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Showing papers in "Journal of analytical and bioanalytical techniques in 2015"


Journal ArticleDOI
TL;DR: Cell proliferation rates, spheroid structures, cellular responses to different anti-cancer drugs, the expression of drug action-related proteins, and the possible correlations among these properties of 3D spheroids on Matrigel are investigated.
Abstract: This study systematically investigated the cell proliferation rates, spheroid structures, cellular responses to different anti-cancer drugs, the expression of drug action-related proteins, and the possible correlations among these properties of 3D spheroids on Matrigel in comparison to 2D monolayer cells, using two cancer cell lines-the prostate cancer cell line, DU145, and the oral cancer cell line, CAL27. Compared to the traditional 2D-cultured cells, 3D-cultured CAL27 cells had enhanced proliferation by approximately 50-70% at various seeding cell densities, whereas 3D-cultured DU145 cells showed reduced proliferation at all tested seeding cell densities by 20-40%. In drug tests, the sensitivity of 3D-cultured DU145 cells relative to 2D-cultured cells showed an obvious drug action mechanism dependency in response to three anticancer drugs, Rapamycin, Docetaxel, and Camptothecin, whereas 3D-cultured CAL27 cells responded more sensitively than 2D-cultured cells to all three tested drugs, Docetaxel, Bleomycin, and Erlotinib, indicating the relative proliferation rate between 3D and 2D cultured cells may be a dominating factor in this case and mitigated the factor of drug action mechanism. The elevated expression of EGFR in 3D-cultured CAL27 was correlated with its more sensitive response to Erlotinib (acting through binding to EGRF) compared to 2D-cultured cells; Similarly, the expression of βIII tubulin in 3D-cultured DU145 cells was found to be increased and correlated with their higher resistance to Doxetaxel compared to 2D-cultured cells.

45 citations


Journal ArticleDOI
TL;DR: An effective analytical method development and its validation can provide significant improvements in precision and a reduction in bias errors and can further help to avoid costly and time consuming exercises.
Abstract: Analytical method development and validation are the continuous and inter-dependent task associated with the research and development, quality control and quality assurance departments. Analytical procedures play a critical role in equivalence and risk assessment, management. It helps in establishment of product-specific acceptance criteria and stability of results.Validation should demonstrate that the analytical procedure is suitable for its intented purpose. Design of experiment is a powerful tool for the method characterization and validation. Analytical professionals should be comfortable to use it to characterize and optimize the analytical method. An effective analytical method development and its validation can provide significant improvements in precision and a reduction in bias errors. It can further help to avoid costly and time consuming exercises.

44 citations


Journal ArticleDOI
TL;DR: The main aim of the review is to give an overview of advancements in solid state fermentation for production of fungal lipase hitherto.
Abstract: Importance of enzymes is ever-growing specifically microbial lipases which are of great industrial significance because of their applications in detergent, food, pharmaceutical, chemical and leather industry. Solid state fermentation (SSF) is an economical alternative for large scale production of enzymes that are produced by fungi. Therefore, production of lipases by solid state fermentation is a good and preferred option than submerged fermentation (SmF). The important factors in fermentation are carbon concentration, nitrogen concentration, pH, growth temperature, fermentation time and moisture content. This review mainly focuses on production of fungal lipase by solid state fermentation using various fungal strains, substrates and fermentation conditions. Enzyme characteristics, industrial application and assay methods of lipase, biomass estimation, enzyme extraction methods and engineering aspects of fermentation are also dealt with briefly. The main aim of the review is to give an overview of advancements in solid state fermentation for production of fungal lipase hitherto.

36 citations


Journal ArticleDOI
TL;DR: A surface-enhanced Raman spectroscopy (SERS)-based method was successfully developed to rapidly measure cocaine in saliva at clinical concentrations, as low as 25 ng/mL, and could be applied to other drugs of interest.
Abstract: Increases in illicit drug use and the number of emergency-room visits attributable to drug misuse or abuse highlight the need for an efficient, reliable method to detect drugs in patients in order to provide rapid and appropriate care. A surface-enhanced Raman spectroscopy (SERS)-based method was successfully developed to rapidly measure cocaine in saliva at clinical concentrations, as low as 25 ng/mL. Pretreatment steps comprising chemical separation, physical separation, and solid-phase extraction were investigated to recover the analyte drug from the saliva matrix. Samples were analyzed using Fourier-transform (FT) and dispersive Raman systems, and statistical analysis of the results shows that the method is both reliable and accurate, and could be used to quantify unknown samples. The procedure requires minimal space and equipment and can be completed in less than 16 minutes. Finally, due to the inclusion of a buffer solution and the use of multiple robust pretreatment steps, with minimal further development this method could also be applied to other drugs of interest.

