This review discusses fluorescent sensors for detecting reactive oxygen species (ROS) and reactive nitrogen species (RNS), which cause inflammation, cancer, neurodegenerative diseases and brain injury [1-3], while some of the species at physiological levels are involved in redox signalling via different post-translational modifications (oxidative eustress) [4]. Though ROS/RNS have been implicated in numerous diseases, they also play a vital role in killing the invading pathogens and microbes [5, 6]. However, excessive generation of ROS/RNS causes misfolding in proteins, damage of cell membranes, amyloid fibril formation, oxidative damage of DNA in living cells by interacting with biomolecules such as proteins, lipids, enzymes, and DNA [2, 7]. Fluorescent probes have emerged as a powerful tool for studying the activities of ROS/RNS. The highly sensitive and specific nature of organic probes allows them to be used as an ideal tool to study ROS-related pathological processes. Approaches other than fluorescent probes can also be utilized to measure ROS/RNS. For example, Rowe SE et al detected ROS with the luminescent probe L-012 from Wako Chemical Corporation in vitro and in cells [8]. Herb M et al used isoluminol and horseradish pereoxidase to measure the extracellular ROS production in cultured macrophages [9]. Please note that updated consensus guidelines on the measurement of reactive oxygen species and oxidative damage in cells and in vivo have been published [10], and should always be consulted.

ROS/RNS represent a group of highly reactive species with diverse biological reactivity. Enzymes produce unique ROS species when it is in demand. When enzymes cannot produce or are genetically impaired, enzyme-dependent ROS production is inhibited [11]. Monitoring the ROS/RNS alone may not be good enough to unravel ROS/RNS implicated pathological processes. Enzyme-activated fluorescent probes can yield valuable information about the production and scavenging of ROS/RNS in a biological system. Commercial kits, such as CellROX from Thermo Fisher Scientific [12-14], are available to measure the ROS level. Ombrato L et al measured the intracellular ROS levels with CellROX Deep Red in a flow cytometer [15]. Silvestre-Roig C et al assayed reactive oxygen species from bone marrow mouse neutrophils undergoing netosis with the cell-permeant 2',7'-dichlorodihydrofluorescein diacetate [16]. Batie M et al measured ROS in HeLa cells using CellROX [17].

Organic dyes have been used extensively in the development of photoluminescent materials, solar cells and chemosensors [18, 19]. Their features include easy synthesis, cheap starting materials, and tunable photophysical properties and easy conjugation with other functional materials. Biomedical diagnostics have benefited from organic dyes since they have been used in drug delivery systems, proteins labels, imaging of neurofibrillary tangles and amyloid fibrils. BODIPY [20], coumarins, cyanine, fluorescein, and rhodamine have been extensively used for ROS/RNS and enzyme imaging [21-24]. For example, Wu J et al detected lipid peroxidation in cells with 5 uM BODIPY-C11 from Invitrogen followed by flow cytometric analysis [25].

Probes with low background fluorescence enable signal amplification upon recognition of a target and can be easily detected. Near-infrared light emitting probes are highly desirable as they avoid the autofluorescence from proteins and cause less damage to living cells.

Photostability is a key characteristic of fluorescence sensing. Fluorescent probes must be stable under physiological conditions, and they should not degrade during imaging due to photobleaching.

Selectivity, the ability to target an analyte of interest from its close analog, is an important parameter. Probes should exploit the unique chemical nature of ROS/RNS or enzymes. Fluorescence probes are ideally suited tools to study the ROS/RNS in-vitro as they are highly sensitive and selective. Sensitivity, the ability to exhibit strong emission change upon interaction with a target, is another important parameter. A probe should produce a strong change in photophysical properties when it interacts with an analyte of interest.

LOD is closely associated with sensitivity. If a probe is highly sensitive towards an analyte, it will have a low LOD. Probe with a low LOD is highly demanded to study enzyme impairment and genetic disorder.

A probe must have a dynamic response range to elucidate pathological processes.

