Blue Fluorescent Product Research Articles

Copper-doped Lanthanide Coordination Polymers as Luminescent Nanoenzyme for Ratiometric Sensing of H2O2 and Glutathione.

Design and fabrication of integrated multifunctional probes with intrinsic catalytic and detection abilities is of great importance to simplify the operation in biosensing application with high sensitivity. Herein, dual-emitting lanthanide coordination polymers (Ln-CPs) were facilely prepared by self-assembly of guanine diphosphate (GDP), terephthalic acid (TA), Tb3+ and Cu2+ designated as Tb/Cu-GDP/TA CPs. The doped Cu2+ endowed CPs with obviously enhanced peroxidase mimicking activity compared with free Cu2+. In the presence of H2O2, the probe catalyzed the oxidation of TA generating a new blue fluorescent product, while the fluorescence of Tb3+ decreased simultaneously. Therefore, a new sensitive ratiometric fluorescent sensor for H2O2 has been developed with a good linear range from 0.01 to 300μM and limit of 1.62nM. Moreover, the proposed platform could be extended to GSH ratiometric assay in the presence of H2O2, and interestingly, the detection performance could be easily adjusted by adding different concentration of H2O2. This work will facilitate the development of luminescent nanoenzymes based on Ln-CPs to construct the simple ratiomatric sensing platform.

A “traffic light” signal ratiometric fluorescence sensor for highly sensitive and selective detection of dopamine

As a neurotransmitter with important biological properties, dopamine (DA) plays a vital role in the nervous system of the human body. In this paper, Tb-dipicolinic acid (DPA), Eu-DPA, and Tb/Eu-DPA with green, red, and pale-yellow fluorescence are synthesized to construct “traffic light” sensor for detecting DA, based on the characteristic reaction of resorcinol (Rs) and DA. Through the energy transfer and quenching effects, the fluorescence signals of the “traffic light” sensor are weakened, while the blue fluorescent product is enhanced. Moreover, the sensing system shows obvious chroma change from green, red, and pale-yellow to blue, respectively. The ratiometric fluorescence signal change and chroma change enable the detection of DA with good sensitivity, accuracy, high selectivity and anti-interference ability. In addition, the method has good application for DA detection in serum samples and injection. More importantly, this study combines the “traffic light” sensor with characteristic reaction of Rs and DA for the first time, which not only broadens the applications of detecting DA in the initial monitoring and early clinical diagnosis of related diseases, but also provides a new idea for designing optical sensors with excellent performance based on characteristic reaction.

Ratiometric fluorescent detection system based on dual-driving catalysis of CuO nanozyme with a classical univariate calibration for the determination of ascorbic acid in serum and fruits

Copper oxide nanoparticles (CuO NPs) as nanozymes have received extensive attention due to their numerous advantages such as water solubility and biomimetic effects. In this work, we combined the inherent oxidase and ascorbate oxidase mimetic activity of CuO NPs to construct a ratiometric fluorescence system for the detection of ascorbic acid (Vitamin C, AA) in serum and fruits. By feat of the dual mimic nanozymatic activity of CuO NPs, the sole substrate o-phenylenediamine (OPD) can be catalyzed to attain yellow fluorescent 2,3-diaminophenazine (oxOPD), while oxidized AA will react with the pristine OPD to form blue fluorescent product. This method has a sensitive response to AA with a good linear relationship in the concentration range of 1–150 μM and low detection limit of 0.16 μM. Practical sample detection of AA in human serum and fruits can be realized reasonably with the recovery rates between 84.83% and 87.28% and 88.53%-113.1%, respectively. More conveniently, the method can be successfully used for scanning measurement analysis through the associated application (ColorAssist) on smartphones and portable UV lamps.

Fluorometric determination of the activity of inorganic pyrophosphatase and its inhibitors by exploiting the peroxidase mimicking properties of a two-dimensional metal organic framework.