35 citations


Journal ArticleDOI
TL;DR: In this article, the chemical composition, molecular structure and physicochemical properties of five Gram-positive bacterial strain: Bacillus cereus, Bacillus subtilis, Sarcina lutea, Staphylococcus aureus, Micococcus luteus were investigated by Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), NMR and intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (IC MALDI TOF MS).
Abstract: The chemical composition, molecular structure and physicochemical properties of five Gram-positive bacterial strain: Bacillus cereus, Bacillus subtilis, Sarcina lutea, Staphylococcus aureus, Micococcus luteus were investigated by Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), NMR spectroscopy and intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (IC MALDI TOF MS). An analysis of FTIR spectra as a function of pH revealed the three major types of cell wall functional groups – carboxyl group, amino group and phosphate group. An analysis of XPS spectra was determinate the major surface components of bacterial cell. 13 C NMR and IC MALDI TOF MS spectra of six bacterial species were registered. Our findings indicate that chemical and structural differences in the cell composition of Gram-positive bacteria can be detected. The obtained results also demonstrate that the combination of FTIR, XPS and NMR spectroscopy with IC MALDI TOF MS technique yields useful information and complements other biochemical and physical methods of microbial cells characteristics.

24 citations


Journal ArticleDOI
TL;DR: In this paper, Fourier transform infrared (FT-IR) and UV spectroscopy results suggest an impact of biofield treatment on the force constant, bond strength and dipole moments of treated drugs such as disulfiram and nicotinic acid that could led to change in their chemical stability as compared to control.
Abstract: Disulfiram is being used clinically as an aid in chronic alcoholism, while nicotinic acid is one of a B-complex vitamin that has cholesterol lowering activity. The aim of present study was to investigate the impact of biofield treatment on spectral properties of disulfiram and nicotinic acid. The study was performed in two groups i.e., control and treatment of each drug. The treatment groups were received Mr. Trivedi’s biofield treatment. Subsequently, spectral properties of control and treated groups of both drugs were studied using Fourier transform infrared (FT-IR) and Ultraviolet-Visible (UV-Vis) spectroscopic techniques. FT-IR spectrum of biofield treated disulfiram showed the shifting in wavenumber of C-H stretching from 1496 to 1506 cm-1 and C-N stretching from 1062 to 1056 cm-1. The intensity of S-S dihedral bending peaks (665 and 553 cm-1) was also increased in biofield treated disulfiram sample, as compared to control. FT-IR spectra of biofield treated nicotinic acid showed the shifting in wavenumber of C-H stretching from 3071 to 3081 cm-1 and 2808 to 2818 cm-1. Likewise, C=C stretching peak was shifted to higher frequency region from 1696 cm-1 to 1703 cm-1 and C-O (COO-) stretching peak was shifted to lower frequency region from 1186 to 1180 cm-1 in treated nicotinic acid. UV spectrum of control and biofield treated disulfiram showed similar pattern of UV spectra. Whereas, the UV spectrum of biofield treated nicotinic acid exhibited the shifting of absorption maxima (λmax) with respect of control i.e., from 268.4 to 262.0 nm, 262.5 to 256.4, 257.5 to 245.6, and 212.0 to 222.4 nm. Over all, the FT-IR and UV spectroscopy results suggest an impact of biofield treatment on the force constant, bond strength, and dipole moments of treated drugs such as disulfiram and nicotinic acid that could led to change in their chemical stability as compared to control.

21 citations


Journal ArticleDOI
TL;DR: The modified standard additions calibration method is suggested, which compensates for matrix effects which may be a serious source of inaccuracy and is a tool that can be used during method development in order to find the most suitable silylation conditions for a given analyte.
Abstract: Substitution of polar functionalized compounds with silylated (e.g., trimethylsilylated) surrogates prior to GC/ MS analysis is a widely used analytical strategy. Calibration is a most demanding step of this strategy. In fact, a calibration function is usually acquired by converting known amounts of the pure analyte to its silylated surrogate using the same conditions employed for processing unknown samples. The cumbersome need of acquiring a new calibration function prevents, to a large extend, the possibility of modifying silylation and instrumental settings on a sample by sample basis as would be appropriate in a number of cases. The modified standard additions calibration method, suggested in this paper, overcomes this difficulty by integrating in a single analytical procedure calibration and sample analysis. Furthermore, the suggested procedure compensates for matrix effects which may be a serious source of inaccuracy and is a tool that can be used during method development in order to find the most suitable silylation conditions for a given analyte. The implementation and benefits of the modified standard additions calibration method are explored in this paper on the basis of a symbolic but enlightening experiment dealing with the very representative GC/MS quantification of biological amino acids via their trimethylsilylated derivatives.

