Enlarging the Stokes Shift by Weakening the π-Conjugation of Cyanines for High Signal-to-Noise Ratiometric Imaging.

The signal-to-noise ratio (SNR) is one of the key features of a fluorescent probe and one that often defines its potential utility for in vivo labeling and analyte detection applications. Here, it is reported that introducing a pyridine group into traditional cyanine-7 dyes in an asymmetric manner provides a series of tunable NIR fluorescent dyes (Cy-Mu-7) characterized by enhanced Stokes shifts (≈230 nm) compared to the parent cyanine 7 dye (<25 nm). The observed Stokes shift increase is ascribed to symmetry breaking of the Cy-Mu-7 core and a reduction in the extent of conjugation. The fluorescence signals of the Cy-Mu-7 dyes are enhanced upon confinement within the hydrophobic cavity of albumin or via spontaneous encapsulation within micelles in aqueous media. Utilizing the Cy-Mu-7, ultra-fast in vivo kidney labeling in mice is realized, and it is found that the liver injury will aggravate the burden of kidney by monitoring the fluorescence intensity ratio of kidney to liver. In addition, Cy-Mu-7 could be used as efficient chemiluminescence resonance energy transfer acceptor for the reaction between H2 O2 and bisoxalate. The potential utility of Cy-Mu-7 is illustrated via direct monitoring fluctuations in endogenous H2 O2 levels in a mouse model to mimic emergency room trauma.

Design of Dual-responsive ROS/RSS Fluorescent Probes and Their Application in Bioimaging.

Intracellular redox homeostasis exerts a tremendous influence on pathophysiological fields. In order to maintain the homeostasis of intracellular redox state, reactive oxygen species (ROS) and reactive sulfur species (RSS) react and transform with each other, and their levels also directly reflect the degree of oxidative stress and disease. To understand the correlation and pathophysiological effects of these two signaling molecules in oxidative stress, unique fluorescence imaging tools are required. Fluorescent probes have become an indispensable detection tool in modern scientific research and disease diagnosis. Currently, there are many fluorescent probes for detecting ROS or RSS. However, the cellular environment is extremely complex. Dual detection fluorescent probes that can monitor more than one species in a biological environment cause our attention. This review describes dual detection fluorescent probes that can detect ROS and RSS simultaneously or sequentially. This will provide strong support for the study of redox homeostasis.

Dual-channel detection of viscosity and pH with a near-infrared fluorescent probe for cancer visualization.

Compared to ordinary cells, tumor cells have a unique microenvironment, characterized by high viscosity, low pH, high reactive oxygen species level and the overexpression of certain proteases. Therefore, viscosity and pH can be used as important parameters for visualizing cancer. We designed a spiro-oxazolidine compound (In-1) for the dual-channel detection of viscosity and pH, with the red channel for detecting viscosity and the blue channel for pH. Interestingly, In-1 can locate different organelles under different conditions. Under physiological conditions, In-1 efficiently targeted lysosomes and showed that the viscosity of lysosomes increases in cancer cells while the pH decreases, which can be used to distinguish and detect cancer cells and normal cells. When we treated HL-7702 cells with CCCP, the probe could effectively target the mitochondria, and the fluorescence intensity in the pH channel decreased. This indicates that In-1 can be used as a powerful tool to simultaneously monitor viscosity and pH in different organelles, and may have a guiding role in diseases caused by mitochondrial and lysosomal microenvironments.

Unique assembly of carbonylpyridinium and chromene reveals mitochondrial thiol starvation under ferroptosis and novel ferroptosis inducer.

To reveal the delicate function of mitochondria, spatiotemporally precise detection tools remain highly desirable. However, current probes with positively charged warheads for targeting mitochondria diffuse out of the mitochondria as the potential of the mitochondrial membrane changes, which directly influences the accuracy of the detection. Herein, we assembled carbonylpyridinium and chromene to afford the probe CM-Mit. Following the ultrafast response to thiol and the dissociation of carbonylpyridinium, the formation of o-quinone methide from CM-Mit was proposed to label proteins, thus avoiding diffusion out of the mitochondria. Therefore, the accurate spatiotemporal detection of thiol in mitochondria was realized. With this excellent probe, ferroptosis inducers were proved to stimulate thiol starvation in mitochondria for the first time in cancer cells. Moreover, CM-Mit was used to screen a compound library developed in-house and the stemona alkaloid analog SA-11 was shown to induce ferroptosis in various cancer cell lines, including a drug-resistant one.

A NIR fluorescent probe tracing norepinephrine exocytosis and depression occurrence at the cellular level.

A NIR fluorescent probe tracing norepinephrine exocytosis and depression occurrence at the cellular level revealed that norepinephrine exocytosis rather than the inherent intracellular concentration was related with depression.

