chemosensors

Review Low Molecular Weight Fluorescent Probes (LMFPs) to Detect the Group 12 Metal Triad

Ashley D. Johnson, Rose M. Curtis and Karl J. Wallace *

The Department of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, MS 39406, USA; [email protected] (A.D.J.); [email protected] (R.M.C.) * Correspondence: [email protected]; Tel.: +1-601-266-4715



Received: 2 March 2019; Accepted: 19 April 2019; Published: 28 April 2019


Abstract: Fluorescence sensing, of d-block elements such as Cu2+, Fe3+, Fe2+, Cd2+, Hg2+, and Zn2+ has significantly increased since the beginning of the 21st century. These particular metal ions play essential roles in biological, industrial, and environmental applications, therefore, there has been a drive to measure, detect, and remediate these metal ions. We have chosen to highlight the low molecular weight fluorescent probes (LMFPs) that undergo an optical response upon coordination with the group 12 triad (Zn2+, Cd2+, and Hg2+), as these metals have similar chemical characteristics but behave differently in the environment.

Keywords: chemosensors; fluorescence; recognition; sensing; group 12 metals (zinc; cadmium; and mercury)

1. Introduction Sensors are used in every aspect of modern life; for example, many modern households have a carbon monoxide sensor or a smoke detector installed. In industry, fiber-optic sensors are used for monitoring process variables, such as temperature, pressure flow, and the level of a liquid. The environment requires sensors to monitor toxic gases and air pollution, and in clinical applications for the detection of medical conditions, i.e., Type 1 diabetes. The field of sensor technology has rapidly been expanding over the last several decades, and the field can be categorized into two broad areas (1) chemical sensors and (2) biosensors. Chemical sensors measure, monitor, and identify chemical compounds, whereas biosensors combine a biological component, for example, microorganisms, tissue, cells, organelles, nucleic acids, enzymes, or antibodies [1]. Both types of sensors have the same purpose; to measure an analyte, either qualitatively or quantitatively. Some excellent reviews have covered recent advances in biosensors, and these have been omitted in this article [2–14]. The word sensor has become more ambiguous over time, and there is no universal definition of the term chemical sensor; the term is highly debated in the scientific community. Therefore, there has been much confusion over the two broad definitions of “chemical sensors” and “biosensor.” One definition states that “chemical sensors are miniaturized devices which can deliver real-time and online information in the presence of a specific compound or ions in complex samples” [15]. This definition itself is not concise, as the term device is also unclear. Interestingly, the International Union of Pure and Applied Chemistry (IUPAC) does not have a specific definition of “chemical sensor” defined in the Compendium of Chemical Terminology: “Gold Book” [16]. The term “biosensor” is also broad, but the IUPAC does define a biosensor “as a device that uses specific biochemical reactions mediated by isolated enzymes, immune systems, tissues, organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals” [16].

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The focus of this review is to highlight how Low Molecular Weight Fluorescent Probes (LMFP), act as chemical sensors. These molecules are defined as a small organic or inorganic molecule that has a molecular weight that is no more than a 1,000 Daltons [17]. The molecules are used to monitor metal ions (chemical analytes). Sensors that contain any biological component as defined by IUPAC or containing proteins and amino acids as part of the receptor (the device) unit are not considered, as the authors argue that a molecular receptor containing a “biological” component should be considered to be a biosensor. Based on our interpretation of a chemical sensor described above, chemical sensors can be described as devices that convert the response into a measurable signal, which can either be related to the concentration of the analyte [18] or provide a binary “on” or “off” signal [15]. The signal can be a change in magnetic or electrical fields [19], resistance [20], capacitance [21], inductance [22], frequency [23] or optical response [24]. Monitoring and measuring metal ions can be carried out using analytical techniques, including atomic absorption spectroscopy (AAS) [25], atomic emission spectroscopy (AES) [26], inductively coupled plasma mass spectrometry (ICP-MS) [27], electrochemical techniques [28], surface plasmon resonance [29], and X-ray spectroscopic methods (e.g., X-ray fluorescence (XRF), X-ray photoelectron spectroscopy (XPS), and X-ray absorption spectroscopy (XAS)) [30]. Each technique has its specific advantages and disadvantages, but all suffer from similar problems, including signal-to-noise ratio, drift, sensitivity, selectivity, specificity, stability, reusability, cost of the specialized technique, the preparation of the samples, and fouling/clogging of instruments. Measuring analytes at low concentrations offers additional challenges too. For example, high sensitivity detection, and false positives can arise. As a consequence, a professionally trained technician is needed to prepare samples, operate (and maintain) instrumentation, and interpret the results. The techniques also need to be tailored to different settings (for different metal ions), and some of these techniques cannot monitor every metal ion, due to interferences. Many of the instruments required are big and bulky, and samples have to be sent back to the lab for analysis, therefore, conducting onsite analysis can be problematic. Nevertheless, these techniques have their uses and will continue to play important roles in the analysis of metal ions. There has been a push to develop analytical methods that are simple, fast, and reliable to detect metal ions in the field. Optical spectroscopy is of particular interest as it provides the basis for sensitive and inexpensive methods of detection. Optical spectroscopy comprises a group of spectroscopic analytical techniques that measure the intensity and wavelength of radiation absorbed and emitted by the sample. Luminescence is a spontaneous emission of radiation from an electronically or vibrationally excited species, not in thermal equilibrium with its environment, the resulting emission is either in the form of fluorescence or phosphorescence. Luminescence covers several techniques that involve the emission of radiation (emissive or non-emissive) by either atoms or molecules, but varies in the manner in which the emission is induced. Optical spectroscopy, for example, UV-Vis and fluorescence, are of particular interest to the sensor community. Fluorescence spectroscopy is particularly attractive, as the scientist can take advantage of manipulating different fluorescent mechanisms. The theoretical aspects of optical spectroscopy and the mechanisms that initiate a recognition event have been covered in reviews, including those by de Silva [31] and Wang [32], which are of particular relevance. The mechanisms involved include—photoinduced electron transfer (PET) [33], intramolecular charge transfer (ICT) [34], the heavy atom (metal) effect [35], the destabilization of nonemissive n-σ* excited states, metal-to-ligand charge transfer (MLCT) [36], Dexter (exchange mechanism), and Förster (coulombic) resonance energy transfer (FRET) [37], excimer/exciplex formation [38], twisted intramolecular charge transfer (TICT), electronic energy transfer (EET), aggregation-induced emission (AIE) [39], excited-state intramolecular proton transfer (ESIPT) [40], and chelation enhancement fluorescence (CHEF) [41]. These mechanisms can be tuned to prepare the fluorescent supramolecular probes for metal ion detection, monitoring, and remediation. In addition to these mechanisms, the underlying principles of molecular recognition and disclosure of analytes have also been extensively reviewed [42–52]. The review articles can be broad, covering a range of metals or probes, or could be very specific, Chemosensors 2019, 7, 22 3 of 48 highlighting the work on one particular cation, such as Fe3+ [53], a specific fluorescent mechanism, such as excimer formation [54], or a specific type of chromophore, for example, squaraine dyes [55]. Alternatively, reviews might highlight the different probes within a given period [56]. In this review, we have highlighted some of the most recent work on the development of low molecular weight fluorescent probes for the group 12 metal ions (Zn2+, Cd2+, and Hg2+). We have chosen to focus on these specific metal ions because they play significant roles in biology and the environment, for better or worse [57–66]; these analytes are optically silent, whereby an outside source is required for monitoring; to the best our knowledge this is the first time a review focuses on this specific triad. Most reviews focus on either the biologically important metal ions (Zn2+, Cu2+, Fe2+, and Fe3+) or environmentally important ions (Hg2+, Pb2+, and Cd2+), that have very little, if any, biological roles. There are a handful of reviews that cover the optical response of a number of different metal ions, but they seem to lack focus. Moreover, the year 2019 is the 150th year since Dimitri Mendeleev has been credited for the development of the periodic law, thus, focusing on a specific group is a modest way to honor the achievement by Mendeleev. The goal of this review is to highlight some of the more recent developments of low molecular weight fluorescent probes that quench the initial fluorescence signal, but the fluorescence signal is switched on upon the addition of metal ions (CHEF) for Zn2+ and Cd2+ ions, or the metal aids in a chemical reaction that produces a new compound that is fluorescent, commonly reported for Hg2+ ion molecular probes. Traditionally, the molecular sensor design has been carried out using two different approaches. The most common method is the intuition method of rational design, from years of experience being in the desired field, using a pen and paper (or chemical drawing software), followed by synthesis, and experimentation. The studies are then usually confirmed (or not) by computational methods, for example density functional theory (DFT) calculations, either to support the fluorescence mechanisms (which cannot be achieved easily by the traditional way) or to propose a binding mode between the analyte and the molecular probe, if a crystal structure cannot be obtained. As computer power is increasing and software is becoming more sophisticated the time required to run a calculation has been reduced. A new strategy in molecular probe design is to initially utilize a computational approach. The advantage of this approach is to investigate the fluorescence mechanisms first, to prepare a more efficient LMFP to improve the limit of detection for the desired analyte. This method has been used successfully for zinc sensors [67–70].

2. Chemosensors for the Group 12 Triad (Zinc, Cadmium, and Mercury) The group 12 elements, zinc, cadmium, and mercury have a valence shell configuration of [Ar]3d104s2, [Kr]4d105s2, and [Xe]4f 145d106s2, respectively. They are known as the “silent ions” as they lack any intrinsic spectroscopic or magnetic properties and, as a consequence, cannot be monitored directly. The chemistries of zinc and cadmium are similar; however, that of mercury is vastly different. All three elements are found in the +2 oxidation state, they all adopt a range of coordination numbers (2, 4, and 6 being the most common) and form tetrahedral and octahedral complexes [71]. Mercury is found predominantly in a linear (coordination 2) or tetrahedral (coordination 4) geometry [72,73]. The 3d sub-shell for zinc is somewhat polarizable, despite its high charge-to-volume ratio [74]. It is well-known that Zn2+ plays pivotal roles in biochemical processes. Over 300 zinc enzymes carry out specific functions and zinc also plays a structural role, helping to stabilize protein binding domains, for example in zinc fingers [75]. Cadmium and mercury have no known biological functions, although there have been reports that cadmium has a limited biological function in marine diatoms [76]. Lane et al. reported that the growth of the diatom Thalassiosira Weissflogii is enhanced when there is a limited supply of zinc, suggesting that under extreme conditions, the presence of cadmium can be biologically beneficial [77,78]. All three group 12 elements form alloys with the other d-block elements [79]. For example, brass is a well-known alloy of copper in which the weight ratio between the metal alloys (and Sn and Pb) gives rise to a specific type of brass [80]. Common brass, used to make the Lincoln cent, is made up of 63% Chemosensors 2019, 7, 22 4 of 48 copper and 37% zinc. Since 1982, a copper-coated zinc alloy has been used instead due to increases in the copper price. Amalgams, such as mercury-containing alloys, are often associated with the teeth and the dentistry fields [81]. Tooth fillings consist of mercury, silver, copper, and tin, as the predominant contributors to amalgam. Cadmium alloys are known, but many have had their alloying element replaced with other metals, due to the fear of leeching into the environment, where it is readily absorbed through the roots and shoots of crops [82]. This impedes the uptake of nutrients required by the plant, disrupting photosynthesis, stunting growth, and resulting in diminished crop yield. Moreover, the bioaccumulation of cadmium in plants, increases the risk of cadmium entering the food chain. This is particularly concerning where extensive agricultural procedures occur, for example, in China [83]. It is often assumed that mercury is “bad”, due to misleading public information. Mercury is found in three broad forms, metallic mercury, inorganic mercury salts, and organic mercury and its form dictates its toxicity [84]. For example, inhaled mercury vapor will accumulate in the body mainly in the central nervous system (CNS) [85]. Ingesting metallic mercury under standard conditions poses a negligible risk of severe toxicity. The toxicity of inorganic mercury salts is dependent on their Ksp 29 1 value. For example, the Ksp for Hg I is 5.2 10 M (+1-oxidation state) and for HgS (+2-oxidation 2 2 × − − state) is 2 10 53 M 1. As salts are non-volatile, inorganic mercury salt poisoning is at a greater risk by × − − oral digestion than inhalation. Additionally, inorganic mercury salts have very low solubility in lipids, so their abilities to cross the blood-brain barrier are significantly reduced [86]. Any ingestion of an inorganic mercury salt that accumulates in the liver, is processed by bile and the kidneys, and then excreted out via urine. Mercury found in its organic form (for example methyl mercury), is the species that the general public refers to as “mercury poising” and is highly toxic [87]. Organic mercurials are formed from elemental mercury through micro-organisms and make their way up the food chain, because methyl mercury is very soluble in lipids and bioaccumulates readily [88]. As mercury is one of the oldest known elements it has extensively been studied and the toxicity of its three forms is well understood. Even though mercury can enter the environment readily, gone are the days of being “mad as a hatter”—mercury poising is rare in the developed world. As there are various ways in which the group 12 triad of metals can enter a biological or environmental surroundings, all three metal ions are toxic above specific concentrations. For example, the World Health Organization (WHO) and the Environmental Protection Agency (EPA) limit for these metal ions in water should not exceed 0.005, 0.001 and 5.0 mg L 1 for Cd2+, Hg2+, and Zn2+ ions, · − respectively [89,90].

2.1. Zinc Probes Zinc is arguably the most frequently studied d-block metal, due to its biological importance [91]. The Zn2+ ions are predominantly located within the confines of proteins and enzymes where they are essential to many biological functions [92]. The human body contains approximately 2–3 g of zinc; the amount found in the blood is kept constant (12–16 µM), however, higher concentrations (mM) are localized in many parts of the body, for example in the breast [93], prostate [94], brain [95], and pancreas [96]. The association constants range from tightly bound (log K > 11) and moderately bound (log K = 7–9) to loosely bound (log K < 7), and affect the bioavailability of Zn2+ ions. Loosely bound zinc is responsible for the dark side of Zn2+ ions. For example, free zinc has an active role in neuronal injury [93], and there have since been numerous reports on the acute toxicity of free zinc and its link to neurodegenerative effects in Alzheimer’s disease [97] and amyotrophic lateral sclerosis (Lou Gehrig’s disease) [98]. Low molecular weight probes that can chelate and monitor Zn2+ ions by an optical response are abound [99–108]. The subject matter appears in many authoritative reviews from investigators that have made seminal contributions in this field [109–123]. Whenever a molecular probe is being designed for a particular analyte, three crucial factors need to be considered; (1) high sensitivity (detection limit), (2) high specificity (distinguishing among metal ions), and (3) high selectivity (for the specific ion pool under investigation). Moreover, it might not be necessary to monitor metal ions at low concentrations, but rather over a dynamic range of Chemosensors 2019, 7, 22 5 of 48 concentrations [124,125]. For example, the concentration of intracellular “free” zinc can be as low as 1 nM, whereby, Zn2+ ions found in synaptic vesicles in the presynaptic bouton of a neuron can be as high as 300 mM [126–128]. Therefore, developing molecular sensors that can work within this range is very challenging. An elegant example of a molecular receptor that can monitor Zn2+ ions over a wide concentration has been prepared by Zhu et al. [129]. The salicylaldehyde motif is a popular organic functional group, often incorporated into molecular probes [130], as it readily undergoes Schiff base reactions, and therefore, introduces the Lewis basic sites Chemosensors 2019, 7, x 5 of 50 to bind metal ions in a stable chelating ring system. Compound 1, a bis(bidentate) iminophenol ligand was synthesizedThe bysalicylaldehyde Chakraborty motif et al. is (Figure a popular1A) organic [ 131], throughfunctional the group, condensation often incorporated of salicaldehyde into and 4-aminobenzylaminemolecular probes [130], in ethanol. as it readily The undergoes X-ray crystal Schiff structure base reactions, shows and that therefore, two resonance-assisted introduces the hydrogenLewis bonding basic (RAHB)sites to bind interactions metal ions in are a present.stable chelating Therefore, ring system. in order Compound for compound 1, a bis(bidentate)1 to act as a iminophenol ligand was synthesized by Chakraborty et al. (Figure 1A) [131], through the molecular probe, Et3N was added to the DMF solution to disrupt the intramolecular hydrogen bonding condensation of salicaldehyde and 4-aminobenzylamine in ethanol.2+ The X-ray crystal structure interactionshows to produce that two the resonance-assisted chelating motif. hydrogen Upon thebonding addition (RAHB) of Zninteractionsions the are absorbancepresent. Therefore, wavelength, in initially seenorder at for 321 compound nm, bathochromically 1 to act as a molecular shifted probe, to 406 Et nm.3N was Unfortunately, added to the DMF the X-raysolution structure to disrupt of the zinc complexthe intramolecular could not be hydrogen solvedas bonding the crystals interaction diffracted to produce weakly, the chelating but the motif. analogous Upon the Co addition2+ complex was solved,of Zn showing2+ ions the a absorbance 2:2 binding wavelength, ratio in the initially solid seen state. at 321 Based nm, onbathochromically the Co2+ structure shifted (ato 406 tetrahedral nm. arrangementUnfortunately, around the the Co X-ray2+ ions) structure and theof the Zn zinc2+ ions comp UV-Vislex could titration not be solved data,the as the authors crystals speculated diffracted that 2+ a 1:1 bindingweakly, stoichiometry but the analogous occurs Co in thecomplex solution was solved, phase. showing Their UV-Vis a 2:2 binding does show ratio in a pseudo-isosbesticthe solid state. 2+ 2+ 2+ Based on the Co structure (a tetrahedral arrangement around the Co ions) and the Zn ions 2+ point at 365UV-Vis nm, titration but yet thedata, binding the authors isotherm speculated saturates that a 1:1 upon binding the additionstoichiometry of two occurs equivalences in the solution of Zn ions. Therefore,phase. Their the UV-Vis author’s does interpretation show a pseudo-isosbestic of a 1:1 binding point at is 365 misleading. nm, but yet the No binding calculated isotherm binding constantssaturates were reported, upon the presumably addition of duetwo equivalences to a number of of Zn different2+ ions. Therefore, species existing the author’s in solution, interpretation rendering it difficultof to a calculate1:1 binding a is precise misleading. binding No calculated constant, binding and their constants claim were of 1:1 reported, binding presumably mode is questionable. due to a The free ligandsnumber (of1) different are essentially species existing non-fluorescent in solution, (φ re=ndering0.025 init difficult DMF) butto calculate on addition a precise of Znbinding2+ ions, a constant, and their claim of 1:1 binding mode is questionable. The free ligands (1) are essentially non- new emission band is observed at 527 nm (λex = 406 nm), Figure1B,C. This increase did not occur in the fluorescent (φ = 0.025 in DMF) but on addition of Zn2+ ions, a new emission band is observed at presence of the other metals studied (Fe3+, Co2+, Cu2+, Ni2+, Cr2+ Mn2+, Cd2+, Hg2+, and Pb2+) and 527 nm (λex = 406 nm), Figures 1B and 1C. This increase did not occur in the presence of the other the authorsmetals attributed studied this(Fe3+ to, Co a2+ CHEF, Cu2+, Ni fluorescence2+, Cr2+ Mn2+, mechanism.Cd2+, Hg2+, and Unfortunately, Pb2+) and the authors no fluorescence attributed this binding to isothermsa wereCHEF reported fluorescence and, mechanism. upon closer Unfortunately, inspection no of fluorescence their titration binding data, isotherms the fluorescence were reported spectra seemed toand, increase upon continuously.closer inspection This of couldtheir titration be due toda anta, aggregatedthe fluorescence fluorescence spectra seemed enhancement, to increase as it is reasonablecontinuously. to assume thatThis othercould be polymeric due to an species aggregated are presentfluorescence in solution, enhancement, and notas it onlyis reasonable in the discrete to 2:2 model,assume which that has other been polymeric suggested species from are their present solid-state in solution, studies. and Thisnot only might in the explain discrete why 2:2 themodel, binding which has been suggested from their solid-state studies. This might explain why the binding constants were not calculated, as they could not be easily modeled. constants were not calculated, as they could not be easily modeled.

(A)

(B) (C)

Figure 1. (A) The proposed coordination structure (based on the Co2+ crystal structure) and (B) the 2+ emission spectrumFigure 1. (A of) The compound proposed1 coordination(DMF 10 µM structure and two (based equivalents on the Co of crystal Et3N, λstructure)ex 422 nm) and and (B) the (C) bar μ λ chart highlightsemission the spectrum emission of compound intensity 1 at(DMF 527 10 nm, M uponand two the equivalents addition of of Et metal3N, exsalts 422 nm) (1.2 and equivalents). (C) bar chart highlights the emission intensity at 527 nm, upon the addition of metal salts (1.2 equivalents). Adapted from [131], with permission from Elsevier. Adapted from [131], with permission from Elsevier.

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Zhang, Zhao, and Zhou prepared chemosensor 2 (Scheme 1), by incorporating a binding motif Zhang, Zhao, and Zhou prepared chemosensor 2 (Scheme1), by incorporating a binding motif to to the dye [132]. The optical studies were carried out in a CH3CN-HEPES buffer (6:4 v/v; pH 7.4). On the dye [132]. The optical studies were carried out in a CH3CN-HEPES buffer (6:4 v/v; pH 7.4). On adding the 20 equivalents of metal salts (Na+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Cd2+, Ag+, Hg2+, Fe2+, Mg2+, adding the 20 equivalents of metal salts (Na+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Cd2+, Ag+, Hg2+, Fe2+, and Cr3+), negligible emission enhancement was seen, except for in Zn2+ ions. The emission spectra of Mg2+, and Cr3+), negligible emission enhancement was seen, except for in Zn2+ ions. The emission LMFP 2 was very weak (φ = 0.06), and was attributed to the PET quenching by the lone pair of spectra of LMFP 2 was very weak (φ = 0.06), and was attributed to the PET quenching by the lone pair electrons residing on the nitrogen atom on the imine group. A 13-fold increase in the fluorescence of electrons residing on the nitrogen atom on the imine group. A 13-fold increase in the fluorescence λ 2+ intensity was seen at 556 nm ( ex = 397 nm) when 20 equivalents of Zn2+ ions were added. The binding intensity was seen at 556 nm (λex = 397 nm) when 20 equivalents of Zn ions were added. The binding 3 −1 constant Ka for a 1:1 binding model was calculated to be approximately 3 × 103 M 1. The stoichiometry constant Ka for a 1:1 binding model was calculated to be approximately 3 10 M− . The stoichiometry was supported by a Job’s plot analysis. As Zn2+ was the only species ×that produced a fluorescent was supported by a Job’s plot analysis. As Zn2+ was the only species that produced a fluorescent signature, the authors investigated the biological imaging of compoun 2, with an array of cell lines. signature, the authors investigated the biological imaging of compoun 2, with an array of cell lines. These included adenocarcinomic human alveolar basal epithelial cells (A549), bronchial epithelial cell These included adenocarcinomic human alveolar basal epithelial cells (A549), bronchial epithelial cell (BEAS-2B), Chinese hamster ovary (CHO), Hela, and human liver cancer cells (HEP G2). Compound (BEAS-2B), Chinese hamster ovary (CHO), Hela, and human liver cancer cells (HEP G2). Compound 2 2 showed different fluorescence emission colors in the cell nucleus and in the cytoplasm. showed different fluorescence emission colors in the cell nucleus and in the cytoplasm.

2+ SchemeScheme 1.1. The proposed binding motifmotif betweenbetween compoundcompound2 2and and Zn Zn2+ ions. The The binding binding environment 1 waswas alsoalso confirmedconfirmed byby 1H NMR spectroscopy.spectroscopy.

ItIt isis well-knownwell-known thatthat UV-visibleUV-visible irradiationirradiation cancan induceinduce isomerizationisomerization ofof organicorganic andand inorganicinorganic compoundscompounds [[108,133].108,133]. This This switching mechanism has been extensively used in molecularmolecular probeprobe designdesign [[134].134]. The spyiropyran functional group is known to undergo a reversible heterolytic cleavage ofof thethe spirospiro C-OC-O bond.bond. A metastable merocyanine is then formed upon cis-trans isomerization. GiordaniGiordani etet al. al. synthesized synthesized a photochromica photochromic compound compound3 containing 3 containing a metal a metal binding binding site, Figuresite, Figure2A[ 135 2A]. They[135].utilized They utilized a methyl a pyridinylmethyl pyridinyl group, often group, used often in zinc used selective in zinc chromophores selective chromophores and incorporated and theincorporated pyridinyl the group pyridinyl onto the group spirochromene onto the spirochr indolicomene nitrogen indolic atom. nitrogen The stability atom. ofThe the stability open-closed of the formopen-closed of the spiropyrene form of the was spiropyrene affected by was the affected electron by withdrawing the electron or withdrawing electron donating or electron groups donating attached togroups it, thus, attached influencing to it, thus, the feasibility influencing of thethe openfeasibility and closed of the form.open and Electron closed donating form. Electron groups favordonating the closedgroups system, favor the whereas closed electron system, withdrawing whereas electr groupson favorwithdrawing the open groups form. Therefore,favor the theopen Giordani form. team incorporated the electron withdrawing NO group into their molecular probe design, to favor the Therefore, the Giordani team incorporated the electron2 withdrawing NO2 group into their molecular openprobe form design, and, to thus, favor making the open the Lewisform and, basic thus, sites ma availableking the to Lewis coordinate basic to sites a metal available center. to Many coordinate metal perchlorate salts were added to a 1 10 4 M CH CN solution of probe 3. The Zn(ClO ) salt produced to a metal center. Many metal perchlorate× − salts 3were added to a 1 × 10−4 M CH3CN solution4 2 of probe a distinctive color at 504 nm, from which the binding isotherm was obtained, Figure2B, although these 3. The Zn(ClO4)2 salt produced a distinctive color at 504 nm, from which the binding isotherm was dataobtained, points Figure were not2B, usedalthough to calculate these data the bindingpoints we constant.re not used A modified to calculate Benesi–Hilderbrand the binding constant. equation A was used but the regression line showed curvature so the calculated binding constant (K = 1.6 104 M 1) modified Benesi–Hilderbrand equation was used but the regression line showed curvature× so the− shouldcalculated be viewedbinding with constant a little (K skepticism. = 1.6 × 104 UponM−1) should the irradiation be viewed of thewith solution, a little skepticism. the absorbance Upon band the 1 disappeared.irradiation of the The solution, switching the was absorbance maintained band over disappeared. 100 second The period. switchingH NMR was studiesmaintained showed over that 100 1 thesecondtrans period.configuration 1H NMR was studies sustained showed when that the the probe trans coordinated configuration to thewas metal sustained ion, confirmed when the byprobeH NMR,coordinated a large to coupling the metal constant ion, confirmed was calculated by 1H NMR, (J = 16 a Hz).large coupling constant was calculated (J = 16 Hz).

