Fluorescence Spectroscopic Approach Research Articles

A highly sensitive micelle-enhanced synchronous spectrofluorimetric determination of the recently approved co-formulated drugs, bilastine and montelukast in pharmaceuticals and human plasma at nanogram levels.

In this study, the simultaneous determination of bilastine and montelukast, two recently approved co-formulated antihistaminic medications, was accomplished using a quick, sensitive, environmentally friendly, and reasonably priced synchronous fluorescence spectroscopic approach for the first time. Enhancement of the method's sensitivity down to nanogram levels was achieved by the addition of sodium dodecyl sulfate (1.0% w/v) as a micellar system. According to the results, bilastine and montelukast's fluorescence was measured at 255.3 and 355.3nm, respectively, using Δλ of 40.0nm and distilled water as a green diluting solvent. With respect to the concentration ranges of bilastine (5.0-300.0ng/ml) and montelukast (50.0-1000.0ng/ml), the method showed excellent linearity (r ≥ 0.9998). The results showed that the suggested method is highly sensitive, with detection limits of 1.42 and 13.74 ng/ml for bilastine and montelukast, respectively. Within-run precisions (intra- and interday) per cent relative standard deviations (RSD) for both analytes were <0.59%. With high percentage recoveries and low percentage RSD values, the designed approach was successfully applied for the simultaneous estimation of the cited medications in their dosage form and human plasma samples. To evaluate the green profile of the suggested method, an analytical GREENNESS metric approach (AGREE) and green analytical procedure index (GAPI) metric tools were used. These two methods for evaluating greenness confirmed that the developed method met the highest number of green requirements, recommending its use as a green substitute for the routine analysis of the studied drugs. The proposed approach was validated according to ICHQ2 (R1) guidelines.

In Vitro Characterization of the Interactions of Human Serum Albumin with Rosuvastatin and Atorvastatin Using Fluorescence Spectroscopy and Molecular Modeling

Human serum albumin (HSA) is one of the most important transporters for drugs in the systemic circulation. In this study, we investigated the interaction of rosuvastatin (ROS) and atorvastatin (ATO) with HSA. Binding of a drug molecule to HSA significantly affects the pharmacokinetics of the drug as it increases drug solubility in plasma, decreases toxicity and protects molecules from oxidation. This study was made using fluorescence spectroscopy and molecular modeling approach. Fluorescence spectra were recorded for two different statins brands at seven different concentrations. The results revealed that both statins (ROS and ATO) cause the fluorescence quenching of the HSA solution. ROS and ATO binds strongly to HSA with the binding constant (Kb) of 1.0246×106 and 0,9018×106, respectively. In addition, it was observed that high concentrations of ATO cause a shift of the emission maximum towards longer wavelengths (red-shift), which may be due to the unfolding of protein chains or denaturation. Furthermore, it was calculated that HSA possesses one binding site for ROS and ATO. Results from molecular docking showed that ROS has a higher affinity for Sudlow site I compared to Sudlow site II and the main binding forces are hydrogen bonds. ATO has nearly equal affinity for both binding sites on HSA, and the main binding forces are hydrophobic interactions.

Simple and affordable synchronous spectrofluorimetric determination of the natural anticancer polyphenols; resveratrol and curcumin in human plasma

In this work, resveratrol and curcumin, two natural polyphenols, were simultaneously determined in human plasma samples using a rapid, sensitive, green, and affordable synchronous fluorescence spectroscopic approach. Several factors affecting the performance of the procedure, including Δλ, pH, diluting solvent, and organized medium, were optimized. Based on the findings, the fluorescence of resveratrol and curcumin was measured at 304 and 443 nm, respectively, with Δλ of 80.0 nm and ethanol as the diluting solvent. Excellent linearity was demonstrated by the approach (r = 0.9999) over the concentration range of 5.00–1000.00 and 2.00–400.00 ng/mL for resveratrol and curcumin, respectively. The obtained detection limits for resveratrol and curcumin were 0.027 and 0.042 ng/mL, respectively, indicating the high sensitivity of the proposed method. Moreover, the method exhibited excellent precision (both inter and intra-day), with %RSD < 1 %. The “green analytical process index” and “Analytical GREEnness” metric tools were used to compare the green profiles of the proposed method to those of the published methods. These two greenness evaluation tools verified that the suggested methodology satisfied the greatest number of green criteria, proposing its usage as a green alternative for the routine analysis of the investigated natural anticancer polyphenols in human plasma.

