Читать книгу Handbook of Aggregation-Induced Emission, Volume 2 - Группа авторов - Страница 43

Metal elements exist widely in nature and have applications in various fields of human daily life. Many metal ions support normal life processes and play an irreplaceable role in the organism. For example, as the second messenger in cells, calcium ions are of great importance in the process of signal transmission. Another example is iron ions, which are converted to each other in the form of ferrous and iron in human body. Inadequate intake of iron ions can cause diseases such as anemia and dysplasia, while excessive intake of iron ions can cause oxidization to damage the body, thereby endangering the human heart and circulatory system. In contrast, some metal ions, even when present in trace amounts in the environment, can cause great harm to living organisms. For example, even traces of chromium(VI) ions enter the human body; it will cause serious damage to human skin, respiratory system, kidneys and other tissues, and even cancer. Therefore, the development of simple and practical metal ion detection methods has always attracted great interests of researchers.

Schiff base compounds have lone‐pair electrons on the nitrogen atom in the structure, so that they can complex with metal ions, thereby changing the overall structure and properties of the compound. Due to the simple synthesis of Schiff base compounds, high yields, rich sources of raw materials, and wide choices, especially their good coordination properties, they have been widely used in the preparation of metal ion complexes. SSB fluorophores are therefore with unique advantages in AIE probes for metal ion detection. Additionally, nitrogen in the imine bond and hydroxyl oxygen atoms contained in SSB have excellent metal coordination ability, and the formed metal ion complex has a large stability constant, which is helpful to reduce the detection limit [8]. Because the coordination ability is influenced dramatically by the hydroxyl oxygen atom, which will experience deprotonation at high pH conditions, by adjusting the pH value, the probe can be recycled, and “logic gate” systems for the detection of both metal ions and pH can be designed accordingly [9–11]. In this section, SSB‐based metal ion probes are classified according to different response mechanisms, and three different types of turn‐off, turn‐on, and ratiometric are included.

Turn‐off metal ion probes have strong fluorescence in their aggregate state and fluorescence quenching after binding with target metal ions. Strong fluorescence comes from the AIE property of the probe, and after binding with metal ions, charge transfer between the metal ion and the probe molecule, i.e. metal‐to‐ligand charge transfer (MLCT), occurs, thus resulting in chelated fluorescence quenching. Most reported typical turn‐off metal ion probes are for Cu²⁺, which is due to the special electronic structure of divalent copper ions. The d9 valence electron layer configuration, which has a single electron, is prone to fluorescence quenching due to MLCT process [12]. Figure 3.3 summarizes representative fluorescent quenching metal ion probes [10,13–16]. Among them, probe 3 is special, the fluorescence of which is first enhanced by combining with zinc ions and then quenched by adding cobalt(II) ions, achieving in detection of both ions in sequence.

Tong et al. prepared a Schiff base‐immobilized hybrid mesoporous silica membrane that can be used for the detection of Cu²⁺ in real‐water samples by immobilizing the Schiff base‐immobilized hybrid mesoporous membrane (SB‐HMM) on the pore surface of mesoporous silica (pore size 3.1 nm) [16]. Fluorescent probe 1 was grafted to the mesoporous silica surface embedded in the porous alumina membrane channels to form SB‐HMM (Figure 3.4a). Probe 1 is not emissive in the homogeneous solution, but SB‐HMM emits strongly due to the aggregation of SSB groups with ESIPT and AIE on the surface of the pores, which enhances fluorescence intensity. The high quantum yield of probe 1 on the surface of SB‐HMM can be used as a fluorescence sensor for Cu²⁺ in aqueous solution and has good sensitivity, selectivity, and reproducibility. SB‐HMM showed significant fluorescence decrease after Cu²⁺ (0–60 μM) was added, which showed good selectivity from other common metal ions (Figure 3.4b). Under the optimal conditions, the detection limit of SB‐HMM is 0.8 μM. In addition, SB‐HMM can be reused for Cu²⁺ sensing after regeneration from an acidic solution, resulting in a reusable dye‐doped fluorescent solid sensor that can be applied for Cu²⁺ sensing in aqueous solutions and other real Cu²⁺‐containing samples (Figure 3.4c).

