Sensitive Spectrophotometric Assay Research Articles

Synergistic redox of dual-shaped silver nanostructures for highly selective sensing of hydrogen peroxide

A novel growth-based plasmonic sensor for hydrogen peroxide (H2O2) determination was developed from dual-shaped silver nanostructures consisting of starch-stabilized yellow silver nanospheres (AgNSs) and citrate-capped red silver nanoprisms (AgNPrs). AgNPrs were enlarged through a unique mechanism of AgNSs dissolution to silver ions which are subsequently reduced to silver metal by H2O2. Citrate-capping maintains the lateral anisotropic growth by protecting the AgNPrs from oxidation while exposing the sensitive edges to Ostwald ripening. This highly unique process addresses the selectivity issues encountered with decomposition-based AgNSs/NPrs sensors. Absorbance values from the AgNSs decomposition and AgNPrs growth facilitated a more sensitive spectrophotometric assay through ratiometric analysis. Increasing the concentration of the AgNSs/NPrs sensor to 80:40 ppm also improved sensitivity of naked eye detection of H2O2 by generating a wide range of color transitions. Furthermore, selectivity, accuracy and precision evaluation suggest that the dual-shaped silver nanostructure sensor is a potential alternative to H2O2 determination by allowing a simple, rapid, highly selective, and sensitive measurement.

A Robust and Sensitive Spectrophotometric Assay for the Enzymatic Activity of Bacterial Adenylate Cyclase Toxins.

Various bacterial pathogens are producing toxins that target the cyclic Nucleotide Monophosphate (cNMPs) signaling pathways in order to facilitate host colonization. Among them, several are exhibiting potent nucleotidyl cyclase activities that are activated by eukaryotic factors, such as the adenylate cyclase (AC) toxin, CyaA, from Bordetella pertussis or the edema factor, EF, from Bacillus anthracis. The characterization of these toxins frequently requires accurate measurements of their enzymatic activity in vitro, in particular for deciphering their structure-to-function relationships by protein engineering and site-directed mutagenesis. Here we describe a simple and robust in vitro assay for AC activity based on the spectrophotometric detection of cyclic AMP (cAMP) after chromatographic separation on aluminum oxide. This assay can accurately detect down to fmol amounts of B. pertussis CyaA and can even be used in complex media, such as cell extracts. The relative advantages and disadvantages of this assay in comparison with other currently available methods are briefly discussed.

Therapeutic diminution of Interleukin-10 with intranasal theophylline administration in hyposmic patients

ObjectiveTo determine changes in nasal mucus Interleukin-10 (IL-10) before and after intranasal theophylline treatment in hyposmic patients, and the relationship of these changes to orally administered theophylline treatment. DesignIL-10 was measured in nasal mucus samples of 17 normal subjects and 39 patients with hyposmia of multiple etiologies by use of a sensitive spectrophotometric ELISA assay. Hyposmia is defined clinically by standardized evaluation of impaired olfactometry, as well as subjectively self reported by the patient. ResultsPrior to treatment, IL-10 levels in nasal mucus were increased in hyposmic patients compared to controls. Following intranasal theophylline administration, over half of treated patients experienced a decrease of nasal mucus IL-10 toward control levels, correlated with a significant improvement in taste and smell function. Patients who increased with intranasal administration, similar to previously reported oral administration, showed no significant improvement in taste or smell function. ConclusionsNasal mucus IL-10 decreased in patients treated with intranasal theophylline. Mechanisms of action include phosphodiesterase and nuclear factor kappa-B inhibition, correlated with an improvement in both olfaction and gustation. IL-10 is induced by therapeutic and systemic administration of theophylline as with oral dosage, but reduced by lower and localized treatment.

A sensitive pH indicator-based spectrophotometric assay for PHB depolymerase activity on microtiter plates.

A continuous spectrophotometric assay for the screening of PHB depolymerase activity in microtiter plates was developed. We evaluated crystalline PHB in the suspension and coated it with the addition of a pH indicator to detect the breakage of the ester bond by proton titration. The reaction rate and the concentration of the recombinant PhaZ1 from Paucimonas lemoignei PHB depolymerase presented a linear correlation. A comparison of the proposed method with the turbidimetric method adapted to the microtiter plates revealed that the use of indicators increases the response signal by at least 5-fold, resulting in increased sensitivity and better signal-to-noise ratio. Furthermore, the proposed method offers a wide range of pH from 5.0 to 9.2 by using different buffer-indicator pairs and was employed for the screening of PHB-depolymerase activity on 140 bacterial strains isolated from Lake Chapala. Eleven strains were positive for PHB-depolymerase activity, which were ACSLRF-27, ACPLRF-6, and ACPLRF-5 (16S rRNA sequence alignment revealed 99-100% similarity with Actinomadura geliboluensis strain A8036, Streptomyces cavourensis strain NRRL 2740, and Streptomyces coelicolor strain DSM 40233, respectively); these that showed the highest activities. In conclusion, the method was successfully applied for finding new strains and for quantifying the PHB depolymerases activity with crystalline PHB.

