A sensitive homogeneous enzyme assay for euchromatic histone-lysine-N-methyltransferase 2 (G9a) based on terbium-to-quantum dot time-resolved FRET

Introduction: Histone modifying enzymes include several classes of enzymes that are responsible for various post-translational modifications of histones such as methylation and acetylation. They are important epigenetic factors, which may involve several diseases and so their assay, as well as screening of their inhibitors, are of great importance. Herein, a bioassay based on terbium-to-quantum dot (Tb-to-QD) time-resolved Förster resonance energy transfer (TR-FRET) was developed for monitoring the activity of G9a, the euchromatic histone-lysine N-methyltransferase 2. Overexpression of G9a has been reported in some cancers such as ovarian carcinoma, lung cancer, multiple myeloma and brain cancer. Thus, inhibition of this enzyme is important for therapeutic purposes. Methods: In this assay, a biotinylated peptide was used as a G9a substrate in conjugation with streptavidin-coated ZnS/CdSe QD as FRET acceptor, and an anti-mark antibody labeled with Tb as a donor. Time-resolved fluorescence was used for measuring FRET ratios. Results: We examined three QDs, with emission wavelengths of 605, 655 and 705 nm, as FRET acceptors and investigated FRET efficiency between the Tb complex and each of them. Since the maximum FRET efficiency was obtained for Tb to QD705 (more than 50%), this pair was exploited for designing the enzyme assay. We showed that the method has excellent sensitivity and selectivity for the determination of G9a at concentrations as low as 20 pM. Furthermore, the designed assay was applied for screening of an enzyme inhibitor, S-(5’-Adenosyl)-L-homocysteine (SAH). Conclusion: It was shown that Tb-to-QD FRET is an outstanding platform for developing a homogenous assay for the G9a enzyme and its inhibitors. The obtained results confirmed that this assay was quite sensitive and could be used in the field of inhibitor screening

Terbium Sensitized Chemiluminescence Method for the Determination of Rabeprazole -Application to Pharmaceutical Analysis and Dissolution Studies

A simple and sensitive chemiluminescence-based method was established for the determination of rabeprazole.The proposed method was based on the enhancing effect of rabeprazole on Ce(IV)-Na2SO3 -Tb(III) chemiluminescence reaction. A possible mechanism was discussed for chemiluminescence system by studying UV-Vis, fluorescence and chemiluminescence spectra. The effects of various chemical parameters were investigated and optimized. Under the optimum conditions, the enhanced chemiluminescence intensity was directly proportional to the concentration of rabeprazole in the range of 0.015-0.2 µg ml-1, with a detection limit of 6 ng ml-1. The proposed method was applied to the analysis of pharmaceutical formulations and human plasma samples and to the dissolution study of rabeprazole tablets with satisfactory results. The results indicated that more than 95% of the labeled amount of rabeprazole was dissolved over 30 min in the basic medium, while only 10% of rabeprazole was released in acidic medium

Strong enhancement of the chemiluminescence of the Cu(II)-H2O2 system on addition of carbon nitride quantum dots, and its application to the detection of H2O2 and glucose

The authors report that carbon nitride quantum dots (CN QDs) exert a strong enhancing effect on the Cu(II)/H2O2 chemilumi-nescent system. Chemiluminescence (CL) intensity is enhanced by CN QDs by a factor of similar to 75, while other carbon nanomaterials have a much weaker effect. The possible mechanism of the effect was evaluated by recording fluorescence and CL spectra and by examining the effect of various radical scavengers. Emitting species was found to be excited-state CNQDs that produce green CL peaking at 515 nm. The new CL system was applied to the sensitive detection of H2O2 and glucose (via glucose oxidase-catalyzed formation of H2O2) with detection limits (3 sigma) of 10 nM for H2O2 and 100 nM for glucose. The probe was employed for glucose determination in human plasma samples with satisfactory results

Facile synthesis of carbon quantum dot/silver nanocomposite and its application for colorimetric detection of methimazole

International audienc

Terbium–To–Quantum Dot Förster Resonance Energy Transfer for Homogeneous and Sensitive Detection of Histone Methyltransferase Activity

International audienceThe development of rapid, simple, and versatile biosensors for monitoring the activity of histone modifying enzymes (HMEs) is needed for the improvement of diagnostic assays, screening of HME inhibitors, and a better understanding of HME kinetics in different environments. Nanoparticles can play an important role in this regard by improving or complementing currently available enzyme detection technologies. Here, we present the development and application of a homogeneous methyltransferase (SET7/9) assay based on time-gated Förster resonance energy transfer (TG-FRET) between terbium complexes (Tb) and luminescent semiconductor quantum dots (QDs). Specific binding of a Tb-antibody conjugate to a SET7/9-methylated Lys4 on a histone H3(1–21) peptide substrate attached to the QD surface resulted in efficient FRET and provided the mechanism for monitoring the SET7/9 activity. Two common peptide-QD attachment strategies (biotin–streptavidin and polyhistidine-mediated self-assembly), two different QD colors (625 and 705 nm), and enzyme sensing with post- or pre-assembled QD–peptide conjugates demonstrated the broad applicability of this assay design. Limits of detection in the low picomolar concentration range, high selectivity tested against non-specific antibodies, enzymes, and co-factors, determination of the inhibition constants of the SET7/9 inhibitors SAH and (R)-PFI-2, and analysis of the co-factor (SAM) concentration-dependent enzyme kinetics of SET7/9 which followed the Michaelis–Menten model highlighted the excellent performance of this TG-FRET HME activity assay

Energy transfer with nanoparticles for in vitro diagnostics

International audienc