Advances in Pyridyl-Based Fluorophores for Sensing Applications

Abstract

Fluorescence sensing plays an important role in high sensitivity, selectivity, and real-time monitoring of biological and environmentally relevant species. Several classes of fluorescent dyes (fluorophores) including rhodamine, BODIPY, 1,8-naphthalimide, and coumarin-among others−when conveniently functionalized with reactive pyridyl receptors, have emerged as effective sensors to detect and quantify chemical species with high accuracy through fluorescent imaging and spectroscopy. Among the sensing targets, monitoring of harmful chemical species, e.g., metal ions (zinc, copper, iron, mercury, cadmium, lead, etc.) and anions (chloride, fluoride, sulfide, thiocyanate, etc.) can be used to understand their physiological and pathological role in live-cells and tissues, as well as to protect human health. This chapter focuses on recent advances in the molecular design of pyridyl-substituted fluorophores, their photophysical properties, and sensing applications.

1. Introduction

Fluorescence detection techniques have become of paramount importance for monitoring biochemical and biological processes, allowing the detection and quantification of levels of chemical species in the human body and in the surrounding environment. Indeed, fluorescence sensing is a highly sensitive technique having numerous parameters that can serve as analytical information, including decay time, energy transfer, and quenching efficiency, in addition to the more conventional measurement of fluorescence intensity or polarization. Through the design of fluorescent dyes (fluorophores), it is possible to obtain molecules and materials that respond to the presence of a target analyte through changes in its physicochemical properties, presenting typically high sensibility and selectivity, quick response time and simplicity of measurement, and quantification of the analyte [1].

When combined with specific receptor units, fluorescent dyes can be extremely useful in several applications such as detection and quantification of chemical species, as well as in understanding their physiological and pathological role in cells and tissues. Receptors based on the pyridyl group are of major importance in ligand design for many of the above applications. The pyridine ring possesses a dipole moment found to be 2.22 D; therefore, it exhibits greater electronegativity as compared with the phenyl ring [2]. The pyridyl groups, such as di-(2-picolyl)amine (DPA), are excellent metal ion binding sites for the construction of fluorescent probes and can be attached to specific fluorophores or integrated into the fluorophore as part of the metal binding group, as found in quinolines. The principal fluorescence mechanisms involved in the design of the chemosensors are schematized in Figure 1 and include:

1. Photoinduced electron transfer (PET, Figure 1a): Originally proposed by A. Prasanna de Silva and coworkers [3], PET involves the use of fluorophore-spacer-receptor-type structures. The spacer is used to separate the fluorophore from the receptor at a certain distance while allowing the intramolecular electron transfer causes the interruption of the fluorophore’s fluorescence. The interaction of the analyte with the receptor causes a change in the redox potential of the receptor and the electron transfer became energetically unfavorable, which leads to the re-establishment of fluorophore’s fluorescence;

2. Intramolecular charge transfer (ICT, Figure 1b): In ICT, the fluorophore can integrate the receptor unit and is characterized by a donor and an electron acceptor group, forming a push−pull system. When the analyte, in particular charged species, interacts with the receptor, causes the strengthening or weakening of the push−pull character, leading to a change in the emission band. This is a characteristic process of ratiometric sensors [4];

3. Resonance energy transfer (FRET, Figure 1c). This mechanism involves the energy transfer from the excited state of a “donor” fluorophore to an “acceptor” fluorophore. In most cases, FRET occurs between two distinct fluorophores with overlapped emission spectrum of the “donor” and the absorption spectrum of the “acceptor” [4].

Figure 1.

Schematic representation of the main fluorescence mechanisms.

Such fluorescence mechanisms have inspired the development of new fluorescent structures and materials for the preparation of optical sensors for analyte detection in real scenarios. This chapter will focus precisely on recent advances in the molecular design of pyridyl substituted fluorophores, their photophysical properties, and sensing applications.

2. Pyridyl groups in fluorescent dyes

2.1 Rhodamine dyes

2.1.1 Molecular design

Xanthene is a heterocyclic organic compound with yellow coloration that contains two benzene rings connected through an oxygen atom and a methylene group (Figure 2). This class of dyes comprises fluorescein, rhodamine, and rhodol derivatives. Rhodamines were first produced in the late nineteenth century. They can be distinguished from other dyes by the presence of N-atoms at positions 3 and 6 of the xanthene core and they are one of the most widely used organic dyes with application in areas such as bioimaging, chemosensing, cosmetics, inks, and textiles. Rhodamine’s photophysical properties are highly dependent on the structural features and substituent groups. The periphery of the xanthene ring can be modified using several strategies and it affects the selectivity in their metal ion-induced signaling pattern. The most common derivatizations are:

1. The derivatization reaction of the carboxylic group at position 2′ of the xanthene leads to spirocyclic derivatives (closed form);

2. Modification at positions 3, 4, 5, and 6. In some cases, the alkylation at positions 3 and 6 can promote a bathochromic shift, which increases with the increase in the degree of alkylation, while in other cases, the functionalization of the amino groups of xanthene moiety can cause the total loss of fluorescence [5];

3. Modifications in the periphery of the phenyl ring at positions 4′ and/or 5′ are difficult to perform, especially when aiming to prepare isomerically pure derivatives from the sequential Friedel–Crafts reaction of an aminophenol with an asymmetric anhydride. This reaction usually led to a mixture of two isomers often difficult to separate and purify. Some of these derivatives are used for labeling molecules of interest [5];

4. Modifications at position 9 are used for the synthesis of dihydro derivatives;

5. Substitution of the xanthene heteroatom (O), for example, by Si can potentiate the absorption and emission capacity in the near-infrared region, fluorescence quantum yield, or fluorescence intensity [6].

Figure 2.

