Fluorescence Polarization Measurements Research Articles

Degradation of Amyloid-β Species by Multi-Copper Oxidases.

Reduction of the production of amyloid-β (Aβ) species has been intensively investigated as potential therapeutic approaches for Alzheimer's disease (AD). However, the degradation of Aβ species, another potential beneficial approach, has been far less explored. To investigate the potential of multi-copper oxidases (MCOs) in degrading Aβ peptides and their potential benefits for AD treatment. We investigated the degradation efficiency of MCOs by using electrophoresis and validated the ceruloplasmin (CP)-Aβ interaction using total internal reflection fluorescence microscopy, fluorescence photometer, and fluorescence polarization measurement. We also investigated the therapeutic effect of ascorbate oxidase (AO) by using induced pluripotent stem (iPS) neuron cells and electrophysiological analysis with brain slices. We discovered that CP, an important MCO in human blood, could degrade Aβ peptides. We also found that other MCOs could induce Aβ degradation as well. Remarkably, we revealed that AO had the strongest degrading effect among the tested MCOs. Using iPS neuron cells, we observed that AO could rescue neuron toxicity which induced by Aβ oligomers. In addition, our electrophysiological analysis with brain slices suggested that AO could prevent an Aβ-induced deficit in synaptic transmission in the hippocampus. To the best of our knowledge, our report is the first to demonstrate that MCOs have a degrading function for peptides/proteins. Further investigations are warranted to explore the possible benefits of MCOs for future AD treatment.

The Study of the Effect of Dimethylsulfoxide (or Diethylsulfoxide) on Quinine Sulfate-DNA Binding by UV-Vis and Steady-State Fluorescence Spectroscopies.

The effect of dimethylsulfoxide (DMSO) and diethylsulfoxide (DESO) on binding between quinine sulfate (QS) and DNA was studied by virtue of UV-Vis absorption, steady-state fluorescence spectroscopies, and fluorescence polarization measurements. The binding constant was determined at three different temperatures and the values of standard Gibbs energy change, enthalpy and entropy of binding were determined. The mechanism of binding and the effect of sulfoxides on this process was revealed. The values of binding constant, fluorescence polarization and iodide quenching studies confirmed that the main binding mode in QS-DNA system is groove binding. Addition of sulfoxides does not change the binding mechanism. Moreover, with addition of sulfoxides binding constant increases due to the removal of water molecules from DNA grooves making them more available for QS molecules.To explain the effect of DMSO and DESO on QS-DNA binding the photophysical properties of QS in aqueous solutions of DMSO and DESO were also studied. On the basis of quantum yield of QS in water, DMSO and DESO the types of intermolecular interactions were discussed. The obtained results show that quantum yield of QS in sulfoxides is lower compared with that in water and aqueous solution of 0.1M H2SO4. QS forms ground state complexes with both DMSO and DESO that are stronger fluorophores compared with free QS molecules.

Reversible Control of RNA Splicing by Photoswitchable Small Molecules.

Dynamics are intrinsic to both RNA function and structure. Yet, the available means to precisely provide RNA-based processes with spatiotemporal resolution are scarce. Here, our work pioneers a reversible approach to regulate RNA splicing within primary patient-derived cells by synthetic photoswitches. Our small molecule enables conditional real-time control at mRNA and protein levels. NMR experiments, together with theoretical calculations, photochemical characterization, fluorescence polarization measurements, and living cell-based assays, confirmed light-dependent exon inclusion as well as an increase in the target functional protein. Therefore, we first demonstrated the potential of photopharmacology modulation in splicing, tweaking the current optochemical toolkit. The timeliness on the consolidation of RNA research as the driving force toward therapeutical innovation holds the promise that our approach will contribute to redrawing the vision of RNA.

Dynamics of transition dipole moment orientation in representative fluorescent proteins.