Liu et al. synthesized SSB AIE Cu²⁺ probes 5 and 6. Compared to the dispersed state, the fluorescence enhancement factors of the two probes in poor solvents are about 300‐ and 34‐folds, respectively. After adding Cu²⁺, the emission intensities of 5 and 6 were significantly reduced [14]. The fluorescence of the solution changed from green to colorless under a 365‐nm UV lamp, and the detection limits for copper ions were 200 ± 23 and 10 ± 0.3 nM. Through dynamic light scattering (DLS) analysis, scanning electron microscopy (SEM), proton nuclear magnetic resonance (¹H‐NMR) titration, electrospray ionization‐mass spectrometry (ESI‐MS), and other analytical methods, it was found that after adding Cu²⁺, the imine bonds in 5 and 6 coordinate with the copper ions, under the strong chelation‐enhanced fluorescence quenching (CHEQ); the rigid probe aggregates are bent and rearranged to form a fluorescent‐quenched complex (Figure 3.5A). After adding excess EDTA, the fluorescence of the system could not be recovered, indicating the irreversibility of the process. Subsequently, 5 and 6 (10 μM) were applied to intracellular Cu²⁺ detection. The probe showed a strong green fluorescence under confocal microscope, and an obvious turn‐off was observed after Cu²⁺ was added, indicating the good cell membrane permeability and intracellular Cu²⁺ detection ability of the probe (Figure 3.5C).

Figure 3.3 Chemical structures of typical turn‐off metal ion probes 1–7.

Figure 3.4 (a) Scheme for immobilization of 4‐chloro‐2‐[(propylimino) methyl]‐phenol (4Cl‐PMP) groups on the surface of mesoporous silica in hybrid mesoporous membranes (HMM, (3‐aminopropyl)triethoxysilane (APTES)‐HMM, SB‐HMM). (b) Fluorescence spectra of SB‐HMM upon the addition of Cu²⁺ (0, 5, 10, 20, 30, 40, 50, and 60 M). The inset shows the changes in the fluorescence intensities of SB‐HMM with and without Cu²⁺ and other metal ions (80 (M): 1, K⁺; 2, Ca²⁺; 3, Fe²⁺; 4, Fe3⁺; 5, Zn²⁺; 6, Ni²⁺; 7, Cd²⁺; 8, Pb²⁺; and 9, Cu²⁺. I₀ and I are the excitation peak intensities of SB‐HMM without and with metal ions, respectively. (c) Regeneration/reuse cycles for SB‐HMM upon the addition of 80 M Cu²⁺.

Source: Reprinted from Ref. [16] (Copyright 2011 Elsevier B.V.).

The fluorescence turn‐on metal ion probe emits no fluorescence or weak fluorescence and fluoresces strongly after interacting with metal ions. Compared with fluorescence turn‐off probes, the background fluorescence is weaker and thus higher signal‐to‐noise ratio and sensitivity, which is more preferable for the detection of metal ions in biological environments [17]. Most SSB turn‐on metal ion probes are mainly reported for Zn²⁺ detection. The specific molecular structures are summarized in Figure 3.6, as shown below. Probes 8–13 are zinc ion detection probes, and probes 14–16 are used for the detection of Al³⁺, Cu²⁺, and Ca²⁺, respectively [8,17–25].

Figure 3.5 (A) Proposed mechanism for AIE and self‐assembly of 5 or 6 by Cu²⁺. (B) Fluorescence spectra of probe 5 upon the addition of 0–10 equiv. Cu²⁺. Inset: Changes of intensity at 534 nm with [Cu²⁺]/[5]. (C) Human esophageal squamous KYSE510: (a) bright‐field image of cells incubated with 10 μM 5 for 30 minutes, (b) fluorescence image of 5, (c) fluorescence image of 5 in the presence 100 μM Cu²⁺ for 15 minutes, (d) bright‐field image of cells incubated with 10 μM 6 for 30 minutes, (e) fluorescence image of 6, (f) fluorescence image of 6 in the presence of 100 μM Cu²⁺ for 15 minutes at 37 °C.

Source: Reprinted from Ref. [14] (Copyright 2017 Elsevier B.V.).

Tong et al. reported a Zn²⁺ fluorescence turn‐on probe 8 based on SSB [24] (Figure 3.7). In a 99% water/DMSO mixed solvent, according to the gradual increase of the Zn²⁺ concentration, the absorption peaks at 310 and 346 nm in the UV absorption spectrum gradually decreased and the newly generated absorption peaks at 333 and 383 nm gradually increased. In the fluorescence titration performed under the same conditions, the initial fluorescence of the solution was very weak. With the addition of zinc ions, a gradually increasing fluorescence peak appeared at 460 nm, and its saturated fluorescence intensity reached 22‐folds compared with the initial intensity. A bright blue fluorescence was observed under UV light. Job's plot and ESI–MS results gave the binding ratio of the probe to the metal ion as 1 : 1, and the binding constant was calculated as 5 × 10⁴ l/mol. The effects of different substituents of salicylaldehyde derivatives were studied by synthesizing various analogues of probe 8. At neutral pH, all substituted derivatives other than 4‐N,N‐diethylamine salicylaldehyde containing a strong electron‐donating group showed fluorescence turn‐on with zinc ions after condensation reaction with 2‐hydrazinopyridine. Among them, probe 8 exhibits the highest fluorescence enhancement and longer fluorescence emission wavelength. Under pH = 7, the detection linear range is 0.1–1 μM, and the detection limit is 30 nM. Immunity experiments show that only paramagnetic Cu²⁺ and Co²⁺ ions cause fluorescence quenching and affect zinc ion detection. Intracellular imaging of zinc ions was performed in HeLa cells, and significant intracellular fluorescence enhancement was observed under a confocal microscope.