Representative examples of rhodamine dyes functionalized with pyridyl groups.

Some studies also focus on the influence of the positional isomers of the pyridine’s nitrogen (ortho-, meta-, and para-) in the sensitivity and selectivity toward analytes, such as metal ions.

2.1.2 Photophysical properties

The excellence of the photophysical properties of rhodamines is one of the main reasons for their success and wide application in several areas. Rhodamines possess high molar absorptivity coefficient (ε), long absorption and emission wavelengths (>500 nm), high fluorescence quantum yield, photostability, and good water solubility [7]. These properties are directly associated with the extensive π-conjugated systems, molecular rigidity, and presence of functional groups.

One of the most interesting features of rhodamine derivatives is the existence of two isomeric forms-spirolactone (closed form) and quinoid (opened form) (Figure 2)-with very different optical properties. The spirolactone form is colorless and nonfluorescent, while the open form is highly fluorescent and has a pink coloration. The open form owes its properties to its extended π-conjugation and the interconversion from the closed to open form allows the rhodamine derivatives to possess an off–on (turn-on) characteristic fluorescence, usually promoted by acid or specific metal ions interactions [8].

2.1.3 Sensing applications

Rhodamines are frequently used in the preparation of highly selective, fast response, and sensitive sensing tools, employed in the detection of contaminants and environmental parameters in air, water, and waste [9]. Figure 2 shows a series of selected examples of rhodamine derivatives/probes, those structural and photophysical features and sensing behaviors will be discussed in the next paragraphs.

One of the most explored rhodamine-based dyes for conjugation with pyridyl derivatives is rhodamine B hydrazide (RhoHyd, Figure 2), being the condensation product between the two moieties involving the terminal NH₂ of RhoHyd. This condensation can be achieved by attaching directly the pyridyl derivatives, through single or double bonds, or by using spacers. Uvdal and co-workers have reported probe 1, which is prepared by appending a hydroxymethyl-pyridine group to RhoHyd [10]. This probe presented specific Hg²⁺-induced color change and fluorescent enhancement in aqueous systems based on a metal binding induced ring-opening process of the spirolactam form. Probe 1 presented a limit of detection (LOD) of 15.7 × 10⁻⁹ mol dm⁻³, and the 1-Hg complex, with 1:1 stoichiometry, was formed by the coordinating atoms •O–N–N–O• from hydroxymethyl-pyridine and RhoHyd - with an association constant of 0.70 × 10⁵ mol⁻¹ dm³. The results also revealed good cell-membrane permeability and applicability of probe 1 for the detection of intracellular Hg²⁺ in living cells with almost no cytotoxicity. The simple change of linking the -NH group of RhoHyd to the hydroxyl-pyridyl derivative using a double bond, allowed the synthesis of probe 2 with even a higher association constant (1.27 × 10⁷ mol⁻¹ dm³), suitable for a pH range from 5 to 9 and capable to detect basal levels of Fe³⁺, as well as the metal ion dynamic changes in live cells at subcellular resolution [11]. The confocal laser scanning microscopy experiments showed two Fe³⁺ pools in mitochondria and endosomes/lysosomes for the first time.

In 2012, a study related to the influence of the number, nature, and size of coordinating entities was reported [12]. The synthesized probe 3 has been reported several times in literature and can be prepared from a condensation reaction between RhoHyd and 2-pyridinecarboxaldehyde [12, 13, 14]. In all cases, the probe was isolated in the ring-closed spirolactam form. Chereddy and co-workers [12] reported that in the presence of 50 × 10⁻³ mol dm⁻³ concentration of Cu²⁺ or Fe³⁺, a clear pink color solution (0.01 mol dm⁻³ Tris HCl:CH₃CN solvent mixture, pH 7.4) was observed with the concomitant appearance of a new peak at 555 nm in the absorption spectra-ring opening mechanism: 57-fold for Fe³⁺ and 53-fold for Cu²⁺. This lack of selectivity of probe 3 was overcome by using a CH₃CN/H₂O binary solution (7:3 v/v) [13]. A 1:1 stoichiometry of the 3-Cu complex was estimated with a binding constant of 2.5 × 10⁴ mol⁻¹ dm³. The UV–vis and fluorescence spectra showed an increase in the absorption maximum band and the depletion of fluorescence intensity, respectively. Besides, this complex proved to be reversible in the presence of KI. In 2017, Stalin and co-workers selected a solvent mixture of CH₃CN/H₂O (2,8, v/v) buffered with 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), pH = 7.2, to perform their studies [14]. In this case, the probe revealed sensitivity (binding constant of 4.25 × 10⁴ mol⁻¹ dm³ and LOD of 0.10 mol dm⁻³) and selectivity toward Cd²⁺ by an intramolecular FRET process induced by the binding to Cd²⁺ ion and significant spectral overlap between the absorption spectrum of 3 with the emission spectrum of the pyridine fragment. Using a hand-held UV lamp, naked-eye detection of Cd²⁺ presence was possible by observing the color change from deep magenta to bright orange. On the other hand, the in situ generated 3–Cd²⁺ complex was able to selectively sense S²⁻, with the remarkable recovery of fluorescence and UV-vis absorption spectra, by means of a displacement approach-formation of a CdS complex. This same chemosensor was later explored by our research group as a probe that could allow discrimination of light-up effects induced by metal ion chelation and variation of pH [15]. The probe synthesis was optimized using a solvent-free approach under microwave irradiation and a crystal suitable for single-crystal X-ray diffraction (SCXRD) proved the isolation of the probe in the expected spirolactam form. The fluorescence properties of the probe were studied and determined that: i) the probe was fluorescent in the pH range 2−4 (max. Value at pH 3; pK_a1 = 2.98 and pK_a2 = 2.89); ii) the presence of Fe³⁺ triggered the opening of the spirolactam ring with the formation of a new and intense fluorescence band at 586 nm (dimethylsulfoxide (DMSO) and DMSO:H₂O (9,1, v/v); and iii) the determined 1:2 (metal: dye) was consistent with the formation of the Fe³⁺ complex with the tridentate probe.