Endogenous cysteine fluorescence monitoring and its deployment in tumour demarcation.

A cysteine-specific fluorescent probe with a wide concentration detection range was used to monitor changes in cysteine levels in HeLa cells under stress and to demarcate the boundary of a xenograft tumour.

Host-guest type multiple site fluorescent probe for GSH detection in living organisms.

The host-guest type molecular size matching strategy, which embodied intramolecular hydrogen bond, aromatic nucleophilic substitution and nucleophilic addition, was utilized to develop a fluorescent probe for GSH specific detection and in situ imaging in organisms.

A sequentially activated bioluminescent probe for observation of cellular H2O2 production induced by cysteine.

We report herein a caged luciferin probe Cy-Hy as a sequentially activated probe to selectively and sensitively sense L-Cys and H2O2. The probe displayed fluorescence and bioluminescence responses toward the two analytes. Utilizing the present probe, cellular excess L-Cys-induced H2O2 up-regulation was observed for the first time in living MDA-MB-231 cells.

Sulphide activity-dependent multicolor emission dye and its applications in in vivo imaging.

Reactive sulfur species (RSS) play pivotal roles in various pathological and physiological processes. There exists an intricate relevance in generation and metabolism among these substances. Although they are nucleophilic, there are still some differences in their reactivity. There are many methods to detect them by using reactive fluorescent probes, but the systematic study of their reactivity is still lacking. In our study, we designed a multiple reaction site fluorescent probe based on benzene conjugated benzopyrylium and NBD. The study revealed that besides both biothiols and hydrogen sulfide, sulfur dioxide (SO2) can cleave the ether bond. There are two reaction forms for GSH with low reactivity: cutting the ether bond and adding the conjugated double bond of benzopyrylium. Nevertheless, Cys/Hcy with higher activity can further rearrange with NBD after cutting the ether bond. In addition, SO2 can not only cleave the ether bond, but also continue to add the conjugated double bond of benzopyrylium. The above processes lead to multicolor emission of the probe, thus realizing the characteristic analysis of different sulfides. Thus the probe can be used for the detection of sulfide in mitochondria, and further for the imaging of sulfide in cells and zebrafish. This effective analysis method will provide a broad application prospect for practical applications.

Substitution-rearrangement-cyclization strategy to construct fluorescent probe for multicolor discriminative analysis biothiols in cells and zebrafish.

Discriminative detection of biothiols (Cysteine, homocysteine and glutathione) is of great significance to clarificate their complex physiological processes, the occurrence and development of related diseases. However, similar structure and reactivity among such species pose huge challenges in developing fluorescent probes to distinguish among of them. In this work, a dual-site probe CTT reacted with the analytes to regulate molecular conjugation through substitution-rearrangement-cyclization strategy, utilizing a multi-channel signal combination mode to realize the distinguishing detection of the three biothiols. Cell and zebrafish imaging experiments sufficiently demonstrated that CTT could semiquantify biothiols, which will provide valuable chemical tool for elucidating the complex biological functions of biothiols.

Rapid Reaction, Slow Dissociation Aggregation, and Synergetic Multicolor Emission for Imaging the Restriction and Regulation of Biosynthesis of Cys and GSH.

Biosynthesis is a necessary process to maintain life. In recent years, research has fully shown that three kinds of biothiols (Cys, Hcy, GSH) mainly play the role in oxidative stress and maintaining cell homeostasis in cells, and that abnormal concentrations will lead to the occurrence of cardiovascular diseases, cancers, etc. Various fluorescent probes have shown unprecedented advantages in detecting their concentrations and studying their biological functions. As a matter of fact, these three kinds of biothiols are generated in the process of biosynthesis in vivo. It is of great significance to understand their biosynthetic pathways and elucidate their synthetic relationships. In this work, to α,ß-unsaturated ketones conjugated ethylenediamine coumarin and pyrandione was introduced boron fluoride and, through its strong electron deficiency effect, afforded a molecule having near-infrared emission and regulated the rigidity of molecules. At the same time, the conjugated double bond is used to respond to molecular rigidity. The rapid response of the probe to biothiols and the slow dissociation aggregation of the probe itself through the response environment could monitor the absence of biothiols in cells. In addition, based on the difference in sensitivity of response of Cys and GSH to the probe, this work studied the interaction between biosynthetic pathways of Cys and GSH in cells through enzyme inhibition for the first time. The relationship of restriction and regulation of biosynthesis in vivo was revealed.

HSA-Lys-161 covalent bound fluorescent dye for in vivo blood drug dynamic imaging and tumor mapping.

