Broad-Specificity Screening of Pyrethroids Enabled by the Catalytic Function of Human Serum Albumin on Coumarin Hydrolysis.

Sensing systems based on cholinesterase and carboxylesterase coupled with different transduction technologies have emerged for pesticide screening owing to their simple operation, fast response, and suitability for on-site analysis. However, the broad spectrum and specificity screening of pyrethroids over organophosphates and carbamates remains an unmet challenge for current enzymatic sensors. Human serum albumin (HSA), a multifunctional protein, can promote various chemical transformations and show a high affinity for pyrethroids, which offer a route for specific and broad-spectrum pyrethroid screening. Herein, for the first time, we evaluated the catalytic hydrolysis function of human serum albumin (HSA) on the coumarin lactone bond and revealed that HSA can act as an enzyme to catalyze the hydrolysis of the coumarin lactone bond. Molecular docking and chemical modifications indicate that lysine 199 and tyrosine 411 serve as the catalytic general base and contribute to most of the catalytic activity. Utilizing this enzymatic activity, a broad specific ratiometric fluorescence pyrethroids sensing system was developed. The binding energetics and binding constants of pesticides and HSA show that pyrethroids bind to HSA more easily than organophosphates and carbamates, which is responsible for the specificity of the sensing system. This study provides a general sensor platform and strategy for screening pesticides and reveals the catalytic activity of HSA on the hydrolysis of the coumarin lactone bond, which may open innovative horizons for the chemical sensing and biomedical applications of HSA.

Natural flavylium-inspired far-red to NIR-II dyes and their applications as fluorescent probes for biomedical sensing.

Fluorescent probes that emit in the far-red (600-700 nm), first near-infrared (NIR-I, 700-900 nm), and second NIR (NIR-II, 900-1700 nm) regions possess unique advantages, including low photodamage and deep penetration into biological samples. Notably, NIR-II optical imaging can achieve tissue penetration as deep as 5-20 mm, which is critical for biomedical sensing and clinical applications. Much research has focused on developing far-red to NIR-II dyes to meet the needs of modern biomedicine. Flavylium compounds are natural colorants found in many flowers and fruits. Flavylium-inspired dyes are ideal platforms for constructing fluorescent probes because of their far-red to NIR emissions, high quantum yields, high molar extinction coefficients, and good water solubilities. The synthetic and structural diversities of flavylium dyes also enable NIR-II probe development, which markedly advance the field of NIR-II in vivo imaging. In the last decade, there have been huge developments in flavylium-inspired dyes and their applications as far-red to NIR fluorescent probes for biomedical applications. In this review, we highlight the optical properties of representative flavylium dyes, design strategies, sensing mechanisms, and applications as fluorescent probes for detecting and visualizing important biomedical species and events. This review will prompt further research not only on flavylium dyes, but also into all far-red to NIR fluorophores and fluorescent probes. Moreover, this interest will hopefully spillover into applications related to complex biological systems and clinical treatments, ranging in focus from the sub-organelle to whole-animal levels.

One Stone, Three Birds: pH Triggered Transformation of Aminopyronine and Iminopyronine Based Lysosome Targeting Viscosity Probe for Cancer Visualization.

The lysosomes of cancer cells have lower pH and higher viscosity than those of normal cells. These features can be used as sensitive and selective markers for cancer diagnosis. In this work, a pH and viscosity dual responsive lysosome targeting fluorescent probe 1 was designed based on the transformation of amino- and imino- forms of pyronine and the twisted intramolecular charge shuttle (TICS) sensing mechanism. Live cancer cells and tumors were effectively distinguished from normal cells and organs through fluorescence imaging of probe 1, which indicated that probe 1 could serve as an effective tool for visualization of tumors at organ level with high selectivity.

Low Polarity-Triggered Basic Hydrolysis of Coumarin as an AND Logic Gate for Broad-Spectrum Cancer Diagnosis.

