Significant Fluorescence Enhancement Research Articles

Ratiometric Fluorescent Probe for Real-Time Detection of β-Galactosidase Activity in Lysosomes and Its Application in Drug-Induced Senescence Imaging.

Senescence is an important biological process, which leads to the gradual degradation of its physiological function and increases morbidity and mortality. Herein, a novel ratiometric fluorescent probe (P1) was constructed by using benzothiazolyl acetonitrile dye as fluorophore, exhibiting significantly enhanced blue-shifted emission to indicate the activity of β-galactosidase (β-gal), a commonly used biomarker for the detection of senescent cells. After incubation with β-gal, the excimer emission of P1 at 620 nm was weakened, while the emission at 533 nm was significantly enhanced, forming an obvious ratiometric probe with high sensitivity and low detection limit (2.7 mU·mL-1). More importantly, probe P1 can locate lysosomes accurately, allowing us to monitor the emergence of living cell senescence in real time. P1 was successfully used to detect β-gal activity in PC-12 cells, Hep G2 cells, and RAW 264.7 cells. It showed strong green fluorescence signal in senescent cells and red fluorescence signal in normal cells, indicating that it can detect endogenous senescence-related β-gal content in living cells. For in vivo drug-induced senescence imaging, after 5 weeks of injection of D-galactose or hydroxyurea, the mice showed significant fluorescence enhancement in specific channels to indicate the activity of β-gal in vivo. At the same time, the senescence of cell-specific organs and skin tissues at the organ level were also detected, which proved that the drug-induced senescence of brain, skin, and muscle tissues was the most serious. These results supported the important application value of P1 in senescence biomedical research.

Aggregation-induced emission carbon dots as Al3+-mediated nanoaggregate probe for rapid and selective detection of tetracycline

Worldwide abuse of tetracycline (TC) seriously threatens environmental safety and human health. Metal-TC complexes formed by residual TC in the environment can also contribute to the spread of antibiotic resistance. Therefore, monitoring of TC residues is still required. Here, we report novel aggregation-induced emission carbon dots (AIE-Cdots) as nanoaggregate probes for the rapid and selective detection of TC residue. Riboflavin precursors with rotational functional groups led to the development of AIE-Cdots. The aggregation of AIE-Cdots was induced selectively for Al3+, amplifying the fluorescence signals owing to the restricted rotation of the side chains on the AIE-Cdot surface. The fluorescence signal of such Al3+-mediated nanoaggregates (Al3+-NAs) was further triggered by the structural fixation of TC at the Al3+ active sites, suggesting the formation of TC-coordinated Al3+-NAs. A linear correlation was observed in the TC concentration range of 0–10 μM with a detection limit of 42 nM. In addition, the strong Al3+ binding affinity of AIE-Cdots produced similar NAs and enhanced fluorescence signals in Al3+–TC mixtures. These AIE-Cdots-based nanoplatforms have a rapid response, good selectivity, and reliable accuracy for detecting TC or aluminum complexes, meeting the requirements for hazardous substance monitoring and removal in environmental applications.

Preferences of interaction sites for proton-induced photophysical signalling with a derivatized rhodamine probe

Chemosensors for selective and sensitive detection of analytes were developed following various structural, functional and operational design methodologies. The interaction between probe and analyte has been vital in chemosensor-based detection technologies. A pertinent question in such a system is to be convincingly addressed, that if the probes incorporating spatially-positioned multiple interaction sites offer preferences towards analyte binding for a favourable and optimal interaction. The derivatized Rhodamine based probe 1 exhibiting proton-induced dual mode signalling here contained several protonation sites. The absorption and fluorescence signal enhancement on protonation of the probe attributed to its proton-mediated spiro-ring opening. Out of several plausible conformations of protonated probe [1.H+], their DFT calculations revealed the proton binding preferentially to the N-donor atom on spiro-ring of 1 instead at other probable sites of protonation such as other hetero- donor atoms on the xanthene core or the carbonyl O- atom on the spiro-ring as the energetically favourable conformation. It was verified with the protonated probe complex's X-ray diffracted single crystal structure.

