both emissionson both emissionsis eﬀective is for effective pH from for 3 pH to 10. from Cellular 3 to 10. experiments Cellular experiments showed that showed this probe that canthis beprobe well can adapted be well to pHadapted in determination to pHin determination between 4 andbetween 8. 4 and 8.

Figure 10. Bispyrene-FluoresceinBispyrene-Fluorescein dyad dyad 1010, ,as as Förster Förster resonance resonance energy energy transfer transfer (FRET) based pH-sensitive probes by Wu [[48].48].

Mitochondrion is a prominent organelle for eukaryotes organisms because of its major role in respiration systems, energy production, enzymatic activity, andand cationscations storage.storage. Monitoring pH fluctuationsfluctuations withinwithin mitochondria mitochondria is anis importantan import challengeant challenge considering considering the importance the importance of the chemical of the ® processeschemical processes involved. involved. Previously, Previously, Carboxy-SNARF Carboxy-SNARF(Seminaphtharhodafluor)® (Seminaphtharhodafluor) was the preferredwas the preferredmitochondrial mitochondrial pH-monitoring pH-monitoring probe due toprobe its passive due to accumulation its passive accumulation [49], but was [49], deemed but insuwas ffideemedciently insufficientlyselective or rapid selective for this or rapid specific for application. this specific Inapplication. order to improve In order mitochondrialto improve mitochondrial pH monitoring, pH monitoring,different approaches different wereapproaches recently were proposed. recently Chenproposed. et al. Chen described et al. adescribed dyad based a dyad on thebased chemical on the chemicalassociation association of cyanine of withcyanine FITC with2, calledFITC 2 “Mito-pH”, called “Mito-pH” (Figure 11(Figure-11)[ 5011-].11 In) [50]. addition In addition to its ability to its abilityto selectively to selectively target mitochondria target mitochondria [51], cyanine [51], hascyanine pH-insensitive has pH-insensitive fluorescent fluorescent properties properties allowing allowinga ratiometric a ratiometric monitoring. monitoring. The ratiometric The ratiometric signal was signal described was described as a linear as response a linear fromresponse pH 6.1from to pH8.4 across6.1 to 8.4in vitro acrossexperiments. in vitro experiments. Yet, Li et al. Yet, described Li et al. adescribe ratiometricd a ratiometric probe by broadening probe by broadening fluorescein fluoresceinabsorbance absorbance with chemical with coupling chemical of coup unsaturatedling of unsaturated indolium (Figure indolium 11- 12(Figure)[52]. 11- As12 previously,) [52]. As previously,such structure such tends structure to accumulate tends to withinaccumulate mitochondria within mitochondria due to its lipophilic due to cationicits lipophilic structure cationic [51]. Thisstructure probe [51]. showed This a probe repetitive showed signal a even repetitive in highly signal ionic even solutions. in highly After ionic full characterization solutions. After of full the characterizationratiometric signal of forthe pH ratiometric varying fromsignal 4 for to 10,pH confocal varying imagingfrom 4 to showed 10, confocal the capacity imaging of showed this probe the capacityto selectively of this reach probe its to target. selectively Chloroquine reach its treatments target. Chloroquine were applied treatments to cells inwere order applied to simulate to cells pH in orderfluctuations, to simulate since pH this fluctuations, compound induces since this intracellular compound alkalization induces intracellular [53]. This novel alkalization probe has [53]. proved This novelits ability probe to has track proved in real-time its ability wide to mitochondrialtrack in real-time pH wide fluctuations. mitochondrial In order pH tofluctuations. increase the In Stokes order to increase the Stokes shift, Qi et al. proposed the hybridization of the fluorescein with a coumarin moiety (Figure 11-13) [54]. Indeed, overlapping of emission and excitation spectra, as in case of fluorescein, may induce the requirement for high-resolution optical devices. Thanks to its hybridization between both fluorophores, the resulting probe was effective in the determination of mitochondrial pH, with maxima wavelengths about 450 and 550 nm for absorption and emission respectively.

Int. J. Mol. Sci. 2020, 21, 9217 8 of 23 shift, Qi et al. proposed the hybridization of the fluorescein with a coumarin moiety (Figure 11-13)[54]. Indeed, overlapping of emission and excitation spectra, as in case of fluorescein, may induce the requirement for high-resolution optical devices. Thanks to its hybridization between both fluorophores, the resulting probe was effective in the determination of mitochondrial pH, with maxima wavelengths aboutInt. J. Mol. 450 Sci. and 2020 550, 21, nmx FOR for PEER absorption REVIEW and emission respectively. 8 of 23

HO O O

O N OH

O O OH H HN N N N O H S HO O O O O O

O O

N OH OH

11 12 13 Figure 11. MitochondrialMitochondrial pH-sensitive probes according to Chen [[50],50], Li [[52],52], and Qi [[54].54]. 3.1.2. Supported Sensors 3.1.2. Supported Sensors For years now, nanoparticles attracted a lot of attention because of their applications in cancer For years now, nanoparticles attracted a lot of attention because of their applications in cancer therapy [55,56]. In addition to physical and chemical advantages inducing flexible and graftable surfaces, therapy [55,56]. In addition to physical and chemical advantages inducing flexible and graftable their biocompatibility allows them to massively penetrate tumor cells. Thus, multifunctional nanoparticles surfaces, their biocompatibility allows them to massively penetrate tumor cells. Thus, were quickly described for therapeutic and diagnostic applications. Therefore, novel pH-sensitive multifunctional nanoparticles were quickly described for therapeutic and diagnostic applications. nanoparticles bearing fluorescein moieties are continuously described. Because pH measurement within Therefore, novel pH-sensitive nanoparticles bearing fluorescein moieties are continuously lysosomes is essential to understand cellular metabolisms, particularly in tumoral cells, Zhang et al. described. Because pH measurement within lysosomes is essential to understand cellular designed novel pH-sensors based on silicon nanodots bearing aptamers and FAM 3 (14) (Figure 12)[57]. metabolisms, particularly in tumoral cells, Zhang et al. designed novel pH-sensors based on silicon The use of the aptamer (AS1411) enhances cellular uptake and specifically targets tumors and lysosomes nanodots bearing aptamers and FAM 3 (14) (Figure 12) [57]. The use of the aptamer (AS1411) due to its strong affinity for nucleolins. Because of native fluorescence properties from these particles, enhances cellular uptake and specifically targets tumors and lysosomes due to its strong affinity for only grafting of FAM 3 was required to create a ratiometric system. The preparation of final nanodots nucleolins. Because of native fluorescence properties from these particles, only grafting of FAM 3 was easily established using simple reactions such as formation of an amide and thiol–Michael click was required to create a ratiometric system. The preparation of final nanodots was easily established chemistry. Using ratiometric calculations between emissions from nanodots and FAM 3, estimations of using simple reactions such as formation of an amide and thiol–Michael click chemistry. Using lysosomal pH were performed. Since aptamers grafted on dots have the ability to recognize malignant ratiometric calculations between emissions from nanodots and FAM 3, estimations of lysosomal pH cells, an observable difference of fluorescence between the imaging of human breast MCF-7 cancer were performed. Since aptamers grafted on dots have the ability to recognize malignant cells, an cells and normal cells MCF10-A was easily observed. Thus, such particles offer an interesting way to observable difference of fluorescence between the imaging of human breast MCF-7 cancer cells and selectively image cancer cells and acquire large amounts of data concerning their lysosomal in-situ normal cells MCF10-A was easily observed. Thus, such particles offer an interesting way to pH variations. selectively image cancer cells and acquire large amounts of data concerning their lysosomal in-situ Whereas aptamers are used as targeting agents in this previous study, Ding et al. proposed their pH variations. own ratiometric pH-sensitive probes specific to cancer cells using folic acid [58]. Due to high density of folate acceptors in cancer cells, gold nanoclusters (AuNCs) covered by bovine serum albumin (BSA), FITC 2, and folic acid, were designed to selectively penetrate their target, providing a ratiometric determination of pHin. Once again, native properties of the used nanoparticles fit perfectly with FITC 2, since a single excitation at 488 nm was enough to induce fluorescence from the clusters and the pH-sensitive probe. Efficient for pH from 6.0 to 7.8, this system was tested over HeLa cells for which pH variations were simulated using ammonium chloride treatments.

