Enhanced oxidase-like activity of g-C3N4 nanosheets supported Pd nanosheets for ratiometric fluorescence detection of acetylcholinesterase activity and its inhibitor

The undesirable enzymatic activity of nanozymes under near neutral p H condition and the traditional single signal output always restrict the analytical application of nanozyme-based biosensors.Herein,graphitic carbon nitride nanosheets supported palladium nanosheets composite （Pd/g-C₃N₄） with both oxidase-like activity and fluorescent property is synthesized.Notably,Pd/g-C₃N₄exhibits enhanced oxidase-like activity compared to Pd NSs under p H 7.4.By c...     

A ratiometric fluorescent probe for iron(III) and its application for detection of iron(III) in human blood serum

A simple and versatile ratiometric fluorescent Fe³⁺ detecting system, probe 1, was rationally developed based on the Fe³⁺-mediated deprotection of acetal reaction. Notably, this reaction was firstly employed to design fluorescent Fe³⁺ probe. Upon treatment with Fe³⁺, probe 1 showed ratiometric response, with the fluorescence spectra displaying significant red shift (up to 132 nm) and the emission ratio value (I₅₂₂/I₃₉₀) exhibiting approximately 2362-fold enhancement. In addition, the probe is highly sensitive (with the detection limit of 0.12 μM) and highly selective to Fe³⁺ over other biologically relevant metal ions. The sensing reaction product of the probe with Fe³⁺ was confirmed by NMR spectra and mass spectrometry. TD-DFT calculation has demonstrated that the ratiometric response of probe 1 to Fe³⁺ is due to the regulation of intramolecular charge transfer (ICT) efficiency. Moreover, the practical utility in fluorescence detection of Fe³⁺ in human blood serum was also conducted, and probe 1 represents the first ratiometric fluorescent probe that can be used to monitor Fe³⁺ level in human blood serum. Finally, probe 1 was further employed in living cell imaging with pancreatic cancer cells, in which it displayed low cytotoxicity, satisfactory cell permeability, and selective ratiometric response to Fe³⁺.     

A new prodrug-derived ratiometric fluorescent probe for hypoxia: high selectivity of nitroreductase and imaging in tumor cell

Based on the hypoxia prodrug moiety of p-nitrobenzyl, a selective ratiometric fluorescent sensor (RHP) for the detection of microenvironment hypoxia was designed and synthesized. RHP can be selectively activated by bioreductive enzymes (NTR) and results in an evident blue to green fluorescent emission wavelength change in both solution phases and in cell lines, which might be the first fluorescent ratiometric probe for hypoxia in solid tumors.     

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.     

A Ratiometric Fluorescent Probe for Imaging of the Activity of Methionine Sulfoxide Reductase A in Cells

Methionine sulfoxide reductase A (MsrA) is an enzyme involved in redox balance and signaling, and its aberrant activity is implicated in a number of diseases (for example, Alzheimer's disease and cancer). Since there is no simple small molecule tool to monitor MsrA activity in real time in vivo, we aimed at developing one. We have designed a BODIPY‐based probe called (S)‐Sulfox‐1, which is equipped with a reactive sulfoxide moiety. Upon reduction with a model MsrA (E. coli), it exhibits a bathochromic shift in the fluorescence maximum. This feature was utilized for the real‐time ratiometric fluorescent imaging of MsrA activity in E. coli cells. Significantly, our probe is capable of capturing natural variations of the enzyme activity in vivo.     

A dual-fluorophore sensor approach for ratiometric fluorescence imaging of potassium in living cells

