A Water-Soluble Multifunctional Probe for Colorimetric Copper Sensing, Lysosome Labelling and Live-Cell Imaging.

We report a water-soluble fluorescence and colorimetric copper probe (LysoBC1); this system can also serve for lysosome labeling and for the dynamic tracking of Cu2+ in living cells. The sensing mechanism takes advantage of the synergic action by the following three components: i) a lysosome targeting unit, ii) the spirolactam ring-opening for the selective copper chelation and iii) the metal-mediated hydrolysis of the rhodamine moiety for fluorescence enhancement. In aqueous environment the molecule acts as a fluorescent reversible pH sensor and as colorimetric probe for Cu2+ at physiological pH; the hydrolysis of the copper targeting unit resulted in a 50-fold increase of the fluorescence intensity. Most importantly, in vitro cell analyses in undifferentiated (SH SY5Y) and differentiated (d-SH SY5Y) neuroblastoma cells, LysoBC1 is able to selectively accumulate into lysosome while the copper binding ability allowed us to monitor intracellular copper accumulation into lysosome.

Hemicyanine-Based Highly Water-Soluble Probe for Extracellular Nitroreductase.

Nitroreductase (NTR) has long been a target of interest for its important role involved in the nitro compounds metabolism. Various probes have been reported for NTR analysis, but rarely able to distinguish the extracellular NTR from intracellular ones. Herein we reported a new NTR sensor, HCyS-NO2, which was a hemicyanine molecule with one nitro and two sulfo groups attached. The nitro group acted as the reporting group to respond NTR reduction. Direct linkage of nitro group into the hemicyanine π conjugate system facilitated the intramolecular electron transfer (IET) process and thus quenched the fluorescence of hemicyanine core. Upon reduction with NTR, the nitro group was rapidly converted into the hydroxylamino and then the amino group, eliminating IET process and thus restoring the fluorescence. The sulfo groups installed significantly increased the hydrophilicity of the molecule, and introduced negative charges at physiological pH, preventing the diffusion into bacteria. Both gram-negative and gram-positive bacteria were able to turn on the fluorescence of HCyS-NO2, without detectable diffusion into cells, providing a useful tool to probe the extracellular reduction process.

Dual-Emissive Iridium(III) Complexes and Their Applications in Biological Sensing and Imaging.

Phosphorescent iridium(III) complexes are widely recognized for their unique properties in the excited triplet state, making them crucial for various applications including biological sensing and imaging. Most of these complexes display single phosphorescence emission from the lowest-lying triplet state after undergoing highly efficient intersystem crossing (ISC) and ultrafast internal conversion (IC) processes. However, in cases where these excited-state processes are restricted, the less common phenomenon of dual emission has been observed. This dual emission phenomenon presents an opportunity for developing biological probes and imaging agents with multiple emission bands of different wavelengths. Compared to intensity-based biosensing, where the existence and concentration of an analyte are indicated by the brightness of the probe, the emission profile response involves modifications in emission color. This enables quantification by utilizing the intensity ratio of different wavelengths, which is self-calibrating and unaffected by the probe concentration and excitation laser power. Moreover, dual-emissive probes have the potential to demonstrate distinct responses to multiple analytes at separate wavelengths, providing orthogonal detection capabilities. In this concept, we focus on iridium(III) complexes displaying fluorescence-phosphorescence or phosphorescence-phosphorescence dual emission, along with their applications as biological probes for sensing and imaging.

Bora-Diaza-Indacene Based Fluorescent Probes for Simultaneous Visualisation of Lipid Droplets and Endoplasmic Reticulum.

Organelle selective fluorescent probes, especially those capable of concurrent detection of specific organelles, are of benefit to the research community in delineating the interplay between various organelles and the impact of such interaction in maintaining cellular homeostasis and its disruption in the diseased state. Although very useful, such probes are synthetically challenging to design due to the stringent lipophilicity requirement posed by different organelles, and hence, the lack of such probes being reported so far. This work details the synthesis, photophysical properties, and cellular imaging studies of two bora-diaza-indacene based fluorescent probes that can specifically and simultaneously visualise lipid droplets and endoplasmic reticulum; two organelles suggested having close interactions and implicated in stress-induced cellular dysfunction and disease progression.

