Photophysical Investigation of a Benzimidazole Derivative and Its Applications in Selective Detection of Fe3+, Thermosensing and Logic Gates

The fluorescence based applications such as chemosensing of Fe3+ ion, temperature sensing, NOR-gate molecular logic operations, and fluorescent ink were achieved using an expired medicine namely Pantoprazole (sensor1) which is a derivative of benzimidazole class. The phenomenon of quenching was a basic principle used in sensing Fe3+. We found that there was a combined effect of dynamic and static processes in quenching of fluorescence of sensor1. To confirm the selectiveness of sensor1, we performed interference experiments with other metal ions. There was no interference between these metal ions and Fe3+. The low LOD value of 1.032 µM suggested that the molecule is highly sensitive towards Fe3+. A high quantum yield of 8.087% increased the possibility of using sensor1 for light applications. There was a 2:1 stoichiometric ratio between sensor1 and Fe3+. The FTIR data analysis confirmed the presence of functional groups S = O, C–O–C, etc. The average particle size found with the DLS method was 130.5 nm. The zeta potential of -12.7 mV indicated that sensor1 is quite stable in solution form. For logic gate application EDTA (Ethylenediaminetetraaceticacid) and Fe3+ were considered as inputs and fluorescence intensity was taken as an output. In thermal sensing application, the high value of activation energy i.e. 908 meV and relative sensitivity of 1.2% 0C−1 affirmed that sensor1 can be used for thermal applications. The sensor1 was also apt for fluorescent ink application. Hence this investigation deduced that sensor1 can be a potential candidate for such applications over other conventional synthesized fluorescent probes.


Introduction
The ascendancy of fluorescent sensors for the detection of metal ions lies in their high rate of selectiveness, sensitivity, non-toxicity, and reversibility. For this reason, there has been much assiduity on research and advancement of such chemosensors. Further, these can also be considered as substitutive for existing expensive, tardy detection methods like Inductive Coupling Plasma Mass Spectroscopy (ICP-MS), Flame Atomic Absorption Spectroscopy, (FAAS) etc. These molecular chemosensors change the color in presence of various metal ions such that detection can be easily done through visual observation. Among these detectable metal ions, Fe 3+ plays a very far-reaching role in many biological and chemical systems. Fe 3+ is essential to manage the homeostatic mechanism of the body. The deficit of Fe 3+ can cause anaemia in humans. Over concentration of Fe 3+ may lead to Parkinson's, Huntington's, and Alzheimer's diseases [1][2][3]. Hence it is essential to design fluorescent chemosensors for the detection of Fe 3+ . Even though there are many reports on Fe 3+ to date, there is always a need for advancement in these sensors in terms of economic procedures for synthesis, non-toxicity, reversibility, etc. [4][5][6].
Temperature plays a vital role in all physical and biological systems. Molecular thermometers are procuring large consideration over conventional temperature detectors such as thermometers, thermistors, thermocouples, etc. This is because these detectors have many shortcomings like low resolution capacity, low sensitivity, less accuracy, etc. The molecular thermometers have many advantages of non-contact measurements, optical stability, fast response, high resolution, non-toxicity, etc. Hence the usage of such molecular thermometers is surging considerably in recent years [7,8].
The idea of using fluorescent molecules for logic gates application was dawned in the early 90 s. Since then there is a lot of development in the field. Based on changes in fluorescence intensities of molecules after adding quenchers, the logic gate operations can be judged. These logic devices can be reliable candidates for applications in medical diagnostics, Photo Dynamic Therapy, recognition of pathophisyological conditions, cryptography, etc. [9,10]. So there is a need for developing economic, accurate molecular logic devices.
Herein, we propose an uncommon idea to reuse an expired medicine namely Pantoprazole (Sensor1) which is highly fluorescent, stable, selective for Fe 3+ metal ion, sensitive towards temperature, and can be applied for logic gate applications. Further, it has been also used for fluorescent ink application. Reworking on such fluorescent waste drugs can be advantageous and proper waste management can be expected. The study can be considered superior to previous reports since chemosensing, temperature sensing, logic gate, and fluorescent ink applications have been achieved by directly using the drug without any irksome synthesis procedures, toxic materials, expensive precursors, and precious time. The sensor1 showed selectivity towards Fe 3+ metal ion, temperature sensitiveness with a sensitivity of 1.21% 0 C −1 , NOR gate logic operations, and fluorescent ink application. Hence the drug can be an acceptable candidate for fluorescence applications.

