Sensitive Fluorescence Emission Research Articles

A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization

Diabetic retinopathy, retinopathy of prematurity and retinal vein occlusion are potentially blinding conditions largely due to their respective neovascular components. The development of real-time in vivo molecular imaging methods, to assess levels of retinal neovascularization (NV), would greatly benefit patients afflicted with these conditions. mRNA hybridization techniques offer a potential method to image retinal NV. The success of these techniques hinges on the selection of a target mRNA whose tissue levels and spatial expression patterns correlate closely with disease burden. Using a model of oxygen-induced retinopathy (OIR), we previously observed dramatic increases in retinal endoglin that localized to neovascular structures (NV), directly correlating with levels of neovascular pathology. Based on these findings, we have investigated Endoglin mRNA as a potential marker for imaging retinal NV in OIR mice. Also of critical importance, is the application of innovative technologies capable of detecting mRNAs in living systems with high sensitivity and specificity. To detect and visualize endoglin mRNA in OIR mice, we have designed and synthesized a novel imaging probe composed of short-hairpin anti-sense (AS) endoglin RNA coupled to a fluorophore and black hole quencher (AS-Eng shRNA). This assembly allows highly sensitive fluorescence emission upon hybridization of the AS-Eng shRNA to cellular endoglin mRNA. The AS-Eng shRNA is further conjugated to a diacyl-lipid (AS-Eng shRNA–lipid referred to as probe). The lipid moiety binds to serum albumin facilitating enhanced systemic circulation of the probe. OIR mice received intraperitoneal injections of AS-Eng shRNA–lipid. Ex vivo imaging of their retinas revealed specific endoglin mRNA dependent fluorescence superimposed on neovascular structures. Room air mice receiving AS-Eng shRNA–lipid and OIR mice receiving a non-sense control probe showed little fluorescence activity. In addition, we found that cells in neovascular lesions labelled with endoglin mRNA dependent fluorescence, co-labelled with the macrophage/microglia-associated marker IBA1. Others have shown that cells expressing macrophage/microglia markers associate with retinal neovascular structures in proportion to disease burden. Hence we propose that our probe may be used to image and to estimate the levels of retinal neovascular disease in real-time in living systems.

Encryption and authentication of security patterns by ecofriendly multi-color photoluminescent inks containing oxazolidine-functionalized nanoparticles

Counterfeiting of confidential documents has been a costly challenge for banks, companies, and customers. Encryption of invisible security marks, such as barcodes, quick response codes, and logos, in national or international confidential documents by high-security anticounterfeiting inks is the most significant solution for counterfeiting problems. Ecofriendly multi-color photoluminescent anticounterfeiting inks based on highly-fluorescent polymer nanoparticles functionalized with new oxazolidine derivatives were developed for the fast and facile encryption of security labels on cellulosic documents, such as paper currency, passport, and certificate. Depending on the polarity of functionalized polymer nanoparticles, a wide range of colors and fluorescence emissions were observed as a result of polar-polar interactions between the oxazolidine molecules and surface functional groups of the nanoparticles. The fluorescent polymer nanoparticles showed spherical, vesicular, and cauliflower-like morphologies resulted from different surface functional groups. Functional polymer nanoparticles displayed high stability and printability on cellulosic substrates due to hydrogen bonding interactions. The highly-fluorescent polymer nanoparticles were also used to prepare anticounterfeiting inks with different colors and fluorescence emissions. All the ecofriendly polymeric anticounterfeiting inks were loaded to stamps with specific marks, and then applied to different confidential documents. Printed labels displayed highly intense fluorescence emission in different colors (green, orange, pink, and purple depending on the matrix polarity) under UV irradiation (365 nm). These water-based multi-color fluorescent anticounterfeiting inks with highly intense, bright, and sensitive fluorescence emission have potential applications in encryption and authentication of security patterns.

A dansyl fluorescent pH probe with wide responsive range in aqueous solution.

A fluorescent pH probe based on diphenylalanine-modified dansyl (FF-Dns) was designed and synthesized. The probe showed sensitive fluorescence emission to pH change over a wide range of 1-13 in aqueous solution. At pH 4-10, FF-Dns displayed the characteristic fluorescent emission of dansyl group (yellow); while protonation of the dimethylamino group caused a very weak fluorescence emission at pH 1-4, and deprotonation of the sulfonamide group caused an emission blue-shift to the green region at pH 11-13. These properties endow FF-Dns with the potential to detect pH variation in physiological environments.

