Time-resolved Fluorescence Analysis Research Articles

Altered excitation energy transfer between phycobilisome and photosystems in the absence of ApcG, a small linker peptide, in Synechocystis sp. PCC 6803, a cyanobacterium

Phycobilisome (PBS) is a large pigment-protein complex in cyanobacteria and red algae responsible for capturing sunlight and transferring its energy to photosystems (PS). Spectroscopic and structural properties of various PBSs have been widely studied, however, the nature of so-called complex-complex interactions between PBS and PSs remains much less explored. In this work, we have investigated the function of a newly identified PBS linker protein, ApcG, some domain of which, together with a loop region (PB-loop in ApcE), is possibly located near the PBS-PS interface. Using Synechocystis sp. PCC 6803, we generated an ApcG deletion mutant and probed its deletion effect on the energetic coupling between PBS and photosystems. Steady-state and time-resolved spectroscopic characterization of the purified ΔApcG-PBS demonstrated that ApcG removal weakly affects the photophysical properties of PBS for which the spectroscopic properties of terminal energy emitters are comparable to those of PBS from wild-type strain. However, analysis of fluorescence decay imaging datasets reveals that ApcG deletion induces disruptions within the allophycocyanin (APC) core, resulting in the emergence (splitting) of two spectrally diverse subgroups with some short-lived APC. Profound spectroscopic changes of the whole ΔApcG mutant cell, however, emerge during state transition, a dynamic process of light scheme adaptation. The mutant cells in State I show a substantial increase in PBS-related fluorescence. On the other hand, global analysis of time-resolved fluorescence demonstrates that in general ApcG deletion does not alter or inhibit state transitions interpreted in terms of the changes of the PSII and PSI fluorescence emission intensity. The results revealed yet–to–be discovered mechanism of ApcG-docking induced excitation energy transfer regulation within PBS or to Photosystems.

Anomalous proton transfer of a photoacid HPTS in nonaqueous reverse micelles.

The proton transfer reaction is one of the fundamental chemical reactions where the reaction dynamics strongly depend on solvent properties such as acidity or basicity. A photoacid 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) shows a sharp decrease of pKa (7.7 → 0.5) upon photoexcitation, and the excited-state proton transfer (ESPT) occurs with ultrafast time constants of 2.5 and 89 ps in bulk aqueous solution. However, the two-step proton transfers via the contact ion pair formation and the proton diffusion are strongly limited inside the nanopools of reverse micelles (RMs). The confinement in small RMs strongly impeded the proton transfer reactions. In this work, we report the ESPT of HPTS confined in methanol-in-oil RMs by steady-state and time-resolved electronic spectroscopy. Interestingly, HPTS shows substantial deprotonation in the excited state only in small RMs, while the ESPT of HPTS does not occur in bulk methanol solution due to the low basicity of aliphatic alcohols. The kinetic analysis of time-resolved fluorescence and transient absorption measurements will compare the proton transfer dynamics of HPTS in the water-in-oil and methanol-in-oil RMs. The ESPT of photoacids, especially in the nonaqueous RMs, can be crucial in understanding many important chemical reactions involving proton transfer in the confined environments of cells and membranes.

Interaction mechanism of pea proteins with selected pyrazine flavors: Differences in alkyl numbers and flavor concentration

