Читать книгу Handbook of Aggregation-Induced Emission, Volume 2 - Группа авторов - Страница 44

Except large amounts of reports for metal ion detection, SSB derivatives were also designed for inorganic species, small organic molecules, as well as macromolecules in biologically and environmentally related analytes. Many reports concern for charged species detection; the probes are usually designed by ionizing AIEgens to increase their solubility in water and rendering them nonfluorescent or weakly fluorescent. In the presence of target molecules with opposite charges, molecular aggregates formed and fluorescence‐enhanced signal was achieved. A series of fluorescent probes with excellent detection properties have been developed by the functionalization of the salicylaldehyde moieties [30] and were successfully applied to the detection of inorganic species [31–38], environmental pollutants [7, 37], biological inorganic molecules [38, 39], amino acids [40, 41], proteins [42–44], enzymes [45–47], and so forth. Herein this section is divided into three aspects: (i) inorganic species, (ii) small biomolecules, and (iii) biomacromolecules to introduce the design and applications for SSB‐based probes in chemical and biological sensing.

Figure 3.10 (a) Chemical structure of probe 17 and binding mode with zinc ion verified by ¹H‐NMR and mass spectra. (b) Fluorescence intensity ratio (F₅₃₀/F₄₇₅) of 17 (10 μM) and 17 with Zn²⁺ (10 μM) in the absence and presence of different metal ions. (c) Absorption spectra of 17 (10 μM) in the presence of 0–50 μM Zn²⁺ in 90% (v/v) ethanol/water (10 mM (4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid) (HEPES), pH 7.0). Inset: Job plot analysis of 17 and Zn²⁺ at a total concentration of 33 μM, indicating the formation of a 1 : 1 metal‐to‐ligand complex. (d) Fluorescence excitation (a) and emission (b) spectra of 17 (10 μM) in the presence of different concentrations of Zn²⁺ in 90% (v/v) ethanol/water (10 mM HEPES, pH 7.0). Inset: fluorescence intensity at 530 and 475 nm and the ratio of F₅₃₀ : F₄₇₅ as a function of Zn²⁺ concentration added to 17.

Source: Reprinted from Ref. [28] (Copyright 2010 John Wiley & Sons, Ltd.).

Figure 3.11 (A) Fluorescence spectra of 18 (20 mM) upon the addition of different concentrations of Al³⁺ (0–500 mM) in 10 mM HEPES buffer at pH 7.0 (containing 0.2% DMSO). Excitation wavelength is set at 370 nm. (B) Calibration curve based on the ratio of fluorescence intensities (I₄₆₁/I₅₃₇) as a function of Al³⁺ concentrations; error bar represents three repeated experiments. (C) Fluorescence intensity ratio (I₄₆₁/I₅₃₇) of 18 (20 mM) upon the addition of 500 mM metal ions in 10 mM HEPES buffer at pH 7.0 (containing 0.2% DMSO). Ions from 1 to 18: blank, Li⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Ba²⁺, Sr²⁺, Fe²⁺, Fe³⁺, Co²⁺, Cu²⁺, Hg²⁺, Ag⁺, Cd²⁺, Mn²⁺, Ga³⁺, and Al³⁺. (D) Confocal fluorescence images of live HeLa cells. (a–d) The cells were incubated with 18 (5.0 mM) for 30 minutes; (e–h) the above cells upon the addition of 200 mM Al³⁺ were then incubated for another 20 minutes; (a) and (e) bright‐field transmission images; (b) and (f) blue channel images at 429–469 nm; (c) and (g) yellow channel images at 511–611 nm; (d) and (h) ratio images generated from (b) and (c) and (f) and (g), respectively. The excitation was set at 405 nm.

Source: Reprinted from Ref. [26] (Copyright 2014 Elsevier B.V.).

