An efficient inclusion complex based fluorescent sensor for mercury (II) and its application in live-cell imaging

doi:10.21203/rs.3.rs-1341774/v1

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An efficient inclusion complex based fluorescent sensor for mercury (II) and its application in live-cell imaging

https://doi.org/10.21203/rs.3.rs-1341774/v1

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published 19 Mar, 2022

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The formation of an inclusion complex between hydroxypropyl-β-cyclodextrin (H-CD) and 4-acetylphenyl-4-(((6-chlorobenzo[d]thiazol-2-yl)-imino)-methyl)-benzoate (L) was investigated by FT-IR, ¹H-NMR, X-ray diffraction (XRD), FT-Raman, scanning electron microscope (SEM) techniques in the solid-state, absorption and emission spectroscopy in the liquid state and the virtual state as molecular docking technique. The binding properties of the inclusion complex (H-CD: L) with cations in deionized water was observed via absorbance and photoluminescence (PL) emission spectroscopy. The fluorescence probe (H-CD: L) inclusion complex (IC) was examined for several heavy metal cations and, identified that the PL emission wavelength of the complex displayed a continuous rise in the fluorescence intensity for Hg²⁺. A linearity range of 1×10^-8- 11×10^-8M and limit of detection value of 2.71×10^-10M was found to be achieved for the detection of Hg²⁺. This outcome proves that the inclusion complex H-CD: L would be a promising material for the development a solid-state fluorescence probe for detecting Hg^2+. It also shows application in real sample analysis and cell imaging.

Hydroxypropyl-Beta-cyclodextrin (H-CD)

Inclusion complex

Colorimetric

Hg2+

fluorescence enhancement

Chemosensor

Currently, heavy metal ion pollution is surpassing the threshold limits in various areas of the world due to the growth of industrialization and modernization, for example, coal, and gold mining [1], oil refining [2], and paint production [3]. In comparison to other heavy metal ions, mercury (Hg²⁺) is the utmost toxic metal pollutant due to its assorted utilisation in numerous industries and horticultural sciences [4, 5]. Non-biodegradable mercury ion prompts extreme tainting of marine resources and soil. Besides, Hg²⁺ gets aggregated in rural and sea-going effluence [6, 7] and causes serious medical and environmental problems, named as myocardial localized necrosis, chemical imbalance, and Minamata sickness [8–11]. As an attempt to ensure human wellbeing, the United States Environmental Protection Agency (USEPA) has proclaimed the Hg²⁺ ion threshold limit value (TLV) in drinking water sources as 2 ppb [12, 13]. In view of, ecological and medical problems related to Hg²⁺, it is exceptionally fundamental to progress a solid sensing strategy with high sensitivity for observing low levels of Hg²⁺ in the environment and biological systems.

Various analytical techniques like electrochemical methods [14], atomic absorption spectroscopy (AAS) [15], inductively coupled plasma spectroscopy (ICPS) [16], and colorimetry [17] have been used for the detection of various heavy metals. The abovementioned analytical techniques need sophisticated and expensive instruments, tedious preparation, and also pre-treatment of the sample. Among them, the spectrofluorimetric technique has attracted much attention due to its facile and effective method of detecting chemical and biological species, high sensitivity, selectivity, quick response, cost-effectiveness, simplicity and a need of low concentration of analytes [18]. Supramolecular IC has gained more attention compared to other fluorophores like coumarin, pyrene and xanthene derivatives, metallic organic frameworks etc., Supramolecular Inclusion complexes were better and preferable fluorogenic probes for sensing the analytes due to their advantages in selectivity, sensitivity, easy sample preparation, real-time analysis.

Cyclodextrin (CD) belong to the family of cyclic oligosaccharides. It is also known as Schrodinger dextrin’s, cycloamyloses and cyclomaltoses. It consists of a macrocyclic ring of glucose subunits joined by α-1,4 glycosidic bonds. Cyclodextrins are obtained from enzymatic hydrolysis of starch. The β-cyclodextrin is a natural oligomer, which has a hydrophobic cavity and an exterior which is highly hydrophilic in nature. The cyclodextrin’s hydrophobic interior surface is torus-shaped structure, which permits guest molecules naturally into the hydrophobic cavity by expelling the molecules of water [19]. The cyclodextrins have the ability to form host-guest complex molecule which leads to a number of advantages in different fields [20, 21]. These cyclodextrins (CDs) have been used in number of industries such as pharmaceutical, food, analytical and catalysis. These CDs acts a solubilizers, diluents, tablet ingredients to enhance the solubility, chemical stability, pharma kinetic properties, and bioavailability of drugs [22]. Hydroxypropyl-beta-cyclodextrin (H-CD) is hydrophilic in nature, and considered as a toxic-free derivative at oral and intravenous doses. Recently, H-CD used to increase the stability, solubility and bio-availability of poorly solvable compounds in drug delivery [23].