A fluorescence chemo sensor approach for determination of finerenone in pharmaceutical formulation and human plasma: Method development and validation

Finerenone, a non-steroidal mineralocorticoid receptor antagonist, has gained recent approval for treating cardiovascular and kidney-related conditions. Herein, an innovative fluorescence chemo sensor was developed for the determination of finerenone in the pharmaceutical dosage form and the plasma matrix. The method is basically based on chemical transformation of finerenone into a fluorescent product through sequential reactions. This transformation occurs through a sequence of steps involving the interaction of finerenone with trimethylamine, resulting in the formation of a nucleophilic intermediate that subsequently reacts with bromoacetyl bromide to form fluorescent product, (S)-N-(2-bromoacetyl)-4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide. The formed fluorescent product exhibits defined emission peak at 338 nm when excited at 248 nm. Significant concentration-dependent fluorescence enhancement was obtained enabling precise finerenone determination in the pharmaceutical formulation and plasma matrix. The method was optimized and validated providing sensitive determination over linearity range of 1–200 ng/mL with a lower limit of detection at 0.280 ng/mL. This strategy provides an efficient, economical substitute and straightforward, more sensitive analytical method for finerenone assessment in various matrices, in contrast to the previously published method, high-performance liquid chromatography-tandem mass spectrometry, which is expensive and time-consuming.

Label-free and highly sensitive detection of microRNA from cancer cells via target-induced cascade amplification generation of lighting-up RNA aptamers

The abnormal expression levels of miRNAs have been proven to be highly related to the generation of various diseases and are also closely associated with the stages and types of disease development. The novel RNA aptamers-based homogenous fluorescent methods were simple, with low background signal and high signal-to-noise ratio, but lacked effective signal amplification technology to achieve sensitive detection of trace miRNA markers. There is an urgent need for combining effective nucleic acid amplification technology with RNA aptamer to achieve highly sensitive and accurate detection of miRNA. For this purpose, a new DNA multi-arm nanostructure-based dual rolling circle transcription machinery for the generation of lighting-up MG RNA aptamers is constructed for label-free and highly sensitive sensing of miRNA-21. In this system, the target miRNA-21 induces a structural transformation of the DNA multi-arm nanostructure probe to recycle miRNA-21 and trigger two independent rolling circle transcription reactions to generate two long RNAs, which can partially hybridize with each other to generate large amounts of complete MG RNA aptamers. These RNA aptamers can associate with organic MG dye to produce significantly enhanced fluorescence signals to accomplish ultrasensitive miRNA-21 detection down to 0.9 fM. In addition, this method exhibits high selectivity to distinguish miRNA-21 even with single nucleotide mismatch, and also has potential application capability to monitor different expression levels of miRNA-21 from different cancer cells. The effective collaboration between MG RNA aptamer and rolling circle transcription reaction makes this fluorescent method show the significant advantages of low background signal, high signal-to-noise ratio and high detection sensitivity. It has great potential to be a promising means to achieve label-free and highly sensitive monitoring of other trace biological markers via a simple change of target sequence.

A red-emitting mitochondria targetable fluorescent probe for detecting viscosity in HeLa, zebrafish, and mice.

Viscosity, an essential parameter of the cellular microenvironment, has the ability to indicate the condition of living cells. It is closely linked to numerous diseases like Alzheimer's disease, diabetes, and cardiovascular disorders. Therefore, it is necessary to design tools to effectively monitor viscosity changes, which could provide promising avenues for therapeutic interventions in these diseases. Herein, we report a novel mitochondria-targeting fluorescent probe GX-VS which was suitable for the detection of viscosity changes in vivo and in vitro. The probe GX-VS had many advantages such as long emission wavelength (650 nm), large Stokes shift (105 nm), significant fluorescence enhancement (59-fold), high sensitivity, good biocompatibility and so on. Biological experiments showed that the probe could target mitochondria and detect viscosity alterations in HeLa cells. Moreover, it has been successfully utilized to monitor viscosity changes induced by lipopolysaccharides (LPS) in inflammatory zebrafishes and living mice, which further underscored the capacity of GX-VS to explore fluctuations in viscosity within living organisms.

