Conceptually innovative fluorophores for functional bioimaging.

Fluorophore chemistry is at the forefront of bioimaging, revolutionizing the visualization of biological processes with unparalleled precision. From the serendipitous discovery of mauveine in 1856 to cutting-edge fluorophore engineering, this field has undergone transformative evolution. Today, the synergy of chemistry, biology, and imaging technologies has produced diverse, specialized fluorophores that enhance brightness, photostability, and targeting capabilities. This review delves into the history and innovation of fluorescent probes, showcasing their pivotal role in advancing our understanding of cellular dynamics and disease mechanisms. We highlight groundbreaking molecules and their applications, envisioning future breakthroughs that promise to redefine biomedical research and diagnostics.

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.

Nanomole Scale Screening of Fluorescent RNA-Methyltransferase Probes Enables the Discovery of METTL1 Inhibitors.

RNA methylation is a metabolic process validated for its association with various diseases, and thus, RNA methyltransferases (MTases) have become increasingly important in drug discovery. Yet, most frequently utilized RNA MTase assays are limited in their throughput and hamper this rapidly evolving field of medicinal chemistry. In this study, we describe a modular nanomole scale building block system that allowed the identification of tailored fluorescent MTase probes to unlock a broad selection of MTase drug targets for fluorescence-based binding assays. Probe candidates were initially prepared on a 4 nanomole scale and could be tested directly from crude reaction mixtures to allow rapid probe identification and optimization. Using an alkyne-azide click late-stage functionalization strategy and in silico protein databank mining, we established a selection of fluorescent probes suitable for relevant drug targets from the METTL and NSUN families, as well as bacterial and viral MTases. Using this concept, a high-throughput screening on the unexplored drug target METTL1 discovered three hit compounds with micromolar potency providing a first-in-class starting point for METTL1 drug discovery.

Diaminonaphthalene Boronic Acid (DANBA): New Approach for Peroxynitrite Sensing Site.

Selective detection of reactive oxygen species (ROS) is vital for studying their role in brain diseases. Fluorescence probes can distinguish ONOO- species from other ROS; however, their selectivity toward ONOO- species depends on the ONOO- recognition group. Aryl-boronic acids and esters, which are common ONOO- recognition groups, are not selective for ONOO- over H2O2. In this study, we developed a diaminonaphthalene (DAN)-protected boronic acid as a new ONOO- recognition group that selectively reacts with ONOO- over H2O2 and other ROS. Three DAN-protected boronic acid (DANBA)-based fluorophores that emit fluorescence over visible to near-infrared (NIR) regions, Cou-BN, BVP-BN, and HDM-BN, and their aryl-boronic acid-based counterparts (Cou-BO, BVP-BO, and HDM-BO), were developed. The DANBA-based probes exhibited enhanced selectivity toward ONOO- over that of their control group, as well as universality in MTT assays and in vitro experiments with PC12 cells. The NIR-emissive HDM-BN was optimized to delineate in vivo ONOO- levels in mouse brains with Parkinson's disease. This DAN-protected boronic acid belongs to a new generation of recognition groups for developing ONOO- probes, and this strategy could be extended to other common hydroxyl-containing dyes to detect ONOO- levels in complex biological systems and processes.

Near-infrared fluorescent probe visual detection of Hg2+ and its application in biological system and ecological system.

Mercury ion (Hg2+), a heavy metal cation with greater toxicity, is widely present in the ecological environment and has become a serious threat to human health and environmental safety. Currently, developing a solution to simultaneously visualize and monitor Hg2+ in environmental samples, including water, soil, and plants, remains a great challenge. In this work, we created and synthesized a near-infrared fluorescent probe, BBN-Hg, and utilized Hg2+ to trigger the partial cleavage of the carbon sulfate ester in BBN-Hg as a sensing mechanism, and the fluorescence intensity of BBN-Hg was significantly enhanced at 650 nm, thus realizing the visualization of Hg2+ with good selectivity (detection limit, 53 nM). In live cells and zebrafish, the probe BBN-Hg enhances the red fluorescence signal in the presence of Hg2+, and successfully performs 3D imaging on zebrafish, making it a powerful tool for detecting Hg2+ in living systems. More importantly, with BBN-Hg, we are able to detect Hg2+ in actual water samples, soil and plant seedling roots. Furthermore, the probe was prepared as a test strip for on-site determination of Hg2+ with the assistance of a smartphone. Therefore, this study offers an easy-to-use and useful method for tracking Hg2+ levels in living organisms and their surroundings.

