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Fluorescein
Kinds of Fluorescein Terms modified by Fluorescein Selected AbstractsEffect of storage media on human periodontal ligament cell apoptosisDENTAL TRAUMATOLOGY, Issue 1 2008Mónica M. Chamorro However, the mechanisms by which different storage conditions alter the functional status of PDL cells have not been determined. The purpose of the present study was to investigate, in vitro, the level of programed cell death or apoptosis in a population of PDL cells following storage under different conditions. Primary human PDL cells were plated into 24-well-culture plates and allowed to attach for 24 h. Cells were then exposed for 1 h to milk, Hank's balanced salt solution (HBSS), Soft Wear contact lens solution or Gatorade at room temperature or on ice. Culture medium was used as a negative control. Apoptosis was evaluated at 24, 48, and 72 h after treatment on quadruplicate samples by using the ST 160 ApopTag Fluorescein Direct In Situ Detection Kit. The total number of cells and the total number of apoptotic cells were counted. The results indicated that at 24 and 72 h, PDL treated with Gatorade and the contact lens solution displayed the highest percentages of apoptotic cells when compared with the other treatment groups at room temperature. Overall, cells treated on ice showed significantly lower levels of apoptosis when compared with treatments at room temperature. In conclusion, the results indicated that apoptosis plays a major role in cell death in cells treated with Gatorade and contact lens solutions in comparison to other storage solutions and that storage on ice can inhibit programed cell death. [source] Integrated on-chip derivatization and electrophoresis for the rapid analysis of biogenic aminesELECTROPHORESIS, Issue 14 2004Nigel P. Beard Abstract We demonstrate the monolithic integration of a chemical reactor with a capillary electrophoresis device for the rapid and sensitive analysis of biogenic amines. Fluorescein isothiocyanate (FITC) is widely employed for the analysis of amino-group containing analytes. However, the slow reaction kinetics hinders the use of this dye for on-chip labeling applications. Other alternatives are available such as o -phthaldehyde (OPA), however, the inferior photophysical properties and the UV ,max present difficulties when using common excitation sources leading to a disparity in sensitivity. Consequently, we present for the first time the use of dichlorotriazine fluorescein (DTAF) as a superior in situ derivatizing agent for biogenic amines in microfluidic devices. The developed microdevice employs both hydrodynamic and electroosmotic flow, facilitating the creation of a polymeric microchip to perform both precolumn derivatization and electrophoretic analysis. The favorable photophysical properties of the DTAF and its fast reaction kinetics provide detection limits down to 1 nM and total analysis times (including on-chip mixing and reaction) of <60 s. The detection limits are two orders of magnitude lower than current limits obtained with both FITC and OPA. The optimized microdevice is also employed to probe biogenic amines in real samples. [source] Highly sensitive and simple fluorescence staining of proteins in sodium dodecyl sulfate-polyacrylamide-based gels by using hydrophobic tail-mediated enhancement of fluorescein luminescenceELECTROPHORESIS, Issue 19-20 2003Chulhun Kang Abstract Fluorescein has an extremely low luminescence intensity in acidic aqueous media. However, when it was bound to proteins, subsequent increase of luminescence intensity took place. Furthermore, when a hydrophobic tail, such as aliphatic hydrocarbons, was introduced to fluorescein, more dramatic increase of luminescence intensity was observed upon binding to proteins. In the present study, by utilizing this luminescence enhancement, three hydrophobic fluorescein dyes (5-dodecanoyl amino fluorescein, 5-hexadecanoyl amino fluorescein, and 5-octadecanoyl amino fluorescein) were examined as noncovalent fluorescent stains of protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Effective incorporation of the dyes to proteins in gels was accomplished either simply by adding dyes at the protein fixation step, or by treating gels with a staining solution after the fixation. The sensitivity of this staining method using the fluorescein derivatives was approximately 1 ng/band for most proteins. For some cases, protein bands containing as low as 0.1 ng were successfully visualized. In addition, the detection sensitivity showed much less protein-to-protein variation than silver staining. This new staining method was also successfully applied to two-dimensional electrophoresis of rat brain proteins. Its overall sensitivity was comparable to that of silver staining. [source] Cationic Conjugated Polyelectrolytes with Molecular Spacers for Efficient Fluorescence Energy Transfer to Dye-Labeled DNA,ADVANCED FUNCTIONAL MATERIALS, Issue 2 2007Y. Woo Abstract Two water-soluble conjugated polyelectrolytes, poly(9,9,-bis(6- N,N,N -trimethylammoniumhexyl)fluorene- alt -1,4-(2,5-bis(6- N,N,N -trimethylammoniumhexyloxy))phenylene) tetrabromide (P1i) and poly((10,10,-bis(6- N,N,N -trimethylammoniumhexyl)-10H-spiro(anthracene-9,9,-fluorene))- alt -1,4-(2,5-bis(6- N,N,N -trimethylammoniumhexyloxy))phenylene) tetrabromide (P2i) are synthesized, characterized, and used in fluorescence resonance energy transfer (FRET) experiments with fluorescein-labeled single-stranded DNA (ssDNA-Fl). P1i and P2i have nearly identical ,-conjugated backbones, as determined by cyclic voltammetry and UV-vis spectroscopy. The main structural difference is the presence of an anthracenyl substituent, orthogonal to the main chain in each of the P2i repeat units, which increases the average interchain separation in aggregated phases. It is possible to observe emission from ssDNA-Fl via FRET upon excitation of P2i. Fluorescein is not emissive within the ssDNA-Fl/P1i electrostatic complex, suggesting Fl emission quenching through photoinduced charge transfer (PCT). We propose that the presence of the anthracenyl "molecular bumper" in P2i increases the distance between optical partners, which decreases PCT more acutely relative to FRET. [source] The safety of intravenous fluorescein for confocal laser endomicroscopy in the gastrointestinal tractALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 5 2010M. B. WALLACE Aliment Pharmacol Ther,31, 548,552 Summary Background, Confocal laser endomicroscopy (CLE) is rapidly emerging as a valuable tool for gastrointestinal endoscopic imaging. Fluorescent contrast agents are used to optimize imaging with CLE, and intravenous fluorescein is the most widely used contrast agent. Fluorescein is FDA-cleared for diagnostic angiography of the retina. For these indications, the safety profile of fluorescein has been well-documented; however, to date, fluorescein is not cleared for use with CLE. Aims, To estimate the rate of serious and total adverse events attributable to intravenous fluorescein when used for gastrointestinal CLE. Methods, We performed a cross sectional survey of 16 International Academic Medical Centres with active research protocols in CLE that involved intravenous fluorescein. Centres using i.v. fluorescein for CLE who were actively monitored for adverse events were included. Results, Sixteen centres performed 2272 gastrointestinal CLE procedures. The most common dose of contrast agent was 2.5,5 mL of 10% sodium fluorescein. No serious adverse events were reported. Mild adverse events occurred in 1.4% of individuals, including nausea/vomiting, transient hypotension without shock, injection site erythema, diffuse rash and mild epigastric pain. The limitation is that only immediate post procedure events were actively monitored. Conclusions, Use of intravenous fluorescein for gastrointestinal CLE appears to be safe with few acute complications. [source] Isothiocyanato Boron Dipyrromethenes,The First BODIPY Analogues of Fluorescein IsothiocyanatePHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2006Nela Malatesti ABSTRACT Two boron complexes of 5-phenyldipyrromethenes bearing isothiocyanate groups on the phenyl ring have been synthesized for the first time. The utility of these new fluorescence probes for labeling biologically relevant proteins is demonstrated on two monoclonal antibodies that bind to antigens overexpressed on cancer cells. Spectral comparison of the two structures reveals significant photophysical differences, including bathochromically shifted excitation and emission bands, increased molar absorptivity and a large increase in fluorescence quantum yield of approximately 10 times. Differences in photophysical parameters are linked to hindered rotation of the phenyl ring in one of the probes. [source] Reduction of Oxidative Stress in Bovine Spermatozoa During Flow Cytometric SortingREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2007P Klinc Contents The goal of the study was to investigate the effect of antioxidant supplementation on the quality of frozen-thawed flow cytometrically sorted bull spermatozoa. Twelve ejaculates from two Holstein Friesian bulls were sorted according to the Beltsville Sperm Sexing Technology. Each ejaculate was divided into three parts and processed as (i) unsorted controls, (ii) according to a standard sorting protocol and (iii) in the presence of different antioxidants (S-AO). Cooling and freezing of the samples were performed in the same way for all three groups, except that antioxidants were added to the TRIS-egg-yolk freezing extender for those semen samples that were already sorted in the presence of antioxidants. The semen quality in frozen-thawed samples was determined by morphology analysis immediately after thawing, motility estimation in a thermo-resistance test after 0, 6, 12 and 24 h incubation at 37°C and Fluorescein isothiocyanate conjugated PNA/propidium iodide (FITC-PNA/PI) staining after 0, 12 and 24 h of incubation at 37°C. There was a significantly higher (p < 0.05) percentage of motile spermatozoa in S-AO samples in comparison to unsorted frozen-thawed control at 0, 6 and 24 h after thawing and compared with normally sorted