Turn-on fluorescent probe based on dicyanoisophorone for bioimaging and rapid detection of peroxynitrite in aqueous media.

A novel dicyanoisophorone-based colorimetric fluorescent probe 3 has been prepared for recognizing peroxynitrite in aqueous conditions. A large bathochromic shift of the absorption band is observed upon titration with ONOO-, inducing a clearly visible solution color change from yellow to pale pink, which makes "naked-eye" detection possible. Moreover, probe 3 can react instantly with ONOO- and is accompanied by a significant fluorescence enhancement at 621 nm while the detection limit is as low as 37 nM. Most importantly, probe 3 exhibits high selectivity and sensitivity towards ONOO- in the presence of other competitive ions in aqueous solution. Probe 3 has also been successfully applied in living MCF-7 cells, and the results suggest that probe 3 could be applied as a potential candidate for the detection of ONOO-.

Corrosion-enabled tryptophan biosensing enhancement on commercially available Mg alloy surfaces.

A novel method enhances tryptophan fluorescence signals 5.45 times using a corrosion-modified magnesium alloy. Corrosion controls and stabilizes the surface morphology, resulting in a significant fluorescence enhancement. This highlights its biosensing potential with long-term stability, crucial for understanding the impact of tryptophan on metabolic and neurological disorders.

Preparation of near-infrared photoacoustic imaging and photothermal treatment agent for cancer using a modifiable acid-triggered molecular platform.

Ratiometric near-infrared fluorescent pH probes with various pKa values were innovatively designed and synthesized based on cyanine with a diamine moiety. The photochemical properties of these probes were thoroughly evaluated. Among the series, IR-PHA exhibited an optimal pKa value of approximately 6.40, closely matching the pH of cancerous tissues. This feature is particularly valuable for real-time pH monitoring in both living cells and living mice. Moreover, when administered intravenously to tumor-bearing mice, IR-PHA demonstrated rapid and significant enhancement of near-infrared fluorescence and photoacoustic signals within the tumor region. This outcome underscores the probe's exceptional capability for dual-modal cancer imaging utilizing near-infrared fluorescence (NIRF) and photoacoustic (PA) modalities. Concurrently, the application of a continuous-wave near-infrared laser efficiently ablated cancer cells in vivo, attributed to the photothermal effect induced by IR-PHA. The results strongly indicate that IR-PHA is well-suited for NIRF/PA dual-modality imaging and photothermal therapy of tumors. This makes it a promising candidate for theranostic applications involving small molecules.

Detecting labile heme and ferroptosis through 'turn-on' fluorescence and lipid droplet localization post Fe2+ sensing.

Iron, a crucial biologically active ion essential for metabolic processes in living organisms, plays a vital role in biological functions, and imbalances in iron levels can lead to various diseases. In this study, we have developed two simple "turn-on" fluorescent probes, NOPy and NOCN, for the quick and selective detection of Fe2+ at nanomolar levels (LOD of 35 nM), accompanied by significant absorption and emission shifts, along with colorimetric demarcation. Both fluorophores exhibit an excellent "turn-on" emission response upon encountering Fe2+ in the cells. Flow cytometry and confocal fluorescence imaging studies demonstrate enhanced fluorescence signals in response to labile iron, efficiently detecting heme during erastin-induced ferroptosis. Interestingly, we also observed that the product formed after Fe2+ sensing localizes within the lipid droplets. These water-soluble and highly sensitive reactive probes, NOPy and NOCN, enable investigations of iron-dependent physiological and pathological conditions. The development of these probes represents an advancement in the field, offering a rapid and selective means for detecting Fe2+ with minimal cytotoxicity.