Potassium is the most abundant intracellular metal in the body, playing vital roles in regulating intracellular fluid volume, nutrient transport, and cell-to-cell communication through nerve and muscle contraction. On the other hand, aberrant alterations in K⁺ homeostasis contribute to a diverse array of diseases spanning cardiovascular and neurological disorders to diabetes to kidney disease to cancer. There is an unmet need for studies of K⁺ physiology and pathology owing to the large differences in intracellular versus extracellular K⁺ concentrations ([K⁺]_intra = 150 mM, [K⁺]_extra = 3–5 mM). With a relative dearth of methods to reliably measure dynamic changes in intracellular K⁺ in biological specimens that meet the dual challenges of low affinity and high selectivity for K⁺, particularly over Na⁺, currently available fluorescent K⁺ sensors are largely optimized with high-affinity receptors that are more amenable for extracellular K⁺ detection. We report the design, synthesis, and biological evaluation of Ratiometric Potassium Sensor 1 (RPS-1), a dual-fluorophore sensor that enables ratiometric fluorescence imaging of intracellular potassium in living systems. RPS-1 links a potassium-responsive fluorescent sensor fragment (PS525) with a low-affinity, high-selectivity crown ether receptor for K⁺ to a potassium-insensitive reference fluorophore (Coumarin 343) as an internal calibration standard through ester bonds. Upon intracellular delivery, esterase-directed cleavage splits these two dyes into separate fragments to enable ratiometric detection of K⁺. RPS-1 responds to K⁺ in aqueous buffer with high selectivity over competing metal ions and is sensitive to potassium ions at steady-state intracellular levels and can respond to decreases or increases from that basal set point. Moreover, RPS-1 was applied for comparative screening of K⁺ pools across a panel of different cancer cell lines, revealing elevations in basal intracellular K⁺ in metastatic breast cancer cell lines vs. normal breast cells. This work provides a unique chemical tool for the study of intracellular potassium dynamics and a starting point for the design of other ratiometric fluorescent sensors based on two-fluorophore approaches that do not rely on FRET or related energy transfer designs.

We report a dual-fluorophore approach for ratiometric fluorescent imaging of K⁺ levels in live cells. Intracellular esterases cleave RPS-1 to detach the K⁺-responsive fluorophore (PS525) from its internal standard (Coumarin 343).     

A New Tetraphenylethylene‐Derived Fluorescent Probe for Nitroreductase Detection and Hypoxic‐Tumor‐Cell Imaging

The fluorescence detection of nitroreductase (NTR) and evaluation of the hypoxia status of tumor cells are vital, not only for clinical diagnoses and therapy, but also for biomedical research. Herein, we report the synthesis and application of a new fluorometric “turn‐on” probe for the detection of NTR ( TPE?NO₂ ) that takes advantage of the aggregation‐induced emission of tetraphenylethylene. TPE?NO₂ can detect NTR at concentrations as low as 5 ng mL^?1 in aqueous solution. The detection mechanism relied on the aggregation and deaggregation of tetraphenylethylene molecules. Moreover, this fluorescent probe can be used to monitor the hypoxia status of tumor cells through the detection of endogenous NTR.     

Ratiometric Fluorescent Nanosensors for Copper(II) Based on Bis(rhodamine)‐Derived PMOs with J‐Type Aggregates

By using pentyl‐linked bis(rhodamine)‐derived tetra‐siloxane (PRh‐Si₄) as the organosilica precursor, highly ordered PRh‐bridged periodic mesoporous organosilicas (PRhPMOs) were prepared. When excited at λ=500 nm, the PRhPMO suspension that contained metal ions showed two separate emission peaks at λ=550 and 623 nm. The first peak, located at λ=550 nm, was due to ring‐opening of the spiro structure in the rhodamine moiety and the second, located at λ=623 nm, originated from fluorescent aggregates of the PRh units embedded in the silica framework of the PRhPMO. By using the different intensity ratios of the two fluorescence signals (FI_550/623), PRhPMOs could be used as turn‐ON type fluorescent ratiometric chemosensors for Cu²⁺. Furthermore, based on the single‐exciton theory, it was deduced that the fluorescent aggregates formed were of the J‐type and had a coplanar configuration. Consequently, PRhPMOs display a longer fluorescence lifetime and greater fluorescent quantum yield than the respective monomers dissolved in solution, which is consistent with the experimental results.     

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.     

Monitoring the pH fluctuation of lysosome under cell stress using a near-infrared ratiometric fluorescent probe

Cell stress responses are associated with numerous diseases including diabetes, neurodegenerative diseases, and cancer. Several events occur under cell stress, in which, are protein expression and organelle-specific pH fluctuation. To understand the lysosomal pH variation under cell stress, a novel NIR ratiometric pH-responsive fluorescent probe (BLT) with lysosomes localization capability was developed. The quinoline ring of BLT combined with hydrogen ion which triggered the rearrangement of π electrons conjugated at low pH medium, meanwhile, the absorption and fluorescent spectra of BLT showed a red-shifts, which gived a ratiometric signal. Moreover, the probe BLT with a suitable pK_a value has the potential to discern changes in lysosomal pH, either induced by heat stress or oxidative stress or acetaminophen-induced (APAP) injury stress. Importantly, this ratiometric fluorescent probe innovatively tracks pH changes in lysosome in APAP-induced liver injury in live cells, mice, and zebrafish. The probe BLT as a novel fluorescent probe possesses important value for exploring lysosomal-associated physiological varieties of drug-induced hepatotoxicity.     

