Recent Progress on the Evolution of Pourbaix Sensors: Molecular Logic Gates for Protons and Oxidants

Recent progress in the area of molecular logic, in particular molecules capable of sensing for acidity and oxidizability, are gathered together in this short review. Originally proposed as AND logic gates that provide a high fluorescence output when simultaneously protonated and oxidized, the concept has been extended from two-input to three-input variants and to include molecules that function as INHIBIT logic gates. Photochemical concepts such as photoinduced electron transfer (PET) and internal charge transfer (ICT) are exploited as favorite design concepts. This review highlights the evolution of Pourbaix sensors with anthracene, pyrazoline, and naphthalimide fluorophores. Future applications abound in various disciplines from corrosion science, material science, geochemistry to cell imaging.


Introduction
The 6th International Conference on Molecular Sensors and Molecular Logic Gates (MSMLG) was held in Dalian, China on June 4 and 5 [1]. The conference presentations were dominated by work on fluorescent molecular sensors for metal cations, and to a lesser extent, on the detection of anions and organic molecules, in solution, luminescent materials, and cell imaging applications [2]. Presentations on the recent developments of molecular logic gates [3] were underrepresented as many in the field did not make the pilgrimage to Dalian. Nonetheless, the conference was an overwhelming success with 193 registered participants [1].
This year, 2018, marks the 25th anniversary since the publication of the first book on fluorescent chemosensors as a symposium series [4]. As comes with technological advancements, it is usually not without criticism and controversy. The analytical chemistry community, as pointed out by Wolfbeis, consider chemical sensors as miniaturized analytical devices that can deliver real-time and online information on the presence of specific compounds or ions in complex samples [5]. In 1997 Desvergne and Czarnik published Fluorescent Chemosensors of Ion and Molecule Recognition and provided a less restrictive definition from an organic chemist's perspective such that a continuous time-measure parameter was not a necessary consideration [6]. A positive consequence has been the creative pursuit of libraries of new molecules for a range of analyte targets. This year also marks the silver anniversary of de Silva's digital leap from fluorescent chemical sensing for a single analyte to multi-input detection with the introduction in 1993 of the cross-fertilization of Boolean algebra with the organic chemistry realm [7]. The current testament in the field of information processing with molecules Molecular Logic-based Computation by de Silva celebrates its 5th birthday [8]. This year is also the 5th anniversary of the concept of 'Pourbaix sensors', the thematic topic of this article [9,10]. the chemosensor community with an interest in the design, synthesis, and function of molecular sensors and molecular logic gates are the target audience. Previous articles have highlighted aspects of design concepts, the mechanisms of operation (photoinduced electron transfer (PET) and internal charge transfer (ICT)) [16][17][18] and some envisioned applications of this new class of chemosensor [19,20]. Pourbaix sensors could, in principle, have applications in various disciplines from living cell imaging, corrosion detection, water quality, medical diagnostics, and environmental analysis. Although this new field is currently at the proof-of-concept stage, we are already considering pragmatic uses. This paper highlights many of our recent advances reported at the 2018 MSMLG conference as well as our latest published results.  The progression from two-input 2 to a three-input AND logic gate 4 required additional spacer and receptor modules resulting in a receptor 1 -spacer 1 -fluorophore-spacer 2 -receptor 2 -spacer 3 -electron donor format [22]. The concept of detecting for a trio (or more) of analytes by a fluorescent response was proposed as a 'lab-on-a-molecule' [23]. Enhanced fluorescence is emitted by 4 in the presence of 'high' Na + , H + and Fe 3+ levels with an Φ f of 0.072 in methanol. The benzo-15-crown-5 ether binds Na + , the tertiary amine H + and the ferrocene is oxidized by Fe 3+ . With reference to 2, PET from the crown receptor to the anthracene fluorophore is a third PET deactivation pathway. The higher Φ f is due to the Na + -bound crown electrostatically retarding the PET process from the excited anthracene to the ferrocenium unit [24]. High Na + , H + and Fe 3+ levels are all associated with steel corrosion thus 4 is a 'lab-on-a-molecule' that could in principle provide an early warning to engineers inspecting the integrity of steel structures in the sea.
