Far-red Fluorescent Probe Research Articles

A colorimetric and far-red fluorescent probe for rapid detection of bisulfite/sulfite in full water-soluble based on biquinolinium and its applications

Bisulfite (HSO3−) and sulfite (SO32−) are involved in numerous physiological processes of living systems. However, high levels of these substances are often correlated to many diseases. Herein, we designed and synthesized a simple full water-soluble colorimetric and far-red fluorescent probe (E)-1-methyl-4-(2-(1-methylquinolin-1-ium-3-yl)vinyl)quinolin-1-ium iodide trifluoromethanesulfonate (DQ) for HSO3−/SO32− detection by coupling 1,4-dimethylquinolinium with 3-quinolinium carboxaldehyde for the first time. The probe DQ showed high selectivity for HSO3− detection via a 1,4-nucleophilic addition reaction with distinct color changes from colorless to purple-red and remarkable far-red fluorescence enhancement in pure aqueous solutions. Specifically, the probe displayed a fast response (<15 s) for bisulfite, which renders it suitable for real time detection of HSO3−. Under the optimized conditions, the far-red fluorescence intensity was linear to the concentrations of HSO3− in the range from 0 to 25 μM and the detection limit was as low as 0.11 μM. Additionally, the probe could be applied to sense HSO3− on paper strips, real sample including vermicelli and sugar and image HSO3− in living cells, which indicated that probe DQ has potential application in food samples and living systems.

A far-red emissive two-photon fluorescent probe for quantification of uracil in genomic DNA

We report a new method for dU detection in genomic DNA combined with UNG excision and fluorescent probe labeling. UNG can remove uracil bases to introduce abasic sites, which can react with NRNO to produce intense fluorescence because of the inhibition of the PET effect. It can also cause the polymerase extension to stop to provide details of dU site information.

A matrix targeted fluorescent probe to monitor mitochondrial dynamics.

Mitochondria are an indispensable organelle for energy production and regulation of cellular metabolism. The structural and functional alterations to mitochondria instigate pathological conditions of cancer, and aging-associated and neurodegenerative disorders. The normal functioning of mitochondria is maintained by quality control mechanisms involving dynamic fission, fusion, biogenesis and mitophagy. Under conditions of mitophagy and neurodegenerative diseases, mitochondria are exposed to different acidic environments and high levels of reactive oxygen species (ROS). Therefore stable molecular tools and methods are required to monitor the pathways linked to mitochondrial dysfunction and disease conditions. Herein, we report a far-red fluorescent probe (Mito-TG) with excellent biocompatibility, biostability, photostability, chemical stability and turn on emission for selective targeting of the mitochondrial matrix in different live cells. The probe was successfully employed for monitoring dynamic processes of mitophagy and amyloid beta (Aβ) induced mitochondrial structural changes.

Rational design of a far-red fluorescent probe for endogenous biothiol imbalance induced by hydrogen peroxide in living cells and mice

Intracellular biothiols are correlated with many diseases such as nerve disorder and Parkinson’s disease likely due to a redox imbalance. In this work, we designed an ultrafast fluorescent probe (Cou-DNBS) for biothiols with a large Stokes shift (131 nm). The probe was constructed through linking the 2,4-dinitrobenzenesulfonyl moiety as the specially recognizing biothiols site to an iminocoumarin fluorophore Cou-NH obtained by fusing an additional benzene ring. The presence of biothiols could ultrafast perform a significant fluorescence emission at 617 nm upon the excitation of 480 with the low limits of detection (2.5 nM for Cys, 1.7 nM for Hcy and 0.84 nM for GSH). HRMS spectra as well as theoretical calculations further evidenced the rationale of recognition mechanism. Furthermore, the probe can successfully visualize endogenous biothiol recovery in living cells damaged by H2O2.

Far-red pentamethine cyanine dyes as fluorescent probes for the detection of serum albumins.