A similar dye with a longer spacer (4) was synthesized via one-pot Schiff base reaction of rhodamine B, ethylenediamine, and isonicotinaldehyde and was characterized by SCXRD, where suitable orange-brown crystals were obtained by slow solvent evaporation methods [16]. The Fe³⁺ recognition mechanism, established by density-functional theory (DFT), involved a PET mechanism between the rhodamine core and pyridine and proved to be reversible by UV/Photoluminescence (PL) and time-resolved photoluminescence (TRPL) in the presence of EDTA (ethylenediaminetetraacetic acid tetrasodium salt). A LOD estimated value of 102.3 × 10⁻⁹ mol dm⁻³ was reported as well as the probe sensitivity in the pH range from 3 to 10 and cellular imaging studies revealed real applicability of the probe in Fe³⁺ detection. Another work showed its selectivity toward SCN⁻ in human embryonic kidney cells, including fluorescence and “naked-eye” detection of nanomolar concentration of the analyte [17]. DFT calculations suggested the existence of non-covalent interactions and long-range electrostatic forces between the analyte and the probe, and a comparison using a fluorescein derivative as a model compound allowed to establish a “lock” and “key” mechanism for the analyte sensing. The probe was used successfully in the quantification of SCN⁻ in real samples such as sheep blood serum and cow milk.

Kan and co-workers reported two probes (5 and 6) prepared by a two-step approach: i) reaction of rhodamine B with ethylenediamine followed by ii) reaction with 2-picolinic acid and pyridine-2,6-dicarbonyl dichloride, respectively [18]. Both probes exhibited excellent selectivity and sensitivity for Fe³⁺ in EtOH/H₂O solution (3:1, v/v, HEPES, 0.5 × 10⁻³ mol dm⁻³, pH = 7.33) and living human breast adenocarcinoma (MCF-7) cells. Probe 5 presented a 1:1 binding stoichiometry and a lower LOD (0.067 × 10⁻⁶ mol dm⁻³) than probe 6, which presented a 1:2 binding stoichiometry. Both were successfully applied in the detection of trace amounts of Fe³⁺ up to 200 × 10⁻⁶ mol dm⁻³- in tap water and real mud water with good recovery efficiency, and once again the turn-on mechanism was observed. Probe 6, reported by Li and co-workers [19], operates under two different Fe³⁺ recognition mechanisms based on the solvent used: i) in acetonitrile (CH₃CN), a Fe³⁺ complex is formed causing the quenching of fluorescence, and ii) in phosphate-buffered saline (PBS), hydrolysis occurred leading to the ring opening and a 75-fold increase in fluorescence intensity, with the formation of dipicolinic acid - a result supported by mass spectrometry (MS). The fluorescent imaging of living cell revealed low cytotoxicity, cell viability, and that the probe could penetrate cell membranes.

Some probes are designed to incorporate selected receptor groups, such as sulfur derivatives-thiourea, sulfonyl, or thiol groups. Sarkar and co-workers [20] prepared a rhodamine-linked pyridyl thiourea probe (7) with distinct cation and anion binding sites. The probe was capable of selectively detecting different analytes: i) in CH₃CN, fluoride was detected by changes in the emission at 518 nm; ii) Al³⁺ detection occurred at concentrations of approximately 10⁻⁵ mol dm⁻³ by colorimetric and ratiometric responses; iii) in aqueous CH₃CN mixture, the probe was capable to distinguish between Al³⁺ and Cu²⁺-possessing higher sensitivity and selectivity toward Al³⁺ at the emission wavelength 558 nm; and iv) the probe could also detect Ag⁺ through an increase in the emission intensity at 416 nm, with a LOD of 2.09 × 10⁻⁴ mol dm⁻³. In 2020, probe 8 based on the linkage of rhodamine B and pyridine-3-sulfonyl chloride was reported [21]. This dye resulted from the combination of an electron-donor group (amino group) for fluorescence and sensitivity enhancement and a recognition group with good ion coordination ability (pyridine-3-sulfonyl chloride). 8 presented fast (280 s) and dual response-absorption and fluorescence-upon addition of Al³⁺, with a LOD of 14.23 × 10⁻⁹ mol dm⁻³. The 8-Al complex could further be used as a sensor for fluoride by fluorescence intensity decrease. The probe was used successfully in the detection of low Al³⁺ concentrations in natural water, living cells, zebrafish, and plant tissues. Other derivatizations can also be used, for example, Duan and co-workers reported a rhodamine-thiospirolactam probe prepared from the reaction of thiooxorhodamine B hydrazide and 2-pyridinecarboxaldehyde (9) [22]. In this case, the N-atom of the spirolactam was replaced by an S-atom, while the carbonyl was converted into a hydrazone linked to the pyridine derivative. The probe presented a color change from colorless to pink and a fluorescence intensity enhancement in the presence of Hg²⁺ even at the ppb level. The thioether probe was compared to its thioamide congener and revealed higher selectivity for Hg²⁺, which was related to its poorer coordination affinity for other interference metal ions.