Acetylphenyl-4-(((6-chlorobenzo[d]thiazol-2-yl)-imino)-methyl)-benzoate (compound L), a crystalline solid is a polycyclic aromatic compound. Compound (L) acts as a fluorescent sensor in a hydrophilic environment. But it exhibits strong fluorescence in the hydrophobic cyclodextrin cavity. It easily binds with mercury metal ions and enhances the fluorescence intensity. In addition, compound (L) has a major role as a fluorescent probe in life cell imaging and real sample analysis. This compound is encapsulated into the H-CD hydrophobic cavity with the release of water molecules.

The key area of the present work is to synthesize H-CD/L solid IC under reflux condition for sensing metal cation. The obtained experimental results showed that the solid IC selectively and sensitively sense mercury metal ion (Hg²⁺) and it was observed through a visible colour change, absorption and fluorescence spectrum. Based on this phenomenon, a new supramolecular sensor (H-CD: L) has been synthesised by using compound L with H-CD and its more effective in photoluminescence (PL) sensing towards Hg²⁺. The H-CD: L solid IC shown considerable color variation, PL enhancement upon the addition of Hg²⁺, which might be used in absorbance and PL sensing for Hg²⁺ in liquid medium. The H-CD: L solid IC could also be applied for real sample analysis and life cell imaging.

2.1. Reagents

Hydroxypropyl-β-cyclodextrin (H-CD) was procured from Sigma Aldrich and used without any further purification. All the chemicals used in this study are of pure analytical grade. Metal chloride salts of Ag⁺, Co²⁺, Cr³⁺, Cs⁺, Hg²⁺, Ba²⁺, Mg²⁺, Fe²⁺, Fe³⁺, K⁺, Na⁺ were obtained from SD Fine-Chem Pvt Ltd., India, metal nitrate salt of Pb²⁺ was obtained from Alfa Aesar, and metal carbonate of was Cd²⁺ obtained from Qualigens and were used without any additional purification. The metal cation solutions (1 ×10^− 8 M) used throughout the experiment were all prepared in double distilled water.

2.2 Apparatus

FT-IR spectrum was used to find out the functional groups and using Thermo Nicolet, Avatar 370. Powder X-ray diffraction (P-XRD) patterns identified from the Rigaku Smart Lab X-ray diffractometer using Cu Kα radiation (λ = 1.5406 Ǻ). Lab RAM HR FT-Raman module, with excitation at 532 nm LASER used to record Raman spectrum. The photophysical studies like the UV-Vis Absorption spectrum were investigated using a Shimadzu UV-visible spectrophotometer (UV-visible 1800). All fluorescence measurements were taken using Shimadzu spectrofluorometer (RF5301PC). All measurements were recorded using PBS buffer solution at pH = 7 medium.

2.3. Synthesis of 4-Acetylphenyl 4-(((6-chlorobenzo[d]thiazol-2-yl) imino) methyl) benzoate

Compound (L) was synthesized via two steps reaction: Schiff base synthesis followed by esterification reaction [24].

STEP-1

The Schiff base was synthesized by reacting equimolar quantities (0.01 mol) of 2-amino-6-chlorobenzothiazole and 4-formylbenzoic acid in 40 ml of pure ethanol. Then 3 drops of glacial acetic acid were added continuously and the mixture was heated under reflux condition for 3 h. Then the mixture was cooled down and the solvent was evaporated under vacuum. The obtained solid product was purified by recrystallization from alcohol to give 4-{[(6-chloro-1,3-benzothiazol-2-yl)-imino]-methyl}-benzoic acid (Intermediate).

STEP-2

To a solution of intermediate (0.02mol) and 1-(4-hydroxyphenyl)-ethan-1-one (0.002mol) in 40 ml of dichloromethane, dimethyl aminopyridine (0.002 mol) in 15ml DCM was added. Then the mixture was cooled down in an ice bath with constant stirring for 10 mins. To this mixture, a solution of N,N′-dicyclohexylcarbodiimide (0.02) mmol was added dropwise. The mixture was stirred for 4 h at room temperature under continuous stirring. the solvent was removed under vacuum and the solid obtained was purified by column chromatography using a mixture of n-hexane/ethyl acetate (8:2) as eluent. The solid obtained was recrystallized from ethanol.

2.4. Preparation of IC (H-CD: L) in an aqueous medium.

The Various concentrations of H-CD (0–12×10^− 3 M) were prepared using double distilled water. The solution of compound (L) (1 ×10^− 3 M) was also prepared using double distilled water/DMSO in the ratio 8:2. Addition of the stock solution of compound (L) to different volumetric flasks followed by the addition of different concentrations of H-CD solutions (0–12×10^− 3 M) which were made up to the mark and then shaken thoroughly. These obtained solutions were used for absorbance and PL emission quantities. All the measurements were taken at room temperature.

