Design and synthesis of large Stokes shift DNA dyes with reduced genotoxicity.

Despite intensive search over the past decades, only a few small-molecule DNA fluorescent dyes were found with large Stokes shifts. These molecules, however, are often too toxic for widespread usage. Here, we designed DNA-specific fluorescent dyes rooted in benzimidazole architectures with a hitherto unexplored molecular framework based on thiazole-benzimidazole scaffolding. We further incorporated a pyrazole ring with an extended sidechain to prevent cell penetration. These novel benzimidazole derivatives were predicted by quantum calculations and subsequently validated to have large Stokes shifts ranging from 135 to 143 nm, with their emission colors changed from capri blue for the Hoechst reference compound to iguana green. These readily-synthesized compounds, which displayed improved DNA staining intensity and detection limits along with a complete loss of capability for cellular membrane permeation and negligible mutagenic effects as designed, offer a safer alternative to the existing high-performance small-molecule DNA fluorescent dyes.

A Reaction-based Ratiometric Fluorescent Probe with Large STOKES Shift for Cu2+ in Neat Aqueous Solution.

A novel reaction-based ratiometric fluorescent probe 1 for Cu2+ using picolinate as the reaction site and hemicyanine as the fluorophore was developed. 1 displayed maximum absorption peak at 355 nm and fluorescence emission peak at 500 nm, with large Stokes shift of 145 nm. Upon reaction with Cu2+, the maximum absorption and fluorescence emission peaks red-shifted to 390 nm and 570 nm respectively, owing to Cu2+-induced hydrolysis of the picolinate moiety in 1. Meanwhile, the solution of 1 turned from green to orange under a 365 nm UV lamp. 1 not only could detect Cu2+ ratiometrically by the ratios of both absorbance (A390 nm/A355 nm) and fluorescence intensity (F570 nm/F500 nm), but also displayed large Stokes shift, fast response, high sensitivity and excellent selectivity over other metal ions in neat aqueous solution.

A dual-function fluorescence 'turn-on' probe that allows Zn (II) bioimaging and quantification of water in the organic solvent.

Herein, a Eugenol-derived fluorescence 'turn-on' probe FLHE was synthesized by condensing 2-((3-(trifluoromethyl)phenyl)amino)benzohydrazide with 5-allyl-2-hydroxy-3-methoxybenzaldehyde. FLHE demonstrated very low fluorescence in the studied organic solvents of varying polarities. However, upon titration with Zn2+ in HEPES buffer (pH = 7.4, 50% ACN, v/v), FLHE showed 40-fold higher fluorescence signals indicating the formation of the FLHE-Zn2+ complex. The fluorescence turn-on phenomenon upon FLHE-Zn2+ complex formation results from a chelation-enhanced fluorescence (CHEF) effect. The FLHE-Zn2+ complexation demonstrated a stokes shift of 156 nm (λex = 350 nm, λem = 506 nm) and an about 33-fold increase in the quantum yield (FLHE, Φ = 0.007; FLHE-Zn2+ complex, Φ = 0.23). The binding constant (Ka) determined by the Benesi-Hildebrand plot for interaction between FLHE and Zn2+ was 5.33 × 103 M-1. FLHE demonstrated a LOD of 31.8 nM for detecting Zn2+ in the environmental samples without interference from other cations and anions. FLHE-based paper strip (FLHE-PS) assay was developed to quantify the Zn2+ ions in water and the water content of organic solvent. FLHE-PS allows the detection of Zn2+ in aqueous solutions with a LOD of 63.2 nM and quantifying water in acetonitrile with a LOD of 0.14%. These results indicate that the FLHE has high applicability for detecting Zn2+ in living cells and environmental samples and detecting the presence of water in the organic solvents.

Golgi-targeted NIR fluorescent probe with large stokes shift for real-time monitoring of nitric oxide in depression model.