Figure 12. Preparation of silicon nanodots 14 bearing aptamers and FITC according to Zhang [57].

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 23

HO O O

O N OH

O O OH H HN N N N O H S HO O O O O O

O O

N OH OH

11 12 13 Figure 11. Mitochondrial pH-sensitive probes according to Chen [50], Li [52], and Qi [54].

3.1.2. Supported Sensors For years now, nanoparticles attracted a lot of attention because of their applications in cancer therapy [55,56]. In addition to physical and chemical advantages inducing flexible and graftable surfaces, their biocompatibility allows them to massively penetrate tumor cells. Thus, multifunctional nanoparticles were quickly described for therapeutic and diagnostic applications. Therefore, novel pH-sensitive nanoparticles bearing fluorescein moieties are continuously described. Because pH measurement within lysosomes is essential to understand cellular metabolisms, particularly in tumoral cells, Zhang et al. designed novel pH-sensors based on silicon nanodots bearing aptamers and FAM 3 (14) (Figure 12) [57]. The use of the aptamer (AS1411) enhances cellular uptake and specifically targets tumors and lysosomes due to its strong affinity for nucleolins. Because of native fluorescence properties from these particles, only grafting of FAM 3 was required to create a ratiometric system. The preparation of final nanodots was easily established using simple reactions such as formation of an amide and thiol–Michael click chemistry. Using ratiometric calculations between emissions from nanodots and FAM 3, estimations of lysosomal pH were performed. Since aptamers grafted on dots have the ability to recognize malignant cells, an observable difference of fluorescence between the imaging of human breast MCF-7 cancer cells and normal cells MCF10-A was easily observed. Thus, such particles offer an interesting way to Int.selectively J. Mol. Sci. image2020, 21 cancer, 9217 cells and acquire large amounts of data concerning their lysosomal in-situ9 of 23 pH variations.

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 23

Whereas aptamers are used as targeting agents in this previous study, Ding et al. proposed their own ratiometric pH-sensitive probes specific to cancer cells using folic acid [58]. Due to high density of folate acceptors in cancer cells, gold nanoclusters (AuNCs) covered by bovine serum albumin (BSA), FITC 2, and folic acid, were designed to selectively penetrate their target, providing a ratiometric determination of pHin. Once again, native properties of the used nanoparticles fit perfectly with FITC 2, since a single excitation at 488 nm was enough to induce fluorescence from the clustersFigureFigure and 12.12.the Preparation pH-sensitive of silicon probe. nanodots Efficient 1414 bearingfor pH aptamersaptamersfrom 6.0 andand to FITC7.8,FITC this accordingaccording system toto ZhangwasZhang tested [[57].57]. over HeLa cells for which pH variations were simulated using ammonium chloride treatments. Whereas most studies are focused on intracellular data, Yang et al. proposed a ratiometric probe Whereas most studies are focused on intracellular data, Yang et al. proposed a ratiometric calledprobe “FITC-FPencalled “FITC-FPen/FPen@AuNC”/FPen@AuNC” for determination for determination of extracellular of extracellular pH (pHex)[59 pH]. Ionic (pH concentrationsex) [59]. Ionic surroundingconcentrations cell surrounding surface is also cell asurface parameter, is also which a parameter, can indicate which metabolic can indicate disorders metabolic such disorders as tumor metastasissuch as tumor [60], metastasis ion transits [60], deregulation ion transits [61 deregulation], or even some [61], virus or even infections some [virus62]. A infections similar chemical [62]. A approachsimilar chemical to the studyapproach from to Ding the study [58] was from proposed Ding [58] here; was BSA proposed protected here; AuNCs, BSA protected were covered AuNCs, by FITCwere andcovered cell penetratingby FITC and peptides cell penetrating (CPP). CPP peptid arees cationic (CPP). peptides,CPP are cationic which are peptides, generally which used are as carriersgenerally for used transporting as carriers compounds for transporting through compounds membranes. through In this membranes. case, the used In this peptide case, (FPen)the used is ensuringpeptide (FPen) agglomeration is ensuring of agglomeration nanosensors atof nanose the cellnsors surface at the whereas cell surface emissions whereas from emissions AuNCs from and FITCAuNCs2 andare providingFITC 2 are theproviding ratiometric the ratiometric determination determination of pHex. of Confocal pHex. Confocal imaging imaging of HeLa of HeLa cells exposedcells exposed to diﬀ erentto different media provedmedia theproved good the sensitivity good sensitivity and low cytotoxicity and low ofcytotoxicity theses nanoparticles of theses (Figurenanoparticles 13). Another (Figure complementary 13). Another complementary strategy was proposed strategy by was Ohgaki proposed et al. using by Ohgaki FITC-poly(ethylene et al. using glycol)-phospholipidFITC-poly(ethylene glycol)-phospholipid derivatives [63]. This derivative small and amphiphilics [63]. This small polymer and has amphiphilic the ability topolymer be inserted has intothe ability plasma to membranebe inserted ofinto cells, plasma and membrane then grafted of FITCcells, and can then be used grafted as probe FITC forcan determinationbe used as probe of pHfor exdetermination. The cell-surface of pH labelingex. The withcell-surface this polymer labeling presented with this many polymer advantages presented such many as its advantages sensitivity, pH-reversibility,such as its sensitivity, and its pH-reversibility, generic application. and its generic application.

Figure 13.13. HeLa extracellular pH determinationdetermination using FITC-FPenFITC-FPen/FPen@AuNC./FPen@AuNC. Reproduced with authorization from Yang [[59].59].

Immobilization of fluorescein for recurrent surface pH determination displays many challenges because it should avoid leaching, degradation, or photobleaching. Preparation of fluorescent pH-sensitive surfaces is also offering additional benefits since it can be used as optic fiber coating or in bioanalytical protocols, such as evanescent wave sensors. Bidmanova et al. investigated a general procedure for immobilization of fluorescein derivatives on surfaces, by using BSA derivatives [64]. Thus, FAM-BSA conjugates were immobilized on glass surface using direct glutaraldehyde cross-linking or after incorporation into ORganically MOdified CERAmics (ORMOCER) (Figure 14). The resulting surfaces have proved to be highly suitable for their mechanical stability, negligible (photo)leaching, and fluorescent pH-sensitivity from 4.0 to 9.0. Due to the ease of use, such protocols

Int. J. Mol. Sci. 2020, 21, 9217 10 of 23

Immobilization of fluorescein for recurrent surface pH determination displays many challenges because it should avoid leaching, degradation, or photobleaching. Preparation of fluorescent pH- sensitive surfaces is also offering additional benefits since it can be used as optic fiber coating or in bioanalytical protocols, such as evanescent wave sensors. Bidmanova et al. investigated a general procedure for immobilization of fluorescein derivatives on surfaces, by using BSA derivatives [64]. Thus, FAM-BSA conjugates were immobilized on glass surface using direct glutaraldehyde cross-linking or after incorporation into ORganically MOdified CERAmics (ORMOCER) (Figure14). TheInt. J. resultingMol. Sci. 2020 surfaces, 21, x FOR have PEER provedREVIEW to be highly suitable for their mechanical stability, negligible 10 of 23 (photo)leaching, and fluorescent pH-sensitivity from 4.0 to 9.0. Due to the ease of use, such protocols may lead to thethe preparationpreparation of aa supportedsupported sensorsensor forfor aa widewide rangerange ofof applicationsapplications includingincluding bioprocessing, biochemical biochemical analyses, analyses, enviro environmentalnmental analysis, analysis, or or healthcare healthcare devices. devices.

