Non-isotopic receptor assay for benzodiazepines using a biotin-labeled ligand and biotin-immobilized microtiter plate.

A non-isotopic receptor assay for benzodiazepine drugs was developed using a biotin-labeled ligand, biotin-1012S. Biotinylated bovine serum albumin (biotin-BSA) was immobilized onto the wall of microtiter plate wells by simple adsorption. Avidin peroxidase conjugate could be extracted from solution owing to its strong interaction with biotin. The amount of avidin peroxidase taken up on the wall was then determined by measuring the enzyme activity. The competition between immobilized biotin on the wall and free biotin for avidin provided the basis for a solid-phase avidin-biotin binding assay. By this binding assay, not only biotin but also biotin-1012S could be measured sensitively. Because 1012S is a ligand with high affinity to benzodiazepine receptors, biotin-1012S could be utilized as a probe ligand for a non-isotopic receptor assay. Based upon the competition between biotin-1012S and various benzodiazepine drugs for the receptor binding sites, a non-isotopic receptor assay was demonstrated.     

Detection of small RNA molecules by a combination of branched rolling circle amplification and bioluminescent pyrophosphate assay

Polyfluorinated compounds (PFCs) are a relatively new and diverse set of compounds analyzed as contaminants in food. Their unique physical-chemical properties dictate the methods used for their analysis. Current analyses of the more volatile PFCs involve gas chromatography–mass spectrometry; liquid chromatography–tandem mass spectrometry is generally used for the less volatile PFCs. Considerations in the analysis of PFCs in foods include contamination from the widespread presence of materials that contain various PFCs, endogenous interfering compounds, and matrix effects. Future opportunities for research on PFCs in food exist, particularly in the areas of biological molecule–PFC interactions and the effects of food processing on these interactions. Future research will be facilitated by the synthesis of a wider variety of analytical standards.     

Classification for dimethylarsenate-decomposing bacteria using a restrict fragment length polymorphism analysis of 16S rRNA genes.

A new monitoring system for bacterial communities involving dimethylarsinic acid (DMAA) decomposition was provided by combining the MPN (Most Probable Number) method and RFLP (restriction-fragment-length polymorphism analysis). The abundance of DMAA decomposing bacteria was estimated by the MPN method using a bacterial culture medium, which included DMAA as the sole carbon source, indicating bacterial cell densities of 1700 cells/ml in Lake Kahokugata and 330 cells/ml in Lake Kibagata. After isolating the dominant bacteria using agar plates, the isolates were classified into some genotype groups by RFLP analysis using 16S rDNA sequences. Classification of the RFLP analysis indicated that 14 isolates of Lake Kahokugata were classified into 6 types, which included 2 dominant types related to genus Pseudomonas, while 8 isolates of Lake Kibagata displayed 6 types including one or two isolates. Moreover, the RFLP types were unique for each lake, suggesting that DMAA decomposing bacteria were specific for the aquatic environment related to the arsenic cycle. The activities of DMAA decomposition mostly matched with the RFLP type category of the isolates. Accordingly, combining the MPN method with the RFLP analysis will play an important role in elucidating the distributions and dynamics of the DMAA-decomposing bacterial community.     

Phytochemical profiling and evaluation of modified resazurin microtiter plate assay of the roots of Trillium govanianum

Abstract

Trillium govanianum Wall. ex D. Don (Melanthiaceae alt. Trilliaceae), is native to the Himalayas. The present study, for the first time, was undertaken to explore the antimicrobial potential, to determine the minimum inhibitory concentration (MIC) values of the methanol extract of the roots of Trillium govanianum and its solid phase extraction (SPE) fractions by using resazurin microtiter assay (REMA) against Gram positive and Gram negative bacterial registered strains and to carry out phytochemical analysis. The remarkable amount of gallic acid equivalent phenolic and quercetin equivalent flavonoid content was manifested by MeOH extract (20.27?±?3.03?mg GAE/g DW and 9.25?±?0.50?mg QE/g DW respectively). The GC/MS analysis revealed the presence saturated and unsaturated components. Considerable level of antibacterial potential against Gram-positive bacteria (MIC: 2.5-0.009?mg/mL) than against Gram-negative bacteria (MIC: 2.5-0.165?mg/mL) were observed. The use of microtiter plates has the advantage of lower cost, fast and quantitative results.     