samples at all times after thawing. The percentage of damaged acrosomes was significantly lower (p < 0.05) in S-AO samples than in the unsorted controls (20.8 ± 6.9% vs 30.3 ± 12.0%). The percentage of morphologically abnormal spermatozoa in this group was significantly lower (p < 0.05) than in the unsorted controls and normally sorted samples (25.8 ± 5.2%, 36.0 ± 12.5% and 35.1 ± 7.4%, respectively). Analysis of frozen-thawed spermatozoa with FITC/PI revealed no significant difference in membrane integrity at 0 and 12 h after sorting, but after 24 h of incubation the S-AO samples had a significantly higher (p < 0.001) percentage of spermatozoa with intact membranes in comparison to unsorted controls and normally sorted semen (40.7 ± 6.3%, 7.8 ± 4.7% and 7.4 ± 4.6%, respectively). The percentage of acrosome-reacted spermatozoa was significantly lower (p < 0.05) in the S-AO samples than in the unsorted controls (14.1 ± 7.5%, 23.4 ± 5.4% and 28.8 ± 6.3% vs 25.9 ± 14.4%, 38.5 ± 16.7% and 79.8 ± 4.1%, for 0, 12 and 24 h after thawing, respectively) and in comparison to normally sorted semen 24 h after thawing (67.3 ± 10.0%). This study demonstrates the highly protective effects of antioxidants on the quality of flow cytometrically sorted frozen-thawed bull spermatozoa. [source] Synthesis and Application of Fluorescein- and Biotin-Labeled Molecular Probes for the Chemokine Receptor CXCR4CHEMBIOCHEM, Issue 7 2008Shinya Oishi Dr. Abstract The design, synthesis, and bioevaluation of fluorescence- and biotin-labeled CXCR4 antagonists are described. The modification of D -Lys8 at an ,-amino group in the peptide antagonist Ac-TZ14011 derived from polyphemusin II had no significant influence on the potent binding of the peptide to the CXCR4 receptor. The application of the labeled peptides in flow cytometry and confocal microscopy studies demonstrated the selectivity of their binding to the CXCR4 receptor, but not to CXCR7, which was recently reported to be another receptor for stromal cell-derived factor 1 (SDF-1)/CXCL12. [source] Luminescent Saccharide Biosensor by Using Lanthanide-Bound Lectin Labeled with FluoresceinCHEMBIOCHEM, Issue 8 2005Yoichiro Koshi A new luminescent biosensor for complicated glycoconjugates was engineered on the basis of a lanthanide-complexed sugar-binding protein (lectin) modified with a fluorophore. By using luminescence resonance energy transfer (LRET), ratiometric luminescent sensing can be carried out and successfully applied to a luminescent assay for an enzymatic trimming of a glycoprotein (see scheme). [source] Toluene diisocyanate enhances human bronchial epithelial cells' permeability partly through the vascular endothelial growth factor pathwayCLINICAL & EXPERIMENTAL ALLERGY, Issue 10 2009H. Zhao Summary Background Toluene diisocyanate (TDI) is a recognized chemical asthmogen; yet, the mechanisms of its toxicity have not been elucidated. Objective To investigate the influence of TDI on the permeability of human bronchial epithelial cell (HBE; HBE135-E6E7) monolayers in vitro, and the expression of vascular endothelial growth factor (VEGF) in these cells. Methods TDI,human serum albumin (HSA) conjugates were prepared by a modification of Son's method. Fluorescein isothiocyanate-labelled dextran and transmission electron microscopy were used to evaluate the effects of TDI,HSA on HBE135-E6E7 permeability. RT-PCR and ELISA were used to evaluate VEGF gene expression and protein release from HBE135-E6E7 cells stimulated by TDI,HSA. A VEGF-neutralizing antibody was used in monolayer permeability experiments to determine the role of the VEGF pathway in this process. Results TDI,HSA significantly increased the permeability coefficients of HBE135-E6E7 monolayers (P<0.01). TDI,HSA treatment significantly increased the expression of VEGF165 and VEGF189 genes (P<0.01). ELISA showed that TDI significantly induces VEGF release from HBE135-E6E7 cells. Cells treated with TDI,HSA and VEGF-neutralizing antibody had significantly lower permeability coefficients than cells treated with TDI,HSA only (P<0.01), but still significantly higher than control cells (P<0.01). Cells treated with TDI,HSA had fewer tight junctions (TJs) than control and HSA-treated cells, and addition of the anti-VEGF antibody did not restore the original number of TJs. Conclusion TDI increases the permeability of HBE cell monolayers, partly through a VEGF-mediated pathway. This suggests the importance of VEGF in TDI-induced pulmonary diseases, but shows that other pathways may be involved in the pathogenic process. [source] Ring-Fluorinated Fluoresceins as an Organic Photosensitizer for Dye-Sensitized Solar Cells Using Nanocrystalline Zinc Oxide.CHEMINFORM, Issue 26 2006Kazumasa Funabiki Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source] Syntheses of Regioisomerically Pure 5- or 6-Halogenated Fluoresceins.CHEMINFORM, Issue 8 2004Guan-Sheng Jiao Abstract For Abstract see ChemInform Abstract in Full Text. [source] Solvent-Free Synthesis of Sulfonephthaleins, Sulfonefluoresceins and Fluoresceins under Microwave Irradiation.CHEMINFORM, Issue 23 2003Simon