17 citations


Journal ArticleDOI
Pranjal Chandra1
TL;DR: The development of electrochemical nanobiosensors composed of nanomaterials and biological receptors (antibody, aptamer, peptide etc) coupled with nanommaterials and electrochemical measurement are likely the most encouraging approach to solve the problems related to sensitivity, rapidity, selectivity, and low cost.
Abstract: Cancer is one of the most leading cause of death worldwide and can take over 200 diverse forms, including lung, prostate, breast, cervical, ovarian, hematologic, colon cancer, and leukemia. It has been found that environmental factors (eg, alcohol, radiation, smoke, and carcinogenic chemical compounds) as well as genetic factors (eg, autoimmune dysfunction and hereditary mutations) are linked with an increased threat in the development and progression of cancer [1]. In addition microorganisms are also reported to be associated with some types of cancer (eg, stomach cancer and cervical cancer etc) [2]. In view of such an important medical condition, several methods have already been discovered to diagnose cancer and many more methods are in the process of development [3]. Conventional clinical approaches to detect cancers are based on biopsy followed by histopathology [4], biomarkers using protein levels or nucleic acid content and its expression in the cancer suspects [5]. Biopsy is the most widely used technique, however, it is an invasive technique and cannot always be used. Furthermore, it cannot be applied when cancer biomarkers are present in an extremely low concentrations in the body fluids and in malignant tissues. Thus, the development of highly sensitive and new techniques of cancer diagnosis is extremely interesting and significant in medical science. Due to high interest in interdisciplinary research in the last decade several nanobiosensors based on spectrophotometric or optical methods, fluorescence immunoassay, chemiluminescence analysis, electrochemistry, radioimmunoassay, capillary electrophoresis and chromatographic analysis have been developed to detect cancer biomarkers (proteomic and genetic markers) and cells [6,7]. The major issues in cancer diagnosis are sensitivity and to develop a miniaturized platforms that can be used as point-of-care medical device and can be applied in the remote areas. The development of electrochemical nanobiosensors composed of nanomaterials and biological receptors (antibody, aptamer, peptide etc) are likely the most encouraging approach to solve the problems related to sensitivity, rapidity, selectivity, and low cost [8-12]. This approach is expected to be very effective for cancer diagnosis due to the combination of conventional bioassay (antibody, aptamer, peptide etc) coupled with nanomaterials and electrochemical measurement. Another advantage of the electrochemical biosensor include its ability to be miniaturized as an onsite medical device, low cost, small, and handy size [13-17]. These features of electrochemical nanobiosensors may serve as a smart alternative to support fast cancer diagnosis, thereby designing better therapeutic strategies which will be extremely helpful in decreasing patient stress.

17 citations


Journal ArticleDOI
TL;DR: It is shown that the LAL assay can be used to determine endotoxemia in fasting and postprandial blood samples from different mammal species including pig, and an essential parameter to overcome this difficulty is the dilution factor that depends on the studied species.
Abstract: Introduction: Lipopolysaccharides (LPS) or so-called endotoxins are potent pro-inflammatory compounds. LPS can be present in the bloodstream in case of septic conditions, leading to measure endotoxemia that is the activity of LPS in plasma. Recent research also reveals a low-grade or so-called metabolic endotoxemia associated with metabolic diseases (e.g. obesity, type 2 diabetes, cardiovascular diseases). In this context, research studies use different experimental models, mostly in humans and rodents. Pig is now emerging as a new animal model in nutritional and metabolic studies. However, information is lacking to date about optimal dilution to be used for sample preparation for the Limulus Amebocyte Lysate test, according to species, especially for pig. Methods: strong>Endotoxemia was measured using the LAL standard reference method. We describe the method of sample preparation and the LAL technique to measure endotoxemia in 4 mammal species: human, mouse, rat and pig. Results: Plasma dilution is necessary to overcome interferences leading to erroneously low or high results. Optimal dilution to avoid interferences in most samples, while maintaining a satisfactory sensitivity, was found to be at least 1/10 for human vs 1/40 for mice, while much higher dilution was mandatory for pigs, namely 1/200. Altogether, mean plasma endotoxemia in all tested samples using the optimal dilution for each species was 0.73 ± 0.05 EU/mL in humans (n=903), 0.9 ± 0.2 EU/mL in rodents (n=295) and 8.5 ±1.3 EU/mL in pigs (n=186), regardless of fasting or postprandial state and/or type of dietary intervention. Conclusion: We show that the LAL assay can be used to determine endotoxemia in fasting and postprandial blood samples from different mammal species including pig. Because its detection is made difficult by interference from other plasma constituents, an essential parameter to overcome this difficulty is the dilution factor that depends on the studied species.

14 citations


Journal ArticleDOI
TL;DR: A highly sensitive method for the quantitation of polysorbate 20 (PS20) and 80 (PS80) in therapeutic peptide formulations demonstrated that even trace levels of PS20 and PS80 could stabilize the peptide against fibrillation and aggregation.
Abstract: A highly sensitive method has been developed for the quantitation of polysorbate 20 (PS20) and 80 (PS80) in therapeutic peptide formulations. A mixed-mode HPLC column was used to separate polysorbates from the peptide and other excipients, and a charged aerosol detector (CAD) was used for the detection. The method was capable of reporting polysorbates as low as 5 ppm, and the sensitivity could be further improved on a needed basis. The method has been used to study the compatibility between polysorbates and m-cresol in the peptide formulation. It was found that both PS20 and PS80 are compatible with m-cresol (at 2.8 mg/ml) when their levels were not greater than 20 ppm. Significant losses of polysorbates were observed when PS20 and PS80 concentrations were above 50 ppm. Furthermore, the agitation study demonstrated that even trace levels of PS20 and PS80 (e.g., 20 ppm) could stabilize the peptide against fibrillation and aggregation.

