Plan of probe MB-MCu2+ ions have paramagnetic nature that result in quenching of fluorescence. Most of the probes show turn off response for Cu2+ ions. Some probes based on picolinate moiety have been reported to showed fluorescence “turn On” response, but these probes have some shortcoming such as short wavelength, poor water solubility, and long response time27. Keeping in mind these shortcomings we have synthesized a new probe MB-M for selective detection of Cu2+ ions. MB-M was based on methylene blue fluorophore which has excellent photophysical properties28,29,30.UV–vis selectivityThe photophysical characteristics of MB-M were investigated in Tris.HCl (10 mmol/L, pH = 7.4) containing 40% MeCN. This solvent system is selected after checking the response of probe MB-M with Cu2+ ions in different ratio as seen from SI-4. Firstly, we have checked the selectivity of MB-M by UV–vis spectrum. It shows absorbance band at 260 nm in absence of analytes. As shown in Fig. 1, when probe MB-M was treated with different ions and amino acid there was no change in absorbance band except with Cu2+ ions. When MB-M solution was treated with Cu2+ ions, the absorbance band present at 260 nm was shifted to 289 nm along with emergence of a new absorbance band at 661 nm which was a characteristic band of methylene blue. Also, the MB-M solution displayed “naked-eyes” detection as the color change from colorless to indigo after the addition of Cu2+ ions (SI-5). These results indicate that Cu2+ ions promote hydrolysis of amide linkage to free the methylene blue derivative. This result also indicate that probe is highly selective for the detection of Cu2+ ions.Figure 1UV–vis selectivity of MB-M (10 μM) with Cu2+ ions (30 μM) and other analytes in 4:6 (v/v) MeCN-Tris. HCl buffer (10 mM), pH 7.4.Fluorescence SelectivityAs shown in Fig. 2, a series of interfering cations and anions (Cu2+, Na+, K+, Cd2+, Hg2+, Zn2+, Mg2+, Fe3+, Al3+, Cl-, l-, F-, CN-, H2O2, NaHS, ONOO-, HSO3-, Hcy, Cys, GSH and NaOCl) as well as amino acids (Cys, Hcy, and GSH) were evaluated in order to check fluorescence selectivity of MB-M. MB-M fluoresced extremely weakly in the absence of any additional analytes. When Cu2 + ions were introduced to the MB-M solution, there was a noticeable emission at 689 nm. No variations in fluorescence were seen with other analytes. This may because the probe’s amide linkage is unable to interact with any analytes and to be hydrolyzed, resulting in the formation of an oxidized derivative of methylene blue with an obvious NIR fluorescence signal. The result also indicates the good selectivity of MB-M toward Cu2+ ions.Figure 2Fluorescence selectivity of MB-M (10 μM) with Cu2+ ions (30 μM) and other analytes in 4:6 (v/v) MeCN-Tris. HCl buffer (10 mM), pH 7.4. The excitation and emission slits are 5 nm and λex = 620 nm.Next, fluorescence titration was carried out with incremental additions of Cu2+ ions to gain additional insight into MB-M’s sensitivity. The probe MB-M did not show emission intensity at 689 nm when excited at 620 nm. As seen in Fig. 3, an emission band at 689 nm was clearly seen when the addition of Cu2+ ions was continued. The increase in emission intensity persisted even when Cu2+ ions concentration reached 6 equiv. After this point, there was no longer any increase in fluorescence intensity. This demonstrated that the complete hydrolysis of amide linkages had taken place. The jobs plots confirm the stoichiometry ratio, which confirm the 1:1 of probe and Cu2+ ions. (SI-6). Additionally, utilizing the 3σ/k method (SI-7), the limit of detection (LOD) for Cu2+ ions are determined as 0.33 µM which satisfies the requirements of the U.S. Environmental Protection Agency (EPA) for drinking water.Figure 3Fluorescence titration of MB-M (10 μM) with Cu2+ ions (0–6 equiv.) in 4:6 (v/v) MeCN-Tris. HCl buffer (10 mM), pH 7.4. The excitation and emission slits are 5 nm and λex = 620 nm.Reversibility of