The design of multifunctional fluorescent probe for detecting Zn2+/ ClO−/H2O and application in high-security level information encryption

In this article, a Schiff base-structured compound (TNB) was synthesized using 3-hydroxy-2-naphthaldehyde hydrazone and 2-hydroxy-5-tert-butylbenzaldehyde as starting materials. The structure of TNB was characterized using NMR spectroscopy, high-resolution MS, and IR spectroscopy. Studies showed that TNB can selectively recognize Zn2+/ClO− in solvent system of H2O/DMSO. Fluorescence and UV–vis spectrophotometry were used to investigate the spectral properties of TNB. TNB displayed a significant fluorescence enhancement effect when bound to Zn2+, while binding to ClO− resulted in fluorescence quenching. Job’s plot and NMR titration were used to study the binding mechanism of TNB with Zn2+/ClO−. Linear standard curves were fitted for different Zn2+/ClO− concentrations corresponding to fluorescence intensity, and detection limits (LOD) were determined as 5.0300 × 10−9 mol/L (Zn2+) and 3.1700 × 10−9 mol/L (ClO−). Furthermore, TNB was used to determine trace water in organic solvents. The fluorescence intensity changes with water content were measured in three organic solvents (THF, DMSO, and acetone). Detection limits (LOD) were calculated as 1.4 × 10−4 % (THF), 8.3 × 10−4 % (DMSO), and 1.2 × 10−3 % (acetone). Using the characteristic reactions of TNB with Zn2+, a two-dimensional code information encryption technology was developed and can be used for high-security information encryption and transmission.

Rhodamine-based Fluorescent Probe With Quick Response and High Selectivity for Imaging Labile Ferrous Iron in Living Cells and Zebrafish.

The transition between its various oxidation states of Iron plays a crucial part in various chemical transformation of cells. Misregulation of iron can give rise to the iron-catalyzed reactive oxygen species disorder which have been linked to a variety of diseases, so it is crucial to monitor the labile iron pool in vivo for clinical diagnosis. According to iron autoxidation and hydrogen abstraction reaction, we reported a novel "off-on" fluorescent probe to response to ferrous (Fe2+) both in solutions and biological systems. The probe responds to Fe2+ with good selectivity toward competing metal ions. What's more, the probe presents significant fluorescent enhancement to Fe2+ in less than 1min, making real-time sensing in biological system possible. The applications of the probe in bioimaging revealed the changes in labile iron pool by iron autoxidation or diverse stimuli.

Introducing fluorescent probe technology for detecting microcystin-LR in the water and cells

BackgroundFor a long time, the environment hazards caused by cyanobacteria bloom and associated microcystins have attracted attention worldwide. Microcystin-LR (MC-LR) is the most widely distributed and most toxic toxin. At present, numerous MC-LR detection methods exist many drawbacks. Therefore, a quick and accurate method for identifying and detecting MC-LR is crucial and necessary. In this work, we strived to introduce a novel fluorescence assay to detect MC-LR in the water and cells. ResultsAccording to the special spatial configuration and physicochemical properties of MC-LR, we designed and constructed six fluorescent probes. The design concepts of the probes were exhaustively elaborated. MC-YdTPA, MC-YdTPE, MC-RdTPA, and MC-RdTPE could show significant fluorescence enhancement in MC-LR solution. Significantly, MC-YdTPA, MC-YdTPE, and MC-RdTPA could also response well in the cells treated with MC-LR, demonstrating these fluorescent probes’ values. The recognition mechanism between probes and MC-LR were also deeply explored: (1) The polyphenylene ring structure of probes may have nested or hydrogen bond weak interaction with the ring structure of MC-LR. (2) The probes can generate a reaction to the hydrogen ions ionized by MC-LR. SignificanceWe proposed the novel ideas for designing MC-LR probes. This research can provide valuable experiences and important assistance in synthesizing MC-LR fluorescent probes. We expect that this work may bring new ideas to develop fluorescent probes for researching MC-LR in vivo and in vitro.

An RNA Methylation-Sensitive AIEgen-Aptamer Reporting System for Quantitatively Evaluating m6A Methylase and Demethylase Activities.