Supramolecular hydrogel of kanamycin selectively sequesters 16S rRNA

As the first example of hydrogelator derived from aminoglycoside antibiotics, the hydrogel of kanamycin indicates that the hydrogel of aminoglycosides preserve the specific interaction with their macromolecular targets (e.g., 16S rRNA), thus illustrating a simple approach to explore and identify possible biological targets of supramolecular nanofibers/hydrogels.     

Evaluation of resazurin microtiter plate assay and HPLC- photodiode array analysis of the roots of Asparagus adscendens

Asparagus adscendens Roxb. (Asparagaceae), is native to the Himalayas. The present study, for the first time, was undertaken to explore the antimicrobial potential, to determine the minimum inhibitory concentration (MIC) values of the methanol extract of the roots of A. adscendens and its solid-phase extraction (SPE) fractions using resazurin microtitre assay against Gram-positive and negative bacterial-registered strains and to carry out HPLC-photodiode array analysis of the SPE fractions. The methanol extract and all SPE exhibited considerable level of antibacterial potential against Gram-positive bacteria (MIC: 2.5–0.009 mg/mL) than against Gram-negative bacteria (MIC: 1.25–2.5 mg/mL). The use of microtitre plates has the advantage of lower cost, fast and quantitative results. Like other Asparagus species, the presence of phenolic compounds in all SPE fractions was evident in the HPLC-PDA data.     

A microtiter plate assay for the detection of inhibitors of the Na +, K + -ATpase

A rapid microtiter plate assay for the detection of inhibitors of the Na⁺, K⁺-ATPase has been developed. The assay is based on the measurement of inorganic phosphate released from the substrate, ATP, and has been designed to be carried out in the individual wells of a microtiter plate. Since the production of inorganic phosphate is determined colorimetrically, multiple samples can be tested simultaneously using a microtiter plate reader. This microtiter plate assay is particularly useful for screening large numbers of samples, such as microbial culture supernatants.     

Spectrophotometric determination of various polyanions with polymeric film optodes using microtiter plate reader

Polycation-sensitive membrane optodes based on the chromoionophore 2′,7′-dichlorofluorescein octadecylester (DCFOE) have previously been developed and used for determination of heparin via a titrimetric method. In this study, it is shown that some other important polyanions such as PPS (pentosan polysulfate), DNA, xanthan, Na-alginate, and carrageenan (food additive) can also be readily determined by using DCFOE-based microtiter plate-format optodes (MPOs) and polycationic titrants that bind these polyanionic species. The optical sensors are prepared with poly(vinyl chloride) (PVC), polyurethane (PU), bis(2-ethylhexyl)sebacate (DOS), and 2′,7′-dichlorofluorescein octadecylester (DCFOE) and exhibit reproducible and sensitive absorbance changes in response to the varying polycationic titrant concentrations. Three different polycations; protamine, poly-l-lysine and poly-l-arginine, are employed as titrants. The method has a detection limit of 1 μg mL⁻¹, and a dynamic range of 1–40 μg mL⁻¹. After the quantitative determinations are successfully demonstrated in buffered solutions, similar titrations are also performed in real samples. The method is validated by recovery studies in these samples. The average polyanion recoveries were quantitative [99.7(±1.3) % for pastry cream with vanillin (protamine titrant); 100.4 (±3.3) % for pastry gel with strawberry(PLA titrant), and 102.9(±2.0) % for pastry gel with strawberry (PLL titrant)].     

Miniaturization of a chloride ion assay for use in a microtiter format

We have miniaturized a common assay for the spectrophotometric determination of chloride in a variety of sample substrates, including biological fluids. The modified assay requires only 2 μL of serum, making it ideal for use with small animal systems for which only small volumes of biological fluids can be obtained. The assay utilized small amounts of inexpensive chemicals, and was shown to provide linear results for chloride up to 150 ppm, and the sensitivity was measured as low as 1 ppm. The assay was employed to measure chloride ion in the serum of alligators, thus allowing for the determination of dehydration status in animals that were exposed to saltwater for prolonged periods of time.     

