Detection of LacZ‐Positive Cells in Living Tissue with Single‐Cell Resolution

The LacZ gene, which encodes Escherichia coli β‐galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ‐positive cells in living organisms or tissues at single‐cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β‐galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single‐cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.     

Colorimetric and ratiometric fluorescent detection of bisulfite by a new HBT-hemicyanine hybrid

A novel HBT-hemicyanine hybrid was prepared. This hybrid not only displays a large red-shifted (Δλ = 125 nm) emission compared to the well known ESIPT dye HBT, but also can be used as a new probe for rapid, colorimetric and ratiometric fluorescent detection of bisulfite with high selectivity and sensitivity in aqueous solution. The detection limit of this probe for HSO₃⁻ was calculated to be about 56 nM with a linear range of 0–25 μM. The potential application of this probe was exampled by detection of bisulfite in real food samples and living cells. Overall, this work not only provided a new ratiometric sensing platform, but also provided a new promising colorimetric and ratiometric fluorescent probe for bisulfite.     

An ESIPT-Based Ratiometric Fluorescent Probe for Highly Sensitive and Rapid Detection of Sulfite in Living Cells

The novel ratiometric fluorescent probe HPQRB with an ESIPT effect based on Michael addition for highly sensitive and fast detection of sulfite in living HepG2 cells is reported. HPQRB can be easily synthesized by a two-step condensation reaction. HPQRB has a large emission shift (Δλ=116 nm), which is beneficial for fluorescence imaging research, and its sulfite-responsive site is based on a rhodamine-like structure with the emission peak at 566 nm, which decreases with increasing sulfite concentration. and its HPQ structure always has an ESIPT effect throughout the reaction process, keeping the emission peak at 450 nm as a self-reference. In particular, HPQRB has high selectivity for sulfite and responds quickly (within 30 s) with a low detection limit (44 nM). Furthermore, HPQRB has been successfully used for fluorescence imaging of sulfite in HepG2 cells, demonstrating the superior ability to detect sulfite under physiological conditions.     

High-Affinity Ratiometric Fluorescence Probe Based on 6-Amino-2,2′-Bipyridine Scaffold for Endogenous Zn2+ and Its Application to Living Cells

Zinc is an essential trace element involved in many biological activities; however, its functions are not fully understood. To elucidate the role of endogenous labile Zn²⁺, we developed a novel ratiometric fluorescence probe, 5-(4-methoxyphenyl)-4-(methylsulfanyl)-[2,2′-bipyridin]-6-amine (6 (rBpyZ)) based on the 6-amino-2,2′-bipyridine scaffold, which acts as both the chelating agent for Zn²⁺ and the fluorescent moiety. The methoxy group acted as an electron donor, enabling the intramolecular charge transfer state of 6 (rBpyZ), and a ratiometric fluorescence response consisting of a decrease at the emission wavelength of 438 nm and a corresponding increase at the emission wavelength of 465 nm was observed. The ratiometric probe 6 (rBpyZ) exhibited a nanomolar-level dissociation constant (K_d = 0.77 nM), a large Stokes shift (139 nm), and an excellent detection limit (0.10 nM) under physiological conditions. Moreover, fluorescence imaging using A549 human lung adenocarcinoma cells revealed that 6 (rBpyZ) had good cell membrane permeability and could clearly visualize endogenous labile Zn²⁺. These results suggest that the ratiometric fluorescence probe 6 (rBpyZ) has considerable potential as a valuable tool for understanding the role of Zn²⁺ in living systems.     

A ratiometric fluorescent probe for thiols based on a tetrakis(4-hydroxyphenyl)porphyrin-coumarin scaffold

In this work, we have designed and synthesized the compound Ratio-HPSSC, based on a tetrakis(4-hydroxyphenyl)porphyrin-coumarin scaffold, as a new ratiometric fluorescent probe for thiols. The ratiometric probe Ratio-HPSSC is highly selective and sensitive to thiols. Importantly, the novel ratiometric probe exhibited a remarkable change in emission color from red to blue. This key feature allows Ratio-HPSSC to be employed for thiol detection by simple visual inspection. Furthermore, we have demonstrated that Ratio-HPSSC is suitable for ratiometric fluorescence imaging of thiols in living cells. We believe that the new ratiometric probe will find interesting applications in chemistry, biology, and medicine.     

Development of a BODIPY-based ratiometric fluorescent probe for hypochlorous acid and its application in living cells

A BODIPY-based ratiometric fluorescent probe for HOCl has been designed based on the transduction of thioether to sulfoxide function. This probe features a marked absorption and emission blue-shift upon the HOCl-promoted rapid transduction, enabling the highly selective and ratiometric detection. In addition, the probe works excellently within a wide pH range of 4–10, addressing the existing pH dependency issue. Living cells studies demonstrate that the probe is cell membrane permeable and can be employed successfully to image endogenous HOCl generation in macrophage cells.     