Materials and Methods
The medicine namely Pantoprazole (sensor1) which has an expiry date of 04/2019 was used for the current study and to conduct experiments. Pantoprazole has a molecular weight of 383.4 g/mol and molecular formula C 16

Detection and Sensing of Fe 3+
Highly stable sensor1 in an aqueous solution was used to detect the Fe 3+ ion. The molecule was excited at 290 nm and it showed emission at 347 nm, which indicates that there might be a π-π * transition [1,6,12]. The solution of sensor1 was prepared with a concentration of 1 × 10 −4 M. Solutions of different metal ions namely Co 2+ , Ca 2+ , Mg 2+ , Zn 2+ , Ba 2+ , K + , Na + , Mn 2+ , Hg 2+ , Pb 2+ , Cu 2+ , cd 2+ ,Ag + ,Li + were prepared with a concentration of 1 × 10 −3 M and titrated against sensor1 separately. There was a minor change in intensity of sensor1 with these ions. We observed minute enhancement in the intensity when Ba 2+ , cu 2+ , Li + , Mn 2+ ions were added to sensor1. The metal ions other than these showed an inconsiderable decrease in intensity. This revealed that sensor1 was not sensitive towards these ions. When Fe 3+ ion was added to sensor1, we observed a sudden decrement in its intensity. This increased the probability of interaction between Fe 3+ and sensor1. Further to affirm the sensitiveness of sensor1 we calculated the detection limit using the formula 3.3(σ/S) [12] where σ is the standard deviation and S is the slope of the calibration curve. The LOD was found to be 1.032 µM.
The selectivity of sensor1 was confirmed by adding Fe 3+ (1 mM) as 7 equivalents with 40 µL each to the solution of sensor1. Each time when we added the Fe 3+ , there was a considerable reduction in the intensity of it (Fig. 1a). But this was not the same for other metal ions. We calculated the degree of reduced or enhanced intensities of sensor1 by each metal ion. Except for Fe 3+ (99.18%) all other ions showed less percentage of quenching efficacies, i.e. we witnessed the increase of fluorescence intensities in cases of Ba 2+ , cu 2+ , Li + , Mn 2+ by 5.45%, 30.41%, 9.71%, 2.15% respectively. There was a quenching effect in cases of Ca 2+ , Mg 2+ , Na + , Hg 2+ , Co 2+ , K + , Zn 2+ , Cd 2+ , Pb 2+ , Ag + by 0.5%, 2.66%, 0.46%, 14.90%, 5.94%, 5%, 14.73%, 3.79%, 4.26%, 3.49% respectively [12]. Hence it was deduced that sensor1 was selective and sensitive for Fe 3+ . Further, we also performed the interference test for sensor1. The solution of sensor1 was added with the solutions of different above mentioned metal ions. We didn't find any interference between Fe 3+ and other metal ions. The histogram shows the comparison of blank and other metal ions (Fig. 1b).
The optical and physical stabilities are the most important properties that fluorescent sensors should have. We examined sensor1 for its physical stability. The solution of sensor1 was kept for observation for about 50 days. The intensity measurement was taken once in 7 days. We didn't observe any major changes in intensity (Fig. 1c). Different increasing concentrations starting from 0.2 mM to 1.2 mM were added to sensor1, there was no shift in emission wavelength of it (Fig. 1d). Hence it can be assumed that the sensor1 can be used for different chemical and physical applications [12].

Job's Plot
To get an insight on stoichiometry between sensor1 and Fe 3+ ion, Job's method was used. The volumetric ratios of sen-sor1 and Fe 3+ were varied from 0.3 to 2.7 ml and 2.7 to 0.3 ml respectively. The total concentration of sensor1 and Fe 3+ was 10 -3 M. The graph of intensity Vs [Fe 3+ ]/([Sen-sor1] + [Fe 3+ ]) was plotted. The graph showed maximum intensity point at 0.3 on X-axis (Fig. 2). Hence there must be a 2:1 stoichiometric ratio between sensor1 and Fe 3+ [13,14].

