Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Fluorometry”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources
PubMed1992

Detection of mutation delta F508 in the cystic fibrosis gene using allele-specific PCR primers and time-resolved fluorometry.

A method to detect the main cystic fibrosis (CF) mutation delta F508 from dried blood spots, whole blood, or saliva using the polymerase chain reaction (PCR) and time-resolved fluorometry (TRF) is described. Samples are treated by boiling in mild alkaline solution, after which two allele-specific PCR reactions are performed. Allele-specific primers and a common biotinylated primer are used in the amplification reactions. To detect the PCR product, an europium-labeled oligonucleotide, complementary to the biotinylated strand of the PCR product, is used in a solution hybridization. Hybridization is done in streptavidin-coated microtitration wells, making the detection easy to perform. After a washing step, the bound label is detected using a time-resolved fluorometer. To analyze function of the assay, 20 dried blood spot samples were tested. PCR amplification of the deletion region combined with gel retardation assay was used as a control method. In the initial testing, 2 samples giving discrepant results in the two assays were found. In addition, 17 samples from known CF patients together with 6 normal control samples were analyzed. Among these patient samples, 10 homozygotes and 6 carriers for mutation delta F508 were found.

Alleles↗
PubMed1995

Potency of antibacterial drugs in milk as analysed by beta-glucuronidase-based fluorometry.

The potency of selected antibacterials on mastitis-causing Escherichia coli, Streptococcus agalactine and Streptococcus uberis in milk, whey and Iso-sensitest broth (ISB) was compared, based on the suppression of bacterial beta-glucuronidase production. The beta-glucuronidase activity in the samples was analysed by substrate-defined fluorometry where the turbidity of milk does not disturb the assay. In ISB, all four E. coli strains were susceptible to enrofloxacin and gentamicin, sulfadoxin-trimethoprim and tetracycline. S. agalactiae and S. uberis strains were susceptible in ISB to most of the antibacterials tested. The antibacterial potency of sulfadoxin-trimethoprim, tetracycline, novobiocin, gentamicin and enrofloxacin on E. coli and S. agalactiae were considerably decreased in milk as compared with that in ISB. However, S. uberis seemed to be more susceptible to antibacterials in milk or whey than in ISB. Regression analysis of the sigmoidal dose-response curves of sulfadoxin-trimethoprim showed that slopes of the linearized lines seemed to become less steep in milk than in the artificial broth medium, indicating a shift of the bactericidic effect in ISB towards a bacteriostatic effect in milk.

Animals↗
PubMed1993

Multifrequency cross-correlation phase fluorometry of chlorophyll a fluorescence in thylakoid and PSII-enriched membranes.

We present here a comparative study on the decay of chlorophyll (Chl) a fluorescence yield in thylakoid membranes and photosystem II (PSII)-enriched samples, measured with multifrequency cross-correlation phase fluorometry. These measurements confirm the general conclusions of Van Mieghem et al. (Biochim. Biophys. Acta 1100, 198-206, 1992), obtained with a flash method, on the effects of reduction of the primary quinone acceptor (QA) on Chl a fluorescence yield of PSII. Different states of the reaction centers of PSII were produced by: (1) pretreatment with sodium dithionite and methyl viologen followed by laser illumination: the doubly reduced QA (QAH2) centers; (2) with laser illumination or pretreatment with diuron: QA- centers; and (3) the addition of micromolar concentration of dichlorobenzoquinone (DCBQ): oxidized QA centers. The data were analyzed with Lorentzian distribution as well as with multiexponential fluorescence decay functions. The analysis with Lorentzian distribution function showed that upon formation of QA-, the major lifetime distribution peak shifted to longer lifetimes: from 0.25 ns to 1.66 ns (pea thylakoid membranes) and from 0.24 ns to 1.31 ns (core PSII). However, when QAH2 was formed, the lifetime distribution peaks shifted back to shorter lifetimes (0.57-0.77 ns) both in thylakoids and PSII membranes. Multiexponential analysis showed three lifetime components: fast (40-400 ps), middle (300-1500 ps) and slow (5-25 ns). When QA- was formed in PSII centers, the amplitude of the fast component decreased, but both the amplitude and the lifetime of the middle component increased severalfold.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorophyll↗
PubMed1994

Site-to-site diffusion in proteins as observed by energy transfer and frequency-domain fluorometry.