Cihelnik Abstract For Abstract see ChemInform Abstract in Full Text. [source] Fixed drug eruption due to sodium fluoresceinCONTACT DERMATITIS, Issue 3 2008E. Di Leo No abstract is available for this article. [source] Discrepancy in measuring CD4 expression on T-lymphocytes using fluorescein conjugates in comparison with unimolar CD4-phycoerythrin conjugates,,CYTOMETRY, Issue 6 2007Lili Wang Abstract Background: Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes. However, the results from the use of these different QFC methods vary considerably in the literature. To better identify the causes of these discrepancies, we measured CD4 expression using FITC and phycoerythrin (PE) conjugates to stain CYTO-TROLÔ Control Cells and T-lymphocytes in whole blood and isolated cell preparations. We further examined pH of the cellular microenvironment as a cause of discordant results obtained with the FITC conjugate. Methods: Calibration with Quantibrite PE-labeled microspheres and the use of unimolar CD4-PE conjugates provided direct measurement of the antibody bound per cell value (ABC) for CD4 expression on normal T-lymphocytes. Calibration for CD4-FITC monoclonal antibody (Mab) labeled CYTO-TROL Control Cells and normal T-lymphocytes was based on molecules of equivalent soluble fluorochrome (MESF) as determined by FITC-labeled microspheres traceable to NIST RM 8640. The MESF value for CD4-FITC Mab was determined that enabled the conversion of the MESF values obtained for CYTO-TROL cells to ABC. We investigated the likely pH change in the fluorescein microenvironments within FITC-labeled Mab and cells stained with FITC-labeled Mab using a pH sensitive indicator. Results: The mean ABC value for T-lymphocytes prepared from fresh whole blood using CD4-PE conjugate (48,321) was consistent with previous results, and it was much higher than the mean ABC using CD4-FITC Mab (22,156). The mean ABC value for CYTO-TROL cells using CD4-PE conjugate (43,090) was also higher than that using CD4-FITC conjugate (34,734), although the discrepancy was not as great. Further studies suggested the discrepancy in CYTO-TROL results may be accounted for by the low pH of the membrane microenvironment, but the greater discrepancy in T-lymphocytes could not be fully explained. Conclusion: CD4 expression on fresh normal whole blood samples and CYTO-TROL cells can be consistently quantified in ABC units using Quantibrite PE quantification beads and unimolar CD4-PE conjugates. Quantification with CD4-FITC conjugate is not as consistent, but may be improved by the use of CD4 T-cells as biological calibrators. This approximation is valid only for surface receptors with consensus ABC values measured by different QFC methods serving as biological standards. Published 2007 Wiley-Liss, Inc. [source] Near-infrared dyes for six-color immunophenotyping by laser scanning cytometryCYTOMETRY, Issue 3 2002Andreas O.H. Gerstner Abstract Background To adequately analyze the complexity of the immune system and reduce the required sample volume for immunophenotyping in general, more measurable colors for the discrimination of leukocyte subsets are necessary. Immunophenotyping by the laser scanning cytometer (LSC), a slide-based cytometric technology, combines cell detection based on multiple colors with their subsequent visualization without the need for physical cell sorting. In the present study, the filter setting of the LSC was adapted for the measurement of the far-red emitting dye cyanine 7 (Cy7), thereby increasing the number of measurable commercially available fluorochromes. Methods The optical filters of the LSC were replaced,photomultiplier (PMT) 3/allophycocyanin (APC): 740-nm dichroic long pass, and 670-/55-nm bandpass; PMT 4/Cy7: 810-/90-nm bandpass. Peripheral blood leukocytes were stained directly by fluorochrome-labeled antibodies or by indirect staining. The tandem dyes of Cy7 (phycoerythrin [PE]-Cy7, APC-Cy7) and the fluorochromes fluorescein isothiocyanate (FITC), PE, PE-Cy5, and APC were tested alone and in different combinations. Results With the new filter combination and tandem fluorochromes, Cy7 was measurable at 488-nm (argon laser) or 633-nm (helium-neon laser) excitation. Resolution was in the range of FITC for PE-Cy7 but approximately 30% lower for APC-Cy7; spillover into the respective donor fluorochrome channel for both tandem dyes was prominent. A six-color panel for leukocyte subtyping was designed. Conclusions With this adaptation, it is possible to measure the tandem conjugates PE-Cy7 and APC-Cy7. This new setup opens the way for six-color immunophenotyping by LSC. Cytometry 48:115,123, 2002. © 2002 Wiley-Liss, Inc. [source] Use of oral fluorescein angiography in the diagnosis of macular oedema within a diabetic retinopathy screening programmeDIABETIC MEDICINE, Issue 12 2001F. M. Razvi Abstract Aims To assess if oral fluorescein angiography (OFA) is a suitable screening method to detect macular oedema in diabetic retinopathy. Methods Eighty-four diabetic patients were included in the study. They were from a consecutive series of patients attending the diabetic