Ratiometric Two-photon Fluorescent Probe Research Articles

Ratiometric two-photon fluorescent probe for imaging hydrogen peroxide during stroke-induced ferroptosis

Ischemic stroke (IS) initiates a programmed cell death through apoptosis, autophagy, and ferroptosis, which results in neuronal damage and brain injury. However, existing methods for in situ detection of ferroptosis are currently insufficient. Therefore, we constructed a series of hydrogen peroxide (H2O2—one of the major ROS) probes (QH2O2, MQH2O2, and BQH2O2) with quinoline derivatives as two-photon fluorophores and boronate as the recognition domain. Experimental results indicated that the linear response range of the probe MQH2O2 to H2O2 increased from < 10–300 μM as compared with the probe QH2O2. Moreover, the linear correlation coefficient also increased from 0.9012 to 0.999, indicating that the ratiometric fluorescent probe has built-in corrections for environmental interference, which allows a more accurate fluorescence analysis. Modification of the probe by connecting triethylene glycol monomethyl ether (BQH2O2) increased the lipophilic index of the probe to 2.01, which enhanced its ability to cross the blood–brain barrier. Therefore, the developed probe is useful for analyzing the H2O2 production during an ischemic stroke and its fluctuation with ferroptosis.

Excellent ratiometric two-photon fluorescent probes for hydrogen sulfide detection based on the fluorescence resonance energy transfer mechanism.

Fluorescence resonance energy transfer (FRET) is an important mechanism to design ratiometric fluorescent probes that are able to detect analytes quantitatively according to the ratio of two well-resolved emission signals. Two-photon (TP) fluorescent probes can realize the detection in living cells and tissues with deeper penetration depth, higher resolution, and lower photodamage in contrast to one-photon fluorescent probes. However, to date, fabricating TP-FRET ratiometric fluorescent probes possessing large two-photon absorption (TPA), high fluorescence quantum yield and perfect FRET efficiency is still challenging. Consequently, to develop excellent TP-FRET ratiometric probes and explore the relationship between their molecular structures and TP fluorescence properties, in this paper, we designed a series of H2S-detecting TP fluorescent probes employing the FRET mechanism based on an experimental probe BCD. Thereafter, we comprehensively evaluated the TP sensing performance of these probes by means of time-dependent density functional theory and quadratic response theory. Furthermore, we determined energy transfer efficiency and fluorescence quantum yield. Significantly, through regulating benzene-fused positions, we successfully improved fluorescence quantum yield and TPA cross-section simultaneously. Large spectral overlap between energy donor emission and acceptor absorption was achieved and near perfect energy transfer efficiency was acquired for all the studied probes. We revealed that these probes exhibit two well-resolved TPA bands, which are contributed by FRET donors and acceptors, respectively. Especially, both the wavelengths and the cross-sections of the two TPA bands agree well with those of energy donors and acceptors, which is the unique TPA spectral profile of FRET probes and has never been previously reported. Moreover, we proposed an excellent TP-FRET probe BCD3 and its product molecule BCD3-H2S, which exhibit large Stokes (141 nm and 88 nm) and emission shifts (5931 cm-1), as well as greatly increased TP action cross-sections (24-fold and 60-fold) in the near-infrared region with respect to BCD and BCD-H2S. Our detailed study can give an insight into the efficient design of novel TP-FRET fluorescent probes.

Maximizing analytical precision: exploring the advantages of ratiometric strategy in fluorescence, Raman, electrochemical, and mass spectrometry detection

