Small-molecule Fluorescent Probes Research Articles

Research progress of organic small probes sensitive to tumor microenvironment

The development, metastasis, and spread of malignant tumors are closely associated with the inherent properties of tumor cells. These tumors often exhibit low pH, low polarity, high viscosity, and hypoxia. Understanding these characteristics is essential for early cancer detection and monitoring treatment effectiveness. Small molecule fluorescent probes have emerged as powerful tools for real-time localization, dynamic detection, and visualization of tumors. In this paper, we review recent progress in the use of these probes to study tumor biology, focusing on their design strategies, response mechanisms, and preclinical applications for detecting pH, viscosity, polarity, and hypoxia.

Reaction-based small-molecule fluorescent probes for endoplasmic reticulum- and mitochondria-targeted biosensing and bioimaging

Reaction-based small-molecule fluorescent probes for endoplasmic reticulum- and mitochondria-targeted biosensing and bioimaging

Red fluorescent AIE bioprobes with a large Stokes shift for droplet-specific imaging and fatty liver diagnosis

Lipid droplets (LDs) as spherical dynamic subcellular organelles, play an important role in various cellular functions such as protein degradation, lipid metabolism, energy storage, signal transduction, and membrane formation. Abnormal function of LDs will lead to a series of diseases and hence monitoring the status of LDs is particularly important. In this study, we synthesized a water-insoluble red fluorescent emitting small molecule fluorescent probe (TPE-TCF), which exhibited aggregation-induced emission (AIE) properties and enabled highly selective real-time imaging of LDs (Pearson’s R value was 0.90). More interestingly, this probe was able to track the dynamic processes of LDs in living cells, including lipophagy, and monitor fatty liver disease in mice. Therefore, TPE-TCF with red fluorescence emission, good biocompatibility, large Stokes shift, AIE properties, LDs imaging, and fatty liver recognition capabilities can be practically used in more LDs-related diseases.

Fluorescent labeling of live-cell surfaceome and its application in antibody-target interaction analysis

BackgroundCell-surface proteins play key roles in the communication between external stimuli and internal signaling. As protein types and expression levels vary in different cells, in-situ visualization of the whole surface proteome (surfaceome) may facilitate the study of their functions in homeostasis maintenance or response to environmental changes (e.g., drug treatment). However, there lacks easily-prepared and universal labeling probes to visualize them in living cells. ResultsWe designed and synthesized a small-molecule fluorescent probe, SRB-NHS, for one-step labeling of surfaceome. Live-cell imaging results exhibited the plasma membrane localization of the fluorescent signal from SRB-NHS and SDS-PAGE/fluorescence scanning results confirmed the covalent labeling of proteins by SRB-NHS, indicating the suitability of SRB-NHS for surfaceome labeling towards different cell lines. SignificanceUpon labeling by SRB-NHS, the cellular internalization of surfaceome was studied under different stimuli (e.g., nutritional deprivation, drug treatments). Intriguingly, specific monitoring of the interaction between antibody drugs and related cell-surface targets can be achieved when the probe is used in combination with fluorescently labeled antibodies and imaged via Förster resonance energy transfer (FRET), offering a new method compatible with various cell lines to monitor the surfaceome or a specific drug-target interaction in situ.

NIR-activatable nitric oxide generator based on nanoparticles loaded small-molecule photosensitizers for synergetic photodynamic/gas therapy

BackgroundTherapeutic approaches that combine conventional photodynamic therapy (PDT) with gas therapy (GT) to sensitize PDT are an attractive strategy, but the molecular structure design of the complex lacks effective guiding strategies.ResultsHerein, we have developed a nanoplatforms Cy-NMNO@SiO2 based on mesoporous silica materials loaded NIR-activatable small-molecule fluorescent probe Cy-NMNO for the synergistic treatment of photodynamic therapy/gas therapy (PDT/GT) in antibacterial and skin cancer. The theoretical calculation results showed that the low dissociation of N-NO in Cy-NMNO enabled it to dissociate effectively under NIR light irradiation, which is conducive to produce Cy and NO. Cy showed better 1O2 generation performance than Cy-NMNO. The cytotoxicity of Cy-NMNO obtained via the synergistic effect of GT and PDT synergistically enhances the effect of photodynamic therapy, thus achieving more effective tumor treatment and sterilization than conventional PDT. Moreover, the nanoplatforms Cy-NMNO@SiO2 realized efficient drug loading and drug delivery.ConclusionsThis work not only offers a promising approach for PDT-GT synergistic drug delivery system, but also provides a valuable reference for the design of its drug molecules.Graphical

Mitochondrial Labeling with Mulberrin-Cy3: A New Fluorescent Probe for Live Cell Visualization.