The mechanism of pea protein (PPI) adsorbing flavor components of cooked beef (pyrazine flavor compounds with different concentrations and different numbers of alkyl groups) was researched by spectroscopic and molecular docking approaches. The binding ability results showed that the concentration of pyrazines and the number of alkyl groups in pyrazine strongly influenced the adsorbing ability of PPI. The binding ability of PPI to the pyrazines decreased in the order 2,3,5-trimethyl-pyrazine (TRP) > 2,5-dimethyl pyrazine (DP) > 2-methylpyrazine (MP). The increase in the concentration of pyrazine flavor compounds promoted the enhancement of the adsorption capacity of PPI. Fluorescence and time-resolved fluorescence analysis showed that different molecular structures caused by the amount of alkyl groups on a pyrazine ring showed different quenching types: MP and DP were mainly subjected to static quenching, and TRP with more alkyl groups was mainly subjected to dynamic quenching. During the adsorption of pyrazine flavor compounds, the structure on the peptide bond of pea protein is destroyed. The conformation of pea protein expanded gradually, and the protein changed from ordered structure to disordered structure with lower ΔH. The interactions between pyrazine and pea protein were spontaneous from thermodynamic analysis. Combined with the results of molecular docking, hydrophobic interactions were dominant in the noncovalent interactions between MP and pea protein, whereas hydrogen bonding was dominant between DP/TRP and pea protein. TRP with more alkyl groups could generate new interactions with PPI (π-π stacking interaction) to improve the adsorption capacity of PPI. Pyrazine with more alkyl groups showed a stronger binding force with pea protein to improve the ability of pea protein to adsorb pyrazine flavor compounds, and they displayed different fluorescence quenching mechanisms. This study could provide a theoretical basis for flavor selection and control in the production of pea protein-based meat analogs and thus improve the overall flavor of plant protein-based meat analog products.

Probing Polarity and pH Sensitivity of Carbon Dots in Escherichia coli through Time-Resolved Fluorescence Analyses.

Intracellular monitoring of pH and polarity is crucial for understanding cellular processes and functions. This study employed pH- and polarity-sensitive nanomaterials such as carbon dots (CDs) for the intracellular sensing of pH, polarity, and viscosity using integrated time-resolved fluorescence anisotropy (FA) imaging (TR-FAIM) and fluorescence lifetime (FLT) imaging microscopy (FLIM), thereby enabling comprehensive characterization. The functional groups on the surface of CDs exhibit sensitivity to changes in the microenvironment, leading to variations in fluorescence intensity (FI) and FLT according to pH and polarity. The FLT of CDs in aqueous solution changed gradually from 6.38 ± 0.05 ns to 8.03 ± 0.21 ns within a pH range of 2-8. Interestingly, a complex relationship of FI and FLT was observed during measurements of CDs with decreasing polarity. However, the FA and rotational correlation time (θ) increased from 0.062 ± 0.019 to 0.112 ± 0.023 and from 0.49 ± 0.03 ns to 2.01 ± 0.27 ns, respectively. This increase in FA and θ was attributed to the higher viscosity accompanying the decrease in polarity. Furthermore, CDs were found to bind to three locations in Escherichia coli: the cell wall, inner membrane, and cytoplasm, enabling intracellular characterization using FI and FA decay imaging. FLT provided insights into cytoplasmic pH (7.67 ± 0.48), which agreed with previous works, as well as the decrease in polarity in the cell wall and inner membrane. The CD aggregation was suspected in certain areas based on FA, and the θ provided information on cytoplasmic heterogeneity due to the aggregation and/or interactions with biomolecules. The combined TR-FAIM/FLIM system allowed for simultaneous monitoring of pH and polarity changes through FLIM and viscosity variations through TR-FAIM.

Bright yellow fluorescent nitrogen and boron doped-CNPs based turn “off–on” probe for highly sensitive and selective detection of glyphosate and hypochlorite

