Probe 32 is an example of an ESIPT-based probe for the measurement of pH fluctuations in living organisms 107. Compound 32 showed blue fluorescence, which rapidly shifted into the green region under slightly basic conditions due to its transition from the enol form to the keto form (Figure 32). This probe was non-toxic and it was used for real-time mitochondrial imaging of Hella cells. In addition, due to the ESIP sensing process, it was applied for the detection of Helicobacter pylori.
Figure 21.
Confocal fluorescence images of MCF–7 cells incubated in high K+ HEPES-buffered solution containing ionophores at different pHs. Fluorescence recognition mechanism and reversibility of probe 3 with Zn2+ ion. Fluorescence image of MCF-7 cells pretreated with probe (10 μM) for 30 min (upper panel) and after incubated with Zn2+ (100 μM) for 1 h (lower panel). Fluorescence image of A549 cells incubated with probe (10 μM) for 30 min (upper panel) and then incubated with 2 μM Mg2+ for 30 min (lower panel). Fluorescence recognition mechanism and reversibility of probe 1 with Hg2+ ion. Fluorescence image of L929 cells incubated with probe (5 μM) after staining for 30 min (upper panel) and after incubated with Hg2+ (50 μM) for another 30 min (lower panel).
Intracellular viscosity is an index affecting physiological processes that affect the transport, diffusion, and interactions between biomolecules. The abnormal viscosity values are related to atherosclerosis, hypertension, diabetes, Alzheimer’s disease, and cancer 83. Wang et al. applied this for highly selective tumor visualization using TICT fluorescent probe 17 (Figure 18) 84.
Because of the low ICT process in 14, the probe showed blue emissive fluorescence output at 467 nm. It was found that the probe displayed a sensitive ratiometric response to tyrosinase with a detection limit of 0.2 U mL−1. The MTT analysis showed moderate cytotoxicity—70% cell viability at 20 μM. In addition, 14 was used to image endogenous tyrosinase in A375 cells in the blue and green channels of a confocal microscope. The illustrated Figure 6 fluorescence compounds 5 and 6 were rationally designed as probes for measurements of intracellular pHs using a very similar strategy 48,49. Both systems are ICT fluorophores in which the electron-accepting part is a nitrogen-containing heterocycle—in 5, it is benzothiazole and in 6, it is benzoeindole.
Fluorescent Probes Based on Fluorescent Resonance Energy Transfer (FRET)
Because of the oxidation of 12, a green emitting 4-amino-1,8-naphthalimide was obtained (Figure 12). The green fluorescence was attributed to the enhanced ICT in the 1,8-naphthalimide fluorophore containing strong electron-donating amines instead of a C-4 carbamate group in the former blue-emitting naphthalimide 12. Using the fluorescent changes at 475 nm and 540 nm, a ratiometric analysis was constructed and applied for the detection of mitochondrial superoxide anions as well as inflammatory D magna. The limit of detection was determined to be 0.370 μM. The cytotoxicity test revealed more than 80% cell viability, suggesting that the probe can be used safely in living cell imaging. The phenomenon, intramolecular charge transfer (ICT), occurs after light absorption of fluorophoric systems in which an electron-donating unit was directly conjugated with an electron-withdrawing one 36,37.
Fluorescent Probe Design
The authors found that the probes 16a-c showed well-pronounced fluorescence emissions at 580–630 nm in a viscose solution such as glycerol due to the hindered TICT process. The observed fluorescence enhancement from low viscose methanol to glycerol solution was by about 19 times. Moreover, all of the three probes showed low cytotoxicity against Hella cells with a calculated viability higher than 80% at 15 μM concentration, and they were used for imaging the fluctuations of the mitochondrial viscosity in Hella cells.
- Aggregation-induced emission (AIE) is a relatively new approach for the rational design of fluorescence probes, which rapidly became very popular in the present time 128,129,130.
- Theranostic agents are macromolecular conjugates including a covalently bonded antitumor drug, imaging marker, and targeting component.
- The maximal fluorescence intensity in 5 was shifted from 515 to 565 nm.
- This caused a proton translocation from the hydroxyl to the carboxylic group.
Figure 57.
Second, on the basis of fluorescence emissions of both the donor and acceptor, ratiometric measurements can be achieved. As was mentioned above, the long-wavelength excitation and the ratiometric signaling reduced the problems of autofluorescence and scattering during fluorescent sensing. The condensation of 3-amino-1,2,4-triazole to the C-4 and C-5 position of the 1,8-naphthalimide fluorophoric system resulted in an interesting architecture suitable for the ratiometric intracellular imaging of pH 50,51,52. The introduced aminotriazole in 1,8-naphthalimide fluorophore has unusually high acidity compared to other 1,8-naphthalimide probes and showed a pKa value in the physiological range of about 6.5. Additionally, the aminotriazole naphthalimides showed increased water solubility, low cytotoxicity, clear cellular location, and membrane permeability making them an excellent platform for pH measurements in living cells 50.