N6-Methyladenosine (m6A) chemical modification determines the fate of the mammalian cellular mRNA to modulate crucial physiological and pathological processes. Dysregulations of m6A methylase and demethylase have been linked to cancer diseases. Therefore, evaluations of enzyme mutants' activities and related inhibitors for discovery of targeted therapeutic strategies are very necessary. Here, we report an RNA methylation-sensitive fluorescent aptamer reporting assay to measure the catalytic activities of m6A enzymes under various conditions. The rationale is that when an RNA aptamer, named A-Pepper, is methylated at a specific adenosine position to generate m6A-Pepper, the latter displays stronger fluorescence than the former upon binding the ligand, which is an aggregation-induced emission-active luminogen. The fluorescence signal enhancement is linearly proportional to the RNA methylation extent, which is equivalent to the methylase activity. On the contrary, the m6A demethylase activity is measured through calculating the fluorescence signal decrease caused by the switching from m6A-Pepper to A-Pepper. The assay has been successfully applied to quantitatively evaluate the mutation and inhibitor effects on the activities of m6A methylases METTL3/METTL14 and demethylase FTO, and the obtained results are well-consistent with those quantified by the expensive and time-consuming golden standard LC-MS/MS. Our work provides a simple tool capable of detecting m6A enzymes' activities and screening their inhibitors in a rapid, quantitative, cost-effective, and high-throughput manner.

Color Routing of the Emission from Magnetic and Electric Dipole Transitions of Eu3+ by Broken-Symmetry TiO2 Metasurfaces.

Manipulation of magnetic dipole emission with resonant photonic nanostructures is of great interest for both fundamental research and applications. However, obtaining selective control over the emission properties of magnetic dipole transitions is challenging, as they usually occur within a manifold of spectrally close emission lines associated with different spin states of the involved electronic levels. Here we demonstrate spectrally selective directional tailoring of magnetic dipole emission using designed photonic nanostructures featuring a high quality factor. Specifically, we employ a hybrid nanoscale optical system consisting of a Eu3+ compound coupled to a designed broken-symmetry TiO2 metasurface to demonstrate directional color routing of the compound's emission through its distinct electric and magnetic-dominated electronic transition channels. Using low numerical aperture collection optics, we achieve a fluorescence signal enhancement of up to 33.13 for the magnetic-dominated dipole transition at 590 nm when it spectrally overlaps with a spectrally narrow resonance of the metasurface. This makes the, usually weak, magnetic dipole transition the most intense spectral line in our recorded fluorescence spectra. By studying the directional emission properties for the coupled system using Fourier imaging and time-resolved fluorescence measurements, we demonstrate that the high-quality-factor modes in the metasurface enable free-space light routing, where forward-directed emission is established for the magnetic-dominated dipole transition, whereas the light emitted via the electric dipole transition is mainly directed sideways. Our results underpin the importance of magnetic light-matter interactions as an additional degree of freedom in photonic and optoelectronic systems. Moreover, they facilitate the development of spectrometer-free and highly integrated nanophotonic imaging, sensing, and probing devices.

Engineering a highly selective leucine aminopeptidase near-infrared fluorescence probe for early diagnosis of diabetic nephropathy

Diabetic nephropathy (DN) is one of the most serious and general complications of diabetes, while schedules for prevention and treatment are unsatisfactory. Leucine aminopeptidase (LAP) as one of the biomarkers of DN is associated with glomerular and/or tubular injury. Imaging of LAP activity in DN disease model in vivo is thus beneficial for early diagnosis and prevention of DN, but such a strategy is still lacking. Herein, an enzyme-activated probe HD-LAP with a NIR fluorescence emission for specific detection of LAP activity in the DN model is designed and synthesized. HD-LAP has a significant fluorescence enhancement after reacted with LAP and shows a NIR fluorescence emission at 704 nm based on intramolecular charge transfer mechanism. Moreover, HD-LAP can be employed to image LAP activity in HK-2 and HepG2 cells. More importantly, HD-LAP is the first example to real-time image LAP in DN mice and clinical serum samples. These results demonstrated that HD-LAP is promising as a powerful tool for the research on LAP associated diabetic diseases in future.

Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging

Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers a major drawback, namely reduced fluorescence signals caused by excessive proteolysis and swelling effects. This caveat results in a lower photon budget and disfavors fluorescence imaging over a large field of view that can cover an entire expanded cell, especially in 3D. In addition, the complex procedures and specialized reagents of expansion microscopy hinder its popularization. Here, we modify expansion microscopy by deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal for point-scanning-based imaging. We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM may be applied for both antibody and lipid staining. TT-ExM displayed enhanced protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures. Importantly, TT-ExM-based lipid staining clearly revealed the complex 3D membrane structures in entire expanded cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase. We also observed lipid-rich chromosome matrices in the mitotic cells. These high-quality 3D images demonstrate the practicality of TT-ExM. Thus, readily available reagents can be deployed in TT-ExM to significantly enhance fluorescence signals and generate high-quality and ultrafine-resolution images under confocal microscopy.