Chemosensors 2019, 7, x 7 of 50

ChemosensorsChemosensors 20192019,, 77,, x 22 77 of of 50 48 (A) (B)

(A) (B)

Figure 2. (A) The proposed binding of Zn2+ and molecular probe 3 (B) The absorption binding

isotherm obtained, upon the addition of Zn(ClO4)2 to a 1 × 10−4 M of 3 in CH3CN solution and Job’s FigureFigure 2. ((AA)) TheThe proposed proposed binding binding of Znof2 +Znand2+ and molecular molecular probe probe3 (B) The3 (B absorption) The absorption binding isothermbinding plot (insert). Unfortunately, the calculated binding constant4 was not calculated using the non-linear isothermobtained, obtained, upon the additionupon the of addition Zn(ClO 4of)2 toZn(ClO a 1 410)2 −to Ma 1 of ×3 10in−4 CH M 3ofCN 3 solutionin CH3CN and solution Job’s plot and (insert). Job’s regression presented. Adapted from [135], with× permission from Elsevier. plotUnfortunately, (insert). Unfortunately, the calculated the binding calculated constant bindin wasg constant not calculated was not usingcalculated the non-linear using the non-linear regression regressionpresented. presented. Adapted from Adapted [135], from with [ permission135], with permission from Elsevier. from Elsevier. Another tridentate coordination moiety was incorporated onto a 1,8 naphthyridine scaffold where a protected L-proline was attached to position two on the naphthlyridine ring system, AnotherAnother tridentatetridentate coordination coordination moiety moiety was was incorporated incorporated onto onto a 1,8 naphthyridinea 1,8 naphthyridine scaffold scaffold where a chemosensor 4 [136]. Upon the addition of various metal salts (Li+, K+ Cd2+, Mn2+, Fe3+, Cu2+, Mg2+, Fe2+, whereprotected a protected L-proline wasL-proline attached was to positionattached two to on position the naphthlyridine two on the ring naphthlyridine system, chemosensor ring system,4 [136]. Ni2+, Co2+, Hg2+, Pb2+, Ag+, and Zn2+) in MeOH,+ + only2+ the Zn2+ 2+ ions3+ showed2+ 2a+ significant2+ 2+ fluorescence2+ 2+ chemosensorUpon the addition 4 [136]. of variousUpon the metal addition salts (Liof various,K Cd metal, Mn salts, Fe (Li+,, CuK+ Cd, Mg2+, Mn,2+ Fe, Fe3+, Ni, Cu2+,, Co Mg2+,, Hg Fe2+, , enhancement2+ + by 2a+ factor of 2.5, whereas 2+all other metal ions quenched emission. The authors NiPb2+, ,Co Ag2+, ,Hg and2+, ZnPb2+,) Ag in MeOH,+, and Zn only2+) in the MeOH, Zn ions only showed the Zn2+ a significantions showed fluorescence a significant enhancement fluorescence by suggested that the CHEF mechanism was the reason for the fluorescence enhancement observed. The enhancementa factor of 2.5, by whereas a factor all otherof 2.5, metal whereas ionsquenched all other emission.metal ions The quenched authors suggestedemission. thatThe the authors CHEF fluorescence signal was saturated after the addition of one equivalent of Zn2+ ions and the calculated suggestedmechanism that was the the CHEF reason mechanism for the fluorescence was the reason enhancement for the fluorescence observed. enhancement The fluorescence observed. signal The was Ka was approximately 6 × 104 M−1. The coordination2+ environment of compound 4 and the Zn2+ fluorescencesaturated after signal the additionwas saturated of one after equivalent the addition of Zn of ionsone equivalent and the calculated of Zn2+ ionsKa was and approximatelythe calculated complex4 was1 investigated by IR spectroscopy and TD-DFT. The2 +natural bond orbital (NBO) K6a was10 Mapproximately− . The coordination 6 × 10 environment4 M−1. The coordination of compound environment4 and the Zn of complexcompound was 4 investigated and the Zn by2+ calculations× suggested that the zinc metal ion preferred to coordinate with the two nitrogen atoms complexIR spectroscopy was investigated and TD-DFT. by TheIR naturalspectroscopy bond orbitaland TD-DFT. (NBO) calculationsThe natural suggested bond orbital that the(NBO) zinc and the oxygen atom (the negative charge density on N(2), N(3), and O(2), were calculated to be calculationsmetal ion preferred suggested to that coordinate the zinc with metal the ion two preferred nitrogen to atoms coordinate and the with oxygen the two atom nitrogen (the negative atoms −0.403, −0.63, and −0.65, respectively), the final coordination site was occupied by an MeOH molecule. andcharge the density oxygen on atom N(2), (the N(3), negative and O(2), charge were density calculated on N(2), to be N(3),0.403, and0.63, O(2), and were0.65, calculated respectively), to be Based on these theoretical calculations and, the IR study, they− suggested− that −the Zn2+ ions had a −the0.403, final −0.63, coordination and −0.65, site respectively), was occupied the by final an MeOHcoordination molecule. site Basedwas occupied on these by theoretical an MeOH calculations molecule. tetrahedral geometry. 2+ Basedand, the on IR these study, theoretical they suggested calculations that the and, Zn theions IR hadstudy, a tetrahedral they suggested geometry. that the Zn2+ ions had a tetrahedral geometry.

The naphthalene moiety has been used by KimKim to prepare two very similar chemosensors, compounds 55 andand 66 [[137,138].137,138]. TheThe hydrazidehydrazide functional functional group group was was utilized, utilized, as as it it readily readily undergoes undergoes a The naphthalene moiety has been used by Kim to prepare two very similar chemosensors, Schia Schiffff base base condensation condensation reaction, reaction, to to prepare prepare a a tridentate tridentate binding binding site site (shown (shownin inred). red). TheThe bindingbinding compounds 5 and 6 [137,138]. The 2hydrazide+ functional group was utilized, as it readily undergoes group is shownshown toto bebe idealideal forfor ZnZn2+ ions and thethe compoundscompounds coordinatedcoordinated inin aa 2:12:1 fashion.fashion. InIn bothboth a Schiff base condensation reaction, to prepare a tridentate binding site (shown in red). The binding examples, the fluorescentfluorescent mechanismmechanism was duedue toto inhibitioninhibition ofof CC=N=N isomerization and ESIPT, uponupon group is shown to2 +be ideal for Zn2+ ions and the compounds coordinated in a 2:1 fashion. In both thethe additionaddition ofof ZnZn2+ ions,ions, which which drastically drastically increased increased the the emission emission intensity, intensity, via via a a CHEF CHEF mechanism. mechanism. examples, the fluorescent mechanism9 was2 due to inhibition of C=N isomerization and ESIPT, upon TheThe calculated β values were were 1 1 × 10109 MM−2− inin a DMSO-buffer a DMSO-buffer and and DMF-buffer DMF-buffer for for compounds compounds 5 and5 and 6, the addition of Zn2+ ions, which drastically× increased the emission intensity, via a CHEF mechanism. 6respectively., respectively. The calculated β values were 1 × 109 M−2 in a DMSO-buffer and DMF-buffer for compounds 5 and 6, respectively.

ChemosensorsChemosensors2019 2019, ,7 7,, 22 x 88 of of 48 50 Chemosensors 2019, 7, x 8 of 50

The chemosensor alone had hydrogen bonding donor and acceptor groups. Kim et al. also TheThe chemosensor chemosensor alone alone had had hydrogen hydrogen bonding bonding donor donor and and acceptor acceptor groups. groups. Kim Kim et et al. al. also also investigated the anion binding ability of compound 6 in DMF [138]. Upon the addition of AcO− and investigatedinvestigated the the anion anion binding binding ability ability of of compound compound6 6in in DMF DMF [138 [138].]. Upon Upon the the addition addition of of AcO AcO− −and and F−, to ligand 6, a 15 nm red shift was observed. The AcO− anion showed the greatest hyperchromic FF−−,, toto ligand ligand6 6,, a a 15 15 nm nm red red shift shift was was observed. observed. The The AcO AcO− −anion anion showed showed the the greatest greatest hyperchromic hyperchromic shift. This was attributed to a stable chelating binding mode between compound 6 and the AcO− shift.shift. ThisThis waswas attributedattributed toto a a stable stable chelating chelating binding binding mode mode between between compound compound6 6and and the the AcO AcO− − anion. The authors proposed a binding stoichiometry of 2:1, whereby, the AcO− molecules were anion.anion. The The authors authors proposed proposed a binding a binding stoichiometry stoichiometry of 2:1, of whereby, 2:1, whereby, the AcO the− molecules AcO− molecules were bound were bound on the same side of the ligand, although no evidence was provided to support their claim. onbound the same on the side same of the side ligand, of the although ligand, although no evidence no evidence was provided was provided to support to theirsupport claim. their claim. A similar binding motif was used as a tridentate binding environment by Erdemir et al. who AA similar similar binding binding motif motif was was used used as as a a tridentate tridentate binding binding environment environment by by Erdemir Erdemir et et al. al. who who prepared a triphenylamine derivative 7 [139]. The absorbance spectrum showed subtle wavelength preparedprepared a a triphenylamine triphenylamine derivative derivative7 7[ 139[139].]. The The absorbance absorbance spectrum spectrum showed showed subtle subtle wavelength wavelength changes which the authors claimed to represent a 1:1 binding stoichiometry with a binding constant changeschanges which which the the authors authors claimed claimed to to represent represent a 1:1 a 1:1 binding binding stoichiometry stoichiometry with with a binding a binding constant constant of 6 6 6of 610 × 10in CHin CHCN.3CN. It isIt is reasonable reasonable to to assume assume that that a a 1:1 1:1 complex complex would would exist exist in in solution. solution. If If this this were were of× 6 × 106 in CH3 3CN. It is reasonable to assume that a 1:1 complex would exist in solution. If this were true, the Benesi–Hildebrand plot would be a straight line, yet the data reported shows a curvature in true,true, the the Benesi–Hildebrand Benesi–Hildebrand plotplot wouldwould bebe a straight line, line, yet yet the the data data reported reported shows shows a acurvature curvature in the double reciprocal plot, indicative of stoichiometries other than a 1:1. The emission spectrum inthe the double double reciprocal reciprocal plot, plot, indicative indicative of of stoichiometries other thanthan aa 1:1.1:1. TheThe emission emission spectrum spectrum 2+2+ 2+2+ showedshowed a a distinctive distinctive enhancement enhancement upon upon the the addition addition of of Cd Cd and and Zn Zn ,, whenwhen a a 1 1µ μMM CHCH3CNCN solutionsolution showed a distinctive enhancement upon the addition of Cd2+ and Zn2+, when a 1 μM CH3 3CN solution was excited at 400 nm. waswas excited excited at at 400 400 nm. nm. Interestingly,Interestingly, the theλ λem forfor CdCd22++ and ZnZn22++ waswas seen seen at 595 nm and 620620 nm,nm, respectively,respectively, aa largelarge Interestingly, the emλem for Cd2+ and Zn2+ was seen at 595 nm and 620 nm, respectively, a large enough difference to discriminate between these two metals. The optical responses between Cd2+2+ and enoughenough di differencefference to to discriminate discriminate between between these these two two metals. metals. The The optical optical responses responses between between Cd Cd2+and and Zn22++ ions are usually difficult to differentiate, due to the similarities of these ions. The authors argue ZnZn2+ ionsions areare usuallyusually didifficultfficultto todi differentiate,fferentiate, due due to to the the similarities similarities of of these these ions. ions. The The authors authors argue argue that the difference in the metals radius (88 and 109 pm for Zn2+2+ and Cd2+2+, respectively) was enough thatthat the the di differencefference in in the the metals metals radius radius (88 (88 and and 109 109 pm pm for for Zn Zn2+and and Cd Cd2+,, respectively) respectively) was was enough enough to influence the binding energy in the maximum wavelength seen. The fluorescence mechanism of toto influence influence the the binding binding energy energy in in the the maximum maximum wavelength wavelength seen. seen. The The fluorescence fluorescence mechanism mechanism of of compound 7 was due to the C=N isomerization which was inhibited upon the coordination of metal compoundcompound7 7was was due due to to the the C C=N=N isomerization isomerization which which was was inhibited inhibited upon upon the the coordination coordination of of metal metal ions, thus, the intensity enhancement was a consequence of CHEF. The fluorescence binding ions,ions, thus, thus, the the intensity intensity enhancement enhancement was awas consequence a consequence of CHEF. of The CHEF. fluorescence The fluorescence binding isotherms binding isotherms showed that the onset of curvature occurred at two equivalents of metal ion, suggesting showedisotherms that showed the onset that of the curvature onset of occurredcurvature at occu tworred equivalents at two equivalents of metal ion, of metal suggesting ion, suggesting that the that the binding constant was not 1:1. bindingthat the constant binding wasconstant not 1:1. was not 1:1.

Lippard has made a career by designing and synthesizing molecules to bind Zn2+ ions selectively. Lippard has made a career by designing and synthesizing molecules to bind Zn2+ ions selectively. OverLippard the years, has his made group a career has by incorporated designing and the synthesizing dipicoylamine molecules functional to bind group Zn2+ on ions to selectively. different Over the years, his group has incorporated the dipicoylamine functional group on to different organic organicOver the sca years,ffolds his [140 group–144 ],has including incorporated the rhodamine the dipicoylamine dyes 8a– functionalc, Figure3 [group145]. on The to rhodaminedifferent organic dye scaffolds [140–144], including the rhodamine dyes 8a–c, Figure 3 [145]. The rhodamine dye itself has itselfscaffolds has been [140–144], used extensivelyincluding the as rhodamine an off–on sensordyes 8a to–c detect, Figure a 3 number [145]. The of metalrhodamine ions [dye146]. itself These has been used extensively as an off–on sensor to detect a number of metal ions [146]. These molecules are moleculesbeen used are extensively colorless inas organican off–on solvents sensor (DMSO, to detect acetone, a number MeOH, of metal and ions CH3 [146].CN) but These under molecules simulated are colorless in organic solvents (DMSO, acetone, MeOH, and CH3CN) but under simulated physiologicalcolorless in conditionsorganic solvents (50 mM PIPES,(DMSO, 100 mMacetone, KCl pHMeOH, 7.0), the and spirolactam CH3CN) opens but uponunder the simulated addition physiological conditions (50 mM PIPES, 100 mM KCl pH 7.0), the spirolactam opens upon the physiological conditions (50 mM PIPES, 100 mM KCl pH 7.0), the spirolactam opens upon the addition of ZnCl2. The absorption band was observed at 569 nm, and the emission wavelength was addition of ZnCl2. The absorption band was observed at 569 nm, and the emission wavelength was

Chemosensors 2019, 7, 22 9 of 48 Chemosensors 2019, 7, x 9 of 50 seenof ZnCl at 5692. The nm. absorption Once the bandconcentration was observed of Zn at2+ 569ions nm, was and greater the emission than 1 mM, wavelength no further was optical seen at 2+ response569 nm. occurred. Once the The concentration φ yield was of calculated Zn ions to was be 0.22 greater for the than zinc 1 mM, complex, no further compared optical to 0.001 response for theoccurred. molecular The probeφ yield 8a. The was Zn calculated2+ ions could to be be 0.22removed for the from zinc the complex, chelating compared motif by the to0.001 addition for theof 2+ N,molecular N N’ N’-tetrakis(2-py probe 8a. Theridinylmethyl)-1,2 Zn ions could beethane removeddiamine from (TPEN) the chelating or ethyle motifnediaminetetra by the addition acetic of acidN,N (EDTA).,N0,N0-tetrakis(2-pyridinylmethyl)-1,2 Compounds 8b and 8c exhibited ethanediamine little zinc binding (TPEN) viaor ethylenediaminetetrathe lactone ring. It was acetic highly acid probable(EDTA). Compoundsthat Zn2+ ions8b didand coordinate8c exhibited with little the zinc DPA binding group, via but the the lactone Zn2+ ring.ions were It was too highly far away probable to 2+ 2+ participatethat Zn ionsin the did coordination coordinate with of the the Lewis DPA group,basic sites, but the in Znthe lactionsone were ring. too Therefore, far away tono participateemission signalin the was coordination observed. ofThis the suggested Lewis basic that sites, the lactone in the lactonegroup did ring. partake Therefore, in the nobinding emission of the signal Zn2+ ion was 2+ withobserved. 8a, but This no evidence suggested was that offered the lactone to substantiate group did this. partake The other in the biological binding ofrelevant the Zn ionsion (Na with+, K8a+, , + + 2+, Cabut2+, noand evidence Mg2+) did was not o generateffered to the substantiate equivalent this. optica Thel response. other biological The authors relevant calculated ions (Na the,K Kt value, Ca 2+ toand be 73 Mg μM,) didwhich not took generate into account the equivalent the zinc opticaldissociati response.on constant The and authors the lactam calculated formation. the Kt Despitevalue to thisbe 73calculatedµM, which value took being into at account the higher the zincconcentration dissociation range, constant in comparison and the lactam to other formation. dipicolylamine Despite molecularthis calculated probes, value it was being still atin thethe higherbiological concentration sensing range range, for neurochemical in comparison toapplications other dipicolylamine [147]. The abilitymolecular for probe probes, 8a itto was track still Zn in2+ ions the biological in Hela cells sensing was rangealso investigated for neurochemical and successfully applications showed [147]. cell The 2+ permeability.ability for probe 8a to track Zn ions in Hela cells was also investigated and successfully showed cell permeability.

FigureFigure 3. 3. LiveLive cell cell imaging imaging of of Hela Hela cells cells after after incubation incubation with with compound compound 8a8a (10(10 μµM)M) and and Hoechst Hoechst 3325833258 nuclear nuclear stain stain (blue): (blue): (A (A) )Bright-field Bright-field transmission transmission image image (B (B) )Hoechst Hoechst 33258 33258 nuclear nuclear stain stain (C(C) )fluorescence fluorescence image image without without Zn Zn2+2 +ionsions (D (D) )Cells Cells incubated incubated with with 8a8a forfor 15 15 minutes, min, washed, washed, and and then 2+ 2+ thentreated treated with with 50 µ 50M μ ZnM Znpyrithione2+ pyrithione for for 20 20 min. minutes. (E) 8a (loadedE) 8a loaded plus Znplus Zn(502+µ (50M)-supplemented μM)-supplemented cells cellstreated treated with with 80 µ 80M μ TPENM TPEN after after 10 min. 10 mins. Scale Scale bar = bar25 =µ M.25 μ AdaptedM. Adapted from from [145 ],[145], with with permission permission from fromAmerican American Chemical Chemical Society. Society.

DipodalDipodal receptors receptors have have been been used used extensively extensively in in molecular molecular recognition recognition [148]. [148]. By By incorporating incorporating pendentpendent sideside armsarms with with the appropriatethe appropriate functional functi groupsonal togroups bind ato guest bind species, a guest the thermodynamic species, the thermodynamicproperties of the properties host-guest of the complex host-guest can becomplex significantly can be significantly enhanced, as enhanced, it wraps itselfas it wraps around itself the aroundguest. the Kim guest. et al. Kim prepared et al. prepared dipodal chemosensor dipodal chemosensor9 from a piperazine9 from a piperazine group with group two with quinoline two quinolinederivatives derivatives attached [attached149]. Many [149]. dipodal Many receptors dipodal receptors are symmetrical are symmetrical but the group but the chose group to synthesize chose to + + 2+ 2+ 3+ 3+ 3+ 2+ synthesizean asymmetric an asymmetric system. On system. the addition On the of addition metal salts of metal (Na ,Ksalts, Mg(Na+, ,K Ca+, Mg,2+ Al, Ca,2+ Ga, Al3+,, Cr Ga3+,, MnCr3+, , 2+ 3+ 2+ 2+ 2+ 2+ 2+ 2+ MnFe2+, ,Fe Fe2+, Fe, Co3+, Co, Ni2+, Ni,2+ Cu, Cu,2+ Zn, Zn2+,, andand Pb Pb2+), significantsignificant optical changes were seen. The The Zn Zn2+ ionsions producedproduced a a significant significant intense broadbroad bandband centered centered around around 500 500 nm, nm, Figure Figure4B. 4B. The The authors authors claimed claimed that thatthe fluorescencethe fluorescence signal signal was turned was turned off, due off, to the due PET to quenchingthe PET quenching mechanism, mechanism, which was subsequentlywhich was 2+ subsequentlyinhibited by theinhibi coordinationted by the coordination of the Zn ions. of the Zn2+ ions. On closer inspection, the broad band was more reminiscent of an excimer band (Figure 4B). It seemed that, in solution, the two naphthalene units in the dipodal system came closer together to produce this band. It just so happened that the Zn2+ ion was the correct size to bring the units together and maximize the excimer signal. Molecular modeling calculations, however, showed that the two fluorophores were significantly further apart, suggesting that this band was not an intramolecular excimer formation. An excimer band might be formed between molecules at concentrations greater than 1 μM [38] and these studies were at 5 μM. Intermolecular excimer formation could have been ruled out by dilution experiments and intramolecular excimer formation could have been ruled out by a model compound with a single fluorophore. Other metals also showed broad bands but of

Chemosensors 2019, 7, x 10 of 50 significantly lower intensity. The emission bands were difficult to assess as the band for Zn2+ ions saturated the other metals’ optical responses. Additionally, it was claimed that the Zn2+ molecular probe was reversible by adding S2−, yet the titration conditions did not seem to be conducive to sulfide chemistry. The studies were in 10 mM bis-tris buffer at pH 7, but at this pH there was an equilibrium between H2S and HS−, and it was not until the pH reached 13 that the sulfide could exist in solution. There is no doubt that the authors showed that upon the addition of Na2S to their receptor, drastic spectral changes were observed (Figure 4C), but the Ksp for ZnS was approximately 2 × 10−25 M-1 [150]. Thus, the onset of ZnS precipitation drove the equilibrium towards the formation of the salt under Chemosensors 2019, 7, 22 10 of 48 their conditions.

(A)

(B) (C)

Figure 4. (A) Proposed binding mode of compound 9 and Zn2+ ions. (B) Fluorescence changes Figureobserved 4. for(A) compound Proposed 9binding(5 µM) uponmode the of additioncompound of di 9ff erentand Zn metal2+ ions. salts ( (3.0B) Fluorescence equivalents) inchanges bis-tris + observedbuffer (10 for mM, compound pH 7.0, λ 9ex (5= μ340M) nm)upon and the (additionC) spectral of different changes ofmetal [Zn( salts9)(NO (3.03)] equivalents)upon the addition in bis-tris of 2 bufferdifferent (10 anions mM, pH added. 7.0, λ Brex −=, 340 BzO nm)−, Cl and− ClO (C−) ,spectral CN−,F− changes,H2PO4 of−,I [Zn(−,N93)(NO−, NO3)]2+− upon, OAc −the, SCN addition− SO4 of− , HSO , pyrophosphate (PPi),- cysteine,− − and− glutathione− − (thiol− − containing− − amino− acids).− Adapted2− and− different4− anions added. Br , BzO , Cl ClO , CN , F , H2PO4 , I , N3 , NO2 , OAc , SCN SO4 , HSO4 , pyrophosphatereprinted from [(PPi),149], cysteine, with permission and glutathione from Elsevier. (thiol containing amino acids). Adapted and reprinted from [149], with permission from Elsevier. On closer inspection, the broad band was more reminiscent of an excimer band (Figure4B). It seemedPhotochromic that, in solution, sensors the have two been naphthalene used in many units inapplications. the dipodal The system development came closer of ratiometric together to 2+ fluorescentproduce this sensors band. Itutilizing just so happened a UV-Visthat switch the Znto senseion wasmetal the ions correct is a sizeunique to bring way theto prepare units together logic gates,and maximize in which a the unimolecular excimer signal. fluorescent Molecular probe modeling was designed calculations, to bind however,a metal that showed could thatbe removed the two byfluorophores a competing were ligand, significantly such as EDTA. further Photoisomerization apart, suggesting thatby UV-Vis this band could was act not as an an intramolecular input and the fluorescenceexcimer formation. emission An could excimer act bandas the might output. be Pu formed et al. betweensynthesized molecules two similar at concentrations molecular switches greater µ µ compoundsthan 1 M[ 3810] and and 11 these, which studies were were utilized at 5 asM. a Intermolecularlogic circuit based excimer on the formation truth table could that have could been be constructedruled out by from dilution four experimentsdifferent inputs and and intramolecular one output (Table excimer 1 and formation Figure could5) [151,152]. have been ruled out by a model compound with a single fluorophore. Other metals also showed broad bands but of significantly lower intensity. The emission bands were difficult to assess as the band for Zn2+ ions saturated the other metals’ optical responses. Additionally, it was claimed that the Zn2+ molecular 2 probe was reversible by adding S −, yet the titration conditions did not seem to be conducive to sulfide chemistry. The studies were in 10 mM bis-tris buffer at pH 7, but at this pH there was an equilibrium between H2S and HS−, and it was not until the pH reached 13 that the sulfide could exist in solution. There is no doubt that the authors showed that upon the addition of Na2S to their receptor, drastic 25 1 spectral changes were observed (Figure4C), but the Ksp for ZnS was approximately 2 10 M [150]. × − − Thus, the onset of ZnS precipitation drove the equilibrium towards the formation of the salt under their conditions. Photochromic sensors have been used in many applications. The development of ratiometric fluorescent sensors utilizing a UV-Vis switch to sense metal ions is a unique way to prepare logic gates, in which a unimolecular fluorescent probe was designed to bind a metal that could be removed by a competing ligand, such as EDTA. Photoisomerization by UV-Vis could act as an input and the fluorescence emission could act as the output. Pu et al. synthesized two similar molecular switches compounds 10 and 11, which were utilized as a logic circuit based on the truth table that could be constructed from four different inputs and one output (Table1 and Figure5)[151,152]. Chemosensors 2019, 7, x 10 of 50

significantly lower intensity. The emission bands were difficult to assess as the band for Zn2+ ions saturated the other metals’ optical responses. Additionally, it was claimed that the Zn2+ molecular probe was reversible by adding S2−, yet the titration conditions did not seem to be conducive to sulfide chemistry. The studies were in 10 mM bis-tris buffer at pH 7, but at this pH there was an equilibrium between H2S and HS−, and it was not until the pH reached 13 that the sulfide could exist in solution. There is no doubt that the authors showed that upon the addition of Na2S to their receptor, drastic spectral changes were observed (Figure 4C), but the Ksp for ZnS was approximately 2 × 10−25 M-1 [150]. Thus, the onset of ZnS precipitation drove the equilibrium towards the formation of the salt under their conditions.

