A Synthetic Fluorescent Mitochondria‐Targeted Sensor for Ratiometric Imaging of Calcium in Live Cells

Ca²⁺ handling by mitochondria is crucial for cell life and the direct measure of mitochondrial Ca²⁺ concentration in living cells is of pivotal interest. Genetically‐encoded indicators greatly facilitated this task, however they require demanding delivery procedures. On the other hand, existing mitochondria‐targeted synthetic Ca²⁺ indicators are plagued by several drawbacks, for example, non‐specific localization, leakage, toxicity. Here we report the synthesis and characterization of a new fluorescent Ca²⁺ sensor, named mt‐fura‐2, obtained by coupling two triphenylphosphonium cations to the molecular backbone of the ratiometric Ca²⁺ indicator fura‐2. Mt‐fura‐2 binds Ca²⁺ with a dissociation constant of ≈1.5 μm in vitro. When loaded in different cell types as acetoxymethyl ester, the probe shows proper mitochondrial localization and accurately measures matrix [Ca²⁺] variations, proving its superiority over available dyes. We describe the synthesis, characterization and application of mt‐fura‐2 to cell types where the delivery of genetically‐encoded indicators is troublesome.     

Stable,Wavelength‐Tunable Fluorescent Dyes in the NIR‐II Region for In Vivo High‐Contrast Bioimaging and Multiplexed Biosensing

Small‐molecule organic fluorophores, spectrally active in the 900–1700 nm region, with tunable wavelength and sensing properties are sought‐after for in vivo optical imaging and biosensing. A panel of fluorescent dyes ( CX ) has been developed to meet this challenge. CX dyes exhibit the wavelength tunability of cyanine dyes and have a rigidified polymethine chain to guarantee their stability. They are chemo‐ and photo‐stable in an aqueous environment and have tunable optical properties with maximal absorbing/emitting wavelength at 1089/1140 nm. They show great potential in high‐contrast in vivo bioimaging and multicolor detection with negligible optical cross talk. Förster resonance energy transfer (FRET) between CX dyes was demonstrated in deep tissue, providing an approach for monitoring drug‐induced hepatotoxicity by detection of OONO^?. This report presents a series of NIR‐II dyes with promising spectroscopic properties for high‐contrast bioimaging and multiplexed biosensing.     

A General Strategy for Development of Near‐Infrared Fluorescent Probes for Bioimaging

Near‐infrared (NIR) fluorescent dyes with favorable photophysical properties are highly useful for bioimaging, but such dyes are still rare. The development of a unique class of NIR dyes via modifying the rhodol scaffold with fused tetrahydroquinoxaline rings is described. These new dyes showed large Stokes shifts (>110 nm). Among them, WR3, WR4, WR5, and WR6 displayed high fluorescence quantum yields and excellent photostability in aqueous solutions. Moreover, their fluorescence properties were tunable by easy modifications on the phenolic hydroxy group. Based on WR6, two NIR fluorescent turn‐on probes, WSP‐NIR and SeSP‐NIR, were devised for the detection of H₂S. The probe SeSP‐NIR was applied in visualizing intracellular H₂S. These dyes are expected to be useful fluorophore scaffolds in the development of new NIR probes for bioimaging.     

Enzyme‐Mediated Endogenous and Bioorthogonal Control of a DNAzyme Fluorescent Sensor for Imaging Metal Ions in Living Cells

Bioorthogonal control of metal‐ion sensors for imaging metal ions in living cells is important for understanding the distribution and fluctuation of metal ions. Reported here is the endogenous and bioorthogonal activation of a DNAzyme fluorescent sensor containing an 18‐base pair recognition site of a homing endonuclease (I‐SceI), which is found by chance only once in 7×10¹⁰ bp of genomic sequences, and can thus form a near bioorthogonal pair with I‐SceI for DNAzyme activation with minimal effect on living cells. Once I‐SceI is expressed inside cells, it cleaves at the recognition site, allowing the DNAzyme to adopt its active conformation. The activated DNAzyme sensor is then able to specifically catalyze cleavage of a substrate strand in the presence of Mg²⁺ to release the fluorophore‐labeled DNA fragment and produce a fluorescent turn‐on signal for Mg²⁺. Thus I‐SceI bioorthogonally activates the 10–23 DNAzyme for imaging of Mg²⁺ in HeLa cells.     