Naphthaldehyde-Based Schiff Base Chemosensor for the Dual Sensing of Cu2+ and Ni2+ Ions.

In this study, a simple Schiff base sensor 1-(((4-nitrophenyl)imino)methyl)naphthalen-2-ol(NNM) has been used for chemosensing of metal ions. The metal sensing properties of sensor NNM have been investigated using UV-visible and fluorescence spectroscopic approaches. The spectral investigations revealed a red shift in absorption spectra and quenching in the emission band of the ligand molecule in the presence of Cu2+ and Ni2+ ions. The binding stoichiometry of sensor NNM for the analyte (Cu2+ and Ni2+ ions) has been investigated by the Job's plot analysis and found to be 1:1 (NNM:Analyte). The data of the Benesi-Hildebrand plot demonstrated that NNM detected Cu2+ and Ni2+ ions in nanomolar quantity. The binding insights among NNM and analytes (Cu2+ and Ni2+ ions) have been confirmed by shifted IR signals. Moreover, the reusabilty of the sensor has been investigated using an EDTA solution. In addition, the sensor NNM also successfully applied to real water samples for the identification and measurement of Cu2+ and Ni2+ ions. Hence, this system could be highly applicable in environmental and biological applications.

Synchronous fluorescence as a green and selective method for the simultaneous determination of finasteride and tadalafil in dosage form and spiked human plasma

Finasteride and tadalafil are combined in a pharmaceutical capsules called Entadfi™, that has received FAD approval. It was indicated for the management of male benign prostatic hyperplasia-related urinary tract issues. In the current study, finasteride and tadalafil concentrations in raw form, laboratory prepared mixtures, pharmaceutical preparation and spiked human plasma were all quantitatively estimated using a sensitive synchronized fluorescence spectroscopic approach united with first derivative. When excited at 260 nm, finasteride display its emission at 320 nm. Yet, when excited at 280 nm, tadalafil displayed its emission at 340 nm. The application of micellar surfactant as sodium dodecyl sulphate (SDS) significantly increased the fluorescence intensity.The overlapping of the fluorescence spectra was entirely eliminated by derivatizing the synchronous spectra to the first derivative, which also made it possible to simultaneously quantify the cited drugs. Without interfering with one another, the first-order synchronous spectra of tadalafil and finasteride at 320 and 330 nm, respectively. The approach revealed linearity alongside an acceptable correlation coefficient for finasteride and tadalafil concentrations over the range of 10 –50 ng/mL. That approach was utilized to estimation of the cited drugs in dosage forms, simultaneously with %recoveries for tadalafil and finasteride of 99.62 ± 0.78 and 100.19 ± 0.60, respectively. Also, four various tools, the national environmental method index, the AGREE evaluation method, the green analytical procedure index and the analytical eco-scale were used to evaluate how environmentally friendly the given approach was. With regard to the metrics of the greenness aspects, the proposed approach appeared to be better than the previously published spectrophotometric methods and HPLC.

Peptide Models of the Cytoplasmic Tail of Influenza A/H1N1 Virus Hemagglutinin Expand Understanding its pH-Dependent Modes of Interaction with Matrix Protein M1.

Influenza A virus hemagglutinin (HA) is a major virus antigen. No cryo-electron microscopy or X-ray data can be obtained for the HA intraviral (cytoplasmic) domain (CT) post-translationally modified with long fatty acid residues bound to three highly conserved cysteines. We recently proposed a model of HA CT of Influenza A/H1N1 virus possessing an antiparallel beta structure based on the experimental secondary structure analysis of four 14-15 amino acid long synthetic peptides, corresponding to the HA CT sequence, with free or acetaminomethylated cysteines. To dispel doubts about possible non-specific "amyloid-like" aggregation of those synthetic peptides in phosphate buffer solution, we have determined the order of oligomers based on blue native gel electrophoresis, membrane filtration, fluorescence spectroscopy and molecular modeling approaches. We have found that unmodified peptides form only low molecular weight oligomers, while modified peptides form both oligomers of low order similar to those found for unmodified peptides and high order conglomerates, which however are not of beta-amyloid-like fold. This study confirms that the beta structure previously detected by circular dichroism spectroscopy analysis is more likely the result of intrinsic propensity of the HA CT amino acid sequence than the consequence of aggregation. The structures of low order oligomers of the synthetic peptides were used for in silico experiments on modeling of HA CT interactions with matrix protein M1 at physiological and acidic pH levels and revealed two different areas of binding. Finally, tripeptides capable of blocking interactions between HA CT and M1 were proposed.