the reactionIn most cases it is seen that Cu2+ ions form complex with probe, and as a result there is an enhancement or decrease in fluorescence intensity. Some of these probe-Cu complexes were used to detect the other RSS species such as hydrogen sulfide, cysteine or EDTA. This was based on that these RSS species could remove Cu2+ ions from complex to free the probe (reverse reaction). To confirm that our synthesized probe MB-M works on the basis of hydrolysis mechanism, we added Cu2+ ions into MB-M solution and found there was strong emission at 689 nm. When a solution of EDTA, H2S or Cysteine was added, as shown in Fig. 4, there were no change in color or emission intensity. This meant that MB-M works on basis of hydrolysis, no reverse mechanism.Figure 4Irreversible reaction of MB-M + Cu2+ (10 μM) with EDTA, Cys and H2S (10 equiv.) in 4:6 (v/v) MeCN-Tris. HCl buffer (10 mM), pH 7.4. The excitation and emission slits are 5 nm and λex = 620 nm.Competitive studyThe ability of MB-M to counteract interference from rival analytes during the measurement of Cu2+ ions was also investigated. According to Fig. 5, the presence of other metal ions in high concentration had no discernible effects on the fluorescent measurement of Cu2+ ions, indicating that the fluorescence response was unaffected by the other ions. All of the aforementioned findings suggested that MB-M may be used for a variety of biological applications because it was an effective tool with high selectively sensing Cu2+ ions.Figure 5Competitive study of MB-M (10 μM) with Cu2+ ions (10 equiv.) in presence of anion in 4:6 (v/v) MeCN-Tris. HCl buffer (10 mM), pH 7.4. The excitation and emission slits are 5 nm, λex = 620 nm.Time and pH dependent fluorescenceThe time-dependent fluorescence studies using the probe MB-M were then carried out. As it can be seen in Fig. 6A, after adding 10.0 equiv. of Cu2+ ions to the MB-M (10 µM) solution, the intensity at 689 nm progressively increased. The intensity reached equilibrium in 5 min., and then remained steady. Furthermore, the effects of pH on probe MB-M fluorescent performance were examined under the excitation of 620 nm to know whether it could be used for detecting Cu2+ ions in a complicated environment. As seen in Fig. 6B, MB-M could respond to Cu2+ ions in the pH range of 2.0 to 7.0 with a notable fluorescence amplification, and the fluorescence remained stable, suggesting that probe MB-M could be used for evaluating Cu2+ ions over a broad pH range. This characteristic is very important for real-time applications.Figure 6A Time and B pH dependent fluorescence (10 μM) with Cu2+ ions (30 μM) in 4:6 (v/v) MeCN-Tris. HCl buffer (10 mM), pH 7.4. The excitation and emission slits are 5 nm and λex = 620 nm.Real sampleTo check the practical ability of probe to daily life, we have applied it to detect the Cu2+ ions in pond water. Pond water is taken from the lake in Zhengzhou university. The sample is prepared by adding 60% pond water and 40% MeCN to it. After MB-M (10 µM) added, the test was performed in 30 mints and results depicted in Fig. 7A. It can be seen that MB-M can be used to detect Cu2+ ions in pond water. We also prepared test strips with probes pre-loaded on paper to increase the practical ability of probe to daily life. It was fascinating to found that only aqueous solutions of Cu2+ ions could result in color changes which is visible by “naked eye” (Fig. 7B). The probe MB-M showed excellent properties as compared to the already reported probes as shown in Table 1.Figure 7(A) Detection of Cu2+ ions in pond water and (B) Test strip for Cu2+ ions detection.Table 1 A comparison table of Probe MB-M with other reported probes.Sensing mechanismFrom obtained spectral data, we thought that the hydrolysis of amide bond to produce oxidized methylene blue may be responsible for the MB-M’s response towards copper ions (Scheme 2). In order to clarify the aforementioned proposal, HR-MS spectrum