Identification of fluorescent-antibody labeled group a streptococci by fluorometry.
[This corrects the article on p. 386 in vol. 14.].
Journal Article↗
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Multifrequency phase and modulation fluorometry.
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Kinetics↗
Fluorometry of selenium in human hair, urine and blood. A single-tube process for submicrogram determination of selenium.
This communication introduces a special lyophilization process for selenium determination by fluorometric methods. It permits a small sample volume, with several modifications including a single test tube process. Samples and standard are lyophilized first, then digested with nitric-perchloric acid mixture in a heated sand bath. Selenium is complexed with 2,3-diaminonaphthalene and extracted by n-hexane in the same test tube. The n-hexane layer is transferred to a cuvette and measured fluorometrically. Selenium concentration in healthy children from Long Island (aged 5-18 years) was: hair 0.765 +/- 0.114 microgram/g (n = 52), urine 28.65 +/- 8.27 micrograms/g creatinine (n = 66), and serum 95.4 +/- 14.4 ng/ml (n = 44). The current literature reflects an increase in the role of selenium in human nutrition. Thus, a simple but reliable method for determination of selenium in biological materials is needed in the clinical and research laboratory.
Adolescent↗
Measurement of metallothionein in rat tissues by a chromatographic method using the fluorometry of thiol groups.
The estimation of metallothionein (MT) levels in a biological sample is required to study the physiological role or the induction of MT, which is a multifunctional protein. MT contains 61 amino acids with 20 homologous cysteines. The high selectivity, sensitivity and stability of N-[4-(dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM) for the determination of SH groups was very useful for the determination of MT. The detection limit of MT by this method was 10 ng/0.5 ml. This method made it possible to measure MT levels in normal tissues in combination with gel filtration chromatography. The age-dependent change in MT levels in normal rat tissues was also investigated by the present method. MT levels in normal rat tissues vary with age. It is thought that this fact is correlated with the biological roles of MT.
Aging↗
Determination of acetaldehyde in human blood by high-performance liquid chromatography using fluorometry.
A method was developed for the measurement of acetaldehyde in human blood by high-performance liquid chromatography (HPLC). The method was based on a pre-column reaction; a fluorescent substance was formed by a coupling reaction between 2 mol of cyclohexan-1,3-dione and 1 mol of acetaldehyde with ammonium acetate. The coupling compound was analyzed by HPLC and the concentration of acetaldehyde was obtained from the calibration curve drawn from the results using standard solutions. The determination was sensitive and reproducible with a range of 0.2-10 microM and a precision (coefficient of variation) of 2.43%.
Acetaldehyde↗
Use of fluorometry for determination of skim milk powder adulteration in fresh milk.
A FAST (fluorescence of advanced Maillard products and Soluble Tryptophan) method for identification of reconstituted milk made from skim milk powder in the fresh milk was developed. Considering milk and skim milk powders variations from different seasons and countries, milk was collected from different dairy farms in different seasons and skim milk powders were collected from different countries to measure the Tryptophan (Trp), advanced Maillard products (AMP) fluorescence values. The results showed that there were differences (P<0.01) between raw and reconstituted milk. The plot of values in each mixed level of raw and reconstituted milk had a correlation coefficient >0.97. The FAST method is a simple, rapid, low-cost and sensitive method enabling the detection of 5% reconstituted milk in fresh milk. The measurement of the Trp, AMP fluorescence values and calculation of the FAST index is a suitable method for large-scale monitoring of fresh milk samples.
Animals↗
On-line estimation of kinetic parameters in anaerobic digestion using observer-based estimators and multiwavelength fluorometry.
Observer-based estimators (OBE) were used for estimation of state variables and kinetic parameters in an anaerobic digestion (AD) process. A simplified first-order model with time-varying kinetic parameters was used to design an OBE for kinetic parameter estimation. This approach was validated on a laboratory-scale anaerobic reactor equipped with a multiwavelength fluorometer for on-line measurements of COD and VFA concentrations in the reactor effluent. The proposed estimators provide continuous adjustment of kinetic parameters and can be used for predictions of state variables between samples acquisition and during sensor failure.
Bacteria, Anaerobic↗
Poor mixing of microdrops with the tear fluid reduces the accuracy of tear flow estimates by fluorometry.
