Significant Fluorescence Enhancement Research Articles

A Catalytic Hairpin Assembly-Based Approach for RNA Imaging in Living Cells.

The advancement of nucleic acid nanotechnology has resulted in broad applications of DNA- and RNA-based molecular sensors for bioanalysis. Catalytic hairpin assembly is such a type of programmable and enzyme-free nucleic acid circuit that has been popularly used in developing biosensors. Genetically encodable fluorogenic RNA-based devices have recently gained a lot of attentions as a powerful tool for intracellular imaging. Combining these two techniques together, we have developed a genetically encodable RNA-based catalytic hairpin assembly circuit for the highly sensitive detection of low-abundance RNAs in living cells. In this system, the binding of one target RNA can catalytically trigger the generation of tens-to-hundreds of fluorogenic RNA reporters and produce a significantly enhanced fluorescence signal. Here, we will introduce the detailed design, optimization, and imaging protocol of this new type of powerful imaging tools.

Pyrene Based Schiff Base for Cascade Fluorescent "On-Off" Detection of Aluminium(III) and Fluoride Ions and its Applications in Live Cells Imaging.

An easy-to-prepare pyrene-based Schiff base PNZ was synthesized by condensing equimolar amount of 1-pyrenebutyric hydrazide with 2-hydroxy-naphthaldehyde, and employed as a fluorescent chemosensor for in-situ cascade detection of aluminium (Al3+) and fluoride (F-) ions. In DMSO:H2O (1 : 1, v/v), the weakly emissive PNZ showed a significant fluorescence enhancement at 455 nm selectively upon the addition of Al3+ due to the complexation-induced formation of a pyrene excimer. Schiff base PNZ and Al3+ formed a complex in 2 : 1 binding ratio with the estimated binding constants of K1:1=9826.01 M-1 and K2:1=3188.49 M-1. The sensing mechanism was explored by performing quantum mechanical calculations and 1H-NMR titration of PNZ with Al3+. The in-situ formed PNZ-Al3+ complex species enabled the fluorescent turn-off detection of F-. Using PNZ and PNZ-Al3+, the concentrations of Al3+ and F- ions can be detected down to 2.89×10-7 M and 1.88×10-7 M, respectively. The cytotoxicity of the PNZ and its ability to bioimage Al3+ and F- ions was examined in the human cervical cancer cell line. Finally, the receptor PNZ was applied for the quantification of Al3+ and F- ions in various real samples, such as tap water, river water, rainwater, mouthwash, and toothpaste.

Unprecedented Photoinduced‐Electron‐Transfer Probe with a Turn‐ON Chemiluminescence Mode‐of‐Action

PeT‐based fluorescent probes were demonstrated to be powerful tools for detection and imaging, owing to their significant fluorescence enhancement in response to specific targets. While numerous examples of fluorescence‐based PeT have been frequently reported, there is not even a single example of a PeT probe that operates via a chemiluminescence mode. Here we report the first PeT‐based turn‐on probe that acts via a chemiluminescent operation mode. We designed, synthesized, and evaluated a novel chemiluminescent probe, featuring a PeT‐based turn‐on mechanism. The probe consists of a phenoxy‐1,2‐dioxetane, linked to an azide unit that acts as a PeT quencher. Upon cycloaddition of a strained cycloalkyne with the azide, a triazole‐dioxetane is formed, which undergoes relatively slow chemiexcitation, resulting in a measurement window with an exceptionally high signal‐to‐noise ratio (over 5000‐fold). The PeT‐dioxetane probe could effectively detect and image two model proteins labeled with strained cycloalkyne units (Myc‐DBCO and Max‐DBCO) through either NHS or maleimide conjugations. Comparative analysis shows that our PeT‐based chemiluminescent probe significantly outperforms a commercially available fluorescent analog. We anticipate that the insights gained from this study will facilitate the development of additional chemiluminescent probes utilizing various PeT‐quenching pathways.