O O = HN O O OH

GA BSA BSA GA BSA GA BSA GA GA BSA GA GA BSA BSA BSA BSA GA GA BSA GA BSA BSA GA BSA GA

Figure 14.14. ImmobilizationImmobilization of of carboxyfluorescein carboxyfluorescein (FAM)-bovine (FAM)-bovine serum serum albumin albumin (BSA) (BSA) on treated on treated glass byglass direct by glutaraldehydedirect glutaraldehyde (GA) cross-linking (GA) cross-linkin or afterg incorporationor after incorporation in ORganically in ORganically MOdified CERAmicsMOdified (ORMOCER),CERAmics (ORMOCER), according to according Bidmanova to Bidmanova [64]. [64]. 3.2. Dual Sensors 3.2. Dual Sensors The development of ratiometric probes is related to the recent improvement of optical devices The development of ratiometric probes is related to the recent improvement of optical devices composing imaging systems. Following this increasing precision, more studies propose to add other composing imaging systems. Following this increasing precision, more studies propose to add other sensitive probes to fluorescein-based compounds. Thus, such dual sensor offers the benefit of two sensitive probes to fluorescein-based compounds. Thus, such dual sensor offers the benefit of two physiochemical data in a single optical read, which is a unique opportunity for the understanding of physiochemical data in a single optical read, which is a unique opportunity for the understanding of biological systems. biological systems. Using AuNCs, Han et al. described the preparation and characterization of a ratiometric probe Using AuNCs, Han et al. described the preparation and characterization of a ratiometric probe 15 providing intracellular dual determinations of Cu2+ concentration and pH [65]. Three different 15 providing intracellular dual determinations of Cu2+ concentration and pH [65]. Three different molecules were grafted on these nanoclusters, firstly FITC 2 as pH-sensitive probe, secondly a molecules were grafted on these nanoclusters, firstly FITC 2 as pH-sensitive probe, secondly a tailor-made specific Cu-ligand (called TPAASH) and thirdly a coumarin derivative as a reference tailor-made specific Cu-ligand (called TPAASH) and thirdly a coumarin derivative as a reference probe (Figure 15). Monitoring of Cu2+ cations is based on the quenching of AuNC emission at 722 nm probe (Figure 15). Monitoring of Cu2+ cations is based on the quenching of AuNC emission at 722 nm (under excitation at 405 nm), following the increase of copper concentration. Therefore, coumarin (under excitation at 405 nm), following the increase of copper concentration. Therefore, coumarin was also grafted on clusters to be used as reference probe since its excitation wavelength is also about was also grafted on clusters to be used as reference probe since its excitation wavelength is also 405 nm and emission is at 472 nm. These novel particles allow the ratiometric determinations of pH about 405 nm and emission is at 472 nm. These novel particles allow the ratiometric determinationsin from 6 to 9, and simultaneously, of Cu2+ concentration up to 11 µM. A few years later, Zhu et al. of pHin from 6 to 9, and simultaneously, of Cu2+ concentration up to 11 µM. A few years later, Zhu et proposed their own version of a dual pH/Cu2+ sensor by grafting FITC 2 and polyethylenimine (PEI) al. proposed their own version of a dual pH/Cu2+ sensor by grafting FITC 2 and polyethylenimine on carbon dots [66]. Due to a ratiometric and linear signal, they were able to determine pH and Cu2+ (PEI) on carbon dots [66]. Due to a ratiometric and linear signal, they were able to determine pH and concentration into yogurt and human serum samples. Cu2+ concentration into yogurt and human serum samples.

Int. J. Mol. Sci. 2020, 21, 9217 11 of 23 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 23

reference probe

H+ Cu2+ pH sensible probe N sensible probe HO O

O OH O S O O N NH HN O N N HN S S N S S Au S 15

FigureFigure 15.15. pHpH andand CuCu22++ sensitivesensitive nanoprobe nanoprobe 1515 accordingaccording to Han [65]. [65].

Oxygen is a keykey componentcomponent for biologicalbiological systems, and especially, forfor mammalmammal cellscells forfor whichwhich metabolism pathwayspathways are are based based on Krebson Krebs cycle. Thus,cycle. dual Thus, sensing dual probes sensing allowing probes the allowing simultaneous the monitoringsimultaneous of oxygenmonitoring concentration of oxygen and concentration pH at the same and time pH areat verythe same important time are in biomedical very important sciences. in Relevantbiomedical publications sciences. Relevant reported publications the creation reported of such dualthe creation sensors of [67 such–69] du basedal sensors on the [67–69] combination based ofon fluoresceinthe combination with O of2-sensitive fluorescein fluorophores, with O2-sensitive such as fluorophores, platinum and such ruthenium as platinum complexes. and ruthenium In order tocomplexes. improve dualIn order monitoring to improve of pH dualin and monitoring O2, Xu et al.of describedpHin and theO2, useXu ofet semiconductingal. described the polymer use of dotssemiconducting16 (Pdots) as polymer a support dots of both 16 sensitive(Pdots) probes,as a support since these of nanomaterialsboth sensitive have probes, an improved since these cell uptakenanomaterials [70]. For have this an system, improved FITC cell2 and uptake a platinum [70]. For porphyrin this system, complex FITC were2 and targeted a platinum as pH porphyrin and O2 sensorscomplex respectively were targeted (Figure as pH 16C). and Once O2 again,sensors the respectively native photophysical (Figure 16C). properties Once again, of the the dots native were employed;photophysical based properties on a FRET of the eff ect,dots this were multimodal employed; sensor based allowson a FRET ratiometric effect, this determination multimodal sensor under excitationallows ratiometric at 405 nm, determination since the fluorescence under excitation of Pdots at 405 (410 nm, to 470since nm) the is fluorescence matching the of absorptionPdots (410 ofto FITC470 nm) (410 is tomatching 500 nm). the Thus, absorption one single of FITC excitation (410 to at 500 405 nm). nm leadsThus, toone emissions single excitation from all sensorsat 405 nm at dileadsfferent to emissions wavelengths from (blue all sensors for Pdots, at different green for wavelengths FITC 2, red for (blue porphyrin). for Pdots, After green calibration, for FITC 2,in red vitro for experimentsporphyrin). onAfter CaSki calibration, cells proved in thatvitro Pdots experi arements well embeddedon CaSki incells the proved cells, allowing that Pdots the improvedare well monitoringembedded ofin boththe physicalcells, allowing parameters the improved using ratiometric monitoring results of (Figure both physical16A,B). parameters using ratiometricA promising results approach, (Figure 16A,B). described by Meier et al., focuses on imaging devices in order to obtain signals from dual sensors using a conventional digital camera [71]. A sensor film was created by the incorporation of microparticles bearing sensors: platinum porphyrin complex for oxygen sensing, FITC 2 for pH sensing and diphenylanthracene as reference. Under light irradiation at 405 nm, emissions in red green blue (RGB) channels can be recorded by a digital camera. After characterizations, calculations and calibrations, the proposed system was even used across in vivo experiments (Figure 17). The dual sensor film was applied on healing wounds where inflamed tissues were directly submitted to abnormal pH and oxygen concentrations. Furthermore, the same team developed a diligent expertise using such systems, by proposing various optimizations, such as a sprayable version [72], or applications concerning chronic wounds [73], water plants [74], and recently, radiation therapy [75].

