ABSTRACT
This work delineates an integrative approach combining spectroscopic and computational studies to decipher the association-induced fluorescence properties of a fluorescent molecular rotor, viz., auramine O (AuO), after interacting with 20-mer duplex DNA having diverse well-matched base pairs. While exploring the scarcely explored sequence-dependent interaction mechanism of AuO and DNA, we observed that DNA could act as a conducive scaffold to the formation of AuO dimer through noncovalent interactions at lower molecular density. The photophysical properties of AuO depend on the nucleotide compositions as described from sequence-dependent shifting in the emission and absorption maxima. Furthermore, we explored such DNA base pair-dependent fluorescence spectral characteristics of AuO toward discriminating the thermodynamically most stable single nucleotide mismatch in a 20-mer sequence. Our results are interesting and could be useful in developing analogues with further enhanced emission properties toward mismatched DNA sequences.
Subject(s)
Benzophenoneidum , DNA , Benzophenoneidum/chemistry , DNA/chemistry , Fluorescent Dyes , Nucleotides , Staining and LabelingABSTRACT
Control of fluorescent molecular assemblies is an exciting area of research with large potential for various important applications, such as, fluorescence sensing/probing, cell imaging and monitoring drug-delivery. In the present contribution, we have demonstrated control on the extent of aggregation of a dye-polyelectrolyte assembly using a macrocyclic host molecule, sulfobutylether-ß-cyclodextrin (SBE-ß-CD). Initially, a cationic molecular rotor based organic dye, Auramine-O (AuO), undergoes aggregation in the presence of an anionic polyelectrolyte, polystyrene sulfonate (PSS), and displays a broad intense new emission band along with large variation in its absorption features and excited-state lifetime. A manipulation of the monomer-aggregate equilibrium of the dye-polyelectrolyte assembly has been achieved by introducing a cyclodextrin based supramolecular host, SBE-ß-CD, which leads to relocation of AuO molecules from polyelectrolyte (PSS) to supramolecular host cavity, owing to the formation of a host-guest complex between AuO and SBE-ß-CD. A reversible control on this manipulation of monomer-aggregate equilibrium is further achieved by introducing a competitive guest for the host cavity i. e., 1-Adamantanol. Thus, we have demonstrated an interesting control on the dye-polyelectrolyte aggregate assembly using a supramolecular host molecule which open up exciting possibilities to construct responsive materials using a repertoire of various host-specific guest molecules.
Subject(s)
Benzophenoneidum/chemistry , Fluorescent Dyes/chemistry , Polystyrenes/chemistry , beta-Cyclodextrins/chemistry , Macromolecular Substances/chemistry , Molecular Structure , Optical Phenomena , Polyelectrolytes/chemistryABSTRACT
G-triplex (G3) has been recognized as a popular intermediate during the folding of G-quadruplex (G4). This has raised interest to anticipate the ultimate formation of G3 by shortening the G4-forming oligonucleotides with the remaining three G-tracts. Some G3 structures have been validated and their stability has been found to be affected by the loop sequences similar to G4s. In this work, however, we first found that an intermolecular parallel G4 structure was preferred in K+ for the oligonucleotide 5'-TGGGTAGGGCGGG-3' (DZ3) containing only three G-tracts. We screened auramine O (AO) as the appropriate fluorophore with a molecular rotor feature to target this G4 structure. AO bound with DZ3 in a 1 : 4 ratio, as confirmed by isothermal titration calorimetry experiments, suggesting the formation of a tetramolecular G4 structure (4erG4). The excimer emission from the labelled pyrene and the DNA melting behavior at various pHs in the presence of Ag+ proved the formation of the 4erG4 structure rather than the prevalent intramolecular G3 folding. This work demonstrates that one should be cautious while putatively predicting a G3 structure from an oligonucleotide containing three G-tracts.