Depression is a debilitating mental illness that poses a serious threat to human health. Nitric Oxide (NO), as an important gasotransmitter, is closely associated with the pathogenesis of depressive disorders. Effective monitoring of NO fluctuation is beneficial for the diagnosis of depression and therapy assessment of antidepressants. Currently, there is a lack of effective methods for rapidly and sensitively identifying NO and elucidating its relationship with depression diseases. Herein, we developed a NIR dye TJ730-based fluorescent probe TJ730-Golgi-NO incorporating benzenesulfonamide as a Golgi-targeted moiety and the thiosemicarbazide group for NO detection. The probe exhibited turn-on fluorescence ability and a large Stokes shift of 158 nm, which shows high sensitivity, selectivity, and rapid response (<1 min) for NO detection. TJ730-Golgi-NO could detect exogenous and endogenous NO in cells stimulated by Glu and LPS, and target Golgi apparatus. Moreover, we disclose a significant increase of NO in the depression model and a weak fluorescence evidenced in the fluoxetine-treated depression mice. This study provides a competent tool for studying the function of NO and helping improve the effective treatment of depression diseases.

A Red-Emission Fluorescent Probe with Large Stokes Shift for Detection of Viscosity in Living Cells and Tumor-Bearing Mice.

Abnormal viscosity is closely related to the occurrence of many diseases, such as cancer. Therefore, real-time detection of changes in viscosity in living cells is of great importance. Fluorescent molecular rotors play a critical role in detecting changes in cellular viscosity. Developing red emission viscosity probes with large Stokes shifts and high sensitivity and specificity remains an urgent and important topic. Herein, a novel viscosity-sensitive fluorescent probe (TCF-VIS1) with a large stokes shift and red emission was prepared based on the 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) skeleton. Due to intramolecular rotation, the probe itself does not fluorescence at low viscosity. With the increase in viscosity, the rotation of TCF-VIS1 is limited, and its fluorescence is obviously enhanced. The probe has the advantages of simple preparation, large Stokes shift, good sensitivity and selectivity, and low cytotoxicity, which make it successfully used for viscosity detection in living cells. Moreover, TCF-VIS1 showed its potential for cancer diagnosis at the cell level and in tumor-bearing mice by detecting viscosity. Therefore, the probe is expected to enrich strategies for the detection of viscosity in biological systems and offer a potential tool for cancer diagnosis.

A Coumarin-Hemicyanine Deep Red Dye with a Large Stokes Shift for the Fluorescence Detection and Naked-Eye Recognition of Cyanide.

In this study, we synthesized a coumarin-hemicyanine-based deep red fluorescent dye that exhibits an intramolecular charge transfer (ICT). The probe had a large Stokes shift of 287 nm and a large molar absorption coefficient (ε = 7.5 × 105 L·mol-1·cm-1) and is best described as a deep red luminescent fluorescent probe with λem = 667 nm. The color of probe W changed significantly when it encountered cyanide ions (CN-). The absorption peak (585 nm) decreased gradually, and the absorption peak (428 nm) increased gradually, so that cyanide (CN-) could be identified by the naked eye. Moreover, an obvious fluorescence change was evident before and after the reaction under irradiation using 365 nm UV light. The maximum emission peak (667 nm) decreased gradually, whilst the emission peak (495 nm) increased gradually, which allowed for the proportional fluorescence detection of cyanide (CN-). Using fluorescence spectrometry, the fluorescent probe W could linearly detect CN- over the concentration range of 1-9 µM (R2 = 9913, RSD = 0.534) with a detection limit of 0.24 µM. Using UV-Vis spectrophotometry, the linear detection range for CN- was found to be 1-27 µM (R2 = 0.99583, RSD = 0.675) with a detection limit of 0.13 µM. The sensing mechanism was confirmed by 1H NMR spectroscopic titrations, 13C NMR spectroscopy, X-ray crystallographic analysis and HRMS. The recognition and detection of CN- by probe W was characterized by a rapid response, high selectivity, and high sensitivity. Therefore, this probe provides a convenient, effective and economical method for synthesizing and detecting cyanide efficiently and sensitively.