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 23

reference probe

H+ Cu2+ pH sensible probe N sensible probe HO O

O OH O S O O N NH HN O N N HN S S N S S Au S 15

Figure 15. pH and Cu2+ sensitive nanoprobe 15 according to Han [65].

Oxygen is a key component for biological systems, and especially, for mammal cells for which metabolism pathways are based on Krebs cycle. Thus, dual sensing probes allowing the simultaneous monitoring of oxygen concentration and pH at the same time are very important in biomedical sciences. Relevant publications reported the creation of such dual sensors [67–69] based on the combination of fluorescein with O2-sensitive fluorophores, such as platinum and ruthenium complexes. In order to improve dual monitoring of pHin and O2, Xu et al. described the use of semiconducting polymer dots 16 (Pdots) as a support of both sensitive probes, since these nanomaterials have an improved cell uptake [70]. For this system, FITC 2 and a platinum porphyrin complex were targeted as pH and O2 sensors respectively (Figure 16C). Once again, the native photophysical properties of the dots were employed; based on a FRET effect, this multimodal sensor allows ratiometric determination under excitation at 405 nm, since the fluorescence of Pdots (410 to 470 nm) is matching the absorption of FITC (410 to 500 nm). Thus, one single excitation at 405 nm leads to emissions from all sensors at different wavelengths (blue for Pdots, green for FITC 2, red for porphyrin). After calibration, in vitro experiments on CaSki cells proved that Pdots are well embeddedInt. J. Mol. Sci. in2020 the, 21 , 9217cells, allowing the improved monitoring of both physical parameters12 using of 23 ratiometric results (Figure 16A,B).

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 12 of 23

Figure 16. Ratiometric visualization of pH (A) and O2 concentration (mg/L) (B) in CaSki cells using described Pdots 16 (C). Reproduced with authorization from Xu [70].

A promising approach, described by Meier et al., focuses on imaging devices in order to obtain signals from dual sensors using a conventional digital camera [71]. A sensor film was created by the incorporation of microparticles bearing sensors: platinum porphyrin complex for oxygen sensing, FITC 2 for pH sensing and diphenylanthracene as reference. Under light irradiation at 405 nm, emissions in red green blue (RGB) channels can be recorded by a digital camera. After characterizations, calculations and calibrations, the proposed system was even used across in vivo experiments (Figure 17). The dual sensor film was applied on healing wounds where inflamed tissues were directly submitted to abnormal pH and oxygen concentrations. Furthermore, the same team developed a diligent expertise using such systems, by proposing various optimizations, such Figure 16. Ratiometric visualization of pH (A) and OO2 concentration (mg/L) (mg/L) ( B) in CaSki cells using as a sprayable version [72], or applications concerning2 chronic wounds [73], water plants [74], and described Pdots 16 (C). Reproduced withwith authorizationauthorization fromfrom XuXu [[70].70]. recently, radiation therapy [75]. A promising approach, described by Meier et al., focuses on imaging devices in order to obtain signals from dual sensors using a conventional digital camera [71]. A sensor film was created by the incorporation of microparticles bearing sensors: platinum porphyrin complex for oxygen sensing, FITC 2 for pH sensing and diphenylanthracene as reference. Under light irradiation at 405 nm, emissions in red green blue (RGB) channels can be recorded by a digital camera. After characterizations, calculations and calibrations, the proposed system was even used across in vivo experiments (Figure 17). The dual sensor film was applied on healing wounds where inflamed tissues were directly submitted to abnormal pH and oxygen concentrations. Furthermore, the same team developed a diligent expertise using such systems, by proposing various optimizations, such as a sprayable version [72], or applications concerning chronic wounds [73], water plants [74], and Figure 17. Recent schematic drawing of the pH and pO2 measurement of skin after radiation therapy recently,Figure radiation 17. Recent therapy schematic [75]. drawing of the pH and pO2 measurement of skin after radiation therapy fromfrom Auerswald Auerswald [75]. [75].

In case of cancer cells, heat production is higher than in healthy cells due to abnormal and aroused In case of cancer cells, heat production is higher than in healthy cells due to abnormal and metabolisms [76]. Thus, the developments of dual sensors monitoring pH and temperature have also aroused metabolisms [76]. Thus, the developments of dual sensors monitoring pH and temperature been investigated. In this context, Rhodamine B is one of the most notorious temperature-sensitive have also been investigated. In this context, Rhodamine B is one of the most notorious ﬂuorescent probes. Liu et al. described the preparation of polystyrene microbeads embedded with temperature-sensitive fluorescent probes. Liu et al. described the preparation of polystyrene rhodamine, which were subsequently coated with FITC 2. This system presents a linear ﬂuorescence microbeads embedded with rhodamine, which were subsequently coated with FITC 2. This system response from rhodamine and temperature from 32 to 38 C. The physicochemical stability and the presents a linear fluorescence response from rhodamine ◦and temperature from 32 to 38 °C. The selective response from each probe are among advantages of such combination. Later, Zhang et al. physicochemical stability and the selective response from each probe are among advantages of such proposed their own dual sensitive system using similar probes, but of a reduced size [77]. In addition combination. Later, Zhang et al. proposed their own dual sensitive system using similar probes, but of a reduced size [77]. In addition to the beneficial nanoscale of this sensor, a europium complex was Figure 17. Recent schematic drawing of the pH and pO2 measurement of skin after radiation therapy included as a reference dye in order to offer a ratiometric determination. Moreover, the surface of from Auerswald [75]. this nanosensor was coated with cationic charges in order to increase its affinity for lysosomes. In case of cancer cells, heat production is higher than in healthy cells due to abnormal and aroused metabolisms [76]. Thus, the developments of dual sensors monitoring pH and temperature have also been investigated. In this context, Rhodamine B is one of the most notorious temperature-sensitive fluorescent probes. Liu et al. described the preparation of polystyrene microbeads embedded with rhodamine, which were subsequently coated with FITC 2. This system presents a linear fluorescence response from rhodamine and temperature from 32 to 38 °C. The physicochemical stability and the selective response from each probe are among advantages of such combination. Later, Zhang et al. proposed their own dual sensitive system using similar probes, but of a reduced size [77]. In addition to the beneficial nanoscale of this sensor, a europium complex was included as a reference dye in order to offer a ratiometric determination. Moreover, the surface of this nanosensor was coated with cationic charges in order to increase its affinity for lysosomes.

Int. J. Mol. Sci. 2020, 21, 9217 13 of 23 to the beneficial nanoscale of this sensor, a europium complex was included as a reference dye in order to offer a ratiometric determination. Moreover, the surface of this nanosensor was coated with cationic charges in order to increase its affinity for lysosomes. Across a series of in vitro experiments using HeLa cells, his dual sensor was able to determine pH ranging from 4.0 to 9.0, and temperature between 25 and 40 ◦C.

