Research Article
Pyrene functionalized luminescent phenylalanine for selective detection of copper (II) ions in aqueous media
https://doi.org/10.21203/rs.3.rs-2319841/v1
This work is licensed under a CC BY 4.0 License
published 09 Jan, 2023
You are reading this latest preprint version
Pyrene
phenylalanine
Fluorescence
Chemosensor
Gel
In the last few decades, molecular recognition and host-guest inclusion have been promising in supramolecular chemistry, which detect many ionic and neutral species based on non-covalent interaction between host and guest moiety.1, 2 It provides a great tool for creating chemosensors to detect various biologically and environmentally crucial metal ions.3–8 Due to their electronic transition, chemosensors binding with the analyte produce a measurable analytical signal.9 In 1867, the first fluorescence-based chemosensor for the detection of Al3+ ions was reported by F. Goppelsroder. Fluorescence-based chemosensors have attractive features, such as selectivity and sensitivity towards various metal ions such as Cu2+, Zn2+, and Ni2+. 10–12 The judicious design of the chemosensors is the crucial factor for tuning selectivity. Recently, various fluorescent chemosensors have been synthesized and utilized for sensing of different metal ions.13 On comparing fluorescence with other competitive methods like ICP-MS, atomic absorbance spectroscopy (AAS),14 Flame atomic absorption spectrometry(FAAS), their easy operation technique, detection of various metals even at their sublethal concentration, fast response, and cost efficiency give us the possibility for their application.15
Fluorescence-based chemosensors are promising for the sensitive and selective testing of heavy metal ions, which act as biological and environmental hazards. In terms of natural abundance, copper is the third most abundant element in the human body and functions in many biological and physiological processes.16, 17 The toxic nature of copper causes dysfunctions in the cellular processes. It results in neurodegenerative diseases such as Wilson’s 18, 19 amyotrophic lateral cases of sclerosis,20, 21 Alzheimer’s,22 etc. Excess amount of copper also affects the plant in terms of victimization of physiological arrest, stunted development, and even death.23–25 Moreover Copper is widely used in several agriculture and industrial applications, that causes copper as a major pollution source in the environment.26, 27 The acceptance level of copper in drinking water is 2mgL− 1 (30µM) recommends by World health organization (WHO).28 In human blood, normal concentrations of copper vary in the range of 100-150g/dl (15.7–23.6 µM).15 Thus, the detection of copper in a highly effective and selective manner is huge importance for environmental and biological sciences.
However, in the field of supramolecular chemistry, self-assembly of low molecular weight species attract great interest due to their application in various fields such as drug delivery, construction of novel soft material, biosensor, gene therapy, etc.29–32 The driving force for the formation of gel through self-assembly are non-covalent interactions such as electrostatic interaction, hydrophobic interaction, π-π interaction, hydrogen bonding interaction, and other supramolecular forces.33, 34 Fluorescent supramolecular gels derived from biologically relevant molecules are currently attracting many researchers worldwide because of their great application potential in several fields.
Pyrene derivatives are widely helpful for developing fluorescence-based chemosensors due to their several properties such as strong fluorescence, long fluorescence lifetime, π-π* stacking, polarity sensitive vibronic emission, high charge carrier mobility, and chemical stability.35–40 in recent years many study have been done using pyrene derivatives to sense various ions such as Al3+, Cu2+, Fe3+, Cr3+.41, 42 Yang et. al develop a pyrene based fluorescent sensor for the detection of Cu2+ ions.43 Here we have designed a fluorescent chemosensor for selective and sensitive detection of Cu2+ ions. It detects Cu2+ through a fluorescence turn-off mechanism in the presence of various other metal ions. 1 also has the property to form a gel through π-π stacking. Moreover, 1 exhibits a promising tool for the detection of Cu2+ ions in natural water samples.
