Substituent effect on fluorescence signaling of the cell permeable HSO₄⁻ receptors through single point to ratiometric response in green solvent

Abstract

Two new 2-(2-aminophenyl)benzimidazole-based HSO₄⁻ ion selective receptors, 6-(4-nitrophenyl)-5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazoline (L₁H) and 6-(4-methoxyphenyl)-5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazoline (L₂H), and their 1 : 1 molecular complexes with HSO₄⁻ were prepared in a facile synthetic method and characterized by physicochemical and spectroscopic techniques along with the detailed structural analysis of L₁H by single crystal X-ray crystallography. Both receptors (L₁H and L₂H) behave as highly selective chemosensor for HSO₄⁻ ions at biological pH in ethanol–water HEPES buffer (1/5) (v/v) medium over other anions such as F⁻, CI⁻, Br⁻, I⁻, AcO⁻, H₂PO₄⁻, N₃⁻ and ClO₄⁻. Theoretical and experimental studies showed that the emission efficiency of the receptors (L₁H and L₂H) was tuned successfully through single point to ratiometric detection by employing the substituent effects. Using 3σ method the LOD for HSO₄⁻ ions were found to be 18.08 nM and 14.11 nM for L₁H and L₂H, respectively, within a very short responsive time (15–20 s) in 100 mM HEPES buffer (ethanol–water: 1/5, v/v). Comparison of the utility of the probes (L₁H and L₂H) as biomarkers for the detection of intracellular HSO₄⁻ ions concentrations under a fluorescence microscope has also been included and both probes showed no cytotoxic effect.

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Introduction

The design and development of selective receptors for the anionic analytes have gained considerable attention in recent years because of the biological significance, potential applications in sensors and for the development of phase transfer reagents.^1,2 Critical physiological processes are being operated through negative ion gradients across lipid bilayer membranes maintained by anion channels.³ The malfunction of this process leads to severe diseases such as cystic fibrosis, nephrolithiasis, osteopetrosis, Angelman syndrome and Bartter's syndrome type III.⁴ Among the various anions, hydrogen sulfate (HSO₄⁻) ions dissociate at high pH to generate toxic sulfate (SO₄²⁻), which causes irritation of the skin and eyes, and even respiratory paralysis.⁵ Despite its crucial roles in biological processes, only few examples of cell permeable sensors for HSO₄⁻ have been reported.⁶ Thus, the design of anion sensors for the hydrogen sulfate ion is important and desirable.

Sensors based on anion-induced changes in fluorescence are particularly attractive due to their simplicity, high degree of specificity and low detection limits.⁷ However, from the experimental point of view, it is well known that the ratiometric responses are more attractive because the ratio between the two emission intensities can be used to measure the analyte concentration and to provide a built-in correction for environmental effects and stability under illumination.⁸ Although there are some reports on single point sensor and ratiometric response, there is no report on the tuning of single point to ratiometric response while keeping the same receptor environment, except for the change in electronic effect, using substituents.

Herein, two newly designed efficient HSO₄⁻ ion-selective receptors, 6-(4-nitrophenyl)-5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazoline (L₁H) and 6-(4-methoxyphenyl)-5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazoline (L₂H) from 2-(2-aminophenyl)benzimidazol (viz. Scheme 1) while keeping same receptor environments for the guest (HSO₄⁻ ions) have been employed to tune the emission efficiency of these new receptors (L₁H and L₂H) through single point to ratiometric detection in a green solvent by exploiting the effects of the substituents within the receptors. The organic moieties (L₁H and L₂H) and the resulting compounds (K[L₁H–HSO₄] and K[L₂H–HSO₄]) have been characterized by physicochemical and spectroscopic techniques along with crystallographic analysis of L₁H by single crystal X-ray diffraction. Both L₁H and L₂H behave as highly selective fluorescent and colorimetric sensors for HSO₄⁻ ions at biological pH in ethanol–water HEPES buffer (1/5; v/v) compared to other anions such as F⁻, CI⁻, Br⁻, I⁻, AcO⁻, H₂PO₄⁻, N₃⁻ and ClO₄⁻. The receptor L₁H behaves as a single point fluorosensor, whereas the receptor L₂H behaves as a ratiometric fluorosensor in identical conditions. Both probes (L₁H and L₂H) were also employed to detect the presence of intracellular bisulphate ions by acquiring the images of HeLa cells under a fluorescence microscope. Comparison of these acquired images showed that the image through ratiometric signaling using L₂H is the better one for cell staining, although both probes have no cytotoxic effect.

