Fluorescence Modification Research Articles

Evaluating the Accuracy of the COMSOL-Based Finite-Element Method for Simulating Plasmon-Modified Fluorescence.

Accurately modeling plasmon-modified fluorescence is important for understanding and guiding the design of experimental nanostructures that reliably enhance fluorescence. They are of particular interest due to their potential to allow localized "hot spots" of high fluorescence enhancement in a reproducible manner. Given the increasingly prevalent use of the COMSOL Multiphysics software package for simulating these phenomena, we investigate its accuracy using an analytically tractable model consisting of a gold nanosphere interacting with either a plane wave or a radiating point dipole. COMSOL simulation results were compared with a formally exact analytical theory. It was found that simulation parameters commonly used for plane-wave scattering do not necessarily produce accurate results for the nanoparticle-plasmon-coupled dipole emission case. Instead, user-input adaptive meshing parameters were found to be helpful in achieving quantitative agreements between COMSOL and analytical theory results for plasmon-modified fluorescence. Our studies suggest convergence to analytically calculated values when a minimum of two additional user-input mesh elements separate the point-dipole position and the nanoparticle surface. This practical insight is expected to aid in the application of COMSOL simulations to planning and interpreting fluorescence modification experiments.

A novel label-free capillary electrophoresis LED-induced fluorescence platform based on catalytic hairpin assembly for sensitive detection of multiple circulating tumor DNA.

Circulating tumor DNA (ctDNA) is a highly promising biomarker for the early diagnosis and treatment of gastric cancer (GC). However, there is still a lack of effective and practical ctDNA detection methods. In this work, a simple and economical capillary non-gel sieving electrophoresis-LED induced fluorescence detection (NGCE-LEDIF) platform coupled with catalytic hairpin assembly (CHA) as the signal amplification strategy is proposed for quantitative detection of PIK3CA E542K and TP53 (two types of ctDNA associated with GC). We have reasonably designed two pairs of programmable oligonucleotide hairpin probes for PIK3CA E542K and TP53. Using a one-pot reaction, the presence of ctDNA triggers the cyclic amplification of CHA, forming numerous thermodynamically stable H1/H2 double-strands. The H1/H2 double-stranded DNA catalyzed by PIK3CA E542K and TP53 can be easily separated by NGCE due to their different lengths, enabling simultaneous detection of both ctDNAs. Under optimal experimental conditions, the detection limits of this strategy for detecting GC-related biomarkers PIK3CA E542K and TP53 are 20.35 pM and 19.61 pM, respectively, and can achieve 730-fold signal amplification. This strategy has a good recovery in the serum matrix. The results of this study show that this strategy has significant advantages such as high selectivity, a simple process, no special instruments and equipment, no need for fluorescence modification of hairpin probes in advance, high automation, low cost, and minimal sample consumption. This provides a powerful method for the detection of trace cancer biomarkers in the serum matrix with good application prospects.

A nitrogen-rich ionic metal-organic vessel: Temperature triggered improved CO2 separation and hydrogen-bond mediated cycloaddition with pore-space-partition induced size-selective Friedel-Crafts alkylation

Herein, we built a two-fold interpenetrated, robust pillar-bilayered framework from anionic [Cd3(µ2-OH)(COO)6] unit and –NH2 hooked nitrogen-rich struts. The activated MOF shows temperature variable CO2 adsorption with minimum uptake loss during multiple cycle and under humid environment. Interestingly, CO2/N2 selectivity displays notable enhancement upon rise in temperature, reaching a value of 490 at 313 K that outperforms some best-performing adsorbents. The MOF catalyses solvent-free and mild-condition CO2 cycloaddition with wide substrate scope, good recyclability and high turnover number. Opposed to common Lewis acid-mediated reaction, pendent –NH2 sites activate the epoxide via H-bonding interaction, as validated from performance comparison of an un-functionalized MOF, and guest-mediated concomitant fluorescence articulation. These hydrogen-bond-donating (HBD) sites further benefit effective Friedel−Crafts (FC) alkylation of indole with β-nitrostyrene, where product is additionally characterized by single-crystal X-ray diffraction. The reaction includes twenty electronically assorted substrates and transpires with high catalyst reusability. The key role of HBD moiety in directing C-C coupling is detailed from correlating the performance of two isostructural MOFs without –NH2 site, judicious choice of the substrates, and fluorescence modification derived framework-electrophile interaction. Importantly, pore-space-partitioning by flanked pyridyl linker induces size-selectivity via restricting the channel diffusion of sterically encumbered indole.