An Efficient Viologen-Based Electron Donor to Nitrogenase.

Nitrogenase catalyzes the reduction of N2 to NH3, supporting all biological nitrogen fixation. Electron donors to this enzyme are ferredoxin or flavodoxin (in vivo) and sodium dithionite (in vitro). Features of these electron donors put a limit on spectrophotometric studies and electrocatalytic applications of nitrogenase. Although it is common to use methyl viologen as an electron donor for many low-potential oxidoreductases, decreased nitrogenase activity is observed with an increasing concentration of methyl viologen, limiting its utility under many circumstances. In this work, we suggest that this concentration-dependent decrease in activity can be explained by the formation of a dimer of the radical cation of methyl viologen (Me2V•+)2 at higher methyl viologen concentrations. In addition, viologens functionalized with positively and negatively charged groups were synthesized and studied using spectroscopy and cyclic voltammetry. A sulfonated viologen derivative, 1,1'-bis(3-sulfonatopropyl)-4,4'-bipyridinium radical {[(SPr)2V•]-}, was found to support full nitrogenase activity up to a mediator concentration of 3 mM, while the positively charged viologen derivative was not an efficient reductant of nitrogenase due to the high standard redox potential. The utility of [(SPr)2V•]- as an electron donor for nitrogenase was demonstrated by a simple, sensitive spectrophotometric assay for nitrogenase activity that can provide accurate values for the specific activity and turnover rate constant under argon. Under N2, the formation of ammonia was confirmed. Because of the observed full activity of nitrogenase and low overpotential, [(SPr)2V•]- should also prove to be valuable for nitrogenase electrocatalysis, including bioelectrosynthetic N2 reduction.

A fast and sensitive activity assay for lytic polysaccharide monooxygenase

BackgroundLytic polysaccharide monooxygenases (LPMO) release a spectrum of cleavage products from their polymeric substrates cellulose, hemicellulose, or chitin. The correct identification and quantitation of these released products is the basis of MS/HPLC-based detection methods for LPMO activity. The duration, effort, and intricate analysis allow only specialized laboratories to measure LPMO activity in day-to-day work. A spectrophotometric assay will simplify the screening for LPMO in culture supernatants, help monitor recombinant LPMO expression and purification, and support enzyme characterization.ResultsBased on a newly discovered peroxidase activity of LPMO, we propose a fast, robust, and sensitive spectrophotometric activity assay using 2,6-dimethoxyphenol (2,6-DMP) and H2O2. The fast enzymatic assay (300 s) consists of 1 mM 2,6-DMP as chromogenic substrate, 100 µM H2O2 as cosubstrate, and an adequate activity of LPMO in a suitable buffer. The high molar absorption coefficient of the formed product coerulignone (ε469 = 53,200 M−1 cm−1) makes the assay sensitive and allows reliable activity measurements of LPMO in concentrations of approx. 0.5–50 mg L−1.ConclusionsThe activity assay based on 2,6-DMP detects a novel peroxidase activity of LPMO. This activity can be accurately measured and used for enzyme screening, production, and purification, and can also be applied to study binding constants or thermal stability. However, the assay has to be used with care in crude extracts, because other enzymes such as laccase or peroxidase will interfere with the assay. We also want to stress that the peroxidase activity is a homogeneous reaction with soluble substrates and should not be correlated to heterogeneous LPMO activity on polymeric substrates.

A Continuous and Sensitive Spectrophotometric Assay for Lipase and Phospholipase A Activities Using α-Eleostearic Acid-Containing Substrates.

To date, several sensitive methods, based on radiolabeled elements or sterically hindered fluorochrome groups, are usually employed to screen lipase and phospholipase A (PLA) activities. Here, a new ultraviolet spectrophotometric assay for lipase or PLA was developed using natural triglycerides or synthetic glycerophosphatidylcholines containing α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) purified from Aleurites fordii seed oil. The conjugated triene present in α-eleostearic acid constitutes an intrinsic chromophore and consequently confers strong UV absorption properties of this free fatty acid as well as of lipid substrates harboring it. The substrate was coated into the wells of a microplate, and the lipolytic activities were measured by the absorbance increase at 272nm due to the transition of α-eleostearic acid moiety from the adsorbed to the soluble state. This continuous assay is compatible with a high-throughput screening method and can be applied specifically to the screening of new potential lipase, PLA1 and PLA2 inhibitors.