In the present work, water-soluble fluorescent nitrogen/boron-doped carbon nano particles (N/B-CNPs) were prepared using o-phenylenediamine and boric acid precursors by a simple one step hydrothermal (HT) method. High Resolution Transmission Electron Microscope (HR-TEM) analysis showed that the N/B-CNPs are spherical in shape with an average size of ∼ 15 nm. The functional and structural composition of N/B-CNPs were confirmed by Fourier Transform Infrared (FTIR), Raman and Nuclear Magnetic Resonance (NMR) spectral studies. N/B-CNPs exhibited excellent photophysical property and photostability at different physiological conditions. N/B-CNPs showed bright yellow fluorescence at 566 nm upon excitation at 400 nm. N/B-CNPs were utilized as a fluorometric probe towards the simultaneous detection of glyphosate and hypochlorite. The fluorescence intensity of N/B-CNPs was selectively quenched in the presence of glyphosate (turn-off) and quenched emission intensity was restored in the presence of hypochlorite (turn-on). The fluorescence quenching mechanism was further ascertained using Stern-Volmer and time resolved fluorescence analysis. The detection probe showed a linear response for glyphosate over the concentrations range of 5.00 to 85.00 µM, with a limit of detection value of 0.088 µM. While in the case of hypochlorite the turn-on assay exhibited good linearity over the range of 5.00 to 50.00 µM with limit of detection as low as 0.12 nM. The N/B-CNPs turn-off/turn-on fluorometric assay exhibited excellent sensitivity and selectivity towards glyphosate and hypochlorite, even in the presence of other interference ions. The practical applicability of the proposed assay was demonstrated by fabricating a paper strip-based sensor and fluorescent thin film. Furthermore, the in vivo toxicity of the N/B-CNPs was evaluated in plant by performing dosage dependent assay.

Early detection of exon 1 huntingtin aggregation in zQ175 brains by molecular and histological approaches.

Huntingtin-lowering approaches that target huntingtin expression are a major focus for therapeutic intervention for Huntington's disease. When the cytosine, adenine and guanine repeat is expanded, the huntingtin pre-mRNA is alternatively processed to generate the full-length huntingtin and HTT1a transcripts. HTT1a encodes the aggregation-prone and highly pathogenic exon 1 huntingtin protein. In evaluating huntingtin-lowering approaches, understanding how the targeting strategy modulates levels of both transcripts and the huntingtin protein isoforms that they encode will be essential. Given the aggregation-propensity of exon 1 huntingtin, the impact of a given strategy on the levels and subcellular location of aggregated huntingtin will need to be determined. We have developed and applied sensitive molecular approaches to monitor the levels of aggregated and soluble huntingtin isoforms in tissue lysates. We have used these, in combination with immunohistochemistry, to map the appearance and accumulation of aggregated huntingtin throughout the CNS of zQ175 mice, a model of Huntington's disease frequently chosen for preclinical studies. Aggregation analyses were performed on tissues from zQ175 and wild-type mice at monthly intervals from 1 to 6 months of age. We developed three homogeneous time-resolved fluorescence assays to track the accumulation of aggregated huntingtin and showed that two of these were specific for the exon 1 huntingtin protein. Collectively, the homogeneous time-resolved fluorescence assays detected huntingtin aggregation in the 10 zQ175 CNS regions by 1-2 months of age. Immunohistochemistry with the polyclonal S830 anti-huntingtin antibody showed that nuclear huntingtin aggregation, in the form of a diffuse nuclear immunostain, could be visualized in the striatum, hippocampal CA1 region and layer IV of the somatosensory cortex by 2 months. That this diffuse nuclear immunostain represented aggregated huntingtin was confirmed by immunohistochemistry with a polyglutamine-specific antibody, which required formic acid antigen retrieval to expose its epitope. By 6 months of age, nuclear and cytoplasmic inclusions were widely distributed throughout the brain. Homogeneous time-resolved fluorescence analysis showed that the comparative levels of soluble exon 1 huntingtin between CNS regions correlated with those for huntingtin aggregation. We found that soluble exon 1 huntingtin levels decreased over the 6-month period, whilst those of soluble full-length mutant huntingtin remained unchanged, data that were confirmed for the cortex by immunoprecipitation and western blotting. These data support the hypothesis that exon 1 huntingtin initiates the aggregation process in knock-in mouse models and pave the way for a detailed analysis of huntingtin aggregation in response to huntingtin-lowering treatments.

Serum estradiol to testosterone ratio as a novel predictor of severe preeclampsia in the first trimester.