FTIR and UV- visible spectra were recorded on a 8400S and Agilent Cary 60 single beam, UV-Visible spectrometer with serial no. - MY19329220, respectively, and fluorescence spectra were measured on a Fluoromax 4CP plus spectrofluorometer with a 10 mm quartz cell at 25 oC. The melting point was measured using an EZ-Melt (automated melting point apparatus). 1H and 13C-NMR spectra were recorded on a JEOL AL 300 FT-NMR at an operating frequency of 500 MHz (1H) and 126 MHz (13C), respectively. Spectrometer operation at 500 MHz and 126 MHz in DMSO-d6 solutions respectively are given in parts per million (ppm) related to Tetramethyl silane (TMS, δ = 0.00ppm), and splitting pattern designated as s (singlet), d (doublet), t(triplet). High-resolution mass spectra (ESI-HRMS) were recorded on SCIEX X500R (TOF-MS) mass spectrometer. For atomic force microscopy NT-MDT, Model: Solvernext was used where samples were produced on tiny glass slides, which were then positioned beneath the device. On an MCR 72 Modular Compact Rheometer, rheological measurements were made utilizing a 25 mm diameter parallel plate geometry with a 1 mm gap. To determine the storage or elastic modulus, G′, and loss or viscous modulus, G′′, the gel sample was put on the lower plate and a stress amplitude sweep experiment was conducted at a constant frequency and 25°C. The frequency sweep measurements were performed at constant stress in the linear viscoelastic range. From each set, vials containing 1 g of compound 1 for rheological experiments.
All chemicals are purchased from a commercial supplier and used without further purification. Lithium hydroxide and L-phenylalanine were purchased from spectrochem. 1-pyrenecarboxyaldehyde was purchased from Sigma-Aldrich, and sodium borohydride was purchased from S. d. fine. Metal salts Ni(NO3)2.6H2O, AgNO3, Pb(NO3)2, Fe(NO3)3.9H2O, Cd(NO3)2.4H2O, KNO3, Co(NO3)2.6H2O, Hg(NO3)2, Ca(NO3)2.4H2O, Cu(NO3)2.3H2O, NaNO3, Zn(NO3)2, were purchased from Himedia.
Spectroscopy measurement: - Alkali, alkaline earth, and transition metal ions were utilized for sensing to investigate variations in the emission and absorption spectra of 1. Nitrate salts of different metals were used to provide the metal ions: Ni(NO3)2.6H2O, AgNO3, Pb(NO3)2, Fe(NO3)3.9H2O, Cd(NO3)2.4H2O, KNO3, Co(NO3)2.6H2O, Hg(NO3)2, Ca(NO3)2.4H2O, Cu(NO3)2.3H2O, NaNO3, Zn(NO3)2. A stock solution of 1 (10µM) was prepared in Water: DMSO (9:1, v/v at room temperature). While to make a stock solution of metal ions (Na+, K+, Pb2+, Ca+ 2, Fe+ 3, Hg+, Ni+ 2, Cu+ 2, Co+ 2, Zn+ 2, Cd+ 2, Ag+ 2) dissolve their nitrate salt in distilled water, (10 mM). Afterward, 3.0 ml of the compound 1 was taken in a quartz cuvette (4 ml, path length, 1 cm) for the titration experiment, and progressively metal ion solutions were added using a micropipette. The maximal excitation and emission wavelengths for fluorescence measurements were 342 nm and 375 nm, respectively, while the wavelength range for absorbance measurements was 200–800 nm.