(A)

(B) (C)

Figure 4. (A) Proposed binding mode of compound 9 and Zn2+ ions. (B) Fluorescence changes observed for compound 9 (5 μM) upon the addition of different metal salts (3.0 equivalents) in bis-tris buffer (10 mM, pH 7.0, λex = 340 nm) and (C) spectral changes of [Zn(9)(NO3)]+ upon the addition of

different anions added. Br-, BzO−, Cl− ClO−, CN−, F−, H2PO4−, I−, N3−, NO2−, OAc−, SCN− SO42−, HSO4−, pyrophosphate (PPi), cysteine, and glutathione (thiol containing amino acids). Adapted and reprinted from [149], with permission from Elsevier.

Photochromic sensors have been used in many applications. The development of ratiometric fluorescent sensors utilizing a UV-Vis switch to sense metal ions is a unique way to prepare logic gates, in which a unimolecular fluorescent probe was designed to bind a metal that could be removed by a competing ligand, such as EDTA. Photoisomerization by UV-Vis could act as an input and the fluorescence emission could act as the output. Pu et al. synthesized two similar molecular switches Chemosensorscompounds2019 ,107, 22 and 11, which were utilized as a logic circuit based on the truth table that could11 of 48 be constructed from four different inputs and one output (Table 1 and Figure 5) [151,152].

Chemosensors 2019, 7, x 11 of 50

The absorption band assigned to the open isomers is seen at 243 nm and a new band at 534 nm is seen upon irradiation in a 2.0 × 10−5 M solution of CH3CN. The purple color is a consequence of the The absorption band assigned to the open isomers is seen at 243 nm and a new band at 534 nm is rigid conjugation of alternated double bonds, across the backbone of the organic molecule, when the seen upon irradiation in a 2.0 10 5 M solution of CH CN. The purple color is a consequence of the molecule is in its closed state. ×This− was shown to be reversible3 and was supported by the difference rigid conjugation of alternated double bonds, across the backbone of the organic molecule, when the in quantum yields (φ) of 0.03 and 0.17 for the open and closed states, respectively. The spectroscopic molecule is in its closed state. This was shown to be reversible and was supported by the difference in signatures were different for the ring opening and closing, and upon interaction with Zn2+ by EDTA, quantum yields (φ) of 0.03 and 0.17 for the open and closed states, respectively. The spectroscopic dual-controlled-switching behavior of compound 10 and 11 was shown, Scheme 2. signatures were different for the ring opening and closing, and upon interaction with Zn2+ by EDTA, dual-controlled-switching behavior of compound 10 and 11 was shown, Scheme2.

2+ Scheme 2.2. TheThe molecular switching behavior of molecular probe 11 withwith thethe interactioninteraction ofof ZnZn2+ ionsions/EDTA/EDTA andand UV-VisUV-Visstimuli. stimuli.

Chemosensors 2019, 7, x 12 of 50 Chemosensors 2019, 7, 22 12 of 48 Table 1. Truth table highlighting the possible binary inputs and the corresponding digital output.

Table 1. Truth table highlighting the possible binary inputs and the corresponding digital output. Input Output* InputIn (1) In (2) In (3) In (4) Output * λem = 515 nm In (1)UV InVis (2) Zn2+ In (3)EDTA In (4) λ = 515 nm UV0 Vis0 0 Zn2+ 0 EDTA 0 em 1 0 0 0 0 0 0 0 0 0 10 01 0 00 0 0 0 00 10 1 00 0 1 0 00 00 0 11 0 0 1 01 01 0 00 1 0 0 11 10 1 00 0 0 0 11 00 0 11 0 0 0 10 01 1 00 1 1 0 00 11 0 11 0 0 1 00 10 1 01 1 0 0 01 01 1 10 1 1 0 1 1 1 0 1 1 1 0 1 0 1 1 0 1 0 1 0 1 1 0 1 0 1 1 0 00 11 1 11 1 0 0 11 11 1 11 1 0 0 * when* when the the emission emission band atat 515 515 nm nm is sevenis seven times times larger larger than thethan initial the bandinitial at ba 421nd nm, at the421 output nm, the is “on”,output this is is “on”,assigned this a is “1”. assigned Otherwise a “1”. a “0” Otherwise is assigned, a i.e.,“0” the is assigned, “off” state. i.e., the “off” state.

Figure 5. The logic circuit representing the Truth Table1 (In 1 (UV), In 2 (Vis), In 3 (Zn 2+), In 4 (EDTA)).

Figure 5. The logic circuit representing the Truth Table 1 (In 1 (UV), In 2 (Vis), In 3 (Zn2+), Zhu et al. systematically changed the linker groups and fluorophores to improve the fluorescent In 4 (EDTA)). output [153]. Zhu employed the PET switch mechanism to quench the fluorescence signal in a number of chemical fluorophores, which include the di(2-dicolyl) amino group (DPA) [154–156]. This functional Zhu et al. systematically changed the linker groups and fluorophores to improve the fluorescent group is known to bind Zn2+ ions and has been attached to anthracene [157], fluorescein [158], and output [153]. Zhu employed the PET switch mechanism to quench the fluorescence signal in a rhodamine [159] groups, directly or via short linker groups, such as the methylene group. Zhu’s number of chemical fluorophores, which include the di(2-dicolyl) amino group (DPA) [154–156]. This team utilized the triazole functionality, to “click” the DPA binding site. Their early molecular probes functional group is known to bind Zn2+ ions and has been attached to anthracene [157], fluorescein relied on fluorophores that were excited around 400 nm, but this visible wavelength region was not [158], and rhodamine [159] groups, directly or via short linker groups, such as the methylene group. conducive if the fluorophore was to be used in fluorescence microscopy for cell imaging, whereby, Zhu’s team utilized the triazole functionality, to “click” the DPA binding site. Their early molecular fluorophores that emit in the near IR region were more desirable. Therefore, they prepared molecular probes relied on fluorophores that were excited around 400 nm, but this visible wavelength region probes 12 to 14 that used the visible region to excite the fluorophore. All of their compounds were was not conducive if the fluorophore was to be used in fluorescence microscopy for cell imaging, evaluated as zinc sensors and studied for their cell imaging capabilities, and all but one was shown whereby, fluorophores that emit in the near IR region were more desirable. Therefore, they prepared to report zinc ion concentration at 50 µM in the Hela cells, by exciting the fluorophores at either molecular probes 12 to 14 that used the visible region to excite the fluorophore. All of their 405 (compound 12 and compound 13) or 488 nm (compound 14). The anilino-DPA group was found compounds were evaluated as zinc sensors and studied for their cell imaging capabilities, and all but to be the key to shift the excitation wavelength for the fluorophores and selectively coordinate the one was shown to report zinc ion concentration at 50 μM in the Hela cells, by exciting the fluorophores Zn2+ ions. at either 405 (compound 12 and compound 13) or 488 nm (compound 14). The anilino-DPA group

Chemosensors 2019, 7, x 13 of 50 Chemosensors 2019, 7, x 13 of 50 was found to be the key to shift the excitation wavelength for the fluorophores and selectively coordinateChemosensorswas found the2019 Zn,to7, 2+ 22be ions. the key to shift the excitation wavelength for the fluorophores and selectively13 of 48 coordinate the Zn2+ ions.

Another dipodal system was developed by Yang et al. who prepared a mixed fluorophore receptorAnotherAnother (coumarin-naphthalene) dipodal dipodal system system was whichwas developed developed containe byd by Yanga veryYang et elaborate al.et al. who who chelating prepared prepared motif, a mixeda mixed compound fluorophore fluorophore 15, Schemereceptorreceptor 3 (coumarin-naphthalene)[160]. (coumarin-naphthalene) The absorbance spectra which which showed contained containe a hypochromic ad verya very elaborate elaborate shift at chelating 335chelating nm and motif, motif, a hyperchromic compound compound15 15, , shiftSchemeScheme at 4303[ 1603nm, [160].]. through The The absorbance absorbance a pseudo-isosbestic spectra spectra showed showed point a at hypochromica 307hypochromic nm. The shift binding shift at at 335 335isotherm nm nm and and was a hyperchromica hyperchromicsigmoidal, whichshiftshift at is 430atindicative 430 nm, nm, through throughof cooperative a pseudo-isosbestic a pseudo-isosbestic binding. The point pointfluorescence at 307at 307 nm. nm. spectrum The The binding binding in EtOH isotherm isotherm (50 μ wasM) was showed sigmoidal, sigmoidal, a which is indicative of cooperative binding. The fluorescence spectrum in EtOH (50 µM)2+ showed a verywhich strong is indicativeemission band of cooperative at 498 nm binding. (λex = 430 The nm). fluorescence There was spectrum a preference in EtOH to the (50 Zn μM) ions, showed in a 2+ very strong emission band at 498 nm (λex = 4303+ nm).2+ There2+ 2+ was a2+ preference2+ + to2+ the Zn2+ ions,+ 3+ in comparisonvery strong to the emission other metal band ions at 498 studied nm ( (Alλex =, 430Ba ,nm). Ca ,There Cu , Hgwas Coa preference, K , Mg ,to Mn the, ZnNa2+, Crions,, in 3+ 2+ 2+ 2+ 2+ 2+ + 2+ 2+ Licomparison+, comparisonPb2+, Cd2+ to, Agto the the+ and other other Fe metal3+ metal), however, ions ions studied studied competition (Al (Al3+, Ba Ba experiments2+, ,Ca Ca2+, Cu, Cu2+ ,showed Hg, Hg2+ Co that2+Co, K +both, Mg,K 2+Cu,, MgMn2+ 2+and, ,Na Mn Fe+, 3+Cr , 3+, + 3+ + 2+ 2+ + 3+ 2+ significantlyNaLi,+, Cr Pb2+,, Li Cdquenched,2+ Pb, Ag,+ Cd andthe , fluorescenceFe Ag3+),and however, Fe intensity,), however,competition when competition addedexperiments to experimentsa [Zn( showed15)]2+ solution. showedthat both that The Cu both binding2+ and Cu Fe 3+ 3+ 2+ and Fe significantly quenched the fluorescence4 −1 intensity, when added to a [Zn(15)] solution. The constantsignificantly was calculated quenched tothe K fluorescencea = 2.0 × 10 intensity, M , using when the added Benesi–Hildebrand to a [Zn(15)]2+ solution.method Theand binding the 4 1 binding constant was calculated to Ka = 2.0 4 10 −1M− , using the Benesi–Hildebrand method and stoichiometryconstant was was calculated confirmed toby Ka aJob’s = 2.0 plot. × 10The× M fluore, usingscence the signal Benesi–Hildebrand was off in the initial method state, and as a the consequencethestoichiometry stoichiometry of the was was C=N confirmed confirmed isomerization by by a aJob’s Job’s and plot. plot. PET The Thequenching. fluore fluorescencescence On signal signaladdition was was offof o inffthe inthe theZn initial2+ initial ions, state, state,the as a 2+ fluorescenceasconsequence a consequence was ofturned of the the C=Non C= viaN isomerization isomerizationthe CHEF mechanism. and and PET The quenching.quenching. limit of detection OnOn additionaddition (LoD) of of thewas theZn calculated Zn2+ions, ions, theto the befluorescence 10fluorescence−7 M. was was turned turned on on via via the the CHEF CHEF mechanism. mechanism. The The limit limit of of detection detection (LoD) (LoD) was was calculated calculated to to be 10 7 M. be −10−7 M.

2+ SchemeScheme 3. 3. ProposedProposed binding binding motif motif between between probe probe 1515 andand Zn Zn2+ ions.ions. Scheme 3. Proposed binding motif between probe 15 and Zn2+ ions. AnotherAnother dual dual fluorescence fluorescence mechanism mechanism used used in in sensor sensor technology technology is is the the PET PET and and ICT ICT quenching quenching 2+ 16 approach,approach,Another which which dual is is inhibite inhibitedfluorescenced by by the themechanism addition used of Zn in2+ sensor ions.ions. technologyMolecular Molecular isprobe probethe PET 16, ,andScheme Scheme ICT quenching4,4, was synthesized by Wang et al. [161] based on a (1,4 diketo-3,6-diphenyl-pyrrolo[3,4-c]pyrrole derivative, synthesizedapproach, by which Wang iset al.,inhibite [161]d based by the on aaddition (1,4 diketo-3,6-diphenyl-pyrrolo[3,4- of Zn2+ ions. Molecular probec]pyrrole 16, Scheme derivative, 4, was whichwhichsynthesized is knownknown by to toWang be be a brilliant aet al.,brilliant [161] red and basedred strongly and on astrong (1,4 fluorescent diketo-3,6-diphenyl-pyrrolo[3,4-ly fluorescent dye. Additionally, dye. Additionally, this organicc]pyrrole this fluorophore organicderivative, fluorophoreis stablewhich tois heat isknown stable with toto high heatbe photostability awith brilliant high photostabilityred and and shows strong aand largely showsfluorescent Stokes a large shift, dye. Stokes which Additionally, shift, makes which it an this makes excellent organic it 16 φ anchoice excellentfluorophore in sensor choice is design. stable in sensor Compoundto heat design. with Compound highshows photostability a weak 16 shows emission and a weak shows band emission at a 630large nm band Stokes ( = at0.07), shift,630 nm in which the (φ = absence 0.07),makes it 2+ 2+ φ inof thean Zn absenceexcellentions, of but choice Zn upon2+ ions, in thesensor but addition upon design. the of Compound Znadditionions of to 16Zn compound shows2+ ions ato weak compound16, aemission blue shift16, banda blue to 560 at shift 630 nm nmto ( 560 (=φ 0.85)=nm 0.07), was observed; no other metal ion showed this enhancement. The optical studies (absorbance and (φ =in 0.85) the wasabsence observed; of Zn2+ no ions, other but metal upon ion the showed addition this of enhancement. Zn2+ ions to compound The optical 16 studies, a blue (absorbance shift to 560 nm andfluorescence),( φfluorescence), = 0.85) was and observed; theand NMR the no studiesNMR other metalstudies showed ion showed that showed no furtherthat this enhancement.no optical further or structuraloptical The optical or changes structural studies occurred (absorbancechanges after 2+ 7 2 the addition of two equivalents of Zn ions. A binding constant of 4.69 10 M− was determined and fluorescence), and the NMR studies showed that no further optical× or structural changes

Chemosensors 2019, 7, x 14 of 50 Chemosensors 2019, 7, 22 14 of 48 occurred after the addition of two equivalents of Zn2+ ions. A binding constant of 4.69 × 107 M−2 was determinedusing the using fluorescence the fluorescence data and the data stoichiometry and the stoichiometry was confirmed was as confirmed a 2:1 metal:probe as a 2:1 complex metal:probe complexformation, formation, through through MS studies. MS studies.

Scheme 4. Proposed binding motif between probe 16 and Zn2+ ions.

2+ Throughout thisScheme section 4. Proposed there have binding been examples motif between of molecular probe 16 probes and Zn for the ions. detection of Zn2+ ions, but very few papers also discuss the counter-ion effect in their systems. Zinc complexes (in Throughout this section there have been examples of molecular probes for the detection of Zn2+ particular ZnCl2) also play an important role, as a moderate strength Lewis acid, in Friedel–Crafts ions,acylations. but very Our few own papers group also has prepared discusspyrene-based the counter- molecularion effect probes in their17 and systems.18 [162]. Zinc Compound complexes17 (in particularwas shown ZnCl to2 simultaneously) also play an important bind the cation role, and as anion,a moderate in a self-assembled strength Lewis process. acid, Self-assembly in Friedel–Crafts acylations.occurred inOur the presenceown group of ZnX has2 salts prepared (X = F− , Clpyrene-based−, Br−,I−, BF 4−molecular, CH3CO2− ,Hprobes2PO4 −,17 ClO and4−, CN18− ,[162]. CompoundNO3−) and 17 ZnSO was4 shown. The optical to simultaneously response was due bind to athe strong cation excimer and anion, band observed in a self-assembled at 410 nm in the process. Self-assemblypresence of ZnCloccurred2 in acetonitrile. in the presence DFT calculations of ZnX2 salts supported (X = F− the, Cl proposed−, Br−, I−, BF structure4−, CH3 (SchemeCO2−, H52).PO The4−, ClO4−, intensity of the excimer signal was dependent on the proximity of the two pyrene units. In absence of CN−, NO3−) and ZnSO4. The optical response was due to a strong excimer band observed at 410 nm the Zn2+ ion, the excimer signal was not observed, as was confirmed by the analogous spectroscopic in the presence of ZnCl2 in acetonitrile. DFT calculations supported the proposed structure (Scheme studies with tetrabutylammonium salts, i.e., the metal was required for the self-assembly process to 5). The intensity of the excimer signal was dependent on the proximity of the two pyrene units. In occur. This suggests that the Zn2+ halide was templating the pyrene units. The geometry around absence of the Zn2+ ion, the excimer signal was not observed, as was confirmed by the analogous the Zn2+ ion was tetrahedral and the pyrene moieties adopted a syn configuration. The fluorescence spectroscopictitrations were studies in good with agreement tetrabutylammonium with the NMR studies, salts, which i.e., showed the metal a 2:1 bindingwas required mode calculated for the self- assemblyby non-linear process regression to occur. with This a suggests calculated that binding the Zn constant2+ halide (K was) of 1.8templating106 M 2thefor pyrene ZnCl . units. This The 21 × − 2 geometryexample around was a good the Zn example2+ ion ofwas size-selective tetrahedral self-assembled and the pyrene recognition moieties and adopted was carried a syn out configuration. at a low Theconcentration, fluorescence to titrations rule out intermolecular were in good excimer agreement formation. with the NMR studies, which showed a 2:1 binding mode calculated by non-linear regression with a calculated binding constant (K21) of 1.8 × 106 M−2 for ZnCl2. This example was a good example of size-selective self-assembled recognition and was carried out at a low concentration, to rule out intermolecular excimer formation.

Chemosensors 2019, 7, x 15 of 50

Chemosensors 2019, 7, 22 15 of 48 Chemosensors 2019, 7, x 15 of 50

Scheme 5. A simultaneous ion pair recognition event. Adapted from [160] with permission from The RoyalScheme Society 5. ofA Chemistry. simultaneous ion pair recognition event. Adapted from [160] with permission from The Royal Society of Chemistry. The Schemenext step 5. Aour simultaneous group took ion was pair to recognitiontether two event.of the Adpyreneapted units, from [160] shown with in permission compound from 17, ontoThe The next step our group took was to tether two of the pyrene units, shown in compound 17, onto an organicRoyal backbone, Society of to Chemistry. prepare a dipodal receptor. Three molecular probes containing an amide an organic backbone, to prepare a dipodal receptor. Three molecular probes containing an amide functional group and a triazole moiety was attached to a benzene scaffold (18a-c ortho, meta and para functionalThe next group step and our agroup triazole took moiety was to was tether attached two of to the a benzenepyrene units, scaff oldshown (18a-c in compoundortho, meta and17, onto para respectively) [163]. respectively) [163]. an organic backbone, to prepare a dipodal receptor. Three molecular probes containing an amide functional group and a triazole moiety was attached to a benzene scaffold (18a-c ortho, meta and para respectively) [163].

2+ 2+ 2+ FluorescenceFluorescence analysis analysis of compounds of compounds 18a and18a 18band with18b differentwith diff metalerent metalions (Zn ions2+, Mg (Zn2+, Cd, Mg2+, Al,3+ Cd, , 3+ 2+ 2+ 2+ 2+ 3+ + Hg2+Al, Ni2+, Hg, Cu2+,, NiFe2+,, Fe Cu3+ and, Fe Na, Fe+) as eitherand Na their) as chloride either their or perc chloridehlorate or perchloratesalts were initially salts were tested, initially withtested, the addition with the of 10 addition equivalents of 10 of equivalents metal salts ofin metalCH3CN salts and in excited CH3CN at 325 and nm. excited The fluorescence at 325 nm. The Fluorescence analysis of compounds 18a and 18b with different metal ions (Zn2+, Mg2+, Cd2+, Al3+, signalfluorescence of the free signalreceptors of theshowed free receptorsthe typical showed monomer the bands typical at monomer380 and 400 bands nm, characteristic at 380 and 400 of nm, 2+ 2+ 2+ 2+ 3+ + the uniqueHgcharacteristic, Ni electronic, Cu of, Fe thetransitions, uniqueFe and in electronic Na the) pyrene-derivedas either transitions their chloride compounds, in the pyrene-derived or perc whichhlorate were salts compounds, assigned were initially to whichthe π -tested,π were* electronwithassigned thetransitions. addition to the π A-ofπ * very10 electron equivalents broad transitions. band of metal at 495 A salts very nm in broad wasCH 3CNalso band and seen, at excited 495 which nm at was 325was alsonm. assigned The seen, fluorescence whichto the was signal of the free receptors showed the typical monomer bands at 380 and 400 nm, characteristic of intramolecularassigned to excimer the intramolecular formation, excimerupon excitation formation, at upon325 nm. excitation On inspection at 325 nm. of Onthe inspectionfluorescence of the the unique electronic transitions in the pyrene-derived compounds, which2+ were assigned to the π-π2*+ spectrum,fluorescence quenching spectrum, was observ quenchinged for was all observedmetals except for all Zn metals2+ (and except to a lesser Zn (anddegree to aCd lesser2+ and degree Mg2+), Cd electron2 +transitions. A very broad band at 495 nm was also seen, which was assigned2+ to the and anda new Mg band), andwas a seen new to band increase was seenat 400 to nm. increase It was at evident 400 nm. that It was the Zn evident2+ salts that showed the Zn significantlysalts showed greaterintramolecularsignificantly fluorescence greater excimer at fluorescence400 formation,nm for 18b at 400upon than nm excitationfor for 18b18a, thanfor at which for32518a nm. ,a for smallOn which inspection increase a small inof increase intensitythe fluorescence in intensitywas 2+ 2+ 2+ observedspectrum,was observed at 400 quenching nm. at The 400 fluorescence nm.was Theobserv fluorescence edintensity for all atmetals intensity 400 nm except atfor 400 18a Zn nm was (and for a consequence 18ato awas lesser a consequencedegree of the Cdtwo pyreneand of the Mg two), 2+ unitsandpyrene in a18a new units adopting band in 18a was anadopting seen anti-orientation, to increase an anti-orientation, at which 400 nm. gave It which was a greater evident gave pyrene–pyrene a that greater the pyrene–pyreneZn salts separation showed separation than significantly was than greater fluorescence at 400 nm for 18b than for 18a, for which a small increase in intensity was availablewas available to 18b. It to was18b shown. It was that shown the binding that the bindingaffinity for affi Zn(ClOnity for4 Zn(ClO)2 was a4 )magnitude2 was a magnitude greater for greater 18a for observed at 400 nm. The fluorescence intensity at 400 nm for 18a was a consequence of the two pyrene than18a forthan 18b, for even18b though, even though the fluorescence the fluorescence change change at 400 atnm 400 was nm not was observed not observed to be to as be dramatic. as dramatic. In In unitsfact this in 18a trend adopting was seen an for anti-orientation, all the other analytes whichstudied, gave a greater we argued pyrene–pyrene that the more separation favorable than chelating was available to 18b. It was shown that the binding affinity for Zn(ClO4)2 was a magnitude greater for 18a than for 18b, even though the fluorescence change at 400 nm was not observed to be as dramatic. In

Chemosensors 2019, 7, x 16 of 50

Chemosensors 2019, 7, 22 16 of 48 fact this trend was seen for all the other analytes studied, we argued that the more favorable chelating motif was the reason for the observed calculated binding constant. To understand the binding mode, motifextensive was IR the and reason molecular for the modeling observed calculatedstudies were binding carried constant. out. It is To known understand that significant the binding infrared mode, extensivespectral changes IR and occur molecular when modeling perchlorate studies coordinate were carrieds with a out.metal It ion is known through that different significant modes infrared [164]. spectralDetermination changes of occur the changing when perchlorate symmetry coordinates of the ClO with4- ion, a metal upon ion binding, through from different Td (ionic) modes to [164 C3v]. Determination(unidentate) and of C the2v (bidentate changing or symmetry bridging), of could the ClOhelp4 −identifyion, upon if the binding, binding frommode T proposedd (ionic) tomodel C3v (unidentate)was consistent and with C2v (bidentateIR data reported. or bridging), Molecular could mechanics help identify calculations if the binding for mode18a and proposed 18b and model their wascorresponding consistent withZn(ClO IR4 data)2 complexes reported. were Molecular carried mechanicsout (Figure calculations 6). Both of the for 18afree andreceptors18b and showed their correspondingvery different π Zn(ClO stacking4) 2interactions.complexes wereThe ortho carried-isomer, out (Figure 18a, showed6). Both that of the the aromatic free receptors scaffold showed of the verymolecular different probesπ stacking participates interactions. in π-stacking The ortho and-isomer, one of18a the, showedpyrene arms that the seemed aromatic to intercalate scaffold of and the molecularform an intramolecular probes participates sandwich in π interaction-stacking and Figure one 6A. of the This pyrene intercalation arms seemed was not to observed intercalate in and the formmeta-isomer, an intramolecular 18b, which sandwich showed the interaction π-π distance Figure between6A. This the intercalation two pyrene was units not to observed be 4.77 Å. in theThe metadistance-isomer, was18b measured, which between showed thethe πpyrene-π distance centroids between (Figure the two6C) and pyrene the unitsCH hydrogens to be 4.77 Å.on Thethe distancetriazole rings was measured orientated between towards the the pyrene center centroids of the molecular (Figure6C) probes. and the This CH hydrogensis in excellent on the agreement triazole ringswith the orientated interactions towards expected the center to generate of the molecularthe excimer probes. band seen This in is the in excellent fluorescence agreement spectrum. with Both the interactionsof the Zn2+ complexes expected to showed generate drastically the excimer diff banderent seen optimized in the fluorescencegeometries. spectrum.The Zn2+ ion Both was of bound the Zn 2in+ complexesa tetrahedral showed arrangement, drastically coordina differentted optimized to the oxygen geometries. atom of The the Zn carbonyl2+ ion was functional bound in group, a tetrahedral and a arrangement,nitrogen atom coordinated in the triazole to the ring, oxygen forming atom a ofseven-membered the carbonyl functional chelating group, ring. andThe adistance nitrogen between atom in thethe triazoletwo pyrene ring, units forming in [Zn( a seven-membered18a)]2+ was 10.8 Å, chelatingwhereas the ring. distance The distance between between the two thepyrene two units pyrene in units[Zn(18a in)] [Zn(2+ was18a )]5.52+ Å.was This 10.8 was Å, whereas in excellent the distanceagreement between with the twofluorescence pyrene units data in in [Zn( which18a )]a2 +broadwas 5.5excimer Å. This band was was in excellent seen at 400 agreement nm for the with 18b the complex; fluorescence the same data inband which was a broadsignificantly excimer less band intense was seenfor the at 16a 400 complex. nm for the 18b complex; the same band was significantly less intense for the 16a complex.

