A Multi‐signal Fluorescent Probe with Multiple Binding Sites for Simultaneous Sensing of Cysteine,Homocysteine, and Glutathione

A novel fluorescent probe was developed by integrating chlorinated coumarin and benzothiazolylacetonitrile and exploited for simultaneous detection of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Featuring four binding sites and different reaction mechanisms for different biothiols, this probe exhibited rapid fluorescence turn‐on for distinguishing Cys, Hcy, and GSH with 108‐, 128‐, 30‐fold fluorescence increases at 457, 559, 529 nm, respectively, across different excitation wavelengths. Furthermore, the probe was successfully applied to the fluorescence imaging of endogenous Cys and GSH and exogenous Cys, Hcy, and GSH in living cells.     

A Multi‐signal Fluorescent Probe with Multiple Binding Sites for Simultaneous Sensing of Cysteine,Homocysteine, and Glutathione

A novel fluorescent probe was developed by integrating chlorinated coumarin and benzothiazolylacetonitrile and exploited for simultaneous detection of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Featuring four binding sites and different reaction mechanisms for different biothiols, this probe exhibited rapid fluorescence turn‐on for distinguishing Cys, Hcy, and GSH with 108‐, 128‐, 30‐fold fluorescence increases at 457, 559, 529 nm, respectively, across different excitation wavelengths. Furthermore, the probe was successfully applied to the fluorescence imaging of endogenous Cys and GSH and exogenous Cys, Hcy, and GSH in living cells.     

A Sensitive and Selective Fluorescent Probe for Cysteine Based on a New Response‐Assisted Electrostatic Attraction Strategy: The Role of Spatial Charge Configuration

A new strategy for fast fluorescent detection of cysteine (Cys), based on a response‐assisted electrostatic attraction, is demonstrated. By utilizing this strategy, we designed and synthesized three fluorescent probes for the specific detection of Cys under actual physiological conditions. The probe m‐ CP , a coumarin fluorophore conjugated with a substituted methyl pyridinium group through an unsaturated ketone unit, showed highly selective and sensitive detection for cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH). The kinetic analysis indicated that the sensing process was highly accelerated (a response time less than 1 min) by the response‐assisted electrostatic attraction. More importantly, control experiments with isomeric probes first demonstrated that the spatial charge configuration of the probe played an important role in Cys‐preferred selectivity and kinetic rate acceleration. Furthermore, the practical utility of the probe m‐ CP in the fluorescent labeling of Cys residues within proteins was demonstrated. Finally, these probes were employed in living cell imaging with HeLa cells, in which it displayed satisfactory cell permeability and enabled us to distinguish active thiols in the cytoplasm, nucleus, and mitochondria.     

Rational Design of an “OFF–ON” Phosphorescent Chemodosimeter Based on an Iridium(III) Complex and Its Application for Time‐Resolved Luminescent Detection and Bioimaging of Cysteine and Homocysteine

Biothiols, such as cysteine (Cys) and homocysteine (Hcy), play very crucial roles in biological systems. Abnormal levels of these biothiols are often associated with many types of diseases. Therefore, the detection of Cys (or Hcy) is of great importance. In this work, we have synthesized an excellent “OFF‐ON” phosphorescent chemodosimeter 1 for sensing Cys and Hcy with high selectivity and naked‐eye detection based on an Ir^III complex containing a 2,4‐dinitrobenzenesulfonyl (DNBS) group within its ligand. The “OFF‐ON” phosphorescent response can be assigned to the electron‐transfer process from Ir^III center and C^N ligands to the DNBS group as the strong electron‐acceptor, which can quench the phosphorescence of probe 1 completely. The DNBS group can be cleaved by thiols of Cys or Hcy, and both the ³M LCT and ³LC states are responsible for the excited‐state properties of the reaction product of probe 1 and Cys (or Hcy). Thus, the phosphorescence is switched on. Based on these results, a general principle for designing “OFF‐ON” phosphorescent chemodosimeters based on heavy‐metal complexes has been provided. Importantly, utilizing the long emission‐lifetime of phosphorescence signal, the time‐resolved luminescent assay of 1 in sensing Cys was realized successfully, which can eliminate the interference from the short‐lived background fluorescence and improve the signal‐to‐noise ratio. As far as we know, this is the first report about the time‐resolved luminescent detection of biothiols. Finally, probe 1 has been used successfully for bioimaging the changes of Cys/Hcy concentration in living cells.     

