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beta-cyclodextrin inclusion complex as a new “turn-off” fluorescent sensor system for sensitive recognition of mercury ion
Journal Pre-proof Curcumin/beta-cyclodextrin inclusion complex as a new “turn-off” fluorescent sensor system for sensitive recognition of mercury ion Samikannu Prabu, Sharifah Mohamad PII:
S0022-2860(19)31637-0
DOI:
https://doi.org/10.1016/j.molstruc.2019.127528
Reference:
MOLSTR 127528
To appear in:
Journal of Molecular Structure
Received Date: 18 October 2019 Revised Date:
29 November 2019
Accepted Date: 3 December 2019
Please cite this article as: S. Prabu, S. Mohamad, Curcumin/beta-cyclodextrin inclusion complex as a new “turn-off” fluorescent sensor system for sensitive recognition of mercury ion, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2019.127528. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Graphical Abstract
Curcumin/Beta-cyclodextrin inclusion complex as a new “turn- off” fluorescent sensor system for sensitive recognition of mercury ion Samikannu Prabu ab* and Sharifah Mohamad ab* a,b
Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603,
Malaysia a,b
University of Malaya Centre for Ionic Liquids, Department of Chemistry, Faculty of Sci-
ence, University of Malaya, Kuala Lumpur 50603, Malaysia *Email:[email protected] (Sharifah Mohamad); [email protected] (S. Prabu)
Abstract The formation of supramolecular complex between the curcumin (CC or probe L) and β-cyclodextrin (β-CD) (CC: β-CD or probe LC) was confirmed using absorption and emission spectroscopy. The binding properties of probe LC with cations in water were observed for the first time via absorption and emission spectroscopies. The selectivity and sensitivity of fluorescence chemosensors have been studied using probe LC. The probe LC showed selective binding to Hg2+ and afforded new absorbance and fluorescence peaks at 379 nm and 502 nm, additionally to the prevailing bands of LC at 432 nm and 535 nm. It additionally showed apparent colour change from yellow to colourlessness and strong fluorescent to weak fluorescent owing to selective binding of Hg2+ ion, which was detected by naked eyes. No noticeable changes of colour and spectra were observed upon the addition of other metal cations such as [Ag+, K+, Na+, Cs+, Ba2+, Fe2+, Mg2+, Pb2+, Mn2+, Ni2+, Cd2+, CO2+, Cr3+, Sn2+ and Zn2+. Hg2+ produced blue shift in absorption spectra and quenching in emission spectra which suggested a likelihood of strong binding of Hg2+ ions with probe LC. The fluorescence response was concentration-dependent and can be well described by the typical Stern–Volmer model. Compared with prior reports, this method is very cheap, selective, sensitive and easily biodegradable of probe LC. Keywords: Curcumin; Beta-cyclodextrin; Inclusion complex; Hg2+; Colorimetric; Chemosensor.
1
1. Introduction In recent years, varied varieties of heavy metal ion pollution issues have attracted people's attention, together with soil contaminations, water pollutions and so on [1–4]. Selective monitoring about the heavy metal ions is of tremendous importance, as they play imperative duties in biological systems and also the environment. Mercury is a highly toxic and widespread global pollutant. Mercury ion (Hg2+) is well-known as an infectious heavy metal ion and causes thoughtful hazard to the human and the environment even when its concentration is low [5–9]. It has wide employment in several industries, like mercury vapor lamps, mercury battery and mercury switch. Mercury ion within the environment is transformed into organomercury species like methyl-mercury (CH3HgX) by aquatic microorganisms, which can accumulate and enter in the human body through the food chain [10–13]. As the continuous prosperity over mercury awareness within human body, it would lead in accordance with much illnesses such namely renal failure, liver damage, psychiatric disorders, and so on [14-15]. Once exposed after absolute environment, ethnical usually suffer severely appropriate in conformity with mercury leak. Thus, mercury is viewed so a precedence-controlled pollutant by way of WHO (World Health Organization) and alternative organizations. Till now, many analytical approaches have been developed for the determination of Hg2+ but, supramolecular complex based fluorogenic chemosensors are better and preferable for recognizing and sensing analytes because of their advantages in terms of simplicity, sensitivity, selectivity, real-time and online detection [16-17]. Curcumin (Scheme S1a) is a natural yellow hydrophobic polyphenol pigment with outstanding photostability and low toxicity [18]. Curcumin is greatly fed on as a seasoning, coloring and retaining agent of food, drugs and cosmetics permanency [19]. Curcumin is hardly used for highly selectively and sensitively sense of heavy metal cations application [20]. The curcumin molecule has two hydroxyl groups at both ends, a conjugation effect occurs from the electron cloud deviation under basic conditions. Also, curcumin is very good chelating agent and it forms complexes with several metal ions like Zn (II), Fe (II) and Cu (II) has also been reported in several
2
studies [21-22]. In addition, curcumin has good fluorescence characteristics and easily degradable. Fluorescent sensor technology has attracted considerable attention as a type of facile and efficient detection method for sensing biological and chemical species because of their simplicity, versatility, high selectivity and sensitivity [23-27]. The recognizing or sensing of heavy metal cationic analytes have recently developed as a key research topic within the field of supramolecular chemistry as a result of heavy metal cations necessary roles in biological, medical and environmental processes [28–31]. The generally enhances the absorbance and fluorescence intensity due the formation of inclusion complex between guest molecules and cyclodextrin. Cyclodextrins (6α-, 7β-, 8γ-cyclodextrin) are well-known doughnut-shaped oligosaccharides linked by α1,4-glycosidic bond to form macrocycles and are capable of forming inclusion complex with hydrophobic guest molecules of suitable shape and size and an outer hydrophilic surface (Scheme S1b) [32]. Beta-cyclodextrin is readily soluble in water to form clear homogeneous solutions and enables them to undergo host–guest interactions with huge number of organic molecules and drugs [33]. Cyclodextrins are at all famous building blocks because supramolecular structures, yet it is used in a number of fields such as medicine, drug, cosmetic, agriculture, analytical chemistry and so on. [34-35]. Many studies on enhancement of solubility, stability, biological application, drug delivery and food application of curcumin with cyclodextrins have been reported [18, 36–42]. In the current study we have focused on curcumin: β-cyclodextrin inclusion complex (probe LC) for cation sensing for the first time. The experimental results showed that probe LC may by selectively and sensitively sense Hg2+ ion through absorption and fluorescence detection along side the color change. The absorption and fluorescence spectral properties of curcumin and inclusion complex were investigated. Based on this phenomenon, a new supramolecular sensitizer (probe LC) has been designed by incorporating curcumin with beta-cyclodextrin and its efficient fluorescence sensing towards Hg2+. The probe LC exhibits substantial color change, blue shift and fluorescence quenching upon complexation with Hg2+, which could be utilized as an absorbance and fluorescent probe for Hg2+ in aqueous solution. The absorbance and 3
fluorescence studies about the complexation of curcumin with β-CD as a chemosensor probe LC (Scheme S1c) for sensing of Hg2+ ion an aqueous solution is proposed.
2. Experimental section 2.1 Reagents β-Cyclodextrin (purity ≥ 99%) and curcumin (purity ≥ 99%) were obtained from Sigma Aldrich and used while not additional purification. Other chemical reagents were of analytical reagent grade and used as received. Metal salts were also obtained from Sigma Aldrich and used while not additional purification. The stock solutions of metal cation (1×10-4M) were prepared using ultrapure water from chloride salts such as Ag+, Cs+, K+, Na+, Ba2+, Cd2+, CO2+, Fe2+, Mg2+, Mn2+, Ni2+, Sn2+, Zn2+, Cr3+, and the nitrate salts of Pb2+. 2.2 Apparatus Spectrophotometric measurements were made with a Shimadzu Ultraviolet-Visible spectroscopy UV-1800 (UV-Vis) recording spectrophotometer equipped with 1 cm quartz cells. Fluorescence spectra (emission spectral measurement) were carried out with Cary Eclipse Fluorescence Spectrophotometer. The Fourier transform infrared (FT-IR) spectra of CC and inclusion complex were recorded on a Perkin-Elmer RX1 FT-IR between 4000 and 400 cm-1 with a resolution of 2 cm-1 at room temperature. The proton nuclear magnetic resonance (1H NMR) spectra were recorded on Lambda JOEL 400 MHz FT-NMR spectrometer and dimethyl sulfoxide (DMSO-D6) had been used as solvent. 2.3 Preparation of inclusion complex in aqueous medium The stock solution of curcumin (1×10-4 M) was prepared using ethanol. The different concentrations of β-CD solution (0,2,4,6,8,10 and 12×10-3 M) were prepared using ultrapure water. Curcumin stock solution was added in a 10 ml volumetric flask and made up to the mark using following concentrations of β-CD solutions 0, 2, 4, 6, 8, 10 and 12×10−3 M respectively and shaken thoroughly. The solutions were prepared just before taking absorbance and emission measurements. All measurements were recorded at room temperature. 2.4 Preparation of solid inclusion complex 4
The solid inclusion complex of β-CD with CC was prepared using coprecipitation method. Meanwhile, 1.0 g of β-CD was dissolved in 30 mL of ultrapure water at room temperature and then this solution was continuous agitation using a magnetic stirrer. On the other hand, 0.3246 g of CC was dissolved in 30 mL of acetone and slowly added to the aqueous β-CD solution. The round bottom flask was covered with aluminum foil and constantly stirred for 48 h at room temperature. The final mixture was refrigerated at 4°C for 24 h. Afterwards, the final mixture was consecutively filtered through a normal filter paper. Finally, the solid inclusion complex was obtained by washed with acetone and water to remove the uncomplexed CC and β-CD respectively. The residue was dried at 50oCfor 48 h and utilized for further studies. 2.5 Preparation of inclusion complex with metal cations in aqueous medium The stock solution of curcumin (1×10-4 M), beta-cyclodextrin (12×10-3 M) and various heavy metal cations ions (1×10-4 M) were prepared by using ultra-pure water. For colorimetrical titrations, the stock solutions (1×10-4 M) of entire metal cations have been prepared in aqueous solution and utilized. To analyze the effect of metal ion through absorption and emission spectra, various heavy metal cation ions were prepared in 10 ml volumetric flask and shaken thoroughly. All the absorption and emission spectra were recorded at room temperature. 3 Results and discussion 3.1 Interaction of CC with β-CD in liquid state: 3.1.1 Absorption spectral characteristics of CC in β-CD medium The formation of inclusion complex between curcumin and beta-cyclodextrin in aqueous solution has been analyzed using absorption spectroscopy. The β-CD containing one or more water molecules was expelled by guest molecules encapsulated in β-CD. The insertion of CC within the β-CD cavity mainly depended on the number of water molecules released in the bulk water. It is reported that the inclusion behavior depends on the individual structural features of the guest and host molecules since the charge fit, shape fit, and size fit effects are the main governing factors on the formation of inclusion complex of cyclodextrin [43].