Unprecedented Photoinduced-Electron-Transfer Probe with a Turn-ON Chemiluminescence Mode-of-Action.

PeT-based fluorescent probes were demonstrated to be powerful tools for detection and imaging, owing to their significant fluorescence enhancement in response to specific targets. While numerous examples of fluorescence-based PeT have been frequently reported, there is not even a single example of a PeT probe that operates via a chemiluminescence mode. Here we report the first PeT-based turn-on probe that acts via a chemiluminescent operation mode. We designed, synthesized, and evaluated a novel chemiluminescent probe, featuring a PeT-based turn-on mechanism. The probe consists of a phenoxy-1,2-dioxetane, linked to an azide unit that acts as a PeT quencher. Upon cycloaddition of a strained cycloalkyne with the azide, a triazole-dioxetane is formed, which undergoes relatively slow chemiexcitation, resulting in a measurement window with an exceptionally high signal-to-noise ratio (over 5000-fold). The PeT-dioxetane probe could effectively detect and image two model proteins labeled with strained cycloalkyne units (Myc-DBCO and Max-DBCO) through either NHS or maleimide conjugations. Comparative analysis shows that our PeT-based chemiluminescent probe significantly outperforms a commercially available fluorescent analog. We anticipate that the insights gained from this study will facilitate the development of additional chemiluminescent probes utilizing various PeT-quenching pathways.

Novel amino-functionalized MOF-based sensor for zinc ion detection in water and blood serum samples

Aquatic systems with low zinc levels can experience a significant decrease in carbon dioxide uptake and limited growth of phytoplankton species. In this study, we describe the use of a new fluorescent sensor based on NH2-MIL-53(Al), and modified with glutaraldehyde and sulfadoxine, for selectively detecting zinc ions in water and blood serum samples. Characterization of the synthesized material was performed using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), Brunauer–Emmett–Teller (BET) surface area analysis, X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM), confirming successful functionalization and preservation of the MOF structure. The sensor’s performance for Zn2+ detection was evaluated by spectrofluorometry, demonstrating a significant fluorescence enhancement upon Zn2+ binding due to the interaction between Zn2+ ions and the sulfonamide groups. With a detection limit as low as 3.14 × 10–2 ppm, the sensor demonstrates high selectivity for Zn2+ over other common metal ions. The sensor’s response is rapid, stable, and reproducible, making it suitable for practical applications. Real sample analysis was conducted in tap water and blood serum samples, with the results compared to those obtained using ICP-OES and a colorimetric test with 5-bromo-PAPS. The comparison confirmed the high accuracy and reliability of the fluorescent sensor in detecting Zn2+ ions in complex matrices. NH2-MIL-53(Al) modified with glutaraldehyde and sulfadoxine shows potential as a selective fluorescent sensor for Zn2+ detection, making it a valuable tool for monitoring the environment and biology.

Solvent-Selective Fluorescence Sensing of Mg2+ and Al3+ Ions by Pincer-Type NNO Schiff Base Ligand: An Experimental and DFT Optimized Approach.

A newly developed dual-functional fluorescence sensing probe (phenylhydrazinyl pyridine) Schiff base (SB) has been designed with good selectivity for distinguishing Mg2+ and Al3+ metal ions in different solvent solutions. SB exhibits quick and visual turn-on fluorescence enhancement in response to Mg2+ and Al3+ detection. The addition of Mg2+ in ACN-HEPES buffer (1 : 1, v/v, pH 7.2) at (λmax=390 nm) and Al3+ in MeOH-HEPES buffer (1 : 1, v/v, pH 7.2) at (λmax=360 nm) resulted in significant enhancement of fluorescence, up to 7-9 times. These low detection limits of 7.1×10-6 M (7.1 μM) and 5.15×10-7 M (0.51 μM) for Mg2+ and Al3+, respectively, have been achieved by this solvent-controlled platform. Due to the sensing potential towards Mg2+, the probe was utilized as an imaging material for breast cancer cells. 1H-NMR studies were utilized to explore SB's sensing mechanism through turn-on fluorescence. Density functional theory (DFT) calculations were utilized to validate optimized SB and its intricate geometries, which govern the sensing mechanism in the solvent environment. Such a probe has extensive potential applications in bioimaging and the assessment of the quality of wastewater.

