Spectrofluorometric Quantification Research Articles

Nitric Oxide Overproduction by cue1 Mutants Differs on Developmental Stages and Growth Conditions.

The cue1 nitric oxide (NO) overproducer mutants are impaired in a plastid phosphoenolpyruvate/phosphate translocator, mainly expressed in Arabidopsis thaliana roots. cue1 mutants present an increased content of arginine, a precursor of NO in oxidative synthesis processes. However, the pathways of plant NO biosynthesis and signaling have not yet been fully characterized, and the role of CUE1 in these processes is not clear. Here, in an attempt to advance our knowledge regarding NO homeostasis, we performed a deep characterization of the NO production of four different cue1 alleles (cue1-1, cue1-5, cue1-6 and nox1) during seed germination, primary root elongation, and salt stress resistance. Furthermore, we analyzed the production of NO in different carbon sources to improve our understanding of the interplay between carbon metabolism and NO homeostasis. After in vivo NO imaging and spectrofluorometric quantification of the endogenous NO levels of cue1 mutants, we demonstrate that CUE1 does not directly contribute to the rapid NO synthesis during seed imbibition. Although cue1 mutants do not overproduce NO during germination and early plant development, they are able to accumulate NO after the seedling is completely established. Thus, CUE1 regulates NO homeostasis during post-germinative growth to modulate root development in response to carbon metabolism, as different sugars modify root elongation and meristem organization in cue1 mutants. Therefore, cue1 mutants are a useful tool to study the physiological effects of NO in post-germinative growth.

Quercetin spectrofluorometric quantification in aqueous media using different surfactants as fluorescence promoters.

Quercetin spectrofluorometric quantification was carried out in aqueous media (pH 7) using micelles of surfactants, namely: CTAB, SDS and TX100 as promoters of quercetin fluorescence, having a critical micelle concentration, CMC, of: 0.94 ± 0.03, 7.7 ± 0.6 and 0.18 ± 0.3 mM, respectively, measured through UV-Vis spectrophotometry. The thermodynamic binding constant, K, for the quercetin-surfactants' micelles supramolecular complex was estimated to be (log(K/M−1)) = 2.87 ± 0.02, 2.78 ± 0.04 and 2.80 ± 0.04, respectively, from fluorescence spectrophotometry. With the aid of these parameters it was possible to construct species distribution diagrams and choose the experimental conditions where quercetin can be quantified in aqueous media from fluorescence measurements. The best lowest limit of detection (0.244 ± 0.092) μM was achieved with CTAB micelles while the best sensitivity (2.919 ± 0.054) M−1 corresponded to SDS.

Fabrication of magnetic Fe3O4@nSiO2@mSiO2–NH2 core–shell mesoporous nanocomposite and its application for highly efficient ultrasound assisted dispersive µSPE-spectrofluorimetric detection of ofloxacin in urine and plasma samples

In this research, a sensitive, simple and rapid ultrasound assisted dispersive micro solid-phase extraction (USAD-µSPE) was developed using a synthesized core–shell magnetic mesoporous nanocomposite (Fe3O4@nSiO2@mSiO2–NH2) as an efficient adsorbent for the preconcentration and spectrofluorometric determination of ofloxacin (OFL) in biological samples. The synthesized adsorbent was characterized using FT-IR spectroscopy, transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), energy dispersive X-ray (EDX) spectroscopy, thermogravimetric analysis (TGA) and Brunauer–Emmett–Teller (BET) analysis. The application of this magnetic nanocomposite as a sensitive solid phase for removal, preconcentration and spectrofluorometric quantification of trace amount of OFL was developed. Influence of various variables including pH, sorbent dosage, desorption solvent properties and sonication time on present method response was studied and optimized. The results showed that using the proposed method OFL can be determined in the linear concentration range of 1.0–500.0µgL−1 with a limit of detection as low as 0.21µgL−1 and relative standard deviation less than 2.5 (%). The results of human urine and blood plasma analysis showed that the method is a good candidate for biological sample analysis purposes.