Subject(s)
DNA/chemistry , G-Quadruplexes , Benzophenoneidum/chemistry , DNA/genetics , Fluorescent Dyes/chemistry , Hydrogen-Ion Concentration , Nucleic Acid Denaturation , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/genetics , Silver/chemistryABSTRACT
In this work, a high-surface-area dual inorganic molecularly imprinted (DIMI) Bi2WO6/CuO/Ag2O photo-catalyst was developed for the selective photocatalytic degradation of methyl green (MG) and auramine O (AO) dyes as target pollutants. The DIMI-Bi2WO6/CuO/Ag2O heterojunction was synthesized by a sono-chemically assisted sol-gel method by coating a layer of molecularly imprinted Ag2O/CuO on the surface of Bi2WO6 nanocubes with MG and AO as the templates. This was followed by calcination for the removal of target molecules and annealing for Ag/Cu oxide preparation. This novel photocatalyst was prepared to overcome the challenge of the co-existing non-target molecules, which has limited the photocatalytic degradation performance. The surface DIMI sites could act as surface defects for accelerating the separation of photogenerated holes and electrons, which led to the increased generation of OH radicals. Moreover, the DIMI sites had increased binding affinity toward MG and AO via the formation of multiple H bonds and electrostatic bonds, which were confirmed by FTIR spectroscopy, PL and EIS studies. The surface DIMI sites led to the increased adsorption and improved local concentration of MG and AO on Bi2WO6/CuO/Ag2O. Consequently, the heterojunction properties of the final DIMI product accelerated the transfer and separation of photogenerated carriers. The high binding affinity of the DIMI sites to MG and AO confirmed the selective recognition, which was tested in the presence of coexisting pollutant dyes. The other characterizations confirmed the successful fabrication and high photocatalytic activity of the high-surface-area DIMI-Bi2WO6/CuO/Ag2O heterostructured composite. In general, the superior interfacial electronic interactions, high migration efficiency of photoinduced charge carriers, and strong visible light absorption of the prepared photocatalyst resulted in good photocatalytic performance.
Subject(s)
Bismuth/chemistry , Light , Molecular Imprinting , Nanotubes/chemistry , Tungsten Compounds/chemistry , Water Pollutants, Chemical/chemistry , Benzophenoneidum/chemistry , Catalysis , Coloring Agents/chemistry , Copper/chemistry , Hydrogen Bonding , Methyl Green/chemistry , Oxides/chemistry , Photolysis/radiation effects , Silver Compounds/chemistry , Spectroscopy, Fourier Transform Infrared , Static Electricity , Surface PropertiesABSTRACT
Microscopy-based tuberculosis (TB) diagnosis i.e. Ziehl-Neelsen screening still remains the primary diagnostic method in resource poor and high TB burden countries, however this method has poor sensitivity (~60%). Bringing three million TB patients who are left undiagnosed under the treatment has been a major focus as part of END-TB strategy across the world. We have developed a portable set-up called 'SeeTB' that converts a bright-field microscope into fluorescence microscope (FM) with minimal interventions. SeeTB, a total internal reflection-based fluorescence excitation system allows visualization of auramine-O stained bacilli efficiently with high signal-to-noise ratio. Along with the device, we have developed a sputum-processing reagent called 'CLR' that homogenizes and digests the viscous polymer matrix of sputum. We have compared the performance of SeeTB system in 237 clinical sputum samples along with FM, GeneXpert and liquid culture. In comparison with culture as gold standard, FM has sensitivity of 63.77% and SeeTB has improved sensitivity to 76.06%. In comparison with GeneXpert, FM has sensitivity of 73.91% while SeeTB has improved sensitivity to 85.51%. However, there is no significant change in the specificity between FM and SeeTB system. In short, SeeTB system offers the most realistic option for improved TB case identification in resource-limited settings.
Subject(s)
Benzophenoneidum/chemistry , Microscopy, Fluorescence/instrumentation , Mycobacterium tuberculosis/isolation & purification , Tuberculosis/diagnosis , Diagnostic Equipment , Diagnostic Tests, Routine , Early Diagnosis , Equipment Design , Humans , Male , Sensitivity and Specificity , Sputum/microbiologyABSTRACT
In this work, the interaction between an Auramine O (AuO) fluorescent molecular rotor and natural DNA, its thermodynamic aspects and the resulting variation of the optical properties upon binding are addressed by a combined spectroscopic (UV-vis and fluorescence) and computational approach. DNA binding causes a shift in the maximum of absorption from 432 nm to 444 nm, a decrease of the extinction coefficient and a dramatic enhancement of fluorescence emission, these results being in agreement with intercalation into the polynucleotide helix. Intercalation is indeed confirmed by the thermodynamic parameters for the binding reaction (in particular, the highly negative ΔH). Theoretical modelling at the TD-DFT level was done on a simplified model system consisting of the AuO molecule intercalated between two DNA base pairs. The evolution of the calculated vertical transitions quantitatively reproduces the experimentally observed hypo- and bathochromic shifts, thus confirming the intercalation hypothesis.