In 2015, Fu and co-workers prepared three novel rhodamine-triazine aminopyridine derivatives, in which the N-atom of the aminopyridine ring was placed in ortho-, meta-, or para- position [23]. These probes’ design took into account the aminopyridine water solubility and the excellent reactivity properties of cyanuric chloride as the connecting bridge. The ortho-derivative (10) presented higher selectivity for Fe³⁺ in water-over other metal ions and amino acids-due to its more suitable space coordination sphere. In the presence of Fe^3+, a new absorption band appeared (562 nm) and the emission intensity at 582 nm increased up to 35-fold, along with the change in color from colorless to pink. This probe possessed a LOD for Fe³⁺ of 4.1 × 10⁻⁸ mol dm⁻³ and an association constant of 1.49 × 10⁶ mol⁻¹ dm³, being a 1:1 stoichiometric structure supported by Job’s plot and MS. Furthermore, the probe revealed to be: i) capable to detect Fe³⁺ in environmental samples using paper-made test kits impregnated with the probe; ii) capable to detect up to 0.3 mol dm⁻³ Fe³⁺ in tap water, and iii) suitable for imaging intracellular Fe³⁺ in HL-7702 cells. Another example was the report from Bhattacharya and co-workers where the three isomers of the pyridine’s nitrogen were compared toward Cu²⁺ and Hg²⁺ sensitivity using probe 11a as the common point [24]. The dye with the pyridine nitrogen at ortho-position was the only isomer that presented selective colorimetric detection of Cu²⁺-in water (pH 7.4), in a medium containing bovine serum albumin and blood serum. The detection mechanism was based on the formation of the Cu²⁺ complex (2:1 stoichiometry) involving the carbonyl oxygen, amido nitrogen, and pyridine nitrogen (see Figure 2). The analytes were detected in different water sources at the ppb level, and the probes could be used for rapid on-site detection by the preparation of portable test strips. A very similar probe to the ortho-derivative 11a, with a methyl substituent in the N-atom attached to the pyridine (11b) was prepared through the condensation of RhoHyd and 2-acetylpyridine and applied in the selective detection of Cu²⁺, again by a turn-on process due to spirolactam ring opening [25]. The probe was suitable for Cu²⁺ detection within a concentration range from 2.0 to 20.0 × 10⁻⁶ mol dm⁻³ and presented a LOD of 0.21 × 10⁻⁶ mol dm⁻³ - a value lower than the maximum concentration established by the World Health Organization (WHO).

In 2014, a study based on the influence of different substituents attached to the N-atom of the xanthene at positions 3 and 6 was reported [26]. The probes were prepared from the condensation of rhodamine 6G with 2-aminoethylpyridine (12), followed by a subsequent nucleophilic substitution (SN₂) reaction with 9-bromomethyl anthracene (13) or with 1-bromo-octane (14). All the probes revealed chromogenic and fluorogenic turn-on spectral responses in the presence of Pb(II) ions and 13 also presented the lowest LOD and reversibility due to the perturbation of the combined PET inhibition and FRET processes associated with its bifluorophoric nature. In the same report, a derivative with two ethyl-substituents at both N-atoms attached to the xanthene core is presented as a selective sensor of Hg²⁺ with a dual mode spectral amplification. The authors have concluded that changes in selectivity and signaling pattern are associated with induced amine rigidity in xanthene. Other positions of the rhodamine dye can also be used for structural modifications. For example, probe 15 based on the modification of the 3′-position of the benzolate in the rhodamine with an amino pyridine substituent was prepared [27]. This probe exhibited high selectivity and sensitivity toward Ni²⁺, possessed a LOD down to 4.6 ppb, and the chelation of the metal ion involved the carboxylate group of the rhodamine moiety and the N-atom of the pyridine moiety.

Another strategy for the design of rhodamine-pyridine probes is by conjugation with other dyes or aromatic rings. In 2016, a rhodamine derivative incorporating a 2-[(1H-pyrrol-2-ylmethyl)-(2-pyridinyl-methyl) amino]- tripodal receptor was reported (16) and used as a sensor for the detection of accumulated Co(II) in Hybanthus enneaspermus plant [28]. The addition of Co(II) to a solution of 16 in THF/H₂O (8:2 v/v, 0.01 mol dm⁻³ HEPES, pH 7.4) promoted the spirolactam ring opening with the formation of a 2:1 complex (probe:Co) with LOD of 4.3 × 10⁻⁹ mol dm⁻³. The complex was reversible in the presence of EDTA and the probe proved to be suitable for in-situ detection of Co(II) in a pH range from 5 to 10. Xu and co-workers designed a multidentate dye 17 with rhodamine-triazole-pyridine units for the detection of Sn²⁺ [29]. In the presence of Sn²⁺ the probe, in CH₃CN:H₂O (99:1, v/v), showed changes in color, from colorless to orange, and in the absorption and fluorescence spectra—appearance of a new band at 560 nm and intensity enhancement at 587 nm, respectively. The recognition mechanism was studied by several techniques and confirmed the formation of stable 5-member or 6-member rings between Sn²⁺ and 17 (1:1 complex).

In 2019, our work group designed a series of pyridyl analogs of rosamines (rhodamine derivatives lacking the carboxylic group at position 2′ of the benzenic ring) and studied the influence of solvent and charge on their photophysical properties [30]. It was found that the structural variation involving the position of the N-atom in the pyridine did not influence the absorption and fluorescence properties of dyes, the same could not be said about the charge - the introduction of a positive charge at the N-atom (18) in the pyridinium analog promoted a significant bathochromic shift in the absorption and fluorescence quenching, both effects associated to d-PET mechanism. Probe 18 showed extinction of color and fluorescence in the presence of EtOH, the same being true for the uncharged derivative. The detection of EtOH was more pronounced for 18 and resulted from the nucleophilic addition of the ethoxide ion to the central 9-position of the xanthene core, the process was reversible with the addition of a weak acid (trifluoroacetic acid, TFA). Two years later, Xie and co-workers reported a pyridine-Si-rhodamine-based probe (19) that could be used as a lysosomal-targeted near-infrared (NIR) fluorescent probe for reactive oxygen species (ROS) [31]. Probe 19 possessed a pyridine-Si-rhodamine moiety as a fluorescent reporter and a borophenylic acid moiety as a reacting group. The probe exhibited good water solubility and, in the presence of hydrogen peroxide (H₂O₂), revealed a significant enhancement in the fluorescence intensity at 680 nm, which could be attributed to the solvent effect and ICT. The response toward other ROS was also evaluated and revealed that the fluorescence enhancement would occur in the order: hypochlorous acid (HClO, 5-fold) < H₂O₂ (14-fold) < hydroxyl radical (•OH, 16-fold). The recognition mechanism, proved by high-resolution MS, indicated the oxidation of phenylboronic acid and was similar with that of phenylboronic acid-based ROS probes. 19 revealed sensitivity to detect ROS in cancer cells and in tumor-bearing mouse xenograft models, being indicative of the probe’s applicability to the study of lysosomal cell death.