Figure 3.6 Chemical structures of typical turn‐on SSB metal ion probes 8–16.

Figure 3.7 (A) Chemical structures of 8 analogues: 8a–f. (B) Fluorescence spectra of 10 mol/l 8 upon the addition of 0–15 mol/l Zn²⁺. Inset: the fluorescence intensity at 460 nm as a function of the Zn²⁺ concentration; excitation was at 364 nm. (C) Fluorescence images of probe 8 before and after adding zinc ion under UV light. The photographs on the right side show 8 in the absence and presence of 8 equiv. Zn²⁺ in a glass cuvette excited by sunlight and UV light (365 nm). (D) Fluorescence images of live HeLa cells. From left to right are bright‐field images, fluorescence images, and overlay images. Top (a–c): cells were incubated with 8 for 20 minutes and washed with TBS twice. Bottom (d–f): cells were incubated with 8 and then with Zn²⁺ (10 mol/l) and pyrithione (10 mol/l) for 20 minutes and washed with TBS twice. Emission was collected at 430–490 nm upon excitation at 405 nm.

Source: Reprinted from Ref. [24] (Copyright 2013 Elsevier B.V.).

The detection range of common metal ion fluorescent probes is generally at the nanomolar and micromolar levels. Few fluorescent probes detect metal ions at the millimolar level. This is because when the metal ion concentration is too high, an aggregation‐caused quenching (ACQ) effect may occur and thus result in an emitting annihilation of classical fluorescent probes. However, the Ca²⁺ concentration related to human diseases is generally in the millimolar range, which begets great challenges for the development of practically applied fluorescent probes for measuring the Ca²⁺ concentration in the human body. Tang's group has developed a calcium ion fluorescent probe 16 with a detection range of 0.6–3.0 mM, which is suitable for the detection of abnormally high blood calcium concentration (Figure 3.8A) [17]. The fluorescence quantum yield of the probe in the pure tetrahydrofuran (THF) solution was 0.23%, and it increased to 10.6% in the solid state. With the increase of Ca²⁺ concentration, the emission of probe 16 at 560 nm was significantly enhanced (Figure 3.8B). Some common metal ions and biological molecules (such as bovine serum albumin [BSA], porcine hemoglobin [PHB], and fetal bovine serum [FBS]) exhibit no interference with the fluorescence spectrum of the probe (Figure 3.8C). UV–vis absorption and high‐resolution mass spectrometry (HRMS) and isothermal titration calorimetry (ITC) determined that the coordination number between probe 16 and calcium ions was 1 : 1. Transmission electron microscopy (TEM) showed that probe 16 formed fibrous aggregates in the presence of calcium ions and irregular aggregates in the absence of calcium ions. The formation of regular aggregates induced by Ca²⁺ significantly enhanced fluorescence. Biological imaging of calcium deposits was performed in the soft tissue of human squamous meningioma and the cracks on the surface of the bovine bone to test the practical applicability of probe 16, and the results were satisfactory (Figure 3.8D, E). Compared with the commonly used calcium ion probe calcein, probe 16 shows advantages of a weak background signal, wash‐free imaging, and higher sensitivity.

Most metal ion probes are tested based on the fluorescence “on–off” mechanism, and the detection result is only related to the fluorescence intensity at a single wavelength, while the ratiometric fluorescence probe takes the advantage of the ratio change of the fluorescence intensity at two wavelengths for analyte detection, which can minimize errors caused by differences in experimental conditions and self‐emission of samples. Figure 3.9 shows a summary of ratiometric metal ion fluorescent probe structures based on SSB. Ratiometric fluorescent probes generally contain a stable fluorescent emission moiety and an active site that can react with metal ions. After reacting with metal ions, a fluorescent emission at another wavelength is generated, and the original emission peak intensity is unchanged or decreased, thereby realizing ratiometric detection [26–29].

