Near-infrared Ratiometric Fluorescent Probe Research Articles

A mitochondrial targeted dual ratiometric near-infrared fluorescent probe based on ICT effect for the detection of SO2 derivative and its bioimaging

SO2 derivatives play an important role in many metabolic processes, excessive ingestion of them can lead to serious complications of various diseases. In this work, a novel dual ratiometric NIR fluorescent probe XT-CHO based on ICT effect was synthesized for detecting SO2 derivative. In the design of the probe, the α, β-unsaturated bond formed between benzopyran and coumarin was used as the reaction site for SO2, meanwhile, the extended π-conjugate system promoted maximum emission wavelength of the probe up to 708 nm. Notably, the probe exhibited high selectivity and sensitivity for detecting SO2, the limit of detection reached 2.13 nM and 58.5 nM in fluorescence spectra and UV–Vis absorption spectra, respectively. The reaction mechanism of SO2 and XT-CHO had been verified by 1H NMR, ESI-MS spectra and DFT calculation. Moreover, the probe was successfully applied in detecting endogenous and exogenous SO2 in living cells and proved possessed the mitochondrial targeted ability.

Ratiometric Near-Infrared Fluorescent Probe Monitors Ferroptosis in HCC Cells by Imaging HClO in Mitochondria.

Hypochlorous acid (HClO) is a typical endogenous ROS produced mainly in mitochondria, and it has strong oxidative properties. Abnormal HClO levels lead to mitochondrial dysfunction, strongly associated with various diseases. It has been shown that HClO shows traces of overexpression in cells of both ferroptosis and hepatocellular carcinoma (HCC). Therefore, visualization of HClO levels during ferroptosis of HCC is important to explore its physiological and pathological roles. So far, there has been no report on the visualization of HClO in ferroptosis of HCC. Thus, we present a ratiometric near-infrared (NIR) fluorescent probe Mito-Rh-S which visualized for the first time the fluctuation of HClO in mitochondria during ferroptosis of HCC. Mito-Rh-S has an ultrafast response rate (2 s) and large emission shift (115 nm). Mito-Rh-S was constructed based on the PET sensing mechanism and thus has a high signal-to-noise ratio. The cell experiments of Mito-Rh-S demonstrated that Fe2+- and erastin-induced ferroptosis in HepG2 cells resulted in elevated levels of mitochondrial HClO and that high concentration levels of Fe2+ and erastin cause severe mitochondrial damage and oxidative stress and had the potential to kill HepG2 cells. By regulating the erastin concentration, erastin induction time, and treatment of the ferroptosis model, Mito-Rh-S can accurately detect the fluctuation of mitochondrial HClO levels during ferroptosis in HCC.

Mitochondrial-Targeted Ratiometric Near-Infrared Fluorescence Probe for Monitoring Nitric Oxide in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a destructive autoimmune disease, where nitric oxide (NO) is closely implicated in the inflammatory processes of RA. Therefore, direct visualization of NO is essential to assess the pathological changes in RA. Herein, a mitochondrial-targeted near-infrared ratiometric fluorescent probe (NFL-NH2), based on the intramolecular charge transfer effect, was synthesized and applied to monitor the changes of NO content in early RA. Specially, probe NFL-NH2 showed a 44-fold fluorescent intensity ratio (I705/I780) response toward NO with a detection limit of 0.536 nM, enabling qualitative and quantitative analysis of NO. Additionally, NFL-NH2 can accurately target mitochondria and sensitively detect exogenous and endogenous NO in RAW 264.7 cells. Notably, in vivo RA monitoring assays demonstrated that NFL-NH2 can rapidly detect NO levels associated with the inflammatory damage degree in RA mice models by ratiometric fluorescence imaging. These results validate that NFL-NH2 holds significant potential for diagnosing NO-mediated RA diseases.