Scheme 1 Schematic representation of the synthesis of the probes L₁H and L₂H, and their corresponding complexes.

Experimental section

Physical measurements

The fluorescence property of the sensor was investigated in water–ethanol (5 : 1, v/v) solvent. The pH study was performed in 100 mM HEPES buffer solution by adjusting pH with HCl or NaOH. The stock solutions (∼10⁻² M) for the selectivity study of the receptors (L₁H and L₂H) towards different anions were prepared with sodium salt of azide, perchlorate, disodium hydrogen arsenate; tetra butyl ammonium salt of chloride, bromide, iodide, acetate, fluoride; dihydrogen phosphate and potassium hydrogen sulphate in water–ethanol (5 : 1, v/v) solvent. In this selectivity study, the amount of these anions was a hundred times greater than that of the receptor used. Fluorescence titration was performed with potassium hydrogen sulphate in water–ethanol (5 : 1, v/v) solvent varying the anion concentration from 0 to 100 μM and concentration of the receptors were fixed at 25 μM throughout the experiment.

Preparation of L₁H and L₂H

Preparation of two receptors, i.e., L₁H and L₂H, was carried out following a common procedure. 2-(2-Aminophenyl)-benzimidazole (2.09 g, 10.0 mmol) and 4-nitrobenzaldehyde (1.51 g, 10.0 mmol) (for L₁H) or 4-methoxybenzaldehyde (1.36 g, 10.0 mmol) (for L₂H) were mixed in dry ethanol (25.0 mL) at room temperature. Then, the reaction mixture was refluxed for 6.0 h. The yellow (L₁H) or brown (L₂H) precipitate of the compounds were obtained from the solution through slow evaporation of the solvent. The two pure recrystallized compounds were then isolated from methanol.

L₁H·C₂₀H₁₄N₄O₂. Anal. found: C, 70.49; H, 4.24; N, 16.51; calc.: C, 70.17; H, 4.12; N, 16.37. IR (cm⁻¹): ν_NH = 3190.26, ν_C=N = 1612.49; ESI-MS: [M + H]⁺, m/z, 343.1480 (100%) (calcd: m/z, 342.11; where M = molecular weight of [L₁H]); ¹H NMR (δ, ppm in DMSO-d₆): 8.201 (d–d, 2H, J₁ = 7, J₂ = 2); 7.986 (d–d, 1H, J₁ = 7.75, J₂ = 1.5); 7.8 (d, 1H, J = 2.5); 7.7 (d, 1H, J = 8); 7.448 (d–d, 2H, J₁ = 7, J₂ = 2); 7.361–7.337 (m, 2H); 7.281–7.165 (m, 3H); 6.884–6.856 (m, 2H). Yield: 90%.

L₂H·C₂₁H₁₇N₃O. Anal. found: C, 76.81; H, 5.15; N, 13.07; calc.: C, 77.03; H, 5.24; N, 12.84. ESI-MS: [M + H]⁺, m/z, 328.1246 (100%) (calcd: m/z, 328.14; where M = molecular weight of [L₂H]); IR (cm⁻¹): ν_NH = 3209.6, ν_C=N = 1608.63 ¹H NMR (δ, ppm in DMSO-d₆): 7.96 (d–d, 1H, J₁ = 7.6, J₂ = 2); 7.651 (d, 1H, J = 8); 7.307–7.077 (m, 7H); 6.866–6.734 (m, 3H); 6.604 (d–d, 1H, J₁ = 7.6, J₂ = 2); 4.401 (s, 1H); 3.601 (s, 1H). Yield: 90%.

Preparation of compounds K[L₁H–HSO₄] and K[L₂H–HSO₄]

The preparation of solid complexes was carried out following a common procedure. To a methanolic solution of L₁H (342 mg, 1.0 mmol) or L₂H (327 mg, 1.0 mmol) (for K[L₁H–HSO₄] or for K[L₂H–HSO₄]), solid potassium hydrogen sulphate (136 mg, 1.0 mmol) was added and the reaction mixture was stirred at ambient temperature for 6.0 h. The solution thus obtained was then kept aside for slow evaporation at room temperature. After a few days, deep yellow crystalline complexes were collected by washing with water and methanol, and then dried in vacuo.