Rapid and Selective Spectrophotometric Assay for Micromolar Level Determination of Hydrogen Peroxide Based on Biocatalysis of Horseradish Peroxidase

Hydrogen peroxide (H2O2) is an essential compound in food, pharmaceutical, clinical, industrial and environmental analysis. H2O2 has been widely used for preservation of raw milk due to its bactericidal properties. However, excess of H2O2 can be deleterious on the nutritional value of milk due to degradation of folic acid. A simple and sensitive spectrophotometric assay was developed for the quantification of H2O2 in milk. This method is based on coupling of Iso-nicotinic acid hydrazide (INH) and Resorcinol (RC) as chromogenic co-substrates in presence of horseradish peroxidase (HRP) and H2O2 to form a red colored product, which has strong absorbance at λmax = 520 nm. The effects of different parameters on the sensitivity of the proposed assay were studied and optimized. Under optimized condition a linearity response for H2O2 are 0.5 μM - 64 μM and 0.5 μM -128 μM for kinetic and fixed time methods, respectively. The detection limit, quantification limit and molar extension co-efficient values for H2O2 assay were found to be 0.87 μM, 2.63 μM and 0.328×105 L/mol/cm, respectively. The Michaelis-Menten (Km) constant and catalytic efficiency were 36.27 μM and 2.0927 ×105 M-1 min-1. The proposed method produced recovery in the range of 99-102.8 % for milk samples and 98.2-101.8 % for rain water samples. Some of the important advantages of the proposed method are, use of inexpensive reagents, simple operation conditions, rapid analysis, wide linear range, high sensitivity, free from interfering substances and precision comparable to the reference methods.

Theophylline increases saliva sonic hedgehog and improves taste dysfunction

ObjectiveTo determine changes in saliva sonic hedgehog (Shh) and in taste dysfunction before and after oral theophylline treatment. DesignShh was measured in parotid saliva of both normal subjects and patients with taste dysfunction of multiple etiologies by use of a sensitive spectrophotometric ELISA assay. Taste dysfunction was defined clinically by both subjective inhibition of taste function (including acuity loss) and impaired gustometry. ResultsTheophylline treatment increased patient saliva Shh and improved taste dysfunction both subjectively and by gustometry. ConclusionsBy use of these systematic clinical measurements theophylline can be demonstrated to increase saliva Shh and improve taste dysfunction. These results are consistent with saliva Shh acting as a taste bud growth factor which stimulates stem cells of taste buds to initiate development and perpetuation of taste bud receptors. Measurements of saliva Shh provide an important marker for the presence of taste dysfunction and in the improvement by theophylline treatment.

Sonic hedgehog is present in parotid saliva and is decreased in patients with taste dysfunction.

To demonstrate that sonic hedgehog (Shh) is present in human parotid saliva and is decreased in human taste dysfunction. Shh was measured in parotid saliva of 27 normal subjects and 81 patients with taste dysfunction of multiple etiologies using a sensitive spectrophotometric ELISA assay. Taste dysfunction was defined clinically both by subjective decreases of taste acuity and flavor perception and by impaired gustometry. Shh was found in parotid saliva of both normal subjects and patients with taste dysfunction. Levels were significantly lower in patients than in normal subjects. Both subjective loss of taste acuity and flavor perception and impaired gustometry was measured in untreated patients. This is the first demonstration of Shh in human saliva. As Shh has been related to taste bud growth and development, its presence in saliva is consistent with its role as a cell signaling moiety involved with stimulation of taste bud stem cells to generate taste receptors. Decreased saliva Shh secretion can be considered a marker of taste dysfunction in patients with multiple pathologies for their dysfunction.

Improved smell function with increased nasal mucus sonic hedgehog in hyposmic patients after treatment with oral theophylline