Development of Luminescent Coelenterazine Derivatives Activatable by β‐Galactosidase for Monitoring Dual Gene Expression

Two bioluminogenic caged coelenterazine derivatives (bGalCoel and bGalNoCoel) were designed and synthesized to detect β‐galactosidase activity and expression by means of bioluminescence imaging. Our approach addresses the instability of coelenterazine by introducing β‐galactose caging groups to block the auto‐oxidation of coelenterazine. Both probes contain β‐galactosidase cleavable caging groups at the carbonyl group of the imidazo–pyrazinone moiety. One of the probes in particular, bGalNoCoel, displayed a fast cleavage profile, high stability, and high specificity for β‐galactosidase over other glycoside hydrolases. bGalN‐oCoel could detect β‐galactosidase activity in living HEK‐293T cell cultures that expressed a mutant Gaussia luciferase. It was determined that coelenterazine readily diffuses in and out of cells after uncaging by β‐galactosidase. We showed that this new caged coelenterazine derivative, bGalNoCoel, could function as a dual‐enzyme substrate and detect enzyme activity across two separate cell populations.     

Real‐Time In Vivo Detection of Cellular Senescence through the Controlled Release of the NIR Fluorescent Dye Nile Blue

In vivo detection of cellular senescence is accomplished by using mesoporous silica nanoparticles loaded with the NIR‐FDA approved Nile blue (NB) dye and capped with a galactohexasaccharide ( S3 ). NB emission at 672 nm is highly quenched inside S3 , yet a remarkable emission enhancement is observed upon cap hydrolysis in the presence of β‐galactosidase and dye release. The efficacy of the probe to detect cellular senescence is tested in vitro in melanoma SK‐Mel‐103 and breast cancer 4T1 cells and in vivo in palbociclib‐treated BALB/cByJ mice bearing breast cancer tumor.     

A ratiometric fluorescent probe for the detection of hypochlorous acid in living cells and zebra fish with along wavelength emission

A ratiometric fluorescence probe, NClO, for the rapid and selective detection of HClO had been designed and synthesized based on a 1,8-naphthalimide derivative. Probe NClO displayed a red emission(λ_(max)= 615 nm). In the presence of HClO, the solution of probe NClO gave off a strong green fluorescence(λ_(em), _(max)= 520 nm) with a rapid response(within seconds). This probe had been applied to image HClO in living cells and zebra fish.     

Ratiometric Fluorescence Imaging of Cellular Polarity: Decrease in Mitochondrial Polarity in Cancer Cells

Mitochondrial polarity strongly influences the intracellular transportation of proteins and interactions between biomacromolecules. The first fluorescent probe capable of the ratiometric imaging of mitochondrial polarity is reported. The probe, termed BOB, has two absorption maxima (λ_abs=426 and 561 nm) and two emission maxima—a strong green emission (λ_em=467 nm) and a weak red emission (642 nm in methanol)—when excited at 405 nm. However, only the green emission is markedly sensitive to polarity changes, thus providing a ratiometric fluorescence response with a good linear relationship in both extensive and narrow ranges of solution polarity. BOB possesses high specificity to mitochondria (R_r=0.96) that is independent of the mitochondrial membrane potential. The mitochondrial polarity in cancer cells was found to be lower than that of normal cells by ratiometric fluorescence imaging with BOB. The difference in mitochondrial polarity might be used to distinguish cancer cells from normal cells.     

Fluorescent Detection of Hypochlorous Acid from Turn‐On to FRET‐Based Ratiometry by a HOCl‐Mediated Cyclization Reaction

Hypochlorous acid (HOCl), a reactive oxygen species (ROS), plays a significant biological role in living systems. However, abnormal levels of HOCl are implicated in many inflammation‐associated diseases. Therefore, the detection of HOCl is of great importance. In this work, we describe the HOCl‐promoted cyclization of rhodamine‐thiosemicarbazides to rhodamine‐oxadiazoles, which is then exploited as a novel design strategy for the development of a new fluorescence turn‐on HOCl probe 2 . On the basis of the fluorescence resonance energy transfer (FRET) signaling mechanism, 2 was further converted into 1 a and 1 b , which represent the first paradigm of FRET‐based ratiometric fluorescent HOCl probes. The outstanding features of 1 a and 1 b include well‐resolved emission peaks, high sensitivity, high selectivity, good functionality at physiological pH, rapid response, low cytotoxicity, and good cell‐membrane permeability. Furthermore, these excellent attributes enable us to demonstrate, for the first time, the ratiometric imaging of endogenously produced HOCl in living cells by using these novel ratiometric probes. We expect that 1 a and 1 b will be useful molecular tools for studies of HOCl biology. In addition, the HOCl‐promoted cyclization reaction of rhodamine‐thiosemicarbazides to rhodamine‐oxadiazoles should be widely applicable for the development of different types of fluorescent HOCl probes.     