Mitochondria, crucial intracellular organelles, are central to energy metabolism, signal transduction, apoptosis, calcium homeostasis, and a myriad of other biological processes, making them a focal point in diverse research fields. The capacity to fluorescently label and visually track mitochondria is crucial for understanding their biological roles. We present mulberrin-Cy3, a novel small molecule fluorescent probe that selectively labels mitochondria in animal cells, including cancer cells, with relative ease. This protocol details the synthesis of mulberrin-Cy3 and its use for visualizing mitochondria in living cells. The synthesis is straightforward and time-efficient, and the labeling method is more accessible than traditional approaches, providing a cost-effective option for mitochondrial visualization at room temperature. The labeling is rapid, with effective labeling achieved within 5 min of incubation. The fluorescent signal is stable and brighter, offering a significant advantage over existing methods. Mulberrin-Cy3 represents a promising mitochondrial labeling compound, providing researchers with a novel experimental tool to explore the complex biological functions of mitochondria. This innovation has the potential to significantly advance our comprehension of mitochondrial dynamics and their role in cellular health and disease.

A Nanofluorescent Probe for Evaluating the Fluctuation of Aminopeptidase N in Nonalcoholic Fatty Liver Disease and Hepatic Fibrosis.

Aminopeptidase N (APN/CD13) is a widely expressed transmembrane ectoenzyme that is crucial for maintaining normal physiological activities. It exhibits abnormal activity closely associated with hepatic fibrosis and nonalcoholic fatty liver disease (NAFLD). Therefore, there is a high demand for noninvasive detection of aminopeptidase N (APN) in the diagnosis and research of related diseases. Here, we developed a small molecule fluorescent probe, Hcy-APN, which is a fluorescent probe with high sensitivity and selectivity for the detection of APN. Furthermore, we synthesized the fluorescent nanoprobe Hcy-APN@MSN by self-assembling Hcy-APN and mesoporous silica nanoparticles in solution using a combination of molecular probe design and nanofunctionalization strategies. The detection limit of this probe was 1.5 ng/mL. Hcy-APN@MSN exhibits more stable spectral characteristics compared to Hcy-APN and is suitable for detecting APN activity in live cells and mice. Hcy-APN@MSN was utilized for in vivo and intracellular imaging of NAFLD and hepatic fibrosis at different stages, as well as for a systematic assessment of APN levels in the liver. The results confirm an elevation in the expression levels of APN in NAFLD and hepatic fibrosis models. Furthermore, we investigated the inhibitory effect of the APN inhibitor bestatin in nonalcoholic fatty liver and hepatic fibrosis disease models, confirming its regulatory effect on APN levels in cells and in vivo in both disease models. Therefore, this study may offer diagnostic possibilities for detecting NAFLD and hepatic fibrosis.

Cancer-Targeting and Viscosity-Activatable Near-Infrared Fluorescent Probe for Precise Cancer Cell Imaging.

Small-molecule fluorescent probes have emerged as potential tools for cancer cell imaging-based diagnostic and therapeutic applications, but their limited selectivity and poor imaging contrast hinder their broad applications. To address these problems, we present the design and construction of a novel near-infrared (NIR) biotin-conjugated and viscosity-activatable fluorescent probe, named as QL-VB, for selective recognition and imaging of cancer cells. The designed probe exhibited a NIR emission at 680 nm, with a substantial Stokes shift of 100 nm and remarkably sensitive responses toward viscosity changes in solution. Importantly, QL-VB provided an evidently enhanced signal-to-noise ratio (SNR: 6.2) for the discrimination of cancer cells/normal cells, as compared with the control probe without biotin conjugation (SNR: 1.8). Moreover, we validated the capability of QL-VB for dynamic monitoring of stimulated viscosity changes within cancer cells and employed QL-VB for distinguishing breast cancer tissues from normal tissues in live mice with improved accuracy (SNR: 2.5) in comparison with the control probe (SNR: 1.8). All these findings indicated that the cancer-targeting and viscosity-activatable NIR fluorescent probe not only enables the mechanistic investigations of mitochondrial viscosity alterations within cancer cells but also holds the potential as a robust tool for cancer cell imaging-based applications.