Figure 6. Fully optimized structures (DFT) in the gas phase of (A) compound 18a,(B) [Zn 18a](ClO ) , · 4 2 (C) compound 18b, and (D) [Zn 18b](ClO ) . Adapted From [163], with permissiom from Taylor & Figure 6. Fully optimized structures· (DFT)4 in2 the gas phase of (A) compound 18a, (B) [Zn·18a](ClO4)2, Francis(C) compound Ltd. 18b, and (D) [Zn·18b](ClO4)2. Adapted From [163], with permissiom from Taylor & Francis Ltd. 2.2. Cadmium Probes As cadmium has essentially no biological use and the bioaccumulation of Cd2+ ions in the environment is a real threat, there is an increasing need to remove cadmium from wastewater from an industrial process. Cadmium has been ranked as one of the top 10 priority lists from the top

Chemosensors 2019, 7, x 17 of 50

2.2. Cadmium Probes

As cadmium has essentially no biological use and the bioaccumulation of Cd2+ ions in the environment is a real threat, there is an increasing need to remove cadmium from wastewater from Chemosensorsan industrial2019, 7 ,process. 22 Cadmium has been ranked as one of the top 10 priority lists from the17 top of 48 275 hazardous materials that have been identified by The Comprehensive Environmental Response, 275Compensation, hazardous materials and Liability that have Act been in the identified USA [165] by, Thetherefore, Comprehensive detection and Environmental remediation Response, of Cd2+ ions Compensation,in the environment and Liability are imperative. Act in the A USA review [165 by], therefore, El-Safty outlined detection the and importance remediation of cadmium of Cd2+ ions in the in theenvironment, environment discussed are imperative. common A review techniques by El-Safty to remediate outlined the Cd importance2+ ions, and of cadmium highlighted in the the environment,chemosensors discussed used commonfor cadmium techniques monitoring to remediate [166]. Cd The2+ ions,chemistry and highlighted and structural the chemosensors similarities of usedcadmium for cadmium is similar monitoring to zinc. [Both166]. the The tetrahedral chemistry an andd octahedral structural similaritiesforms are known. of cadmium The type is similar of Lewis 2+ to zinc.basic Bothatom theplays tetrahedral a role in the and geometry. octahedral Fo formsr example, are known. Parkin Theet al. type showed of Lewis that Zn basic ions atom preferred plays a 2+ a roletetrahedral in the geometry. environment For example,when the Parkindonor etatom al. showedwas sulfur, that whereas Zn2+ ions the preferred Cd complex a tetrahedral showed a 2+ 2+ environmentsix-coordinate when environment the donor atom [167]. was Selenolato sulfur, whereascomplexes the are Cd known2+ complex to coordinate showed ato six-coordinate Zn and Cd in 2+ environmenta distorted [tetrahedral167]. Selenolato environment complexes [168]. are A known review to published coordinate by toSigel Zn 2and+ and Martin Cd2+ statein a distortedthat the Cd tetrahedralion could environment be found in [168 4,]. 5, A reviewand 6 coordination published by Sigelenvironments, and Martin 20%, state 8%, that and the Cd56%2+ ionof the could time, be foundrespectively in 4, 5, [169]. and 6 coordination environments, 20%, 8%, and 56% of the time, respectively [169]. 2+ Therefore,Therefore, it is it notis not surprising surprising that that the th LMFPe LMFP reported reported for for the the detection detection of of Cd Cd2+ ions ions is is similarsimilar to to thethe probesprobes for for zinc zinc ions. ions. Additionally, Additionally, the the fluorescence fluorescence mechanism mechanism is is alike. alike. For For example, example, Pu Pu et al.et al. synthesizedsynthesized a closely a closely related related molecule molecule to probesto probes10 and10 and11 and11 and subtly subtly changed changed their their binding binding motif motif to to 2+ prepareprepare a sensor a sensor for for Cd 2Cd+ ions ions [170 [170].]. A rhodamine-basedA rhodamine-based conjugated conjugated dyad dyad probe probe (19 )(19 has) has been been synthesized synthesized by Stalinby Stalin and and coworkers coworkers (Scheme(Scheme6)[ 1716) ].[171]. The authorsThe authors claimed claimed that the that FRET th mechanisme FRET mechanism between thebetween rhodamine the rhodamine and pyridine and 2+ conjugatedpyridine dyadconjugated is responsible dyad is responsible for the emission for the observed emission upon observed the coordination upon the coordination of Cd2+ ions of Cd in the ions in the organic–aqueous system (CH3CN:HEPES buffer 2:8, v/v, pH 7.2, 10 mM). However, upon organic–aqueous system (CH3CN:HEPES buffer 2:8, v/v, pH 7.2, 10 mM). However, upon inspection of theirinspection molecular of probe, their itmolecular is difficult probe, to justify it is the difficul authorst to claim. justify For the FRET authors to occur, claim. several For criteriaFRET to must occur, be met:several (i) thecriteria fluorescence must be emissionmet: (i) the spectrum fluorescence of the donor emission molecule spectrum must of overlap the donor the absorption molecule ormust excitationoverlap spectrum the absorption of the acceptoror excitation chromophore, spectrum of (ii) th thee acceptor two fluorophores chromophore, (donor (ii) and the acceptor)two fluorophores must be in(donor close proximityand acceptor) to one must another be in (typically close proximity 1 to 10 nm), to (iii)one theanother transition (typically dipole 1 orientations to 10 nm),of (iii) the the donortransition and acceptor dipole mustorientations be approximately of the donor parallel and acceptor to each must other, be and approximately (iv) the fluorescence parallel lifetimeto each other, of theand donor (iv) molecule the fluorescence must be of lifetime sufficient of durationthe donor to mole allowcule the must FRET be to occur.of sufficient The pyridine duration unit to alone allow is the notFRET usually to consideredoccur. The pyridine a fluorophore. unit alone It is moreis not reasonable usually considered to assume a thatfluorophore. the mechanism It is more is an reasonable off–on fluorescenceto assume mechanism, that the mechanism based on theis an spirolactam off–on fluore ringscence opening. mechanism, However, based the authors on the didspirolactam show high ring 2+ selectivityopening. and However, sensitivity the towardsauthors Cddid2 +showions inhigh the selectivity presence ofand other sensitivity competing towards metal Cd ions, ions which in the waspresence indicated of byother the competing appearance metal of a ions, new which fluorescence was indicated band at by 590 the nm, appearance due to Cd of 2a+ newcoordination. fluorescence band at 590 nm, due to Cd2+ coordination. Spectroscopic studies8 were used to determine a LoD of 1 × Spectroscopic studies were used to determine a LoD of 1 10− M, which is well below the WHO −8 × 2+ acceptable10 M, limitwhich of is 1.85 well mM below for Cd the2+ WHOions in acceptable drinking water. limit of Both 1.85 the mM association for Cd constantions in drinking and binding water. ratioBoth were the determined association fromconstant the absorptionand binding spectra. ratio were The determined absorption titrationfrom the was absorption used to spectra. calculate The 4 −1 an associationabsorption titration constant was of 4 used104 toM calculate1 and, from an association the Job’s plot constant data, theof 4 binding × 10 M ratio and, was from found the toJob’s × − be 1:1.plot Indata, the the presence binding of ratio sulfide was ionsfound CdS to be was 1:1. formed, In the presence which resulted of sulfide in ions decomplexation CdS was formed, and thewhich disappearanceresulted in ofdecomplexation the emission band and observedthe disappearance at 590 nm. of The the results emission described band observed above suggest at 590 that nm. the The results described above suggest that the sens2 or was recycled during the detection of S2− ions. sensor was recycled during the detection of S − ions.

Scheme 6. The proposed binding of Cd 2+ ions and chemosensor 19. Scheme 6. The proposed binding of Cd2+ ions and chemosensor 19. A FRET molecular probe incorporating rhodamine was synthesized by Goswami et al. Chemosensor 20 was shown to be selective for Cd2+ ions, with the sensor acting as an ‘off–on’

FRET sensor capable of detecting Cd2+ ions in a mixed organic–aqueous solvent system. Additionally, a distinctive color change was seen, typical of rhodamine dyes. A quinoline–benzothiazole functional group (FRET donor) was tethered to the rhodamine molecule (FRET acceptor), via a nonconjugated Chemosensors 2019, 7, x 18 of 50

A FRET molecular probe incorporating rhodamine was synthesized by Goswami et al. Chemosensor 20 was shown to be selective for Cd2+ ions, with the sensor acting as an ‘off–on’ FRET sensor capable of detecting Cd2+ ions in a mixed organic–aqueous solvent system. Additionally, a Chemosensors 2019, 7, 22 18 of 48 distinctive color change was seen, typical of rhodamine dyes. A quinoline–benzothiazole functional group (FRET donor) was tethered to the rhodamine molecule (FRET acceptor), via a nonconjugated spacer,spacer, Scheme Scheme7 [7 172[172].]. AsAs thethe emissionemission spectraspectra ofof thethe quinolinequinoline benzothiazole group group overlapped overlapped significantlysignificantly with with the the absorption absorption spectra spectra of theof ring-openedthe ring-opened rhodamine rhodamine B dye, B itdye, was it possible was possible to observe to theobserve transfer the of transfer energy of from energy donor from to acceptor,donor to acceptor, once the sensoronce the interacted sensor interacted with the Cdwith2+ theions. Cd Both2+ ions. the quantumBoth the yieldquantum and FRETyield and efficiency FRET ofefficiency the sensor of the were sensor calculated were calculated from fluorescence from fluorescence studies carried studies out incarried MeOH out/H Oin (1:4,MeOH/Hv/v, pH2O 7.2,(1:4, 10mM v/v, pH HEPES 7.2, bu10mMffer solution) HEPES buffer and were solution) found and to be wereφ = 0.37found and to 48%, be φ 2 respectively. = 0.37 and These48%, respectively. experimental These conditions experimental were optimized conditions for were the water optimized content. for Theythe water reported content. that They reported that when the percentage of water was increased above 80%, the intensity ratio (I585/I470) when the percentage of water was increased above 80%, the intensity ratio (I585/I470) of the emission bandof the decreased emission band significantly, decreased whereas significantly, less water whereas content less showedwater content modest showed changes modest in the changes emission in the emission intensity. From the fluorescence titration, Aich and coworkers found that the lowest intensity. From the fluorescence titration, Aich and coworkers found that the lowest concentration of 2+ −7 concentration2+ of Cd ions detectable by the sensor7 was 3 × 10 M and the association constant Cd ions detectable by the sensor was 3 10− M and the association constant (determined using 5 −1 (determined using a nonlinear least-squares× fit) was5 found1 to be 1 × 10 M . The other metal+ ions2+ a nonlinear least-squares fit) was found to be 1 10 M− . The other metal ions studied (K , Ca , studied (K+, Ca2+, Ni2+, Mn2+, Zn2+, Cu2+, Fe3+, Cr×3+, Hg2+, and Pb2+) did not show the same optical Ni2+, Mn2+, Zn2+, Cu2+, Fe3+, Cr3+, Hg2+, and Pb2+) did not show the same optical response. The response. The 1:1 binding mode was confirmed both in solution and in the solid state. The X-ray 1:1 binding mode was confirmed both in solution and in the solid state. The X-ray crystal structure crystal structure confirmed that the Cd2+ binds in an octahedral environment with five of the donor confirmed that the Cd2+ binds in an octahedral environment with five of the donor groups from groups from compound 20 and a chloride ion in the remaining coordination site. compound 20 and a chloride ion in the remaining coordination site.

2+ Scheme 7. The binding mode of Cd ions to chemosensor 20 MeOH/H2O (1:4, v/v, pH 7.2, 10mM HEPES buffer solution). Scheme 7. The binding mode of Cd2+ ions to chemosensor 20 MeOH/H2O (1:4, v/v, pH 7.2, 10mM HEPES buffer solution). As Cd2+ ions are hazardous to the environment the preparation of portable sensing kits is attractive.As Cd Goswami2+ ions are immersed hazardous thin to layerthe environment chromatography the preparation (TLC) plates of soaked portable with sensing20 in MeOHkits is 4 solutionattractive. (2 Goswami10− M), removedimmersed excess thin MeOHlayer chromatography and placed the probe(TLC) coated plates plates soaked into with a MeOH 20 in solutionMeOH × 3 Chemosensorsof cadmium 2019 ions, 7−,4 x (2 10 M), which showed distinctive color changes (Figure7). 19 of 50 solution (2 × 10 M),× removed− excess MeOH and placed the probe coated plates into a MeOH solution of cadmium ions (2 × 10−3 M), which showed distinctive color changes (Figure 7).

Figure 7. ((AA)) Photographs Photographs of of TLC TLC plates plates after after the the immersion immersion of of molecular molecular probe probe 2020 intointo a MeOH solution (i) afterafter thethe additionaddition of of Cd Cd2+2+ ionsions ((iiii)) underunder ambientambient light; light; and and ( B(B)) under under hand-held hand-held UV-light UV-light at at365 365 nm, nm, (i) ( afteri) after the the addition addition of Cd of2 Cd+ ions,2+ ions, (ii) ( underii) under hand-held hand-held UV-light UV-light at 365 at nm, 365 Adaptednm, Adapted from [from172], [172],with permission with permission from Americanfrom American Chemical Chemical Society. Society.

Another popular fluorescent mechanism that is being used in molecular probe design is aggregation induced emission (AIE), a similar mechanism to excimer formation. Wang et al. employed this by tethering a hydrophobic tetraphenylethene (TPE) moiety (hydrophobic part) onto a β-cyclodextrin (hydrophilic part), via a click reaction to give compound 21. Therefore, by varying the ratios of water and organic solvent the mechanism could be turned on and off. The triazole moiety was used as the Lewis basic site to coordinate the Cd2+ ions [171]. In solutions containing ≤50% water, the sensor gave no significant emission when the probe was excited at 330 nm. However, upon the addition of Cd2+ ions, a noticeable emission band emerged around 476 nm. This could be attributed to the overlap of the tetraphenylethene groups as a result of the 2:1 coordination between the sensor and Cd2+ ions. This same behavior was observed as the water content of the solvent system was increased up to 98%. In higher concentrations of water, the solution of the sensor becomes strongly emissive at 476 nm, and this emission was visible with the naked eye, Figure 8. This strong emission was due to the formation of aggregates in solution, owing to the decreased solubility of the sensor. Zhang et al. observed that in a solution of DMSO/H2O (1:1, v/v) the sensor only responded to Cd2+ ions and no other metal ions and they were able to detect Cd2+ ions at concentrations as low as 1 × 10−8 M.

Figure 8. (A) The proposed binding mode of chemosensor 21 and Cd2+ ions and (B) the emission turn- on in 50 μM solution of 21 with a different ratio of DMSO-H2O (λex = 330 nm). Aggregation could be seen by the naked-eye once the water content was greater than 90%. Adapted from [173] with permission from The Royal Society of Chemistry.

Chemosensors 2019, 7, x 19 of 50

Figure 7. (A) Photographs of TLC plates after the immersion of molecular probe 20 into a MeOH solution (i) after the addition of Cd2+ ions (ii) under ambient light; and (B) under hand-held UV-light at 365 nm, (i) after the addition of Cd2+ ions, (ii) under hand-held UV-light at 365 nm, Adapted from [172], with permission from American Chemical Society. Chemosensors 2019, 7, 22 19 of 48 Another popular fluorescent mechanism that is being used in molecular probe design is aggregation induced emission (AIE), a similar mechanism to excimer formation. Wang et al. Another popular fluorescent mechanism that is being used in molecular probe design is aggregation employed this by tethering a hydrophobic tetraphenylethene (TPE) moiety (hydrophobic part) onto induced emission (AIE), a similar mechanism to excimer formation. Wang et al. employed this by a β-cyclodextrin (hydrophilic part), via a click reaction to give compound 21. Therefore, by varying tethering a hydrophobic tetraphenylethene (TPE) moiety (hydrophobic part) onto a β-cyclodextrin the ratios of water and organic solvent the mechanism could be turned on and off. The triazole moiety (hydrophilic part), via a click reaction to give compound 21. Therefore, by varying the ratios of water was used as the Lewis basic site to coordinate the Cd2+ ions [171]. In solutions containing ≤50% water, and organic solvent the mechanism could be turned on and off. The triazole moiety was used as the the sensor gave no significant emission when the probe was excited at 330 nm. However, upon the Lewis basic site to coordinate the Cd2+ ions [171]. In solutions containing 50% water, the sensor gave addition of Cd2+ ions, a noticeable emission band emerged around 476 nm.≤ This could be attributed no significant emission when the probe was excited at 330 nm. However, upon the addition of Cd2+ to the overlap of the tetraphenylethene groups as a result of the 2:1 coordination between the sensor ions, a noticeable emission band emerged around 476 nm. This could be attributed to the overlap of and Cd2+ ions. This same behavior was observed as the water content of the solvent system was the tetraphenylethene groups as a result of the 2:1 coordination between the sensor and Cd2+ ions. increased up to 98%. In higher concentrations of water, the solution of the sensor becomes strongly This same behavior was observed as the water content of the solvent system was increased up to 98%. emissive at 476 nm, and this emission was visible with the naked eye, Figure 8. This strong emission In higher concentrations of water, the solution of the sensor becomes strongly emissive at 476 nm, and was due to the formation of aggregates in solution, owing to the decreased solubility of the sensor. this emission was visible with the naked eye, Figure8. This strong emission was due to the formation Zhang et al. observed that in a solution of DMSO/H2O (1:1, v/v) the sensor only responded to Cd2+ of aggregates in solution, owing to the decreased solubility of the sensor. Zhang et al. observed that in 2+ ions and no other metal ions and they were able to detect Cd2+ ions at concentrations as low as a solution of DMSO/H2O (1:1, v/v) the sensor only responded to Cd ions and no other metal ions and 1 × 10−8 M. they were able to detect Cd2+ ions at concentrations as low as 1 10 8 M. × −

Figure 8. (A) The proposed binding mode of chemosensor 21 and Cd2+ ions and (B) the emission

turn-onFigure 8. in (A 50) TheµM proposed solution ofbinding21 with mode a di ffoferent chemosensor ratio of DMSO-H 21 and Cd2O(2+ ionsλex and= 330 (B) nm). the emission Aggregation turn- couldon in be50 seenμM solution by the naked-eye of 21 with once a different the water ratio content of DMSO-H was greater2O (λ thanex = 330 90%. nm). Adapted Aggregation from [173 could] with be permissionseen by the from naked-eye The Royal once Society the water of Chemistry. content was greater than 90%. Adapted from [173] with permission from The Royal Society of Chemistry. Many LMFPs have been adsorbed (physisorption or chemisorption) onto surfaces to improve sensitivity. This is done to increase the number of binding sites, by incorporating a number of the desired molecular probes onto a single platform. Commonly used scaffolds are mesoporous silica materials. These materials are rigid and porous and have been used to support a number of molecular sensors. An advantage of these materials is that they are optically transparent in the visiable range, therefore, the signal generated is genuinely from the receptor and the analyte. These types of materials have been used in sensor applications and for remediation purposes [119,129,174,175]. A reusable fluorescent sensor capable of detecting Cd2+, Hg2+, and Pb2+ ions in aqueous solutions was synthesized by Zhu et al. by physidorption, of a 2,2-dipicoylamine modified naphthalimide fluorophore, 22, onto the surface of a silica microsphere [176]. It is inherently hydrophilic and capable of metal detection and adsorption (Scheme8), as indicated by increases in its fluorescence intensity. Control studies indicated that, in the absence of the silylpropylamine linkers on the microspheres, no adsorption of metal ions had occurred. Under optimal conditions (low concentration of metal ions and 22 at pH 4.5–10), compound 22 had an adsorptivity greater than 95%. In acidic media (pH < 4.5), the Chemosensors 2019, 7, x 20 of 50

Many LMFPs have been adsorbed (physisorption or chemisorption) onto surfaces to improve sensitivity. This is done to increase the number of binding sites, by incorporating a number of the desired molecular probes onto a single platform. Commonly used scaffolds are mesoporous silica materials. These materials are rigid and porous and have been used to support a number of molecular sensors. An advantage of these materials is that they are optically transparent in the visiable range, therefore, the signal generated is genuinely from the receptor and the analyte. These types of materials have been used in sensor applications and for remediation purposes [119,129,174,175]. A reusable fluorescent sensor capable of detecting Cd2+, Hg2+, and Pb2+ ions in aqueous solutions was synthesized by Zhu et al. by physidorption, of a 2,2-dipicoylamine modified naphthalimide fluorophore, 22, onto the surface of a silica microsphere [176]. It is inherently hydrophilic and capable of metal detection and adsorption (Scheme 8), as indicated by increases in its fluorescence intensity. Control studies indicated that, in the absence of the silylpropylamine linkers on the microspheres, no adsorption of metal ions had occurred. Under optimal conditions (low concentration of metal ions

Chemosensorsand 22 at pH2019 4.5–10),, 7, 22 compound 22 had an adsorptivity greater than 95%. In acidic media (pH20 < of4.5), 48 the adsorption capability of the sensor was greatly diminished, which allowed the desorption of metal ions from the sensor. Presumably, the nitrogen atoms become protonated, removing the ability adsorptionof the molecular capability probe of to the form sensor a Lewis was acid-base greatly diminished, adduct, before which the acidic allowed medium the desorption removes the of metal metal ionsions. from This the allows sensor. microspheres Presumably, coated the nitrogen with 22 atoms to be recycled becomeprotonated, multiple times, removing by washing the ability them of with the molecularan acidic solution, probe to then form readjusting a Lewis acid-base the pH adduct,by adding before base. the Fluorescence acidic medium studies removes determined the metal that ions. the ThisLoD allows of 22 microsphereswas in the nM coated concentration with 22 to range be recycled with multiplean association times, constant by washing of 2.3 them ×10 with7 M− an1 for acidic Cd2+ solution,ions. then readjusting the pH by adding base. Fluorescence studies determined that the LoD of 22 7 1 2+ was inInterestingly, the nM concentration this molecular range probe with anwas association able to remove constant Cd2+ of (and 2.3 Hg10 2+M and− forPb2+ Cd) fromions. tap water, 2+ × 2+ 2+ lab wastewater,Interestingly, and this simulated molecular biological probe was fluid. able Samples to remove were Cd spiked(and with Hg 2.0 mgandL Pb−1 Cd)2+ fromsalts tapand water, lab wastewater, and simulated biological fluid. Samples were spiked with 2.0 mg L 1 Cd2+ 99%, 98%, and 96% of the metal was recovered from tap water, wastewater, and biological· − fluid, saltsrespectively, and 99%, indicating 98%, and 96%that the of the molecular metal was probe recovered is a good from candidate tap water, for wastewater,cadmium remediation, and biological but fluid,it remains respectively, to be seen indicating if the probe that thecan molecular selectively probe remove is a metals good candidate from a mixture for cadmium of metal remediation, ions in the butsamples. it remains to be seen if the probe can selectively remove metals from a mixture of metal ions in the samples.

Scheme 8. Chemosensor 22, attached to silica, is able to undergo a fluorescence turn on, upon the addition of Cd2+, Hg2+, and Pb2+. The removal of the metal was achieved by the addition of acid. Scheme 8. Chemosensor 22, attached to silica, is able to undergo a fluorescence turn on, upon the Lvaddition and Shen of Cd synthesized2+, Hg2+, and Pb a ratiometric2+. The removal porphyrin-based of the metal was fluorescent achieved by probe the addition (23) for of detecting acid. Cd2+ ions in aqueous systems from pH 6.5 to 10 [177]. The incorporation of sulfonic groups enhances Lv and Shen synthesized a ratiometric porphyrin-based fluorescent probe (23) for detecting Cd2+ the probe’s water solubility of the porphyrin ring system. The 2,20-dipyridylamine (DPA) possesses ions in aqueous systems from pH 6.5 to 10 [177]. The incorporation of sulfonic groups enhances the remarkable binding ability towards Cd2+ ions; therefore two DPA groups were attached to coordinate probe’s water solubility of the porphyrin ring system. The 2,2’-dipyridylamine (DPA) possesses the Cd2+ ions. The porphyrin fluorophore serves as an ideal signaling unit owing to its large conjugated remarkable binding ability towards Cd2+ ions; therefore two DPA groups were attached to coordinate π plane. Upon the coordination of DPA to Cd2+ ions, a ratiometric fluorescent change to the emission the Cd2+ ions. The porphyrin fluorophore serves as an ideal signaling unit owing to its large spectra of the probe was observed. The fluorescence mechanism is based on an intramolecular charge conjugated π plane. Upon the coordination of DPA to Cd2+ ions, a ratiometric fluorescent change to transfer (ICT). Compound 23 shows a good solubility in aqueous solutions without the addition of an organic solvent. Spectroscopic studies were carried out in HEPES buffer solution (pH 7.4). Upon the addition of Cd2+ ions to a solution of the probe, the emission wavelength shifted from 653 to 611 nm (φ = 0.15). The noticeable hypsochromic shift of 42 nm was indicative of a reduction in the electron donating ability of the DPA moieties, upon Cd2+ ions coordination, as the Cd2+ ion is electron-withdrawing. Moreover, as the Cd2+ ions are coordinated to the DPA molecule through the nitrogen atom, the porphyrin ring system is perturbed subtly, therefore, decreasing the conjugation, and shifting the wavelength in the blue direction. Direct fluorimetric titration as a function of Cd2+ ion concentration was used to calculate a dissociation constant (Kd) value of 31 µM, and a Job’s plot showed a 1:2 binding ratio between the sensor and Cd2+ ions. Often signaling systems that depend on fluorescence changes (increases or decreases) at a fixed wavelength, suffer from uncertainty in absolute fluorescence measurements; however, a ratiometric fluorescence response enhances the reliability of the signaling from a quantitative viewpoint. Through linear regression of the calibration plot ([F611/F653] vs [Cd2+]), the detection limit was calculated to be 0.032 µM. Furthermore, the Cd2+ ions could be Chemosensors 2019, 7, x 21 of 50 the emission spectra of the probe was observed. The fluorescence mechanism is based on an intramolecular charge transfer (ICT). Compound 23 shows a good solubility in aqueous solutions without the addition of an organic solvent. Spectroscopic studies were carried out in HEPES buffer solution (pH 7.4). Upon the addition of Cd2+ ions to a solution of the probe, the emission wavelength shifted from 653 to 611 nm (φ = 0.15). The noticeable hypsochromic shift of 42 nm was indicative of a reduction in the electron donating ability of the DPA moieties, upon Cd2+ ions coordination, as the Cd2+ ion is electron-withdrawing. Moreover, as the Cd2+ ions are coordinated to the DPA molecule through the nitrogen atom, the porphyrin ring system is perturbed subtly, therefore, decreasing the conjugation, and shifting the wavelength in the blue direction. Direct fluorimetric titration as a function of Cd2+ ion concentration was used to calculate a dissociation constant (Kd) value of 31 μM, and a Job’s plot showed a 1:2 binding ratio between the sensor and Cd2+ ions. Often signaling systems that depend on fluorescence changes (increases or decreases) at a fixed wavelength, suffer from Chemosensorsuncertainty2019 in, 7absolute, 22 fluorescence measurements; however, a ratiometric fluorescence response21 of 48 enhances the reliability of the signaling from a quantitative viewpoint. Through linear regression of the calibration plot ([F611/F653] vs [Cd2+]), the detection limit was calculated to be 0.032 μM. removedFurthermore, from the the Cd DPA2+ ions groups could by be the removed addition from of EDTA. the DPA As compoundgroups by the23 showed addition good of EDTA. aqueous As solubilitycompound studies 23 showed in Hela good cells aqueous and A549 solubility (adenocarcinomic studies in human Hela cells alveolar and basal A549 epithelial) (adenocarcinomic cells, the twohuman cancer alveolar lines remainedbasal epithelial) 87% viable, cells, implyingthe two cancer that the lines sensor remained exhibited 87% minimal viable, cytotoxicityimplying that and the is likelysensor safe exhibited for use minimal in bioimaging. cytotoxicity and is likely safe for use in bioimaging.