of MB-M solution after the addition of copper ions was checked. There was a new peak at 284.1212 which was consistent with the mass of oxidized methylene (SI-8). This illustrate that Cu2+ ions can promote hydrolysis of amide linkage in MB-M to form oxidized methylene blue. The sensing mechanism was further verified by taking 1HNMR of compound after the addition of copper ions SI-9. The spectrum showed that oxidized methylene blue is formed after the hydrolysis.Scheme 2Proposed response mechanism.Cell imagingThe cytotoxicity of MB-M towards HepG-2 cells was assessed using an MTT test before the research of fluorescence imaging in live cells. Figure 8 revealed that more than 90% cell survival at a concentration of 20 µM of MB-M.Figure 8The cytotoxicity of MB-M towards HepG-2 cells.We then investigated MB-M’s applicability in biological systems. Only probe MB-M (20 µM) was used to incubate HepG-2 cells in the control group, and they displayed negligible fluorescence (Fig. 9A–C). On the other hand, the intensity of the intracellular fluorescence was dramatically increased when it was exposed to MB-M (20 µM) and Cu2+ ions (100 µM) (Fig. 9D–F). As the concentration of Cu2+ ions were increased, the fluorescence was increased (Fig. 9G–L). This phenomenon show that MB-M is cell membrane permeable and may use as a potential sensor for Cu2+ ions detection in living cells.Figure 9Fluorescence of MB-M (20.0 μM, 30 min) loaded living HepG-2 cells (A–C) and further treated by Cu2+ (100 to 300 μM, 30 min) (D–L). Bright fields (A, D, G and J), fluorescence images (B, E, H and K), and overlay images (C, F, I and L). λex:638 nm; λem:660–720 nm (B, E, H and K).Additionally, we used the probe MB-M in the cuproptosis investigation (Fig. 10). We aim to investigate whether HepG-2 cells can experience cuproptosis and develop treatment resistance of Hepatocellular Carcinoma (HCC) utilizing disulfiram-copper as inducer. Upon being incubated for two hours with varying concentrations of Disulfiram (3, 5, 10 μM) and 30 μM Cu2+ , the majority of cells exhibited swelling and flotation in contrast to those that were alone incubated with Cu2 + ions. However, compared to the control group that only included Cu2+ ions, the fluorescence intensity of the cuproptosis group was significantly higher. It has been proposed that disulfiram helped cells absorb an excessive amount of copper, which ultimately caused the cells to die.Figure 10Fluorescence of Probe MB-M (10.0 μM, 30 min) loaded living HepG-2 cells (A–C) and further treated by Cu2+ (30 μM, 30 min) (D–F). Fluorescence images of probe after treating with different concentration of disulfiram (3, 5 and 10 uM) and Cu2+ (30 μM) ions (G-O). Merge is overlay images of bright and red channel. λex: 638 nm; λem: 660–720 nm.Theoretical calculation researchMethodAll the DFT calculations were performed at B3LYP/6-31Gg (d, p) level. Interaction between probe BM-M and Cu2+ ions was determined by performing non-covalent interaction (NCI) and quantum theory of atoms in molecule (QTAIM) analysis. The sensitivity of probe towards Cu2+ ions was examined by analyzing the electronic properties of the complex through natural bond orbital (NBO), electron density difference (EDD) and frontier molecular orbital (FMO) analysis. NCI and RDG plots were used to evaluate the Van der Waals and covalent and repulsive interactions. The study of topological parameters was established through QTAIM analysis. The charge transfer study by NBO, EDD and FMO analysis showed the high sensitivity of probe towards Cu2+ ions.Computational procedureThe DFT simulations were performed using Gaussian 09 software35 and for all the geometric optimization and interactions analysis B3LYP/6-31G(d,p) level of DFT was utilized. B3LYP is considered a reliable functional for interaction studies36. The values of interaction energies were computed using the Eq. (1).$$ {\text{E }} = {\text{ E}}_{{{\text{complex}}}} {-} \, \left( {{\text{E}}_{{{\text{sensor}}}} + {\text{ E}}_{{{\text{analyte}}}} } \right) $$