A Native‐Chemical‐Ligation‐Mechanism‐Based Ratiometric Fluorescent Probe for Aminothiols

Thiol‐containing amino acids (aminothiols) such as cysteine (Cys) and homocysteine (Hcy) play a key role in various biological processes including maintaining the homeostasis of biological thiols. However, abnormal levels of aminothiols are associated with a variety of diseases. The native chemical ligation (NCL) reaction has attracted great attention in the fields of chemistry and biology. NCL of peptide segments involves cascade reactions between a peptide‐α‐thioester and an N‐terminal cysteine peptide. In this work, we employed the NCL reaction mechanism to formulate a Förster resonance energy transfer (FRET) strategy for the design of ratiometric fluorescent probes that were selective toward aminothiols. On the basis of this new strategy, the ratiometric fluorescent probe 1 for aminothiols was judiciously designed. The new probe is highly selective toward aminothiols over other thiols and exhibits a very large variation (up to 160‐fold) in its fluorescence ratio (I₄₅₈/I₆₀₃). The new fluorescent probe is capable of ratiometric detection of aminothiols in newborn calf and human serum samples and is also suitable for ratiometric fluorescent imaging of aminothiols in living cells.     

Ratiometric fluorescence detection of bleomycin based on proximity-dependent fluorescence conversion of DNA-templated silver nanoclusters

We design a ratiometric fluo rescent sensing platform for bleomycin(BLM) by using proximity-dependent DNA-templated silver nanoclusters(DNA-AgNCs) probe.This ratiometric sensing system is constructed with DNA-AgNCs as single fluorophore.The proposed strategy is based on the two following facts:(1) a covert DNA can approach and transform the DNA-AgNCs with green emission(G-DNA-AgNCs) into red emission through hybridization reaction.(2) The specific cleavage of the convert DNA by BLM in the presence of Fe(Ⅱ) inhibits the discoloration of G-DNA-AgNCs.Thus,benefiting from the specific recognition of BLM and unique properties of G-DNA-AgNCs,a hignly-sensitive ratiometric sensor for BLM has been successfully developed.The detection limit is as low as 30 pmol/L.This label-free fluorescence probe possesses advantages of convenient synthetic process and low cost.Moreover,this ratiometric method has been applied to the detection of BLM in human serum samples,illustrating a promising tool for analysis of BLM in cancer therapy.     

A Single‐Wavelength‐Emitting Ratiometric Probe Based on Phototriggered Fluorescence Switching of Graphene Quantum Dots

Ratiometric fluorescent probes are of great importance in research, because a built‐in correction for environmental effects can be provided to reduce background interference. However, the traditional ratiometric fluorescent probes require two luminescent materials with different emission bands. Herein a novel ratiometric probe based on a single‐wavelength‐emitting material is reported. The probe works by regulating the luminescent property of graphene quantum dots with UV illumination as activator. The ratiometric sensor shows high sensitivity and specificity for iron ions. Moreover, the ratiometric sensor was successfully employed to monitor ferritin levels in Sprague Dawley rats with chemical‐induced acute liver damage. The proposed single‐wavelength ratiometric fluorescent probe may greatly broaden the applicability of ratiometric sensors in diagnostic devices, medical applications, and analytical chemistry.     

Development of FRET-based ratiometric fluorescent Cu2+ chemodosimeters and the applications for living cell imaging

Coumarin-rhodamine-based compounds 1a,b were rationally designed and synthesized as novel FRET ratiometric fluorescent chemodosimeters. Ratiometric chemodosimeters 1a,b exhibit several favorable features, including a large variation in the emission ratio, well-resolved emission peaks, high sensitivity, high selectivity, low cytotoxicity, and good cell membrane permeability. Importantly, these excellent attributes enable us to demonstrate ratiometric imaging of Cu(2+) in living cells by using these novel ratiometric fluorescent chemodosimeters.     

