On-line Fluorescence Detection Research Articles

Analysis of tamoxifen–DNA adducts in endometrial explants by MS and 32P-postlabeling

The nonsteroidal antiestrogen tamoxifen increases the risk of endometrial cancer; however, the mechanism for the induction of these tumors is not known. Recently, Sharma et al. [Biochem. Biophys. Res. Commun. 307 (2003) 157], using high performance liquid chromatography (HPLC) with online postcolumn photochemical activation and fluorescence detection, reported the presence of ( E)-α-(deoxyguanosin- N 2-yl)tamoxifen in DNA from human endometrial explants incubated with tamoxifen. Inasmuch as the methodology used by these investigators does not allow unambiguous characterization of tamoxifen–DNA adducts, we have used two additional techniques (HPLC coupled with electrospray ionization tandem mass spectrometry and 32P-postlabeling analyses) to assay for the presence of tamoxifen–DNA adducts in the human endometrial explant DNA. Tamoxifen–DNA adducts were not detected by either method.

Rapid Analysis of Factor V Leiden Mutation by Membrane Mediated Restriction Digestion and Ultra-Thin Layer Gel Electrophoresis

Mutation of blood coagulation factor V at the protein C cleavage site (Leiden mutation) results in activated protein C resistancy and concomitant blood clotting. Individuals that carry this mutation have significantly increased risk of thrombic episodes. Using polymerase chain reaction/ restriction fragment length polymorphism (PCR/RFLP) analysis, single nucleotide polymorphisms (SNP) such as the factor V Leiden mutation can be readily detected. In this paper, we discuss the applicability of membrane mediated restriction endonuclease digestion in sub-microliter volumes, in conjunction with ultra-thin layer agarose gel electrophoresis for rapid genotyping of factor V Leiden. The amplified and restriction enzyme digested DNA fragments were fluorescently labeled with ethidium bromide during the electrophoresis separation process. Combining membrane mediated restriction digestion with ultra-thin layer agarose gel electrophoresis separation and sensitive on-line laser-induced fluorescence detection enabled completion both the digestion and electrophoresis analysis procedures in less than 15 minutes for up to 96 samples, thus, genotyping of factor V Leiden mutation with this new protocol can be accomplished in a sensitive and high throughput manner.

On-line analysis of CdSe nanoparticle formation in a continuous flow chip-based microreactor

The online analysis of cadmium selenide nanoparticle formation in continuous-flow microfluidic reactors is described. The as-produced particles exhibit sharp excitonic absorption and emission peaks (∼30–40 nm) with relatively high quantum efficiencies (∼10%). The mean size and dispersity of the particles, determined using on-line fluorescence detection, may be controlled by varying the reaction temperature and/or the flow rate. The microfluidic approach provides considerable control over nucleation/growth processes and is a promising strategy for the direct production of near-monodisperse nanoparticles without recourse to further size selection.

Determination of total folate in plant material by chemical conversion into para-aminobenzoic acid followed by high performance liquid chromatography combined with on-line postcolumn derivatization and fluorescence detection.

A procedure involving chemical conversion of all forms of folate present in plant material into para-aminobenzoic acid (PABA) and a liquid chromatographic-fluorimetric determination with on-line postcolumn derivatization is reported. All folates are cleaved with liberation of PABA by hydrogen peroxide followed by acid hydrolysis using concentrated hydrochloric acid (37%) at 110 degrees C for 6 h. The reaction yield for individual folates conversion to PABA ranged from 44.4 to 97.3%. PABA could be determined sensitively by on-line postcolumn derivatization with fluorescamine, the detection limit for PABA being 3.02 nM. On the basis of this principle, a method for the determination of total folate in plant material, including a purification step on an affinity column, is presented, which offers a sufficient sensitivity and selectivity for routine analysis of total folate in natural samples. The total folate contents of tomatoes, carrots, white cabbage, and spinach were determined, and the results were quite comparable to the data reported. The recovery of PABA and the comparison of total folate analysis in spinach on different occasions (over 6 months) are also reported. The method is reliable, universal for all folates, including polyglutamate and monoglutamate forms, and eliminates the need for a deconjugation step and multiple conversion reactions.

Separation and characterization of NOM by high-performance liquid chromatography and on-line three-dimensional excitation emission matrix fluorescence detection.