Subject(s)
Benzophenoneidum/chemistry , DNA/chemistry , Models, Chemical , Spectrum Analysis , Fluorescence , Molecular Dynamics SimulationABSTRACT
UV and NIR-responsive monoolein cubic phase was prepared by including poly(ethyleneimine) (PEI)/cinnamic acid (CA) conjugate and gold nanoparticle (GNP) within its structure. UV irradiation elevated significantly the release % of Auramine O loaded in cubic phase containing PEI/CA, possibly because of the trans-to-cis isomerization of CA. NIR irradiation also increased significantly the release % of FITC-dextran loaded in cubic phase containing PEI/CA. The release % of the dye loaded in cubic phase containing PEI/CA and GNP was elevated more markedly by NIR irradiation, possibly due to the phase transition of cubic phase and the disassembling of PEI/CA assembly.
Subject(s)
Cinnamates/chemistry , Glycerides/chemistry , Metal Nanoparticles , Polyethyleneimine/chemistry , Benzophenoneidum/administration & dosage , Benzophenoneidum/chemistry , Dextrans/administration & dosage , Dextrans/chemistry , Drug Liberation , Fluorescein-5-isothiocyanate/administration & dosage , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/chemistry , Fluorescent Dyes/administration & dosage , Gold , Infrared Rays , Ultraviolet RaysABSTRACT
Self-aggregation of organic molecules is rarely seen with macrocyclic hosts like ß-cyclodextrin, as they preferentially involve the formation of inclusion complexes with the guest molecule. In this contribution, we report the self-aggregation of a guest molecule induced by negatively charged sulfated ß-cyclodextrin (SCD) to yield highly emissive aggregates of a recently projected amyloid marker dye, Auramine O (AuO). The SCD templated AuO aggregates display very different photophysics when compared to its reported behavior in a wide range of various chemical and biological environment but show a remarkable similarity with the recently reported photophysical behavior of AuO in human insulin fibrillar media, thus providing important insights into the molecular form of AuO responsible for its amyloid sensing ability. The self-assembled AuO aggregates formed in the presence of SCD display a significantly long excited-state lifetime, suggesting the retardation of the torsional relaxation of dye in the aggregated state, which otherwise leads to a very short excited-state lifetime for the monomeric form of the dye in the isolated form. Detailed time-resolved emission spectra (TRES) measurements show a dynamic Stokes shift suggesting excitonic migration within the AuO aggregates. The supramolecular aggregate assembly displays remarkable sensitivity to important external stimuli like temperature or ionic strength of the medium, pitching for its possible application in designing stimuli-responsive sensing schemes for important analytes.
Subject(s)
Benzophenoneidum/chemical synthesis , Cyclodextrins/chemistry , Sulfates/chemistry , Benzophenoneidum/chemistry , Macromolecular Substances/chemical synthesis , Macromolecular Substances/chemistry , Molecular StructureABSTRACT
Residues of harmful dyes such as Auramine O (AO) in herb and food products threaten the health of people. So, fast and sensitive detection techniques of the residues are needed. As a powerful tool for substance detection, terahertz (THz) spectroscopy was used for the quantitative determination of AO by combining with an improved partial least-squares regression (PLSR) model in this paper. Absorbance of herbal samples with different concentrations was obtained by THz-TDS in the band between 0.2THz and 1.6THz. We applied two-dimensional correlation spectroscopy (2DCOS) to improve the PLSR model. This method highlighted the spectral differences of different concentrations, provided a clear criterion of the input interval selection, and improved the accuracy of detection result. The experimental result indicated that the combination of the THz spectroscopy and 2DCOS-PLSR is an excellent quantitative analysis method.