Many other examples of rhodamine-pyridyl derivatives can be found in literature for selectively sensing several analytes, such as picric acid [32].

2.2 BODIPY dyes

2.2.1 Molecular design

The 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, also known as boron dipyrrin or boron dipyrromethene (BODIPY), is one of the most popular families of organic fluorophores that have found numerous practical applications as fluorescence probes for bioimaging and sensing, laser dyes, and as bright pigments in various fields of technology, e.g. in solar fuel generation, in photovoltaic devices, in light-harvesting arrays for antenna systems, and in photocatalysis, among others. Their main credits are due to the excellent photophysical and spectral properties they possess, including insensitivity to solvent polarity and pH, high photostability with high absorption coefficients, and high fluorescence quantum yield, allowing them to be excited at rather long wavelength (~500 nm) [33, 34]. When compared with rhodamine and fluorescein dyes, the BODIPY fluorophore is smaller and more insensitive to environmental conditions, while Förster radius R_o has about the same value [35].

From the molecular design point of view, the BODIPY dye (Figure 3) can be functionalized at the pyrrolic ring, at the central meso-position, and at the boron atom [36]. By introducing substituent groups into the different positions of the BODIPY scaffold, as well as by varying the conjugation length with appropriate spacer or π-linker, the spectroscopic, (photo)physical, and chemical characteristics of the final molecule can be fine-tuned according to the intended application.

Figure 3.

Representative examples of BODIPY dyes functionalized with pyridyl and polypyridyl groups.

2.2.2 Photophysical properties

The BODIPY typically exhibits a weak absorption band in 350–450 nm region and a strong absorption band in the 450–580 nm, corresponding to π → π* transitions. It shows a strong fluorescence spectrum in the visible region and the fluorescence quantum yields are typically higher than 0.8. The BODIPY dye shows fluorescence lifetime (s) on nanosecond scale, which is independent of the excitation and emission wavelengths, suggesting simple emission from the locally excited state [35].

2.2.3 Sensing applications

Several BODIPY derivatives having very attractive photophysical properties and photochemical stability have found very useful applications as fluorescent platforms for sensing applications. The introduction of the pyridyl or polypyridyl groups at the periphery of the BODIPY core can lead to a large variety of chemosensors for detecting anions, cations, amino acids, etc. Figure 3 shows a series of selected examples of these BOPIDY derivatives with different sensing behaviors.

Y. Wu and co-workers [37] reported one of the most notable examples of meso-functionalized BODIPY for detection of Zn²⁺ by preparing the probe (20) through the combination of 1,3,5,7-tetramethyl-boron dipyrromethene with the DPA receptor. This probe works in an aqueous solution, it exhibits λ_abs/λ_em = 491/509 nm with Ф_F = 0.077 and has the advantage of being very selective for Zn²⁺, with the fluorescence emission of zinc-binding being pH independent in the range of pH 3−10.

Another example of a meso-functionalized BODIPY comes from the X. You’s group [38], through the preparation of the BODIPY derivative (21) containing a tridentate 2-N-(2-pyridylmethyl)amino-phenol ligand. The probe, which is almost nonfluorescent because of the PET quenching process from the meso-electron-donating substituent to the excited BODIPY unit, upon addition of Hg²⁺, the fluorescence intensity increased remarkably, showing a very high sensitivity (detection limit ≤2 ppb), a rapid response time (≤5 seconds), and high selectivity for Hg²⁺ over other metal cations.

Developed by G. T. Sfrazzetto and co-workers [39], probe 22 contains a tetratia-aza-crown receptor and an alkyl-pyridinium moiety to get water solubility and selectivity for target mitochondria. The probe was found to be highly selective to detect Cu⁺ in solution and in living cells through an emission quenching response, which is attributed to the PET process between the BODIPY core and the Cu⁺ chelated tetrathia-aza crown receptor.

Through a benzyl pyridinium cleavable unit at meso position of BODIPY, probe 23 was developed for detection of HOCl. Upon addition of HOCl, it exhibits a fast-responsive rate and a dramatic red fluorescence increase (λ_em = 614 nm, 170-fold) with high selectivity and sensitivity (LOD = 60 × 10⁻⁹ mol dm⁻³) [40].

The dyad 24 featuring two BODIPY fluorophores linked by a N,N′-(pyridine-2,6-diylbis(methylene))-dianiline substituent showed a highly selective fluorescent turn-on response in the presence of Hg²⁺ [41]. Through theoretical calculations, it was possible to predict the photophysical properties of the 24-Hg²⁺ complex, both the reductive and oxidative PETs are prohibited, thus justifying its strong fluorescence emission observed experimentally.