Molecules of fluorescent proteins (FPs) exhibit distinct optical directionality. This optical directionality is characterized by transition dipole moments (TDMs), and their orientation with respect to the molecular structures. Although our recent observations of FP crystals allowed us to determine the mean TDM directions with respect to the framework of representative FP molecules, the dynamics of TDM orientations within FP molecules remain to be ascertained. Here we describe the results of our investigations of the dynamics of TDM directions in the fluorescent proteins eGFP, mTurquoise2 and mCherry, through time-resolved fluorescence polarization measurements and microsecond time scale all-atom molecular dynamics (MD) simulations. The investigated FPs exhibit initial fluorescence anisotropies (r0) consistent with significant differences in the orientation of the excitation and emission TDMs. However, based on MD data, we largely attribute this observation to rapid (sub-nanosecond) fluorophore motions within the FP molecular framework. Our results allow improved determinations of orientational distributions of FP molecules by polarization microscopy, as well as more accurate interpretations of fluorescence resonance energy transfer (FRET) observations.

Label-free and Dye-free Fluorescent Sensing of Tetracyclines Using a Capture-Selected DNA Aptamer

Tetracyclines are a group of important antibiotics with a common four-ring scaffold. While most tetracyclines are currently used only in animals, their leaching into the environment and residues in food have caused health concerns. Aptamers are an attractive way to detect tetracyclines, and all previously reported aptamers for tetracyclines were obtained by immobilizing target molecules. In this work, we selected a few DNA aptamers by immobilizing the DNA library using oxytetracycline as the target. We obtained new aptamers with no overlapping sequences compared to the previously reported ones, and a representative sequence named OTC5 had a dissociation constant of 147 nM measured by isothermal titration calorimetry. Similar binding affinities were also observed with tetracycline and doxycycline. Because tetracyclines are fluorescent and their fluorescence intensity was enhanced by binding to the aptamers, a label-free and dye-free fluorescent biosensor was developed with a detection limit of 25 nM oxytetracycline. The sensor was able to detect targets in milk after extraction. Fluorescence polarization measurement showed that this aptamer is insensitive to sodium concentration but requires magnesium. Finally, a strand-displacement biosensor was designed, and it has a detection limit of 1.2 μM oxytetracycline.

Membrane Interactivity of Capsaicin Antagonized by Capsazepine.

Although the pharmacological activity of capsaicin has been explained by its specific binding to transient receptor potential vanilloid type 1, the amphiphilic structure of capsaicin may enable it to act on lipid bilayers. From a mechanistic point of view, we investigated whether capsaicin and its antagonist capsazepine interact with biomimetic membranes, and how capsazepine influences the membrane effect of capsaicin. Liposomal phospholipid membranes and neuro-mimetic membranes were prepared with 1,2-dipalmitoylphosphatidylcholine and with 1-palmitoyl-2-oleoylphosphatidylcholine and sphingomyelin plus cholesterol, respectively. These membrane preparations were subjected to reactions with capsaicin and capsazepine at 0.5–250 μM, followed by measuring fluorescence polarization to determine the membrane interactivity to modify the fluidity of membranes. Both compounds acted on 1,2-dipalmitoylphosphatidylcholine bilayers and changed membrane fluidity. Capsaicin concentration-dependently interacted with neuro-mimetic membranes to increase their fluidity at low micromolar concentrations, whereas capsazepine inversely decreased the membrane fluidity. When used in combination, capsazepine inhibited the effect of capsaicin on neuro-mimetic membranes. In addition to the direct action on transmembrane ion channels, capsaicin and capsazepine share membrane interactivity, but capsazepine is likely to competitively antagonize capsaicin’s interaction with neuro-mimetic membranes at pharmacokinetically-relevant concentrations. The structure-specific membrane interactivity may be partly responsible for the analgesic effect of capsaicin.