Benzothiazole based cyanine dyes with bridged groups in the pentamethine chain were studied as potential far-red fluorescent probes for protein detection. Spectral-luminescent properties were characterized for unbound dyes and in the presence of serum albumins (bovine (BSA), human (HSA), equine (ESA)), and globular proteins (β-lactoglobulin, ovalbumin). We have observed that the addition of albumins leads to a significant increase in dyes fluorescence intensity. However, the fluorescent response of dyes in the presence of other globular proteins was notably lower. The value of fluorescence quantum yield for dye bearing a sulfonate group complexed with HSA amounted to 42% compared with 0.2% for the free dye. The detection limit of HSA by this dye was greater than 0.004 mg ml−1 which indicates the high sensitivity of dye to low HSA concentrations. Modelling of structure of the dyes complexes with albumin molecules was performed by molecular docking. According to these data, dyes could bind to up to five sites on the HSA molecule; the most preferable are the haemin-binding site in subdomain IB and the dye-binding site in the pocket between subdomains IA, IIA and IIIA. This work confirms that pentamethine cyanine dyes could be proposed as powerful far-red fluorescent probes applicable for highly sensitive detection of albumins.

Quantitative kinetics of intracellular singlet oxygen generation using a fluorescence probe

Singlet oxygen (1O2) is a type of reactive oxygen species involved in numerous physiological activities. We previously reported that 1O2-specific oxidation products are increased in patients with prediabetes, suggesting that measurement of 1O2 may be an important indicator of physiological and pathological conditions. The turnover in the generation and quenching of 1O2 is extremely rapid during biological activities owing to it high reactivity and short lifetime in solution. However, the dynamic changes in 1O2 generation in living cells have not been fully explored. In this study, we investigated whether the kinetics of 1O2 generation can be quantified using a far-red fluorescent probe for mitochondrial 1O2, Si-DMA, following addition of the 1O2 generator, endoperoxide, to mammalian cells. The kinetics of Si-DMA fluorescence intensity dose-dependently increased following treatment of mammalian living cells with endoperoxide. Alternatively, treatment with 1O2 quenchers decreased the fluorescence intensities following endoperoxide treatment. Our results indicate that the kinetics of intracellular 1O2 can be readily obtained using Si-DMA and time-lapse imaging, which provides new insights into the mechanism of 1O2 generation in mammalian cells and the exploration of 1O2 generators and quenchers.

A novel colorimetric and far-red emission ratiometric fluorescent probe for the highly selective and ultrafast detection of hypochlorite in water and its application in bioimaging.

Hypochlorous acid (HOCl)/hypochlorite (OCl-), an important reactive oxygen species, plays a number of important roles in various physiological processes. However, abnormal levels of OCl- can cause many serious diseases, such as atherosclerosis, rheumatoid arthritis, cardiovascular disease, and cancer. The development of efficient methods to monitor OCl- in biological systems is therefore of particular importance. Thus, we herein report a novel isophorone-based fluorescent probe, i.e., DCOH-FR-OCl, for OCl- detection. This probe was found to exhibit colorimetric and far-red ratiometric fluorescence response signals, excellent selectivity and sensitivity for OCl- (detection limit, 88.06 nM), a remarkably large Stokes shift (158 nm), an ultrafast response (completed within 3 s), perfect photostability, and good water solubility (100% in aqueous media). DCOH-FR-OCl, as we know, is the first probe that can detect OCl- by using two different response signals at an ultrafast speed with a large Stokes shift in fully aqueous media. Furthermore, DCOH-FR-OCl can be successfully employed in the real-time imaging of endogenous and exogenous OCl- in living cells.

Construction of a novel far-red fluorescence light-up probe for visualizing intracellular peroxynitrite

Peroxynitrite (ONOO-) is an important reactive oxygen species (ROS), which can react with a variety of biologically active species and cause many diseases, such as cancer, neurodegenerative disorders. Herein, we develop a novel far-red fluorescent probe DCM-KA, which is equipping with α-ketoamide moiety caged DCM-NH2. The probe exhibits fluorescence off-on response to ONOO- over other biological interfering analytes by ONOO--induced α-ketoamide deprotection reaction. More importantly, the probe is biocompatible and has been successfully utilized to visualize endogenous ONOO- production in macrophage cell line J774A.1.