Perovskite Probe-Based Machine Learning Imaging Model for Rapid Pathologic Diagnosis of Cancers.

Accurately distinguishing tumor cells from normal cells is a key issue in tumor diagnosis, evaluation, and treatment. Fluorescence-based immunohistochemistry as the standard method faces the inherent challenges of the heterogeneity of tumor cells and the lack of big data analysis of probing images. Here, we have demonstrated a machine learning-driven imaging method for rapid pathological diagnosis of five types of cancers (breast, colon, liver, lung, and stomach) using a perovskite nanocrystal probe. After conducting the bioanalysis of survivin expression in five different cancers, high-efficiency perovskite nanocrystal probes modified with the survivin antibody can recognize the cancer tissue section at the single cell level. The tumor to normal (T/N) ratio is 10.3-fold higher than that of a conventional fluorescent probe, which can successfully differentiate between tumors and adjacent normal tissues within 10 min. The features of the fluorescence intensity and pathological texture morphology have been extracted and analyzed from 1000 fluorescence images by machine learning. The final integrated decision model makes the area under the receiver operating characteristic curve (area under the curve) value of machine learning classification of breast, colon, liver, lung, and stomach above 90% while predicting the tumor organ of 92% of positive patients. This method demonstrates a high T/N ratio probe in the precise diagnosis of multiple cancers, which will be good for improving the accuracy of surgical resection and reducing cancer mortality.

Labeling selectivity of lipid droplets fluorescent probes: Twisted intramolecular charge transfer (TICT) vs intramolecular charge transfer (ICT).

Fluorescence imaging technology is a versatile and essential tool in the field of biomedical research. To obtain excellent imaging results, the precise labeling of fluorescent probes is an important prerequisite. Nevertheless, the labeling selectivity of most fluorescent probes is not satisfactory, new design concepts are desperately needed. In this context, two isomeric lipid droplets (LDs) fluorescent probes Lipi-Cz-1 and Lipi-Cz-2 have been sophisticatedly developed with TICT and ICT-emitting characteristic, respectively. The more environmentally sensitive TICT-emitting Lipi-Cz-1 exhibits a significantly enhanced labeling selectivity in LDs imaging compared to the ICT-emitting Lipi-Cz-2, sufficiently illustrating the effectiveness of TICT-emitting characteristic in improving labeling selectivity. Additionally, Lipi-Cz-1 displays high photostability and biocompatibility. These advantages enable Lipi-Cz-1 to be finely applied in multimode fluorescence imaging, e.g. time-lapse 3D confocal imaging to monitor changes of the number and size of LDs during starvation, two-photon 3D imaging to compare the variations of LDs in various liver tissues, and STED super-resolution imaging to visualize the nanoscale LDs with the resolution of 65 nm. Overall, these imaging findings validate the effectiveness of the new strategy for improving the labeling selectivity.

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.

Ratiometric fluorescent probe with AIE characteristics for hypochlorite detection and biological imaging.

It is very important and highly valuable to detect ClO- in samples and living cells with accuracy and speed. In this work, a novel fluorescent probe NA was prepared from 4-bromo-1,8-naphthalic anhydride by acylation reaction and Suzuki coupling reaction and used for the detection of ClO-. Thiomethyl serves as the recognition group for probe NA, while naphthalimide serves as fluorescent chromophore. The probe exhibited an extremely pronounced blue shift from yellow to blue fluorescence within 1 min after the addition of hypochlorite (ClO-). The probe demonstrates high sensitivity to ClO- with a limit of detection (LOD) of 1.22 µM. Also, probe NA demonstrates excellent selectivity and immunity to interference. Additionally, simple fluorescent test strips containing probe NA were prepared in this study, enabling rapid detection of ClO- in water samples. And NA had been effectively used to image endogenous and exogenous ClO-fluorescence in living cells. The results suggest that probe NA has significant potential for portable detection and biological applications.

