Fluorescein Isothiocyanate Isomer Research Articles

In vitro and in vivo assessment of delivery of hydrophobic molecules and plasmid DNAs with PEO–PPO–PEO polymeric micelles on cornea

The stability and bio-distribution of genes or drug complexes with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO–PPO–PEO, Pluronic F-68) polymeric micelles (PM) are essential for an effective nanosized PM delivery system. We used Förster resonance energy transfer (FRET) pairs with PM and measured the FRET ratio to assess the stability of PM in vitro and in vivo on the cornea. The FRET ratio reached a plateau at 0.8 with 3% PM. Differential scanning calorimetry measurement confirmed the complex formation of FRET pairs with PM. Confocal imaging with the fluorophores fluorescein isothiocyanate isomer I (FITC) and rhodamine B base (RhB) also showed the occurrence of FRET pairs in vitro. The fluorophores were mixed with 3% PM solution or the FITC-labeled PEO–PPO–PEO polymers (FITC-P) were mixed with RhB-labeled plasmids (RhB–DNA). In addition, the in vitro corneal permeation of FRET pair complexes with PM reached a 0.8 FRET ratio. One hour after eye drop administration, FRET pairs colocalized in the cytoplasm, and surrounded and entered the nuclei of cells in the cornea, and the polymers were located in the corneal epithelial layers, as detected through anti-PEG immunohistochemistry. Furthermore, fluorescence colocalization in the cytoplasm and cell nucleus of the corneal epithelium was confirmed in tissues where RhB or RhB–DNA complexed with FITC-P was found to accumulate. We demonstrate that at a concentration of 3%, PM can encapsulate FRET pairs or RhB–DNA and retain their integrity within the cornea 1 h after administration, suggesting the feasibility and stability of PEO–PPO–PEO polymers as a vehicle for drug delivery.

Multi-wavelength fluorescence detection of submicromolar concentrations using a filter-free fluorescence sensor

In this paper, we report on the development of a filter-free fluorescence sensor for the detection of multiple fluorescence wavelengths from a solution of fluorescent dyes with submicromolar concentrations. The filter-free fluorescence sensor has a photo-gate structure, and the intensities of excitation light and fluorescence can be calculated from the photocurrents for the different gate voltages. This sensor makes it possible to measure various wavelengths without the need for optical filters or mirrors. The separation ability of excitation light and fluorescence in the developed sensor was more than 1000:1 for an LED light source. Fluorescence measurements were carried out for fluorescein isothiocyanate isomer (FITC) and sulforhodamine 101 acid chloride (Texas Red) dyes. Based on our calculations, the excitation light and fluorescence from submicromolar concentrations of FITC, Texas Red, and a mixed solution were successfully separated and simultaneously detected.

Fluorescent, magnetic dual-responsive molecularly imprinted polymers for the selective detection of moxidectin in animal samples

A simple and effective approach is presented to fabricate fluorescent and magnetic dual-responsive molecularly imprinted polymers for selective recognition of moxidectin. Magnetic gelatin was prepared to provide not only easy magnetic separation, but also significant amino functionalities, allowing surface modification with fluorescein isothiocyanate isomer I and acrylic acid (AA). Based on the double bond provided by AA, molecularly imprinted polymers could be directly coated onto the surface of modified magnetic gelatin. The dual-responsive imprinting polymers feature a high adsorption capacity (87.1 mg g−1), selective fluorescence response toward moxidectin (imprinting factor = 3.6), rapid magnetic separation, and good reproducibility. Subsequently, the dual-responsive composites were successfully applied as sorbents for selective determination of moxidectin. Upon optimization, a linear range of 10–1000 ng mL−1 and a detection limit of 6 ng mL−1 were achieved. The results agree well with those obtained by the classic HPLC–FLD method (r = 0.9935). This developed strategy may find its potential application in rapid, simple, sensitive, and selective determinations of target molecules in complex samples.

Layered Double Hydroxide Nanotransporter for Molecule Delivery to Intact Plant Cells.

