A chemosensor selection for the fluorescence identification of tryptophan (Trp) amino acids in aqueous solutions with nanomolar detection

Sensors and Actuators B 246 (2017) 563–569

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A chemosensor selection for the fluorescence identification of tryptophan (Trp) amino acids in aqueous solutions with nanomolar detection Kundan Tayade a , Mahendra Sonawane a , Pritam Torawane a , Amanpreet Singh b , Narinder Singh b,∗ , Anil Kuwar a,∗ a b

School of Chemical Sciences, North Maharashtra University, Jalgaon, MS, India Department of Chemistry, Indian Institute of Technology, Ropar, Punjab, India

a r t i c l e

i n f o

Article history: Received 24 November 2016 Received in revised form 5 February 2017 Accepted 20 February 2017 Keywords: Receptor Amino acid Single crystal X-ray

a b s t r a c t A new disulfide-based, imine-linked fluorescent receptor 1 has been synthesized for the highly selective gratitude of tryptophan (Trp) among the all amino acids investigated in aqueous solutions via synergistic effects of intermolecular hydrogen bonding and electrostatic interactions. The photophysical properties of the receptor 1 were evaluated by UV/Vis absorption and fluorescence spectroscopic methods. Receptor 1 selectively recognized tryptophan (Trp) amino acid in DMSO/water with a detection limit down to 47.6 nM. The mechanism of binding was fully validated by computational studies. The theoretical calculations revealed the role of ␲-␲ stacking as well as hydrogen bonding in binding of tryptophan with receptor. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Chemosensors are molecules competent of connecting a given ionic and biomolecular species selectively such that the attaching occasion induces a measurable signal, mainly frequently a change in the spectral properties of a chromophore or fluorophore. In one meticulous plan, reported receptors live in the binding location for the goal of ionic and biomolecular species within the receptor and transitions occurs upon aggressive dislocation of this reporter molecule from the binding site by the guest molecules [1–5]. Even though some powerful analytical methods such as Gas chromatography, High Performance Liquid Chromatography, Gel permission chromatography are currently used to monitor low levels of these biomolecules, they have some drawbacks such as being time-consuming and expensive [6–8]. Thus, cheap and simple methods for monitoring these biomolecules are in high demand. Still, only a little fluorescent chemosensors for amino acids have been explained thus distant [9–11]. Amino acids, the input components of proteins, are small molecules with a variety of functional side chain groups, which consequence in different roles of amino acids in biochemical processes [12,13]. In this family unit, lysine is directly related to the

∗ Corresponding authors. E-mail addresses: [email protected] (N. Singh), [email protected] (A. Kuwar). http://dx.doi.org/10.1016/j.snb.2017.02.121 0925-4005/© 2017 Elsevier B.V. All rights reserved.

Krebs–Henseleit cycle and polyamine synthesis, and an suitable amount of lysine in the diet is essential for the metabolic functions and weight gain of animals, histidine is necessary for the expansion and restore of tissue as well as for the organize of essential trace elements communication in biological bases, tryptophan participates a critical element in life development such as protein biosynthesis, animal growth, and plant development [14–16]. In addition, the lack of some amino acids causes various irregularities, for example, shortage of tryptophan results in slow growth, dry mouth, nausea, blurred, edema, hair depigmentation, lethargy, liver damage, muscle and fat loss [17,18]. As the effect of growing notice waged to human fitness, diagnosis and treatment of illness, a lot of attempts have been straighter to the growth of new processes toward amino acid investigation. Although there are many systems for their recognition [19–21], still there is scope for development of new fluorescent and colorimetric chemosensors that can operate able in semi aqueous or purely aqueous medium. As a result, the curiosity in developing visual chemosensors purposely distinguishing an objective amino acid has developed progressively and the ability to easily and quickly obtain fingerprints for tryptophan amino acid by chemosensors will still comprise a get through. The amino acid containing aromatic rings such as Tryptophan, phenylalanine and tyrosine have affinity to interact with organic receptor through ␲-␲ stacking, which results in change in photo physical properties of receptor [22–24]. These types of non-covalent interactions