Acetaminophen Has Lipid Composition-Dependent Membrane Interactivity That Could Be Related to Nephrotoxicity but Not to Analgesic Activity and Hepatotoxicity

Objective: Although acetaminophen is one of the most widely used over-the-counter drugs, the mechanisms by which this classical drug exerts analgesic, hepatotoxic, and nephrotoxic effects remain unclear. We hypothesized that acetaminophen might act on cellular membranes of nerves, liver, and kidneys. In order to verify this hypothesis, we studied the interactivity of acetaminophen with biomimetic lipid bilayer membranes by comparing with structurally related phenacetin. Methods: Liposomal membranes (unilamellar vesicles suspended in the buffer of pH 7.4) were prepared with phospholipids and cholesterol to mimic the membrane lipid composition of neuronal cells, hepatocytes, and nephrocytes. They were subjected to reactions with acetaminophen and phenacetin at clinically relevant concentrations, followed by measuring fluorescence polarization to determine their membrane interactivity to modify membrane fluidity. Results: Acetaminophen and phenacetin interacted with neuro-mimetic and hepato-mimetic membranes to increase membrane fluidity at 10–100 μM. Both drugs were more effective in fluidizing hepato-mimetic membranes than neuro-mimetic membranes. Although the relative membrane-interacting potency was phenacetin >> acetaminophen in neuro-mimetic and hepato-mimetic membranes, such membrane effects conflicted with their relative analgesic and hepatotoxic effects. Acetaminophen and phenacetin strongly interacted with nephro-mimetic membranes to increase membrane fluidity at 2–100 μM and 0.1–100 μM, respectively. Phenacetin interacted significantly with nephro-mimetic membranes at lower concentrations (<2 μM) than acetaminophen, which was consistent with their relative nephrotoxic effects. Conclusion: In comparison with phenacetin, lipid composition-dependent membrane interactivity of acetaminophen could be related to nephrotoxicity but not to analgesic activity and hepatotoxicity.

The Polar Lipid Fraction E from Sulfolobus acidocaldarius Can Be Used as Liposomal Drug Stabilizing Agents to Reduce the Leakage of the Antivascular Drug Combretastatin A4 Disodium Phosphate from Tetraether/Diester Hybrid Archaeosomes

Liposomes have many advantages as therapeutic capsules over free drugs such as small molecule drugs and nucleic acids. Cholesterol is commonly used as a membrane stabilizing agent in liposomal drugs (e.g., mRNA-lipid nanoparticle COVID-19 vaccines). However, due to the vulnerability of cholesterol to oxidation and the etiological role of cholesterol in many disorders, it is desirable to find an alternative means to stabilize liposomal membranes for drug delivery. In this study, we demonstrated that the polar lipid fraction E (PLFE), which contains exclusively bipolar tetraether macrocyclic lipids, isolated from the thermoacidophilic archaeon S. acidocaldarius can greatly stabilize the liposomal formulation of the anti-vascular drug, combretastatin A4 disodium phosphate (CA4P). Stability was assessed by determining the leakage rate constant k of entrapped CA4P fluorometrically. We found that, at 37 °C, PLFE decreases the k value monotonically from 1.54 × 10−2 s−1 for 100% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) liposomes to 3.4 × 10−5 s−1 for 100% PLFE archaeosomes, a change of k by two orders of magnitude. The changes in k of CA4P leakage are correlated well with the changes in liposomal CA4P’s cytotoxicity against MCF-7 breast cancer cells. We further showed that the reduction in spontaneous leakage of entrapped CA4P by PLFE can be attributed to the increased membrane surface charge and the increased membrane order and packing tightness in liposomes, as reflected by the zeta potential (−6.83 to −41.1 mV from 0 to 100 mol% PLFE) and diphenylhexatriene (DPH) fluorescence polarization (0.13 to 0.4 from 0 to 100 mol% PLFE) measurements. Moreover, we showed that PLFE slows down CA4P leakage more than cholesterol in POPC liposomes. These results together suggest that PLFE lipids can serve as an effective stabilizing agent for liposomal drugs and could potentially be useful for the optimization of liposomal CA4P for cancer treatment.