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.     

A colorimetric and ratiometric fluorescent turn-on fluoride chemodosimeter and application in live cell imaging: high selectivity via specific SiO cleavage in semi aqueous media and prompt recovery of ESIPT along with the X-ray structures

A ratiometric fluorescent turn-on probe for fluoride ion, based on modulation of the excited-state intramolecular proton transfer (ESIPT) process by chemodosimetric desilylation pathway is reported. The probe SNBT (silyl protected hydroxynaphthalene benzothiazole moiety) shows a significant increase of ratiometric absorption band at 440 nm and emission band at 477 nm by the deprotection of fluoride mediated silyl bond cleavage in CH₃CN–H₂O (8/2, v/v, 25 °C). The test strips based on SNBT and F⁻ are fabricated, which can act as a convenient and efficient F⁻ test kits. Furthermore, the biological application shows that it can be very useful as a selective fluoride probe in the fluorescence imaging of living cells.     

Development of ratiometric fluorescent pH sensors based on chromenoquinoline derivatives with tunable pKa values for bioimaging

In this work, we first studied the pH-dependent characteristic of chromenoquinoline. Based on this, we then designed and synthesized two novel chromenoquinoline derivatives that can act as fluorescent pH sensors. The pK_a values of two novel chromenoquinoline derivatives can be modulated from 2.32 to 4.38 and 6.27 by introducing EDG on the backbone of chromenoquinoline. Furthermore, we demonstrate that the sensor 4 can be used as a ratiometric fluorescent pH sensor for fluorescence imaging in living cells.     

Cytotoxicity of Amino-BODIPY Modulated via Conjugation with 2-Phenyl-3-Hydroxy-4(1H)-Quinolinones

The combination of cytotoxic amino-BODIPY dye and 2-phenyl-3-hydroxy-4(1H)-quinolinone (3-HQ) derivatives into one molecule gave rise to selective activity against lymphoblastic or myeloid leukemia and the simultaneous disappearance of the cytotoxicity against normal cells. Both species′ conjugation can be realized via a disulfide linker cleavable in the presence of glutathione characteristic for cancer cells. The cleavage liberating the free amino-BODIPY dye and 3-HQ derivative can be monitored by ratiometric fluorescence or by the OFF-ON effect of the amino-BODIPY dye. A similar cytotoxic activity is observed when the amino-BODIPY dye and 3-HQ derivative are connected through a non-cleavable maleimide linker. The work reports the synthesis of several conjugates, the study of their cleavage inside cells, and cytotoxic screening.     

A highly selective ratiometric fluorescent probe for H2S based on new heterocyclic ring formation and detection in live cells

A 3-indolylacrylate derivative, 3-IA, prepared by connecting an ethyl acrylate in 3-position of indole has been synthesised and characterised. Ethyl acrylate moiety acts as the Michael acceptor towards H₂S, and the resultant addition product then participates in intramolecular cyclisation with the ester group at 2-position to form another new heterocyclic ring. Blue fluorescence of 3-IA turned into green in presence of H₂S, leading to ratiometric behaviour of the fluorescent sensor with large stokes shift of 55 nm. Probe 3-IA has excellent selectivity towards H₂S over other biothiols and other competing anions. Density function theory/time-dependent density function theory calculations were carried out to validate the reaction mechanism and the electronic properties of 3-IA. Importantly, the ratiometric probe 3-IA shows great promise in H₂S detection by simple visual fluorescent inspection in filter paper-based protocol. The probe shows its excellent ability to detect H₂S in different natural water samples. Furthermore, we have employed our probe to detect H₂S for ratiometric imaging in live Vero cell.     

