Selective Fluorescence Imaging Research Articles

Development of a urea-bond cleavage reaction induced by nitric oxide for fluorescence imaging.

Nitric oxide (NO) is a multifunctional signalling molecule with indispensable roles in physiological processes, but its abnormal production is implicated in various disease conditions. Detecting NO is crucial for interrogating its biological roles. Although many o-phenylenediamine-based fluorescent probes have been developed, only a small fraction has been employed in vivo. Moreover, these probes largely require direct modifications of the fluorophore backbones to render NO responsiveness, which restricts the general applicability of o-phenylenediamine-based probe designs to other types of fluorophores. Here, we report the rational development, optimization, and application of a NO-induced urea-bond cleavage reaction for selective fluorescence detection and imaging of NO in living systems. Through rational design and extensive screening, we identified a 2-aminophenylurea-derived functionality that can react with NO through N-nitrosation, acyltriazole formation, and hydrolysis to induce the cleavage of the urea bond and release of the amino-containing coumarin fluorophore. By caging different amino-containing fluorophore scaffolds with the 2-aminophenylurea-derived functionality, we modularly developed a series of NO fluorescent probes with different excitation and emission profiles for the detection of NO in aqueous solutions and live cells. Among these probes, the near-infrared probe has been demonstrated to enable in vivo fluorescence visualization of elevated endogenous levels of NO in a murine inflammation model. Overall, this study provides a NO-induced urea-bond cleavage reaction and establishes the utility of this reaction for the general and modular development of NO fluorescent probes, thus opening new opportunities for studying and manipulating NO in biological systems.

A fluorescent ratiometric potassium sensor based on IPG4-silica microparticles for selective detection and fluorescence imaging of potassium cations.

Potassium cations play many important roles in living organisms, especially in electro-physiology, since they are involved in neurotransmission and muscle contractions. We report the synthesis of a ratiometric fluorescent microsensor for potassium (K+) detection, based on the fluorescent probe ION potassium green 4. Potassium-sensitive fluorescent microparticles were obtained by using silica as the core material. We obtained silica-based microsensors with sizes in the micrometer range, spherical shapes, good monodispersity, optimal selectivity and a sensitivity range of 0 to 40 mM. The microsensors also proved to be non-toxic in cell cultures and suitable for fluorescence imaging, offering new possibilities for non-invasive optical detection, quantification and in situ monitoring of K+ variations in cell culture systems.

Treatment evaluation of Rheumatoid arthritis by in situ fluorescence imaging of the Golgi cysteine

Rheumatoid arthritis (RA) is a long-term systemic inflammatory disease that causes severe joint pain. Golgi stress caused by redox imbalance significantly involves in acute and chronic inflammatory diseases, in which cysteine (Cys), as a representative reducing agent, may be an effective biomarker for RA. Hence, in order to achieve RA early detection and drugs evaluation, based on our previous work about innovative Golgi-targeting group, we established a phenylsulfonamide-modified fluorescence probe, Golgi-Cys, for the selective fluorescence imaging of Cys in Golgi apparatus in vivo. By application of Golgi-Cys, the Cys changes under Golgi stress in cells were elucidated. More importantly, we found that the probe can be effectively utilized for the RA detection and treatment evaluation in situ.

Developing fluorescence-improved fluoran dyes and applying for a copper selective and sensitive fluorescent probe and cell imaging

Developing fluoran dyes for fluorescent material is significant but challenging because they are notorious for terrible luminescent efficiency. In this manuscript, two fluoran dyes (F3 and F4) were synthesized by the introduction of thiazole and indazole into the fluoran skeleton, which showed colored and fluorescent spirolactone opening form in a wide pH range (F3: 1–10, F4: 1–13). In addition, the absorption and emission spectra of methyl esterification products F3Me and F4Me showed large Stokes shifts (over 75 nm) in water and achieved an improved fluorescence quantum yield of 30.1% in CHCl3. Based on the dye F4Me, a new fluorescent probe F4HP was developed, which showed high selectivity and high sensitivity (LOD of 2.0 nM) to copper ions in H2O/CH3CN (v/v, 3/2) system. Moreover, the sensing experiment was allowed in the pH range of 4.0–7.5. Furthermore, the F4HP exhibits suitable cell cytotoxicity and can be applied for imaging intracellular copper ions by confocal fluorescence imaging technology.