eye-screening clinic, with retinopathy at the macula requiring ophthalmology assessment. All patients were subsequently examined in the eye hospital, by ophthalmologist slit lamp biomicroscopy assessment as the gold standard, followed by oral fluorescein angiography. Results This study indicates a sensitivity of 92% and specificity of 81%. Only 4.8% of patients developed a minor reaction to oral fluorescein; 84.5% of images were of good quality. Conclusions Oral fluorescein angiography is an efficient and highly sensitive tool for the detection of macular oedema. It can be used as an adjunct in the diabetic screening service to identify patients with oedema within a disc diameter of the macula. Ultimately it will ensure that only necessary and smaller numbers of patients are referred to ophthalmologists. Diabet. Med. 18, 1003,1006 (2001) [source] Dry film microchips for miniaturised separationsELECTROPHORESIS, Issue 24 2009Rosanne M. Guijt Abstract In this work microfluidic devices were made from the dry film photoresist Ordyl SY330, characterised by optical and electron microscopy and used for electrophoretic separations. A simple and fast microfabrication process was developed for the fabrication of channels that are 50,,m wide and 30,,m in height, requiring only the use of an office laminator, a hot plate, an exposure source and mask and an electric drill to make four microdevices in less than 1,h. The optical properties of the photoresist were studied and the resist showed significant absorbance below 370,nm and 570,630,nm, and had an optical transmission of 80% between 400 and 550,nm. Fluorescence emission over the region of maximum transmission was low allowing these devices to be used for fluorescence detection at 488/512,nm. Electrophoretic separation of APTS and three derivatised sugars was performed in 20,mM phosphate buffer, pH 2.5 with efficiencies of the three sugars of 40,000 plates (2,100,000,plates/m) within 30,s at a field strength of 500,V/cm. The simple fabrication process also allowed microchannels to be easily filled with chromatography particles before sealing, avoiding the challenging task of slurry packing, and the potential of these devices for liquid chromatography was demonstrated by the extraction of fluorescein onto anion exchange particles. [source] High-sensitivity detection of biological amines using fast Hadamard transform CE coupled with photolytic optical gatingELECTROPHORESIS, Issue 17 2007Kevin L. Braun Abstract Here, we report the first utilization of Hadamard transform CE (HTCE), a high-sensitivity, multiplexed CE technique, with photolytic optical gating sample injection of caged fluorescent labels for the detection of biologically important amines. Previous implementations of HTCE have relied upon photobleaching optical gating sample injection of fluorescent dyes. Photolysis of caged fluorescent labels reduces the fluorescence background, providing marked enhancements in sensitivity compared to photobleaching. Application of fast Hadamard transform CE (fHTCE) for fluorescein-based dyes yields a ten-fold higher sensitivity for photolytic injections compared to photobleaching injections, due primarily to the reduced fluorescent background provided by caged fluorescent dyes. Detection limits as low as 5,pM (ca. 18,molecules per injection event) were obtained with on-column LIF detection using fHTCE in less than 25,s, with the capacity for continuous, online separations. Detection limits for glutamate and aspartate below 150,pM (1,2,amol/injection event) were obtained using photolytic sample injection, with separation efficiencies exceeding 1×106,plates/m and total multiplexed separation times as low as 8,s. These results strongly support the feasibility of this approach for high-sensitivity dynamic chemical monitoring applications. [source] Design, characterization, and utilization of a fast fluorescence derivatization reaction utilizing o -phthaldialdehyde coupled with fluorescent thiolsELECTROPHORESIS, Issue 7 2007Suminda Hapuarachchi Abstract We have developed a chemical derivatization scheme for primary amines that couples the fast kinetic properties of o -phthaldialdehyde (OPA) with the photophysical properties of visible, high quantum yield, fluorescent dyes. In this reaction, OPA is used as a cross-linking reagent in the labeling reaction of primary amines in the presence of a fluorescent thiol, 5-((2-(and-3)- S -(acetylmercapto)succinoyl)amino)fluorescein (SAMSA fluorescein), thereby incorporating fluorescein (,,=,78,000,M,1, quantum yield of 0.98) into the isoindole product. Detection is based on excitation and emission of the incorporated fluorescein using the 488,nm laser line of an Ar+ laser rather than the UV-excited isoindole, thereby eliminating the UV light sources for detection. Using this method, we have quantitatively labeled biologically important primary amines in less than 10,s. Detection limits for analysis of glutamate, glycine, GABA, and taurine were less than 2,nM. We present the characterization of OPA/SAMSA-F reaction and the potential utility of the derivatization reaction for