The initial rapid fall in tear film fluorescence after instilling a 1 microliter drop of fluorophore could be a result of stimulated tear flow or of distribution of the dye throughout the tear fluid present in the conjunctival sac. An attempt was made to decide this by comparing the fall in fluorescence of two different fluorophores instilled at an interval of several minutes. In fact, very few patterns of initial rapid loss were found, but it was noted that there was usually a strong gradient of fluorescence from side to side across the cornea that took several minutes to dissipate. It seems that lateral mixing of a microdrop with the tear fluid is not instantaneous and that caution should be exercised in interpreting such measurements to estimate the total volume of tear fluid.
Conjunctiva↗
Viscosity and order in erythrocyte membranes studied with nanosecond fluorometry.
The viscosity and the order in the interior of human erythrocyte membranes were investigated by the fluorescence depolarization technique in the nanosecond region with 1,6-diphenyl-1,3,5-hexatriene (DPH). After pulsed excitation with a polarized light, the fluorescence anisotropy ratio of DPH in membranes rapidly decreased and gave a final value (r infinity). The rate of initial decrease and the value of r infinity related to the viscosity in the interior of the membranes and a wobbling angle of DPH which reflects a size of range for the phospholipid motion relating to the order of membrane structure. For normal human erythrocyte membranes the viscosity and the wobbling angle were obtained to be 0.82 poise and 42 degrees, at 37 degrees C. Similar values were obtained for spectrin-free membranes. Hardened membranes by the cross-linking of the cytoskeletal proteins with glutaraldehyde showed a small wobbling angle of 37 degrees, but the viscosity of them was unchanged.
Blood Viscosity↗
Fluorescence spectral properties of the anticancer drug topotecan by steady-state and frequency domain fluorometry with one-photon and multi-photon excitation.
Topotecan is an antitumor agent with activity against a variety of cancers. We examined the steady-state and time-resolved fluorescence spectral properties of topotecan with one- and two-photon excitation. Topotecan was found to display a high two-photon cross section near 20 GM for wavelengths within the fundamental output of a Ti:sapphire laser, 800-880 nm. In frozen solution the anisotropies of topotecan are near the theoretical maxima for one-photon and two-photon excitation with colinear electronic transitions. The intensity and anisotropy decays of topotecan fluorescence were found to be homogeneous (single exponentials) in phosphate-buffered saline and propylene glycol. The steady-state and time-resolved data indicate that topotecan binds to a double-helical DNA oligomer d(AT)10 resulting in increased anisotropies and multiexponential intensity and anisotropy decays. Subnanosecond components in the anisotropy decay of the DNA-topotecan complex suggest loose binding of the drug to DNA. Loose binding of topotecan to DNA is also revealed by accessibility of topotecan to collisional quenching by iodide.
Antineoplastic Agents↗
Fluorometers dissected. Fluorometry offers lower detection limits for various applications
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Journal Article↗
Solid-phase PCR with hybridization and time-resolved fluorometry for detection of HLA-B27.
BACKGROUND: Preactivated solid surfaces provide new possibilities for multiple consecutive reactions in a microtiter plate format. In this study, a combination of PCR and subsequent hybridization in the same microtiter well was applied for the detection of HLA-B27 alleles. METHODS: A multiplex solid-phase PCR to amplify the HLA-B27 alleles together with beta-actin as an amplification control gene was performed on the NucleoLink (Nunc) surface. PCR was followed by hybridization and detection with time-resolved fluorescence. For the covalent capture of the PCR primers onto the solid support via a 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride-mediated reaction, different 5'-end modifications of oligonucleotides were tested [amination, phosphorylation, and a poly(dT)10 linker]. RESULTS: For covalent immobilization of the primers, amination of the 5' end combined with use of the poly(dT)10 linker was superior. At least 19.5% of the primer added per well was attached via a stable bond. When the standard time-resolved, fluorescence-based HLA-B27 detection system was compared with the newly developed method in a sample series of 82 genomic DNAs and the corresponding dried-blood spots, all results were in full agreement. CONCLUSIONS: The new solid-phase PCR approach can be applied for multiple-target DNA detection. PCR followed by hybridization can be accomplished in a few hours using precoated strips and dried-blood spot PCR templates.
5' Untranslated Regions↗
[Study on the EMF-temperature co-effects on protein conformation by fluorometry].