Synthesis of New Bodipy Hydrazide Fluorescent Probes for the Detection of Carbonylated Proteins Generated by Oxidative Stress.

Oxidative stress is a cellular disorder implicated in various severe diseases and redox biology and represents an important field of research for the last decades. One of the major consequences of oxidative stress is the carbonylation of proteins, which is also a reliable marker to assess protein oxidative modifications. Accumulation of carbonylated proteins has been associated with aging and age-related diseases and can ultimately causes cell death. Detection of these oxidative modifications is essential to understand and discover new treatments against oxidative stress. We describe the design and the synthetic pathway of new BODIPY fluorescent probes functionalized with hydrazide function for protein carbonyl labeling to improve existing methodologies such as 2D-Oxi electrophoresis. Hydrazide BODIPY analogues show very good fluorescent properties such as NIR emission up to 633 nm and quantum yield up to 0.88. These new probes were validated for the detection and quantification of carbonylated proteins with 2D-Oxi electrophoresis using mouse muscle protein extracts, as well as both flow cytometry and microscopy using oxidant stressed C2 C12 cells.

Surface-engineered erythrocyte membrane-camouflage fluorescent bioprobe for precision ovarian cancer surgery.

PURPOSE: Fluorescence imaging-guided surgery has been used in oncology. However, for tiny tumors, the current imaging probes are still difficult to achieve high-contrast imaging, leading to incomplete resection. In this study, we achieved precise surgical resection of tiny metastatic cancers by constructing an engineering erythrocyte membrane-camouflaged bioprobe (AR-M@HMSN@P). METHODS: AR-M@HMSN@P combined the properties of aggregation-induced emission luminogens (AIEgens) named PF3-PPh3 (P), with functional erythrocyte membrane modified by a modular peptide (AR). Interestingly, AR was composed of an asymmetric tripodal pentapeptide scaffold (GGKGG) with three appended modulars: KPSSPPEE (A6) peptide, RRRR (R4) peptide and cholesterol. To verify the specificity of the probe in vitro, SKOV3 cells with overexpression of CD44 were used as the positive group, and HLF cells with low expression of CD44 were devoted as the control group. The AR-M@HMSN@P fluorescence imaging was utilized to provide surgical guidance for the removal of micro-metastatic lesions. RESULTS: In vivo, the clearance of AR-M@HMSN@P by the immune system was reduced due to the natural properties inherited from erythrocytes. Meanwhile, the A6 peptide on AR-M@HMSN@P was able to specifically target CD44 on ovarian cancer cells, and the electrostatic attraction between the R4 peptide and the cell membrane enhanced the firmness of this targeting. Benefiting from these multiple effects, AR-M@HMSN@P achieved ultra-precise tumor imaging with a signal-to-noise ratio (SNR) of 15.2, making it possible to surgical resection of tumors < 1 mm by imaging guidance. CONCLUSION: We have successfully designed an engineered fluorescent imaging bioprobe (AR-M@HMSN@P), which can target CD44-overexpressing ovarian cancers for precise imaging and guide the resection of minor tumors. Notably, this work holds significant promise for developing biomimetic probes for clinical imaging-guided precision cancer surgery by exploiting their externally specified functional modifications.

pH-Responsive Aminobenzocoumarins as Fluorescent Probes for Biological Acidity.

Regulation of pH plays an essential role in orchestrating the delicate cellular machinery responsible for life as we know it. Its abnormal values are indicative of aberrant cellular behavior and associated with pathologies including cancer progression or solid tumors. Here, we report a series of bent and linear aminobenzocoumarins decorated with different substituents. We investigate their photophysical properties and demonstrate that the probes display strong pH-responsive fluorescence "turn on" behavior in highly acidic environments, with enhancement up to 300-fold. In combination with their low cytotoxicity, this behavior enabled their application in bioimaging of acidic lysosomes in live human cells. We believe that these molecules serve as attractive lead structures for future rational design of novel biocompatible fluorescent pH probes.

