Handbook of Aggregation-Induced Emission, Volume 2. Группа авторов
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), λex = 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.).
3.2.3 Ratiometric pH Probes
As one of the key parameters, pH plays a crucial role in all life forms including external environment as well as cellular functions. Small changes in the pH of the environment may even affect the lives of many plants and animals. In addition, pH is a key factor in pharmaceuticals, food, and drinking water. For intracellular pH, the fluctuation has a significant effect on cell growth, enzyme activity, and ion transport. pH is also one of the important parameters to distinguish cancer cells from normal cells. Therefore, monitoring pH is critical to maintaining our living environment and improving the quality of our life.
The hydroxyl groups of SSB experience deprotonation according to the increase of medium pH; thus, most SSB AIE fluorophores show significant fluorescent wavelength change, usually blue‐shifted as pH increases [49–53]. Therefore, SSB is of unique advantage for designing ratiometric fluorescent pH probes. In particular, fluorescent pH probes with a ratiometric response manner are highly preferred for pH monitoring in complex samples because of their visible fluorescence color change and better resistance to variations of sensor concentration and external environment. Till date, a number of ratiometric fluorescent pH probes have been designed based on SSB and successfully applied in test paper‐based detection [50] and in bioimaging [49, 52].
A representative work is the first SSB ratiometric fluorescent pH probe designed and reported by Tong's group in 2011 and applied in the imaging of pH variation in living cells [49]. As shown in Figure 3.21a and b, 4‐carboxylaniline‐5‐chlorosalicylaldehyde Schiff base (34) changed the fluorescence color from orange to green (λem = 559/516 nm) when pH of the solution increased from 3.43 to 9.56. The pKa1 of 34 is 4.8 for deprotonation of the carboxyl group resulting in a decrease of fluorescent intensity of orange light. The pKa2 of 34 is 7.4 for deprotonation of the hydroxyl group and caused the enhancement of green fluorescence according to further increase of the pH. The fluorescence intensity ratio I516/I559 changed dramatically from 5.0 to 7.0, indicating that 34 could be a sensitive pH probe at the range of 5.0–7.0. Figure 3.21c demonstrates the ratiometric fluorescent imaging of H+ concentration variation in HepG2 cells by probe 34, showing that the SSB molecule 35 deserves in detecting pH variation in living cells.
A series of works designing SSB‐based ratiometric pH probes were then reported in the following years. Molecular structures of the probes (shown in Figure 3.22a) and their applications as test papers were reported with good contrast (Figure 3.22b). Tang and coworkers [52] ameliorated the molecular structure, endowing the optimal detection range as 6.86–8.01, which covers the pH range of blood and intracellular fluid of healthy individuals and achieved satisfied results in cell imaging (Figure 3.22c, d). Compound 36 colocalized well on mitochondria compared with MitoTracker Deep‐Red FM; Pearson's coefficient was obtained as high as 0.92. The plot of the ratiometric fluorescence intensity of 36 in HeLa cells as a function of pH indicated a satisfactory linearity of R = 0.93, showing that SSB‐based ratiometric pH probes perform satisfactorily in real environmental samples as well as for intracellular pH evaluation.
3.2.4 Bioimaging
Specific subcellular organelle imaging is of great significance as visualization of organelles and their morphology or functional changes is essential for the study of biological processes such as metabolism and diseases at the cellular level, as well as clinical diagnosis, drug development, or medical intervention. Among numerous advanced imaging technologies, fluorescence imaging has been recognized as one of the most powerful tools in biological systems due to the high sensitivity and in situ and real‐time observation, noninvasive testing, and cost‐effective performance. In the past decades, AIE fluorophores have achieved great progresses in specific cell imaging [54]. Despite the significant advantage as “light‐up bioprobe” of most common AIEgen conjugates [55], due to the ESIPT characteristic, probes based on SSB show another unique benefit in bioimaging such as no self‐absorption, large Stokes shift, high contrast ratio, and excellent photobleaching resistance. Furthermore, because the 3, 4, and 5 positions on the benzene ring of the salicylaldehyde molecule are easily functionalized by chemical modification, live‐cell SSB‐based fluorescent probes for specific localization of mitochondria, lysosomes, lipid droplets (LDs), and other