Synergistically Activated Aggregation-Induced Emission Probe for Precise In Situ Staining of Lipids in Atherosclerotic Plaques.

Visualizing the localization and distribution of lipids within arteries is crucial for studying atherosclerosis. However, existing lipid-specific probes face challenges such as strong hydrophobicity and nonspecific staining of lipophilic organelles or tissues, making them impractical for the precise identification of atherosclerotic plaques. To address this issue, we design a synergistically activated probe, Cbz-Lys-Lys-TPEB, which responds to cathepsin B (CTB) and H2O2 for the in situ generation of aggregation-induced emission luminogens (AIEgens). This enables specific staining of lipids within arteries and precise imaging of atherosclerotic plaques. The probe combines a tetraphenylethene building block with a hydrophilic peptide sequence (Cbz-Lys-Lys) and phenylboric acid module, providing excellent water solubility and fluorescence quenching in a molecular dispersion state. Upon interaction with H2O2 and CTB within plaques, the hydrophilic Cbz-Lys-Lys-TPEB probe is specifically cleaved and converted into hydrophobic AIEgens, leading to rapid aggregation and significant fluorescence enhancement. Interestingly, the in situ-liberated AIEgens display distinct lipid binding ability, effectively tracking the location and distribution of lipids in plaques. This synergistic target-activated AIEgen liberation strategy demonstrates significant feasibility for the reliable and accurate identification of atherosclerotic plaques, holding tremendous potential for clinical diagnosis and risk stratification of atherosclerosis.

Early Diagnosis of Liver Injury and Real-Time Evaluation of Photothermal Therapy Efficacy with a Viscosity-Responsive NIR-II Smart Molecule.

The early diagnosis of liver injury and in situ real-time monitoring of tumor therapy efficacy are important for the enhancement of personalized precision therapy but remain challenging due to the lack of reliable in vivo visualization tools with integrated diagnostic, therapeutic, and efficacy monitoring functions. Herein, a smart second near-infrared window (NIR-II) molecule (BITX-OH) is rationally designed for diagnosis and therapy by vinyl-bridging hydroxyl diphenyl xanthine unit and benzo[cd]indolium skeleton. BITX-OH exhibits high selectivity and sensitivity toward viscosity, exhibiting a significant enhancement (1167-fold) in NIR-II fluorescence at 962nm. With the assistance of BITX-OH and NIR-II fluorescence imaging, early diagnosis and therapeutic evaluation of non-alcoholic fatty liver (NAFL), as well as in-site real-time monitoring of hepatic fibrosis (HF) in live mice have been successfully achieved, which is at least several hours earlier than the typical clinical test. Notably, BITX-OH displays excellent photothermal conversion efficiency when exposed to an 808nm laser, which can induce tumor ablation and increase viscosity, thereby enhancing NIR-II fluorescence for the real-time evaluation of photothermal therapy (PTT). This viscosity-based "self-monitoring" strategy provides a convenient and reliable platform for timely obtaining therapeutic feedback to avoid over- or under-treatment, thus enabling personalized precision therapy.

Bimetallic metal organic frameworks-based nanozyme for colorimetry/fluorescence dual-mode uremic toxin detection

Chronic kidney disease (CKD) can greatly increase the mortality risk for clinical patients, with a major challenge being the accurate detection of uremic toxins. Metal organic frameworks (MOFs)-based colorimetry/fluorescence dual-mode probe have shown great potential for bio-detection. However, one of the challenges for MOFs-based nanoprobe is to effectively improve the activity of mimetic enzymes for colorimetric assays while maintaining their outstanding luminescence response for fluorescence sensing. Here, we developed a novel bimetallic Zr/Ce-MOFs, which possesses high catalytic activity and excellent luminescence properties for dual-mode sensing of uremic toxins. The co-existence of Ce and Zr in the MOFs improves the catalytic activity for colorimetric detection of phosphate ions (one type of uremic toxins) and the strong coordinate covalent interaction between phosphate ions and Zr/Ce leads to a significant fluorescence enhancement of the MOFs. Furthermore, we achieved high sensitivity phosphate ions analysis in serum samples. This strategy provides an easy and reliable platform for the detection of uremic toxins in complicated physiological environment.

