Showing papers on "Fluorescence spectrometry published in 1999"

Fluorescence spectroscopy of single biomolecules.

Abstract: Recent advances in single-molecule detection and single-molecule spectroscopy at room temperature by laser-induced fluorescence offer new tools for the study of individual macromolecules under physiological conditions. These tools relay conformational states, conformational dynamics, and activity of single biological molecules to physical observables, unmasked by ensemble averaging. Distributions and time trajectories of these observables can therefore be measured during a reaction without the impossible need to synchronize all the molecules in the ensemble. The progress in applying these tools to biological studies with the use of fluorophores that are site-specifically attached to macromolecules is reviewed.

A simple and precise method for measuring ammonium in marine and freshwater ecosystems

Abstract: The accurate measurement of ammonium concentrations is fundamental to understanding nitrogen biogeochemistry in aquatic ecosystems. Unfortunately, the commonly used indophenol blue method often yie...

PCR Detection of Bacteria in Seven Minutes

Abstract: A portable, real-time polymerase chain reaction (PCR) instrument, consisting of an array of microfabricated silicon reaction chambers with integrated optical detectors, analyzed samples of 5 to 500 bacteria cells in as little as 7 minutes. The analysis included cell lysis, PCR, detection of the PCR product with a target-specific fluorescence energy transfer probe, and automated alerting of positive signals by the software. Thermal cycle times of only 17 seconds, the fastest that could be achieved on the instrument, yielded good productivity at each cycle and indicated that still faster analysis could potentially be performed. The short run times were achieved because of efficient heating and cooling of the reactions, precision in which the target temperatures were reached, and the sensitive, dedicated mini-optical detection systems.

C60 and Water-Soluble Fullerene Derivatives as Antioxidants Against Radical-Initiated Lipid Peroxidation

Abstract: C60, vitamin E, and three C60 derivatives (polar 1 and water-soluble C3/D3C60s) were examined for their antioxidant effects on prevention of lipid peroxidation induced by superoxide and hydroxyl radicals The protection effect on lipid peroxidation was found to be in the sequence: C60 ≥ vitamin E > 1 > none, for liposoluble antioxidants, and C3C60 ≫ D3C60 > none, for water-soluble ones Fluorescence quenching of PyCH2COOH (Py = pyrene) by both C3- and D3C60s shows that the Stern−Volmer constant, KSV, is about the same for both quenchers in aqueous solution Upon addition of liposomes, the fluorescence quenching becomes more efficient: 5-fold higher in KSV for C3C60 than for D3C60 When Py(CH2)nCOOH (n = 1, 3, 5, 9, or 15) was incorporated in lipid membranes, the KSVs all were small and nearly equal for D3C60 but were quite large and different for C3C60 with the sequence: n = 1 < 3 < 5 < 9 < 15 The better protection effect of C3C60 on lipid peroxidation than that of D3C60 is attributed to its stronger

Photodetection of early human bladder cancer based on the fluorescence of 5-aminolaevulinic acid hexylester-induced protoporphyrin IX: a pilot study.

Abstract: Exogenous administration of 5-aminolaevulinic acid (ALA) is becoming widely used to enhance the endogenous synthesis of protoporphyrin IX (PpIX) in photodynamic therapy (PDT) and fluorescence photodetection (PD). Recently, results have shown that the chemical modification of ALA into its more lipophilic esters circumvents limitations of ALA-induced PpIX like shallow penetration depth into deep tissue layers and inhomogeneous biodistribution and enhances the total PpIX formation. The present clinical pilot study assesses the feasibility and the advantages of a topical ALA ester-based fluorescence photodetection in the human bladder. In this preliminary study 5-aminolaevulinic acid hexylester (h-ALA) solutions, containing concentrations ranging from 4 to 16 mM, were applied intravesically to 25 patients. Effects of time and drug dose on the resulting PpIX fluorescence level were determined in vivo with an optical fibre-based spectrofluorometer. Neither local nor systemic side-effects were observed for the applied conditions. All conditions used yielded a preferential PpIX accumulation in the neoplastic tissue. Our clinical investigations indicate that with h-ALA a twofold increase of PpIX fluorescence intensity can be observed using 20-fold lower concentrations as compared to ALA.