Electron-donating and -withdrawing groups discriminate the fluorometric sensing of phosgene.

Phosgene, diphosgene, and chlorine are called choking agents due to their acute toxicity to the respiratory system by directly attacking through inhalation and causing acute hypoxia, asphyxia and death. For these reasons, small-molecule fluorescent probes have been developed for their detection to ensure public safety. In this regard, two thiophene-based chemodosimetric fluorescent probes TCAO ((Z)-3-(4-(dimethylamino)phenyl)thiophene-2-carbaldehyde oxime) and HMBT ((Z)-4-(2-((hydroxyimino)methyl)thiophen-3-yl)benzonitrile) were designed, synthesized and characterized using 1H-NMR, 13C NMR, FT-IR spectroscopy and HRMS methods. Probe TCAO exhibited higher selectivity and sensitivity for detecting phosgene than probe HMBT. The electron-donating group (EDG) and electron-withdrawing group (EWG) play a crucial role in detecting phosgene. TCAO, bearing EDG, exhibited a fluorescence 'turn-on' response by the NGP-assisted conversion of aldoxime to the cyano group in the presence of phosgene; whereas HMBT, bearing EWG, did not show any fluorometric response. Therefore, further studies were conducted on TCAO, and the quantum yield changed from Φ = 0.043 to Φ = 0.155 in the presence of phosgene. The limit of detection for TCAO was estimated to be as low as 51 nm. In addition, onsite monitoring for visual detection was performed using the easy-to-handle paper-strip method.

Design, synthesis and application of polarity- and pH-sensitive dual-responsive small molecule fluorescent probe for early cancer diagnosis

Cancer is one of the deadliest and most complex diseases globally. It is well known that cancer cells exhibit a unique intracellular microenvironment characterized by low pH and low polarity compared to normal cells. Thus, dual-responsive fluorescent probe are more suitable for such complex and variable physiological environments than single-response probe. In this study, we designed and synthesized a dual-responsive fluorescent probe, CCT, which responds to both pH and polarity based on intramolecular charge transfer (ICT) and photoelectron transfer (PET) mechanisms.We synthesized probe CCT by combining naphthylimide, a coumarin unit, and morpholine through a six-step process. The PET mechanism between morpholine and naphthylimide renders probe CCT pH-sensitive. Additionally, the direct linkage of the polarity-sensitive units, coumarin and naphthylimide, creates a larger polarity-sensitive system. When both low pH and low polarity conditions are present, the fluorescence intensity of probe CCT is significantly higher than under separate conditions. This increased fluorescence intensity matches the low pH and low polarity microenvironment of cancer cells, facilitating bioimaging.Fluorescence imaging results demonstrate that probe CCT effectively distinguishes between cancer and normal cells, suggesting its potential as a powerful tool for early cancer diagnosis.

Recent Developments in Small-Molecule Fluorescent Probes for Cellular Senescence

Cellular senescence is a recently emerged research topic in modern biology. Often described as a double-edged sword, it encompasses numerous essential biological processes, including beneficial effects such as wound healing and embryonic development, as well as detrimental contributions to chronic inflammation and tumor development. Consequently, there is an increasing need to unravel the intricate networks of senescence and develop reliable detection methods to distinguish it from related phenomena. To address these challenges, a variety of detection methods have been developed. In particular, small-molecule fluorescent probes offer distinct advantages such as suitability for real-time live cell monitoring and in vivo imaging, superior tunable properties, and versatile applications. In this review, we explored recent advancements in the development of small-molecule fluorescent probes toward monitoring cellular senescence by targeting various senescence-related biological phenomena. These phenomena include the upregulation of senescence-associated enzymes, perturbation of the subcellular environment, and increased endogenous ROS levels. Moreover, multi-senescence biomarker-targeting approaches are also discussed to improve their sensitivities and specificities for the detection of cellular senescence. With recent advances in senescence probe development, current challenges in this field are also discussed to facilitate further progress.