Neo-construction of a SO2-tunable near-infrared ratiometric fluorescent probe for high-fidelity diagnosis and evaluation hazards of Cd2+-induced liver injury

Cadmium-contaminated water and food are seriously hazardous to the human health, especially liver injury. To understand the entanglement relationship between cadmium ion (Cd2+)-induced liver injury and the biomarker sulfur dioxide (SO2), a reliable bioanalytical tool is urgently needed, detecting SO2 to diagnose and evaluate the extent of liver injury in vivo. Herein, based on the Förster resonance energy transfer (FRET) mechanism, a novel SO2-tunable NIR ratiometric fluorescent probe (SMP) was developed, it was used to diagnose and treat liver injury induced by Cd2+ in biosystems. Specifically, it was constructed by conjugating a NIR dicyanoisophorone with a NIR benzopyranate as the donor and acceptor, respectively, and the ratiometric response of SO2- regulated by the Michael addition reaction. In addition, SMP exhibits rapid reaction time (<15 s), two well-resolved emission peaks (68 nm) with less cross-talk between channels for high imaging resolution, superior selectivity, and low limit of detection (LOD=80.3 nM) for SO2 detection. Impressively, SMP has been successfully used for intracellular ratiometric imaging of Cd2+-induced SO2 and diagnostic and therapeutic evaluation in liver injury mice models with satisfactory results. Therefore, SMP may provide a powerful molecular tool for revealing the occurrence and development relationship between SO2 and Cd2+-induced liver injury. Environmental ImplicationCadmium ions are one of the well-known toxic environmental pollutants, which are enriched in the human body through inhalation of cadmium-contaminated air or from the food chain, leading to damage in various organs, especially liver injury. Therefore, we developed a novel fluorescent probe that can specifically detect SO2 in Cd2+-induced liver injury, which is critically important for the diagnosis and evaluation of Cd2+-induced liver injury diseases. The specific detection of SO2 of this probe has been successfully demonstrated in live HepG2 cells and Cd2+-induced liver injury mice.

A ratiometric near-infrared fluorescent probe with a large Stokes shift for rapid and sensitive detection of hydrogen sulfide in food samples and imaging in biological system

Hydrogen sulfide (H2S) has multiple contributions to living organisms and is also linked to the freshness of most high-protein foodstuffs. Therefore, developing a highly sensitive technology for on-site detection of H2S is promising and crucial. In this paper, a novel ratiometric near-infrared fluorescent probe 1,1,3-trimethyl-2-(4-(5,5,7-trimethyl-4,5,6,7-tetrahydro-3H-4,6-methanobenzo[d]imidazol-2-yl)styryl)− 1H-benzo[e]indol-3-ium iodide (TIBI) was designed and synthesized from α-pinene derivative 4,6,6-trimethylbicyclo[3.1.1]heptane-2,3-dione for the specific detection of H2S. TIBI displayed several excellent merits during the detection of H2S including low detection limit (19.43 nM), short response time (50 s), and wide pH detection range (59). The emission wavelength of TIBI blue-shifted from 690 nm to 460 nm accompanying with the fluorescence color changed from red to blue at the presence of H2S, and its fluorescence intensity ratio (I460 nm/I690 nm) enhanced up by 540 folds. The TIBI-loaded test strips could successfully achieved the real-time and convenient monitoring of H2S produced from food spoilage. Besides, probe TIBI could be also applied for imaging H2S level in living cells and zebrafish.

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.

Ratiometric near-infrared fluorescent probe for nitroreductase activity enables 3D imaging of hypoxic cells within intact tumor spheroids.