14 citations


Journal ArticleDOI
TL;DR: Steiner et al. as discussed by the authors presented a forensic toxicology drug testing laboratory at the Tripler Army Medical Center in Honolulu, HI 96859, USA with the goal of developing a drug testing system for the forensic toxicological drug testing lab.
Abstract: Wes E Steiner1* and William A English2 1Department of Chemistry and Biochemistry, Eastern Washington University, 226 science Building, Cheney, WA 99004, USA 2Forensic Toxicology Drug Testing Laboratory, Tripler Army Medical Center, 1 Jarrett White Road, Honolulu, HI 96859, USA *Corresponding Author: Wes E Steiner, Department of Chemistry and Biochemistry, Eastern Washington University, 226 science, Building, Cheney, USA, Tel: (509) 359-6521, Fax: (509) 359-6973; E-mail: wsteiner@ewu.edu

Journal ArticleDOI
Ramos Tm, Costa Ff, Pinto Isb, Pinto Sm, Abreu Lr 
TL;DR: In this article, the influence of SCC on physicochemical properties and protein fractions of milk was evaluated using microfluidic electrophoresis for separation and quantification of milk proteins.
Abstract: The objective of this study was to evaluate the influence of somatic cell count (SCC) on the physicochemical properties and protein fractions of milk. Milk was collected and analyzed for somatic cell count, fat, lactose, acidity, total solids, ash, total nitrogen, soluble nitrogen at pH 4.6, and soluble nitrogen in trichloroacetic acid (TCA) 12%. Milk was divided into four groups according to the value of SCC, each constituting a treatment, as follows: Treatment 1 ( 1 million cells/ml). The electrophoretic profile of milk was also evaluated using microfluidic electrophoresis for separation and quantification of milk proteins. An increase in the concentration of SCC resulted in a significant increase in the amount of fat, soluble nitrogen and soluble protein (casein) fractions, and a reduction of α-casein, β-casein, and κ-casein. There was a higher proteolytic activity associated with high SCC. Changes in protein fractions of milk caused by high SCC had strong implications regarding the potential of milk as raw material for manufacturing products as the industrial yield of milk is mainly associated with the casein fraction.

Journal ArticleDOI
TL;DR: A rapid, precise and selective RP-LC method was developed for simultaneous determination of the widely used oral antidiabetic; metformin hydrochloride (MTF), with some commonly prescribed oral antidisabetics, namely; sitagliptin phosphate (SIT), pioglitazone hydrochlorides (PGZ), gliclazide (GLZ), glibenclamide (GLB) and repaglinide (RPG) as mentioned in this paper.
Abstract: A rapid, precise and selective RP-LC method was developed for simultaneous determination of the widely used oral antidiabetic; metformin hydrochloride (MTF), with some commonly prescribed oral antidiabetics, namely; sitagliptin phosphate (SIT), pioglitazone hydrochloride (PGZ), gliclazide (GLZ), glibenclamide (GLB) and repaglinide (RPG). The chromatographic separation carried out using gradient elution mode with acetonitrile: 0.05M potassium dihydrogen phosphate (MKP) and 0.01M sodium octane sulphonate (SOS) (pH 3.55) at flow rate 0.85 ml/min on Kromasil 100-C18, (30 × 0.4 cm, 10 μm) at 40°C. UV detection was carried out at 220 nm. The method was validated according to ICH guidelines. Linearity, accuracy and precision were satisfactory over the concentration ranges (μg/ ml) of 0.05-205 for MTF, 0.05-100 for PGZ, GLB and SIT, 0.1-100 for RPG and 1-100 μg/ml for GLZ. The correlation coefficients were >0.99 for all analytes. Limits of quantification (LOQs) found were 0.002, 0.003, 0.009, 0.012, 0.007 and 0.024 μg/ml for MTF, SIT, PGZ, GLZ, GLB and RPG respectively. The developed method is specific and accurate for quality control and routine analysis of the cited drugs in their pharmaceutical preparations. It is recommended for application in the quality control of the herbal antidiabetic products to detect possible counterfeits.

Journal ArticleDOI
TL;DR: In this paper, a poly(brilliant blue) modified carbon paste electrode was fabricated for the detection of dopamine in the presence of large excess of ascorbic acid and uric acid in phosphate buffer solution of pH 7.4.
Abstract: Poly (brilliant blue) modified carbon paste electrode was fabricated for the detection of dopamine in the presence of large excess of ascorbic acid and uric acid in phosphate buffer solution of pH 7.4. The redox peaks obtained at poly (brilliant blue) modified carbon paste electrode shows good electrocatalytic activity towards the oxidation of dopamine. From the study of scan rate variation the electrode process was found to be adsorption controlled. The limit of detection of dopamine was found to be 6.7 × 10-7 M. and the simultaneous study shows the good result with peak to peak separation between dopamine and other two analytes ascorbic acid and uric acid by both cyclic voltammetry and differential pulse voltammetric techniques

Journal ArticleDOI
TL;DR: In this paper, a modified carbon paste electrode (MCPE) was used to determine the electrochemical behavior of folic acid (FA) in 0.2M PBS at pH 7.4 with the scan rate of 50 mVs-1.
Abstract: Sodium alpha olefin sulphonate (SAOS) was used for the modification of carbon paste electrode (CPE) to determine the electrochemical behavior of folic acid (FA) in 0.2M phosphate buffer solution (PBS) at pH 7.4 with the scan rate of 50 mVs-1. The effects of scan rate, concentration and simultaneous determination of FA at modified carbon paste electrode (MCPE) were studied. The effect of interference of dopamine was carried out and real sample analysis of FA was studied at MCPE. From the scan rate and concentration shows that, the overall electrode process was found to be diffusion-controlled at SAOSMCPE and detection limit was found to be 28.8 μM. The modified electrode (SAOSMCPE) exhibits good electrocatalytic activity towards the determination of folic acid when compared to BCPE. The same method can also be applied for other drug analysis.