Uptake Assays in Xenopus laevis Oocytes Using Liquid Chromatography-mass Spectrometry to Detect Transport Activity

Xenopus laevis oocytes are a widely used model system for characterization of heterologously expressed secondary active transporters. Historically, researchers have relied on detecting transport activity by measuring accumulation of radiolabeled substrates by scintillation counting or of fluorescently labelled substrates by spectrofluorometric quantification. These techniques are, however, limited to substrates that are available as radiolabeled isotopes or to when the substrate is fluorescent. This prompted us to develop a transport assay where we could in principle detect transport activity for any organic metabolite regardless of its availability as radiolabeled isotope or fluorescence properties. In this protocol we describe the use of X. laevis oocytes as a heterologous host for expression of secondary active transporters and how to perform uptake assays followed by detection and quantification of transported metabolites by liquid chromatography-mass spectrometry (LC-MS). We have successfully used this method for identification and characterization of transporters of the plant defense metabolites called glucosinolates and cyanogenic glucosides ( Jørgensen et al., 2017 ), however the method is usable for the characterization of any transporter whose substrate can be detected by LC-MS.

Interference-free spectrofluorometric quantification of aristolochic acid I and aristololactam I in five Chinese herbal medicines using chemical derivatization enhancement and second-order calibration methods

A rapid interference-free spectrofluorometric method combined with the excitation-emission matrix fluorescence and the second-order calibration methods based on the alternating penalty trilinear decomposition (APTLD) and the self-weighted alternating trilinear decomposition (SWATLD) algorithms, was proposed for the simultaneous determination of nephrotoxic aristolochic acid I (AA-I) and aristololactam I (AL-I) in five Chinese herbal medicines. The method was based on a chemical derivatization that converts the non-fluorescent AA-I to high-fluorescent AL-I, achieving a high sensitive and simultaneous quantification of the analytes. The variables of the derivatization reaction that conducted by using zinc powder in acetose methanol aqueous solution, were studied and optimized for best quantification results of AA-I and AL-I. The satisfactory results of AA-I and AL-I for the spiked recovery assay were achieved with average recoveries in the range of 100.4–103.8% and RMSEPs <0.78ngmL−1, which validate the accuracy and reliability of the proposed method. The contents of AA-I and AL-I in five herbal medicines obtained from the proposed method were also in good accordance with those of the validated LC-MS/MS method. In light of high sensitive fluorescence detection, the limits of detection (LODs) of AA-I and AL-I for the proposed method compare favorably with that of the LC-MS/MS method, with the LODs <0.35 and 0.29ngmL−1, respectively. The proposed strategy based on the APTLD and SWATLD algorithms by virtue of the “second-order advantage”, can be considered as an attractive and green alternative for the quantification of AA-I and AL-I in complex herbal medicine matrices without any prior separations and clear-up processes.

Low-dose rotenone exposure induces early senescence leading to late apoptotic signaling cascade in human trabecular meshwork (HTM) cell line: An in vitro glaucoma model.

This study aimed to determine whether the prolonged exposure of the human trabecular meshwork (HTM) cell line to a low dose (1 nM) of rotenone could simulate a glaucomatous-like condition and serve as a cellular model for its etiological analysis. Under 1-nM rotenone exposure for 24-72 h, HTM cells showed a decrease in cell viability as assessed by an MTT assay and showed mitochondrial dysfunction as assessed by measuring H2 DCFDA fluorescence; a decrease in ATP level was also observed. Flow cytometric analysis showed an increase in cellular size and granularity. Elevated AF showed initial senescence. LF staining with SBB and its spectrofluorometric quantification confirmed growth arrest. An accumulation of cytoplasmic myocilin, IL-6, and MMP-9 at 72 h of exposure supported glaucomatous induction. TEM revealed morphological changes in mitochondria and nuclei of treated cells. Signaling cascades were assessed by immunoblotting and immunocytochemical analysis. This study showed a shift in status of the cells from initial senescence to induction of apoptosis in the HTM cell line due to continuous low-dose exposure to rotenone; however, at 72 h, both senescence and apoptotic features are apparent in these cells. This is the first report that reveals the potential of a prolonged low-dose exposure of rotenone to simulate senescence in the HTM cell line to cause a glaucomatous condition.

