Functional Fluorescent Probes Research Articles

Functional fluorescent probe based on amide condensation of coumarin and melatonin for the turn-on detection of calcium ion in vitro and living cell

Due to a physiological significant role of calcium ion, it is essential to develop ways of detecting and monitor the level of Ca2+ ions in living cell. Herein, we rationally designed and synthesized a functional blue fluorescent compound CM based on amide condensation of coumarin and melatonin. Interestingly, it could exhibit an “On-OFF” fluorescence response for Ca2+ in an aqueous environment with 80-fold enhancement in bright blue emission, which is also free from interference of other co-existing cations of high concentration. A detection limit of 0.28 ppm was obtained, and the binding ratio of CM and Ca2+ is further investigated by Job’s plot. After coordination of CM with calcium ion, a significant “Tyndall Effect” can be observed, and blue emissive nanowires induced by aggregation of complex of CM and Ca2+ were further investigated by Dynamic Light Scattering, analysis of Zeta potential, and confocal imaging. The molecular docking of CM with melatonin receptor 1A is also conducted. MTT assay reveals that CM ligand and the corresponding Ca2+ complex are non-cytotoxic. Moreover, CM shows good membrane permeability, and the relative cellular uptake mechanism is mainly dominated by an energy-dependent process. CM and its complex of Ca2+ can be further used as intracellular luminescent imaging probes and their localization in cell organelles is also revealed.

Initial Experience of NIR-II Fluorescence Imaging-Guided Surgery in Foot and Ankle Surgery

Optical imaging in the second near-infrared (NIR-II; 900–1880 nm) window is currently a popular research topic in the field of biomedical imaging. This study aimed to explore the application value of NIR-II fluorescence imaging in footandankle surgeries. A lab-established NIR-II fluorescence surgical navigation system was developed and used to navigate foot and ankle surgeries which enabled obtaining more high-spatial-frequency information and a higher signal-to-background ratio (SBR) in NIR-II fluorescence images compared to NIR-I fluorescence images; our result demonstrates that NIR-II imaging could provide higher-contrast and larger-depth images to surgeons. Three types of clinical applicationscenarios (diabetic foot, calcaneal fracture, and lower extremity trauma) were included in this study. Using the NIR-II fluorescence imaging technique, we observed the ischemic region in the diabetic foot before morphologicalalterations, accurately determined the boundary of the ischemic region in the surgical incision, and fully assessed the blood supply condition of the flap. NIR-II fluorescence imaging can help surgeons precisely judge surgical margins, detect ischemic lesions early, and dynamically trace the perfusion process. We believe that portable and reliable NIR-II fluorescence imaging equipment and additional functional fluorescent probes can play crucial roles in precision surgery.

A Dual Functional Fluorescent Probe Based on Phenothiazine for Detecting Hg2+ and ClO- and its Applications.

For the efficient detection of Hg2+ and ClO-, a double-analyte-responsive fluorescent probe PTB was successfully synthesized by combining N-butyl-3-formyl phenothiazine with hydrazine benzothiazole, and designing a specific reaction site for recognizing two analytes (Hg2+ and ClO-) in a compound. It was shown that probe PTB successfully formed a stable complex with Hg2+ in the coordination ratio of 2:1 by using the strong sulfur affinity of Hg2+, which resulted in a remarkable "turn-off" effect, with a quenching efficiency of 92.5% and four reversible cycles of Hg2+ fluorescence detection. For the fluorescence detection of Hg2+, the response time is fast (≤ 2min) and the detection limit is low (7.8nM), showing extremely high sensitivity, and the performance is obviously better than that of the reported fluorescent probes for detecting Hg2+. In particular, probe PTB has low toxicity and good biocompatibility, and has been successfully used for imaging of Hg2+ in living cells. Moreover, probe PTB uses thioether bond and carbon-nitrogen double bond as reaction sites to detect ClO-, which has large Stokes Shift (149nm), good selectivity, high quenching efficiency (96.5%) and fast time response (about 10s), and successfully detects ClO- in actual water samples. The dual functional fluorescent probe PTB is sensitive for Hg2+ and ClO-. It has been successfully used for making pH fluorescent test paper and imaging detection of exogenous Hg2+ in VSMC cells with low toxicity.