Benzimidazole-based imine-linked chemosensor: chromogenic sensor for Mg2+ and fluorescent sensor for Cr3+

We synthesized an imine-linked, benzimidazole-based chemosensor that can be used for chromogenic recognition of Mg²⁺ and fluorescent recognition of Cr³⁺. The chemosensor shows sensitive, selective, and ratiometric recognition of Cr³⁺ through concurrent quenching at one wavelength and enhancement of fluorescence intensity at another wavelength. It can also be used to detect Mg²⁺ via UV–vis absorption spectroscopy. DFT calculations support these phenomena. The sensor can be used to strain microbe cells without breakage.     

Naphthalimide-rhodamine based fluorescent probe for ratiometric sensing of cellular pH

The pH values of lysosomes in cancer cells is slightly lower than that in normal cells, which can be used to distinguish cancer cells from normal cells. According to this, a naphthalimide-rhodamine based fluorescent probe(hereafter referred to as RBN) with a pK_a of 4.20 was designed and synthesized for ratiometric sensing of cellular pH via fluorescence resonance energy transfer(FRET), which can respond to different pH precisely through ratiometric fluorescence intensity(Ⅰ_(577)/Ⅰ_(540)). RBN can be employed to distinguish cancer cells from normal cells on the basis of different fluorescent response, in particular, RBN showed excellent water solubility and low cell toxicity, all these are quite significant for potential application in cancer diagnose and therapy.     

Environmentally Sensitive Color-Shifting Fluorophores for Bioimaging

We introduce color-shifting fluorophores that reversibly switch between a green and red fluorescent form through intramolecular spirocyclization. The equilibrium of the spirocyclization is environmentally sensitive and can be directly measured by determining the ratio of red to green fluorescence, thereby enabling the generation of ratiometric fluorescent probes and biosensors. Specifically, we developed a ratiometric biosensor for imaging calcium ions (Ca²⁺) in living cells, ratiometric probes for different proteins, and a bioassay for the quantification of nicotinamide adenine dinucleotide phosphate.     

A FRET-based fluorescent Zn2+ sensor: 3D ratiometric imaging,flow cytometric tracking and cisplatin-induced Zn2+ fluctuation monitoring

Monitoring labile Zn²⁺ homeostasis is of great importance for the study of physiological functions of Zn²⁺ in biological systems. Here we report a novel ratiometric fluorescent Zn²⁺ sensor, CPBT, which was constructed based on chelation-induced alteration of FRET efficiency. CPBT was readily cell membrane permeable and showed a slight preferential localization in the endoplasmic reticulum. With this sensor, 3D ratiometric Zn²⁺ imaging was first realized in the head of zebra fish larvae via Z-stack mode. CPBT could track labile Zn²⁺ in a large number of cells through ratiometric flow cytometric assay. More interestingly, both ratiometric fluorescence imaging and flow cytometric assay demonstrated that the labile Zn²⁺ level in MCF-7 cells (cisplatin-sensitive) decreased while that in SKOV3 cells (cisplatin-insensitive) increased after cisplatin treatment, indicating that Zn²⁺ may play an important role in cisplatin induced signaling pathways in these cancer cells.

A Zn²⁺ sensor exhibiting 3D ratiometric imaging and flow cytometric ability was constructed based on the FRET mechanism, and cisplatin-induced endogenous labile Zn²⁺ fluctuations were monitored in real time.     

Gasotransmitter Regulation of Phosphatase Activity in Live Cells Studied by Three‐Channel Imaging Correlation

Enzyme activity in live cells is dynamically regulated by small‐molecule transmitters for maintaining normal physiological functions. A few probes have been devised to measure intracellular enzyme activities by fluorescent imaging, but the study of the regulation of enzyme activity via gasotransmitters in situ remains a long‐standing challenge. Herein, we report a three‐channel imaging correlation by a single dual‐reactive fluorescent probe to measure the dependence of phosphatase activity on the H₂S level in cells. The two sites of the probe reactive to H₂S and phosphatase individually produce blue and green fluorescent responses, respectively, and resonance energy transfer can be triggered by their coexistence. Fluorescent analysis based on the three‐channel imaging correlation shows that cells have an ideal level of H₂S to promote phosphatase activity up to its maximum. Significantly, a slight deviation from this H₂S level leads to a sharp decrease of phosphatase activity. The discovery further strengthens our understanding of the importance of H₂S in cellular signaling and in various human diseases.