Subject(s)
Benzophenoneidum/analysis , Benzophenoneidum/chemistry , Coloring Agents/analysis , Coloring Agents/chemistry , Terahertz Spectroscopy/methods , Least-Squares AnalysisABSTRACT
In the present study, we designed hydrogels for dual drug release: the hydrogels that covalently contained the polymeric micelles that possess different drug release properties. The hydrogels that were formed from polymeric micelles possessing a tightly packed (i.e., well-entangled) inner core exhibited a higher storage modulus than the hydrogels that were formed from the polymeric micelles possessing a loosely packed structure. Furthermore, we conducted release experiments and fluorescent observations to evaluate the profiles depicting the release of two compounds, rhodamine B and auramine O, from either polymeric micelles or hydrogels. According to our results, (1) hydrogels that covalently contains polymeric micelles that possess different drug release properties successfully exhibit the independent release behaviors of the two compounds and (2) fluorescence microscopy can greatly facilitate efforts to evaluate drug release properties of materials.
Subject(s)
Benzophenoneidum/chemistry , Drug Liberation , Hydrogels/chemistry , Micelles , Polyesters/chemistry , Polyethylene Glycols/chemistry , Rhodamines/chemistry , Hydrogels/chemical synthesis , Microscopy, FluorescenceABSTRACT
Cellulose (Cel) and sugarcane bagasse (SB) were oxidized with an H3PO4-NaNO2 mixture to obtain adsorbent materials with high contents of carboxylic groups. The oxidation reactions of Cel and SB were optimized using design of experiments (DOE) and response surface methodology (RSM). The optimized synthesis conditions yielded Cox and SBox with 4.8mmol/g and 4.5mmol/g of carboxylic acid groups, respectively. Cox and SBox were characterized by FTIR, TGA, PZC and solid-state 13C NMR. The adsorption of the model cationic dyes crystal violet (CV) and auramine-O (AO) on Cox and SBox in aqueous solution was investigated as a function of the solution pH, the contact time and the initial dye concentration. The adsorption of CV and AO on Cox was described by the Elovich equation and the pseudo-first-order kinetic model respectively, while the adsorption of CV and AO on SBox was described by the pseudo-second-order kinetic model. Adsorption isotherms were well fitted by the Langmuir and Konda models, with maximum adsorption capacities (Qmax) of 1117.8mg/g of CV and 1223.3mg/g of AO on Cox and 1018.2mg/g of CV and 682.8mg/g of AO on SBox. Desorption efficiencies were in the range of 50-52% and re-adsorption capacities varied from 65 to 81%, showing the possibility of reuse of both adsorbent materials.
Subject(s)
Benzophenoneidum/chemistry , Benzophenoneidum/isolation & purification , Cellulose/chemistry , Gentian Violet/chemistry , Gentian Violet/isolation & purification , Saccharum/chemistry , Adsorption , Anti-Infective Agents/chemistry , Anti-Infective Agents/isolation & purification , Coloring Agents/chemistry , Coloring Agents/isolation & purification , Hydrogen-Ion Concentration , Kinetics , Water Pollutants, Chemical/chemistry , Water Pollutants, Chemical/isolation & purificationABSTRACT
Present work reports the synthesis of semi-Interpenetrating Network Polymer (semi-IPN) using Gelatin-Gum xanthan hybrid backbone and polyvinyl alcohol in presence of l-tartaric acid and ammonium persulphate as the crosslinker-initiator system. Reaction parameters were optimized with Response Surface Methodology (RSM) in order to maximize the percent gel fraction of the synthesized sample. Polyvinyl alcohol, l-Tartaric acid, ammonium persulphate, reaction temperature, time and pH of the reaction medium were found to make an impact on the percentage gel fraction obtained. Incorporation of polyvinyl alcohol chains onto hybrid backbone and crosslinking between the different polymer chains were confirmed through techniques like FTIR, SEM-EDX and XRD. Semi-IPN was found to be very efficient in the removal of cationic dyes rhodamine-B (70%) and auramine-O (63%) from a mixture with an adsorbent dose of 700 mg, initial concentration of rhodamine-B 6 mgL-1 and auramine-O 26 mgL-1, at an time interval of 22-25 h and 30 °C temp. Further to determine the nature of adsorption Langmuir and Freundlich adsorption isotherm models were studied and it was found that Langmuir adsorption isotherm was the best fit model for the removal of mixture of dyes. Kinetic studies for the sorption of dyes favored the reaction mechanism to occur via a pseudo second order pathway with R2 value about 0.99.