Boron and nitrogen co-doped carbon dots as the dual functional fluorescent probe for Fe3+ and pH detection

Carbon dots doped with boron and nitrogen atoms (B, N-CDs) were designed and prepared by hydrothermal strategy using citric acid, urea and borax as raw materials. The structure and optical properties of B, N-CDs were characterized by various characterization technologies. The results showed that the B, N-CDs exhibited good mono-dispersion, excellent fluorescence stability, and high fluorescence intensity (quantum yield up to 32 %). A dual-functional Fe3+ and pH detection platform based on B, N-CDs was constructed. The Fe3+ sensing method based on B, N-CDs fluorescence quenching showed an excellent linear relationship between 0.044–70 μM (R2 = 0.994), with a detection limit as low as 0.044 μM. Then, this detection method was successfully applied to the high-accuracy detection of Fe3+ in water samples and iron supplement tablets. The study of the quenching mechanism showed that the fluorescence of B, N-CDs was quenched by Fe3+ in two ways: static quenching and dynamic quenching. Moreover, B, N-CDs can also be used as a pH fluorescence sensor in the ranges of pH 2.0–4.8 and 5.2–7.6. The developed sensing system based on B, N-CDs has high selectivity and sensitivity for the detection of Fe3+, and a wide detection range for pH.

Switchable and Functional Fluorophores for Multidimensional Single-Molecule Localization Microscopy.

Multidimensional single-molecule localization microscopy (mSMLM) represents a paradigm shift in the realm of super-resolution microscopy techniques. It affords the simultaneous detection of single-molecule spatial locations at the nanoscale and functional information by interrogating the emission properties of switchable fluorophores. The latter is finely tuned to report its local environment through carefully manipulated laser illumination and single-molecule detection strategies. This Perspective highlights recent strides in mSMLM with a focus on fluorophore designs and their integration into mSMLM imaging systems. Particular interests are the accomplishments in simultaneous multiplexed super-resolution imaging, nanoscale polarity and hydrophobicity mapping, and single-molecule orientational imaging. Challenges and prospects in mSMLM are also discussed, which include the development of more vibrant and functional fluorescent probes, the optimization of optical implementation to judiciously utilize the photon budget, and the advancement of imaging analysis and machine learning techniques.

Rational Design of pH-Independent and High-Fidelity Near-Infrared Tunable Fluorescent Probes for Tracking Leucine Aminopeptidase In Vivo.

Accurate detection of target analytes and generation of high-fidelity fluorescence signals are particularly critical in life sciences and clinical diagnostics. However, the majority of current NIR-I fluorescent probes are vulnerable to pH effects resulting in signal distortion. In this work, a series of fluorescence-tunable and pH-independent probes are reported by combining optically tunable groups of unsymmetric Si-rhodamines and introducing the methoxy instead of the spiro ring on the benzene ring at position 9. To validate the concept, the leucine aminopeptidase response site was introduced into Si-2,6OMe-NH2 with the best optical properties to synthesize Si-LAP for monitoring the intrahepatic LAP in vivo. Therefore, the design approach may provide a new and practical strategy for designing innovative functional fluorescent probes and generating high-stability and high-fidelity fluorescent signals.