Organic Fluorophores with Large Stokes Shift for the Visualization of Rapid Protein and Nucleic Acid Assays.

Organic small-molecule fluorophores, characterized by flexible chemical structure and adjustable optical performance, have shown tremendous potential in biosensing. However, classical organic fluorophore motifs feature large overlap between excitation and emission spectra, leading to the requirement of advanced optical set up to filter desired signal, which limits their application in scenarios with simple settings. Here, a series of wavelength-tunable small-molecule fluorescent dyes (PTs) bearing simple organic moieties have been developed, which exhibit Stokes shift up to 262 nm, molar extinction coefficients ranged 30,000-100,000 M-1 cm-1, with quantum yields up to 54.8 %. Furthermore, these dyes were formulated into fluorescent nanoparticles (PT-NPs), and applied in lateral flow assay (LFA). Consequently, limit of detection for SARS-CoV-2 nucleocapsid protein reached 20 fM with naked eye, a 100-fold improvement in sensitivity compared to the pM detection level for colloidal gold-based LFA. Besides, combined with loop-mediated isothermal amplification (LAMP), the LFA system achieved the visualization of single copy level nucleic acid detection for monkeypox (Mpox).

A Reaction-Based ESIPT Fluorescent Probe for the Detection of Hg2+ with Large Stokes Shift.

A novel reaction-based fluorescent probe 1 for Hg2+ was designed and synthesized. 1 was almost nonfluoresent due to inhibition of the ESIPT process between hydroxy group and imid carbonyl oxygen by diphenylphosphinothioate group. After reacting with Hg2+, the fluorescence intensity of 1 exhibited significant enhancement owing to recovery of the ESIPT process via Hg2+-promoted desulfurization-hydrolysis of the diphenylphosphinothioate moiety and cleavage of the P-O bond. 1 not only showed rapid response, high sensitivity, excellent selectivity for Hg2+ over other metal ions, but also could detect Hg2+ with large Stokes shift (165 nm), which was attributed to the ESIPT process. Moreover, the reaction mechanism was fully validated by absorption spectra, fluorescence spectra, fluorescence color as well as ESI-MS analysis. 1 is the reaction-based ESIPT fluorescent probe for the detection of Hg2+ with large Stokes shift, rapid response, high sensitivity and selectivity.

A near-infrared fluorescent probe with remarkably large stokes shift for specifical imaging of peroxynitrite fluctuations in Hela cells.

Peroxynitrite (ONOO-), an endogenous reactive nitrogen species, plays an important role in maintaining intracellular homeostasis. Abnormal levels of ONOO- in cells could cause protein oxidation which is confirmed that related with Alzheimer's diseases, so accurate monitoring of ONOO- in cells is crucial. Herein, a novel fluorescent probe (XPC) based on dicyanomethylene-4H-benzothiopyran was developed by regulating its intramolecular charge transfer (ICT) effect to detect ONOO-. Once reaction with ONOO-, the fluorescence of XPC was turned on and the emission wavelength could reach up to 750 nm. Furthermore, XPC exhibited satisfactory performances for ONOO- such as large Stokes shift (200 nm), good sensitivity (Limit of detection = 13 nM), high selectivity to ONOO- over other a reactive nitrogen species (RNS)/reactive oxygen species (ROS). More importantly, XPC was successfully applied for monitoring the fluctuations of ONOO- in living cells.

γ-Glutamyltranspeptidase-Activated Near-Infrared fluorescent probe for visualization of Drug-Induced liver injury.