Siva and ChellapaChellapa synthesized a ratiometric benzimidazole tagged coumarin probe 24.24. Compound 24 could selectively detect Cd2+ and F ions in a CH CN/H O (1:9, v/v) HEPES-buffered Compound 24 could selectively detect Cd2+ and F− ions in a CH3CN/H22O (1:9, v/v) HEPES-buffered solutionssolutions atat pHpH 7.52,7.52, SchemeScheme9 [9 178[178].]. The The UV-Vis UV-Vis absorption absorption spectrum spectrum showed showed three three distinct distinct bands bands at 2+ 292,at 292, 342, 342, and and 401 401 nm. nm. The The addition addition of of Cd Cd2+ions ions led led to to a hyperchromica hyperchromic shift shift at at 292 292 and and 343 343 nm, nm, and and a hypochromica hypochromic shift shift at at 401 401 nm, nm, through through an an isosbestic isosbestic point point at at 367 367 nm, nm,indicative indicative ofof metalmetal complexescomplexes inin solution.solution. Upon exciting probe 24 at 340 nm, two distinct emission bands at 410 and 530 nmnm werewere 2+ observed. The addition ofof CdCd2+ ionsions led led to to a a blue blue emission emission band band at at 410 410 nm nm and and an an increase increase in emission intensity,intensity, at at 530 530 nm, nm, through through an isoemissive an isoemissive point 518point nm. 518 From nm. these From spectral these changes spectral the changes association the constant was calculated to be 3.5 103 M 1, using a modified Benesi Hildebrand method and the LoD association constant was calculated× to be− 3.5 × 103 M−1, using a modified− Benesi−Hildebrand method was determined to be 1.5 10 10 M. The addition of the Cd2+ ions inhibited the intermolecular charge and the LoD was determined× − to be 1.5 × 10−10 M. The addition of the Cd2+ ions inhibited the transferintermolecular (ICT) between charge transfer the lone (ICT) pair between on the secondary the lone pair amine on inthe the secondary imidazole amine ring in system the imidazole and the carbonylring system group and on the the carbonyl coumarin group unit. on It is the reasonable coumarin that unit. the It molecular is reasonable probe that would the chelatemolecular the probe metal ionswould in achelate tridentate the metal fashion, ions via in the a tridentate three Lewis fashio basicn, sitesvia the (Scheme three 9Lewis, red bonds).basic sites Additionally, (Scheme 9, thered Chemosensorsauthorsbonds). carriedAdditionally, 2019, 7 out, x an extensivethe authors anion carried study out which an showedextensive very anion little study spectral which changes showed for a numbervery22 littleof of50 tetrabutylammoniumspectral changes for a salts number (TBAX, of tetrabutylammonium where X = F−, Cl−, Br −salts,I−, (TBAX, OAc−, NO where3−,H X3 PO= F4−,− Cl, CN−, Br−).−, FluorideI−, OAc−, wasNO3− the, H only3PO4− anion, CN−). to Fluoride produce was a small the changesonly anion in the to fluorescenceproduce a small spectrum, changes which in the was fluorescence also shown spectrum,to inhibit the which ICT mechanism,was also shown by forming to inhibit a hydrogen the ICT mechanism, bonding interaction by forming with a the hydrogen NH group bonding in the interactionbenzimidazole with group. the NH group in the benzimidazole group.

Scheme 9. Proposed binding of molecular probe 24 and Cd2+ ions.

Scheme 9. Proposed binding of molecular probe 24 and Cd2+ ions.

Another LMFP coumarin dyad, 25, was prepared by Kumar and Ahmed [179]. The molecular probe served as, both, a colorimetric sensor and a fluorescent sensor, through chelation-enhanced fluorescence (CHEF), Figure 9. Upon the addition of Cd2+ ions in a HEPES-buffered solution (20 mM, CH3CN:H2O, 3:7, v/v, pH 7.0) of probe 25, the solution changed from yellow to colorless, whereas there were no color changes observed upon the addition of the other metal ions studied (Na+, K+, Ba2+, Ca2+, Mg2+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Ag+, Al3+, Hg2+, Hg+, and Sn2+). On the addition of Cd2+ ions to a solution of 25, there was a blue shift in the absorbance band from 387 nm to 370 nm, whereas the addition of other metal ions resulted in no notable changes to the absorbance spectra. Titration studies were carried out to further study the interactions between 25 and Cd2+ ions. The gradual changes in the absorbance bands suggests that a new species was produced during the titration. A Job’s plot was used to verify the formation of a 1:1 complex between the sensor and cadmium ions and a Benesi–Hilderbrand plot was used to calculate an association constant of 1.8 × 106 M−1. Furthermore, the LoD was calculated to be 6.3 × 10−8 M. The fluorescence data of the sensor with different metal ions was analogous to the absorbance data, as there were only notable spectroscopic changes in the presence of Cd2+ ions. Under the same solution conditions as the absorbance experiments, 25 was non-fluorescent. However, in the presence of Cd2+ ions, a gradual increase in the emission band at 495 nm was observed and the absolute fluorescence quantum yield (φ) increased by a factor of nine, upon coordination with the Cd2+ ions. A fluorescence lifetime decay was observed from 0.217 to 1.97 ns, after the addition of Cd2+ ions, indicative of the strong coordination between the sensor and Cd2+ ions. In an attempt to prepare a workable probe, test strips were prepared, which changed color upon the immersion into a Cd2+ solution (5 × 10−4 M), Figure 9. Interestingly, the authors used their probe to monitor the amount of Cd2+ in lipstick, hair dye, and makeup cream as Cd2+ is a common ion found in the preparation of these cosmetics. Spectrofluorometry and AAS analysis showed the amount of Cd2+ ions in the samples to be very similar (Table 2).

Chemosensors 2019, 7, 22 22 of 48

Another LMFP coumarin dyad, 25, was prepared by Kumar and Ahmed [179]. The molecular probe served as, both, a colorimetric sensor and a fluorescent sensor, through chelation-enhanced fluorescence (CHEF), Figure9. Upon the addition of Cd 2+ ions in a HEPES-buffered solution (20 mM, CH3CN:H2O, 3:7, v/v, pH 7.0) of probe 25, the solution changed from yellow to colorless, whereas there were no color changes observed upon the addition of the other metal ions studied (Na+,K+, Ba2+, Ca2+, Mg2+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Ag+, Al3+, Hg2+, Hg+, and Sn2+). On the addition of Cd2+ ions to a solution of 25, there was a blue shift in the absorbance band from 387 nm to 370 nm, whereas the addition of other metal ions resulted in no notable changes to the absorbance spectra. Titration studies were carried out to further study the interactions between 25 and Cd2+ ions. The gradual changes in the absorbance bands suggests that a new species was produced during the titration. A Job’s plot was used to verify the formation of a 1:1 complex between the sensor and cadmium ions and a Benesi–Hilderbrand plot was used to calculate an association constant of 1.8 106 M 1. Furthermore, the LoD was calculated to be 6.3 10 8 M. The fluorescence data × − × − of the sensor with different metal ions was analogous to the absorbance data, as there were only notable spectroscopic changes in the presence of Cd2+ ions. Under the same solution conditions as the absorbance experiments, 25 was non-fluorescent. However, in the presence of Cd2+ ions, a gradual increase in the emission band at 495 nm was observed and the absolute fluorescence quantum yield (φ) increased by a factor of nine, upon coordination with the Cd2+ ions. A fluorescence lifetime decay was observed from 0.217 to 1.97 ns, after the addition of Cd2+ ions, indicative of the strong coordination betweenChemosensors the 2019 sensor, 7, x and Cd2+ ions. In an attempt to prepare a workable probe, test strips were prepared,23 of 50 which changed color upon the immersion into a Cd2+ solution (5 10 4 M), Figure9. × −

(A) (B)

Figure 9. (A) Proposed binding between compound 25 and Cd2+ ions and (B) photographs of colorimetricFigure 9. (A (left) Proposed) and fluorometric binding between (right) test compound kits. The filter25 and papers Cd2+ wereions coatedand (B with) photographs25 (0.1 mM) of andcolorimetric then immersed (left) and into fluorometric a HEPES-bu ff(rightered) solution test kits. (20 The mM, filter CH papers3CH-H were2O 3:7, coatedv/v, pHwith 7). 25 Adapted (0.1 mM) fromand [then179] withimmersed permission into a fromHEPE theS-buffered Centre Nationalsolution (20 de lamM, Recherche CH3CH-H Scientifique2O 3:7, v/v, (CNRS) pH 7). andAdapted the Royalfrom Society[179] with of Chemistry. permission from the Centre National de la Recherche Scientifique (CNRS) and the Royal Society of Chemistry. Interestingly, the authors used their probe to monitor the amount of Cd2+ in lipstick, hair dye, and 2+ makeupTable cream 2. asThe Cd calculatedis a common concentration ion found of inCd the2+ ions preparation in cosmetics of these and cosmetics. personal Spectrofluorometrycare samples by 2+ and AASfluorometric analysis and showed atomic the absorp amounttion spectroscopy of Cd ions (AAS) in the [179]. samples to be very similar (Table2).

Amount of Cd2+ in mol L−1) Sample Fluorometric with probe 25 AAS Lipstick 2.07 × 10−6 1.96 × 10−6 Hair color 1.12 × 10−6 1.06 × 10−6 Makeup cream 7.92 × 10−6 7.20 × 10−6

Many molecular probes designed to bind cadmium can also bind zinc ions and it is often difficult to distinguish between the two. The binding ratio between the metal and the probe can be enough to discriminate between two metal ions. For example, LMWP 26 was synthesized by Rajak et al. Scheme 10 [180]. Fluorene–pyridine were coupled together via an imine bond. Compound 26 served as both a colorimetric sensor, producing noticeable color changes in the presence of both zinc and cadmium, and a turn-on fluorescence sensor. As Cd2+ ions were added to a CH3CN:H2O solution (8:2, v/v, 10 mM HEPES, pH 7.4) of 26, the solution changed from pale yellow to deep orange, whereas a deep pink color was observed in the presence of Zn2+ ions. The UV-Vis titration data showed the emergence of several new bands, upon the addition of Cd2+ ions to a solution of the sensor, with a noticeable difference to that of the Zn2+ ion titration. Fluorescence titrations further highlighted the probe’s selectivity for Cd2+ and Zn2+ ions. Decomplexation of the [CdCl(26)] complex and subsequent emission turn off could be accomplished with the addition of S2- ions to a solution of the complex. The fluorescence turn-off also served as a means of distinguishing between Zn2+ and Cd2+ ions, as decomplexation of the Zn2+ ion’s complex did not occur in the presence of S2− ions. A Job’s plot verified the binding stoichiometry of the sensor for Cd2+ ions as 1:1 with an association constant of 6.4 × 104 M−1, calculated from a Benesi–Hildebrand plot using the data from UV-Vis titrations. The weak fluorescence band seen around 430 nm is due to the lone pair on the nitrogen atom on the imine group quenching the intensity. However, on chelation of Cd2+ ions with the sensor, the PET process is prevented and the molecule becomes less flexible, as the free rotation of the azomethine carbon is also restricted. The coordination environment was further validated by studying the 1H NMR titration of Cd2+ ions into a solution of the sensor which showed a clear disappearance of the phenolic proton and a downfield shift of the azomethine proton. From the fluorescence titration, an LoD of 0.53 μM for Cd2+ ions was calculated and a fluorescence quantum yield of 0.996 was observed, whereas the fluorescence Chemosensors 2019, 7, 22 23 of 48

Table 2. The calculated concentration of Cd2+ ions in cosmetics and personal care samples by fluorometric and atomic absorption spectroscopy (AAS) [179].

Amount of Cd2+ in mol L 1) Sample · − Fluorometric with Probe 25 AAS Lipstick 2.07 10 6 1.96 10 6 × − × − Hair color 1.12 10 6 1.06 10 6 × − × − Makeup cream 7.92 10 6 7.20 10 6 × − × −

Many molecular probes designed to bind cadmium can also bind zinc ions and it is often difficult to distinguish between the two. The binding ratio between the metal and the probe can be enough to discriminate between two metal ions. For example, LMWP 26 was synthesized by Rajak et al. Scheme 10[ 180]. Fluorene–pyridine were coupled together via an imine bond. Compound 26 served as both a colorimetric sensor, producing noticeable color changes in the presence of both zinc and 2+ cadmium, and a turn-on fluorescence sensor. As Cd ions were added to a CH3CN:H2O solution (8:2, v/v, 10 mM HEPES, pH 7.4) of 26, the solution changed from pale yellow to deep orange, whereas a deep pink color was observed in the presence of Zn2+ ions. The UV-Vis titration data showed the emergence of several new bands, upon the addition of Cd2+ ions to a solution of the sensor, with a noticeable difference to that of the Zn2+ ion titration. Fluorescence titrations further highlighted the probe’s selectivity for Cd2+ and Zn2+ ions. Decomplexation of the [CdCl(26)] complex and subsequent emission turn off could be 2 accomplished with the addition of S − ions to a solution of the complex. The fluorescence turn-off also served as a means of distinguishing between Zn2+ and Cd2+ ions, as decomplexation of the Zn2+ 2 ion’s complex did not occur in the presence of S − ions. A Job’s plot verified the binding stoichiometry of the sensor for Cd2+ ions as 1:1 with an association constant of 6.4 104 M 1, calculated from a × − Benesi–Hildebrand plot using the data from UV-Vis titrations. The weak fluorescence band seen around 430 nm is due to the lone pair on the nitrogen atom on the imine group quenching the intensity. However, on chelation of Cd2+ ions with the sensor, the PET process is prevented and the molecule becomes less flexible, as the free rotation of the azomethine carbon is also restricted. The coordination environment was further validated by studying the 1H NMR titration of Cd2+ ions into a solution of the sensor which showed a clear disappearance of the phenolic proton and a downfield shift of the Chemosensorsazomethine 2019, proton.7, x From the fluorescence titration, an LoD of 0.53 µM for Cd2+ ions was calculated24 of 50 and a fluorescence quantum yield of 0.996 was observed, whereas the fluorescence quantum yield of quantum26 was yield calculated of 26 towas be calculated 0.042. As Zn to2 be+ and 0.042. Cd 2As+ produced Zn2+ and diCdff2+erent produced optical different inputs with optical one inputs output, a withbinary one logicoutput, gate a binary function logic could gate be function produced coul Tabled be3). produced This selective Table behavior 3). This mimicsselective an behavior OR binary mimicslogic an gate OR (Figure binary 10 lo).gic gate (Figure 10).

SchemeScheme 10. 10. SchematicSchematic representation representation the the binding binding behavior behavior of ofprobe probe 2626 andand Cd Cd2+ and2+ and Zn2+ Zn ions.2+ ions.

Figure 10. Logic schemes prepared from the binary inputs/outputs in the Truth tables [180].

Table 3. Truth table highlighting the binary inputs and the corresponding digital output [180].

(A) (B) (C) Input Output* Input Output* Input Output*

Zn2+ Cd2+ λem 485 or 495 nm Zn2+ EDTA λem 485 nm Cd2+ S2− λem 495 nm 0 0 0 0 0 0 0 0 0 1 0 1 1 0 1 1 0 1 0 1 1 0 1 0 0 1 0 1 1 1 1 1 0 1 1 0 * (A) OR logic gate, the output “1” is assigned to Zn2+ and Cd2+ when the emission bands appears at 485 and 495 nm, respectively and “0” in the absence of metal i.e., no emission. (B) and (C) logic gates operate as an INHIBIT function (a combination of an AND function plus an additional event, in this xample NOT). Addition of the metal produces the output “1” and the inverter (EDTA for Zn2+ or S2− for Cd2+) switches off the emission, output “0”. A simple cleft-shaped fluorescent chemosensor, 27, was prepared by Qian and Yang, Scheme 11 [181]. Originally prepared to bind group 1 and group 2 metal ions, NH4+ ions and heavy metal salts in methanol and at high concentrations (1.9 × 10−2 M) by Maass and Vögtle [182–184], the molecular cleft did not discriminate between different metal ions. The fluorescence spectra for 10 μM of compound 27 (20 mM Tris-HCl, pH 7.4) with Cd2+ ions showed a significant fluorescence band at 417 nm. The other metals studied did not show any response (Ba2+, Mn2+, Hg2+, Ni2+, Ca2+, Cu2+, Co2+, Pb2+, Mg2+, Zn2+, Cd2+, Fe2+, Fe3+, Cr3+, Ag+, Li+, Na+, and K+). The PET mechanism quenched the initial fluorescence intensity, via the lone pairs on the nitrogen atom. On the addition of Cd2+ ions, the flexible molecular cleft became rigid and the fluorescence increased, due to CHEF, and the π-π* transitions gave rise to the emission band observed. The association constant calculated by the nonlinear least square analysis was found to be 5.3 × 107 M−1.

Chemosensors 2019, 7, x 24 of 50

quantum yield of 26 was calculated to be 0.042. As Zn2+ and Cd2+ produced different optical inputs with one output, a binary logic gate function could be produced Table 3). This selective behavior mimics an OR binary logic gate (Figure 10).

Chemosensors 2019, 7, 22 24 of 48 Scheme 10. Schematic representation the binding behavior of probe 26 and Cd2+ and Zn2+ ions.

Figure 10. Logic schemes prepared from the binary inputs/outputs in the Truth tables [180].

TableFigure 3. Truth10. Logic table schemes highlighting prepared the binaryfrom the inputs binary and inputs/outputs the corresponding in the digitalTruth tables output [180]. [180 ]. (A) (B) (C) Table 3. Truth table highlighting the binary inputs and the corresponding digital output [180]. Input Output * Input Output * Input Output * 2+ 2+ (A) 2+ (B) 2+ (C)2 Zn Cd λem 485 or 495 nm Zn EDTA λem 485 nm Cd S − λem 495 nm Input Output* Input Output* Input Output* 0 0 0 0 0 0 0 0 0 2+ 2+ λ 2+ λ 2+ 2− λ 1Zn 0Cd em 485 1 or 495 nm 1Zn EDTA 0 em 485 1 nm Cd 1 S 0em 495 nm 1 00 10 1 0 00 10 0 0 0 0 1 0 0 11 10 1 1 11 10 1 0 1 0 1 1 0 *(A) OR0 logic1 gate, the output 1 “1” is assigned 0 to Zn2+ and1 Cd2+ when 0 the emission 0 bands1 appears 0 at 485 and 495 nm,1 respectively1 and “0” in 1 the absence of metal1 i.e., no1 emission. (B 0 ) and (C) logic1 gates operate1 as an0 INHIBIT function (a combination of an AND function plus an additional event, in this xample NOT). Addition of the metal * (A) OR logic gate, the output “1” is assigned to2 +Zn2+ and2 Cd2+2 +when the emission bands appears at produces the output “1” and the inverter (EDTA for Zn or S − for Cd ) switches off the emission, output “0”. 485 and 495 nm, respectively and “0” in the absence of metal i.e., no emission. (B) and (C) logic gates operate as an INHIBIT function (a combination of an AND function plus an additional event, in this A simple cleft-shaped fluorescent chemosensor, 27, was prepared by Qian and Yang, xample NOT). Addition of the metal produces the output “1” and the inverter (EDTA for Zn2+ or S2− Scheme 11[ 181]. Originally prepared to bind group 1 and group 2 metal ions, NH + ions and for Cd2+) switches off the emission, output “0”. 4 heavy metal salts in methanol and at high concentrations (1.9 10 2 M) by Maass and Vögtle [182–184], × − the molecularA simple cleftcleft-shaped did not fluorescent discriminate chemosensor, between diff 27erent, was metal prepared ions. by The Qian fluorescence and Yang, Scheme spectra 11 for 10[181].µM ofOriginally compound prepared27 (20 to mM bind Tris-HCl, group 1 pH and 7.4) group with 2 Cdmetal2+ ions ions, showed NH4+ ions a significant and heavy fluorescence metal salts bandin methanol at 417 nm. and The at high other concentrations metals studied (1.9 did × not10−2 showM) by any Maass response and Vögtle (Ba2+ ,[182–184], Mn2+, Hg the2+, Nimolecular2+, Ca2+ , Cucleft2+, did Co2 +not, Pb discriminate2+, Mg2+, Zn 2between+, Cd2+, Fedifferent2+, Fe3 +metal, Cr3+ ,ions. Ag+ ,The Li+ ,fluorescence Na+, and K+ ).spectra The PET for mechanism10 μM of 2+ quenchedcompound the 27 initial (20 mM fluorescence Tris-HCl, pH intensity, 7.4) with via Cd the loneions pairsshowed on a the significant nitrogen fluorescence atom. On the band addition at 417 of 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ Cdnm.2+ Theions, other the flexible metals molecularstudied did cleft not becameshow any rigid response and the (Ba fluorescence, Mn , Hg increased,, Ni , Ca due, Cu to, CHEF,Co , Pb and, 2+ 2+ 2+ 2+ 3+ 3+ + + + + theMgπ-,π Zn* transitions, Cd , Fe gave, Fe rise, Cr to the, Ag emission, Li , Na band, and observed. K ). The ThePET associationmechanism constant quenched calculated the initial by Chemosensorsfluorescence 2019 intensity,, 7, x via the lone pairs on the nitrogen 7atom.1 On the addition of Cd2+ ions,25 ofthe 50 the nonlinear least square analysis was found to be 5.3 10 M− . flexible molecular cleft became rigid and the fluorescence× increased, due to CHEF, and the π-π* transitions gave rise to the emission band observed. The association constant calculated by the nonlinear least square analysis was found to be 5.3 × 107 M−1.

Scheme 11. Proposed binding mode of 27 and Cd2+ ions.

Scheme 11. Proposed binding mode of 27 and Cd2+ ions. Liu and Wang took advantage of the encapsulating and adsorbing capabilities of cationic surfactant hexadecyl trimethyl ammonium bromide (CTAB) and the selectivity of thymine-squaraine Liu and Wang took advantage of the encapsulating and adsorbing capabilities of cationic based fluorescent chemosensor 28, to develop a system capable of sensing and capturing Cd2+ ions, surfactant hexadecyl trimethyl ammonium bromide (CTAB) and the selectivity of thymine-squaraine Figure 11[ 185]. The thymine moiety of 28 efficiently bound to Cd2+ ions, while the squaraine moiety based fluorescent chemosensor 28, to develop a system capable of sensing and capturing Cd2+ ions, served as a signaling unit whose spectroscopic performances could be tuned by changing its solution Figure 11 [185]. The thymine moiety of 28 efficiently bound to Cd2+ ions, while the squaraine moiety environments. The CTAB micelles could induce aggregation of the squaraine dyes, reducing the served as a signaling unit whose spectroscopic performances could be tuned by changing its solution likelihood of any intermolecular electron transfer. On the addition of Cd2+ ions, the fluorescence environments. The CTAB micelles could induce aggregation of the squaraine dyes, reducing the intensity of compound 28 increased. Optimal conditions for the system were observed when the likelihood of any intermolecular electron transfer. On the addition of Cd2+ ions, the fluorescence concentration of CTAB was less than 1 mM. At higher concentrations, the CTAB micelles made it intensity of compound 28 increased. Optimal conditions for the system were observed when the difficult for the Cd2+ ions to access the thymine moiety via an adsorption process into the cell. Under concentration of CTAB was less than 1 mM. At higher concentrations, the CTAB micelles made it these conditions, a 5-fold increase in the emission band at 660 nm was observed for compound 28 difficult for the Cd2+ ions to access the thymine moiety via an adsorption process into the cell. Under these conditions, a 5-fold increase in the emission band at 660 nm was observed for compound 28 in the presence of Cd2+ ions. There was no notable emission change observed for any other metal ions that were studied (K+, Zn2+, Li+, Fe3+, Hg2+, Fe2+, Ag+ Co2+ Na+ Ca2+, and Cu2+). The sensor was also found to be very sensitive, demonstrating an LoD in the nM range. The binding constant was determined using a Benesi–Hildebrand plot and found to be 2 × 103 M−1. Additionally, a Job’s plot revealed that the binding stoichiometry between the squaraine moiety and Cd2+ ions was 1:1. Competition studies did not highlight any notable inhibition, due to the presence of other metal ions. However, the presence of some anionic and non-ionic surfactants (sodium dodecyl sulfate (SDS), Dodecylbenzene sulfonic acid sodiym salt (SDBS), and Triton X-100) did diminish the probe’s ability to detect Cd2+ ions. The micelle was found to be stable to photobleaching, as decomplexation did not occur when the soft material was exposed to constant irradiation for 6 hours.