Eu-doped carbon quantum dot as a selective probe for visualizing and monitoring sulfite in biological systems.

The detection of SO3 2- in complex environments and its visualization at the cellular level are critical for understanding its role in biological processes. In this study, we developed an Eu-doped long-wavelength fluorescent carbon quantum dot (CD2) and investigated the detection mechanism, interference effects and cellular imaging applications of the fluorescent probe CD2. The results show that the addition of SO3 2- induces an electronic rearrangement that restores CD2 to its original structure, leading to a rapid increase in fluorescence intensity. Selectivity experiments showed that CD2 has excellent selectivity to SO3 2-, with minimal interference from common anions. In addition, CD2 shows good biocompatibility for cellular imaging applications, as evidenced by the high cell viability observed in HeLa cells. Using confocal microscopy, we detected a significant enhancement of red fluorescence in HeLa cells after addition of exogenous SO3 2-, demonstrating the potential of CD2 as a probe for monitoring cellular SO3 2- levels. These findings highlight the promise of CD2 as a selective SO3 2- detection probe in complex environments and its utility in cellular imaging studies. Further studies are necessary to fully exploit the potential of CD2 in various biological and biomedical applications.

An innovative probe based on aluminum ion-triggered aggregation induced emission effect for ratiometric fluorescence visual detection of fluoroquinolone

Based on the aggregation-induced emission effect triggered by Al3+, a novel probe was constructed for the ratiometric fluorescence visualization detection of fluoroquinolones (FQs) residues. In this probe, Al3+ cased the glutathione-modified copper nanoclusters (GSH-CuNCs) to aggregate by the interaction with surface carboxyl groups, restricting the intramolecular vibration and rotation of GSH-CuNCs and reducing the non-radiative relaxation of excited states. Significant enhancement of fluorescence and quantum yield were achieved. In the presence of FQs, Al3+/GSH-CuNCs coordinated with FQs to form a polymer that inhibited intramolecular proton transfer in the excited state of FQs, thereby enhancing the intrinsic fluorescence of FQs, while the fluorescence intensity of Al3+/GSH-CuNCs was not disturbed. The probe solution changed from red to blue (or green) under UV lamp as the concentration of FQs increased, providing the possibility for semi-quantitative visual analysis. It also showed sensitive spectral responses to FQs with detection limits of 7.9 (ciprofloxacin), 6.8 (gatifloxacin), and 9.5 nM (enrofloxacin), respectively. The application potential in the detection of FQs residues in poultry eggs was verified by real sample test. Additionally, the probe has been extended to the development of fluorescent test paper and the smartphone detection platform, contributing to rapid, portable, visual quantitative analysis of FQs.

Lighting Up Fluorescence: Precise Recognition of Halogenated Solvents Through Effective Fluorescence Detection Using Chalcone Derivatives as a D-A-D-A-type Fluorescent Chemosensor.

In this paper, we report a D-A-D-A-type fluorescence sensor, FX, composed of triphenylamine and pyrazine units as electron donors, pyridine units, and α-β unsaturated carbon-based structures as electron acceptors. FX exhibits typical ICT characteristics. As a dual-emission material, FX undergoes acid-base-induced color changes and displays mechanofluorochromic properties in the solid state. In solution, FX, as an AIE material, shows significant fluorescence enhancement behavior in most halogenated solvents. Notably, it achieves a high quantum yield of 0.672 in a chloroform solution. We utilized this phenomenon to quantitatively detect chloroform through fluorescence titration analysis, with a detection limit of 0.061%. Additionally, we developed a test paper to verify the practical applicability of the sensor for detecting halogenated solvents. The fluorescence enhancement behavior was confirmed through DFT calculations. The results indicate that FX is not only a multifunctional dual-state emission material but also provides valuable references for the fluorescence detection of halogenated solvents.