5
The absorption maximum of curcumin is 429 nm, which was attributed to the polyunsaturated systems, conjugated and benzene ring of CC [40]. There is an intramolecular hydrogen bond formed between the methoxy group and the phenolichydroxyl (OH) group of the CC. Two absorption bands were observed for CC nearly at 429 nm and 251 nm. The strong absorption band at 429 nm could be assigned to a combination of π →π* and n→ π* transitions. The weak absorption band at 251 nm corresponds to a π→ π* transition [44]. As shown in Fig. 1a, on increasing the concentration of β-CD from 0M to 0.012 M, the absorption spectrum of CC is slightly bathochromic shift at longer wavelength (429 nm 432 nm) with a gradual increase within the absorbance. This behavior has been attributed to the enhanced dissolution of the CC molecule by complexation through the hydrophobic interaction between CC and β-CD. The above results suggest that inclusion complex is formed between CC and β-CD (Scheme S1c). The formation of an inclusion complex is completed at 1.0×10−4 M (Fig. 1c). Thus, the β-CD effects on the absorption spectra of CC could be ascribed to the formation of inclusion complex through the hydrophobic interaction only. In UV-Visible spectra, the binding constant of CC: β-CD inclusion complex has been calculated by studying the changes in the absorption intensity maxima with β-CD concentration. In the case of 1:2 inclusion complex, formed between CC and βCD, the equilibrium is often written as:
+ − ⇌ : −
(1)
The equilibrium constant for the above equilibrium is given by equation (2) [: ]
= [][ ]
(2)
The stoichiometric ratios and binding constant “K” of the inclusion complex may be determined using Benesi–Hildebrand equation [45] assuming the formation of a 1:2 host–guest inclusion complex by UV spectroscopy. The Benesi-Hildebrand relation for such equilibrium is given in Eq. (3).
=
∆
+
(3)
[] ∆ []
6
where A-A0 is the difference between the absorbance of CC in the presence and absence of β-CD, ∆ε is the difference between the molar absorption coefficient of CC and the inclusion complex (CC: β-CD), [β − CD] , [CC]0 are the initial concentrations of β-CD and CC respectively, whereas k is the binding constant. The absorption intensity maxima at 429 nm was taken to draw the Benesi–Hildebrand plot 1/A-A0 versus 1/ [β-CD]2. It gives a straight line as shown in Figs. 1b. The good linear correlations coefficient (R2=0.9973) was obtained; it confirms the formation of a 1:2 inclusion complex between CC: 2β-CD [46]. The binding constant value “K” value was calculated from slope of the Benesi–Hildebrand plot according to Eq (4), and it is found to be 46.168×103 M−1 at 303 K.
=
(4)
! [" ]
3.1.2 Fluorescence spectral characteristics of CC in β-CD medium The fluorescence emission spectra of curcumin with various concentrations of β-CD are shown in Fig. 2a. When guest molecules in aqueous solution are encapsulated in cyclodextrins, emission spectra may be influenced which indicates the formation of inclusion complexes between guest and host molecules [47]. The maximum absorption wavelength of the CC has been fixed with excitation fluorescence spectroscopy. Curcumin shows a broad structureless fluorescence band in water at 571 nm, with fixed excitation at 429 nm. The CC exhibits a large hypsochromic shift in its spectrum from 571 nm to 535 nm with a concomitant increase in fluorescence intensity on the addition of β-CD. The hypsochromic shift (~36 nm) in the emission spectrum confirms the binding of curcumin to the hydrophobic cavity of β-CD. The hypsochromic shift observed is in consistency with the fact that CC experiences a less polar environment in the hydrophobic cavity of β-CD. The hypsochromic changing concerning the fluorescence maxima yet the rise of the fluorescence depth suggests that the construction regarding an inclusion complex of CC with β-CD. The above spectral exchange effects suggest as the form of an inclusion complex between CC and β-CD (Scheme S1c). The binding constant (K) for the formation of 1:2 inclusion complex has been found evaluating the changes in the fluorescence intensity of emission maxima with β-CD concentration using the modified Benesi-Hildebrand equation [45]. 7
##
=
∆
+
(5)
[] ∆ []
where I and Io are the fluorescence intensities of CC in the absence and presence of β-CD, respectively. Fig. 2b depicts the plot of 1/[I-Io] versus 1/[β–CD]2. The good linearity of the HB plot with the correlation co-efficient of R2 = 0. 9925 indicates the 1:2 inclusion complex formation CC with 2β-CD. The binding constant “K” value was calculated from the slope of the Benesi–Hildebrand plot by using the equation (6) and it is found to be 56.769 x 103 M-1at 303 K.
=
(6)
! [#"# ]
3.1.3 Spontaneity of inclusion complexation reaction The thermodynamic parameters (∆G) for the inclusion process can be calculated from the binding constant value “K” by the following equation at 303K. ∆G = -RT ln K
(7)
where ∆G is the Gibbs free energy change, R is the gas constant (J/mol·K), T is the temperature in Kelvin and “K” is the binding constant in mol−1.The binding constant “K” in the excited state (56.769×103 M-1) is higher than the “K” value in the ground state (46.168×103 M−1). ∆G values of the complexation process are found to be −27.04 kJmol−1 and −27.55 kJmol−1 for ground and excited state respectively. The negative values of ∆G indicate the spontaneity of inclusion complexation reaction at 303 K. 3.2 Interaction of CC with β-CD in solid state: 3.2.1 Fourier - transform infra-red spectral analysis Fig S1 depicts the FT-IR spectra for β-CD, CC and inclusion complex. The FT-IR spectrum of β-CD (Fig S1a) was characterized by a strong absorption band at 3293 cm-1 (stretching vibration of -OH), 2925 cm-1 (stretching vibration of -C-H), 1415 cm1
(stretching vibration of -OH deformation), 1152 cm-1 (stretching vibration of asym-
metry -C-O-C), 1020 cm-1 (stretching vibration of symmetry -C-O-C), 947 cm-1 (stretching vibration of skeletal vibration involving α-1,4 linkage) and 851 cm-1 (Breath of glucose ring). The FT-IR spectrum of CC (Fig S1b) was consider by an absorption of band of at 3493.45 cm-1 (phenol group, intramolecular H-bond), 3006.12
8
cm-1 (aliphatic C-H stretching), 2927.90 cm-1 (aromatic C-H stretching), 1626.24 cm-1 (-CO stretching), 1601.42 cm-1 (aromatic C=C stretching), 1426.95 cm-1 (aliphatic OH stretching), 1266.36 cm-1 (aromatic C-O stretching), 1026.29 cm-1 (-C-O-C stretching) and 959.74 cm-1 ( C=C bending). The FT-IR spectrum of the solid inclusion complex exhibited no features similar to pure CC and β-CD. The stretching frequencies of aliphatic C-H stretching, aliphatic -OH, -CO and bending -C=C of the complex product appeared at 3000.17 cm-1, 1425.63 cm-1, 1623.37 cm-1 and 959.74 cm-1 respectively, as shown in Fig. S1c. However, those specific stretching frequencies are getting only slight shifts from pure CC. The stretching frequencies of phenol group intramolecular H-bond, aromatic C-H, aromatic C=C, aromatic C-O and -C-O-C of the complex product appeared at 3392.42 cm-1, 2947.37 cm-1, 1585.09 cm-1, 1296.53 cm-1 and 1034.73 cm-1 respectively. Hence, the large shifts in stretching frequencies of pure CC became inclusion complex. These changes indicated that the aromatic part of CC is involved in an inclusion complexation on the hydrophobic cavity of β-CD. 3.2.2 Nuclear Magnetic Resonance Spectral Analysis The 1H NMR spectra of β-CD, CC and inclusion complex were revealed in the Fig. S2. Dissimilarities in the chemical shift’s value of CC and β-CD and inclusion complex were listed Table 1. The positive and negative signs meant an upfield and downfield shifts. The 1H NMR spectrum of β-CD contains nine variabilities of protons (H1- H9) as shown in Fig. S2a. It can be seen from the 1H NMR spectrum of inclusion complex (Fig. S2c), a downfield and upfield shift were happening for H1, H5 and H7 protons and H2, H3, H4, H6, H8 and H9, respectively. The above chemical shifts were changes owing to CC inclusion complex with β-CD. The CC contains ten variabilities of protons (H1- H10) as shown in Fig. S2b. The ∆δ values of the H8, H9 and H10 protons in the inclusion complexes have relatively small effects. Even though, the ∆δ values of the H1, H2, H3, H4, H5, H6a, b and H7 protons displayed large effects. The above small chemical shift changes owing to H8, H9 and H10 protons were located outside of the β-CD cavity. The above large chemical shift changes owing to H1, H2, H3, H4, H5, H6a, b and H7 protons were located inside of the β-CD cavity. The above re-
9
sults suggest that the strong supramolecular inclusion complex formation between CC and β-CD. The
13
C NMR spectra of β-CD, CC and inclusion complex were shown in
