A facile synthesis of a novel 4-hydroxyl-3-azo coumarin based colorimetric probes for detecting Hg²⁺ and a fluorescence turn-off response of 3CBD to Fe³⁺ in aqueous environment

Abstract

Two azo dyes, (E)-3-(benzo[d]thiazol-2-yldiazenyl)-4-hydroxy-2H-chromen-2-one (3CBD) and (E)-4-hydroxy-3-(quinolin-2-yldiazenyl)-2H-chromen-2-one (3CQD), were designed and synthesized using facile methods. The structures were validated through FTIR and NMR spectroscopy. The photophysical property analyses were further studied using UV-Vis and fluorescence spectrophotometers. Consequently, the absorption and emission spectra of 3CBD confirmed its selectivity of Hg²⁺ and turn-off response to Fe³⁺. On the other hand, the absorption spectra analysis of 3CQD demonstrated selectivity in the presence of Hg²⁺. The colorimetric investigations demonstrated a significant visual response specifically for Hg²⁺, enabling real-time analysis in the corresponding solutions. The presence of other coexisting metal ions does not interfere with the detection of the target metal ion. The fluorescence studies of the two probes revealed that 3CBD was highly fluorescent, which was significantly quenched by Fe³⁺, upon excitation at 340 nm. Utilizing Job plot analyses, it was determined that the complexes 3CBD–Hg²⁺ and 3CQD–Hg²⁺ exhibit a binding stoichiometry of 1 : 1. The association constants for these complexes were measured to be 7.48 × 10⁵ and 9.12 × 10⁵ M⁻¹, respectively, indicating a strong association between both probes and their respective metal ions. Both chemosensors exhibited comparable limits of detection (LOD) and limits of quantification (LOQ) of 0.03 μM and 0.10 μM, respectively. Reversible studies confirmed that only chemosensor 3CQD could serve as a secondary sensor for EDTA. The theoretical studies calculated using Density Functional Theory (DFT) program at B3LYP/6-31G** (Spartan '10 package) level.

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1. Introduction

Chemosensing techniques have been gaining momentum in the last few decades in the field of analytical chemistry, due to their simplicity and practicality in applications.^1–4 The increase in human population density over past decades, triggered the technological advancement in many fields of science, to respond to basic needs essential for survival.^5,6 Consequently, these technological advancements come at the cost of environmental pollution, which leads to new diseases and many other implications in the ecosystem.^6–10 Industrial revolutions have been at the center of environmental pollution, especially in the areas such as agriculture for synthetic fertilizers^9,11,12 and pesticides,^8,13,14 food technology,^15–17 film industries^9,18–20 and many others.^6,9,21,22 In essence, the ever-increasing presence of dissolved cations and anions in environmental streams, has become the main source of primary consumption of toxic substances. The presence of dissolved substances in environmental stream have become a serious concern with regard to health hazards, raising concerns even on United Nation agendas, the sustainable development goals.²³ Heavy metals in particular, have been the primary concerns, due to their adverse effects on the ecosystem, mostly on human wellbeing, once their concentration is above threshold.^21,24,25 Moreover, heavy metals such as dissolved elemental mercury are some of the most toxic cations to human physiological systems, with effects which can lead to sudden deaths.^26,27

Furthermore, mercury(II) ion, a heavy metal, poses significant environmental toxicity,^28,29 even at low concentrations, mercury(II) can cause severe harm to both animals and the human body, usually leading to damage to vital organs like the brain and the potential development of chronic diseases, ultimately resulting in death. Mercury(II) enters the environment through various means, including municipal and industrial wastes, mining activities, battery usage, and fuel combustion.³⁰ Given the evident threat mercury poses to the environment, its ongoing monitoring, control, and detection remain crucial areas of focus.^4,28 Based on these considerations, the development of highly sensitive and selective colorimetric sensors for mercury(II) has gained significant attention. These sensors offer advantages such as simplicity, cost-effectiveness, and ease of operation compared to conventional elemental analytical techniques like Atomic Absorption Spectroscopy (AAS) and Inductive Coupled Plasma-Mass Spectroscopy (ICP-MS).^31–33