Photochromic reaction and fluorescence of dithienylethenes in the solid state

Abstract: Photochromic cyclization reaction efficiency and fluorescence of two dithienylethenes in two solid states, colloidal solutions and amorphous films, were studied. Colloidal solutions of dithienylethenes were prepared by dilution of concentrated methanol solution into hot water. The diameter of particles of the colloidal solutions was about 1 μm according to observation by a scanning electron microscope. Photochromic cyclization reaction efficiency of colloidal solution of cis-1,2-dicyano-1,2-dithienylethene (1) was as high as that of hexane solution of 1. An amorphous film of 1 also shows high efficiency. On the other hand, neither colloidal solution nor polycrystal of 2,3-bis(2,4,5-trimethyl-3-thienyl)maleic anhydride (2) was photochromic; only an amorphous film of 2 was colored very slightly by UV irradiation. For the dithienylethenes in the solid states, long-wavelength fluorescence which can be assigned to the colored closed-ring form was observed, while deeply colored hexane solution did not give any long-wavelength fluorescence. Fluorescence spectra and fluorescence decay curves indicated the existence of efficient energy transfer from the open-ring form to the closed-ring form of dithienylethenes in the solid states.

Two-Beam Cross-Correlation: A Method To Characterize Transport Phenomena in Micrometer-Sized Structures

Abstract: To determine flow properties, namely, the velocity and angle of the flow in microstructured channels, an experimental realization based on fluorescence correlation spectroscopy is described. For this purpose, two micrometer-sized spatially separated volume elements have been created. The cross-correlation signal from these has been recorded and evaluated mathematically. In addition to previous results, two-beam cross-correlation allows for fast and easy determination of even small (down to 200 μm/s) flow velocities, as well as simultaneous measurement of diffusion properties of single dye molecules within a rather short detection time of 5−100 s and an error rate of less than 20%. The spatial flow resolution is around 1−2 μm, limited by the diameter of the volume element. Furthermore, vectorial flow data can be obtained and evaluated. A discussion of the theoretical background and an experimental verification of the theoretical results is performed. The feasibility of fast and easy data processing is show...

Sunscreen application by photosensitive patients is inadequate for protection

Abstract: Photosensitive patients often comment that sunscreen products seem of little benefit. We used fluorescence spectroscopy to assess quantitatively their sunscreen application technique. A dose-response relationship for sunscreen skin surface thickness and fluorescence intensity was determined for an intrinsically fluorescent sunscreen, Neutrogena sun protection factor (SPF) 15. Ten women with long-standing photosensitivity conditions were asked to apply this sunscreen in the manner they would normally on a bright sunny day. Fluorescence measurements were taken from all unclothed body areas, comprising 17 sites of the head, neck, upper and lower limbs. Geometric regression analysis of the dose-response data showed a high level of correlation (r = 0.99) between sunscreen thickness and fluorescence intensity, allowing fluorescence measurements to be converted to an equivalent sunscreen thickness. The overall median sunscreen thickness was 0.5 mg/cm2, with median thicknesses of individual sites ranging from 0 to 1.2 mg/cm2. The most frequently missed sites were the posterior neck, lateral neck, temples and ears, all of which had median thicknesses of 0 mg/cm2. Hence, photosensitive patients fail to apply sunscreen in some prominently exposed sites, and use average thicknesses far less than the manufacturers' recommendation (2 mg/cm2). The level of protection is much lower than anticipated from the stated SPF of the product.

Interaction of a novel red-region fluorescent probe, Nile Blue, with DNA and its application to nucleic acids assay

Abstract: A novel fluorimetric method was developed for the rapid determination of DNA and RNA based on their quenching effect on the cationic red-region fluorescent dye Nile Blue (NB). In the investigation of the interaction of NB with DNA by steady-state polarization measurements, thermal denaturing study, determination of absorption and fluorescence characteristics, salt effect study and electrophoresis experiments, the results supported the suggestion that NB served as an intercalator to the stack base pairs of nucleic acids. Further evidence showed that the quenching could be ascribed to the static quenching mode. A binding constant of about 106 M–1 and a binding site size of about three base pairs were obtained by spectral methods. Under optimum conditions, the calibration curves for the determination of calf thymus DNA (CT DNA) and yeast RNA were linear over the ranges 3.0 ng mL–1–2.0 µg mL–1 and 27 ng mL–1–10 µg mL–1, respectively. The detection limits were 3.0 ng mL–1 for CT DNA and 27 ng mL–1 for RNA. The relative standard deviation (n = 6) was within 2.1% in the middle of the linear range. Interferences from some interesting co-existing substances in the determination of DNA were also examined.

Platelet membrane fluidity individuals at risk for Alzheimer's disease: a comparison of results from fluorescence spectroscopy and electron spin resonance spectroscopy.