Versatile Metal-Free Arylation of BODIPY and Bis(BF2) Chromophores by Using Arylazosulfones in a Sunflow System.

BODIPYs have a well-established role in biological sciences as chemosensors and versatile biological markers due to their chemical reactivity, which allows for fine-tuning of their photophysical characteristics. In this work, we combined the unique reactivity of arylazo sulfones with the advantages of a "sunflow" reactor to develop a fast, efficient, and versatile method for the photochemical arylation of BODIPYs and other chromophores. This approach resulted in red-shifted emitting fluorophores due to extended electronic delocalization at the 3- and 5-positions of the BODIPY core. This method represents an advantageous approach for BODIPY functionalization compared to existing strategies.

Alared: Solvatochromic and Fluorogenic Red Amino Acid for Ratiometric Live-Cell Imaging of Bioactive Peptides.

To fill the need for environmentally sensitive fluorescent unnatural amino acids able to operate in the red region of the spectrum, we have designed and synthesized Alared, a red solvatochromic and fluorogenic amino acid derived from the Nile Red chromophore. The new unnatural amino acid can be easily integrated into bioactive peptides using classical solid-phase peptide synthesis. The fluorescence quantum yield and the emission maximum of Alared-labeled peptides vary in a broad range depending on the peptide's environment, making Alared a powerful reporter of biomolecular interactions. Due to its red-shifted absorption and emission spectra, Alared-labeled peptides could be followed in living cells with minimal interference from cellular autofluorescence. Using ratiometric fluorescence microscopy, we were able to track the fate of the Alared-labeled peptide agonists of the apelin G protein-coupled receptor upon receptor activation and internalization. Due to its color-shifting environmentally sensitive emission, Alared allowed for distinguishing the fractions of peptides that are specifically bound to the receptor or unspecifically bound to different cellular membranes.

Fluorogenic in-situ Labelling of Gelatin Polymer in Aqueous Solution and Hydrogel.

Gelatin polymers made from partially degraded collagen are important biomaterials, but their in-situ analysis suffers from uncontrollable covalent labelling and poor spatio-temporal imaging resolution. Herein, three tetrazolate-tagged tetraphenylethylene fluorophores (TPE-TAs) are introduced for practical fluorogenic labelling of gelatin in aqueous phase and hydrogels. These probes with aggregation-induced emission characteristics offer negligible background and elicit turn-on fluorescence by simply mixing with the gelatin in aqueous phase, giving a detection limit of 0.15 mg/L over a linear dynamic range up to 100 mg/L. This method does not work for collagens and causes minimal interference with gelatin properties. Mechanistic studies reveal a key role for multivalent electrostatic interactions between the abundant basic residues in gelatin (e.g., lysine, hydroxylysine, arginine) and anionic tetrazolate moieties of the lipophilic fluorophore synergistically in spatially rigid macromolecular encapsulation to achieve fluorogenic labelling. The AIE strategy by forming non-covalent fluorophore-gelatin complexes was developed for novel hydrogels that exhibited reversible fluorescence in response to dynamic microstructural changes in the hydrogel scaffold upon salting-in/out treatments, and enabled high spatio-temporal imaging of the fiber network in lyophilized samples. This work may open up avenues for in-situ imaging analysis and evaluation of gelatin-based biomaterials during processes such as in vivo degradation and mineralization.

N-Aryl-DABCO Salts as an Unprecedented Sensing Platform for the Detection of Thiols and Selenols.