ROS-activated selective fluorescence imaging of cancer cells via the typical LDH-based nanozyme

The detection and treatment based on tumor microenvironment (TME) are essential in cancer therapy. Currently, nanomaterials with mimic-enzyme property have attracted tremendous interest and the regulation of the sensing and catalytical activity inside the living cells remains a challenge. Herein, a reactive oxygen species (ROS) driven nanozyme, with selective activated fluorescence imaging and multiple enzyme activity (including peroxidase, superoxide dismutase and catalase), was achieved via the confinement of CDs on the ultrathin CuAl-LDH (denoted as CDs/LDHzyme). This unique CDs/LDHzyme demonstrate H2O2-responsive turn-on fluorescence via the self-regulating electron transfer process between Cu2+ and CDs. Furthermore, generated Cu+ through this electron transfer process can be beneficial for the POD-like activity, resulting an outstanding catalytic efficiency with the maximum initial velocity (Vmax) as high as 2.002 × 10−7 M s−1, which is 229.9% higher than the natural peroxidase enzyme (Horseradish Peroxidase, HRP). Structure characterization and energy level calculation can further prove the mechanism of this self-regulating electron transfer process. These findings open an avenue to enable the utilization of nanozyme as a remedial nanoplatform to the selective fluorescence imaging and nanozyme-initiated chemodynamic therapy (NCDT).

Rational Construction of Protein-Mimetic Nano-Switch Systems Based on Secondary Structure Transitions of Synthetic Polypeptides.

The manipulation of the flexibility/rigidity of polymeric chains to control their function is commonly observed in natural macromolecules but largely unexplored in synthetic systems. Herein, we construct a series of protein-mimetic nano-switches consisting of a gold nanoparticle (GNP) core, a synthetic polypeptide linker, and an optically functional molecule (OFM), whose biological function can be dynamically regulated by the flexibility of the polypeptide linker. At the dormant state, the polypeptide adopts a flexible, random-coiled conformation, bringing GNP and OFM in close proximity that leads to the "turn-off" of the OFM. Once treated with alkaline phosphatase (ALP), the nano-switches are activated due to the increased separation distance between GNP and OFM driven by the coil-to-helix and flexible-to-rigid transition of the polypeptide linker. The nano-switches therefore enable selective fluorescence imaging or photodynamic therapy in response to ALP overproduced by tumor cells. The control over polymer flexibility represents an effective strategy to manipulate the optical activity of nano-switches, which mimics the delicate structure-property relationship of natural proteins.

Highly selective fluorescent probe in aqueous solution based on coumarin Schiff base for detecting Cu2+ and specific biosensing of glutathione in mitochondria

The concentration change of overexpressed mitochondrial glutathione (mGSH) in cancer cells can effectively induce cell apoptosis, therefore the development of mGSH -activated turn-on fluorescent probe for cancer cells is extremely beneficial for chemotherapy research. Herein, coumarin Schiff base fluorescent probe N'-((7-(diethylamino)-2-oxo-2H-chromen-3-yl) methylene) picolinohydrazide (NCMP) was prepared, which exhibited selective fluorescence quenching upon the addition of Cu2+. The conjugate NCMP-Cu2+ complex could be used to selectively detect GSH, presenting an “ON-OFF-ON” fluorescence response pattern. The detection limits were 1.98×10-8 M for Cu2+ and 8.20×10-8 M for GSH, respectively. The binding mode of Cu2+ ion with NCMP was ascertained by Job plot, ESI-MS, FT-IR and DFT methods. Probes NCMP-Cu2+ was almost non-toxic, which could be employed for selective fluorescence imaging of GSH in zebrafish and mitochondria of living cells. Consequently, this developed sensor platform has potential application value for future cancer research and biomedical diagnostics.

