Handbook of Aggregation-Induced Emission, Volume 2. Группа авторов

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СКАЧАТЬ 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 (λ_em = 559/516 nm) when pH of the solution increased from 3.43 to 9.56. The pK_a1 of 34 is 4.8 for deprotonation of the carboxyl group resulting in a decrease of fluorescent intensity of orange light. The pK_a2 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 organelles can be rationally designed by chemical modification with the target‐reactive functional groups. In general, since fluorescence probes based on SSB generally have a relatively high positive electric potential, most SSB probes prefer to accumulating and lighting up mitochondria through charge interaction due to the high negative mitochondrial membrane potential (MMP). Thus, SSB‐based bioprobes are widely applied in imaging ions as well as biomolecules in mitochondria such as H⁺, S²⁻, and esterase [38, 47, 52]. Moreover, some researches have also reported the application potential of SSB probes in imaging‐guided selective cancer cell recognition and therapy [56, 57].

Figure 3.21 (a) Schematic of deprotonation processes of compound 34. (b) pH‐dependence of the emission spectra of 34 (60 μM) in phosphate buffer and ratiometric calibration curve of I₅₁₆/I₅₅₉. (c) Confocal fluorescence images of H⁺ in HepG2 cells after incubation with 34 (60 μM) and nigericin.

      Source: Reprinted from Ref. [49] (Copyright 2011 Royal Society of Chemistry).

Figure 3.22 (a) Chemical structures of some SSB probes with ratiometric pH‐responsive fluorescence. (b) Color changes (top: under room light; bottom: under a 360‐nm UV light) of compound 35‐based test papers at different pH values.

      Source: Reprinted with permission from Ref. [50] (Copyright 2015 Royal Society of Chemistry).

      (c) Photograph of 36 in water/ethanol (fw = 99%, v/v) with different pH values under a 360‐nm UV light. (d) Confocal fluorescence images of HeLa cells incubated with 36 (50 μmol/l) and different pH buffers.

      Source: Panels (c) and (d) are adapted with permission from Ref. [52] (Copyright 2018 American Chemical Society).

The mitochondrion is one of the most important organelles in eukaryotic cells. Mitochondrial metabolism provides the energy required for the metabolism of the entire cell; meanwhile, they are involved in processes such as cell differentiation, cell communication, and cell apoptosis [58]. Most of SSB‐based bioprobes accumulate in mitochondria after passing through cytoplasm membrane may be because of an ~1.3 mV zeta‐potential of SSB derivatives [47] that makes it liable to be attracted by the ~−180 mV extreme negative potential of mitochondria. Modified by mitochondrial targeted groups, triphenylphosphonium (TPP) and pyridinium, Liu and coworkers developed SSB‐based probes 39 and 40, which perform excellent mitochondrial imaging specifically [56, 59]. Moreover, such a modification often gives SSB fluorophores some additional attractive functions. For example, as illustrated in Figure 3.23b, as TPP is sensitive to negative charge, owing to the more negative mitochondrial membrane potential of cancer cells than normal cells, 39 can selectively accumulate in cancer cell mitochondria and light up its fluorescence. Furthermore, this probe also shows high cytotoxicity toward HeLa cells as it could trigger mitochondrial dysfunctions. Thus, chemotherapy with 40 is specifically targeted to mitochondria of cancer cells and monitored by itself. Images of HeLa cells costained with MitoTracker/LysoTracker shown in Figure 3.23c demonstrate that 39 could accumulate on mitochondria with high efficiency, while for normal NIH‐3T3 cell lines, the mitochondria and lysosomes were all lit up without specificity. After incubating tetramethylrhodamine ethyl ester (TMRE), a mitochondrial membrane potential indicator, with 39 pretreated HeLa cells, the fluorescence intensity of the cells decreased, indicating that 39 can cause a reduction in the mitochondrial membrane СКАЧАТЬ