2.5. Preparation of the solid IC (H-CD: L)

The solid IC of compound L with H-CD was synthesized using reflux condition method. Firstly, 0.077g of H-CD (1mmol) in 50 mL of deionized water was continuously agitated using a magnetic stirrer at normal room temperature. Next, 0.022 g of compound (L) (1mmol) was dissolved in 10 mL of deionized water/DMSO in the ratio 8:2 and added to the abovementioned aqueous H-CD solution. The mixture was poured into a round bottom flask and kept for reflux condition and continuously stirred for 24 h at 40°C. The obtained complex was dried at 2°C for 48 h. Subsequently, the obtained compound was filtered through a Whatman filter paper. Then the obtained IC was washed many times with double distilled water to expel unreacted compounds. Then the obtained IC was dried in the oven for 48 hrs at 50°C and used for future studies.

2.6. Spectrofluorometric determination of Hg²⁺ ion in the IC

The stock solution of the IC (1 ×10^− 3 M) and a suitable aliquot of mercury metal ion Hg²⁺ (1 ×10^− 8 M) were dissolved by using deionized water and it was transferred into 10ml standard volumetric flask and diluted to the mark. The solutions were mixed thoroughly and the photoluminescence (PL) intensity was measured at 308 nm on exciting the sample at 291 nm.

2.7. Cell Incubation

L929 cells is an established and well-characterized cancer cell line that has demonstrated high reproducible results. L929 were cultured in modified Eagle’s medium (MEM) supplemented with fetal bovine serum at 37°C consisting 5% CO₂ in an air incubator. L929 cell lines were cultured on a 96-well plated culture medium with a concentration of 1 ×10⁵ cell per well. The cultured cells were further treated with H-CD: L complex (0, 25, 50, 75, 100 µg concentrations in PBS buffer) for 3–4 hours with MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. After the incubation, the cells were washed with PBS buffer 3–5 times, and DMSO was added to solvate the formazan crystals to confirm that the non-availability of the H-CD: L in the additional region. The H-CD: L treated cells were further treated with Hg²⁺ ions in various concentrations (control and 50 µM) for 10 minutes and the obtained cells were subjected to fluorescence imaging. The confocal microscope images were observed at an excitation wavelength of 570 nm and the images were captured using a fluorescence microscope.

3.1. Solvatochromic studies for the compound L

3.1.1. Influence of various organic solvents on PL excitation and emission spectra

The absorbance and the PL spectra were obtained in different solvents of varying polarity (Fig. 1a, b). Table 1 gives the spectroscopic data of compound L in various solvents.

Table-1 Spectroscopic data of compound L in various solvents.

Organic Solvents	l_a (nm)	l_f (nm)	ῡ_a (cm^− 1)	ῡ_f (cm^− 1)	Δῡ(cm^− 1)	(ῡ_a+ῡ_f)/2
Toluene	314	630	31847	15873	15974	23860
1,4 Dioxane	315	633	31746	15797	15998	23771
Ethyl acetate	316	644	31645	15527	16117	23586
DCM	319	654	31446	15290	16156	23368
DMSO	320	660	31350	15151	16198	23250
Acetonitrile	321	668	31152	14970	16182	23061
Butanol	326	673	31055	14858	16298	22957
Propanol	328	674	30487	14836	15651	22662
Ethanol	330	676	30303	14792	15510	22547
Methanol	334	678	29940	14749	15190	2234

Maximum UV-visible absorption and PL wavelength of compound L were identified in the range 314–334 and 630–678 nm respectively in the selected solvents. On enhancing solvent polarity considerable redshifts was observed. A significant red shift ranging upto 48 nm was observed in the PL emission spectra of compound L. This is an indication to affirm the influence of solvent polarity on the energy of the singlet excited state. On increasing the solvent polarity Stoke’s shift values enhanced from 15974 cm^− 1 to 16298 cm^− 1 for n-hexane to butanol respectively. This remarkable bathochromic redshift reveals that in polar solvents, the molecule acquires higher stabilty in a singlet excited state than in the ground state.

Solvent functions F_1, F_2, F₃ are employed in the Bakhshiev [25], Lippert-Mataga [26], and Kawski- Chamma- Viallet [27] equations respectively.

Table.2 Values obtained for solvent polarity functions through diverse correlation methods.