We report measurements of the site-to-site diffusion coefficients in proteins and model compounds, which were measured using time-dependent energy transfer and frequency-domain fluorometry. The possibility of measuring these diffusion coefficients were shown from simulations, which demonstrate that donor (D)-to-acceptor (A) diffusion alters the donor frequency response, and that this effect is observable in the presence of a distribution of donor-to-acceptor distances. For decay times typical of tryptophan fluorescence, the simulations indicate that D-A diffusion coefficients can be measured ranging from 10(-7) to 10(-5) cm2/s. This possibility was verified by studies of a methylene-chain linked D-A pair in solutions of varying viscosity. The D-A diffusion was also measured for two labeled peptides and two proteins, melittin and troponin I. In most cases we used global analysis of data sets obtained with varying amounts of collisional quenchers to vary the donor decay time. Unfolding of troponin I results in more rapid D-A diffusion, whereas for melittin more rapid diffusion was observed in the alpha-helical state but over a limited range of distances.

Diffusion↗
PubMed1996

Fluorescence study of the conformational properties of recombinant tick anticoagulant peptide (Ornithodorus moubata) using multifrequency phase fluorometry.

Steady-state and multifrequency phase fluorometry were used to characterize the conformational state and conformational dynamics of recombinant tick anticoagulant peptide (Ornithodorus moubata) (TAP). The TAP contains two tryptophan residues at positions 11 and 37. The fluorescence emission varies sigmoidally as a function of pH with a pKa of 6.01 +/- 0.07. This pH dependency suggests that tryptophan fluorescence is quenched by His43 at low pH. This is confirmed by modification of the histidine with diethylpyrocarbonate. At pH 9 the fluorescence decay is well described by a sum of three exponentials (0.52, 1.9 and 5.4 ns), which decrease all three at pH 4 (0.25, 1.61 and 4.4 ns). From the reactivity of the fluorescence lifetimes toward N-bromosuccinimide and from the calculation of the accessibility we can attribute the long lifetime to Trp11, the short one to Trp37 and the middle one to both. The anisotropy decay was resolved into two components of 3.85 ns and 0.27 ns at pH 4 and 4.5 ns and 0.6 ns at pH 9. The long anisotropy decay time corresponds to the rotational correlation time of the protein, the short one to local mobility of the tryptophan residues.

Animals↗
PubMed1995

Development of a sensitive chemiluminometric assay for the detection of beta-galactosidase in permeabilized coliform bacteria and comparison with fluorometry and colorimetry.

We developed a chemiluminometric assay of beta-galactosidase in coliform bacteria, using a phenylgalactose-substituted 1,2-dioxetane derivative as a substrate. Permeabilization of cells is required to ensure the efficient cellular uptake of this compound. By this method, one coliform seeded in 100 ml of sterile water can be detected after a 6- to 9-h propagation phase followed by a 45-min enzyme assay in the presence of polymyxin B. Compared with fluorometry and colorimetry, chemiluminometry afforded 4- and 1,000-fold increases in sensitivity and 1- and 6-h increases in the speed of detection, respectively.

Colorimetry↗
PubMed1966

Identification of fluorescent-antibody labeled group A streptococci by fluorometry.

A quantitative system, amenable to automation, for determining the presence of group A streptococci in broth culture is described. After separation from the broth, the cells' protein coats are removed by digestion with 0.1% trypsin, and they are then stained with anti-A fluorescent antibody (FA). Excess FA is removed, and bound FA is put into solution by dissociation with demineralized distilled water. The amount of FA bound to the cells is quantitated by fluorometry of the solution. The level of nonspecific staining is measured by staining the cells with fluorescein-conjugated normal rabbit globulin absorbed with group A cells, dissociating, and quantifying, as above. The two quantities are subtracted to measure specific binding of FA to group A cells. A clinical trial showed 92% agreement with microscopists.

Bacterial Proteins↗
PubMed2000

Time-resolved fluorometry PCR assay for rapid detection of herpes simplex virus in cerebrospinal fluid.