Drug-induced liver injury (DILI), induced by overdose or chronic administration of drugs, has become the leading cause of acute liver failure. Therefore, an accurate diagnostic method for DILI is critical to improve treatment efficiency. The production of γ-glutamyltranspeptidase (GGT) is closely related to the progression of drug-induced hepatotoxicity. KL-Glu exhibits a prominent GGT-activated NIR fluorescence (734 nm) with a large Stokes shift (137 nm) and good sensitivity/selectivity, making it favorable for real-time detection of endogenous GGT activity. Using this probe, we evaluated the GGT up-regulation under the acetaminophen-induced liver injury model. Moreover, KL-Glu was successfully used to assess liver injury induced by the natural active ingredient triptolide and the effective amelioration upon treatment with N-acetyl cysteine (NAC) or Glutathione (GSH) in cells and in vivo by fluorescent trapping the fluctuation of GGT for the first time. Therefore, the fluorescent probe KL-Glu can be used as a potential tool to explore the function of GGT in the progression of DILI and for the early diagnosis and prognostic evaluation of DILI.

Development of europium(III) complex fluorescent probe for hydrogen sulfide detection and its application in water samples.

Hydrogen sulfide (H2 S) is a crucial endogenous signaling component in organisms that is involved in redox homeostasis and numerous biological processes. Modern medical research has confirmed that hydrogen sulfide plays an important role in the pathogenesis of many diseases. Herein, a fluorescent probe Eu(ttbd)3 abt based on europium(III) complex was designed and synthesized for the detection of H2 S. Eu(ttbd)3 abt exhibited significant quenching for H2 S at long emission wavelength (625 nm), with rapid detection ability (less than 2 min), high sensitivity [limit of detection (LOD) = 0.41 µM], and massive Stokes shift (300 nm). Additionally, this probe showed superior selectivity for H2 S despite the presence of other possible interference species such as biothiols. Furthermore, the probe Eu(ttbd)3 abt was successfully applied to detect H2 S in water samples.

A Turn-On Lipid Droplet-Targeted Near-Infrared Fluorescent Probe with a Large Stokes Shift for Detection of Intracellular Carboxylesterases and Cell Viability Imaging.

Carboxylesterases (CEs) play important physiological roles in the human body and are involved in numerous cellular processes. Monitoring CEs activity has great potential for the rapid diagnosis of malignant tumors and multiple diseases. Herein, we developed a new phenazine-based "turn-on" fluorescent probe DBPpys by introducing 4-bromomethyl-phenyl acetate to DBPpy, which can selectively detect CEs with a low detection limit (9.38 × 10-5 U/mL) and a large Stokes shift (more than 250 nm) in vitro. In addition, DBPpys can also be converted into DBPpy by carboxylesterase in HeLa cells and localized in lipid droplets (LDs), emitting bright near-infrared fluorescence under the irradiation of white light. Moreover, we achieved the detection of cell health status by measuring the intensity of NIR fluorescence after co-incubation of DBPpys with H2O2-pretreated HeLa cells, indicating that DBPpys has great potential applications for assessing CEs activity and cellular health.

Organic Anion Transporting Polypeptide 3A1 (OATP3A1)-Gated Bio-Orthogonal Labeling of Intracellular Proteins.

Organic anion transporting polypeptides (OATPs) were found to readily deliver membrane impermeable, tetrazine bearing fluorescent probes into cells. This feature was explored in OATP3A1 conditioned bio-orthogonal labeling schemes of various intracellular proteins in live cells. Confocal microscopy and super-resolution microscopy (STED) studies have shown that highly specific and efficient staining of the selected intracellular proteins can be achieved with the otherwise non-permeable probes when OATP3A1 is present in the cell membrane of cells. Such a transport protein linked bio-orthogonal labeling scheme is believed to be useful in OATP3A1 activity-controlled protein expression studies in the future.

Mapping the Complete Photocycle that Powers a Large Stokes Shift Red Fluorescent Protein.