A Space-Dependent 'Enzyme-Substrate' Type Probe based on 'Carboxylesterase-Amide Group' for Ultrafast Fluorescent Imaging Orthotopic Hepatocellular Carcinoma.

Fast and selective fluorescence imaging for a biomarker to related-disease diagnosis remains a significant challenge due to complex physical environment. Human carboxylesterase (CE) is expected to be a potential biomarker of hepatocellular carcinoma (HCC) to improve the accuracy of diagnosis. However, existing probes for CE has slow response rate and low selectivity. Herein, the amide group is selected as CE-responsive sites based on the "substrate-hydrolysis enzymatic reaction" approach. From a series of off-on probes with leave groups in the amide unit, probe JFast is screened with the optimal combination of rapid response rate and high selectivity toward CE. JFast requires only 150s to reach the maximum fluorescence at 676nm in the presence of CE and free from the interference of other esterase. Computational docking simulations indicate the shortest distance between the CE and active site of JFast . Cell and in vivo imaging present that the probe can turn on the liver cancer cells and tumor region precisely. Importantly, JFast is allowed to specifically image orthotopic liver tumor rather than metastatic tumor and distinguish human primary liver cancer tissue from adjacent ones. This study provides a new tool for CE detection and promotes advancements in accurate HCC diagnosis.

Enhancing the Selectivity of Leucine Aminopeptidase Near-Infrared Fluorescent Probes for Assisting in Surgical Tumor Resection

Selective fluorescence imaging of analytes is a challenge for monitoring diseases as homologues interfere with the imaging agents. Leucine aminopeptidase (LAP), a kind of protease, is related to tumor pathogenesis. The known LAP fluorescent probes based on leucine recognition have limited selectivity. Herein, a selective t-butyl-alanine recognition unit for LAP through the ligand regulation strategy is prepared as a new near-infrared (NIR) fluorescent probe (DCM-LAP) having a large Stokes shift of 214 nm and a high sensitivity with a detection limit of 168 mU/L. DCM-LAP has an enhanced response toward LAP with NIR fluorescence at 656 nm based on intramolecular charge transfer. The probe is selective without being interfered with by biological enzymes including the aminopeptidase N (APN). DCM-LAP can image LAP activity in living cells. It can also visualize the cell invasion and migration processes. DCM-LAP is employed in the real-time imaging of LAP in tumor-bearing nude mice and guides in the accurate resection of breast tumors. It also distinguishes tumor tissues from normal with a high tumor-to-normal ratio (9.8). The DCM-LAP probe can thus assist in the investigations of LAP-associated clinical disease.

Three birds one stone: an enzyme-activatable theragnostic agent for fluorescence diagnosis, photodynamic and inhibitor therapies.

AX11890, an inhibitor of overexpressed enzyme, KIAA1363, in some breast cancers, was conjugated with a benzo[a]phenothiazinium photosensitizer to develop a tumor micro-environment-responsive photosensitizer NBS-L-AX. In normal cells, the special geometry of NBS-L-AX causes the fluorescence and photodynamic therapeutic (PDT) effect of NBS-L to be quenched. In cancer cells, when allowed to interact with the enzyme KIAA1363, the geometry of NBS-L-AX changes such that it becomes fluorescent and photodynamically active. Thus, the material of NBS-L-AX serves as an activated imaging and PDT treatment agent for breast cancers. In addition, NBS-L-AX also shows a selective inhibition effect against breast cancer cells.

Rational design of a turn-on near-infrared fluorescence probe for the highly sensitive and selective monitoring of carboxylesterase 2 in living systems.