The specific combination of human serum albumin and fluorescent dye will endow superior performance to a coupled fluorescent platform for in vivo fluorescence labeling. In this study, we found that lysine-161 in human serum albumin is a covalent binding site and could spontaneously bind a ketone skeleton quinoxaline-coumarin fluorescent dye with a specific turn-on fluorescence signal for the first time. Supported by the abundant drug binding domains in human serum albumin, drugs such as ibuprofen, warfarin and clopidogrel could interact with the fluorescent dye labeled human serum albumin to feature a substantial enhancement in fluorescence intensity (6.6-fold for ibuprofen, 4.5-fold for warfarin and 5-fold for clopidogrel). The drug concentration dependent fluorescence intensity amplification realized real-time, in situ blood drug concentration monitoring in mice, utilizing ibuprofen as a model drug. The non-invasive method avoided continuous blood sample collection, which fundamentally causes suffering and consumption of experimental animals in the study of pharmacokinetics. At the same time, the coupled fluorescent probe can be efficiently enriched in tumors in mice which could map a tumor with a high-contrast red fluorescence signal and could hold great potential in clinical tumor marking and surgical resection.

Thiol "Click" Chromene Ring Opening and Subsequent Cascade Nucleophilic Cyclization NIR Fluorescence Imaging Reveal High Levels of Thiol in Drug-Resistant Cells.

As the structural unit of natural products, chromene derivatives show a wide range of biological activity and pharmacological activity due to their unique photophysical and chemical properties. Ten years ago, our research group discovered the "thiol-chromene" click reaction, which achieved the selective detection of thiols through the change of the optical spectrum. Afterward, we attempted to develop various chromene-based fluorescent probes for imaging including near-infrared (NIR) probe, ratiometric probe, and multifunctional probe. However, how to integrate the fluorophore and reaction sites into the chromene-based skeleton remains challenging. In this work, we connected the chromene motif with the NIR fluorophore methylene blue utilizing a carbamate spacer to provide a new fluorescent probe (CM-NIR), which is triggered by thiols to open the pyran ring followed by attacking the carbamate by phenolate to releases the methylene blue. This novel cascade mechanism avoids the formation of para-quinone methides, which proved to be toxic to normal cells. CM-NIR also showed the specific imaging of thiols in living cells and mice. More importantly, the thiols level in drug-resistant cancer cells was found to be significantly higher than that in the corresponding cancer cell, which indicated that the thiols level may have an important role in cancer cells developing drug resistance.

Specific Fluorescent Probe Based on "Protect-Deprotect" To Visualize the Norepinephrine Signaling Pathway and Drug Intervention Tracers.

In recent years, increased social pressure and other factors have led to a surge in the number of people suffering from depression: studies show that quite a few people will experience major depression in their lifetime. Currently, it is widely believed that the internal cause of major depression is reduced levels of norepinephrine (NE) in brain tissue. Norepinephrine is very similar in structure and chemical properties to the other two catecholamine neurotransmitters, epinephrine (EP) and dopamine (DA). These three neurotransmitters are synthesized sequentially through enzymatic reactions in the biological system. Therefore, design of a norepinephrine-specific fluorescent probe is very challenging. In this work, we utilized a "protect-deprotect" strategy: longer emission wavelength cyanine containing water-soluble sulfonate was protected by a carbonic ester linking departing group thiophenol; the ß-hydroxy ethyl amine moiety of norepinephrine may react with the carbonic ester via nucleophilic substitution and intramolecular nucleophilic cyclization to release the fluorophore. The process realized the specific red fluorescence detection of norepinephrine. Imaging of the norepinephrine nerve signal transduction stimulated by potassium ion was studied. More importantly, real-time fluorescence imaging of norepinephrine levels in the brain of rats stimulated by antidepressant drugs was studied for the first time.

A practical pH-compatible fluorescent sensor for hydrazine in soil, water and living cells.

The excellent water solubility of hydrazine (N2H4) allows it to easily invade the human body through the skin and respiratory tract, thereby damaging human organs and the central nervous system. To realize the monitoring of N2H4 effectively, first, coumarin was used to construct an inner alicyclic ring as the reaction site, extending the conjugation and strengthening the rigidity of the probe Co-Hy to improve its luminescence performance and enhance its ability to resist acids and alkalis. Second, we introduced a carboxyl group at the ortho position of the inner alicyclic ring to improve the water solubility of Co-Hy, and its strong electron pulling effect increased the activity of the reaction site. Spectroscopy experiments showed that Co-Hy featured excellent water solubility, high pH resistance (pH 4-11), excellent selectivity, fast analysis speed (within 5 minutes), and a low detection limit toward N2H4 (69 nM, 2.2 ppb). In addition, test-strip, spray, and cell-imaging experiments confirmed the outstanding application potential of Co-Hy for convenient N2H4 analysis in a variety of environments.