Notably, chemosensing probes that are capable of selectively discriminating target analytes in aqueous solution are highly on demand. To date, a few aqueous based sensing probes have been reported in literature, however, most are haunted by cross-detection problem.^34–37 Thus, the detection of heavy metal cations in aqueous environments are still high on the priority list, due to their prospective of developing field-based sensing kits,^14,38–41 which can be readily used anywhere, including rural areas. In addition, aqueous solution-based sensing probes can be essential in physiological systems, for monitoring the presence and concentration of specific cations of concern, and be able to regulate them. The mode of cation detection is largely based on the design of the molecular framework, normally via several interaction mechanisms,^8,42–45 which in many cases characterized by functional groups^46–50 and coordination interaction.^51–54

Commonly used binding units in colorimetric chemosensors include bipyridines, Schiff bases, guanidine, rhodamine dyes, and azo dyes.^55–57 Among these, azo dyes have gained prominence due to their straightforward synthesis, distinctive photophysical properties, and strong affinity for various heavy metal ions.^58–60 In this study, we present the synthesis of two novel coumarin-based azo probes, namely (E)-3-(benzo[d]thiazol-2-yldiazenyl)-4-hydroxy-2H-chromen-2-one (3CBD) which exhibits selectivity for Hg²⁺ under UV-Vis and Fe³⁺ in fluorescence, and (E)-4-hydroxy-3-(quinolin-2-yldiazenyl)-2H-chromen-2-one (3CQD) which specifically detects Hg²⁺ in UV-Vis, both in aqueous solutions.

2. Experiment

2.1 Materials

All reagents and solvents employed for the synthesis of the chemosensors were procured from Sigma Aldrich and Merck and utilized without further purification. Metal solutions of 0.01 M were prepared from nitrates salts (Ag⁺, Na⁺, Al³⁺, Ca²⁺, Ba²⁺, Fe²⁺, Fe³⁺, Cr³⁺, Hg²⁺, Cu²⁺, Co²⁺, Cd²⁺, Zn²⁺, Li⁺, Pb²⁺ and Ni²⁺). UV-Vis and emission spectroscopy measurements were carried out using a PerkinElmer Lambda 35 UV-Vis spectrometer and a PerkinElmer LS 45 fluorescent spectrometer. The spectroscopic analyses were performed in a standard 3.0 ml quartz cuvette with a path length of 1 cm. Fourier Transform Infrared (FT-IR) data was collected using a PerkinElmer FT-IR 180 spectrometer, employing KBr discs. Proton Nuclear Magnetic Resonance (¹H NMR) and Carbon-13 Nuclear Magnetic Resonance (¹³C NMR) analyses were conducted on a Bruker Advance DPX 400 Spectrometer operating at 400 MHz. The NMR experiments were carried out in CDCl₃ and d₆-DMSO solvents at room temperature, with tetramethyl silane (TMS) serving as the internal reference.

2.2 Synthesis and characterization 3BD and 3QD

The chemosensors 3CBD and 3CQD were synthesized in a one-step reaction according to Scheme 1. The procedure involved the dropwise addition of their respective diazonium salt solutions (0.01 mol) to a coupling 4-hydroxy coumarin solution (0.01 mol) in a methanol/water mixture (30 ml) under ice conditions for 0.5 h with continuous stirring. The products were obtained after the addition of concentrated ammonia solution,^61–64 filtered, and recrystallized in ethanol to yield a brown 3CBD precipitate and an orange 3CQD precipitate. The orange solid of 3CBD was obtained in a yield of 92%. Its FT-IR ν_max (cm⁻¹) was found to be 3393 (O–H), 3302 (≡C–H), 1683 (C=O), and 1601 (C=C) (Fig. S4†). The ¹H NMR (CDCl3, 400 MHz) showed peaks at δ = 15.53 (s, OH), 9.11 (d, J = 8.46 Hz, 1H), 8.29 (d, J = 8.84, 1H), 8.24 (d, 1H), 7.84 (d, 1H), 7.73 (d, 1H), 7.69 (d, 1H), 7.38 (d, 1H), and 7.32 (d, 1H) (Fig. S1 and S3†). The brown solid of 3CQD was obtained in a yield of 92%. Its FT-IR ν_max (cm⁻¹) was found to be 3393 (O–H), 3302 (≡C–H), 1683 (C=O), and 1601 (C=C) (Fig. S5†). The ¹H NMR (CDCl₃, 400 MHz) showed peaks at δ = 15.53 (s, OH), 9.11 (d, J = 8.46 Hz, 1H), 8.29 (d, J = 8.84, 1H), 8.24 (d, 1H), 7.84 (d, 1H), 7.73 (d, 1H), 7.69 (d, 1H), 7.38 (d, 1H), and 7.32 (d, 1H) (Fig. S1 and S2†).