Fig.S3. Dissimilarities in the chemical shifts of CC and β-CD and inclusion complex states were listed Table 2. The β-CD (Fig. S3a) exhibits six variabilities of carbons (C1–C6). It can be seen from the 13C NMR spectrum of inclusion complex (Fig. S3c), all the carbons chemical shift values were shifted with diminished intensity. These significant changes owing to an encapsulation of CC into β-CD. As shown in Fig. S3b, CC was displays thirteen variabilities of carbons (C1–C13). Whereas in the
13
C
NMR spectrum of inclusion complex; a small and great chemical shift were obtained for C10a, b and C11 and C1, C2, C3, C4, C5, C6, C7, C8, C9, C12 and C13 respectively. The above results suggest that the noticeable changes of chemical shift in C1, C2, C3, C4, C5, C6, C7, C8, C9, C12 and C13 carbons indicated the CC inclusion complex with β-CD. The small changes of chemical shift in C10a, b and C11 carbons were suggest that the influence of CC molecule. 3.3 Analytical application of CC: β-CD complex as chemosensor 3.3.1 Selectivity study of CC: β-CD complex as chemosensor 3.3.1.1 Colorimetric sensing study The colorimetric sensing study by naked eye is the simplest way to easily observe the high selectivity of probe LC to different heavy metal cations. Fig. 3 depicts the photographs of probe LC after addition of different mono, di and trivalent metal cations of biological and ecological importance, viz., Na+, K+, Ag+, Cs+, Mg2+, Mn2+, Ba2+, Co2+, Ni2+, Pb2+, Hg2+, Zn2+, Cd2+, Fe2+, Sn2+and Cr3+. The Probe LC chemosensor showed a highly sensing and selective recognition behavior toward Hg2+ ion by changing the color of the solution from yellow (432 nm) to colorless (379 nm). This selective color change can be used for the “naked eye” for the recognition of Hg2+. 3.3.1.2 Absorption spectroscopy response of chemosensor probe LC Based on the above colorimetric sensing study results, detailed electronic spectroscopic studies were carried out to investigate the selective sensing of probe LC toward Hg2+.The absorption spectra of probe LC in the presence and absence of various mono-, di and trivalent heavy metal cations of biological and ecological importance, 10
viz., Na+, K+, Ag+, Cs+, Mg2+, Mn2+, Ba+, Co2+, Ni2+, Pb2+, Hg+, Zn2+, Cd2+, Fe2+, Sn2+ and Cr3+are shown in Fig. 4a.The order of change of absorption intensities of probe LC is Hg2+< Fe2+< Mn2+< K+< Ag+< Mg2+< Cd2+< Zn2+< Pb2+< Co2+< Cr3+< Cs+< Na+< Sn2+< Ni2+< Ba2+< probe LC. By comparison, probe LC shows a very high selectivity toward Hg2+. As can be seen in this figure, the absorption maximum of probe LC and other metal cations except Hg2+ at λmax = 432 nm. upon addition of Hg2+cation to probe LC chemosensor underwent dramatic change of an absorption intensities as well as broad peak of on n-π* appeared at 379 nm (a blue shift of 53 nm from 432 nm) and very clear isosbestic point is observed at 394 nm, which confirmed the existence of a well-defined stoichiometric complex. The spectrum appearance of the large blueshift from 482 to379 nm, suggest that strongly an interaction of Hg2+ with the deprotonated aliphatic oxygen of probe LC to form the complex probe LC: Hg2+. [48] As shown in Fig 4b, the absorbance response (A0/A) of probe LC at equimolar concentration (1×10−4 M) of various mono-, di and trivalent metal cations at 432nm. This illustrated clearly that Hg2+ exhibited good response than other mono-, di and trivalent metal cations such as Na+, K+, Ag+, Cs+, Mg2+, Mn2+, Ba+, Co2+, Ni2+, Pb2+, Zn2+, Cd2+, Fe2+, Sn2+ and Cr3+. The comparative diagram of various mono, di and tri heavy metal cations absorption intensity is shown in (Fig. 4c). A significant decrease of absorption intensity of probe LC with Hg
2+
ions was observed. These results illus-
trated that probe LC could be an effective interact with Hg2+. 3.3.1.3 Fluorescence spectroscopic response of chemosensor probe LC The fluorescence responses of probe LC has been explored in presence and absences of various mono-, di and trivalent heavy metal cations of biological and ecological importance, viz., Na+, K+, Ag+, Cs+, Mg2+, Mn2+, Ba+, Co2+, Ni2+, Pb2+, Hg+, Zn2+, Cd2+, Fe2+, Sn2+ and Cr3+ are shown in Fig. 5a. In good agreement with the findings based on the above absorption study, the host-guest system CC: β–CD (probe LC) shows rather strong fluorescence emission in the range from 311 nm to 323 nm in an aqueous water medium. The strong fluorescence emission intensity of probe LC is observed at 512 nm. However, based on addition of 2 equivalent metal cations, the addition of Hg2+ resulted in a significant diminution of the fluorescence emission intensity 512 nm and blue shift to 502 nm. 11
As shown by the fluorescence emission spectra, adding Hg2+ to probe LC lead to an obvious “on-off” fluorescence response, while the other heavy metal cations do not lead to any significant fluorescence quenching under same spectroscopic conditions. The above observations clearly suggest that the excellent selectivity of the probe LC towards Hg2+only. Fig. 5b shows percentage of quenching of probe LC on addition of various metal cations. The quenching of Hg2+ cation is 73.88%, when compared to probe LC and other metal cations. The Comparative diagram of various mono, di and tri heavy metal cations fluorescence emission intensity is shown in Fig. 5c. A significant diminution of fluorescence emission intensity of probe LC with Hg
2+
ion was observed.
The order of change of fluorescence intensities of probe LC is Hg2+< Fe2+< Cr3+< K+< Mg2+< Ba2+< Cs+< Co2+< Zn2+
plex formation. These results clearly establish the very high sensing ability of probe LC for Hg2+ cation in water. 3.3.2.2 Sensing and Binding Studies using Fluorescence Spectroscopy Sensitivity is a very significant parameter to estimate the presentation of a fluorescent chemosensor. In good agreement with the findings by the above absorption studies, probe LC exhibited specific fluorescence intensity responses towards Hg2+ under similar conditions. Fig. 7a depicts the fluorescence emission spectra of probe LC with the Hg2+ion. The effect of increasing the Hg2+ concentration causes decrease the fluorescence intensity without any shift in the peak. The quenching effects of probe LC by Hg2+ have been studied, and the quenching process was attributed to the formation of a complex between probe LC and Hg2+.Thus, it is appraised here that the probe LC act as a turn off fluorescence for Hg2+ ions compared to other metal cations. The modified Benesi-Hildebrand plot of 1/(F0-F) as a function of 1/[ Hg2+] as shown in Fig. 7c. The binding constant (Kb) between probe LC with Hg2+ was calculated from fluorescence spectrophotometer by using a modified Benesi-Hildebrand equation [45, 55-57] stated below, $$%&'
=
$%() $%&'
where, I
min,
+
* [+',-. ] $
%() $%&'
I and Imax are the emission intensities of probe LC in the absence
of Hg2+ ion, at varying Hg2+ concentration, and at a concentration of complete saturation respectively. K is the binding constant and [Hg2+] is the concentration of Hg2+. The modified Benesi-Hildebrand plot is giving a good linear correlation coefficient R2= 0.9919 for Hg2+ ion. The association constant (Ka) between probe LC and the Hg2+ion was found to be 2.11×10-2 M−1at 25◦C. Relative fluorescence emission intensities at 516 nm with the function of MnO4- ion concentration is observed and given in Fig. 5d. The low concertation of Hg2+ ion is high fluorescence emission intensity (~100.72) and high concentration of Hg2+ion is very low fluorescence emission intensity (~30.06). These results indicate the well-defined stoichiometric complex formation between probe LC and Hg2+ through quenching mechanism. 3.3.2.3 Stern-Volmer analysis
13
The mechanism of quenching process was carried out by using Stern-Volmer law. The linear plotting of relative fluorescence intensities (I0/I) versus quencher concentration of Hg2+ion follows a linear trend according to a typical Stern−Volmer equation expression: I0/I = 1+ Ksv [Hg2+]
(8)
Here I and I0 are fluorescence intensities in the presence and absence of added Hg2+cations. The slope of this line is Ksv. These results obtained are also consistent with the static quenching. The Stern-Volmer plot gives a good linear correlation coefficient R2= 0.9905 for Hg2+ion concentration in the range from 0–45 µM in Fig. 7b. The linear equation was calculated as I0/I = 0.9948 + 0.0165 c, with KSV value of 0.0113. Probe LC was found to have a detection limit of 5.02 µM and LOQ 16.73 µM. The experimental parameters for the sensing of Hg2+ using Probe LC fluorescence chemosensor are given Table 3. 3.4 Binding stoichiometry of metal ions complex The stoichiometry ratio of the inclusion complex of CC and Hg2+ ions were confirmed by using jobs plot of continuous variation method. We are calculating ∆A from the absorbance of metal complex (Amc) was subtracted from probe LC (Alc).