Quantum Yield
The quantum yield was calculated using the single point method. Tryptophan was chosen as a reference with absorbance 3.009 at 270 nm and emission at 337 nm. The literature quantum yield of Tryptophan was found to be 0.15 at 270 nm. The quantum yield was calculated by the following equation [15][16][17], where Q R is the quantum yield of standard reference,I and I R are integrated fluorescence intensities of sample and reference respectively. A R and A are the absorbance of reference and sample. n and n R are RIs of sample and reference respectively. The calculated quantum yield was found to be 8.087%.

FTIR Analysis
The presence of functional groups such as S = O, N-H, C-O-C, etc. In sensor1 was confirmed by performing FTIR analysis (Fig. 3). The peak at 3368.75 cm −1 may be attributed to the stretching vibrations of the N-H group. The absorption peaks at 2922.37 cm −1 , 1616.82 cm −1 ,1551.86 cm −1 represent the stretching modes of C-H, C = N, C = C bonds [1,12]. The bands in the range 1460 cm −1 to 1017 cm −1 represent the vibrations of C-N, C-O, C-S, and C-H bonds respectively. The peak at 1337.33 cm −1 represents the stretching of the S = O group. Peaks in the range 916 cm −1 to 713 cm −1 represented the bending vibrations of C = C, C-H bonds [18,19].

Quenching Analysis
The solution of sensor1 was prepared with a concentration of 1 × 10 −4 M. The fluorescence intensity of sensor1 was highly quenched with the addition of Fe 3+ . (1 × 10 -3 ) Initiating with 40 µL seven equivalents were added to sensor1, we observed considerable decrement in intensity. To predict the type of quenching we took the help of the Stern-Volmer relation which defines the dependency of intensity with change in concentration. The plot of F 0 /F Vs [Fe 3+ ] was plotted and was analyzed by the following equation [20], where F 0 and F are fluorescence intensity without and with quencher i.e. Fe 3+ . Ksv is the Stern-Volmer constant and [Q] is the concentration of quencher. This plot showed a bend towards the Y axis, which indicated that the lessening of intensity was not only due to collision between sensor1 and Fe 3+ (Fig. 4a). There may be also a likeliness of complex formation in the excited state. Added, to confirm our assumption that quenching was not only due to the dynamic process, the graph of [( F0 F )-1]/[Q] Vs [Fe 3+ ] was plotted (Fig. 4b). This graph accounts for modified S-V equation,  Vs F F0 was a straight line with a nearly unit correlation coefficient (Fig. 4c). The slope of the graph was taken as K SV value and was found to be 8.76 × 10 3 M −1 . This study suggested that there is a combined effect of dynamic and static processes which is responsible for quenching of the intensity of sensor1 [21,22]. This combined effect is between the functional groups C-O-C or S = O etc. of sensor1 and Fe 3+ . Fe 3+ has shown a high chelating effect towards these functional groups, hence considerable quenching was observed [23]. There is also a possibility of non-radiative transfer of energy or electrons from excited states of sensor1 to half filled 3d 5 states of Fe 3+ [1]. The mechanism is represented pictorially in Fig. 4d.

Physical Properties of Sensor1
To predict the size of the Sensor1, Dynamic Light Scattering (DLS) measurements were performed. The molecule had an average size of 130.5 nm. The experiment was performed at a scattering angle of 90 0 and in mono-dispersed distribution form. 10% of total particles had approximately a size of 121.9 nm, 50% had 163.4 nm with a mean size of 169.4 nm (Fig. 5a). Further, to know the flocculation rate (Stability of the solution) of the suspension, we measured the zeta potential. The value of -12.7 mV, indicated that the particles are stable and they take a longer time to agglomerate in solution form [24][25][26] (Fig. 5b). The energy bandgap of the sensor1 was determined using Tauc's method (Fig. 5c). Exponent value n = 0.5 was taken which is meant for the indirect type of transition. The band gap was estimated to be 3.89 eV [27][28][29]. Added, to confirm the melting point and to gather the thermal information of the material we performed DSC measurements. There was negligible mass loss up to the temperature of 150 0 C. But the curve in the range 150 0 C-215 0 C shows that there was an abrupt release of energy and weight loss of the sensor1. We observed the least mass loss in the range 240-300 0 C. The DSC peaks are shown in Fig. 5d [30,31].