Solvents	F₁ (ԑ,n)^a	F₂ (ԑ,n)^b	F₃ (ԑ,n)^c	ԑ^d	n^e
Toluene	0.01531	0.03339	0.34568	2.37	1.487
1,4 Dioxane	0.02150	0.04361	0.30848	2.22	1.422
Ethyl acetate	0.20072	0.49254	0.54878	6.08	1.372
DCM	0.21835	0.59496	0.49958	9.08	1.424
DMSO	0.26398	0.84210	0.74329	47.24	1.477
Acetonitrile	0.31538	0.8600	0.666	38.50		1.354
Butanol	0.26856	0.745	0.6573	19.84		1.426
Propanol	0.27249	0.7712	0.67212	21.80		1.424
Ethanol	0.29976	0.8170	0.6545	27.33		1.37
Methanol	0.30870	0.856	0.65200	35.10		1.33
^aLippert-Mataga solvent function; ^bBakhshiev solvent function; ^cKawski-Chamma-Viallet solvent function;

ԑ = dielectric; ^en= refractive index.

3.1.2. Determination of the experimental and theoretical ground state and excited state dipole moments

Linear correlations for solvent polarity methods proposed by Bilot-Kawski, Lippert-Mataga, Bakhshiev, Kawski-Chamma-Viallet, and Reichardt (Fig. 2) were used to obtain the experimental value of singlet excited state dipole moment. Stoke’s shift (υ_A-υ_F) and its mean (υ_A + υ_F) / 2 were plotted against polarity functions F₁(ε,n), F₂(ε,n), F₃(ε,n).

Table.3 Statistical analysis of the correlations of various solvent spectral shifts of Compound L

Correlations	Compound	Slope	Correlation factor ‘r’	Number of data
Lippert-Mataga	L	869.11	0.9721	6
Bakhshiev	L	317.96	0.9636	7
Ground-State dipole moment µ_g = 3.53D

Excited-State dipole moment µ_e = 4.71D

The ratio between the excited state and the ground state dipole moment is (µ_e/µ_g) = 1.33D

Calculated excited-state dipole moment (µ_e) from Lippert-Mataga equation = 5.06D

Calculated excited-state dipole moment (µ_e) from Bakhshiev’s equation = 4.45D

Calculated excited-state dipole moment (µ_e) from Kawski-Chamma-Viallet’s equation = 4.67D

3.2. Interaction of H-CD with L in the aqueous state

3.2.1. UV-visible absorption spectral analysis of compound L in H-CD

Development of a supramolecular IC with compound L and hydroxypropyl beta-cyclodextrin has been identified and analysed by using a UV-Visible absorption spectrum. The inclusion of guest molecule (compound L) into host molecule (H-CD), which reduce the number of water molecules. The complex behaviour depends on the particular structural (shape fit, charge fit, size fit) features of guest (compound L) and host molecules. The absorbance of compound L increases while adding different concentrations of H-CD (0-0.012M) (Fig. 3a). In general, for different concentrations of H-CD, the absorbance range increases in the UV spectrum due to improved dissolution and encapsulation of compound L into H-CD non-polar cavity via hydrophobic interaction. Then the equilibrium equation for solid IC formed between guest and host molecule (H-CD: L) can be written as,

H-CD + L ⇌ H-CD…… L (3)

(4)

By employing the Benesi–Hildebrand [25] equation for the inclusion host-guest complex (H- CD: L) determines the binding constant ‘K’ and stoichiometric ratios, where, A is the absorbance of compound L in the presence of H-CD and A₀ is the absorbance of compound L in the absence of H-CD, molar absorption coefficient (Δ_ε), [L] and [H-CD] are the initial concentration of compound L and H-CD respectively. Figure 3 (b, c) shows the plot of 1/A-A₀ vs 1/ [ H-CD]² for compound L, a better linear range observed confirms the successful formation of the solid IC (H-CD). The binding constant ‘K’ was found to be 0.03 M^− 1.

3.2.2. PL spectral properties of compound L in H-CD medium

The interaction of H-CD and compound L was analysed using PL spectroscopy. In the emission spectra of compound L (Fig. 4a), the photoluminescence intensity increases gradually with the variation in H-CD concentration from 0 to 12x10^− 3 M. The obtained results show that compound L is encapsulated into the H-CD hydrophobic cavity to form H-CD: L solid IC. The fluorescence emission spectrum was found to be at 309 nm. The binding constant ‘K’ for the solid IC was calculated with the difference in maximum emission intensity by varying the H-CD concentration using the Benesi- Hildebrand equation [28] (Fig. 4b, c).

(5)

where [H-CD]₀² represents the H-CD concentration, I and I₀ were the PL intensities of compound L in the absence and presence of H-CD respectively, I\(\mathbf{\text{'}}\) is the limiting intensity. The binding constant ‘K’ found to be 0.0335 M^− 1.