3.3. Bacterial Growth Monitoring aqueous physicochemical data, such as pH, is also a significant opportunity to understand bacterial growth. Indeed, due to their capacity to transform and adapt their environment through their quick proliferations and the production of extracellular compounds, ionic variations cause pH changes in a few hours [78]. The modification of pH along incubation periods has been widely used to monitor microorganism growths in various media, including in blood cultures. In fact, it is usually used to detect the presence of bacterial contamination in medical [79], food [80], and water [81] sectors, which is becoming more of a relevant issue because of emergence of bacterial resistance [82]. Thus, pH-sensitivity around the neutral domain of fluorescein perfectly fits with microbial culture, since most of in vitro experiments using common strains are conducted at physiological pH. For example, Si et al. designed nanoparticles bearing fluoresceinamine (FANPs) for an accurate real-time detection of Escherichia coli growth (Figure 18)[83]. Resultant non-toxic and fluorescent polystyrene nanoparticles, obtained after grafting FA 4 by carbodiimide coupling, were incorporated in in vitro cultures, where emitting fluorescence was directly linked to medium pH. Following the proliferation of Escherichia coliInt. J., Mol. the Sci. pH 2020 is, 21,decreasing x FOR PEER REVIEW which directly reduce the fluorescence of the14 particles. of 24 Thus, the simple fluorescence from culture suspensions is sufficient to determine if there is any growthCommented [M1]: We would remove word “A”. Please or inhibition of the strain. This system was also challenged using cultures exposed to diffconfirmerent it. concentrations of antibiotics, where pH (and fluorescence) kinetics were precise enough to determine Commented [f2]: Approved and corrected with a new version bacterial behaviors. Thus, such particles can easily be used for the screening of potential growthjust below inhibitors or for the determination of bacterial minimal inhibitory concentration.

Figure 18. FluorescenceFigure 18. Fluorescence extinguishments extinguishments due due to to the the acidiﬁcationacidification of medium of medium by the bybacterial the bacterial growth growth in a1 and c1. Reproducedin a1 and c1. Reproduced with authorization with authorization from from Si Si [83 [83]]. .

In their view, Wang et al. focused on pH monitoring in case of bacterial growth on Petri dishes In theircultures view, [84] Wang. Since et their al. focusedcreation in on 1887, pH Petri monitoring dishes are still in the case most of widely bacterial used support growth for on in Petri dishes cultures [84].vitro Since cultures their due to creation their flexibility in 1887, fitting Petri the broad dishes microbial are diversity. still the In mostaddition widely to agarose used and support for in vitro culturesappropriate due to nutriments, their flexibility Wang et fitting al. proposed the broad to add microbial silicone nanoparticles diversity. bearing In addition FA 4 and to agarose and porphyrin moieties in the preparation. These nanosensors are physicochemical stable, easy to appropriate nutriments,incorporate within Wang agar et preparation, al. proposed are not to internalized add silicone by bacteria nanoparticles, and are based bearing on ratiometric FA 4 and porphyrin moieties in themethod. preparation. Thus, this study These offers nanosensors an effective protocol are physicochemical to design pH-sensitive stable, Petri easydishes, to which incorporate can within agar preparation,be used are to visualize not internalized bacterial growth by bacteria, or determine and resultant are based pH variations on ratiometric due to bacterial method. behaviors Thus, this study (Figure 19). offers an effective protocol to design pH-sensitive Petri dishes, which can be used to visualize bacterial growth or determine resultant pH variations due to bacterial behaviors (Figure 19).

Figure 19. Bacterial and pH imaging using ratiometric signal from nanoparticles included in agar from Wang [84].

Some strains have the ability to adhere on surfaces and form a biofilm throughout the production of an extracellular matrix promoting the survival, progression and growth synchronization of the colony. Bacterial biofilms are a major medical concern because of their ability to colonize indwelling devices, most importantly catheters and implants [85]. In order to study the metabolisms and mechanisms implicated in such organized structures, in vitro biofilm cultures have showed their utility, as well as the development of suitable fluorescent probes. Gashti et al. described the preparation of a pH-monitoring microfluidic platform adapted for biofilm studies [86]. To reach this goal, FITC 2 based silver core-silica shell nanoparticles were prepared, and then, covalently linked to glass substrates using click chemistry. This microfluidic system has many advantages, such as physicochemical stability, lack of leakage of/from nanoparticles (NPs) and real-time pH measurement. For example, dynamic pH responses from an oral biofilm of Streptococcus salivarius were monitored following exposition to different glucose concentrations

Int. J. Mol. Sci. 2020, 21, 9217 14 of 23 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 14 of 23

hv hv hv' hv'' hv' hv'' HO O OH Silicon NP O Bacterial Incubation HN O

FigureFigure 19.19. BacterialBacterial and and pH pH imaging imaging using using ratiometric ratiometric signal signal from from nanoparticles nanoparticles included included in agar in fromagar Wangfrom Wang [84]. [84].

SomeSome strainsstrains have have the the ability ability to adhere to adhere on surfaces on surfaces and form and a biofilmform a throughout biofilm throughout the production the ofproduction an extracellular of an matrix extracellu promotinglar thematrix survival, promoting progression the and survival, growth synchronizationprogression and of the growth colony. Bacterialsynchronization biofilms of are the a majorcolony. medical Bacterial concern biofilms because are a major of their medical ability concern to colonize because indwelling of their devices, ability mostto colonize importantly indwelling catheters devices, and most implants importantly [85]. In ordercatheters to study and implants the metabolisms [85]. In order and mechanismsto study the implicated in such organized structures, in vitro biofilm cultures have showed their utility, as well as the metabolismsInt. and J. Mol. mechanisms Sci. 2020, 21, x FOR PEERimplicated REVIEW in such organized structures, in vitro biofilm15 of 24cultures have developmentshowed their of utility, suitable as fluorescent well as the probes. development Gashti et al. of described suitable thefluor preparationescent probes. of a pH-monitoring Gashti et al. microfluidic(Figure platform 20). Offering adapted bacterial for biofilm biofilm studiesmonitoring, [86 such]. To described reach thissystem goal, has also FITC advantages2 based linke silverd core-silica described theto preparationmicrofluidic scales of wherea pH-monitoring liquids consummations micro fluidicare reduced, platform cultures adaptedimproved duefor tobiofilm laminar studies [86]. shellTo reach nanoparticles thisflows goal, and were reactionFITC prepared, kinetics2 based optimized and silver then, [87,88] core-silica covalently. Moreover, shell linkedthis studynanoparticles to is glass also suggesting substrates were that, usingprepared, due to clickthe and chemistry. then, Thiscovalently microfluidic linkeduse of nanoparticles systemto glass has substrates as many platform advantages, forusing fluorescent click such probchemistry.es, as additional physicochemical This sensitive microfluidic probes stability, may system be lack also ofhas leakage many integrated to create a multi-modal microfluidic system and to increase income of information. ofadvantages,/from nanoparticles such as physicochemical (NPs) and real-time stability, pH measurement. lack of leakage For of/from example, nanoparticles dynamic pH (NPs) responses and fromreal-time an oral pH biofilm measurement. of Streptococcus For salivariusexample,were dyna monitoredmic pH followingresponses exposition from an to dioralfferent biofilm glucose of concentrationsStreptococcus salivarius (Figure 20were). O monitoredffering bacterial following biofilm exposition monitoring, to different such described glucose system concentrations has also advantages(Figure 20). linkedOffering to microfluidicbacterial biofilm scales monitoring, where liquids such consummations described system are reduced,has also advantages cultures improved linked dueto microfluidic to laminar flowsscales and where reaction liquids kinetics consummation optimizeds are [87 ,88reduced,]. Moreover, cultures this improved study is alsodue suggestingto laminar that,flows due and to reaction the use ofkinetics nanoparticles optimized as platform[87,88]. Moreov for fluorescenter, this study probes, is additionalalso suggesting sensitive that, probes due to may the beuse also of integratednanoparticles to create as platform a multi-modal for fluorescent microfluidic probes, system additional and to increase sensitive income probes of may information. be also integrated to create a multi-modal microfluidic system and to increase income of information. Formatted: Snap to grid