Ratiometric strategy are an invaluable method that helps to detect and quantify analytes. This approach relies on measuring changes in the ratio of two or more signals to improve the accuracy and sensitivity of the results. Ratiometric strategies are widely used in a variety of fields including biomedical, environmental monitoring and food safety. It is particularly popular when traditional single-signal based detection methods are not feasible, especially when interfering substances severely affect the detection. In addition, ratiometric methods have the potential to improve the accuracy and reliability of analyte detection, leading to better results in a variety of complex environments. The article provides a comprehensive review of ratiometric strategy, focusing on ratiometric fluorescent nanoprobes for the visual detection of analytes. This paper also discusses the design of ratiometric two-photon fluorescent probes for biomedical imaging, the synthesis of ratiometric surface-enhanced Raman scattering nanoprobes for the imaging of intracellular analytes, the development of ratiometric molecularly imprinted electrochemical sensors for detection of electroactive species, and the use of isotopically-labeled internal standards in matrix-assisted laser desorption/ionization for ratiometric analysis. The article not only discusses each technique in detail, including its principles, advantages, potential applications, and limitations, but also highlights recent advances in each method and possible future directions.

A simple ratiometric fluorescent probe for two-photon imaging of carbon monoxide in living cells and zebrafish

Carbon monoxide (CO) is an important gas signaling molecule and has been widely involved in regulating important life processes. Effective monitoring of CO in living systems is critical. Combined with the accuracy of ratio detection and the advantages of two-photon imaging, a simple ratiometric two-photon fluorescent probe RTFP was rationally designed and synthesized using 7-(diethylamino)-4-hydroxycoumarin as a two-photon fluorophore and allyl carbonate as the reactive unit. Probe RTFP exhibited excellent selectivity and sensitivity towards CO, and was successfully applied to image endogenous CO in living cells and zebrafish.

New ICT-Based Ratiometric Two-Photon near Infrared Probe for Imaging Tyrosinase in Living Cells, Tissues, and Whole Organisms

Melanoma is a type of highly malignant and metastatic skin cancer. In situ molecular imaging of endogenous levels of the melanoma biomarker tyrosinase (TYR) may decrease the likelihood of mortality. In this study, we proposed the weakly fluorescent probe 1-(4-(2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl)phenyl)-3-(4-hydroxybenzyl)urea (DCM-HBU), which releases a strong red-shifted fluorescent signal after a TYR-mediated oxidation followed by hydrolysis of the urea linkage. The large Stokes shift of the dye is owed to the recovery of the intramolecular charge transfer (ICT) effect. The resulting probe derivate shows a highly ratiometric fluorescence output. Furthermore, the simultaneous excitation by two near-infrared (NIR) photons of the released derivative of dicyanomethylene-4H-pyran (DCM-NH2) fluorophore could avoid the usual drawbacks, such as cellular absorption, autofluorescence, and light scattering, due to an usually short wavelength of the excitation light on biological systems, resulting in images with deeper tissue penetration. In addition, the probe is useful for the quantitative sensing of TYR activity in vivo, as demonstrated in zebrafish larvae. This new ratiometric two-photon NIR fluorescent probe is expected to be useful for the accurate detection of TYR in complex biosystems at greater depths than other one-photon excited fluorescent probes.

Monitoring cell viability in N-nitrosodiethylamine induced acute hepatitis and detection of hydrazine in solution and gas phase with Dual-function fluorescent probes.

The highly toxic N-nitrosodiethylamine (NDEA) and hydrazine (N2H4) caused severe environmental contamination and serious health risks. Herein, we designed the two-photon ratiometric fluorescent probe (Nap-2), emission maximum shifted from 466nm to 571nm, to monitor cell viability of NDEA induced acute hepatitis via esterase activity detection. Furthermore, the probe Nap-2 evaluate the hydrazine (N2H4) content in the solution and gas phase. It is worth mentioning that we used NDEA induced acute hepatitis in the mice and evaluated the negative correlation of esterase activity in the tissue cells and serum with Nap-2. The probe Nap-2 exhibited that acute hepatitis induced by NDEA decreased cell viability. Furthermore, we made convenient test papers using Nap-2 to detect N2H4 in solution and gas phase. After adding N2H4, the fluorescence color changed from blue to yellow and was visible to the naked eye. This work provides a convenient tool and method for evaluating the toxicity of NDEA induced acute hepatitis and detecting N2H4 in the environment.

Monitoring glutathione dynamics in DNA replication (S-phase) using a two-photon reversible ratiometric fluorescent probe.