Abstract: Rationale: Previous fluorescence studies employing 1,6-diphenyl-1,3,5-hexatriene (DPH) have revealed an increase in the fluidity of platelet membranes from individuals with Alzheimer’s disease (AD) and their first-degree relatives. This biophysical alteration has been reported to be relatively specific for the hydrocarbon core of platelet membranes, where DPH preferentially localizes; this effect is not reflected by the fluorescent reporter triethylamino-DPH, which labels membranes at the lipid-aqueous interface. Objective: The goal of this study was to explore the validity and reproducibility of these findings using an independent biophysical technique, electron spin resonance (ESR) spectroscopy. Methods: Platelet membranes prepared from first-degree relatives of patients with AD were labeled with DPH, or the spin-labeled fatty acid probes 5-doxylstearate (5-DS) and 12-doxylstearate (12-DS). These spin labeled probes provide an index of structural order at the respective depths of their nitroxide moieties in the membrane. The resulting preparations were examined by fluorescence and ESR spectroscopy. Results: Increased platelet membrane fluidity (PMF), as determined by the fluorescence anisotropy of DPH, was associated with only a modest reduction in the order parameter derived for 5-DS labeled membranes. In contrast, the mean order parameters derived from the paired samples labeled with 12-DS differed substantially from each other, and revealed decreased order (increased fluidity) in the hydrocarbon 12-C region where DPH preferentially localizes. Conclusions: These results provide an independent validation of the biophysical alterations of platelet membranes that are manifested by a subgroup of patients with AD and their first-degree relatives.

Enhanced synchronous spectrofluorometric determination of tetracycline in blood serum by chemometric analysis. Comparison of partial least-squares and hybrid linear analysis calibrations.

Abstract: Tetracycline has been determined in human serum samples by a combination of: (1) synchronous fluorescence spectra of whole sera treated with Mg2+, and (2) the multivariate calibration methods of partial least-squares (PLS-1) and a variant of the recently introduced hybrid linear analysis (HLA), which does not require the knowledge of pure-component spectra. The calibration set was designed with 50 sera spiked with concentrations of tetracycline in the range 0.0−4.0 μg mL-1. Studies concerning validation, precision, accuracy and figures of merit (selectivity, sensitivity and limit of determination) were also carried out. A novel wavelength-selection procedure was applied to minimize the effect of nonmodeled interferents present in serum samples containing bilirubin, triglycerides, and salicylate. Overall, the performance of the newly developed HLA approach seems to be better than that of PLS-1.

Quantification of bacterial polyhydroxyalkanoic acids by Nile red staining.

Abstract: The fluorescence properties of one chemically and seven biologically produced polyhydroxyalkanoic acids were investigated as film castings and in living cells respectively after staining with Nile red. All these polyesters show a similar fluorescence behaviour, revealing a clear fluorescence maximum at an excitation wavelength between 540 nm and 560 nm and an emission wavelength between 570 nm and 605 nm. This could be shown by the use of two-dimensional fluorescence spectroscopy and flow cytometry. The examination of native poly(3-hydroxybutyric acid), poly(3HB), granules isolated from cells of Ralstonia eutropha H16 showed that the addition of 6.0 μg Nile red is necessary for total staining of 1.0 mg granules. The fluorescence intensity at an excitation wavelength of 550 nm and an emission wavelength of 600 nm showed high correlation to the poly(3HB) concentration of grana suspensions at different grana concentrations. These results and the staining of cell suspensions during cultivation experiments revealed that Nile red has a high potential for the quantitative determination of hydrophobic bacterial polyhydroxyalkanoic acids.

Terbium and Rhodamine as Labels in a Homogeneous Time-resolved Fluorometric Energy Transfer Assay of the β Subunit of Human Chorionic Gonadotropin in Serum

Abstract: Background: Fluorescence resonance energy transfer (FRET) is a powerful tool in analytical chemistry. The aim of the present work was to use FRET to design a homogeneous immunoassay. Methods: We used a highly fluorescent terbium (Tb3+) chelate (donor) and the organic fluorochrome rhodamine (acceptor) combined with time-resolved detection of the acceptor emission in homogeneous assay format for the measurement of the β subunit of human chorionic gonadotropin (βhCG) in serum. We used two antibodies labeled with Tb3+ and rhodamine, respectively, recognizing different epitopes on βhCG. The close proximity between the labels in the immunocomplex permitted energy transfer between the pulse-excited Tb3+ donor (decay time >1 ms) and the acceptor rhodamine (decay time of 3.0 ns). The prolonged emission of donor-excited acceptor (energy transfer) was measured after the short-lived background and acceptor emissions had decayed. The emission of donor-excited rhodamine was measured at a wavelength of where the emission of unbound donor is minimal. Results: The energy transfer signal was directly proportional to the βhCG concentration in the sample. The limit of detection was 0.43 μg/L, and the assay was linear up to 200 μg/L. Total assay imprecision in the range 10–185 μg/L was between 7.5% and 2.8%. Conclusions: Although less sensitive than heterogeneous, dissociation-enhanced europium-based separation assays, the presented assay format has advantages such as speed and simplicity, which make the assay format ideal for assays requiring a high throughput.