Scheme 1 Reaction scheme of 3CQD and 3CBD.

3. Results and discussions

3.1 Effect of metal ions on photophysical properties of 3CBD and 3CQD

3.1.1. UV vis absorption assay: metal screening/selectivity. The binding affinity of both 3CQD and 3CBD towards various cations, including Ag⁺, Na⁺, Al³⁺, Ca²⁺, Ba²⁺, Fe²⁺, Fe³⁺, Cr³⁺, Hg²⁺, Cu²⁺, Co²⁺, Cd²⁺, Zn²⁺, Li⁺, Pb²⁺, and Ni²⁺, was investigated using UV-Vis spectroscopy. In its unbound state, 3CQD exhibited an absorption band at 450 nm in an aqueous ethanol solution, and significant spectral changes were observed upon the addition of 0.5 molar equivalents of Hg²⁺ and Co²⁺ (Fig. 1A). The incremental addition of Hg²⁺ to the 3CQD solution led to a decrease in the absorption band at 450 nm and the appearance of two peaks at 397 nm and 506 nm (Fig. 1B). This was accompanied by a noticeable colour change from yellow to pink. However, no spectral or colour changes were observed when aliquots of Co²⁺ were added to 3CQD (Fig. 2).

Fig. 1 (A) Absorption spectra changes of 3CQD (1.56 × 10⁻⁴ M) in the presence of 0.5 molar equivalence of different metal ions in water : ethanol (2 : 1); (B) absorption spectra of 3CQD (1.56 × 10⁻⁴ M) in 2 : 1 water : ethanol solution upon stepwise addition of Hg²⁺ 4 molar equivalences.

Fig. 2 Colour changes observed in solution of 3CQD (1.56 × 10⁻⁴ M) in the presence of 0.5 equivalence of different metal ions in 2 : 1 water : ethanol solution. Metal ion concentration 0.01 M.

In the case of 3CBD, its spectral pattern exhibited an absorption band at 464 nm in an aqueous acetonitrile solution. Upon the addition of 1 molar equivalence of Fe²⁺, Cu²⁺, and Hg²⁺, new absorbance peaks were observed at 464 nm, 468 nm, and 537 nm, respectively (Fig. 3A). The gradual addition of Hg²⁺ to the 3CBD solution led to the actual formation of the absorption band at 537 nm and the disappearance of the initially existing 3CBD peak at 430 nm (Fig. 3B). This was accompanied by a significant colour change from yellow to pink (Fig. 4). The selectivity of Hg²⁺ over the other competitive cations is ascribed to the chemistry of the cationic mercury with regard to its atomic size, the chemical environment influenced by the solvent used, in relation to the complementary receptors of the ligands, matching the binding/coordinating cavities, which is specific, sensitive and selective only to a particular cation, Hg²⁺ in this case.

Fig. 3 (A) Absorption spectra changes of 3CBD (1.58 × 10⁻⁴ M) in the presence of 1 molar equivalence of different metal ions in water: acetonitrile; (B) absorption spectra of 3CBD (1.58 × 10⁻⁴ M) in 1 : 1 (water : acetonitrile) upon stepwise addition of 4 molar equivalence of Hg²⁺.

Fig. 4 Colour changes observed in solution of 3CBD (1.58 × 10⁻⁴ M), in the presence of 1 molar equivalence of different metal ions in 1 : 1 water : acetonitrile. Metal ion concentration 0.01 M.

3.1.2. Competition studies of 3CBD and 3CQD. To evaluate the selectivity of 3CQD for Hg²⁺ over other interfering metal cations, competition experiments were conducted. A solution of 3CQD containing 0.5 molar equivalence of a competing ion was prepared, and then 0.5 molar equivalence of Hg²⁺ was added while monitoring the absorbance. The results, shown in the bar diagram (Fig. 5), indicate that none of the tested metal ions interfered with the detection of Hg²⁺.