A review: Red/near-infrared (NIR) fluorescent probes based on nucleophilic reactions of H2 S since 2015.

The topics of human health and disease are always the focus of much attention. Hydrogen sulfide (H2 S), as a double-edged sword, plays an important role in biological systems. Studies have revealed that endogenous H2 S is important to maintain normal physiological functions. Conversely, abnormal levels of H2 S may contribute to various diseases. Due to the importance of H2 S in physiology and pathology, research into the effects of H2 S has been active in recent years. Fluorescent probes with red/near-infrared (NIR) emissions (620-900 nm) are more suitable for imaging applications in vivo, because of their negligible photodamage, deep tissue penetration, and maximum lack of interference from background autofluorescence. H2 S, an 'evil and positive' molecule, is not only toxic, but also produces significant effects; a 'greedy' molecule, is not only a strong nucleophile under physiological conditions, but also undergoes a continuous double nucleophilic reaction. Therefore, in this tutorial review, we will highlight recent advances made since 2015 in the development and application of red/NIR fluorescent probes based on nucleophilic reactions of H2 S.

Mutual correlation evaluation of Cys and Hcy in serum through reaction activity regulated fluorescence quantification.

We report herein a strategy involving using a single fluorescent probe with the assistance of two pH conditions to simultaneously detect cysteine and homocysteine quantitatively. The concentrations of Cys and Hcy in human serum samples were found to display a positive correlation, which might promote a further understanding of thiol-related diseases.

Fluorescent Imaging of Resveratrol Induced Subcellular Cysteine Up-Regulation.

Even through many physiological pathways of resveratrol have been established since its association with the "French paradox" in 1992, its exact pathological roles are still ambiguous and disputable. To understand how resveratrol exerts its effects would not only enlighten the pathogenesis study of related diseases, but also promote the development of more potent molecules. Focusing on the reported cellular directly and indirectly ROS scavenging processes of resveratrol, we evaluated the activation effect of the inherent antioxidation thiols system in subcellular level by two cysteine (Cys) specific fluorescent probes mitochondria targetable Mito-1 and lysosomes targetable Ly-1. We found that 50 µM resveratrol treatment could induce distinct Cys up-regulation in both mitochondria and lysosomes which might be a general biological phenomenon in various cell lines for the first time.

Heat Stroke in Cell Tissues Related to Sulfur Dioxide Level Is Precisely Monitored by Light-Controlled Fluorescent Probes.

Heat stroke (HS) can cause serious organism damage or even death. Early understanding of the mechanism of heat cytotoxicity can prevent or treat heat stroke related diseases. In this work, probe Ly-NT-SP was synthesized, characterized, and used for sulfur dioxide (SO2) detection in lysosomes. PBS solutions of probe Ly-NT-SP at pH 5.0 present a marked broad emission band in the green zone (535 nm). After UV irradiation, the spiropyran group in Ly-NT-SP isomerizes to the merocyanine form (Ly-NT-MR), which presented a weak red-shifted emission at 630 nm. In addition, photocontrolled isomerization of Ly-NT-SP to Ly-NT-MR generated a C═C-C═N+ fragment able to react, through a Michael addition, with SO2 to yield a highly emissive adduct with a marked fluorescence in the green channel (535 nm). In vitro studies showed a remarkable selectivity of photoactivated Ly-NT-MR to SO2 with a limit of detection as low as 4.7 µM. MTT viability assays demonstrated that the Ly-NT-SP is nontoxic to HeLa cells and can be used to detect SO2 in lysosomes. Taking advantage of this, the sensor is successfully applied to image increasing SO2 values in lysosomes during heat shock for the first time. Moreover, we also confirmed that the increased SO2 can protect the small intestine against damage induced by heat shock through regulating oxidative stress in cells and mice.

The chronological evolution of small organic molecular fluorescent probes for thiols.

Abnormal concentrations of biothiols such as cysteine, homocysteine and glutathione are associated with various major diseases. In biological systems, the structural similarity and functional distinction of these three small molecular thiols has not only required rigorous molecular design of the fluorescent probes used to detect each thiol specifically, but it has also inspired scientists to uncover the ambiguous biological relationships between these bio-thiols. In this minireview, we will discuss the evolution of small organic molecular fluorescent probes for the detection of thiols over the past 60 years, highlighting the potent methodologies used in the design of thiol probes and their particular applications in the semi-quantification of cellular thiols and real-time labelling. At the same time, the present challenges that limit their further application will be discussed. We hope that this minireview will promote future research to enable deeper insight into the crucial role of thiols in biological systems.