Journal ArticleDOI
TL;DR: The study showed that the choice of columns and detectors influenced the identification of the compounds, which could serve as baseline data for this species in searching for lipid biomarkers for aquatic pollution monitoring studies.
Abstract: GC-MS is an important instrument in lipid profiling and lipid biomarker identification. The choice of columns and detectors is important in analysis and identification of the biomolecules. In the present study, GC-MS fingerprinting of fatty acids (FAs) in different tissues of freshwater mollusc Lamellidens marginalis was carried out in GC-MS with two different columns, TR-FAME and TR-WaxMS, to examine if choice of columns and detectors influence the identifications of the lipids. These sentinel organisms are used in aquatic pollution monitoring studies as they accumulate toxic environmental contaminants to levels well above those present in the surrounding environment thus providing information on the spatio-temporal pollution trends. FAs identified in different tissues include the saturated FAs palmitic acid (C16:0), margaric acid (C17:0), stearic acid (C18:0), monounsaturated FAs (MUFAs) oleic acid (C18:1), eicosenoic acid (C20:1) and polyunsaturated fatty acid (PUFAs) linoleic acid (C18:2). More numbers of FAs were detected in TR-FAME column than TR-WaxMS column. Several non-fatty acid compounds like butylated hydroxyl toluene (BHT) and dibutyl phthalate were also identified in the FAME (fatty acid methyl ester) preparation using NIST (MS) library. It was interesting to note that the FA peaks (6/14) were outnumbered by the non-fatty acid peaks (8/14). Such compounds could be taken as unidentified FAs unless powerful detectors like MS are available and can lead to erroneous inferences. The study showed that the choice of columns and detectors influenced the identification of the compounds. The information generated on fatty acid profiles of this bivalve could serve as baseline data for this species in searching for lipid biomarkers for aquatic pollution monitoring studies.

Journal ArticleDOI
TL;DR: In this article, the authors developed and validated a simple and relatively economical analytical method for detecting complex sugar mixtures in fermentation broth based on High-Performance Thin-Layer Chromatography (HPTLC).
Abstract: Carbohydrates are the principal sources of nutrient and energy in large-scale submerged fermentation processes. A method for detection and quantification of sugar levels are very advantageous because they can be considered as key indicators in determining the yields and the productivity of the process. For this reason, the objective of this study is to develop and validate a simple and relatively economical analytical method for detecting complex sugar mixtures in fermentation broth based on High-Performance Thin-Layer Chromatography (HPTLC). HPTLC is a widely used, fast and accurate method of separating complex mixtures. The proposed method involved the chromatographic separations of xilo-, galacto-, fructo-oligosaccharides mixtures at different molecular weights, tri- and disaccharides (raffinose, sucrose, lactose), and the corresponding monosaccharides (xylose, fructose, galactose) on HPTLC plates, using different eluent mixtures and elution conditions. The documentation of plates was performed using TLC visualization device and the images of plates were processed using a digital processor. HPTLC methods development using instrumental techniques as OPLC (Over Pressure Liquid Chromatography) and AMD (Automated Multiple Development) has been also described, as to simultaneously monitor several samples in the same elution, with significant time and solvent savings. Four different carbohydrates complex mixtures were analyzed using HPTLC techniques, as to optimize the quality of the separation among components. The methods set up were then applied for quantitative determination of sugars. As a model of submerged fermentations, a strain of Bifidobacterium spp. was used, a saccharolytic bacterium with probiotic activities in the human gut, able to anaerobically ferment complex sugar mixtures. Results could be easily extended to other fermentation processes.

Journal ArticleDOI
TL;DR: Sodium dodecyl sulphate/poly(brilliant blue)/multi walled carbon nanotube modified carbon paste electrode was fabricated for the electroanalysis of dopamine in the presence of ascorbic acid and uric acid in phosphate buffer solution of pH 7.4 as mentioned in this paper.
Abstract: Sodium dodecyl sulphate/poly(brilliant blue)/multi walled carbon nanotube modified carbon paste electrode was fabricated for the electroanalysis of dopamine in the presence of ascorbic acid and uric acid in phosphate buffer solution of pH 7.4. The key parameters such as sensitivity, selectivity, antifouling property and stability were achievedby the modified electrode. The redox peaks obtained at modified electrode shows good electrocatalytic activity towards the oxidation of dopamine. From the effect of scan rateand concentration the electrode phenomenon was confirmed to be adsorption-controlled process. The lower limit of detection of dopamine was 2.69 × 10-7M, and the simultaneous analysis shows a good result with peak to peak separation between dopamine and other two analytes ascorbic acid and uric acid by both cyclic voltammetry and differential pulse voltammetric techniques.

