The synthesis of MH-5 was completed through a three-step reaction process. Initially, the starting compounds, 2,3,3-trimethyl-1-propyl-3H-indol-1-ium iodide and 2,3,5,6,8,9-hexahydrobenzo[b][1, 4, 7, 10]tetraoxacyclododecine-12-carbaldehyde [53, 54], were synthesized in the first and second steps. In the last step, these compounds were combined to yield MH-5. The structure of MH-5 was confirmed using NMR (both 1H and 13C NMR) and Q-TOF LC/MS mass spectrometry, with corresponding spectra provided in Figs. S1–S3 of the Electronic Supplementary Material. The synthetic pathway for MH-5 was illustrated in Scheme 1.

Scheme 1.

Synthesis of the sensor, MH-5

Spectroscopic studies of MH-5 with cations

To determine the selectivity of MH-5, the color changes observable to the naked eye were initially tested by exposing MH-5 (5.0 μM) to various metal ions (5.0 μM), including Li+, Ag+, Al3+, Fe3+, K+, Ca2+, Co2+, Cd2+, Hg2+, Cu2+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Cr3+, and Zn2+ in DMSO-PBS buffer (10 mM, pH 7.25, 1:9 v/v). Among all tested ions, only Li+ triggered a noticeable color shift in MH-5 from pink to pale pink (see Electronic Supplementary Material Fig. S4). Correspondingly, UV–Vis spectra of MH-5 with these metal ions were obtained under conditions similar as to those of the naked eye detection tests. MH-5 alone (5.0 μM) showed a peak absorption at 550 nm (ε = 7.80 × 104 M−1 cm−1). However, when Li+ was added, the absorbance at 550 nm decreased significantly (ε = 2.58 × 104 M−1 cm−1 at a 1:1 MH-5/Li+ ratio). In contrast, other ions including Ag+, Al3+, Fe3+, K+, Ca2+, Co2+, Cd2+, Hg2+, Cu2+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Cr3+, and Zn2+ exhibited minimal responses, as shown by MH-5 analysis (Fig. 1a).

The metal ion selectivity of MH-5 was further evaluated using fluorescence spectroscopy. As shown in Fig. 1b, MH-5 exhibited a strong emission peak at 602 nm when excited at 530 nm, and the fluorescence quantum yield was calculated to be 0.096 [57]. Upon the addition of the metal ions tested, there was a slight quenching in fluorescence intensity at 602 nm; however, a substantial quenching effect (83%) was observed specifically with Li+, resulting color change under UV-lamb (see Electronic Supplementary Material Fig. S5). The quantum yield value also changed to 0.034. These findings from naked eye observation, absorption and fluorescence spectroscopy indicate that MH-5 serves as a highly selective colorimetric and on–off fluorescent sensor for detecting Li+.

Fig. 1

a Absorbance and b fluorescence spectra of 5.0 µM MH-5 to the metal ions tested (5.0 µM of Ag+, Al3+, Fe3+, K+, Ca2+, Co2+, Cd2+, Hg2+, Cu2+, Li+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Cr3+, and Zn2+) in DMSO-PBS buffer (10 mM, pH = 7.25, v/v, 1:9)

The time response of a fluorescence sensor is a crucial parameter for assessing its practical applicability. The kinetics of the interaction between MH-5 and Li+ were examined in DMSO-PBS buffer solution (10 mM, pH 7.25, 1:9 v/v). As presented in Fig. S6 of the Electronic Supplementary Material, the emission intensity of MH-5 at 602 nm decreased rapidly upon the addition of 1.0 equivalent of Li+, reaching a minimum value within 2 min and remaining stable over long reaction times. This rapid response highlights the potential of MH-5 as an efficient sensor, providing time-saving advantages and enhancing its applicability in practical settings.

In order to understand the optimal working condition of MH-5 and MH-5/Li+, the effect of pH on selectivity was studied in the range of 3.5–10 pH. MH-5 showed no fluorescence response to hydrogen ions within a pH range of 3.5 to 9.0, indicating strong stability over a wide pH spectrum. In the presence of Li+, fluorescence quenching was observed at 602 nm, with the fluorescence signal remaining stable from pH 3.5 to 8.0. These findings suggest that MH-5 is suitable for Li+ detection within a pH range of 3.5 to 8.0, supporting its potential applicability in biological systems (see Electronic Supplementary Material Fig. S7).