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are highly sensitive toward micro environment therefore can be used for development of highly sensitive sensors. Earlier Keeping these things in mind we have designed a disulphide based amine conjugated with 2,3-dihydroxybenzaldehyde through Schiff base linkage. Two hydroxyl groups provide hydrogen bonding interactions that can be used for selectively binding with suitable analyte. Here, we report the synthesis and characterization of a 3,3’-((1E,1’E)-((disulfanediylbis(2,1phenylene))bis(azanylylidene))bis(methanylylidene))bis(benzene1,2-diol) (1) and develop it for selective sensing of tryptophan. Further sensing of amino acids has been examined by using UV–vis and fluorescence titration experiments in aqueous solvent. The selective and sensitive nature of 1 toward tryptophan amino acid arises from the non-covalent interactions between imine, hydroxyl and sulphur containing functional groups.

Scheme 1. Synthesis of receptor 1.

the structure was solved with the olex2.solve structure solution program using Charge Flipping and refined with the olex2.refine refinement package using Gauss-Newton minimization.

2. Experimental section

2.4. Spectroscopic studies

2.1. General information

The amino acids recognition studies of 1 were performed at ambient temperature. All solutions were shaken properly to ensure consistency before recording the absorption and emission spectra. The amino acids recognition ability was studied by adding a fixed amount of different amino acids (1 equivalent, 20 ␮L, 1 × 10−3 M, c = 1 mM, in H2 O) to the standard solution of receptor 1 (c = 0.01 mM, 1 × 10−5 M, 2 mL, in DMSO). The fluorescence titration experiment was carried out to determine the association constant (Ka ) of the receptor 1 with the selective amino acids. The titration experiment was accomplished through a stepwise addition of amino acids solution (0.02 mL, 1 mM, guest in water) to a solution of receptor 1 (2 mL, 0.01 mM, host in DMSO) in the cell. The fluorescence intensity was recorded at ␭exc /␭em = 300/432 nm alongside a reagent blank. The excitation and emission slits were both set to 5.0 nm. All the experiments were repeated three times and mean of these values were taken, the variation of results has indicated using error bars (Fig. 6).

All reactions were carried out using oven-dried glassware beneath a slight positive pressure of nitrogen unless otherwise specified. Where necessary, solvents were purified prior to use. All chemicals were purchased from Sigma Aldrich, India. All reactions were magnetically stirred and monitored by thin-layer chromatography (TLC). 1 H NMR (400 MHz) and 13C NMR (100 MHz) spectra were determined on a Bruker AVANCE II 400 spectrometer. Chemical shifts for 1 H NMR are reported in parts per million (ppm), calibrated to the solvent peak set. Fluorescence measurements were made with a HORIBA JOBIN YVON, Fluoromax-4 Spectrofluorometer equipped with a xenon lamp. UV–vis absorption spectra were recorded on a Shimadzu UV-2450 spectrophotometer. 2.2. Synthesis of receptor 1 Receptor 1 was synthesized by refluxing 2,2 -disulfanediyldianiline (0.248 g, 1 mmol) and 2,3dihydroxybenzaldehyde (0.276 g, 2 mmol) in ethanolic medium (50 mL) for four hour. After cooling, the receptor 1 obtained as red crystal powder was collected by filtration and subsequently washed with cold ethanol, followed by recrystallization with ethanol. Crystals suitable for X-ray diffraction determination were obtained by slowly evaporating an ethanol solution of receptor 1 at room temperature. Yield 90%, mp > 250 ◦ C. 1 H NMR (400 MHz, DMSO-d6 ): d = 6.79 (t, 2H, Ar-H, J = 8 Hz), 6.97(d, 2H, Ar-H, J = 8 Hz), 7.08 (d, 2H, Ar-H, J = 8 Hz), 7.23 (d, 2H, Ar-H, J = 8 Hz), 7.29 (t, 2H, Ar-H, J = 8 Hz), 7.42 (d, 2H, Ar-H, J = 8 Hz), 7.56 (d, 2H, Ar-H, J = 8 Hz), 8.88(s, 2H, CH N), 9.12(s, 2H, Ar-OH), 12.86 (s, 2H, Ar-OH) ppm. 13 C NMR (100 MHz, DMSO-d ,): ␦ = 117.8, 117.9, 118.9, 119.1, 6 119.4, 123.0, 126.2, 127.5, 127.7, 130.5, 145.5, 148.9, 163.6 ppm. LC–MS(ESI) (C26 H20 N2 S2 O4 ) [M + H+ ] m/z Calcd 489.09 found 489.40. 2.3. X-Ray structure description Data of single crystal of receptor was collected on Bruker diffractometer. A suitable crystal was selected and mounted on a diffractometer. The crystal was kept at 298 K during data collection. The data collection crystal was an orange colour rectangular plate. The data collection strategy was set up to measure a quadrant of reciprocal space with a redundancy factor of 3.9, which means that 90% of these reflections were measured at least 3.9 times. Phi and omega scans with a frame width of 2.0◦ were used. Data integration was done with Denzo and scaling and merging of the data was done with Scalepack. Merging the data and averaging the symmetry equivalent reflections resulted in a Rint value of 0.036. Using Olex2,