A far-red FRET fluorescent probe for ratiometric detection of l-cysteine based on carbon dots and N-acetyl-l-cysteine-capped gold nanoparticles.

A novel far-red fluorescence resonance energy transfer (FRET) fluorescent probe for ratiometric detection of l-cysteine (l-Cys) has been designed. The system was established a FRET assembly by positively charged carbon dots (CDs) and negatively charged N-acetyl-l-cysteine capped gold nanoparticles (NAC-AuNPs). The fluorescence of CDs at 539 nm could be effectively quenched in the presence of NAC-AuNPs owing to FRET process, while the emission of NAC-AuNPs at 630 nm was appeared. Subsequently, the interactions between l-Cys and NAC-AuNPs resulted in the decreased emission intensity of NAC-AuNPs, but the emission intensity of CDs kept almost constant due to the continuous FRET efficiency. The ratio of emission intensities at 539 and 630 nm (I539/I630) exhibited a linear correlation to the l-Cys concentration in the range of 1.0-110 μM with the detection limit of 0.16 μM. Moreover, this far-red ratiometric sensor also revealed excellent selectivity toward l-Cys over other amino acids, which showed very high potential in the practical application for diagnosing of cysteine-related disease.

A far-red fluorescent probe for sensing laccase in fungi and its application in developing an effective biocatalyst for the biosynthesis of antituberculous dicoumarin.

A far-red fluorescent probe has been developed for sensing fungal laccase. The probe was used to determine that Rhizopus oryzae had a high level endogenous laccase amongst 24 fungal strains. The Rhizopus oryzae was then used as a biocatalyst for the preparation of dicoumarin resulting in significant inhibition of Mycobacterium tuberculosis H37Ra.

A mitochondria-targetable colorimetric and far-red fluorescent probe for the sensitive detection of carbon monoxide in living cells

A simple colorimetric and far-red fluorescent probe was developed for the first time to visualize the gasotransmitter carbon monoxide in subcellular mitochondria.

A carbonothioate-based far-red fluorescent probe for the specific detection of mercury ions in living cells and zebrafish.

The detection of ionic mercury (Hg2+) is very important because it is a highly toxic environmental pollutant that could cause serious diseases and threaten human health. Herein, we designed a new carbonothioate-based far-red fluorescent probe, CBRB, with a seminaphthorhodafluor dye as the fluorophore for the detection of Hg2+. The CBRB probe by itself exhibited very weak fluorescence due to the enhanced photo-induced electron transfer (PET) effect and inhibited the intramolecular charge transfer (ICT) process caused by the carbonothioate moiety. Upon addition of Hg2+, a tremendous fluorescence enhancement was achieved, attributed to the removal of the carbonothioate group via a specific mercury-promoted desulfurization reaction. Moreover, the probe displayed a large Stokes shift (about 105 nm) and was used to quantitatively measure the concentration of Hg2+ for concentrations ranging from 0 to 1 μM (DL = 3.6 nM). In addition, CBRB in our experiments responded exclusively to Hg2+, even in the presence of high concentrations other ions. Gratifyingly, this probe was successfully used to monitor Hg2+ in environmental water samples and to image Hg2+ in living cells as well as in zebrafish.

A far-red fluorescent probe for selective G-quadruplex DNA targeting

The development of rapid and simple approaches for detection of G-quadruplex DNA structures has attracted significant attention to disclose their diverse physiological and pathological functions. Thiazole orange (TO) is a common fluorescence probe used for the detection of G-quadruplexes. However, it still suffers from some common problems like non-selective for G-quadruplex and emission in the lower wavelength region of spectrum, thus hampering its further applications. Probes with turn-on fluorescence in the far-red region are highly sought-after due to minimal auto-fluorescence and cellular damage. In this paper, we described a far-red fluorescent probe (L-1) by introducing an amine group into styrylquinolinium scaffold. The experimental results indicated that L-1 exhibited significant fluorescence enhancement when treated with G-quadruplexes but retained weak fluorescence in the presence of duplex DNAs. In addition, this probe also displayed higher binding affinity for parallel G-quadruplexes. The characteristics of L-1 were further investigated with UV–vis spectrophotometry, fluorescence, circular dichroism, KI quenching, FID assay and molecular docking to validate optical photophysical properties, as well as the selectivity, sensitivity and detailed binding mode toward G-quadruplex DNAs.