A copper(II)-based two-dimensional metal-organic framework with nanosheet structure (CuBDC NS) that possesses peroxidase (POx) mimicking activity was prepared. In the presence of hydrogen peroxide, the system catalyses the oxidation of terephthalic acid to a blue-fluorescent product (excitation = 315nm; emission = 425nm). Pyrophosphate has a very strong affinity for Cu2+ ionand blocks the POx-mimicking activity of theCuBDC NS. If, however, inorganic pyrophosphatase is present, the POx mimicking activity is gradually restored because pyrophosphate is hydrolyzed. The findings were used to design a method for the determination of the activity of inorganic pyrophosphataseby fluorometry. Fluorescence increases linearly in the 1-50mU·mL-1inorganic pyrophosphatase activity range. The limit of detection is 0.6mU·mL-1 (S/N= 3). Graphical abstract A copper(II)-based two-dimensional metal-organic framework (CuBDC NS) is described that possesses POx-mimicking activity. Inorganic pyrophosphate (PPi) was hydrolyzed to phosphate in the presence of inorganic pyrophosphatase (PPase). Hence, it cannot coordinate with Cu2+ in CuBDC NS, its structure was well-conserved to catalyses the oxidation of terephthalic acid (H2BDC) to produce a blue fluorescent product (oxBDC) in the presence of hydrogen peroxide (H2O2).

A copper(II)/cobalt(II) organic gel with enhanced peroxidase-like activity for fluorometric determination of hydrogen peroxide and glucose.

A bimetallic organic gel was prepared by mixing the bridging ligand 2,4,6-tri(4-carboxyphenyl)-1,3,5-triazine with Cu(II) and Co(II) ions at room temperature. The resulting metal-organic gel (MOG) shows enhanced peroxidase-like activity, most likely due to the synergetic redox cycling between Co(III)/Co(II) and Cu(II)/Cu(I) pairs. These accelerate interfacial electron transfer and generation of hydroxy radicals. The MOG can catalyze the reaction of H2O2 with terephthalic acid (TPA), producing a blue fluorescence product with the maximum excitation/emission at 315/446nm. The enzyme mimic was used to design a fluorometric method for H2O2 that has a 81nM detection limit. H2O2 is also formed by glucose oxidase-assisted oxidation of glucose by oxygen, and an assay for glucose was worked out based on the above method. It has a 0.33μM detection limit. This study may open up a new avenue to design and synthesize nanomaterial-based biomimetic catalysts with multiple metal synergistically enhanced catalytic activity for potential applications in biocatalysis, bioassays and nano-biomedicine. Graphical abstracts Schematic presentation of the synergic catalytic effect of Cu(II)/Co(II) bimetallic organic gel promoted by the redox cycle between Co(III)/Co(II) and Cu(II)/Cu(I) pairs. The bimetallic organic gel can catalyze the reaction of H2O2 with terephthalic acid,thereby producing a blue-fluorescent product.

Sensitive fluorometric determination of platelet-derived growth factor BB and avian influenza A virus DNA via dual signal amplification using the hybridization chain reaction and glucose oxidase assisted recycling

A method is described for fluorometric determination of platelet-derived growth factor BB (PDGF-BB) and avian influenza A (H1N1) virus DNA. It is based on the use of the hybridization chain reaction (HCR) and of glucose oxidase (GOx) assisted dual-recycling amplification. A silver coated glass slide (SCGS) serves as an ideal material for separation. A signal DNA/initiator triggers the HCR and generates a cascade of hybridization to form a nicked double-helix polymer. Upon addition of the analytes (PDGF-BB or H1N1 DNA) and capture DNA immobilized on the SCGS, the nicked double-helix polymer binds on the surface of the SCGS through formation of a [capture DNA/analyte/signal DNA] sandwich structure. The GOx-biotin-streptavidin (SA) complexes were then attached to the nicked double-helix polymer through SA-biotin interaction. After cleavage by DNase I, the bound GOx is transferred into the buffer. Glucose is added and enzymatically oxidized to produce H2O2. The H2O2 formed oxidizes the substrate 3-(p-hydroxyphenyl)-propanoic acid to give a blue fluorescent product (with excitation/emission maxima at 320/416nm) under the catalysis of horseradish peroxidase. Under optimal conditions, fluorescence increases linearly in the 0.5 to 70pmol·L-1 PDGF-BB concentration range, and the detection limit is 191 fmol·L-1. For the H1N1 virus DNA, the respective data are 2.5 to 300pmol·L-1 and 826 fmol·L-1. Graphical abstract Schematic presentation for detection of analytes (PDGF-BB or H1N1 virus DNA) based on the dual-signal amplification of Hybridization Chain Reaction (HCR) and glucose oxidase (GOx) using silver coated glass slide (SCGS) as separation material.