Ternary Cobalt (II)-Metformin-Glycine/Histidine/Proline Complexes: Multispectroscopic DNA, HSA, and BSA Interaction and Cytotoxicity Studies.

The synthesized water-soluble ternary complexes [Co(met)(gly)(Cl)2] (1), [Co(met)(hist)(Cl)2] (2), and [Co(met)(pro)(Cl)2] (3), (met = metformin, gly = glycine, hist = histidine, and pro = proline) were evaluated using spectro-analytical techniques, and the stereochemistry of the complexes was determined to be octahedral. UV-Vis absorption, competitive DNA-binding experiments using ethidium bromide (EB) by fluorescence, fluorescence emission studies, viscosity studies, and gel electrophoresis techniques were all employed to explore the binding characteristics of the cobalt (II) complexes with CT-DNA and groove-binding mechanism established. The salt-dependent association of the complexes to CT-DNA was investigated using UV-Vis spectrophotometric analysis. The association of the cobalt (II) complexes with BSA and HSA was explored by utilizing UV-Vis absorption and fluorescence spectroscopy approaches. The findings show that the complexes exhibit adequate capacity to quench BSA and HSA fluorescence and that the binding response is mostly a static quenching mechanism. The cytotoxicity of the complexes has also been appraised with the human breast adenocarcinoma cell lines (MCF-7) and (MDA-MB-231) by utilizing the MTT assay. For each cell line, the IC50 values were computed. In both cell lines, all the complexes were active.

An eco-friendly matrix-augmented fluorescence spectroscopic approach for the analysis of mitoxantrone, an oncogenic therapy; application to the dosage form and biological matrices.

Mitoxantrone (MXN) is a synthetic anthracenedione oncogenic therapy. It is often prescribed as an anticancer agent to manage a variety of cancers. A green, fast, and easy fluorimetric technique for the assay of MXN as a topoisomerase type II enzyme suppressor. An investigation of MXN's fluorescence behavior in various media and solvents constituted the basis for this new technique. Methanol was shown to enhance the intrinsic fluorescence considerably. After excitation at 610 nm, the highest fluorescence intensity was found at 675 nm. Various experimental parameters, such as media, solvents, and pH levels, were tested and adjusted. ICH (International Conference on Harmonization) guidelines were followed when validating procedures. It was possible to achieve linearity in the 0.02-1.50 μg ml-1 with the method. The sensitivity (in terms of limit of detection and limit of quantification) was 0.003 and 0.008 μg ml-1 , indicating low toxicity. As a result, the current technology has a remarkable recovery for detecting residues in diverse bodily fluids. Also, the quantum yield was estimated for the designed system. Finally, the method was rated by eco-scale scoring.

Probing the binding mechanism of the verbascoside and human serum albumin by fluorescence spectroscopy and molecular docking approach

Probing the binding mechanism of the verbascoside and human serum albumin by fluorescence spectroscopy and molecular docking approach

Electrophysiology and Fluorescence Spectroscopy Approaches for Evaluating Gamete and Embryo Functionality in Animals and Humans.

This review has examined two of the techniques most used by our research group for evaluating gamete and embryo functionality in animal species, ranging from marine invertebrates to humans. Electrophysiology has given access to fundamental information on some mechanisms underpinning the biology of reproduction. This technique demonstrates the involvement of ion channels in multiple physiological mechanisms, the achievement of homeostasis conditions, and the triggering of profound metabolic modifications, often functioning as amplification signals of cellular communication. Fluorescence spectrometry using fluorescent probes to mark specific cell structures allows detailed information to be obtained on the functional characteristics of the cell populations examined. The simple and rapid execution of this methodology allowed us to establish a panel helpful in elucidating functional features in living cells in a simultaneous and multi-parameter way in order to acquire overall drafting of gamete and embryo functionality.