Fig. 5 Absorption responses of 3CQD (1.56 × 10⁻⁴ M) upon addition of 0.5 molar equivalent of various metal ions (red bar) and upon addition of 0.5 molar equivalent of Hg²⁺ with 0.5 molar equivalent other metal ions (blue bars). The experiments were performed in 2 : 1 water : ethanol solution. Metal ion concentration 0.01 M.

Similar experiments were carried out to assess the selectivity of 3CBD for Fe²⁺, Cu²⁺, and Hg²⁺. Surprisingly, none of the competing metal ions, including Fe²⁺ and Cu²⁺, were able to compete with Hg²⁺ (Fig. 6). This suggests that even in the presence of Fe²⁺ or Cu²⁺, 3CBD consistently forms a complex with Hg²⁺, making it a highly selective chemosensor for Hg²⁺.

Fig. 6 Absorption responses of 3CBD (1.58 × 10⁻⁴ M) upon addition of 1 molar equivalent of various metal ions (red bar) and upon addition of 1 molar equivalent of Hg²⁺ with 1 molar equivalent other metal ions (blue bars). The experiments were performed in 1 : 1 water : acetonitrile solution. Metal ion stock solutions of 0.01 M.

3.1.3. Determining binding stoichiometry, association constant, and limit of detection. The binding stoichiometry and association constants (K_s) of the complexes 3CQD–Hg²⁺ and 3CBD–Hg²⁺ were determined using the Jobs plot with the continuous variation method⁶⁵ and the Benesi–Hildebrand equation. Absorbance was plotted against the molar fraction of Hg²⁺. The 3CQD–Hg²⁺ complex exhibited maximum absorbance at a molar fraction of 0.5, indicating a 1 : 1 stoichiometry of complex formation (Fig. 7A). To evaluate the association constants (K_s), the absorbance data were further analyzed. The 1/A_o − A values, where A is the absorption intensity in the presence of different concentrations of Hg²⁺ and A_o is the absorption intensity in the absence of Hg²⁺, were plotted against 1/Hg²⁺. The data were fitted linearly to the Benesi–Hildebrand equation, from which the association constants (K_a) were obtained using the slope and intercept of the line (Fig. 7B).

Fig. 7 (A) A Job's plot for 3CQD–Hg²⁺ binding in 2 : 1 water : ethanol with a constant total concentration of 1.56 × 10⁻⁴ M; (B) Benesie–Hildebrand plot of 1/(A_o − A) against 1/[Hg²⁺] to calculate the binding constant between 3CQD and Hg²⁺ and the limit of detection of 3CQD.

Similarly, the 3CBD–Hg²⁺ complex exhibited maximum absorbance at a molar fraction of 0.5, indicating a 1 : 1 stoichiometry of complex formation (Fig. 8A), and the association constant for Hg²⁺ binding with 3CBD was determined to be 7.43 × 10⁵ M⁻¹ (Fig. 8B). Furthermore, the limits of detection (LOD) and quantification (LOQ) for both probes in detecting Hg²⁺ were determined using the same method. Surprisingly, both probes exhibited similar LOD and LOQ values of 0.03 μM and 0.10 μM, respectively.⁶⁶

Fig. 8 (A) A Job's plot for 3CBD–Hg²⁺ binding in 1 : 1 water : acetonitrile solution with a constant total concentration of 1.58 × 10^{− 4} M; (B) Benesie–Hildebrand plot of 1/(A_o − A) against 1/[Hg²⁺] to calculate the binding constant between 3CBD and Hg²⁺ as well as the limit of detection of 3CBD.

3.1.4. Reversibility studies of 3CQB. The reversibility of the binding mechanisms is a crucial aspect for the practical application of the sensing system, as it enables reusability. Therefore, the reversibility of 3CQD was examined in the presence of ethylenediaminetetraacetic acid (EDTA), a versatile ligand. Upon the addition of an excess of EDTA to the solution of 3CQD–Hg²⁺ complex in ethanol: water, the absorbance of the complex reverted back to the original absorbance of 3CQD (Fig. 9). This observed reversal could be attributed to the dissociation of the carboxylic acid groups of EDTA ligand, which release protons to protonate the lone pair of electrons involved in Hg²⁺ binding.⁶⁷

Fig. 9 Reversibility studies of the binding process between 3CQD–Hg²⁺ upon addition of 0.5 molar equivalence of EDTA.