Acid-Promoted D-A-D Type Far-Red Fluorescent Probe with High Photostability for Lysosomal Nitric Oxide Imaging.

To accurately monitor the variations of lysosomal nitric oxide (NO) under physiological condition remains a great challenge for understanding the biological function of NO. Herein, we developed a new chemotype probe, namely, MBTD, for acid-promoted and far-red fluorescence imaging of lysosomal NO in vitro and ex vivo. MBTD was rationally designed by incorporating o-phenylenediamino (OPD) moiety into the donor-acceptor-donor (D-A-D) type fluorophore based on a dual intramolecular charge transfer (ICT) mechanism. Compared to previously reported OPD-based NO probes, MBTD displays several distinct advantages including large stroke shift, huge on-off ratio with minimal autofluorescence, and high NO specificity. Particularly, MBTD exhibits an acid-promoted response to NO with high acid tolerance, which greatly improves the spatial resolution to lysosomal NO by excluding the background noise from other nonacidic organelles. Furthermore, MBTD displayed much longer-lived and more stable fluorescence emission in comparison with the commercialized NO probe. MBTD was employed for ratiometric examination of the exogenous or endogenous NO of macrophages. More importantly, MBTD was able to detect the variation of lysosomal NO level in an acute liver injury mouse model ex vivo, implying the potential of MBTD for real-time monitoring the therapeutic efficacy of drug candidates for the treatment of acute liver injury. MBTD as a novel type of NO probe might open a new avenue for precisely sensing lysosomal NO-related pathological and therapeutic process.

The live cell DNA stain SiR-Hoechst induces DNA damage responses and impairs cell cycle progression

SiR-Hoechst (SiR-DNA) is a far-red fluorescent DNA probe being used widely for time-lapse imaging of living cells that is reported to be minimally toxic at concentrations as high as 10–25 µM. However, measuring nuclear import of Cyclin B1, inhibition of mitotic entry, and the induction of γH2AX foci in cultured human cells reveals that SiR-Hoechst induces DNA damage responses and G2 arrest at concentrations well below 1 µM. SiR-Hoechst is useful for live cell imaging, but it should be used with caution and at the lowest practicable concentration.

Far-Red Fluorescent Probe for Imaging of Vicinal Dithiol-Containing Proteins in Living Cells Based on a pKa Shift Mechanism.

Vicinal dithiol-containing proteins (VDPs) play fundamental roles in intracellular redox homeostasis and are responsible for many diseases. In this work, we report a far-red fluorescence turn-on probe MCAs for VDPs exploiting the pKa shift of the imine functionality of the probe. MCAs is composed of a merocyanine Schiff base as the fluorescent reporter and a cyclic 1,3,2-dithiarsenolane as the specific ligand for VDPs. The imine pKa of MCAs is 4.8, and it exists predominantly in the Schiff base (SB) form at physiological pH. Due to the absence of a resonating positive charge, it absorbs at a relatively short wavelength and is essentially nonfluorescent. Upon selective binding to reduced bovine serum albumin (rBSA, selected as the model protein), MCAs was brought from aqueous media to the binding pockets of the protein, causing a large increase in pKa value of MCAs (pKa = 7.1). As a result, an increase in the protonated Schiff base (PSB) form of MCAs was observed at the physiological pH conditions, which in turn leads to a bathochromically shifted chromophore (λabs = 634 nm) and a significant increase in fluorescence intensity (λem = 657 nm) simultaneously. Furthermore, molecular dynamics simulations indicate that the salt bridges formed between the iminium in MCAs and the residues D72 and D517 in rBSA resist the dissociation of proton from the probe, thus inducing an increase of the pKa value. The proposed probe shows excellent sensitivity and specificity toward VDPs over other proteins and biologically relevant species and has been successfully applied for imaging of VDPs in living cells. We believe that the present pKa shift switching strategy may facilitate the development of new fluorescent probes that are useful for a wide range of applications.