Preeclampsia (PE) is the most common medical complication during pregnancy and the second leading cause of maternal death worldwide. However, a better predictive model of PE remains to be explored. A total of 15 severe preeclampsia (sPE) and 75 healthy control patients were included in this study. Patient data was obtained from September 2019 to September 2021. Nuchal translucency (NT) and crown-rump length (CRL) of the fetus were acquired by ultrasound. Maternal blood samples were collected at 11+0 to 13+6 weeks of gestation. Chemiluminescent immunoassays were used to detect serum testosterone (T) and estradiol (E2) levels. Time-resolved fluorescence analysis was used to examine the levels of serum pregnancy-associated plasma protein A (PAPPA) and β-human chorionic gonadotrophin (β-HCG) protein. The sPE group exhibited increased T levels, and decreased E2 levels and E2/T ratios from 11 to 14weeks of gestation, compared with the control group. E2 and the E2/T ratio showed positive linear correlation with CRL in pregnant women. Body-mass-index (BMI), T, and E2 were determined to be the main factors that affected the occurrence of sPE at the 12-week gestation period time point. The receiver operating characteristic (ROC) curve revealed that the AUC of the E2/T ratio was .717. The imbalanced T and E2 levels in the patients had a specific intrinsic relevance with sPE, which suggests them as novel predictors of the sPE.

Molecular co-localization of multiple drugs in a nanoscopic delivery vehicle for potential synergistic remediation of multi-drug resistant bacteria

Anti-microbial resistant infection is predicted to be alarming in upcoming years. In the present study, we proposed co-localization of two model drugs viz., rifampicin and benzothiazole used in anti-tuberculosis and anti-fungal agents respectively in a nanoscopic cationic micelle (cetyl triethyl ammonium bromide) with hydrodynamic diameter of 2.69 nm. Sterilization effect of the co-localized micellar formulation against a model multi-drug resistant bacterial strain viz., Methicillin resistant Staphylococcus aureus was also investigated. 99.88% decrease of bacterial growth in terms of colony forming unit was observed using the developed formulation. While Dynamic Light Scattering and Forsters Resonance Energy Transfer between benzothiazole and rifampicin show co-localization of the drugs in the nanoscopic micellar environment, analysis of time-resolved fluorescence decays by Infelta-Tachiya model and the probability distribution of the donor–acceptor distance fluctuations for 5 μM,10 μM and 15 μM acceptor concentrations confirm efficacy of the co-localization. Energy transfer efficiency and the donor acceptor distance are found to be 46% and 20.9 Å respectively. We have also used a detailed computational biology framework to rationalize the sterilization effect of our indigenous formulation. It has to be noted that the drugs used in our studies are not being used for their conventional indication. Rather the co-localization of the drugs in the micellar environment shows a completely different indication of their use in the remediation of multi-drug resistant bacteria revealing the re-purposing of the drugs for potential use in hospital-born multi-drug resistant bacterial infection.

Alternative processing of human HTT mRNA with implications for Huntington's disease therapeutics.

Huntington disease is caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT) that is translated into a polyglutamine stretch in the huntingtin protein (HTT). We previously showed that HTT mRNA carrying an expanded CAG repeat was incompletely spliced to generate HTT1a, an exon 1 only transcript, which was translated to produce the highly aggregation-prone and pathogenic exon 1 HTT protein. This occurred in all knock-in mouse models of Huntington's disease and could be detected in patient cell lines and post-mortem brains. To extend these findings to a model system expressing human HTT, we took advantage of YAC128 mice that are transgenic for a yeast artificial chromosome carrying human HTT with an expanded CAG repeat. We discovered that the HTT1a transcript could be detected throughout the brains of YAC128 mice. We implemented RNAscope to visualize HTT transcripts at the single molecule level and found that full-length HTT and HTT1a were retained together in large nuclear RNA clusters, as well as being present as single transcripts in the cytoplasm. Homogeneous time-resolved fluorescence analysis demonstrated that the HTT1a transcript had been translated to produce the exon 1 HTT protein. The levels of exon 1 HTT in YAC128 mice, correlated with HTT aggregation, supportive of the hypothesis that exon 1 HTT initiates the aggregation process. Huntingtin-lowering strategies are a major focus of therapeutic development for Huntington's disease. These approaches often target full-length HTT alone and would not be expected to reduce pathogenic exon 1 HTT levels. We have established YAC128 mouse embryonic fibroblast lines and shown that, together with our QuantiGene multiplex assay, these provide an effective screening tool for agents that target HTT transcripts. The effects of current targeting strategies on nuclear RNA clusters are unknown, structures that may have a pathogenic role or alternatively could be protective by retaining HTT1a in the nucleus and preventing it from being translated. In light of recently halted antisense oligonucleotide trials, it is vital that agents targeting HTT1a are developed, and that the effects of HTT-lowering strategies on the subcellular levels of all HTT transcripts and their various HTT protein isoforms are understood.