3.2 Fluorescence assay

In its unbound state, 3CBD exhibited a maximum emission at 410 nm (λ_ex = 340 nm). To demonstrate its potential as a fluorescent chemosensor, a fluorescence titration was performed by adding 3.5 molar equivalence of various cations (Ag⁺, Na⁺, Al³⁺, Ca²⁺, Ba²⁺, Fe²⁺, Fe³⁺, Cr³⁺, Hg²⁺, Cu²⁺, Co²⁺, Cd²⁺, Zn²⁺, Li⁺, Pb²⁺, and Ni²⁺) to a solution of 3CBD in ethanol: water at pH 7.0. Among the tested cations, only the addition of Fe³⁺ resulted in a decrease in fluorescence emission (Fig. 10). Interestingly, 3CQD exhibited low emission intensity in its ground state, indicating the absence of a highly conjugated system due to the delocalization of the lone pair of oxygen in the hydroxyl group attached to the coumarin moiety. None of the tested metal ions were able to activate the intramolecular charge transfer process (ICT) to restore conjugation, which would trigger a “turn-on” response in the emission spectra through chelation-enhanced fluorescence of the molecule.⁶⁸

Fig. 10 Emission spectra of 3CBD (1.58 × 10⁻⁴ M) in the presence of the aliquot (6.67 × 10⁻⁴ M) of different metal ions. The experiments were conducted in ethanol : water solution, excited at 340 nm.

3.2.1. Fluorescence titration of 3CBD with Fe³⁺. Fluorescence titration experiments were performed by progressively adding Fe³⁺ ions, which resulted in a gradual decrease in the emission intensity at 412 nm, as depicted in Fig. 11. Subsequently, the limit of detection, limit of quantification, and the association constants for the binding of Fe³⁺ with 3CBD were calculated using the Benesi–Hildebrand equation based on the obtained emission intensity results.

Fig. 11 Emission spectra of 3CBD (1.58 × 10⁻⁴ M) in the presence of the aliquot 60 molar equivalence of different metal ions.

3.2.2. Competition studies. The selectivity of 3CBD towards Fe³⁺ was assessed through competition experiments. In these experiments, 3.5 molar equivalents of various competing metal ions were added to the probe, followed by the addition of an equivalent amount of Fe³⁺, while monitoring the changes in emission. Among the tested cations (Na⁺, Al³⁺, Ca²⁺, Ba²⁺, Fe²⁺, Fe³⁺, Cr³⁺, Hg²⁺, Cu²⁺, Co²⁺, Cd²⁺, Zn²⁺, Li⁺, Pb²⁺, and Ni²⁺, only Ag⁺) demonstrated significant interference with the detection of Fe³⁺, as illustrated in Fig. 12.

Fig. 12 Fluorescence responses of 3CBD (1.58 × 10⁻⁴ M) upon addition of 127 molar equivalence of various metal ions (blue bar) and upon addition of Fe³⁺ with 127 molar equivalents of other metal ions (red bars) in water: acetonitrile solution. Metal ion stock solutions (0.01 M). Excitation of 340 nm.

3.3 Computation calculations

To gain further insights into the electronic transitions, the structures of the uncomplexed forms (3CBD and 3CQD) as well as the complexed forms (3CBD–Hg²⁺ and 3CQD–Hg²⁺) of the sensors were calculated in gas phase using molecular mechanics (MMFF) and semi empirical (PM3) methods with Spartan '10 molecular modelling software. The calculations revealed a significant decrease in the energy gap (HOMO–LUMO) from the uncomplexed to the complexed forms, as shown in Table 1. This decrease in the energy gap is consistent with the observed spectral changes of both 3CBD and 3CQD upon complexation with Hg²⁺, indicating a red shift.⁶⁹ The energy levels of both the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) decreased upon the addition of Hg²⁺ to 3CBD and 3CQD, which is indicative of stable orbital sets (LUMO and HOMO). The substantial reduction in the band gap between HOMO and LUMO of the chemosensors is attributed to the chemosensor-to-metal charge transfer between 3CBD and Hg²⁺, as well as 3CQD and Hg²⁺. These observations suggest that the complexation of both chemosensors with Hg²⁺ results in the formation of stable complexes.⁶⁷