L-phenylalanine (0.1651g; 1mmol) and LiOH (0.041g; 1mmol) were dissolved in methanol (10 ml), and previously dissolved 1-pyrenecarboxyaldehyde (0.230g; 1mmol) in hot methanol was added, drop-wise. The reaction mixture was refluxed for 5–6 hr. at 65 oC followed by cooled to room temperature, NaBH4 (0.0378g 1mmol) was added portion wise and stirred at room temperature for 4–5 hours. The solvent was removed under vacuum, and the crude product was dissolved in distilled water (10 ml), it was brought to a pH of 5–6 by adding 1N dil. HCl. The resultant solid was filtered off and dried in a vacuum. Light yellow-colored solid compound 1 formed (Scheme 1). Yield (0.311 g, 82%); Mp. 222oC; FT-IR (KBr) : 3437(br), 3042(m), 2856, 1617(s), 1386, 1119, 843, 708 Wave number [cm− 1]; 1HNMR (500 MHz, DMSO-d6) δ 8.32–7.93 (m, 9H), 7.20–7.14 (m, 5H), 4.46 (d, J = 13.0 Hz, 1H) 4.27 (d, J = 12.0 Hz, 1H), 3.53 (t, J = 6.5 Hz, 2H), 2.98–2.94 (m, 1H), 2.87–2.89 (m, 1H);13C NMR (126 MHz, DMSO-d6) δ 174.67, 138.27, 133.17, 130.76, 130.34, 130.14, 129.26, 128.64, 128.02, 127.39, 127.31, 127.23, 126.99, 126.19, 125.11, 125.07, 124.62, 123.89, 62.23, 49.02, 38.42; HRMS (ESI-TOF)+ Calculated [M + H]+ m/z 380.1651, Found [M + H]+ 380.1631.
The desired chemosensor 1 was synthesized by utilizing L-phenylalanine and 1-pyrenecarboxyaldehyde (PyCHO). The complete characterization has been done by FT-IR, UV-vis, 1HNMR, and 13CNMR, HRMS. 1 shows excellent solubility in DMSO. The idea for designing this type of probe is based on fluorescent receptor moiety. Here we have aimed to design a molecule that possesses a free binding site in the form of -COOH group. This free-binding site can be applicable for a multifunctional application by providing a binding site for interaction with different cations, anions, and neutral species.
1.2. Sensing properties of 1 towards Cu 2+ ion.
The UV-vis absorption spectrum of chemosensor 1 in DMSO/water (1:9, v/v, r.t. 25˚C) (10µM) is characterized. 1 showed absorbance values at, λmax, nm (ε); 266 nm (2.7 ×104), 276 nm (4.0 ×104), 313 nm (1.5 ×104), 327 nm (2.6 × 104), 324 nm (3.6 × 104). (Figure S4) These various absorptions are referred for the typical absorption peak of a pyrene-containing compound and the multiple absorptions correspond to various π-π* transitions in pyrene fluorophore. Further to investigate the effect of the cation binding on the absorption behavior of 1, various alkali, alkaline, and transition metal ions (Na+, K+, Ca+ 2, Fe+ 3, Co+ 2, Ni+ 2, Cu+ 2, Zn+ 2, Cd+ 2, Ag+ 2, Hg+, Pb2+) were added to a solution of 1. Interaction of 1 with metal ions does not show any remarkable change in the absorption spectrum of 1 except Cu2+ ion. The addition of Cu2+ ion with 1, shows a decrease in its absorbance. (Fig. 1a). Further, to explore the association properties of 1 with Cu2+ ion. UV-Visible titration experiment of 1 with the addition of several aliquots of Cu2+ ion has been done. As a result, the formation of several isosbestic points (262, 269, 271, 279, 305,334, 348) gives a site for the possibility of the several binding modes between Cu2+ ion and 1 (Figure S5).
To gain insight into the selectivity of 1 towards various metal ions through fluorescence upon excitation at 342nm. Neat 1 exhibited a characteristic emission band for pyrene monomer at 375 nm, 394 nm, and 416 nm.44, 45 (Figure S6) While on the addition of metal ions (Na+, Pb2+, K+, Cd+ 2, Ca+ 2, Fe+ 3, Ag+ 2, Co+ 2, Ni+ 2, Zn+ 2, Hg+) (100mM) into a solution of 1 (10µM) in Water: DMSO (9:1, v/v) doesn’t exhibit any considerable change in the emission spectra. Only Cu2+ ion was able to make a significant change in the emission spectra of 1. (Fig. 1b) Fluorescence lifetime measurement of 1 was measured at monomer emission i.e., 375nm, exhibits a decay time is 1.43 ns. (Fig. 2) Addition of 10 eq. of Cu2+ ions show quenching in the emission intensity of 1 up to 85% in the view of the paramagnetic nature of Cu2+ ions. These findings suggest that 1 shows excellent selectivity towards Cu2+ ions. A fluorescence titration experiment is carried out to examine the effect on the intensity of the emission of 1 by adding several aliquots of Cu2+ ions. On addition of several aliquots of Cu2+ (1µL) to the solution of 1 immediately tends to “turn off” fluorescence at 375nm (29%), 394nm (26%), and 416 nm (24%). (Figure S7) Increasing Cu2+ concentration (2–15 µL) lead to a continuous decrease in the emission intensity at 375nm (85%), 394nm (86%), and 416 nm (74%).
