Fluorescence-based adenylyl cyclase assay adaptable to high throughput screening

The second messenger cAMP has been implicated in numerous cellular processes such as glycogen metabolism, muscle contraction, learning and memory, and differentiation and development. Genetic evidence suggests that the enzyme that produces cAMP, adenylyl cyclase (AC), may be involved in pathogenesis in many of these cellular processes. In addition, these data suggest that membrane-bound ACs may be valuable targets for therapeutics to treat pathogenesis of these processes. The development of a robust real-time adenylyl cyclase assay that can be scalable to high-throughput screening could help in the development of novel therapeutics. Here we report a novel fluorescence-based cyclase assay using Bodipy FL GTPgammaS (BGTPgammaS). The fluorescence of the Bodipy moiety of BGTPgammaS was dramatically enhanced by incubation with the minimal catalytic core of wild-type-AC (wt-AC) and a mutant with decreased purine selectivity (mut-AC), in an AC activation-dependent manner. No increase in fluorescence was observed using Bodipy FL ATPgammaS (BATPgammaS) as substrate for either wt-AC or mut-AC. Using BGTPgammaS, forskolin, Gsalpha.GTPgammaS and the divalent cation Mn(2+) potently enhanced the rate of fluorescence increase in a concentration-dependent manner. The fluorescence enhancement of the Bodipy moiety was inhibited by known inhibitors of AC such as 2'deoxy,3'AMP and 2',5'-dideoxy-3'ATP. Furthermore, the fluorescence assay is adaptable to 96-well and 384-well multiplate format and is thus applicable to high throughput screening methodologies.     

CE assay of methylmalonyl-coenzyme-a mutase activity

Methylmalonyl-coenzyme A mutase (MCM) is a 5'-deoxyadenosylcobalamin-linked mitochondrial enzyme that catalyzes the isomerization of L-methylmalonyl-coenzyme A to succinyl-coenzyme A. We described a method for methylmalonyl-CoA and succinyl-CoA separation by CE, suitable for the evaluation of MCM activity. The working conditions for optimal separation were obtained in order to achieve the best resolution in the shortest analysis time. The optimization of buffer composition together with other variables, such as injection time, separation voltage, migration time, and capillary temperature, resulted in a solution of 30 mM NaH2PO4 containing 15 mM SDS, pH 3.2. Separations were carried out in an uncoated fused-silica capillary (55 cm, 50 microm id) at -25 kV, reading at 254 nm. The method performance was evaluated by measuring total and holo-MCM activity in biological matrices such as rat liver and human peripheral blood lymphocytes (PBL). The mean MCM activity was expressed in nmol/h/mg protein of tissue/cell extract and was calculated from the amount of reaction product formed. The rapidity of analysis and utmost precision (repeatability and within-laboratory reproducibility) point out the potentialities of the proposed method for the differential diagnosis of methylmalonic acidemia, in relation to protein or coenzyme defects.     

Product identification and adenylyl cyclase activity in chloroplasts of Nicotiana tabacum

In view of the ongoing debate on plant cyclic nucleotide metabolism, especially the functional presence of adenylyl cyclase, a novel detection method has been worked out to quantify the reaction product. Using uniformly labelled (15)N-ATP as a substrate for adenylyl cyclase, a qualitative and quantitative liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) method was developed to measure de novo formed (15)N-adenosine 3',5'-cyclic monophosphate. Adenylyl cyclase activity was observed in chloroplasts obtained from Nicotiana tabacum cv. Petit Havana and the kinetic parameters and influence of various metabolic effectors are discussed in their context.     

Structure and dynamics of photoactivatable adenylyl cyclase

Russian Chemical Bulletin - The full-atom 3D model of the dimer of the photoregulated adenylyl cyclase is constructed. It contains a molecule of flavin mononucleotide chromophore in the...     

Immunochromatographic assay for the detection of ochratoxin A

The detection of mycotoxins—toxic contaminants of fungal origin—is an important problem in the food and feed quality control. An immunochromatographic system was developed for the detection of ochratoxin A (OTA), which is one of the priority contaminants in grain. Monoclonal antibodies against OTA and their conjugates with colloidal gold nanoparticles were prepared. The detection is based on the competition of OTA in a sample and an OTA-protein conjugate immobilized on a test strip for the binding to anti-bodies on the colloidal particle surface. The method was tested in the analysis of plant extracts (maize and barley extracts). It was shown that OTA can be detected in a medium with a high content of an organic solvent (up to 35% of methanol). The disappearance of the line in the test zone is visually detected at OTA concentrations starting from 50 ng/mL. In the case of the video-digital detection of changes in the color intensity of the test zone, the limit of detection of OTA is 5 ng/mL. The duration of the assay is 10 min.     