By using high-performance size exclusion chromatography (HPSEC) and reversed-phase high-performance liquid chromatography with on-line three-dimensional excitation emission matrix fluorescence detection, we measured fluorescence properties of natural organic matter (NOM) as a function of molecular size (MS) and polarity. The work was carried out with Suwannee River Fulvic Acid, Aldrich Humic Acid, and a naturally occurring river NOM sample. Significant differences in fluorescence maximum pattern were found as NOM was separated chromatographically based on MS and polarity. There existed a strong relationship between MS, fluorescence pattern, and polarity. Humic-, fulvic-, and protein-like fluorescence fractions had distinct hydrophilic/hydrophobic nature. The results suggest that HPSEC may be better for characterizing major fulvic-like fluorescence and smaller MS fractions but not those having humic- and protein-like fluorescence and larger MS, which may be strongly adsorbed onto the HPSEC column because of their hydrophobic nature. This study has significant implications for further understanding the nature of NOM and its complexation with trace metals.

Chromatographic behavior of cadmium in an ion-pair reversed-phase micro HPLC system and its application to the determination of bio-available cadmium in soil samples.

A novel reversed phase ion-pair micro HPLC system with on-line fluorescence detection has been developed systematically and studied for the determination of cadmium in its bio-available fractions of soil samples. In this system, a micro ODS column of 1.0 mm i.d.x150 mm length and a mobile phase containing 6 mmol L(-1) 8-hydroxyquinoline 5-sulphonic acid (HQS), 3 mmol L(-1) cetyltrimethylammonium bromide (CTMABr), 10 mmol L(-1) acetic acid-acetate buffer (pH 5.1) as well as 50% acetonitrile at 50 microL min(-1) flow rate were employed to determine cadmium with a 2 microL flow cell through its fluorescence at 518 nm under 338 nm excitation. Furthermore, the composition of Cd-HQS chelate formed on the column was confirmed to be [Cd(HQS)(2)](2-) through a log-log plot method, and then combined with the ion-pair reagent by the electrostatic force under the chromatographic condition proposed. With such a method, the detection limit of cadmium was 8.48 ng mL(-1) (3sigma) with 1 microL sample injection, and the linear range for the determination of cadmium was 30-800 ng mL(-1) (R(2)=0.992). This method has been successfully applied to determination of cadmium in its bio-available fractions of BCR-483 and soil samples without interference from other coexistent metal ions. The RSD (n=6) was less than 7.3%. The results were in agreement with the indicative value for BCR-483 and those for the soil samples obtained by ICP-MS with a pretreatment of bis(1,1,3,3-tetramethylbutyl) phosphinic acid extraction.

Validation of an algorithm for automatic quantification of nucleic acid copy numbers by real-time polymerase chain reaction

Real-time quantitative polymerase chain reaction (PCR) with on-line fluorescence detection has become an important technique not only for determination of the absolute or relative copy number of nucleic acids but also for mutation detection, which is usually done by measuring melting curves. Optimum assay conditions have been established for a variety of targets and experimental setups, but only limited attention has been directed to data evaluation and validation of the results. In this work, algorithms for the processing of real-time PCR data are evaluated for several target sequences ( p53, IGF-1, PAI-1, Factor VIIc) and compared to the results obtained by standard procedures. The algorithms are implemented in software called SoFAR, which allows fully automatic analysis of real-time PCR data obtained with a Roche LightCycler instrument. The software yields results with considerably increased precision and accuracy of quantifications. This is achieved mainly by the correction of amplification-independent signal trends and a robust fit of the exponential phase of the signal curves. The melting curve data are corrected for signal changes not due to the melting process and are smoothed by fitting cubic splines. Therefore, sensitivity, resolution, and accuracy of melting curve analyses are improved.

Trace determination of nitrated polycyclic aromatic hydrocarbons using liquid chromatography with on-line electrochemical reduction and fluorescence detection

An efficient clean-up procedure coupled with a high performance liquid chromatography (HPLC) with on-line electrochemical (EC) reduction and fluorescence detection (FLD) was developed to quantify nitrated polycyclic aromatic hydrocarbons (NPAHs) in the airborne particulate. In this process, NPAHs were extracted ultrasonically followed by analysis by using a reversed phase column with an aqueous eluent containing 70% aqueous acetonitrile and sodium monocholoroacetate as a buffer solution. The extraction efficiencies were above 83% for 1-nitropyrene and 1,3-dinitropyrene (1,3-DNP) 1,6-DNP, and 1,8-DNP, and calibration graphs were linear with very good correlation coefficients ( r>0.999) and the detection limits were in the range of 1.0–2.2 pg for dinitropyrenes and nitropyrene. The proposed method provides a relatively simple and convenient procedure for determining the NPAHs samples in airborne particulate.