Quaternary N-aryl-DABCO salts were introduced for the first time as a highly selective sensing platform for thiols and selenols. By employing this platform, a highly sensitive coumarin based "off-on" fluorescent probe was designed and synthesized. The probe possesses a good solubility in water, low background fluorescence, and, most importantly, demonstrates high selectivity to aryl thiols and selenols over their aliphatic counterparts and other common nucleophiles. A dramatic increase in fluorescence intensity is achieved through the selective cleavage of the quaternized DABCO-ring, yielding a piperazine derivatives with a high fluorescence quantum yield (~72 %). Moreover, stability of the probe to the most used reducing agents DTT and TCEP was demonstrated. The limits of detection for p-thiocresol and phenyl selenide were evaluated to be 22 nM and 6 nM, respectively.

Azaphosphinate Dyes: A Low Molecular Weight Near-Infrared Scaffold for Development of Photoacoustic or Fluorescence Imaging Probes.

Near-infrared (NIR) dyes are desirable for biological imaging applications including photoacoustic (PA) and fluorescence imaging. Nonetheless, current NIR dyes are often plagued by relatively large molecular weights, poor water solubility, and limited photostability. Herein, we provide the first examples of azaphosphinate dyes which display desirable properties such as low molecular weight, absorption/emission above 750 nm, and remarkable water solubility. In PA imaging, an azaphosphinate dye exhibited a 4.1-fold enhancement in intensity compared to commonly used standards, the ability to multiplex with existing dyes in whole blood, imaging depths of 2.75 cm in a tissue model, and contrast in mice. An improved derivative for fluorescence imaging displayed a >10-fold reduction in photobleaching in water compared to the FDA-approved indocyanine green dye and could be visualized in mice. This new dye class provides a robust scaffold for the development of photoacoustic or NIR fluorescence imaging agents.

Xanthene-Based Dyes for Photoacoustic Imaging and their Use as Analyte-Responsive Probes.

Developing imaging tools that can report on the presence of disease-relevant analytes in multicellular organisms can provide insight into fundamental disease mechanisms as well as provide diagnostic tools for the clinic. Photoacoustic imaging (PAI) is a light-in, sound-out imaging technique that allows for high resolution, deep-tissue imaging with applications in pre-clinical and point-of-care settings. The continued development of near-infrared (NIR) absorbing small-molecule dyes promises to improve the capabilities of this emerging imaging modality. For example, new dye scaffolds bearing chemoselective functionalities are enabling the detection and quantification of disease-relevant analytes through activity-based sensing (ABS) approaches. Recently described strategies to engineer NIR absorbing xanthenes have enabled development of analyte-responsive PAI probes using this classic dye scaffold. Herein, we present current strategies for red-shifting the spectral properties of xanthenes via bridging heteroatom or auxochrome modifications. Additionally, we explore how these strategies, coupled with chemoselective spiroring-opening approaches, have been employed to create ABS probes for in vivo detection of hypochlorous acid, nitric oxide, copper (II), human NAD(P)H: quinone oxidoreductase isozyme 1, and carbon monoxide. Given the versatility of the xanthene scaffold, we anticipate continued growth and development of analyte-responsive PAI imaging probes based on this dye class.

Effect of Aggregated Lysozyme on Fluorescence Properties of Rose Bengal.

Protein aggregates cause abnormal states and trigger various diseases, including neurodegenerative disorders. This study examined whether the xanthene dye derivative Rose Bengal could track a series of conformational changes in protein aggregates. Using lysozyme as a model protein, aggregated proteins were prepared by heating under acidic conditions. The absorption spectra, steady-state fluorescence spectra, fluorescence quantum yield, fluorescence lifetime, and phosphorescence lifetime of a solution containing Rose Bengal in the presence of aggregated lysozyme were measured to identify their spectroscopic characteristics. The absorption spectrum of Rose Bengal changed significantly during the formation of agglomerates in heated lysozyme. Additionally, the fluorescence intensity decreased during the initial stages of the aggregation process with an increase in heating time, followed by an increase in intensity along with a red-shift of the peak wavelength. The decrease in quantum yield with a fixed fluorescence lifetime supported the formation of a nonfluorescent ground-state complex between Rose Bengal and the aggregated lysozyme. Based on the characteristic changes in absorption and fluorescence properties observed during the aggregation process, Rose Bengal is considered an excellent indicator for the sensitive discernment of aggregated proteins.