The detection of inorganic species such as CN⁻, F⁻, UO₂²⁺, S²⁻, and ClO⁻ is of great significance in environment, biology, and industry. Cyanide (CN⁻) is one of the most powerful poisons. It affects vascular, vision, cardiac, endocrine, and metabolic functions and causes fatal damage to the nervous system. The fluoride ion (F⁻) is very useful in the treatment for osteoporosis and orthodontics. However, excess F⁻ leads to fluorosis, which results in the increment in the bone density. Taking the advantage of hydrogen bonding with the hydroxyl group of SSB, the detection of such basicity anions like CN⁻ and F⁻ is facile by SSB fluorescent probes with high sensitivity and satisfactory selectivity. As shown in Figure 3.12a and b, after adding CN⁻, probe 20 undergoes deprotonation due to the basicity of CN⁻. Cyclization reaction occurred under the catalysis of CN⁻, and the corresponding benzoxazole formed gradually in this process, therefore lighting up the fluorescence [34]. The detection limit is 5.92 × 10⁻⁷ M, lower than the WHO guideline of CN⁻ in drinking water (1.9 μM). The competitive experiments reveal high sensing selectivity and sensitivity of 20 for CN⁻ over other anions (Figure 3.12c). Test papers were also prepared for the practical application of cyanide detection. By influencing hydrogen bonding, SSB‐based fluorine ion fluorescent probes were also reported. Figure 3.12d lists some SSB probes for F⁻ detection. The possible processes and mechanisms are proposed, as demonstrated in Figure 3.12e. The addition of F⁻ resulted in a blue‐shifted emission, which was increasing gradually with an increasing concentration of F⁻. This change was explained by the disruption of the existing six‐membered hydrogen bonding, involving the hydroxyl group and imine nitrogen, which allow the ESIPT process through deprotonation of the phenolic hydroxyl group. Excellent experimental results of detection limits and selectivity over other anions were also obtained in real sample detection (Figure 3.12f, g).

Chen et al. reported a fluorescence turn‐on SSB probe 25 for uranyl ion (UO₂²⁺) detection with high efficiency [32]. As one of the radioactive metal elements, uranium is an important raw material for the nuclear industry. At present, uranium‐based nuclear power facilities have gradually increased their proportion in power generation facilities in various countries. Metal uranium is extremely radioactive and chemically toxic, with a half‐life of hundreds of millions of years, which can cause lasting disturbances and damage to the immune, reproduction, and hematopoietic systems of the organism. Uranyl ion (UO₂²⁺) is the most commonly existing formation of uranium in natural water; therefore, the detection of UO₂²⁺ in water is of great significance for the assessment of water pollution. As illustrated in Figure 3.13a, 25 undergoes a complex interaction and forms aggregates with the addition of UO₂²⁺, exhibiting a fluorescence enhancement at 540 nm, which was linearly related to the concentration of UO₂²⁺ in the range of 1–25 ppb. The limit of detection was achieved as low as 0.2 ppb with a relative standard deviation (RSD) of 1.3% (Figure 3.13b, c). Such a detection method was successfully utilized in quantifying UO₂²⁺ in fuel processing wastewaters. Other fluorescent probes based on salicylaldehyde azine derivatives for the detection of sulfur(38) (S²⁻), hypochlorite(37) (ClO⁻), and so on were also developed by the modification of a metal complex of SSB.

In addition to necessary metal ions and nonmetal ions, a number of biologically small molecules are also important components to maintain the normal metabolic activities of complex biological organisms. For instance, pyrophosphate ion (PPi) is an important biologically related inorganic species, which plays an important role in the synthesis of DNA, RNA, and proteins and in life activities such as signal transduction. Studies have shown that the occurrence of arteriosclerosis and osteoarthritis is closely related to abnormal pyrophosphate levels in human body. In recent years, the analysis and detection of pyrophosphate have attracted increasing attention of scientists. Pyrophosphate has a strong coordination with Cu²⁺. Most fluorescent detection methods for pyrophosphate are based on this principle. Due to the superior coordination ability of SSB and Cu²⁺, SSB–copper(II) complex is therefore very suitable for the design and synthesis of pyrophosphate probes. As Figure 3.14a shows, Tong and coworkers developed a facile PPi fluorescence turn‐on probe 26 of copper(II) complex [39]. The AIE fluorescence of 26 was completely quenched due to the coordination of Cu²⁺. The complexation of PPi with Cu²⁺ resulted in the release of free 26, which reaggregated in the solution and recovered the orange fluorescence. Figure 3.14b reveals good selectivity of the probes with other common anions. The fluorescent signal enhancement showed excellent linear relationship with a PPi concentration range of 0–15 μM, and the detection limit was obtained as 0.064 μM.