Liquid Chromatography with On-Line Electrochemical Derivatization and Fluorescence Detection for the Determination of Phenols

A new methodological approach for the determination of monosubstituted phenols is described. After liquid chromatographic separation of the analytes, an on-line electrochemical derivatization is carried out and the reaction products are detected fluorometrically. Phenols are oxidized in the electrochemical cell to form fluorescent dimers and higher oligomers, which were identified by on-line electrochemistry/mass spectrometry. Major advantages of the proposed method include enhanced selectivity and sensitivity. Without prior enrichment of the analytes, limits of detection down to 2 x 10(-9) M (20 fmol) may be reached for selected phenols, e.g., for 4-octylphenol, 4-ethylphenol, and 4-(1-indanyl)phenol. Only readily available instrumentation is required for these measurements.

Photo‐Conversion of Methane into Higher Hydrocarbons Using 355 NM Laser Radiation

A laser‐based technique for the conversion of methane into higher hydrocarbons and hydrogen has been developed. This technique involves the photo‐dissociation of methane using a high power pulsed ultraviolet laser at 355 nm. The reaction products, such as CH, CH2, C2H2, atomic and molecular hydrogen, are characterized by real‐time laser induced fluorescence for the first time, to our knowledge. In addition to this online fluorescence detection of the species, gas chromatography is also applied to analyze the stable hydrocarbon products generated due to photo‐dissociation of methane. Another interesting result of the laser excitation of methane is the observation of Stimulated Raman lines (Stokes and Antistokes) observed in the 150–850 nm region, which is a manifestation of the inherent characteristics of the laser radiation such as high directionality and high intensity.

Phospholipase A2-Catalyzed Membrane Leakage Studied by Immobilized Liposome Chromatography with Online Fluorescent Detection

Unilamellar liposomes composed of phosphatidylcholine with an entrapped self-quenching fluorescent dye, calcein, were immobilized in chromatographic gel beads by avidin–biotin binding. Bee venom phospholipase A2 (PLA2) was applied in a small amount onto the immobilized liposome column. The release of calcein from the immobilized liposomes resulting from the catalyzed hydrolysis of the phospholipids was detected online by immobilized liposome chromatography (ILC) using a flow fluorescent detector. The PLA2-catalyzed membrane leakage of the immobilized liposomes as studied with ILC was found to be affected by the gel pore size used for immobilization, by liposome size, and as expected by the concentration of calcium, but was unaffected by the flow rate of ILC. The largest PLA2-induced calcein release from the liposome column was detected on large unilamellar liposomes immobilized on TSK G6000PW or Sephacryl S-1000 gel in the presence of 1 mM Ca2+ in the aqueous mobile phase. Comparison with the PLA2-catalyzed membrane leakage in free liposome suspensions, we conclude that the fluorescent leakage from liposomes hydrolyzed by PLA2 can be rapidly and sensitively detected by ILC runs using large amount of immobilized liposomes with entrapped fluorescent dye.

Quantification of antiandrogen effect determined by Lightcycler technology

During the last decade, the possible effects of xenobiotics on male reproductive health have resulted in great concern. More recently, evidence of antiandrogen effect in vivo by certain chemicals has been reported. The classical Hershberger in vivo assay determining organ weight changes can be improved by measuring hormone levels as well as determining changes in gene expression of androgen-responsive genes. A real-time RT-PCR method using LightCycler technology (Roche) suitable for quantitative determination of gene expression is described. The technique combines rapid thermocycling with online fluorescence detection of PCR product formation. In this study, investigation of expression of prostate specific binding protein polypeptide C3 (PBP C3) and testosterone-repressed prostatic message 2 (TRPM-2) in the ventral prostate was performed in 60-days-old castrated Wistar rats treated daily with testosterone with or without addition of flutamide or vinclozolin for 7 days in total. We show that we can quantify the level of gene expression by use of LightCycler technology, supported by changes in reproductive organ weights as well as in hormone levels, and that analysis of gene expression levels is an even more sensitive endpoint.

Quantitative real-time RT-PCR detection of breast cancer micrometastasis using a multigene marker panel.