Chemosensors 2019, 7, 22 25 of 48

in the presence of Cd2+ ions. There was no notable emission change observed for any other metal ions that were studied (K+, Zn2+, Li+, Fe3+, Hg2+, Fe2+, Ag+ Co2+ Na+ Ca2+, and Cu2+). The sensor was also found to be very sensitive, demonstrating an LoD in the nM range. The binding constant was determined using a Benesi–Hildebrand plot and found to be 2 103 M 1. Additionally, a Job’s × − plot revealed that the binding stoichiometry between the squaraine moiety and Cd2+ ions was 1:1. Competition studies did not highlight any notable inhibition, due to the presence of other metal ions. However, the presence of some anionic and non-ionic surfactants (sodium dodecyl sulfate (SDS), Dodecylbenzene sulfonic acid sodiym salt (SDBS), and Triton X-100) did diminish the probe’s ability to detect Cd2+ ions. The micelle was found to be stable to photobleaching, as decomplexation did not Chemosensorsoccur when2019, 7, thex soft material was exposed to constant irradiation for 6 h. 26 of 50

Figure 11. The proposed intermolecular interaction between compound 28 and the Cd2+ ions encapsulated in the micelle. Adapted and reprinted from [183], with permission from Elsevier. Figure 11. The proposed intermolecular interaction between compound 28 and the Cd2+ ions encapsulatedTwo turn-“on” in the micelle. fluorescent Adapted sensors and compoundsreprinted from29a [183],and with29b werepermission synthesized from Elsevier. by Xu and coworkers, 2+ 2+ Scheme 12[ 186]. Both compounds could bind both Zn and Cd ions in a CH3OH/H2O solution Two turn-“on” fluorescent1 sensors compounds 29a and 29b were synthesized by Xu and (1:1, v/v, Tris, 10 mmol L− , pH 7.4). These molecular probes displayed a weak fluorescence emission coworkers, Scheme 12 [186].· Both compounds could bind both Zn2+ and Cd2+ ions in a CH3OH/H2O band, however, in the presence of Zn2+ and Cd2+ ions, an eight-fold increase in the emission intensity, solution (1:1, v/v, Tris, 10 mmol⋅L−1, pH 7.4). These molecular probes displayed a weak fluorescence due to CHEF mechanism, was observed at 430 nm. Data obtained from the fluorescence titrations and emission band, however, in the presence of Zn2+ and Cd2+ ions, an eight-fold increase in the emission Job’s plots, suggested a 1:1 complex formation. This was supported by NMR studies. The calculated intensity, due to CHEF mechanism, was observed at 430 nm. Data obtained from the fluorescence association constants of compounds 29a and 29b to Cd2+ ions were 1.5 107 M 1and 1.0 106 M 1, titrations and Job’s plots, suggested a 1:1 complex formation. This was supported× by− NMR studies.× − respectively. Competition studies were carried out using other metal ions but there were no notable The calculated association constants of compounds 29a and 29b to Cd2+ ions were 1.5 × 107 M−1and 1.0 interferences observed in the fluorescence spectra. There were however interferences from biologically × 106 M−1, respectively. Competition studies were carried out using other metal ions but there were relevant anions, namely adenosine triphosphate (ATP), adenosine diphosphate (ADP), and other no notable interferences observed in the fluorescence spectra. There were however interferences from phosphate ions. Complete quenching was observed when phosphate ions were introduced to a solution biologically relevant anions, namely adenosine triphosphate (ATP), adenosine diphosphate (ADP), of the complex demonstrating the complex’s ability to serve as an on–off sensor for phosphate ions. and other phosphate ions. Complete quenching was observed when phosphate ions were introduced to a solution of the complex demonstrating the complex’s ability to serve as an on–off sensor for phosphate ions.

ChemosensorsChemosensors2019 2019, 7, 7 22, x 27 of26 50 of 48

Scheme 12. The proposed interaction between the molecular sensors and the metal ions. The binding 2+ 2+ cavityScheme size 12. was The ideal proposed to chelate interaction Cd betweenor Zn theions. molecu Whenlar sensors the phosphate and the metal anion ions. was The added, binding the fluorescencecavity size intensitywas ideal was to switchedchelate Cd off2+. or Zn2+ ions. When the phosphate anion was added, the fluorescence intensity was switched off. Kim and Harrison synthesized a tripodal receptor containing quinoline and pyridyl aminophenol 2+ moietiesKim (compound and Harrison30)[187 synthesized]. It was shown a tripodal to prefer toreceptor coordinate containing with Zn quinolineions in CHand3CN pyridyl solution 2+ butaminophenol had a preference moieties for Cd(compoundions in 30 aqueous) [187]. It solutions. was shown Optical to prefer investigations to coordinate were with carried Zn2+ outions in in the + + 2+ 2+ 2+ 2+ 3+ 3+ 3+ + presenceCH3CN of solution 19 diff erentbut had nitrate a preference metal salts for Cd (Na2+ ions,K in, aqueous Zn , Cd solutions., Mg ,Optical Ca , Alinvestigations, Ga , In were, Pb , 2+ 3+ 2+ 2+ 3+ 2+ 2+ 2+ + Hgcarried, Cr out, Mn in the, Fepresence, Fe of, Co19 different, Ni , nitrate Cu , Agmetal) atsalts 10 (NaµM+, in K+ CH, Zn32+CN, Cd solution.2+, Mg2+, Ca The2+, Al addition3+, Ga3+, of 2+ ZnIn3+ions, Pb+, lead Hg2+ to, Cr a3+ large, Mn2+ fluorescence, Fe2+, Fe3+, Co enhancement2+, Ni2+, Cu2+, (Agφ +=) at0.001 10 μ toMφ in= CH0.24)3CN at solution. 510 nm. The The addition only other metalof Zn ion2+ ions that lead produced to a large an emissionfluorescence intensity enhancement increase (φ was = 0.001 Cd2 +tobut φ = 0.24) to a lesser at 510extent, nm. The approximately only other onemetal third ion of that the produced magnitude an seen emission for Zn intensity2+ (φ= increase0.039). The was other Cd2+ but metals to a lesser did not extent, lead approximately to an increase in fluorescentone third intensity,of the magnitude but several seen did for interfere Zn2+ (φ= with0.039). the The probe’s other abilitymetals todid bind not tolead Zn to2+ an. Similar increase optical in fluorescent intensity, but several did interfere with the probe’s ability to bind to Zn2+. Similar optical investigations were undertaken in a CH3CN:H2O (1:1, v/v) HEPES-buffered solution (10 mM, pH 7.4). Theinvestigations fluorescence were enhancement undertaken seen in in athe CH presence3CN:H2O of (1:1, Cd 2+v/v)remained HEPES-buffered the same, solution but the fluorescence(10 mM, pH 7.4). The fluorescence enhancement 2seen+ in the presence of Cd2+ remained the same, but the enhancement seen in the presence of Zn was only 10% of that seen in CH3CN. Other metal ions 2+ didfluorescence not lead to enhancement an increase in seen intensity, in the presence but Zn2+ of, Cr Zn3+,was Cu2 +only, Co 210%+, Ni of2 +that, Hg seen2+ interfered in CH3CN. with Other Cd 2+ metal ions did not lead to an increase in intensity, but Zn2+, Cr3+, Cu2+, Co2+, Ni2+, Hg2+ interfered with binding. The differences in fluorescence intensities between the two solvent systems suggested that the Cd2+ binding. The differences in fluorescence intensities between the two solvent systems suggested coordination environments for Cd2+ ions were similar in both. The Zn2+ ion could adopt an equilibrium that the coordination environments for Cd2+ ions were similar in both. The Zn2+ ion could adopt an between tetrahedral and octahedral geometries. The binding environment for [Cd(NO ) (30)] was equilibrium between tetrahedral and octahedral geometries. The binding environment3 2 for confirmed by solid-state studies (Figure 12) and supported by NMR studies. The weak fluorescence [Cd(NO3)2(30)] was confirmed by solid-state studies (Figure 12) and supported by NMR studies. The 2+ seenweak for fluorescence30 was a consequence seen for 30 of was PET a consequence quenching, which of PET was quenching, inhibited which upon was the inhibited coordination upon of the Zn ions. The binding constants, in CH CN were calculated to be 1.4 106 M 1 and 5.6 105 M 1, for coordination of Zn2+ ions. The binding3 constants, in CH3CN were calculated− to be 1.4 × 106 M−1 and− 2+ 2+ × × Zn5.6, × and 105 Cd M−1, respectively,for Zn2+, and but Cd it2+ isrespectively, unclear how but these it is binding unclear constants how these were binding obtained, constants as no bindingwere 2+ 2+ isothermsobtained, were as no reported. binding isotherms It was also were claimed reported. that 30 It wasbound also Cd claimedions that more 30 favorably bound Cd than2+ ions Zn moreions infavorably an organic-aqueous than Zn2+ ions system. in an No organic-aqueous binding constants system. were No reported binding toconstants back up were this claim,reported only to aback single fluorescenceupChemosensors this claim, 2019 spectrum only, 7, x a single showed fluorescence that the intensity spectrum was showed greater that for the Cd intensity2+ ions. was greater for Cd2+28 ions. of 50

Figure 12. X-Ray crystal structure of [Cd(NO ) (30)]. Adapted from [187] with permission from The 3 2 RoyalFigure Society 12. X-Ray of Chemistry. crystal structure of [Cd(NO3)2(30)]. Adapted from [187] with permission from The Royal Society of Chemistry.

Hou and Wang developed a phenylene-acetylene based compound 31 that was capable of selectively detecting Fe2+ by colorimetric methods and Cd2+ and Zn2+ via ratiometric fluorescent detection [188]. Optical studies were carried out in the presence of 23 different chloride or perchlorate metal salts (Li+, Ba2+, Ca2+, K+, Li+, Mg2+, Mn2+, Na+, Cr3+, Au3+, Zr4+, Rh3+, Pb2+, Ag+, Co2+, Cu2+, Ni2+, Hg2+, Cd2+, Zn2+, Fe2+, Fe3+, Pt2+). In an EtOH:H2O (10 μM, 1:1, v/v) solution, 31 showed two π-π* absorption bands at 286 nm and 327 nm, and the addition of Fe2+ ion led to the appearance of a new peak at 573 nm, with a corresponding color change from colorless to purple-red. The other metals did not produce any significant changes in the absorption spectra, and their presence did not interfere with the probe’s response to Fe2+. Job’s plot and ESI-MS data indicated that the 31:Fe2+ complex had a 2:1 binding stoichiometry and the association constant was calculated to be 3.4 × 107 M−1. The limit of detection for the method was found to be 2.0 × 10−8 M. When excited at 340 nm, 31 showed an emission at 408 nm and the addition of Cd2+ ions led to a decrease in the intensity of the emission at 408 nm, the appearance of a new emission at 475 nm, and a change in fluorescent color from blue to cyan. The addition of Zn2+ ions led to a decrease in the intensity of the emission at 408 nm, the appearance of a new emission at 498 nm, and a change in fluorescent color from blue to yellow-green. No other metals showed a significant response, and only Fe2+ interfered with the probe’s response to Cd2+ and Zn2+. The ratios of the two emissions (I475/I408 and I498/I408) could be used to calculate the LoD for Cd2+ and Zn2+ of 2.6 × 10−8 M and 2.0 × 10−9 M, respectively. Job’s plot and ESI- MS data indicated that the 31:Cd2+ complex had a 1:1 binding stoichiometry and the association constant was calculated to be 0.2 × 106 M−1. The 31:Zn2+ complex had a 2:1 binding stoichiometry, and an association constant of 0.9 × 106 M−1. 1H NMR data indicated that the metals bound to the nitrogen atoms; the proposed sensing mechanism for the probe toward Fe2+, Cd2+, and Zn2+ is presented in Scheme 13.

Chemosensors 2019, 7, 22 27 of 48

Hou and Wang developed a phenylene-acetylene based compound 31 that was capable of selectively detecting Fe2+ by colorimetric methods and Cd2+ and Zn2+ via ratiometric fluorescent detection [188]. Optical studies were carried out in the presence of 23 different chloride or perchlorate metal salts (Li+, Ba2+, Ca2+,K+, Li+, Mg2+, Mn2+, Na+, Cr3+, Au3+, Zr4+, Rh3+, Pb2+, Ag+, Co2+, Cu2+, 2+ 2+ 2+ 2+ 2+ 3+ 2+ Ni , Hg , Cd , Zn , Fe , Fe , Pt ). In an EtOH:H2O (10 µM, 1:1, v/v) solution, 31 showed two π-π* absorption bands at 286 nm and 327 nm, and the addition of Fe2+ ion led to the appearance of a new peak at 573 nm, with a corresponding color change from colorless to purple-red. The other metals did not produce any significant changes in the absorption spectra, and their presence did not interfere with the probe’s response to Fe2+. Job’s plot and ESI-MS data indicated that the 31:Fe2+ complex had a 2:1 binding stoichiometry and the association constant was calculated to be 3.4 107 M 1. The limit of × − detection for the method was found to be 2.0 10 8 M. When excited at 340 nm, 31 showed an emission × − at 408 nm and the addition of Cd2+ ions led to a decrease in the intensity of the emission at 408 nm, the appearance of a new emission at 475 nm, and a change in fluorescent color from blue to cyan. The addition of Zn2+ ions led to a decrease in the intensity of the emission at 408 nm, the appearance of a new emission at 498 nm, and a change in fluorescent color from blue to yellow-green. No other metals showed a significant response, and only Fe2+ interfered with the probe’s response to Cd2+ and Zn2+. 2+ The ratios of the two emissions (I475/I408 and I498/I408) could be used to calculate the LoD for Cd and Zn2+ of 2.6 10 8 M and 2.0 10 9 M, respectively. Job’s plot and ESI-MS data indicated that the × − × − 31:Cd2+ complex had a 1:1 binding stoichiometry and the association constant was calculated to be 0.2 106 M 1. The 31:Zn2+ complex had a 2:1 binding stoichiometry, and an association constant of × − 0.9 106 M 1. 1H NMR data indicated that the metals bound to the nitrogen atoms; the proposed × − sensingChemosensors mechanism 2019, 7, x for the probe toward Fe2+, Cd2+, and Zn2+ is presented in Scheme 13. 29 of 50

Scheme 13. The binding modes of probe 31 and the didifferentfferent metal ions.ions.

2.3. Mercury Probes Out of the three metal ions in group 12, mercury in the form form of of organic organic mercury mercury is the the most most toxic. toxic. Therefore, designingdesigning molecular molecular chemosensors chemosensors for mercuryfor mercury ions isions a very is importanta very important endeavor, endeavor, especially inespecially aqueous in systems, aqueous as certainsystems, biological as certain species biological readily species convert readily metallic convert mercury metallic into organic mercury mercury. into Thereorganic have mercury. been manyThere di havefferent been types many of fluorescentdifferent types sensors of fluorescent for mercury, sensors recent for examples mercury, include recent examples include quantum dots [189], biosensors [8], carbon dots [190], carbon nanoparticles [191], gold and silver nanoparticles[192,193], but the focus in this final section is on LMFP sensors. Highly selective off–on optical sensor 32 was synthesized by Ahmed et al. for the detection of mercuric ions in an organic-aqueous, HEPES-buffered (CH3CN:H2O, 1:2, v/v, pH 7.2) system [194], Compound 32 drastically changed color from yellow (340 nm) to purple (550 nm), upon the addition of Hg2+ ions and was also found to be highly fluorescent, with an emission band at 430 nm, when the coordination complex was formed in situ. The weak fluorescence emission of 32 was a consequence of PET quenching which was inhibited upon by the coordination of the Hg2+ ions. The binding constant was calculated to be 1.9 × 103 M−1 using the Benesi–Hildebrand linear regression analysis, which showed a straight line. However on closer inspection of the regression line, the data presented was not a Benesi–Hildebrand plot. The authors plotted an intensity ratio versus concentration of Hg2+ ions, therefore, the binding constant reported should be viewed with a little skepticism. The coordination environment was confirmed by IR and NMR studies, as compound 32 had distinct functional groups (carbonyl and hydrazide) that could act as IR labels, the stretching frequencies shifted when coordinated to Hg2+ ion, the tridentate binding motif was further supported by DFT calculations. The Hg2+ ion was removed from the binding site by the addition of KI ions to precipitate HgI2, which turned off the emission signal. Therefore, this “off–on–off” approach could be translated into binary code to build an INHIBIT logic gate (a combination function consisting of and AND gate plus one other event, in this example a NOT event), Figure 13.

Chemosensors 2019, 7, 22 28 of 48

quantum dots [189], biosensors [8], carbon dots [190], carbon nanoparticles [191], gold and silver nanoparticles [192,193], but the focus in this final section is on LMFP sensors. Highly selective off–on optical sensor 32 was synthesized by Ahmed et al. for the detection of mercuric ions in an organic-aqueous, HEPES-buffered (CH3CN:H2O, 1:2, v/v, pH 7.2) system [194], Compound 32 drastically changed color from yellow (340 nm) to purple (550 nm), upon the addition of Hg2+ ions and was also found to be highly fluorescent, with an emission band at 430 nm, when the coordination complex was formed in situ. The weak fluorescence emission of 32 was a consequence of PET quenching which was inhibited upon by the coordination of the Hg2+ ions. The binding constant was calculated to be 1.9 103 M 1 using the Benesi–Hildebrand linear regression analysis, × − which showed a straight line. However on closer inspection of the regression line, the data presented was not a Benesi–Hildebrand plot. The authors plotted an intensity ratio versus concentration of Hg2+ ions, therefore, the binding constant reported should be viewed with a little skepticism. The coordination environment was confirmed by IR and NMR studies, as compound 32 had distinct functional groups (carbonyl and hydrazide) that could act as IR labels, the stretching frequencies shifted when coordinated to Hg2+ ion, the tridentate binding motif was further supported by DFT calculations. The Hg2+ ion was removed from the binding site by the addition of KI ions to precipitate HgI2, which turned off the emission signal. Therefore, this “off–on–off” approach could be translated into binary code to build an INHIBIT logic gate (a combination function consisting of and AND gate Chemosensorsplus one other 2019, event,7, x in this example a NOT event), Figure 13. 30 of 50

(A)

(B)

Input Output In 1 (Hg2+) In 2 (KI) Fluorescence enhancement at 429 nm 0 0 0 0 1 0 1 0 1 1 1 0

(C)

Figure 13. Molecular probe 32, highlights the proposed binding environment and color and fluorescence Figure 13. Molecular probe 32, highlights the proposed binding environment and color and changes (A) chemical scheme, (B) circuit diagram corresponding to the Hg2+ and KI inputs and (C) truth fluorescence changes (A) chemical scheme, (B) circuit diagram corresponding to the Hg2+ and KI tabled [194]. inputs and (C) truth tabled [194].

Aggregation-induced emission (AIE) has become a popular mechanism exploited for sensors often used in chemodosimeter and chemoreactand type molecular probes. A recent review covers the use of chemodosimeters (non-reversible covalent bonds and chemoreactands with reversible covalent bonds), whereby, the optical properties were different from the bound and unbound chemical adducts [53]. Chatterjee and coworkers have prepared chemodosimeter 33 to detect organic and inorganic Hg2+ species, Figure 14 [193]. The probe functions based on a mercury ion-promoted transmetalation reaction between tetraphenylethylene mono-boronic acid 33 and Hg2+ ions. For aggregation to occur, the correct working concentration needed to be established, which was found to be 30 μM (H2O-THF (90:10)). On the addition of HgCl2 to probe 33, transmetalation occurred to form compound 33a. As a consequence of the chemical transformation, the planarity of 33a-HgCl was restored and the onset of AIE occurred. The AIE aggregation was supported by transmission electron microscopy (TEM) and dynamic light scattering (DLS) experiments, which showed average nanoaggregates of 36 nm but, after the addition of HgCl2 to the 30 μM solution of 33, these grew to 690 nm. No other metal was shown to transmetalate compound 33, therefore, the molecular chemodosimeter was very specific for Hg2+ ions. Interestingly, methylmercury ion was also shown to undergo transmetalation, forming 33b, which also aggregated and turned on emission. As methylmercury ion readily bioaccumulates in fish, the chemodosimeter was tested using zebrafish embryos. In the presence of methylmercury ion, fluorescence microscopic images showed that a distinctive blue glow occurred. This elegant experiment showed that transmetallation of the C-B bond to C-Hg bond, and aggregation can occur in vivo, making this a highly attractive approach to monitor organomercury species, in higher order fish species.

Chemosensors 2019, 7, 22 29 of 48

Aggregation-induced emission (AIE) has become a popular mechanism exploited for sensors often used in chemodosimeter and chemoreactand type molecular probes. A recent review covers the use of chemodosimeters (non-reversible covalent bonds and chemoreactands with reversible covalent bonds), whereby, the optical properties were different from the bound and unbound chemical adducts [53]. Chatterjee and coworkers have prepared chemodosimeter 33 to detect organic and inorganic Hg2+ species, Figure 14[ 193]. The probe functions based on a mercury ion-promoted transmetalation reaction between tetraphenylethylene mono-boronic acid 33 and Hg2+ ions. For aggregation to occur, the correct working concentration needed to be established, which was found to be 30 µM (H2O-THF (90:10)). On the addition of HgCl2 to probe 33, transmetalation occurred to form compound 33a. As a consequence of the chemical transformation, the planarity of 33a-HgCl was restored and the onset of AIE occurred. The AIE aggregation was supported by transmission electron microscopy (TEM) and dynamic light scattering (DLS) experiments, which showed average nanoaggregates of 36 nm but, after the addition of HgCl2 to the 30 µM solution of 33, these grew to 690 nm. No other metal was shown to transmetalate compound 33, therefore, the molecular chemodosimeter was very specific for Hg2+ ions. Interestingly, methylmercury ion was also shown to undergo transmetalation, forming 33b, which also aggregated and turned on emission. As methylmercury ion readily bioaccumulates in fish, the chemodosimeter was tested using zebrafish embryos. In the presence of methylmercury ion, fluorescence microscopic images showed that a distinctive blue glow occurred. This elegant experiment showed that transmetallation of the C-B bond to C-Hg bond, and aggregation can occur in vivo, making this a highly attractive approach to monitor organomercury species, in higher order fish species. Chemosensors 2019, 7, x 31 of 50

(A)

(B)

FigureFigure 14.14. Schematic representation representation of of the the se sensingnsing process process of of chemodosimeter chemodosimeter 3232 onon the the addition addition of 2+ 2+ + + ofHg Hg ionion and and CH CH3Hg3Hg. Reprinted. Reprinted (adapted) (adapted) with with permission permission from [[195].195]. ((AA))Chemical Chemical Scheme,Scheme, ((BB)) proposedproposed fluorescence fluorescence aggregation aggregation

AnotherAnother chemodosimeter, chemodosimeter, has has been been developed developed by Ou by et Oual. [196] et al. who [196 employed] who employeda protection– a protection–deprotectiondeprotection strategy to strategy monitor to mercury. monitor This mercury. approach This approachtakes advantage takes advantage of the distinctly of the distinctly different dielectronicfferent electronic properties properties of two diff oferent two molecules. different molecules. For example, For protected example, aldehydes protected and aldehydes ketones and can 2+ ketonesbe deprotected can be deprotected with Hg2+ withsalts. Hg As nosalts. other As nometal other ion metal causes ion a causes selective a selective cleavage cleavage under under these conditions, the molecular probes are highly selective for Hg2+ ions. Thioacetal-modified pyrene sensor 34 was prepared and was shown to be cleaved by Hg2+ ions, to form pyrenecarboxaldehyde 34a, Scheme 14. Compound 34 undergoes an off-on fluorescence response when Hg2+ ions are added. The Hg2+ ions deprotect the thioacetals to form the aldehyde functional group, confirmed by 1H NMR as the aldehyde proton appeared at 10.82 ppm. The UV-Vis and fluorescence experiments were used to verify that one equivalent of Hg2+ ions were needed to fully convert 34 to 34a.

Scheme 14. Chemodosimeter 34.

The pyridine ring is a versatile moiety and is often found in molecular receptors. Tetrahydrodibenzo phenanthridines, contain a pyridine functional group with five fused six- membered ring systems with connectivity, shown in Figure 15. This arrangement of ring systems are found in natural products and biologically active compounds [197,198]. Two bis(dithioester) substituted phenanthridines, compound 35 and compound 36, were synthesized by Sundaramurthy et al., who showed that they could coordinate with Hg2+ ions [199]. Mono-sulfur derivative 35 demonstrated a strong fluorescence turn on signal in an aqueous solution, in the presence of Hg2+ ions, whereas tetra-sulfur derivative 36 showed a strong fluorescence quenching

Chemosensors 2019, 7, x 31 of 50

(A)

(B)

Figure 14. Schematic representation of the sensing process of chemodosimeter 32 on the addition of

Hg2+ ion and CH3Hg+. Reprinted (adapted) with permission from [195]. (A)Chemical Scheme, (B) proposed fluorescence aggregation

Another chemodosimeter, has been developed by Ou et al. [196] who employed a protection– deprotection strategy to monitor mercury. This approach takes advantage of the distinctly different Chemosensors 2019, 7, 22 30 of 48 electronic properties of two different molecules. For example, protected aldehydes and ketones can be deprotected with Hg2+ salts. As no other metal ion causes a selective cleavage under these conditions,these conditions, the molecular the molecular probes are probes highly are selective highly for selective Hg2+ ions. for HgThioacetal-modified2+ ions. Thioacetal-modified pyrene sensor pyrene 34 wassensor prepared34 was and prepared was shown and was to shownbe cleaved to be by cleaved Hg2+ ions, by Hg to2+ formions, pyrenecarboxaldehyde to form pyrenecarboxaldehyde 34a, Scheme34a, 14. Scheme Compound 14. Compound 34 undergoes34 undergoes an off-on an fluorescence off-on fluorescence response response when Hg when2+ ions Hg are2+ added.ions are The added. Hg2+The ions Hg deprotect2+ ions deprotect the thioacetals the thioacetals to form tothe form aldehyde the aldehyde functional functional group, group,confirmed confirmed by 1H NMR by 1H as NMR the aldehydeas the aldehyde proton proton appeared appeared at 10.82 at 10.82ppm. ppm.The UV The-Vis UV-Vis and fluorescence and fluorescence experiments experiments were were used used to to verifyverify that thatone oneequivalent equivalent of Hg of2+ Hg ions2+ ionswere were needed needed to fully to fully convert convert 34 to34 34ato. 34a.

SchemeScheme 14. Chemodosimeter 14. Chemodosimeter 34. 34.

