This reactive recognition mechanism was confirmed using mass spectroscopic analysis (Figure 35). Gunnlaugsson et al. used the “fluorophore–spacer–receptor” format in compounds 24–26 to obtain fluorescent probes for the visualization of microdamage in bones such as cracks and scratches 98. Probes 24–26 possessed phenyliminodiacetate as a receptor unit that can bind Ca2+ ions selectively and completely quenched the fluorophore emissions. These effects were applied for the visualization of cracks and scratches on bones that generated Ca2+ sites on the bone surface capable of binding with the exposed probes 24–26, leading to a bright fluorescent emission because of the hindered PET. It was found that the blue-emitting probe 26 was masked by the autofluorescence from the bone matrix, while the binding of both 24 and 25 gave significant fluorescence arising from the 4-amino-1,8-naphthalimide fluorophore (Figure 25).
- Similar to the ESIPT, the FRET signaling output has some benefits in the construction of fluorescent probes for biomedical applications.
- The structure of the present review can be summarized according to Scheme 1.
- ICT probe based on cation interaction with an electron-donating or electron-withdrawing group.
- Confocal laser fluorescence images of probe 16a in HeLa cells.
Molecular logic gates are intelligent sensory molecules that analyze more than one analyte by mimicking electronic logical elements in the computer. These molecules are able to perform different sensor functions simultaneously and can calculate autonomously a composite result, which is why some scientists define Worldtradex scam them as molecular-level labs 28. The information processing by Boolean Logic from a molecular device can be particularly useful for the rapid screening of medical conditions in which the analysis is not performed by a physician, but by the molecular device itself.
Figure 57.
For example, for the mouse treated with Lipo-66-NH2, the fluorescence signal appeared in almost the whole body but mainly in the liver and the tumor site, at 24 h post-injection. In contrast, for the mice treated with the molecular fluorophore 66-NH2, the fluorescence mainly resided in the kidneys and was cleared out of the body more quickly (Figure 62) 172. Preparation of pH-sensitive fluorescent 48-PAA-b-PnBA micelles and internalization in living A549 cells visualized by fluorescent microscopy. The cells were incubated for 24 h with fluorescent micelles in different concentrations (0.155 mg/mL—upper image; 0.250 mg/mL—lower image). Fluorescence images of A375 after 30 min of incubation with probe 14 (upper panel). Cells exhibit strong blue emissions compared to green channel suggesting that the probe was not significantly catalyzed by endogenous tyrosinase.
Figure 48.
Moreover, this probe exhibited lysosome-targeting ability, good biocompatibility, and excellent photostability. It was successfully applied to map lysosomal pH both in vitro and in vivo. Furthermore, the lysosomal pH changes were monitored during the caudal fin regeneration of a fish model (medaka larvae) using the fluorescent signaling properties of 42. It was observed that during the regeneration, lysosomal acidification is required to promote autophagic activity for cell debris degradation. These results could be used as a platform for the visualization of various lysosome-involved biological processes such as stress and inflammatory responses.
- Ex vivo NIR imaging has confirmed significantly increased retention of the conjugate in mice brains relative to other organs with an increase of up to eightfold in signal intensity.
- The acidic microenvironment of cancer cells ensures the effective drug release induced by the breakage of the acylhydrazone bond, which results in the fluorescence recovery of the conjugated polymer, contributing to the monitoring of the drug release process.
- This improvement is even more pronounced (492-fold) when the above conjugate is combined with temozolomide—a standard drug for the treatment of glioblastoma.
- However, after a reaction with formaldehyde, the fluorescence intensity was enhanced 14 times due to the ICT-activated fluorescence, after the formation of an electron-withdrawing Schiff base instead of the former electron-donating amine group.
- Confocal fluorescence imaging of MCF-7 cells incubated with the nanoprobe 29 (50 μg/mL) and then treated with LPS (1 µg/mL) and IFN-γ (100 ng/mL).
- Based on this knowledge, Meng et al. prepared NIR fluorescent probe 20 (Figure 21) in which an effective TICT process quenched the fluorescence emission at 680 nm after decreased viscosity as a result of increased temperature 87.
For example, when a cation interacts with the electron-donor group in an ICT system, it reduces the electron-donating ability of the donor. Due to the resulting reduction in conjugation, a blue shifting of the absorption is predicted together with a reduction in the extinction coefficient (Figure 1, left side). In principle, the fluorescence spectra are shifted in the same direction as the corresponding absorption spectra. Additionally, changes in the quantum yields and lifetimes are also expected, as the observed effects strongly depend on the charge and the size of the cation. In this review, we discussed the main mechanisms and approaches for the rational design of fluorescent probes, finding applications in medical diagnosis and drug delivery systems. In addition, an overview focusing on recent achievements in the development of fluorescence probes and molecular logic devices with potential applications in biology and medicine was presented.