3. Results and discussion 3.1. Synthesis and characterization Receptor1 was synthesized by simple condensation reaction of 2,2 -disulfanediyldianiline with 2,3-dihydroxybenzaldehyde in the ethanolic medium under reflux condition (Scheme 1). The red coloured precipitate was separated out after filtration; the receptor was characterized by 1 H NMR, 13 C NMR and LC–MS spectroscopic methods (Fig. S1–3, SI). The spectral investigation gave consistent data along with the molecular structure of 1. Finally, the structure of 1 was confirmed using single crystal X-ray crystallography shown in Fig. 1.The crystallographic data are listed in Table S1. The CIF file for receptor 1 was deposited in the Cambridge Structure Database with CCDC No 1470572. Perspective views of the crystal structure of 1, with labelling and packing diagram are shown in Fig. 1 and S4, (SI). Crystal of receptor 1 suitable for X-ray diffraction was obtained by slow evaporation of an ethanol solution of compound responding to the formula C26 H20 N2 O4 S2 . The crystal was tetragonal in molecular setting with I41 /a space group and there were eight molecules present in a unit cell (Z = 8). Hydrogen bonding of the phenolic–OH was major advantageous feature in host-guest complexation. Invariability, phenolic hydrogen atom formed an intramolecular hydrogen bond to the N atom of the azomethine group, giving a six member ring. This interaction was usually characterized in terms of phenolic oxygen to imine nitrogen separation [25,26]. This distance varies little between the two molecules. In all free receptor structure, the molecular involvement was via intramolecular hydrogen bonding. The receptor 1 exhibits intramolecular hydrogen bond-

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Fig. 2. A comparisons of UV–vis absorbance spectra of receptor 1 before and after addition of tryptophan.

Fig. 1. (a) Paerspective view of the receptor 1, Displacement ellipsoids are drawn at the 40% probability level and H atoms are represented by circles of arbitrary size. (b) Pbattern of intermolecular and intramolecular hydrogen bonding.

ing, where the −H atom of the phenolic hydroxyl group formed a strong O–HN intramolecular hydrogen bond with ON distance 1.38 and 1.40 Å respectively, which was in the middle of the expected range of such hydrogen bonds. The longer bond distance of receptor 1 undergoes a solvent assisted keto-tautomerization leading to a 2,3-dihydroxybenzaldehyde derivative of more hydrogen bonding environment in presence of tryptophan and exhibit blue shifting with little quenching of fluorescence intensity due to intramolecular charge transfer (ICT) process [27,28]. As shown in figure, receptor 1 is showing interesting intramolecular hydrogen bonding between four different molecules of ligand; here oxygen acts as hydrogen bond donor as well as hydrogen bond acceptor atom. Similarly OH hydrogen attached to second hydroxyl group form intermolecular hydrogen bonding with nitrogen. The supramolecular network supposed to break out upon interaction with tryptophan, which results in change in emission profile of receptor. The bond angles and bond lengths are shown in Tables S2 and S3. Hydrogen bonding parameters also incorporated in supporting information (Table S4). 3.2. Binding studies through UV–vis and fluorescence spectroscopy techniques After synthesis and characterization, the fluorescent receptor 1 was applied for the selective optical sensing of amino acids in phosphate buffer (pH = 7.0). The absorption spectrum of receptor 1 with