Subject(s)
Coloring Agents/isolation & purification , Polymers/chemistry , Textile Industry , Waste Disposal, Fluid/methods , Adsorption , Benzophenoneidum/chemistry , Benzophenoneidum/isolation & purification , Coloring Agents/chemistry , Gelatin/chemistry , Hydrogen-Ion Concentration , Kinetics , Polymers/chemical synthesis , Polysaccharides, Bacterial/chemistry , Polyvinyl Alcohol/chemistry , Rhodamines/chemistry , Rhodamines/isolation & purification , Spectroscopy, Fourier Transform Infrared , Tartrates/chemistry , Temperature , Water Pollutants, Chemical/chemistry , Water Pollutants, Chemical/isolation & purification , X-Ray DiffractionABSTRACT
Designing extrinsic fluorescence sensors for amyloid fibrils is a very active and important area of research. Recently, an ultrafast molecule rotor dye, Auramine O (AuO), has been projected as a fluorescent amyloid marker. It has been claimed that AuO scores better than the most extensively utilized gold-standard amyloid probe, Thioflavin-T (ThT). This advantage arises from the fact that AuO, in addition to its usual emission band (â¼500 nm), also displays a large red-shifted emission band (â¼560 nm), exclusively in the presence of human insulin fibril medium and not in the native protein or buffer media. On the contrary, for ThT, the emission maximum (â¼490 nm) largely remains unchanged while going from protein to fibril. This otherwise unknown large red-shifted emission band of AuO, observed in the presence of human insulin fibrils, was tentatively attributed to a species formed upon fast proton dissociation from excited AuO. It was proposed that because of the long excited-state lifetime (â¼1.8 ns) of AuO upon association with human insulin fibrils, this fast proton dissociation from excited AuO could be observed, which is otherwise not observed in buffer or native protein media, owing to its very short excited-state lifetime (â¼1 ps). Herein, we show that despite the long excited-state lifetime of AuO in other fibrillar media (human serum albumin and lysozyme), the new red-shifted emission band at 560 nm is not observed, thus possibly suggesting a different origin of the red-shifted emission band of AuO in human insulin fibril medium. We convincingly show that this red-shifted band of AuO (â¼560 nm) could be observed under conditions that promote dye aggregation, such as a premicellar concentration of surfactants and polyelectrolytes. These AuO aggregates display strong emission wavelength dependence of transient decay traces, similar to that for AuO in human insulin fibril medium. Detailed time-resolved emission spectral (TRES) measurements suggest that the AuO/premicellar surfactant and AuO/human insulin fibril system share similar features, such as a dynamic red-shift in TRES and an isoemissive point in the time-resolved area-normalized emission spectra, suggesting that the characteristic red-shifted emission band of AuO in human insulin fibril medium may arise from AuO aggregates.
Subject(s)
Amyloid/chemistry , Artifacts , Benzophenoneidum/chemistry , Fluorescent Dyes/chemistry , Insulins/chemistry , Staining and Labeling/methods , Acrylic Resins/chemistry , Benzothiazoles , Humans , Kinetics , Muramidase/chemistry , Polymers/chemistry , Protein Aggregates , Protons , Serum Albumin, Human/chemistry , Sodium Dodecyl Sulfate/chemistry , Sulfonic Acids/chemistry , Thiazoles/chemistryABSTRACT
Recently, Auramine O (AuO) has been projected as a fluorescent fibril sensor, and it has been claimed that AuO has an advantage over the most extensively utilized fibril marker, Thioflavin-T (ThT), owing to the presence of an additional large red-shifted emission band for AuO, which was observed exclusively for AuO in the presence of fibrillar media and not in protein or buffer media. As fibrils are very rich in ß-sheet structure, a fibril sensor should be more specific toward the ß-sheet structure so as to produce a large contrast between the fibril form and native protein form, for efficient detection and in vitro mechanistic studies of fibrillation. However, in this report, we show that AuO interacts significantly with the native form of bovine serum albumin (BSA), which is an all-α-helical protein and lacks the ß-sheet structure, which are the hallmarks of a fibrillar structure. This strong interaction of AuO with the native form of BSA leads to a large emission enhancement of AuO for the native protein itself, and leads to a low contrast between the BSA protein and its fibrils. More importantly, the large red-shifted emission band of AuO, reported in the presence of human insulin fibrils, and which was projected as its major advantage over ThT, is not observed in the presence of BSA fibrils as well as fibrils from other proteins, such as lysozyme, human serum albumin, and ß-lactoglobulin. Thus, our results provide information on the universal applicability of the distinctive and claimed-to-be-advantageous photophysical features reported for AuO in human insulin fibrils towards fibrils from other proteins. Time-resolved fluorescence measurements also support the proposition of a strong interaction of AuO with native BSA. Additionally, tryptophan emission of the protein has been explored to further elucidate the binding mechanism of AuO with native BSA. Evaluation of thermodynamic parameters revealed that the binding of AuO with native BSA involved positive enthalpy and entropy changes, suggesting dominant contributions from hydrophobic and electrostatic interactions toward the association of AuO with native BSA. Molecular docking calculations have been performed to identify the principal binding location of AuO in native BSA.