This encourages us to synthesize perylenediimide 23 as an efficient PET-based probe (Figure 24) 97. In addition, the observed pKa value of 6.35 in 23 made it a suitable probe for pH determination in the physiological range. Probe 23 showed good cell permeability in L929 cell lines and according to the MTT analysis; it was low https://worldtradex.blog/ cytotoxic. An approach for the construction of thermosensitive fluorescence probes was based on the viscosity-sensing TICT probes due to the high dependence of viscosity toward temperature. The lack of oxygen in tissues is known as hypoxia. With hypoxia stress, excessive reactive oxygen species (ROS) are produced, which results in cell damage and death.
The general advantage of the reactive probes is their selectivity that was induced from a specific reaction with the analyte. A typical example of a reactive ICT probe is presented in Figure 8. Probe 8 represents a conjugated system containing two electron-accepting fragments—indole and thiocarbonyl units 53. The thiocarbonyl was introduced as a selective Hg2+ receptor moiety due to the thiophilic characteristics of that cation.
Fluorescent Probes Based on Twisted Intramolecular Charge Transfer (TICT)
Imaging of LO2 cells treated with probe 10 (10 μM) for 30 min under hypoxic conditions with 1% O2. A549 cells incubated with 8 (10 μM) for 30 min, and continually incubated with Hg2+ (100 μM) for 60 min. Structure of 68 and release process of drug molecule and fluorescent dye upon light activation 174. Illustration of the response of 66 toward hypoxia in tumor-bearing mice model. Typical fluorescence images of HepG2 xenograft tumor-bearing mice upon intravenous injection of 66-NH2 (in PBS containing 1 % DMSO), Lipo-66 (in PBS), and Lipo-66-NH2 (in PBS) for 4 h and 24 h. Copyright (2019) Ivyspring International Publisher.
Figure 2.
Illustration of 43 and 44 and their LDs-specific cell and atherosclerosis imaging. Perylene-3,4,9,10-tetracarboxylic diimide-based probe 23. Construction of Worldtradex forex broker NE probe 9 containing hydroxyl fluorophores and self-immolation group. ICT probe based on cation interaction with an electron-donating or electron-withdrawing group. An NHS ester allows the functionalization of an amino moiety in a variety of molecules, including DNA and RNA oligonucleotides, as well as peptides or proteins.
- The obtained ESIPT fluorescence of 34 in the presence of F− was centered at 586 nm (Figure 34).
- HepG2 cells were incubated with 35 (10.0 μM) for 15 min prior to incubating with HOCl (20 equiv.) for 15 min.
- As a receptor fragment in probe 12, a triphenylphosphonium unit was introduced in which the diphenylphosphinate part trigged selective oxidation in the presence of superoxide anions.
- That is why the intracellular imaging of these ions could be an excellent diagnostic tool.
- In the presence of NaOCl, an emission at 585 nm with a small Stokes shift (25 nm) was observed, suggesting the interruption of the ESIPT process.
This system showed a fluorescent signal at 728 nm due to the strong ICT occurring from the electron-donating nitrogen in the chelate moiety to the chromenylium-cyanine fluorophore 38. In presence of Hg2+, the fluorescence of 1 was blue-shifted to 663 nm due to the lowered ICT efficiency as a result of the Hg2+ chelating process in which the lone electron pair on the nitrogen participated. This observation has several advantages for the monitoring of living organisms and it was successfully applied for real-time detection of Hg2+ in living cells. First, probe 1 emitted in a near-infrared (NIR) region where the biomolecules have weak light absorption and fluorescence that reduce background interference such as auto-fluorescence, scattering, and absorption by biomolecules. In addition, the NIR has a good penetrating ability, allowing deep penetration in living systems.
The performed imaging of L929 cells demonstrated low toxicity and high sensitivity and selectivity for detection of intracellular Cu2+ by probe 38. Monitoring intracellular selenocysteine (Sec) is of significant interest for studying Sec metabolism and its changes in disease-relevant concentrations. The 1,8-naphthalimide 13 was prepared as a selective probe for Sec imaging by ratiometric fluorescence response 72. The 1,8-naphthalimide probe 13 was a blue-emitting ICT system with acrylate moiety at position C-4 (Figure 13). Based on the observed changes, a ratiometric analysis with a detection limit of 12 nM selenocysteine was observed. Furthermore, probe 13 was applied for the high specificity and selectivity rapid quantification of exogenous and endogenous Sec in living Hela cells.