A dual functional fluorescent probe based on naphthalimide for detecting Cu2+ and pH and its applications

Cu2+ and pH play a key role in life systems and it is necessary to develop an effective detection method. In view of the significant advantages of the fluorescent probe, we have synthesized a dual functional fluorescent probe NCNB based on naphthalimide for detecting Cu2+ and pH. The probe NCNB is sensitive (LOD = 44.8 nM) and fast for Cu2+ (within 150 s). It can be used not only for qualitative and quantitative detection of Cu2+ in actual water samples, but also for making pH fluorescent test paper with good effect. In addition, the probe NCNB has been successfully used for imaging detection of exogenous Cu2+ in Caco-2 cells with low toxicity.

A novel functional fluorescent probe based on a pyrene derivative for the detection of multiple pollutants

In regards to environmental protection, the detection of metal ions and pesticide residues remains meaningful and challenging. Herein, we report a novel multifunctional fluorescent probe based on a pyrene derivative, 4-[(pyren-1-ylimino)methyl]benzene-1,3-diol (PBD), that can recognize Al3+, Cr3+, Fe3+ and mesotrione with quantification of the sample being tested. In addition, the PBD probe can detect Hg2+ in a “turn-off” fluorescent mode. It is worth noting that the hydrolysis of carbon–nitrogen double bonds in the PBD Schiff base structure is targeted and catalyzed by Fe3+, Al3+ and Cr3+ cations, while mesotrione can also promote the hydrolysis of Schiff bases through the formation of intermolecular hydrogen bonds. Consequently, the PBD probe with weak fluorescence was converted to 1-aminopyrene in a localized state, showing stronger fluorescence. In addition, chelation may lead to fluorescence quenching of the PBD probe in the presence of Hg2+. The quantitative determination of Fe3+, Al3+, Cr3+ and mesotrione is achieved in which the limit of detection (LOD) is calculated to be 0.52, 0.30, 0.32 and 0.70 μM, respectively. Hg2+ detection is realized with an LOD of 1.93 μM. Moreover, the sensitive PBD probe is utilized to determine the amount mesotrione in actual water samples, demonstrating its potential practical applications. In addition, the multifunctional PBD probe can be employed for intracellular imaging of Fe3+ and Hg2+ based on its low cytotoxicity and certain water solubility. The present work describes a very competitive fluorescent probe for the detection of different types of pollutants, which is relevant to future research regarding environmental contaminant monitoring.

Dense and Acidic Organelle-Targeted Visualization in Living Cells: Application of Viscosity-Responsive Fluorescence Utilizing Restricted Access to Minimum Energy Conical Intersection

Cell-imaging methods with functional fluorescent probes are an indispensable technique to evaluate physical parameters in cellular microenvironments. In particular, molecular rotors, which take advantage of the twisted intramolecular charge transfer (TICT) process, have helped evaluate microviscosity. However, the involvement of charge-separated species in the fluorescence process potentially limits the quantitative evaluation of viscosity. Herein, we developed viscosity-responsive fluorescent probes for cell imaging that are not dependent on the TICT process. We synthesized AnP2-H and AnP2-OEG, both of which contain 9,10-di(piperazinyl)anthracene, based on 9,10-bis(N,N-dialkylamino)anthracene that adopts a nonflat geometry at minimum energy conical intersection. AnP2-H and AnP2-OEG exhibited enhanced fluorescence as the viscosity increased, with sensitivities comparable to those of conventional molecular rotors. In living cell systems, AnP2-OEG showed low cytotoxicity and, reflecting its viscosity-responsive property, allowed specific visualization of dense and acidic organelles such as lysosomes, secretory granules, and melanosomes under washout-free conditions. These results provide a new direction for developing functional fluorescent probes targeting dense organelles.

A Functional Fluorescent Probe for Zn2+ and Cu2+ Detection in Food Products Based on Tetraphenylethylene Derivative

A Functional Fluorescent Probe for Zn2+ and Cu2+ Detection in Food Products Based on Tetraphenylethylene Derivative

Organelle Interaction and Drug Discovery: Towards Correlative Nanoscopy and Molecular Dynamics Simulation.