Subject(s)
Benzophenoneidum/metabolism , Serum Albumin, Bovine/metabolism , Animals , Benzophenoneidum/chemistry , Benzothiazoles , Binding Sites , Cattle , Circular Dichroism , Humans , Hydrophobic and Hydrophilic Interactions , Insulin/chemistry , Insulin/metabolism , Molecular Docking Simulation , Protein Binding , Protein Structure, Tertiary , Serum Albumin, Bovine/chemistry , Static Electricity , Temperature , Thermodynamics , Thiazoles/chemistry , Thiazoles/metabolismABSTRACT
A sensitive and selective assay of DNA is developed by utilizing a signal transduction strategy with the rational redesign of the hairpin structured G-quadruplex molecular switch (G4-MS) assembled using auramine O (AO). By monitoring the changes of the fluorescent signal, we could identify and further quantitatively determine the target DNA in the samples.
Subject(s)
Benzophenoneidum/chemistry , DNA/analysis , Fluorescent Dyes/chemistry , G-Quadruplexes , Nucleic Acid Hybridization , Humans , Molecular StructureABSTRACT
The simultaneous and competitive ultrasound-assisted removal of Auramine-O (AO), Erythrosine (Er) and Methylene Blue (MB) from aqueous solutions were rapidly performed onto copper-doped zinc sulfide nanoparticles loaded on activated carbon (ZnS:Cu-NP-AC). ZnS:Cu nanoparticles were studied by FESEM, XRD and TEM. First, the effect of pH was optimized in a one-at-a-time procedure. Then the dependency of dyes removal percentage in their ternary solution on the level and magnitude of variables such as sonication time, initial dyes concentrations and adsorbent dosage was fully investigated and optimized by central composite design (CCD) under response surface methodology (RSM) as well as by regarding desirability function (DF) as a good and general criterion. The good agreement found between experimental and predicted values supports and confirms the suitability of the present model to predict adsorption state. The applied ultrasound strongly enhanced mass transfer process and subsequently performance. Hence, a small amount of the adsorbent (0.04 g) was capable to remove high percentage of dyes, i.e. 100%, 99.6% and 100% for MB, AO and Er, respectively, in very short time (2.5 min). The experimental equilibrium data fitting to Langmuir, Freundlich, Temkin and Dubinin-Radushkevich models showed that the Langmuir model applies well for the evaluation and description of the actual behavior of adsorption. The small amount of proposed adsorbent (0.015 g) was applicable for successful removal of dyes (RE>99.0%) in short time (2.5 min) with high adsorption capacity in single component system (123.5 mg g(-1) for MB, 123 mg g(-1) for AO and 84.5 mg g(-1) for Er). Kinetics evaluation of experiments at various time intervals reveals that adsorption processes can be well predicated and fitted by pseudo-second-order and Elovich models.