Fluorescent probes and degraders of the sterol transport protein Aster-A.

Our understanding of sterol transport proteins (STPs) has increased exponentially in the last decades with advances in the cellular and structural biology of these important proteins. However, small molecule probes have only recently been developed for a few selected STPs. Here we describe the synthesis and evaluation of potential proteolysis-targeting chimeras (PROTACs) based on inhibitors of the STP Aster-A. Based on the reported Aster-A inhibitor autogramin-2, ten PROTACs were synthesized. Pomalidomide-based PROTACs functioned as fluorescent probes due to the intrinsic fluorescent properties of the aminophthalimide core, which in some cases was significantly enhanced upon Aster-A binding. Most PROTACs maintained excellent binary affinity to Aster-A, and one compound, NGF3, showed promising Aster-A degradation in cells. The tools developed here lay the foundation for optimizing Aster-A fluorescent probes and degraders and studying its activity and function in vitro and in cells.

Preparation of Fluorescent "on-off-on" Carbon Quantum Dots and Their Application to the Detection of Fe3+ and Ascorbic Acid and Information Encryption.

Carbon quantum dots are a new type of fluorescent carbon-based nanomaterials, and their excellent properties have provoked a strong research interest. Herein, blue-fluorescent carbon quantum dots (k-CQDs) were successfully synthesized by a simple one-step hydrothermal method using chitosan and ethylenediaminetetraacetic acid as precursors. It was found that Fe3+ could quench the fluorescence of k-CQDs by a dynamic quenching mechanism that increased the positive charge in solution. Due to ascorbic acid (AA) can reduce Fe3+ to Fe2+, the positive charge in solution was reduced and the fluorescence of k-CQDs was restored. Based on the mechanism of the fluorescence "on-off-on", k-CQDs were used for the detection of Fe3+ and AA with strong antijamming capability. The LOD for Fe3+ concentrations in the ranges of 0 to 30 µM and 30 to 100 µM were 0.3 µM and 0.76 µM, respectively. The LOD for AA concentrations in the ranges of 0 to 82.5 µM and 82.5 to 172.5 µM were 3.93 µM and 1.63 µM, respectively. Spiking recoveries of Fe3+ in tap water, AA in orange juice and tomato juice were 87.93 ∼ 101.13%, 86.77 ∼ 105.15% and 86.43 ∼ 103.80%, respectively. Meanwhile, k-CQDs also showed good potential for anti-counterfeiting encryption.

Recent advances in hypoxia-activated compounds for cancer diagnosis and treatment.

Hypoxia, as a prevalent feature of solid tumors, is correlated with tumorigenesis, proliferation, and invasion, playing an important role in mediating the drug resistance and affecting the cancer treatment outcomes. Due to the distinct oxygen levels between tumor and normal tissues, hypoxia-targeted therapy has attracted significant attention. The hypoxia-activated compounds mainly depend on reducible organic groups including azo, nitro, N-oxides, quinones and azide as well as some redox-active metal complex that are selectively converted into active species by the increased reduction potential under tumor hypoxia. In this review, we briefly summarized our current understanding on hypoxia-activated compounds with a particular highlight on the recently developed prodrugs and fluorescent probes for tumor treatment and diagnosis. We have also discussed the challenges and perspectives of small molecule-based hypoxia-activatable prodrug for future development.

Design and synthesis of hemicyanine-based NIRF probe for detecting Aß aggregates in Alzheimer's disease.