A novel fluorescent probe for highly sensitive and selective detection of cysteine and its application in cell imaging

A new fluorescent probe (ABPA) based on 1,8-naphthalimide, which designed on the “receptor1-fluorophore-spacer-receptor2” model and employed conjugated addition/cyclization sequence mechanism for Cys detection was reported. The probe shows high selectivity and sensitivity for cysteine (Cys) over other amino acids including the similar structured homocysteine (Hcy) and glutathione (GSH). It displays a highly sensitive fluorescence emission to Cys with a detection limit of 0.12μM (S/N=3), which is far below the normal level of Cys (30–200μM) in living cells. Furthermore, ABPA was successfully applied to bioimage intracellular Cys in living cells with low cytotoxicity.

Multiple Stimuli-Responsive Fluorescence Behavior of Novel Polyamic Acid Bearing Oligoaniline, Triphenylamine, and Fluorene Groups.

Multiple stimuli-responsive fluorescent materials have gained increasing attention for their fundamental investigation and intelligent applications. In this work, we report design and synthesis of a novel polyamic acid bearing oligoaniline, triphenylamine, and fluorene groups, which served as sensitive units and fluorescence emission unit, respectively. The resulting polymer exhibits multiple stimuli-responsive fluorescence switching behavior triggered by redox species, pH, electrochemical, and pressure stimuli. Every fluorescence switching mechanism upon each stimulus was studied in detail. The interactions and energy transfer between sensitive units and emission unit are largely responsible for this fascinating fluorescent switching behavior. This work provides a deep understanding of the optical switching essence upon these stimuli, opening the way for the development of new fluorescent sensing applications.

Ultrasensitive and Rapid Determination of Folic Acid Using Ag Nanoparticles Enhanced 1, 10-Phenantroline-Terbium (III) Sensitized Fluorescence.

A novel spectrofluorimetric probe based on Ag nanoparticle (AgNPs)-enhanced terbium (III) (Tb) fluorescence was introduced for the sensitive determination of folic acid (FA). The effect of gold and silver nanoparticles in different size was investigated on the well-known Tb sensitized fluorescence emission of 1, 10-phenantroline (Phen). The greatest fluorescence intensity was observed in the presence of AgNPs with a diameter of ~6nm maybe due to their highest surface area. Furthermore, it's discovered that FA can form Tb-Phen -FA ternary complexes and cause a notable diminution in this enhanced fluorescence system. Based on this finding, a high sensitive and selective method was developed for the determination of FA. Effects of various parameters like Ag NPs, Phen and Tb(3+) concentration and pH of media were investigated. In the optimum circumstances, the fluorescence emission of AgNPs-Phen-Tb collection was declined linearly by increasing the concentration of FA in the range of 0.5 to 110nmolL(-1). Limits of detection and quantification were achieved to be 0.21 and 0.62nmolL(-1), respectively. The method has good linearity, recovery, reproducibility and sensitivity, and was adequately exploited to follow FA content in pharmaceutical, fortified flour and human urine samples.

The synthesis and luminescent properties of novel water-soluble CaF2:Tb3+ nanoparticles

Novel water-soluble CaF2:Tb3+ nanoparticles were synthesized using benzoimidazole-5-carboxylic acid (Ligand) as surfactant. The particle size of the nanoparticles is below 10nm. The investigation of fluorescence properties showed that there exists energy transfer from the surface coating of nanoparticles to Tb3+ leading to the sensitized fluorescence emission of Tb3+. The luminescence lifetimes of Tb3+ were 937.56μs (37.41%) and 2.78ms (62.59%). It is more important that Cu2+ and Fe3+ can give rise to emission quenching to the Tb3+ of nanoparticles, especially Cu2+. In the region of 2.5×10−5–5×10−4M, the higher the concentration of Cu2+ or Fe3+ is, the stronger fluorescence quenching to the Tb3+ is. Additionally, the possible mechanisms of the luminescence quenching were also discussed.

A coumarin-based fluorescent probe for thiols and its application in cell imaging

A new coumarin-based fluorescent probe CAA containing an acrylamide group has been designed and synthesized for the detection of biothiols. The probe CAA shows high selectivity and sensitivity for cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) over other amino acids. A fluorescence turn-on response is induced by a Michael-type reaction. It shows a highly sensitive fluorescence emission to Cys, Hcy, and GSH, with detection limits of 192nM for Cys, 158nM for Hcy, and 155nM for GSH (S/N=3). Furthermore, confocal fluorescence microscopy imaging using RAW264.7 macrophages demonstrates that CAA can be an effective fluorescent probe for thiols in living cells.