Logic Gate Application
To investigate logic gate application with sensor1, we conducted an experiment including sensor1, Fe 3+ , and EDTA. Concentrations with 1 × 10 −4 M, 1 × 10 −3 M and 1 × 10 −3 M of sensor1, Fe 3+ , and EDTA (A mixture of THF(Tetrahydrofuran) and water in a 1:1 ratio) were prepared respectively. The intensity of sensor1 at 347 nm emission wavelength was recorded. The intensity dropped considerably when Fe 3+ (40 µl) were added to the solution of sensor1. However, when the chelating agent EDTA was added, we observed a bit of enhancement in emission intensity. But this increment was inconsiderable and was below the threshold value. Hence we considered it as a '0' output value. When Fe 3+ was added again to this solution we noticed a further drop in intensity. The threshold value was set to 666 a.u. Fe 3+ and EDTA was considered as inputs and the resulting emission intensity was taken as output. The intensity above threshold value was taken as output value '1' and the intensity below threshold value was taken as '0' output value. The results resembled the outputs of the 'NOR' logic gate (Fig. 6). Thus we can assume that sensor1 can be used for the application of molecular logic gates, keypads, switches, etc. [10,32,33].

Application in Temperature Sensing
For the appraisal of temperature sensing behavior of sensor1, we observed the intensities of sensor1 at temperatures ranging from 25 0 to 70 0 C. Interestingly sensor1 was very much sensitive towards increasing temperatures owing to the fact that it can be used as a molecular thermometer in the physiological temperature range. As the temperature rose from 25 0 to 70 0 C we observed a decrement of 54.55% intensity with a sensitivity of 1.21% 0 C −1 . We also noticed that there was negligible or no shift in emission wavelength (347 nm) with the increment in temperature (Fig. 7a). The intensity showed good linear relation with temperature, I = -18.87 T + 2415.96 with a correlation coefficient of 0.973.Where 'I' can be indicated as fluorescence intensity and 'T' as temperature (Fig. 7b). The domination of non-radiative decays, enhancement of mobility, and collision rate of molecules at higher temperatures could be the reason for the reduction in the intensity. For the affirmation of reversibility of sensor1, consecutive heating and cooling experiments were performed. The sensor1 solution was first cooled to 15 0 C and the corresponding intensity was noted down. Further, the temperature was raised to 50 0 C and again the intensity was recorded. This procedure was repeated for five cycles and the Intensity Vs number of cycles graph was plotted [34]. The results were obtained as intended (Fig. 7c).
To look over the sensitiveness of the sensor1 towards temperature, we used the Arrhenius equation for finding activation energy. The expression goes as T,F 0 , and F have the usual meaning. A is a dimensionless constant, E a is activation energy and K B is Boltzmann's constant. The graph of ln (F 0 /F-1) Vs 1/K B T gave a straight line with an activation energy value of 908 meV which was much higher than previously reported values (Fig. 7d). Hence we can say that sensor1 is more sensitive towards temperature than any other reported sensors.Thus reversibility, sensitiveness, non-toxicity, wide range of temperature for detection makes sensor1 a good candidate for the application of molecular thermosensing [35,36].

As Fluorescent Ink
Satisfying PL stability, solubility, high fluorescence, and non-venomous properties of sensor1 made us use it for fluorescent ink application. In recent periods traditional inks which are used in information storage and encryption, document labeling, etc. are being replaced by fluorescent inks, hence are gaining more attention. For this application, the aqueous solution of sensor1 with a concentration of 1 M was prepared and the word 'INK' was written on Whatman filter paper (Fig. 8). The characters were invisible under daylight and visible under 350 nm UV light. This investigation made sensor1 apt for fluorescent ink application [3,[37][38][39].

Conclusion
This research work can be completed with a deduction that the highly fluoregenic waste/expired medicine namely Pantoprazole (sensor1) can be directly reused for fluorescence applications like Fe 3+ sensing, logic gate application, temperature sensing, and fluorescent ink application. The physical stabilities of sensor1 were confirmed by zeta potential and DSC studies. Further, to get a deep insight into molecule FTIR analysis, DLS measurements were performed. The 2:1 stoichiometry between molecule and Fe 3+ suggested the formation of a non-fluorescent complex between these two. The lower value of LOD i.e. 1.032 µM confirmed sensor1 was sensitive towards Fe 3+ . The activation energy of 908 meV implied that sensor1 can be used for thermal sensing applications. As there is no synthesis procedure involved in this method, this can be too economic and less time consuming. Hence this report can be considered as superior to previous reports in which tardy and expensive methods are involved.