The inter-organelle interactions, including the cytomembrane, endoplasmic reticulum, mitochondrion, lysosome, dictyosome, and nucleus, play the important roles in maintaining the normal function and homeostasis of cells. Organelle dysfunction can lead to a range of diseases (e.g., Alzheimer’s disease (AD), Parkinson’s disease (PD), and cancer), and provide a new perspective for drug discovery. With the development of imaging techniques and functional fluorescent probes, a variety of algorithms and strategies have been developed for the ever-improving estimation of subcellular structures, organelle interaction, and organelle-related drug discovery with accounting for the dynamic structures of organelles, such as the nanoscopy technology and molecular dynamics (MD) simulations. Accordingly, this work summarizes a series of state-of-the-art examples of the recent progress in this rapidly changing field and uncovering the drug screening based on the structures and interactions of organelles. Finally, we propose the future outlook for exciting applications of organelle-related drug discovery, with the cooperation of nanoscopy and MD simulations.

A Novel Functional Fluorescent Probe Based on a Pyrene Derivative for Detection of Multi-Pollutants

A Novel Functional Fluorescent Probe Based on a Pyrene Derivative for Detection of Multi-Pollutants

Dual-Channel Imaging of Amyloid-β Plaques and Peroxynitrite To Illuminate Their Correlations in Alzheimer's Disease Using a Unimolecular Two-Photon Fluorescent Probe.

Alzheimer's disease (AD) involves multiple pathological factors that mutually cooperate and closely contact to form interaction networks for jointly promoting the AD progression. Therefore, the comonitoring of different factors is particularly valuable for elucidating their level dynamics and complex interactions. However, such significant investigations remain a major challenge due to the lack of unimolecular fluorescent probes capable of simultaneous and discriminative visualization of multiple targets. To address this concern, as proof of principle, we rationally designed a unimolecular fluorescent probe to discriminate and simultaneously profile amyloid-β (Aβ) plaques and peroxynitrite (ONOO-), which are both the pronounced AD pathological factors. Herein, a novel ONOO- reaction trigger was installed onto an Aβ plaque binding fluorophore to generate a dual functional fluorescent probe, displaying completely separate spectral responses to Aβ plaques and ONOO- with high selectivity and sensitivity. With this probe, for the first time, we comonitored the distribution and variation of Aβ plaques and ONOO- through two independent fluorescence channels, demonstrating their close apposition and tight correlation during AD course in live cell and mouse models through two-photon imaging mode. Notably, Aβ aggregates induce the neuronal ONOO- generation, which conversely facilitates Aβ aggregation. The two critical events, ONOO- stress and Aβ aggregation, mutually amplify each other through positive feedback mechanisms and jointly promote the AD onset and progression. Furthermore, by coimaging of the level dynamics of Aβ plaques and ONOO-, we found that the cerebral ONOO- is a potential biomarker, which emerges earlier than Aβ plaques in transgenic mouse models. Overall, the dual-channel responsive performance renders this probe as a powerful imaging tool to decipher Aβ plaque-ONOO- interactions, which will facilitate AD-associated molecular pathogenesis elucidation and multitarget drug discovery.

γ‐Glutamyltranspeptidase (GGT)‐Activatable Fluorescence Probe for Durable Tumor Imaging

Abstractγ‐Glutamyltranspeptidase (GGT) is overexpressed in several types of cancer. Existing GGT‐targeting fluorescence probes can image these cancers, but the fluorescent hydrolysis product leaks from the target cancer cells during prolonged incubation or fixation. Here, we present a functionalized fluorescence probe for GGT, 4‐CH2F‐HMDiEtR‐gGlu, which is designed to generate an azaquinone methide intermediate during activation by GGT; this intermediate reacts with intracellular nucleophiles to generate a fluorescent adduct that is trapped inside the cells, without loss of the target enzyme activity. Application of the probe to patient‐derived xenograft (PDX) mice enabled in vivo cancer imaging for a prolonged period and was also compatible with fixation and immunostaining of the cancer tissue.