Multiplex Profiling of miR-122 for Preclinical and Clinical Evaluation of Drug-Induced Liver Injury by a Full-Scale Platform.

Diagnostic and monitoring for drug-induced liver injury (DILI) predominantly rely on serum aminotransferases. However, owing to their widespread expression across multiple organs, a significant challenge emerges from the absence of reliable biomarkers for DILI diagnosis. Herein, we introduce a concept for DILI detection, circumventing the nonspecific elevation and delayed release of aminotransferases and then straightforwardly focusing on the core feature of DILI, abnormal gene expression caused by drug overdose. The developed full-scale platform integrates the properties of spherical nucleic acids with elaborately designed fluorescence in situ hybridization sequences, enabling the sensitive and specific profiling of drug-overdosed miR-122 expression alterations across molecular, cellular, organismal, and clinical scales and effectively bypassing the phenotypic features of disease. Furthermore, the diagnostic efficacies of serum and total RNA extracted from both mouse and human blood samples for DILI diagnosis were analyzed using the receiver operating characteristic curve and principal component analysis. We anticipate that this universal platform holds potential in facilitating DILI diagnosis, therapeutic evaluation, and prognosis.

Visualizing the Cell-Matrix Interactions and Cytoskeleton of Disseminated Tumor Cells.

Tumor cells often leave the primary tumor mass and get settled in a foreign tissue years before the development of overt metastases, exhibiting the highly inefficient nature of metastatic colony formation. In fact, the tumor cells that disseminate into distant organs and subsequently invade the parenchyma of these organs rarely proceed to found actively growing metastatic colonies. Instead, the majority of these tumor cells undergo prolonged proliferative arrest unless they are swiftly eliminated by the immune system. Together, these observations indicate that the proliferative capacity of the disseminated tumor cells (DTCs) serves as a key determinant of the efficiency of metastasis, highlighting the need to better understand the mechanism governing the proliferation of these cells. Recent studies are unveiling the importance of the interactions between DTCs and the microenvironment of the host tissue in regulating the proliferation of DTCs. However, the details of such interactions remain to be fully delineated. Here I describe the methods for visualizing and analyzing the interactions between DTCs and the extracellular matrix (ECM) components of the host tissue as well as the cytoskeleton of the DTCs that support these interactions. The methods described here will facilitate the study of how DTCs interact with the ECM of their host tissue, which will be crucial for elucidating the mechanism that underlies the regulation of DTC proliferation by the DTC-ECM interactions.

Quantifying Membrane Alterations with Tailored Fluorescent Dyes: A Rapid Antibiotic Resistance Profiling Methodology.

Accurate detection of bacterial antibiotic sensitivity is crucial for theranostics and the containment of antibiotic-resistant infections. However, the intricate task of detecting and quantifying the antibiotic-induced changes in the bacterial cytoplasmic membrane, and their correlation with other metabolic pathways leading to antibiotic resistance, poses significant challenges. Using a novel class of 4-aminophthalimide (4AP)-based fluorescent dyes with precisely tailored alkyl chains, namely 4AP-C9 and 4AP-C13, we quantify stress-mediated alterations in E. coli membranes. Leveraging the unique depth-dependent positioning and environment-sensitive fluorescence properties of these dyes, we detect antibiotic-induced membrane damage through single-cell imaging and monitoring the fluorescence peak maxima difference ratio (PMDR) of the dyes within the bacterial membrane, complemented by other methods. The correlation between the ROS-induced cytoplasmic membrane damage and the PMDR of dyes quantifies sensitivity against bactericidal antibiotics, which correlates to antibiotic-induced lipid peroxidation. Significantly, our findings largely extend to clinical isolates of E. coli and other ESKAPE pathogens like K. pneumoniae and Enterobacter subspecies. Our data reveal that 4AP-Cn probes can potentially act as precise scales to detect antibiotic-induced membrane damage ("thinning") occurring at a subnanometer scale through the quantification of dyes' PMDR, making them promising membrane dyes for rapid detection of bacterial antibiotic resistance, distinguishing sensitive and resistant infections with high specificity in a clinical setup.

Lanthanide metal-organic frameworks as ratiometric fluorescent probes for real-time monitoring of PFOA photocatalytic degradation process.