∆A = Alc-Amc A mole fraction of metal ions Hg
(9) 2+
against the change in absorbance (∆A) as
shown in Fig 8. It is observable that the maximum values ∆A for Hg2+ ions appear at around 0.6 M fraction which indicates that the formation of a 2:1 stoichiometric complex between probe LC and Hg2+ ion. 4 The possible binding mechanism of quenching effect by Hg2+ To determine and monitor the interaction between probe LC and Hg2+, the UV– Vis spectra and fluorescence emission spectra were examined. As illustrated in Fig. 9A, the absorption spectrum of pure curcumin is characterized by sharp peak at 429 nm, due to the enol form and mono anion forms [58]. In addition, it is interesting to find that the absorption maximum of enol form, mono anion form with trans-geometry of curcumin solution is observed as peak at 420–430 nm and enolic proton is more active than the phenolic proton [59-61]. The strong π(HOMO) → π(LUMO) transition of curcumin was found to be 429 nm in a solution that masked the weak electronic di14
pole forbidden n → p band. This band is in agreement for CU in the enol form [62]. Upon addition of curcumin solution to the solution of Beta cyclodextrin, increase an absorption intensity and a slight red shift of the absorption spectra (from 429 nm to 433 nm) was observed due the inclusion complex formation between curcumin and beta cyclodextrin (probe LC). Upon adding Hg2+ to the solution of probe LC, a large blue shift of the absorption spectra (from 433 nm to 379 nm) was observed due to interaction between Hg2+ and probe LC as a mercury chelator, but in the presence of oxalate ion due to its high affinity toward Hg2+ ion. At the same time, the color of the reaction mixture changed from yellow to colourless as shown in Fig 9C. So, probe LC could be used as a visual tool for an appropriate detection of Hg2+ without using any very expensive instruments, which is superior over other analytical methods. The fluorescence titration of probe LC against Hg2+ was carried out and spectral change is presented in Fig 9b. The fluorescence spectrum of pure curcumin is measured in excitation wavelength at λex = 425 nm for enol forms of curcumin [61]. The fluorescence emission spectrum of pure curcumin was exhibiting a single broad band with a peak at 571 nm. The fluorescence intensity of probe LC was observed at 535 nm, with enhancements of fluorescence intensity and also blue shift. This behavior indicated that curcumin molecule is entrapped into the β-CD cavity to form 1:2 inclusion complex. Thus, the variation in the fluorescence emission spectrum of curcumin is due to Excited State Intra-Molecular Hydrogen Transfer (ESIHT), since an unusually longer wavelength emission of curcumin in solution has been found to be due to ESIHT [62]. After adding Hg2+, probe LC solution has a large blue shift (from 535 to 502 nm) and quenching of fluorescence intensity, indicating the strong binding between the Probe LC and Hg2+.Thus, in accordance with the 2:1 stoichiometry, curcumin is likely to chelate Hg2+ via its O(-) atom and OH group of β-CD. Based on the UV–Vis spectra and fluorescence analysis, it is confirmed that the O(-) atom of curcumin coordinate with Hg2+ ion. The inclusion complex (probe LC) system exhibited high selectivity and sensitivity for Hg2+and the supramolecular sensing process was shown in Figure 10. 5 Analysis of real water samples and recovery test
15
The proposed chemosensor was applied to the determination of Hg2+ in drinking water and tap water samples. Drinking water and tap water samples were analyzed without any pretreatment. All the water samples were spiked with inclusion complex at single concentration level and then analyzed. The results are shown in Table 4. The recoveries of Hg2+ were 104% and 92% for drinking water and tap water samples respectively. The recovery of the proposed sensor is satisfactory. The present chemosensor may be useful for the determination of Hg2+ in real samples. 6 Conclusion In this article, an efficient, low cost, simple, and visual measurement system that utilizes CC: β-CD for specific recognition toxic Hg2+ ion was successfully developed. A simple probe LC shows a noticeable change in the absorbance and fluorescence emission spectral (turn-off fluorescence) behavior in the presence of toxic Hg2+ ion. Curcumin contained one aliphatic hydroxy group and two phenolic hydroxy groups and the presence of beta cyclodextrin and toxic Hg2+ ion led to the formation of cationic complex. Also, the deprotonation of aliphatic hydroxy group in curcumin resulted in spectral and colour changes. The LOD and LOQ of the proposed chemo sensor (probe LC: Hg2+) are 5.02 µM and 16.73 µM, respectively. Even more remarkably, this technique can provide a novel and eco-friendly approach for in situ identification of toxic Hg2+ion in environmental sample. The proposed sensor was used to analyze Hg2+ in real water samples with satisfactory results. Reference 1.
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Table 1 Variation in the 1H NMR chemical shifts (δ/ppm) of CC and β-CD protons in free and complexed states determined in DMSO-d6 at 298 K
Substance
β-CD
CC
Protons Free (δ, ppm)
Complex (δ, ppm)
∆δ (ppm)
H1
5.72
5.70
-0.02
H2
4.78
4.80
0.02
H3
3.61
3.77
0.16
H4
3.61
3.77
0.16
H5
4.77
4.55
-0.22
H6
2.30
2.33
0.03
H7
3.60
3.58
-0.02
H8
3.58
3.60
0.02
H9
3.62
3.63
0.01
H1
9.55
9.70
0.15
H2
6.79
6.83
0.04
H3
6.69
6.75
0.06
H4
7.48
7.40
0.08
H5
2.46
2.50
0.04
H6a, b
7.27
7.23
-0.04
H7
7.09
7.05
-0.04
H8
3.79
3.82
0.03
H9
7.60
7.57
-0.03
H10
6.01
6.03
0.02
Table 2 Variation in the 13C NMR chemical shifts (δ/ppm) of CC and β-CD protons in free and complexed states determined in DMSO-d6 at 298 K
Substance
Protons C1
CC
β-CD
Free ppm)
(δ, Complex (δ, ppm)
∆δ (ppm)
149.86
152.45
2.59
C2
111.79
114.81
3.02
C3
124.18
127.16
2.98
C4
141.25
144.43
3.18
C5
116.20
120.39
4.19
C6
150.22
152.30
2.08
C7
56.18
58.69
2.51
C8
148.79
148.25
-0.54
C9
126.84
125.24
-0.60
C10a, b
183.74
183.24
0.50
C11
101.41
101.26
-0.15
C12
121.59
122.80
1.21
C13
145.21
144.06
-1.15
C1
110.4
101.41
8.99
C2
74.4
72.93
1.47
C3
74.1
72.57
1.53
C4
83.1
82.06
1.04
C5
79.6
73.58
6.02
C6
62.5
60.44
0.06
Table 3: Experimental parameters for the sensing of Hg2+ using Probe LC fluorescence chemosensor
Name of the parameters
Values
λ excitation (nm)
430
λ emission (nm)
502
Linear range (µM)
0-45
Correlation coefficient (r)
0.9905
LOD (µM)
5.02
LOQ (µM)
16.73
Table 4 Determination of Hg2+ in real water samples. Added [Hg2+] (M)
Found total [Hg2+]a (M)
Recovery (%)
Drinking Water
1.00 x10-4
1.04±0.03
104
Tap Water
1.00 x10-4
0.92±0.01
92
Real water samples
a
Mean ± standard deviation (n= 3).
Figure. 1. (a) Absorption spectra of CC (1x10−4 M) in different β-CD concentrations: (1) 0M (2) 0.002M (3) 0.004M (4) 0.006M (5) 0.008M (6) 0.010M and (7) 0.012M; (b) Benesi–Hildebrand plot of 1 / (A − A0) vs. 1 / [β − CD] and (c) Absorption intensity of CC changes at 429 nm.
Figure. 2. (a) Fluorescence spectra of CC (1X10−4 M) in different β-CD concentrations: (1) 0M (2) 0.002M (3) 0.004M (4) 0.006M (5) 0.008M (6) 0.010Mand (7) 0.012M; (b) Benesi–Hildebrand plot of 1 / (I − I0) vs. 1 / [β − CD] and (c) Fluorescence intensity of CC changes at 571 nm.
Figure. 3. Color changes of sensor probe LC with different metal cations
Figure. 4. (a) UV-vis absorption responses of β-CD/CC (probe LC) (12x10-3 M/1x104
M) in the presence and absence of chloride salts of different metal cations (1x10-4
M); (b) Absorbance response (A0/A) of β-CD/CC (probe LC) (12x10-3 M/1x10-4 M) at equimolar concentration ([cation] = 1×10 -4 M) with various metal cations at 432 nm; (c) Comparison diagram of Probe LC with various metal cations at 432 nm.
Figure. 5. (a) Fluorescence responses of β-CD/CC (probe LC) (12x10-3 M/1x10-4 M) in the presence and absence of chloride salts of different metal cations (1x10-4 M); (b) Bar diagram showing % quenching of probe LC with various metal cations; (c) Comparison diagram of Probe LC with various metal cations at 517 nm.
Figure. 6. (a) Absorption titration spectra of probe LC upon addition of Hg2+ ion concentrations from 0 to 10 µM); (b) Benesi–Hildebrand plot of 1 / (A−A0) vs. 1 / [Hg2+]; (c) Relative absorption intensities of probe LC at 432 nm as a function of [Hg2+].
Figure. 7. (a) The fluorescence spectra changes of probe LC upon addition of 0–100 µM of Hg2+ ion in water solvent; (b) Stern–Volmer plot for the quenching of probe LC with Hg2+ ion; (c) Binding constant of probe LC with Hg2+ was based on a BenesiHildebrand plot and (c) Trend of probe LC emission as a function of [Hg2+] at 520 nm. (λex = 430 nm)
Figure. 8. Job's plot analysis for the complexation between CC: β-CD with Hg2+
Figure. 9. (A) Absorption titration spectra of (a) Pure curcumin (b) Probe LC (CC: βCD) (c) Probe LC + Hg2+ (d) Pure CC + Hg2+; (B) Fluorescence spectra of (a) Pure curcumin (b) Probe LC (CC: β-CD) (c) Probe LC + Hg2+ (d) Pure CC + Hg2+; (C) Color changes of sensor probe LC with Hg2+ metal cation.
Figure 10 Proposed mechanistic pathway for probe LC for the sensing of Hg2+ ion based on complexation.
Research Highlights
Inclusion complex (Probe LC) was confirmed using absorbance, fluorescence, FT-IR and NMR spectroscopy. Probe LC can act as “naked- eye” indicator for Hg2+. The probe LC exhibits selective and sensitive detection of Hg2+ ion through fluorescence quenching mode. 2:1 Binding stoichiometry between probe LC and Hg2+ ion.
Curcumin/Beta-cyclodextrin inclusion complex as a new “on -on- off” fluorescent sensor system for sensitive recognition of mercury ion Samikannu Prabu ab* and Sharifah Mohamad ab* a,b
Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603,
Malaysia a,b
University of Malaya Centre for Ionic Liquids, Department of Chemistry, Faculty of Science,
University of Malaya, Kuala Lumpur 50603, Malaysia
Supplementary information Figures Scheme. S1. Structures of (a) Curcumin (b) β-cyclodextrin and (c) probe LC. Figure. S1. FT-IR spectra of (a) β-CD, (b) CC (c) solid inclusion complex Figure. S2. 1H NMR spectra of (a) β-CD, (b) CC (c) solid inclusion complex Figure. S3. 13C NMR spectra of (a) β-CD, (b) CC (c) solid inclusion complex
Scheme. S1.
Scheme. S1. Structures of (a) Curcumin (b) β-cyclodextrin and (c) probe LC.
Figure S1
Figure S1. FT-IR spectra of (a) β-CD, (b) CC (c) solid inclusion complex
Figure S2
Figure S2. 1H NMR spectra of (a) β-CD, (b) CC (c) solid inclusion complex
Figure S3
Figure S3.
13
C NMR spectra of (a) β-CD, (b) CC (c) solid inclusion complex
Declaration of Interest Statement The authors declare that there is no conflict of interests regarding the publication of this paper.
