ESIPT-based Fluorescent Probe Research Articles

An ESIPT-Based Fluorescent Probe for Wash-Free Cell Membrane Imaging.

The cell membrane tracking is important for studying the membrane function and diagnosing membrane-related diseases, so the development of fluorescent molecule specifically lighting up cell membrane is highly desirable. In this work, we designed and readily prepared a fluorescent dye (BOHI) that can target cell membrane. The pH-dependent emission spectra reveal that BOHI shows fluorescence based on the excited-state intramolecular proton transfer (ESIPT) under acidic and neutral conditions, while the blue-shifted emission from the phenolate anion of BOHI was observed when pH ≥ 9. Among various solvents, the weakest fluorescence emission was observed in H2O, which is favorable to wash-free imaging. The fluorescence intensity of BOHI is greatly affected by surfactants. The anionic surfactant can induce intense green fluorescence, while very weak fluorescence was observed in the solution of cationic surfactant. Hela cells images show that BOHI can specifically targets and lights up cell membrane.

Study on ESIPT process of fluorescence probe BT-ITC for detecting H2S in DMF/H2O mixed solution

A probe molecule named BT-ITC with an isothiocyanate group was synthesized and evaluated for its H2S sensing ability based on the excited state intramolecular proton transfer (ESIPT) process J K Kim, S Y Bong, R Park, J Park, D Ok Jang 2022 An ESIPT-based fluorescent turn-on probe with isothiocyanate for detecting hydrogen sulfide in environmental and biological systems, Spectrochimica Acta Part A 278 121333. The BT-ITC molecule has a large Stokes shift, short response time, and low detection limit. The end result of the reaction between the molecules BT-ITC and H2S is the BTC-NH2 molecule. The hydrogen bond of the Enol structure BTC-NH2 molecule in excited-state (S1 state) is strengthened, compared to the ground-state (S0 state). The calculated absorption and fluorescence spectra were in good agreement with the experimental values. The frontier molecule orbitals (FMOs), the potential energy curve (PEC), and scatter plots of reduced density gradient (RDG) were calculated. According to aforementioned, it was discovered that the charges of BTC-NH2 molecule were rearranged via an electron transition brought on by photoexcitation. This changes the acidity and alkaline of the proton donor and acceptor, and thus promotes the ESIPT process.

Debut of a NIR ESIPT-based fluorescent probe with synergistic effects for boosting high-contrast imaging of β-galactosidase in ovarian cancer

Long-wavelength emission with high contrast toward species of interest is highly desired for fluorescence imaging in living objects. Herein, we proposed an exquisite enzyme-regulated “aggregation-induced emission (AIE) and intramolecular charge transfer (ICT)” strategy to optimize the signal-to-noise ratio for highly sensitive detection of enzymes. Benefiting from the synergistic effect, probes ABT-Gal-1 and ABT-Gal-2 exhibited faint background signals but dramatically enhanced near-infrared fluorescence upon exposure toward β-galactosidase (β-gal). Notably, both probes could be accessible in aqueous media. Preferably, the fluorescence intensity of ABT-Gal-1 exhibited a satisfactory linear relationship in the wide concentration range of 0.5–30 U/L with a very low detection limit to 0.24 U/L. Further, by monitoring endogenous β-gal in ovarian cancer cells, a precious merit of resistance to multiple washes was identified, which was facilitated by the nanoaggregate formation in situ upon activation. This work proposes a guiding strategy for manipulating various lighting mechanisms to develop attractive probes and, equally importantly, it provides an ideal practical tool for elucidating the biological role of β-gal in biological scenarios.

An ESIPT-Based Fluorescent Probe for Aqueous Cu+ Detection through Strip, Nanofiber and Living Cells.