External Quality Assessment of Maternal Serum Levels of Alpha-Fetoprotein, Free Beta-Human Chorionic Gonadotropin, and Unconjugated Estriol in Detecting Down Syndrome and Neural Tube Defects in the Second Trimester of 87 Maternal Serum Samples, Based on 105-139 Days.

BackgroundWe aimed to insure the accuracy and reproducibility of alpha-fetoprotein (AFP), free beta-human chorionic gonadotropin (free β-hCG), and unconjugated estriol (uE3) concentrations for the screening for trisomy 21 (T21) and neural tube defects (NTD) in the second trimester. We conducted an external quality assessment of 6 laboratories, using maternal serum specimens.Material/MethodsSerum specimens collected from 87 women of singleton pregnancies (4 with T21, 5 with NTD, and 78 with normal fetuses) were divided into 6 equivalent-volume fractions and transported to 6 laboratories (A, B, C, D, E, and F). All laboratories used the time-resolved fluorescence analyzer and supporting reagents to measure concentrations of AFP, free β-hCG, and uE3. The screening efficacies of T21 and NTD were compared with the certified or accredited status of the participants’ quality systems.ResultsConcentrations of AFP measured by laboratory F were low compared with those determined by the other 5 laboratories, and the differences were significant (P<0.01). There was no statistically significant difference in the free β-hCG and uE3 concentrations measured by the 6 laboratories (P>0.05). The correlation coefficients for the 3 multiples of the median values were all >0.900. The McNemar paired chi-squared test showed the differences in the positivity and detection rates were not statistically significant (P=1.000).ConclusionsAFP, freeβ-hCG, and uE3 values measured by the other 5 laboratories were comparable with those of laboratory A, with good linear correlation. When used in the maternal prenatal screening of T21 and NTD, the test results met the clinical requirements.

Bovine serum albumin functionalized blue emitting Ti3C2 MXene quantum dots as a sensitive fluorescence probe for Fe3+ ion detection and its toxicity analysis

In the present work, an improved class of protein functionalized fluorescent 2D Ti3 C2 MXene quantum dots (MXene QDs) was prepared using a hydrothermal method. Exfoliated 2D Ti3 C2 sheets were used as the starting precursor and transport protein bovine serum albumin (BSA) was used to functionalize the MXene QDs. BSA-functionalized MXene QDs exhibited excellent photophysical property and stability at various physiological parameters. High-resolution transmission electron microscopy analysis showed that the BSA@MXene QDs were quasispherical in shape with a size of ~2 nm. The fluorescence intensity of BSA@MXene QDs was selectively quenched in the presence of Fe3+ ions. The mechanism of fluorescence quenching was further substantiated using time-resolved fluorescence and Stern-Volmer analysis. The sensing assay showed a linear response within the concentration range 0-150 μM of Fe3+ ions with excellent limit of detection. BSA@MXene QDs probe showed good selectivity toward ferric ions even in the presence of other potential interferences. The practical applicability of BSA@MXene QDs was further tested in real samples for Fe3+ ion quantification and the sensor had good recovery rates. The cytotoxicity studies of the BSA@MXene QDs toward the human glioblastoma cells revealed that BSA@MXene QDs are biocompatible at lower doses and showed significant cytotoxicity at higher dosages.