(1)
Here Ecomplex, Esensor and Eanalyte represents the energy of complex, probe and analyte respectively. NCI analysis was performed using Multiwfn 3.837 and VMD38 to identify the nature of non-covalent interactions between adsorbate and adsorbent surface. In non-covalent interaction studies, QTAIM analysis perform a crucial role by identifying all the intermolecular as well as intramolecular interactive forces. In, QTAIM analysis, The strength and the type of interactive forces between the atoms were characterized through total electron density (ρ), potential energy density (V(r)), total energy density (H(r)), kinetic energy density (G(r)), Laplacian of the electron density (∇2ρ)39. NBO and EDD analysis were employed to study charge transfer. The sensitivity of probe towards the analyte was further determined by FMO analysis where HOMO–LUMO energy gap was computed by using Eq. (2)40$$ {\text{E}}_{{{\text{gap}}}} = {\text{ E}}_{{{\text{LUMO}}}} – {\text{ E}}_{{{\text{HOMO}}}} $$
(2)
Geometric optimizationThe optimized geometry of the probe MB-M consists of fused benzene and thiomorpholine ring that are connected to morpholine ring through carbonyl group. The optimized geometry of the bare probe is given in Fig. 11 a. The interaction of Cu2+ ions was studied at four different positions of probe as shown in Fig. 11b–e. The interaction energy values of the Cu@probe complexes were found to be −21.247 kcal/mol (at site A), −30.569 kcal/mol (at site B), −27.671 kcal/mol (at site C) and −30.655 kcal/mol (at site D). Among all the studied configurations of the complexes the maximum value of adsorption energy (as indicated by larger negative value) was observed to be −30.655 kcal/mol at site D where Cu2+ ions showed interaction with the oxygen of carbonyl group.Figure 11(a) Optimized geometry of probe, (b) Optimized geometry of Cu@probe at site A, (c) Optimized geometry of Cu@probe at site B, (d) Optimized geometry of Cu@probe at site C, (e) Optimized geometry of Cu@probe at site D, (f) QTAIM analysis of Cu@probe complex.NCI analysisThe type of interacting forces between probe and analyte were critically analyzed by 3D isosurface and 2D RDG plot of the complex. The blue and green color in the isosurface and RDG plot represents hydrogen bonding/ion–dipole and weak Van der Waals interactions while red color indicates repulsive forces between the atoms. In 2D RDG plot, (λ2)ρ is plotted against RDG (s) by considering following equation$$ s = \frac{1}{{2\left( {3\pi^{2} } \right)^{\frac{1}{3}} }}\frac{\nabla \rho }{{\rho^{4/3} }} $$
(3)
The value of (λ2)ρ describes the kind of interactions present in the respective compound. The value of (λ2)ρ > 0 describes repulsive forces whereas the value of (λ2)ρ < 0 describes attractive forces between analyte and probe41. The NCI isosurface and RDG plot of the Cu@probe is shown in Fig. 12 a and b. For NCI analysis, complex with high interaction energy was selected to observe the nature of interactions. The resulting 2D RDG and 3D isosurfaces plots contained red and green color spikes and patches which arise due intra-atomic non-covalent interactions of probe atoms. However, dark blue patches can be clearly seen between analyte (Cu) and O and H-atoms of probe. The absence of blue spikes in 2D RDG plot shows that interactions between probe atoms and Cu2+ ions are shared shell instead of closed shell interactions. The findings of NCI analysis are consistent with interaction energy where interaction energy values lie in chemisorption range.Figure 12(a) NCI isosurface of Cu@probe complex, (b) 2D RDG plot of Cu@probe complex, (c) 3D EDD isosurface of Cu@probe complex, (d) HOMO–LUMO orbital density of probe, (e) HOMO–LUMO orbital density of Cu@probe complex.QTAIM analysisIn order to determine the kinds of bonds and non-covalent interactions between analyte and probe, Bader et