PurposeWe previously demonstrated the presence of sonic hedgehog (Shh) in nasal mucus in normal subjects and in patients with smell loss (hyposmia). Nasal mucus Shh levels were found significantly diminished in untreated hyposmic patients of multiple etiologies. Since treatment with oral theophylline has been previously associated with improvement in smell function we wished to study if such treatment increased nasal mucus Shh as well as improved smell function in patients with hyposmia. MethodsForty-four patients with hyposmia of several etiologies were evaluated for changes in hyposmia by subjective measurements of smell, taste and flavor perception and by olfactometry. Measurements of nasal mucus Shh were made in relationship to each set of sensory measurements. Patients were treated with oral theophylline at doses of 200–800mg for periods of 2–10months with sensory function, nasal mucus Shh and serum theophylline levels evaluated at these time intervals. Nasal mucus Shh measurements were made with a sensitive spectrophotometric ELISA assay and theophylline with a fluorometric assay. ResultsThere was consistent, significant improvement in subjective responses in smell, taste and flavor perception and in olfactometry associated with increased nasal mucus Shh and serum theophylline after theophylline treatment. ConclusionsImprovement in smell function and in nasal mucus Shh was positively correlated in a dose-response relationship after treatment with oral theophylline. Results are consistent with a successful role for theophylline in improvement of smell function in hyposmic patients of multiple etiologies associated with increased nasal mucus Shh which can act as a biochemical marker for smell function.

Sensitive Spectrophotometric Assay of Two Phosphodiesterase Type 5-Inhibitors in Pure and Dosage forms Using Potassium Permanganate

Sensitive Spectrophotometric Assay of Two Phosphodiesterase Type 5-Inhibitors in Pure and Dosage forms Using Potassium Permanganate

Increasing Nasal Mucus Sonic Hedgehog (Shh) Improves Human Olfaction

Sonic hedgehog (Shh) has been shown to act as a cell signaling and growth factor moiety in many tissues in humans and animals. Shh has not been associated with smell function. We therefore wondered if Shh played a role in human smell function and, if so, if it were present in nasal mucus. Thus, we measured Shh in nasal mucus in 14 normal subjects and in 44 patients with smell loss (hyposmia) of several etiologies by use of a sensitive spectrophotometric ELISA assay. Shh was found in nasal mucus in both normal subjects [7538±1105 pg/ml (M±SEM)] and in hyposmic patients (149±2 pg/ml) with levels in patients significantly lower than in normal subjects. These changes occurred in patients with losses of smell function related to post‐influenza‐like infection, allergic rhinitis and head injury. Treatment with oral theophylline 200‐800 mg daily for 2‐10 months significantly increased Shh levels in nasal mucus in hyposmic patients (49191±1710 pg/ml) and increased smell function significantly in over 60% of the patients. This is the first demonstration of Shh in nasal mucus. Its stimulation by theophylline resulted in improved smell function among hyposmic patients of multiple etiologies.

Activity of Leucine Aminopeptidase of Telchin licus licus: An Important Insect Pest of Sugarcane

The enzymatic activity of leucine aminopeptidase (EC 3.4.11.1) from the intestinal tract of sugarcane giant borer (Telchin licus licus) was assayed by using a simple and sensitive spectrophotometric assay that uses L-leucyl-2- naphthylamide as substrate. In this assay, L-leucyl-2-naphthylamide is hydrolyzed to produce 2-naphthylamine and Lleucine. The product 2-naphthylamine reacts with Fast Black K and can be monitored using a continuous spectrophotometric measurement at 590 nm. The data on the kinetic parameters indicates that the Km for the L-leucyl-2- naphthylamide at pH 7.0 was found to be lower than those found for other LAP substrates. The Km and Vmax for the LAP were determined to be 84.03 µM and 357.14 enzymatic units mg(-1), respectively. A noticeable difference of LAP activity between the two insect orders tested was observed. This method could be used to screen for natural LAP inhibitors.

Sensitive and Selective Spectrophotometric Assay of Lead in Aqueous Solution

Sensitive and Selective Spectrophotometric Assay of Lead in Aqueous Solution

Sensitive spectrophotometric assay of psychoactive drug in pharmaceutical formulations and in biological fluids using redox reaction

Sensitive spectrophotometric assay of psychoactive drug in pharmaceutical formulations and in biological fluids using redox reaction

Membrane proteins in four acts: Function precedes structure determination

Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/proton translocation. (1) Crystal structures of the eight subunit hetero-oligomeric trans-membrane dimeric cytochrome b 6 f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of 17 monotopic and polytopic hetero-subunits. (II) β-Barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B 12 binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins [1]. A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a “fishing pole” model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83 Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained [2]. A crystal structure of the N-terminal translocation domain of colicin E3 complexed to OmpF established the role of OmpF as an import channel for colicin nuclease cytotoxins. (IV) α-Synuclein, associated with the etiology of Parkinson’s Disease, is an example of a protein, which is soluble and disordered in solution, but which can assume an ordered predominantly α-helical conformation upon binding to membranes. When subjected in its membrane-bound form to a trans-membrane electrical potential, α-synuclein can form voltage-gated ion channels. Summary of methods to assay functions/activities: (i) sensitive spectrophotometric assay to measure electron transfer activities; (ii) hydrophobic chromatography to deplete lipids, allowing reconstitution with specific lipids for studies on lipid–protein interactions; (iii) microbiological screen to assay high affinity binding of colicin receptor domains to Escherichia coli outer membrane receptors; (iv) electrophysiology/channel analysis (a) to select channel-occluding ligands for co-crystallization with ion channels of OmpF, and (b) to provide a unique description of voltage-gated ion channels of α-synuclein.