Process design for augmentation and spectrofluorometric quantification of neutral lipid by judicious doping of pathway intermediate in the culture of marine Chlorella variabilis for biodiesel application

Over the past few years microalgae have emerged as the most promising feedstock for biodiesel production. However, enhancing lipid content remains a major scientific challenge. Many studies irrationally rely upon nitrogen starvation for improving lipid content at the cost of biomass. In this study, strategic enhancement of neutral lipid without compromising with biomass production was achieved in marine Chlorella variabilis by supplementing the culture with triacylglycerol pathway intermediate, citric acid (1gL−1 pure and 0.5gL−1 orange peel extract as a low cost substitute). The microalga accumulated 17% and 34% (on dry cell weight basis) more neutral lipid, when cultivated in with citric acid and orange peel extract as supplements respectively, without any adverse effect on growth. The medium supplemented with orange peel extract stimulated better growth than that supplemented with citric acid. Gas chromatography studies validated that both the supplemented media yielded desired fatty acid composition for biodiesel production.

Trilateral '3P' Mechanics of Stabilized Layersomes Technology for Efficient Oral Immunization.

The main objective of the present work is to investigate the potential of polyelectrolyte stabilized layersomes as vaccine adjuvants for oral immunization. Herein, bovine serum albumin (BSA) loaded vesicular nanoformulations were prepared via thin film hydration method. The effect of various explanatory variables on responses were examined by a 6-factor, 3-level Box-Behnken design using JMP statistical software. An improved stability of vesicular nanoformulations in simulated biological media revealed that alternate layer-by-layer coating using polyacrylic acid and polyallylamine hydrochloride improved the robustness of vesicular structure. The formulation was lyophilized to improve long-term storage stability using trehalose as cryoprotectant. As evident from in process stability analysis, the chemicals, process and lyophilization conditions had no detrimental effect on the conformational stability and integrity of antigen. Mathematical modelling and DDsolver analysis confirmed that the release profile of vesicular nanoformulations altered from Baker-Lonsdale to Higuchi fashion, following integration of layer-by-layer coating. Confocal microscopy and spectrofluorometric quantification demonstrated a selective, dose and time dependent accumulation of nanoformulations in antigen presenting cells. As evident from in vivo immunization study, layersomes produced higher humoral (mean IgG in serum), mucosal (mean sIgA titre in biological fluids) and cellular immune response (IL-2 and IFN-y level) compared to free antigen. The observed effects might be attributed to improved protection, permeation and presentation of antigen using layersomes. In conclusion, polyelectrolyte stabilized vesicular nanoformulations contribute promising toolbox for mass immunization using human/veterinary vaccine or other protein/peptides delivery especially by oral route.

Exposure of primary porcine urothelial cells to benzo(a)pyrene: in vitro uptake, intracellular concentration, and biological response