Large Stokes shift (LSS) red fluorescent proteins (RFPs) are highly desirable for bioimaging advances. The RFP mKeima, with coexisting cis- and trans-isomers, holds significance as an archetypal system for LSS emission due to excited-state proton transfer (ESPT), yet the mechanisms remain elusive. We implemented femtosecond stimulated Raman spectroscopy (FSRS) and various time-resolved electronic spectroscopies, aided by quantum calculations, to dissect the cis- and trans-mKeima photocycle from ESPT, isomerization, to ground-state proton transfer in solution. This work manifests the power of FSRS with global analysis to resolve Raman fingerprints of intermediate states. Importantly, the deprotonated trans-isomer governs LSS emission at 620 nm, while the deprotonated cis-isomer's 520 nm emission is weak due to an ultrafast cis-to-trans isomerization. Complementary spectroscopic techniques as a table-top toolset are thus essential to study photochemistry in physiological environments.

A fluorescent probe for monitoring sulfite in living cells with large Stokes shift and rapid response.

Sulfite (SO32-) is considered as a monitor of a wide range of physiological processes. However, cells and tissues are adversely affected when the body ingests high level of sulfite. Here, we designed and synthesized a "turn on" fluorescent probe ImiSft-1 with 2-cyano-N-methylacetamide as the specific recognition site of SO32-. This probe predominantly achieved high response intensity to SO32- and desirable properties such as large Stokes shift (∼180 nm), fast response time (within 15 s), and high sensitivity (LOD = 0.12 µM). Importantly, the probe was highly selective for sulfite from other bio-species including biological thiols. Other functional properties included broad pH adaptability (5.0-10.0) and low cytotoxicity. Given these advantages and the fluorescence imaging in living MCF-7 cells, it was demonstrated that probe ImiSft-1 could monitor the changes of sulfite concentration in living cells.

A NIR fluorescence probe for monitoring Cys upregulation induced by balsam pear polysaccharide and imaging in zebrafish.

In this work, we introduced the acrylate recognition group into dicyanoisophorone derivative DCI-C-OH to construct the NIR fluorescent probe DCI-C-Cys with a large Stokes shift (240 nm). DCI-C-Cys could specifically respond to Cys, resulting in a 22-fold increase in fluorescence intensity at 702 nm. Meanwhile, the probe has the advantages of good water solubility, high sensitivity (93 nM), and excellent biocompatibility. Moreover, DCI-C-Cys successfully monitored endogenous and exogenous Cys in HepG2 cells and zebrafish. Most importantly, we found that balsam pear polysaccharide could lead to the increase of intracellular Cys levels, which might be conducive to the further study of the antioxidant mechanism of balsam pear polysaccharide.

LSSmScarlet2 and LSSmScarlet3, Chemically Stable Genetically Encoded Red Fluorescent Proteins with a Large Stokes' Shift.

Red fluorescent proteins with a large Stokes' shift (LSSRFPs) are genetically encoded and efficiently excited by 488 nm light, allowing simultaneous dual-color one- and two-photon fluorescence imaging and fluorescence correlation spectroscopy in combination with green fluorescent proteins FPs. Recently, based on the conventional bright mScarlet RFP, we developed the LSSRFP LSSmScarlet. LSSmScarlet is characterized by two pKa values at pH values of 1.9 and 5.8. In this study, we developed improved versions of LSSmScarlet, named LSSmScarlet2 and LSSmScarlet3, which are characterized by a Stokes' shift of 128 nm and extreme pH stability with a single pKa value of 2.2. LSSmScarlet2 and LSSmScarlet3 had 1.8-fold faster and 3-fold slower maturation than LSSmScarlet, respectively. In addition, both LSSRFPs were 1.5- to 1.6-fold more photostable and more chemically resistant to denaturation by guanidinium chloride and guanidinium thiocyanate. We also compared the susceptibility of the LSSmScarlet2, LSSmScarlet3, and other LSSRFPs to the reagents used for whole-mount imaging, expansion microscopy, and immunostaining techniques. Due to higher pH stability and faster maturation, the LSSmScarlet3-LAMP3 fusion was 2.2-fold brighter than LSSmScarlet-LAMP3 in lysosomes of mammalian cells. The LSSmScarlet3-hLAMP2A fusion was similar in brightness to LSSmScarlet-hLAMP2A in lysosomes. We successfully applied the monomeric LSSmScarlet2 and LSSmScarlet3 proteins for confocal imaging of structural proteins in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet2 protein at a resolution of 1.41 Å. Site-directed mutagenesis of the LSSmScarlet2 protein demonstrated the key role of the T74 residue in improving the pH and chemical stability of the LSSmScarlet2 protein.