Journal ArticleDOI
TL;DR: Transgenic cassava lines with cyanide content three folds less than the cyanidecontent in the wild type relatives were produced and RNAimediated downregulation of expression of cytochrome P450 genes responsible for biosynthesis of cyano-glycosides was confirmed by the authors.
Abstract: Being the fourth most important crop in the developing countries surpassed only by maize, rice and sugarcane as a source of calories, cassava (Manihot esculenta Crantz) is doubtlessly a famine reserve crop due to its drought tolerance, ability to grow on infertile soils and its ability to recover from disease and pest attacks. However, this important tuber crop has a fair share of demerits, among which is the fact that all parts of the plant contain toxic levels of cyanogenic glycosides, which have to be removed by laborious processing before cassava can be safely consumed. Conventional methods for removal of cyanogenic glycosides in cassava have seldom been successful for decades. Genetic engineering holds the key to overcoming majority of these limitations in order to produce cassava plants in which desirable traits are optimized and undesirable traits downregulated. The objective of this study was to determine the levels of cyanogenic compounds in three Kenyan cassava genotypes along with an exotic model cultivar in which cyanogenic glycosides had been downregulated via RNA interference approach in an earlier study by the authors. Cassava roots from transgenic and wild type genotypes were harvested, peeled, cut to pieces and washed three times with cold water, following cyanogenic compounds were extraction by homogenization in acid extraction medium. The supernatant obtained after centrifugation of the homogenate was analyzed for cyanogenic compounds content by spectrophotometric procedures. From this study, transgenic cassava lines with cyanide content three folds less than the cyanide content in the wild type relatives were produced. This confirmed RNAimediated downregulation of expression of cytochrome P450 genes responsible for biosynthesis of cyano-glycosides previously undertaken by the authors in an earlier study.

Journal ArticleDOI
TL;DR: In this article, an HPLC-UV method was set-up to allow the simultaneous quantification of the reduced-GSH, oxidised-GSSG and nitroso-GSNO glutathione species.
Abstract: In the present work, an HPLC-UV method was set-up to allow the simultaneous quantification of the reduced- GSH, oxidised-GSSG and nitroso-GSNO glutathione species. Chromatographic separation was achieved on YMC ODS-A C18 column (150 × 4.6 mm, 5 μm), coupled to a Guard-c precolumn (YMC-Pack, 10 × 1-4,0 mm). The eluted compounds were detected at 215 nm by UV-detector, by keeping the column oven at room temperature while the auto-sampler temperature was maintained at 4°C. A fractional factorial design has been applied for the optimization of the mobile phase resulting in baseline separated peaks within 6 minutes. In-house validation was evaluated by linearity, limits of detection (LODs), limits of quantification (LOQs), reproducibility, repeatability and recovery. The detection and quantification limits obtained for standard solutions were below 0.2 μM and 0.6 μM, respectively (RSD values below 2%). The developed method was applied to the measurement of GSH, GSSG and GSNO in human pulmonary cells (A549) exposed to limonene, limonene oxide solubilized into the culture medium and to NO2 as gas phase. Results show an increase in GSH levels, without significant changes in GSSG, when cells were exposed to limonene oxide, while cells exposed to NO2 resulted in a significant increase of GSNO amount. Detection limits were of 1 μM for the glutathione species measured in A549 cells, with RSD values below 2.5%. In conclusion, the present HPLC-UV method can be readily used to measure in a rapid, simultaneous and accurate way the status of GSH, GSSG and GSNO in human cells, their simultaneous quantification helping to better predict the potential impact of chemicals on human health.

Journal ArticleDOI
TL;DR: In this article, surface-enhanced Raman scattering (SERS) has long since found wide application in the field of heterogeneous catalysis for both chemical and photochemical reactions, which allows the in-situ detection of reactants or products or by-products in reactions that take place at the surface of nanostructured metals like silver, gold and copper.
Abstract: Surface-enhanced Raman scattering (SERS) has long since found wide application in the field of heterogeneous catalysis for both chemical and photochemical reactions. Actually, this technique allows the in-situ detection of reactants or products or by-products in reactions that take place at the surface of nanostructured metals like silver, gold and copper. Silver nanoparticles, when activated by adsorption of chloride anions, are able to catalyze reactions for adsorbed molecules, in addition to promote higher SERS enhancements. Bimetallic nanoparticles, made of silver as SERS-active metal and another metal with strong performance in heterogeneous catalysis like palladium, or metal oxide such as titania, extremely active in photoreactions, allow monitoring the time evolution of different reactions by observing the changes observed in the SERS bands of the adsorbed species.

Journal ArticleDOI
TL;DR: In this paper, a carbon paste electrode was modified by electropolymerisation of 1 mM Glycine in 0.2 M Acetate buffer solution (ABS) at pH-5.
Abstract: The carbon paste electrode was modified by electropolymerisation of 1 mM Glycine in 0.2 M Acetate buffer solution (ABS) at pH-5. The voltammetric response of Paracetamol (PA) at Poly(Glycine) Modified carbon paste electrode (MCPE) shows excellent electrocatalytic activity when compared to bare carbon paste electrode (BCPE) at sweep rate of 100 mV/s-1. From the study of scan rate variation the electrode process was found to be diffusion controlled. The concentration effect of paracetamol was studied. The simulations determination of PA, DA and AA in their sample mixture was analyzed by using both cyclic voltammetric and differential pulse voltammetric techniques.

