Nm Of Probe Research Articles

Near-infrared absorption and emission probes with optimal connection bridges for live monitoring of NAD(P)H dynamics in living systems

Two NAD(P)H-biosensing probes consisting of 1,3,3-trimethyl-3H-indolium and 3-quinolinium acceptors, linked by thiophene, A, and 3,4-ethylenedioxythiophene, B, bridges are detailed. We synthesized probes C and D, replacing the thiophene connection in probe A with phenyl and 2,1,3-benzothiadiazole units, respectively. Probe E was prepared by substituting probe A's 3-quinolinium unit with a 1-methylquinoxalin-1-ium unit. Probe solutions are non-fluorescent but in the presence of NADH, exhibit near-infrared fluorescence at 742.1 nm and 727.2 nm for probes A and B, respectively, and generate absorbance signals at 690.6 nm and 685.9 nm. In contrast, probes C and D displayed pronounced interference from NADH fluorescence at 450 nm, whereas probe E exhibited minimal fluorescence alterations in response to NAD(P)H. Pre-treatment of A549 cells with glucose in the presence of probe A led to a significant increase in fluorescence intensity. Additionally, subjecting probe A to lactate and pyruvate molecules resulted in opposite changes in NAD(P)H levels, with lactate causing a substantial increase in fluorescence intensity, conversely, pyruvate resulted in a sharp decrease. Treatment of A549 cells with varying concentrations of the drugs cisplatin, gemcitabine, and camptothecin (5, 10, and 20 µM) led to a concentration-dependent increase in intracellular fluorescence intensity, signifying a rise in NAD(P)H levels. Finally, fruit fly larvae were treated with different concentrations of NADH and cisplatin illustrating applicability to live organisms. The results demonstrated a direct correlation between fluorescence intensity and the concentration of NADH and cisplatin, respectively, further confirming the efficacy of probe A in sensing changes in NAD(P)H levels within a whole organism.

Ratiometric fluorescent probes based on through-bond energy transfer of cyanine donors to near-infrared hemicyanine acceptors for mitochondrial pH detection and monitoring of mitophagy.

Two ratiometric near-infrared fluorescent probes have been developed to selectively detect mitochondrial pH changes based on highly efficient through-bond energy transfer (TBET) from cyanine donors to near-infrared hemicyanine acceptors. The probes consist of identical cyanine donors connected to different hemicyanine acceptors with a spirolactam ring structure linked via a biphenyl linkage. At neutral or basic pH, the probes display only fluorescence of the cyanine donors when they are excited at 520 nm. However, acidic pH conditions trigger spirolactam ring opening, leading to increased π-conjugation of the hemicyanine acceptors, resulting in new near-infrared fluorescence peaks at 740 nm and 780 nm for probes A and B, respectively. This results in ratiometric fluorescence responses of the probes to pH changes indicated by decreases of the donor fluorescence and increases of the acceptor fluorescence under donor excitation at 520 nm due to a highly efficient TBET from the donors to the acceptors. The probes only show cyanine donor fluorescence in alkaline-pH mitochondria. However, the probes show moderate fluorescence decreases of the cyanine donor and considerable fluorescence increases of hemicyanine acceptors during the mitophagy process induced by nutrient starvation or under drug treatment. The probes display rapid, selective, and sensitive responses to pH changes over metal ions, good membrane penetration, good photostability, large pseudo-Stokes shifts, low cytotoxicity, mitochondria-targeting, and mitophagy-tracking capabilities.

Pyridoanthrone-based chromo-fluorogenic amphiphiles for selective CN− detection and their bioimaging application

Cyanide anion (CN−) is one of the most hazardous chemicals for humans but is inevitably used in various purposes. In this study, we prepared fluorescent pyridoanthrone-based probes 1 and 3 with an N-octyl viologen pendant to detect this toxic anion. Among various anions, the presence of CN− only turned over a large fluorescence enhancement of these viologen probes, giving an LOD of 1 and 10 nM for probes 1 and 3, respectively. These probes also enabled for ‘naked-eye detection’ thanks to the generation of vivid pink color in the presence of this anion. Other probes (2, 4 and 5) with no N-octyl viologen pendant were also capable of selectively sensing CN−, but clearly inferior to probes 1 and 3 in terms of detection sensitivity. The sensitive and fast CN− detection was ascribed to the efficient addition reaction of each N-octyl viologen probe with CN−, leading to the generation of a highly fluorescent hydrolyzed product, isolated as a pyridoanthrone amine derivative. Due to the presence of hydrophobic and aromatic-aromatic interactions as well as H-bonding between probe molecules, the probes organized into self-assemblies with the positive surfaces in aqueous solution, allowing fast addition reactions between the probe and CN− to produce color and fluorescence emission. Probe 1 proved effective at detecting CN− content in HeLa cells and could be used as a solid state for inexpensive and convenient detection of CN−. The current system was fully studied by various analytical methods such as UV–vis, fluorescence, and 1H NMR spectroscopy, DLS, FE-SEM images, TEM, DFT calculations, HRMS and X-ray crystallography.