Bimetallic complexation for significant fluorescence enhancement of faecal pigment towards water quality testing

Systematic spectroscopic investigations revealed bimetallic complexation of faecal pigments (FP) in waterthrough their multiple binding sites. FP-Zn(II)/Gd(III) complexes showed narrow emission (FWHM ∼ 26 nm) with maximum intensity compared to different bimetallic complexes in water. The fluorescence intensity of bimetallic Zn(II)/Gd(III) complexes of FP increased ∼3–5 fold in aqueous media compared to the FP-Zn(II) complexes, i.e. well-known as Schlesinger’s test. The optimum Zn(II): Gd(III) stoichiometry was observed at 3:2 for maximum FP fluorescence enhancement. The bimetallic FP-Zn(II)/Gd(III) complexes also exhibited higher fluorescence lifetimes and quantum yield than FP-Zn(II) complexes. The photophysical investigations demonstrated that bimetallic Zn(II)/Gd(III) complexation increased the rigidity of chromophores present in FP. Consequently, a decrease in non-radiative decay rate constants (knr) by ∼ 1.5 times was observed for bimetallic complexes compared to FP-Zn(II) in water. Hence, a detection limit of the nanomolar concentration range of FP could be achieved in the aqueous media. The effect of diverse interfering factors (humic acid, water hardness, pH) on the fluorescence behaviour of FP-Zn(II)/Gd(III) complexes was also investigated with an approach mimicking real water samples. Finally, a simple non-extraction analytical approach for sensitive detection of FP in water was demonstrated while also considering the fluorescence matrix interference problem.

An aggregation-induced fluorescence probe for H2S and its application in living cells

Hydrogen sulphide (H2S) plays an important role in the regulation of a number of important physiological and pathological processes. Therefore, the development of new fluorescent probes for H2S is of great importance and has recently attracted much attention. In this paper, a novel coumarin-based fluorescent probe named (1E)-1-(1-(6-chloro-2-oxo-2H-chromen-3-yl)ethylidene)thiosemicarbazide (1), which contained a CN moiety as a specific reaction site for nucleophilic addition of H2S, was prepared. Upon addition of H2S, probe 1 exhibited significant fluorescence enhancement with a detection limit as low as 0.54 μM, accompanied by a colour change from cyan to colourless. The mechanism was confirmed by ESI-MS, Job’s plot, DFT and the probe 1 was successfully applied to the imaging of H2S in HeLa cells.

Flexible electrospun test strips functionalized with a graphene oxide/doxorubicin complex for dopamine detection

A novel flexible test strip has been developed for the selective and sensitive detection of dopamine (DA) using an electrospun polyacrylonitrile nanofiber membrane as a substrate and doxorubicin (DOX)-functionalized graphene oxide (GO) as a fluorescent ligand. Employing the Langmuir-Blodgett technique, the fluorescent probe of GO/DOX was controllably and flatly applied onto the surface of nanofiber membranes. The adsorption behavior of DA and DOX on GO in solution and on the surface of solid test strips has been investigated, respectively. The results showed that DA exhibited a higher affinity for GO, both in solution and on the surface of test strips, compared to DOX. Due to the competitive adsorption of DOX and DA onto GO, the addition of DA to the GO/DOX complex led to a significant fluorescence enhancement, attributed to the replacement of the GO-supported DOX with DA of a higher adsorption affinity to GO, enabling the detection of DA. This nanofiber-based method proves to be simple, sensitive, selective, and effectively minimizes interference from common biomolecules and small molecules in serum. Thus, this portable, cost-effective, and selective nanofiber strip holds great promise for early diagnosis of neurodegenerative diseases by measuring DA levels.