Imaging Neurotransmitters with Small-Molecule Fluorescent Probes.

Neurotransmitters play a crucial role in regulating communication between neurons within the brain and central nervous system. Thus, imaging neurotransmitters has become a high priority in neuroscience. This minireview focuses on recent advancements in the development of fluorescent small-molecule fluorescent probes for neurotransmitter imaging and applications of these probes in neuroscience. Innovative approaches for probe design are highlighted as well as attributes which are necessary for practical utility, with a view to inspiring new probe development capable of visualizing neurotransmitters.

Recent Advances in Organic Small-Molecule Fluorescent Probes for the Detection of Zinc Ions (Zn2+).

Zinc(II) ions (Zn2g) play crucial roles in the growth, propagation, and metabolism of animals, plants, and humans. Abnormal concentrations of Zn2+ in the environment and living organisms pose potential risks to environmental protection and human health. Therefore, it is imperative to develop rapid, reliable and in-situ detection methods for Zn2+in both environmental and biological contexts. Furthermore, effective analytical methods are required for diagnosing diseases and understanding physiological metabolic mechanisms associated with Zn2+concentration levels. Organic small-molecule fluorescent probes offer advantages such as fast, reliable, convenient, non-destructive detection capabilities and have significant application potential in Zn2+ detection and bioimaging; thus garnering extensive attention. Over the past two years alone, various organic small-molecule probes for Zn2+ based on different detection mechanisms and fluorophores have been rapidly developed. However, these probes still exhibit several limitations that need further resolution. In light of this context, we provide a comprehensive summary of the detection mechanisms, performance characteristics, and application scope of Zn2+ fluorescence probes since year 2022 while highlighting their advantages. We also propose solutions to address existing issues with these probes and outline future directions for their advancement. This review aims to serve as a valuable reference source offering insights into the development of advanced organic small-molecule-based fluorescence probes specifically designed for detecting Zn2+.

Activatable NIR Fluorescence Probe for Epinephrine Detection and Bioimaging Based on Anionic Heptamethine Cyanine.

Epinephrine (EP) is an essential catecholamine in the human body. Currently, most EP detection methods are not suitable for in vivo detection due to material limitations. An organic small molecule fluorescent probe based on a chemical cascade reaction for the detection of EP was designed. Anionic heptamethine cyanine dye was selected as a fluorescent dye because of its NIR fluorescence emission with excellent biocompatibility. The secondary amine of EP nucleophilically attacks the carbonate of the probe with its stronger nucleophilicity and further undergoes intramolecular nucleophilic cyclization to release the fluorophore. Other substances containing only primary amines or no β-OH lack reaction competitiveness due to their weaker nucleophilicity or inability to undergo further cyclization. The fluorescence recovery of the probe was linearly related to the EP concentration of 2-75 μmol/L. The detection limit was 0.4 μmol/L. The recovery rate was 94.78-111.32%. Finally, we successfully achieved bioimaging of EP in living cells and EP analogue in nematodes.

Development and Application of Cationic Nile Blue Probes in Live-Cell Super-Resolution Imaging and Specific Targeting to Mitochondria.

Mitochondria are essential organelles involved in various metabolic processes in eukaryotes. The imaging, targeting, and investigation of cell death mechanisms related to mitochondria have garnered significant interest. Small-molecule fluorescent probes have proven to be robust tools for utilizing light to advance the study of mitochondrial biology. In this study, we present the rational design of cationic Nile blue probes carrying a permanent positive charge for these purposes. The cationic Nile blue probes exhibit excellent mitochondrial permeability, unique solvatochromism, and resistance to oxidation. We observed weaker fluorescence in aqueous solutions compared to lipophilic solvents, thereby minimizing background fluorescence in the cytoplasm. Additionally, we achieved photoredox switching of the cationic Nile blue probes under mild conditions. This enabled us to demonstrate their application for the first time in single-molecule localization microscopy of mitochondria, allowing us to observe mitochondrial fission and fusion behaviors. Compared to conventional cyanine fluorophores, this class of dyes demonstrated prolonged resistance to photobleaching, likely due to their antioxidation properties. Furthermore, we extended the application of cationic Nile blue probes to the mitochondria-specific delivery of taxanes, facilitating the study of direct interactions between the drug and organelles. Our approach to triggering cell death without reliance on microtubule binding provides valuable insights into anticancer drug research and drug-resistance mechanisms.