The ability to accurately diagnose cancer is the cornerstone of early cancer treatment. The mitochondria in cancer cells maintain a higher pH and lower polarity relative to that in normal cells. A probe that reports signals only when both conditions are met may provide a reliable method for cancer detection with reduced false positives. Here, we construct an AND logic gate fluorescent probe using mitochondrial microenvironments as inputs. Utilizing the hydrolysis of a coumarin scaffold, the probe generates fluorescence signals ("ON") only when high pH (>7.0) and low polarity conditions exist simultaneously. Additionally, the higher mitochondrial membrane potential in cancer cells provides an additional level of selectivity because probe has increased affinity for cancer cell mitochondria. These capabilities endow the probe with a high contrast fluorescence diagnosis ability of cancer at cellular and tissue levels (as high as 51.9 fold), which is far exceeding the clinic threshold of 2.0 fold.

Engineering a lipid droplet targeting fluorescent probe with a large Stokes shift through ester substituent rotation for in vivo tumor imaging.

Great progress has been made with lipid droplet targeting fluorescent probes in a wide range of biomedical fields. However, the Stokes shifts of most fluorescent probes are relatively small, leading to strong biological background fluorescence, poor signal-to-noise ratios, self-quenching in the commonly used microscopes and the need for in vivo imaging systems. In this manuscript, the ester substituent rotation of fluorophores was supposed to result in a large Stokes shift via steric hindrance effects and resonance effects. A lipid droplet targeting fluorescent probe 1 was achieved by simply appending a 4-substituted ester group onto the classic coumarin fluorophore. Probe 1 exhibited large Stokes shifts (122 to 184 nm) in both high polarity and weak polarity solvents with good lipophilicity and polarity responsive ability (3500 fold fluorescence enhancement). Probe 1 was suitable for washing-free imaging of lipid droplets in living cells with excellent specificity and rapidity (<2 min). Probe 1 was applied for distinguishing cancer cells from normal cells by taking advantage of the abnormalities of lipid droplets of cancer cells. Due to its huge Stokes shift, probe 1 can be used for in vivo tumor imaging by the excitation of a blue laser, which is important for biomedical research.

Carbon-Dipyrromethenes: Bright Cationic Fluorescent Dyes and Potential Application in Revealing Cellular Trafficking of Mitochondrial Glutathione Conjugates.

Boron-dipyrromethenes (Bodipys), since first reported in 1968, have emerged as a fascinating class of dyes in the past few decades due to their excellent photophysical properties including bright fluorescence, narrow emission bandwidth, resistance to photobleaching, and environment insensitivity. However, typical Bodipys are highly lipophilic, which often results in nonfluorescent aggregates in aqueous solution and also severely limits their bioavailability to cells and tissues. In this work, based on a simple one-atom B → C replacement in the Bodipy scaffold, we present a new class of carbon-dipyrromethenes (Cardipys for short) fluorescent dyes with tunable emission wavelengths covering the visible and near-infrared regions. These Cardipys not only retain the excellent photophysical properties of conventional Bodipys but also show improved water solubility and photostability due to their cationic character. Moreover, the cationic character also makes them extremely easy to penetrate the cell membrane and specifically accumulate into mitochondria without resorting to any mitochondria-targeted groups. Interestingly, several Cardipys bearing active styryl groups could serve as fluorescent indicators to map cellular trafficking of the glutathione conjugates produced within mitochondria under the catalysis of glutathione S-transferase (GST), thus showing potential in either exploring the detoxification mechanism of the mitochondrial GST/GSH system or evaluating the drug resistance of cancer cells that is closely related with GST activity.

Fluorescent Carbon Dots for in Situ Monitoring of Lysosomal ATP Levels.

Monitoring the ATP levels in lysosomes in situ is crucial for understanding their involvement in various biological processes but remains difficult due to the interference of ATP in other organelles or the cytoplasm. Here, we report a lysosome-specific fluorescent carbon dot (CD), which can be used to detect ATP in acidic lysosomes with "off-on" changes of yellow fluorescence. These CDs were successfully applied in real-time monitoring of the fluctuating concentration of lysosomal ATP induced by drug stimulation (e.g., chloroquine, etoposide, and oligomycin). Because of the excellent specificity, these CDs are promising agents for drug screening and medical diagnostics through lysosomal ATP monitoring.

Simultaneous Detection of Human Serum Albumin and Sulfur Dioxide in Living Cells Based on a Catalyzed Michael Addition Reaction.

