The necessary building block for phytoplankton growth is phosphate (PO₄^3-) [1,2]. Global primary productivity of which is directly impacted by phosphorus bioavailability. In addition to restricting nitrogen fixation, phosphorus may also be a significant factor limiting primary output [3,4]. One of the key markers of pollution research is the measurement of the phosphate concentration of water bodies in the environment [5,6]. The overabundance of phosphorus released in industrial and agricultural effluent in recent years has caused the surrounding water to become eutrophic [7]. As a result, the amount of dissolved oxygen in the water drops and algae and other planktons proliferate quickly. Algae, planktons, and other aquatic life eventually declined in number or vanished entirely [8]. Thus determination phosphate ion in aqueous media is of tremendous relevance to the fields of agriculture, medicine, and the environment. Therefore, for a thorough understanding of the biogeochemical process and environmental preservation, simple and precise methods for determining the amount of phosphorus in water are necessary both theoretically and practically [9-13].

Thus, existing analytical approaches appear to be unfeasible, especially in the light of the financial status, literacy levels and analytical skill sets of common man in far flung areas. Therefore, there is an urgent need to develop rapid and cost-effective tests for naked-eye and onsite detection. Supra-molecular analytical chemistry provides an alternate approach to these standard tests. This branch of chemistry makes available intelligent organic and inorganic molecular architectures referred to as “Receptors” [14-16]. Herein, we present a molecular receptor tool to address this issue. This tool is advantageous and attractive in being common man-friendly and cost-effective.

Methodology

Exploring or designing a molecular system for sensing and naked-eye detection of phosphate anion is indeed a challenging task owing to the rigorous requirements. High sensitivity for a given interaction through fast display, ability to trace minimal analyte (phosphate) concentrations in aqueous media, and tolerance to external constraints are the pre-requisites. Any molecular approach which could overcome these restrictions can be investigated for possible applications. In order to assemble the required receptor design, we searched and worked with a highly conjugated, Y-shaped organic π-DA intramolecular charge transfer (ICT) system (Figure 1). This possess an amine moiety (–NH₂) as the donor along with CN and electron deficient aromatic ring as the two acceptors labeled as A1 and A2. Since in this molecule two acceptor moieties are in direct contact with a single donor moiety through extensive and alternate single and double bonds, we anticipate a strong pull-push effect [17]. If phosphate anion comes in contact with donor site, a strong ICT modulation could happen and prompt ion sensing is plausible. Importantly our proposed receptor, 2-amino-3-((2, 4-dinitrobenzylidene) amino) maleonitrile is a yellow solid and highly stable towards heat and light, hence could be practicable for real time applications.

Figure 1: Molecular structure of receptor with different intramolecular charge transfer (ICT) pathways directed from a single donor (D) to two acceptors labeled A₁ and A₂.

Ion Sensing Study

absorption band at 405 nm. Interaction of various common anions with receptor was investigated through spectrophotometric titrations in the form of sodium salts solutions prepared in distilled water. Among the anions tested (F^- , Cl^- , Br^- , I^- , CN^- , BO₃^- , SO₄^2-, NO₃^-, PO₄^3-, CH3COO^-), only phosphate species resulted in selective manipulation of its charge transfer absorption properties. Addition of this PO₄^3- is anticipated to show proton abstraction from the polarized -N-H site. This in turn results in the formation of negatively charged species bearing high charge density on nitrogen, in turn facilitating CT transitions. The same is clearly visualized with a fascinating colorimetric change of receptor solution from yellow to violet (Figure 2).

Figure 2: Absorption titration of receptor with upon addition of various equivalents of phosphate anion. Here molecule is taken in CH3CN and phosphate anion as sodium salt solution in water. Naked-eye colorimetric changes of receptor molecule in presence of phosphate anion also shown.

The instantaneously observed colorimetric changes are attributed to a strong red shift (170 nm) of 405 nm absorption peak, which is quite rare for any ion recognition event. This signifies the admirable sensitivity of the proposed molecular receptor towards phosphate recognition in aqueous media. Here continuous additions of PO₄^3- resulted in the decreasing intensity of 405 nm absorption and a concomitant increase of 575 nm band through a clear isosbestic point at 460 nm (Figure 2). These spectral changes and colorimetric behavior clearly reflects a typical Bronsted acid-base equilibrium or proton transfer signaling mechanism involving receptor as a proton donor and phosphate anions as acceptor [20]. The proposed mechanism can be assured due to the fact that above spectral changes was replicated by the addition of sodium hydroxide solution to the receptor. The similar mechanism has been previously proposed by Kaloo, M.A et al (2013) in their molecular recognition studies with similar ion sensors [18].