tryptophan is shown in Fig. 2. Receptor 1 exhibits peak maxima at 342 and 300 nm. The longer wavelength band at 342 nm may be assigned to transitions associated with phenol ring. The addition of tryptophan into receptor 1, there had no change in receptor 1. This is fact that the UV–vis spectra of receptor 1 remained unchanged even upon addition of tryptophan which illustrated that it did not bound with tryptophan in ground state and receptor 1 need to be excited for binding. Next, the fluorescence spectra of receptor 1 were recorded in the absence and presence of various amino acids in phosphate buffer (pH = 7.0) (Fig. 3). The fluorescent receptor 1 showed a strong emission peak at 432 nm, when excited at 300 nm. The addition of various amino acids caused selective changes in the fluorescence profile of receptor 1 in the presence of tryptophan. The addition of tryptophan results in the fluorescence quenching along with a blue-shift in the fluorescent emission from at 432 nm to 382 nm. No significant change in the fluorescence profile of receptor was observed in the presence of other tested amino acids. The results clearly supported the high selectivity of receptor 1 towards tryptophan. The fluorescence intensity of receptor 1 at 432 nm was enhanced steadily and blue-shifted with little quenching to 382 nm with the successive incremental addition of tryptophan (Fig. 4). The conformationally flexible sulphur contain of receptor 1 created a shape-complementary cavity to accommodate tryptophan selectively to form the host-guest complex through multiple intermolecular hydrogen bonds, which block the free rotation of the sulphur chain and also the intermolecular charge transfer (ICT) occurred between the encapsulated tryptophan and the fluorophoric unit of receptor 1 resulted in the fluorescence little quenching along with the blue-shift [29]. The fluorescence intensity at 382 nm was plotted as a function of the tryptophan concentration (Fig. 4) revealed that the emission of the receptor was found to be linearly proportional to the tryptophan concentration. The fluorescence titration data of receptor 1 was used to calculate the detection limit and the binding constant (Ka ). Detection limit of receptor 1 was calculated with 3␴ method on the basis of 3* SD/S (where SD is the standard deviation of the blank solution and S is the slope of the calibration curve) [30] which was found to be 47.6 nM within the linear range of 0–5 ␮M. The binding constants for a 1:1 stoichiometry were estimated using linear fittings of emission titration data, through Benesi–Hildebrand(B–H) method and were found to be Ka = 1.42 × 103 M−1 (Fig. 4). To analyses the binding behavior of receptor 1 with tryptophan, 1 H NMR of receptor was recorded in presence of tryptophan. As shown in Fig. S6, proton signal in aromatic region get broad due to induction of ␲-␲ stacking interaction between indole ring of tryptophan and aromatic unit of receptor 1.

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Fig. 3. Modulation of fluorescence responses of receptor 1 (0.1 mM) upon addition of 1 equiv. of various amino acids, ␭ex = 300 nm.

Fig. 4. (a) Fluorescence titration spectra of receptor 1 (0.1 mM) with tryptophan (1 mM). (b): Calibration curve for calculation of detection limit. (c) Benesi-Hildebrand plot for determining the affinity constant (Ka ) of receptor 1.Tryptophanamino acid complexation, Ka = 1.42 × 103 M−1 , where F = (F−F0 ).

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Fig. 5. DFT (GGA-DFT package of DMol3) computed probable structure of receptor 1 and its 1. tryptophan complex.

Fig. 6. Competitive fluorescence responses of 1 in the presence of various amino acids.