A microfluidic protease activity assay based on the detection of fluorescence polarization

This article describes a fluorescence polarization (FP)-based protease assay on a microfluidic device that is compatible with fast and reproducible analyses of protease activities. The optical systems were arranged for simultaneously measuring fluorescence intensities of vertical and horizontal polarization planes, and the binding of tetramethylrhodamine (TMR) labeled-biotin with streptavidin was utilized for optimizing FP detection in continuously flowing solutions within 74-μm wide, 12-μm deep microchannels of a glass chip. In developing off-chip FP-based assays for proteinase K, trypsin, papain and elastase, TMR conjugated-casein protein (TMR-α-casein) was employed as a universal substrate. After optimization of the hydrodynamic flow control to allow complete mixing of TMR-α-casein and short proteolysis time as possible, and of buffer composition to minimize protein sticking problems, the developed assay was transferred to the microfluidic chip by monitoring FP changes of TMR-α-casein in the main microchannel. The results indicate that the proposed device would serve as an integrated microfluidic platform with automated injection of reacting species, diffusion-controlled mixing, reaction and detection for protease activities without the need to separate the products.     

In-column fiber-optic laser-induced fluorescence detection for CE

A highly sensitive in-column fiber-optic LIF detector for CE has been constructed and evaluated. In this detection system, a 457-nm diode-pumped solid-state blue laser was used as the excitation light source and an optical fiber (40 mum od) was used to transmit the excitation light. One end of the optical fiber was inserted into the separation capillary and was in situ positioned at the detection window. The other end of the fiber was protruded from the capillary to capture the excitation light beam from the blue laser. Fluorescence emission was collected by a 40 x microscope objective, focused on a spatial filter, and passed through a yellow color filter before reaching the photomultiplier tube. The present CE-fluorescence detection is a simple and compact optical system. It reduces the laser scattering effect from the capillary and fiber as compared to the conventional LIF detection for CE. Its utility was successfully demonstrated by the separation and determination of D-penicillamine labeled with naphthalene-2,3-dicarboxaldehyde. The detection limit was 0.8 nM (S/N = 3). The present detection scheme has been proven to be attractive for sensitive fluorescence detection for CE.     

High-voltage paper electrophoretic assay for guanyl cyclase.

A simple, sensitive and rapid technique is described, permitting separation of cGMP from GMP, GDP and GTP by the use of unidirectional high-voltage paper electrophoresis. The recovery of labeled cGMP in the assay of guanyl cyclase, by this procedure is 85-90%; the blank values (no enzyme) are negligible.     

An electrochemical approach for detection of DNA methylation and assay of the methyltransferase activity

This work develops an electrochemical approach for rapid detection of the genomic DNA methylation level, assay of methyltransferase activity, and evaluation and screening of the inhibitors of methyltransferase. This method may be a help for the discovery of anticancer drugs.     

A label-free and sensitive fluorescent assay for one step detection of protein kinase activity and inhibition

In this paper, a label-free, highly sensitive and simple assay for one step detection of protein kinase (PKA) activity and inhibition that avoids the fluorescent dye process has been established. The detection was based on the fluorescence (FL) quenching of peptide-Ag nanoclusters (Ag NCs) caused by antibody modified Au nanoparticles (anti-Au NPs) via fluorescence resonance energy transfer (FRET). With PKA and adenosine 5′-triphosphate (ATP) introduced, the substrate peptide of Ag NCs could react with PKA via targeted phosphorylation, and followed by the linking interactions between peptide-Ag NCs and anti-Au NPs. According to the fluorescence quenching of Ag NCs, the activity of protein kinase can be facilely monitored in the range of 0.1–2000 mU/μL with high sensitivity. The detection limit for PKA is 0.039 mU/μL. We further explored the inhibitory effect of H-89 for protein kinase activity. The developed method was also applied to the investigation of drug-induced PKA activation in HeLa cells, which provides a promising means for screening of kinase-related drugs and the clinical diagnosis of disease.     

Magnetic beads-based electrochemiluminescence assay for rapid and sensitive detection of telomerase activity

Telomerase is a potential cancer marker. We developed a new and robust telomerase activity assay which combines the modified telomere repeat amplification protocol (TRAP) with magnetic beads-based electrochemiluminescence (ECL) detection. The high performance of this assay is related to the determination of telomerase activity from single cell levels, and ECL intensity is linear over the range of 1–1000 HeLa cell equivalents. The proposed telomerase assay offers a highly cost- and time-effective alternative to presently available telomerase assays, which are limited by tedious and complicated post-PCR detection.     