Calibration Curve

For quantification of phosphate anion concentration in water samples, standard calibration curve representing absorption ratiometric response (A575 nm/A405 nm) of receptor at 575 nm and 405 nm wavelengths with respect to the phosphate anion concentrations was developed [19]. A near- linear correlation obtained, demonstrates its potential utility for determination of phosphate anion in water samples up to 5-10 μM concentrations (Figure 3) with the help of this formula (LOD = 3σ/m); here ‘σ’ refers to standard deviation, ‘m’ refers to the slope of calibration curve [20].

Figure 3: Calibration curve constructed for the estimation of phosphate anion in water samples. 

Reversibility of Molecular receptor-anion Interaction

We further tried to explore the possibility of reversibility of the phosphate anion interaction. Here it was observed that upon addition of Ca²⁺ to the resulting assembly of anion-receptor interaction, immediate yellow color appeared. (Figure 2). Same holds true for Mg²⁺ with lower sensitivity compared to that of Ca²⁺. The equilibrium is driven by the formation of insoluble calcium and magnesium phosphates. Similarly addition of a proton (H⁺) source (sulphuric acid) drives the equilibrium back towards neutral receptor, simply by protonating the negatively charged species (Figure 2). These hypotheses are verified by the results of the absorption titration profile (Figure 4).

Figure 4: Reversibility of receptor-phosphate ion interaction by addition of Calcium ion. 

Construction of Boolean Logic Applications

Keeping in view the above explored recognition performance of molecular receptor was investigated for construction of some complex logical arithmetic applications via special combinations of phosphate anion and acid as inputs and absorption signals as out puts. The inputs and outputs were coded by binary digits. Here zero (0) is coded for ‘OFF’ while as one (1) is coded for ‘ON’. Since the absorption signal output produced by the phosphate anion (labeled here as InCH) in the solution can be efficiently reversed by the acidic input (labeled here as InH), such an optical switching performance of molecule by H⁺ produces a molecular inhibition (INH) function. InH acts as an inhibitor w.r.t to the response driven by InCH at 575 nm. Upon setting absorption response of molecule at 403 nm (A403 nm) as another absorption output, an implication (IMP) logic gate can be constructed. Thus, a complementary IMP/INH function can be comprehended. The logic circuit for the same has been presented in figure 5a. The switching behaviour of molecule between two states could be efficiently replicated multiple times by alternate additions of various combinations of chemical inputs. Such type of behaviour could be realized in the form of circuit deciphering “Writing-Reading-Erasing-Reading” behaviour at single molecular level. In such type of a logic mimic, let we have absorption signal output, A575 nm as “ON” state while as A403 nm as “OFF” state. Besides we will have InCH and InH as chemical inputs for the Set (S) and Reset (R) processes. In the presence of chemical input (S=1), means system writes and memorizes binary state “1” which could be erased by reset chemical input (R=1), resulting in writing and memorization of binary state “0”. Such reversible and reconfigurable successions could be envisaged in the form of feedback loop indicative of data storage attribute with “Writing-Reading-Erasing-Reading” [21] logic functions anticipated w.r.t output at 575 nm (Figure 5b). The significance of the proposed system could be experienced in terms of its potential bi-stability behaviour, “ON–OFF” state and therefore “Multi- Write” aptitude, which is important for the data storage systems.

Figure 5: (a) Complementary IMP/INH functions and truth table. (b) Feedback loop displaying writing-Reading-Erasing-Reading behavior.

In summary, we have explored and developed an interesting example of high conjugated CT molecular receptor for sensing of phosphate anion in water samples. This receptor was then used as an in-situ, near-real time tool for the naked-eye diagnosis of phosphate concentration in water. Since our receptor molecule can be reversed by the addition of calcium ion and protic acids, this makes it reusable and economic approach for detection of phosphate in water. Owing to the highly sensitive nature, strong absorption changes and reversibility, molecular receptor was explored for logic arithmetic applications like molecular memory which can mimic “Writing- Reading-Erasing-Reading” functions.

Acknowledgments

We are highly thankful to Bashir Ahmed GDC Bhaderwah for his help in the construction of logic gates and circuits.

Conflicts of Interest

Authors declare no conflicts of Interest.

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