Journal ArticleDOI
TL;DR: The work presents off-line combination of capillary electrophoresis of microbial clumping with spectrometric detection and describes and optimization of the environmental conditions having an impact on reproducibility and quality of one-dimensional intact-cell MALDI TOF spectra.
Abstract: Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed as a technique of choice for quick classification of bacterial cells and precise characterization of microorganisms. The evaluated MALDI MS procedure was confronted with automated biochemical method using the VITEK 2 Compact system employed as a reference method. Moreover, the investigation aimed at describing and optimization of the environmental conditions having an impact on reproducibility and quality of one-dimensional intact-cell MALDI TOF spectra. The work presents off-line combination of capillary electrophoresis of microbial clumping with spectrometric detection.

Journal ArticleDOI
TL;DR: In this paper, the effects of pH, contact time, initial concentration of MB and temperature on adsorption, as well as the thermodynamics and kinetics of MB were investigated.
Abstract: The activated carbon samples (SC and PC) were prepared from papyrus plant by chemical activation with sulfuric acid and phosphoric acid, respectively. The SC and PC were then modified with nitric acid to give the oxidized sulfuric carbon (OSC) and oxidized phosphoric carbon (OPC) and utilized for removing Methylene Blue (MB) from aqueous solution with initial dye concentration (5-650) mg/L and a temperature of 25-45°C. The effects of pH, contact time, initial concentration of MB and temperature on adsorption, as well as the thermodynamics and kinetics were investigated. The experimental results showed that: (1) The adsorption capacity of MB was dependent upon pH and maximum adsorption occurs at pH 8. (2) The adsorption process was in good agreement with the Langmuir adsorption isotherm model. (3) The adsorption kinetics followed a pseudo-second order kinetic model and intra particle diffusion was involved in the adsorption process. (4) Thermodynamic results indicated that the adsorption process was spontaneous and endothermic in nature. The proposed adsorbent was successfully applied for the removal of Methylene Blue (MB) from aqueous solution with a percent recovery of the sample SC 98.9%, PC 98.3%, OSC 97.5%, OPC 95.05% when using 0.1-1M HCl.

Journal ArticleDOI
TL;DR: This work has developed a methodology for bacterial cell sorting using the semi-automated autoMACS® Pro Separator for the first time, and produced a complete method for sorting of bacteria displaying 15-mer peptides on their cell surface using this device, including downstream bioinformatic analysis of candidates for binding to a target of interest.
Abstract: Biopanning by bacterial display has many advantages over yeast and phage display, including the speed to discovery of affinity reagents and direct amplification of bound cells without the need to elute and reinfect. However, widespread use is limited, in part due to poor performance achieved using manual Magnetic-Activated Cell Sorting (MACS) methods, and an absence of widely-available, low cost, high-performance sorting alternatives. Here, we have developed a methodology for bacterial cell sorting using the semi-automated autoMACS® Pro Separator for the first time, and have produced a complete method for sorting of bacteria displaying 15-mer peptides on their cell surface using this device, including downstream bioinformatic analysis of candidates for binding to a target of interest. Two autoMACS® programs designed for isolation of target cells with low frequency were evaluated and adapted to bacterial biopanning, using protective antigen (PA) of Bacillus anthracis as the model system. In contrast to manual MACS, the bacterial display library was preferentially enriched by autoMACS® sorting, yielding several promising candidates after only three rounds of biopanning and bioinformatic analysis. Individual candidates were evaluated for relative binding to fluorescently-labeled PA target or streptavidin negative control using Fluorescence-Activated Cell Sorting (FACS). The top thirteen peptide candidates from the autoMACS® sort demonstrate binding to PA with low cross-reactivity to streptavidin, while only two of eighteen candidates from the manual sort showed binding to PA, and both demonstrated greater cross-reactivity to streptavidin. Overall, the autoMACS® platform quickly harvested higher affinity peptide candidates with demonstrated specificity to the PA target. Peptide candidates produced with this method contained the previously reported PA consensus WXCFTC, further validating this method and the commercially available autoMACS® platform as the first low cost, semi-automated biopanning approach for bacterial display that is widely accessible and more reliable than the MACS/FACS standard protocol.

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TL;DR: In this article, a method for the determination of finasteride using sodium 1,2-naphthoquine-4- sulfonate (NQS) has been developed.
Abstract: A rapid, simple and sensitive method for the determination of finasteride using sodium 1,2-naphthoquine-4- sulfonate (NQS) has been developed. The method is based on the fact that a brown product can be formed by the reaction between finasteride and sodium NQS in a buffer solution of a pH 13.0. Beer’s law is obeyed in the range 2-14 μg/ml of finasteride at maximum wavelength of 447 nm. The linear regression equation of the calibration curve is y=0.062857x+0.069857, with a linear regression correlation coefficient of 0.999. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.03 and 0.09 μg/ml, respectively. The method has been successfully applied to the determination of finasteride in pharmaceutical formulation. The results were in good agreement with those obtained with the official high performance liquid chromatography (HPLC) method. The proposed method is useful for routine analysis of finasteride in quality control laboratories.