Exciton confined states in conjugated polymers studied by spectroscopic fluorescence polarization measurements

Spectroscopic fluorescence polarization (P) measurements have been used to investigate exciton dynamics in conjugated polymers. We apply photoluminescence anisotropy to ensembles of non-interacting organic semiconductor molecules to explore exciton migration. An experimental observation shows linearly decreasing P values as the emission wavelength increases in partially oxidized poly[2-methoxy-5-(2-ethyloxy)-1,4-phenylenevinylene] (MEH-PPV) ensemble molecules. We discuss the origin of the experimental data with a computational simulation and P values for single chromophore perylene diimide dye molecules. We propose that the physical mechanism responsible for this behavior is the presence of exciton confined and blocking states at blue-shifted emission sites, which arise from excitons in partially oxidized parts of MEH-PPV.

Accurate Targeting and Controllable Release of Hybrid Liposome Containing a Stretchable Copolymer

AbstractDeveloping an intelligent drug delivery/release system, which can transport drugs to the target tissues precisely and release drugs timely, is an important challenge in chemotherapy. A multistage sensitive drug delivery system is designed by inserting a folate (FA) modified lipid and a pH/temperature dual‐sensitive amphiphilic copolymer into a liposome bilayer. The stretchable copolymer plays a role in protection on FA ligand for more accurate targeting. Then, the stretch ability of the copolymer in the liposome bilayer is verified by using the Langmuir–Blodgett film technique. The interaction between the 1,2‐dipalmitoyl‐sn‐glycerol‐3‐phosphocholine (DPPC) monolayer and hybrid liposomes is found to increase, indicating the FA ligand is exposed due to the copolymer shrinking with increasing temperature. Fluorescence polarization measurements demonstrate that the insertion of the copolymer improves the stability of the liposome and offers pH‐controllability for drug release. As a result, the drug leakage of the hybrid liposome is restrained significantly at pH 7.4, while at an acidic pH, the drug release is accelerated. The designed pH/temperature dual‐sensitive copolymer is expected to provide more precise targeting and environmentally controlled drug release to drug delivery systems based on liposomes.

Different Grp94 components interact transiently with the myocilin olfactomedin domain in vitro to enhance or retard its amyloid aggregation

The inherited form of open angle glaucoma arises due to a toxic gain-of-function intracellular misfolding event involving a mutated myocilin olfactomedin domain (OLF). Mutant myocilin is recognized by the endoplasmic reticulum (ER)-resident heat shock protein 90 paralog, glucose regulated protein 94 (Grp94), but their co-aggregation precludes mutant myocilin clearance by ER-associated degradation. When the Grp94-mutant myocilin interaction is abrogated by inhibitors or siRNA, mutant myocilin is efficiently degraded. Here we dissected Grp94 into component domains (N, NM, MC) to better understand the molecular factors governing its interaction with OLF. We show that the Grp94 N-terminal nucleotide-binding N domain is responsible for accelerating OLF aggregation in vitro. Upon inhibiting the isolated N domain pharmacologically or removing the Pre-N terminal 57 residues from full-length Grp94, OLF aggregation rates revert to those seen for OLF alone, but only pharmacological inhibition rescues co-aggregation. The Grp94-OLF interaction is below the detection limit of fluorescence polarization measurements, but chemical crosslinking paired with mass spectrometry analyses traps a reproducible interaction between OLF and the Grp94 N domain, as well as between OLF and the Grp94 M domain. The emerging molecular-level picture of quinary interactions between Grp94 and myocilin points to a role for the far N-terminal sequence of the Grp94 N domain and a cleft in the M domain. Our work further supports drug discovery efforts to inhibit these interactions as a strategy to treat myocilin-associated glaucoma.

Concentration of FAD as a marker for cervical precancer detection.