Enhanced Weathering and Corrosion Resistance of Eu-Doped ZnO Solid Solution Material by Fluorescence Modification

Enhanced Weathering and Corrosion Resistance of Eu-Doped ZnO Solid Solution Material by Fluorescence Modification

Tunable ultraviolet laser induces high signal-to-noise ratio in intrinsic fluorescence titration for protein- ligand interaction studies

Protein intrinsic fluorescence emitted by tryptophan residues is widely used for macromolecular mechanism studies, such as the protein binding by ligands and enzyme kinetics. However, modifications in fluorescence intensity can be weak using the intrinsic fluorescence in many proteins containing few tryptophan residues, or with their weak involvement in the binding. A low signal to noise ratio is a main drawback, due to the weak intensity of Xenon lamp illumination in the spectrofluorometer at the ultraviolet (UV) range, in addition to the small intrinsic fluorescence modifications with ligand binding, in many cases. We report herein the montage and the utilization of a laser light source in a spectrofluorometer in the UV range for the titration of protein intrinsic fluorescence. A significantly stronger modification of the protein intrinsic fluorescence was obtained with the titrations represented by E. coli Arginyl tRNA synthetase and human 17β-hydroxysteroid dehydrogenase type 7 with their natural ligands using a wavelength-tunable UV laser source. Also, a higher signal to noise ratio was obtained with the laser source. Finally, a similar KD was obtained with the titration using the two light sources. These results are supported by the direct binding study of equilibrium dialysis, showing that the laser source yields a reliable binding analysis. Thus, the utilization of a laser source in the intrinsic fluorescence titration and related usage can give a significant opening for the wider applications of the spectroscopy and photo technology to many new proteins with limited intrinsic fluorescence signals, while their mechanisms are critical to understand biochemistry and will facilitate medical applications. This study includes fundamental, applied, and interdisciplinary areas of physics.

Functional correlation of ATP1A2 mutations with phenotypic spectrum: from pure hemiplegic migraine to its variant forms

BackgroundMutations in ATP1A2, the gene encoding the α2 subunit of Na+/K+-ATPase, are the main cause of familial hemiplegic migraine type 2 (FHM2). The clinical presentation of FHM2 with mutations in the same gene varies from pure FHM to severe forms with epilepsy and intellectual disability, but the correlation of these symptoms with different ATP1A2 mutations is still unclear.MethodsTen ATP1A2 missense mutations were selected according to different phenotypes of FHM patients. They caused pure FHM (FHM: R65W, R202Q, R593W, G762S), FHM with epilepsy (FHME: R548C, E825K, R938P), or FHM with epilepsy and intellectual disability (FHMEI: T378N, G615R, D718N). After ouabain resistance and fluorescence modification, plasmids carrying those mutations were transiently transfected into HEK293T and HeLa cells. The biochemical functions were studied including cell survival assays, membrane protein extraction, western blotting, and Na+/K+-ATPase activity tests. The electrophysiological functions of G762S, R938P, and G615R mutations were investigated in HEK293T cells using whole-cell patch-clamp. Homology modeling was performed to determine the locational distribution of ATP1A2 mutations.ResultsCompared with wild-type pumps, all mutations showed a similar level of protein expression and decreased cell viability in the presence of 1 µM ouabain, and there was no significant difference among the mutant groups. The changes in Na+/K+-ATPase activity were correlated with the severity of FHM phenotypes. In the presence of 100 µM ouabain, the Na+/K+-ATPase activity was FHM > FHME > FHMEI. The ouabain-sensitive Na+/K+-ATPase activity of each mutant was significantly lower than that of the wild-type protein, and there was no significant difference among all mutant groups. Whole-cell voltage-clamp recordings in HEK293T cells showed that the ouabain-sensitive pump currents of G615R were significantly reduced, while those of G762S and R938P were comparable to those of the wild-type strain.ConclusionsATP1A2 mutations cause phenotypes ranging from pure FHM to FHM with epilepsy and intellectual disability due to varying degrees of deficits in biochemical and electrophysiological properties of Na+/K+-ATPase. Mutations associated with intellectual disability presented with severe impairment of Na+/K+-ATPase. Whether epilepsy is accompanied, or the type of epilepsy did not seem to affect the degree of impairment of pump function.