Multi‐Fluorinated Azido Coumarins for Rapid and Selective Detection of Biological H2S in Living Cells

Hydrogen sulfide (H₂S) is an endogenously produced gaseous signaling molecule with multiple biological functions. In order to visualize the endogenous in situ production of H₂S in living cells in real time, here we developed multi‐fluorinated azido coumarins as fluorescent probes for the rapid and selective detection of biological H₂S. Kinetic studies indicated that an increase in fluorine substitution leads to an increased rate of H₂S‐mediated reduction reaction, which is also supported by our theoretical calculations. To our delight, tetra‐fluorinated coumarin 1 could react with H₂S fast (t_1/2≈1 min) and selectively, which could be further used for continuous enzymatic assays and for visualization of intracellular H₂S. Bioimaging results obtained with 1 revealed that d ‐Cys could induce a higher level of endogenous H₂S production than l ‐Cys in a time‐dependent manner in living cell.     

An Ultrasensitive Cyclization‐Based Fluorescent Probe for Imaging Native HOBr in Live Cells and Zebrafish

Bromine has been reported recently as being the 28^th essential element for human health. HOBr, which is generated in vivo from bromide, is a required factor in the formation of sulfilimine crosslinks in collagen IV. However, to date, no method for the specific detection of native HOBr in vivo has been reported. Herein, we develop a simple small molecular probe for imaging HOBr based on a specific cyclization catalyzed by HOBr. The probe can be easily synthesized in high yield through a Suzuki cross‐coupling reaction. The probe exhibits ultrahigh sensitivity at the picomole level, in addition to specificity for HOBr and real‐time response. Importantly, without Br^? stimulation, this probe reports native HOBr levels in HepG2 cells. Thus, the probe is a promising new tool for imaging endogenous HOBr and may provide a means for finding new physiological functions of HOBr in living organisms.     

Target‐Triggered NIR Emission with a Large Stokes Shift for the Detection and Imaging of Cysteine in Living Cells

Background autofluorescence from biological systems generally reduces the sensitivity of a fluorescent probe for imaging biological targets. Addressing this challenge requires the development of fluorescent probes that produce emission in the near‐infrared region. Herein, we report the design and synthesis of a fluorescent probe that generates an NIR emission with a large Stokes shift upon the selective response to Cys over Hcy and GSH. The probe is designed to consist of two Cys‐sensing sites, an acrylate ester and an aldehyde installed ortho to each other. The reaction of the probe with Cys triggers an excited state intramolecular proton transfer process upon photo‐excitation, thereby producing an NIR emission with a large Stokes shift. Accordingly, this probe hold great promise for the selective detection of Cys in biological systems. We further demonstrate the capacity of this probe for Cys imaging in living cells.     

Smart Poly(N‐isopropylacrylamide) Containing Iridium(III) Complexes as Water‐Soluble Phosphorescent Probe for Sensing and Bioimaging of Homocysteine and Cysteine

Homocysteine (Hcy) and cysteine (Cys) are two important kinds of amino acids in human bodies. Herein, we synthesized an iridium(III) complex‐functionalized poly(N‐isopropylacrylamide) and its hydrogel, which could be used as the excellent phosphorescent bioprobe for sensing Hcy and Cys. Their detection can be realized in aqueous system through the variations in absorption and photoluminescence spectra. Furthermore, living cell imaging experiments demonstrate that the phosphorescent bioprobe is membrane permeable and can monitor the changes of Hcy and Cys within living cells. In addition, the probe is also thermoresponsive, and its photoluminescence intensified with increasing temperature. These results suggests that this bioprobe has promising application in biomedical fields.     

A Photoacoustic Probe for the Imaging of Tumor Apoptosis by Caspase‐Mediated Macrocyclization and Self‐Assembly

Photoacoustic (PA) imaging shows promise in the sensitive detection of caspase‐3 activated in early tumor apoptosis in response to chemotherapy; smart PA probes are thus in high demand. Herein, we report the first smart PA probe ( 1‐RGD ) responsive to caspase‐3, enabling real‐time and high‐resolution imaging of tumor apoptosis. 1‐RGD is designed to leverage the synergetic effect of active delivery and caspase‐3 activation. It is selectively recognized by active caspase‐3 to trigger peptide substrate cleavage and biocompatible macrocyclization‐mediated self‐assembly, leading to an amplified PA imaging signal and prolonged retention in apoptotic tumor cells. Strong, high‐resolution PA images are obtained in chemotherapy‐induced apoptotic tumors in living mice after intravenous administration with 1‐RGD , facilitating sensitive reporting of caspase‐3 activity and distribution within tumor tissues.     