Imidazo[1,5-a]pyridine-Based Fluorescent Probes: A Photophysical Investigation in Liposome Models.

Imidazo[1,5-a]pyridine is a stable scaffold, widely used for the development of emissive compounds in many application fields (e.g., optoelectronics, coordination chemistry, sensors, chemical biology). Their compact shape along with remarkable photophysical properties make them suitable candidates as cell membrane probes. The study of the membrane dynamics, hydration, and fluidity is of importance to monitor the cellular health and to explore crucial biochemical pathways. In this context, five imidazo[1,5-a]pyridine-based fluorophores were synthesized according to a one-pot cyclization between an aromatic ketone and benzaldehyde in the presence of ammonium acetate and acetic acid. The photophysical features of prepared compounds were investigated in several organic solvents and probes 2-4 exhibited the greatest solvatochromic behavior, resulting in a higher suitability as membrane probes. Their interaction with liposomes as artificial membrane model was tested showing a successful intercalation of the probes in the lipid bilayer. Kinetic experiments were carried out and the lipidic phase influence on the photophysical features was evaluated through temperature-dependent experiments. The results herein reported encourage further investigations on the use of imidazo[1,5-a]pyridine scaffold as fluorescent membrane probes.

Discovery of an Ultra-rapid and Sensitive Lysosomal Fluorescence Lipophagy Process.

Non-invasive dynamic tracking of lysosomes and their interactions with other organelles is important for the study of lysosomal function and related diseases. However, many fluorescent dyes developed so far to target lysosomes cannot be used to monitor these processes due to the high concentrations required for imaging, long cell penetration times, and non-ideal photostability. In this regard, we synthesized three lysosomal targeting probes with large Stokes shifts, good stability, and high brightness. The Q-P-ARh dye, developed by us for the first time, can stain lysosomes at ultra-low concentrations (1.0 nM) without affecting the physiological functions of the lysosomes. More importantly, its excellent anti-interference ability and ultrafast lysosomal staining ability (within 1.0 min) clearly monitored the entire dynamic process of lipophagy. Ultimately, this method can greatly contribute to the study of autophagy pathways. This novel fluorescence platform shows great promise for the development of biological probes for application in pathological environments.

In-vitro and in-vivo monitoring of gold(III) ions from intermediate metabolite of sodium aurothiomalate through water-soluble ruthenium (II) complex-based luminescent probe.

Real-time monitoring of drug metabolism in vivo is of great significance to drug development and toxicology research. The purpose of this study is to establish a rapid and visual in vivo detection method for the detection of an intermediate metabolite of the gold (I) drug. Gold (I) drugs such as sodium aurothiomalate (AuTM) have anti-inflammatory effects in the treatment of rheumatoid arthritis. Gold(III) ions (Au3+) are the intermediate metabolite of gold medicine, and they are also the leading factor of side effects in the treatment of patients. However, the rapid reduction of Au3+ to Au+ by thiol proteins in organisms limits the in-depth study of metabolism of gold drugs in vivo. Here we describe a luminescence Au3+ probe (RA) based on ruthenium (II) complex for detecting Au3+ in vitro and in vivo. RA with large Stokes shift, good water solubility and biocompatibility was successfully applied to detect Au3+ in living cells and vivo by luminescence imaging, and to trap the fluctuation of Au3+ level produced by gold (I) medicine. More importantly, the luminescent probe was used to the detection of the intermediate metabolites of gold (I) drugs for the first time. Overall, this work offers a new detection tool/method for a deeper study of gold (I) drugs metabolite.