Sensitized luminescence of water-dispersible CaF2: RE3+ (RE=Eu, Tb, Ce/Tb) nanoparticles through surfactant coating ligands

A facile route has been established for the synthesis of PVP/CaF2: RE3+ and PVP/CaF2: Ce3+ nanoparticles (RE=Eu, Tb, Ce/Tb; PVP=Poly(N-vinyl-2-pyrrolidone)) heated at 70°C in ethanol at ambient pressure. The as-prepared nanoparticles were shown to be well dispersed in water. The nanoparticles were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and transmission electron microscopy (TEM). The investigation of fluorescence properties showed that there exists energy transfer from the surface coating of PVP to Eu3+ and Tb3+ leading to the sensitized fluorescence emission of Eu3+ and Tb3+. Co-doping the PVP/CaF2: Tb3+ nanoparticles with Ce3+ results in significant increase in the emission intensity of Tb3+ and this has been attributed to the sensitization of Ce3+ and PVP on Tb3+.

Conjugated Polymers: Conjugated Polymer Nanoparticles with Ag+‐Sensitive Fluorescence Emission: A New Insight into the Cooperative Recognition Mechanism (Part. Part. Syst. Charact. 11/2013)

Conjugated Polymers: Conjugated Polymer Nanoparticles with Ag⁺‐Sensitive Fluorescence Emission: A New Insight into the Cooperative Recognition Mechanism (Part. Part. Syst. Charact. 11/2013)

Conjugated Polymer Nanoparticles with Ag+‐Sensitive Fluorescence Emission: A New Insight into the Cooperative Recognition Mechanism

Hybrid fluorescent nanoparticles (NPs) were developed on the basis of a series of chemically close polyfluorene‐based conjugated polymers and an amphiphilic polymer. NPs constructed from polyfluorene with 2,1,3‐benzothiadiazole units incorporated in the backbone of polymer chains displayed Ag+‐sensitive fluorescence emission features with high selectivity, which was demonstrated to be attributable to Ag+‐induced aggregation and fluorescence quenching. The negatively charged sulfonic acid groups of amphiphilic polymer components located on the surfaces of the hybrid NPs and therefore provided binding sites for metal ions, which was found indispensable for the observed silver ion sensing features of the as‐prepared hybrid NPs. Additionally, it was demonstrated that the benzothiadiazole‐containing conjugated polymer components not only acted as the fluorescence signaling source but also participated in the metal binding/recognition processes. Thus, a mechanism of cooperative recognition‐based aggregation‐induced fluorescence static quenching and Ag+‐triggered fluorescence dynamic quenching responsible for the observed Ag+ sensing results was confirmed.

Energy Transfer Studies in Binary Dye Solution Mixture of Coumarin 440 + Coumarin 540 and its LifetimeCalculations

The sensitized fluorescence emission from the bimixture [Coumarin 440 (donor) + Coumarin 540 (acceptor)] has been measured as a function of dye concentration using a fluorescence spectrophotometer. The concentration of Coumarin 440 (donor) is kept constant at 5×10-5M while the concentration of Coumarin 540 (acceptor) is varied. The spectroscopic parameters for this bimixture have been calculated from the concentration dependence of peak fluorescence intensity curves. Also the data obtained from the Horiba JobinYvon Time Resolved Fluorescence Spectrophotometer was analyzed for the determination of lifetimes at various emission peaks. A comparative study between theoretically calculated lifetimes and experimentally obtained lifetimes shows a good agreement. The results indicate that the energy transfer process between unlike molecules can be studied by lifetime measurements (which could also be determined from the fluorescence emission spectral studies). Many attempts have been made in studies aimed to improve dye lasers efficiency and to extend their spectral range of operation. Energy transfer dye lasers (ETDL) have generated considerable interest as they are useful in obtaining enhanced laser output, wide tuning range, reduced concentration and pump threshold requirements and ultra-short pulse width in contrast to their single dye counterpart [1-7]. Energy Transfer Dye Lasers using numerous donor-acceptor dye pairs have been reported by various investigators duringthe last five decades. Fluorescence energy transfer is the transfer of the excited state energy from a donor (D) to acceptor (A) [8–19]. This transferoccurs without the appearance of photon and is primarily a resultof dipole–dipole interaction between the donor and the acceptor.The rate of energy transfer depends upon the extent of overlapof the emission spectrum of the donor with the absorptionspectrum of the acceptor, the relative orientation of the donorand acceptor transition dipoles and the distance between thesemolecules. The non-radiative energy transfer occurs as a result ofdipole–dipole coupling between the donor and the acceptor,anddoes not involve the emission and reabsorption of photons. In contrast to these trivial factorsnonradiative energy transfer depends upon the molecular detailsof donor–acceptor pairs.Non-radiative energy transfer is effective overdistance ranging of 50A°.The intervening of solvent or other macromoleculeshas little effect on the efficiency of the energy transfer, whichdepends primarily on the D–A distances[18].Dye lasers have some limitations as the dye solution used as an active medium absorbs energy from the excitation source in a very limited range and so the emission band also has these limitations. If a dye laser has to be used as an ideal source its spectral region needs to be extended. In order to extend the spectral region of operation mixtures of different dye solutions/dye molecules embedded in solid matrices arebeingused.The use of such energy transfer in dye lasers is also helpful in minimizing the photoquenching effects and thereby, increasing the laser efficiency. In the present studies the energy transfer mechanism has been investigated in the ethanol solution mixture, i.e. Coumarin 440 and Coumarin 540 from their absorption and emission spectra. The dependence of lifetimes of the dye molecules on their

