A nanoplatform based on metal-organic frameworks and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity and inhibition.

A nanoplatform based on metal-organic frameworks (MOFs) and lambda exonuclease (λ exo) for the fluorimetric determination of T4 polynucleotide kinase (T4 PNK) activity and inhibition is described. Fe-MIL-88 was selected as the nanomaterial because of its significant preferential binding ability to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) and its quenching property. The synthesized Fe-MIL-88 was characterized by transmission electron microscope, scanning electron microscope, and X-ray photoelectron spectroscopy. In the presence of T4 PNK, FAM-labeled dsDNA (FAM-dsDNA) is phosphorylated on its 5'-terminal. λ exo then recognizes and cleaves the phosphorylated strand yielding FAM-labeled ssDNA (FAM-ssDNA). The fluorescence of the produced FAM-ssDNA is quenched due to Fe-MIL-88's absorbing on FAM-ssDNA. On the contrary, in the absence of T4 PNK, the phosphorylation and cleavage processes cannot take place. Therefore, the fluorescence of FAM-dsDNA still remains. The fluorescence intensity is detected at the maximum emission wavelength of 524 nm using the maximum excitation wavelength of 488 nm. The assay of T4 PNK based on the fluorescence quenching of FAM-ssDNA achieves a linear relationship in the range 0.01-5.0 U mL-1 with a detection limit of 0.0089 U mL-1 in buffer. The assay exhibits excellent performance for T4 PNK activity determination in a complex biological matrix. The results also reveal the ability of the assay for T4 PNK inhibitor screening. Graphical abstract Schematic presentation of a nanoplatform based on Fe-MIL-88 and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity. FAM-ssDNA, FAM-labeled single-stranded DNA; cDNA, complementary DNA; λ exo, lambda exonuclease;T4 PNK, T4 polynucleotide kinase.

Nitrogen doped graphene quantum dots as a fluorescent probe for mercury(II) ions.

A highly selective fluorescent probe for Hg2+ is reported. It consists of nitrogen doped graphene quantum dots (NGQDs) that are nearly spherical in shape, have an average diameter of 2.7 nm and excitation-independent emission. The blue fluorescence of the NGQDs (with maximum excitation/emission at 378/447 nm) is quenched by Hg2+ due to both dynamic and static quenching. The probe has a wide detection range (2.5 µM - 800 µM) and a limit of detection of 2.5 µM. The dynamic and static quenching constants are 417 M-1 and 63500 M-1, respectively. The probe was used to quantfy Hg2+ in spiked real water samples with satisfactory results. Graphical abstract ᅟ.

Solvent effect on the relative quantum yield and fluorescence quenching of a newly synthesized coumarin derivative.

We estimated the relative florescence quantum yield (Φ) of 8-methoxy-3-[1-(4,5-dicarbomethoxy-1,2,3-triazoloacetyl)]coumarin [8MDTC] using a single-point method with quinine sulfate in 0.1 M of sulfuric acid used as a standard reference. The fluorescence lifetimes, radiative and non-radiative decay rate constants are calculated. Relative quantum yields were found to be less in the non-polar solvents, indicating that the solute exhibits less fluorescence in a non-polar environment. The fluorescence quenching of [8MDTC] by aniline was studied at room temperature by examining the steady state in five different solvents in order to explore various possible quenching mechanisms. The experimental results show a positive deviation in Stern-Volmer plots in all solvents. Ground state complex and sphere of action static quenching models were used to interpret the results. Many quenching rate parameters were calculated using these models. The values of these parameters suggest that the sphere of action static quenching model agrees well with the experimental results. Further, a finite sink approximation model was used to check whether these bimolecular reactions were diffusion limited or not. The values of the distance parameter R' and the diffusion coefficient D were determined and are compared with the values of the encounter distance R and diffusion coefficient D calculated using the Stokes-Einstein equation.

Spectroscopic investigations on the effect of N-acetyl-L-cysteine-capped CdTe Quantum Dots on catalase.

Quantum dots (QDs) are recognized as some of the most promising semiconductor nanocrystals in biomedical applications. However, the potential toxicity of QDs has aroused wide public concern. Catalase (CAT) is a common enzyme in animal and plant tissues. For the potential application of QDs in vivo, it is important to investigate the interaction of QDs with CAT. In this work, the effect of N-Acetyl-L-cysteine-Capped CdTe Quantum Dots with fluorescence emission peak at 612 nm (QDs-612) on CAT was investigated by fluorescence, synchronous fluorescence, fluorescence lifetime, ultraviolet-visible (UV-vis) absorption and circular dichroism (CD) techniques. Binding of QDs-612 to CAT caused static quenching of the fluorescence, the change of the secondary structure of CAT and the alteration of the microenvironment of tryptophan residues. The association constants K were determined to be K288K=7.98×10(5)Lmol(-1) and K298K=7.21×10(5)Lmol(-1). The interaction between QDs-612 and CAT was spontaneous with 1:1 stoichiometry approximately. The CAT activity was also inhibited for the bound QDs-612. This work provides direct evidence about enzyme toxicity of QDs-612 to CAT in vitro and establishes a new strategy to investigate the interaction between enzyme and QDs at a molecular level, which is helpful for clarifying the bioactivities of QDs in vivo.

Static and dynamic model fluorescence quenching of laser dye by carbon tetrachloride in binary mixtures.

The fluorescence quenching of laser dye namely 4,4(‴)-Bis (2-butyloctyl-oxy)-p-quaterphenyl [BIBUQ] by carbon tetrachloride has been studied in different solvent mixtures of 1-4 dioxane (DN) and acetonitrile (AN) at room temperature. The quenching is found to be appreciable and a positive deviation from linearity was observed in the Stern-Volmer plot in all the solvent mixtures. Various parameters for the quenching process have been determined by sphere of action static quenching model and finite sink approximation model. The magnitudes of these rate parameters indicate that positive deviation in the Stern-Volmer (S-V) plot is both due to static and dynamic processes.

Solvent effect on the relative quantum yield and fluorescence quenching of 2DAM.

The relative quantum yield of diethyl 2-acetamido-2-((3-oxo-3H-benzo[f]chromen-1-yl)methyl)malonate [2DAM] is estimated using single point method with quinine sulfate as standard reference. The quantum yield varies between 0.1161 and 0.3181 depending on the nature of the solvent. The rates of radiative and non radiative decay constants are also calculated. The fluorescence quenching of [2DAM] by aniline is studied at room temperature, by steady state, in five different solvents namely acetonitrile (AN), 1,4 dioxane (DX), 1,2 dichloroethane (DCE), tetrahydrofuran (THF) and toluene (TOL), in order to explore various possible quenching mechanisms. The experimental results show a positive deviation in Stern Volmer plots for all solvents. Various parameters for the quenching process are determined by ground state complex, sphere of action static quenching model and finite sink approximation model. The magnitudes of these rate parameters indicate that positive deviation in the Stern Volmer (SV) plot is due to both static and dynamic processes. Further, finite sink approximation model is used to check whether these bimolecular reactions were diffusion limited or not. The values of distance parameter R' and diffusion co efficient D are determined and then compared with the values of encounter distance R and diffusion coefficient D calculated using Stokes-Einstein equation.