The redox regulator glutathione (GSH) migrates to the nucleus to give a safeguard to DNA replication in the S-phase. The fluctuation of GSH dynamics in the cell cycle process may help to understand cancerogenesis or other abnormalities in DNA replication. For the first time, we attempted to track the time-dependent S-phase change using the newly developed ratiometric fluorescent probe Nu-GSH. This probe is highly chemoselective towards glutathione and shows an emission intensity shift from 515 nm to 455 nm. It has shown fluorescence reversibility from blue to green channels while scavenging reactive oxygen species H2O2. Both ratiometric fluorescence images and FACS analysis have provided quantitative information on the GSH levels in the nucleoli during DNA replication in the S-phase. Furthermore, GSH fluctuation reciprocated the decay of the S-phase on a time scale. Additionally, its two-photon ability guaranteed its capability to study GSH dynamics in live cells/tissues noninvasively. We envision that the probe Nu-GSH can be used to get high-throughput quantitative information on glutathione dynamics and give an opportunity to monitor its perturbation during the course of cell division.

Construction of a ratiometric two-photon ER-targeting fluorescent probe for the imaging of peroxynitrite in living systems

Peroxynitrite (ONOO-) could deplete ER-associated Ca2+ and cause ER stress, and thereby precise and in situ detection of ONOO- at ER is highly important for the study of its biological roles. Herein, we present a ratiometric two-photon ER-targeting fluorescent probe (NAB) for the detection of ONOO- in living cells and brain tissues. NAB utilized 4-bromomethylphenyl borate as a recognition site for ONOO-, and utilized p-toluenesulfonamide as ER-targeting group. After the response of NAB to ONOO-, the 4-bromomethylphenyl borate converted to hydroxyl, resulting in the change of the fluorescence color from blue to yellow. The probe NAB could show highly sensitive response to ONOO- within 6 s, and exhibited excellent selectivity to ONOO-. The probe NAB mainly located at ER and showed excellent ER-targeting property. Ratiometric two-photon imaging demonstrated that the probe NAB could selectively detect exogenous ONOO- at ER, and also could be applied to image metformin-induced ONOO- in living HepG2 cells. Furthermore, NAB can effectively detect the changes of ONOO- level in brain tissues, and revealed that the endogenous ONOO- level at ER showed increase in hippocampus of the depressive mouse.

Tracking Autophagy Process with a TBET and AIE-Based Ratiometric Two-Photon Viscosity Probe

Autophagy is a cellular self-degrading process that plays a key role in cellular health and functioning. Since autophagy disorder is related to many diseases, it is highly important to detect autophagy. This study aimed to establish a dual-sensing mechanism-based ratiometric viscosity-sensitive lysosome-targeted two-photon fluorescent probe Vis-sun to track the autophagy process (the increase in lysosome viscosity during autophagy) by combining through bond energy transfer (TBET) and aggregation-induced emission (AIE). The introduction of TBET not only overcame the interference of background signals but also achieved the baseline separation of two emission peaks, thus reducing the crosstalk between emissions, as well as the noninvasive bio-sensing of biological targets and long-term real-time tracer imaging by introducing AIE. In vitro experiments showed that the fluorescence intensity at 485 nm decreased gradually on increasing the volume ratio of water to tetrahydrofuran (Vwater/VTHF), while the fluorescence intensity at 605 nm increased significantly. Also, the fluorescence signal was maximized when the water content reached 100%. At the same time, the probe exhibited a significant dependence on the ambient viscosity. Therefore, the dynamic monitoring of lysosome viscosity during autophagy and the in situ imaging of autophagy fluctuations during stroke-induced neuroinflammation were successfully achieved by implementing Vis-sun lysosome anchoring with morpholine.

A two-photon ratiometric fluorescent probe for real-time imaging and quantification of NO in neural stem cells during activation regulation.