Time-resolved fluorescence imaging technique for rat brain tumors analysis

The paper presents a new approach to assessing the state of tissues that differ in phenotype and in the degree of immunocompetent cells activity using photosensitizers (PS) and time-resolved fluorescence analysis methods. The main attention is paid to the detection of differences between tumor cells and tumor-associated macrophages (TAM) using spectroscopic and microscopic methods by the fluorescent kinetics signal and the difference in the accumulation of PS (the accumulation is several times greater in macrophages). The results of the PS photoluminescence study were obtained using two different techniques: time-resolved spectroscopy and time-resolved fluorescence microscopy (FLIM). Time-resolved spectroscopic analysis of the PS fluorescence lifetime was performed on adult female rats with induced C6 glioma in vivo. 5-ALA-induced Pp IX, which is widely used in clinical practice for carrying out effective conduction photodiagnostics and PDT, was used as the PS.

A Time-Resolved-Fluorescence Lateral Flow Assay for Rapid Detection of Cholyglycine Acid for the Diagnosis of Liver Diseases

Liver disease is a great danger to human health. The determination of blood level of Cholyglycine acid (CG) is a vital biomarker for the assessment of liver function in clinic, which is contribute to the diagnosis of liver diseases. Thus, establishing accuracy, rapid and convenient method for the detection of glycolic acid is of great significance. In this study, a time-resolved-fluorescence (TRF) lateral flow assay for rapid detection of CG was development. The analytical detection limit (mean of zero-2 SD) was 0.06 μg/mL The method showed good linearity in the range of 0.2–40 g/mL and was not affected by biomolecules with similar structure to CG. The analytical mean recovery of control was between 90–110% and the imprecision of intra- and inter-assay of CVs was less than 10%. No significant matrix effect was observed in saline, serum, plasma or whole blood. A good correlation was found with the homogeneous enzyme immunoassay (HEIA) assay (slopes 1.0463, y-intercepts 0.2721 μg/mL, R = 0.989, n = 50, P &lt; 0.001). The CG TRF analysis could provide reproducible and quantitative information about the state of liver in a few minutes, which is suitable for the detection of liver diseases in point-of-care-testing (POCT) conditions.

Can di-4-ANEPPDHQ reveal the structural differences between nanodiscs and liposomes?

The potential-sensitive di-4-ANEPPDHQ dye is presently gaining popularity in structural studies of the lipid bilayer. Within the bilayer, dye environmental sensitivity originates from the excitation induced charge redistribution and is usually attributed to solvent relaxation. Here, di-4-ANEPPDHQ is utilized to compare the structure of neutral and negatively charged lipid bilayers between two model systems: the nanodiscs and the liposomes. Using the well-established approach of measuring solvatochromic shifts of the steady-state spectra to study the bilayer structural changes has proved insufficient in this case. By applying an in-depth analysis of time-resolved fluorescence decays and emission spectra, we distinguished and characterized two and three distinct emissive di-4-ANEPPDHQ species in the liposomes and the nanodiscs, respectively. These emissive species were ascribed to the dual emission of the dye rather than to solvent relaxation. An additional, long-lived component present in the nanodiscs was associated with a unique domain of high order, postulated recently. Our results reveal that the di-4-ANEPPDHQ steady-state fluorescence should be interpreted with caution. With the experimental approach presented here, the di-4-ANEPPDHQ sensitivity was improved. We confirmed that the bilayer structure is, indeed, altered in the nanodiscs. Moreover, molecular dynamic simulations showed a distribution of the probe in the nanodiscs plane, which is sensitive to lipid composition. In POPC nanodiscs, probe frequently interacts with MSP, while in POPC-POPG nanodiscs, such interactions are rare. We did not observe, however, any impact of those interactions on the probe fluorescence.

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model.

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users' needs.

Electron spin contrast of high-density and perfectly aligned nitrogen-vacancy centers synthesized by chemical vapor deposition

Nitrogen-vacancy (NV) centers in diamonds provide a high-sensitivity magnetometer. We investigated the spin properties of high-density and perfectly aligned NV centers grown using the chemical vapor deposition method by varying the nitrogen concentration. Basically, the optical initialization and spin-dependent fluorescence are determined by intersystem crossing (ISC) in the excited state. In this study, we found that nitrogen impurities lead to a non-radiative and spin-mixing decay working as a competitive pathway against the ISC, using time-resolved fluorescence analysis. Our results will elucidate NV–defect interaction, which significantly contributes to the quality of the quantum material.