We have introduced a time-resolved fluorometry (TRF)-based microwell hybridization assay for PCR products in detection of herpes simplex virus (HSV) in cerebrospinal fluid (CSF) specimens. TRF is a sensitive nonradioactive detection technique which involves the use of lanthanide chelates as fluorescent labels. We used PCR primers from the glycoprotein D genes of HSV type 1 (HSV-1) and HSV-2. The biotinylated PCR products were collected on streptavidin-coated microtitration wells and hybridized with short oligonucleotide probes, europium labeled for HSV-1 and samarium labeled for HSV-2. The TRF results were obtained as counts per second and as signal-to-noise (S/N) ratios. The sensitivity of the assay was 0.1 infectious units (PFU) of HSV in CSF specimens, and the S/N values increased with the virus amount, up to 68.5 for 10(3) PFU of HSV-1 and to 58.5 for 10(3) PFU of HSV-2, allowing semiquantitation of HSV in CSF. The primers and probes recognized all the studied 48 HSV wild-type samples, with S/N ratios of 12.4 to 190 (HSV-1) and 5.1 to 248 (HSV-2). We tested CSF specimens, 100 for each HSV type, which were HSV PCR negative by Southern blot and 22 CSF specimens which were HSV-1 or -2 PCR blot positive. In the TRF test, the mean S/N ratio for the HSV-1-negative CSF was 1.37 (standard deviation [SD] = 0.513) and for the HSV-2-negative CSF it was 1.03 (SD = 0.098). The HSV-1 blot-positive CSF yielded S/N ratios of 3.6 to 85.9, and the HSV-2 blot-positive CSF yielded ratios from 1.9 to 13. Using the mean S/N ratio for negative CSF specimens + 3 SD as the cutoff yielded all the previously HSV-positive specimens as TRF positive. The TRF PCR assay for HSV in CSF specimens is a rapid and sensitive method, improves interpretation of PCR results, and is well suited for automation.

Central Nervous System Viral Diseases↗
PubMed2006

Patch fluorometry: shedding new light on ion channels.

Patch fluorometry has emerged as a new approach to the study of the structure-function relationship in membrane-embedded functional ion channels. Simultaneous fluorescent and electrical recordings are achieved from a small number of channels in a cell-free membrane patch, yielding high recording sensitivities. Further improvement of this approach should permit direct observation of the gating motion of a single-channel protein.

Animals↗
PubMed1983

Monitoring free flap perfusion by serial fluorometry.

An efficient method to evaluate the patency of microvascular anastomoses and to predict flap viability is crucial when free flaps are used in reconstructive surgery. We report a study of 60 neurovascular island flaps in rats where the nutrient artery and/or vein were transected and anastomosed. Fluorescein dye was injected immediately after anastomosis, and the uptake and elimination of dye were quantified at 5-minute intervals with the fiberoptic perfusion fluorometer. Analysis of dye delivery clearly identified six hypofluorescent flaps presumed to be ischemic. In five of these flaps, revision was attempted. Three evidenced improved fluorescein uptake and elimination on reinjection of dye. These flaps survived, whereas the other two necrosed. Of the 54 flaps deemed patent after the first injection, two evidenced poor fluorescence on reinjection 3 hours later. These flaps necrosed and evidenced venous thrombosis (which probably occurred during the 3 hours between the first and second injections). Fiberoptic fluorometry can be employed to assess a free flap immediately after vascular anastomosis and throughout the critical hours postoperatively. Assessment of uptake and elimination after single or serial injections reflects flap perfusion and predicts viability with a high degree of accuracy.

Animals↗
PubMed1998

The frequency of an inactivating point mutation (566C-->T) of the human follicle-stimulating hormone receptor gene in four populations using allele-specific hybridization and time-resolved fluorometry.

We have described previously in the Finnish population an inactivating point mutation (566C-->T) in the human FSH receptor (FSHR) gene. In women, this mutation causes hypergonadotropic ovarian failure with arrest of follicular maturation and infertility, whereas in men, there is variable suppression of spermatogenesis, but no absolute infertility. To determine whether the same FSHR mutation occurs in other populations, its frequency was determined in Finland, Switzerland, Denmark, and the Chinese population of Singapore. The mutation was screened for using genomic DNA extracted from whole blood or dried blood spots. Exon 7 of the FSHR gene was first amplified using a pair of biotinylated primers. The PCR products were then immobilized on streptavidin-coated microtitration wells and hybridized using short allele-specific oligonucleotide probes labeled with europium. Time-resolved fluorometry was used for europium signal detection. To test the reliability of this method, 40 isolated DNA samples and 35 dried blood spot samples were blindly tested for the 566C-->T FSHR mutation. The analyses yielded identical results with denaturing gradient gel electrophoresis and allele-specific restriction enzyme digestion of the same samples, thus demonstrating the reliability of the tested method. Automation of this procedure allows the screening of large numbers of samples, which was subsequently carried out to investigate the frequency of the 566C-->T mutation in the study populations. A total of 4981 samples from the above-mentioned 4 countries were analyzed. The frequency of the 566C-->T mutation was 0.96% for all Finnish samples (n=1976), with a strong enrichment of the mutant allele in the northeastern part of the country. Only 1 mutation carrier was identified in the samples from Switzerland (n=1162), whereas none was found in samples from Denmark (n=1094) and the Singapore Chinese (n=540). These results suggest that the 566C-->T mutation of the FSHR gene is enriched in Finland, but is uncommon in other populations.