Developing a novel tool capable of real-time monitoring and accurate quantification of NO is critical to understanding its role in physiological and pathological processes. Herein, a two-photon ratiometric fluorescent probe (NOP) was developed for real-time imaging and quantification of NO based on fluorescence resonance energy transfer-photoinduced electron transfer (FRET-PET). In this developed probe, coumarin (CM) and naphthalimide with o-phenylenediamine (NPM) were rationally designed as a fluorescent donor and acceptor, respectively, to enable a ratiometric fluorescence response to NO. The developed NO probe demonstrated good detection linearity with the concentration of NO in the range of 0.100–200 μM, with a detection limit of 19.5 ± 1.00 nM. Considering the advantages of high selectivity, good accuracy and rapid dynamic response (<15 s), the developed NO probe was successfully applied for real-time imaging and accurate quantification of NO in neural stem cells (NSCs) and different regions of mouse brain tissue with a penetration depth of 350 μm. Using this powerful tool, it was found that NO regulated the activation and differentiation of quiescent NSCs (qNSCs). In addition, NO-induced differentiation of qNSCs into neurons was found to be dose-dependent: 50.0 μM NO caused about 50.0% of qNSCs to differentiate into neurons. Moreover, different regions of the mouse brain were observed to be closely related to the concentration of NO, and the concentration of NO in the DG region was found to be lower than that in the S1BF, CA1, LD and CPu of the Alzheimer's disease (AD) mouse brain. The symptoms of AD mice were significantly improved through the treatment with NO-activated NSCs in the DG region.

Development of a ratiometric nitric oxide probe with baseline resolved emissions by an ESIPT and rhodol ring opened-closed integrated two-photon platform.

In recent years, reflecting the degree of cellular inflammation through in situ monitoring of nitric oxide using fluorescence sensing has received much attention due to many merits such as non-invasiveness and easy operation. In particular, two-photon excitation microscopy can significantly improve the imaging resolution and visualization time. In the meantime, a ratiometric-based nitric oxide fluorescent sensor can avoid the interference of many factors, including light source intensity, solvent scattering degree, solvent color, solvent viscosity, probe distribution, and instrument performance, and improve the accuracy of the result. However, the mutual interference of two emission peaks is still an issue restricting the development of this field. In this work, the Rh-NO-F dye obtained by modifying the rhodol dye with benzothiazole exhibited excited state intramolecular proton transfer (ESIPT) in the closed ring state. In the open ring state, however, the emission wavelength can be significantly red-shifted by increasing the degree of dye conjugation. By introducing o-phenylenediamine, the recognition domain of NO, we successfully designed and synthesized a ratiometric two-photon NO fluorescent probe, Rh-NO-P, which showed a 154 nm increase in the maximum emission wavelength before and after the response and almost no interference between the two emission peaks. Confocal imaging showed that the probe could achieve in situ detection of exogenous NO fluctuations in cells. The probe was also successfully applied to detect the changes in NO content during wound healing in mice.

Chlorination-induced pKa decrease: an improved strategy to design ratiometric hypochlorite fluorescent probes with ideally high selectivity.

Based on the selective ClO--triggered chlorination reaction and the subsequent pKa decrease of phenols, a new strategy was developed for rationally designing ratiometric ClO- fluorescent probes with high selectivity. By investigating the fluorescence responses of 6-cyano-2-naphthol toward ClO- and the pKa-dependent response mechanism, we developed a rapid, sensitive and selective two-photon ratiometric fluorescent probe, Naph-DFOB, to detect ClO-. This probe displayed a ratiometric fluorescence change (from 509 nm to 628 nm) toward ClO- and was successfully applied to image intracellular ClO- in living cells with two-photon excitation. Using Naph-DFOB as a useful tool, the investigation of lipopolysaccharide (LPS)-induced acute lung injury in a mouse model was effectively performed.

Two-photon fluorescence imaging and ratiometric quantification of mitochondrial monoamine oxidase-A in neurons.

Herein, we designed and developed a single two-photon ratiometric fluorescence probe (TMF2P) for selective and accurate determination of mitochondrial MAO-A in live neurons. It was discovered that the increases in MAO-A levels under oxidative stress resulted in an elevated influx of Ca2+ flow into mitochondria through the transient receptor potential melastatin 2 (TRPM2) channels.