Plasmonics for advance single-molecule fluorescence spectroscopy and imaging in biology

The elucidation of complex biological processes often requires monitoring the dynamics and spatial organization of multiple distinct proteins organized on the sub-micron scale. This length scale is well below the diffraction limit of light, and as such not accessible by classical optical techniques. Further, the high molecular concentrations found in living cells, typically in the micro- to mili-molar range, preclude single-molecule detection in confocal volumes, essential to quantify affinity constants and protein-protein reaction rates in their physiological environment. To push the boundaries of the current state of the art in single-molecule fluorescence imaging and spectroscopy, plasmonic materials offer encouraging perspectives. From thin metallic films to complex nano-antenna structures, the near-field electromagnetic coupling between the electronic transitions of single emitters and plasmon resonances can be exploited to expand the toolbox of single-molecule based fluorescence imaging and spectroscopy approaches. Here, we review two of the most current and promising approaches to study biological processes with unattainable level of detail. On one side, we discuss how the reduction of the fluorescence lifetime of a molecule as it approaches a thin metallic film can be exploited to decode axial information with nanoscale precision. On the other, we review how the tremendous progress on the design of plasmonic antennas that can amplify and confine optical fields at the nanoscale, powered a revolution in fluorescence correlation spectroscopy. Besides method development, we also focus in describing the most interesting biological application of both technologies.

Multi-spectroscopic, thermodynamic and molecular docking studies to investigate the interaction of eplerenone with human serum albumin.

The binding of small molecular drugs with human serum albumin (HSA) has a crucial influence on their pharmacokinetics. The binding interaction between the antihypertensive eplerenone (EPL) and HSA was investigated using multi-spectroscopic techniques for the first time. These techniques include ultraviolet-visible (UV-vis) spectroscopy, Fourier-transform infrared (FTIR), native fluorescence spectroscopy, synchronous fluorescence spectroscopy and molecular docking approach. The fluorescence spectroscopic study showed that EPL quenched HSA inherent fluorescence. The mechanism for quenching of HSA by EPL has been determined to be static in nature and confirmed by UV absorption and fluorescence spectroscopy. The modified Stern-Volmer equation was used to estimate the binding constant (Kb ) as well as the number of bindings (n). The results indicated that the binding occurs at a single site (Kb = 2.238 × 103 L mol-1 at 298 K). The enthalpy and entropy changes (∆H and ∆S) were 58.061 and 0.258 K J mol-1 , respectively, illustrating that the principal intermolecular interactions stabilizing the EPL-HSA system are hydrophobic forces. Synchronous fluorescence spectroscopy revealed that EPL binding to HSA occurred around the tyrosine (Tyr) residue and this agreed with the molecular docking study. The Förster resonance energy transfer (FRET) analysis confirmed the static quenching mechanism. The esterase enzyme activity of HSA was also evaluated showing its decrease in the presence of EPL. Furthermore, docking analysis and site-specific markers experiment revealed that EPL binds with HSA at subdomain IB (site III).

Addition of Mercury Causes Quenching of NIR Fluorescence Emission Spectra of a Photoactivatable PAiRFP1 Protein.

Biliverdin (BV) containing far-red light photoactivatable near-infrared fluorescent protein (NIR-FP) named PAiRFP1 has been developed by directed molecular evolution from one bathy bacteriophytochrome of Agrobacterium tumefaciens C58 called Agp2 or AtBphP2. Usually, the fluorescence intensity of the NIR emission spectra of PAiRFP1 tends to increase upon repeated excitation by far-red light. This study aimed at exploring the role of PAiRFP1 and its mutants, such as V386A, V480A, and Y498H, as NIR biosensors for the detection of Hg2+ ions in the buffer solutions. In this study, we used PCR-based site-directed reverse mutagenesis, fluorescence spectroscopy, and molecular modeling approaches on PAiRFP1 and its mutants. It was found that PAiRFP1 and its mutants experienced strong quenching of NIR fluorescence emission spectra upon the addition of different concentrations (0-3μM) of mercuric chloride (HgCl2). We hypothesized that PAiRFP1 and its variants have some potential to be used as NIR biosensors for the in vitro detection of Hg2+ ions in biological media. Moreover, we also hypothesized that PAiRFP1 would be the best tool to use as a NIR biosensor to detect Hg2+ ions in living organisms because of its higher signal-to-noise (SNR) ratio than other infra-red fluorescent proteins.