The bioelectromagnetic effects are wildly concerned for a long time, and related researches are conducted in all kinds of directions in recent years. The EMF-temperature co-effects are more interesting nowadays. By studying the effects of EMF co-operated with temperature on a protein, an irreversible protein denaturation is found under the processing of EMF, and this denaturation is also Arrhenius-rule-obeyed. In addition, a protein denaturation model under the EMF-temperature co-effects is built. In this paper the EMF-temperature co-effects are explained in a way of molecular reacting kinetics, and the athermal effects of EMF are also discussed to some degree.
Electromagnetic Fields↗
Fluorometry in the Department of Medical Chemistry.
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Chemistry Techniques, Analytical↗
Frequency-domain lifetime fluorometry of double-labeled creatine kinase.
Myofibril-bound creatine kinase EC 2.7.3.2 (CK), a key enzyme of muscle energy metabolism, has been selected for studies of conformational changes that underlie the cellular control of enzyme activity. For fluorescence spectroscopy measurements, the CK molecule was double-labeled with IAF (5-iodoacetamidofluorescein) and ErITC (erythrosin 5'-isothiocyanate). Measurement of fluorescence resonance energy transfer (FRET) from fluorescein to erythrosin was used to obtain information about the donor-acceptor pair distance. Frequency-domain lifetime measurements evaluate the donor-acceptor distance in the native CK molecule as 7.8 nm. The Förster radius equals 5.3 nm with the resolution range from 0.2 to 1.0 nm. Erythrosin-fluorescein labeling (EFL) was tested for artificial conformational changes of the CK molecule with high-salt concentration treatment. The transition distance, defined by His-97 and Cys-283 and derived from a 3D model equals 0.766 nm for the open (inactive) form and 0.277 nm for the closed (reactive) form of the CK molecule. In this way, the resolution range of the used spectroscopy method is significant, concerning the difference of 0.489 nm. Nevertheless, the CK enzyme activity, assessed by the hexokinase-coupled assay, was diminished down to 1 % of the activity of the native enzyme. EFL is suitable for description of conformational behavior implied from the regulation of creatine kinase. However, the observed inhibition restricts EFL to studies of conformational changes during natural catalytic activity.
Adenosine Triphosphate↗
[Use of Doppler fluorometry for the assessment of microcirculation in patients with severe mechanical trauma].
Described in the paper are issues related with studying the micro-hemocirculation in victims with mechanical shock-genesis trauma by using the laser Doppler fluorimetry. The results are indicative of a nature of changes occurred in the microcirculation system in such victims with different-severity traumatic shock. It is concluded that, in principle, it is possible to use the method in question within the diagnostics of the impaired microcirculation in patients with shock-genesis lesions at disease exacerbation.
Adult↗
[Study on the double fluorescers fluorometry and its application].
Butylrhodamine B (BRB) was produced and entered into aqueous phase when pH=8.7 buffer solution was added to toluene extractive solution of GaCl4BRB+. And then BRB2 x TBF was extractred by tetrabromofluorescein (TBF). N-butanol (toluene:n-butanol 1:1) was added to toluene extractive solution of BRB2 x TBF. The sensitivity was raised because of the assistant effect of BRB2 x TBF. The detection limit was 0.1 microg/L. Beer's law was obeyed in the range of 0-0.15 mg/L with a relative standard deviation of 3.9% for 0.0027% gallium in metal and the recoveries were 94.7%-105.2%.
English Abstract↗
Fluorometry of citrate in serum, with use of citrate (pro-3S)-lyase.
We describe a procedure for enzymatic assay of citrate in human serum. The citrate is degraded to acetate and oxaloacetate with citrate oxaloacetate-lyase (pro-3S-CH2-COO- yields acetate) (EC 4.1.3.6). Some oxaloacetate loses CO2 to form pyruvate. Addition of malate and lactate dehydrogenases (EC 1.1.1.37 and 1.1.1.27) permits determination of the oxaloacetate and pyruvate generated, and thus of the citrate concentration. The decrease in NADH concentration is measured fluorometrically. Results obtained for 30 consecutive human sera by this procedure were compared to the procedure in which the citrate is converted to pentabromoacetone. There was no statistically significant difference in values obtained by the two procedures. The range of values (mean plus or minus 2 SD) found for sera from 25 blood donors by this procedure was 12.8-27.2 mg/liter (mean, 19.0 mg/liter). Serum citrate as measured by both procedures during a glucose tolerance test was decreased from initial values under the influence of administered glucose (and endogenous insulin). Insulin concentrations were also measured during these glucose-tolerance tests. Citrate concentrations remain subnormal after the glucose and insulin concentrations return to their initial values. This accords with published reports.
Acetone↗