Fluorescent molecular probes that report nitroreductase activity have promise as imaging tools to elucidate the biology of hypoxic cells and report the past hypoxic history of biomedical tissue. This study describes the synthesis and validation of a "first-in-class" ratiometric, hydrophilic near-infrared fluorescent molecular probe for imaging hypoxia-induced nitroreductase activity in 2D cell culture monolayers and 3D multicellular tumor spheroids. The probe's molecular structure is charge-balanced and the change in ratiometric signal is based on Förster Resonance Energy Transfer (FRET) from a deep-red, pentamethine cyanine donor dye (Cy5, emits ∼660 nm) to a linked near-infrared, heptamethine cyanine acceptor dye (Cy7, emits ∼780 nm). Enzymatic reduction of a 4-nitrobenzyl group on the Cy7 component induces a large increase in Cy7/Cy5 fluorescence ratio. The deep penetration of near-infrared light enables 3D optical sectioning of intact tumor spheroids, and visualization of individual hypoxic cells (i.e., cells with raised Cy7/Cy5 ratio) as a new way to study tumor spheroids. Beyond preclinical imaging, the near-infrared fluorescent molecular probe has high potential for ratiometric imaging of hypoxic tissue in living subjects.

Real-time visualization the pH fluctuations of living cells with a ratiometric near-infrared fluorescent probe

In situ real-time quantitative monitoring pH fluctuation in complex living systems is vitally significant. In the current work, a ratiometric near-infrared (NIR) probe (MCyOH) was developed to confront this challenge. MCyOH exhibited good sensitivity, photostability, reversibility, and an ideal pKa (pKa = 6.65). Ratiometric character of MCyOH is beneficial to accuracy detect the pH fluctuations in living cells under different stimulation. The observations showed that intracellular pH was decreased when HepG2 cells under oxidative stress or starvation conditions. In particular, HepG2 cells was acidulated after addition of ethanol, however, the acidification phenomenon was attenuated or disappeared when HepG2 cells preincubated with disulfiram or fomepizole. Finally, MCyOH was successfully applied to observe the increasement of intracellular pH when HepG2 cells treated with fomepizole individually. Overall, MCyOH would be a practical candidate to explore pH-associated physiological and pathological varieties.

Near-infrared colorimetric and ratiometric fluorescent probe for dual-channel imaging of mitochondria and cysteine in the oxidative stress model

Mitochondria, known as the “cellular powerhouse”, play a variety of fundamental roles in various physiological and pathological processes, whose oxidative damage is usually emerging as a crucial induction factor that not only induces apoptosis but may also contribute to epilepsy and some other related diseases. Therefore, it is of notable significance to construct a high-discrimination method for informing the diagnosis and treatment of related diseases. Employing Cys-triggered conjugate addition cyclization-elimination strategy, we rationally constructed a mitochondria-targeted colorimetric and ratiometric near-infrared (NIR) fluorescent probe, named Mito-Pa, for Cys-triggered dual-channel fluorescence imaging of mitochondrial oxidative damage during apoptosis. Taking advantage of good membrane permeability and low cytotoxicity, probe Mito-Pa was successfully applied to visualize the mitochondria during apoptotic and the fluctuation of endogenous and exogenous Cys, or generated upon lipopolysaccharide-induced oxidative stress, in various living systems, including cancer cells, zebrafish, and mice. Thus, our proposed probe Mito-Pa would provide fresh insight for a better understanding of the ultimate relationship of Cys with mitochondrial oxidative damage, enabling easy access to accurately monitor the occurrence and progress of related diseases.

A novel ratiometric NIR fluorescent probe based on tanshinone IIA with double excitation for the detection of hydrazine

Hydrazine (N2H4) is a highly reactive chemical substance which is widely used in various industrial fields. Overexposure to hydrazine can bring potential risk to environmental safety and human health due to its toxicity and carcinogenicity. In this study, a near-infrared ratiometric fluorescent probe (TPE) with dual-excitation and dual-emission mode was designed and synthesized for the sensing of hydrazine. TPE displayed a highly sensitive and selective response to hydrazine with low detection limit (58 nM), short response time (3 min) and wide pH range (5–10). The sensing mechanism was investigated by 1H NMR titration, HRMS and DFT calculations. In view of the excellent properties, TPE was successfully applied for the detection of hydrazine in multiple systems including environmental water, food and gaseous samples. In addition, TPE was also successfully utilized for the real-time sensing of hydrazine in living cells and zebrafishes.