3.3. Characterisation of compound (L) with H-CD in solid-state

3.3.1. FT-IR spectral analysis

FT-IR spectral analysis was employed to study the hydrophobic host-guest complex interactions between H-CD and compound L. The spectrum of hydroxy-propyl-beta cyclodextrin, compound L, and IC (H-CD: L) is depicted in Fig. 5. The characteristic absorption frequency range for H-CD was observed at 3345 cm^− 1 (O-H stretching band), 2897 cm^− 1(C-H stretching band), 1640 cm^− 1 (bending O-H band), 1418 cm^− 1 (deformation O-H), 1151 cm^− 1 (C-O-C stretching band), 1014 cm^− 1(C-C-O stretching band), 947 cm^− 1 (α-1,4 linkage skeletal band), 854 cm^− 1 (C-O, C-C, C-C-H from anomeric band) [29].

4-Acetylphenyl4-(((6-chlorobenzo[d]thiazol-2-yl)-imino)-methyl)-benzoate IR frequency range was observed at 3405 cm^− 1(stretching N-H band), 3090 cm^− 1 (aromatic C-H stretching band), 2933 cm^− 1(asymmetric C-H stretching band), 1721cm^− 1(stretching O-C = O vibration),1690 cm^− 1(C = N stretching band),1679 cm^− 1(stretching C = O band), 1266 cm^− 1 (stretching C-N band), 690 cm^− 1 (stretching C-S band) and bending band of ν(C-H) at 1374 cm^− 1, and halo compound ν(C-Cl) observed at 821cm^− 1 respectively. The range of ν(OH), ν(CH₂) in H-CD 3345 cm^− 1, 2897.23 cm^− 1 was shifted to 3302 cm^− 1, 2919 cm^− 1 in the solid IC. Bands at 1721cm^− 1, 1690 cm^− 1,1679 cm^− 1, 1266 cm^− 1, 821cm^− 1, 690 cm^− 1 which corresponds to stretching vibration of ν(O-C = O), ν(C = N), ν(C = O), ν(C-N), ν(C-Cl), ν(C-S), and bending vibration of ν(C-H) at 1374 cm^− 1 of 4-acetylphenyl benzoate appeared in the IC. This result proves that the developed solid IC was confirmed.

3.3.2 Raman spectral analysis

Raman spectroscopy is employed to describe the width and intensity of the molecular structures [30]. The Raman spectrum of compound L, H-CD, and H-CD: L complex were characterized in Fig. 6 and the assignments of peaks are tabulated in Table 4.

The vibrations of ν (C = C), δ (C-C), δ (C-H), and ν(C-N) appeared at 1614cm^− 1, 1582.66 cm^− 1, 1470 cm^− 1, and 1285 cm^− 1 respectively. The ring deformation of ν (C-C) and C-NH out of plane bending peaks appear at 1334 cm^− 1 and 236 cm^− 1 respectively Raman spectrum of hydroxypropyl beta-cyclodextrin, ν(O-H) vibration was observed at 3323 cm^− 1 because of the high number of intramolecular and intermolecular hydrogen bonds. The ν (C-H) vibration of CH₂ showed at 2935 cm^− 1 and the vibration of δ (O-H) appeared at 1393 cm^− 1. The δ(C-H) vibrations appeared at 1468, 1340, 1263 cm^− 1. The vibration of ν (C-O) were found to be 1051, 1042 cm^− 1. The deformation in-plane glucose ring was identified at 761 and 489 cm^− 1 respectively.

The H-CD ν (O-H) vibration 3323 cm^− 1 was shifted to a lower wavenumber 3266 cm^− 1 in the solid IC. Similarly, the ν (C = C) vibration and the C-NH out of plane bending vibration in compound L shifts also shifts to a lower wavenumber. The obtained results clearly explained that the compound L is incorporated in the cavity of H-CD.

Table.4: FT Raman band assignments of compound L, H-CD, and the solid IC (H-CD: L)

Compound L (cm^− 1)	Complex (cm^− 1)	Assigned peak	H-CD (cm^− 1)	Complex (cm^− 1)	Assigned peak
1614	1605	ν (C = C)	3323	3266	ν (O-H)
1582	1582	ν (C = C)	2935	2934	ν (C-H)
1472	1472	ν (C-C); δ (C-C)	1468	1418	δ (C-H)
1334	1333	ν (C-C); Ring deformation	1393	1363	δ (O-H)
1285	1285	ν (C-H)	1340	1301	δ (C-H)
1246	1246	ν (C-C); δ (C-H)	1263	1238	δ (C-H)
1144	1144	δ (C-H); ν (C-C)	1134	1125	ν (C-C)
1020	1020	ν (C-C)	1051	1046	ν (C-O)
236	195	C-NH out of plane bending	942	941	ν (C-O)
			855	855	Breath of glucose ring
			761	762	In-plane deformation
			580	580	ν (O-H)
			489	480	In plane deformation of glucose ring.