The Thepyridine pyridine ring ring isis a versatilea versatile moiety moiety and is oftenand foundis often in molecular found in receptors. molecular Tetrahydrodibenzo receptors. Tetrahydrodibenzophenanthridines, phenanthridines, contain a pyridine contain functional a grouppyridine with functional five fused grou six-memberedp with five ring fused systems six- with memberedconnectivity, ring systems shown with in Figure connectivity, 15. This shown arrangement in Figure of 15. ring This systems arrangement are found of ring in natural systems products are foundand in biologically natural products active compoundsand biologically [197,198 ac].tive Two compounds bis(dithioester) [197,198]. substituted Two bis(dithioester) phenanthridines, Chemosensors 2019, 7, x 32 of 50 substitutedcompound phenanthridines,35 and compound compound36, were synthesized 35 and bycompound Sundaramurthy 36, etwere al., whosynthesized showed that by they 2+ could coordinate with Hg ions [199]. Mono-sulfur derivative 35 demonstrated2+ a strong fluorescence Sundaramurthy2+ et al., who showed that they could coordinate with Hg2+ ions [199]. Mono-sulfur towards Hg ions. The binding stoichiometry of both sensors toward2+ Hg ions was 1:1, according to derivativeturn on 35 signal demonstrated in an aqueous a strong solution, fluorescence in the presence turn ofon Hg signalions, in whereasan aqueous tetra-sulfur solution, derivative in the 36 the UV-Vis absorption spectra, fluorescence measurements,2+ and computation (DFT) studies. The LoD showed a strong2+ fluorescence quenching towards Hg ions. The binding stoichiometry of both sensors presencefor the mono-sulfur of Hg ions, derivative whereas tetra-sulfur was found derivative to be as 36low showed as 0.91 a strongnM for fluorescence Hg2+ ions, quenchingwhile the toward Hg2+ ions was 1:1, according to the UV-Vis absorption spectra, fluorescence measurements, bis(dithioester) derivative demonstrated an LoD of 0.04 nM. The spectroscopic changes were also and computation (DFT) studies. The LoD for the mono-sulfur derivative was found to be as low as seen in paper-based test strips as seen in Figure 15. 0.91 nM for Hg2+ ions, while the bis(dithioester) derivative demonstrated an LoD of 0.04 nM. The spectroscopic changes were also seen in paper-based test strips as seen in Figure 15.

FigureFigure 15. Changes 15. Changes in the in fluorescence the fluorescence of probes of probes 35 and35 and36 in36 paperin paper strips strips with with different different metal metal ions ions 2+ (A) probe(A) probe 35 (B35) probe(B) probe 36, and36, andat different at different concentration concentration (nM) (nM) of Hg of2+ Hg ions ions(C) probe (C) probe 35 and35 and(D) probe (D) probe 36. Adapted36. Adapted from from [199] [199 with] with permission permission from from the the CentreCentre NationalNational de de la la Recherche Recherche Scientifique Scientifique (CNRS) (CNRS)and and the Royalthe Royal Society Society of Chemistry. of Chemistry.

Anthracene-based,Anthracene-based, reusable reusable fluorescent fluorescent sensor sensor 37 was37 was synthesized synthesized via a via one-pot a one-pot reaction reaction imine imine condensationcondensation by Misra by Misra et al. et [200]. al. [200 As]. anthracene As anthracene is hydrophobic, is hydrophobic, it was it wasexpected expected that thatcompound compound 37 37 would aggregate when the solvent mixture had a high content of water. By changing the THF:H O would aggregate when the solvent mixture had a high content of water. By changing the THF:H2O 2 ratio,ratio, it was it was found found that that the theoptimal optimal working working condition condition required required 70% 70% water. water. A strong A strong emission emission band band around 518 nm was observed (20 μM) with a calculated φ of 0.614. As the water content was increased, the molecules came closer together to restrict intramolecular rotation around the imine bond and, therefore, increased the fluorescence emissions, Scheme 15. In solutions where the water content was low, very weak emission bands were observed in the fluorescence spectra (φ = 0.002). When Hg(NO3)2 was added to the aggregates, significant optical quenching was seen in the fluorescence emission, due to the heavy atom effect. This was a consequence of perturbating the intersystem crossing from the first singlet state (S1) to the lowest triplet state (T1), thereby, increasing the spin-orbit coupling from the aromatic scaffold of the molecular probe to the external heavy atom [201,202], upon the coordination of the Hg2+ ions to 37, through chelation. Therefore, the Stern–Volmer analysis showed the quenching constant (Ksv) to be 2.5 × 105 M−1 and the LoD was found to be in the ppb concentration range (3 ppb). Moreover, analysis of municipal water and river water from the local area, using probe 37 detected Hg2+ ions.

Chemosensors 2019, 7, 22 31 of 48 around 518 nm was observed (20 µM) with a calculated φ of 0.614. As the water content was increased, the molecules came closer together to restrict intramolecular rotation around the imine bond and, therefore, increased the fluorescence emissions, Scheme 15. In solutions where the water content was low, very weak emission bands were observed in the fluorescence spectra (φ = 0.002). When Hg(NO3)2 was added to the aggregates, significant optical quenching was seen in the fluorescence emission, due to the heavy atom effect. This was a consequence of perturbating the intersystem crossing from the first singlet state (S1) to the lowest triplet state (T1), thereby, increasing the spin-orbit coupling from the aromatic scaffold of the molecular probe to the external heavy atom [201,202], upon the coordination of the Hg2+ ions to 37, through chelation. Therefore, the Stern–Volmer analysis showed the quenching 5 1 constant (Ksv) to be 2.5 10 M and the LoD was found to be in the ppb concentration range (3 ppb). × − Moreover, analysis of municipal water and river water from the local area, using probe 37 detected HgChemosensors2+ ions. 2019, 7, x 33 of 50

Scheme 15. Proposed aggregation behavior of 37 in water.

The rhodamine fluorophorefluorophore has been used as a molecularmolecular probe for a variety of metal ions over the years and xanthenexanthene derivativesderivatives have previously been highlighted in this review. Compound 38 2+ 2+ was shownshown toto distinguishdistinguish betweenbetween HgHg2+ and Cu2+ ions.ions. It It was was prepared prepared by by Gao Gao and Zheng who 2+ showed that either in the absence oror presencepresence ofof UVUV light,light, aa distinctiondistinction couldcould bebe mademade betweenbetween HgHg2+ 2+ and Cu2+ ions,ions, scheme Scheme 16 16 [203].[ 203]. Optical Optical studies studies were were ca carriedrried out out in in the the presence presence of of 16 16 different different metal 2+ 2+ + + 3+ 2+ + 2+ 2+ 2+ 3+ 2+ 2+ 2+ 2+ nitrate salts (Hg2+, ,Cu Cu2+, Ag, Ag+, K,K+, Al, Al3+, Ca,2+ Ca, Na,+, Na Ba2+,, Ba Co2+,, CoCd2+,, Fe Cd3+, Mg, Fe2+, ,Mn Mg2+, Zn, Mn2+, Ni,2+ Zn, Pb2+,) Ni in a, Pb2+) in a IPA:H O solvent (25 µM, 8:1, v/v). Probe 38 was initially colorless, indicative of the closed IPA:H2O solvent2 (25 μM, 8:1, v/v). Probe 38 was initially colorless, indicative of the closed lactam 2+ lactammoiety. moiety. However, However, on the addition on the addition of one equivalent of one equivalent of Hg2+ ions, of Hg a newions, band a new at 557 band nm at was 557 observed nm was observed(colorless (colorlessto pink), indicating to pink), indicating that the spirolactam that the spirolactam ring was in ring the was open in form, the open which form, is very which common is very commonfor these formolecular these molecular probes. Compound probes. Compound 38 shows 38a veryshows weak a very emission weak intensity, emission intensity,but an emission but an 2+ emission band was seen at 578 nm,2+ when Hg ions were added to the solution (λex = 550 nm). This band was seen at 578 nm, when Hg ions were added to the solution (λex = 550 nm). This phenomenon phenomenonwas not seen wasin the not presence seen in theof the presence other ofmetal the othercations. metal Copper(II) cations. is Copper(II) well-known is well-knownto quench the to quenchfluorescence the fluorescence emission, therefore, emission, it therefore, was notit surprisi was notng surprising that an emission that an emission signal was signal not was seen not on seen the 2+ 2+ onaddition the addition of Cu2+ ofions, Cu butions, on the but addition on the additionof Hg2+ ions, of Hga newions, absorption a new absorptionband was seen band at was559 nm seen and at 2+ 559a visible nm and color a visiblechange color from change colorless from to colorlesspurple was to purpleobserved. was The observed. coexistence The coexistence of Hg2+ and of Cu Hg2+ inand the 2+ Cusolutionin theshowed solution a decrease showed in a decreasethe emission in the intens emissionity of intensity the band of theat 559 band nm, at indicating 559 nm, indicating that the 2+ 2+ thatpresence the presence of Hg2+ ofinterfered Hg interfered with Cu with2+ detection. Cu detection. One way One to deliberately way to deliberately make their make mixed their system mixed systemmonitor monitor one of the one ions of theover ions the overother, the is to other, take isadvantage to take advantage of solubility of solubilityproducts. products.Bromide ions Bromide were 7 2+ ions were used to precipitate HgBr (K−7sp = 3 10 ), enabling the detection2+ of Cu ions. used to precipitate HgBr2 (Ksp = 3 ×2 10 ), enabling× − the detection of Cu ions.

Scheme 16. The coordination of 38 to Hg2+ ions.

As mercury is classified as a “soft” metal, mercury ions forms many stable ionic compounds that are insoluble [204,205]. Thermodynamic stability is further enhanced when “soft” Lewis bases are present in the receptor, for example sulfur atoms [206]. The carbonyl group (C=O) in the lactam ring in rhodamine probes could be readily converted to the analogous thio (C=S) group, by using

Chemosensors 2019, 7, x 33 of 50

Scheme 15. Proposed aggregation behavior of 37 in water.

The rhodamine fluorophore has been used as a molecular probe for a variety of metal ions over the years and xanthene derivatives have previously been highlighted in this review. Compound 38 was shown to distinguish between Hg2+ and Cu2+ ions. It was prepared by Gao and Zheng who showed that either in the absence or presence of UV light, a distinction could be made between Hg2+ and Cu2+ ions, scheme 16 [203]. Optical studies were carried out in the presence of 16 different metal nitrate salts (Hg2+, Cu2+, Ag+, K+, Al3+, Ca2+, Na+, Ba2+, Co2+, Cd2+, Fe3+, Mg2+, Mn2+, Zn2+, Ni2+, Pb2+) in a IPA:H2O solvent (25 μM, 8:1, v/v). Probe 38 was initially colorless, indicative of the closed lactam moiety. However, on the addition of one equivalent of Hg2+ ions, a new band at 557 nm was observed (colorless to pink), indicating that the spirolactam ring was in the open form, which is very common for these molecular probes. Compound 38 shows a very weak emission intensity, but an emission band was seen at 578 nm, when Hg2+ ions were added to the solution (λex = 550 nm). This phenomenon was not seen in the presence of the other metal cations. Copper(II) is well-known to quench the fluorescence emission, therefore, it was not surprising that an emission signal was not seen on the addition of Cu2+ ions, but on the addition of Hg2+ ions, a new absorption band was seen at 559 nm and a visible color change from colorless to purple was observed. The coexistence of Hg2+ and Cu2+ in the solution showed a decrease in the emission intensity of the band at 559 nm, indicating that the presence of Hg2+ interfered with Cu2+ detection. One way to deliberately make their mixed system monitor one of the ions over the other, is to take advantage of solubility products. Bromide ions were Chemosensors 2019, 7, 22 32 of 48 used to precipitate HgBr2 (Ksp = 3 × 10−7), enabling the detection of Cu2+ ions.

2+ SchemeScheme 16. 16.The The coordination coordination of of 3838 to toHg Hg2+ ions. ions.

As mercuryAs mercury is classified is classified as a “soft”as a “soft” metal, metal, mercury mercury ions ions forms forms many many stable stableionic compounds ionic compounds that that are insolubleare insoluble [204,205 [204,205].]. Thermodynamic Thermodynamic stability stability is is further further enhanced enhanced when when“soft” Lewis “soft” bases Lewis are bases are present inpresent the receptor, in the receptor, for example for example sulfur sulfur atoms atoms [206 [206].]. TheThe carbonyl carbonyl group group (C=O) (C in=O) the inlactam the lactamring ring in in rhodamine probes could be readily converted to the analogous thio (C=S) group, by using rhodamine probes could be readily converted to the analogous thio (C=S) group, by using Lawesson’s Chemosensors 2019, 7, x 34 of 50 reagent. Zhou, Hu and Yao synthesized thiooxo-rhodamine B hydrazine compounds 39 and 40, which 2+ containLawesson’s a number reagent. of sulfur Zhou, atoms Hu and to helpYao synthe the molecularsized thiooxo-rhodamine probe to selectively B hydrazine coordinate compounds to Hg 39 over otherand metal 40, which ions [contain203]. Botha number molecular of sulfur probes atoms to are help colorless the molecular but addition probe to selectively of Hg2+ ions coordinate leads to a significantto Hg2+ increase over other in absorptionmetal ions [203]. at 560 Both nm, molecula due to ther probes spirolactam are colorless ring-opening. but addition No of optical Hg2+ ions changes wereleads seen to in a thesignificant presence increase of the in otherabsorption metals at 560 (Na nm,+,K due+, to Mg the2+ spirolactam, Ca2+, Al3 ring-opening.+, Cr3+, Ba2+ No, Hg optical2+, Mn 2+, + + 2+ 2+ 3+ 3+ 2+ 2+ 2+ Pb2+,changes Cd2+, Niwere2+, seen Zn2 +in, Cothe 2presence+, Fe3+) of in the a EtOH other /metalsHEPES (Na solution, K , Mg (5, CaµM, 1:1,Al ,v Cr/v pH , Ba 7.4), Hg but, Mn Ag+, and Pb2+, Cd2+, Ni2+, Zn2+, Co2+, Fe3+) in a EtOH/HEPES solution (5 μM 1:1, v/v pH 7.4) but Ag+ and Cu2+ Cu2+ ions increased in intensity at 560 nm. Probe 40 selectively detected Hg2+ ions via an “off–on” ions increased in intensity at 560 nm. Probe 40 selectively detected Hg2+ ions via an “off–on” response response in solution. The Job’s plot and UV-Vis titration data indicated that the stoichiometry between in solution. The Job’s plot and UV-Vis titration data indicated that the stoichiometry between 40 and 2+ 40 andHg Hg2+ ionsions was was1:2. This 1:2. is This unsurprising is unsurprising as there as is therefree rotation is free rotationaround the around bisthiophene the bisthiophene bond, so the bond, 2+ so thetwo two rhodamine rhodamine units units could could adapt adapt an ananti-conformation anti-conformation with withthe theHg2+ Hg ions ionsbound bound in each in each 2+ coordinationcoordination environment. environment. By By comparison, comparison, compoundcompound 3939 boundbound the the Hg Hg2+ ionsions in a in 1:1 a 1:1ratio. ratio. The The associationassociation constants constants for 39 forand 39 40andwith 40 with mercury mercury were were calculated calculated using using the Benesi-Hildebrandthe Benesi-Hildebrand method and wasmethod found and towas be found 4 10to 5beM 4 ×1 10and5 M−1 1.3 and 1.310 ×11 10M11 M2−,2, respectively. respectively. Interestingly, Interestingly, the thefluorescence fluorescence × − × − mechanismmechanism for for the the two two molecular molecular probes probes was was very very different. different. Molecular Molecular probe 39 probe was shown39 was to exhibit shown to exhibita FRET a FRET mechanism, mechanism, due to due overlap to overlap between between the emission the emission spectra ofspectra the bisthiophene of the bisthiophene donor and the donor absorption spectra of the rhodamine B acceptor. Compound 40 did not show FRET and the and the absorption spectra of the rhodamine B acceptor. Compound 40 did not show FRET and the fluorescence emission was due to the ring-opening of the spriolactam. The authors also demonstrated fluorescence emission was due to the ring-opening of the spriolactam. The authors also demonstrated that compounds 39 and 40 could monitor the levels of Hg2+ ions in municipal water. that compounds 39 and 40 could monitor the levels of Hg2+ ions in municipal water.

4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) possesses unique spectroscopic signatures 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) possesses unique spectroscopic and thesignatures BODIPY and moiety the BODIPY has been moiety used has extensively been used inextensively molecular in probemolecular design probe [207 design–209 [207–209].]. Qiu, Zhang, and YinQiu, synthesized Zhang, and a Yin BODIPY-based synthesized a probe BODIPY-based with a dipyridylamino probe with a dipyridylamino (DPA) receptor (DPA) (compound receptor41 ), for 2+ 2+ 2+ which(compound the optical 41 response), for which was the di opticalfferent response for Cu was, Hg different, and for Pb Cu2+ions, Hg [2+210, and]. OnPb2+ the ions addition [210]. On of the Cu2+,the Hg addition2+, and Pbof the2+ ions, Cu2+, theHg2+ optical, and Pb response2+ ions, the was optical different response for each was metaldifferent ion. for Optical each metal studies ion. were carriedOptical out instudies the presence were carried of aout number in the presence of different of a number perchlorate of different metal perchlorate salts (Ag+ metal, Ba2 +salts, Ca (Ag2+,+ Cd, 2+, 2+ Ba2+,2 Ca+ 2+, Cd2+ 2+, Co2+2+, Cu+2+, Fe2+2+, Hg2+,+ K+, Mg2+2+, Na2++, Ni2+2,+ Pb2+, Zn2+) in a 2.0 μM CH3CN solution. The Co , Cu , Fe , Hg ,K , Mg , Na , Ni , Pb , Zn ) in a 2.0 µM CH3CN solution. The presence 2+ 2+ of Hgpresence2+ and Pb of2 Hg+ in theand solution,Pb in the lead solution, to a blue lead shiftto a blue in the shif maximumt in the maximum absorbance absorbance peak from peak 595from nm to 595 nm to 572 nm and a visible color change from blue to purple. The presence of Cu2+ ions in the 572 nm and a visible color change from blue to purple. The presence of Cu2+ ions in the solution led solution led to a blue shift in the maximum absorbance from 595 nm to 512 nm, and a visible color to a blue shift in the maximum absorbance from 595 nm to 512 nm, and a visible color change from change from blue to fuchsia. Compound 41 showed a single weak emission, when it was excited at blue to365 fuchsia. nm. The Compoundpresence of Hg412+showed and Pb2+ aions single led to weak a new emission, emission band when at it 587 was nm excited and a change at 365 from nm. The 2+ 2+ presencedark ofred Hg fluorescenceand Pb to orangeions led fluorescence. to a new emission The presence band of atCu 5872+ ions nm led and to aan change emission from band dark at red 545 nm and a change from dark red fluorescence to green fluorescence. Competition studies have showed that the presence of Cu2+ ions in the solution quenched the Hg2+ and Pb2+ ions fluorescent responses. The presence of other metal ions did not influence the Cu2+ response. Both Hg2+ and Pb2+ ions could be removed from the binding site of 41, when EDTA was added to the solution. A Job’s plot and ESI-MS data indicated a 1:1 binding stoichiometry, and 1H NMR data indicated that the probe’s fluorescent and colorimetric responses were due to π delocalization between the N-pyridyl and BODIPY groups, caused by the coordination of the metal ions to the BODIPY and DPA groups. As Hg2+, Pb2+ and Cu2+ produced different emission intensities it was possible to construct OR, NOT, and AND logic circuits, where the inputs were the metal ions and the output was the emission intensity at 587 nm. If the emission intensity was greater than a chosen arbitrary unit (150 au), then “1” was assigned for (i) both Hg2+ and Pb2+, (ii) Hg2+, or (iii) Pb2+ on their own were present, meaning that the gate was opened. If, Cu2+ was present alone, with Hg2+ or Pb2+ or both Hg2+ and Pb2+, it led to

Chemosensors 2019, 7, 22 33 of 48

fluorescence to orange fluorescence. The presence of Cu2+ ions led to an emission band at 545 nm and a change from dark red fluorescence to green fluorescence. Competition studies have showed that the presence of Cu2+ ions in the solution quenched the Hg2+ and Pb2+ ions fluorescent responses. The presence of other metal ions did not influence the Cu2+ response. Both Hg2+ and Pb2+ ions could be removed from the binding site of 41, when EDTA was added to the solution. A Job’s plot and ESI-MS data indicated a 1:1 binding stoichiometry, and 1H NMR data indicated that the probe’s fluorescent and colorimetric responses were due to π delocalization between the N-pyridyl and BODIPY groups, caused by the coordination of the metal ions to the BODIPY and DPA groups. As Hg2+, Pb2+ and Cu2+ produced different emission intensities it was possible to construct OR, NOT, and AND logic circuits, where the inputs were the metal ions and the output was the emission intensity at 587 nm. If the emission intensity was greater than a chosen arbitrary unit (150 au), then “1” was assigned for (i) both Hg2+ and Pb2+, (ii) Hg2+, or (iii) Pb2+ on their own were present, meaning that the gate was opened.Chemosensors If, Cu2+ 2019was, 7, xpresent alone, with Hg2+ or Pb2+ or both Hg2+ and Pb2+, it led to35 of a 50 situation whereby the intensity was lower than 150 au, therefore a “0” was assigned, meaning the gate was a situation whereby the intensity was lower than 150 au, therefore a “0” was assigned, meaning the closed.gate Therefore, was closed. the Therefore, combinations the combinations of the three of ditheff erentthree different metal ions metal gave ions risegave to rise di fftoerent different logic gates (OR, NOT,logic andgates AND), (OR, NOT, Figure and 16AND),. Figure 16.

(A)

Input (1) Input (2) Input (3) Output 0 0 0 0 1 0 0 1 0 1 0 1 0 0 1 0 1 1 0 1 1 0 1 0 0 1 1 0 1 1 1 0

(B)

(C)

2+ 2+ 2+ μ FigureFigure 16. Truth 16. Truth table table and and logic logic circuit circuit input input (1), (1),(2), and (2), (3) and Pb (3), Hg Pb, and2+, Cu Hg2 ions+, and (20 CuM, 2CH+ ions3CN), (20 µM, respectively, and output is the fluorescence intensity at 587 nm [210]. (A) molecular probe, (B) truth CH CN), respectively, and output is the fluorescence intensity at 587 nm [210]. (A) molecular probe, 3 table and (C) circuit. (B) truth table and (C) circuit. Gao and Chen synthesized a Schiff-base probe with coumarin and benzimidazole fluorophores (42), which was capable of simultaneously detecting Hg2+ and Cu2+ ions, by two different mechanisms [211]. Compound 42 produced a fluorescence emission enhancement, when bound to Hg2+ ions, whereas, the Cu2+ ions quenched the emission band. A weak fluorescence intensity at 426 nm (φ = 0.009) was seen for 42 in a 10 μM HEPES:DMSO (20 mM, 9:1, v/v) solution. A significant enhancement was seen in the presence of Hg2+ ions (φ = 0.152), whereas in the presence of Cu2+ ions, a red shift to 448 nm was seen, but the emission band was reduced in intensity (φ = 0.005). Job’s plot analysis and fluorescence titration data both indicated a 1:1 binding ratio for both the Cu2+ and Hg2+ complexes. The structure of the complexes was supported by HRMS, IR, and NMR studies. The Hg2+ ion bound

Chemosensors 2019, 7, 22 34 of 48

Gao and Chen synthesized a Schiff-base probe with coumarin and benzimidazole fluorophores (42), which was capable of simultaneously detecting Hg2+ and Cu2+ ions, by two different mechanisms [211]. Compound 42 produced a fluorescence emission enhancement, when bound to Hg2+ ions, whereas, the Cu2+ ions quenched the emission band. A weak fluorescence intensity at 426 nm (φ = 0.009) was seen for 42 in a 10 µM HEPES:DMSO (20 mM, 9:1, v/v) solution. A significant enhancement was seen in the presence of Hg2+ ions (φ = 0.152), whereas in the presence of Cu2+ ions, a red shift to 448 nm was seen, but the emission band was reduced in intensity (φ = 0.005). Job’s plot analysis and 2+ 2+ fluorescenceChemosensorsChemosensors 20192019 titration,, 77,, xx data both indicated a 1:1 binding ratio for both the Cu and Hg complexes.3636 ofof 5050 The structure of the complexes was supported by HRMS, IR, and NMR studies. The Hg2+ ion bound inin aa tridentatetridentatetridentate fashion,fashion,fashion, butbut thethethe molecularmolecularmolecular probesprobprobeses underwentunderwent hydrolysishydrolysis viavia aa mercury-promotedmercury-promotedmercury-promoted hydrolysishydrolysis (Scheme(Scheme 1717),17),), soso thethe fluorescence fluorescencefluorescence emissionemisemissionsion waswas duedue toto the the coumarin-carboxaldehyde coumarin-carboxaldehyde beingbeing formed.formed. Fluorescence Fluorescence titrationtitration datadatadata werewerewere usedusedused tototo determine determinedetermine the thethe probe’s probe’sprobe’s sensitivity sensitivitysensitivity to toto Hg HgHg2+2+2+ and andand Cu CuCu22+2++ ions,ions, andand thethe Benesi-HildebrandBenesi-HildebrandBenesi-Hildebrand equation equation used used to to calculatecalculate thethe associationassociation constantsconstants for for thethe probeprobe 22++ 2+2+ 4 4 −1 1 4 4−1 1 towardstowards CuCu2+ ionsionsions andand and HgHg Hg2+ ions,ions,ions, gavegave gave 77 7 ×× 1010104 MMM−1 −andandand 99 ×× 9 1010410 MM−1M,, respectively.respectively.− , respectively. × ×

SchemeScheme 17.17. TheThe coordination coordination and and subsequent subsequent hydrolysis hydrolysis ofof molecularmolecular probeprobe 4242...