K[L₁H–HSO₄]·C₂₀H₁₅KN₄O₆S. Anal. found: C, 50.04; H, 3.25; N, 11.91; calc.: C, 50.19; H, 3.16; N, 11.71. ESI-MS in methanol: [I²⁻ + 2H + Na]⁺, m/z, 463.16 (obsd with 6% abundance) (calcd: m/z, 463.06); [I²⁻ + K + Na + H]⁺, m/z, 505.0080 (obsd with 12% abundance) (calcd: m/z, 505.06); where I²⁻ = L₁ + HSO₄⁻. IR (cm⁻¹): ν_S=O = 1114.86; 1H NMR (δ, ppm in DMSO-d₆): 8.201 (d–d, 2H, J₁ = 7, J₂ = 2); 8.0 (d–d, 1H, J₁ = 7.75, J₂ = 1.5); 7.82 (d, 1H, J = 2.5); 7.72 (d, 1H, J = 7.5); 7.480–7.458 (m, 2H); 7.39–7.175 (m, 5H); 6.906–6.875 (m, 2H). Yield: 75%.

K[L₂H–HSO₄]·C₂₁H₁₈KN₃O₅S. Anal. found: C, 54.15; H, 3.99; N, 9.25; calc.: C, 54.41; H, 3.92; N, 9.07. ESI-MS in methanol: [I²⁻ + 2H + Na]⁺, m/z, 447.92 (obsd with 11% abundance) (calcd: m/z, 448.089); [I²⁻ + K + Na + H]⁺, m/z, 486.2086 (obsd with 35% abundance) (calcd: m/z, 486.09; where I²⁻ = L₂ + HSO₄⁻; IR (cm⁻¹)): ν_S=O = 1111.00, ¹H NMR (δ, ppm in DMSO-d₆): 7.96 (d–d, 1H, J₁ = 7.6, J₂ = 2); 7.645 (d, 1H, J = 8); 7.306–7.077 (m, 7H); 6.867–6.731 (m, 3H); 6.65 (d–d, 1H, J₁ = 7.6, J₂ = 1.6); 4.401 (s, 1H); 3.601 (s, 1H). Yield: 75%.

X-ray data collection and structural determination

Single crystals suitable for single crystal X-ray crystallography were obtained from the methanolic solution of L₁H on slow evaporation at room temperature. X-ray single crystal data were collected using Mo K_α (λ = 0.7107 Å) radiation on a SMART APEX II diffractometer equipped with a CCD area detector. Data collection, data reduction and structure solution/refinement were carried out using the software package of SMART APEX II. The crystallographic data, selected bond lengths and bond angles are tabulated in Tables 1 and 2. A total of 26 495 reflections were measured, out of which 3542 were independent, and 3202 were observed [I > 2σ(I)]. The structure was solved by direct methods using SHELXS-97,⁹ and refined by full-matrix least-squares refinement methods based on F² using SHELXL-97. All non-hydrogen atoms were refined anisotropically, and all calculations were performed using WingX Package.¹⁰ The important crystal and refinement parameters are given in Table 1. The resulting crystals were found suitable for structural studies.

Table 1 Crystal data and refinement details for L₁H

Empirical Formula	C20H15N4O2
Formula weight	343.36
Crystal system	Orthorhombic
Space group	Pca21
a (Å)	12.0792(5)
b (Å)	9.1171(4)
c (Å)	15.1341(7)
α = β = γ	90^°
Volume (Å^3)	1666.68(13)
Temperature (K)	296(2)
Z	4
ρcalc (g cm^−3)	1.368
μ (mm^−1)	0.092
F(000)	716
θ range (deg)	2.80–26.78
Reflections collected	26495
Reflections independent	3542
Final R indices [I > 2σ(I)]	3202
R indices (all data)	0.0302
Goodness-of-fit on F^2	1.033

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Table 2 Selected bond distances (Å) and bond angles (°) for L₁H

Bond length (Å)
C22–N3	1.389(2)
C23–N3	1.3220(18)
C23–N2	1.3697(19)
N2–C11	1.386(2)
N2–C4	1.4570(17)
C7–N1	1.471(2)

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Bond angles (°)
N3–C23–N2	112.68(13)
N3–C23–C3	128.21(13)
N2–C23–C3	119.10(12)
C23–N2–C11	107.34(11)
C23–N2–C4	126.13(13)
C11–N2–C4	126.27(13)
N3–C22–C12	130.12(14)
N3–C22–C11	110.30(13)
N4–C4–N2	108.20(12)
N4–C4–C21	112.33(12)
N2–C4–C21	111.87(11)