In vivo selective fluorescence imaging of carboxylesterase 2 (CES2) remains a great challenge because existing fluorescence probes can potentially suffer from interference by other hydrolases. In addition, some fluorescent probes that have been separately reported for measuring CES2 activity in vitro are affected by autofluorescence and absorption of the biological matrix due to their limited emission wavelength or short Stokes shift. Herein, based on the substrate preference and catalytic performance of CES2, a novel and NIR fluorescent probe was developed, in which a hemi-cyanine dye ester derivative was used as the basic fluorescent group. In the presence of CES2, the probe was hydrolyzed to expose the fluorophore CZX-OH (λabs ∼ 675 nm, λem ∼ 850 nm), which led to a notable red-shift in the fluorescence (∼175 nm) spectrum. Confocal imaging of cells and live mice demonstrated that the fluorescent signal of this probe was related to the real activities of CES2 in cancer cells. All these results will powerfully promote the screening of CES2 regulators and the analysis of CES2-related physiological and pathological processes.

Synthesis of First Coumarin Fluorescent Dye for Actin Visualization.

Selective fluorescence imaging of actin protein hugely depends on the fluorescently labeled actin-binding domain (ABD). Thus, it is always a challenging task to image the actin protein (in vivo or in vitro) directly with an ABD-free system. To overcome the limitations of actin imaging without an ABD, we have designed a facile and cost-effective red fluorescent coumarin dye 7-hydroxy-4-methyl-8-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-ylimino)methyl-2H-chromen-2-one (CTC) for actin binding. The selective binding of the dye was investigated using the gut and eye of the model organism Drosophila melanogaster and C2C12 and SCC-9 cell lines. Our result suggests two major advantages of CTC over the dyes presently used for imaging actin proteins. First, the dye can bind to actin efficiently without any secondary intermediate. Second, it is much more stable at room temperature and exhibits excellent photostability. To the best of our knowledge, this is the first fluorescent dye that can bind to the actin protein without employing any secondary intermediate/actin-binding domain. These findings could pave the way for many biologists and physicists to successfully employ the CTC dye for imaging and tracking actin proteins by fluorescence microscopy in various in vivo and in vitro systems.

Radical nucleophilic substitution/cyclization: A novel strategy for selective and ultrafast fluorescence imaging of cysteine levels in ferroptosis process

Diphenylisoindolo[2,1-a]quinoline can be used to detect cysteine among homocysteine, glutathione, and other 19 natural amino acids. Unlike other reported probes, the response mechanism involves sulfhydryl radical nucleophilic substitution and cyclization, and thus the differences in ring-formation kinetics enable high selectivity. After treated with Cys, the response process was completed rapidly and the maximum fluorescence intensity (at 496 nm) was reached extremely fast (<1 s) when excited at 380 nm in MeCN-PBS buffer (10.0 mM, pH = 7.4, 3:7 (v/v)). The quantum yield after the reaction was increased almost 7 times to be 0.02 from 0.003. Fluorescence intensity displayed a good quantitative linear relationship in the range 1–10 μM Cys with a detection limit of 270 nM. Furthermore, the probe was demonstrated for real-time monitoring of intracellular cysteine levels within HepG2 cells in ferroptosis process.

Yellow-Emitting Carbon Dots for Selective Fluorescence Imaging of Lipid Droplets in Living Cells.

This study shows a one-pot preparation of carbon dots by a solvothermal method in ethylene glycol. The carbon dots show yellow-colored fluorescence emission in water. The carbon dots showed distinct preference to be present in the hydrophobic environment which was evident from their efficient transfer from aqueous phase to organic phase. They were also found to locate themselves in the vesicle bilayer and micelle core. This inherent lipophilic character of these carbon dots has been successfully utilized for the selective imaging of lipid droplets inside the living cells. The selective imaging of lipid droplets was confirmed by similar staining patterns with other staining dyes and the starvation study.