The total amount of Cu2+ required to concentrate quench in the emission intensity of 1 is 15µM (5 eq.) to the concentration of 1. The fluorescence quenching of 1 with Cu2+ ion is explored by the classical Stern-Volmer equation.
I0/I = 1 + Ksv [Q]
Where, I0 and I are the emission intensity of neat 1 and 1 with Cu2+, respectively. [Q] represents the concentration of Cu2+ ion, and Ksv represents the Stern-Volmer quenching constant. The plot of I0/I vs varying concentration of Cu2+ ion represents good linearity (R2 = 0.9725) with the calculated Ksv value of 2.65 X 105 M− 1. The obtained Ksv value strongly suggests the quenching behavior of 1 concerning Cu2+ ions. (Figure S8) The quantum yield of 1 for the monomer emission was determined to be 0.23 and after the addition of Cu2+ is 0.06 with quinine sulfate monohydrate (ϕ = 0.54 in 0.5M sulfuric acid) being used as a standard (see ESI for details).
The selectivity of any probe towards a particular metal ion is the most desirable thing. Therefore, to investigate the selectivity of 1 towards Cu2+ ions, we examine the emission characteristics of 1 with Cu2+ ions in the presence of other competing metal ions. As a result, we found that none of the competitive metals can alter the emission quenching caused by the interaction of Cu2+ with 1. (Fig. 3a) These results specify that 1 can be utilized as a selective sensor for Cu2+ ions in presence of other competing metal ions. Now to get insight into the binding stoichiometry of 1 towards Cu2+ for the formation of complex analyzed by jobs plot method. As a result, we found 2:1 stoichiometry between 1 and Cu2+ ions complex. (Fig. 3b) A proposed binding mechanism for the formation of a 2:1 complex of Cu2+ ions with 1 (Figure S13). Where Cu2+ coordinates with the oxygen atom of carboxylic moiety.
The binding constant between 1 with Cu2+ ion was calculated by using the Benesi-Hildebrand equation.
1/I-I0 = 1/ Ka [Cu2+] [Imax – I0] + 1/ Imax -I0
I represent the emission intensity of the 1-Cu2+ complex, which varies with the concentration of Cu2+. I0 represents the emission intensity of 1, and Imax is the fluorescence intensity of the 1-Cu2+ complex at the maximum concentration of Cu2+ ions. Binding constant Ka was evaluated by plot between 1/(I0-I) vs 1/[Cu2+]. Plot between 1/(I0-I) vs 1/[Cu2+] shows good linearity (R2 = 0.9822) with the value of binding constant Ka = 5 x 104 M− 1. (Figure S9)
The sensitivity of 1 has been observed by adding varying concentrations of Cu2+ (10− 11 to 10− 3 M) to 1 (10 µM) solution. A plot between I0/I vs. [Cu2+] shows a significant change at 10− 8 M Cu2+ concentration (Figure S10). Therefore, for accuracy in the detection limit, further addition of varying concentrations of Cu2+ ion (1 x 10− 8 to 9 x 10− 8 M) to the solution of 1. Again, a plot between I0/I vs. [Cu2+] displays a linearity relationship (R2 = 0.9813), and the limit of detection (LOD) is 20 ppb or 2 x 10− 8 M (Figure S11). The LOD value is lower than the concentration of Cu2+ recommended by WHO 16µM in the drinking water. Therefore, it exhibits that chemosensor 1 could be potentially used as a sensor for detecting Cu2+ ions.