2+ AnotherAnother exampleexample ofof molecularmolecularmolecular chemodosimeterchemodosimchemodosimetereter thatthatthat cancancan selectivelyselectivelyselectively monitormonitormonitor HgHgHg2+2+ ionsions isis μ compoundcompound 4343 [[212].[212].212]. A A solution solution of of compoundcompound 4343 inin 10 10 µμMM HEPES HEPES (10 (10 mM,mM, pHpH 7)7) hadhad aa maximummaximummaximum absorptionabsorption band bandband at atat 315 315315 nm, nm,nm, and andand when whenwhen excited excitedexcited at 371 atat nm,371371 anm,nm, weak aa weakweak emission emissionemission band was bandband observed waswas observedobserved at 450 nm. atat Interestingly,450450 nm.nm. Interestingly,Interestingly, the authors thethe failed authorsauthors to mention failedfailed to whyto mentionmention the excitation whywhy thethe wavelength excitationexcitation used wavelengthwavelength in their studies usedused inin was theirtheir so farstudiesstudies removed waswas fromsoso farfar the removedremoved maximum fromfrom absorption thethe maximummaximum band. absoabso An increaserptionrption band.band. in absorption AnAn increaseincrease and in emissionin absorptionabsorption intensity andand 2+ wasemissionemission seen intensity whenintensity Hg waswasions seenseen were whenwhen added HgHg2+2+ ions toions43 were.were The addedadded fluorescence toto 4343.. TheThe quantum fluorescencefluorescence yield of quantumquantum43 was yield calculatedyield ofof 4343 φ 2+ withwaswas calculatedcalculated respect to withwith rhodamine respectrespect toBto and rhodaminerhodamine found to BB andand be φ foundfound= 0.027. toto bebe The φ == 0.027.0.027. addition TheThe of additionaddition one equivalence ofof oneone equivalenceequivalence of Hg 2+ φ ledofof HgHg to a2+ led ten-foldled toto aa increaseten-foldten-fold inincreaseincrease fluorescent inin fluorescentfluorescent intensity (intensityintensityφ = 0.22), ((φ which == 0.22),0.22), was whichwhich accompanied waswas accompaniedaccompanied by a change byby in aa 2+ 2+ + fluorescencechangechange inin fluorescencefluorescence from colorless fromfrom to colorlesscolorless blue. No toto other blue.blue. metal NoNo otherother cation metalmetal showed cationcation the showedshowed same response thethe samesame (Zn responseresponse, Hg ,(Zn(Zn Na2+2+,, 2+ 2+ + 2+ 2+ 3+ 2+ 3+ 2+ 3+ 2+ PbHgHg2+2+,, NaNa Co++,, PbPb,K2+2+,, , CoCo Ni2+2+,, K,K Cu++,, NiNi2+2+,, Al, CuCu2+2+,,, Mn AlAl3+3+,, MnMn, Cr2+2+,, Cr,Cr Mg3+3+,, MgMg, Fe2+2+,, FeFe,3+3+ Cd,, CdCd2+2+).).). ESI-MS ESI-MSESI-MS and andand NMR NMRNMR studiesstudiesstudies showedshowed 2+ thatthat thethe fluorescencefluorescencefluorescence emissionemissionemission waswaswas dueduedue tototo thethethe cleavage cleavagecleavage of ofof the thethe carbonothioate, carbonothioate,carbonothioate, via viavia a aa Hg HgHg2+2+-promoted-promoted-promoted hydrolysis,hydrolysis, thereby,thereby, formingformingforming 7-hydroxy-4-methylcoumarin7-hydroxy-4-methylcoumarin7-hydroxy-4-methylcoumarin dye,dye, whichwhich waswaswas highlyhighlyhighly fluorescent.fluorescent.fluorescent. ThisThis 2+ “simple”“simple”“simple” molecule moleculemolecule was waswas used usedused to toto monitor monitormonitor deliberately deliberatelydeliberately spiked spikedspiked tap taptap water waterwater with withwith Hg HgHg2+2+,,, toto show show that that it it could could bebe usedused inin aa real-worldreal-world application.application.

The imine functional group can undergo hydrolysis under acidic conditions [213]. This is a The imine functional group can undergo hydrolysis under acidic conditions [213]. This is a well- well-knownThe imine and functional thoroughly group investigated can undergo mechanism, hydrolysis whereby under acidic the rate conditions determine [213]. step This is the is a initial well- known and thoroughly investigated mechanism, whereby the rate determine step is the initial nucleophilicknown and additionthoroughly to theinvestigated carbon-nitrogen mechanism, double wh bond.ereby Itthe has rate also determine been shown step that is metalthe initial ions nucleophilic addition to the carbon-nitrogen double bond. It has also been shown that metal ions can cannucleophilic accelerate addition the hydrolysis to the carbon-nitrogen mechanism via double a substrate-metal bond. It has association, also been shown whereby that the metal metal ions ion can is accelerateaccelerate thethe hydrolysishydrolysis mechanismmechanism viavia aa substrsubstrate-metalate-metal association,association, wherebywhereby thethe metalmetal ionion isis coordinatedcoordinated toto thethe nitrogennitrogen atom,atom, enhancingenhancing thethe substratesubstrate forfor nucleophilicnucleophilic attackattack [214].[214]. CompoundCompound 4444 waswas preparedprepared byby DuanDuan etet al.al. [215].[215]. ItIt waswas shownshown thatthat compoundcompound 4444 worksworks asas aa turn-turn- onon sensorsensor throughthrough HgHg2+2+ promotedpromoted hydrolysis,hydrolysis, inin whichwhich thethe imineimine isis cleavedcleaved toto formform thethe coumarincoumarin aldehydealdehyde fluorophorefluorophore andand aa 5-aminoisophthalic5-aminoisophthalic acidacid methylmethyl ester.ester. TheThe cleavagecleavage diddid notnot occuroccur inin thethe presencepresence ofof otherother metalmetal ionsions (Na(Na+,+, KK++,, MgMg2+2+,, CaCa2+2+,, CdCd2+2+,, MnMn2+2+,, FeFe2+2+,, CoCo2+2+,, NiNi2+2+,, CuCu2+2+,, ZnZn2+2+,, AgAg++,, PbPb2+2+,, HgHg2+2+)) −6 inin aa 22 ×× 1010−6 MM CHCH33CN:HCN:H22OO 8:28:2 (v/v)(v/v) solutionsolution atat physiologicalphysiological pHpH (0.1(0.1 MM KClOKClO44 buffer,buffer, pHpH 7.3).7.3). CompoundCompound 4444 displayeddisplayed aa weakweak fluorescencefluorescence signalsignal atat 553030 nm.nm. TheThe lonelone pairpair onon thethe nitrogennitrogen atomatom PET,PET, quenchedquenched thethe emissionemission intensity.intensity. OnOn thethe additionaddition ofof HgHg2+2+ ions,ions, aa 20-fold20-fold increaseincrease inin emissionemission waswas seenseen atat 490490 nm.nm. TheThe emissionemission atat 490490 nmnm waswas assignedassigned toto thethe coumarincoumarin aldehydealdehyde fluorophore,fluorophore, andand 2+ thethe ratioratio betweenbetween thethe intensityintensity atat 490490 nmnm andand 530530 nmnm (I(I490490/I/I530530)) waswas usedused toto detectdetect HgHg2+ ions,ions, viavia aa

Chemosensors 2019, 7, 22 35 of 48 coordinated to the nitrogen atom, enhancing the substrate for nucleophilic attack [214]. Compound 44 was prepared by Duan et al. [215]. It was shown that compound 44 works as a turn-on sensor through Hg2+ promoted hydrolysis, in which the imine is cleaved to form the coumarin aldehyde fluorophore and a 5-aminoisophthalic acid methyl ester. The cleavage did not occur in the presence of other metal ions (Na+, K+, Mg2+, Ca2+, Cd2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Ag+, Pb2+, Hg2+) in a 2 10 6 M × − CH3CN:H2O 8:2 (v/v) solution at physiological pH (0.1 M KClO4 buffer, pH 7.3). Compound 44 displayed a weak fluorescence signal at 530 nm. The lone pair on the nitrogen atom PET, quenched the emissionChemosensors intensity. 2019, 7, x On the addition of Hg2+ ions, a 20-fold increase in emission was seen at 49037 nm. of 50 The emission at 490 nm was assigned to the coumarin aldehyde fluorophore, and the ratio between the ratiometric approach. The LoD of Hg2+ was calculated to be2 +in the nanomolar concentration range. intensity at 490 nm and 530 nm (I490/I530) was used to detect Hg ions, via a ratiometric approach. The LoDFluorescence of Hg2+ was was calculated significantly to be inturned the nanomolar on as 44concentration broke down range.into two Fluorescence separate molecules, was significantly one of turnedwhich, on the as coumarin44 broke aldehyde down into is twohighly separate fluorescent. molecules, It was onereasoned of which, that 44 the could coumarin be used aldehyde to monitor is highlyHg2+ ions fluorescent. in the human It was breast reasoned adenocarcinoma that 44 could cell be used(MCF-7) to monitor line, which Hg2 was+ ions observed in the human when the breast cells adenocarcinomawere incubated cell with (MCF-7) Hg2+ ions. line, which was observed when the cells were incubated with Hg2+ ions.

3. Multivariate Analysis 3. MultivariateMolecular recognition Analysis between low molecular weight fluorescent probes (LMFPs) and metal cations has dominatedMolecular the recognition field for many between years. low Sensor molecular arrays, using weight statistical fluorescent analysis probes and pattern-recognition(LMFPs) and metal algorithmscations has are dominated now becoming the field the for main many approach years. Sens to measureor arrays, and using monitor statistical a number analysis of analytesand pattern- in arecognition single experiment, algorithms and are a recentnow becoming review written the main by approach Jolliffe, highlights to measure some and monitor recent examples a number in of fluorescentanalytes in sensing a single arrays experiment, [216]. Here, and a we recent give areview couple written of recent by examplesJolliffe, highlights on this new some sensing recent approach,examples to in highlight fluorescent the sensing direction arrays of research [216]. Here, in the we field. give a couple of recent examples on this new sensingMinami approach, et al. to used highlight commercially the direction available of research dyes in (alizarin the field. red, bromopyrogallol red, and pyrogallolMinami red) et that al. containedused commercially the catechol available moiety [dyes217]. (alizarin The catechol red, functionalbromopyrogallol groups red, reacted and withpyrogallol 3-nitrophenylbornoic red) that contained acid (3-NPBA)the catechol to formmoiety a dynamic[217]. The covalent catechol bond. function Theal metal groups ion reacted competed with with3-nitrophenylbornoic the boronic acid for acid the (3-NPBA) catechol moiety; to form thus,a dynamic upon coordinationcovalent bond. of The the metal ion ion, competed 3-NPBA was with displaced.the boronic The acid metal-dye for the complexcatechol generatedmoiety; thus, diff erentupon colors,coordination depending of the on metal the dye ion, and 3-NPBA the metal, was Schemedisplaced. 18. The metal-dye complex generated different colors, depending on the dye and the metal, SchemeThe coordination18. of the catechol dyes and 3-NPBA resulted in blue shifts in the absorption spectra, and the addition of metal ions led to a red shift, due to the displacement of 3-NPBA and the formation of a dye-metal complex. FAB-MS and titration isotherms indicated that both the dye:3-NPBA and dye:metal complexes had a 1:1 stoichiometry. Extensive univariant work gave an understanding of 2+ 2+ 2+ the interaction and the Kassoc for selected metal ions (Cu , Ni and Zn ), which were in the range of 5 102 M 1 to > 106 M 1. The array utilized a 384 well microplate for high throughput screening. × − − The response of the sensor array in the presence of 11 metals was investigated (50 mM HEPES buffer solution containing 10 mM NaCl interferant, 40 µM catechol dye, 4 mM 3-NPBA, and 10 µM metals) and was capable of discriminating between Ni2+, Cu2+, Zn2+, Cd2+, Co2+, Fe2+, Hg2+, Al3+, Pb2+, Ga3+, and Ca2+. LDA analysis indicated that 95.2% of variance was sufficient to discriminate between the different metals, and the cross-validation method indicated a 100% correct classification within each cluster, Figure 17A. A potential semi-quantitative analysis to determine the concentration of the

Scheme 18. The displacement approach by which the dyes formed a dynamic bond with the boronic acid derivative which was then displaced upon the coordination of the metal ion.

The coordination of the catechol dyes and 3-NPBA resulted in blue shifts in the absorption spectra, and the addition of metal ions led to a red shift, due to the displacement of 3-NPBA and the

Chemosensors 2019, 7, x 37 of 50 ratiometric approach. The LoD of Hg2+ was calculated to be in the nanomolar concentration range. Fluorescence was significantly turned on as 44 broke down into two separate molecules, one of which, the coumarin aldehyde is highly fluorescent. It was reasoned that 44 could be used to monitor Hg2+ ions in the human breast adenocarcinoma cell (MCF-7) line, which was observed when the cells were incubated with Hg2+ ions.

3. Multivariate Analysis

ChemosensorsMolecular2019 ,recognition7, 22 between low molecular weight fluorescent probes (LMFPs) and36 ofmetal 48 cations has dominated the field for many years. Sensor arrays, using statistical analysis and pattern- recognition algorithms are now becoming the main approach to measure and monitor a number of metals was demonstrated using a mixture of Cu2+, Zn2+, and Ni2+, and a regression analysis using analytes in a single experiment, and a recent review written by Jolliffe, highlights some recent a support vector machine (SVM) algorithm (Figure 17B). The results of the semi-quantitative assay, examples in fluorescent sensing arrays [216]. Here, we give a couple of recent examples on this new where the three different metals were tested at a concentration range of 0–3.7 ppm with 20 repetitions at sensingeach concentration, approach, to showedhighlight that the a direction plot of the of actual research vs. predicted in the field. concentrations was highly accurate. TheMinami concentration et al. rangeused overcommercially which the available results were dyes obtained (alizarin (Ni 2red,+ < 30bromopyrogallol ppm, Cu2+ < 2.5 red, ppm, and pyrogallolZn2+ < 6 red) ppm), that the contained method’s potentialthe catechol for high-throughput moiety [217]. The screening, catechol and function the factal thatgroups the assayreacted gave with 3-nitrophenylbornoicno response in the presence acid (3-NPBA) of othermetals to form common a dynamic in environmental covalent bond. water The sources, metal ion such competed as K+, Na with+, 2+ theMgChemosensors boronic, indicated acid2019, 7for that, x the the assaycatechol developed moiety; by thus, Sasaki up andon coworkerscoordination could of bethe a usefulmetal toolion, for3-NPBA real38 time of 50was displaced.testing, in The environmental metal-dye complex water sources. generated different colors, depending on the dye and the metal, Schemeformation 18. of a dye-metal complex. FAB-MS and titration isotherms indicated that both the dye:3-NPBA and dye:metal complexes had a 1:1 stoichiometry. Extensive univariant work gave an Kassoc 2+ 2+ 2+ understanding of the interaction and the for selected metal ions (Cu , Ni and Zn ), which were in the range of 5 × 102 M-1 to > 106 M-1. The array utilized a 384 well microplate for high throughput screening. The response of the sensor array in the presence of 11 metals was investigated (50 mM HEPES buffer solution containing 10 mM NaCl interferant, 40 μM catechol dye, 4 mM 3- NPBA, and 10 μM metals) and was capable of discriminating between Ni2+, Cu2+, Zn2+, Cd2+, Co2+, Fe2+, Hg2+, Al3+, Pb2+, Ga3+, and Ca2+. LDA analysis indicated that 95.2% of variance was sufficient to discriminate between the different metals, and the cross-validation method indicated a 100% correct classification within each cluster, Figure 17A. A potential semi-quantitative analysis to determine the concentration of the metals was demonstrated using a mixture of Cu2+, Zn2+, and Ni2+, and a regression analysis using a support vector machine (SVM) algorithm (Figure 17B). The results of the semi- quantitative assay, where the three different metals were tested at a concentration range of 0–3.7 ppm with 20 repetitions at each concentration, showed that a plot of the actual vs. predicted concentrations was highly accurate. The concentration range over which the results were obtained (Ni2+ < 30 ppm, Cu2+ < 2.5 ppm, Zn2+ < 6 ppm), the method’s potential for high-throughput screening, and the fact that the assay gave no response in the presence of other metals common in environmental water sources, such as K+, Na+, Mg2+, indicated that the assay developed by Sasaki and coworkers could be a useful tool forScheme real 18.timeThe testing, displacement in environmental approach by water which sources. the dyes formed a dynamic bond with the boronic Schemeacid derivative 18. The displacement which was then approach displaced by upon which the the coordination dyes formed of the a dynamic metal ion. bond with the boronic

acid(A) derivative which was then displaced upon the coordination(B) of the metal ion.

The coordination of the catechol dyes and 3-NPBA resulted in blue shifts in the absorption spectra, and the addition of metal ions led to a red shift, due to the displacement of 3-NPBA and the

Figure 17. (A) Linear discriminant analysis (LDA) score plots for the response of the sensor array to 11 Figure 17. (A) Linear discriminant analysis (LDA) score plots for the response of the sensor array to analytes (and control) in a HEPES buffer (50 mM) solution, with sodium chloride (10 mM). The [metal 11 analytes (and control) in a HEPES buffer (50 mM) solution, with sodium chloride (10 mM). The ion] = 10 mM. 20 repetitions were measured for each analyte. (B) The cross-validation routine shows [metal ion] = 10 mM. 20 repetitions were measured for each analyte. (B) The cross-validation routine 100% correct classification LDA results of the semi-quantitative assay, toward three different metal ions shows 100% correct classification LDA results of the semi-quantitative assay, toward three different (Cu2+, Zn2+ and Ni2+); concentration range of 0–3.7 ppm.%. Adapted from [218] with permission from metal ions (Cu2+,Zn2+and Ni2+); concentration range of 0–3.7 ppm.%. Adapted from [218] with The Royal Society of Chemistry. permission from The Royal Society of Chemistry.

Our group has carried out multivariate pattern recognition approaches to the binding between coumarin–enamine probes (compounds 45a–e) and ten divalent metal ions [219]. The coordination of the metal ions with compounds 45b–e, through the nitrogen atom, showed optical responses in a univariant approach, but the data were limited in terms of discrimination. To capture this wealth of information and to be able to carry out the discrimination of metal cations as proposed, a multivariate approach was adopted. A multimode microwell plate reader was utilized, allowing for rapid and automated acquisition of multivariate data. The sensor array comprised compounds 45b to 45f. Compound 45a did not possess a binding site on the aromatic ring system, so it was used as a reference.

Chemosensors 2019, 7, 22 37 of 48

Our group has carried out multivariate pattern recognition approaches to the binding between coumarin–enamine probes (compounds 45a–e) and ten divalent metal ions [219]. The coordination of the metal ions with compounds 45b–e, through the nitrogen atom, showed optical responses in a univariant approach, but the data were limited in terms of discrimination. To capture this wealth of information and to be able to carry out the discrimination of metal cations as proposed, a multivariate approach was adopted. A multimode microwell plate reader was utilized, allowing for rapid and automated acquisition of multivariate data. The sensor array comprised compounds 45b to 45f. ChemosensorsCompound 201945a, 7did, x not possess a binding site on the aromatic ring system, so it was used as a reference.39 of 50

The absorbanceabsorbance response response of thisof this array array of four of sensorsfour sensors at 330, 380,at 330, 400, 380, and 430400, nm, and and 430 fluorescence nm, and λ λ fluorescenceintensity at 330 intensity/450 nm, at 330330/450/528 nm,nm, 330330/528/580 nm,nm, 380330/580/450 nm,nm, 380380/450/528 nm,nm, and380/528 380/ 450nm, nmand ( 380/450ex/ em) were determined concurrently. The sensing array was exposed to a panel of analytes comprising nm (λex/λem) were determined concurrently. The sensing array was exposed to a panel of analytes the chloride salts of—Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Hg2+, Mg2+, Ni2+, Pb2+, and Zn2+, and all comprising the chloride salts of—Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Hg2+, Mg2+, Ni2+, Pb2+, and Zn2+, and all measurements were conducted in the same same solvent solvent (D (DMSO)MSO) as as the the univariate univariate analysis analysis for for consistency. consistency. The sensor arrayarray waswas exposed exposed to to each each metal metal multiple multiple times, times, generating generating a cluster a cluster of 18 of replicates 18 replicates for each for analyte. This approach generated a very large multivariate data set (4 sensors by 10 analytes by each analyte. This approach generated a very large multivariate data set× (4 × sensors by× 10 × analytes 18 replicates = 720 data points, each described by nine instrumental variables). Linear discriminant by ×18 × replicates = 720 data points, each described by nine instrumental variables). Linear discriminantanalysis (LDA) analysis was used (LDA) to organizewas used the to high-dimensionalityorganize the high-dimensionality data and to extract data and the mostto extract relevant the mostinformation, relevant ultimately information, providing ultimately the providing information the necessary information to classify necessary the to ten classify analytes the considered. ten analytes considered.The well-established LDA algorithm computed a linear combination of the original variables that maximizedThe well-established the separation LDA between algorithm multiple computed analytes, a linear while combination minimizing of the the separation original variables between thatreplicate maximized measurements the separation of the samebetween analyte multiple [218 ].an Inalytes, doing while so, the minimizing algorithm the generated separation a new between set of replicatevariables, measurements called factors, whichof the weresame returnedanalyte [218]. in order In doing of decreasing so, the relativealgorithm information generated content. a new set Data of variables,reduction wascalled then factors, accomplished which were by retainingreturned onlyin order the firstof decreasing two or three relative LDA factorsinformation and dropping content. Datathe higher-order reduction was ones. then This accomplished produced a transformed by retaining dataset, only the in which first two each or point three was LDA associated factors withand droppinga pair or athe triplet higher-order of numbers, ones. respectively, This produced typically a transformed referred to asdataset, the factor in which scores each of those point points. was associatedThese factor with scores a pair could or bea triplet used asof coordinatesnumbers, resp in plottingectively, the typically transformed referred points to as in the either factor a two- scores or ofthree-dimensional those points. These plot. factor scores could be used as coordinates in plotting the transformed points in eitherIt was a two- shown or that,three-dimensional the first two factors plot. obtained from the LDA analysis, out of the ten present in the system, accounted for 80.7% of the total information content of the original system. A two-dimensional score plot (Figure 18) was obtained that showed clear clustering of the data—replicate measurements of samples of each divalent metal ion were classified as similar and grouped together by the LDA analysis. In short, the pattern-based approach was capable of differentiating almost all ten divalent metal cations, using the additional information collected through patterning and multivariate analysis. However, this analysis failed to definitively separate the ten metal ions, this was due to similarities in geometry that was adopted upon the binding of the molecular probe.

(A) (B)

Figure 18. (A) Two-dimensional LDA score plot for the analysis of probes 45a–e binding 10 divalent metal chlorides. (B) A three-dimensional LDA score plot captured 90% of all information available from the pattern recognition system, significantly improving on the separation of difficult cases that were imperfectly discriminated, using the two-dimensional results. Confidence ellipsoids were drawn at 95% confidence. Adapted from [218] with permission from The Royal Society of Chemistry.

Chemosensors 2019, 7, x 39 of 50

The absorbance response of this array of four sensors at 330, 380, 400, and 430 nm, and fluorescence intensity at 330/450 nm, 330/528 nm, 330/580 nm, 380/450 nm, 380/528 nm, and 380/450 nm (λex/λem) were determined concurrently. The sensing array was exposed to a panel of analytes comprising the chloride salts of—Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Hg2+, Mg2+, Ni2+, Pb2+, and Zn2+, and all measurements were conducted in the same solvent (DMSO) as the univariate analysis for consistency. The sensor array was exposed to each metal multiple times, generating a cluster of 18 replicates for each analyte. This approach generated a very large multivariate data set (4 × sensors by 10 × analytes by 18 × replicates = 720 data points, each described by nine instrumental variables). Linear discriminant analysis (LDA) was used to organize the high-dimensionality data and to extract the most relevant information, ultimately providing the information necessary to classify the ten analytes considered. The well-established LDA algorithm computed a linear combination of the original variables that maximized the separation between multiple analytes, while minimizing the separation between replicate measurements of the same analyte [218]. In doing so, the algorithm generated a new set of variables, called factors, which were returned in order of decreasing relative information content. Data reduction was then accomplished by retaining only the first two or three LDA factors and dropping the higher-order ones. This produced a transformed dataset, in which each point was associated with a pair or a triplet of numbers, respectively, typically referred to as the factor scores

Chemosensorsof those2019 points., 7, 22 These factor scores could be used as coordinates in plotting the transformed38 of points 48 in either a two- or three-dimensional plot.

(A) (B)

FigureFigure 18. ( A18.) Two-dimensional(A) Two-dimensional LDA LDA score score plot plot for the for analysisthe analysis of probes of probes45a– 45ae binding–e binding 10 divalent 10 divalent metalmetal chlorides. chlorides. (B) A(B three-dimensional) A three-dimensional LDA LDA score score plot plot captured captured 90% 90% of all of information all information available available fromfrom the patternthe pattern recognition recognition system, system, significantly significantly improving improving on the on separationthe separation of di offfi cultdifficult cases cases that that werewere imperfectly imperfectly discriminated, discriminated, using theusing two-dimensional the two-dimensional results. Confidenceresults. Confidence ellipsoids wereellipsoids drawn were at 95%drawn confidence. at 95% confidence. Adapted from Adapted [218] withfrom permission[218] with permission from The Royal from SocietyThe Royal of Chemistry. Society of Chemistry. 4. Conclusions and Future Prospects

The chemical sensors that have been highlighted throughout this review showcase recent low molecular weight fluorescent probes designed to target the group 12 triad (Zn2+, Cd2+, and Hg2+). During the last few years there has been a great deal of work on the design, synthesis, and detection of these metal ions. Much of the work presented here is focused on the traditional approach of sensor design, where the spectroscopic response is univariant in nature i.e., one LMFP targets one metal. As small molecule recognition becomes more popular, it is becoming more challenging to prepare novel compounds, but as computer power improves significantly, monitoring and discriminating metal ions in a complex environment seems to be the direction in which the sensor community is moving. Groups still publish their findings on LMFP and, whilst writing this review and taking a closer look at the data, it is clear that there are some general omissions or misleading statements. One of the biggest challenges is to work in 100% aqueous system, however, the systems are often in a mixture of organic-aqueous systems, and more often than not, only a fraction is water. Nevertheless, it has been claimed that a particular probe works in water. Additionally, there is often little or no discussion on the aqueous chemistry of the metal ion which influences the coordination. Moreover, many reports often omit a serious discussion on speciation, in their particular system, claiming that only a 1:1 probe-metal exists, when the data often suggest that other species might exist. More often than not, the counter-anion is omitted and authors make the assumption that it plays no role in binding, but only exists to balance the charge, when many counter ions can form bridging species. This is particularly important if a system is to work in an aqueous system, as anions might not exist in their conjugate base form, depending on the pH, which might influence the speciation. Additionally, care must be taken in reporting equilibrium constants using old linear regression methods, for example, the Benesi–Hilderbrand double reciprocal plot and Job’s plot analysis. Even though they are easy to use they are significantly over used and misinterpreted. For example, subtle curvature in the Benesi–Hilderbrand plot is often mistaken as indicative of a 1:1 binding, but in fact it more often than not implies a more complicated equilibria occurring in the system. There are many problems associated with these regression calculations. Computer power has significantly improved over the years and software now exists for non-linear regression analysis to calculate more precise binding constants, including those involving complex equilibria. Chemosensors 2019, 7, 22 39 of 48

Author Contributions: The authors comprise a graduate student (A.D.J.), an undergraduate student (R.M.C.) and the principle investigator (K.J.W.). Each student contributed significantly to the review. All three authors extensively carried out the literature search and wrote specific sections. A.D.J. wrote the section pertaining to the cadmium section, R.M.C. wrote the section on mercury, and K.J.W. put together the remaining sections and looked after the overall management of the manuscript. Funding: This research received no external funding. Acknowledgments: The financial support for this work was provided, in part, by the NSF grant OCE-0963064 and the Eagle Scholars Program for Undergraduate Research (Eagle SPUR) at the University of Southern Mississippi. The authors would also like to thank the department of chemistry and biochemistry and the honors college for their continued support and Peter Cragg at University of Brighton, School of Pharmacy and Biomolecular Sciences for constructive criticism Conflicts of Interest: The authors declare no conflict of interest.

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