Computational design of ratiometric two-photon fluorescent Zn2+ probes based on quinoline and di-2-picolylamine moieties

To improve two-photon absorption (TPA) response of a newly synthesized probe, a series of ratiometric two-photon fluorescent Zn2+ sensors based on quinoline and DPA moieties have been designed. The one-photon absorption, TPA, and emission properties of the experimental and designed probes before and after coordination with Zn2+ are investigated employing the density functional theory in combination with response functions. The design consists of two levels. In the first level of design, five probes are constructed through using several electron acceptors or donors to increase accepting or donating ability of the fluorophores. It shows that all the designed probes have stronger TPA intensities at longer wavelengths with respect to the experimental probe because of the increased intra-molecular charge transfer. Moreover, it is found that the probe 4 built by adding an acyl unit has the largest TPA cross section among the designed structures due to the form of longer conjugated length and more linear backbone. One dimethylamino terminal attached along the skeleton can improve TPA intensity more efficiently than two side amino groups. Therefore, in the second level of design, a new probe 7 is formed by both an acyl unit and a dimethylamino terminal. It exhibits that the TPA cross sections of probe 7 and its zinc complex increase dramatically. Furthermore, the fluorescence quantum yields of the designed probes 4 and 7 are calculated in a new way, which makes use of the relation between the computed difference of dipole moment and the measured fluorescence quantum yield. The result shows that our design also improves the fluorescence quantum yield considerably. All in all, the designed probes 4 and 7 not only possess enhanced TPA intensities but also have large differences of emission wavelength upon Zn2+ coordination and strong fluorescence intensity, which demonstrates that they are potential ratiometric two-photon fluorescent probes.

An enzyme-activated two-photon ratiometric fluorescent probe with lysosome targetability for imaging β-glucuronidase in colon cancer cells and tissue

Colon cancer is a malignant neoplasm with high mortality that has seriously threatened human life. Accumulating evidence reveals that the β-glucuronidase (GLU), a lysosomal exoglycosidase enzyme, plays important roles in the pathological progression of colon cancer. Unfortunately, understanding the pathological roles of GLU remains a challenge due to the lack of effective detection methods for visualization the fluctuations of GLU in tissues. In this paper, based on hydrolysis function of GLU, an enzyme-activated ratiometric two-photon (TP) fluorescent probe (RN-GLU) was designed. RN-GLU was synthesized by introducing a glucopyranuronic acid methyl ester as the recognition group and 1, 8-naphthalimide as a TP fluorophore. In the presence of GLU, the trigger group was removed made an ICT process occurred induced enhancement of fluorescence ratio (I553 nm/I441 nm, 214-fold). Probe RN-GLU displayed low detection limit (1.2 × 10−2 μg/L) and rapid detection to GLU in vitro through a ratiometric response mode. Meanwhile, RN-GLU exhibited high selectivity for GLU and showed nearly no response to other relevant biological species. The imaging results demonstrated that RN-GLU could be applied for ratiometric monitoring of endogenous GLU levels in HCT116 cells with good lysosome targetable ability. Due to its two-photon excitation, RN-GLU could monitor GLU in colon cancer tumor tissue with good penetration ability (imaging depth of 200 μm). RN-GLU could be developed as a potential method for evaluating and confirming the functions of GLU in colon cancer diagnosis and complex biosystem.

Two-photon ratiometric fluorescent probe for imaging of hypochlorous acid in acute lung injury and its remediation effect

Acute lung injury (ALI) is a pulmonary inflammatory disease with high morbidity and mortality rates. However, owing to the unknown etiology and rapid progression of the disease, the diagnosis of ALI is full of challenges with no effective treatment. Since the inflammatory response and oxidative stress played vital roles in the development of ALI, we herein developed the largest emission cross-shift (△λ = 145 nm) two-photon ratiometric fluorescent probe of TPRS-HOCl with high selectivity and short response time toward hypochlorous acid (HOCl) for exploring the relevance between the degree of ALI and HOCl concentration in the development process of the disease. In addition, the inhibition effect of HOCl during different treatment periods was also evaluated. Moreover, the tendency of imaging results was basically in accordance with that of hematoxylin and eosin (H&E) staining and the treatment effect became better in the early stage when using N-acetylcysteine (NAC), demonstrating the sensitivity of TPRS-HOCl toward ALI response. Thus, TPRS-HOCl has great potential to diagnose ALI in the early stage and guide for effective treatment.