AUTHOR CONTRIBUTION FORM
TITLE OF THE MANUSCRIPT: Curcumin/Beta-cyclodextrin inclusion complex as a new “turn- off” fluorescent sensor system for sensitive recognition of mercury ion Samikannu Prabu ab* and Sharifah Mohamad ab* a,bDepartment of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia a,bUniversity of Malaya Centre for Ionic Liquids, Department of Chemistry, Faculty of Sci-ence, University of Malaya, Kuala Lumpur 50603, Malaysia *Email:[email protected] (Sharifah Mohamad); [email protected] (S. Prabu)
Author contributions: The above authors are equal contribution for in this manuscript Samikannu Prabu
29/11/2019
Sharifah Mohamad
29/11/2019
S0022-2860(19)31637-0
DOI:
https://doi.org/10.1016/j.molstruc.2019.127528
Reference:
MOLSTR 127528
To appear in:
Journal of Molecular Structure
Received Date: 18 October 2019 Revised Date:
29 November 2019
Accepted Date: 3 December 2019
Please cite this article as: S. Prabu, S. Mohamad, Curcumin/beta-cyclodextrin inclusion complex as a new “turn-off” fluorescent sensor system for sensitive recognition of mercury ion, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2019.127528. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Graphical Abstract
Curcumin/Beta-cyclodextrin inclusion complex as a new “turn- off” fluorescent sensor system for sensitive recognition of mercury ion Samikannu Prabu ab* and Sharifah Mohamad ab* a,b
Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603,
Malaysia a,b
University of Malaya Centre for Ionic Liquids, Department of Chemistry, Faculty of Sci-
ence, University of Malaya, Kuala Lumpur 50603, Malaysia *Email:[email protected] (Sharifah Mohamad); [email protected] (S. Prabu)
Abstract The formation of supramolecular complex between the curcumin (CC or probe L) and β-cyclodextrin (β-CD) (CC: β-CD or probe LC) was confirmed using absorption and emission spectroscopy. The binding properties of probe LC with cations in water were observed for the first time via absorption and emission spectroscopies. The selectivity and sensitivity of fluorescence chemosensors have been studied using probe LC. The probe LC showed selective binding to Hg2+ and afforded new absorbance and fluorescence peaks at 379 nm and 502 nm, additionally to the prevailing bands of LC at 432 nm and 535 nm. It additionally showed apparent colour change from yellow to colourlessness and strong fluorescent to weak fluorescent owing to selective binding of Hg2+ ion, which was detected by naked eyes. No noticeable changes of colour and spectra were observed upon the addition of other metal cations such as [Ag+, K+, Na+, Cs+, Ba2+, Fe2+, Mg2+, Pb2+, Mn2+, Ni2+, Cd2+, CO2+, Cr3+, Sn2+ and Zn2+. Hg2+ produced blue shift in absorption spectra and quenching in emission spectra which suggested a likelihood of strong binding of Hg2+ ions with probe LC. The fluorescence response was concentration-dependent and can be well described by the typical Stern–Volmer model. Compared with prior reports, this method is very cheap, selective, sensitive and easily biodegradable of probe LC. Keywords: Curcumin; Beta-cyclodextrin; Inclusion complex; Hg2+; Colorimetric; Chemosensor.
1
1. Introduction In recent years, varied varieties of heavy metal ion pollution issues have attracted people's attention, together with soil contaminations, water pollutions and so on [1–4]. Selective monitoring about the heavy metal ions is of tremendous importance, as they play imperative duties in biological systems and also the environment. Mercury is a highly toxic and widespread global pollutant. Mercury ion (Hg2+) is well-known as an infectious heavy metal ion and causes thoughtful hazard to the human and the environment even when its concentration is low [5–9]. It has wide employment in several industries, like mercury vapor lamps, mercury battery and mercury switch. Mercury ion within the environment is transformed into organomercury species like methyl-mercury (CH3HgX) by aquatic microorganisms, which can accumulate and enter in the human body through the food chain [10–13]. As the continuous prosperity over mercury awareness within human body, it would lead in accordance with much illnesses such namely renal failure, liver damage, psychiatric disorders, and so on [14-15]. Once exposed after absolute environment, ethnical usually suffer severely appropriate in conformity with mercury leak. Thus, mercury is viewed so a precedence-controlled pollutant by way of WHO (World Health Organization) and alternative organizations. Till now, many analytical approaches have been developed for the determination of Hg2+ but, supramolecular complex based fluorogenic chemosensors are better and preferable for recognizing and sensing analytes because of their advantages in terms of simplicity, sensitivity, selectivity, real-time and online detection [16-17]. Curcumin (Scheme S1a) is a natural yellow hydrophobic polyphenol pigment with outstanding photostability and low toxicity [18]. Curcumin is greatly fed on as a seasoning, coloring and retaining agent of food, drugs and cosmetics permanency [19]. Curcumin is hardly used for highly selectively and sensitively sense of heavy metal cations application [20]. The curcumin molecule has two hydroxyl groups at both ends, a conjugation effect occurs from the electron cloud deviation under basic conditions. Also, curcumin is very good chelating agent and it forms complexes with several metal ions like Zn (II), Fe (II) and Cu (II) has also been reported in several
2
studies [21-22]. In addition, curcumin has good fluorescence characteristics and easily degradable. Fluorescent sensor technology has attracted considerable attention as a type of facile and efficient detection method for sensing biological and chemical species because of their simplicity, versatility, high selectivity and sensitivity [23-27]. The recognizing or sensing of heavy metal cationic analytes have recently developed as a key research topic within the field of supramolecular chemistry as a result of heavy metal cations necessary roles in biological, medical and environmental processes [28–31]. The generally enhances the absorbance and fluorescence intensity due the formation of inclusion complex between guest molecules and cyclodextrin. Cyclodextrins (6α-, 7β-, 8γ-cyclodextrin) are well-known doughnut-shaped oligosaccharides linked by α1,4-glycosidic bond to form macrocycles and are capable of forming inclusion complex with hydrophobic guest molecules of suitable shape and size and an outer hydrophilic surface (Scheme S1b) [32]. Beta-cyclodextrin is readily soluble in water to form clear homogeneous solutions and enables them to undergo host–guest interactions with huge number of organic molecules and drugs [33]. Cyclodextrins are at all famous building blocks because supramolecular structures, yet it is used in a number of fields such as medicine, drug, cosmetic, agriculture, analytical chemistry and so on. [34-35]. Many studies on enhancement of solubility, stability, biological application, drug delivery and food application of curcumin with cyclodextrins have been reported [18, 36–42]. In the current study we have focused on curcumin: β-cyclodextrin inclusion complex (probe LC) for cation sensing for the first time. The experimental results showed that probe LC may by selectively and sensitively sense Hg2+ ion through absorption and fluorescence detection along side the color change. The absorption and fluorescence spectral properties of curcumin and inclusion complex were investigated. Based on this phenomenon, a new supramolecular sensitizer (probe LC) has been designed by incorporating curcumin with beta-cyclodextrin and its efficient fluorescence sensing towards Hg2+. The probe LC exhibits substantial color change, blue shift and fluorescence quenching upon complexation with Hg2+, which could be utilized as an absorbance and fluorescent probe for Hg2+ in aqueous solution. The absorbance and 3
fluorescence studies about the complexation of curcumin with β-CD as a chemosensor probe LC (Scheme S1c) for sensing of Hg2+ ion an aqueous solution is proposed.
2. Experimental section 2.1 Reagents β-Cyclodextrin (purity ≥ 99%) and curcumin (purity ≥ 99%) were obtained from Sigma Aldrich and used while not additional purification. Other chemical reagents were of analytical reagent grade and used as received. Metal salts were also obtained from Sigma Aldrich and used while not additional purification. The stock solutions of metal cation (1×10-4M) were prepared using ultrapure water from chloride salts such as Ag+, Cs+, K+, Na+, Ba2+, Cd2+, CO2+, Fe2+, Mg2+, Mn2+, Ni2+, Sn2+, Zn2+, Cr3+, and the nitrate salts of Pb2+. 2.2 Apparatus Spectrophotometric measurements were made with a Shimadzu Ultraviolet-Visible spectroscopy UV-1800 (UV-Vis) recording spectrophotometer equipped with 1 cm quartz cells. Fluorescence spectra (emission spectral measurement) were carried out with Cary Eclipse Fluorescence Spectrophotometer. The Fourier transform infrared (FT-IR) spectra of CC and inclusion complex were recorded on a Perkin-Elmer RX1 FT-IR between 4000 and 400 cm-1 with a resolution of 2 cm-1 at room temperature. The proton nuclear magnetic resonance (1H NMR) spectra were recorded on Lambda JOEL 400 MHz FT-NMR spectrometer and dimethyl sulfoxide (DMSO-D6) had been used as solvent. 2.3 Preparation of inclusion complex in aqueous medium The stock solution of curcumin (1×10-4 M) was prepared using ethanol. The different concentrations of β-CD solution (0,2,4,6,8,10 and 12×10-3 M) were prepared using ultrapure water. Curcumin stock solution was added in a 10 ml volumetric flask and made up to the mark using following concentrations of β-CD solutions 0, 2, 4, 6, 8, 10 and 12×10−3 M respectively and shaken thoroughly. The solutions were prepared just before taking absorbance and emission measurements. All measurements were recorded at room temperature. 2.4 Preparation of solid inclusion complex 4
The solid inclusion complex of β-CD with CC was prepared using coprecipitation method. Meanwhile, 1.0 g of β-CD was dissolved in 30 mL of ultrapure water at room temperature and then this solution was continuous agitation using a magnetic stirrer. On the other hand, 0.3246 g of CC was dissolved in 30 mL of acetone and slowly added to the aqueous β-CD solution. The round bottom flask was covered with aluminum foil and constantly stirred for 48 h at room temperature. The final mixture was refrigerated at 4°C for 24 h. Afterwards, the final mixture was consecutively filtered through a normal filter paper. Finally, the solid inclusion complex was obtained by washed with acetone and water to remove the uncomplexed CC and β-CD respectively. The residue was dried at 50oCfor 48 h and utilized for further studies. 2.5 Preparation of inclusion complex with metal cations in aqueous medium The stock solution of curcumin (1×10-4 M), beta-cyclodextrin (12×10-3 M) and various heavy metal cations ions (1×10-4 M) were prepared by using ultra-pure water. For colorimetrical titrations, the stock solutions (1×10-4 M) of entire metal cations have been prepared in aqueous solution and utilized. To analyze the effect of metal ion through absorption and emission spectra, various heavy metal cation ions were prepared in 10 ml volumetric flask and shaken thoroughly. All the absorption and emission spectra were recorded at room temperature. 3 Results and discussion 3.1 Interaction of CC with β-CD in liquid state: 3.1.1 Absorption spectral characteristics of CC in β-CD medium The formation of inclusion complex between curcumin and beta-cyclodextrin in aqueous solution has been analyzed using absorption spectroscopy. The β-CD containing one or more water molecules was expelled by guest molecules encapsulated in β-CD. The insertion of CC within the β-CD cavity mainly depended on the number of water molecules released in the bulk water. It is reported that the inclusion behavior depends on the individual structural features of the guest and host molecules since the charge fit, shape fit, and size fit effects are the main governing factors on the formation of inclusion complex of cyclodextrin [43].