For better distribution in the lysosomes, a morpholine linker was also bonded to 38. In the absence of Cu2+, this probe emitted yellow-green fluorescence from the 1,8-naphthalimide unit. The authors found that the presence of Cu2+ induced the hydrolysis of rhodamine from spirolactam to ring-opened xanthene form, which was confirmed by ESI-MS analysis. The rhodamine spirolactam form is colorless and absorbed at the UV region. However, the formed ring-opened xanthene has absorption in the spectral region of the yellow-green fluorescence of the 1,8-naphthalimide unit.
Figure 44.
This nano-system showed a strong red fluorescence centered at 650 nm. In the presence of peroxynitrite, the peripheral ferrocene units were oxidized and a PET process from the periphery to the focal carbon dot occurred. As a result, the former fluorescence was quenched efficiently by 80%. The fluorescence intensity of nanoprobe 29 decreased linearly in the range of 4 nM to 0.12 μM ONOO−.
Fluorescent resonance energy transfer (FRET) is another important photophysical technique that provides a useful fluorescent signal for sensing and imaging application, especially in biological media 18,117,118. This process occurs by the nonradioactive, resonant transfer of energy from an excited donor fluorophore to a closely placed acceptor molecule in the ground state. It is important to understand that this is not the result of emissions from the donor being absorbed by the acceptor. FRET is observable due to a donor–acceptor dipole–dipole interaction. That is why the general requirement for FRET is the spectral overlap between the fluorescence spectrum of the donor and the absorption spectrum of the acceptor that generates enough energy for the dipole–dipole coupling.
Fluorescent Probes as a Tool in Diagnostic and Drug Delivery Systems
Many fluorescent probes have poor photostability and can be consumed by other active substances. Hence, through fluorescent dye screening and rational design strategy, chemists can cleverly construct functional fluorescent probes with great photostability and chemical stability. In addition, enhancing sensitivity against analytes is another crucial issue to accelerate the progress of clinical application, in order to avoid competition from the cellular microenvironment.
Figure 32.
The general photophysics of ESIPT is represented in Figure 31. Fluorescence imaging with endogenous O2•− using probe 12 (2 μM) for 30 min. RAW264.7 cells were treated with PMA/LPS and stained with 12. Moreover, based on the ratiometric pH response to the tumor microenvironment, the condensed aminotriazolyl-1,8-naphthalimide 7 was successfully used to image the pH of subcutaneous tumor xenografts 51. Fluorescence images of MCF-7 cells pretreated with probe 5 (5 μM) for 2 h and after incubated using PBS with various pH values for 20 min. The images are collected at 470–545 nm (upper row) and 545–650 nm (lower row).
Fluorescent Probes Based on Twisted Intramolecular Charge Transfer (TICT)
A spacer holds the fluorophore and receptor close to each other, but in separated conjugated systems. After excitation of the fluorophore, an https://worldtradex.online/ electron transfer occurs from the HOMO of the electron-donating receptor to the low-lying HOMO of the electron-accepting fluorophore. When a cation binds to the recognition moiety, the energy of the HOMO of the receptor is lowered so that the photoinduced electron transfer cannot happen from the HOMO of the donor to the fluorophore (Figure 22). In other words, the presence of a guest is signalized with the enhanced quantum yield of fluorescence, and respectively, fluorescence intensity.
Recently, a bright idea to apply the PET process in a fluorescent probe 31 for the monitoring of voltage in neurons and neural signals in real time was reported (Figure 30) 103,104. Based on the fluorescence properties of NIR TICT probe 18, Zhan et al. developed, for the first time, a fluorescent method for the early exploration of idiopathic pulmonary fibrosis through changes in viscosity (Figure 19) 85. Confocal laser fluorescence images of probe 16a in HeLa cells. Cells incubated with the probe (10 μM) for 20 min (upper panel) and incubated with monensin (10 μM) for 30 min and probe 16a (10 μM) for another 20 min (lower panel). Proposed sensing mechanism for Sec activation of 13. The in situ fluorescence changes of Hela cells stimulated to Na2SeO3 (5 μM) for different times (1, 2, 6, 12 h), and then incubated with 13 (5 μM).
The potency of the conjugate was positively influenced by the addition of temozolomide; although, temozolomide itself did not have any effect in reducing the cell number due to the inherited resistance of these cell lines to temozolomide treatment. Self-assembly of the FRET-based probe 40 into water-soluble micelles in an aqueous solution. Photostability comparison of the stained HeLa cells by using (left panel) pure BODIPY-labeled actin and (right panel) probe 40 micelles internalized in HeLa cells. Images were taken after different time intervals of continually intense excitation. Specific requirements for the detection of FRET signals.