Constructed on the benzothiazole-oxanthracene structure, a fluorescent probe RBg for Cu+ was designed under the ESIPT mechanism and synthesized by incorporating amide bonds as the connecting group and glyoxal as the identifying group. Optical properties revealed a good sensitivity and a good linear relationship of the probe RBg with Cu+ in the concentration range of [Cu+] = 0-5.0 μmol L-1. Ion competition and fluorescence-pH/time stability experiments offered further possibilities for dynamic Cu+ detection in an aqueous environment. HRMS analysis revealed a possible 1:1 combination of RBg and Cu+. In addition, colorimetric Cu+ detection and lysosome-targeted properties of the probe RBg were analyzed through RBg-doped PVDF nanofiber/test strips and RBg-Mito/Lyso trackers that were co-stained in living HeLa cells, enabling the probe's future applications as real-time detection methods for dynamic Cu+ tracking in the lysosomes and Cu+ detection under diversified conditions.

Rational development of an ESIPT-based fluorescent probe with large Stokes shift for imaging of hydrogen sulfide in live cells

It is crucial to monitor hydrogen sulfide (H2S) because H2S plays a vital role in the regulation of many physiology and pathology processes. Many evidences indicate that endogenous H2S is closely associated with many diseases such as inflammation and cancers. Herein, we reported a novel fluorescent probe BTDI to monitor the fluctuation of H2S based on the excited-state intramolecular proton transfer (ESIPT) mechanism both ex vivo and in vivo. The selectivity of BTDI for H2S is significantly higher than that for biothiols and other potential anions. After the probe responded to H2S, the nucleophilic addition reaction of the H2S with probe BTDI resulted the shifting of maximum emission peak from 630 nm to 542 nm and the fluorescent signals change from red to green emission along with a large Stokes shift (240 nm). Moreover, BTDI can be successfully applied to detect extracellular and endogenous H2S in living cells through fluorescent cell-imaging, which provides a promising tool for the specific recognition of H2S in complex biological systems.

Theoretical study on the optical properties of an ESIPT-based fluorescent probe for phosgene.

A fluorescent probe Pi with the excited-state intramolecular proton transfer (ESIPT) properties was synthesized and used to detect the phosgene in solution and gas phases. However, the detection mechanism of the fluorescent probe needs to be further studied. Herein, the density functional theory (DFT) and time-dependent density functional theory (TD-DFT) methods were adopted to explore the molecular structures and electronic spectra properties of probe and its product Pio after reacting with phosgene. Through analysis for molecular structure parameters and infrared vibrations accompanied with the hydrogen bond of Pi, it is confirmed that the intramolecular hydrogen bond of Pi is enhanced under light excitation, which illustrates the occurrence of ESIPT reaction combined with the scanned potential energy curves. It can be seen from the simulated spectra that Pi shows double fluorescence through ESIPT process, while the fluorescent product Pio exhibits the single fluorescence due to the disappearance of intramolecular hydrogen bond. Through the study on the structure and optical properties of Pi and Pio, it can be helpful to deeply understand the intrinsic mechanism of the detection of phosgene by the Pi molecule probe, which also supplies a reference to the further study about the fluorescence probe.

A novel ESIPT-based fluorescent probe with dual recognition sites for the detection of hydrazine in the environmental water samples and in-vivo bioimaging

Hydrazine (N2H4), an important chemical intermediate, has been widely used in industrial production and agricultural life, but it has also caused environmental pollution. A novel ESIPT-based fluorescent probe with dual recognition sites, 2-(benzothiazole-2-yl)-1,4-imphenyl bis 4-bromobutyric acid (BRBA), was developed to selectively detect N2H4 under complex conditions. BRBA exhibits accurate detection for N2H4 with a good linear relationship ranging from 0 to 150 μM, and the LOD can reach 0.1 μM. Importantly, taking advantage of low cytotoxicity and a large Stokes shift, BRBA can be utilized to monitor environmental water samples and successfully applied to imaging HeLa cells and zebrafish.