Dual-response fluorescent probe for lipid droplets and hypochlorous acid and imaging study in inflammation and atheromatous plaques

Simultaneously detecting two biomarkers can significantly enhance disease diagnosis accuracy and deepen our understanding of disease pathogenesis. Developing a single fluorescent probe with two independent response units for this purpose is crucial. In line with this, a novel fluorescent probe (TPA-DT-DNH) had been proposed, featuring triphenylamine and C=N groups sensitive to lipid droplets (LDs) and hypochlorous acid (HClO), respectively. Spectral results highlighted that only the co-presence of LDs and HClO could “turn-on” the fluorescence of TPA-DT-DNH, when the concentration of one substance remained constant, the fluorescence intensity linearly increased with the concentration of the other substance. The imaging results demonstrated that TPA-DT-DNH can differentiate between normal cells and cells rich in LDs when HClO was present, and can detect HClO in HeLa cells. Furthermore, dynamic changes in the inflammation process induced by LPS can be visualized in both cells and zebrafish. Additionally, significant fluorescence enhancement was observed in atheromatous plaque.

Bidirectional Polymerization-Transcription Amplification-Encoded Dual-Color Fluorescent Biosensor for Label-Free and Primer-Free Detection of Multiple piRNAs.

PIWI-interacting RNAs (piRNAs) are a type of endogenous noncoding RNAs with a length of 24-31 nucleotides, and they can specifically bind with PIWI proteins to form the piRNA/PIWI complexes for regulating multiple physiological and pathological processes. Herein, we develop a bidirectional polymerization-transcription amplification-encoded dual-color fluorescent biosensor for label-free and primer-free measurements of multiple piRNAs. The designed hairpin probe contains a palindromic tail, and it can serve as the target recognition unit, polymerization primer, and transcription template. In the presence of target piRNAs, the hairpin probes are opened to expose a palindromic sequence that can trigger bidirectional polymerization and transcription reaction with the assistance of KF polymerase and T7 RNA polymerase for the production of numerous RNA aptamers. The aptamers subsequently bind with the corresponding fluorophores (DFHBI-1T/MG) to form the RNA aptamer-fluorophore complexes for the generation of enhanced fluorescence signals. This biosensor can sensitively detect piR-36026 with a limit of detection (LOD) of 82.08 aM and piR-36743 with a LOD of 44.44 aM. Moreover, it can quantify cellular piRNAs with single-cell sensitivity and distinguish cancer cells from normal cells. Furthermore, it has the capability of distinguishing the expression of piRNAs in the tissues of breast cancer patients and healthy individuals. By simply altering the target recognition site of the hairpin probe, this biosensor can be extended to detect various piRNAs, offering a powerful platform for piRNA-related clinical diagnostics and therapeutics.

Mitochondria-targeted NIR molecular probe for detecting viscosity of gland damage and SO2 in actual samples

Glandular damage can be caused by various factors, including disease, trauma, or other abnormalities within the organism. The viscosity of the gland is one of the important indicators to measure the degree of damage. Sulfur dioxide (SO2) is widely used as an important food additive due to its preservative and bleaching properties, but its overuse has serious negative impacts on the environment, so it is urgent to develop a simple detection method. Herein, we designed and synthesized a mitochondria-targeted near-infrared (NIR) fluorescence probe (BDC) for the detection of viscosity and SO2. BDC consisted of a donor-π-acceptor (D-π-A) structure and extended double bonds bridging rotor, which enabled sensitive response to viscosity and intense fluorescence emission. The TICT (twisted intramolecular charge transfer) of BDC was inhibited with an increase in viscosity, accompanied by a significant enhancement of red fluorescence signal with emission wavelength beyond 800 nm. Notably, BDC was able to noninvasively and sensitively monitor the viscosity changes in the glands of non-obese diabetic (NOD) mice model. BDC was utilized for monitoring SO2 in food and environmental samples through Michael addition reactions, providing a straightforward tool for SO2 detection in food safety and environmental monitoring.