Highly selective “turn-on” fluorescence determination of mercury ion in food and environmental samples through novel anthracene and pyrene appended Schiff bases

Novel pyrene- and anthracene-based Schiff base derivatives (A1 and P1) were designed and prepared for selective “turn-on” fluorescence determination of Hg2+ ions in food and environmental samples. Structural characterization of novel compounds was performed by standard spectroscopic methods and photophysical properties were exanimated by UV–vis, steady-state fluorescence, time-resolved fluorescence, 3D-fluorescence and excitation-emission matrix (EEM) analysis. Investigation of binding and interaction mechanism clearly indicated that A1 and P1 can sensitively and selectively detect Hg2+ ions in real samples via inhibition of PET processes after 1:1 complex formation. In order to perform the determination of Hg2+ ions in food and environmental samples, experimental conditions such as pH, buffer, sensor concentration and measurement time were optimized. The detection limits of presented methods were determined as 12.22 nM (2.45 μg L−1) and 8.32 nM (1.67 μg L−1) in the linear range of 0.005 μM–45 μM and 0.008 μM–38 μM for A1 and P1, respectively. After validation with ICP-MS and spike/recovery test, A1 and P1 were successfully used for fluorescence analysis of Hg2+ ions in food and environmental samples. In addition, the paper-based test kit was prepared with pyrene appended Schiff base (P1) which, could be applied successfully for monitoring Hg2+ ions.

Direct energy transfer from photosystem II to photosystem I confers winter sustainability in Scots Pine

Evergreen conifers in boreal forests can survive extremely cold (freezing) temperatures during long dark winter and fully recover during summer. A phenomenon called “sustained quenching” putatively provides photoprotection and enables their survival, but its precise molecular and physiological mechanisms are not understood. To unveil them, here we have analyzed seasonal adjustment of the photosynthetic machinery of Scots pine (Pinus sylvestris) trees by monitoring multi-year changes in weather, chlorophyll fluorescence, chloroplast ultrastructure, and changes in pigment-protein composition. Analysis of Photosystem II and Photosystem I performance parameters indicate that highly dynamic structural and functional seasonal rearrangements of the photosynthetic apparatus occur. Although several mechanisms might contribute to ‘sustained quenching’ of winter/early spring pine needles, time-resolved fluorescence analysis shows that extreme down-regulation of photosystem II activity along with direct energy transfer from photosystem II to photosystem I play a major role. This mechanism is enabled by extensive thylakoid destacking allowing for the mixing of PSII with PSI complexes. These two linked phenomena play crucial roles in winter acclimation and protection.

Rapid Screening for EGFR Inhibitor in Rhei Radix et Rhizoma by HTRF Assay Coupled with HPLC Peak Fractionation.

In this paper, an HPLC peak fractionation approach combined with homogeneous time-resolved fluorescence analysis is proposed for screening epidermal growth factor receptor inhibitors from Rhei Radix et Rhizoma. With this approach, the amount of sample used for a single HPLC run is sufficient for performing a multiple assay due to the miniaturization ability of the homogeneous time-resolved fluorescence technology. This allows for improving the stability and repeatability of the activity assay for each fraction. From a total of 26 fractions collected from the Rhei Radix et Rhizoma extract, 13 fractions exhibit inhibitory activity against the epidermal growth factor receptor. The structures of activity compounds were determined by HPLC-LTQ-Orbitrap MS, revealing the presence of gallic acid, rhein, and emodin with IC50 values of 21.5, 5.29, and 10.2 µM, respectively. The ligand epidermal growth factor receptor interactions were explored by molecular docking simulations, and the inhibitory effects of the three compounds on A549 cell growth were tested in vitro by an MTT assay. This study demonstrates the suitability of the present screening method for drug discovery in natural products.