Microplate based assay for thrombin detection using an RNA aptamer as affinity ligand and cleavage of a chromogenic or a fluorogenic peptide substrate

The authors report on a microplate based thrombin assay by using an RNA aptamer (Toggle-25) as the affinity ligand. The aptamer is nuclease-resistant because it contains nucleobases modified with 2′-deoxyfluoro saccharides. The aptamer is coated on the surface of the wells of microplates where it captures thrombin from a sample. Following a washing step, a chromogenic or a fluorogenic peptide substrate is added which is cleaved by the captured thrombin to form a yellow product (with absorbance at 405 nm) or a blue fluorescent product (with an emission maximum at 444 nm) after incubation at 37 °C for 2 h. The assay enables thrombin to be determined with a 10 fM detection limit when using the fluorogenic substrate. The assay is selective in that several other proteins do not interfere. The assay can be applied to the determination of thrombin in diluted human serum and plasma.

Lysozyme as sensitive reporter for fluorometric and PCR based detection of E. coli and S. aureus using magnetic microbeads

We describe the use of lysozyme as a sensitive reporter for detection of bacteria. The assay comprises the following steps: First, specific primers are used for targeting the rDNA of E. coli and S. aureus, respectively, and the polymerase chain reaction is applied to amplify bacterial DNA. Then, the target DNA binds to the capture probe immobilized on magnetic beads, with lysozyme acting as signal reporter that catalyzes the hydrolysis of an enzyme substrate to form the blue fluorescent product 4-methylumbelliferone. Coupled with multiple lysozyme substrates, this fluorometric detecting method has a detection limit of 50 CFUa <...mL(-1) bacteria. The optical changes may also be detected by colorimetry and monitored with bare eyes. The method was selective over other bacteria and was successfully applied to the determination of E. coli and S. aureus in human serum, which provides a perspective for lysozyme application and species-specific pathogenic bacteria detection.

Rapid titration of retroviral vectors using a β-lactamase protein fragment complementation assay

The availability of rapid and quantitative titration assays for retroviral vectors is important, especially in the context of clinical applications. In this report, we describe a novel assay to titrate lentiviral and gamma retroviral vectors. This rapid assay is based on protein fragment complementation involving the N-terminal (Bla1) and the C-terminal (Bla2) fragments of TEM-1 β-lactamase (BLAK). The Bla1 protein fragment is incorporated in the vector's envelope during vector production. Bla1-bearing vectors are titrated on Bla2-expressing cells. Upon transduction, Bla1 and Bla2 heterodimerize and restore BLAK's enzymatic function. The enzymatic activity of BLAK is quantified by flow cytometry using the green fluorescent CCF2/AM substrate, which is converted into a blue fluorescent product. The enzymatic conversion of the CCF2/AM substrate was found to be directly related to vector entry, as a neutralizing antibody completely blocked the conversion. The titers obtained using this rapid assay correlated well with the titers measured by functional transduction assays. The whole assay can be finished within 8 h. Thus, it is considerably less time consuming compared with other transduction-based titration assays for lentiviral and gamma retroviral vectors.