The change in emission intensity at 475 nm shows that amino acids are interacting with the receptor through hydrogen bonding which disturb the original confirmation of receptor and hence the Photophysical profile also changed. However tryptophan which has an aromatic unit also, can interact though ␲-␲ stacking interactions as revealed form theoretical calculation. The unique selectivity of tryptophan over other amino acids is due to involvement of aromatic stacking interactions.

3.3. DFT calculations To acquire additional close into the structure of receptor 1 and electronic message between its individual chromophores, DFT calculations on receptor 1 were performed using the GGA-DFT package of DMol3 with density functional theory. All calculations run through generalized gradient approximations GGA with basic set double numeric plus polarization (DNP). Geometry of

structures was minimized to calculate HOMO-LUMO gap. From optimized structures it was perceived that upon addition of tryptophan to receptor 1, binding energy becomes more negative, which prove the binding affinity of receptor 1 with tryptophan. Here amine hydrogens of tryptophan form hydrogen bond with sulphur of receptor 1, similarly COOH hydrogen form hydrogen bond with sulphur. Also aromatic ring of tryptophan stacked with aromatic ring of phenolic ring of receptor 1. All these non-covalent interactions provide stability to host-guest complex [31,32]. Upon complexation with tryptophan, the calculated interaction energy (Eint = Ecomplex Ereceptor Etryptophan )of −372.84 kcal mol−1 indicates the formation of a stable complex between receptor 1 and tryptophan. Also, there is an increase in the stability ofthe whole system. The band gap between HOMO and LUMO of receptor 1 alone becomes lowered for the 1. tryptophancomplex due to the possible charge transfer process occurring between receptor 1 and tryptophan (Fig. 5 and S5, SI).

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3.4. Comparative binding studies

References

To check the practical applicability of receptor 1, the competitive experiments were performed. One important criteria for a chemosensor is to give a specific response for the selective detection of target analytes (i.e. tryptophan) over a wide range of potentially competing amino acids, and to avoid the cross sensitivity. The competition experiments were conducted by recording the fluorescence spectra of 1 in the presence of 1.0 equiv. of tryptophan mixed with 1 equiv. of other amino acids mentioned above, respectively. Upon addition of tryptophan to the solution of receptor 1, new emission band were arises at 382 nm and 475 nm, where as another analytes show only emission at 475 nm. The bar diagram plotted by comparing ratio of fluorescence intensity at 382 nm and 475 nm (F382 /F475 ), which clearly confirmed the specificity of tryptophan for receptor. No significant variation in the fluorescence was found by comparison with the same amount of tryptophan solution in the presence and absence of other amino acids, and the relative error was less than ±5% (Fig. 6). These results point out that the recognition of tryptophan by receptor 1 is not notably interfered by other simultaneous amino acids. Therefore, the receptor 1 exhibits a high selectivity and specificity toward tryptophan, and opens a new door for the analytical application of 1 in real sample analysis. To justify the novelty and importance of presented work, the comparison of receptor 1 with existing literature has been tabulated in supporting information as Table S6. As shown in table our methods have advantages over existing literature such as working in aqueous medium, lower detection limit and shift in emission maxima at lower wavelength.

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3.5. Real sample analysis To find out the practical application of probe 1 in competing environment, spike/recovery experiment was performed in human serum samples. Human serum samples were prepared by using literature method.9 Human blood (2 mL) was centrifuged at 2000 rpm for 30 min at room temperature. Acetonitrile (1.2 mL) was added, and then different concentrations of tryptophan were spiked. After vortexing for 30 s, the mixture was centrifuged at 10000 rpm for 10 min. The supernatant was used for analysis. A known concentration of tryptophan was added in different samples. Emission intensity was recorded and concentration of tryptophan was determined by comparing the value with calibration curve. As shown in table, high recovery percentage (Table S5) of tryptophan reveals that another biomolecules present in human serum did not interfere the detection. 4. Conclusion In conclusion, a fluorescent dipodal material bearing imine linkages, sulphur and hydroxyl groups has been synthesized and crystal was tetragonal in molecular setting with I41 /a space group and there were eight molecules present in a unit cell (Z = 8). The receptor 1were investigated for their recognition properties towards various amino acids, and the system was found to be highly selective and sensitive to tryptophan, with a detection limit of 47.6 nM in DMSO. The mechanism of binding was fully validated using theoretical studies and NMR spectra. The non-covalent interactions present in ligands are sensitive toward microenvironment which used for selective sensing of tryptophan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2017.02.121.