Pourbaix sensors 5 and 6 are our first examples with a naphthalimide fluorophore ( Figure 2) [25,26]. An example by Gan and co-workers has much the same hallmarks of 6, but with a piperazine bridge and carboxyferrocene connected at the 4-amino terminus and an oxyalkyl chain at the imide position [27]. It was tested under acidic and oxidizing conditions, but only as separate inputs, so the synergetic effect from both inputs was not captured in the fluorescence output. Compared to the anthracene prototypes 1-4, the naphthalimide prototypes 5-8 have longer emission maxima and are solubility in aqueous methanol. Molecule 6 is designed in the same manner as 1 and 2 with a fluorophore-spacer 1 -receptor-spacer 2 -electron donor format. The emission peak maximum of 6 is observed at 526 nm with a four-fold switching enhancement and an Φ f of 0.04 which is double the emission of the anthracene models 1 and 2.
Rearrangement of the modules, as exemplified by 5, in the order 'electron-donor-spacer 1 -fluorophore-spacer 2 -receptor' improved device performance to yield a 16-fold switching enhancement and a Φ f of 0.09. A sister molecule with piperazine instead of methylpiperazine as the proton receptor switches 6-fold with an Φ f of 0.06 [28]. The shorter distance for charge transfer between the ferrocene and the excited state fluorophore is a favorable factor as is the ππ* ICT state, which causes a dipole moment in the photoexcited state [29]. Consequently, charge separation within the excited naphthalimide fluorophore yields a negative charge build-up at the imide carbonyl end and a positive charge build up at the 4-amino end [30]. binds Na + , the tertiary amine H + and the ferrocene is oxidized by Fe 3+ . With reference to 2, PET from the crown receptor to the anthracene fluorophore is a third PET deactivation pathway. The higher Φf is due to the Na + -bound crown electrostatically retarding the PET process from the excited anthracene to the ferrocenium unit [24]. High Na + , H + and Fe 3+ levels are all associated with steel corrosion thus 4 is a 'lab-on-a-molecule' that could in principle provide an early warning to engineers inspecting the integrity of steel structures in the sea.  [25,26]. An example by Gan and co-workers has much the same hallmarks of 6, but with a piperazine bridge and carboxyferrocene connected at the 4-amino terminus and an oxyalkyl chain at the imide position [27]. It was tested under acidic and oxidizing conditions, but only as separate inputs, so the synergetic effect from both inputs was not captured in the fluorescence output. Compared to the anthracene prototypes 1-4, the naphthalimide prototypes 5-8 have longer emission maxima and are solubility in aqueous methanol. Molecule 6 is designed in the same manner as 1 and 2 with a fluorophore-spacer1-receptor-spacer2-electron donor format. The emission peak maximum of 6 is observed at 526 nm with a four-fold switching enhancement and an Φf of 0.04 which is double the emission of the anthracene models 1 and 2.
Rearrangement of the modules, as exemplified by 5, in the order 'electron-donor-spacer1fluorophore-spacer2-receptor' improved device performance to yield a 16-fold switching enhancement and a Φf of 0.09. A sister molecule with piperazine instead of methylpiperazine as the The 'electron-donor-spacer 1 -fluorophore-spacer 2 -receptor' arrangement was further tested by designing two pairs of regioisomers as exemplified by 7 and 8 [31]. We observed that 7 functions as a H + , Fe 3+ -driven AND logic gate with a 10-fold fluorescence enhancement and a Φ f of 0.03 in 1:1 (v/v) methanol/water. Regioisomer 8 did not response to H + and Fe 3+ inputs with an elevated fluorescence signal. Rather Zhang has reported a novel ferrocenyl-naphthalimide in water reminiscent to a Pourbaix sensor [32]. However, the adjacent thiosulfoxide prevents the piperazine nitrogen atom from acting as a proton receptor. Rather the molecular device is presented as a multi-channel dual probe for Cu 2+ or Hg 2+ . The yellow solution turns colorless in the presence of either metal cation, and simultaneously results in a fluorescent 'turn-on' response with Cu 2+ with a Φ f of 0.59 and a fluorescent 'turn-off' response with Hg 2+ in 7:3 (v/v) acetonitrile/water. The molecule was used in intracellular fluorescent imaging studies and found to be non-toxic and to give a promising response on Cu 2+ detection.