An ESIPT-based fluorescent turn-on probe with isothiocyanate for detecting hydrogen sulfide in environmental and biological systems

A probe with an isothiocyanate group was synthesized and evaluated for its H2S sensing ability. Upon addition of H2S, the probe exhibited ratiometric properties during absorption with a red-shift. The probe exhibited fluorescent off–on responses towards H2S via the ESIPT process, due to the conversion of isocyanate into amine. UV–vis, fluorescence, and 1H NMR spectroscopic analyses were performed to investigate the sensing mechanism. The probe has a large Stokes shift, short response time, and low detection limit. It can be used to estimate H2S levels within the range of 0–36 nM. The practical applicability of the probe was demonstrated using water samples and living cells.

An ESIPT-based fluorescent probe with large Stokes shift for peroxynitrite detection in HeLa cells and zebrafish

The quick coupled reaction of nitric oxide (NO) with superoxide free radicals (·O2−) produces peroxynitrite (ONOO−), it is a powerful biological oxidant. And according to interact with proteins, lipids, nucleic acids and other organisms, the ONOO− eventually leads to all sorts of diseases, for instance, Alzheimer's disease, diabetes, cancer, inflammation. To fully understand the multiple pathological processes of ONOO− in vivo, it is imperative to exploit effective tools to realize the precise, fast and sensitive detection of endogenous ONOO−. We described a novel fluorescent probe named YV in this work, covering the synthesis, characterization and biological application. YV can detect ONOO− by means of fluorescence imaging. Because of the borate group cleavage, YV exhibited a fluorescence intensity change toward ONOO−. It was, the principle of ESIPT that resulted in fluorescence enhancement. The probe YV shows a series of advantages such as high selectivity, fast response, significant Stokes shift and low detection limit. Fluorescence imaging can be used to identify variations in ONOO− in Hela cells and zebrafish. YV will be a robust imaging tool for detecting endogenous ONOO−.

Engineering an ESIPT-based fluorescent probe for dual-channel (Vis/ NIR) ratiometric monitoring of intracellular sulfur dioxide by single wavelength excitation

ESIPT-based probes have large stokes shift and dual emission, and can quantify the analytes through dual-channel LSCM imaging under one excitation wavelength without of the need of two lasers or corresponding parameter corrections. To exploit these advantages of ESIPT mechanism and achieve a big breakthrough in their excitation-emission wavelength and solubility which of the old probes dissatisfied the requirements of biology experiments, we challenged to develop a favorable and practical SO2 probe. In this work, the benzopyrylium moiety which had both a built-in SO2 site and good water solubility, was conjugated with 10-hydroxybenzo[h]quinoline to construct ESIPT-based probe NIR-PT with dual emission (enol form:544 nm; keto form: 733 nm). NIR-PT could ratiometricly visualize SO2 levels in aqueous solution with 460 nm as a excitation. Moreover, we have successfully applied NIR-PT to visualize SO2 levels in cells and mice under oxidative stress.

An ESIPT-based fluorescent probe for the detection of multiple analytes and a facile approach to discriminate between arsenate and pyrophosphate in water

A simple ESIPT-based fluorescent probe for the detection of the multiple analytes such as chromium (Cr3+), arsenate (HAsO42−), and pyrophosphate (PPi, P2O74−) has been reported.

A new simple ESIPT-based fluorescent probe for rapid detection of cysteine with high sensitivity and specificity and bioimaging in living cells

Based on Excited-State Intramolecular Proton Transfer (ESIPT) fluorophore and an acrylate moiety as Michael addition and leaving site, a simple but colorimetric turn-on fluorescent probe FVCA was designed and synthesized. Upon addition of Cys to the solution of FVCA, the absorption spectra changed from 375 nm to 460 nm (Δ85 nm) and afforded visible color change from colorless to yellow. Meanwhile, the fluorescence emission spectra emerged at 554 nm with remarkable changes, accompanying with an obvious fluorescence color change from colorless to bright green. In addition, FVCA exhibited rapid response (∼ 60 s), high sensitivity (detection limit: 8.37 nM), large strokes shift (∼ 94 nm) and excellent selectively to Cys over other biological mercaptans. Moreover, FVCA had strong anti-interference ability for Cys without any interference from other biologically-relevant thiols even Hcy and GSH, even obvious visible and fluorescence color changes. More, the sensing mechanism was proved to be the ESIPT-based addition-cyclization reaction process by 1H NMR, HRMS and TD-DFT. Importantly, FVCA can be used to image exogenous and endogenous Cys in living A549 cells with low toxicity.