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 Reversible Fluorescent Probe for Real‐Time Live‐Cell Imaging and Quantification of Endogenous Hydropolysulfides

The chemical biology of reactive sulfur species, including hydropolysulfides, has been a subject undergoing intense study in recent years, but further understanding of their “intact” function in living cells has been limited owing to a lack of appropriate analytical tools. In order to overcome this limitation, we developed a new type of fluorescent probe that reversibly and selectively reacts to hydropolysulfides. The probe enables live‐cell visualization and quantification of endogenous hydropolysulfides without interference from intrinsic thiol species such as glutathione. Additionally, real‐time reversible monitoring of oxidative‐stress‐induced fluctuation of intrinsic hydropolysulfides has been achieved with a temporal resolution on the order of seconds, a result which has not yet been realized using conventional methods. These results reveal the probe's versatility as a new fluorescence imaging tool to understand the function of intracellular hydropolysulfides.     

Development of a Two‐Photon Fluorescent Probe for Imaging of Endogenous Formaldehyde in Living Tissues

Investigation of the physiological and pathological functions of formaldehyde (FA) are largely restricted by a lack of useful FA imaging agents, in particular, those that allow detection of FA in the context of living tissues. Herein, we present the rational design, synthesis, and photophysical property studies of the first two‐photon fluorescent FA probe, Na‐FA . Importantly, the highly desirable attributes of the probe Na‐FA (such as a very large turn‐on signal (up to 900‐fold), a low detection limit, and a very fast onset imparted by the unique design aspects of the probe), make it possible to monitor endogenous FA in living tissues for the first time. Furthermore, sodium bisulfite was identified as a simple and convenient inhibitor of FA within biological environments.     

Near-Infrared mitochondria-targeted fluorescent probe for cysteine based on difluoroboron curcuminoid derivatives

A highly selective dual-channel NIR fl uorescent probe (DFB1) based on curcuminoid difl uoroboron is developed for discrimination Cys over GSH, Hcy and other amino acids in mitochondria of living cells.     

A Small‐Molecule FRET Reporter for the Real‐Time Visualization of Cell‐Surface Proteolytic Enzyme Functions

Real‐time imaging of cell‐surface‐associated proteolytic enzymes is critical to better understand their performances in both physiological and pathological processes. However, most current approaches are limited by their complexity and poor membrane‐anchoring properties. Herein, we have designed and synthesized a unique small‐molecule fluorescent probe, which combines the principles of passive exogenous membrane insertion and Förster resonance energy transfer (FRET) to image cell‐surface‐localized furin‐like convertase activities. The membrane‐associated furin‐like enzymatic cleavage of the peptide probe leads to an increased fluorescence intensity which was mainly localized on the plasma membrane of the furin‐expressed cells. This small‐molecule fluorescent probe may serve as a unique and reliable reporter for real‐time visualization of endogenous cell‐surfaceassociated proteolytic furin‐like enzyme functions in live cells and tissues using one‐photon and two‐photon microscopy.     

Dual‐Reactable Fluorescent Probes for Highly Selective and Sensitive Detection of Biological H2S

Hydrogen sulfide (H₂S) is an important endogenous signaling molecule with a variety of biological functions. Development of fluorescent probes for highly selective and sensitive detection of H₂S is necessary. We show here that dual‐reactable fluorescent H₂S probes could react with higher selectivity than single‐reactable probes. One of the dual‐reactable probes gives more than 4000‐fold turn‐on response when reacting with H₂S, the largest response among fluorescent H₂S probes reported thus far. In addition, the probe could be used for high‐throughput enzymatic assays and for the detection of Cys‐induced H₂S in cells and in zebrafish. These dual‐reactable probes hold potential for highly selective and sensitive detection of H₂S in biological systems.     

Stretchable Electrochemical Sensor for Real‐Time Monitoring of Cells and Tissues

Stretchable electrochemical sensors are conceivably a powerful technique that provides important chemical information to unravel elastic and curvilinear living body. However, no breakthrough was made in stretchable electrochemical device for biological detection. Herein, we synthesized Au nanotubes (NTs) with large aspect ratio to construct an effective stretchable electrochemical sensor. Interlacing network of Au NTs endows the sensor with desirable stability against mechanical deformation, and Au nanostructure provides excellent electrochemical performance and biocompatibility. This allows for the first time, real‐time electrochemical monitoring of mechanically sensitive cells on the sensor both in their stretching‐free and stretching states as well as sensing of the inner lining of blood vessels. The results demonstrate the great potential of this sensor in electrochemical detection of living body, opening a new window for stretchable electrochemical sensor in biological exploration.     