Fluorescent probes with high pKa values based on traditional, near-infrared rhodamine, and hemicyanine fluorophores for sensitive detection of lysosomal pH variations.

Sterically hindered fluorescent probes (A-C) have been developed by introducing 2-aminophenylboronic acid pinacol ester to a traditional, A, a near-infrared rhodamine dye, B, and a near-infrared hemicyanine dye, C, forming closed spirolactam ring structures. Probe A was non-fluorescent under basic pH conditions whereas probes B and C were moderately fluorescent with fluorescence quantum yields of 9% and 5% in pH 7.4 PBS buffer containing 1% ethanol, respectively. With all probes increasing acidity leads to significant increases in fluorescence at 580 nm, 644 and 744 nm for probes A, B and C with fluorescence quantum yields of 26%, 21% and 10% in pH 4.5 PBS buffer containing 1% ethanol, respectively. Probes A, B and C were calculated to have pKa values of 5.81, 5.45 and 6.97. The difference in fluorescence under basic conditions is ascribed to easier opening of the closed spirolactam ring configurations due to significant steric hindrance between the 2-aminophenylboronic acid pinacol ester residue and an adjacent H atom in the xanthene derivative moiety in probe B or C. The probes show fast, reversible, selective and sensitive fluorescence responses to pH changes, and are capable of sensing lysosomal pH variations in living cells.

Fluorescent probes based on π-conjugation modulation between hemicyanine and coumarin moieties for ratiometric detection of pH changes in live cells with visible and near-infrared channels

We report two ratiometric fluorescent probes based on π-conjugation modulation between coumarin and hemicyanine moieties for sensitive ratiometric detection of pH alterations in live cells by monitoring visible and near-infrared fluorescence changes. In a π-conjugation modulation strategy, a coumarin dye was conjugated to a near-infrared hemicyanine dye via a vinyl connection while lysosome-targeting morpholine ligand and o-phenylenediamine residue were introduced to the hemicyanine moieties to form closed spirolactam ring structures in probes A and B, respectively. The probes show only visible fluorescence of the coumarin moiety under physiological and basic conditions because the hemicyanine moieties retain their closed spirolactam ring structures. However, decrease of pH to acidic condition causes spirolactam ring opening, and significantly enhances π-conjugation within the probes, thus generating new near-infrared fluorescence peaks of the hemicyanine at 755 nm and 740 nm for probes A and B, respectively. Moreover, the probes display ratiometric fluorescence response to pH with decreases of the coumarin fluorescence and increases of the hemicyanine fluorescence when pH changes from 7.4 to 2.5. The probes are fully capable of imaging pH changes in live cells with good ratiometric responses in visible and near-infrared channels, and effectively avoid fluorescence blind spots under neutral and basic pH conditions – an issue that typical intensity-based pH fluorescent probes run into. The probe design platform reported herein can be easily applied to prepare a variety of ratiometric fluorescent probes for detection of biological thiols, metal ions, reactive oxygen and nitrogen species by introducing appropriate functional groups to hemicyanine moiety.

A CuO-functionalized NMOF probe with a tunable excitation wavelength for selective detection and imaging of H2S in living cells.

Recently, fluorescent nanoscale metal-organic frameworks (NMOFs) have been proven to be useful probes for the detection and imaging of active biomolecules in living cells. However, the excitation wavelengths of these NMOF fluorescence probes are mostly in the ultraviolet region, which unavoidably results in reduced cell activity, limited tissue penetration depth and inevitable biological background interference. Herein, to solve this problem, a CuO functionalized NMOF probe with a tunable excitation wavelength based on Förster resonance energy transfer (FRET) for selective detection and imaging of the third important gaseous signaling molecule hydrogen sulfide (H2S) in living cells as an example is presented. In the energy transfer system, NMOF confines the luminophore organic dye thiazole orange within its intrinsic porous matrix as the energy donor, in which the excitation wavelength of the NMOF can be tuned simply from UV to Vis through the choice of dye molecules, and the H2S-responding site copper oxide nanoparticle (CuO NP) is the acceptor. After the surface functionalization of CuO NPs onto the NMOF, the fluorescence of the NMOF can be efficiently quenched based on the FRET. When H2S appeared, the fluorescence of the nanoprobe is recovered due to the interruption of FRET. This facile yet powerful strategy not only provides an instantaneous fluorescence probe for selective H2S detection in living cells but also offers a valuable approach for using porous NMOFs to sense other biological species.