Multicolor Two-Photon Microscopy Imaging of Lipid Droplets and Liver Capsule Macrophages In Vivo.

Lipid droplets (LDs) store energy and supply fatty acids and cholesterol. LDs are a hallmark of chronic nonalcoholic fatty liver disease (NAFLD). Recently, studies have focused on the role of hepatic macrophages in NAFLD. Green fluorescent protein (GFP) is used for labeling the characteristic targets in bioimaging analysis. Cx3cr1-GFP mice are widely used in studying the liver macrophages such as the NAFLD model. Here, we have developed a tool for two-photon microscopic observation to study the interactions between LDs labeled with LD2 and liver capsule macrophages labeled with GFP in vivo. LD2, a small-molecule two-photon excitation fluorescent probe for LDs, exhibits deep-red (700 nm) fluorescence upon excitation at 880 nm, high cell staining ability and photostability, and low cytotoxicity. This probe can clearly observe LDs through two-photon microscopy (TPM) and enables the simultaneous imaging of GFP+ liver capsule macrophages (LCMs) in vivo in the liver capsule of Cx3cr1-GFP mice. In the NAFLD mouse model, Cx3cr1+ LCMs and LDs increased with the progress of fatty liver disease, and spatiotemporal changes in LCMs were observed through intravital 3D TPM images. LD2 will aid in studying the interactions and immunological roles of hepatic macrophages and LDs to better understand NAFLD.

Construction and evaluation of near-infrared fluorescent probes for imaging lipid droplet and lysosomal viscosity

Microenvironmental viscosity is a crucial parameter for biological systems, and its abnormal fluctuations are closely associated with various functional disorders and diseases. However, it is still important and urgent to develop improved near-infrared fluorescent probes for micro-viscosity with dual-organelle targeting properties, low background noise, and high sensitivity. Herein, two BODIPY-based small-molecule fluorescent probes were designed and synthesized, which were explored for their viscosity- and polarity-responsive properties, and were further applied to imaging sub-cellular viscosity in living cells. Interestingly, BSZ-Ph and BSZ-R displayed near-infrared fluorescence (more than 650 nm) and were sensitive to environmental viscosity and polarity due to the introduction of a benzothiazole at the 2-position and electron-rich aniline groups at the 5-position of the BODIPY core, respectively. The fluorescence intensity increased exponentially with the viscosity changes. Furthermore, the probe BSZ-Ph could successfully target lipid droplets and image cellular viscosity changes by treating lipopolysaccharides (LPS) and nystatin. Comparatively, the probe BSZ-R could successfully target the dual organelles of lipid droplets and lysosomes and image cellular viscosity changes by treating LPS and monensin. Therefore, in this work, we reported two new BODIPY-based near-infrared fluorescent probes, BSZ-Ph and BSZ-R, for cellular viscosity imaging, which could target lipid droplets and the dual organelles of lysosomes and lipid droplets, respectively. The study could provide a reference for the future development of fluorescent probes for viscosity in lipid droplets and lysosomes.

Advancements in Small Molecule Fluorescent Probes for Superoxide Anion Detection: A Review.

Superoxide anion (O2•-), a significant reactive oxygen species (ROS) within biological systems, plays a widespread role in cellular function regulation and is closely linked to the onset and progression of numerous diseases. To unveil the pathological implications of O2•- in these diseases, the development of effective monitoring techniques within biological systems is imperative. Small molecule fluorescent probes have garnered considerable attention due to their advantages: simplicity in operation, heightened sensitivity, exceptional selectivity, and direct applicability in monitoring living cells, tissues, and animals. In the past few years, few reports have focused on small molecule fluorescence probes for the detection of O2•-. In this small review, we systematically summarize the design and application of O2•- responsive small molecule fluorescent probes. In addition, we present the limitations of the current detection of O2•- and suggest the construction of new fluorescent imaging probes to indicate O2•- in living cells and in vivo.