Figure 3.12 (a) Chemical structures of cyanide probes 20 and 21. (b) Proposed sensing mechanism of sensor 20 for the detection of cyanide. (c) Fluorescence spectra (λ_ex = 347 nm) and photographs of fluorimetric (excitation at 365 nm) responses of 20 (50 μM) before and after the addition of various anions (100 equiv.) in water with cetyltrimethylammonium bromide (CTAB).

Source: Panels (b) and (c) are reprinted from Ref. [34] (Copyright 2017 Royal Society of Chemistry).

(d) Chemical structures of fluoride probes 22, 23, and 24. (e) The possible processes and mechanisms involved in the SSB probe with fluorine ions. (f) Linear relationship of 22 with the concentration of F⁻ ions. (g) Column diagrams of the fluorescence intensity of 22 with tetrabutylammonium (TBA) salts at λ_max 486 nm; red bars represent the addition of various anions to the blank solution, and black bars represent the subsequent addition of F⁻ (2 equiv.) to those respective solutions (22 + A⁻ + F⁻).

Source: Reprinted from Ref. [31] (Copyright 2016 Elsevier B.V.).

Figure 3.13 (a) Design rationale of the fluorescence turn‐on detection of UO₂²⁺ based on AIE characteristics of 25. (b) Fluorescence spectra (λ_ex = 370 nm) of 25 (30 mM at pH 10.3) in the presence of different amounts of UO₂²⁺. (c) Linear relationship of 25 with the addition of different amounts of UO₂²⁺.

Source: Adapted with permission from Ref. [32] (Copyright 2014 Elsevier B.V.).

Figure 3.14 (a) Schematic illustration of the PPi detection mechanism of 26 copper(II) complex. (b) Fluorescent intensity response at 570 nm of 26 copper(II) complex with different anions in a 20% DMSO aqueous solution.

Source: Reprinted from Ref. [39] (Copyright 2015 Royal Society of Chemistry).

Cysteine (Cys) as one of the abundantly existing amino acids plays an indispensable role in physiological activities such as metabolism in complex biological organisms. However, how to improve the selectivity of the probe for cysteine and effectively distinguish it from homocysteine and glutathione in actual detection is a challenge for the fluorescence detection of Cys. Tong et al. reported an SSB‐based fluorescence turn‐on probe 27 for the detection of cysteine over homocysteine and glutathione [40]. As represented in Figure 3.15a, the cyclization reaction between Cys and the acryloyl ester on 27 results in the following hydrolyzation of 27 to produce SSB fluorophore with both AIE and ESIPT. Due to the selectivity of the cyclization reaction efficiency, this method is more selective for cysteine than for homocysteine and glutathione. The linear range of cysteine detection in the buffer is 0.5–30 μM, and the detection limit is 0.46 μM. This method was also applied for the effective quantitation of Cys in FBS.

Figure 3.15 (a) Cyclization reaction of 27 with Cys followed by hydrolysis to give the final SSB fluorophore. (b) Fluorescence spectra of 27 in the presence of different amounts of Cys in PBS buffer. Inset: photographs of 27 before and after the addition of Cys under a 365‐nm UV lamp and the fluorescence intensity at 558 nm as a function of Cys concentration. (c) Fluorescence intensity of 27 in the presence of Cys, Hcy, GSH, and other amino acids.

Source: Reprinted from Ref. [40] (Copyright 2015 Royal Society of Chemistry).

The detection and quantification of biological macromolecules including proteins, enzymes, and polysaccharides are of crucial importance to life science, biotechnology, as well as health care of human beings such as clinical diagnostic examinations and treatment monitoring. Taking advantage of the AIE and ESIPT effects, diverse sensing systems based on SSB fluorophores were facilely set up. Tong's group has established fluorescent SSB probes for the detection of proteins and enzymes with large Stokes shift in the past decade. For instance, Figure 3.16a demonstrates a noncovalently labeled fluorescence turn‐on detection method specifically to a highly cationic protein, protamine [42]. When the pH of the solution was at 9.16, probe 28 dissolved well in water due to the dissociation of the carboxyl group and thus exhibited extreme weak fluorescence. Adding protamine to the solution formed 28‐protamine aggregates based on electrostatic interactions, and the protamine concentration was measured by detecting the fluorescence enhancement signal of the aggregates (Figure 3.16b, c). The detection limit was as low as 43 ng/ml. The probe was also employed to study the electrostatic association between protamine and heparin.