In a similar approach, dyad 25 consisting of a 2,2′-(ethane-1,2-diylbis(oxy))bis(N,N-bis(pyridine-2-ylmethyl)-aniline receptor, which was covalently connected through aromatic amides with two BODIPY fluorophores, was found to selectively detect both Hg²⁺ and Cd²⁺ ions [42]. In this case, the receptor has been designed to effectively wrap around a metal ion and, at the same time, make the dye water-soluble for its operation in aqueous environment. This probe exhibited LOD values of 38 × 10⁻⁹ mol dm⁻³ for aqueous Hg²⁺ and a 77 × 10⁻⁹ mol dm⁻³ for aqueous Cd²⁺.

The functionalization of the BODIPY dye at the δ-position has been also highly explored and one of the most representative examples is the distyryl-substituted BODIPY dye 26 developed by E. U. Akkaya’s group [43]. This compound contains the DPA receptor combined with six triethylene glycol (TEG) groups to provide water solubility. It presents λ_abs /λ_em = 680/726 nm and the gradual addition of Zn²⁺ ions to this compound results in a blueshift to 625 nm with a concomitant increase in emission intensity, in aqueous solutions, resulting from the coordination of Zn(II) ions to the DPA receptor (see Figure 3). Other similar BODIPY probes for Zn²⁺ include: (i) the BODIPY functionalized with a N,N-di-(2-picolyl)ethylenediamine (DPEN) receptor [44], which can detect Zn²⁺ cation through fluorescence enhancement and also detect pyrophosphate anion through a fluorescence quenching and (ii) the BODIPY featuring a DPEN and a methyl acetate group for monitoring and quantifying levels of Zn²⁺ in living cells and detecting intracellular Zn²⁺ released from intracellular metalloproteins [45].

Probe 27 is another interesting example of a BODIPY functionalized at δ-position with a 1-(furan-2-yl)-N-((pyridin-2-yl)methyl)methanamine group [46]. The probe is almost nonfluorescent, but upon addition of Cu²⁺, a large bathochromic shift in the absorption and fluorescence spectra and induced fluorescence amplification at ∼600 nm was observed, showing great potential for imaging and sensing of Cu²⁺ in living cells. On the other hand, by modifying BODIPY with a 4-aminostyryl group [47], a probe for Cu²⁺ with a large Stokes shift, high photostability, and high quantum yield was obtained for monitoring in vivo Cu²⁺ imaging in live mice.

2.3 1,8-Naphthalimide dyes

2.3.1 Molecular design

1,8-Naphthalimide (NI) core is considered as one of the most versatile fluorophore units due to its synthetic versatility and unique photophysical properties. The aromatic NI core, an electron acceptor, along with the N-imide site can be easily modified (Figure 4), which allows the introduction of an enormous variety of structural units and functional groups in the main core. Regarding their photophysical properties, naphthalimide structures are strongly influenced by the nature of the substituent. The functionalization at C-4 with donor moieties, such as amine or hydroxyl groups, induces a red-shifted ICT band with marked solvatochromic effect. These characteristics encourage the use of NI as probes as the changes in spectroscopic properties, such as absorption, dichroism, and fluorescence can all be used to monitor their binding to different analytes. The NI and its derivatives have immense potential in the development of new fluorescent probes, laser dyes, optoelectronic materials, and bioimaging but also present high antitumor and antiviral activities [48].

Figure 4.

Representative examples of 1,8-naphthalimide dyes functionalized with pyridyl groups.

2.3.2 Photophysical properties

The spectroscopic properties of 1,8-naphthalimides are strongly dependent on the C-4 substituent group. To increase the fluorescent quantum yield, the substituent group at the 4-position should be an electron-donating group. Other features that contribute for 1,8-naphthalimides extensive use are related with their extraordinary thermal and chemical stability.

2.3.3 Sensing applications

Several NI derivatives have been used as fluorescent platforms in distinct sensing applications. The introduction of one or more pyridyl units in the periphery of the NI core led to a large variety of chemosensors for different analytes. In Figure 4, a series of NI derivatives having different structural characteristics and sensing behaviors are shown.

The first example, probe 28, uses a 1,8-naphthalimide unit as a receptor, and 1-(2-pyridyl)piperazine as a receptor to design a turn-on fluorescent probe for Fe³⁺ [46]. In this example, the sensor was achieved by mild reaction and simple post process and found to have excellent selectivity and sensitivity to Fe³⁺. The chelation with Fe³⁺ over other cations caused a 15.8-fold fluorescence enhancement, which could be explained by the fact that the N-atoms in pyridine and piperazine moieties provided the binding sites for Fe³⁺ and enhancing the fluorescence by blocking the PET process. The maximal fluorescence intensity was linearly proportional to the Fe³⁺ concentration (60–140 × 10⁻⁶ mol dm⁻³), a LOD of 81 × 10⁻⁹ mol dm⁻³ and the probe worked in a pH range of 5.0–8.0. A 1:1 complex was formed reversibly between the probe and Fe³⁺. Moreover, tests were performed with other metal cations and it was verified a negligible influence on the fluorescence spectrum of probe 28/Fe³⁺. This result indicated that probe 28 had good anti-interference ability and was a reliable high sensitivity fluorescent probe for Fe³⁺.

In the next example, a fluorescent ion-imprinted probe (FIIS) for rapid and convenient detection of Cu²⁺ ions was fabricated. Probe 29 represents a fluorescent polymerizable ligand, 4-(2-aminomethyl)pyridine-N-allylnaphthalimide [49]. The design of this probe took into consideration to increase the fluorescence quantum yield of 1,8-naphthalimides and at the same time introduced a chelating unit, the substituent group at the 4-position should be an electron-donating group. Taking these considerations into account, 2-aminomethyl pyridine was chosen as the C-4 substituent group in the synthesis of this fluorescent functional monomer (F). The FIIS was prepared by surface functionalization of PVDF membrane with a thin layer of Cu²⁺ ion-imprinted polymer using the synthesized ligand as the fluorescent functional monomer. The intensity of fluorescence emission of FIIS decreased linearly with the increase of Cu²⁺ ions concentration in the range of 0-70.0 × 10⁻⁶ mol dm⁻³. The results of selectivity tests indicated that FIIS had a high specific recognition ability for Cu²⁺ and its application in the determination of Cu²⁺ in real water samples revealed a LOD for Cu²⁺ ions in the range of 0.11–0.14 × 10⁻⁶ mol dm⁻³.