High-throughput sensing microtiter plate for determination of biogenic amines in seafood using fluorescence or eye-vision

A new optical sensing microplate was developed for rapid screening for the presence of biogenic amines (BAs) in seafood samples with high sensitivity. The deposition of a sensing spot (containing a chameleon dye (Py-1) in a polymeric cocktail) on the bottom of the wells of a standard microplate renders the plate a new sensing tool for a rapid and parallel detection of up to 96 (real) samples. This sensing microplate enables (1) a semi-quantitative readout of analyte concentration by eye-vision, (2) a rapid fluorescence readout of 96 samples with standard instrumentation in less than two minutes (unlike chromatographic and electrophoretic methods), (3) a statistically robust data evaluation (with 8-12 replicates) and (4) a rapid parallel sample preparation with standard 8 or 12-channel micropipettes. On reaction with biogenic amines, the dye shows a significant visible color change from blue over green to red color. The appearance of red color favorably coincides with the concentration of BAs that can induce symptoms of poisoning. The linear ranges of fluorescence calibration data for six biogenic amines cover the clinical toxicological relevant range of BAs that is too low to be detected by the human nose. The LODs range from 0.16 to 0.56 μg mL(-1), with correlation coefficients (r(2)) between 0.985 and 0.999. Finally, the evolution of spoilage of four fish samples (monitored by determination of their BA status) and the increase of their total amine content were found to agree well with previous data on time-dependent evolution of BAs in fish.     

Rapid and highly sensitive detection of Escherichia coli O157:H7 in food with loop-mediated isothermal amplification coupled to a new bioluminescent assay

Testing for bioluminescent pyrophosphate is a convenient method of DNA detection without complex equipments, but it is insufficiently sensitive and offers no particular time advantage over other rapid detection methods. The shortcomings of the traditional bioluminescent pyrophosphate method have been addressed by using 2-deoxyadenosine-5-(α-thio)-triphosphate (dATPαS) instead of dATP for LAMP, thus reducing the high background signal and generating a constant background value. In this study, LAMP coupled to a novel bioluminescent pyrophosphate assay was developed to detect E. coli O157:H7. The new method has a limit of detection of <10 copies/μL or 5 CFU/mL; its sensitivity is higher than that of the conventional LAMP assay. Moreover, a food-borne pathogen can be detected when a single DNA template is included in the LAMP assay, making it 100 times more sensitive than the traditional LAMP method. Three hundred food samples were tested with this assay and the accuracy of detection was verified with a culture method and MALDI Biotyper. The assay only took 90–120 min and detected <10 copies of the pathogen. This method had the advantages of rapidity, sensitivity, and simplicity, so it is very competitive for the rapid and highly sensitive detection of food-borne pathogens.     

Automating a 96-well microtiter plate assay for identification of AGEs inhibitors or inducers: application to the screening of a small natural compounds library

Advanced glycation end-products (AGEs) are involved in the pathogenesis of numerous affections such as diabetes and neurological diseases. AGEs are also implied in various changes in tissues and organs. Therefore, compounds able to break them or inhibit their formation may be considered as potential drugs, dietary supplements, or bioactive additives. In this study, we have developed a rapid and reliable (Z′ factor calculation) anti-AGEs activity screening based on the overall fluorescence of AGEs. This method was successfully evaluated on known AGEs inhibitors and on a small library of natural compounds, yielding coherent results when compared with literature data.     

Development of a fluorescence-based microtiter plate method for the measurement of glutathione in yeast

The present work was aimed to the development of a fluorescence assay using the universal 96-well microplate format, for the measurement of reduced glutathione (GSH) in yeast cells. The method relies upon the reaction between GSH and a highly selective fluorogenic probe, i.e. naphthalene-2,3-dicarboxaldehyde (NDA). The optimization of the method included the extraction step of GSH from cultured yeast cells in a cold perchloric acid solution, derivatization conditions (10-min reaction at pH 8.6 and at 20 ± 2 °C in darkness) and stability studies of the resulting fluorescent adduct. Full selectivity was observed versus other endogenous thiols (except for γ-glutamylcysteine), glutathione disulfide (GSSG) and enzymatic reducing reagents of GSSG. Linearity was verified in the range 0.3-6.5 μM (R² > 0.98) and limits of quantification and detection were 0.3 and 0.05 μM, respectively. Relative standard deviation corresponding to repeatability (n = 3) and inter-day precision (n = 5) were 2.8 and 6.1%, respectively. Mean GSH recovery from cell extracts was 95%. The method appeared highly correlated (R² = 0.96) with a previously reported HPLC method.The method was then applied to the monitoring of GSH in the yeast strain Kluyveromyces lactis during its growth period and in the presence of an inhibitor of GSH biosynthesis. The method presents the main advantage of a high throughput for the measurement of biological samples. The extent of the method to the study of the redox couple GSSG/GSH by including an enzymatic reduction step and the enhancement of the fluorescence signal using cyclodextrins were discussed.     