Electrochemical Sensor for Lead Cation Sensitized with a DNA Functionalized Porphyrinic Metal-Organic Framework.

An efficient electrochemical sensor was presented for lead cation detection using a DNA functionalized iron-porphyrinic metal-organic framework (GR-5/(Fe-P)n-MOF) as a probe. The newly designed probe showed both the recognition behavior of GR-5 to Pb(2+) with high selectivity and the excellent mimic peroxidase performance of (Fe-P)n-MOF. In the presence of Pb(2+), GR-5 could be specifically cleaved at the ribonucleotide (rA) site, which produced the short (Fe-P)n-MOF-linked oligonucleotide fragment to hybridize with hairpin DNA immobilized on the surface of screen-printed carbon electrode (SPCE). Because of the mimic peroxidase property of (Fe-P)n-MOF, enzymatically amplified electrochemical signal was obtained to offer the sensitive detection of Pb(2+) ranging from 0.05 to 200 nM with a detection limit of 0.034 nM. In addition, benefiting from the Pb(2+)-dependent GR-5, the proposed assay could selectively detect Pb(2+) in the presence of other metal ions. The SPCE based electrochemical sensor along with the GR-5/(Fe-P)n-MOF probe exhibited the advantages of low-cost, simple fabrication, high sensitivity and selectivity, providing potential application of on-site and real-time Pb(2+) detection in complex media.

Teth137, a Conserved Factor of Unknown Function from Thermoanaerobacter ethanolicus JW200, Represses the Transcription of the adhE Gene In Vitro

Teth137, a 13.7kD protein of unknown function from Thermoanaerobacter ethanolicus JW200, is encoded by 360 nucleotides and has been obtained by DNA-coupled column previously. However, no function study of Teth137 has been published. Homologous modeling of Teth137 shows the protein is comprised of a helix-turn-helix motif which is a typical DNA-binding domain. Therefore, it is speculated Teth137 is a DNA-binding protein and involved in transcription of the adhE gene (encodes alcohol dehydrogenase E). To investigate the function of Teth137, recombinant Teth137 is overexpressed in Escherichia coli JM109 and purified by DEAE column. Purified Teth137 exhibits the affinity with the adhE promoter region in gel electrophoresis mobility shift assay (GEMSA). Teth137 at the concentration of 48μM retards the migration of 5nM of probe in the presence of the competitor DNA. Mutant analysis indicates that S69, T70, P71 and T72 are critical to protein-DNA interface; Gly substitutions at these residues results in the loss of the binding ability with the adhE promoter region. Moreover, T. ethanolicus JW200 RNA polymerase, σ subunit and template plasmid are prepared for in vitro transcription assay to detect the regulation function of Teth137. The results of the in vitro transcription show that the transcription of 5nM of the template plasmid is inhibited by 48μM of Teth137.

Prevalence of Seasonal Influenza Viruses and Pandemic H1N1 Virus in Beijing from 2008 to 2012

Prevalence of Seasonal Influenza Viruses and Pandemic H1N1 Virus in Beijing from 2008 to 2012

Disruption of an SP2/KLF6 Repression Complex by SHP Is Required for Farnesoid X Receptor-induced Endothelial Cell Migration

The farnesoid X receptor (FXR) signaling pathway regulates bile acid and cholesterol homeostasis. Here, we demonstrate, using a variety of gain- and loss-of-function approaches, a role of FXR in the process of cell motility, which involves the small heterodimeric partner (SHP)-dependent up-regulation of matrix metalloproteinase-9. We use this observation to reveal a transcriptional regulatory mechanism involving the SP/KLF transcription factors, SP2 and KLF6. Small interference RNA-based silencing studies in combination with promoter, gel shift, and chromatin immunoprecipitation assays indicate that SP2 and KLF6 bind to the matrix metalloproteinase-9 promoter and together function to maintain this gene in a silenced state. However, upon activation of FXR, SHP interacts with SP2 and KLF6, disrupting the SP2/KLF6 repressor complex. Thus, together, these studies identify a mechanism for antagonizing Sp/KLF protein repression function via SHP, with this process regulating endothelial cell motility.