dynamic chemical monitoring of biologically relevant analytes using CE. [source] A simple light-emitted diode-induced fluorescence detector using optical fibers and a charged coupled device for direct and indirect capillary electrophoresis methodsELECTROPHORESIS, Issue 9 2006David Arráez-Román Abstract We constructed a simple fluorescence detector for both direct and indirect CE methods using a blue light-emitted diode (470,nm) as excitation source, a bifurcated optical fiber as a waveguide, and a CCD camera as a detector. The connection of all the components is fairly easy even for nonexperts and the use of a CCD camera improves the applicability of this detector compared to the others using PMTs because it permits the recording of 2-D electropherograms or phosphorescence measurements. This detector provides a compact, low cost, and rapid system for the determination of native fluorescence compounds which have high quantum yields by CE with direct fluorescence detection, showing an LOD of 2.6×10,6,M for fluorescein; the determination of fluorescence derivative compounds by CE with direct fluorescence detection, showing an LOD of 1.6×10,7,M for FITC-labeled 1,6-diaminohexane; and nonfluorescence compounds by CE with indirect fluorescence detection with an LOD of 2.7×10,6,M for gallic acid. [source] Determination of biogenic amines in HeLa cell lysate by 6-oxy-(N -succinimidyl acetate)-9-(2',methoxycarbonyl) fluorescein and micellar electrokinetic capillary chromatography with laser-induced fluorescence detectionELECTROPHORESIS, Issue 4 2006Liwei Cao Abstract An MEKC-LIF method using 6-oxy-(N -succinimidyl acetate)-9-(2'-methoxy-carbonyl) fluorescein (SAMF) newly synthesized in our lab as a labeling reagent for the separation and determination of eight typical biogenic amines was proposed. After careful study of the derivatization condition such as pH value, reagent concentration, temperature, and reaction time, derivatization reaction was accomplished as quickly as 10,min with stable yield. Optimal separation of SAMF-labeled amines was achieved with a running buffer (pH,9.3) containing 30,mM boric acid, 25,mM SDS, and 20%,v/v ACN. The proposed method allowed biogenic amines to be determined with LODs as low as 0.25,2.5,nmol/L and RSD values from 0.4 to 4.5%. The present method has been successfully used to monitor biogenic amines in HeLa cells and fish samples. This study exploits the potential of MEKC-LIF with SAMF labeling as a tool for monitoring biogenic amines involved in complex physiological and behavioral processes in various matrices. [source] Rapid and sensitive determination of phosphorus-containing amino acid herbicides in soil samples by capillary zone electrophoresis with diode laser-induced fluorescence detectionELECTROPHORESIS, Issue 23 2005Eva Orejuela Abstract A straightforward and sensitive method has been developed for the analysis of phosphorus-containing amino acid herbicides (glufosinate and aminomethylphosphonic acid, the major metabolite of glyphosate) in soil samples. For this purpose, the analytical features of two indocyanine fluorescent dyes, sulfoindocyanine succinimidyl ester (Cy5) and 1-ethyl-1-[5-(N -succinimidyl-oxycarbonyl)pentyl]-3,3,3,3-tetramethyl-indodicarbocyanine chloride, as labeling reagents for the determination of these herbicides by CZE with diode LIF detection were investigated. Practical aspects related to the labeling chemistry and CZE separation showed that the two probes behave similarly, Cy5 being the best choice for the determination of these herbicides on account of its higher sensitivity. The optimum procedure includes a derivatization step of the pesticides at 25°C for 30,min and direct injection to CZE analysis, which is conducted within about 14,min using ACN in the running buffer. The lowest detectable analyte concentration ranged from 0.025 to 0.18,µg/L with a precision of 3.6,5.4%. These results indicate that indocyanine fluorescence dyes are useful as rapid and sensitive labels for the determination of these herbicides when compared with typical fluorescein dyes such as FITC and 5-(4,6-dichloro- s -triazin-2-ylamino) fluorescein, because they provide faster labeling reactions even at room temperature and the excess of reagent practically does not interfere the determination. Finally, the Cy5 method was successfully applied to soil samples without a preliminary clean-up procedure, and the herbicides were measured without any interference from coexisting substances. The recoveries of these compounds in these samples at fortification levels of 100,500,ng/g were 90,93%. [source] Single cell manipulation, analytics, and label-free protein detection in microfluidic devices for systems nanobiologyELECTROPHORESIS, Issue 19 2005Wibke Hellmich Abstract Single cell analytics for proteomic analysis is considered a key method in the framework of systems nanobiology which allows a novel proteomics without being subjected to ensemble-averaging, cell-cycle, or cell-population effects. We are currently developing a single cell analytical method for protein fingerprinting combining a structured