Competition studies were also performed to assess the selective sensing behavior of MH-5 toward Li+ in the presence of competing metal ions, including Ag+, Al+3, Fe3+, K+, Ca2+, Co2+, Cd2+, Hg2+, Cu2+, Li+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Cr3+, and Zn2+. Upon adding 5.0 equivalents of these competing metal ions, a fluorescence quenching at 602 nm (indicated by blue bars) was observed following the subsequent addition of 5.0 equivalents of Li+. These findings indicate that MH-5 can function as an effective Li+ sensor even in the presence of various competing cations (Fig. 2).

Fig. 2

Fluorescence responses of 5.0 µM MH-5 to the presence of 50.0 µM metal ions tested (black bars) and the subsequent addition of Li+ (blue bars) in DMSO-PBS buffer (10 mM, pH = 7.25, v/v, 1:9); the bars represent the fluorescence intensity at 602 nm (excitation wavelength was 530 nm)

Spectroscopic studies of MH-5 with anions

The remarkable photophysical characteristics of MH-5 were further examined using absorbance and emission spectroscopy in the presence of the following anions in their tetrabutylammonium salts from CN−, Br−, AcO−, Cl−, SO32−, I−, SO42−, OH−, NO3−, NO2−, ClO4−, HPO42−, CO32−, and H2PO4− in DMSO-PBS buffer (10 mM, pH = 7.25, v/v, 1:9). As previously noted, MH-5 (5.0 µM) showed an absorption band with a molar absorptivity of ε = 7.80 × 104 M−1 cm−1 at 550 nm. Upon the addition of CN− to the MH-5 solution, the color changed from pink to colorless (see Electronic Supplementary Material Fig. S8), and the absorbance intensity at 550 nm nearly disappeared (Fig. 3a). In contrast, the color and UV–VİS spectrum of MH-5 remained largely unchanged in the presence of other tested anions.

The fluorescence spectra of MH-5 were obtained both in the absence and presence of CN− in a DMSO-PBS buffer (10 mM, pH 7.25, 1:9 v/v). As illustrated in Fig. 3b, MH-5 (5.0 μM) exhibited strong fluorescence at 602 nm upon excitation at 530 nm, as mentioned above. While the addition of various anions resulted in a slight quenching of fluorescence intensity at 602 nm, a pronounced quenching effect (92%) was specifically observed with CN−, accompanied by a visible color change under UV light (see Electronic Supplementary Material Fig. S9). The quantum yield value also changed to 0.019. These results from naked eye observation, absorption, and fluorescence spectroscopy demonstrate that MH-5 functions as a highly selective colorimetric and on–off fluorescent sensor for CN− detection.

Fig. 3

a Absorbance and b fluorescence spectra of 5.0 µM MH-5 to anions tested (5.0 µM of CN−, Br−, AcO−, Cl−, SO32−, I−, SO42−, OH−, NO3−, NO2−, ClO4−, HPO42−, CO32−, and H2PO4−) in DMSO-PBS buffer (10 mM, pH = 7.25, v/v, 1:9)

The kinetic of the MH-5/CN− interaction system was also studied in DMSO-PBS buffer solution (10 mM, pH 7.25, v/v, 1:9). As shown in Fig. S10 of the Electronic Supplementary Material, the emission intensity of MH-5 at 602 nm rapidly decreased upon the addition of 1.0 equivalent of CN−, reaching its minimum value within 5 min and remaining stable with extended reaction time. This property of MH-5 underscores its suitability as a highly efficient sensor, offering time-saving advantages and enhancing its utility for practical applications.