Determination of glucose using a coupled-enzymatic reaction with new fluoride selective optical sensing polymeric film coated in microtiter plate wells

The determination of glucose in beverages is demonstrated using newly developed fluoride selective optical sensing polymeric film that contains aluminum (III) octaethylporphyrin (Al[OEP]) ionophore and the chromoionophore ETH7075 cast at the bottom of wells of a 96-well polypropylene microtiter plate. The method uses a dual enzymatic reaction involving glucose oxidase enzyme (GOD) and horseradish peroxidase (HRP), along with an organofluoro-substrate (4-fluorophenol) as the source of fluoride ions. The concentration of fluoride ions after enzymatic reaction is directly proportional to the glucose level in the sample. The method has a detection limit of 0.8 mmol L⁻¹, a linear range of 0.9-40 mmol L⁻¹ and a sensitivity of 0.125 absorbance/decade of glucose concentration. Glucose levels in several beverage samples determined using the proposed method correlate well with a reference spectrophotometric enzyme method based on detection of hydrogen peroxide using bromopyrogallol red dye (BPR). The new method can also be used to determine H₂O₂ concentrations in the 0.1-50 mmol L⁻¹ range using a single enzymatic reaction involving H₂O₂ oxidation of 4-fluorophenol catalyzed by HRP. The methodology could potentially be used to detect a wide range of substrates for which selective oxidase enzymes exist (to generate H₂O₂), with the high throughput of simple microtiter plate detection scheme.     

Design of a coupled bioluminescent assay for a recombinant pyruvate kinase from a thermophilic Geobacillus

A simple and rapid method using coupled bioluminescent assay was developed to determine level of ADP. ADP is involved in many biological reactions and ADP assay can be used for assaying some reactions universally by monitoring ADP formation or depletion. ADP analysis involves incubation of ADP or extracts containing ADP with pyruvate kinase (PK) and PEP. The ATP formed by this reaction is determined by measuring the intensity of the initial light flash produced when luciferin-luciferase preparation injected into the reaction mixture. In regard to the main role of the PK in this assay, the gene of PK from a Geobacillus species has been cloned in expression vector pET28a (+), sequenced and overexpressed in Escherichia coli. Recombinant protein was purified using Ni-NTA column and then the purified PK was used in a coupled bioluminescent assay for ADP measurement. Kinetic properties of PK are determined according to a bioluminescent assay using firefly luciferase.     

Molecular docking and 3D-QSAR study of pyranmycin derivatives against 16S rRNA A site

To understand pharmacophore properties of pyranmycin derivatives and to design novel inhibitors of 16S rRNA A site, comparative molecular field analysis (CoMFA) approach was applied to analyze three-dimensional quantitative structure–activity relationship (3D-QSAR) of 17 compounds. AutoDock 3.0.5 program was employed to locate the orientations and conformations of the inhibitors interacting with 16S rRNA A site. The interaction mode was demonstrated in the aspects of inhibitor conformation, hydrogen bonding and electrostatic interaction. Similar binding conformations of these inhibitors and good correlations between the calculated binding free energies and experimental biological activities suggest that the binding conformations of these inhibitors derived from docking procedure were reasonable. Robust and predictive 3D-QSAR model was obtained by CoMFA with q² values of 0.723 and 0.993 for cross-validated and non-cross-validated, respectively. The 3D-QSAR model built here will provide clear guidelines for novel inhibitors design based on the Pyranmycin derivatives against 16S rRNA A site.     

Microtiter plate assay for phosphate using a europium-tetracycline complex as a sensitive luminescent probe

A new luminescent europium probe is presented for the determination of phosphate (P) in microtiter plate format. The assay is based on the quenching of the luminescence of the europium-tetracycline (EuTc) 1:1 complex by phosphate using a reagent concentration of 20.8 μmol/L. The probe is excited at 400 nm and displays a large Stokes’ shift of 210 nm. The emission maximum is located at 616 nm. The system works best at neutral pH 7 and is therefore suitable for phosphate determination in biological and biochemical systems. The linear range of the calibration plot is from 5 × 10⁻⁶ mol/L to 7.5 × 10⁻⁴ mol/L of phosphate, and the limit of detection is 3 μmol/L.