3.2.3 Nuclear Magnetic Resonance Spectral Analysis (¹H NMR)

¹H NMR is one of the significant methods used to decide the sub-atomic interaction among host and guest particles [31, 32]. Essentially, ¹H NMR results depends on the perception of specific line enlargement or chemical shift (Δδ) relocation of guest (compound L) and host (H-CD) protons. Indeed, that critical compound shift happens just for hydrogens (H) present on the inward surface protons (H3 and H5) of the H-CD, and not exactly for the external environment protons (H1, H2, H4, and methylene H6), which allows one to build up clearly, that these progressions are the product of solid IC (H-CD: L) development and not the non-specific interaction among the two molecules [33, 34]. ¹H NMR spectroscopy has been utilized for quite a long time to acquire cyclodextrin IC binding mode data, and as of recently, it is one of the major strategies for solid IC study [35, 36, 37].

As for the compound L, it couldn't be disintegrated in deionized water by any means. Hence, a few scientists, Chirag et al chose a solvent DMSO for ¹H NMR spectra when they met the comparative issue [38, 39]. The ¹H NMR spectra of guest molecule, host molecule and IC (H-CD: L) are shown in Fig. 7. The chemical shifts of H-CD free protons and H-CD: L complex are shown in Table 5, the chemical shifts were measured by using the following equations:

Δδ = Δδ (IC) – Δδ (H-CD)

Δδ = Δδ (IC) – Δδ (L) (6)

In this arrangement, positive(+ ve) and negative(-ve) chemical shifts show upfield and downfield shifts, respectively. Table 5 shows that the protons H3 and H5, placed inside the hydrophobic environment of H-CD, obviously move towards the upfield direction, related with the minor upfield shifts of protons H2 and H4, which are present in exterior of the cavity. This confirms that the encapsulation between the compound L and the interior environment of the H-CD cavity.

Table 5

Change in ¹H NMR chemical shift values of compound L and H-CD in free and IC states (H-CD: L) determined in DMSO-d6 at 298 K
Substance	H-Protons	Free (δ, ppm)	IC (δ, ppm)	Δδ(ppm)
H-CD	1 2 3 4 5 6	5.16 3.767 3.925 3.400 3.626 3.50	5.09 3.736 3.856 3.35 3.520 3.47	-0.07 -0.031 -0.069 -0.05 -0.106 -0.03
Compound L	a b c d e f g h i	7.55 7.74–7.73 8.12–8.06 8.35–8.32 7.83–7.75 8.27–8.24 7.50–7.49 7.97–7.95 2.46	7.45 7.65 8.04 8.24 7.65 8.20 7.47 7.93 2.40	-0.1 -0.09 -0.08 -0.11 -0.18 -0.04 -0.03 -0.04 -0.06

3.2.4 X-ray diffraction (XRD)

X-ray diffractometry (XRD) is used to study the physical properties (i.e., crystalline or amorphous) of the host-guest solid IC molecules. H-CD, compound L, and H-CD: L (solid IC), XRD patterns were depicted in Fig. 8. The XRD patterns of H-CD, showed no obvious characteristic diffraction peaks. It exhibits one broad diffraction peak band around 20°, indicating that the H-CD is in an amorphous nature [40]. The compound L exhibits sharp peaks indicating the crystalline nature of the guest molecule. The sharp peaks in compound L and broad diffraction peaks in host molecule H-CD are shown in H-CD: L (IC). This confirms that the compound L is encapsulated in the H-CD cavity and as well as it suggests that the solid IC H-CD: L is amorphous.

3.2.5 Scanning electron microscope (SEM)

Scanning electron microscope (SEM) is one of the qualitative methods, used to analyse the morphological features of the host (H-CD), guest molecules (compound L) and their solid IC (H-CD: L). The morphological structure of H-CD, compound L and solid IC (H-CD: L) are shown in Fig. 9. Typical amorphous structures of H-CD and crystal structure of compound L are originated in various sizes. The solid IC (H-CD: L) is found to be an amorphous moiety and homogeneous in nature. Hence, the differences in shape and size of H-CD: L compared to the parent compound, confirms the development of the H-CD: L solid IC.