5
The absorption maximum of curcumin is 429 nm, which was attributed to the polyunsaturated systems, conjugated and benzene ring of CC [40]. There is an intramolecular hydrogen bond formed between the methoxy group and the phenolichydroxyl (OH) group of the CC. Two absorption bands were observed for CC nearly at 429 nm and 251 nm. The strong absorption band at 429 nm could be assigned to a combination of π →π* and n→ π* transitions. The weak absorption band at 251 nm corresponds to a π→ π* transition [44]. As shown in Fig. 1a, on increasing the concentration of β-CD from 0M to 0.012 M, the absorption spectrum of CC is slightly bathochromic shift at longer wavelength (429 nm 432 nm) with a gradual increase within the absorbance. This behavior has been attributed to the enhanced dissolution of the CC molecule by complexation through the hydrophobic interaction between CC and β-CD. The above results suggest that inclusion complex is formed between CC and β-CD (Scheme S1c). The formation of an inclusion complex is completed at 1.0×10−4 M (Fig. 1c). Thus, the β-CD effects on the absorption spectra of CC could be ascribed to the formation of inclusion complex through the hydrophobic interaction only. In UV-Visible spectra, the binding constant of CC: β-CD inclusion complex has been calculated by studying the changes in the absorption intensity maxima with β-CD concentration. In the case of 1:2 inclusion complex, formed between CC and βCD, the equilibrium is often written as:
+ − ⇌ : −
(1)
The equilibrium constant for the above equilibrium is given by equation (2) [: ]
= [][ ]
(2)
The stoichiometric ratios and binding constant “K” of the inclusion complex may be determined using Benesi–Hildebrand equation [45] assuming the formation of a 1:2 host–guest inclusion complex by UV spectroscopy. The Benesi-Hildebrand relation for such equilibrium is given in Eq. (3).
=
∆
+
(3)
[] ∆ []
6
where A-A0 is the difference between the absorbance of CC in the presence and absence of β-CD, ∆ε is the difference between the molar absorption coefficient of CC and the inclusion complex (CC: β-CD), [β − CD] , [CC]0 are the initial concentrations of β-CD and CC respectively, whereas k is the binding constant. The absorption intensity maxima at 429 nm was taken to draw the Benesi–Hildebrand plot 1/A-A0 versus 1/ [β-CD]2. It gives a straight line as shown in Figs. 1b. The good linear correlations coefficient (R2=0.9973) was obtained; it confirms the formation of a 1:2 inclusion complex between CC: 2β-CD [46]. The binding constant value “K” value was calculated from slope of the Benesi–Hildebrand plot according to Eq (4), and it is found to be 46.168×103 M−1 at 303 K.
=
(4)
! [" ]
3.1.2 Fluorescence spectral characteristics of CC in β-CD medium The fluorescence emission spectra of curcumin with various concentrations of β-CD are shown in Fig. 2a. When guest molecules in aqueous solution are encapsulated in cyclodextrins, emission spectra may be influenced which indicates the formation of inclusion complexes between guest and host molecules [47]. The maximum absorption wavelength of the CC has been fixed with excitation fluorescence spectroscopy. Curcumin shows a broad structureless fluorescence band in water at 571 nm, with fixed excitation at 429 nm. The CC exhibits a large hypsochromic shift in its spectrum from 571 nm to 535 nm with a concomitant increase in fluorescence intensity on the addition of β-CD. The hypsochromic shift (~36 nm) in the emission spectrum confirms the binding of curcumin to the hydrophobic cavity of β-CD. The hypsochromic shift observed is in consistency with the fact that CC experiences a less polar environment in the hydrophobic cavity of β-CD. The hypsochromic changing concerning the fluorescence maxima yet the rise of the fluorescence depth suggests that the construction regarding an inclusion complex of CC with β-CD. The above spectral exchange effects suggest as the form of an inclusion complex between CC and β-CD (Scheme S1c). The binding constant (K) for the formation of 1:2 inclusion complex has been found evaluating the changes in the fluorescence intensity of emission maxima with β-CD concentration using the modified Benesi-Hildebrand equation [45]. 7
##
=
∆
+
(5)
[] ∆ []
where I and Io are the fluorescence intensities of CC in the absence and presence of β-CD, respectively. Fig. 2b depicts the plot of 1/[I-Io] versus 1/[β–CD]2. The good linearity of the HB plot with the correlation co-efficient of R2 = 0. 9925 indicates the 1:2 inclusion complex formation CC with 2β-CD. The binding constant “K” value was calculated from the slope of the Benesi–Hildebrand plot by using the equation (6) and it is found to be 56.769 x 103 M-1at 303 K.
=
(6)
! [#"# ]
3.1.3 Spontaneity of inclusion complexation reaction The thermodynamic parameters (∆G) for the inclusion process can be calculated from the binding constant value “K” by the following equation at 303K. ∆G = -RT ln K
(7)
where ∆G is the Gibbs free energy change, R is the gas constant (J/mol·K), T is the temperature in Kelvin and “K” is the binding constant in mol−1.The binding constant “K” in the excited state (56.769×103 M-1) is higher than the “K” value in the ground state (46.168×103 M−1). ∆G values of the complexation process are found to be −27.04 kJmol−1 and −27.55 kJmol−1 for ground and excited state respectively. The negative values of ∆G indicate the spontaneity of inclusion complexation reaction at 303 K. 3.2 Interaction of CC with β-CD in solid state: 3.2.1 Fourier - transform infra-red spectral analysis Fig S1 depicts the FT-IR spectra for β-CD, CC and inclusion complex. The FT-IR spectrum of β-CD (Fig S1a) was characterized by a strong absorption band at 3293 cm-1 (stretching vibration of -OH), 2925 cm-1 (stretching vibration of -C-H), 1415 cm1
(stretching vibration of -OH deformation), 1152 cm-1 (stretching vibration of asym-
metry -C-O-C), 1020 cm-1 (stretching vibration of symmetry -C-O-C), 947 cm-1 (stretching vibration of skeletal vibration involving α-1,4 linkage) and 851 cm-1 (Breath of glucose ring). The FT-IR spectrum of CC (Fig S1b) was consider by an absorption of band of at 3493.45 cm-1 (phenol group, intramolecular H-bond), 3006.12
8
cm-1 (aliphatic C-H stretching), 2927.90 cm-1 (aromatic C-H stretching), 1626.24 cm-1 (-CO stretching), 1601.42 cm-1 (aromatic C=C stretching), 1426.95 cm-1 (aliphatic OH stretching), 1266.36 cm-1 (aromatic C-O stretching), 1026.29 cm-1 (-C-O-C stretching) and 959.74 cm-1 ( C=C bending). The FT-IR spectrum of the solid inclusion complex exhibited no features similar to pure CC and β-CD. The stretching frequencies of aliphatic C-H stretching, aliphatic -OH, -CO and bending -C=C of the complex product appeared at 3000.17 cm-1, 1425.63 cm-1, 1623.37 cm-1 and 959.74 cm-1 respectively, as shown in Fig. S1c. However, those specific stretching frequencies are getting only slight shifts from pure CC. The stretching frequencies of phenol group intramolecular H-bond, aromatic C-H, aromatic C=C, aromatic C-O and -C-O-C of the complex product appeared at 3392.42 cm-1, 2947.37 cm-1, 1585.09 cm-1, 1296.53 cm-1 and 1034.73 cm-1 respectively. Hence, the large shifts in stretching frequencies of pure CC became inclusion complex. These changes indicated that the aromatic part of CC is involved in an inclusion complexation on the hydrophobic cavity of β-CD. 3.2.2 Nuclear Magnetic Resonance Spectral Analysis The 1H NMR spectra of β-CD, CC and inclusion complex were revealed in the Fig. S2. Dissimilarities in the chemical shift’s value of CC and β-CD and inclusion complex were listed Table 1. The positive and negative signs meant an upfield and downfield shifts. The 1H NMR spectrum of β-CD contains nine variabilities of protons (H1- H9) as shown in Fig. S2a. It can be seen from the 1H NMR spectrum of inclusion complex (Fig. S2c), a downfield and upfield shift were happening for H1, H5 and H7 protons and H2, H3, H4, H6, H8 and H9, respectively. The above chemical shifts were changes owing to CC inclusion complex with β-CD. The CC contains ten variabilities of protons (H1- H10) as shown in Fig. S2b. The ∆δ values of the H8, H9 and H10 protons in the inclusion complexes have relatively small effects. Even though, the ∆δ values of the H1, H2, H3, H4, H5, H6a, b and H7 protons displayed large effects. The above small chemical shift changes owing to H8, H9 and H10 protons were located outside of the β-CD cavity. The above large chemical shift changes owing to H1, H2, H3, H4, H5, H6a, b and H7 protons were located inside of the β-CD cavity. The above re-
9
sults suggest that the strong supramolecular inclusion complex formation between CC and β-CD. The
13
C NMR spectra of β-CD, CC and inclusion complex were shown in
Fig.S3. Dissimilarities in the chemical shifts of CC and β-CD and inclusion complex states were listed Table 2. The β-CD (Fig. S3a) exhibits six variabilities of carbons (C1–C6). It can be seen from the 13C NMR spectrum of inclusion complex (Fig. S3c), all the carbons chemical shift values were shifted with diminished intensity. These significant changes owing to an encapsulation of CC into β-CD. As shown in Fig. S3b, CC was displays thirteen variabilities of carbons (C1–C13). Whereas in the
13
C
NMR spectrum of inclusion complex; a small and great chemical shift were obtained for C10a, b and C11 and C1, C2, C3, C4, C5, C6, C7, C8, C9, C12 and C13 respectively. The above results suggest that the noticeable changes of chemical shift in C1, C2, C3, C4, C5, C6, C7, C8, C9, C12 and C13 carbons indicated the CC inclusion complex with β-CD. The small changes of chemical shift in C10a, b and C11 carbons were suggest that the influence of CC molecule. 3.3 Analytical application of CC: β-CD complex as chemosensor 3.3.1 Selectivity study of CC: β-CD complex as chemosensor 3.3.1.1 Colorimetric sensing study The colorimetric sensing study by naked eye is the simplest way to easily observe the high selectivity of probe LC to different heavy metal cations. Fig. 3 depicts the photographs of probe LC after addition of different mono, di and trivalent metal cations of biological and ecological importance, viz., Na+, K+, Ag+, Cs+, Mg2+, Mn2+, Ba2+, Co2+, Ni2+, Pb2+, Hg2+, Zn2+, Cd2+, Fe2+, Sn2+and Cr3+. The Probe LC chemosensor showed a highly sensing and selective recognition behavior toward Hg2+ ion by changing the color of the solution from yellow (432 nm) to colorless (379 nm). This selective color change can be used for the “naked eye” for the recognition of Hg2+. 3.3.1.2 Absorption spectroscopy response of chemosensor probe LC Based on the above colorimetric sensing study results, detailed electronic spectroscopic studies were carried out to investigate the selective sensing of probe LC toward Hg2+.The absorption spectra of probe LC in the presence and absence of various mono-, di and trivalent heavy metal cations of biological and ecological importance, 10