Figure 3.16 (a) Design principle of the fluorescence turn‐on detection of protamine based on AIE characteristics of 28 and its application in detecting the interaction between protamine and heparin. (b) Fluorescence spectra of 28 in the presence of different amounts of protamine (from 0 to 30 mM). The inset shows the photographs of the solution of 28 in the absence (a) and presence (b) of protamine under a 365‐nm UV light. (c) Kinetic behavior of the fluorescence intensity (peaks in fluorescence spectra) of 28 with the addition of different amounts of protamine.

Source: Adapted with permission from Ref. [42] (Copyright 2010 Royal Society of Chemistry).

Another type of a series of SSB probe is to detect hydrophobic proteins such as BSA and human serum albumin (HSA) in aqueous solution. Figure 3.17a shows a ratiometric fluorescent probe 29 for the detection of hydrophobic proteins (casein) or proteins with hydrophobic pockets (BSA, HSA) through hydrophobic interaction [44]. Probe 29 emits a blue fluorescence at 436 nm due to the deprotonation of the hydroxyl group when it dissolves in water at pH 7.4. When binding to the hydrophobic pocket of a protein such as BSA, the OH group recovers generating a new red‐shifted emission enhancement at 518 nm, resulting in an obvious fluorescent color distinction that can be easily distinguished by naked eye (Figure 3.17b, c). The fluorescence intensity ratio, I₅₁₈/I₄₃₆, was linearly related to the concentrations of a series of hydrophobic proteins. The detection limits for BSA, HSA, and casein based on IUPAC (C_DL = 3 Sb/m) were 16.2, 10.5, and 5.7 mg/ml, respectively.

Enzymes are biomacromolecules that accelerate or catalyze biological or chemical reactions, and most enzymes belong to an important class of proteins in essence. There is no doubt that enzymes play a critical part in nearly all metabolic processes so that the evaluation of enzyme activity is of great significance. A mostly general and direct idea for the enzyme activity detection probe design is to modify the functional group with a substrate of the enzyme to block the probe fluorescence. As shown in Figure 3.18a and d, substituting ortho‐hydroxyl groups of salicylaldehyde to destroy the ESIPT process and quench fluorescence is a very simple and effective way to design fluorescence light‐up enzyme probes. Probes 30 and 31 are two examples following this design principle for β‐galactosidase and esterase activity evaluation, respectively [45, 47]. The probes perform good linear relationship at the range of 0–0.1 U/ml for β‐galactosidase and 0.01–0.15 U/ml for esterase, with the detection limits for β‐galactosidase and esterase as 0.014 and 0.005 U/ml, respectively. Such probes also perform well in imaging enzymes in live cells with low cytotoxicity.

Figure 3.17 (a) Synthesis and schematic presentation of the ratiometric fluorescence change of 29 upon binding to the hydrophobic pocket of BSA. The emission wavelength of 29 changes from 436 to 518 nm. (b) Photographs of 29 before and after the addition of various kinds of proteins under a UV lamp (365 nm). (c) Fluorescence spectra of 29 (10 mM) upon the addition of various concentrations of BSA in 10 mM PBS buffer at pH = 7.4. Excitation wavelength was set at 363 nm. (d) Ratiometric calibration curve of I₅₁₈/I₄₃₆ as a function of BSA concentrations.

Source: Reprinted from Ref. [44] (Copyright 2013 Royal Society of Chemistry).

There is also an indirect approach to detect enzymes like β‐lactamase, an important bacterial enzyme for some kinds of bacteria resistant to β‐lactam antibiotics (penicillins, cephalosporins, etc.), by cleaving the amide group with high catalytic efficiency [46]. The detection method contains three steps for fluorescence lighting‐up. As Figure 3.19a illustrates, β‐lactamase reacts with the lactam of its substrate (cefazolin sodium) to produce a secondary amine, initiating a spontaneous elimination reaction and affording a thiol compound. The thiol could further react with the sulfonate group of probe 32, releasing the SSB derivative with both AIE and ESIPT characteristics. The fluorescent signal enhancement relates linearly in the range of 0–10 mU/ml, and the detection limit was 0.5 mU/ml. This indirect method was also successfully applied to testing paper fabrication and achieved good analytical performance.