A new near-infrared ratiometric fluorescent probe using conjugated dicyanoisophorone and quinoline with double vinylene linkers for sensing of pH and its application

pH plays a crucial role in multiple biological processes, therefore, the development of pH imaging and measurement using the fluorescent probes in biological systems is highly active and has an urgent demand. In this work, a new ratiometric near-infrared (NIR) fluorescent probe DCIQ based on conjugated dicyanoisophorone and quinoline with double vinylene linkers was obtained for pH sensing. The remote conjugated quinoline and middle phenol used as an acceptor for protons may induce dual-modal colorimetric and fluorescence changes as a function of tiny variations in pH in the range of 6–8. Compared to previously reported probes, the proposed DCIQ probe was advantageous in terms of near-infrared emission signal (734 nm), large Stockes shift (173 nm), high selectivity, and good sensitivity. In addition, DCIQ could be as potential probe for viscosity by the fluorescence change. Moreover, the DCIQ-immobilized test papers exhibited reversible colorimetric changes as a function of variations in the environmental acid/base (>4 cycles) through naked-eye colorimetric detection. The desirable pKa (7.24) indicated that DCIQ probe could be used as a molecular tool for readily entering living cells with low cytotoxicity. Importantly, the DCIQ probe was successfully demonstrated for real-time detection of pH fluctuation in HeLa cells through ratiometric fluorescence images.

A lysosome-targeting ratiometric fluorescent probe used to detect Nitroxyl (HNO) in a Parkinson's disease model

Parkinson's disease (PD) is a neurodegenerative disease common in middle-aged and older individuals. At present, the exact cause is unknown, and the diagnosis is cumbersome, which seriously hinders the early diagnosis of patients. During the same time, during PD, there are conditions for the formation of nitroxyl (HNO). In addition, it has been shown that HNO has toxic effects on dopaminergic neurons and can lead to depletion of Glutathione (GSH), which is associated with features of PD. Therefore, HNO could be used as a potential biomarker for PD. Unfortunately, studies on changes in HNO content during PD are rarely reported. Here, we synthesized the first near-infrared ratiometric fluorescent probe HX-HNO for detecting HNO content in a PD model. The probe has excellent selectivity and sensitivity, and can detect HNO produced in various biochemical processes. Most importantly, in a PD model generated by rotenone, HX-HNO detected elevated levels of HNO. The visualization of HNO by HX-HNO provides a powerful tool for the early diagnosis of PD.

A NIR ratiometric fluorescence probe for rapid, sensitive detection and bioimaging of hypochlorous acid

Hypochlorous acid (HClO) is a condition where there is not enough oxygen in body tissues due to an imbalance between oxygen supply and consumption for cellular functions. In order to comprehend the biological functions of HClO within cells, the development of an effective and selective detection method is of great crucial. In this paper, a near-infrared ratiometric fluorescent probe (YQ-1) for detecting HClO was exploited based on a benzothiazole derivative. YQ-1′s fluorescence shifted from red to green with a large blue shift (165 nm) in the presence of HClO, and the solution's color changed from pink to yellow. YQ-1 quickly detected HClO (within 40 s) with a low detection limit (4.47 × 10−7 mol/L) and was not affected by other interferences. The mechanism of YQ-1′s response to HClO was confirmed by HRMS, 1H NMR and density functional theory (DFT) calculations. Moreover, due to its low toxicity, YQ-1 successfully utilized for fluorescence imaging for HClO both endogenous and exogenous in cells.