The effect of pH on CN− detection by MH-5 was evaluated through fluorescence spectroscopy. Fluorescence spectra of MH-5, both in the presence and absence of CN−, were recorded across a pH range of 3.5 to 10 (see Electronic Supplementary Material Fig. S11). The sensor showed no response to hydrogen ions within a pH range of 3.5 to 9.0, indicating strong stability over a wide pH spectrum. In the presence of CN−, fluorescence quenching was observed at 602 nm, with the fluorescence signal remaining stable from pH 3.5 to 10.0. These findings suggest that MH-5 is suitable for CN− detection within a pH range of 3.5 to 9.0, supporting its potential applicability in biological systems.

Competition studies were also performed to assess the selective sensing behavior of MH-5 toward CN− in the presence of competing anions, including Br−, AcO−, Cl−, SO32−, I−, SO42−, OH−, NO3−, NO2−, ClO4−, HPO42−, CO32−, H2PO4−, PO43−, and SCN−. Upon adding 5.0 equivalents of these competing anions, a fluorescence quenching at 602 nm (indicated by blue bars) was observed following the subsequent addition of 5.0 equivalents of CN−. These findings indicate that MH-5 can function as an effective CN− sensor even in the presence of various competing anions (Fig. 4).

Fig. 4

Fluorescence responses of 5.0 µM MH-5 to the presence of 50.0 µM anions tested (black bars) and the subsequent addition of CN− (blue bars) in DMSO-PBS buffer (10 mM, pH = 7.25, v/v, 1:9); the bars represent the fluorescence intensity at 602 nm (excitation wavelength was 530 nm)

Proposed binding mode and sensing mechanisms

To elucidate the binding mode of MH-5 with Li+ and CN−, the binding stoichiometries for MH-5 with Li+ and with CN− were initially examined. Various analytical techniques were employed to accurately determine the stoichiometry of interactions of MH-5 with Li+ and CN−, respectively. Job’s plot, derived from UV–Vis spectroscopic measurements, exhibited a maximum absorption when the molar fraction of Li+ and CN− reached approximately 0.5 (see Electronic Supplementary Material Fig. S12 and Fig. S13). This result suggests a 1:1 binding stoichiometry between MH-5 and both Li+ and CN−.

UV–Vis titration and fluorescence titration were also applied to study the binding stoichiometry between MH-5 and Li+ and CN−. As depicted in Fig. S14 and S15 of the Electronic Supplementary Material, the absorption intensity of MH-5 (5.0 µM) in DMSO-PBS buffer (10 mM, pH = 7.25, v/v, 1:9) at 550 nm gradually decreased upon the addition of Li+ and CN− at varying concentrations, reaching saturation upon the addition of 1.0 equivalent of each ion. This behavior suggests a 1:1 stoichiometric interaction between MH-5 and both Li+ and CN−. Furthermore, clear isosbestic points were observed at 337 nm for Li+ and 354 nm for CN−, indicating a shift from the free form of MH-5 to the complexed forms MH-5 + Li+ and MH-5 + CN−as binding interactions occur [58]. Fluorescence titration experiments also supported the 1:1 stoichiometry between MH-5 and both Li+ and CN−. As seen in Fig. 5a and 5b, the titration curve (a plot from fluorescence intensities versus Li+ or CN− at various concentrations) decreased linearly, reaching saturation at 1:1 ratio of MH-5 between Li+ and CN−. This outcome is consistent with a 1:1 binding stoichiometry for both ions. MALDI-TOF mass results also supported a 1:1 ratio between MH-5 and CN− (see Electronic Supplementary Material Fig. S16).

Fig. 5

Fluorescence titration of 5.0 µM MH-5 with increasing concentrations (0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, 2.7, 3.0, 3.3, 3.6, 3.9, 4.2, 4.5, 4.8, 5.0, 7.5, and 10 µM, respectively) in DMSO-PBS buffer (10 mM, pH = 7.25, v/v, 1:9) of Li+ (a) and CN− (b)

FT-IR spectroscopy was used to elucidate the sensing mechanism of MH-5 for Li+ ions (Fig. 6). Upon addition of Li+, the peaks of -OH and -C–O–C at 3488 cm−1 and 1103 cm−1, respectively, shifted. These findings indicate that Li+ ion interacts with the crown ether portion of MH-5 [59].