.We report the ex vivo results of an in-house fabricated portable device based on polarized fluorescence measurements in the clinical environment. This device measures the polarized fluorescence and elastic scattering spectra with 405-nm laser and white light sources, respectively. The dominating fluorophore with 405-nm excitation is flavin adenine dinucleotide (FAD) with a fluorescence peak around 510 nm. The measured spectra are highly modulated by the interplay of scattering and absorption effects. Due to this, valuable information gets masked. To reduce these effects, intrinsic fluorescence was extracted by normalizing polarized fluorescence spectra with polarized elastic scattering spectra obtained. A number of fluorophores contribute to the fluorescence spectra and need to be decoupled to understand their roles in the progression of cancer. Nelder–Mead method has been utilized to fit the spectral profile with Gaussian to decouple the different bands of contributing fluorophores (FAD and porphyrin). The change in concentration of FAD during disease progression manifests in the change in ratio of total area to FWHM of its Gaussian profile. Receiver operating characteristic (ROC) curve analysis has been used to discriminate different grades of cervical precancer by using the ratio as input parameter. The sensitivity and specificity for discrimination of normal samples from CIN I (cervical intraepithelial neoplasia) are 75% and 54%, respectively. Further, the normal samples can be discriminated from CIN II samples with 100% and 82% sensitivity and specificity, respectively, and the CIN I from CIN II samples can also be discriminated with 100% sensitivity and 90% specificity, respectively. The results show that the change in the concentration of (FAD) can be used as a marker to discriminate the different grades of the cancer and biochemical changes at an early stage of the cancer can also be monitored with this technique.

The Neuronal Tau Protein Blocks In Vitro Fibrillation of the Amyloid-β (Aβ) Peptide

Two pathophysiological characteristics in brains of Alzheimer's disease are accumulation of aggregated Amyloid-β (Aβ) peptides and hyperphosphorylated tau proteins as extracellular senile plaques and intracellular neurofibrillary tangles (NFTs), respectively. Both types of deposits develop both independently and with combined pathological reciprocal effects. The interplay between native full-length tau protein (tau-441) and Aβ40 from a molecular point of view was addressed in this study by using a combination of techniques such as transmission electron microscopy (TEM), atomic force microscopy (AFM), NMR, circular dichroism and fluorescence spectroscopy. The interaction between native, non-phosphorylated tau-441 protein and different stages of the Aβ peptide aggregation in vitro were characterized. Tau-441 protein prevents Aβ40 fibrillation in a concentration-dependent manner (C. Wallin, Y. Hiruma, S. Wärmländer, I. Huvent, J. Jarvet, J. P. Abrahams, A. Gräslund, G. Lippens, J. Luo, JACS. 2018;140(126):8138-8146), monitored by Thioflavin T (ThT) kinetics. The attenuation of Aβ40 fibrillation was confirmed with TEM imaging, as an aggregated Aβ40 sample showed typical amyloid fibrils while more amorphous structures were found for samples incubated in the presence of tau-441. To test whether tau-441 influences the Aβ40 aggregation at an early stage with monomeric Aβ40, NMR and fluorescence polarization measurements were conducted. However, native tau-441 proteins did not display any specific monomeric interaction. In contrast, tau-441 displays a two magnitudes higher affinity towards fibrils compared to monomers, suggesting reduced fibril-induced secondary nucleation processes. The decrease in Aβ40 aggregation is most likely due to an interaction with low molecular weight oligomers and fibrils rather than soluble monomeric Aβ40 peptides. The interaction between Aβ40/tau-441 and resulting prevention of the Aβ40 fibrillation may indicate an important interplay between two misfolding proteins in Alzheimer's disease.