An ESIPT-based fluorescent probe with fast-response for detection of hydrogen sulfide in mitochondria

Excited-state intramolecular proton transfer (ESIPT) has recently received considerable attention due to its dual fluorescent changes and large Stokes shift. Hydrogen sulfide (H2S) is a gas signal molecule that plays important roles in modulating the functions of different systems. Herein, by modifying 2-(2́-hydroxyphenyl) benzothiazole (HBT) scaffold, a novel near-infrared mitochondria-targeted fluorescent probe HBTP-H2S has been rationally designed based on excited-state intramolecular proton transfer (ESIPT) effect. The nucleophilic addition reaction of the H2S with probe HBTP-H2S caused the break of the conjugated skeleton, resulting the shifting of maximum emission peak from 658 nm to 470 nm. HBTP-H2S showed fast-response response time, good selectivity and a large Stokes shift (188 nm) toward H2S. Most importantly, inspired by the inherent advantages of the probe, HBTP-H2S was successfully employed to monitor mitochondrial H2S in HepG2 cells.

ESIPT-based fluorescent probe for bioimaging and identification of group IIIA ions in live cells and zebrafish

Aluminum (Al), gallium (Ga), indium (In) are three essential elements in group IIIA of the periodic table, which all share similar chemical properties and are also vital in many aspects of bio- and environmental systems. Proper control of their levels is thus necessary as overexposure to them has been linked to onsets of many diseases. Fluorescence based molecular probes have always been the driving horse for detecting vital ions including group IIIA ions. However, only a few such probes have been reported so far and all of them are faced with one or more shortcomings such as not very high sensitivity, incapability to detect multiple ions simultaneously, and poor cell penetration abilities due to emitted fluorescence at shorter wavelengths. To meet those challenges, we herein presented the successful development and application of a novel group IIIA ions fluorescent probe, NBD-hnap, in live RAW264.7 cell and zebrafish models, especially the imaging of ocular tumor cell OCM-1 (human choroid melanoma cells). NBD-hnap was synthesized by a simple conjugation of NBD and hnap molecules under suitable conditions. Subsequent experimental analysis and theoretical calculations confirmed that NBD-hnap forms a 1:1 chelate with each of three selected group IIIA ions. Further evaluation proved that NBD-hnap can realize highly sensitive [LODs of 113, 82 and 150 nM for Al(III), Ga(III), and In(III) respectively in aqueous solutions] and highly selective (over a dozen of interfering cations) through an ESIPT-based fluorescent sensing mechanism with strong far-red emission around 640 nm. Those value merits make NBD-hnap superior to other group IIIA ion probes reported before and NBD-hnap is thus expected to find wider and greater applications in the near future.

A new ESIPT-based fluorescent probe for the highly sensitive detection of amine vapors

A new ESIPT-based fluorescent probe has been developed as a rapid, highly sensitive, and selective sensor for amine vapors.

Two-photon ESIPT-based fluorescent probe using 4-hydroxyisoindoline-1,3-dione for the detection of peroxynitrite.

Excited-state intramolecular proton transfer (ESIPT)-based fluorophores with two-photon excitation fluorescence (TPEF) are rare. Our aim with this research was to develop ESIPT-based fluorophores exhibiting TPEF. Herein, we used 4-hydroxyisoindoline-1,3-dione as a scaffold to develop a two-photon fluorescent probe BHID-Bpin, for the detection of peroxynitrite (ONOO-). BHID-Bpin exhibits excellent selectivity, sensitivity, and fast response towards ONOO- in PBS buffer solution (10 mM, pH = 7.40). Additionally, BHID-Bpin displays high photo-stability under two-photon irradiation at 750 nm. Furthermore, the probe can image endogenous ONOO- in HeLa cells and exogenous ONOO- in rat hippocampal slices at a depth of 110 μm.

