Abiotic Fluorescent Receptors for Detection of Pb2+ in Aqueous Media as well as Living Cells: Mechanistic and Sensing Aspects

Substantial utility of lead in numerous industries (storage, batteries, gasoline, cable manufacture, paint and ammunition), has resulted in recurring of environmental contamination. Lead being one of the highly abundant elements in nature, ranks third among the list of toxic substances. Owing to its ever-increasing demand and allied toxicity (memory loss, irritability, anemia, muscle paralysis, and mental retardation), development of novel synthetic receptors suitable to track Pb2+ in vitro as well as in vivo has been an exciting area of research in the past few decades. In this review, emphasis has been diverted to summarize and briefly discuss major contributions made towards development of fluorescent abiotic receptors capable of sensing ionic lead (Pb2+) under aqueous environments as well as in living cells.


Introduction
Lead (Pb) presents one of the most abundant among heavy metals, widely distributed and mobilized in the environment due to its extensive usage in industries like batteries, gasoline, cable, pigments and in our daily lives [1][2][3]. Low-level environmental exposure to lead is associated with numerous sources (industrial processes, water pipes, petrol, paint, solder in canned foods) and pathways (water, food, air, household dust, street dirt, soil, etc.) [4][5][6]. Owing to the vulnerability of Pb 2+ at low concentrations, occupational and environmental exposures remain a serious problem [7][8][9]. Due to public health campaigns, a decline in its commercial usage has resulted, and hence lead poisoning has become rare. In spite of this, chronic exposure to low levels of lead is still a public health issue, especially among some minorities and socioeconomically disadvantaged groups.
Various instrumentation based methods, such as X-ray absorption spectroscopy (XAS), inductively coupled plasmaoptical emission spectroscopy (ICP-OES), anodic stripping voltammetry (ASV), chromatography, atomic absorption spectrometry (AAS), inductively coupled plasma atomic emission spectrometry (ICP-AES), inductively coupled plasma mass spectrometry (ICP-MS), cold vapor AAS or flame AAS-ETA (electrothermal atomization) and photometry are used for Pb 2+ determination at ppm and ppb levels in various matrices [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29]. Although these conventional methodologies are accurate, but are allied with tedious sample pre-treatment, sophistication, time consumption, expertise, and usually lack convenience for routine analysis of bulk of samples. In addition, such instrumental basis totally lacks potential to imagine Pb 2+ in live cells or bio systems. Another most crucial drawback of these methods is that it assesses the total lead content in a given sample, and not the ionic form. To monitor lead in ionic form (Pb 2+ ) at nano gram (ng) or sub-ng level in a wide variety of systems (in vitro and/or in vivo), synthetic molecular recognition based approaches (receptors) are highly expedient [30][31]. This can be assigned to the tendency of such abiotic molecular systems to perform in situ monitoring in real time, amid high selectivity and sensitivity. Most importantly, abiotic receptors are usually reusable, easy to operate and realize high sample through output. Among wide variety of chemical receptors, fluorescent ones are highly sensitive, lower in cost, easy to access, versatile, offer sub-ng spatial resolution with submicron visualization and sub millisecond temporal resolution [32][33][34]. Most importantly; wide number of parameters can be tuned with fluorescent based methods in order to optimize the convenient readout. In addition to this, complex analytical setbacks could be removed by controlling the excitation and emission wavelengths, time window of signal collection, and polarization of the excitation beam or of the emitted light [35][36][37].
Till date, substantial progress has been made in the development of fluorescent receptors for Pb 2+ . Existing molecular systems (organic/inorganic molecules) exploit synthetic strategies as well as use of surfactants, multi-peptide self-assembled biomolecular saffolds, polymers, nano-material (functional nanoparticles), DNAzymes, etc. However in this review, our focus will be solely on the single molecule based abiotic fluorescent receptors displaying sensing of Pb 2+ both under aqueous as well living systems.