Sensitive fluorescent polarization immunoassay by optimizing synchronization mismatch condition

Fluorescence polarization (FP) is a one of the measurement techniques to study molecular interactions. We previously developed our own FP measurement system based on synchronization detection that uses a liquid crystal layer and an image sensor. The measurement cycle was fixed to 100 without any theoretical considerations, however, the influence of the synchrony and measurement cycles for FP values should be considered. In the present paper, we carried out an experimental and theoretical investigation into the influence of the synchrony between liquid crystal operation and image sampling for FP values of our system. When there was synchronization mismatch, the experimental FP values obtained using fluorescein ethylene glycol solution and the theoretical FP values changed according to the number of measurement cycles. Additionally, we measured the FP immunoassay for a physiologically active substance under synchronization mismatch. The synchronization mismatch influenced the measurement performance of the system, indicating that optimization of the number of image samplings was necessary to improve the measurement performance. For instance, the Mismatch 0.99 case, the measurement cycle should be 50 cycle judging from its dynamic range and R-square (R2). From the investigation, we obtained theoretical and experimental knowledge on FP response to facilitate further applications of our FP system.

Mapping Various Cooking Oil using Fluorescence Polarization

In this report we have succeeded to map various cooking oils using change of fluorescence polarization. Various cooking oils consisted of several vegetables oils and animal oils (chicken oil and lard) were used in the experiment, and some oils were measured in two different times. The change of polarization angle &amp;tetha; was measured as the difference between linear polarized green pointer laser as incoming light and fluorescence light using a pair of polarizers. The direct measurement of fluorescence polarization gives a new unique result of critical polarizer’s angle φc that can group vegetable cooking oils into group 1 (at φc = 10o for VCO, olive, and soybean), group 2 (at φc = 20o for palm, corn and rice bran), group 3 (at φc = 30o for sunflower and canola), and also animals cooking oils into group 4 (at φc = 20o for chicken oil), and group 5 (at φc = 40o for lard). Mostly cooking oils can be distinguished using modified maps. The large difference φc and &amp;tetha; of lard from vegetable oils provides an advantage to develop for testing halal oil due to lard contamination. The capability of this method has benefits, at least, as a complement and simple method in comparison to other expensive sophisticated instruments such as fluorescence spectroscopy or GCMS methods with their derivation’s instruments.

Inhibition of copper-induced lipid peroxidation by sinapic acid and its derivatives in correlation to their effect on the membrane structural properties

The efficiencies of sinapic acid and its derivatives syringic acid, syringaldehyde, three sinapoyl esters (ethyl, propyl, butyl sinapates), 4-vinylsyringol and sinapine were investigated for prevention of lipid peroxidation in correlation with their interactions with model lipid membrane systems. Significant antioxidant activities of propyl and butyl sinapates were seen by fluorimetric assay in phosphatidylcholine liposomes as model membrane using C11-BODIPY581/591 lipophilic fluorescent probe. The sinapic acid esters also had the highest impact on membrane structural properties, as observed by differential scanning calorimetry and fluorescence polarisation measurements. The greatest protection of phospholipids from peroxidation by these esters correlated well with their polarity and insertion into the lipid bilayer.

2D polarization imaging as a low-cost fluorescence method to detect \u03b1-synuclein aggregation ex vivo in models of Parkinson\u2019s disease

A hallmark of Parkinson’s disease is the formation of large protein-rich aggregates in neurons, where α-synuclein is the most abundant protein. A standard approach to visualize aggregation is to fluorescently label the proteins of interest. Then, highly fluorescent regions are assumed to contain aggregated proteins. However, fluorescence brightness alone cannot discriminate micrometer-sized regions with high expression of non-aggregated proteins from regions where the proteins are aggregated on the molecular scale. Here, we demonstrate that 2-dimensional polarization imaging can discriminate between preformed non-aggregated and aggregated forms of α-synuclein, and detect increased aggregation in brain tissues of transgenic mice. This imaging method assesses homo-FRET between labels by measuring fluorescence polarization in excitation and emission simultaneously, which translates into higher contrast than fluorescence anisotropy imaging. Exploring earlier aggregation states of α-synuclein using such technically simple imaging method could lead to crucial improvements in our understanding of α-synuclein-mediated pathology in Parkinson’s Disease.