Detecting hydrogen peroxide resulting from NADPH activation is an important stage to study the oxidative stress and inflammation related diseases. Hydrogen peroxide has been implicated in important cellular processes, including apoptosis, cell growth, and proliferation. Superoxide formed from NADPH is converted to hydrogen peroxide either spontaneously or by superoxide mutase. Because of its reactivity and membrane permeability, H₂O₂ is selected as an analyte among other oxidants to quantitate the extracellular release of ROS in phagocytes. Several chromogenic and fluorogenic reagents have been used to detect H₂O₂ in biological systems. Reaction of H₂O₂ with aryl boronic acid yields the corresponding phenols. Based on this mechanism aryl boronic acid-substituted fluorophores have been developed to detect the H₂O₂. Initially, Lo and Chu designed a boronate-based probe for detection of H₂O₂ [76]. Chang et al studied extensively the intercellular detection of H₂O₂ using aryl boronate-based fluorophores. Probe HP1 contains two boronic esters at the 3' and 6' position of Xanthenone scaffold [26]. HP1 generates fluorescein upon reacting with H₂O₂. It exhibits a > 500 fold higher response for H₂O₂ over similar ROS such as t-butyl hydroperoxide, O₂^-, NO, or OCl^-. Similar to HP1, probes HP2-HP6 are producing fluorescent derivatives upon reacting with H₂O₂ [27, 28]. B. Tang et al developed a near-infrared fluorescent probe (Cy-O-EB) for monitoring the glutathione/hydrogen peroxide changes in vivo based on switching on-off a five-membered ring involved in ebselen. This probe facilitates the selenium-nitrogen chemical switch that allows reversible monitoring redox status changes during apoptosis. It shows linear response between relative fluorescence intensity and concentrations of H₂O₂ from 0 to 200 µM. S. M. Cohen et al synthesized a new class of fluorescent probes for detecting endogenous H₂O₂ in biological systems [30]. Fluorescein (FBBBE) and coumarin (CBBE)-based probes were sensitive for the detection of endogenous H₂O₂ in biologically relevant micromolar concentration range. M. Kodera et al developed a metal complex (MBFh2) based fluorescent probe for the detection of hydrogen peroxide [31]. This probe comprises a non-heme iron complex and a non-fluorescent 3,7-dihydroxyphenoxazine derivative. Upon reaction with H₂O₂, the probe generates a fluorescent derivative, resorufin, via intramolecular oxidation reaction. D. H. Choi et al developed a reversible probe based on diketopyrrolopyrrole-tellurophene conjugate (DPPT3) for H₂O₂ using the redox properties of tellurium atom. After the addition of H₂O₂, the probe exhibits strong fluorescence enhancement along with a color change from light blue to light purple. The detection limit of this probe was found to be 6 µM.

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Figure 1. Fluorescent probes for hydrogen peroxide.

The superoxide anion radical, a product of a one-electron reduction of molecular oxygen, damages biological membranes and tissues directly or acts as a precursor for more ROS. Thus in the presence of H₂O₂, it generates hydroxyl radicals, while in the presence of nitric oxide, peroxynitrite is formed. SR1 has been used for the fluorometric detection of superoxidase dismutase activity [33]. Superoxide radical oxidizes the probe by deleting hydrogen to yield the compound 2-(2-pyridil)-benzothiazole which has strong fluorescence. Similar to SR1, SR2 also produces strong fluorescent derivative upon reacting with superoxide radicals [34]. Superoxide concentration can be quantified by monitoring its reaction with SR3, either by recording absorbance of the final reaction product at a wavelength of 470 nm or by measuring its fluorescence emission at 550 nm using an excitation wavelength of 470 nm [35]. Another important feature of this probe is the significant rate constant obtained between SR3 and superoxide ion (1.5 ± 0.3 X 105 M^-1 S^-1) during kinetic stopped-flow measurements, suggesting that SR3 can be used to quantitate superoxide without significant interference from other nonspecific reactions that may occur on much slower time scales. B.Tang et al designed Vanillin with 8-amino quinolone (SR4) for the detection of superoxide radical [36]. SR4 was oxidized by superoxide anion radical to form a compound with quinoid structure, which had no fluorescence, the decrease of fluorescence intensity was exploited to determine superoxide radical indirectly. They have also designed a mitochondrial-targeted two-photon fluorescent probe for imaging of superoxide ((MF-DBZH) [36]. MF-DBZH consists of a fluorene, superoxide responsive benzothiazoline, and mitochondrial reactive triphenylphosphonium moiety. Superoxide anion radical mediated dehydrogenation of benzothiazoline results expansion of π-conjugation of the probe. Thus, the sensor molecule generates a robust green emission upon reacting with superoxide radicals. B.Kalyanaraman et al designed membrane permeable hydropropidine for detecting superoxide radicals in live cells [37]. A reaction between superoxide radical and hydropropidine cation generates characteristic product, 2-hydroxypropidium. One electron-oxidizing species (peroxynitrates, peroxidases) produces corresponding propidium dication and homo and heterodimeric products upon reacting with hydropropidine cation. Y. Fujita et al developed molecular probe rhodamine b hydrazide for bioimaging of hydroxyl radicals in plant cells [38]. The probe reacts with intracellular hydroxyl radicals, resulting in its conversion to rhodamine b. Thermo Fisher Scientific MitoSOX for measuring mitochondrial ROS can be used in live-cell imaging [9, 77] or assayed through flow cytometry [78]. Ratiometric glutathione redox potential indicator, glutaredoxin 1 (GRX1)-redox-sensitive green fluorescent protein (roGFP)2 has also been used [79, 80]. The cellular production of reactive oxygen species can also be assessed through the superoxide-specific oxidation of dihydroethidium (DHE) to ethidium, which in turn binds to DNA and RNA and generates fluorescence [81, 82]. For example, Nortley R et al incubated human and mouse brain slices with 8 uM dihydroethidium for 40 minutes to visualize ROS-producing cells [82]. Superoxide can also be detected by ESR spectroscopy through the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine [83].