Laser-induced fluorescence technique for the quantification of mixing in impinging jets

Abstract: A laser-induced fluorescence (LIF) technique is implemented to measure concentration profiles and mixing performance in two impinging jet geometries. Experimental results are reported for steady and unsteady (transitional) mixing of initially segregated miscible fluids. Flow structures are visualized by imaging concentration distributions at five vertical planes throughout the mixers. Mixing is quantified for each Reynolds number examined by calculating the overall intensity of segregation. Mixing performance varies substantially as a function of Reynolds number. Optimum operating conditions are identified for both impinging jet geometries. The results demonstrate the ability of laser-induced fluorescence to quantitatively capture small- and large-scale flow structures and accurately and reproducibly quantify mixing performance in real time for industrially-relevant mixing devices. The LIF technique proves to be an accurate and versatile method to quantify mixing performance of miscible fluids.

Detection of bioaerosols using multiwavelength UV fluorescence spectroscopy

Abstract: This article describes the development of an aerosol generation apparatus to investigate the fluorescence spectra of bioaerosols. The experimental system was set up in a Biosafety Level II Laboratory. The system included an aerosol generator, chamber, aerosol monitoring instrumentation, and laser-induced-fluorescence detection system. The aerosol generators, chamber, and monitors were housed in an enclosure with the exhaust vented through a double HEPA filtration system. A Hospitak nebulizer using aqueous suspensions generated aerosols of bacteria. Aerosols of pollens were generated using a small-scale dry powder generator. The aerosol chamber, with four windows for optical access, was designed with the aid of a computational fluid dynamics code to optimize the generation of aerosol beams with a well-defined geometry for reproducible fluorescence measurements. Aerosol concentrations and aerodynamic diameters in the chamber were determined using a filter, an impinger, a modified Andersen impactor, and an A...

Spectrofluorimetric determination of piroxicam in the presence and absence of β-cyclodextrin

Abstract: The complexation between β-cyclodextrin (β-CD) and piroxicam (PX) was investigated by both fluorescence and absorption spectrometry. A 1:2 guest:host stoichiometry for the complex was established, and its association constant was calculated by applying a non-linear regression method to the changes brought about by the presence of β-CD in both the fluorescence and absorbance spectra of PX. During the study of the influence of the pH on the fluorescence emission of the complex, an efficient enhancement of the signals at acidic pH was observed. This suggests that the protonated form of PX is included more effectively than the ionized form in the β-CD cavity. Based on the results obtained, spectrofluorimetric methods for the determination of PX were developed. The best limits of detection and quantification were obtained using β-CD at an acidic pH. The dynamic range in this latter case was 0.02–1 µg ml–1. The method was applied satisfactorily to the determination of piroxicam in a pharmaceutical preparation.

Use of Real-Time PCR and Fluorimetry To Detect Lamivudine Resistance-Associated Mutations in Hepatitis B Virus

Abstract: Very rapid amplification of DNA by PCR in small volumes can be continuously monitored by the detection of the binding of probes with a rapid cycler with built-in fluorometric detection. Primers were designed to amplify approximately 100 bp of the polymerase gene of hepatitis B virus (HBV) spanning codon 550, where mutations associated with resistance to lamivudine invariably occur. Four hybridization probes were synthesized: one was 3′ labelled with fluorescein and hybridized upstream of codon 550. The others were 5′ labelled with Cy5 and 3′ labelled with biotin and spanned codon 550. The Cy5-labelled oligonucleotides contained either wild-type (ATG) or mutant (GTG or ATT) sequences. A Cy5-labelled probe and either the fluorescein-labelled probe or Sybr Green 1 (a compound that fluoresces when bound to double-stranded DNA) were included in each PCR. After completion of the amplification by using a LightCycler (Idaho Technology), the temperature at which the Cy5 probe melted from the product was determined in a melt program that took ca. 3 min. Pre- and posttreatment samples from eight patients (five chronic and three transplant) who failed lamivudine treatment were amplified, and the presence of mutations in codon 550 was determined by ABI sequencing and by using the LightCycler; in some cases PCR products were also cloned, and multiple clones were sequenced. Concordant results were obtained in all cases. We found the LightCycler to be better at resolving the sequences of genomic mixtures; for example, two samples showed a sequence at codon 550 of (A/G)T(G/T), which was found by fluorimetry to be mixtures of GTG and ATT but no ATG, and this finding was confirmed by the sequencing of clones. However, this approach was not more sensitive than population sequencing for the detection of the presence of mixtures. Overall, this pilot study has demonstrated an approach that could be an extremely rapid and economical method for the detection of lamivudine resistance-associated mutations in HBV.