Circular orientation fluorescence emitter imaging (COFEI) of rotational motion of motor proteins

Single-molecule fluorescence polarization technique has been utilized to detect structural changes in biomolecules and intermolecular interactions. Here we developed a single-molecule fluorescence polarization measurement system, named circular orientation fluorescence emitter imaging (COFEI), in which a ring pattern of an acquired fluorescent image (COFEI image) represents an orientation of a polarization and a polarization factor. Rotation and pattern change of the COFEI image allow us to find changes in the polarization by eye and further values of the parameters of a polarization are determined by simple image analysis with high accuracy. We validated its potential applications of COFEI by three assays: 1) Detection of stepwise rotation of F1-ATPase via single quantum nanorod attached to the rotary shaft γ; 2) Visualization of binding of fluorescent ATP analog to the catalytic subunit in F1-ATPase; and 3) Association and dissociation of one head of dimeric kinesin-1 on the microtubule during its processive movement through single bifunctional fluorescent probes attached to the head. These results indicate that the COFEI provides us the advantages of the user-friendly measurement system and persuasive data presentations.

Fluorescence polarization immunoassay for rapid screening of the pesticides thiabendazole and tetraconazole in wheat.

Fluorescence polarization immunoassays (FPIAs) for thiabendazole and tetraconazole were first developed. Tracers for FPIAs of thiabendazole and tetraconazole were synthesized and the tracers' structures were confirmed by HPLC-MS/MS. The 4-aminomethylfluorescein-labeled tracers allowed achieving the best assay sensitivity and minimum reagent consumption in comparison with aminofluorescein-labeled and alkyldiaminefluoresceinthiocarbamyl-labeled tracers. Measurements of fluorescence polarization were performed using a portable device. The developed FPIA methods were applied for the analysis of wheat. Fast and simple sample preparation technique earlier developed by authors for pesticides was adapted for thiabendazole and tetraconazole. The limits of detection of thiabendazole and tetraconazole in wheat were 20 and 200μg/kg, and the lower limits of quantification were 40 and 600μg/kg, respectively. The recovery test was performed by two methods-FPIA and HPLC-MS/MS. The results obtained by FPIA correlated well with those obtained by HPLC-MS/MS (r2 = 0.9985 for thiabendazole, r2 = 0.9952 for tetraconazole). Average recoveries of thiabendazole and tetraconazole were 74 ± 4% and 72 ± 3% by FPIA, and average recoveries of thiabendazole and tetraconazole were 86 ± 2% and 74 ± 1% by HPLC-MS/MS (n = 15). Graphical abstract ᅟ.

Binding characteristics of imidazolium-based ionic liquids with calf thymus DNA: Spectroscopy studies

In this work, the interaction of 1-butyl-3-methylimidazolium methanesulfonate ([C4mim][MS]) and 1-hexyl-3-methylimidazolium methanesulfonate ([C6mim][MS]) with calf thymus deoxyribonucleic acid (ctDNA) were investigated firstly by fluorescence spectra and UV absorption spectra, respectively. Fluorescence of [Cnmim][MS] (n = 4, 6) was quenched by ctDNA through static quenching. Groove binding between [Cnmim][MS] (n = 4, 6) and ctDNA was proved by UV absorption spectra, single-stranded and double-stranded DNA studies, iodide quenching studies and fluorescence polarization measurements. Binding constants of ctDNA with [Cnmim][MS] (n = 4, 6) were calculated at different temperatures (285 K, 298 K, 310 K), which indicated that the binding force of [C6mim][MS]-ctDNA system was larger than that of [C4mim][MS]-ctDNA system, and the binding force increased with increasing IL-alkyl chain length. The thermodynamic parameter results suggested that there existed electrostatic and hydrophobic interactions between the ctDNA with [Cnmim][MS] (n = 4, 6), and the hydrophobic interaction played the major role (at the concentration of study) in the interaction process of ctDNA with [Cnmim][MS] (n = 4, 6). These results may help us to reveal the interaction mechanism of ionic liquids (ILs) with ctDNA and demonstrate the effect of alkyl chain length of the cation on the binding constant of ILs-ctDNA interaction.