Alleles↗
PubMed1980

Quantitative determination of clobazam in serum and urine by gas chromatography, thin layer chromatography and fluorometry.

The procedures available for determination of clobazam (Frisium, Hoechst) are gas chromatography, fluorometry, and thin-layer chromatography. The study presents detailed descriptions of analytical procedures appropriate for routine determinations in serum and urine, and results from human trials. Moreover, the physicochemical properties of clobazam, viz., solubility, distribution, and protein binding are given.

Anti-Anxiety Agents↗
PubMed1999

Diagnosis of enterovirus and rhinovirus infections by RT-PCR and time-resolved fluorometry with lanthanide chelate labeled probes.

Detection of enteroviruses and rhinoviruses has traditionally been based on laborious and time-consuming virus isolation. Recently, rapid and sensitive assays for detecting enterovirus and rhinovirus genomic sequences by reverse transcription-polymerase chain reaction (RT-PCR) have been introduced. An RT-PCR assay is described that amplifies both enteroviral and rhinoviral sequences, followed by liquid-phase hybridization carried out in a microtiter plate format. In the hybridization assay, amplicons are identified by enterovirus- or rhinovirus-specific probes carrying lanthanide chelate labels, which can be detected simultaneously by time-resolved fluorometry. The sensitivity and specificity of the RT-PCR-hybridization method were evaluated with a representative collection of enteroviruses and rhinoviruses and tested further its applicability to the clinical setting with cerebrospinal fluid samples and nasopharyngeal aspirates. The RT-PCR assay amplified all enteroviruses and rhinoviruses tested, and all but one amplicon gave a positive result in the subsequent hybridization assay. The RT-PCR-hybridization method was more sensitive than virus isolation for the detection of enteroviruses and rhinoviruses in the clinical samples. High sensitivity, rapidity, and easy performance make the assay suitable for the routine diagnosis of enterovirus and rhinovirus infections.

Animals↗
PubMed2001

[Determination of trace mercury in the liquorice by cold atomic fluorometry].

By applying the nondispersive apparatus with reduction and gasification, and the wet dissolving method, the trace mercury in liquorice is determined by cold atomic fluorometry. We studied the conditions that can affect the determined results. We compared two methods for cleaning the glass ware, and contrasted the working curve method with addition plotting method. We also studied the applicability of this experimental method. The recovery is 86.9%-110.8%, and the error is less than 13.1%. It proves the method is efficient and satisfactory.

Drug Contamination↗
PubMed1979

Rapid fluorometry of estrogens in nonpregnancy urine, with use of chloroform extraction and purification by anion-exchange chromatography.

We describe a new method of extraction and purification of estrogens in low concentration in urine, involving a rapid enzymic hydrolysis of a 10-mL sample, automatic extraction (in a tube) with CHCl3/ethyl acetate, purification by chromatography on a disposable "mini-column" of AG 1-X2, and fluorometry by continuous flow according to Itrich's procedure [Acta Endocrinol. (Copenhagen) 35, 34 (1960)]. Conditions of hydrolysis, extraction, and purification were studied. Within-day precision (CV) was 7.5%, the mean between-day precision 8.5%. The sensitivity was 6 microgram of total estrogens per liter of urine. The specificity was assessed particularly by comparison with the results obtained by Schöller et al. [Acta Endocrinol. (Copenhagen) 57, suppl. 107 (1966)] and by gas-chromatography-mass-spectrometry. The normal limits as calculated by this technique were identical to values reported by others. The technique is rapid; results are obtained in 3 h. It is quite suitable for routine determinations: 100 assays can be done by a team of three technicians in one working day.

Chloroform↗
PubMed1989

Constant infusion fluorometry to predict flap survival.

Prediction of surgical flap survival in a dog model was evaluated by using the technique of fluorometry. The model provides a graded variation of flap ischemia with tissue necrosis occurring at the distal end. The indicator sodium fluorescein was administered via the saphenous vein at a constant infusion rate for 20 minutes. Quantitative fluorescence measurements were obtained with surface illumination of the flap at 132 sites for 15 flaps in the five animals. Wash-in slopes and wash-out clearances, both measures of tissue perfusion, were compared with the survival of flap regions observed at seven days. The sensitivity and specificity of fluorometric wash-in and wash-out measurements were compared in their ability to predict flap survival. The study showed sensitivities of 100% and 95% and specificities of 97% and 86%, respectively for the two methods. The wash-in procedure with constant infusion may be preferable to the wash-out technique since it requires less time to complete. In addition, it avoids bolus injections thus reducing the risk of anaphylactoid reactions.

Animals↗