A Redox‐Activatable Fluorescent Sensor for the High‐Throughput Quantification of Cytosolic Delivery of Macromolecules

Efficient delivery of biomacromolecules (e.g., proteins, nucleic acids) into cell cytosol remains a critical challenge for the development of macromolecular therapeutics or diagnostics. To date, most common approaches to assess cytosolic delivery rely on fluorescent labeling of macromolecules with an “always on” reporter and subcellular imaging of endolysosomal escape by confocal microscopy. This strategy is limited by poor signal‐to‐noise ratio and only offers low throughput, qualitative information. Herein we describe a quantitative redox‐activatable sensor (qRAS) for the real‐time monitoring of cytosolic delivery of macromolecules. qRAS‐labeled macromolecules are silent (off) inside the intact endocytic organelles, but can be turned on by redox activation after endolysosomal disruption and delivery into the cytosol, thereby greatly improving the detection accuracy. In addition to confocal microscopy, this quantitative sensing technology allowed for a high‐throughput screening of a panel of polymer carriers toward efficient cytosolic delivery of model proteins on a plate reader. The simple and versatile qRAS design offers a useful tool for the investigation of new strategies for endolysosomal escape of biomacromolecules to facilitate the development of macromolecular therapeutics for a variety of disease indications.     

AgHalo: A Facile Fluorogenic Sensor to Detect Drug‐Induced Proteome Stress

Drug‐induced proteome stress that involves protein aggregation may cause adverse effects and undermine the safety profile of a drug. Safety of drugs is regularly evaluated using cytotoxicity assays that measure cell death. However, these assays provide limited insights into the presence of proteome stress in live cells. A fluorogenic protein sensor is reported to detect drug‐induced proteome stress prior to cell death. An aggregation prone Halo‐tag mutant (AgHalo) was evolved to sense proteome stress through its aggregation. Detection of such conformational changes was enabled by a fluorogenic ligand that fluoresces upon AgHalo forming soluble aggregates. Using 5 common anticancer drugs, we exemplified detection of differential proteome stress before any cell death was observed. Thus, this sensor can be used to evaluate drug safety in a regime that the current cytotoxicity assays cannot cover and be generally applied to detect proteome stress induced by other toxins.     

Rational Design of a Dual‐Reactivity‐Based Fluorescent Probe for Visualizing Intracellular HSNO

Thionitrous acid (HSNO), the smallest S‐nitrosothiol, is emerging as a potential key intermediate in cellular redox regulation linking two signaling molecules H₂S and NO. However, the chemical biology of HSNO remains poorly understood. A major hurdle is the lack of methods for selective detection of HSNO in biological systems. Herein, we report the rational design, synthesis, and evaluation of the first fluorescent probe TAP‐1 for HSNO detection. TAP‐1 showed high selectivity and sensitivity to HSNO in aqueous media and cells, providing a useful tool for understanding the functions of HSNO in biology.     

A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O‐GlcNAc Transferase

Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high‐throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead‐based strategy to measure the group‐transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O‐GlcNAc transferase (OGT) as a model system through detailed Michaelis–Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high‐throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group‐transfer enzymes.     

A Reversible Fluorescent Probe for Real-Time Quantitative Monitoring of Cellular Glutathione

The ability to monitor and quantify glutathione (GSH) in live cells is essential in order to gain a detailed understanding of GSH-related pathological events. However, owing to their irreversible response mechanisms, most existing fluorescent GSH probes are not suitable for this purpose. We have developed a ratiometric fluorescent probe (QG- 1 ) for quantitatively monitoring cellular GSH. The probe responds specifically and reversibility to GSH with an ideal dissociation constant (K_d) of 2.59 mm and a fast response time (t_1/2=5.82 s). We also demonstrate that QG- 1 detection of GSH is feasible in a model protein system. QG- 1 was found to have extremely low cytotoxicity and was applied to determine the GSH concentration in live HeLa cells (5.40±0.87 mm ).     

HKOH‐1: A Highly Sensitive and Selective Fluorescent Probe for Detecting Endogenous Hydroxyl Radicals in Living Cells

The hydroxyl radical (^.OH), one of the most reactive and deleterious reactive oxygen species (ROS), has been suggested to play an essential role in many physiological and pathological scenarios. However, a reliable and robust method to detect endogenous ^.OH is currently lacking owing to its extremely high reactivity and short lifetime. Herein we report a fluorescent probe HKOH‐1 with superior in vitro selectivity and sensitivity towards ^.OH. With this probe, we have calibrated and quantified the scavenging capacities of a wide range of reported ^.OH scavengers. Furthermore, HKOH‐1r, which was designed for better cellular uptake and retention, has performed robustly in detection of endogenous ^.OH generation by both confocal imaging and flow cytometry. Furthermore, this probe has been applied to monitor ^.OH generation in HeLa cells in response to UV light irradiation. Therefore, HKOH‐1 could be used for elucidating ^.OH related biological functions.