Novel bifluorescent Zn(II)–cryptolepine–cyclen complexes trigger apoptosis induced by nuclear and mitochondrial DNA damage in cisplatin-resistant lung tumor cells

Four novel bifluorescent Zn(II)–cryptolepine–cyclen complexes, namely [Zn(BQTC)]Cl2 (Zn(BQTC)), [Zn(BQA) (Cur)Cl] (Zn(BQACur)), [Zn (TC)]Cl2 (Zn(TC)), and [Zn (AP) (Cur)Cl] (Zn(APCur)), bearing curcumin (H-Cur), cyclen (TC), 1,10-phenanthrolin-5-amine (AP), and novel cryptolepine–cyclen derivatives (BQTC and BQA) were prepared for cell nucleus- and mitochondria-specific imaging. MTT assay results indicated that Zn(BQTC) and Zn(BQACur) exhibit stronger anticancer activity against cisplatin-resistant A549R lung tumor cells than ZnCl2, Zn(TC), Zn(APCur), H-Cur, TC, AP, BQTC, and BQA. Due to the dual fluorescence characteristic of Zn(BQTC), selective fluorescence imaging of the nucleus and mitochondria of A549R cancer cells was conducted. Further, Zn(BQTC), obtained by the functionalization of Zn(TC) with cryptolepine derivative substituents, efficiently inhibited DNA synthesis, thus resulting in high cytotoxicity (selective for A549R lung tumor cells) accompanied by DNA impairment in nuclear and mitochondrial fractions. Additionally, Zn(BQTC) caused severe damage to the mitochondrial DNA (mtDNA) and nuclear DNA (nDNA), sequentially disrupted mitochondrial and nuclear functions, and promoted the DNA damage–induced apoptotic signaling pathway and adenosine triphosphate depletion (ATP). Thus, Zn(BQTC) can be used as an anticancer drug by targeting mtDNA and nDNA. Most importantly, Zn(BQTC) showed higher efficacy in inhibiting cancer growth (55.9%) in A549R tumor-bearing mice than Zn(TC) (31.2%) and cisplatin, along with a promising in vivo safety profile. These results demonstrate the applicability of the developed novel bifluorescent Zn(II)–cryptolepine–cyclen complexes as promising DNA-targeting anticancer agents for cancer treatment.

Fabrication and characterization of pore-selective silver-functionalized honeycomb-patterned porous film and its application for antibacterial activity

Pore-selective silver (Ag)-functionalized honeycomb-patterned (HCP) polystyrene (PS) films were fabricated via a modified breath figure method using an interfacial chemical reaction and its application for an antibacterial film using the role of micro-trapping pit was systematically studied. PS polymer solution containing ferrocene which acts as a reducing agent was cast under humid conditions containing silver nitrate (AgNO3) to induce an interfacial chemical reaction between ferrocene and AgNO3 at the water droplet/polymer solution interface. The confinement of Ag+ to the humidity is possible due to the formation of the water complex of Ag+ such as Ag+(H2O)n stabilized by the Lennard–Jones potential and electrostatic interactions. Ag+ may be carried to the polymer solution surface via the water humidity despite being a heavy metal. Scanning electron microscopy and elemental mapping analysis revealed that the pores of the obtained films were selectively coated by Ag. The contact angle of the HCP porous films was greatly decreased by the pore selective Ag functionalization, which acts as a micro-trapping pit for the antibacterial activity. The antibacterial activity of the film against Escherichia coli and Staphylococcus aureus was assessed by the micro-dilution method, selective fluorescence imaging, and crystal violet assay. The study of BF for a functional HCP film by accompanying an interfacial chemical reaction for the pore-selective Ag functionalization shows that specific metal or inorganic materials could be pore-selectively functionalized by a similar method under several conditions, thus potentially giving the HCP films various applications.

Selective Fluorescence Imaging of Cancer Cells Based on ROS-Triggered Intracellular Cross-Linking of Artificial Enzyme.

Inside living cells, regulation of catalytic activity of artificial enzymes remains challenging due to issues such as biocompatibility, efficiency, and stability of the catalyst, by which the practical applications of artificial enzymes have been severely hindered. Here, an artificial enzyme, PTT-SGH, with responsiveness to reactive oxygen species (ROS), was obtained by introducing a catalytic histidine residue to pentaerythritol tetra(3-mercaptopropionate) (PTT). The artificial enzyme formed large aggregates in cells via the intracellular ROS-mediated oxidation of thiol groups. The process was significantly facilitated in tumor cells because of the higher ROS concentration in the tumor microenvironment. The catalytic activity of this artificial enzyme was intensively enhanced through deprotonation of cross-linked PTT-SGH, which showed typical esterase activities. Selective fluorescence imaging of tumor cells was achieved using the artificial enzyme to trigger the cleavage of the ester bond of the caged fluorophore inside living cells.