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Cell preparation and in vitro cellular imaging

Human cervical cancer cells, i.e., HeLa cell lines, were used throughout the study. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL) supplemented with 10% FBS (Gibco BRL) and 1% antibiotic mixture containing penicillin, streptomycin and neomycin (PSN, Gibco BRL) at 37 °C in a humidified incubator with 5% CO₂. For the experimental study, cells were grown to 80–90% confluence, harvested with 0.025% trypsin (Gibco BRL) and 0.52 mM EDTA (Gibco BRL) in PBS (phosphate-buffered saline, Sigma Diagnostics) and then plated at the desire cell concentration and allowed to re-equilibrate for 24 h before any treatment. Cells were rinsed with PBS and incubated with DMEM-containing L₁H or L₂H (10 μM, 1% DMSO) for 15 min at 37 °C. All experiments were conducted in DMEM containing 10% FBS and 1% PSN antibiotic mixture. The imaging system was composed of a fluorescence microscope (ZEISS Axioskop 2 plus) with an objective lens [10×].

Cell cytotoxicity assay

To test the cytotoxicity of L₁H and L₂H, MTT [3-(4,5-dimethylthiazol-2-yl)-2,S-diphenyl tetrazolium bromide] assay was performed as per a previously reported procedure.¹¹ After treatments of the probe (5, 10, 25, 50, and 100 μM), 10 μl of MTT solution (10 mg mL⁻¹ PBS) was added in each well of a 96-well culture plate, and incubated at 37 °C for 8 h. All of the mediums were removed from the wells and replaced with 100 μl of acidic isopropanol. The intracellular formazan crystals (blue-violet) formed were solubilized with 0.04 N acidic isopropanol, and the absorbance of the solution was measured at 595 nm with a microplate reader. The data obtained are shown as means ± S.D. of three independent experiments. The cell cytotoxicity was calculated based on a cell viability of 100%.

Theoretical calculation

The gradient-corrected DFT level, involving the hybrid 3-parameter fit of exchange and correlation functionals of Becke (B3LYP), which includes the correlation functional of Lee, Yang, and Parr (LYP), was used. The standard split valence basis sets 6-31G(d) and 6-31G(d) were applied for other atoms. Natural population analysis (NPA) (implemented in Gaussian 09 program) at B3LYP/6-31G(d) level was carried out to compute the charge on each atom.

Results and discussion

Synthesis and characterization

The organic moieties (L₁H and L₂H) were synthesized by condensing an ethanolic solution of 2-(2-aminophenyl)-benzimidazole with benzaldehyde derivatives (for L₁H, 4-nitrobenzaldehyde and for L₂H, 4-methoxybenzaldehyde) in 1 : 1 mole ratio (Scheme 1). They were characterized by physicochemical and spectroscopic techniques. In addition, the solid state structure of L₁H was confirmed by single crystal X-ray crystallography after collecting the single crystals of L₁H from the methanolic solution. The molecular view of L₁H with atom labeling scheme is shown in Fig. 1, which shows that L₁H crystallizes in the orthorhombic space group Pca2₁. The crystallographic data and bond parameters are tabulated in Tables 1 and 2. The bond distance of C23–N2 (1.3697 Å) is longer than that of C23–N3 (1.3220 Å); however, both these values are significantly shorter than that of C4–N2 (1.4570 Å) or C4–N4 (1.4530 Å).

Fig. 1 Molecular view of L₁H with atom numbering scheme.

