Small-molecule Fluorescent Probes Research Articles

Biotinylated Viscosity Sensitive Cell Membrane Probe for Targeted Imaging and Precise Visualization of Tumor Cells and Tumors.

Cancer is a global health challenge that urgently requires more sensitive and effective cancer detection methods. Fluorescence imaging with small molecule fluorescent probes has shown great promise for cancer detection but most of the developed probes lack active tumor cell targeting, which makes them unable to selectively target tumors, thereby reducing the accuracy of in vivo tumor detection. Herein, we report a novel probe Bio-S that combines a viscosity-sensitive and cell membrane targetable fluorescent group with biotin for targeted imaging and precise visualization of tumor cells and tumors. Bio-S exhibits sensitive fluorescence changes for viscosity at ∼660 nm and excellent cell membrane localization and imaging ability (red fluorescence, wash-free, and long-term imaging). Moreover, compared with the nonbiotinylated control probe C6-S, the biotinylated Bio-S can specifically target tumor cell membranes, thereby achieving much higher selectivity and sensitivity in distinguishing tumor cells from normal cells. Mice imaging experiments show that tail vein injection of Bio-S can target tumors and monitor lung cancer metastasis at the in vivo level. Therefore, this work provides an effective new strategy and tool for tumor-targeted detection and precise diagnosis.

A novel small-molecule fluorescent probe caused by minimal structural modifications for specific staining of the cell nuclear membrane.

The nuclear membrane is a double-layered structure that physically protects the cell's DNA from the chemical reactions occurring in other parts of the cell. In this study, we present the first brand-new small-molecule fluorescent probe that selectively stains the nuclear membrane, allowing for the visualization of nuclear morphology without interfering with the DNA's activity.

Classification of self-assembled fluorescent probes and their application in cancer diagnosis.

The high sensitivity, high selectivity, real-time monitoring capability, non-destructiveness, and versatility of small molecule fluorescent probes make them indispensable and powerful tools in bioscience research. Self-assembling fluorescent probes are a novel type of material that spontaneously assemble fluorescent dyes with specific molecules into nanoscale structures. Compared with ordinary small molecule fluorescent probes, self-assembled fluorescent probes have higher stability, selectivity, sensitivity, and temporal stability in detection. In recent years, the incidence and mortality of cancer have increased year by year, which has brought great challenges to the safety of human life, and traditional diagnostic methods such as nuclear magnetic resonance, ultrasound diagnosis, and X-ray tomography are time-consuming and have low resolution. The boundary between normal tissue and cancer tissue cannot be accurately distinguished during surgical resection, resulting in the possibility of recurrence after surgery. Fluorescent probes can quickly and efficiently diagnose and label cancerous tumor cells, which is of great significance for cancer discovery and treatment. In this paper, we review the classification of self-assembled fluorescent probes (molecular self-assembled fluorescent probes, nanomaterial self-assembled fluorescent probes and biological macromolecule self-assembled fluorescent probes) that are used in identifying and imaging cancerous tumor tissues. Furthermore, we discuss the current problems faced by self-assembled fluorescent probes through the specific identification and monitoring of enzymes with abnormal contents, active substances and low pH in the tumor microenvironment, hoping to give readers more inspiration.

Visualization of Hg2+ Stress on Plant Health at the Subcellular Level Revealed by a Highly Sensitive Fluorescent Sensor.

The presence of Hg2+ causes substantial stress to plants, adversely affecting growth and health by disrupting cell cycle divisions, photosynthesis, and ionic homeostasis. Accurate visualization of the spatiotemporal distribution of Hg2+ in plant tissues is crucial for the management of Hg pollution; however, the related research is still at its early stage. Herein, a small-molecule amphiphilic fluorescent probe (termed LJTP2) was developed for the specific detection of Hg2+ with a high sensitivity (~16 nM). Fluorescent imaging applications with LJTP2 not only detected the dynamic distribution of Hg2+ within plant cells at the subcellular level but also enabled the understanding of cell membrane health under Hg2+ stress. This study introduces a valuable imaging tool for elucidating the molecular mechanism of Hg2+ stress in plants, demonstrating the potential of the application of small-molecule fluorescent probes in plant science.

Activatable Molecular Probes With Clinical Promise for NIR-II Fluorescent Imaging.

The second near-infrared window (NIR-II) fluorescence imaging has been widely adopted in basic scientific research and preclinical applications due to its exceptional spatiotemporal resolution and deep tissue penetration. Among the various fluorescent agents, organic small-molecule fluorophores are considered the most promising candidates for clinical translation, owing to their well-defined chemical structures, tunable optical properties, and excellent biocompatibility. However, many currently available NIR-II fluorophores exhibit an "always-on" fluorescence signal, which leads to background noise and compromises diagnostic accuracy during disease detection. Developing NIR-II activatable organic small-molecule fluorescent probes (AOSFPs) for accurately reporting pathological changes is key to advancing NIR-II fluorescence imaging toward clinical application. This review summarizes the rational design strategies for NIR-II AOSFPs based on four core structures (cyanine, hemicyanine, xanthene, and BODIPY). These NIR-II AOSFPs hold substantial potential for clinical translation. Furthermore, the recent advances in NIR-II AOSFPs for NIR-II bioimaging are comprehensively reviewed, offering clear guidance and direction for their further development. Finally, the prospective efforts to advance NIR-II AOSFPs for clinical applications are outlined.