A Versatile Visual Molecular Imprinting-Driven Switchable Nanozyme Activity-Based Trimodal Assay and Logic Gate Circuits of Ethyl Carbamate.

Concerns regarding the hazard of the carcinogenic ethyl carbamate (EC) have driven attempts to exploit efficient, timely, straightforward, and economic assays for warning early food safety. Here, we proposed a novel molecularly imprinted polymer Co@MOF-MIP, with a high peroxidase (POD)-like activity and a bright blue fluorescence emission, to develop a versatile visual assay for colorimetric, fluorescent, and photothermal trimodal detection and logic gate outputting of EC. Briefly, the POD-like activity of Co@MOF-MIP made it to decompose H2O2 into ·OH for oxidizing colorless 3,3',5,5'-tetramethylbenzidine (TMB) into a blue oxTMB, resulting in a 660 nm irradiated photothermal effect and bursting the blue fluorescence of Co@MOF-MIP via inner filter effect, observing a decreased fluorescence signal together with an increased colorimetric and 660 nm irradiated photothermal signals. However, EC could specifically fill the imprinted cavities of Co@MOF-MIP to block the catalytic substrates TMB and H2O2 out of Co@MOF-MIP for further reacting with the inside catalytic center of Co2+, resulting in the transformation suppressing of TMB into oxTMB, yielding an EC concentration-dependent trimodal responses in fluorescence signal enhancement, colorimetric, and 660 nm irradiated photothermal signal decreases. Assisted by the portable devices such as smartphones and hand-held thermal imagers, a visual onsite portable trimodal analytical platform was proposed for EC fast and accurate detection with the low detection limits of 1.64, 1.24, and 1.78 μg/L in colorimetric, fluorescent, and photothermal modes, respectively. Interestingly, these reactive events could be programmed by the classical Boolean logic gate analysis to offer a novel promising avenue for the big data Internet of Things monitoring and warning early residual EC in a more intelligent, dynamical, fast, and accurate manner, safeguarding food safety.

Precise AIE-Based Ternary Co-Assembly for Saccharide Recognition and Classification.

Saccharides are involved in nearly all life processes. However, due to the complexity and diversity of saccharide structures, their selective recognition is one of the most challenging tasks. Distinct from conventional receptor designs that rely on delicate and complicated molecular structures, here a novel and precise ternary co-assembled strategy is reported for achieving saccharide recognition, which originates from a halogen ions-driven aggregation-induced emission module called p-Toluidine, N, N'-1-propen-1-yl-3-ylidene hydrochloride (PN-Tol). It exhibits ultra-strong self-assembly capability and specifically binds to 4-mercaptophenylboronic acid (MPBA), forming highly ordered co-assemblies. Subsequent binding of various saccharides results in heterogeneous ternary assembly behaviors, generating cluster-like, spherical, and rod-like microstructures with well-defined crystalline patterns, accompanied by significant enhancement of fluorescence. Owing to the excellent expandability of the PN module, an array sensor is constructed that enables easy classification of diverse saccharides, including epimer and optical isomers. This strategy demonstrates wide applicability and paves a new avenue for saccharide recognition, analysis, and sequencing.