Fig. 6

FT-IR spectra of MH-5 (black line) and MH-5 + Li+ (blue line)

The sensing mechanism between MH-5 and CN− was elucidated through 13C NMR titration experiments. In these experiments, CN− (dissolved in DMSO-d6) was added to a DMSO-d6 solution of MH-5, allowing for detailed observation of binding interactions. As illustrated in Fig. 7, the peak at 180 ppm belonging to the carbon numbered as C1 in the molecule shifted to 81.2 ppm after CN− was added. This is probably due to the conversion of C1 from sp2 hybridization to sp3 hybridization [60]. Moreover, a new peak at 118.2 ppm appeared, corresponding to CN− [61]. These results indicate a nucleophilic attack by CN− on the carbon atom (C1) of the indoline moiety [62, 63].

Fig. 7

13C NMR spectra of MH-5 (red line) and in the presence of CN− (black line)

We also studied the reversibility of the binding of Li+ and CN− by MH-5 in DMSO-PBS buffer (10 mM, pH = 7.25, v/v, 1:9), respectively. Ethylenediaminetetraacetic acid (EDTA) was used to remove Li+ ion from the MH-5 + Li+ [51]. As depicted in Fig. S17 of the Electronic Supplementary Material, the addition of EDTA led to a rapid increase in the fluorescence peak at 602 nm, indicating that MH-5 can detect Li+ through a reversible mechanism. On the other hand, trifluoroacetic acid (TFA) was used to remove the CN− ion from MH-5 + CN− [64]; however, as shown in Fig. S18 of the Electronic Supplementary Material, no significant change was observed in the fluorescence spectrum of MH-5 + CN−. These results suggest an irreversible interaction between MH-5 and CN−.

Based on the results obtained, the binding mechanisms of MH-5 between Li+ and CN− were proposed as shown in Scheme 2. CN− detection was achieved through its nucleophilic addition to the indolium group of the sensor [65, 66], while Li+ was detected by its coordination with oxygen atoms in the crown ether moiety [47, 67]. In both detection mechanisms, the binding interactions with Li+ and CN− induced fluorescence quenching in the fluorescence of MH-5.

Scheme 2.

Proposed response mechanisms of MH-5 toward Li+ and CN−

To further confirm the binding mechanism between MH-5 and Li+/CN−, optimized structures of MH-5, MH-5 + Li+, and MH-5 + CN− were obtained by density functional theory (DFT) calculations at the B3LYP/6-311G (d,p) level using the Gaussian 09 program [68]. As seen in Fig. 8, the orbital spatial distributions were found as − 8.60 eV/ − 5.63 eV (HOMO → LUMO) for MH-5, − 11.83 eV/ − 8.03 eV (HOMO → LUMO) for MH-5 + Li+, and − 5.59 eV/ − 0.94 eV (HOMO-1 → LUMO) for MH-5 + CN−. The energy gaps of MH-5, MH-5 + Li+, and MH-5 + CN− were found as 2.97 eV, 3.82 eV, and 4.65 eV, respectively. The energy gaps of MH-5 + Li+ and MH-5 + CN− were higher than that of MH-5. The increasing energy gap between the HOMO and LUMO induces a blue shift in the absorbance wavelength for both Li+ and CN− [37, 69]. These results support the proposed mechanisms illustrated in Scheme 2.

Fig. 8

Energy diagrams of HOMO and LUMO orbitals: a MH-5, b MH-5 + Li+, c MH-5 + CN−

Detection limit studies

The detection limits for MH-5 with Li+ and CN− ions were determined through absorption and fluorescence analyses across a concentration range of MH-5/Li+ or CN− from 0.1 to 5.0 µM. Calibration curves were generated by applying linear regression to the relationship between Li+ or CN− concentrations and the corresponding absorption or fluorescence intensities. Detection limits were then calculated using the formula 3σ/k [70,71,72], where σ represents the standard deviation of blank signals from MH-5 (with n = 10) and k denotes the slope of the calibration curves.