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response towards fluoride ion, Sens. Actuators B Chem. 202 (2014) 1333–1337. K. Tayade, S.K. Sahoo, B. Bondhopadhyay, V.K. Bhardwaj, N. Singh, A. Basu, R. Bendre, A. Kuwar, Highly selective turn-on fluorescent sensor for nanomolar detection of biologically important Zn2+ based on isonicotinohydrazide derivative: APPLICATION in cellular imaging, Biosens. Bioelectron. 61 (2014) 429–433. M.S. Wheal, L.T. Palmer, Chloride analysis of botanical samples by ICP-OES, J. Anal. At. Spectrom. 25 (2010) 1946–1952. A. Kuwar, R. Patil, A. Singh, S.K. Sahoo, J. Marek, N. Singh, A two-in-one dual channel chemosensor for Fe3+ and Cu2+ with nanomolar detection mimicking the IMPLICATION logic gate, J. Mater. Chem. C 3 (2015) 453–460. K. Tayade, B. Bondhopadhyay, H. Sharma, A. Basu, V. Gite, S. Attarde, N. Singh, A. Kuwar, Photochemical & photobiological sciences ‘turn-on ’ fluorescent chemosensor for zinc(II) dipodal ratiometric receptor: application in live cell imaging, Photochem. Photobiol. Sci. 13 (2014) 1052–1057. A. Singh, A. Singh, N. Singh, D.O. Jang, Selective detection of Hg (II) with benzothiazole-based fl uorescent organic cation and the resultant complex as a ratiometric sensor for bromide in water, Tetrahedron (2016) 1–7. H. Sharma, N. Singh, D.O. Jang, A benzimidazole/benzothiazole-based electrochemical chemosensor for nanomolar detection of guanine, RSC Adv. 5 (2015) 6962–6969. J. Singh, A. Singh, N. Singh, Urea based organic nanoparticles for selective determination of NADH, RSC Adv. 4 (2014) 61841–61846.

Biographies Mr. KundanTayade is currently a Ph.D. student in North Maharashtra University, Jalgaon, India. He had completed his master from the same University. His current main research is design fluorescent probes and chemical sensors.

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Dr. Mahendra Sonawane is completed a Ph.D from North Maharashtra University, Jalgaon, India. He did his MSc degree from the same University. His current research is focused on design of fluorescent based chemosensors. Mr. Pritam Torawane is currently a Ph.D student in North Maharashtra University, Jalgaon, India. He did his MSc degree from the same University. His current research is focused on design of fluorescent based chemosensors. Mr. Amanpreet Singh is currently a Ph.D. student in Indian Institute of technology, Ropar, India. He had completed his master from the Dr B. R. Ambedkar National Institute of Technology, Jalandhar, India. His current main research is design of fluorescent probes for biomolecules. Dr. Narinder Singh completed his Ph.D. degree from Guru Nanak Dev University, Amritsar. After spending 3 years as a postdoctoral fellow in abroad, he joined the Indian Institute of Technology, Ropar (IIT Ropar) as an Assistant Professor in Department of Chemistry. He has published more than 175 research papers in International peer reviewed journals (ACS, RSC, Elsevier, Wiley, etc.). His areas of research include super-molecular and materials chemistry for the application of Chemo- and Biosensors, Photo-detectors, Synthesis of new organic receptors. Dr. Anil S. Kuwar is currently working as an Assistant Professor at North Maharashtra University, Jalgaon, India. He had completed his Ph.D. from the North Maharashtra University. His current main research is supramolecular chemistry, organic light emitting diode and bioinorganic chemistry.