Figure 20. S.Figure salivarius 20. S. salivariusbiofilm biofilm pH response pH response under under expositions to glucose to glucose solutions solutions (A) and imaging (A) and of imaging ofCommented [M3]: Please add scale bar for Figure 20B. localized acidificationlocalized acidification (red) and (red) biofilm and biofilm accumulation accumulation (green) (green) ((BB)).. Reproduced Reproduced with with authorization authorization from from Gashti [86]. Commented [f4]: Corrected Gashti [86]. Figure 20. S.In salivarius comparison biofilm with previous pH response reported under studies expositions in this review, to glucosetargeted orsolutions optimized (A probes) and areimaging of In comparisonrarely developed with previous for imaging reported specific bacteri studiesal organelles. in this review, Due to their targeted small size or optimizedand their potential probes are rarely localized acidification (red) and biofilm accumulation (green) (B). Reproduced with authorization developed formobility imaging during specific imaging bacterial experimentations, organelles. bacteria Due do to not their fit perfectly small size with and specific their organelle potential mobility from Gashti [86]. during imagingmonitoring. experimentations, Furthermore, diacetate bacteria derivatives do not (FDA fit perfectly and CFDA with5) are specific perfect candidates organelle for monitoring. monitoring bacterial enzymatic activity, explaining why recent studies are mainly using fluorescein Furthermore, diacetate derivatives (FDA and CFDA 5) are perfect candidates for monitoring bacterial In comparisonat its purpose with [89 previous–93]. However, reported some studies work also in used this these review, fluorescein targeted derivatives or optimized for their probes are enzymatic activity,pH-sensitivity. explaining why recent studies are mainly using fluorescein at its purpose [89–93]. rarely developed for imaging specific bacterial organelles. Due to their small size and their potential However, someA work wide alsodiversity used of bacterial these fluoresceinspecies exists across derivatives all biological for systems. their pH-sensitivity. Each local microbiome mobility duringis directly imaging linked toexperimentations, the properties of its bacteria surroundings, do sincenot fit only perfectly bacteria supporting with specific local organelle A wide diversity of bacterial species exists across all biological systems. Each local microbiome is monitoring. physicochemicalFurthermore, properties diacetate are able derivatives to fully grow. (FDAThus, using and pH CFDA-sensitivity 5 )of arefluorescein perfect along candidates in for directly linkedvivo to assays the properties can be used ofas a its way surroundings, to determine the sincesuitable only environment bacteria for supporting each strain. For local example, physicochemical monitoring bacterial enzymatic activity, explaining why recent studies are mainly using fluorescein properties areaciduric able to bacteria fully are grow. generally Thus, involved using in pH-sensitivity buccal microbiome, of fluorescein as described in along the studyin vivo below.assays can be at its purposeAmong [89–93]. buccal strains, However, Streptococci some mutans work, Bifidobacterium also used dentium these, and B. fluorescein longum have been derivatives observed for their used as a way to determine the suitable environment for each strain. For example, aciduric bacteria pH-sensitivity.in case of caries lesions, often linked to local acidification. In order to compare their survival rate in acidic environment, Nakajo et al. proposed protocols using CFDA 5 [94]. Using pH sensitivity of fluorescein, the difference between pHin and pHex offers a way to observe bacterial ability to protect itself against acidification. In this case, it appeared that Bifidobacterium strains are the most stable species in acidic environment explaining why they are predominant in case of caries lesions. Using similar protocols based on comparison between intra and extracellular pH, bacterial life cycle can be also monitored since substrate consummation, and resulting productions of metabolites may induce variations in both environments. For example, Bouix et al. described a protocol to monitor pHin variations following the malolactic fermentation (MLF) of Oenococcus oeni in wine [95]. Using diacetate derivatives of carboxyfluorescein 3 and Oregon Green 7, pHin variations from 3 to 6 were accurately observed using flow cytometry analysis along different growth phases. It appeared that fermentation occurs during the exponential phase, where malic acid is co-excreted as lactic acid,

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 15 of 23

A wide diversity of bacterial species exists across all biological systems. Each local microbiome is directly linked to the properties of its surroundings, since only bacteria supporting local Int. J. Mol. Sci. 2020, 21, 9217 15 of 23 physicochemical properties are able to fully grow. Thus, using pH-sensitivity of fluorescein along in vivo assays can be used as a way to determine the suitable environment for each strain. For example, aciduricare generally bacteria involved are generally in buccal involved microbiome, in buccal as described microbiome, in the study as described below. Among in the buccalstudy strains,below. AmongStreptococci buccal mutans strains,, Bifidobacterium Streptococci dentiummutans,, Bifidobacterium and B. longum havedentium, been and observed B. longum in casehave of been caries observed lesions, inoften case linked of caries to locallesions, acidification. often linked In to orderlocal acidification. to comparetheir In order survival to compare rate in their acidic survival environment, rate in acidicNakajo environment, et al. proposed Nakajo protocols et al. using proposed CFDA 5protocols[94]. Using using pH CFDA sensitivity 5 [94]. of fluorescein,Using pH thesensitivity difference of fluorescein,between pH thein and difference pHex off betweeners a way pH toin observe and pH bacterialex offers a ability way to to observe protect bacterial itself against ability acidification. to protect itselfIn this against case, it acidification. appeared that InBifidobacterium this case, it appearedstrains are that the Bifidobacterium most stable species strains in are acidic the environment most stable speciesexplaining in acidic why theyenvironment are predominant explaining in casewhy ofthey caries are lesions.predominant in case of caries lesions. Using similar protocols based on comparison between intra and extracellular pH, bacterial life cycle can be also monitored since substrate consu consummation,mmation, and resulting productions of metabolites may induce variations in bothboth environments.environments. For For example, Bouix et al. de describedscribed a a protocol to monitor pH in variationsvariations following following the the malolactic malolactic fermentation fermentation (MLF) (MLF) of of OenococcusOenococcus oeni oeni inin wine wine [95]. [95]. Using diacetate derivative derivativess of carboxyfluorescein carboxyfluorescein 3 and Oregon Green 7,, pHpHinin variations from 3 to 6 were accurately observed using flow flow cytometry analysis along different different growth phases. It appeared that fermentation occurs during during the the exponential exponential phas phase,e, where where malic malic acid acid is is co co-excreted-excreted as as lactic lactic acid, acid, carbon dioxide, and proton. This This production production induces induces an an increase of pHin andand stimulates stimulates the the ATP synthase pathway. This This study study shows shows that that pH mo monitoringnitoring using fluorescein fluorescein derivatives provides valuable insights into the cellular mechanisms at play in pathogenesis.pathogenesis.