Here we report a powerful method that facilitates the transport of biologically active materials across the cell wall barrier in plant cells. Positively charged delaminated layered double hydroxide lactate nanosheets (LDH-lactate-NS) with a 0.5‒2 nm thickness and 30‒60 nm diameter exhibit a high adsorptive capacity for negatively charged biomolecules, including fluorescent dyes such as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate isomer I(FITC) and DNA molecules, forming neutral LDH-nanosheet conjugates. These neutral conjugates can shuttle the bound fluorescent dye into the cytosol of intact plant cell very efficiently. Furthermore, typical inhibitors of endocytosis and low temperature incubation did not prevent LDH-lactate-NS internalization, suggesting that LDH-lactate-NS penetrated the plasma membrane via non-endocytic pathways, which will widen the applicability to a variety of plant cells. Moreover, the absence of unwanted side effects in our cytological studies, and the nuclear localization of ssDNA-FITC suggest that nano-LDHs have potential application as a novel gene carrier to plants.

Hierarchical mesoporous silicas templated by PE-b-PEO-b-PLA triblock copolymer for fluorescent drug delivery

Hierarchical mesoporous silicas for fluorescent drug-delivery molecule templated by PE-b-PEO-b-PLA triblock copolymer.

An efficient core-shell fluorescent silica nanoprobe for ratiometric fluorescence detection of pH in living cells.

Intracellular pH plays a vital role in cell biology, including signal transduction, ion transport and homeostasis. Herein, a ratiometric fluorescent silica probe was developed to detect intracellular pH values. The pH sensitive dye fluorescein isothiocyanate isomer I (FITC), emitting green fluorescence, was hybridized with reference dye rhodamine B (RB), emitting red fluorescence, as a dual-emission fluorophore, in which RB was embedded in a silica core of ∼40 nm diameter. Moreover, to prevent fluorescence resonance energy transfer between FITC and RB, FITC was grafted onto the surface of core-shell silica colloidal particles with a shell thickness of 10-12 nm. The nanoprobe exhibited dual emission bands centered at 517 and 570 nm, under single wavelength excitation of 488 nm. RB encapsulated in silica was inert to pH change and only served as reference signals for providing built-in correction to avoid environmental effects. Moreover, FITC (λem = 517 nm) showed high selectivity toward H(+) against metal ions and amino acids, leading to fluorescence variation upon pH change. Consequently, variations of the two fluorescence intensities (Fgreen/Fred) resulted in a ratiometric pH fluorescent sensor. The specific nanoprobe showed good linearity with pH variation in the range of 6.0-7.8. It can be noted that the fluorescent silica probe demonstrated good water dispersibility, high stability and low cytotoxicity. Accordingly, imaging and biosensing of pH variation was successfully achieved in HeLa cells.

Uptake and bioconversion of stereoisomeric dipeptide prodrugs of ganciclovir by nanoparticulate carriers in corneal epithelial cells

Purpose: The objective of this study is to investigate cellular uptake of prodrug-loaded nanoparticle (NP). Another objective is to study bioconversion of stereoisomeric dipeptide prodrugs of ganciclovir (GCV) including L-Val-L-Val-GCV (LLGCV), L-Val-D-Val-GCV (LDGCV) and d-Val-l-Val-GCV (DLGCV) in human corneal epithelial cell (HCEC) model.Methods: Poly(D,L-lactic-co-glycolic acid) (PLGA) NP encapsulating prodrugs of GCV were formulated under a double emulsion method. Fluorescein isothiocyanate isomer–PLGA conjugates were synthesized to fabricate biocompatible fluorescent PLGA NP. Intracellular uptake of FITC-labeled NP was visualized by a fluorescent microscope in HCEC cells.Results: Fluorescent PLGA NP and non-fluorescent NP display similar hydrodynamic diameter in the range of 115–145 nm with a narrow particle size distribution and zeta potentials around −13 mV. Both NP types showed identical intracellular accumulation in HCEC cells. Maximum uptake (around 60%) was noted at 3 h for NP. Cellular uptake and intracellular accumulation of prodrugs are significantly different among three stereoisomeric dipeptide prodrugs. The microscopic images show that NPs are avidly internalized by HCEC cells and distributed throughout the cytoplasm instead of being localized on the cell surface. Following cellular uptake, prodrugs released from NP gradually bioreversed into parent drug GCV. LLGCV showed the highest degradation rate, followed by LDGCV and DLGCV.Conclusion: LLGCV, LDGCV and DLGCV released from NP exhibited superior uptake and bioreversion in corneal cells.