Selective Fluorescence Imaging of Cancer Cells Based on ROS‐Triggered Intracellular Cross‐Linking of Artificial Enzyme

AbstractInside living cells, regulation of catalytic activity of artificial enzymes remains challenging due to issues such as biocompatibility, efficiency, and stability of the catalyst, by which the practical applications of artificial enzymes have been severely hindered. Here, an artificial enzyme, PTT‐SGH, with responsiveness to reactive oxygen species (ROS), was obtained by introducing a catalytic histidine residue to pentaerythritol tetra(3‐mercaptopropionate) (PTT). The artificial enzyme formed large aggregates in cells via the intracellular ROS‐mediated oxidation of thiol groups. The process was significantly facilitated in tumor cells because of the higher ROS concentration in the tumor microenvironment. The catalytic activity of this artificial enzyme was intensively enhanced through deprotonation of cross‐linked PTT‐SGH, which showed typical esterase activities. Selective fluorescence imaging of tumor cells was achieved using the artificial enzyme to trigger the cleavage of the ester bond of the caged fluorophore inside living cells.

A biocompatible ruthenium-based composite fluorescent probe using bovine serum albumin as a scaffold for ethylene gas detection and its fluorescence imaging in plant tissues

The detection of phytohormone ethylene is significant for extending the shelf-life of postharvest and controlling plant maturity. In this work, we report a biocompatible fluorescent probe for trace detection of the phytohormone ethylene based on the mechanism of the olefin metathesis reaction. The probe was prepared by simply incubating the ruthenium-based complex (FPRu) with bovine serum albumin (BSA). Then the structure of the probe was characterized by Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR) and circular dichroism circular dichroism (CD). The probe possesses a satisfactory detection of limit (LOD) for ethylene (1.17 ppm) and a decent linear range (4–250 ppm). Moreover, the probe-based test paper could also detect ethylene vapor with the naked eye, completing the visualization effect. Ethylene concentration monitoring at plant maturity could be done in solution and vapor. In particular, due to the protection of the hydrophobic pocket of BSA, the ruthenium-based fluorescent probe was also effectively applied to the fluorescence imaging in plant tissues. This work provides a new strategy to achieve highly selective detection and fluorescence imaging of ethylene in plant tissues without complicated operations and expensive instruments, which is of great significance for plant biology, agriculture and the food industry.

A "Double-Locked" and Enzyme/pH-Activated Theranostic Agent for Accurate Tumor Imaging and Therapy.

Theranostic agents for concurrent cancer therapy and diagnosis have begun attracting attention as a promising modality. However, accurate imaging and identification remains a great challenge for theranostic agents. Here, we designed and synthesized a novel theranostic agent H6M based on the “double-locked” strategy by introducing an electron-withdrawing nitro group into 1-position of a pH-responsive 3-amino-β-carboline and further covalently linking the hydroxamic acid group, a zinc-binding group (ZBG), to the 3-position of β-carboline to obtain histone deacetylase (HDAC) inhibitory effect for combined HDAC-targeted therapy. We found that H6M can be specifically reduced under overexpressed nitroreductase (NTR) to produce H6AQ, which emits bright fluorescence at low pH. Notably, H6M demonstrated a selective fluorescence imaging via successive reactions with NTR (first “key”) and pH (second “key”), and precisely identified tumor margins with a high S/N ratio to guide tumor resection. Finally, H6M exerted robust HDAC1/cancer cell inhibitory activities compared with a known HDAC inhibitor SAHA. Therefore, the NTR/pH-activated theranostic agent provided a novel tool for precise diagnosis and efficient tumor therapy.