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