Highly Sensitive Fluorescent Analysis of Dynamic Glycan Expression on Living Cells Using Glyconanoparticles and Functionalized Quantum Dots

A double signal amplification strategy was designed for highly sensitive and selective in situ monitoring of carbohydrate on living cells. The double signal amplification included the multiplex sandwich binding of functionalized quantum dots (QDs) to both glycan groups on the cell surface and glyconanoparticles and a cadmium cation sensitized fluorescence emission of Rhod-5N. Using the sialic acid-phenylboronic acid recognition system as a model, the 3-aminophenylboronic acid functionalized QDs (APBA-QDs) were synthesized by covalently binding APBA to mercaptopropionic acid capped CdS QDs, and the glyconanoparticles, polysialic acid stabilized gold nanoparticles (PSA-AuNPs), were prepared by a one-pot procedure. The APBA-QDs first recognized the sialic acid (SA) groups on BGC-823 human gastric carcinoma (BGC) cells and then the PSA on AuNPs, which were further used to bind more APBA-QDs on the cell surface for signal amplification. After the bound QDs were dissolved to release the Cd(2+), a Cd(2+)-sensitized fluorescence method was developed for the detection of BGC cells in a linear range from 5.0 × 10(2) to 1.0 × 10(7) cells mL(-1) with a limit of detection down to 210 cells mL(-1) (8 cells in 40 μL of solution) and the dynamic monitoring of SA expression variation on the cell surface. The monitoring result was identical with that from flow cytometric analysis. This approach showed high specificity and acceptable reproducibility. This strategy provided a promising platform for highly sensitive cytosensing and cytobiologic study.

How myosin VI coordinates its heads during processive movement

A processive molecular motor must coordinate the enzymatic state of its two catalytic domains in order to prevent premature detachment from its track. For myosin V, internal strain produced when both heads of are attached to an actin track prevents completion of the lever arm swing of the lead head and blocks ADP release. However, this mechanism cannot work for myosin VI, since its lever arm positions are reversed. Here, we demonstrate that myosin VI gating is achieved instead by blocking ATP binding to the lead head once it has released its ADP. The structural basis for this unique gating mechanism involves an insert near the nucleotide binding pocket that is found only in class VI myosin. Reverse strain greatly favors binding of ADP to the lead head, which makes it possible for myosin VI to function as a processive transporter as well as an actin-based anchor. While this mechanism is unlike that of any other myosin superfamily member, it bears remarkable similarities to that of another processive motor from a different superfamily--kinesin I.

Fluorescence resonance energy transfer analysis of apolipoprotein E C-terminal domain and amyloid β peptide (1-42) interaction

The potential neurotoxicity of soluble forms of amyloid beta peptide (Abeta) as a key factor in early pathogenesis of Alzheimer's disease is being recognized. In addition, there is growing evidence of the essential role of apolipoprotein E (apoE) in amyloid formation, although molecular details of apoE/Abeta interaction are poorly understood. We employed apoE C-terminal (CT) domain comprising residues 201-299 to identify binding location of Abeta(1-42) by fluorescence resonance energy transfer (FRET) and quenching analyses. Native tryptophan (Trp) residues in the apoE CT domain served as FRET donor, whereas N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) covalently attached to a unique cysteine residue substituted at position 4 of Abeta(1-42) (AEDANS-F4C-Abeta(1-42)) served as FRET acceptor. Fluorescence analysis verified that the oligomerization behavior of AEDANS-F4C-Abeta(1-42) was not abrogated by covalent attachment of AEDANS and that apoE CT domain/AEDANS-F4C-Abeta(1-42) association results in formation of a soluble complex. A large decrease in Trp fluorescence emission was noted in mixtures containing apoE CT domain and AEDANS-F4C-Abeta(1-42), accompanied by appearance of sensitized fluorescence emission of AEDANS as a result of intermolecular FRET. An average distance of separation of 22.6 Angstroms between donors and acceptor was calculated. Fluorescence quenching by potassium iodide (KI) did not reveal significant differences in apoE CT domain Trp microenvironment in the absence or the presence of Abeta(1-42). A twofold increase in quenching constant was noted for KI quenching of AEDANS fluorescence emission in the presence of apoE CT domain, indicative of alterations in Abeta conformation upon interaction with apoE CT domain. We propose intermolecular FRET analysis as a discriminating approach to examine apoE/Abeta interaction, a potentially critical factor in early events involved in amyloid formation.