Investigating the binding mechanism of topiramate with bovine serum albumin using spectroscopic and computational methods.

Various spectroscopic techniques involving fluorescence spectroscopy, circular dichroism (CD), and computational approaches were used to elucidate the molecular aspects of interaction between the antiepileptic drug topiramate and the multifunctional transport protein bovine serum albumin (BSA) under physiological conditions. Topiramate quenched BSA fluorescence in a static quenching mode, according to the Stern-Volmer quenching constant (Ksv ) data derived from fluorescence spectroscopy for the topiramate-BSA complex. The binding constant was also used to calculate the binding affinity for the topiramate-BSA interaction. Fluorescence and circular dichroism experiments demonstrate that the protein's tertiary structure is affected by the microenvironmental alterations generated by topiramate binding to BSA. To establish the exact binding site, interacting residues, and interaction forces involved in the binding of topiramate to BSA, molecular modeling and simulation approaches were used. According to the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) calculations, the average binding energy between topiramate and BSA is -421.05 kJ/mol. Topiramate was discovered to have substantial interactions with BSA, changing the structural dynamic and Gibbs free energy landscape patterns.

Advanced microscope studies of the cell polarization instructing activity of Ras proteins

Ras gene mutations are detected in around 30% of human cancers. These mutations have been linked with polarity disruption in epithelial cells, a hallmark for cancer. Still, the cell depolarization mechanism remains unknown. Here, we investigate polarized cells by means of advanced fluorescence microscopy and spectroscopy approaches, such as superresolution STED microscopy, fluorescence correlation spectroscopy, and spectral imaging, to explore the differences between apical and basal membranes regarding lipid organization and Ras-lipid interactions.

Three-dimensional excitation-emission matrix (EEM) fluorescence approach to probing the binding interactions of polystyrene microplastics to bisphenol A

Microplastics (MPs) are readily to bind with contaminants, hence becoming the mobile composite pollutions with the migration of MPs in the environment. However, the binding mechanisms between MPs and various contaminants are still unclear due to the scarcity of investigation methods. In this work, three-dimensional excitation-emission matrix fluorescence spectroscopy approach is developed to probe the binding interactions between polystyrene MPs and a typical hydrophobic comtaminant bisphenol A (BPA). The binding affinity and thermodynamic parameters of the binding interactions are accurately and rapidly determined. Roles of environmental conditions in the binding interactions of MPs and BPA are discriminated. Results show that the binding strength of MPs to BPA is relatively higher under neutral (logK=4.96, pH 7) and weak acidic (logK=4.63, pH 6) conditions. The binding strength significantly grows with the increasing temperature from 4 to 35 °C. The binding process is barely influenced by ionic strength, but being promoted in the presence of divalent ions (Ca2+ and Mg2+). The binding process of MPs to BPA is solely driven by entropy. Under neutral condition, hydrophobic forces dominate the binding interactions between MPs and BPA, with the assistance of weak hydrogen bonds. The predominant hydrophobic interactions are weakened under acidic conditions, resulting in lower binding strength. Under alkaline conditions, both hydrophobic forces and hydrogen bonds are weakened, while electrostatic repulsions are magnified, leading to the obviously decreased binding strength. The results benefit to a better understanding of the composition pollutions of MPs and hydrophobic pollutants in aquatic environments.

Surfactant flooding for enhanced oil recovery – Insights on oil solubilization through optical spectroscopic approach