γ-Glutamyltranspeptidase (GGT)-Activatable Fluorescence Probe for Durable Tumor Imaging.

γ-Glutamyltranspeptidase (GGT) is overexpressed in several types of cancer. Existing GGT-targeting fluorescence probes can image these cancers, but the fluorescent hydrolysis product leaks from the target cancer cells during prolonged incubation or fixation. Here, we present a functionalized fluorescence probe for GGT, 4-CH2 F-HMDiEtR-gGlu, which is designed to generate an azaquinone methide intermediate during activation by GGT; this intermediate reacts with intracellular nucleophiles to generate a fluorescent adduct that is trapped inside the cells, without loss of the target enzyme activity. Application of the probe to patient-derived xenograft (PDX) mice enabled in vivo cancer imaging for a prolonged period and was also compatible with fixation and immunostaining of the cancer tissue.

An ICT-based fluorescent probe guided by theoretical calculation for selectively mapping endogenous GSH in living cells.

Glutathione (GSH) is one of the most essential bio-thiols to maintain the redox balance of organisms which is strongly associated with many physiological processes. Detecting the concentration and mapping the distribution of GSH in the living system is significant to study many related diseases. In this work, we have successfully constructed an ICT-based model to guide the design and synthesis of GSH specific fluorescent probe CF1. A serials spectroscopy test demonstrated that the response of CF1 towards GSH owned large stokes shift (~167nm) and an excellent linear relationship (0-120μM, R2=0.9961). Furthermore, CF1 was successfully applied to image endogenous GSH in different cell lines with high sensitivity. This work is instructive for the oriented synthesis of ICT-based functional fluorescent probe and the further visualization of intracellular targets in the living system.

Development of dual function polyamine-functionalized carbon dots derived from one step green synthesis for quantitation of Cu2+ and S2- ions in complicated matrices with high selectivity.

The study of biologically important Cu2+ and S2- ions has drawn great attention in the recent years since an abnormal level of these ions is an indication for health impairment. Therefore, a reliable strategy for effective fluorescence determination of Cu2+ and S2- ions was developed. Simply, the method based on economical plant-dependent thermolysis procedure for efficient green synthesis of water dispersible luminescent polyamine-based carbon dots (PA@C-dots) utilizes Vitis vinifera juice as precursor with a high quantum yield (32.1%) and good photo-stability. The fluorescent PA@C-dots were characterized by different spectroscopical, physical, and structural techniques. Furthermore, the synthesized PA@C-dots can be used as an efficient dual functional fluorescent probe for the sensitive and selective estimation of Cu2+ and S2- ions. The incorporation of Cu2+ ions and their adsorption on the surface of PA@C-dot skeleton leads to the respectable fluorescence quenching of C-dots (turn-off mode). The Cu2+-PA@C-dot was found to be sensitive to S2- ions. The addition of S2- recovers the fluorescence (turn-on mode) of Cu2+-PA@C-dots, thanks to its capacity for withdrawing Cu2+ from the shell of PA@C-dots. Fluorescence quenching in the range of 0.07-60μM Cu2+ was obtained with LOD and LOQ of 0.02 and 0.066μM, respectively. Sulfide detection provides linearity in the range of 0.8 to 95μM with LOD and LOQ of 0.24 and 0.79μM, respectively. The optimal excitation and emission wavelengths for all experiments are 435nm and 498nm, respectively. Experiment results elucidate that the proposed method is suitable for Cu2+and S2- ion detection in environmental water samples. Graphical abstract Green synthesis of polyamine-functionalized nanoprobe by thermolysis method from plant source as bifunctional sensing platform for determination of Cu2+ and S2- in environmental water samples.