A highly selective NIR ratiometric fluorescent probe for in situ detection of metabolized hydrazine in living cells

Hydrazine (N2H4) is a great concern to human health and its detection in living cells is of growing importance. In this report, we employed a hydrogen bonding-anchored sensing strategy to develop a near-infrared (NIR) fluorescent probe CDM-1, which is capable of detecting N2H4 with ratiometric and colorimetric responses. The introduction of a chloro-substitution to the dicyanoisophorone scaffold improved the stability of the probe through Cl…CO n→π * interactions. Meanwhile, this substitution also provided a lower pKa value of the phenol CDM, allowing the generation of ratiometric changes after CDM-1 reacted with N2H4 under physiological conditions (pH = 7.4). Notably, we took advantage of utilizing both the hydrogen-bonding interaction and the 6-exo addition to target two anchors on N2H4 through “N” and “H” atoms, which enabled high selectivity and reactivity with a low limit of detection value at 0.3 ppb. CDM-1 has been successfully loaded onto paper strips to detect N2H4 vapor. Furthermore, the probe demonstrated its potential biological value in monitoring both the exogenous and endogenous N2H4, particularly as a metabolite of the anti-tuberculosis drug, Isoniazid (INH), in living cells.

A phenyl-substituted hemicyanine with maintained pKa as ratiometric near-infrared fluorescent probe for in vitro and in vivo pH determinations

In this study a new ratiometric pH probe, PHE-C, was designed and synthesized with the aim of enlarging the wavelength difference (Δλfl) between the two peaks of the typical hemicyanine fluorophore without significantly affecting its pKa value. The two emission maxima at 682 and 730 nm, associated with the respective protonation states of the hydroxyl group in PHE-C, have a larger Δλfl (48 nm) than that of unsubstituted hemicyanine. Importantly, the pKa of PHE-C (6.55) is the same as that of the unsubstituted counterpart. Consequently, the ratio of the intensities of the 682 and 730 nm emission bands is linearly related to pH in the range of 5.5–7.5, which is appropriate for monitoring small changes in pH of weakly acidic physiological environments. Moreover, PHE-C possesses good photostability, sensitivity and reversibility, and it displays the same response as pH meter when determining the pH in bovine serum solutions, indicating that it can be utilized in complex biological environments. Finally, the results of studies aimed at assessing the applicability to monitoring pH changes in living cells and mice demonstrate that the new probe is a highly sensitive ratiometric probe for in vivo pH determinations.

A near-infrared ratiometric fluorescent probe for hydrazine and its application for gaseous sensing and cell imaging

Hydrazine (N2H4) is a widely used raw material in the chemical industry, but at the same time hydrazine has extremely high toxicity. Therefore, the development of efficient detection methods is crucial for monitoring hydrazine in the environment and evaluating the biological toxicity of hydrazine. This study reports a near-infrared ratiometric fluorescent probe (DCPBCl2-Hz) for the detection of hydrazine by coupling a chlorine-substituted D-π-A fluorophore (DCPBCl2) to the recognition group acetyl. Due to the halogen effect of chlorine substitution, the fluorophore has an elevated fluorescence efficiency and a lowered pKa value and is suitable for physiological pH conditions. Hydrazine can specifically react with the acetyl group of the fluorescent probe to release the fluorophore DCPBCl2, so the fluorescence emission of the probe system significantly shifted from 490 nm to 660 nm. The fluorescent probe has many advantages, such as good selectivity, high sensitivity, large Stokes shift, and wide applicable pH range. The probe-loaded silica plates can realize convenient sensing gaseous hydrazine with content down to 1 ppm (mg/m3). Subsequently, DCPBCl2-Hz was successfully applied to detect hydrazine in soils. In addition, the probe can also penetrate living cells and allow the visualization of intracellular hydrazine. It can be anticipated that probe DCPBCl2-Hz will be a useful tool for sensing hydrazine in biological and environmental applications.

Lysosome-targeted high resolution near-infrared fluorescent probe for low physiological pH imaging in vivo

Low physiological pH have an essential role in keeping the digestive function of stomach, avoiding infection and affecting the absorption effect of oral drugs, monitoring the minor fluctuations of low physiological pH is of great concern. An imidazopyridine-based ratiometric near-infrared (NIR) fluorescent probe SPO is rationally designed and synthesized for low physiological pH imaging. SPO exhibits high-resolution characteristics, including large Stokes shift (237 nm), remarkable emission shift (110 nm) and good fluorescence quantum yield (Φ = 0.68). It also possesses good reversibility, high selectivity and excellent photostability, and profits to monitoring of continuous pH changes. Moreover, ratiometric fluorescence imaging showed that the probe could conveniently and sensitively track the lysosomal pH fluctuation in living cells. The pKa value of SPO (pKa = 3.0) permits the accurate monitoring of the minor fluctuations of low physiological pH, and SPO was used for noninvasive monitoring gastric pH in living mice successfully.