A concern when analysing heavy metal ions in water is the formation of a metal salt precipitate at higher pH levels [11]. A strategy for ensuring precipitation does not occur during analysis in water is to work at high proton concentrations of say pH 2 [33]. Example 9 was designed as an H + -driven fluorescent pH indicator for use in water (Figure 3) [34]. As the molecule emits in the UV and blue visible region of the electromagnetic spectrum, it is susceptible to quenching by an inner filter effect by Fe 3+ . A line break was not observed in the Job's plot analysis ruling a metal-ligand interaction. Probe 9 has a pK a of 7.9 and a fluorescent quantum yield of 0.62 in water. Addition of 11 mM Fe 3+ at pH 3 results in near complete quenching of the fluorescent. The fluorescent output in response to H + and Fe 3+ is an example of two-input INHIBIT logic and provides a way of avoiding hydrolysis, thus ensuring a more reliable measurement.
visible region of the electromagnetic spectrum, it is susceptible to quenching by an inner filter effect by Fe 3+ . A line break was not observed in the Job's plot analysis ruling a metal-ligand interaction. Probe 9 has a pKa of 7.9 and a fluorescent quantum yield of 0.62 in water. Addition of 11 mM Fe 3+ at pH 3 results in near complete quenching of the fluorescent. The fluorescent output in response to H + and Fe 3+ is an example of two-input INHIBIT logic and provides a way of avoiding hydrolysis, thus ensuring a more reliable measurement.  Triarylpyrazolines have been in the molecular engineer's toolkit for some time as fluorescent pH indicators [35], metal ion chemosensors [36][37][38], and cell imaging probes [39]. The rigid heterocyclic framework and facile aryl-substitution allows for fine-tuning of the photophysical properties [40]. Pyrazoline-based chemosensors are usually designed with a PET process originating from a moiety at the 5-position and an ICT state modulated by the choice of aryl substituents on the phenyl rings at the 1and 3-positions. Quenching of the fluorescent occurs at 10 −1 M acid due to protonation of the N2 atom. We capitalized on this known deactivation pathway to demonstrate H + -driven ternary 'off-on-off' logic with 10 [41] and H + , Fe 3+ -driven INHIBIT logic with 11 [42] (Figure 3). The latter logic result comes from an enhanced fluorescent signal at low proton and high oxidant threshold levels. While the H + -driven indicator 10 has a fluorescence quantum yield of 0.38 and a fluorescence enhancement of 20 [41], the redox-enabled pH-disabled logic gate 11 registers a remarkable 120-fold fluorescent enhancement, but with only 0.012 quantum yield output [42] in aqueous methanol. Experiments with stronger oxidants and inert electrodes should enhance the fluorescence output.
Zhu reports a dicyanomethylene-4H-pyran 12 with conjugated dimethylaniline and ferrocene moieties that in THF emits at 610 nm on addition of one equivalent of Fe 3+ [43]. It is suggested that the fluorescence is regulated by both PET and ICT mechanisms. The study includes cyclic voltammetry and time-resolved fluorescence measurements in addition to DFT calculations. Examined as a redox-fluorescent switch, the fluorescence enhancement exceeds 100-fold. Regrettably, though, the authors do not report the effect of acid on the fluorescence modulation, and consequently, the built-in two-input logic gate features.
A H + , Fe 3+ driven INHIBIT logic gate 13 by Wu uses absorbance at 405 nm in acetonitrile as the output channel [44].
A modular design is evident according to an 'electron-donor-fluorophore-spacer-receptor' construction.
The prototype consists of a dimethylaniline moiety (receptor) attached to a terpyridyl Pt complex (fluorophore) linked to a ferrocenyl acetylide (electron donor). A virtual spacer via the carbon-carbon bond between the dimethylaniline and terpyridyl units keeps these two modules electronically decoupled. Photoinduced electron transfer from the dimethylaniline unit to the ferrocenyl acetylide regulates the emission output.
A three-input combinatorial INHIBIT logic gate 14 was demonstrated by James and co-workers using a ferrocene-naphthalimide-boronic acid conjugate (Figure 4) [45]. A high fluorescence output was observed in the presence of 'high' Fe 3+ , 'low' sodium L-ascorbate and 'low' F − . Minimal fluorescence output is observed with no added chemical inputs on excitation at 403 nm in THF. One equivalent of Fe 3+ is sufficient to oxidize the ferrocene moiety and to cause a 100-plus fluorescent enhancement centred at 512 nm. Fluoride acts as a nucleophile to form a tetrahedral boronate anion, which causes a delayed fluorescence enhancement, possibly due to an ion pair interaction between Fe 3+ and F − .