The application of portable microchip electrophoresis for the screening and comparative analysis of synthetic cathinone seizures

Variation in the chemical composition of illicit tablets and powders is common among samples within a given drug seizure. Using microchip electrophoresis (ME), multiple tablets can be screened in a cost-effective and timely manner. This method could be used in conjunction with reporting methods that focus solely on statistical sampling to infer homogeneity or otherwise of a larger subset of tablets. Some frequently observed synthetic cathinones, often present in illicit tablets seized in New Zealand, were chosen for analysis. An ME device (Agilent Bioanalyzer 2100) was used to electrophoretically separate synthetic cathinones. The background electrolyte was composed of a 50mM sodium tetraborate buffer with 50mM sodium dodecyl sulphate at pH 9.66. Analytes were derivatised prior to analysis for 3min at 90°C, employing fluorescein isothiocyanate isomer I (FITC). A characteristic fluorescent profile was obtained for each tablet, in terms of the number of constituents, relative peak height ratios and migration times. The repeatability of the developed method was assessed for a wide range of tablets and relative standard deviations of 0.4–5.2% and 1.6–5.5% were calculated for migration times and peak height ratios, respectively. The use of microchip tablet profiles in the forensic case comparison of illicit drug seizure samples in realistic scenarios is discussed.

Metal-enhanced fluorescence using aggregated silver nanoparticles

A simple strategy to prepare aggregated Ag nanoparticle composites in solution by self-assembly is developed. Sodium citrate is used as the reduction agent to produce negatively charged Ag nanoparticles which are then aggregated using cationic polyacrylamide (PAM) to form self-assembled Ag-PAM microcomposites. In these microcomposites, the degree of aggregation of Ag nanoparticles can be controlled by changing the Ag/PAM ratio. This special structural feature can be applied in the study of metal-enhanced fluorescence. Confocal laser scanning microscopy and time-resolved fluorescence measurements are used to examine the fluorescence enhancement phenomenon of the Ag-PAM microcomposites. The microcomposites considerably enhanced the fluorescence intensity of fluorescein isothiocyanate isomer (FITC).

A microwave-assisted fluorescent labeling method for the separation and detection of amphetamine-like designer drugs by capillary electrophoresis

A microwave-assisted fluorescence labeling method for use in CE–LIF (capillary electrophoresis–laser induced fluorescence) is described. Six amphetamine-like designer drugs, namely, o-, m-, p-chloro- and o-, m-, p-fluoro-amphetamine derivatives, were synthesized and used as model compounds. FITC (fluorescein isothiocyanate isomer I) and a blue-laser were used as the fluorescent labeling reagent and excitation source, respectively. When a microwave oven was used, the reaction was complete within ∼5min, while the classical method required at least 20h (usually, an overnight reaction). A mimic oral fluid sample was obtained by spiking oral fluid from a volunteer with the six standards, and after liquid–liquid extraction and microwave-derivatization, it was possible to process the analytes by CE–LIF within a period of ∼10min; the wavelength of the blue-laser used was 473nm. For comparison, data obtained using classical methods, including CZE–UV (capillary zone electrophoresis–UV absorbance detection), sweeping-MEKC–UV (micellar electrokinetic chromatography–UV absorbance detection) and LC–Q-TOFMS (liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry) are also reported.

Electrokinetic microchip-based sample loading for surface plasmon resonance detection

In this reported work, a microfluidic-based fluid electrokinetic force has been used for surface plasmon resonance or surface plasmon resonance image (SPRI) detection. In this device, a modified rectangular microarray of Au instead of previous reported square or circular gold segments was designed and microfabricated with gold segment geometries and characterised by surface morphology studies. A voltage drop of maximal 400 V/cm was applied without gold erosion to drive fluorescein isothiocyanate isomer I in a high-conductivity medium on the bare gold surface layer, enabling SPRI detection.