Nanofiber fluorescence coating for evaluation of complex solid-/gas-multi-phase and nano-/micro- multi-scale nanocomposite foam structure

Dual fluorescence design based on chemical coating and physical staining was successfully applied in revealing the multi-phase and multi-scale structure simultaneously in nanocomposite foams for the first time (that is nanometer-scale solid phase fillers selectively located chemical coating fluorescence, micrometer-scale solid phase cell walls selectively located physical staining fluorescence and air phase bubbles selectively located no fluorescence). The hybrid fluorescence modification method enables us to selectively locate different fluorescence dyes on nanofiber surface and in polymer matrix. Furthermore, quantitative analysis of the three-dimensional (3D) nanofiber network structure in nanocomposite foams is studied in-depth via numerical reconstruction. The evolution of 3D nanofiller network structure in multi-phase and multi-scale nanocomposite foams is demonstrated via both experimental visualization and numerical calculation. It is found that the average shortest nanofiber distance decreases with increasing cell wall thickness and with decreasing air content. Therefore, the dual fluorescence design exploring 3D functional filler network structure in complicated multi-phase and multi-scale nanocomposite foams greatly helps researchers study the structure-performance relationship of multi-functional materials with nanoscale functional fillers.

Intracellular MicroRNA Imaging with MoS2-Supported Nonenzymatic Catassembly of DNA Hairpins.

Amplification strategies for low-level microRNA detection in living cells are pivotal for gene diagnosis and many cellular bioprocesses. In this work, we develop an amplification strategy for microRNA-21 (miRNA-21) imaging in living cells with MoS2-supported catassembly of DNA hairpins. The MoS2 nanosheet with low cytotoxicity serves as the nanocarrier and excellent fluorescence quencher, which can transfer fluorescent metastable hairpin DNA into the cells easily in a nondestructive manner and significantly reduce background signals. The three-branched catalyzed hairpin assembly (TB-CHA) probes contain three types of designed DNA molecular beacons with the modification of Cy3 in the terminal. In the presence of miRNA-21, the catalyzed hairpin assembly (CHA) reaction would be triggered and a "Y"-shaped three-branched duplex nanostructure would be formed, which would release from the surface of the MoS2 nanosheet due to the reduced affinity between the DNA duplex and MoS2 nanosheet. The multisite fluorescence modification and the circular reaction of TB-CHA probes allowed a significant fluorescence recovery in a live-cell microenvironment. The ultrasensitive detection of miRNA-21 is achieved with a detection limit of 75.6 aM, which is ∼5 orders of magnitude lower than that of a simple strand displacement-based strategy (detection limit: 8.5 pM). This method offers great opportunities for the ultrasensitive live-cell detection of miRNAs and helps in gaining a deeper understanding of the physiological functions of miRNAs in cancer research and life processes.

Fluorescence Proteomic Technology to Analyze Peripheral Blood Mononuclear Cells in Chronic Chagas Disease.

The thiol moieties of cysteinyl residues in proteins undergo a number of modifications including nitrosylation, oxidation, persulfidation, sulfenylation, and others. These protein modifications may influence gain as well as loss of function in biological and disease conditions. Herein, we describe a quantitative approach that combines accurate, sensitive fluorescence modification of cysteinyl-S-nitrosyl (SNOFlo) groups that leaves electrophoretic mobility unaffected and offers the measurement of changes in S-nitrosylation (SNO) status relative to protein abundance. This approach has been useful in evaluating the global protein abundance and SNO profile of Chagas seropositive individuals that were categorized in clinically asymptomatic (C/A) and clinically symptomatic (C/S) subgroups and compared to normal healthy (N/H) controls. Through analyzing the proteome datasets with different bioinformatics and statistics tools, potential pathologic mechanisms in disease progression are identified. We also propose a panel of protein biomarkers that have a potential to identify the infected individuals at risk of developing clinical Chagas disease.

BODIPY Peptide Labeling by Late-Stage C(sp3 )-H Activation.