The third example, probe 30, was published by Wu and co-workers and presented the successful design and synthesis of a simple fluorescent and colorimetric probe [50]. The design involved the functionalization in the N-imide site but also in positions 3 and 4 of the NI core. This probe exhibited an excellent selective fluorescence response for the simultaneous detection of Zn²⁺ and Al³⁺ with a single excitation wavelength in the same solvent system. The LOD of probe 30 for Zn²⁺ and Al³⁺ were 14.4 × 10⁻⁶ and 74.0 × 10⁻⁶ mol dm⁻³, respectively. In addition, the solution of probe 30 with Zn²⁺ exhibited a dramatic color change from bright green to bright blue, light, and dark blue with Al³⁺, which could be easily detected by naked eye under UV.

The fourth example represents a ratiometric and selective fluorescent probe (31) for Cu²⁺. This probe was easily synthesized by conjugating 2-(aminomethyl)pyridine and N-butyl-4-bromo-5-nitro-1,8-naphthalimide [51]. The design and synthesis took into consideration was the mechanism of ICT since this mechanism had been widely exploited for cation sensing. Another aspect that was taken into consideration was the use of a tetradentate receptor site with nitrogen and pyridyl donors since there were strong pieces of evidence that these receptors were very useful for binding Cu²⁺ ions [52]. The capture of Cu²⁺ by the receptor resulted in the reduction of the electron-donating ability of the two amino groups of the naphthalene ring; thus, the receptor showed a 50 nm blue shift of fluorescence emission and provided high selectivity for Cu²⁺ over other heavy and first transition metal ions. The fluorescence of the probe at 525 nm remains unaffected between pH 4.7−13. This probe presents high sensitivity and selectivity toward Cu²⁺ ions, allows the detection of Cu²⁺ ratiometrically, and forms a 1:1 complex (see Figure 4) [51].

In the last example, Lee and co-workers designed a pyrene-appended naphthalimide, probe 32, as a ratiometric fluorescence probe that can detect Zn²⁺ ion in physiological conditions [53]. In this approach, the pyrene unit acts as a reference fluorophore emitting an unaffected fluorescence intensity for Zn²⁺ and the naphthalimide-dipicolylamine moiety acts as a Zn²⁺ sensing unit providing a fluorescence change based on a PET mechanism. This probe displayed a ratiometric change in the fluorescent intensities at 385 and 530 nm, which corresponds to the emissions of pyrene and naphthalimide units, for Zn²⁺ allowing for a precise quantitative analysis. This ratiometric change could be also visualized by a fluorescent color change from blue to green. The probe presented a rapid detection of Zn²⁺ ions in a 1:1 ratio with high sensitivity, even in the presence of other competitive metal ions, and with a LOD of 10.5 × 10⁻⁹ mol dm⁻³. Moreover, this probe was able to detect Zn²⁺ ions in the pH range of 4–11 and it could be efficiently recycled by treating it with EDTA.

2.4 Coumarin dyes

2.4.1 Molecular design

Coumarins are a large family of compounds containing the 2H-chromen-2-one motif. This platform has been widely used in the design of fluorescent chemosensors because of its small size, excellent biocompatibility, strong and stable fluorescence emission, and good structural flexibility. Hence, this scaffold is an important unit in the development of fluorescent chemosensors with different applications in different fields, such as molecular recognition, molecular imaging, bioorganic, analytical, and materials chemistry, as well as in biology and medical science. Most coumarins were synthesized or designed de novo rather than via post-functionalization of the coumarin skeleton. The synthetic transformation of coumarins into other heterocyclic compounds and larger fused heterocycles with a coumarin moiety has also been developed [54]. In addition, the benzene subunit of the coumarin ring system is not as reactive as the unsubstituted benzene ring, while the 3 and 4 positions are highly reactive.

2.4.2 Photophysical properties

Although the coumarin unit exhibits a very weak fluorescence, the introduction of proper substituents originates new coumarin derivatives with significant fluorescence in the visible light range. Hundreds of coumarin dyes have been developed as active components due to their improved quantum yields, tunable emission wavelengths, and the fact that they are very responsive to the polarity of their microenvironments. The previously published results on the photophysical properties of fluorescent coumarins have revealed important structure-property relationships, which have also been important to guide the design of fluorescent chemosensors.

2.4.3 Sensing applications

A vast variety of coumarin-derived fluorescent chemosensors were built by combining the coumarin moiety with other functional receptors. Herein we present a series of coumarin derivatives in which the receptor is a pyridyl moiety (Figure 5).

Figure 5.

Representative examples of coumarin dyes functionalized with pyridyl groups.

L. Wang and co-workers reported two ratiometric probes, 33a and 33b, to be employed in the quantitative determination of pH value in acidic pH zone. The development of such ratiometric probes, employing the ratio of two emissions at different wavelengths as the detecting signal, allows for more accurate analysis [55]. The reported probes were strategically designed with a 7-diethylamino-coumarin moiety as the fluorophore and pyridine as the receptor. Both probes exhibited a fluorescence ratiometric response to acidic pH. For probe 33a, upon decreasing the pH from 8.35 to 2.36, the fluorescence emission spectra exhibited a large red shift from 529 to 616 nm, and the emission ratio changed dramatically from 8.58 to 0.09. The emission ratio also displayed good linearity with the pH in the range of 4.0 to 6.5, which is valuable for the quantitative determination of pH values in this acidic pH window. Similar behavior was observed for probe 33b. By performing some NMR experiments and theoretical calculations they conclude that the ratiometric response of the probes to acidic pH was due to H⁺ binding with the nitrogen of the pyridine receptor and the induced enhancement of the ICT process.