Real-time RT-PCR is a relatively new technology that uses an online fluorescence detection system to determine gene expression levels. It has the potential to significantly improve detection of breast cancer metastasis by virtue of its exquisite sensitivity, high throughput capacity and quantitative readout system. To assess the utility of this technology in breast cancer staging, we determined the relative expression levels of 12 cancer-associated genes (mam, PIP, mamB, CEA, CK19, VEGF, erbB2, muc1, c-myc, p97, vim and Ki67) in 51 negative-control normal lymph nodes and in 17 histopathology-positive ALNs. We then performed a receiver operating characteristic (ROC) curve analysis to determine the sensitivity and specificity levels of each gene. Areas under the ROC curve indicated that the most accurate diagnostic markers were mam (99.6%), PIP (93.3%), CK19 (91.0%), mamB (87.9%), muc1 (81.5%) and CEA (79.4.0%). mam was overexpressed in 16 of 17 lymph nodes known to contain metastatic breast cancer at levels ranging from 22- to 2.8 x 10(5)-fold above normal mean expression, whereas PIP was overexpressed from 30- to 2.2 x 10(6)-fold above normal in 13 lymph nodes. Real-time RT-PCR analysis of pathology-negative LN from breast cancer patients revealed evidence of overexpression of PIP (6 nodes), mam (3 nodes) and CEA (1 node) in 8 of 21 nodes (38%). Our results provide evidence that mam, PIP, CK19, mamB, muc1 and CEA can be applied as a panel for detection of metastatic and occult micrometastatic disease.

Fluorescent labeling of a narrow polydispersity high molecular weight polystyrene

Narrow polydispersity polystyrene with a molecular weight of 1.03×106 has been functionalized with anthracene groups using the metallocene technique. Side reactions such as chain coupling and bond scission can be limited by working in THF at –75°C. Gel permeation chromatography combined with on-line viscometric, refractive index and fluorescence detection allows us to verify that degradation did not exceed 1 bond scission/180 000 monomer units. The degree of grafting increases with the reaction time but remains modest (< 5% after 7 h) at low temperature.

Experimental photodynamic laser therapy for rheumatoid arthritis with a second generation photosensitizer.

Photodynamic laser therapy has been shown to be a new method for the treatment of synovitis in various animal models. Its principle is the accumulation of a photosensitizing drug in the inflamed synovium which is destroyed by photoactivation of the drug. In the present animal study we demonstrate the effect of a second-generation photosensitizer and suggest a concept for light dosimetry within the joint. We used 38 inbred rabbits for the IgG-induced arthritis model; 2 mg/kg of the benzoporphyrin derivative monoacid ring-A (BPD-MA) Verteporfin were administered 3 h before irradiation, which was performed using a 690-nm diode laser coupled to quartz glass fiber with a cylinder diffusor tip at a total light energy of either 180 or 470 J. During irradiation specific fluorescence of BPD-MA was monitored using a spectroscopy unit. The effect of the photodynamic laser therapy was documented grossly and histologically after 1 week. Within the 470 J-group a complete necrosis of the inflamed synovium was observed. The bradytrophic structures of the joint, however, remained unchanged. Throughout the 180 J-group the extent of necrosis was minor. During irradiation the tissue fluorescence of BPD-MA showed a dose-dependent decrease. Using BPD-MA as a photosensitizer a highly selective and minimal invasive synoviorthesis can be performed. At a dose of 2 mg/kg the histological effect depends on the light dose. For optimum efficacy a total energy of 470 J seems favorable. Online fluorescence detection can be used to monitor the effect of light administration. For dosimetry therefore an online tissue fluorescence detection may represent a technical solution.

Determination of Harmane and Harmine in Human Blood Using Reversed-Phased High-Performance Liquid Chromatography and Fluorescence Detection

A number of tremorogenic β-carboline alkaloids have been found in common plant-derived foodstuffs, beverages, and inhaled substances. Because of their natural presence in the food chain, there is a growing concern regarding the potential risks of certain essential tremors associated with the long-term, low-level dietary exposure to these alkaloids. The purpose of this study was to develop an effective analytical method to determine blood levels of two major β-carboline derivatives, harmane and harmine. Human blood was extracted with ethyl acetate and methyl-t-butyl ether (2:98) under an alkaline condition. After evaporation of organic solvent, the samples were reconstructed in methanol. The samples were fractionated on a 250 × 4.6-mm C18 reversed-phase column with an isocratic mobile system consisting of 17.5 mM potassium phosphate buffer (ph 6.5) and methanol (30:70), followed by an on-line fluorescence detection. The method had the detection limit to determine 206 and 81 pg/ml of harmane and harmine, respectively, in 10 ml of human blood. The intraday precision (C.V.) at 25 ng/ml was less than 6.7 and 3.4% for harmane and harmine, respectively. The interday precision was 7.3% for harmane and 5.4% for harmine. The method has proven sensitive, reproducible, and thus useful for both laboratory and clinical studies of β-carboline toxicities.