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Figure 2. Fluorescent probes for superoxide radicals.

The hydroxyl radical, highly ROS, is primarily responsible for cellular disorders and cytotoxic effects that can be responsible for the oxidative damage of DNA, protein or lipids. Electron spin resonance (ESR) has been widely used for detecting hydroxyl radicals. However, poor sensitivity and low spatial resolution of ESR make it difficult to detect hydroxyl radical at a single cell level. On the other hand, a fluorometric detection using fluorescent probes will be suitable for this purpose concerning the sensitivity as well as spatial resolution. Several fluorescent probes for detecting hydroxyl radical have been reported. Among them, the ones based on fluorophores linked nitroxides are one of the most successful. Presence of nitroxide radical quenches fluorescence emission of the fluorophore. The mechanism involved in this detection technique is that hydroxyl radicals react with dimethyl sulfoxide to produce methyl radicals, which then combine with nitroxide moiety of the fluorophores to increase the fluorescence intensity. Anthracene-based HR1 is highly selective for hydroxyl radical and exhibits enhanced fluorescence upon reaction with hydroxyl radicals [39]. Tang et al designed a nitroxide-linked BODIPY probe (HR2) for the detection of hydroxyl radicals [40]. HR2 rapidly responded to hydroxyl radicals, with a detection limit of 18 pM. When HR3 is converted to its corresponding O-methyl hydroxylamine by hydroxyl radical, it results in the loss of ESR signal intensity and concurrent with an enhancement in fluorescence emission [41]. Naphthalene based hydroxyl radical probe HR4 sensing mechanism is similar to HR1 [84]. This probe was able to detect the radical hydroxyl concentration down to 1 X 10^-7 mol L-1. T. Yi et al developed a ratiometric fluorescent probe (HR5) for the detection of hydroxyl radicals based on the naphthalimide derivative [43]. The absorbance of the naphthalimide based probe at 371 nm and 461 nm increased upon addition hydroxyl radicals. Similarly, the fluorescence emission intensity of the probe at 552 nm was enhanced. The detection limit of the naphthalimide based probe for hydroxyl radicals was found to be 2.0 X 10^-7. M. J. Tae et al designed a rhodamine fluorescent probe for hydroxyl radicals based on the oxidative C-H abstraction reaction of rhodamine cyclic hydrazide (HR6) [44]. Upon treatment with hydroxyl radical, rhodamine-based probe shows strong emission at 550 nm and pink-red color. The probe showed excellent selectivity for hydroxyl radical at pH 4-10.

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Figure 3. Fluorescent probes for hydroxyl radicals.