Construction of a dual “off-on” near-infrared fluorescent probe for bioimaging of HClO in rheumatoid arthritis

Hypochlorous acid (HClO) serves as a critical biomarker in inflammatory diseases such as rheumatoid arthritis (RA), and its real-time imaging is essential for understanding its biological functions. In this study, we designed and synthesized a novel probe, RHMB, which ingeniously integrates rhodamine B and methylene blue fluorophores with HClO-specific responsive moieties into a single molecular framework. Upon exposure to HClO, RHMB exhibited significant dual-channel fluorescence enhancement characterized by high sensitivity (LODs of 2.55 nM and 14.08 nM), excellent selectivity, and rapid response time (within 5 s). Notably, RHMB enabled reliable imaging of both exogenous and endogenous HClO in living cells and in zebrafish, employing a unique duplex-imaging turn-on approach that highlighted its adaptability across various biological contexts. Furthermore, RHMB effectively monitored HClO fluctuations in an RA mouse model and assessed the therapeutic efficacy of diclofenac (Dic) in alleviating RA symptoms. These findings underscore the potential of RHMB as an invaluable tool for elucidating the biological roles of HClO in various diseases.

Enhanced Recognition of a Herbal Compound Epiberberine by a DNA Quadruplex-Duplex Structure.

The small molecule epiberberine (EPI) is a natural alkaloid with versatile bioactivities against several diseases including cancer and bacterial infection. EPI can induce the formation of a unique binding pocket at the 5' side of a human telomeric G-quadruplex (HTG) sequence with four telomeric repeats (Q4), resulting in a nanomolar binding affinity (KD approximately 26 nM) with significant fluorescence enhancement upon binding. It is important to understand (1) how EPI binding affects HTG structural stability and (2) how enhanced EPI binding may be achieved through the engineering of the DNA binding pocket. In this work, the EPI-binding-induced HTG structure stabilization effect was probed by a peptide nucleic acid (PNA) invasion assay in combination with a series of biophysical techniques. We show that the PNA invasion-based method may be useful for the characterization of compounds binding to DNA (and RNA) structures under physiological conditions without the need to vary the solution temperature or buffer components, which are typically needed for structural stability characterization. Importantly, the combination of theoretical modeling and experimental quantification allows us to successfully engineer Q4 derivative Q4-ds-A by a simple extension of a duplex structure to Q4 at the 5' end. Q4-ds-A is an excellent EPI binder with a KD of 8 nM, with the binding enhancement achieved through the preformation of a binding pocket and a reduced dissociation rate. The tight binding of Q4 and Q4-ds-A with EPI allows us to develop a novel magnetic bead-based affinity purification system to effectively extract EPI from Rhizoma coptidis (Huang Lian) extracts.

Ultra-sensitive and fast nitroreductase-responsive fluorescence probe for real-time bioimaging of hypoxia in tumor cells and zebrafish wound inflammation

Hypoxia is intertwined with the occurrence and progression of tumors and inflammation. To date, various fluorescent probes have been constructed for monitoring degrees of hypoxia in tumor cells, however, efficiently evaluating the degrees of hypoxia caused by inflammation in real-time has been rarely exhibited via fluorescence imaging technique. Hence, an ultra-sensitive and fast-response fluorescent probe R19-NTR using rhodamine 19 as the fluorescent reporter and nitro aromatic groups as the recognition unit was performed to estimate the degrees of hypoxia through assessing the changes in the level of nitroreductase. The probe R19-NTR was basically non-fluorescent on account of the intramolecular photo-induced electron transfer process. Upon selectively reacted with nitroreductase, the R19-NTR featured an ultrafast response (< 1 min), and a significant fluorescence enhancement (ca. 114-fold). Encouraged by these outstanding properties, R19-NTR has been utilized for monitoring the degree of hypoxia in tumor cells and zebrafish wound inflammation. The results revealed that tumor cells under normoxic and different degrees of hypoxic conditions can be distinguished successfully, as well as the differentiation between tumor cells and normal cells. Moreover, the in vivo visualization disclosed that the endogenous nitroreductase of 6–10 days post fertilization zebrafish embryos was mainly focused on the liver and intestine. In particularly, the elevation of hypoxia degrees in zebrafish wound inflammation caused by severed tails were successfully visualized for the first time, as well as the variation of hypoxia degrees in gradually dying zebrafish embryos. The present research may provide a promising methodology for the diagnosis of inflammation-related diseases and tumors.