viz., Na+, K+, Ag+, Cs+, Mg2+, Mn2+, Ba+, Co2+, Ni2+, Pb2+, Hg+, Zn2+, Cd2+, Fe2+, Sn2+ and Cr3+are shown in Fig. 4a.The order of change of absorption intensities of probe LC is Hg2+< Fe2+< Mn2+< K+< Ag+< Mg2+< Cd2+< Zn2+< Pb2+< Co2+< Cr3+< Cs+< Na+< Sn2+< Ni2+< Ba2+< probe LC. By comparison, probe LC shows a very high selectivity toward Hg2+. As can be seen in this figure, the absorption maximum of probe LC and other metal cations except Hg2+ at λmax = 432 nm. upon addition of Hg2+cation to probe LC chemosensor underwent dramatic change of an absorption intensities as well as broad peak of on n-π* appeared at 379 nm (a blue shift of 53 nm from 432 nm) and very clear isosbestic point is observed at 394 nm, which confirmed the existence of a well-defined stoichiometric complex. The spectrum appearance of the large blueshift from 482 to379 nm, suggest that strongly an interaction of Hg2+ with the deprotonated aliphatic oxygen of probe LC to form the complex probe LC: Hg2+. [48] As shown in Fig 4b, the absorbance response (A0/A) of probe LC at equimolar concentration (1×10−4 M) of various mono-, di and trivalent metal cations at 432nm. This illustrated clearly that Hg2+ exhibited good response than other mono-, di and trivalent metal cations such as Na+, K+, Ag+, Cs+, Mg2+, Mn2+, Ba+, Co2+, Ni2+, Pb2+, Zn2+, Cd2+, Fe2+, Sn2+ and Cr3+. The comparative diagram of various mono, di and tri heavy metal cations absorption intensity is shown in (Fig. 4c). A significant decrease of absorption intensity of probe LC with Hg
2+
ions was observed. These results illus-
trated that probe LC could be an effective interact with Hg2+. 3.3.1.3 Fluorescence spectroscopic response of chemosensor probe LC The fluorescence responses of probe LC has been explored in presence and absences of various mono-, di and trivalent heavy metal cations of biological and ecological importance, viz., Na+, K+, Ag+, Cs+, Mg2+, Mn2+, Ba+, Co2+, Ni2+, Pb2+, Hg+, Zn2+, Cd2+, Fe2+, Sn2+ and Cr3+ are shown in Fig. 5a. In good agreement with the findings based on the above absorption study, the host-guest system CC: β–CD (probe LC) shows rather strong fluorescence emission in the range from 311 nm to 323 nm in an aqueous water medium. The strong fluorescence emission intensity of probe LC is observed at 512 nm. However, based on addition of 2 equivalent metal cations, the addition of Hg2+ resulted in a significant diminution of the fluorescence emission intensity 512 nm and blue shift to 502 nm. 11
As shown by the fluorescence emission spectra, adding Hg2+ to probe LC lead to an obvious “on-off” fluorescence response, while the other heavy metal cations do not lead to any significant fluorescence quenching under same spectroscopic conditions. The above observations clearly suggest that the excellent selectivity of the probe LC towards Hg2+only. Fig. 5b shows percentage of quenching of probe LC on addition of various metal cations. The quenching of Hg2+ cation is 73.88%, when compared to probe LC and other metal cations. The Comparative diagram of various mono, di and tri heavy metal cations fluorescence emission intensity is shown in Fig. 5c. A significant diminution of fluorescence emission intensity of probe LC with Hg
2+
ion was observed.
The order of change of fluorescence intensities of probe LC is Hg2+< Fe2+< Cr3+< K+< Mg2+< Ba2+< Cs+< Co2+< Zn2+
plex formation. These results clearly establish the very high sensing ability of probe LC for Hg2+ cation in water. 3.3.2.2 Sensing and Binding Studies using Fluorescence Spectroscopy Sensitivity is a very significant parameter to estimate the presentation of a fluorescent chemosensor. In good agreement with the findings by the above absorption studies, probe LC exhibited specific fluorescence intensity responses towards Hg2+ under similar conditions. Fig. 7a depicts the fluorescence emission spectra of probe LC with the Hg2+ion. The effect of increasing the Hg2+ concentration causes decrease the fluorescence intensity without any shift in the peak. The quenching effects of probe LC by Hg2+ have been studied, and the quenching process was attributed to the formation of a complex between probe LC and Hg2+.Thus, it is appraised here that the probe LC act as a turn off fluorescence for Hg2+ ions compared to other metal cations. The modified Benesi-Hildebrand plot of 1/(F0-F) as a function of 1/[ Hg2+] as shown in Fig. 7c. The binding constant (Kb) between probe LC with Hg2+ was calculated from fluorescence spectrophotometer by using a modified Benesi-Hildebrand equation [45, 55-57] stated below, $$%&'
=
$%() $%&'
where, I
min,
+
* [+',-. ] $
%() $%&'
I and Imax are the emission intensities of probe LC in the absence
of Hg2+ ion, at varying Hg2+ concentration, and at a concentration of complete saturation respectively. K is the binding constant and [Hg2+] is the concentration of Hg2+. The modified Benesi-Hildebrand plot is giving a good linear correlation coefficient R2= 0.9919 for Hg2+ ion. The association constant (Ka) between probe LC and the Hg2+ion was found to be 2.11×10-2 M−1at 25◦C. Relative fluorescence emission intensities at 516 nm with the function of MnO4- ion concentration is observed and given in Fig. 5d. The low concertation of Hg2+ ion is high fluorescence emission intensity (~100.72) and high concentration of Hg2+ion is very low fluorescence emission intensity (~30.06). These results indicate the well-defined stoichiometric complex formation between probe LC and Hg2+ through quenching mechanism. 3.3.2.3 Stern-Volmer analysis
13
The mechanism of quenching process was carried out by using Stern-Volmer law. The linear plotting of relative fluorescence intensities (I0/I) versus quencher concentration of Hg2+ion follows a linear trend according to a typical Stern−Volmer equation expression: I0/I = 1+ Ksv [Hg2+]
(8)
Here I and I0 are fluorescence intensities in the presence and absence of added Hg2+cations. The slope of this line is Ksv. These results obtained are also consistent with the static quenching. The Stern-Volmer plot gives a good linear correlation coefficient R2= 0.9905 for Hg2+ion concentration in the range from 0–45 µM in Fig. 7b. The linear equation was calculated as I0/I = 0.9948 + 0.0165 c, with KSV value of 0.0113. Probe LC was found to have a detection limit of 5.02 µM and LOQ 16.73 µM. The experimental parameters for the sensing of Hg2+ using Probe LC fluorescence chemosensor are given Table 3. 3.4 Binding stoichiometry of metal ions complex The stoichiometry ratio of the inclusion complex of CC and Hg2+ ions were confirmed by using jobs plot of continuous variation method. We are calculating ∆A from the absorbance of metal complex (Amc) was subtracted from probe LC (Alc).
∆A = Alc-Amc A mole fraction of metal ions Hg
(9) 2+
against the change in absorbance (∆A) as
shown in Fig 8. It is observable that the maximum values ∆A for Hg2+ ions appear at around 0.6 M fraction which indicates that the formation of a 2:1 stoichiometric complex between probe LC and Hg2+ ion. 4 The possible binding mechanism of quenching effect by Hg2+ To determine and monitor the interaction between probe LC and Hg2+, the UV– Vis spectra and fluorescence emission spectra were examined. As illustrated in Fig. 9A, the absorption spectrum of pure curcumin is characterized by sharp peak at 429 nm, due to the enol form and mono anion forms [58]. In addition, it is interesting to find that the absorption maximum of enol form, mono anion form with trans-geometry of curcumin solution is observed as peak at 420–430 nm and enolic proton is more active than the phenolic proton [59-61]. The strong π(HOMO) → π(LUMO) transition of curcumin was found to be 429 nm in a solution that masked the weak electronic di14
pole forbidden n → p band. This band is in agreement for CU in the enol form [62]. Upon addition of curcumin solution to the solution of Beta cyclodextrin, increase an absorption intensity and a slight red shift of the absorption spectra (from 429 nm to 433 nm) was observed due the inclusion complex formation between curcumin and beta cyclodextrin (probe LC). Upon adding Hg2+ to the solution of probe LC, a large blue shift of the absorption spectra (from 433 nm to 379 nm) was observed due to interaction between Hg2+ and probe LC as a mercury chelator, but in the presence of oxalate ion due to its high affinity toward Hg2+ ion. At the same time, the color of the reaction mixture changed from yellow to colourless as shown in Fig 9C. So, probe LC could be used as a visual tool for an appropriate detection of Hg2+ without using any very expensive instruments, which is superior over other analytical methods. The fluorescence titration of probe LC against Hg2+ was carried out and spectral change is presented in Fig 9b. The fluorescence spectrum of pure curcumin is measured in excitation wavelength at λex = 425 nm for enol forms of curcumin [61]. The fluorescence emission spectrum of pure curcumin was exhibiting a single broad band with a peak at 571 nm. The fluorescence intensity of probe LC was observed at 535 nm, with enhancements of fluorescence intensity and also blue shift. This behavior indicated that curcumin molecule is entrapped into the β-CD cavity to form 1:2 inclusion complex. Thus, the variation in the fluorescence emission spectrum of curcumin is due to Excited State Intra-Molecular Hydrogen Transfer (ESIHT), since an unusually longer wavelength emission of curcumin in solution has been found to be due to ESIHT [62]. After adding Hg2+, probe LC solution has a large blue shift (from 535 to 502 nm) and quenching of fluorescence intensity, indicating the strong binding between the Probe LC and Hg2+.Thus, in accordance with the 2:1 stoichiometry, curcumin is likely to chelate Hg2+ via its O(-) atom and OH group of β-CD. Based on the UV–Vis spectra and fluorescence analysis, it is confirmed that the O(-) atom of curcumin coordinate with Hg2+ ion. The inclusion complex (probe LC) system exhibited high selectivity and sensitivity for Hg2+and the supramolecular sensing process was shown in Figure 10. 5 Analysis of real water samples and recovery test
15
The proposed chemosensor was applied to the determination of Hg2+ in drinking water and tap water samples. Drinking water and tap water samples were analyzed without any pretreatment. All the water samples were spiked with inclusion complex at single concentration level and then analyzed. The results are shown in Table 4. The recoveries of Hg2+ were 104% and 92% for drinking water and tap water samples respectively. The recovery of the proposed sensor is satisfactory. The present chemosensor may be useful for the determination of Hg2+ in real samples. 6 Conclusion In this article, an efficient, low cost, simple, and visual measurement system that utilizes CC: β-CD for specific recognition toxic Hg2+ ion was successfully developed. A simple probe LC shows a noticeable change in the absorbance and fluorescence emission spectral (turn-off fluorescence) behavior in the presence of toxic Hg2+ ion. Curcumin contained one aliphatic hydroxy group and two phenolic hydroxy groups and the presence of beta cyclodextrin and toxic Hg2+ ion led to the formation of cationic complex. Also, the deprotonation of aliphatic hydroxy group in curcumin resulted in spectral and colour changes. The LOD and LOQ of the proposed chemo sensor (probe LC: Hg2+) are 5.02 µM and 16.73 µM, respectively. Even more remarkably, this technique can provide a novel and eco-friendly approach for in situ identification of toxic Hg2+ion in environmental sample. The proposed sensor was used to analyze Hg2+ in real water samples with satisfactory results. Reference 1.