Dual‐Emission Channels for Simultaneous Sensing of Cysteine and Homocysteine in Living Cells

The development of a fluorescent probe to distinguish between cysteine (Cys) and homocysteine (Hcy) is always a challenge owing to their structural similarity, and the simultaneous detection of Cys and Hcy by utilizing different emission channels is especially difficult. In this work, we designed and synthesized a new fluorescent probe to differentiate between Cys and Hcy on the basis of a coumarin derivative with a chlorine atom and an α,β‐unsaturated aldehyde. Cys and Hcy induced different cascade reactions with the probe, which led to different products with distinct photophysical properties. The nonfluorescent probe responded to Cys and emitted strong blue fluorescence, whereas it reacted with Hcy and generated yellow fluorescence without interference from glutathione. In addition, the probe was successfully applied to distinguish between Cys and Hcy in living cells.     

A Lysosome‐Compatible Near‐Infrared Fluorescent Probe for Targeted Monitoring of Nitric Oxide

The requirement for nitric oxide (NO) of lysosomes has motivated the development of a sophisticated fluorescent probe to monitor the distribution of this important biomolecule at the subcellular level in living cells. A near‐infrared (NIR) fluorescent Si‐rhodamine (SiRB)‐NO probe was designed based on the NO‐induced ring‐opening process of Si‐rhodamine. The probe exhibits fast chromogenic and fluorogenic responses, and high sensitivity and selectivity toward trace amounts of NO. Significantly, the spirolactam in Si‐rhodamine exhibits very good tolerance to H⁺, which in turn brings extremely low background fluorescence not only in the physiological environment but also under acidic conditions. The stability of the highly fluorescent product in acidic solution provides persistent fluorescence emission for long‐term imaging experiments. To achieve targeted imaging with improved spatial resolution and sensitivity, an efficient lysosome‐targeting moiety was conjugated to a SiRB‐NO probe, affording a tailored lysosome‐targeting NIR fluorescent Lyso‐SiRB‐NO probe. Inheriting the key advantages of its parent SiRB‐NO probe, Lyso‐SiRB‐NO is a functional probe that is suited for monitoring lysosomal NO with excellent lysosome compatibility. Imaging experiments demonstrated the monitoring of both exogenous and endogenous NO in real time by using the Lyso‐SiRB‐NO probe.     

Benzothiazole‐Pyimidine‐Based BF2 Complex for Selective Detection of Cysteine

Due to the similar structure and reactivity of cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), the simultaneous discrimination of Cys over Hcy and GSH by a single fluorescent sensor is still a great challenge. In this work, a benzothiazole‐pyimidine‐based boron difluoride complex ( BPB ) was developed as a new fluorescent sensor for Cys. The sensor exhibits a highly selective “turn‐on” response to cysteine over Hcy, GSH and other amino acids in aqueous solution at physiological pH. The observed pseudo‐first‐order rate constant for the reaction of BPB with Cys was calculated to be about 0.062 min⁻¹. The detection limit of this sensor for Cys was determined to be 332 nm, and bioimaging of exogenous Cys by this sensor was successfully applied in living cells, thus indicating that this sensor holds great potential for biological applications.     

Development of an AND Logic‐Gate‐Type Fluorescent Probe for Ratiometric Imaging of Autolysosome in Cell Autophagy

The concomitant detection of two biological events facilitates the highly selective and sensitive analysis of specific biological functions. In this article, we report an AND logic‐gate‐type fluorescent probe that can concurrently sense two biological events in living cells: H₂O₂ accumulation and acidification. The probe exhibits a unique fluorescence sensing mechanism, in which a xanthene fluorophore is oxidatively transformed to a xanthone derivative by H₂O₂, thereby resulting in a clear dual‐emission change. This transformation is significantly accelerated under weak acidic conditions, which enables the selective and sensitive detection of H₂O₂ production in an acidic cellular compartment. This unique sensing property was successfully applied to the ratiometric fluorescence imaging of autolysosome formation in selective mitochondrial autophagy (mitophagy), which highlights the utility of this novel probe in autophagy research.