Figure 3.18 (a) Fluorescent light‐up probe 30 for β‐galactosidase detection. (b) Fluorescence spectra of 30 (100 μM) in the presence of various concentrations of β‐galactosidase in the PBS buffer solution and calibration curve of the fluorescence intensities (I₅₄₅) versus β‐galactosidase concentrations. Insets from left to right: photographs of 30 (100 μM) without or with β‐galactosidase under UV light (365 nm). (c) Imaging β‐galactosidase activity in cells. Images of probe 30 (50 μM) in C6/LacZ cells and HeLa cells for two hours at 37 °C.

Source: Panels (a–c) are adapted permission from Ref. [45] (Copyright 2015 Royal Society of Chemistry).

(d) Fluorescent light‐up probe 31 for sensing of esterase. (e) Fluorescence spectra of 31 (100 μM) in the presence of various concentrations of esterase (0–1.0 U/ml) in a 10 mM PBS buffer solution and calibration curve of the fluorescence intensities (I₅₈₀) versus esterase concentrations at pH 7.4, 37 °C. Insets from left to right: photographs of 31 (100 μM) without or with esterase under UV light (365 nm). (f) Confocal fluorescent image of MCF‐7 cells with incubation of 31 (50 μM) for 10 minutes or preincubated with a 10 mM inhibitor for 20 minutes and then treated with 31 (50 μM) for 10 minutes.

Source: Panels (d–f) are reprinted from Ref. [47] (Copyright 2017 American Chemical Society).

Figure 3.19 (a) An indirect approach for fluorescence light‐up detection of β‐lactamase using probe 32. (b) Photographs of test papers under a UV lamp (365 nm) for the detection of β‐lactamase at various concentrations (0–7.0 mU/ml) in the PBS solution containing cefazolin sodium (4.8 mM). (c) The corresponding fluorescence intensity of spots read by Image J software versus the concentrations of β‐lactamase. (d) Calibration curve for β‐lactamase detection.

Source: Reprinted from Ref. [46] (Copyright 2018 Royal Society of Chemistry).

A polysaccharide SSB sensor for facile, sensitive, and selective heparin detection has also been fabricated [48]. Heparin is a mucopolysaccharide composed of D‐β‐glucuronic acid and N‐acetylglucosamine to form a repeating disaccharide unit. Its skeleton has many anionic groups (such as carboxyl and sulfonic acid groups, etc.), making heparin highly negatively charged. As a medicinal anti‐hemagglutinating agent as well as a special antidote, heparin is hence of great significance for analytical detection. As shown in Figure 3.20a, salicylazine 33 is modified with two positively charged tertiary amine groups, which can be combined with negatively charged heparin through a charge–charge interaction. The emission of probe 33 in the Tris‐HCl buffer solution at pH 7.0 was extremely weak may be and its emission at 530 nm increased rapidly upon the addition of heparin (Figure 3.20b), which was due to the aggregation through electrostatic interactions. When the concentration of heparin reached 22 μg/ml, a fluorescence enhancement of about 40‐folds had been detected. The linear range is 0.2–14 μg/ml, the detection limit is 57.6 ng/ml, and the response time is as short as 2 minutes. Figure 3.20c also represents good selectivity of probe 33 for heparin from other polysaccharides such as chondroitin sulfate (ChS), hyaluronic acid (HA), and dextran (DeX).

Figure 3.20 (a) Design principle of the fluorescence turn‐on detection of heparin based on AIE characteristics of 33. (b) Fluorescence spectra of 33 in the presence of different amounts of heparin (from 0 to 22 μg/ml), λ_ex = 391 nm. (c) The fluorescence intensity of 33 in the presence of different amounts of HA, DeX, ChS, and heparin.

Source: Adapted with permission from Ref. [48] (Copyright 2013 Elsevier B.V.).