Abiotic fluorescent receptors
Chang et al. synthesized and developed a new fluorescent receptor (Figure 1), and studied it for probing Pb 2+ in living cells [38]. Highly selective fluorescent "turn on" response due to photo induced electron transfer (PET) process was achieved in presence of competing ions under simulated physiological conditions (HEPES, pH 7) [39]. The stochiometry of the complex was determined to be 1:1. Receptor 1 could detect Pb 2+ in drinking water up to 15 ppb. Thus possessing sensitivity to Environmental Protection Agency (EPA) levels of lead poisoning [40]. The binding constant (Kd) between Pb 2+ and 1 was obtained as 23 ± 4 µM. The developed receptor was successfully probed to track Pb 2+ in live cell imaging. The designed receptor could also respond to the changes in the intracellular Pb 2+ levels within mammalian cells.  [41]. Molecule 2 showed significant increase in the fluorescence intensity as a result of inhibition of PET in methanol solvent. The signal readout was established to occur via 1:1 complex formation. The detection limit for Pb 2+ was calculated to be 4.14 × 10 -4 M -1 . Most importantly proposed receptor design could penetrate living membrane without harming cells, and was further utilized for detection of Pb 2+ during live cell imaging.  (Figure 3) [42,43]. Receptor 3 offered selective recognition of Pb 2+ under physiological conditions (20 mM HEPES buffer). The fluorescent signaling was achieved through inhibition of PET process. Receptor displayed good photostability and sensitivity toward EPA limits of lead poisoning in drinking water (15 ppb). Besides, compound 3 is applicable to probe intracellular lead contents in live cells. The dissociation constant of complex between probe and Pb 2+ was fund to be 10 µM.  was calculated to be 1.63 × 10 -7 M competing metal ions in aqueous solvent system (HEPES=20 mM, pH 7.4). Under such conditions, receptor could successfully detect Pb 2+ in cultured cells. The fluorescence signaling from receptor 5 for Pb 2+ was attributed to a large Chelation enhanced Fluorescence (CHEF) [46]. The detection limit of 5 for Pb 2+ was found to be 1.5 ppb (sensitivity below US-EPA levels of lead poisoning in drinking water, 15 ppb). The Jobs studies revealed 1:1 binding mode between 5 and Pb 2+ . The association constant, K a for coordination of Pb 2+ with receptor was fund to be 6.3 x 10 4 M -1 .  Figure  6) [47]. The sensor design is based on dianthracene-cyclenbased 'clickates', and receptor could detect Pb 2+ through fluorescence enhancement signal transduction. A large CHEF effect was responsible for the fluorescence enhancement in presence of Pb 2+ . The receptor 6 could more selectively detect Pb 2+ in aqueous solvent system (HEPES=50 mM, pH 7.4). Contrary to 6, other receptor design 7 showed a relatively small Chelation-enhanced Quenching (CHEQ) effect even in presence of Cu 2+ and Hg 2+ also [48]. The stochiometry of the complexation between 6 and Pb 2+ was revealed to be 1:1. The binding constant (log K= 10.7 ± 0.5) showed a strong binding affinity of 6 for Pb 2+ . Importantly, 6-7 were reported to be the first equal-equivalent responding receptors for Pb 2+ in aqueous solution. In addition, owing to the selectivity and strong binding affinity of receptor 6, it was successfully utilized for the detection of Pb 2+ in living cells as well as in fetal calf serum.
Weng et al. devised and synthesized a dual functional molecular receptor (Figure 7) based on rhodamine backbone. Receptor displays a fluorescence "turn on" response selectively in presence of Pb 2+ [49]. Authors introduced an acyclic diethyl iminodiacetate moiety on to it for achieving binding of Pb 2+ . The designed receptor 8 could sense Pb 2+ and Cu 2+ in water through two different approaches in aqueous media, HEPES buffer with only 1% CH3CN (v/v). The mechanism of Pb 2+ recognition was proposed to proceed through rhodamine spirolactam ring-opening mechanism. The probe showed high binding affinity only toward Pb 2+ even in the presence of other biologically relevant species with low detection limits of 2.5 × 10 -7 M. A 1:1 stochiometry was demonstrated to exist between Pb 2+ and receptor during complex formation. The corresponding binding constant was 4 . Importantly, receptor successfully displayed the "turn on" response with Pb 2+ in living cells.   (Figure 8) [50]. Receptor 9 showed remarkable detection ability in a wide pH range between 4-10 in a mixed solvent (MeOH/H2O, 2:1 v/v, HEPES=100 mM, pH=7.0). The binding of Pb 2+ with 9 was proposed to increase the rigidity of compound thereby generating efficient CHEF. The complexation was proposed to be of 2:1 stochiometry between receptor and Pb 2+ . Receptor 9 was successfully utilized for the determination of Pb 2+ in aqueous solution of bovine serum albumin protein as well as in different water samples. In addition, response of 9 for Pb 2+ was successfully reversed by using disodium salt of ethylenediaminetetraacetate (EDTA). The limit of detection was calculated to be 8 nM (lower than the lead toxic level defined by US-EPA). Lan et al. synthesized cyclen-functionalized perylenediimide based receptors 10 and 11 (Figure 9). The receptor displayed a highly selective Pb 2+ sensing through fluorescence enhancement response in a neutral HEPES buffer solution. The sensing studies were examined in 10 mM HEPES/DMSO (v/ v=90/10, pH=7.2) [51]. The recognition mechanism involved 1:2 and 1:1 binding stochiometry between Pb 2+ and receptors, 10 and 11 respectively. The binding constants for such complexation were calculated to be (log K=10.6 ± 0.5 and 8.0 ± 0.3) respectively. The presence of aliphatic chain on one side of molecule instead of crown ring resulted in successful penetration of receptor 11 into the living cells. Such characteristic features made the receptor suitable for imaging applications.

Conclusions
This review summarizes the foremost progress made in the development of fluorescent receptors for detection of Pb 2+ in aqueous as well as living cells. Emphasis has been given mainly to reports diverted towards practical applications, with lower detection limits, increased selectivity, and hence reliable in terms of quality assurance. Selected references are being mentioned which will provide the ground work for an individual to know quickly the proposed and developed strategies, and their applications for Pb 2+ recognition. In this direction, some of the crucial design considerations, such as choice of fluorophore, selectivity, and mechanism of recognition are momentarily discussed.
Research till date has been conducted nearly in the academic arena, and little attention is paid towards incorporation of long wavelength fluorophores. Thus there exists a plenty of room for further advances, mostly in advancing applications towards sensitivity and selectivity in diverse environmental and biological matrices. It is these facets that require the next stage of innovation.
We hope this review inspires researchers with unique analytical, synthetic, material and physical chemistry tools, to delve into this existing area of research, and to those who have something to do with or interest in the biology or medical sciences.