3.4 Characterisation of compound (L) with H-CD in the theoretical state

Molecular docking technique

Binding cavity of the H-CD was docked into compound L. Then the obtained results were represented in Fig. 10. The 3D diagram of compound L and H-CD were individually exposed in Fig. 10(a, b). The calculated binding energy between the compound L and H-CD was found to be -5.9 kcal mol^− 1. The compound L accepted a solid conformation and it was wrapped by H-CD. This encapsulation helped compound L to anchor to the binding site of H-CD. Docking studies were performed using Auto dock Vina software and PyMol software was used for visualization. The above molecular simulations provided a rational explanation of the interactions between the compound L and H-CD. Further, provided that reasonable data about the mode of action of the H-CD: L solid IC.

3.5. Analytical application of H-CD: L probe as a fluorescence sensor for Hg²⁺ detection

3.5.1. Ability of metal sensing towards IC

During the formation of IC, the PL techniques showed a continual enhancement in the intensity of H-CD: L complex which acts as an important factor for chemosensory applications. Hence, to estimate the IC chemical sensing ability, the PL emission spectra of IC were recorded using different heavy metal ions to identify the metal sensing behaviour of the IC. Figure 11 (a, b) displays that the PL emission spectra of IC in the metal free (absence) and the presence of various metal ions such as Ag⁺, CO²⁺, Cr³⁺, Cs⁺, Hg²⁺, Ba²⁺, Mg²⁺, Fe²⁺, Fe³⁺, K⁺, Na⁺. It was observed that on addition of heavy metal cations to IC (H-CD: L) produced no noteworthy change, whereas on addition of Hg²⁺ showed an enhanced PL intensity.

3.5.2. Selectivity study for the IC towards Hg²⁺

PL spectra of H-CD: L IC + heavy metal ions (as Ag⁺, CO²⁺, Cr³⁺, Cs⁺, Hg²⁺, Ba²⁺, Mg²⁺, Fe²⁺, Fe³⁺, K⁺, Na⁺) were analysed. Figure 11c shows that the PL intensity increased on the addition of Hg²⁺ on the fluorescent probe (H-CD: L complex) and not influenced by the presence of other heavy metal ions. This result shows that the IC probe has a very high selective on Hg²⁺. Later, the H-CD: L IC used as a chemosensor for Hg²⁺ sensing in the aqueous medium. PL emission spectra of IC with various concentrations of Hg²⁺ were analysed to obtain the sensitivity of fluorescent probe. Figure 11d displays the PL spectrum of various concentrations of an analyte. The emission intensity was found to be 317 nm on the addition of the analyte. The limit of quantitation was calculated using (10 σ)/s and was found to be 8.13×10^− 10 M. The linear dynamic range of H-CD: L complex + analyte is 1×10^− 8 M − 11×10^− 8 M and the detection of limit was found to be 2.71×10^− 10 M (Fig. 11e). The plausible mechanism for the PL enhancement of IC fluorescent probe with Hg²⁺ ions is shown in Scheme 2.

Table 6

Proposed IC method compared with reported methods.
Reported Methods	Reagents	Linear range	LOD	Ref.
Voltammetric	2-Mercapto benzothiazole (2-MBT)	5×10^− 8 − 8×10^− 7 M	1×10^− 8 M	[41]
Spectrophotometry	Meloxicam	1.0–15.0 mM	0.296 mM	[42]
Spectrophotometry	Azocarmine G	0.3–50.0 nM	0.125 nM	[43]
Spectro fluorimetry	β-CD: 2H1NA complex	1×10^− 4- 2×10^− 4 M	2.4×10^− 5 M	[30]
Spectro fluorimetry	H-CD: L	1×10^− 8-11×10^− 8M	2.71 ×10^− 10 M	This work

3.6. Detection of Hg²⁺ in live cells

Biosensing of target molecules to selectively observe guest species in living cells are of excessive significance for biological applications [44]. The cell viability and cytotoxicity assay were investigated in L929 cells by using MTT assay as shown in Fig. 12a. The cell viability of H-CD: L showed greater than 90% upon addition of H-CD: L with different concentrations ranging from 0-100 µg/ml (24hrs). The results confirmed that H-CD: L is non-toxic and bio-compatible. The synthesized H-CD: L and H-CD: L + analyte act as an excellent biological probe which was confirmed by fluorescence stability and cytotoxicity. Figure 12b explains the fluorescence image of H-CD: L using L929 cells is considered as a further application. The fluorescence images of L929 cells were incubated with H-CD: L and H-CD: L + analyte. The fluorescence images were obtained by using the confocal microscope.

3.7 Real water sample analysis

The proposed chemosensor H-CD: L was used for the detection of Hg²⁺ in drinking water, tap water and lake water samples. Different waters were investigated without any pre-treatment. All the water samples tests were spiked with H-CD: L IC at fixation level and investigated. The outcomes are displayed in Table 7. The recovery range of Hg²⁺ was found to be 97–101%. The developed fluorescent probe has been successfully applied for the detection of Hg²⁺ in real examples.