Late-stage BODIPY diversification of structurally complex amino acids and peptides was accomplished by racemization-free palladium-catalyzed C(sp3 )-H activation. Transformative fluorescence modification proved viable by triazole-assisted C(sp3 )-H arylation in a chemo- and site-selective fashion, providing modular access to novel BODIPY peptide sensors.

BODIPY Peptide Labeling by Late‐Stage C(sp3)−H Activation

AbstractLate‐stage BODIPY diversification of structurally complex amino acids and peptides was accomplished by racemization‐free palladium‐catalyzed C(sp3)−H activation. Transformative fluorescence modification proved viable by triazole‐assisted C(sp3)−H arylation in a chemo‐ and site‐selective fashion, providing modular access to novel BODIPY peptide sensors.

Three-dimensional direct visualization of silica dispersion in polymer-based composites.

The uniform dispersion of silica fillers or other neutral fillers in the polymer matrix is significant for fabricating high-performance polymer-based composites. However, there is a long-standing challenge to provide a comprehensive, wide-area and real 3D distribution map to achieve direct visualization for the dispersion state of neutral silica. Herein, we propose a novel strategy for modifying silica fillers with commercial fluorophores to form fluorescent fillers in a standard manner. Through fluorescence imaging technology, we successfully observed the 2D-planar and 3D-spatial dispersion states of silica fillers in the polymer matrix. This success not only provides a visualized evaluation method for the spatial dispersion of an oxide filler, but also offers great potential in the further establishment of industrialized standards for the polymer-based composite industry.

High-Resolution Bubble Printing of Quantum Dots.

Semiconductor quantum dots (QDs) feature excellent properties, such as high quantum efficiency, tunable emission frequency, and good fluorescence stability. Incorporation of QDs into new devices relies upon high-resolution and high-throughput patterning techniques. Herein, we report a new printing technique known as bubble printing (BP), which exploits a light-generated microbubble at the interface of colloidal QD solution and a substrate to directly write QDs into arbitrary patterns. With the uniform plasmonic hot spot distribution for high bubble stability and the optimum light-scanning parameters, we have achieved full-color QD printing with submicron resolution (650 nm), high throughput (scanning rate of ∼10-2 m/s), and high adhesion of the QDs to the substrates. The printing parameters can be optimized to further control the fluorescence properties of the patterned QDs, such as emission wavelength and lifetime. The patterning of QDs on flexible substrates further demonstrates the wide applicability of this new technique. Thus, BP technique addresses the barrier of achieving a widely applicable, high-throughput and user-friendly patterning technique in the submicrometer regime, along with simultaneous fluorescence modification capability.

Protein Cysteinyl-S-Nitrosylation: Analysis and Quantification.

The thiol moiety of cysteine residues can undergo a number of biologic modifications including oxidation, sulfenylation, nitrosylation, persulfidation, metalation, and other modifications. These modifications can control biological function, including gain as well as loss of function. Herein, we focus attention on the proteomic analysis of S-nitrosylation in health and disease. We describe a novel quantitative approach that combines accurate, sensitive fluorescence modification of cysteinyl-S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo), and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. We present several studies where suitability of this approach for investigating endogenous S-nitrosylation is addressed.

Salicylaldehyde-functionalized block copolymer nano-objects: one-pot synthesis via polymerization-induced self-assembly and their simultaneous cross-linking and fluorescence modification

Salicylaldehyde-functionalized nano-objects are prepared via RAFT-mediated polymerization-induced self-assembly. Their simultaneous stabilization and fluorescence modification can be achieved by one-step reaction.

Tyrosine-Specific Chemical Modification with in Situ Hemin-Activated Luminol Derivatives

Tyrosine-specific chemical modification was achieved using in situ hemin-activated luminol derivatives. Tyrosine residues in peptide and protein were modified effectively with N-methylated luminol derivatives under oxidative conditions in the presence of hemin and H2O2. Both single and double modifications of the tyrosine residue occurred in the reaction of angiotensin II with N-methylated luminol derivative 9. Tyrosine-specific chemical modification of the model protein bovine serum albumin (BSA) revealed that the surface-exposed tyrosine residues were selectively modified with 9. We succeeded in the functionalization of several proteins using azide-conjugated compound 18 using alkyne-conjugated probes by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or dibenzocyclooctyne (DBCO)-mediated copper-free click chemistry. This tyrosine-specific modification was orthogonal to conventional lysine modification by N-hydroxysuccinimide (NHS) ester, and dual functionalization by fluorescence modification of tyrosine residues and PEG modification of lysine residues was achieved without affecting the modification efficiency.