J. Portilla and co-workers reported the coumarin probe (34) bearing a 7-hydroxy −4-methylcoumarin unit for selective detection of Mg²⁺ [56]. The design also includes the 2-pyridylhydrazone substituent as a chelating unit as well as a phenolic hydroxyl group in the fluorophore unit. In addition, the 2-pyridylhydrazone substituent has the C=N donor system that can quench the fluorescence of the fluorophore by PET process and C=N isomerization. The coordination of probe 34 to Mg²⁺ probably disrupts these processes and increases its structural rigidity producing a fluorescence enhancement. The binding mode of the complex probe 34- Mg²⁺ was studied by several spectroscopic methods and revealed the formation of a 1:1 complex (see Figure 5). The probe showed good binding ability toward Mg²⁺, low interference from Ca^2+, and a LOD of 105 × 10⁻⁹ mol dm⁻³ in ethanol-water solution.

Another example of a simple coumarin-pyridyl probe was presented by K. Xu and co-workers. The study presents two probes, but we will focus on probe 35 [57]. This probe contains C=N bond to enhance the ability of binding metal ions and contribute to extending the system conjugation. As a result, free probe displays a weak fluorescence due to C=N isomerization, but when a metal ion binds to the chelating unit, the isomerization process is disrupted and there is a fluorescence enhancement. The probe was synthesized for the sequential detection of Zn²⁺ ion and phosphate anion (PA) in DMF (dimethylformamide)/HEPES buffer medium. The binding of Zn²⁺ resulted in a pronounced fluorescence enhancement, accompanied by a noticeable color change in the naked eye. The detection limits of probe 35 toward Zn²⁺ was 1.03 × 10⁻⁷ mol dm⁻³. Probe 35-Zn²⁺ complex was then used as a probe for detecting phosphate anion, showing an off–on–off fluorescence switching response with Zn²⁺ and phosphate anion.

Probe 36 was published by K. J. Wallace, with the intention of synthesizing a planar molecule with a high degree of conjugation, which could be easily perturbed to produce a spectroscopic response, taking advantage of intramolecular hydrogen bonding [58]. The design also took into consideration that electron-withdrawing functional groups attached to a carbonyl moiety will pull electron density away from the carbon atom, consequently making this region more electrophilic and susceptible to rapid nucleophilic attack. This probe can undergo the Michael addition of cyanide at the α,β-unsaturated carbonyl, and demonstrated its selectivity for CN⁻ over 12 common anions with LOD of approximately 4 ppb [59].

The next examples were designed by F. Yu and co-workers and represent two-photon fluorescence probes, probes 37 and 38, possessing coumarin derivatives, for selective and sensitive detection of Zn²⁺ [60]. Both probes exhibited excellent analytical properties for Zn²⁺ detection including rapid response, high sensitivity, and good selectivity. In each probe, the coumarin moiety acts as a fluorophore and 2-hydrazinopyridine unit as a metal ion coordination site. Upon addition of Zn²⁺, solutions of the weakly emissive probes 37 or 38 become strongly fluorescent with emission at 543 nm (probe 37 ca. seven times and probe 38 ca. four times) in HEPES buffer. In addition, the two-photon properties of these coumarin derivatives make them applicable to detect Zn²⁺ in biological systems.

2.5 Other pyridyl-based fluorophores

In quinoline dyes, for example, the pyridyl group is part of the fluorophore, as well as an integral part of the metal-binding group. This fluorophore can be conveniently functionalized with several substituent groups for sensing essentially Zn²⁺ including 6-methoxy-(8-p-toluenesulfonamido) group [61], DPA group [62], among others. Other pyridyl-based fluorophores include: (i) the triazole-pyridine system featuring two pyridine receptors, which behave as an interesting ICT chemosensor for cations and anions [63]; (ii) the 7-nitrobenzo-2-oxo-1,3-diazole dye comprising two pyridines for Zn(II) detection [64] and (iii) the carbazole derivative integrating pyridine units exhibiting fluorescence switching by acid/base exposing [65].

3. Conclusions

The optical properties of dyes as well as their sensitivity and selectivity toward analytes are highly dependent not only on the fluorophore backbone but also on its substituents and the solvent in which the detection occurs.

Throughout the chapter, several classes of fluorescent dyes-rhodamines, BODIPY’s, 1,8-naphthalimides, and coumarins-functionalized with reactive pyridyl receptors were examined. The presented examples explored the strategies used for structural optimization to improve sensing abilities using the principal fluorescence sensing mechanisms. In coming years, new developments are expected toward better sensitivity and selectivity of the probes, to improve their application in the detection and quantification of important analytes in the fields of health and environment.

Acknowledgments

This work received financial support from PT national funds (FCT/MCTES, Fundação para a Ciência e a Tecnologia, and Ministério da Ciência, Tecnologia e Ensino Superior) through the project UIDB/50006/2020, UIDP/50006/2020, PTDC/QUI-QIN/28142/2017, EXPL/QUI-OUT/1554/2021 and PARSUK for the Portugal-UK Bilateral Research Fund (BRF 2022). A. M. G. Silva and A. Leite thank FCT for funding through program DL 57/2016 - Norma transitória.

Conflict of interest

The authors declare no conflict of interest.