As vital important bioactive species, human serum albumin (HSA) and sulfur dioxide (SO2) are essential molecules in the organisms and act a pivotal part in many biological events. Although studies have shown that SO2-induced HSA radicals can cause oxidative damage, the underlying mechanism of the synergistic effect of HSA and SO2 in various diseases is obscure, mainly because of the lack of powerful tools that can simultaneously detect HSA and SO2 in living systems. In this work, we report a novel single-site, double-sensing fluorescent probe 1 for the simultaneous detection of HSA and SO2. The probe is based on our finding that HSA can catalyze a Michael addition reaction between the probe and SO2, which induces a change in fluorescence. Probe 1 can effectively entered the endoplasmic reticulum and can be used to image exogenously introduced and de novo synthesis of HSA in endoplasmic reticulum. Furthermore, the simultaneous detection of HSA and SO2 was realized for the first time with probe 1. More important, we observed that HSA still retains its activity to catalyze the Michael addition reaction of 1 and SO2 in living cells, which may provide a significant boost in the study of the role of HSA in medicine and pharmacy.

Hydrogen-Bond-Induced Emission of Carbon Dots for Wash-Free Nucleus Imaging.

Carbon dots (CDs) are emerging as powerful tools for biosensing and bioimaging because of their intrinsic properties such as abundant precursors, facile synthesis, high biocompatibility, low cost, and particularly robust tunability and stability. In this work, a new type of CDs was prepared from m-phenylenediamine and folic acid by hydrothermal method. Interestingly, the as-prepared CDs show blue emission in non-hydrogen-bonding solution, whereas robust green emission in hydrogen-bonding solution. Based on this phenomenon, a novel fluorescence sensing mechanism named as hydrogen-bonding-induced emission (HBIE) was proposed. The HBIE-CDs have large Stokes shift (141 nm) in water, good biocompatibility, and ultrasmall size, which facilitates their translocation into living cells. Very importantly, the as-prepared HBIE-CDs show strong affinity toward nucleic acid without interference from other biological species. After binding with DNA/RNA through hydrogen bond, as high as 6-fold green fluorescence enhancement of HBIE-CDs was observed. Since the nucleus is rich in DNA/RNA, these HBIE-CDs were successfully used for rapid and, especially, wash-free subcellular in situ imaging of the nucleus in living cells in a fluorescence turn on mode, which has a great practicability to be used for nucleus imaging in bioanalytical studies and clinical applications.

Far-Red to Near-Infrared Carbon Dots: Preparation and Applications in Biotechnology.

As novel fluorescent nanomaterials, carbon dots (CDs) exhibit excellent photostability, good biocompatibility, and high quantum yield (QY). Their superior properties make them promising candidates for biomedical assays and therapy. Among them, the red-emission (>600 nm) CDs have attracted increasing attention in the past years due to their little damage to the biological matrix, deep tissue penetration, and minimum autofluorescence background of biosamples. This Review, summarizes the recent progress of far-red to near-infrared (NIR) CDs from the preparation and their biological applications. The challenges in designing far-red and NIR CDs and their further applications in biomedical fields are also discussed.

Retrosynthesis of Tunable Fluorescent Carbon Dots for Precise Long-Term Mitochondrial Tracking.

Mitochondria play a significant role in many cellular processes. Precise long-term tracking of mitochondrial status and behavior is very important for regulating cell fate and treating mitochondrial diseases. However, developing probes with photostability, long-term tracking capability, and tunable long-wavelength fluorescence has been a challenge in mitochondrial targeting. Carbon dots (CDs) as new fluorescent nanomaterials with low toxicity and high stability show increasing advantages in bioimaging. Herein, the mitochondria tracking CDs (MitoTCD) with intrinsic mitochondrial imaging capability and tunable long-wavelength fluorescence from green to red are synthesized where the lipophilic cation of rhodamine is served as the luminescent center of CDs. Due to the excellent photostability, superior fluorescence properties and favorable biocompatibility, these MitoTCD are successfully used for mitochondrial targeting imaging of HeLa cells in vitro and can be tracked as long as six passages, which is suitable for long-term cell imaging. Moreover, these MitoTCD can also be used for zebrafish imaging in vivo.

A rhodol-hemicyanine based ratiometric fluorescent probe for real-time monitoring of glutathione dynamics in living cells.