microfluidic device with latest optical laser technology for single cell manipulation (trapping and steering), free-solution electrophoretical protein separation, and (label-free) protein detection. In this paper we report on first results of this novel analytical device focusing on three main issues. First, single biological cells were trapped, injected, steered, and deposited by means of optical tweezers in a poly(dimethylsiloxane) microfluidic device and consecutively lysed with SDS at a predefined position. Second, separation and detection of fluorescent dyes, amino acids, and proteins were achieved with LIF detection in the visible (VIS) (488,nm) as well as in the deep UV (266,nm) spectral range for label-free, native protein detection. Minute concentrations of 100,fM injected fluorescein could be detected in the VIS and a first protein separation and label-free detection could be achieved in the UV spectral range. Third, first analytical experiments with single Sf9 insect cells (Spodoptera frugiperda) in a tailored microfluidic device exhibiting distinct electropherograms of a green fluorescent protein-construct proved the validity of the concept. Thus, the presented microfluidic concept allows novel and fascinating single cell experiments for systems nanobiology in the future. [source] Analytical potential of 6-oxy-(N -succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein for the determination of amino compounds by capillary electrophoresis with laser-induced fluorescence detectionELECTROPHORESIS, Issue 10 2005Liwei Cao Abstract The analytical potential of a fluorescein analogue, 6-oxy-(N -succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein (SAMF), for the first time synthesized in our laboratory, as a labeling reagent for the labeling and determination of amino compounds by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was investigated. Biogenic monoamines and amino acids were chosen as model analytes to evaluate the analytical possibilities of this approach. The derivatization conditions and separation parameters for the biogenic amines were optimized in detail. The derivatization was performed at 30°C for 6 min in boric acid buffer (pH 8.0). The derivatives were baseline-separated in 15 min with 25 mM boric acid running buffer (pH 9.0), containing 24 mM SDS and 12.5% v/v acetonitrile. The concentration detection limit for biogenic amines reaches 8×10,11 mol·L,1 (signal-to-noise ratio = 3). The application of CE in the analysis of the SAMF-derivatized amino acids was also exploited. The optimal running buffer for amino acids suggested that weak acidic background electrolyte offered better separation than the basic one. The proposed method was applied to the determination of biogenic amines in three different beer samples with satisfying recoveries varying from 92.8% to 104.8%. Finally, comparison of several fluorescein-based probes for amino compounds was discussed. With good labeling reaction, excellent photostability, pH-independent fluorescence (pH 4,9), and the resultant widely suited running buffer pH, SAMF has a great prospect in the determination of amino compounds in CE. [source] A polymeric master replication technology for mass fabrication of poly(dimethylsiloxane) microfluidic devicesELECTROPHORESIS, Issue 9 2005Hai-Fang Li Abstract A protocol of producing multiple polymeric masters from an original glass master mold has been developed, which enables the production of multiple poly(dimethylsiloxane) (PDMS)-based microfluidic devices in a low-cost and efficient manner. Standard wet-etching techniques were used to fabricate an original glass master with negative features, from which more than 50 polymethylmethacrylate (PMMA) positive replica masters were rapidly created using the thermal printing technique. The time to replicate each PMMA master was as short as 20 min. The PMMA replica masters have excellent structural features and could be used to cast PDMS devices for many times. An integration geometry designed for laser-induced fluorescence (LIF) detection, which contains normal deep microfluidic channels and a much deeper optical fiber channel, was successfully transferred into PDMS devices. The positive relief on seven PMMA replica masters is replicated with regard to the negative original glass master, with a depth average variation of 0.89% for 26 ,m deep microfluidic channels and 1.16% for the 90 ,m deep fiber channel. The imprinted positive relief in PMMA from master-to-master is reproducible with relative standard deviations (RSDs) of 1.06% for the maximum width and 0.46% for depth in terms of the separation channel. The PDMS devices fabricated from the PMMA replica masters were characterized and applied to the separation of a fluorescein isothiocyanate (FITC)-labeled epinephrine sample. [source] Microaffinity purification of proteins based on photolytic elution: Toward an efficient microbead affinity chromatography on a chipELECTROPHORESIS, Issue 3 2005Woo-Jae Chung Abstract A bead affinity chromatography system, which was based on the photolytic elution method, was integrated into a glass-silicon