Reversibility of 1-Cu+ 2 with EDTA
The chemical reversibility phenomenon of the binding tendency of 1 with Cu2+ ions was also examined by the addition of EDTA solution to the 1 + Cu2+ system. As results indicate, the addition of EDTA solution replaces the Cu+ 2 ion from 1 + Cu+ 2 complex by regaining the emission intensity of 1 due to the great stability of EDTA-Cu2+ complex, which confirms the regeneration of 1 from 1 + Cu2+ complex, and hence 1 can be useful in the binding process. These findings suggest the chemical reversibility of the binding of 1 to Cu2+ ions (Figure S12).
Rheology study
- The gel has great relevance for application; therefore, we examine the strain recovery rheology studies for the viscoelastic characteristics (storage modulus G′ > loss modulus G′′) and mechanical strength of a gel. The mechanical characteristics of gel materials have been investigated using rheology. 1g of 1 was measured for the storage modulus (G′) and loss modulus (G′′) about applied stress and frequency. Figure 3 represents the variation in G′ and G″ to the applied frequency on 1. This is observed that at any applied frequency, both G′ and G′′ are essentially frequency independent, and at any given point, G′ is bigger than G′′ over the entire frequency range, confirming the actual gel-like nature of the material. (Fig. 4a)
The shear-thinning tendency of gel was characterized by plotting a shear rate vs viscosity rheology plot. In the shear rate vs viscosity plot, the power-law equation was fit to the linear region as per the formula
η = Kγn−1
η is for viscosity, n and K are for shear-thinning coefficient and γ is for shear rate.
As we saw in the viscosity graph, it also confirms that the viscosity of 1 increases with increasing stress. (Fig. 4b) This result indicates the formation of hard gel on increasing the stress on 1. Moreover, to explore the thermal stability and photoluminescence of synthesized 1 as a gel, to check the thermal stability of gel, heat up to 100˚C in an oil bath and no effect was observed, this indicates the high thermal stability of 1. Although 1 also exhibits blue emission on long-range wavelength. (Fig. 5)
AFM STUDY
AFM studies were carried out to investigate the surface properties for 1. The 2D and 3D morphology of 1 exhibit uniformly distributed, with root mean square roughness (Rq) = 8.010 nm, average roughness (Ra) = 6.344 nm, and maximum profile peak height = 19.802 nm. (Fig. 6a,6c) It is observed that particles of 1 are uniformly distributed and comprised of the sphere shape. The formation of non-covalent interaction forms this sphere shape, i.e., Intermolecular hydrogen bonding between -COOH moiety in 1 (Fig. 6b). Here the interaction between -COOH moiety is more dominant than the π-π interaction between pyrene moiety. It was also observed by the fluorescence spectra of 1 where a significant pyrene excimer peak was not observed.
Water samples preparation for Cu2+ detection
- To analyze the copper content in different water samples, tap water (TW), distilled water (DW), River water (RW), and Pond water (PW) were collected. The collected water was pretreated by boiling and filtration to avoid insoluble impurities present, before being used to detect copper content analysis by 1. Water samples were filtered to separate the insoluble substance from them. To remove dissolved salts like chlorine content from water samples, they were boiled for one hour and then cooled at room temperature.
Analysis of Cu2+ in various natural water samples
- To explore the applicative study of 1 in an environmental approach, we explore the recognition ability of 1 in the vicinity of Cu2+ ions in natural water samples. High sensitivity and selectivity of 1 towards detection of Cu2+ without any significant interference of other competitive metal ions and its LOD value of 2 x 10− 8 M, provide a main driving force to use 1 for the determination of Cu2+ in natural water samples from different-different resources.