Glutathione (GSH) plays crucial roles in various physiological and pathological processes. The fluctuation of the GSH level is closely associated with a variety of diseases and cellular functions. Hence, it is important to real-time monitor the fluctuation of GSH in living cells. In this work, we presented a rhodol-hybridized hemicyanine fluorophore (RdH) as a selective, rapid-response, ratiometric, and reversible fluorescent probe for intracellular GSH (t1/2 = 89 ms, Kd = 1.42 mM). The imaging assays in living cells revealed that RdH could be used to real-time monitor GSH dynamics in A549 cells under a laser scanning confocal microscope by ratiometric fluorescence changes.

Highly fluorescent organic polymers for quenchometric determination of hydrogen peroxide and enzymatic determination of glucose.

Strongly fluorescent polymers (FPs) were prepared from citric acid and ethylenediamine via a hydrothermal approach. The FPs display low toxicity, water solubility, a quantum yield of 91%, good photostability and stability in the physiological pH range. Ferric ions are found to quench the fluorescence which is best measured at excitation/emission wavelengths of 350/440 nm. Because ferric ions (Fe3+) can quench the fluorescence of FPs, a fluorometric method was developed for fast detection of Fe3+ and within 1 min. FPs can also be used indirectly for the detection of hydrogen peroxide because of its fast Fenton reaction with Fe2+ to generate of Fe3+. The detection limits are 8 µM for Fe(III) and 0.6 µM for H2O2. On the basis of the glucose oxidase catalyzed of glucose and the Fenton reaction, the FPs can also be used to quantify glucose with a linear response in the 0.5-10 µM concentration range. Graphical abstract A new type of polymer with high fluorescence quantum yield was prepared. It is shown to enable the fast detection of ferric ions, hydrogen peroxide and glucose based on quenching and on the glucose oxidase and Fenton reactions.

Copper-Catalyzed Radical Cascade Cyclization To Access 3-Sulfonated Indenones with the AIE Phenomenon.

An efficient copper-catalyzed radical cascade cyclization strategy was developed, by which a wide variety of 3-sulfonyl substituted indenones were prepared in one pot via reaction of 2-alkynylbenzonitriles with sulfonyl hydrazides in the presence of TBHP and CuI under mild reaction conditions. Much more importantly, the 3-sulfonyl indenones, synthesized through our newly developed copper-catalyzed radical cascade cyclization strategy, were found to own typical aggregation-induced emission (AIE) properties, showing orange to red emission with large Stokes shift (more than 135 nm). In addition, such newly found AIEgens could be successfully used in live cell imaging, exhibiting excellent biocompatibility and application potential.

Phosphorus Radical-Initiated Cascade Reaction To Access 2-Phosphoryl-Substituted Quinoxalines.

An effective radical cascade cyclization strategy was developed, by which a wide range of 2-phosphoryl-substituted quinoxalines were prepared in one pot via reaction of ortho-diisocyanoarenes with diarylphosphine oxides in the presence of AgNO3 under mild reaction conditions.

Construction of a selective fluorescent probe for GSH based on a chloro-functionalized coumarin-enone dye platform.

Glutathione (GSH), the most abundant intracellular biothiol, protects cellular components from damage caused by free radicals and reactive oxygen species (ROS), and plays a crucial role in human pathologies. A fluorescent probe that can selectively sense intracellular GSH would be very valuable for understanding of its biological functions and mechanisms of diseases. In this work, a 3,4-dimethoxythiophenol-substituted coumarin-enone was exploited as a reaction-type fluorescent probe for GSH based on a chloro-functionalized coumarin-enone platform. In the probe, the 3,4-dimethoxythiophenol group functions not only as a fluorescence quencher through photoinduced electron transfer (PET) to ensure a low background fluorescence, but also as a reactive site for biothiols. The probe displays a dramatic fluorescence turn-on response toward GSH with the long-wavelength emission (600 nm) and significant Stokes shift (100 nm). The selectivity of the probe toward GSH over cysteine (Cys), homocysteine (Hcy), and other amino acids was demonstrated. Assisted by laser-scanning confocal microscopy, we have demonstrated that the probe could specifically sense GSH over Cys/Hcy in human renal cell carcinoma SiHa cells.