More than 90% of all bladder cancers are transitional cell carcinomas arising from the cells lining the inside of the hollow organ (uroepithelium). Cell cultures from primary urinary bladder epithelial cells (PUBEC) of pigs were established to assess the uptake, intracellular concentration, and subcellular distribution of the environmental pollutant benzo(a)pyrene (BaP). During treatment of the cells with 0.5μM BaP for up to 24h, intracellular concentration of BaP increased without saturation but with marked differences between various PUBEC pools. Analysis of BaP uptake by laser scanning microscopy indicated that BaP is rapidly partitioned into the cell membrane, while only a slight but significant increase in BaP fluorescence intensity was observed in the cytosol and nucleus. Spectrofluorometric quantification of BaP in PUBEC using ex situ calibration revealed a strong accumulation of BaP, leading to intracellular concentrations ranging from 7.28 to 35.70μM in cells exposed to 0.5μM BaP and from 29.9 to 406.64μM in cells exposed to 10μM BaP. These results were confirmed by gas chromatographic mass spectrometric analysis. Apoptotic cell nuclei were assessed by TUNEL analysis to see whether BaP exposure at the given concentrations results in a toxic effect. While apoptotic cells were barely detectable in control epithelial cells, there was a marked elevation in apoptosis in the BaP-exposed cells. In conclusion, a comprehensive study on uptake and quantification of BaP in epithelial cells from pig bladder is reported for the first time. The study may be helpful in understanding the pattern of BaP uptake and distribution in bladder and its possible implication in bladder cancer development.

Assessment of an in vitro whole cigarette smoke exposure system: The Borgwaldt RM20S 8-syringe smoking machine

BackgroundThere have been many recent developments of in vitro cigarette smoke systems closely replicating in vivo exposures. The Borgwaldt RM20S smoking machine (RM20S) enables the serial dilution and delivery of cigarette smoke to exposure chambers for in vitro analyses. In this study we have demonstrated reliability and robustness testing of the RM20S in delivering smoke to in vitro cultures using an in-house designed whole smoke exposure chamber.ResultsThe syringe precision and accuracy of smoke dose generated by the RM20S was assessed using a methane gas standard and resulted in a repeatability error of ≤9%. Differential electrical mobility particle spectrometry (DMS) measured smoke particles generated from reference 3R4F cigarettes at points along the RM20S. 53% ± 5.9% of particles by mass reached the chamber, the remainder deposited in the syringe or connecting tubing and ~16% deposited in the chamber. Spectrofluorometric quantification of particle deposition within chambers indicated a positive correlation between smoke concentration and particle deposition. In vitro air-liquid interface (ALI) cultures (H292 lung epithelial cells), exposed to whole smoke (1:60 dilution (smoke:air, equivalent to ~5 μg/cm2)) demonstrated uniform smoke delivery within the chamber.ConclusionsThese results suggest this smoke exposure system is a reliable and repeatable method of generating and exposing ALI in vitro cultures to cigarette smoke. This system will enable the evaluation of future tobacco products and individual components of cigarette smoke and may be used as an alternative in vitro tool for evaluating other aerosols and gaseous mixtures such as air pollutants, inhaled pharmaceuticals and cosmetics.

Bacterial growth kinetics estimation by fluorescence in situ hybridization and spectrofluorometric quantification

The aim of this study was to develop a specific and rapid method to identify and quantify relevant bacterial populations in mixed biomass by spectrofluorometric quantification (SQ) of whole cells hybridized with fluorescently labelled oligonucleotide probes targeting mature 16S ribosomal RNA (rRNA). Probe targeting the precursor of rRNA synthesis was also employed because it was being suggested as more indicative of the activity state of the micro-organisms. Original fluorescence in situ hybridization protocol was modified to be applied to liquid samples and the fluorescence emission from the Cy3-labelled cells was measured by spectrofluorometry. The method was calibrated on an exponentially growing cell suspension of Acinetobacter johnsonii and was successfully applied to generate kinetic data. No substantial difference in the estimated maximum specific growth rate (mu(max)) values was found between the SQ method and the classical method, using absorbance at 420 nm (6.2 d(-1)). The preliminary validation tests showed their direct applicability to target enriched cultures. This study demonstrated the validity of the SQ method to easily quantify the concentration and to determine the growth rate of specific micro-organisms present in mixed cultures. The proposed method can be directly utilized for quantification and kinetic characterization of microbial enrichments. It has the advantage of being easily applicable using simple, inexpensive equipment suitable for routine analysis.

Aluminum inhibits apoplastic flow of high–molecular weight solutes in root apices of Zea mays L.