The peaks obtained in ¹H NMR spectrum of L₁H and L₂H have been identified, and they are in accordance with the structural formula of L₁H and L₂H in the solution state (Fig. S1 and S2†). The ESI mass spectrum of the compound L₁H in methanol shows a peak at m/z 443.1480 with 100% abundance, which is assignable to [M + H]⁺ (calculated value at m/z, 443.12), where M is the molecular weight of L₁H (Fig. S3†). The ESI mass spectrum of the compound L₂H in methanol shows a peak at m/z 328.1246 with 100% abundance, which is assignable to [M + H]⁺ (calculated value at m/z, 328.14), where M is the molecular weight of L₂H (Fig. S4,†). The IR spectra of L₁H and L₂H show the characteristic stretching of the N–H and C=N bonds (Fig. S5 and S6†). L₁H and L₂H undergo non-covalent hydrogen bonding interaction with HSO₄⁻ ions, which results in enhancement of the fluorescence intensity (Scheme 2). To establish the mechanism of the formation of the adduct with HSO₄⁻ ions, the species, formulated as K[L₁H–HSO₄] and K[L₂H–HSO₄], were isolated in solid state from the reaction of one mole potassium hydrogen sulphate with one mole of the organic moiety (L₁H or L₂H) in methanol under stirring condition. The complexes are soluble in methanol, DMSO, and acetonitrile. The peaks obtained in ¹H NMR spectrum of K[L₁H–HSO₄] and K[L₂H–HSO₄] have been identified, and they are in accordance with the structural formula of L₁H and L₂H in the solution state (Fig. S7 and S8†). The ESI mass spectrum of the compound K[L₁H–HSO₄] in methanol shows a peak at m/z 463.16 and 505.0080 (Fig. S9†), which is assignable to [I²⁻ + 2H + Na]⁺ and [I²⁻ + K + Na + H]⁺, respectively, where I²⁻ = L₁⁻ + HSO₄⁻. The ESI mass spectrum of the K[L₂H–HSO₄] in methanol shows a peak at m/z, 447.9254 and 486.2086 (Fig. S10†), which is assignable to [I² + 2H + Na]⁺ and [I²⁻ + K + Na + H]⁺, respectively, where I²⁻ = L₂⁻ + HSO₄⁻. The IR spectra of K[L₁H–HSO₄] and K[L₂H–HSO₄] show the characteristic stretching of S=O at 1114 and 1111 cm⁻¹, respectively (Fig. S11 and S12†). All these data confirm the composition of K[L₁H–HSO₄] and K[L₂H–HSO₄].

Scheme 2 Schematic representation of the plausible mechanism of hydrogen sulfate sensing.

¹H NMR titration

In order to strengthen the above pathway of bonding of HSO₄⁻ ions with the receptors, ¹H NMR titration was performed by concomitant addition of HSO₄⁻ ions to the DMSO-d₆ solution of L₁H and L₂H (Fig. S13 and S14, ESI†). Significant spectral changes for L₁H and L₂H were observed upon addition of HSO₄⁻ ions. In case of L₁H, 5 min after the addition of HSO₄⁻ ions, the peaks due to the proton in N–Hⁱ appeared with the peak of H^h proton at δ = 6.884–6.856 ppm (2H, m). It is noteworthy that they were remarkably affected and became equivalent to one hydrogen of H^h due to the disappearance of Hⁱ from N–Hⁱ. Moreover, the multiplet peaks appeared at δ = 7.361–7.337 ppm (2H, m), which is assignable to split up of H^e and H^e' protons, as the peak for H^e shifted downfield due to the bonding of HSO₄⁻ ions with H^e. In case of L₂H titration, the peak at 4.401 ppm, due to the proton of N–H, disappeared after HSO₄⁻ addition. All other protons of L₁H and L₂H remained unaffected after interaction with HSO₄⁻ ions (Tables S1 and S2, ESI†).

Spectral characteristics

Emission study. On excitation of L₁H at 400 nm and L₂H at 390 nm, a weak emission at 485 nm in both was observed in water–ethanol (5 : 1) solvent mixture (Fig. S15 and S16†). Fluorescence quantum yields (F) were estimated by integrating the area under the fluorescence curves with the equation:
where A is the area under the fluorescence spectral curve and OD is the optical density of the compound at the excitation wavelength. The standard used for the measurement of fluorescence quantum yield was anthracene (ϕ = 0.29 in ethanol). The emission intensities of the organic molecule in the presence of various concentrations of HSO₄⁻ ions were measured. The fluorescence spectrum properties of L₁H (25 μM) and L₂H (25 μM) were investigated in ethanol–water (1 : 5, v/v) HEPES buffer (0.1 M, pH = 7.4) at 25 °C as a function of added [HSO₄⁻] (Fig. 2a and b). L₂H showed a fluorescence of higher intensity at 430 nm. After addition of HSO₄⁻ the fluorescence intensity band shows a ratiometric enhancement at 485 nm (Fig. 2b). The ratiometric signaling of fluorescence output at two different wavelengths plotted as a function of concentration of HSO₄⁻ indicates that the fluorescence intensity ratio at wavelengths 485 nm and 430 nm (I₄₈₅/I₄₃₀) gradually increases with increase in the concentration of HSO₄⁻ ions (Fig. S17†), and after a certain time it levels up to produce a sigmoidal curve. Note that the fluorescence emission band of L₁H at 485 nm is very weak at room temperature. Addition of HSO₄⁻ (25 μM) to L₁H (25 μM) in HEPES buffer solution at pH 7.4 afforded a 120 times single point enhancement in fluorescence intensity (Fig. 2a). In the absence of HSO₄⁻ anion, the fluorescence intensity of L₁H is very low. However, in presence of HSO₄⁻, fluorescence intensity greatly increased due to bonding interaction between the deprotonated N⁻ atom (as pK_a of NH ≈ 6.9, resulting experimental pH of the medium) of imidazole moiety with the proton of the added HSO₄⁻ ion. This is also supported by the computational study of the probes (L₁H and L₂H) and their corresponding adducts with HSO₄⁻ (Fig. S18 and S19†). The computational study indicates that the closeness of the HSO₄⁻ ion with L₁H is to a great extent comparable to that of the HSO₄⁻ ion with L₂H as the theoretical bond distances of CH⋯O (of HSO₄⁻) (2.258 Å) and N⋯H (of HSO₄⁻) (1.898 Å) in [L₁H–HSO₄]⁻ adducts are shorter than those of CH⋯O (of HSO₄⁻) (2.371 Å) and N⋯H (of HSO₄) (1.958 Å) in [L₂H–HSO₄]⁻ adducts. The electronic effect of the substituents (–NO₂ in L₁H and –OMe in L₂H) present in the para-position of the phenyl ring plays the key role in the tuning of the fluorescence signaling from single point response to ratiometric response.