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Table 1 Variation in the 1H NMR chemical shifts (δ/ppm) of CC and β-CD protons in free and complexed states determined in DMSO-d6 at 298 K
Substance
β-CD
CC
Protons Free (δ, ppm)
Complex (δ, ppm)
∆δ (ppm)
H1
5.72
5.70
-0.02
H2
4.78
4.80
0.02
H3
3.61
3.77
0.16
H4
3.61
3.77
0.16
H5
4.77
4.55
-0.22
H6
2.30
2.33
0.03
H7
3.60
3.58
-0.02
H8
3.58
3.60
0.02
H9
3.62
3.63
0.01
H1
9.55
9.70
0.15
H2
6.79
6.83
0.04
H3
6.69
6.75
0.06
H4
7.48
7.40
0.08
H5
2.46
2.50
0.04
H6a, b
7.27
7.23
-0.04
H7
7.09
7.05
-0.04
H8
3.79
3.82
0.03
H9
7.60
7.57
-0.03
H10
6.01
6.03
0.02
Table 2 Variation in the 13C NMR chemical shifts (δ/ppm) of CC and β-CD protons in free and complexed states determined in DMSO-d6 at 298 K
Substance
Protons C1
CC
β-CD
Free ppm)
(δ, Complex (δ, ppm)
∆δ (ppm)
149.86
152.45
2.59
C2
111.79
114.81
3.02
C3
124.18
127.16
2.98
C4
141.25
144.43
3.18
C5
116.20
120.39
4.19
C6
150.22
152.30
2.08
C7
56.18
58.69
2.51
C8
148.79
148.25
-0.54
C9
126.84
125.24
-0.60
C10a, b
183.74
183.24
0.50
C11
101.41
101.26
-0.15
C12
121.59
122.80
1.21
C13
145.21
144.06
-1.15
C1
110.4
101.41
8.99
C2
74.4
72.93
1.47
C3
74.1
72.57
1.53
C4
83.1
82.06
1.04
C5
79.6
73.58
6.02
C6
62.5
60.44
0.06
Table 3: Experimental parameters for the sensing of Hg2+ using Probe LC fluorescence chemosensor
Name of the parameters
Values
λ excitation (nm)
430
λ emission (nm)
502
Linear range (µM)
0-45
Correlation coefficient (r)
0.9905
LOD (µM)
5.02
LOQ (µM)
16.73
Table 4 Determination of Hg2+ in real water samples. Added [Hg2+] (M)
Found total [Hg2+]a (M)
Recovery (%)
Drinking Water
1.00 x10-4
1.04±0.03
104
Tap Water
1.00 x10-4
0.92±0.01
92
Real water samples
a
Mean ± standard deviation (n= 3).
Figure. 1. (a) Absorption spectra of CC (1x10−4 M) in different β-CD concentrations: (1) 0M (2) 0.002M (3) 0.004M (4) 0.006M (5) 0.008M (6) 0.010M and (7) 0.012M; (b) Benesi–Hildebrand plot of 1 / (A − A0) vs. 1 / [β − CD] and (c) Absorption intensity of CC changes at 429 nm.
Figure. 2. (a) Fluorescence spectra of CC (1X10−4 M) in different β-CD concentrations: (1) 0M (2) 0.002M (3) 0.004M (4) 0.006M (5) 0.008M (6) 0.010Mand (7) 0.012M; (b) Benesi–Hildebrand plot of 1 / (I − I0) vs. 1 / [β − CD] and (c) Fluorescence intensity of CC changes at 571 nm.
Figure. 3. Color changes of sensor probe LC with different metal cations
Figure. 4. (a) UV-vis absorption responses of β-CD/CC (probe LC) (12x10-3 M/1x104
M) in the presence and absence of chloride salts of different metal cations (1x10-4
M); (b) Absorbance response (A0/A) of β-CD/CC (probe LC) (12x10-3 M/1x10-4 M) at equimolar concentration ([cation] = 1×10 -4 M) with various metal cations at 432 nm; (c) Comparison diagram of Probe LC with various metal cations at 432 nm.
Figure. 5. (a) Fluorescence responses of β-CD/CC (probe LC) (12x10-3 M/1x10-4 M) in the presence and absence of chloride salts of different metal cations (1x10-4 M); (b) Bar diagram showing % quenching of probe LC with various metal cations; (c) Comparison diagram of Probe LC with various metal cations at 517 nm.
Figure. 6. (a) Absorption titration spectra of probe LC upon addition of Hg2+ ion concentrations from 0 to 10 µM); (b) Benesi–Hildebrand plot of 1 / (A−A0) vs. 1 / [Hg2+]; (c) Relative absorption intensities of probe LC at 432 nm as a function of [Hg2+].
Figure. 7. (a) The fluorescence spectra changes of probe LC upon addition of 0–100 µM of Hg2+ ion in water solvent; (b) Stern–Volmer plot for the quenching of probe LC with Hg2+ ion; (c) Binding constant of probe LC with Hg2+ was based on a BenesiHildebrand plot and (c) Trend of probe LC emission as a function of [Hg2+] at 520 nm. (λex = 430 nm)
Figure. 8. Job's plot analysis for the complexation between CC: β-CD with Hg2+
Figure. 9. (A) Absorption titration spectra of (a) Pure curcumin (b) Probe LC (CC: βCD) (c) Probe LC + Hg2+ (d) Pure CC + Hg2+; (B) Fluorescence spectra of (a) Pure curcumin (b) Probe LC (CC: β-CD) (c) Probe LC + Hg2+ (d) Pure CC + Hg2+; (C) Color changes of sensor probe LC with Hg2+ metal cation.
Figure 10 Proposed mechanistic pathway for probe LC for the sensing of Hg2+ ion based on complexation.
Research Highlights
Inclusion complex (Probe LC) was confirmed using absorbance, fluorescence, FT-IR and NMR spectroscopy. Probe LC can act as “naked- eye” indicator for Hg2+. The probe LC exhibits selective and sensitive detection of Hg2+ ion through fluorescence quenching mode. 2:1 Binding stoichiometry between probe LC and Hg2+ ion.
Curcumin/Beta-cyclodextrin inclusion complex as a new “on -on- off” fluorescent sensor system for sensitive recognition of mercury ion Samikannu Prabu ab* and Sharifah Mohamad ab* a,b
Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603,
Malaysia a,b
University of Malaya Centre for Ionic Liquids, Department of Chemistry, Faculty of Science,
University of Malaya, Kuala Lumpur 50603, Malaysia
Supplementary information Figures Scheme. S1. Structures of (a) Curcumin (b) β-cyclodextrin and (c) probe LC. Figure. S1. FT-IR spectra of (a) β-CD, (b) CC (c) solid inclusion complex Figure. S2. 1H NMR spectra of (a) β-CD, (b) CC (c) solid inclusion complex Figure. S3. 13C NMR spectra of (a) β-CD, (b) CC (c) solid inclusion complex
Scheme. S1.
Scheme. S1. Structures of (a) Curcumin (b) β-cyclodextrin and (c) probe LC.
Figure S1
Figure S1. FT-IR spectra of (a) β-CD, (b) CC (c) solid inclusion complex
Figure S2
Figure S2. 1H NMR spectra of (a) β-CD, (b) CC (c) solid inclusion complex
Figure S3
Figure S3.
13
C NMR spectra of (a) β-CD, (b) CC (c) solid inclusion complex
Declaration of Interest Statement The authors declare that there is no conflict of interests regarding the publication of this paper.
AUTHOR CONTRIBUTION FORM
TITLE OF THE MANUSCRIPT: Curcumin/Beta-cyclodextrin inclusion complex as a new “turn- off” fluorescent sensor system for sensitive recognition of mercury ion Samikannu Prabu ab* and Sharifah Mohamad ab* a,bDepartment of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia a,bUniversity of Malaya Centre for Ionic Liquids, Department of Chemistry, Faculty of Sci-ence, University of Malaya, Kuala Lumpur 50603, Malaysia *Email:[email protected] (Sharifah Mohamad); [email protected] (S. Prabu)
Author contributions: The above authors are equal contribution for in this manuscript Samikannu Prabu
29/11/2019
Sharifah Mohamad
29/11/2019