Alzheimer's disease (AD), a progressive neurodegenerative disorder, has garnered increased attention due to its substantial economic burden and the escalating global aging phenomenon. Amyloid-ß deposition is a key pathogenic marker observed in the brains of Alzheimer's sufferers. Based on real-time, safe, low-cost, and commonly used, near-infrared fluorescence (NIRF) imaging technology have become an essential technique for the detection of AD in recent years. In this work, NIRF probes with hemicyanine structure were designed, synthesized and evaluated for imaging Aß aggregates in the brain. We use the hemicyanine structure as the parent nucleus to enhance the probe's optical properties. The introduction of PEG chain is to improve the probe's brain dynamice properties, and the alkyl chain on the N atom is to enhance the fluorescence intensity of the probe after binding to the Aß aggregates as much as possible. Among these probes, Z2, Z3, Z6, X3, X6 and T1 showed excellent optical properties and high affinity to Aß aggregates (Kd = 24.31 âˆ¼ 59.60 nM). In vitro brain section staining and in vivo NIRF imaging demonstrated that X6 exhibited superior discrimination between Tg mice and WT mice, and X6 has the best brain clearance rate. As a result, X6 was identified as the optimal probe. Furthermore, the docking theory calculation results aided in describing X6's binding behavior with Aß aggregates. As a high-affinity, high-selectivity, safe and effective probe of targeting Aß aggregates, X6 is a promising NIRF probe for in vivo detection of Aß aggregates in the AD brain.

A new fluorescent probe for the visualization of progerin.

Hutchinson-Gilford progeria syndrome (HGPS) or progeria is a rare genetic disease that causes premature aging, leading to a drastic reduction in the life expectancy of patients. Progeria is mainly caused by the intracellular accumulation of a defective protein called progerin, generated from a mutation in the LMNA gene. Currently, there is only one approved drug for the treatment of progeria, which has limited efficacy. It is believed that progerin levels are the most important biomarker related to the severity of the disease. However, there is a lack of effective tools to directly visualize progerin in the native cellular models, since the commercially available antibodies are not well suited for the direct visualization of progerin in cells from the mouse model of the disease. In this context, an alternative option for the visualization of a protein relies on the use of fluorescent chemical probes, molecules with affinity and specificity towards a protein. In this work we report the synthesis and characterization of a new fluorescent probe (UCM-23079) that allows for the direct visualization of progerin in cells from the most widely used progeroid mouse model. Thus, UCM-23079 is a new tool compound that could help prioritize potential preclinical therapies towards the final goal of finding a definitive cure for progeria.

Near-infrared Absorption and Emission Probes with Optimal Connection Bridges for Live Monitoring of NAD(P)H Dynamics in Living Systems.

Two NAD(P)H-biosensing probes consisting of 1,3,3-trimethyl-3H-indolium and 3-quinolinium acceptors, linked by thiophene, A, and 3,4-ethylenedioxythiophene, B, bridges are detailed. We synthesized probes C and D, replacing the thiophene connection in probe A with phenyl and 2,1,3-benzothiadiazole units, respectively. Probe E was prepared by substituting probe A's 3-quinolinium unit with a 1-methylquinoxalin-1-ium unit. Solutions are non-fluorescent but in the presence of NADH, exhibit near-infrared fluorescence at 742.1 nm and 727.2 nm for probes A and B, respectively, and generate absorbance signals at 690.6 nm and 685.9 nm. In contrast, probes C and D displayed pronounced interference from NADH fluorescence at 450 nm, whereas probe E exhibited minimal fluorescence alterations in response to NAD(P)H. Pre-treatment of A549 cells with glucose in the presence of probe A led to a significant increase in fluorescence intensity. Additionally, subjecting probe A to lactate and pyruvate molecules resulted in opposite changes in NAD(P)H levels, with lactate causing a substantial increase in fluorescence intensity, conversely, pyruvate resulted in a sharp decrease. Treatment of A549 cells with varying concentrations of the drugs cisplatin, gemcitabine, and camptothecin (5, 10, and 20 µM) led to a concentration-dependent increase in intracellular fluorescence intensity, signifying a rise in NAD(P)H levels. Finally, fruit fly larvae were treated with different concentrations of NADH and cisplatin illustrating applicability to live organisms. The results demonstrated a direct correlation between fluorescence intensity and the concentration of NADH and cisplatin, respectively, further confirming the efficacy of probe A in sensing changes in NAD(P)H levels within a whole organism.