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TL;DR: In this paper, the authors proposed to functionalize activated carbon with oxygen and nitrogen containing groups to improve removal of Cd(II) ions from aqueous solutions, and the batch technique sorption studies were conducted and the factors controlling the adsorption process were tested.
Abstract: This study plans to functionalize activated carbon with oxygen and nitrogen containing groups to improve removal of Cd(II) ions from aqueous solutions. Activated carbon (AC) was treated with nitric acid giving MC-HNO3 that was pursued by modifying with EDTA to form MC-EDTA. AC, MC-HNO3 and MC-EDTA were analyzed using FTIR, SEM, TEM, Boehm titration, point of zero charge and N2 adsorption-desorption analysis. The batch technique sorption studies of Cd(II) onto sorbents were conducted and the factors controlling the adsorption process of Cd(II) were tested. Langmuir and Freundlich models were used to analyze the isotherm data. The equilibrium data fitted well Langmuir isotherm for all adsorbents. Pseudo-first order, pseudo-second order, intraparticle diffusion and the Boyd equations were used to analyze the kinetic data. The rate constants, equilibrium capacities and related correlation coefficients (R2) for each kinetic model were evaluated and discussed. The second order model is the best fit model for the three sorbents. Though intraparticle diffusion has essential role in rate-controlling step in the adsorption process of Cd(II) onto the investigated sorbents, film diffusion is also governing this process. The thermodynamic parameters ΔG°, ΔH° and ΔS° for the sorption processes of Cd(II) onto the adsorbents were estimated, and spontaneity of adsorption was deduced from the negative sign of ΔG°. Desorption study highlighted the cost saving due to the easiness of regeneration for both MC-HNO3 and MC-EDTA.

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TL;DR: In this paper, an improvement of analytical methods for intramolecular carbon isotope compositions (δ13C) of pyruvate was presented, which can be used to categorize pyruve into 13C distribution patterns, which might indicate different production processes or raw materials.
Abstract: This paper presents the improvement of analytical methods for intramolecular carbon isotope compositions (δ13C) of pyruvate. Decarboxylated by H2O2, pyruvate yields acetic acid and CO2. Headspace solid phase micro-extractiongas chromatography-pyrolysis-gas chromatography-combustion-isotope ratio mass spectrometry (HS-SPME-GCPy- GC-C-IRMS) was used to measure the intramolecular δ13C values of acetic acid. δ13C value of CO2 can be later calculated using mass balance equation. The method’s consistency was confirmed by comparison of the δ13C value of CO2 from calculation to its direct measurement. Results of this study confirmed the method improvement because pyruvate 13C intramolecular distribution patterns were obtained. Two intramolecular 13C distribution patterns for commercial chemical reagents were found using this developed method. Intramolecular 13C distribution patterns for pyruvate were found for application in dietary supplements. Its origin was inferred. The method presented herein is expected to be a useful tool for categorization of pyruvate into different intramolecular 13C distribution patterns, which might indicate different production processes or raw materials.

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TL;DR: In this paper, total reflection X-ray fluorescence (TXRF) was used for fast and reliable quantitative analysis of heavy metals in plants used for accumulation studies, and the results showed that direct analysis of suspended powdered samples may be used as a fast and simple method for screening.
Abstract: This work is to demonstrate the usefulness of total reflection X-ray fluorescence (TXRF) for fast and reliable quantitative analysis of heavy metals in plants used for accumulation studies. A model study of beans germination in lead contaminated environment under controlled laboratory conditions was realized. Metal accumulation in different parts of the plant was evaluated. Two different sample preparation procedures for TXRF analysis were considered: microwave acid digestion and direct analysis of suspended powdered sample. Quantitative determination of macro, micro, and trace elements was performed. Root showed the highest accumulation of lead, followed by stem, leaves and crops. Results showed that direct analysis of suspended powdered samples may be used as a fast and simple method for screening.

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TL;DR: The new method is presented, applicable for use with specific or bulk peptidyl-tRNA, any Pth enzyme, and a range of reaction conditions including solvent additives, to characterize Pth activity, determine enzyme kinetic parameters, screen for inhibitors, and determine inhibitory parameters.
Abstract: The emerging importance of Peptidyl-tRNA hydrolase (Pth) enzymes necessitates the need for a widely applicable functional assay to further studies of this important enzyme family. Previously reported methods for monitoring Pth function suffer from limitations of cost, time, substrate availability, and application compatibility. Herein we present a new method for the rapid and precise characterization of Pth activity. The method is applicable for use with specific or bulk peptidyl-tRNA, any Pth enzyme, and a range of reaction conditions including solvent additives. The method also allows for semi-automated quantitative assessment of peptidyl-tRNA cleavage. No specialized equipment, harmful reagents, or time-consuming techniques are required. We use the new method to characterize Pth activity, determine enzyme kinetic parameters, screen for inhibitors, and determine inhibitory parameters.