Singlet oxygen (¹O₂), an excited state of molecular oxygen, is a highly reactive species that can perturb the biological process through oxidizing the biomolecules. Although various singlet oxygen sensors have been reported, it is still challenging to detect singlet oxygen generated in the biological process because of its short lifetime. Most of the singlet oxygen reporters have been reported based on anthracene scaffold which reacts rapidly with ¹O₂ to form thermo stable endoperoxide. A decrease in the absorbance at 355 nm is used as a measure of ¹O₂ concentration. The method is not sensitive because the detection strategy is based on absorbance changes. Nagano et al designed singlet oxygen probe based on fluorescein using diphenyl anthracene as a reporter (SO1) [46]. SO1 exhibits weak fluorescence in the absence of ¹O₂ as a consequence of effective Photoelectron transfer (PET) from diphenyl anthracene to xanthene skeleton. Under the basic condition, SO1 generates strong fluorescence upon reacting with singlet oxygen. Ma et al developed probe SO2 for the detection of singlet oxygen that contains an anthracene and electron rich tetrathiafulvalene [45]. As a result of PET from tetrathiafulvalene from anthracene, probes do not have the strong fluorescence emission. Singlet oxygen-induced oxidative transformation of anthracene moiety causes inhibition of PET and probe become strong emitter. The probe SO3 has a low fluorescence quantum yield due to photoinduced electron transfer (PET) between tetrathiafulvalene and anthracene units [47]. After reaction with singlet oxygen, more than 500 fold increase in fluorescence was observed. W. Nam et al developed a series of fluorescent probes (StPBF) for the detection of singlet oxygen [48]. Asymmetrically substituted 1,3-diarylisobenzofurans based probes undergo the (2+4) cycloaddition reaction with ¹O₂, generating ratiometric fluorescent responses. E. Lemp et al designed ¹O₂ sensitive probes by combining a ¹O₂ trap plus a naphthoxazole moiety linked directly or through an unsaturated bond to the oxazole ring (FN4) [49]. Photooxidation caused by ¹O₂ produces strong new fluorescent chemical entity whose emission intensity is two orders of magnitude higher than the molecular probe.

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Figure 4. Fluorescent probes for singlet oxygen.

Peroxynitrite, a strong oxidizing agent, is formed in vivo from a diffusion-controlled reaction between superoxide and nitric oxide. Excess production of peroxynitrite is implicated in many human diseases including ischemic reperfusion injury, rheumatoid arthritis, septic shock, multiple sclerosis, atherosclerosis, stroke, and inflammatory bowel disease. K. Han et al designed a near-IR reversible fluorescent probe containing an organoselenium functional group (PN1) that can be used for detecting of peroxynitrite oxidation and reduction events under physiological conditions [50]. The fluorescence of PN1 is quenched as a result of PET between the modulator and the transducer, but oxidation of selenium prevents the PET, causing the fluorescence emission to be “turn on.” Based on the enzymatic catalytic cycle, selenoxide can be successfully and quickly reduced to selenide by GSH, at which point the probe begins to function. PN2 has been developed based on a specific reaction between ketone and peroxynitrite [51]. This probe successfully detected peroxynitrite generated in murine macrophage cells activated by phorbol 12-myristate 13-acetate (PMA), and lipopolysaccharides (LPS). Detection mechanism of PN3 is based on the oxidation of rhodamine B hydrazide, non-fluorescent substance, by peroxynitrite to give rhodamine B like fluorescence emission [52]. This probe can be used in the range of 7.5X10^-8 - 3.0 X 10^-6 mol L^-1 with a detection limit of 2.4 X 10^-8 mol L^-1. Yang et al developed peroxynitrite selective probe PN4, which contains a ketone unit linked to a dichlorofluorescein through an ether linkage. Upon reaction with peroxynitrite in situ, the ketone unit of the PN4 would generate a dioxirane that would selectively oxidize the phenyl ring to afford dienone product and, more importantly, release the fluorescent molecule [53]. In potassium phosphate buffer (pH 7.3), the addition of this probe with 15 equivalents of peroxynitrite causes 7-8 fold increase in emission intensity. P. Malingappa et al designed rhodamine b phenyl hydrazide (RBPH) bearing a spirolactam to sense peroxynitrite in vivo [85]. This probe is highly sensitive due to phenyl hydrazide which undergoes oxidative cleavage upon reacting with peroxynitrite. This probe was able to detect the peroxynitrite instantaneously with a detection limit of 1.4 nM. Y.-M. Wang et al developed a dansyl derivative (Ds-DAB) for peroxynitrite detection [54]. Ds-DAB produces dansyl acid (5-(dimethylamino)-1naphthalene sulfonic acid) and benzotriazole as major products upon reacting with peroxynitrite. The reaction of peroxynitrite with secondary amine is to form nitrosamine by direct nucleophilic nitrosation. Protonation and followed by hydrolysis yield the fluorescent dansyl acid along with benzotriazole.

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Figure 5. Fluorescent probes for peroxynitrite.