Near-Infrared Two-Photon J-Aggregation-Induced Organic Fluorescent Dots with Large Stokes-Shift for Ratiometric Imaging of Hypochlorous Acid in Living Cells and Brains of AD Mice.

Alzheimer's disease (AD) is characterized by multiple toxicity from various biomarkers, and it is very important to monitor the fluctuation of biomarker level in brain tissues to track its early onset, illness progression, and therapeutic effect. Hypochlorous acid (HOCl) is confirmed to be a reliable biomarker to extend the scope of diagnosis. However, the practical applications of the developed conjugated small molecule fluorescent probes for detecting HOCl are often restricted to a large extent by their low solubility, aggregation-caused quenching (ACQ), unsatisfactory fluorescence brightness, small Strokes-shift, and lack of self-correcting ratiometric emitters. In this study, a J-aggregation-induced organic fluorescent dot (PBT dots), is reported which is facilely developed by co-assembling newly designed organic molecule PBT with polymer DSPE-PEG, for ClO- imaging in living brain-derived Endothelial (bEnd.3) cells and brain tissues of AD mice. The developed two-photon PBT dots show NIR emission at 715nm, large Stokes-shift of 245nm, quick response within 2s, ratiometric sensing properties, and favorable blood-brain barrier (BBB) penetrate ability. The results demonstrate that HOCl level is elevated in AD mouse brain, and PBT dot holds promise as an imaging probe to understand and reveal AD pathologies.

Robust Fluorescent Nanoprobe for Rapid Evaluation of the Selenium Supplementation Effect and Imaging.

At present, an increasing number of people pay more attention to selenium-enriched food, but the quality of the selenium-enriched food varies. Therefore, there is an urgent need to develop a new tool to assess the effects of selenium supplementation in foods by rapidly detecting the levels of the metabolite selenium selenocysteine (Sec). In this work, a fluorescent nanoprobe CS-Sec was designed, synthesized, and characterized for Sec detection and imaging in living biosystems, which exhibited the advantages of good biocompatibility, excellent water solubility, high sensitivity, high selectivity, and rapid response (2.5 min) for Sec detection and imaging in vitro and in vivo and evaluation of selenium supplementation in selenium-rich foods. Specifically, CS-Sec was constructed by grafting alkyne groups on organic small-molecule fluorescent probes with azide groups on azido chitosan by click chemistry. A 2,4-dinitrophenyl ether (DNB) with a strong intramolecular charge transfer (ICT) effect was selected as a response group and fluorescence-quenching group, which had excellent chemical specificity toward Sec. In addition, CS-Sec has high selectivity and sensitivity toward Sec over other analytes, and an excellent limit of detection (LOD) is as low as 15 nM. Impressively, CS-Sec has been successfully used to detect and image the concentration of Sec in living HepG2 cells and mouse models with exciting results, indicating that the newly constructed CS-Sec can provide a robust molecule tool for the rapid evaluation of the selenium supplementation effect and imaging in the future.

Redefining Molecular Probes for Monitoring Subcellular Environment: A Perspective.

The development of small-molecule fluorescent probes has revolutionized the monitoring of in vivo physicochemical parameters, offering unprecedented insights into biological processes. In this perspective, we critically examine recent advances and trends in the design and application of fluorescent probes for real-time in vivo monitoring of subcellular environments. Traditional concepts such as membrane potential, microviscosity, and micropolarity have been superseded by more biologically relevant parameters like membrane voltage, tension, and hydration, enhancing the accuracy of physiological assessments. This redefinition not only presents an evolved concept with broader applications in monitoring subcellular dynamics but also addresses the unmet needs of subcellular biology more effectively. We also highlight the limitations of commonly used probes in providing specific information about the redox environment, noting their nonspecificity to oxidants and the influence of various chemical interactions. These probes typically rely on free radical mechanisms and require metal catalysts to react with hydrogen peroxide. They include naphthalimide, fluorescein, BODIPY, rhodamine, cyanine cores to cover the UV-vis-near-infrared window. The motif of this perspective is to provide critical insights into trending fluorescent-based systems employed in real-time or in vivo physicochemical-responsive monitoring, thus aiming to inform and inspire further research in creating robust and efficient fluorescent probes for comprehensive in vivo monitoring applications.

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.

RETRACTION: Diagnostic Value for Methylation in Cervical Cancer Based on a Small-Molecule Fluorescent Probe Targeting DNMT1.

RETRACTION: Diagnostic Value for Methylation in Cervical Cancer Based on a Small-Molecule Fluorescent Probe Targeting DNMT1.

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.

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

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.