A mitochondria-targetable fluorescent probe for dual-channel NO imaging assisted by intracellular cysteine and glutathione.

A mitochondria-specific fluorescent probe for NO (1) was synthesized by the direct conjugation of a pyronin dye with one of the amino groups of o-phenylenediamino (OPD). The probe could selectively detect NO over dehydroascorbic acid (DHA), ascorbic acid (AA), and methylglyoxal (MGO) as well as the reactive oxygen/nitrogen species (ROS/RNS) with the significant off-on response due to the production of a red-emission triazole 2. In the presence of cysteine/glutathione (Cys/GSH), 2 could be further transformed into a green-emission aminopyronin 4 and a red-emission thiopyronin 5, respectively. Assisted by intracellular Cys and GSH, the probe demonstrated its potential to monitor mitochondrial NO in a dual-channel mode.

Simultaneous fluorescence sensing of Cys and GSH from different emission channels.

A chlorinated coumarin-hemicyanine dye with three potential reaction sites was exploited as fluorescent probe for biothiols. The Cys-induced substitution-rearrangement-cyclization, Hcy-induced substitution-rearrangement, and GSH-induced substitution-cyclizatioin cascades lead to the corresponding amino-coumarin, amino-coumarin-hemicyanine, thiol-coumarin with distinct photophysical properties, enabling Cys and GSH to be selectively detected from different emission channels at two different excitation wavelengths.

Constructing a fluorescent probe for specific detection of cysteine over homocysteine and glutathione based on a novel cysteine-binding group but-3-yn-2-one.

A but-3-yn-2-one-based 7-diethylaminocoumarin dye was exploited as a fluorescent probe to specifically detect Cys over Hcy/GSH in pure PBS buffer. The probe itself is nonfluorescent due to the donor-excited photoinduced electron transfer (d-PET) process. The Cys-induced Michael addition-rearrangement cascade reaction leads to an amino-substituted product with strong fluorescence due to inhibiting C[double bond, length as m-dash]C isomerization induced fluorescence quenching by a produced intramolecular N-HO hydrogen bond. The Hcy (or GSH)-induced Michael addition reaction leads to a thiol-substituted product (or ), which lacks any intramolecular hydrogen-bonding interaction, and thus displays very poor fluorescence due to the efficient C[double bond, length as m-dash]C isomerization induced fluorescence quenching. Even in the presence of Hcy (or GSH), the probe could also detect Cys with the obvious fluorescence enhancement. Assisted by using a laser scanning confocal microscope, we demonstrated that the probe could selectively image Cys in the human renal cell carcinoma 786-0 cells.

A Silicon-Rhodamine-Based Heavy-Atom-Free Photosensitizer for Mitochondria-targeted Photodynamic Therapy.

Silicon-xanthene derivatives (SiXs) have gained popularity in the field of bioimaging due to their advantageous far-red to near-infrared (NIR) absorption and emission wavelengths, notable brightness (ε × Φ), inherent mitochondrial targeting properties and high photo-stability, making them an excellent candidate for photodynamic therapy (PDT). Nevertheless, the utilization of SiXs as photosensitizers (PSs) for PDT in cancer treatment remains largely unexplored, primarily due to their limited capacity to generate cytotoxic reactive oxygen species (ROS). However, the potential of SiXs in PDT warrants further investigation. In this study, utilizing the spin-orbit charge transfer-induced intersystem crossing (SOCT-ISC) mechanism, we reported one novel heavy-atom-free, mitochondria-targeted, silicon-rhodamine-based photosensitizer (SiR-PXZ), which demonstrated excellent biocompatibility, minimal dark toxicity, favorable water-solubility and stability, and considerable singlet oxygen quantum yield under 660 nm light irradiation (ΦΔ = 0.16 in air-saturated PBS). Moreover, SiR-PXZ could be rapidly taken up by the mitochondria and efficiently induced apoptosis of cancer cells with an IC50 value of 1.2 µM. The in vivo studies showed that SiR-PXZ exhibited excellent anti-tumor effects, making it potentially valuable for clinical application. This study offers a source of ideas for the construction of SiXs-based photosensitizers for photodynamic cancer treatment in the future.