Nitric oxide (NO), generated by nitric oxide synthase, has been regarded as an important signaling molecule and plays vital roles in various physiological as well as pathological processes. Endogenous NO has facilitated multiple processes in the various physiological systems, such as the cardiovascular system, immune system, and central nervous system. NS1 can sense NO at wide pH range, and it's susceptible towards NO, which is highly desirable for the determination of NO in biological systems [55]. Moreover, its ability to detect NO released from inducible type NO synthase in a biological system also demonstrated. NS2 was used for the real-time detection of NO with a good temporal and spatial resolution [56]. NS2 was treated into activated rat aortic smooth muscle cells, where the ester bonds are hydrolyzed by intracellular esterase, generating fluorescent derivative. The fluorescence intensity in the cells is enhanced in a NO concentration-dependent manner. The detection limit of NO by NS2 was 5 nM. Lippard et al designed a highly selective and sensitive probe NS3 for NO [57]. The mechanism of turn-on emission of NS3 by NO requires NO-induced reduction of Cu(II) to Cu(I), forming NO+. Spectroscopic and product analyses of the reaction of the NS3 with NO indicated that the N-nitrosated fluorescein ligand is the species responsible for fluorescence turn-on. NS4 is operating on PET mechanism to detect NO in a biological sample. NO was successfully detected using flow injection with spectrofluorimetry and the linear range was from 1.1 X 10^-7 to 5.0 X 10^-6 [58]. In the presence of NO, vicinal diaminobenzo acridine is transformed triazole-based fluorescent derivative. Y. Xiao et al designed a ratiometric fluorescent probe (BRP-NO) for the detection of NO [86]. In their design, they have combined BODIPY and tetramethylrhodamine for efficient energy transfer cassette. BRP-NO exhibits characteristic BODIPY absorption spectra. In the presence of NO, rhodamine spirolactam is opened up and generates a fluorescent rhodamine structure. Upon excitation of the BODIPY moiety at 488 nm, probe emits at 590 nm due to fluorescence resonance energy transfer (FRET). J. Cui et al developed colorimetric chemosensor (DAN) for NO based on 1,8-naphthalimide [87]. They have incorporated o-phenylenediamine moiety to 1,8-naphthalimide scaffold to detect the NO. Upon reaction of DAN with NO, triazol form of 1,8-naphthalimide was produced, which inhibits the PET effect of 3-amino together with ICT of 4-amino in 1,8-naphthalimide. H. Liu et al designed a highly water-soluble red light emitting fluorescent probe based on BODIPY (BDP-NO) for detection nitric oxide [61]. Two o-phenylenediamine moieties are functionalized at 2,6 position of BODIPY to detect the NO. The detection mechanism is based on the reaction of the o-phenylenediamine group of the probe with NO to turn on the probe via a benzotriazole derivative. Fluorescent probes based on the fusion of NO-binding domains with fluorescent proteins have been designed [88].

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Figure 6. Fluorescent probes for nitric oxide.

Hypochlorite, a strong oxidative species, is produced by the mammalian immune system. It exists in equilibrium with hypochlorous acid at physiological pH. However, the highly reactive nature of hypochlorite is causing inflammation and neurodegenerative-related diseases. Endogenous hypochlorite is produced from the reaction of chloride and hydrogen peroxide catalyzed by the enzyme Myeloperoxidase. Many research groups have used selective oxidation of S, Se, and Te by hypochlorite to design the fluorescent sensors. Based on PET mechanism, probes HA1, HA2, and HA3 have been reported for the detection of hypochlorite [62-64]. Upon reaction with hypochlorite, S, Se, and Te are oxidized to corresponding to oxides and shutting down the PET process in probes. Thus, probes have become a strong indicator of hypochlorite. In the case of HA4, oxidation induced by hypochlorite convert the p-methoxyphenol group to a corresponding benzoquinone, increasing emission intensity [65]. X.-Q. Yu et al designed mitochondrial targeting fluorescent probes based on rhodamine (HA5 Rh-TPP) for the detection of hypochlorite [66]. Rhodamine spirolactam bearing benzoyl acetohydrazide was used to target hypochlorite. In the presence of hypochlorite, oxidation-hydrolysis of benzoyl acetohydrazide lead to the opening of the spirolactam ring. Z. Li et al developed a BODIPY derivative with hydroxymethyl group at meso position (HA6) for detection of hypochlorite [89]. Upon reaction with hypochlorite, hydroxymethyl group was transformed to a formyl group, which exhibits a highly specific and rapid turn off response. H. Jiang et al reported hypochlorite sensing probes based on selenoxide elimination reaction [68]. In the presence of hypochlorite, probes underwent the oxidation of selenium and followed by elimination of selenoxide, which produces the fluorescent coumarin derivatives.

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Figure 7. Fluorescent probes for hypochlorite.