Cyanine-coumarin composite NIR dye based instantaneous -response probe for biothiols detection and oxidative stress assessment of mitochondria

Developing near-infrared (NIR) dye based functional fluorescent probes to analysis the bioactive substances contributes to the understanding of cellular physiopathology. Although there are many fluorescent probes have been developed, the probes can instantaneous response to biothiols is still lacking. In this study, we have designed and synthesized a novel composite NIR dye (Hcyc), which was composed by cyanine and coumarin. Hcyc was further applied to construct a biothiols responsive probe (Hcyc-NO) to assess the oxidative stress status of mitochondria in living cells. The optical properties showed that Hcyc has long absorption and emission wavelength in different solvent system (>720 nm). Interestingly, Hcyc-NO could respond to GSH, Cys, Hcy rapidly (<5 s), the absorption peak of red shifted from 556 nm to 723 nm and fluorescence intensity instantaneously enhanced at 751 nm after adding biothiols. In addition, Hcyc-NO prossesses others excellent sensing properties such as high selectivity and sensitivity. Confocal imaging revealed that Hcyc-NO (1 μM) could target mitochondria and selective image biothiols (GSH, Cys and Hcy) in vitro. Furthermore, the probe could rapidly reflect oxidative stress induced by lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) in vitro and in vivo. These results indicate that Hcyc is an excellent NIR dye, and the probe Hcyc-NO may be an effective tool for instantaneous detection of biothiols and real-time assessing the oxidative stress of cells.

Optical Imaging of Glucose Uptake and Mitochondrial Membrane Potential to Characterize Her2 Breast Tumor Metabolic Phenotypes.

With the large number of women diagnosed and treated for breast cancer each year, the importance of studying recurrence has become evident due to most deaths from breast cancer resulting from tumor recurrence following therapy. To mitigate this, cellular and molecular pathways used by residual disease prior to recurrence must be studied. An altered metabolism has long been considered a hallmark of cancer, and several recent studies have gone further to report metabolic dysfunction and alterations as key to understanding the underlying behavior of dormant and recurrent cancer cells. Our group has used two probes, 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl) amino]-2-deoxyglucose (2-NBDG) and tetramethyl rhodamine ethyl ester (TMRE), to image glucose uptake and mitochondrial membrane potential, respectively, to report changes in metabolism between primary tumors, regression, residual disease, and after regrowth in genetically engineered mouse (GEM)-derived mammospheres. Imaging revealed unique metabolic phenotypes across the stages of tumor development. Although primary mammospheres overexpressing Her2 maintained increased glucose uptake ("Warburg effect"), after Her2 downregulation, during regression and residual disease, mammospheres appeared to switch to oxidative phosphorylation. Interestingly, in mammospheres where Her2 overexpression was turned back on to model recurrence, glucose uptake was lowest, indicating a potential change in substrate preference following the reactivation of Her2, reeliciting growth. Our findings highlight the importance of imaging metabolic adaptions to gain insight into the fundamental behaviors of residual and recurrent disease. IMPLICATIONS: This study demonstrates these functional fluorescent probes' ability to report metabolic adaptations during primary tumor growth, regression, residual disease, and regrowth in Her2 breast tumors.

Naphthalimide-sulfonamide fused dansyl-sulfonamide fluorescent probe for tracking glutathione of lysosome with a dual-emission manner

Developing glutathione-specific fluorescent probes with novel fluorescence-responsive manners can enrich the applications of probes in living cells. In this work, we designed and synthesized a dual fluorophores (naphthalimide and dansyl) co-modified fluorescent probe, which displayed a high selectivity toward glutathione (GSH). In living cells, this probe could specifically visualize the GSH of lysosome and accompanied by two different fluorescence emissions. One is green emission that can be assigned to the nucleophilic substitution product (GSH-linked naphthalimide) while the other is orange emission that can be derived from the cleavage product (dansyl). Especially, such dual-emission bioimaging in living cells had a spatiotemporal and synchronous manner. It is expected to provide an alternative strategy for designing the functional fluorescent probes to be applied in multi-color bioimaging in future.