As depicted in Fig. S19 and Fig. S20 of the Electronic Supplementary Material, the calibration curves exhibit strong linear correlations between CN− concentrations and both absorbance and fluorescence intensities. The correlation coefficients are high, with R2 = 0.9971 for the absorbance measurements (y = − 0.063x + 0.310) and R2 = 0.9979 for the fluorescence measurements (y = − 76.697x + 430.04). The calculated detection limits were 0.355 µM for absorbance-based analysis and 0.154 µM for fluorescence-based analysis. Absorbance and fluorescence-based determination limit calculations were also performed for Li+. As shown in Fig. S21 and Fig. S22 of the Electronic Supplementary Material, the calibration curves show good linear relationships between Li+ concentration and absorbance/fluorescence intensities, with good correlation coefficients (R2 = 0.9982, y = − 0.0512x + 0.3914 from the absorbance measurements, R2 = 0.9991, y = − 94.41x + 541.7 from the fluorescence measurements). The detection limits were determined to be 0.189 µM for the absorbance-based study and to be 0.150 µM for the fluorescence-based study. The calculated detection limits for both Li+ and CN− are below the concentration thresholds for these ions permitted in drinking water [4, 73].

Biological application of MH-5 in detecting Li +and CN −

The cytotoxic effect of the MH-5 material at different concentrations on HT-29 and L9-29 cell lines was investigated using the Alamar Blue assay [55, 56]. The MH-5 material induced 21.24% and 19.65% cell death rates in cancerous and healthy cell lines, respectively (Fig. 9). These cell death rates suggest that the material has relatively low or moderate toxicity. Cell death around 20% does not indicate high toxicity; however, it still shows an effect that warrants attention.

Fig. 9

Cell viability (%) of the MH-5 on HT-29 and L9-29 cell lines. The error bars represent the mean ± SE of three independent measurements, with significance set at p < 0.05

Even though we tested the toxicology of MH-5 on two different cell lines, HT-29 was chosen for cell imaging. The ability to detect Li+ in live HT-29 was investigated using fluorescence microscopy. Live HT-29 cells incubated with 10 µM MH-5 showed weak fluorescence (Fig. 10b). From the merged image of the cell lines, it can be seen that MH-5 molecules can easily pass through the cell membrane (Fig. 10c) Then, the cells were loaded with Li+. For Li+ loading conditions, live HT-29 cells were incubated with 10 µ LiNO3 at 37 °C for 4 h and then 10 µM MH-5 was added to the culture medium and incubated at 37 °C for 30 min. As seen in Fig. 10e, HT-29 cells incubated with Li+ did not show fluorescence. The same experiments were carried out for CN− ions. The cells were loaded with CN−. For CN− loading conditions, live HT-29 cells were incubated with 10 µ CN− at 37 °C for 4 h, and then 10 µM MH-5 was added to the culture medium and incubated at 37 °C for 30 min. As seen in Fig. 10h, HT-29 cells incubated with CN− did not show fluorescence. In general, it can be concluded that Li+ and CN− ions in living cells are important molecules in selectivity by inhibiting the fluorescence properties of cell lines that are easily stained by the MH-5 chemosensor without damaging the cell lines.

Fig. 10

Fluorescence images of Li+ and CN− with MH-5 in HT-29 cell lines. a Bright-field image of HT-29 cells incubated with MH-5 (10 µM). b The fluorescence image of HT-29 cells with MH-5. c Merged image of a and b. d Bright-field image of HT-29 cells pre-incubated with LiNO3 (10 μM) and then incubated with MH-5 (10 μM). e The fluorescence image of HT-29 cells with Li+ and MH-5. f Merged image of d and e. g Bright-field image of HT-29 cells pre-incubated with CN− (10 μM) and then incubated with MH-5 (10 μM). h The fluorescence image of HT-29 cells with CN− and MH-5. i Merged images of g and h

Comparison with previously reported sensors

The sensing ability of the present sensor toward CN− and Li+ ions was also compared with others reported sensors in the literature, which is shown in Table S1. From the comparison, it is evident that the present sensor MH-5 has several advantages such as simple synthesis route, fast response time, low detection limit, and bio-imaging applications.