On-line X-ray fluorescence detection for capillary electrophoresis separations

In these investigations, capillary electrophoresis with on-line X-ray fluorescence detection (CE-XRF) has been demonstrated for the first time. The insertion of a polyethylene sample cell between fused-silica capillary segments enabled continuous XRF detection during electrophoresis with minimal additional band broadening. Detection limits in the 10−4 M range are currently feasible for the CE-XRF separation of metal complexes, and design advances will enhance detectability to the 10−5–10−6 M range, permitting studies of important environmental and biological samples.

Continuous extraction with acidified subcritical water of arsenic, selenium and mercury from coal prior to on-line derivatisation-atomic fluorescence detection

Acidified subcritical water is proposed for the continuous extraction of minor pollutants (namely, selenium, arsenic and mercury) from coal prior to continuous derivatisation (by hydride formation for Se and As, and cold vapor formation for Hg) and determination by atomic fluorescence. Coal samples (3 g) were subjected to a 15 min static extraction followed by a 90 min dynamic extraction with water modified with 4% (v/v) HNO3 for both steps. An in-depth study of the variables affecting the continuous leaching step, as well as those referring both to preconcentration (for mercury), derivatisation and detection (all of them) was performed. The linear ranges of the calibration curves for all analytes were at the ng ml–1 level, with correlation coefficients, r2, better than 0.999 for Se and As and better than 0.99 for Hg. The method was validated by using a bituminous coal reference material (NIST SRM 1635). The good precision of the method [RSDs (n = 6) of 12.0, 4.7 and 6.5% for As, Se and Hg, respectively], together with its safety and rapidity make it a good alternative for the determination of these analytes in coal.

Analysis of recombinant cytokines in human body fluids by immunoaffinity capillary electrophoresis.

An immunoaffinity capillary electrophoresis (ICE) system for rapidly quantifying recombinant cytokines in human body fluids has been developed. Cytokines within biological fluids were labeled with a red light emitting fluorochrome and injected into the capillary. Selected cytokines were captured by immobilized antibodies on the internal surface of the capillary, and held while unbound materials were purged. The cytokines were then eluted electrophoretically in acidic buffer. Individual cytokine peaks were detected by on-line laser-induced fluorescence detection coupled to a computerized fiber-optic spectrometer, and analyzed by integration of the eluted peaks. The comparison of the results of ICE to routine assays used for these cytokines demonstrates that ICE provides a fast and accurate procedure for defining these cytokines in complex biological samples. Immunoaffinity separations can be used for any material to which a specific antibody can be raised, making this procedure applicable to a wide range of molecules of biomedical interest.

Real-time quantitative RT-PCR after laser-assisted cell picking.

The present study describes a technique for quantitation of mRNA in a few isotypic cells obtained from an intact organ structure by combining laser-assisted cell picking and real-time PCR. The microscopically controlled lasering of selected cells in stained tissue sections was applied to lung alveolar macrophages, which are unique in that they can alternatively be gathered as a pure cell population from intact lungs by bronchoalveolar lavage as a reference technique. TNF-alpha was chosen as the transcriptionally inducible target gene to be quantified in alveolar macrophages of control rat lung, as well as low- and high-challenge lungs stimulated by endotoxin and IFN-gamma nebulization. Online fluorescence detection for quantitation of the number of amplified copies was based on 5' nuclease activity of Taq polymerase cleaving a sequence-specific dual-labeled fluorogenic hybridization probe. A pseudogene-free sequence of PBGD served as an internal calibrator for comparative quantitation of target. A quick procedure and minimized loss of template were achieved by avoiding RNA extraction, DNase digestion and nested-PCR. Using this approach, we demonstrated dose-dependent manifold upregulation of the ratio of TNF-alpha mRNA copies per one copy of PBGD mRNA in alveolar macrophages of the challenged lungs. The quantitative data obtained from laser-picked alveolar macrophages were well matched with those of lavaged alveolar macrophages carried out in parallel. We suggest that this new combination of laser-assisted cell picking and real-time PCR has great promise for quantifying mRNA expression in a few single cells or oligocellular clusters in intact organs, allowing assessment of transcriptional regulation in defined cell populations.