Liquid–liquid interface assisted synthesis of multifunctional and multicomponent hydrogel particles

We present a new method to synthesize hydrogel particles by exploiting the interface formed between two immiscible liquids. Spherical monomer droplets, of varying diameters, could be suspended at the planar interface formed between two immiscible liquids. While suspended, polymerization could proceed, after which the hydrogel particles that were formed could be collected. Using this approach, we were able to synthesize particles containing various monomers/co-monomers including: N-isopropylacrylamide, 2-hydroxyethyl methacrylate, and N-(3-aminopropyl)methacrylamide hydrochloride. The approach also allowed for the facile encapsulation of various inorganic nanoparticles and small molecules, including: Au nanoparticles, Ag nanoparticles, magnetic cobalt (Co) nanoparticles, tris(4-(dimethylamino)phenyl)methylium chloride (crystal violet) and fluorescein isothiocyanate isomer I (FITC). The benefit of this approach is the ability to load polymer particles with a wide variety of moieties without the need to optimize any reaction conditions. So long as the species to be encapsulated in the particle are soluble in water, and minimally soluble in the solvents used to form the interface, they will be incorporated in the polymerized particle.

Flaky Tail Mouse Denotes Human Atopic Dermatitis in the Steady State and by Topical Application with Dermatophagoides pteronyssinus Extract

The barrier abnormality, a loss-of-function mutation in the gene encoding filaggrin (FLG), which is linked to the incidence of atopic dermatitis (AD), is a recently discovered but important factor in the pathogenesis of AD. Flaky tail (Flg(ft)) mice, essentially deficient in filaggrin, have been used to investigate the role of filaggrin on AD. However, the relevancy of Flg(ft) mice to human AD needs to be determined further. In this study, we observed the clinical manifestations of Flg(ft) mice in the steady state and their cutaneous immune responses against external stimuli, favoring human AD. Under specific pathogen-free conditions, the majority of Flg(ft) mice developed clinical and histological eczematous skin lesions similar to human AD with outside-to-inside skin barrier dysfunction evaluated by newly devised methods. In addition, cutaneous hapten-induced contact hypersensitivity as a model of acquired immune response and a mite extract-induced dermatitis model physiologically relevant to a human AD were enhanced in Flg(ft) mice. These results suggest that the Flg(ft) mouse genotype has potential as an animal model of AD corresponding with filaggrin mutation in human AD.

Effect of surface functionality of magnetic silica nanoparticles on the cellular uptake by glioma cells in vitro

The cellular uptake efficiency of nanoparticles depends mainly on the surface characteristics of these materials. In this study, amine- and ester-terminated polyamidoamine (PAMAM) dendrimers were used to functionalize fluorescein-doped magnetic mesoporous silica nanoparticles (FMNPs) to evaluate the effect of surface functionality on cellular uptake by glioma cells. The successful synthesis of these materials was confirmed by transmission electron microscopy (TEM), thermogravimetric analysis (TGA) and zeta potential titration. Neither of these materials showed acute cytotoxicity and it was demonstrated that the different surface functionalities regulated the ability of the nanoparticles to cross cell membranes. In addition to serving as an imaging agent, fluorescein isothiocyanate isomer I (FITC) was used as a model drug to evaluate the drug release rates of these drug delivery systems.