al. proposed the QTAIM analysis42. Bond critical points which usually describe the nature of non-covalent interactions have been elaborated using different parameters in QTAIM, such as total electron density (ρ), potential energy density (V(r)), total energy density (H(r)), kinetic energy density (G(r)), Laplacian of the electron density (∇2ρ). The value of electron density less than 0.1 indicates non-covalent interactions while value of electron density greater than 0.1 indicates the covalent nature of bond43. H(r) and ∇2ρ values greater than zero indicate non-covalent contact and less than zero show covalent contact44. Similar to this, a ratio of -V(r) to G(r) less than 1 represents a non-covalent interaction while greater than 1 denotes covalent interaction45. The QTAIM geometry of the complex is displayed in Fig. 11f. Three BCPs 1, 2 and 3 were observed in the Cu@probe complex with ρ value of 191.351, 117.801 and 0.376 respectively, indicating the shared shell interactions sensor atoms and Cu2+ ions atom. The BCP 1 appeared between the oxygen of carbonyl and Cu2+ ions with H(r) and ∇2ρ values of −0.2415419007E + 06 and −0.9661013778E + 06 (less than zero) respectively, also demonstrating covalent interactions between oxygen and Cu2+ ions. In addition, the BCP 3 generated between hydrogen of benzene ring and Cu2+ ions with H(r) and ∇2ρ values of −0.4304606513E + 01 and −0.1718000329E + 02 respectively, reveal the existence of covalent interaction. The values of H(r) and ∇2ρ was found to be −0.1088779546E + 06 and −0.4354914032E + 06 (less than zero) respectively.NBO and EDD analysisNBO analysis was executed to evaluate the charge transfer mechanism between the probe and analyte. The complexation of probe with Cu2+ ions resulted in the transfer of charge towards Cu2+ ions from the probe. The observed value of charge transfer from probe towards Cu2+ ions was found to be 0.046 e-. EDD analysis was performed to further determine the area of charge density between sensor and Cu2+ ions. The accumulation and depletion of charge density between the interacting fragments can be determined by the appearance of light blue and purple color patches respectively in the isosurface of the complex. The appearance of light blue patch on the oxygen atom of carbonyl and purple color patch on Cu2+ ions indicated the transfer of charge density from sensor towards Cu2+ ions (as shown in Fig. 12c). The study of charge transfer mechanism by NBO and EDD analysis indicated the existence of strong interactive force between sensor and Cu2+.FMO analysisThe sensitivity of probe towards analyte can be defined by evaluating the electronic properties of probe before and after complex formation with the respective analyte46. High sensitivity of probe for the analyte is a consequence of significant reduction in the magnitude of HOMO–LUMO gap after complexation of probe with analyte47. The energies of HOMO and LUMO of bare probe were found to be −4.695 eV and −0.151 eV respectively and the calculated value of HOMO–LUMO energy gap was 4.544 eV. After the complexation of probe with Cu2+ ions, the observed energies of HOMO and LUMO were −3.407 eV and −0.546 eV respectively, with the HOMO–LUMO energy gap of 2.861 eV. The significant reduction in the energy gap after complex formation depicts high sensitivity of probe for Cu2+ ions. Along with the reduction in HOMO–LUMO energy gap value, the shifting of orbital density of bare probe and its complex with Cu2+ ions have been presented in Fig. 12,e. In the case of free probe, the orbital density of HOMO is spread over entire molecule while the orbital density of LUMO is shifted towards benzothiazine part of the compound. In the case of Cu@probe complex, the orbital of HOMO is scattered over carbonyl and Cu2+ ions whereas the orbital density of LUMO is also spread over the probe surface.
An activatable NIR turn-on fluorescent probe for copper (II) ion and live cell imaging