Subject(s)
Charcoal/chemistry , Coloring Agents/chemistry , Copper/chemistry , Nanoparticles/chemistry , Sulfides/chemistry , Ultrasonics/methods , Zinc Compounds/chemistry , Adsorption , Analysis of Variance , Benzophenoneidum/chemistry , Erythrosine/chemistry , Hydrogen-Ion Concentration , Kinetics , Methylene Blue/chemistry , Nanoparticles/ultrastructure , TemperatureABSTRACT
SETTING: Tuberculosis control projects, Damien Foundation Bangladesh. OBJECTIVES: To compare transmitted fluorescence (Olympus CX21™/FRAEN FluoLED™) with epi-fluorescence (Zeiss Primostar iLED™) light-emitting diode microscopes (LED-FM) and various auramine staining and destaining/counterstaining techniques for the detection of acid-fast bacilli. DESIGN: Multicentre blinded reading of routine smears on both types of microscopes using different staining techniques in multiple phases. LED-FM rechecking of discordant series with and without restaining to calculate operating characteristics. RESULTS: Among 64 874 smears, both instruments detected 9.6% positives. Compared to the standard technique, the stronger auramine-O formulation did not perform better. Thiazine red counterstaining tended to yield more false-positive as well as false-negative errors. Combined destaining/counterstaining (sensitivity 93%, positive predictive value [PPV] 98%) proved significantly less effective. Both destaining with 1% hydrochloric acid (HCl) and 10% alcohol and the standard 0.5% HCl and 70-95% alcohol were equally accurate (sensitivity 95-96%, PPV 99%). The sturdiness of the microscopes in field conditions was sub-optimal: only 5/16 instruments did not break down. CONCLUSIONS: Both microscopes performed equally well. The standard staining technique is as good as the more complicated and expensive variations. A destaining solution containing only 10% alcohol works perfectly well. The inferior quality of LED-FM microscope components is an obstacle to FM expansion.
Subject(s)
Microscopy, Fluorescence/methods , Sputum/microbiology , Tuberculosis/diagnosis , Benzophenoneidum/chemistry , False Negative Reactions , False Positive Reactions , Organic Chemicals/chemistry , Predictive Value of Tests , Reproducibility of Results , Sensitivity and Specificity , Staining and LabelingABSTRACT
Although conjugation is well known as an important contributor to color, there is scant recognition concerning involvement of imine and iminium functions in the physiological effects of this class of dyes and pigments. The group includes the dyes methylene blue, rhodamine, malachite green, fuchsin, crystal violet, auramine and cyanins, in addition to the pigments consisting of pyocyanine, phthalocyanine and pheophytin. The physiological effects consist of both toxicity and beneficial aspects. The unifying theme of electron transfer-reactive oxygen species-oxidative stress is used as the rationale in both cases. Toxicity is frequently prevented or alleviated by antioxidants. The apparent dichotomy of methylene blue action as both oxidant and antioxidant is rationalized based on similar previous cases. This mechanistic approach may have practical benefit. This review is important in conveying, for the first time, a unifying mechanism for toxicity based on electron transfer-reactive oxygen species-oxidative stress arising from imine-iminium.
Subject(s)
Coloring Agents/toxicity , Electron Transport , Imines/toxicity , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolism , Animals , Benzophenoneidum/chemistry , Benzophenoneidum/toxicity , Coloring Agents/chemistry , Disease Models, Animal , Humans , Imines/chemistry , Indoles/chemistry , Indoles/toxicity , Isoindoles , Methylene Blue/chemistry , Methylene Blue/toxicity , Pheophytins/chemistry , Pheophytins/toxicity , Pyocyanine/chemistry , Pyocyanine/toxicity , Rhodamines/chemistry , Rhodamines/toxicity , Rosaniline Dyes/chemistry , Rosaniline Dyes/toxicity , Trityl Compounds/chemistry , Trityl Compounds/toxicityABSTRACT
Both auramine-O (AuO) and thioflavin-T (ThT) behave as fluorescent molecular rotors, meaning that their (non)radiative properties are markedly affected by the intramolecular rotation of the molecule. In this article, steady-state and time-resolved fluorescence of AuO and ThT were measured in three alcohols, 1-propanol, 1-butanol, and 1-pentanol, over a wide range of temperatures (86-260 K). These solvents are glass-forming liquids, and their viscosity and dielectric relaxation time increase by more than 10 orders of magnitude as the temperature is lowered from room temperature to ~100 K. Accordingly, the fluorescence nonradiative rates constants of AuO and ThT in these solvents decrease by about 3 orders of magnitude at the latter temperature range. We found very good correspondence between the temperature dependence of the nonradiative rate constant, k(nr), of both molecules and the dielectric relaxation rate of the solvents. The k(nr) values of AuO are twice those of ThT along the whole temperature range. The temperature dependence of k(nr) is consistent with the nonradiative model suggested by Glasbeek and co-workers.