A bio-barcode assay for on-chip attomolar-sensitivity protein detection

Functionalized nanoparticles hold great promise in realizing highly sensitive and selective biodetection. We report a single disposable chip which is capable of carrying out a multi-step process that employs nanoparticles--a bio-barcode assay (BCA) for single protein marker detection. To illustrate the capability of the system, we tested for the presence of prostate specific antigen (PSA) in buffer solution and goat serum. Detection was accomplished at PSA concentrations as low as 500 aM. This corresponds to only 300 copies of protein analytes using 1 microL total sample volume. We established that the on-chip BCA for PSA detection offers four orders of magnitude higher sensitivity compared to commercially available ELISA-based PSA tests.     

A new microbial assay for the toxicity detection of contaminated soils

A comparative study to detect toxicity prior to bioremediation treatment was set in order to investigate dehydrogenase activity inhibition of a common soil bacterium caused by soil contaminated with Cu, Pb, and As. A spectrophotometric test with Pseudomonas fluorescens strain ATCC 13525 utilising the 2,3,5-triphenyl tetrazolium chloride (TTC) reduction by microbial dehydrogenase has been adapted for this purpose. Soil samples are incubated for 48 hours at 30 +/- 1 degrees C in 18-ml tubes in the presence of TTC as an artificial electron-acceptor. The reduced TTC forms a reddish colour substance named triphenyl formazan (TPF), which can be extracted from the microbial cells and measured colorimetrically. The rapid response of biological activity in microorganisms and the reported sensitivity to the toxicants in the contaminated samples are reflected by the TTC reduction method, which is a sensitive tool for toxicity screening of contaminated sites, routine monitoring of bioremediation processes, as well as for feasibility studies of bioremediation treatments, in order to assess whether a specific pollutant or any other substance at a site location could inhibit the microbiological processes.     

Simultaneous LIF and retro-reflected beam interference detection for CE

This Short Communication describes a novel optical detection method for CE, based on the combination of LIF detection and retro-reflected beam interference detection. By the use of a side-illuminated laser beam, an on-column multifunctional detection for CE has been developed, and some key elements in the scheme have been optimized. In addition, two miscellaneous samples including fluorescent dyes, carbohydrates and amino acids have been determined to evaluate its performance. Without any additional pretreatment, sufficient LOD and linear ranges have been achieved for most analytes. Its performance in retro-reflected beam interference detection is better than those mentioned in a former report, and its fluorescent sensitivity is comparable with the achievable sensitivity by conventional LIF systems, all of which together combine high sensitivity and universal analysis to a certain extent.     

Light-emitting diode-compatible probes for indirect detection of anions in CE

A range of compounds were evaluated as probes for the indirect detection of inorganic ions using CE and light-emitting diodes (LEDs) as the light source. Emphasis was placed on examining probes likely to absorb strongly in the UV-Vis region near 350-430 nm as compounds, which absorb at longer wavelengths tend to be bulkier and adsorb onto the capillary wall. These probes should act as a replacement for the very effective but carcinogenic probe chromate. Two probes were identified and evaluated: p-nitrophenol and 4-hydroxy-3,5-dinitrobenzoic acid. The former showed the most potential with low-mobility anions, while the later had a moderate electrophoretic mobility and was more suitable for a wider mobility range of analytes. However, neither could match the efficiencies and LOD of chromate for the separation of the fast inorganic ions such as chloride, nitrate and sulphate. Nevertheless, application of the 4-hydroxy-3,5-dinitrobenzoic acid system to the determination of oxalate in Bayer liquors showed excellent sensitivity and selectivity.     

A high-throughput screening assay for hydroxynitrile lyase activity

A high-throughput screening assay for hydroxynitrile lyase activity accepting a wide range of HNL-substrates is presented, which is useful either for enzyme fingerprinting or screening of huge variant libraries generated in metagenome or directed evolution approaches.     

A novel dot-blot DNAzyme-linked aptamer assay for protein detection

In this work, a novel dot-blot DNAzyme-linked aptamer assay (DLAA) for protein detection is developed with thrombin as a model protein. A peroxidase-like DNAzyme which serves as the catalytic label is tethered to a 15-mer thrombin-binding aptamer to form a label-free DNAzyme-linked aptamer probe. Based on specific interaction of the aptamer with target protein immobilized on nitrocellulose membrane, a DNAzyme layer is introduced onto the membrane. The DNAzyme can catalyze the H₂O₂-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine to produce a colored insoluble product that is apt to be adsorbed onto the nitrocellulose membrane. As a result, blue dots appear on the membrane, in contrast to the colorless background. As the concentration of thrombin increases, the color of dots gets deep. Such a protein concentration-dependent color change can be quantified via an image-processing software, with a detection limit of 0.6 μM. Furthermore, this assay has been applied successfully to the detection of thrombin in biological samples (e.g., human serum), indicating its practicality for bioanalysis.