Reversible probes can be used to monitor pathological and physiological processes in in vitro as they switch into off-on states for multiple cycles. Chang et al developed a fluorescent sensor (RP1) for imaging reversible redox cycles in living cells [69]. This probe features a reversible response to many oxidation or reduction events, a >50-fold fluorescence dynamic range, and excitation and emission profiles in the visible region to minimize cellular damage and autofluorescence. T. Nagano et al developed a reversible near-infrared fluorescence probe (RP2) for ROS based on oxidation-reduction nature of the tellurium (Te) [70]. RP2 is converted to oxidized form by various ROS, while the produced oxidized form is quickly reduced in the presence of glutathione to regenerate RP2. This redox-induced reversible NIR-fluorescence response of RP2 was allowed to detect the endogenous production of ROS.

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Figure 8. Fluorescent probes for reversible redox cycles.

Enzyme-activated fluorescent probes (EAFP) provide the opportunity to monitor the response of cancer cells to therapy in real time. The strategy involved in the enzyme sensing process is that samples being tested are treated with EAFPs which react with target enzymes and this is followed by analysis (imaging, SDS-PAGE, etc.) or purification of the target using tags attached to EAFPs. In contrast with ROS probes, EAFPs have the advantage that they can offer direct biochemical evidence about the enzymes. Bertossi R et al developed a sulfatase enzyme-responsive fluorescent probe (EAPF1) which can be used to assay the mycobacterial strain assignments based on genetically conserved mycobacterial sulfatases [71]. Moreover, this probe was used in the context of an in-gel assay to discriminate mycobacterial species and strains very rapidly (5-15 min); Mycobacterial species generated fluorescent bands that varied in intensity and position on the gel, and the pattern can be used to identify the particular mycobacterial strain. S. Nishimoto et al developed EAFP2 for hypoxia cells [72].

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Figure 9. Fluorescent probes for enzymes.

EAFP2 consists of an indolequinone unit and a rhodol fluorophore in which the indolequinone unit has a quenching nature of fluorescence as well as a hypoxia-sensitive moiety. Consequently, while fluorescence emission of rhodol in the EAFP2 conjugation was effectively suppressed, enzymatic treatment under hypoxic conditions resulted in an intense fluorescence in a hypoxia-selective manner as a result of a one-electron reductive bond dissociation of EAFP2 to release rhodol fluorophore. M. Li et al developed aminopeptidase N responsive ratiometric fluorescence probes based on 1, 8-naphthalimide (EAFP3) [73]. Aminopeptidase induced cleavage of the amide bond between amino acids and fluorophore releases 1,8-naphthalimide in a non-reversible manner to emit the green fluorescence. Kinetic assays showed that Km values for these probes were lower than that of the commercial available probe L-Leu-p-nitroanilide. It indicates that these probes may bind more strongly to APN than L-Leu-p-nitroanilide. EAFP3's practical feasibility to detect aminopeptidase was successfully proved in live cancer cells. Q. Zhu et al designed a monoamine oxidase (MAO) sensitive fluorescent probe based on fluorescein (EAFP4) [74]. In their design, the 3-aminopropoxy group, a substrate of MAOs, is connected on one phenolic hydroxyl group of fluorescein and the other is connected to different functional groups to achieve high selectivity and cell permeability. Under the reaction with MAO's, a propylamino group will be oxidized to give strong fluorescent derivative. LOD of this probe were 3.5 and 6.0 µg mL^-1 for MAO-A and MAO-B respectively. Promega MAO-Glo™ Assay utilizes a luminogenic MAO substrate, which Silva MC et al used to measure monoamine oxidase activity [90]. B. Liu et al developed a green light-emitting probe AIE-Lyso-1 for the in situ visualization of lysosomal esterase activity [75]. AIE-Lyso-1 was designed by combining aggregation-induced emission (AIE) and excited state intramolecular proton transfer (ESIPT) mechanism. After reaction with esterase to hydrolysis the acetyl groups, the corresponding derivative would be brighter by intramolecular hydrogen bond formation to facilitate the ESIPT process and aggregation. The detection limit for lysosomal esterase was found to be 2.4 X10^-3 U mL^-1. This probe has the potential for diagnosis of lysosomal esterase deficiency related diseases.

In this review, fluorescent probes based on synthetic organic fluorophores have been discussed in the context of the detection of ROS/RNS and enzymes. The enzyme activated fluorescent probes could potentially be used to image exogenous or endogenous expression. Both ROS/RNS and enzyme activated fluorescent probes will immensely help cancer, inflammation-related disease therapy.