A novel colorimetric and ratiometric NIR fluorescent sensor for glutathione based on dicyanomethylene-4H-pyran in living cells

Glutathione (GSH) plays a critical role in maintaining oxidation-reduction homeostasis in biological systems. Considering the detection of GSH by fluorescence sensors is limited by either the short wavelength emission or the poor photostability, a highly stable colorimetric and ratiometric NIR fluorescent sensor (DCM-S) for GSH detection has been constructed on the basis of dicyanomethylene-4H-pyran (DCM) chromophore. The specific disulfide bond is incorporated via a carbamate linker as the GSH responsive group, which simultaneously blue-shifts and quenches the fluorescence. Upon addition of GSH, DCM-S exhibits outstanding colorimetric (from yellow to red) and ratiometric fluorescent response with the 6-fold enhancement of NIR fluorescence at 665 nm in quantum yield. More importantly, the GSH-treated DCM-S (DCM-NH₂ actually) possesses 20-fold longer fluorescence half-life period as well as much better photostability than the FDA-approved ICG. Finally, the ratiometric detection of GSH is also successfully operated in the living cell imaging, exhibiting NIR fluorescence and large Stokes shift (215 nm) with nearly no background fluorescence interference. As a consequence, DCM-S can be utilized as colorimetric and ratiometric NIR fluorescent sensor for GSH, with a great potential in the development of GSH-induced drug delivery system.     

Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor

Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm that can be used for ratiometric imaging. The intensity ratio responds with an apparent pK(a) of 6.6 and a >10-fold dynamic range. Furthermore, pHRed has a pH-responsive fluorescence lifetime that changes by ~0.4 ns over physiological pH values and can be monitored with single-wavelength two-photon excitation. After characterizing the sensor, we tested pHRed's ability to monitor intracellular pH by imaging energy-dependent changes in cytosolic and mitochondrial pH.     

Recent Progress of Small-Molecule Ratiometric Fluorescent Probes for Peroxynitrite in Biological Systems

Peroxynitrite (ONOO⁻) as a major reactive oxygen species plays important roles in cellular signal transduction and homeostatic regulation. Precise detection of ONOO⁻ in biological systems is vital for exploring its physiological and pathological function. Among numerous detection methods, fluorescence imaging technology using fluorescent probes offers some advantages, including simple operation, high sensitivity and selectivity, as well as real-time and nondestructive detection. In particular, ratiometric fluorescent probes, in which the built-in calibration of the two emission bands prevents interference from the biological environment, have been extensively employed to monitor the fluctuation of bioactive species. In this review, we will discuss small-molecule ratiometric fluorescent probes for ONOO⁻ in live cells or in vivo, which involves chemical structures, response mechanisms, and biological applications. Moreover, the challenges and future prospects of ONOO⁻-responsive ratiometric fluorescent probe are also proposed.     

Fluorescent detection of hypochlorous acid from turn-on to FRET-based ratiometry by a HOCl-mediated cyclization reaction

Hypochlorous acid (HOCl), a reactive oxygen species (ROS), plays a significant biological role in living systems. However, abnormal levels of HOCl are implicated in many inflammation-associated diseases. Therefore, the detection of HOCl is of great importance. In this work, we describe the HOCl-promoted cyclization of rhodamine-thiosemicarbazides to rhodamine-oxadiazoles, which is then exploited as a novel design strategy for the development of a new fluorescence turn-on HOCl probe 2. On the basis of the fluorescence resonance energy transfer (FRET) signaling mechanism, 2 was further converted into 1a and 1b, which represent the first paradigm of FRET-based ratiometric fluorescent HOCl probes. The outstanding features of 1a and 1b include well-resolved emission peaks, high sensitivity, high selectivity, good functionality at physiological pH, rapid response, low cytotoxicity, and good cell-membrane permeability. Furthermore, these excellent attributes enable us to demonstrate, for the first time, the ratiometric imaging of endogenously produced HOCl in living cells by using these novel ratiometric probes. We expect that 1a and 1b will be useful molecular tools for studies of HOCl biology. In addition, the HOCl-promoted cyclization reaction of rhodamine-thiosemicarbazides to rhodamine-oxadiazoles should be widely applicable for the development of different types of fluorescent HOCl probes.     

Photoinduced electron transfer (PET) based Zn2+ fluorescent probe: transformation of turn-on sensors into ratiometric ones with dual emission in acetonitrile

Ratiometric sensors for the detection of metal ions have gained increasing attention due to its self-calibration tendency for the environmental effects. In this context, we have synthesized and characterized a dual emitting ratiometric Zn(2+) probe (1) having acridinedione as a fluorophore and N,N-bis(2-pyridylmethyl)amine (BPA) as a receptor unit. Existence of two different conformation of the molecule with photoinduced electron transfer (PET) from amine moiety to the acridinedione fluorophore leads to dual emission, namely locally excited (425 nm) and anomalous charge transfer emission (560 nm) in aprotic solvents. In the presence of one equivalent of Zn(2+), a 15-fold fluorescence enhancement in the locally excited state together with the quenching of charge transfer emission is observed. The intensity changes at the two emission peaks allow a ratiometric detection of Zn(2+) under PET signaling mechanism. The utilization of PET process for the ratiometric fluorescence change will further signify the importance of PET mechanism in sensing action. Addition of Zn(2+) to 1 in acetonitrile/water mixtures shows a single emission peak with fluorescence enhancement.