Identification of chemical species of fluorescein isothiocyanate isomer–I (FITC) monolayers on platinum by doubly resonant sum‐frequency generation spectroscopy

AbstractDoubly resonant sum‐frequency generation (DR‐SFG) spectroscopy of fluorescein‐4‐isothiocyanate isomer‐I (FITC) monolayers on platinum was performed. Vibrational spectra of the monolayers for the IR wavenumber of 1750–1450 cm−1 were measured with visible probes ranging from 431 to 582 nm. Two vibrational bands at 1643 and 1610 cm−1 were observed, and their DR‐SFG excitation profiles displayed different shapes. By rinsing the monolayers with an alkaline solution, the smaller wavenumber band disappeared and the larger wavenumber band gained intensity. On the basis of the spectral response to the rinsing, we concluded that the FITC molecules existed on platinum as deprotonated and protonated forms; the former corresponds to the 1643 cm−1 band and the latter to the 1610 cm−1 band. The deprotonated form was assigned to an anionic surface species, and the protonated form to a neutral surface species by comparing the DR‐SFG excitation profiles with electronic absorption spectra of the protolytic forms of fluorescein in an aqueous solution (Sjöback R et al., Spectrochimica Acta A 1995; 51: L7–L21). The results demonstrate that the measurement of DR‐SFG excitation profiles is a useful technique to identify chemical species of monolayers on metal surfaces. Copyright © 2008 John Wiley & Sons, Ltd.

A standardized pre-treatment method of biofilm flocs for fluorescence microscopic characterization

The availability of epifluorescence microscopy opens the door for the visualization of biofilm flocs via the fluorescence staining technique. However, one of the main problems in the application of the method is a missing standardized protocol for sample preparation. In this study, different pre-treatment methods for the simultaneous staining of different sludge flocs components e.g. extracellular polymeric substances (EPS) and microorganisms (MO) were compared. A method was chosen, which remains the shape of sludge flocs from a municipal wastewater treatment plant and allows to stain major parts of the flocs thoroughly: For this the surrounding wastewater was removed by centrifugation. Then the flocs were resuspended in sterile tap water, immobilized by drying on a diagnostic slide and fixated with formaldehyde. The staining of the different compounds was performed with tetramethylrhodamine isothiocyanate-labeled lectin Concanavalin A (TRITC-Con A; glycoconjugates), fluoresceinisothiocyanate isomer I (FITC; amino groups) and 4,6-diamidino-2-phenylindoldihydrochloride (DAPI; nucleic acids). This staining method was successfully applied for protein- and carbohydrate-quantification using gelatin mixtures of dextran and bovine serum albumin (BSA). The pre-treatment and staining methods were appropriate to compare the composition of different biofilm flocs such as nitrifying activated sludge from a municipal wastewater treatment plant and a pure culture of the bacterial strain Pseudomonas putida (ATCC 17514) as revealed by the results of conventional analytical methods.

Development of an integrated direct-contacting optical-fiber microchip with light-emitting diode-induced fluorescence detection

In this paper, one poly(dimethylsiloxane) (PDMS) sandwich microchip integrated with one direct-contacting optical fiber was fabricated by using a thin-casting method. This novel integrated PDMS sandwich microchip included top glass plate, PDMS membrane replica with microfluidic networks and optical fiber, flat PDMS membrane and bottom glass plate. As the tip of excitation optical fiber completely contacted with the separation microchannel in this integrated microchip, it not only increased the excitation light intensity to achieve the high sensitivity, but also reduced the diameter of excitation beam to obtain high resolution. In addition, we found that this rigid PDMS sandwich microchip structure effectively prevented PDMS microchannel distortion from rigid optical fiber, and provided a substantial convenience for microchips manipulating. A blue light-emitting diode (LED) was applied as excitation source by using optical fiber to couple excitation light into its direct-contacting microchannel for fluorescence detection. The performances of this integrated PDMS sandwich microchip was demonstrated by separating the mixture of sodium fluorescein (SF) and fluorescein isothiocyanate isomer I (FITC), and showed a higher sensitive and resolution than those obtained from the conventional integrated optical-fiber PDMS microchip with a 100-μm distance between fiber tip and separation microchannel. Additionally, the reproducibility of this integrated microchip with LED-induced fluorescence detection was also examined by separation of a mixture of FITC-labeled amino acids.