AbstractUsing an aluminum (Al)‐sensitive maize cultivar, we investigated the influence of Al on the apoplastic solute bypass flow and its relationship with Al‐induced (1 h, 50 μM) callose formation and root growth. We selected the fluorescent probes 8‐hydroxypyrene‐1,3,6‐trisulfonic acid, trisodium salt (MW 524) (HPTS) and dextran‐Texas Red (TR) conjugates (MW 3,000, 10,000, and 40,000) to monitor their apoplastic transport. Confocal laser–scanning microscopy (CLSM) analysis and spectrofluorometric quantification showed Al‐induced callose formation in peripheral root cells within 1 h. Pretreatment of plants with the callose synthesis inhibitor 2‐deoxy‐D‐glucose (DDG) reduced the callose formation by half. Uptake experiments with both HPTS and dextrans showed uniform dye distribution in control root apices. After Al treatment for 1 or 2 h, which inhibited root growth by 32% or 50%, respectively, the dyes accumulated in the epidermal and outer cortical cell layers, especially in the 1–2 mm apical root zone. Al treatment reduced the export of the dyes out of the apical 1 cm treatment zone. This was due to strong sorption of HPTS but not of dextrans by Al‐loaded cell walls. Aluminum treatment reduced loading into the xylem sap particularly of higher–molecular weight dextrans. Pretreatment of roots with DDG and presence of 50 mM mannitol during the Al treatment partially forestalled the inhibitory effect of Al on the dye transport, but only slightly reduced the Al‐induced growth inhibition. Exudation experiments revealed that xylem water flow remained unaffected by the Al treatment of the root tips. The results with dextran suggest that Al binding in cell walls of the root apex inhibits apoplastic bypass flow of higher–molecular weight solutes, which might contribute to Al‐induced inhibition of root growth.

Specific spectrofluorometric quantification of d-penicillamine in bulk and dosage forms after derivatization with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole

A specific and sensitive fluorometric method is proposed for the determination of d-penicillamine in pure form and pharmaceuticals. The method is based on coupling between d-penicillamine and 4-fluoro-7-nitrobenzo-2-oxa-1,3-dizole (NBD-F) to produce the corresponding fluorescent NBD-penicillamine. The optimum conditions for the reaction were carefully investigated and incorporated into the procedure. The reaction was proceeded in borate buffer (pH 7.2) containing 20% of ethanol to give intense fluorescence having its excitation and emission wavelengths at 465 and 530 nm, respectively. The fluorescence intensity was a linear function of the drug concentration over the range of 0.6–3 μg ml −1. Regression analysis of the concentration fluorescence intensity showed good correlation ( r = 0.9975) with a detection limit of 2 × 10 −3 μg ml −1 (1.3 × 10 −8 M). The mean of percentage recoveries of added authentic drug was 100.00 ± 1.05. The method was applied successfully to the determination of this drug in pharmaceutical dosage form. The mean recovery for the commercial capsules was 98.1 ± 0.54%. The results were sufficiently accurate, precise and comparable with those obtained from the official method. The method is specific for the intact drug, and can be adopted in the presence of possible interferences.

Spectrofluorometric quantification of neutral and polar lipids in zooplankton using Nile red

This study defines the conditions (wavelength selection and sensitivity) for spectrofluorometric quantification of neutral and polar lipids using Nile red (NR). The fluorescence intensity of the probe with standard lipids was examined in relation to lipid concentration and composition. Standards used included commercial triolein (TO) and phosphatidylcholine (PC), triacylglycerols (TG) and phospholipids (PL) from the krill Euphausia superba and Meganyctiphanes norvegica, and wax esters (WE) and PL from the copepods Paraeuchaeta antarctica and Drepanopus pectinatus. At any wavelength tested, polar and neutral lipids could be detected and fluorescence overlapped resulting in an interference of 10% to 20%. This interference between polar and neutral lipids was quantified for the standardisation. Wavelength selection for maximum emission with neutral and polar lipid classes achieved maximal sensitivity. Relationships between fluorescence intensity and lipid concentration were shown to be linear and specific to the lipid used. Large errors occurred when calibrations were performed with commercial lipids or with lipids obtained from other plankton species. Therefore, only the use of specific lipids as standards provided simultaneous and absolute quantification of neutral and polar lipids (relative errors were lower than 5%). The presence of PL in the sample (a ratio as low as 1:20) improved fluorescence of NR with TG by enhancing the solubility of the lipid microemulsion in water.