microchip to purify specific target proteins. CutiCore® beads, which were coupled with a photo-cleavable ligand, such as biotin and an RNA aptamer, were introduced into a filter chamber in the microchip. The protein mixture containing target protein labeled with fluorescein isothiocyanate (FITC) was then passed through the packed affinity beads in the microchamber by pressure-driven flow. During the process, the adsorbed protein on the bead was monitored by fluorescence. The concentrated target protein on the affinity bead was released by simple irradiation with UV light at a wavelength of 360 nm, and subsequently eluted with the phosphate buffer flow. The eluted target protein was quantitatively detected via the fluorescence intensity measurements at the downstream of the capillary connected to the outlet of the microchip. The microaffinity purification allowed for a successful method for the identification of specific target proteins from a protein mixture. In addition, the feasibility of this system for use as a diagnosis chip was demonstrated. [source] Integrated on-chip derivatization and electrophoresis for the rapid analysis of biogenic aminesELECTROPHORESIS, Issue 14 2004Nigel P. Beard Abstract We demonstrate the monolithic integration of a chemical reactor with a capillary electrophoresis device for the rapid and sensitive analysis of biogenic amines. Fluorescein isothiocyanate (FITC) is widely employed for the analysis of amino-group containing analytes. However, the slow reaction kinetics hinders the use of this dye for on-chip labeling applications. Other alternatives are available such as o -phthaldehyde (OPA), however, the inferior photophysical properties and the UV ,max present difficulties when using common excitation sources leading to a disparity in sensitivity. Consequently, we present for the first time the use of dichlorotriazine fluorescein (DTAF) as a superior in situ derivatizing agent for biogenic amines in microfluidic devices. The developed microdevice employs both hydrodynamic and electroosmotic flow, facilitating the creation of a polymeric microchip to perform both precolumn derivatization and electrophoretic analysis. The favorable photophysical properties of the DTAF and its fast reaction kinetics provide detection limits down to 1 nM and total analysis times (including on-chip mixing and reaction) of <60 s. The detection limits are two orders of magnitude lower than current limits obtained with both FITC and OPA. The optimized microdevice is also employed to probe biogenic amines in real samples. [source] Analysis of amino acids in individual human erythrocytes by capillary electrophoresis with electroporation for intracellular derivatization and laser-induced fluorescence detectionELECTROPHORESIS, Issue 3 2004Hua Zhang Abstract A method for monitoring amino acids in single erythrocytes is described. For intracellular derivatization, reagent fluorescein isothiocyanate (FITC) was introduced into living cells by electroporation. For an 8 ,m erythrocyte, the analytes were diluted by a factor of only 1.6. After completion of the derivatization reaction, a single cell was injected into the separation capillary tip and lysed there. The derivatized amino acids were separated by capillary electrophoresis, followed by laser-induced fluorescence detection. Nine amino acids were quantitatively determined, with amounts of amino acids ranging from 3.8,32 amol/single cell. [source] Highly sensitive and simple fluorescence staining of proteins in sodium dodecyl sulfate-polyacrylamide-based gels by using hydrophobic tail-mediated enhancement of fluorescein luminescenceELECTROPHORESIS, Issue 19-20 2003Chulhun Kang Abstract Fluorescein has an extremely low luminescence intensity in acidic aqueous media. However, when it was bound to proteins, subsequent increase of luminescence intensity took place. Furthermore, when a hydrophobic tail, such as aliphatic hydrocarbons, was introduced to fluorescein, more dramatic increase of luminescence intensity was observed upon binding to proteins. In the present study, by utilizing this luminescence enhancement, three hydrophobic fluorescein dyes (5-dodecanoyl amino fluorescein, 5-hexadecanoyl amino fluorescein, and 5-octadecanoyl amino fluorescein) were examined as noncovalent fluorescent stains of protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Effective incorporation of the dyes to proteins in gels was accomplished either simply by adding dyes at the protein fixation step, or by treating gels with a staining solution after the fixation. The sensitivity of this staining method using the fluorescein derivatives was approximately 1 ng/band for most proteins. For some cases, protein bands containing as low as 0.1 ng were successfully visualized. In addition, the detection sensitivity showed much less protein-to-protein variation than silver staining. This new staining method was also successfully applied to two-dimensional electrophoresis of rat brain proteins. Its overall sensitivity was comparable to that of silver staining. [source] | |||||||||||||||||||||||||||||||||||||