The assessment of perfluorooctanoic acid (PFOA) photocatalytic degradation usually involves tedious pre-treatment and sophisticated instrumentation, making it impractical to evaluate the degradation process in real-time. Herein, we synthesized a series of lanthanide metal-organic frameworks (Ln-MOFs) with outstanding fluorescent sensing properties and applied them as luminescent probes in the photocatalytic degradation reaction of PFOA for real-time evaluation. As the catalytic reaction proceeds, the fluorescence color changes significantly from green to orange-red due to the different interaction mechanisms between the electron-deficient PFOA and smaller radius F- with the ratiometric fluorescent probe MOF-76 (Tb: Eu = 29:1). The limit of detection (LOD) was calculated to be 0.0127 mM for PFOA and 0.00746 mM for F-. In addition, the conversion rate of the catalytic reaction can be read directly based on the chromaticity value by establishing a three-dimensional relationship graph of G/R value-conversion rate-time (G/R indicates the ratio between green and red luminance values in the image.), allowing for real-time and rapid tracking of the PFOA degradation. The recoveries of PFOA and F- in the actual water samples were 99.3-102.7% (RSD = 2.2-4.4%) and 100.7-105.3% (RSD = 3.9-6.8%), respectively. Both theoretical calculations and experiments reveal that the detection mechanism was attributed to the photoinduced electron transfer and energy transfer between the analytes and the probe. This method simplifies the sample analysis process and avoids the use of bulky instruments, and thus has great potential on the design and development of quantitative time-resolved visualization methods to assess catalytic performance and reveal mechanisms.

Syntheses and Applications of Coumarin-Derived Fluorescent Probes for Real-Time Monitoring of NAD(P)H Dynamics in Living Cells across Diverse Chemical Environments.

Fluorescent probes play a crucial role in elucidating cellular processes, with NAD(P)H sensing being pivotal in understanding cellular metabolism and redox biology. Here, the development and characterization of three fluorescent probes, A, B, and C, based on the coumarin platform for monitoring of NAD(P)H levels in living cells are described. Probes A and B incorporate a coumarin-cyanine hybrid structure with vinyl and thiophene connection bridges to 3-quinolinium acceptors, respectively, while probe C introduces a dicyano moiety for replacement of the lactone carbonyl group of probe A which increases the reaction rate of the probe with NAD(P)H. Initially, all probes exhibit subdued fluorescence due to intramolecular charge transfer (ICT) quenching. However, upon hydride transfer by NAD(P)H, fluorescence activation is triggered through enhanced ICT. Theoretical calculations confirm that the electronic absorption changes upon the addition of hydride to originate from the quinoline moiety instead of the coumarin section and end up in the middle section, illustrating how the addition of hydride affects the nature of this absorption. Control and dose-response experiments provide conclusive evidence of probe C's specificity and reliability in identifying intracellular NAD(P)H levels within HeLa cells. Furthermore, colocalization studies indicate probe C's selective targeting of mitochondria. Investigation into metabolic substrates reveals the influence of glucose, maltose, pyruvate, lactate, acesulfame potassium, and aspartame on NAD(P)H levels, shedding light on cellular responses to nutrient availability and artificial sweeteners. Additionally, we explore the consequence of oxaliplatin on cellular NAD(P)H levels, revealing complex interplays between DNA damage repair, metabolic reprogramming, and enzyme activities. In vivo studies utilizing starved fruit fly larvae underscore probe C's efficacy in monitoring NAD(P)H dynamics in response to external compounds. These findings highlight probe C's utility as a versatile tool for investigating NAD(P)H signaling pathways in biomedical research contexts, offering insights into cellular metabolism, stress responses, and disease mechanisms.

Sulfur-based fluorescent probes for biological analysis: A review.