Percolating plasmonic networks for light emission control.

Optical nanoantennas have revolutionised the way we manipulate single photons emitted by individual light sources in a nanostructured photonic environment. Complex plasmonic architectures allow for multiscale light control by shortening or stretching the light wavelength for a fixed operating frequency, meeting the size of the emitter and that of propagating modes. Here, we study self-assembled semi-continuous gold films and lithographic gold networks characterised by large local density of optical state (LDOS) fluctuations around the electrical percolation threshold, a regime where the surface is characterised by large metal clusters with fractal topology. We study the formation of plasmonic networks and their effect on light emission from embedded fluorescent probes in these systems. Through fluorescence dynamics experiments we discuss the role of global long-range interactions linked to the degree of percolation and to the network fractality, as well as the local near-field contributions coming from the local electro-magnetic fields and the topology. Our experiments indicate that local properties dominate the fluorescence modification.

Fluorescence modification of the AAAA (4A) loop: toward a probe of the structural dynamics of the i-motif of the retinoblastoma gene

Systematic modification of the 4A loop region of the Rb gene with (Py)A fluorophore units allows discrimination of the fluorescence signals corresponding to structural dynamics from single-stranded to i-motif structures.

Cellular prion protein imaging analysis with aptamer-labeled Ru(bpy) 3 2+ -doped silica nanoparticles

A new type of highly selective aptamer-labeled fluorescent silica nanoparticles [Apt-tris(2,2′-bipyridyl)ruthenium(II)@SiO2 NPs] were prepared through the reverse microemulsion method by using prolonged fluorescence lifetime ruthenium complexes of tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy) 3 2+ ) as the source of the fluorescence for cellular prion protein imaging. Investigations showed that the newly prepared Ru(bpy) 3 2+ @SiO2 NPs possessed superior advantages of strong fluorescence, low toxicity, and easy surface modification for bioconjugation. Cell imaging experiments indicated that Apt-Ru(bpy) 3 2+ @SiO2 NPs had great tendency to human bone marrow neuroblastoma cells (SK-N-SH cells), since they can express large amount of prion protein on the surface of the cell, while in HeLa cells this phenomenon disappeared for the reason that HeLa cells cannot express prion proteins.

Molecular recognition of a model globular protein apomyoglobin by synthetic receptor cyclodextrin: effect of fluorescence modification of the protein and cavity size of the receptor in the interaction

Labelling of proteins with some extrinsic probe is unavoidable in molecular biology research. Particularly, spectroscopic studies in the optical region require fluorescence modification of native proteins by attaching polycyclic aromatic fluoroprobe with the proteins under investigation. Our present study aims to address the consequence of the attachment of a fluoroprobe at the protein surface in the molecular recognition of the protein by selectively small model receptor. A spectroscopic study involving apomyoglobin (Apo-Mb) and cyclodextrin (CyD) of various cavity sizes as model globular protein and synthetic receptors, respectively, using steady-state and picosecond-resolved techniques, is detailed here. A study involving Förster resonance energy transfer, between intrinsic amino acid tryptophan (donor) and N, N-dimethyl naphthalene moiety of the extrinsic dansyl probes at the surface of Apo-Mb, precisely monitor changes in donor acceptor distance as a consequence of interaction of the protein with CyD having different cavity sizes (β and γ variety). Molecular modelling studies on the interaction of tryptophan and dansyl probe with β-CyD is reported here and found to be consistent with the experimental observations. In order to investigate structural aspects of the interacting protein, we have used circular dichroism spectroscopy. Temperature-dependent circular dichroism studies explore the change in the secondary structure of Apo-Mb in association with CyD, before and after fluorescence modification of the protein. Overall, the study well exemplifies approaches to protein recognition by CyD as a synthetic receptor and offers a cautionary note on the use of hydrophobic fluorescent labels for proteins in biochemical studies involving recognition of molecules.