Designing an ESIPT-based fluorescent probe for imaging of hydrogen peroxide during the ferroptosis process

Hydrogen peroxide (H2O2), depending on its levels, plays a crucial role in either modulating various biological processes as a signal molecule, or mediating oxidative damage as a toxin. Therefore, monitoring intracellular H2O2 levels is pivotal for exploring its physiological and pathological roles. Using a modified 2-(2′-hydroxyphenyl) benzothiazole (HBT) as the fluorophore, and a pinacol phenylborate ester as the responsive group, herein we developed an excited-state intramolecular proton transfer (ESIPT)-based probe BTFMB. The probe exhibited turn-on fluorescence response, large Stokes shift (162 nm) and low detection limit (109 nM) toward H2O2, and was successfully applied for monitoring exogenous and endogenous production of H2O2, and identifying accumulation of H2O2 during the ferroptosis process.

An ESIPT-based ratiometric fluorescent probe for the discrimination of live and dead cells.

Cell death can destroy homeostasis and is a hallmark of many pathological conditions. Discrimination of live and dead cells is a crucial task for the biological, medical and pharmaceutical studies. Herein, we constructed an ESIPT-based fluorescent probe (BTE) on the basis of the different esterase activity in live and dead cells. Under excitation, the probe BTE showed the blue emission peaked at 465nm, while it mainly displayed green emission peaked at 543nm after it was hydrolyzed by esterase. Imaging of the cells treated by H2O2 and ultraviolet (UV) radiation demonstrated that the probe BTE is effective in the detection of the health of cells, could help us to better understand cell death and its effects in a range of diseases and treatments.

An NIR ESIPT-based fluorescent probe with large stokes shift for specific detection of Cys and its bioimaging in cells and mice

ESIPT-based (Excited-State Intramolecular Proton Transfer) fluorescent probes have been popular because of their large stokes shift and environmental sensitivity. In this work, by coupling dicyanoisophorone and benzothiazole with toluene, we built a cysteine (Cys) fluorescent probe containing a near-infrared ESIPT fluorophore, and an acryloyl group as a nucleophilic addition and leaving site. It was found that only Cys enabled nucleophilic addition and release of hydroxy toluene in conjugated system, with a turn-on NIR emission based on ESIPT effect of benzothiazole. The probe SYP exhibited an excellent selectivity for Cys without any interference from H2S and other thiols (glutathione and homocysteine), attributed to the formation of the seven-membered heterocyclic compound favored kinetically. More, the sensing mechanism was proved to be the ESIPT process by 1H-NMR and MS titration experiments. Of course, the probe displayed its practical application by imaging endogenous Cys in cells and mice. This work innovatively proposed a method for designing a near-infrared fluorescent probe specifically for Cys based on the ESIPT mechanism.

Rational design of an ESIPT-based fluorescent probe for selectively monitoring glutathione in live cells and zebrafish.

Glutathione (GSH), an extremely important antioxidant, is a major participant in maintaining redox homeostasis and tightly associated with various clinical diseases. Thus, accurate and rapid detection of intracellular GSH is imperative to elucidate its role in physiological and pathological processes. Herein, by modifying 2-(2'-hydroxyphenyl) benzothiazole (HBT) scaffold, we developed an excited-state intramolecular proton transfer (ESIPT)-based fluorescent probe BTFMD for tracking GSH, which exhibited good selectivity, excellent water solubility, a large Stokes shift (181nm) and fast response rate (within 10min). Furthermore, the probe was successfully applied for imaging of endogenous GSH in live cells and zebrafish, and probing into the role of GSH in the development of cancer and Parkinson's disease.