Detection enhancement in nano-channels using micro-machined silicon groove

The present paper reports on an experimental study of the possibility to use a micro-machined detection groove to enhance the detection sensitivity in flat-rectangular nano-channels for ultra-rapid liquid chromatography separations. Transversally running detection grooves with three different axial widths (respectively, 2, 4 and 6 μm) and one depth (4.75 μm) were tested in glass and silicon channels for the whole range of detectable fluorescein isothiocyanate isomer I, FITC, concentrations. The groove with the most square-like cross-section (i.e., 4 μm wide and 4.75 μm deep) yielded the best combination of detection gain and minimal additional band broadening. In a 1 cm long channel, the effective plate loss caused by the 4 μm wide groove would only be of the order of 20%, while the gain in S/N-ratio was of the order of a factor of 5. The detection groove concept yields larger gains in silicon channel substrates than in glass channel substrates, due to the larger stray light losses occurring in the latter.

Optimization of the separation and on-line sample concentration of phenethylamine designer drugs with capillary electrophoresis–fluorescence detection

Five 2C-series of phenethylamine designer drugs, including 2,5-dimethoxy-4-ethylthio-phenethylamine (2C-T-2), 2,5-dimethoxy-4-( n)-propylthiophenethylamine (2C-T-7), 4-chloro-2,5-dimethoxyphenethylamine (2C-C), 4-bromo-2,5-dimethoxy-phenethylamine (2C-B), 2,5-dimethoxy-4-iodo-phenethylamine (2C-I), were synthesized and standard GC/MS and fluorescence spectra are reported for them. A mixture of the five drugs was separated and detected by means of capillary electrophoresis (CE) with native fluorescence and light emitting diode (LED)-induced fluorescence (LIF) detection, respectively, for comparison. In the former case, exciting at a wavelength of 300 nm from a Xe lamp was used. The detection limits were found to be only in the range of ∼10 −4 M by the micellar electrokinetic chromatography (MEKC) mode but were improved to ∼10 −7 M when the sweeping-MEKC mode was used. For a highly sensitive analysis, LED-induced fluorescence detection was examined by derivatizing the compounds with a fluorescent dye, fluorescein isothiocyanate isomer I (FITC). A blue-LED (∼2 mW) was used as the fluorescence excitation source. The detection limits were improved to ∼10 −7 and ∼10 −8 M, respectively, when the MEKC and stacking-MEKC modes were applied. A mimic urine sample was obtained by spiking urine from a volunteer with the five standards, and after liquid–liquid extraction, the sample was examined by means of the MEKC–LIF mode. The extraction procedures used for the urine sample and the CE conditions for the separation were optimized.

Pharmacokinetics and Biodisposition of Poly(vinyl alcohol) in Rats and Mice

Poly(vinyl alcohol) (PVA) of various molecular weight (MW=10,560-116,600) was successfully labeled with fluorescein isothiocyanate isomer I (FITC) according to the method of de Belder and Granath. A high-performance size-exclusion chromatographic procedure was developed for the quantitative analysis of FITC-labeled poly(vinyl alcohol) (F-PVA) in biological samples. F-PVA (80 K) disappeared slowly from the blood circulation according to the first-order kinetics (t1/2=7 h) after intravenous injection to rats. A dose-independent behavior of F-PVA (80 K) was observed in the blood circulation, in the tissue distribution and in the urinary and fecal excretions. This suggested that PVAs are eliminated exclusively by the mechanisms that do not involve saturable transport processes. Furthermore, it was found that PVAs are very stable in the body because no degradation product was detected in the urine and feces. 125I-labeled poly(vinyl alcohol) (125I-PVA) was prepared by introducing tyramine residues to the hydroxyl groups of PVA molecules by the 1,1'-cabonyldiimidazole (CDI) activation method. 125I-PVA (80 K) was retained in the blood circulation for several days after intravenous injection to mice. Although the tissue distribution of PVAs was small, a significant accumulation into the liver and the spleen was observed. Fluorescence microscopic examination of paraffin section of the liver revealed that F-PVA (80 K) was endocytosed by the liver parenchymal cells. 125I-PVA (80 K) captured by liver was slowly transported via the bile canaliculi and gall bladder to the intestine and excreted in the feces. It was suggested, therefore, a long time is necessary for 125I-PVA (80 K) to be excreted perfectly from the body.