Fig. 2 (a) Fluorescence spectra of L₁H (25 μM) as a function of externally added HSO₄⁻ [0–30 μM] in ethanol–water (1 : 5, v/v) HEPES buffer (0.1 M, pH = 7.4) at 25 °C [λ_em = 485 nm, λ_ex = 400 nm]. (b) Fluorescence spectra of L₂H (25 μM) as a function of externally added HSO₄⁻ [0–30 μM] in ethanol–water (1 : 5, v/v) HEPES buffer (0.1 M, pH = 7.4) at 25 °C [λ_em: 485 nm, λ_ex = 390 nm].

There was almost no interference for the detection of HSO₄⁻ in the presence of 100 equivalents of tetrabutylammonium salt of chloride, bromide, iodide and acetate; sodium salt of azide, sulphide, and cyanide; dihydrogen phosphate; dihydrogen arsenate; and potassium salt of nitrate and sulphate. Job's plot analysis (Fig. 3a and b) revealed that L₁H and L₂H both bonded with HSO₄⁻ ions to form the adducts in 1 : 1 molar ratio. The binding constant values, calculated from the emission intensity data, were found to be 3.25 × 10⁵ M^−1/2 for L₁H and 1.48 × 10⁵ for L₂H (Fig. 4a and b) using the modified Benesi–Hildebrand equation:^12,13

1/(F_x − F₀) = 1/(F_max − F₀) + (1/K[C])(1/(F_max − F₀)

where F₀, F_x, and F_∞ are the emission intensities of organic moiety considered in the absence of HSO₄⁻ ions, at an intermediate HSO₄⁻ concentration, and at a concentration causing complete interaction, respectively; K is the association constant and [C] is [HSO₄⁻]. The average fluorescence lifetime measurements of L₁H and L₂H, in presence and absence of HSO₄⁻ ion in water–ethanol (5 : 1) medium indicates the gradual increase with increase in [HSO₄⁻] (Fig. 5a and b). The average lifetimes are calculated to be 8.32 ns for L₁H, 9.42 ns for the mixture of L₁H : HSO₄⁻ (1 : 0.5) and 10.75 ns for the mixture of L₁H : HSO₄⁻ in 1 : 1 molar ratio. The average lifetime for L₂H is 8.26 ns for L₂H : HSO₄⁻ (1 : 0.5) is 8.58 ns and in case of L₂H : HSO₄⁻ (1 : 1), is 11.79 ns. The strong binding of HSO₄⁻ ions with organic moiety is also due to the binding constant value. According to the equations: τ ⁻¹ = k_r + k_nr and k_r = Φ_f/τ,¹⁴ the radiative rate constant k_r and total non-radiative rate constant k_nr of the organic moieties (L₁H, L₂H), K[L₁H–HSO₄], and K[L₂H–HSO₄] were listed in Tables S3 and S4.† The data suggest that the fluorescent enhancement is ascribed to the decrease in ratio of k_nr/k_r from 181.40 for L₁H to 1.8 for K[L₁H–HSO₄], and from 14.4 for L₂H to 1.0839 for K[L₂H–HSO₄].