Spectrofluorometric quantification of neutral and polar lipids suggests a food‐related recruitment bottleneck for juveniles of a deposit‐feeding polychaete population

A growing body of evidence suggests that juveniles of species that deposit feed as adults are more susceptible to food limitation than are conspecific adults. To quantify ontogenetic variations in the nutritional condition of the depositfeeding polychaete Pseudopolydora kempi japonica, I modified existing spectrofluorometric assays of neutral and polar lipids. The hydrophobic fluorochrome Nile red fluoresces yellow‐orange when bound to neutral lipids and red‐orange when bound to polar lipids. The ratio of neutral lipids (energy storage) to polar lipids (cell membranes) provides an index of nutritional condition that is scaled to body size. For individual P. kempi japonica ranging in length from 2 to 12 mm, I quantified the florescence from each lipid class and analyzed sizedependent variations in the neutral/polar lipid ratio. P. kempi japonica showed a size‐dependent increase in the neutral/polar lipid ratio up to a body length of −6–8 mm; little size‐dependent change in the ratio occurred beyond this size. Ontogenetic changes in both particle‐size selection and the assimilated diet of P. kempi japonica display size‐dependent patterns that are similar to the lipid data. Together, the three lines of evidence suggest that P. kempi japonica juveniles experience a food‐related recruitment bottleneck until they grow to −6–8 mm. Once juveniles grow beyond this size, they appear to forage like adults and achieve adult levels of neutral lipid. Because deposit‐feeding benthos ingest a low‐quality and nutritionally dilute diet that will be difficult for small juveniles to digest, ontogenetic niche shifts and food‐related recruitment bottlenecks during the juvenile stage may be common in the population ecology of species that deposit feed as adults.

Automated high-performance liquid chromatographic separation with spectrofluorometric detection of a malondialdehyde-thiobarbituric acid adduct in plasma

An automated HPLC separation method for the detection of malondialdehyde (MDA) in plasma samples of a healthy population is described. The procedure involves a first step of acidic hydrolysis and complex formation with thiobarbituric acid (TBA), then a protein precipitation step, and finally the separation of TBA-MDA adducts through a complete HPLC apparatus with spectrofluorometric quantification. This procedure is very useful for a routine laboratory because of its rapidity ( t R = 1.7 min), simplicity and applicability, and the method gives very good results with respect to linearity (0.1–12 μmol/l), precision (within-assay C.V.%: 8, between-assay C.V.%: 10) and accuracy (recovery average: 91.66%).

Carbohydrate exposure of human promyelocytic HL60 cells and histiocytic U937 cells during phagocytic differentiation assessed with fluoresceinated lectins

The display of carbohydrate structures was measured in promyelocytic HL60 cells and in histiocytic U937 cells induced to differentiate to phagocytic cellsin vitro during three to seven days of cultivation in the presence of dimethylsulfoxide (DMSO). It was assessed by micro-or spectrofluorometric quantification of the binding of fluorescent lectins. Changes in the cell size and the association and uptake of IgG-or complementopsonized yeast cells (Saccharomyces cerevisiae) were used as signs of phagocyte differentiation. The binding of wheat germ agglutinin (WGA), concanavalin A (Con A),Ricinus communis agglutinin-I (RCA-I) andUlex europaeus agglutinin-I (UEA-I) varied due to the presence of DMSO during cultivation, and without DMSO also on the number of days in culture and the type of cell.