Chromatographic Quantification of Argpyrimidine, a Methylglyoxal-Derived Product in Tissue Proteins: Comparison with Pentosidine

Methylglyoxal (MG), an α-dicarbonyl compound, can be produced in vivo by several metabolic pathways and the Maillard reaction. It reacts rapidly with proteins to form advanced glycation end products or AGEs. We previously isolated and characterized a blue fluorescent product of the reaction between MG and arginine, which we named argpyrimidine. We found that argpyrimidine was stable to acid hydrolysis, which allowed us to hydrolyze tissue proteins with 6 N HCl and quantify argpyrimidine by high-performance liquid chromatography. Here we report argpyrimidine concentrations in human lens and serum proteins as determined by HPLC. We have also measured pentosidine, a fluorescent AGE derived from pentose sugars, and compared the concentrations of pentosidine and argpyrimidine. We found two- to threefold higher argpyrimidine concentrations in diabetic serum proteins than in nondiabetic controls (9.3 ± 6.7 vs 4.4 ± 3.4 pmol/mg). We found a significant correlation (P = 0.0001) between serum protein argpyrimidine and glycosylated hemoglobin. Argpyrimidine concentrations were approximately seven times greater in brunescent cataractous lenses than in aged noncataractous lenses. Pentosidine concentrations in serum and lens proteins were much lower than argpyrimidine concentrations; in general, argpyrimidine levels were 10–25 times higher than pentosidine. Results from our study confirm that MG-mediated arginine modifications occur in vivo and provide a method for assessing protein–arginine modification by MG in aging and diabetes.

β-Lactamase as a Marker for Gene Expression in Live Zebrafish Embryos

In this report we describe the development of a sensitive assay for gene expression in zebrafish embryos using β-lactamase as a reporter gene. We show that injection of a green fluorescent substrate for β-lactamase allows the detection of reporter gene expression in live embryos. The β-lactamase enzyme catalyzes the hydrolysis of the substrate, thereby disrupting fluorescence resonance energy transfer from the donor to the acceptor dye in the molecule. As a result, a blue fluorescent product is produced and retained specifically in cells within which the enzyme is expressed. β-Lactamase is therefore suitable for monitoring spatially restricted patterns of gene expression in the early embryo. We suggest that this new reporter system provides a major advancement in sensitivity over the existing methods for monitoring gene expressionin vivoduring early embryogenesis.

ChemInform Abstract: Kinetics of the Oxidation of Reserpine with Peroxodisulphate.

AbstractIn acidic media the Rauwolfia alkaloid (I) is oxidized to give the blue fluorescent product (III) via the yellowish green fluorescent dehydroreserpine (II) at two first‐order (or pseudo‐ first‐order) steps (activation energy 44 kJ/mol; mechanism including nucleophilic displacement).

Studies of the Chemiluminescence of Several Xanthene Dyes. VI. A Kinetic Analysis of a Blue-fluorescent Product in the Chemiluminescent Reaction of Eosin Y with Hydrogen Peroxide

Abstract In the present study, we found that the absorbance at 395 nm of the chemiluminescent reaction system of eosin Y with hydrogen peroxide initially increased to a maximum, and then gradually decreased. From the finding that the absorption spectrum of a chromatographically-isolated blue-fluorescent product (ME) showed a peak at the same wavelength (395 nm), we could confirm that the increment of the absorbance at 395 nm (Δ395) was mainly due to the generation of ME; we then extended the investigation of the mechanism for the chemiluminescent reaction by a kinetic analysis of ME. The observed value of Δ395 could be approximately calculated by the expression of [ME], which was derived from a kinetic scheme proposed in the previous paper. The ratio of the generation rates of ME in different media, which was calculated from the observed values of Δ395, was in good agreement with that estimated from the curves of the chemiluminescence emission intensity versus the time measured in the media. These results gave additional evidence in support of the view that the blue-fluorescent species is the primary excited product in the reaction.

Photochemical generation of stable fluorescent compounds (photofluorescence)

Abstract

The effect of hydrogen peroxide and copper(II) on reduced and oxidized diphosphopyridine nucleotide

Alkaline hydrogen peroxide in the presence of copper(II) ions has a multiple effect upon reduced and oxidized diphosphopyridine nucleotide: (a) reduced diphosphopyridine nucleotide is oxidized to diphosphopyridine nucleotide, ( b) diphosphopyridine nucleotide is split at the nicotinamide ribose bond, and ( c) nicotinamide is oxidized to give a blue fluorescent product. Neither N-methylpyridine nor N 1 -methylnicotinamide salts are split by hydrogen peroxide. Both free nicotinamide and pyridine are oxidized to fluorescent products.