Fig. 3 (a) Job's plot of L₁H and (b) L₂H showing maxima at 1:1 molar ratio.

Fig. 4 (a) Binding constant (K) value of 3.25 × 105 M⁻¹ for L₁H determined from the intercept/slope of the plots. (b) Binding constant (K) value of 1.48 × 105 M⁻¹ for L₂H determined from the intercept/slope of the plots.

Fig. 5 (a) Time resolved fluorescence decay of L₁H (10 mM) and (b) L₂H (10 mM) in the absence and presence of added HSO₄⁻ ions (5 mM and 10 mM) in 100 mM HEPES buffer (ethanol/water: 1/5, v/v) [λ_em: 485 nm].

Absorption study. The UV-Vis spectrum of L₁H showed the characteristic absorption bands at ca. 226 nm, 262 nm, 290 nm, 300 nm, and 346 nm, which are attributable to intramolecular π–π* and n–π* transitions. During the titration, by adding the solution of HSO₄⁻ ions to the colourless solution of L₁H in ethanol–water (1 : 5, v/v) in HEPES buffer (0.1 M, pH 7.4) at 25 °C, the peak at 346 nm gradually decreased, and a new peak around 390 nm was generated through an isosbestic point at 364 nm (Fig. 6a) due to the formation of a complex of the receptor with HSO₄⁻ ion in the solution state. Similarly, UV-Vis spectrum of L₂H showed the characteristic absorption bands at 225 nm, 291 nm, and 350 nm. In a similar type of titration, by addition of the solution of HSO₄⁻ ion to the colourless solution of L₂H in ethanol–water (1 : 5, v/v) in HEPES buffer (0.1 M, pH 7.4) at 25 °C, the peak at 350 nm was red-shifted to a new peak at 390 nm (Fig. 6b) through an isosbestic point at 365 nm due to the formation of an adduct of [L₂H–HSO₄]⁻ in the solution state.

Fig. 6 (a) Changes in the absorption spectrum of L₁H (25 μM) upon addition of 0–30 μM of HSO₄⁻ in ethanol–water (1 : 5, v/v) HEPES buffer (0.1 M, pH 7.4) at 25 °C. (b) Changes in the absorption spectra of L₂H (25 μM) upon addition of 0–30 μM of HSO₄⁻ in ethanol–water (1 : 5, v/v) HEPES buffer (0.1 M, pH 7.4) at 25 °C.

Selectivity. The fluorescence response of organic moiety towards the different anions were investigated with 100 times concentrations of Cl⁻, Br⁻, I⁻, F⁻, CN⁻, OAc⁻, NO₃⁻, S²⁻, SO₄²⁻, H₂PO₄⁻, H₂AsO₄⁻ (Fig. S20 and S23†). This study indicates that both L₁H and L₂H have excellent selectivity for HSO₄⁻ ions over other anions.

Effect of pH. The fluorescence intensity of the organic moieties L₁H and L₂H was measured at various pH values in HEPES buffer (0.1 M) at 25 °C by adjusting the pH using HCl or NaOH in the presence and absence of HSO₄⁻ ions (Fig. 7a and b†). Here, we demonstrate that the fluorescence intensity of both organic compounds does not vary in the pH range of 5.0–12.0 in absence of HSO₄⁻ ions; however, in presence of HSO₄⁻ ions, both moieties exhibited a pH dependency in the fluorescence intensity over the pH range 5.0 to 8.0. It is also noteworthy that the fluorescence intensity of the organic moiety in presence of HSO₄⁻ ions is higher than that in the absence of HSO₄⁻ ions due to the formation of adducts of HSO₄⁻ ions with the deprotonated receptors (L₁⁻ and L₂⁻; after deprotonation of the nitrogen atom of –NH in the imidazole ring) through H-bonding. At a higher pH range (pH 8.0–12.0), the gradual decrease in the fluorescence intensity is due to the decreasing formation probability of the adducts of HSO₄⁻ ions with the deprotonated receptors (L₁⁻ and L₂⁻) as there is a tendency of HSO₄⁻ ions to be deprotonated. As a result of this observation, both probes are very effective as sensors in analytical and bioanalytical studies, which were carried out at biological pH 7.4 in ethanol–water (1 : 5, v/v) HEPES buffer (0.1 M) at 25 °C.