A Highly Stable Two-Photon Ratiometric Fluorescence Probe for Real-Time Biosensing and Imaging of Nitric Oxide in Brain Tissues and Larval Zebrafish

A Highly Stable Two-Photon Ratiometric Fluorescence Probe for Real-Time Biosensing and Imaging of Nitric Oxide in Brain Tissues and Larval Zebrafish

Two-Photon, Ratiometric, Quantitative Fluorescent Probe Reveals Fluctuation of Peroxynitrite Regulated by Arginase 1.

Peroxynitrite, a transient reactive oxygen species (ROS), is believed to play a deleterious role in physiological processes. Herein, we report a two-photon ratiometric fluorescent probe that selectively reacts with peroxynitrite yielding a >200-fold change upon reaction. The probe effectively visualized fluctuations in peroxynitrite generation by arginase 1 in vivo and in vitro. This provides evidence that arginase 1 is a critical regulator of peroxynitrite.

Two-photon ratiometric fluorescent probe based on NBD-amine functionalized semiconducting polymer nanoparticles for real-time imaging of hydrogen sulfide in living cells and zebrafish

The thiolysis of 7-nitro-1,2,3-benzoxadiazole amine (NBD-A) paves the way for specific sensing of H2S over biothiols and real-time imaging in living organisms. Rational fabrication of NBD-A-based probe with ratiometric mode and two-photon excitation is highly appealing to achieve high quality bioimaging. In this work, the NBD-A molecules are assembled with poly(9,9-dioctylfluorenyl-2,7-diyl) polymer nanoparticles, defined as NBD@PFO, to construct two-photon ratiometric probes for H2S detection through the fluorescence resonance energy transfer (FRET). For the construction of NBD@PFO nanohybrids, polymer nanoparticles are employed as the NBD-A molecular vehicle, energy donor and two-photon absorber, while NBD-A is served as the response unit and energy acceptor. Taking advantages of NBD-A and polymer nanoparticles, the obtained NBD@PFO probes exhibit high selectivity, fast response (<5 s), ratiometric detection and two-photon excitation. Our results indicate that NBD@PFO nanohybrids are successfully applied for monitoring of H2S concentration in living cells and zebrafish, exhibiting great potential of polymer nanoparticles to improve the imaging capability of an organic small molecular probe.

Developing Push-Pull Hydroxylphenylpolyenylpyridinium Chromophores as Ratiometric Two-Photon Fluorescent Probes for Cellular and Intravital Imaging of Mitochondrial NQO1.

This work highlights the use of push-pull hydroxylphenylpolyenylpyridinium fluorophores coupled with trimethyl lock quinone to engineer the ratiometric two-photon probes for cellular and intravital imaging of mitochondrial NAD(P)H:quinone oxidoreductase 1 (NQO1), a critical antioxidant enzyme responsible for detoxifying quinones. As a typical representative, QBMP showed favorable binding with NQO1 with a Michaelis constant of 12.74 μM and exhibited a suite of superior properties, including rapid response (4 min), large Stokes shift (162 nm), ultralow detection limit (0.9 nM), favorable two-photon cross section for the released fluorophore (70.5 GM), and deep tissue penetration (225 μm) in fixed brain tissues. More importantly, this probe was successfully applied for distinguishing different NQO1-expressing cancer and normal cells, revealing decreased NQO1 activity in a cellular Parkinson's disease model, screening NQO1 inducers as neuroprotective agents, and imaging of NQO1 in live mouse brain.