The earlier process was used to well prepare the stock solution for analysis of the water samples. Afterward, fluorescence spectra of 1 with different water samples were recorded. Here we take 2.5 ml of water samples (Pond water (PW), Distilled water (DW), Lake Water (LW), and Tap water (TW)) in a quartz cuvette with the addition of 250 µL of 1 (100 mM; Water: DMSO; 9:1, v/v at room temperature). Impressively, we found quenching (̴ 43%) in the case of pond water to distilled water. (Fig. 7a) Furthermore, the addition of 20 µL Cu2+ion to the solution of 1 and water sample (250 µL + 2.5 mL), gives further quenching in the emission spectra.
The linearity response of 1, to the addition of various natural water samples (100–500µl) subsequently in 2.5 ml of 1 (10µM), a pithy decrease in the emission of 1 exhibited good linearity (Fig. 7b). This result strongly suggests that 1 works as a fluorescence sensor detect Cu2+ ions in biological and environmental contexts. Moreover, the quenching in the spectrum of 1 with the addition of pond water with 20 µL Cu2+ ion (10 ppb), respectively. It suggests that the present concentration of Cu2+ ion in pond water at that stage is < 10 ppb. This indicates that pond water contains a much higher amount of Cu2+ as compared to tap water. Therefore, we can say that 1 feasibly works better to detect Cu2+ ions in a real water sample.
In this article, we have reported the synthesis of a new (pyren-1-ylmethyl)-L-phenylalanine, fluorescent compound 1 for selective and sensitive sensing of Cu2+ ions. The mechanism for Cu2+ ions detection is based on a fluorescent “turn off” mechanism. The above study showed that 1 has a specific fluorescence quenching towards Cu2+ ions in a Water - DMSO solution (9:1, v/v, r.t. 25˚C) in the existence of the several competitive metal ions (K+, Na+, Ca+2, Co+2, Fe+3, Ni+2, Zn+2, Cu+2, Hg+, Cd+2, Ag+2, Pb2+). The LOD value of the chemosensor for specific detection of Cu2+ ions reached 2 x 10-8 M showing that 1 was highly applicable for quantitative analysis of Cu2+ ions. In natural water analysis, 1 showed excellent results for the detection of Cu2+, exhibiting its potential as an ideal chemosensor. Moreover, 1 also exhibits properties of the gel, and even at high temperature and high-pressure this gel exhibits excellent stability, which increases its applicative potential in the environmental application.
Acknowledgments
MDP acknowledges the Institute of Eminence (IoE)-Banaras Hindu University for the funding. AT acknowledges Banaras Hindu University, Varanasi, for providing the facility for research work. KKN and TM sincerely acknowledges UGC-CSIR New Delhi for research fellowships.
Authors' contributions - All authors contributed to the study conception and design. Arpna Tamrakar; Experiment design, Conceptualization, Data curation, Visualization validation, Resources, Kamlesh Kumar Nigam; Experiment design, Conceptualization, Data curation, Visualization validation, Resources, Tarkeshwar Maddeshiya; Experiment design, Conceptualization, Data curation, Visualization validation, Resources, Mrituanjay D Pandey; Conceptualization, Data curation, Visualization validation, Resources, Supervision
Funding:
This work is funded by the Institute of Eminence (IoE)-Banaras Hindu University.
Data Availability All authors conform that the data of this article is available inside the article.
Ethics Approval Not applicable.
Consent to Participate Not applicable.
Consent for Publication Not applicable.
Conflicts of Interest The authors declare they have no competing interests.
Scheme 1 is available in the Supplementary Files section.
No competing interests reported.
#In the memory of Dr. D. Kumar (Department of Chemistry, Ewing Christian College, Prayagraj), Deceased on April 02, 2022.
Scheme 1: -Synthetic Scheme for 1.
published 09 Jan, 2023
Editorial decision: Major revision
19 Dec, 2022
Reviews received at journal
13 Dec, 2022
Reviewers agreed at journal
07 Dec, 2022
Reviewers invited by journal
07 Dec, 2022
Editor assigned by journal
07 Dec, 2022
Submission checks completed at journal
01 Dec, 2022
First submitted to journal
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You are reading this latest preprint version