3.4. Other Other Applications Applications Following thethe principle principle of of drug drug vectorization, vectorization, any componentany component or organelle or organelle from biological from biological systems systemsmay be targetedmay be by targeted fluorescein by derivativesfluorescein inderivatives order to monitor in order its pHto monitor variations. its ForpH example,variations. Li etFor al. example,recently described Li et al. recently a novel described pH-sensitive a novel probe pH-sen targetingsitive bones probe [ 28targeting]. It appears bones that [28]. pH It appears is a relevant that pHdatum is a concerningrelevant datum bones co homeostasisncerning bones [96] homeostasis or abnormal [96] issues, or suchabnormal as metastasis issues, such [97]. as To metastasis create an [97].efficient To bonecreate pH-sensitive an efficient probe, bone FITC pH-sensitive2 was conjugated probe, FITC to alendronate, 2 was conjugated a bisphosphonate to alendronate, compound a bisphosphonatepresenting a significant compound affinity presenting for hydroxyapatite a significant (HAp) affinity (17) (Figurefor hydroxyapatite 21). The resulting (HAp) probe (17) was (Figure able 21).to selectivity The resulting target probe bones, was without able to interfering selectivity with target other bones, calcium without compounds interfering or organs. with other Nude calcium mouse compoundsmodels were or tested organs.in vivo Nudeand mouse pH variations models fromwere 6.8tested to 7.4 in werevivo observable.and pH variations The use offrom a ratiometric 6.8 to 7.4 wereversion observable. would provide The use a powerful of a ratiometric diagnostic version tool, andwould this provide probe is a a powerful remarkable diagnostic example oftool, fluorescein and this probevectorization is a remarkable for pH monitoring. example of fluorescein vectorization for pH monitoring.

Figure 21. BoneBone pH-sensitive pH-sensitive probe 17 according to Li [28]. [28].

The design of ﬂuoresceinfluorescein based ratiometric se sensorsnsors was applied to the development of novel imaging tools. tools. Mathew Mathew et al. et described al. described a novel a nanoprobe novel nanoprobe for imaging for imagingand monitoring and monitoring pH variations pH Caenorhabditis elegans withinvariations Caenorhabditis within elegans, a nematode, a usually nematode used usually as in vi usedtro model as in vitroorganismmodel [98]. organism Monitoring [98]. C. elegans intestinalMonitoring intracellular intestinal intracellular pH of natural pH of strains natural of strains C. elegans of was quitewas challenging, quite challenging, because because of the of presencethe presence of a of protecting a protecting and and surrounding surrounding cuticle, cuticle, and and selective selective intestinal intestinal uptake uptake [99]. [99]. To To overcome overcome these issues, nanocolloidal silica particles bearing FITC 2 and rhodamine patterns were prepared. Thanks to the intrinsic properties of the nanoparticles, the ﬂuorophores are physicochemically stable and able to bypass the selective intestinal barrier. The resulting probe was successfully challenged by Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 16 of 23 these issues, nanocolloidal silica particles bearing FITC 2 and rhodamine patterns were prepared.

Int.Thanks J. Mol. to Sci. the2020 intrinsic, 21, 9217 properties of the nanoparticles, the fluorophores are physicochemically 16stable of 23 and able to bypass the selective intestinal barrier. The resulting probe was successfully challenged by using two different mutants (eat-3 and N2), considering that first strain presents a lower pH due usingto fragmented two different mitochondria. mutants (Thus,eat-3 and thisN2 study), considering proposes a that new first and strain non-invasive presents way a lower to monitor pH due the to fragmentedpHin of C. elegans mitochondria., which would Thus, interest this study all proposesfurther studies a new using and non-invasive this specie as way model to monitor organism the [100]. pHin of C.Besides elegans, whichthe medicinal would interest applications all further described studies above, using thispH speciemonitoring as model is also organism a useful [100 tool]. in odontology.Besides The the medicinaldental microbiome applications is a symbiotic described partner above, pHwith monitoring our organism is also [101]. a usefulHowever, tool the in odontology.proliferation of The aciduric dental bacteria microbiome within is a a sturdy symbiotic biofilm partner can metabolize with our organism sugars into [101 organic]. However, acids, thewhich proliferation may lead to of demineralization aciduric bacteria withinof enamel a sturdy [102]. biofilm In order can to metabolizeprevent any sugars stubborn into damage organic to acids, the whichdentition, may such lead toas demineralizationcaries, the healthcare of enamel specialis [102].ts Incan order be toprovided prevent with any stubborna tool to damage evaluate to the dentition,production such of asorganic caries, theacids healthcare from dental specialists microb caniome be provided under withsugary a tool environment. to evaluate theFurthermore, production ofconventional organic acids procedures, from dental using microbiome pH paper under for exam sugaryple, environment.are not readily Furthermore, applicable to conventional the dental procedures,topography. usingThus, pH Sharma paper et for al. example, worked areon notan optical readily system applicable involving to the dentalfluorescein topography. 1 in order Thus, to Sharmamonitoring et al. dental worked pH [103]. on an Fluorescein optical system 1 fits involvingperfectly the fluorescein requirements1 in orderof this to use monitoring case thanks dental to its pHregulatory [103]. Fluorescein approval (FDA,1 fits perfectlyEMA) for the internal requirements use in humans. of this use The case main thanks attention to its regulatorywas focused approval on the (FDA,creation EMA) of the for fiber internal optic-based use in humans. dental Theprobe main and attention post data was processing. focused on Based the creation on a ratiometric of the fiber optic-basedcalculation using dental anionic probe andand postdianionic data processing.forms of fluorescein, Based on athis ratiometric device allowed calculation the monitoring using anionic of anddental dianionic pH across forms pilot of fluorescein, studies, whereas this device acidic allowed production the monitoring was observable of dental following pH across sucrose pilot studies, rinse whereas(Figure 22). acidic production was observable following sucrose rinse (Figure 22).

Figure 22.22.Dental Dental pH pH monitoring monitoring using usin ﬂuoresceing fluorescein ﬂuorescence fluorescence under expositionunder exposition to glucose to solutions, glucose fromsolutions, Sharma from [103 Sharma]. [103].

Obviously, thethe simplest simplest use use of fluoresceinof fluorescein pH-sensitivity pH-sensitivity should should be acidic be acidic or basic or titrations basic titrations around neutralaround domain.neutral domain. Whereas Whereas colorimetric colorimetric titrations intitrations biological in mediumbiological require medium further require calibrations further duecalibrations to irregular due nativeto irregular absorptions, native absorptions, fluorescent titrationsfluorescent are titrations convenient are toconvenient use. An exampleto use. An of titrationexample wasof titration described was by described Burton et by al. Burton in order et toal. determinein order to sarcosine determine concentration sarcosine concentration in urine [104 in]. Previouslyurine [104]. debated, Previously sarcosine debated, is nowadays sarcosine recognized is nowadays as an accurate recognized marker as of an development accurate marker of prostate of cancersdevelopment [105– 107of ].prostate In order cancers to reduce [105–107]. the cost In of order conventional to reduce techniques the cost of based conventional on chromatography, techniques fluorescentbased on chromatography, titration was established fluorescent using titration enzymatic was established transformation using of enzymatic sarcosine transformation in formaldehyde, of whichsarcosine is used in formaldehyde, as a reactant forwhich the is production used as a reacta of formicnt for acid. the Therefore,production fluorescentof formic acid. intensity Therefore, from fluoresceinfluorescent isintensity directly from proportional fluorescein to is initial directly amount proportional of sarcosine to initial (Figure amount 23). Despiteof sarcosine an expected (Figure lower23). Despite sensitivity an expected of this protocol lower insensitivity comparison of tothis chromatographic protocol in comparison systems, associating to chromatographic enzymatic transformationsystems, associating with enzymatic fluorescence transformation titration should with be fluorescence considered fortitration its effi shouldcacy, effi beciency, considered practical, for andits efficacy, flexible efficiency, procedures. practical, and flexible procedures.