Surfactant flooding is a proven method employed to improve the recovery of crude oil from subsurface reservoirs but the knowledge about microemulsion formation, factors related to their stability, and the trapping mechanism of crude oil components in a microemulsion is still not clear for designing of injection fluid for specific crude type. The interaction mechanisms of four crude oils with surfactant-brine solutions were studied in this work for better insight into the molecular level mechanisms during surfactant flooding. UV–visible absorption and fluorescence spectra of the separated maltene and asphaltene fractions indicate the structural difference of the components in the molecular level, particularly, the size of the fused aromatic rings (FAR) present within individual bulk crude sample. Moreover, the FTIR spectra of the separated asphaltenes from the crude samples clearly showed the subtle difference in the functional groups present in respective FAR structures. Well-characterized compositionally different crude oils were mixed with four different surfactant-brine solutions (anionic – SDS, AOS, cationic - CTAB, and neutral - TX-100) and examined using UV–visible absorption and fluorescence spectroscopic approach. Detailed analysis of the acquired absorption and fluorescence spectra of the crude oil-surfactant-brine solutions distinctly demonstrate the entrapment of different crude oil components within the micelles formed by surfactants of different charges while the synchronous fluorescence spectra show the entrapment of specific size FARs within the specific micellar microenvironment. The results exhibit that the anionic surfactant entraps the smaller size (2–4) FARs whereas cationic and neutral surfactants entrap small as well as medium size (2–6) FARs within the formed micelles in the microemulsions. The present spectroscopic study signifies the importance of molecular-level interaction mechanisms for customized designing of surfactant solutions for maximizing crude oil recovery from petroleum reservoirs.

Hidden intermediates in Mango III RNA aptamer folding revealed by pressure perturbation

Fluorescent RNA aptamers have the potential to enable routine quantitation and localization of RNA molecules and serve as models for understanding biologically active aptamers. In recent years, several fluorescent aptamers have been selected and modified to improve their properties, revealing that small changes to the RNA or the ligands can modify significantly their fluorescent properties. Although structural biology approaches have revealed the bound, ground state of several fluorescent aptamers, characterization of low-abundance, excited states in these systems is crucial to understanding their folding pathways. Here we use pressure as an alternative variable to probe the suboptimal states of the Mango III aptamer with both fluorescence and NMR spectroscopy approaches. At moderate KCl concentrations, increasing pressure disrupted the G-quadruplex structure of the Mango III RNA and led to an intermediate with lower fluorescence. These observations indicate the existence of suboptimal RNA structural states that still bind the TO1-biotin fluorophore and moderately enhance fluorescence. At higher KCl concentration as well, the intermediate fluorescence state was populated at high pressure, but the G-quadruplex remained stable at high pressure, supporting the notion of parallel folding and/or binding pathways. These results demonstrate the usefulness of pressure for characterizing RNA folding intermediates.

Monitoring the binding and insertion of a single transmembrane protein by an insertase

Cells employ highly conserved families of insertases and translocases to insert and fold proteins into membranes. How insertases insert and fold membrane proteins is not fully known. To investigate how the bacterial insertase YidC facilitates this process, we here combine single-molecule force spectroscopy and fluorescence spectroscopy approaches, and molecular dynamics simulations. We observe that within 2 ms, the cytoplasmic α-helical hairpin of YidC binds the polypeptide of the membrane protein Pf3 at high conformational variability and kinetic stability. Within 52 ms, YidC strengthens its binding to the substrate and uses the cytoplasmic α-helical hairpin domain and hydrophilic groove to transfer Pf3 to the membrane-inserted, folded state. In this inserted state, Pf3 exposes low conformational variability such as typical for transmembrane α-helical proteins. The presence of YidC homologues in all domains of life gives our mechanistic insight into insertase-mediated membrane protein binding and insertion general relevance for membrane protein biogenesis.

Comment on "Emissive H-Aggregates of an Ultrafast Molecular Rotor: A Promising Platform for Sensing Heparin".

For heparin sensing, Mudliar and Singh developed fluorescence and absorption spectroscopic approaches by utilizing emissive H-aggregates of thioflavin T (ThT) formed upon heparin binding. It has been proposed that the methods work not only in pure aqueous solution but also in complex biological media such as human serum. However, the optical features used to detect and quantify heparin are very sensitive to the ionic strength of the solution and completely vanish at 1.45 mM NaCl. Curiously, the authors were able to determine the heparin content of 1% serum samples containing the same level of electrolyte. In addition, the experimental conditions employed for heparin detection in serum samples were substantially modified, reducing the optical path length from 1 to 0.1 cm and increasing the dye concentration by an unknown measure. ThT shows a concentration-dependent tendency for aqueous aggregation, which markedly modifies its absorption and fluorescence properties. The authors have failed to verify that spectral characteristics of the ThT-heparin system observed in pure aqueous medium remain unchanged at higher dye concentrations and in the presence of serum components. Taking these issues into consideration, the heparin detection scheme offered for serum samples cannot be reproduced.