Table.7. Real water sample analysis

Sample

Spiked concentration (nM)

Proposed method

(nM)

RSD (%)

AAS method

(nM)

RSD (%)

Tap water

0.0

2.5

5.0

30.10

32.6

35.1

1.00

1.71

0.87

30.26

32.76

35.26

0.80

1.78

0.95

Wastewater

0.0

2.5

5.0

18.18

20.68

23.18

0.66

1.50

0.78

18.15

20.65

23.15

0.76

1.38

0.85

River water

0.0

2.5

5.0

22.12

24.62

27.12

1.20

0.72

1.01

21.18

23.68

26.18

1.21

0.88

1.05

The formation of solid IC between 2-hydroxypropyl beta-cyclodextrin and compound L was identified through different states (i.e., solid, liquid and virtual state). The binding constants ‘K’ of the synthesized H-CD: L IC was investigated through UV-visible and PL spectral analysis. The solid IC was identified and confirmed by using XRD, Raman, SEM, NMR techniques. Molecular docking studies were performed to prove the successful formation of the IC theoretically. The sensing behaviour of the H-CD: L IC towards Hg²⁺ was identified using PL spectroscopy. The estimated LOD was found to be 2.71×10^− 10 M. The intracellular uptake of Hg²⁺ by H-CD: L IC was tested in the L929 cancer cell line which proved the complex to be non-toxic and biocompatible.

Acknowledgment

The researchers and authors express their gratitude to the Major research project (MRP DSC-1830), Center for Research, CHRIST (Deemed to be University), Bangalore, Karnataka, India for providing the required financial support for completing this research work.

Author Declarations

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Competing Interests

The authors have no competing interests with the work presented in this manuscript.

Ethics approval/declarations-Not applicable

Consent to participate-Not applicable

Consent for publication-Not applicable

Code availability-Not applicable

Availability of data and material/ Data availability- Not applicable

Authors' contributions Keerthana P: Conceptualization, Methodology, Hanna Abbo: Methodology, Anila Rose Cherian: Conceptualization, Methodology, Salam Titinchi: Writing- Reviewing and Editing, Anitha Varghese: Writing- Reviewing and Editing

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Schemes 1 & 2 are available in the Supplementary Files section.

Scheme1.png
Scheme 1. Schematic representation of the inclusion complex between Hydroxypropyl betacyclodextrin and Compound L.
Scheme2.png
Scheme 2. The plausible mechanism for the PL enhancement of probe H-CD: L with Hg²⁺.

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published 19 Mar, 2022

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Editorial decision: Accept for Publication
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An efficient inclusion complex based fluorescent sensor for mercury (II) and its application in live-cell imaging

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Experimental Section

2.1. Reagents

2.2 Apparatus

2.3. Synthesis of 4-Acetylphenyl 4-(((6-chlorobenzo[d]thiazol-2-yl) imino) methyl) benzoate

2.4. Preparation of IC (H-CD: L) in an aqueous medium.

2.5. Preparation of the solid IC (H-CD: L)

2.6. Spectrofluorometric determination of Hg2+ ion in the IC

2.7. Cell Incubation

3. Results And Discussion

3.1. Solvatochromic studies for the compound L

3.1.1. Influence of various organic solvents on PL excitation and emission spectra

3.1.2. Determination of the experimental and theoretical ground state and excited state dipole moments

3.2. Interaction of H-CD with L in the aqueous state

3.2.1. UV-visible absorption spectral analysis of compound L in H-CD

3.2.2. PL spectral properties of compound L in H-CD medium

3.3. Characterisation of compound (L) with H-CD in solid-state

3.3.1. FT-IR spectral analysis

3.3.2 Raman spectral analysis

3.2.3 Nuclear Magnetic Resonance Spectral Analysis (1H NMR)

3.2.4 X-ray diffraction (XRD)

3.2.5 Scanning electron microscope (SEM)

3.4 Characterisation of compound (L) with H-CD in the theoretical state

3.5. Analytical application of H-CD: L probe as a fluorescence sensor for Hg2+ detection

3.5.1. Ability of metal sensing towards IC

3.5.2. Selectivity study for the IC towards Hg2+

3.6. Detection of Hg2+ in live cells

3.7 Real water sample analysis

4. Conclusion

Declarations

References

Scheme

Supplementary Files

Status:

Journal Publication

Version 1

2.6. Spectrofluorometric determination of Hg²⁺ ion in the IC

3.2.3 Nuclear Magnetic Resonance Spectral Analysis (¹H NMR)

3.5. Analytical application of H-CD: L probe as a fluorescence sensor for Hg²⁺ detection

3.5.2. Selectivity study for the IC towards Hg²⁺

3.6. Detection of Hg²⁺ in live cells