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Figure 23. Titration of sarcosine using fluorescein according to Burton [104]. Figure 23. TitrationTitration of sarcosine using fluore fluoresceinscein according to Burton [[104].104]. While BCEG 6 is one of most sensitive pH fluorescent probes around the neutral domain, a new While BCEG 6 is one of most sensitive sensitive pH pH fluorescen fluorescentt probes around the neutral domain, a new fluorescein-like derivative called SNARF® ® (18, Figure 24) represents a recent breakthrough. With a fluorescein-likefluorescein-like derivativederivative called called SNARF SNARF®( 18(18, Figure, Figure 24 24)) represents represents a recent a recent breakthrough. breakthrough. With With a pKa a pKa similar to BCEG, this probe offers a double fluorescent emission signal at 580 and 640 nm for pKasimilar similar to BCEG, to BCEG, this probe this probe offers aoffers double a double fluorescent fluorescent emission emission signal at signal 580 and at 640 580 nm and for 640 one nm single for excitationone single atexcitation 488nm. Thisat 488nm. double This signal double allows signal the estimationallows the ofestimation pH using of apH ratiometricin using a ratiometric calculation one single excitation at 488nm. This double signal allows the estimationin of pHin using a ratiometric calculation without any supplementary probe. For example, Golda-VanEeckhoutte et al. recently withoutcalculation any without supplementary any supplementary probe. For example,probe. For Golda-VanEeckhoutte example, Golda-VanEeckhoutte et al. recently et showed al. recently that SNARFshowed® that18 was SNARF more® 18 effi wascient more for the efficient determination for the determination of pH in phytoplankton, of pHin in phytoplankton, than BCECF 6 [than108]. showed that SNARF® 18 was more efficient for the determinationin of pHin in phytoplankton, than BCECF 6 [108]. By using an acetate ester analog, the pH sensor is accumulating inside cells under the BCECFBy using 6 [108]. an acetate By using ester an analog, acetate theester pH analog, sensor the is pH accumulating sensor is accumulating inside cells inside under cells the activityunder the of activity of esterase. The intracellular probe concentration is based on enzymatic kinetics, leading to activityesterase. of The esterase. intracellular The intracellular probe concentration probe concen is basedtration on enzymaticis based on kinetics, enzymatic leading kinetics, to fluorescence leading to fluorescence fluctuations independently from pH variations. The intrinsic ratiometric property from® fluorescencefluctuations independentlyfluctuations independently from pH variations. from pH variations. The intrinsic The ratiometric intrinsic ratiometric property fromproperty SNARF from SNARF® overcomes this issue. Thus, this derivative offers an easy and reliable solution for SNARFovercomes® overcomes this issue. Thus,this issue. this derivative Thus, this off ersderivative an easy andoffers reliable an easy solution and forreliable monitoring solution of pH forin inmonitoring phytoplankton, of pH whichin in phytoplankton, allows observation which of cellular allows metabolic observation processes of cellular responding metabolic to the processes variation monitoring of pHin in phytoplankton, which allows observation of cellular metabolic processes responding to the variation in oceans. respondingin oceans. to the variation in oceans. O O N O N O

O O OH OH HOOC HOOC 18 18 ® FigureFigure 24.24. Chemical structure of Seminaphtharhodafluor,Seminaphtharhodafluor, SNARFSNARF® 18. Figure 24. Chemical structure of Seminaphtharhodafluor, SNARF® 18. 4. Conclusions 4. Conclusions 4. ConclusionsThis review is intended to provide an overview of recent advances in the field of fluorescein This review is intended to provide an overview of recent advances in the field of fluorescein derivatives,This review their fluorescentis intended pH-sensitive to provide properties,an overview and of applications recent advances in biological in the systems.field of fluorescein Despite its derivatives, their fluorescent pH-sensitive properties, and applications in biological systems. Despite derivatives,discovery in their 1871, fluorescent fluorescein andpH-sensitive its derivatives properties attracted, and lasting applications interest in from biol theogical scientific systems. community Despite its discovery in 1871, fluorescein and its derivatives attracted lasting interest from the scientific itsover discovery the past decades.in 1871, fluorescein The development and its ofderivatives new fluorescein attracted derivatives lasting interest remains from relevant the sincescientific the community over the past decades. The development of new fluorescein derivatives remains relevant propertiescommunity of over members the past of decades. this family The of development probes perfectly of new fit fluorescein with changes derivatives in pH around remains the relevant neutral since the properties of members of this family of probes perfectly fit with changes in pH around the sinceand physiological the properties domains. of members New of fluorescein-based this family of probes dyads perfectly for the fit creation with changes of optimized in pH oraround targeted the neutral and physiological domains. New fluorescein-based dyads for the creation of optimized or probesneutral haveand beenphysiological one of main domains. recent New focus. fluorescei Nanoparticlesn-based bearing dyads fluorescein for the creation moieties, of optimized increasing itsor targeted probes have been one of main recent focus. Nanoparticles bearing fluorescein moieties, targetedstability andprobes offering have a been broad one spectrum of main of applications,recent focus. representsNanoparticles one ofbearing the most fluorescein recent advances moieties, in increasing its stability and offering a broad spectrum of applications, represents one of the most increasingthis field. Chemicalits stability combinations and offering or a grafting broad mostlyspectrum used of FITCapplications, or FAM derivatives,represents one but of some the novelmost recent advances in this field. Chemical combinations or grafting mostly used FITC or FAM recentfluorescein advances derivatives in this have field. been Chemical described combinations in order to modulate or grafting photophysical mostly used properties, FITC or improve FAM derivatives, but some novel fluorescein derivatives have been described in order to modulate derivatives,measurements but accuracies, some novel and fluorescein expand the fieldsderivatives of application. have been Results described and findings in order gathered to modulate within photophysical properties, improve measurements accuracies, and expand the fields of application. photophysical properties, improve measurements accuracies, and expand the fields of application.

Int. J. Mol. Sci. 2020, 21, 9217 18 of 23 this review undoubtedly suggest that ﬂuorescein will continue to be an essential pH-sensitive probe in the future.

Author Contributions: Writing—original draft preparation, F.L.G.; writing—review and editing, F.L.G.; V.M.; A.G.; M.R.; D.P. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the French National Research Agency under the program CHARMMMAT ANR-11-LABX-0039-grant. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

AuNC Gold nanoclusters BCECF 20,70-bis-(2-carboxyethyl)-carboxyfluorescein BSA Bovine serum albumin CFDA Carboxyfluorescein di-acetate FA Fluoresceinamine FAM/CF Carboxyfluorescein FDA Fluorescein di-acetate FITC Fluorescein isothiocyanate FRET Förster resonance energy transfer NP Nanoparticles pHin/ex Intra/extra cellular pH SNARF Seminaphtharhodafluor

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