A Highly Selective Economical Sensor for 4-Nitrophenol

Herein, an inexpensive commercially available sensor is presented for the detection of 4-nitrophenol (4NP) pollutant. Sodium fluorescein (NaFl) is used as a sensor to detect trace amounts of 4NP in acetonitrile (MeCN). The photophysical properties of NaFl were studied in two different solvents, MeCN (aprotic) and water (protic), with varying concentrations of different nitroaromatics using UV-visible absorption and fluorescence spectrophotometry. In an aqueous medium, photophysical properties of NaFl did not change in the presence of nitroaromatics. However, examination of the photodynamics in MeCN demonstrated that NaFl is extremely sensitive to 4NP (limit of detection: 0.29 µg/mL). This extreme specificity of NaFl towards 4NP when dissolved in MeCN, as compared to other nitroaromatics, is attributed to hydrogen bonding of 4NP with NaFl in the absence of water, resulting in both static and dynamic quenching processes. Thus, NaFl is demonstrated as a simple, inexpensive, sensitive, and robust optical turn off sensor for 4NP.

Ligand modulation of the conformational dynamics of the A2A adenosine receptor revealed by single-molecule fluorescence

G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 2296.31 on TM6 was measured using fluorescence correlation spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150–350 ns), intermediate (50–60 μs) and slow (200–300 μs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y2887.53, and Y2907.55) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 2296.31 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

Do equilibrium and rate constants of intramicellar reactions depend on micelle size ?

We have studied the combined static and dynamic quenching of pyrene by methyl viologen in sodium alkyl sulfate micelles varying in volume by a factor of more than 4. Size...

Ligand Modulation of the Conformational Dynamics of the A2A Adenosine Receptor Revealed by Single-Molecule Fluorescence

ABSTRACTG protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra-and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A Receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 229 on TM6 was measured using Fluorescence Correlation Spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150-350 ns), intermediate (50-60 μs) and slow (200-300 μs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y288, and Y290) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 229 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

Spectral Properties of Laser Dyes at varying Temperature

Spectral properties of laser dyes 4-methyl-7-(4-morpholinyl)-2 H pyrano [2,3-b] pyridin-2-one (LD – 425) and 6,7,8,9-tetrahydro-6,8,9-trimethyl-4-(trifluoromrthyl)-2H-pyrano[2,3b][1,8]naphthyridin-2-one (LD – 489) have been investigated in the temperature range 250C - 650C by steady state and transient methods. Fluorescence intensity decreases with increase in temperature with fluorescence band maxima shifted towards shorter wavelength in both the dyes. Further, fluorescence lifetime has decreased with increase in temperature for LD - 425, whereas remained constant with change in temperature for LD - 489. The possible deactivation mechanisms are discussed. The quenching effect of these dyes in the presence of aniline at different temperature has been studied and the Stern-Volmer (S-V) plots are non-linear showing positive deviation. It has been observed that dynamic quenching constant increases with temperature, whereas static quenching constant is independent of temperature.

Investigations on sensing of picric acid in aqueous medium via fluorescence quenching of quinine sulfate

This research describes systematic investigations on sensing of high explosives such as picric acid (PA), RDX, NTO, and trinitrotoluene (TNT) in aqueous medium via fluorescence quenching of quinine sulfate (QS). Although all the explosives exhibit fluorescence quenching of QS, highest response is observed for PA. Fluorescence quenching of [Formula: see text][Formula: see text]50% (in contrast to pristine QS) at [Formula: see text][Formula: see text]390 nm is observed for 10 nm (2.29 [Formula: see text]g of PA dissolved in 20 [Formula: see text]l of distilled water). The analysis of the Stern–Volmer (SV) plot implies dominance of static quenching mechanism in comparison to dynamic quenching mechanism. Furthermore, the effect of operational temperature on fluoresce quenching response for PA has been investigated, and values of enthalpy, entropy, and Gibbs free energy of interaction at various temperatures are estimated. The temperature-dependent studies reveal that fluorescence quenching is due to formation of strong hydrogen bonds, complemented by computational analysis.

Multimode binding and stimuli responsive displacement of acridine orange dye complexed with p-sulfonatocalix[4/6]arene macrocycles

Pictorial presentation of the different aspects as displayed by the AOH+–SCXn systems in regard to multi-mode binding, dynamic quenching and stimuli responsive fluorescence “turn ON”, demonstrating very rich supramolecular photochemistry.

Absolutely “off–on” fluorescent CD-based nanotheranostics for tumor intracellular real-time imaging and pH-triggered DOX delivery

Absolutely “off–on” fluorescent CDs-based nanotheranostics was designed for the tumor intracellular real-time imaging and pH-triggered DOX delivery, via both static quenching and dynamic quenching mechanisms.

Human plasma dynamically quenches the fluorescein at the initial point of Oxygen Radical Absorption Capacity (ORAC) Assay

Objective: Oxygen Radical Absorbance Capacity (ORAC) assay measures the quenching of fluorescent probe by peroxyl radicals. Antioxidants present in biological systems block the quenching of fluorescence probe. We experienced dynamic quenching of fluorescein in ORAC assay by human plasma while plasma ORAC assay was optimized. Therefore, for the first time, we report the quenching of fluorescein by plasma at the initial point of ORAC assay. Results: Aqueous whole and non-protein fractions of plasma were used in the analysis. Since both fractions showed a similar pattern of quenching at the initial stage, quenched percentage of fluorescein was calculated and added to each sample in subsequent analysis. Addition of extra 20% fluorescein allowed plasma samples to quench the required amount of fluorescein and follow normal decay curves afterwards. Further, change of fluorescein quenching (ΔF/F 0 ) disclosed a dose dependent linear relationship with plasma (R 2 =0.8). It can be speculated that dynamic quenching exhibited by human plasma biomolecule/s at the initial stage would be of non-protein aqueous phase molecule/s. We suggest initiating further studies to detect, identify and quantify the fluorescein quenching biomolecules present in human plasma for further improvements in plasma ORAC assay.