Rational Design of NIR-II G-Quadruplex Fluorescent Probes for Accurate In Vivo Tumor Metastasis Imaging.

Accurate in vivo imaging of G-quadruplexes (G4) is critical for understanding the emergence and progression of G4-associated diseases like cancer. However, existing in vivo G4 fluorescent probes primarily operate within the near-infrared region (NIR-I), which limits their application accuracy due to the short emission wavelength. The transition to second near-infrared (NIR-II) fluorescent imaging has been of significant interest, as it offers reduced autofluorescence and deeper tissue penetration, thereby facilitating more accurate in vivo imaging. Nonetheless, the advancement of NIR-II G4 probes has been impeded by the absence of effective probe design strategies. Herein, through a "step-by-step" rational design approach, we have successfully developed NIRG-2, the first small-molecule fluorescent probe with NIR-II emission tailored for in vivo G4 detection. Molecular docking calculations reveal that NIRG-2 forms stable hydrogen bonds and strong π-π interactions with G4 structures, which effectively inhibit twisted intramolecular charge transfer (TICT) and, thereby, selectively illuminate G4 structures. Due to its NIR-II emission (940 nm), large Stokes shift (90 nm), and high selectivity, NIRG-2 offers up to 47-fold fluorescence enhancement and a tissue imaging depth of 5 mm for in vivo G4 detection, significantly outperforming existing G4 probes. Utilizing NIRG-2, we have, for the first time, achieved high-contrast visualization of tumor metastasis through lymph nodes and precise tumor resection. Furthermore, NIRG-2 proves to be highly effective and reliable in evaluating surgical and drug treatment efficacy in cancer lymphatic metastasis models. We are optimistic that this study not only provides a crucial molecular tool for an in-depth understanding of G4-related diseases in vivo but also marks a promising strategy for the development of clinical NIR-II G4-activated probes.

Rapid detection of hypobromous acid by a tetraphenylethylene-based turn-on fluorescent AIE probe and its applications

BackgroundSimilar to hypochlorous acid (HClO), hypobromous acid (HBrO) is one of the most notable reactive oxygen species (ROS). Overexpression of HBrO is linked to various diseases causing organ and tissue loss. Due to HBrO's role in the oxidation of micropollutants, real-time monitoring of HBrO in water-based systems is essential. Tetraphenylethylene (TPE)-based organic aggregation-induced emission luminophores (AIEgens) are an emerging category of fluorescent probe materials that have attracted considerable attentions. However, AIE probes are rarely applied to detect HBrO. Developing faster, more precise, and more sensitive AIE probes is thus crucial for detecting biological and environmental HBrO. ResultsA small molecule fluorescent probe 4-(1,2,2-triphenylvinyl)benzamidoxime (SWJT-21) was synthesized for the sensitive and selective detection of hypobromous acid (HBrO) based on aggregation-induced emission (AIE). The amidoxime unit of SWJT-21 would undergo an oxidation reaction with HBrO, leading to a structure differentiation between the probe and the product, and therefore the turn-on fluorescence by the AIE effect. The probe could recognize hypobromous acid rapidly (less than 3 s) in high aqueous phase (99 % water) with a turn-on fluorescence response. It was determined that the limit of detection for HBrO was 5.47 nM. Moreover, SWJT-21 demonstrates potential as a test strip for the detection of HBrO. SWJT-21 was also successfully used for the monitoring of HBrO in water samples and for the detection of endogenous/exogenous HBrO in living cells and zebrafish. SignificanceA special AIE fluorescence turn-on probe SWJT-21 based on tetraphenylethylene was designed for detecting HBrO in the environmental and biological systems. This probe has an extremely low detection limit of 5.47 nM and is able to detect HBrO in 99 % aqueous phase in less than 3 s.