Fig. 7 (a) Fluorescence response to pH of L₁H (25 μM) in absence and in presence of HSO₄⁻ (one equivalent) at different pH in 100 mM HEPES buffer (ethanol–water: 1/5) at 25 °C. (b) Fluorescence response to pH of L₂H (25 μM) in the absence and presence of HSO₄⁻ (one equivalent) at different pH values in 100 mM HEPES buffer (ethanol–water: 1 : 5) at 25 °C.

Analytical figure of merit. To calculate the detection limit, calibration curves (Fig. 8a and b) in the lower concentration region (0–5 μM) were obtained. From the slope of the curve(s) and the standard deviation of seven replicate measurements of the zero level (σ_zero) the detection limit was estimated using the equation, 3σ/S.¹⁵ From this study, the detection limit of L₁H and L₂H for HSO₄⁻ ions were calculated to be 18.8 nM and 14.11 nM, respectively.

Fig. 8 (a) Calibration curve in the nanomolar range with error bars for calculating the LOD of HSO₄⁻ by L₁H in 100 mM HEPES buffer (ethanol–water: 1 : 5) at 25 °C. (b) Calibration curve in the nanomolar range with error bars for calculating the LOD of HSO₄⁻ by L₂H in 100 mM HEPES buffer (ethanol–water: 1 : 5) at 25 °C.

Cell imaging. To examine the utility of the probes in biological systems, they were applied to human cervical cancer HeLa cell lines. Here, HSO₄⁻ ions and L₁H or L₂H were allowed to uptake by the cells of interest and the images of the cells were recorded by fluorescence microscopy after excitation at ∼400 and 405 nm (Fig. 9). In addition, the in vitro study showed that 50 μM of L₁H and L₂H was not cytotoxic to the cell for up to 8.0 h (Fig. S24 and S25†). The results indicate that both probes have significant potentiality for both in vitro and in vivo application as HSO₄⁻ sensors as well as imaging in different ways as same manner for live cell imaging can be followed instead of fixed cells. In this study, it is also observed that the clarity of the image is significantly better by employing L₂H than L₁H due to the ratiometric signaling.

Fig. 9 Phase contrast (1 and 1′) and fluorescence images of HeLa cells after incubation with LH in presence of (2 and 2′) 0 μM, (3 and 3′) 5 μM and (4 and 4′) 10 μM of HSO₄⁻ for 30 min at 37 °C.

Conclusion

In conclusion, two new 2-(2-aminophenyl)benzimidazol-based HSO₄⁻ ion-selective receptors (L₁H and L₂H), and their 1 : 1 molecular adducts with HSO₄⁻ were synthesized and characterized using physicochemical and spectroscopic techniques along with single crystal X-ray crystallography of L₁H for detailed structural analysis. Both receptors (L₁H and L₂H) behave as highly selective fluorescent sensor for HSO₄⁻ ions at biological pH in green solvent than other anions by the naked eye. Here, the new receptors (L₁H and L₂H) have the same environments to accept the guest (HSO₄⁻ ions), but the emission efficiency of the receptors has been tuned successfully through single point to ratiometric detection in green solvent by exploiting substituent effects (−R effect of –NO₂ group, and +R effect of OMe group) within the receptors. The limit of detection for HSO₄⁻ ions (by 3σ method) were calculated to be 18.08 nM and 14.11 nM for L₁H and L₂H, respectively. Both probes could be used as biomarkers for the detection of intracellular HSO₄⁻ ions in HeLa cells as they are both non-cytotoxic agents. However, L₂H is a better candidate compared to L₁H for acquiring the fluorescence image (Fig. 9), although they have the same receptor environments for the HSO₄⁻ ions. From this study, it may be concluded that substituents present in the appropriate position of the receptor control the mode of signaling (ratiometric or single point) of the sensor, and the ratiometric signaling is better than the single point signaling as expected.

Acknowledgements

Financial assistance from CSIR, New Delhi, India is gratefully acknowledged. M. Mukherjee wishes to thank to UGC, New Delhi, India for offering the fellowship. The authors are grateful to USIC, The University of Burdwan, for the single crystal X-ray diffractometer facility under PURSE program. We sincerely acknowledge Prof. Samita Basu and Mr Ajay Das, Chemical Science Division, SINP, Kolkata for enabling TCSPC instrument and Prof. B. Mukhopadhyay, IISER, Kolkata to record some ¹H NMR spectra and ESI Mass Spectra.

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Footnote

† Electronic supplementary information (ESI) available. CCDC 968396. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra03551h

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