A Novel Near-Infrared Ratiometric Fluorescence Probe for Hg2+ Based on Quinoline-Fused Rhodamine Dye.

As one of the most toxic metals, Mercury ions cause serious environmental pollution and threaten the health of living organisms. Hence, we designed and synthesised a new near-infrared (NIR) ratiometric fluorescent probe toward monitoring of Hg2+ based on quinoline-fused rhodamine dye. Owing to the specific spirolactam ring-opening reaction, the probe exhibits a ratiometric fluorescent change after treatment of Hg2+ with increased emission in NIR and significantly reduced emission in visible region. The specific response mechanism and dual-channel fluorescence change allow the probe to have remarkable detection selectivity, fast response and high detection sensitivity. Moreover, with the properties of excellent cell permeability and low cytotoxicity, probe can be applied as detection tool for mercury ions with dual-channel ratiometric fluorescence imaging in living cell.

A facile ratiometric near-infrared fluorescent probe using conjugated 1,8-naphthalimide and dicyanoisophorone with a vinylene linker for detection and bioimaging of hypochlorite.

A new ratiometric near-infrared fluorescent probe 3 for detecting ClO- using conjugated 1,8-naphthalimide and dicyanoisophorone with a vinylene linker was reported. Probe 3 exhibited a ratiometric signal (I705/I535), large Stokes shift (205 nm), high selectivity and sensitivity, low detection limit (0.738 μM), rapid response (within 3 s) and good biocompatibility. The sensing mechanism involved the oxidation of the olefin double bond by ClO- to release the initial N-butyl-4-hydroxyl-3-formyl-1,8-naphthalimide 1, followed by inhibition of an ICT process from the electron-donor 4-hydroxyl-1,8-naphthalimide to dicyanoisophorone. At the same time, the probe 3-loaded test strips were applied in sensing of ClO- with moderate "naked-eye" color changes. Additionally, probe 3 has been successfully used for ratiometric bioimaging of ClO- in HeLa cells with low cytotoxicity.

Bioimaging of glutathione variation for early diagnosis of hepatocellular carcinoma using a liver-targeting ratiometric near-infrared fluorescent probe.

Reliable biomarkers are crucial for early diagnosis of diseases and precise therapy. Biological thiols (represented by glutathione, GSH) play vital roles in the antioxidant defense system for maintaining intracellular redox homeostasis in organisms. However, the aberrant variation in the cellular concentration of GSH correlates with diverse diseases including cancer. Here, a ratiometric near-infrared fluorescent probe CyO-Disu is constructed for the specific sensing of GSH variation in live cells and mice models of hepatic carcinoma (HCC). CyO-Disu features three key elements, a response moiety of bis(2-hydroxyethyl) disulfide, a near-infrared fluorescence signal transducer of heptamethine ketone cyanine, and a targeting moiety of D-galactose. By virtue of its liver-targeting capability, CyO-Disu was utilized for evaluating GSH fluctuations in primary and metastatic hepatoma living cells. To evaluate the efficacy of CyO-Disuin vivo, orthotopic HCC and pulmonary metastatic hepatoma mice models were employed for GSH imaging using two-dimensional and three-dimensional fluorescence molecular tomographic imaging systems. The bioimaging results offered direct evidence that GSH displayed varied concentrations during the progression of HCC. Therefore, the as-synthesized probe CyO-Disu could serve as a potential powerful tool for the early diagnosis and precise treatment of HCC using GSH as a reliable biomarker.