A far-red fluorescent probe based on a phospha-fluorescein scaffold for cytosolic calcium imaging.

The far-red emissive fluorescent probe CaPF-1 based on a phospha-fluorescein scaffold enables the detection of cytosolic calcium ions in living cells. The probe can be excited in the red region (λabs = 636 nm) and exhibits a sufficiently high fluorescence turn-on response in the far-red region (λem = 663 nm) upon complexation with calcium ions. The hydrophilic and anionic characteristics of this phospha-fluorescein fluorophore allowed the cytosolic localization of CaPF-1. Moreover, it was possible to visualize histamine-induced calcium oscillation in HeLa cells using CaPF-1.

Far-red fluorescent probes for canonical and non-canonical nucleic acid structures: current progress and future implications.

The structural diversity and functional relevance of nucleic acids (NAs), mainly deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are indispensable for almost all living organisms, with minute aberrations in their structure and function becoming causative factors in numerous human diseases. The standard structures of NAs, termed canonical structures, are supported by Watson-Crick hydrogen bonding. Under special physiological conditions, NAs adopt distinct spatial organisations, giving rise to non-canonical conformations supported by hydrogen bonding other than the Watson-Crick type; such non-canonical structures have a definite function in controlling gene expression and are considered as novel diagnostic and therapeutic targets. Development of molecular probes for these canonical and non-canonical DNA/RNA structures has been an active field of research. Among the numerous probes studied, probes with turn-on fluorescence in the far-red (600-750 nm) region are highly sought-after due to minimal autofluorescence and cellular damage. Far-red fluorescent probes are vital for real-time imaging of NAs in live cells as they provide good resolution and minimal perturbation of the cell under investigation. In this review, we present recent advances in the area of far-red fluorescent probes of DNA/RNA and non-canonical G-quadruplex structures. For the sake of continuity and completeness, we provide a brief overview of visible fluorescent probes. Utmost importance is given to design criteria, characteristic properties and biological applications, including in cellulo imaging, apart from critical discussion on limitations of the far-red fluorescent probes. Finally, we offer current and future prospects in targeting canonical and non-canonical NAs specific to cellular organelles, through sequence- and conformation-specific far-red fluorescent probes. We also cover their implications in chemical and molecular biology, with particular focus on decoding various disease mechanisms involving NAs.

A far-red ratiometric fluorescent probe for SO2 derivatives based on the ESIPT enhanced FRET platform with improved performance

A ratiometric fluorescent probe (L-HF3) based on an efficient FRET platform constructed by 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) and 3-hydroxyflavone (HF) fluorophores for detection of sulfur dioxide derivatives (HSO3−/SO32−) was developed. The probe featured very large (pseudo) Stokes shifts, good selectivity and high sensitivity, high FRET efficiency and high resolution of the emission bands. The probe was also mitochondria-targeted and could detect exogenous and endogenous bisulfite in living cells.

A highly selective and ultrasensitive ratiometric far-red fluorescent probe for imaging endogenous peroxynitrite in living cells

Peroxynitrite (ONOO−) is involved in various physiological and pathological processes. However the detailed mechanisms of ONOO− in the biological systems remain unclear, probably due to the lack of specific and sensitive methods for detecting ONOO− in living cells. Herein, based on the intramolecular charge transfer (ICT) mechanism, we have developed a highly specific and ultrasensitive ratiometric far-red fluorescent probe RFR-PN for monitoring ONOO− in living cells.Experimental results demonstrated probe RFR-PN possesses high specificity toward ONOO− than other various bioactive molecules. Probe RFR-PN could quantitatively and sensitively detect ONOO− with the detection limit of 0.9 nM by the ratiometric fluorescence method. Surprisingly, the fast response of probe RFR-PN for ONOO− (<5 s) was achieved, and it is essential for the real-time detection of ONOO− in the living systems. Moreover, probe RFR-PN was successfully applied to the fluorescence imaging of endogenous ONOO− in living cells.