Application of sulphanilamides disazo dyes with Tropaeolin O for simple, rapid and sensitive spectrophotometric assay of medicines

A rapid, simple and sensitive spectrophotometric method for the determination of some sulphanilamides is described. The method is based on the formation of blue coloured disazo dyes by the diazotization of sulphonamides viz. sulphanilamide (SA), sulphamerazine (SMR), sulphamethazine (SMZ), sulphadimethoxine (SDM), sulphamethoxazole (SMX), sulphadiazine (SDA), sulfathiazole (STZ), sulphaguanidine (SGN), sulphamonomethoxine (SMM), sulphamethoxypyridazine (SMP) in 0.5 M hydrochloric acid media at ice bath followed by the azocoupling reaction with acid monoazo dye Tropaeolin O (TrO) at pH = 10.5. Formed products are stable for 10 h at room temperature. Effective molar absorptivities at absorbance maxima 595 nm for disazo dyes were ∼10 4 M −1 cm −1. Stoichiometric ratios of the components of disazo dyes were determined by means of mole ratio and continuous variations methods. Linear ranges for sulphanilamides determination were 0.4–14.0 μg ml −1. The methods were successfully approved at suphanilamides determination in model solutions and commercial pharmaceutical preparations.

Sensitive spectrophotometric assay of simvastatin in pharmaceuticals using permanganate

Two simple, sensitive, selective and inexpensive spectrophotometric methods are described for the determination of simvastatin (SMT) in bulk drug and in tablets using permanganate as the oxidimetric reagent. In method A, SMT is treated with a measured excess of permanganate in acetic acid medium and the unreacted oxidant is measured at 550 nm, whereas in method B the reaction is carried out in alkaline medium and the resulting manganate is measured at 610 nm. In method A, the amount of permanganate reacted corresponds to the SMT content and the absorbance is found to decrease linearly with the concentration; and in method B, the absorbance increases with concentration. The working conditions of assays were optimized, and the methods were validated according to the current ICH guidelines. Under optimum conditions, SMT could be assayed in the concentration ranges, 1.47 - 17.67x10-5 and 2.27 - 27.18 x10-6 mol/L by method A and method B, respectively. The calculated molar absorptivities are 3.2 x 10³ and 2.5 x 10(4) L/mol/cm for method A and method B, respectively with corresponding Sandell sensitivity values of 0.0387 and 0.0178 μg/cm². The limits of detection (LOD) and quantification (LOQ) have also been reported. Accuracy and precision for the assay were determined by calculating the intra-day and inter-day at three concentrations; the intra-day RSD was < 2% and the accuracy was better than 2.15 % (RE). The methods were applied successfully for the determination of SMT in tablet dosage form with a high percentage of recovery, good accuracy and precision, and without measurable interference by the excipients. The accuracy was further ascertained from placebo and synthetic mixture analysis and also from the spike-recovery method.

Simple and sensitive spectrophotometric assay of ofloxacin in pharmaceuticals based on ion-pair reaction

Two simple, sensitive, economical and extraction-free spectrophotometric methods have been developed for the determination of ofloxacin (OFX) in pure form and in tablets. The methods are based on the interaction of OFX with two sulphonphthalein dyes, namely, bromothymol blue (method A) and bromophenol blue (method B) in dichloromethane medium to form stable, yellow-colored ion-pair complexes peaking at 410 nm. Under the optimum conditions, OFX could be assayed in the concentration ranges, 1.25-20 and 1.0-16 ?g mL-1 OFX by method A and method B, respectively, with correlation coefficient of 0.999 in both methods. The apparent molar absorptivity values are calculated to be 1.74 x 104 and 2.18 x 104, L moL-1 cm-1, for method A and method B, respectively, with corresponding Sandell sensitivity values of 0.021 and 0.017 ?g cm-2. The limits of detection (LOD) and quantification (LOQ) are also reported. The stoichiometry of the reaction was found to be 1:1 in both cases and the conditional stability constant (Kf) of the complexes have also been reported. The intra-day and inter-day variation was assessed. The methods were applied to determine OFX from marked tablet formulations. Statistical analysis proved that the proposed methods were both accurate and precise.