Chemosensors 2018, 6, x 6 of 9 secure fastening of the molecular logic gate to the amino-terminated beads by peptide coupling chemistry [50]. The millimeter-sized sensor glows fluorescent green in the presence of 200 µM H + and 20 µM Fe 3+ , which can be reversibly switched to the initial 'off' state on addition of hydroxide and EDTA. The beads were observed to be robust and stable when stored in methanol over numerous months, which bodes well for real-time continuous measurements.

Conclusions
Chemosensors that function as molecular logic gates responsive to pH and redox potential could have practical applications in corrosion science, medical diagnostics, catalysis, environmental monitoring, and cell biology [19,20]. With the proof-of-concept now established, one obvious direction is the testing of Pourbaix sensors in living cells. Many of the presented examples, notably with a naphthalimide fluorophore, are naturally suited as fluorescent cellular imaging probes and as potential cancer therapy agents [51,52]. Other avenues to consider are the development of solidbound fluorescent Pourbaix sensors [49] and luminescent-based nanomaterial platforms (e.g., carbon quantum dot sensors [53,54]).
For those interested in a challenge, current limitations to be addressed include: (1) enhanced aqueous solubility without the need for a co-solvent; (2) improved fluorescent quantum yields (Φf ≥ 0.1); (3) longer emission wavelengths towards the infra-red region; and (4) prototypes responsive to various oxidants.
To conclude, the 7th International Conference on Molecular Sensors and Molecular Logic Gates (MSMLG) will be held in 2020 in Rio, Nevada, America. Our host will be Anthony Czarnik.  The molecule 15 is a logic gate array equivalent to wiring together three-input AND, three-input INHIBIT and two-input OR logic gates [46]. On first inspection, the molecular device appears as a three-input logic gate driven by Na + , H + and Fe 3+ as with 4. A bright green fluorescence with an Φ f of 0.0193 is observed in the presence of 'high' levels of all three analytes. However, a green fluorescence output is also observed in the presence of 'high' H + and Fe 3+ and 'low' Na + levels. Despite the favourable photogenerated electric field effect at the 4-amino position of the excited naphthalimide fluorophore [29], the greater separation distance between the crown ether receptor and the fluorophore is not enough to retard PET. The net result is that of eight possible truth table entries, two combinations of inputs yield a green emission output.
Many of the logic gates reviewed are soluble in an organic solvent, or require a co-solvent for solubility in water. Hence, the attachment of organic molecules onto solid supports is a convenient way of circumventing the issue of insolubility in water. Attachment onto a solid support opens up many applications too, such as molecular computational identification (MCID), the labelling and identification of small objects [47]. PASS 1 and YES logic tags with anthracene and 4-aminonaphthalimindes have recently featured on polyethyleneglycol-polystyrene (Tentagel) beads and silica particles [48]. Our first Pourbaix sensor on a polymer bead 16 retains the modular skeleton of 5 [49], but is extended with a benzoic acid moiety, which functions as a linker unit allowing for secure fastening of the molecular logic gate to the amino-terminated beads by peptide coupling chemistry [50]. The millimeter-sized sensor glows fluorescent green in the presence of 200 µM H + and 20 µM Fe 3+ , which can be reversibly switched to the initial 'off' state on addition of hydroxide and EDTA. The beads were observed to be robust and stable when stored in methanol over numerous months, which bodes well for real-time continuous measurements.

Conclusions
Chemosensors that function as molecular logic gates responsive to pH and redox potential could have practical applications in corrosion science, medical diagnostics, catalysis, environmental monitoring, and cell biology [19,20]. With the proof-of-concept now established, one obvious direction is the testing of Pourbaix sensors in living cells. Many of the presented examples, notably with a naphthalimide fluorophore, are naturally suited as fluorescent cellular imaging probes and as potential cancer therapy agents [51,52]. Other avenues to consider are the development of solid-bound fluorescent Pourbaix sensors [49] and luminescent-based nanomaterial platforms (e.g., carbon quantum dot sensors [53,54]).
For those interested in a challenge, current limitations to be addressed include: (1) enhanced aqueous solubility without the need for a co-solvent; (2) improved fluorescent quantum yields (Φ f ≥ 0.1); (3) longer emission wavelengths towards the infra-red region; and (4) prototypes responsive to various oxidants.
To conclude, the 7th International Conference on Molecular Sensors and Molecular Logic Gates (MSMLG) will be held in 2020 in Rio, Nevada, America. Our host will be Anthony Czarnik.
Funding: This research received no significant external funding.