Development of multipurpose laboratory XIEES spectrometer and its application to surface XAFS analysis of Al2O3 films.

A new laboratory spectrometer for X-ray Induced Electron Emission Spectroscopy (XIEES) has been developed. This spectrometer is suitable for measurements of X-ray Absorption Fine Structure (XAFS) spectrum in surface sensitive fluorescence and electron emission detection modes as well as in standard transmission mode. It is the advantage of this spectrometer that a wide range of x-ray energy scanning between 0.48 and 41 keV is available. At the Cu K-edge, a energy resolution of about 2 eV has been achieved. The spectrometer has been used to analyze the Al2O3 (40 A and 500 A)/Si(100) with total electron yield measurement.

Quenching of fluorescence of Rhodamine 610 in binary and ternary mixture solutions

The sensitized fluorescence emission from bimixtures, Coumarin 500 (donor) + Rhodamine 610 (acceptor) and Rhodamine 610 (donor) + Nile Blue 590 (acceptor) and also from the ternary mixture, Coumarin 500 (donor) + Rhodamine 610 (inter mediator) + Nile Blue 590 (acceptor), have been measured as a function of dye concentration and excitation wavelength using a fluorescence spectrophotometer. The spectroscopic parameters for the bimixtures have been estimated from the concentration dependence of the peak fluorescence intensity curves.

A study of intramolecular energy transfer in Cm(III) complexes with aromatic ligands by time-resolved laser fluorescence spectroscopy

Sensitized fluorescence emission of f-element ions was studied for the humate and fulvate complexes of Cm(III) by time-resolved laser fluorescence spectroscopy. Complexation with chelating aromatic carboxylic acids, which may be regarded as complexing functional groups of humic substances, e.g. phthalic and salicylic acids, was investigated in parallel for comparison. Excitation and emission spectra, together with lifetimes of excited states, were investigated for the Cm(III) complex species as well as for the individual pure ligans. The fluorescence enhancement was found to be weak in the Cm(III)-phthalate and strong in the Cm(III)-salicylate. The latter is comparable with the Cm(III)-fulvate. The efficiency of energy transfer was calculated from the excitation at 308 nm. The results are discussed in terms of energy overlapping between triplet states of ligands and excited levels of Cm(III).

Electric Field Effects on Sensitized Fluorescence Emission From Nematic Liquid Crystal Cells

Abstract Remarkable electric field effects were observed with sensitized fluorescence intensities of a perylene derivative (BPDC), when rectangular AC waves (up to 10 V0-p ) were applied to nematic liquid crystal cells containing a sensitizer (C307 or AHDA) and an acceptor (BPDC) as the guest molecules. Measurements of the fluorescence lifetime of AHDA indicated that the rates of energy transfer from AHDA to BPDC were independent of the electric fields. The electric field effects completely changed the sign when the intensity variation detected at vertical direction was compared with that at the horizontal direction with respect to the liquid crystal cell. The observed electric field effects on the sensitized fluorescence were consequently suggested to come from the orientation control of transition dipole moment of BPDC through the molecular alignment of the liquid crystal. The suggestion was confirmed by the measurement of dichroic absorption of the donor-acceptor systems.

Changes in the distribution of platelet membrane proteins revealed by energy transfer

The redistribution of platelet membrane proteins in response to platelet activation was studied. To investigate this process we prepared a variety of platelet ligands, including di- and tetrameric concanavalin A, IgG, thrombin, wheat-germ agglutinin and other lectins. These ligands were conjugated either with acceptor (rhodamine isothiocyanate) or donor (fluoresceine isothiocyanate) fluorophore. Platelets exposed to various combinations of ligand species were stimulated with different aggregating agents, and changes in sensitized fluorescence emission or donor quenching were recorded. Energy transfer was observed with thrombin, dimeric concanavalin A after addition of thrombin and various combinations of dimeric concanavalin A with other membrane ligands. The preincubation of platelets with colchicine prevented energy transfer between appropriate ligand pairs and platelet activator. Our studies show that nonradiative energy transfer can be used to analyze redistribution of membrane receptor sites in platelets.