Tong et al. developed a dual‐wavelength ratio fluorescent probe 17 for Zn²⁺ based on rhodamine B salicylaldehyde fluorene derivative [28]. In general, salicylaldehyde hydrazone structure can generate a strong fluorescence enhancement response with zinc ions, but the fluorescence intensity is low in the absence of metal ions. In order to achieve the effect of the dual‐wavelength ratiometric response, the probe needs to have a similar quantum yield before and after the reaction with the metal ions. Rhodamine dyes have a longer excitation wavelength, higher quantum yield, and suitable water solubility. Combining 4‐N,N‐diethylamine salicylaldehyde hydrazone structure with rhodamine dyes, ratiometric zinc ion probe 17 was designed and synthesized. By exciting the fluorescence at the iso-absorption point at 400 nm, the maximum emission wavelength is red-shifted by 17 nm before and after the probe 17 binding with zinc ions, and the iso-emission point appears at 500 nm (Figure 3.10b, c). Under the UV lamp, a clear fluorescent color change of the solution from dark blue-green to yellow-green was achieved and observed by naked eye. The fluorescence intensity ratio at 530 and 475 nm is linear in the Zn²⁺ concentration range of 0–10 μM and is independent of other interfering ions. The minimum detection limit is 0.05 μM. Other probes containing rhodamine and salicylaldehyde hydrazone for Zn²⁺ detection were also synthesized, and the salicylaldehyde structure is found to be indispensable for ratiometric detection of zinc ions due to the different emissions of the probe and probe–zinc complex, as shown in Figure 3.10a.

Figure 3.8 (A) Illustration of the light‐up detection of Ca²⁺ with probe 16. (B) Photoluminescence (PL) spectra of 16 treated with CaCl₂ at different concentrations in phosphate‐buffered saline (PBS) buffer solution (pH = 7.4). (C) PL spectra of 16 in PBS buffer upon the addition of various metal ions and biological molecules. (D) Confocal laser scanning microscopy (CLSM) images of bovine bone microcracks by staining with calcein and 16: (a–c) whole‐projection images (z stack) and (d–f) 3D images of the microcrack stained with calcein; (g–i) whole‐projection images (z stack) and (j–l) 3D images of the microcrack stained with 16. (E) CLSM images of CaCl₂ embedded in calcium deposits in psammomatous meningioma slice: (d) the bright‐field images; (e) the fluorescence images; and (f) the merged images.

Source: Reprinted from Ref. [17] (Copyright 2018 American Chemical Society).

A ratiometric fluorescent probe 18 using 3‐hydroxyflavones and salicylaldehyde hydrazone Al³⁺ ion was reported [26]. The fluorescence color of the probe 18 solution changed from yellow to white after the addition of aluminum ions due to the emission intensity at 461 nm being increased but the intensity at 537 nm decreased as shown in Figure 3.11A. I₄₆₁/I₅₃₇ has a linear relationship with the concentration of aluminum ions (Figure 3.11B). The lowest detection limit measured under the experimental conditions is 0.29 μM (7.8 ppb), and the fluorescence response is stable in the pH range of 3.0–10.0. DLS showed that the average diameter of aggregates decreased significantly before and after the interaction with aluminum ions, indicating that aluminum ions and probe 18 could form a water‐soluble complex, but there were still aggregates of probe 18 in the solution, thus showing that a white color fluorescence originated from the mixed color of the probe aggregate and the complex. The coordination ratio between probe 18 and the aluminum ion is 1 : 1 based on Job's plot; NMR titration indicates that both hydroxyl groups in the probe molecule participate in the coordination with the aluminum ion. Selectivity experiments with other metal ions show that a significant increase in I₄₆₁/I₅₃₇ was only found in the presence of Al³⁺ (Figure 3.11C). HeLa cells were selected for intracellular experiments, and the fluorescence signal was only observed in the yellow light channel. With the addition of aluminum ions, the yellow light signal was basically unchanged under the confocal microscope, and the fluorescence of the blue light channel gradually increased, showing that probe 18 can successfully perform ratio imaging of aluminum ion in cells (Figure 3.11D).

Figure 3.9 (a) Chemical structures of ratiometric fluorescent probes 17–19 based on SSB. (b) The proposed 1 : 2 metal‐to‐ligand ratio binding model of probe 19 with Zn²⁺. (c) Absorption and ratiometric fluorescence spectrum response of probe 19 with Zn²⁺.

Source: Reprinted from Ref. [27] (Copyright 2009 Elsevier B.V.).