Table 1 The summary of HOMO–LUMO orbitals and the energy gaps of 3CBD and 3CQD in the absence and presence of and Hg²⁺

Compound	C=O bond length (Å)	N=N bond length (Å)	C=O̲ (Mulliken charge) (electrostatic charge)	Coumarin–N̲=N (Mulliken charge) (electrostatic charge)	N=N̲–R (Mulliken charge) (electrostatic charge)	C=N̲ (Mulliken charge) (electrostatic charge)
3CBD	1.212	1.227	−0.340, −0.525	+0.058, +0.015	+0.004, −0.026	−0.080, −0.385
3CBD–Hg^2+	1.264	1.247	−0.365, −0.717	+0.118, −0.046	+0.084, +0.100	+0.081, −0.641
3CQD	1.209	1.232	−0.308, −0.501	+0.040, +0.370	+0.001, −0.295	−0.041, −0.632
3CQD–Hg^2+	1.242	1.250	−0.352, −0.827	+0.279, +0.285	+0.133, −0.142	+0.181, −0.660

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The chemical interaction between the chemosensors and Hg²⁺ induced structural distortion. For example, in the case of 3CBD, complexation with Hg²⁺ resulted in lengthening of the carbonyl and azide bonds by 0.052 Å and 0.020 Å, respectively. A similar trend was observed in 3CQD, with the carbonyl bond length increasing by 0.0033 Å and the azide bond length increasing by 0.018 Å. These changes indicate the interaction of these functional groups with Hg²⁺. The presence of electron-donating groups such as carbonyls in the complexation with cations affects the stability of the complex. This increased electron-accepting property of the system leads to a reduction in the band gap and consequently a bathochromic effect or a decrease in emission.

3.4 Proposed binding mechanisms

Molecular modelling was utilized to validate the complexation of 3CBD with Hg²⁺ (Fig. 13A) and 3CQD with Hg²⁺ (Fig. 13B). The presence of the azo functionality in both compounds creates a more electron-dense region that readily accommodates the dication. Upon complexation, it is evident that the rest of the molecule becomes electron-deficient as electrons are drawn towards the Hg²⁺ ion, particularly in the azo region. The hydroxyl group in 3CBD and the carbonyl group in 3CQD both contribute to the electron density in the complexation region, facilitating the accommodation of Hg²⁺ (Table 2).

Fig. 13 (A) 3CBD–Hg²⁺ complex; (B) 3CQD–Hg²⁺ both at PM3 level using Spartan '10 V1.10.

Table 2 Comparative study on the proposed method and the existing fluorogenic Hg²⁺ probes

Method	LOD	References
3CBD	0.10 μM	Current work
3CQD	0.10 μM	Current work
	9.45 × 10^−3 μM	7
	1.1 × 10^−1 μM	70
	—	71
	9.45 × 10^−3 μM	72
	4.89 μM	73
	—	74

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4. Conclusion

In conclusion, the synthesis and characterization of two highly stable and sensitive chemosensors, 3CBD and 3CQD, were successfully performed. The sensitivity of these chemosensors towards various cations was evaluated in ACN–H₂O and EtOH–H₂O solutions. Remarkably, both chemosensors exhibited a strong affinity for Hg²⁺ ions, which was confirmed through UV-Vis titration and visual observations of a distinct colour change from yellow to light pink. The binding stoichiometry of the chemosensors with Hg²⁺ was determined to be predominantly 1 : 1 using the job's plot method. The selectivity of the chemosensors relies primarily on charge–charge interactions and the involvement of electron-rich groups such as C=O and N=N in the binding process.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the Department of Chemistry, Faculty of Science, Nelson Mandela University, Port Elizabeth, South Africa and Department of Physics, Chemistry and Materials Science, Faculty of Agriculture, Engineering and Natural Sciences, University of Namibia, Windhoek, Namibia.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3ra04047j

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