The widespread adoption of small-molecule fluorescence detection methodologies in scientific research and industrial contexts can be ascribed to their inherent merits, including elevated sensitivity, exceptional selectivity, real-time detection capabilities, and non-destructive characteristics. In recent years, there has been a growing focus on small-molecule fluorescent probes engineered with sulfur elements, aiming to detect a diverse array of biologically active species. This review presents a comprehensive survey of sulfur-based fluorescent probes published from 2017 to 2023. The diverse repertoire of recognition sites, including but not limited to N, N-dimethylthiocarbamyl, disulfides, thioether, sulfonyls and sulfoxides, thiourea, thioester, thioacetal and thioketal, sulfhydryl, phenothiazine, thioamide, and others, inherent in these sulfur-based probes markedly amplifies their capacity for detecting a broad spectrum of analytes, such as metal ions, reactive oxygen species, reactive sulfur species, reactive nitrogen species, proteins, and beyond. Owing to the individual disparities in the molecular structures of the probes, analogous recognition units may be employed to discern diverse substrates. Subsequent to this classification, the review provides a concise summary and introduction to the design and biological applications of these probe molecules. Lastly, drawing upon a synthesis of published works, the review engages in a discussion regarding the merits and drawbacks of these fluorescent probes, offering guidance for future endeavors.

A cyanine based fluorescent probe for detecting hypochlorite in vitro and in vivo.

Hypochlorite (ClO-) is recognized as a bioactive substance that plays a crucial role in various physiological and pathological processes. The increase of ClO- content in cells is a key factor in the early atherosclerosis lesions, which are closely linked to cardiovascular and cerebrovascular diseases. Therefore, the development of an efficient and sensitive method for detecting hypochlorite in tap water, serum, and living cells, including animal model in vivo is of paramount importance. In this study, a novel fluorescent probe (Cy-F) based on the cyanine group was designed for the specific detection of ClO-, demonstrating exceptional selectivity, high sensitivity, and rapid response. The probe successfully detected ClO- in tap water and serum with a limit of detection (LOD) of 2.93 × 10-7 M, showcasing excellent anti-interference capabilities. Notably, the probe exhibited good biocompatibility, low biological toxicity, and proved effective for detecting and analyzing ClO- in live cells and zebrafish. This newly developed probe offers a promising approach and valuable tool for detecting ClO- with biosafety considerations, paving the way for the design of functional probes tailored for future biomedical applications.

Water-soluble near-infrared AgInS2 quantum dots for Ca2+ detection and bioimaging.

Calcium ions (Ca2+) are key players in intracellular signaling as second messengers and play a pivotal role in various physiological processes. In this study, near-infrared water-soluble AgInS2 quantum dots (AIS QDs) for Ca2+ detection were synthesized by a one-step hydrothermal method. The fluorescence quantum yield (PL QY) of the quantum dots was as high as 23.99 %. With low cytotoxicity and good fluorescence properties, as well as short reaction time, the ternary AIS QDs have excellent synthesis efficiency and quantum yield, which are advantageous for Ca2+ detection and bioimaging applications. The fluorescence quenching of the quantum dots showed a clear linear relationship with calcium ion concentration in the range of 0-250 µM (detection limit: 0.65 µM). Confocal imaging experiments demonstrate the excellent biofluorescence imaging capability of AIS QDs. By tuning the Ag/In molar ratio, AIS QDs can achieve fluorescence emission in the near-infrared wavelength band (620-700 nm), and the near-infrared fluorescence imaging has deeper tissue penetration, less tissue absorption and photodamage, and lower interference of spontaneous fluorescence, which further expands the potential of QDs for bioimaging applications.

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.

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.

Spectroscopic Study of a Novel Binaphthyl Amine Fluorescent Probe for Chiral Recognition of D/L-Lysine.

Lysine plays a crucial role in promoting development, enhancing immune function, and improving the function of central nervous system tissues. The two configurational isomers of amino acids have significantly different effects. Currently, methods for chiral recognition of lysine have been reported; however, previous detection methods have drawbacks such as expensive equipment and complicated detection processes. Fluorescence analysis, on the other hand, boasts high sensitivity, strong selectivity, and simple operation. In this study, we synthesized four novel Binaphthyl-Amine (BINAM)-based fluorescent probes capable of specifically identifying the L-configuration of lysine among the twenty amino acids that constitute human proteins. The enantiomeric fluorescence enhancement ratio (ef or ΔIL/ΔID) reached up to 15.29, demonstrating high enantioselectivity. In addition, we assessed the probe's recognition capabilities under varying pH levels, reaction times, and metal ion conditions, along with its limit of detection (LOD) and quantum yield. Our results suggest that this probe serves as a highly stable tool for the detection of chiral lysine.